Clinical Immunology_ Principles and Practice ( PDFDrive )

1014 Part seven Organ-Specific Inflammatory Disease


most frequent and significant gut manifestations, and these are agammaglobulinemia infrequently develop overt GI disease (they
discussed in detail below. However, a variety of inherited condi- lack B cells but have functionally intact T-cell populations). Even
tions of broad lymphocyte or innate immune cell dysfunction persons with selective IgA deficiency have little in the way of GI
can also lead to gut disease. The study of monogenic diseases disease, including infections. In fact, the T-cell dysfunction that
that are associated with inflammatory diseases of the gut have often accompanies CVID may contribute substantially to sus-
come to the attention of investigators, as they may be models ceptibility to GI disease.
for the mechanisms that could contribute to the pathophysiology In terms of an increased GI infection risk, autoimmune
of more genetically complex conditions, such as Crohn disease gastritis-induced achlorhydria could increase small-bowel
and UC. 68 exposure to swallowed commensals and pathogens escaping this
innate gastric barrier to infection. In addition, although the loss
Common Variable Immunodeficiency of secreted IgA alone is not enough to meaningfully increase
CVID is a syndrome of hypogammaglobulinemia (low levels of susceptibility to intestinal infections, perhaps the additional loss
IgG and IgA and/or IgM) accompanied by recurrent sinopul- of IgG and IgM and presence of T-cell dysfunction is more
monary infections (Chapter 34). Patients have no isohemag- permissive, but no mechanism for this has been established.
glutinins and cannot mount an adequate antibody response to Mucosal nodular lymphoid hyperplasia, characterized by disor-
many vaccine antigens. In general, Ig replacement therapy ganized secondary lymphoid nodules with poorly formed germinal
improves the sinopulmonary infection rate but does not affect centers, is likely related to the inability of B cells to undergo
other complications, such as autoimmune disease and GI class switching when presented with antigen in situ. Last, CVID
symptoms, including intestinal infections. Case series of patients enteropathy is characterized clinically by severe malabsorption
with CVID have shown that up to 60% of patients experience and histologically by blunted villi and increased intraepithelial
69
GI symptoms and that endoscopic and histological abnormalities lymphocytes, and epithelial apoptosis is associated with excess
70
can occur in the majority of patients with CVID. When consider- Th1 cytokine secretion (IL-12 and IFN-γ). It remains unknown
ing the differential diagnostic possibilities for the GI complications why this inflammatory lesion occurs in the first place.
of CVID, it is helpful to separate them into infectious, immune-
mediated, and neoplastic processes. Among the infectious agents, Diagnosis
Giardia lamblia, nontyphoidal Salmonella, and Campylobacter The workup of GI complications of CVID often takes place as
jejuni are frequently seen, but Cryptosporidium may also be found, symptoms are usually first episodic but may eventually turn into
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and C. difficile and viral agents (CMV) may be encountered. In chronic, progressive complaints. The key to successful diagnosis
addition to the higher rate of gastric H. pylori infection, small- is the initial search for treatable infectious causes. This requires
intestinal bacterial overgrowth (SIBO) is present in up to 30% stool assay and culture for bacterial (especially Campylobacter)
of patients with CVID. The immune-mediated GI complications and protozoal pathogens. In addition, hydrogen breath testing
of CVID include idiopathic enteropathy (villous atrophy, increased can help detect SIBO to provide another antibiotic-responsive
intraepithelial lymphocytes/microscopic colitis, nodular lymphoid etiology of the symptoms.
hyperplasia) (Fig. 75.1F) manifesting as severe malabsorption Negative results for infectious causes require endoscopy for
(see below) and, much less frequently, macroscopic ulcerating biopsy of the proximal small intestine and colon. Routine histology
69
disease resembling UC or Crohn disease. CVID-associated will detect the features of enteropathy, including blunted villi
autoimmune disease involving the GI tract also includes type II and increased IELs, which are often interpreted as celiac disease.
gastritis that can lead to achlorhydria and vitamin B 12 deficiency Unlike celiac disease, there usually is a lack of plasma cell infiltrate
(“pernicious anemia”) and even autoimmune hepatitis and and crypt hyperplasia, as well as a preserved brush border and
primary biliary cirrhosis. Last, neoplastic complications of Goblet cells, and there is often an increase in epithelial cell
72
intestinal lymphoma and gastric adenocarcinoma (related to apoptosis, particularly in the colon. In fact, some of the villus
achlorhydric autoimmune gastritis) have been reported. changes in CVID enteropathy can be mimicked by SIBO, which
should be investigated as described above. If the diagnosis of
Presentation celiac disease must be considered, then genetic testing for celiac
The most common symptom of GI complications of CVID is susceptibility HLA alleles should be performed; the absence of
episodic diarrhea that can progress to chronic diarrhea, regardless combinations of A and B alleles for HLA-DQ2 or -DQ8 that
of the etiology. Weight loss and evidence of vitamin or mineral confer risk will rule this out. However, even if celiac disease gene
deficiencies may occur, with underlying maldigestion or mal- alleles are detected, it still does not indicate that this is a gluten-
absorption. In addition, patients may present with abdominal driven celiac disease lesion but will require a trial of GFD
pain related to splenomegaly (with or without portal hyperten- nonetheless. The functional significance of any histological lesion
sion) or ascites (portal hypertension secondary to hepatic nodular in the small intestine can be evaluated by a measure of steatorrhea
regenerative hyperplasia [NRH] as a known complication of (fecal fat excretion) and small-bowel absorption (d-xylose
CVID). Fever, together with intestinal obstruction and GI bleeding, absorption test), values that can be used to track improvement
may indicate the development of small-bowel lymphoma. Patients following treatments.
with CVID may report increased frequency of GERD symptoms
as well as dyspepsia, but these complaints are nonspecific and Treatment
have been difficult to link with immune defects or autoimmune None of the GI complications of CVID is treated by
complications. intravenous or subcutaneous immunoglobulin (IV/SCIG) (initial
treatment might alleviate GI symptoms, it does not treat overt
Immune Pathophysiology gut inflammation and ulcers), and in fact, these complications
73
It is not clear if lack of Igs alone causes susceptibility to gut typically occur in patients who have adequate trough levels of
infections and inflammation because patients with X-linked IgG. Patients with any bacterial or protozoal pathogens should be

CHaPter 75 Immunological Diseases of the Gastrointestinal Tract 1015


treated with a recommended course of conventional antibiotics, (especially Salmonella spp. and C. difficile) occurs in CGD, an
including those positive for the C. difficile toxin. SIBO should be idiopathic CGD-associated IBD also develops: In the mouth,
treated, and recurrent SIBO may need cycling antibiotic regimens. granulomatous stomatitis and dental abscesses cause pain and
The treatment of the idiopathic enteropathy is very challenging. difficulty eating; in the esophagus, dysphagia, chest pain, and
Although this seems to be a late complication in a subset of vomiting may result from narrowing by strictures or stenosis
patients, it can be fatal. In the early stages, it may be responsive and dysmotility related to granulomatous inflammation and
to a short course of oral corticosteroids, either prednisone or fibrosis; in the stomach, loss of motility and capacity caused by
budesonide. Case reports attest to the efficacy of infliximab, but thickened walls and narrowed lumen leads to vomiting, epigastric
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this cannot be routinely advised. It is possible that immunosup- pain, and weight loss as a result of feeding difficulty; in the small
pressants may be used to control the inflammatory response and large intestines, diarrhea (including protein losing enter-
underlying the small-bowel mucosal damage, but this should opathy), bowel obstruction (large granulomata compromising
only be done in a closely observed clinical setting, with monitoring the size of the lumen), rectal bleeding, and tenesmus may result
for infections. At all times the patient’s nutritional status should from active colitis/enteritis with mucosal ulceration, anal fissures,
be maintained, initially using the oral route but administering and perianal abscesses. In addition, because of the transmural
parenteral nutrition to compliment oral nutrition and when nature of the granulomatous inflammation, penetrating complica-
oral feedings are not adequately absorbed and exacerbating the tions, such as fistulae and abscesses, can occur. Feeding difficulties
diarrhea. and the chronic inflammatory state itself predispose to growth
Portal hypertension may complicate CVID, typically from delay that often affects pediatric patients with CGD.
the nodular regenerative hyperplasia of the liver; however, little Hepatic abscesses represent another frequent complication
78
fibrosis occurs. It needs to be clarified in individual patients in CGD, occurring in up to 45% of patients. These patients
with splenomegaly (≈20%) that portal hypertension is not caused most often present with fever as well as abdominal pain, fatigue,
by excessive splenic vein flow associated with splenomegaly and, less often, abdominal tenderness and hepatomegaly on
(induced by antibody-mediated autoimmune cytopenias) that examination. The erythrocyte sedimentation rate (ESR) and the
might be ameliorated by splenectomy. In any scenario, the alkaline phosphatase level are elevated in half the affected
management of such late complications requires an especially individuals. However, a high level of suspicion, especially in the
experienced team of internists, surgeons, and nutritionists. setting of fever with or without abdominal pain, should instigate
a search for hepatic abscesses.
KeY COnCePts Immune Pathophysiology
Common Variable Immunodeficiency (CVID) Given the defects in ability to kill intracellular bacteria and fungi,
pathogens, and possibly commensals alike, it is thought that the
• The majority of gastrointestinal (GI) complications of CVID are infectious
and generally do not respond to intravenous or subcutaneous immu- exuberant granulomatous response is caused by delayed antigenic
noglobulin (IV/SCIG) therapy (compared with sinopulmonary suppurative clearance or persistent infection. In this way, granulomata continue
infections). to multiply and grow while other inflammatory pathways that
• CVID enteropathy is a rare immune-mediated complication of CVID normally deal with the microbes or are induced by cytokines
that also does not respond to IV/SCIG. are activated. The end results of granulomatous inflammation
• CVID enteropathy is often confused with celiac disease because of are most evident in tissues rich in macrophages and reticuloen-
similar villus damage on biopsy, but additional features (lack of plasma dothelial cells, such as the gut lamina propria, liver, lymph nodes,
cells, increased epithelial apoptosis, absence of celiac gene risk alleles)
can help differentiate them. and spleen.
• CVID enteropathy has no established therapy though judicious use
of short courses of oral steroids, or conventional immunosuppression Diagnosis
may relieve the malabsorption and diarrhea temporarily. The symptoms and signs will dictate the initial diagnostic
examinations. For diarrheal complaints, stool culture and
examination for C. difficile toxin are required; in the setting of
Chronic Granulomatous Disease hypoalbuminemia, fractional fecal excretion of α 1 antitrypsin
CGD results from defects in the nicotinamide adenine dinucleotide can detect protein-losing enteropathy (>50 mg/24 hour) as a
phosphate (NADPH)–oxidase complex that impair the ability result of either diffuse mucosal inflammation or lymphangiectasia.
of phagocytic cells to produce the reactive oxygen species required For complaints of dysphagia, vomiting, or epigastric pain, upper
to kill bacteria and fungi within intracellular phagolysosomes endoscopy can help document macroscopic and microscopic
(Chapter 22). Patients with CGD have recurrent infections of involvement with granulomatous inflammation. Radiological
the skin, lungs, liver, and bone, and nearly half develop gut studies using oral contrast may be helpful in showing a narrowed
inflammatory complications affecting areas anywhere from the lumen, stricturing, and motility and mucosal abnormalities of
mouth to the anus. Interestingly, the frequency of GI disease in the esophagus and stomach but cannot provide histological
CGD is higher in the X-linked gp91 phox defect, but the most confirmation. However, radiological imaging studies may be the
recently described p40 phox defect occurred in a young male patient primary diagnostic tools to evaluate obstructive symptoms from
who presented with granulomatous colitis alone. 75-77 the small intestine, including barium small-bowel studies and
CT or magnetic resonance enterography. Images can show lumen
Presentation narrowing; bowel wall thickening; mucosal abnormalities, includ-
The most typical GI complaints in patients with CGD are ing ulceration; and penetrating complications, such as fistulization.
abdominal pain and diarrhea (with or without rectal bleeding). Finally, to evaluate lower abdominal and perianal pain and rectal
GI symptoms usually begin in the first decade of life, sometimes bleeding, anoscopy, colonoscopy, and pelvic CT or magnetic
preceding the diagnosis of CGD. Although infectious diarrhea resonance imaging (MRI) will help diagnose granulomatous

1016 Part seven Organ-Specific Inflammatory Disease


inflammation of the colon and its complications, including the most frequent isolate, S. aureus) is generally appropriate
perirectal and perianal abscesses. In the case of hepatic abscess although percutaneous aspiration of hepatic abscesses maybe
detection (generally 1–6 cm), CT, MRI, and ultrasonography helpful. 83
have similar sensitivity (≈60%). Active abscesses appear to be
solid, hypoechoic lesions on ultrasonography or postcontrast
ring–enhancing lesions on CT and MRI. KeY COnCePts
The GI histological diagnosis hinges on the presence of Chronic Granulomatous Disease (CGD)
noncaseating granulomata, both gross and microscopic. These
granulomata are often seen against a background of acute • Half of patients with CGD develop gastrointestinal (GI) involvement
inflammation (acute focal colitis, crypt abscesses, cryptitis) as with granulomatous inflammation.
well as chronic inflammation (lymphocytic infiltrate, Paneth • Symptoms are caused by both obstructive complications of stricture
formation and mucosal inflammation.
cell metaplasia in the colon) that can be mild to severe. The • A resemblance to Crohn disease is suggested by granulomatous
histological picture may resemble Crohn disease except that the inflammation, transmural bowel involvement, and underlying innate
granulomata of CGD are well defined, often large collections of immune defect, but unlike in Crohn disease, there are also prominent
epithelioid histiocytes that can expand the mucosa (and even skin and lung infections and periodic acid-Schiff–positive lipid-laden
deform the overlying epithelium to make it look flattened in the macrophages in the lamina propria.
case of the villous mucosa of the small intestine). Like Crohn • Complicated hepatic abscesses require specialized medical
intervention.
disease, the inflammation can affect the three layers of the gut
wall, but unlike in Crohn disease, CGD biopsy specimens also
show prominent lipid-laden macrophages that have periodic
acid-Schiff (PAS)–positive cytoplasmic granules. GI Complications Occurring in Other Primary
Immunodeficiency States
Treatment Severe combined immunodeficiency (SCID) covers a wide
Current clinical practice for CGD includes using prophylactic phenotype of low-to-absent T cells, NK cells, and dysfunctional
antimicrobials to prevent infections, typically trimethoprim- B cells reflecting the mechanisms of the genetic defect (Chapter
sulfamethoxazole for bacterial and itraconazole for fungal 35). Recurrent infectious diarrhea and thrush are typical GI
84
infections. Some clinicians also use IFN-γ (subcutaneous, three- conditions before a diagnosis of SCID in newborns and neonates.
times-weekly dosing) to prevent infections, although this is not After allogeneic hematopoietic stem cell transplantation (HSCT),
79
a universal practice. Obviously, discovery of infectious etiologies GI graft-versus-host disease (GvHD) is a possibility. For many
of diarrhea should be treated appropriately. of the monogenic immunodeficiency diseases that confer sus-
Once infections are ruled out and granulomatous inflammation ceptibility to GI inflammation, the success of HSCT to correct
of the GI tract is established, with or without complications, the secondary effect on the GI tract will depend entirely on
such as stricturing of the bowel, treatment with corticosteroids whether the gene defect affects the myeloid or lymphoid cells
is indicated; beginning doses up to 1 mg/kg/day tapering over primarily. If the gut stromal or epithelial cells also depend on
12–20 weeks to maintenance doses of 2.5–5 mg every other day normal function of the defective gene, then bone marrow
has been reported to induce rapid alleviation of symptoms. Use transplantation will not likely relieve the GI effects.
of sulfasalazine for colitis may have limited benefit in some The rare X-linked recessive Wiskott-Aldrich syndrome
patients. Isolated reports of successful use of cyclosporine and (Chapter 35) results from Wiskott-Aldrich syndrome (WAS) gene
infliximab have indicated that these agents are best reserved for mutations, a signaling protein largely restricted to hematopoietic
refractory cases because of the potential for infectious side effects. cells, and leads to a syndrome of eczema, thrombocytopenia,
Similarly both granulocyte–colony-stimulating factor (G-CSF) and infections related to combined immunodeficiency (impaired
and granulocyte macrophage–colony-stimulating factor (GM-CSF) antibody responses and T-cell function). An interesting aspect
have been used with therapeutic benefit in GI complications of of the gut complication in this immunodeficiency is that patients
CGD because of their success in the granulomatous colitis of may develop a noninfectious colitis resembling UC (confluent
80
glycogen-1-β storage disease. Although there are no data to mucosal inflammation with ulceration, crypt abscesses, and no
suggest that IFN-γ worsens established disease, it also may not granulomata) with the bleeding exacerbated by the thrombo-
85
prevent it as >40% of one cohort developed GI manifestations cytopenia. Patients may respond to mesalamine drugs, but use
of granulomatous inflammation after starting it; in contrast, of steroids and immunosuppressive must be done cautiously
isolated reports have attributed alleviation of GI inflammation because of exacerbating infection risk. Successful bone marrow
81
to IFN-γ. IL-1 receptor blockade, based on preclinical efficacy transplantation can treat the colitis as well.
data in CGD, may offer limited success. 82 Early-onset IBD is a severe complication of rare mutations
Finally, it cannot be overstated that surgical drainage of that affect IL-10 production or the IL-10 receptor. Patients present
complicating abscesses and resection of fibrotic or refractory with colitis within weeks of birth that has features of Crohn
strictures need to be pursued, when indicated. Although there disease. Diagnosis is made by genetic testing as well as by assay
can be considerable postoperative complications because of of peripheral blood mononuclear cells for absence of IL-10
ongoing fistula formation and wound breakdown, these problems production or absent IL-10 signaling demonstrated by decreased/
can be managed by administration of corticosteroids. In addition, absent induction of phospho-STAT3 following the addition of
86
judicious use of endoscopic therapy to dilate narrowed esophageal IL-10. The only cure is with HSCT.
or pyloric regions is an option for symptomatic strictures. The hyper-IgM syndrome (type 1) (Chapter 34) is an X-linked
Treatment of hepatic abscesses has been shown to be amenable condition resulting from mutations in CD40 ligand leading
to nonsurgical management by adding corticosteroids to the to defective antibody class switching (hence high [or normal]
78
antibiotic regimen. Empiric antibiotic coverage (at least to cover IgM with low IgA and IgG) and NK- and T-cell cytotoxicity.

CHaPter 75 Immunological Diseases of the Gastrointestinal Tract 1017


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CHaPter 75 Immunological Diseases of the Gastrointestinal Tract 1019.e1


MUL t IPL e -CHOIC e QU est IO ns

1. A 73-year-old Caucasian female who is undergoing workup and reports a history of irritable bowel syndrome (IBD). She
for chronic diarrhea and weight loss has been incidentally says that her primary physician performed a “celiac test but
found to have a gastrin level of 2500 pg/mL and have serum it was negative.” Your first choice of tests is:
immunoglobulin G (IgG) antibody positive for Helicobacter A. Esophagogastroduodenoscopy with biopsies of the duo-
pylori. There is no history of peptic ulcer disease, pyrosis, or denum and duodenal bulb
kidney stones, and yet she has been referred for evaluation B. Genetic human leukocyte antigen (HLA) testing for DQ2
for gastrinoma. The first test you order is: and DQ8 risk alleles
A. Esophagogastroduodenoscopy with biopsy C. Repeat testing for serum IgA antibodies to tissue trans-
B. Abdominal computed tomography (CT) glutaminase, including total serum IgA level
C. Serologic testing for celiac disease D. Monitor anti–tissue transglutaminase levels while the patient
D. Secretin stimulation test is on a gluten-free diet
E. Fecal testing for H. pylori antigen
3. Which of the following is characteristic of ulcerative
2. A 22-year-old Caucasian female is referred for chronic colitis?
abdominal pain and bloating. She reports that a cousin has A. Presence of antiflagellin antibodies
celiac disease, and she says that “every time that I eat bread, B. Inflammation of the full thickness of the bowel wall
I feel bad and get diarrhea.” She has maintained her weight C. Tobacco use increases the frequency
and has no other specific symptoms. She is on no medications D. Surgery can be curative

76









Inflammatory Hepatobiliary Diseases



Carlo Selmi, Michael P. Manns, M. Eric Gershwin






Inflammatory hepatobiliary disease generically refers to chronic Environmental factors have also been linked to AIH develop-
autoimmune diseases associated with predominant hepatic or ment. Specifically, infections such as hepatitis C virus (HCV) may
biliary clinical manifestations, with no evidence of infectious trigger a process of molecular mimicry leading to exaggerated
disease. Based on specific cellular targets, we can distinguish immune responses against self-components with structural
autoimmune hepatitis (AIH) targeting hepatocytes from primary homology. In support of this hypothesis, it has been demonstrated
biliary cholangitis (PBC) (also known as primary biliary cirrhosis) that HCV shares high amino-acid sequence homology with the
and primary sclerosing cholangitis (PSC), both of which target autoantigenic target of antiliver/kidney microsomal-1 (LKM-1)
the biliary tract. Cirrhosis is the common evolution of inflam- autoantibodies, and that 10% of HCV patients are seropositive for
matory hepatobiliary diseases, regardless of the target tissue, anti-LKM-1. Nonviral environmental factors include medications,
eventually leading to liver failure. However, pathogenesis and in particular antibiotics (nitrofurantoin and minocycline), statins,
therapeutics may vary in between the spectrum of inflammatory and anti-tumor necrosis factor (TNF)-α agents (adalimumab and
1,2
hepatobiliary diseases. This chapter describes the main char- infliximab). However, the disease is slightly different from classical
acteristics of AIH, PBC, and PSC, with particular attention to AIH and usually does not require long-term immunosuppressive
clinical manifestations, pathogenesis, autoantibodies, and thera- treatment. 2
peutic options. From an immunological standpoint, liver damage is likely
orchestrated by CD4 lymphocytes, which can differentiate into
AUTOIMMUNE HEPATITIS T-helper cell-1 (Th1), Th2, and Th17 cells (Chapter 16). These
effector cells initiate a cascade of immune reactions largely
Epidemiology determined by the cytokines they produce: (i) Th1 cells secrete
Autoimmune hepatitis (AIH) is a severe liver disease affecting mainly interleukin (IL)-2 and interferon (IFN)-γ; IFN-γ is
almost all age groups worldwide. Although an accurate estimate considered the main orchestrator of tissue damage since it not
of its incidence is not available, it is believed be around 1 per only stimulates CD8 cells but also enhances the expression of
100 000 person-years, supposedly with higher incidence in HLA class I, induces the expression of HLA class II molecules
3
Scandinavia. AIH preferentially affects females, with a male:female on hepatocytes, and activates monocytes/macrophages, which
ratio of 1 : 4 and a two-peak incidence during adolescence as in turn release IL-1 and TNF-α; (ii) Th2 cells produce IL-4,
well as in the age range 30–45 years. 2 IL-10, and IL-13, cytokines that induce the maturation of B cells
into plasma cells, with consequent production of autoantibodies;
Pathogenesis (iii) Th17 cells, which arise in the presence of transforming
AIH is a chronic inflammatory disease of unknown etiology growth factor (TGF)-β and IL-6 and produce IL-17, IL-22, TNF-α,
7
resulting from the immune-mediated destruction of hepatocytes and the chemokine CCL- 20. Th17 cells, which are crucial in
secondary to a breakdown of immune tolerance against liver PBC, are being evaluated also in AIH, as an increased number
4
tissues. Both genetics and environmental factors contribute to of Th17 cells has been reported in the peripheral blood and liver
the development of the disease. Genetics certainly represents a of patients with AIH compared with healthy controls. 8
strong risk factor for the development of AIH, as susceptibility
to AIH is strongly influenced by the large and highly polymorphic Clinical Features and Diagnosis
human leukocyte antigen (HLA) region, which contains several AIH is a complex disease with various manifestations, predomi-
5
genes related to the adaptive immune system (Chapter 5). AIH nantly in the liver, but it is not limited to this organ. The onset
has been associated with polymorphisms in the genes encoding is most frequently insidious, whereas 20–30% of patients present
the HLA class II DRB1 alleles, which have also been confirmed with an acute icteric hepatitis, which is consistently associated
in genome-wide association studies (GWAS). In particular, with peripheral hypergammaglobulinemia. Two types of AIH
expression of HLA-DRB1*03:01 and HLA-DRB1*04:01 alleles are distinguished primarily based on the autoantibody patterns:
confers susceptibility to AIH-1 in European and North American i.e., AIH type 1, with antinuclear and/or antismooth muscle
populations, whereas the DRB1*04:05 and DRB1*04:04 alleles antibodies; and AIH type 2, with antiliver/kidney/microsomal
6
are linked to AIH susceptibility in Japan, Argentina, and Mexico. type 1 antibody and/or antiliver cytosol type 1 antibody. Type
Interestingly, DRB1*13:02 and DQB1*03:01 alleles were found 1 AIH (AIH-1) can affect people of any age and sex, and it is
2
to be protective. Moreover, certain HLA alleles have been associ- associated with expression of HLA-DRB1*03:01. AIH-1 patients
ated with AIH clinical manifestations, response to treatment, are more likely to be male, to present with high immunoglobulin
and prognosis. G (IgG) levels, and to be positive for antinuclear antibodies/
1021

1022 Part seven Organ-Specific Inflammatory Disease


anti-SMA. Also, AIH-1 may deteriorate despite corticosteroid TABLE 76.1 Diagnostic Criteria for
2
treatment and is more likely to progress to liver transplantation. autoimmune Hepatitis
Type 2 AIH (AIH-2) affects primarily girls and young women,
and it has been linked to alleles encoding the DR3 (DRB1*0301) Criteria Points
and DR7 (DRB1*07:01) molecules, and to positivity for anti-LMK Sex
antibodies. 2 Male +2
Blood tests usually show marked elevation of aminotransfer- Female 0
ases, usually with an increase in the cholestatic pattern (increased Ratio of alkaline phosphatase vs. AST/ALT
alkaline phosphatase, bilirubin). Furthermore, serum globulins, >3.0 +3
particularly of the γ type, are commonly increased in AIH cases, 1.5–2.0 +2
+1
1.0–1.5
regardless of the histological stage. <1.0 0
Lastly and most importantly, serum autoantibodies are invari- Autoantibodies (ANA, SMA, LKM-1) titer
ably positive (defined as titer >1 : 80) in patients with AIH. >1 : 80 +3
Antinuclear antibodies (ANAs) and/or antismooth-muscle (SMAs, 1 : 80 +2
directed against actin) autoantibodies are more frequently detected 1 : 40 +1
in AIH-1, whereas anti-LKM-1 antibodies (directed at cytochrome <1 : 40 0
AMA
P450 2D6 or UDP-glucuronosyltransferases) predominate in Positive −4
AIH-2. Negative 0
Without treatment, the clinical course of AIH is characterized Seropositivity for other autoantibodies +2
by high mortality, with 5- and 10-year survival rates estimated Viral hepatitis markers
as 50% and 10%, respectively. However, the use of corticosteroids Negative +3
has dramatically improved the course of the disease, with a 10-year Positive −3
2
survival rate exceeding 90%. Complications associated with AIH History of drug use −4
Yes
are similar to those of other progressive liver diseases, as chronic No +1
hepatitis can evolve to cirrhosis and ultimately to hepatocellular Average alcohol consumption (g/day)
carcinoma (HCC), despite the use of immunosuppressive therapy. <25 +2
At the time of diagnosis, approximately 30% of adult patients >60 −2
have histological evidence of cirrhosis; when appropriately treated, Presence of genetic factors (HLA, DR3 or DR4) +1
however, only a small number of patients develop cirrhosis during Presence of other autoimmune disorders +2
Liver histology
follow-up if inflammation is resolved. The occurrence of HCC Interface hepatitis +3
in patients with AIH is a rare event and develops only in long- Predominant lymphocytic infiltrate +1
standing cirrhosis. In the absence of solid data on large numbers Rosetting of liver cells +1
of cases, the incidence of primary liver neoplasia should be None of the above −5
regarded as similar to other nonviral cases of cirrhosis. Biliary changes −3
There is no single diagnostic test for AIH; therefore diagnosis Other changes −3
is based upon several indicative clinical, serological, biochemical, Response to therapy +2
Complete
and histological findings. The presence of other causes of liver Relapse +3
disease must also be excluded. A set of diagnostic criteria has
been established, and subsequently updated, with high sensitivity A score >15 or >17 indicates a definite diagnosis of autoimmune hepatitis pre- or
and specificity (Table 76.1). Factors taken into account in this posttreatment, respectively. On the other hand, scores of 10–15 and 12–17 indicate a
probable diagnosis, pre- or posttherapy, respectively. AMA, antimitochondrial
system include sex, plasma biochemical variables, serum auto- autoantibodies; ANA, antinuclear antibodies; HLA, human leukocyte antigen; LKM-1,
antibodies, liver histology, possible cofactors (drugs, alcohol, antiliver–kidney microsomal antibodies; SMA, antismooth-muscle antibodies.
viruses), and response to medical treatment. 9
Serum Autoantibodies positivity is not uncommon in sera from patients with viral or
Autoantibodies represent a critical feature of the disease and other autoimmune liver diseases, as well as in as many as 15%
12
may guide the diagnosis. In 2004 the International Autoimmune of healthy subjects, especially in older age groups. Similarly to
Hepatitis Group established procedures and reference guidelines most autoantibodies, ANA positivity, pattern, or titer does not
for more reliable serum autoantibody testing to overcome the reflect various AIH phenotypes; nor can these factors predict
10
lack of standardization. Not only serum ANA, SMA, and LKM the disease’s natural history.
should be evaluated, but other sets of autoantibodies should Serum SMAs are autoantibodies reacting with different proteins
also be tested in suspected cases, including those against liver- (actin, tubulin, vimentin, desmin, cytokeratins) of the cytoskeletal
cytosol type 1 (LC1), perinuclear antineutrophil cytoplasmic components (microfilaments, microtubuli, intermediate fila-
antibodies (pANCA), soluble liver antigen/liver-pancreas antigen ments). Their presence characterizes both autoimmune (AIH-1,
11
(SLA/LP), and the asialoglycoprotein receptor. Finally, other celiac disease) and viral diseases (chronic hepatitis C, infectious
less specific autoantibodies are also detected in a subgroup of mononucleosis). When detected at high titers (>1 : 80), they are
patients, i.e., anticardiolipin, chromatin, and Saccharomyces considered a sensitive marker for AIH-1, being found in up to
cerevisiae, but these are of limited clinical significance. 80% of cases. A recent study showed that anti-SMA-T/G (peri-
ANAs were the first autoantibodies observed in AIH sera tubular/glomerular staining) positive subjects with normal liver
more than 50 years ago and remain the most sensitive marker function are at low risk of progression to AIH, whereas positive
of AIH, producing most frequently a homogeneous or speckled SMA and raised ALT (>55 IU/L) indicate higher risk, with a
pattern. However, the test is not specific for AIH, since ANA positive predictive value of 22%. 13

CHaPter 76 Inflammatory Hepatobiliary Diseases 1023


Serum autoantibodies against LKM-1 are the main serological
markers of AIH-2 and recognize the proximal renal tubule and Therapy
hepatocellular cytoplasm. The 50-kDa autoantigen was identified In contrast to other autoimmune liver diseases, immunosup-
as the cytochrome P450 2D6 (CYP2D6). Interestingly, the sequence pressants are the treatment of choice for AIH, based on the good
14
between amino acids 316 and 327, which is most likely exposed response in terms of biochemistry, histology, and survival.
on the surface of the molecule, appears to be a region capable Corticosteroids, in particular prednisone, represent the first-line
of differentiating LKM-1 activity in AIH and HCV and may of treatment in monotherapy or in combination with azathioprine,
represent a key target for autoimmunity. The mechanisms of inducing remission (i.e., normal transaminases and IgG levels)
9
onset remain enigmatic, and solid evidence of a causative role in over 80% of patients, regardless of the presence of cirrhosis.
of hepatitis C virus cross-reactivity is still awaited. Similarly, the Once achieved, remission can be maintained with azathioprine
pathogenic role of anti-LKM-1 antibodies and their prognostic alone. Relapses after therapy discontinuation are common since
significance are debated, despite the development of AIH-2 in only 20% of patients remain in sustained remission. It should
animal models after immunization with human CYP2D6 or with be noted, however, that subgroups of patients manifest disease
adenoviruses in mice transgenic for human CYP2D6. Finally, two progression (approximately 10%) or are intolerant to standard
other types of serum anti-LKM have been described in patients therapy (13%). In such patients, other drugs have been anecdotally
with ticrynafen-associated hepatitis (anti-LKM-2, directed against tried, including methotrexate, cyclophosphamide, tacrolimus,
CYP2C9) and in 10% of patients with type 2 AIH (anti-LKM-3, ursodeoxycholic acid, cyclosporine, and mycophenolate mofetil,
directed against uridine-diphosphate glucuronosyl transferase 1A the latter two being those most frequently reported as alternative
[UGT1A]), either alone or in combination with LKM-1 antibodies. medications. Liver transplantation is the ultimate treatment for
Anti-SLA/LP antibodies are detectable by radioimmunoassay AIH patients presenting with acute liver failure or end-stage
and enzyme-linked immunosorbent assay, but not by immuno- chronic liver disease and for those with hepatocellular carcinoma
fluorescence, and are directed against various epitopes of a UGA that meet the transplant criteria. Although liver transplantation
tRNA suppressor. Serum anti-SLA/LP antibodies are occasionally for AIH is very successful, it should be noted that AIH may recur
9
found in patients with AIH who are negative for ANA, SMA, after transplant. Patients with AIH undergoing liver transplant
and anti-LKM and are cumulatively detected in 10–30% of cases have overall 5- and 10-year survival rates of 90% and 75%,
of AIH-1 and -2. Recent data indicate that anti-SLA/LP antibodies respectively, although infectious complications and disease
are also detectable in subgroups of pediatric patients with recurrence are common. 15
autoimmune cholangitis or in adult patients with HCV infection
when tested with sensitive methods.
Anti-LC1 antibodies are detected by indirect immunofluo- KeY COnCePts
rescence in sera from up to 50% of patients with type 2 AIH
and less frequently in type 1 AIH or chronic hepatitis C. • Autoimmune hepatitis (AIH) is a severe autoimmune disease associated
Importantly, however, anti-LC1s are the only detectable markers with high morbidity and mortality, especially due to the development
in 10% of AIH cases. The LC1 autoantigen is the liver formimi- of cirrhosis and possibly hepatocellular carcinoma.
notransferase cyclodeaminase, an enzyme involved in folate • Autoantibodies represent a distinctive feature of AIH and can also aid
in the definition of AIH subsets.
metabolism. Interestingly, serum anti-LC1 antibodies correlate • Liver biopsy is helpful, and histology remains the gold standard for
with AIH severity and progression. grading and staging, particularly to determine the response to therapy.
Antibodies to the asialoglycoprotein receptor are observed
in up to 90% of all patients with AIH and often coexist with
other autoantibodies while lacking specificity for the disease.
Similar to anti-LC1, however, antiasialoglycoprotein titers are CLInICaL PearLs
associated with more florid inflammatory disease activity and
with less effective treatment responses. • Autoimmune hepatitis (AIH) may cause an increase in aminotrans-
Finally, antibodies to neutrophil cytoplasmic antigens (pANCA) aminases and in cholestatic markers.
can be detected by indirect immunofluorescence in sera from • Autoantibody testing is helpful. Antinuclear antibodies (ANAs), SMAs,
and antiliver/kidney microsomal (LKM) are the most important, but
patients with AIH-1; they also can be detected in a subgroup of other sets of autoantibodies also should be tested in suspected cases,
patients with PSC or chronic viral hepatitis. in particular anti-LC1, perinuclear antineutrophil cytoplasmic antibodies
(pANCA), soluble liver antigen/liver-pancreas antigen (SLA/LP), and
Histology the asialoglycoprotein receptor antibody.
The role of liver histology in the management of AIH remains • Diagnostic criteria are available and provide good sensitivity and
critical, and all patients with suspected cases should undergo a specificity.
liver biopsy. In fact, although no typical feature can prove the
diagnosis, histology remains the gold standard for grading and
staging, particularly to determine the response to therapy.
Common findings include periportal hepatitis with lymphocyte tHeraPeUtIC PrInCIPLes
and plasma cell infiltrate and piecemeal necrosis. Fibrosis usually • Glucocorticoids represent the cornerstone of autoimmune hepatitis
is observed, and bridging necrosis ultimately indicates advanced (AIH) therapy as monotherapy or in combination with azathioprine.
disease evolving into frank cirrhosis. Importantly, the presence • Other immunosuppressants, i.e., methotrexate, tacrolimus, cyclosporine,
of granulomas, bile duct damage, or iron or copper accumulation and mycophenolate mofetil, have been used in patients who are
should not be overlooked, since these signs point toward other intolerant or refractory to standard therapy.
diagnoses. On the other hand, steatosis is a nonspecific finding • Liver transplantation is very successful; however, AIH may recur after
transplant.
that does not rule out AIH. 9

1024 Part seven Organ-Specific Inflammatory Disease


PRIMARY BILIARY CHOLANGITIS more frequent in PBC than in controls, with E. coli being the
main etiological agent. The mechanisms linking infections and
Nomenclature autoimmune biliary tract damage are suspected to involve
Recently the nomenclature for primary biliary cirrhosis has been molecular mimicry. Besides E. coli, a number of other bacteria
shifted toward primary biliary cholangitis (PBC) to correct the have been examined as cross-reactive agents in PBC, including
inaccurate description and remove the cirrhosis stigma as well Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus aureus,
as all the misunderstandings, disadvantages, and discriminations Neisseria meningitidis, Salmonella minnesota, Mycobacterium
emanating from this misnomer. This change was necessary after gordonae, and Trypanosoma brucei. Novosphingobium aromaticiv-
the dramatic improvement in PBC diagnosis, prognosis, and orans, a ubiquitous xenobiotic-metabolizing gram-negative
treatment. Currently, an early diagnosis of PBC can be established bacterium, has been proposed to be the ideal candidate for PBC
with the more accurate measurements of markers of cholestasis development because it contains proteins with the highest degree
and with improvement in the detection of the classic serological of homology with the major epitope of PDC-E2 while having
hallmark, antimitochondrial antibodies (AMAs). In parallel, the ability to metabolize organic compounds and estrogens.
treatment was improved with the introduction of orthotopic Moreover, N. aromaticivorans can elicit antibody reactivity in
liver transplantation and ursodeoxycholic acid (UDCA, 13–15 mg/ PBC patients but not in controls. Regarding viral infections, a
kg daily) therapy. At present, two of three patients diagnosed novel human beta-retrovirus was found in perihepatic lymph
with primary biliary cirrhosis and treated with UDCA have an nodes and other biological samples from patients with PBC;
24
expected survival equal to that of the general population, and however, the data have remained inconclusive. Xenobiotics are
only a minority will ever develop cirrhosis. 16 foreign compounds that may trigger an autoimmune response
to self-proteins, which may modify their molecular structures
Epidemiology or complex to self or nonself proteins to generate neoantigens.
PBC is a chronic autoimmune cholestatic disease characterized In this view, it was shown that 2-nonynoic acid, an organic
by high-titer serum AMA and immune-mediated destruction compound found in nail polish, was able to elicit AMA production
1
of the small- and medium-sized intrahepatic bile ducts. It affects in patients with PBC once they were attached to the major
women more frequently, with a female:male ratio of 9 : 1; the mitochondrial epitope backbone. 25
average age at diagnosis is within the fifth and sixth decades of The complex pathogenesis of PBC, however, is not fully
life. Geographically, it presents with widely variable prevalence; explained by genetics and environmental factors; as suggested
the highest rate has been described in Minnesota, with a point for most other autoimmune diseases, epigenetic modifications
prevalence of 402 per million in the general population. 17,18 could represent the link between genetic and environmental
factors influencing the onset and evolution of PBC. Epigenetic
Pathogenesis dysregulation results in the overexpression of certain genes in
26
As with essentially every autoimmune disease, genetics and several key immune cells. However, conclusive data implicating
environmental factors interact to lead to PBC. Genetic factors epigenetic modifications in the pathogenesis of PBC are not yet
play a significant role in PBC susceptibility, as suggested by the available.
fact that it is more frequent in relatives of affected individuals;
the term “familial PBC” has been coined to indicate families that Clinical Features and Diagnosis
have more than one case. In our experience, 6% of cases have a In the early phase of disease, symptoms of PBC are mainly
19
first-degree relative that is also affected, while AMA may be fatigue and pruritus, while physical findings may include skin
positive in first-degree relatives and offspring of patients with hyperpigmentation, hepatosplenomegaly, and (rarely) xanthe-
PBC without any sign of disease, thus indirectly suggesting the lasmas. End-stage symptoms are indistinguishable from those
20
existence of a strong genetic predisposition. More importantly, of other types of liver cirrhosis and include ascites, jaundice,
the concordance rate observed among monozygotic twins for hepatic encephalopathy, and upper digestive bleeding. Fatigue is a
PBC is 63%, among the highest reported in autoimmunity, nonspecific symptom present in 70% of PBC patients and is often
reinforcing the idea that genetic risk factors play an important overlooked, particularly in middle-aged women. The severity of
21
role. As for other autoimmune disorders, genetic factors are fatigue is independent of the stage of PBC or its other features
not limited to a single gene but to a complex multigene trait. (pruritus or severe cholestasis), and it appears not to depend on
The relevance of the multifactorial genetic basis in PBC has been psychiatric factors. Pruritus is similarly frequent, being present
reinforced by recent observations including the high concordance in 70% of patients with PBC and jaundice. Pruritus might long
21
rate among monozygotic (identical) twins as well as by reports precede jaundice onset and typically worsens at night, after contact
that lymphocytes from women with PBC preferentially use one with wool, or in warm climates. Portal hypertension is frequently
of their X chromosomes, rather than the usual random X chromo- found in patients with PBC and, importantly, does not imply
some inactivation observed in healthy women. 22,23 PBC has been the presence of liver cirrhosis. Over half of untreated patients
associated with the HLA-DRB1*08 allele but also with two eventually develop portal hypertension over a 4-year period.
protective alleles, HLA-DRB1*11 and -DRB1*13. Polymorphisms Prevention and treatment for PBC-associated portal hypertension
in HLA class II loci were confirmed to play a key role by GWAS. are the same as for other chronic liver diseases. Metabolic bone
Notably, this study demonstrated that genes coding for disease is present in PBC when comparing sex- and age-matched
interleukin-12 and its receptor also confer risk for PBC. 24 healthy individuals. Hyperlipidemia is common in up to 85% of
However, it can be hypothesized that other factors may patients with PBC; both elevated serum cholesterol and triglyceride
contribute to genetics as complementary modes of pathogenetic levels are encountered. Interestingly, however, such alterations
import, including epigenetics, exposure to environmental factors, are not accompanied by a proportionally increased incidence
etc. Infections and xenobiotics play a key role in increasing the of cardiovascular events or atherosclerosis and do not correlate
susceptibility to PBC. Urinary tract and vaginal infections are with disease stage. Autoimmune diseases may overlap with PBC,

CHaPter 76 Inflammatory Hepatobiliary Diseases 1025



TABLE 76.2 Diagnostic Criteria for Serum Autoantibodies
Primary Biliary Cholangitis
AMAs are highly specific for PBC and can be detected in nearly
Parameters 100% of patients when sensitive diagnostic methodologies based
Elevated ALP > 2 × ULN or GGT > 5 × ULN on recombinant antigens are used. In most clinical settings,
24
AMA positivity however, indirect immunofluorescence techniques are used for
Chronic granulomatous cholangitis on liver biopsy
initial screening of cases and might provide falsely positive or
Diagnosis defined in the presence of at least 2 out of 3 criteria. negative results. AMAs are directed against components of the
ALP, alkaline phosphatase; AMA, antimitochondrial antibodies; ULN, upper limit of 2-oxoacid dehydrogenase (2-OADC) family of enzymes within
normal; GGT, γ-glutamyltransferase.
the mitochondrial respiratory chain, most frequently the E2- and
E3-binding protein (E3BP) components of the pyruvate dehy-
most commonly Sjögren syndrome, Raynaud phenomenon, drogenase complex and the E2 components of the 2-oxo glutarate
autoimmune thyroid disease, scleroderma, and systemic lupus dehydrogenase and branched-chain 2-oxo acid dehydrogenase
erythematosus, whereas the prevalence of rheumatoid arthritis complexes. For all three antigens, epitopes contain the motif
does not differ when PBC cases are compared with healthy DKA, with lipoic acid covalently bound to the lysine (K) residue.
controls. Patients affected by both PBC and scleroderma have a The role of lipoic acid in epitope recognition by AMA is unclear.
less aggressive liver disease, which suggests an active interaction A direct pathogenic role of AMA is debatable, since no clinical
between the two conditions. As with other types of cirrhosis, correlation can be found and animal models developing serum
end-stage PBC can be complicated by the occurrence of HCC, AMA do not develop PBC-like liver lesions. Autoantibodies other
30
and patients with intense nodular liver structure on ultrasound than AMA can be found in 76% of PBC patients. ANA can be
should be monitored by computed tomography. Importantly, found in 50% of PBC patients, with the most common patterns
PBC is not associated with cholangiocarcinoma (CCA) or breast being “nuclear rim” or “multiple nuclear dots.” The pattern is
cancer. 1 based on the recognition by the autoantibodies of gp210 and
The diagnosis of PBC is based on the presence of two out nucleoporin 62 (within the nuclear pore complex) and Sp100
of three internationally accepted criteria, i.e., detectable serum and promyelocytic leukemia protein (PML) (possibly also cross-
AMA (titer >1 : 40), increased enzymes indicating cholestasis (i.e., reacting with small ubiquitin-like modifiers, SUMO), respec-
31
alkaline phosphatase) for longer than 6 months, and a compatible tively. ANA-positive patients are more frequently AMA-negative,
27
or diagnostic liver histology (Table 76.2). In a large number possibly because of the lack of a masking effect of these latter
of cases (20–60%), the diagnosis of PBC is established in the antibodies. Similarly, the pathogenic role of ANA in PBC remains
absence of symptoms indicating a liver condition or cholestasis, enigmatic, although cross-sectional and longitudinal data
and the proportion of asymptomatic cases at diagnosis has been demonstrate an association between ANA positivity and a worse
32
steadily increasing over the past decade. At presentation, PBC is prognosis. Finally, patients with PBC and limited systemic
suspected if a biochemical cholestatic pattern (increased plasma sclerosis have detectable serum anticentromere antibodies in
alkaline phosphatase or γ-glutamyltransferase) is present with 10–15% of cases. Monitoring autoantibody titers does not cor-
no similar increase in plasma aminotransferase levels. Serum relate with PBC severity or clinical outcomes, whereas a change
IgMs are typically elevated in PBC cases, with no correlation in the sp100 autoantibody level may have prognostic utility with
with AMA titers or levels of other Ig subtypes. Once cirrhosis respect to the development of fibrosis on liver biopsy.
has developed, biochemical alterations are similar to those with
other types of cirrhosis. The progression of PBC varies widely, Histology
and the factors influencing the severity and progression of the PBC histology is classified into four stages (Fig. 76.1). Stage I
disease are largely unknown. Having symptoms at presentation manifests with portal tract inflammation with predominantly
is considered the major factor determining survival rates of lymphoplasmacytic infiltrates, resulting in vanishing septal and
patients with PBC. In fact, asymptomatic PBC is accompanied interlobular bile ducts (diameter <100 µm). At this stage, bile
by 10-year survival rates similar to those of the general popu- duct obliteration and granulomas (possibly found at all stages)
lation. On the other hand, 67% of precirrhotic patients will are strongly suggestive of PBC. In stage II a periportal inflam-
develop liver cirrhosis over a 7-year observation period, while matory infiltrate is observed, and signs of cholangitis, granulomas,
70% of asymptomatic patients will develop symptoms. Patients and florid proliferation of ductules are typical. Stage III dem-
with symptomatic PBC show a more rapid progression to late- onstrates septal or bridging fibrosis, with ductopenia (over half
stage disease and a worse prognosis than their asymptomatic of the visible interlobular bile ducts having vanished) and copper
counterparts, with survival times among symptomatic subjects deposition in periportal and paraseptal hepatocytes visible. Stage
28
within 6–10 years. Older age at diagnosis and signs of advanced IV corresponds to frank cirrhosis. The observation of eosinophils
disease (clinical, histological, or biochemical) are associated with in the portal tract is considered a specific finding in PBC
a worse prognosis. The establishment of accurate prognostic histology.
models to predict survival in patients with PBC is of obvious Novel noninvasive biomarkers are under evaluation for predict-
importance in clinical practice. The model based on the Mayo ing liver histology, and in this view the aminotransferase to platelet
29
score is the only one validated and is the most widely utilized ; ratio index (APRI) and fibrosis index on the basis of the four
it is calculated based on clinical (age, presence of ascites) and factors (FIB-4) scores are statistically different between groups
biochemical variables as represented by cholestasis (bilirubin and may be a predictor of advanced disease in PBC. 33
levels) and liver function (prothrombin time, albumin). However,
this model has its limitations, as it is a static representation of Therapy
a dynamic entity and has a lower accuracy for patients with PBC treatment is currently based on UDCA, which is the only
early disease. approved drug for this rare disease. Its mechanism of action is

1026 Part seven Organ-Specific Inflammatory Disease


e.g., cytotoxic T lymphocyte antigen-4 (CTLA-4) Ig (abatacept)
35
or the CD40-antagonist FFP104, are under investigation. When
the disease has already progressed and bile is accumulating,
obeticholic acid (OCA), an analog of CDCA with a much higher
affinity to the farnesoid X receptor, has been shown to decrease
bile synthesis, promote secretion, and induce liver regeneration
in animal models. In a phase III trial, OCA administered with
UDCA or as monotherapy for 12 months resulted in a decrease
in alkaline phosphatase and total bilirubin levels that differed
significantly from the changes observed with placebo. 35
Ultimately, UDCA represents the cornerstone therapy of PBC.
Doses ranging from 13 to 15 mg/kg are currently used for
optimum bile enrichment and in 50% of patients normalize
alkaline phosphatase. Other immunosuppressive treatments
should be started only in combination with UDCA.
Liver transplantation is the ultimate treatment for end-stage
A
PBC, with survival rates of 92% and 85% at 1 and 5 years after
transplant, respectively. Recurrence is common, and its rates seem
to be influenced by certain immunosuppressive regimens. The
use of UDCA in cases of recurrence is safe and recommended.

KeY COnCePts
• Primary biliary cholangitis (PBC) is a chronic autoimmune cholestatic
disease affecting preferentially women in the fifth to sixth decades
of life.
• Genetics plays a strong role in PBC pathogenesis, as suggested by
familial clustering and the high concordance in monozygotic twins.
• Antimitochondrial antibodies (AMAs) are highly specific for PBC and
can be detected in nearly 100% of patients.


CLInICaL PearLs
B
• Fatigue and pruritus represent the most frequently observed symptoms,
FIG 76.1 Histological Findings in Early Stages of Primary which may be disabling for patients.
Biliary Cirrhosis, i.e., Nonsuppurative Destructive Cholangitis, • Primary biliary cholangitis (PBC) can be frequently associated with
Following Hematoxylin and Eosin Staining. (A) Mixed lym- other autoimmune diseases, e.g., Sjögren syndrome and systemic
sclerosis.
phocytic and plasma cell periductular inflammation with bile • PBC patients should be observed for metabolic bone disease.
duct infiltration and granulomatous reaction (square). Magnification
× 200. (B) Detail of bile duct disruption with lymphocytic and
plasmacellular periductular and intraepithelial infiltration. Magnifica-
tion × 400. tHeraPeUtIC PrInCIPLes
• Ursodeoxycholic acid (UDCA) (13–15 mg/kg) remains the cornerstone
therapy for primary biliary cholangitis and is the only approved drug.
incompletely understood. Depending on the various phases of • Glucocorticoids may be effective in early phases of the disease.
the disease, various therapies might be effective. • Biological therapies are under investigation, e.g., ustekinumab and
During the early phase of the disease, glucocorticoids might abatacept.
be effective; however, safety concerns about the long-term use • Obeticholic acid appears to be effective in reducing cholestatic
biomarkers.
of such medications arise. Budesonide, due to its high first-pass
metabolism, has minimal systemic adverse effects; at 6–9 mg daily,
it has been demonstrated superior to UDCA both in terms of PRIMARY SCLEROSING CHOLANGITIS
histology and biochemical markers. Other immunosuppressants,
such as methotrexate and azathioprine, have also been suggested, Primary sclerosing cholangitis (PSC) is a progressive cholestatic
and there is evidence supporting the use of the latter in PBC liver disease of unknown etiology presenting with autoimmune
with autoimmune hepatitis overlap syndrome. In recent years, features and associated with significant morbidity and mortality.
because of better understanding of PBC pathogenesis, new Unlike PBC, PSC can affect all tracts of the biliary tree, including
targeted therapies have been tested. Considering a possible role the extrahepatic bile ducts.
of the IL-17/23 axis, ustekinumab, a monoclonal antibody against
the p40 subunit of IL-23, has been tested. Although associated Epidemiology
with a modest decrease in alkaline phosphatase after 28 weeks The prevalence of PSC is approximately 10 in 100 000 in northern
4,5
of therapy, ustekinumab did not otherwise change alkaline Europe and the United States, while it is far less common in
34
phosphatase. Other therapies targeting T-cell costimulation, southern Europe and Asia. Recent data from Olmsted County,

CHaPter 76 Inflammatory Hepatobiliary Diseases 1027


Minnesota, reported a disease prevalence of 20.9 per 100 000 normal until late stages. Autoantibodies are of limited use in
36
men and 6.3 per 100 000 women. Epidemiological data indicate the diagnosis of PSC due to low sensitivity and specificity. Only
that annual incidence rates are not increasing over time despite a limited percentage of patients (33%) has positive pANCA,
earlier ages at diagnosis, and survival is possibly longer, similar which usually is found in IBD patients without PSC.
to what was observed in PBC. Imaging represents a useful diagnostic tool, as it may find
In contrast to the vast majority of autoimmune diseases, PSC the unique strictured and dilated tracts within the intrahepatic
is more commonly diagnosed in men, with a female:male ratio or extrahepatic bile ducts. Among the imaging techniques,
estimated as 1 : 2, with a preference for 30- to 40-year-olds. PSC endoscopic retrograde cholangiopancreatography (ERCP) and
has a strong association with inflammatory bowel disease. magnetic resonance cholangiopancreatography (MRCP) are
Approximately 60–80% of patients with PSC present with currently considered equal for sensitivity, but their results are
inflammatory bowel disease, of which 87% have ulcerative colitis influenced by the operator’s skill and experience.
and 13% have Crohn disease. 37
Histology
Pathogenesis Although not necessary for establishing the diagnosis, liver
The etiopathogenesis of PSC is unknown, but evidence is growing histology is essential when staging PSC or when the small-duct
that (auto) immune-mediated mechanisms play a role. The notion variant, an overlap syndrome, or CCA is suspected. The histologi-
of an autoimmune pathogenesis is supported by the frequent cal picture varies widely, from minimal alterations to cirrhosis
association with inflammatory bowel disease (IBD), the presence with portal inflammation, concentric “onion skin” periductal
of serum autoantibodies, and the reported HLA associations. fibrosis, and periportal fibrosis developing into septal and bridging
Genetic risk determines PSC susceptibility, including HLA necrosis. Fig. 76.2 illustrates the histological findings in two
haplotypes on chromosome 6p21, the inhibitory HLA-C2 killer- representative cases of early and advanced PSC.
immunoglobulin receptor (KIR) ligand, and HLA-C1 homozygos-
ity. Also, a positive and negative association with HLA-HLDRB1*15
and -DRB1*07, respectively, has been reported. A recent study
on Italian patients with PBC did not find any association with
DRB1*03, *04 or *13:01 alleles typically detected in Northern
38
Europe. Family and twin studies are not available for PSC;
however, a GWAS has reported the HLA region as the only major
association, albeit in a minority of patients and sometimes
overlapping with IBD-associated genes. The innate immune
system and microorganisms (possibly derived from an IBD-
affected gut) may participate in the onset and/or perpetuation
of disease. It has been proposed that cholangiocytes are first
activated by bacterial stimuli in the presence of gut-specific
chemokines and endothelial cell adhesion molecules in the tissue
microenvironment. Also, gut-primed T cells can migrate into
the portal tracts and peribiliary spaces to form focal lesions.
Finally, chronic inflammation and progressive fibrosis of the
biliary epithelium lead to chronic cholestasis secondary to A
vanishing bile ducts, ultimately causing biliary cirrhosis.
Clinical Features and Diagnosis
PSC symptoms generally are nonspecific and include abdominal
pain, jaundice, and fever in the case of bacterial cholangitis; at
more advanced stages, symptoms include the characteristics of
decompensated cirrhosis or neoplasia. Commonly, PSC is further
complicated by episodic bacterial cholangitis, especially in the
setting of biliary strictures. Lastly, subgroups of patients manifest
the “small duct” variant or overlap syndrome.
The median time span from diagnosis to liver-related death
39
or liver transplantation can be estimated at 18 years, and the
prognosis is influenced by the possible onset of CCA, which
may be difficult to detect because biliary structures are already
altered. It is important to distinguish small-duct PSC, as the
natural history is relatively benign and only a minority (12%)
of patients develop classical PSC. 40 B
Due to the nonspecific symptoms, PSC currently is diagnosed
most commonly in the absence of symptoms and during routine FIG 76.2 Histological Findings in Primary Sclerosing Chol-
blood tests in healthy individuals or patients with IBD. In labora- angitis. (A) Early disease and periductular fibrosis. Magnification
tory testing, PSC is characteristically accompanied by a biochemi- x 200, hematoxylin and eosin staining. (B) Advanced disease
cal cholestatic pattern, as indicated by elevated serum alkaline with cirrhosis and bile duct substitution by fibrous scar (square).
phosphatase and γ-glutamyltransferase. Liver function tests are Magnification × 200, Masson staining.

1028 Part seven Organ-Specific Inflammatory Disease


ANA and/or SMA positivity and/or hypergammaglobulinemia; (ii)
Therapy serum AMA negativity by immunofluorescence; (iii) biochemical
The treatment of PSC includes medical and endoscopic measures and/or histological features of cholestatic and hepatocellular
as well as liver transplantation. UDCA has been investigated in injury; and (iv) exclusion of chronic viral, metabolic, or toxic
several clinical trials, with conflicting results. Overall, the available liver disease. This definition possibly includes PBC with atypical
evidence suggests that UDCA fails to induce a substantial change presentation, small-duct PSC, idiopathic adulthood ductopenia,
in the course of PSC, despite remaining the most prescribed AIH with bile duct damage, concurrent AIH and small-duct
drug. However, it appears that high-dose UDCA (20 mg/kg/day) PSC, and various transitional stages of the classic diseases.
might reduce the rate of progression and possibly prevent the Consensus is awaited on this issue, and standardization of
development of colon cancer in patients with PSC and Ulcerative diagnostic criteria for overlap syndromes also has not been
colitis (UC). Endoscopic measures are indicated to treat com- achieved.
plicated PSC through the opening of short- and long-segment
stenosis of the common bile duct and short-segment stenosis OVERLAP SYNDROMES
of the hepatic ducts near to the bifurcation. The treatment can
be repeated over time once restenosis ensues, and resulting survival As many as 18% of patients with autoimmune liver disease also
rates are higher than those of patients not treated endoscopically. present with features of a second autoimmune liver disease. An
Finally, PSC represents an important indication for liver trans- even higher percentage of patients has an additional autoimmune
plantation since patients are commonly younger than those with disease, e.g., rheumatoid arthritis, Sjögren syndrome, or systemic
other autoimmune liver diseases. Recurrence of disease is common sclerosis. These patients are considered to have overlap syndromes.
and affects 20–40% of transplanted patients during prolonged Patients with hepatobiliary overlap syndromes usually present
follow-up. It is still debated whether medical treatment with with both hepatocellular and cholangiocellular injury with
UDCA in fact produces a longer survival. biochemical and histological features of AIH and PBC or PSC.
When not treated, these patients show a progressive course toward
liver cirrhosis and failure. More specifically, AIH–PBC overlap
syndrome is found in 10% of adults with AIH or PBC, and
KeY COnCePts AIH–PSC overlap syndromes are found in 6–8% of children,
adolescents, and young adults with AIH or PSC. Besides the
• Primary sclerosing cholangitis (PSC) is a chronic autoimmune cholestatic existence of overlaps, in rare cases transitions are also possible
disease that can affect all tracts of the biliary tree, including the
extrahepatic bile ducts. from PBC to AIH, from AIH to PBC, or from AIH to PSC. The
• Unlike other patients with autoimmune diseases, the majority are pathogenesis of the overlap syndromes is poorly understood,
male. and few data are available regarding the clinical characteristics
• PSC can be complicated by cholangiocarcinoma. and outcomes of such patients. Thus the clinical management
of overlap syndromes is based on single diseases, and medical
treatment remains empiric. Thus UDCA is used for chronic
cholestasis, and immunosuppressants (steroids and azathioprine)
CLInICaL PearLs are used for AIH; for end-stage disease, liver transplantation is
indicated.
• Primary sclerosing cholangitis (PSC) is frequently associated with IBD
(60–80% of patients). IgG4-RELATED CHOLANGITIS
• PSC is not associated with any specific autoantibodies; however,
perinuclear antineutrophil cytoplasmic antibodies (pANCA) may be There have been numerous reports of a new clinical entity coined
positive. autoimmune pancreatitis/IgG4-associated sclerosing cholangitis
• Imaging is helpful in detecting PSC, which manifests with strictured with peculiar clinical and therapeutic characteristics. IgG4-related
and dilated bile ducts, both intra- and extrahepatic.
sclerosing cholangitis is more frequent in men than in women,
and usually at an older age. The clinical features of the disease
appear similar to those with CCA or PSC. IgG4-related sclerosing
cholangitis has been associated with autoimmune pancreatitis,
tHeraPeUtIC PrInCIPLes and the coexistence of both manifestations is suggestive of IgG4-
related disease. The differential diagnosis with PSC may be
• Ursodeoxycholic acid (UDCA) (20 mg/kg) may be effective in slowing difficult; however, elevated IgG4 serum concentration (up to
primary sclerosing cholangitis (PSC) progression.
• Endoscopic procedures are indicated to treat stenoses. 10% of PSC patients may have elevated levels) and longer stenosis
• Liver transplantation is highly effective, and PSC represents an important found by imaging may support the diagnosis. Histology is
indication for such procedure, particularly due to the younger age of important, as it may discover an IgG4-positive lymphoplasmacytic
affected patients. tissue infiltrate. Overall, the prognosis is considered to be good;
however, some cases of portal hypertension and liver cirrhosis
have been reported.
Corticosteroids induce a remarkable improvement in clinical
AUTOIMMUNE CHOLANGITIS and biochemical manifestations, and a dosage of 0.6 mg/kg/day
of prednisolone should be started for 2–4 weeks and gradually
The term autoimmune cholangitis was first introduced to indicate tapered over 2–3 months. Immunosuppressant agents have been
AMA-negative PBC, possibly with serum ANA. However, more used effectively in anecdotal cases. The optimal treatment is still
recently this category has been expanded to include (i) serum debated.

CHaPter 76 Inflammatory Hepatobiliary Diseases 1029



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CHaPter 76 Inflammatory Hepatobiliary Diseases 1029.e1


MUL t IPL e CHOIC e QU est IO ns

1. Autoimmune liver diseases: 4. Autoimmune hepatitis:
A. In all cases affects women more than men A. Is associated with serum ANCA
B. Are categorized by the type of cells targeted B. Is associated with serum ANA
C. Are invariably associated with IBD C. Is associated with serum AMA
D. Are invariably ANA positive D. Is associated with serum hypogammaglobulinemia
2. Primary biliary cholangitis: 5. Primary sclerosing cholangitis:
A. Is predominant in men A. Is associated with IBD
B. Is predominant in children B. Is diagnosed most efficiently with liver histology
C. Is predominant in women C. Is associated with serum ANA
D. Affects equally both sexes D. Is associated with serum AMA
3. Primary biliary cholangitis is associated more frequently with:
A. Rheumatoid arthritis
B. Fybromialgia
C. Infertility
D. Sjogren syndrome

77









Immunotherapy of Cancer



Anu Sharma, Matthew Campbell, Cassian Yee, Sangeeta Goswami,
Padmanee Sharma





The ability of the immune system to recognize and eradicate KEY CONCEPTS
cancer was first postulated in the 19th century; however, proof
of principle remained elusive until later. It was noted that some Three Phases of Immune Interaction With
patients with sarcoma underwent spontaneous regression of their Tumor Cells
tumors upon incidental development of a skin infection with • Elimination: Tumor cells are detected and destroyed by the immune
Streptococcus pyogenes, which was conjectured to elicit an immune system.
response against the infection as well as against cancer cells. • Equilibrium: Tumor cells are not eliminated by the immune system.
After this observation, a mixture of heat-killed S. pyogenes and • Escape: Tumor cells escape immune control.
Serratia marcescens (Coley’s toxin) was used to treat patients
with sarcoma, which resulted in complete tumor regression in
1
some patients. These early studies with Coley’s toxin prompted
many clinical trials aimed at stimulating the immune response The continuous interaction of cancer cells with the immune
to eradicate cancer, including the use of Bacille Calmette-Guérin system may favor the outgrowth of less immunogenic tumors that
2
(BCG) as a treatment for superficial bladder cancer. However, can escape immune control, termed the escape phase. Tumor cell
incomplete understanding of the mechanistic details of immune escape can occur through different mechanisms including (i) loss
responses led to failure of many early clinical trials. With recent of antigens and/or MHC expression; (ii) increased resistance to the
advances in our understanding of basic principles that guide cytotoxic effects of immunity through induction of antiapoptotic
immune responses, specifically T-cell responses, immunotherapy mechanisms; (iii) development of defects in antigen processing/
is now established as one of the pillars of cancer treatment. presentation; (iv) loss of crucial genes, such as genes related to the
One important principle that has reshaped our thinking of interferon (IFN) signaling pathway, which makes tumor cells more
cancer immunology is related to immunosurveillance. Cancer resistant to immune-mediated killing by IFN; or (v) recruitment
immunosurveillance relies on immune cells, including T cells, of immunosuppressive cells to the tumor microenvironment,
which can recognize antigens, including mutated proteins that which can inhibit antitumor immune responses and contribute
generate novel antigens. T cells recognize these antigens when to the escape of edited tumor variants. 5
they are bound to self-MHC (major histocompatibility complex)
molecules.
In the early 2000s, the hypothesis of cancer immunosurveil- KEY CONCEPTS
lance was redefined to state that the immune system controls Mechanisms of Tumor Cell Immune Escape
both the tumor quality (immunogenicity) and the quantity
(tumor load), thus eliminating tumors and sculpting the immu- • Loss of antigen or major histocompatibility complex (MHC)
expression
nogenic phenotypes of tumors in immunocompetent hosts. The • Resistance to cytotoxicity
interaction between the host immune system and the tumor • Defects in tumor antigen processing/presentation
cells has been proposed in three phases, including elimination, • Loss of crucial genes involved in the immune response
equilibrium, and escape (the three E’s). In the elimination phase, • Recruitment of immunosuppressive cells to the tumor microenviron-
cancer cells are detected and destroyed by the adaptive and innate ment
arms of the immune system before becoming clinically apparent.
These data helped to frame the importance of the immune system
in cancer development and revived the theory of cancer Besides increasing our understanding of immunosurveillance,
immunosurveillance. 3 we have gained new insights regarding mechanisms that regulate
Cancer cells that evade elimination enter the equilibrium T-cell responses, which have led to the development of an entirely
phase, where T cells keep the remaining cancer cells in check new field known as immune checkpoint therapy. The current
without eliminating them. This phase may extend throughout chapter will discuss (i) Activation and regulation of T-cell
3
the host’s lifetime. Clinical examples of the equilibrium phase responses, (ii) Immune checkpoint therapy, (iii) Other inhibitory
include the duration between successful treatment of the primary immune checkpoints, (iv) Immune checkpoint therapy
tumor and relapse, the existence of disease-free state despite with clinical benefits in solid tumors and hematological malignan-
micrometastasis, and the development of donor-derived tumors cies, (v) Early and late-phase trials with immune checkpoint
after organ transplant. 4 therapy in other tumors, (vi) Immune costimulatory molecules,

1033

1034 ParT EighT Immunology of Neoplasia




Immune Other inhibitory
checkpoint therapy immune checkpoints
Anti-CTLA-4 TIM-3
Anti-PD-1/PD-L1 LAG-3
VISTA

Immune
Oncolytic virus costimulatory
immunotherapy molecules
ICOS
IMLYGIC (T-VEC)
4-1BB
OX40
Cancer Adoptive
vaccines cell therapy
Preventative vaccines Viral specific T cells
Therapeutic vaccines CAR T cells
T cells


Cytokine Monoclonal
therapy antibodies
IFN-α2b Conjugated mAbs
IL-2 Bispecific mAbs
Naked mAbs


Fig 77.1 Anticancer immunotherapeutic strategies.



(vii) Adoptive cell transfer, (viii) Monoclonal antibodies,
(ix) Cytokine therapy, (x) Cancer vaccines, (xi) Oncolytic virus
immunotherapy, (xii) Clinical challenges in immunotherapy,
and (xiii) Perspectives on future developments. Various anticancer
immunotherapeutic strategies are illustrated in Fig. 77.1.
APC
ACTIVATION AND REGULATION OF
T-CELL RESPONSES MHC CD80/CD86
Peptide
T-cell activation involves complex interactions involving both Signal 1 TCR Signal 2
T-cell receptor (TCR) signaling and CD28 costimulation (Fig. CD28
77.2) (Chapter 12). T-cell receptor interacts with foreign antigen
in the context of self-MHC, which provides “signal 1.” However,
signal 1 by itself is insufficient to enable T-cell activation.
“Signal 2” is provided when CD28 receptor, which is constitutively T cell
expressed on T cells, binds to B7-1 (CD80) and B7-2 (CD86)
molecules that are expressed on antigen-presenting cells (APCs)
but not on tumor cells. Therefore tumor cells alone are unable
to start the process of T-cell activation. T cells require fragments
of tumor cells to be phagocytosed by APCs such as dendritic T cell activation
cells, with eventual antigen processing and presentation by Fig 77.2 T cell activation. The “signal 1” for T cell activation, occurs
the APCs. T cells then interact with the APCs to receive both following the recognition of MHC-peptide complex on an antigen-
signals 1 and 2 for appropriate T-cell activation, which leads to presenting cell (APC) by the T cell receptor (TCR) on a T cell. The
cytokine production and proliferation as well as active killing “signal 2” for T cell activation is provided by binding of B7 molecules
of tumor cells. (CD80/ 86) on the APC to CD28 on the T cells. Following this interaction,
Early research in the mid-1990s demonstrated that T-cell T cells are activated and perform various effector functions.
activation was a complex event, which enabled proliferation and
functional differentiation but also induced inhibitory pathways high homology to CD28 and binds to B7 (CD80 and CD86)
that eventually could attenuate and terminate T-cell responses. molecules with much higher affinity than CD28. Two research
The first intrinsic T-cell inhibitory molecule to be described groups independently showed that activation of T cells results
was cytotoxic lymphocyte antigen-4 (CTLA-4), which is in induction of CTLA-4, which accumulates at the T cell–APC
expressed on T cells only after T-cell activation. CTLA-4 has interface and eventually outcompetes CD28 for binding to B7,

ChaPTEr 77 Immunotherapy of Cancer 1035


Fig 77.3 Regulation of T cell response. Upon T cell activa-
tion, T cells traffic to tumor leading to the cell surface
expression of immune checkpoints such as CTLA-4, which
bind with a higher affinity to the B7 molecules, thereby
downregulating T cell responses. Targeting CTLA-4 with
anti-CTLA-4 antibodies blocks this inhibitory interaction and APC APC
CD28 is again free to interact with the B7 molecules, further
amplifying T cell responses against the tumor. CD28 CD80/CD86
Signal 1 Signal 1 Signal 2
CTLA-4 Inhibitory
signal
anti-CTLA-4
CTLA-4
T cell T cell




T cell inactivation T cell activation



6,7
thereby abrogating T-cell responses. Based on these data, two ligands, PD-L1 and PD-L2, which appear to be equally capable
researchers proposed to enhance antitumor immune responses of suppressing T-cell responses. 11
by blocking the CTLA-4 inhibitory pathway (Fig. 77.3). This Antibodies targeting the PD-1/PD-L1 interaction have shown
approach, termed immune checkpoint therapy, has proven to beneficial clinical responses in multiple tumor types. Nivolumab
be a paradigm shift in cancer therapy. was the first mAb targeting PD-1 to show significant clinical
activity in patients with unresectable or metastatic melanoma,
IMMUNE CHECKPOINT THERAPY non–small-cell lung carcinoma (NSCLC), metastatic RCC, and
classical Hodgkin lymphoma. 11
Anti-CTLA-4 Therapy Given the considerable activity of agents targeting immune
CTLA-4 is an immune checkpoint that is expressed on the surface checkpoints, numerous early studies have been conducted across
of activated T cells to restrain T-cell responses. In other words, several solid tumors. Presented in Table 77.1 is the published
unrestricted T-cell responses can damage normal tissues and literature for agents that have been US Food and Drug Administra-
cells; therefore CTLA-4 plays an important role in controlling tion (FDA) approved or tested in the phase III setting in mela-
T-cell responses and in preventing damage to self. This mechanism noma, RCC, and NSCLC.
protects normal cells, but in the setting of cancer, this mechanism
also protects cancer cells. Preclinical studies elegantly demon- KEY CONCEPTS
strated that blocking CTLA-4 enhances antitumor immune
responses. 8 Primary Targets of Immune Checkpoint Therapy
Ipilimumab was the first fully human monoclonal (IgG1) • CTLA-4: interferes with T-cell costimulatory signal
antibody (mAb) against CTLA-4 to enter clinical trials in the • PD-1: interferes with T-cell receptor signaling
late 1990s. Phase I and II trials demonstrated remarkable clinical
responses and tumor regression in cancer patients, including
patients with melanoma, renal cell carcinoma (RCC), prostate The blockade of CTLA-4 and PD-1/PD-L1 inhibitory interac-
9
cancer, urothelial carcinoma, and ovarian cancer. In both early tions has been extensively explored clinically. Nevertheless, these
and late-phase trials, ipilimumab has shown consistent activity interactions represent only a fraction of the potential targets
against melanoma. The success of anti-CTLA-4 in eliciting durable that can serve to improve antitumor immune responses. Addi-
clinical responses and in potentially curing some patients with tional inhibitory pathways have been identified and are currently
cancer led to the establishment of a new field termed immune being targeted to improve antitumor immune responses. A few
checkpoint therapy, which has expanded with the identification of these are discussed in the sections that follow.
of additional T-cell inhibitory pathways. 10
T-Cell Immunoglobulin and Mucin Domain 3
Anti-PD-1/PD-L1 Therapy T-cell immunoglobulin and mucin-domain containing-3 (TIM-3)
Programmed death 1 (PD-1) belongs to the CD28/CTLA-4 family is expressed by dendritic cells, monocytes, and T cells. TIM-3 is
and is expressed on the surface of activated T cells, B cells, postulated to bind to its presumed ligand galectin-9, which is
monocytes, dendritic cells (DC) and natural killer (NK) cells. expressed on tumors. Studies in murine models indicate that
Its role in T-cell inhibition was first demonstrated in early 2000. TIM-3 is coexpressed with PD-1 on T cells. Dual blockade of
Unlike CTLA-4, PD-1 appears to inhibit T-cell responses by TIM-3 and PD-1 has been shown to improve antitumor immune
12
interfering with TCR signaling, which is distinctly different from responses and reduce tumor growth in preclinical studies.
CTLA-4 and its ability to inhibit T-cell responses by competing In a phase I/II trial, the safety and efficacy of MBG453, an
with CD28 for binding to B7 molecules. PD-1 interacts with antibody against TIM-3, is being evaluated as a single agent

1036 ParT EighT Immunology of Neoplasia



TABLE 77.1 FDa approval of immune Checkpoint inhibitors in the Treatment of Cancer
approval
Cancer Type Drug Dose, Frequency, Duration Disease State Line of Treatment Year
Melanoma Ipilimumab 3 mg/kg IV Q3wk × 4 doses Unresectable/Metastatic Any
until PD/tox
Melanoma Pembrolizumab 2 mg/kg IV Q3wk until PD/tox Unresectable/Metastatic Refractory 2014
Melanoma Nivolumab 3 mg/kg IV Q2wk until PD/tox Unresectable/Metastatic Post ipilimumab 2014
Melanoma Pembrolizumab 2 mg/kg IV Q3wk until PD/tox Unresectable/Metastatic Initial 2015
Melanoma Nivolumab 3 mg/kg IV Q2wk until PD/tox Unresectable/Metastatic Initial 2015
Melanoma Ipilimumab plus Ipi 3 mg/kg IV Q3wk ×4 + Nivo Unresectable/Metastatic Initial 2015
Nivolumab 1 mg/kg IV Q3wk ×4 followed by BRAF V600 WT
Nivo Q3wk ×4 until PD/tox
Melanoma Ipilimumab 3 mg/kg IV Q3wk × 4 doses 12 Surgically resected, Adjuvant 2015
until PD/tox high risk for recurrence
Kidney Nivolumab 3 mg/kg IV Q3wk until PD/tox Advanced Post 1 antiangiogenic therapy 2015
NSCLC All PD-L1 + Pembrolizumab 2 mg/kg IV Q3wk until PD/tox Advanced Post platinum 2015
NSCLC All Nivolumab 3 mg/kg IV Q2wk until PD/tox Advanced Post platinum, Post EGFR/ALK 2015
for pt with mutation
NSCLC Atezolizumab 1200 mg/kg IV Q3wk until PD/tox Advanced Post platinum, Post EGFR/ALK 2016
for pt with mutation
Urothelial Atezolizumab 1200 mg IV Q3wk until PD/tox Unresectable/Metastatic Post platinum 2016
Hodgkin lymphoma Nivolumab 3 mg/kg IV Q3wk until PD/tox Post autologous SCT, 2016
posttransplant brentuximab
vedotin
Head and Neck Pembrolizumab 200 mg IV Q3wk until PD/tox Unresectable/Metastatic On or post platinum 2016
Squamous
Head and Neck Nivolumab 3 mg/kg IV Q2wk until PD/tox Unresectable/Metastatic On or post platinum 2016
Squamous

ALK, anaplastic lymphoma kinase; EGFR, epidermal growth factor receptor; FDA, US Food and Drug Administration; IV, intravenous; ipi, ipilimumab; nivo, nivolumab; NSCLC,
non–small-cell lung carcinoma; PD, disease progression; pt, patients; SCT, stem cell transplant; tox, toxicity; wk, week; WT, wild type.


and in combination with anti-PD-1 in patients with advanced A randomized phase III trial in patients with metastatic
malignancies (ClinicalTrials.gov). melanoma was conducted whereby patients received either
anti-CTLA-4 antibody (ipilimumab), with or without a peptide
Lymphocyte Activation Gene 3 (gp100) vaccine, or vaccine treatment alone. The observed median
Lymphocyte activation gene-3 (LAG-3) is expressed on B cells, overall survival (OS) was 10 months for patients who received
some T cells, and NK cells. LAG-3 is postulated to bind to MHC ipilimumab plus gp100 vaccine, 10.1 months for patients who
13
class II, which inhibits T-cell responses. Immuntep (IMP321), received ipilimumab alone, and 6.4 months for patients who
a recombinant soluble fusion protein of LAG-3, is currently being received only the gp100 vaccine. Importantly, approximately 20%
tested in two or more clinical trials (ClinicalTrials.gov). A phase of patients who received ipilimumab had long-term survival of
I study is also testing another anti-LAG-3 (BMS-986016) as greater than 3 years. These data led to the FDA approval of
monotherapy or in combination with anti-PD-1 (nivolumab) ipilimumab for metastatic melanoma in 2011. 15
in patients with advanced solid tumors (ClinicalTrials.gov). Another phase III trial in patients with previously untreated
metastatic melanoma demonstrated that OS was significantly
V-Domain Ig Suppressor of T-Cell Activation longer for patients who received ipilimumab plus dacarbazine
V-domain Ig suppressor of T-cell activation (VISTA) is a negative chemotherapy compared with patients who received dacarbazine
16
immune checkpoint ligand, which shares homology with PD-L1 alone. Additionally, ipilimumab versus placebo was investigated
and is primarily expressed on hematopoietic cells. Preclinical in patients with high-risk stage III melanoma after complete
studies with VISTA blockade have shown improved antitumor resection, where ipilimumab was given every 3 weeks for
immune responses, leading to impeded tumor growth and 4 doses, followed by every 3 months for up to 3 years. Median
14
improved survival. Clinical trials with anti-VISTA mAbs are recurrence-free survival was 46.5% in patients treated with
ongoing and include a phase I study with JNJ-61610588, a fully ipilimumab versus 34.8% for patients who received placebo,
human IgG1 anti-VISTA mAb (ClinicalTrials.gov). which led to the FDA approval of ipilimumab in the adjuvant
17
setting. Ipilimumab is approved in the metastatic as well as
IMMUNE CHECKPOINT THERAPY WITH in the adjuvant setting for the treatment of melanoma. Impor-
tantly, to provide an accurate estimate of long-term survival
CLINICAL BENEFIT IN SOLID TUMORS AND following treatment with ipilimumab, a pooled analysis was
HEMATOLOGICAL MALIGNANCIES performed from 10 prospective and two retrospective studies,
including the two phase III trials mentioned above. The data
Melanoma demonstrated long-term survival of approximately 20% of
In recent years, immunotherapy has dramatically changed the patients, with survival of 10 years noted for some patients.
landscape of melanoma treatment: Since 2011, four immuno- The results were independent of ipilimumab dosing or prior
therapies have been approved by the FDA. therapy. 18

ChaPTEr 77 Immunotherapy of Cancer 1037


Antibodies against another immune checkpoint inhibitor, KEY CONCEPTS
PD-1 (nivolumab), have also been explored for the treatment
of patients with metastatic melanoma. A phase III study in patients US Food and Drug Administration-Approved
with advanced melanoma who progressed after ipilimumab Checkpoint Inhibitors in Melanoma
therapy demonstrated an objective response rate (ORR) of 31.7% • Ipilimumab: metastatic melanoma, used in the adjuvant setting
in patients treated with nivolumab compared with an ORR of • Nivolumab: first-line treatment for metastatic melanoma; treatment
10.6% in patients treated with chemotherapy alone. 19 of refractory unresectable or metastatic melanoma
Another randomized clinical trial in patients with previously • Pembrolizumab: first-line treatment for metastatic melanoma
untreated metastatic melanoma without BRAF mutation showed • Ipilimumab plus nivolumab: first-line treatment for patients with previ-
an improved OS in patients treated with nivolumab (72.9%) ously untreated, unresectable, or metastatic melanoma
compared with patients treated with dacarbazine chemotherapy
(42.1%) at 1 year. Subgroup analysis demonstrated that the
nivolumab-treated patients had improved OS regardless of chemotherapy. OS and PFS were significantly better with
20
expression of the ligand for PD-1 (PD-L1) on tumor cells. nivolumab compared with docetaxel, regardless of PD-L1 expres-
Based on these two phase III trials, nivolumab was FDA approved sion on tumor cells. This led to FDA approval of nivolumab for
25
for the treatment of patients with refractory unresectable or the treatment of patients with squamous NSCLC. This approval
metastatic melanoma and also for the first-line treatment of was subsequently expanded to all subtypes of NSCLC, based on
patients with metastatic melanoma. another phase III study that demonstrated a median OS of 12.2
Another anti-PD-1 mAs, pembrolizumab, was investigated months for patients with nonsquamous NSCLC who received
in a phase II study that randomized patients with metastatic nivolumab treatment compared with 9.4 months for patients
melanoma who were previously treated with ipilimumab and a who received docetaxel. 26
BRAF inhibitor. Patients received either pembrolizumab 2 mg/ Similarly, another phase III study evaluated pembrolizumab
kg every 3 weeks, pembrolizumab 10 mg/kg every 3 weeks, or versus docetaxel for the treatment of patients with previously
chemotherapy. The 6-month progression-free survival (PFS) was treated advanced NSCLC (both squamous and nonsquamous).
significantly higher in patients treated with pembrolizumab Patients enrolled in this trial were selected to have at least 1%
compared with patients treated with chemotherapy (34% in of tumor cells positive for the expression of PD-L1. The median
2 mg/kg cohort, 38% in 10 mg/kg cohort, and 16% in chemo- OS observed with pembrolizumab was 10.4 months with the
21
therapy cohort). The FDA approved pembrolizumab at a dose 2 mg/kg dose and 12.7 months with the 10 mg/kg dose versus
of 2 mg/kg intravenous every 3 weeks for the treatment of 8.5 months with docetaxel. In patients with at least 50% of tumor
refractory melanoma. Pembrolizumab versus ipilimumab was cells expressing PD-L1, the OS was 14.9 months with pembro-
then investigated in another phase III trial, where patients were lizumab 2 mg/kg and 17.3 months with pembrolizumab 10 mg/
randomized to two pembrolizumab regimens (10 mg/kg dose kg versus 8.2 months after treatment with docetaxel. Based on
every 2 weeks or every 3 weeks) and an ipilimumab regimen these findings, the FDA approved pembrolizumab as single-agent,
(3 mg/kg dose every 3 weeks for 4 cycles), with PFS and OS as second-line therapy for patients with NSCLC whose tumors
the primary endpoints. The 6-month PFS was 47.3% with express PD-L1. 27
pembrolizumab treatment every 2 weeks compared with 46.4% Another phase III trial investigated pembrolizumab versus
with pembrolizumab treatment every 3 weeks, and 26.5% with chemotherapy in previously untreated patients with advanced
ipilimumab treatment. One-year estimates of survival were 74.1% NSCLC, with PD-L1 expression on at least 50% of tumor cells
for patients receiving pembrolizumab every 2 weeks, 68.4% for and without epidermal growth factor receptor (EGFR) or
those receiving pembrolizumab every 3 weeks, and 58.2% for anaplastic lymphoma kinase (ALK) mutations. The study
those receiving ipilimumab, leading to the FDA approval of demonstrated statistically significant improvements in OS for
pembrolizumab as a first-line treatment for patients with meta- patients randomized to pembrolizumab compared with chemo-
static melanoma. 22 therapy; the study also showed a significant improvement in
Another phase III trial compared ipilimumab, nivolumab, median PFS of 10.3 months in the pembrolizumab group versus
28
and the combination in patients with untreated, unresectable, 6.0 months in the chemotherapy group. This study led to the
or metastatic melanoma with PFS and OS as coprimary endpoints. approval of pembrolizumab as first-line therapy for the treatment
Treatment with combination therapy was statistically superior, of patients with metastatic NSCLC whose tumors express PD-L1.
leading to a median PFS of 11.5 months versus 6.9 months for Several other PD-1/PD-L1 checkpoint inhibitors are in late-
nivolumab alone versus 2.8 months for ipilimumab alone. stage clinical testing for patients with lung cancer.
However, the data on the OS were not sufficiently mature to
present. Based on these findings, the FDA approved combination
therapy with ipilimumab plus nivolumab as first-line treatment
for patients with previously untreated, unresectable, or metastatic KEY CONCEPTS
melanoma, regardless of PD-L1 expression on tumor cells. 23 FDA-Approved Checkpoint Inhibitors in Non–
Lung Cancer Small-Cell Lung Cancer
Similar to melanoma, NSCLC is also characterized by high • Nivolumab: treatment of all subtypes of non–small-cell lung cancer
24
mutational load, and immune checkpoint therapy has led to (NSCLC)
durable antitumor response in NSCLC. • Pembrolizumab: single-agent, second-line therapy for NSCLC expressing
In a phase III study, patients with advanced squamous NSCLC PD-L1
who progressed during or after first-line chemotherapy were • Pembrolizumab first-line therapy for metastatic NSCLC expressing
PD-L1
randomly assigned to receive nivolumab versus docetaxel

1038 ParT EighT Immunology of Neoplasia


chemotherapy. The approval was based on data from a nonran-
Renal Cell Carcinoma domized trial in patients with HNSCC who failed to respond
36
Antiangiogenic therapies have been the established treatment to platinum-based chemotherapy. Following treatment, pem-
for patients with metastatic RCC. However, patients eventually brolizumab demonstrated an ORR of 16%. The median OS
develop innate or adaptive resistance to antiangiogenic therapy, observed with pembrolizumab was 9.6 months, compared with
which could be attributed partly to changes in the immune the historical median OS of only 6 months. In this trial, responses
29
microenvironment, proving a strong rationale to investigate were seen in patients with human papillomavirus (HPV)-positive
the clinical effects of immune checkpoint therapy in RCC patients. tumors (24%) as well as in patients with HPV-negative tumors
A phase III study in patients with advanced or metastatic (16%), respectively.
RCC, who had received at least one prior treatment, demonstrated Another phase III trial investigated nivolumab versus standard
improved OS of 25 months after treatment with nivolumab single-agent systemic therapy (methotrexate, docetaxel, or cetux-
compared with OS of 19.6 months in patients treated with imab) in patients with recurrent or metastatic HNSCC after
30
everolimus. Based on this trial, the FDA approved nivolumab platinum-based chemotherapy. This study demonstrated that
as second-line therapy for patients with metastatic RCC. New treatment with nivolumab resulted in longer OS of 7.5 months
immune-based treatment strategies for treating RCC patients compared with an OS of 5.1 months with standard single-agent
37
are in development. therapy. These findings led to the FDA approval of nivolumab
in the treatment of platinum-refractory recurrent HNSCC.
Bladder Cancer
Platinum-based chemotherapy has been the established first-line Hodgkin Lymphoma
treatment for patients with metastatic bladder cancer; no new A phase I study of nivolumab in previously treated patients
therapies have been established in the past 30 years, including with relapsed or refractory Hodgkin lymphoma demonstrated
no standardized second-line treatment because of the dismal an objective response of 87%, including 17% of patients with a
objective response rates of 10% or less with tested chemotherapies. complete response and 70% of patients with a partial response;
However, preclinical studies have demonstrated clinical benefits the remaining 13% had stable disease. The Reed-Sternberg cells
31
with immune checkpoint therapy in bladder tumors. A single- that define Hodgkin lymphoma have frequent amplification of
arm phase II study investigated the role of an anti-PD-L1 antibody chromosome 9p24.1, which leads to expression of PD-1 ligand in
(atezolizumab), to block one of the ligands that interacts with Reed-Sternberg cells and provides strong rationale for immune
38
PD-1, in patients with locally advanced or metastatic urothelial checkpoint therapy that blocks the PD-1/PD-L1 axis. Findings
bladder cancer whose disease had progressed after previous from this study led to FDA approval of nivolumab for patients
treatment with platinum-based chemotherapy. Compared with with previously treated relapsed or refractory Hodgkin lymphoma.
the historical control ORR of 10%, treatment with atezolizumab
resulted in a significantly improved ORR of 15% for all treated
32
patients, regardless of PD-L1 expression. This study led to FDA EARLY AND LATE-PHASE TRIALS WITH
approval of atezolizumab in patients with locally advanced or CHECKPOINT THERAPY IN OTHER TUMORS
metastatic urothelial bladder cancer whose cancers had progressed
during or after treatment with platinum-based chemotherapy. Pancreatic Cancer
A phase I/II study investigated nivolumab in patients with Pancreatic cancer has a poor prognosis with limited treatment
locally advanced or metastatic urothelial cancer whose disease options. A poor understanding of the immune microenvironment
had progressed after previous treatment with platinum-based in pancreatic cancer has been a roadblock in developing effective
chemotherapy. This study demonstrated that nivolumab mono- treatment strategies. We have generated preliminary data to
33
therapy led to an ORR of 24.4%. Another, larger phase II study indicate that pancreatic tumors have immune infiltrates and
assessed the clinical activity and safety of nivolumab in 270 differentially express inhibitory immune checkpoints compared
patients with metastatic bladder cancer who had progressive with tumors that respond to checkpoint therapy, such as mela-
39
disease despite first-line platinum-based chemotherapy. This noma and bladder cancer. Therefore treatment of pancreatic
study reported an ORR of 19.6% with nivolumab therapy. 34 cancer with combination strategies that include immune
Additionally, in another phase I/II study that treated patients checkpoint therapy is being explored.
with metastatic bladder cancer who progressed after platinum- Early-phase trials with ipilimumab have demonstrated delayed
40
based chemotherapy, patients who received combination treatment responses in patients with advanced pancreatic cancer. Immune
with ipilimumab 3 mg/kg plus nivolumab 1 mg/kg had an ORR checkpoint therapies that are under development for treating
of 38.5% versus an ORR of 26% for patients who received ipili- pancreatic cancer include pembrolizumab, nivolumab with or
mumab at 1 mg/kg plus nivolumab at 3 mg/kg; patients without ipilimumab, and durvalumab with or without treme-
35
who received nivolumab alone had an ORR of 24.4%. These limumab (ClinicalTrials.gov).
early findings with higher-dose ipilimumab provide evidence
for improved responses with combination immune checkpoint Prostate Cancer
therapy. Despite advances in the treatment of metastatic castration-
resistant prostate cancer (mCRPC), novel immune-based strategies
Head and Neck Cancer that can provide durable and long-term responses in patients
Head and neck cancer is one of the cancer types for which new are still needed. Sipuleucel-T (autologous cellular immunotherapy)
immune-based cancer treatments are currently in development. is the only approved immunotherapy for patients with prostate
41
In 2016, pembrolizumab was FDA approved for the treatment cancer. However, the exact mechanism of action of sipuleucel-T
of patients with recurrent or metastatic head and neck squamous- remains to be elucidated, and clinical metrics and/or biomarkers
cell carcinoma (HNSCC) that continued to progress despite to evaluate responses with sipuleucel-T are currently lacking.

ChaPTEr 77 Immunotherapy of Cancer 1039


Several early-phase clinical trials have evaluated different doses, costimulatory signal for T-cell proliferation and survival. We
+
schedules, and combinations of immune checkpoint inhibitors have shown that ICOS effector T cells increase in patients after
45
in patients with mCRPC. A phase I/II trial (CA184–107) of anti-CTLA-4 (ipilimumab) treatment. An increased frequency
+
ipilimumab as a single agent or in combination with radiotherapy of ICOS CD4 T cells was also identified as a pharmacodynamic
46
in patients with previously treated mCRPC demonstrated a biomarker of anti-CTLA-4 therapy. Another study with a
42
decline in prostate-specific antigen (PSA) in 15% of patients. different anti-CTLA-4 antibody (tremelimumab) also reported
+
However, a phase III clinical trial (CA184–043) of bone-directed an increase in ICOS CD4 T cells in blood samples obtained
47
radiotherapy followed by either ipilimumab or placebo in patients from patients with breast cancer. Collectively, these findings
with mCRPC failed to demonstrate an improvement in OS. 43 indicated a biologically relevant role for ICOS in antitumor
Further studies are warranted to investigate the potential immune responses elicited by anti-CTLA-4 therapy.
effects of combining immune checkpoint therapy with other Furthermore, preclinical studies demonstrated that combina-
treatment strategies, including other immunotherapies, chemo- tion therapy with anti-CTLA-4 plus an agonist to target the
therapy, radiotherapy, and hormonal agents, in patients with ICOS/ICOSL pathway led to a significant improvement in T-cell
48
prostate cancer in different treatment settings (metastatic hor- responses and tumor rejection. These data led multiple phar-
mone-naïve or metastatic castration-resistant disease). maceutical companies to pursue the development of an anti-ICOS
antibody, including a recent trial with GSK3359609, an anti-ICOS
IMMUNE COSTIMULATORY MOLECULES antibody for treating patients with advanced solid tumors
(NCT02723955; ClinicalTrials.gov).
Multiple ligand-receptor interactions play a pivotal role in shaping ICOS previously was shown to be expressed on T-follicular
49
T-cell responses, including those that positively regulate T-cell helper cells (Tfh cells). A clinical trial is ongoing with administra-
function, thus making them potential targets for cancer immu- tion of an anti-ICOS antibody as treatment for patients with
notherapy. Many such costimulatory molecules are being relapsed or refractory peripheral T-cell lymphoma follicular
investigated in preclinical or early-phase clinical studies. The variant or angioimmunoblastic T-cell lymphoma (ClinicalTrials.
two major families of costimulatory receptors expressed by T gov).
cells are the immunoglobulin-like (Ig) superfamily (including
inducible T-cell costimulator [ICOS]) and the tumor necrosis 4-1BB (CD137)
factor receptor (TNFR) superfamily (including OX40 and 4-1BB (CD137) is an activation-induced costimulatory molecule
44
4–1BB). These costimulatory molecules are suitable candidates that is expressed on activated T cells, NK cells, dendritic cells,
for the development of targeted therapeutics. A schematic eosinophils, mast cells, endothelial cells, and some tumor
overview of the costimulatory and coinhibitory ligand-receptor cells. Anti-4-1BB mAs have demonstrated improved antitumor
interactions is illustrated in Fig. 77.4. T-cell responses, with rejection of established syngeneic tumor
cell lines in preclinical models. Several early-phase trials with
Inducible T-Cell Costimulator anti-4-1BB antibodies have been initiated, and they have dem-
ICOS is another member of the CD28/CTLA-4 immunoglobulin onstrated antitumor responses, including partial remission and
superfamily, whose expression is increased upon T-cell activation. stabilization of disease in a subset of patients with advanced
ICOS can be expressed on activated effector T cells as well as malignancies; however, immune-related adverse events (irAEs)
on activated T regulatory cells (Tregs). ICOS interacts with its have led to reevaluation of the dose and schedule of treatment
50
ligand, ICOSL, which is expressed on APCs, and provides a key with anti-4-1BB agonists. Two separate phase I clinical studies







Macrophage
PD-L1/
MHC-II VISTA PD-L2


Coinhibitory VISTA
interactions Dendrictic cell LAG-3 receptor PD-1 PD-L1/
PD-L2

PD-1/PD-L1 PD-1 TIM-3 Galectin 9 or
B7 CTLA-4 other receptor
B7 CD28
MHC-peptide complex TCR Tumor cell
OX40 L OX40 T cell
Costimulatory 4-1BB L 4-1BB
ICOS
ICOS L
interactions
Fig 77.4 Costimulatory and coinhibitory ligand–receptor interactions: A schematic overview
of the costimulatory and coinhibitory interactions between a T cell and dendritic cell; T cell and
a macrophage and a T cell and a tumor cell in the tumor microenvironment.

1040 ParT EighT Immunology of Neoplasia


to assess the safety and immunoregulatory activity of urelumab
(BMS-663513) in patients with advanced and/or metastatic Adoptive Immunotherapy With Genetically
tumors and relapsed/refractory B-cell non-Hodgkin lymphoma Modified Lymphocytes
are under investigation (ClinicalTrials.gov). In early 2000, studies demonstrated that genetically engineered
lymphocytes transduced with a retrovirus encoding a TCR to
OX40 (CD134) recognize melanoma-melanocyte antigen (MART-1) could
OX40 (CD134) is expressed on activated T cells, including mediate tumor regression. 53
activated effector and Tregs. Engagement of OX40 with its ligand To improve the T-cell antigen specificity, a new class of
enhances proliferation and survival of T cells. Preclinical studies “chimeric antigen receptor” (CAR) was developed. This model
with OX40 have shown increased antitumor responses and overall was capable of overcoming limitations associated with central
survival. A trial with the murine anti-OX40 agonist 9B12 mAb and peripheral tolerance, generating efficient T cells. The advan-
in patients with advanced cancers was well tolerated and dem- tage of CAR compared with conventional TCR is that the antigen
onstrated regression of at least one metastatic lesion in 12 out need not be processed and presented by MHC. Importantly, this
51
of 30 patients. Humanized antibodies against OX40 are under approach can be used in all patients who express the same tumor
clinical development. A phase I first-in-human study to evaluate antigen, regardless of HLA type. 53,54
the anti-OX40 antibody GSK3174998 as monotherapy and in
combination with anti-PD-1 therapy (pembrolizumab) in patients Clinical Development of Chimeric Antigen Receptor
with advanced solid tumors is ongoing (ClinicalTrials.gov). In T Cells
addition, a phase Ib dose escalation study of the anti-OX40 Chimeric antigen receptor T cells (CAR T cells) are defined as
antibody MOXR0916 plus the anti-PD-L1 antibody atezolizumab T cells that are genetically modified to express an artificial
is ongoing in patients with locally advanced or metastatic solid construct, consisting of a synthetic TCR targeted at a predeter-
tumors (ClinicalTrials.gov). mined antigen expressed on tumor cells.
Coupling the CAR construct, which typically consists of a
ADOPTIVE CELL TRANSFER single-chain variable fragment (scFv; directed against a known
tumor antigen), with the aid of a linker and a spacer to the
Several clinical studies have demonstrated that the reinfusion transmembrane domain and a CD3ζ signaling domain, which
of autologous lymphocytes in cancer patients, isolated from their provides “signal 1” necessary for T-cell activation, paved its way
own tumor or peripheral blood, could inhibit tumor growth, as first-generation CAR T cells. Preclinical studies using first-
and these initial studies formed the basis for adoptive T-cell generation CAR T cells directed against CD19 expressing B-cell
therapy. 52 malignancies and HER2/Neu showed promising results. 55
For adoptive immunotherapy, the isolation of antigen-specific After this success, the second-generation CAR T cells were
T cells from the peripheral blood or tumor is followed by clonal developed by coupling the intracellular portion of the construct
expansion and transfusion back into the tumor-bearing host. with additional costimulatory signaling domains such as CD28
Adoptive T-cell transfer has achieved tumor regression and or 4–1BB, which provided “signal 2” for T-cell activation and
improved survival by increasing the number of reactive T further improved the efficacy of CAR T cells.
cells, providing long-term immune protection and guaranteeing
antigen specificity. The downside to this method is both the KEY CONCEPTS
expense and the expertise needed to perform this labor-intensive
therapy. Assuming that the T cells are already primed to the Chimeric Antigen Receptor (CAR) T Cells
tumor and are antigen-specific, they can be polyclonally activated Synthetic Receptor
ex vivo before being reinfused back into the patient. Suppressing
the immune system before adoptive transfer is important for • Single-chain variable fragment
• Linker and a spacer to the transmembrane domain
improving the antitumor efficacy. Significant progress has been • CD3ζ signaling domain
made to establish adoptive T-cell therapy as a standard-of-care • Additional costimulatory signaling domains
method for the treatment of cancer, and multiple studies are
ongoing. 53
Further optimization led to the development of the
Adoptive Transfer of Tumor-Specific Cytotoxic T Cells third-generation CAR T cells, which incorporated two-tandem
Studies in murine tumor models have demonstrated that the costimulatory domains by coupling ICOS, CD27, CD28, 4–1BB,
lymphocytes in transplantable tumors are capable of recognizing or OX40 (e.g., CD28/4–1BB/CD3ζ or CD28/OX40/CD3ζ), hence
tumor cells in vitro, and the adoptive transfer of these syngeneic demonstrating varying degrees of in vitro and in vivo T-cell activa-
tumor-infiltrating lymphocytes (TIL) could mediate tumor tion, proliferation, and cytokine (interleukin [IL]-2) production. 55
regression in certain cancers. In vitro studies demonstrated that The armored CAR T represent the next-generation CAR T
TIL obtained from melanoma patients could specifically recognize cells, which incorporate not only the two-costimulatory domains
autologous tumors. Further studies based on this observation but also an additional transgene for cytokines (e.g., IL-12) or
demonstrated that transfer of autologous TIL could mediate ligands (e.g., CD40L or 4–1BBL); this helps these CAR T cells
objective regression in patients with metastatic melanoma and to survive and/or disrupt an immunosuppressive tumor micro-
53
across other cancers. An alternative source of endogenous environment. 53,55 The key concepts of the four generations of
tumor-reactive T cells can be found in the peripheral blood of CAR T cells are illustrated in Fig. 77.5.
patients. Strategies to isolate and expand these rare circulating CTL019 is the first investigational CAR T to receive
T cells are being developed for further investigation in clinical breakthrough-therapy status by the FDA for the treatment of
trials to target a broader population of patients. 54 adult and pediatric relapsed/refractory acute lymphoblastic

ChaPTEr 77 Immunotherapy of Cancer 1041


CAR T Cells development of phage/yeasts or ribosome display technology helped
generate mAbs from any species and facilitated selection based
1st 2nd 3rd on specificity, stability, and affinity.
generation generation generation Armored The mounting success of a mAb as a therapeutic agent is based
V L V L V L V L on three characteristics, including (i) an Fc moiety that mediates
antibody-dependent cellular cytotoxicity (ADCC) and complement-
dependent cytotoxicity (CDC); (ii) an Fab moiety that promotes
high specificity and affinity for antigen binding; and (iii) a molecular
V H
V H
V H
V H
mass of about 150 kDa, which extends the circulatory half-life of
the mAb up to 21 days. Importantly, the mechanism of tumor
cell killing by mAbs involves (i) binding to specific receptors on
tumor cells via the Fab portion of the antibody and triggering
cytotoxicity via the Fc portion of the antibody or (ii) binding to
specific receptors on tumor cells via the Fab portion of the antibody
Cytokine and blocking important signaling pathways via the Fab portion
transgene
CD3ζ of the antibody or a combination of both mechanisms.
CD28 CD28 CD28 Over the past 15 years, antibody-based immunotherapy has
been established as a successful strategy for treating patients
Additional with hematological malignancies and some solid tumors. To
Linker costimulators enhance the effector functions, mAbs have been conjugated to
Spacer CD3ζ (ICOS, 4-1BB, radioisotopes, chemotherapeutic agents, bacterial toxins, cytokines,
OX40)
60
Intracellular and enzymes ; therefore mAbs are classified as follows:
domain
Single chain Naked mAbs
variable fragments CD3ζ CD3ζ
Cytokine transgene These mAbs are not attached to any drug or radioactive material
Fig 77.5 Different generations of CAR T cell constructs. The and bind to specific molecules or antigens on tumor cells, which
1st generation CAR T comprising of a T cell signaling domain; can block important signaling pathways, such as EGFR, or enable
the 2nd generation, with one costimulation domain and a T cell the immune system to destroy the tumor cells by ADCC or a
60
signaling domain; the 3rd generation, with two costimulation combination of both. ADCC is an important mechanism for
domains and a T cell signaling domain; and the armored CAR T tumor cell killing that is mediated by the interaction between the
cell with similar construct to third generation but with an additional Fc region of an antibody and FcγRIIIa receptors on the surface
pro-inflammatory cytokine transgene. of immune cells. mAbs may bind to a cell surface target via its
Fab region and engage the effector cell expressing FcγRIIIa with
the Fc region of the mAb, leading to subsequent killing of the
leukemia (NCT02228096; ClinicalTrials.gov). CTL019 is a second- tumor cell. ADCC is thought to be a key mechanism of activity
generation CAR T cell that comprises an anti-CD19 extracellular of the FDA-approved mAbs rituximab and trastuzumab. 60
scFv and CD3-ζ and 4–1BB intracellular signaling domains. Rituximab, a chimeric mAb targeted against the B-cell surface
Adoptive transfer of CAR T cells has demonstrated significant antigen CD20, was the first mAb to be FDA approved for thera-
promise in targeting hard-to-treat hematologic malignancies and peutic use against B-cell non-Hodgkin lymphoma (NHL),
solid tumors. Although the safety concerns related to cytokine CD20-positive NHL, and chronic lymphocytic leukemia (CLL).
release syndrome still need to be fully addressed, multiple CAR Trastuzumab (Herceptin) is another such mAb directed against
T cells are in clinical development. 56 human EGFR-2, a tyrosine kinase membrane receptor, which is
overexpressed on approximately 30% of breast cancer cells in a
Adoptive Transfer of Viral-Specific T Cells subset of breast cancer patients. Trastuzumab monotherapy has
The adoptive transfer of T cells isolated or engineered to have demonstrated prolonged disease stability in a significant number
specificity and reactivity for viral DNA can efficiently treat of advanced-metastatic breast cancer patients. 61
Epstein–Barr virus (EBV)-associated posttransplant lympho- Other successful mAbs approved by the FDA for patients with
57
proliferative disease. It is known that CD8 and CD4 virus-specific solid tumors include bevacizumab targeting vascular endothelial
T cells in chronic infections are characterized by the loss of T-cell growth factor (VEGF) (as first-line and second-line treatment of
function due to the upregulation of inhibitory molecules such metastatic colon cancer in combination with 5-FU chemotherapy;
as PD-1 and TIM-3; therefore the adoptive transfer of autologous in combination with carboplatin and paclitaxel chemotherapy as
T cells with high-affinity TCRs for viral proteins in combination first-line treatment of advanced NSCLC patients who have not
with inhibitors of PD-1 or TIM-3 could be an ideal approach yet been treated with chemotherapy; as a single agent in patients
for the targeted therapy of virally induced malignancies. 58 with glioblastoma whose tumors have progressed after treatment;
and for treating mRCC patients in combination with IFN-α);
MONOCLONAL ANTIBODIES cetuximab targeting EGFR (as a single agent in HNSCC patients
with failure to platinum-based therapy; in combination with
The establishment of hybridoma technology in 1975 enabled the radiotherapy for regionally advanced HNSCC; and as palliative
efficient development of mAbs. Immortalized B lymphocytes treatment of pretreated metastatic EGFR-positive colorectal
from immunized hosts were fused with nonimmunoglobulin cancer); panitumumab targeting EGFR (as a single agent for
murine myeloma cells, forming hybridomas, which produced treating pretreated EGFR-positive metastatic colorectal cancer);
59
large amounts of antibodies with high reactivity. Later the alemtuzumab targeting CD52 (as a single agent for treating CLL);

1042 ParT EighT Immunology of Neoplasia


and ofatumumab targeting CD20 (for the treatment of CLL human IL-10 (AM0010) is being investigated in patients with
patients refractory to fludarabine and alemtuzumab). advanced solid tumors. 66
However, certain properties of cytokines pose significant
Conjugated/Tagged/Labeled/Loaded mAbs clinical challenges in achieving antitumor responses. Some
These mAbs target a molecule or antigen expressed on tumors cytokines are pleiotropic and act on several immune cells and
and are coupled to a cytotoxic or radioactive agent to enable mediate opposite effects. High-dose IL-2 is known to promote
delivery of a toxic substance directly to the tumor site. Treatment cytotoxic activity of effectors (CD8 T cells and NK cells) and
with a radiolabeled mAb is termed radioimmunotherapy (RIT). differentiation of CD4 T cells into T helpers. However, IL-2
Ibritumomab tiuxetan is a radiolabeled mAb against the CD20 can also preferentially expand Tregs due to a higher affinity to
antigen expressed on B lymphocytes and is FDA approved to the IL-2 receptor (CD25) on these cells. Sometimes multiple
treat different lymphomas. A randomized phase III trial comparing cytokines have the same functional effect, leading to redundancy
ibritumomab tiuxetan to rituximab in patients with relapsed or of cytokine signaling; therefore the therapeutic manipulation of
refractory NHL demonstrated a higher overall response rate (80% cytokines can be challenging, as the modification of one cytokine
versus 56%) and complete response (CR) rate (30% versus 16%) can be compensated by another cytokine. 65
for ibritumomab tiuxetan, but similar time to disease progression
for both treatment groups. 62 CANCER VACCINES
A different approach that uses mAbs coupled to chemothera-
peutic agents is known as antibody-drug conjugates (ADC). Efforts to produce effective anticancer vaccines have been ongoing
Brentuximab vedotin (Zevalin) is a mAb conjugated to a chemotoxic for decades. The two known categories are preventive cancer
drug called monomethyl auristatin E (MMAE), targeting the cell vaccines and therapeutic cancer vaccines.
membrane antigen CD30 on lymphocytes. A phase II clinical study Preventive vaccines consist of those that target infectious
(NCT00848926; ClinicalTrials.gov) with brentuximab vedotin in organisms that can cause cancer such as strains of HPV, which
patients with refractory Hodgkin lymphoma demonstrated contribute to the development of some head/neck cancers, anal
complete remission in 34% of patients, partial remission in 40% cancers, and cervical cancers. The three FDA-approved vac-
63
of the patients, and tumor regression in 94% of patients. Bren- cines include Cervarix HPV (against HPV-16,-18, -31,-33,-45),
tuximab vedotin is FDA approved to treat Hodgkin and anaplastic Gardasil (against HPV-6,-11,-16,-18), and Gardasil 9 (against
large-cell lymphoma. Trastuzumab emtansine (T-DM1) is another HPV-6,-11,-16,-18,-31,-33,-45,-52,-58). 67
example of an antibody-drug conjugate, which is FDA approved For the treatment of established cancers, sipuleucel-T was
for the treatment of HER2-positive breast cancer. 61 the first vaccine to be FDA approved. It is a cell-based vaccine
consisting of autologous peripheral blood mononuclear cells
Bispecific Monoclonal Antibodies (PBMC), which are collected from each patient and activated
These mAbs are formed by coupling two different mAbs together ex vivo with a recombinant fusion protein PA2024 (consisting
and allowing the construct to bind to two separate proteins of a prostate antigen and prostatic acid phosphatase fused to
simultaneously, thus enabling the mAb to direct the immune GM-CSF) before reinfusion into the patient. A phase III clinical
system to act against the tumor. trial demonstrated significant improvement in median OS after
Blinatumomab is one such example where one part of the treatment with sipuleucel-T in asymptomatic men with castrate-
mAb binds to the CD19 protein expressed on B-lineage acute resistant prostate cancer. Treatment with sipuleucel-T also resulted
lymphoblastic leukemia cells, while the second part of the mAb in an improvement in the rate of 3-year survival in 31.7% of
67
binds to the CD3 protein found on T cells, thereby linking these patients compared with 23.0% for those receiving placebo. The
two cell types to activate the T cell to exert cytotoxicity on the exact mechanism of action for sipuleucel-T remains to be defined.
+
CD19 tumor cells. Blinatumomab was first approved by the
FDA in 2014, based on the observations of a phase II trial in ONCOLYTIC VIRUS IMMUNOTHERAPY
patients with relapsed or refractory acute lymphoblastic leukemia
in which 40% of patients achieved a CR or complete response Oncolytic viruses represent a new class of therapeutic agents
with partial hematologic recovery (CRh). 64 that promote antitumor immune responses, which depend on
mechanisms that elicit selective tumor cell killing and induction of
CYTOKINE THERAPY systemic antitumor immunity. A variety of native and genetically
modified viruses are being developed and clinically investigated
Cytokines are low-molecular-weight proteins or glycoproteins as oncolytic agents. 68
that mediate cell-to-cell communication and play a pivotal role IMLYGIC (talimogene laherparepvec, T-VEC) was the first
in regulating biological activities, including innate immunity, oncolytic viral therapy to receive FDA approval for the treatment
adaptive immunity, inflammation, and hematopoiesis. of unresectable cutaneous and subcutaneous and nodal lesions
IL-2 and IFN-α have been used previously to treat patients recurrent after surgery in melanoma patients. IMLYGIC dem-
with metastatic melanoma and RCC. However, due to the onstrated improved median OS in a phase III study in patients
69
nonspecific activation of the immune response and the toxicities with advanced melanoma. IMLYGIC is a genetically modified
associated with these cytokines, as well as the development of herpes simplex virus type designed to selectively replicate within
novel immunotherapy agents as discussed above, the use of the tumor cells and induce lysis.
high-dose IL-2 and IFN-α has diminished in clinical practice.
Additional cytokines, including granulocyte macrophage–colony- CLINICAL CHALLENGES IN IMMUNOTHERAPY
stimulating factor (GM-CSF), IL-7, IL-12, IL-15, IL-18, and IL-21,
have also been investigated in clinical studies for patients with Multiple challenges that remain for immunotherapy agents include
65
advanced cancers. Currently, the antitumor activity of PEGylated resistance mechanisms and immune-related adverse events.

ChaPTEr 77 Immunotherapy of Cancer 1043


IFNγ receptor, which initiates signaling through the Janus kinase/
Resistance Mechanisms signal transducer and activator of transcription (JAK/STAT)
73
Durable clinical responses with immune checkpoint therapy have pathway to regulate gene expression. Data from two independent
been well documented; some of these responses last for years. studies have established that the loss of genes involved in the
74
However, many patients do not respond to initial therapy or IFNγ signaling pathway may lead to both primary and adaptive
75
relapse with disease after initially responding to treatment. resistance to immune checkpoint therapy.
Resistance mechanisms may involve the following: (i) activation Previously published data from a study in patients with bladder
+
hi
of oncogenic pathways (WNT/B-catenin signaling, loss of PTEN cancer demonstrated increased frequency of a CD4 ICOS T-cell
45
expression), (ii) loss of interferon-gamma (IFNγ) signaling genes, subset that produced IFNγ following anti-CTLA-4 therapy. We
(iii) recruitment of immunosuppressive cells such as Treg, further hypothesized that tumor cells harboring mutations in
myeloid-derived suppressor cells, M2 macrophages, and expression the IFNγ-signaling pathway are not susceptible to the IFNγ
of other inhibitory immune checkpoints, and (iv) composition produced by these T cells, which may result in resistance to
74
of gut microbiome. anti-CTLA-4 therapy. Evaluation of genomic alterations of
IFNγ-pathway genes in melanoma patients treated with ipilim-
umab demonstrated that the tumors of the nonresponders had
KEY CONCEPTS loss of IFNγ-pathway genes at significantly higher frequencies
Mechanisms of Tumor Resistance to Immune- compared with the responders.
Mediated Therapy Furthermore, mice bearing melanoma tumors with knockdown
of IFNγ-receptor, resulted in impaired tumor rejection when
• Activation of oncogenic pathways treated with anti-CTLA-4 therapy.
• Loss of interferon-γ signaling genes in the tumor cells Similarly, another study compared paired tumor biopsies that
• Recruitment of immunosuppressive cells (e.g., Treg cells, myeloid-
derived suppressor cells) were collected from four melanoma patients before treatment
• Expression/secretion of inhibitory molecules with anti-PD-1 (pembrolizumab) and at the time of disease
75
• Composition of the gut microbiome relapse. In two out of the four patients whose disease relapsed
after initial responses to pembrolizumab therapy, mutations were
detected in the genes encoding interferon-receptor–associated
Activation of Oncogenic Pathways Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), two critical
Recent research has identified an intricate interplay between components of the IFNγ signaling pathway.
oncogenic pathways and antitumor immunity. Taken together, these findings indicate that the loss of IFNγ
In one such study, the whole exome sequencing and gene signaling limits the effectiveness of the immune system to
expression profiling of melanoma biopsies revealed that tumor-cell eliminate tumor cells and induces resistance to immune check-
intrinsic activation of the WNT/β-catenin pathway correlates with point therapy.
70
absence of T cells in the tumor microenvironment. Further
investigation in a genetically engineered autochthonous mouse Immunosuppressive Tumor Microenvironment
melanoma model confirmed that increased oncogenic β-catenin In addition to the above-mentioned genomic alterations in tumor
+
+
signaling results in a failure to recruit CD8α and CD103 DC cells, other resistance mechanisms within the tumor microen-
populations in the tumor due to repressed expression of the vironment include Treg cells, myeloid-derived suppressor cells
chemokine CCL4. Consequently, mice with tumors expressing active (MDSCs), M2 macrophages, expression of other inhibitory immune
β-catenin responded poorly to anti-CTLA-4 and anti-PD-L1 therapy, checkpoints (TIM-3, LAG-3, VISTA), recruitment of inhibitory
compared with mice with tumors lacking β-catenin expression. 70 molecules/ligands (indoleamine dioxygenase [IDO], carcinoem-
Apart from the activation of active β-catenin, the loss of PTEN bryonic antigen-related cell adhesion molecule 1 [CEACAM1],
expression is a frequent event in melanoma, especially in tumors PD-L1), and the secretion of inhibitory cytokines (IL-10, IL-35,
with BRAF mutations. PTEN is a negative regulator of the PI3K-AKT transforming growth factor [TGF]-β); these may have a direct
pathway, and the complete loss of PTEN is associated with increased negative effect on effector T-cell function and hence contribute
signaling through this pathway, correlating with shorter OS in toward immunosuppression and resistance to immunotherapy. 76,77
71
patients with advanced melanoma. A recent study in a melanoma
mouse model demonstrated that the loss of PTEN expression can Gut Microbiome
reduce the therapeutic activity of immune checkpoint therapy. Two independent preclinical studies reported that the composition
The study additionally demonstrated that a selective PI3K-β-isoform of the gut microbiome can contribute to the difference in
inhibitor could induce synergy with immune checkpoint therapy responses to immunotherapy in cancer patients. 78,79
72
in the preclinical model. Similarly, findings in human melanoma One study compared the tumor growth in mice with distinct
specimens confirmed that the loss of PTEN correlates with exclusion gut microbiota and identified that mice that harbored the com-
of CD8 T cell in the tumor and increased PI3K activation. Impor- mensal Bifidobacterium species had enhanced spontaneous
tantly, patients with tumors lacking PTEN expression demonstrated antitumor immunity compared with mice with a different gut
poor clinical responses to anti-PD-1 therapy compared with patients microbiota composition. Importantly, the direct administration
with retained PTEN expression. 72 of the Bifidobacterium species into mice with established mela-
noma tumors improved tumor-specific immunity and response
Loss of Interferon γ Signaling to anti-PD-L1 therapy. 78
It is well established that IFNγ, a critical cytokine produced Similarly, another study in tumor-bearing mice and cancer
by T cells and other immune cells, plays a role in promoting patients demonstrated that the efficacy of anti-CTLA4 therapy is
innate and adaptive immune responses and in inhibiting associated with T-cell responses specific for the microbiota species
tumor cell proliferation. IFNγ functions by binding to the Bacteroides fragilis. The study reported that antibiotic-treated and

1044 ParT EighT Immunology of Neoplasia



TABLE 77.2 Frequency of immune-related adverse Events in Phase iii Clinical Trials With
immune Checkpoint Therapies across Diseases

any Toxicity
all grades %/
Drug/ Disease/No. of pts grade ≥3 % Diarrhea Colitis Pneumonitis hepatitis rash Endocrine
Ipilimumab 33/5 8/5 NA 4/0 44/2 8/4
Melanoma 108
{Hodi FS, 2010 #10}
Ipilimumab Adjuvant
Melanoma Resected/ 99/55 49/10 16.3/8 NA 25/8 34/13 38/8
471{Eggermont AM, 201 #59}
Ipilimumab
MCRPC 393{Kwon ED, 2014 #56} 98/59 51/17 7/5 NA 12/4 17/1 5/2
Tremelimumab 96/52 51/18 NA included with NA 1/1 33/2 8/3
Melanoma 325{Ribas A, 2013 #58} diarrhea
Ipilimumab/ 311 99/56 33/6 12/9 2/0 7/2 35/0 11/2
Nivolumab/ 313 99/44 19/2 1/1 1/0 6/3 22/0 14/1
Combination/ 313 100/69 44/9 12/8 28/3 30/5
Melanoma
{Larkin J, 2015 #1}
Related to Tx
Pembro Q2wk / 278 80/13 17/3 2/1 0/0 1/1 15/0 17/1
Pembro Q3wk / 277 73/10 15/1 4/3 1/0 2/2 13/0 13/1
Ipilimumab / 256 73/20 23/3 8/7 0/0 1/0 15/1 7/2
Melanoma
{Robert C, 2015 #9}
Nivolumab
NSCLC Squamous 272 58/7 8/0 1/1 5/1 2/0 4/0 2/ 0
{Brahmer J, 2015 #43} 69/10 8/1 1/1 4/1 1/1 13/1 8/0
NSCLC Non-Squamous 292
{Borghaei H, 2015 #42}
Pembro
2 mg/kg / 339 63/15 7/1 1/1 5/2 1/<1 9/<1 14/1
10 mg/kg 343 66/16 6/1 1/<1 4/2 1/0 13/<1 16/1
NSCLC All{Herbst RS, 2015 #45}
Nivolumab
MRCC / 406{Motzer RJ, 2015 #31} 79/19 12/1 NA 4/1 NA 10/<1 NA
MCRPC, metastatic castrate-resistant prostate cancer; NSCLC, non–small-cell lung cancer; MRCC, metastatic clear-cell renal cell carcinoma; pembro, Pembrolizumab; NA, not
available; pts, patients.


germ-free tumor bearing mice did not respond to anti-CTLA-4 immune checkpoint antibodies can be associated with distinctive
81
therapy compared with mice harboring Bacteroides fragilis. inflammatory tissue damage, which has been termed immune-
Additionally, the study reported that ipilimumab-treated patients related adverse events (irAEs). Table 77.2 summarizes the adverse
with metastatic melanoma tend to have Bacteroides fragilis as a events associated with anti-CTLA-4 and anti-PD-1, respectively,
significant component of their microbiome. 79 in phase III clinical trials. The spectrum of irAEs related to
Collectively, these studies demonstrate the importance of gut anti-CTLA4 and anti-PD-1/PD-L1 immunotherapy includes
microbiota and its role in facilitating the anticancer effects of common reactions involving the gastrointestinal tract (diarrhea,
immune checkpoint therapy. colitis), skin (rash, dermatitis), liver (transaminitis), or endocrine
axis (hypophysitis, thyroiditis).
Immune-Related Adverse Events The combination of nivolumab (anti-PD-1) and ipilimumab
The clinical use of immunotherapeutic strategies is expanding (anti-CTLA-4) has a much higher incidence of irAEs compared
rapidly, and the side effect profile of immunotherapeutic agents is with monotherapy with either antibody. For instance, a phase
80
distinct from that of conventional anticancer agents. Therefore III clinical trial in patients with advanced melanoma receiving
it is important to recognize and manage the irAEs in the growing nivolumab or ipilimumab, or a combination of both antibodies,
patient population treated with immunotherapy. Early recogni- demonstrated grade 3/4 treatment-related adverse events in 16%
tion and treatment of symptoms, time to discontinuation of of patients treated with nivolumab, in 27% of patients treated
therapy in case of an adverse event, and adherence to established with ipilimumab, and in 55% of patients treated with the
treatment algorithms are essential to prevent patient morbidity combination. 82
and mortality. The optimal management of irAEs is based on clinical experi-
ence, and many of these irAEs are transient and responsive to
irAEs With Immune Checkpoint Immunotherapy steroid therapy. Various immunomodulatory or immunosup-
83
Immune checkpoint blockade with antibodies targeting CTLA-4 pressive agents such as azathioprine and mycophenolate mofetil
and PD-1/PD-L1 has been extensively evaluated in the clinical (MMF) have also been effective in the management of steroid-
setting. In a subset of patients, nevertheless, treatment with these refractory irAEs. 84

ChaPTEr 77 Immunotherapy of Cancer 1045


Recent findings from a retrospective study involving two clinical
trials with ipilimumab in patients with prostate cancer identified Biomarkers
CD8 T-cell clonal expansion within the systemic circulation as To identify potential biomarkers that are relevant to antitumor
85
a potential correlative biomarker of irAEs. These findings immune responses, clinical trials are being designed to obtain
highlight a potential biomarker to predict, and provide early tumor tissues to gain a better understanding of the tumor
intervention for, toxicities that occur in patients who receive microenvironment and of immunological changes that occur
10
ipilimumab therapy. over time. Several biomarkers have been associated with clinical
outcomes to immune checkpoint therapy, including high levels
irAEs With Adoptive T-Cell Therapy of PD-L1 expression, infiltration of effector T cells, and mutational
The adoptive transfer of T cells is generally well tolerated, but landscape. 70,93,94 However, it is unlikely that a single biomarker
it may be associated with the occurrence of life-threatening irAEs. will predict clinical outcome to immunotherapy; therefore, it
Serious irAEs after adoptive T-cell therapy may occur when T will be necessary to integrate multiple biomarkers in immune
cells targeting differentiation antigens in the tumor also recognize monitoring studies. Additionally, immune monitoring studies
these antigens on normal cells. For example, patients with should include gene expression studies, epigenetics studies, flow
melanoma who receive adoptive T-cell therapy with T cells that cytometry and CYTOF studies, immunohistochemical studies,
target differentiation antigens such as MART-1 and gp100 may mutational load studies, neoantigen studies, and microbiome
develop vitiligo and uveitis as irAEs, since these antigens are also studies. Sufficient access to longitudinal patient samples will be
expressed on normal cells. 86,87 Additionally, patients with meta- required to conduct these types of studies.
static RCC who were treated with carbonic anhydrase-IX
88
(CAIX)-specific CAR T cells developed liver toxicity. In addition, Combination Therapy
fatal irAEs were reported in cancer patients who received anti- Immune checkpoint therapy has resulted in durable clinical
MAGE-A3 TCR-transduced T cells. 89 responses, but in only a fraction of patients. On the basis of the
Adoptive T-cell therapy with CAR T cells may also be associ- encouraging results with monotherapy, combination treatment
ated with another potentially life-threatening toxicity termed strategies are being explored extensively. 95
as cytokine release syndrome (CRS). The hallmark of CRS is Because anti-CTLA-4 and anti-PD-1 antibodies have distinct
elevated circulating levels of cytokines—including IL-6, TNF-α, mechanisms of action, preclinical studies evaluated combination
and interferon-γ—resulting in fever, rigors, hypotension, and treatment with anti-CTLA-4 and anti-PD-1/PD-L1, which
hypoxia. 90,91 demonstrated improved antitumor immune responses. 31,96
Treatment of irAEs associated with adoptive T-cell therapy, A detailed study in preclinical tumor models demonstrated
such as uveitis and colitis, can often be managed with topical that treatment with a combination of anti-CTLA-4 and anti-PD-1
or systemic corticosteroids; as for CAR T-cell therapy–related antibodies improves antitumor immunity and synergistically
CRS, it is generally managed with either high-dose corticosteroids eradicates tumors, and this synergy relies on the interdependence
or agents such as tocilizumab (an IL-6 receptor-blocking between IL-7 and IFNγ signaling in T cells; the lack of either
antibody). 92 pathway abrogates the therapeutic effects of this combination
therapy. 31
PERSPECTIVES ON FUTURE DEVELOPMENTS In 2015 the FDA approved the first combination immuno-
therapy of nivolumab (anti-PD-1) plus ipilimumab (anti-CTLA-4)
Recent success with immunotherapy in the management of cancer for patients with previously untreated and unresectable metastatic
has given credence to the long-held belief that the immune system melanoma. 82,97 Patients receiving this combination had an objec-
can be used to treat cancer. However, the current immunothera- tive response rate of 50%. Importantly, this combination was
peutic strategies form only the tip of the iceberg in terms of effective irrespective of the PD-L1 expression. Several other
potential targets and combinations that can serve to improve combination therapies are currently being tested in clinical trials
clinical responses. A large number of additional agonistic and (ClinicalTrials.gov).
antagonistic mAbs that target immune checkpoints, as well as Combination treatments with immune checkpoint therapy
costimulatory molecule to potentiate T cell–mediated immune plus targeted therapy (angiogenesis inhibitors, tyrosine kinase
responses, are in various stages of clinical development; these inhibitors, BRAF inhibitors) also hold promise for improved
include antibodies against ICOS, OX40, 4–1BB, LAG-3, and VISTA efficacy. A phase I trial (NCT01472081; ClinicalTrials.gov)
(ClinicalTrials.gov). Furthermore, intense clinical investigation investigating the combination of nivolumab with sunitinib (a
is currently ongoing for the development of effective anticancer receptor tyrosine-kinase inhibitor) demonstrated tumor regression
98
vaccines and adoptive cell transfer strategies (ClinicalTrials.gov). in patients with metastatic RCC. The safety and efficacy of
another tyrosine-kinase inhibitor (lenvatinib) in combination
with pembrolizumab (anti-PD-1) are currently being explored
in a phase I/II study (ClinicalTrials.gov).
Another interesting combination currently being evaluated
ON ThE hOriZON BOX is radiotherapy plus immunotherapy. Several studies have
• Develop optimal patient selection strategy for cancer demonstrated that radiotherapy supports tumor-specific immu-
immunotherapy nity by enhancing antigen presentation and increasing the vulner-
• Develop the most appropriate timing for cancer immunotherapy ability of tumor cells to T cell–mediated attack. 99,100 Therefore
• Maximize cancer therapy effectiveness through strategies for: combination therapy with radiation plus immune checkpoint
• Developing combined immunotherapeutic approaches therapy or other immunotherapeutic strategies is warranted.
101
• Optimally integrating standard anticancer modalities with
immunotherapy A phase I trial of hypofractionated radiotherapy in combination
with MEDI4736 (an anti-PD-L1 antibody) and tremelimumab

1046 ParT EighT Immunology of Neoplasia


(anti-CTLA-4) is being tested in patients with metastatic mela- 16. Robert C, Thomas L, Bondarenko I, et al. Ipilimumab plus dacarbazine
noma and lung, breast, and pancreatic cancer (ClinicalTrials. for previously untreated metastatic melanoma. N Engl J Med
gov). 2011;364(26):2517–26.
17. Eggermont AM, Chiarion-Sileni V, Grob JJ, et al. Adjuvant ipilimumab
versus placebo after complete resection of high-risk stage III melanoma
CONCLUSIONS (EORTC 18071): a randomised, double-blind, phase 3 trial. Lancet
Oncol 2015;16(5):522–30.
Cancer immunotherapy has established itself as a critical pillar 18. Schadendorf D, Hodi FS, Robert C, et al. Pooled Analysis of Long-Term
in cancer therapy. It has resulted in durable clinical responses Survival Data From Phase II and Phase III Trials of Ipilimumab in
in patients whose tumors were refractory to conventional Unresectable or Metastatic Melanoma. J Clin Oncol
anticancer therapies. Nonetheless, additional research is required 2015;33(17):1889–94.
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and also to assess new strategies in this emerging field with in patients with advanced melanoma who progressed after anti-CTLA-4
significant untapped potential. Current challenges include rational treatment (CheckMate 037): a randomised, controlled, open-label, phase
development and optimization of combination strategies, 3 trial. Lancet Oncol 2015;16(4):375–84.
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ChaPTEr 77 Immunotherapy of Cancer 1048.e1


MULT i PLE-C h O i CE QUEST i ONS

1. The immunotherapy regimen associated with the “maximum A. Programmed death 1 (PD-1)/PD-L1
response” or the most patients with significant reduction in B. PD-L1/CD80
tumor burden based on phase III clinical trials completed to C. Inducible T-cell costimulator (ICOS)/ICOS-L
date is ———? D. CD68/cytotoxic T lymphocyte antigen-4 (CTLA-4)
A. Nivolumab 3. A 28-year-old female is diagnosed with cutaneous melanoma
B. Ipilimumab on her right shoulder. After surgical resection and imaging,
C. Nivolumab plus ipilimumab she is diagnosed with stage III melanoma. She is interested
D. Pembrolizumab
in receiving adjuvant immune checkpoint therapy to reduce
2. A 64-year-old patient was diagnosed with a large 11.5-cm the risk of relapse at 3 years. Which immune checkpoint
mass in the left kidney. The patient underwent nephrectomy, inhibitor has received US Food and Drug Administration
which revealed a pT3aN0cM0 clear-cell renal cell carcinoma. (FDA) approval for this indication?
After 2 years of observation, he was found to have numerous A. Nivolumab
pulmonary metastases. He was treated with sunitinib (Sutent) B. Ipilimumab
for 15 months and was later found to have disease progression. C. Atezolizumab
His physician is interested in treating him with nivolumab. D. Pembrolizumab
Which pathway does nivolumab target?

78









Lymphoid Leukemias



Moshe E. Gatt, Shai Izraeli






Leukemias are a group of hematological malignant clonal diseases promoted the hypothesis that at least two genetic hits are required
2
arising in bone marrow. This chapter focuses on the two most for the development of ALL (Fig. 78.1). The first occurs during
common lymphoid leukemias: acute lymphoblastic leukemia fetal lymphopoiesis, most likely because of a “developmental
(ALL) and chronic lymphocytic leukemia (CLL). The immuno- accident” of the V(D)J recombination machinery, and results
logical aspects of both diseases are extensively discussed, with a in clonal proliferation of a preleukemic clone. Intrauterine
special emphasis on the clinical and laboratory features of each metastasis of this clone from one twin to the other via their
disorder. shared placental circulation is responsible for the concordant
leukemia. Additional genetic hits in the preleukemic cells occur
ACUTE LYMPHOBLASTIC LEUKEMIA after birth and are required for the development of full-blown
leukemia. The initial findings in identical twins with leukemia
ALL manifests with the clonal proliferation and accumulation have been extended to sporadic ALL. In at least 70% of the
of malignant lymphoid progenitors. ALL is a developmental patients, the preleukemic clone can be detected molecularly
disease. Most childhood ALLs arise as a “developmental accident” in the neonatal blood samples collected after birth (known
during normal fetal lymphopoiesis. Studies of chromosomal as Guthrie cards). More recently, careful molecular analysis of
translocations in ALL cells have identified key genes involved in the cord blood of normal infants has demonstrated that the
normal lymphopoiesis and hematopoiesis. Conversely, basic occurrence of a preleukemic clone carrying a leukemia-defining
studies of the development of the immune system and the immune chromosomal translocation might be common. However, only
receptors have provided important tools for the diagnosis and 1% of children born with such a preleukemic clone will develop
management of ALL. These achievements in basic and clinical leukemia, implying the impracticality of molecular screen for
research have led to the remarkable transformation of ALL from early diagnosis of childhood ALL.
a uniformly fatal disorder to a disease that is curable in more
1
than 80% of children. Adults, however, have not fared as well.
KEY CONCEPTS
Epidemiology and Etiology Environmental Factors in the Epidemiology of
ALL is the most common malignancy of childhood. One of Childhood Acute Lymphoblastic Leukemia (ALL):
every 2000 children will develop leukemia by 15 years of age. Roles for Infection and Immunity?
In contrast, ALL accounts for <20% of leukemias in adults. In
developed countries, incidence peaks at 2–5 years of age. This “Common” B-cell precursor ALL at the preschool age is the most common
low age at peak of incidence is characteristic of affluent societies. 2 type of ALL in the suburban regions of affluent countries. The causes
for this phenomenon are unknown. A popular hypothesis suggests a
Most ALLs are sporadic. Less than 5% are associated with modified immune response to delayed infections during infancy.
hereditary or constitutional syndromes. Children with Down
syndrome have an approximately 20-fold increased risk of
developing ALL. Other diseases associated with increased risk The causes of the relatively rare postnatal leukemogenic genetic
include inherited genomic instability syndromes, such as ataxia– hits are unknown. As the risk of B-cell precursor ALL during
telangiectasia, Bloom syndrome, and Li-Fraumeni syndrome. early childhood is markedly increased by higher socioeconomic
Conversely, ALL is more common in patients with congenital status and a suburban style of living in which the exposure of
B-cell immune deficiencies (Chapter 34). These include X-linked children to infectious pathogens is typically delayed beyond the
agammaglobulinemia (XLA), selective immunoglobulin A (IgA) neonatal period, Greaves hypothesized that many childhood cases
deficiency, and common variable immunodeficiency (CVID). are the consequence of an abnormally late immunological
2
Interestingly, some cases of CVID associated with leukemia have response to common infections. One proposed mechanism is
been recently shown to be caused by germline mutations in that growth inhibitory factors, such as interferon (IFN) or
3
IKZF1, which encodes the lymphoid transcription factor Ikaros. transforming growth factor-β (TGF-β), secreted during this
Germline mutations in additional hematopoietic and B-cell immune response provide a survival advantage to a preleukemic
6
5
4
developmental genes, such as ETV6, , RUNX1, and PAX5, also clone setting the stage for additional leukemogenic mutations.
predispose to ALL. Recently a more direct suggestion for the involvement of infection
Although ALL is not hereditary, there is a markedly increased (or the response to infection) in the pathogenesis of ALL has
risk of leukemias in identical twins, and studies in these twins have been provided. Pax5 heterozygous mice developed preB-cell ALL
shed light on the etiology of childhood ALL. This phenomenon has only after exposure to common mouse pathogens. 7
1049

1050 ParT EighT Immunology of Neoplasia


often contains a translocation that fuses the E2A(TCF3) gene
Immunological and Molecular Classification of ALL on chromosome 19 with the PBX1 gene on chromosome 1.
Immunological Classification Leukemic cells that express both cytoplasmic and surface Ig
ALL subtypes are typically identified by their immunophenotype. heavy chains have been designated transitional preB-cell ALL.
They are positioned by the lymphoid developmental stage at The surface µ chains on these leukemic cells are noncovalently
which the leukemic cell appears to have been arrested (Table 78.1). associated with surrogate light chain (VpreB λ5[λ14.1]), CD79a,
and CD79b.
B-Cell Precursor Leukemias
B-cell precursor ALLs are the most common childhood leukemias.
ProB-cell ALL is characterized by expression of CD19 and CD34 CLiNiCaL PEarLS
without CD10. This is the most common leukemia of infants Mature Versus Precursor B-Cell Acute
that contains rearrangements of the MLL(KMT2A) gene on Lymphoblastic Leukemia (ALL)
chromosome 11q23. It is associated with a poor outcome.
Leukemic blast cells of early preB-cell ALL resemble normal Since the treatment of mature B-cell leukemia (the leukemic form of
B-lymphoid cell precursors. They express CD19, CD22, and Burkitt lymphoma) is vastly different from the treatment of B-cell precursor
ALL, it is critical to distinguish between the two. Burkitt leukemia is
CD79a. CD10 and terminal deoxynucleotidyl transferase (TdT) characterized by mature B-cell phenotype and by the presence of
are detectable in 90% of cases. CD34 is detected in >75% of chromosomal translocations involving the cMyc gene (Chapter 79).
cases. Early preB-cell ALL is the most prevalent type of ALL and
is thus often called “common ALL” (cALL).
In preB-cell ALL lymphoblasts accumulate cytoplasmic Mature B-cell ALL is the leukemic form of Burkitt lymphoma
immunoglobulin (Ig) heavy chains but have no detectable surface (Chapter 79). As treatment is dramatically different from that
Igs. This subtype also expresses CD19, CD22, and CD79a. It for B-cell precursor ALL, this subtype must be specifically ruled
out as part of the immunophenotypic evaluation of ALL. Mature
B-cell ALL cells express surface Ig µ heavy chains in association
with either κ or λ light chains. In most cases, cells have L3
2 hit morphology and express CD19, CD22, and CD20. CD10 and
nd
st
1 hit mutation in cell of ALL CD23 are frequently expressed, whereas CD34 is absent.
transformed clone
T-Cell ALL
T-cell ALL is more prevalent in less affluent countries, which is
likely a reflection of a lower incidence of the common B-lineage
early childhood peak. In affluent countries, T-cell ALL occurs in
latent period 10–15% of children with ALL. It is also more common in adults.
In many treatment protocols, T-cell ALL is considered to have
a less favorable prognosis compared with B-cell precursor ALL.
In T-cell ALL, the cells express surface CD7 and cytoplasmic
-9 months Birth 3 years CD3 (cCD3) antigens. More than 90% of the T lymphoblasts
Fig 78.1 A Model for the Development of Childhood Acute express CD2, CD5, and TdT. Three stages of immunophenotypic
+
+
Lymphoblastic Leukemia (ALL). The first acquired genomic differentiation can be determined: early (CD7 , cCD3 , surface
−
−
−
+
−
hit (e.g., chromosomal translocation or change in chromosomal CD3 , CD4 , and CD8 ), intermediate (cCD3 , surface CD3 ,
−
+
+
+
+
copy number) occurs during fetal hematopoiesis and results in CD4 , CD8 , and CD1 ), and late (surface CD3 , CD1 , and
+
+
clonal proliferation of a preleukemic clone. This event is common, either CD4 or CD8 ). However, immunophenotype does not
occurring in up to one in every 20 children. Additional genetic conform to any of these maturation stages in as many as 25%
aberrations occurring after birth are required for the development of T-lineage ALL cases. T-cell receptor (TCR) proteins are
of ALL. These events are rare and estimated to occur in about heterogeneously expressed in T-lineage ALL. In approximately
5
1% of children born with a preleukemic clone. two-thirds of cases, membrane CD3 and TCR proteins are absent.
TABLE 78.1 immunophenotypic Classification of acute Lymphoblastic Leukemia (aLL)
LEUKOCYTE aNTigEN EXPrESSiON (% OF CaSES POSiTiVE) FrEQUENCY (%)
Subtype CD19 cCD22 CD79a CD10 CD7 CD2 cCD3 cig µ sig µ sig κ/λ Children adults
Pre-preB 100 >95 a >95 0 0 0 0 0 0 0 5 10
Early preB 100 >95 a >95 95 5 <5 0 0 0 0 60–65 50–55
PreB 100 100 a 100 >95 0 0 0 100 0 0 20–25 10
Transitional preB 100 100 a 100 50 0 0 0 100 100 0 1–3 ?
B 100 100 a 100 50 0 0 0 >95 >95 >95 2–3 4
PreT <5 0 0–20 45 100 0 100 0 0 0 1 5
T <5 0 0–20 45 100 95 100 a 0 0 0 10–15 15–20
c, cytoplasmic; cIg µ, cytoplasmic immunoglobulin µ chain; sIg µ, surface immunoglobulin µ chain; sIg κ/λ, surface immunoglobulin κ or λ chains.
a Detectable on the cell-surface membrane in some cases.

ChaPTEr 78 Lymphoid Leukemias 1051



TABLE 78.2 Frequencies of Major, Chromosomal Translocations
Clinically important genetic aberrations in
Childhood and adult acute Lymphoblastic Chromosomal translocations can be divided into two general
subtypes. The first involves translocation of a gene into the
Leukemia (aLL) proximity of a strong regulatory region. This often results in the
genetic aberration Children adults marked overexpression of the oncogene. Often these translocations
B-Cell Lineage are mediated by the V(D)J recombination machinery (Chapter
Hyperdiploidy (>50 30% 9% 4) and can be therefore viewed as an unfortunate developmental
Chromosomes) “accident” caused by the physiological lymphocyte specific
Hypodiploid (<45 chromosomes) 1% 2% genomic instability. Examples include the activation of the MYC
Amplified 21q 2% 2% oncogene by t(8;14) in Burkitt lymphoma and of the SCL (TAL1)
TEL-AML1 (t12;21) 25% 3% gene by t(1;14) or by rearrangement with the STIL gene on
MLL rearrangements 9% 13% chromosome 1p32 in T-cell ALL. The second involves creation
BCR-ABL 4% 33%
E2A-PBX1 5% 4% of novel fusion proteins consisting of parts of the genes that
“Ph like” including CRLF2 8% 25% participate in the chromosomal translocation. For example
MYC rearrangements 2% 5% t(12;21) fuses the TEL (ETV6) gene on chromosome 12 with
the AML1 (RUNX1) gene on chromosome 21.
T-Cell Lineage
Notch1 mutations 60% 70% Amplifications and Deletions
TAL1 (SCL) cluster 58% 33% Amplifications and deletions of small chromosomal regions are
HOX11 (TLX1) cluster 3% 33% another type of structural aberration that is often detected in
HOX11L2 (TLX3) cluster 20% 5%
LYL1 cluster 12% 37% leukemias. For example, deletions of the INK4A locus or of
MLL-ENL 2% 2% PAX5 are commonly detected in T- and B-cell precursor ALLs,
NUP214-ABL 6% (?) respectively.
Oncogenic-Activating Mutations
Oncogenic-activating mutations in ALL are reported with an
increasing frequency. The Notch pathway, for example, is normally
In half these cases, however, TCR proteins (TCRβ, TCRαα, or involved in T-cell development. This pathway is activated by
both) are present in the cytoplasm. When membrane CD3 and acquired mutations in >60% of T-cell ALL.
TCR chains are expressed, the αβ form of the TCR predominates.
Only a minority of cases express TCR γδ proteins. Genes Involved in Leukemogenesis Often Play Key Roles in
The expression of myeloid-associated antigens, such as CD13, Normal Development
CD15, CD33, or CD65, in leukemic lymphoblasts confounded Many of the genes modified by chromosomal translocations,
leukemia classification in early studies. However, the diagnosis amplification, deletions, or point mutations are active during
of B-lineage ALL should be made when leukemic cells express normal lymphoid or hematopoietic development (Table 78.3).
cytoplasmic Ig, CD79a, or CD19 plus CD22, regardless of myeloid- The acquired aberrations promote malignant transformation by
associated antigen expression. Likewise, the diagnosis of T-lineage either overexpression or dysregulated expression (in the wrong cell
ALL should be made when leukemic cells express CD7 plus or in the wrong developmental stage) of the developmental gene.
either surface or cytoplasmic CD3. An immature immunophe- A recent discovery involving the IL7Rα serves as an example.
+
− +
notype of T-cell ALL, early T phenotype (ETP) ALL, has been Loss of function mutations in this receptor causes T B NK severe
8
described and is similar to normal ETP precursors. True combined immunodeficiency (Chapter 35). Activating mutations
10
biphenotypic leukemias usually coexpress B- or T-cell markers are found in 10% of T-cell ALL. An example involving both
with cytoplasmic myeloperoxidase. 9 misexpression and activation of an immune receptor is the aber-
rant activation of the receptor to thymic stromal lymphopoietin
Genetic and Molecular Classification (TSLP). This receptor, created by heterodimerization of IL7Rα
Virtually every leukemic cell contains acquired alterations in and CRLF2, is normally expressed in dendritic cells (DCs) and
multiple genes. These alterations often manifest gross numerical some CD4 T cells. It is involved in allergy and inflammation.
or structural aberrations that frequently define a specific clinical Activation of this receptor in B-lineage ALL is multistaged.
subtype of ALL. Common and/or clinically significant genetic First, chromosomal rearrangements cause the misexpression
aberrations typically found in ALL are summarized in Table 78.2. of CRLF2 in a B-cell precursor. This is followed by activat-
ing mutations in the TSLP receptor components—CRLF2,
Numerical Chromosomal Aberrations IL7Rα, or the downstream signaling enzymes JAK2 or JAK1.
10
Deviation from the normal chromosomal modal number is called The aberrant expression and activation of the TSLP receptor
aneuploidy and is the most common chromosomal aberration occurs in 10% of sporadic B-cell ALLs and in 50% of Down
in cancer. High hyperdiploid ALL (Fig. 78.2A), containing 50–60 syndrome ALLs. It is associated with a poor prognosis. It may
chromosomes, is the most common type of B-lineage ALL in also be amenable to targeted therapy. Conversely, the acquired
children and is associated with about 90% cure rate. There is genetic aberration may block the normal developmental func-
typically an excess of specific chromosomes, most commonly tion of the involved gene(s). A good example is the genomic
chromosomes 6, 10, 14, 17, 18, 21, and X. Hypodiploid ALL deletions or inactivating mutations in B-cell differentiation
(see Fig. 78.2B), containing <45 chromosomes, is much rarer genes, such as PAX5, EBF, or IKZF1, detected in about 50% of
and associated with a very poor prognosis. B-lineage ALLs.

1052 ParT EighT Immunology of Neoplasia











1 2 3 4 5






6 7 8 9 10 11 12





13 14 15 16 17 18




19 20 21 22 X Y
UN
A










1 2 3 4 5








6 7 8 9 10 11 12






13 14 15 16 17 18








19 20 21 22 X Y
B
Fig 78.2 Chromosomal Aneuploidy in Acute Lymphoblastic Leukemia (ALL). A typical karyotype
of (A) hyperdiploid and (B) hypodiploid ALL. (Courtesy of B. Stark and D. Betts.)

ChaPTEr 78 Lymphoid Leukemias 1053



TABLE 78.3 Example of hematopoietic Recent therapies with BCR signaling inhibitors effective in CLL
11
genes involved in the Pathogenesis or lymphomas may prove useful for pre-BCR ALLs. The E2A
of Leukemia gene is rarely fused with the HLF gene in the t(17;19) transloca-
tion. This is an extremely rare translocation that is associated
gene(s) Normal hematopoietic Leukemic with diffuse intravascular coagulation (DIC), hypercalcemia, and
Names Development involvement an extremely poor prognosis. 12
SCL (TAL1) Hemangioblast specification T-ALL Several genetic lesions have prognostic significance and may
Erythropoiesis and be included in risk stratification in future treatment protocols.
megakaryopoiesis Examples are deletions of Ikaros (IKZF1) and CRLF2 aberrations.
LMO1/2 Similar to SCL T-cell ALL A novel subgroup of ALLs has been termed “Philadelphia-like
NOTCH1 T lymphocytes T-cell ALL ALL” because of gene expression that is similar to BCR-ABL. It
HOX11 Spleen T-cell ALL is generally associated with markedly poor prognosis. 13,14 These
E2A T and B lymphocytes BCP-ALL
PAX5 B lymphocytes BCP-ALL, B-NHL leukemias are characterized by activation of either the ABL or
SLP-65 B lymphocytes BCP-ALL the JAK kinase pathway and are caused by many types of fusion
TEL Bone marrow hematopoietic BCP-ALL, T-cell ALL genes or aberrant expressed receptors. Their proper diagnosis
stem cells rarely myeloid is important because of the potential therapeutic activity of either
malignancies ABL or JAK inhibitors.
RUNX1 Definite hematopoiesis BCP-ALL, AML (M0-M1)
(AML1, Megakaryopoiesis and T Hereditary FPD/AML T-Lineage ALL
CBFA2) lymphocytes
CBFB Same as RUNX1 AML (M4e) Although multiple genetic and molecular subtypes of T-cell ALL
C/EBP 1-3 Myeloid cells AML (M1, M2) have been recently described, their clinical significance is presently
8
PU.1 Myeloid and lymphoid stem AML unclear, except for the bad prognosis of ETP ALL. Most of the
cells genetic aberrations in T-cell ALL results in the abnormal expres-
GATA1 Erythropoiesis, AML (M7) associated sion of transcription factors. Examples include SCL (TAL1),
megakaryopoiesis, and with trisomy 21 which forms a complex with LMO1 or LMO2, HOX11L2 (TLX3)
mast cells
FLT3 Hematopoiesis and AML and ALL or HOX11 (TLX1), LYL1, and MYB. Other abnormalities include
lymphopoiesis MLL–ENL fusion and amplification of the ABL oncogene.
MLL Hematopoiesis stem cells AML and ALL Cooperating mutations are activating mutations in NOTCH1
IL7R T lymphocytes ALL or interleukin-7R (IL-7R) and inactivating mutations in the E3
ligase FBW7 or the phosphatase PTEN.

Clinical Features
Major, Clinically Relevant, Molecular Subtypes of ALL The clinical signs and symptoms relate to the replacement of
(See Table 78.2.) bone marrow cells by leukemic blasts and to the infiltration of
extramedullary sites. Pallor, fatigue, petechiae, bleeding, or fever
B-Lineage ALL may be caused by the pancytopenia. Bone pain and arthralgias,
Hyperdiploid ALL and ALL cells TEL/AML1 gene translocation the onset of a limp and refusal to walk, and even frank arthritis
comprise the majority of “common ALL” leukemias that occur are not uncommon. The musculoskeletal symptoms may be
typically in young children and rarely in adults. Both are associated confused with osteomyelitis or juvenile rheumatoid arthritis,
with an extremely good prognosis. In contrast, the much less which may delay the diagnosis. Uncommonly, central nervous
common hypodiploidy (<45 chromosomes) and the internal system (CNS) involvement may present as signs and symptoms
amplification of chromosome 21q (iAMP 21q) are associated of increased intracranial pressure, such as headaches and pap-
with a poor prognosis. illedema; or as cranial nerve palsies, nuchal rigidity, and rarely
Another aberration with a poor prognosis is the BCRF- hyperphagia and obesity caused by infiltration of the hypothala-
ABL fusion protein created by the t(9;22) “Philadelphia chromo- mus. Overt testicular leukemia manifests as painless testicular
some.” Its frequency is low in children (3–5%) and much higher enlargement. Mediastinal involvement, common in T-cell ALL,
in adults (at least 30%). The prognosis dramatically improved may cause dyspnea and superior vena cava syndrome.
by treatment with BCR–ABL1 inhibitors. Clinical laboratory findings often include anemia and throm-
The MLL(KMT2A) gene located on chromosome 11q23 is bocytopenia. Approximately 20% of children present with
involved in fusion translocations with >80 different partner genes. leukocyte counts >50 000/µL. Importantly, approximately 40%
The most common translocation in ALL fuses the MLL with the of children have leukocyte counts of <10 000/µL, and leukemic
AF4 gene on chromosome 4. It is characteristic of infant leukemia blasts may or may not be seen on peripheral smears. Therefore
and is associated with a poor prognosis. the diagnosis of leukemia may occasionally be missed in routine
The clinical significance of the E2A (TCF3)-PBX1 fusion automated blood count. Elevated serum lactate dehydrogenase
protein caused by t(1;19) translocation, occurring in <5% of activity and uric acid and phosphorous concentrations are
childhood ALL, has greatly improved by more intensive chemo- common in patients with a large leukemic cell burden.
therapy. The change in the prognostic significance of this particular The diagnosis of ALL is established by bone marrow examina-
translocation with improved therapy exemplifies the general tion. The normal bone marrow contains <5% blasts. A minimum
principle that the prognostic impact of a clinical or biological of 25% lymphoblasts on differential examination of the bone
parameter is highly dependent on the specific treatment protocol. marrow aspirate is necessary for the diagnosis of ALL. Most
E2A-PBX1 ALLs also represents a subtype of ALL with activation children with ALL have a hypercellular marrow with blasts
of signaling downstream to the preB-cell receptor (pre-BCR). constituting 60–100% of the nucleated cells.

1054 ParT EighT Immunology of Neoplasia


15
Traditionally, CNS leukemia has been defined as the presence analysis. In most centers, karyotype by classic cytogenetics (see
of at least five leukocytes per microliter of cerebrospinal fluid Fig. 78.2) is performed. However, because of the lack of meta-
(CSF) and the detection of leukemic blast cells, by the presence phases, the yield of karyotypic analysis of ALL in multicenter
of cranial nerve palsy, or by retinal involvement, as detected by protocols is often <70%. Furthermore, normal karyotypes are
ophthalmoscopy. Although overt CNS leukemia is relatively rare, sometimes derived from the normal cells in bone marrow rather
submicroscopic CNS involvement is present at diagnosis in at than the leukemia blasts. This drawback can be overcome by
least half the patients in the absence of any neurological symp- interphase fluorescent in-situ hybridization (FISH), a technique
toms. Thus CNS-directed therapy is routinely included in ALL that does not require metaphases. All clinically relevant structural
therapy. and numerical chromosomal aberrations can be detected with
The differential diagnosis of ALL includes neoplastic and the use of commercially available FISH probes (Fig. 78.3). Fusion
nonneoplastic diseases. Because children with ALL present with translocations, such as BCR–ABL, TEL–AML1, and MLL–AF4,
a variety of nonspecific symptoms, several pediatric nonmalignant can all be detected by using reverse transcription–polymerase
conditions may be confused with leukemia. Since treatment with chain reaction (RT-PCR).
steroids may mask the presence of ALL, serious consideration The elucidation of the human genome and the invention of
of the diagnosis of ALL must be given before starting treatment the genomic technologies are in the process of revolutionizing
with steroids to any pediatric nonmalignant disorder. Bone the diagnostics of leukemias. New methodologies of next-
marrow examination is recommended in case of uncertainty. generation genome sequencing (NGS) are likely to transform
Idiopathic thrombocytopenic purpura (ITP) is a common both our understanding of leukemia biology and the diagnostic
cause of bruising and petechiae in children. ITP is characterized approach. Routine use of NGS mutation panels and copy number
by the absence of any other hematological abnormalities. Bone genomic arrays are likely to replace routine cytogenetic analysis
marrow should be examined if anemia or hepatosplenomegaly in the near future.
are present.
Infectious mononucleosis may present with fever, malaise,
adenopathy, splenomegaly, rash, and lymphocytosis. The atypical
lymphocytes may morphologically resemble the leukemic
lymphoblasts. Rarely, flow cytometry may be necessary to dis-
tinguish between the activated atypical lymphocytes and the
immature leukemic lymphoblasts.
Leukemoid reactions, observed in sepsis, acute hemolysis, and
other disorders, are usually easy to distinguish from ALL by
morphological examination of peripheral blood smear. Since
occasionally ALL presents with pancytopenia, aplastic anemia
is also on the differential diagnosis list. A B

CLiNiCaL PEarLS
Acute Lymphoblastic Leukemia (ALL) and der(21)
Rheumatoid Disorders

• ALL can mimic juvenile idiopathic arthritis (JIA; Chapter 53) and other
musculoskeletal disorders.
• Because leukemic blasts may be absent from the peripheral blood,
bone marrow examination should be considered in any child with JIA, der(12)
especially prior to commencing steroid therapy.

As many as 10% of children with ALL are first evaluated at
pediatric rheumatology clinics. Fever, arthralgias, arthritis, or a
limp accompanied by anemia, mild splenomegaly, and lymph-
adenopathy frequently can be confused with juvenile idiopathic
arthritis (Chapter 53) or osteomyelitis. These patients may be C
treated with antibiotics and antiinflammatory agents for several
weeks to months before the diagnosis of ALL is finally made. Fig 78.3 Molecular Cytogenetic Techniques for the Diagnosis
Bone marrow examination should be seriously considered in of Chromosomal Translocations in Acute Lymphoblastic
such patients. Leukemia (ALL). (A) and (B) display interphase fluorescent in-situ
As leukemic lymphoblasts are seen as small round blue hybridization (FISH) with probes to the AML1 (RUNX1) gene on
cells when stained with hematoxylin and eosin, they may be chromosome 21 (red) and to the TEL (ETV6) gene on chromosome
rarely be confused with metastatic small round cell pediatric 12 (green). (A) displays a normal cell, (B) displays a leukemic
tumors, including neuroblastoma, rhabdomyosarcoma, and cell that has undergone a fusion TEL-AML1 translocation (arrow),
retinoblastoma. and (C) displays the same translocation as depicted in B, but
on metaphase chromosomes. It uses a molecular cytogenetic
Special Diagnostic Tests technique called spectral karyotyping (arrows). Classic cytogenetic
The classification and the risk stratification for treatment protocols analysis often misses this translocation. (Courtesy of Dr. L.
of ALL is based on detailed immunophenotyping and genotyping Trakhtenbrot.)

ChaPTEr 78 Lymphoid Leukemias 1055



Principles of Therapy 1 TABLE 78.4 Major Prognostic Factors in
acute Lymphoblastic Leukemia (aLL)
a
Supportive therapy is given before the initiation of leukemia
specific therapy. This includes hydration, treatment, and preventive Prognostic
therapy of hyperuricemia; blood and platelet transfusion; and Factor good Prognosis Worse Prognosis
treatment of emergencies, such as respiratory insufficiency Age at Age 1 year to <10 years <1 year; >10 year
associated with mediastinal leukemia. It is highly recommended diagnosis (children) (children)
that children and adults with ALL be treated in specialized centers >60 years (adult)
as a part of clinical prospective studies. Such clinical trials have Peripheral <50 000 cells/µL >100 000 cells/µL
led to the dramatic improvement in the outcome of patients blood WBC
with ALL achieved over the last several decades. Response to Early response to therapy Slow response to
Negative MRD at the end
therapy.
therapy
Typical remission induction regimens include a glucocorticoid of induction High MRD
(prednisone, prednisolone, or dexamethasone), vincristine, Genetic Hyperdiploidy (>50 chr.); BCR/ABL MLL/AF4
l-asparaginase, and, in many protocols, anthracycline. The abnormalities TEL/AML1 (ETV6/ Hypodiploidy <45 chr.
rate of complete remission now ranges from 97% to 99% in RUNX1)
children and from 75% to 90% in adults. Intensification therapy a The most important prognostic factor is the treatment protocol. Thus the prognostic
uses either high doses of multiple agents not used during the significance of various clinical and laboratory variables may differ between protocols.
induction phase or repeats the induction regimen. Regimens Here, the significant parameters common to most studies are listed.
used for children include high-dose methotrexate with or WBC, white blood cells; MRD, minimal residual disease; chr., chromosomes.
without 6-mercaptopurine; high-dose l-asparaginase given for
an extended period; an epipodophyllotoxin plus cytarabine; or
a combination of dexamethasone, vincristine, l-asparaginase, The most significant prognostic factor is the initial response
doxorubicin, and thioguanine, with or without cyclophosphamide. to therapy. A rapid clearance of leukemic cells from blood or
Another integral component of many protocols is reinduc- bone marrow confers a favorable prognosis. More recently, the
tion therapy. This treatment employs drugs similar to those level of minimal residual disease after the induction of clinical
used during the initial phase of induction therapy and has remission has emerged as a powerful tool for gauging treatment
improved the outcomes of both children and adults with ALL. response and predicting outcome.
Maintenance therapy consist a combination of methotrexate
administered weekly and mercaptopurine administered daily. Where Immunology Meets Oncology—Minimal
Some protocols add intermittent pulses of vincristine and Residual Disease
dexamethasone. Modern treatment protocols have led to morphological complete
CNS prophylactic therapy consisting of cranial irradiation remission in the majority of patients, which is defined as <5%
plus intrathecal chemotherapy, introduced after the induction blasts in bone marrow examination. If treatment is discontinued
of complete remission, became one of the cornerstones of ALL at that stage, in most patients the disease will eventually relapse.
therapy in the 1970s. More recently, because of concerns about Indeed, all prospective clinical studies have shown that ALL
neurotoxicity and the occurrence of brain tumors, intensive should be treated for at least 2 years. These facts indicate that
intrathecal and systemic chemotherapy are used instead for most at the completion of remission induction not all clonogenic
patients. malignant lymphoblasts have been destroyed, even though most
Allogeneic stem cell transplantation (SCT) is reserved for of the patients are in clinical and morphological remission. Indeed,
10
relapsed or refractory leukemia and for patients with a very- by this criterion, patients may have as many as 10 undetectable
high-risk leukemia that is likely to demonstrate a slow response neoplastic cells when in remission. Since, by definition, leukemic
to therapy. With modern therapy, including targeted therapy cells must constitute at least 1–5% of the nucleated cells in bone
with imatinib for BCR–ABL, there is almost no indication for marrow to be detected by microscopic examination, morphologi-
SCT in first remission. With improvements in the prevention cal examination is clearly inadequate for evaluation of the quality
of transplant-related toxicities, suitable marrow donors are not of remission in patients with ALL. Therefore more sensitive
only human leukocyte antigen (HLA)–identical siblings or techniques for the detection of rare leukemic cells are required.
single-antigen mismatched family members but also matched This is the rationale behind the recent incorporation of modern
unrelated donors and, in some situations, two- and three-antigen techniques of detection of minimal residual disease (MRD) into
mismatched family members. treatment protocols of childhood ALL.
During the last 2 decades, two general methodologies have
Prognostic Factors been developed for the sensitive detection of submicroscopic
Modern therapy for ALL is based on adjustment of the intensity residual leukemic cells. These methodologies could not have
of therapy to the risk assessment of the relapse hazard (Table been developed without elucidation of the scientific basis of the
78.4). The two major clinical parameters of prognostic significance developmental phenotype of immune cells (Chapters 7 and 8) and
are age at diagnosis and leukocyte count. A presenting age between of the elaborate process of Ig gene rearrangements (Chapter 4).
9
1 and 9 years and a leukocyte count <50 × 10 /L are favorable The most widely studied DNA-based MRD methodology is
prognostic factors. In adults, the outcome of therapy worsens based on the identification of clonospecific rearrangements of
16
with increasing age and leukocyte count. Patients >60 years of Ig genes or TCRs (Ig/TCR-PCR). This approach exploits the
9
age and/or a leukocyte counts >100 × 10 /L have a particularly physiological process of somatic rearrangement of Ig and TCR
poor response to treatment. Females fare somewhat better gene loci that occur during the early differentiation of B cells
compared with males. The prognostic significance of the major and T cells. Thus any single T or B lymphocyte carries a unique
genetic aberrations has been described above. rearrangement that is not shared by any other lymphoid cell.

1056 ParT EighT Immunology of Neoplasia


-4
Since leukemia is clonal (i.e., it originates from one lymphoid of leukemic blasts to below 10 cells within the first 2–4 weeks
cell), all of the leukemic cells of a particular person carry the of therapy is detected in ≈40% of children with ALL and is
same Ig and/or TCR rearrangements. Because leukemic cells are associated with an extremely good prognosis. Conversely, the
genetically unstable, they often (>90% of the cases) carry multiple presence of >0.1% blasts after 2 or 3 months of therapy defines
rearrangements, a fact that facilitates the usefulness of using a very-high-risk group. These initial findings have led the BFM-
these rearrangements as a clonal marker for MRD detection. AEIOP study group to initiate a prospective study on children
The major advantages of this technique are the exquisite sensitivity with ALL that utilize the MRD level determined by Ig/TCR-PCR
-5
(at least 10 ), reliability, reproducibility, and its applicability to for risk classification. This study, which involved 3184 patients,
>90% of children with ALL. The application of NGS techniques confirmed the strength of PCR MRD over genetic classification
17
is likely to lower the costs and complexity of this approach. as a prognostic marker. At present, MRD studies have been
Current strategies for flow cytometric detection of MRD rely incorporated into most treatment protocols. Patients with high
on combinations of leukocyte markers that do not normally MRD are stratified into a high-risk arm and receive more intensive
occur in cells of the peripheral blood and bone marrow. Such chemotherapy.
leukemia-associated phenotypes can be identified by multiple
color staining techniques (Fig. 78.4). Flow cytometric analysis Course and Prognosis
of these immunophenotypes allows the detection of one leukemic With current protocols, the cure rate of children with ALL is
-4
cell among 10 or more normal cells. The advantages of flow ≈80%. More than 90% of the children with low-risk leukemia
cytometry for MRD detection are adequate sensitivity, and the and ≈70% of those with high-risk leukemia are cured. The cure
presence of immunophenotyping facilities in most major centers rate for adults is significantly lower.
that facilitate timely performance of the analysis on fresh cells Most relapses occur during treatment or within the first 2
at a reasonable cost. There are, however, a few disadvantages. It years after completion of therapy. However, late relapses (even 10
requires a flow cytometry operator with a high level of expertise, years after diagnosis) are increasingly seen. The clinical relapse
and it is more difficult to standardize compared with PCR. It can occur in an extramedullary site, most often the CNS and
may be difficult to distinguish between regenerating B-cell the testes. Leukemic relapse occasionally occurs at other sites,
progenitors and leukemic blasts. Thus flow MRD is most reliable including the eye, ear, ovary, uterus, bone, muscle, tonsil, kidney,
and sensitive at very early stages (up to 4 weeks) of therapy. mediastinum, pleura, and paranasal sinuses. The prognosis of
Many clinical studies of patients enrolled on ALL treatment relapse depends on the time from diagnosis (earlier is worse),
protocols have revealed strikingly similar results. Fast clearance leukocyte count, immunophenotype, and genotype (similar to


Clinical remission

Diagnosis Week 6 Week 20
10 4 10 4 10 4
10 3 10 3 10 3
CD10 10 2 CD10 10 2 CD10 10 2

10 1 10 1 10 1

10 0 10 0 10 0
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
CD38 CD38 CD38
10 4 10 4 10 4

10 3 10 3 10 3
CD10 10 2 CD10 10 2 CD10 10 2

10 1 10 1 10 1
10 0 10 0 10 0
10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4 10 0 10 1 10 2 10 3 10 4
CD38 CD38 CD38
Fig 78.4 Different Kinetics of Leukemia Cytoreduction Revealed by Minimal Residual
Disease (MRD) Studies With Flow Cytometry. The left panels illustrate the leukemia-specific
immunophenotype (CD10 , CD38 ) determined at diagnosis in two children with ALL. This
+
−
phenotype is not found in normal bone marrow. Bone marrow samples were collected during
clinical remission from both patients. In one patient (top panels), 0.04% of mononuclear cells
expressed the leukemia-specific phenotype at week 6. MRD was undetectable by week 20. In
the other patient (bottom panels), a profound remission (MRD <0.01%) was achieved by week
6 and maintained at week 20.

ChaPTEr 78 Lymphoid Leukemias 1057


the prognostic factors at diagnosis). Isolated extramedullary ON ThE hOriZON
relapse carries a better prognosis. Although some relapses can
be treated with chemotherapy only, most are being treated Translational Research of Acute Lymphoblastic
with SCT. The most important prognostic factor that has Leukemia (ALL)
emerged is the MRD level before transplantation. More than • Whole genomic analyses will discover all leukemia genetic abnormalities,
half the late relapses in patients with “good prognosis” ALL are enabling further personalization of treatment.
curable. The prognosis of early relapses and relapsed T-cell ALL • Novel therapies targeting specific leukemia-associated abnormalities
is grim. will be implemented.
• Immunotherapy utilizing antibodies directed against lymphoid antigens
Treatment Sequelae and conjugated either to toxins or to T-cell engaging molecules or
modified T cells will be routinely implemented in treatment of high-risk
In the 1990s, improvements in supportive care reduced the rate ALL.
of early death to <2%. However, the death rate among older
patients during remission induction therapy remains as high as
30% because of hematological, hepatic, and cardiac toxicities.
Long-term toxicity is of major concern for children cured
from ALL. Aseptic necrosis of various bones has emerged as a CHRONIC LYMPHOCYTIC LEUKEMIA
common late toxicity of glucocorticoids, especially in adolescent
girls treated with dexamethasone. The rate of long-term neuro- Chronic lymphocytic leukemia (CLL) is an often indolent
toxicity has been reduced as a result of replacement of cranial lymphoproliferative neoplasm of mature peripheral circulating
irradiation with high-dose and intrathecal methotrexate. However B cells. It is the most common leukemia in adults living in
intensive methotrexate therapy can also have late neurodegenera- countries in the Western hemisphere. CLL originates from a
tion effects. The dose of anthracyclines used in most ALL protocols clonal lymphoid evolved mature stem cell that can be identified
is unlikely to produce severe cardiomyopathy. by its distinct B-cell Ig gene rearrangement. Both clinically and
Secondary cancers are a major concern. The development of at the molecular level, it is a heterogeneous disease. Some patients
therapy-related acute myeloid leukemia (AML) has been linked have an indolent course, whereas other have a more rapid and
to the use of topoisomerase II inhibitors (teniposide and eto- aggressive disease. During its progression, CLL may be associated
poside), and the risk is apparently dependent on the treatment with significant immune deficiencies and autoimmune phenom-
schedule and the concomitant use of other agents. Children who ena that can complicate its course and treatment. These abnor-
received cranial irradiation at 6 years of age or younger are most malities may be profound and thus alter the nature of the disease.
susceptible to the development of brain tumors. They are attributed to the clonal nature of its B-cell origin. Less
Long-term follow-up studies reported from Scandinavia of than 5% of patients have T-cell CLL. Understanding the molecular
children cured of ALL confirm excellent long-term outcomes, pathways involving BCR moieties enables focusing of novel and
as judged by their socioeducational achievements and by repro- targeted chemoimmunotherapy.
ductive success resulting in healthy offspring. 18
Epidemiology
Current Controversies and Future Perspectives The extensive use of automated peripheral blood lymphocyte
The high cure rates in patients with ALL (nearly 90% of children counts has increased the rate of diagnosis of asymptomatic
and 50% of adults) attest to the steady progress that has been patients with CLL. The incidence rate of CLL increases logarithmi-
made in treating this disease. A further increase in cure rates cally from age 35 years, with a median age at the time of diagnosis
will require efforts to maximize the efficacy and minimize the of 65 years. There is a male predilection, and the disease appears
toxicity of current therapy. Currently MRD measurement can to have geographical and ethnic variations in incidence. In the
reliably identify the extremely good-risk and extremely poor-risk United States, CLL is uncommon in people of Asian descent.
patients. However, about half the patients belong to an “intermedi- Because many of these patients may never require tissue diagnosis
ate risk” group in which most of the relapses occur. Optimization or inpatient treatment, cases among them are not likely to be
of diagnosis and treatment of this group of patients is one of recorded in a tumor registry, thus making the true annual
the current major challenges. incidence of the disease higher than previously thought (6.8 per
22
Advanced genomic technologies carry the promise of discover- 100 000 population). With sensitive techniques, a monoclonal
ing the full spectrum of leukemogenic pathways and the identifica- population of B lymphocytes that is indistinguishable in immu-
tion of targets for new therapies. Genomic analysis has also nophenotype from CLL cells may be found in the blood of 3.5%
19
discovered marked subclonal heterogeneity. Most of the relapses of persons >40 years of age. 23
apparently arise from a minor subclone present at diagnosis. The presence of B lymphocytes <5000/µL showing clonality
The identification and specific targeting of these resistant cells is defined as monoclonal B-lymphocytosis (MBL). MBL is an
is a major future challenge. indolent disorder that may progress to frank CLL at a rate of
An exciting development is the advent of immunotherapy. 1–2% per year, and almost all patients with CLL begin with
Blinatumomab, a novel bispecific antibody engaging autologous MBL. This state is divided into low or high MBL (below or above
T cells against CD19 positive leukemic cells, has been recently 500/µL clonal lymphocytes in the peripheral blood). Low MBL
20
approved for therapy. New cell therapy based on autologous is more abundant and rarely progresses to frank CLL, whereas
24
T cells engineered to express anti-CD19 receptor coupled to high MBL may progress at the above-mentioned rate. Some
intracytoplasmic stimulatory molecules (CAR-T cells) have also patients with MBL will experience autoimmune phenomenon,
shown a tremendous efficacy against highly resistant B-cell as later described for CLL. However, the importance of the MBL
21
precursor ALL. This is therefore an exciting period in novel clone is not fully understood and does not necessarily imply a
treatments of ALL beyond chemotherapy. future progression to CLL. 24

1058 ParT EighT Immunology of Neoplasia



Central lymphoid tissue Peripheral lymphoid tissue
Precursor B cell Peripheral B cell
Bone marrow Extrafollicular area Follicular area Marginal zone MALT and BM

Mantle Marginal zone
cell memory B cell
Progenitor B Unmutated CLL
cell
Intestine


Pre-B
cell Centrocyte
Naïve peripheral
B cell
FDC
GC
Immature
B cell

Longed lived
Centroblast plasma cell
Mutated CLL

Somatic mutations
Fig 78.5 B-Cell Development and Neoplasia. B-cell lymphoproliferative disorders are related
to different stage of normal B-cell development. Maturation starts at bone marrow. The more
mature cells exit to the peripheral blood but do not experience the somatic hypermutation in the
germinal center (GC), hence giving rise to the unmutated chronic lymphocytic leukemia (CLL)
form. When the B cells mature and differentiate within the GC into the hypermutated centrocytes,
they will pathologically become the mutated form of CLL. Other forms of B-cell neoplasms may
arise at other stages. ProB and preB cells give rise to acute lymphoblastic leukemia; intermediate
stages give rise to mantle zone lymphoma; and late and more mature stages give rise to marginal
zone lymphomas and plasma cell–derived multiple myelomas.





The etiology of CLL is still unknown. However, as with other bind autoantigens) are normally eliminated. Once the centrocytes
forms of malignancy, there is increasing evidence for the role of are selected, they become plasma or memory B cells (Fig. 78.5).
inherited factors in its development. Family surveys show a genetic The sequence and structure of Igs expressed by CLL cells
predisposition in first-degree relatives, who also demonstrate from various patients have been found to be similar, suggesting
increased susceptibility to other lymphoproliferative disorders, a common pathogenetic antigen, such as an autoantigen-provoking
25
including other lymphomas. Anticipation, the phenomenon clonal expansion. These findings suggest that antigenic stimulus
of earlier onset, and a more severe phenotype in successive plays a role in the promotion of CLL proliferation.
generations have been reported in families of patients with CLL. CLL may originate from a clone with few or no V domain
mutations or from a more mature clone where these domains
Pathogenesis and the Biology of are hypermutated. These differences in the extent of V domain
Leukemic Lymphocytes mutation suggest differing entities with two different develop-
CLL is now viewed as two related entities, both of which originate mental histories. Both originate from antigen-stimulated mature
from B lymphocytes but differ in their activation state and in B lymphocytes. However, in the CLL with mutated Ig V domains,
the maturation state of the cellular subgroup. 23. the proliferating B cell may have traversed the germinal centers,
Normal B lymphocytes mature in bone marrow (Chapter 7). whereas in CLL with unmutated Ig V domains, the malignant
In the process, they undergo rearrangement of Ig V(D)J gene B cell may derive from a naïve, pre–germinal center B cell.
segments to create the code for an Ig molecule that serves as the The mutational status of the V domains strongly correlates
BCR for antigen (Chapter 4). When an antigen of adequate with prognosis in that patients with an unmutated clone have
affinity engages the receptor, the cell enters a germinal center a much worse prognosis compared with patients with mutated
located in a lymphoid follicle. There, as a centroblast, it rapidly clones. These patients may differ also in their association with
divides and the V domains of its Ig undergo somatic hypermuta- specific genetic aberrations. 11q22-23 (the ataxia–telangiectasia
tion. Cells with receptors that have enhanced antigen-binding mutated gene) or 17p13 (the p53 gene) deletions are associated
affinity proliferate in the presence of the antigen, whereas with poor outcome and with an unmutated V domain profile.
centrocytes with receptors that no longer bind the antigen (or These genes regulate apoptosis and resistance to chemotherapy.

ChaPTEr 78 Lymphoid Leukemias 1059


13q14 deletion or a normal karyotype is associated with a mutated express exhaustion markers, including programmed death protein
profile and better prognosis. These chromosomal aberrations 1 (PD-1), at higher levels. Accordingly, CLL cells express high
have independent prognostic significance (unrelated to the levels of PD-1 ligand (PD-L1). In addition, NK cells have reduced
26
mutational status). effector activities that are associated with low expression levels
Surface membrane antigens typically found on B-cell CLL of the activating receptors NK-cell p30-related protein (NKp30)
cells include CD19, CD21, and CD23. Expression of membrane and NK group 2 member D (NKG2D). Together, these findings
IgM, IgD, and CD79b is reduced and is thus consistent with a provide an explanation for ability of CLL cells to evade immune-
phenotype matching that of mature, activated B lymphocytes. mediated destruction.
The pathological features of biopsy specimens of lymph nodes CLL has been classically characterized by the accumulation
23
are those of a small lymphocytic lymphoma. The coexpression of mature B cells that evade apoptosis, with high levels of the
of CD5, a T cell–associated antigen, is a phenotypic characteristic BCL2 antiapoptotic protein. Contradicting this dogma is the
+
and part of the disease defining criteria. CD5 B cells can be measurement of CLL kinetics that has shown CLL cells to prolifer-
28
found in the peripheral blood of normal adults, suggesting that ate at a high dynamic rate of up to 1% of the clone per day.
+
specific subsets of CD5 B cells from the mantle zone may be In addition, proliferation-related genes (e.g., c-MYC and E2F1)
the normal counterparts of B-cell CLL. The low expression of have been found to be upregulated, especially in the unmutated
27
the BCR is the hallmark of CLL cells and contributes to impair- CLLs. This finding suggests that CLL is not solely an accumula-
ments in the activation of the cell following BCR stimulation. tive disease but also has a proliferative element.
BCR signaling in CLL has been extensively studied and has
shed considerable light on the pathogenesis of the disease, thus Clinical Features of CLL
aiding in the design of targeted therapies. The BCR is a multimeric
complex containing the antigen-specific surface Ig and the two CLiNiCaL PEarLS
membrane-bound signal transduction elements CD79A and Chronic Lymphocytic Leukemia (CLL):
CD79B. Binding of antigen to the BCR induces activation of Clinical Manifestations
upstream kinases, which, in turn, activate other kinases by means
of their cytoplasmic moieties, including SYK and the Src kinase. • Absolute blood lymphocytosis >5000 /mm sustained over a period
3
These kinases further activate the Bruton tyrosine kinase (BTK), of 4 weeks (to exclude transient lymphocytosis related conditions as
PI3K, and other downstream pathways, including phospholipase viral infections)
C gamma 2 (PLC-γ2), calcium signaling, protein kinase C (PKC), • At least 30% lymphocytes in a normo- or hypercellular marrow
nuclear factor (NF)-κB signaling, mitogen-activated protein • Phenotypically monoclonal lymphocytes
• They express mature B-cell markers (e.g., CD19) and CD5
26
kinases (MAPKs), and nuclear transcription. The upstream • Morphologically mature-appearing lymphocytes
kinases BTK and PI3K are currently targeted by specific agents, • Most patients having some degree of lymphadenopathy on physical
and other kinases and pathways are being explored for novel examination
therapies. • Progression and the need for treatment depend on lymphocyte doubling
The CD38 surface molecule supports B-cell interactions and time, bulky symptomatic disease, anemia, thrombocytopenia, auto-
differentiation. Under certain circumstances, CD38 also augments immune phenomenon
signaling of BCR, delivering signals that regulate the apoptosis
of B cells. Expression of CD38 correlates with expression of The clinical diagnosis of CLL requires an absolute lymphocytosis
9
unmutated V domains and suggests a bad prognosis. Another with a threshold of >5000 × 10 /L mature-appearing lymphocytes
molecule that influences the BCR is the ζ-associated protein 70 in the blood smear, persistence of the lymphocytosis for >4 weeks,
(ZAP70). High levels of this receptor associated protein tyrosine and a distinct immunophenotype (as described above). Approxi-
kinase (usually found in T and natural killer [NK] cells, but not mately a quarter of patients with CLL are asymptomatic at
in normal B cells) are detected in the majority of unmutated diagnosis. The clinical characteristics at presentation include
CLLs and correlate with a poor prognosis. lymphadenopathy (87%), splenomegaly (54%), hepatomegaly
The microenvironment may play a role in the pathogenesis (14%), high white blood cell (WBC) count, anemia, and throm-
of CLL. Interactions with stromal cells rescue CLL cells from bocytopenia (20%). Very high WBC counts are rare but may
apoptosis in vitro. Activated T cells support the growth of CLL develop along the course of the disease. Hyperleukocytosis, which
cells; cytokines, such as IL-4 and vascular endothelial growth causes leukostasis and necessitates emergency treatment, is
factor (VEGF), and chemokines, such as CXCL12, support the extremely rare. Other organs that are involved include other
expansion of CLL clones. When compared with blood-derived lymphoid tissues and rarely solid organs or skin.
cells, expression profiling of CLL cells in the patient’s lymph A prognostic evaluation of the patient with CLL begins with
nodes showed BCR and NF-κB activation promoting cell prolifera- study of the patient’s blood and bone marrow. Lymph node
tion, thus indicating the strong effects of the tumor microenviron- biopsy is not necessary but, if performed, may reveal the diagnosis
27
ment. Mesenchymal stromal cells are also commonly found in of small lymphocytic lymphoma (SLL), which is considered a
secondary lymphatic tissues of patients with CLL. There they different manifestation of the same disease. The differential
provide survival and migration signals to CLL cells, as well as diagnosis includes other low-grade lymphoproliferative disorders,
protection from apoptosis and modulation of antigen presenta- such as a leukemic phase of lymphoma and mantle cell lymphoma
26
tion. Endothelial cell and follicular dendritic cells (FDCs) may (usually negative for CD23); hairy cell leukemia (CD5 and CD21
also play a role. negative and CD103 and CD25 positive); T-cell leukemia (other
Interaction with and evasion from the normal immune system T-cell markers, such as CD3, CD4, and CD7); and prolymphocytic
26
has been shown to be of significance. The number of circulating leukemia (PLL), which is distinguished by morphologically
T cells, oligoclonal in both the CD4 and the CD8 compartments, immature-appearing cells and the presence of FMC7 and CD79b
is increased. The function of these T cells is impaired, and they on the cell surface. T-cell CLL is rare (<5%).

1060 ParT EighT Immunology of Neoplasia



TABLE 78.5 Major Factors associated With Prognosis of Chronic Lymphocytic
Leukemia (CLL) 30,4,5,6,7,9,31,33,34
Median Used in Clinical
Definition Survival (Years) Practice
Rai stage 0 Leukocytosis 12.5 Yes
1 Leukocytosis and lymphadenopathy 8.4
2 Lymphocytosis plus hepatosplenomegaly 6.9
3 Lymphocytosis plus anemia (<11 gr/dL) 1.5
4 Lymphocytosis plus thrombocytopenia (<100 000 × 10 /L) 1.5
9
Binet Leukocytosis and lymphadenopathy Age-matched Yes
Stage A Lymphadenopathy of more than two involved areas 7
Stage B Anemia or thrombocytopenia 2
Stage C
β 2 microglobulin Normal 9.7 Yes
Elevated 4.5
CD 38 >30% 2.9 to <10 years Yes
Early-stage CLL <30% 9 to >26 years
Chromosomal aberrations 17p- 2.7 Sometimes
(FISH analysis) 11q- 6.5
Trisomy 12 9.5
Normal karyotype 9.3
13q- 11.1
Zeta-associated protein 70 (ZAP-70) >20% 7.5 If available
Early-stage CLL <20% Not reached
Mutational status Unmutated 5.7 to <9.9 years If available
Mutated 10.2 to >24 years




The oldest staging systems of CLL risk stratification rely on Unmutated Mutated
30
29
measurement of disease bulk as reported by Rai and Binet CD5 CD38 CD5
(Table 78.5). These staging systems are very useful for identifying
patients who will need treatment at the time of diagnosis, but CD19 CD19
not for predicting who will subsequently need treatment. About
33% of patients never require treatment and have a long survival. 17p- Normal
In another third, an initial indolent phase is followed by disease 11q- 13q-
progression. The remaining third of patients exhibit an aggressive CD23 CD23
disease at the onset and need immediate treatment. ZAP70
The most powerful predictors for rapid and aggressive progres- CD20 CD20
sion are the mutational status of the V domains and chromosomal β2m
abnormalities, as described above (Fig. 78.6; see Table 78.5). In
contrast to genomic aberrations and serum markers, such as High risk phenotype Low risk phenotype
CD38, the mutational profile has the advantage of remaining Fig 78.6 Risk Markers and Stratification in Chronic Lympho-
constant during disease evolution. NGS has identified new genetic cytic Leukemia (CLL). CLL is one disease with two different
31
markers that have altered prognosis for patients with CLL. entities. It commonly expresses normal levels of the B-cell surface
TP53, ATM, NOTCH1, and SF3B1 mutations indicate a worse antigens CD19 and CD23, low levels of CD20, and coexpression
prognosis. of the pan T-cell antigen CD5. The unmutated genotype correlates
with a worse prognosis, featuring positive CD38 surface antigen,
Treatment intracellular ZAP70, chromosomal aberrations as 17p- and 11q-,
As CLL remains an incurable tumor, treatment may be delayed and high levels of soluble β 2 microglobulin. Conversely, the
and the patient monitored until becoming symptomatic (see mutated form of CLL has a better prognosis, lacks these phe-
Table 78.4). Indications for therapeutic intervention include the notypic features, and has fewer chromosomal aberrations,
development of symptoms, a worsening anemia and/or throm- including 13q-.
bocytopenia, autoimmune cytopenias, progressive splenomegaly,
progressive lymphadenopathy, or a lymphocyte doubling time
of ≤6 months. No prospective data yet exist to support the early
treatment of asymptomatic patients with adverse prognostic analogues (most commonly fludarabine) with or without
features. However, this group warrants close monitoring. cyclophosphamide have been shown to induce higher response
Chlorambucil, alone or combined with corticosteroids, has rates, with some patients achieving complete remission. Benda-
been the most commonly used drug. It is advantageous in relieving mustine is a novel agent that contains both alkylating properties
symptoms, even in advanced disease. However, several randomized and a purine-like benzimidazole ring. It has been shown to be
controlled trials have failed to demonstrate improved survival, effective in CLL and less toxic than the fludarabine and cyclo-
and hardly any patient achieves complete remission (CR). Purine phosphamide combination. 32

ChaPTEr 78 Lymphoid Leukemias 1061



ThEraPEUTiC PriNCiPLES others), and immune checkpoint inhibitors. Some immunological
Chronic Lymphocytic Leukemia (CLL) Is Incurable, novel approaches include expanded autoreactive activated T cells
(chimeric antigen receptor–modified T cells [CAR-T]), which
but It Is Possible to Ameliorate Symptoms is showing promise. 39
• High rates of complete remissions and encouraging survival curves Allogeneic hematopoietic stem cell transplantation (allo-
for high-risk patients HSCT) is the only curative treatment for CLL. Allogeneic HSCT
• Watch and wait approach in the case of asymptomatic patients with relies on myeloablative doses of chemoradiotherapy, which makes
early-stage CLL the treatment unacceptably risky for the majority of patients
• Supportive care (immunoglobulins, blood supplements and erythro- with CLL. In nonmyeloablative, or reduced-intensity, approaches,
poietin, treatment of infections) rates of engraftment are similar to fully ablative conditioning
• Steroids with or without chemotherapy, especially for autoimmune
phenomenon regimens, but with lower rates of early toxicity. Early evidence
• Conventional chemotherapy (alkylating agents, such as chlorambucil suggests that the graft-versus-leukemia (GVL) effect is present.
and cyclophosphamide; purine analogues, such as fludarabine and Patients with deletion 17p (p53 involvement) with an extremely
32
cladribine; other lymphoma regimens; bendamustine) poor prognosis are physically fit candidates for allo-HSCT, even
• Targeted therapy with or without chemotherapy (i.e., rituximab, ofa- as newer agents (i.e., ibrutinib, idelalisib, and venetoclax) are
tumumab, obinutuzumab (all anti-CD20 antibodies) and alemtuzumab showing high efficacy in this high-risk population. Studies
(anti-CD52 antibody)
• B-cell receptor pathway inhibitors—BTK (ibrutinib), PI3K-d (idelalisib), involving autologous transplantation and high-dose chemotherapy
and BCL2 antagonists (venetoclax) for CLL have a limited survival advantage.
• Allogeneic (mostly with reduced intensity) stem cell transplantation With treatment advances, the overall survival of patients with
CLL has improved across the globe as a result of targeted therapy,
even in high-risk and in patients with relapsed and refractory
CLL. Thus classic prognostication (as presented in Table 78.5)
The CD52 antigen is present on lymphocytes (B, T, and NK is becoming less accurate as treatment improves.
cells), monocytes, and some granulocytes. The humanized
anti-CD52 monoclonal antibody (mAb; CAMPATH-1, alemtu- Immunological Aspects of CLL
zumab) has demonstrated high activity in previously treated
33
patients and those with refractory CLL. Nevertheless, because CLiNiCaL PEarLS
of significant B- and T-cell depletion, patients are exposed to Chronic Lymphocytic Leukemia (CLL):
severe infectious complications, particularly reactivation CMV Immunologic Manifestations
infection.
The anti-CD20 mAb rituximab (Mabthera) as a single agent • Panhypogammaglobulinemia
has shown limited efficacy in CLL, possibly because of the weak • A monoclonal immunoglobulin peak, usually of the immunoglobulin
M type
receptor expression on CLL cells. However, in combination with • Downregulation of T-cell function and aberrant cytokine production
chemotherapy, particularly with fludarabine and cyclophospha- • Defects in the complement system
32
mide or bendamustine, it appears to act synergistically and to • High risk of recurrent infections—encapsulated bacteria and opportu-
achieve high rates of response, including molecular complete nistic infections
remission and prolongation of disease-free and overall survival. • Autoimmune-associated phenomena:
A newer fully human mAb targeting CD20 (ofatumumab) has • Autoimmune hemolytic anemia
a very potent effect as a single agent in both rituximab-naïve • Autoimmune thrombocytopenia
• Pure red cell aplasia and autoimmune neutropenia
34
and rituximab-treated patients. It targets a different epitope • Other autoimmune disorders (myositis, vasculitis, pemphigus vulgaris,
on the CD20 antigen. A second-generation anti-CD20 antibody, acquired angioedema, glomerulonephritis)
obinutuzumab, was added to chlorambucil in the treatment of
the older adult population. When compared with chlorambucil,
alone or in combination with rituximab and chlorambucil, CLL is characterized by multiple immune deficiencies and
32
obinutuzumab was shown to be superior and relatively safer. autoimmune phenomena. It is reasonable to hypothesize that
Other investigational agents showing efficacy include lenalidomide immune incompetence and autoimmunity are two sides of the
(an immune modulator related to thalidomide), albeit with an same coin.
interestingly initial flair-up phenomenon of disease-enlarged
lymph nodes. The Pathophysiological Rationale
A major breakthrough in treatment was achieved through CLL cells secrete TGF-β, which is a potent inhibitor of B-cell
drugs targeting the BCR pathway. A BTK inhibitor, ibrutinib, proliferation. They also release high levels of circulating IL-2
has shown an impressive progression-free survival in relapsing receptor, which downregulates the T-helper (Th) cell function.
patients and as first-line treatment 35,36 for patients with resistant Unlike normal cells, activated B cells, both B-cell CLL cells and
or high-risk 17p-deleted CLL. In combination with rituximab, anergic normal B cells, fail to present soluble antigen and
another pathway inhibitor, the PI3Kδ inhibitor idelalisib had alloantigens. Moreover, the T cells in patients with CLL often
an efficacy superior to rituximab alone in frail, older patients demonstrate profound abnormalities of their antigen receptor
37
who were relapsing. Furthermore, clinical trials with small (TCR) repertoire and appear dysfunctional in terms of cytokine
40
molecules targeting BCL2, venetoclax, have shown promising secretion. This cytokine imbalance may be the cause of upregula-
38
results, even in patients with relapsing and highly resistant CLL. tion of the BCL-2 antiapoptotic activity. T-cell dysfunction could
Under investigation are various novel agents targeting other also explain the higher incidence of autoimmune complications,
surface molecules (CD23, CD79b, and other CD20 inhibitors), such as autoimmune hemolytic anemia, among patients receiving
pathway inhibitors (SYK, PI3K, second-generation BTK, and purine analogues therapy, which induce T-cell depletion. Certain

1062 ParT EighT Immunology of Neoplasia


T-cell subsets appear to prevent the development of autoreactive represses the emergence of fludarabine-induced AIHA, but the
B cells. When these are absent (e.g., after treatment with purine latter may also be seen with other chemotherapies (i.e., benda-
42
analogues), autoreactive B-cell clones may easily emerge and mustine). Autoimmune phenomena in patients treated with
expand. purine analogues (mostly fludarabine-related) are of a more
severe nature.
Immunological Deficiencies Other rare entities are reported as paraneoplastic autoimmune
Patients with CLL are extremely sensitive to a number of infec- disorders with connective tissue disease manifestations, such
tious agents. A monoclonal Ig peak, usually of the IgM type, is as polymyositis, dermatopolymyositis, and focal myositis or
found in 5% of patients with CLL, and a small amount of a as vasculitis, pemphigus vulgaris, and acquired angioedema.
monoclonal component can be identified in the serum or urine These autoimmune disorders are related to T-cell dysfunc-
of 60% of patients. Hypogammaglobulinemia occurs in at least tion and may be associated with purine analogue treatment.
60% of B-cell CLL cases and may include all three classes (IgG, Paraneoplastic pemphigus also occurs in patients with CLL
IgA, and IgM). The pathogenesis of hypogammaglobulinemia and may be triggered by chemotherapy and radiotherapy.
in B-cell CLL is poorly understood, as this phenomenon is rare Glomerulonephritis and nephrotic syndrome are seldom
in other B-cell malignancies except multiple myeloma. Low reported but, when present, are related to different mecha-
Ig levels correlate with recurrent infections of encapsulated nisms, such as cryoglobulins and antineutrophil cytoplasmic
organisms. In patients who receive intravenous immune globulin antibodies (ANCAs).
(IVIG), there is a decrease in the incidence of major bacterial Therapy of autoimmune phenomena includes high-dose
41
infections. steroids and disease control. In patients refractory to or
Infections are a major cause of morbidity and mortality in relapsing after steroid therapy, more aggressive treatment is
patients with CLL. Impaired humoral and cellular immunities, warranted. High-dose Igs offers transient amelioration in some
defects in the complement systems, and variable neutropenia, patients. Splenectomy or splenic irradiation, cytotoxic agents,
depending on marrow infiltrates, all contribute to the high rate or cyclosporine may represent valid rescue approaches. In
of infections. Opportunistic infections are initially uncommon cases where AIHA has been triggered by fludarabine, further
as the result of the relative preservation of cellular immunity exposure is hazardous. Rituximab may be an alternative
early in the disease. Infection risk increases following purine agent for the treatment CLL-associated autoimmune diseases,
analogue therapy because of the side effects of myelosuppression including rare autoimmune phenomena, such as pemphigus
and marked lymphopenia with T-cell depletion. The addition and PRCA.
of rituximab, the anti–B cell marker CD20 antibody, to nucleoside
analogue-based therapy does not appear to increase the risk of Other Malignancies
early or late infections but may increase the rate of neutropenia. Second malignancies (hematological and solid tumors) are not
Active immunization with vaccines is hampered by the patient’s uncommon in CLL. The most common hematological malignancy
inability to generate or retain a long and significant immune is the Richter transformation to diffuse large B-cell lymphoma,
response. which occurs in ≈5% of patients, as well as other high-grade
lymphoproliferative diseases. Dermatological tumors, such as
Autoimmune Phenomena basal cell carcinoma, are the most frequent of the solid tumors
Autoimmune-associated features are common in CLL. These encountered in patients with CLL, and these malignancies are
32
manifestations primarily affect hematopoietic cells. For example, more likely to be locally aggressive and metastatic. The patho-
the most common known cause of autoimmune hemolytic anemia genesis of these second cancers is not fully understood, and
41
(AIHA) is CLL. Positive result of direct antiglobulin test (direct although disease-related genetic factors (i.e., 17p deletion, notch
Coomb test) has been reported to be as high as in 7–35% of mutation) are a major determinant, it is probably multifactorial
patients with CLL, and AIHA itself occurs in 10–25% of patients and includes Epstein-Barr virus (EBV) infection and BCR
during the course of their disease, twice as often in patients with configuration to respond to multiple autoantigens and immune/
unmutated genes as in those with mutated ones. Autoantibodies inflammatory stimuli present in the microenvironment. 32,43
against red blood cells (RBCs) are warm-reactive polyclonal IgG.
They are not secreted by the malignant clone, but rather by CONCLUSIONS
41
normal B cells. Cold agglutinins are rare. AIHA is thought to
arise from the imbalance among lymphocyte subsets, contributed CLL is a common indolent lymphoid neoplasm with a wide
to by therapy, resulting in the emergence of the autoimmune clinical heterogeneity. It is suspected and diagnosed more
clone. It is usually observed in advanced stages of the disease, commonly because of routine blood tests. Diagnosis is made
correlates with a poor prognosis, and has a close relationship with simple immunophenotyping. Cytogenetics and molecular
with the activity of the CLL. After therapy, the autoimmune diagnostic techniques are needed to determine the prognosis.
antibodies may remit in 70% of the treated patients. The complications of CLL appear to be unique to this neoplasm
Idiopathic thrombocytopenic purpura (ITP) is observed in and are part of a failing immune system with T-cell and B-cell
about 2–3% of cases and presents as increased megakaryocytes in dysregulation causing both deficiencies predisposing patients to
bone marrow. It should be distinguished from immune thrombo- recurrent infections and autoimmune diseases. New molecular
cytopenia induced by marrow infiltration, which is very common and protein markers are key to finding novel effective targeted
41
in up to 50% of patients at presentation. Two-thirds of patients therapies.
with CLL-associated ITP also have AIHA (Evan syndrome). Pure
red cell aplasia (PRCA) and autoantibodies against neutrophils Please check your eBook at https://expertconsult.inkling.com/
are only rarely observed but are part of the CLL-related autoim- for self-assessment questions. See inside cover for registration
munity repertoire. Interestingly, the addition of cyclophosphamide details.

ChaPTEr 78 Lymphoid Leukemias 1063


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CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in

ChaPTEr 78 Lymphoid Leukemias 1063.e1


MULT i PLE-C h O i CE QUEST i ONS

1. Which of the following cell surface antigens is not consistent C. Determination of secreted immunoglobulins
with the diagnosis of acute lymphoblastic leukemia (ALL)? D. Determination of clonal V(D)J rearrangements
A. CD3 E. Serial bone marrow biopsies
B. CD19 3. CRLF2:
C. Surface Immunoglobulin M (IgM) A. Is a signaling cytoplasmic molecule
D. CD22 B. Is part of the receptor to thymic stromal lymphopoietin
E. Intracytoplasmic IgM
C. Is a major driver of T-acute lymphoblastic leukemia
2. The detection of minimal residual disease of ALL is based D. Modulate the antileukemic effects of mast cells
on: E. Inhibits the growth of leukemic cells
A. Bone marrow magnetic resonance imaging
B. Positron emission tomography with computed tomography