Principal component analysis yields results comparable to those of an elaborate Boolean strategy: simplifying the assessment of measurable residual disease in chronic lymphocytic leukemia patients.

INTRODUCTION: Measurable residual disease (MRD) is becoming increasingly important in the chronic lymphocytic leukemia (CLL) context. It is of independent prognostic significance in terms of favorable progression-free and overall survival. The standardized methods used to assess CLL MRD are based on flow cytometry and real-time quantitative PCR. We here present a nine-color assay for CLL MRD with the ROR-1 marker antigen as recommended by the European Research Initiative (ERIC) on CLL; the sensitivity is at least 10-5. MATERIALS AND METHODS: We used 54 samples to develop a new principal component analysis (PCA) method based on the Kaluza© "radar" presentation mode. We used a Navios flow cytometer (Beckman Coulter©). RESULTS: We confirmed the linearity of our method over more than five dilutions. The specificity limit was 1.3×10-6 and the lower limit of detection was 3.6×10-6. Compared to the Boolean method, the sensitivity, specificity, and positive and negative predictive values of our PCA method were 100%. When MRD was detectable, PCA and Boolean assays were in agreement (linear regression, R2 = 0.99). CONCLUSION: We developed a new PCA-based method for detection of CLL MRD. Our method is comparable to that of the consensus method in terms of sensitivity, and it is also much easier and faster.

A fixed-duration immunochemotherapy approach in CLL: 5.5-year results from the phase 2 ICLL-07 FILO trial.

In previously untreated, medically fit patients with chronic lymphocytic leukemia (CLL), research is focused on developing fixed-duration strategies to improve long-term outcomes while sparing patients from serious toxicities. The ICLL-07 trial evaluated a fixed-duration (15-month) immunochemotherapy approach in which after obinutuzumab-ibrutinib induction for 9 months, patients (n = 10) in complete remission (CR) with bone marrow (BM) measurable residual disease (MRD) <0.01% continued only ibrutinib 420 mg/day for 6 additional months (I arm), whereas the majority (n = 115) received up to 4 cycles of fludarabine/cyclophosphamide-obinutuzumab 1000 mg alongside the ibrutinib (I-FCG arm). Primary analysis at month 16 showed that 84 of 135 (62.2%) patients enrolled achieved CR with a BM MRD <0.01%. Here, we report follow-up at median 63 months. Peripheral blood (PB) MRD was assessed 6 monthly beyond the end of treatment using a highly sensitive (10-6) flow cytometry technique. In the I-FCG arm, the PB MRD <0.01% rate (low-level positive <0.01% or undetectable with limit of detection ≤10-4) in evaluable patients was still 92.5% (74/80) at month 40 and 80.6% (50/62) at month 64. No differences in the PB MRD status were apparent per to the IGHV mutational status. In the overall population, 4-year progression-free and overall survival rates were 95.5% and 96.2%, respectively. Twelve deaths occurred overall. Fourteen serious adverse events occurred beyond the end of treatment. Thus, our fixed-duration immunochemotherapy approach produced deep and sustained PB MRD responses, high survival rates, and low long-term toxicity. A randomized trial is needed to compare our immunochemotherapy approach with a chemotherapy-free strategy. This trial was registered at www.clinicaltrials.gov as #NCT02666898.

Immuno-regulatory malignant B cells contribute to Chronic Lymphocytic Leukemia progression.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous B cell neoplasm ranging from indolent to rapidly progressive disease. Leukemic cell subsets with regulatory properties evade immune clearance; however, the contribution of such subsets during CLL progression is not completely elucidated. Here, we report that CLL B cells crosstalk with their immune counterparts, notably by promoting the regulatory T (Treg) cell compartment and shaping several helper T (Th) subsets. Among various constitutively- and BCR/CD40-mediated factors secreted, tumour subsets co-express two important immunoregulatory cytokines, IL10 and TGFß1, both associated with a memory B cell phenotype. Neutralizing secreted IL10 or inhibiting the TGFß signalling pathway demonstrated that these cytokines are mainly involved in Th- and Treg differentiation/maintenance. In line with the regulatory subsets, we also demonstrated that a CLL B cell population expresses FOXP3, a marker of regulatory T cells. Analysis of IL10, TGFß1 and FOXP3 positive subpopulations frequencies in CLL samples discriminated 2 clusters of untreated CLL patients that were significantly different in Tregs frequency and time-to-treatment. Since this distinction was pertinent to disease progression, the regulatory profiling provides a new rationale for patient stratification and sheds light on immune dysfunction in CLL.

miRNA profile at diagnosis predicts treatment outcome in patients with B-chronic lymphocytic leukemia: A FILO study.

During many years, chemo-immunotherapy fludarabine-cyclophosphamide-rituximab (FCR) was the gold standard for first line treatment of medically fit patients with symptomatic B-chronic lymphocytic leukemia (CLL). Over the last decade, targeted biotherapies have revolutionized the treatment of B-CLL patients and almost entirely supplanted FCR. However, no biomarker still exists to predict the complete remission (CR) with undetectable minimal residual disease (uMRD) in bone marrow (BM), which remains the best predictive factor for survival. MicroRNAs represent a class of molecular biomarkers which expression is altered in B-CLL. Our study aimed at identifying before treatment blood miRNAs that predict treatment outcome in previously untreated B-CLL patients (NCT01370772, https://clinicaltrials.gov/ct2/show/NCT01370772). Using hierarchical clustering of miRNA expression profiles discriminating 8 patients who achieved CR with BM uMRD from 8 patients who did not achieve CR and displayed detectable BM MRD, we identified 25 miRNAs differentially expressed before treatment. The expression of 11 miRNAs was further validated on a larger cohort (n=123). Based on the dosage of 5 miRNAs at diagnosis, a decision tree was constructed to predict treatment outcome. We identified 6 groups of patients with a distinct probability of being CR with BM uMRD to FCR treatment, ranging from 72% (miR-125b, miR-15b and miR-181c high) to 4% (miR-125b and miR-193b low). None of the patients displaying high expression levels of miR-125b, miR-15b and miR-181c relapsed during study follow-up. In contrast, patients with low miR-15b and high miR-412, or with low miR-125b and miR-193b, demonstrated significant low PFS. RNA sequencing of blood at diagnosis identified that patients relapsing after treatment are characterized by significant enrichment of gene signatures related to cell cycle, MYC target genes, metabolism and translation regulation. Conversely, patients achieving CR with BM uMRD displayed significant enrichment in genes related to communication between CLL cells and the microenvironment, immune system activation and upregulation of polycomb PRC2 complex target genes. Our results suggest that blood miRNAs are potent predictive biomarkers for FCR treatment efficacy and might be implicated in the FCR efficacy in B-CLL patients, providing new insight into unmet need for the treatment of B-CLL patients and identifying pathways predictive of patients' remission. Clinical trial registration: ClinicalTrials.gov, identifier NCT01370772.

Humoral response to mRNA anti-COVID-19 vaccines BNT162b2 and mRNA-1273 in patients with chronic lymphocytic leukemia.

Immunocompromised individuals such as patients with chronic lymphocytic leukemia (CLL) are at risk of impaired immune responses to vaccination. The objective of our study was to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses in patients with CLL after the first, second, and third doses of the BNT162b2 or mRNA-1273 vaccines and after a single dose for patients with confirmed previous COVID-19. In all, 530 patients were included in the study. Patients received 2 doses at a 4-week interval and a third dose if they were seronegative after the second dose. Response rate was 27% after dose 1 and 52% after dose 2. Post-dose 2 treatment-naïve patients had the highest response rate (72%) followed by patients previously treated by chemoimmunotherapy (60%). Among patients receiving therapy, those receiving Bruton tyrosine kinase inhibitor alone (22%) or in combination with anti-CD20 monoclonal antibodies or venetoclax (0%) had the poorer response rate whereas patients who received venetoclax monotherapy achieved a significantly higher response rate (52%). A multivariable analysis identified age older than 65 years, ongoing CLL treatment, and gamma globulin ≤6 g/L as independent predictors of the absence of seroconversion. Post-dose 2 seronegative patients had a global response rate of 35% after dose 3. This study provides an argument for the use of a third dose and for prophylactic SARS-CoV-2 neutralizing monoclonal antibodies.

Angiogenic factors could help us to define patients obtaining complete response with undetectable minimal residual disease in untreated CLL patients treated by FCR: results from the CLL2010FMP, a FILO study.

Angiogenesis is in a constant balance between pro and anti-angiogenic factors. Neoangiogenesis, implicated in metastatic spreading is characterized in solid cancers, but fairly new in chronic lymphocytic leukemia (CLL). We hypothesize that secretion of angiogenic factors could be correlated to the pathogenesis of CLL, and therefore predict the outcome of patients. We investigated concentrations of 22 cytokines and chemokines in 137 non-del 17p B-CLL patients, treated with a fludarabine-cyclophosphamide-rituximab (FCR)-based regimen. We constructed a biomarker index defining different risk groups based on lymphocyte count, the intensity of CD20 antigen on CD19+ cells, Ang-2, and PDGF-BB plasma concentrations at diagnosis. Four groups were defined, exhibiting specific molecular signatures and correlated with progression-free survival of patients. Our results suggest that we can determine at diagnosis of non-del 17p B-CLL patients, those with a very high probability of progression-free survival, independently of IGVH mutational status and residual disease at the end of treatment.

Multicentric MFI30 study: Standardization of flow cytometry analysis of CD30 expression in non-Hodgkin lymphoma.

CD30 transmembrane receptor, a member of the tumor necrosis factor receptor family, is expressed in different lymphomas. Brentuximab vedotin (BV), a CD30 monoclonal antibody (Ab)-drug conjugate, is effective in CD30-positive lymphomas. However, the response to BV is not always correlated to CD30 expression detected by immunohistochemistry (IHC). The objectives of this study were to standardize and evaluate CD30 intensity by flow cytometry (FCM) in non-Hodgkin's lymphomas. Twelve centers analyzed 161 cases on standardized cytometers using normalized median fluorescence intensity (nMFI30) of three different Abs, of which one clone can recognize the same epitope as BV. FCM distinguished four groups of cases: negative group (n = 110) which showed no expression with the three clones; high positive group (n = 13) which gave nMFI30 > 5% with all tested clones; dim positive group (n = 17) which showed nMFI30 > 1% with all tested clones and <5% for at least one; discordant group (n = 21) with positive and negative expression of the different clones. In consistency with the literature, CD30 was positive in all anaplastic large cell lymphomas, in some diffuse large B-cell lymphomas (DLBCL), and in other rare lymphomas. FCM results were concordant with those of IHC in 77% of cases. Discrepancies could be explained by clones-related differences, microenvironment, or intracytoplasmic staining. Interestingly, FCM was more sensitive than IHC in 11% of cases, especially in DLBCL. Multicenter standardized FCM of specific CD30 could improve case detection and extend the treatment of BV to various CD30-positive lymphomas.

Prognostic value of high-sensitivity measurable residual disease assessment after front-line chemoimmunotherapy in chronic lymphocytic leukemia.

Measurable residual disease (MRD) status is widely adopted in clinical trials in patients with chronic lymphocytic leukemia (CLL). Findings from FILO group trials (CLL2007FMP, CLL2007SA, CLL2010FMP) enabled investigation of the prognostic value of high-sensitivity (0.7 × 10-5) MRD assessment using flow cytometry, in blood (N = 401) and bone marrow (N = 339), after fludarabine, cyclophosphamide, and rituximab (FCR)-based chemoimmunotherapy in a homogeneous population with long follow-up (median 49.5 months). Addition of low-level positive MRD < 0.01% to MRD ≥ 0.01% increased the proportion of cases with positive MRD in blood by 39% and in bone marrow by 27%. Compared to low-level positive MRD < 0.01%, undetectable MRD was associated with significantly longer progression-free survival (PFS) when using blood (72.2 versus 42.7 months; hazard ratio 0.40, p = 0.0003), but not when using bone marrow. Upon further stratification, positive blood MRD at any level, compared to undetectable blood MRD, was associated with shorter PFS irrespective of clinical complete or partial remission, and a lower 5-year PFS rate irrespective of IGHV-mutated or -unmutated status (all p < 0.05). In conclusion, high-sensitivity (0.0007%) MRD assessment in blood yielded additional prognostic information beyond the current standard sensitivity (0.01%). Our approach provides a model for future determination of the optimal MRD investigative strategy for any regimen.

A fixed-duration, measurable residual disease-guided approach in CLL: follow-up data from the phase 2 ICLL-07 FILO trial.

Trials assessing first-line, fixed-duration approaches in chronic lymphocytic leukemia (CLL) are yielding promising activity, but few long-term data are available. We report follow-up data from a phase 2 trial (ICLL07 FILO) in previously untreated, medically fit patients (N = 135). Patients underwent obinutuzumab-ibrutinib induction for 9 months; then, following evaluation (N = 130 evaluable), those in complete remission and with bone marrow measurable residual disease (BM MRD) <0.01% (n = 10) received ibrutinib for 6 additional months; those in partial remission and/or with BM MRD ≥0.01%, the majority (n = 120), also received 4 cycles of immunochemotherapy (fludarabine/cyclophosphamide-obinutuzumab). Beyond end of treatment, responses were assessed every 3 month and peripheral blood MRD every 6 months. At median follow-up 36.7 months from treatment start, progression-free and overall survival rates (95% confidence interval) at 3 years were 95.7% (92.0% to 99.5%) and 98% (95.1% to 100%), respectively. Peripheral blood MRD <0.01% rates were 97%, 96%, 90%, 84%, and 89% at months 16, 22, 28, 34, and 40, respectively. No new treatment-related or serious adverse event occurred beyond end of treatment. Thus, in previously untreated, medically fit patients with CLL, a fixed-duration (15 months), MRD-guided approach achieved high survival rates, a persistent MRD benefit beyond the end of treatment, and low long-term toxicity. This trial was registered at www.clinicaltrials.gov as #NCT02666898.

Diagnosis and Treatment of Chronic Lymphocytic Leukemia: Recommendations of the French CLL Study Group (FILO).

As a result of significant recent developments, the management of patients with chronic lymphocytic leukemia (CLL) is changing, and new therapeutic options will continue to emerge in the near future. The recommendations of the French Innovative Leukemia Organization (FILO-CLL) group presented here are intended to provide practical recommendations for physicians taking care of CLL patients, taking into account the availability of both biological tests and therapies in daily practice in France at the time of publication. This text details the documented information and guidelines on diagnosis, indications for treatment, infectious complications and therapeutic strategies in frontline and relapsed CLL as well as in particular conditions such as autoimmune cytopenia or Richter syndrome.

Early treatment with FCR versus watch and wait in patients with stage Binet A high-risk chronic lymphocytic leukemia (CLL): a randomized phase 3 trial.

We report a randomized prospective phase 3 study (CLL7), designed to evaluate the efficacy of fludarabine, cyclophosphamide, and rituximab (FCR) in patients with an early-stage high-risk chronic lymphocytic leukemia (CLL). Eight hundred patients with untreated-stage Binet A disease were enrolled as intent-to-treat population and assessed for four prognostic markers: lymphocyte doubling time <12 months, serum thymidine kinase >10 U/L, unmutated IGHV genes, and unfavorable cytogenetics (del(11q)/del(17p)/trisomy 12). Two hundred and one patients with ≥2 risk features were classified as high-risk CLL and 1:1 randomized to receive either immediate therapy with 6xFCR (Hi-FCR, 100 patients), or to be observed according to standard of care (Hi-W&W, 101 patients). The overall response rate after early FCR was 92.7%. Common adverse events were hematological toxicities and infections (61.0%/41.5% of patients, respectively). After median observation time of 55.6 (0-99.2) months, event-free survival was significantly prolonged in Hi-FCR compared with Hi-W&W patients (median not reached vs. 18.5 months, p < 0.001). There was no significant overall survival benefit for high-risk patients receiving early FCR therapy (5-year OS 82.9% in Hi-FCR vs. 79.9% in Hi-W&W, p = 0.864). In conclusion, although FCR is efficient to induce remissions in the Binet A high-risk CLL, our data do not provide evidence that alters the current standard of care "watch and wait" for these patients.

[A primitive plasma cell leukemia with immunoglobulin (Ig) E]. / À propos d'un cas de leucémie plasmocytaire primitive à IgE.

We report here a case of primitive plasma cell leukemia with immunoglobulin (Ig) E. IgE myeloma is an exceptional variant of multiple myeloma, with a very poor prognosis. Its biological diagnosis requires specific analyzes in order to detect IgE gammopathy. Plasma cell leukemia (PCL) is also a very rare and very severe form of multiple myeloma. There are two variants: primitive PCL (pPCL) occurring de novo and secondary PCL (sPCL), evolution of a preexisting myeloma. Its diagnosis is essentially biological since it is defined by a blood plasmocytosis greater than 2 G/L or 20% of the leucocytes.

Obinutuzumab and ibrutinib induction therapy followed by a minimal residual disease-driven strategy in patients with chronic lymphocytic leukaemia (ICLL07 FILO): a single-arm, multicentre, phase 2 trial.

BACKGROUND: In patients with chronic lymphocytic leukaemia, achievement of a complete response with minimal residual disease of less than 0·01% (ie, <1 chronic lymphocytic leukaemia cell per 10 000 leukocytes) in bone marrow has been associated with improved progression-free survival. We aimed to explore the activity of induction therapy for 9 months with obinutuzumab and ibrutinib, followed up with a minimal residual disease-driven therapeutic strategy for 6 additional months, in previously untreated patients. METHODS: We did a single-arm, phase 2 trial in 27 university hospitals, general hospitals, and specialist cancer centres in France. Eligible patients were at least 18 years old and previously untreated, and had immunophenotypically confirmed B-cell chronic lymphocytic leukaemia; an Eastern Cooperative Oncology Group (ECOG) performance status score of less than 2; a Binet stage C according to IWCLL 2008 criteria or Binet stage A and B with active disease; no 17p deletion or absence of p53 mutation; and were considered medically fit. In the first part of the study (induction phase), all participants received eight intravenous infusions of obinutuzumab 1000 mg over six 4-weekly cycles and oral ibrutinib 420 mg once per day for 9 months. In part 2, after assessment on day 1 of month 9, patients with a complete response and bone marrow minimal residual disease of less than 0·01% received only oral ibrutinib 420 mg once per day for 6 additional months. Patients with a partial response, or with a complete response and bone marrow minimal residual disease of 0·01% or more, received 6 months of four 4-weekly cycles of intravenous fludarabine, cyclophosphamide, and obinutuzumab 1000 mg, alongside continuing ibrutinib 420 mg once per day. The primary endpoint was the proportion of patients achieving a complete response with bone marrow minimal residual disease less than 0·01% on day 1 of month 16 assessed by intention to treat (ITT). This trial is registered with ClinicalTrials.gov (number NCT02666898) and is still open for follow-up. FINDINGS: Between Oct 27, 2015, and May 16, 2017, 135 patients were enrolled. After induction treatment (day 1 of month 9), 130 patients were evaluable, of which ten (8%) achieved a complete response with bone marrow minimal residual disease of less than 0·01% and were assigned to ibrutinib, and 120 (92%) were assigned to ibrutinib plus fludarabine, cyclophosphamide, and obinutuzumab. After minimal residual disease-guided treatment (day 1 of month 16), 84 (62%, 90% CI 55-69) of 135 patients (ITT population) achieved a complete response with bone marrow minimal residual disease of less than 0·01%. The most common haematological adverse event was thrombocytopenia (in 45 [34%] of 133 patients at grade 1-2 in months 1-9 and in 43 [33%] of 130 patients at grade 1-2 in months 9-15). The most common non-haematological adverse events were infusion-related reactions (in 83 [62%] patients at grade 1-2 in months 1-9) and gastrointestinal disorders (in 62 [48%] patients at grades 1 and 2 in months 9-15). 49 serious adverse events occurred, most frequently infections (ten), cardiac events (eight), and haematological events (eight). No treatment-related deaths occurred. INTERPRETATION: Obinutuzumab and ibrutinib induction therapy followed by a minimal residual disease driven strategy is safe and active in patients with previously untreated chronic lymphocytic leukaemia. With longer follow-up, including assessing the evolution of minimal residual disease, if response is maintained, this strategy could be an option in the first-line setting in patients with chronic lymphocytic leukaemia, although randomised evidence is needed. FUNDING: Roche, Janssen.

Prevalence of BTK and PLCG2 mutations in a real-life CLL cohort still on ibrutinib after 3 years: a FILO group study.

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.