Precision Medicine Approach Based on Molecular Alterations for Patients with Relapsed or Refractory Multiple Myeloma: Results from the MM-EP1 Study.

BACKGROUND: Despite that cytogenetic and molecular analysis of tumor cells can rapidly identify recurring molecular abnormalities, no personalized therapy is currently available in the setting of relapsed/refractory multiple myeloma (r/r MM). METHODS: MM-EP1 is a retrospective study aimed at comparing a personalized molecular-oriented (MO) versus a non-molecular-oriented (no-MO) approach in r/r MM. Actionable molecular targets and their associated therapies were the BRAF V600E mutation and BRAF inhibitors; t(11;14)(q13;q32) and BCL2 inhibitors; and t(4;14)(p16;q32) with FGFR3 fusion/rearrangements and FGFR3 inhibitors. RESULTS: One hundred three highly pretreated r/r MM patients with a median age of 67 years (range 44-85) were included. Seventeen (17%) patients were treated using an MO approach with BRAF inhibitors (vemurafenib or dabrafenib, n = 6), BCL2 inhibitor (venetoclax, n = 9), or FGFR3 inhibitor (erdafitinib, n = 2). Eighty-six (86%) patients received non-MO therapies. Overall response rate was 65% in MO patients versus 58% in the non-MO group (p = 0.053). Median PFS and OS were 9 and 6 months (HR = 0.96; CI95 = 0.51-1.78; p = 0.88) and 26 and 28 months (HR = 0.98; CI95 = 0.46-2.12; p = 0.98), respectively, in MO and no-MO patients. CONCLUSION: Despite the low number of patients treated with an MO approach, this study highlights the strengths and weakness of a molecular-targeted approach for the treatment of multiple myeloma. Widespread biomolecular techniques and improvement of precision medicine treatment algorithms could improve selection for precision medicine in myeloma.

Sequence analyses of relapsed or refractory diffuse large B-cell lymphomas unravel three genetic subgroups of patients and the GNA13 mutant as poor prognostic biomarker, results of LNH-EP1 study.

Advances in molecular profiling of newly diagnosed diffuse large B-cell lymphoma (DLBCL) have recently refine genetic subgroups. Genetic subgroups remain undetermined at the time of relapse or refractory (RR) disease. This study aims to decipher genetic subgroups and search for prognostic molecular biomarkers in patients with RR-DLBCL. From 2015 to 2021, targeted next-generation sequencing analyses of germline-matched tumor samples and fresh tissue from RR-DLBCL patients were performed. Unsupervised clustering of somatic mutations was performed and correlations with patient outcome were sought. A number of 120 patients with RR-DLBCL were included in LNH-EP1 study and a molecular tumor landscape was successfully analyzed in 87% of patients (104/120 tumor samples). The median age was 67.5 years (range 27.4-87.4), median number of previous treatments was 2 (range 1-9). The most frequently mutated genes were TP53 (n = 53 mutations; 42% of samples), CREBBP (n = 39; 32%), BCL2 (n = 86; 31%), KMT2D (n = 39; 28%) and PIM1 (n = 54; 22%). Unsupervised clustering separated three genetic subgroups entitled BST (enriched in BCL2, SOCS1, and TNFRSF14 mutations); TKS (enriched in TP53, KMT2D, and STAT6 mutations); and PCM (enriched in PIM1, CD79B, and MYD88 mutations). Median overall survival (OS) was 11.0 (95% confidence interval [CI]: 8.1-12.6) months. OS was not significantly different between the three genetic subgroups. GNA13 mutant was significantly associated with an increased risk of death (hazard ratio: 6.6 [95% CI: 2.1-20.6]; p = .0011) and shorter OS (p = .0340). At the time of relapse or refractory disease, three genetic subgroups of DLBCL patients were delineated, which could help advance precision molecular medicine programs.

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor.

BACKGROUND: hPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages. METHODS: Two hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other's (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations. RESULTS: A hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors. CONCLUSION: The present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

Molecular Landscape of Therapy-related Myeloid Neoplasms in Patients Previously Treated for Gynecologic and Breast Cancers.

Definition of therapy-related myeloid neoplasms (TRMN) is only based on clinical history of exposure to leukemogenic therapy. No specific molecular classification combining therapy-related acute myeloid leukemia and therapy-related myelodysplastic syndromes has been proposed. We aimed to describe the molecular landscape of TRMN at diagnosis, among 77 patients with previous gynecologic and breast cancer with a dedicated next-generation sequencing panel covering 74 genes. We investigated the impact of clonal hematopoiesis of indeterminate potential-associated mutations (CHIP-AMs defined as presence at TRMN stage of mutations described in CHIP with a frequency >1%) on overall survival (OS) and the clinical relevance of a modified genetic ontogeny-based classifier that categorized patients in 3 subgroups. The most frequently mutated genes were TP53 (31%), DNMT3A (19%), IDH1/2 (13%), NRAS (13%), TET2 (12%), NPM1 (10%), PPM1D (9%), and PTPN11 (9%). CHIP-AMs were detected in 66% of TRMN patients, with no impact on OS. Yet, patients with CHIP-AM were older and had a longer time interval between solid tumor diagnosis and TRMN. According to our modified ontogeny-based classifier, we observed that the patients with TP53 or PPM1D mutations had more treatment lines and complex karyotypes, the "MDS-like" patients were older with more gene mutations, while patients with "De novo/pan-AML" mutations were younger with more balanced chromosomal translocations. Median OS within each subgroup was 7.5, 14.5, and 25.2 months, respectively, with statistically significant difference in multivariate analysis. These results support the integration of cytogenetic and molecular markers into the future TRMN classification to reflect the biological diversity of TRMN and its impact on outcomes.

Clonal heterogeneity of acute myeloid leukemia treated with the IDH2 inhibitor enasidenib.

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.

Discovery of AG-120 (Ivosidenib): A First-in-Class Mutant IDH1 Inhibitor for the Treatment of IDH1 Mutant Cancers.

Somatic point mutations at a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer a novel gain of function in cancer cells, resulting in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite. Elevated 2-HG levels are implicated in epigenetic alterations and impaired cellular differentiation. IDH1 mutations have been described in an array of hematologic malignancies and solid tumors. Here, we report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1 mutant enzyme that exhibits profound 2-HG lowering in tumor models and the ability to effect differentiation of primary patient AML samples ex vivo. Preliminary data from phase 1 clinical trials enrolling patients with cancers harboring an IDH1 mutation indicate that AG-120 has an acceptable safety profile and clinical activity.

B-cell tumor development in Tet2-deficient mice.

The TET2 gene encodes an α-ketoglutarate-dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell-specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2-/- malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA-specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda-deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A-induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.

A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model.

D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.

Quantitation of isocitrate dehydrogenase (IDH)-induced D and L enantiomers of 2-hydroxyglutaric acid in biological fluids by a fully validated liquid tandem mass spectrometry method, suitable for clinical applications.

A recent update of the hallmarks of cancer includes metabolism with deregulating cellular energetics. Activating mutations in isocitrate dehydrogenase (IDH) metabolic enzymes leading to the abnormal accumulation of 2-hydroxyglutaric acid (2-HGA) have been described in hematologic malignancies and solid tumours. The diagnostic value of 2-HGA levels in blood to identify IDH mutations and its prognostic significance have been reported. We developed a liquid chromatography tandem mass spectrometry method allowing a rapid, accurate and precise simultaneous quantification of both L and D enantiomers of 2-HGA in blood samples from acute myeloid leukaemia (AML) patients, suitable for clinical applications. The method was also develop for preclinical applications from cellular and tissues samples. Deuterated (R,S)-2-hydroxyglutaric acid, disodium salt was used as internal standard and added to samples before a solid phase extraction on Phenomenex STRATA™-XL-A (200mg-3mL) 33µm cartridges. A derivatization step with (+)- o,o'-diacetyl-l-tartaric anhydride permitted to separate the two resulting diastereoisomers without chiral stationary phase, on a C18 column combined to a Xevo TQ-MS Waters mass spectrometer with an electrospray ionization (ESI) source. This method allows standard curves to be linear over the range 0.34-135.04µM with r(2) values>0.999 and low matrix effects (<11.7%). This method, which was validated according to current EMA guidelines, is accurate between-run (<3.1%) and within-run (<7.9%) and precise between-run (<5.3CV%) and within-run (<6.2CV%), and is suitable for clinical and preclinical applications.

Serum 2-hydroxyglutarate production in IDH1- and IDH2-mutated de novo acute myeloid leukemia: a study by the Acute Leukemia French Association group.

PURPOSE: Mutated isocitrate dehydrogenases (IDHs) 1 and 2 produce high levels of 2-hydroxyglutarate (2-HG). We investigated whether, in acute myeloid leukemia (AML), serum 2-HG would predict the presence of IDH1/2 mutations at diagnosis and provide a marker of minimal residual disease (MRD). PATIENTS AND METHODS: Serum samples from 82 patients at diagnosis of de novo AML (IDH1/2 mutated, n = 53) and 68 patients without AML were analyzed for total 2-HG and its ratio of D to L stereoisomers by mass spectrometry. We measured 2-HG levels and molecular markers of MRD (WT1 and NPM1) in serial samples of 36 patients with IDH1/2 mutations after induction therapy. RESULTS: In patients with AML with IDH1/2 mutations, 2-HG serum levels were significantly higher than in patients with IDH1/2 wild type (P < .001). Area under the receiver operating characteristic curve was 99%. The optimum diagnostic cutoff between IDH1/2 mutated and normal was 2 µmol/L (sensitivity, 100%; specificity, 79%). Quantification of the D/L stereoisomers increased specificity (100%; 95% CI, 83% to 100%) compared with total 2-HG (P = .031). In patients with IDH2 R172 mutations, 2-HG levels were higher relative to those with other IDH1/2 mutations (P < .05). During follow-up, serum 2-HG levels showed strong positive correlation with WT1 and NPM1 (P < .001). After induction therapy, total 2-HG serum levels < 2 µmol/L were associated with better overall (P = .008) and disease-free survival (P = .005). CONCLUSION: Serum 2-HG is a predictor of the presence of IDH1/2 mutations and outcome in these patients. Discrimination between D/L stereoisomers improved specificity.

Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation.

A number of human cancers harbor somatic point mutations in the genes encoding isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These mutations alter residues in the enzyme active sites and confer a gain-of-function in cancer cells, resulting in the accumulation and secretion of the oncometabolite (R)-2-hydroxyglutarate (2HG). We developed a small molecule, AGI-6780, that potently and selectively inhibits the tumor-associated mutant IDH2/R140Q. A crystal structure of AGI-6780 complexed with IDH2/R140Q revealed that the inhibitor binds in an allosteric manner at the dimer interface. The results of steady-state enzymology analysis were consistent with allostery and slow-tight binding by AGI-6780. Treatment with AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human acute myelogenous leukemia cells in vitro. These data provide proof-of-concept that inhibitors targeting mutant IDH2/R140Q could have potential applications as a differentiation therapy for cancer.

TET2, a tumor suppressor in hematological disorders.

The TET family of proteins has been described a few years ago. Only recently, their roles in DNA modification, through the oxidation of methyl-cytosine, and in normal and malignant development, through the description of TET2 as a tumor suppressor have been documented. The conjunction of these findings has prompted large efforts to understand the biology of these novel entities. Here, we summarize the recent results implicating TET2 in hematological malignancies suggesting that further studies are required to fully understand the role of DNA methylation alterations during transformation.

TET2 inactivation results in pleiotropic hematopoietic abnormalities in mouse and is a recurrent event during human lymphomagenesis.

Loss-of-function mutations affecting one or both copies of the Ten-Eleven-translocation (TET)2 gene have been described in various human myeloid malignancies. We report that inactivation of Tet2 in mouse perturbs both early and late steps of hematopoiesis including myeloid and lymphoid differentiation in a cell-autonomous manner, endows the cells with competitive advantage, and eventually leads to the development of malignancies. We subsequently observed TET2 mutations in human lymphoid disorders. TET2 mutations could be detected in immature progenitors endowed with myeloid colony-forming potential. Our results show that the mutations present in lymphoid tumor cells may occur at both early and later steps of lymphoid development and indicate that impairment of TET2 function or/and expression predisposes to the development of hematological malignancies.