Long-term safety and outcome of fludarabine, cyclophosphamide and mitoxantrone (FCM) regimen in previously untreated patients with advanced follicular lymphoma: 12 years follow-up of a phase 2 trial.

Fludarabine combinations are very affective in follicular lymphoma (FL) with high rates of complete response and prolonged survival. However, late toxicities could be a concern. The aim of the present study was to analyze the long-term impact on survival, relapse and late toxicities of a trial of treatment with fludarabine, mitoxantrone and cyclophosphamide (FCM regimen) for untreated patients with advanced stage FL. One hundred and twenty patients enrolled in a phase 2 trial of treatment with FCM regimen between 2000 and 2003 were evaluated. After a median follow-up of 12 years, 52 patients eventually relapsed/progressed with 10 year progression-free survival (PFS) of 46 %. Ten patients showed histological transformation to aggressive lymphoma with a risk of transformation of 2 and 9 % at 5 and 10 years, respectively. Three patients developed therapy-related myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML) and seven solid neoplasms with an overall risk of 3 and 8 % at 5 and 10 years, respectively. Twenty-six patients eventually died during the follow-up. Overall survival at 10 years was 83 %. In conclusion, FCM regimen allows excellent long-lasting response in previously untreated patients with FL. The incidence of late events including histological transformation and secondary neoplasia is low but not negligible.

Superiority of bortezomib, thalidomide, and dexamethasone (VTD) as induction pretransplantation therapy in multiple myeloma: a randomized phase 3 PETHEMA/GEM study.

The Spanish Myeloma Group conducted a trial to compare bortezomib/thalidomide/dexamethasone (VTD) versus thalidomide/dexamethasone (TD) versus vincristine, BCNU, melphalan, cyclophosphamide, prednisone/vincristine, BCNU, doxorubicin, dexamethasone/bortezomib (VBMCP/VBAD/B) in patients aged 65 years or younger with multiple myeloma. The primary endpoint was complete response (CR) rate postinduction and post-autologous stem cell transplantation (ASCT). Three hundred eighty-six patients were allocated to VTD (130), TD (127), or VBMCP/VBAD/B (129). The CR rate was significantly higher with VTD than with TD (35% vs 14%, P = .001) or with VBMCP/VBAD/B (35% vs 21%, P = .01). The median progression-free survival (PFS) was significantly longer with VTD (56.2 vs 28.2 vs 35.5 months, P = .01). In an intention-to-treat analysis, the post-ASCT CR rate was higher with VTD than with TD (46% vs 24%, P = .004) or with VBMCP/VBAD/B (46% vs 38%, P = .1). Patients with high-risk cytogenetics had a shorter PFS and overall survival in the overall series and in all treatment groups. In conclusion, VTD resulted in a higher pre- and posttransplantation CR rate and in a significantly longer PFS although it was not able to overcome the poor prognosis of high-risk cytogenetics. Our results support the use of VTD as a highly effective induction regimen prior to ASCT. The study was registered with http://www.clinicaltrials.gov (NCT00461747) and Eudra CT (no. 2005-001110-41).

Clinical applicability and prognostic significance of molecular response assessed by fluorescent-PCR of immunoglobulin genes in multiple myeloma. Results from a GEM/PETHEMA study.

Minimal residual disease monitoring is becoming increasingly important in multiple myeloma (MM), but multiparameter flow cytometry (MFC) and allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) techniques are not routinely available. This study investigated the prognostic influence of achieving molecular response assessed by fluorescent-PCR (F-PCR) in 130 newly diagnosed MM patients from Grupo Español Multidisciplinar de Melanoma (GEM)2000/GEM05 trials (NCT00560053, NCT00443235, NCT00464217) who achieved almost very good partial response after induction therapy. As a reference, we used the results observed with simultaneous MFC. F-PCR at diagnosis was performed on DNA using three different multiplex PCRs: IGH D-J, IGK V-J and KDE rearrangements. The applicability of F-PCR was 91·5%. After induction therapy, 64 patients achieved molecular response and 66 non-molecular response; median progression-free survival (PFS) was 61 versus 36 months, respectively (P = 0·001). Median overall survival (OS) was not reached (NR) in molecular response patients (5-year survival: 75%) versus 66 months in the non-molecular response group (P = 0·03). The corresponding PFS and OS values for patients with immunophenotypic versus non-immunophenotypic response were 67 versus 42 months (P = 0·005) and NR (5-year survival: 95%) versus 69 months (P = 0·004), respectively. F-PCR analysis is a rapid, affordable, and easily performable technique that, in some circumstances, may be a valid approach for minimal residual disease investigations in MM.

Long-term prognostic significance of response in multiple myeloma after stem cell transplantation.

For establishing the true effect of different response categories in patients with multiple myeloma (MM) treated with autologous stem cell transplantation, we evaluated, after a median follow-up of 153 months, 344 patients with MM who received a transplant between 1989 and 1998. Overall survival (OS) at 12 years was 35% in complete response (CR) patients, 22% in near complete response (nCR), 16% in very good partial response (VGPR), and 16% in partial response (PR) groups. Significant differences in OS and progression-free survival were found between CR and nCR groups (P = .01 and P = .002, respectively), between CR and VGPR groups (P = .0001 and P = .003), or between CR and PR groups (P = .003 and P = < 10(-5)); no differences were observed between the nCR and VGPR groups (P = .2 and P = .9) or between these groups and the PR group (P = .1 and P = .8). A landmark study found a plateau phase in OS after 11 years; 35% patients in the CR group and 11% in the nCR+VGPR+PR group are alive at 17 years; 2 cases had relapsed in the nCR+VGPR+PR group. In conclusion, MM achieving CR after autologous stem cell transplantation is a central prognostic factor. The relapse rate is low in patients with > 11 years of follow-up, possibly signifying a cure for patients in CR.

Benefit from autologous stem cell transplantation in primary refractory myeloma? Different outcomes in progressive versus stable disease.

BACKGROUND: Several studies of autologous stem cell transplantation in primary refractory myeloma have produced encouraging results. However, the outcome of primary refractory patients with stable disease has not been analyzed separately from the outcome of patients with progressive disease. DESIGN AND METHODS: In the Spanish Myeloma Group 2000 trial, 80 patients with primary refractory myeloma (49 with stable disease and 31 with progressive disease), i.e. who were refractory to initial chemotherapy, were scheduled for tandem transplants (double autologous transplant or a single autologous transplant followed by an allogeneic transplant). Patients with primary refractory disease included those who never achieved a minimal response (≥ 25% M-protein decrease) or better. Responses were assessed using the European Bone Marrow Transplant criteria. RESULTS: There were no significant differences in the rates of partial response or better between patients with stable or progressive disease. However, 38% of the patients with stable disease at the time of transplantation remained in a stable condition or achieved a minimal response after transplantation versus 7% in the group with progressive disease (P=0.0017) and the rate of early progression after transplantation was significantly higher among the group with progressive disease at the time of transplantation (22% versus 2%; P=0.0043). After a median follow-up of 6.6 years, the median survival after first transplant of the whole series was 2.3 years. Progression-free and overall survival from the first transplant were shorter in patients with progressive disease (0.6 versus 2.3 years, P=0.00004 and 1.1 versus 6 years, P=0.00002, respectively). CONCLUSIONS: Our results show that patients with progressive refractory myeloma do not benefit from autologous transplantation, while patients with stable disease have an outcome comparable to those with chemosensitive disease.

Allogeneic stem cell transplantation after reduced intensity conditioning in patients with relapsed or refractory Hodgkin's lymphoma. Results of the HDR-ALLO study - a prospective clinical trial by the Grupo Español de Linfomas/Trasplante de Médula Osea (GEL/TAMO) and the Lymphoma Working Party of the European Group for Blood and Marrow Transplantation.

BACKGROUND: Although Hodgkin's lymphoma is a highly curable disease with modern chemotherapy protocols, some patients are primary refractory or relapse after first-line chemotherapy or even after high-dose therapy and autologous stem cell transplantation. We investigated the potential role of allogeneic stem cell transplantation in this setting. DESIGN AND METHODS: In this phase II study 92 patients with relapsed Hodgkin's lymphoma and an HLA-identical sibling, a matched unrelated donor or a one antigen mismatched, unrelated donor were treated with salvage chemotherapy followed by reduced intensity allogeneic transplantation. Fourteen patients showed refractory disease and died from progressive lymphoma with a median overall survival after trial entry of 10 months (range, 6-17). Seventy-eight patients proceeded to allograft (unrelated donors, n=23). Fifty were allografted in complete or partial remission and 28 in stable disease. Fludarabine (150 mg/m(2) iv) and melphalan (140 mg/m(2) iv) were used as the conditioning regimen. Anti-thymocyte globulin was additionally used as graft-versus-host-disease prophylaxis for recipients of grafts from unrelated donors. RESULTS: The non-relapse mortality rate was 8% at 100 days and 15% at 1 year. Relapse was the major cause of failure. The progression-free survival rate was 47% at 1 year and 18% at 4 years from trial entry. For the allografted population, the progression-free survival rate was 48% at 1 year and 24% at 4 years. Chronic graft-versus-host disease was associated with a lower incidence of relapse. Patients allografted in complete remission had a significantly better outcome. The overall survival rate was 71% at 1 year and 43% at 4 years. CONCLUSIONS: Allogeneic stem cell transplantation can result in long-term progression-free survival in heavily pre-treated patients with Hodgkin's lymphoma. The reduced intensity conditioning approach significantly reduced non-relapse mortality; the high relapse rate represents the major remaining challenge in this setting. The HDR-Allo trial was registered in the European Clinical Trials Database (EUDRACT, https://eudract.ema.europa.eu/) with number 02-0036.

Minerval induces apoptosis in Jurkat and other cancer cells.

Minerval is an oleic acid synthetic analogue that impairs lung cancer (A549) cell proliferation upon modulation of the plasma membrane lipid structure and subsequent regulation of protein kinase C localization and activity. However, this mechanism does not fully explain the regression of tumours induced by this drug in animal models of cancer. Here we show that Minerval also induced apoptosis in Jurkat T-lymphoblastic leukaemia and other cancer cells. Minerval inhibited proliferation of Jurkat cells, concomitant with a decrease of cyclin D3 and cdk2 (cyclin-dependent kinase2). In addition, the changes that induced on Jurkat cell membrane organization caused clustering (capping) of the death receptor Fas (CD95), caspase-8 activation and initiation of the extrinsic apoptosis pathway, which finally resulted in programmed cell death. The present results suggest that the intrinsic pathway (associated with caspase-9 function) was activated downstream by caspase-8. In a xenograft model of human leukaemia, Minerval also inhibited tumour progression and induced tumour cell death. Studies carried out in a wide variety of cancer cell types demonstrated that apoptosis was the main molecular mechanism triggered by Minerval. This is the first report on the pro-apoptotic activity of Minerval, and in part explains the effectiveness of this non-toxic anticancer drug and its wide spectrum against different types of cancer.

Busulfan 12 mg/kg plus melphalan 140 mg/m2 versus melphalan 200 mg/m2 as conditioning regimens for autologous transplantation in newly diagnosed multiple myeloma patients included in the PETHEMA/GEM2000 study.

BACKGROUND: The aim of this study was to compare the long-term safety and efficacy of oral busulfan 12 mg/kg plus melphalan 140 mg/m(2) and melphalan 200 mg/m(2) as conditioning regimens for autologous stem cell transplantation in newly diagnosed patients with multiple myeloma in the GEM2000 study. DESIGN AND METHODS: The first 225 patients received oral busulfan 12 mg/kg plus melphalan 140 mg/m(2); because of a high frequency of veno-occlusive disease, the protocol was amended and a further 542 patients received melphalan 200 mg/m(2). RESULTS: Engraftment and hospitalization times were similar in both groups. Oral busulfan 12 mg/kg plus melphalan 140 mg/m(2) resulted in higher transplant-related mortality (8.4% versus 3.5%; P=0.002) due to the increased frequency of veno-occlusive disease in this group. Response rates were similar in both arms. With respective median follow-ups of 72 and 47 months, the median progression-free survival was significantly longer with busulfan plus melphalan (41 versus 31 months; P=0.009), although survival was similar to that in the melphalan 200 mg/m(2) group. However, access to novel agents as salvage therapy after relapse/progression was significantly lower for patients receiving busulfan plus melphalan (43%) than for those receiving melphalan 200 mg/m(2) (58%; P=0.01). CONCLUSIONS: Conditioning with oral busulfan 12 mg/kg plus melphalan 140 mg/m(2) was associated with longer progression-free survival but equivalent survival to that achieved with melphalan 200 mg/m(2) but this should be counterbalanced against the higher frequency of veno-occlusive disease-related deaths. This latter fact together with the limited access to novel salvage therapies in patients conditioned with oral busulfan 12 mg/kg plus melphalan 140 mg/m(2) may explain the absence of a survival difference. Oral busulfan was used in the present study; use of the intravenous formulation may reduce toxicity and result in greater efficacy, and warrants further investigation in myeloma patients. (Clinicaltrials.gov identifier: NCT00560053).

Inhibition of c-Myc down-regulation by sustained extracellular signal-regulated kinase activation prevents the antimetabolite methotrexate- and gemcitabine-induced differentiation in non-small-cell lung cancer cells.

Non-small-cell lung cancer (NSCLC) is characterized by severe resistance to chemotherapy. Here, we demonstrate that A549 adenocarcinoma cells permanently differentiate with the antimetabolites methotrexate (MTX) and gemcitabine (GE) when blocking the resistance mechanism that normally counteracts this process. MTX (1-10 microM) and GE (1 microM) induced growth arrest accompanied by sustained extracellular signal-regulated kinase (ERK1/2) phosphorylation and moderate reduction of c-Myc levels after 96 h, whereas only a low percentage of the cells differentiated. Combination with the mitogen-activated protein kinase kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis-(methylthio)butadiene (U0126) reduced MTX- or GE-induced ERK1/2 over-phosphorylation, nearly abolished c-Myc expression, and provoked radical morphological changes in all cells. Besides the appearance of multilamellar bodies and intracellular cytokeratin reorganization, modulation of molecular markers occurred in a manner consistent with differentiation (gelsolin, +300%; surfactant protein A and C, -70%). Similar to U0126, c-Myc inactivation with specific small interfering RNA initiated differentiation only in the presence of MTX, demonstrating that inhibition of the mitogen-activated protein kinase/ERK pathway alone or down-regulation of c-Myc is not sufficient to induce this process. It is noteworthy that withdrawal of antitumoral drugs and U0126 neither reversed differentiation nor reactivated proliferation. Our results reveal that maintenance of a certain threshold of c-Myc expression through sustained ERK1/2 activation represents a molecular mechanism that confers resistance to antimetabolite-induced differentiation in A549 cells, and provide a novel molecular basis for therapeutic strategies based on irreversible differentiation of cancer cells using conventional chemotherapeutic antimetabolites in combination with inhibitors of the MEK/ERK pathway or c-Myc.

dup(1)(q21q32) as a sole cytogenetic event is associated to a leukemic transformation in myelodysplastic syndromes.

Duplication of the long arm of chromosome 1 (1q) has been detected accompanied with other chromosome abnormalities in Myelodysplastic Syndromes (MDS). However, as a sole karyotypic change, it is rarely observed. We present here two patients affected of a MDS that showed a dup(1)(q21q32) as a sole cytogenetic change in their bone marrow cells. Complementary methodologies confirmed the duplication of chromosome 1q and, did not show additional cryptic chromosome abnormalities. One patient acquired a secondary trisomy 8 and the other one progressed toward an acute leukemia with no additional cytogenetic alterations.

High clinical and molecular response rates with fludarabine, cyclophosphamide and mitoxantrone in previously untreated patients with advanced stage follicular lymphoma.

BACKGROUND: Purine analogs have demonstrated significant activity in patients with follicular lymphoma. The aim of this study was to analyze the efficacy and toxicity of a fludarabine combination as first-line treatment in patients with advanced-stage disease. DESIGN AND METHODS: This is a phase II trial including 120 patients (< or =65 years) treated with 6 cycles of fludarabine, cyclophosphamide and mitoxantrone (FCM). Molecular response was assessed by q-PCR in peripheral blood. RESULTS: Of 119 patients with an assessable response, complete response was achieved in 99 (83%) partial response in 13 (11%) and 7 (6%) did not respond. After treatment, 37 out of 46 (81%) patients achieved molecular response. After a median follow-up of 3.9 years, 32 patients have relapsed. The 5-year progression-free survival was 58% (95% confidence interval: 47-69). Variables associated with a shorter progression-free survival were a poor performance status (ECOG> or =2), > or =2 extranodal sites and high beta2-microglobulin. Sixteen episodes of grade 3-4 infections were observed. Two patients died during therapy (of progressive multifocal leukoencephalopathy and bronchoaspiration respectively). No late toxicity has been observed. Twelve patients died during follow-up (9 after relapse, 2 during chemotherapy, 1 in complete remission after surgery for meningioma). The overall survival at 5 years was 89%. ECOG > or =2 and high beta2-microglobulin were associated with a shorter survival. CONCLUSIONS: FCM results in high complete and molecular response rates, with prolonged response duration in younger patients with advanced-stage follicular lymphoma. The combination of FCM with rituximab as front-line treatment warrants further investigation.

Long-term hematological reconstitution and clinical evaluation of autologous peripheral blood stem cell transplantation after cryopreservation of cells with 5% and 10% dimethylsulfoxide at -80 degrees C in a mechanical freezer.

We report the long-term evaluation over 12 years of a simplified technique for stem-cell cryopreservation at -80 degrees C without rate-controlled freezing and with 5% (n=251) or 10% (n=47) DMSO as the sole cryoprotectant. Platelet recovery was greater in the 5% DMSO group while long-term hematological recovery did not differ. Factors influencing a faster hematological recovery were infusion of more than 2.7x10(6)/Kg of CD34+ cells, 10% DMSO cryopreservation and G-CSF. We confirm that the procedure is feasible with reduction in infusion-related toxicity from 60% using 5% DMSO. Differences in hematological reconstitution were not clinically significant if a minimum of 1.5x10(6)/Kg CD34+-cells were infused.

Effect of chemotherapy with alkylating agents on the yield of CD34+ cells in patients with multiple myeloma. Results of the Spanish Myeloma Group (GEM) Study.

BACKGROUND AND OBJECTIVES: Although alkylating agents are clearly beneficial in multiple myeloma (MM), their deleterious effect on bone marrow hematopoietic progenitor cells usually precludes their use as front-line therapy in patients scheduled to undergo autologous stem cell transplantation (ASCT). We analyzed the impact of first-line chemotherapy with alkylating agents on stem cell collection in MM patients. DESIGN AND METHODS: Seven hundred and eighty-nine patients included in the Spanish multicenter protocol GEM-2000 underwent mobilization therapy after four courses of alternating VBMCP/VBAD chemotherapy. RESULTS: The mobilization regimens consisted of standard or high-dose granulocyte colony-stimulating factor (G-CSF) in 551 (70%) patients, and chemotherapy and G-CSF in 206 (26%) patients. The CD34+ cell yield was lower than 4x10(6)/kg in 388 patients (49%), and equal or greater than 4x10(6)/kg in 401 patients (51%). Multivariate analysis indicated that advanced age (p<0.0001) and longer interval between diagnosis and mobilization (p=0.012) were the two variables associated with a lower CD34+ cell yield. Significant differences in CD34+ cell yield were not observed between the mobilization regimens. Of the 789 patients included in the protocol, 726 (92%) underwent the planned ASCT, whereas 25 (3%) patients did not because of the low number of CD34+ cells collected. Following ASCT, 0.5x10(9) neutrophils/L could be recovered after 11 days (median time; range, 5-71 days) and 20x10(9) platelets/L could be recovered after 12 days (median time; range, 6-69 days). INTERPRETATION AND CONCLUSIONS: A short-course of therapy with alkylating agents according to the GEM-2000 protocol was associated with an appropriate CD34+ cell collection, and allowed the planned ASCT to be performed in the majority of MM patients.

Pathogenic study of anti-CD20 infusion-related severe refractory shock in diffuse large B-cell lymphoma.

Although rituximab is an effective and safe therapy for B-cell lymphoid malignancies, a few cases of severe infusion-related reactions have been reported. Severe refractory distributive shock is an infrequent side-effect of treatment with rituximab and, to our knowledge, there are no reports describing its pathogenesis in a case of fatal outcome in detail. We present for the first time a case of fatal rituximab infusion-related refractory distributive shock in a patient with CD5+ diffuse large B-cell lymphoma (DLBCL) and analyse the pathogenic mechanisms involved. We have compared measurements obtained from the patient that experienced lethal refractory shock with the four subsequent DLBCL patients treated with rituximab, either at diagnosis or upon relapse, at our center. Serum cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70] and complement components C3 and C4 were analysed, both pretreatment, and 3 h and 9 h after the onset of infusion. When compared with the control subjects, the potential risk factors for rituximab toxicity displayed by the patient that suffered refractory shock included C4 hypercomplementemia, IFN-gamma and IL-10 hypercytokinemia, as well as a high tumor burden. The refractory shock was distributive with most cytokines (IFN-gamma, TNF-alpha, IL-2, IL-4, IL-6 and IL-8) peaking 3 h after infusion and coinciding with the onset of the shock. Furthermore, the concentrations of IL-10 were persistently elevated. In conclusion, the cytokine pattern was similar to that observed in patients with rapid onset septic shock and serum cytokines reached levels markedly higher than previously described in other cases of severe rituximab infusion-related toxicity.

Comparison of intensive chemotherapy, allogeneic or autologous stem cell transplantation as post-remission treatment for adult patients with high-risk acute lymphoblastic leukemia. Results of the PETHEMA ALL-93 trial.

BACKGROUND AND OBJECTIVES: The optimal post-remission therapy for adults with high-risk acute lymphoblastic leukemia (ALL) is not well established. This multicenter randomized trial by the Spanish PETHEMA Group was addressed to compare three options of post-remission therapy in adults with high-risk ALL: chemotherapy, allogeneic stem cell transplantation (SCT) and autologous SCT. DESIGN AND METHODS: A total of 222 valid high-risk ALL patients entered the trial. All received a standard five-drug/five-week induction course. Patients in complete remission with an HLA-identical family donor were assigned to allogeneic SCT (n=84) and the remaining were randomized to autologous SCT (n=50) or to delayed intensification followed by maintenance chemotherapy up to 2 years in complete remission (n=48). RESULTS: Overall, 183 patients achieved complete remission (82%). With a median follow-up of 70 months, the median disease-free survival and overall survival were 17 and 23 months, respectively. The 5-year disease-free survival and overall survival were 35% (95% CI, 30%-41%) and 34% (95% CI, 28%-39%), respectively. Patients allocated to the chemotherapy, allogeneic and autologous SCT were comparable in the main pre-treatment ALL characteristics and the rate of response to therapy. Intention-to-treat analysis showed no differences between patients according to whether they had or did not have a donor in disease-free survival (39%, 95% CI 30-48% vs. 33%, 95% CI 23-41%) and overall survival (44%, 95% CI 35-52% vs. 35%, 95% CI 25-44%), as well as for autologous SCT vs. chemotherapy comparisons (disease-free survival: 40%, 95% CI 28-52% vs. 51%, 95% CI 37-67%; overall survival: 43%, 95% CI 29-58% vs. 52%, 95% CI 39-65%). No differences were observed when the analysis was made on the basis of the treatment actually performed. INTERPRETATION AND CONCLUSIONS: This study failed to prove that, when a family donor is available, allogeneic SCT produces a better outcome than autologous SCT or chemotherapy in adults with high-risk ALL.

[Prevalence and prognostic significance of myeloid markers in adults with high-risk acute lymphoblastic leukemia]. / Prevalencia y significado pronóstico de los marcadores mieloides en adultos con leucemia aguda linfoblástica de alto riesgo.

BACKGROUND AND OBJECTIVE: The prognostic value of myeloid antigen expression in adult acute lymphoblastic leukemia (ALL) is controversial. The objective of this study was to evaluate the frequency and prognostic significance of myeloid antigen expression in adults with high risk ALL. PATIENTS AND METHOD: Between June 1993 and July 2002, 222 adults patients with high-risk ALL were treated according to the PETHEMA LAL 93 protocol. The frequency of myeloid antigen expression, its association with other clinical and biologic variables and the prognostic significance in terms of complete remission (CR) rate, event free survival (EFS) and overall survival (OS) were analyzed. RESULTS: Myeloid antigen expression was present in 96 out of 222 patients (43%). No association was observed between myeloid antigen expression and the main clinical and biologic characteristics of ALL. Response to treatment was slower in patients expressing myeloid antigens, but no differences were found in CR achievement, EFS or OS. The probability of EFS at 10 years for ALL patients without and with myeloid antigen expression was 35% and 34%, respectively, while the probability of OS at 10 years was 30% and 33%, respectively. This absence of differences in EFS and OS probabilities was also observed when only slow responding patients were analyzed. CONCLUSIONS: In this study, myeloid antigen expression did not have prognostic influence in adult patients with high risk ALL.

Radiation therapy overcomes adverse prognostic role of cyclooxygenase-2 expression on Reed-Sternberg cells in early Hodgkin lymphoma.

PURPOSE: To analyze the role of radiation therapy (RT) on the adverse prognostic influence of cyclooxygenase-2 (COX-2) expression on Reed-Sternberg (RS) cells, in the setting of early Hodgkin lymphoma (HL) treated with ABVD (adriamycin, vinblastine, bleomycin, dacarbazine). METHODS AND MATERIALS: In the present study we retrospectively investigated the prognostic value of COX-2 expression in a large (n=143), uniformly treated early HL population from the Spanish Network of HL using tissue microarrays. Univariate and multivariate analyses were done, including the most recognized clinical variables and the potential role of administration of adjuvant RT. RESULTS: Median age was 31 years; the expression of COX-2 defined a subgroup with significantly worse prognosis. Considering COX-2(+) patients, those who received RT had significantly better 5-year progression-free survival (PFS) (80% vs 54% if no RT; P=.008). In contrast, COX-2(-) patients only had a modest, nonsignificant benefit from RT in terms of 5-year PFS (90% vs 79%; P=.13). When we compared the outcome of patients receiving RT considering the expression of COX-2 on RS cells, we found a nonsignificant 10% difference in terms of PFS between COX-2(+) and COX-2(-) patients (P=.09), whereas the difference between the 2 groups was important (25%) in patients not receiving RT (P=.04). CONCLUSIONS: Cyclooxygenase-2 RS cell expression is an adverse independent prognostic factor in early HL. Radiation therapy overcomes the worse prognosis associated with COX-2 expression on RS cells, acting in a chemotherapy-independent way. Cyclooxygenase-2 RS cell expression may be useful for determining patient candidates with early HL to receive consolidation with RT.

Differential Impact of Relative Dose-Intensity Reductions in Diffuse Large B-Cell Lymphoma Treated with R-CHOP21 or R-CHOP14.

DLBCL is an aggressive lymphoma treated with R-CHOP. Recently, attempts have been made to improve the outcome by increasing both dose-density and intensity but there have been no benefits in terms of survival. When treating malignancies RDI is important to consider but there is little published information on DLBCL. The purpose of this study was to analyze the differential prognostic impact of RDI in two cohorts of DLBCL patients treated with R-CHOP21 or R-CHOP14. From January 2001 to August 2013 we included DLBCL patients homogenously treated with R-CHOP21 or R-CHOP14, with or without radiotherapy, at University Hospital Son Espases, Hospital Son Llatzer of Palma and Hospital del Mar of Barcelona (N = 157). In order to avoid selection bias the patients were retrospectively identified from the Pathology Department and Pharmacy registries. Median follow-up was 68 months. There was no difference in the response or survival between the two cohorts. In the R-CHOP21 group, both a reduction higher than 15% in RDI (RR 7.41) and R-IPI (RR 2.99) were independently associated with OS. However, a reduction higher than 15% in RDI (RR 4.41) was only noted for PFS. In the R-CHOP14 group, NCCN-IPI (RR 7.09) and B-symptoms (RR 5.37) for OS; AA stage III-IV (RR 6.26) and bulky disease (RR 4.05) for PFS. There was a trend towards a higher rate of RDI reduction observed in the R-CHOP14 group but it only made an impact in the R-CHOP21 group. We conclude that R-CHOP21 and R-CHOP14 are equivalent regimens in terms of response and survival, but only if RDI reductions are avoided. For patients receiving R-CHOP21 we recommend using clinical and support measures in order to avoid RDI reductions.

Feasibility and results of autologous stem cell transplantation in de novo acute myeloid leukemia in patients over 60 years old. Results of the CETLAM AML-99 protocol.

BACKGROUND AND OBJECTIVES: The benefits of high-dose cytarabine, anthracyclines and hematopoietic stem cell transplantation in the treatment of acute myeloid leukemia (AML) are greater in younger rather than in older patients. We assessed the proportion of patients over 60 years with de novo AML who qualified for intensive therapy and determined the feasibility and results of autologous stem cell transplantation (ASCT) in first complete remission (CR). DESIGN AND METHODS: Induction therapy included idarubicin, cytarabine and etoposide. Patients who achieved CR received one cycle of mitoxantrone and cytarabine and ASCT as consolidation therapies. RESULTS: Over a 4-year period, 258 patients were registered of whom 135 (52%) were enrolled for intensive treatment. The CR rate was 61%, advanced age (p=0.033) and unfavorable cytogenetics (p=0.015) emerged as independent negative prognostic factors for CR. The 2-year overall survival (OS) was 23 % (CI 14%-30%) and was poorer in patients with unfavorable cytogenetics (p=0.035), age over 70 years (p=0.019) or leukocytosis (p=0.006). Only 27% of the potential candidates underwent ASCT. The probability of 2-year leukemia-free survival after consolidation was 39% (CI 6%-71%) for these patients and 22% (CI 6% - 39%) for candidate patients not undergoing ASCT (p=0.07). INTERPRETATION AND CONCLUSIONS: Over 25% of the patients 60 to 70 years with de novo AML benefit from standard intensive treatment. In these patients, ASCT has a tolerable toxicity and may have a positive impact on leukemia-free survival.

Fluorescence resonance energy transfer-based real-time polymerase chain reaction method without DNA extraction for the genotyping of F5, F2, F12, MTHFR, and HFE.

Blood samples are extensively used for the molecular diagnosis of many hematological diseases. The daily practice in a clinical laboratory of molecular diagnosis in hematology involves using a variety of techniques, based on the amplification of nucleic acids. Current methods for polymerase chain reaction (PCR) use purified genomic DNA, mostly isolated from total peripheral blood cells or white blood cells (WBC). In this paper we describe a real-time fluorescence resonance energy transfer-based method for genotyping directly from blood cells. Our strategy is based on an initial isolation of the WBCs, allowing the removal of PCR inhibitors, such as the heme group, present in the erythrocytes. Once the erythrocytes have been lysed, in the LightCycler(®) 2.0 Instrument, we perform a real-time PCR followed by a melting curve analysis for different genes (Factors 2, 5, 12, MTHFR, and HFE). After testing 34 samples comparing the real-time crossing point (CP) values between WBC (5×10(6) WBC/mL) and purified DNA (20 ng/µL), the results for F5 Leiden were as follows: CP mean value for WBC was 29.26±0.566 versus purified DNA 24.79±0.56. Thus, when PCR was performed from WBC (5×10(6) WBC/mL) instead of DNA (20 ng/µL), we observed a delay of about 4 cycles. These small differences in CP values were similar for all genes tested and did not significantly affect the subsequent analysis by melting curves. In both cases the fluorescence values were high enough, allowing a robust genotyping of all these genes without a previous DNA purification/extraction.