David Polsky, MD, PhD, Professor of Dermatology and Pathology;
Alfred W. Kopf, MD, Professor of Dermatologic Oncology; Director, Pigmented Lesion Section; The Ronald O. Perelman Dept of Dermatology; New York University School of Medicine; NYU Langone Medical Center

Q: Summer sun season is upon us. What role, if any, does sun exposure have in the causation of cutaneous: 1) Actinic keratosis? 2) Squamous cell carcinoma? 3) Basal cell carcinoma? 4) Malignant melanoma? How completely should humans try to eliminate sun exposure lifelong?
A: The potential health effects (both benefits and harms) of sun exposure have been the subject of much controversy over the last 100 years. In the early 20th century when dietary sources of vitamin D were more limited, sun exposure was thought to promote both bone health and optimal health overall. Early epidemiologic studies, and later laboratory investigations demonstrated a definitive causal link between ultraviolet light exposure (i.e. the damaging wavelengths from sunlight) and skin tumors. Unfortunately, the genie was out of the bottle, and everybody wanted to be tan due to the mistaken belief that it was a sign of good health.
Interestingly, the contribution of sun exposure in different skin cancers varies.
Actinic keratosis: These extremely common skin growths are potentially pre-cancerous pre-cursors of squamous cell carcinoma. They occur on the same skin sites as squamous cell carcinoma (described below) and have a high rate of ultraviolet light-induced mutations.
Squamous cell carcinoma: This malignant tumor has the most direct relationship between sunlight and cancer. These growths occur on the face, arms and hands; skin sites that have received high cumulative levels of sun exposure. In addition, epidemiologic studies found a strong association of squamous cell carcinoma and outdoor occupations. Ultraviolet light-induced mutations are seen in more than 80% to 90% of these growths.
Basal Cell Carcinoma: This skin cancer is also linked to sun exposure, but not as directly as squamous cell carcinoma. The rate of ultraviolet light-induced mutations is lower in basal cell carcinoma than squamous cell carcinoma, and some studies suggest that skin cells’ ability to repair ultraviolet light-induced DNA damage is critical in preventing basal cell carcinoma. In contrast to squamous cell carcinoma, intensive sun exposure in childhood and adolescence may play an important role in basal cell carcinoma.
Cutaneous melanoma: While most cases are clearly caused by sunlight, melanoma is a heterogeneous disease. Some forms are associated with long-term chronic sun exposure; the more common form is associated with indoor occupations and intermittent, intense sun exposure (i.e. sunburns) in childhood and/or adulthood. Rare forms that occur on mucosal surfaces and the palms and soles are unlikely to be associated with sun exposure. Besides sun exposure related risk factors, having a large number of moles and/or atypical/dysplastic moles/nevi is even more important in identifying individuals with an increased chance of developing melanoma.
So how completely should humans try to eliminate sun exposure lifelong, and what are the most effective methods? Clearly sun exposure contributes to all forms of skin cancer so individuals, especially those with fair skin should protect themselves. Patients with light skin and hair color have higher risks because they have relatively low levels of the skin pigment melanin that protects skin cells from sun-induced DNA damage. Patients with lower levels of melanin sustain more DNA damage per minute of sun exposure than darker skinned individuals. To reduce skin damage caused by ultraviolet light, protective clothing should be the major strategy (e.g. long sleeves, long pants, hat, etc.). Nowadays, specialized lightweight sun protective clothing is available for different types of activities (e.g. water sports, golf, hiking, etc.). For skin that cannot be covered by clothing, use a high SPF, broad-spectrum sunscreen. SPF means sun protection factor, a measure of how well a sunscreen blocks ultraviolet B radiation, the wavelengths that cause sun burn. While SPF numbers roughly translate into how many times longer you can stay in the sun without burning, most people apply less than half the amount used to develop the numbers. Also sunscreens may lose effectiveness throughout the day. Experts typically recommend an SPF of at least 30 to insure a minimal level of protection. ‘Broad spectrum’ sunscreens have ingredients that also block ultraviolet A radiation which contributes to skin cancer and photoaging but doesn’t cause sunburns. Apply sunscreen generously in the morning and at midday to insure adequate sun protection throughout the day. Finally, planning outdoor activities to avoid the midday sun between 10AM and 4PM, is an effective strategy but may be impractical.
Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Lisandra E. West, PhD, Senior Scientific Knowledge Engineer, CollabRx.

Q: How might current thinking on tumor evolution/heterogeneity and sequential mutational profiling inform optimal therapy selection?
A: I’d like to share some learnings from ASCO, 2016:
To help think about tumor evolution over the course of time and treatment of cancer, I’m a fan of the palm tree analogy (made by Charles Swanton, ASCO 2016). Early genetic alterations that occur in the developing tumor are “trunk” genetic events that are present in every cancer cell in the tumor. Trunk driver mutations may be predictive of drug sensitivity or intrinsic resistance. As the tumor continues to grow some cells will develop additional mutations that will be shared by their progeny cells within the tumor. To continue the palm tree analogy, this results in a branching effect, with each branch representing a sub-clonal population of tumor cells that have new acquired mutations not shared with the original trunk cells or cells in branches that evolved earlier. Swanton cites an earlier study (Landau et al, Cell, 2013) that demonstrated that acquisition of subclonal driver mutations (occurring in the tree branches) results in significantly reduced survival over time as compared to patients whose tumors lack subclonal drivers.
How does subclonal heterogeneity (many tree branches) affect response to targeted therapy? Swanton cited a study (Pearson et al, Cancer Discovery, (2016)) that showed that the best response to FGFR inhibitor therapy occurred in patients whose tumors were homogenous (having few or no branches) for FGFR amplification. Non-responding tumors had sub-clonal heterogeneous FGFR amplification AND presence of FGFR non-amplified tumor cells. This work indicates that targeting trunk or early clonal events is important because response to targeted therapy is better when a greater proportion of cancer cells in the patient share genetic alterations. Interestingly, the overlap of mutations from two different metastatic sites biopsied from the same patient showed that percentage of mutations shared between sites decreases after therapy (venn diagrams presented by Alexandra Snyder, MD, at ASCO 2016). That is to say, there is higher diversity in mutational profiles from different metastatic sites after treatment. This indicates cancer evolution over the course of treatment(s), and leads to questions about whether molecular data from a single biopsy site can be sufficient to inform treatment decision making as cancer progresses.
Thus, therapy drives tumor heterogeneity, which then fosters polyclonal cancer therapy resistance. In a study of patients with metastatic CRC treated with EGFR antibodies, liquid biopsy and sequencing demonstrated that KRAS, NRAS, BRAF, and EGFR mutations were present in post-treatment circulating tumor DNA, whereas they had been absent in pretreatment cfDNA (Bettegowda et al, Science Translational Medicine (2014)). Treatment with a PI3K inhibitor has been shown to select for sub-clones that harbor inactivating PTEN mutations which confer resistance to PIK3CA inhibitor treatment (Juric et al, Nature (2013)). Chemotherapy damages DNA and causes mutations in tumor cells that persist after treatment. These cells go on to form sub-clonal populations that are resistant to drug treatment. In a heterogeneous tumor, drug resistant subclones take over and become the dominant cancer clones that ultimately cause cancer recurrence. Quite remarkably (at least to me), Swanton suggested consideration of treating a patient to stable disease, versus maximal tumor response, based on the idea that treating to maximal tumor response may provoke the rise of a resistant and untreatable subclonal cancer cell population – to the detriment of the patient.
An emerging mechanism fueling tumor diversity and subclonal evolution is genomic DNA cytosine deamination catalyzed by members of the AID/APOBEC family of DNA deaminases which induce genomic damage through their DNA deaminating activity. Deregulation of APOBEC enzymes causes a general mutator phenotype that gives rise to the non-random somatic mutations observed in cancer, ultimately manifesting as diverse and heterogeneous tumor subclones (Mcgranahan et al, Science Translational Medicine (2015); De Bruin et al, Science (2014)). Systemic drug treatment provides selective pressure that gives clones harboring resistance-conferring mutations a competitive growth advantage. We are beginning to understand that it will be necessary to predict and target tumor evolution over the course of treatment. This will require measuring (through ongoing liquid biopsies?) cancer’s subclonal structure that mitigates drug resistance.
Importantly we are beginning to find evidence that lots of mutations in cancer (mutational load/burden) or heterogeneity itself may represent an “Achilles heel” in cancer (Charles Swanton, ASCO 2016). Mutational burden is emerging as a predictive biomarker for sensitivity to checkpoint inhibitor immunotherapy in several cancer types including non-small cell lung cancer and melanoma. Detecting mutational burden, monitoring tumor evolution, and timing and sequencing of immunotherapy with targeted therapies seem likely to be areas of intense investigation moving forward in cancer research.
Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

John R. Adler, Jr., MD, CEO & Editor-in-Chief Cureus.com;
Dorothy & TK Chan Professor, Emeritus, Stanford University

Q: CollabRx, and its “sister” not for profit organization “Cancer Commons”, are dedicated to rapid dissemination and implementation of the best evidence to improve cancer care. The National Academy of Medicine has called for the building of rapid learning communities. How can your company and journal “CUREUS” help facilitate these efforts?
A: CollabRx is dedicated to rapid dissemination and implementation of the best evidence to improve cancer care. But where does one find such evidence? Meanwhile, the National Academy of Medicine has called for the building of rapid learning communities. Yet what is the vehicle for such physician interaction? The answer to both of the above questions is peer-reviewed journals, which are the foundation for most scientific communication. Unfortunately the processes inherent to traditional medical journals undermine these very objectives, i.e. the “rapid” dissemination and “rapid” learning of critical knowledge. I would like to argue here that our reflexive reverence for conventional processes for curating peer reviewed knowledge can and is holding back better cancer treatment.
Modern medicine prides itself for being grounded in the scientific method, and looks to science as the only credible mechanism for improving cancer care. However, it is also important to remind our collective selves that the first step in the scientific method starts with observation, and in the case of cancer, much of this is clinical observation. Because most clinicians are ill equipped to take “bedside” observations and turn them into practical new cancer therapies, (a complex task often requiring lots of money and the backing of large organizations), it is essential that these “in-the-trenches” physicians be able to clearly communicate their discoveries to big cancer research institutions and Pharma. Traditionally this communication happened through peer-reviewed journals, whose review processes function as a safe guard against the publication and dissemination into the mainstream of fallacious science. I would like to suggest that something new, and better, might be afoot.
Burdened by their long legacies as vehicles for academic promotion and tenure, most journals subject observational clinical science to almost the same level of scientific, statistical and documentary scrutiny as that of very complex and socially highly consequential research. Yearning for the fame that comes with publishing big scientific studies, few such “luxury” journals (as nicknamed by Nobel Laureate Barry Schekman) truly value humble clinical observations, or “small science”, as I like to term it. This innate bias represents a hurdle to the documentation and dissemination of clinical science within oncology, and by virtue of such, it undermines the advancement of cancer care itself. Recognizing this impediment to scientific communication, Cureus, a new generation medical journal was created 4 years ago which seeks to blend the traditions of peer reviewed journalism with many contemporary tools that have recently emerged from within the consumer internet. By virtue of design, technology and philosophy, Cureus has sought to streamline the publication of small science without compromising scientific quality. It accomplishes this goal by combining accelerated prepublication peer review with a never-ending “exhaustive” post publication “crowd sourced” peer review, a process referred to as SIQ (Scholarly Impact Quotient) “scoring”. To further break down barriers to the documentation of innovative clinical science, Cureus service is free for both authors and readers, many of which find the increasingly cost of subscription or publishing fees prohibitive. Just maybe, future “cures” for cancer will be reported and authenticated in Cureus, thereby helping CollabRx and the National Library of Medicine to better live up to their own missions.
Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Ruth L. Katz, MD, Professor of Pathology, Director Image Analysis Lab; Chief research Cytopathology, University of Texas, MD Anderson Cancer center, Houston, Texas.

Q: Liquid biopsy is “all the rage” in the cancer diagnostic space these days. You have worked with Circulating Tumor Cells (CTCs) for quite some time. Beyond R and D, is there a practical clinical usefulness for CTC at this time, what is it and how does one go about using it well?
A: Yes, I believe there are several important practical clinical applications for CTCs beyond R&D, including the early diagnosis of lethal cancers, such as lung cancer. If discovered at an early stage in the disease and treated appropriately with video-assisted transthoracic resection or stereotactic body radiation therapy (SBRT), the early diagnosis of lung cancer would result in a much improved survival rate. If there is a sensitive and specific CTC test for lung cancer, that can be performed as an adjunct to an indeterminate nodule on spiral CT scan or as a primary screening test in high risk individuals and that can be validated and replicated in laboratories worldwide, it would have a major impact on the devastating morbidity and mortality that lung cancer currently wreaks.
Unfortunately, while CTCs are a subject of intense interest to the scientific community, and a current avid pursuit of many researchers, with thousands of publications appearing in the last few years, there is still a state of confusion regarding their reliability as a biomarker for the presence of early cancers, as well as to what constitutes the best platform for the isolation and enumeration of CTCs. Numerous techniques to detect CTCs have been described ranging from different EpCAM capture antibody methods via magnetic beads, with subsequent positive staining for Cytokeratin’s (CK8,CK18, CK19) and negative staining for CD45, to filter methods that rely on isolation of CTCs by size and morphology. Other methods include negative selection of CTCs by depletion of leukocytes from the blood stream. All the aforementioned methods have their pros and cons, with lack of sensitivity being the most prominent disadvantage, as early in dissemination CTCs adopt an EMT phenotype and most cells will escape detection via EPCAM. At MD Anderson Cancer Center, our lab has devised a sensitive and specific CTC test for lung cancer, that relies on a gradient enrichment step, followed by a multi-probe DNA FISH test that has been designed to detect genetic aberrations common in lung cancers irrespective of histologic subtype, including small cell and non-small cell lung cancers. The objective of CTC detection is for an unambiguous test that is specific for rare CTC detection, without inadvertent detection of accompanying background lymphocytes and mononuclear cells. Using a method known as FICTION, in which we perform dual staining for combinations of cytokeratin, SNAIL1, ALDH1 and CD45 followed by FISH for multiple different DNA probes, we have shown that genetically abnormal cells undergo a constant phenotypic evolution or lineage plasticity throughout different time points after tumor resection. In addition there is also genetic heterogeneity amongst the CTCs. Therefore to overcome this phenotypic variance, we have focused on an antigen independent or label-free assay, in which only aneuploid cells with gains of extra genetic material can be scored as CTCs. We have defined a threshold of CTCs, above which a sample can be called positive, and in this way we have been able to detect needle biopsy confirmed lung cancer < 1 cm on spiral CTC scan We also demonstrate that CTCs disappear over time in patients undergoing removal of their tumors either by surgery or by SBRT.
Other important applications involve detecting CTCs that carry ligands which can be targeted by specific antibodies that are conjugated to deliver a lethal dose of the conjugate. For example, in small cell lung cancer (SCLC) an antibody drug- conjugate targeted to a novel protein called delta-like protein 3 (DLL3) (Rovalpituzumab tesirine (Rova-T),Stemcentrx) may bind selectively to the CTCs of SCLC and deliver a drug that kills these cells, while not affecting the normal blood cells. Although not yet approved as a companion diagnostic for CTCs, it would seem that the next logical step for exploiting the presence CTCs would be making drugs of this class that would be effective in fighting both small cell and non-small cell lung cancer. This would take CTCs into the realm of application.
In summation, I would say that CTCs may imminently be used in practical application both in the early detection of lethal cancers as well as the creation of drugs targeting CTCs themselves. There is still work to be done, and we should bear in mind that in order to be acceptable to laboratory professionals, as well as useful to the general public, tests for CTCs for lung cancer should be fairly easy to perform and replicate, and should be validated in a CLIA approved laboratory. We are on the cusp of an exciting new phase in oncology and precision medicine.
Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Razelle Kurzrock, MD, Chief, Division of Hematology and Oncology,UCSD School of Medicine; Senior Deputy Director, Clinical Science; Director, Center for Personalized Cancer Therapy; Director, Clinical Trials Office, UCSD Moores Cancer Center, San Diego, California

Q: How is it determined that an investigational cancer drug is ready for entrance into Phase 1 clinical trials? And, how is that transition accomplished?
A: Phase 1 oncology studies encompass clinical trials wherein a new drug or combination of drugs is given to patients for the first time. These studies may include: (i) new combinations of FDA-approved or investigational drugs; and (ii) a first-in-human drug.
For this commentary, I will concentrate on first-in-human phase 1 studies. The main objectives of first-in-human studies include elucidating: (i) safety/toxicity; (ii) pharmacokinetics; (iii) optimal dose; and (iv) response signals.
These studies are designed with dose-escalation steps. The most efficient and, hence, most popular design is termed 3 + 3. Three patients are entered on a dose level, and depending on the side effects or lack thereof, the next cohort receives a higher dose (if there is no toxicity greater than grade 2) or there is an expansion of the current dose (if there is > grade 3 toxicity). The degree to which the doses are escalated can be predetermined by a variety of mathematical schemes.
How is a drug readied for Phase 1? It must go through multiple steps (outlined below) that often take 7 to 8 years. Once the steps are complete, the drug can be granted an Investigational New Drug (IND) by the FDA and given to humans.
The process starts with target discovery and then documenting in vitro and in vivo efficacy. But there is much more. Here are a few examples of the studies that must be done.

• Formulation/drug stability
• Pharmacology/pharmacokinetic in animals
• ADME (absorption, distribution, metabolism, excretion)
• P450 inhibition or induction
• Short and long-term safety—often in 2 species
  • Single dose toxicity
  • Repeat dose toxicity
• Teratogenicity in animals
• Carcinogenicity in animals
• Establish manufacturing processes meeting FDA guidelines

A key step is determining the starting dose for the first patient. While a primary concern is prevention of toxicity, an initial dose that is too conservative is also undesirable and can lead to a large proportion of patients being treated at sub-therapeutic doses.
Measures such as the no-observed-adverse-effect-level (NOAEL), maximum tolerated dose and lethal dose (LD) are determined in animals. Well-established algorithms convert these numbers to a suggested safe dose in humans. Sometimes a measure such as 10% of the LD10 (one tenth of the lethal dose to 10% of mice) is used, especially for cytotoxics, to define the first dose in humans. There is abundant evidence that Phase 1 oncology trials are exceedingly safe, with deaths that are even possibly due to drug being in the 1.5 % range. In contrast, ~50% of patients with cancer who enroll on Phase 1 trials will have succumbed to their disease by nine months.
Developing a new drug, from target discovery through preclinical to clinical testing and then to FDA approval, costs about one billion dollars and takes 10 to 15 years. For every 5,000 to 10,000 compounds that enter the pipeline, only one receives approval. And even drugs that reach clinical trials have only about a 15% chance of being approved. Drug development is a long and expensive process.

Disclosure
Dr. Kurzrock has research funding from Genentech, Merck Serono, Pfizer, Sequenom, Foundation Medicine, and Guardant, as well as consultant fees from Sequenom and Actuate Therapeutics and an ownership interest in Novena, Inc. and Curematch, Inc.
Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.