The spectrum of EWSR1-rearranged neoplasms at a tertiary sarcoma centre; assessing 772 tumour specimens and the value of current ancillary molecular diagnostic modalities.

Background EWSR1 rearrangements were first identified in Ewing sarcoma, but the spectrum of EWSR1-rearranged neoplasms now includes many soft tissue tumour subtypes including desmoplastic small round cell tumour (DSRCT), myxoid liposarcoma (MLPS), extraskeletal myxoid chondrosarcoma (EMC), angiomatoid fibrous histiocytoma (AFH), clear cell sarcoma (CCS) and myoepithelial neoplasms. We analysed the spectrum of EWSR1-rearranged soft tissue neoplasms at our tertiary sarcoma centre, by assessing ancillary molecular diagnostic modalities identifying EWSR1-rearranged tumours and reviewing the results in light of our current knowledge of these and other Ewing sarcoma-like neoplasms.Methods We retrospectively analysed all specimens tested for EWSR1 rearrangements by fluorescence in situ hybridisation (FISH) and/or reverse transcription-PCR (RT-PCR) over a 7-year period.Results There was a total of 772 specimens. FISH was performed more often than RT-PCR (n=753, 97.5% vs n=445, 57.6%). In total, 210 (27.9%) specimens were FISH-positive for EWSR1 rearrangement compared to 111 (14.4%) that showed EWSR1 fusion transcripts with RT-PCR. Failure rates for FISH and RT-PCR were 2.5% and 18.0%. Of 109 round cell tumours with pathology consistent with Ewing sarcoma, 15 (13.8 %) cases were FISH-positive without an identifiable EWSR1 fusion transcript, 4 (3.7%) were FISH-negative but RT-PCR positive and 4 (3.7%) were negative for both. FISH positivity for DSRCT, MLPS, EMC, AFH and CCS was 86.3%, 4.3%, 58.5%, 60.0% and 87.9%, respectively. A positive FISH result led to diagnostic change in 40 (19.0%) EWSR1-rearranged cases. 13 FISH-positive cases remained unclassifiable.Conclusions FISH is more sensitive for identifying EWSR1 rearrangements than RT-PCR. However, there can be significant morphologic and immunohistochemical overlap between groups of EWSR1-rearranged neoplasms, with important prognostic and therapeutic implications. FISH and RT-PCR should be used as complementary modalities in diagnosing EWSR1-rearranged neoplasms, but as tumour groups harbouring EWSR1 rearrangements are increasingly characterised and because given translocations involving EWSR1 and its partner genes are not always specific for tumour types, it is critical that these are evaluated by specialist soft tissue surgical pathologists noting the morphologic and immunohistochemical context. As RT-PCR using commercial primers is limited to only the most prevalent EWSR1 fusion transcripts, the incorporation of high-throughput sequencing technologies into the standard diagnostic repertoire to assess for multiple molecular abnormalities of soft tissue tumours in parallel (including detection of newly characterised Ewing sarcoma-like tumours) might be the most effective and efficient means of ancillary diagnosis in future

ALL-1/MLL1, a homologue of Drosophila TRITHORAX, modifies chromatin and is directly involved in infant acute leukaemia

Rearrangements of the ALL-1/MLL1 gene underlie the majority of infant acute leukaemias, as well as of therapy-related leukaemias developing in cancer patients treated with inhibitors of topoisomerase II, such as VP16 and doxorubicin. The rearrangements fuse ALL-1 to any of \u3e50 partner genes or to itself. Here, we describe the unique features of ALL-1-associated leukaemias, and recent progress in understanding molecular mechanisms involved in the activity of the ALL-1 protein and of its Drosophila homologue TRITHORAX

Risk factors associated with short-term complications in mandibular fractures: the MANTRA study—a Maxillofacial Trainee Research Collaborative (MTReC)

Abstract Introduction Complications following mandibular fractures occur in 9–23% of patients. Identifying those at risk is key to prevention. Previous studies highlighted smoking, age and time from injury to presentation as risk factors but rarely recorded other possible confounders. In this paper, we use a collaborative snapshot audit to document novel risk factors and confirm established risks for complications following the treatment of mandibular fractures. Methods The audit was carried out by 122 OMFS trainees across the UK and Ireland (49 centres) over 6 months, coordinated by the Maxillofacial Surgery Trainees Research Collaborative. Variables recorded included basic demography, medical and social history, injury mechanism and type, management and 30-day outcome. Results Nine hundred and forty-seven (947) patients with fractured mandibles were recorded. Surgical management was carried out in 76.3%. Complications at 30 days occurred 65 (9%) of those who were managed surgically. Risk factors for complications included male sex, increasing age, any medical history, increasing number of cigarettes smoked per week, increasing alcohol use per week, worse oral hygiene and increased time from injury to presentation. Discussion We have used a large prospective snapshot audit to confirm established risk factors and identify novel risk factors. We demonstrate that time from injury to presentation is confounded by other indicators of poor health behaviour. These results are important in designing trial protocols for management of mandibular fractures and in targeting health interventions to patients at highest risk of complications. </jats:sec

Fludarabine, cyclophosphamide and mitoxantrone in relapsed or refractory chronic lymphocytic leukemia and low grade non-Hodgkin’s lymphoma

A regimen combining fludarabine, cyclophosphamide and mitoxantrone (FCM) was used to treat 29 patients with relapsed or refractory chronic lymphocytic leukemia (CLL, N= 24) and low-grade non-Hodgkin’s lymphoma (NHL, N= 5) based on evidence suggesting synergism between the 3 drugs. Patients were treated with mitoxantrone 5 mg/m(2) IV day 1 only, fludarabine 25 mg/m(2) IV for 3 days or 24 mg/m(2) orally for 5 days, cyclophosphamide 250 mg/m(2) IV for 3 days or 150 mg/m(2) orally for 5 days inclusive. Eighteen patients had previously received fludarabine and most were heavily pretreated with 40% having >2 prior treatments. A median number of 4 FCM courses ( range of 1 - 9) were given. The response rate was 78.5%: 32% complete remission, 25% nodular partial remission, 21.5%, partial remission. Median duration of response was 19 months and median survival was 42 months. Sixteen patients (57%) developed neutropenia to <0.5 x 10(9)/ l and 12 (43%) infectious complications. Four patients developed large cell lymphoma ( Richter’s syndrome) and 2 acute myeloid leukemia. FCM is a useful combination for relapsed or refractory CLL and low grade NHL with high response rates and long duration of response. The role of FCM as first line therapy deserves study as well as its combination with the monoclonal antibody Rituximab

Utility of sarcoma-specific fusion gene analysis in paraffin-embedded material for routine diagnosis at a specialist centre.

Aims Diagnosis of soft tissue sarcomas can be difficult. It can be aided by detection of specific genetic aberrations in many cases. This study assessed the utility of a molecular genetics/cytogenetics service as part of the routine diagnostic service at the Royal Marsden Hospital. Methods A retrospective audit was performed over a 15-month period to evaluate the diagnostic usefulness for soft tissue sarcomas with translocations of fluorescence in situ hybridisation (FISH) and reverse-transcriptase PCR (RT-PCR) in paraffin-embedded (PE) material. Results were compared with histology, and evaluated. Results Molecular investigations were performed on PE material in 158 samples (total 194 RT-PCR and 174 FISH tests), of which 85 were referral cases. Synovial sarcoma, Ewing sarcoma and low-grade fibromyxoid sarcoma were the most commonly tested tumours. Myxoid liposarcoma showed the best histological and molecular concordance, and alveolar rhabdomyosarcoma showed the best agreement between methods. FISH had a higher sensitivity for detecting tumours (73%, compared with 59% for RT-PCR) with a better success rate than RT-PCR, although the latter was specific in identifying the partner gene for each fusion. In particular, referral blocks in which methods of tissue fixation and processing were not certain resulted in higher RT-PCR failure rates. Conclusions FISH and RT-PCR on PE tissue are practical and effective ancillary tools in the diagnosis of soft tissue sarcomas. They are useful in confirming doubtful histological diagnoses and excluding malignant diagnoses. PCR is less sensitive than FISH, and the use of both techniques is optimal for maximising the detection rate of translocation-positive sarcomas

Delayed response to fludarabine in lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia.

We retrospectively reviewed time to response and incidence of delayed responses in 13 patients with lymphoplasmacytic lymphoma/Waldenstrom's macroglobulinemia (LPL/WM) treated with fludarabine with or without cyclophosphamide. During follow-up post-treatment, seven delayed responses (54%) were observed, improving the initial overall response rate of 61% to a final response rate of 77%

Interstitial insertion of retinoic acid receptor-alpha gene in acute promyelocytic leukemia with normal chromosomes 15 and 17

Abstract The translocation t(15;17)(q22;q21) is seen exclusively in patients with acute promyelocytic leukemia (APL) and in the promyelocytic blast crisis of chronic myeloid leukemia (CML). This translocation juxta- poses the promyelocytic leukemia (PML) gene on chromosome 15 and the retinoic acid receptor-alpha (RARA) gene on chromosome 17, resulting in the formation of a chimeric mRNA transcript. We describe a patient with the microgranular variant form of APL, with no detectable cytogenetic abnormality of either chromosomes 15 or 17, who nevertheless had juxtaposition of PML and RARA genes and expressed a chimeric transcript. Conventional cytogenetics showed the karyotype 46,XY,d- er(3)t(3;8)(p25;q12). Fluorescent in situ hybridization (FISH) with paints for chromosomes 8, 15, and 17 confirmed the presence of structurally intact chromosomes 15 and 17 and trisomy for chromosome 8q. Nevertheless, FISH using cosmid probes for PML and RARA showed their juxtaposition on one chromosome 15 homolog. Both genes were also present on their normal homologs; in addition, part of the RARA gene was still present on the remaining chromosome 17. DNA analysis by Southern blotting, performed with a variety of probes including PML, RARA and retinoic acid receptor-beta (RARB), showed a rearrangement in PML. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed the existence of hybrid transcripts of 276, 455 bp and 623 bp, from PML- RARA on the der(15) chromosome, consistent with alternate exon splicing of the long form of the transcript occurring in 50% to 60% of patients with APL. Our results show that APL patients with cytogenetically normal chromosomes 15 and 17 may, nevertheless, have involvement of both PML and RARA genes defining a subgroup of APL, t(15;17)- negative/PML-RARA-positive which is analogous to Philadelphia chromosome-negative/BCR-ABL-positive CML. In this case, the presence of chimeric transcripts suggests that treatment with all-trans RA may be warranted in APL, even in the absence of detectable cytogenetic change, showing the usefulness of RT-PCR or FISH to aid diagnosis

Cytogenetics of long-term survivors of ETV6-RUNX1 fusion positive acute lymphoblastic leukemia.

This study describes the cytogenetics of 33 children with ETV6-RUNX1 positive acute lymphoblastic leukemia (ALL) who had been in continuous complete remission for a minimum of 8.8 years [median event-free survival (EFS) 10.9 years]. The results were compared with a published series of 16 fusion positive patients treated on the same childhood ALL trial, who had relapsed (median EFS, 2.3 years). Interphase fluorescence in situ hybridization (FISH) at diagnosis showed deletion of the second ETV6 signal from all fusion positive cells in 45% of the long-term survivors but in none of the relapsed patients, whereas patients with mixed populations with retained or lost second signals were more frequent among those who had relapsed (69%) than the long-term survivors (21%). Interphase populations with two fusion signals in 18% of the long-term survivors and 31% of relapsed patients were smaller in the long-term survivors (median, 4% of total cells) than in the relapsed patients (median, 84%). The additional copy of chromosome 21 in 30% of long-term survivors and in 69% of relapsed patients was a derived chromosome 21 in 20% and 55% of patients, respectively. Metaphase FISH for 26 long-term survivors and 15 relapsed patients revealed complex karyotypes in both groups. Variant translocations involved different chromosome arms between the long-term survivors and relapsed patients. It appears that the two groups have some distinguishing cytogenetic features at the time of diagnosis, which may provide pointers to relapse that are worthy of more detailed study

Delineation of the minimal region of loss at 13q14 in multiple myeloma

Previous studies have focused on the incidence and prognostic implications of 13q14 deletions in multiple myeloma (MM), but none has sought to delineate the minimal common deleted region (CDR). In an effort to do so, dual-color interphase fluorescence in situ hybridization (FISH) was applied on 82 myeloma cases, initially by use of three probes for 13q14 (RBI, D13S319, and DIMS). Deletions were detected in 29/82 (35.4%) cases, and all except one were monoallelic. Subsequently, contiguous YACs, PACs, and a BAC spanning the 13q14-q21 region were employed for deletion mapping in addition to a 13q telomere probe. Large deletions extending to the 13q34 region were found in 55% of the deleted cases, whereas an additional 13.8% showed loss of both 13q34 and 13q 14 regions with retention of 13q21. A CDR of approximately 350 kb was identified at 13q 14 with the proximal border approximately 120 kb centromeric from D13S319, encompassing an area rich in expressed sequence tagged sites and containing DLEU1, DLEU2, and RFP2 genes. Direct sequencing of the RFP2 gene revealed no mutations in six patients and four MM cell lines harboring deletions of the CDR. However, a role for RFP2 in the pathogenesis of MM cannot yet be excluded, given that alternative mechanisms such as haploinsufficiency remain possible. (C) 2003 Wiley-Liss, Inc