Timothy Yeatman - US grants

p53 gene /protein; metastasis; colon neoplasms; gene mutation; neoplasm /cancer genetics; genetic susceptibility; cooperative study; restriction fragment length polymorphism; oncoproteins; microarray technology; human tissue;

DESCRIPTION: (Applicant's Description) The goal of this grant proposal is to allow the principal investigator to mentor and train new physician scientists within the framework of pursuing translational research. The environment created will be invaluable to developing independent investigators who can truly bridge the gap between basic science and clinical application of novel therapeutics. Because the principal investigator (PI) was recently appointed as Program Leader for the Gastrointestinal Tumor Program at the H. Lee Moffitt Cancer Center, he has the authority to restructure the program with a central focus on colorectal cancer and Src oncogenesis. With this focus, basic laboratory research projects are now directly linked to new clinical trials. The PI's research has recently identified the novel finding of mutations in the Src proto-oncogene in advanced human colorectal cancer patients which are activating and transforming. This observation provides the rationale for pursuing Src-directed novel therapeutic agents such as tyrosine kinase inhibitors and angiogenesis inhibitors for the treatment of colorectal cancer. To support this effort, the PI has recently received a renewal of his funding through the American Cancer Society as a Research project Grant (MPG 97751; 1/99 - 1/02) entitled "Src Activation in Colon Cancer." We have several basic science collaborators who have provided us with a number of novel compounds which specifically inhibit Src or related tyrosine kinases in vitro and we are developing significant relationships with several pharmaceutical companies (Novartis, Zeneca, Sugen) which already have similar agents in the pipeline for clinical trials. The career plan presented in this application provides a direct relationship between the laboratory and the clinical application of novel therapeutics. The environment is ideal for the candidate interested in pursuing a career engaged in translational research. The surgical residency and the Society of Surgical Oncology approved surgical oncology fellowship will provide a continuous stream of candidates for this comprehensive laboratory and clinical research experience.

PROJECT SUMMARY/ABSTRACT The objective of this combined UH2/UH3 application is to develop a cost-effective, rapid, test that can be rapidly translated to the clinic and ultimately be used to re-purpose a class of FDA-approved colorectal cancer (CRC) EGFR inhibitor (EGFRi) therapeutics (cetuximab and panitumumab). To date, only negative genetic predictors (mutant KRAS/NRAS) of EGFRi response have been employed clinically, currently restricting EGFRi use to wild-type RAS/RAF subpopulations, and more recently, just to ?left-sided? lesions. We recently reported a new prognostic role for APC that relates to the number of alleles mutated and to the association with other mutant genes such as KRAS and TP53 (Nat Commun, 2016). Further analysis also revealed that mutant APC (A) genotypes, in combination with mutant TP53 (P), are strongly correlated with a gene expression signature measuring cetuximab sensitivity (CTX-S). These data led to the provocative hypothesis that mutant APC + TP53 (AP) genotypes together---more so than either mutant gene alone (A_ / _P), or wild-type AP (_ _)---may have a new role in positively-predicting EGFRi outcomes (Nat Commun, Under Review, 2018). This hypothesis is based on 3 key observations we have recently made in our cohort and in TCGA data: (1) CTX-S scores are significantly higher in: AP > A_ / _P > _ _ tumors in both WT and MUT KRAS tumors; (2) AP mutations are more frequent in Left (cetuximab-sensitive) > Right (cetuximab-resistant) CRC; (3) AP mutations are almost non-existent in MSI tumors (highly cetuximab-resistant). These findings have two potentially high-impact clinical implications: (1) they re-define the patient selection strategy by further restricting EGFRi therapies to wild-type RAS/RAF patients harboring AP mutations, thereby increasing response rates; (2) they could expand the therapeutic opportunity to treat a substantial number of previously-excluded mutant KRAS/NRAS patients who have AP mutations in both left and right CRCs. If the test for these mutations is developed and clinically validated, the utilization of these drugs could be expanded to ~25% more patients, including more first-line patients. For the vast majority of CRC patients, AP mutations are not assessed in current practice. Unlike KRAS/NRAS oncogenes, both A and P are tumor suppressor genes that can have a multitude of inactivating mutations that must be detected. Thus, there is an opportunity to change clinical practice and standards of care to ultimately improve CRC outcomes with a new test. In the LabCorp CAP/CLIA environment, we plan to develop a new highly sensitive, specific and cost-effective targeted DNA-sequencing ?assay? to detect mutations in the coding regions of the principal genes APC, TP53, KRAS, BRAF, NRAS from formalin fixed paraffin embedded (FFPE) tissue samples. In the UH2 Phase of this proposal, a new FFPE targeted DNA sequencing assay, with greater potential to accurately detect mutations at low allelic frequencies, will be analytically validated with the following approaches so that it can be offered in a CAP/CLIA laboratory for testing clinical samples: (1) A variety of cell lines, both native and engineered, will be used to ensure analytic sensitivity, specificity, and reproducibility. (2) ~100 formalin fixed paraffin embedded (FFPE) samples of variable age, quality, tumor heterogeneity, grade, and stage matched to the highest quality, ?gold standard? fresh frozen (FF) samples from the same originating tumor will be used to assess assay performance. In the UH3 Phase, we plan to perform clinical validation of the test developed in the UH2 Phase to provide evidence that the presence of AP mutations may predict EGFRi responses translating into improved clinical outcomes. Here, we will demonstrate that assay of APC and TP53 genotypes along with those already performed as SOC (KRAS/NRAS/BRAF) can be clinically validated (i.e. used to predict cetuximab sensitivity/response) using human samples derived from: (1) a retrospective CRC observational study relating CRC genetics to predicted cetuximab sensitivity. (2) a historical NCI trial (CALGB 80203) where patients were treated with cetuximab but were not sequenced.

PROJECT SUMMARY/ABSTRACT Colorectal cancer (CRC) is a major cause of cancer deaths which is curable if detected early. Unfortunately, many CRC tumors are diagnosed at more advanced stages where cure rates remain low with 20% five-year survival. The epidermal growth factor receptor (EGFR) is a major FDA-approved therapeutic target in metastatic CRC that may be underutilized. To date, it is still difficult to identify patients who may benefit from EGFRi therapy. In fact, the only identified biomarkers for EGFRi therapy are mutations in downstream genes that activate the RAS pathway (KRAS (40%), NRAS (5%), and possibly BRAF (10%)). Unfortunately, these biomarkers are negative predictors that are only ~50% accurate in predicting non-responders. Thus, accurate prediction of EGFRi sensitivity is still problematic. We have recently employed a novel hybrid approach using a predictive cetuximab sensitivity (CTX-S) gene expression signature score in 468 CRC tumors with coincident global gene expression and exome sequencing data to identify mutations beyond those in KRAS/BRAF/NRAS that might predict EGFRi responders. We found that tumors harboring the combination of APC + TP53 mutations are predicted to be the most sensitive to cetuximab (CTX). Whereas most CRC tumors (70%) have truncating mutations in APC and mutations in TP53 are present in 50% of CRC tumors, co-mutation of both genes is present in ~25% of CRC and heretofore has never been rigorously measured in clinical trials. Thus, we hypothesize co- mutated APC + TP53 as a new composite biomarker to identify CTX sensitive tumors that would allow greater response rates in WT RAS/RAF patients and potentially extend therapy to some mutant KRAS patients. To understand and validate the full potential of this novel biomarker, here we seek to determine the cellular impact of the APC and TP53 mutations that increase the tumor sensitivity to EGFR inhibition. Further, we seek to identify additional early/intermediate biomarkers of response correlating with the presence of these mutations. Finally, we will determine if these mutations alter the gene expression?based consensus molecular subtype (CMS). Tumors with CMS2 are predicted to have greater sensitivity to EGFR inhibition and appear to strongly correlate with APC + TP53 mutations in our human datasets. We seek to determine if mutations in APC + TP53 can convert molecular subtypes CMS1,3,4 to CMS2, coincident with enhancing EGFRi sensitivity. This application will provide the necessary pre-clinical validation of novel molecular biomarkers to best predict which CRC tumors will be responsive to EGFRi, and to support subsequent future clinical validation that may lead to expanded application of an under-used, FDA-approved targeted therapy.

PROJECT SUMMARY/ABSTRACT Metastatic colorectal cancer (CRC) has very poor prognosis with median survival of less than 36 months. The epidermal growth factor receptor (EGFR) is a major therapeutic target in metastatic CRC. EGFR inhibitors (EGFRi) such as cetuximab and panitumumab are FDA approved only in patients with WT KRAS/NRAS. MUTANT ?RAS? patients have been historically thought to be non-responsive, and more recently, right-sided patients have been considered insensitive as a group, limiting the utility of this drug class. Moreover, while FDA approved for first-line therapy, these agents are seldom used. Using a novel approach fusing gene expression and DNA sequencing, our laboratory recently reported the identification of MUTANT APC + TP53 as a strong predictor of EFGRi sensitivity for WT RAS, but also? surprisingly---for MUTANT KRAS patients. Therefore, we have developed the provocative hypothesis that some MUTANT KRAS CRC patients might benefit from EGFRi. Here we propose an early phase ?signal finding? clinical trial to prospectively validate the potential of MUTANT APC + TP53 as biomarkers to predict cetuximab sensitivity, and repurpose/expand the utility of EGFRi to a molecular subset of MUTANT KRAS patients that have been historically overlooked. Moreover, some of these patients may harbor right-sided tumors. For this purpose, two specific aims have been proposed: AIM 1. To perform a ?signal-finding?, single agent cetuximab, 3rd-line clinical trial to prospectively assess the potential for APC(A) + TP53(P) mutations to predict EGFRi sensitivity in MUTANT KRAS patients. Based on early historical trials, few if any mutant KRAS patients responded to cetuximab. However, no patients were selected based on mutational profiles. Here we propose, based on intriguing pre-clinical data, that molecular subpopulations harboring APC + TP53 mutations may benefit from EGFRi. A minimally-positive result from a ?signal finding? trial, requiring only a few responding/stable patients, would lead to a larger randomized trial. Given the fact that there are really no effective therapies for mutant KRAS patients, particularly in the 3rd line, a cost-effective exponential statistical design has been proposed. AIM 2. Evaluation of an ultrasensitive cfDNA duplex sequencing assay for predicting initial response and monitoring disease progression and minimal residual disease in metastatic CRC. Substantially expanding the utility of an already FDA approved drug?particularly to the ?undruggable? MUTANT KRAS genotype---would be considered a breakthrough moment for 40% of metastatic CRC patients, with a major impact on clinical practice. Success in this ?signal finding? trial would lead to more definitive trials in the future that would change clinical practice.