Cancer: From Genomics to Pharmaceutics

CANCER:FROM GENOMICS TOPHARMACEUTICSEDITORSZeynep Karakaş - Meryem Sedef ErdalMüge Sayitoğlu - Merva Soluk Tekkeşin100th ANNIVERSARY OF THE REPUBLIC BOOKSCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TOPHARMACEUTICSEditorsProf. Dr. Zeynep KARAKAS¸˙Istanbul University Istanbul Faculty of Medicine, Department of Pediatrics, Division of Hematology/Oncology,˙Istanbul, Turkiye ¨Assoc. Prof. Dr. Meryem Sedef ERDAL˙Istanbul University Faculty of Pharmacy, Department of Pharmaceutical Technology, ˙Istanbul, Turkiye ¨Prof. Dr. M¨uge SAY˙ITOGLU ˘˙Istanbul University Aziz Sancar Institute of Experimental Medicine, Genetics Department, ˙Istanbul, Turkiye ¨Prof. Dr. Merva SOLUK TEKKES¸˙IN˙Istanbul University Faculty of Dentistry, Department of Oral Pathology, ˙Istanbul, Turkiye ¨Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Published byIstanbul University PressIstanbul University Central Campus, 34452 Beyazıt/Fatih, ˙Istanbul - T¨urkiyewww.iupress.istanbul.edu.trCANCER: FROM GENOMICS TO PHARMACEUTICSEditors: Zeynep Karakas¸, Meryem Sedef Erdal, M¨uge Sayitoglu, Merva Soluk Tekkes¸in ˘E-ISBN: 978-605-07-1621-4DOI: 10.26650/B/LSB28LSB48LSB56.2024.019Istanbul University Publication No: 5353Published Online in August, 2024It is recommended that a reference to the DOI is included when citing this work.This work is published online under the terms of Creative Commons Attribution-NonCommercial 4.0International License (CC BY-NC 4.0)https://creativecommons.org/licenses/by-nc/4.0/This work is copyrighted. Except for the Creative Commons version published online, the legalexceptions and the terms of the applicable license agreements shall be taken into account.This book was published as part of the Istanbul University Press’ 100th Anniversary of theRepublic book project.iiCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSCONTENTSFOREWORD iPREFACE iiACKNOWLEDGEMENTS iiiCHAPTER 1NEW GENOMIC ERA: WHAT WE HAVE LEARNED FROM THE CANCER GENOME?Eda SUN, M¨uge SAY˙ITOGLU ˘ 1CHAPTER 2CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSISKhusan KHODZHAEV, Ozden HATIRNAZ ¨ 13CHAPTER 3EXOSOMES IN CANCER DEVELOPMENT AND METASTASISMerve C¸ igdem ˘ OZGEL, S¸eref Bu ¨ gra TUNC¸ ER ˘ 32CHAPTER 4METHYLATION BIOMARKERS AND LIQUID BIOPSY STUDIES IN CANCERSeval TURNA, Semra DEMOKAN 84CHAPTER 5THE ROLE OF MIRNAS IN CONTROL OF K-RAS GENE EXPRESSIONB¨us¸ra KURT GULTAS¸LAR, Ebru Esin Y ¨ OR¨ UKER ¨ 130CHAPTER 6GERMLINE PREDISPOSITION TO CHILDHOOD CANCERSTugc¸e SUDUTAN, Y¨ucel ERB ˘˙ILG˙IN 147CHAPTER 7BIG DATA / MULTIOMIC APPROACHES IN CANCER RESEARCHSeda SUSG ¨ UN, Barıs¸ SALMAN, Sibel Aylin U ¨ GUR ˘ ˙IS¸ER˙I 176CHAPTER 8NEW THERAPEUTIC APPROACHES IN GENOME˙Ildeniz USLU- BIC¸ AK, B¨us¸ra YAS¸A-C¸ EV˙IK, Selc¸uk SOZER TOKDEM ¨ ˙IR 191CHAPTER 9TARGETING STRATEGIES WITH NEW DRUG DELIVERY SYSTEMS IN CANCERTHERAPYYavuz Selim C¸ EL˙IK, Burcu MESUT, Yıldız OZSOY ¨ 240Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CHAPTER 10CURRENT ADVANCES IN MEDICINAL CHEMISTRY OF ANTICANCER AGENTSEfe Dogukan D ˘˙INCEL, Nuray ULUSOY GUZELDEM ¨ ˙IRC˙I 263CHAPTER 11THE ROLE OF PHARMACOGENOMICS IN CHEMOTHERAPEUTIC SUSCEPTIBILITY ANDTOXICITYOzge Sultan ZENG ¨ ˙IN, Mahmoud ABUDAYYAK, G¨ul OZHAN ¨ 283Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

FOREWORDI am honored to present to you this book, collaboratively written by doctoral studentsand advisors on current research issues at the institute. This work commemorates the100th Anniversary of the Republic of Turkey and to the ˙Istanbul University’s Institute ofGraduate Studies in Health Sciences, which earned the ORPHEUS (Organisation of PhD inBiomedicine and Health Sciences European System) label in its 41st year. I would like tothank the rectorate of ˙Istanbul University for their contribution to this project’s inception andto the ˙Istanbul University Press for bringing it to fruition. As an institution that prioritizesthe goal of raising scientists and competent people in their fields, I would like to expressmy gratitude to the authors who have enriched our book with important topics and theever-expanding body of knowledge in the field.Prof. Dr. Zeynep Karakas¸iCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

PREFACECancer is a devastating disease that affects millions of people worldwide, causing significantmorbidity and mortality. With the development of new technologies and the abundanceof genomic data available, researchers can better understand the genetic causes of cancerand develop targeted therapies. Recent research on cancer genome data has alteredour understanding of the hallmarks of cancer due to the discovery of novel malignanttransformation mechanisms. The integration and analysis of big genomic data have providednew insights into the evolution of cancer, metastasis mechanisms, and germline predispositionto cancer. Results of international genome projects opened a new window to transcribedgenomic regions and the noncoding RNA world. Additionally genome editing approaches arenow in use in clinics and giving scientists the ability to change the genetic material not onlyfor Mendelian type of genetic disorders as well as cancer.This book, entitled Cancer: From Genomics to Pharmaceutics, is a part of the ‘100 e-booksproject on the 100th Anniversary of the Republic of Turkey’ designed by ˙Istanbul University.The aim of the book is to provide a comprehensive overview of the latest developments incancer research, focusing on the intersection of genomics and pharmaceuticals as well as toincrease the academic co-operation between PhD candidates and supervisors that all chaptersare co-written by PhD candidates and their supervisors.This book is intended for researchers, clinicians, students, and anyone interested in thelatest developments in cancer research. It is designed to provide a comprehensive overviewand to serve as a valuable resource for those working in the field.We hope that this book will contribute to the recent knowledge and attention of cancerresearch.Assoc. Prof. Dr. Meryem Sedef ErdalProf. Dr. Muge Sayito ¨ glu˘Prof. Dr. Merva Soluk Tekkes¸iniiCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

ACKNOWLEDGEMENTSWe would like to express our sincere thanks to Prof. Dr. Fatma Savran Oguz and Assoc. Prof. ˘Dr. Nurcan Orhan as assistant directors of Institute of Graduate Studies in Health Science(June 2022-August 2023), Prof. Dr. Merva Soluk Tekkes¸in, Assoc. Prof. Dr. Meryem SedefErdal and Prof. Dr. Ays¸e Evrim Bayrak as members of Institute Director Board for theircontribution to the formation and preparation of the books.iiiCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSCHAPTER 1NEW GENOMIC ERA: WHAT WE HAVE LEARNEDFROM THE CANCER GENOME?Eda SUN1,2, Muge SAY ¨˙ITOGLU ˘ 31PhD Candidate, ˙Istanbul University, Institute of Graduate Programs in Health Sciences, Genetics PhD Program,˙Istanbul, T¨urkiye2˙Istanbul University, Aziz Sancar Institute of Experimental Medicine, Genetics Department, ˙Istanbul, T¨urkiyeE-mail: [email protected]., ˙Istanbul University, Aziz Sancar Institute of Experimental Medicine, Genetics Department, ˙Istanbul,T¨urkiyeE-mail: [email protected]: 10.26650/B/LSB28LSB48LSB56.2024.019.001ABSTRACTCancer is a complex genetic disease and is the second leading cause of death in the world. With the publication ofthe first publication of the human genome project in 2001, cancer studies have also accelerated. As the outputs of thesestudies; it is possible to predict metastasis-recurrence by following the clones developing in clonal evolution withhigh-depth readings, cancer-specific mutation signatures, chromotripsis-chromoplexy and kataegis which explain thepunctuated phase carcinogenesis theory different from multistep carcinogenesis and driver and passenger mutationsdue to the reasons for different responses to the same treatment and prognosis were described. Together with what wehave learned, the empty pieces of the puzzle have been started to complete and both understanding the carcinogenesisprocess and planning the precision treatment have been paved. With this open road, the way of planning treatmentin the new genomic era began to transform. In this chapter, we will discuss what we have learned from the cancergenome that supports this transformation.Keywords: Cancerogenesis, Next-generation sequencing, genetic instability, cancer evolutionCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

2 NEW GENOMIC ERA: WHAT WE HAVE LEARNED FROM THE CANCER GENOME?1. IntroductionGenetic events affecting the fate and the characteristics of the cells accumulate and leadto cancer development in years. Malignant transformation, progression and metastasis aredirected by single nucleotide variations, chromosomal variations (insertions, deletions, otherstructural variations) and epigenetics events. The concept of genetic changes that causecancer was first reported by a Boveri in the 1910s that it could be caused by chromosomalanomalies (1). In the mid-1980s, scientists identified two master terms of genes that causecancer, as oncogenes and tumor suppressor genes. These genes are associated with genomicchanges, which are now well-defined as identified by Macconaill and Garraway (2). Afterthat, along with the sequencing of the genome in the 2000s, cancer researchers opened thefield of cancer genome with their studies. By sequencing the cancer genome, the manycomplexities involved in the origin of cancer, the cancer-causing genes that vary betweenand within tumor types, and the mechanisms that contribute to tumorigenesis have begunto be elucidated. Comprehensive cancer genomic profiling allowed us to understand notonly the mechanisms of malignant transformation in the somatic tissue but also germlinepredisposition genes in the families. Whole genome array (single nucleotide variation (SNV),expression or methylation) and next generation sequencing (NGS) techniques offered anopportunity to understand the detailed genomic background of cancers. Enormous amount ofgenomic knowledge in different types of cancers already accumulated (The Cancer GenomeAtlas (TCGA), the Cancer Genome Project (CGP), and the International Cancer GenomeConsortium, St Jude ect.). (3-5) (https://www.sanger.ac.uk/group/cancer-genomeproject/,https://www.cancer.gov/ccg/research/genome-sequencing/tcga and https://www.stjude.org/research/translational-innovation/pediatric-cancer-genome-project.html).However, there are a lot of unknown answers due to the heterogeneous nature of cancer.NGS approaches including whole genome sequencing (WGS), whole exome sequencing(WES), targeted sequencing and transcriptome sequencing demonstrated the utility of thesomatic and germline genomic data into clinical practice. Deep sequencing approachesare now used for molecular risk classification of patients, for detection of tumor burden,for selection of individualized therapies, to understand possible side effects and therapyresponse, for identification of potential therapeutic targets, prediction of relapse, tracingminimal residual disease and identification of germline predisposition to cancer. Pan-cancergenetic markers that are shared between multiple solid or hematological cancers lead toproduction of commercial kits and NGS based cancer panels are perfectly implemented in theclinical practice in many countries and provide us fast and affordable tests to help oncologistsat diagnosis and during the follow up and treatment periods.Cancers have special acquired characteristics that are long established called “hallmarksof the cancer” such as; most of the cancers arise from somatic mutations, that give advantagefor clonal proliferation, multistep and polygenic events accumulate in the cells, malignantCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Eda SUN, M¨uge SAY˙ITOGLU ˘ 3transformation takes years to evolve to cancer until diagnosis, germline predisposition is arare event in cancer ect. The fact that the new genomic knowledge has changed many ofthese marks and re-shaped the perspective of cancer genetics (Figure 1). Moreover, novelmechanisms and a new terminology had been described in the field such as driver-passengermutations, mutation signature, chromothripsis, kataegis and chromoplexy, clonal metastasis.This chapter is aimed to discuss and outline what we have learned cancer genome in the newgenomic era and some of them will be discussed in detailed in this book (see at Chapter 2;clonal evolution, Chapter 6; germline predisposition).Figure 1: Genomic hallmarks of cancer1.1. Driver and passenger mutationsIt has been shown that in order for a cell to acquire a malignant character, its DNA mustbe changed, the emergence of mutations or epimutations. Mutations that give this drivingfirst step force occur with mutations in genes that disrupt cell division/apoptosis, genomeintegrity or gene expression pattern. These mutations are called “driver” mutations (6).Driver mutations train driver, the following mutations are the ”passenger” mutations in thewagon. Passenger mutations do not play a role in cancer progression but contribute to cancercell formation by impairing genomic instability. This multistep process suggests that cancer isboth multicellular and an accumulation of overlapping genetic events. Mutation profiles varyCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

4 NEW GENOMIC ERA: WHAT WE HAVE LEARNED FROM THE CANCER GENOME?between the individuals within the same cancer and between different histological subtypesor clones.Driver mutations are divided into two functional categories according to how they lead tooncogenesis. They can be either proto-oncogenes or tumor suppressor genes. Proto-oncogenesare disease causing genes that result from gain of function mutations, activation that providesa division regardless of cell proliferation requirement. Looking from the cell membranetowards the nucleus: EGFR as growth factor receptors in lung adenocarcinomas; ABL assignal transduction protein in CML; MYC as transcription factor in Burkitt Lymphoma andBCL2 as an anti-apoptotic gene in CLL, all elements in a signal transduction pathway canbe proto-oncogenes. Heterozygous mutation of these elements is sufficient to play a role incarcinogenesis (7). BCR::ABL chromosomal translocation in CML; BRAF V600E mutation inmelanoma or HER2 gene amplification in breast cancer are the most common proto-oncogeneactivation (8). Tumor suppressor genes are either gatekeeper genes that directly control celldivision or caretaker genes that are responsible for DNA repair and the integrity of the genome(9). Homozygous mutations of these genes are required. In other words, the brake is brokenin the loss of the first allele but there is still a susceptible carrier, and as a second hit thehandbrake is broken in the loss of the second allele and it leads to cancer (10).Until now millions of variations have been identified successfully, but the functionalimpacts still remain to be lighted. Distinguishing the driver and the passenger mutationsin a certain type of cancer is challenging. Although it is variable, mathematical modelsshowed that 5-8 driver mutations are required for cancer development (11) but the level ofpassenger mutations are quite high (hundreds of). Multiple and early driver events are notcommon but lead to clonal sweeps and are associated with intratumor heterogeneity and worseclinical outcome. Whereas the passenger mutations arise randomly at each cell division andare not under selection like drivers (12). Moreover, driver mutations in the subclones mayconfer a fitness advantage to the mutated cells and change the cell fate. Many of the drivermutations are still unknown yet, but identification of driver mutations is important for noveldrug development for specific cancers. It is crucial to identify the genetic variation in thesomatic tissue is driver or passenger? At that point, comparison of frequency and/or thefunction-based methods are used to determine driver and passenger variations. Each of themhas different advantages (13).1.2. Mutation SignaturesMutation signature concept was firstly introduced in 2012 in breast cancer patients’ WGSdata. Mutational signatures are the unique combinations of mutation types produced byvarious mutational processes. Gene variations that are arising due to endogenous factorsand environmental exposures accumulating the physiological imprints of DNA. Since theprobability of coexistence indicates certain cancers, databases were created by analyzing theCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Eda SUN, M¨uge SAY˙ITOGLU ˘ 5genetic background of patients in large-scale cancer data. Whole genome studies from sporadiccancers revealed 21 different types of mutation signatures resulted from approximately 4600WGS and 19000 WES data (COSMIC database (https://cancer.sanger.ac.uk/signatures/).These combinations are both cancer specific genetic signatures and coexistences. Mutationsignatures are non-random mostly single base, indel, rearrangement and copy numbersubstitutions. The idea of mutation signatures came with association of smoking at least17 types of human cancer. One question that has remained is how this tobacco smokingcauses these different types of cancer. How smoking tobacco causes these different typesof cancer was the starting point of the idea. More than 5000 cancer patients revealedthe mutational signatures associated with tobacco smoking (14). Mutational signatures arepatterns of mutations imprinted on the genomes of cells. More than 20 different mutationsignatures were identified in a study of 5000 patients, and at least 4 of them were associatedwith smoking tobacco cigarettes. One of these associated signatures matches experimentalevidence indicating direct DNA damage. This creates a replication stress and a basic sites,producing C>T mutations, C>G transversions or G/C deletions (15). This replication damagecould create single base substitution signatures, doublet base substitutions, small insertionsand deletions and copy number signatures (16). As a second step, the mutational clock starts tobe ticking accelerated in every single cell of our body. They tick with different rates in differentcell types, and they accumulate mutations throughout life and initiating carcinogenesis. As theclinical aspect of mutational signatures; BRCA1-deficient or BRCA2-deficient patients withhomologous recombination deficiency can be referred to PARP-related treatments (17,18),and patients with mismatch repair deficiency can be referred to MMR deficient tumors tocheckpoint blockade treatments (19,20).1.3. Clonal evolution & clonal monitoringAs same as the Darwinian theory of variation, clonality model represents cell lineagesderived from a single ancestral cell of tumor evolution (21,22). In other words, it is theabnormal proliferation of a group of cells from a single cell. Even if whole cells have thesame ancestor, the origin of clones may accumulate additional driver variations in the ongoingprocess. This leads to the formation of different subclones and intra-tumor heterogeneity (23).The subclones formed by the acquisition of new anomalies in each division with genomicinstability are called clonal evolution. Determination of existing subclones is important bothfor the decision of accurate treatment landscape (24) and for the follow-up of the disease.Recurrence of cancer after relapse can be triggered by several mechanisms (Table 1).The first concept is the founding clone acquires new mutations, expands, and becomes thepredominant clone at relapse. The second is a non-founding clone or subclone that resistschemotherapy, acquires new mutations, expands, and becomes the predominant clone atrelapse. The third is an ancestral, pre-diagnostic clone evolves and becomes the predominantCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

6 NEW GENOMIC ERA: WHAT WE HAVE LEARNED FROM THE CANCER GENOME?clone at relapse. The last one is treatment that triggers the emergence of a new clone that wasnot previously present and causes a second pathology (25,26).Table 1: Recurrence of cancer after relapse can be triggered by several mechanisms5 Recurrence of cancer after relapse can be triggered by several mechanisms (Table 1). The first concept is the founding clone acquires new mutations, expands, and becomes the predominant clone at relapse. The second is a non-founding clone or subclone that resists chemotherapy, acquires new mutations, expands, and becomes the predominant clone at relapse. The third is an ancestral, prediagnostic clone evolves and becomes the predominant clone at relapse. The last one is treatment that triggers the emergence of a new clone that was not previously present and causes a second pathology (25,26). Table 1: The cause of clonal evolution Changes the gene expression pattern Malignant phenotype occurs Environment Infections Stem-cells Genomic alterations Translocations Mutations Amplifications Deletions Reduced apoptosis Increased proliferation Decreased differentiation Minimal residual disease (MRD) is very few amounts of cancer cells that remain in the body after treatment. MRD is used either for effectiveness of the treatment protocol or for monitoring the remissions against the risk of recurrence. MRD follow-up is very important because it is very important with the clonal change sequence in AML (27). Since AML patients are in adulthood, mutation in DNMT3A, a DNA repair gene, develops spontaneously in the preleukemic period. The preleukemic clone acquires the NPM1 mutation and continues as the founder clone. As a third step, the clone acquires the FLT3-ITD mutation, becomes the dominant clone and the patient develops AML. In the remission period, the patient may relapse because the DNTMT3A (R882H) mutation is present in the patients, and the 5-year survival remains at 5% (27). Knowing when the patient will relapse is very important in order to direct the patient to alternative treatments. For this reason, since the markers used in MRD follow-up are important. It is difficult to determine individual markers with NGS, but it is necessary for the patient's earnings. Clonal evolution of cancer is discussed in detail in Chapter 2. 1.4. Cancer evolution is not always stochastic, it could be punctuated By sequencing the cancer genome, somatic hypermutation regions, which are an existing system in the genome for the immunological response, but have been observed differently in the cancer genome, have been identified. Mutation storms clustered in these regions, especially cytosine-rich regions (C>T, C>G or C>A) have been identified. These variations rise in 10% of ER-positive breast carcinomas (28). The protein families of activation-induced deaminase (AID) and apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) are comprised in this process, which is referred to as \"kataegis\" that means thunder (29). Unlike chromothripsis, kataegis is a good prognostic marker for breast cancers. Somatic hypermutation, kataegis mutation clusters were also defined by whole genome sequencing in esophagus cancer (30,31). Minimal residual disease (MRD) is very few amounts of cancer cells that remain in thebody after treatment. MRD is used either for effectiveness of the treatment protocol or formonitoring the remissions against the risk of recurrence. MRD follow-up is very importantbecause it is very important with the clonal change sequence in AML (27). Since AMLpatients are in adulthood, mutation in DNMT3A, a DNA repair gene, develops spontaneouslyin the preleukemic period. The preleukemic clone acquires the NPM1 mutation and continuesas the founder clone. As a third step, the clone acquires the FLT3-ITD mutation, becomesthe dominant clone and the patient develops AML. In the remission period, the patient mayrelapse because the DNTMT3A (R882H) mutation is present in the patients, and the 5-yearsurvival remains at 5% (27).Knowing when the patient will relapse is very important in order to direct the patientto alternative treatments. For this reason, since the markers used in MRD follow-up areimportant. It is difficult to determine individual markers with NGS, but it is necessary for thepatient’s earnings. Clonal evolution of cancer is discussed in detail in Chapter 2.1.4. Cancer evolution is not always stochastic, it could be punctuatedBy sequencing the cancer genome, somatic hypermutation regions, which are an existingsystem in the genome for the immunological response, but have been observed differently in thecancer genome, have been identified. Mutation storms clustered in these regions, especiallycytosine-rich regions (C>T, C>G or C>A) have been identified. These variations rise in 10%of ER-positive breast carcinomas (28). The protein families of activation-induced deaminase(AID) and apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC)are comprised in this process, which is referred to as ”kataegis” that means thunder (29).Unlike chromothripsis, kataegis is a good prognostic marker for breast cancers. Somatichypermutation, kataegis mutation clusters were also defined by whole genome sequencing inesophagus cancer (30,31).Cancer may not always occur in a long and multi-step process like the known cancertheory. Carcinogenesis may be occurring in punctuated phase (32). It may have occurredcatastrophically in a single incident. Complex rearrangements can occur when DNA that startsCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. 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Eda SUN, M¨uge SAY˙ITOGLU ˘ 7to degrade just as the cell is about to initiate apoptosis can be patched randomly. The resultingdouble chain breaks and rearrangements may cause loss of tumor suppressor, fusion gene anddouble minute formation (33). Patching after fractures is called “chromothripsis” which meansfragmentation of the chromosome. Chromothripsis was first described by the result of thesequencing of a CLL patient that showed 42 genomic rearrangements involved in chromosome4q (32). This process occurs in 2%–3% of human cancers, with an elevated prevalence inbone cancers, pediatric medulloblastoma, and neuroblastoma (34,35). Chromothripsis usuallyprogresses with p53 mutation and is clinically associated with a poor prognostic marker(34,36).A series of rearrangements involving multiple chromosomes and often translocationshas been termed chromoplexy. Chromoplexy explained by a whole-genome sequencing of7 prostate cancer tumors showed complex chains of balanced rearrangements, involvingboth intra-chromosomal and inter-chromosomal events (37). Baca et. al. studied 57prostate tumors and they identified 5596 somatic rearrangements (38). Forty percent of theserearrangements were complex and prolonged rearrangements both consist of translocationsand deleted fragments which is different from chromothripsis. These chains have recentlybeen termed “chromoplexy” by the means of weaved genomic rearrangements occurring.1.5. Genomic Instability of CancerGenome instability is the accumulation of genomic alterations that cause cell cyclederegulation that prevent tumor cell proliferation during its lifecycle. Genome instabilitycan occur by 3 different mechanisms: 1) loss of function of DNA repair genes by base pairalterations, 2) Microsatellite instability (MSI), and 3) Chromosome instability (CIN).Checkpoint blockage inhibitor therapy is based on an estimation somatic point mutationthat is the number of mutations per million bases. This concept estimates the value oftumor mutation burden (TMB). There is a strong correlation between response to checkpointblockade immunotherapy and high TMB in solid tumors (39,40). Additionally, DNA DamageResponse and Repair (DDR) genes are in association with high TMB. They cause the increaseimmunogenetic by increasing specific neoantigen load (41,42).MSI is another genomic instability concept that inactivating variations in mismatch repairgenes (MMR), consisting of the 1-6 nucleotide repeats (43). Cancer patients can be eithermicrosatellite stable (MSS), low microsatellite instability (MSI-L) or high microsatelliteinstability (MSI-H) (44). Studies clarify with the many patients that high TMB and MSI-Hresulting in the producing new antibodies (45). Also, MSI-H detected cervix carcinoma andadrenocortical carcinoma also carry the high mutation burden (46). Identification of TMBhas high cost with the screening of the entire genome and has controversial analysis. On theother hand, MSI detection can be either immunohistochemistry, PCR or NGS. Determiningthe MSI-TMB correlation is important in terms of directing patients to treatments accordingCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSto their burden they carry.2. ConclusionGrowing amount of big cancer data and its integrative analysis improved the cancerdiagnosis, follow-up, treatment selection and response, development of novel targetedtherapies, population based and familial risks. New molecular mechanisms that we learnedshowed us alternative fast forward mechanisms are possible for malignant transformationother than multistep carcinogenesis which takes 20 to 30 years to appear. Cancer associatedmutations give cells selective growth advantage and affect cell fate, survival and genomemaintenance. It is also a fact that interpatient, intratumor and inter-metastatic heterogeneityis probably related to the large mutation spectrum of somatic cancer tissue and germline hostvariations. Cancer genome knowledge also showed us actionable variations and utilization ofthis data could be useful for prevention or early detection of cancer.REFERENCES1. Harris H. Concerning the origin of malignant tumours by Theodor Boveri. Translatedand annotated by Henry Harris. J Cell Sci 2008; 121 Suppl 1: v-vi.2. Macconaill LE, Garraway LA. Clinical implications of the cancer genome. J Clin Oncol2010; 28(35): 5219-28.3. Zhang J, Bajari R, Andric D, Gerthoffert F, Lepsa A, Nahal-Bose H,et al. TheInternational Cancer Genome Consortium Data Portal. Nat Biotechnol 2019; 37(4):367-9.4. Cancer Genome Atlas Research N. Comprehensive genomic characterization defineshuman glioblastoma genes and core pathways. Nature 2008; 455(7216): 1061-1068.5. Pleasance ED, Cheetham RK, Stephens PJ, McBride DJ, Humphray SJ, Greenman CD,et al. A comprehensive catalogue of somatic mutations from a human cancer genome.Nature 2010; 463(7278): 191-6.6. Haber DA, Settleman J. Cancer: drivers and passengers. Nature 2007; 446(7132):145-146.7. Brown G. Oncogenes, Proto-Oncogenes, and Lineage Restriction of Cancer Stem Cells.Int J Mol Sci 2021; 22(18).8. Botezatu A, Iancu IV, Popa O, Plesa A, Manda D, Huica I, et al. Mechanisms ofOncogene Activation. New Aspects in Molecular and Cellular Mechanisms of HumanCarcinogenesis: InTech; 2016.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Eda SUN, M¨uge SAY˙ITOGLU ˘ 99. Liu Y, Hu X, Han C, Wang L, Zhang X, He X, et al. Targeting tumor suppressor genesfor cancer therapy. Bioessays 2015; 37(12): 1277-86.10. Burkhart DL, Sage J. Cellular mechanisms of tumour suppression by the retinoblastomagene. Nat Rev Cancer 2008; 8(9): 671-82.11. Stratton MR, Campbell PJ, Futreal PA. The cancer genome. Nature 2009; 458(7239):719-24.12. Reiter JG, Baretti M, Gerold JM, Makohon-Moore AP, Daud A, Iacobuzio-DonahueCA, et al. An analysis of genetic heterogeneity in untreated cancers. Nat Rev Cancer2019; 19(11): 639-50.13. Pon JR, Marra MA. Driver and passenger mutations in cancer. Annu Rev Pathol 2015;10: 25-50.14. Alexandrov LB, Ju YS, Haase K, Van Loo P, Martincorena I, Nik-Zainal S, et al.Mutational signatures associated with tobacco smoking in human cancer. Science 2016;354(6312): 618-22.15. Morganella S, Alexandrov LB, Glodzik D, Zou X, Davies H, Staaf J, et al. Thetopography of mutational processes in breast cancer genomes. Nat Commun 2016;7: 11383.16. Koh G, Degasperi A, Zou X, Momen S, Nik-Zainal S. Mutational signatures: emergingconcepts, caveats and clinical applications. Nat Rev Cancer 2021; 21(10): 619-37.17. Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, et al. Specifickilling of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase.Nature 2005; 434(7035): 913-7.18. Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, et al. Inhibition ofpoly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med2009; 361(2): 123-34.19. Mandal R, Samstein RM, Lee KW, Havel JJ, Wang H, Krishna C, et al. Genetic diversityof tumors with mismatch repair deficiency influences anti-PD-1 immunotherapyresponse. Science 2019; 364(6439): 485-91.20. Le DT, Durham JN, Smith KN, Wang H, Bartlett BR, Aulakh LK, et al. Mismatchrepair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017;357(6349): 409-13.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

10 NEW GENOMIC ERA: WHAT WE HAVE LEARNED FROM THE CANCER GENOME?21. Vendramin R, Litchfield K, Swanton C. Cancer evolution: Darwin and beyond. EMBOJ 2021; 40(18): e108389.22. Swanton C. Intratumor heterogeneity: evolution through space and time. Cancer Res2012; 72(19): 4875-82.23. Greaves M, Maley CC. Clonal evolution in cancer. Nature 2012; 481(7381): 306-13.24. Mardis ER. The Impact of Next-Generation Sequencing on Cancer Genomics: FromDiscovery to Clinic. Cold Spring Harb Perspect Med 2019; 9(9).25. Grimwade D, Ivey A, Huntly BJ. Molecular landscape of acute myeloid leukemia inyounger adults and its clinical relevance. Blood 2016; 127(1): 29-41.26. Ramos NR, Mo CC, Karp JE, Hourigan CS. Current Approaches in the Treatment ofRelapsed and Refractory Acute Myeloid Leukemia. J Clin Med 2015; 4(4): 665-95.27. Morita K, Wang F, Jahn K, Hu T, Tanaka T, Sasaki Y, et al. Clonal evolution of acutemyeloid leukemia revealed by high-throughput single-cell genomics. Nat Commun 2020;11(1): 5327.28. Nik-Zainal S, Alexandrov LB, Wedge DC, Van Loo P, Greenman CD, Raine K, et al.Mutational processes molding the genomes of 21 breast cancers. Cell 2012; 149(5):979-93.29. Rebhandl S, Huemer M, Greil R, Geisberger R. AID/APOBEC deaminases and cancer.Oncoscience 2015; 2(4): 320-33.30. Nones K, Waddell N, Wayte N, Patch AM, Bailey P, Newell F, et al. Genomic catastrophesfrequently arise in esophageal adenocarcinoma and drive tumorigenesis. Nat Commun2014; 5: 5224.31. Dai JY, Wang X, Buas MF, Zhang C, Ma J, Wei B, et al. Whole-genome sequencing ofesophageal adenocarcinoma in Chinese patients reveals distinct mutational signaturesand genomic alterations. Commun Biol 2018; 1: 174.32. Stephens PJ, Greenman CD, Fu B, Yang F, Bignell GR, Mudie LJ, et al. Massive genomicrearrangement acquired in a single catastrophic event during cancer development. Cell2011; 144(1): 27-40.33. Forment JV, Kaidi A, Jackson SP. Chromothripsis and cancer: causes and consequencesof chromosome shattering. Nat Rev Cancer 2012; 12(10): 663-70.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Eda SUN, M¨uge SAY˙ITOGLU ˘ 1134. Molenaar JJ, Koster J, Zwijnenburg DA, van Sluis P, Valentijn LJ, van der Ploeg I, etal. Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesisgenes. Nature 2012; 483(7391): 589-93.35. Rausch T, Jones DT, Zapatka M, Stutz AM, Zichner T, Weischenfeldt J, et al. Genomesequencing of pediatric medulloblastoma links catastrophic DNA rearrangements withTP53 mutations. Cell 2012; 148(1-2): 59-71.36. Magrangeas F, Avet-Loiseau H, Munshi NC, Minvielle S. Chromothripsis identifies arare and aggressive entity among newly diagnosed multiple myeloma patients. Blood2011; 118(3): 675-8.37. Berger MF, Lawrence MS, Demichelis F, Drier Y, Cibulskis K, Sivachenko AY, et al.The genomic complexity of primary human prostate cancer. Nature 2011; 470(7333):214-20.38. Baca SC, Prandi D, Lawrence MS, Mosquera JM, Romanel A, Drier Y, et al. Punctuatedevolution of prostate cancer genomes. Cell 2013; 153(3): 666-77.39. Fabrizio DA, George TJ, Jr., Dunne RF, Frampton G, Sun J, Gowen K, et al. Beyondmicrosatellite testing: assessment of tumor mutational burden identifies subsets ofcolorectal cancer who may respond to immune checkpoint inhibition. J GastrointestOncol 2018; 9(4): 610-7.40. Devarakonda S, Rotolo F, Tsao MS, Lanc I, Brambilla E, Masood A, et al. TumorMutation Burden as a Biomarker in Resected Non-Small-Cell Lung Cancer. J ClinOncol 2018; 36(30): 2995-3006.41. Chae YK, Davis AA, Raparia K, Agte S, Pan A, Mohindra N, et al. Association of TumorMutational Burden With DNA Repair Mutations and Response to Anti-PD-1/PD-L1Therapy in Non-Small-Cell Lung Cancer. Clin Lung Cancer 2019; 20(2): 88-96 e86.42. Mouw KW, Goldberg MS, Konstantinopoulos PA, D’Andrea AD. DNA Damage andRepair Biomarkers of Immunotherapy Response. Cancer Discov 2017; 7(7): 675-93.43. Garrido-Ramos MA. Satellite DNA: An Evolving Topic. Genes (Basel) 2017; 8(9).44. Bonneville R, Krook MA, Chen HZ, Smith A, Samorodnitsky E, Wing MR, et al.Detection of Microsatellite Instability Biomarkers via Next-Generation Sequencing.Methods Mol Biol 2020; 2055: 119-32.45. Chalmers ZR, Connelly CF, Fabrizio D, Gay L, Ali SM, Ennis R, et al. Analysis of100,000 human cancer genomes reveals the landscape of tumor mutational burden.Genome Med 2017; 9(1): 34.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

12 NEW GENOMIC ERA: WHAT WE HAVE LEARNED FROM THE CANCER GENOME?46. Bonneville R, Krook MA, Kautto EA, Miya J, Wing MR, Chen HZ, et al. Landscape ofMicrosatellite Instability Across 39 Cancer Types. JCO Precis Oncol 2017; 2017.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSCHAPTER 2CANCER EVOLUTION: FROM MULTISTEPCARCINOGENESIS TO CHROMOTHRIPSISKhusan KHODZHAEV1,2, Ozden HATIRNAZ ¨ 3,41PhD Candidate., ˙Istanbul University Institute of Graduate Studies in Health Sciences, ˙Istanbul, T¨urkiye2˙Istanbul University, Aziz Sancar Institute of Experimental Medicine, Genetics Department, ˙Istanbul, T¨urkiyeE-mail: [email protected]. Dr., ˙Istanbul University, Aziz Sancar Institute of Experimental Medicine, Genetics Department, ˙Istanbul,T¨urkiye4Prof. Dr., Acıbadem Mehmet Ali Aydınlar University, School of Medicine, Department of Medical Biology,˙Istanbul, T¨urkiyeE-mail: [email protected]: 10.26650/B/LSB28LSB48LSB56.2024.019.002ABSTRACTThe combination of environmental exposure and genetic background are the main factors in cancer development.Cancer is not only a multifactorial but also a multistep disorder. Studies show that cancer develops in time in amultistage manner and is diagnosed years after the first hit. There are also different hypotheses on tumor evolution,such as; the linear, branching, neutral and punctuation models. They all agree on the multistep development ofcancer but differ in how the mutational burden and sub-clones occur. During the multistep tumor development, thealtered tissue is exposed to different factors, resulting in alterations. Some of these mutations are known as drivermutations and are held responsible for tumoral cell survival, whereas passenger mutations occur coincidentally andmostly lead to tumor heterogeneity. In addition to single variations or small alterations, complex structural variationsare commonly detected in tumors. These complex structural alterations may be caused by genetic variations relatedto genomic stability or therapy-related exposure that are leading to catastrophic genome wide events. These eventscan also interfere with the therapy response and disease prognosis. Identifying these complex genomic variations isalready being used in the stratification of tumors, and they may even take part in the following therapy response andsupport decision making in therapy selection.Keywords: Multistep carcinogenesis, tumor evolution, structural variants, chromoagenesisCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

14 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSIS1. IntroductionIt was only in 1902 when Boveri hypothesized that even the most malignant tumors couldform from normal tissue cells. This abnormal behavior is derived from the cell itself ratherthan the surroundings (1). Today, this phenomenon is called “clonal expansion”. On the otherhand, Peter Nowell hypothesized that most cancers originate from a single neoplastic cell,and by additional somatic alterations, the cell gains unstoppable proliferation and survival(2). This hypothesis might have been true if the cancer progression was only a linear event.However, it is now known that tumors possess highly heterogeneous sub-clones and throughoutthe cancer evolution new variants occur.In biology, the term evolution describes the changes in heritable characteristics ofbiological populations in successive generations. Similarly, cells are also subjected to differentfactors and selection, leading to an evolutionary process. From the zygotic stage, the cellsare regularly exposed to mutagens resulting in somatic variations that accumulate in the cells,which may or may not affect their fate. We can determine these variations in so-calledhealthy populations and also in cancer tissues. Most of these variations are neutral passengervariations, meaning they mainly occur in non-coding or non-regulatory regions and do notaffect the phenotype. On the other hand, a few driver variations are more than enough forcarcinogenesis to start.This chapter will focus on how cancer progression occurs, which mechanisms are involvedand what are the consequences of this progression over the cell. Hence, understandingmultistep carcinogenesis can lead us to the right pathway.1.1. Multistep carcinogenesisCancer is a multifactorial disorder. Genetic, epigenetic, and environmental factors playa combined role in cancer development, which is an extended process that may take years todetect.Colorectal cancer (CRC) is the third most prevalent cancer and the second cause ofcancer-associated mortality worldwide (3). CRC is a suitable model for explaining themultistep process of cancer development. Although CRC can achieve promising results inearly diagnosis, more than 50% of the patients are diagnosed at later stages and developmetastasis to different organs with a high mortality rate (4). This fact is primarily due to thelong-term differentiation and mutation accumulation in the epithelial cells of the colon.1.2. From normal cell to colorectal cancerThe colon (large intestine) is the end of the gastrointestinal (GI) tract. The surface ofthe intestinal system is covered with intestinal epithelial cells (IEC) that secrete mucosa tothe luminal surface. These cells are renewed every other 4-5 days by stem cells that arelocated in the crypts, and the aged IEC are discarded by apoptotic signals (5). Multiplesignaling pathways regulate this process. WNT, EGFR/MAPK, and NOTCH pathways takeCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 15part in the maintenance of the stem cell compartment, and BMP and TGF-beta regulate thedifferentiation (5, 6). The high division rate in IEC also leads to the accumulation of a highnumber of variations. Other than these, additional risk factors (like diet and lifestyle) mayincrease the transformation of the IEC. The first studies to define clonal expansion in CRC triedto correlate the tumor size and their molecular profiles (7). These studies were followed by thedetermination of adenomatous polyposis coli (APC) gene variations in early phase polyps andactivating Kirsten rat sarcoma (K-RAS) mutations in the advanced adenocarcinoma samples,and TP53 variations leading to a transition into malignant colorectal tumors (7, 8) (Figure 1).Figure 1: Multistep carcinogenesis in colon cancer. With the loss of a second allele of the tumorsuppressor (either a germline or a somatic driver mutation) the hyperproliferation starts and additionaldriver mutations can lead to a malignant transformation. The figure is created with BioRender.com.The most common altered gene in CRC is APC, a critical element of WNT signaling.APC is a tumor suppressor, and it was first discovered in a hereditary cancer syndromecalled familial adenomatous polyposis (FAP) (9). The FAP patients were shown to possessheterozygous germline variations in APC, and individual cells probably lost the second alleledue to different factors. Most sporadic CRC cases were also shown to lose both allelesof the APC (10, 11). The disruption of APC leads to constitutive activation of the WNTpathway, leading the epithelial cells to constant self-renewal. Deregulated WNT signalingcreates a benign outgrow in the colon’s luminal surface, known as an adenoma. However, asmall fraction of these adenomas can turn into aggressive tumors with the accumulation ofadditional driver variations which may affect critical cellular pathways like mitogen-activatedprotein kinase (MAPK), TP53, and transforming growth factor (TGF)-beta.As illustrated in Figure 1 MAPK pathway is hit by protooncogene activating variations inCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

16 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSISKRAS, BRAF, or PIK3CA genes. MAPKs are one of the main signaling pathways that takepart in different cellular functions, including cell proliferation and apoptosis. It is reportedthat more than 40% of all MAPK signaling pathway members were altered in colorectalcancers (12). One of the major altered genes in the MAPK pathway is KRAS. KRAS belongsto the RAS gene family, which are small G-binding proteins and germline variations lead toRASopathies that are a group of cancer predisposition syndromes. Moreover, their somaticvariations lead to malignant transformation in different cancers. KRAS is a protooncogene andaltered in 10-25% of all cancers, once activated it can trigger more than 20 different proteinsthat support the malignant transformation (13). More than 40% of all CRCs possess KRASvariation (14). Patients with KRAS mutations show poor prognosis and higher metastasis rate(15, 16). Moreover, deregulation of KRAS also affects the upstream of the pathway and thesepatients develop resistance to tyrosine kinase inhibitors (17).The tumor progression will not be completed just by the loss of a tumor suppressor geneor oncogene activation. In the beginning, the immunohistochemistry (IHC) and later, thegenome-wide analysis showed that TP53 is frequently mutated in CRCs (18). TP53 is themost studied and the most well-known tumor suppressor protein in all cancers. TP53 wasshown to be mutated in around 60% of all CRCs either by loss of function (LOF) as a tumorsuppressor gene or gain of function mutations (GOF) to accumulate oncogenic properties(19). TP53 is responsible for the stability of the genome. During cellular stress, TP53 isactivated to induce cell cycle arrest and apoptosis. In CRC, the frameshift mutations ormissense mutations either inhibit the tumor suppressor activity of TP53 and block it to startthe transcription of tumor suppressor genes or promote tumor development and growth byactivating oncogene transcripts (19). Additionally, the frequency of TP53 variations is foundto be higher in tumors with high microsatellite instability (MSI), which is an indication ofmismatch repair pathway (MMR) defect (20).As described above, the accumulation of variations takes time before a malignant tumorarises. Previously, it was reported that this process takes decades (21, 22). However, ina recent study, the evolutionary process of 38 cancers was reconstructed by whole genomesequencing (WGS). They reported that the mutational spectrum was significantly changedin more than 40% of the samples, and the driver mutations occurred many years before thediagnostic stage but not decades (23). Although today, with the advancement in genomesequencing technology, we have more knowledge on the genomic infrastructure of a cell, it isnow even more complex to understand how, and in which step these events occur and whichevents trigger which one.2. Tumor Evolution and Tumor HeterogeneityA cell represents eight hallmarks to form a malignant tumor (24). These involvecell signaling, growth suppression, metabolic deregulation, avoiding the immune system,Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 17resistance to cell death, gaining replicative immortality, activating metastasis and accessingvasculature, genome instability and tumor-promoting inflammation. Although the cancer cellspresent common hallmarks, the evolution that leads to these deregulations can cause variationsin an individual tumor’s cellular, histological and genetic features (intratumoral heterogeneity,Figure 2) or between tumors from different patients (intertumoral heterogeneity).Figure 2: Tumor heterogeneity. The tumor mutational burden can start as a single germline and/orsomatic variation and with environmental exposure or other genetic effects that trigger new variationsto occur. At the time of the diagnosis, a tumor possesses at least 3-5 driver and many more passengervariations.Studies showed that various cancers share specific mutational profiles. Moreover,mutagenic factors lead to similar mutation types, leaving a fingerprint in the tissue theyare exposed to. Since the discovery of this fact, scientists have tried to figure out themutational signatures either by in vitro experiments or mathematical models. The termmutational signature refers to unique combinations of mutation types that are generatedby different mutational processes (25). Primary studies of parallel sequencing aimed todetect individual point mutations, copy number variations, or structural variants in a tumor.The improvement of the next-generation sequencing technology and the development ofnew tools for bioinformatic analysis allowed researchers to analyze independent mass dataas one. The collective work of the International Cancer Genome Consortium (ICGC)and The Cancer Genome Atlas facilitated the Pan-Cancer Analysis of Whole Genomes(PCAWG) for the meta-analysis of more than 2500 whole-cancer genomes and matchingtissue DNA controls. Today the mutational signatures are cataloged in the COSMIC database(https://cancer.sanger.ac.uk/signatures/) which is regularly updated (latest version is v3.3,Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

18 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSISupdated on June 2022). The database provides detailed information on each different variantclass (Single base substitutions (SBS), double-strand breaks (DSB), small insertions/deletions(ID), and copy number variations (CN)), a set of mutational signatures are defined. Thereare also different catastrophic events occurring in the cancer genome like; whole genomedoubling, chromosomal chromoplexy and chromothripsis which can drive tumor evolutionand several tumor evolution models will be explained in detail in the following section.2.1. Tumor evolution modelsSince human patients cannot be biopsied at several time points throughout cancerprogression, cancer evolution can be inferred from the single time point sample analysis.Similar to the phylogenetic study of species, with this approach, one can study the phylogenyof cells within a tumor sample where each group of cells can have a unique mutation setand mutations shared between groups of cells. This approach, however, will give the pictureof ”living branch points,” and this picture will not represent intermediate clones that wereeliminated during cancer progression. Hence there are several proposed models of cancerevolution: linear, branching, neutral and punctuated evolution (26).The linear tumor evolution model is described as a linear acquisition of mutations wherenew driver mutations are accumulated, which gives the advantage to outcompete all othersub-clones. In this model, the phylogenetic analysis should reveal a single major sub-clone.One of the first studies which supported this model was genotyping G6PD (27) and methylationanalyses (28) of cancer samples. This model was also supported by the work conducted byFaeron and Vogelstein, where they showed a gradual accumulation of mutations in coloncancer which leads to more advanced cancer (8).In contrast to the linear model, branching evolution proposes a model where severalsub-clones diverge from a common ancestral clone. Since each sub-clone has a fitness,they proliferate and expand simultaneously, so when cells are analyzed at a single timepoint depending on the fitness of sub-clones, several major sub-clones are detected. Thenext-generation sequencing (NGS) studies performed on leukemia (29), breast cancer (30),melanoma (31), liver cancer (32), and brain cancer (32) samples, among other cancer types,support this model of cancer evolution.Neutral tumor evolution is an extreme branching evolution model where random mutationsoccur throughout cancer progression, which do not have an impact on the fitness of cancercells. This model could be valid at least for some cancer types, where highly branchedphylogeny can be drawn using subclone specific mutations. A study performed on the TCGAcohort showed that one-third of the cohort supported this model (33).The punctuated model has similarities with the branching model in that this model alsohas several branches, with the exception that this branching occurs at the early stages of cancerprogression, and unlike the linear model, these changes occur at concise periods. So, thisCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 19model proposes that mutational events occur as ”bursts,” which give rise to several subclones.However, these subclones rapidly die out, leaving few dominant subclones. Punctuatedevolution primarily involves DNA copy number aberrations and chromosomal structuralrearrangements.The structural rearrangements can occur at a single chromosome level or whole genome.Although the gene amplification phenomenon has been known for more than 40 years (34)the use of NGS provided a high resolution to determine these structural events that takeplace genome-wide. Simple structural variants can occur in a single gene region where twobreakpoints occur and lead to a loss or a gain in that location, causing an unbalanced genomicchange. Whereas when the break occurs in both strands of the DNA and rescue, there won’tbe any information gain or loss which will lead to a balanced rearrangement. On the otherhand, there are also complex structural variants. The breakage occurs in multiple regionsinvolving close by regions and creating a single derivative chromosome or diverse derivativechromosomes. These events primarily result from multiple DNA breakage cycles, followedby fusions and new cycles of DNA breakage (35). The following section will focus on thesecomplex structural variants.2.2. ChromoanagenesisMechanisms have been proposed to describe such localized catastrophic rearrangementevents: chromothripsis, chromoanasynthesis and chromoplexy. These events were suggestedto be termed as chromoanagenesis (Figure 3) (36, 37).Chromothripsis describes a single event where chromosomal segments are broken intoseveral pieces and randomly reassembled (38). This event was first reported in cancer; laterstudies also reported this phenomenon in other diseases. Numerous mechanisms have beensuggested triggering chromothripsis: mitotic errors, premature chromosome condensation,p53 defects, viral integration, and telomere attrition. One possible mechanism which explainsthe localized nature of chromothripsis is the formation of a micronucleus and subsequentdamage to chromosome(s) entrapped in the micronucleus (39). Micronuclei can be formeddue to chromosome segregation failure of cellular stress factors occurring throughout thecell cycle (40). Peter Le et al. designed an experimental setup for modeling chromosomesegregation failure, studied different types of genomic rearrangement events, and demonstratedone of the mechanisms of chromothripsis (41).The chromoanasynthesis was first described by Liu et al. in 2011 (42). This single eventcan lead to local DNA copy number changes or chromosomal rearrangements. Mechanismssuch as DNA replication defects were proposed, where multiple template switching eventsmay occur (43, 44). Formation of the micronucleus is also a possible mechanism forchromoanasynthesis which may be caused by asynchronous and defective DNA replication inthe micronucleus (45).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

20 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSISThe term chromoplexy was first suggested by Sylvan C Baca et al. in 2013 (46).The authors studied prostate cancer and identified numerous translocations and deletions.Chromothripsis usually involves rearrangement of several chromosomes which is caused byDNA double strand breaks. Chromoplexy, unlike chromothripsis and chromoanasynthesis,does involve little or no copy number alterations (47).Figure 3: Three different types of chromoanagenesis2.2.1. ChromothripsisThe first documented complex chromosomal rearrangements (CCRs) report was a casewith mental retardation and dysmorphic features where chromosomal translocations involvingthree chromosomes were seen (48). Another study identified “complex translocations”involving three or four chromosomes (49). These rearrangements were termed complexchromosomal rearrangements, where several and not reciprocal exchanges between twochromosomes are involved (50). Stephens et al. reported 42 rearrangements in chromosome 4and rearrangements affecting chromosomes 1, 12, and 15 in CML sample (38). A subsequentstudy performed by these authors in small-cell lung cancer cell lines revealed rearrangedchromosome 8 and double-minute fragments. They also noted that rearrangement eventsinvolved single homologous chromosomes. Authors suggested the term chromothripsis (fromthe Greek ”chromosome” and ”shuttering”) (38).2.2.2. Detection techniques of chromothripsisComplex chromosomal rearrangement events can be detected by different types oftechniques. Paired and next-generation sequencing methods provide the possibility ofdetection not only of the precise breakpoints, but also nucleotide changes in theseregions. Another technique is array comparative genomic hybridization (aCGH). Copynumber analysis with this method allows the detection of deletions and duplication andtheir precise genomic location. The limitation of this method is its inability to detectCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 21balanced chromosomal translocations or the order of chromosomal segments in a derivativechromosome (51). Another method of detecting chromosomal rearrangements is fluorescencein situ hybridization (FISH). Multicolor FISH, where chromosome specific probes areconjugated with probes of different fluorescence, can be used to detect chromosomal segmentsin a derivative chromosome and double minutes (38). FISH technique is also used to identifythe precise location of the breakpoint with the use of locus-specific probes (51). Conventionalkaryotyping can also be used for the identification of chromosomal translocations. However,the nature of CCRs makes it challenging to identify rearrangements of different sizes and thenumber of chromosomes involved.2.2.3. Mechanisms of chromothripsisSeveral mechanisms have been suggested to explain chromothripsis. The most acceptedmechanism is DNA damage in micronuclei, where during chromosome segregation, laggingchromosomes can be trapped in a nuclear envelope outside of the main nucleus. Thesemicronuclei can facilitate the abnormal condensation of chromosomes and access ofcytoplasmic nucleases, leading to DNA breaks (52, 53). Crasta et al., with the use ofmulticolor fluorescence chromosome painting (SKY) have shown that multiple chromosomalfragments can be formed in micronuclei (54). Ly et al. have shown that missegregatedchromosome can be entrapped in the micronucleus, and when DNA breaks are occurred andDNA is repaired with NHEJ (non-homologous end joining), the resulting chromosome willshow typical characteristics of chromothripsis (55). The other possible mechanism is theabortion of programmed cell death. This mechanism was first suggested in a report of aLi-Frumeni syndrome patient with Sonic Hedgehog medulloblastoma (56), where impairedDNA damage repair could lead to the escape of cells with damaged DNA from apoptosis. Thismechanism of chromothripsis was also detected in acute and chronic lymphocytic leukemia(57). Another proposed mechanism is the formation of dicentric chromosomes, which maybe formed due to telomere shortening or DNA damage. Dicentric chromosomes can formchromatin bridges where two centromeres are pulled in opposing directions during mitosis.When aberrant nuclear envelopes are formed in the presence of a chromatin bridge, thisstructure is destroyed by cytoplasmic exonuclease TREX1 (58). This event will lead to DNAfragment losses and the formation of double minutes. This mechanism can be exemplifiedin the carriers of t(15;21) Robertsonian translocations, who have the risk of iAMP21 ALL2700 times higher than in the population. In this ALL subtype, amplification of a region onchromosome 21 is initiated by chromothripsis (59). Exogenous factors can also be linked todamage and subsequent chromosomal rearrangements. Morishita et al. have irradiated a spotwithin nuclei of oral squamous cell carcinoma and authors have detected a chromothripsis-likechromosomal rearrangement event on one of the monoclonal cell lines. Exogenous factors likeviral infections can cause DNA pulverization. Sch¨utze et al. studied the association betweenCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

22 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSISchromothripsis and HPV (60). Authors have reported that in one cultured human foreskin,keratinocytes infected with HPV at passage 40 showed high intrachromosomal rearrangementsof chromosome 8 (of note is the gain of MYC at 8q24.21) and a single translocation betweenchromosome 8 and 4 (60).3. Tumor evolution and cancer treatmentCancer treatment has many aspects, including surgery, radiotherapy and systemicchemotherapy. Almost in all chemotherapy applications, an exceptional decrease in tumorsize is detected. On the other hand, due to mutational burden obtained during tumor evolutioncan lead to an inevitable resistance to the treatment and recurrence occurs. Tumor mutationalburden (TMB) is an index representing the number of mutations per megabase (muts/Mb)in a given cancer type (61). Different cut-off values were suggested for different cancertypes ranging from 10 to 37 muts/Mb (62, 63). High TMB is a predictive biomarker forimmunotherapy (64), which can be explained by the assumption that cancer cells with highmutational burden will express neoantigens which the immune system can detect. BesidesTMB, the expression level of PD-L1 and microsatellite instability (MSI) are also predictors ofimmunotherapy response. Since high TMB can be detected without the biomarkers, detectingthe extent of tumor burden is important for patients who may benefit from immunotherapy(65, 66). TMB can be determined with the use of different sequencing techniques. Wholeexome sequencing would be optimal for the assessment of mutational burden, but becauseof the cost and time needed, targeted NGS panels were also suggested as an alternative (67,68). However, there are still concerns about the use of panels with a limited number of genes,which poses a lack of standardization on selecting genes of interest. Another challenging topicis whether the recurrent or driver mutations should be included in the estimation of TMB.Besides tumor mutational burden interspersed throughout the genome, the detection offocal catastrophic events like chromothripsis are helpful in predicting cancer progressionand therapy response. In contrast to the gradual accumulation of mutational burden andsubsequent evolution of tumorigenesis, chromothripsis provides a chance for developingcancer cells through a rapid accumulation of hundreds of rearrangements (59, 69, 70). Ina study conducted on a cohort of 2658 paired normal-tumor samples consisting of differentcancer types, Isidro Cortes-Ciriano et al. used whole genome sequencing and reported that ´29% of samples had high confidence chromothripsis events (71). In another study, a cohort of18394 cases consisting of 132 cancer types showed an overall 5% chromothripsis like patterns(CTLP) (72). This study also showed that the most frequently affected chromosome was17, which contains the TP53 gene, and the frequency was observed as 48% in CTLP casescompared to 19% cases in non-CTLP. Faiyaz Notta et al. reported that 65.4% of pancreaticcancer samples harbored at least one chromothripsis event and that 11% of these events affectedchromosome 18, which resulted in the loss of the SMAD4 gene (69). Further analyses showedCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 23that SMAD4 loss was accompanied by copy gain of GATA6 which is implicated in pancreaticcancer development. In osteosarcoma, occurrence of chromothripsis was observed in 33.1%of cases, and most affected chromosomes were reported as chromosome 8 (11.5%) and 17(9.2%) (73).Chromothripsis was reported to appear both at early and later stages of carcinogenesis,depending on the cancer type. For example, multiple glioblastoma biopsies revealed thatchromothripsis was present in a subset of samples suggesting that this event appearedat later stages and was clone specific (74). Analysis of prostate tumors showed thatchromothripsis was present at different stages, suggesting that this event may occur at anystage of carcinogenesis (75). However, some cancer types, like acute myeloid leukemia(AML), were reported to harbor chromothripsis at pre-neoplastic stage suggesting this eventmight contribute to the initiation of tumorigenesis (76). Analysis of chronic myeloid leukemia(CML) samples representing different timepoints showed that chromothripsis appeared laterin cancer progression which indicates that this event was not an initiating factor. Nevertheless,it contributed to increasing tumor aggressiveness (77). Furthermore, in some cancers likepancreatic (69) and breast cancer (78) where chromothripsis was detected in primary samplespersisted through cancer progression to metastasis, suggesting this event may have occurredearly in the cancer development. These studies suggest that chromothripsis can be an earlyevent or a contributing factor to cancer development/progression in some cancer types.The extent of the association of chromothripsis and prognosis may differ dependingon the cancer type. For example, in epithelial glioblastoma it was associated withintermediate prognosis (79) and in uveal melanomas (80), osteosarcoma (73), AML (76), acutelymphoblastic leukemia (ALL) (81), chronic lymphoid leukemia (CLL) (82) chromothripsiswas linked to poor prognosis. In (83, 84)addition, chromothripsis was associated with therapyresistance in multiple melanomas (84) and glioma (85).Treatment strategies in cancers with chromothripsis primarily depend on the affectedgenomic region in cancer cells. For example, glioblastoma patients with MDM2 containingdouble minutes generated as a result of chromothripsis, can benefit from MDM inhibitors(85). It is also important to distinguish chromothripsis events, especially in childhood cancersarising from germline cancer-predisposing aberrations (like TP53 mutation in Li-FraumeniSyndrome) (86). Li-Fraumeni Syndrome associated malignancies have a higher rate ofchromothripsis compared to overall incidence (56). Since therapy options like radiotherapy,which rely on DNA damage, may cause the development of secondary malignancies in patientswith cancer predisposition syndromes (87), these patients might benefit from treatment optionsnot relying on apoptosis inductions through DNA damage.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICS4. ConclusionSince the discovery of catastrophic events like chromothripsis in 2011, the model oftumorigenesis through gradual mutation accumulation was challenged. This concept ofmassive mutation acquisition through catastrophic events shed light on the role of biologicalprocesses of nuclear envelope formation, chromosome segregation, cytoplasmic nucleases likeTREX1 and DNA damage repair in carcinogenesis. These focal events also may point to thepotential role of the affected genomic region where affected genes may elucidate the etiologyof tumorigenesis. Further studies focusing on the role of chromothripsis in different cancertypes will help in identifying its role in that cancer, will be used as a candidate biomarker fordiagnosis of cancer subtype, indicator of prognosis and decision making in targeted therapyselection.REFERENCES1. Boveri T. Uber mehrpolige Mitosen als Mittel zur Analyse des Zellkerns: Stuber; 1902. ¨2. Nowell PC. The clonal evolution of tumor cell populations. Science.1976;194(4260):23-8.3. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in185 countries. CA Cancer J Clin. 2018;68(6):394-424.4. Tauriello DV, Calon A, Lonardo E, Batlle E. Determinants of metastatic competency incolorectal cancer. Mol Oncol. 2017;11(1):97-119.5. Clevers H. The intestinal crypt, a prototype stem cell compartment. Cell.2013;154(2):274-84.6. Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell.2012;149(6):1192-205.7. Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M,et al. Genetic alterations during colorectal-tumor development. N Engl J Med.1988;319(9):525-32.8. Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell.1990;61(5):759-67.9. Nishisho I, Nakamura Y, Miyoshi Y, Miki Y, Ando H, Horii A, et al. Mutationsof chromosome 5q21 genes in FAP and colorectal cancer patients. Science.1991;253(5020):665-9.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 2510. Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell.1996;87(2):159-70.11. Wood LD, Parsons DW, Jones S, Lin J, Sjoblom T, Leary RJ, et al. The genomiclandscapes of human breast and colorectal cancers. Science. 2007;318(5853):1108-13.12. Slattery ML, Mullany LE, Sakoda LC, Wolff RK, Samowitz WS, Herrick JS. TheMAPK-Signaling Pathway in Colorectal Cancer: Dysregulated Genes and TheirAssociation With MicroRNAs. Cancer Inform. 2018;17:1176935118766522.13. Dinu D, Dobre M, Panaitescu E, Birla R, Iosif C, Hoara P, et al. Prognosticsignificance of KRAS gene mutations in colorectal cancer–preliminary study. J MedLife. 2014;7(4):581-7.14. Simanshu DK, Nissley DV, McCormick F. RAS Proteins and Their Regulators in HumanDisease. Cell. 2017;170(1):17-33.15. Silver JM, Rawlings CE, 3rd, Rossitch E, Jr., Zeidman SM, FriedmanAH. Ganglioglioma: a clinical study with long-term follow-up. Surg Neurol.1991;35(4):261-6.16. Dienstmann R, Mason MJ, Sinicrope FA, Phipps AI, Tejpar S, Nesbakken A, et al.Prediction of overall survival in stage II and III colon cancer beyond TNM system: aretrospective, pooled biomarker study. Ann Oncol. 2017;28(5):1023-31.17. Karapetis CS, Khambata-Ford S, Jonker DJ, O’Callaghan CJ, Tu D, Tebbutt NC, et al.K-ras mutations and benefit from cetuximab in advanced colorectal cancer. N Engl JMed. 2008;359(17):1757-65.18. Giannakis M, Mu XJ, Shukla SA, Qian ZR, Cohen O, Nishihara R, et al.Genomic Correlates of Immune-Cell Infiltrates in Colorectal Carcinoma. Cell Rep.2016;15(4):857-65.19. Nakayama M, Oshima M. Mutant p53 in colon cancer. J Mol Cell Biol.2019;11(4):267-76.20. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature.1997;386(6625):623-7.21. Jones S, Chen WD, Parmigiani G, Diehl F, Beerenwinkel N, Antal T, et al. Comparativelesion sequencing provides insights into tumor evolution. Proc Natl Acad Sci U S A.2008;105(11):4283-8.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

26 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSIS22. Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Diaz LA, Jr., Kinzler KW.Cancer genome landscapes. Science. 2013;339(6127):1546-58.23. Gerstung M, Jolly C, Leshchiner I, Dentro SC, Gonzalez S, Rosebrock D, et al. Theevolutionary history of 2,658 cancers. Nature. 2020;578(7793):122-8.24. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discov. 2022;12(1):31-46.25. Alexandrov LB, Stratton MR. Mutational signatures: the patterns of somatic mutationshidden in cancer genomes. Curr Opin Genet Dev. 2014;24(100):52-60.26. Davis A, Gao R, Navin N. Tumor evolution: Linear, branching, neutral or punctuated?Biochim Biophys Acta Rev Cancer. 2017;1867(2):151-61.27. Linder D, Gartler SM. Glucose-6-phosphate dehydrogenase mosaicism: utilization as acell marker in the study of leiomyomas. Science. 1965;150(3692):67-9.28. Noguchi S, Motomura K, Inaji H, Imaoka S, Koyama H. Clonal analysis of human breastcancer by means of the polymerase chain reaction. Cancer Res. 1992;52(23):6594-7.29. Gawad C, Koh W, Quake SR. Dissecting the clonal origins of childhood acutelymphoblastic leukemia by single-cell genomics. Proc Natl Acad Sci U S A.2014;111(50):17947-52.30. Yates LR, Gerstung M, Knappskog S, Desmedt C, Gundem G, Van Loo P, et al. Subclonaldiversification of primary breast cancer revealed by multiregion sequencing. Nat Med.2015;21(7):751-9.31. Harbst K, Lauss M, Cirenajwis H, Isaksson K, Rosengren F, Torngren T, et al.Multiregion Whole-Exome Sequencing Uncovers the Genetic Evolution and MutationalHeterogeneity of Early-Stage Metastatic Melanoma. Cancer Res. 2016;76(16):4765-74.32. Ling S, Hu Z, Yang Z, Yang F, Li Y, Lin P, et al. Extremely high genetic diversity in asingle tumor points to prevalence of non-Darwinian cell evolution. Proc Natl Acad SciU S A. 2015;112(47):E6496-505.33. Williams MJ, Werner B, Barnes CP, Graham TA, Sottoriva A. Identification of neutraltumor evolution across cancer types. Nat Genet. 2016;48(3):238-44.34. Alt FW, Kellems RE, Bertino JR, Schimke RT. Selective multiplication of dihydrofolatereductase genes in methotrexate-resistant variants of cultured murine cells. J Biol Chem.1978;253(5):1357-70.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 2735. Li Y, Roberts ND, Wala JA, Shapira O, Schumacher SE, Kumar K, et al. Patterns ofsomatic structural variation in human cancer genomes. Nature. 2020;578(7793):112-21.36. Holland AJ, Cleveland DW. Chromoanagenesis and cancer: mechanisms andconsequences of localized, complex chromosomal rearrangements. Nat Med.2012;18(11):1630-8.37. Pellestor F, Gatinois V. Chromoanagenesis: a piece of the macroevolution scenario. MolCytogenet. 2020;13:3.38. Stephens PJ, Greenman CD, Fu B, Yang F, Bignell GR, Mudie LJ, et al. Massive genomicrearrangement acquired in a single catastrophic event during cancer development. Cell.2011;144(1):27-40.39. Hatch EM, Fischer AH, Deerinck TJ, Hetzer MW. Catastrophic nuclear envelope collapsein cancer cell micronuclei. Cell. 2013;154(1):47-60.40. Liu S, Kwon M, Mannino M, Yang N, Renda F, Khodjakov A, et al. Nuclear envelopeassembly defects link mitotic errors to chromothripsis. Nature. 2018;561(7724):551-5.41. Ly P, Brunner SF, Shoshani O, Kim DH, Lan W, Pyntikova T, et al. Chromosomesegregation errors generate a diverse spectrum of simple and complex genomicrearrangements. Nat Genet. 2019;51(4):705-15.42. Liu P, Erez A, Nagamani SC, Dhar SU, Kolodziejska KE, Dharmadhikari AV, et al.Chromosome catastrophes involve replication mechanisms generating complex genomicrearrangements. Cell. 2011;146(6):889-903.43. Lee JA, Carvalho CM, Lupski JR. A DNA replication mechanism forgenerating nonrecurrent rearrangements associated with genomic disorders. Cell.2007;131(7):1235-47.44. Hastings PJ, Ira G, Lupski JR. A microhomology-mediated break-induced replicationmodel for the origin of human copy number variation. PLoS Genet. 2009;5(1):e1000327.45. Guo X, Ni J, Liang Z, Xue J, Fenech MF, Wang X. The molecular origins andpathophysiological consequences of micronuclei: New insights into an age-old problem.Mutat Res Rev Mutat Res. 2019;779:1-35.46. Baca SC, Prandi D, Lawrence MS, Mosquera JM, Romanel A, Drier Y, et al. Punctuatedevolution of prostate cancer genomes. Cell. 2013;153(3):666-77.47. Shen MM. Chromoplexy: a new category of complex rearrangements in the cancergenome. Cancer Cell. 2013;23(5):567-9.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

28 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSIS48. Nuzzo F, Marini A, Baglioni C, Ford CE, De Carli L, Piceni Sereni L. A case of multoplechromosomal rearrangements with persistence of foetal haemoglobin. Cytogenetics.1968;7(3):169-82.49. Fredga K, Hall B. A complex familial translocation involving chromosomes 5, 9 and 13.Cytogenetics. 1970;9(4):294-306.50. Pai GS, Thomas GH, Mahoney W, Migeon BR. Complex chromosome rearrangements.Report of a new case and literature review. Clin Genet. 1980;18(6):436-44.51. Balajee AS, Hande MP. History and evolution of cytogenetic techniques: Current andfuture applications in basic and clinical research. Mutat Res Genet Toxicol EnvironMutagen. 2018;836(Pt A):3-12.52. Terradas M, Martin M, Genesca A. Impaired nuclear functions in micronuclei results ingenome instability and chromothripsis. Arch Toxicol. 2016;90(11):2657-67.53. Terzoudi GI, Karakosta M, Pantelias A, Hatzi VI, Karachristou I, Pantelias G. Stressinduced by premature chromatin condensation triggers chromosome shattering andchromothripsis at DNA sites still replicating in micronuclei or multinucleate cellswhen primary nuclei enter mitosis. Mutat Res Genet Toxicol Environ Mutagen.2015;793:185-98.54. Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y, et al. DNA breaksand chromosome pulverization from errors in mitosis. Nature. 2012;482(7383):53-8.55. Ly P, Teitz LS, Kim DH, Shoshani O, Skaletsky H, Fachinetti D, et al. Selective Ycentromere inactivation triggers chromosome shattering in micronuclei and repair bynon-homologous end joining. Nat Cell Biol. 2017;19(1):68-75.56. Rausch T, Jones DT, Zapatka M, Stutz AM, Zichner T, Weischenfeldt J, et al. Genomesequencing of pediatric medulloblastoma links catastrophic DNA rearrangements withTP53 mutations. Cell. 2012;148(1-2):59-71.57. Pei J, Jhanwar SC, Testa JR. Chromothripsis in a Case of TP53-Deficient ChronicLymphocytic Leukemia. Leuk Res Rep. 2012;1(1):4-6.58. Maciejowski J, de Lange T. Telomeres in cancer: tumour suppression and genomeinstability. Nat Rev Mol Cell Biol. 2017;18(3):175-86.59. Li Y, Schwab C, Ryan S, Papaemmanuil E, Robinson HM, Jacobs P, et al. Constitutionaland somatic rearrangement of chromosome 21 in acute lymphoblastic leukaemia. Nature.2014;508(7494):98-102.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 2960. Schutze DM, Krijgsman O, Snijders PJ, Ylstra B, Weischenfeldt J, Mardin BR, et al.Immortalization capacity of HPV types is inversely related to chromosomal instability.Oncotarget. 2016;7(25):37608-21.61. Chalmers ZR, Connelly CF, Fabrizio D, Gay L, Ali SM, Ennis R, et al. Analysis of100,000 human cancer genomes reveals the landscape of tumor mutational burden.Genome Med. 2017;9(1):34.62. Marabelle A, Fakih M, Lopez J, Shah M, Shapira-Frommer R, Nakagawa K, et al.Association of tumour mutational burden with outcomes in patients with advanced solidtumours treated with pembrolizumab: prospective biomarker analysis of the multicohort,open-label, phase 2 KEYNOTE-158 study. Lancet Oncol. 2020;21(10):1353-65.63. Schrock AB, Ouyang C, Sandhu J, Sokol E, Jin D, Ross JS, et al. Tumor mutationalburden is predictive of response to immune checkpoint inhibitors in MSI-high metastaticcolorectal cancer. Ann Oncol. 2019;30(7):1096-103.64. Strickler JH, Hanks BA, Khasraw M. Tumor Mutational Burden as a Predictorof Immunotherapy Response: Is More Always Better? Clin Cancer Res.2021;27(5):1236-41.65. Sholl LM, Hirsch FR, Hwang D, Botling J, Lopez-Rios F, Bubendorf L, et al. ThePromises and Challenges of Tumor Mutation Burden as an Immunotherapy Biomarker:A Perspective from the International Association for the Study of Lung Cancer PathologyCommittee. J Thorac Oncol. 2020;15(9):1409-24.66. Wu Y, Xu J, Du C, Wu Y, Xia D, Lv W, et al. The Predictive Value of Tumor MutationBurden on Efficacy of Immune Checkpoint Inhibitors in Cancers: A Systematic Reviewand Meta-Analysis. Front Oncol. 2019;9:1161.67. Budczies J, Allgauer M, Litchfield K, Rempel E, Christopoulos P, Kazdal D, et al.Optimizing panel-based tumor mutational burden (TMB) measurement. Ann Oncol.2019;30(9):1496-506.68. Quy PN, Kanai M, Fukuyama K, Kou T, Kondo T, Yamamoto Y, et al.Association Between Preanalytical Factors and Tumor Mutational Burden Estimatedby Next-Generation Sequencing-Based Multiplex Gene Panel Assay. Oncologist.2019;24(12):e1401-e8.69. Notta F, Chan-Seng-Yue M, Lemire M, Li Y, Wilson GW, Connor AA, et al. A renewedmodel of pancreatic cancer evolution based on genomic rearrangement patterns. Nature.2016;538(7625):378-82.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

30 CANCER EVOLUTION: FROM MULTISTEP CARCINOGENESIS TO CHROMOTHRIPSIS70. Nones K, Waddell N, Wayte N, Patch AM, Bailey P, Newell F, et al. Genomic catastrophesfrequently arise in esophageal adenocarcinoma and drive tumorigenesis. Nat Commun.2014;5:5224.71. Cortes-Ciriano I, Lee JJ, Xi R, Jain D, Jung YL, Yang L, et al. Comprehensive analysisof chromothripsis in 2,658 human cancers using whole-genome sequencing. Nat Genet.2020;52(3):331-41.72. Cai H, Kumar N, Bagheri HC, von Mering C, Robinson MD, Baudis M.Chromothripsis-like patterns are recurring but heterogeneously distributed features in asurvey of 22,347 cancer genome screens. BMC Genomics. 2014;15:82.73. Smida J, Xu H, Zhang Y, Baumhoer D, Ribi S, Kovac M, et al. Genome-wide analysisof somatic copy number alterations and chromosomal breakages in osteosarcoma. Int JCancer. 2017;141(4):816-28.74. Abou-El-Ardat K, Seifert M, Becker K, Eisenreich S, Lehmann M, Hackmann K, etal. Comprehensive molecular characterization of multifocal glioblastoma proves itsmonoclonal origin and reveals novel insights into clonal evolution and heterogeneity ofglioblastomas. Neuro Oncol. 2017;19(4):546-57.75. Kovtun IV, Murphy SJ, Johnson SH, Cheville JC, Vasmatzis G. Chromosomalcatastrophe is a frequent event in clinically insignificant prostate cancer. Oncotarget.2015;6(30):29087-96.76. Rucker FG, Dolnik A, Blatte TJ, Teleanu V, Ernst A, Thol F, et al. Chromothripsis islinked to TP53 alteration, cell cycle impairment, and dismal outcome in acute myeloidleukemia with complex karyotype. Haematologica. 2018;103(1):e17-e20.77. Bassaganyas L, Bea S, Escaramis G, Tornador C, Salaverria I, Zapata L, et al. Sporadicand reversible chromothripsis in chronic lymphocytic leukemia revealed by longitudinalgenomic analysis. Leukemia. 2013;27(12):2376-9.78. Tang MH, Dahlgren M, Brueffer C, Tjitrowirjo T, Winter C, Chen Y, et al. Remarkablesimilarities of chromosomal rearrangements between primary human breast cancersand matched distant metastases as revealed by whole-genome sequencing. Oncotarget.2015;6(35):37169-84.79. Korshunov A, Chavez L, Sharma T, Ryzhova M, Schrimpf D, Stichel D, et al. Epithelioidglioblastomas stratify into established diagnostic subsets upon integrated molecularanalysis. Brain Pathol. 2018;28(5):656-62.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Khusan KHODZHAEV, Ozden HATIRNAZ ¨ 3180. van Engen-van Grunsven AC, Baar MP, Pfundt R, Rijntjes J, Kusters-VandeveldeHV, Delbecq AL, et al. Whole-genome copy-number analysis identifies new leads forchromosomal aberrations involved in the oncogenesis and metastastic behavior of uvealmelanomas. Melanoma Res. 2015;25(3):200-9.81. Forero-Castro M, Robledo C, Benito R, Abaigar M, Africa Martin A, Arefi M, etal. Genome-Wide DNA Copy Number Analysis of Acute Lymphoblastic LeukemiaIdentifies New Genetic Markers Associated with Clinical Outcome. PLoS One.2016;11(2):e0148972.82. Salaverria I, Martin-Garcia D, Lopez C, Clot G, Garcia-Aragones M, Navarro A, etal. Detection of chromothripsis-like patterns with a custom array platform for chroniclymphocytic leukemia. Genes Chromosomes Cancer. 2015;54(11):668-80.83. Lee KJ, Lee KH, Yoon KA, Sohn JY, Lee E, Lee H, et al. Chromothripsis in TreatmentResistance in Multiple Myeloma. Genomics Inform. 2017;15(3):87-97.84. Erson-Omay EZ, Henegariu O, Omay SB, Harmanci AS, Youngblood MW,Mishra-Gorur K, et al. Longitudinal analysis of treatment-induced genomic alterationsin gliomas. Genome Med. 2017;9(1):12.85. Brunelli M, Eccher A, Cima L, Trippini T, Pedron S, Chilosi M, et al. Next-generationrepeat-free FISH probes for DNA amplification in glioblastoma in vivo: Improvingpatient selection to MDM2-targeted inhibitors. Cancer Genet. 2017;210:28-33.86. Kuhlen M, Borkhardt A. Cancer susceptibility syndromes in children in the area ofbroad clinical use of massive parallel sequencing. Eur J Pediatr. 2015;174(8):987-97.87. Evans DG, Birch JM, Ramsden RT, Sharif S, Baser ME. Malignant transformation andnew primary tumours after therapeutic radiation for benign disease: substantial risks incertain tumour prone syndromes. J Med Genet. 2006;43(4):289-94.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

CANCER: FROM GENOMICS TO PHARMACEUTICSCHAPTER 3EXOSOMES IN CANCER DEVELOPMENT ANDMETASTASISMerve C¸ igdem ˘ OZGEL ¨ 1,2, S¸eref Bugra TUNC¸ ER ˘31PhD Candidate., ˙Istanbul University Institute of Graduate Studies in Health Sciences, ˙Istanbul, T¨urkiye2˙Istanbul University, Oncology Institute, Division of Cancer Genetics, Department of Basic Oncology, ˙Istanbul,T¨urkiyeE-mail: [email protected]. Prof. ˙Istanbul University, Oncology Institute, Division of Cancer Genetics, Department of Basic Oncology,˙Istanbul, T¨urkiyeE-mail: [email protected]: 10.26650/B/LSB28LSB48LSB56.2024.019.003ABSTRACTNaturally formed extracellular capsules with a diameter of 40–100 nm are known as exosomes of endosomalorigin. They are present in all body fluids and are secreted by the majority of body cells. Exosomes are vesicleswith a lipid bilayer membrane around them and are made up of proteins, lipids, RNA, and DNA. It can also becreated as a vehicle for delivering functional biomolecules, such as pharmaceuticals, to target locations to controlangiogenesis, metastasis, apoptosis, and inflammation. Because they are related to the cell they come from, thecontents of exosomes are known as circulating biomarkers. Exosomes provide communication between differentcells in different places through the transfer of cell contents between cells. Additionally, they serve crucial rolesin neurotransmitter release by neurons, tissue repair, immunological response, and surveillance, and cells utilizeexosomes in pathological situations like cancer and viral infections and the spread of those infections. Cancermetastasis is a multi-step process that begins with the infiltration of tumor cells locally and concludes with theinvasion of the tumor cells into the lymphatic or circulatory systems. Cancer cells must access distant organs fromthe blood. All phases of this mechanism require exosomes. This book chapter looks at how exosomes impact thegrowth and metastasis of tumors in different types of cancer.Keywords: Exosome, metastasis, lung cancer, endometrial cancer, bone sarcomas, breast cancer, ovariancancer, pancreatic cancer, prostate cancer, cervical cancerCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Merve C¸ igdem ˘ OZGEL, S¸eref Bu ¨ gra TUNC¸ ER ˘ 331. Introduction1.1. ExosomesIn 1981, Trams et al. discovered exosomes for the first time in images of healthy andmalignant cells taken under an electron microscope. It was isolated by Johnstone et al. in1986. The identification of exosomes is one of the most important developments in cellbiology over the last 30 years or so (1-3).Exosomes are endosomal-derived extracellular vesicles that are present in nature and rangein size from 40 to 100 nm (4). Almost every cell in the body releases these (5). They arepresent in every body fluid, including blood, urine, amniotic fluid, breast milk, serum, andplasma (6). Exosomes are vesicles that precipitate under a centrifugal force of 100,000 g(7). Microvesicles or microparticles are described as larger vesicles (greater than 100 nm) orvesicles that precipitated at 10,000 g (8, 9). The genesis and growth outward from the cellmembrane of ectosomes and drained vesicles serve as distinguishing characteristics (10).Exosomes are produced by endosomes within the cytosol of cells, and some of theextracellular and cellular endocytic cargo may be integrated into tiny vesicles produced byearly endosomes budding inward, giving rise to a new category of cytoplasmic vesicles knownas multivesicular bodies (MVB) (11).Membrane proteins keep their direction constant on the cell membrane by encapsulatingthe internal endosomal components into tiny vesicles because invagination of the endosomalmembrane is required for MVB formation (12). MVBs either fuse with lysosomes to beginthe process of degrading them or with the plasma membrane to release these internal vesicles,known as exosomes, into the extracellular environment (11, 13).Exosomes are produced ten times more frequently by cancer cells than by normal cells,and it has been shown that these exosomes from tumors help cells communicate by carryingproteins, lipids, RNAs, growth factors, and chemokines (14-16).1.2. Exosomal CargoesExosomes are lipid bilayer membranes that contain a payload of biological moleculeslike proteins, lipids, RNA, and DNA. Cholesterol, ganglioside, sphingomyelin, (hexosyl)ceramide, phosphatidylserine, and phosphatidylethanolamine are examples of exosomal lipids(17). miRNAs/miRs, tRNAs, rRNAs, mRNAs, circRNAs, lncRNAs, lincRNAs, cfDNAs,and mtDNAs are the most common exosomal nucleic acids (18). Exosome compositionis highly correlated with the pathophysiology and origin of secreting cells. Depending onthe requirements of the cells, the amount of protein in some intermediaries can incorporatedefinite signaling molecules, like HSP70/90 family members, plasma membrane receptors(for example, EGFR), cytokines, cytoskeleton molecules, and other cytosolic ingredients likesurvivin, Ca2+, and ubiquitin (18). The composition of the lipid membrane and the exosomecontent may be influenced by exosome activity as well as the kind of cell that produces them(19).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

34 EXOSOMES IN CANCER DEVELOPMENT AND METASTASISAll exosomes contain heat-shock proteins, cytoskeletal proteins, and proteins from thetetraspanin family (20, 21). Tetraspanins, specifically CD9 and CD63, are membraneproteins that are utilized to allocate intermediaries from another extracellular capsule (4).In addition to these common proteins, exosomes also contain proteins unique to the cellsfrom which they originated (4). Exosomes produced by cancer cells, for instance, includeseveral different tumor antigens in contrast to exosomes produced by dendritic cells, whichhave surface antigens like CD80 and CD86 (4). Sphingomyelin, ceramide, cholesterol, andglycerolphospholipids are more prevalent in the lipid layer surrounding exosomes than in thecell membrane, indicating that the lipid composition of exosomes differs from the lipid contentof the cell membrane (4). Exosome stability maintenance and cell release are made easier bythis lipid composition (Figure 1) (22).Figure 1: Exosome structure. TEM; Tetraspanin-rich microdomains (19) (Permission was obtainedfrom Xiangxuan Zhao).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Merve C¸ igdem ˘ OZGEL, S¸eref Bu ¨ gra TUNC¸ ER ˘ 351.3. Exosome Secretion and UptakeEarly endosomes that arise by invaginating through the cell membrane are the source ofexosomes (19). Early endosomes continuously gather varying amounts of cargo from thecytosol in response to certain signals, which causes them to develop into late endosomes, orMVBs with a significant number of intraluminal vesicles (ILVs) (Figure 2) (23). A major factorin the development of ILVs is an endosome sorting complex (ESCRT), which is necessary forthe machinery dependent on transport complexes (11). In order to release intrinsic ILV intothe extracellular medium and create exosomes, MVBs can go to the plasma membrane andfuse with it (19). MVBs can also interact with lysosomes, where their materials are brokendown and reused for other purposes (19).Specific signaling pathways play a major role in controlling how MVB moves and fuseswith the plasma membrane (19). It has been demonstrated that the MVB fusion processat the plasma membrane involves SNAREs and the Rab GTPases family, including Rab27aand Rab27b (24, 25). Evidence suggests that the generation of MVB is independent ofthe presence of the ESCRT complex (19). For illustration, a significant number of ILVscan continue to develop and subsequently migrate to the membrane of the cell for additionalexosome secretion even after all ESCRTs have been silenced (with Tspanins and ceramide) (26,27). By blocking inactive sphingomyelinase 2 (nSMase2), leading to creates ceramide fromsphingomyelin, the modestly sized chemical GW4682 can lessen the synthesis of exosomesfrom ESCRT-independent (28).When these small biologic carrier molecules deliver exosomes to recipient cells throughbody fluids like blood, they recognize and conjugate with the membrane receptors, activatingcertain signaling networks in the cells of interest (19). Exosomes are able to enter cellsof interest through additional mechanisms, including clathrin-dependent endocytosis, lipidraft (Caveolae/caveolin-1)-mediated endocytosis, and macropinocytosis/phagocytosis (29).Exosomes are transported into cells by MVBs, which engage in interactions with an organellein the cytoplasm of cells containing degradative enzymes enclosed in a membrane to regenerateexosomal ingredients or release their contents into the cell to serve as secondary messengers(19). By means of transcellular transport, exosomes can also be re-secreted from cells (19).Because exosomes transport certain molecules, likely proteins and lipids, that are similarto the source of cells, such as MHC or oncoproteins, they may interact with receptors presenton the membrane of target cells and result in a reaction that results in biological modifications(30, 31). Exosomes can therefore engage with recipient cells through these proceduresto release cargo or trigger signaling cascades that may eventually lead to modifications incellular activity (32).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

36 EXOSOMES IN CANCER DEVELOPMENT AND METASTASISFigure 2: Exosome release and uptake. (i) To produce an early endosome structure, specific regionsof the cell membrane may invaginate with the payload. In the meantime, with the help of ESCRTs andanother associated proteins, the endosomal lipid bilayer can encapsulate a variety of desirable payloadsto independently construct diverse ILVs encased within late endosomes (MVBs), so early endosomescan continue collecting different cargoes from the cytoplasm. To break down and recycle their contents,MVBs can travel to lysosomes and interact with them, or alternatively, they can join with the plasmamembrane to create exosomes, which are then released from cells to carry ILVs. (ii) Blood and otherbody fluids can carry exosomes to target cells. Intermediaries work by either directly transferring signalsafter attaching to the recipient cells’ surface receptors or by being drawn into the target cells via variousendocytosis pathways to produce endosomes once more. Exosomes can interact with lysosomes todigest and recycle their contents inside target cells, release their contents into the cytoplasm to undertakevarious signal transduction processes, or refuse with the plasma membrane to accomplish transcellulartransport (19) (Permission was obtained from Xiangxuan Zhao).1.4. Functions of ExosomesExosomes participate in cellular communication between various cell types and differentregions by transporting cellular information between cells (32). Despite the fact that exosomesare thought to have substantial effects under pathological circumstances, including cancer, theyhave been detected in any kind of cell as well as in a variety of biologic circumstances (33).Exosomes are therefore an essential intermediary for the functioning of cells in physiologicallyhealthy circumstances (32). As an example, these intermediaries are crucial for immunologicalreactions and surveillance (34), neurotransmitter secretion by nerve cells (35), or woundhealing (36). These intermediaries are also used by cells in abnormal situations like viralcontamination and effusion (37) or carcinoma (38). The exosome and cargo can also beemployed as circulating biomarkers from a therapeutic standpoint because they can resembleand correlate their profiles with the cell of origin (39).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Merve C¸ igdem ˘ OZGEL, S¸eref Bu ¨ gra TUNC¸ ER ˘ 37These intermediaries are also possible to acquire as vehicles for the transportation oftherapeutics, such as proteins, lipids, mRNA, miRNA, and DNA, and another useful biologicmolecules, to specific locations in order to affect angiogenesis, metastasis, programmed celldeath, inflammation, as well as other processes (40). Exosomes have been demonstrated innumerous studies to be efficient therapeutic carriers for a variety of disorders, including cancer(41), and diseases associated with neurosurgery (42), and Parkinson’s disease (43).Exosome release and uptake have been linked to a number of tumorigenesis-relatedpathways (32). These include extracellular remodeling, the exchange of oncogenes andoncoproteins, the ejection of pro-inflammatory substances, and also the modification of thestromal cell phenotype to produce a pre-metastatic niche (38).1.5. Exosomes as BiomarkersAn important component of tumor treatment has always been early tumor diagnosis (44).The prognosis for benign tumors treated through clinical means is typically favorable (44).These intermediaries are believed to eventually serve as biomarkers for disease identificationand prognosis since they are available in a variety of bodily fluids that consist of saliva, urine,and blood (45).Extracellular vesicles have been found to be more prevalent in the blood in certainusual liver diseases, like liver cancer and conditions connected to the hepatitis virus (46).Exosomal miRNAs specific to adenocarcinomas, such as miR-181p, miR-30a, miR-30e,and miR-361p, along with those specific to squamous cell carcinomas, such as miR-10b,miR-15b, and miR-320b, have been found in lung cancer tumors (44). It is believed thatthese miRNAs can serve as early-stage diagnostic indicators for lung cancer (47). Theseintermediaries are produced by hypoxic bladder tumor cells had considerably higher levels oflncRNA-urothelial cancer-associated 1 (lncRNA-UCA1) expression, and patients with bladdercancer had similarly elevated levels of lncRNA-UCA1 expression in their serum (44). ThislncRNA’s potential as a detection biological marker for bladder carcinoma is highlighted asa result (48). A prevalent malignancy among men is prostate cancer (44). Invasive biopsy isstill the highest norm for detection, although this procedure is quite likely to result in infectionand bleeding (44). Prostate-specific antigen (PSA) levels in the blood are not an adequateindicator of recurrence (44). Exosomes in the urine are regarded as a particularly quick andeasy way to test for prostate cancer, though (44). Additionally, the acidic environment of thisdisease causes the tumor to generate more exosomes, which allows patients to express highquantities of CD81 and PSA nanovesicles (49). In one study, individuals’ urine extracellularvesicles following prostate tumor excision were shown to have 2–26 times less glucuronicacid, D-ribose 5 phosphate, and isobutyryl L-carnitine than they did before surgery (50). Abone marrow biopsy can still result in significant pain and problems for patients, even if thesuccess rate of treatment for acute lymphoblastic leukemia (ALL) has reached 80–90% (44).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

38 EXOSOMES IN CANCER DEVELOPMENT AND METASTASISExosomes generated by ALL cells have higher equalities of CD19 relative to healthy cells,which may be utilized to detect ALL, according to research by Johnson et al (51).Furthermore, exosome synthesis and release may be impacted by cancer risk factors(44). By triggering the transcription of the tumor suppressor-activated pathway 6 (TSAP6),radiation can activate p53 and cause the release of exosomes (52). In Wu et al.’s research(53) it has been discovered that tobacco may cause the secretion of extracellular vesiclesfrom the lungs, > 90% of which are exosomes. Anomalies in the number and structure ofcentrosomes are common in a variety of cancer types (44). According to studies, pancreaticductal adenocarcinoma cells with extra centrosomes produce more exosomes due to lysosomaldysfunction and the presence of extra centrosomes (54, 55).Additionally, exosomes can act as a prognostic indicator for treatments (44). The amountof GOLM1NAA35 chimeric RNA in patients’ sputum was utilized as a biological marker ina planned investigation of people with squamous cell carcinoma of the esophagus to assesstherapeutic reaction, repetition, and early diagnosis (56). The exosomes in gastric cancerpatients’ malignant ascites have been shown to encourage the adenocarcinoma of the stomachin human cells (AGS) (44). Exosomes isolated from cancerous ascites dramatically decreasedthe median lifespan of animals in abdominal xenograft models employing AGS cells (44).Exosomes from malignant ascites also have high expression levels of miR-196, miR-92, andmiR-1307 (57). According to research by Manier et al.(58), exosomes containing miR-18aand miR-let-7b were found in the serum of 156 individuals with multiple myeloma who hadrecently been diagnosed and were receiving standard care. Patient progression-free survival(PFS) and overall survival durations were substantially correlated with the levels of thesemiRNAs (44). Fibroblast growth factor 11 can be targeted by exosomal miR-24-3p producedby nasopharyngeal cancer cells to inhibit T cell activity (44). Exosomal miR-243p has beenlinked to PFS, and studies have revealed that patients with lower levels of this miRNA intheir serum have longer PFS (59-61). In comparison to patients with osteosarcoma who hada good chemotherapeutic reaction, Xu et al. (62) discovered that individuals with a weakchemotherapy reaction had significantly lower levels of miR-133a, miR-124, miR-199-3p,and miR-385 and also discovered that serum exosomes had significantly higher levels ofmiR-135b, miR-148a, miR-27a, and miR-9. These results specify that diverse exosomal RNAparticles can act as direct biological indicators for osteosarcoma malignancies having a rangeof chemotherapy sensitivity (44).1.6. Relationship Between Exosome and Tumor MicroenvironmentFor a long time, exosomes were believed to be metabolic waste products from cells (44).Exosomes are critical for cellular communication, as researchers have learned through agrowing body of study on this topic (63-65). Exosomes have also been shown to have animpact on invasion, tumor growth, spread and angiogenesis (66, 67). The development of theCancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Merve C¸ igdem ˘ OZGEL, S¸eref Bu ¨ gra TUNC¸ ER ˘ 39tumor microenvironment (TME), which consists of tumor vessels, extracellular matrix (ECM),and additional cancer-free cells (68, 69) like stromal cells, fibroblasts, and inflammatory cells,has also been reported to be supported by exosomes (70). Consequently, it is believed thatTME-specific therapy may be successful (71, 72).These intermediaries may potentially affect in the TME cells’ proliferation anddifferentiation (44). The development of osteoclasts and the activity of bone resorptioncan both be promoted by exosomes produced from osteosarcoma (44). Exosomes from cancercells that express CD171 have been demonstrated to be able to increase the invasiveness,motility, and proliferation of glioblastoma cells (73). Exosomes produced by MDA-MB-231cells in breast cancer can target SRC kinase signal inhibitor 1 to enhance osteoclastproliferation and differentiation by transferring miR-20a-5p to bone marrow macrophages(74). It has been discovered that breast cancer MCF7 cells actively recruit exosomes frommesenchymal stem cells (MSC) that have differentiated into adipocytes (44). Later on, it wasdiscovered that this promoted MCF7 cell proliferation and migration as well as protected cellsagainst chemotherapeutic drug-induced or serum deprivation-induced apoptosis in vitro (75).Exosomes produced by cancer cells can encourage the transformation of normal fibroblastsor MSCs into cancer-associated fibroblasts (CAFs), which can stimulate the tumor cells toinvade and spread around the body (76, 77). Additionally, it has been proposed that the tumorprotein p53 may cause sensory nerves attached to tumors via extracellular vesicles to undergoadrenergic trans-differentiation (78).1.7. Role of Exosomes in Tumor ProgressionExosomes increase tumor growth and invasion in TME and function as a communicationtool (44). The first is that exosomes released by tumor stromal cells have unique impactson tumor cells (44). Lazar et al. (79) discovered, for instance, that melanoma cells’ fattyacid oxidation increases in the presence of adipocyte exosomes, which promotes increasedmigration and invasion. Then, it has been suggested that the exosomes that tumor cells producecan influence stromal cells to encourage the invasion and spread of tumor cells (44). Onetypical CAF biomarker is α -smooth muscle actin (44). Exosomal miR-1247-3p directly targetsβ 1,4-galactosyltransferase III in extremely metastatic hepatocellular carcinoma, activating the1 integrin/NF-?B signaling pathway in fibroblasts (44). By secreting inflammatory chemicals,including IL6 and IL8, activated CAFs further accelerate the development of cancer (80).Additionally, exosomal miR-105 from basal cell carcinomas inhibits the expression of MAXinteracting protein 1 (MXI), an inhibitor of MYC transcriptional activity, in CAFs, resultingin MYC activation-dependent gene expression and encouraging tumor cell development (81).McAtee et al. (82) discovered that treatment of prostate stromal cells with tumor exosomesgreatly promoted their migration in a manner reliant on the catalytic activity of Hyal1,explaining why high Hyal1 levels encourage the progression of prostate cancer. Becker et al.Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

40 EXOSOMES IN CANCER DEVELOPMENT AND METASTASIS(83) discovered that extracellular vesicles from tumors can lead fibroblasts to develop intomyofibroblasts, which release matrix metalloproteinase (MMP) and modify the ECM.These intermediaries released by cancerous cells have the ability to alter vascularendothelial cells’ characteristics and encourage the development of new blood vessels (44).For instance, in the situation of hypoxia in tumor cells, MSC-derived extracellular vesiclescontrol the development of vessels through a variety of miRNAs, including miR-126, miR-214,and miR-296 (84). Exosomes rich in miR-23a from nasopharyngeal cancer can encourage thedevelopment, migration, and production of microtubules in human umbilical vein endothelialcells (85). Loss of p85α expression in breast cancer may cause stromal fibroblasts to developCAF-like characteristics (44). Exosomes produced by p85α-deficient fibroblasts may speed upthe development of cancer by inducing the epithelial-mesenchymal transition (EMT), whichis a result of the canonical Wnt pathway (86).1.8. Exosome and MetastasisMany steps are involved in the spread of cancer cells; they start with local infiltrationbefore moving into the bloodstream or lymphatic system and eventually spreading to otherparts of the body. Cancer cells must enter and exit distant organs from the bloodstream. Allphases of this process require exosomes (44).According to the “seed and soil” idea of Stephen Paget from 1889, malignant cells(referred to as seeds) interact with their host environment (referred to as soil) and allow for theprogression of the cancer (87). The interaction between cancer cells and TME has since beenthe subject of intensive research in the field of cancer, which has produced reliable evidence(88).Numerous cells, such as macrophages, fibroblasts, and endothelial cells, come together toproduce the TME, a complex cellular milieu (32). It plays a significant part in remodelingcancer cells, modifying their activity, and controlling the growth of tumors and the spreadof metastatic disease (32). Additionally, stromal cells and cancer cells both emit varioussubstances that may cause changes in distant tissues and create the ideal conditions for thedevelopment of a pre-metastatic niche (PMN) (89, 90). Exosomes have been recognizedas one of the critical elements in this instance for the production of tumor and stromal cellmetastasis and regulation of an oncogenic phenotype (66, 91, 92).An essential component of tumor development and metastasis is intercellularcommunication (93). It has been demonstrated that exosomes released by malignancies canencourage the development of PMNs (44). Metastatic niches are created at distant metastasissites by extracellular vesicles released by tumor cells (83). Fibroblasts, endothelial cells,and ECM make up the majority of the niche’s matrix environment prior to transfer (44). Inaddition to producing growth hormones and inducing inflammation, fibroblasts also expressfibronectin and MMP (94).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.

Merve C¸ igdem ˘ OZGEL, S¸eref Bu ¨ gra TUNC¸ ER ˘ 411.8.1. OrganotropismAccording to published research on organotropism, all evidence has pointed to a functionfor exosomes in triggering metastases in particular organs; however, there is no informationavailable to show what causes particular organotropism (32). With the discovery of Hoshinoet al. (70), this knowledge was altered. According to their findings, integrins on the exosomesproduced by cancer play a crucial role in determining where metastatic cells will eventuallyend up (32). They used intermediaries from several tumor cell lines, particularly thosethat have metastasized to the brain, lung, or liver, to study the biological distribution ofthese intermediaries and their role in metastatic functioning when injected into animals (32).Exosomes from various cell lines were injected into the organ region where each cancermetastasized most frequently (32). Additionally, this uptake was restricted to a certainpopulation of cells in each organ, including the brain endothelial cells, liver Kupffer cells, andlung fibroblasts and epithelial cells (32). Migration and inflammatory genes were upregulatedas a result of this integrin-specific exosomes uptake (70). A proteomic investigation thatrevealed different patterns of integrin expression on the surface of intermediaries dependingon the host organ revealed that ITGα6β4 and ITGα6β1 steered exosomes to lung metastasis,ITGβ3 to brain metastasis, and ITGα?β5 to liver metastasis (32). Mice exhibited lessexosome uptake and fewer metastases when these integrins were inhibited (70). Otherexosome-associated integrins related to organotropism include ITGα4 for lymph nodes, ITGβ1for the brain, ITGα4β1, and ITGα?β3 for bone metastases (95).1.8.2. Neo-AngiogenesisAngiogenesis is a crucial component of the growth of cancer because it enhances thesupply of nutrients and oxygen to the expanding cellular mass (89). Hypoxia, which canaffect gene expression and phenotypic alterations, is closely related to this process (96). Theimportance of intermediaries from disease in regulating the neo-angiogenic process, whichhelps tumor cells move to new sites and increase nutrient supply, has therefore been highlightedin a number of investigations (32).Additionally, exosomal miRNAs have been linked to angiogenesis linked to metastasis aswell as the induction of vascular permeability (32).1.8.3. Pro-Metastatic Phenotype InductionThe surface membranes of exosomes include different RNAs and proteins that are part oftheir cargo (97). Consequently, the release or contact of these compounds in target cells mayresult in modifications to cell behavior and the formation of more aggressive phenotypes (33).1.8.4. Stromal-Tumor InteractionsBoth tumor-derived exosomes and exosomes generated by cells otherwise malignant onesmay communicate with tumor cells, altering their biological function and possibly eventriggering more aggressive circumstances (33).Cancer: from Genomics to Pharmaceutics, edited by Zeynep Karakas, et al., Istanbul University Press, 2024. ProQuest Ebook Central, http://ebookcentral.proquest.com/lib/uitm-ebooks/detail.action?docID=31789562.Created from uitm-ebooks on 2025-12-02 14:42:18. Copyright © 2024. Istanbul University Press. All rights reserved.