Survey of 548 oncogenic fusion transcripts in thyroid tumors supports the importance of the already established thyroid fusions genes

Neoplasms frequently present structural chromosomal aberrations that can alter the level of expression of a protein or to the expression of an aberrant chimeric protein. In the thyroid, the PAX8‐PPARG fusion is present in the neoplastic lesions that have a follicular architecture—follicular thyroid carcinoma (FTC) and follicular variant of papillary thyroid carcinoma (FVPTC), and less frequently in follicular thyroid adenoma (FTA), while the presence of RET/PTC fusions are largely restricted to papillary thyroid carcinoma (PTC). The ability to detect fusion genes is relevant for a correct diagnosis and for therapy. We have developed a new fusion gene microarray‐based approach for simultaneous analysis of all known and predicted fusion gene variants. We did a comprehensive screen for 548 known and putative fusion genes in 27 samples of thyroid tumors and three positive controls—one thyroid cancer cell line (TPC‐1) and two PTCs with known CCDC6‐RET (alias RET/PTC1) fusion gene, using this microarray. Within the thyroid tumors tested, only well known, previously reported fusion genes in thyroid oncology were identified. Our results reinforce the pathogenic role played by RET/PTC1, RET/PTC3, and PAX8‐PPARG fusion genes in thyroid tumorigenesis. © 2012 Wiley Periodicals, Inc.     

Soft tissue tumors characterized by a wide spectrum of kinase fusions share a lipofibromatosis‐like neural tumor pattern

Gene fusions resulting in oncogenic activation of various receptor tyrosine kinases, including NTRK1‐3, ALK, and RET, have been increasingly recognized in soft tissue tumors (STTs), displaying a wide morphologic spectrum and therefore diagnostically challenging. A subset of STT with NTRK1 rearrangements were recently defined as lipofibromatosis‐like neural tumors (LPFNTs), being characterized by mildly atypical spindle cells with a highly infiltrative growth in the subcutis and expression of S100 and CD34 immunostains. Other emerging morphologic phenotypes associated with kinase fusions include infantile/adult fibrosarcoma and malignant peripheral nerve sheath tumor‐like patterns. In this study, a large cohort of 73 STT positive for various kinase fusions, including 44 previously published cases, was investigated for the presence of an LPFNT phenotype, to better define the incidence of this distinctive morphologic pattern and its relationship with various gene fusions. Surprisingly, half (36/73) of STT with kinase fusions showed at least a focal LPFNT component defined as >10%. Most of the tumors occurred in the subcutaneous tissues of the extremities (n = 25) and trunk (n = 9) of children or young adults (<30 years old) of both genders. Two‐thirds (24/36) of these cases showed hybrid morphologies with alternating LPFNT and solid areas of monomorphic spindle to ovoid tumor cells with fascicular or haphazard arrangement, while one‐third (12/36) had pure LPFNT morphology. Other common histologic findings included lymphocytic infiltrates, staghorn‐like vessels, and perivascular or stromal hyalinization, especially in hybrid cases. Mitotic activity was generally low (<4/10 high power fields in 81% cases), being increased only in a minority of cases. Immunoreactivity for CD34 (92% in hybrid cases, 89% in pure cases) and S100 (89% in hybrid cases, 64% in pure cases) were commonly present. The gene rearrangements most commonly involved NTRK1 (75%), followed by RET (8%) and less commonly NTRK2, NTRK3, ROS1, ALK, and MET.     

An evaluation and recommendation of the optimal methodologies to detect RET gene rearrangements in papillary thyroid carcinoma

To recommend a reliable and clinically realistic RET/PTC rearrangement detection assay for papillary thyroid carcinoma (PTC), we compared multiplex quantitative polymerase chain reaction (qPCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). RET/PTC rearrangement was detected using either RET break‐apart FISH followed by multicolor FISH to confirm CCDC6/RET or NCOA4/RET fusions, or by multiplex qPCR to detect 14 RET/PTC subtypes with simultaneous RET mRNA expression. RET protein expression was detected by IHC. The specificity and sensitivity of multiplex qPCR and IHC were calculated using break‐apart FISH as a reference. Among 73 PTC patients with sufficient tissue available for FISH and multiplex qPCR, 10 cases were defined as RET/PTC positive by both assays, including eight CCDC6/RET and two NCOA4/RET fusions with relatively high RET mRNA. In addition, multiplex qPCR identified another two CCDC6/RET fusion positive cases, but with low RET mRNA expression. IHC staining identified 11 RET positive cases among 39 patients with available samples. In comparison to FISH, multiplex qPCR displayed 100% sensitivity and 97% specificity to detect RET/PTC fusions, while IHC was neither sensitive nor specific. Our data reveal that both multiplex qPCR and FISH assays are equally applicable for detection of RET/PTC rearrangements. Break‐apart FISH methodology is highly recommended for the wider screening of RET rearrangements (regardless of partner genes), while multiplex qPCR is preferred to identify all known fusion types using one assay, provided mRNA expression is also measured. IHC analysis could potentially provide an additional method of fusion detection dependent on further optimization of assay conditions and scoring cutoffs. © 2014 Wiley Periodicals, Inc.     

Novel ZC3H7B‐BCOR,MEAF6‐PHF1, and EPC1‐PHF1 fusions in ossifying fibromyxoid tumors—molecular characterization shows genetic overlap with endometrial stromal sarcoma

PHF1 gene rearrangements have been recently described in around 50% of ossifying fibromyxoid tumors (OFMT) including benign and malignant cases, with a small subset showing EP400‐PHF1 fusions. In the remaining cases no alternative gene fusions have been identified. PHF1‐negative OFMT, especially if lacking S100 protein staining or peripheral ossification, are difficult to diagnose and distinguish from other soft tissue mimics. In seeking more comprehensive molecular characterization, we investigated a large cohort of 39 OFMT of various anatomic sites, immunoprofiles and grades of malignancy. Tumors were screened for PHF1 and EP400 rearrangements by FISH. RNA sequencing was performed in two index cases (OFMT1, OFMT3), negative for EP400‐PHF1 fusions, followed by FusionSeq data analysis, a modular computational tool developed to discover gene fusions from paired‐end RNA‐seq data. Two novel fusions were identified ZC3H7B‐BCOR in OFMT1 and MEAF6‐PHF1 in OFMT3. After being validated by FISH and RT‐PCR, these abnormalities were screened on the remaining cases. With these additional gene fusions, 33/39 (85%) of OFMTs demonstrated recurrent gene rearrangements, which can be used as molecular markers in challenging cases. The most common abnormality is PHF1 gene rearrangement (80%), being present in benign, atypical and malignant lesions, with fusion to EP400 in 44% of cases. ZC3H7B‐BCOR and MEAF6‐PHF1 fusions occurred predominantly in S100 protein‐negative and malignant OFMT. As similar gene fusions were reported in endometrial stromal sarcomas, we screened for potential gene abnormalities in JAZF1 and EPC1 by FISH and found two additional cases with EPC1‐PHF1 fusions. © 2013 Wiley Periodicals, Inc.     

Novel PPP1CB‐ALK fusion in spindle cell tumor defined by S100 and CD34 coexpression and distinctive stromal and perivascular hyalinization

A novel group of S100‐ and CD34‐positive spindle cell tumors with distinctive stromal and perivascular hyalinization harboring recurrent gene fusions involving kinases including RAF1, BRAF, NTRK1/2/3, and RET have been recently reported. To our knowledge, no such cases harboring ALK rearrangements have been identified. We report a previously healthy 41‐year‐old male with a 12‐cm intramuscular shoulder mass. The tumor was composed of bland‐appearing spindled to epithelioid cells, arranged in a patternless pattern in a background of loose myxoid stroma containing striking amianthoid‐like stromal collagen and perivascular rings. In accordance with the previously reported tumors, the tumor cells showed diffuse immunopositivity with S100 and CD34, while lacking SOX10 expression. Targeted RNA‐based next‐generation sequencing identified a novel serine/threonine‐protein phosphatase PP1‐beta‐catalytic subunit (PPP1CB)‐ALK fusion gene. Although ALK break‐apart was not detected by FISH, likely due to a paracentric inversion of chromosome 2, the presence of the fusion was confirmed by Sanger sequencing showing a 10‐bp linker between exon 6 of PPP1CB and intron 19 of ALK while maintaining reading frame. Subsequent ALK‐1 immunostain exhibited diffuse cytoplasmic staining in the tumor cells. Our case expands the molecular genetic spectrum of the distinctive group of spindle cell tumors with CD34/S100+ immunophenotype, supporting the important role of various kinases as drivers of oncogenesis. Awareness of this entity including its unique morphologic and immunophenotypic features as well as its interchangeable kinase gene fusions is crucial for correct classification and potential targeted therapy, particularly in aggressive subsets.     

NTRK3 kinase fusions in Spitz tumours

Oncogenic fusions in TRK family receptor tyrosine kinases have been identified in several cancers and can serve as therapeutic targets. We identified ETV6–NTRK3, MYO5A–NTRK3 and MYH9–NTRK3 fusions in Spitz tumours, and demonstrated that NTRK3 fusions constitutively activate the mitogen‐activated protein kinase, phosphoinositide 3‐kinase and phospholipase Cγ1 pathways in melanocytes. This signalling was inhibited by DS‐6051a, a small‐molecule inhibitor of NTRK1/2/3 and ROS1. NTRK3 fusions expand the range of oncogenic kinase fusions in melanocytic neoplasms and offer targets for a small subset of melanomas for which no targeted options currently exist. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

NTRK testing: First results of the QuiP‐EQA scheme and a comprehensive map of NTRK fusion variants and their diagnostic coverage by targeted RNA‐based NGS assays

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next‐generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA‐/DNA‐based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study. Nine institutions took part in the FISH‐based and 18 in the NGS‐based round robin test, the latter additionally subdivided into low‐input and high‐input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin‐fixed and paraffin‐embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS‐based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISH‐ and tNGS‐based NTRK testing can be well established in a routine diagnostic setting. Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA‐based tNGS panels.     

The two genes generating RET/PTC3 are localized in chromosomal band 10q11.2

PCR analysis of DNA from a selected panel of human-rodent somatic cell hybrids and fluorescent in situ hybridization (FISH) analysis allowed us to localize the human ELEI gene. This previously uncharacterized gene is fused with the tyrosine kinase (tk) domain of the RET proto-oncogene to generate the oncogenic sequence RET/PTC3, thus providing a third example of RET oncogenic activation in papillary thyroid carcinomas. ELEI was localized to band 10q11.2, the subband where RET also maps, at a minimum distance of more than 500 kb from the proto-oncogene. The fusion event corresponding to the rearrangement reciprocal to that leading to the formation of RET/PTC3 was also identified and characterized. The karyotype of two RET/PTC3 positive tumors did not show any evidence of chromosome 10 abnormalities. The data indicate that a cytogenetically undetectable paracentric inversion within 10q11.2 generates RET/PTC3.     

High‐throughput diagnostic profiling of clinically actionable gene fusions in lung cancer

Molecular profiling of non‐small cell lung cancers (NSCLC) has a strong impact on clinical decision making and current oncological therapies. Besides detection of activating mutations in EGFR, analysis of ALK and ROS1 gene rearrangements has come into focus for targeted therapies. Targeted massive parallel sequencing (MPS) has been established for routine diagnostic profiling of the most prevalent oncogenic mutations in NSCLC, but not for the detection of gene rearrangements yet. Here, we present and evaluate an MPS‐based panel sequencing approach which simultaneously detects ALK, ROS1, and RET fusions as well as somatic mutations in a single multiplex assay using formalin‐fixed paraffin‐embedded (FFPE) tissue. To this end, we first evaluated sensitivity and specificity of the fusion assay retrospectively by employing it to a set of 50 NSCLC with known gene fusions (n = 35) and with no gene fusions (n = 15). The sensitivity and specificity of the MPS assay for the detection of known fusions was 100%. In a second prospective phase, we implemented the approach of parallel mutation and gene fusion detection in our routine diagnostic workflow to assess performance of the test in a diagnostic outreach setting. Our prospective screening of 109 NSCLC samples revealed four gene fusions all of which were confirmed by FISH. In conclusion, our approach facilitates simultaneous high‐throughput detection of gene fusions and somatic mutations in NSCLC samples and is able to replace conventional methods. © 2015 Wiley Periodicals, Inc.     

RET oncogene activation in papillary thyroid carcinoma.

The RET proto-oncogene encodes a cell membrane tyrosine-kinase receptor protein whose ligands belong to the glial cell line-derived neurotrophic factor. RET functions as a multicompetent receptor complex that includes alphaGFRs and RET. Somatic rearrangements of RET designated as RET/PTC (from papillary thyroid carcinoma) were identified in papillary thyroid carcinoma before RET was recognized as the susceptibility gene for MEN2. There are now at least at least 15 types of RET/PTC rearrangements involving RET and 10 different genes. RET/PTC1 and RET/PTC3 are by far the most common rearrangements. All of the rearrangements are due to DNA damage and result in the fusion of the RET tyrosine-kinase (RET-TK) domain to the 5'-terminal region of heterologous genes. RET/PTC rearrangements are very common in radiation-induced tumors but have been detected in variable proportions of sporadic (i.e., non-radiation associated) papillary carcinomas. It is estimated that up to approximately half the papillary thyroid carcinomas in the United States and Canada harbor RET/PTC rearrangements, most commonly RET/PTC-1, followed by RET/PTC-3 and occasionally RET/PTC-2. The cause of these rearrangements in sporadic papillary carcinomas is not known, but the close association between their presence and the papillary carcinoma phenotype indicates that they play a causative role in tumor development. The proposed mechanisms of RET/PTC-induced tumorigenesis and the clinical and pathologic implications of RET/PTC activation are discussed.     

Targeted next generation sequencing of MLH1‐deficient,MLH1 promoter hypermethylated,and BRAF/RAS‐wild‐type colorectal adenocarcinomas is effective in detecting tumors with actionable oncogenic gene fusions

Oncogenic gene fusions represent attractive targets for therapy of cancer. However, the frequency of actionable genomic rearrangements in colorectal cancer (CRC) is very low, and universal screening for these alterations seems to be impractical and costly. To address this problem, several large scale studies retrospectivelly showed that CRC with gene fusions are highly enriched in groups of tumors defined by MLH1 DNA mismatch repair protein deficiency (MLH1d), and hypermethylation of MLH1 promoter (MLH1ph), and/or the presence of microsatellite instability, and BRAF/KRAS wild‐type status (BRAFwt/KRASwt). In this study, we used targeted next generation sequencing (NGS) to explore the occurence of potentially therapeutically targetable gene fusions in an unselected series of BRAFwt/KRASwt CRC cases that displayed MLH1d/MLH1ph. From the initially identified group of 173 MLH1d CRC cases, 141 cases (81.5%) displayed MLH1ph. BRAFwt/RASwt genotype was confirmed in 23 of 141 (~16%) of MLH1d/MLH1ph cases. Targeted NGS of these 23 cases identified oncogenic gene fusions in nine patients (39.1%; CI95: 20.5%‐61.2%). Detected fusions involved NTRK (four cases), ALK (two cases), and BRAF genes (three cases). As a secondary outcome of NGS testing, we identified PIK3K‐AKT‐mTOR pathway alterations in two CRC cases, which displayed PIK3CA mutation. Altogether, 11 of 23 (~48%) MLH1d/MLH1ph/BRAFwt/RASwt tumors showed genetic alterations that could induce resistance to anti‐EGFR therapy. Our study confirms that targeted NGS of MLH1d/MLH1ph and BRAFwt/RASwt CRCs could be a cost‐effective strategy in detecting patients with potentially druggable oncogenic kinase fusions.     

ALK oncoproteins in atypical inflammatory myofibroblastic tumours: novel RRBP1‐ALK fusions in epithelioid inflammatory myofibroblastic sarcoma

ALK oncogenic activation mechanisms were characterized in four conventional spindle‐cell inflammatory myofibroblastic tumours (IMT) and five atypical IMT, each of which had ALK genomic perturbations. Constitutively activated ALK oncoproteins were purified by ALK immunoprecipitation and electrophoresis, and were characterized by mass spectrometry. The four conventional IMT had TPM3/4‐ALK fusions (two cases) or DCTN1‐ALK fusions (two cases), whereas two atypical spindle‐cell IMT had TFG‐ALK and TPM3‐ALK fusion in one case each, and three epithelioid inflammatory myofibroblastic sarcomas had RANBP2‐ALK fusions in two cases, and a novel RRBP1‐ALK fusion in one case. The epithelioid inflammatory myofibroblastic sarcoma with RRBP1‐ALK fusion had cytoplasmic ALK expression with perinuclear accentuation, different from the nuclear membranous ALK localization in epithelioid inflammatory myofibroblastic sarcomas with RANBP2‐ALK fusions. Evaluation of three additional uncharacterized epithelioid inflammatory myofibroblastic sarcomas with ALK cytoplasmic/perinuclear‐ accentuation expression demonstrated RRBP1‐ALK fusion in two cases. These studies show that atypical spindle‐cell IMT can utilize the same ALK fusion mechanisms described previously in conventional IMT, whereas in clinically aggressive epithelioid inflammatory myofibroblastic sarcoma we identify a novel recurrent ALK oncogenic mechanism, resulting from fusion with the RRBP1 gene. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.