Eva González-Barca

Gene expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy

Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell-like (GCB) and activated-B cell-like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

Identification of Leptomeningeal Disease in Aggressive B-Cell Non-Hodgkin's Lymphoma: Improved Sensitivity of Flow Cytometry

PURPOSE Here, we evaluate the sensitivity and specificity of a new 11-parameter flow cytometry (FCM) approach versus conventional cytology (CC) for detecting neoplastic cells in stabilized CSF samples from newly diagnosed aggressive B-cell non-Hodgkins lymphoma (B-NHL) at high risk of CNS relapse, using a prospective, multicentric study design. PATIENTS AND METHODS Moreover, we compared the distribution of different subpopulations of CSF leukocytes and the clinico-biologic characteristics of CSF+ versus CSF-, patients, in an attempt to define new algorithms useful for predicting CNS disease. RESULTS Overall, 27 (22%) of 123 patients showed infiltration by FCM, while CC was positive in only seven patients (6%), with three other cases being suspicious (2%). CC+/FCM+ samples typically had more than 20% neoplastic B cells and/or >or= one neoplastic B cell/microL, while FCM+/CC- samples showed lower levels (P < .0001) of infiltration. Interestingly, in Burkitt lymphoma, presence of CNS disease by FCM could be predicted with a high specificity when increased serum beta2-microglobulin and neurological symptoms coexisted, while peripheral blood involvement was the only independent parameter associated with CNS disease in diffuse large B-cell lymphoma, with low predictive value. CONCLUSION FCM significantly improves the sensitivity of CC for the identification of leptomeningeal disease in aggressive B-NHL at higher risk of CNS disease, particularly in paucicellular samples.

R-ESHAP as salvage therapy for patients with relapsed or refractory diffuse large B-cell lymphoma: the influence of prior exposure to rituximab on outcome. A GEL/TAMO study

The findings of this study suggest that combined therapy wih rituximab, etoposide, cytarabine, cisplatinum and methylprednisolone may be effective prior to autologous stem cell transplantation in patients with refractory or relapsed diffuse large B-cell lymphoma. See related perspective article on page 1776. Background The role of re-treatment with rituximab in aggressive B-cell lymphomas still needs to be defined. This study evaluated the influence of prior exposure to rituximab on response rates and survival in patients with diffuse large B-cell lymphoma treated with rituximab plus etoposide, cytarabine, cisplatinum and methylprednisolone (R-ESHAP). Design and Methods We retrospectively analyzed 163 patients with relapsed or refractory diffuse large B-cell lymphoma who received R-ESHAP as salvage therapy with a curative purpose. Patients were divided into two groups according to whether rituximab had been administered (n=94, “R+” group) or not (n=69, “R-” group) prior to R-ESHAP. Results Response rates were significantly higher in the R- group in the univariate but not in the multivariate analysis. In the analysis restricted to the R+ group, we observed very low complete remission and overall response rates in patients with primary refractory disease (8% and 33%, respectively), as compared to those in patients who were in first partial remission (41% and 86%) or who had relapsed disease (50% and 75%) (p<0.01 in both cases). Overall, 60% and 65% of patients in the R+ and R- groups, respectively, underwent stem-cell transplantation after the salvage therapy. With a median follow-up of 29 months (range, 6–84), patients in the R+ group had significantly worse progression-free survival (17% vs. 57% at 3 years, p<0.0001) and overall survival (38% v 67% at 3 years, p=0.0005) than patients in the R- group. Prior exposure to rituximab was also an independent adverse prognostic factor for both progression-free survival (RR: 2.0; 95% CI: 1.2–3.3, p=0.008) and overall survival (RR: 2.2; 95% CI: 1.3–3.9, p=0.004). Conclusions R-ESHAP was associated with a high response rate in patients who were not refractory to upfront rituximab-based chemotherapy. However, the survival outcome was poor for patients previously exposed to rituximab, as compared to in those who had not previously been treated with rituximab.

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.

MYC protein expression and genetic alterations have prognostic impact in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.

MYC alterations influence the survival of patients with diffuse large B-cell lymphoma. Most studies have focused on MYC translocations but there is little information regarding the impact of numerical alterations and protein expression. We analyzed the genetic alterations and protein expression of MYC, BCL2, BCL6, and MALT1 in 219 cases of diffuse large B-cell lymphoma. MYC rearrangement occurred as the sole abnormality (MYC single-hit) in 3% of cases, MYC and concurrent BCL2 and/or BCL6 rearrangements (MYC double/triple-hit) in 4%, MYC amplifications in 2% and MYC gains in 19%. MYC single-hit, MYC double/triple-hit and MYC amplifications, but not MYC gains or other gene rearrangements, were associated with unfavorable progression-free survival and overall survival. MYC protein expression, evaluated using computerized image analysis, captured the unfavorable prognosis of MYC translocations/amplifications and identified an additional subset of patients without gene alterations but with similar poor prognosis. Patients with tumors expressing both MYC/BCL2 had the worst prognosis, whereas those with double-negative tumors had the best outcome. High MYC expression was associated with shorter overall survival irrespectively of the International Prognostic Index and BCL2 expression. In conclusion, MYC protein expression identifies a subset of diffuse large B-cell lymphoma with very poor prognosis independently of gene alterations and other prognostic parameters.

Serious Complications of Bacteremia Caused by Viridans Streptococci in Neutropenic Patients with Cancer

We prospectively studied 485 episodes of bacteremia in neutropenic patients with cancer. Viridans streptococci caused a total of 88 episodes (18%). Ten (11%) of these 88 cases were associated with serious complications: acute respiratory distress syndrome (ARDS) plus septic shock (5 cases), ARDS (3), and septic shock (2). Streptococcus mitis was the species most frequently isolated (7 of 10 episodes). Four viridans streptococci showed a diminished susceptibility to penicillin (MICs ranged from 0.25 to 4 microg/mL), and 5 strains were resistant to ceftazidime (MICs ranged from 2 to >32 microg/mL). Patients with viridans streptococcal bacteremia (VSB) who developed serious complications were compared with patients with VSB without complications. Severe oral mucositis (70% vs. 32.5%, respectively; P=.036), high-dose chemotherapy with cyclophosphamide (60% vs. 25%, respectively; P=.043), and allogeneic bone marrow transplantation (40% vs. 10%, respectively; P=.040) were the only variables found to be significantly associated with the development of complications. Neither a specific species of viridans streptococci nor resistance to penicillin was associated with the occurrence of complications. The mortality rate was higher in case patients than in control patients (80% vs. 17.5%, respectively; P<.001). Serious complications associated with VSB occur mainly in patients receiving high-dose chemotherapy with cyclophosphamide before allogeneic bone marrow transplantation who develop severe oral mucositis; these complications are associated with a high mortality rate.

Gastrointestinal Involvement in Mantle Cell Lymphoma: A Prospective Clinic, Endoscopic, and Pathologic Study

The frequency of gastrointestinal (GI) tract involvement in mantle cell lymphoma (MCL) at diagnosis is reported to be below 30%. To investigate the actual frequency of GI involvement by MCL, upper and lower endoscopy was prospectively performed on 13 untreated MCL patients at diagnosis. Multiple biopsies from endoscopically normal and abnormal gastric and colonic mucosa were studied with immunohistochemistry (IHC) for CD20, CD5, and cyclin D1, as well as fluorescence in situ hybridization (FISH) for t(11;14) and polymerase chain reaction (PCR) for immunoglobulin heavy chain gene. Abnormal mucosa was identified in 38% of cases by upper endoscopy (mainly mild nonspecific gastritis) and in 54% of cases by lower endoscopy (mostly micropolyps). Histologically, infiltration by MCL was demonstrated in the stomach in 77% of cases and in the colon in 77% of cases. As a whole, 92% of patients showed upper or lower GI tract infiltration by MCL. Histologic evidence of MCL involvement was present in all cases with endoscopically abnormal mucosa, but it was also observed in two-thirds of cases with endoscopically unremarkable mucosa. Positive cyclin D1 IHC was seen in all instances displaying CD20 and CD5-positive lymphoid infiltrates, whereas t(11;14) was demonstrated by FISH in 63.5% and PCR was clonal in 64% of those instances. In conclusion, the great majority of MCL patients showed GI tract involvement at the time of diagnosis, not uncommonly in the form of minute lymphoid infiltrates. IHC for cyclin D1 was significantly more sensitive than FISH t(11;14) or PCR for immunoglobulin heavy chain gene to confirm MCL in this setting.

AICAR induces apoptosis independently of AMPK and p53 through up-regulation of the BH3-only proteins BIM and NOXA in chronic lymphocytic leukemia cells

5-Aminoimidazole-4-carboxamide riboside or acadesine (AICAR) induces apoptosis in chronic lymphocytic leukemia (CLL) cells. A clinical study of AICAR is currently being performed in patients with this disease. Here, we have analyzed the mechanisms involved in AICAR-induced apoptosis in CLL cells in which it activates its only well-known molecular target, adenosine monophosphate-activated protein kinase (AMPK). However, AMPK activation with phenformin or A-769662 failed to induce apoptosis in CLL cells and AICAR also potently induced apoptosis in B lymphocytes from Ampkα1(-/-) mice, demonstrating an AMPK-independent mechanism of cell death. Importantly, AICAR induced apoptosis irrespective of the tumor suppressor TP53 or ataxia telangiectasia mutated (ATM) status via induction of the mitochondrial pathway. Apoptosis was preceded by an increase in mRNA and protein levels of proapoptotic BCL-2 family proteins of the BH3-only subgroup, including BIM, NOXA, and PUMA in CLL cells. Strikingly, B lymphocytes from Noxa(-/-) or Bim(-/-) mice were partially protected from the cytotoxic effects of AICAR. Consistently, B cells from Noxa(-/-)/Bim(-/-) mice resisted induction of apoptosis by AICAR as potently as B lymphocytes overexpressing transgenic BCL-2. These findings support the notion that AICAR is an interesting alternative therapeutic option for CLL patients with impaired p53 function and resistance to conventional chemotherapy.

Safety and efficacy of cyclophosphamide, adriamycin, vincristine, prednisone and rituximab in patients with human immunodeficiency virus‐associated diffuse large B‐cell lymphoma: results of a phase II trial

Immunochemotherapy with cyclophosphamide, adriamycin, vincristine, prednisone and rituximab (R‐CHOP) is the standard treatment in non‐immunosuppressed patients with diffuse large B‐cell lymphoma (DLBCL), but its adequacy has not been definitively established in patients with human immunodeficiency virus (HIV)‐related lymphoma. This phase II trial aimed to evaluate the safety and efficacy of six cycles of R‐CHOP in patients with HIV‐related DLBCL and to determine whether response to highly active antiretroviral therapy (HAART) had prognostic impact. Patients were eligible if they had performance status <3 and absence of active opportunistic infections. Eighty‐one patients were enrolled, 57 in stages III or IV, International Prognostic Index (IPI) 0 or 1 (n = 26), 2 (n = 19), 3 (n = 20) and 4 or 5 (n = 16), and median CD4 lymphocyte count of 0·158 × 109/l. The main adverse events were neutropenia (48% of cycles) and infections (10% of cycles), which were fatal in seven patients. Complete response was achieved in 55 (69%) patients, with an estimated 3‐year disease‐free survival of 77% and 3‐year overall survival of 56%. IPI score and virological response to HAART were the prognostic parameters for response and survival. In HIV‐related DLBCL R‐CHOP is feasible, safe and effective. The prognosis depends on lymphoma‐related parameters and on the response to HAART.