Ana Ferrer

Fludarabine, Cyclophosphamide, and Mitoxantrone as Initial Therapy of Chronic Lymphocytic Leukemia: High Response Rate and Disease Eradication

Purpose: Fludarabine, cyclophosphamide, and mitoxantrone (FCM) results in a high response rate in previously treated patients with chronic lymphocytic leukemia (CLL). The aim of this study was to investigate FCM as frontline therapy in CLL. Experimental Design: Sixty-nine patients under the age of 65 years with active CLL were treated. Patients received six cycles of fludarabine 25 mg/m2 i.v. × 3 days, cyclophosphamide 200 mg/m2 i.v. × 3 days, and mitoxantrone 6 mg/m2 i.v. × 1 day. Treatment outcome was correlated with clinical and biological variables. The clinical significance of eradicating minimal residual disease (MRD) was also analyzed. Results: The overall response, MRD-negative complete response (CR), MRD-positive CR, nodular partial response (PR), and PR rates were 90%, 26%, 38%, 14%, and 12%, respectively. Severe (grades 3 or 4) neutropenia developed in 10% of the patients. Major and minor infections were reported in 1% and 8% of cases, respectively. Median response duration was 37 months. Patients with del(17p) failed to attain CR. Patients achieving MRD-negative CR had a longer response duration and overall survival than patients with an inferior response. Low serum lactate dehydrogenase levels, low ZAP-70 expression, and mutated IgVH genes predicted longer response duration. Finally, both low ZAP-70 and CD38 expression in leukemic cells correlated with MRD-negativity achievement. Conclusion: FCM induces a high response rate, including MRD-negative CRs in untreated patients with active CLL. Treatment toxicity is acceptable. Both high ZAP-70 and increased CD38 expression predict failure to obtain MRD-negative response. Patients in whom MRD can be eradicated have longer response duration and overall survival than those with inferior response. These results indicate that FCM can be an ideal companion for chemoimmunotherapy of patients with CLL.

Cytogenetic aberrations and their prognostic value in a series of 330 splenic marginal zone B-cell lymphomas: a multicenter study of the Splenic B-Cell Lymphoma Group.

We conducted a retrospective collaborative study to cytogenetically characterize splenic marginal zone lymphoma (SMZL) and ascertain the prognostic value of chromosomal aberrations. Of 330 cases, 72% displayed an aberrant karyotype, 53% were complex, and 29% had a single aberration. The predominant aberrations were gains of 3/3q and 12q, deletions of 7q and 6q and translocations involving 8q/1q/14q. CD5 expression was detected in 39 of 158 cases (25%). The cytogenetic makeup of the CD5(+) group differed significantly from that of the CD5(-) group. Cases with unmutated IGHV were significantly associated with deletions of 7q and TP53. A strong association was noted between usage of the IGVH1-2 and deletion 7q, 14q alterations, and abnormal karyotype. On univariate analysis, patients with more than or equal to 2 aberrations, 14q alterations, and TP53 deletions had the shortest survival; 7q deletion did not affect survival. On multivariate analysis, cytogenetic aberrations did not retain prognostic significance; the parameters negatively affecting survival were hemoglobin and age. In conclusion, the cytogenetic profile of SMZL is distinct from other B-cell lymphomas. Complexity of the karyotype, 14q aberrations, and TP53 deletions are poor prognostic indicators and may be considered together with other clinicobiologic parameters to ascertain the prognosis of SMZL.

Nuclear Survivin Expression in Mantle Cell Lymphoma Is Associated with Cell Proliferation and Survival

Survivin is a member of the inhibitor of apoptosis protein family that is expressed in G2/M phase. Survivin is overexpressed and associated with parameters of poor prognosis in different human tumors. The role of survivin in the pathogenesis of mantle cell lymphoma (MCL) was examined in a series of typical and blastoid tumors. Survivin was detected as a nuclear pattern in a variable number of tumor cells. Mitotic figures were always positive with a strong delineation of the chromosomes. Western blot analysis confirmed the presence of survivin only in nuclear fractions. Protein expression detected by immunohistochemistry correlated with mRNA levels analyzed by quantitative real-time reverse transcription-polymerase chain reaction (P < 0.0001). Survivin expression levels were higher in blastoid MCL variants (P < 0.0001) and were associated with the proliferative activity (P = 0.001), but not with the ploidy status of the tumors. The number of apoptotic cells was independent of survivin or Ki-67 expression. Overall survival was significantly shorter in patients with high survivin expression. However, in a multivariate analysis, proliferative index was a better predictor of survival than survivin score. These findings indicate that survivin is commonly expressed in MCL with a nuclear and mitotic pattern. The expression levels are strongly associated with the proliferative activity of the tumors and the survival of the patients, suggesting a potential role in cell cycle regulation and tumor progression.

cdc25a and the splicing variant cdc25b2, but not cdc25B1, ‐B3 or ‐C, are over‐expressed in aggressive human non‐Hodgkin's lymphomas

cdc25 is a family of phosphatases that activate the cyclin‐dependent kinases at different points of the cell cycle. cdc25A and ‐B, but not ‐C, have been shown to have oncogenic potential. Three different splicing variants of the cdc25B gene, cdc25B1, ‐B2 and ‐B3, have also been identified. Experimental studies suggest that cdc25B2 may be more active in vivo than cdc25B3 and ‐B1, but the relative expression of these splicing variants in human tumors is not known. In this study, we have analyzed the expression of cdc25A, ‐B1, ‐B2, ‐B3 and ‐C mRNA in 9 non‐neoplastic lymphoid samples, 89 non‐Hodgkin`s lymphomas and 9 hematological cancer cell lines by semi‐quantitative RT‐PCR. cdc25A, ‐B and ‐C protein expression was examined by Western blot. Normal peripheral blood lymphocytes and reactive tissues expressed cdc25B1 and ‐B3 mRNA and very low or undetectable levels of cdc25A, ‐B2 and ‐C. High levels of cdc25A and cdc25B2 were found in 35% and 39% of the tumors, respectively, and they were more frequently observed in aggressive than in indolent lymphomas. cdc25B1 and ‐B3 splice variants were detected in virtually all tumors, and no significant differences were found between high‐ and low‐grade lymphomas. cdc25A and ‐B protein expression was also higher in aggressive than in indolent lymphomas. cdc25C expression was relatively low in virtually all cases. In conclusion, these findings suggest that cdc25A and ‐B2, but not cdc25B1, ‐B3 and ‐C, are over‐expressed in a relatively large number of malignant lymphomas and may participate in the pathogenesis of aggressive variants. Int. J. Cancer (Pred. Oncol.) 89:148–152, 2000.

Leukemic involvement is a common feature in mantle cell lymphoma

The reported incidence of peripheral blood involvement in patients with mantle cell lymphoma (MCL) ranges from 13% to 77%. The aim of the study was to analyze the prevalence and the biologic and clinical significance of leukemic involvement in a series of patients with MCL.

Incidence and Prognostic Impact of Secondary Cytogenetic Aberrations in a Series of 145 Patients with Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a mature B‐cell neoplasm with an aggressive behavior, characterized by the t(11;14)(q13;q32). Several secondary genetic abnormalities with a potential role in the oncogenic process have been described. Studies of large MCL series using conventional cytogenetics, and correlating with proliferation and survival, are scarce. We selected 145 MCL cases at diagnosis, displaying an aberrant karyotype, from centers belonging to the Spanish Cooperative Group for Hematological Cytogenetics. Histological subtype, proliferative index and survival data were ascertained. Combined cytogenetic and molecular analyses detected CCND1 translocations in all cases, mostly t(11;14)(q13;q32). Secondary aberrations were present in 58% of patients, the most frequent being deletions of 1p, 13q and 17p, 10p alterations and 3q gains. The most recurrent breakpoints were identified at 1p31‐32, 1p21‐22, 17p13, and 1p36. Aggressive blastoid/pleomorphic variants displayed a higher karyotypic complexity, a higher frequency of 1p and 17p deletions and 10p alterations, a higher proliferation index and poor survival. Gains of 3q and 13q and 17p13 losses were associated with reduced survival times. Interestingly, gains of 3q and 17p losses added prognostic significance to the morphology in a multivariate analysis. Our findings confirm previous observations indicating that proliferation index, morphology and several secondary genetic alterations (3q gains and 13q and 17p losses) have prognostic value in patients with MCL. Additionally, we observed that 3q gains and 17p losses detected by conventional cytogenetics are proliferation‐independent prognostic markers indicating poor outcome.

Incidence and clinical significance of Bcl-2/IgH rearrangements in follicular lymphoma

Bcl-2 / IgH rearrangement is the molecular hallmark of follicular lymphoma (FL) which is present in 70-90% of the cases at diagnosis. The clinical significance of this feature is controversial. The aim of this study was to analyze the bcl-2 / IgH rearrangement by means of a PCR technique, and to correlate molecular findings with clinical characteristics and outcome. Sixty-nine patients (median age, 53 years; male/female ratio: 35/34) diagnosed with FL in a single institution were included in the study. A total of 77 DNA samples were analyzed, 54 were obtained from lymph node biopsy and 23 from peripheral blood or bone marrow. Bcl-2 / IgH rearrangement was assessed for both the major breakpoint region (MBR ) and the minor cluster region (mcr ) breakpoints by a PCR technique. Thirty-nine out of sixty patients (65%) with assessable samples were found to have a bcl-2 / IgH rearrangement in the MBR breakpoint, whereas bcl-2 / IgH rearrangement in mcr was observed in one patient (2%) and no rearrangement at MBR or mcr in the remaining 20 patients (33%). Regarding the initial characteristics, patients with bcl-2 / IgH rearrangements at MBR or mcr were younger (<65 years) than those with no rearrangement at these sites (p =0.0001). No differences were found according to bcl-2 / IgH rearrangement in terms of complete response rate, time to treatment failure and overall survival. In our series bcl-2 / IgH rearrangement at MBR or mcr, which was found in 67% of the patients, was not correlated with response to treatment, survival nor time-to-treatment-failure.

c-myc mRNA expression and genomic alterations in mantle cell lymphomas and other nodal non-Hodgkin’s lymphomas

Cyclin D1 is a weak oncogene that cooperates with c-myc activation in the development of B cell lymphomas in transgenic animals. Cyclin D1 is constantly overexpressed in human mantle cell lymphomas (MCL). However, the status of c-myc gene in these tumors is not known. We have examined the c-myc mRNA expression and genomic alterations, including mutational analysis of exon 1, intron 1, and exon 2 regulatory elements, in a series of 33 MCL, 22 typical and 11 blastoid variants. In addition, c-myc alterations were also examined in 56 nodal non-Hodgkin’s lymphomas (NHL). c-myc mRNA overexpression was found in 38% (11/29) of MCL with a slightly higher frequency in blastoid variants (5/10, 50%) than in typical cases (6/19, 31%). Genetic alterations were only found in one blastoid MCL showing a three-fold c-mycgene amplification. In other nodal NHL, c-myc overexpression was found in 24% (7/29) of indolent tumors but in 70% (19/27) of aggressive variants. c-myc Genetic alterations detected in these cases were gene rearrangement and hypermutations in one Burkitt’s lymphoma, and individual point mutations in intron 1 or exon 2 in 1/19 (5%) indolent and 7/16 (44%) aggressive variants. These results indicate that c-myc is overexpressed in a subset of MCL, but structural gene alterations are less frequent than in other nodal NHL.

Follicular lymphoma in early stages: high risk of relapse and usefulness of the Follicular Lymphoma International Prognostic Index to predict the outcome of patients.

Abstract:  Background and objectives: Patients with follicular lymphoma (FL) in advanced stages are currently deemed incurable with standard treatments. However, FL is considered to be eradicable in the small group of patients presenting with localized disease. The objective of this study was to analyze the clinical features and the outcome of a series of patients with FL in early stages with a long follow‐up.Patients and methods: A total of 48 patients (25m/23f; median age: 50 yr) diagnosed consecutively with FL in Ann Arbor stage I (25 cases) or II (23) at a single institution with a median follow‐up of 9.5 yr were included in the study. Main biological and clinical characteristics at diagnosis, including Follicular Lymphoma International Prognostic Index (FLIPI) were analyzed; treatment and response were assessed and analyzed for prognosis.Results: The histologic subtypes were: FL type I, 20 cases (42%); type II, 24 (50%); type III, three (6%); and unclassifiable, one (2%). Distribution according to FLIPI was: low risk (36 cases) and intermediate risk (five cases). Treatment mainly consisted of combination chemotherapy (CHOP in 34 cases) plus involved‐field radiotherapy in 26 cases. Forty patients (89%) achieved a complete response (CR), three (7%) a partial response, and two (4%) were non‐responders; the remaining three patients did not receive therapy. No initial variable predicted CR achievement. About 57% of the patients in CR eventually relapsed with a relapse risk of 46% at 10 yr. Intermediate‐risk FLIPI predicted failure‐free survival. Histologic transformation was observed in six patients with a 10‐yr risk of transformation of 13%. Twelve patients died during follow‐up, in two cases as a result of unrelated causes. Overall survival (OS) at 10 yr was 79%. The FLIPI was the sole variable predicting OS.Conclusions: Although the majority of patients with localized FL achieve CR, the risk of relapse is high. The FLIPI is of prognostic value in these patients.

Activation of mitochondrial apoptotic pathway in mantle cell lymphoma: high sensitivity to mitoxantrone in cases with functional DNA-damage response genes

Mantle cell lymphoma (MCL) is a mature B-cell proliferation characterized by the presence of translocation t(11;14)(q13;q32), an aggressive clinical course, and poor response to chemotherapy. The majority of drugs currently used in the treatment of lymphoproliferative disorders induce cell death by triggering apoptosis, but few data concerning drug-induced apoptosis in MCL have been reported. We have analysed the mechanisms of drug-induced cell death in four cell lines with the t(11;14) and in primary cells from 10 patients with MCL. Mitoxantrone, a topoisomerase II inhibitor, induced a strong cytotoxic effect in three cell lines (JVM-2, REC-1, and Granta 519), and in primary MCL cells. This cytotoxic effect due to apoptosis induction was observed despite the presence of either p53 or ATM abnormalities. However, no cytotoxic effect was detected after incubation with DNA-damaging agents in the NCEB-1 cell line, carrying p53 and ATM alterations, despite the presence of functional mitochondrial machinery. These results support that mitoxantrone can be effective in the treatment of MCL but that this activity requires the integrity of functional DNA-damage response genes.