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PURPOSE Clinical calculators and nomograms have been endorsed by the American Joint Committee on Cancer (AJCC), as they provide the most individualized and accurate estimate of patient outcome. Using molecular and clinicopathologic variables, a third-generation clinical calculator was built to predict recurrence following resection of stage I-III colon cancer. METHODS Prospectively collected data from 1,095 patients who underwent colectomy between 2007 and 2014 at Memorial Sloan Kettering Cancer Center were used to develop a clinical calculator. Discrimination was measured with concordance index, and variability in individual predictions was assessed with calibration curves. The clinical calculator was externally validated with a patient cohort from Washington University's Siteman Cancer Center in St Louis. RESULTS The clinical calculator incorporated six variables: microsatellite genomic phenotype; AJCC T category; number of tumor-involved lymph nodes; presence of high-risk pathologic features such as venous, lymphatic, or perineural invasion; presence of tumor-infiltrating lymphocytes; and use of adjuvant chemotherapy. The concordance index was 0.792 (95% CI, 0.749 to 0.837) for the clinical calculator, compared with 0.708 (95% CI, 0.671 to 0.745) and 0.757 (0.715 to 0.799) for the staging schemes of the AJCC manual's 5th and 8th editions, respectively. External validation confirmed robust performance, with a concordance index of 0.738 (95% CI, 0.703 to 0.811) and calibration plots of predicted probability and observed events approaching a 45° diagonal. CONCLUSION This third-generation clinical calculator for predicting cancer recurrence following curative colectomy successfully incorporates microsatellite genomic phenotype and the presence of tumor-infiltrating lymphocytes, resulting in improved discrimination and predictive accuracy. This exemplifies an evolution of a clinical calculator to maintain relevance by incorporating emerging variables as they become validated and accepted in the oncologic community.

The widespread use of tumor DNA mismatch repair (MMR) protein immunohistochemistry in gastrointestinal tract (GIT) carcinomas has unveiled cases where the MMR protein status differs between synchronous/metachronous tumors from the same patients. This study aims at examining the frequency, patterns and molecular etiologies of such inter-tumoral MMR discordances. We analyzed a cohort of 2159 colorectal cancer (CRC) patients collected over a 5-year period and found that 1.3% of the patients (27/2159) had ≥ 2 primary CRCs, and 25.9% of the patients with ≥ 2 primary CRCs (7/27) exhibited inter-tumoral MMR discordance. We then combined the seven MMR-discordant CRC patients with three additional MMR-discordant GIT carcinoma patients and evaluated their discordant patterns and associated molecular abnormalities. The 10 patients consisted of 3 patients with Lynch syndrome (LS), 1 with polymerase proofreading-associated polyposis (PAPP), 1 with familial adenomatous polyposis (FAP), and 5 deemed to have no cancer disposing hereditary syndromes. Their MMR discordances were associated with the following etiologies: (1) PMS2-LS manifesting PMS2-deficient cancer at an old age when a co-incidental sporadic MMR-proficient cancer also occurred; (2) microsatellite instability-driven secondary somatic MSH6-inactivation occurring in only one—and not all—PMS2-LS associated MMR-deficient carcinomas; (3) “compound LS” with germline mutations in two MMR genes manifesting different tumors with deficiencies in different MMR proteins; (4) PAPP or FAP syndrome-associated MMR-proficient cancer co-occurring metachronously with a somatic MMR-deficient cancer; and (5) non-syndromic patients with sporadic MMR-proficient cancers co-occurring synchronously/metachronously with sporadic MMR-deficient cancers. Our study thus suggests that inter-tumoral MMR discordance is not uncommon among patients with multiple primary GIT carcinomas (25.9% in patients with ≥ 2 CRCs), and may be associated with widely varied molecular etiologies. Awareness of these patterns is essential in ensuring the most effective strategies in both LS detection and treatment decision-making. When selecting patients for immunotherapy, MMR testing should be performed on the tumor or tumors that are being treated.

Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.

BACKGROUND: RAS mutations are prognostic for patients with metastatic colorectal cancer (mCRC). We investigated clinical, pathologic and survival differences based on RAS exon for patients with colorectal liver metastases (CRLM). METHODS: This retrospective, single-center study included patients with R0/R1 resection of CRLM from 1992–2016. Patients with unresected extrahepatic disease or liver-first resection were excluded. Overall survival (OS) and recurrence-free survival (RFS) were assessed and stratified by mutation status and location. Fisher’s exact test, Wilcoxon Rank Sum test, and Log-rank test were used, where appropriate. RESULTS: 938 mCRC patients were identified with median age of 57 (range 19–91). Of the 445 patients with KRAS mutations, 407 (91%) had a mutation in exon 2, 14 (3%) exon 3, and 24 (5%) exon 4. Median OS was 71.4 months (95%CI:66.1–76.5). Patients with KRAS mutations had worse OS compared to KRAS wild-type patients (median 55.5 versus 91.3 months, p

The aim of this study was to determine whether intraprocedural 18F-FDG PET/CT can be used as a predictor of local tumor progression after percutaneous ablation of colorectal liver metastases. Methods: In this institutional review board–approved prospective study, 39 patients (19 men and 20 women; median age, 56 y) underwent split-dose 18F-FDG PET/CT-guided ablation followed by immediate biopsy and contrast-enhanced CT imaging of the ablation zone. Binary categorization of biopsy tissues was performed on the basis of the presence of only nonviable coagulation necrosis or viable tumor cells. Minimum ablation margin measurements from contrast-enhanced CT imaging were categorized as 0 mm, 1–4 mm, 5–9 mm, or greater than or equal to 10 mm. SUVs were obtained from PET/CT imaging, and SUV ratios were calculated from 3-dimensional regions of interest located in the ablation zone and surrounding normal liver. All predictive variables (biopsy, minimum margin distance, and SUV ratio) were evaluated as predictors of time to local tumor progression identified on imaging using competing-risks regression models (uni- and multivariate analyses). Results: A total of 62 consecutive ablations were evaluated. The mean SUV ratio was significantly higher for viable tumor–positive immediate postablation biopsies (n = 10) than for tumor-negative biopsies (n = 52) (85.8 ± 92.2 vs. 42.3 ± 45.5) (P = 0.03) and for a minimum margin size of less than 5 mm (n = 15) than for a minimum margin size of greater than or equal to 5 mm (n = 47) (78.5 ± 99.1 vs. 38.3 ± 78.5) (P = 0.01). After a median follow-up period of 22.5 (range, 7–52) months, 23 of 62 ablated tumors showed local tumor progression (37.1%). The local tumor progression rate was significantly higher for viable tumor–positive biopsies (8/10) than for negative biopsies (15/52) (80% vs. 29%) (P = 0.001) and for a minimum margin size of less than 5 mm (9/15) than for a minimum margin size of greater than or equal to 10 mm (2/15) (60% vs. 13%) (P = 0.02) but not 5–9 mm (37.5%; 12/32) (P = 0.5). In a competing-risks analysis, biopsy results (P = 0.07) and the minimum margin size (P = 0.08) were borderline significant, but the SUV ratio was not (P = 0.22). However, for negative biopsy ablations, the minimum margin size and SUV ratio were predictive imaging factors for local tumor progression; subdistribution hazard ratios were 0.564 (0.325–0.978) (P = 0.04) and 1.005 (1.001–1.009) (P = 0.005), respectively. Conclusion: The SUV ratio and minimum margin size can independently predict colorectal metastasis local tumor progression after liver ablation when there are no viable tumor cells on immediate postablation biopsies.

The incidence of esophagogastric cancer is rapidly rising, but only a minority of patients derive durable benefit from current therapies. Chemotherapy as well as anti-HER2 and PD-1 antibodies are standard treatments. To identify predictive biomarkers of drug sensitivity and mechanisms of resistance, we implemented prospective tumor sequencing of patients with metastatic esophagogastric cancer. There was no association between homologous recombination deficiency defects and response to platinum-based chemotherapy. Patients with microsatellite instability–high tumors were intrinsically resistant to chemotherapy but more likely to achieve durable responses to immunotherapy. The single Epstein–Barr virus–positive patient achieved a durable, complete response to immunotherapy. The level of ERBB2 amplification as determined by sequencing was predictive of trastuzumab benefit. Selection for a tumor subclone lacking ERBB2 amplification, deletion of ERBB2 exon 16, and comutations in the receptor tyrosine kinase, RAS, and PI3K pathways were associated with intrinsic and/or acquired trastuzumab resistance. Prospective genomic profiling can identify patients most likely to derive durable benefit to immunotherapy and trastuzumab and guide strategies to overcome drug resistance. Significance: Clinical application of multiplex sequencing can identify biomarkers of treatment response to contemporary systemic therapies in metastatic esophagogastric cancer. This large prospective analysis sheds light on the biological complexity and the dynamic nature of therapeutic resistance in metastatic esophagogastric cancers. Cancer Discov; 8(1); 49–58. ©2017 AACR. See related commentary by Sundar and Tan, p. 14. See related article by Pectasides et al., p. 37. This article is highlighted in the In This Issue feature, p. 1

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5+ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5+ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.

A plethora of tests are routinely ordered and interpreted by pathologists to assist the management of colorectal cancer patients. Many of these tests are immunohistochemistry assays using antibodies against prognostically relevant proteins, some of which predict therapeutic response. This review focuses on tissue DNA-based tests. It presents novel methodologies for assessing well-established biomarkers, updates the expanding spectrum of genetic alterations that are associated with resistance to inhibition of epidermal growth factor receptor signaling, and briefly discusses emerging actionable alterations that may translate into new therapeutic options for colorectal cancer patients. The utility of next-generation sequencing is emphasized.

BRAF V600E colorectal cancers are insensitive to RAF inhibitor monotherapy due to feedback reactivation of receptor tyrosine kinase signaling. Combined RAF and EGFR inhibition exerts a therapeutic effect, but resistance invariably develops through undefined mechanisms. In this study, we determined that colorectal cancer progression specimens invariably harbored lesions in elements of the RAS–RAF–MEK–ERK pathway. Genetic amplification of wild-type RAS was a recurrent mechanism of resistance in colorectal cancer patients that was not seen in similarly resistant melanomas. We show that wild-type RAS amplification increases receptor tyrosine kinase-dependent activation of RAS more potently in colorectal cancer than in melanoma and causes resistance only in the former. Currently approved RAF inhibitors inhibit RAF monomers but not dimers. All the drug-resistant lesions we identified activate BRAF V600E dimerization directly or by elevating RAS-GTP. Overall, our results show that mechanisms of resistance converge on formation of RAF dimers and that inhibiting EGFR and RAF dimers can effectively suppress ERK-driven growth of resistant colorectal cancer. Cancer Res; 77(23); 6513–23. ©2017 AACR.

Perineural invasion (PNI) is an ominous event strongly linked to poor clinical outcome. Cells residing within peripheral nerves collaborate with cancer cells to enable PNI, but the contributing conditions within the tumor microenvironment are not well understood. Here, we show that CCR2-expressing inflammatory monocytes (IM) are preferentially recruited to sites of PNI, where they differentiate into macrophages and potentiate nerve invasion through a cathepsin B–mediated process. A series of adoptive transfer experiments with genetically engineered donors and recipients demonstrated that IM recruitment to nerves was driven by CCL2 released from Schwann cells at the site of PNI, but not CCL7, an alternate ligand for CCR2. Interruption of either CCL2–CCR2 signaling or cathepsin B function significantly impaired PNI in vivo. Correlative studies in human specimens demonstrated that cathepsin B–producing macrophages were enriched in invaded nerves, which was associated with increased local tumor recurrence. These findings deepen our understanding of PNI pathogenesis and illuminate how PNI is driven in part by corruption of a nerve repair program. Further, they support the exploration of inhibiting IM recruitment and function as a targeted therapy for PNI. Cancer Res; 77(22); 6400–14. ©2017 AACR.

Microsatellite instability (MSI), a well-established driver pathway in colorectal carcinogenesis, can develop in both sporadic and hereditary conditions via different molecular alterations in the DNA mismatch repair (MMR) genes. MMR protein immunohistochemistry (IHC) is currently widely used for the detection of MMR deficiency in solid tumors. The IHC test, however, can show varied staining patterns, posing challenges in the interpretation of the staining results in some cases. Here we report a case of an 80-year-old female with a colonic adenocarcinoma that exhibited an unusual “null” IHC staining pattern with complete loss of all 4 MMR proteins (MLH1, MSH2, MSH6, and PMS2). This led to subsequent MLH1 methylation testing and next generation sequencing which demonstrated that the loss of all MMR proteins was associated with concurrent promoter hypermethylation of MLH1 and double somatic truncating mutations in MSH2. These molecular findings, in conjunction with the patient's age being 80 years and the fact that the patient had no personal or family cancer history, indicated that the MMR deficiency was highly likely sporadic in nature. Thus, the stringent Lynch syndrome type surveillance programs were not recommended to the patient and her family members. This case illustrates a rare but important scenario where a null IHC phenotype signifies complex underlying molecular alternations that bear clinical management implications, highlighting the need for recognition and awareness of such unusual IHC staining patterns.

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically defined tumor types, coupled with an expanding portfolio of molecularly targeted therapies, demands flexible and comprehensive approaches to profile clinically relevant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative using a comprehensive assay, MSK-IMPACT, through which we have compiled tumor and matched normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel noncoding alterations, and mutational signatures that were shared by common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

// Efsevia Vakiani 1 , Ronak H. Shah 2 , Michael F. Berger 1, 2 , Alvin P. Makohon-Moore 3 , Johannes G. Reiter 4 , Irina Ostrovnaya 5 , Marc A. Attiyeh 3 , Andrea Cercek 6 , Jinru Shia 1 , Christine A. Iacobuzio-Donahue 1, 3 , David B. Solit 2 and Martin R. Weiser 7 1 Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA 2 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA 3 The David Rubenstein Pancreatic Cancer Research Center, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA 4 Program for Evolutionary Dynamics, Harvard University, Cambridge, MA, 02138, USA 5 Department of Epidemiology and Biostatistics, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA 6 Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA 7 Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY, 10065, USA Correspondence to: Efsevia Vakiani, email: vakianie@mskcc.org Keywords: next-generation sequencing, colorectal cancer, anastomotic recurrence Received: October 25, 2016 Accepted: April 07, 2017 Published: April 18, 2017 ABSTRACT Purpose: Anastomotic recurrences (AR) occur in 2–10% of colorectal carcinoma cases after resection of primary tumor (PT). Currently, there are no molecular data investigating their genetic profile and multiple theories exist about their pathogenesis. The aim of our study was to compare the genomic profile of AR to that of the patients’ corresponding matched PT and, when available, to a distant metastasis (DM). Experimental Design: Thirty-six tumors from 14 patients were genotyped using a capture-based, next-generation assay to define the mutational status of 341 cancer-associated genes. All patients had R0 resection of their PT and AR occurred 1.1−7.0 years following PT resection. A DM or a second AR was analyzed in 8 patients. All tumors were microsatellite stable except in one patient with Lynch syndrome. Results: A total of 254 somatic mutations were detected including 138 mutations in the microsatellite stable (MSS) cases. The most commonly mutated genes were APC , KRAS , TP53 , PIK3CA , ATM and PIK3R1 . In all patients with MSS tumors the AR and PT shared between 50−100% of mutations, including mutations in key driver genes, consistent with these tumors being clonally related. Genetic events private to DM were not detected in AR and phylogenetic analysis showed that ARs were more closely related to PT than DM. In the Lynch syndrome patient the PT and AR showed distinct somatic mutations consistent with independent primaries. Conclusions: ARs are clonally related to PT in sporadic colorectal carcinomas and do not appear to represent seeding of the anastomotic site by distant metastases.

The-Cancer-Genome-Atlas data on colorectal carcinoma have provided a comprehensive view of the tumor’s genomic alterations and their tumorigenic roles. Tumor morphology, however, has not been fully integrated into the analysis. The aim of this study was to explore relevant associations between tumor morphology and the newly characterized genomic alterations in colorectal carcinoma. Two-hundred-and-seven colorectal carcinomas that had undergone whole exome sequencing as part of The-Cancer-Genome-Atlas project and had adequate virtual images in the cBioPortal for Cancer Genomics http://www.cbioportal.org/ constituted our study population. Upon analysis, a tight association between “microsatellite instability-high histology” and microsatellite instability-high (p

Perineural invasion (PNI) is an ominous form of cancer progression along nerves associated with poor clinical outcome. Glial derived neurotrophic factor (GDNF) interacts with cancer cell RET receptors to enable PNI, but downstream events remain undefined. We demonstrate that GDNF leads to early activation of the GTPase Cdc42 in pancreatic cancer cells, but only delayed activation of RhoA and does not affect Rac1. Depletion of Cdc42 impairs pancreatic cancer cell chemotaxis toward GDNF and nerves. An siRNA library of guanine nucleotide exchange factors was screened to identify activators of Cdc42. ARHGEF7 (β-Pix) was required for Cdc42 activation and chemotaxis toward nerves, and also colocalizes with RET under GDNF stimulation. Cdc42 enables PNI in an in vitro dorsal root ganglia coculture model, and controls the directionality of migration but does not affect cell speed or cell viability. In contrast, Rac1 was necessary for cell speed but not directionality, while the RhoA was not necessary for either cell speed or directionality. Cdc42 was required for PNI in an in vivo murine sciatic nerve model. Depletion of Cdc42 significantly diminished the length of PNI, volume of PNI, and motor nerve paralysis resulting from PNI. Activated Cdc42 is expressed in human salivary ductal cancer cells invading nerves. These findings establish the GDNF–RET–β-Pix–Cdc42 pathway as a directional regulator of pancreatic cancer cell migration toward nerves, highlight the importance of directional migration in PNI, and offer novel targets for therapy. Implications: Cdc42 regulates cancer cell directional migration toward and along nerves in PNI.

Most patients with KRASG12C–mutant non–small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRASG12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRASG12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRASG12C colorectal cancer. Significance: The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors. See related commentary by Koleilat and Kwong, p. 1094. This article is highlighted in the In This Issue feature, p. 1079

Importance Diagnosing the site of origin for cancer is a pillar of disease classification that has directed clinical care for more than a century. Even in an era of precision oncologic practice, in which treatment is increasingly informed by the presence or absence of mutant genes responsible for cancer growth and progression, tumor origin remains a critical factor in tumor biologic characteristics and therapeutic sensitivity. Objective To evaluate whether data derived from routine clinical DNA sequencing of tumors could complement conventional approaches to enable improved diagnostic accuracy. Design, Setting, and Participants A machine learning approach was developed to predict tumor type from targeted panel DNA sequence data obtained at the point of care, incorporating both discrete molecular alterations and inferred features such as mutational signatures. This algorithm was trained on 7791 tumors representing 22 cancer types selected from a prospectively sequenced cohort of patients with advanced cancer. Results The correct tumor type was predicted for 5748 of the 7791 patients (73.8%) in the training set as well as 8623 of 11 644 patients (74.1%) in an independent cohort. Predictions were assigned probabilities that reflected empirical accuracy, with 3388 cases (43.5%) representing high-confidence predictions (>95% probability). Informative molecular features and feature categories varied widely by tumor type. Genomic analysis of plasma cell-free DNA yielded accurate predictions in 45 of 60 cases (75.0%), suggesting that this approach may be applied in diverse clinical settings including as an adjunct to cancer screening. Likely tissues of origin were predicted from targeted tumor sequencing in 95 of 141 patients (67.4%) with cancers of unknown primary site. Applying this method prospectively to patients under active care enabled genome-directed reassessment of diagnosis in 2 patients initially presumed to have metastatic breast cancer, leading to the selection of more appropriate treatments, which elicited clinical responses. Conclusions and Relevance These results suggest that the application of artificial intelligence to predict tissue of origin in oncologic practice can act as a useful complement to conventional histologic review to provide integrated pathologic diagnoses, often with important therapeutic implications.

Aim:\ud Significant recent changes in management of locally advanced rectal cancer include preoperative staging, use of extended neoadjuvant therapies, and minimally invasive surgery (MIS). This study was aimed at characterizing those changes and associated short‐term outcomes.\ud \ud Method:\ud We retrospectively analysed treatment and outcome data from patients with T3/4 or N+ locally advanced rectal cancer ≤15 cm from the anal verge who were evaluated at a comprehensive cancer center in 2009–2015.\ud \ud Results:\ud In total, 798 patients were identified and grouped into five cohorts based on treatment year: 2009‐2010, 2011, 2012, 2013, and 2014‐2015. Temporal changes included increased reliance on MRI staging, from 57% in 2009‐2010 to 98% in 2014‐2015 (p < 0.001); increased use of total neoadjuvant therapy, from 17% to 76% (p < 0.001); and increased use of MIS, from 33% to 70% (p < 0.001). Concurrently, median hospital stay decreased (from 7 to 5 days; p < 0.001), as did the rates of grade III‐V complications (from 13% to 7%; p < 0.05), surgical site infections (from 24% to 8%; p < 0.001), anastomotic leak (from 11% to 3%; p < 0.05), and positive circumferential resection margin (from 9% to 4%; p < 0.05). TNM downstaging increased from 62% to 74% (p = 0.002).\ud \ud Conclusion:\ud Shifts toward MRI‐based staging, total neoadjuvant therapy, and MIS occurred between 2009 and 2015. Over the same period, treatment responses improved, and lengths of stay and the incidence of complications decreased.

Several molecular tests are performed routinely for the management of the upper GI adenocarcinomas and colorectal cancer (CRC), including testing for microsatellite instability, KRAS and BRAF mutations and HER2 gene amplifications. Other molecular biomarkers are emerging as clinically important as new resistance mechanisms are identified and/or as new targeted therapies are developed. In gastrointestinal stromal tumors (GISTs), molecular tests are critical to classify GISTs in distinct groups that can be matched to specific therapies. Although the majority of molecular tests aim at the detection of sporadic genetic alterations, germline testing for pathogenic genetic aberrations is also very important in patients with a family history and/or early onset of cancer. This chapter discusses established and emerging molecular biomarkers in CRC, upper GI adenocarcinomas, and GISTs. It also summarizes common hereditary GI cancer syndromes and the genetic alterations that underlie them.

BackgroundThe Memorial Sloan Kettering Cancer Center (MSK) colon cancer recurrence nomogram is a risk calculator that provides patients and clinicians with individualized prediction of recurrence following curative resection of colon cancer. Although validated on multiple separate cohorts, the nomogram requires periodic updating as patient care changes over time. The aim of this study was to evaluate the nomogram’s accuracy in a contemporary cohort and modify the tool to reflect improvements in outcome related to advances in colon cancer therapy.MethodsA contemporary patient cohort was compiled, including consecutive colon cancer patients undergoing curative resection for stage I–III colon adenocarcinoma at MSK from 2007 to 2014. The nomogram’s predictive accuracy was assessed by concordance index and calibration plots of predicted vs actual freedom from recurrence at 5 years after surgery.ResultsData from a total of 999 eligible patients with complete records were used for validation. Median follow-up among survivors was 37 months. The concordance index was 0.756 (95% confidence interval = 0.707 to 0.805), indicating continued discriminating power, but the calibration plot revealed that the nomogram overestimated recurrence risk. Recalibration of the nomogram by estimating a new baseline freedom-from-recurrence function restored the nomogram’s accuracy.ConclusionThe updated nomogram retains the original nomogram’s variables but includes a lower baseline estimation of recurrence risk, reflecting improvements in outcomes for all stages of colon cancer, likely resulting from advances in imaging and integration of multiple treatment modalities.

BACKGROUND Ovarian metastases from colorectal cancer (OM-CRC) often are unresponsive to chemotherapy and are associated with poor survival. To the authors' knowledge, the clinicopathologic and genomic predictors of OM-CRC are poorly characterized and optimal clinical management remains unclear. METHODS Women with a histopathological diagnosis of OM-CRC who were treated at Memorial Sloan Kettering Cancer Center from 1999 to 2015 were identified. Next-generation somatic mutation profiling (Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets [MSK-IMPACT]) was performed on 38 OM-CRC cases, including 21 matched tumor pairs/trios. Regression models were used to analyze variables associated with progression-free survival and overall survival (OS). RESULTS Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS), SMAD family member 4 (SMAD4), and neurotrophic receptor tyrosine kinase 1 (NTRK1) mutations were more frequent in cases of OM-CRC than in instances of CRC occurring without OM. SMAD4 and lysine methyltransferase 2D (KMT2D) mutations were associated with reduced OS. Matched multisite tumor sequencing did not identify OM-specific genomic alterations. Of the 195 patients who underwent oophorectomy for OM-CRC (median age, 49 years with a progression-free survival of 9.4 months and an OS of 23 months from oophorectomy), 76% had extraovarian metastasis (EOM). In multivariable analysis, residual disease after surgery (R2 resection) was associated with worse survival. Patients with EOM were less likely to achieve R0/R1 surgical resection status (complete macroscopic resection without clinical/radiological evidence of disease) (48% vs 94%). However, if R0/R1 resection status was achieved, both patients with (35.9 months vs 12 months) and without (43.2 months vs 14.5 months) EOM were found to have better OS. Among 114 patients with R0/R1 resection status, 23 (20%) had no disease recurrence, including 10 patients (9%) with > 3 years of follow-up. CONCLUSIONS Loss-of-function alterations in SMAD4 are frequent and predictive of worse survival in patients with OM-CRC. Similar to oligometastatic CRC to the lung or liver, surgical resection of OM-CRC is associated with a better outcome only if all macroscopic metastatic disease is resected. Cancer 2016. © 2016 American Cancer Society.

Immune checkpoint blockade targeting the programmed death-1 (PD-1) pathway has shown efficacy in several types of cancers including mismatch-repair-deficient colorectal carcinoma. In some tumor types, programmed death-ligand 1 (PD-L1) expression detected by immunohistochemistry has shown utility as a predictive marker for response to anti-PD-1 therapies. This utility, however, remains to be determined in colorectal carcinoma. In addition, although tumor-infiltrating lymphocytes have been associated with better prognosis in colorectal carcinoma, the prognostic value of PD-1 expression in these lymphocytes and its interaction with PD-L1 expression still await investigation. To address these questions, we performed a pilot study to evaluate the patterns of PD-L1 and PD-1 immunohistochemical expression on colorectal carcinoma cells and their tumor-infiltrating lymphocytes, respectively. Using tissue microarray, we found that 5percnt; (19sol;394) of colorectal carcinomas exhibited high tumor PD-L1 expression, and 19percnt; (76sol;392) had elevated numbers of PD-1-positive tumor-infiltrating lymphocytes. PD-L1 levels correlated with PD-1 levels (Plt;0.001), and mismatch-repair-deficient tumors had significantly higher rates of high PD-L1 and PD-1 expression when compared with mismatch-repair-proficient tumors (18percnt; vs 2percnt; and 50percnt; vs 13percnt;, respectively; Plt;0.001 for both). Staining intensity was also stronger for both markers in mismatch-repair-deficient tumors. Furthermore, we observed that among patients with mismatch-repair-deficient colorectal carcinoma, PD-1sol;PD-L1 expression stratified recurrence-free survival in an inter-dependent manner: an association between high PD-1-positive tumor-infiltrating lymphocytes and improved recurrence-free survival (Pequals;0.041) was maintained only when the tumors had low-level PD-L1 expression (Pequals;0.006); patients whose tumors had both high PD-1-positive tumor-infiltrating lymphocytes and high PD-L1 expression had a significantly worse recurrence-free survival (Plt;0.001). Thus, our results not only provide a foundation for further assessment of PD-L1 immunohistochemistry as a predictive marker for anti-PD-1 therapy in colorectal carcinoma, they also shed light on the prognostic impact of tumor-infiltrating lymphocytes in different subsets of mismatch-repair-deficient colorectal carcinomas.

Background and Objectives Achieving a pathologic complete response (pCR) after neoadjuvant therapy has been associated with better prognosis in rectal cancer patients. The objective of this study was to investigate the relationship between distance to the anal verge (DTAV) and pCR. Methods Review of a prospectively maintained database of patients with locally advanced rectal cancer who received neoadjuvant treatment was completed. Uni- and multivariate analysis assessed the association between DTAV and pCR after neoadjuvant therapy. Results Of 827 included patients, 20% had a pCR. We found that pCR rates were 11% for tumors 10 cm from the anal verge (P = 0.002). Multivariate analysis also showed a strong association between DTAV and pCR (P = 0.008). The bimodal distribution of pCR resulted in a lower odds ratio of pCR for tumors 8 cm from the anal verge. Conclusions Patients with low tumors ( 8 cm), were less likely to have a pCR. Further investigation is warranted to determine if these observations are related to tumor biology or possibly differences in radiation technique. J. Surg. Oncol. 2016;114:637–641. © 2016 Wiley Periodicals, Inc.

ARID1A is a chromatin remodeling gene that is mutated in a number of cancers including colorectal carcinoma (CRC). Loss of ARID1A has been associated with an adverse outcome in some types of cancer. However, literature data have not been consistent. Major limitations of some outcome studies include small sample size and heterogeneous patient population. In this study, we evaluated the prognostic value of ARID1A in a homogeneous group of early stage CRC patients, a population where prognostic markers are particularly relevant. We collected a retrospective series of 578 stage I or II CRCs. All patients underwent surgery with curative intent and without neoadjuvant or adjuvant therapy. ARID1A expression was analyzed by immunohistochemistry using tissue microarray. We found ARID1A loss in 49 of 552 analyzable tumors (8.9%). Compared with the ARID1A-retained group, cases with ARID1A loss were associated with female sex (P < .001), mismatch-repair protein deficiency (P < .001), poor differentiation (P < .001), lymphovascular invasion (P = .001), and higher pT stage (P = .047). However, at a median follow-up of 49 months, ARID1A loss did not correlate with overall, disease-specific, or recurrence-free survival. This is the first systematic analysis to evaluate the prognostic significance of ARID1A in stage I/II CRCs, and our data indicate that ARID1A loss lacks prognostic significance in this population despite its association with other adverse features. Such data are clinically relevant, as efforts are ongoing in identifying markers that can detect the small but significant subset of early stage CRCs that will have a poor outcome.

Background: Emerging evidence suggests differences in colorectal cancer in HIV-infected patients (HIV+) compared with HIV− patients. Microsatellite instability (MSI), occurring in a subset of colorectal cancer, is present at a higher rate in certain cancers in HIV+ patients. Colorectal cancer with MSI share some characteristics with those reported for HIV+ colorectal cancer. On this premise, we studied clinical and pathologic features of HIV+ colorectal cancer and evaluated for MSI using matched HIV− colorectal cancer controls. Methods: Two nested, matched cohorts were identified from a hospital-based cohort of colorectal cancer patients. HIV+ colorectal cancers were identified and random control patients were matched for selected characteristics. Mismatch repair protein (MMR) IHC was performed as the detection method for MSI. Variables were compared between cases and controls using fixed-effects logit modeling to account for matching. Results: We included 184 colorectal cancer samples (38 HIV+, 146 HIV− control). Median patient age at colorectal cancer onset was 55. When compared with HIV− colorectal cancer, HIV+ patients were more likely to have smoked (P = 0.001), have right-sided colorectal cancer (37% vs. 14%; P = 0.003), and tumor-infiltrating lymphocytes (TIL) above 50/10 high-power fields (21% vs. 7%). There was no difference in MMR protein expression (P = 0.6). HIV+ colorectal cancer patients had reduced overall survival (P = 0.02) but no difference in progression-free survival. Conclusions: HIV+ patients developed colorectal cancer at a lower median age than population estimates, had a higher frequency of right-sided disease, and increased TILs, suggesting potential biologic differences compared with uninfected patients. Impact: Clinicopathologic differences in colorectal cancer of HIV+ persons may have implications for tumor pathogenesis. Cancer Epidemiol Biomarkers Prev; 25(7); 1098–104. ©2016 AACR.

Nerves enable cancer progression, as cancers have been shown to extend along nerves through the process of perineural invasion, which carries a poor prognosis. Furthermore, the innervation of some cancers promotes growth and metastases. It remains unclear, however, how nerves mechanistically contribute to cancer progression. Here, we demonstrated that Schwann cells promote cancer invasion through direct cancer cell contact. Histological evaluation of murine and human cancer specimens with perineural invasion uncovered a subpopulation of Schwann cells that associates with cancer cells. Coculture of cancer cells with dorsal root ganglion extracts revealed that Schwann cells direct cancer cells to migrate toward nerves and promote invasion in a contact-dependent manner. Upon contact, Schwann cells induced the formation of cancer cell protrusions in their direction and intercalated between the cancer cells, leading to cancer cell dispersion. The formation of these processes was dependent on Schwann cell expression of neural cell adhesion molecule 1 (NCAM1) and ultimately promoted perineural invasion. Moreover, NCAM1-deficient mice showed decreased neural invasion and less paralysis. Such Schwann cell behavior reflects normal Schwann cell programs that are typically activated in nerve repair but are instead exploited by cancer cells to promote perineural invasion and cancer progression.

Targeted therapy for metastatic colorectal carcinoma consists of anti-EGFR therapy for patients with RAS/RAF wild-type tumors. However, the response rate remains low, suggesting the presence of alternative drivers possibly also representing potential therapeutic targets. We investigated receptor tyrosine kinase (RTK) alterations and MAP2K1 (MEK1) mutations in a large cohort of colorectal carcinoma patients studied by Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets and The Cancer Genome Atlas, focusing on amplifications, fusions, and hotspot mutations in RTK genes and MAP2K1. RTK gene amplifications were confirmed with FISH and immunohistochemical (IHC) staining. Among 751 colorectal carcinoma cases with next-generation sequencing data, 7% and 1% of colorectal carcinoma harbored RTK alterations and MAP2K1 hotspot mutations (n = 7), respectively. RTK-altered cases had fewer concurrent RAS/RAF mutations (P = 0.003) than RTK/MAP2K1 wild-type colorectal carcinoma. MAP2K1-mutated colorectal carcinoma showed no RAS/RAF mutations. ERBB2 (n = 32) and EGFR (n = 13) were the most frequently altered RTKs, both activated by amplification and/or hotspot mutations. Three RTK fusions were identified: NCOA4-RET, ERBB2-GRB7, and ETV6-NTRK3. Only 1 of 6 patients with an RTK or MAP2K1 alteration who received anti-EGFR and/or anti-ERBB2 therapy demonstrated stable disease; the rest progressed immediately. Overall, RTK alterations and MAP2K1 mutations occur in approximately 8% of colorectal carcinoma. In spite of the usual absence of RAS/RAF mutations, response to anti-EGFR and/or anti-ERBB2 therapy was poor in this limited group. Larger studies are warranted to further define these kinase alterations as novel therapeutic targets in colorectal carcinoma and as negative predictors of response to anti-EGFR therapy. Implications: Targetable kinase alterations were identified in a subset of advanced colorectal carcinoma patients, preferentially associated with wild-type RAS/RAF, and may predict poor response to standard anti-EGFR therapy. Mol Cancer Res; 14(3); 296–301. ©2015 AACR.

4602 Background: Thermal ablation (TA) is used as a local cure for selected colorectal liver metastases (CLM) with minimal risk. A critical limitation of TA has been early local tumor progression (LTP). The goal of this study is to establish the role of ablation zone (AZ) biopsy in predicting LTP. Methods: This institutional review board-approved prospective study included patients with CLM of 5cm or less in maximum diameter, with confined liver disease or stable, limited extrahepatic disease. Both radiofrequency(RF) and microwave(MW) ablation modalities were used. A biopsy of the center and margin of the AZ was performed immediately after ablation. The applicators were also examined for the presence of viable tumor cells. All samples containing morphologically identified tumor cells were further interrogated with immunohistochemistry to determine the proliferative and viability potential of the detected tumor cells. Ablation margin size was evaluated on the first CT scan performed 4–8 weeks after ablation and was confirmed by 3D assessment with Ablation Confirmation Software (Neuwave™). Variables were evaluated as predictors of time to LTP with the competing-risks model (uni- and multivariate analyses). Results: Between November 2009 and February 2019, 102 patients with 182 CLMs were enrolled. Mean tumor size was 2.0 cm (range, 0.6–4.8 cm). MW was used in 95/182 (52%) tumors and RF in 87/182 (48%). Median follow-up was 19 months. Technical effectiveness was evident in 178/182 (97%) ablated tumors on the first contrast material–enhanced CT at 4–8-weeks post-ablation. The cumulative incidence of LTP at 12 months was 19% (95% confidence interval [CI]: 14, 27). Samples from 64 (35%) of the 178 technically successful cases contained viable tumor. At univariate analysis, tumor size, minimal margin size, and biopsy results were significant in predicting LTP. In a multivariate model, margin size of less than 5 mm (P < .001; hazard ratio [HR], 4.3), and positive biopsy results (P = .02; HR, 1.8) remained significant. LTP within 12 months after TA was noted in 3% (95% CI: 1, 6) of tumor-negative biopsy CLMs with margins of at least 5 mm. Conclusions: Biopsy and pathologic examination of the AZ predicts LTP regardless of TA modality used. This can optimize ablation as a potential local cure for patients with limited CLM.

The anti-HER2 antibody trastuzumab is standard care for advanced esophagogastric (EG) cancer with ERBB2 (HER2) amplification or overexpression, but intrinsic and acquired resistance are common. We conducted a phase II study of afatinib, an irreversible pan-HER kinase inhibitor, in trastuzumab-resistant EG cancer. We analyzed pretreatment tumor biopsies and, in select cases, performed comprehensive characterization of postmortem metastatic specimens following acquisition of drug resistance. Afatinib response was associated with coamplification of EGFR and ERBB2. Heterogeneous 89Zr-trastuzumab PET uptake was associated with genomic heterogeneity and mixed clinical response to afatinib. Resistance to afatinib was associated with selection for tumor cells lacking EGFR amplification or with acquisition of MET amplification, which could be detected in plasma cell-free DNA. The combination of afatinib and a MET inhibitor induced complete tumor regression in ERBB2 and MET coamplified patient-derived xenograft models established from a metastatic lesion progressing on afatinib. Collectively, differential intrapatient and interpatient expression of HER2, EGFR, and MET may determine clinical response to HER kinase inhibitors in ERBB2-amplified EG cancer. Significance: Analysis of patients with ERBB2-amplified, trastuzumab-resistant EG cancer who were treated with the HER kinase inhibitor afatinib revealed that sensitivity and resistance to therapy were associated with EGFR/ERBB2 coamplification and MET amplification, respectively. HER2-directed PET imaging and cell-free DNA sequencing could help guide strategies to overcome the emergence of resistant clones. See related commentary by Klempner and Catenacci, p. 166. This article is highlighted in the In This Issue feature, p. 151

Intrahepatic cholangiocarcinomas are histologically heterogenous. Using a cohort of 184 clinically defined, resected intrahepatic cholangiocarcinomas, we retrospectively classified the histology into 4 subtypes: large duct (LD), small duct (SD) (predominantly tubular [SD1] or predominantly anastomosing/cholangiolar, [SD2]), or indeterminate. Then, we tested the 4 subtypes for associations with risk factors, patient outcomes, histology, and immunophenotypic characteristics. SD was the most common (84%; 24% SD1 and 60% SD2) with lower proportions of LD (8%), and indeterminate (8%). Primary sclerosing cholangitis was rare (2%), but correlated with LD (P=0.005). Chronic hepatitis, frequent alcohol use, smoking, and steatosis had no histologic association. LD was associated with mucin production (P0.001), perineural invasion (P=0.002), CA19-9 staining (P0.001), CK7, CK19, CD56 immunophenotype (P=0.005), and negative albumin RNA in situ hybridization (P0.001). SD was histologically nodular (P=0.019), sclerotic (P0.001), hepatoid (P=0.042), and infiltrative at the interface with hepatocytes (P0.001). Albumin was positive in 71% of SD and 18% of LD (P=0.0021). Most albumin positive tumors (85%) lacked extracellular mucin (P0.001). S100P expression did not associate with subtype (P0.05). There was no difference in disease-specific or recurrence-free survival among the subtypes. Periductal infiltration and American Joint Committee on Cancer eighth edition pT stage predicted survival by multivariable analysis accounting for gross configuration, pT stage, and histologic type. pT2 had worse outcome relative to other pT stages. Significant differences in histology and albumin expression distinguish LD from SD, but there is insufficient evidence to support further subclassification of SD.

Mutations of Hepatocyte-Nuclear-Factor-1-Homeobox-A gene and loss of Liver-Fatty-Acid-Binding-Protein are well documented in hepatocellular adenoma. However, the role of Hepatocyte-NuclearFactor-1-Homeobox-A mutations in hepatocellular carcinoma remains to be determined. In this study, all hepatocellular neoplasms evaluated by our institutional Memorial Sloan Kettering-Integrated Mutational Profiling of Actionable Clinical Targets assay or the Cancer Genome Atlas sequencing, and cases reported in the literature, were queried for Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations. Together, 11 of 672 (1.6%) hepatocellular carcinomas harbored Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations. The single case from our institution (n=153) was extremely well differentiated, arising in a background of adenomatosis. Both the adenoma and carcinoma component contained the same 2 somatic Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations (p. E32* and L214Q), with loss of Liver-Fatty-Acid-Binding-Protein. From the literature, 2 of 146 (1.4%) hepatocellular carcinomas had Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations, and both arose in a background of adenomatosis. Information on pre-existing adenoma for the remaining cases (8/373, from The Cancer Genome Atlas) was not available. Hepatocyte-Nuclear-Factor-1-Homeobox-A mutations in carcinomas were associated with negative viral hepatitis status (p=0.004), mutually exclusive with Catenin-Beta-1 hotspot mutations, and trended to occur more in females (p=0.06) and without cirrhosis (p=0.03). Grade was not associated with Hepatocyte-Nuclear-Factor-1-Homeobox-A status (p=0.28). Somatic Hepatocyte-Nuclear-Factor-1Homeobox-A mutations occur in approximately 1–2% of hepatocellular carcinoma, often in a background of adenomatosis. Our findings suggest that malignant transformation of Hepatocyte-Nuclear-Factor-1Homeobox-A mutated hepatocellular adenoma occurs, albeit infrequently. Hepatocellular adenomas with Hepatocyte-Nuclear-Factor-1-Homeobox-A mutation or adenomatosis with loss of Liver-Fatty-Acid-Binding-Protein warrant thorough sampling and examination.

This study aimed to compare common histologic markers at the invasive front of colon adenocarcinoma in terms of prognostic accuracy and interobserver agreement. Consecutive patients who underwent curative resection for stages I to III colon adenocarcinoma at a single institution in 2007 to 2014 were identified. Poorly differentiated clusters (PDCs), tumor budding, perineural invasion, desmoplastic reaction, and Crohn-like lymphoid reaction at the invasive front, as well as the World Health Organization (WHO) grade of the entire tumor, were analyzed. Prognostic accuracies for recurrence-free survival (RFS) were compared, and interobserver agreement among 3 pathologists was assessed. The study cohort consisted of 851 patients. Although all the histologic markers except WHO grade were significantly associated with RFS (PDCs, tumor budding, perineural invasion, and desmoplastic reaction: P

Importance Treatment of locally advanced rectal (LARC) cancer involves chemoradiation, surgery, and chemotherapy. The concept of total neoadjuvant therapy (TNT), in which chemoradiation and chemotherapy are administered prior to surgery, has been developed to optimize delivery of effective systemic therapy aimed at micrometastases. Objective To compare the traditional approach of preoperative chemoradiation (chemoRT) followed by postoperative adjuvant chemotherapy with the more recent TNT approach for LARC. Design, Setting, and Participants A retrospective cohort analysis using Memorial Sloan Kettering Cancer Center (MSK) records from 2009 to 2015 was carried out. A total of 811 patients who presented with LARC (T3/4 or node-positive) were identified. Exposures Of the 811 patients, 320 received chemoRT with planned adjuvant chemotherapy and 308 received TNT (induction fluorouracil- and oxaliplatin-based chemotherapy followed by chemoRT). Main Outcomes and Measures Treatment and outcome data for the 2 cohorts were compared. Dosing and completion of prescribed chemotherapy were assessed on the subset of patients who received all therapy at MSK. Results Of the 628 patients overall, 373 (59%) were men and 255 (41%) were women, with a mean (SD) age of 56.7 (12.9) years. Of the 308 patients in the TNT cohort, 181 (49%) were men and 127 (49%) were women. Of the 320 patients in the chemoRT with planned adjuvant chemotherapy cohort, 192 (60%) were men and 128 (40%) were women. Patients in the TNT cohort received greater percentages of the planned oxaliplatin and fluorouracil prescribed dose than those in the chemoRT with planned adjuvant chemotherapy cohort. The complete response (CR) rate, including both pathologic CR (pCR) in those who underwent surgery and sustained clinical CR (cCR) for at least 12 months posttreatment in those who did not undergo surgery, was 36% in the TNT cohort compared with 21% in the chemoRT with planned adjuvant chemotherapy cohort. Conclusions and Relevance Our findings provide additional support for the National Comprehensive Cancer Network (NCCN) guidelines that categorize TNT as a viable treatment strategy for rectal cancer. Our data suggest that TNT facilitates delivery of planned systemic therapy. Long-term follow-up will determine if this finding translates into improved survival. In addition, given its high CR rate, TNT may facilitate nonoperative treatment strategies aimed at organ preservation.

Reliable assessment of the BRAF mutation status is becoming increasingly important in the clinical management of colorectal carcinomas (CRC). The aim of this study was to investigate the use of a recently developed mutation-specific antibody (VE1; SpringBio, Pleasanton, CA) to detect the BRAF V600E protein in paraffin tissue. We analyzed by immunohistochemistry (IHC) 117 cases that had been evaluated for BRAF mutation using a MALDI-TOF mass spectrometry-based assay. Immunohistochemical staining was evaluated without the knowledge of the genetic data and was considered positive when there was distinct homogenous cytoplasmic staining in the tumor cells. The analyzed cases included 4 polyps, 63 primary CRC, and 50 metastatic CRC. Forty-five of the 46 (97.8%) cases that were positive by IHC had a BRAF V600E mutation by genetic analysis; the 1 discordant case was notably of signet ring cell type. Similarly, 66 of the 67 (98.5%) cases that were negative by IHC were also negative by genetic analysis. Four cases that showed weak cytoplasmic staining and/or nuclear staining in the tumor cells were considered to be IHC equivocal; by genetic analysis, 2 of the 4 were positive and 2 were negative. The overall sensitivity and specificity of IHC for the detection of a BRAF V600E mutant tumor was 93.7% and 95.6%, respectively. Our results support the use of VE1 IHC for identification of colorectal neoplasms harboring the BRAF V600E mutation. Difficulties in immunohistochemical interpretation may arise in a small number of cases and in those cases molecular testing is required.

The PI3K/AKT/mTOR pathway is activated through multiple mechanisms in colorectal carcinoma. Here, the clinicopathologic and molecular features of AKT1 E17K–mutated colorectal carcinoma in comparison with PIK3CA-mutated colorectal carcinoma are described in detail. Interestingly, in comparison with PIK3CA mutants, AKT1 E17K was significantly associated with mucinous morphology and concurrent BRAF V600E mutation. Among PIK3CA mutants, exon 21 mutations were significantly associated with BRAF V600E mutation, MSI-H status, and poor differentiation, while exon 10 mutations were associated with KRAS/NRAS mutations. Three of four AKT1 mutants with data from both primary and metastatic lesions had concordant AKT1 mutation status in both. Both AKT1- and PIK3CA-mutant colorectal carcinoma demonstrated frequent loss of PTEN expression (38% and 34%, respectively) and similar rates of p-PRAS 40 expression (63% and 50%, respectively). Both patients with AKT1 E17K alone had primary resistance to cetuximab, whereas 7 of 8 patients with PIK3CA mutation alone experienced tumor shrinkage or stability with anti-EGFR therapy. These results demonstrate that AKT1 E17K mutation in advanced colorectal carcinoma is associated with mucinous morphology, PIK3CA wild-type status, and concurrent RAS/RAF mutations with similar pattern to PIK3CA exon 21 mutants. Thus, AKT1 E17K mutations contribute to primary resistance to cetuximab and serve as an actionable alteration. Implications: This first systematic study of AKT1 and PIK3CA hotspot mutations and their association with cetuximab resistance and BRAF V600E mutation has important ramifications for the development of personalized medicine, particularly in identifying patient candidates for PI3K or AKT inhibitors. Mol Cancer Res; 13(6); 1003–8. ©2015 AACR.

Strategies to enhance tumor immunogenicity may expand the role of immunotherapy beyond the mismatch repair-deficient subtype. In this pilot study, biopsies were performed at baseline and after four cycles of FOLFOX in eight patients receiving neoadjuvant chemotherapy for stage II/III locally advanced rectal cancer. Immunostaining was performed for T cell subsets (CD3+, CD8+, CD45RO+); macrophages (CD163+); T regulatory cells (FOXP3+); and expression of MHC class I, PD-1 and PD-L1. Changes in cell number or intensity were quantified and correlated with treatment response. Pretreatment patterns of immune infiltrates were mixed and did not correlate with treatment response. Posttreatment increases in T cell infiltrates (CD3+, CD8+ and CD45RO+) and MHC-I expression were observed in five patients. CD163+ cell numbers increased in four patients. FOXP3+ cells numbers increased in two patients, decreased in two other patients and remained unchanged in three patients. PD-1 scores increased in seven patients, and PD-L1 scores increased in four patients. Changes in tumor T cell responses did not correlate with treatment response. Changes in FOXP3+ cells were associated with treatment response in some patients: two patients with increases in FOXP3+ cells had poor responses, whereas the patient with the greatest reduction in FOXP3+ cells had a complete response. The patient with a complete clinical response had a much higher increase in MHC-I expression than other patients. These results suggest that chemotherapy can increase immune activity in the tumor microenvironment and could potentially be utilized to prime immune responses prior to immunomodulatory treatments.

The aim of this study was to determine whether intraprocedural (18)F-FDG PET/CT can be used as a predictor of local tumor progression after percutaneous ablation of colorectal liver metastases. Methods: In this institutional review board–approved prospective study, 39 patients (19 men and 20 women; median age, 56 y) underwent split-dose (18)F-FDG PET/CT-guided ablation followed by immediate biopsy and contrast-enhanced CT imaging of the ablation zone. Binary categorization of biopsy tissues was performed on the basis of the presence of only nonviable coagulation necrosis or viable tumor cells. Minimum ablation margin measurements from contrast-enhanced CT imaging were categorized as 0 mm, 1–4 mm, 5–9 mm, or greater than or equal to 10 mm. SUVs were obtained from PET/CT imaging, and SUV ratios were calculated from 3-dimensional regions of interest located in the ablation zone and surrounding normal liver. All predictive variables (biopsy, minimum margin distance, and SUV ratio) were evaluated as predictors of time to local tumor progression identified on imaging using competing-risks regression models (uni- and multivariate analyses). Results: A total of 62 consecutive ablations were evaluated. The mean SUV ratio was significantly higher for viable tumor–positive immediate postablation biopsies (n = 10) than for tumor-negative biopsies (n = 52) (85.8 ± 92.2 vs. 42.3 ± 45.5) (P = 0.03) and for a minimum margin size of less than 5 mm (n = 15) than for a minimum margin size of greater than or equal to 5 mm (n = 47) (78.5 ± 99.1 vs. 38.3 ± 78.5) (P = 0.01). After a median follow-up period of 22.5 (range, 7–52) months, 23 of 62 ablated tumors showed local tumor progression (37.1%). The local tumor progression rate was significantly higher for viable tumor–positive biopsies (8/10) than for negative biopsies (15/52) (80% vs. 29%) (P = 0.001) and for a minimum margin size of less than 5 mm (9/15) than for a minimum margin size of greater than or equal to 10 mm (2/15) (60% vs. 13%) (P = 0.02) but not 5–9 mm (37.5%; 12/32) (P = 0.5). In a competing-risks analysis, biopsy results (P = 0.07) and the minimum margin size (P = 0.08) were borderline significant, but the SUV ratio was not (P = 0.22). However, for negative biopsy ablations, the minimum margin size and SUV ratio were predictive imaging factors for local tumor progression; subdistribution hazard ratios were 0.564 (0.325–0.978) (P = 0.04) and 1.005 (1.001–1.009) (P = 0.005), respectively. Conclusion: The SUV ratio and minimum margin size can independently predict colorectal metastasis local tumor progression after liver ablation when there are no viable tumor cells on immediate postablation biopsies.

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically-defined tumor types, coupled with an expanding portfolio of molecularly-targeted therapies, demands flexible and comprehensive approaches to profile clinically significant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative utilizing a comprehensive assay, MSK-IMPACT, through which we have compiled matched tumor and normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel non-coding alterations, and mutational signatures that were shared among common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

Metastatic colorectal cancers (mCRCs) are clinically heterogeneous, but the genomic basis of this variability remains poorly understood. We performed prospective targeted sequencing of 1,134 CRCs. We identified splice alterations in intronic regions of APC and large in-frame deletions in CTNNB1, increasing oncogenic WNT pathway alterations to 96% of CRCs. Right-sided primary site in microsatellite stable mCRC was associated with shorter survival, older age at diagnosis, increased mutations, and enrichment of oncogenic alterations in KRAS, BRAF, PIK3CA, AKT1, RNF43, and SMAD4 compared with left-sided primaries. Left-sided tumors frequently had no identifiable genetic alteration in mitogenic signaling, but exhibited higher mitogenic ligand expression. Our results suggest different pathways to tumorigenesis in right- and left-sided microsatellite stable CRC that may underlie clinical differences.

Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5+ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5+ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.

Malignant mesothelioma (MM) is a rare, aggressive tumor often associated with asbestos exposure and characterized by complex genetic abnormalities, including deletions of chromosome 22. A gene fusion involving EWSR1 and YY1 gene on 14q32 has been reported in 2 patients over the age of 60 with peritoneal MM. However, the incidence of EWSR1 rearrangements in MM and the spectrum of its fusion partners remain unknown. We recently encountered 2 MM cases with EWSR1-ATF1 fusions and sought to investigate the prevalence and clinicopathologic features associated with this abnormality. As both index cases occurred as intra-abdominal tumors in young adults, we searched our files for pleural and peritoneal MM occurring in adults younger than age of 40. All cases were tested by fluorescence in situ hybridization using custom bacterial artificial chromosomes probes for EWSR1, FUS, and ATF1 genes. When available, immunohistochemistry for BAP1 was performed. A total of 25 MM from patients aged 40 or less were screened, either from peritoneum (n=13) or pleura (n=12), with a median age of 31 (range: 7 to 40 y). Two additional ATF1-rearranged tumors were identified at pleural and peritoneal sites with EWSR1 and FUS as fusion partners, respectively, for a total of 4 cases (16%, 4/25). The fusion-positive cases displayed classic epithelioid morphology, immunoreactivity for cytokeratins and WT1, and negativity for S100. BAP1 expression was retained in the 3 fusion-positive cases with available material, and in 80% (12/15) of the fusion-negative cases. Our results expand the spectrum of tumor types harboring EWSR1/FUS-ATF1 gene fusions to include a subgroup of conventional epithelioid MM. Other features of this unique MM subset include young age at presentation, lack of asbestos exposure and retained BAP1 expression.

BACKGROUND RAS and PIK3CA mutations in metastatic colorectal cancer (mCRC) have been associated with worse survival. We sought to evaluate the impact of RAS and PIK3CA mutations on cumulative incidence of metastasis to potentially curable sites of liver and lung and other sites such as bone and brain. METHODS We performed a computerized search of the electronic medical record of our institution for mCRC cases genotyped for RAS or PIK3CA mutations from 2008 to 2012. Cases were reviewed for patient characteristics, survival, and site-specific metastasis. RESULTS Among the 918 patients identified, 477 cases were RAS wild type, and 441 cases had a RAS mutation (394 at KRAS exon 2, 29 at KRAS exon 3 or 4, and 18 in NRAS). RAS mutation was significantly associated with shorter median overall survival (OS) and on multivariate analysis independently predicted worse OS (HR, 1.6; P

AT-rich interactive domain-containing protein 1A (ARID1A), a chromatin remodeling gene recently discovered to be a tumor suppressor in ovarian cancers, has been found to be mutated at low frequencies in many other tumors including colorectal carcinoma (CRC). An association between ARID1A alteration and DNA mismatch repair (MMR) deficiency has been implicated; understanding this association may facilitate the understanding of the role of ARID1A in the various tumors. In this pilot study, we analyzed the immunohistochemical expression of ARID1A in a consecutive series of 257 CRCs that fulfilled a set of relaxed criteria for Lynch syndrome screening; 59 (23%) were MMR deficient by immunohistochemistry (44 MLH1/PMS2 deficient, 9 MSH2/MSH6 deficient, 4 MSH6 deficient, and 2 PMS2 deficient). ARID1A loss was observed in 9% (22/257) of the cohort: 24% of MMR-deficient tumors (14/59, 13 of the 14 being MLH1/PMS2 deficient) and 4% of MMR-normal tumors (8/198) (P.05). MLH1 (mutL homolog 1) promoter hypermethylation was observed in 10 of the 13 MLH1/PMS2-deficient/ARID1A-loss tumors, indicating an association between ARID1A loss and sporadic microsatellite unstable CRCs. Among the MMR-deficient cases, ARID1A loss correlated with old age (P = .04), poor tumor differentiation (P.01), medullary histology (P.01), and an increased rate of nodal and distant metastasis (P = .03); these patients also trended toward a worse 5-year overall survival. Among MMR-normal tumors, no differences in clinicopathological features were detected between the groups stratified by ARID1A. In conclusion, our results suggest that ARID1A loss may be linked to a specific subset of sporadic microsatellite unstable CRCs that may be medullary but is more likely to present with metastatic disease, warranting further investigation.

BACKGROUND The validity of the KRAS mutation as a predictor of recurrence-free survival (RFS) or overall survival (OS) is unclear. The current study investigated whether the presence of the KRAS mutation decreased RFS or OS in patients with colorectal cancer who underwent liver resection. METHODS Patients with resected colorectal liver metastases who received adjuvant hepatic arterial infusion plus systemic therapy and for whom KRAS data was available were evaluated. Correlation between KRAS and clinical factors was done using the Fisher exact test. Kaplan-Meier methods were used to estimate the median RFS and OS. RESULTS A total of 169 patients were evaluated, 118 of whom had KRAS wild-type (WT) and 51 had KRAS mutated (MUT) tumors. The 3-year RFS rate was 46% for patients with KRAS WT (95% confidence interval [95% CI], 35%-56%) and 30% (95% CI, 16%-44%) for patients with KRAS MUT (P =.005). The 3-year OS rate was 95% (95% CI, 87%-98%) and 81% (95% CI, 62%-95%), respectively, for patients with KRAS WT and KRAS MUT (P =.07). On multivariate analysis, KRAS remained a significant predictor of RFS (hazard ratio, 1.9). The 3-year cumulative recurrence rate by site of metastases was as follows: 2% versus 13.4% for bone (P≤.01), 2% versus 14.5% for brain (P =.05), 33.2% versus 58% for lung (P≤.01), and 30% versus 47% for liver (P =.10) in patients with KRAS WT versus KRAS MUT. CONCLUSIONS In the current study, among patients with resected colorectal liver metastases who were treated with adjuvant hepatic arterial infusion plus systemic therapy, patients with KRAS MUT were found to have a significantly worse 3-year RFS (30%) compared with KRAS WT (46%) p=.005. The cumulative incidence of bone, brain, and lung metastases was significantly higher for patients with KRAS MUT compared with those with KRAS WT. Cancer 2014;120:3965–3971. © 2014 American Cancer Society.

BACKGROUND BRAF mutations occur in 5% to 11% of patients with metastatic colorectal cancer (mCRC) and have been associated with poor prognosis. The current study was undertaken to determine the clinicopathologic characteristics, PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) mutation frequency, and outcomes after metastasectomy in patients with BRAF-mutant mCRC. METHODS Data from 1941 consecutive patients with mCRC who underwent KRAS/BRAF mutation testing between 2009 and 2012 at a single institution were identified to identify BRAF-mutant mCRC cases (92 cases). BRAF wild-type mCRC cases from 2011 (423 cases) served as a control group. RESULTS BRAF-mutated mCRC was found to be significantly associated with older age at diagnosis, female sex, right-sided location, poorly differentiated morphology, and mucinous histology compared with wild-type cases. BRAF-mutant cases more frequently progressed from stage III disease (32% vs 17%; P = .003) and among those patients with stage III disease, T4 disease was more common (48% vs 27%; P = .05). PIK3CA was found to be co-mutated in 5% of BRAF-mutant tumors versus 17% of KRAS-mutant tumors (P

3585 Background: We assembled a large series of consecutive patients with unresectable metastatic colorectal cancer (mCRC) to identify genomic biomarkers of response and survival. Methods: Patients with unresectable mCRC treated at Memorial Sloan Kettering with genomic tumor profiling between 2014 and 2017 were included. Patients who underwent upfront metastasectomy or received neoadjuvant/conversion chemotherapy were excluded. Clinical information was retrieved from electronic medical records, and we evaluated associations between genomic profiles with progression free survival (PFS) on first-line chemotherapy and overall survival (OS). Categorical data were analyzed by Fisher exact test and time-to-event data were analyzed by Cox proportional hazards models. Results: Of 1453 mCRCs profiled in this period, 471 patients met the study criteria. Median age was 59 years (range, 18 to 95), and 73% of patients were stage IV at diagnosis. Most tumors (91%) were microsatellite stable (MSS). The most frequent first-line regimen was FOLFOX +/- bevacizumab (66%). Among MSS patients treated with oxaliplatin-containing regimens (n = 305), 7% harbored alterations in genes associated with DNA damage response (DDR) (BRCA1, BRCA2, ATM, PALB2). DDR gene alterations were not associated with PFS (P = 0.94) nor were different quartiles of large-state transitions (P = 0.54). Genomic alterations that significantly varied by duration of response included BRAF (16%, 10%, and 5% for PFS < 6 months, 6-12 months, and > 12 months, respectively) and APC (62%, 74%, and 80% for PFS < 6 months, 6-12 months, and > 12 months, respectively). APC mutation, single or dual, was associated with significantly longer PFS (HR 0.67) and OS (HR 0.59) in multivariate analysis versus no WNT pathway alteration or alterations in other WNT pathway genes (RNF43, AXIN2, CTNNB1). Conclusions: In unresectable mCRC patients, mutations in APC were associated with better outcomes; absence of an APC alteration or the occurrence of other WNT pathway alterations was associated with shorter survival. Somatic alterations in DDR genes were not associated with outcomes in mCRC patients receiving oxaliplatin-containing regimen.

IMPORTANCE: The watch-and-wait (WW) strategy aims to spare patients with rectal cancer unnecessary resection. OBJECTIVE: To analyze the outcomes of WW among patients with rectal cancer who had a clinical complete response to neoadjuvant therapy. DESIGN, SETTING, AND PARTICIPANTS: This retrospective case series analysis conducted at a comprehensive cancer center in New York included patients who received a diagnosis of rectal adenocarcinoma between January 1, 2006, and January 31, 2015. The median follow-up was 43 months. Data analyses were conducted from June 1, 2016, to October 1, 2018. EXPOSURES: Patients had a clinical complete response after completing neoadjuvant therapy and agreed to a WW strategy of active surveillance and possible salvage surgery (n = 113), or patients underwent total mesorectal excision and were found to have a pathologic complete response (pCR) at resection (n = 136). MAIN OUTCOMES AND MEASURES: Kaplan-Meier estimates were used for analyses of local regrowth and 5-year rates of overall survival, disease-free survival, and disease-specific survival. RESULTS: Compared with the 136 patients in the pCR group, the 113 patients in the WW group were older (median [range], 67.2 [32.1-90.9] vs 57.3 [25.0-87.9] years, P

Aberrant Wnt signaling can drive cancer development. In many cancer types, the genetic basis of Wnt pathway activation remains incompletely understood. Here, we report recurrent somatic mutations of the Drosophila melanogaster tumor suppressor-related gene FAT1 in glioblastoma (20.5%), colorectal cancer (7.7%), and head and neck cancer (6.7%). FAT1 encodes a cadherin-like protein, which we found is able to potently suppress cancer cell growth in vitro and in vivo by binding β-catenin and antagonizing its nuclear localization. Inactivation of FAT1 via mutation therefore promotes Wnt signaling and tumorigenesis and affects patient survival. Taken together, these data strongly point to FAT1 as a tumor suppressor gene driving loss of chromosome 4q35, a prevalent region of deletion in cancer. Loss of FAT1 function is a frequent event during oncogenesis. These findings address two outstanding issues in cancer biology: the basis of Wnt activation in non-colorectal tumors and the identity of a 4q35 tumor suppressor.

Immunohistochemical staining for DNA mismatch repair proteins may be affected by various biological and technical factors. Staining variations that could potentially lead to erroneous interpretations have been recognized. A recently recognized staining variation is the significant reduction of staining for MSH6 in some colorectal carcinomas. The frequency and specific characteristics of this aberrant MSH6 staining pattern, however, have not been well analyzed. In this study of 420 colorectal carcinoma samples obtained from patients fulfilling the Revised Bethesda Guidelines, we detected 9 tumors (2%) showing extremely limited staining for MSH6 with positive staining present in5% of the tumor cells. Our analyses showed that these tumors belonged to two distinct categories: (1) MLH1 and/or PMS2 protein-deficient carcinomas (n=5, including 1 with a pathogenic mutation in PMS2); and (2) MLH1, PMS2 and MSH2 normal but with chemotherapy or chemoradiation therapy before surgery (n=4). To test our hypothesis that somatic mutation in the coding region microsatellite of the MSH6 gene might be a potential underlying mechanism for such limited MSH6 staining, we evaluated frameshift mutation in a (C)(8) tract in exon 5 of the MSH6 gene in seven tumors that had sufficient DNA for analysis, and detected mutation in four; all four tumors belonged to the MLH1/PMS2-deficient group. In conclusion, our data outline the main scenarios where significant reduction of MSH6 staining is more likely to occur in colorectal carcinoma, and suggest that somatic mutations of the coding region microsatellites of the MSH6 gene is an underlying mechanism for this staining phenomenon in MLH1/PMS2-deficient carcinomas.