Quantitative PCR analysis for Bcl-2/IgH in a phase III study of Yttrium-90 Ibritumomab Tiuxetan as consolidation of first remission in patients with follicular lymphoma.

PURPOSE The randomized First-Line Indolent Trial (FIT) was conducted in patients with advanced follicular lymphoma (FL), to evaluate the safety and efficacy of yttrium-90 ((90)Y) ibritumomab tiuxetan given as consolidation of complete or partial remission. This study of minimal residual disease was undertaken in parallel, to determine the rate of conversion from bcl-2 polymerase chain reaction (PCR) -detectable to -undetectable status and the corresponding effect on progression-free survival (PFS). PATIENTS AND METHODS Blood samples from 414 patients ((90)Y-ibritumomab, n = 208; control, n = 206) were evaluated using real-time quantitative polymerase chain reaction (RQ-PCR); 186 were found to have the bcl-2 rearrangement and were thus eligible for inclusion in the RQ-PCR analysis. Results Overall, 90% of treated patients converted from bcl-2 PCR-detectable to -undetectable disease status, compared with 36% in the control group. Treatment significantly prolonged median PFS in patients converting to bcl-2 PCR-undetectable status (40.8 v 24.0 months in the control group; P < .01, hazard ratio [HR], 0.399). In patients who had bcl-2 PCR-detectable disease at random assignment, treatment significantly prolonged median PFS (38.4 v 8.2 months in the control group; P < .01, HR, 0.293). CONCLUSION Eradication of PCR-detectable disease occurred more frequently after treatment with (90)Y-ibritumomab tiuxetan and was associated with prolongation of PFS.

Mutation of the Wilms' tumor 1 gene is a poor prognostic factor associated with chemotherapy resistance in normal karyotype acute myeloid leukemia: the United Kingdom Medical Research Council Adult Leukaemia Working Party.

PURPOSE: To determine the clinical relevance of Wilms' tumor 1 (WT1) gene mutations in acute myeloid leukemia (AML) with normal karyotype (NK). PATIENTS AND METHODS: Exons 7 and 9 of WT1 were screened in samples from 470 young adult NK AMLs using a combination of direct sequencing and high-resolution capillary electrophoresis. RESULTS: Overall, 51 mutations were detected in 47 cases (10%): 46 frameshift mutations with insertion/deletion of one to 28 base pairs in exon 7 (n = 45) or exon 9 (n = 1), with a median mutant level of 45% (range, 8% to 86%), and five substitutions in exon 9: D396N (n = 3), H397Y (n = 1) and H397Q (n = 1). Patients with WT1 mutations had an inferior response to induction chemotherapy compared with wild-type cases (complete remission rate, 79% v 90%, odds ratio [OR] = 3.02; 95% CI, 1.17 to 7.82; P = .02), a higher rate of resistant disease (15% v 4%; OR = 9.33; 95% CI, 2.38 to 36.6; P = .001), an increased cumulative incidence of relapse (67% v 43%, hazard ratio [HR] = 3.02; 95% CI, 1.69 to 5.38; P = .0008), with a reduction in both relapse-free survival (22% v 44%; HR = 2.16; 95% CI, 1.32 to 3.55; P = .005) and overall survival (26% v 47%; HR = 1.91; 95% CI, 1.23 to 2.95; P = .007) at 5 years. In multivariate analysis, which included FLT3 internal tandem duplication and NPM1 mutation status, the presence of a WT1 mutation remained an independent adverse prognostic factor. CONCLUSION: WT1 mutations are a negative prognostic indicator in NK AML and may be suitable for the development of targeted therapy.

Reliable detection of clonal IgH/Bcl2 MBR rearrangement in follicular lymphoma: methodology and clinical significance.

The prognostic significance of IgH/Bcl2 rearrangement in follicular lymphoma (FL) remains contentious; polymerase chain reaction (PCR) methodology and tissue source variability may account for some inconsistencies. As IgH/Bcl2 major breakpoint region (MBR) sequences may be found in normal blood, an MBR+ result by conventional PCR in blood/bone marrow may not indicate FL. To establish tumour MBR status, 190 lymphoid tissue samples with histologically evident FL (and therefore >1% tumour cells) were examined by real-time quantifiable PCR; 50% (95/190) had clonal MBR IgH/Bcl2 (MBR was considered clonal when >1%). Overall survival (median = 11.5 years) of MBR+ and MBR- patients was not significantly different.

JH probe real-time quantitative polymerase chain reaction assay for Bcl-2/IgH rearrangements.

Follicular lymphoma (FL) characteristically bears the t(14;18)(q32;q21). However, only approximately 75% of the consequent Bcl-2 breakpoints lie within the major breakpoint region (MBR) or the minor cluster region (mcr). While these can be quantified by cluster region-specific real-time quantitative polymerase chain reaction (RQ-PCR), a significant proportion of cases are left requiring a customized approach. Therefore, an RQ-PCR assay for the quantification of Bcl-2/IgH breakpoints has been developed that uses germline JH TaqMan probes and germline JH primers in combination with customized forward primers. Validation of this approach by comparison with an established MBR RQ-PCR showed both techniques to be concordant across a wide range of copy numbers with a sensitivity of five copies per 10(5) cells. In addition, to generate standard curves equating to diverse Bcl-2/IgH rearrangements, a strategy for using placental DNA as a surrogate standard was devised. The performance of the assay in detecting molecular evidence of disease in sequential biopsies from five patients (three with atypical Bcl-2/IgH breakpoints identified by long-range or inverse PCR, one MBR+ and one mcr+) was tested. This alternative approach represents a sensitive and specific means of quantifying common and atypical Bcl-2/IgH rearrangements and maximizes the number of patients with FL suitable for molecular monitoring.

The relative role of peripheral blood and bone marrow for monitoring molecular evidence of disease in follicular lymphoma by quantitative real-time polymerase chain reaction.

Peripheral blood (PB) and bone marrow (BM) are used interchangeably for t(14;18) (IgH/BCL-2) molecular monitoring in follicular lymphoma (FL) and detection of rearrangement after treatment has been correlated to increased risk of relapse. To determine the relative value of each tissue, MBR t(14;18) was quantified by real-time polymerase chain reaction in 52 simultaneous paired PB and BM samples from 38 FL patients. In total, 79% of sample pairs taken in remission (n = 19) or when no morphological disease was evident in the BM (n = 29) had t(14;18) copy number within one log difference and the median difference was small. These findings suggest that, in remission, PB may be adequately monitored. In general, however, higher copy number was detected in BM than in the corresponding PB sample.