[Mutation testing for non-small-cell lung cancer]. / Mutasjonstesting ved ikke-småcellet lungekreft.

BACKGROUND: Epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) are a relatively new class of drugs for treatment of non-small-cell lung cancer. The national professional group for lung cancer, The Norwegian Lung Cancer Group, recommends that patients with non-small-cell lung cancer are tested for mutations in the EGFR gene. Here, we report the experience collected after the introduction of such testing in Norway in 2010. MATERIAL AND METHOD: Information on the number of patients tested, gender distribution, histopathological data and analysis results have been collected from the molecular-pathology laboratories at the university hospitals in Tromsø, Trondheim, Bergen and Oslo for the period from May 2010 to May 2011. RESULTS: During this period, altogether 1,058 patients with lung cancer were tested for mutations in the EGFR gene, equal to approximately half of all those who were diagnosed with non-small-cell lung cancer. A mutation was detected in 123 patients (11.6 per cent). There was a higher proportion of mutation-positive women than men (17.6 per cent, compared to 6.3 per cent, p < 0.001), and a lower proportion with squamous cell carcinoma than for other histopathological subtypes (3.0 per cent, compared to 12.9 per cent, p < 0.001). Of a total of 80 cytological tests, nine (11.3 per cent) were positive. INTERPRETATION: In light of the relatively high mutation frequency and a considerable number of positives in the group with squamous cell carcinoma, we recommend to continue the practice of mutation-testing all patients with non-small-cell lung cancer.

A hepatocyte nuclear factor-4 alpha gene (HNF4A) P2 promoter haplotype linked with late-onset diabetes: studies of HNF4A variants in the Norwegian MODY registry.

Variants in hepatocyte nuclear factor (HNF)-4alpha cause maturity-onset diabetes of the young, type 1 (MODY1) and may also be risk factors for type 2 diabetes. We sequenced the HNF4A gene of 95 MODY3-negative probands from the Norwegian MODY Registry. We found three novel coding variants in exon 8 of HNF4A: G326R, T339I, and W340X. In intron 7, we noted a single nucleotide polymorphism in the binding site of a previously published primer pair, which in some cases caused allelic drop out when amplifying exon 8. We also detected two novel sequence variants of the P2 promoter region, of which P2 -192C>G showed linkage with diabetes in two families (maximal logarithm of odds score of 3.1 and 0.8, respectively). This variant and a surrounding haplotype restricted by 3.7 Mb was also found in two Danish MODY pedigrees. The age of onset was higher in the P2 -192C>G carriers (median 45 years) compared with that reported for other MODY1 individuals. We could not support a biological role of the P2 promoter variant by in vitro transfection assays. In conclusion, we have identified three novel HNF4A mutations and a 3.7-Mb haplotype, including the HNF4A P2 promoter, which was linked with diabetes.

Tumor versus stromal cells in culture--survival of the fittest?

Two of the signature genetic events that occur in human gliomas, EGFR amplification and IDH mutation, are poorly represented in experimental models in vitro. EGFR amplification, for example, occurs in 40 to 50% of GBM, and yet, EGFR amplification is rarely preserved in cell cultures derived from human tumors. To analyze the fate of EGFR amplified and IDH mutated cells in culture, we followed the development over time of cultures derived from human xenografts in nude rats enriched for tumor cells with EGFR amplification and of cultures derived from patient samples with IDH mutations, in serum monolayer and spheroid suspension culture, under serum and serum free conditions. We observed under serum monolayer conditions, that nestin positive or nestin and SMA double positive rat stromal cells outgrew EGFR amplified tumor cells, while serum spheroid cultures preserved tumor cells with EGFR amplification. Serum free suspension culture exhibited a more variable cell composition in that the resultant cell populations were either predominantly nestin/SOX2 co-expressing rat stromal cells or human tumor cells, or a mixture of both. The selection for nestin/SMA positive stromal cells under serum monolayer conditions was also consistently observed in human oligodendrogliomas and oligoastrocytomas with IDH mutations. Our results highlight for the first time that serum monolayer conditions can select for stromal cells instead of tumor cells in certain brain tumor subtypes. This result has an important impact on the establishment of new tumor cell cultures from brain tumors and raises the question of the proper conditions for the growth of the tumor cell populations of interest.

Tyrosine-kinase mutations in c-KIT and PDGFR-alpha genes of imatinib naïve adult patients with gastrointestinal stromal tumours (GISTs) of the stomach and small intestine: relation to tumour-biological risk-profile and long-term outcome.

BACKGROUND: The identification of activating mutations in either c-KIT cell surface growth factor receptor or platelet-derived growth factor receptor alpha (PDGFRA) has lead the way for the development of novel agents that selectively inhibit key molecular events in gastrointestinal stromal tumour (GIST) pathogenesis. The aim of this study was to investigate the role of c-KIT and PDGFRA gene mutations in primary resectable, imatinib naïve GISTs located in the stomach and small intestine. METHODS: All adult patients with GIST located in either stomach or small intestine who underwent surgical resection without prior imatinib (Glivec) treatment were included. DNA extraction and mutational analysis were performed. Mutational analyses were performed for c-KIT (exons 9, 11, 13, and 17) and the PDGFRA genes (exons 12, 14 and 18). Clinical and pathological parameters were analyzed in relation to the mutations in c-KIT and PDGFRA. RESULTS: A total of 38 patients who underwent surgery for GIST located in either the stomach (n = 24) or in the small intestines (n = 14) were included. Mutations were found in 31 of 38 (81.6 %) patients, with 24 (63.2 %) located in c-KIT and 7 (18.4 %) in the PDGRFA exons, respectively. Seven patients (18.4 %) were wildtype (WT). The most common mutation was in c-KIT exon 11. Incidentally found GISTs were significantly smaller (size >5 cm in 15 % for incidental vs. 71 % for symptomatic; OR of 13.4, 95 % CI 2.3-76.5; P = 0.001) and had lower mitotic rate (0 % for incidental vs. 44 % of the symptomatic; OR 0.52, 95 % CI 0.36-0.75; P = 0.005). Accordingly, the Fletcher grade was significantly better for incidental cases, with most having very low or low risk (85 %) in contrast to 19 of 25 (76 %) symptomatic cases showing moderate to high-risk features (OR 17.4, 95 % CI 2.98-101.7; P < 0.001). However, the distribution of c-KIT, PDGFRA and WT was not differently distributed between incidental and symptomatic GISTs. Long-term survival up to 25 years (median: 8 years) was best determined by Fletcher risk-score in the multivariate model (HR 14.1, 95 % CI 1.7-114.5; p = 0.013). CONCLUSIONS: Long-term survival in resected GISTs of the stomach and small intestine is best determined by Fletcher risk-score. Mitotic activity appears related to tumour size and young age at onset. Mutational status did not influence the clinical or tumour-specific features in this cohort.

Improved diagnostic segregation of mantle cell lymphoma by determination of cyclin D1/D3 expression ratio in formalin-fixed tissue.

Mantle cell lymphomas (MCLs) are associated with a characteristic t(11;14)(q13;q32) chromosomal translocation. This causes the CCND1 gene on chromosome 11 to be co-localized with the immunoglobulin heavy chain gene on chromosome 14, resulting in increased expression of cyclin D1. The cyclin D1/D3 expression ratio, as an approach to segregate MCLs from other small B-cell lymphomas, has not previously been evaluated in formalin-fixed, paraffin-embedded tissue. We found that mean cyclin D3 expression was lower in MCLs (P < 0.05) than in chronic lymphocytic leukemias (CLLs), follicular lymphomas (FLs), marginal zone/mucosa-associated lymphoid tissue lymphomas (MALTs), multiple myelomas (MMs), and reactive lymph nodes. As expected, mean cyclin D1 expression was increased in MCL (P < 0.05), but in several cases the expression of cyclin D1 did overlap with the level observed in CLLs, FLs, MALTs, MMs, and reactive lymph nodes. The cyclin D1/D3 expression ratio, however, did fully separate MCLs from FLs, CLLs, and reactive lymph nodes. The mean expression ratio was also significantly different between MCL and MALT (P < 0.05), but 3 MCL cases had values overlapping those of some MALTs. The expression ratio was not significantly different between MCL and MM. In conclusion, the cyclin D1/D3 expression ratio gave an improved segregation of MCLs from CLLs, FLs, MALTs, and reactive lymph nodes, as compared with determination of cyclin D1 alone in formalin-fixed, paraffin-embedded tissue.