Identification of novel GNAS mutations in intramuscular myxoma using next-generation sequencing with single-molecule tagged molecular inversion probes.

BACKGROUND: Intramuscular myxoma (IM) is a hypocellular benign soft tissue neoplasm characterized by abundant myxoid stroma and occasional hypercellular areas. These tumors can, especially on biopsy material, be difficult to distinguish from low-grade fibromyxoid sarcoma or low-grade myxofibrosarcoma. GNAS mutations are frequently involved in IM, in contrast to these other malignant tumors. Therefore, sensitive molecular techniques for detection of GNAS aberrations in IM, which frequently yield low amounts of DNA due to poor cellularity, will be beneficial for differential diagnosis. METHODS: In our study, a total of 34 IM samples from 33 patients were analyzed for the presence of GNAS mutations, of which 29 samples were analyzed using a gene-specific TaqMan genotyping assay for the detection of GNAS hotspot mutations c.601C > T and c602G > A in IM, and 32 samples using a novel next generation sequencing (NGS)-based approach employing single-molecule tagged molecular inversion probes (smMIP) to identify mutations in exon 8 and 9 of GNAS. Results between the two assays were compared for their ability to detect GNAS mutations with high confidence. RESULTS: In total, 23 of 34 samples were successfully analyzed with both techniques showing GNAS mutations in 12 out of 23 (52%) samples. The remaining 11 samples were analyzed with either TaqMan assay or smMIP assay only. The TaqMan assay revealed GNAS mutations in 16 out of 29 samples (55%), with six samples c.601C > T (p.R201C; 38%) and ten samples c.602G > A (p.R201H; 62%) missense mutations. The smMIP assay identified mutations in 16 out of 28 samples (57%), with five samples c.601C > T (p.R201C; 31%) and seven samples c.602G > A (p.R201H; 44%) missense mutations. In addition, four samples (25%) revealed novel IM-associated mutations, including c.601C > A (p.R201S), c.602G > T (p.R201L), c.602G > C (p.R201P) and c.680A > G (p.Q227R). Combining the results of both tests, 23 out of 34 sporadic IM samples (68%) showed a GNAS mutation. CONCLUSIONS: Both the TaqMan and the smMIP assay a show a high degree of concordance in detecting GNAS hotspot mutations in IM with comparable sensitivity. However, since the NGS-based smMIP assay permits mutation detection in whole exons of GNAS, a broader range of GNAS mutations can be identified by the smMIP approach.

RAS testing in metastatic colorectal cancer: excellent reproducibility amongst 17 Dutch pathology centers.

In 2013 the European Medicine Agency (EMA) restricted the indication for anti-EGFR targeted therapy to metastatic colorectal cancer (mCRC) with a wild-type RAS gene, increasing the need for reliable RAS mutation testing. We evaluated the completeness and reproducibility of RAS-testing in the Netherlands. From 17 laboratories, tumor DNA of the first 10 CRC cases tested in 2014 in routine clinical practice was re-tested by a reference laboratory using a custom next generation sequencing panel. In total, 171 CRC cases were re-evaluated for hotspot mutations in KRAS, NRAS and BRAF. Most laboratories had introduced complete RAS-testing (65%) and BRAF-testing (71%) by January 2014. The most employed method for all hotspot regions was Sanger sequencing (range 35.7 - 49.2%). The reference laboratory detected all mutations that had been found in the participating laboratories (n = 92), plus 10 additional mutations. This concerned three RAS and seven BRAF mutations that were missed due to incomplete testing of the participating laboratory. Overall, the concordance of tests performed by both the reference and participating laboratory was 100% (163/163; κ-static 1.0) for RAS and 100% (144/144; κ-static 1.0) for BRAF. Our study shows that RAS and BRAF mutations can be reproducibly assessed using a variety of testing methods.

Is there a role for antigen selection in mantle cell lymphoma? Immunogenetic support from a series of 807 cases.

We examined 807 productive IGHV-IGHD-IGHJ gene rearrangements from mantle cell lymphoma (MCL) cases, by far the largest series to date. The IGHV gene repertoire was remarkably biased, with IGHV3-21, IGHV4-34, IGHV1-8, and IGHV3-23 accounting for 46.3% of the cohort. Eighty-four of 807 (10.4%) cases, mainly using the IGHV3-21 and IGHV4-34 genes, were found to bear stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences and were placed in 38 clusters. Notably, the MCL stereotypes were distinct from those reported for chronic lymphocytic leukemia. Based on somatic hypermutation (SHM) status, 238/807 sequences (29.5%) carried IGHV genes with 100% germ line identity; the remainder (569/807; 70.5%) exhibited different SHM impact, ranging from minimal (in most cases) to pronounced. Shared replacement mutations across the IGHV gene were identified for certain subgroups, especially those using IGHV3-21, IGHV1-8, and IGHV3-23. Comparison with other entities, in particular CLL, revealed that several of these mutations were "MCL-biased." In conclusion, MCL is characterized by a highly restricted immunoglobulin gene repertoire with stereotyped VH CDR3s and very precise SHM targeting, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Hence, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases.

Multiplex ligation-dependent probe amplification for the detection of chromosomal gains and losses in formalin-fixed tissue.

Molecular analysis on formalin-fixed paraffin-embedded tissue is of increasing importance in diagnostic histopathology and tumor research. Multiplex ligation-dependent probe amplification (MLPA) is a technique that can be used for detection of copy number alterations of up to 45 different DNA sequences in one experiment. It can be performed on partially degraded DNA, which makes this technique very suitable for analysis of formalin-fixed lesions. We tested the reliability of MLPA by analyzing DNA isolated from formalin-fixed melanomas that were previously characterized by comparative genomic hybridization (CGH), and additionally the applicability of MLPA was tested by analyzing 29 routinely processed melanocytic lesions. MLPA appears to be a reliable and efficient method to evaluate DNA copy number changes as 86% of the loci tested revealed concordant CGH results. Discordance mainly involved alterations that were detected by MLPA and not by CGH probably due to a combination of lower resolution of CGH and occasionally false positive MLPA results. For application of MLPA in a diagnostic setting, different probes on a specific region of interest should be used to prevent false positive MLPA results. In a research setting as well as in a diagnostic setting, MLPA is a fast technique to screen large numbers of formalin-fixed lesions for DNA gains and losses.

Distinct chromosomal aberrations in sinonasal mucosal melanoma as detected by comparative genomic hybridization.

Sinonasal mucosal melanomas are the most frequent mucosal melanomas and arise from melanocytes located in the nasal cavity and the paranasal sinuses. The melanoma types, cutaneous melanoma, uveal melanoma, and mucosal melanoma, differ in etiology, geographic distribution, and clinical behavior. Genetic alterations have been previously studied in cutaneous and uveal melanomas but, to the best of our knowledge, not in mucosal melanomas. Comparative genomic hybridization (CGH) was performed on 14 routinely processed sinonasal mucosal melanomas. Furthermore, ploidy analysis was performed on 11 tumors to provide complementary data on the DNA index. The CGH profiles of sinonasal mucosal melanomas show remarkably consistent alterations: chromosome arm 1q is gained in all tumors and gains of 6p and 8q are present in 93 and 57%, respectively. Comparison of CGH data with both the common variants of cutaneous melanoma and uveal melanoma revealed that sinonasal mucosal melanomas harbor a distinct pattern of chromosomal abnormalities. Ploidy analysis also showed that diploid tumors exhibit gains of 1q and alterations of chromosome 6 (3 of 3 cases tested), whereas clear-copy gains and high-copy gains were seen only in triploid and tetraploid tumors (6 of 8 cases tested). This indicates that alteration of chromosomes 1 and 6 may precede polyploidization and formation of clear-copy gains and high-copy gains.

LAF4, an AF4-related gene, is fused to MLL in infant acute lymphoblastic leukemia.

Infant acute lymphoblastic leukemia (ALL) with MLL gene rearrangements is characterized by a proB phenotype and a poor clinical outcome. We analyzed an infant proB ALL with t(2;11)(p15;p14) and an MLL rearrangement on Southern blot analysis. Rapid amplification of cDNA ends-polymerase chain reaction (PCR) and reverse transcriptase-PCR identified the LAF4 gene mapped on chromosome region 2q11.2-q12 as a fusion partner of the MLL gene. The LAF4 gene was identified previously by its high sequence homology to the AF4 protein and encodes a protein of 1,227 amino acids. The t(4;11)(q21;q23), creating the MLL-AF4 chimeric transcripts, is the predominant 11q23 chromosome translocation in infant ALL and is associated with an extremely poor prognosis. Our findings further suggest that fusion of MLL to one of the AF4 family members (AF4/LAF4/AF5Q31) might determine a proB-cell phenotype in infant leukemia.

Allelic imbalance in the diagnosis of benign, atypical and malignant Spitz tumours.

To test the diagnostic usefulness of allelic imbalance (AI) analysis based on routinely paraffin-embedded tissue, a series of 55 benign Spitz naevi, Spitz tumours with uncertain malignant potential, and malignant Spitzoid melanomas was investigated. Laser microdissection was used to ensure representative sampling of lesional cells and to investigate AI in separate tumour areas of four melanomas. AI was found in 2/12 (17%) typical Spitz naevi, 3/9 (33%) atypical Spitz tumours, 12/17 (65%) atypical Spitz tumours suspicious for melanoma and 15/17 (88%) Spitzoid melanomas. Additional immunohistochemical staining for Ki-67 using the MIB-1 antibody revealed positive deeply situated lesional cells in 0/6 (0%) Spitz naevi, 1/8 (13%) atypical Spitz tumours, 5/14 (35%) atypical Spitz tumours suspicious for melanoma, and 7/14 (50%) Spitzoid melanomas, respectively. Two of the melanomas examined for AI in separate tumour areas showed intratumoural genetic heterogeneity. In view of the finding of AI and deeply situated Ki-67 positive cells not only in melanomas but also in Spitz tumours with uncertain malignant potential, these approaches appear to have no direct diagnostic applicability for the distinction between benign and malignant Spitz tumours. Further molecular studies will be required to determine whether Spitz tumours and Spitzoid melanomas are unrelated entities, or whether there is a true spectrum of tumour progression.