ALBERT

Description: Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B cell receptor immunoglobulins (BcR IG). Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR IG stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR IG stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. In order to address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29,856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed 'satellites', were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Description: Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B cell lymphocytosis (MBL), characterized by the presence of monoclonal CLL-like B cells in the peripheral blood, yet at lower numbers than those required for the diagnosis of CLL. MBL is distinguished into low-count (LC-MBL) and high-count (HC-MBL), based on the number of circulating CLL-like cells. While the former does not virtually progress into a clinically relevant disease, the latter may evolve into CLL at a rate of 1% per year. In CLL, genomic studies have led to the discovery of recurrent gene mutations that drive disease progression. These driver mutations may be detected in HC-MBL and even in multipotent hematopoietic progenitor cells from CLL patients, suggesting that they may be essential for CLL onset. Using whole-genome sequencing (WGS) we profiled LC-MBL and HC-MBL cases but also CLL patients with stable lymphocytosis (range: 39.8-81.8*109 CLL cells/l) for 〉10 years (hereafter termed indolent CLL). This would refine our understanding of the type of genetic aberrations that may be involved in the initial transformation rather than linked to clinical progression as is the case for most, if not all, CLL driver mutations. To this end, we whole-genome sequenced CD19+CD5+CD20dim cells from 6 LC-MBL, 5 HC-MBL and 5 indolent CLL cases; buccal control DNA and polymorphonuclear (PMN) cells were analysed in all cases. We also performed targeted deep-sequencing on 11 known driver genes (ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, FBXW7) in 8 LC-MBL, 13 HC-MBL and 7 indolent CLL cases and paired PMN samples. Overall similar mutation signatures/frequencies were observed for LC/HC-MBL and CLL concerning i) the entire genome; with an average of 2040 somatic mutations observed for LC-MBL, 2558 for HC-MBL and 2400 for CLL (186 for PMN samples), as well as ii) in the exome; with an average of non-synonymous mutations of 8.9 for LC-MBL, 14.6 for HC-MBL, 11.6 for indolent CLL (0.9 for PMN samples). Regarding putative CLL driver genes, WGS analysis revealed only 2 somatic mutations within NOTCH1, and FBXW7 in one HC-MBL case each. After stringent filtering, 106 non-coding variants (NCVs) of potential relevance to CLL were identified in all MBL/CLL samples and 4 NCVs in 2/24 PMN samples. Seventy-two of 110 NCVs (65.5%) caused a potential breaking event in transcription factor binding motifs (TFBM). Of these, 29 concerned cancer-associated genes, including BTG2, BCL6 and BIRC3 (4, 2 and 2 samples, respectively), while 16 concerned genes implicated in pathways critical for CLL e.g. the NF-κB and spliceosome pathways. Shared mutations between MBL/CLL and their paired PMN samples were identified in all cases: 2 mutations were located within exons, whereas an average of 15.8 mutations/case for LC-MBL, 8.2 for HC-MBL and 9 for CLL, respectively, concerned the non-coding part. Finally, 16 sCNAs were identified in 9 MBL/CLL samples; of the Döhner model aberrations, only del(13q) was detected in 7/9 cases bearing sCNAs (2 LC-MBL, 3 HC-MBL, 2 indolent CLL). Targeted deep-sequencing analysis (coverage 3000x) confirmed the 2 variants detected by WGS, i.e. in NOTCH1 (n=1) and FBXW7 (n=1), while 4 subclonal likely damaging variants were detected with a VAF 10 years display similar low genomic complexity and absence of exonic driver mutations, assessed both with WGS and deep-sequencing, underscoring their common low propensity to progress. On the other hand, HC-MBL comprising cases that may ultimately evolve into clinically relevant CLL can acquire exonic driver mutations associated with more dismal prognosis, as exemplified by subclonal driver mutations detected by deep-sequenicng. The existence of NCVs in TFBMs targeting pathways critical for CLL prompts further investigation into their actual relevance to the clinical behavior. Shared mutations between CLL and PMN cells indicate that some somatic mutations may occur before CLL onset, likely at the hematopoietic stem-cell level. Their potential oncogenic role likely depends on the cellular context and/or microenvironmental stimuli to which the affected cells are exposed. Disclosures Stamatopoulos: Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses. Ghia:Adaptive: Consultancy; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria, Research Funding.

Description: The IGHV4-34 gene is very frequent (~10%) in the B cell receptor immunoglobulin (BcR IG) gene repertoire of chronic lymphocytic leukemia (CLL). Over 30% of IGHV4-34 CLL cases can be assigned to different subsets with stereotyped BcR IG. The largest is subset #4 which represents ~1% of all CLL and ~10% of IGHV4-34 CLL and is considered a prototype for indolent disease. The BcR IG of a great majority (~85%) of IGHV4-34 CLL cases carry a significant load of somatic hypermutation (SHM), often with distinctive SHM patterns. This holds especially true for stereotyped subsets and is suggestive of particular modes of interactions with the selecting antigen(s). In detail, subsets #4 and #16, both involving IgG-switched cases (IgG-CLL), exhibit the greatest sequence similarity in SHM profiles, whereas they differ in this respect from IgM/D subsets #29 and #201. Prompted by these observations, here we explored the extent that these subset-biased SHM profiles in different IGHV4-34 stereotyped subsets were reflected in distinct demographics, clinical presentation, genomic aberrations and outcomes. Within a multi-institutional series of 20,331 CLL patients, 1790 (8.8%) expressed IGHV4-34 BcR IG. Following established bioinformatics approaches for the identification of BcR IG stereotypy, 573/1790 IGHV4-34 CLL cases (32%) were assigned to stereotyped subsets; of these, 340 cases (19% of all IGHV4-34 CLL and 60% of stereotyped IGHV4-34 cases) belonged to subsets #4, #16, #29 and #201, all concerning IGHV-mutated CLL (M-CLL). Clinicobiological information was available for 275/340 patients: #4, n=150; #16, n=44; #29, n=39; and #201, n=42. Comparisons between subsets revealed no differences in gender and age distribution. Interestingly, however, 36-43% of each subset cases were young for CLL (defined as patients aged ≤55 years), which is higher compared to general CLL cohorts, where young patients generally account for ~25% of cases. In contrast, significant differences were identified between subsets regarding: (i) disease stage at diagnosis, with 〉90% of IgG subsets #4 and #16 diagnosed at Binet stage A versus 83% in subset #201 and 74% in subset #29 (p=0.029); (ii) CD38 expression, ranging from 1% in subset #4 to 10% in subset #201 (p=0.013); (iii) the distribution of del(13q), peaking at a remarkable 92% in subset #29 versus only 37% in subset #16 (p

Description: Introduction: CLL is a heterogeneous disease in terms of response to treatment, with some patients reaching complete and prolonged remissions, while others relapsing early and requiring several lines of treatments. This highly variable course is partly explained by the existence of a heterogenic panel of genetic alterations (mutations, chromosomal abnormalities) that allow the development of drug-resistant aggressive CLL subclones. Therefore, a functional characterization of the cytogenetic alterations associated to CLL drug resistance may provide new means of improving the current therapeutic strategies. We and others have already reported that the gain of 2p (2p+) is recurrent in CLL. However, the candidate gained gene(s) on the 2p remain to be identified. Previously data: we have observed that the 2p gain is frequent in previously untreated CLL Binet stages B/C (21/132, 15.9%), and is associated with bad prognostic factors, such as 11q deletion (p=0.0008) and unmutated IGHV (p=0.02). Using a SNP-array approach, we have identified a minimally gained region of 1.28Mb on 2p16.1-15. This region included the gene CRM1/XPO1 (Chromosome Region Maintenance 1/Exportin-1), a gene also recurrently mutated in CLL. A qPCR assessment confirmed that XPO1 was overexpressed in the 2p+/CLL patients (1.4-fold increase compared to 2p-/CLL; p=0.02). The objective of our work was to identify the potential role of XPO1 in CLL drug resistance by using the selective XPO1 inhibitor Selinexor (KPT-330, provided by Karyopharm Therapeutics), which is currently in Phase II human clinical trials in hematological and solid cancers. Methods: We have analyzed 36 2p+/CLL and we have searched for XPO1 mutations in 436 CLL samples. CLL drug resistance associated to XPO1 overexpression/mutation was assessed by measuring the rate of programmed cell death (PCD) on cells from 2p- and wildtype (wt) XPO1/CLL (n=20), 2p+/XPO1 wt/CLL (n=8) and on XPO1 mut/CLL (n=6). After 24 hours treatment with Fludarabin + Cyclophosphamid + Rituximab (FCR), Ibrutinib (Ibru), Idelalisib + Rituximab (Ide+R) and Selinexor, cells were stained with Annexin-V and propidium iodide and PCD was assessed by flow cytometry. KPT-301 was used as a negative control. For the inhibition assay, the inhibitor Q-VD-Oph was added 30 min before inducing cell death. Mitochondrial membrane depolarisation was assessed using tetramethyllrhodamine ethyl ester probe and flow cytometry analysis. Results: (i) Using a FISH approach, we fully confirmed the gain of XPO1 in 2p+/CLL samples. Additionally, we found that the XPO1 gain was often subclonal, suggesting that it tends to arise late in leukemic development. Longitudinal FISH analyses, performed on 8 2p+/CLL-treated patients, showed a similar or increasing percentage of cells carrying XPO1 gain at relapse, when compared to diagnosis; (ii) XPO1 was mutated in 23/436 (5.3%) CLL and in 2/30 (6.7%) 2p+/CLL; (iii) Selinexor induced PCD in 2p-/XPO1 wt/CLL (35% of PCD). The results were similar in all tested CLL, independently of prognostic factors (del13q, tri12, del11q, del17p, IGHV status), while sparing the non leukemic cells from patients or B cells from healthy donors; (iv) Selinexor induced CLL PCD through a caspase-dependant apoptotic pathway, as evidenced by inhibition of cell death by Q-VD-Oph, and cleavage of the caspase-3. Selinexor also induced mitochondrial depolarization and was associated with upregulation and activation of the pro-apopototic Bax protein; (v) XPO1 mut/CLL were significantly resistant to PCD induced by Selinexor (p=0.003). In contrast, the mutations in XPO1 had no effect in FCR and Ibru PCD induction; (vi) 2p+/CLL cells were resistant to PCD induced by all tested drugs: FCR (p=0.01), Ibru (p=0.003), Ide+R (p=0.004) and Selinexor (p=0.0001). Conclusion: Our data show that 2p+/CLL is associated to FCR, Ibru and Ide+R drug resistance. Strikingly, Selinexor, a new XPO1 inhibitor, is unable to induce PCD in 2p+ and/or XPO1 mut CLL, which strongly suggests a key role for XPO1 in the CLL drug resistance associated to the 2p gain. Altogether, our work provide substantial progress in the understanding of the role of XPO1 in CLL drug resistance and suggests that the assessment of the 2p gain and the mutations in XPO1 will be considered before to decide a CLL therapy. As 2p gain could be observed in other B malignancies, it is tempting to extend these recommendations to all Selinexor treatments. Disclosures Choquet: Janssen: Consultancy; Roche: Consultancy. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria.

Description: Immunoglobulin (IG) G-switched chronic lymphocytic leukemia (G-CLL) is highly enriched for 3 stereotyped CLL subsets, utilizing either the IGHV4-34 gene, namely mutated subsets #4 and #16, or the IGHV4-39 gene, namely unmutated subset #8. These subsets, collectively accounting for ~30% of all G-CLL, are not represented within the common IgM/D variant, thus prompting speculations about distinct ontogenetic origin and/or immune triggering, as well as raising questions regarding the timing of class-switch recombination (CSR) in relation to malignant transformation. Considering the above, we sought to investigate the potential existence of B cells expressing clonotypic mu transcripts within the bulk of IgG-switched CLL cells in cases assigned to the aforementioned subsets. Using high-throughput next-generation sequencing (NGS, MiSeq Illumina), we interrogated the IgM+ B-cell repertoire of CLL subset #4 (n=8), subset #16 (n=1) and subset #8 (n=2) for the presence of clonotypic mu transcripts. PCR amplicons were generated from cDNA using a set of IGHV4/IGHM primers. The paired-end Illumina protocol allowed sequencing of the complementarity determining region 3 (CDR3) twice/read, thus increasing the accuracy of results. For 3/8 subset #4 cases multiple blood samples of the same time point were analyzed as reproducibility controls. A purpose-built bioinformatics algorithm was developed for raw NGS data processing, which included: (i) quality filtering of reads; (ii) merging of paired-end reads via local alignment; (iii) preparation of filtered-in fasta sequences for submission to the IMGT/HighV-QUEST tool; and, (iv) IMGT/HighV-QUEST metadata mining for subset-specific B-cell receptor (BcR) IG rearrangements. Subset-specific CDR3 motifs were defined according to established criteria. Overall, 7,125,958 IGHV-IGHD-IGHJ-IGHC rearrangements (189,988-673,835/sample) were included in the search for stereotyped motifs, corresponding to 1,056,967 distinct clonotypes (i.e. BcR IG rearrangements with a particular IGHV gene and amino acid CDR3 sequence) (7,163-123,276/sample, median=76,109). Regarding subset #4, 7/8 cases exhibited mu transcripts of subset #4-specific IG rearrangements ("subset #4 M-clonotypes"); by definition, these rearrangements utilized the IGHV4-34/IGHJ6 genes and had identical CDR3 length (20 amino acids), however their CDR3 amino acid composition varied (2-75 distinct subset #4 M-clonotypes/sample, median=8). In 5/7 cases these subset #4 M-clonotypes were characterized by CDR3s that were identical and/or highly similar (≤2 amino acid differences, ≥ 90% identity) to the CDR3 of the IgG-switched CLL clone. The M-clonotypes expressing CDR3s identical to those of the IgG-switched CLL clone represented the most expanded subset #4 M-clonotype within the sample, while the less expanded, "satellite" clonotypes may represent subclones that were selected against due to lower affinity with the driving antigen. The possibility that these “satellite” clonotypes derive from PCR and/or sequencing error cannot be a priori excluded, however replicate sample analysis produced identical subset #4 M-clonotypes in all cases tested, thus raising confidence in the accuracy of the data. Analysis of the subset #16 case yielded similar results, i.e. 2 subset #16 M-clonotypes, one of which was identical to the IgG-switched clonotypic BcR IG. Both subset #8 cases also carried subset #8 M-clonotypes, yet only one case exhibited an M-clonotype with a CDR3 identical to that of the respective G-CLL clone. Interestingly, this M-clonotype was accompanied by many highly similar, less expanded “satellite” clonotypes (n=109), raising the possibility that SHM may be occurring in (pre-)CLL clones carrying truly unmutated IGHV genes, but pass unnoticed due to negative selection. Although their actual frequency cannot be conclusively determined due to the inherent limitations of PCR-based NGS analysis, subset-specific rearrangements represented a very minor fraction of the sequenced IGHV4/IGHM clonotypes in all cases tested (median frequency 0.04%). Overall, our findings suggest that while CLL clones are primed prior to CSR for malignant transformation on the basis of their BcR IG features, G-CLL quickly transits through CSR either because full-blown malignant transformation occurs at a later time point, or because CSR offers a selective advantage to the malignant clone. Disclosures No relevant conflicts of interest to declare.

Description: In myeloma, plasma exchange (PE) has been suggested to prevent rapidly progressive kidney failure by reducing exposure to nephrotoxic light chains. We carried out a randomized controlled multi-centre trial comparing PE or no PE in 104 patients of whom 101 met the inclusion, exclusion criteria and 4 were lost to follow-up. We compared baseline characteristics as well as renal outcomes and performed a futility analysis to determine the sample size necessary for potential statistical significance for the changes noted. Thirty-nine patients were randomized to the control group and 58 to the PE group with a 6-month follow-up. The baseline characteristics of these 2 groups were similar including serum creatinine, dialysis dependence, age, gender, serum calcium, serum albumin, 24 -hour urine for protein levels and Durie-Salmon myeloma staging. Thirteen (33.3%) of the control group and 19 (33.3%) of the PE group died within 6 months of follow up. Ten patients (31%) in the control and 10 patients (21%) in the PE arm were dialysis dependent at 6 months. Seven patients (47%) came off dialysis in the control and 13 patients (59%) in the PE arm with the mean number of dialysis days from 0–6 months being 45.7±67.6 in the control versus 29.2±56.1 in the PE arm at 6 months. The mean serum creatinine in the control group was 314.6±256.1 μmol/L versus 215.4±215.3 μmol/L in the PE group and the composite end point of death, dialysis or serum creatinine 〉254 μmol/L occurred in 12 (30.8%) in the control and 11 (19.3%) in the PE arm. The futility analysis to indicate the per group sample size necessary to achieve statistical significance at 6 months for the difference we observed was infinite for cumulative mortality, 805 for dialysis dependence, 2418 for coming off dialysis, 321 for number of dialysis days, 132 for creatinine difference of 100 μmol/L and for the composite outcome of death, dialysis or creatinine〉354 μmol/L, 737. We did not observe a statistically significant difference in mortality or renal morbidity for PE versus no PE in patients with myeloma and rapidly progressive kidney failure.

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: Key Points Aberrant neutrophil maturation is associated with reduced effector functions in β-thalassemia. PU.1, the key regulator of terminal neutrophil maturation, is dysregulated in β-thalassemia.

Description: The arrays of proteins adsorbed from plasma onto a series of polystyrene copolymeric latexes were analyzed by enzyme-linked immunosorbent assay (ELISA) of washed beads and immunoblotting of proteins desorbed from the beads and separated by polyacrylamide gel electrophoresis (PAGE). Beads were prepared by continuous emulsion polymerization in the absence of surfactant. Coomassie brilliant blue staining of gel electropherograms of desorbed proteins indicated that the presence of small amounts of comonomers (1 to 10 mole %) significantly influenced the composition of the adsorbed protein layer. Immunoblotting revealed that fibrinogen, fibronectin, and vitronectin were adsorbed by all surfaces investigated. C3 and Clq adsorption varied significantly with copolymer composition. The ELISAs revealed that although the concentrations of vitronectin and fibronectin in plasma are similar, the extent of vitronectin adsorption from 70% to 85% plasma was greater by two orders of magnitude than fibronectin adsorption. Vitronectin adsorbed on carboxylic acid-containing copolymers reacted more strongly with a conformationally sensitive antivitronectin monoclonal antibody (MoAb) than vitronectin adsorbed to polystyrene and was more susceptible to cleavage by plasma proteases(s). The results show that vitronectin is a major protein adsorbed from concentrated plasma and that small changes in the chemical composition of a copolymer profoundly affects the extent and nature of protein adsorption from complex mixtures such as plasma.

Description: To characterize early B-cell precursors in humans, we correlated immunoglobulin heavy chain (IgH) gene rearrangement status with the CD34, CD19, and CD10 cell surface markers. Highly purified adult bone marrow (BM) cell fractions were obtained by two successive rounds of flow cytometric cell sorting, and IgH rearrangements were analyzed by polymerase chain reaction (PCR) amplification. Complete VDJH rearrangements were observed in the CD34+ CD19+ fraction, but not in the more immature CD34+ CD19− fraction. About one quarter of these rearrangements had an open reading frame, thus potentially permitting the synthesis of a μ chain. Partial DJH rearrangements were detected in both CD34+ CD19+ and CD34+ CD19− subsets, although they were less abundant in the latter. When triple labeling was used to better characterize the CD34+ CD19− population, DJH rearrangements were found to be present in the CD34+ CD10+ CD19− fraction, but not in the more primitive CD34+ CD10− CD19−. These results indicate that IgH gene rearrangements occur in CD34+ BM cells and that they initiate in immature progenitors expressing the CD10, but not yet the CD19 surface antigen. Finally, the presence of IgH gene rearrangements in CD34+ BM cells provides a useful marker of clonality to evaluate the possible involvement of these cells in various B-cell lymphoid malignancies.