Ana Muntañola

Landscape of somatic mutations and clonal evolution in mantle cell lymphoma

Significance This is a comprehensive whole-genome/whole-exome analysis of mantle cell lymphoma (MCL). We sequenced 29 MCL cases and validated the findings by target sequencing of 172 additional tumors. We identified recurrent mutations in genes regulating chromatin modification and genes such as NOTCH2 that have a major impact on clinical outcome. Additionally, we demonstrated the subclonal heterogeneity of the tumors already at diagnosis and the modulation of the mutational architecture in the progression of the disease. The identification of new molecular mechanisms may open perspectives for the management of MCL patients. Mantle cell lymphoma (MCL) is an aggressive tumor, but a subset of patients may follow an indolent clinical course. To understand the mechanisms underlying this biological heterogeneity, we performed whole-genome and/or whole-exome sequencing on 29 MCL cases and their respective matched normal DNA, as well as 6 MCL cell lines. Recurrently mutated genes were investigated by targeted sequencing in an independent cohort of 172 MCL patients. We identified 25 significantly mutated genes, including known drivers such as ataxia-telangectasia mutated (ATM), cyclin D1 (CCND1), and the tumor suppressor TP53; mutated genes encoding the anti-apoptotic protein BIRC3 and Toll-like receptor 2 (TLR2); and the chromatin modifiers WHSC1, MLL2, and MEF2B. We also found NOTCH2 mutations as an alternative phenomenon to NOTCH1 mutations in aggressive tumors with a dismal prognosis. Analysis of two simultaneous or subsequent MCL samples by whole-genome/whole-exome (n = 8) or targeted (n = 19) sequencing revealed subclonal heterogeneity at diagnosis in samples from different topographic sites and modulation of the initial mutational profile at the progression of the disease. Some mutations were predominantly clonal or subclonal, indicating an early or late event in tumor evolution, respectively. Our study identifies molecular mechanisms contributing to MCL pathogenesis and offers potential targets for therapeutic intervention.

Diffuse Large B-Cell Lymphoma: Clinical and Biological Characterization and Outcome According to the Nodal or Extranodal Primary Origin

PURPOSE To study the main clinicobiologic features, response, and outcome of patients with diffuse large B-cell lymphoma (DLBCL) according to the primary site, lymph node, or different extranodal organs of the disease. PATIENTS AND METHODS We included 382 patients consecutively diagnosed with DLBCL in a single institution during a 13-year period. Morphology, immunophenotyping, proliferation index, differentiation profile, bcl-2/JH rearrangement, and clinical characteristics were analyzed according to the primary site of the lymphoma. RESULTS Sites of the disease were: lymph node, 222 cases (58%); Waldeyers ring (WR), 42 (11%); and extranodal sites, 118 (31%), including GI tract in 45 cases. Primary extranodal cases, particularly GI, showed a bcl-6 expression more frequently than nodal cases. Patients with primary WR or GI lymphomas presented with early-stage disease, no marrow infiltration, normal serum lactate dehydrogenase, and low- to low/intermediate-risk international prognostic index (IPI) more frequently than the remainder. Complete response (CR) rate was 63%, with WR and GI lymphomas having a higher CR rate (85% and 80%, respectively) than the other groups. In the whole series, 5-year overall survival (OS) was 52%. Patients with WR or GI lymphomas showed better OS (5-year OS: 77% and 68%, respectively) than patients with nodal or other extranodal sites. In the multivariate analysis, IPI, bulky disease, and beta2-microglobulin were the main variables to predict OS; no nodal or extranodal site maintained their prognostic value. CONCLUSION In the present series, the primary site of disease was associated with particular clinicopathologic features and outcome, though the latter largely depended on other factors.

Observation versus antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia

The effectiveness of antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia (ET) is not proven. In this study, the incidence rates of arterial and venous thrombosis were retrospectively analyzed in 300 low-risk patients with ET treated with antiplatelet drugs as monotherapy (n = 198) or followed with careful observation (n = 102). Follow-up was 802 and 848 person-years for antiplatelet therapy and observation, respectively. Rates of thrombotic events were 21.2 and 17.7 per 1000 person-years for antiplatelet therapy and observation, respectively (P = .6). JAK2 V617F-positive patients not receiving antiplatelet medication showed an increased risk of venous thrombosis (incidence rate ratio [IRR]: 4.0; 95% CI: 1.2-12.9; P = .02). Patients with cardiovascular risk factors had increased rates of arterial thrombosis while on observation (IRR: 2.5; 95% CI: 1.02-6.1; P = .047). An increased risk of major bleeding was observed in patients with platelet count greater than 1000 x 10(9)/L under antiplatelet therapy (IRR: 5.4; 95% CI: 1.7-17.2; P = .004). In conclusion, antiplatelet therapy reduces the incidence of venous thrombosis in patients with JAK2-positive ET and the rate of arterial thrombosis in patients with associated cardiovascular risk factors. In the remaining low-risk patients, this therapy is not effective as primary prophylaxis of thrombosis, and observation may be an adequate option.

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.

Rituximab, Fludarabine, Cyclophosphamide, and Mitoxantrone: A New, Highly Active Chemoimmunotherapy Regimen for Chronic Lymphocytic Leukemia

PURPOSE The addition of monoclonal antibodies to chemotherapy has significantly improved treatment of chronic lymphocytic leukemia (CLL). Based on excellent results with the chemotherapy-only regimen fludarabine, cyclophosphamide, and mitoxantrone (FCM), we built a new chemoimmunotherapy combination--rituximab plus FCM (R-FCM). We report a phase II clinical trial consisting of an initial treatment with R-FCM followed by rituximab maintenance. PATIENTS AND METHODS Seventy-two untreated CLL patients age 70 years or younger received rituximab 500 mg/m(2) on day 1 (375 mg/m(2) the first cycle), fludarabine 25 mg/m(2) IV on days 1 to 3, cyclophosphamide 200 mg/m(2) on days 1 to 3, and mitoxantrone 6 mg/m(2) IV on day 1, given at 4-week intervals with up to six cycles supported with colony-stimulating factor. Patients achieving response received maintenance with rituximab 375 mg/m(2) every 3 months for 2 years. RESULTS The overall response, minimal residual disease (MRD) -negative complete response (CR), MRD-positive CR, and partial response rates were 93%, 46%, 36%, and 11%, respectively. Severe neutropenia developed in 13% of patients. Major and minor infections were reported in 8% and 5% of cycles, respectively. Advanced clinical stage, del(17p), or increased serum beta2-microglobulin levels correlated with a lower CR rate. CONCLUSION R-FCM is highly effective in previously untreated CLL, with an 82% CR rate and a high proportion of MRD-negative CRs (46%). Treatment toxicity is acceptable. Parameters correlating with a lower response rate were advanced clinical stage, high serum beta2-microglobulin levels, and del(17p). Based on these results, R-FCM warrants further investigation in randomized clinical trials.

Improving survival in patients with chronic lymphocytic leukemia (1980-2008): the Hospital Clinic of Barcelona experience.

Whether advances in treatment are prolonging survival of patients with chronic lymphocytic leukemia (CLL) is unclear. We analyzed presentation patterns and survival over time in 929 patients followed from 1980 to 2008 at the Hospital Clinic of Barcelona. The 5- and 10-year relative survival (adjusted for the expected survival in the general population) was estimated in patients seen in 2 periods of time: 1980-1994 (n = 451) and 1995-2004 (n = 365). We found that CLL shortens life expectancy in all age groups independently of clinical features at diagnosis. Nevertheless, survival is improving, particularly in some groups of patients. Thus, relative survival was significantly higher in the 1995-2004 cohort than in the 1980-1994 group both at 5 years (incidence rate ratio [IRR] = 0.46; P = .004) and 10 years (IRR = 0.65; P = .007) from diagnosis. The improved survival was largely due to a decrease in CLL-attributable mortality in patients younger than 70 years in Binet stage B or C at diagnosis (IRR = 0.40; P = .001 at 5 years; IRR = 0.33; P < .001 at 10 years). These results suggest that newer treatments are changing the prognosis of CLL, particularly in younger patients with advanced disease, whereas no improvement is yet observed in older subjects or those with lower-risk disease.

Expanded and highly active proliferation centers identify a histological subtype of chronic lymphocytic leukemia ("accelerated" chronic lymphocytic leukemia) with aggressive clinical behavior.

Background The concept of “accelerated” chronic lymphocytic leukemia is frequently used by both pathologists and clinicians. However, neither histological criteria to define this form of chronic lymphocytic leukemia nor its clinical correlates and prognostic impact have been formally defined in large series of patients. Design and Methods Tissue biopsies from 100 patients with chronic lymphocytic leukemia were analyzed for the size of proliferation centers and their proliferation rate as assessed by mitosis count and Ki-67 immunostaining. Histological patterns were correlated with main clinico-biological features and outcome. Results A suspicion of disease transformation was the main reason for carrying out tissue biopsy, which was performed at a median time of 14 months (range, 0 to 204 months) after the diagnosis of chronic lymphocytic leukemia. The biopsy showed histological transformation to diffuse large B-cell lymphoma in 22 cases. In the remaining 78 patients, the presence of expanded proliferation centers (broader than a 20x field) and high proliferation rate (either >2.4 mitoses/proliferation center or Ki-67 >40%/proliferation center) predicted a poor outcome and were selected to define a highly proliferative group. Thus, 23 patients with either expanded proliferation centers or high proliferation rate were considered as having “accelerated” chronic lymphocytic leukemia. These patients displayed particular features, including higher serum lactate dehydrogenase levels and more frequently elevated ZAP-70 than “non-accelerated” cases. The median survival from biopsy of patients with “non-accelerated” chronic lymphocytic leukemia, “accelerated” chronic lymphocytic leukemia and transformation to diffuse large B-cell leukemia was 76, 34, and 4.3 months, respectively (P<0.001). Conclusions The presence of expanded and/or highly active proliferation centers identifies a group of patients with “accelerated” chronic lymphocytic leukemia characterized by an aggressive clinical behavior.

ZAP-70 Expression in Normal Pro/Pre B Cells, Mature B Cells, and in B-Cell Acute Lymphoblastic Leukemia

Purpose: The ZAP-70 gene is normally expressed in T and natural killer cells, where it is required for the T-cell receptor (TCR) signaling. More recently, it has been described that ZAP-70 contributes to the B-cell development at early stages of B-cell differentiation in mice. The purpose was to investigate the presence of ZAP-70 in normal pro/pre B cells and mature B cells and in tumoral cells from B-acute lymphoblastic leukemias (B-ALL). Experimental Design: ZAP-70 expression was ascertained by flow cytometry, immunofluorescence, Western blot, and quantitative reverse transcription-PCR. Analysis of ZAP-70 and other signaling proteins of the pre-TCR/TCR was done by Western blot. Results: ZAP-70 was expressed in pro/pre B cells but not in normal mature B cells derived from bone marrow, peripheral blood, or tonsil. Among tumoral cells, ZAP-70 was expressed in 56% of B-ALLs with pro/pre B-cell phenotype and in 4 of 6 Burkitt/ALL lymphomas. In B-ALL cells, expression of CD38 protein correlated with ZAP-70 expression (P = 0.05). Mutational analysis of the ZAP-70 gene revealed the absence of mutations in cases lacking ZAP-70 expression. Moreover, other elements of the pre-TCR/TCR signaling pathway, like LAT and Lck, were also found in B-ALL cells. Conclusions: Among normal B-cell subsets, ZAP-70 was found expressed in normal pro/pre B cells but not in a significant proportion of normal B cells with mature phenotype. Moreover, the presence of ZAP-70 in B-ALLs probably reflects their cellular origin. The lack of ZAP-70 expression in normal mature B cells suggests that its expression in mature-derived neoplasms with different cellular origin, such as Burkitts lymphoma and chronic lymphocytic leukemia, might be due to an aberrant phenomenon.

Abdominal Computed Tomography Predicts Progression in Patients With Rai Stage 0 Chronic Lymphocytic Leukemia

PURPOSE Whether computed tomography (CT) should be routinely included in the diagnostic work-up in patients with chronic lymphocytic leukemia (CLL) has not yet been determined. The aim of this study was to analyze the prognostic significance of abdominal CT in patients with CLL in Rai clinical stage 0. PATIENTS AND METHODS Abdominal CT was performed at diagnosis in 140 patients consecutively diagnosed with CLL in Rai stage 0 disease. RESULTS An abnormal abdominal CT was found in 38 patients (27%). Abnormal CT correlated with increased bone marrow infiltration (P = .024), high lymphocyte count (P = .001), increased ZAP-70 expression (P = .003), and short lymphocyte doubling time (LDT; P = .007). Patients with abnormal CT progressed more frequently and had a shorter time to progression than those with normal CT (median, 3.5 years v not reached, respectively; P < .001) and required earlier treatment intervention. In a multivariate analysis, only high ZAP-70 expression (relative risk = 3.60) and an abnormal abdominal CT (RR = 2.71) correlated with disease progression. CONCLUSION In this series, an abnormal abdominal CT was a strong predictor of progression in patients with early-stage CLL. The inclusion of CT scans in the initial work-up of patients with early clinical stage on clinical grounds can, therefore, provide relevant clinical information.

Rituximab maintenance after first-line therapy with rituximab, fludarabine, cyclophosphamide, and mitoxantrone (R-FCM) for chronic lymphocytic leukemia

The effectiveness of rituximab maintenance therapy in the treatment of chronic lymphocytic leukemia has been investigated in a phase 2 clinical trial that included an initial treatment with rituximab 500 mg/m2 on day 1 (375 mg/m2 the first cycle), fludarabine 25 mg/m2 on days 1 to 3, cyclophosphamide 200 mg/m2 on days 1 to 3, and mitoxantrone 6 mg/m2 on day 1 (R-FCM), for 6 cycles, followed by a maintenance phase with rituximab 375 mg/m2 every 3 months for 2 years. Sixty-seven patients having achieved complete response (CR) or partial response (PR) with R-FCM were given maintenance therapy. At the end of maintenance, 40.6% of patients were in CR with negative minimal residual disease (MRD), 40.6% were in CR MRD-positive, 4.8% remained in PR, and 14% were considered failures. Six of 29 patients (21%) who were in CR MRD-positive or in PR after R-FCM improved their response upon rituximab maintenance. The 4-year progression-free survival (PFS) and overall survival rates were 74.8% and 93.7%, respectively. MRD status after R-FCM induction was the strongest predictor of PFS. Maintenance with rituximab after R-FCM improved the quality of the response, particularly in patients MRD-positive after initial treatment, and obtained a prolonged PFS. This trial was registered at www.clinicaltrialsregister.eu as identifier #2005-001569-33.