Melanoma and Skin Cancer TRDG: Meetings and Minutes

Higher among women and whites. 13% of high school students. Dr. Heckman studied 500 women by survey and phone interview. She found evidence for some addictive symptoms, anxiety and some information that tanning decreased negative feelings. The FDA recommends regular screening for indoor tanners. Screening is increasing but only 30%. Dr. Heckman spoke about tanning dependence. “Tanorexia” is a lay term. A comment was made that in Asia there are patterns in the other direction to have fair skin. UV exposed keratinocytes produce beta-endorphins. Orek and Bartek, Cell 2007. Naltrexone can reduce dependence and cause withdrawal symptoms. Dopamine and striatal area related to addiction. There is only minor genetic difference in people who are tanning dependent. Correlates with tanning: sun burning, smoking, lower skin protection, trouble paying for tanning. Dr. Heckman edited a book on indoor tanning in 2012. She spoke about interventions to modify cancer risk behaviors. She spoke about tailored internet intervention to help people visualize skin damage. 1234 participants nationally to take survey and randomized to intervention during the summer. She noted an issue of some individuals enrolling multiple times: reduced sample size to 965. UV exposure and sunburn decreased over time in the intervention group. The use of sun screen increased and indoor tanning decreased. Intervention module use correlated with observed effects. She is doing a follow-up study focused on dissemination and implementation (supported by an NIH R01 grant). She plans to recruit more broadly and use social media. Changing the intervention to increase engagement; mobile version, peer interaction. Will be looking at longer-term outcomes, costs and is using an online consent form. There was discussion about exercise as a potential intervention. Dr. Heckman is involved with Society of Behavioral Medicine for position statement published in a journal called Translational Behavioral Medicine. In PA, tanning is prohibited in those who are under 17 (some minors). Dr. Heckman mentioned “Don’t Fry Day” that has been going on for 8 years as a national campaign. Collapse

P29S mutant melanomas are resistant to various inhibitors including Vemurafenib, Dabrafenib, Trametinib or a MEK inhibitor. Dr. Chernoff spoke about “Next-gen” Pak inhibitors. He showed Rac1 P29S phenotypes in zebrafish in comparison with BRAF or KRAS mutant mRNA injections. He adds drugs to the water to inhibit signaling in the zebrafish. p-ERK disappears with a Pak or a MEK inhibitor. The inhibitors also block p-PAK in the zebrafish. There are however issues with adding inhibitors for extended time during zebrafish development but there is a window for reversing oncogene effects. Dr. Chernoff is collaborating with Dr. Meenhard Herlyn at the Wistar Institute  to treat a panel of cell lines with BRAF or RAC1 -/+ NRAS mutations. He is using PAK inhibitors to show specificity to RAC1 mutants while with Vemurafenib the RAC1 lines were resistant. Dr. Chernoff is also looking at cell lines from Yale in collaboration with Dr. Ruth Halaban. His approach involves contrasting the PAK inhibitor with the RAF inhibitor. Dr. Chernoff spoke about melanoma models including a Zebrafish transgenic model: transgenic Kita:Gal4 with p53-/- and mitf-/- where he is looking at RAC1 expression in this background. He is also developing conditional knock-in RAC1 alleles in mice and working on a CRISPR-Cas9 knockin mice for genome editing and cancer modeling (Dr. Feng Zhang).  Dr. Chernoff mentioned there is on target toxicity to the heart due to PAC2 with first generation inhibitors. Collapse

A validation set had 104 cases and similar results. PD-L1 not on the list. The 28 genes were assessed by RT-PCR so the focus was on mRNA expression. The assay performance was assessed and seemed better in predicting metastases by class II for stages >1. There is utility of sentinel node biopsy. The gene set does not supplant sentinel node biopsy. There was discussion about what to do with gene information as far as adjuvant therapy. There is need for prospective trial. The curves have a tail that may be of interest for the poor prognostic category and so there may be a difference between the patients who metastasize early or there may be a difference in response among the more aggressive tumors. There is a lot of clinical data at FCCC and discussion about directions. There is an interferon-controlled trial for stage III at FCCC. There was discussion about liquid biopsy and whether they can predict metastasis prior to scans. Guardant is doing tests and there is a melanoma CTC test. It may be worthwhile to look at blood samples from patients with stage II and III melanoma versus class I and class II gene expression profile and then if possible look at liquid biopsy pre-operatively as well as post-operatively to see of any measurement in blood might predict metastasis before CT scans. Some pilot data regarding feasibility could support an IIT, and fresh blood could be obtained using the opportunity blood collection protocol. BRAF mutation may be low hanging fruit but guardant is looking at many other genes. There was discussion about looking for cfDNA from patients with dysplastic moles. Collapse

UV-B induces an inflammatory microenvironment. Macrophages are recruited into the skin after UV-B. Isolated a melanoma cell line from mice and mixed with macrophages that have been activated by UV. Is IFN-gamma playing an active role? If anti-IFN-g injected this inhibited tumor growth. He showed that human melanoma-associated macrophages make IFN-gamma. Dr. Zaidi has been pursuing the hypothesis that IFN-gamma secreting melanoma associated macrophages can be used as prognostic biomarkers. UV irradiation induces influx of macrophages in the skin of normal human volunteers. Neutrophils are also recruited but they exit in adults. In neonatal mice the infiltration is only macrophages. Dr. Kerry Campbell brought up issue of lymphocytes in the adult mice may be counteracting the macrophages. Dr. Zaidi spoke about immune checkpoints. CTLA4 or PD-1 are T-cell brakes. CD28 is an activating receptor, along with OX40, GITR, CD137, CD27, and HVEM. CTLA4 is inhibitory along with PD-1. TIM-3, BTLA, VISTA and LAG3 are other inhibitory receptors. Ipilumumab has an 11% response rate; Nivolumab has a 32% response rate; Ipilumumab plus Nivolumab have a 61% response rate in metastatic melanoma. However, predicting response is the challenge. CTLA4 is the highest induced gene ~200-fold following UV-B irradiation in melanocytes. In collaboration with Dr. Jedd Wolchok at MSKCC Dr. Zaidi showed CTLA4 expression in human melanoma cells and tissues. He is interested in regulation of CTLA4 after UV-B and what is its function? Provided evidence that CTLA4 is induced by IFN-gamma treated melanocytes. PD-L1 is also induced. Question by Dr. Kerry Campbell about B7 ligand induction. IRF1 binds to CTLA4 promoter in an IFN-gamma dependent fashion. Dr. Zaidi did co-culture experiment to assess function of induced CTLA4 in UV-B irradiated melanoma cells. T-cells express B7. Showed hi CTLA4-expressors were protected. Keratinocytes do not make CTLA4 in response to IFN-gamma. Second hypothesis that CTLA4 expression on melanoma cells can be a predictive biomarker of response to ipilumumab. He is working with the biorepository to get tissues. Future directions include further testing of the hypotheses. Dr. Von Mehren raised a question about correlations between macrophages and TILs. Nevi are transformed but senescent and then deletion of PTEN or CDKN2A leads to tumor progression. There was discussion about tissue from surgery including lymph nodes and resected metastases. There was discussion about melanoma CTCs and circulating macrophages. There was discussion of combination of checkpoint inhibitor plus BRAF inhibitor. Dr. Zaidi is working on IFN-gamma inhibition for therapy as well as targeting the macrophages. Has HGF mice that produce sarcomas by 18 months and melanomas by 10-12 months after UV or faster if crossed with CDKN2A mice. Collapse

The participants of the meeting spoke about their translational research interests. Dr. Sujana Movva is a medical oncologist who sees melanoma patients and opens up clinical trials. Some fellows work with her to help review databases or molecular profiles. She also sees patients with sarcoma. Dr. Anthony Olszanski serves on the NCCN panel and lectures on melanoma. The patient population he sees currently is 50:50 between GI and melanoma. He mentioned that we are well-positioned in Philadelphia—with potential to grow. He mentioned that Jefferson’s melanoma program is headed by Dr. Mastrangelo and at U. Penn Dr. Lynn Schuchter leads the clinical effort and has done so for years. At CINJ Dr. Howard Kaufmann leads melanoma efforts and is interested in immunotherapy. The program at FCCC has opportunity. Dr. El-Deiry mentioned Dr. Meenhard Herlyn and the SPORE as well as P01 in melanoma at the Wistar Institute and a strong melanoma program at Penn State University. It wouldn’t be too difficult to build bridges should anyone from FCCC be interested. There was discussion that we need to assess where we are with databases, tissues in the biosample repository. It was mentioned that FCCC is getting a Nanostring machine in the near future and the capability of this technology to measure RNA expression from paraffin-embedded archival tissues. There was mention that Dr. Matt Zuk left and went to Florida; was doing dermatology, screening, and role not filled. Dr. Gil Yosipovich is Dermatology lead at Temple. Dr. Jeffrey Farma has worked to build referral base. Dr. Sanjay Reddy is doing 2-3 surgeries per week in melanoma. The clinical volume is here at FCCC and retention of patients is here due to availability of trials. Drs. Olszanski and Movva have opened all available ECOG trials. 1609 was the most interesting in looking at adjuvant interferon versus two doses of ipilumumab. This trial completed accrual by November 2014 and is expected to be an influential study. Another trial looking at ipilumumab plus interferon did not enroll and will close. FCCC also had an ipilumumab plus avastin study for advanced disease. Immune checkpoint including anti-PD1 has moved into first line therapy for metastatic melanoma. FCCC is opening an ipilumumab versus nivolumab (BMS) that is going to the IRB soon for stage III. Another study is TVEC pembrolizumab; plasmapheresis-based TIL therapy. Getting studies here has been challenging although there has been tremendous growth versus 4-5 years ago when there was only one trial in melanoma. There is currently interest in pan-RAF inhibitors. TEVA med is blocking BRAF and CRAF and EGFR. A study referred to as 14054 has 3 arms for metastatic disease in phase I and includes the pan-RAF inhibitor MLN2480 plus an mTOR inhibitor in the first arm (Dr. Olszanski mentioned experience with a patient on this arm who required hospitalization due to Steven Johnson Syndrome). The second arm involves the combination of Alisertib (aurora kinase inhibitor) plus MLN2480 and the 3^rd arm involves combination of pan-RAF inhibitor MLN2480 with paclitaxel. FCCC has not opened vemurafenib or dabrafenib trials in the adjuvant setting because of concerns they may make disease worse. With vemurafenib>dabrafenib keratoacanthomas/squamous cell cancers of the skin have been concerns and especially would increase risks in the adjuvant setting. Drs. Olszanski and Farma have worked with Dr. Kerry Campbell doing flow cytometry. They have been looking at primary tumors or lymph node metastases for signatures looking at immune markers. They have data on 10 patients. Age of patients was older and there is some issue about a control group. It was suggested to ask Kerry to come and speak about the data, in terms of where it stands and what are next steps. It was mentioned that the vast majority of tumor tissue samples in the biosample repository are from stage I, II, and III patients and much less so from patients with stage IV disease. There is need to address this to capture more stage IV tissues for a variety of scientific questions. Dr. Farma mentioned that everyone with stage III, and IV has been getting a 50 gene test with abstracts submitted at SSO, ASCO, and other meetings describing the initial experience in 45 pts. Patients in this cohort have demographics and treatment data. Dr. Alfonso Bellacosa mentioned he is collaborating with 2 people who are doing NGS; Nick Hayword at Royal Brisbane Hospital, and another person in Tuscany. He mentioned they are willing to share data and to look at specific genes in their data. He also mentioned the Ca bioportal at MSKCC collaboration plus other data.  Drs. Farma and Movva mentioned a Melanoma Dx test through a company (Castle Bioscience). The company wants tissue, interested in having FCCC sign up for a registry. Dr. El-Deiry spoke about the kinome assay James Duncan technology that has been developed and potential applications in melanoma. There was a discussion about whether we should spend more time analyzing non-responders. Dr. Olszanski mentioned a patient who was a responder to ipilumumab and when disease relapsed it wouldn’t respond to pembrolizumab. The patient’s tumor was rechallenged with ipilumumab and didn’t respond. Dr. Movva spoke about interrogating signatures from responders and non-responders. There was discussion about newer technologies for serum DNA, and circulating tumor cells and scenarios where they may be used to monitor disease status as well as to analyze resistance mechanisms or prognostic markers. There was a general discussion about what can we do at FCCC that would be unique or develop a niche. Dr. Movva mentioned mucosal melanomas (it was mentioned that there is a strong focus on uveal melanomas at MSKCC)—do we have tissue, NRAS in cancer code? Dr. El-Deiry spoke about hyperspectral imaging technology available in his lab at FCCC. This uses hundreds of wavelengths to provide spectral signatures of preneoplastic or neoplastic lesions that can be followed over time and/or with therapy. He mentioned previous collaboration with Dr. Keith Flaherty years ago when Dr. Nadia Khan was a resident and they did imaging of melanoma patients when the plexicon compound that later became vemurafenib was being tested at U. Penn. The machine is now here at FCCC and available for clinical protocols as a low risk type of measurement that collects data on lesions in an unbiased manner (spectroscopy). There was additional discussion regarding potential opportunities with imaging sentinel lymph nodes using isotopic or fluorescence/near infrared dyes. Drs. Margie Clapper and Harvey Hensley were mentioned as potential collaborators and that there is also interest in intra-operative imaging of minimal residual disease particularly in the peritoneal cavity. Dr. Reddy mentioned that we see plenty of patients at FCCC with stage I and II melanoma. There was discussion that in some patients there are questions regarding management of lymph node disease. There may be opportunities with sub-ungal melanomas. Dr. El-Deiry spoke about potential directions to consider as far as the biology of the melanomas and intra-operative sampling of circulating tumor cells. Dr. Farma mentioned that some patients have Bulky stage III disease and Dr. Reddy mentioned that during surgery depending on the location of the tumor it may be feasible to sample the blood proximal or distal to a tumor. It would also be relatively easy to measure CTCs during tumor manipulation. Dr. von Mehren mentioned that it would be of interest to know the variability in CTC counts and that it would be of interest to look at disease recurrence/DFS and make correlations. Dr. El-Deiry mentioned it would be feasible to look at prognostic markers on CTCs and there is technology to do genomics on limiting numbers of cells including Nanostring. Dr. Bellacosa spoke about TDG in connection with genomic stability as an emzyme that demethylates the DNA. TDG protects cells from mutations and activates transcription through demethylation. He looked at melanoma. A mouse knockout of the TDG gene was lethal and this pointed to effects on neural crest. He mentioned that melanoma in general has low levels of TDG so what happens if you reduce levels of TDG? He mentioned that it is counterintuitive to reduce levels of a protective gene. He pursued this as an experimental strategy and thought he would see increased tumor phenotype with reducing TDG but actually observed that melanoma cells stopped growing. The melanoma cells with TDG knockdown started looking like melanocytes. He used a lentivirus vector to lower level of TDG through gene knockdown. Dr. Bellacosa performed high-throughput screening looking for TDG inhibitors, and has a few. Two of them recapitulate growth suppression on tumor cells including an anti-helminthic agent and another compound called Jaglone (black walnut extract). Nothing grows around black walnuts and it has been proposed as a chemoprotective agent. MITF is completely suppressed by Jaglone. He has submitted 2 patents to inhibit TDG for cancer therapy. He is doing a bigger high-throughput screen and making analogues in collaboration with the Moulder Center. He mentioned carboxycytosine is a biomarker that goes up and that Dr. Astsaturov has PDX’s in melanoma. There was some discussion about whether Jaglone itself or even black walnut extract might be translatable because there is always a balance in translational research between the feasible in the near future versus the desirable optimized lead or analogue that may take many more years to develop. There was some discussion of the desirability to get some in vivo data, and perhaps to use PDX models that use therapy resistant tumors from patients (i.e. resistance to BRAF inhibitors or immune checkpoint therapies). There was a brief discussion about vorinostat in melanoma and the issue of epigenetic priming to immune checkpoint therapy. There may be some interest within the group to explore further in terms of developing innovative translational trials. Collapse