Russell Saal

A ≥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 CBFβ/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 ≥1 log10 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 log10 rise. A ≥1 log10 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.

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.

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

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‐γ 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‐γ. Responsiveness of B‐CLL MHC class I mRNA to IFN‐γ 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 β2m and β2m‐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

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.

Autografting followed by rituximab for chemosensitive mantle cell lymphoma: A pilot study and literature review

Mantle cell lymphoma (MCL) is rarely cured with either conventional-dose chemotherapy or autografting. Recent evidence suggests that anti-CD20 monoclonal antibody therapy (rituximab) in combination with chemotherapy may improve the response rate. We report a pilot study of autografting using busulfan-melphalan conditioning followed by rituximab in 9 patients (median age 52 years) with chemosensitive MCL. Rituximab was given for 4 doses of 375 mg/m(2) between 4 and 10 weeks post-transplant. Three of 5 patients autografted after induction therapy remain alive in clinical and molecular complete remission at 33-50 months post-transplant. Only 1 of 4 patients autografted after relapse remains in complete remission. Two of the 3 patients with persistent marrow molecular positivity post-autograft became negative after rituximab therapy. Molecular negativity was first observed in 2 patients only after rituximab therapy. Overall, 2 patients have relapsed and the remaining 3 died of late-onset respiratory failure, probably reflecting infection and/or aggressive conditioning in an older patient population. These preliminary results, together with a review of the literature, suggest that the combination of autografting and rituximab may lead to durable molecular remissions in patients with chemosensitive MCL. Further studies are required to clarify whether the administration of rituximab: (1) is optimal pre- or post-autograft and (2) impacts on the incidence of infection and idiopathic pneumonitis in this context.

Prognostic utility of spontaneous erythroid colony formation and JAK2 mutational analysis for thrombotic events in essential thrombocythaemia

Background:  Thrombotic events in essential thrombocythaemia (ET) are difficult to predict with current risk stratification based on age and prior history of thrombosis.

A simple method for synthesis of B-cell clonospecific probes

Summary. A sensitive PCR‐based method was developed to produce B‐cell clonogenic probes without the need for sequencing and specific oligonucleotide synthesis. Specificity and sensitivity were assessed and found to be comparable to that achieved using established methods. Possible applications include the detection of MRD, bone marrow involvement with lymphoma, and the contamination of autologous bone marrow or peripheral blood progenitor cell harvests with malignant cells carrying IgH rearrangements.

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.