Histology of the vertebral artery-dural junction: relevance to posterolateral approaches to the skull base.

OBJECTIVE: The far-lateral and extreme-lateral infrajugular transcondylar-transtubercular exposure (ELITE) and extreme-lateral transcondylar transodontoid (ELTO) approaches provide access to lesions of the foramen magnum, inferolateral to mid-clivus, and ventral pons and medulla. A subset of pathologies in this region require manipulation of the vertebral artery (VA)-dural interface. Although a cuff of dura is commonly left on the VA to avoid vessel injury during these approaches, there are varying descriptions of the degree of VA-dural separation that is safely achievable. In this paper the authors provide a detailed histological analysis of the VA-dural junction to guide microsurgical technique for posterolateral skull base approaches. METHODS: An ELITE approach was performed on 6 preserved adult cadaveric specimens. The VA-dural entry site was resected, processed for histological analysis, and qualitatively assessed by a neuropathologist. RESULTS: Histological analysis demonstrated a clear delineation between the intima and media of the VA in all specimens. No clear plane was identified between the connective tissue of the dura and the connective tissue of the VA adventitia. CONCLUSIONS: The VA forms a contiguous plane with the connective tissue of the dura at its dural entry site. When performing posterolateral skull base approaches requiring manipulation of the VA-dural interface, maintenance of a dural cuff on the VA is critical to minimize the risk of vascular injury.

Structural Variation Detection by Proximity Ligation from Formalin-Fixed, Paraffin-Embedded Tumor Tissue.

The clinical management and therapy of many solid tumor malignancies depends on detection of medically actionable or diagnostically relevant genetic variation. However, a principal challenge for genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed, paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology for generating long-range DNA sequence data as is required to detect genomic structural variation or long-range genotype phasing. We have developed a high-throughput chromosome conformation capture approach for FFPE samples that we call Fix-C, which is similar in concept to Hi-C. Fix-C enables structural variation detection from archival FFPE samples. This method was applied to 15 clinical adenocarcinoma- and sarcoma-positive control specimens spanning a broad range of tumor purities. In this panel, Fix-C analysis achieves a 90% concordance rate with fluorescence in situ hybridization assays, the current clinical gold standard. In addition, novel structural variation undetected by other methods could be identified, and long-range chromatin configuration information recovered from these FFPE samples harboring highly degraded DNA. This powerful approach will enable detailed resolution of global genome rearrangement events during cancer progression from FFPE material and will inform the development of targeted molecular diagnostic assays for patient care.

Functional screening in Drosophila identifies Alzheimer's disease susceptibility genes and implicates Tau-mediated mechanisms.

Using a Drosophila model of Alzheimer's disease (AD), we systematically evaluated 67 candidate genes based on AD-associated genomic loci (P < 10(-4)) from published human genome-wide association studies (GWAS). Genetic manipulation of 87 homologous fly genes was tested for modulation of neurotoxicity caused by human Tau, which forms neurofibrillary tangle pathology in AD. RNA interference (RNAi) targeting 9 genes enhanced Tau neurotoxicity, and in most cases reciprocal activation of gene expression suppressed Tau toxicity. Our screen implicates cindr, the fly ortholog of the human CD2AP AD susceptibility gene, as a modulator of Tau-mediated disease mechanisms. Importantly, we also identify the fly orthologs of FERMT2 and CELF1 as Tau modifiers, and these loci have been independently validated as AD susceptibility loci in the latest GWAS meta-analysis. Both CD2AP and FERMT2 have been previously implicated with roles in cell adhesion, and our screen additionally identifies a fly homolog of the human integrin adhesion receptors, ITGAM and ITGA9, as a modifier of Tau neurotoxicity. Our results highlight cell adhesion pathways as important in Tau toxicity and AD susceptibility and demonstrate the power of model organism genetic screens for the functional follow-up of human GWAS.

Gliosarcoma arising from an oligodendroglioma (oligosarcoma).

Gliosarcoma, a biphasic tumor with both mesenchymal and glial elements, is typically considered a variant of astrocytoma (glioblastoma), WHO Grade IV. A 57-year-old man presented with altered mental status and was found to have a large right frontal mass. Biopsy and subsequent subtotal resection revealed a WHO Grade II oligodendroglioma with classic histological features, expression of IDH1 R132H mutant protein, and chromosome 1p19q co-deletion. Fifteen months later, the patient developed recurrent tumor composed of intersecting fascicles of spindled cells with necrosis and a high mitotic index. The recurrent tumor stained for both mesenchymal and glial elements, consistent with the diagnosis of gliosarcoma, and showed retained IDH1 R132H expression. By FISH analysis, the gliosarcoma showed no evidence of 1p19q co-deletion. We performed SNP arrays and detailed SNP analysis of both the oligodendroglioma and the gliosarcoma. This demonstrated loss of heterozygosity (LOH) of chromosomes 1 and 19 in the gliosarcoma with retention of the same full-length chromosomes 1 and 19 found intact in the oligodendroglioma. Not surprisingly, the gliosarcoma harbored multiple additional alterations, consistent with clonal evolution. There have been only rare reports of sarcomatous transformation of oligodendroglioma ("oligosarcoma") and most were published prior to the development of modern genetic modalities. Here we present a case with detailed genetic evidence that suggests that mesenchymal metaplasia sarcomatous transformation is possible in classic oligodendrogliomas with 1p19q codeletions.

A common 8q (MYC) amplification detected in a multifocal anaplastic astrocytoma by SNP array karyotyping.

The distinction of multifocal versus multicentric gliomas can conceivably have important therapeutic implications. We present a 27-year-old man with two radiologically distinct non-enhancing infiltrative masses in the anterior frontal lobe and the posterior temporoparietal region. No intervening disease was evident on MRI modalities; the lesions were stable over a period of many months. He underwent two separate resections a few months apart. Given the question of whether his tumors represented two de novo primary multicentric tumors or one multifocal tumor, single nucleotide polymorphism (SNP) array karyotyping and in situ hybridization studies were performed on both tumors. The two tumor profiles looked remarkably similar, histologically and genetically: both were anaplastic astrocytomas with a common 33Mb gain/ amplification of 8q23.3-q24.3, including MYC amplification, suggesting a monoclonal origin. The temporoparietal neoplasm showed several additional genetic alterations. This case illustrates that even with today's advanced neuroimaging modalities, extensive radiologically invisible tumor may be present between seemingly separate sites of glioma involvement. Thus modern global genomic studies of such tumors may help distinguish whether multiple tumors represent one extensive neoplasm with microscopically invasive disease or multiple genetically distinct tumors.

Molecular classification of adult renal epithelial neoplasms using microRNA expression and virtual karyotyping.

Oncocytoma, chromophobe renal cell carcinoma (chRCC), and the eosinophilic variant of clear cell RCC (ccRCC) are morphologically similar tumors with significantly different clinical courses. These renal tumor subtypes show characteristic structural genetic changes; however, the mRNA expression patterns of oncocytoma and chRCC are strikingly similar. MicroRNAs (miRNA) are small RNA molecules that regulate the expression of many genes and have been shown to be useful for tumor classification and identification. The miRNA expression was analyzed from formalin-fixed paraffin-embedded tissue in 5 cases each of oncocytoma, ccRCC, papillary RCC, chRCC, and 4 normal kidney tissues using microarrays. Affymetrix single-nucleotide polymorphism arrays were used to detect chromosomal imbalances in each of the tumors. Eighteen miRNAs were significantly different among the 4 tumor types. The microRNA miR-21, a known oncogenic miRNA, was found to be upregulated in papillary and clear cell carcinomas. Four miRNAs could differentiate oncocytomas from chRCCs and the 3 could differentiate papillary RCC from ccRCC, including miR-126, a known vasculogenic miRNA. Of the 18 differentially expressed miRNAs, only 2 correlated with copy number changes in the chromosomal region harboring these genes. One tumor, originally diagnosed as an oncocytoma by morphology, showed a virtual karyotype and miRNA expression pattern consistent with chromophobe carcinoma. Further investigation of the tumor showed vascular invasion. Our study suggests that miRNA expression can be used to differentiate the common subtypes of renal epithelial neoplasms but further validation is necessary. In addition, the lack of correlation between miRNA expression and virtual karyotype suggests a non-copy-number-related mechanism for miRNA gene expression regulation in renal neoplasia.

KRAS mutation detection in colorectal cancer by a commercially available gene chip array compares well with Sanger sequencing.

BACKGROUND: Binding of a ligand to the epidermal growth factor receptor (EGFR) stimulates various intracellular signaling pathways resulting in cell cycle progression, proliferation, angiogenesis and apoptosis inhibition. KRAS is involved in signaling pathways including RAF/MAPK and PI3K and mutations in this gene result in constitutive activation of these pathways, independent of EGFR activation. Seven mutations in codons 12 and 13 of KRAS comprise around 95% of the observed human mutations, rendering monoclonal antibodies against EGFR (e.g. cetuximab and panitumumab) useless in treatment of colorectal cancer. METHODS: KRAS mutation testing by two different methodologies was compared; Sanger sequencing and AutoGenomics INFINITI® assay, on DNA extracted from colorectal cancers. RESULTS: Out of 29 colorectal tumor samples tested, 28 were concordant between the two methodologies for the KRAS mutations that were detected in both assays with the INFINITI® assay detecting a mutation in one sample that was indeterminate by Sanger sequencing and a third methodology; single nucleotide primer extension. CONCLUSIONS: This study indicates the utility of the AutoGenomics INFINITI® methodology in a clinical laboratory setting where technical expertise or access to equipment for DNA sequencing does not exist.

Myxoid liposarcoma: a case report of a sentinel metastasis to the parotid gland with molecular confirmation.

Myxoid liposarcoma is a subtype of liposarcoma with a predilection for the deep soft tissues of the extremities that accounts approximately for 10% of all adult soft tissue sarcomas. We report a case of a metastatic myxoid liposarcoma to the parotid gland, with fine-needle aspiration cytology correlation and molecular characterization. The lesion was diagnosed in a 53-year-old Hispanic male who presented with a left posterior thigh mass. A core needle biopsy established the diagnosis of myxoid liposarcoma. The patient underwent limb-sparing, wide local excision of the malignancy and later presented with an initial metastatic lesion to the parotid gland. The diagnosis of metastatic myxoid liposarcoma was rendered by fine-needle aspiration cytology with cell block preparation, and molecular confirmation. Although myxoid/round cell liposarcomas are classically described as having minimal pleomorphism on cytologic material, we encountered significant pleomorphism in our case. Therefore, a diagnosis of myxoid/round cell liposarcoma should still be a diagnostic consideration even if markedly pleomorphic cells are seen in fine-needle aspiration biopsies.

Detection of myxoid liposarcoma-associated FUS-DDIT3 rearrangement variants including a newly identified breakpoint using an optimized RT-PCR assay.

Myxoid/round cell liposarcoma is characterized by the recurrent translocations t(12;16)(q13;p11) and, less commonly, t(12;22)(q13;q12), which fuse FUS or EWSR1, respectively, to DDIT3 on chromosome 12. Although a number of different variant breakpoints have been described, greater than 90% of all cases have one of the three different FUS-DDIT3 fusions, which may have clinical significance. To identify the individual breakpoints, a sequence-specific assay such as reverse transcription-PCR (RT-PCR) is needed. In this study, we optimized primer design to develop an RT-PCR assay for the detection of the most common translocations in formalin-fixed paraffin-embedded tissue specimens. We compared our assay with primers previously published for testing formalin-fixed paraffin-embedded specimens and achieved the most consistent results with our primers. We obtained RNA from 32 MLS cases, of which 27 carried one of the three common FUS-DDIT3 chimeric transcript types. Four of the negative cases were from very small biopsies with very low RNA concentration. One case was consistently negative by RT-PCR, but showed a FUS rearrangement by fluorescent in situ hybridization, suggesting that it may harbor one of the rarer FUS-DDIT3 chimeric types. In addition to the common fusions, our assay also identified a novel FUS-DDIT3 fusion between exon 9 of FUS and exon 3 of DDIT3 in one of the cases.

Mast cell burden and reticulin fibrosis in the myeloproliferative neoplasms: a computer-assisted image analysis study.

The mast cell has been associated with fibrosis in many different tissues, organs, and different disease processes including hematopoietic malignancies. Mast cells are often increased in the bone marrow of patients with primary bone marrow disorders, and patients with systemic mastocytosis often have a second concomitant neoplastic disease of the bone marrow. The goals of the current study were to determine the role the mast cell has in the pathogenesis of myeloproliferative neoplasms (MPN) and to correlate the mast cell burden with the degree of reticulin fibrosis. We used computer-assisted image analysis of bone marrow core biopsies stained for mast cell tryptase from patients with myeloproliferative neoplasms [31 cases: 12 chronic myelogenous leukemia (CML), 6 primary myelofibrosis (PMF), 4 essential thrombocythemia (ET), 4 polycythemia vera (PV), and 5 chronic myeloproliferative disorder, unclassifiable (CMPD-U)]. Although the number of cases of some subtypes of MPN was small, the results suggested that PMF and ET each had significantly more mast cells than both CML and control cases (P<0.01 and 0.05, respectively, Mann-Whitney test). CMPD-U and PV showed no significant differences from the control cases, but the CML cases had significantly fewer mast cells than our control cases (P=0.02, Mann-Whitney test). In addition, the quantity of mast cells seen in the bone marrows of MPN patients correlated with reticulin fibrosis (P=0.04, Mann-Whitney test). Our studies highlight the different mast cell quantities in different myeloproliferative neoplasms and suggest a direct role for the mast cell in intramedullary fibrosis. Further studies are warranted to confirm our observation and to study the mechanisms by which mast cells contribute to fibrosis in the MPN setting.

Polymorphisms in TGFbeta and TNFalpha are associated with the myelodysplastic syndrome phenotype.

CONTEXT: Myelodysplastic syndromes (MDSs) are characterized by ineffective hematopoiesis, excessive apoptosis, and the aberrant expression of a number of cytokines. The genes encoding these cytokines are significantly polymorphic. It is unknown whether these cytokine polymorphisms are associated with, and may therefore be playing a role in the pathogenesis of, MDS. OBJECTIVE: To determine if certain polymorphisms in the tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) cytokines are overrepresented in a cohort of patients with MDSs. DESIGN: DNA was isolated from the peripheral blood or bone marrow aspirate of 21 patients with MDS. The genotypes for 4 different polymorphisms, 2 in TNFalpha and 2 in TGFbeta1, were determined using single-specific-primer polymerase chain reaction. The allele and genotype frequencies were compared with similar populations in the National Cancer Institute SNP500 database. RESULTS: In our MDS population, the -308A/A genotype of the TNFalpha gene and the TGFbeta1 allele +29T and genotype +29T/T, each associated with higher levels of expression, were overrepresented in our MDS population. CONCLUSIONS: Polymorphisms associated with increased expression in the cytokines TNFalpha and TGFbeta1 are overrepresented in the MDS population suggesting that increased TNF-alpha and TGF-beta1 activity may contribute to the susceptibility and/or pathogenesis of MDS. Further studies with larger sample sizes are warranted to confirm our observation.