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Using a massively parallel (next-generation) DNA sequencing strategy, a member of our group recently discovered that a subset of ALK-negative inflammatory myofibroblastic tumors harbors rearrangements involving ROS1 (v-ros avian UR2 sarcoma virus oncogene homolog 1), Expression of ROS1 in inflammatory myofibroblastic tumors has not previously been evaluated.Archival tumor samples from 30 inflammatory myofibroblastic tumors were evaluated, including 21 ALK-positive tumors (10 previously confirmed to harbor ALK rearrangements) and 9 ALK-negative tumors (including 2 known to harbor ROS1 rearrangements).Archival samples from 60 other spindle cell neoplasms in the differential diagnosis with inflammatory myofibroblastic tumor were also evaluated, including 10 cases each of desmoid fibromatosis; myofibroblastic sarcoma; gastrointestinal stromal tumor, spindle cell type; leiomyosarcoma; follicular dendritic cell sarcoma; and dedifferentiated liposarcoma (5 morphologically low grade and 5 with the so-called ‘inflammatory MFH’ pattern).Immunohistochemistry was performed on 4-μm-thick formalin-fixed paraffin-embedded whole tissue sections following pressure cooker antigen retrieval (0.001 M citrate buffer; p H 6.0), using a rabbit anti-ROS1 monoclonal antibody (0 dilution; 40 min incubation; clone D4D6; Cell Signaling Technology, Danvers, MA).Immunohistochemistry was performed on whole tissue sections following pressure cooker antigen retrieval using a rabbit anti-ROS1 monoclonal antibody.The results were scored as ‘positive’ or ‘negative,’ and the pattern of staining was recorded.PCR and RT-PCR were then performed using primers that flank the breakpoints in the known YWHAE-ROS1 and TFG-ROS1 fusions.In brief, DNA and RNA were extracted from formalin-fixed paraffin-embedded tumor samples using the Qiagen All Prep DNA/RNA kit according to the manufacturer’s protocol.

FISH analysis was performed on interphase nuclei on 5-μm-thin sections of formalin-fixed paraffin-embedded tissue from one case that was found to be positive for ROS1 by immunohistochemistry (see Results), according to the standard protocols.The purpose of this study was to determine whether immunohistochemistry for ROS1 could predict ROS1 rearrangement in inflammatory myofibroblastic tumor.In total, 30 inflammatory myofibroblastic tumors were evaluated, including 21 ALK-positive tumors (10 confirmed to harbor ALK rearrangements, with TPM3, CLTC, RANPB2, and FN1 fusion partners) and 9 ALK-negative tumors (including 2 known to harbor ROS1 rearrangements).Next-generation DNA sequencing was performed on one case found to be positive for ROS1 by immunohistochemistry (with previously unknown genotype).In brief, hematoxylin and eosin-stained slides were reviewed to evaluate tumor content and viability and an area enriched for 20% tumor was circled.