Background Pancreatic neuroendocrine tumors (PANETs) are rare, slow growing cancers that often present with local and distant metastasis upon detection. PANETS contain distinct karyotypes, epigenetic dysregulation, and recurrent mutations in MEN1 , ATRX , and DAXX ( MAD+ ); however, the molecular basis of disease progression remains uncharacterized. Methods We evaluated associations between aneuploidy and the MAD+ mutational state of 532 PANETs from 11 published genomic studies and 19 new cases using a combination of exome, targeted panel, shallow WGS, or RNA-seq. We mapped the molecular timing of MAD+ PANET progression using cellular fractions corrected for inferred tumor content. Results In 287 PANETs with mutational data, MAD+ tumors always exhibited a highly recurrent signature of loss of heterozygosity (LOH) and copy-number alterations affecting 11 chromosomes, typically followed by genome doubling upon metastasis. These LOH chromosomes substantially overlap with those that undergo non-random mis-segregation due to ectopic CENP-A localization to flanking centromeric regions in DAXX -depleted cell lines. Using expression data from 122 PANETs, we found decreased gene expression in the regions immediately adjacent to the centromere in MAD+ PANETs. Using 43 PANETs from AACR GENIE, we inferred this signature to be preceded by mutations in MEN1 , ATRX , and DAXX . We conducted a meta-analysis on 226 PANETs from 8 CGH studies to show an association of this signature with metastatic incidence. Our study shows that MAD+ tumors are a genetically diverse and aggressive subtype of PANETs that display extensive chromosomal loss after MAD+ mutation, which is followed by genome doubling. Conclusions We propose an evolutionary model for a subset of aggressive PANETs that is initiated by mutation of MEN1 , ATRX , and DAXX , resulting in defects in centromere cohesion from ectopic CENP-A deposition that leads to selective loss of chromosomes and the LOH phenotype seen in late-stage metastatic PANETs. These insights aid in disease risk stratification and nominate potential therapeutic vulnerabilities to treat this disease.

Uterine carcinosarcoma, also known as Malignant Mixed Mullerian Tumour, is a high-grade biphasic neoplasm composed of sarcomatous elements thought to originate via transdifferentiation from high-grade endometrial carcinoma. To identify molecular factors contributing to the histogenesis of this tumour, we analyzed DNA extracted from matched carcinoma and sarcoma components from 12 cases of carcinosarcoma by a molecular inversion probe microarray to assess genomic copy number alterations (CNAs) and allelic imbalances. Widespread CNAs were identified in tumours with serous histology in the carcinoma component (9/12), while the remaining three cases with endometrioid carcinoma were near-diploid. Quantification of the extent of genomic aberrations revealed a significant increase in sarcoma relative to carcinoma in tumours with well-delineated histologic components. Focal amplification of 13q31.3 was identified in 6/12 profiled tumours, of which four harboured the aberration exclusively in the sarcoma component. This result was verified by fluorescence in situ hybridization against GPC5, the only gene situated within the minimal region of amplification. In a validation cohort composed of 97 carcinosarcomas and other uterine sarcomas, amplification of GPC5 (GPC5/CEP13 ratio >= 2.2) was identified in 11/97 (11.3%) cases (9/64 carcinosarcoma, 1/3 rhabdomyosarcoma, 1/21 leiomyosarcoma, 0/8 adenosarcoma, 0/1 undifferentiated endometrial sarcoma) and an additional 4 (2.8%) cases had low level gains (GPC5/CEP13 ratio >= 1.5 but <2.2). The functional relevance of Glypican-5, the gene product of GPC5, in regulating differentiation and lineage commitment was demonstrated in an endometrial carcinoma cell line in vitro. In conclusion, we identified GPC5 amplification as a molecular event mediating epithelial-mesenchymal transdifferentiation in a subset of uterine carcinosarcomas.

Neuroendocrine tumours (NETs) are cancers of neuroendocrine often presenting with diverse genomic landscapes. We explored the molecular profiles of gastrointestinal (GINET) and pancreatic NETs (PNET) by profiling 45 NETs combining exome and RNA sequencing, shallow whole genome sequencing, and fluorescent in situ hybridization. In addition to expected somatic mutations and copy number alterations, we found that PNETs contained a highly consistent copy neutral loss-of-heterozygosity (CN-LOH) profile affecting a greater percentage of the genome than any cancer analyzed to date. Our data indicates that onset of extreme autozygosity may be triggered by the loss of DAXX/ATRX, and subsequent biallelic loss of MEN1. We confirmed this molecular timing model using targeted clinical sequencing data from 23 PNETs made available by the AACR GENIE project. Against this background of CN-LOH, several chromosomal regions consistently retained heterozygosity, suggesting selection for crucial allele-specific components specific to PNET progression and potential new therapeutic targets.

Introduction: Fluorescence in situ hybridization (FISH) is the standard procedure for the detection of anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangement in non-small-cell lung carcinoma (NSCLC) but is expensive and time consuming. We tested three antibodies to ALK, using various detection systems, and hypothesized that ALK immunohistochemistry (IHC) may represent a cost-effective and efficient means of screening for ALK rearrangement in NSCLC.Methods: We screened 377 stage I or II NSCLC cases in a tissue microarray by FISH and IHC (5A4 [Leica Biosystems Newcastle Ltd, Newcastle upon Tyne, UYnited Kingdom] by Nichirei's N-Histofine ALK detection kit [Nichirei Biosciences inc., Tokyo, Japan], 5A4 by Novocastra with ADVANCE [Dako Canada inc., Burlington, Ontario, Canada], D5F3 by Cell Signaling Technology with ADVANCE [Cell Signalling Technologies inc., Danvers, MA], and DAKO clone ALK1 with FLEX [Dako Canada inc., Burlington, Ontario, Canada] and ADVANCE). IHC was scored as 0, 1+, 2+, or 3+. Possibly positive or positive cases were further analyzed by IHC and FISH on whole section.Results: Tissue microarray results were available on 377 cases by IHC and 273 cases by FISH. Eleven cases were positive or possibly positive by either IHC or FISH, and three cases were positive or possibly positive by both methods. Three cases were ALK-positive by FISH on whole section validation. There was no correlation between semiquantitative IHC score (1+, 2+, 3+) and ALK rearrangement by FISH. D5F3 (Cell Signaling by ADVANCE) and 5A4 (Novocastra by ADVANCE) showed the greatest combination of sensitivity (100%) and specificity (87.5% for 5A4 by Novocastra and 75% for D5F3 by Cell Signaling), and produced no false--negative results.Conclusions: IHC is a reliable screening tool for identification of ALK rearrangement in NSCLC and is antibody dependent. D5F3 Cell Signaling) and 5A4 (Novocastra) can be used with FISH for identification of IHC-positive cases to reduce screening costs.

Hyalinizing clear-cell carcinoma (HCCC) is a rare, low-grade salivary gland tumor with distinctive clear-cell morphology and pattern of hyalinization as well as focal mucinous differentiation. However, histological overlap exists with other salivary gland tumors, such as epithelial-myoepithelial carcinoma (EMCa), salivary myoepithelial carcinoma, and mucoepidermoid carcinoma (MEC). The potential relationship between HCCC and its morphological mimics has not been yet investigated at the genetic level. In this study, we conducted a molecular analysis for the presence of rearrangements in MAML2, commonly seen in MECs, and EWSR1, involved in "soft tissue myoepithelial tumors" (SMET) by fusion with POU5F1, PBX1, or ZNF444. Fluorescence in situ hybridization (FISH) was performed on 23 HCCC cases for abnormalities in MAML2, EWSR1, FUS, POU5F1, PBX1, and ZNF444. FISH for MAML2 was negative in all cases (0 of 14), including those with mucinous differentiation (0 of 7). An EWSR1 rearrangement was identified in 18 of 22 HCCCs (82%), while no break-apart signals were seen in FUS, POU5F1, PBX1, or ZNF444. 3'RACE on an EWSR1 rearranged HCCC identified an EWSR1-ATF1 fusion, which was confirmed by RT-PCR. ATF1 involvement was further confirmed by FISH analysis in 13 of 14 EWSR1-rearranged HCCC cases (93%). In contrast, all control cases tested, including among others 5 EMCa and 3 MEC with clear cells, were negative for EWSR1 and ATF1 rearrangements. The presence of EWSR1-ATF1 fusion in most HCCCs reliably separates these tumors from its histological mimics. The distinction from MEC is particularly important, as conventional MEC grading schemes overgrade these indolent HCCCs, potentially impacting on treatment.

BACKGROUND: Fluorescence in situ hybridization (FISH) results from fine needle aspirates (FNA) of B-cell non-Hodgkin lymphomas (NHLs) were reviewed to 1) investigate the value added by using specific gene rearrangement probes to lymphoma diagnosis, prognosis, and subtyping; and 2) evaluate the prevalence of cytogenetic alterations other than specific translocations. METHODS: FISH results from assays performed on cytospin preparations from NHL FNAs over a 6-year period (2003-2009) were selected. Immunophenotyping, clinical data, and cytomorphologic data were reviewed according to the current World Health Organization (WHO) classification system. Hybridized probes, the purpose for the assay (subtyping or prognosis), and the cytogenetic abnormalities observed were retrieved from cytology reports. Data was categorized according to specific rearrangements and other chromosomal abnormalities. RESULTS: Successful results were obtained in 284 (95.3%) of 298 cases from 282 patients. Abnormalities were found in 216 (76%) cases and 68 (24%) did not show alteration. Among cases submitted for subtyping, 198 showed FISH-positive results, and specific gene rearrangements were found in 122 (61.6%) cases as follows: follicular 82, mantle cell 21, marginal zone 3, "dual hit" 13, and Burkitt lymphoma 3. In 21 cases, abnormalities were useful for prognosis. Nonspecific alterations alone or in combination with translocations were found in 98 cases. CONCLUSIONS: FISH performed on cytospin preparations was useful for confirmation of specific subclasses of NHL and may also provide valuable prognostic information. Cytogenetic abnormalities other than specific translocations were frequently found and could provide supportive evidence for a definitive diagnosis of lymphoma in FNA. Cancer (Cancer Cytopathol) 2010;118:250-8. (C) 2010 American Cancer Society.

Fluorescence in situ hybridization (FISH) results from fine needle aspirates (FNA) of B-cell non-Hodgkin lymphomas (NHLs) were reviewed to 1) investigate the value added by using specific gene rearrangement probes to lymphoma diagnosis, prognosis, and subtyping; and 2) evaluate the prevalence of cytogenetic alterations other than specific translocations. FISH results from assays performed on cytospin preparations from NHL FNAs over a 6-year period (2003-2009) were selected. Immunophenotyping, clinical data, and cytomorphologic data were reviewed according to the current World Health Organization (WHO) classification system. Hybridized probes, the purpose for the assay (subtyping or prognosis), and the cytogenetic abnormalities observed were retrieved from cytology reports. Data was categorized according to specific rearrangements and other chromosomal abnormalities. Successful results were obtained in 284 (95.3%) of 298 cases from 282 patients. Abnormalities were found in 216 (76%) cases and 68 (24%) did not show alteration. Among cases submitted for subtyping, 198 showed FISH-positive results, and specific gene rearrangements were found in 122 (61.6%) cases as follows: follicular 82, mantle cell 21, marginal zone 3, “dual hit” 13, and Burkitt lymphoma 3. In 21 cases, abnormalities were useful for prognosis. Nonspecific alterations alone or in combination with translocations were found in 98 cases. FISH performed on cytospin preparations was useful for confirmation of specific subclasses of NHL and may also provide valuable prognostic information. Cytogenetic abnormalities other than specific translocations were frequently found and could provide supportive evidence for a definitive diagnosis of lymphoma in FNA. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.

CONTEXT:The development of trastuzumab has led to new therapeutic strategies for patients with breast cancer. Trastuzumab has been shown to only be effective in those patients whose cancers are HER2 positive by either immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH).OBJECTIVE:To determine the accuracy of HER2 testing in 2 provinces in Canada.DESIGN:Patients with metastatic breast cancer diagnosed between 1999 and 2002 who were HER2 positive in the local laboratories were included in this study. All cases were retested centrally, by IHC (using 4 methods) and FISH. In addition, 205 locally reported HER2-negative metastatic breast cancer cases were retested centrally.RESULTS:Concordance between the 505 local IHC-positive cases with central IHC testing varied between 79.3% and 89.6% depending on the IHC method and scoring method used. HER2 gene amplification by FISH was present in 86.1% of local IHC-positive cases. Concordance between the 205 local IHC-negative tumors with central testing ranged between 94.8% and 100% for IHC and was 98.5% for FISH.CONCLUSIONS:This study demonstrated that locally reported HER2-negative results were highly accurate, but less accuracy was demonstrated with local HER2-positive results. These results emphasize the need for participation in a quality assurance program.

Given the important prognostic and predictive utility of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB2) [human epidermal growth factor receptor-2 (HER2/neu)] in breast cancer, it is recommended that ErbB2 testing be performed on all invasive breast cancers at the time of diagnosis. A consensus, however, has not yet been reached as to the optimal method of evaluating ErbB2 status. Immunohistochemistry to detect protein overexpression and fluorescence in situ hybridization (FISH) to detect ErbB2 gene amplification are the most frequently used methods. As no one detection method fulfills all necessary requirements of reliability, reproducibility, and ease of use, we developed a novel approach in the form of a simple assay we refer to as protein and gene double staining (PGDS) which simultaneously evaluates protein overexpression and gene amplification by combining immunohistochemistry with chromogenic in situ hybridization (CISH). A total of 134 invasive breast carcinomas, including 81 cases with a full-face section and 53 cases included in a tissue microarray (TMA), were assessed by PGDS, and the results were correlated with ErbB2 gene amplification status as determined by FISH. ErbB2 gene copy number determined by CISH analysis in the PGDS assay showed excellent concordance with that of FISH (correlation coefficient 0.82; P<0.001 with full-face section cases, and 0.98; P<0.001 with cases in a TMA). The overall concordance rate for gene amplification status between PGDS and FISH was 90.12% in cases with a full-face section and 92.45% with TMA cases. Perfect correlation was seen between the PGDS assay and FISH in cases that were considered either nonamplified or highly amplified by the dual assay. Of the 17 cases that showed low amplification by PGDS, 5 were classified as nonamplified by FISH. Correction for chromosome 17 copy number in the FISH assessment contributed to the discordance between CISH and FISH results. This newly developed PGDS method represents a novel approach to ErbB2 status determination that combines the assessment of both protein overexpression and gene amplification in one simple assay. It is likely that this assay will aid in immunohistochemical calibration and will also increase the sensitivity and specificity of ErbB2 testing.