Blinatumomab after R-CHOP bridging therapy for patients with Richter transformation: a phase 2 multicentre trial.

Richter transformation (RT) is an aggressive lymphoma occurring in patients with chronic lymphocytic leukaemia. Here we investigated the anti-CD3/anti-CD19 T-cell-engager blinatumomab after R-CHOP (i.e. rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) in patients with untreated RT of diffuse large B-cell lymphoma histology (NCT03931642). In this multicentre phase 2 study, patients without complete response (CR) after two cycles of R-CHOP were eligible to receive an 8-week blinatumomab induction via continuous vein infusion with stepwise dosing until 112 µg/day. The primary endpoint was the CR rate after blinatumomab induction and secondary endpoint included safety, response duration, progression-free and overall survival. Thirty-nine patients started the first cycle of R-CHOP, 25 of whom received blinatumomab. After blinatumomab induction, five (20%) patients achieved CR, four (16%) achieved partial response, and six (24%) were stable. Considering the entire strategy, the overall response rate in the full-analysis-set was 46% (n = 18), with CR in 14 (36%) patients. The most common treatment-emergent adverse events of all grades in the blinatumomab-safety-set included fever (36%), anaemia (24%), and lymphopaenia (24%). Cytokine release syndrome (grade 1/2) was observed in 16% and neurotoxicity in 20% of patients. Blinatumomab demonstrated encouraging anti-tumour activity (the trial met its primary endpoint) and acceptable toxicity in patients with RT.

Faster clinical decisions in B-cell acute lymphoblastic leukaemia: A single flow cytometric 12-colour tube improves diagnosis and minimal residual disease follow-up.

Assessing minimal residual disease (MRD) in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) is essential for adjusting therapeutic strategies and predicting relapse. Quantitative polymerase chain reaction (qPCR) is the gold standard for MRD. Alternatively, flow cytometry is a quicker and cost-effective method that typically uses leukaemia-associated immunophenotype (LAIP) or different-from-normal (DFN) approaches for MRD assessment. This study describes an optimized 12-colour flow cytometry antibody panel designed for BCP-ALL diagnosis and MRD monitoring in a single tube. This method robustly differentiated hematogones and BCP-ALL cells using two specific markers: CD43 and CD81. These and other markers (e.g. CD73, CD66c and CD49f) enhanced the specificity of BCP-ALL cell detection. This innovative approach, based on a dual DFN/LAIP strategy with a principal component analysis method, can be used for all patients and enables MRD analysis even in the absence of a diagnostic sample. The robustness of our method for MRD monitoring was confirmed by the strong correlation (r = 0.87) with the qPCR results. Moreover, it simplifies and accelerates the preanalytical process through the use of a stain/lysis/wash method within a single tube (<2 h). Our flow cytometry-based methodology improves the BCP-ALL diagnosis efficiency and MRD management, offering a complementary method with considerable benefits for clinical laboratories.

Feasibility of Optical Genome Mapping from Placental and Umbilical Cord Sampled after Spontaneous or Therapeutic Pregnancy Termination.

Optical genome mapping (OGM) is an alternative to classical cytogenetic techniques to improve the detection rate of clinically significant genomic abnormalities. The isolation of high-molecular-weight (HMW) DNA is critical for a successful OGM analysis. HMW DNA quality depends on tissue type, sample size, and storage conditions. We assessed the feasibility of OGM analysis of DNA from nine umbilical cord (UC) and six chorionic villus (CV) samples collected after the spontaneous or therapeutic termination of pregnancy. We analyzed quality control metrics provided by the Saphyr system (Bionano Genomics) and assessed the length of extracted DNA molecules using pulsed-field capillary electrophoresis. OMG data were successfully analyzed for all six CV samples. Five of the UC samples did not meet the Saphyr quality criteria, mainly due to poor DNA quality. In this regard, we found that DNA quality assessment with pulsed-field capillary electrophoresis can predict a successful OGM analysis. OGM data were fully concordant with the results of standard cytogenetic methods. Moreover, OGM detected an average of 14 additional structural variants involving OMIM genes per sample. On the basis of our results, we established the optimal conditions for sample storage and preparation required for a successful OGM analysis. We recommend checking DNA quality before analysis with pulsed-field capillary electrophoresis if the storage conditions were not ideal or if the quality of the sample is poor. OGM can therefore be performed on fetal tissue harvested after the termination of pregnancy, which opens up the perspective for improved diagnostic yield.

Cytogenetics in the management of chronic lymphocytic leukemia: Guidelines from the Groupe Francophone de Cytogénétique Hématologique (GFCH).

Chromosomal abnormalities are frequent in chronic lymphocytic leukemia (CLL), and most have prognostic value. In addition to the four well-known abnormalities (13q, 11q and 17p deletions, and trisomy 12), other recurrent aberrations have been linked to the disease outcome and/or drug resistance. Moreover, the complex karyotype has recently emerged as a prognostic marker for patients undergoing immunochemotherapy or targeted therapies. Here, we describe the main chromosomal abnormalities identified in CLL and related disorders (small lymphocytic lymphoma and monoclonal B-cell lymphocytosis) by reviewing the most recent literature and discussing their detection and clinical impact. Lastly, we provide technical guidelines and a strategy for the cytogenetic assessment of CLL.

Cytogenetics in the management of myeloproliferative neoplasms, mastocytosis and myelodysplastic/myeloproliferative neoplasms: Guidelines from the Group Francophone de Cytogénétique Hématologique (GFCH).

Myeloproliferative neoplasms, mastocytosis, myeloid/lymphoid neoplasms with hypereosinophilia and tyrosine kinase gene fusions, and myelodysplastic/myeloproliferative neoplasms are clonal hematopoietic cancers that, with the exception of certain entities, have an indolent course. In addition to their increasingly important role in the diagnosis of these entities, as shown by the recent classification of hematolymphoid tumors in the 5th edition of the World Health Organization and the International Consensus Classification of myeloid neoplasms and acute leukemias, identification of the profile of acquired genetic abnormalities is essential for adapting patient management and early detection of patients at high risk of progression. Alongside molecular abnormalities, cytogenetic abnormalities play an important role in the diagnosis, prognosis and follow-up of these diseases. Here, we review the recent literature on the impact of chromosomal abnormalities in these different entities and provide updated cytogenetic recommendations and guidelines for their management.

Cytogenetics in the management of mature T-cell and NK-cell neoplasms: Guidelines from the Groupe Francophone de Cytogénétique Hématologique (GFCH).

Mature T-cell and natural killer (NK)-cell neoplasms (MTNKNs) are a highly heterogeneous group of lymphomas that represent 10-15 % of lymphoid neoplasms and have usually an aggressive behavior. Diagnosis can be challenging due to their overlapping clinical, histological and immunophenotypic features. Genetic data are not a routine component of the diagnostic algorithm for most MTNKNs. Indeed, unlike B-cell lymphomas, the genomic landscape of MTNKNs is not fully understood. Only few characteristic rearrangements can be easily identified with conventional cytogenetic methods and are an integral part of the diagnostic criteria, for instance the t(14;14)/inv(14) or t(X;14) abnormality harbored by 95 % of patients with T-cell prolymphocytic leukemia, or the ALK gene translocation observed in some forms of anaplastic large cell lymphoma. However, advances in molecular and cytogenetic techniques have brought new insights into MTNKN pathogenesis. Several recurrent genetic alterations have been identified, such as chromosomal losses involving tumor suppressor genes (SETD2, CDKN2A, TP53) and gains involving oncogenes (MYC), activating mutations in signaling pathways (JAK-STAT, RAS), and epigenetic dysregulation, that have improved our understanding of these pathologies. This work provides an overview of the cytogenetics knowledge in MTNKNs in the context of the new World Health Organization classification and the International Consensus Classification of hematolymphoid tumors. It describes key genetic alterations and their clinical implications. It also proposes recommendations on cytogenetic methods for MTNKN diagnosis.

Optical genome mapping for prenatal diagnosis: A prospective study.

PURPOSE: Cytogenetic analysis provides important information for prenatal decision-making and genetic counseling. Optical genome mapping (OGM) has demonstrated its performances in retrospective studies. In our prospective study, we assessed the quality of DNA obtained from cultures of amniotic fluid (AF) and chorionic villi (CV) and evaluated the ability of OGM to detect all clinically relevant aberrations identified by standard methods. METHODS: A total of 37 prenatal samples from pregnancies with a fetal anomaly on ultrasound were analyzed prospectively by OGM between January 1, 2021 and June 31, 2022. OGM results were interpreted blindly and compared to the results obtained by standard techniques. RESULTS: OGM results were interpretable in 92% of samples. We observed 100% concordance between OGM and karyotype and/or chromosomal microarray results. In addition, OGM identified a median of 30 small (<100 kb) structural variations per case with the involvement of 12 OMIM genes, of which 3 were OMIM morbid genes. CONCLUSION: This prospective study showed OGM performed well in detecting genomic alterations in cell cultures from prenatal samples. The place of OGM in relation to CMA or exome sequencing remains to be defined in order to optimize the prenatal diagnostic procedure.

Genetic events associated with venetoclax resistance in CLL identified by whole-exome sequencing of patient samples.

Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported though remain poorly understood. Here, we analyze longitudinal tumor samples from 11 patients with disease progression while receiving venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at the posttreatment time point. We found the previously described acquired BCL2-G101V mutation in only 4 of 11 patients, with 2 patients showing a very low variant allele fraction (0.03%-4.68%). Whole-exome sequencing revealed acquired loss(8p) in 4 of 11 patients, of which 2 patients also had gain (1q21.2-21.3) in the same cells affecting the MCL1 gene. In vitro experiments showed that CLL cells from the 4 patients with loss(8p) were more resistant to venetoclax than cells from those without it, with the cells from 2 patients also carrying gain (1q21.2-21.3) showing increased sensitivity to MCL1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to the combination of MCL1 inhibitor and venetoclax. Differential gene expression analysis comparing bulk RNA sequencing data from pretreatment and progression time points of all patients showed upregulation of proliferation, B-cell receptor (BCR), and NF-κB gene sets including MAPK genes. Cells from progression time points demonstrated upregulation of surface immunoglobulin M and higher pERK levels compared with those from the preprogression time point, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for patients with venetoclax-resistant CLL.

Optical Genome Mapping in Routine Cytogenetic Diagnosis of Acute Leukemia.

Cytogenetic aberrations are found in 65% of adults and 75% of children with acute leukemia. Specific aberrations are used as markers for the prognostic stratification of patients. The current standard cytogenetic procedure for acute leukemias is karyotyping in combination with FISH and RT-PCR. Optical genome mapping (OGM) is a new technology providing a precise identification of chromosomal abnormalities in a single approach. In our prospective study, the results obtained using OGM and standard techniques were compared in 29 cases of acute myeloid (AML) or lymphoblastic leukemia (ALL). OGM detected 73% (53/73) of abnormalities identified by standard methods. In AML cases, two single clones and three subclones were missed by OGM, but the assignment of patients to cytogenetic risk groups was concordant in all patients. OGM identified additional abnormalities in six cases, including one cryptic structural variant of clinical interest and two subclones. In B-ALL cases, OGM correctly detected all relevant aberrations and revealed additional potentially targetable alterations. In T-ALL cases, OGM characterized a complex karyotype in one case and identified additional abnormalities in two others. In conclusion, OGM is an attractive alternative to current multiple cytogenetic testing in acute leukemia that simplifies the procedure and reduces costs.

Mast cell density and its clinical relevance in Waldenström's macroglobulinemia.

The presence of numerous mast cells (MCs) mixed with tumor cells in the bone marrow (BM) is a hallmark of the diagnosis of Waldenström's macroglobulinemia (WM). MCs have been shown to support lymphoplasmacytic cell growth, but there is thus far no demonstration of the prognostic impact of BM MC density in WM. We investigated BM MC density by sensitive and specific digital quantification, allowing the analysis of a large area infiltrated by BM tumor cells. A total of 65 WM patients were investigated, including 54 at diagnosis and 11 at relapse. Tryptase and CD20 immunohistochemisty staining was performed on contiguous sections of deparaffinized BM trephine biopsies. After numerization of each section, the BM surface area was manually marked out, excluding the bone framework and adipocytes to limit the analyses to only hematopoietic tissue. MCs were assessed using a digital tool previously used to quantify immune-cell infiltrates on tumor-tissue sections. Deep next-generation sequencing and allele-specific PCR were used to explore the MYD88 and CXCR4 mutational status. MC density was heterogeneous among the WM patients. An optimal MC density threshold (> 56 MC.mm-2) was defined according to ROC curve analysis of overall survival. A higher MC density (> 56 MC.mm-2) was associated with greater BM involvement by WM lymphoplasmacytic cells and less hepatosplenic involvement (p = 0.023). Furthermore, MC density significantly correlated with a higher ISSWM score (p = 0.0003) in symptomatic patients. Patients with a higher MC density showed shorter median OS (56.5 months vs. nonreached, p = 0.0004), even in multivariate analysis after controlling for other predictive variables, such as age, ISSWM score, and CXCR4 mutational status. In conclusion, MC density can be accurately measured in WM patients using a specific digital tool on well-outlined hematopoietic tissue surfaces. High MC density is associated with aggressive features and a poor clinical outcome, emphasizing the need for further investigation of the involvement of MCs in the pathophysiology of WM.

Reduced telomere length in amniocytes: an early biomarker of abnormal fetal development?

Telomeres protect chromosome ends and control cell division and senescence. During organogenesis, telomeres need to be long enough to ensure the cell proliferation necessary at this stage of development. Previous studies have shown that telomere shortening is associated with growth retardation and congenital malformations. However, these studies were performed in newborns or postnatally, and data on telomere length (TL) during the prenatal period are still very limited. We measured TL using quantitative PCR in amniotic fluid (AF) and chorionic villi (CV) samples from 69 control fetuses with normal ultrasound (52 AF and 17 CV) and 213 fetuses (165 AF and 48 CV) with intrauterine growth retardation (IUGR) or congenital malformations diagnosed by ultrasound. The samples were collected by amniocentesis at the gestational age (GA) of 25.0 ± 5.4 weeks and by CV biopsy at 18.1 ± 6.3 weeks. In neither sample type was TL influenced by GA or fetal sex. In AF, a comparison of abnormal versus normal fetuses showed a significant telomere shortening in cases of IUGR (reduction of 34%, P < 10-6), single (29%, P < 10-6) and multiple (44%, P < 10-6) malformations. Similar TL shortening was also observed in CV from abnormal fetuses but to a lesser extent (25%, P = 0.0002; 18%, P = 0.016; 20%, P = 0.004, respectively). Telomere shortening was more pronounced in cases of multiple congenital anomalies than in fetuses with a single malformation, suggesting a correlation between TL and the severity of fetal phenotype. Thus, TL measurement in fetal samples during pregnancy could provide a novel predictive marker of pathological development.

Telomere Status of Advanced Non-Small-Cell Lung Cancer Offers a Novel Promising Prognostic and Predictive Biomarker.

Telomere length appears to correlate with survival in early non-small-cell lung cancer (NSCLC), but the prognostic impact of telomere status in advanced NSCLC remains undetermined. Our purpose was to evaluate telomere parameters as prognostic and predictive biomarkers in advanced NSCLC. In 79 biopsies obtained before treatment, we analyzed the telomere length and expression of TERT and shelterin complex genes (TRF1, TRF2, POT1, TPP1, RAP1, and TIN2), using quantitative PCR. Non-responders to first-line chemotherapy were characterized by shorter telomeres and low RAP1 expression (p = 0.0035 and p = 0.0069), and tended to show higher TERT levels (p = 0.058). In multivariate analysis, short telomeres were associated with reduced event-free (EFS, p = 0.0023) and overall survival (OS, p = 0.00041). TERT and TRF2 overexpression correlated with poor EFS (p = 0.0069 and p = 0.00041) and OS (p = 0.0051 and p = 0.007). Low RAP1 and TIN2 expression-levels were linked to reduced EFS (p = 0.00032 and p = 0.0069) and OS (p = 0.000051 and p = 0.02). Short telomeres were also associated with decreased survival after nivolumab therapy (p = 0.097). Evaluation of telomere status in advanced NSCLC emerges as a useful biomarker that allows for the selection of patient groups with different clinical evolutions, to establish personalized treatment.

Real-world outcomes following venetoclax therapy in patients with chronic lymphocytic leukemia or Richter syndrome: a FILO study of the French compassionate use cohort.

The BCL2 inhibitor venetoclax is transforming the management of patients with chronic lymphocytic leukemia (CLL), given its high efficacy in relapsed/refractory CLL as observed in both early-phase and randomized clinical trials. The present study aimed to determine whether venetoclax is effective and well tolerated in patients with CLL or Richter's syndrome (RS) in a real-world setting and to highlight factors impacting survival. Data from a venetoclax French compassionate use program were collected for 67 patients (60 with CLL and 7 with RS). Most patients presented adverse genetic features, such as TP53 disruption (74%) or complex karyotype (58%). Tumor lysis syndrome was observed in 14 (22%) patients, and 16 (24%) patients were hospitalized for grade III/IV infection. In the CLL cohort, ORR was 75 %, 1-year PFS was 61% (95% CI = 47-72%) and 1-year OS 70% (95% CI = 56-80%). No impact of TP53 disruption was noted while complex karyotype was identified as a predictor of both inferior PFS (HR = 3.46; 95% CI = 1-12; log-rank p = 0.03) and OS (HR = 3.2; 95% CI = 0.9-11.4, log-rank p = 0.047). Among the seven patients with RS, two achieved an objective response to venetoclax; however, the median OS was only 1.1 month. The well-balanced safety/efficacy profile of venetoclax is confirmed in this real-world setting. Complex karyotype should be evaluated as a predictive factor of survival for patients treated by venetoclax.