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.

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.

Application of self-quenched JH consensus primers for real-time quantitative PCR of IGH gene to minimal residual disease evaluation in multiple myeloma.

Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of >or=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.

Molecular characteristics and gene segment usage in IGH gene rearrangements in multiple myeloma.

BACKGROUND AND OBJECTIVES: Analysis of IgH rearrangements in B-cell malignancies has provided clinical researchers with a wide range of information during the last few years. However, only a few studies have contributed to the characterization of these features in multiple myeloma (MM), and they have been focused on the analysis of the expressed IgH allele only. Comparison between the expressed and the non-functional IgH alleles allows further characterizion of the selection processes to which pre-myeloma cells are submitted. DESIGN AND METHODS: We analyzed a cohort of 84 untreated MM patients in order to characterize their functional VDJH and non-functional DJH rearrangements. The pattern of mutations and gene segment usage for both types of rearrangements was analyzed by polymerase chain reaction and sequencing. RESULTS: VH3 and VH1 family members were over- and under-represented, respectively. VH3-30 and VH3-15 segments were the most frequently used, whereas VH4-34 was found only in non-functional or heavily mutated VDJH rearrangements. DH2 and DH3 family members were over-represented in both VDJH and DJH repertoires, while the DH1 family was under-represented only in the productive VDJH rearrangements. Finally, DH3-22 and DH2-21 gene segments were found to be over-represented in the functional repertoire while segments commonly used by less mature B-cell malignancies, such as DH6-19 or DH3-3, were under-represented. INTERPRETATION AND CONCLUSIONS: Data reported here help to identify the clonogenic MM cell as a post-germinal center B cell that has undergone selection processes during the germinal center reaction.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.

Gene scanning of VDJH-amplified segments is a clinically relevant technique to detect contaminating tumor cells in the apheresis products of multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.

Low frequency of the TEL/AML1 fusion gene in acute lymphoblastic leukaemia in Spain.

We report on a series of Spanish patients with acute lymphoblastic leukaemia in whom the t(12;21) [TEL/AML1] translocation could not be identified with two sensitive techniques: reverse transcript-polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH). 101 cases were analysed: 38 children (29 B-cell precursor; nine T-cell precursor) and 63 adults (48 B-cell precursor; 15 T-cell precursor). Specific RT-PCR to amplify the TEL/AML1 fusion transcript was negative in all 101 cases. Moreover, all 38 paediatric samples were also negative by interphase FISH analysis for the presence of the TEL/AML1 fusion. These results suggest the existence of geographic/race variations in the genotype of acute lymphoblastic leukaemia (ALL).

The identification of a novel role for BRCA1 in regulating RNA Polymerase I transcription

The unrestrained proliferation of cancer cells requires a high level of ribosome biogenesis. The first stage of ribosome biogenesis is the transcription of the large ribosomal RNAs (rRNAs); the structural and functional components of the ribosome. Transcription of rRNA is carried out by RNA Polymerase I (Pol-I) and its associated holoenzyme complex. Here we report that BRCA1, a nuclear phosphoprotein, and a known tumour suppressor involved in variety of cellular processes such as DNA damage response, transcriptional regulation, cell cycle control and ubiquitylation, is associated with rDNA repeats, in particular with the regulatory regions of the rRNA gene. We demonstrate that BRCA1 interacts directly with the basal Pol-I transcription factors; upstream binding factor (UBF), selectivity factor-1 (SL1) as well as interacting with RNA Pol-I itself. We show that in response to DNA damage, BRCA1 occupancy at the rDNA repeat is decreased and the observed BRCA1 interactions with the Pol-I transcription machinery are weakened. We propose, therefore, that there is a rDNA associated fraction of BRCA1 involved in DNA damage dependent regulation of Pol-I transcription, regulating the stability and formation of the Pol-I holoenzyme during initiation and/or elongation in response to DNA damage.