bluuebunny/crossref_metadata_2025 huggingface dataset:
Advanced-stage colon cancer prior to the screening age: Association with age, race, and insurance status.
Authors: You Y. N. , Xing Y. , Feig B. W. , Rodriguez-Bigas M. A. , Skibber J. M. , Chang G. J. , Cormier J. N.
Published: February 2017

377

Background: Stage-at-diagnosis is a major determinant of outcome in colon cancer. Population-based screening is currently only recommended after age 50, but the number of colon cancers diagnosed before 50 is increasing. We aimed to identify risk factors associated with advanced-stage disease (stage III or IV) among colon cancers diagnosed before 50.

Methods: Between 2003 and 2007, 248,716 colon adenocarcinomas (25,383 diagnosed before age 50) were reported to the National Cancer Database. Patients with familial adenomatous polyposis were excluded. Among the cases diagnosed before 50, independent predictors for presenting at advanced stages (stage III or IV) were identified using multivariate logistic regression.

Results: 10.2% of all colon cancers occur prior to age 50. Stage III or IV disease accounted for 62.2% of the cases before age 50, and 46.9% of those diagnosed at or after age 50 (p<0.001). Among colon cancers diagnosed before 50, independent risk factors for advanced-stage disease included: very young age groups (age 18-30 and age 30-40 vs. age 40-50), African American race (vs. Caucasian), and having Medicaid, non-Medicare government insurance or no insurance (vs. private insurance), after adjusting for gender, tumor location, geographic region, and treatment facility type (Table).

Conclusions: In the unscreened population of patients before age 50, disparities in age, race, and insurance are associated with the stage at presentation. The risk factors identified help alert the treating clinicians toward patients most vulnerable to delays in their cancer diagnosis.

[Table: see text]

No significant financial relationships to disclose.
Oncogenic mutations in MAPK cascade as novel molecular biomarkers for treatment of gastric cancer patients with EGFR inhibitors.
Authors: Corso G. , Marrelli D. , Pascale V. , Garosi L. , Velho S. , Seruca R. , Roviello F.
Published: February 2017

39

Background: Mitogen-activated protein kinase (MAPK) cascade and phosphatidylinositol 3-kinase (PI3K) survival pathways are frequently activated in the progression of gastrointestinal malignancies. In this study, we aimed to determine the frequency of gene mutations in members of these pathways-EGFR, KRAS, BRAF, PIK3CA and MLK3 in a series of 63 gastric carcinomas with high levels of microsatellite instability.

Methods: Gene mutation analysis was performed by PCR amplification followed by direct sequencing. In selected tumor cases, EGFR expression was evaluated by immunohistochemistry. Association studies between molecular data and clinicopathologic characteristics were performed.

Results: Mutations in EGFR (3'-UTR polyA repeat), KRAS, PIK3CA and MLK3 genes occurred in 30 (47.6%), 11 (17.5%), 9 (14.3%) and 2 (3.2%) of the MSI gastric cancer (GC) cases, respectively. No BRAF or EGFR hotspot mutations were identified. Overall, mutations in at least one of these genes were found in 55.6% (35/63) of gastric carcinomas. From those mutant cases 40.0% (14/35) of them had concomitant gene mutations, always involving EGFR polyA deletions. Interestingly, we observed significant associations between oncogenic mutations and female gender (p=0.046) old age of diagnosis (p=0.001) and intestinal subtype (p=0.043).

Conclusions: Our results show that MSI gastric carcinoma frequently shows activation of EGFR-MAPK and PI3K pathways. Within all alterations found, deletions of the A13 repeats of EGFR were common, suggesting this molecular event as an important biomarker for stratification of GC patients for treatment with EGFR inhibitors.

No significant financial relationships to disclose.
Retrospective review of colorectal cancer specimens in individuals younger than age 50 for microsatellite instability testing and DNA mismatch repair enzyme expression.
Authors: Durie N. , Staszewski H. , Palaia T. , Ragolia L. , Nugent W.
Published: February 2017

392

Background: Data from studies performed at tertiary referral centers suggest that the yield of patients with the underlying defect in hereditary non-polyposis colon cancer (HNPCC) could be improved by screening every colon cancer for the phenotypic expression of the MMR defect by immunohistochemistry (IHC) and/or an assay for microsatellite instability (MSI). We propose to determine the incidence of microsatellite unstable colon cancers and the incidence of absence of expression of DNA MMR enzymes in de-identified colorectal cancer specimens from patients under the age of 50 who have presented to Winthrop University Hospital over the past ten years. This incidence will be compared with those suggested by recent large retrospective studies in tertiary care centers.

Methods: Immunoperoxidase staining for MLH1, MSH2, MSH6 and PMS2 were performed on formalin-fixed tissue. MSI assays were performed on microdissected DNA from paraffin-embedded tissue blocks. All cases were tested with five mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and two pentanucleotide repeat markers (Penta C and Penta D). Tumor samples in which two or more altered monoclonal repeat markers were found out of five were classified as MSI-H.

Results: Screening for expression of DNA mismatch repair enzymes and MSI in an enriched selected (by age <50) population of colorectal cancer patients resulted in detection of 7/51 (14%) specimens that were MSI-H and 7/61 (12%) that were “positive” for lack of expression of at least one DNA mismatch repair enzyme.

Conclusions: Our results are similar to those reported in the literature in unselected series of patients with colorectal cancer. IHC staining or MSI analysis alone may be insufficient in selecting those colorectal cancer patients that should be referred for genetic testing for HNPCC. However, screening of an unselected population with both these modalities should have a low but clinically significant yield. Since other research have shown that traditional criteria such as the Bethesda guidelines are inadequate, we support the use of both measures prospectively in all colorectal cancer patients.

No significant financial relationships to disclose.
Optimising magnetic resonance imaging for preoperative staging and surgical planning in colon cancer at 1.5 tesla and 3.0 tesla.
Authors: Hunter C. J. , Scurr E. , Jeyadevan N. , Collins D. J. , Abulafi M. , Tekkis P. P. , Georgiou P. A. , Leach M. O. , Brown G.
Published: February 2017

395

Background: The aim of this study was to determine best technique for future preoperative staging and operative planning in colon cancer using MRI at 1.5T, and feasibility at 3.0T.

Methods: 39 scans were performed in 4 healthy volunteers comparing bowel distension using different oral contrast techniques; no oral preparation, 1L of water, 1L of poly ethylene glycol (Klean-Prep), and 1L of 2.1% barium sulphate and 3.2% sorbitol solution (E-Z-Cat) at 1.5T. Bowel distension was scored blindly by 2 experienced radiologists. A further 35 scans compared image quality of T2-weighted pulse sequences; 2D-TrueFISP, 3D-TrueFISP, HASTE, 2D-TSE and 3D-TSE. Scans were scored for movement artefact, colon wall and colon mesentery visualisation. Signal to noise ratio (SNR) was calculated. Inter-observer agreement was assessed using Cohen's Kappa. Equivalent optimised protocols were derived at 3.0T and tested for feasibility.

Results: Inter-observer agreement for scoring was good (kappa = 0.61-0.67). Sorbitol-barium sulphate gave significantly better small bowel distension than no oral preparation, water and Klean-Prep (mean score 3.8 vs 1.6, 2.2 and 1.9, p≤0.007). 2D TrueFISP sequences gave the highest mean score for scan quality, significantly better than 3D-TSE, 2D-TSE, 3D-TrueFISP and HASTE (mean 10.6 vs 3.2, 7.8, 7.7 and 7.4, p≤0.012). 2D-TSE sequences obtained best SNR, significantly greater than obtained using 3D-TSE (p=0.045). 3.0T sequences were comparable or superior.

Conclusions: Colon imaging using MRI requires fast sequences and adequate bowel distension. Our observations indicate that 2D-TrueFISP and 2D-TSE sequences with 1L Sorbitol-Barium sulphate orally give the best T2-weighted images of the colon wall and adjacent structures for cancer staging at 1.5T; equivalent 3.0T protocols are possible.

No significant financial relationships to disclose.
Final results of a University of Chicago phase II consortium trial of sorafenib (SOR) in patients (pts) with imatinib (IM)- and sunitinib (SU)-resistant (RES) gastrointestinal stromal tumors (GIST).
Authors: Campbell N. P. , Wroblewski K. , Maki R. G. , D'Adamo D. R. , Chow W. A. , Gandara D. R. , Antonescu C. , Stadler W. M. , Vokes E. E. , Kindler H. L.
Published: February 2017

4

Background: GIST pts who develop resistance to IM and SU have few therapeutic options. SOR inhibits KIT, VEGFR, PDGFR-β, and BRAF kinases. In preclinical models, SOR has activity against several IM-RES mutations that are resistant to SU (Heinrich. ASCO 2009).

Methods: We performed a multi-center, phase II trial of SOR in unresectable, KIT-expressing GIST pts who had disease progression on IM by RECIST. After FDA approval of SU for IM-RES GIST, the study was amended in 2/07 to require progression after both IM and SU. Pts received SOR 400 mg orally twice daily. CT scans were obtained Q2 28-day cycles. The primary endpoint was objective response rate. A Simon minimax 2-stage design required 1 response in 18 pts to proceed to a second stage, and 4 responses in 32 IM/SU RES pts for further investigation.

Results: 38 pts (6 IM-RES, 32 IM/SU-RES) enrolled 1/06-9/09 at 6 centers. Median follow-up for survivors: 31 months (mo). Pt characteristics: male 55%; median age 57 (range 42-85); PS 0/1/2: 47%/47%/6%. Median cycles: 4 (range 1-37). 63% pts had at least 1 dose reduction. Partial response (PR): 13% (1 IM-RES, 4 IM/SU-RES); stable disease (SD): 55% (3 IM-RES, 18 IM/SU-RES). Disease control rate (PR + SD): 68%. Median progression-free survival: 5.2 mo (95% CI: 3.4, 7.4). Median overall survival 11.6 mo (95% CI: 8.8, 18.0); 1-year survival 50%; 2-year survival 29%. Three pts remain on trial receiving study drug (1 PR at 34 mo; 2 SD at 18 and 37 mo). Grade 3/4 toxicities (% pts): hand-foot syndrome 45%, hypertension 21%, diarrhea 8%, hypophosphatemia 8%, GI bleed 5%, rash 5%, thrombosis 3%, GI perforation 3%, fatigue 3%, anemia 3%.

Conclusions: SOR is active in IM-and SU-resistant GIST. Some pts treated with SOR experience prolonged disease control. SOR is well-tolerated in GIST pts, but dose reductions are often required. SOR warrants further investigation in GIST. Supported by NCI grant N01-CM-62201.

[Table: see text]
Assay result variability during determination of mismatch repair deficiency status using immunohistochemistry: A transatlantic comparative study.
Authors: Hutchins G. , Handley K. , Baehner F. L. , Lopatin M. , Yaziji H. , Lee M. , Seymour M. T. , Kerr D. J. , Gray R. G. , Quirke P.
Published: February 2017

403

Background: Colorectal cancer patients with deficient mismatch repair (dMMR) have significantly fewer recurrences and may respond less well to chemotherapy. Immunohistochemical (IHC) determination of MMR status is therefore recommended to identify patients in whom adjuvant therapy is not indicated, but little is known regarding the variability of assay results. We aimed to define MMR IHC assay variability in formalin-fixed, paraffin-embedded (FFPE) material from the QUASAR-1 colorectal cancer trial contained within heterogeneity-prone tissue microarrays (TMAs)

Methods: TMA sections of FFPE material from the QUASAR-1 trial were distributed to 2 independent laboratories (Leeds, UK and Vitro Molecular Laboratories [VML], FL, USA) for MMR IHC assays. Serial TMA sections were stained with MLH1/MSH2 using techniques blinded to the other laboratory. Each stained section was double-scored independently and results compared to determine inter-assay variability.

Results: Matched MMR data were available for 1224 cases of MLH1 and 1223 cases of MSH2. Of these, loss of expression of MMR (dMMR) was reported in 160 cases (13.1%) by VML and 179 cases (14.6%) by Leeds. 140 (11.4%) were dMMR in both labs, 20 cases (1.6%) by VML alone and 39 (3.2%) by Leeds alone. Discordant dMMR status was observed in 56/166 (34%) dMMR cases identified with MLH1 and 10/34 (29%) cases identified with MSH2. Kappa coefficients for inter-assay agreement were 0.798 (95% CI = 0.748 - 0.848) for MMR status overall, 0.778 (95% CI = 0.722 - 0.835) for MLH1 and 0.823 (95% CI = 0.714 - 0.932) for MSH2. 85% (33/39) of discordant Leeds dMMR colon cancers were in the right colon where dMMR is more common, compared to 47% of VML discordant dMMR cases.

Conclusions: Independent determination of MMR status by IHC on TMAs is associated with excellent inter-assay agreement. The reasons for MMR case discordance are under further investigation. These results further support routine MMR testing by IHC.

No significant financial relationships to disclose.
Measuring the cost-effectiveness (CE) of therapies treating metastatic colorectal cancer (mCRC).
Authors: Lakdawalla D. , Penrod J. , Maclean R. , Humphrey J. S. , Seabury S.
Published: February 2017

405

Background: Traditional CE approaches often produce results that appear to be at odds with patients' (pts) behavior. Patients with terminal disease often value gains in survival much more highly than is suggested by standard CE estimates. In the context of metastatic cancer, CE classifies as cost-ineffective many therapies that pts appear to value highly. This study assesses whether CE analyses conflict with estimates of pt value for mCRC therapy. We infer pt value from the willingness to pay (WTP) of cancer pts for therapy out-of-pocket (OOP), and compare these estimates to the value implied by traditional CE. In contrast to traditional CE approaches, this study uses real-world data on pts' own WTP for therapy OOP.

Methods: Revealed preference analysis is performed on retrospective claims data, including the pt OOP spending component, to infer the value metastatic cancer pts (N=13,938) place on 29 different therapies for CRC, breast, lung or head and neck cancer. The primary outcome measure is the average value placed by pts on therapy. Secondary outcomes are: utilization of therapy, total expenditures on cancer drugs, and the price elasticity of demand for therapy.

Results: Utilization of therapy displays a negative but inelastic relationship with OOP spending on drugs (the estimated price elasticity is -0.007, with p<0.01). The estimated annual WTP OOP for treatment of mCRC specifically was $207,555, approximately 19 times the average annual cost of therapy ($10,775). Approximately 99% of mCRC pts value the therapy more than the cost. The analysis implies that the average mCRC pt values a statistical life year at approximately $282,000. In comparison, traditional CE analyses employed by regulators assign much lower values for a statistical life-year; for example, the UK's NICE uses a threshold of <$50,000 per year.

Conclusions: Traditional economic valuations of medical treatment perform poorly in the terminal care setting, because they fail to recognize that pts place higher value on care provided at the end of life. As a result, these methods vastly understate the CE of treatment for mCRC and other terminal diseases. Use of pts' own OOP spending behavior appears to produce different conclusions for the value of therapy.

[Table: see text]
Evaluation of the derivation of human colon adenocarcinoma from Lgr5 colon stem cells.
Authors: Chiu V. K. , Paty P. , Chiu T. K. , Le Rolle A. , Shia J. , Zeng Z. , Jarnagin W. R. , Weiser M. R. , Rafii S.
Published: February 2017

410

Background: Human colon stem cells share phenotypic hallmarks of self-renewal and proliferation that are associated with tumor cells. Lgr5 is a marker of colon stem cells and not colon differentiated cells. In mice, deletion of the adenomatous polyposis coli gene in colon stem cells but not in colon differentiated cells, rapidly gives rise to macroscopic adenomas. We investigated the role of Lgr5 colon stem cells in human colon tumors in relation to tumor cell of origin, tumor progression, tumor recurrence, and overall survival.

Methods: Using in situ hybridization, we determined the histological distribution of Lgr5 mRNA in human colon specimens at different stages of tumor development and tumor recurrence after chemotherapy. Using gene expression analysis, we analysed Lgr5 mRNA expression levels in human normal colon (n = 33), normal liver (n = 13), colon adenomas (n = 45), primary colon adenocarcinomas (n = 170), liver metastates (n = 48) and lung metastates (n = 20). We examined the correlation between Lgr5 mRNA expression, K-ras mutation status and overall survival in stage IV colon adenocarcinomas.

Results: Human normal colon Lgr5 mRNA was always expressed at basal level and restricted to human colon stem cells. In contrast, we observed an all (Lgr5(+)) or none (Lgr5(-)) expression in human colon adenomas, adenocarcinomas and liver metastases. When present Lgr5 mRNA expression was increased 3-10 fold compared to normal colon. The Lgr5 gene expression analysis provided similar results with increased expression in 66% of human colon adenomas, 62% of primary colon adenocarcinomas, 72% of colon liver metastases and 55% colon lung metastases when compared to normal colon. We have determined that 22.5% (18/80) of Lgr5(+)and 46.4% (26/56) of Lgr5(-) colon adenocarcinoma specimens have K-ras mutations. Kaplan-Meier estimates of median overall survival in Lgr5(+) and Lgr5(-) Stage IV colon adenocarcinomas were 20 months and 15 months, respectively.

Conclusions: Human colon adenocarcinomas are derived predominantly from Lgr5 colon stem cells. Lgr5(+) colon adenocarcinomas required less frequent K-ras mutation for tumor progression then Lgr5(-) colon adenocarcinomas.

No significant financial relationships to disclose.
EGFR expression in invasive anal carcinoma.
Authors: Paliga A. A. , Onerheim R. , Gologan A. , Spatz A. , Vuong T.
Published: February 2017

412

Background: Squamous cell anal carcinoma (SCAC) treatment remains unchanged since the institution of chemoradiation over 4 decades ago. Epidermal growth factor receptor (EGFR) is a protein often expressed in aggressive cancers and that is the target of the monoclonal antibody: cetuximab. Concurrent cetuximab and radiation has been particularly effective treating squamous cell carcinoma of the head and neck. Like head and neck cancer, anal cancer is an epithelial tumor of the alimentary tract that is radioresponsive and is associated with HPV infection. The rarity of this cancer limits its evaluation for biological markers. This study set out to thoroughly characterize EGFR expression by immunohistochemistry in 101 invasive SCAC tissue samples.

Methods: One hundred and one pretreatment paraffin embedded invasive SCAC biopsies, obtained from the Montreal area between 1999 and 2009, were tested for EGFR expression by immunohistochemistry. All samples were confirmed to harbor invasive anal carcinoma on H&E slide preparations. Corresponding cancerous areas were identified on paraffin tissue blocks and cut out for tissue microarray analysis. Samples were immunostained with an EGFR antibody (clone SPM 341) on the Discovery XT Autostainer (Ventana), and staining was assessed by light microscopy by two pathologists. A semiquantitative combination score combining staining intensity with the percent of cells staining gave a final score: just detectable or weak (1+); moderate (2+); strong/intense (3+).

Results: Of 101 patient biopsies, 82 samples had sufficient material for interpretation. Of these samples, 72/82 (90%) stained positive for EGFR, while 41/82 (50%) samples displayed at least moderate to strong staining.

Conclusions: This is the largest cohort of SCAC tissue samples tested to date for EGFR expression and it confirms that the vast majority of invasive SCAC overexpress EGFR. EGFR likely plays a role in anal cancer tumor-genesis and progression. Testing of EGFR inhibitors in this patient population is justified.

No significant financial relationships to disclose.
Use of gene expression profiling to determine prognosis and therapeutic targets for patients with appendiceal carcinoma.
Authors: Kim M. K. , Blazer D. G. , Stewart J. H. , Guy C. , Shen P. , Levine E. , Hsu S. D.
Published: February 2017

418

Background: Appendiceal carcinomas are rare neoplasms that often present as peritoneal surface malignancies. The tumors are treated similarly to primary colorectal metastases, despite having different biological environments. The aim of this study is to utilize genomic analyses to understand the underlying biology of appendiceal carcinoma in order to better guide prognosis and therapy.

Methods: Forty-one metastatic human tumors to the peritoneum (26 appendiceal carcinoma, 15 colorectal carcinoma) were subjected to global gene expression analysis. Unsupervised hierarchical clustering was used to differentiate phenotypes based on differential gene expression. Phenotypes were further analyzed to find differences in oncogenic pathway deregulation and findings were validated using gene specific inhibitors in vitro.

Results: Unsupervised hierarchical clustering revealed three distinct phenotypes, two consisting of predominantly low grade appendiceal samples (Cluster 1 and Cluster 2) and one consisting of predominantly colorectal samples (Cluster 3). Cluster 1 consisted of patients with good prognosis and Cluster 2 consisted of patients with poor prognosis (p = 0.046). Further characterization of Cluster 1 and Cluster 2 with oncogenic pathway signatures identified increased deregulation of the EGFR pathway in the poor prognosis cluster. Similarly, colon cancer cell lines with the poor prognosis phenotype were found to be more sensitive to gefitinib (EGFR inhibitor) (p = 0.038).

Conclusions: The use of geneexpression profiling can be used to identify prognosis and novel therapeutic targets for patients with appendiceal carcinoma. These methods can serve as models to understand the biology of appendiceal carcinoma and to assist in the design of future trials for patients with appendiceal cancer.

No significant financial relationships to disclose.
The significance of HPV status in patients with anal cancer: A comparative technical analysis.
Authors: Doll C. M. , Roldán G. B. , Klimowicz A. , Gustafson K. , Petrillo S. K. , Chan A. K. , Lees-Miller S. P. , Magliocco A. M.
Published: February 2017

425

Background: HPV status is an important prognostic factor for outcome in head and neck cancers, with improved survival noted in HPV-positive vs -negative tumors. However, this effect has not been studied in anal cancer. Challenges exist in defining reliable and methods of detecting the presence of high-risk HPV virus or the expression of HPV-related proteins. Purpose: To determine p16 expression and HPV16 status, in association with HPV subtyping, in pre-treatment anal cancers, and to correlate marker status with clinical outcome.

Methods: This retrospective study evaluated anal cancer patients treated between 1992-2005 with definitive RT or CRT at a single institution (Tom Baker Cancer Centre, Calgary, Canada). HPV subtyping, p16 protein expression via conventional immunohistochemistry (IHC) and automated quantitative IHC (AQUA), and HPV16 in-situ hybridization (CISH) was performed in a subset of patient tumors with sufficient pretreatment tumor specimen. The correlation between results using these different techniques and association of HPV markers with clinical outcome was evaluated.

Results: 89 patients were identified; M:F 1:2; median age 57 years. Median tumor size was 3.5 cm. All patients were treated with radical external beam RT with curative intent; 81% received concurrent chemotherapy. Clinical CR was observed in 73% of patients at 3 months post-treatment. Median OS was 82 months. Of tumor specimens analyzed for HPV subtype, 78% (28/36) were HPV16. Using conventional IHC (DAB), 80% (28/35) of tumors over-expressed p16 (score 3). HPV16 CISH was positive in 81% (34/42). P16 AQUA™ score correlated with HPV16 subtyping, CISH for HPV16, and p16 DAB. With HPV16 subtyping as the gold standard for HPV16 infection, false positive and false negative rates were 8% and 4% with DAB, and 10% and 13% with CISH. In univariate analysis only p16 AQUA score > 244 (upper 15%) was associated with PFS (p= 0.006-95% CI 8.4 [1.8-38.6]) and OS (p= 0.013-95% CI 4.5 [1.4-15]).

Conclusions: p16 expression via quantitative IHC correlates with other methods, including HPV16 subtyping, and appears more reliable for defining HPV16 status than CISH. Only quantitative IHC identified a subset of patients with worse outcome.

No significant financial relationships to disclose.
Polymorphisms of thymidylate synthase as prognostic factor in rectal cancer.
Authors: Arrazubi V. , Suarez J. , Guerrero D. , Cambra K. , Gomez Dorronsoro M. L. , Arias F. , Balen E. , Teijeira L. , Vera R.
Published: February 2017

433

Background: Neoadjuvant fluoropyrimidine-based chemotherapy (ChT) plus radiotherapy (Rt) is a standard approach for locally advanced rectal cancer. Polymorphisms of thymidylate synthase (TS), the target for fluoropyrimidines, are recognized prognostic factors in colon cancer. The aim of this study was to evaluate the prognostic value of the polymorphisms of TS in rectal cancer after neoadjuvant ChT plus Rt.

Methods: We studied one-hundred consecutive patients with stage II/III rectal cancer between November 2001 and March 2009. Patients underwent surgery 6-8 weeks after neoadjuvant Rt (5,040 cGy) plus fluoropyrimidine-based ChT. DNA was extracted from paraffin embedded biopsies. TS1494del6 and 5′-28bp repeat +G/C SNP polymorphisms were determined.

Results: Sixty-seven percent were men and median age was 67 years. ypT stage was: T0 9%, T1 2%, T2 27%, T3 60% and T4 2%; 32% had locoregional adenopathies. The median follow-up was 45 months and relapse occurred in 20% of patients. Polimorphisms could be determined in 98% of pt: -6bp/-6bp 10%, - 6bp/+6bp 39%, +6bp/+6bp 51% and 2R/2R 72%, 2R/3R 21%, 3R/3R 6%. The grade of pathological tumour regression was not associated with polymorphisms. Relapses occurred in 40% of patients -6bp/-6bp, 22% of patients -6bp/+6bp and 21% of patients +6bp/+6bp. The difference in disease- free survival (DFS) between the first and the third groups was stadistically significative (p=0.049). No relation between 5′-28bp repeat +G/C SNP polymorphism and DFS was found.

Conclusions: Our data suggest that the TS1494del6 polimorphism may be an important prognosis factor in rectal cancer receiving neoadjuvant chemoradiotherapy.

No significant financial relationships to disclose.
Contribution of the epithelial-mesenchymal (EMT) phenotype to the sensitivity of colorectal cancer cell lines to the p21-activated kinase inhibitor, PF-3758309.
Authors: Im E. , Pitts T. M. , Kulikowski G. , Tan A. , Tentler J. J. , Murray B. , Carley W. , Smeal T. , Guo C. , Eckhardt S. G.
Published: February 2017

438

Background: Increased expression of p21–activated kinase (PAK) family proteins has been observed in a range of malignancies including those of breast, ovarian, colorectal (CRC), and pancreatic origin. PAK (PAK 1-6) proteins are critical mediators of cell proliferation, motility, transcription, and translation. In initial studies, we demonstrated that the PAK4 inhibitor, PF-3758309, exhibited robust single agent efficacy against selected CRC cell lines, with IC50s of less than 0.015 uM, whereas some cell lines were relatively resistant to the agent, with IC50s of greater than 1 uM. In subsequent gene array analyses, CRC cell lines sensitive or resistant to PF-3758309 exhibited overexpression of core genes associated with a mesenchymal or epithelial phenotype, respectively. Thus, the goal of this study was to assess the functional consequences of altering the expression of EMT-associated genes and to seek rational combination partners in CRC.

Methods: CRC cell lines that were sensitive to PF-3758309 (IC50=0.015 uM) were transfected with selected short hairpin- (sh)RNAs or micro-(mi)RNAs that are known to regulate EMT. Semi-quantitative RT-PCR and immunoblotting were performed to confirm target knockdown. The transfected cell lines were then exposed to increasing concentrations of PF-3758309 to determine the functional role of these genes in conferring responsiveness to PF-3758309.

Results: Sensitive CRC cell lines were transfected with shRNAs to Zeb1, vimentin, and caldesmon, genes that are associated with a mesenchymal phenotype. Interestingly, when the cell lines were exposed to increasing concentrations of PF- 3758309 they demonstrated a ′right shift′ towards a more resistant phenotype. Likewise, transfection with miRNA 200c, a known suppressor of Zeb1, resulted in a similar shift towards resistance.

Conclusions: These data suggest that the EMT phenotype may play a functional role in determining CRC sensitivity to the PAK4 inhibitor PF-3758309, and in addition, a rational combination targeting the epithelial phenotype with epidermal growth factor receptor inhibitors, may be warranted.

[Table: see text]
The expression of frizzled-3 receptor in colorectal cancer and colorectal adenoma.
Authors: He W. , Wong S. C. , Ma B. , NG S. S. , Lam M. Y. , Chan C. M. , Au T. C. , Chan J. K. , Chan A. T.
Published: February 2017

444

Background: Wnt signaling is well known for its role in colorectal cancer (CRC) formation through transcriptional activities of nuclear β-catenin. Although activation of Wnt signaling depends on specific Wnt/Frizzled receptors (FZD) combinations, the specificity of the interaction and the role of FZD in that particular interaction are still unknown. Among the 10 Wnt receptors of the FZD protein family, FZD-3 is involved in neurodevelopmental abnormalities and gastric cancer carcinogenesis. However, the expression of FZD-3 in CRC is not clear. Therefore in this study, we examined the expression of FZD-3 in CRC cell lines and CRC patient tissues with various pathological stages. The information obtained will be important for us to understand the role of FZD-3 in the development of CRC.

Methods: FZD-3 mRNA expression was studied in CRC metastatic SW620, primary SW480 and normal CCD18co cell lines using quantitative real-time polymerase chain reaction with primers and a Taqman minor grove binder probe (Applied Biosystems, Foster City, USA). Moreover, paraffin-embedded specimens of 40 CRC patient tissues, 25 colorectal adenoma (CA) tissues were retrieved from the Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region for FZD-3 immunostaining using an anti-FZD-3 antibody (Catalog no: MAB1001, R&D systems Inc., Minneapolis, USA) in an automatic Ventana Benchmark XT immunostainer (Ventana Medical Systems Inc., Tucson, USA).

Results: FZD-3 mRNA was up-regulated in metastatic SW620 cell line (fold-change: 622) and in primary SW480 cell line (fold-change: 820) when compared to that in normal CCD18co cell line. Furthermore, immunostaining showed that FZD-3 protein was expressed in 100% (40/40) of CRC specimens and 84% (21/25) of CA specimens. Detailed analysis showed that FZD-3 protein was significantly up-regulated in CRC, CA when compared to their adjacent normal colorectal epithelial tissues (p < 0.0005, Wilcoxon matched pairs test).

Conclusions: This study provided evidence that FZD-3 is involved in CRC carcinogenesis and it is a potential therapeutic target in CRC.

No significant financial relationships to disclose.
Clinical outcome in patients with metastatic colorectal cancer harboring KRAS p.G13D mutation treated with cetuximab.
Authors: Bando H. , Yoshino T. , Shinozaki E. , Yuki S. , Nishina T. , Kadowaki S. , Yamazaki K. , Tsuchihara K. , Fujii S. , Yamanaka T.
Published: February 2017

448

Background: Metastatic colorectal cancer patients with KRAS codon 12 or 13 mutated tumors are presently excluded from treatment with cetuximab (Cmab). On the other hand, a few patients who have mutated KRAS status occasionally respond to Cmab. The tumors of those patients predominantly had codon 13 mutation, and all codon 13 responder have mutation of p.G13D. We now compared the efficacy of Cmab among patients with p.G13D- mutant, other KRAS mutant and KRAS wild-type colorectal cancer.

Methods: The patients from 9 Japanese institutions were retrospectively collected and analyzed. All patients were refractory to fluoropyrimidine, oxaliplatin and irinotecan, and were treated with Cmab and irinotecan combination regimen. Response rate (RR), progression-free survival (PFS) and overall survival (OS) were calculated respectively according to KRAS status.

Results: Ninety four patients were treated with combination therapy. Among 94 cases, 7 cases were p.G13D-mutant KRAS, 23 cases were other mutant KRAS and 63 cases were wild-type KRAS. Baseline characteristics by each subset were well-balanced. While one partial response (PR) and 4 stable diseases (SD) cases were found in 7 p.G13D-mutated cases, no PR was found in other KRAS mutated cases. Median PFS of the patients with p.G13D-mutant, other KRAS mutant and KRAS wild-type were 4.5 months (95%CI 1.7-), 2.3 months (95%CI 1.9-4.3), 4.6 months (95%CI 3.5-6.5) respectively. And median OS of the patients with p.G13D- mutant, other KRAS mutant and KRAS wild-type were 9.3months (95%CI 8.5- 11.8), 7.4 months (95%CI 4.5-9.4), 12.2 months (95%CI 8.7-19.8) respectively. Although statistical significance was not found between the two mutated groups, there are trends that the patients with p.G13D-mutant may have received better clinical benefits from Cmab than the patients with other KRAS mutant.

Conclusions: Cmab may have therapeutic benefit in the patients with KRAS p.G13D-mutant colorectal cancer although further evaluation is warranted.

No significant financial relationships to disclose.
MicroRNA profile and KRAS status in metastatic colorectal cancer.
Authors: Valladares-Ayerbes M. , Blanco M. , Reboredo M. , Lorenzo-Patiño M. J. , Arnal F. , Haz Conde M. , Medina Villaamil V. , Santamarina Caínzos I. , Antón Aparicio L. M. , Calvo Martinez M.
Published: February 2017

453

Background: microRNAs (miRNAs) are small non-coding RNA with regulatory functions. miRNAs expression is deregulated in cancer. Our objective is to establish a miRNA expression profile as a prognostic and predictive signature in Colorectal Cancer (CRC). In this cohort, patients with metastatic (m) CRC have been included. The expression of miRNAs have been analyzed and correlated with clinical and pathologic parameters, including KRAS status, disease free survival (DFS), and overall survival (OS).

Methods: Tissue samples of mCRC were obtained as formalin-fixed paraffin-embedded (FFPE) specimens from the archives of our institution, after obtaining informed consent of patients. Genomic DNA was extracted using Qiaamp DNA mini kit (Qiagen). For the mutational testing of KRAS gene, we use the TheraScreen KRAS PCR kit (Qiagen). miRNA-enriched RNA was extracted using High Pure miRNA Isolation Kit (Roche). miRNA expression was analyzed by quantitative reverse transcription PCR (RT-qPCR) using LNA technology (Exiqon) in a LightCycler 480 (Roche) device. REST2009 (Qiagen), SPSS 19 (IBM), and Prism (GraphPad) software was used to data analysis.

Results: One-hundred and forty mCRC patients have been included. To date, we have extracted DNA and performed KRAS mutational testing for 127 FFPE specimens. From these, 72 (56.7%) were wt while the remaining 55 (43.3%) were mt. We have performed a miRNA expression studies in 28 tumors. miRNAs let-7a, miR-17, miR-21, and miR-200c were analyzed. 5S rRNA and U6 snRNA were amplified as reference genes. miR-21, miR-17, and let-7a were found upregulated (p<0.05) in mCRC samples versus healthy colon. However, for all 4 miRNAs analyzed, there were no significant differences of expression between wt and mt tumor samples for KRAS gene.

Conclusions: The expression of 4 miRNAs analyzed is not correlated with mutations in KRAS gene. However, 3 of 4 miRNAs analyzed were found upregulated in tumors. The results for the association of the expression of miRNAs analyzed with the response to treatment and other clinical parameters will be presented during the symposium. Supported in part by SERGAS, Xunta de Galicia, Grant PS08/77.

No significant financial relationships to disclose.
In silico and ex vivo approaches identify a role for toll-like receptor 4 in the biology of sporadic colorectal cancer.
Authors: Sussman D. A. , Santaolalla R. , Clarke J. , Abreu M. T.
Published: February 2017

460

Background: Several lines of evidence suggest that the host response to bacteria plays a role in the development of colorectal cancer (CRC). Work on innate immune signaling has shown that toll-like receptor-4 (TLR4) is required for colitis-associated neoplasia in a mouse model, suggesting that TLRs may be a critical link between colonic bacteria and the development of colon cancer. The objective of the present study was to test the hypothesis that dysregulated TLR4 expression occurs in a subset of sporadic colorectal cancers.

Methods: Bioinformatic and immunohistochemical approaches were used to test the hypothesis. Publicly available, online microarray data sets of gene expression data (Oncomine and Gene Expression Omnibus) were searched for information relevant to CRC. Searches were included if data sets contained human patient data with paired samples, ≥ 20 subjects per study, and use of standardized microarray platforms. Excluded searches contained patients with genetic cancer syndromes or colitis. Associations were investigated between TLR4 expression and oncogenic pathways. Staining of human CRCs for TLR4 was performed in a consecutive series of 10 patients at the University of Miami. Intensity of staining for TLR4 was calculated using immunofluorescence and NIH's ImageJ software. 5 different areas per 10x-field were quantified per tumor.

Results: Heat maps were generated from the microarray data and demonstrated an increased expression of TLR4 in colon adenomas and CRCs when compared to matched samples of normal colonic mucosa. In silico, expression of TLR4 was positively correlated with expression of COX-2, β-catenin, and EGFR. By IHC, 30% of CRCs have increased TLR4 expression compared with histologically normal epithelium obtained from the tumor margins. ImageJ analysis revealed that CRC's could be segregated based on fluorescent intensity into negative, intermediate, and strongly positive samples.

Conclusions: TLR4 receptor expression defines a subset of patients with sporadic CRC and is associated with expression of key oncogenic pathways. Further study is needed to establish the relationship between TLR4 and CRC outcomes.

No significant financial relationships to disclose.
Inactivation of O6 methylguanine DNA methyltransferase and nitrogen oxide in colorectal cancer.
Authors: Coudry R. A. , Assaf L. , Crespo R. , Santos E. M.
Published: February 2017

461

Background: O6Methylguanine DNA methyltransferase (MGMT or AGT) activity plays an important role in colorectal carcinogenesis. Inactivation of MGMT can occur by promoter CpG island hypermetilation or through inhibition caused by nitrogen oxide (NO) and species derived from it. NO is synthesized from L-arginine by nitric oxide syntases (NOS) and plays a critical role in various biological processes and many disorders including cancer.

Methods: We evaluated the expression of NOS1, NOS2, NOS3 and MGMT protein by immunohistochemistry. Plasma concentrations of nitrate/nitrite were analyzed by spectrophotometry. Methylation in the promoter region of MGMT was investigated by pyrosequencing.

Results: Methylation of MGMT and loss of MGMT expression was detected in 68.8% and 32.6% of tumors, respectively. There was no significant association between MGMT methylation and protein expression with NOS1, NOS2 and NOS3 or nitrate/nitrite. Significant association was found between MGMT expression and methylation status (p < 0.001). NOS2 immunoreactivity was observed in the cytoplasm of the malignant cells (95.7%) and was positively associated with lymph node involvement (p = 0.046) and advanced stage (p = 0.048). Nitrite also showed association with lymph node involvement (p = 0.010).

Conclusions: Loss of expression of MGMT protein is frequently caused by promoter hypermethylation at the MGMT gene and both are not associated with NOS enzymes or nitrate/nitrite. NOS2 overexpression and plasma concentration of nitrite may serve as a predictor for the prognosis of patients with CRC.

No significant financial relationships to disclose.
Genetic profiles of hereditary nonpolyposis colorectal cancer.
Authors: Therklidsen C. , Jonsson G. , Bernstein I. , Nilbert M.
Published: February 2017

469

Background: With the aim to identify genetic markers of hereditary nonpolyposis colorectal cancer (HNPCC), we applied tiling BAC array-based comparative genomic hybridization (aCGH) to 46 HNPCC-associated colorectal cancer.

Methods: 32 k iling BAC arrays were used to generate high-density genomic profiles. Tumors were selected through a case-control design with half of the tumors derived from individuals with disease-predisposing mismatch repair gene mutations and the reminder from phenotypic HNPCC families without identified mutations. In addition, an equal number of sporadic tumors were used for comparison.

Results: Tumors with disease-predisposing germline mutations showed frequent gains of chromosomes 1p (39%), 17 (43%), 19 (57%) and 22q (30%). HNPCC associated tumors without mutations did as a group have more complex alterations with the most frequent changes being gains of 20q (70%), 19 (35%), 17 (26%) and loss of 18 (39%). Gains of 1p and 20q and loss of chromosome 18 were identified as significant discriminators between HNPCC tumors with/without germline MMR gene mutations.

Conclusions: The aCGH profiles of HNPCC-associated colorectal cancer suggest that specific gains and losses may be used to distinguish between tumors with/without germline mismatch repair gene mutations.

No significant financial relationships to disclose.
Effect of changes in the glycolytic metabolism on tumor progression and dissemination in gastric cancer.
Authors: Zieker D. , Koenigsrainer I. , Beckert S. , Glatzle J. , Loeffler M. , Taichman R. S. , Koenigsrainer A.
Published: February 2017

47

Background: Metastases are a frequent finding in gastric cancer patients and are associated with a poor prognosis. Recent data indicate that there might be a link between metabolic changes in the glycolytic system and dissemination in gastric cancer. The enhanced expression of the glycolytic enzyme phosphoglycerate kinase 1 (PGK1) and its signalling targets CXCR4 and CXCL12 seem to play a crucial role in enabling gastric tumours to develop dissemination.

Methods: Microarray analysis was conducted investigating human specimens from consecutive gastric cancer patients with dissemination versus gastric cancer patients without dissemination. Subsequently siRNA-knock- down concerning PGK1 and CXCR4 and transfection (overexpression) of PGK1 was performed. Further, our thesis was investigated in a metastatic nude mouse model simulating human gastric cancer behavior by orthotopic tumour implantation. Tumour growth was detected by PET-MR-imaging and section of the animals.

Results: Microarray analysis revealed a significant overexpression of PGK1, CXCR4 and CXCL12 in gastric cancer specimens with dissemination. Further siRNA-knock-down of PGK1 and CXCR4 showed a significant co-regulation on expression and protein level in vitro. The transfection (overexpression) of PGK1 also revealed a significant upregulation of its signalling targets CXCR4 and CXCL12 on expression and protein level. In addition the transfected cells showed a 50-fold distinctive property in the invasion assay compared to cancer cells without PGK1 overexpression. The in-vivo results obtained in the mouse model showed that elevated PGK1 expression increased invasive and metastatic behavior of implanted gastric tumours significantly and PET-MR-imaging and section correlated very well comparing tumour growth and metastasis.

Conclusions: The overexpression of the glycolytic enzyme PGK1 and its signalling targets in gastric cancer might be a promising regulation-pathway promoting dissemination. This data may provide new prognostic markers and/or potential therapeutic targets to prevent migration of gastric carcinoma cells and has intriguing connection to an old hypothesis advocated by Otto Warburg for tumour metabolism.

No significant financial relationships to disclose.
Patterns of failure in patients with resectable synchronous liver metastases from rectal cancer (SLMRC): An analysis of the outcomes with and without pelvic radiotherapy.
Authors: Butte J. M. , Gonen M. , Ding P. , Goodman K. A. , Wong W. D. , Jarnagin W. R. , Weiser M. R. , D'Angelica M. I.
Published: February 2017

473

Background: The role of pelvic radiation (RT) remains undefined in the management of patients with resectable SLMRC. A retrospective study of patients with SLMRC treated with and without pelvic RT was performed to determine recurrence patterns (distant vs. pelvic) and survival.

Methods: Data from consecutive patients with SLMRC (disease free interval ≤ 12 months) undergoing complete resection of the rectal primary and liver metastases between 1990 and 2008 were identified from a prospective database. Demographics, tumor and treatment related variables were correlated with recurrence patterns. Competing risks analysis was used to determine the risk of pelvic and extra pelvic recurrence.

Results: A total of 185 patients underwent complete resection of a rectal primary and liver metastases. One hundred eighty (97%) received CH during their treatment course and 91 patients (49%) received pelvic RT either before (65, 71.4%) or after (26, 28.6%) rectal resection. The 5-year disease-specific survival was 51% for the entire cohort with a median follow-up of 44 months for survivors. Survival was associated with negative liver margin (p<0.001), absence of LVI (p<0.01), and favorable clinical risk score (p=0.001). Overall, 130 patients (70%) recurred; 18 (10%) having pelvic recurrence as any part of their relapse and 7 (4%) having isolated pelvic recurrence. Recurrence pattern did not correlate with survival. Competing risks analysis demonstrated that the risk of any pelvic recurrence was significantly lower than extra pelvic recurrence (p<0.001), independent of the use of RT (Table).

Conclusions: Pelvic recurrence after complete resection of SLMRC is uncommon and significantly exceeded by extra pelvic failures. Given the low pelvic recurrence risk in the cohort of patients who did not undergo pelvic RT, these data suggest that with proper patient selection, CH and surgical resection without pelvic RT is appropriate for patients with known SLMRC.

[Table: see text]

No significant financial relationships to disclose.