ALK fusion and its association with other driver gene mutations in Finnish non-small cell lung cancer patients.

Screening of anaplastic lymphoma tyrosine kinase (ALK) gene fusions in non-small cell lung cancer (NSCLC) patients enables the identification of the patients likely to benefit from ALK-targeted therapy. Our aim was to assess the prevalence of ALK fusion in Finnish NSCLC patients, which has not been reported earlier, and to study the presence of ALK fusion in relation to clinicopathological characteristics and other driver gene mutations. A total of 469 formalin-fixed paraffin-embedded tumor tissue specimens from Finnish NSCLC patients were screened for ALK fusion by immunohistochemistry (IHC). For confirmation of IHC results, fluorescence in situ hybridization (FISH) was conducted for 171 specimens. Next-generation sequencing was performed for all ALK-positive specimens to characterize the association of ALK fusion with mutations in targeted regions of 22 driver genes. Of the 469 tumors screened, 11 (2.3%) harbored an ALK fusion, including nine adenocarcinomas and two large cell carcinomas. The IHC results for all 11 ALK-positive and 160 random ALK-negative specimens were confirmed by FISH. ALK fusion was significantly associated with never/ex-light smoking history (P<0.001) and younger age (P=0.004). Seven ALK-positive tumors showed additional mutations; three in MET, one in MET and CTNNB1, two in TP53, and one in PIK3CA. Our results show that ALK fusion is an infrequent alteration in Finnish NSCLC patients. Although the majority of ALK-positive cases were adenocarcinomas, the fusion was also seen in large cell carcinomas. Further studies are needed to elucidate the clinical significance of the coexistence of ALK fusion with MET, TP53, CTNNB1, and PIK3CA mutations.

Mutated ephrin receptor genes in non-small cell lung carcinoma and their occurrence with driver mutations-targeted resequencing study on formalin-fixed, paraffin-embedded tumor material of 81 patients.

Non-small cell lung carcinoma (NSCLC) is the most common subtype of lung cancer. The oncogenic potential of receptor tyrosine kinases (RTKs) is widely known and they are potential targets for tailored therapy. Ephrin receptors (Ephs) form the largest group of RTKs. Nevertheless, Ephs are not widely studied in NSCLC so far. The aim of our study was to investigate novel mutations of Eph genes (EPHA1-8, EPHB1-4, EPHB6) and their association with clinically relevant mutations in BRAF, EML4-ALK, EGFR, INSR, KDR, KRAS, MET, PDGFRA, PDGFRB, PIK3, PTEN, RET, and TP53 in NSCLC patients. Targeted resequencing was conducted on 81 formalin-fixed, paraffin-embedded NSCLC tumor specimens. We analyzed missense and nonsense mutations harbored in the coding regions of the selected genes. We found 18 novel mutations of Ephs in 20% (16 of 81) of the patients. Nearly half of these mutations occurred in the protein kinase domain. The mutations were not mutually exclusive with other clinically relevant mutations. Our study shows that Ephs are frequently mutated in NSCLC patients, and occur together with other known mutations relevant to the pathogenicity of NSCLC.

Targeted resequencing reveals ALK fusions in non-small cell lung carcinomas detected by FISH, immunohistochemistry, and real-time RT-PCR: a comparison of four methods.

Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements occur in a subgroup of non-small cell lung carcinomas (NSCLCs). The identification of these rearrangements is important for guiding treatment decisions. The aim of our study was to screen ALK gene fusions in NSCLCs and to compare the results detected by targeted resequencing with results detected by commonly used methods, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and real-time reverse transcription-PCR (RT-PCR). Furthermore, we aimed to ascertain the potential of targeted resequencing in detection of ALK-rearranged lung carcinomas. We assessed ALK fusion status for 95 formalin-fixed paraffin-embedded tumor tissue specimens from 87 patients with NSCLC by FISH and real-time RT-PCR, for 57 specimens from 56 patients by targeted resequencing, and for 14 specimens from 14 patients by IHC. All methods were performed successfully on formalin-fixed paraffin-embedded tumor tissue material. We detected ALK fusion in 5.7% (5 out of 87) of patients examined. The results obtained from resequencing correlated significantly with those from FISH, real-time RT-PCR, and IHC. Targeted resequencing proved to be a promising method for ALK gene fusion detection in NSCLC. Means to reduce the material and turnaround time required for analysis are, however, needed.

Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS.

The development of tyrosine kinase inhibitor treatments has made it important to test cancer patients for clinically significant gene mutations that influence the benefit of treatment. Targeted next-generation sequencing (NGS) provides a promising method for diagnostic purposes by enabling the simultaneous detection of multiple mutations in various genes in a single test. The aim of our study was to screen EGFR, KRAS, and BRAF mutations by targeted NGS and commonly used real-time polymerase chain reaction (PCR) methods to evaluate the feasibility of targeted NGS for the detection of the mutations. Furthermore, we aimed to identify potential novel mutations by targeted NGS. We analyzed formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens from 81 non-small cell lung carcinoma patients. We observed a significant concordance (from 96.3 to 100%) of the EGFR, KRAS, and BRAF mutation detection results between targeted NGS and real-time PCR. Moreover, targeted NGS revealed seven nonsynonymous single-nucleotide variations and one insertion-deletion variation in EGFR not detectable by the real-time PCR methods. The potential clinical significance of these variants requires elucidation in future studies. Our results support the use of targeted NGS in the screening of EGFR, KRAS, and BRAF mutations in FFPE tissue material.

The hypermethylation of the O6-methylguanine-DNA methyltransferase gene promoter in gliomas--correlation with array comparative genome hybridization results and IDH1 mutation.

The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.

Identification of susceptibility loci at 7q31 and 9p13 for bipolar disorder in an isolated population.

We performed a linkage analysis on 23 Finnish families with bipolar disorder and originating from the North-Eastern region of Finland, using the Illumina Linkage Panel IV (6K) Array with an average intermarker spacing of 0.65 cM across the genome. We detected genome-wide significant evidence for linkage of mood disorder (bipolar disorder type I, II, or not otherwise specified, manic type of schizoaffective psychosis, cyclothymia, or recurrent depression) to chromosomes 7q31 (LOD = 3.20) and 9p13.1 (LOD = 4.02). Analyzing the best markers on the complete set of 179 Finnish bipolar families supported the findings on chromosome 9p13 (maximum LOD score of 3.02 at position 383 Mb, immediately upstream of the centromere). This region harbors several interesting candidate genes, including contactin associated protein-like 3 (CNTNAP3) and aldehyde dehydrogenase 1 (ALDH1B1). For the 7q31 locus, only one extended pedigree and ten families originating from the same late settlement region in North-Eastern Finland provided evidence for linkage, suggesting that a gene predisposing to bipolar disorder is enriched in that region. Candidate genes of interest in this locus include potassium-voltage-gated channel, member 2 (KCND2) and calcium-dependent activator protein for secretion 2 (CADPS2). The loci on the centromeric region of 9p13 and the telomeric region of 7q31 may represent susceptibility loci for mood disorder in the Finnish population.

Genetics of maximal walking speed and skeletal muscle characteristics in older women.

The aim of this study was to examine whether maximal walking speed, maximal isometric muscle strength, leg extensor power and lower leg muscle cross-sectional area (CSA) shared a genetic effect in common. In addition, we wanted to identify the chromosomal areas linked to maximal walking speed and these muscle characteristics and also investigate whether maximal walking speed and these three skeletal muscle characteristics are regulated by the same chromosomal areas. We studied 217 monozygotic (MZ) and dizygotic (DZ) female twin pairs aged 66 to 75 years in the Finnish Twin Study on Aging study. The DZ pairs (94) were genotyped for 397 microsatellite markers in 22 autosomes and X-chromosome. Genetic modeling showed that, muscle CSA, strength, power and walking speed shared a genetic effect in common which accounted for 7% of the variation in CSA, 51% in strength, 37% in power and 35% in walking speed. The results of an explorative multipoint linkage analysis suggested that the highest LOD score found for each phenotype was 2.41 for walking speed on chromosome 13q22.1, 2.14 for strength on chromosome 15q14, 2.84 for power on chromosome 8q24.23, and 2.93 for muscle CSA on chromosome 20q13.31. Also a suggestive LOD score, 2.68, for muscle CSA was found on chromosome 9q34.3. The chromosomal areas of a suggestive linkage for strength and power partly overlapped LOD scores higher than 1.0 being seen for these phenotypes on chromosome 15. The present study was the first genome-wide linkage analysis to be conducted for these multifactorial and clinically important phenotypes underlying functional independence in older women.