The utility of FOXO1 fluorescence in situ hybridization (FISH) in formalin-fixed paraffin-embedded specimens in the diagnosis of alveolar rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive neoplasm with unique t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations, resulting in PAX3/FOXO1 and PAX7/FOXO1 fusion genes, in approximately 80% of cases. These translocations and their gene fusions have not been identified in other neoplasms, making their identification an attractive target for applying ancillary diagnostic techniques such as fluorescence in situ hybridization (FISH). We report our experience with a dual-color break-apart FISH probe for the detection of FOXO1 (13q14) rearrangements in neoplasms within the differential diagnosis of ARMS, using routinely processed formalin-fixed, paraffin-embedded tissues. A total of 52 sarcomas were analyzed including ARMS (n = 25), embryonal rhabdomyosarcomas (n = 8), neuroblastoma (n = 1), desmoplastic small round cell tumors (n = 2), Ewing sarcoma/primitive neuroectodermal tumors (EWS/PNET; n = 15), and round cell liposarcoma (n = 1). Cytogenetics and/or reverse transcription polymerase chain reaction data were available on a subset of the ARMS (n = 11) and EWS/PNET cases (n = 5). A minimum of 100 interphase tumor nuclei were evaluated for the presence of intact or translocated signals. FOXO1 gene rearrangements were identified in 88% (22/25) of ARMS (mean: 91% positive cells/case; range: 50% to 100%), whereas no rearrangements were detected in the other neoplasms examined (mean: 1.4% positive cells/case; range: 0% to 4%). FOXO1 (13q14) FISH on formalin-fixed, paraffin-embedded tissues samples showed excellent concordance with reverse transcription polymerase chain reaction and cytogenetic analyses in ARMS cases, demonstrated excellent specificity (100%) when applied to potential mimickers such as EWS/PNET, and played an important role in the differential diagnosis of small round cell tumors.

The utility of fluorescence in situ hybridization (FISH) in the diagnosis of myxoid soft tissue neoplasms.

Diagnosing myxoid soft tissue neoplasms can be challenging because of overlapping histologic features. Distinct chromosomal translocations have been identified in several myxoid sarcomas, including t(12;16)(q13;p11) FUS-DDIT3 in myxoid liposarcoma, t(7;16)(q34;p11) FUS-CREB3L2 in low-grade fibromyxoid sarcoma, and t(9;22)(q31;q12) EWSR1-NR4A3 in extraskeletal myxoid chondrosarcoma. These recurrent chromosomal alterations are attractive targets for diagnostic studies. To that end, dual-color, break-apart fluorescence in situ hybridization (FISH) probes spanning the genomic regions of EWSR1 (22q12), DDIT3 (12q13), and FUS (16p11) (Vysis, Downer's Grove, IL) were evaluated in formalin-fixed, paraffin-embedded tissues from myxoid neoplasms, including intramuscular myxoma (n=10), myxoid liposarcoma (n=18), low-grade fibromyxoid sarcoma (n=10), extraskeletal myxoid chondrosarcoma (n=13), and myxofibrosarcoma (n=8). Of the myxoid liposarcomas, 18/18 cases had a rearrangement of the DDIT3 gene, with 17/18 (94.4%) showing both DDIT3 and FUS gene rearrangements. A FUS gene rearrangement was identified in 7/10 (70%) of low-grade fibromyxoid sarcomas, with no changes involving EWSR1 or DDIT3. An EWSR1 translocation was seen in 6/13 (46.2%) of extraskeletal myxoid chondrosarcomas, without changes in DDIT3 or FUS genes. The remaining neoplasms studied showed no rearrangements involving DDIT3, FUS, or EWSR1 genes. In conclusion, interphase FISH using DDIT3 and FUS probes identifies the characteristic translocation in myxoid liposarcoma. FUS and EWSR1 probes are useful in confirming the diagnosis of low-grade fibromyxoid sarcoma and extraskeletal myxoid chondrosarcoma, respectively. The specificity of the probes is documented as none of the non-translocation-associated myxoid tumors showed genomic abnormalities with the probes tested. FISH is capable of providing specific ancillary information useful in this often difficult differential diagnosis.

Extranodal marginal zone B-cell lymphomas of the ocular adnexa: multiparameter analysis of 34 cases including interphase molecular cytogenetics and PCR for Chlamydia psittaci.

Extranodal marginal zone B-cell lymphomas of MALT type (MALT lymphomas) show site-dependent variations in their morphologic, phenotypic, and/or cytogenetic findings. This report describes a comprehensive analysis of 34 ocular adnexa MALT lymphomas, including interphase fluorescence in situ hybridization for MALT lymphoma-associated cytogenetic abnormalities and polymerase chain reaction for Chlamydia psittaci, which has recently been suggested to be associated with ocular adnexa lymphomas. A typical morphologic pattern was identified in 79% of cases, while overtly monocytoid cytology (12%), predominantly plasmacytic features (9%), and lymphoepithelial lesions (3%) were uncommon. Aberrant CD43 or CD5 expression was also uncommon (12% and 3%, respectively). Plasmacytic differentiation (41%) was associated with stage IV disease (P=0.036) and gains of chromosomes 3 and/or 18q (P=0.021) (79%). +3 was more frequent in the orbit than in lacrimal gland or conjunctiva (P=0.005). Each of 31 cases was negative for MALT1 translocations. IGH translocations were identified in 3 cases (10%), although the translocation partner gene could not be identified. Polymerase chain reaction assays targeting species-specific regions within the C. psittaci omp1 and omp2 genes were negative in each of 30 cases. This study identifies the characteristic morphologic, phenotypic, and cytogenetic findings in ocular adnexa MALT lymphoma, including a subset differing from those arising at other anatomic sites. The frequent presence of +3 and/or +18q suggests that these abnormalities may contribute to lymphomagenesis. The lack of C. psittaci in this series, in contrast to some prior reports, indicates that there may also be geographic heterogeneity in the pathogenesis of ocular adnexa MALT lymphoma.

Fluorescence in situ hybridization analysis of immunoglobulin heavy chain translocations in plasma cell myeloma using intact paraffin sections and simultaneous CD138 immunofluorescence.

Fluorescence in situ hybridization (FISH) studies are much more sensitive than classical cytogenetics for identification of karyotypic abnormalities in plasma cell myeloma. However, FISH analysis of bone marrow samples is often challenging because of a large number of admixed non-neoplastic hematopoietic elements. In this report, we describe a novel method using FISH analysis of intact paraffin sections of formalin-fixed, bone marrow clot preparations with simultaneous CD138 tyramine signal amplification (TSA)-mediated immunofluorescence. We studied 22 cases of plasma cell myeloma for translocations involving the immunoglobulin heavy chain locus that are of known diagnostic and/or prognostic significance. All cases were analyzed using dual color, break-apart immunoglobulin heavy chain probe and dual color, dual fusion probes for t(11;14)(q13;q32) and t(4;14)(p16;q32). TSA-mediated fluorochrome deposition in CD138+ cells was unaltered by protease pretreatment. Translocations were identified in 10 cases, including five with t(11;14)(q13;q32) and three with t(4;14)(p16.3;q32). When present, abnormalities were identified in a large percentage of CD138+ cells (47 to 93%, median 84%). This technique allows for efficient molecular cytogenetic analysis of plasma cell myeloma using routinely archived paraffin-embedded material.

The incidence of topoisomerase II-alpha genomic alterations in adenocarcinoma of the breast and their relationship to human epidermal growth factor receptor-2 gene amplification: a fluorescence in situ hybridization study.

Clinical and in vitro evidence supports the concept that human epidermal growth factor receptor-2 ( HER2 ) gene amplification prediction of response to anthracycline-based chemotherapy in breast cancer is not a direct effect of HER2 overexpression, but the result of coamplification of topoisomerase II-alpha ( TOP2A ). We investigated the relationship of TOP2A to HER2 genomic alterations by fluorescence in situ hybridization (FISH) and the correlations with polysomic states for chromosome 17 (CEP17). One hundred thirty-eight cases of breast cancer HER2 gene amplified by 2-color FISH ( HER2 /CEP17) were reevaluated with a 3-color probe set ( HER2 /CEP17/ TOP2A ) to investigate the frequency of coamplification and deletion of TOP2A . TOP2A was never amplified in the absence of HER2 amplification and was coamplified with HER2 in 68 (50%) of 137 cases; HER2 gene copy number was higher than the TOP2A copy number ( P < .01). Of the 137 cases with HER2 amplification, 23 (16%) showed a monoallelic deletion of TOP2A . Of the 43 cases not amplified for HER2 , 27 (63%) were CEP17 eusomic, 13 (30%) polysomic, and 3 (7%) monosomic. Of the HER2 nonamplified cases, 2 (5%) showed monoallelic deletion of both the HER2 and TOP2A . The current study demonstrates the complex interrelationship between the HER2 and TOP2A genes in breast cancer. The clinical implications of TOP2A amplification and deletion in breast cancer need to be further defined. If TOP2A gene dosage can be confirmed to correlate with tumor responsiveness to anthracycline-based therapy in the clinical setting, FISH testing for TOP2A status may be warranted to aid in the selection of the most appropriate therapy.

Expression profile and molecular genetic regulation of cyclin D1 expression in epithelioid sarcoma.

Epithelioid sarcoma is a distinctive, aggressive soft tissue tumor typically presenting as a subcutaneous or deep dermal mass in the distal extremities of young adults. Molecular genetic data of well-characterized cases of epithelioid sarcoma are sparse. A recent cytogenetic study of epithelioid sarcoma by conventional metaphase comparative genomic hybridization reported recurrent gains at chromosome 11q13, a region containing many genes, including the cyclin D1 gene. Cyclin D1 is a positive cell cycle regulator that is overexpressed in a variety of neoplasms, including mantle cell lymphoma and breast carcinoma. The objective of this study was to examine cyclin D1 expression in epithelioid sarcoma. Of 24 cases evaluated, 23 (96%) displayed cyclin D1 nuclear expression using immunohistochemical evaluation. Eight cases, which expressed cyclin D1 by immunohistochemistry, were evaluated by fluorescence in situ hybridization (FISH) and RNA in situ hybridization (RISH) for amplification of the cyclin D1 gene and messenger RNA (mRNA) expression, respectively. Seven of eight cases showed a typical eusomic state. One case showed pseudoamplification due to aneusomy/polysomy. There was no evidence of cyclin D1 gene amplification or messenger RNA overexpression detected by FISH or RNA in situ hybridization analyses, respectively. Our data clearly demonstrate that cyclin D1 protein is upregulated in epithelioid sarcoma, suggesting a role for this cell cycle regulator in the pathogenesis of epithelioid sarcoma. The high level of cyclin D1 protein expression in epithelioid sarcoma appears to be regulated by translational and/or post-translational mechanisms.

Chromosome 1p allelic loss by fluorescence in situ hybridization is not observed in dysembryoplastic neuroepithelial tumors.

Differentiation of dysembryoplastic neuroepithelial tumor (DNT) from cystic low-grade oligodendroglioma, particularly in a limited biopsy orfragmented specimen, may be impossible. Research has shown that allelic loss of chromosome 1p is a relatively common finding in oligodendrogliomas. Little is known about chromosome 1p status in DNT. We retrospectively evaluated 14 DNTs for loss of heterozygosity (LOH) on chromosome 1p by fluorescence in situ hybridization (FISH) and compared the results with 1p FISH analysis in 57 low-grade oligodendrogliomas (World Health Organization grade II). The 14 DNTs arose in 8 females and 6 males (mean age, 20.9 years at the time of surgery). All 14 DNTs were 1p intact by FISH analysis. The 57 low-grade oligodendrogliomas arose in 31 males and 26 females (mean age, 43.2 years). LOH on chromosome 1p was present in 31 (54%) of 57 tumors; the remaining 26 tumors were 1p intact. LOH on chromosome 1p is not a feature of DNTs. LOH on chromosome 1p may be a useful differential diagnostic feature (favoring oligodendroglioma) in a subset of cases in which specimen fragmentation or size raises the differential diagnosis of DNT vs oligodendroglioma.