Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach: a multi-institutional research study.

BACKGROUND: Gene fusion events resulting from chromosomal rearrangements play an important role in initiation of lung adenocarcinoma. The recent association of four oncogenic driver genes, ALK, ROS1, RET, and NTRK1, as lung tumor predictive biomarkers has increased the need for development of up-to-date technologies for detection of these biomarkers in limited amounts of material. METHODS: We describe here a multi-institutional study using the Ion AmpliSeq™ RNA Fusion Lung Cancer Research Panel to interrogate previously characterized lung tumor samples. RESULTS: Reproducibility between laboratories using diluted fusion-positive cell lines was 100%. A cohort of lung clinical research samples from different origins (tissue biopsies, tissue resections, lymph nodes and pleural fluid samples) were used to evaluate the panel. We observed 97% concordance for ALK (28/30 positive; 71/70 negative samples), 95% for ROS1 (3/4 positive; 19/18 negative samples), and 93% for RET (2/1 positive; 13/14 negative samples) between the AmpliSeq assay and other methodologies. CONCLUSION: This methodology enables simultaneous detection of multiple ALK, ROS1, RET, and NTRK1 gene fusion transcripts in a single panel, enhanced by an integrated analysis solution. The assay performs well on limited amounts of input RNA (10 ng) and offers an integrated single assay solution for detection of actionable fusions in lung adenocarcinoma, with potential savings in both cost and turn-around-time compared to the combination of all four assays by other methods.

Multicenter evaluation of an automated, multiplex, RNA-based molecular assay for detection of ALK, ROS1, RET fusions and MET exon 14 skipping in NSCLC.

The current study assessed the performance of the fully automated RT-PCR-based Idylla™ GeneFusion Assay, which simultaneously covers the advanced non-small cell lung carcinoma (aNSCLC) actionable ALK, ROS1, RET, and MET exon 14 rearrangements, in a routine clinical setting involving 12 European clinical centers. The Idylla™ GeneFusion Assay detects fusions using fusion-specific as well as expression imbalance detection, the latter enabling detection of uncommon fusions not covered by fusion-specific assays. In total, 326 archival aNSCLC formalin-fixed paraffin-embedded (FFPE) samples were included of which 44% were resected specimen, 46% tissue biopsies, and 9% cytological specimen. With a total of 179 biomarker-positive cases (i.e., 85 ALK, 33 ROS1, 20 RET fusions and 41 MET exon 14 skipping), this is one of the largest fusion-positive datasets ever tested. The results of the Idylla™ GeneFusion Assay were compared with earlier results of routine reference technologies including fluorescence in situ hybridization, immunohistochemistry, reverse-transcription polymerase chain reaction, and next-generation sequencing, establishing a high sensitivity/specificity of 96.1%/99.6% for ALK, 96.7%/99.0% for ROS1, 100%/99.3% for RET fusion, and 92.5%/99.6% for MET exon 14 skipping, and a low failure rate (0.9%). The Idylla™ GeneFusion Assay was found to be a reliable, sensitive, and specific tool for routine detection of ALK, ROS1, RET fusions and MET exon 14 skipping. Given its short turnaround time of about 3 h, it is a time-efficient upfront screening tool in FFPE samples, supporting rapid clinical decision making. Moreover, expression-imbalance-based detection of potentially novel fusions may be easily verified with other routine technologies without delaying treatment initiation.

Protein atlas of fibroblast specific protein 1 (FSP1)/S100A4.

Fibroblast specific protein 1 (FSP1)/S100A4 is a calcium binding protein which has been linked to epithelial-mesenchymal transition, tissue fibrosis, pulmonary vascular disease, metastatic tumour development, increased tumour cell motility and invasiveness. This protein is reported to be also expressed in newly formed and differentiated fibroblasts and has been used in various studies to demonstrate epithelial-mesenchymal transition (EMT). We aimed to characterize S100A4 positive cells in different human tissue compartments, with the focus on fibroblasts/myofibroblast. We found S100A4 expression in a wide range of cells. Fibroblasts/myofibroblasts showed a broad spectrum of staining intensity, ranging from negative to strong expression of S100A4, with the strongest expression in smooth muscle actin positive myofibroblasts. Cells of haematopoietic lineage, namely CD4 and CD8 positive T-lymphocytes, but not B-lymphocytes expressed S100A4. All investigated monocytes, macrophages and specialised histiocytes were positive for S100A4. Even some epithelial cells of the kidney and bladder were positive for S100A4. Expression was also found in the vasculature. Here, cells of the subendothelial space, tunica adventitia and some smooth muscle cells of the tunica media were positive for S100A4. In summary, S100A4 is expressed in various cell types of different lineage and is not, as originally believed, specific for fibroblasts (FSP). Results attained under the premise of specificity of FSP1/S100A4 for fibroblasts, like the founding research on EMT type 2 in kidney and liver, therefore need to be reinterpreted.

Analysis of selected oncogenes (AKT1, FOS, BCL2L2, TGFbeta) on chromosome 14 in granulosa cell tumors (GCTs): a comprehensive study on 30 GCTs combining comparative genomic hybridization (CGH) and fluorescence-in situ-hybridization (FISH).

In previous studies, we have demonstrated a number of cytogenetic alterations in granulosa cell tumors (GCTs), especially on chromosomes X, 12, 14, and 22. However, little is known about specific loci on 14q, which could play an important role in tumor pathology. Therefore, we assessed four important genes in 30 GCTs using fluorescence-in situ-hybridization (FISH). Comparative genomic hybridization (CGH) was performed on paraffin-embedded material. Then, we applied FISH with gene-specific DNA probes for AKT1 (14q32.32), FOS (14q24.3), BCL2L2 (14q11.2-q12), and TGFbeta3 (14q24), and tried to find a correlation between CGH, FISH, tumor stage, and survival. In CGH, 7 of 30 cases (23.3%) showed complete gains on chromosome 14. FISH of the four loci revealed gains of hybridization signals in 8 of 30 cases (26.6%), indicating trisomy of the whole chromosome arm. The same aberration was detected by FISH in 2 of 30 cases (6.6%), which were negative using CGH. One case (1 of 30; 3.3%) was found to have a gain on chromosome 14 by CGH, which could not be confirmed by FISH. A correlation with tumor stage or survival could not be established. Our results suggest that GCTs may be characterized by trisomy of chromosome 14. A specific oncogene that could play a particular role in the tumorigenesis of GCTs was not identified on chromosome 14.

Lung adenocarcinoma with BRAF G469L mutation refractory to vemurafenib.

BRAF V600E is an emerging drug target in lung cancer, but the clinical significance of non-V600 BRAF mutations in lung cancer and other malignancies is less clear. Here, we report the case of a patient with metastatic lung adenocarcinoma with BRAF G469L mutation refractory to vemurafenib. We calculated a structure model of this very rare type of mutated BRAF kinase to explain the molecular mechanism of drug resistance. This information may help to develop effective targeted therapies for cancers with non-V600 BRAF mutations.

Simultaneous analysis of HER2 gene and protein on a single slide facilitates HER2 testing of breast and gastric carcinomas.

This study sought to evaluate a new combined gene and protein detection platform in the context of HER2 evaluation in breast and gastric carcinomas. HER2 immunohistochemistry (IHC) and dual color in situ hybridization (Dual ISH) were combined on a single slide. Results were compared with conventional HER2 IHC and fluorescence ISH. Results from the gene and protein assay were reliable and highly reproducible for both breast and gastric carcinomas. Concordance was found between conventional HER2 IHC and ISH testing and the gene and protein assay in the same laboratory (>95% for Dual ISH; lower for IHC because of different antibody clones), between IHC and Dual ISH performed on the same slide (>92%), and in the gene and protein assays between laboratories (>96%). This cost- and time-effective method provides fast and definitive results (IHC confirmed by means of Dual ISH) to aid in rapid treatment decisions. It can also be applied to other gene and protein combinations.

Mutational spectrum and therapy response of metastasized GIST in Central Switzerland - a population-based study.

BACKGROUND: Until now, no population-based studies investigated the mutational status of primary GIST (PT) and corresponding metastases and correlated these data with response to Imatinib or Sunitinib therapy. PATIENTS AND METHODS: In a retrospective observation study, all metastatic GISTs of the last 15years of our institution were investigated for mutations in c-kit and in PDGFRα gene in each PT and corresponding metastasis. Correlation with clinical outcome and response to Imatinib or Sunitinib therapy was performed. RESULTS: In 13 PT c-kit mutations in exon 9 (3), exon 11 (7) and exon 13 (1), 2 wild type genotypes, and no PDGFRα mutation were detected. In three metastases a switch from heterozygosity to homozygosity and one additional exon 13 mutation was observed. All 10 persons with available follow-up received Imatinib as first-line chemotherapy. Five of them (3 exon 9 mutations, 1 wild type, 1 additional exon 13 mutation) stopped Imatinib due to tumour progression. In three cases, Sunitinib as second-line chemotherapy was ended due to the same reasons. CONCLUSIONS: Our data support previous observations, that PDGFRα mutations play no important role in metastasized GISTs. The influence of Imatinib and Sunitinib therapy in metastasized GISTs with wild type genotype and c-kit exon 9 mutations needs further investigation.

KRAS and BRAF mutations in ovarian tumors: a comprehensive study of invasive carcinomas, borderline tumors and extraovarian implants.

OBJECTIVE: Mutations of BRAF, a downstream mediator of K-RAS, have been described in serous borderline tumors of the ovary. Data concerning other types of ovarian tumors are scarce. Therefore, we assessed KRAS and BRAF mutation in a series of more than 100 different ovarian tumors. METHODS: Paraffin-embedded material, including invasive carcinomas, borderline tumors, benign lesions and implants, was used. BRAF codon 600 in exon 15 and K-RAS codon 12 in exon 2 were analysed. RESULTS: 92 cases (92%), including all serous carcinomas (100%), did not show a mutation of BRAF. Eight cases (8.0%), including five serous borderline tumors (31.25%), contained a mutation. In all serous borderline tumors, codon 600 was affected. The remaining three cases were invasive carcinomas of endometrioid (mutation on codon 600), mucinous (mutation on codon 600) and clear cell (mutation on codon 615) subtype. There was no BRAF mutation in mucinous borderline tumors. Regarding K-RAS, 89 cases (87.25%) did not show an aberration. The 11 positive borderline tumors (10.7%) were of serous (22.2%) and of mucinous type (46.6%). There was a KRAS mutation in a serous and a mucinous invasive carcinoma each. BRAF and K-RAS mutations were mutually exclusive and not seen in implants. CONCLUSION: Mutation of either K-RAS or BRAF is frequent in borderline tumors but is not found in invasive serous carcinomas and is very rare in other invasive subtypes. This supports the notion of different pathological pathways. For the development of extraovarian implants, further studies are observed.