ALBERT

Description: Blastic plasmacytoid dendritic cell neoplasm is a clonal disease derived from precursors of plasmacytoid dendritic cells (pDC). It is a rare neoplasm involving the skin which may or may not be associated from the outset with a leukemic component. The disease invariably progresses to aggressive leukemic dissemination, leading to a differential diagnosis with acute leukemia. In 2004, we set up a French network to recruit biological data at diagnosis. Diagnosis was according to recommendations (Swerdlow et al, 2008), with, in addition, a mandatory panel of pDC markers (Garnache-Ottou et al, 2009) detected by flow cytometry or by immunohistochemistry on infiltrated blood, bone marrow or cutaneous lesions. In total, 109 cases of BPDCN were included in 35 hospitals (2000-2013). BPDCN is more prevalent in men (sex ratio 4.4/1) and in elderly subjects (median age: 63 years; 7 patients were

Description: Introduction Mantle-cell lymphoma (MCL) is of poor prognosis, with a median survival of about 5 years. Besides the t(11;14) translocation, several secondary genetic abnormalities have been shown to correlate with prognosis. However, most studies have analysed patients with heterogeneous treatment, mostly with anthracyclin-based regimens. In 2004, the European MCL Network started the randomized MCL Younger trial comparing R-CHOP followed by high-dose radiochemotherapy and autologous stem cell transplantation (ASCT) versus alternating R-CHOP/R-DHAP followed by a high-dose cytarabine conditioning regimen and ASCT in previously untreated MCL stage II-IV patients up to the age of 65y. The R-CHOP/R-DHAP arm showed improved time to treatment failure (TTF) and, potentially, overall survival (OS) (Hermine et al., ASH 2010, ASH 2012). Our aim was to revisit the prognostic value of some gene copy number alterations (GCNA) in this randomized trial and to determine whether high-dose cytarabine could counteract some of those factors. Methods The inclusion criteria for this biological study were: confirmed histological diagnosis of MCL, the availability of diagnostic tumor DNA and complete clinical data. When no frozen biopsy was available, peripheral samples with more than 50% tumor cells were considered eligible for GCNA analysis. CDKN1B, CDK2, and MDM2 were analyzed using quantitative multiplex PCR of short fluorescent fragments (QMPSF) (Jardin et al., BJH 2009), 6q25-q26, CDK4, and the 13q14 locus were analyzed by multiplex ligation-dependent probe amplification (MLPA) (MRC-Holland CLL kit), and MYC, CDKN2A, ATM, RB1 and TP53 were assessed by both methods. The analyses of the prognostic value of GCNA was adjusted for clinical prognostic factors summarized in the quantitative MIPI score (age, performance status, LDH, and WBC). The rate of proliferating tumor cells (Ki-67 index) was centrally assessed by the reference pathologists of the European MCL Pathology Panel according to published guidelines (Klapper et al., J Hematopathol 2009). Outcome variables were TTF from treatment start to stable disease, progression, or death from any cause, and OS from trial registration to death from any cause. Results Of 135 patients fulfilling the inclusion criteria (median age 56 years), 49%, 26%, and 25% of patients were of low, intermediate, and high MIPI risk . The most frequent amplification involved MYC (18%), whereas the most frequent deletion involved the 13q14 locus (36%), including RB1 in 26%. As expected, CDKN2A and TP53 deletions were frequently found (25% and 22%, respectively). ATM alterations mostly consist of deletion (25%), but amplification was found in 3 of 129patients. The frequencies of GCNA did not differ according to the type of sample analyzed i.e. tumor biopsies (n=79) vs. high tumor load peripheral blood or bone marrow samples (n=56). The Ki-67 index was higher in patients with CDKN2A or RB1 deletion compared to patients without, but was not different between patients with or without TP53 deletion. Only TP53 gene status was associated with MCL cytology, with more frequent deletion in blastoid forms (4/8) than in classic MCL (11/81, 14%). In univariable analyses, deletions of CDKN2A, 13q14, RB1, CDKN1B, and TP53 were associated with shorter TTF and OS, whereas GCNA of 6q25-q26, MYC, ATM, CDK2, CDK4, and MDM2 were not prognostic. In multivariable analyses, adjusting for MIPI score, CDKN2A and TP53 deletions showed independent prognostic impact with hazard ratios of 2.4 (p=0.001) and 2.3 (p=0.004) for TTF and 2.3 (p=0.007) and 2.4 (p=0.007) for OS. This effect was observed in both treatment arms (Figure 1). In addition, there was an interacting effect of CDKN2A (p16) deletion and TP53 deletion on TTF (p=0.004). Conclusions The introduction of high-dose cytarabine in first-line treatment of younger MCL patients did not erase the adverse prognostic value of TP53 and CDKN2A deletions observed with previous regimens. Moreover, our study identified a small patient group of very bad prognosis which could benefit of more aggressive regimens or new targeted drugs combination. Figure 1: TTF (left) and OS (right) according to CDKN2A/TP53 deletion status in patients of the R-CHOP/R-DHAP study arm; nom=not deleted, del=deleted Figure 1:. TTF (left) and OS (right) according to CDKN2A/TP53 deletion status in patients of the R-CHOP/R-DHAP study arm; nom=not deleted, del=deleted Figure 2 Figure 2. Disclosures Feugier: Roche: Honoraria. Haioun:Roche, Celgene, Takeda, Pfizer, Janssen,: Honoraria.

Description: Introduction: In a recent French FRALLE versus LALA comparison, we have demonstrated a large benefit in outcome when adolescents and young adults were treated in a pediatric rather than an adult ALL protocol (Boissel JCO 2003). Similar provocative results have been observed in three other pediatric versus adult ALL studies (Stock ASH 2000, de Bont Leukemia 2004, Testi ASH 2004). One explanation may be the larger amounts of steroids, vincristine (VCR), and L-asparaginase (L-aspa) administered in pediatric patients. The GRAALL-2003 study was thus designed to offer a pediatric approach in adults with Ph-negative ALL until 60 years of age. Methods: Treatment included a 5-drug induction, high dose-intensity consolidation blocks, delayed intensification, and 2-year maintenance. The comparison with the former LALA-94 protocol showed a 8.6-fold, 3.7-fold, and 16-fold increase in cumulative doses of prednisone, VCR, and L-aspa, respectively. One difference with childhood ALL therapy remained indication of allogeneic SCT in first CR which was offered to all patients with high-risk factor and a donor. In addition, induction was reinforced with a hyper-cyclophosphamide sequence (HyperC) in case of poor early response (cortico- and/or chemoresistant ALL). In the present report, 212 GRAALL-2003 patients with Philadelphia-negative ALL aged 15–55 years with a median follow-up of 18 months were compared to 712 patients previously treated in the LALA-94 trial. Results: Cohorts were comparable in terms of prognostic factors. CR rate was significantly higher in GRAALL patients (93 vs 88%, P=0.02) due to a reduction in resistant disease (0.5 vs 8%, P

Description: TLX1 is a homeodomain transcription factor generally associated with a favorable outcome in T-cell acute lymphoblastic leukemia (T-ALL). However, the molecular mechanisms of TLX1 deregulation remain unclear and various transcript levels in the absence of 10q24 abnormalities have been reported. A reproducible and accurate delineation of TLX1+ T-ALL will be necessary for proper therapeutic stratification. We have studied 264 unselected T-ALLs (171 adults and 93 children) and show that T-ALLs expressing high levels of TLX1 (n = 35, 13%), defined as a real-time quantitative polymerase chain reaction (RQ-PCR) level of TLX1 greater than 1.00 ABL, form a homogeneous oncogenic group, based on their uniform stage of maturation arrest and oncogenetic and transcriptional profiles. Furthermore, TLX1-high T-ALLs harbor molecular TLX1 locus abnormalities in the majority (31/33), a proportion largely underestimated by standard karyotypic screening. T-ALLs expressing TLX1 at lower levels (n = 57, 22%) do not share these characteristics. Prognostic analysis within the adult LALA94 and GRAALL03 prospective protocols demonstrate a better event-free survival (P = .035) and a marked trend for longer overall survival (P = .059) for TLX1-high T-ALLs, while the expression of lower levels of TLX1 does not impact on prognosis. We propose that TLX1+ T-ALLs be defined as cases expressing TLX1/ABL ratios greater than 1 and/or demonstrating TLX1 rearrangement. Therapeutic modification should be considered for those patients.

Description: During the years of 2005 to 2008, the MILE (Microarray Innovations in LEukemia) study research program was performed in 11 laboratories across three continents: 7 from the European Leukemia Network (ELN, WP13), 3 from the US and 1 in Singapore. The first stage was designed as biomarker discovery phase to generate whole-genome gene expression profiles (GEP) from recognized categories of clinically relevant leukemias and myelodysplastic syndromes (MDS). These were C1: mature B-ALL with t(8;14), C2: pro-B-ALL with t(11q23)/MLL, C3: c-ALL/pre-B-ALL with t(9;22), C4: T-ALL, C5: ALL with t(12;21), C6: ALL with t(1;19), C7: ALL with hyperdiploid karyotype, C8: c-ALL/pre-B-ALL without specific genetic abnormalities, C9: AML with t(8;21), C10: AML with t(15;17), C11: AML with inv(16)/t(16;16), C12: AML with t(11q23)/MLL, C13: AML with normal karyotype or other abnormalities, C14: AML with complex aberrant karyotype, C15: CLL, C16: CML, C17: MDS, and C18: non-leukemic and healthy bone marrow samples as controls and were compared to conventional diagnostic assays (“Gold Standard”). Data from the completed MILE Stage I included 2143 retrospectively collected adult and pediatric samples tested with HG-U133 Plus 2.0 microarrays (Affymetrix). In total only 47 analyses (2.1%) failed technical quality criteria. Cross-validation accuracy (average of three 30-fold cross-validations) of the final 2096 MILE Stage I samples was 92.1% concordant with the center-specific “Gold Standard” diagnosis (average call rate 99.4%). In nine classes the sensitivity was ≥94.3%: C2, C3, C4, C5, C9, C10, C11, C15, and C16. Lower sensitivities were observed for C7, C8, C14, and C17; which can largely be explained by the biological heterogeneity and non-standardized “Gold Standard” definitions for these entities. Yet, it is notable that all these classes showed specificities above 98.1%. In order to assess the clinical utility of microarray-based diagnostics a prospective Stage II was subsequently performed using a customized microarray representing 1480 probe sets. Overall, 1156 high quality GEP have been generated in MILE Stage II and represent an independent and blinded test set for the algorithms developed. A focused classification scheme aimed at accurately addressing only acute leukemias resulted in a 95.5% median sensitivity and a 99.5% median specificity for the 14 classes included in the classifier (C1 – C14, n=696). Lower accuracies were observed for the interface of C7–C8 in ALL, as well as C12 and C14 in AML. Interestingly, during the process of discrepant results analyses, it was observed that for 7.5% (n=52) of acute leukemias microarray results were correctly diagnosing samples as compared to the initial “Gold Standard” diagnoses entered into the study database, either because of erroneous entries into case report forms (24%) or subsequent re-testing of left-over material following the suggested diagnosis from the microarray (76%). In addition, predicted accuracies for CLL, CML and MDS in Stage II were 99.2%, 95.2%, and 81.5%, respectively. In conclusion, the MILE research study confirms in a final cohort of 3252 patients that microarrays accurately classify acute and chronic leukemia samples into known diagnostic and prognostic sub-categories. This final report underlines that the standardized method of gene expression profiling with low technical failure rate and simplified standard operating procedures may improve current “Gold Standards” as an adjunct to conventional diagnostic algorithms and potentially offers a reliable diagnostic/prognostic tool for many patients who don’t have access to a state-of-the-art “Gold Standard” workup. Our gene expression database, intended to be submitted to the public domain, will further contribute to research that aims to elucidate the molecular understanding of leukemias.

Description: PAX5 is a transcription factor central to B-cell differentiation, frequently mutated in pediatric B-ALL (38.9% in 192 B-ALL, Nature,2007;446:758). PAX5 mutations consist in whole or partial gene deletions or amplifications, fusion gene or point mutations. Adult B-ALL differs in many regards from pediatric B-ALL as demonstrated by differences in prognosis and genetic abnormalities (ETV6-RUNX1 almost absent from adult; BCR-ABL1 rare in children). We screened the occurrence of PAX5 mutations in a series of 119 adult patients with B-lineage ALL prospectively treated in the GRAALL-2003 (pediatric-like protocol for BCR-ABL1 negative patients) or GRAAPH-2003 (imatinib-containing protocol for BCR-ABL1 positive patients), with a median follow-up of 667 days. PAX5 copy number was evaluated using quantitative PCR of each exon performed in hexaplicate (10 exons). PAX5 point mutations of the paired and transactivation domains (exons 2, 3, 7, 8 and 9) were evaluated by sequence analysis. PAX5 rearrangement was evaluated by caryotype and FISH analysis. Globally, PAX5 mutations were identified in 35 cases (30%). Isolated complete deletion of 1 allele was found in 13 cases (11%). Hypomorphic PAX5 alleles were detected in 22 cases (19%) consisting either of partial deletion (13 cases), partial amplification (1), point mutations (7 cases, frequently associated with complete deletion of the remaining allele in 5 cases) or fusion gene (1 PAX5-ELN). In addition, 2 cases of whole PAX5 amplification were found (2%). The remaining 82 cases had two normal PAX5 alleles (68%). BCR-ABL1 fusion gene was significantly associated with PAX5 mutations (Pearson Chi2, p=0.045) occurring in 40% of patients with PAX5 mutations and 18.3% without. PAX5 mutations were rare in the early stages of B-ALL as estimated by the immunological EGIL classification (only 2/35 PAX5 mutant cases [5.7%] vs. 16/82 [19.5%] in normal PAX5 B-ALL cases blocked at the B-I stage). Frequencies of CD10+ and CD20+ blasts were significantly higher in PAX5 mutant cases (Mann-Whitney, p=0.022 and p=0.038, respectively). As PAX5 is essential to the immunoglobulin heavy chain DHJH to VHDHJH transition, we analyzed IGH rearrangement. No difference was detected regarding the frequency of IGH rearrangement (78% in normal vs. 74% in mutants) or the rearranged VH. Survival was not significantly different between the two groups of patients (log rank, p=0.45). In conclusion, PAX5 mutations were a frequent event in adult B-ALL, associated with BCR-ABL1 fusion gene but were not associated with a significant clinical evolution.

Description: Background: In recent years, oncogenic understanding of T-ALL has led to the identification of multiple molecular markers. However, each molecular abnormality accounts for a small proportion of cases and risk stratification at diagnosis still relies on age, clinical presentation, and early response to therapy. NOTCH1 and/or FBXW7 mutations have been recently reported to be a recurrent abnormality in T-ALL, both leading to activation of the NOTCH pathway. Studies in pediatric series have suggested a favorable outcome for NOTCH1 mutated T-ALL, but this has not been evaluated in large series of adult cases. Furthermore, FBXW7 prognostic impact remains unknown in both populations. Methods: In order to evaluate the incidence of these mutations and their prognostic impact in adults, we performed a retrospective analysis of 141 patients (median age, 28 years) with T-ALL treated within the LALA-94 (N=87) or the more recent GRAALL-2003 (N=54) French trials. These patients were representative of the overall population since their outcome did not differ from the overall T-ALL cases treated in either LALA-94 (estimated 3-year OS, 41%) or GRAALL-2003 (estimated 3-year OS, 66%) trial. Furthermore, the patients from the LALA-94 and the GRAALL-2003 trials did not differ with respect to sex ratio, age, WBC, and initial complete remission rate (92% and 98%, respectively). Exons 26 (HD N-terminal), 27 (HD C-terminal), 28 (juxtamembrane domain) and 34 (transactivation domain TAD and the PEST domain) of NOTCH1 and exons 9, 10 and 12 of WD40 domain of FBXW7 were sequenced. Results: We identified 101 cases with NOTCH1 and/or FBXW7 mutations (72%) and 40 wild type (WT) samples (28%). NOTCH1 was mutated in 88 patients (59 HD only, 15 HD+PEST, 9 PEST only, 5 other). FBXW7 was mutated in 34 patients, alone in 13 cases or in association with NOTCH1 mutations in 21 cases. There was no significant correlation between NOTCH1 and/or FBXW7 mutations and immunophenotypic or oncogenetic features. There was a trend for a higher WBC and more frequent CNS involvement in patients demonstrating WT NOTCH1 and FBXW7. Similarly, high-risk MRC/ECOG criteria (age〉35y and/or WBC〉100G/L) were found in 56% of patients from the mutated subgroup versus 73% in the WT subgroup (P=0.06). The prognostic impact of NOTCH1 mutations alone did not reach statistical significance on multivariate analysis (P=0.09). On the other hand, multivariate analysis showed that the GRAALL-2003 trial and the presence of NOTCH1 and/or FBXW7 mutations were the only factors associated with a longer EFS (P=0.001 and 0.035, respectively). Median EFS was 36 months for patients with NOTCH1 and/or FBXW7 mutations versus 17 months for WT patients. Conclusions: These data demonstrate that NOTCH pathway activation by either NOTCH1 or FBXW7 mutation identifies a large group of T-ALL adult patients (72%) with a favorable outcome that could be used for treatment stratification.

Description: From January 2000 to July 2006, 580 BCR-ABL negative patients with HR/VHR-ALL (200 T-ALL and 380 BCP-ALL (age≥10 or WBC≥50 or CNS+ or MLL-R) were included in the FRALLE 2000-BT trials. Induction regimen is prednisone (PRED) prephase + IT MTX, VCR, L-Aspa, DNR 120mg/m2 cumulated dose or DNR 160mg/m2 + cyclophosphamide 1g/m2 (T-ALL and D21 M2M3 marrow or MLL-R BCP-ALL). MRD at EOI is quantitatively determined by DNA-based PCR for Ig/TCR rearrangements either competitive PCR with GeneScan analysis or RQ-PCR with clone specific primers (sensitivity ranges 0.5×10−3–10−3, and 10−3–10−5, respectively). MRD status at EOI are available for 425 out of 552 CR (77%). MRD results are reported as negative, positive

Description: Ph+ ALL accounts for approximately one third of ALL cases in patients aged 55 years or older. The median survival of older Ph+ ALL patients is one year, with practically no long-term survivor (Blood, 98, Supp1 p319a, 2001). Imatinib has demonstrated remarkable, although transient, activity in relapsed and refractory Ph+ ALL, which prompted the GRAALL to implement a treatment protocol associating imatinib and chemotherapy in previously untreated elderly patients: ALL patients aged 55 years or older are treated with steroids during one week and Ph+ve cases are then offered a specific therapy including an induction treatment with steroids, cyclophosphamide, daunorubicin and vincristine, followed, irrespective of response to induction chemotherapy, by imatinib, 600 mg daily, combined with intermittent steroids during 2 months. Patients in complete response are then given 10 blocks of alternating chemotherapy, including 2 additional two-month blocks of imatinib, for a total treatment duration of 2 years. Therapy of occult central nervous system leukemia includes 5 intrathecal injections of methotrexate and cranial irradiation. The study is intended to include 30 patients and its main objective is to improve overall one-year survival to 70%. Results are compared with those obtained in 21 Ph+ ALL elderly patients treated according to our previous protocol. Since January 2003, 21 patients aged 58 to 78 years (median: 64.7 years) were included in the AFR09 protocol. Their median follow-up is 3 months. 15/19 patients are in complete response after induction chemotherapy vs 6/21 in the historical controls given similar induction regimen but with no steroids before chemotherapy (p=0.002). The projected overall survival is 95% at 9 months vs 62% in the control group (p=0.08, log-rank test). The 9-month projected event-free survival is 83% vs 10% (p

Description: Patients with T-cell acute lymphoblastic leukemias (T-ALLs) within the Leucémies Aiguës Lymphoblastiques de l'Adulte-94 (LALA-94) prospective trial were treated with a 4-drug per 4-week induction, with intermediate-dose cytarabine and mitoxantrone salvage treatment for patients not achieving complete remission (CR) in 1 course. Only the latter received allografts, if possible, thus providing an informative setting for assessing early response. Representative patients with T-ALL (91 patients) were classified into surface T-cell receptor (TCR)–expressing T-ALL patients (TCRαβ+ or TCRγδ+), pre-αβ T-ALL patients (cTCRβ+, TCR–), and immature (IM) cTCRβ–, TCR– T-ALL patients; 81 patients underwent genotyping for SIL-TAL1, CALM-AF10, HOX11, and HOX11L2. Overall, CR was obtained in 81 (89%) patients; relapse rate was 62% at 4 years and overall survival (OS) rate was 38%. CR rate was significantly lower in IM T-ALL patients after 1 course (45% vs 87%; P 〈 .001) and after salvage (74% vs 97%; P = .002), with the latter inducing a higher rate of CR (9 [64%] of 14) than initial induction. Once CR was obtained, cumulative relapse rates were similar for IM, pre-αβ, and TCR+ T-ALL patients (P = .51), but were higher in HOX11L2 (83%) and SIL-TAL1 (82%) T-ALL patients compared with other genetic subgroups (48%; P = .05). This was associated with an inferior OS for HOX11L2 T-ALLs (13% vs 47% in HOX11L2-T-ALLs; P = .009). The majority of patients with HOX11 T-ALL underwent allografting, predominantly in second CR, but were not associated with a superior OS. Both TCR and genotypic stratification can therefore contribute to risk-adapted management of adult T-ALLs.