A p53 score derived from TP53 CRISPR/Cas9 HMCLs predicts survival and reveals a major role of BAX in the response to BH3 mimetics.

ABSTRACT: To establish a strict p53-dependent gene-expression profile, TP53-/- clones were derived from TP53+/+ and TP53-/mut t(4;14) human myeloma cell lines (HMCLs) using CRISPR/Cas9 technology. From the 17 dysregulated genes shared between the TP53-/- clones from TP53+/+ HMCLs, we established a functional p53 score, involving 13 genes specifically downregulated upon p53 silencing. This functional score segregated clones and myeloma cell lines as well as other cancer cell lines according to their TP53 status. The score efficiently identified samples from patients with myeloma with biallelic TP53 inactivation and was predictive of overall survival in Multiple Myeloma Research Foundation-coMMpass and CASSIOPEA cohorts. At the functional level, we showed that among the 13 genes, p53-regulated BAX expression correlated with and directly affected the MCL1 BH3 mimetic S63845 sensitivity of myeloma cells by decreasing MCL1-BAX complexes. However, resistance to S63845 was overcome by combining MCL1 and BCL2 BH3 mimetics, which displayed synergistic efficacy. The combination of BH3 mimetics was effective in 97% of patient samples with or without del17p. Nevertheless, single-cell RNA sequencing analysis showed that myeloma cells surviving the combination had lower p53 score, showing that myeloma cells with higher p53 score were more sensitive to BH3 mimetics. Taken together, we established a functional p53 score that identifies myeloma cells with biallelic TP53 invalidation, demonstrated that p53-regulated BAX is critical for optimal cell response to BH3 mimetics, and showed that MCL1 and BCL2 BH3 mimetics in combination may be of greater effectiveness for patients with biallelic TP53 invalidation, for whom there is still an unmet medical need.

Diagnosis and prognosis are supported by integrated assessment of next-generation sequencing in chronic myeloid malignancies. A real-life study.

Next-generation sequencing (NGS) is used to investigate the presence of somatic mutations. The utility of incorporating routine sequencing to guide diagnosis and therapeutic decisions remains unclear. We report the findings of an observational, multicenter study that aimed to assess the impact of somatic mutation testing by NGS in a reallife setting of chronic myeloid malignancies. A total of 177 patients were enrolled, partitioned into two overlapping groups. In group A (n=94), the indication was to search for clonal hematopoiesis, in a context of suspected myelodysplastic syndrome or myeloproliferative neoplasia. In group B (n=95), the theranostic impact of somatic mutations was studied. A panel of 34 genes was used on DNA extracted from blood or bone marrow samples. Within group A, the detection of clonal hematopoiesis supported the diagnosis of chronic myeloid malignancies for 31 patients while the absence of clonal hematopoiesis ruled out the suspected diagnosis in 47 patients. Within group B, NGS identified prognostically relevant somatic mutations in 32 patients, which had a therapeutic impact in 18 cases. By determining the presence or absence of somatic mutations, the application of NGS in daily practice was found to be useful for an integrated final diagnosis in 83% of the patients. Moreover, the search for somatic mutations had a prognostic impact that led to treatment modification in 19% of the cases. This study outlines the fact that adequate implementation of new investigations may have a significant positive medico-economic impact by enabling appropriate management of patients.

Epidemiology, clinical picture and long-term outcomes of FIP1L1-PDGFRA-positive myeloid neoplasm with eosinophilia: Data from 151 patients.

FIP1L1-PDGFRA-positive myeloid neoplasm with eosinophilia (F/P+ MN-eo) is a rare disease: robust epidemiological data are lacking and reported issues are scarce, of low sample-size and limited follow-up. Imatinib mesylate (IM) is highly efficient but no predictive factor of relapse after discontinuation has yet been identified. One hundred and fifty-one patients with F/P+ MN-eo (143 males; mean age at diagnosis 49 years; mean annual incidence: 0.18 case per million population) were included in this retrospective nationwide study involving all French laboratories who perform the search of F/P fusion gene (study period: 2003-2019). The main organs involved included the spleen (44%), skin (32%), lungs (30%), heart (19%) and central nervous system (9%). Serum vitamin B12 and tryptase levels were elevated in 74/79 (94%) and 45/57 (79%) patients, respectively, and none of the 31 patients initially treated with corticosteroids achieved complete hematologic remission. All 148 (98%) IM-treated patients achieved complete hematologic and molecular (when tested, n = 84) responses. Forty-six patients eventually discontinued IM, among whom 20 (57%) relapsed. In multivariate analysis, time to IM initiation (continuous HR: 1,01 [0.99-1,03]; P = .05) and duration of IM treatment (continuous HR: 0,97 [0,95-0,99]; P = .004) were independent factors of relapse after discontinuation of IM. After a mean follow-up of 80 (56) months, the 1, 5- and 10-year overall survival rates in IM-treated patients were 99%, 95% and 84% respectively. In F/P+ MN-eo, prompt initiation of IM and longer treatment durations may prevent relapses after discontinuation of IM.

FLT3 ligand plasma levels have no impact on outcomes after allotransplant in acute leukemia.

OBJECTIVE: This study was designed to assess the impact on outcomes of early soluble Fms-like tyrosine kinase 3 ligand concentrations (sFLc) in patients receiving an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: This was a prospective monocentric study including all allo-HSCT patients included in the previous FLAM/FLAL study (Peterlin et al., 2019). Blood samples collected before the start of conditioning then post-transplant were frozen, stored and tested by ELISA. The parameters considered were hematopoietic recoveries, Leukemia Free Survival and Overall Survival, acute and chronic GVHD, grade 3 or 4 acute and/or extensive chronic GVHD-free and relapse-free survival (GRFS). RESULTS: Forty-one patients were included, a total of 179 samples were assayed for sFLc. There was no impact of sFLc levels (<=median vs> median) on acute and chronic GVHD incidences, LFS, OS nor GRFS. CONCLUSION: At variance with induction results for AML (Peterlin et al., 2019) endogenous sFLc do not appear to be a prognostic marker at the time of or after allo-HSCT. Even though the results are negatives, this is, to the best of our knowledge, the only prospective series specifically addressing the question of sFLc impact after allo-HSCT in acute leukemias.

A novel complete blood count-based score to screen for myelodysplastic syndrome in cytopenic patients.

The diagnosis of myelodysplastic syndromes (MDS) is often challenging, time- and resource-consuming. A thorough analysis of complete blood count (CBC) parameters could, however, help to screen for MDS among other causes of cytopenia. To test this hypothesis, 109 newly-diagnosed MDS patients and 399 cytopenic patients older than 50 years with confirmed absence of MDS were enrolled in a prospective study. Multiparametric analysis highlighted three CBC parameters that were significantly different between the two cohorts: mean corpuscular volume, absolute neutrophil count and median neutrophil complexity and width of dispersion of the events measured (Ne-WX), which were used to define an MDS-CBC score. This score enables the prediction of MDS with 86% sensitivity and 88% specificity. The MDS-CBC score excluded MDS in 89% of cytopenic controls. Moreover, high score values at MDS diagnosis significantly correlated with decreased event-free (P = 0·02) and overall survival (P = 0·01). The power of this score was confirmed in an independent validation cohort (MDS n = 34, cytopenic controls n = 28). The MDS-CBC score is an easy and fast tool to exclude or suspect MDS in unselected patients with cytopenia of unknown reasons at the time of analysis, by prompting blood smear examination. It may thus improve allocation of further MDS-specific work-up in patients with cytopenia at the time of CBC assessment.

The doubling potential of T lymphocytes allows clinical-grade production of a bank of genetically modified monoclonal T-cell populations.

BACKGROUND AIMS: To produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited. METHODS: We used clinical-grade conditions and two donors who were homozygous and identical for all HLA-alleles except HLA-DP. After mixed lymphocyte culture and transduction, we obtained 14 HLA-DP-specific T-cell clones transduced with the HSV-TK suicide gene. Clones were then selected on the basis of their specificity and functional characteristics and evaluated for their doubling potential. RESULTS: After these steps of selection the clone NAT-DP4(TK), specific for HLA-DPB1*04:01/04:02, which produced high levels of interferon-γ (IFNγ), tumor necrosis factor (TNF), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was fully sequenced. It has two copies of the HSV-TK suicide transgene whose localizations were determined. Four billion NAT-DP4(TK) cells were frozen after 50 PDs. Thawed NAT-DP4(TK) cells retain the potential to undergo 50 additional PDs, a potential very far beyond that required to produce a biological effect. This PD potential was confirmed on 6/16 additional different T-cell clones. This type of well-defined clone can also support a second genetic modification with CAR constructs. CONCLUSION: The possibility of choosing rare donors and exploiting the natural proliferative potential of T lymphocytes may dramatically reduce the clinical and immunologic complexity of adoptive transfer protocols that rely on the use of third-party T-cell populations.

"Double-hit" chronic lymphocytic leukemia: An aggressive subgroup with 17p deletion and 8q24 gain.

Chronic lymphocytic leukemia (CLL) with 17p deletion (17p-) is associated with a lack of response to standard treatment and thus the worst possible clinical outcome. Various chromosomal abnormalities (including unbalanced translocations, deletions, ring chromosomes and isochromosomes) result in the loss of 17p and one copy of the TP53 gene. The objective of the present study was to determine whether the type of chromosomal abnormality leading to 17p- and the additional aberrations influenced the prognosis in a series of 195 patients with 17p-CLL. Loss of 17p resulted primarily from an unbalanced translocation (70%) with several chromosome partners (the most frequent being chromosome 18q), followed by deletion 17p (23%), monosomy 17 (8%), isochromosome 17q [i(17q)] (5%) and a ring chromosome 17 (2%). In a univariate analysis, monosomy 17, a highly complex karyotype (≥5 abnormalities), and 8q24 gain were associated with poor treatment-free survival, and i(17q) (P = .04), unbalanced translocations (P = .03) and 8q24 gain (P = .001) were significantly associated with poor overall survival. In a multivariate analysis, 8q24 gain remained a significant predictor of poor overall survival. We conclude that 17p deletion and 8q24 gain have a synergistic impact on outcome, and so patients with this "double-hit" CLL have a particularly poor prognosis. Systematic, targeting screening for 8q24 gain should therefore be considered in cases of 17p- CLL.

Is allogeneic stem cell transplantation for myelofibrosis still indicated at the time of molecular markers and JAK inhibitors era?

OBJECTIVE: The role of allogenic stem cell transplantation (ASCT) is still debated in myelofibrosis (MF). METHODS: A retrospective analyzed was performed to compare the outcome of 71 patients with intermediate-2 or high-risk Dynamic International Prognosis Scoring System+ (DIPSS+) primary (PMF) or secondary (SMF) myelofibrosis with an indication of ASCT as they ultimately underwent the procedure (n=34) or not (n=37). RESULTS: Five-year overall survival (OS) was not statistically different between both groups (allograft: 52% vs no allograft: 34%, P=.12). However, progression to myelodysplastic syndrome or acute myeloid leukemia at 5 years was significantly lower in transplanted patients (14% vs 50%, P=.01). In univariate analysis, 5-year OS was significantly higher for transplanted vs non-transplanted patients with unfavorable karyotype (75% vs 0%, P=.001), SMF (71% vs 20%, P=.001) or high DIPSS+ score (46% vs 15%, P=.03). There was also a trend for better 5-year OS in allografted patients with high JAK2V617F burden (>65%) (75% vs 8%, P=.07). Interestingly, the survival of patients who did not proceed to ASCT was dramatically increased by the use of ruxolitinib. CONCLUSIONS: Not all intermediate-2/high-risk DIPSS+ MF patients benefit from ASCT, especially since the introduction of JAK2 inhibitors.

PRIMA-1Met induces myeloma cell death independent of p53 by impairing the GSH/ROS balance.

The aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 µM to more than 200 µM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 µM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo.

RITA (Reactivating p53 and Inducing Tumor Apoptosis) is efficient against TP53abnormal myeloma cells independently of the p53 pathway.

BACKGROUND: The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells. METHODS: A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines. RESULTS: Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥ 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤ 19%). CONCLUSION: These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.

Cytogenetics in the management of multiple Myeloma: The guidelines from the Groupe Francophone de Cytogénétique Hématologique (GFCH).

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow. Despite considerable advances in the treatment, MM is considered an incurable chronic disease with a very heterogeneous prognosis, mostly depending on genomic alterations whose complexity evolves over time. The cytogenetic analysis of MM is performed on CD138+ sorted PCs, in order to detect the following high risk cytogenetic abnormalities: t(4;14), 17p/TP53 deletion, 1q21 gain/amplification, 1p32 deletion, as well as t(11;14) because of its therapeutic implication. This minimal panel can be enlarged to detect other recurrent abnormalities, according to the prognostic score chosen by the laboratory. Although the knowledge of the genetic landscape of MM is evolving rapidly with improved molecular technologies, risk scores remain to be refined as they require more time for consensual validation. The GFCH present here the overview of genomics alterations identified in MM and related PCs diseases associated with their prognostic factor, when available, and recommendations from an expert group for identification and characterization of those alterations. This work is the update of previous 2016 recommendations.

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.

Added value of molecular karyotype in childhood acute lymphoblastic leukemia.

Background: Thanks to an improved therapeutic regimen in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), 5 year-overall survival now exceeds 90%. Unfortunately, the 25% of children who relapse have an initial poor prognosis, potentially driven by pre-existing or emerging molecular anomalies. The latter are initially and essentially identified by cytogenetics. However, some subtle alterations are not visible through karyotyping. Methods: Single nucleotide polymorphisms (SNP) array is an alternative way of chromosomal analysis allowing for a more in-depth evaluation of chromosomal modifications such as the assessment of copy number alterations (CNA) and loss of heterozygosity (LOH). This method was applied here in retrospective diagnosis/relapse paired samples from seven children with BCP-ALL and in a prospective cohort of 38 newly diagnosed childhood cases. Results: In the matched study, compared to the initial karyotype, SNP array analysis reclassified two patients as poor prognosis cases. Modulation during relapse was seen for 4 CNA and 0.9 LOH. In the prospective study, SNP reclassified the 10 patients with intermediate karyotype as 7 good prognosis and 3 poor prognosis. Ultimately, in all the children tested, SNP array allowed to identify additional anomalies compared to conventional karyotype, refine its prognostic value and identify some druggable anomalies that could be used for precision medicine. Overall, the anomalies detected could be segregated in four groups respectively involved in B-cell development, cell proliferation, transcription and molecular pathways. Conclusion: SNP therefore appears to be a method of choice in the integrated diagnosis of BCP ALL, especially for patients initially classified as intermediate prognosis. This complementary method of both cytogenetics and high throughput sequencing allows to obtain further classified information and can be useful in case of failure of these techniques.

A new cytokine-based dynamic stratification during induction is highly predictive of survivals in acute myeloid leukemia.

The aim of this study was to assess the potential impact of the kinetics of serum levels of seven cytokines during induction in acute myeloid leukemia (AML) patients. Indeed, the role of cytokines, in the pathophysiology and response to therapy of AML patients, remains under investigation. Here, we report on the impact of peripheral levels of two cytokines, the Fms-like tyrosine kinase 3 ligand (FL) and interleukin-6 (IL-6), evaluated during first-line intensive induction. A new risk stratification can be proposed, which supersedes the ELN 2017 classification to predict survivals in AML patients by examining the kinetic profile of these cytokines during the induction phase. It segregates three groups of, respectively, high-risk, characterized by a stagnation of low FL levels, intermediate risk, with dynamic increasing FL levels and high IL-6 at day 22, and favorable risk with increasing FL levels but low IL-6 at day 22.