BRAF alterations are frequent in cerebellar low-grade astrocytomas with diffuse growth pattern.

Cerebellar low-grade astrocytomas with a diffuse pattern of growth are uncommon, comprising World Health Organization (WHO) grade II diffuse astrocytomas (DA) and a minority of WHO grade I pilocytic astrocytomas (PA), so-called PA, "diffuse variant." Among 106 cerebellar low-grade astrocytomas (WHO grade I and II) operated on at the Mayo Clinic (1984-2010), we identified 19 such cases: 8 PA, "diffuse variant," 5 DA, and 6 that we were unable to classify further (low-grade astrocytomas, subtype indeterminate). We characterized these tumors using immunohistochemistry and currently available molecular markers (IDH1/2 mutations and BRAF mutation/fusion gene status) and investigated whether the markers could be used to aid the diagnostic process in combination with the clinical and pathologic features. KIAA1549-BRAF fusion was detected in 4 PA, "diffuse variant," 2 DA, and 2 low-grade astrocytomas, subtype indeterminate, indicating that these tumors were molecularly consistent with PA, the most common subtype of the series. A BRAF V600E mutation was detected in 1 PA, "diffuse variant" case; an IDH1 R132G mutation was found in 1 DA case. These results suggest that KIAA1549-BRAF fusion status and IDH1/2 and BRAF V600E mutational analyses may assist in the histologic classification of this diagnostically challenging group of tumors and result in a more accurate and objective combined molecular and histologic classification.

Nodular fasciitis: a novel model of transient neoplasia induced by MYH9-USP6 gene fusion.

Nodular fasciitis (NF) is a relatively common mass-forming and self-limited subcutaneous pseudosarcomatous myofibroblastic proliferation of unknown pathogenesis. Due to its rapid growth and high mitotic activity, NF is often misdiagnosed as a sarcoma. While studying the USP6 biology in aneurysmal bone cyst and other mesenchymal tumors, we identified high expression levels of USP6 mRNA in two examples of NF. This finding led us to further examine the mechanisms underlying USP6 overexpression in these lesions. Upon subsequent investigation, genomic rearrangements of the USP6 locus were found in 92% (44 of 48) of NF. Rapid amplification of 5'-cDNA ends identified MYH9 as the translocation partner. RT-PCR and direct sequencing revealed the fusion of the MYH9 promoter region to the entire coding region of USP6. Control tumors and tissues were negative for this fusion. Xenografts of cells overexpressing USP6 in nude mice exhibited clinical and histological features similar to human NF. The identification of a sensitive and specific abnormality in NF holds the potential to be used diagnostically. Considering the self-limited nature of the lesion, NF may represent a model of 'transient neoplasia', as it is, to our knowledge, the first example of a self-limited human disease characterized by a recurrent somatic gene fusion event.

HMGA2 expression analysis in cytological and paraffin-embedded tissue specimens of thyroid tumors by relative quantitative RT-PCR.

The distinction between benign and malignant thyroid tumors in some cytological and histological specimens remains challenging. The aim of this study was to evaluate the use of High Mobility Group A2 (HMGA2) mRNA expression to distinguish benign from malignant thyroid tumors in cytological and histological specimens. RNA samples from 170 thyroid formalin-fixed paraffin-embedded (FFPE) tissues and 226 fine needle aspiration (FNA) specimens were analyzed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The FFPE tissues included 34 follicular adenomas, 10 Hürthle cell adenomas (HA), 6 hyperplastic nodules, 4 atypical adenomas, 44 classic papillary thyroid carcinomas (PTC), 29 follicular variant of PTC, 23 follicular thyroid carcinomas, 17 Hürthle cell carcinomas (HC), and 3 anaplastic thyroid carcinomas. The FNA specimens included 55 follicular adenomas, 34 HA, 20 hyperplastic nodules, 8 Hashimoto thyroiditis, 32 PTC, 24 follicular variant of PTC, 30 follicular thyroid carcinomas, 21 HC, and 2 anaplastic thyroid carcinomas. HMGA2 mRNA levels were expressed as relative fold change after normalizing with a calibrator. HMGA2 expression in thyroid carcinomas (16.8-fold for FFPE and 18.2-fold for FNA) was significantly higher than in benign lesions (0.8-fold for FFPE and 0.8-fold for FNA). HMGA2 expression in HC was relatively low (1.8-fold for FFPE and 8.5-fold for FNA) compared with the other types of carcinomas. HMGA2 expression values of 4.5-fold and 5.9-fold were used as cutoff points for FFPE and FNA (excluding HA and HC), respectively, to separate benign and malignant thyroid tumors, with 97.5% clinical specificity and 79.8% sensitivity for FFPE, and 95.2% clinical specificity and 88.6% sensitivity for the FNA specimens. Conventional RT-PCR supported the qRT-PCR results. Detection of HMGA2 mRNA expression by qRT-PCR may be a useful tool to assist in the diagnosis of well-differentiated thyroid carcinomas. The 1-step qRT-PCR method is a sensitive, accurate, and reliable technique for gene expression analysis of thyroid tumors.

Diagnostic utility of IMP3 expression in thyroid neoplasms: a quantitative RT-PCR study.

The capability of molecular markers to differentiate between benign and malignant well-differentiated thyroid tumors remains unclear. The aim of this study was to evaluate the use of insulin-like growth factor II mRNA binding protein-3 (IMP3) mRNA expression to distinguish benign from malignant thyroid tumors. RNA samples from 80 formalin-fixed, paraffin-embedded thyroid tissues, including 22 usual papillary thyroid carcinomas (PTCs), 18 follicular variants of PTC, 5 follicular thyroid carcinomas, 33 follicular adenomas, and 2 hyperplastic nodules, were used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. IMP3 mRNA expression levels in thyroid tumors were expressed as relative fold change (fold) after normalization with normal thyroid RNA. The results showed that thyroid carcinomas including PTC, follicular variants of PTC, and follicular thyroid carcinomas have significantly higher IMP3 expression levels with 48.3, 35.3, and 43.8 fold, respectively, compared with benign thyroid lesions (2.8 fold). Using the IMP3 expression value of 5 fold as a cutoff point to separate benign and malignant thyroid tumors, IMP3 qRT-PCR analysis had a 91.4% clinical specificity and 86.7% clinical sensitivity for the diagnosis of well-differentiated thyroid carcinomas. Conventional RT-PCR and immunohistochemical analysis for IMP3 in a subset of cases supported the qRT-PCR results. These results indicate that detection of IMP3 mRNA expression levels by qRT-PCR may be a useful molecular marker to assist in the diagnosis of well-differentiated thyroid carcinomas.

Carboxypeptidase M: a biomarker for the discrimination of well-differentiated liposarcoma from lipoma.

The discrimination between well-differentiated liposarcomas/atypical lipomatous tumors and lipomas can be diagnostically challenging at the histological level. However, cytogenetic identification of ring and giant rod chromosomes supports the diagnosis of well-differentiated liposarcoma/atypical lipomatous tumor. These abnormal chromosomes are mainly composed of amplified genomic sequences derived from chromosome 12q13-15, and contain several genes, including MDM2, CDK4 (SAS), TSPAN31, HMGA2, and others. MDM2 is consistently amplified in well-differentiated liposarcomas/atypical lipomatous tumors, and up to 25% in other sarcomas. As part of a large genomic study of lipomatous neoplasms, we initially found CPM to be consistently amplified in well-differentiated liposarcomas/atypical lipomatous tumors. To further explore this initial finding, we investigated the copy number status of MDM2 and CPM by fluorescent in situ hybridization (FISH) on a series of 138 tumors and 17 normal tissues, including 32 well-differentiated liposarcoma/atypical lipomatous tumors, 63 lipomas, 11 pleomorphic lipomas, 2 lipoblastomas, 30 other tumors and 17 normal fat samples. All 32 well-differentiated liposarcoma/atypical lipomatous tumors showed amplification of MDM2 and CPM, usually >20 copies per cell. The other tumors lacked MDM2 and/or CPM amplification. Chromogenic in situ hybridization confirmed the above results on a subset of these tumors (n=27). These findings suggest that identification of CPM amplification could be used as an alternative diagnostic tool for the diagnosis of well-differentiated liposarcoma/atypical lipomatous tumors.