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Supplementary Methods, Figure Legend, Table 1 from Mapping Geographic Zones of Cancer Risk with Epigenetic Biomarkers in Normal Breast Tissue

Purpose: We previously found that an epithelial-to-mesenchymal transition (EMT)–based gene expression signature was highly correlated with the first principal component (PC1) of 326 colorectal cancer tumors and was prognostic. This study was designed to improve these signatures for better prediction of metastasis and outcome.Experimental Design: A total of 468 colorectal cancer tumors including all stages (I–IV) and metastatic lesions were used to develop a new prognostic score (ΔPC1.EMT) by subtracting the EMT signature score from its correlated PC1 signature score. The score was validated on six other independent datasets with a total of 3,697 tumors.Results: ΔPC1.EMT was found to be far more predictive of metastasis and outcome than its parent scores. It performed well in stages I to III, among microsatellite instability subtypes, and across multiple mutation-based subclasses, demonstrating a refined capacity to predict distant metastatic potential even in tumors with a “good” prognosis. For example, in the PETACC-3 clinical trial dataset, it predicted worse overall survival in an adjusted multivariable model for stage III patients (HR standardized by interquartile range [IQR] = 1.50; 95% confidence interval, 1.25–1.81; P = 0.000016, N = 644). The improved performance of ΔPC1.EMT was related to its propensity to identify epithelial-like subpopulations as well as mesenchymal-like subpopulations. Biologically, the signature was correlated positively with RAS signaling but negatively with mitochondrial metabolism. ΔPC1.EMT was a “best of assessed” prognostic score when compared with 10 other known prognostic signatures.Conclusions: The study developed a prognostic signature score with a propensity to detect non-EMT features, including epithelial cancer stem cell–related properties, thereby improving its potential to predict metastasis and poorer outcome in stage I–III patients. Clin Cancer Res; 22(3); 734–45. ©2015 AACR.

Supplementary Figure 1 from Mapping Geographic Zones of Cancer Risk with Epigenetic Biomarkers in Normal Breast Tissue

Supplementary Data from DNA Copy-Number Alterations Underlie Gene Expression Differences between Microsatellite Stable and Unstable Colorectal Cancers

Supplementary Figures and Tables

Purpose: Colorectal cancer prognosis is currently predicted from pathologic staging, providing limited discrimination for Dukes stage B and C disease. Additional markers for outcome are required to help guide therapy selection for individual patients.Experimental Design: A multisite single-platform microarray study was done on 553 colorectal cancers. Gene expression changes were identified between stage A and D tumors (three training sets) and assessed as a prognosis signature in stage B and C tumors (independent test and external validation sets).Results: One hundred twenty-eight genes showed reproducible expression changes between three sets of stage A and D cancers. Using consistent genes, stage B and C cancers clustered into two groups resembling early-stage and metastatic tumors. A Prediction Analysis of Microarray algorithm was developed to classify individual intermediate-stage cancers into stage A–like/good prognosis or stage D–like/poor prognosis types. For stage B patients, the treatment adjusted hazard ratio for 6-year recurrence in individuals with stage D–like cancers was 10.3 (95% confidence interval, 1.3-80.0; P = 0.011). For stage C patients, the adjusted hazard ratio was 2.9 (95% confidence interval, 1.1-7.6; P = 0.016). Similar results were obtained for an external set of stage B and C patients. The prognosis signature was enriched for downregulated immune response genes and upregulated cell signaling and extracellular matrix genes. Accordingly, sparse tumor infiltration with mononuclear chronic inflammatory cells was associated with poor outcome in independent patients.Conclusions: Metastasis-associated gene expression changes can be used to refine traditional outcome prediction, providing a rational approach for tailoring treatments to subsets of patients. (Clin Cancer Res 2009;15(24):7642–51)

Purpose: About 15% of colorectal cancers harbor microsatellite instability (MSI). MSI-associated gene expression changes have been identified in colorectal cancers, but little overlap exists between signatures hindering an assessment of overall consistency. Little is known about the causes and downstream effects of differential gene expression.Experimental Design: DNA microarray data on 89 MSI and 140 microsatellite-stable (MSS) colorectal cancers from this study and 58 MSI and 77 MSS cases from three published reports were randomly divided into test and training sets. MSI-associated gene expression changes were assessed for cross-study consistency using training samples and validated as MSI classifier using test samples. Differences in biological pathways were identified by functional category analysis. Causation of differential gene expression was investigated by comparison to DNA copy-number data.Results: MSI-associated gene expression changes in colorectal cancers were found to be highly consistent across multiple studies of primary tumors and cancer cell lines from patients of different ethnicities (P < 0.001). Clustering based on consistent changes separated additional test cases by MSI status, and classification of individual samples predicted MSI status with a sensitivity of 96% and specificity of 85%. Genes associated with immune response were up-regulated in MSI cancers, whereas genes associated with cell-cell adhesion, ion binding, and regulation of metabolism were down-regulated. Differential gene expression was shown to reflect systematic differences in DNA copy-number aberrations between MSI and MSS tumors (P < 0.001).Conclusions: Our results show cross-study consistency of MSI-associated gene expression changes in colorectal cancers. DNA copy-number alterations partly cause the differences in gene expression between MSI and MSS cancers.

Purpose: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors.Experimental Design: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings.Results: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P = 0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR.Conclusions: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.

Estrogen imprinting is used to describe a phenomenon in which early developmental exposure to endocrine disruptors increases breast cancer risk later in adult life. We propose that long-lived, self-regenerating stem and progenitor cells are more susceptible to the exposure injury than terminally differentiated epithelial cells in the breast duct. Mammospheres, containing enriched breast progenitors, were used as an exposure system to simulate this imprinting phenomenon in vitro. Using MeDIP-chip, a methylation microarray screening method, we found that 0.5% (120 loci) of human CpG islands were hypermethylated in epithelial cells derived from estrogen-exposed progenitors compared with the non–estrogen-exposed control cells. This epigenetic event may lead to progressive silencing of tumor suppressor genes, including RUNX3, in these epithelial cells, which also occurred in primary breast tumors. Furthermore, normal tissue in close proximity to the tumor site also displayed RUNX3 hypermethylation, suggesting that this aberrant event occurs in early breast carcinogenesis. The high prevalence of estrogen-induced epigenetic changes in primary tumors and the surrounding histologically normal tissues provides the first empirical link between estrogen injury of breast stem/progenitor cells and carcinogenesis. This finding also offers a mechanistic explanation as to why a tumor suppressor gene, such as RUNX3, can be heritably silenced by epigenetic mechanisms in breast cancer. [Cancer Res 2008;68(6):1786–96]

Two well-characterized EGFR inhibitor (EGFRi) therapies (cetuximab, panitumumab) have been FDA approved for metastatic colorectal cancer (CRC). Unfortunately, the extended RAS/RAF testing required in the drug labels, identifies only non-responders, and only ~50% of treated patients will respond to therapy. Thus, there is an unmet need to develop additional biomarkers to identify EGFRi sensitive patients. Using an innovative hybrid approach fusing gene expression and DNA sequencing, we recently reported that combined mutations in APC and TP53 were strongly correlated with a validated gene expression signature measuring cetuximab sensitivity in CRC human tumors (Yang et al. Cancer Epidemiol Biomarkers Prev. 2019), suggesting a potential positive biomarker role of APC + TP53 mutations. This finding prompted us to develop a DNA test designed to positively select EGFRi-responsive patients as augmentation to currently used negative predictors for CRC patients. By leveraging the TwinStrand Duplex Sequencing (DS) technology, we recently developed an ultrasensitive custom 6-gene panel (APC, TP53, KRAS, BRAF, NRAS, and HRAS) CLIA-certified assay for FFPE CRC tissues. The assay has been analytically validated using reference cell lines (n=10) which were individually sequenced to >3,000x Duplex depth. The 6-gene DS assay yielded exceptionally high assay performance: (1) accuracy (passed, r2 = 0.98 for Duplex VAF vs. target VAF for Positive Control mutations); (2) sensitivity (passed, all mutations >1/5,000 were detected in all Positive Control replicates; 1 mutation at 1/5,000 was detected in 2/3 replicates), (3) specificity (passed, 2 mutations detected in the Negative Control at 1 count in 1 replicate; VAF 1/500). Importantly, the validation analysis on fresh frozen (FF):FFPE paired tissues from 21 CRC patients shows: (1) The 6-gene DS assay performed on FFPE samples faithfully reproduces assay results on FF samples; (2) The ultrasensitive DS assay can accurately detect additional “new” mutations at low allelic frequencies compared to a standard NGS method. TwinStrand DS identified 17 mutations not identified by standard NGS; 13 of these new mutations had < 10% VAF. Furthermore, preliminary Kaplan-Meier analysis of the third-line EGFRi data shows that while the statistical significance was not achieved due to the small simple size (n=28), patients harboring combined APC and TP53 mutations(AP) (vs non-AP patients) tended to have longer progression free survival (6.65 vs 3.95 months, P=0.064) and overall survival (17.80 vs 10.65 months, P=0.14). Additional data acquisition and analysis is ongoing. Thus, there is an opportunity to change clinical practice and standards of care to ultimately improve CRC outcomes if this new, robust, sensitive test is clinically-validated. Citation Format: Mingli Yang, Michael J. Schell, Lance Pflieger, Jesse Salk, Timothy J. Yeatman. Development of an APC and TP53-based duplex sequencing assay to positively predict colorectal cancer response to EGFR inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4343.

Background Early cancer detection could identify tumors at a time when outcomes are superior and treatment is less morbid. This prospective case-control sub-study (from NCT02889978 and NCT03085888) assessed the performance of targeted methylation analysis of circulating cell-free DNA (cfDNA) to detect and localize multiple cancer types across all stages at high specificity. Participants and methods The 6689 participants [2482 cancer (>50 cancer types), 4207 non-cancer] were divided into training and validation sets. Plasma cfDNA underwent bisulfite sequencing targeting a panel of >100 000 informative methylation regions. A classifier was developed and validated for cancer detection and tissue of origin (TOO) localization. Results Performance was consistent in training and validation sets. In validation, specificity was 99.3% [95% confidence interval (CI): 98.3% to 99.8%; 0.7% false-positive rate (FPR)]. Stage I–III sensitivity was 67.3% (CI: 60.7% to 73.3%) in a pre-specified set of 12 cancer types (anus, bladder, colon/rectum, esophagus, head and neck, liver/bile-duct, lung, lymphoma, ovary, pancreas, plasma cell neoplasm, stomach), which account for ∼63% of US cancer deaths annually, and was 43.9% (CI: 39.4% to 48.5%) in all cancer types. Detection increased with increasing stage: in the pre-specified cancer types sensitivity was 39% (CI: 27% to 52%) in stage I, 69% (CI: 56% to 80%) in stage II, 83% (CI: 75% to 90%) in stage III, and 92% (CI: 86% to 96%) in stage IV. In all cancer types sensitivity was 18% (CI: 13% to 25%) in stage I, 43% (CI: 35% to 51%) in stage II, 81% (CI: 73% to 87%) in stage III, and 93% (CI: 87% to 96%) in stage IV. TOO was predicted in 96% of samples with cancer-like signal; of those, the TOO localization was accurate in 93%. Conclusions cfDNA sequencing leveraging informative methylation patterns detected more than 50 cancer types across stages. Considering the potential value of early detection in deadly malignancies, further evaluation of this test is justified in prospective population-level studies.

Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. The RAS pathway is activated in more than 55% of CRC and has been targeted for therapeutic intervention with MEK inhibitors. Unfortunately, many patients have de novo resistance, or can develop resistance to this new class of drugs. We have hypothesized that much of this resistance may pass through SRC as a common signal transduction node, and that inhibition of SRC may suppress MEK inhibition resistance mechanisms. CRC tumors of the Consensus Molecular Subtype (CMS) 4, enriched in stem cells, are difficult to successfully treat and have been suggested to evade traditional chemotherapy agents through resistance mechanisms. Here, we evaluate targeting two pathways simultaneously to produce an effective treatment by overcoming resistance. We show that combining Trametinib (MEKi) with Dasatinib (SRCi) provides enhanced cell death in 8 of the 16 tested CRC cell lines compared to treatment with either agent alone. To be able to select sensitive cells, we simultaneously evaluated a validated 18-gene RAS pathway activation signature score along with a 13-gene MEKi resistance signature score, which we hypothesize predict tumor sensitivity to this dual targeted therapy. We found the cell lines that were sensitive to the dual treatment were predominantly CMS4 and had both a high 18-gene and a high 13-gene score, suggesting these cell lines had potential for de novo MEKi sensitivity but were subject to the rapid development of MEKi resistance. The 13-gene score is highly correlated to a score for SRC activation, suggesting resistance is dependent on SRC. Our data show that gene expression signature scores for RAS pathway activation and for MEKi resistance may be useful in determining which CRC tumors will respond to the novel drug combination of MEKi and SRCi.

Epithelial-to-mesenchymal transition (EMT) is a process linked to metastasis and drug resistance with non-coding RNAs (ncRNAs) playing pivotal roles. We previously showed that miR-100 and miR-125b, embedded within the third intron of the ncRNA host gene MIR100HG, confer resistance to cetuximab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, in colorectal cancer (CRC). However, whether the MIR100HG transcript itself has a role in cetuximab resistance or EMT is unknown.The correlation between MIR100HG and EMT was analyzed by curating public CRC data repositories. The biological roles of MIR100HG in EMT, metastasis and cetuximab resistance in CRC were determined both in vitro and in vivo. The expression patterns of MIR100HG, hnRNPA2B1 and TCF7L2 in CRC specimens from patients who progressed on cetuximab and patients with metastatic disease were analyzed by RNAscope and immunohistochemical staining.The expression of MIR100HG was strongly correlated with EMT markers and acted as a positive regulator of EMT. MIR100HG sustained cetuximab resistance and facilitated invasion and metastasis in CRC cells both in vitro and in vivo. hnRNPA2B1 was identified as a binding partner of MIR100HG. Mechanistically, MIR100HG maintained mRNA stability of TCF7L2, a major transcriptional coactivator of the Wnt/β-catenin signaling, by interacting with hnRNPA2B1. hnRNPA2B1 recognized the N6-methyladenosine (mMIR100HG and hnRNPA2B1 interact to control the transcriptional activity of Wnt signaling in CRC via regulation of TCF7L2 mRNA stability. Our findings identified MIR100HG as a potent EMT inducer in CRC that may contribute to cetuximab resistance and metastasis by activation of a MIR100HG/hnRNPA2B1/TCF7L2 feedback loop.

Simple Summary Colorectal cancer (CRC) is a major cause of cancer deaths. Cetuximab is an FDA-approved, underutilized therapeutic targeting the epidermal growth factor receptor (EGFR) in metastatic CRC. To date, despite selection of patients with wild-type RAS, it is still difficult to identify patients who may benefit from EGFR inhibitor (e.g., cetuximab) therapy. Our aim is to molecularly classify CRC patients to better identify subpopulations sensitive to EGFR targeted therapy. APC and TP53 are two major tumor suppressor genes in CRC whose mutations contribute to tumor initiation and progression and may identify cetuximab-sensitive tumors. Recently, it has been suggested that the consensus molecular subtype (CMS) classification may be used to help identify cetuximab-sensitive patients. Here, we report an analysis of multiple CRC tumor/PDX/cell line datasets using combined APC and TP53 mutations to refine the CMS classification to better predict responses to cetuximab to improve patient outcomes. Abstract Recently, it was suggested that consensus molecular subtyping (CMS) may aide in predicting response to EGFR inhibitor (cetuximab) therapies. We recently identified that APC and TP53 as two tumor suppressor genes, when mutated, may enhance cetuximab sensitivity and may represent easily measured biomarkers in tumors or blood. Our study aimed to use APC and TP53 mutations (AP) to refine the CMS classification to better predict responses to cetuximab. In total, 433 CRC tumors were classified into CMS1-4 subtypes. The cetuximab sensitivity (CTX-S) signature scores of AP vs. non-AP tumors were determined across each of the CMS classes. Tumors harboring combined AP mutations were predominantly enriched in the CMS2 class, and to a lesser degree, in the CMS4 class. On the other hand, AP mutated CRCs had significantly higher CTX-S scores compared to non-AP CRCs across all CMS classes. Similar results were also obtained in independent TCGA tumor collections (n = 531) and in PDMR PDX/PDO/PDC models (n = 477). In addition, the in vitro cetuximab growth inhibition was preferentially associated with the CMS2 cell lines harboring A/P genotypes. In conclusion, the AP mutation signature represents a convenient biomarker that refines the CMS classification to identify CRC subpopulations predicted to be sensitive to EGFR targeted therapies.

In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test.

Background Over half of colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors, has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms that are not well understood. This study aims to investigate MEK inhibitor-associated resistance signaling and identify subpopulation(s) of CRC patients who may be sensitive to biomarker-driven drug combination(s). Methods We classified 2250 primary and metastatic human CRC tumors by consensus molecular subtypes (CMS). For each tumor, we generated multiple gene expression signature scores measuring MEK pathway activation, MEKi “bypass” resistance, SRC activation, dasatinib sensitivity, EMT, PC1, Hu-Lgr5-ISC, Hu-EphB2-ISC, Hu-Late TA, Hu-Proliferation, and WNT activity. We carried out correlation, survival and other bioinformatic analyses. Validation analyses were performed in two independent publicly available CRC tumor datasets (n = 585 and n = 677) and a CRC cell line dataset (n = 154). Results Here we report a central role of SRC in mediating “bypass”-resistance to MEK inhibition (MEKi), primarily in cancer stem cells (CSCs). Our integrated and comprehensive gene expression signature analyses in 2250 CRC tumors reveal that MEKi-resistance is strikingly-correlated with SRC activation (Spearman P –320), which is similarly associated with EMT (epithelial to mesenchymal transition), regional metastasis and disease recurrence with poor prognosis. Deeper analysis shows that both MEKi-resistance and SRC activation are preferentially associated with a mesenchymal CSC phenotype. This association is validated in additional independent CRC tumor and cell lines datasets. The CMS classification analysis demonstrates the strikingly-distinct associations of CMS1-4 subtypes with the MEKi-resistance and SRC activation. Importantly, MEKi + SRCi sensitivities are predicted to occur predominantly in the KRAS mutant, mesenchymal CSC-like CMS4 CRCs. Conclusions Large human tumor gene expression datasets representing CRC heterogeneity can provide deep biological insights heretofore not possible with cell line models, suggesting novel repurposed drug combinations. We identified SRC as a common targetable node–-an Achilles’ heel–-in MEKi-targeted therapy-associated resistance in mesenchymal stem-like CRCs, which may help development of a biomarker-driven drug combination (MEKi + SRCi) to treat problematic subpopulations of CRC.

Background: EGFR is a major therapeutic target for colorectal cancer. Currently, extended RAS/RAF testing identifies only nonresponders to EGFR inhibitors (EGFRi). We aimed to develop a mutation signature that further refines drug-sensitive subpopulations to improve EGFRi outcomes. Methods: A prespecified, 203-gene expression signature score measuring cetuximab sensitivity (CTX-S) was validated with two independent clinical trial datasets of cetuximab-treated patients with colorectal cancer (n = 44 and n = 80) as well as an in vitro dataset of 147 cell lines. The CTX-S score was then used to decipher mutated genes that predict EGFRi sensitivity. The predictive value of the identified mutation signature was further validated by additional independent datasets. Results: Here, we report the discovery of a 2-gene (APC+TP53) mutation signature that was useful in identifying EGFRi-sensitive colorectal cancer subpopulations. Mutant APC+TP53 tumors were more predominant in left- versus right-sided colorectal cancers (52% vs. 21%, P = 0.0004), in microsatellite stable (MSS) versus microsatellite instable (MSI) cases (47% vs. 2%, P < 0.0001), and in the consensus molecular subtype 2 versus others (75% vs. 37%, P < 0.0001). Moreover, mutant APC+TP53 tumors had favorable outcomes in two cetuximab-treated patient-derived tumor xenograft (PDX) datasets (P = 0.0277, n = 52; P = 0.0008, n = 98). Conclusions: Our findings suggest that the APC and TP53 combination mutation may account for the laterality of EGFRi sensitivity and provide a rationale for refining treated populations. The results also suggest addition of APC+TP53 sequencing to extended RAS/RAF testing that may directly increase the response rates of EGFRi therapy in selected patients. Impact: These findings, if further validated through clinical trials, could also expand the utility of EGFRi therapies that are currently underutilized.

Aim: To analyze trends in Utah melanoma diagnosis and study the impact of rurality. Patients & methods: State-wide melanoma incidence was calculated using Surveillance, Epidemiology, and End Results data (2005–2013). A subset of 5199 patients treated in an integrated healthcare system was further stratified for urban or rural residence. Results: Early-stage tumors accounted for most of the increase in melanoma incidence over time. Age-adjusted melanoma incidence rate was higher in rural counties (46.7 vs 39.4). Anatomic site and stage did not differ between rural and urban patients. Rural patients were more commonly diagnosed by a local primary care provider. Conclusion: Rurality had an impact on melanoma diagnosis in the specialty and location of the diagnosing provider.

EGFR inhibitor (EGFRi) therapies (cetuximab and panitumumab) have been approved for metastatic CRC patients who harbor wild-type KRAS/NRAS. Unfortunately, only ~50% of treated patients will respond to therapy. While FDA approved for first-line therapy, these agents are seldom used, significantly limiting their utility. Thus, there is an unmet need to develop additional biomarkers to identify EGFRi sensitive patients. Using an innovative hybrid approach fusing gene expression and DNA sequencing, we recently reported that combined mutations in APC and TP53, two major tumor suppressor genes, were strongly correlated with a validated gene expression signature measuring cetuximab sensitivity (CTX-S) in 468 CRC human tumors (Yang et al. Cancer Epidemiol Biomarkers Prev. 2019). Further analysis reported that APC and TP53 mutations predict cetuximab sensitivity across consensus molecular subtypes (CMS1-4) (Thota et al. Cancers. 2021). While APC truncated mutations are known to mediate WNT pathway activation, one of key features of CMS2 CRCs reported to be associated with cetuximab response, little is known about TP53 mutations with cetuximab sensitivity. To investigate a contributive role of mutant TP53 in vitro, we stably transfected expression plasmids of three hotspot mutations (R175H, R248W, R273H) of TP53 in APC-mutated CRC cells such as SW48 cells harboring WT KRAS/NRAS. Multiple stable clones for each of three TP53 mutations (empty vector as a control) were selected for biochemical and RNASeq analyses. We found that the three oncogenic mutations of TP53 enhanced cetuximab-mediated apoptosis under low serum culture conditions (2% FBS). Interestingly, the enhanced drug effect was associated with altered p53 pathway signatures as assessed by RNASeq. In support of the role of TP53 mutations, siRNA-knockdown of TP53 in SW48 cells also demonstrated an increased sensitivity to cetuximab treatments. Collectively, these in vitro analysis supports TP53 mutations, in combination with APC mutations, as predictive mutation biomarkers of cetuximab sensitivity in CRC. Citation Format: Mingli Yang, Heiman Wang, Thomas B. Davis, Lance Pflieger, Ramya Thota, W. Jack Pledger, Timothy J. Yeatman. Hotspot mutations of TP53 sensitize APC-mutated colorectal cancer cells to cetuximab in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5366.

PURPOSE: A telehealth oncology practice was created to care for patients in rural communities to improve access to health care, decrease financial burdens, and save time. PATIENTS AND METHODS: Patients with cancer at Sevier Valley Hospital in Richfield, Utah, were treated with a real-time video-based telehealth program under the care of an oncologist at a tertiary medical center. Data on financial savings, travel hours, mileage avoided, carbon emissions reduced, and revenue retained by Sevier Valley Hospital were collected from 2015 to 2018. RESULTS: From 2015 to 2018, 119 patients with cancer in Richfield, Utah, were treated with telehealth for oncology visits, accounting for 1,025 patient encounters. On average, patients saved 4 hours and 40 minutes and 332 miles roundtrip per encounter. In total, patients’ savings were estimated to be $333,074. Carbon emissions were reduced by approximately 150,000 kg. Of new patient referrals, 59% were for solid tumors (70 of 119 referrals; 42 metastatic and 28 nonmetastatic cancers), and 41% were hematology consultations (49 of 119 referrals; 28 classical and 21 malignant hematologic conditions). We estimate that Sevier Valley Hospital retained $3,605,500 in revenue over this 4-year period. CONCLUSION: Using a telehealth program in rural Utah, patients with cancer benefited from substantial time and monetary savings. The local medical center was able to retain revenue it would have otherwise lost to outsourcing cancer care. Recent regulatory changes to address the COVID-19 pandemic should increase the number of patients with cancer treated via telehealth nationwide.

It is rare that stage IV colon cancer is cured with chemotherapy. Here we report the long-term survival of a patient who presented with highly advanced disease characterized by a papillary architecture as well as porta hepatis lymph nodes but responded extremely well to FOLFIRI/bevacizumab. His original tumor underwent comprehensive genomic testing that included whole genome DNA sequencing, targeted sequencing, and RNA sequencing. These genetic results suggest the patient’s tumor harbored mutations in APC, KRAS, and TP53 as well as in PIK3CB. Moreover, the RNA-seq data suggested that the tumor belonged to the consensus molecular subtype 4, the “inflamed, immune phenotype,” with increased angiogenesis. Deep sequencing of highly responsive cancers may yield molecular insights into mechanisms underpinning a remarkable response.

Colorectal cancers (CRCs) are hard-wired to RAS/RAF/MEK/ERK pathway oncogenic signaling. However, the promise of targeted therapeutic inhibitors (KRASi/BRAFi/MEKi/ERKi), has been tempered by disappointing clinical activity, likely due to complex resistance mechanisms including intrinsic and adaptive resistance. Here we report a central role of SRC in mediating intrinsic and adaptive “bypass”-resistance to MEK inhibition in cancer stem cells (CSCs). The SRC oncogene is a well-studied non-receptor tyrosine kinase that can regulate multiple signaling pathways involved in the control of cell proliferation, survival, differentiation, adhesion, invasion and motility. Previously, our laboratory was the first to document that human CRC truncating mutations in the negative regulatory domain of SRC result in SRC activation (Nat Genet 1999). Now our integrated and expansive gene expression signature analyses in >2000 human CRCs reveal that MEKi bypass-resistance is strikingly-correlated with SRC activation, which is associated clinically with regional metastasis, disease recurrence and poor overall survival, and associated biologically with EMT and “stemness”. In support of this, SRC-associated MEKi resistance is strongly related to the CMS4 subtype that has a mesenchymal CSC phenotype. The finding is further supported by in vitro analyses demonstrating that CSCs vs. non-CSCs display greater levels of MEKi-resistance, which is attenuated by a SRC inhibitor. These results support a novel notion that SRC may serve as a common targetable node in MEKi resistance mechanisms, permitting effective cancer stem cell targeting. These pre-clinical data, based on strikingly-correlated pathway analyses of thousands of human tumors, support the “fast-track” development of a biomarker-driven (MEKi + SRCi) drug combination targeting problematic SRC-mediated, mesenchymal CSCs. Citation Format: Mingli Yang, Thomas B. Davis B. Davis, Michael V. Nebozhyn, Andrey Loboda, Heiman Wang, Michael J. Schell, Lance Pflieger, Ramya Thota, W. Jack Pledger, Timothy J. Yeatman. A novel role of SRC in mediating bypass resistance to MEK inhibition in colorectal cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1083.

Introduction: The intrinsic heterogeneity of colorectal cancer (CRC) makes it difficult to determine which patients will benefit from adjuvant or systemic therapy and which patients will not require further treatment beyond surgical resection. This had led to a generalized ‘one size fits all’ approach to therapy. Such heterogeneity also makes it a great challenge to predict which patients might respond to targeted therapies. Precision medical diagnostics now hold promise to address these new challenges.Areas covered: The authors first review various molecular/genomic classification studies of colorectal cancer, including the prognostic role of MSI (microsatellite instability), CIN (chromosomal instability), and driver genes such as KRAS, NRAS, BRAF, PIK3CA and TP53, as well as our recent finding revealing a new prognostic role of APC and partnering mutations with KRAS and TP53. The authors then discuss prediction of treatment response for either adjuvant therapy or systemic targeted therapy (e.g. ...

PTPRS is the most commonly mutated receptor tyrosine phosphatase in colorectal cancer (CRC). PTPRS has been shown to directly affect ERK and regulate its activation and nuclear localization. Here we identify that PTPRS may play a significant role in developing adaptive resistance to MEK/ERK inhibitors (MEKi/ERKi) through SRC activation. Moreover, we demonstrate a new clinical approach to averting adaptive resistance through the use of the SRC inhibitor, dasatinib. Our data suggest the potential for dasatinib to enhance the efficacy of MEKi and ERKi by preventing adaptive resistance pathways operating through SRC.

e16191 Background: Biliary tract cancers are aggressive tumors with limited treatment options. Several ongoing clinical trials are currently exploring role of immune therapy in advanced BTC. Programmed death-ligand 1 (PD-L1), tumor mutational burden (TMB), and microsatellite instability (MSI) are commonly used immune related-biomarkers. Herein, we analyzed the TMB, MSI and PD-L1 expression in advanced BTC. Methods: We retrospectively evaluated the association of TMB, MSI and PD-L1 expression with survival and related treatment outcomes. Results: The immune mediated biomarkers were reported in a total of 62 BTC patients (pts). The mean age of the pts is 73 (range 32-83) years, with predominant females (59%) and Caucasians (82%). The most common histology noted was intrahepatic cholangiocarcinoma (67%) followed by extrahepatic cholangiocarcinoma (18%) and gallbladder carcinoma (15%). Approximately, 40% received 2 or more lines of therapy while 20% of patients didn’t receive any treatment and over. The treatment regimen included gemcitabine-based regimen (35%), 5FU based regimen (18%), immune therapy (15%) and targeted therapy (6%). The TMB was low in 52 pts (83%), intermediate 7 pts (11%) and high in 2 pts (3%). MSI is stable in 55 pts (89%) and PD-L1 expression is negative in 29 pts (47%) and positive in 23 pts (37%). Conclusions: Our data suggests the BTC’s have in general low TMB, are microsatellite stable and have low PD-L1 expression. The potential prognostic and predictive value of these immune related- biomarkers need to be validated in larger studies.

Colorectal cancer (CRC) is a highly heterogeneous disease, for which prognosis has been relegated to clinicopathologic staging for decades. There is a need to stratify subpopulations of CRC on a molecular basis to better predict outcome and assign therapies. Here we report targeted exome-sequencing of 1,321 cancer-related genes on 468 tumour specimens, which identified a subset of 17 genes that best classify CRC, with APC playing a central role in predicting overall survival. APC may assume 0, 1 or 2 truncating mutations, each with a striking differential impact on survival. Tumours lacking any APC mutation carry a worse prognosis than single APC mutation tumours; however, two APC mutation tumours with mutant KRAS and TP53 confer the poorest survival among all the subgroups examined. Our study demonstrates a prognostic role for APC and suggests that sequencing of APC may have clinical utility in the routine staging and potential therapeutic assignment for CRC., APC is a well-known tumour suppressor that is frequently inactivated in colorectal cancer. Here, the authors sequence more than 1000 cancer genes in 468 colorectal cancers and show that mutation signatures can be used to classify the tumours and that multiple mutations in APC are associated with a poor prognosis.

267 Background: Intermountain Healthcare has used telehealth since 2015 to expand oncology care for patients living in rural Utah communities. Telehealth services have historically not been widely available to patients living in non-rural locations due to restrictions in reimbursement policies. Changes during the COVID-19 pandemic, including the 1135 waiver authority and the Coronavirus Preparedness and Response Supplemental Appropriations Act, allows for reimbursement of provider professional fees for electronic visits occurring at any location including a patient’s home. This has allowed greater access to telehealth care for patients with cancer, many of whom are at increased risk of contracting SARS-CoV-2. Methods: Patient visits from Intermountain Healthcare’s two largest oncology sites, Intermountain Medical Center and Dixie Regional Medical Center, were evaluated from the electronic health record (iCentra). Four types of encounters were considered “televisits”: scheduled video visits (SVVs), alternative video visits (AVVs), telehealth visits, and phone visits. All visits were synchronous. Patients were seen from home except for telehealth visits where patients were seen remotely from a rural Intermountain site. SVVs occurred over Intermountain’s Connect Care software platform. AVVs utilized a non-Intermountain software such as FaceTime, Google Duo, Skype, or Webex. Visits were analyzed from December 1, 2019 to June 1, 2020. Results: From December 2019 to May 2020, there were 1,798 televisits performed. Three-month televisit totals increased from 175 from December-February to 1,623 from March-May (Table). Intermountain Medical Center increased televisits from 8 to 514 over these same time periods. Conclusions: Televisits have been utilized to maintain continuity of oncology care while minimizing patients’ exposure to healthcare facilities. On May 15, 2020, Governor Gary Herbert issued an executive order de-escalating Utah COVID-19 risk phase from moderate to low-risk. This may be associated with the decrease in televisits from April to May. [Table: see text]

Left-sided colorectal cancer (CRC) tumors have been reported to be more favorably associated with better survival than right-sided tumors. A molecular basis of the laterality of prognosis, however, is still poorly understood. We have recently developed a multi-gene mutation classification system from targeted exome sequencing of 1,321 cancer-related genes on 468 CRC tumor specimens (Schell et al, Nat Commun, 2016). We found that among 17 significantly mutated genes, four (BRAF, APC, KRAS, TP53) demonstrated pairwise, statistically significant, correlations, with APC-partnering mutations playing a central role in predicting overall survival. APC might assume 0, 1, or 2 identified truncating mutations, each with a striking differential impact on survival. Tumors lacking any APC mutation carried a worse prognosis than single mutation tumors, but the triply-mutated tumors (APC, KRAS, TP53) with two APC mutations conferred substantially worse survival. Thus, we hypothesized that APC-partnering mutations might also have a significant role in the sidedness effect on CRC outcome. We carried out various Left (L) vs Right (R) survival analyses on 464 tumors that had the primary tumor location data. When individual driver mutations (BRAF, APC, KRAS, TP53) were analyzed separately, although a statistically significant association with overall survival was seen in left-sided tumors for APC mutations and BRAF (V600E) for all patients and for MSS patients, no significant survival difference was observed when each of four driver mutations was (left vs right) compared. However, when APC-partnering mutations (i.e. APC WT; APC only; APC/KRAS; APC/TP53; APC/KRAS/TP53) were analyzed, left-sided APC/KRAS tumors were found to be associated with dramatically better survival than their right-sided counterparts (HR=0.37 (0.17-0.82), logrank p=0.0078 for all patients; HR=0.059 (0.021-0.162), logrank p=0.0001 for MSS patients). Analysis of APC hit mutation (0-hit, 1-hit, 2-hit) groups reveals that left-sided APC 2-hit tumors had significantly better survival than right-sided counterparts in all patients and MSS patients. However, surprisingly, we also identified that several mutation subgroups conferred better survival in right-sided tumors than left-sided tumors. For example, APC-only mutation tumors were associated with better survival than their left-sided counterparts (logrank p=0.030) in MSS patients (n=403); Right-sided all wild-type (BRAF, APC, KRAS, TP53) tumors conferred much better survival (HR=0.12 (0.038-0.368), logrank p=0.0135) in APC WT tumors (n=35). In conclusion, our findings identified a novel role of APC-partnering mutation subgroups associated with the sidedness effect, suggesting that routine clinical APC and TP53 mutation assessment, in addition to BRAF and KRAS, may refine and improve predicting outcomes in left vs right-sided CRC patients. Citation Format: Mingli Yang, Michael Schell, Andrey Loboda, Michael Nebozhyn, W. Jack Pledger, Timothy Yeatman. A novel role of APC-partnering mutations in the sidedness of colorectal cancer survival [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5789.

e19152 Background: Cancer treatment is becoming more complex, necessitating subspecialty expertise and multidisciplinary approaches to treatment planning. Simultaneously, there is increasing demand to provide care as close to home as possible. While tumor boards have long been an institutional backbone to providing high-quality multidisciplinary care in tertiary facilities, connecting several hospitals and dozens of cancer specialists in a large integrated healthcare system is unique and potentially transformational for smaller facilities and communities. Methods: Using highly-secure, network firewall-protected Cisco Telepresence and WebEx capabilities, 11 disease specific tumor boards (Breast, GI, Sarcoma, GU, Thoracic, Head/Neck, Melanoma, Neuro, Heme, Hepatobiliary, Gyn) were organized across Intermountain Healthcare’s 24 geographically and medically diverse hospitals spanning over 500 miles. Meetings for each of these disease-specific tumor boards have been held at least every 1-2 weeks, at set times and days since July 2019. Cases are submitted to the appropriate tumor board by individual providers from anywhere in the system. Submitted cases are reviewed by a designated subspeciality leader. Cases are either added to the system-wide agenda, or at times, the clinical decision can be resolved immediately. Included cases’ records including pathology, radiology and pertinent medical history are obtained for display and discussion. After each tumor board, recommendations and conclusions are recorded by nurse navigators for future review and consultation. Results: From July 2019 to February 2020, 1,598 patient cases were discussed. Just as relevant, 293 unique oncology providers (surgeons, medical oncologists, radiation oncologists, genetic counselors, nurse navigators, and therapists) participated in tumor board discussions. These deliberations provided insight, experience and recommendations directly related to patient care. Conclusions: Our system-wide, disease-specific, multi-disciplinary tumor boards are useful in connecting oncology providers and subspecialists. This effort has led to better collaboration, coordination and delivery of high-quality cancer care to patients throughout a large healthcare system that includes thousands of patients and dozens of cancer providers in smaller/rural communities. In addition, provider engagement has improved. Work is ongoing to prospectively evaluate the effects on treatment decisions and clinical outcomes.

4094 Background: The Consensus Molecular Subtypes (CMS) of colorectal cancer (CRC) have prognostic and predictive value in identifying patients that derive benefit from EGFR targeted therapies. The CMS2 cohort was specifically noted to predict response to cetuximab. Besides CMS classification, we recently reported a two-gene mutation signature of APC and TP53 (AP) that predicts potential response to cetuximab. In this study, we hypothesize AP mutations, in addition to CMS cohorts, predict cetuximab sensitivity. Methods: A prespecified and validated 203 gene expression signature score measuring cetuximab sensitivity (CTX S-score) was used as a surrogate for response to cetuximab sensitivity. A cohort of 458 patients with colorectal cancer was accrued between October 2006 and September 2011. The population classified into CMS cohorts, and CTX-S scores were determined across each of the cohorts based on AP mutation status. Results: Among 458 tumor samples sequenced, AP mutations were identified as significantly associated with higher CTX-S scores. Among the CMS 1-4 cohorts identified, AP mutations were noted in 13 of 77 (17%) patients in CMS1 cohort, 87 of 116 (75%) patients in CMS2 cohort, 15 of 64 (23%) patients in CMS3 cohort, 46 of 112 (41%) patients in CMS4 cohort, indicating that AP mutations are dominant in CMS2 cohort. Further CTX-S score comparisons across CMS cohorts based on AP status show that AP mutated tumors have higher CTX-S scores than non-AP mutated tumors—irrespective of the CMS cohorts (p

e17604 Background: MRI-targeted biopsy is increasingly utilized over standard 12-core transrectal ultrasound (TRUS) biopsy for men with MRI-visible prostate lesions. Some clinicians defer biopsy for PI-RADS v2 category 1 and 2 lesions per the PRECISION Study (Kasivisvanathan et. Al, NEJM, 2018). The aim of this study was to independently validate the accuracy of PI-RADS v2 in detecting prostate cancer (PCa) when applied to MRI/US fusion-guided biopsies in an independent cohort of 156 patients from a large integrated community health system. Methods: Men undergoing MRI/US fusion-guided biopsy from 2016-2020 in the Intermountain Healthcare system were consecutively analyzed in this retrospective study. MRI were interpreted from four abdominal fellowship trained radiologists all with at least 5 years of experience. Fusion biopsies were performed by two urologists. Men were stratified into groups based on their PI-RADS v2 category 1-5. Biopsies were considered positive when Gleason ≥3+3. Results: A total of 156 men had 258 lesions for which they underwent MRI/US fusion-guided biopsies in the Intermountain Healthcare system from 2016 to 2020. The PCa detection rate for PIRADSv2 category 1-2 was 29.8%, category 3 32.6%, and category 4-5 37.6%. PIRADS v2 category 1, 2, 3, 4, and 5 yielded any PCa in 25, 15.9, 23.8, 53.1, and 66.7%, respectively (Table). PIRADS v2 category 1-2, 3, and 4-5 yielded any PCa in 16.8%, 23.8%, and 57.7%, respectively. Conclusions: PI-RADS v2 categories generally correlate with PCa detection rates, however, to avoid biopsy, the test must be both sensitive and specific, with low false negative rates. In our institution, we show that PI-RADS 1, 2, and 3 do not rule out the presence of PCa, and therefore should not be used as the sole factor in determining the need for prostate lesion biopsy. [Table: see text]

566 Background: Hepatobiliary (HB) tumors are aggressive tumors with emerging evidence for increasing sensitivity to immune checkpoint inhibitors (ICI). Tumor mutation burden (TMB) was found to be a quantitative biomarker associated with production of neoantigens within the tumor and predict the sensitivity to immune therapy. Herein, we explore the TMB, microsatellite instability (MSI) and PD-L1 expression as a potential biomarker of response to immune therapy in HB tumors. Methods: We retrospectively assessed all patients with hepatobiliary malignancies who have undergone next generation sequencing (NGS) between October 2009 and June 2019. We then analysed the tumor mutation burden and PD-L1 of these tumors and also identified frequency of patients with no clinically actionable mutations. Results: In our total 61 patients with HB tumors predominantly were male (62.3%) with mean age of 63 years. Thirty-four patients had hepatocellular carcinoma, 22 patients had cholangiocarcinoma and 5 patients had gallbladder carcinoma. The most common risk factors were smoking status, cirrhosis, alcohol consumption and hepatitis C virus. The mean TMB reported was 3.2 (1.16 – 7.35). MSI was identified in 13 patients and one was indeterminate. Only 17 patients had PD-L1 positive. At least, 37 patients had one clinically actionable mutation while 24 patients had no clinically actionable mutations. Mean overall survival was 16.6 months, but no statistically significant difference was found by high PD-L1 (3 vs 3.7 months, p=0.3) expression. Conclusions: Our data suggests the TMB in HB tumors is low in general irrespective of their underlying risk factors. We also noted more than half had microsatellite stable tumors and PD-L1 expression. Future larger studies are needed to evaluate TMB, MSI and PD-L1 as a potential biomarker in hepatobiliary tumors to help select patients that will benefit from immune therapy.

165 Background: The Guardian Research Network (GRN) is a nationwide consortium of integrated health systems, who share their electronic health records (EHR) to democratize clinical trial access through improvements in “process”. The GRN is a unique-in-class, free-to join, non-exclusive consortium leveraging the digital EHR---including labs, medications, demographics and non-discrete data (all text) data--- mining nightly for clinical trial candidates. Using a suite of NLP and AI tools, the GRN dramatically improves the efficiency of the clinical research staff, by electronically searching all records for the I/E criteria for trials. The GRN uses a central IRB, one contract and legal review, promising to revolutionize the trial accrual process and speed drug development. Methods: With a database of > 1.0M patients, the GRN reviews all active cancer records nightly from > 110 member hospitals to produce lists of trial candidates. Comprehensive electronic screens were filtered by manual reviews to rapidly find best candidates. Results: Our data collected over 10 mo suggest comprehensive electronic queries examining hundreds of thousands of records daily eliminate > 90% of ineligible patients in minutes. Manual review further refines eligible list. This is vastly different from current opportunistic screening approaches that examine only a tiny fraction of potential trial candidates (last week's new patients). Conclusions: The GRN has executed an all-inclusive approach to trial accrual, embedding a scalable database search technology within an integrated trial network. The novel approach seeks to exponentially expand operational capabilities of CTOs with limited staff, review all eligible patients, and solve for a large unmet need for democratizing trial access in the community. [Table: see text]

Colorectal cancer (CRC) growth and progression is frequently driven by RAS pathway activation through upstream growth factor receptor activation or through mutational activation of KRAS or BRAF. Here we describe an additional mechanism by which the RAS pathway may be modulated in CRC. PTPRS, a receptor-type protein tyrosine phosphatase, appears to regulate RAS pathway activation through ERK. PTPRS modulates ERK phosphorylation and subsequent translocation to the nucleus. Native mutations in PTPRS, present in ~10% of CRC, may reduce its phosphatase activity while increasing ERK activation and downstream transcriptional signaling.

We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD, and the most up-regulated gene in SC was versican (VCAN) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

Background Senescence-associated genes (SAGs) are responsible for the senescence-associated secretory phenotype, linked in turn to cellular aging, the aging brain, and the pathogenesis of cancer. Objective We hypothesized that senescence-associated genes are overexpressed in older patients, in higher grades of glioma, and portend a poor prognosis. Methods Forty-seven gliomas were arrayed on a custom version of the Affymetrix HG-U133+2.0 GeneChip, for expression of fourteen senescence-associated genes: CCL2 , CCL7 , CDKN1A , COPG , CSF2RB , CXCL1 , ICAM - 1 , IGFBP -3, IL - 6 , IL - 8 , SAA4 , TNFRSF - 11B , TNFSF - 11 and TP53 . A combined "senescence score" was generated using principal component analysis to measure the combined effect of the senescence-associated gene signature. Results An elevated senescence score correlated with older age (r=0.37; P =.01) as well as a higher degree of malignancy, as determined by WHO, histological grade (r=0.49; P P =.06). Gliosarcomas showed the highest scores. Six genes independently correlated with either age ( IL - 6 , TNFRSF - 11B , IGFBP - 3 , SAA4 , and COPG ), prognosis ( IL - 6 , SAA4 ), or the grade of the glioma ( IL - 6 , IL - 8 , ICAM - 1 , IGFBP - 3 , and COPG ). Conclusion We report: 1) a novel molecular signature in human gliomas, based on cellular senescence, translating the concept of SAG to human cancer; 2) the senescence signature is composed of genes central to the pathogenesis of gliomas, defining a novel, aggressive subtype of glioma; and 3) these genes provide prognostic biomarkers, as well as targets, for drug discovery and immunotherapy.

The RAS pathway is known to be commonly activated and a key determinant of colorectal cancer (CRC) biology, with mutational activation of RAS/RAF genes observed in ~50% of CRC patients. The relationship between gene mutation and expression is, however, poorly understood. We analyzed how the expression of RAS/RAF genes vs. their respective mutations impacted outcomes. The relationship to other rarely mutated RAS/RAF genes (HRAS, ARAF, RAF1 (i.e. c-RAF)) was also examined in these tumors. While KRAS expression was positively associated with KRAS mutations (r=0.26, p Citation Format: Mingli Yang, Michael J. Schell, Timothy J. Yeatman. BRAF expression is associated with poor survival in colorectal cancers independent of BRAF (V600E) mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3170.

The Notch signaling pathway plays a significant role in differentiation, proliferation, apoptosis, and stem cell processes. It is essential for maintenance of the normal colon crypt and has been implicated in colorectal cancer oncogenesis. Downregulation of the Notch pathway through gamma-secretase inhibitors (GSIs) has been shown to induce apoptosis and enhance response to chemotherapy in a variety of malignancies. In this study, we analyzed the effect of MRK-003 (Merck), a potent inhibitor of gamma-secretase, on oxaliplatin-induced apoptosis in colon cancer. Unexpectedly, gamma-secretase inhibition reduced oxaliplatin-induced apoptosis while GSI treatment alone was shown to have no effect on growth or apoptosis. We determined that the underlying mechanism of action involved an increase in protein levels of the anti-apoptotic Bcl-2 family members Mcl-1 and/or Bcl-xL which resulted in reduced Bax and Bak activation. Blocking of Mcl-1 and/or Bcl-xL through siRNA or the small molecule inhibitor obatoclax restored the apoptotic potential of cells treated with both oxaliplatin and MRK-003. Moreover, obatoclax synergized with MRK-003 alone to induce apoptosis. Our findings warrant caution when treating colon cancer with the combination of GSIs and chemotherapy, whereas other drug combinations, such as GSIs plus obatoclax, should be explored.

Alpha-fetoprotein (AFP)-secreting hepatocellular carcinomas (HCC) represent a genetically distinct subset of tumors often associated with a worse prognosis. However, the molecular mechanisms that underlie these phenotypic differences remain poorly understood. HCC tumor samples from 27 patients were profiled using the Affymetrix 133 Plus 2.0 GeneChips. GeneGO Metacore software was used to identify altered biologic pathways. Expression validation was confirmed by RT-PCR. Manipulation of miR-675 by overexpression and antagomir-mediated knockdown was carried out with subsequent evaluation of effects on cell behavior by cell cycle, proliferation, invasion, and growth in soft agar assays. We identified a strong relationship between primary tumor H19 gene expression and elevated serum AFP. H19 has recently been identified to encode microRNA-675 (miR-675), and we confirmed the relationship in an independent sample of patients. Pathway analyses of the effect of miR-675 overexpression in hepatoma cells revealed a predominant upregulation of cell adhesion and cell cycle initiation pathways. We have demonstrated that miR-675 mediates increases in proliferation and an accumulation of cells with tetraploid DNA content associated with a repression of Rb. We also demonstrated that overexpression of miR-675 alters cellular morphology, reduces invasive potential, and increases anchorage-independent growth capacity. These findings are consistent with a mesenchymal-to-epithelial transition, associated with a reduction in the expression of the key EMT mediator, Twist1. Expression of the miR-675 in hepatocellular carcinoma links a dramatic upregulation of proliferative and growth capacity with inhibition of motility in HCC cells.

De novo and acquired resistance, which are largely attributed to genetic alterations, are barriers to effective anti-epidermal-growth-factor-receptor (EGFR) therapy. To generate cetuximab-resistant cells, we exposed cetuximab-sensitive colorectal cancer cells to cetuximab in three-dimensional culture. Using whole-exome sequencing and transcriptional profiling, we found that the long non-coding RNA MIR100HG and two embedded microRNAs, miR-100 and miR-125b, were overexpressed in the absence of known genetic events linked to cetuximab resistance. MIR100HG, miR-100 and miR-125b overexpression was also observed in cetuximab-resistant colorectal cancer and head and neck squamous cell cancer cell lines and in tumors from colorectal cancer patients that progressed on cetuximab. miR-100 and miR-125b coordinately repressed five Wnt/β-catenin negative regulators, resulting in increased Wnt signaling, and Wnt inhibition in cetuximab-resistant cells restored cetuximab responsiveness. Our results describe a double-negative feedback loop between MIR100HG and the transcription factor GATA6, whereby GATA6 represses MIR100HG, but this repression is relieved by miR-125b targeting of GATA6. These findings identify a clinically actionable, epigenetic cause of cetuximab resistance.

Background The KRAS gene is mutated in about 40 % of colorectal cancer (CRC) cases, which has been clinically validated as a predictive mutational marker of intrinsic resistance to anti-EGFR inhibitor (EGFRi) therapy. Since nearly 60 % of patients with a wild type KRAS fail to respond to EGFRi combination therapies, there is a need to develop more reliable molecular signatures to better predict response. Here we address the challenge of adapting a gene expression signature predictive of RAS pathway activation, created using fresh frozen (FF) tissues, for use with more widely available formalin fixed paraffin-embedded (FFPE) tissues. Methods In this study, we evaluated the translation of an 18-gene RAS pathway signature score from FF to FFPE in 54 CRC cases, using a head-to-head comparison of five technology platforms. FFPE-based technologies included the Affymetrix GeneChip (Affy), NanoString nCounter™ (NanoS), Illumina whole genome RNASeq (RNA-Acc), Illumina targeted RNASeq (t-RNA), and Illumina stranded Total RNA-rRNA-depletion (rRNA). Results Using Affy_FF as the “gold” standard, initial analysis of the 18-gene RAS scores on all 54 samples shows varying pairwise Spearman correlations, with (1) Affy_FFPE (r = 0.233, p = 0.090); (2) NanoS_FFPE (r = 0.608, p

Background The Notch signalling pathway is activated in a variety of malignancies and has been implicated in colorectal cancer progression. One of the first steps in the Notch pathway activation is mediated by γ-secretase, a proteolytic enzyme which produces an activated intracellular Notch (ICN). RO4929097 is a selective inhibitor of γ-secretase. We tested the activity of RO4929097 in patients with metastatic, refractory colorectal cancer. Patients and methods Patients with metastatic colorectal cancer who had received at least two prior lines of systemic chemotherapy were enrolled on the study. Patients were treated with RO4929097 at its recommended phase II dose of 20 mg daily, 3 days on and 4 days off continuously. Cycle length was 28 days. Imaging was performed every two cycles. Archival tissue specimens were stained immunohistochemically for components of the notch pathway: Notch1, ICN and the downstream target HES1. Results Thirty-seven patients were enrolled of whom 33 were evaluable for toxicity and response. Immunohistochemical analysis of archival tissues demonstrated positive staining for the notch receptor as well as intracellular notch and the downstream gene HES1 in the majority of patients. Nevertheless, no objective radiographic responses were observed in this group and only six patients had stable disease as their best response. Median PFS was 1.8 months and median overall survival (OS) was 6.0 months. Conclusion In this study of RO4929097 in patients with refractory metastatic colorectal cancer, no radiographic responses were seen and time to progression was short, which suggests that RO4929097 at the study dose and schedule has minimal single agent activity in this malignancy.

Personalized cancer care means that a treatment is delivered to the patient in a very directed fashion. No longer is a one-size fits all approach acceptable. More importantly, the standard approach using non-targeted chemotherapy, treating many patients to help an unknown few, is rapidly falling in disfavor to therapies paired with unique biomarkers. Recent success in targeting patient subpopulations harboring RAF mutations has shown promise for accelerating the drug development process. Moreover, this success suggests the need for increased focus on cancer diagnostics. Biomarker discovery, however, has been a substantial challenge, limited by the availability of a large number of high quality, well-annotated specimens. Here, we describe the development of one of the world’s largest molecular databases of human cancer that will have a wide spectrum of utility in the years to come as the clinical data mature. The database supports a broad initiative in personalized medicine called “Total Cancer Care”. This represents a successful private/public partnership between and an academic medical center, a large network of community hospitals, and a large pharmaceutical company.

Aims Despite continual efforts to develop prognostic and predictive models of colorectal cancer by using clinicopathological and genetic parameters, a clinical test that can discriminate between patients with good or poor outcome after treatment has not been established. Thus, the authors aim to uncover subtypes of colorectal cancer that have distinct biological characteristics associated with prognosis and identify potential biomarkers that best reflect the biological and clinical characteristics of subtypes. Methods Unsupervised hierarchical clustering analysis was applied to gene expression data from 177 patients with colorectal cancer to determine a prognostic gene expression signature. Validation of the signature was sought in two independent patient groups. The association between the signature and prognosis of patients was assessed by Kaplan–Meier plots, log-rank tests and the Cox model. Results The authors identified a gene signature that was associated with overall survival and disease-free survival in 177 patients and validated in two independent cohorts of 213 patients. In multivariate analysis, the signature was an independent risk factor (HR 3.08; 95% CI 1.33 to 7.14; p=0.008 for overall survival). Subset analysis of patients with AJCC (American Joint Committee on Cancer) stage III cancer revealed that the signature can also identify the patients who have better outcome with adjuvant chemotherapy (CTX). Adjuvant chemotherapy significantly affected disease-free survival in patients in subtype B (3-year rate, 71.2% (CTX) vs 41.9% (no CTX); p=0.004). However, such benefit of adjuvant chemotherapy was not significant for patients in subtype A. Conclusion The gene signature is an independent predictor of response to chemotherapy and clinical outcome in patients with colorectal cancer.