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PURPOSE To characterize the relationship between tumor-infiltrating lymphocytes (TIL), tumor mutational burden (TMB), and genetic alterations in microsatellite stable (MSS), microsatellite instability (MSI), or mutant POLE/POLD1 colon cancer. MATERIALS AND METHODS Four hundred ninety-nine resected stage I-III colon tumors treated between 2014 and 2019 were assessed for TIL; somatic mutations, copy number alterations, and structural changes in > 400 oncogenes; and MSI status. RESULTS Of the 499 tumors analyzed, 313 were MSS, 175 were MSI, and 11 had POLE/POLD1 pathogenic mutations. Both the percentage of tumors with a high level of TIL (≥ 4 lymphocytes per high-power field) and the median TMB differed significantly between the three phenotypes: MSS, 4.5% and 6 mutations/Mb; MSI, 68% and 54 mutations/Mb; POLE/POLD1, 82% and 158 mutations/Mb ( P < .05). Within each phenotype, TMB did not vary significantly with TIL level. Among MSI tumors, the median number of frameshift indels was significantly higher in tumors with high levels of TIL (20 v 17; P = .018). In the MSS group, significantly higher proportions of tumors with high levels of TIL had mutations in the transforming growth factor-β (36% v 12%; P = .01), RAS (86% v 54%; P = .02), and Hippo (7% v 1%; P = .046) pathways; in contrast, TP53 alterations were associated with low levels of TIL (74% v 43%; P = .01). CONCLUSION The association between TIL, TMB, and genetic alterations varies significantly between MSI, MSS, and mutant POLE/POLD1 colon tumors. These differences may help explain tumoral immunity and lead to predictors of response to immunotherapy.

Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS‐mt) and KRAS‐wild‐type (KRAS‐wt) patients. We found that KRAS‐mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment., Oncogenic KRAS is associated with poor outcomes in locally advanced rectal cancer (LARC) but the underlying biologic mechanisms are not fully understood. We investigated differences in gene expression profiles between KRAS mutant and KRAS wild‐type LARC tumors. We found that KRAS mutant tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix.

This diagnostic study explores the diagnostic accuracy of differentially expressed miRNAs between stage I and stage II or III rectal cancers., Key Points Question Can the expression levels of microRNA (miRNA) in the primary tumor distinguish locally advanced disease among nonmetastatic rectal cancers? Findings This diagnostic study of 2 independent cohorts found that a set of 19 miRNAs can effectively distinguish locally advanced disease (stage II or III) among nonmetastatic rectal cancers. Within this set of miRNAs, 3 miRNA-mRNA functional interactions were identified by integrative transcriptome analysis. Meaning These results suggest that the identified miRNAs in this study have potential to serve as diagnostic biomarkers in rectal cancer., Importance Accurate clinical staging is important in rectal cancer because it determines the appropriate treatment and prognosis. Despite the use of multiple diagnostic imaging tools, it is sometimes difficult to clinically distinguish stage I tumors from stage II or III locally advanced disease. Identification of differentiating microRNAs (miRNAs) between these 2 groups may improve the clinical diagnostic power and provide insight into the biology of tumor progression. Objectives To investigate differences in the expression of miRNAs in stage I vs stage II or III rectal cancers and integrate matched mRNA profiling data to identify possible functional roles of these miRNAs. Design, Setting, and Participants The primary tumor specimens from patients who were enrolled in 2 prospective clinical trials between March 24, 2004, and November 16, 2012 (American College of Surgeons Oncology Group [ACOSOG] Z6041 and Timing of Rectal Cancer Response to Chemoradiation [TIMING]) were sequenced to arrive at a set of 127 cases (41 stage I and 86 stage II or III tumors) with matched miRNA and messenger RNA (mRNA) profiling data. These findings were also evaluated in an independent cohort of 127 patient specimens (29 stage I and 98 stage II or III tumors) from The Cancer Genome Atlas Rectum Adenocarcinoma (TCGA-READ) that also had matched miRNA and mRNA data. Data analysis was performed from September 1, 2019, to September 1, 2020. Main Outcomes and Measures Alterations in miRNA expression between stage I and stage II or III tumors and their potential gene targets. Results A total of 254 pretreatment rectal adenocarcinoma specimens were analyzed in this study as 2 distinct cohorts: 127 samples in the ACOSOG/TIMING (stage I group: 27 [66%] male; mean [SD] age, 64.4 [10.8] years; stage II or III group: 47 [55%] male; mean [SD] age, 57.0 [11.4] years), and another 127 samples from TCGA-READ (stage I group: 17 [59%] male; mean [SD] age, 63.6 [12.0] years; stage II or III group: 48 [49%] male; mean [SD] age, 64.5 [11.4] years). A total of 19 miRNAs were overexpressed in stage II or III vs stage I tumors in both cohorts. This miRNA signature had an excellent discriminative value for distinguishing stage II or III from stage I rectal tumors (area under the curve, 0.88; 95% CI, 0.83-0.94 in ACOSOG/TIMING cohort and area under the curve, 0.84; 95% CI, 0.77-0.91 in the TCGA-READ cohort). Integrative analysis revealed 3 miRNA-mRNA pairs that exhibited significant correlations in both cohorts: miR-31-5p-SATB2, miR-143-3p-KLF5, and miR-204-5p-EZR. Conclusions and Relevance This diagnostic study found that many of the dysregulated miRNAs in stage II or III vs stage I rectal cancers have biological implications for tumor progression. The results of this study suggest that these miRNAs could assist as diagnostic biomarkers to better identify patients with locally advanced rectal cancer.

Purpose To validate the finding of an association between single nucleotide polymorphisms (SNPs) and toxicity during chemoradiotherapy (CRT) in rectal cancer patients, in an independent population. Methods and Materials The cohort consisted of 165 patients who received CRT for rectal cancer from 2006 to 2012. Prospectively recorded toxicity information, graded according to the Common Terminology Criteria for Adverse Events version 3.0, was retrieved from the medical record. Additionally, a subset of 52 patients recorded their gastrointestinal symptoms weekly during CRT, using the 7-item Bowel Problems Scale. Deoxyribonucleic acid was extracted from normal tissue in the proctectomy specimens and screened for 3 SNPs: XRCC1 R399Q, XPD K751Q, and TGFβ1 R25P. Univariable and multivariable logistic regression models were constructed. Results The median radiation dose was 50.4 Gy, and all patients received concurrent chemotherapy. Toxicities measured by the Common Terminology Criteria for Adverse Events were closely associated with patient-reported outcomes for the patients who completed the 7-item Bowel Problems Scale. Grade ≥3 toxicity occurred during CRT in 14 patients (8%). All 14 patients had either XRCC1 R399Q or TGFβ1 R25P polymorphisms. The TGFβ1 R25P polymorphism was significantly associated with grade ≥3 toxicity (odds ratio [OR] 3.47, P =.04) and, in patients who completed the Bowel Problems Scale, with grade ≥4 toxicity (OR 5.61, P =.02). The latter finding persisted in a multivariable logistic regression model controlling for ethnicity, age, and sex (adjusted OR 1.83, P =.02). Conclusions We have validated the correlation between the TGFβ1 R25P SNP and acute toxicity during CRT in an independent cohort using both clinician- and patient-reported toxicity. The information from our study could be used as a basis to formulate a prospective trial testing the utility of this SNP as a biomarker of acute toxicity during neoadjuvant treatment in locally advanced rectal cancer.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation, and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic, or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation, and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to individual patients. We established a biorepository of 32 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic, or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and theirex vivoresponses to clinically relevant chemotherapy and radiation treatment correlate well with responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive rectal cancer followed by metastasis to lung and liver. Importantly, engrafted tumors closely reflected the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based,in vitroplatform coupled with endoluminal propagation in animals.

Locally advanced rectal cancer (LARC) is treated with chemoradiation prior to surgical excision, leaving residual tumors altered or completely absent. Integrating layers of genomic profiling might identify regulatory pathways relevant to rectal tumorigenesis and inform therapeutic decisions and further research. We utilized formalin-fixed, paraffin-embedded pre-treatment LARC biopsies (n=138) and compared copy number, mRNA, and miRNA expression with matched normal rectal mucosa. An integrative model was used to predict regulatory interactions to explain gene expression changes. These predictions were evaluated in vitro using multiple colorectal cancer cell lines. The Cancer Genome Atlas (TCGA) was also used as an external cohort to validate our genomic profiling and predictions. We found differentially expressed mRNAs and miRNAs that characterize LARC. Our integrative model predicted the upregulation of miR-92a, miR-182, and miR-221 expression to be associated with downregulation of their target genes after adjusting for the effect of copy number alterations. Cell line studies using miR-92a mimics and inhibitors demonstrate that miR-92a expression regulates IQGAP2 expression. We show that endogenous miR-92a expression is inversely associated with endogenous KLF4 expression in multiple cell lines, and that this relationship is also present in rectal cancers of TCGA. Our integrative model predicted regulators of gene expression change in LARC using pre-treatment FFPE tissues. Our methodology implicated multiple regulatory interactions, some of which are corroborated by independent lines of study, while others indicate new opportunities for investigation.

The study was undertaken to determine if microsatellite instability (MSI) status influences the composition of the tumor microbiome in primary colon carcinoma. 426 unique primary, stage I-III colon biopsy or resection specimens (234 right-sided, 192 left-sided) were sequenced by MSK-IMPACT, a large panel next generation sequencing (NGS) assay. MSI status [150 MSI-H; 276 microsatellite stable (MSS)] was determined using the MSISensor algorithm, which is clinically validated to detect MSI status from NGS data. We have developed a technique for identifying the presence of microbial species in tumor tissue being tested with NGS by analysis of non-human DNA read sequences. The method has been validated and determined to have comparable accuracy to reference microbial tests for both viruses and bacteria. We identified 13 bacterial species that are enriched in MSI-H colon carcinoma compared to MSS (odds ratio, 95% confidence interval greater than 1 with Bonferonni adjustment) (Table 1). We also observed that MSI-H is more common in right-sided tumors compared to left-sided tumors (53% vs. 13%, p=1.0E-4). To understand if sidedness of the tumor was a significant confounder, we performed stratified analysis of bacteria enrichment for right and left sided colon carcinoma. 8 of the 13 species remained significantly enriched in MSI-H tumors independent of sidedness; 9 of 13 in right-sided tumors; and 12 of 13 in left-sided tumors. Non-significant confidence intervals are underlined in Table 1. While Fusobacterium nucleatum has previously been reported to be enriched in MSI-H colon cancer, the broader spectrum of species enriched in MSI-H colon carcinoma we observed in our study has not been reported. The different microbiome suggests that either these bacteria play a role in development of MSI-H tumors or a unique immune environment exists in MSI-H tumors. The relationship between this unique microbiome and MSI status is the focus of further investigation. Table 1:Bacteria enriched in MSI-H colon cancerMSI-HMSI-HMSI-HMSSMSSMSSBacteria speciesLocationPositiveNegativeTotalPositiveNegativeTotalOdds ratioBonferroni adjusted 95% confidence intervalFusobacterium nucleatumTotal/right/left54/44/1096/81/15150/125/2538/20/18238/89/149276/109/1673.5/2.4/5.5(1.6-7.7)/(1.3-4.4)/(2.2-14.1)Bacteroides fragilisTotal/right/left47/35/12103/90/13150/125/2537/16/21239/93/146276/109/1672.9/2.3/6.4(1.3-6.5)/(1.2-4.4)/(2.6-15.9)Prevotella intermediaTotal/right/left27/22/5123/103/20150/125/2514/10/4262/99/163276/109/1674.1/2.1/10.2(1.4-12.5)/(1.01-4.7)/(2.5-41.1)Prevotella melaninogenicaTotal/right/left20/18/2130/107/23150/125/256/3/3270/106/164276/109/1676.9/5.9/4.8(1.5-31.9)/(1.7-20.8)/(0.8-30.0)Odoribacter splanchnicusTotal/right/left26/20/6124/105/19150/125/2513/5/8263/104/159276/109/1674.2/4.0/6.3(1.4-13.3)/(1.4-11.0)/(2.0-20.0)Selenomonas sputigenaTotal/right/left33/31/2117/94/23150/125/2522/15/7254/94/160276/109/1673.3/2.1/2.0(1.3-8.4)/(1.04-4.1)/(0.4-10.2)Bacteroides xylanisolvensTotal/right/left32/25/7118/100/18150/125/2521/8/13255/101/154276/109/1673.3/3.2/4.6(1.3-8.6)/(1.4-7.3)/(1.6-13.0)Prevotella denticolaTotal/right/left20/16/4130/109/21150/125/259/3/6267/106/161276/109/1674.6/5.2/5.1(1.2-17.2)/(1.5-18.3)/(1.3-19.6)Leptotrichia buccalisTotal/right/left17/13/4133/112/21150/125/256/5/1270/104/166276/109/1675.8/2.4/31.6(1.2-27.2)/(0.8-7.0)/(3.4-296.4)Prevotella sp. oral taxon 299Total/right/left14/12/2136/113/23150/125/252/1/1274/108/166276/109/16714.1/11.5/14.4(1.2-161.7)/(1.5-89.7)/(1.3-165.6)Porphyromonas gingivalisTotal/right/left13/10/3137/115/22150/125/252/0/2274/109/165276/109/16713.0/19.9/11.3(1.1-150.8)/(1.1-343.0)/(1.8-71.1)Streptococcus anginosusTotal/right/left19/16/3131/109/22150/125/2510/4/6266/105/161276/109/1673.9/3.9/3.7(1.1-14.1)/(1.2-11.9)/(0.9-15.7)Fretibacterium fastidiosumTotal/right/left15/13/2135/112/23150/125/256/3/3270/106/164276/109/1675.0/4.1/4.8(1.03-24.3)/(1.1-14.8)/(0.8-30.0) Citation Format: Chad Michael Vanderbilt, Ajaratu Keshinro, Chin-Tung Chen, Esther Babady, Michael F. Berger, Ahmet Zehir, Marc Ladanyi, Jaclyn F. Hechtman, Zsofia K. Stadler, Jinru Shia, Martin R. Weiser. Unique tumor microbiome in microsatellite instability high (MSI-H) colon carcinoma [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 6095.

192 Background: Rectal cancers are clinically different from colon cancers and have not yet been molecularly characterized due to the scarcity of pretreatment specimens. Discovery of molecular determinants of response to chemotherapy and radiation in patients with locally advanced rectal cancer (LARC, stage II and III) are needed to select those who can avoid surgery and benefit from watch-and-wait strategies. Methods: We profiled 371 pre-treatment specimens using targeted-exome sequencing of 468 cancer genes (n = 325), whole-exome sequencing (n = 100), and RNA-sequencing (n = 113). The targeted-sequencing cohort included patients with stage I (n = 44), II (n = 41), III (n = 176), and IV (n = 64) disease. Primary tumors were divided into lower (LR: 0-4 cm to anal verge, n = 62), middle (MR: 4-8 cm, n = 115), and upper rectum (UR: 8-12 cm, n = 107). We examined molecular determinants of complete response (CR) and relapse free survival (RFS) in LARC patients treated with chemoradiotherapy only (CRT: n = 39), induction chemotherapy + CRT (INCT: n = 87) and consolidation chemotherapy after CRT (CCNT: n = 63). Results: Among MSS cases, oncogenic gene and signaling pathway alterations did not vary by clinical stage. WNT pathway alterations, driven by APC mutations, were more frequent in the UR (89% UR v 86% MR v 60% LR, p < 0.001) while RTK/RAS alterations were more frequent in the LR (54% UR v 69% MR v 72% LR, p < 0.03). A set of genes enriched in mTOR signaling, G2M checkpoint, EMT transition, and DNA repair were overexpressed in the UR (FDR < 0.1). The 5-yr RFS rate for LARC was 75% (CI: 68%-82%) and 24% of the cases had a CR (n = 45). MSI cases had a higher rate of CR compared to MSS cases (50% v 23%, p = 0.07) and none relapsed (n = 8). KRAS-altered MSS tumors exhibited worse RFS in cases treated with CNCT (5-yr: 74% v 97%, p = 0.01), but not in cases treated with INCT (5-yr: 67% v 72%, p = 0.7). Conclusions: WNT alterations are more frequent in the UR while RTK/RAS alterations are more frequent in the LR, suggesting differences in tumor biology between proximal and distal rectal cancer. Further, we report correlations between distinct molecular profiles and response to treatment paradigms that could guide the design of future clinical trials.

Background Sorafenib is the standard systemic therapy for unresectable or recurrent hepatocellular carcinoma (HCC) but adds minimal increase in survival. Therefore, there is a great need to develop novel therapies for advanced or recurrent HCC. One emerging field of cancer treatment involves oncolytic viruses that specifically infect, replicate within, and kill cancer cells. In this study, we examined the ability of GLV-1h68, a recombinant vaccinia virus derived from the vaccine strain that was used to eradicate smallpox, to kill sorafenib-resistant (SR) HCC cell lines. Methods Four SR HCC cell lines were generated by repeated passage in the presence of sorafenib. Median inhibitory concentration was determined for all cell lines. The infectivity, viral replication, and cytotoxicity of GLV-1h68 were assayed for both parental and SR HCC cells. Results Infectivity increased in a time and concentration-dependent manner in all cell lines. All cell lines supported efficient replication of virus. No difference between the rates of cell death between the parental and SR cell lines was observed. Conclusion Our results demonstrate that the oncolytic vaccinia virus GLV-1h68 kills both parental and SR HCC cell lines efficiently. This study indicates that patients who have failed treatment with sorafenib remain viable candidates for oncolytic therapy.

602 Background: In liquid biopsy, analyses of circulating tumor DNA (ctDNA) in blood have been used as a tool in cancer screening, identifying tumor mutations and monitoring treatment responses/recurrences. Aberrant methylation of some genes have been associated with cancer. Two genes, BCAT1 and IKZF1, are highly methylated and specific to colorectal cancer (CRC) in blood. Detection of these two methylated genes in post-surgery samples also correlates with higher chance of recurrence. Here we investigated the presence of methylated BCAT1 and IKZF1 at multiple time-points for each patient. Methods: Plasma was extracted from blood within 4 hours of the blood collection. ctDNA were extracted from plasma and bisulphite converted using commercially available kits. Real-time qPCR was used to analyze the converted DNA in three replicates and deemed positive if at least one replicate detected either methylated BCAT1 or IKZF1. Quantification of both genes were determined using separate gene-specific standard curves. Results: Nine Stage II/III rectal cancer (RC) patients was enrolled into the study, with one pre-treatment blood draw and several subsequent draws collected throughout the treatment cycles. five patients tested positive for at least one of the methylated genes prior to treatment. Four of these five patients showed an immediate drop in detection for the assayed genes after one cycle of chemotherapy and either remains at low concentration or is undetectable through the rest of the treatment cycles. These patients either have partial or complete response to the treatment regimen. One patient with continuous high levels of methylated BCAT1 and IKZF1 post-treatment have recurred two months after surgery. Conclusions: Methylated BCAT1 and/or IKZF1 were detected in over 50% of the Stage II/III RC patients pre-treatment. This assay is sensitive to the effect of chemotherapy regimen. A significant number of these patients showed a partial/complete response to their treatment regimen and/or reduction in tumor burden, which is reflected in the loss of detection of these two genes. This study shows that this assay may be a viable tool to pair with Imaging for assessment of patient response to treatment regimen.

BackgroundThe response of rectal cancers to neoadjuvant chemoradiation (CRT) is variable, but tools to predict response remain lacking. We evaluated whether KRAS and TP53 mutations are associated with pathologic complete response (pCR) and lymph node metastasis after adjusting for neoadjuvant regimen.MethodsRetrospective analysis of 229 pretreatment biopsies from patients with stage II/III rectal cancer was performed. All patients received CRT. Patients received 0-8 cycles of FOLFOX either before or after CRT, but prior to surgical excision. A subset was analyzed to assess concordance between mutation calls by Sanger Sequencing and a next-generation assay.ResultsA total of 96 tumors (42%) had KRAS mutation, 150 had TP53 mutation (66%), and 59 (26%) had both. Following neoadjuvant therapy, 59 patients (26%) achieved pCR. Of 133 KRAS wild-type tumors, 45 (34%) had pCR, compared with 14 of 96 (15%) KRAS mutant tumors (p=.001). KRAS mutation remained independently associated with a lower pCR rate on multivariable analysis after adjusting for clinical stage, CRT-to-surgery interval and cycles of FOLFOX (OR 0.34; 95% CI 0.17-0.66, p&nbsp

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

The c-Met proto-oncogene encodes a receptor tyrosine kinase (TK) that promotes invasive tumor growth and metastasis. Recent studies show that the presence of c-Met gene amplification is predictive for selective c-Met TK inhibitors in gastric cancer and lung cancer. In this study, we utilized a highly quantitative PCR/ligase detection reaction technique to quantify c-Met gene copy number in primary colorectal cancer (CRC) (N=247), liver metastases (N=147), and paired normal tissues. We identified no differences in c-Met gene copy number between normal colonic mucosa and liver tissue. However, mean c-Met gene copy number was significantly elevated in CRC compared with normal mucosa (P0.001), and in liver metastases compared with normal liver (P0.001). Furthermore, a significant increase in c-Met was seen in liver metastases compared with primary CRC (P0.0001). c-Met gene amplification was observed in 2% (3/177) of localized cancers, 9% (6/70) of cancers with distant metastases (P0.02), and 18% (25/147) of liver metastases (P0.01). Among patients treated by liver resection, there was a trend toward poorer 3-year survival in association with c-Met gene amplification (P=0.07). Slight increases in c-Met copy number can be detected in localized CRCs, but gene amplification is largely restricted to Stage IV primary cancers and liver metastases. c-Met gene amplification is linked to metastatic progression, and is a viable target for a significant subset of advanced CRC.

Background: Treatment for locally advanced rectal cancer (LARC) consists of neoadjuvant chemoradiotherapy (NCRT) followed by radical excision. Patients with tumors carrying a mutant KRAS are less likely to respond to NCRT compared to KRAS wild type tumors. We hypothesized that an RNA-based signature differentiating KRAS mutant and wild type patients could serve as an indicator of the biological process associated with response to NCRT. We found that the RNA-based signature is enriched for stromal and immune genes. Furthermore, the stromal component of the signature is a predictor of response to NCRT. Methods: Tumors from 120 LARC patients enrolled in a multicenter phase 2 trial studying response to NCRT were tested for KRAS status by Sanger Sequencing or Memorial Sloan Kettering (MSK)-IMPACT assay and gene expression was quantified by sequencing. Colorectal cancer (CRC) patients from MSK (n = 95) and TCGA (n = 261), previously annotated for KRAS mutation status and gene expression, were used for validation. A KRAS-inducible mouse model and CRC patient-derived xenografts (PDXs) were utilized to determine the cell of origin for the gene expression signature. Stromal enrichment was assessed with the ESTIMATE stromal gene signature. Immunohistochemistry (IHC) was completed for Periostin (POSTN), a stromal marker from the RNA-signature. Variant Allele Frequency (VAF) was used to measure the abundance of KRAS, TP53 and Adenomatous Polyposis Coli (APC) mutant alleles in tumors, and was quantified by targeted exome sequencing with the MSK-IMPACT assay. Results: Analysis of the KRAS-associated gene signature showed significant stromal inactivation in KRAS mutant patients. The signature was validated in the MSK and TCGA cohorts. The stromal signature was recapitulated in a KRAS inducible mouse model. Human CRC PDXs in mouse indicated that the signature arose from murine stroma and not human epithelium. Consistent with the stromal signature, IHC for POSTN, a stromal marker, was significantly lower in the KRAS mutant tumors compared with the KRAS wild type tumors (p Conclusions: This study shows that a KRAS mutation in CRC is associated with a lower expression of a stromal signature and that this signature is derived from the tumor microenvironment. This study indicates that CRC KRAS mutant tumors and a stromal subtype are closely related. Understanding this relationship may play a key role in elucidating the mechanism by which a KRAS mutant tumor is resistant to standard therapy. Citation Format: Raphael Pelossof, Moshe Elkatebts, Oliver Chow, Lauren Fairchild, Kevin O’Rourke, Jesse J. Smith, Chin-Tung Chen, Samuel Brook, Maurizio Scaltriti, Jinru Shia, Philip Paty, Christina Leslie, Scott Lowe, Jose Baselga, Julio Garcia-Aguilar. KRAS mutation status is associated with stromal inactivation in colorectal cancer and predicts poor response to neoadjuvant chemoradiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4078.

628 Background: KRAS mutation in colorectal cancer (CRC) is characterized by an altered transcriptional profile when compared to wild-type KRAS tumors. The list of differentially expressed genes overlaps significantly with a stromal fibroblast activation (SFA) signature present across multiple carcinomas. We have reported low expression of SFA genes in KRAS mutant CRC compared to KRAS wild type tumors. Here we sought to confirm the variation of the SFA signature with KRAS mutation and infer its origin in the stromal component of the tumor using experimental models. Methods: The SFA signature was assessed in an inducible-KRAS murine CRC model using RNA-sequencing, and in a CRC cell line with and without a transduced KRAS mutant vector by microarray analysis. Finally, RNA-sequencing of CRC patient-derived xenografts (PDXs) was used to determine whether the SFA signature was being expressed in the tumor epithelium or the surrounding stroma by leveraging the ability to align sequenced reads to the mouse and human genomes separately. Results: The SFA signature was identified in the inducible-KRAS mouse model, matching human cohort observations of decreased SFA gene expression in KRAS mutant CRC. On the other hand, KRAS transduction did not recapitulate the SFA signature in a CRC cell line, suggesting that the presence of stroma may be required for the expression of the SFA signature. Finally, RNA-seq reads for SFA signature genes in CRC PDXs immediately after implantation aligned primarily to the human genome but in later passages of the same PDXs aligned only to the mouse genome. These data suggest that the SFA transcriptional program is associated with the stroma rather than the epithelial tumor cells. Conclusions: KRAS mutation in CRC is associated with a gene expression signature derived from the tumor stroma. These findings suggest that KRAS mutation in the epithelial tumor cells may impact the tumor microenvironment in CRC.

A main limitation of therapies that selectively target kinase signaling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of EGFR, is effective in a subset of KRAS wild type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here, we show for the first time that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance but resistant cells remained sensitive to combinatorial inhibition of EGFR and MEK (Figure 1). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60 % (6/10) of the cases (Table 1). KRAS mutant alleles were detectable in the blood of cetuximab treated patients as early as 10 months prior to radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months prior to radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

BackgroundThe early detection of cardiotoxicity and congestive heart failure (CHF) from anthracyclines and anti-HER2 agents is currently limited to measuring changes in left ventricular ejection fraction (LVEF) at arbitrary time points. This approach has limited sensitivity and specificity and has led to the investigation of putative biomarkers such as cardiac Troponin I (TnI), a highly specific marker of myocardial damage and C-reactive protein (CRP), a sensitive inflammatory marker. In a pre-planned analysis we investigated these as biomarkers of cardiotoxicity within a prospective study testing the feasibility of ddAC- followed by weekly P with T and L.Materials and MethodsPatients (pts) with HER2+ EBC enrolled at MSKCC and DF/HCC and received ddAC (A 60mg/m2 + C 600mg/m2) x 4 → weekly P (80mg/m2) x 12 + T + L (1000mg/day). T+L continued for a total of 1yr. At baseline pts had LVEF ≥50%. Pts with unstable angina, CHF, recent MI, uncontrolled arrhythmia, grade 3 QT prolongation were excluded. LVEF was assessed by MUGA scan at mths 0, 2, 6, 9 and 18. TnI and CRP were measured every 2 wks right before treatment (Rx) during ddAC-PTL, then at mths 6, 9 and 18. TnI was categorized as “undetectable” (< 0.06 ng/ml; MSKCC, 0.31ng/ml). Elevated CRP was defined as (>0.8mg/dl; MSKCC, >0.3mg/dl; DF/HCC). Investigators were blinded to these results until pts completed 18mth follow-up (F/U).ResultsFrom Apr 07- Apr 08, 95 pts were enrolled; 39/95 (41%) withdrew due to PTL toxicities (incl. 3 with asymptomatic LVEF (aLVEF) declines and 3 with CHF). Final biomarker results were available in 84 pts (88%) and 11 pts (12%) continue on study. During Rx, minimal elevations in TnI occurred in 55 pts (65%). One pt had ↑TnI above normal range with AC#4; MUGA 1 wk later was unchanged (LVEF 75%), but she died from sepsis during subsequent Rx without evidence of CHF. Elevations in TnI occurred only during chemoRx and no pt had a ↑TnI during TL or at 18mth F/U. Of 55pts with elevated TnI, 25 (45%) had aLVEF declines (3 ↓ ≥16%, 10 ↓ 10-15%, 12 ↓ 5 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3088.

METHODS: In 63 patients with CRC liver metastases, we quantified MET mRNA from microdissected tumor and adjacent normal liver by Real-Time RT-PCR. Using random primed cDNA and genespecific PCR, specimen threshold cycle was determined and standard curves generated. Samples were normalized using 18S RNA. Tumor MET gene overexpression was defined as mRNA levels above the median of normal adjacent liver. Overall survival (OS) was compared by Kaplan-Meier and Log-rank. Median follow-up for survivors was 44 months.

Abstract #2108 Background: DD q 2 weekly (w) AC → P + T x 1 year (y) has an acceptable safely profile w/ congestive heart failure (CHF) rate of 1/70 pts (Dang, JCO 2008). Lapatinib (L) is effective in HER2 (+) BC. We conducted a pilot study of dd AC → w P + T + L to determine its feasibility and cardiac safety. Methods: Enrolled pts had HER2 (+) BC; LVEF > 50%. Rx consisted of AC at 60/600 mg/m2 x 4 q 2 w (w/ pegfilgrastim 6 mg day 2) → P at 80 mg/m2 x 12 q w + T x 1 y; L (1000 mg daily beginning w/ P + T and continued x 1 y). MUGA is obtained at baseline and at months (mo) 2, 6, 9, and 18. Rx is considered feasible if 1) > 80% pts can complete the PTL phase without a dose delay or reduction and 2) the cardiac event rate (CHF or cardiac death) is < 4%. Pts can remain on-Rx w/ one dose reduction of L (1000 mg → 750 mg) for a G 3 event or < G 3 toxicity (unacceptable). Results: From March 2007 to April 2008, we enrolled 95 pts. Median (med) age was 45 years (range, 28-73). At a med follow-up of 7 months, 90 are evaluable. Of the 90 pts, 34 (37%) withdrew from study during the PTL phase; 29 for a 2nd event of G 3 or unacceptable < G 3 toxicities (15 G 3 diarrhea, 4 G 1/2 diarrhea, 1 G 3 rash, 2 G 2 rash, 1 G 3 dyspnea and also had G 3 diarrhea, 1 G 3 ↑QTc also had G 3 diarrhea, 1 G 3 ↑ALT also had G 3 diarrhea, 1 G 3 paronychia, 1 G 3 pneumonitis, 1 asymptomatic LVEF ↓, 1 myocarditis) and 5 for other reasons (2 personal reason, 1 PCP pneumonia, 1 progression, 1 P hypersensitivity). Overall, 25/90 (27%) pts had G 3 diarrhea and 31/90 (34%) pts required a dose reduction of lapatinib. Med LVEF at baseline is 67% (N=95), at mo 2 is 68% (N=90), at mo 6 is 65% (N=53), and mo 9 is 65% (N=28). To date there are no patient drop-outs due to significant LVEF declines after dd AC; one patient dropped during PTL out due to an asymptomatic LVEF decline. Discussion: L at 1000 mg/day is not feasible combined w/ weekly P and T by protocol stipulation (> 20% pts required L dose reduction) primarily due to excessive G 3 diarrhea. These results have led to the modification of Design 2 (Arm D) of ALTTO. We will report updated results. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2108.