Fludarabine, Cyclophosphamide and Rituximab:an effective chemoimmunotherapy combination with high remission rates for chronic lymphocytic leukaemia

BACKGROUND: Combined Fludarabine and Cyclophosphamide is now standard first-line therapy in chronic lymphocytic leukaemia (CLL) and the addition of Rituximab improves outcome.

METHODS: We adopted a modified Fludarabine, Cyclophosphamide and Rituximab (FCR) protocol in treating 39 patients (median age 57 years) with progressive or advanced CLL. Depending on CR, treatment was given for four or six cycles.

RESULT: Twenty-six patients were treatment naïve and 13 were pre-treated. Twelve patients had progressive Binet stage A, 16 stage B and 11 stage C disease. The overall response rate (ORR) was 100%, with 75% achieving CR. Neutropenia was the major toxicity in 71/187 (38%) of the cycles. There were five deaths, two from infection and three from progressive disease. Twenty-six of 31 patients have maintained their post-treatment disease status for a median of 17 months (2-41).

CONCLUSION: We conclude that FCR is a feasible, well-tolerated and effective treatment for patients with CLL.

Treatment of relapse after allogeneic bone marrow transplantation with reduced intensity conditioning (FLAG +/- Ida) and second allogeneic stem cell transplant

Acute leukaemias in relapse after allogeneic stem cell transplantation (SCT) respond poorly to donor leucocyte infusions (DLI) compared with chronic myeloid leukaemia (CML), at least in part because of faster disease kinetics. Fludarabine-containing 'non-myeloablative' chemotherapy followed by further allo SCT may offer more rapid and effective disease control. We report 14 patients with relapse after allo SCT for acute leukaemia [seven acute myeloid leukaemia (AML), five acute lymphoblastic leukaemia (ALL)] or refractory anaemia with excess blasts in transformation (RAEB-t, n = 2) treated with fludarabine, high-dose cytosine arabinoside (ara-C) and granulocyte colony-simulating factor (G-CSF) with (n = 10) or without (n = 2) idarubicin (FLAG +/- Ida) or DaunoXome (FLAG-X) (n = 2) and second allo SCT from the original donor. Donors were fully human leucocyte antigen (HLA) -matched in 13 cases with a single class A mismatch in one. Actuarial overall survival was 60% and disease-free survival was 26% at 58 months. Remissions after the second SCT were longer than those after the first bone marrow transplantation (BMT) in eight of the 13 assessable patients to date. Haematopoietic recovery was rapid. Transplants were well tolerated with no treatment-related deaths. The major complication was graft-versus-host disease (GvHD, acute >/= grade II-2 cases, chronic - eight cases, two limited, six extensive) although there have been no deaths attributable to this. FLAG +/- Ida and second allo SCT is a safe and useful approach and may be more effective than DLI in the treatment of acute leukaemias relapsing after conventional allo SCT.

FLAG-idarubicin and allogeneic stem cell transplantation for Ph-positive ALL beyond first remission

We describe a single centre experience of eight consecutive patients with relapsed or refractory Ph+ ALL treated with the FLAG/idarubicin regimen followed by BMT or PBSCT. Following FLAG/idarubicin, one achieved a partial response and seven CR. All patients subsequently received allogeneic transplants: one sibling BMT, three matched unrelated (MUD) BMT and four sibling PBSCT. Two patients received second transplants with PBSC from their original BM donors following FLA/Ida with no further conditioning. Three patients are alive in CR 9, 24 and 32 months after transplant. Seven of eight patients had a cytogenetic response following FLAG/Ida induction and one of seven became bcr-abl negative. All eight patients had a complete cytogenetic response following transplant. Four of five assessable patients became p190 bcr-abl negative after transplant; three of these subsequently relapsed. Both patients with the p210 bcr-abl transcript remained bcr-abl positive in CR after transplant. FLAG/Ida was well tolerated and appears to be effective in inducing remission in relapsed Ph+ ALL. The use of FDR-containing chemotherapy without further conditioning prior to PBSCT deserves further study in heavily pre-treated patients and, in patients with relapsed ALL following BMT, may be a safer option than DLI (donor lymphocyte infusion) by avoiding the associated risk of aplasia.

Combination of transient lymphodepletion with busulfan and fludarabine and peptide vaccination in a phase I clinical trial for patients with advanced melanoma.

Taking advantage of homeostatic mechanisms to boost tumor-specific cellular immunity is raising increasing interest in the development of therapeutic strategies in the treatment of melanoma. Here, we have explored the potential of combining homeostatic proliferation, after transient immunosuppression, and antigenic stimulation of Melan-A/Mart-1 specific CD8 T-cells. In an effort to develop protocols that could be readily applicable to the clinic, we have designed a phase I clinical trial, involving lymphodepleting chemotherapy with Busulfan and Fludarabine, reinfusion of Melan-A specific CD8 T-cell containing peripheral blood mononuclear cells (exempt of growth factors), and Melan-A peptide vaccination. Six patients with advanced melanoma were enrolled in this outpatient regimen that demonstrated good feasibility combined with low toxicity. Consistent depletion of lymphocytes with persistent increased CD4/CD8 ratios was induced, although the proportion of circulating CD4 regulatory T-cells remained mostly unchanged. The study of the immune reconstitution period showed a steady recovery of whole T-cell numbers overtime. However, expansion of Melan-A specific CD8 T-cells, as measured in peripheral blood, was mostly inconsistent, accompanied with marginal phenotypic changes, despite vaccination with Melan-A/Mart-1 peptide. On the clinical level, 1 patient presented a partial but objective antitumor response following the beginning of the protocol, even though a direct effect of Busulfan/Fludarabine cannot be completely ruled out. Overall, these data provide further ground for the development of immunotherapeutic approaches to be both effective against melanoma and applicable in clinic.

Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.

PURPOSE: The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. PATIENTS AND METHODS: We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. RESULTS: We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). CONCLUSION: The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

Functional analysis of the ATM-p53-p21 pathway in the LRF CLL4 trial: blockade at the level of p21 is associated with short response duration.

PURPOSE: This study sought to establish whether functional analysis of the ATM-p53-p21 pathway adds to the information provided by currently available prognostic factors in patients with chronic lymphocytic leukemia (CLL) requiring frontline chemotherapy. EXPERIMENTAL DESIGN: Cryopreserved blood mononuclear cells from 278 patients entering the LRF CLL4 trial comparing chlorambucil, fludarabine, and fludarabine plus cyclophosphamide were analyzed for ATM-p53-p21 pathway defects using an ex vivo functional assay that uses ionizing radiation to activate ATM and flow cytometry to measure upregulation of p53 and p21 proteins. Clinical endpoints were compared between groups of patients defined by their pathway status. RESULTS: ATM-p53-p21 pathway defects of four different types (A, B, C, and D) were identified in 194 of 278 (70%) samples. The type A defect (high constitutive p53 expression combined with impaired p21 upregulation) and the type C defect (impaired p21 upregulation despite an intact p53 response) were each associated with short progression-free survival. The type A defect was associated with chemoresistance, whereas the type C defect was associated with early relapse. As expected, the type A defect was strongly associated with TP53 deletion/mutation. In contrast, the type C defect was not associated with any of the other prognostic factors examined, including TP53/ATM deletion, TP53 mutation, and IGHV mutational status. Detection of the type C defect added to the prognostic information provided by TP53/ATM deletion, TP53 mutation, and IGHV status. CONCLUSION: Our findings implicate blockade of the ATM-p53-p21 pathway at the level of p21 as a hitherto unrecognized determinant of early disease recurrence following successful cytoreduction.