Sporadic Colorectal Adenocarcinoma Research Articles

Ring Finger 43 Hot-spot Frameshift Mutation G659V in Colorectal Cancer Patients: Report from a Tertiary Cancer Care Hospital in North India.

The Ring Finger 43 (RNF43) is a tumor suppressor gene that negatively regulates the Wnt/β-catenin signaling. The p.G659fs is a recurrent RNF43 C-terminal truncating variant frequent in colorectal cancer (CRC) patients. We aimed to identify this hotspot variant in CRC patients and assessed the relationship between the mutation, clinical characteristics, and tumor β-catenin localization. Formalin-fixed, paraffin-embedded tissue samples of upfront, surgically resected, sporadic colorectal adenocarcinoma cases were selected. The p.G659fs mutation was determined by capillary sequencing with sequence-specific primers. Tissue microarray and immunohistochemistry were employed to analyze nuclear β-catenin expression and the expression of mismatch repair (MMR) proteins, respectively. In addition, clinical details were retrieved from the hospital medical records and data were analyzed. The RNF43 p.G659fs mutation was observed in 8% of CRC patients. In total, 25% of tumors showed a loss of immunostaining for one or more MMR proteins and 14.6% of tumors showed positive nuclear β-catenin staining. The p.G659fs variant was significantly enriched in MMR-deficient tumors (P = 0.04). Importantly, no correlation was observed between the variant and nuclear β-catenin localization (P = 0.48), indicating a Wnt-independent role of this variant in CRC tumors. To the best of our knowledge, this is the first study from North India to show the involvement of RNF43 p.G659fs variant in CRC patients. The mutation correlated with MMR protein deficiency and seems to be conferring tumorigenicity independent of the Wnt pathway.

Double Negativity for Expression of YAP1 and CDX2 Defines an Aggressive Type of Colitis-associated Cancer.

Patients with inflammatory bowel disease have markedly increased risk for developing colitis-associated colorectal adenocarcinoma (CAC). There is no established prognostic biomarker for CAC. A retrospective study was performed on a cohort of 57 CACs. Expression of caudal type homeobox transcription factor 2 (CDX2) and YES-associated protein 1 (YAP1) expression was correlated with clinicodemographic and histopathological features. Neither YAP1 nor CDX2 expression alone was significantly associated with tumor invasion beyond the muscularis propria or lymph node status. However, a subgroup of CAC with double negativity for expression of YAP1 and CDX2 was more frequently found in younger patients, and more frequently associated with higher pathological tumor stage and nodal metastasis. Furthermore, a positive correlation between CDX2 and YAP1 expression was identified in CAC and sporadic colorectal adenocarcinoma. Our study demonstrates that double negativity for expression of YAP1 and CDX2 defines a subgroup of CAC with early onset and aggressive clinical features.

Prognostic value of serum inflammatory markers in colorectal cancer.

To date, there have been many studies on postoperative complications and oncologic outcomes, but the findings remain controversial. The purpose of the study was to determine the prognostic value of serum inflammatory markers as pretreatment in curatively resected colorectal cancer. The effects of serum inflammatory marker levels on postoperative morbidity were also examined. Prospectively collected data from 1590 patients with primary sporadic colorectal adenocarcinoma who underwent curative resection between September 2012 and December 2013 at our institution were retrospectively analyzed. White blood cell count (× 103/mcl), neutrophil/lymphocyte ratio, C-reactive protein, erythrocyte sedimentation rate (ESR), and Glasgow prognostic score (GPS) were evaluated. GPS was determined as follows: GPS of 2, elevated CRP level (> 1.0mg/dl) and hypoalbuminemia (< 3.5g/dl); GPS of 1, elevated CRP or hypoalbuminemia; and GPS of 0, neither elevated CRP nor hypoalbuminemia. The median follow-up period for the 1590 patients was 59months. The overall postoperative morbidity rate was 24.8%. In multivariate analysis, female sex (P =0.006), rectal cancer other than colon cancer (P <0.001), CEA level > 5ng/ml (P <0.001), ESR > 27mm/h (P =0.014), and a GPS of 1 or 2 (P <0.001) were independent risk predictors for morbidity. A GPS of 1 or 2 (P <0.001) and postoperative morbidity (P <0.001) significantly affected both overall survival and disease-free survival in multivariate analyses. A GPS of 1 or 2 is a significant risk factor for postoperative morbidity and a prognostic factor for long-term survival.

Prognostic impact of changes in base excision repair machinery in sporadic colorectal cancer

Objectiveto evaluate the prognostic value of base excision repair proteins in sporadic colorectal cancer. MethodsPre-treatment tumor samples from 72 patients with sporadic colorectal adenocarcinoma were assessed for APC, MPG, Polβ, XRCC1 and Fen1 expression by immunohistochemistry. The associations of molecular data were analyzed in relation to clinical features and TNM staging as a prognosis predictor and disease-free survival. ResultsHigher levels of MPG, Polβ and XRCC1, but not Fen1, were associated with unfavorable pathological outcomes, such as poor cellular differentiation, advanced TNM stages, presence of lymphatic and perineural invasions and metastatic lymph nodes. MPG and Polβ overexpression were associated with right-sided CRC. However, only MPG high expression is associated with shorter disease-free survival in CRC patients. ConclusionsOur results suggest that increased expression of MPG, Polβ and XRCC1 are more likely to evolve to poor pathological outcomes, but only the elevated expression of MPG protein predicts recurrence. The BER proteins appear to be suitable candidates to refine the TNM current staging of colorectal cancer.

Low acyl-CoA synthetase 5 expression in colorectal carcinomas is prognostic for early tumour recurrence

It has been shown that the metabolism of long chain fatty acids is involved in colorectal carcinogenesis. Acyl-CoA synthetases (ACSL) activate free fatty acids by synthesis of acyl-CoA thioesters. ACSL isoform 5 (ACSL5) is involved in enterocytic differentiation and maturation by regulating both pro-apoptotic and anti-proliferative effects. Whilst impaired expression of ACSL5 has been associated with sporadic colorectal carcinogenesis, little is known about ACSL5 as a prognostic factor. Aim of this retrospective study was to characterize the prognostic impact of ACSL5 expression levels in sporadic colorectal adenocarcinomas. A total of 72 patients with a median follow-up of 54 months was included. Using a standardized immunohistochemical approach, colorectal adenocarcinomas with low (n=41; group 1) or high (n=31; group 2) ACSL5 levels were identified. In a one-year follow-up, tumour recurrence was significantly increased in group 1 (p=0.0279). The finding was independent of the TNM- and UICC-stage in the surgical resections. Frequency of lymph node metastasis and mortality was not different between the groups. In a long-time follow-up no differences were found between the ACSL5 groups. The data indicate that ACSL5 could be an independent prognostic factor for early recurrence of sporadic colorectal adenocarcinoma.

Dietary risk factors for colorectal cancer in Brazil: a case control study.

BackgroundHigh meat intake and low consumption of vegetables, fruits and whole grains have been associated with increased risk of colorectal cancer in some relevant cohort studies conducted in distinct ethnic populations. The role of the dietary pattern on the risk of sporadic colorectal adenocarcinoma (SCA) in Brazil is unknown; therefore, it was the aim of the present study.MethodsThe dietary patterns of 169 patients with SCA and 101 controls were analysed by food frequency recall. Crude odds ratios were calculated and given within 95 % confidence intervals.ResultsPatients reported higher average intakes of beef (32.0 ± 1.8 versus 23.7 ± 1.6, P = 0.0069), chicken (18.1 ± 0.9 versus 12.2 ± 0.8, P = 0.0002), and pork (8.9 ± 0.9 versus 3.4 ± 0.5, P < 0.0001). These individuals had a 1.025, 1.069, and 1.121-fold increased risk of SCA.Similar consumption of fish, vegetables, fruits and whole grains was reported by patients and controls.ConclusionsMeat consumption is greater in patients with SCA in the Brazilian population. Considering the study population – characterized by ethnic heterogeneity –, the environmental factor related to food habits may be associated with higher incidence of this disease in Brazil.

Association between multidrug resistance 1 gene polymorphisms and genetic susceptibility to sporadic colorectal adenocarcinoma

Objective To investigate the association between genetic polymorphism in exon 26 C3435T and exon 21 G2677T/A of the multidrug resistance 1 (MDR1) gene and the sporadic colorectal adenocarcinoma.Methods A total of 123 patients with colorectal adenocarcinoma and 86 healthy subjects as controls were included.Snapshot SNP typing was used to determine the allele and genotype frequencies of MDR1 C3435T,and G2677T/A.Results There was no significant difference in the distribution of MDR1 C3435T and G2677T/A alleles and genotypes frequencies between control group and case group.When stratified by clinicopathological features such as lesion distribution,differentiation degree and Duke' s stage in the patients with colorectal adenocarcinoma,the statistically significant differences were found in the frequency of C3435T genotype between patients with colon adenocarcinoma (CC 22.2％,CT 63.4％,and TT 14.8 ％) and rectal adenocarcinoma (CC 50.7％,CT 31.9％,and TT 17.4％) (P ＜ 0.01).The frequency of 3435T allele was decreased significantly in patiens with rectal adenocarcinoma (33.3％) as compared with colon adenocarcinoma (46.3％,P ＜0.05).And the frequency of 2677GT genotype was decreased significantly in patients with moderately-differentiated (26.9％) as compared with well-differentiated (46.4％) and poorly-differentiated (47.1％,P ＜ 0.05).Furthermore,the frequency of 2677GT genotype was increased significantly in patients with colon adenocarcinoma (46.3％) as compared with rectal adenocarcinoma (24.6％,P ＜ 0.05),and the frequency of 2677G allele was decreased significantly in patiens with poorly differentiated adenocarcinoma (3.7％) as compared with moderately-differentiated (28.5％) and well-differentiated adenocarcinoma (11.0％,P ＜ 0.05).Conclusion MDR1 C3435T and G2677T/A polymorphisms were found to be not associated with colorectal adenocarcinoma. Key words: Colorectal carcinoma; Multidrug resistance gene; Polymorphism

C936T polymorphism of the VEGF gene in relation to the risk and the clinical and biological characteristics of sporadic colorectal adenocarcinoma.

BackgroundOne of the main glycoproteins responsible for angiogenesis is the vascular endothelial growth factor. It is believed that C936T polymorphism, located in the VEGF gene, is correlated with susceptibility towards development of sporadic colorectal adenocarcinoma. The aim of this study was to identify the frequencies of the genotypes of C936T polymorphism of the VEGF gene in patients with sporadic colorectal adenocarcinoma, in comparison with controls, and whether this correlates with the degree of tumor invasion, lymph node involvement and occurrence of metastases at the time of the diagnosis. The analysis was done on 261 patients with sporadic colorectal adenocarcinoma and 261 controls. The genotypes of C936T polymorphism were evaluated by means of the polymerase chain reaction and enzyme digestion, using peripheral blood samples.ResultsThe occurrences of genotype 936CC were similar in the two groups (80.5% versus 78.5%, p = 0.2288). In relation to tumor location, lymph node involvement, infiltration and tumor metastasis, no statistically significant results were obtained (p = 0.3116, p = 0.8485, p = 0.9408 and p = 0.2861, respectively).ConclusionC936T polymorphism of the VEGF gene did not influence the occurrence of sporadic colorectal adenocarcinoma development and did not correlated with the degree of tumor invasion, lymph node involvement and occurrence of metastases.

Molecular markers for colon diagnosis, prognosis and targeted therapy.

Colorectal adenocarcinoma (CRC), the second leading cancer-related death in the United States, remains a global public health issue. Sporadic CRC is considered the result of sequential mucosal changes from normal colonic mucosa to adenocarcinoma. Efforts in understanding the molecular pathways leading to CRC tumorigenesis may lead to identifying novel, individually tailored therapeutic targets for patients. In this review, we focus on well-published prognostic and predictive markers in CRC and examine their role in clinical practice.

Expression of MACC1 and MET in Inflammatory Bowel Disease-associated Colonic Neoplasia

Metastasis-associated in colon cancer-1 (MACC1), a newly identified regulator of HGF-MET signaling, may participate into the key steps of sporadic colorectal adenocarcinoma development. Given there are many pathogenetic distinctions between colitis-associated colorectal cancer (CAC) and sporadic colorectal adenocarcinomas, the potential roles of MACC1 in CAC carcinogenesis remain unknown. For the first time, we evaluated the expressions of MACC1 and MET in IBD-associated colitis, dysplasia, and adenocarcinoma. Expression was investigated by immunohistochemistry in tissue microarrays consisting of 13 normal colon, 11 active colitis, 9 dysplasia, 51 conventional CAC, 5 mucinous adenocarcinoma, and 1 signet ring cell adenocarcinoma specimens. The expression level of MACC1 or MET was evaluated with H-score system. MACC1 expression was significantly higher in IBD-associated dysplasia than that in corresponding inflammatory or normal colonic tissue, and its level was further elevated from dysplasia to conventional CAC. Higher MACC1 expression was seen in a patient with CAC who had multifocal dysplasia or synchronous carcinoma. MACC1 overexpression (H-score >100) was seen in 67% of conventional CAC but in 0% of dysplasia and 0% of inflammation or normal colon. There was no difference of MACC1 expression found among well, moderately and poorly differentiated CAC. MET expressions in inflammation, dysplasia, and conventional CAC were statistically similar. No parallel expression of MACC1 and MET was detected in this study. MACC1 and MET expression was not increased in mucinous or signet ring cell carcinoma, 2 distinct variants of CAC. Stepwise increase of MACC1 expression from IBD-associated colitis to dysplasia to adenocarcinoma suggests that MACC1 is strongly associated with conventional CAC tumorigenesis in a manner independent of MET. MACC1 may serve as a potential marker for early diagnosis of conventional CAC.

Comparison of clinicopathologic features of young and older patients with sporadic colorectal adenocarcinoma

Comparison of clinicopathologic features of young and older patients with sporadic colorectal adenocarcinoma

Comparison of clinicopathologic features of young and older patients with sporadic colorectal adenocarcinoma

Amac. Yasli ve genc yas hastalarda kolorektal kanserlerin ozellikleriyle ilgili halen suregelen tartismalar vardir. Bu calismada yasin sporadik kolorektal kanserlerin klinikopatolojik karakteristikleri uzerine etkisi retrospektif olarak incelendi. Yontem. Ocak 2001-Eylul 2005 arasinda adenokarsinom nedeniyle Kocaeli Universitesi Tip Fakultesi Genel Cerrahi Klinigi’nde kolektomi yapilan toplam 170 olgu retrospektif olarak incelendi. Bulgular. Yuz yetmis olgudan 40’i (%23,5) elli yas ve altinda, 130’u (%76,5) elli yasin ustunde idi. Elde edilen sonuclar istatistiksel olarak degerlendirildi. Yasli ve genc hasta gruplari arasinda klinik ozellkler, histopatolojik ozellikler, uygulanan cerrahi yontemler veya ameliyat sonrasi komplikasyonlar acisindan fark bulunmadi. Sonuc. Bu calismaya gore sporadik kolerektal adenokarsinomlu genc ve yasli hastalar arasinda kilnik ozellikler, histopatolojik ozellikler, uygulanan cerrahi yontemler veya ameliyat sonrasi komplikasyonlar acisindan fark yoktur. Literaturlerde belirtilen daha once saptanmis olan farklar gentetik varyasyonlarla iliskili olabilir.

Genomic era diagnosis and management of hereditary and sporadic colon cancer.

The morbidity and mortality attributable to heritable and sporadic carcinomas of the colon are substantial and affect children and adults alike. Despite current colonoscopy screening recommendations colorectal adenocarcinoma (CRC) still accounts for almost 140000 cancer cases yearly. Familial adenomatous polyposis (FAP) is a colon cancer predisposition due to alterations in the adenomatous polyposis coli gene, which is mutated in most CRC. Since the beginning of the genomic era next-generation sequencing analyses of CRC continue to improve our understanding of the genetics of tumorigenesis and promise to expand our ability to identify and treat this disease. Advances in genome sequence analysis have facilitated the molecular diagnosis of individuals with FAP, which enables initiation of appropriate monitoring and timely intervention. Genome sequencing also has potential clinical impact for individuals with sporadic forms of CRC, providing means for molecular diagnosis of CRC tumor type, data guiding selection of tumor targeted therapies, and pharmacogenomic profiles specifying patient specific drug tolerances. There is even a potential role for genomic sequencing in surveillance for recurrence, and early detection, of CRC. We review strategies for diagnostic assessment and management of FAP and sporadic CRC in the current genomic era, with emphasis on the current, and potential for future, impact of genome sequencing on the clinical care of these conditions.

Expression of MACC1and MET in IBD-Associated Colorectal Cancer

MACC1, a regulator of the MET signaling pathway, is best known as a marker of poor prognosis and distant metastasis of colorectal adenocarcinoma (CRC) but also participates in the transition from colorectal adenoma to CRC. Despite evidence that CRCs complicating IBD are pathogenetically distinct from sporadic CRCs, the potential role of MACC1 in …

Clinicopathologic and molecular features of sporadic early-onset colorectal adenocarcinoma: an adenocarcinoma with frequent signet ring cell differentiation, rectal and sigmoid involvement, and adverse morphologic features

AbstractRecent literature suggests an increasing incidence of colorectal carcinoma in young patients. We performed a histologic, molecular, and immunophenotypic analysis of patients with sporadic early-onset (≤40 years of age) colorectal carcinoma seen at our institution from the years 2000–2010 and compared these tumors to a cohort of consecutively resected colorectal carcinomas seen in patients >40 years of age. A total of 1160 primary colorectal adenocarcinomas were surgically resected for the years 2000 through 2010. Of these, 75 (6%) were diagnoses in patients ≤40 years of age of which 13 (17%) demonstrated abnormalities in DNA mismatch repair, 4 (5%) were in patients with known germline genetic disorders (two patients with familial adenomatous polyposis, one patient with juvenile polyposis, and one patient with Li-Fraumeni syndrome), and three patients (4%) had long-standing chronic inflammatory bowel disease. The sporadic early-onset colorectal carcinoma group comprised a total of 55 patients (55/1160, 5%) and were compared with a control group comprising 73 consecutively resected colorectal carcinomas with proficient DNA mismatch repair in patients >40 years of age. For the early-onset colorectal carcinoma group, most cases (33/55, 60%) were diagnosed between the age of 35 and 40 years of age. Compared with the control group, the early-onset colorectal carcinoma group was significantly different with respect to tumor location (P<0.007) with 80% (44/55 cases) identified in either the sigmoid colon (24/55, 44%) or rectum (20/55, 36%). Morphologically, early-onset colorectal carcinomas more frequently displayed adverse histologic features compared with the control colorectal carcinoma group such as signet ring cell differentiation (7/55, 13% vs 1/73, 1%, P=0.021), perineural invasion (16/55, 29% vs 8/73, 11%, P=0.009) and venous invasion (12/55, 22% vs 4/73, 6%, P=0.006). A precursor adenomatous lesion was less frequently identified in the early-onset colorectal carcinoma group compared with the control group (19/55, 35% vs 39/73, 53%, P=0.034). Of the early-onset colorectal carcinomas, only 2/45 cases (4%) demonstrated KRAS mutations compared with 11/73 (15%) of the control group colorectal adenocarcinomas harboring KRAS mutations, although this difference did not reach statistical significance (P=0.13). BRAF V600E mutations were not identified in the early-onset colorectal carcinoma group. No difference was identified between the two groups with regard to tumor stage, tumor size, number of lymph node metastases, lymphatic invasion, tumor budding, mucinous histology, or tumor-infiltrating lymphocytes. Both groups had similar recurrence-free (P=0.28) and overall survival (P=0.73). However, patients in the early-onset colorectal carcinoma group more frequently either presented with or developed metastatic disease during their disease course compared with the control colorectal carcinoma group (25/55, 45% vs 18/73, 25%, P=0.014). In addition, 8/55 patients (15%) in the early-onset colorectal carcinoma group developed local recurrence of their tumor while no patients in the control colorectal carcinoma group developed local recurrence (P<0.001), likely due to the increased incidence of rectal carcinoma in the patients with early-onset colorectal carcinoma. Our study demonstrates that colorectal carcinoma is not infrequently diagnosed in patients ≤40 years of age and is not frequently the result of underlying Lynch syndrome or associated with other cancer-predisposing genetic conditions or chronic inflammatory conditions. These tumors have a striking predilection for the distal colon, particularly the sigmoid colon and rectum and are much more likely to demonstrate adverse histologic factors, including signet ring cell differentiation, venous invasion, and perineural invasion.

KRAS mutation detection in Tunisian sporadic coloractal cancer patients with direct sequencing, high resolution melting and denaturating high performance liquid chromatography

The Kirsten Rat Sarcoma (KRAS) oncogene has been introduced recently as a genetic biomarker for metastatic sporadic colorectal cancer prior to anti-EGFR treatment. Identifying patients with KRAS mutations that not respond to EGFR targeted therapies require sensitive, rapid and efficacious routine technique. We have attempted to evaluate the efficiency of three conventional methods: direct sequencing, HRM and DHPLC, to detect mutations in codon 12 and 13 of the KRAS exon2 gene. For this first Tunisian study on KRAS, we detected 45.83% of altered KRAS gene among 48 formalin-fixed paraffin-embedded sporadic colorectal adenocarcinoma patients. The use of HRM-sequencing allowed as enlarging the detected KRAS exon 2 mutations (22/48) in comparison with direct sequencing (17/48). DHPLC was used to confirm results when consensus was not observed between HRM and direct sequencing. This study brings an interesting data concerning an inter-method validation between sequencing and HRM in the investigation of sporadic colorectal cancer biomarker. It also shows that KRAS mutations occur at similar frequencies in Tunisian patients as in other populations; and suggests that the same genes are at play in sporadic CRC cancer, despite ethnic, geographical and environmental differences between countries.

Gene polymorphisms involved in folate and methionine metabolism and increased risk of sporadic colorectal adenocarcinoma

This pilot study has compared the polymorphic genotype frequencies of methylenetetrahydrofolate reductase (MTHFR A1298C and C677T), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G), and thymidylate synthase (TS 2R/3R) in 113 patients with sporadic colorectal adenocarcinoma (SCA) and 188 healthy blood donors, used as matched controls. The aim was to assess the role of these genotypes in the increased risk of SCA among the southeastern Brazilian population. Carriers of genotype MTRR 66GG, or the combined variants MTHFR 1298AC + CC plus 677CT + TT, or MTHFR 677CT + TT plus MTR 2756AG + GG, or MTHFR 1298AC + CC plus 677CT + TT plus MTR 2756AG + GG, or yet, MTHFR 1298AC + CC plus 677CT + TT plus MTRR 66AG + GG, respectively, showed an increased risk of the order of 1.99-, 3.26-, 2.22-, 10.92-, and 14.88-fold of developing SCA when compared with carriers of the other studied polymorphic genotypes, whether in isolation or in combination. In addition, individuals with the MTHFR 677CT + TT or the MTR 2756AG + GG genotypes had a 2.12- and a 1.42-fold increased risks of SCA onset before 50 years of age. African-Brazilians with the MTRR 66GG genotype had a 1.98-fold increased risk of SCA while individuals with the MTR 2756AG + GG and the MTHFR 677CT + TT genotypes showed a 2.11- and a 1.62-fold increased risk of undifferentiated and advanced tumors at diagnosis, respectively. Carriers of genotype MTHFR 1298AC + CC or MTHFR 1298AC + CC plus MTRR 66AG + GG had a 1.42- and a 3.07-fold increased risk of rectal tumor, respectively. Additionally, carriers of MTHFR 677CT + TT or MTHFR 677CT + TT plus TS 2R/3R + 3R/3R had a 1.55- and a 5.39-fold increased risk for colon tumor, respectively, in comparison with carriers of the wild genotypes. These data suggest that all polymorphisms coding for folate and methionine-dependent enzymes, particularly when present in combination with other polymorphisms, have consistent roles in the increased risk of SCA among the southeastern population of Brazil.

BDNF/TrkB Content and Interaction with Gastrin-Releasing Peptide Receptor Blockade in Colorectal Cancer

Objective: Neurotrophin and neuropeptide pathways are emerging targets in cancer. Here we show that brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, are present in colorectal cancer and that BDNF levels are increased in tumors compared to nontumor tissue. In addition, we investigate the role of BDNF in influencing the response of colorectal cancer cells to inhibition of gastrin-releasing peptide receptors (GRPR). Methods: Fresh-frozen sporadic colorectal adenocarcinoma specimens and adjacent nonneoplastic tissue from 30 patients, as well as paraffin-embedded colorectal cancer samples from 21 patients, were used in this study. Cell proliferation and mRNA and protein levels were examined in HT-29 or SW620 cells treated with a GRPR antagonist, human recombinant BDNF (hrBDNF), a Trk antagonist K252a, or cetuximab. Results: Expression of BDNF and TrkB was detected in tumor samples and cell lines. BDNF levels were higher in tumor samples compared to nonneoplastic tissue. BDNF expression and secretion were increased by GRPR blockade in HT-29 cells through a mechanism dependent on epidermal growth factor receptors. Treatment with hrBDNF prevented the effect of GRPR blockade on cell proliferation, whereas a Trk inhibitor reduced proliferation. Conclusions: BDNF and TrkB are present in colorectal cancer and might contribute to resistance to GRPR antagonists.

Aberrant methylation of hMLH1 and p16INK4a in Tunisian patients with sporadic colorectal adenocarcinoma

The methylation of CpG islands in the promoters is associated with loss of protein via repression of gene transcription. Several studies have demonstrated that tumour suppressor and DNA repair genes are often aberrantly hypermethylated in colorectal cancer. The present study was conducted to examine whether the methylation profile of p16INK4a and hMLH1 (human mutL homologue 1) promoters was associated with clinical features and patients' survival in CRC (colorectal carcinoma). Aberrant methylation of p16INK4a and hMLH1 promoters was found in 47.2 and 53.4% of tumours respectively. For adjacent non-tumoral mucosa, p16INK4a was fully unmethylated in 30% of the cases, whereas hMLH1 was predominantly unmethylated (76%). Methylation of p16INK4a correlated with gender and tumour size (P=0.005 and 0.035 respectively), whereas those of hMLH1 significantly correlated with overall survival (P log rank=0.007). Concomitant methylation of p16INK4a and hMLH1 was associated with TNM (tumour, lymph node and metastases) stage and tumour size (P=0.024 and 0.021 respectively). Our data show that loss of hMLH1 expression through aberrant methylation could be used as a marker of poor prognosis in CRC.

Abstract 822: KRAS and BRAF mutation analysis using formalin fixed and paraffin embedded colorectal tissue and fine needle aspiration biopsies from thyroid

Abstract Point mutations in codons 12, 13 and 61 of the KRAS gene and in codon 600 of the BRAF gene can be identified in neoplastic tissue from many different organs. KRAS mutations are present in approximately 40% of sporadic colorectal adenocarcinoma and this analysis is important to determine response to anti-EGFR targeted therapies. BRAF mutations are present in approximately 40% of the papillary thyroid carcinoma and 10% sporadic colorectal adenocarcinoma. BRAF mutation V600E is associated with more aggressive thyroid tumors and its detection is important for thyroid cancer prognosis. Detection of BRAF mutation in colorectal cancer is also useful for characterizing sporadic tumors. A method developed in-house to identify mutations in KRAS (codos 12 and 13) was validated using formalin-fixed paraffin-embeded tissue (FFPE) from 31 surgical specimens diagnosed with metastatic colorrectal cancer. Similarly, a method for BRAF mutation analysis (codon 600) was developed and validaded using 39 smears from fine needle aspiration biopsies (FNAB) of the thyroid. Cytology analysis was performed to confirm the presence of neoplastic or suspected lesions. DNA was extract from 1-4 slides containing the FFPE tissue sections or smears from the FNAB. Mutation analyses were performed by Sanger sequencing and results were compared with pyrosequencing. Automatic analysis of nucleotide sequences using the SeqScape software (Applied Biosystems) was also implemented. There was 85.7% and 100% concordance between Sanger sequencing and pyrosequencing for KRAS and BRAF mutation analysis, respectively. Precision intra- and inter-assay analysis were concordant, with the exception of one sample with KRAS mutation. Sensitivity was evaluated using pools containing different proportions of mutated and non-mutated DNA from cell lines. Data analysis by visual inspection resulted in the detection of the mutant allele in samples with at least 10% of mutated DNA. However, KRAS and BRAF mutations analysis using SeqScape software detected the mutant allele in pools containing at least 30% and 25% of mutated DNA, respectively. This is in accordance with the overall sensitivity observed by standard sequencing analysis. In conclusion, KRAS and BRAF mutation analysis can be successfully performed using small amount of tissue sections from FFPE specimens and smears from FNAB. This material has the advantage of requiring small amounts of sample and also allows cytology review of the tissue content prior to molecular analysis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 822.