Lung Cancer/Mesothelioma Cancer TRDG: Meetings and Minutes

There are exclusive drivers in lung cancer: KRAS EGFR, BRAF, ROS, MET, etc. Smokers have more KRAS mutations (a lot more) whereas EGFR, and ALK are much more in non-smokers. EGFR exon 19 and 21 mutations account for half of mutations in never smokers. Dr. Clapper described estrogen and hydrocarbon intermediates and showed the lung can metabolize estrogen. 4-OH estrogen derivatives are carcinogenic with higher levels in tumors versus normal lung tissue. Dr. Clapper presented evidence that 4-OHE is carcinogenic, can transform mammary epithelial cells in culture and 4-OHE can cause tumors in mouse models e.g. uterine adenocarcinoma. 4-OHE is more concentrated in breast cancers than benign lesions. 4-OHE can induce DNA damage in the PTEN gene in endometrial carcinoma cells immortalized by T-antigen. Mutations occur in the phosphatase domain of PTEN and it can cause mutations in EGFR. Dr. Clapper is exploring human bronchial epithelial cells and transforming with E2 or 4-OHE looking at DNA damage, gene specific effects among other directions. She is exploring effects in various preclinical experimental mouse models. There was additional discussion about efforts in collaboration with Steve Kelsen at Temple (transplant program) on banked lung tissue that is not used for transplant, looking at metabolite profiles from the healthy donors versus patients with lung cancer. Dr. Clapper also mentioned developing inhibitors as chemopreventive agents. She spoke about e-cigarettes and their use by high school students. There are 7000 flavors. Collapse

Lung cancer contributes to most cancer mortality and hasn’t changed much in decades. He spoke about a national lung cancer screening trial comparing CT to chest X-ray. The study enrolled 54,000 individuals with smoking history, age 55-74, and followed them. Over an 8-year period there was a 20% reduction in lung cancer mortality and 7% reduction in overall mortality. Number needed to screen was 320. There are many ongoing CT scan trials around the world. The field is working to refine guidelines for screening based on tobacco exposure, occupation (radon exposure). USPFT recommends screening individuals aged 55-80 with >30 pack year smoking history.  Screening is not recommended beyond 15 years after quitting smoking. 1 low dose CT is like 15 chest X-rays. A flight to San Francisco is equivalent to half a low dose CT. Dr. Kumar discussed key elements of screening programs and the current program at Fox Chase Cancer Center. LDCT is done on the same day as the provider visit at which time appropriate follow-up is arranged. Patient get referred through a thoracic navigator who follows the patients. Patients are referred for a smoking cessation program run by the same practitioners. The future will go towards a risk prediction model. Age, tobacco, COPD, FH or personal history of lung cancer, BMI, low level of education all contribute to risk. A positive finding on a CT scan tends to have more of an effect to get patients to stop smoking than a negative scan. Dr. Kumar spoke about research directions looking at quality of screening projects, further approaches for early detection including risk prediction models, combining CT with bronchoscopy, exhaled breath tests, CTCs, a molecular test looking at an auto-antibody test to predict on nodules. Epithelial airway brushings are being looked at in terms of a genomic classifier; there is an FDA-approved test. There was discussion about why 80% of smokers do not get lung cancer and only 20% do. There is interest but no known results as far as a genetic basis for risk. There was discussion about racial and ethnic differences and opportunities at Temple. There is a 2-3% annual risk of other cancers that includes lung cancer in patients with Head & Neck cancer. There was discussion about opportunities for research for patients at risk including prevention targeting cytochrome P450 (Clapper), potential use of CTCs or liquid biopsy—discussion of gene expression changes. There is a need to store more metastatic tumor samples and blood samples. Collapse

BAP1 is a deubiquitinase that governs transcriptional events through chromatin remodeling or a coactivator of transcription. Not all peritoneal mesotheliomas are like pleural. Some are radiation related and are less complex in their genomic changes than the asbestos related mesotheliomas. There is very low prevalence of BAP1 in the germ-line of patients with sporadic mesotheliomas. Recent results were published by Dr. Testa (https://www.ncbi.nlm.nih.gov/pubmed/26719535). There is a mouse model: chronic IP injection of asbestos to assess in vivo carcinogenic susceptibility in GEM models. Compared BAP1 +/+ and +/- mice. Results were recently published (https://www.ncbi.nlm.nih.gov/pubmed/26896281). Dr. Testa did some experiments with NF2+/- mice from Tyler Jacks as a faithful GEM model of asbestos-induced malignant mesothelioma but didn’t work in different mouse background (FVB). The +/- mice got mesothelioma within a year. The tumors had frequent loss of second allele. P16/ARF was lost in 75% of the mouse tumors. P53 inactivation was rare as it is in human. Akt, PAK and ERK was activated in the tumors.  Hippo, FAK and PAK are downstream of NF2 (Merlin protein). P16, p19 or both as +/- had reduced survival in the double heterozygotes after exposure to asbestos. When NF2/p16/p19 +/- mice were created these mice had much reduced survival after asbestos. There was upregulation of c-MET and p-MET in the TKO mice in the malignant mesothelioma tumors. Dr. Testa spoke about a Verastem trial using premetrexate to treat bulk tumors and then FAK inhibitor to get the stem cells. He recently published a relevant paper related to this translational effort (https://www.ncbi.nlm.nih.gov/pubmed/24848258). Dr. Testa developed a mouse model designed to recapitulate BAP1-related malignant mesothelioma susceptibility. BAP1+/- mice have shortened survival after asbestos exposure. 85% of liver metastases from uveal melanomas have BAP1 mutations. There are mesotheliomas that occur spontaneously in BAP1+/- mice and knock-in mutant mice also have reduced survival after asbestos exposure. Dr. Testa is collaborating with Rebecca Simmons (U. Penn) through a superfund grant and also doing chemoprevention with Flaxseed with a collaborator at U. Penn (https://www.ncbi.nlm.nih.gov/pubmed/26678224). Dr. Testa described additional research directions where he has been looking at fungal remediated asbestos versus ambler asbestos in NF2/CDKN2A heterozygous mutant mice. NALP3 inflammasome sensing of asbestos Jurg Tschopp Science 2008 paper. Disrupting inflammasome prevents IL1-beta release which promotes mesothelial cell proliferation. Asc+/- had delayed MM development after asbestos exposure. Results were recently published (https://www.ncbi.nlm.nih.gov/pubmed/26935421). There are further efforts with anti-inflammatory agents and also incorporation of CRISPR approaches in experimental design. Collapse

He spoke about a Pembrolizumab + revlimid approved concept and working with a clinical fellow on the protocol. He discussed this as a phase I study in metastatic disease beyond 1st line with correlative studies; may get post Bx; nanostring. Ohio State interested (Carbone); Dr. Kerry Campbell is looking at PD1 in T-cell subsets and NK cells that is unique to FCCC, Dr. Edna Cukierman is interested in correlations with desmoplasia—immune restrictive part of the stroma-myofibroblastic activity—not area coverage of desmoplasia—little stroma that is superactive will repel the immune cells; immune monitoring lab; revlimid allows more immune trafficking, inhibits T-regs; not clear yet if desmoplasia impacts on PD1 or PD-L1; Dr. Cukierman wants to reprogram the fibrosis and this includes epigenetic changes. Dr. Borghaei mentioned an ECOG pembrolizumab maintenance trial, further discussion of the Immune monitoring lab, an alisertib erlotinib phase II protocol and an immuno-oncology clinic to manage toxicities. Dr. Cukierman mentioned philanthropic support to correlate immune signatures, cholesterol levels with desmoplastic levels and also that periostin is a major protein that changes in desmoplastic cells. Dr. Borghaei further discussed preclinical and translational directions involving EGFR and Aurk. Ari-4175 Aricef is a good immune modulator. PD-L1 and response (nivolumab plus ipilumumab) collaboration with MSKCC to figure our differences between responders and non-responders. There was discussion of KRAS/p53 model, other FAP inhibitors and a TEM7R antibody he made as a fellow and localizes to tumor cells. Dr. Borghaei spoke about KRAS mutations as occurring in 30% of NSCLC. EGFR/aurK co-inhibition is effective from synthetic lethal (Igor Astsaturov et al). Erlotinib + MLN8237 drug (Baynyukova) were mentioned including effects on A549 KRAS mutant xenografts, effects on signaling pathways downstream of Ras. Dr. Borghaei has done some RPPA analysis with MDACC showing decreased Akt family and he currently holds the IND for combination therapy in the phase I/II setting. Dr. Borghaei spoke about ARI-4175, an inhibitor of dipep peptidase, oral, targets FAP. Inhibition of FAP has immune modulatory activity. He has in vivo data in HCT116 xenografts using Cetuximab, drug or combination therapy. ARI-4175 increases spleen weight due to increased plasma cells; measure Ig. The drug may be augmenting ADCC. Alex MacFarlane has characterized a myeloid population of cells that do not appear to be suppressive. IL-1R mice do not unpregulate these cells. Collapse

Gender disparity in SE Asia 15% men versus 83% women are among the people who get lung cancer who are never smokers. Dr. Clapper suspected that estrogen may be playing a role in the gender bias. The lung makes estrogen and this is elevated in the tumors. Association with decreased survival, stimulation of cell proliferation and acceleration of tumors in p53fl/fl mice. Clinically HRT increases mortality from lung cancer (primarily in NSCLC). Tamoxifen also conferred protection towards lung cancer in a cohort of breast cancer patients. FCCC has a large cohort of dual primaries from the cancer registry. 250 have breast and also developed lung cancer. ER-alpha overexpressed in breast cancer while ER-beta is overexpressed in the lung tumors when they occur in different patients. Looked at whether radiotherapy might be a factor although more modern radiation is less of an issue. She has been looking at estrogen metabolites, free radicals and mutations. She has done smoking exposure experiments in mice for up to 20 weeks and performed microarrays on lung RNA. Was looking at early changes. 3, 8, and 20 weeks. Cyp1b1 was differentially expressed at all time points where it was increased. Dr. Cukierman was interested in collagen 3a1 levels that decreased. Dr. Clapper described promoter control of cyp1b1 including by ER, steroids, melatonin. She spoke about estrogen metabolism to 4-catechol estrogen that is an ER agonist and mutagen, as well as a detoxification pathway. 4-OHE is carcinogenic in vivo and increases anchorage independent growth of mammary epithelial cells. Induces uterine adenocarcinoma tumors in mice. She described detection of estrogen and metabolites by mass spec from mice. 4-OHE1 levels were dramatically elevated in lung of smoke-exposed animals. Used cyp1b1-/- mice that are resistant to DMBA-induced carcinogenesis. There was discussion about whether it would be useful to look at p53 and actual mutations associated with 4-OHE1 as a carcinogen-specific signature. There was discussion about whether bladder cancer may occur as well and whether there may be changes in cyp1b1 expression. There was mention of an upcoming bladder cancer trial with interest in correlatives. She pursued experiments in the oral cavity as well and saw effects on motility with cyp1b1 KD and also noted a downregulation in beta-catenin with upregulation of E-cadherin. She looked at cyp enzyme expression and estrogen metabolites in normal-tumor lung pairs from human, various OHE species such as 4-OHE (carcinogenic) and 2-OHE (protective) and measured ratios. Looked in urine as well. Dr. Clapper is collaborating with Dr. Alice Ma on biomarkers in the Asian population in Philly. Found more production of 4-OHE among the Asian women. There was discussion about estrogen effects in mouse models with EGFR mutation. She spoke about nasal epithelium sampling with a brush to look at field effect in bronchial epithelium for gene expression profiling with a collaborator in Boston. There are correlations between nasal epithelium and bronchial epithelium from smokers and non-smokers. She spoke about the Percepta Test that is CLIA approved and commercially available. There was discussion about use of test in the clinic to help with lung nodules. Dr. Clapper spoke about e-cigrettes and possibly doing something there. There are now 1900 flavors. The flavors are heated and inhaled and there is little info on safety. Dr. Clapper has adapted the FCCC smoking machine to test effects of e-cig smoke. She mentioned nicotine is carcinogenic according to the literature. She measured levels of cotinine as a nicotine delivery measure after tobacco vs e-cig vapor to mimic human exposures. She also described effects on detoxification enzymes within the lung. She described various future directions in mouse models, mechanistic studies of 4-OHE, estrogen metabolite profiling. There was discussion about whether there may be opportunities in breast cancer, estrogen and smoking. Dr. Cukierman spoke about assessing desmoplasia and modeling. Dr. Clapper has a compound that inhibits cyp1b1 and is interested in more screening. Other strategies might involve tamoxifen and also the protective 2-OHE. Collapse

There are 3200 new cases of mesothelioma diagnosed in the US each year, usually at an advanced stage; 70-80% with history of asbestos exposure for pleural, less in peritoneal mesothelioma. <5% of heavily asbestos exposed individuals develop mesothelioma. MM tumors frequently lose NF2/Merlin. Marc Ladanyi at MSKCC put together a figure with changes that include BAP1, CDKN2A, NF2, RICTOR, p53, LATS2, LATS1 in hippo pathway with NF2. There was discussion about synthetic lethal screening for BAP1 as a shared interest with Frank Rauscher at Wistar. Dr. Testa spoke about various downstream pathways from NF2 including Pak, Fak, Mst-LATS-YAP-miR29, mTORC-Tsc1-Akt-PI3K. He mentioned that PAK inhibitor IPA-3 works at micromolar range and inhibits viability of mesothelioma cell lines In mice loss of NF2 and CDKN2A (INK4a/Arf) accelerates asbestos induced MM development and reduces mouse survival. These NF2/CDKN2A Mice are being crossed to BAP1 mice; already has cell lines and has been testing using tail vein injections for metastasis assays. Dr. Testa discussed work with Verastem on their novel FAK inhibitor. NF2 loss leads to increased FAK signaling. VS-4718 inhibits FAK phosphorylation. Human and mouse NF2-deficient mesothelioma cells are especially sensitive to VS-4718. Re-expression of NF2 abolishes sensitivity to VS-4718. VS-4718 inhibits aldefluor(+) MM cells whereas conventional chemo agents stimulate. Verastem also has a dual FAK and PI3K inhibitor that worked better than individual inhibitors. A superfund grant looking at flak-seed derivative NK cells, macrophages after asbestos exposure. Collapse

Dr. Borghaei reported that clinical volumes in lung cancer are as good or better than before Dr. Cory Langer left FCCC. Dr. Ranee Mehra has published in NEJM (Ceritinib in ALK-Rearranged Non–Small-Cell Lung Cancer, 2014). There are currently 22 lung cancer trials at FCCC. FCCC gets referrals from throughout the region including from Penn and Johns Hopkins for specific FCCC trials. This includes the ALK trials and BMS immunotherapy trials at FCCC that are unique in Philadelphia. We treat T790M mutant lung cancer. It was mentioned that Jefferson just started a lung center in their pulmonary division. FCCC has recruited a 4th physician Dr. Jessica Bauman who will be joining from DFCI in Head & Neck and thoracic malignancies with expertise in communications and palliative care. It was mentioned that at FCCC, the pulmonary docs are very busy but they are very collaborative and willing to work with others to capture specimens. There have been some donors in the last year in support of lung cancer research at FCCC. There was discussion about the Microtron with PET/CT imager and potential opportunities for research. Dr. Margie Clapper is looking at the contribution of estrogen to lung cancer in males & females. She has performed gene expression profiling after animal exposure to tobacco smoke and found that expression of the cytochrome P450 enzyme CIP1B1 involved in estrogen metabolism is greatly altered by tobacco smoke. She has been working on estrogen metabolite profiling in lung tissue and urine and this has been providing insights into female lung cancer incidence rates. CIP1B1 leads to changes in estrogen metabolism that contribute to lung cancer. Dr. Grace Ma at Temple has a cohort of 400K Chinese women on the US east coast; got urine and is analyzing estrogen metabolites looking for evidence of passive smoke exposure. The Chinese make twice as much of carcinogenic estrogen detected in the urine. Question from Dr. Erica Golemis about whether there are risks of other cancers. Dr. Clapper believes there is a connection between breast and lung cancer and estrogen. Dr. Clapper says soy consumption may protect the Chinese from the breast cancer. It was mentioned by Dr. Clapper there is a need to capture these patients who appear to develop both lung and breast cancer that may have links to estrogen metabolism—need registry of women with the two tumors. There is a collaboration agreement with Temple pulmonary with research opportunities with Dr. Criner. Dr. Clapper has a collaborator in Italy who exposes animals to cigarette smoke and who has looked at the ability of aspirin to inhibit lung tumors; only works in females, and it appears that aspirin is active versus NSAIDs. She would like to look at estrogen connections and mentioned that mammary glands from animals exposed to smoke have a terminal end bud stage block that with aspirin is relieved and they differentiate. There are questions about interactions with hormone replacement therapy. It is felt this there is a niche at FCCC. Dr. Hoss Borghaei spoke about an interest in testing KRAS/p53 mice in smoking experiments and Dr. Margie Clapper mentioned her ongoing experiments with e-cigarettes. Dr. Clapper is working with Dr. Tom Gould who gets the brains of the smoke-exposed mice and studies flavors that contribute to the addiction. “If you like chocolate chip cookies you want chocolate flavored e-cigarettes.” In terms of potential interventions, Dr. Clapper is developing a CIP1B1 inhibitor and is interested in humans studies with a goal to alter an estrogen metabolite profile regardless of an exposure. In collaboration with Dr. Edna Cukierman they have shown that the available inhibitor can inhibit proliferation and motility and it is not expected to be toxic based on its prior use. Dr. El-Deiry asked about programmatic efforts and or historical efforts to develop multi-investigator projects. There is a Lung working group that meets regularly and that has been making progress but also has been looking for focus. This is interest in the area of early detection/intervention. There is a need for study of a high-risk population and current excitement with the interaction with the Temple lung center and the potential to study premalignant tissue. Dr. Borghaei mentioned that there are programmatic themes that can be developed into programmatic grants, but there have been some challenges. Dr. Clapper spoke of interest at FCCC in a lung cancer risk assessment program but that historically this couldn’t be offered as a clinical service. Now FCCC has a low-dose CT screening program and for patients at high risk this is paid for by Medicare. Dr. Rohit Kumar submitted a grant to expand the population to screen. Temple recruited a new thoracic surgeon from Dartmouth who is interested in outcomes research that the group wants to reach out to. There have been issues with getting tissues from the OR at FCCC for ongoing research. There are 3 surgeons who operate on lung cancer patients at FCCC. Some of the obstacles appear to be in an efficient way to consent the patients as well as issues with how rapidly tissue is processed in pathology. Drs. Kumar and Krishnamurthy mentioned that transbronchial needle biopsies are a reasonable source of tissue, although it was discussed that this is limiting in terms of success of PDX models and that fresh tissue from the OR is still very desirable for research. Dr. Golemis asked whether birth control affects lung cancer risk? This is being studied in a WHO trial. Collapse

The participants of the meeting initially introduced themselves giving a few words about their translational research interests. Dr. Yanis Boumber is a medical oncologist who treats lung cancer in the clinic. He mentioned a musashi project involving NSCLC metastasis with Dr. Erica Golemis as mentor with several grants including K08, DOD CDA, R21 being resubmitted. His work has been ACS-IRG supported. He explained that musashi is an RNA-binding protein and stem cell marker regulating translation of TGf-beta, smad3 and claudin 7. He mentioned a project with Synta with HSP90 conjugate for difficult to treat cancers and suggested this is highly effective as a single agent in a SCLC model. Drug conjugate is conjugated with cytotoxics such as irinotecan, concentrates in tumor, undergoes cleavage in the tumor and that a clinical trial is expected to open in 2016. Dr. Mark Hallman is a radiation oncologist with expertise in thoracic, lymphoma, and GU malignancies and has recent clinical training with stereotatic dosing. He has a background in basic science and would like to liaison with Rad Onc. He mentioned that EGFR signaling in the proximal tubule is different after injury. He reminded the group that the Microtron with many opportunities for research. He mentioned an interest in using radiosensitizers with deeper tissue penetration. This Microtron technology was piloted in China. Construction is starting at FCCC. The institution is also getting an MR/PET. Trials are being written. Activates carbon and oxygen within tissue and this can be measured by PET to measure the dose. Can look at perfusion of a tumor using the PET. Will have small animal PET. He mentioned several photosensitizers in hypoxic tissues including 5-ALA and acridine orange. Dr. Edna Cukierman is interested in stroma and lung fibrosis. She has been approached by a company interested in tumor-stroma interactions and did an siRNA screen. She also got surgical samples and harvested fibroblasts. Her goal is to inhibit stromal activation as a way to treat cancer. She has frozen lung cancer fibroblasts and an assay running that measures stromal activation. Desmoplasia is the outcome in NSCLC.  Gene expression profiling of fibroblast signatures in lung. Interested in collaborations with Drs. Hoss Borghaei and Margie Clapper and the Criner group. Dr. Erica Golemis is working with ganetespib and did an siRNA screen for sensitizers. Hits included anti-mullerian hormone. EMT signature relevant to pathway. Interested in biomarkers of ganetepsib and collaborations with Drs. Ranee Mehra and Margie Clapper. Dr. Ranee Mehra is a clinical oncologist who treats lung cancer and head and neck cancer. She is doing a phase I NCCN trial of multi-agent therapy including vorinostat + cisplat/pemetrexate + radiation looking at HDAC expression in non-squamous NSCLC. Runn-in of vorinostat, get tissue and then more tissue after chemotherapy. She has been looking for changes in TS expression. HDAC inhibitors may synergize w/ radiation and also being tested in other tumor types. Vorinostat plus chemotherapy is toxic and so dose is lower. HDAC1 expression, TS correlates. Can pursue cooperative group or work w/ new HDAC inhibitor. There was mention of immunotherapy trials in lung cancer that Dr. Hoss Borghaei is involved with. Dr. Ranee Mehra is driving Alk trials, first in human; Alk trials with newer better inhibitors. 5% of lung cancer. Crizotinib, Suritinib is approved. Electinib is not approved, some 3rd generation inhibitor is in development. Pfizer drug is Alk/ROS inhibitor. MGH and Colorado are working on resistance mechanism. Alk mutations all over the place. Field moving towards TKI plus PD1 to improve TTP. Dr. Rohit Kumar is a member of the pulmonary division and is happy to collaborate, work to provide core biopsies from procedures he does frequently. Dr. Walter Scott is a thoracic surgeon. He has a research interest in exhaled breath to diagnose lung cancer in collaboration with an Israeli company. There was mention that Dr. Kathy Alpaugh has interest in CTCs. SCLC has more CTCs. Dr. El-Deiry spoke about efforts of his group to isolate and phenotype CTCs using new technologies including acoustic separation. He mentioned a goal to culture isolated cells, interest in genomics, and PDX models. He also mentioned an interest in analyzing tumor DNA as another opportunity that may help monitor tumors as well as resistance to therapy. Dr. Ranee Mehra has an interest in using circulating tumor DNA in disease response monitoring as an early marker of treatment response on clinical trials or approved therapies. She particularly pointed out that immune checkpoint inhibitors often lead to what appear to be bigger tumors after two months prior to tumor shrinkage by 4 months. This would seem to be a good opportunity for a tumor marker like circulating tumor DNA that may give an early indication of response to therapy. This was acknowledged by the group to be a great idea and should be pursued. There was discussion about research using a smoking machine and interest in prevention applications.  FAP studies in house being targeted by Drs. Hoss Borghaei and Igor Astsaturov. Dr. Yanis Boumber is doing more with targeting EGFR; mentioned clovis compounds. The Tyler Jacks lung cancer model is here at FCCC. Collapse