Chairman and Director Chief of Lymphoma Director, Clinical and Translational Cancer Research

		John Theurer Cancer Center at Hackensack University Medical Center

While it is recognized that cancer is a collection of complex and diverse pathological and biological entities, at the genetic level, cancer arises from a clone that has accumulated enough requisite somaticallyacquired modified genes that force normal cells to grow abnormally. A mutation is a single base change in the DNA, like a “tax” that must be paid every time a cell replicates. While such spontaneous mutations normally arise stochastically at a very low frequency, they become fairly common over time in an adult body. For the most part, however, they have no clear effect on the affected cell behavior due to either redundancy in the cell and/or the silent nature of the mutation (ie, bystander or passenger mutations).

In contrast, oncogenesis results form the cumulative effect of driver mutations, which appear to occur at a surprisingly small number in any cancer cell subtype—perhaps three to six in most cases. The development of the original clone and its progression to disseminated disease has previously been thought to be a linear process driven by serially acquired new mutations that continue to give growth advantage to the cell. Such changes&mdash;which include base pair substitutions, small insertions or deletions, chromosomal rearrangements, and gains and losses in gene copy number&mdash;occur either spontaneously or due to selection pressure (eg, acquired chemoresistance).

While traditional gene-focused characterization studies using PCR-amplification and conventional sequencing (Sangerbased capillary sequencing methodology) have shed light on cancer cell biology, identifying frequently mutated genes across different cancer subtypes, they were limited by cost and throughput. The advances in technological tools for massively parallel, high-throughput sequencing of DNA has enabled the comprehensive characterization of somatic mutations in large number of tumor samples, illustrating an emerging shift toward a potential new classification of cancers, as I have previously discussed in this column (Hoadley KA, et al. 

. 2014;158[4]:929-944). In particular, whole genome sequencing and whole exome sequencing have revealed the extraordinary and unanticipated diversity of cancer, not only among patients, but also within a patient through impressive clonal heterogeneity (not just a linear evolution of the original clone).

	Although a mutational landscape review for each tumor subtype is well beyond the scope of this column, the ability to identify which mutations are likely to be “drivers” in cancer pathogenesis and to elucidate how mutated genes affect the biology of a given tumor are fundamental current challenges that might provide hope in cancer genomics in the future.

	First, the number of mutations varies enormously among cancer subtypes: for example, the mutation rates for pediatric and hematological cancers show the lowest mutation rates (about 1 mutation/Mb for chronic lymphocytic leukemia), while much higher (10-15 times) mutation rates are seen in melanoma and lung cancer, likely reflecting exposure to environmental mutagens. Not only the mutation rate but also the spectrums of mutations vary among cell types based on exposure to carcinogens and particularly, mutations in mismatch repair genes. These factors likely explain in part why hematological cancers, particularly in pediatric patients, are more sensitive to chemotherapy compared with other tumor types, as well as hematologic cancers in elderly patients. On the other hand, in the era of checkpoint inhibitors, such as those used in melanoma, patients with more genetically complex tumors seem to be more immunogenic, and hence respond better to immunotherapy.

	Second, what is the role of chance versus environmental/ behavioral factors versus genetic predisposition in the development of cancer? One of the most unexplained issues in oncology is why some tissue types give rise to most human cancers (as reflected in breast, colon, lung, and prostate cancers), as opposed to some very rare tumors, such as sarcomas. A report earlier this year suggested that the lifetime risk of cancers among different tissue types is strongly correlated with the total number of divisions of the normal self-renewing cells maintaining that specific tissue’s homeostasis (Tomasetti C, Vogelstein B. Science. 2015;347[6217]:78-81). Such mutations could be explained in large part by how many stem-cell divisions—each of which associated with the risk of random mutations&mdash;have accumulated by a certain age or over a lifetime of stem-cell divisions. This report also suggested that only one-third of the variation in cancer risk among tissues is attributable to environmental factors or inherited predispositions.

	Not surprisingly, this assertion generated a huge reaction online and across the medical community with the perception that the majority of cancer-associated mutations are due to “bad luck,” through random mutations arising during DNA replication in normal, noncancerous stem cells. Numerous authors argued about those conclusions, particularly regarding the difficulty to assess the actual number of stem-cell divisions for each organ or tissue and the risk to discourage prevention measures against cancer. Tomasetti and Vogelstein replied with an interesting analogy using a car journey: the biggest factor being “the longer the trip (ie, the longest renewal of stem cells), the higher the risk of accidents,” with bad driving (environmental/behavioral factors) or poor maintenance of the car (inherited genetic factors) both increasing the risk of problems during that journey.

	Though knowing the mutational landscape appears essential to better understanding and stratification of patients and a required effort toward precision medicine, these mutations are often too many/too complex to stop or control. Understanding the hierarchy among such mutations seems a prerequisite to using them as real target. A recent report suggests that interfering with a single gene can turn colorectal cancer cells into normal cells in experimental models (Dow LE, et al. 

. 2015;161[7]:1539-1552). The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority (up to 90%) of human colorectal cancer cells. A mouse model where APC could be conditionally suppressed showed regression of established tumors through widespread tumor-cell differentiation. Though this research is only preclinical, it is very encouraging and could apply to other gastrointestinal tumors.

	Though some argue that high throughput approach to cancer has not delivered, I maintain that we are at a new dawn and should embrace customized Cura Personalis!

Articles

The ability to identify which mutations are likely to be "drivers" in cancer pathogenesis and to elucidate how mutated genes affect the biology of a given tumor are fundamental current challenges that might provide hope in cancer genomics in the future.

The Evolving Story of Mutational Landscape in Cancer and Its Implications: Bad Luck or Great Hope?

	The University of Texas Health Science Center at San Antonio

Moving through a crowd of people, if one pays attention to individuals, it’s easy to discern different characteristics that make each person unique. Likewise, scientists have long conceptualized variations in cells comprising the different tissue compartments in the human body. Yet, even in seemingly uniform cell populations within subcompartments, cell-cell heterogeneity is becoming more evident, far beyond what was previously expected.

We can attribute this new awareness to the advent of innovative technologies that enable us to maneuver and manipulate single cells in fine-tuned microfluidic channels and to increasingly sensitive methods that probe metabolites, macromolecules, and cellular nano-physical structures. At present, researchers from both academic and corporate organizations are aggressively pursuing to optimize and develop more sensitive methods. Indeed, the era of noninvasive single cell biopsy is at hand!

Why does paying attention to single cells matter? Classically, biological research has focused on the analysis of pooled cells, from the thousands to the millions. Paradoxically, these conventional assays requiring the use of pooled cells suffer from limits of detection, and as a result, important alterations in a subset of effector cells within a cell population are overlooked, their relevance diluted in a sea of averages.

Normally, the alterations in cell physiology and molecular parameters (eg, cell biophysical features and gene expression profiles) are important in maintaining homeostasis and biological processes, such as development. But the aberrant changes that occur in a small population of cells or even in a single initiating cell can be pivotal for the development and progression of diseases, such as cancer and the acquisition of resistance to therapy.

		Single-cell isolation and characterization using CIM and DEPArray systems, respectively. Individual PSA/PSMA-positive prostate epithelial cells have been isolated from urine or needle biopsy washes for downstream analysis using BioMark or AFM. The same approaches will be used to isolate epithelial cancer cells and the associated stromal fibroblasts from endocervical brushes and to isolate circulating rare cells from blood samples of cancer patients.

Here at The University of Texas Health Science Center at San Antonio, we have developed the Single-Cell Biopsy and Characterization Core (SBCC), which recently received prestigious funding from the Cancer Prevention and Research Institute of Texas (CPRIT).

The SBCC will implement and develop a single cell manipulation and analysis pipeline to provide: 1) clinical sample processing for single-cell isolation and banking; 2) high-throughput single-cell analysis platforms; and 3) data integration by single-cell specialized bioinformatics.

Furthermore, we have developed and are continuously optimizing new methods to isolate single cells from limited clinical materials, including: 1) exfoliated cancer cells from urine, as well as endocervical and oral brushes; 2) circulating rare cells in blood (eg, singular circulating tumor cells [CTCs], clustered CTCs, circulating macrophages, and circulating adipose stromal cells); and 3) epithelial cancer cells and stromal fibroblasts from needle biopsy washes.

A major advantage for single-cell biopsy is that small amounts of clinical materials are used in cancer detection without interference with standard pathology diagnostics. Furthermore, the development of noninvasive methodologies, such as liquid biopsies, will allow us to actively monitor disease development or progression in high-risk patient populations. Intratumor heterogeneity can also be interrogated by single-cell analysis. Hidden within the milieu of clonal tumor cells, sometimes stroma, and inflammatory cells, there might lie a population of tumor initiating cells with distinct physical characteristics and molecular expression profiles. These cells could emerge as recurrent tumors after the elimination of the clonal tumor cell population by first-line therapeutic intervention of the primary tumor. Characterizing this phantom menace by single-cell analysis can lead to stratification of patients at risk of recurrence or developing drug resistance.

, BioMark/microfluidic chip system for single-cell expression analysis of candidate genes and microfluidic chip.

Expression heat map and representative amplification curves and melting point peaks from a single-cell PCR assay. Single-cell protein detection can be similarly analyzed using the proximity ligation assay.

With a view to developing and enhancing our single-cell analysis pipeline and serving the research community, we have acquired manual immunofluorescence- micromanipulator workstations and a DEPArray unit (Silicone Biosystems, Inc), which uses dielectrophoresis to separate single cells or subpopulation of cells in suspension within a microelectronic array 

, the Biomark HD MX/HX system (Fluidigm, Inc) is used to determine single-cell expression of gene panels in a 48 x 48 microfluidic chip format, which can assay the expression of 48 genes in 48 single cells on one chip (4 nanoliter per reaction; total, 2304 reactions). A 96 x 96 loading platform is also available. The Catalyst Atomic Force Microscope unit (Bruker, Inc) has the capacity to collect quantitative nanophysical/mechanical data (eg, cell adhesiveness and elasticity), along with tapping, force, and contact modes of single cells. With the help of this state-of-the-art facility, the interphase between clinical sampling and single-cell analysis is currently within our grasp.

The development of such noninvasive tools requiring minimal clinical samples will help us to examine heterogeneity within tumors, which can be a determinant of cancer growth, metastatic spread, and drug response. Detecting and characterizing populations of culprit cells will greatly enhance our capabilities to actively monitor cancer progression, predict clinical outcome, and design personalized therapy.

Even in seemingly uniform cell populations within subcompartments, cell-cell heterogeneity is becoming more evident, far beyond what was previously expected.

The Era of Interrogating Single Cells in Cancer Biopsy Is Now

Three years ago, the Intellisphere Oncology Specialty Group launched the Giants of Cancer Care® program to recognize the contributions of oncology leaders whose efforts have truly advanced the care of patients with cancer. It has been our privilege to salute those who have contributed so richly to the remarkable ongoing progress in oncology.

The inaugural Giants class recognized the accomplishments of 12 luminaries in the field, with 16 recipients being honored in the second year. This year, the Giants family welcomed another 12 remarkable members at a ceremony in Chicago on May 29.

This year’s Giants are a dynamic and diverse group, each taking a unique path to the pinnacle of the field. The 2015 award winners were recognized in Breast Cancer, Community Outreach/Education, Gastrointestinal Cancer, Genitourinary Cancer, Gynecologic Malignancies, Immuno-Oncology, Lymphoid Neoplasms, Lung Cancer, Melanoma, Myeloid Neoplasms, Scientific Advances, and Supportive/Palliative Care.

A critical component of the Giants program is that the winners are selected by their peers. After an open nomination process, the final voting is conducted by the Giants of Cancer Care selection committee, an advisory panel comprising 28 esteemed oncologists.

Giants of Cancer Care are individuals who propel the field forward, establishing the building blocks for future advances. Thus, it is critical that the Giants be selected by those individuals who are inspired by their work and are following the trail that they have blazed. As the Isaac Newton quote says, “If I have seen further than others, it is by standing upon the shoulders of giants.” It is in this spirit that our Giants coverage this month includes not only the accomplishments of this year’s winners, but also peer testimonials recognizing the inspirational work.

And so, please join us in honoring and celebrating this year’s Giants. The celebration will continue beyond this issue with more detailed profiles of award winners in future editions. For additional coverage of the 2015 Giants and all of the previous winners, go to 

It has been our privilege to salute those who have
contributed so richly to the remarkable ongoing progress in oncology.

Tradition of Greatness Continues With 3rd Annual Giants Class

The treatment landscape for advanced melanoma is expanding dramatically. Just a few years ago, we had little to offer patients. Nothing showed a survival benefit. But over the past few years, the FDA has approved a handful of new agents that improve treatment response and overall and progression-free survival. Although a number of patients have had amazing responses to immunotherapy, there still are a number of them who do not respond to these agents. In addition, targeted therapy can only be used in select patient populations, and responses are not as durable as desired. Thankfully, preclinical research into novel agents and clinical trials are flourishing.

Advances in molecular biology are leading the way in targeting multiple signals in the pathways to treatment resistance. Precision medicine is now on the horizon as bench researchers investigate better biomarkers that can offer better patient selection for specific agents and combinations.

At the Laura and Isaac Perlmutter Cancer Center at NYU Langone Medical Center, we have a very active group of researchers focusing both on preclinical work and clinical trials. I am the proud recipient of the Melanoma Research Alliance and ASCO’s Young Investigator Awards.

One of the grants will focus on developing new strategies to overcome resistance to treatments targeted at inhibiting BRAF mutations that commonly cause certain cancers like melanoma. This direction is critically important because approximately half of patients with melanoma have an activating BRAF V600 mutation. Even though new therapies that target BRAF became FDA approved in 2013, resistance occurs after approximately 12 months of treatment with these agents.

Through this grant, we also will investigate in a multiphase trial whether resistance to targeted therapy can be overcome by adding an antibody targeting ERBB3 to BRAF and MEK inhibitors. This trial will examine the safety, tolerability, and preliminary efficacy of the combination of dabrafenib, trametinib, and MM-121. We will particularly focus on progression-free survival, objective response rate, overall survival, and adverse events.

Part and parcel of the study will be a comprehensive review of pre- and posttreatment tumor serial biopsies. MM-121 is a fully human anti-ERBB3 monoclonal antibody. Additionally, we hope to learn whether specific biomarkers can help to better identify a subset of patients most likely to benefit from the combination of targeted therapies. Thus, we will be able to offer patients more personalized treatment recommendations up front.

Dabrafenib, a small-molecule, selective BRAF inhibitor, and trametinib, a small-molecule, selective inhibitor of MEK1 and MEK2, were first approved by the FDA as single agents for advanced melanoma in early 2013. Last year, the FDA approved each drug for combo regimens.

Currently, I am the only researcher looking at the addition of the ERBB3 antibody to combat inhibition, while a number of fellow researchers are evaluating ways to overcome resistance to BRAF/ MEK inhibition.

A second area of my research concerns developing treatment strategies for specific melanoma populations, especially those with wildtype melanoma—in other words, those without BRAF, NRAS, and KIT somatic mutations. Once appropriate funding is secured, I will pursue this course of research, potentially opening up the development of novel therapies and clinical trials for the wild-type melanoma patient population.

This past April, researchers presented findings at the AACR Annual Meeting from two key melanoma trials. In the head-to-head randomized phase III KEYNOTE 006 trial presented at AACR, investigators compared pembrolizumab with ipilimumab, finding that the PD-1 inhibitor pembrolizumab extended progression-free survival and overall survival with fewer systemic side effects than the CTLA-4 inhibitor ipilimumab, the current standard of care. Based on this trial, the drug manufacturer, Merck, plans to secure FDA approval of pembrolizumab for frontline, single-agent therapy for advanced melanoma.

A phase I trial, also presented this past at AACR, evaluated ipilimumab and the PD-1 inhibitor nivolumab in combination, testing different doses and combinations. It showed impressive results. In the trial arm of patients getting concurrent dosing, 1-year overall survival was 85% and two-year survival was 79%. Yet even though concurrent dosing achieved far better outcomes, investigators remain concerned about toxicities, largely gastrointestinal and liver complications. Clearly, this is an exciting time for melanoma research and development. For example, we know for a fact that aberrant intracellular signaling pathways and cell cycle disruption permit melanoma to progress. As we continue to better understand immune biology and its role in melanoma and other tumor types, we may soon be able to screen for many more biomarkers in the upfront setting, and immediately use agents most likely to generate a response. New knowledge about the many different mechanisms involved in resistance will lead to additional targeted therapies in the varied signaling pathways. New combinations are being tested in scores of clinical trials. I am hopeful that we will have even more to offer patients as results become available.

Melissa A. Wilson, MD, PhD, discusses how targeted therapies and combination protocols are revolutionizing the treatment paradigm of advanced melanoma.

July 2015 | Oncology & Biotech News

The Evolving Story of Mutational Landscape in Cancer and Its Implications: Bad Luck or Great Hope?

The Era of Interrogating Single Cells in Cancer Biopsy Is Now

Tradition of Greatness Continues With 3rd Annual Giants Class

Targeted Therapies, Combination Protocols Are Revolutionizing Treatment for Advanced Melanoma