The impact of SF3B1 mutations in CLL on the DNA-damage response.

Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.

Ibrutinib-induced lymphocytosis in patients with chronic lymphocytic leukemia: correlative analyses from a phase II study.

Ibrutinib and other targeted inhibitors of B-cell receptor signaling achieve impressive clinical results for patients with chronic lymphocytic leukemia (CLL). A treatment-induced rise in absolute lymphocyte count (ALC) has emerged as a class effect of kinase inhibitors in CLL and warrants further investigation. Here we report correlative studies in 64 patients with CLL treated with ibrutinib. We quantified tumor burden in blood, lymph nodes (LNs), spleen and bone marrow, assessed phenotypic changes of circulating cells and measured whole-blood viscosity. With just one dose of ibrutinib, the average increase in ALC was 66%, and in>40% of patients the ALC peaked within 24 h of initiating treatment. Circulating CLL cells on day 2 showed increased Ki67 and CD38 expression, indicating an efflux of tumor cells from the tissue compartments into the blood. The kinetics and degree of the treatment-induced lymphocytosis was highly variable; interestingly, in patients with a high baseline ALC the relative increase was mild and resolution rapid. After two cycles of treatment the disease burden in the LN, bone marrow and spleen decreased irrespective of the relative change in ALC. Whole-blood viscosity was dependent on both ALC and hemoglobin. No adverse events were attributed to the lymphocytosis.

Treatment of older patients with mantle-cell lymphoma.

BACKGROUND: The long-term prognosis for older patients with mantle-cell lymphoma is poor. Chemoimmunotherapy results in low rates of complete remission, and most patients have a relapse. We investigated whether a fludarabine-containing induction regimen improved the complete-remission rate and whether maintenance therapy with rituximab prolonged remission. METHODS: We randomly assigned patients 60 years of age or older with mantle-cell lymphoma, stage II to IV, who were not eligible for high-dose therapy to six cycles of rituximab, fludarabine, and cyclophosphamide (R-FC) every 28 days or to eight cycles of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) every 21 days. Patients who had a response underwent a second randomization to maintenance therapy with rituximab or interferon alfa, each given until progression. RESULTS: Of the 560 patients enrolled, 532 were included in the intention-to-treat analysis for response, and 485 in the primary analysis for response. The median age was 70 years. Although complete-remission rates were similar with R-FC and R-CHOP (40% and 34%, respectively; P=0.10), progressive disease was more frequent with R-FC (14%, vs. 5% with R-CHOP). Overall survival was significantly shorter with R-FC than with R-CHOP (4-year survival rate, 47% vs. 62%; P=0.005), and more patients in the R-FC group died during the first remission (10% vs. 4%). Hematologic toxic effects occurred more frequently in the R-FC group than in the R-CHOP group, but the frequency of grade 3 or 4 infections was balanced (17% and 14%, respectively). In 274 of the 316 patients who were randomly assigned to maintenance therapy, rituximab reduced the risk of progression or death by 45% (in remission after 4 years, 58%, vs. 29% with interferon alfa; hazard ratio for progression or death, 0.55; 95% confidence interval, 0.36 to 0.87; P=0.01). Among patients who had a response to R-CHOP, maintenance therapy with rituximab significantly improved overall survival (4-year survival rate, 87%, vs. 63% with interferon alfa; P=0.005). CONCLUSIONS: R-CHOP induction followed by maintenance therapy with rituximab is effective for older patients with mantle-cell lymphoma. (Funded by the European Commission and others; ClinicalTrials.gov number, NCT00209209.).

Standard-dose anti-CD20 antibody rituximab has efficacy in chronic lymphocytic leukaemia: results from a Nordic multicentre study.

OBJECTIVES: This prospective multicentre study was conducted to assess the efficacy of the monoclonal anti-CD20 antibody rituximab in patients with chronic lymphocytic leukaemia (CLL). Secondary objectives were defined as the tolerability and feasibility of rituximab in patients with CLL. METHODS: Twenty-four heavily pretreated patients with CLL were treated with a standard dose of 375 mg m-2 of rituximab given once weekly for four doses. RESULTS: The overall response rate was 35% and all the responses were partial as defined by the revised NCI criteria. In 17 (85%) of 20 patients with initially measurable peripheral lymph nodes the size of lymph nodes decreased by at least 50%, while an improvement of the bone marrow infiltration was observed only in two (11%) of 18 evaluable patients. The median duration of the overall response was 12.5 wk. Rituximab was relatively well tolerated. Although side-effects were common (75%) they were usually mild or moderate. There was only one grade 3 adverse event and no grade 4 events. CONCLUSIONS: Standard-dose rituximab has activity in heavily pretreated patients with CLL, although the response is mainly limited to the lymph nodes and of short duration. Since rituximab has in vitro synergism with chemotherapeutic agents and is well tolerated by CLL patients, it is reasonable to investigate rituximab in combination with other treatments.

A Danish population-based analysis of 105 mantle cell lymphoma patients: incidences, clinical features, response, survival and prognostic factors.

This study presents the first large clinical analysis of 105 unselected mantle cell lymphoma (MCL) patients diagnosed from 1992 to 2000 in a well-defined Danish population. The annual incidences were 0.7/100000 for men and 0.2/100000 for women, with no significant change during the study period. Of 97 evaluable cases, 43% achieved a complete response (CR) after initial therapy. The median disease-free (DFS) and overall survival (OS) rates were 15 and 30 months, respectively. In multivariate analysis, splenomegaly (P=0.002), anaemia (P=0.0001) and age (P=0.002), but not the international prognostic index (IPI) and the Ann Arbor staging system, had an independent impact on survival. Moreover, in a sub-analysis of 45 younger MCL patients (<65 years), a trend towards an OS plateau of 58% was observed in cases without splenomegaly and anaemia (n=29). Thus, in contrast to previously suggested prognostic factors, these variables may prove useful for clinical decisions in a significant subset of MCL patients.

Recruitment of CD34+ cells during large-volume leukapheresis.

Mobilized peripheral blood stem and progenitor cells (PBPCs) are increasingly used to restore hematopoiesis after myeloablative treatment. To obtain a sufficient number of CD34(+) cells, many studies have focused on the improvement of the collection technique during the leukapheresis procedure (LP), and so-called large-volume leukapheresis (LVL) procedures have been developed. Such procedures can be performed by extending the duration of the LP and/or by increasing the inlet flow rate. However, no previous studies have compared the efficiency of these procedures. In the present study, we compared the kinetics of PBPCs recruitment (including CD34(+) cell subsets), the PBPCs yield, and the collection efficiency as well as the overall feasibility of the procedures during a single LVL performed by standard (group I) (median 85 ml/min; range 50-97 ml/min) and high inlet flow rates (group II) (median 130 ml/min; range 110-150 ml/min). Seven patients with hematological malignancies were enrolled and allocated to each group. The patients' blood volumes (BV) were processed four times. The apheresis product (AP) was collected in four separate bags, which were changed every time one BV had been processed. The CD34(+) cell number and CD34(+) cell subsets were assessed in the four collection bags and in peripheral blood (PB) before every time one BV had been processed and after the leukapheresis. The CD34(+) cell yield exceeded the pre-apheresis CD34(+) cell number per ml BV in 6 out of 7 patients in group I and in 3 out of 7 patients in group II. In group II, the recruitment of CD34(+) cells from the bone marrow (BM) to PB starts in the second collection period--as early as 30-60 min after initiating the procedure. No exhaustion in the recruitment was observed in the two groups for at least 5 h during the leukapheresis, and all CD34(+) cell subsets were recruited at a steady rate. However, the collection efficiency in group II was only half of that in group I. In addition, we experienced many technical problems during the leukapheresis in group II. Thus, in 4 out of 7 patients in this group, it was not possible to perform the maximal inlet flow rate because of catheter problems. In conclusion, due to the technical problems associated with the high inlet flow rate procedure and the fact that the relative number of CD34(+) cells harvested and recruited during the leukapheresis was higher in group I than II and, also reflected an approximately two-fold higher collection efficiency, we recommend that LVL be performed by standard inlet flow rate.

Soluble CD40 ligand induces selective proliferation of lymphoma cells in primary mantle cell lymphoma cell cultures.

Interaction between CD40 and the CD40 ligand (CD40L) is critical for the survival and proliferation of B cells during immunopoiesis. However, the role of CD40L in the pathogenesis of malignant lymphomas is ambiguous. Primary mantle cell lymphoma (MCL) cells were cultured in the presence of recombinant human CD40L trimer (huCD40LT), and a significant time- and dose-dependent induction of DNA synthesis was observed in thymidine incorporation assays (n = 7, P <.04). The maximal rate of DNA synthesis was reached at huCD40LT doses of 100 ng/mL and above after 4 days of culture, but a significant increase of DNA synthesis was detected already at doses of 1 ng/mL (P =.03). HuCD40LT never inhibited the basal level of DNA synthesis. These findings established 400 ng/mL of huCD40LT for 4 days as standard conditions in the system. Under these conditions, huCD40LT significantly increased the proportion of cells in the S/G(2)/M phases of the cell cycle in 4 of 7 studied cases, while the fraction of apoptotic cells remained unchanged (n = 7). HuCD40LT also induced expression of CD80/B7-1, CD86/B7-2, and CD95/Fas and up-regulated the expression of HLA-DR (n = 6). With the use of bromodeoxyuridine incorporation in triple-color flow cytometric analysis, it was found that huCD40LT induced cell-cycle progression in light chain-restricted cells only, of which a median of 14% (range, 0.5% to 29%; n = 4) returned to G(0/1) phase DNA content after bromodeoxyuridine incorporation, demonstrating completion of at least one cell cycle in the presence of huCD40LT. Thus, primary clonal MCL cells are activated and can proliferate in the presence of huCD40LT as a single agent.

S-phase induction by interleukin-6 followed by chemotherapy in patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma.

Interleukin-6 (IL-6) has in vitro demonstrated growth regulatory effects on tumor cells from patients with chronic lymphocytic leukemia (CLL) and lymphoma. The proliferation rate of these cells is usually very low and this is thought to be one of the reasons for the lack of a curative potential of cytostatic chemotherapy in CLL and low grade NHL. Recombinant human (rh) IL-6 might increase the in vivo proliferation rate leading to a higher sensitivity for chemotherapy. We tested this hypothesis by administering rhIL-6 to 9 CLL patients and 3 NHL patients in doses of 2.5 micrograms/kg, 5 micrograms/kg and 10 micrograms/kg s.c. daily for 5 days followed by CHOP chemotherapy on the last day of rhIL-6 injection. Six patients had two treatment cycles. The proportion of cells in S-phase was determined by the bromodeoxyuridine labeling index (LI). Three patients achieved a partial remission, one patient had progressive disease and the remaining patients demonstrated no change. Two patients, who received 10 micrograms/kg/day rhIL-6, demonstrated a significant increase in LI, one of these was first observed in the second treatment cycle. A significant decrease was seen in two patients receiving 2.5 micrograms/kg and 5 micrograms/kg respectively. Immunophenotypic assessment demonstrated that rhIL-6 increased the expression of CD20 in all CLL patients with a reversal after cessation of rhIL-6. We conclude that rhIL-6, in the dosage and schedule used in this study, did not increase the proportion of the cells in S-phase and that the growth stimulatory effects of rhIL-6 in CLL in vivo probably are insignificant. However, the role of rhIL-6 in CLL as inducer of increased CD20 expression prior to anti-CD20 antibody treatment remains to be determined.

Linear reduction of clonal cells in stem cell enriched grafts in transplanted multiple myeloma.

In 30 patients with multiple myeloma who were scheduled for peripheral blood stem-cell transplantation, a quantitative analysis of the stem cells following enrichment by anti-CD34 was carried out. To detect the cells of the specific myeloma clone, polymerase chain reaction (PCR) was performed using unique allele-specific oligo primers for the immunoglobulin heavy chain rearrangement. The clonogenic cells before and after stem-cell enrichment, were quantified by a limiting dilution assay and a highly sensitive semi-nested PCR combined with a real-time quantitative PCR. In order to accomplish a statistically adequate end-point analysis, a large number of PCR analyses (40 per sample) were performed. By this technique the lowest detection limit observed was one myeloma cell per 106 cells. Myeloma cells were detected in 29/30 samples from the CD34-enriched fraction. The CD34 selection procedure resulted in a median 28-fold enrichment of CD34+ haemopoietic precursor cells. The stem-cell selection reduced the median concentration of clonal cells per million total cells by half, with a highly significant linear relationship between the number of myeloma cells before and after stem cell enrichment. The median depletion of clonal cells by the overall procedure was 2.15 log units, corresponding to a reduction of the total quantity of clonal cells reinfused into the patients by at least 99.3%. We conclude that CD34+ cell enrichment led to a reliable tumour cell depletion of the order of 2 log, which may not be sufficient since the total number of tumour cells in the leukapheresis product was 7.2 log (median).