Colorectal and SI Cancer TRDG: Meetings and Minutes

Obesity is linked to various cancers and contributes to mortality. Dietary fat reduction improved long-term breast cancer outcomes; study in 1990’s. Insulin and IGF1 drive cell growth. Metabolic syndrome has been associated with increased incidence and mortality from colorectal cancer although some inconsistency in the literature re degree of effect. There is no information on association with those at genetic risk. Dr. Esnaola spoke about SEER data from Medicare where he looked at a cohort of >36,000 patients with CRC stages I-IV, age at diagnosis of 66 and above, gathered various socio-demographic data and looked at various codes for metabolic syndrome or its components. He looked at OS, PFS as a function of primary variable of metabolic syndrome (MetS) versus its components. He used billing data and made the point that obesity is usually undercoded. He saw decreased MetS as a function of older age. Hispanic>black>white prevalence of MetS. He saw rising prevalence of MetS beginning at 1999. OS seemed better with MetS, although not much of an effect. No effect by stage.

DFS also did not see much difference. The results were recently published (https://www.ncbi.nlm.nih.gov/pubmed/23280333). He looked at hyperinsulinemia; C-peptide and IGFBP. High insulin and reduced IGFBP associated with higher mortality.

Diabetes was associated with worse outcomes (both OS and DFS) for stages I-III but not a big difference in stage IV. Dyslipidemia appeared to be associated with better outcomes (OS and DFS) in CRC by stage although not a big effect by stage IV. Dr. Esnaola discussed some directions going forward based on the results and their implications for screening different populations at risk. Collapse

He mentioned that you accumulate more genetic events based on exposures that can impact epigenetic load; sees methylation differences in lipid, CHO metabolism and insulin signaling. Now trying to validate in independent populations. He has an R21 with Dr. Margie Clapper looking for epigenetic signatures in mouse colitis. Also has a submission with Dr. David Weinberg and Nestor Esnaola. He mentioned he would like to work backwards in time to see who is predisposed to colon cancer and to try to differentiate them. In terms of translation, his work would be applicable to people coming in for colonoscopies, in terms of what fraction are on the path to developing colon cancer. Dr. Margie Clapper discussed two potential opportunities for projects. One project involves work on lesions that are flat that harbor p53 mutation and those that are polypoid and harbor beta-catenin mutation in colitis; morphological subtypes arise via different mechanisms and respond differently to therapy. She mentioned that loss of p53 versus mutation of beta-catenin drives the morphological subtypes. In colitis p53 is an early event in ulcerative colitis. She is planning to study an intervention with a p53 conformation modifying small molecule to prevent progression. Dr. Clapper is also working with groups to develop imaging probes for early detection and this work involves human tissue analysis. She mentioned MMP-based probes for early detection in the min mouse model. Initial work with probes that are too broad-based led to the direction of an MMP7-specific probe. Dr. Jean-Pierre Issa mentioned that he is working very actively in 2-3 areas relevant to this discussion. One area involves the interaction between the microbiome and the methylator phenotype in colon cancer. This is exciting work and Dr. Issa is collaborating with a group in Florida. He mentioned the observations are reproducible in mouse models and that changes are detectable in normal mucosa. He further mentioned that CIMP(+) and (-) colon tumors have different microbiomes. Another area of investigation Dr. Issa is pursuing uses CRC lines for drug screening versus epigenetics; epigenetic targeting with drugs. He mentioned that at JHU combination of Aza-C plus irinotecan is being looked at in the clinic in colon cancer. Dr. Issa is also interested in figuring out relationships between aging, methylation and cancer. He mentioned that preventing methylation prevents cancer in the min mouse model and that caloric restriction will prevent methylation. He is studying the induction of inflammation and has foundation grant for this. He is working on Tet proteins and interactions with DNA methylation. Dr. Sergei Grivennikov in studying inflammatory cytokines and has genetic mouse models with cytokine KO. He is interested in the role of the microbiome in inflammation and in CRC. A therapeutic approach he is developing involves the impact of modulation of inflammation plus 5-FU. He is interested in immune cells, cytokines and the TME. IL17 is induced by microbes and is associated with poor prognosis. He is looking at the role of IL23 and IL1 in IL17 induction and looking at tumor growth, invasion and metastasis. He is also interested in the recruitment of myeloid cells and neutrophils, angiogenesis and invasion. Dr. Jon Chernoff presented a direction he is interested in pursuing in collaboration with Dr. James Duncan. They are interested in the mechanism of resistance in KRAS colon cancer looking at the basal kinome in 3-D organoid and PDX models. They plan to treat PDX’s with inhibitors and ask about reprogramming response. At this point the translation to a human end-point would involve tissues. He will reach out to clinicians in order to develop more therapeutic/interventional applications. Dr. Alfonso Bellacosa is working on genome and epigenome stability. He is focusing on TDG that demethylates the DNA and asking whether TDG explain the methylator phenotype in CRC. He is collaborating with Dr. Issa in studying the Tet/TDG axis in CRC. He mentioned that Tet genes are mutated at a low frequency in CRC and that CRCs have elevated levels of a metabolite. He is modeling this in mice and through collaboration with Dr. Clapper is crossing min mice with tet or TDG mutant mice. With Tet1 inactivation, min adenomas are bigger and with Tet1/TDG KO bigger adenomas and hemorrhagic features. He is looking at DNA methylation profiles. This is R01-funded work that is coming up for renewal and he wants to ID critical methylated genes. Dr. Bellacosa is pursuing another angle that applies to various cancers including CRC. He found that TDG is low in CRC and other cancers but certain levels of expression are needed as acute downregulation of TDG leads to arrest, senescence and reduced xenograft formation. He has screened for TDG inhibitors and ID’d two candidates for repurposing. Dr. El-Deiry discussed several directions that may be developed into SPORE projects. These include a direction to bring Quinacrine plus capecitabine (oral 5-FU) in combination therapy for patients with metastatic colorectal cancer. Quinacrine has single agent anti-tumor efficacy and appears to affect multiple target pathways including NF-kB, STAT3, Mcl-1, works in mutant p53 and mutant KRAS or BRAF expressing tumors, and has an anti-angiogenic effect. Thus this is a translational project with correlative science including use of CTCs for PD biomarker analysis. A second project Dr. El-Deiry is developing in the context of a SPORE involves targeting a subgroup of MSI-high CRCs with BRCA2 mutation and c-MET overexpression. This is a relatively new direction that arose from collaboration with Caris Life Sciences and potentially important as the MSI-high tumors can present as aggressive metastatic tumors that can be resistant to therapy including 5-FU. Given the prevalence of these molecular changes Dr. El-Deiry estimates ~4000 patients each year in the US may fall in this subgroup with advanced stage. The project has several directions including the testing of the sensitivity of MSI-high colorectal cancer cell lines to PARP inhibitors, c-MET inhibitors, or mitomycin as single agents or in combination. He is currently pursuing a validation set and preliminarily has found the prevalence of BRCA2 mutations seems to be present. He would like to investigate a mouse model with deletion of BRCA2 in an MMR-deficient background and would like to unravel the connections as far as signaling between BRCA2 deficiency and c-MET. He would like to determine the impact of the BRCA2 mutation/c-MET overexpression on prognosis versus MSI-high without BRCA2 mutation/c-MET overexpression. Finally he is working to develop a clinical protocol using a c-MET inhibitor plus a PARP inhibitor -/+ mitomycin for patients with MSI-high CRC. Dr. El-Deiry mentioned a 3^rd direction that is focused on 5-FU toxicity and variations in 5-FU metabolism in patients. This has a preclinical component looking at p53 regulation of 5-FU metabolizing genes including DPYD that has been recently linked to the EMT. In the clinic Dr. El-Deiry is interested in developing predictive assays for 5-FU toxicity using lymphocytes or circulating serum RNA that can be correlated with 5-FU PK monitoring. Dr. El-Deiry said he will reach out to others who expressed interest in a potential SPORE effort including some who could not make this meeting. These include Drs. David Weinberg, David Wiest, Glenn Rall. He mentioned he will work on further organizing including reaching out to potential core directors, clinical collaborators, and the NCI to begin to assess the level of interest from their perspective. Collapse

He spoke about bacteria that attach to the tumor and he is trying to sequence loosely attached versus firmly attached bacteria. Dr. Margie Clapper spoke about sugar substitutes and microbiota. She leads the Cancer Prevention and Control Program at FCCC. Her work is focused on prevention primarily in the risk arena using in vivo models, cell culture, and human specimens. She is working with several models including the APC and min mouse models. She studies sporadic CRC and inflammatory bowel disease, more the latter with interest in early detection and early intervention. Morphological subtypes of flats and polypoid lesions don’t arise in the same way. The min model at FCCC develops colon tumors (other min models develop adenomas) and this hasn’t been looked at for microbiota effects. Dr. Clapper has collaborated with Dr. Harry Cooper for 17 years on inflammatory colon cancer models. She mentioned there is data to be mined on gene expression profiling. Dr. Clapper serves as a contractor with the NCI and has access to new agents that come through NCI, and can investigate Tasks that are funded through the contracts. She has an interest in molecular in vivo imaging that is now focused on early detection. To explore this she has been using MMP protease activity and bioactivatable probes with interest in clinical development with Visen that was in the works. She mentioned FCCC was poised to do this research in the clinic and involves endoscopy and MRI. She hopes to do this imaging research in parallel with endoscopy to be able to find smaller lesions. Dr. Clapper also has a reporter mouse of beta-catenin signaling and can use it to intervene with preventive agents. She has a grant to look at folate and the contribution to folate induced colon cancer. Looking for driver of multiple colon tumors. Dr. Elin Sigurdson said investigators can get tissue from the OR and she can foresee injection pre-operatively and then imaging resected specimens. There was discussion that there should be follow-up on the status of the IND. There was some discussion that folate levels are different in different countries. There was also mention that currently at FCCC there is no tracking of ischemic time on banked tissue. Dr. Harry Cooper mentioned he has collaborated from pathology with Dr. Clapper and stated that we don’t get a lot for resected CRCs—probably 100 specimens per year that get banked along with normal mucosa, both snap frozen and formalin fixed. Dr. Michael Hall mentioned that folate is protective in large epidemiologic studies while Dr.  Clapper is finding it may be harmful. The risk assessment group at FCCC is interested in hereditary GI-related cancers and is doing population science based research. Blood is collected from 80% of high risk probands but Dr. Hall mentioned we don’t have too many tumor specimens, mostly 7 tubes of blood, half to biosample repository. He has worked with Dr. Enders looking at mitotic checkpoint proteins and their dysregulation. Some of that work is continuing with Dr. Erica Golemis. Dr. Hall is collaborating with Foundation Medicine and is doing tumor-based testing on Variants of Unknown Significance. How much is in germline, how to manage that situation. Epidemiologic and psychosocial aspects; survey work; patient’s understanding of disease. Participates in mutation-specific penetrance study; info accumulated over the last 10 years. Web-based formats for providing genetic info back to patients. Dr. El-Deiry mentioned collaborative studies over the last couple of years involving sequencing of oncogenes, TSGs, and other drug resistance genes in metastatic sites from colorectal cancer. This work was presented at the 2014 ASCO meeting and in general is hypothesis generating and can lead to clinical protocol development. He gave several examples including the use of Her2 targeted therapy for CRC with lung metastasis if tumors overexpress Her2 (and the data shows there is an enrichment of Her2 expression in lung metastases from CRC although the numbers are low ~4%). Another example involves CRC with peritoneal metastases where expression of Topo1 and ERCC1 in the peritoneal metastases would predict sensitivity to irinotecan and resistance to oxaliplatin. This kind of profiling data suggests the possibility of enrolling patients with these profiles to combination chemotherapy with FOLFIRI (vs FOLFOX that they may not do as well with). The selection of subgroups with peritoneal metastases for therapy has not been done before and may lead to being able to show that for some patients FOLFIRI is the regimen of choice. Dr. Jon Chernoff mentioned a project with Dr. James Duncan on KRAS signaling. Dr. Chernoff and Duncan are looking at KRAS in colon cancer effects on the kinome as the disease develops and what happens to the kinome when patients get a MEK inhibitor. There is a clinical trial aspect. They want to use the technology on clinical samples not to guide therapy but to test specimens. They don’t need inhibitor-treated patients at the present time and are looking to characterize the basal kinome. To do this they need a couple of mg of protein and the tumor sample would have to be frozen in the OR at -80 degrees. Dr. Chernoff is looking for a clinical collaborator and has been collaborating with Champions Oncology including PDX mouse models treated with MEK inhibitors. He is planning to generate PDX mice with KRAS driven colorectal tumors. Dr. El-Deiry mentioned laboratory efforts focused on developing therapeutics targeting mutant p53, TRAIL Receptors, hypoxia, and stem cells He mentioned an interest in personalized therapeutics, profiling metastases. He mentioned recent efforts to examine immune checkpoint proteins in sub-types of colorectal cancer and that it may be of interest for Dr. Chernoff to look at the kinome in primary and metastatic tumors. Dr. Elin Sigurdson has a background in metastasis research. She chairs the NCI GI Steering Committee for Rectal cancer. The cooperative groups report to the rectal task force pulling information from SWOG, and others and plan trials and if CTEP has money they get funded. For Rectal cancer this has led to the PROSPECT trial. She mentioned one of the issues is to better plan local excision and colostomy avoidance. She mentioned serving on a colon panel as well for metastatic disease. There are very few trials with correlative science and a big push nationally to do sequencing and then have a target for which there is an agent to get randomized to standard therapy versus targeted therapy while getting standard therapy initially. The first buckets are KRAS and BRAF. Dr. Joshua Meyer spoke about a trial looking at patients who are candidates for pre-operative chemo-RT and taking tissue and doing Foundation Medicine to sequence and correlate with response. He mentioned the plan to enroll 40 patients and look at ~300 genes that Foundation does. Currently discussing whether they can provide access to germ-line information that is obtained from the normal part of the samples. Dr. El-Deiry mentioned the an interest in creating PDX models from circulating tumor cells and performing genomic analysis, heterogeneity analysis as well as drug sensitivity testing in subgroups of colorectal cancer. He mentioned earlier work while at Penn State looking at heterogeneity of CTC numbers in patients with advanced colorectal cancer and work in collaboration with surgical colleagues that relates to CTC numbers around a tumors inflow versus outflow. Such directions have potential to ultimately impact on surgical technique and further analysis of this type may also unravel heterogeneity in the biology of the tumors in vivo as far as which ones produce many cells or a few which would have potential implications for chemotherapy use as well. It may be possible in the future to sample CTCs near a tumor’s outflow by interventional radiology and this may have impact on the plan of care. Collapse