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We do not know the precise figure for solid organ tumors diagnosed each year in Spain and it is therefore difficult to calculate whether there has been a decrease in cancer diagnoses as a consequence of the pandemic. Some indirect data suggest that the pandemic has worsened the stage at which some non-hematological neoplasms are diagnosed. Despite the lack of robust evidence, oncology patients seem more likely to have a poor outcome when they contract COVID-19. The antibody response to infection in cancer patients will be fundamentally conditioned by the type of neoplasia present, the treatment received and the time of its administration. In patients with hematological malignancies, the incidence of infection is probably similar or lower than in the general population, due to the better protective measures adopted by the patients and their environment. The severity and mortality of COVID-19 in patients with hematologic malignancies is clearly higher than the general population. Since the immune response to vaccination in hematologic patients is generally worse than in comparable populations, alternative methods of prevention must be established in these patients, as well as actions for earlier diagnosis and treatment. Campaigns for the early diagnosis of malignant neoplasms must be urgently resumed, post-COVID manifestations should be monitored, collaboration with patient associations is indisputable and it is urgent to draw the right conclusions to improve our preparedness to fight against possible future catastrophes.

CAR-T cell therapy represents a therapeutic revolution in the prognosis and treatment of patients with certain types of hematological cancer. However, they also pose new challenges in the healthcare, regulatory and financial fields. The aim of the RET-A project was to undertake a strategic reflection on the management of CAR-T therapies within the Spanish National Health System, to agree on recommendations that will help to better deal with the new context introduced by these cell therapies in the present and in the future. This think tank involved 40 key agents and opinion leaders. The experts identified three great challenges for implementing advanced therapies in Spain: therapeutic individualisation, with a multidisciplinary approach; acceleration of access times, by minimizing bureaucracy; and increase in the number of centers qualified to manage the CAR-T therapies in the NHS. The experts agreed on the ideal criteria for designating those qualified centers. They also agreed on a comprehensive CAR-T care pathway with the timings and roles which would ideally be involved in each part of the process., This study was funded by Gilead Sciences, Inc.

Mixed acute rejection is a clinicopathological entity that is difficult to accurately diagnose, and so may be under-reported. Allografts are lost more often than in either humoral or cellular rejection. The diagnosis requires both histological and immunological studies on renal biopsy and blood specimens from the transplant recipient to provide the required rescue therapy to abolish the allogeneic response against the graft. We present a clinical case report of an active mixed acute rejection driven by a

Multiparametric Flow Cytometry (MFC) for Immunophenotyping is a very useful tool for diagnosis of Waldenstrom’s macroglobulinemia (WM). It can be used for the distinction between different entities that can be responsible for an IgM monoclonal component, including IgM monoclonal gammopathy undetermined significance (MGUS), asymptomatic WM, and symptomatic WM, as well as other close B-cell lymphoproliferative disorders, such as chronic lymphocytic leukemia, marginal zone lymphoma and IgM multiple myeloma. The identification and quantification of a clonal cell population with a typical WM phenotype is almost diagnostic of this disorder. Clonal WM lymphocytes are characterized by the constant expression of pan-B markers (CD19, CD20, CD22, CD24) and surface immunoglobulin, and are usually positive for FMC7, CD25, CD27, CD45RA, and BCL-2. Moreover, these lymphocytes are almost always negative for CD103, CD11c, CD10 antigens, and frequently negative for CD5 and CD23. Plasma cells present in WM are characterized by an immunophenotype indistinguishable from normal plasma cells, although they belong to the tumor clone. Immunophenotyping also helps to evaluate the response of the disease to the therapy, and it is probably a better monitoring parameter that the M-component, the usual way to evaluate the disorder outcome. In this work, all these concepts are reviewed in detail to help in the comprehension of the use of MFC in WM.

Fluorescence in situ hybridisation (FISH) is an effective technique for the cytogenetic analysis of Waldenstrom macroglobulinemia (WM), but the potential impact of molecular cytogenetics on disease evolution and as a prognostic marker is still unknown. Deletion of the long arm of chromosome 6 (6q-) is the most frequent cytogenetic abnormality in WM. This study analysed the prevalence of this aberration in 102 WM patients, and correlated it with disease characteristics. The incidence of 6q21 deletion was 7% by conventional cytogenetics and 34% when analysed by FISH (54% when cytoplasmic immunoglobulin M-FISH was used). Patients with deletion of 6q displayed features of adverse prognosis, such as higher levels of beta2-microglobulin and monoclonal paraprotein and a greater tendency to display anaemia and hypoalbuminemia. Interestingly, there was a correlation between the presence of 6q deletion and the International Staging System prognostic index (incidence of 6q- among patients stratified in stages 1, 2 and 3 was 24%, 42% and 67% respectively). Those patients diagnosed with smouldering WM who displayed the abnormality showed a trend to an earlier requirement of treatment. Finally, the survival analysis did not show differences between the two groups of patients, probably due to the short follow up of our series.

Recent developments have led to remarkable improvements in the assessment and treatment of patients with multiple myeloma (MM). New technologies have become available to precisely evaluate the biology and extent of the disease, including information about cytogenetics and genetic abnormalities, extramedullary manifestations and minimal residual disease. New, more effective drugs have been introduced into clinical practice, which enable clinicians to significantly improve the outcome of patients but also pose new challenges for the prevention and management of their specific side effects. Given these various new options and challenges, it is important to identify the minimal requirements for diagnosis and treatment of patients, as access to the most sophisticated advances may vary depending on local circumstances. Here, we propose the minimal requirements and possible options for diagnosis, monitoring and treatment of patients with multiple myeloma.

Of 167 newly diagnosed acute promyelocytic leukaemia patients, 83 patients were long (L)-form (50%), eight variable (V)-form (5%) and 76 short (S)-form (45%). The V-form and S-form groups presented a significantly higher percentage of patients with white blood cell counts > 10 × 109/l (P

The human immunoglobulin lambda (IGL) locus contains seven J-Clambda gene regions of which only J-Clambda1, J-Clambda2 J-CA3 and J-Clambda7 encode the four Iglambda isotypes, ie Mcg, Ke-Oz-, Ke-Oz+, and Mcp, respectively. We used isotype-specific DNA probes for detection of IGL gene rearrangements in 212 B cell malignancies: 76 precursor B cell acute lymphoblastic leukemias (precursor B-ALL), 74 Iglambda+ chronic B cell leukemias (CBL), 34 Iglambda+ non-Hodgkin lymphomas (B-NHL), and 28 Iglambda+ multiple myelomas (MM). The J-Clambda3 gene region was most frequently involved (50%), followed by J-Clambda2 (38%) and J-Clambda1 (9%). There was no involvement of the J-Clambda4 and J-Clambda5 gene regions. Rearrangements to J-Clambda6 (n= 4) were exclusively found in precursor B-ALL (19% of all IGL rearrangements in precursor B-ALL) and only a single J-Clambda7 recombination was detected in an Iglambda+ B-NHL. In the group of Iglambda+ malignancies, a significant shift was observed from predominant J-Clambda3 usage (54%) in mature surface Iglambda+ malignancies (CBL and B-NHL) to 60% J-Clambda2 usage in Iglambda+ secreting MM. The distribution of IGL isotype rearrangements found in MM resembled the Iglambda isotype protein expression reported in MM patients. Based on these extensive Southern blot data, we suggest that a rapid and efficient detection of clonal IGL gene rearrangements can be obtained when a single Bg/II digest is used in combination with the IGLJ2 probe, which detects clonality in >95% of cases with an Iglambda+ malignancy. Higher percentages (>98%) can be reached by including a second digest (HindIII) that reduces the chance of comigration of rearranged and germline bands. In case of precursor B-ALL we recommend including the IGLJ6 probe for the detection of rearrangements to J-Clambda6.

Contains fulltext : 107785.pdf (Publisher’s version ) (Open Access) PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

The frequencies of human leukocyte antigen (HLA) class I and class II specificities and haplotypic associations were determined in 1940 unrelated donors from Castilla y Leon and compared with other Iberian, Mediterranean and European populations. Specificities were determined using polymerase chain reaction reverse sequence-specific oligonucleotide or polymerase chain reaction sequence-specific primer techniques. In the analysis, 19, 29 and 13 specificities were found for HLA-A, -B and -DRB1, respectively, with HLA-A*02 (26%), -A*01 (11%), -B*44 (16%), -B*35 (10%), -DRB1*07 (16%) and -DRB1*13 (14%) showing the highest frequencies. In addition, 10 common HLA-A-B-DRB1 haplotypic associations were observed, A*01-B*08-DRB1*03 (3%) and A*29-B*44-DRB1*07 (3%) being the most frequent ones. These findings indicate that the population of Castilla y Leon is genetically equidistant from the Portuguese and other Spanish populations and shares a common origin with other Iberian populations, in which European, Mediterranean and North African genetic components are present; this is in agreement with the historical and genetic background of the population. These data contribute to a better understanding of the genetic structure of the Iberian Peninsula and provide a healthy control population from our region that should be useful for the study of disease associations.

Contains fulltext : 52126.pdf (Publisher’s version ) (Closed access) The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Melphalan-prednisone (MP) has been the gold standard treatment for more than 30 years in elderly multiple myeloma (MM) patients. In order to assess whether the combination of dexamethasone with melphalan (MD) could improve on the efficacy of MP, we have carried out a randomized trial comparing both treatment approaches. A total of 201 patients/=70 years old were included in the study. The overall response rate was similar after six cycles (MP: 67.9%versus MD: 64.5%) and after 12 cycles (MP: 49.4%versus MD: 46.1%). However, the proportion of complete responses (CR) was higher in the MD arm, particularly after 12 cycles (MD: 22.4%versus MP: 9.1%; P0.05). There was no significant difference in event-free survival (MP: 15.9 months versus MD: 23.3 months). The median overall survival in both arms was almost identical (MP: 29.4 months versus MD: 27.2 months; P = 0.63). No significant differences in haematological toxicity were observed, but non-haematological toxicity was significantly higher in the MD arm. According to these results MP remains as the gold standard for treatment of MM and should be the reference for comparison of new therapeutic approaches involving novel agents.

Although multiple myeloma (MM) is predominantly a disease of the elderly, few studies have focused on the identification of prognostic factors in this group of patients. Four hundred twenty five MM patients65 years were uniformly treated with chemotherapy (MP or VCMP/VBAD). Multivariate analysis identified 4 factors with independent unfavorable prognostic influence: high percentage of S-phase bone marrow plasma cells (2.5%); elevated beta(2) microglobulin (B2M) (4 mg/L); age80 years old; and LDH serum levels (above normal limit). The S-phase value was the most powerful independent prognostic factor to discriminate subgroups of patients with different prognosis. Thus, 3 main risk categories could be identified according to S-phase values:/=1%, 1-3% and3%, with median survivals of 34, 22 and 12 months, respectively (p0.0001). Our study also proved the value for elderly patients of the recently developed International Score System (ISS) based on B2M and albumin. Furthermore, the number of S-phase cells helped to subdivide the ISS III Group identifying a subset of patients with very poor prognosis defined by an additional high S-phase, who displayed a median survival of only 8 months. These results demonstrate that elderly patients can be accurately classified according to prognosis, which may be particularly valuable when comparing the efficacy of new treatment strategies. Moreover, our results underline the high prognostic value of proliferative activity of PC, a parameter that should be considered in routine laboratory investigations of MM.

Of 167 newly diagnosed acute promyelocytic leukaemia patients, 83 patients were long (L)-form (50%), eight variable (V)-form (5%) and 76 short (S)-form (45%). The V-form and S-form groups presented a significantly higher percentage of patients with white blood cell counts10 x 10(9)/l (P0.05). The S-form cases displayed a significantly higher number of cases with M3v microgranular features (P = 0.005) and CD34 expression (P0.0001). There were no differences between the three isoforms in complete remission (CR) rate (overall CR 90%), but the 3-year disease-free survival was lower for V-form cases than it was for L- and S-form cases (62% vs. 94% and 89%, P = 0.056). We conclude that the V-form and S-form types are associated with some negative prognostic features at diagnosis. However, our data were only able to demonstrate an association with adverse prognosis in the V-form type and, moreover, as the number of cases was limited, needs to be confirmed in large, uniformly treated series.

We report on a series of Spanish patients with acute lymphoblastic leukaemia in whom the t(12;21) [TEL/AML1] translocation could not be identified with two sensitive techniques: reverse transcript-polymerase chain reaction (RT-PCR) and fluorescence in-situ hybridization (FISH). 101 cases were analysed: 38 children (29 B-cell precursor; nine T-cell precursor) and 63 adults (48 B-cell precursor; 15 T-cell precursor). Specific RT-PCR to amplify the TEL/AML1 fusion transcript was negative in all 101 cases. Moreover, all 38 paediatric samples were also negative by interphase FISH analysis for the presence of the TEL/AML1 fusion. These results suggest the existence of geographic/race variations in the genotype of acute lymphoblastic leukaemia (ALL).

In the present article, two new types of PML/RARA junctions are described. Both were identified in diagnostic samples from two t(15;17)(q22;q21)-positive acute promyelocytic leukemia (APL) patients who failed to achieve complete remission. By using different sets of primers, reverse transcriptase polymerase chain reaction (RT-PCR) of PML/RARA junctions showed atypical larger bands compared with those generated from the three classical PML breakpoints already described. Sequence analysis of the fusion region of the amplified cDNAs allowed us to determine the specificity of these fragments in both patients. This analysis showed two new hybrid transcripts that were 53 and 306 base pairs (bp) longer than that expressed by the NB4 cell line (PML breakpoint within intron 6), and are the result of the direct joining of RARA exon 3 with PML exon 7a (patient 2) or the 5' portion of PML exon 7b (patient 1), respectively. In patient 1, RT-PCR analysis of the reciprocal RARA/PML junction showed a smaller transcript than that expected in bcr1 cases, while in patient 2 no amplified fragment was obtained. Cytogenetic analysis and/or fluorescence in situ hybridization (FISH) showed that both patients had the t(15;17) translocation. The clinical and hematological profiles expressed by the two patients carrying these unexpected types of PML/RARA rearrangement did not differ significantly from that commonly seen in other APLs with the exception of the poor outcome. Genes Chromosomes Cancer 27:35-43, 2000.

The aims of the present study were: (a) to analyze the incidence of leukemia-associated phenotypes within patients with acute lymphoblastic leukemia; (b) to determine the frequency and the causes of phenotypic changes in relapse samples, and (c) to analyze the clinical impact of the immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia (ALL). A total of 72 ALL cases (T lineage: 16, B lineage: 56 patients) were included in the study and a total of 323 complete remission follow-up bone marrow samples were analyzed; 78% of the cases displayed leukemia-associated phenotypes at diagnosis (T-lineage ALL: 100%, and B-lineage ALL: 72%). The overall distribution of the different types of leukemia-associated phenotypes was: lineage “infidelity” 56%, asynchronous antigen expression 23%, antigen over-expression 18%, tissue-restricted phenotypes 15%, and “rate” phenotypes 5%. The incidence of DNA aneuploidy was 15%, most of the cases corresponding to B-lineage leukemias. From these patients 75% displayed more than one aberrant phenotype. The presence of more than one blast cell population was detected in 28% of the cases.

In this study the incidence of DNA aneuploidy in a large series of untreated multiple myeloma (MM) patients was assessed in order to determine its clinical and prognostic significance. A total of 156 MM patients were included in the study. DNA measurements were performed in all cases at diagnosis using two different flow cytometry methods: (1) propidium iodide (PI) staining on isolated nuclei, and (2) CD38/PI double staining on whole cells. The DNA ploidy status was correlated with the most relevant clinical and haematological disease characteristics. From the 156 cases analysed, 91 (58%) were aneuploid (56% hyperdiploid and 2% hypodiploid). The correlation between the two techniques on the detection of DNA aneuploidy was excellent, although CD38/PI double staining would be preferable in cases with5% of DNA aneuploid plasma cells (PC). Upon comparing the clinical and haematological disease characteristics of hyperdiploid versus diploid cases, the former group was characterized by a lower age, reduced incidence of anaemia, lower beta 2M levels, higher proliferative activity within the residual normal haemopoietic cells, increased expression of CD56 antigen in PC, and higher proportion of PB CD4+ T cells. In contrast, diploid cases had a higher expression of the CD10, CD20 and CD15 antigens and greater numbers of PB CD56+CD3- NK cells (P0.05). Circulating PC were identified in six cases, all being diploid. Overall survival was significantly longer in hyperdiploid compared to diploid MM (P = 0.02). These results show that over 50% of MM patients are aneuploid, almost all of them being hyperdiploid. This characteristic is associated with better prognosis.

Patients with multiple myeloma (MM) experience osteolytic bone destruction resulting in skeletal-related events (SREs: pathologic fracture, spinal cord compression, surgery or radiation to bone). Severity of bone destruction correlates with significant morbidity and is associated with decreased survival. Osteoclast activity is enhanced in MM and is primarily regulated by RANK Ligand (RANKL) and osteoprotegerin. Denosumab is a fully human monoclonal antibody which binds RANKL, preventing activation of RANK and inhibiting osteoclast differentiation, activation and survival. Bone resorption and cancer-induced bone destruction are thereby reduced. Denosumab was superior to zoledronic acid (ZA) in 3 large randomized trials in patients with breast, prostate, or solid tumors-only analysis. Denosumab is approved for the prevention of SREs in patients with bone metastases from solid tumors in many regions worldwide. The current trial will evaluate the efficacy and safety of denosumab compared with ZA in preventing skeletal complications in patients with MM. The primary endpoint will determine if denosumab is non-inferior to ZA in prevention of the first on-study SRE. If denosumab is found to be non-inferior to ZA, superiority in time to first on-study SRE and time to first-and-subsequent SRE will be assessed as secondary endpoints. Patients will be randomly assigned 1:1 to receive subcutaneous (SC) denosumab 120 mg and intravenous (IV) placebo or IV ZA 4 mg and SC placebo every 4 weeks (Q4W). Randomization will be stratified by planned autologous transplantation (yes/no), anti-myeloma therapy (novel therapy-based vs non-novel therapy-based), disease stage (International Staging System 1, 2, or 3), previous SRE (yes/no), and region (Japan vs other). Daily supplements of calcium ≥500 mg and vitamin D ≥400 IU will be strongly recommended. Patient screening and enrollment is underway. In this event-driven study, data cutoff for the primary efficacy analysis is planned for when approximately 800 patients are anticipated to experience an on-study SRE. The trial is sponsored by Amgen Inc. and registered with ClinicalTrials.gov (NCT01345019). Disclosure A. Palumbo: Advisory Board Member: Celgene and Janssen-Cilag Honoraria: Celgene, Janssen-Cilag, Bristol-Myers Squibb, Millenium, Merck, Onyx and Amgen Consultancy: Celgene and Janssen-Cilag B.G. Durie: Advisory Board Member: Celgene Corporation, Millenium Pharmaceuticals, and Onyx Pharmaceuticals N. Raje: Advisory Board Member: Celgene, Millenium, Amgen Corporate-sponsored Research: Eli-Lilly, Acetylon R. Garcia Sanz: Honoraria: Amgen, Jansen Cilag, Celgene, Novartis O. Sezer: Advisory Board Member: Amgen, Janssen E. Terpos: Advisory Board Member: Amgen, Novartis W. Willenbacher: Advisory Board Member: Amgen Y. Qian: Employee and Stock Ownership: Amgen A. Balakumaran: Employee and Stock Ownership: Amgen All other authors have declared no conflicts of interest.

Introduction Plasma Cell Leukemia (PCL) is characterized by a high number of plasma cells in peripheral blood (>2 × 109/L) and more than 20% of circulating plasmocytes on differential count. Primary PCL or secondary form, after previous multiple myeloma (MM), represent the most aggressive forms of clonal gammapathy with very poor outcome from both conventional chemotherapy and autologous or allogeneic transplant. For this reasons new effective treatment approaches are required. The proteasome inhibitor Bortezomib has been shown to be effective in advanced MM with a rapid response but few reports have been published regarding its potential role on PCL. We report here the clinical results of a serie of PCL patients that received proteasome inhibitor, Bortezomib (Velcade®), as induction therapy. Patients and Clinical Results We describe 10 patients with PCL treated with Bortezomib. Median age was 58 y (30–70). 4 cases were primary, de novoPCL and 6 cases were secondary forms of relapsed or progressive MM. Patients with secondary PCL had received a median of 3 previous lines of therapy including autologous transplantation (5 cases). Bortezomib at standard dose iv: 1.3 mg/m2 days 1,4, 8,11 every 21 days were used alone in 2 patients and in combination with high dose of Dexamethasone +/− Adryamicin in the rest. 3 cases recived the BDD scheme (Bortezomib iv, Pegylated Doxorrubicin iv and oral Dexamethasone). 9 patients were evaluable for drug efficacy with 7 cases showing some grade of favourable response with the first cycle, including 3 cases of CR. 4 patients have died: 1 during first weeks and 3 for progression, 2–3 months after initial response. 3 cases received allogeneic transplant with related donor and reduced conditioning and are alive 12, 14 and 30 months postransplant. The median follow up since PCL diagnosis of 6 alive patients is 14 months (3–39 months). 3 patients received maintenance therapy that included low dose thalidomide. Grade 2 thrombopenia, neutropenia and gastrointestinal symptoms were the main secondary toxicity. No complicated tumoral lysis syndrome were observed. Conclusions and Comments Bortezomib has been shown to be effective by several mechanisms in patients with MM resistant to multiple previous treatments and as induction therapy alone or in combination with other drugs. The favourable responses observed in these cases of PCL; including de novo patients, and the biological ex vivo results in PCL cell lines, suggest the potential efficacy of this agent combined with dexamethasone and doxorubicin. Although more experience is necessary these data support future trials with Bortezomib combined with other drugs for example BDD scheme in the induction treatment of PCL. The role of allogeneic intensification therapy has also to be investigated in young patients after control of PCL with this approach. For maintenance of response, the role of this drug or other new drugs as thalidomide or lenalidomide has to be investigated.

The outcome of patients (pts) with WM, a rare B-cell malignancy, varies widely. The international community has convened a panel to develop a prognostically meaningful staging system for survival after treatment initiation. Toward establishing IPSS, we performed multivariate analyses with bootstrap validation in a series of 587 pts (median age 67, range: 28 to 95, M/F ratio: 1.7) diagnosed between September 1979 and December 2001. Diagnostic and treatment criteria fulfilled recommendations of the 2nd international WM workshop. Front-line treatment was initiated at diagnosis in 69% and 4 mo to 164 mo later in the remaining pts. Criteria for initiation of therapy included cytopenia, constitutional symptoms, organomegaly, hyperviscosity, IgM-related disorders, which pertained to 51%, 44%, 35%, 31% and 13%, respectively. Treatment regimens comprised alkylating agents, fludarabine and rituximab in 369, 195 and 23 subjects, respectively. Baseline parameters included age>65 yr in 57%, Hb≤11.5 g/dL in 65%, platelet count ≤100 x109/L in 9%, granulocyte count ≤1.5 x109/L in 9%, B2M >3 mg/L in 56%, albumin ≤3.5 g/dL in 36% and monoclonal protein >7.0 g/dL in 7%. With a median follow-up of 64 mo (range, 6 mo–182 mo), the median survival after treatment initiation was 87 mo (95%CI 79–103) regardless of the type of therapy applied (p=0.3). Using results of univariate survival analyses, recursive partitioning and martingale residuals analyses, 7 adverse characteristics for inclusion in multivariate analyses were identified during an expert meeting: age >65 yr, platelet count ≤ 100x109/L, B2M >3 mg/L, M-protein >7.0 g/dL, granulocytes ≤1.5 x109/L, Hb ≤11.5 g/dL and albumin ≤3.5 g/dL. The Cox proportional hazard model with stepwise selection selected the first 6 covariates. Bootstrap resampling (500 replicates) validated the selection of the first 4 covariates in at least 80% of the replicates. Selection of at least one of the last 2 covariates in more than 80% of the replicates indicated a correlation between these 2 covariates, and validated the inclusion of Hb only. Using the combination of age, Hb, platelet count, B2M and M-protein, low risk was defined by the presence of ≤1 adverse characteristic except age, high risk by the presence of >2 adverse characteristics; the remaining patients with 2 adverse characteristics or age >65yr had intermediate risk, comprising 27%, 38% and 35% of patients with 5-yr survival rates of 87%, 68% and 36% (p

Introduction and objectives : Plitidepsin is a cyclic depsipeptide originally isolated from the marine tunicate, Aplidium albicans. It appears very potent against multiple myeloma (MM) cells. Specifically, it was observed that it was active against a broad panel of 35 human MM cell lines, which included MM cells resistant to conventional anti-MM agents and novel agents (i.e. thalidomide, bortezomib). Plitidepsin also induced cell death in primary MM tumor cells freshly isolated from patients resistant to thalidomide or its analogs and/or proteasome inhibitors. Phase I has been completed exploring 4 different schedules of administration. Muscle and liver (transaminases and/or alkaline phosphatase) toxicities were the main DLTs. Hematological toxicity was not observed at the recommended dose. The aim of this trial was to explore the activity of plitidepsin in patients with previously treated refractory/relapsed MM. Material and Methods: This is a non-randomized two-stage Phase II, multicenter, clinical and pharmacokinetic trial, with Aplidin® (APLD) 5 mg/m2 as a 3 h intravenous infusion every 2 weeks, with antiemetic and antihistaminic prophylaxis. In the first stage 16 patients evaluable for efficacy were included. At least one response was requested in order to proceed with the second stage, in which a total of 37 patients will be included. Results: Between June’04 and June’05, 19 patients have been enrolled and data are available for 18 patient (7 men and 11 women, median age was 65.7y, range 48–82). Patients were previously relapsed/refractory. Prior treatments included: stem cell transplantation 64.7%, thalidomide 35.28% and bortezomib 17.64%. The median previous chemotherapy lines received were 3, range 1–6. The APLD median number of cycles received 4, range 1–16. Thirteen patients are currently evaluable for efficacy. One patient (7.7%) achieved a partial response (PR) with a 70% reduction in M-component lasting 8 months. Stable disease lasting between 3–5 months was observed in 3 patients (23.0%). In 2 patients (15.38%) a stabilization lasting 2.5 months was stated and the remaining 7 patients (53.8%) progressed. NCI-CTC grade 3–4 related toxicities were reported for n=17 patients and were mainly fatigue in 2 patients (11.8%), myalgia in 1 patient (5.9%), elevation of CPK in 1 patient (5.9%) and transient transaminases increase in 9 patients (52.9%). Significant hematological toxicity did not occurred in spite of 2 patients included and treated with thrombopenia grade 3–4 and 2 patient with neutropenia grade 3. Conclusions: First stage data shows that APLD presents hints of activity in patients with refractory/relapsed MM, with acceptable toxicity profile, thus meeting the criteria for proceeding to second stage recruitment. The absence of significant hematological toxicity is a well known feature of this drug and is being confirmed in this trial.

Acute promyelocytic leukaemia (APL) is characterised by the t(15;17) leading to the fusion of the PML gene with the retinoic acid receptor a (RARa) gene. APL is associated with favourable prognosis, however, approximately 15–20% of patients ultimately relapse. With regards to pre-treatment characteristics, is now clear that long-established prognostic factors such as age and leukocyte count have a major impact on outcome. However, none of these parameters is robust enough, in order to adjust treatments strategies according to the risk of relapse. The quantification of PML-RARa fusion gene transcripts (leukemia-specific chimeric mRNA) can add important prognostic information useful for risk group stratification. However, few clinical studies have been carried out and there are discrepancies concerning the prognostic significance of the quantification of fusion gene transcripts of newly diagnosed patients. In this study we tested the PML- RARa fusion gene transcripts level in 126 newly diagnosed patients with t (15;17) in order to determine their correlation with disease characteristics at presentation and to identify a subset of patients at high risk of relapse. The presence of the PML- RARa fusion gene transcripts was analysed from 1mg of RNA by real-time quantitative RT-PCR (RQ-PCR) based on TaqMan probes and the 7700 Sequence Detector, performed according to the “Europe Against Cancer Program” (Leukemia2003, 17:2323; 2318–2357). Results: In 121 out of 126 patients, evaluable data were obtained: 73 (60,3%) cases had bcr-1, 4 (3,3%) cases had bcr-2 and 44 (36,4%) cases had bcr-3 fusion types. The expression of the fusion gene was reported as the PML- RARa normalized quantities (NQ, number of PML- RARa copies per 1x104 ABL copies as gene control). The median of NQ was 3030 and the expression was highly variable (range of 826 to 9605). The NQ was not significantly associated with disease characteristic at diagnosis, such as age, percentage of blasts cells, fusion types and platelet counts. Nevertheless, patients with NQ above 3000 had lower white cell counts (7,4±12,1 vs 24,2±39,3x109/L, p=0,03). Therefore, the fusion transcript copy number per leukemic cell was higher in patients with lower leukocyte count. Moreover, in survival analysis, the NQ did not correlate with disease free survival or overall survival. Conclusions: Our data confirms the wide range of differences in expression activity between individual patients. The NQ did not correlate with the clinical and biological disease characteristics except for white cell counts. Neither did it correlate with principal prognostic parameters (response and survival).

Multiple myeloma (MM) remains as an incurable disease although new therapies can achieve a high rate of complete remissions (CR). Unfortunately, most patients ultimately relapse due to the persistence of minimal residual disease (MRD), and only a minority could be cured. Detection and quantification of these cells is an important tool for monitoring these patients and predicting a potential relapse. Here we analyze by RQ-PCR the MRD in MM patients achieving CR in order to classify them into different risk categories. MATERIAL AND METHODS: 38 MM patients uniformly treated according to the GEM-2000 (Spanish group for Myeloma) protocol, and that have achieved CR following PBSCT were included in the study. 22 were IgG, 9 IgA, 6 B-J and 1 non-secretory (κ/λ 21/16). 27 were male & 11 female with a median age of 58 (range 48–65). Bone marrow samples obtained at diagnosis and 3 months after transplant were analyzed. Complete (VDJH) and incomplete (DJH) Ig rearrangements were amplified with the Biomed-2 strategy (Leukemia2003;17:2257). PCR clonal products were sequenced on an ABI Prism 377 Sequence detector. VH, DH and JH segments were identified by comparing with germinal sequences on V-Base and BLAST databases. An ASO primer at the N-region was designed for each patient with the OLIGO 6.0 software. RQ-PCR was then performed on an ABI Prism 7700 using the ASO specific forward primer, a JH reverse intronic primer (JH1–6) and a TaqMan probe (JH1,2,4,5, JH3 or JH6) to amplify the patient specific rearrangement. Sample quality and quantity was controlled evaluating the standard curve of the albumin gene amplification. MRD was calculated according to ΔCT method. RESULTS: In 14 out of the cases included in the study, MRD investigation was not possible because the N-region was not longer enough to design the ASO primer (n=3), poor quality in the diagnostic sample to obtain the standard curve (n=8) or low plasma cell infiltration at diagnosis to obtain correct dilutions (n=3). The remaining 24 patients were classified into different risk groups according to the MRD level obtained 3 months after transplantation with a cut-off point of 0.01% tumor cells. Thus, progression free survival (PFS) was longer in those patients with MRD< 10−4 (p=0.03, figure 1A). By contrast, upon comparing the impact on PFS of immofixation (IFX) in these 24 patients that were in CR (defined by conventional electrophoresis criteria), it was observed that patients with IFX (−) didn’t showed a different outcome from those IFX (+) (figure 1B). CONCLUSION: In summary, although RQ-PCR is a labor and time-consuming technique, it is an useful tool for monitoring MRD in MM. The level of 10−4 can contribute to classify patients into 2 groups (high and low MRD) with different risk of relapse that can be used to design specific therapies.

It is assumed that patients with primary refractory myeloma are the most likely to benefit from early HDT. In four series the CR rate was between 8 and 40% and the overall survival ranged from 4 to 6 years. However‚ the number of patients with progressive disease vs those with “stable disease” was not given in published series. The aim of our study was to investigate the efficacy in terms of response up-grading and survival of early HDT in patients with primary refractory myeloma. From October 1999 to December 2003 patients with MM younger than 70 years were given 6 courses of VBMCP/VBAD chemotherapy. Patients with refractory disease were scheduled to receive a tandem transplant‚ the first procedure intensified with busulphan-12 mg/kg-/melphalan-140 or melphalan-200 and the second with the CVB -cyclophosphamide‚ etoposide and BCNU- or with a dose-reduced intensity “allo” conditioned with fludarabine/melphalan-140‚ depending on sibling donor availability. Response and progression were defined according to the EBMT criteria. Forty-nine patients with primary refractory disease were identified. Twenty patients showed progression after their initial chemotherapy while 29 patients had “non-responding‚ non-progressive disease”. Eighty percent of the patients achieved some degree of response after the first autologous transplant (CR or near-CR: 8%‚ PR: 56%‚ MR: 16%) while 10% did not respond and 10% died from the procedure. Twenty-four patients were given a second transplant (autologous: 17‚ allogeneic: 7). Forty-six percent of the 17 patients who received a second autologous transplant upgraded their response (CR: 6%‚ PR: 17%‚ MR: 23%) while 41% had progressive disease or “no-change” and 13% died from the procedure. Three of the seven patients who underwent a dose-reduced intensity “allo” responded (2 CR‚ 1 PR) while two had progressive disease and two died from transplant-related complications. The median survival of the whole series of 49 patients was 32 months. Patients who had progressive disease after the initial chemotherapy had a significantly shorter survival than those who showed “non-responsive‚ non-progressive disease” (median 21 months vs. not reached‚ p=0.003). Finally‚ patients with “non-responding‚ non-progressive disease” had similar survival than those with chemosensitive disease intensified with HDT in the GEM trial. Conclusions. A high-dose therapy approach in patients with primary refractory myeloma results in a high overall response‚ but the CR rate is low‚ patients with progressive disease to the initial chemotherapy have short survival despite intensive therapy‚ and patients with “non-responding‚ non-progressive disease” have similar survival than chemosensitive patients. Whether the good outcome of the latter patients is mainly due the effect of HDT or to the natural history of a more indolent disease remains to be determined.

8021 Background: It has been claimed that patients with primary refractory myeloma benefit from early HDT. However, the reported series have 2 shortcomings: 1) patients with “unresponsive-progressive disease” vs those “non-responding-non progressing” were not analyzed separately and 2) some included patients had MR or even PR according to the EBMT criteria. Aim: Response and survival after early HDT in the two populations of truly primary refractory MM Methods: From Oct 1999 to Dec 2004, 829 patients with MM received 6 cycles of VBMCP/VBAD and at least one transplant. 79 of the 829 patients were refractory to VBMCP/VBAD. These resistant patients were scheduled to receive a tandem transplant, the first with Bu-12/MEL-140 or MEL-200 and the second “autologous” with CVB or “allo- RIC” (if donor available) with Fluda/MEL-140. Response and progression were defined by the EBMT criteria. Results: 32 of the 79 primary refractory patients had progressive disease under the initial chemotherapy while 47 were “unresponsive, non-progressive”. The prognostic features, including cytogenetics, were similar in both groups. 70% of the patients responded to the first HDT (CR/nCR 8%, PR 48%, MR 13%). 37 patients were given a second transplant (26 “auto”, 11 “allo”). 41% who received a second “auto” up-graded their response (CR 9%, PR 14%, MR 18%) while 42% who underwent “allo-RIC” increased their response (CR 28%, PR 14%). Median survival of the whole series was 3 years. Patients progressing while on therapy had a shorter survival than the “no-change” group (median 2 yrs vs not reached, p=0.00002). Finally, the 47 “non-responsive, non-progressors” patients had similar survival than the 718 with chemosensitive disease intensified with HDT. Conclusions: 1) HDT in patients with primary refractory MM results in a low CR rate, 2) patients progressing while on initial therapy have a short survival despite the intensive approach and 3) patients with “non-responding, non-progressive” disease have similar survival than chemosensitive patients. Whether this good outcome is due to the impact of HDT or to the natural history of a more indolent disease remains to be determined. No significant financial relationships to disclose.