Gilles Salles

Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients

Whereas the chimeric type I anti-CD20 Ab rituximab has improved outcomes for patients with B-cell malignancies significantly, many patients with non-Hodgkin lymphoma (NHL) remain incurable. Obinutuzumab (GA101) is a glycoengineered, humanized anti-CD20 type II Ab that has demonstrated superior activity against type I Abs in vitro and in preclinical studies. In the present study, we evaluated the safety, efficacy, and pharmacokinetics of GA101 in a phase 1 study of 21 patients with heavily pretreated, relapsed, or refractory CD20(+) indolent NHL. Patients received GA101 in a dose-escalating fashion (3 per cohort, range 50/100-1200/2000 mg) for 8 × 21-day cycles. The majority of adverse events (AEs) were grades 1 and 2 (114 of 132 total AEs). Seven patients reported a total of 18 grade 3 or 4 AEs. Infusion-related reactions were the most common AE, with most occurring during the first infusion and resolving with appropriate management. Three patients experienced grade 3 or 4 drug-related infusion-related reactions. The best overall response was 43%, with 5 complete responses and 4 partial responses. Data from this study suggest that GA101 was well tolerated and demonstrated encouraging activity in patients with previously treated NHL up to doses of 2000 mg. This trial is registered at www.clinicaltrials.gov as NCT00517530.

Next-Generation Sequencing in Diffuse Large B-Cell Lymphoma Highlights Molecular Divergence and Therapeutic Opportunities: a LYSA Study.

Purpose: Next-generation sequencing (NGS) has detailed the genomic characterization of diffuse large B-cell lymphoma (DLBCL) by identifying recurrent somatic mutations. We set out to design a clinically feasible NGS panel focusing on genes whose mutations hold potential therapeutic impact. Furthermore, for the first time, we evaluated the prognostic value of these mutations in prospective clinical trials. Experimental Design: A Lymphopanel was designed to identify mutations in 34 genes, selected according to literature and a whole exome sequencing study of relapsed/refractory DLBCL patients. The tumor DNA of 215 patients with CD20+de novo DLBCL in the prospective, multicenter, and randomized LNH-03B LYSA clinical trials was sequenced to deep, uniform coverage with the Lymphopanel. Cell-of-origin molecular classification was obtained through gene expression profiling with HGU133+2.0 Affymetrix GeneChip arrays. Results: The Lymphopanel was informative for 96% of patients. A clear depiction of DLBCL subtype molecular heterogeneity was uncovered with the Lymphopanel, confirming that activated B-cell–like (ABC), germinal center B-cell like (GCB), and primary mediastinal B-cell lymphoma (PMBL) are frequently affected by mutations in NF-κB, epigenetic, and JAK–STAT pathways, respectively. Novel truncating immunity pathway, ITPKB, MFHAS1, and XPO1 mutations were identified as highly enriched in PMBL. Notably, TNFAIP3 and GNA13 mutations in ABC patients treated with R-CHOP were associated with significantly less favorable prognoses. Conclusions: This study demonstrates the contribution of NGS with a consensus gene panel to personalized therapy in DLBCL, highlighting the molecular heterogeneity of subtypes and identifying somatic mutations with therapeutic and prognostic impact. Clin Cancer Res; 22(12); 2919–28. ©2016 AACR. See related commentary by Lim and Elenitoba-Johnson, p. 2829

Identification of an alternative CD20 transcript variant in B-cell malignancies coding for a novel protein associated to rituximab resistance

Human CD20 is a B-cell lineage-specific marker expressed by normal and leukemic B cells from the pre-B to the plasma-cell stages and is a target for rituximab (RTX) immunotherapy. A CD20 reverse transcriptase-polymerase chain reaction (PCR) on B-cell lines cDNA yielded a short PCR product (DeltaCD20) corresponding to a spliced mRNA transcript linking the exon 3 and exon 7 ends. We established here that this novel, alternatively spliced CD20 transcript is expressed and detectable at various levels in leukemic B cells, lymphoma B cells, in vivo tonsil- or in vitro CD40L-activated B cells, and Epstein-Barr virus (EBV)-transformed B cells, but not in resting CD19(+)- or CD20(+)-sorted B cells from peripheral blood or bone marrow of healthy donors. The truncated CD20 sequence is within the reading frame, codes a protein of 130 amino acids ( approximately 15-17 kDa) lacking large parts of the 4 transmembrane segments, suggesting that DeltaCD20 is a nonanchored membrane protein. We demonstrated the translation into a DeltaCD20 protein which is associated with the membrane CD20 protein and showed its involvement in RTX resistance. Study of patient samples before and after RTX resistance or escape confirms our in vitro findings.

High-dose cytarabine does not overcome the adverse prognostic value of CDKN2A and TP53 deletions in mantle cell lymphoma

We revisited the prognostic value of frequently detected somatic gene copy number alterations (CNAs) in mantle cell lymphoma (MCL) patients treated first line with immunochemotherapy and autologous stem cell transplantation (ASCT), with or without high-dose cytarabine, in the randomized European MCL Younger trial. DNA extracted from tumor material of 135 patients (median age, 56 years) was analyzed by multiplex ligation-dependent probe amplification and/or quantitative multiplex polymerase chain reaction of short fluorescent fragments. As expected, MYC (18%) was the more frequently gained, whereas RB1 (26%), ATM (25%), CDKN2A (p16) (25%), and TP53 (22%) were the more frequently deleted. Whether adjusted for MCL International Prognostic Index (MIPI) or not, deletions of RB1, CDKN2A, TP53, and CDKN1B were associated with shorter overall survival (OS), similarly in both treatment arms, whereas CNAs in MYC, ATM, CDK2, CDK4, and MDM2 had no prognostic value. Additive effects were seen for CDKN2A (hazard ratio, 2.3; P = .007, MIPI-adjusted) and TP53 deletions (hazard ratio, 2.4; P = .007), reflected in a dismal outcome with simultaneous deletions (median OS, 1.8 years) compared with single deletions (median OS, 4.3 and 5.1 years) or without these deletions (median OS, 7 years), again similarly in both treatment arms. The additive prognostic effects of CDKN2A and TP53 deletions were independent of the Ki-67 index. Despite immunochemotherapy, high-dose cytarabine, and ASCT, younger MCL patients with deletions of CDKN2A (p16) and TP53 show an unfavorable prognosis and are candidates for alternative therapeutic strategies. This trial was registered at www.clinicaltrials.gov as #NCT00209222.

Telomeric damage in early stage of chronic lymphocytic leukemia correlates with shelterin dysregulation

Cells of B-cell chronic lymphocytic leukemia (B-CLL) are characterized by short telomeres despite a low proliferative index. Because telomere length has been reported to be a valuable prognosis criteria, there is a great interest in a deep understanding of the origin and consequences of telomere dysfunction in this pathology. Cases of chromosome fusion involving extremely short telomeres have been reported at advanced stage. In the present study, we address the question of the existence of early telomere dysfunction during the B-CLL time course. In a series restricted to 23 newly diagnosed Binet stage A CLL patients compared with 12 healthy donors, we found a significant increase in recruitment of DNA-damage factors to telomeres showing telomere dysfunction in the early stage of the disease. Remarkably, the presence of dysfunctional telomeres did not correlate with telomere shortening or chromatin marks deregulation but with a down-regulation of 2 shelterin genes: ACD (coding for TPP1; P = .0464) and TINF2 (coding for TIN2; P = .0177). We propose that telomeric deprotection in the early step of CLL is not merely the consequence of telomere shortening but also of shelterin alteration.

Whole exome sequencing of relapsed/refractory patients expands the repertoire of somatic mutations in diffuse large B-cell lymphoma.

Despite the many efforts already spent to enumerate somatic mutations in diffuse large B‐cell lymphoma (DLBCL), previous whole‐genome and whole‐exome studies conducted on patients of mixed outcomes failed at characterizing the 30% of patients who will relapse or resist current immunochemotherapies. To address this issue, we performed whole‐exome sequencing of normal/tumoral DNA pairs in 14 relapsed/refractory (R/R) patients subclassified by full‐transcriptome arrays (six activated B‐cell like, three germinal center B‐cell like, and five primary mediastinal B‐cell lymphomas), from the LNH‐03 LYSA clinical trial program. Aside from well‐known DLBCL features, gene and pathway level recurrence analyses proposed several interesting leads including TBL1XR1 and activating mutations in IRF4 or in the insulin regulation pathway. Sequencing‐based copy number analysis defined 23 short recurrently altered regions involving genes such as REL, CDKN2A, HYAL2, and TP53. Moreover, it highlighted mutations in genes such as GNA13, CARD11, MFHAS1, and PCLO as associated with secondary variant allele amplification events. The five primary mediastinal B‐cell lymphomas (PMBL), while unexpected in a R/R cohort, showed a significantly higher mutation rate (P = 0.003) and provided many insights on this classical Hodgkin lymphoma related subtype. Novel genes such as XPO1, MFHAS1, and ITPKB were found particularly mutated, along with various cytokine‐based signaling pathways. Among these analyses, somatic events in the NF‐κB pathway were found preponderant in the three DLBCL subtypes, confirming its major implication in DLBCL aggressiveness and pinpointing several new candidate genes.

Immunoglobulin heavy chain/light chain pair measurement is associated with survival in diffuse large B-cell lymphoma

Abstract Elevated serum free light chains (FLCs) have been associated with an unfavorable prognosis in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to determine the clinical relevance of a quantitative assessment of intact circulating immunoglobulin (Ig), using serum Ig heavy chain/light chain pair (HLC) measurements in patients with DLBCL. FLC and HLC were measured in 409 serum samples of patients with DLBCL included in the LNH03-B clinical trial program of the Groupe d’Etudes des Lymphomes de l’Adulte (GELA). Patients with an abnormal IgMκ/IgMλ ratio or an abnormal FLC ratio more frequently displayed adverse clinical characteristics. Patients with abnormal IgMκ/IgMλ ratios had inferior progression-free survival (PFS) and overall survival (OS) as compared to patients with a normal ratio in the overall cohort (5-year PFS 44.9% vs. 69.3%, p = 0.0003 and 5-year OS 50.8% vs. 78.1%, p = 0.0003) and in the R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) cohort (5-year OS 43.5% vs. 70.3%, p = 0.003). In multivariate analysis, including elevated FLC/HLC and International Prognostic Index (IPI), an abnormal IgMκ/IgMλ ratio (hazard ratio [HR] = 1.54, 95% confidence interval [CI] 1.03–2.3, p = 0.03) remained predictive of shorter progression-free survival. Gene expression profile experiments and immunohistochemistry indicate that this measurement is at least partially related to tumor cell secretion. Both elevated serum FLCs and an abnormal IgMκ/IgMλ ratio are associated with unfavorable outcomes in patients with DLBCL treated by R-CHOP.

Prevalence of Anticentromere F Protein Autoantibodies in 347 Patients with Non-Hodgkin's Lymphoma

Abstract: An association between autoimmunity and hematological malignancies has been reported including the detection of antinuclear autoantibodies (ANAs) in patients suffering from non‐Hodgkins lymphoma (NHL), with a high prevalence of ANAs directed to components of the mitotic apparatus or the mitosis‐associated proteins. Previous studies have demonstrated that one of the targets of such ANAs could be the CENP‐F protein, especially in some carcinomas. The prevalence and specificity of anti‐CENP‐F autoantibodies (aAbs) thus were analyzed in 347 patients with different histological subgroups of NHL before any treatment of NHL, along with 150 controls. The detection of these aAbs was performed using two techniques: a radioimmunological assay (RIA) and an indirect immunofluorescence technique (IIF). Twenty‐five (7.2%) NHL patients and 2 (1.3%) control patients displayed anti‐CENP‐F aAbs using RIA. This difference between the two groups was found to be significant (P < 0.01), with a higher prevalence of aAbs in the follicular (13%) and in the marginal zone B and MALT (10.2%) lymphoma subgroups. By IIF, 10 (2.9%) patients with NHL displayed aAbs with a CENP‐F or CENP‐F‐like pattern, whereas none of the control group did. In conclusion, these data demonstrate that a significant incidence of anti‐CENP‐F aAbs is observed, before any treatment, in some histological subgroups of NHL patients. In addition to the usefulness of anti‐CENP‐F aAbs as a marker for some NHL subgroups, prospective studies may be important to evaluate the predictive value of anti‐CENP‐F aAbs for the development of carcinomas.

Biological and Clinical Relevance of Associated Genomic Alterations in MYD88 L265P and non-L265P–Mutated Diffuse Large B-Cell Lymphoma: Analysis of 361 Cases

Purpose: MYD88 mutations, notably the recurrent gain-of-function L265P variant, are a distinguishing feature of activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL), leading to constitutive NFκB pathway activation. The aim of this study was to examine the distinct genomic profiles of MYD88-mutant DLBCL, notably according to the presence of the L265P or other non-L265P MYD88 variants. Experimental Design: A cohort of 361 DLBCL cases (94 MYD88 mutant and 267 MYD88 wild-type) was submitted to next-generation sequencing (NGS) focusing on 34 genes to analyze associated mutations and copy number variations, as well as gene expression profiling, and clinical and prognostic analyses. Results: Importantly, we highlighted different genomic profiles for MYD88 L265P and MYD88 non-L265P–mutant DLBCL, shedding light on their divergent backgrounds. Clustering analysis also segregated subgroups according to associated genetic alterations among patients with the same MYD88 mutation. We showed that associated CD79B and MYD88 L265P mutations act synergistically to increase NFκB pathway activation, although the majority of MYD88 L265P–mutant cases harbors downstream NFκB alterations, which can predict BTK inhibitor resistance. Finally, although the MYD88 L265P variant was not an independent prognostic factor in ABC DLBCL, associated CD79B mutations significantly improved the survival of MYD88 L265P–mutant ABC DLBCL in our cohort. Conclusions: This study highlights the relative heterogeneity of MYD88-mutant DLBCL, adding to the fields knowledge of the theranostic importance of MYD88 mutations, but also of associated alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy. Clin Cancer Res; 23(9); 2232–44. ©2016 AACR.

Personalized risk prediction for event-free survival at 24 months in patients with diffuse large B-cell lymphoma.

We recently defined event‐free survival at 24 months (EFS24) as a clinically relevant outcome for patients with DLBCL. Patients who fail EFS24 have very poor overall survival, while those who achieve EFS24 have a subsequent overall survival equivalent to that of the age‐ and sex‐matched general population. Here, we develop and validate a clinical risk calculator (IPI24) for EFS24. Model building was performed on a discovery dataset of 1,348 patients with DLBCL and treated with anthracycline‐based immunochemotherapy. A multivariable model containing age, Ann Arbor stage, normalized serum LDH, ALC, ECOG performance status, bulky disease, and sex was identified. The model was then applied to an independent validation dataset of 1,177 DLBCL patients. The IPI24 score estimates the probability of failing to achieve the EFS24 endpoint for an individual patient. The IPI24 model showed superior discriminatory ability (c‐statistic = 0.671) in the validation dataset compared to the IPI (c‐statistic = 0.649) or the NCCN‐IPI (c‐statistic = 0.657). After recalibration of the model on the combined dataset, the median predicted probability of failing to achieve EFS24 was 36% (range, 12–88%), and the IPI24 showed an EFS24 gradient in all IPI groups. The IPI24 also identified a significant percentage of patients with high risk disease, with over 20% of patients having a 50% or higher risk of failing to achieve EFS24. The IPI24 provides an individual patient level probability of achieving the clinically relevant EFS24 endpoint. It can be used via electronic apps. Am. J. Hematol. 91:179–184, 2016.