A rapid RT-PCR screening assay incorporating multiplexed validated control genes for CBF rearrangements at diagnosis in AML.

AIMS: Our objective was to establish a multiplexed assay using the Biomed 1 primers to detect AML1-ETO transcripts and 10 different CBFB-MYH11 transcripts, using BCR and ABL transcripts as controls. METHODS: Control genes were systematically tested for characteristics of optimal controls. The final assay was validated on 50 AML patient samples. RESULTS: Testing confirmed that the designated control gene criteria were fulfilled. Of 50 patient samples tested, four RT-PCR results were discordant with the cytogenetic result. In three cytogenetically negative cases, RT-PCR detected cryptic CBF rearrangements (one AML1-ETO and two CBFB-MYH11). The fourth case was inv(16) positive but negative by RT-PCR; however, the control gene result revealed suboptimal RNA quality. CONCLUSIONS: We have described a robust multiplex RT-PCR assay that incorporates experimentally validated control genes that are important for accurate interpretation. The assay is more sensitive than cytogenetics in the detection of CBF AML. Application to large patient cohorts will determine the prognostic significance of cryptic CBF rearrangements compared with their cytogenetic counterparts.

B cell chronic lymphocytic leukaemia cells have reduced capacity to upregulate expression of MHC class I in response to interferon-gamma.

AIMS: An important consideration in the design of a tumour vaccine is the ability of tumour-specific cytotoxic T lymphocytes (CTL) to recognise unmanipulated tumour cells in vivo. To determine whether B-CLL might use an escape strategy, the current studies compared B-CLL and normal B cell MHC class I expression. METHODS: Flow cytometry, TAP allele PCR and MHC class I PCR were used. RESULTS: While baseline expression of MHC class I did not differ, upregulation of MHC class I expression by B-CLL cells in response to IFN-gamma was reduced. No deletions or mutations of TAP 1 or 2 genes were detected. B-CLL cells upregulated TAP protein expression in response to IFN-gamma. Responsiveness of B-CLL MHC class I mRNA to IFN-gamma was not impaired. CONCLUSIONS: The data suggest that MHC class I molecules might be less stable at the cell surface in B-CLL than normal B cells, as a result of the described release of beta(2)m and beta(2)m-free class I heavy chains from the membrane. This relative MHC class I expression defect of B-CLL cells may reduce their susceptibility to CTL lysis in response to immunotherapeutic approaches.

Prognostic value of ZAP-70 expression in chronic lymphocytic leukemia as assessed by quantitative polymerase chain reaction and flow cytometry.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is a disorder in which the tempo of disease progression is highly variable, and prognostic markers that can be utilized at diagnosis are regarded as clinically important. Currently, there are several prognostic factors, such as immunoglobulin heavy chain (IgVH) mutational status, and ZAP-70 protein expression in neoplastic B-cells, that have demonstrated significant discriminative power in the prognostication of CLL. They are, however, largely unavailable in the routine diagnostic laboratory setting. METHODS: In this study, we characterized the IgVH status and ZAP-70 expression by molecular techniques in a cohort of 108 patients with CLL, and correlated these results with three different methods of ZAP-70 expression by flow cytometry. We then assessed the results of these methods in terms of prognostic power as characterized by time to first treatment (TTFT). RESULTS: By comparing three different flow cytometry methods using receiver­operator curve (ROC) analysis, we identified that by utilizing a corrected mean fluorescence intensity (CorrMFI) algorithm for assessing ZAP-70 expression, there was good correlation with both IgVH mutational status, and ZAP-70 expression as assessed by qPCR. We were also able to show that ZAP-70 expression, as assessed by both qPCR and the CorrMFI method, was prognostic of TTFT. CONCLUSIONS: While confirmation in a larger patient cohort, with longer follow-up is required, we believe that the CorrMFI represents the most promising method currently available in a routine diagnostic setting for the assessment of ZAP-70 expression in CLL patients.

WT1 expression as a marker of minimal residual disease predicts outcome in acute myeloid leukemia when measured post-consolidation.

WT1 levels may be a useful predictor of leukemia free survival (LFS) following treatment of acute myeloid leukemia (AML). We report a retrospective study in which levels of WT1 expression from patients with de novo AML were measured from bone marrow and peripheral blood at diagnosis, post-induction, post-consolidation and relapse. We demonstrate that higher levels of WT1 in peripheral blood at diagnosis are associated with poorer LFS independent of age and cytogenetic risk-group (n=85, p=0.028). When measured at post-consolidation, the presence of detectable WT1 is associated with poorer LFS in univariate analysis of both peripheral blood (p=0.024) and bone marrow (p=0.019). In a multivariate analysis including age and cytogenetic risk, the association remained significant for bone marrow (p=0.016) with a trend observed for peripheral blood (p=0.06). These findings have formed the basis for ongoing research.

A >or=1 log rise in RQ-PCR transcript levels defines molecular relapse in core binding factor acute myeloid leukemia and predicts subsequent morphologic relapse.

Core binding factor acute myeloid leukemia (CBF AML), with t(8;21)(q22;q22), inv(16)(p13q22) or t(16;16)(p13;q22) and the associated fusion gene transcripts AML1/ETO or CBFbeta/MYH11, has a favourable clinical prognosis although significant numbers of patients still suffer relapse. We examined the prognostic utility of serial bone marrow minimal residual disease (MRD) monitoring by RQ-PCR in a cohort of patients with CBF AML with long term clinical follow-up. Twenty-nine patients were evaluated with a median follow of 34 months. Twelve relapses occurred at a median of 11 months (range 4 - 17) from diagnosis. RQ-PCR levels at diagnosis, post-induction chemotherapy and post-consolidation were not predictive of outcome. However, a >or=1 log(10) rise at any stage in transcript level relative to the level from a remission bone marrow sample correlated with inferior leukemia free survival (LFS) and imminent morphologic relapse (hazard ratio 8.6). Relapses occurred a median of 60 days (range 45 - 272) after a log(10) rise. A >or=1 log(10) rise in transcript levels strongly predicts subsequent morphologic relapse in CBF AML and therefore defines molecular relapse. Our data support a simple RQ-PCR model for prediction of impending relapse which has the potential for widespread clinical applicability. Prospective identification of high risk patients will enable clinical trials to assess the efficacy of treatment initiated at molecular relapse.

The Hyper-CVAD chemotherapy regimen has an adverse long-term impact on the ability to mobilize peripheral blood stem cells, which can be readily circumvented by using the early cycles for mobilization.

The Hyper-CVAD chemotherapy regimen is being increasingly applied to a number of haematological malignancies. We assessed the impact of Hyper-CVAD on peripheral blood stem cell (PBSC) yields and examined the optimal timing of PBSC collection when using this regimen. Seventy-four consecutive patients were identified in whom an attempt was made to collect PBSC, usually on recovery from cycle A or B. Where PBSC collection was attempted after cycle 3B, only 18% (3/17) of patients successfully mobilized. Fifty-seven patients were mobilized on recovery from cycle 1B (n = 13), 2A (n = 22), 2B (n = 14) or 3A (n = 8). Compared with cycle 2A, 1B was not superior in achieving the minimum of > or =2 x 10(6)/kg CD34+ cells (100% vs. 77%, p = 0.13), but was superior in terms of total CD34+ yield (21.4 vs. 3.2 x 10(6)/kg, p < 0.001), achieving the target CD34+ cell count of > or =5 x 10(6)/kg (92% vs 36%, p = 0.002), and obtaining both a minimum (92% vs. 18%, p < 0.001) and target (77% vs. 0%, p < 0.001) graft with a single apheresis. There were no significant differences in PBSC yields following cycles 2A, 2B and 3A. Hyper-CVAD has substantial stem cell toxicity which can be readily circumvented by using the early chemotherapy cycles for mobilization.