Fluorescence In Situ Hybridization for MDM2 Amplification as a Routine Ancillary Diagnostic Tool for Suspected Well-Differentiated and Dedifferentiated Liposarcomas: Experience at a Tertiary Center

Background. The assessment of MDM2 gene amplification by fluorescence in situ hybridization (FISH) has become a routine ancillary tool for diagnosing atypical lipomatous tumor (ALT)/well-differentiated liposarcoma and dedifferentiated liposarcoma (WDL/DDL) in specialist sarcoma units. We describe our experience of its utility at our tertiary institute. Methods. All routine histology samples in which MDM2 amplification was assessed with FISH over a 2-year period were included, and FISH results were correlated with clinical and histologic findings. Results. 365 samples from 347 patients had FISH for MDM2 gene amplification. 170 were positive (i.e., showed MDM2 gene amplification), 192 were negative, and 3 were technically unsatisfactory. There were 122 histologically benign cases showing a histology:FISH concordance rate of 92.6%, 142 WDL/DDL (concordance 96.5%), and 34 cases histologically equivocal for WDL (concordance 50%). Of 64 spindle cell/pleomorphic neoplasms (in which DDL was a differential diagnosis), 21.9% showed MDM2 amplification. Of the cases with discrepant histology and FISH, all but 3 had diagnoses amended following FISH results. For discrepancies of benign histology but positive FISH, lesions were on average larger, more frequently in “classical” (intra-abdominal or inguinal) sites for WDL/DDL and more frequently core biopsies. Discrepancies of malignant histology but negative FISH were smaller, less frequently in “classical” sites but again more frequently core biopsies. Conclusions. FISH has a high correlation rate with histology for cases with firm histologic diagnoses of lipoma or WDL/DDL. It is a useful ancillary diagnostic tool in histologically equivocal cases, particularly in WDL lacking significant histologic atypia or DDL without corresponding WDL component, especially in larger tumors, those from intra-abdominal or inguinal sites or core biopsies. There is a significant group of well-differentiated adipocytic neoplasms which are difficult to diagnose on morphology alone, in which FISH for MDM2 amplification is diagnostically contributory

Angiomatoid fibrous histiocytoma: comparison of fluorescence in situ hybridization and reverse transcription polymerase chain reaction as adjunct diagnostic modalities

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue neoplasm of intermediate biologic potential and uncertain differentiation, most often arising in the extremities of children and young adults. Although it has characteristic histologic features of a lymphoid cuff surrounding nodules of ovoid cells with blood-filled cystic cavities, diagnosis is often difficult due to its morphologic heterogeneity and lack of specific immunoprofile. Angiomatoid fibrous histiocytoma is associated with recurrent chromosomal translocations, leading to characteristic EWSR1-CREB1, EWSR1-ATF1, and, rarely, FUS-ATF1 gene fusions; fluorescence in situ hybridization (FISH), detecting EWSR1 or PUS rearrangements, and reverse transcription-polymerase chain reaction (RT-PCR) for EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts have become routine ancillary tools. We present a large comparative series of FISH and RT-PCR for AFH. Seventeen neoplasms (from 16 patients) histologically diagnosed as AFH were assessed for EWSR1 rearrangements or EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts. All 17 were positive for either FISH or RT-PCR or both. Of 16, 14 (87.5%) had detectable EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts by RT-PCR, whereas 13 (76.5%) of 17 had positive EWSR1 rearrangement with FISH. All 13 of 13 non-AFH control neoplasms failed to show EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts, whereas EWSR1 rearrangement was present in 2 of these 13 cases (which were histopathologically myoepithelial neoplasms). This study shows that EWSR1-CREB1 or EWSR1-ATF1 fusions predominate in AFH (supporting previous reports that PUS rearrangement is rare in AFH) and that RT-PCR has a comparable detection rate to FISH for AFH. Importantly, cases of AFH can be missed if RTPCR is not performed in conjunction with FISH, and RT-PCR has the added advantage of specificity, which is crucial, as EWSR1 rearrangements are present in a variety of neoplasms in the histologic differential diagnosis of AFH, that differ in behavior and treatment (C) 2015 Elsevier Inc. All rights reserved

1997; 82-1

© F e r r a t a S t o r t i F o u n d a t i o n 65 that encompasses true biphenotypic leukemias as well as others which are ALL or AML with atypical expression of a single marker from another lineage. Cases described as phenotypic switch probably represent examples of biphenotypic acute leukemias originating from a primitive stem cell with the potential to differentiate along the lymphoid or myeloid lineage, with the pathway taken being determined by the therapy used. We have proposed a scoring system aimed at distinguishing cases of bona fide biphenotypic acute leukemia from those with aberrant expression of a marker from another lineage, eg. Ly+AML and My+ALL. This system is based on the number and degree of specificity of the markers (lymphoid and myeloid) expressed by the leukemic cells. 2 Morphology and cytochemistry In one third of the cases, the blasts resemble lymphoblasts (L1 or L2 morphology). Cytochemical staining with Sudan black B (SBB), MPO and ␣-naphthyl esterase (ANAE) are negative (< 3%+ blasts). The remaining cases can be classified as AML on the basis of standard morphology and cytochemistry because they show more than 3% blasts positive with SBB or MPO, or display a pattern of ANAE activity typical of monoblasts (diffuse, strong and sensitive to NaF). According to FAB criteria most of the latter cases are either M1 or M2 or, rarely, M4 and M5. We have not yet seen a case with biphenotypic features which corresponds to the M3, M6 or M7 subtypes. It is not unusual to identify two distinct blast populations in the same patient, one of small size with a high nucleus/cytoplasm ratio resembling lymphoblasts and the other larger with more abundant cytoplasm with or without granulation ( Immunological markers Diagnosis of biphenotypic acute leukemia is based on immunophenotyping. According to the lymphoid and myeloid markers expressed by the blasts, four groups can be distinguished. The most common are those in which the blasts coexpress myeloid and B-lymphoid, less often T-lymphoid antigens. Trilineage differentiation with expression of B, T and myeloid markers is rare and coexistence Acute biphenotypic leukemia of blasts expressing only B and T cell markers is very uncommon. Most cases are terminal deoxynucleotidyl transferase (TdT) positive and express early hemopoietic markers such as CD34 and class-II HLA-DR determinants. In cases of biphenotypic acute leukemia classified as ALL by light microscopy morphology and cytochemistry (SBB-, MPO-), MPO activity can be demonstrated using sensitive techniques on unfixed cells at the ultrastructural level 3 or with the anti-MPO monoclonal antibody. 5 These findings support the myeloid commitment of the blasts. The fact that MPO activity cannot be detected by light microscopy cytochemistry is related to the small amount of this enzyme, which is only seen by electron microscopy in very small granules and in cell membranes. Alternatively, it is possible that the blasts contain the enzyme as a proenzyme form, which nevertheless is detectable with the monoclonal antibody. In cases of biphenotypic acute leukemia presenting with morphological and cytochemical features of AML, there is a high incidence (c.50%) of rearrangement of the Ig-heavy chain gene and/or T cell receptor chain genes, further confirming the lymphoid commitment of the blasts at a genomic level (simultaneously with the myeloid features) 2 Cytogenetics There is no single chromosomal abnormality which is uniquely associated with biphenotypic acute leukemia. Our own data and those of others demonstrate that structural chromosome abnormalities are frequent and that there is a high incidence of the Philadelphia chromosome t(9;22), 7 Clinical features Biphenotypic acute leukemias may affect adults or children, particularly infants under 2 years old. They may present as de novo or, rarely, they become apparent during a relapse following anti-AML or ALL therapy. The WBC is often high and most cases have a varying proportion of circulating blasts. 8 There are no uniform criteria about whether to treat these cases as ALL or AML when they are diagnosed only by standard morphology and cytochemistry, or whether to use an approach which combines drugs that are effective for ALL and AML, 9 followed by bone marrow or mobilized peripheral stem cell transplantation in complete remission. 10 Extensive data on response to therapy and clinical outcome are not available; however, our impression based on cases treated at the RMH and from single cases reported in the literature is that of a poor outcome in both children and adults. This may be related to the underlying chromosome abnormality. Conclusions In summary, biphenotypic acute leukemia is an uncommon type of leukemia which probably arises in a multipotent progenitor cell with the capability of differentiating along both myeloid and lymphoid lineages. This is supported by: i) immunological, cytochemical and molecular involvement of genes and/or protein products present in lymphoid and myeloid cells; ii) cytogenetic findings such as the presence of the Philadelphia chromosome or the MLL gene at 11q23, and iii) the documented phenomenon of in vivo and in vitro phenotypic switch in some of the cases. Data are emerging that biphenotypic acute leukemia has a poor prognosis and thus is likely to require a more intensive treatment approach to achieve long-term complete remissions, probably obtainable only with high-dose therapy followed by an allo-or autograft, which may eradicate the disease permanently. © F e r r a t a S t o r t i F o u n d a t i o

The comparative utility of fluorescence in situ hybridization and reverse transcription-polymerase chain reaction in the diagnosis of alveolar rhabdomyosarcoma.

Fluorescence in situ hybridization (FISH) for FOXO1 gene rearrangement and reverse transcription-polymerase chain reaction (PCR) for PAX3/7-FOXO1 fusion transcripts have become routine ancillary tools for the diagnosis of alveolar rhabdomyosarcomas (ARMS). Here we summarize our experience of these adjunct diagnostic modalities at a tertiary center, presenting the largest comparative series of FISH and PCR for suspected or possible ARMS to date. All suspected or possible ARMS tested by FISH or PCR for FOXO1 rearrangement or PAX3/7-FOXO1 fusion transcripts over a 7-year period were included. FISH and PCR results were correlated with clinical and histologic findings. One hundred samples from 95 patients had FISH and/or PCR performed. FISH had higher rates of technical success (96.8 %) compared with PCR (88 %). Where both tests were utilized successfully, there was high concordance rate between them (94.9 %). In 24 histologic ARMS tested for FISH or PCR, 83.3 % were translocation-positive (all for PAX3-FOXO1 by PCR) and included 3 histologic solid variants. In 76 cases where ARMS was excluded, there were 3 potential false-positive cases with FISH but none with PCR. PCR had similar sensitivity (85.7 %) and better specificity (100 %) in aiding the diagnosis of ARMS, compared with FISH (85 and 95.8 %, respectively). All solid variant ARMS harbored FOXO1 gene rearrangements and PAX3-FOXO1 ARMS were detected to the exclusion of PAX7-FOXO1. In comparative analysis, both FISH and PCR are useful in aiding the diagnosis of ARMS and excluding its sarcomatous mimics. FISH is more reliable technically but has less specificity than PCR. In cases where ARMS is in the differential diagnosis, it is optimal to perform both PCR and FISH: both have similar sensitivities for detecting ARMS, but FISH may confirm or exclude cases that are technically unsuccessful with PCR, while PCR can detect specific fusion transcripts that may be useful prognostically

Desmoplastic small round cell tumor evaluation of reverse transcription-polymerase chain reaction and fluorescence in situ hybridization as ancillary molecular diagnostic techniques

Desmoplastic small round cell tumor (DSRCT) is a rare, biologically aggressive soft tissue neoplasm of uncertain differentiation, most often arising in the abdominal and pelvic cavities of adolescents and young adults with a striking male predominance. Histologically, it is characterized by islands of uniform small round cells in prominent desmoplastic stroma, and it has a polyimmunophenotypic profile, typically expressing WT1 and cytokeratin, desmin, and neural/neuroendocrine differentiation markers to varying degrees. Tumors at other sites and with variant morphology are more rarely described. DSRCT is associated with a recurrent t(11;22)(p13;q12) translocation, leading to the characteristic EWSR1-WT1 gene fusion. Fluorescence in situ hybridization (FISH), to detect EWSR1 rearrangement, and reverse transcription-polymerase chain reaction (RT-PCR) to assess for EWSR1-WT1 fusion transcripts are routine diagnostic ancillary tools. We present a large institutional comparative series of FISH and RT-PCR for DSRCT diagnosis. Twenty-six specimens (from 25 patients) histologically diagnosed as DSRCT were assessed for EWSR1 rearrangement and EWSR1-WT1 fusion transcripts. Of these 26 specimens, 24 yielded positive results with either FISH or RT-PCR or both. FISH was performed in 23 samples, with EWSR1 rearrangement seen in 21 (91.3%). RT-PCR was performed in 18 samples, of which 13 (72.2%) harbored EWSR1-WT1 fusion transcripts. The sensitivity of FISH in detecting DSRCT was 91.3%, and that of RT-PCR was 92.8% following omission of four technical failures. Therefore, both methods are comparable in terms of sensitivity. FISH is more sensitive if technical failures for RT-PCR are taken into account, and RT-PCR is more specific in confirming DSRCT. Both methods complement each other by confirming cases that the other method may not. In isolation, FISH is a relatively non-specific diagnostic adjunct due to the number of different neoplasms that can harbor EWSR1 rearrangement, such as Ewing sarcoma. However, in cases with appropriate morphology and a typical pattern of immunostaining, FISH is confirmatory of the diagnosis

Long remissions in hairy cell leukemia with purine analogs: A report of 219 patients with a median follow-up of 12.5 years

BACKGROUND: Both pentostatin and cladribine have efficacy in hairy cell leukemia (HCL), but it is not known which agent achieves better results. METHODS: We reviewed a series of 219 patients with HCL, with median follow-up from diagnosis of 12.5 years (range 1.0 -34.6 yrs), treated with either pentostatin (n = 185) or cladribine (n = 34), to compare these agents and assess the potential for cure. RESULTS: Overall response to pentostatin was 96% with a complete response (CR) in 81% and a median disease-free survival (DFS) of 15 years. Response to first-line cladribine was 100% with a CR in 82% and DFS of 11+ years. The relapse rates at 5 years and 10 years were 24% and 42%, respectively, with pentostatin, and 33% and 48% with cladribine. Survival at 10 years was respectively 96% and 100%. CR rates decreased with each sequential relapse through 69% to 45% (P < or = 0.001). Patients achieving CR after first-line treatment had a significantly longer DFS (P = 0.00007) than those achieving a partial response; a similar result was seen after second-line therapy (P = 0.00001). DFS also declined with sequential treatment (P = 0.00005). CONCLUSION: We have shown equivalent efficacies for both agents in the treatment of HCL, with DFS showing no plateau. True cure in HCL remains elusive, but the addition of monoclonal antibodies may be beneficial. Our results suggest that achieving CR should remain the main goal of treatment

Comparative genomic hybridization and BUB1B mutation analyses in childhood cancers associated with mosaic variegated aneuploidy syndrome

We previously demonstrated that constitutional BUB1B mutations cause mosaic variegated aneuploidy, a condition characterized by constitutional aneuploidies and childhood cancer predisposition. To further investigate the role of BUB1B in cancer predisposition we performed comparative genomic hybridization analysis in an embryonal rhabdomyosarcoma from an MVA case with biallelic BUB1B mutations, revealing aneuploidies typical of sporadic E-RMS, with gain of chromosomes 3, 8, 13 and loss of chromosomes 9, 14, X. To investigate whether somatic BUB1B mutations occur in sporadic childhood cancers we screened 30 Wilms tumours, 10 acute lymphoblastic leukemias, nine rhabdomyosarcomas and 11 rhabdomyosarcoma cell lines for BUB1B mutations. We identified seven exonic and six intronic variants. Six of the exonic variants were synonymous and one resulted in a non-synonymous conservative missense alteration that was also present in a control. These data suggest that the genetic progression in rhabdomyosarcoma from MVA and non-MVA cases may be similar, but that somatic BUB1B mutations are unlikely to be common in sporadic childhood cancers known to be associated with MVA