Oncogenomics: From Basic Research to Precision Medicine

Japan

Preface

Franco Dammacco and Franco Silvestris

Precision medicine, perhaps more aptly referred to as personalized medicine (PM), recognizes the individual variability that underlies the prevention and treatment of diverse disease states. It is an innovative concept and its novel approaches are the subject of growing interest. PM integrates recognition of the established clinical and pathological features of a disease with its molecular profiling to thus guide diagnostic, prognostic, and therapeutic strategies that precisely address the patient’s particular condition. The development of PM has been strongly supported by basic and technological progress in the omics sciences, including next-generation genome sequencing and the availability of computational tools that allow the rapid analysis of large datasets.

Although potentially applicable to all areas of medicine, the most meaningful application of PM has been and continues to be in oncology, given the relentless, increasing prevalence of cancer worldwide, its very high mortality despite recent, remarkable therapeutic advancements, and the steadily improving understanding of the oncogenic mechanisms that trigger the onset of several tumor types. The ultimate goal of oncological clinical care, while still on the remote horizon, is a pharmacogenomics-based strategy in which, rather than the cytotoxic chemotherapies currently indiscriminately administered to all patients with the same tumor type, a molecularly targeted therapy is delivered based on the use of rationally designed drugs, such that each patient receives the right drug at the right dose.

With this gradual shift in the treatment paradigm, researchers and clinicians involved in oncology are being confronted with an enormous amount of information that needs to be carefully weighed and interpreted if findings with immediate clinical implications are to be correctly distinguished from those requiring further in-depth analysis. While clinical judgment remains invaluable, the development of novel therapies will rely on the unique knowledge provided by informatics. In addition, the establishment of an open-access data source, in which the consistent flow of emerging knowledge can be collected and then retrieved as needed, has been proposed by several stakeholders. Although this implies unlimited access to detailed molecular information, which inevitably raises ethical and societal concerns, the creation of a controlled Clinical Practice Research Datalink is expected to be an essential tool leading to the success of PM.

Recognition of the clinical utility of (sub)classifying tumors in the achievement of PM has been accompanied by a renewed appreciation of the complexity of cancer. Thus, PM has been rightly viewed as a disruptive innovation, the consequences of which range from the need for new educational models and curricula in medical schools, such that students gain an understanding of the molecular mechanisms of various diseases and are able to interpret -omics data, to the training of clinicians in the choice of the appropriate test according to its positive predictive value, sensitivity, and specificity. On the patient end, the cultural shift brought about by PM necessitates a broader discussion of the ethical and legal use of a patient’s freely accessible, openly stored molecular information. Moreover, as PM becomes the norm in health care, it will deeply change the patient–doctor relationship, including a greater reliance on biomarker tests developed by the diagnostics industry and on molecularly targeted therapies designed in conjunction with the pharmaceutical industry.

The extent to which PM, with its molecularly targeted agents, is able to better treat certain tumors than is currently the case with standard care is being investigated in randomized trials and through comparisons with historical data. The encouraging successes have been accompanied by disappointing failures that have revealed the many problems that remain to be addressed before molecularly targeted approaches are clinically implemented on a wider scale. These include, but are not limited to, intratumor heterogeneity, unexplained drug resistance, unreliable methods for monitoring drug responses and disease recurrence, and insufficient experience in the use of new drug combinations. Preclinical testing in well-established models is a prerequisite for clinical trials in adult and pediatric patients. Finally, despite the new insights in basic and clinical oncology that have emerged from research into PM and associated fields such as the omics sciences, the crucial problem will be to reconcile the resulting innovations with issues related to safety, clinical efficacy, cost-effectiveness, and ethical and societal concerns.

Because data related to molecular oncology must be retrieved from hundreds of papers published in a vast array of journals, we felt that the time was right for a comprehensive, user-friendly textbook that, drawing on international expertise, would serve as a valuable source of information and continuing education for anyone (basic and clinical oncologists, molecular biologists, general practitioners, advanced medical students, and the oncology readership in more general terms) seeking to keep abreast of the developments in the exciting and rapidly evolving area of PM.

The 46 chapters that make up this volume have been divided into the following seven sections: (1) Molecular medicine: a novel approach to cancer investigation; (2) Oncogenomics: circulating biomarkers in clinical oncology; (3) Gastrointestinal tumors: molecular diagnosis and treatment: (4) Perspectives in breast cancer genomics; (5) Lung cancer: role of genomics in clinical practice; (6) Genomics in genitourinary cancer; and (7) Genomics in neuroendocrine tumors, melanoma, and sarcoma.

Thus, this volume is a valuable record of current knowledge and important considerations in the fields of oncogenomics and PM. We are deeply indebted to the diligent and thoughtful authors who accepted our invitation to contribute chapters to this book and thus made it possible to publish an up-to-date source of information within a reasonable space of time. Our equally heartfelt thanks go to Elsevier, the publisher of this volume, and to Timothy Bennett, Editorial Project Manager, for his sage advice, professional skill, and commendable promptness in bringing this volume to fruition in a timely manner.

Part I

Molecular Medicine: A Novel Approach to Cancer Investigation

Outline

Chapter 1 From the Double Helix to Oncogenomics and Precision Cancer Medicine

Chapter 2 Intratumor Heterogeneity

Chapter 3 The Role of Proteomics in Cancer Research

Chapter 4 Next-Generation Sequencing in Clinical Practice

Chapter 5 Cancer Epigenetics

Chapter 6 Cancer Stem Cells

Chapter 7 Cancer Stem Cells, Apoptosis Pathways and Mechanisms of Death Resistance

Chapter 8 Targeting the Hedgehog and Notch Signaling Pathways in Cancer Stem Cells

Chapter 9 Cancer Stem Cells in Multiple Myeloma and the Development of Novel Therapeutic Strategies

Chapter 1

From the Double Helix to Oncogenomics and Precision Cancer Medicine

An Evolving Story

Franco Dammacco and Franco Silvestris,    Department of Biomedical Sciences & Human Oncology, University of Bari Aldo Moro, Bari, Italy

Abstract

With the completion of the Human Genome Project in June 2000 and in step with the development of next generation -omics, a new era of medicine has started. In particular, the application and expansion of the molecular profiling of tumors have ushered in the development of personalized treatments, collectively referred to as precision cancer medicine (PCM), a field that is steadily growing. An increasing number of targetable molecules are being produced by the pharmaceutical industry, and their efficacy is strictly related to their specificity for reliable and well-characterized oncogenic pathways. The US Food and Drug Administration has therefore recommended that the development of these agents should be accompanied by companion diagnostics. The genomic revolution is also changing the way clinical trials of targeted drugs for cancer patients are being planned and conducted. The most common master protocols include basket trials, umbrella trials, and platform trials. Due to their flexibility and characteristics across molecularly defined tumor subtypes, novel drugs are included or excluded depending on their efficacy. Two examples on the use of precision diagnosis and treatment are described. The first one concerns patients with advanced non-small cell lung carcinoma, characterized by actionable or druggable oncogenic alterations and the sometimes remarkable improvement in objective response rates, progression-free survival, and overall survival compared with platinum-based chemotherapy. The second successful example of PCM is malignant melanoma. In parallel with a better understanding of the molecular mechanisms driving melanoma progression and drug resistance, the standard of care for this tumor has shifted from poorly effective chemotherapy to more optimistic targeted approaches. However, despite these encouraging results, a wider clinical application of PCM is still hampered by several drawbacks that will require major efforts before they can be overcome. It is hoped that, through the continuous and progressive refinement of its applications, PCM will eventually result in significant progress in the prevention, diagnosis, and treatment of tumors.

Keywords

Human genome; master protocols; oncogenomics; precision medicine; targeted drug

Abbreviations

ALK anaplastic lymphoma kinase

CDx companion diagnostics

EGFR epidermal growth factor receptor

FDA Food and Drug Administration

MoAb monoclonal antibody

NSCLC non-small cell lung cancer

ORR objective response rate

OS overall survival

PCM precision cancer medicine

PD-1 programmed death 1

PD-L1 programmed death ligand 1

PFS progression-free survival

PMI precision medicine initiative

ROS1 rat osteosarcoma

TKI tyrosine kinase inhibitor

VEGF vascular endothelial growth factor

***

"Bethink you of the seed

whence you have sprung; for you were not created

to lead the life of brutes,

but virtue and knowledge to pursue"

(The Divine Comedy by Dante Alighieri [1265-1321], Hell, XXVI canto)

An irrepressible impulse to pursue knowledge is innate to humankind and was emphatically expressed by Dante Alighieri in his imagining of Ulysses urging his traveling companions to follow his lead in steering his ship beyond the Pillars of Hercules, where they would face the unknown. Approximately 700 years later, the same strong curiosity drives the worldwide scientific community.

The Double Helix and the Human Genome Project

In a one-page paper published in Nature on April 25, 1953, James D. Watson and Francis H. Crick first described the double helix, the twisted-ladder structure of DNA. This extraordinary discovery was a milestone in the history of science and laid the foundations of modern molecular biology. The authors were awarded the 1962 Nobel Prize in Physiology or Medicine.

Less than 50 years later, on June 26, 2000, during a press teleconference, US President Bill Clinton, Prime Minister Tony Blair of the United Kingdom (via satellite), Dr. Francis Collins, Director of the National Human Genome Research Institute, and Dr. Craig Venter, President and Chief Scientific Officer of Celera Genomics Corporation, jointly announced the completion of the first survey within the Human Genome Project. The final, high-quality, reference genome sequence was generated and then completed by the end of 2002, more than 2 years ahead of schedule (Collins, Morgan, & Patrinos, 2003; Venter et al., 2001).

Complete mapping of the human genome, the result of an immense research endeavor, was described by US President Clinton as the most important, most wondrous map ever produced by humankind. In fact it was a genetic revolution, marking a turning point in all fields of biology and the beginning of a new era in research. The significance of this work was already predicted in 1986 by the 1975 Nobel laureate Renato Dulbecco, who expressed the following, at the time visionary, idea of sequencing the human genome: We are at a turning point in the study of tumor virology and cancer in general. If we wish to learn more about cancer, we must now concentrate on the cellular genome. We are back to where cancer research started, but the situation is drastically different because we have new knowledge and crucial tools, such as DNA cloning. We have two options: either to try to discover the genes important in malignancy by a piecemeal approach, or to sequence the whole genome of a selected animal species.… I think that it will be far more useful to begin by sequencing the cellular genome. The sequence will make it possible to prepare probes for all the genes and to classify them for their expression in various cell types at the level of individual cells by means of cytological hybridization. The classification of the genes will facilitate the identification of those involved in progression. In which species should this effort be made? If we wish to understand human cancer, it should be made in humans because the genetic control of cancer seems to be different in different species. Research on human cancer would receive a major boost from the detailed knowledge of DNA (p. 1055).

The complete sequencing of the DNA blueprint of Homo sapiens (3 billion DNA letters) has been (rightly) acknowledged as one of the greatest scientific undertakings of all times. Its full achievement can be considered (albeit perhaps somewhat exaggeratedly) as the biological equivalent of the moon landing or the splitting of the atom. Although completion of the Human Genome Project has important therapeutic implications for all types of disease, the most obvious and immediate ones are in the field of oncology because of the increasing prevalence of malignant tumors in the world’s aging population, the general trend to greater longevity, the high lethality of most tumors, the limited success of chemotherapy for the majority of advanced neoplasias, and the strong demand for clinically applicable scientific advancements in this field. Complete sequencing of the genes of several cancers, as planned in the Cancer Genome Project, will improve our knowledge of the molecular mechanisms responsible for neoplastic transformation (Green, Watson, & Collins, 2015). Indeed, the expanded molecular profiling of the tumors so far studied has already revealed a large number of potentially actionable targets.

Precision Medicine Initiative and Precision Cancer Medicine

In parallel with the complete sequencing of the human genome, there has been a rapid development of related technologies, including the various -omics (genomics, proteomics, regulomics, transcriptomics, and metabolomics), for biomedical analysis (Fig. 1.1). This has been supported by new computational tools for collecting and analyzing large data sets, commonly known as big data. Together, they have allowed the design of molecularly targeted therapies that in later iterations are expected to revolutionize the treatment of cancer (Savage & Antman, 2002). The first convincing example of this innovative approach was the tyrosine kinase inhibitor (TKI) imatinib, first administered to patients with chronic myeloid leukemia and subsequently shown to be effective in gastrointestinal stromal tumors.

Figure 1.1 Omics sciences show new molecular landscapes in cancer research and treatment. CGH, comparative genomic hybridization; DNA, deoxyribonucleic acid; Methyl-DIP, methylated DNA immunoprecipitation; miRNAs, microribonucleic acid; mRNA, messenger ribonucleic acid; SELDI-TOF, surface-enhanced laser desorption/ionization time of flight; SNP, single-nucleotide polymorphism.

In addition to the clear clinical benefits deriving from personalized targeted therapy, this innovative approach received a substantial political endorsement with the launching of the Precision Medicine Initiative (PMI) by US President Barack Obama. In his State of the Union Address held on January 20, 2015, Obama announced key actions to accelerate progress in the program, beginning with an allocation of $215 million. The concept underlying the PMI is that prevention and treatment must take into account individual variability, an interpretation considered as visionary (Collins & Varmus, 2015). As spelled out in its mission statement, PMI seeks to enable a new era of medicine through research, technology, and policies that empower patients, researchers, and providers to work together toward development of individualized care. Precision medicine is, therefore, a thoroughly innovative strategy, one that recognizes the roles played by individual variations in genes, environment, and lifestyle in effective disease prevention and treatment planning.

However, in the absence of a clear definition and unequivocal interpretation, the terms targeted, individualized, and personalized medicine are often used interchangeably and, in many contexts, mistakenly. Schleidgen, Klingler, Bertram, Rogowski, and Marckmann (2013), in an attempt to remove this ambiguity, conducted a systematic review of the literature and thereby arrived at the following definition: Personalized medicine seeks to improve stratification and timing of health care by utilizing biological information and biomarkers on the level of molecular disease pathways, genetics, proteomics as well as metabolomics (p. 55).

Nonetheless, extension of the striking benefit of targeted medicine achieved with imatinib in chronic myeloid leukemia to all, or at least the majority, of human tumors was illusory and led to a rapid shift from the concept of tailored medicine to the more attractive and perhaps more feasible precision cancer medicine (PCM). The most cogent consequences of this shift have been (1) a research shift from the morphologic to the genetic characterization of the tumor in each single patient; (2) a better prognostic definition of each tumor; (3) the identification of new molecular markers, thus allowing an earlier diagnosis; and (4) the development of intelligent drugs that, with increasing progress in molecular oncology, are expected to more precisely reach the specific cellular targets of neoplastic cells, thus allowing the pursuit of an ever more personalized medicine.

Oncogenomics in the Fight Against Cancer

At the dawn of the 20th century, the German scientist Paul Ehrlich (1854–1915), a Nobel laureate in 1908, advanced the concept of a magic bullet to indicate drugs capable of acting directly on the programmed cell-structural targets of tumors. He is therefore considered the founder of chemotherapy and was among the first to envision targeted medicine as a strategy to destroy or neutralize a specific target, whether an infectious agent or a neoplastic cell, without harming normal tissues (Strebhardt & Ullrich, 2008). However, despite progressive refinements in chemotherapy and significant improvements in the treatment of many types of tumors, no single chemotherapeutic agent or combination thereof has been able, with rare exceptions, to achieve cure.

At variance with classic chemotherapy, in which nonspecific characteristics of all rapidly proliferating cells, including normal cells, are exposed to the actions of the drug(s), targeted therapy is specifically aimed at the mechanisms related to the expression of the oncogenes and oncosuppressor genes underlying the onset of neoplastic transformation. Yet, until significant advances in the molecular characterization of tumors are made, PCM should be restricted to rationally designed clinical trials. Announcements on commercial websites of personalized cancer care through the marketed molecular characterization of the patient’s tumor are highly deceptive, if not criminal. The obvious danger is the endorsement of drugs of unproven benefit and the provision of information that magnifies the benefits of PCM (Gray et al., 2015) while ignoring its limitations.

An important advancement, following the final guidance issued by the US Food and Drug Administration (FDA) on August 6, 2014, was the recommendation to the pharmaceutical and biotechnology industries to simultaneously develop companion diagnostics (CDx) for the corresponding drugs developed for PCM-based therapy. In step with the emergence of next-generation -omics, CDx tests have become important, often indispensable, tools to identify the specific biomarkers that justify the use of novel targeted therapies. However, the increasing number of biomarkers and the consequent adoption of the corresponding targeted therapies are inevitably resulting in diagnostic and therapeutic algorithms of ever greater complexity (Rosenbaum & Weisman, 2017). The success of PCM relies on the availability of suitable molecular CDx, but the accuracy of these tests is crucial for the correct identification of patients likely to benefit from the therapy while avoiding treatment-related toxicity.

This statement is exemplified by the administration of erlotinib as the first-line treatment for patients with advanced epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC) (Cheng, Palma, Scudder, Poulios, & Liesenfeld, 2017). It was calculated that aggregate treatment costs for patients tested with laboratory-developed tests were roughly $7.3 million more expensive than the cost of treating patients who underwent FDA-approved CDx tests, due to the remarkably higher drug- and adverse-event-related costs for patients who were misclassified and therefore incorrectly treated with the targeted therapy or with chemotherapy (Cheng et al., 2017).

Master Protocols and Clinical Trials of Targeted Drugs

The genomic revolution has led to major changes in the way new controlled clinical trials of targeted drugs for adult and pediatric cancer patients are planned and carried out (Hunter & D'Agostino, 2015; Simon, 2017). Here, the general features of master protocols are described (Table 1.1). Specific examples related to the various tumor types can be found in many of the following chapters.

Table 1.1

Adapted from Renfro, L. A., & Sargent, D. J. (2017). Statistical controversies in clinical research: Basket trials, umbrella trials, and other master protocols: A review and examples. Annals of Oncology, 28, 34–43; and Simon, R. (2017). Critical review of umbrella, basket and platform designs for oncology clinical trials. Clinical Pharmacology & Therapeutics, http://dx.doi.org/10.1002/cpt.814 [Epub ahead of print].

Among the controlled clinical trials that have emerged with targeted therapies are (1) umbrella trials, in which patients with a primary tumor arising from a specific anatomic site are enrolled, but each patient is treated according to the molecular characterization of his/her tumor; (2) basket trials, in which patients with multiple tumor types are enrolled, their eligibility depending on the biomarker status of their tumor; and (3) platform trials, which are randomized trials including multiple experimental arms and a single control arm. Novel drugs, tested across molecularly characterized tumor subtypes, are included or excluded depending on their efficacy or inefficacy. A Bayesian inference model is usually followed (Rashdan & Gerber, 2016).

The successful adoption of master protocols requires the joint cooperation of pharmaceutical industries, university institutions, and academic societies; experts in oncology and molecular biology; biostatisticians; and international cooperative groups/registries (Renfro & Sargent, 2017). Moreover, all master protocols in oncology are subjected to continuous refinements, in step with further developments of next-generation sequencing and the discovery of multiple driving mutations and biomarkers.

NSCLC and Melanoma: Successful Examples of Precision Cancer Medicine

Here we provide just two examples of how precision diagnosis and treatment are remarkably improving the objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) of cancer patients: in those with advanced NSCLC (Reck & Rabe, 2017; Schallenberg, Merkelbach-Bruse, & Buettner, 2017) and melanoma. Many other examples can be found elsewhere in this volume. Those presented in the following were chosen because they well demonstrate that despite advancements in chemotherapy, lung tumors and melanoma are still responsible for 1.6 and 1.7 million deaths annually worldwide, respectively (GLOBOCAN, 2012).

NSCLC

Our discussion is largely based on the excellent review article by Reck and Rabe (2017). In addition to immunocytochemical and/or immunohistochemical analyses, the molecular testing of NSCLC has become routine and the subclassification of these tumors has allowed a more effective therapeutic assignment. Molecular profiling includes detection of mutations in the genes encoding EGFR and BRAF V600E, translocations in the genes encoding anaplastic lymphoma kinase (ALK) and rat osteosarcoma (ROS1), and the genetic expression of programmed death ligand 1 (PD-L1) (Folch, Costa, Wright, & VanderLaan, 2015; Travis et al., 2013). Additional oncogenic alterations are likely to be discovered in the near future. Chromosomal instability, genome duplications, and subclonal mutations must also be searched for and characterized, as these abnormalities may not only have prognostic implications but will also stimulate the development of new therapeutic agents (Jamal-Hanjani et al., 2017).

Patients with advanced NSCLC lacking actionable oncogenic alterations are commonly given platinum-based chemotherapy, which in 25%–35% results in an ORR, defined as a tumor reduction of at least 30% (Eisenhauer et al., 2009); however, median survival is roughly 10 months, and the 1-year OS rate is 30%–40% (Novello et al., 2016). The folate antimetabolite pemetrexed is administered as maintenance therapy in patients with nonsquamous-cell NSCLC (Li et al., 2012). The inclusion of bevacizumab, a monoclonal antibody (MoAb) against vascular endothelial growth factor (VEGF), with platinum-based chemotherapy improves both the ORR and the PFS compared with chemotherapy alone, but combination therapy also has a high rate of adverse and often severe events (Soria et al., 2013).

Additional and more recent trials testing the anti-VEGF-receptor-2 antibody ramucirumab with docetaxel or the combination of nintedanib, an antiangiogenic TKI, with docetaxel versus docetaxel alone demonstrate the involvement of a proangiogenic pathway in rapidly progressing or refractory tumors, as the efficacies of nintedanib and ramucirumab were greater in these patients than in those receiving first-line chemotherapy. Similarly, the combined treatment of previously treated patients with bevacizumab and paclitaxel was better than paclitaxel alone as measured by a better PFS (Garon et al., 2014; Reck et al., 2014).

The identification of targetable mutations in the EGFR and the good sensitivity of the respective tumors to EGFR TKIs are important examples of the successful application of PCM to NSCLC. EGFR mutations occur in the tumors of ≤20% of Caucasian patients but in roughly 50% of those of East Asian patients (Dearden, Stevens, Wu, & Blower, 2013). A careful assessment of randomized trials of the EGFR TKIs gefitinib, erlotinib, and afatinib showed that, compared with first-line chemotherapy, these molecules induce significant improvements in both ORR and PFS and lower the occurrence of adverse events (Lee et al., 2015).

However, despite the impressive responses to EGFR TKIs, tumor progression occurs in many patients 9–12 months after treatment. Thus, additional targetable oncogenic alterations have been studied, particularly the secondary exon 20 T790M missense mutation, detected in up to 60% of NSCLC patients. The third-generation EGFR TKI osimertinib targets the T790M mutation as well as primary activating EGFR mutations (Yu et al., 2013). Patients with T790M mutations who suffered tumor progression after EGFR TKI treatment have an ORR of approximately 60% and a median PFS of roughly 10 months. In patients with tumors characterized by a T790M mutation that progressed after EGFR TKI therapy, osimertinib was able to induce a significant prolongation in the PFS to a median 10 months and a highly significant increase in the ORR from 31% to 71% (Mok et al., 2017) compared with platinum-based chemotherapy

ALK and ROS1 translocations are of low frequency (≤7% and ≤2%, respectively) in NSCLC (Bergethon et al., 2012; Kwak et al., 2010), but they are targetable by the TKI crizotinib. Both previously treated and untreated patients whose tumors carry these translocations are more effectively treated with crizotinib than with chemotherapy (Solomon et al., 2014). However, because patients with ALK translocations become resistant to crizotinib during therapy, they are additionally treated with the second-generation ALK TKIs ceritinib and alectinib, both of which yield an ORR of up to 56% and a median PFS of 5.7–8.0 months (Peters et al., 2017; Soria et al., 2017). In patients with tumors carrying the ROS1 translocation, an ORR of 72% and a median PFS of 19 months following crizotinib therapy have been reported (Shaw et al., 2014).

BRAF mutations are also of low frequency (~2%) in NSCLC, but roughly half are of the V600E type. In a study of these patients, combined treatment with dabrafenib (a BRAF inhibitor) plus trametinib (a MEK inhibitor) yielded an ORR of 63% and a median PFS of ~10 months (Planchard et al., 2016). In another study, the addition of a second BRAF inhibitor, such as vemurafenib, resulted in an ORR of 42% and a median PFS of 7.3 months (Hyman et al., 2015).

The basis of immunotherapy in patients with NSCLC is that a major mechanism by which tumors escape immune system recognition and rejection is the tumor-induced suppression of specific T-cell activation, a phenomenon related to the action of inhibitory pathways (so-called immune checkpoints). This knowledge has been exploited in the development of specific antibodies capable of interacting with cytotoxic T-lymphocyte–associated antigen 4 or with PD-1 or PD-ligand 1 (PD-L1) (Pardoll, 2012; Postow, Callahan, & Wolchok, 2015). Compared with chemotherapy, anti-PD-1 or anti-PD-L1 antibodies have been shown to induce a considerable improvement in the OS of previously treated patients with advanced NSCLC (Borghaei et al., 2015; Herbst et al., 2016), although important adverse events occur in almost one-third of them.

The detection of PD-L1 expression on tumor cells and immune cells is a useful criterion to select patients expected to be particularly responsive to immunotherapy, although inconsistencies have been reported (Topalian, Taube, Anders, & Pardoll, 2016). Nonetheless, the FDA and the European Medicines Agency recommend that initial treatment with pembrolizumab, an anti-PD-1 receptor MoAb, be restricted to patients with tumors characterized by high PD-L1 expression. In a study of randomly assigned untreated patients with advanced NSCLC in which the tumor cells exhibited high-level (tumor proportion score ≥50%) expression of PD-L1, pembrolizumab was able to induce a higher ORR and to prolong both PFS and OS compared to platinum-based chemotherapy while reducing by half the occurrence of grade 3 or 4 adverse events (Reck et al., 2016).

A recent phase 3 study compared the anti-PD-L1 antibody durvalumab with placebo as consolidation therapy in patients with stage III NSCLC who did not show disease progression after two or more cycles of platinum-based chemoradiotherapy. Patients receiving durvalumab had a significantly longer PFS than those treated with placebo. The safety features were roughly similar between the two groups (Antonia et al., 2017).

Melanoma

The molecular assessment and treatment of this tumor also provide an important example of the profitable applications of molecular medicine in cancer diagnosis and therapy. Whereas melanoma prevalence and mortality rates almost tripled in the last three decades of the 20th century, the mortality of melanoma patients in most age-sex groups has stabilized or even declined in the past 10–15 years. Especially for patients with metastatic melanoma, the prognosis was poor, with a median OS of 7.5 months and a 5-year survival of about 6% (Barth, Wanek, & Morton, 1995). However, based on the molecular diagnosis of melanoma, in which the pathogenic involvement of several driver genes, such as BRAF, NRAS, and c-KIT, was identified, different subtypes of the disease correlating with clinical patterns, including superficial cutaneous, nodular, malignant lentigo or acral melanoma, have been recognized. The underlying pathogenic mutations of these tumors are directly actionable by the TKIs vemurafenib, dabrafenib, and trametinib.

The introduction of these small molecules for the treatment of metastatic melanoma occurred in 2010. The results of third-line clinical trials showed a doubling of the length of the OS in treated patients. Encouraging results were also obtained in the treatment of wild-type melanomas by immunotherapy with Ipilimumab. This anti-CTLA-4 MoAb was the first immune-checkpoint inhibitor able to produce a durable clinical benefit in a small subset of patients (Hodi et al., 2010).

In parallel with the growing knowledge of the molecular mechanisms driving melanoma progression and drug resistance, the standard of treatment for metastatic melanoma has shifted from chemotherapy, the efficacy of which was largely disappointing, to efficient targeted/immunotherapeutic approaches. In patients with advanced melanoma harboring BRAFV600 mutations, targeted strategies combining BRAF with MEK inhibitors as well as checkpoint-inhibitor combinations, such as anti-PD-1 and anti-CTLA-4 MoAbs, have almost quadrupled OS (Fig. 1.2), with >50% of patients surviving >5 years (Ascierto et al., 2016; Long et al., 2017; Robert et al., 2014; Wolchok et al., 2017).

Figure 1.2 Drugs employed in the therapy of malignant melanoma from 1975 to 2010 have not resulted in a significant improvement of the patients’ overall survival. On the contrary, a remarkable improvement in the prognosis of this tumor has been observed following the introduction in the past 7 years of the B-Raf enzyme inhibitor vemurafenib, monoclonal antibodies directed against the immune checkpoint cytotoxic T-lymphocyte antigen (CTLA)-4 such as ipilimumab, as well as against programmed cell death (PD)-1 and PD ligand 1 (PD-L1) such as pembrolizumab and nivolumab, MEK inhibitor drugs such as trametinib, and inhibitors of mutant BRAF such as dabrafenib.

Whether the combined use of checkpoint inhibitors with TKIs in metastatic melanoma or with chemotherapy in NSCLC is effective and has an acceptable safety profile remains to be established. A recent study showed that the combination of pembrolizumab and chemotherapy in NSCLC induced a significantly better ORR than achieved with chemotherapy alone (Langer et al., 2016).

Precision Cancer Medicine: Uncertainties and Drawbacks

The major advancements contributing to the development of personalized therapies were summarized previously and, in the case of advanced NSCLC, drew largely on the overview provided by Reck and Rabe (2017). As made clear by this discussion, a prerequisite of effective personalized treatment is the molecular and genomic profiling of individual lung cancer patients. Compared with the largely established platinum-based chemotherapy, further supported by maintenance treatment with pemetrexed, subsequent lines of therapy, and radiotherapy, significantly better results are being obtained with personalized treatments in terms of ORR and PFS, albeit less often in terms of OS. However, the expectation that, following the launch of PMI, cancer would be defeated or at least properly controlled was no doubt overly optimistic and is still far from being met. Moreover, in the field of medical oncology as a whole, PMI has a number of drawbacks that should be emphasized:

1. A large majority of tumors are characterized by an array of molecular alterations; it is therefore unlikely that a single drug specifically targeting one of them will be able to induce a clinically important and sustained effect (Hunter, 2016; Prasad, Fojo, & Brada, 2016).

2. Although new multitargeted drugs directed at one or more signaling pathways are being proposed with increasing frequency, signaling pathways are likely to be only partially inhibited by these agents. However, the simultaneous administration of two or more of these agents will no doubt be unfeasible due to the enhanced toxicity. A dose reduction would limit toxicity but would probably result in less inhibition of the targeted signaling pathways (Tannock & Hickman, 2016).

3. Although the molecular analysis of tumor samples has become increasingly routine and the cost has decreased accordingly, the identification of multiple potential targets will inevitably imply the administration of different targeted agents, with foreseeable high costs and more severe adverse events (Tannock & Hickman, 2016).

4. Intratumor heterogeneity is a major obstacle to the significant success of molecularly chosen agents. A more comprehensive understanding of the molecular features of individual tumors is essential if PCM is to be pursued as an effective therapeutic approach.

5. For most patients with metastatic cancer, the benefits induced by a targeted therapy are limited and short-lived. Given that the clinical blockade of oncogenes usually results in transient effectiveness, patients should be monitored for the clonal evolution of their tumors because this may provide insights into the design of therapies that also evolve over time (Amirouchene-Angelozzi, Swanton, & Bardelli, 2017).

6. An important and discouraging limitation of PCM is the development of resistance in a highly heterogeneous disease such as cancer. This emphasizes the importance of precision prevention research and, thereby, the accelerated development of cancer prevention agents (Brahme & Szabo, 2017).

7. With the exception of a few molecules (e.g., trastuzumab and vemurafenib), the large majority of the new targeted drugs are far from being the standard of care.

8. More effective, molecularly driven cancer therapies will to a large extent depend on a deeper understanding of the molecular, cellular, and environmental factors giving rise to organ-specific tumorigenesis (Schneider, Schmidt-Supprian, Rad, & Saur, 2017).

Conclusions

PCM is still a young discipline, and it is currently difficult to foresee its actual impact on tumor prevention, diagnosis, and therapy. Nonetheless, the hope persists that through continuous and progressive streamlining of its applications, PCM will eventually result in significant progress in the treatment of many cancers. Indeed, advances in PCM are already leading to important medical breakthroughs, including new medical treatments, closely tailored according to the genetic profile of an individual’s tumor. Typical examples are patients with breast, lung, and colorectal cancers, as well as melanomas and leukemias, who routinely undergo molecular testing and, on the basis of the corresponding results, are candidates for selected treatments that improve survival and reduce exposure to the adverse effects of conventional chemotherapy. The growing amount of data has been accompanied by rapid technological advancements that are remarkably improving our ability to interpret the largely undefined interactions between genes and proteins, normal and pathological cells, and innate and environmental factors. The complete sequencing of the human genome has been a crucial landmark and, at the same time, the starting point of a deeply innovative scientific revolution, one that is expected to fuel biological research for several decades to come.

References

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Chapter 2

Intratumor Heterogeneity

Biological and Clinical Implications

Rekha Gyanchandani, Nolan Priedigkeit, Ahmed Basudan and Adrian V. Lee,    Women’s Cancer Research Center, Department of Pharmacology & Chemical Biology, UPMC Hillman Cancer Center, Magee Womens Research Institute, University of Pittsburgh, Pittsburgh, PA, United States

Abstract

Tumors are composed of heterogeneous populations of cells that differ in size, morphology, genetic profile, gene expression, and growth rate. This intratumor heterogeneity (ITH) contributes to tumor progression, metastasis, and resistance to therapy. The clinical implications of such heterogeneity are profound, given that the identification of distinct subpopulations within a tumor may predict disease outcome or treatment response. Recent advances in molecular analysis technologies have identified tremendous ITH and clonal evolution. While these studies have enhanced our understanding of ITH, the utilization of this knowledge to guide decision-making in routine clinical practice seems distant. In this chapter, we discuss the different levels at which solid tumors display ITH including genetic, transcriptomic, proteomic, spatial, and phenotypic heterogeneity. We highlight the clinical implications of such heterogeneity, current limitations in the field and potential strategies to overcome ITH and improve therapies.

Keywords

Heterogeneity; genetic; transcriptomic; proteomic; spatial; phenotypic; clonal evolution; tumor progression; resistance; prognosis

Acknowledgments

We acknowledge funding support from the Breast Cancer Research Foundation, Susan G. Komen Scholar Award (SAC110021), Fashion Footwear Association of New York (FFANY), Nicole Meloche Memorial Breast Cancer Research Foundation, Shear Family Foundation, and Magee-Womens Research Institute and Foundation. NP was supported by a training grant from the NIH/NIGMS (2T32GM008424-21) and an individual fellowship from the NIH/NCI (5F30CA203095).

Introduction

Intratumor heterogeneity (ITH) refers to the variability within a tumor, which is characterized by the presence of different cell populations with distinct genetic and phenotypic traits. These cell populations may differ in their morphology, genetic makeup, molecular profile, signaling pathways, proliferation, migration, metastatic potential, and response to anticancer drugs. Two models have been proposed to explain the origin of ITH: clonal evolution and the cancer stem cell (CSC) hypothesis (Shackleton, Quintana, Fearon, & Morrison, 2009). We have recently reviewed the cellular and evolution of breast cancer (Zhang, Lee, & Rosen, 2017). As per the clonal evolution model, tumor cells accumulate genetic alterations due to inherent genomic instability (Nowell, 1976). These changes confer a selective advantage resulting in clonal expansion and diversification within the tumor. On the other hand, the CSC model postulates that a small fraction of tumor cells with self-renewal and differentiation capacity can drive tumorigenesis and ITH (Al-Hajj, Wicha, Benito-Hernandez, Morrison, & Clarke, 2003; Lapidot et al., 1994). In addition to these models, the interaction of tumor cells with the surrounding microenvironment plays an important role in furthering phenotypic heterogeneity and tumor evolution (Holzel, Bovier, & Tuting, 2013).

The genetic and phenotypic heterogeneity within a tumor can impact clinical diagnosis, prognosis, treatment response, and resistance. Current clinical practice often relies on a single core biopsy specimen for initial diagnosis, and then analysis of a surgical resection to assess clinical and histopathological features that may predict prognosis and/or response to therapy. In both cases, a small part of the tumor is analyzed. Knowing that there is substantial subclonal diversity within a tumor, one small region may not be representative of the whole tumor (R. Gyanchandani, Lin, et al., 2016). In addition, a biopsy provides only a single snapshot in time, while in reality a tumor is a constantly evolving entity. This maybe particularly problematic for aggressive metastatic tumors, where treatment regimens are selected based on an initial evaluation of the primary tumor (Priedigkeit et al., 2017). Given clonal diversity, distinct subpopulations within a heterogeneous tumor can show different sensitivities to anticancer drugs (Kreso et al., 2013; Onuchic et al., 2016; Sharma et al., 2010). Treatments can enrich for resistant subclones that have a survival advantage over the bulk population of cancer cells and cause tumor regrowth. Hence, it is important to characterize ITH to develop more effective therapies and better informed clinical decision-making. With the availability of high-throughput genomic technologies, a number of studies have shed light on the subclonal tumor architecture using multiregion and single cell approaches (Gerlinger et al., 2012; Navin et al., 2010; Navin et al., 2011; Nik-Zainal et al., 2012; Schwarz et al., 2015; Shah et al., 2009; Xu et al., 2012; Yachida et al., 2010; Yu et al., 2014; Zhang, Fujimoto et al., 2014; Zhang, Mercado-Uribe et al., 2014). However, these technologies are currently limited by high costs, detection limits, and sequencing artifacts. Although, a large body of evidence is beginning to describe the extent of ITH and its biological and clinical implications in cancer, the integration of this knowledge in clinical decision making is lacking. Here, we discuss the different levels at which tumors display ITH including genetic, transcriptomic, proteomic, spatial, and phenotypic heterogeneity. We highlight the clinical implications of such heterogeneity, current limitations in the field, and potential strategies to overcome ITH and improve therapies.

Levels of Intratumor Heterogeneity

Genetic Heterogeneity

Genomic instability is a hallmark of cancer, which drives single nucleotide variants, chromosomal rearrangements and aneuploidy. Defects in cell division including aberrant DNA replication and loss of DNA repair mechanisms can enhance genomic instability and tumorigenesis (Fujiwara et al., 2005; Gordon, Resio, & Pellman, 2012; Lv et al., 2012). In the context of this genomic instability, multiple rounds of cell division and selective pressures induced by cytotoxic therapies, immune evasion, and metastatic selection, tumor cells continue to acquire new mutations throughout their life histories in order to survive and are thus subject to Darwinian selection (Nowell, 1976). Alterations that confer a growth advantage in the context of these pressures lead to preferential expansion of distinct clones, generating a substantial genetic diversity within a single tumor (Burrell, McGranahan, Bartek, & Swanton, 2013). With the advent of deep massively parallel sequencing (MPS), it is possible to comprehensively characterize DNA copy number changes and variant allele frequencies in a given tumor sample. The analysis of the tumor cell fraction carrying a mutation can then be used to build phylogenetic trees and infer the evolutionary history of cancers (for review of methodologies please see Schwartz & Schaffer, 2017). In fact, several models of clonal evolution have been proposed to describe the diverse subclonal architecture observed in tumors, including linear, branched, convergent, parallel, punctuated, and neutral evolution (Davis, Gao, & Navin, 2017; McGranahan & Swanton, 2017). Also, multiple modes of evolution may be operative within a single tumor at any given time. The linear model, which was first proposed in colorectal cancer, follows a stepwise clonal selection theme and provides a somewhat simplistic view of tumor progression (Fearon & Vogelstein, 1990). In contrast, branched evolution, which is more commonly observed in cancer, shows independent emergence of subclones with distinct genetic alterations (Gerlinger et al., 2012; Haffner et al., 2013; Schwarz et al., 2015; Yates & Gerstung, 2015; Zhang, Fujimoto et al., 2014; Zhang, Mercado-Uribe et al., 2014). In convergent evolution, unique and different mutations converge to convey the same phenotypic output, while in parallel evolution, distinct subclones derived from the same parental clone acquire similar alterations (Fisher et al., 2014; Yates & Gerstung, 2015). In punctuated evolution, subclonal alterations are acquired in short bursts early on in tumor progression and a few dominant clones undergo stable expansion (Baca et al., 2013). Unlike the above discussed modes of evolution, neutral evolution is defined by the absence of a selection process, where heterogeneity is driven purely by stochastic events (Williams, Werner, Barnes, Graham, & Sottoriva, 2016). Another level of complexity operative in the subclonal architecture within a tumor is the cooperativity and competition among the different subclones (Cleary, Leonard, Gestl, & Gunther, 2014; Marusyk et al., 2014). In a recent study by the Polyak group, a minor subclone with less fitness was shown to support the growth of all other clones in a noncell-autonomous manner by secreting IL11 into the tumor milieu (Marusyk et al., 2014). These findings highlight an important lesson in comprehensively characterizing ITH in tumors, as they suggest that defining and targeting a minor population of cells in a given tumor may be, counterintuitively, a compelling strategy for therapy.

Several bioinformatics tools have been developed to enable the deconvolution of clonal compositions in bulk tumors using deep sequencing data. ABSOLUTE was one of the earliest computational methods that could infer tumor purity and ploidy from the analysis of somatic mutations in a single bulk tumor sample (Carter et al., 2012). Other methods have refined the inference of these clonal compositions by the use of multiregion bulk tumor samples. In a cohort of 104 triple-negative breast cancers (TNBCs), Shah et al. used PyClone tool to identify tumor subpopulations from allele frequency measurements for 2414 somatic mutations (Shah et al., 2012). Oesper et al. introduced THetA (Tumor Heterogeneity Analysis) algorithm to estimate normal and tumor admixtures from DNA copy number data (Oesper, Mahmoody, & Raphael, 2013). Similarly, Ha et al. developed a novel probabilistic model called TITAN to estimate the proportion of cells harboring specific copy number aberrations and loss of heterozygosity (LOH) events (Ha et al., 2014). We have recently reported on a method for epigenomic deconvolution (EDec) and highlighted metabolic coupling between different cell types in breast cancer (Onuchic et al., 2016). Over the past decade there has been a significant increase in the number of studies employing multiregion sequencing, which have extended our understanding of ITH. In an initial study, macro-dissected breast tumors with nuclei isolation using fluorescence-activated cell sorting (FACS) were subjected to array comparative genomic hybridization (aCGH) analysis (Navin et al., 2010). Several clonal subpopulations were identified within a single macro-dissected region or existing in multiple regions. Zhang et al. performed multiregion whole-exome sequencing (WES) on 11 localized lung adenocarcinomas and found that presence of a larger subclonal mutation fraction in the primary tumor may be associated with increased likelihood of postsurgical relapse in these patients (Zhang, Fujimoto et al., 2014; Zhang, Mercado-Uribe et al., 2014). Schwarz et al. examined whole-genome copy number profiles in 135 spatially and temporally separated samples from 14 patients with high-grade serous ovarian cancer (HGSOC) (Schwarz et al., 2015). Subclonal tumor populations were identified in pretreatment biopsies, which underwent expansion during chemotherapy, suggesting a role in clinical relapse.

Genome-wide studies have also examined ITH in paired primary and metastatic tumors. In a study by Gerlinger et al., mutational heterogeneity was identified in multiple samples from primary clear cell renal cell carcinoma (ccRCC) and associated metastases (Gerlinger et al., 2012). While, VHL was mutated in all analyzed regions as a truncal driver event, other tumor suppressor genes (SETD2, PTEN, and KDM5C) showed multiple distinct and spatially separated inactivating mutations, suggesting convergent evolution. In another study, 32 somatic mutations were identified in a lobular breast cancer metastasis (Shah et al., 2009). Five of those 32 mutations were highly prevalent in the primary tumor, while six were present at low frequencies. These findings suggest that mutational heterogeneity can occur early in cancer and undergo significant evolution with disease progression. Similar findings were shown by Yachida et al. in pancreatic cancer, where preexisting clonal populations in the primary carcinoma gave rise to distant metastases, but these clones genetically evolved from the original nonmetastatic clone (Yachida et al., 2010). Further, Nik-Zainal et al. developed algorithms that showed that subclonal diversification was prominent in 21 breast cancers, where a few clonal mutations were present in most cells within a tumor and most mutations occurred only in a small fraction of cells (Nik-Zainal et al., 2012). Although the available bioinformatic tools provide a powerful approach to assess ITH in bulk tumors, the accuracy of deconvolution is limited by the depth of sequencing, detection limit, and error rate of the sequencing platform. Also, the resolution of deconvolution is limited by the type and number of regions analyzed.

Single cell sequencing is the most unequivocal approach for the assessment of ITH as it allows direct analysis of the cells present within the distinct clonal subpopulations. In a study by Navin et al., analysis of 100 single cells in a polygenomic breast tumor identified three distinct clonal subpopulations that seemed to follow a punctuated mode of evolution (Navin et al., 2011). Similarly, additional analysis of 100 single cells from a monogenomic primary tumor and matched liver metastasis indicated that a single clonal expansion formed the primary tumor and the metastasis. Xu et al. performed single-cell exome sequencing on 25 cells from a ccRCC tumor and showed that mutations that had different allele frequencies within the population also had different types of genetic lesions (Xu et al., 2012). Rare mutations showed an increased percentage of transitions and the common mutations showed increased transversions. Single-cell sequencing analysis of a case of colon cancer identified two independent clones; the major tumor clone with APC and TP53 mutations, whereas the minor clone harbored CDC27 and PABPC1 mutations. The findings suggest that the minor clone may also play important protumorigenic roles at the individual level (Yu et al., 2014). Although, single-cell sequencing is a promising new approach to infer clonal heterogeneity, it is limited by the high cost, and the low amount of input material, making whole-genome amplification a necessity. Hence, the method is prone to allelic drop out and amplification errors. To counter this issue, Wang et al., recently developed a promising approach called NucSeq