Impact of KRAS and BRAF mutations on treatment efficacy and survival in high-grade gastroenteropancreatic neuroendocrine neoplasms.

High-grade gastroenteropancreatic neuroendocrine neoplasms (HG GEP-NEN) typically disseminate early. Treatment of metastatic disease has limited benefit and prognosis is generally discouraging. Data on the clinical impact of mutations in HG GEP-NEN are scarce. There is an unmet need for reliable biomarkers to predict treatment outcome and prognosis in metastatic HG GEP-NEN. Patients with metastatic HG GEP-NEN diagnosed at three centres were selected for KRAS-, BRAF mutation and microsatellite instability (MSI) analyses. Results were linked to treatment outcome and overall survival. After pathological re-evaluation, 83 patients met inclusion criteria: 77 (93%) GEP neuroendocrine carcinomas (NEC) and six (7%) GEP neuroendocrine tumours (NET) G3. NEC harboured higher frequency of mutations than NET G3. Colon NEC harboured a particular high frequency of BRAF mutations (63%). Immediate disease progression on first-line chemotherapy was significantly higher for NEC with BRAF mutation (73%) versus wild-type (27%) (p = .016) and for colonic primary (65%) versus other NEC (28%) (p = .011). Colon NEC had a significant shorter PFS compared to other primary sites, a finding independent of BRAF status. Immediate disease progression was particularly frequent for BRAF mutated colon NEC (OR 10.2, p = .007). Surprisingly, BRAF mutation did not influence overall survival. KRAS mutation was associated with inferior overall survival for the whole NEC population (HR 2.02, p = .015), but not for those given first-line chemotherapy. All long-term survivors (>24 m) were double wild-type. Three NEC cases (4.8%) were MSI. Colon NEC with BRAF mutation predicted immediate disease progression on first-line chemotherapy, but did not affect PFS or OS. Benefit of first-line platinum/etoposide treatment seems limited for colon NEC, especially for BRAF mutated cases. KRAS mutations did not influence treatment efficacy nor survival for patients receiving first-line chemotherapy. Both frequency and clinical impact of KRAS/BRAF mutations in digestive NEC differ from prior results on digestive adenocarcinoma.

Mutation Spectrum in Liquid Versus Solid Biopsies From Patients With Advanced Gastroenteropancreatic Neuroendocrine Carcinoma.

PURPOSE: Gastroenteropancreatic neuroendocrine carcinomas (GEP-NEC) are rare and have a poor prognosis. Most GEP-NEC are diagnosed with metastatic disease, with only minor biopsies available for molecular diagnostics. We assessed the applicability of liquid biopsies for molecular profiling of GEP-NEC. MATERIALS AND METHODS: We performed massive parallel sequencing of 76 cancer-related genes in circulating tumor DNA from 50 patients with advanced GEP-NEC and compared findings to previous analyses of solid tumor biopsies from the same patients. Plasma samples were collected before therapy, and the median time span between blood and tissue sampling was 25 days. RESULTS: We detected 178 somatic mutations in the liquid biopsies, 127 (71%) were also detected in the solid biopsies, whereas 51 (29%) were unique to the liquid biopsies. In the same 76 genes, we previously detected 199 somatic mutations (single nucleotide variants) in solid biopsies, of which 127 (64%) were also now detected in liquid biopsies. In exploratory subgroup assessments, concordance was higher in patients with liver metastases (P = 1.5 × 10-5) and increasing with level of liver involvement (P = 1.2 × 10-4). The concordance was similar between GEP-NEC with different primary sites, except being lower in esophageal cases (P = .001). Concordance was not associated with tumor mutation burden. Tumor tissue mutations also detected in liquid biopsies was lower for MSI (40%) versus MSS tumors (70%; P = 7.8 × 10-4). We identified potentially targetable mutations in plasma of 26 (52%) of patients with GEP-NEC; nine patients (18%) had potentially targetable mutation detected only in liquid biopsies. CONCLUSION: Liquid biopsy analyses may be an applicable alternative to solid biopsies in GEP-NEC. Liquid biopsies may add additional mutations compared with tumor biopsies alone and could be useful for biomarker assessment in clinical trials for these patients.

The level of claudin-7 is reduced as an early event in colorectal carcinogenesis.

BACKGROUND: Compromised epithelial barriers are found in dysplastic tissue of the gastrointestinal tract. Claudins are transmembrane proteins important for tight junctions. Claudins regulate the paracellular transport and are crucial for maintaining a functional epithelial barrier. Down-regulation of the oncogenic serine protease, matriptase, induces leakiness in epithelial barriers both in vivo and in vitro. We found in an in-silico search tight co-regulation between matriptase and claudin-7 expression. We have previously shown that the matriptase expression level decreases during colorectal carcinogenesis. In the present study we investigated whether claudin-7 expression is likewise decreased during colorectal carcinogenesis, thereby causing or contributing to the compromised epithelial leakiness of dysplastic tissue. METHODS: The mRNA level of claudin-7 (CLDN7) was determined in samples from 18 healthy individuals, 100 individuals with dysplasia and 121 colorectal cancer patients using quantitative real time RT-PCR. In addition, immunohistochemical stainings were performed on colorectal adenomas and carcinomas, to confirm the mRNA findings. RESULTS: A 2.7-fold reduction in the claudin-7 mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001). Reductions in the claudin-7 mRNA levels were also detected in mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01) and carcinomas (p < 0.01), compared to a control sample from the same individual. The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings. CONCLUSIONS: Our results show that the claudin-7 mRNA level is decreased already as an early event in colorectal carcinogenesis, probably contributing to the compromised epithelial barrier in adenomas.

Collagen mRNA levels changes during colorectal cancer carcinogenesis.

BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6) and type VII collagen (alpha1) during colorectal cancer carcinogenesis. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels for alpha1(IV), alpha 4(IV), alpha 6(IV), and alpha1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals. In addition, corresponding tissue was examined from healthy volunteers (n = 20). mRNA levels were normalized to beta-actin. Immunohistochemical analysis of the distributions of type IV and type VII collagens were performed on normal and affected tissues from colorectal cancer patients. RESULTS: The alpha1(IV) and alpha1(VII) mRNA levels were statistically significantly higher in colorectal cancer tissue (p < 0.001) as compared to corresponding tissue from healthy controls. This is an early event as tissue from adenomas also displayed a higher level. There were small changes in the levels of alpha 4(IV). The level of alpha 6(IV) was 5-fold lower in colorectal cancer tissue as compared to healthy individuals (p < 0.01). The localisation of type IV and type VII collagen was visualized by immunohistochemical staining. CONCLUSION: Our results suggest that the down-regulation of alpha 6(IV) mRNA coincides with the acquisition of invasive growth properties, whereas alpha1(IV) and alpha1(VII) mRNAs were up-regulated already in dysplastic tissue. There are no differences in collagen expression between tissues from healthy individuals and normal tissues from affected individuals.

Intraoperative, radio-guided sentinel lymph node mapping in 110 nonsmall cell lung cancer patients.

BACKGROUND: Sentinel lymph node identification has been tested in lung cancer patients with conflicting results. The present study was designed to assess the sensitivity, negative predictive value, and accuracy of intraoperative sentinel lymph node mapping by means of a radio-guided method in patients with nonsmall cell lung cancer to find the most appropriate definition of sentinel lymph node and to evaluate the usefulness of different particle sizes of radiocolloid. METHODS: One hundred ten patients with clinically N0 nonsmall cell lung cancer were enrolled in the pilot study of intraoperative sentinel node identification. Four quadrants of the peritumoral tissue were injected with 2 mL of 0.5 mCi technetium-99m suspension. Four radiocolloids of different particle size were used. After complete lymphadenectomy, all resected lymph nodes were examined with hematoxylin-eosin staining. All sentinel nodes negative for metastases by routine staining were searched further for metastatic deposits with both serial sections and immunohistochemistry for cytokeratins. RESULTS: The radio-guided method had a high identification rate, a high sensitivity, and a high negative predictive value (100%, 87%, and 93%, respectively) when immunohistochemistry was considered. When standard hematoxylin and eosin staining was applied, sensitivity and negative predictive value of sentinel lymph node labeling was lower (74% and 89%, respectively). No significant differences were found in either the sensitivity or negative predictive value among the colloid solutions of different particle size used in radio labeling, although smaller particles have shown a tendency to produce better results. CONCLUSIONS: The radio-guided technique provides efficient sentinel lymph node identification in lung cancer. Further studies are warranted to confirm the clinical utility of this strategy.