ALBERT

Description: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous category of lymphoid tumors that comprises different clinical forms not fully recognized in the WHO classification. In this regard, extranodal (EN) DLBCLs have particular clinicobiological features and outcome, sometimes related to the specific site where the lymphoma arises. Nowadays, rituximab plus chemotherapy (CT) is the gold-standard in the treatment of DLBCL. However, the superiority of rituximab-CT (R-CT) over CT alone has not been addressed for all the clinical subsets of the disease and, in fact, the clinical role of the new therapies might be different for primary nodal or EN DLBCLs. The aim of this study was to assess the impact of rituximab in patients suffering from nodal or EN DLBCL. Two-hundred and thirty non-immunocompromised patients (112M/118F; median age, 61 years) diagnosed with CD20+DLBCL in a single institution between 1997 and 2006 (five years before and after establishing R-CT as the standard treatment in DLBCL) and treated with adriamycin-containing regimens were the subject of the present study. The series included 148 primary nodal and 82 EN DLBCL. Patients with primary CNS lymphoma were excluded and lymphomas arising at Waldeyer ring were considered as nodal DLBCL. The main EN sites were GI (n=26), bone (n=13), soft tissue (n=13), lung/pleura (n=9), liver (n=9), and other (n=12). Main clinico-biological and evolutive variables were analyzed. One hundred nineteen patients received only CT and 111 R-CT. Eighty-seven cases with available information were assigned to germinal center B-cell-like (GCB) (41%) or non-GCB (59%) groups according to the Hans method (Blood2004;103:275–82) based on CD10, BCL6 and MUM1 expression. Main initial features, including the primary nodal or EN origin, international prognostic index (IPI), and GCB/non-GCB categories were similar for CT and R-CT groups. No correlation was observed between the GCB/non-GCB groups and the primary site of the tumor, although nodal lymphomas more frequently expressed MUM1 than EN (69% vs. 31%, respectively; p=0.01). CR rate and 5-year overall survival (OS) according to the treatment arm (CT vs. R-CT) is detailed for the whole series and for the nodal and EN groups in the table and OS curves depicted in the figure. In the whole series, variables predicting poor OS in the multivariate analysis were high-risk IPI (RR 2.5; p

Description: The outcome of patients (pts) with PTCL receiving conventional therapy is dismal. Because of this, there is an increasing interest to investigate intensive treatments in these patients. The aim of this study was to analyze the toxicity, response and outcome of a phase II trial that includes high-dose chemotherapy (CT) plus ASCT as first-line treatment for pts with PTCL. Forty-one pts (30M/11F; median age: 47 yrs.) diagnosed with PTCL (excluding cutaneous and anaplastic ALK+), in stages II-IV and 0.1). Four-year OS was 57% (95%CI: 31–83%) and 71% (95%CI: 37–100%), respectively (p〉0.1). In summary, in this series of patients with PTCL a relatively high CR rate was obtained with high-dose CHOP/ESHAP followed by ASCT. Toxicity was manageable. The contribution of ASCT to pts outcome is debatable because of the absence of significant differences in OS and EFS of patients in CR transplanted vs. those not transplanted. Novel strategies aimed at increasing the CR rate in these patients warrant investigation.

Description: The outcome of patients (pts) with PTCL receiving conventional therapy is dismal. Because of this, there is an increasing interest to investigate intensive treatments in these pts. The aim of the study is to analyze the interim results, in terms of toxicity, response and outcome, of a phase II trial that includes high-dose chemotherapy (CT) plus ASCT as 1st-line treatment for pts with PTCL. Thirty-four pts (23M/11F; median age: 47 yrs.) diagnosed with PTCL (excluding cutaneous and anaplastic ALK+), in stages II–IV and ≤65 yrs, who have already finished planned therapy, are the subject of this analysis. Nine pts (26%) presented with primary extranodal disease, 24 (71%) were in stage IV, and 13 (38%) had bone marrow involvement. Fifty percent of the pts had high/intermediate or high-risk IPI, whereas 53% were in the groups 3 or 4 according to the Italian Index for PTCL. Pts received intensive CT (3 courses of high-dose CHOP [cyclophosphamide 2000 mg/m2 day 1, adriamycin 90 mg/m2 day 1, vincristine 2 mg day 1, prednisone 60 mg/m2/day, days 1 to 5, with mesnum and G-CSF], alternating with 3 courses of standard ESHAP). Responders (CR or PR) were submitted to ASCT. Median follow-up of surviving pts was 3.9 yrs (range, 0.5–7.6). Twenty four pts (71%) received the planned 6 courses of CT. Response rate after CT was: CR, 12 cases (35%); PR, 8 (23%); failure, 14 (42%), including one pt who died because of sepsis. Hematological toxicity of CT mainly consisted of neutropenia (grades 3–4 in 87 and 62% after high-dose CHOP and ESHAP, respectively) and thrombocytopenia (grades 3–4 in 63 and 68%, respectively). Severe infection requiring hospitalization was observed in 38 and 15% of courses of high-dose CHOP or ESHAP, respectively. Only 14 of the 20 candidates (70% of all candidates and 41% of all pts) received ASCT due to lack of stem-cell mobilization (3 cases), previous toxicity (2) and pt decision (1). No differences in the outcome were seen among these 20 pts according to whether or not they could eventually receive ASCT. No major toxicity was observed after ASCT. The overall response after the whole treatment was: CR, 14 cases (41%), PR, 5 (15%), failure, 15 (44%). Two of 14 pts in CR and the 5 pts in PR eventually progressed. Four-year failure-free survival (FFS) was 30%, whereas 4-year disease-free survival for pts achieving CR was 63%. Nineteen pts have died during follow-up, with a 4-yr overall survival (OS) of 38% (95%CI: 21–55%). Most patients died because of PTCL progression, but 2 pts died in CR due to a secondary acute lymphoblastic leukemia and to a lung cancer, respectively. IPI and Italian Index for PTCL were able to predict FFS and OS. In summary, in this series of patients with PTCL a relatively high CR rate was obtained with high-dose CHOP/ESHAP followed by ASCT. Toxicity was manageable. However, the prognosis of patients with PTCL, particularly of those not achieving CR, is still very unfavorable. Figure Figure

Description: Abstract 3247 Background: Ph+ALL is a rare subgroup of pediatric ALL with very high risk of treatment failure and reported long-term overall survival (OS) of only 30–40%. Standard treatment includes allogeneic SCT in first complete remission (CR1). Imatinib has been lately incorporated to first-line treatment regimens. In a recent study with pediatric Ph+ALL patients, imatinib given continuously at a dose of 340 mg/m2/day in combination with chemotherapy resulted in a significant improvement in early outcome (Schultz KR et al, J Clin Oncol 2009). Aim: to describe the Spanish Cooperative Group SHOP results in Ph+ALL with imatinib given continuously at an intermediate dose in combination with intensive chemotherapy and compare the toxicities and outcome of patients treated in the imatinib cohort -SHOP 05- with those treated without imatinib -SHOP 94 and SHOP 99- (historical controls). Patients and Methods: children with Ph+ALL aged 1–18 years treated in 39 institutions in Spain were enrolled in three consecutive SHOP-ALL trials (SHOP 94/SHOP 99/SHOP 05) from February-1994 to April-2010. Imatinib, at a dose of 260 mg/m2/d was added to an intensive chemotherapy regimen since day-15 of induction treatment in SHOP 05. Allogeneic SCT from matched sibling donor (MSD) or matched unrelated donor (MUD) was scheduled in CR1 in all trials. Toxicities were graded following WHO criteria. Survival analysis was determined by the Kaplan-Meier estimate. Results: a total of 47 patients out of 1436 pediatric ALL were enrolled during the study period (SHOP 94: 8, SHOP 99: 23, SHOP 05: 16). Four patients enrolled in SHOP 99 trial were excluded from the analysis for protocol violation (imatinib treatment in CR1 in a pre-imatinib protocol). Among 43 evaluable patients, 16 were treated with continuous intermediate-dose imatinib as per SHOP 05 protocol whereas 27 received a similar chemotherapy backbone treatment without imatinib. There were 22 boys (51.2%) and median age was 6.8 years, range 1.2–15. Median WBC (109/L) was 41, range 2.8–481.2. Three patients (7.3%) had CNS involvement at diagnosis. Main clinical characteristics at presentation were comparable among the three trials. All patients in the imatinib cohort (SHOP 05) and 24 out of 27 patients in the non-imatinib cohort (SHOP 94/99) achieved CR1 after induction treatment, with 3 refractory patients in the non-imatinib cohort. Imatinib in combination with intensive chemotherapy was in general well tolerated. There were no induction deaths in our series. One patient in the imatinib group died in CR1 due to an infection prior to SCT. There were more grade III-IV transaminase (ALT) elevations in consolidation in patients receiving imatinib (7 out of 16) than in those not receiving it (2 out of 19). Hepatic failure or bilirrubin elevation greater than grade I were not observed in these cases and ALT elevations were transient without imatinib discontinuation or dose-adjustment. Hematological toxicities were similar in patients treated with or without imatinib, and no unexpected treatment delay was observed. No pleural effusions were reported. Thirty patients underwent an allogeneic SCT in first CR, 17 out of 27 (63%) patients in trials SHOP 94/99 (non-imatinib cohort) and 13 out of 16 (81%) patients in trial SHOP 05 (imatinib cohort). Median time to SCT was 6.4 months (range 3–14.8). Transplant related mortality was 19.5%. Ten patients relapsed (non imatinib cohort: 9, imatinib cohort: 1), at a median time of 17.6 months (range 8.3–35.2). Median follow-up of the trials SHOP 94/99 (pre-imatinib) and SHOP 05 (imatinib) was 113 and 30 months respectively. Event-free survival (EFS) at 30 months was 33.3% (+/−9.1%) and 75.2 % (+/−12.6) respectively (p = 0.032) and overall survival (OS) at 30 months was 38.46% (+/−9.5%) and 84.6 (+/−10%) respectively (p=0.029). Conclusion: Intermediate dose of imatinib (260 mg/m2/day) given concomitantly with chemotherapy and followed by allogeneic SCT (MSD or MUD) had an acceptable toxicity profile and markedly improved early EFS and OS in pediatric Ph+ALL. Longer follow-up is needed to assess the effect of imatinib on long-term EFS. Disclosures: Off Label Use: Imatinib is a tyrosine-kinase inhibitor approved for the treatment of chronic myeloid leukemia. Its use in acute lymphoblastic leukemia Philadelphia positive has not been approved yet although preliminar results show promising results.

Description: Introduction Despite the remarkable improvement in survival of children with acute leukemia in the last decades, some patients still have a poor outcome. FMS-like tyrosine Kinase 3 (FLT3) is a tyrosine-kinase receptor with a key role in hematopoiesis whose mutations and overexpression have emerged as negative prognostic biomarkers in childhood leukemia. Infant leukemias (those diagnosed at age50 chromosomes) and of MLL-rearranged cases presenting either as ALL or acute myeloblastic leukemia (AML), as these are the subgroups with highest expression of FLT3 reported in the literature, although other leukemia subtypes were also represented. The mRNA expression levels of FLT3, nucleoside transporters (NT) (hENT1, hENT2, hCNT1, hCNT3) and Ara-C ME, dCK and CNII, were quantified using real-time PCR and analyzed with the 2-ΔΔCt method, with non-neoplastic samples used as controls for the relative quantification. Direct nucleoside and Ara-C uptake measurements were performed using [5,6-3H]-nucleosides. The role FLT3 might play in the expression of NT and ME genes as well as on the activity of Ara-C transporters in these cell lines was addressed by repressing FLT3 function with its specific inhibitor PKC412 (sold only for research purposes). Results We included 56 patients (68% males) diagnosed with acute leukemia. The median age was 5.3 years (range 0-16.2), with 3 cases of infant leukemia. Fifty cases (89%) were precursor B-ALL (24 hyperdiploid cases, 5 MLL rearranged, 3 BCR-ABL+, 4 E2A-PBX1+, 5 TEL-AML1+, 9 other subtypes), 5 cases were AML (4 MLL positive cases and one case with mutated FLT3) and one case was a T-ALL harboring FLT3 mutation. As expected, the FLT3 expression was higher in cases with ALL and MLL rearrangement and, to a less extent, in ALL with hyperdiploidy. Interestingly, a significant positive correlation was found between FLT3 and hENT1 expression (mRNA levels) with all patient samples (figure 1). hENT1 expression and cytarabine-mediated uptake was significantly repressed when MV4-11 and SEM cell lines were exposed to the FLT3 inhibitor PKC412 (figures 2 & 3) without affecting hENT2, CNT1 and CNT3 expression and activity. Conclusions Our results show a strong correlation between FLT3 and the Ara-C transporter hENT1 in pediatric leukemia patients. This observation was consistent with the in vitro evidence that FLT3 inhibition resulted in hENT1 repression and down-regulation of Ara-C uptake in leukemic derived cell lines. Taken together, our data suggest that FLT3 regulates hENT1, thereby modulating the ability of cancer cells to incorporate Ara-C and promote its cytotoxic action. As FLT3 inhibitors are currently being tested as mono or combined therapy with Ara-C in several clinical trials, based upon our observations we suggest that a better schedule design might eventually be needed when dealing with treatments involving FLT3 inhibitors and Ara-C, thereby improving the outcome of this subset of patients. Disclosures: Off Label Use: The presentation include the results of in vitro studies with the FLT3 inhibitor PKC412. This drug was only used for in vitro studies, exclusively for research purposes.

Description: Acute myeloid leukemia with multilineage dysplasia (AML-MD) is recognized as a major AML category in the WHO classification, closely related to myelodysplasia and associated with poor prognosis. The biological background of this entity, however, has not been extensively assessed. In this context, we analyzed the gene expression profile in a series of patients with AML-MD from our institution. First, the transcriptional signature of diagnostic samples from 19 patients with AML-MD (age: 71, 30–93; 8M/11F) was analyzed, including 11 cases of intermediate-risk cytogenetics and 8 adverse karyotype samples. In a second step, we selected from the previous analysis those AML-MD patients with normal karyotype (NK AML-MD, n=8) and compared their genomic profile with 11 additional samples corresponding to normal karyotype AML without underlying multilineage dysplasia (NK AML). Overall gene expression was examined with oligonucleotide HGU133 Plus 2.0 arrays (Affymetrix), and gene expression measures were normalized using RMA methodology from the Affy package (Bioconductor project). Unsupervised two-dimensional cluster analysis of highly variable genes was performed with the dChip v1.3 software. In addition, supervised analysis to identify genes with significant differential expression was done with the Limma package (Bioconductor), which employs Bayesian statistics adjusted for multiple testing. Unsupervised analysis among AML-MD patients identified two major groups, mainly clustered according to cytogenetics: Group 1 (n=10) contained 9 patients with intermediate-risk cytogenetics whereas Group 2 (n=9) showed predominance of high-risk karyotype (78%; p=0.006). Supervised analysis allowed the identification of a cluster of 92 genes differentially expressed according to cytogenetics category, such as several ribosomal constituents and genes involved in translation (RPS20, EIF3S3, LOC400055), which were overexpressed in intermediate-risk AML-MD, or genes involved in the immune response (FCGR3A-3B, IL1R2, PLXNC1, FCAR, CLEC4D-4E, TNFRSF10C, C5R1), found to be highly expressed in AML-MD with high-risk cytogenetics. In a further analysis, gene expression profiling of NK AML-MD was compared to NK AML. Interestingly, unsupervised analysis revealed a clear distinction between both subgroups, with 1861 genes differentially expressed. Moreover, a subset of 34 genes, selected according to a high index of variable expression (log fold change〉1.0), showed a characteristic pattern in normal karyotype AML-MD. Among these, transcription factor TAL1, RNA-binding protein MSI2, and erythrocyte membrane protein RHAG were found to be overexpressed in NK AML-MD, while other genes such as RASSF4 and PIK3CD, involved in Ras signaling, or the transcription factor CEBPD were significantly underexpressed in this AML category. In conclusion, analysis of gene expression profile of AML-MD supports the biological heterogeneity of this AML category, partially associated with underlying cytogenetic abnormalities. Moreover, the striking distinctive profile observed in NK AML-MD suggests that multilineage dysplasia is associated to a specific transcriptional signature, distinguishable from normal cytogenetics AML lacking dysplastic features.

Description: Key Points In intermediate-risk AML, effect of FLT3 burden is modulated by NPM1 mutation, especially in patients with a low ratio. Combined evaluation of NPM1 mutation and FLT3-ITD burden might contribute to identify patients who benefit from early allogeneic HSCT.

Description: Relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) has a dismal outcome, and no effective targeted immunotherapies for T-ALL exist. The extension of chimeric antigen receptor (CAR) T cells (CARTs) to T-ALL remains challenging because the shared expression of target antigens between CARTs and T-ALL blasts leads to CART fratricide. CD1a is exclusively expressed in cortical T-ALL (coT-ALL), a major subset of T-ALL, and retained at relapse. This article reports that the expression of CD1a is mainly restricted to developing cortical thymocytes, and neither CD34+ progenitors nor T cells express CD1a during ontogeny, confining the risk of on-target/off-tumor toxicity. We thus developed and preclinically validated a CD1a-specific CAR with robust and specific cytotoxicity in vitro and antileukemic activity in vivo in xenograft models of coT-ALL, using both cell lines and coT-ALL patient–derived primary blasts. CD1a-CARTs are fratricide resistant, persist long term in vivo (retaining antileukemic activity in re-challenge experiments), and respond to viral antigens. Our data support the therapeutic and safe use of fratricide-resistant CD1a-CARTs for relapsed/refractory coT-ALL.

Description: B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric cancer, and high-hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD is an initiating oncogenic event affiliated to childhood B-ALL, the mitotic and chromosomal defects associated to HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated to chromosome alignment defects at the metaphase plate leading to robust chromosome segregation defects and non-modal karyotypes. Mechanistically, biochemical, functional and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness and mis-localization of the chromosome passenger complex proteins Aurora B Kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and impaired spindle assembly checkpoint (SAC) thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated to defective condensin complex, AURKB and SAC.

Description: Post-remission therapy in patients with acute myeloid leukemia (AML) is assigned according to the predictable biological risk of the disease, mainly based on cytogenetics. Nonetheless, optimal post-remission strategy for the intermediate-risk subtype, given the prognostic heterogeneity of this category, is currently undefined. Analysis of potentially relevant molecular features within this subgroup might contribute to clarify the role of autologous stem cell transplantation (autoHSCT) in these patients. Thirty seven patients (age: 53, 15–66; 51% female) diagnosed with intermediate-risk de novo AML during the period 1994–2006 who received an autoHSCT in first complete response were included in the study. Pre-transplant therapy was similar in all patients, consisting of standard induction chemotherapy (ICE, n=8, IDICE, n=29) and one cycle of high-dose ara-C-based consolidation chemotherapy. Internal tandem duplication of flt-3 (flt-3 ITD) and exon 12 NPM1 mutations were studied by either PCR or RT-PCR following standard methods. Gene expression profiling was examined in 28 patients with oligonucleotide HGU133 Plus 2.0 arrays (Affymetrix). Gene expression measures were normalized using RMA methodology (Affy package), and dChip v1.3 and Limma software (Bioconductor) were used for unsupervised and supervised analyses. In order to identify genes with prognostic value, a supervised analysis based on patients’ outcome (relapsed patients vs. long-term responders, i.e. 〉2-year duration) was performed. The combined results of NPM mutation and flt-3 ITD defined three subgroups of patients with different outcome: group 1 (n=12), constituted by patients with mutated NPM1 without flt-3 ITD; group 2 (n=20), which included patients with neither NPM1 mutation or flt-3 ITD; and group 3 (n=5), defined by flt-3 ITD regardless NPM1 mutational status. Thus, 5-year survival of these 3 subgroups of patients was 91%±9%, 52%±12%, and 20%±18%, respectively (p=0.02; see figure). Preliminary results of multiple gene profile comparisons between subgroups of patients with different outcome disclosed a cluster of genes with differential expression. Thus, in the most significant balanced comparison, 1238 genes were found to vary significantly in the unsupervised analysis, and 109 differentially expressed genes were identified in the supervised analysis. Interestingly, overexpression of genes such as TNF, RETN, CFLAR, SLC16A7, ENG, CD48, PLCR1, and SULTB1 correlated with a high relapse risk, whereas increased expression of YY1, FBXL12 and EXOSC6 were associated with a favorable outcome. In conclusion, presence of NPM1 mutation and flt-3 ITD are strong predictors of the outcome after autoHSCT in patients with intermediate-risk AML. Furthermore, genome-wide analysis may contribute to further define gene clusters with prognostic significance in patients with cytogenetically intermediate-risk AML receiving autoHSCT as consolidation therapy. Figure Figure