FAK loss reduces BRAFV600E-induced ERK phosphorylation to promote intestinal stemness and cecal tumor formation.

BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a 'just-right' level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vil1-Cre;BRAFLSL-V600E/+;Ptk2fl/fl mice, Fak deletion maximized BRAFV600E's oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a 'just-right' ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.

[A Case of BRAF Mutant Cecal Cancer Treated with Encorafenib, Binimetinib, and Cetuximab Triple Therapy].

A Japanese woman in her early 70's presented to our hospital with abdominal pain and nausea. Abdominal computed tomography showed irregular wall thickening of the ileocecal region and small intestine dilatation. Colonoscopy revealed a tumor lesion at the ileocecal valve and adenocarcinoma was detected in the biopsy specimen. Accordingly, the diagnosis was cecal cancer and bowel obstruction. Right hemicolectomy was performed as palliative surgery, and laparotomy findings revealed peritoneal dissemination. The final staging was pT4a, pN2b, pM1c, pStage Ⅳc, harboring a BRAFV600E mutation. Rapid postoperative tumor progression occurred, leading to multiple liver metastases and ascites. Encorafenib, binimetinib, and cetuximab triple therapy was started as a second line regimen. The therapy was extremely effective. CA19-9 level decreased to within normal range, and the liver tumor size was visibly diminished. After receiving treatment for 2 months in outpatient care, she had to discontinue the treatment due to carcinomatous peritonitis. Unfortunately, she died 6 months after initial diagnosis. BRAF-mutated colon cancer is associated with poor prognosis. In Japan, encorafenib, binimetinib, and cetuximab triple therapy is a new BRAF targeting regimen approved in 2020. We report this clinical course in hopes of eventually achieving better outcomes for patients with this aggressive disease.

Surgical Resection for Local and Lateral Lymph Node Recurrence of MSI-high Cecal Cancer with the BRAF V600E Mutation.

An 84-year-old female underwent open right hemicolectomy with D3 lymph node dissection for cecal cancer, pathologically identified as pT4aN2M0 Stage IIIc and BRAF mutation-positive. Due to early recurrence of abdominal wall and right lateral lymph nodes, the patient was treated with FOLFOXIRI+Bevacizumab. Imaging after 5 courses of chemotherapy found tumor shrinkage and no new metastases. The patient did not tolerate chemotherapy well, and tumor resection was performed. Microsatellite instability (MSI) testing using multiplex polymerase chain reaction (PCR) fragment analysis revealed MSI-high status. The patient is currently recurrence-free without chemotherapy at 1 year postoperatively. BRAF-mutated colorectal cancer has a poor prognosis, and may require resection of the metastatic or recurrent tumor after comprehensive evaluation.

[miRNA Expression Profile in Ileocecal Adenocarcinoma Cells Infected with Cryptosporidium]. / Cryptosporidium ile Enfekte Edilmis Ileoçekal Adenokarsinom Hücrelerindeki miRNA Ekspresyon Profili.

Cryptosporidium spp. is an opportunistic protozoan transmitted by fecal-oral route via oocysts. The agent may cause severe infection especially in individuals with suppressed immune system, due to its intracellular location and ability to cause auto-infection. MicroRNAs (miRNAs) are non-translated endogenous RNA molecules with an average of 22 nucleotides in length that regulate the expression of genes involved in important biological functions such as proliferation, differentiation, apoptosis and immune response. Recent studies have focused on the role of miRNAs in pathogenesis of infectious diseases and their potential to be used as biomarkers. The aim of this study was to determine the miRNA profile of human ileocecal adenocarcinoma (HCT-8) cells at 24 hours of infection with Cryptosporidium spp. In the study, the HCT-8 cell line was infected with Cryptosporidium spp. that were isolated from infected human stool samples and RNA was isolated from the cells 24 hours after infection. After this process, cDNA synthesis was performed and the expression of 95 human miRNA profiles were investigated by polymerase chain reaction (PCR) method. Fold changes of expression were determined by comparison with Cryptosporidium spp. uninfected cell lines. Sequence information of miRNAs and their target genes were performed via TargetScanHuman7.1 and miRDB websites, while gene ontology (GO) pathways of target genes were analyzed with the mirPath v.3 program. It was detected that the expression of 10 miRNAs were upregulated and 11 of them were downregulated compared with the control group. It was observed that, this 21 differentially expressed miRNAs were mainly associated with apoptosis, mitotic cell cycle, and immune response. Hsa-miR-612, hsa-miR-6763-5p, hsa-miR-188-5p, hsa-miR-664b-3p, hsa-miR-210-3p, hsa-let-7e-5p hsa-let-7b-3p, hsa-miR-4787-3p, hsa-miR-548ab, hsa-miR-3714 and hsamiR-4803 were found to be associated with apoptosis; and hsa-miR-612, hsa-miR-664b-3p, hsa-miR210-3p, hsa-let-7e-5p, hsa-let-7b-3p, hsa-miR-548ab, and hsa-miR4803 were found to be associated with mitotic cell cycle. The balance of proliferation and apoptosis is very significant in the development of infection and cancer. It is thought that determination of the effect of miRNAs on proliferation-apoptosis balance could provide information related to the etiopathogenesis and prognosis of infections, and on the role of microorganisms in carcinogenesis. In this study, 12 differentially expressed miRNAs were found to be associated with immune response. This may emphasize the role of miRNAs in the prevention and treatment of infections. It was concluded that, miRNAs could be used in the diagnosis, treatment and prevention of infections with the determination of miRNA's role in the infection mechanism as a result of the increasing number of studies.

Multifocal in situ mantle cell neoplasia of the ileocecal region: a case report with simultaneous nodal and extranodal involvement.

In situ mantle cell neoplasia (ISMCN) is a rare entity of disputed clinical significance. We report an additional case, unusual by its presentation in the large intestine and its multifocal involvement of several nodal and extranodal sites. The diagnosis was made in a 46-year-old male patient from a surgical specimen resected for cecal adenocarcinoma. Gross examination showed multiple small polypoid lesions surrounding the ileocecal valve, corresponding to lymphoid aggregates with hyperplastic follicles. Numerous cyclin D1/SOX11+ lymphoid cells, harboring the t(11;14)(q13;q32) translocation, were present in the inner layers of mantle zones. The same lesions were found in the ileum, the appendix, and the regional lymph nodes. The final diagnosis was multifocal ISMCN of the ileocecal region, with both nodal and extra-nodal involvement. A simple surveillance was decided. Our observation expands the clinical spectrum of the disease and underlines the necessity to closely examine even normal-appearing reactive lymphoid tissues.

Molecular and presymptomatic analysis of a Moroccan Lynch syndrome family revealed a novel frameshift MLH1 germline mutation.

Lynch syndrome (LS) is an autosomal dominant disorder characterized by an increased risk of extracolonic cancers and early age of onset. It is associated with germline mutations in the DNA mismatch repair (MMR) genes. We report a case of a patient with colorectal cancer referred to our medical genetics department for molecular analysis and genetic counseling. The proband is a 64-year-old woman diagnosed with a tumor of the cecum. Histopathological examination showed a moderately differentiated mucinous adenocarcinoma categorized by pT3 N0. Analysis of her pedigree revealed three siblings who had colon cancer, as well as one relative with brain cancer. Based on these findings, molecular genetic investigation was found to be necessary in order to identify the disease-causing mutation. Immunohistochemistry staining of MMR proteins was performed on the tumor sample of the index proband. Mutational analysis of the MLH1/MSH2 genes was carried out. Analysis was extended to the family members and the general population. This led to the identification of a heterozygous frameshift duplication in the MLH1 gene at position 910 (c.910dupG). Three siblings had inherited the mutation from their mother, two of whom were asymptomatic at the time of diagnosis. To the best of our knowledge, this is a novel pathogenic duplication that has not been reported in the databases and literature. The outcome of the present case suggests that this mutation was the primary cause of LS in the family.

Site-specific Differences in Colonic Adenocarcinoma: KRAS Mutations and High Tumor Budding Are More Frequent in Cecal Adenocarcinoma.

Recent literature indicates that adenocarcinomas of the cecum differ with respect to molecular alterations compared with noncecal proximal colon adenocarcinomas and that cecal tumor site may be a prognostically relevant variable. We compared molecular alterations, histopathologic features, and disease-specific survival in a series of 328 colonic adenocarcinomas identified over a 2-year period and stratified by tumor location (cecum, right colon, and left colon). Overall, cecal adenocarcinomas demonstrated the highest frequency of molecular abnormalities with 74% harboring either a KRAS exon 2 or 3 mutation, a BRAF mutation, or DNA mismatch repair protein deficiency. KRAS mutations were more frequently seen in the cecum compared with all other tumor sites (P=0.03). KRAS mutations were identified in 46% of cecal adenocarcinomas compared with only 25% of adenocarcinomas of the right colon (P=0.004). Cecal adenocarcinomas more frequently displayed adverse histopathologic features, in particular high tumor budding (31%), compared with tumors of the right colon (18%; P=0.04) and tumors of the left colon (17%; P=0.02). Overall stage was the most important independent predictor of disease-specific survival in the multivariable analysis; however, cecal tumor site and high tumor budding were also predictive of poor survival, particularly in patients with stage III or IV tumors. In conclusion, cecal adenocarcinomas are characterized by a high frequency of KRAS mutations compared with noncecal right colon tumors, frequently display high tumor budding, and may be a prognostically relevant variable, particularly in patients with stage III or IV disease.

[A Case of Cecal Cancer Associated with Preoperatively Diagnosed Lynch Syndrome Caused by EPCAM Deletion].

A 43-year-old woman underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy for endometrioid adenocarcinoma of the uterine body(Stage ⅢC)at 40 years of age. Screening of the adenocarcinoma samples for Lynch syndrome by immunohistochemistry for mismatch repair proteins indicated a loss of MSH2/MSH6 proteinexpression . A genetic test revealed a deletion of about 20 kb, including exons 8 and 9 of the EPCAM gene. Colonoscopy revealed a type 1 tumor in the cecum. The risk of developing metachronous colorectal cancer and postoperative survival according to the extent of colectomy(total colectomy versus segmental colectomy)and her marked obesity were considered collectively. The patient subsequently selected total colectomy with ileorectal anastomosis. Pathological findings revealed mucinous carcinoma(Stage Ⅱ). Patients with Lynch syndrome caused by deletion of EPCAM are not usually at a high risk of uterine body cancer, but the risk of developing uterine body cancer should be noted when the range of EPCAM deletion extends near to MSH2, as inthis case.

[Long-Term Survival of a Patient with KRAS Mutated Colon Cancer Successfully Treated with Regorafenib].

A 65-year-old woman underwent iliocecal resection for cecal cancer. During post-operative follow-up, she was diagnosed with metastasis to the abdominal wall and a curative resection was performed. After 12 courses of adjuvant chemotherapy with a modified combination of folinic acid, 5-fluorouracil, and oxaliplatin (mFOLFOX6), recurrence was noted in the lung. A curative resection was successfully performed and she was subsequently treated with bevacizumab and a combination of folinic acid, 5-fluorouracil, and irinotecan (FOLFIRI). One year after surgical resection, recurrence in the remnant lung was diagnosed. Because of the KRAS mutation, she could not be treated with anti-epidermal growth factor antibodies. The metastatic lung tumor continued to enlarge. Therefore, we selected regorafenib as third-line chemotherapy. After treatment with regorafenib, the size of the target lesion decreased significantly.

Are all RAS mutations the same? Coexisting KRAS and NRAS mutations in a caecal adenocarcinoma and contiguous tubulovillous adenoma.

Mutations of the human Kirsten rat sarcoma viral oncogene homologue (KRAS) and the highly homologous human neuroblastoma RAS viral oncogene homologue (NRAS) are associated with resistance to antiepidermal growth factor receptor therapies in patients with colorectal cancer. In this report, we describe a caecal adenocarcinoma that contains both KRAS c.35G>T (G12V) and NRAS c.34G>A (G12S) mutations. The adenocarcinoma arises from a contiguous high-grade tubulovillous adenoma, which also carries the identical KRAS and NRAS mutations, supporting their common origin. While KRAS mutations are common in colorectal cancers, NRAS mutations are relatively rare and the coexistence of multiple RAS mutations is not documented, presumably reflecting similar functions of wild-type and mutant forms of RAS. Recent experimental evidence has suggested that KRAS and NRAS may in fact mediate distinct biological processes in the colon, and this unusual case potentially illustrates the hypothesis clinically. Characterisation of the diverse and divergent functions of RAS family members and mutant forms of RAS in the colon form important considerations for the development of RAS-targeting therapeutics.

Mixed adenoneuroendocrine carcinoma of gastrointestinal tract: report of two cases.

Mixed adenoneuroendocrine carcinoma (MANEC) is a rare tumor of the gastrointestinal tract that consists of a dual adenocarcinomatous and neuroendocrine differentiation, each component representing at least 30% of the tumor. To date, only seven cases have been reported in the cecum, and less than 40 in the stomach. Our first case was diagnosed in a 74-years-old female as a polypoid lesion of the cecum with direct invasion in the transverse colon, without lymph node metastases. The second case was diagnosed in the stomach of a 46-years-old male as a polypoid tumor of the antral region that invaded the pancreas and presented metastases in 22 regional lymph nodes. The metastatic tissue was represented by the glandular component. In both cases, the tumor consisted of a moderately-differentiated tubular adenocarcinoma (with mucinous component in Case 1) intermingled with neuroendocrine carcinoma. Ki67 index was lower than 20% in Case 1, respectively higher than 20% in Case 2. The neuroendocrine component was marked by synaptophysin and neuron specific enolase, being negative for Keratins 7/20. The neuroendocrine component represented 60% in Case 1, and 40% in Case 2, respectively. The glandular components were marked by carcinoembryonic antigen, maspin and keratin 20/7 (Case 1/2). Both cases were proved to be microsatellite stable. Independently by the localization and tumor stage, MANECs appear to be highly malignant tumors, with high risk for distant metastases. The aggressiveness seems to depend on the endocrine component, independent of its proportion. The neuroendocrine component could be a dedifferentiated adenocarcinoma with a neuroendocrine phenotype.

A randomised, open-label phase II trial of afatinib versus cetuximab in patients with metastatic colorectal cancer.

PURPOSE: This randomised phase II trial aimed to compare efficacy of the irreversible ErbB family blocker, afatinib, with cetuximab in patients with KRAS wild-type metastatic colorectal adenocarcinoma (mCRC) with progression following oxaliplatin- and irinotecan-based regimens. Efficacy in patients with KRAS mutations was also evaluated. PATIENTS AND METHODS: Patients with KRAS wild-type tumours were randomised 2:1 to afatinib (40 mg/day, increasing to 50 mg/day if minimal toxicity) or cetuximab weekly (400 mg/m2 loading dose, then 250 mg/m2/week) according to number of previous chemotherapy lines. All patients with KRAS-mutated tumours received afatinib. Primary end-points were objective response (OR) for the wild-type group and disease control for the KRAS-mutated group. Secondary end-points were progression-free survival (PFS) and overall survival (OS). RESULTS: Patients with KRAS wild-type tumours (n=50) received afatinib (n=36) or cetuximab (n=14). Unconfirmed and confirmed ORs were 3% and 0% for afatinib versus 20% and 13% for cetuximab (odds ratio: 0.122 [P=0.0735] and <0.001, respectively). Median PFS was 46.0 and 144.5 days for afatinib and cetuximab, respectively. Median OS was 355 days with afatinib but not reached for cetuximab. In the KRAS-mutated group (n=41), five (12%) patients achieved confirmed disease control (stable disease; P=0.6394 [comparison versus 10%]); no ORs were reported. Median PFS and OS were 41.0 and 173days, respectively. Most frequent treatment-related adverse events were diarrhoea and rash across groups. CONCLUSIONS: The efficacy of afatinib was inferior to cetuximab in patients with KRAS wild-type mCRC. In patients with KRAS-mutated tumours, disease control was modest with afatinib. Afatinib had a manageable safety profile.

MicroRNA-31 expression in colorectal serrated pathway progression.

MicroRNAs have been increasingly recognized as useful biomarkers for colorectal cancers (CRC). We have recently observed that microRNA-31 (miR-31) expression is associated with BRAF mutation and prognosis in CRC. Moreover, high miR-31 expression is frequently detected in sessile serrated adenomas compared with hyperplastic polyps (HPs). These results suggest that miR-31 may contribute to the progression of serrated lesions. At a follow-up colonoscopy, we observed the case of a 75-year-old man with a 7-mm flat-elevated lesion in the cecum and diagnosed the lesion as an early invasive carcinoma with serrated features. Tissue specimens were obtained from the representative areas to compare the molecular alterations in the carcinoma component with those in the HP component. Higher miR-31 expression was observed in the carcinoma component (57-fold increase) and the HP component (8-fold increase) compared with the paired normal mucosa, suggesting that miR-31 may be one of the key molecules in serrated pathway progression.

Genetic mechanisms in interval colon cancers.

BACKGROUND AND AIM: The factors underlying the development of interval colon cancers are not well defined and are likely heterogeneous. We sought to determine whether there are distinct molecular properties associated with interval colon cancers. METHODS: Colon cancers diagnosed within 5 years of a complete and well-prepped colonoscopic examination were identified over a 7-year period at a single institution. The clinical and pathological features of the tumors were defined. Analysis of DNA mismatch repair (MMR) and genotyping of a panel of oncogenes associated with colon cancer were performed. RESULTS: Forty-two interval colon cancers were diagnosed at an average age of 70 years. 69 % of tumors were located in the right colon. 41 % of tumors exhibited DNA microsatellite instability (MSI). Loss of staining of DNA MMR proteins by immunohistochemistry (IHC) was confirmed in 82 % of the MSI-positive tumors. Among tumors with abnormal MSI and IHC, 54 % exhibited somatic methylation of the MLH1 promoter, but the remaining 43 % exhibited molecular features indicative of underlying Lynch syndrome (LS). The frequency of somatic mutations in the KRAS, BRAF, NRAS, and PIK3CA oncogenes was similar between interval cancer cases and controls. CONCLUSIONS: Interval colon cancers are not distinguished by the activation of the KRAS, NRAS, BRAF, or PIK3CA oncogenic pathways. However, MSI pathway defects are present in a significant proportion of interval colon cancers. Underlying LS may explain nearly half of these MSI-positive cases, and the remaining cases appear to represent sporadic serrated pathway tumors.

Allelic imbalance at an 8q24 oncogenic SNP is involved in activating MYC in human colorectal cancer.

BACKGROUND: The rs6983267 at 8q24.21 has been established as a significant cancer-related single nucleotide polymorphism (SNP). The risk allele showed similarity to the binding site of transcription factor TCF4/LEF1 that activates transcription of MYC. However, little is known about the role of this SNP in increasing MYC activity in colorectal cancers (CRCs). METHODS: The genotypes of rs6983267 in peripheral blood and primary cancers, MYC activity and copy number (CN) alteration were examined in 107 CRCs. Next, we plotted the number of cancers cell lines exhibiting specific G/T genotypes in 746 cancer cell lines of the Sanger Institute database. Then we validated the relationship between the 8q24 SNP status and clinicopathologic parameters in 68 CRCs with loss of heterozygosity (LOH). RESULTS: The MYC module activity was activated by either transcription in the risk allele (G) or by amplification in the non-risk allele (T). Then, we confirmed that the CN amplification dominantly occurred in the non-risk allele, whereas CN neutral LOH, which indicated uniparental disomy (UPD) was more frequently observed for the risk allele. Finally, we confirmed that risk allele dominant cases, either by amplification or by UPD, indicated a more malignant clinical phenotype than non-risk allele dominant cases. CONCLUSIONS: The development of CRC requires MYC activation through retention of the risk allele, or amplification of the non-risk allele at the oncogenic SNP in the site of primary tumor.