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: 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 heavy chain variable gene (IGHV) replacement or "VH replacement" (VHR) modifies a rearranged IGHV-D-J sequence by replacing the original IGHV gene with another. This process leaves a detectible "footprint" at the IGHV-D junction of the existing sequence. Roughly 33% of chronic lymphocytic leukemia (CLL) cases exhibit stereotyped B cell receptors (BCRs) often characterized by signature VH CDR3 amino acids. Various mechanisms have been put forth to account for stereotypy in CLL. An overarching hypothesis is that the stereotyped BCRs are antigen driven. Within this concept, a variety of mechanisms could lead to the signatures including somatic mutations and addition/deletion of nucleotides at junctional regions. Here we explore the possibility that VHR provides another mechanism to account for some of the stereotyped rearrangements and some of their signature VH CDR3 amino acid residues in CLL. We examined IG sequences of 26,642 CLL cases and ~16 million healthy controls (HC) to find relic footprints as indicators of VHR. This was done using the VHRFA program developed by Lin Huang et al (PLoS ONE, 2013), as well as our own program which duplicates the VHRFA results but is better able to process large numbers of sequences. The frequency of VHR was similar in CLL and HC (11.6 and 11.9%, respectively). Focusing solely on CLL sequences to define a relationship between VHR and stereotypy, we found highly significant differences in VHR frequencies between stereotyped (n=8,568) and non-stereotyped cases (n=18,074), with stereotyped cases exhibiting VHR at a greatly reduced frequency (7.7% vs. 13.5%, respectively). When comparing VHR frequencies between stereotyped cases and non-stereotyped cases that used the same IGHV, we found that the number of subsets with low VHR exceeded those with elevated VHR ~2:1, accounting for the overall VHR in stereotyped cases being lower than non-stereotyped cases. Further restricting comparisons of stereotyped subsets to non-stereotyped cohorts by matching VH CDR3 length led to similar conclusions. Within stereotyped cases there was a wide distribution of VHR, ranging from 55.6% to 0.1%. Restricting VH CDR3 lengths to "short" (5 - ≤13), "medium" (13.1 - ≤20) and "long" (20.1 - ≤28), the corresponding VHR increased monotonically with length (1.1, 8.2, and 11.9% respectively). Notably, subsets showing elevated VH replacement included better prognosis subsets, #4, 77 and 201 (23.8, 22.1, and 28.6%, respectively). Among low VHR frequency subsets were those associated with worse prognosis, #1, 2, 5, 6, 8, 9 and 10 (VHR frequencies: 0.2, 0.1, 0.9, 2.3, 7.7, 9.0 %, respectively). This was most strikingly exhibited by subsets #1 and #2, both of which comprise patients with poor clinical courses. Each of these sets of sequences displayed virtually no examples of VHR (0.2 and 0.1%, respectively). This might be predicted because these two subsets have relatively short VH CDR3 lengths (subset #1: 13 aa; subset #2: 9 aa), based on the length association mentioned above. Detailed analyses of the presence of footprints and the position of these in the rearranged IGHV-D-J indicated that for some subsets, certain signature VH CDR3 amino acids could be the result of VHR. For example in subset #201, sequence analysis suggests that VHR is responsible for an arginine and for a glutamine in the 5' portion of the VH CDR3. Similarly, VHR may craft the characteristic glutamine on the 5' end of the subset #6 VH CDR3. Thus, our studies indicate that, as a whole, CLL IGHV-D-J sequences use VHR at a frequency comparable to that of normal B cells and significantly less than that of non-stereotyped rearrangements. However, certain stereotyped cases are dramatically enriched for evidence of VHR. Moreover among these cases, the footprints found in the VH CDR3s of stereotyped cases can be shown to directly code for signature amino acids in VH CDR3s. Finally, stereotyped cases with high levels of VHR tend to be those with better clinical courses, whereas those worse outcome stereotyped cases exhibit less evidence for this process. This latter finding is consistent with the concept that VHR is one of the molecular mechanisms used by developing B cells to edit BCRs having high affinity for autoantigens. Since many CLL BCRs are autoreactive, including those found to have high levels of VHR such as subset #4, this implies a fundamental defect in tolerance mechanisms in those normal B cells that eventually became leukemic. Disclosures Agathangelidis: Gilead: Research Funding. Hadzidimitriou:Janssen: Honoraria, Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

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: 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.

Description: B-PLL is defined by the presence of prolymphocytes in peripheral blood exceeding 55% of lymphoid cells. The diagnosis, mainly based on clinical and morphological data, can be difficult because of overlap with other B-cell malignancies. Because of the rarity of the disease, only case reports and small series describe its cytogenetic features. Few prognostic markers have been identified in this aggressive leukemia usually resistant to standard chemo-immuno therapy. We report here the cytogenetic and molecular findings in a large series of B-PLL. We also studied the in vitro response to novel targeted drugs on primary B-PLL cells. The study included 34 cases with a diagnosis of B-PLL validated by morphological review performed by three independent expert cytologists. The diagnosis of mantle cell lymphoma was excluded by karyotype (K) and FISH using CCND1, CCND2 and CCND3 probes. Median age at diagnosis was 72 years [46-88]. K was complex (≥3 abnormalities) in 73%, and highly complex (HCK≥5) in 45%. Combining K and FISH data, the most frequent chromosomal aberrations were: translocation targeting the MYC gene [t(MYC)] (21/34, 62%), 17p deletion including TP53 gene (13/34, 38%), trisomy 18/18q (10/33, 30%), 13q14 deletion (10/34, 29%), trisomy 3 (8/33, 24%), trisomy 12 (8/34, 24%) and 8p deletion (7/31, 23%). Whole-Exome Sequencing analysis of paired tumor-control DNA was performed in 16 patients. The most frequently mutated genes were TP53(6/16, 38%), associated with del17p in all, MYD88 (n=4), BCOR (n=4), MYC (n=3), SF3B1 (n=3), FAT1 (n=3), SETD2 (n=2), CHD2 (n=2), CXCR4 (n=2), BCLAF1 (n=2) and NFASC (n=2). Distribution of the chromosomal aberrations is shown in Fig 1. The main group of patients (21/34, 62%) had a t(MYC) that was associated with a higher % of prolymphocytes (86 vs 76, p=0.03), CD38 expression (90% vs 15%,p

Description: The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is biased and characterized by the existence of subsets of cases with closely homologous (“stereotyped”) complementarity-determining region 3 (CDR3) sequences. In the present series, 201 (21.9%) of 916 patients with CLL expressed IGHV genes that belonged to 1 of 48 different subsets of sequences with stereotyped heavy chain (H) CDR3. Twenty-six subsets comprised 3 or more sequences and were considered “confirmed.” The remaining subsets comprised pairs of sequences and were considered “potential”; public database CLL sequences were found to be members of 9 of 22 “potential” subsets, thereby allowing us to consider them also “confirmed.” The chance of belonging to a subset exceeded 35% for unmutated or selected IGHV genes (eg, IGHV1-69/3-21/4-39). Comparison to non-CLL public database sequences showed that HCDR3 restriction is “CLL-related.” CLL cases with selected stereotyped immunoglobulins (IGs) were also found to share unique biologic and clinical features. In particular, cases expressing stereotyped IGHV4-39/IGKV1-39-1D-39 and IGHV4-34/IGKV2-30 were always IgG-switched. In addition, IGHV4-34/IGKV2-30 patients were younger and followed a strikingly indolent disease, contrasting other patients (eg, those expressing IGHV3-21/IGLV3-21) who experienced an aggressive disease, regardless of IGHV mutations. These findings suggest that a particular antigen-binding site can be critical in determining the clinical features and outcome for at least some CLL patients.

Description: The immunoglobulin heavy chain variable (IGHV) gene repertoire in CLL is biased and uniquely characterized by the existence of subsets of cases with “stereotyped” heavy chain complementarity-determining region 3 (HCDR3) sequences. As previously shown, HCDR3 stereotypy may have important pathogenetic and clinical implications, at least for certain subsets of CLL patients. However, the detection of stereotypy has so far been hindered, mainly due to the lack of suitable computational tools. We developed new methods for identification of sequence patterns within HCDR3 amino acid (AA) sequences using a sophisticated combinatorial pattern discovery algorithm. Included in the analysis were HCDR3 sequences from 2,845 CLL patients from our collaborating institutions, as well as 5,344 non-CLL sequences from public databases, for a total of 8,189. The identified patterns were subjected to a multiple and strict filtering process, based on the following criteria: sequence relatedness; location (offset) within HCDR3; and HCDR3 AA length. Clustering of sequences based on the filtered HCDR3 patterns revealed that the CLL IG repertoire can be distinguished in two broad categories: the first includes cases with heterogeneous B cell receptors (BCRs) (non-clustered cases) while the second is characterized by remarkable BCR stereotypy (clustered cases). In particular, 783/2,845 CLL sequences (27.5%) were placed in 339 ground-level clusters. Common sequences among these ground-level clusters allowed their progressive grouping in clusters at higher levels of hierarchy. High-level clusters were characterized by striking IGHV repertoire restriction, with only six IGHV genes (1–69, 1–3, 1–2, 3–21, 4–34, 4–39) accounting for 〉80% (382/459) of cases. While ground-level clusters provided a high-resolution picture of HCDR3 stereotypy, high-level clusters were considerably larger in size (up to 86 sequences each) and able to capture and describe more distant sequence relationships in the form of more widely shared sequence patterns. In particular, they were defined by patterns characterized by just a few critically positioned residues, reminiscent of the receptors expressed by cells participating in innate immune responses: due to this remarkable structural conservation, we consider them to be BCR “archetypes”. To test the hypothesis that sequence relatedness between IGHV genes may have structural and functional meaning for the IGHV repertoire in CLL, we also constructed sequence distance trees of functional human IGHV genes. Examination of the tree for the IGHV3 gene subgroup revealed a branching that was reflected in the repertoires of clustered vs. non-clustered CLL sequences. In particular, IGHV3-21, the foremost example of a gene with a propensity to be used in clustered rearrangements in CLL, belongs to a branch clearly distinct from other branches that include, for instance, the IGHV3-23, IGHV3-30, IGHV3-33 and IGHV3-7 genes, which were essentially absent from the repertoire of CLL sequences in high-level clusters; a similar case was also evident among IGHV1 subgroup genes. On these grounds, we argue that CLL cases with clustered (i.e. stereotyped) vs. non-clustered (i.e. heterogeneous) BCRs could derive from different progenitor cell populations evolutionarily adapted to particular antigenic challenges. In particular, prompted by the fact that clustered cases were found to express a limited set of highly conserved BCRs, we propose that in such cases the clonogenic progenitors may originate from a B cell population intermediate between a true innate immune system and the conventional adaptive B cell immune system, similar to what has been previously suggested for mouse B1 cells.

Description: Background : Expression of ZAP-70 protein has been shown to be correlated with mutational status of immunoglobulin heavy-chain variable region (IgVH) genes, a major prognostic factor in CLL. We investigated whether the detection of ZAP-70 protein by flow cytometric analysis using unconjugated and conjugated monoclonal antibodies (mAbs) could be applied securely in the workup of patients with CLL. Methods: Flow cytometric analysis of ZAP-70 protein was performed using the method described by Crespo et al (N Engl J Med2003;348:1764) with minor modifications. Both fresh and cryopreserved mononuclear cells from CLL patients and healthy donors were fixed and permeabilized using Fix and Perm kit (Caltag Laboratories), incubated with anti-ZAP-70 mAb (clone 2F3.2, Upstate Biotechnology) and then revealed with goat antimouse FITC mAb (Immunotech). Finally cells were incubated with CD3-APC, CD56-APC and CD19-PC5. We also tested 3 mAbs conjugated to various fluorochromes: 2F3.2-FITC (Upstate), 1E7.2-PE (eBioscience), 1E7.2-PE or -Alexa 488 (Caltag). ZAP-70 protein detection in B-cells was expressed either as a percentage of its expression in the T and NK-cells or as a ratio (R) of T-cell mean cell fluorescence (MCF) to B-cell MCF. Western blotting of protein lysates from purified B-cells was carried out to control results obtained by cytometry in 55 samples. Mutational status was defined using a cutoff of 98%. Results: In 13 healthy donors, the mean percentage of ZAP-70 protein expression obtained by flow cytometry with unconjugated mAb (clone 2F3.2) was 4.69% ± 1.93 [range 2–9%] and the R ratio was 6.64 ± 1.54 and 〉 4.8. In 83 B-CLL samples, ZAP-70 expression was determined using the same method and compared to IgVH mutational status. Results in table below show a 75% concordance between gene mutations and ZAP-70 expression when considering a percentage of positive B-cells 〉 20%. A better concordance (81%) is obtained with a threshold T-cell MCF/ B-cell MCF at 4 determined by Youden’s index. To note the high concordance (90%) between unmutated status and ZAP-70 + expression (19/21). Comparison with at least 1 of the 3 conjugated mAbs has been performed for 63 samples, with discordant results in our laboratories. 62 mutated IgVH samples 21 unmutated IgVH samples ZAP-70 B-Cells + ≤ 20 % : 43 〉 20% : 19 T-cell MCF/B-cell MCF ≥ 4 : 48 〈 4 : 19 Conclusions: Our data document the concordance between IgVH gene mutational status and ZAP-70 protein expression measured by flow cytometry, particularly in ZAP-70 negative samples. We found that the indirect method of labelling with unconjugated anti-ZAP-70 mAb remains until now, in our hands, the gold standard method compared to the available dyes conjugate mAbs.