In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Detection of minimal residual disease (MRD) in chronic lymphocytic leukaemia (CLL) is becoming increasingly important as treatments improve. An internationally harmonised four-colour (CLR) flow cytometry MRD assay is widely used but has limitations. The aim of this study was to improve MRD analysis by identifying situations where a less time-consuming CD19/CD5/κ/λ analysis would be sufficient for detecting residual CLL, and develop a six-CLR antibody panel that is more efficient for cases requiring full MRD analysis. In 784 samples from CLL patients after treatment, it was possible to determine CD19/CD5/κ/λ thresholds that identified cases with detectable MRD with 100% positive predictive value (PPV). However, CD19/CD5/κ/λ analysis was unsuitable for predicting iwCLL/NCI response status or identifying cases with no detectable MRD. For the latter cases requiring a full MRD assessment, a six-CLR assay was designed comprising CD19/CD5/CD20 with (1) CD3/CD38/CD79b and (2) CD81/CD22/CD43. There was good correlation between four-CLR and six-CLR panels in dilution studies and clinical samples, with 100% concordance for detection of residual disease at the 0.01% (10(-4)) level (n=59) and good linearity even at the 0.001-0.01% (10(-5)-10(-4)) level. A six-CLR panel therefore provides equivalent results to the four-CLR panel but it requires fewer reagents, fewer cells and a much simpler analysis approach.

Despite very similar gene expression profiles, the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is heterogeneous. Immunoglobulin VH (IgVH) mutational status and expression of B-cell receptor (BCR) signaling mediators have been associated with disease progression. However, the consequences of BCR engagement on cell survival and evolution of the disease remain unclear. We show here that B-CLL cell survival is dependent on the threshold of BCR stimulation induced by immobilized antibody, in contrast to soluble anti-M F(ab) ¶2 antibody, which leads to apoptosis. Measurement of metabolic activity and apoptotic response discriminated two subgroups. ''Nonresponders'' showed low metabolic activity and unmodified apoptotic response upon BCR stimulation. In contrast,''responders'' exhibited increased metabolic activity and inhibition of spontaneous apoptosis. This survival advantage was associated to a BCR-dependent activation profile leading to induction of cyclin D2/cyclindependent kinase 4 (cdk4) expression and G 1 cell cycle progression. The ability to respond to BCR ligation correlated with an unfavorable clinical course and allowed to define an additional group of patients among IgVH-mutated cases exhibiting a risk of progression. Remarkably, we show that Zap70 expression was neither mandatory nor sufficient to generate downstream survival signals and cyclin D2/cdk4 up-regulation. In conclusion, BCR engagement has a significant effect on B-CLL cell survival, activation, and G 1 progression. Furthermore, our results provide new insights in the physiopathology of progressive IgVH-mutated cases.

Background: Mucous membrane pemphigoid (MMP) still represents a potentially life-and sight-threatening disease. In a subset of patients with severe MMP, conventional immunosuppressants are ineffective or contraindicated.Observations: Twenty-five patients with severe refractory MMP, including 5 with mucous membranedominant epidermolysis bullosa acquisita, received 1 or 2 cycles of rituximab (375 mg/m 2 weekly for 4 weeks). Twenty-one of the patients were receiving concomitant therapy with dapsone and/or sulfasalazine therapy, which was maintained during rituximab cycles. Complete responses in all affected sites (ocular and/or extraocular) were obtained in 17 patients (68%) by a median time of 12 weeks after the first cycle, and 5 additional patients responded completely after a second cycle, yielding an 88% complete response rate. In all but 1 of the 10 patients with ocular lesions, their eyes became noninflammatory within a mean of 10 weeks. Among the 3 patients (12%) who developed severe infectious complications, 2 (8%) died; they had been receiving concomitant conventional immunosuppressants and high-dose corticosteroids and were hypogammaglobulinemic. Treatment with immunosuppressants was discontinued for all other patients, and no other infection was observed. Ten patients experienced relapse after a mean of 4 (range, 1-16) months after achieving complete responses.Conclusions: Rituximab appears to have rapid and dramatic efficacy in patients with severe, refractory MMP. The occurrence of severe infections in patients receiving concomitant conventional immunosuppressants supports using rituximab without other immunosuppressants. Controlled prospective studies are warranted to define an optimal treatment protocol.

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells

Immunocompromised individuals such as chronic lymphocytic leukemia (CLL) patients are at risk of impaired immune responses to vaccination. The objective of our study was to evaluate SARS-CoV-2-specific antibody responses in CLL patients, after the first, second and third doses of the BNT162b2 and mRNA-1273, and after a single dose for patients with confirmed prior COVID-19. Five hundred and thirty patients were included in the study. Patients received 2 doses at a 4-week interval, and a third dose if seronegative after the second dose. Response rate was 27% post-dose 1 and 52% post-dose 2. Post-dose 2 treatment-naïve patients had the highest response rate (72%) followed by patients previously treated by chemoimmunotherapy (60%). Among patients on therapy, patients on BTKi alone (22%) or in combination with anti-CD20 monoclonal antibodies or venetoclax (0%) had the poorer response rate whereas patients on venetoclax monotherapy achieved a significantly higher response rate (52%). A multivariate analysis identified as independent predictors of the absence of seroconversion: age >65 years, ongoing CLL treatment and gamma-globulins ≤6g/L. Post-dose 2 seronegative patients had a global post-dose 3 response rate of 35%. This study provides an argument for the use of a third dose and for prophylactic SARS-CoV-2 neutralizing monoclonal antibodies.

Mutational analyses performed following acquired ibrutinib resistance have suggested that chronic lymphocytic leukemia (CLL) progression on ibrutinib is linked to mutations in Bruton tyrosine kinase (BTK) and/or phospholipase Cγ2 (PLCG2) genes. Mutational information for patients still on ibrutinib is limited. We report a study aimed to provide a “snapshot” of the prevalence of mutations in a real-life CLL cohort still on ibrutinib after at least 3 years of treatment. Of 204 patients who initiated ibrutinib via an early-access program at 29 French Innovative Leukemia Organization (FILO) centers, 63 (31%) were still on ibrutinib after 3 years and 57 provided a fresh blood sample. Thirty patients had a CLL clone ≥0.5 × 109/L, enabling next-generation sequencing (NGS); BTK and PLCG2 mutations were detected in 57% and 13% of the NGS samples, respectively. After median follow-up of 8.5 months from sample collection, the presence of a BTK mutation was significantly associated with subsequent CLL progression (P = .0005 vs no BTK mutation). Our findings support that mutational analysis should be considered in patients receiving ibrutinib who have residual clonal lymphocytosis, and that clinical trials are needed to evaluate whether patients with a BTK mutation may benefit from an early switch to another treatment.

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCRdependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases. [Cancer Res 2009;69(16):6387-95]