Cancer: Principles and Practice of Oncology 9th Edition - Cap12 Biologia da Medicina do Câncer

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RAJU KUCHERLAPATI

CHAPTER 12 BIOLOGY OF PERSONALIZED CANCER MEDICINE

It is well established that cancer is a genetic disease. At the genetic level cancer cells are different from their precursor cells. It is understood that a series of genetic changes are nec-essary for a normal cell to begin the process of transformation, eventually leading to cancer. Cancers can be generally classi-fi ed into sporadic cancers and those that result from a genetic predisposition. Some individuals in the population inherit spe-cifi c mutations in particular genes that predispose them to cer-tain types of cancers. Although these individuals are born with a mutation in a predisposition gene, they do not develop tumors until later in life; and it is now well understood that, besides the inherited predisposition gene mutation, additional genetic changes are required for the cells in these individuals to become tumors. In sporadic cases a randomly acquired somatic mutation or another type of genetic change in a gene that is critical for the normal regulation of growth in the appropriate cell type might initiate a series of events that even-tually leads to tumor formation. In addition to genetic muta-tions, changes in copy number of individual genes or subsets of genes; chromosomal aberrations including translocations, insertions, deletions and inversions; and changes in expression patterns of genes as well as epigenetic changes also play criti-cal roles in tumor susceptibility, tumor initiation, and progres-sion. Understanding all of the important genetic and genomic changes in each cancer type will help in accurate diagnosis and prognosis and increase the ability to stratify the patient popu-lations to help assess the most optimal treatments for each patient. The use of such genetic and genomic information to determine treatment decisions is referred to as personalized medicine. Knowledge about the genetic and genomic changes that accompany cellular transformation and the events that are critical for initiation and maintenance of the cancerous state is increasing at a rapid pace. Because most cancers are clonal in origin and because it is possible to obtain an ade-quate amount of tumor material from tumor biopsies or resec-tions, it is now possible to examine and document the genetic and genomic changes in tumor cells very accurately. As a result, understanding of the genetic and genomic changes in cancer is signifi cantly greater than many other human diseases. This knowledge allows implementation of the principles of personalized medicine into clinical management of cancer patients. This chapter will consider examples of how cancer genetics and genomics are affecting the ability to manage can-cer patients.

CANCER PREDISPOSITIONThere is a large body of evidence that indicates that certain families have a higher incidence of a particular cancer. Epidemiological studies reveal that family members descen-dant from individuals who developed cancer, especially at a younger age, have a higher risk of developing cancer. This was

followed by studies of families where the predisposition to develop cancer was found to be inherited. Another line of evi-dence that reveals the genetic basis for cancer came from stud-ies of twins. Monozygotic twins have the same genetic compo-sition, while dizygotic twins have a 50% probability of sharing an identical copy of any gene. The fact that there is a higher concordance of cancer incidence in monozygotic twins but not in dizygotic twins provides additional critical evidence for the genetic basis of cancer.1 Studies during the past half century have established not only the familial predisposition of cancer but also to identifi ed several genes that are involved in cancer predisposition. That knowledge, in turn, helps to explain the genetic processes and mechanisms that lead to cancer and to develop tests to identify individuals at risk for certain types of cancers.

There are several examples of gene mutations that are responsible for cancer predisposition. Cancers that show a familial predisposition include childhood retinoblastoma, col-orectal cancer, early onset breast cancer, and several types of renal cancer, among many others. One cancer type where there is a large amount of information about familial predisposition is colorectal cancer (CRC).2 CRC is one of the most common cancers, and it is estimated that as many 875,000 new cases of CRC are diagnosed every year in the world. The greater acces-sibility of the colon and rectum to detect or follow the cancer as well as the relative ease of obtaining biopsies facilitate study of this cancer.

CRC can be classifi ed into familial cases and sporadic cases. Various estimates of the relative proportions of these two cat-egories of CRC have been made and in some estimates the familial cases represent 10% of all CRCs, while other esti-mates place this number to be 25% or more.3 Several genes that are involved in familial cases have been identifi ed and are discussed in the sections that follow.

FAMILIAL ADENOMATOUS POLYPOSIS

Individuals with familiar adenomatous polyposis (FAP) are born normally and develop hundred to thousands of benign colonic polyps during their early adulthood. Unless these tumors are detected and treated (usually by surgery), one or more of them may develop into adenocarcinomas that can metastasize. Family studies revealed that this predisposition was inherited in an autosomal dominant fashion, and individ-uals who have inherited the susceptibility allele almost always exhibit the phenotype (near 100% penetrance). Linkage anal-ysis revealed that the gene for FAP is located on human chro-mosome 5. Positional cloning has enabled the cloning of the gene that was designated as adenomatous polyposis coli (APC).4,5 APC is a classic tumor suppressor gene, and inactivation or

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142 Molecular Biology of Cancer

modifi cation of both copies of the gene is necessary for the initiation of CRC development.

APC plays an important role not only in the relatively rare cases of FAP but also in a majority of sporadic CRC. Most of the sporadic colorectal tumors are also the result of the inacti-vation of both copies of the APC gene, but unlike FAP, where one copy is already mutated in the germline, in sporadic cases both copies are sequentially mutated in somatic colonic epi-thelial cells.6 This observation explains the earlier onset of tumors and the abundance of tumors in FAP patients.

Since FAP is inherited in an autosomal dominant fashion and since individuals with FAP are born with a mutation in the APC gene, when an individual with FAP is identifi ed it is recognized that all of the immediate family members are at risk to carry the mutant allele. The siblings of the affected individual are at 50% risk, and other relatives would also be at risk depending on the nature of familial relationship. Because the diagnosis of FAP is unambiguous, genetic testing is not always conducted. It is most desirable to sequence the germline DNA of the affected individual to identify the muta-tion, inform the relatives of their risk, and recommend the testing for the specifi c mutation as appropriate.

LYNCH SYNDROMEAnother predisposition to CRC is Lynch syndrome (LS), named after Henry Lynch who fi rst described this syndrome.7 LS is also inherited as an autosomal dominant disorder, and individuals who inherit a disease allele develop CRC at an ear-lier age than sporadic CRC but later than FAP patients. The manifestation of colorectal neoplasms in individuals with this syndrome is less severe than in FAP patients, with the develop-ment of a few tumors later in life.

It was noted that the LS tumors have a unique feature in that they show genetic instability as revealed by expansion or contraction of the length of microsatellites.8–10 Based on these observations these tumors are classifi ed as microsatellite insta-ble (MSI�). This knowledge of microsatellite instability plays an important role in discovering the genes important for LS. The fi rst gene that was responsible for a subset of LS cases was found to be a human gene that has homology to a bacterial gene that is necessary for repairing single nucleotide mis-matches and small insertions or deletions that result from errors in DNA replication.11,12 This gene was designated Mut S homolog 2 (MSH2). MSH2 encodes a protein that is required for recognition and repair of DNA mismatches. It was later discovered that mutations in other genes that encode members of the mismatch repair complex also cause LS. In addition to MSH2 the genes that encode other members of this complex that are now known to be involved are Mut L homolog 1 (MLH1), Mut S homolog 6 (MSH6), postmeiotic segregation 2 (PMS2), and Mut L homolog 3 (MLH3). Mutations in all of these genes are now implicated in colon cancer susceptibility.11,13–18

Relatives of LS patients are also at increased risk to carry the mutant gene and, therefore, for CRC. As is the case for FAP it would be desirable to establish the specifi c mutation that causes LS and test the immediate relatives to establish or rule out the presence of the specifi c mutation in their germline. The presence or absence of mismatch repair proteins, espe-cially MLH1, can also be detected by immunological methods, and individuals whose tumors do not have a detectable level of MLH1 are excellent candidates for testing of their germline; if a germline mutation in the gene is detected, informing that patient’s immediate relatives and testing them for the specifi c mutation may be warranted.

The precise incidence of LS in the population has been dif-fi cult to assess. Because individuals with LS develop fewer

tumors and later in life than those with FAP, they are more dif-fi cult to distinguish from sporadic cases, and, therefore, testing for LS has not become routine. According to some studies the incidence of LS among patients with colorectal neoplasms is as high as 3%.19 These results suggest that examination of all colorectal tumors for its MSI status and testing for mismatch repair gene mutations in individuals whose tumors are MSI� may help identify individual at risk with a greater effi ciency.20,21

OTHER POLYPOSIS SYNDROMESThere are other syndromes that are relatively rare that predis-pose individuals to CRC risk. These include Peutz-Jeghers syn-drome, juvenile polyposis, and Cowden’s disease. Genes that are involved in these syndromes have been identifi ed and include LKB1, SMAD4, BMPR1A, and PTEN. Although all of the syndromes mentioned above are inherited in a domi-nant fashion, mutations in MYH, a homolog of an excision repair gene in Escherichia coli, cause a tumor predisposition but in a recessive fashion (de la Chapelle22 gives a detailed review).

ASSOCIATION STUDIESMost of the genetic mutations in genes described above result in CRC with high penetrance. This raises the question if there are other genes where mutations or variants cause a predispo-sition to CRC but with lower penetrance. Studies of sibling pairs that are concordant or discordant for CRC as well as association studies have identifi ed regions of the genome or single nucleotide polymorphisms (SNP) that may be important in CRC susceptibility. One such polymorphism is that located on human chromosome 8. In an initial study Zanke et al.23 examined a large cohort of individuals who had large bowel cancer and an equal number of controls for associations with genes or variants in the genome. In this study they identifi ed SNPs at 8q24, which shows signifi cant association with sus-ceptibility to colon cancer. This region was also shown to be responsible for susceptibility to several other cancers.24,25 Additional follow-up studies confi rmed and extended these observations, implicating other regions of the genome in sus-ceptibility to colon cancer among “sporadic” cancer cases.26 Like several other SNP variants that have been shown to be associated with complex diseases, the SNPs at 8q24 also lie in a region that is not known to harbor any genes. However, Pomerantz et al.27 were able to show that this variant is func-tionally important in regulating the expression of the cellular oncogene c-myc that is located a few hundred kilobases away from the variant. This group also showed that variants at 8q24 that are known to be involved in other solid tumors also act through their action on the myc oncogene.28 These results sug-gest that it might be possible to identify individuals within the general population that show susceptibility to several different solid tumors. Identifi cation of such susceptible individuals may, in turn, help in more careful monitoring or other inter-ventions, which may lead to prevention of the cancers.

BREAST CANCERSusceptibility to early onset breast cancer has been extensively studied. It was recognized that certain families have a high incidence of breast and ovarian cancers. Careful examination of these families revealed that the predisposition to these can-cers is inherited in an autosomal dominant fashion. Genetic linkage analysis revealed that, at least in some families, this


Chapter 12: Biology of Personalized Cancer Medicine 143

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trait is linked to markers on human chromosome 17.29 When positional cloning approaches became available, it was deter-mined that mutations in a gene on this chromosome were found to be responsible for this predisposition.30,31 That gene was designated BRCA1 (BReast CAncer-1). In other families a sec-ond gene, BRCA2, located on chromosome 13, was found to be involved in the cancer predisposition. Women who inherit a mutation either in BRCA1 or BRCA2 are at high risk for devel-opment of breast or ovarian cancer. Mutations in these genes can be inherited or they could result from new mutational events. If an individual with mutations in either BRCA1 or BRCA2 has been identifi ed, it would be important to assess if other members in their family are at risk. If inherited, since the mutations are dominant acting, each of the immediate rela-tives (siblings) would have a 50% risk of carrying the same mutation and therefore would also be at high risk for develop-ing cancer. Individuals with known pathogenic mutations may elect prophylactic mastectomy and oophorectomy or careful surveillance to detect tumors at their earliest stage.

Testing individuals at high risk for breast cancer or colon cancer is a common practice. If an individual tests positive for a pathogenic mutation, it is prudent to test for the presence of the same mutation in that individual’s immediate relatives and manage them based on the results.

It is estimated that only a fraction of women that carry a pathogenic mutation in BRCA1 or BRCA2 are detected. Detection of these mutations well in advance of the time at which they would develop their fi rst breast or ovarian tumor would have signifi cant positive implications for their health. A relatively simple way of identifying individuals at risk is through the use of family history. Several family history tools are available, and one that was developed by the surgeon gen-eral of the United States is easily accessible and free of charge on the U.S. Department of Health and Human Services Web site (search for “My Family Health Portrait Tool”). Algorithms that can assess relative risk have been developed, and depend-ing on these risk predictions, appropriate individuals may be recommended to undergo genetic testing.

EARLY DETECTIONIt is well established that the long-term survival of patients, whose tumors were diagnosed at early stages, are signifi cantly greater than those whose tumors are detected at a later stage. For example, the long-term survival of patients whose colonic tumors were detected at stage I is 95%, while it is only 5% when the tumors are detected at stage IV.

There are different methods for early detection of can-cers. For colon cancer detection, colonoscopies are recom-mended for all individuals over the age 50. Palpation of the prostate is a routine procedure during annual medical exam-inations. Mammograms are useful in detecting at least a sig-nifi cant portion of breast tumors. Some of these methods are expensive and patient compliance is not adequately high. Alternative strategies for detection of cancer at early stages are in development.

One approach to identify such markers was used by Faca et al.32 In this study, a murine model for pancreatic cancer was used as a starting point. These mice reliably develop pancre-atic tumors during specifi c periods of their life. Plasma samples from these mice at different stages of tumor development were sampled and analyzed for their protein composition using proteomic approaches. Several proteins that were found to be overexpressed during early stages of cancer were examined in the blood from 30 newly diagnosed patients with pancreatic cancer and an appropriate set of 30 controls. This approach enabled them to identify a panel of fi ve proteins that was able to discriminate pancreatic cancer cases from matched controls

in blood specimens obtained as much as 12 months prior to the diagnosis of pancreatic cancer. Similar approaches enabled identifi cation of protein markers that are important for ovar-ian and colon cancers.33,34

The reason why tumor-specifi c markers can be detected in the circulating system prior to clinical diagnosis of disease is not well understood. It is possible that some tumor cells die, releasing their contents into circulation. Alternatively, some subsets of tumor cells escape their original site and are in cir-culation. Such cells are referred to as circulating tumor cells (CTC). Escape of tumor cells from their source of origin and entering the circulation is, of course, an important step in metastasis. Methods to detect CTC have advanced signifi -cantly in the past few years. It has been long known that tumor cells may express novel proteins on their cell surface. Although antibodies against many of these proteins are available, purifi -cation of circulating tumors cells proved to be difficult. However, many of the solid tumors are derived from epithelial cells, and such cells are not normally part of the circulation. Therefore, epithelial cell markers can be used as capture agents. This approach, together with the development of novel fl ow cells that allow for slow and gentle movement of cells through a substrate coated with the appropriate antibodies, is now shown to be a suitable method for capturing these rare circulating tumor cells.35 The availability of intact cells will allow more detailed molecular examination of tumor cells, which could prove to be powerful in early diagnosis. It is important, however, to understand how well these circulating cells refl ect the state of the tumor at its initial location. The ability to isolate circulating tumor cells has signifi cant impli-cations for our ability to understand the nature of the tumor and to devise appropriate interventions for the patients.

TUMOR CLASSIFICATION AND PATIENT STRATIFICATION

Assessment of the molecular origins of tumors and the molec-ular profi les of the tumors has important implications for accurate diagnosis, determination of the prognosis, and treat-ment decisions for a patient. Methodologies for assessing such features are rapidly evolving.

Early methods of tumor classifi cation were based on cyto-genetic methods in hematological malignancies. The fi rst of chromosomal translocations that was identifi ed as important in human cancer is the t(9;22)(q34;q11) associated with chronic myelogenous leukemia (CML).36 Because the discov-ery was made from investigators from Philadelphia, this rear-ranged chromosome has been designated the Philadelphia chromosome. During the past 40 years hundreds of such trans-locations have been described in many different malignancies, and more than 300 different genes have been implicated in these abnormalities. For example, in acute myeloid leukemia 267 balanced rearrangements have been described.37 These translocations sometimes result in inappropriate activation of genes or the creation of novel fusion genes, with novel func-tions resulting in cancer. The nature of these translocations is critical for accurate diagnosis and in several cases for targeted therapies. Although a majority of these translocations are described in hematological malignancies, several such translo-cations have been described in solid tumors, and it is now clear that such translocations are also common in most, if not all, solid tumors.38,39 As is the case for hematological malig-nancies, specifi c translocations would not only help classify the tumors but also may provide novel targets for drug devel-opment. One such example is the EML-ALK4 translocation in lung cancer.38

Diagnosis of tumors can also be made on the basis of the gene expression profi les of the tumor. An example is the



distinction between Burkitt’s lymphoma and large B-cell lym-phoma. These two disorders are treated differently, and, there-fore, it is important to distinguish between them accurately. The diagnosis of Burkitt’s lymphoma is largely based on mor-phologic fi ndings, immunological data, and cytogenetic fea-tures. Diffuse large B-cell lymphoma and Burkitt’s lymphoma have some overlapping clinical features. In addition, Burkitt’s lymphoma has the t(8;14) translocation that results in the activation of the myc oncogene, but this translocation is also present in a subset of diffuse large B-cell lymphoma. Examination of Burkitt’s lymphoma cases and large B-cell lymphoma samples by global gene expression profi le analysis enabled the identifi cation of a panel of genes whose expres-sion profi les can distinguish the two categories with a very high level of accuracy.40

Some novel tests based on the patterns of gene expression profi ling have been developed, and intensive efforts for addi-tional tests are currently under way. An example of a type of test that was developed was one to predict the recurrence of cancer in women with tamoxifen-treated, node-negative breast cancer.41 RNA extracted from paraffi n-embedded sec-tions of breast tumors from women who were enrolled in a clinical trial to study the effects of tamoxifen treatment were examined for expression of a panel of 21 genes. Based on the analysis of the data the investigators were able to translate the data into a recurrence score. It is likely that similar efforts with either gene expression profi les or protein expression profi les of tumors will result in identifi cation of marker sets that can be used for accurate diagnosis and prognosis of many tumor types.

Efforts to use comprehensive genomic data for tumor diag-nosis and stratifi cation are proving to be extremely valuable. An example is the examination of human glioblastomas.42 In this study several groups of investigators examined tumor samples from human glioblastomas and the corresponding normal DNA from the same patients for changes in gene and genomic copy number, gene expression profi les, and the muta-tional status of a large number of genes. Based on this compre-hensive analysis this group of investigators was able to classify the tumors and assess the pathways that are deregulated in this cancer type. Similar types of efforts are under way for many other cancer types.

Other investigations have focused on a comprehensive examination of genetic changes in tumor cells and tumor tis-sue. These efforts have examined all of the coding regions of the tumor genome and in some cases the complete genomes of tumors.43–46

TREATMENTAs the understanding of the genetic and genomic changes that are responsible for tumor progression increases, it is becoming possible to understand the particular genetic changes and bio-chemical pathways that are modifi ed in tumor cells. This knowledge, in turn, helps determine which patients are most likely to benefi t from which drugs. There are several excellent examples of this feature and others are emerging rapidly. Some of these are briefl y described below.

One of the fi rst examples of a targeted therapy is for CML. In 1960 Nowell and Hungerford47 described a marker chromosome in a human leukemia. In 1973 Rowley48 defi ned this marker chromosome, designated the Philadelphia chromosome, to be the result of a translocation involving human chromosomes 9 and 22. It was later shown that this specifi c translocation results in a novel fusion gene product that involves a break point cluster region (BCR) and an oncogene that is homologous to the Abelson murine leuke-

mia virus (ABL). The fusion product, designated BCR-ABL, is a tyrosine kinase and is expressed only in these tumor cells. It has been shown that the formation of this fusion gene is sufficient for the cells to become transformed. Mutational analysis of the fusion gene revealed that the loss of function of this protein leads to loss of its oncogenic activity. These observations led to the development of a spe-cifi c inhibitor for this fusion protein. The drug, originally designated STI571, now imatinib, was found to be effi ca-cious in preclinical studies. Clinical studies with this drug revealed that the drug that is orally administered is rela-tively safe and as many as 98% of the CML patients showed hematologic response in as little as 4 weeks of drug admin-istration.49,50 Additional studies showed that the drug’s effects favorably compare with standard therapy for newly diagnosed CML patients. This is the current choice of drug for CML that is diagnosed to have the Philadelphia chro-mosome.

A second example of targeted therapy is for a subset of breast cancer patients. A subset of breast cancers (20% to 30%) are known to have amplifi cation of a growth factor receptor gene, ERBB2 (HER2). Women whose breast cancers have a high level of expression of this gene have a shortened survival. It has been shown that the amplifi cation of the HER2 gene is directly involved in the pathogenesis. Therefore, it was considered that development of inhibitors of this pro-tein, which is expressed on the surface of the tumor cells, might provide an approach to treat these breast cancer patients. Several antibodies directed against this target were found to bind this target with high affi nity and inhibit their proliferation. A humanized murine monoclonal antibody was developed as a therapeutic agent. This drug, trastu-zumab, was found to be relatively safe, and its administra-tion resulted in better disease-free progression and higher rates of overall response compared to chemotherapy alone. Combination of chemotherapy and trastuzumab improved the outcomes even more.51,52 Based on these clinical trails the drug was approved and is currently the standard of therapy for patients whose breast tumors have amplification of HER2.

The role of genetic changes in drug response was well described in non–small cell lung cancer (NSCLC). Until the early part of this century the most widely used treatment for NSCLC has been chemotherapy, which results in a small increase in survival and is associated with several adverse effects. It was known that these lung cancer cells express higher levels of epidermal growth factor (EGFR) as com-pared to normal lung cells. EGFR is a member of a class of transmembrane signaling proteins. In the presence of its ligand, epidermal growth factor, the receptor dimerizes, resulting in phosphorylation of tyrosine in the intracellular domain and leading to a cascade of events that promote cell growth. One of the drugs that was developed to inhibit this tyrosine kinase activity is gefi tinib. Treatment of patients with gefi tinib resulted in variable responses among individu-als with lung cancer. In early clinical trials individuals from Japan had better responses than those from the United States, and female never-smokers were better responders among both ethnic groups. In the United States the response rates were less than 20%, and the response did not correlate with EGFR levels as measured by immunohistochemistry. To understand the basis for this variable response, one group hypothesized that mutations in a receptor tyrosine kinase may be responsible for drug response.53 To test this hypoth-esis they obtained tumor DNA samples from tumors prior to treatment and examined the DNA for mutations in the acti-vation loops of 47 receptor kinase genes. A small number of tumors had heterozygous mutations in the EGFR gene. A



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more comprehensive examination revealed heterozygous missense and deletion mutations in a region corresponding to proximity of the adenosine triphosphate (ATP) binding cleft and the target of the drug gefi tinib. Tumor DNA from patients who responded to the drug, obtained prior to initia-tion of treatment, had a mutation in EGFR in all responders, while only 1 of 61 nonselected patients had such a mutation. They were also able to show that lung caner cell lines that carried one of the mutations responded to low doses of gefi -tinib and also inhibited the autophosphorylation of the EGFR protein.

A second group of investigators reasoned that since the drug targets EGFR, mutations in that gene might be respon-sible for the differential effect of the drug.54 To test this hypothesis they examined the biopsy samples from patients who later showed responses to gefi tinib. These studies revealed that of the nine samples examined, eight had genetic changes in the region of the gene corresponding to the intracellular domain of the protein. The nature of the mutations detected were also point mutations, leading to a change in amino acid and deletions that resulted in loss of a few amino acids of the protein. No such mutations were detected in other tumor types, and the changes were found to be somatic. Using tran-sient transfection of the mutant version of the gene into mam-malian cells revealed that the mutations led to hyperactivation of the protein and to better responses to the drug at lower concentrations.

Both of these studies and another study published soon after55 revealed that in NSCLC, the target of the drug gefi tinib acquires certain somatic mutations, some of which result in activation of the protein. It is those patients whose EGFR has acquired one of these activation mutations who respond to the drug. These observations paved the way for the development of a molecular diagnostic test to identify patients who might be better responders to the tyrosine kinase inhibitors gefi tinib and erlotinib.

To clinically assess if selection of patients whose lung tumors had an activation mutation would respond better to a tyrosine kinase inhibitor, several clinical trials were conducted. Although the trail designs and the number of patients in each trial varied, the general schema of several clinical trials was to examine the tumor DNA for EGFR mutations and assess their response rates and progression-free survival as well as long-term survival benefi ts of the drug.

In one study Mok et al.56 compared the effectiveness of chemotherapy versus gefi tinib treatment in patients with lung cancer. In a phase 3 study, they randomly assigned treatment-naive patients to receive gefi tinib or chemotherapy. They observed that in the cohort who had EGFR mutations both the response rates as well as progression-free survival were better in the mutation-positive group. Interestingly, in the mutation-negative cohort chemotherapy yielded better response rates and better progression-free survival. These trial results suggest that patient stratifi cation based on EGFR status and treatment with a tyrosine kinase inhibitor for mutation-positive patients and chemotherapy for mutation-negative patients would yield optimal response rates.

DEVELOPMENT OF RESISTANCE TO TYROSINE KINASE

INHIBITORSDespite the fact that individuals whose tumors have an acti-vation mutation in EGFR respond better to tyrosine kinase inhibitors and such treatment leads to longer survival, all patients appear to relapse and tumor begins to grow again.

To understand the basis for this relapse Kobayashi et al.57 obtained a biopsy sample from a relapsed patient and exam-ined the status of the EGFR gene in the sample prior to treatment and the sample after relapse. Using direct DNA sequencing and sequencing of cDNA prepared from the tumor RNA sample, they observed that the relapsed tumor contained a novel mutation in the EGFR gene that resulted in a T790M mutation. Using molecular biological methods the investigators showed that the presence of this mutation in the background of an original responsive mutation ren-dered the EGFR protein 100 times more resistant to the drug. When they tested cells carrying the resistant allele with four commercial EGFR inhibitors, they found that an irre-versible inhibitor of EGFR strongly inhibited EGFR func-tion at relatively low concentrations. This observation sug-gested that periodic genetic monitoring of the tumor prior to treatment and at the time of relapse might help in the choice of the most appropriate drug or treatment for the patient.

ALTERNATIVE MECHANISMS OF RESISTANCE

It was observed that some individuals whose tumors have activating mutations in the EGFR gene do not respond to tyrosine kinase inhibitors. It is noted that some of these tumors have mutations in KRAS.58–60 Activation of the EGFR-mediated signaling pathway involves the activation of the RAS-Map kinase pathway. Thus the product of RAS acts downstream of EGFR. Therefore, it is reasonable that inde-pendent activation of the downstream target renders the sta-tus of EGFR irrelevant to growth phenotype of these cells. These results form the basis for testing NSCLC for both EGFR and KRAS mutations. Patients who are most likely to respond to EGFR tyrosine kinase inhibitors would be those whose tumors have an activating mutation in EGFR and are wild type for KRAS.

Role of KRAS Mutations in Other Epidermal Growth Factor Receptor Inhibitors

Patients with colorectal cancer who failed chemotherapy were administered an antibody against EGFR, cetuximab. Although treatment with this monoclonal antibody resulted in improved overall survival, the disease progressed in more than 50% of the patients. Since a subset of colorectal tumors were known to have activating mutations in KRAS, a down-stream component of EGFR signaling cascade, it is reason-able to assume that the KRAS mutations might render the treatment with cetuximab ineffective. This hypothesis was directly tested in a clinical trial. Patients who did not receive prior EGFR inhibitor treatment were randomized to receive cetuximab plus best supportive care or best sup-portive alone. Tumor samples, when available, were assessed for the status of KRAS. Approximately 40% of both groups had KRAS activation mutations. Patients with wild type KRAS tumors had better overall survival, better progression-free survival, and signifi cantly better response rates than sup-portive care alone.61 Similar results were obtained with the use of another monoclonal antibody against EGFR, panitu-mumab.62 Based on these studies the U.S. Food and Drug Administration has changed the label for both drugs to require genetic testing of the KRAS prior to administration of either of these antibody drugs.



BRAF INHIBITORSThe RAS-Map kinase pathway is activated in a number of dif-ferent tumor types. Efforts to identify small molecule inhibi-tors that can effectively inhibit this pathway have been under way for many years. There are different genetic changes that could result in the activation of this pathway.

EGFR amplifi cation, mutations in the EGFR gene, muta-tions in a member of the RAS gene family, or certain muta-tions in BRAF are some of the examples of how this activation is accomplished. Efforts to target BRAF have been signifi cantly successful.

Melanomas are capable of metastasis, and there are few effective therapies for metastatic melanoma. It was discovered that in melanomas as many as 40% to 60% may carry an acti-vation mutation in BRAF. Interestingly, nearly 90% of the RAF mutations in this cancer involve codon 600, which results in a substitution of glutamic acid to valine (V600E). Two drugs, PLX4032 and PLX4720 (Plexxikon, Roche Pharmaceuticals, South San Francisco, California), effectively inhibit this modi-fi ed protein in in vitro studies. Based on these encouraging data, clinical trials were conducted to assess the clinical effi cacy of one of these drugs. In the initial dose escalation study the testing for the genetic status of BRAF was not a requirement, while in an extension study only those patients with an activa-tion mutation in BRAF were included. In the dose escalation study a substantial number of patients were positive for the BRAF mutation. A substantial number (61%) of the patients responded to the drug, and all of the individuals whose tumors had BRAF mutations were among the responders, while patients without the mutation did not show a response.63

In the extension phase of the study only patients with the V600E mutation were treated with drug and remarkably 81% of them responded. The responses were durable and involved all metastatic sites of the tumor. The overall survival in this population is being assessed in a phase 3 trial. If these trials are successful it would benefi t the patients to conduct testing for the status of BRAF and treat those who have the BRAF activa-tion with this inhibitor. BRAF mutations are also detected in other tumor types, and the mutations detected in these tumors include V600E. Therefore, it is possible that this or other BRAF inhibitors will fi nd widespread use in many tumor types.

The importance of genetic testing prior to treatment with BRAF inhibitors is underscored by studies that indicate that treatment of BRAF-negative melanoma patients with an RAF inhibitor may result in activation of the MAP kinase pathway and is therefore contraindicated.64,65

THE FUTUREAs the knowledge about the genetic changes that lead to the initiation and progression of cancer increases, so too the ability to choose the most appropriate drug to which the tumor will respond is also increasing. Understanding of the genetic and genomic changes in cancer has been fueled by new high-throughput technologies and large-scale approaches. During the past few years the ability to detect copy number changes, chromosomal aberrations, gene expression profi le changes, global methylation changes, and DNA sequence changes has dramatically improved. More recently a signifi cant reduction in the cost of DNA sequenc-ing is also fueling efforts to sequence large sets of genes, whole exomes, and even whole genome sequencing from tumors and, when available, their corresponding normal samples. All of these results are increasing the understanding of the biology of cancer, but they are also increasing the abil-ity to stratify patients and to choose the most appropriate

drug or treatment based on the genetic composition of the tumor and the patient.

An illustrative example of the types of information that can be obtained from such large-scale studies can be found in the results on human glioblastomas published by two groups.66,67 The Cancer Genome Atlas Research Network42 analyzed copy number changes, expression profi le changes, and methylation changes and sequenced a subset of genes in the genome in a large number of glioblastoma multiforme and compared them with normal samples obtained from the same patients. Parsons et al.43 examined the coding sequence of more than 22,000 genes in 22 samples. They also examined copy number changes and expres-sion profi les in these tumor samples. These studies identifi ed a number of genes that were not previously implicated in glio-magenesis, most notably NF1 and IDH1. The results also pro-vided important clues about how glioblastoma multiforme acquire resistance to the alkylating agent temozolomide. Similar types of studies in other cancers have been published.

CHANGING FACE OF PERSONALIZED MEDICINE

As the information about critical genetic and genomic changes in many different cancers is accumulating, it is becoming possible to incorporate this information into patient treatment. The types of changes can be illustrated from the changes in treatment of lung cancer. The importance of EGFR and KRAS testing in making decisions about the suitability of tyrosine kinase inhibitors was described earlier in this chapter. A subset of lung tumors have mutations in the BRAF gene and, based on the results from clinical studies of melanoma patients, highly specifi c BRAF inhibitors might be the choice treatment for these patients. A subset of tumors have ErbB2 amplifi cation, which is a common event in breast cancers, and patients whose breast tumors have this amplifi -cation respond well to trastuzumab. Clinical trials to evalu-ate the effi cacy of using trastuzumab in lung cancer patients with ErbB2 amplifi cation are under way. A subset of lung tumors also has activation mutations in one of several genes, leading to an activation of the PI3K pathway. There are sev-eral drugs, some of which are already approved for clinical use, that are effective inhibitors of this pathway, and these are excellent candidates with which to treat this group of patients. Another subset of patients has a unique transloca-tion that involved the EML and ALK4 genes. It has been shown that lung cancer patients with tumors that bear this translocation respond well to a new tyrosine kinase inhibitor crizotinib.66

Other NSCLC tumors have amplifi cation in the onco-gene MET. Inhibitors against this target and its ligand hepatocyte growth factor are in development, and some of them are being tested for their effi cacy to treat lung cancer patients in clinical trials. Therefore, it is highly likely that in the near future tumors from patients diagnosed with NSCLC will be tested for the status of a battery of genes that include EGFR, RAS, RAF, ErbB2, Met, and the pres-ence of the EML-ALK4 translocation to determine the most optimal treatment for each patient. Such efforts are already in place at several academic medical centers. A list of exam-ples of genetic changes that could affect treatment decisions is presented in Table 12.1.

SUMMARYThe understanding of the genetic and genomic differences among individuals that affect cancer susceptibility and the



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genetic and genomic changes that are critical for the initia-tion and progression of cancer is increasing at a rapid pace. The rapid decrease in the cost of DNA sequencing and other genomic analysis is fueling this increase in knowledge. This new knowledge is helping in accurate prediction, early detection, and prognosis of cancer. Genetic differences

among individuals and somatic changes during the develop-ment of cancer are also important in determining the appro-priate treatment strategies for each patient. The use of genetic and genomic information is referred to as personal-ized medicine, which has the ability to transform the prac-tice of oncology.

TABLE 12.1

EXAMPLES OF GENETIC CHANGES TO FACILITATE TREATMENT DECISIONS

Genetic Change Indication

BCR-ABL translocation Chronic myelogenous leukemiaErbB2 amplifi cation Certain breast cancersEGFR mutations Sensitivity to tyrosine kinase inhibitors in NSCLCEGFR mutations Resistance to tyrosine kinase inhibitors in NSCLCK-RAS Resistance to tyrosine kinase inhibitors in NSCLCK-RAS Resistance to EGFR antibodies in colon cancerB-RAF Sensitivity to B-RAF inhibitors in melanomaEML-ALK4 translocation Sensitivity to ALK4 inhibitorsRAS-MAPK pathway members Sensitivity to drugs that inhibit RAS-MAPK pathwayPTEN, AKT and PI3 kinase Sensitivity to drugs that inhibit the AKT and PI kinase pathwaysBRCA1 and BRCA2 Sensitivity to PARP inhibitorsHGF and MET mutations or amplifi cation Sensitivity to HGF and MET inhibitorsMutations in genes in the angiogenesis pathway Sensitivity to angiogenesis inhibitors

EGFR, epidermal growth factor receptor; NSCLC, non–small cell lung cancer; PARP, poly(adenosine diphosphate-ribose) polymerase; PI3, phosphatidylinositol 3; HGF, hepatocyte growth factor; MET, mesenchymal-to-epithelial transition.

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Cancer: Principles and Practice of Oncology 9th Edition - Cap12 Biologia da Medicina do Câncer

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