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Background/Purpose The median survival for patients with metastatic colorectal cancer (mCRC) has progressively increased over the past decades. Since the introduction of 5-fluorouracil (5-FU)-based chemotherapy, followed by hepatic resection of metastases, and more recently the adoption of newer chemotherapeutic regimens associated with targeted therapy, the gains are getting more substantial. Despite the recognition of the potential for long-term survival after surgical resection of metastatic disease, long-term survival data to determine the potential curative role of chemotherapy alone is lacking. Methods We performed a retrospective review of 2751 patients who presented with mCRC at The MD Anderson Cancer Center from 1990 through 2003. Patients alive at 5 years who achieved complete response with chemotherapy and were not submitted to any surgical or interventional procedures directed to the metastatic sites were included in the analysis. Results The 5-year overall survival rate for all patients with mCRC during this period was 10.8%. Among these long-term survivors, 2.2% achieved a sustained complete response after chemotherapy (all 6 with fluoropyrimidines and 2 with irinotecan) as the only treatment modality and were without evidence of disease until the last follow-up visit (median of 10.3 years). This number corresponds to 0.24% (6 of 2541) of all patients with mCRC included in this review. Conclusion Cure with chemotherapy alone is possible for a very small number of patients with metastatic colorectal cancer. Improved therapies are increasing complete response rates, but the impact of modern chemotherapy on durable complete responses will require additional follow up.

Purpose: This study was designed to determine whether DNA methylation biomarkers are associated with recurrence and survival in colon cancer patients. Experimental Design: A retrospective analysis of 82 patients who received curative surgical resection for American Joint Committee on Cancer (AJCC) high-risk stage II or III colon cancer (1999–2007) was conducted. DNA methylation status was quantitatively evaluated by the pyrosequencing method. We preselected three tumor suppressor genes and one locus of interest; CHFR, ID4, RECK, and MINT1. Mean methylation levels of multiple CpG sites in the promoter regions were used for analysis; 15% or more was defined as methylation positive. The association of recurrence-free survival (RFS) and overall survival (OS) with methylation status was analyzed by the log-rank test, Kaplan–Meier method, and Cox proportional hazards model. Results: Methylation levels of ID4, MINT1, and RECK did not correlate with RFS or OS. CHFR was methylation positive in 63% patients. When methylation status was dichotomized (negative or low: Conclusions: The extent of CHFR promoter methylation correlates with RFS, indicating it is a promising epigenetic marker for recurrence. Clin Cancer Res; 17(13); 4531–40. ©2011 AACR.

BACKGROUND: It is hypothesized that BRAF mutant cancers represent a discrete subset of metastatic colorectal cancer (CRC) defined by poorer survival. This study investigates whether BRAF mutant CRC is further defined by a distinct pattern of metastatic spread and explores the impact of BRAF mutation and microsatellite instability (MSI) on prognosis in metastatic CRC. METHODS: By using prospective clinical data and molecular analyses from 2 major centers (Royal Melbourne Hospital and The University of Texas MD Anderson Cancer Center), patients with known BRAF mutation status were analyzed for clinical characteristics, survival, and metastatic sites. RESULTS: The authors identified 524 metastatic CRC patients where BRAF mutation status was known; 57 (11%) were BRAF mutant tumors. BRAF mutant tumors were significantly associated with right-sided primary tumor, MSI, and poorer survival (median, 10.4 months vs 34.7 months, P < .001). A distinct pattern of metastatic spread was observed in BRAF mutant tumors, namely higher rates of peritoneal metastases (46% vs 24%, P = .001), distant lymph node metastases (53% vs 38%, P = .008), and lower rates of lung metastases (35% vs 49%, P = .049). In additional survival analyses, MSI tumors had significantly poorer survival compared with microsatellite stable tumors (22.1 months vs 11.1 months, P = .017), but this difference was not evident in the BRAF mutant population. CONCLUSIONS: The pattern of metastatic spread observed in this study further defines BRAF mutant CRC as a discrete disease subset. The authors demonstrated that, unlikely early stage disease, MSI is associated with poorer survival in metastatic CRC, although this is driven by its association with BRAF mutation. Cancer 2011;. © 2011 American Cancer Society.

BACKGROUND: Patient outcomes following resection of colorectal liver metastases (CLM) after second-line chemotherapy regimen is unknown. METHODS: From August 1998 to June 2009, data from 1099 patients with CLM were collected prospectively. We retrospectively analyzed outcomes of patients who underwent resection of CLM after second-line (2 or more) chemotherapy regimens. RESULTS: Sixty patients underwent resection of CLM after 2 or more chemotherapy regimens. Patients had advanced CLM (mean number of CLM ± standard deviation, 4 ± 3.5; mean maximum size of CLM, 5 ± 3.2 cm) and had received 17 ± 8 cycles of preoperative chemotherapy. In 54 (90%) patients, the switch from the first regimen to another regimen was motivated by tumor progression or suboptimal radiographic response. All patients received irinotecan or oxaliplatin, and the majority (42/60 [70%]) received a monoclonal antibody (bevacizumab or cetuximab) as part of the last preoperative regimen. Postoperative morbidity and mortality rates were 33% and 3%, respectively. At a median follow-up of 32 months, 1-year, 3-year, and 5-year overall survival rates were 83%, 41%, and 22%, respectively. Median chemotherapy-free survival after resection or completion of additional chemotherapy administered after resection was 9 months (95% confidence interval, 4-14 months). Synchronous (vs metachronous) CLM and minor (vs major) pathologic response were independently associated with worse survival. CONCLUSIONS: Resection of CLM after a second-line chemotherapy regimen was found to be safe and was associated with a modest hope for definitive cure. This approach represents a viable option in patients with advanced CLM. Cancer 2011;. © 2011 American Cancer Society.

BACKGROUND: Preoperative chemoradiation for rectal cancer can decrease the number of evaluable lymph nodes. Hence, the prognostic role of lymph node evaluation in patients with rectal cancer who receive preoperative chemoradiation is unclear. The authors of this report evaluated the prognostic impact of the number of lymph nodes examined in patients with rectal cancer who had negative lymph nodes based on the pathologic extent of disease (ypN0) after they received preoperative chemoradiation. METHODS: Between 1990 and 2004, 372 patients with nonmetastatic rectal adenocarcinoma received preoperative chemoradiation followed by mesorectal excision and had ypN0 disease. The median radiation dose was 45 gray, and 68% of patients received adjuvant chemotherapy. RESULTS: Patients had a median of 7 lymph nodes examined after preoperative chemoradiation. Compared with patients who had ≤7 lymph nodes examined, patients who had >7 lymph nodes had higher 5-year rates of freedom from relapse (86% vs 72%; log-rank P = .005) and cancer-specific survival (95% vs 86%; log-rank P = .0004), but no significant difference was observed in the overall survival rate (87% vs 81%; log-rank P = .07). Multivariate Cox proportional models demonstrated that patients who had >7 lymph nodes examined had a significantly lower risk of relapse (hazard ratio [HR], 0.39; P = .003) and death from rectal cancer (HR, 0.45; P = .04) but a similar risk of all-cause mortality (HR, 0.75; 95% CI, 0.46-1.20; P = .23) compared with patients who had ≤7 lymph nodes examined. CONCLUSIONS: The number of lymph nodes examined was associated independently with disease relapse and cancer-specific survival in patients with rectal cancer who had ypN0 disease after receiving preoperative chemoradiation. Hence, the authors concluded that the number of negative lymph nodes examined may be a prognostic factor in patients with rectal cancer who receive preoperative chemoradiation. Cancer 2011;. © 2011 American Cancer Society.

In the version of this article originally published, an author was missing from the author list. Alexander J. Lazar should have been included between Jorge M. Blando and James P. Allison. The author has been added to the list, and the author contributions section has been updated to include Alexander J. Lazar's contribution to the study. The error has been corrected in the print, PDF and HTML versions of the manuscript.

Pancreatic heterotopia has been described at several abdominal and intrathoracic locations, most commonly in the stomach and upper part of the small intestine. Its occurrence in the rectum is unusual, and malignant transformation in the rectum has not been reported. We report a case of ductal adenocarcinoma arising in a rectal pancreatic heterotopia in a 42-year-old woman with delayed local recurrence. The tumor was composed of well to moderately differentiated ductal adenocarcinoma infiltrating through the full thickness of the anorectal wall with extension into the vaginal septum. A focus of ectopic pancreas consisting of exocrine acini and small ducts adjacent to the tumor with some ducts showing mild to severe dysplasia reminiscent of pancreatic intraepithelial neoplasia was also observed. Although our literature search found 31 documented reports of tumors arising in the heterotopic pancreas, the present case is the first case of ductal adenocarcinoma arising in a focus of pancreatic heterotopia in the rectum.

Controversy currently exists about the optimum preoperative treatment platform for locoregionally advanced esophageal cancer, namely, preoperative chemoradiotherapy (preoperative C/RT) or preoperative chemotherapy alone. We therefore reviewed sequential phase II/III trials performed at a single institution to assess the impact of preoperative chemotherapy versus preoperative C/RT strategies.In all, 157 esophageal cancer patients were sequentially enrolled in phase II/III trials at the University of Texas M.D. Anderson Cancer Center from March 27, 1990, to March 8, 2005. The treatment approaches included preoperative chemotherapy, n = 76 (INT 113 and ID90-01); preoperative C/RT, n = 81 (ID96-189 and DM98-349). Analysis was by intention to treat. Factors evaluated included demographics, preoperative staging, type of surgery, pathology, adjuvant therapies, and long-term outcome.Adenocarcinoma predominated (85%), with cT3 (73%) and cN1 (43%). No significant difference was noted between groups in demographics or perioperative mortality. More patients with preoperative C/RT were staged with endoscopic ultrasound (52% versus 9%, p0.001). Preoperative C/RT demonstrated increased pathologic complete response (28% versus 4%, p0.001) and overall survival (3 years, 48% versus 29%, p = 0.04). Preoperative C/RT was a significant independent predictor of improved overall survival (hazard ratio 0.58, 95% confidence interval: 0.37 to 0.90, p = 0.015) and disease-free survival (hazard ratio 0.55, 95% confidence interval: 0.35 to 0.85, p = 0.007) in multivariable regression.In sequential phase II/III trials involving locoregionally advanced esophageal cancer patients, preoperative C/RT was associated with improved overall and disease-free survival rates (p = 0.046 and p = 0.015, respectively) and increased pathologic complete response (p0.001) compared with preoperative chemotherapy.

Progress in the treatment of hepatic colorectal metastases (HCRM) demands pathologic indicators of therapy response. We observed that a majority of residual tumor cells are seen at the tumor-normal interface (TNI) in resected HCRM specimens and hypothesized that tumor thickness at the TNI correlates with radiologic and pathologic response and recurrence-free survival (RFS).This study included 103 patients with HCRM resected after preoperative chemotherapy with or without bevacizumab. Imaging response was assessed by response evaluation criteria in solid tumors (RECIST) and recently described CT morphology criteria by Chun et al. The pathologic response was categorized as complete (no tumor cells), major (50% residual tumor cells), or minor (or =50% residual tumor cells). The maximum thickness of uninterrupted layers of tumor cells was measured perpendicular to the TNI by 2 pathologists independently, followed by consensus review for discrepant cases. For specimens containing1 tumor, the average tumor thickness at the TNI was used.Sixty-five patients received oxaliplatin-based chemotherapy, 38 received irinotecan-based chemotherapy, and 75 received concurrent bevacizumab. A complete pathologic response was seen in 9 patients, a major response in 44, and a minor response in 50. Median tumor thickness at the TNI was 2.8 mm (interquartile range, 0.5 to 6 mm). Tumor thickness correlated better with radiologic response as determined by Chun et al (P0.0001) than by RECIST criteria (Spearman r=0.35, P0.001). Tumor thickness correlated with pathologic response (Spearman r=0.80, P0.0001). Greater thickness predicted shorter recurrence-free survival, and this correlation remained in multivariate analysis (P=0.015). Tumor thickness was smaller in patients treated with bevacizumab than in patients not given bevacizumab (P=0.03).Tumor thickness measured at the TNI is potentially a new prognostic factor for therapy response and survival outcome in patients with resected HCRM.

The optimal duration, safety, and benefit of preoperative chemotherapy in patients with colorectal liver metastases (CLM) are unclear. We evaluated the association between the duration of preoperative chemotherapy with 5-fluorouracil (5-FU), leucovorin, oxaliplatin (FOLFOX) ± bevacizumab, pathologic response, and hepatotoxicity after hepatic resection for CLM. A total of 219 patients underwent hepatic resection following FOLFOX with or without bevacizumab and were divided into 2 groups according to the chemotherapy duration: 1–8 cycles (short duration [SD]; N = 157) and ≥9 cycles (long duration [LD]; N = 62). The frequency of complete or major pathologic response, sinusoidal injury, and major postoperative morbidity were compared. Treatment consisting of ≥9 cycles was not associated with an increase in complete or major pathologic response (SD vs. LD, 57% vs. 55%; P = .74). The incidence of sinusoidal injury was higher in the LD group (26% vs. 42%; P = .017). The incidence of liver insufficiency was higher in the LD group (4% vs. 11%; P = .035). Sinusoidal injury did not predict postoperative liver insufficiency; multivariate analysis revealed ≥9 cycles was the only independent predictor of postoperative liver insufficiency (P = .031; odds ratio = 3.90). Chemotherapy including bevacizumab was associated with a significantly higher frequency of complete or major response in both SD and LD groups. Extended preoperative chemotherapy increases the risk of hepatotoxicity in CLM without improving the pathologic response. The type of chemotherapy (FOLFOX with bevacizumab) has more impact on pathologic response than the duration of chemotherapy.

e16051Background: Preoperative chemoradiotherapy (CRT) is one standard option for localized esophageal or gastroesophageal junction (GEJ) cancer patients but an optimal concurrent chemotherapy comb...

3595Background: AT-rich interactive domain 1A (ARID1A) is a component of the SWI/SNF chromatin remodeling complex that regulates gene expression. Inactivating mutations of ARID1A have been reported...

e15600Background: The CMS provide a representation of CRC heterogeneity. Upregulated immune pathways are suggested in CMS1 through lymphocyte (lymph) reactivity and CMS4 with an inflammatory respon...

Purpose Oxaliplatin-based chemotherapy can cause hepatic sinusoidal injury, with resultant sinusoidal damage and portal hypertension. We sought to explore the relationship between oxaliplatin induced hepatic sinusoidal injury, increases in spleen size, and the subsequent development of thrombocytopenia. Patients and Methods We retrospectively assessed the relationship between chemotherapy exposure, changes in spleen size (determined by volumetric measurements), and platelet counts in 136 patients treated with adjuvant fluorouracil and oxaliplatin (FOLFOX) or fluoropyrimidine for stage II or III colorectal adenocarcinoma. Hepatic sinusoidal injury and changes in spleen size were graded in a separate population of 63 patients with metastatic colorectal cancer receiving fluoropyrimidine and oxaliplatin before liver resection. Results Spleen size increased in 86% of patients treated with adjuvant FOLFOX (P < .001), with a ≥ 50% increase in 24% of patients. Spleen size did not significantly increase in patients treated with adjuvant fluoropyrimidine. Increases in spleen size correlated with cumulative oxaliplatin dose (P = .003). Patients with splenic enlargement ≥ 50% had higher rates of thrombocytopenia in the first year after completion of chemotherapy (27% v 5%; P = .003). In patients with hepatic metastases treated with preoperative fluoropyrimidine and oxaliplatin, increases in spleen size was a predictor of higher histologic grades of sinusoidal injury in both univariate (P = .03) and multivariate (P = .02) analyses. Conclusion Increases in spleen size correlate with increasing grade of hepatic sinusoidal injury and can serve as a simple method for identifying patients at risk for this toxicity. Oxaliplatin-induced increases in spleen size should be recognized as a potential etiology of persistent thrombocytopenia after oxaliplatin treatment.

BACKGROUND: The use of platinum-based chemoradiation for esophageal cancer is routine, but it is unclear which class of cytotoxic are optimum. It was hypothesized that chemoradiotherapy with fluoropyrimidine, taxane, and camptothecin would have preserved or improved efficacy with no compromise in safety. METHODS: Patients with histologically confirmed, resectable esophageal carcinoma were eligible. In addition to other tests, a baseline endoscopic ultrasonography (EUS) was obtained. Patients were medically fit and had near–normal organ functions. Patients received docetaxel and irinotecan, plus 5–fluorouracil as induction therapy and then the same cytotoxics with 50.4 grays of radiotherapy followed by an attempted surgery. Pathologic complete response (pathCR) at a rate of ≥20% was the primary endpoint. The pathCR and R0 resection were correlated with overall survival (OS). Safety was documented. RESULTS: Fifty-five patients were enrolled. Seven were women, and the median age was 56 years. Fifty-three (96%) patients had EUST3, and 41 (75%) had EUSN1 disease. Forty-three (78%) patients underwent surgery, 20% achieved a pathCR, and 76.4% underwent an R0 resection. The median survival (n = 55 patients) was 43.3 months (range, 19-75 months). Baseline clinical parameters were not found to be predictive of OS; however, patients with a pathCR (P = .005) and who underwent R0 resection (P ≤ .0001) had an improved OS. There was 1 treatment-related postsurgical death reported. Grade 3 or 4 toxicity (graded according to the National Cancer Institute Common Toxicity Criteria [version 2.0]) was observed in 62% of patients. CONCLUSIONS: The results of the current study documented that this 3-drug, noncisplatin-based chemoradiotherapy was feasible, safe, and active but not better than the published cisplatin-based chemoradiotherapy. A fluoropyrimidine and another cytotoxic (from any class) may be adequate to establish a baseline chemoradiotherapy regimen to combine biologics. Cancer 2010. © 2010 American Cancer Society.

We designed this Phase II trial to assess the efficacy and safety of the addition of bevacizumab to concurrent neoadjuvant capecitabine-based chemoradiation in locally advanced rectal cancer.Between April 2004 and December 2007, 25 patients with clinically staged T3N1 (n = 20) or T3N0 (n = 5) rectal cancer received neoadjuvant therapy with radiotherapy (50.4 Gy in 28 fractions over 5.5 weeks), bevacizumab every 2 weeks (3 doses of 5 mg/kg), and capecitabine (900 mg/m(2) orally twice daily only on days of radiation), followed by surgical resection a median of 7.3 weeks later.Procedures included abdominoperineal resection (APR; 6 patients), proctectomy with coloanal anastamosis (8 patients), low anterior resection (10 patients), and local excision (1 patient). Eight (32%) of 25 patients had a pathologic complete response, and 6 (24%) of 25 had10% viable tumor cells in the specimen. No patient had Grade 3 hand-foot syndrome, gastrointestinal toxicity, or significant hematologic toxicity. Three wound complications required surgical intervention (one coloanal anastamostic dehiscence requiring completion APR and two perineal wound dehiscences after initial APR). Five minor complications occurred that resolved without operative intervention. With a median follow-up of 22.7 months (range, 4.5-32.4 months), all patients were alive; one patient has had a recurrence in the pelvis (2-year actuarial rate, 6.2%) and 3 had distant recurrences.The addition of bevacizumab to neoadjuvant chemoradiation resulted in encouraging pathologic complete response without an increase in acute toxicity. The impact of bevacizumab on perineal wound and anastamotic healing due to concurrent bevacizumab requires further study.

640 Background: In advanced gastrointestinal (GI) malignancies, genetic profiling is often performed with the goal of facilitating enrollment of patients into clinical trials. While multigene genetic profiling has become the standard of care in many practices, the data on success rate of identifying actionable genomic alterations remain limited. In this study, we aimed to characterize the rate of actionable mutations using larger ( > 150 genes) and smaller ( < 150 genes) panels across different GI malignancies. Methods: We reviewed all reports of formalin-fixed paraffin-embedded clinical specimens sent for next-generation sequencing (NGS-using assays of at least 45 genes) for patients with advanced GI malignancies between 2012-2017 at MD Anderson Cancer Center. Actionable mutations were defined as those matching or informing the use of targeted therapies available in clinical trials, or FDA-approved. These were determined by a precision oncology support team (pct.mdanderson.org), using available literature and functional genomic screens. Novel actionable mutations were defined as those not used in current testing guidelines for GI malignancies. Results: Out of 11968 detected mutations, 3832(32.0%) were deemed to be actionable mutations and the remainder were either in non-actionable genes, deemed benign, or variants of unknown significance. Therefore, 1987 (65.1%) of assays had actionable mutations. When limited to novel actionable mutations, the rate fell to 21.5% (659/3052). Compared to CRC, other GI malignancies were 1.65 times more likely to have a novel actionable mutation (95% CI 1.35-2.00, p< .001). The use of larger and smaller panels did not differ in detecting novel actionable mutations, but larger panels resulted in a 3.5-fold higher number of mutations not deemed clinically actionable. Conclusions: Despite incorporation of NGS in oncology practice for GI malignancies, the success rate of detecting novel actionable mutations beyond those in the current guidelines remains low. Using assays with larger gene numbers does not seem to improve this detection rate. Future studies are required to evaluate the success rate of clinical interventions when actionable alterations are present.

127 Background: Patients with localized esophageal adenocarcinoma (L-EAC) who are not suitable for surgery receive definitive chemoradiation. However, there are no biomarkers or imaging variables to predict clinical complete response (cCR; negative bx and physiologic PET post chemoradiation) or prognosticate favorable overall survival (OS). We analyzed tumor metabolic activity by standardized uptake value (SUV) or total lesion glycolysis (TLG). Methods: 266 patients with L-EAC, who declined or were unsuitable for surgery, were analyzed. Cox proportional hazards regression model for OS was analyzed using categorized SUV (low; < 6.5, moderate; 6.5-12.9, or high; ≥12.9) or TLG (low; < 24.2, moderate; 24.2-82.6, or high; ≥82.6). Logistic regression model for cCR was analyzed using categorized SUV (low; < 5.4 or high; ≥5.4) or TLG (low; < 27.0 or high; ≥27.0). Results: Mean SUV and TLG were 12.8 ± 10.7 and 209 ± 376.8, respectively. Both SUV and TLG were significantly associated with the length of the tumor (p < 0.0001) and clinical stage (p < 0.0001). Higher SUV and TLG were significantly associated with shorter OS than low SUV and TLG (moderate SUV; HR 1.79, CI 1.19-2.69, high SUV; HR 2.82, 1.90-4.18, moderate TLG; HR 1.82, 1.14-2.90, high TLG; 3.16, 2.11-4.74). 68 patients (28%) achieved cCR and remained free of recurrence. In the multivariate logistic regression model, low SUV and low TLG highly predicted cCR without recurrence (low SUV; OR 3.15, 1.38–7.19, low TLG; OR 4.79, 2.28-10.08). Conclusions: Tumor metabolic activity is highly associated with prognosis and response to chemoradiation in patients with L-EAC not undergoing surgery. Further refinements (addition of biomarkers) could allow personalized care of these patients.

653 Background: PI3K pathway is a known modulator of anti-tumor immune response and is frequently activated in CRC through genetic alterations such as PTEN loss (PTENloss) and PIK3CA mutations (PIK3CAmut). This study aims to determine the impact of these alterations on immune cell infiltration, priming and activation in early stage CRC patients (pts). Methods: Immune infiltrates and checkpoints were evaluated using quantitative immunohistochemistry (IHC) on primary CRC (N = 59) for both center of tumor (CT) and invasive margin (IM). Pts were evaluated by presence or absence of either PTENloss or PIK3CAmut (collectively termed PI3K pathway alterations). Microsatellite unstable (MSI) and stable (MSS) tumors were analyzed separately. Clinicopathologic data was examined for potential associations with PI3K pathway alterations. Separately, mRNA data (Agilent) was obtained for immune related genes from an internal cohort with PTEN and PIK3CA annotation (N = 73). Results: 59 pts comprised IHC cohort (40 MSS, 19 MSI); 23 pts (39%) had PTENloss or PIK3CAmut. In Agilent cohort, 16 of 73 pts (22%) had PI3K pathway alterations. In MSS CRC, these alterations were more common in CMS1 (p = 0.03), on right side (p = 0.048) and with peritumoral lymphocytes (p = 0.031). MSS pts with PI3K pathway alterations had higher PD1 protein expression (p = 0.04), 2.1 and 2.3 times increased density of CD3+ (p = 0.01) and CD8+ (p = 0.04) cells respectively, and higher Granzyme B protein expression (p = 0.04) in the CT. These pts also had higher PDL1 gene expression (p = 0.046). MSS CRC pts with PIK3CAmut similarly had 2 times more PDL1 protein expression in epithelial cells of the IM (p = 0.01). Alternate checkpoints were also increased in pts with PI3K pathway alterations, including higher protein expression of LAG3 in CT (P = 0.046) and higher gene expression of CTLA4, TIM3, and TIGIT (P < 0.05 for all). Conclusions: PI3K pathway activated MSS CRC is associated with increased immune engagement, but also upregulation of key immune checkpoints in early stage tumors resulting in an ineffective immune response. Combination of PI3K pathway inhibition with immunotherapy merits investigation in this subset of pts.

The high interobserver variability in grading dysplasia in Barrett's esophagus demands a biomarker that can be applied in routine surgical pathology practice. Immunohistochemistry for phosphorylated histone H3 is a reliable marker of identifying mitotic figures and has not been evaluated in Barrett's esophagus-associated neoplastic lesions. We retrospectively studied the expression of phosphorylated histone H3 in 88 endoscopic biopsy samples of Barrett's esophagus without dysplasia (n=19), indefinite for dysplasia (n=11), low-grade dysplasia (n=27), high-grade dysplasia (n=19), or adenocarcinoma (n=12) from a sample of 54 patients. The samples were included after consensus diagnosis of two gastrointestinal pathologists on the hematoxylin–eosin (HE)-stained sections. Anti-phosphorylated histone H3-labeled mitotic figures were counted per 10 consecutive high-power fields (HPFs) in three distinct regions: surface epithelium, upper 2/3, and lower 1/3 of the crypts. Anti-phosphorylated histone H3-labeled mitotic counts for the three compartments of the crypts and the total scores for Barrett's esophagus, indefinite for dysplasia, low-grade dysplasia, high-grade dysplasia, and adenocarcinoma were compared using the Mann–Whitney U test. For each compartment, the number of anti-phosphorylated histone H3-positive nuclei was higher in low-grade dysplasia than in Barrett's esophagus without dysplasia or indefinite for dysplasia (P

Inclusion of histologic classification into the risk assessment of adenocarcinoma arising from Barrett's esophagus (BE) has placed surgical pathologist in the center of clinical care and research endeavors. Recent advances in endoscopic diagnostic and therapeutic modalities demand additional proficiency in diagnosis and grading of dysplasia. The objectives of this study are as follows: (1) Discuss the definition of BE and features differentiating BE vs intestinal metaplasia involving cardia. (2) Describe the morphological approach of diagnosing and grading of dysplasia and differentiation of high-grade dysplasia from intramucosal carcinoma. (3) Role of special stains in diagnosis of BE and dysplasia. (4) Brief review the literature on histologic and endoscopic factors associated with progression of BE to adenocarcinoma. (5) Discuss the biomarkers in progression of BE to adenocarcinoma. The following conclusions from this review are important and should be applied in routine practice. Because of the controversy in defining BE, the histologic type of columnar mucosa and presence or absence of intestinal metaplasia should be specified in pathology report. The major limitations of appropriately diagnosing and grading dysplasia include technical problems related to biopsy processing and staining, presence of acute inflammation, and high interobserver variations among pathologists. The extent of high-grade dysplasia, high-grade dysplasia with certain endoscopic abnormalities, and low-grade dysplasia, when diagnosed with consensus by 2 or 3 gastrointestinal pathologists, has higher risk of progression to adenocarcinoma. All nonhistologic markers are still in the investigational phase and have not yet been validated in phase 3/4 prospective trials.

Barrett’s esophagus (BE)/Barrett’s metaplasia (BM) is a recognized precursor of esophageal adenocarcinoma (EA) with an intermediary stage of dysplasia. The low yield and high cost of endoscopic screening of patients with BE underscores the need for novel biomarkers, such as microRNA (miRNA), which have emerged as important players in neoplastic progression for risk assessment of developing dysplasia/adenocarcinoma. Recently, we reported highly elevated levels of miRNA-196a (miR-196a) in EA and demonstrated its growth-promoting and anti-apoptotic functions. Here, we evaluated miR-196a as a marker of BE progression to low-grade dysplasia, high-grade dysplasia, and EA using microdissected paraffin-embedded tissues from 11 patients. Higher levels of miR-196a were observed in EA, BE, and dysplastic lesions compared with normal squamous mucosa, and in high-grade dysplasia compared with BE and low-grade dysplasia. Using frozen tumor tissues from 10 additional patients who had advanced EA, we evaluated the correlation of miR-196a with its in silico-predicted targets, keratin 5 (KRT5), small proline-rich protein 2C (SPRR2C), and S100 calcium-binding protein A9 (S100A9), which are down-regulated during BE progression. MiR-196a levels inversely correlated with the predicted target mRNA levels in EA. We confirmed that miR-196a specifically targets KRT5, SPRR2C, and S100A9 3′ UTRs using miR-196a-mimic and luciferase reporter-based assays. In conclusion, this study identified miR-196a as a potential marker of progression of BE and KRT5, SPRR2C, and S100A9 as its targets.

Activated nuclear factor-kappa B (NF-kappaB) in the pretreatment cancer tissue of patients with localized esophageal adenocarcinoma (LEA) undergoing preoperative chemoradiation is associated with poor prognosis. It is known that constitutively activated NF-kappaB prior to any therapy portends poor prognosis, and it is also known that activated NF-kappaB in the treated specimen is associated with poor prognosis. However, the prognosis of patients who have treatment-induced activation of NF-kappaB (meaning their cancers activate NF-kappaB during or after therapy) is not been reported. We hypothesized that the treatment-induced activation of NF-kappaB would impart poor prognosis similar to that imparted by constitutively activated NF-kappaB cancer. Patients with LEA who had undergone preoperative chemoradiation plus surgery and had pre- and post-therapy cancer tissue available were selected. Pre- and post-therapy cancer tissues were stained by immunohistochemistry for nuclear staining of NF-kappaB. The overall survival (OS) and disease-free survival were assessed and compared for patients who had intrinsic constitutively activated NF-kappaB cancer with those who had induced activation of NF-kappaB only post-therapy. A total of 41 patients with LEA were investigated. Twenty-five patients had NF-kappaB positive cancer at baseline, and 16 had NF-kappaB negative cancer at baseline but became positive post-therapy. There was no difference in the location, histology grade, clinical stage, or the curative resection (RO) resection rate in the two populations. OS (P = 0.71), disease-free survival (P = 0.86), and median survivals (Converters: 24 months [95% confidence intervals: 7.78 to 40.22]vs. Nonconverters: 34.13 months [95% confidence intervals: 3.54 to 64.27]) were not different between the two groups. Our data suggest that activation of NF-kappaB in response to stress/injury of therapy leads to poor OS. These results need to be confirmed in a larger number of patients. It may be that only pre-therapy evaluation of NF-kappaB is insufficient to assess prognosis of patients with LEA. Additional implications include that when effective anti-NF-kappaB therapies become available, they may have to be considered in patients whose cancers do not have constitutively activated NF-kappaB or cancer may have to be monitored during therapy with biomarker assessments.

BACKGROUND: Previous microarray expression profiling studies have shown that genes located on chromosome region 1q21-23 were down-regulated in the progression of Barrett esophagus to esophageal adenocarcinoma and that, among patients with the latter condition, these genes were differentially expressed between responders and nonresponders of preoperative chemoradiation therapy. It was unclear whether this difference was due to loss of heterozygosity (LOH) or to other genetic alterations at this locus. METHODS: The status of chromosome 1q LOH was retrospectively evaluated in formalin-fixed, paraffin-embedded pretreatment biopsy specimens from 33 patients with locally advanced esophageal adenocarcinoma who were treated with preoperative neoadjuvant therapy, and the findings were correlated with clinicopathologic features and overall survival duration. LOH was determined by polymerase chain reaction analysis of 7 dinucleotide microsatellite markers that spanned chromosomal region 1q21-23. RESULTS: Allelic loss of chromosome 1q with any marker was found in 66% of tumors; 58% of tumors demonstrated loss of chromosome 1q21, and 45% of tumors demonstrated loss of chromosome 1q23. Patients with loss of chromosome 1q21.3 had shorter overall survival duration than patients without loss of chromosome 1q21.3 (P = .02). The difference in survival with loss of the chromosome 1q21.3 region was also found to be significant in a subset of patients with incomplete pathologic response to preoperative therapy (P = .024). CONCLUSIONS: Loss of chromosome 1q was a frequent finding in esophageal adenocarcinoma cases, and loss of the 1q21.3 region was associated with shorter overall survival duration. Cancer 2009. © 2009 American Cancer Society.

BACKGROUND AND STUDY AIMS: The CO 2 laser is a surgical tool that is widely used because of its predictable penetration depth and minimal collateral damage due to efficient absorption of CO 2 laser energy by tissue water. Until recently, endoscopic use was limited by lack of an efficient, flexible delivery system. The aim of the current study was to evaluate the performance, efficacy, and safety of a novel, photonic band-gap CO 2 laser configured for esophageal mucosal ablation. MATERIALS AND METHODS: This was an endoscopic experimental study in a porcine survival model. Initial evaluation was done on ex vivo tissue followed by endoscopic studies at 7-, 10-, 15-, and 20-W power and at 0-, 1-, 2-, 5-, and 10-mm distances, using continuous and pulsed currents, to determine optimal performance settings. In an IACUC-approved protocol, six pigs underwent circumferential ablation of the distal 6 cm of the esophagus at 10W continuous current. The animals were monitored for 2 or 4 weeks to evaluate delayed effects. Prior to euthanasia, the proximal esophagus was ablated to evaluate the homogeneity of ablation and depth of injury immediately after single and repeat ablation. RESULTS: The animals resumed normal diets within 24 hours and experienced no dysphagia or weight loss. Pathology at 2 and 4 weeks revealed complete re-epithelialization with minimal histologic injury. A single application of the laser produced complete transepithelial ablation of a mean of 83.3 % of the surface area (range 55 % - 100 %); depth of injury was to the muscularis mucosa in five pigs and to the superficial submucosa in one pig. With ablation, sloughing, and re-ablation, a mean of 95 % transepithelial ablation was achieved (range 80 % -100 %) with similar depth of injury. CONCLUSIONS: Using a novel, flexible CO 2 laser, homogeneous ablation was achieved with predictable penetration and minimal deep tissue injury. These results warrant further evaluation of the laser in Barrett’s esophagus, as it may overcome the limitations of current technologies including perforation, stricture, and inhomogeneity.

BACKGROUND: In patients with esophageal cancer who receive chemoradiation, tools to predict/prognosticate outcome before administering therapy are lacking. The authors evaluated initial standardized unit value (iSUV) of 18F-fluoro-2-deoxy-D-glucose positron emission tomography and its association with overall survival and the degree of pathologic response after surgery. METHODS: The authors analyzed 161 patients with esophageal adenocarcinoma who had chemoradiation followed by surgery. The log-rank test, univariate Cox proportional hazards model, Kaplan-Meier survival plot, and Fisher exact test were used to analyze dichotomized iSUV and its association with overall survival and pathologic response. RESULTS: The median age of 161 patients was 61 years (range, 26-80 years) and the majority of patients had lower esophageal or gastroesophageal junction involvement. All patients received fluoropyrimidine and, most commonly, a taxane or platinum compound with concomitant radiation. The median radiation dose was 45 grays (Gy) (range, 45 Gy-50.4 Gy). The median iSUV for all patients was 10.1 (range, 0-58). Using the Fisher exact test, iSUV was not found to be associated with the location of the primary cancer. iSUV higher than the median (10.1) was associated with a better pathologic response (P = .06). Patients with primary cancer with iSUV >10.1 had a lower risk for death (hazards ratio of 0.56) compared with those with iSUV ≤10.1. Higher iSUV was nonsignificantly associated with improved survival (P = .07). CONCLUSIONS: Data from the current study suggest that lower iSUV is associated with poor survival and lower probability of response to chemoradiation. iSUV needs to be further evaluated because it may be used to complement other imaging or biomarker assessments to individualize therapy. Cancer 2009. © 2009 American Cancer Society.

Purpose The primary goal of this study was to evaluate whether pathologic response to chemotherapy predicts patient survival after preoperative chemotherapy and resection of colorectal liver metastases (CLM). The secondary goal of the study was to identify the clinical predictors of pathologic response. Patients and Methods A retrospective review was performed of 305 patients who underwent preoperative irinotecan- or oxaliplatin-based chemotherapy, followed by resection of CLM. Pathologic response was systematically evaluated and reported as the mean of the percentage of cancer cells remaining within each tumor. Univariate and multivariate analyses were performed to identify the predictors of pathologic response and survival. Results Cumulative 5-year overall survival rates by pathologic response status were as follows: 75% complete response (no residual cancer cells), 56% major response (1% to 49% residual cancer cells), and 33% minor response (≥ 50% residual cancer cells; complete v major response, P = .037; major v minor response, P = .028). Multivariate analysis revealed that only surgical margin status (P = .050; hazard ratio [HR], 1.77) and pathologic response (major response: P = .034; HR, 4.80; minor response: P = .007; HR, 6.93) were independent predictors of survival. Multivariate analysis of the predictors of pathologic response revealed that carcinoembryonic antigen level ≤ 5 ng/mL, tumor size ≤ 3 cm, and chemotherapy with fluoropyrimidine plus oxaliplatin and bevacizumab were independent predictors of pathologic response. Conclusion Pathologic response predicts survival after preoperative chemotherapy and resection of CLM. Degree of pathologic response represents a new outcome end point for prognosis after resection of CLM.

SUMMARY. Thromboembolic event (TEE) is the most common complication and a second cause of mortality in cancer patients. Multiple hypotheses for occurrence of TEE have been proposed. There are no reports on the frequency/impact of TEE in localized gastroesophageal cancer patients. We hypothesized that TEE at baseline and during chemoradiotherapy (CTRT) in gastroesophageal cancer patients would have an impact on overall survival (OS) of these patients. All consecutive patients with gastroesophageal cancer undergoing CTRT from 2001 to 2004 were eligible for this analysis. Baseline and subsequent TEEs were documented and correlated with patient characteristics and OS. One hundred ninety-eight patients were analyzed. TEEs were documented in 9.6% of the patients. At baseline, TEEs were documented in 4.0% of the patients. During CTRT, TEEs were documented in 6.1% of the patients. Pulmonary embolism (43.5%) and lower extremity venous thromboses (39%) were the most frequent TEEs. Median OS for patients with a TEE occurring at anytime was 17.7 versus 32.0 months for patients who never developed a TEE (P = 0.014). TEEs at baseline correlated with poor median survival: 13.1 versus 30.7 months for those without a TEE (P = 0.029). In a multivariable analysis, TEE at baseline and/or during CTRT was an independent predictor of OS (hazard ratio, 1.818; P = 0.040). Our data are the first to document the frequency of TEE in gastroesophageal cancer patients undergoing CTRT, and that TEE is an independent prognosticator of OS. Active research to prevent and treat TEEs is needed to improve survival of patients with localized gastroesophageal cancer.

Background This report describes the clinical evaluation of a novel, low-cost, high-resolution endoscopic microscope for obtaining fluorescent images of the cellular morphology of the epithelium of regions of the esophagus with Barrett's metaplasia. This noninvasive point imaging system offers a method for obtaining real-time histologic information during endoscopy. Objective The objective of this study was to compare images taken with the fiberoptic endoscopic microscope with standard histopathologic examination. Design Feasibility study. Setting The University of Texas M.D. Anderson Cancer Center Department of Gastroenterology. Patients, Interventions, and Main Outcome Measurements The tissue samples studied in this report were obtained by endoscopic resection from patients with previous diagnoses of either high-grade dysplasia or esophageal adenocarcinoma. Results Three distinct tissue types were observed ex vivo with the endoscopic microscope: normal squamous mucosa, Barrett's metaplasia, and high-grade dysplasia. Squamous tissue was identified by bright nuclei surrounded by dark cytoplasm in an ordered pattern. Barrett's metaplasia could be identified by large glandular structures with intact nuclear polarity. High-grade dysplasia was visualized as plentiful, irregular glandular structures and loss of nuclear polarity. Standard histopathologic examination of study samples confirmed the results obtained by the endoscopic microscope. Limitations The endoscopic microscope probe had to be placed into direct contact with tissue. Conclusions It was feasible to obtain high-resolution histopathologic information using the endoscopic microscope device. Future improvement and integration with widefield endoscopic techniques will aid in improving the sensitivity of detection of dysplasia and early cancer development in the esophagus.

Suppression of annexin A1 (ANXA1), a mediator of apoptosis and inhibitor of cell proliferation, is well documented in various cancers but the underlying mechanism remains unknown. We investigated whether decreased ANXA1 expression was mediated by microRNAs (miRNAs), which are small, non-coding RNAs that negatively regulate gene expression. Using Sanger miRBase, we identified miR-584, miR-196a and miR-196b as potential miRNAs targeting ANXA1. Only miRNA-196a showed significant inverse correlation with ANXA1 mRNA levels in 12 cancer cell lines of esophageal, breast and endometrial origin (Pearson's correlation -0.66, P=0.019), identifying this as the candidate miRNA targeting ANXA1. Inverse correlation was also observed in 10 esophageal adenocarcinomas (Pearson's correlation -0.64, P=0.047). Analysis of paired normal/tumor tissues from additional 10 patients revealed an increase in miR-196a in the cancers (P=0.003), accompanied by a decrease in ANXA1 mRNA (P=0.004). Increasing miR-196a levels in cells by miR-196a mimics resulted in decreased ANXA1 mRNA and protein. In addition, miR-196a mimics inhibited luciferase expression in luciferase plasmid reporter that included predicted miR-196a recognition sequence from ANXA1 3'-untranslated region confirming that miR-196a directly targets ANXA1. miR-196a promoted cell proliferation, anchorage-independent growth and suppressed apoptosis, suggesting its oncogenic potential. This study demonstrated a novel mechanism of post-transcriptional regulation of ANXA1 expression and identified miR-196a as a marker of esophageal cancer.

BACKGROUND. Lymphovascular invasion (LVI) and/or lymph node metastases (LNM) adversely influence the overall survival (OS) of patients with T1 esophageal adenocarcinoma. Although endoscopic therapy may be adequate for patients with T1a cancer, patients with T1b cancer require esophagectomy/lymphadenectomy. The authors hypothesized that LVI status would subclassify T1b cancers and facilitate new therapeutic strategies. METHODS. Ninety-nine consecutive patients with T1 adenocarcinoma were analyzed after they underwent esophagectomy/lymphadenectomy. LNM was assessed in all patients, and LVI was assessed in 89 patients. OS was correlated with pathologic cancer stage in association with LVI and LNM. RESULTS. The 5-year OS rate for patients with T1a tumors (88%) was superior to that for patients with T1b tumors (62%; P = .001). The 5-year OS rate for patients who had cancers without LVI (85%) was superior to the rate for patients who had cancers with LVI (36%; P = .0001). It is noteworthy that, for cancers without LVI, the 5-year OS rate for patients with T1b tumors (77%) was similar to the rate for patients with T1a tumors (90%; P = .08), but it was superior to the rate for patients with T1b tumors that had LVI (27%; P = .006). The presence of LVI and/or LNM resulted in worse 5-year OS (≤37%) compared with the lack of LVI and/or LNM (88%; P < .001). The rate of LNM for patients who had T1b tumors without LVI still was 19%, and the relapse rate was 16%. CONCLUSIONS. The current results demonstrated that LVI distinguishes the biologic behavior of early esophageal cancer, and patients who have T1b cancer without LVI have a clinical biology similar to that of patients with T1a cancer. If LNM before surgery can be diagnosed with high sensitivity by better endoscopic techniques and/or molecular biomarkers, then a new therapeutic paradigm for T1b cancers could emerge. Further research is needed on patients with T1b esophageal adenocarcinoma. Cancer 2008. © 2008 American Cancer Society.

The current American Joint Committee on Cancer (AJCC) esophageal cancer staging for nodal status is difficult to interpret and is based solely on lymph node location relative to the primary tumor's esophageal location. Recent reports suggest that the number of lymph nodes involved is also an important factor. We reviewed our esophageal experience to propose an improved nodal staging system.In all, 1,027 patients with resected esophageal cancer from 1970 to 2005 were reviewed. Lymph nodes stations were assigned according to AJCC criteria. Overall survival was assessed by Kaplan-Meier analysis. The impact of location, number of involved lymph nodes, and use of preoperative chemotherapy or radiation therapy, or both, was assessed.Nonregional nodal involvement (n = 17) was associated with decreased survival compared with regional (n = 441) or celiac nodal (n = 73) involvement (3-year: 0% versus 24% and 23%; p0.001). The number of involved lymph nodes was strongly associated with survival (3-year: 0 nodes = 63%, 1 to 3 nodes = 31%, more than 3 nodes = 13%; p0.001), and multivariable Cox proportional-hazards analysis suggested that the location and number of involved lymph nodes were independent predictors of survival (p0.001). We propose a modified nodal staging system that designates celiac nodes as regional and includes number of involved nodes: pN0, no nodes (3 years = 63%, n = 496); pN1-regional, 1 to 3 nodes (3 years = 32%, n = 292); pN2-regional, more than 3 nodes (3 years = 14%, n = 222); pN3-nonregional node (3 years = 0%, n = 17 [p0.0001]). This modified nodal staging system better predicts survival than the current AJCC nodal staging system in which survival for pN1 (3 years = 24%) and pM1a (3 years = 23%) do not differ (p = 0.67). The use of induction before surgical resection did not alter the predictive effect of the new nodal staging system.Modification of the AJCC nodal classification system to incorporate the number of involved lymph nodes with regional and nonregional node location simplifies and better predicts long-term survival than does the current AJCC nodal system.

Background Endoscopic resection is increasingly utilized for treating early stage esophageal cancer, and endoscopic ultrasound (EUS) frequently guides treatment selection. Studies report greater than 80% sensitivity and 90% specificity, but our experience suggests less accuracy at the gastroesophageal (GE) junction. The objective of this study is to determine the accuracy of EUS for depth of GE junction cancer and the potential treatment implications. Methods A retrospective review of a prospective database was performed for patients from 1995 to 2014 with GE junction esophageal cancer that underwent EUS staging and resection (surgical or endoscopic) without neo-adjuvant therapy. Patient, tumor, EUS, and pathologic characteristics were examined. Results For the 181 patients that met criteria, the median age was 66 years, 17% were female, 91% white, and 98% had adenocarcinoma. Concordance between EUS (u) T and pathologic (p) T was 48%, with 23% under-staged and 29% over-staged. The EUS was accurate in the following: uT0 6% (1 of 18); uT1a 56% (23 of 41); uT1b 58% (41 of 71); uT2 10% (2 of 21); and uT3 70% (21 of 30). Inaccurate EUS depth had potential to lead to over-treatment in 38% (27 of 71) of uT1b and 76% (16 of 21) of uT2. In 50% of pT1a tumors, EUS depth was T1b or greater. Logistic regression revealed tumor length (continuous variable) to be associated with inaccurate uT ( p = 0.016). Accurately staged tumors were significantly longer than inaccurately staged tumors (2.7 vs 1.7 cm, p = 0.011). Conclusions Early to intermediate GE junction tumors are frequently over-staged. This highlights the importance of diagnostic endoscopic resection for determining accurate tumor depth and selecting correct therapy.

Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use -- https://www.nature.com/authors/policies/license.html#terms Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMS) with distinguishing features: CMS1 (MSI Immune, 14%), hypermutated, microsatellite unstable, strong immune activation; CMS2 (Canonical, 37%), epithelial, chromosomally unstable, marked WNT and MYC signaling activation; CMS3 (Metabolic, 13%), epithelial, evident metabolic dysregulation; and CMS4 (Mesenchymal, 23%), prominent transforming growth factor β activation, stromal invasion, and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intra-tumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC - with clear biological interpretability - and the basis for future clinical stratification and subtype-based targeted interventions.

Preoperative chemoradiation in neoadjuvant setting is the standard of care for loco-regional esophageal adenocarcinoma (EAC). However, less than 30% of patients develop complete pathological response indicating need for newer therapeutic strategies. Cyclin dependent kinase 9 (CDK9) is found to be over expressed in EAC compared to Barrett’s esophagus. Our previous studies demonstrated strong antitumor effects on inhibition of CDK9 in EAC both in vitro and in vivo. Here we report augmented tumor regression in irradiated xenografts on combination with Flavopiridol, a well-established clinically used CDK inhibitor, compared to single modality treatments. In vitro studies indicate that Flavopiridol could radiosensitize FLO-1 and SKGT4 EAC cells with sustained 53BP1 foci especially in SKGT4 cells. Flavopiridol and BAY1143572, a more selective inhibitor of CDK9, could radiosensitize additional EAC radiation sensitive and resistant cell lines (SKGT4-R, OE-33 and OE-33-R) as analyzed by MTT assay, apoptosis, formation of 53BP1 foci and clonogenic assay. Flavopiridol and BAY1143572 showed a radio-synergistic action by downregulating MCL-1 and Axl. In conclusion, the radio-sensitizing capacities of CDK9 inhibitors presented here suggest that their adjuvant administration might improve EAC therapy. Note: This abstract was not presented at the meeting. Citation Format: Omkara Lakshmi M. Veeranki, Rashmi Dokey, Alicia Mejia, Zhimin Tong, Jianhu Zhang, Yawei Qiao, Pankaj Kumar Singh, Riham Katkhuda, Barbara Minoo, Jaime Rodriguez Canales, Steven Lin, Sunil Krishnan, Dipen Maru. Role of CDK9 inhibition as a sensitizer to radiation in esophageal adenocarcinoma: In vitro and in vivo efficacy study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5843. doi:10.1158/1538-7445.AM2017-5843

Background: Side of primary tumor has prognostic and predictive significance in metastatic colorectal cancer (mCRC). RAS/BRAF wild type (WT) left (L) sided tumors have improved outcomes with anti-EGFR therapy while right (R) sided tumors do worse. We aimed to identify mutations (MTS) in RAS/BRAF WT patients (pts) which may explain the differing response. Methods: Using a 46 gene panel, we compared MT frequencies by side in 1880 mCRC pts. Overall survival (OS) was summarized with Kaplan-Meier curves, the log rank test, and Cox models. Microsatellite unstable pts were excluded from univariate OS analysis. Results: RAS mutant (MT) pts were more likely to be APC MT (OR 1.64, P Given the association of tumor location and OS, we stratified pts by side and compared APC MT/WT pts. Improved OS with APC MTS was independent of side (L-HR 0.70, P=0.0011; R-HR 0.62, P=0.0012), but pts with R APC WT tumors stood out as an extreme risk group with the worst OS of all L/R and APC MT/WT combinations even in RAS/BRAF WT pts (P When stratifying APC MT by genomic location, only MTS in the mutation cluster region (n=686) that contains axin and β-catenin binding sites remained prognostic in multivariate models (HR 0.63, P Conclusion: APC WT R sided pts represents a group with poor prognosis regardless of RAS/BRAF MT status. Given the difference in CTNNB1 MT rate and importance of MTS in axin/ β-catenin binding sites, WNT signaling differences between L and R sided tumors may be important to explore further. Baseline Characteristics of Patients with APC/RAS/BRAF Wild Type Right Sided TumorsBaseline CharacteristicRight Sided APC/RAS/BRAF WT (N=88, 15.2%)Other Right Sided Tumors (N=492, 84.8%)P-ValueMedian Age (IQR)56 (47-61)56 (48-64)0.23GenderFemale46 (52.3%)241 (49.0%)0.56Male42 (47.7%)251 (51.0%)MSI-H8 (10.5%)25 (6.5%)0.22HistologyAdenocarcinoma50 (56.8%)366 (74.4%)0.0018Mucinous / Signet38 (43.2%)123 (25.0%)Other03 (0.6%)Average # of Mutations Per Patient (+/- SD)1.38+/- 2.013.10 +/- 1.79 Citation Format: Jonathan M. Loree, Krittiya K. Korphaisarn, Michael Lam, Van K. Morris, Kanwal P. Raghav, Michael J. Overman, Cathy Eng, Arvind Dasari, Bryan K. Kee, David Fogelman, Robert A. Wolff, Kenna Shaw, Russell Broaddus, Mark J. Routbort, Rajyalakshmi Luthra, Dipen M. Maru, David G. Menter, Funda Meric-Bernstam, Scott Kopetz. APCWT/RASWT/BRAFWT tumors represent an under recognized poor prognostic group of right sided colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4742. doi:10.1158/1538-7445.AM2017-4742

Chronic infection and associated inflammation have long been suspected to promote human carcinogenesis. Recently, certain gut bacteria, including some in the Fusobacterium genus, have been implicated in playing a role in human colorectal cancer (CRC) development. However, the Fusobacterium species and subspecies involved and their oncogenic mechanisms remain to be determined. We sought to identify the specific Fusobacterium spp. and ssp. in clinical CRC specimens by targeted sequencing of Fusobacterium 16S ribosomal RNA gene. Five Fusobacterium spp. were identified in clinical CRC specimens. Additional analyses confirmed that Fusobacterium nucleatum ssp. animalis was the most prevalent F. nucleatum subspecies in human CRCs. We also assessed inflammatory cytokines in CRC specimens using immunoassays and found that expression of the cytokines interleukin-17A and tumor necrosis factor-alpha was markedly increased but interleukin-21 decreased in the colorectal tumors. Furthermore, the chemokine (C-C motif) ligand 20 was differentially expressed in colorectal tumors at all stages. In in vitro co-culture assays, F. nucleatum ssp. animalis induced CCL20 expression in CRC cells and monocytes. It also stimulated the monocyte/macrophage activation and migration. Our observations suggested that infection with F. nucleatum ssp. animalis in colorectal tissue could induce inflammatory response and promote CRC development. Further studies are warranted to determine if F. nucleatum ssp. animalis could be a novel target for CRC prevention and treatment. Citation Format: Xiangcang Ye, Rui Wang, Rajat Bhattacharya, Delphine R. Boulbes, Fan Fan, Ling Xia, Adoni Harish, Nadim J. Ajami, Matthew C. Wong, Daniel P. Smith, Joseph F. Petrosino, Susan Venable, Wei Qiao, Veera Baladandayuthapani, Dipen Maru, Lee M. Ellis. Fusobacterium nucleatum subspecies animalis influences pro-inflammatory cytokine expression and monocyte activation in human colorectal tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2674. doi:10.1158/1538-7445.AM2017-2674

3574 Background: A limited understanding of the immune microenvironment of mismatch repair-proficient metastatic colorectal cancer (mCRC) impedes efforts to develop effective immunotherapy treatments for the majority of CRC patients. Liver metastatic disease is common and associated with poor outcomes. While T-cell infiltration of liver metastases positively correlates with survival, most mCRC patients do not benefit from checkpoint-blockade therapy. Tissue associated macrophages (TAMs) have been associated with an immune suppressive environment, but their prognostic role in mCRC is largely unknown. Methods: Comprehensive analysis of gene expression and immunohistochemistry (IHC) in 25 microsatellite stable (MSS) untreated liver metastatectomy (LM) specimens was performed. Clinical outcomes including recurrence, immunologic data, and tumor microsatellite status were evaluated and correlated. Results: Principal component analysis of immune and cancer pathway related genes were performed and compared with recurrence status. All samples were confirmed MSS. There were distinct differences in gene expression between patients who remained disease free and those who recurred. Among immune related genes CXCL5, IRF4, IL6R, TNF, CTLA4, ICOS, and ARG1 were relatively over-expressed in non-recurrent tumors, while PPARG, AIRE, and EPCAM were over-expressed in recurrent tumors (FDR 0.2, p < 0.05). Cibersort analysis predicts a significantly higher number of M2 versus M1 macrophages regardless of recurrence status (p < 0.05), with an approximate M1:M2 ratio of 1:2 and a higher total number of M1/M2 macrophages in tumors that recur. On IHC, an average of 29% of cells per sample expressed macrophage marker CD68. Relatively fewer CD3, CD4, and CD8 T cells were observed with average infiltration rates of 7.4%, 3.6%, and 2.6% respectively. Conclusions: CRC liver metastases demonstrate evidence of a large TAM population with significant M2 component and a smaller T cell population. A greater number of TAMs appear to correlate with recurrence, while a more immunogenic phenotype correlates with lower recurrence risk.

e15143 Background: TP53 mutations (mut) are prevalent in colorectal cancer (CRC) patients (pts) and can result in oncogenic gain of function effect as well as loss of tumor suppressor function depending on the mut. Methods: The Cancer Genome Atlas (TCGA) data of 220 CRC pts with tumors sequenced and gene copy numbers (CN) annotated was obtained. Pts with CMS subtype data also available were included (n = 167). Chi square test was used to compare frequency of TP53 mut and CN change among CMS subtypes. TP53 mRNA and protein levels were compared by TP53 mut and CN change using independent sample t test. Results: 86 (51.5%) pts were TP53 mutant, with 35.3% missense and 16.2% loss of function (LOF) mut. LOF mut included 14 non-sense and 13 frameshift mut, and occurred mostly in codons 213 and 306. Missense mut mostly affected codons 175, 248 and 273 in 10, 9 and 8 pts respectively. TP53 mut were most frequent in CMS2 and CMS4 subtypes (62.7 and 62.5% respectively). TP53 mut in CMS2 were mostly missense and CMS 4 pts mostly had LOF mut. TP53 CN loss was seen in 74.7% CMS 2, 68.8% CMS 4, 43.5% CMS 3 and 6.9% CMS1 pts. 84.9% pts with TP53 mut had CN loss vs 28.4% of wild type pts (p < 0.001). Frequency of TP53 CN loss did not vary significantly by type of TP53 mut. Pts with TP53 missense mut had higher p53 mRNA and protein levels than those with LOF mut. Pts with TP53 CN loss had lower mRNA levels but no change in protein levels than those with normal CN. Conclusions: The higher TP53 mRNA and protein levels with missense mut suggest possible gain of function. Research on interactions of TP53 change with wnt, myc, and TGF beta pathway genes may reveal synthetic lethal treatment combinations. [Table: see text]

SMAD4 is an essential mediator in the transforming growth factor-β pathway. Sporadic mutations of SMAD4 are present in 2.1-20.0% of colorectal cancers (CRCs) but data are limited. In this study, we aimed to evaluate clinicopathologic characteristics, prognosis, and clinical outcome associated with this mutation in CRC cases. Data for patients with metastatic or unresectable CRC who underwent genotyping for SMAD4 mutation and received treatment at The University of Texas MD Anderson Cancer Center from 2000 to 2014 were reviewed. Their tumors were sequenced using a hotspot panel predicted to cover 80% of the reported SMAD4 mutations, and further targeted resequencing that included full-length SMAD4 was performed on mutated tumors using a HiSeq sequencing system. Using The Cancer Genome Atlas data on CRC, the characteristics of SMAD4 and transforming growth factor-β pathway mutations were evaluated according to different consensus molecular subtypes of CRC. Among 734 patients with CRC, 90 (12%) had SMAD4 mutations according to hotspot testing. SMAD4 mutation was associated with colon cancer more so than with rectal cancer (odds ratio 2.85; p

586 Background: The impact of intratumor heterogeneity on prognosis in metastatic colorectal cancer (mCRC) is unclear, however relative variant allele frequency (rVAF) of key mutations within a tumor may impact outcomes. Therefore, we sought to determine whether rVAF of RAS ( KRAS & NRAS) mutant (mt) clones impacts overall survival (OS) in mCRC patients (pts). Methods: Using a next generation sequencing panel of 201 cancer related genes, we tested 200 mCRC tumors / matched normals. Mutations, indels, and copy number variant (CNV) information were obtained. An rVAF of RAS clones was determined by dividing RAS mt VAF by the VAF of the mutated gene with the highest allele frequency. This truncal gene served as a marker of the total malignant population in a specimen. Pts were stratified at an rVAF of 50%. OS was compared with Kaplan-Meier curves, the log-rank test, and Cox regression. We assessed the impact of CNV on our findings by correcting the rVAF for CNVs in RASand truncal mutations. Results: Of 200 pts, 15% had RAS mt rVAF < 50%, 40.5% had rVAF ≥ 50%, and 44.5% were RAS wild type (WT). Age, gender, MSI status, histology, and stage at diagnosis were similar between groups. More RAS WT pts had BRAF mutations (19.1% vs 1.2% and 3.3%, P< 0.0001), left sided (78.7% vs 56.8% and 60%, P= 0.02), or poorly differentiated tumors (27.3% vs 8.6% and 13.3%, P= 0.003) compared to pts with rVAF ≥ 50% or rVAF < 50%, respectively. Mean coverage was 807x for RAS and 602x for truncal mutations. OS was better in pts with an rVAF < 50% compared to pts with rVAF ≥ 50% regardless of whether rVAF was corrected for CNV (HR 0.6; 95% CI 0.39-0.93, P =0.029) or not (HR 0.48; 95% CI 0.31-0.82, P= 0.010). mOS for pts with WT, rVAF < 50% and rVAF ≥ 50% tumors were 65.8, 55.7, and 38.6 months ( P= 0.0025). In multivariate models controlling for stage at diagnosis and BRAF mutation, pts with rVAF < 50% (HR 1.75; 95% CI 1.03-2.97, P = 0.04) and rVAF ≥ 50% (HR 2.46; 95% CI 1.66-3.65, P< 0.0001) had worse OS compared to WT pts. When rVAF was used as a continuous variable, every 1% increase in rVAF RAS mt resulted in a 1% increased hazard of death ( P

Current algorithms for surveillance of patients with esophageal adenocarcinoma (EAC) after chemoradiation and surgery (trimodality therapy [TMT]) remain empiric. The authors hypothesized that the frequency, type, and timing of relapses after TMT would be highly associated with surgical pathology stage (SPS), and therefore SPS could be used to individualize the surveillance strategy. Between 2000 and 2010, 518 patients with EAC were identified who underwent TMT at The University of Texas MD Anderson Cancer Center and were frequently surveyed. Frequency, type, and timing of the first relapse (locoregional and/or distant) were tabulated according to SPS. Standard statistical approaches were used. The median follow-up time after esophageal surgery was 55.4 months (range, 1.0-149.2 months). Disease relapse occurred in 215 patients (41.5%). Higher SPS was associated with a higher rate of relapse (0/I vs II/III, P≤.001; 0/I vs II, P=.002; SPS 0/I vs III, P≤.001; and SPS II vs III, P=.005) and with shorter time to relapse (P

Background BRAF mutations occur in 5% to 10% of metastatic colorectal cancers and are biomarkers associated with a poor prognosis. However, the outcomes with standard chemotherapy over sequential lines of therapy in a large cohort of patients with BRAF -mutant tumors have not been described. Patients and Methods We searched the M.D. Anderson Cancer Center databases for patients with colorectal cancer and identified BRAF mutations between December 2003 and May 2012. Patients were analyzed for clinical characteristics, PFS, overall survival, and chemotherapeutic agents used. Survival was estimated according to the Kaplan–Meier method. Results Among the 1567 patients tested for BRAF mutations at our institution, 127 (8.1%) had tumors with BRAF mutations. The 71 patients who presented with metastatic disease received a median of 2 lines of chemotherapy. For the first 3 lines of chemotherapy, median PFS was 6.3 months (n = 69 patients; 95% confidence interval [CI], 4.9-7.7 months), 2.5 months (n = 58 patients; 95% CI, 1.8-3.0 months), and 2.6 months (n = 31 patients; 95% CI, 1.0-4.2 months), respectively. Median PFS was not affected by the backbone chemotherapeutic agent in the first-line setting, whether oxaliplatin-based or irinotecan-based (6.4 months vs. 5.4 months, respectively; P = .99). Conclusion PFS is expectedly poor for patients with BRAF -mutated metastatic colorectal cancer. Despite the ascertainment bias present (with testing preferentially performed in patients suitable for clinical trials in refractory disease), these data provide historic controls suitable for future study design and support the idea that novel therapeutic options are essential in this population.

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely, shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1, verteporfin, significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1+. Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties, suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population. Cancer Res; 74(15); 4170–82. ©2014 AACR.

Pathologic complete response (pCR) to neoadjuvant chemoradiation for esophageal cancer is associated with improved outcomes. We evaluated whether a nomogram designed to predict who would have a pCR after trimodality therapy could also predict outcome after definitive chemoradiation.Patients in this retrospective, single-institution analysis had received chemoradiation without surgery for esophageal cancer from 1998 through 2010; 333 such patients had complete information on all variables required for the pCR nomogram: sex; T status (by endoscopic sonography); tumor grade; tumor avidity on positron emission tomography (PET); and esophagogastroduodenoscopy (EGD)-directed biopsy results after chemoradiation. We used multivariate Cox regression to test potential associations between clinical outcomes [overall survival (OS), locoregional recurrence, and distant metastasis] and patient or treatment factors and the pCR nomogram score; the component variables of the nomogram were not reintroduced into the multivariate analysis.The median follow-up time for all patients (median age 66 years) was 18.2 months (30.7 months for those alive at the time of analysis). Patients with nomogram scores ≤125 (median for all patients) had significantly worse outcomes than patients with scores125: median OS time 19.7 vs. 48.2 months; disease-free survival (DFS) time 6.1 vs. 31.1 months; locoregional failure-free survival time 17.7 months vs. not reached; and distant metastasis-free survival time 11.7 months vs. not reached (all P0.001). Multivariate Cox regression analysis indicated that nomogram score independently predicted each survival outcome, along with other patient and disease factors.The pCR nomogram score predicted survival outcomes in patients receiving definitive chemoradiation for esophageal cancer. Although this nomogram requires further validation, it may prove useful for stratifying patients for clinical trials designed to intensify treatments for patients at the highest risk of relapse.