Clonality analysis of lymphoid proliferations using the BIOMED-2 clonality assays: a single institution experience.

BACKGROUND: Clonality determination in patients with lymphoproliferative disorders can improve the final diagnosis. The aim of our study was to evaluate the applicative value of standardized BIOMED-2 gene clonality assay protocols for the analysis of clonality of lymphocytes in a group of different lymphoid proliferations. MATERIALS AND METHODS: With this purpose, 121 specimens from 91 patients with suspected lymphoproliferations submitted for routine diagnostics from January to December 2011 were retrospectively analyzed. According to the final diagnosis, our series comprised 32 cases of B-cell lymphomas, 38 cases of non-Hodgkin's T-cell lymphomas and 51 cases of reactive lymphoid proliferations. Clonality testing was performed using the BIOMED-2 clonality assays. RESULTS: The determined sensitivity of the TCR assay was 91.9%, while the sensitivity of the IGH assay was 74.2%. The determined specificity of the IGH assay was 73.3% in the group of lymphomas and 87.2% in the group of reactive lesions. The determined specificity of the TCR assay was 62.5% in the group of lymphomas and 54.3% in the group of reactive lesions. CONCLUSIONS: In the present study, we confirmed the utility of standardized BIOMED-2 clonality assays for the detection of clonality in a routine diagnostical setting of non-Hodgkin's lymphomas. Reactions for the detection of the complete IGH rearrangements and reactions for the detection of the TCR rearrangements are a good choice for clonality testing of a wide range of lymphoid proliferations and specimen types while the reactions for the detection of incomplete IGH rearrangements have not shown any additional diagnostic value.

Correlation of t(14;18) translocation breakpoint site with clinical characteristics in follicular lymphoma.

BACKGROUND: t(14;18)(q32;q21) translocation is an important genetic feature of follicular lymphoma resulting in antiapoptotic B-cell lymphoma 2 (BCL2) protein overexpression. On chromosome 18 breakpoint-site variation is high but does not affect BCL2. Breakpoint most commonly occurs at major breakpoint region (MBR) but may happen at minor cluster region (mcr) and between MBR and mcr at 3'MBR and 5'mcr. The aim of this study was to analyze the correlation of t(14;18)(q32;q21) breakpoint site with clinical characteristics in follicular lymphoma. PATIENTS AND METHODS: We included patients diagnosed with follicular lymphoma who received at least 1 cycle of systemic treatment and had the t(14;18)(q32;q21) translocation detected by polymerase chain reaction (PCR) at MBR, mcr or 3'MBR prior to first treatment. Among patients with different breakpoints, sex, age, disease grade, stage, B-symptoms, follicular lymphoma international prognostic index (FLIPI), presence of bulky disease, progression free survival and overall survival were compared. RESULTS: Of 84 patients, 63 had breakpoint at MBR, 17 at mcr and 4 at 3'MBR. At diagnosis, the MBR group had a significantly lower disease stage than the mcr group. Although not significant, in the MBR group we found a higher progression-free survival (PFS) and overall survival (OS), lower grade, age, FLIPI, and less B-symptoms. CONCLUSIONS: Compared to patients with mcr breakpoint, those with MBR breakpoint seem to be characterised by more favourable clinical characteristics. However, a larger study would be required to support our observation.

Sensitivity and reproducibility of conventional qualitative and quantitative PCR assays for detection of the t(14;18)(q32;q21) chromosomal translocation in biopsy material from patients with follicular lymphoma.

Follicular lymphoma (FL) is characterized by the t(14;18)(q32;q21) chromosomal translocation which can be detected by polymerase chain reaction (PCR) in approximately 70% of cases. The aim of our retrospective study was to evaluate the sensitivity and the reproducibility of both conventional qualitative and quantitative PCR assays for detection of the t(14;18)(q32;q21) chromosomal translocation in biopsy material. Fifty-seven formalin-fixed, paraffin-embedded tumor lymph node (LN) specimens from 50 patients with FL were included in the study. Qualitative PCR was performed with primer sets specific for the MBR, far3'-MBR and the mcr regions, respectively. Quantitative PCR was performed using the LightCycler instrument and the LightCycler - t(14;18) Quantification Kit (MBR). The overall detection rate of the t(14;18) in our study (52.6%) was in accordance with the literature. Of the t(14;18)-positive cases, 49.1% had breakpoints within the MBR and only 3.5% had breakpoints within the mcr. The most sensitive method was LightCycler-based PCR with a detection rate of 47.4%, followed by MBR1,2 assay (43.9%). We observed good agreement between qualitative MBR1,2 and quantitative LightCycler-based assay with a slightly higher detection rate of the quantitative method. The sensitivities of both methods were in accordance with results from other studies. Since LightCycler-based assay detects only breakpoints within the MBR, qualitative PCR should be employed in routine diagnostic settings for detection of breakpoints within the mcr and far3'-MBR regions.

Diagnostic value of immunoglobulin κ light chain gene rearrangement analysis in B-cell lymphomas.

Analysis of the immunoglobulin κ light chain (IGK) gene is an alternative method for B-cell clonality assessment in the diagnosis of mature B-cell proliferations in which the detection of clonal immunoglobulin heavy chain (IGH) gene rearrangements fails. The aim of the present study was to evaluate the added value of standardized BIOMED-2 assay for the detection of clonal IGK gene rearrangements in the diagnostic setting of suspected B-cell lymphomas. With this purpose, 92 specimens from 80 patients with the final diagnosis of mature B-cell lymphoma (37 specimens), mature T-cell lymphoma (26 specimens) and reactive lymphoid proliferation (29 specimens) were analyzed for B-cell clonality. B-cell clonality analysis was performed using the BIOMED-2 IGH and IGK gene clonality assays. The determined sensitivity of the IGK assay was 67.6%, while the determined sensitivity of the IGH assay was 75.7%. The sensitivity of combined IGH+IGK assay was 81.1%. The determined specificity of the IGK assay was 96.2% in the group of T-cell lymphomas and 96.6% in the group of reactive lesions. The determined specificity of the IGH assay was 84.6% in the group of lymphomas and 86.2% in the group of reactive lesions. The comparison of GeneScan (GS) and heteroduplex pretreatment-polyacrylamide gel electrophoresis (HD-PAGE) methods for the analysis of IGK gene rearrangements showed a higher efficacy of GS analysis in a series of 27 B-cell lymphomas analyzed by both methods. In the present study, we demonstrated that by applying the combined IGH+IGK clonality assay the overall detection rate of B-cell clonality was increased by 5.4%. Thus, we confirmed the added value of the standardized BIOMED-2 IGK assay for assessment of B-cell clonality in suspected B-cell lymphomas with inconclusive clinical and cyto/histological diagnosis.

Are there geographical differences in the frequency of SYT-SSX1 and SYT-SSX2 chimeric transcripts in synovial sarcoma?

Synovial sarcoma (SS) is characterized by the t(X;18)(p11.2;q11.2) chromosomal translocation, which results in generating either SYT-SSX1, SYT-SSX2 or, infrequently, SYT-SSX4 fusion gene. The ratio of SYT-SSX1:SYT-SSX2 fusions is close to 2:1 in the majority of studies, and SYT-SSX2 fusion has been only rarely observed in biphasic SS. In the present study, we compared two series of patients with SS, Slovenian (37 cases) and Dutch (14 cases), with respect to clinical, pathological and molecular findings. The two groups did not differ with regard to clinicopathological features. Whereas the frequency of different SYT-SSX fusions in the Dutch group was similar to that reported in the literature, we found an unexpectedly high number of tumors with SYT-SSX2 fusion in the Slovenian group. The ratio of SYT-SSX1:SYT-SSX2 fusion was 7:18 for monophasic and 2:7 for biphasic tumors in the Slovenian group. This distribution differs significantly from that observed in the Dutch group in the present study (P = 0.041) as well as from data reported in the recent large multi-institutional study on 243 patients (P = 0.0001). Our findings indicate possible geographical differences in the frequency of two SYT-SSX fusion transcripts in patients with synovial sarcoma.