ALK-positive inflammatory myofibroblastic tumor harboring ALK gene rearrangement, occurring after allogeneic stem cell transplant in an adult male.

Inflammatory myofibroblastic tumor arose as a defined neoplasm from the disparate group of tumors (both neoplastic and inflammatory) originally described as inflammatory pseudotumors. The morphologic features are well described, and 50-60% of cases are associated with fusions of the anaplastic lymphoma kinase (ALK) gene. We describe an inflammatory myofibroblastic tumor in the lower abdominal wall of an adult male, which occurred 88days after he received an allogeneic stem cell transplant for T-lymphoblastic lymphoma, and which was positive for ALK immunohistochemistry and showed ALK gene rearrangement by fluorescence in situ hybridization. Two other cases are reported in the post-stem cell transplant setting, but both occurred in children and did not have molecular analysis performed. The etiology remains unclear, but may be due to immune dysregulation caused by any combination of prior chemotherapy, radiotherapy and immune suppression. These neoplasms should be considered as a rare consequence of allogeneic stem cell transplantation and referral to a specialist sarcoma center for further management may be required.

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.

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 FUS 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 FUS 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 RT-PCR 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.

ALK translocation is associated with ALK immunoreactivity and extensive signet-ring morphology in primary lung adenocarcinoma.

BACKGROUND: ALK rearrangement is particularly observed in signet-ring sub-type adenocarcinoma. Since fluorescence in situ hybridization (FISH) is not suitable for mass screening, we aimed to characterize the predictive utility of tumour morphology and ALK immunoreactivity to identify ALK rearrangement, in a primary lung adenocarcinoma dataset enriched for signet-ring morphology, compared with that of other morphology. METHODS: 7 adenocarcinomas from diagnostic archives reported with signet-ring morphology were assessed and compared with 11 adenocarcinomas without signet-ring features over the same time period. Growth patterns were reviewed, ALK expression was assessed by standard immunohistochemistry using ALK1 clone and Envision detection (Dako), and ALK rearrangement was assessed by FISH (Abbott Molecular). Associations between groups and predictive utility of tumour morphology and ALK expression using FISH as gold standard were calculated. RESULTS: 2 excision lung biopsy cases with pure (100%) signet-ring morphology and solid patterns demonstrated diffuse moderate cytoplasmic ALK immunoreactivity (2+) and harboured ALK rearrangements (p=0.007), unlike 5 mixed-signet-ring and 11 non-signet-ring adenocarcinomas, which showed negative or 1+ immunoreactivity; and did not harbour ALK rearrangements (p>0.1). ALK expression was not associated with ALK copy number. 6 of 7 cases with signet ring morphology stained for TTF-1. Pure signet-ring morphology and moderate ALK expression were both associated with ALK rearranged tumours. CONCLUSION: ALK rearrangement is strongly associated with ALK immunoreactivity, and was seen only in tumours with pure signet-ring morphology and solid growth pattern. Tumour morphology, growth pattern and ALK immunoreactivity appear to be good indicators of ALK rearrangement, with TTF-1 positivity aiding in proving primary pulmonary origin.

Incidence of MLL rearrangement in acute myeloid leukemia, and a CALM-AF10 fusion in M4 type acute myeloblastic leukemia.

To determine the incidence of the mixed lineage leukemia (MLL) gene rearrangements in acute myeloid leukemia (AML) without cytogenetically-detected 11q23 abnormalities, we screened 64 cases of AML at diagnosis for MLL rearrangement by FISH. Three cases (4.7%) had a MLL rearrangement detected; one was shown to have a cryptic t(11;22)(q23;q11) and another to have a t(9;11)(p21-22;q23) which had been missed by the conventional cytogenetic study. No 11q23 structural abnormality was visible in the third case. Twenty-six of the 64 cases were further studied by Southern blotting and DNA hybridization, and four of these cases (15%) were found to have MLL rearrangement: in three of these, FISH had not detected any abnormality. FISH was also used to confirm MLL involvement in eight cases of AML that had a cytogenetic abnormality at 11q23; in one of these, Southern blot did not show a rearrangement. The survival of patients with MLL abnormalities identified by cytogenetics, FISH and/or DNA analysis was significantly worse than that of patients without MLL abnormalities (event-free survival p = 0.016) although two patients with a t(9;11)(p21-22;q23) were long-term survivors, consistent with this particular translocation having a better prognosis. One further case with a cytogenetic abnormality close to 11q23 was studied; it was found to have a t(10;11)(p13;q21), and the breakpoints were shown by FISH to involve the Clathrin Assembly Lymphoid Myeloid (CALM) gene at 11q21 and the AF10 gene at 10p13. Our data confirm the value of combining cytogenetic, FISH and molecular analyses to define the incidence and precise nature of MLL and 11q23 abnormalities in AML.