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: Background Cytogenetic abnormalities are of key importance for predicting clinical course and response to therapy in patients with chronic lymphocytic leukemia (CLL). Trisomy 12 (tri12), the third most frequent chromosomal aberration in CLL patients (10-20%), is associated with an intermediate prognostic risk but represents a clinical heterogeneous entity. Recently, next generation sequencing have revealed recurrent mutations in genes that were unknown to be involved in CLL pathogenesis, including NOTCH1, MYD88, SF3B1, XPO1 and BIRC3. In patients harboring tri12, NOTCH1 mutations have been shown to be present in up to 25% of cases and to confer unfavorable outcome explaining in part the clinical heterogeneity of tri12 patients. To better understand the genetic basis and prognosis of tri12 patients, we performed a multicenter retrospective study combining extensive mutational and cytogenetic analysis. Methods Patients carrying tri12 were identified using fluorescence in situ hybridization (FISH) and/or chromosome banding (CB). Main clinical and biological characteristics were collected and included in univariate analysis of prognostic factors, comprising age, Binet stage (A vs. B-C), splenomegaly, lymphocyte doubling time (LDT), LDH, beta2microglobulin (B2M), CD38 expression, IGHV mutational status, percentage of interphase nuclei positive (INP) for tri12, additional FISH (del13q, del11q, del17p) or chromosomal aberrations and presence of complex karyotype (〉 2 CB abnormalities). Search for mutations was performed by Sanger direct sequencing for TP53 (exons 5-10), NOTCH1 (exon 34), MYD88 (exons 14-16), SF3B1 (exons 14-16) and XPO1 (exons 14-15). Primary and secondary endpoints were time to first treatment (TFT), response to therapy, time to next treatment (TNT) and overall survival (OS). Results The study population comprised a total of 177 untreated patients including 112 and 75 patients with stage A and B-C CLL, respectively. The median age at diagnosis was 62 years old (range, 31-87) and 33% of patients were female. B2M was superior to 4 mg/L in 30/92 (32%) patients and LDH elevated in 65%. CD38 expression was positive (〉30%) in 58% and IGHV status was unmutated in 60%. Among the whole study population, all patients were positive for tri12 by FISH and 158/165 by CB. Tri12 was associated by CB with tri19 in 21 patients (13.2%), tri18 in 12 patients (7.5%), tri3 in 1 patient (

Description: The benefit of radiotherapy (RT) following chemotherapy in limited-stage DLBCL remains controversial. Before the Rituximab era, 4 randomized trials have been reported with conflicting results (ECOG 1484 and SWOG 8736, GELA 93-1 and 93-4 studies). More recently, the German Unfolder study prematurely closed the R-CHOP without RT arm in bulky limited-stage DLBCL due to an excess of relapse. In 2005, we conducted a randomized trial in patients with non-bulky (defined by a tumor size 50% but a persistent positive FDG-PET) after C4, 2 additional cycles of R-CHOP followed by RT (even if not initially allocated) were recommended. The primary objective was EFS at one year after the last randomization, and secondary objectives were the impact of interim FDG-PET on EFS and the toxicity of RT. From May 2005 to December 2013, 313 patients were randomized and 301 patients are currently evaluable. Median age was 56 yr (20-75). There were 181 males and 120 females: 106 patients (35%) were older than 60 yr. Most patients had normal LDH (82%), PS=0 (80%), and no B symptoms (96% of cases). Modified IPI score was as follows: IPI =0 (n=170), IPI=1 (n=113), IPI=2 (n=16), IPI=3 (n=2). Main tumor sites were cervical (n=159), Waldeyer’s ring and sinus (n=36), inguinal (n=29), axillary (n=25), mediastinum (n=21). Extra-nodal sites were observed in 121 patients (40%). One hundred and fifty patients were randomized in the R-CHOP arm and 151 in the R-CHOP + RT arm. After 4 cycles, 253 patients (84%) were in CR and 43 in PR (14%). Three patients had stable disease. Thirty-four patients (79%) out of the 43 partial responders received 2 additional cycles of R-CHOP followed by RT (including 12 patients not initially allocated to RT arm). At the end of treatment, CR and PR rate were 94% and 3%, respectively. Seven (4%) out of the 151 patients randomized in the RT arm declined radiation. With a median follow-up of 51 months (2-110), there were 20 relapses: 12 in the R-CHOP arm and 8 in the R-CHOP+RT arm (p=ns). Sixteen patients died. Causes of death were as follows: relapses (n=9), toxic (n=1), secondary malignancies (n=3), unknown (n=3). Median time of relapse was 21 months (2-110 months). EFS and OS are not statistically different between the two arms. In an intent to treat analysis, 5y-EFS is 87% n the R-CHOP arm versus 91% in the R-CHOP + RT arm (HR=0.55), p=0.13, and 5yr-OS is 90% in the R-CHOP arm versus 95% in the R-CHOP + RT arm (HR=0.60), p=0.32. For patients in complete response after the 4 cycles of R-CHOP (84% of the patients), 5yr-EFS is 89% in the R-CHOP arm versus 91% in the R-CHOP + RT arm (HR=0.59), p=0.24. In this prospective study, the results demonstrate that in non-bulky limited-stage DLBCL, R-CHOP alone (4 to 6 cycles) induces very high CR rate with a very good overall survival and a very low relapse rate. With the current follow-up, the addition of radiotherapy is not significantly superior to R-CHOP alone and should be reserved to the minority of patients who do not reach CR after R-CHOP. Disclosures Gyan: Roche: Research Funding.

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: Microarray analysis can identify differentially expressed genes associated with distinct clinical and therapeutically relevant classes of both pediatric and adult leukemias. Recently, the MILE (Microarray Innovations in Leukemia) research study program has been launched in 10 centers: 7 from the European Leukemia Network (ELN, WP13) and 3 from the US. In this study, which will include 4,000 patients, the clinical accuracy of gene expression profiles of 16 acute and chronic leukemia subclasses, MDS, and non-leukemia as control will be assessed as compared to current routine diagnostic workup. Each center is trained on an identical microarray protocol and uses the same laboratory equipment, kits, and reagents for target preparation (Affymetrix HG-U133 Plus 2.0). First, the intra- and inter-laboratory comparability was investigated using 2 different cell line samples, MCF-7 and HEPG2, with different amounts of starting material (1 μg and 5 μg input for cDNA synthesis). Also, each center prepared in parallel total RNA and processed replicate samples from three leukemia pts (AML with t(8;21), CML, and CLL). We found a high reproducibility among the different centers: unsupervised analyses accordingly group the two different cell lines distinct from the three types of leukemia samples. In hierarchical clustering and principal component analysis the non-leukemia samples are clearly distinct from the leukemia samples and no clustering of the individual centers can be seen. Remarkably, for the replicates of the leukemia samples the squared correlation coefficients of gene expression range between 0.975 and 0.997 for CML, between 0.975 and 0.998 for CLL, and between 0.970 and 0.999 for the AML with t(8;21). Secondly, the samples were analyzed by a classification algorithm. The algorithm was trained on a database that contains gene expression profiles of 〉1,600 leukemia patients and cell lines and can distinguish 16 different classes of leukemia, MDS, and non-leukemia. Several methods are used to form linear classifiers for all 18 * (18 – 1)/2 = 153 class pairs. The average cross-validation accuracy is 91% or higher. Miscalls are predominantly seen in the distinction between MDS and AML with normal karyotype. The accuracy of resubstitution (application of the classifier to the data forming the classifier) is 100%. For the new data accurate predictions for the non-leukemia cell lines, AML with t(8;21), and CLL were observed. Interestingly, the CML in blast crisis is predicted as AML with other abnormalities. This may be due to the fact that the classifier was trained on CML in chronic phase only. In conclusion, for the first time an international multi-center research study demonstrates a very high reproducibility of microarray analyses performed at different centers for the same leukemia samples. This lays the foundation for an international clinical research initiative evaluating the application of microarrays in the diagnosis and classification of hematological malignancies.

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: 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: Purpose: The use of rituximab, a chimeric monoclonal antibody to CD20, has led to significant improvement in the treatment of B-cell non-Hodgkin's lymphoma and mature B-cell ALL. CD20 is expressed in 30 to 50% of adult BCP-ALL patients. Although some single arm studies suggested that adding rituximab to chemotherapy could improve the outcome of these patients, no randomized study has been reported so far. Methods: To evaluate the potential benefit of adding rituximab, we conducted a multicenter randomized trial comparing the pediatric-inspired GRAALL protocol to the same regimen plus rituximab, in patients aged 18-59 years old with newly diagnosed CD20-positive Ph-negative BCP-ALL enrolled in the GRAALL 2005 trial. CD20 positivity was defined as expression of CD20 in more than 20% of leukemia blasts. Rituximab (375 mg/m2) was given during induction (day 1 and 7), salvage reinduction when needed (day 1 and 7), consolidation blocks (6 infusions), late intensification (day 1 and 7) and first year of maintenance (6 infusions) for a total of 16 to 18 infusions. Allogeneic stem cell transplantation (SCT) was offered in first complete remission (CR) to patients with one or more conventional high-risk criteria and a donor. The primary study objective was event-free survival (EFS). A study sample size of 220 patients was estimated in order to detect a 20% gain in EFS at 2 years (two-sided test, power 85%, type 1 error 5%). A sensitivity analysis was performed after censoring patients allografted in first CR at transplant time. This trial was registered at http://www.clinicaltrials.gov as #NCT00327678. Results: From 2005 to 2014, 220 patients from 56 centers were randomized. Eleven patients had non-eligibility criteria (n=5 Ph+ ALL; n=3 CD20-negative ALL; n=1 HIV infection) or withdrew their consent (n=2) and were accordingly excluded from this modified ITT analysis that dealt with 209 patients (105 in the rituximab arm and 104 in the control arm). Median age was 40 years. Both randomization arms were well balanced for pretreatment characteristics including age, ECOG status, WBC, and central nervous system (CNS) involvement (6% of the whole cohort). After induction ± salvage reinduction, CR rate was 92% and 91% in rituximab and control arm, respectively. In patients who reached CR after first induction and were evaluated for Ig/TCR minimal residual disease level (MRD), the rates of patients with MRD

Description: Many somatic genetic abnormalities have been identified in T-cell acute lymphoblastic leukemia (T-ALL) but each individual abnormality accounts for a small proportion of cases; therapeutic stratification consequently still relies on classical clinical markers. NOTCH1 and/or FBXW7 mutations both lead to activation of the NOTCH1 pathway and are among the most frequent mutations in T-ALL. We screened 141 adult diagnostic T-ALL samples from patients treated on either the Lymphoblastic Acute Leukemia in Adults (LALA)-94 (n = 87) or the GRAALL-2003 (n = 54) trials. In 88 cases (62%) there were demonstrated NOTCH1 mutations (42% heterodimerization [HD], 10% HD+proline glutamate serine threonine [PEST], 6% PEST, 2% juxtamembrane mutations, 2% transactivation domain [TAD]) and 34 cases (24%) had FBXW7 mutations (21 cases had both NOTCH1 and FBXW7 mutations); 40 cases (28%) were wild type for both. There was no significant correlation between NOTCH1 and/or FBXW7 mutations and clinico-biologic features. Median event-free survival (EFS) and overall survival (OS) were 36 versus 17 months (P = .01) and not reached versus 32 months (P = .004) in patients with NOTCH1 and/or FBXW7 mutations versus other patients, respectively. Multivariate analysis showed that the presence of NOTCH1/FBXW7 mutations was an independent good prognostic factor for EFS and OS (P = .02 and P = .01, respectively). These data demonstrate that NOTCH1 pathway activation by either NOTCH1 or FBXW7 mutation identifies a large group of patients with a favorable outcome that could justify individual therapeutic stratification for T-ALL.