Notch2 Increases the Resistance to Venetoclax-Induced Apoptosis in Chronic Lymphocytic Leukemia B Cells by Inducing Mcl-1

Chronic lymphocytic leukemia (CLL) has experienced a clinical revolution—thanks to the discovery of crucial pathogenic mechanisms. CLL is still an incurable disease due to intrinsic or acquired resistance of the leukemic clone. Venetoclax is a Bcl-2 inhibitor with a marked activity in CLL, but emerging patterns of resistance are being described. We hypothesize that intrinsic features of CLL cells may contribute to drive mechanisms of resistance to venetoclax. We analyzed the expression of Interferon Regulatory Factor 4 (IRF4), Notch2, and Mcl-1 in a cohort of CLL patients. We evaluated CLL cell viability after genetic and pharmaceutical modulation of Notch2 expression in patients harboring trisomy 12. We tested venetoclax in trisomy 12 CLL cells either silenced or not for Notch2 expression or in combination with an inhibitor of Mcl-1, AMG-176. Trisomy 12 CLL cells were characterized by low expression of IRF4 associated with high levels of Notch2 and Mcl-1. Notch2 and Mcl-1 expression determined protection of CLL cells from spontaneous and drug-induced apoptosis. Considering the involvement of Mcl-1 in venetoclax resistance, our data demonstrated a contribution of high levels of Notch2 and Mcl-1 in a reduced response to venetoclax in CLL cells carrying trisomy 12. Furthermore, reduction of Mcl-1 expression by silencing Notch2 or by treatment with AMG-176 was able to restore the response of CLL cells to venetoclax. The expression of Notch2 identifies a subset of CLL patients, mainly harboring trisomy 12, characterized by high levels of Mcl-1. This biological mechanism may compromise an effective response to venetoclax.

A Review on the Therapeutic Role of TKIs in Case of CML in Combination With Epigenetic Drugs

Chronic myeloid leukemia is a malignancy of bone marrow that affects white blood cells. There is strong evidence that disease progression, treatment responses, and overall clinical outcomes of CML patients are influenced by the accumulation of other genetic and epigenetic abnormalities, rather than only the BCR/ABL1 oncoprotein. Both genetic and epigenetic factors influence the efficacy of CML treatment strategies. Targeted medicines known as tyrosine-kinase inhibitors have dramatically improved long-term survival rates in CML patients during the previous 2 decades. When compared to earlier chemotherapy treatments, these drugs have revolutionized CML treatment and allowed most people to live longer lives. Although epigenetic inhibitors’ activity is disrupted in many cancers, including CML, but when combined with TKI, they may offer potential therapeutic strategies for the treatment of CML cells. The epigenetics of tyrosine kinase inhibitors and resistance to them is being studied, with a particular focus on imatinib, which is used to treat CML. In addition, the use of epigenetic drugs in conjunction with TKIs has been discussed. Resistance to TKIs is still a problem in curing the disease, necessitating the development of new therapies. This study focused on epigenetic pathways involved in CML pathogenesis and tumor cell resistance to TKIs, both of which contribute to leukemic clone breakout and proliferation.

Comprehensive analysis of mutations and clonal evolution patterns in a cohort of patients with cytogenetically normal acute myeloid leukemia

Background Relapsed acute myeloid leukemia (AML) is associated with the acquisition of additional somatic mutations which are thought to drive phenotypic adaptability, clonal selection and evolution of leukemic clones during treatment. We performed high throughput exome sequencing of matched presentation and relapsed samples from 6 cytogenetically normal AML (CN-AML) patients treated with standard remission induction chemotherapy in order to contribute with the investigation of the mutational landscape of CN-AML and clonal evolution during AML treatment. Result A total of 24 and 32 somatic variants were identified in presentation and relapse samples respectively with an average of 4.0 variants per patient at presentation and 5.3 variants per patient at relapse, with SNVs being more frequent than indels at both disease stages. All patients have somatic variants in at least one gene that is frequently mutated in AML at both disease presentation and relapse, with most of these variants are classic AML and recurrent hotspot mutations including NPM1 p.W288fs, FLT3-ITD, NRAS p.G12D and IDH2 p.R140Q. In addition, we found two distinct clonal evolution patterns of relapse: (1) a leukemic clone at disease presentation acquires additional mutations and evolves into the relapse clone after the chemotherapy; (2) a leukemic clone at disease presentation persists at relapse without the addition of novel somatic mutations. Conclusions The findings of this study suggest that the relapse-initiating clones may pre-exist prior to therapy, which harbor or acquire mutations that confer selective advantage during chemotherapy, resulting in clonal expansion and eventually leading to relapse.

Can the New and Old Drugs Exert an Immunomodulatory Effect in Acute Myeloid Leukemia?

Acute myeloid leukemia (AML) is considered an immune-suppressive neoplasm capable of evading immune surveillance through cellular and environmental players. Increasing knowledge of the immune system (IS) status at diagnosis seems to suggest ever more attention of the crosstalk between the leukemic clone and its immunologic counterpart. During the last years, the advent of novel immunotherapeutic strategies has revealed the importance of immune dysregulation and suppression for leukemia fitness. Considering all these premises, we reviewed the “off-target” effects on the IS of different drugs used in the treatment of AML, focusing on the main advantages of this interaction. The data reported support the idea that a successful therapeutic strategy should consider tailored approaches for performing leukemia eradication by both direct blasts killing and the engagement of the IS.

Chronic Myelomonocytic Leukemia Gold Jubilee

Chronic myelomonocytic leukemia (CMML) was named 50 years ago to describe a myeloid malignancy whose onset is typically insidious. This disease is now classified by the World Health Organisation as a myelodysplastic syndrome (MDS)-myeloproliferative neoplasm (MPN) overlap disease. Observed mostly in ageing people, CMML is characterized by the expansion of monocytes and, in many cases, granulocytes. Abnormal repartition of circulating monocyte subsets, as identified by flow cytometry, facilitates disease recognition. CMML is driven by the accumulation, in the stem cell compartment, of somatic variants in epigenetic, splicing and signaling genes, leading to epigenetic reprogramming. Mature cells of the leukemic clone contribute to creating an inflammatory climate through the release of cytokines and chemokines. The suspected role of the bone marrow niche in driving CMML emergence and progression remains to be deciphered. The clinical expression of the disease is highly diverse. Time-dependent accumulation of symptoms eventually leads to patient death as a consequence of physical exhaustion, multiple cytopenias and acute leukemia transformation. Fifty years after its identification, CMML remains one of the most severe chronic myeloid malignancies, without disease-modifying therapy. The proliferative component of the disease that distinguishes CMML from severe MDS has been mostly neglected. This review summarizes the progresses made in disease understanding since its recognition and argues for more CMML-dedicated clinical trials.

Case Report: Very Late, Atypical Extra-Medullary Relapse in a Patient With Acute Promyelocytic Leukemia (APL) Rescued With a Transplant-Free Approach

Relapses of acute promyelocytic leukemia (APL) beyond 7 years from the first molecular remission are exceptional, and it is unclear whether these relapses represent a new, therapy-related leukemia rather than a delayed relapse of the original leukemic clone. The increase extra-medullary relapses (ER) in the era of all-trans retinoic acid (ATRA) therapy suggests a potential correlation between ATRA therapy and ER, and several potential explanations have been proposed. The gold standard post-remission approach, particularly for patients in late relapse, has not yet been established. The benefit of a transplant approach has been questioned in this setting because continuing ATRA-arsenic trioxide (ATO) might be curative. Here we report on the case of an APL patient who relapsed 9 years after achieving her first molecular complete remission (mCR) and who showed an atypical isolated localization at nodal sites, including the into- and peri-parotid glands. Genomic PML/RARa breakpoint analysis detected the same bcr3 PML/RARa hybrid gene in DNA purified from bone marrow and lymph nodes, suggesting that the relapse was because of the reemergence of the initial clone. This case shows that APL, treated with ATRA and cytotoxic drugs, may still emerge in extra-medullary sites even after a very prolonged mCR and could be salvaged with an ATO-based protocol, not including a transplant approach.

Comprehensive analysis of mutations and clonal evolution patterns in a cohort of patients with cytogenetically normal acute myeloid leukemia

Background Relapsed acute myeloid leukemia (AML) is associated with the acquisition of additional somatic mutations which are thought to drive phenotypic adaptability driving clonal selection during treatment and evolution of leukemic clones. We performed high throughput exome sequencing of matched presentation and relapsed samples from 6 cytogenetically normal AML (CN-AML) patients treated with standard remission induction chemotherapy in order to investigate the mutational landscape of CN-AML and clonal evolution during AML treatment. Result A total of 24 and 32 somatic variants were identified in presentation and relapse samples respectively with an average of 4.0 variants per patient at presentation and 5.3 variants per patient at relapse, with SNVs were more frequent than indels at both disease stages. All patients have somatic variants in at least one gene that frequently mutated in AML at both disease presentation and relapse, with most of these variants are classic AML and recurrent hotspot mutations including NPM1 p.W288fs, FLT3-ITD, NRAS p.G12D and IDH2 p.R140Q. In addition, we found two clonal evolution patterns of relapse: (1) a leukemic clone at disease presentation acquires additional mutations and evolves into the relapse clone after the chemotherapy; (2) a leukemic clone at disease presentation persists at relapse without the addition of novel somatic mutations. Conclusion The findings from this study suggest that the relapse-initiating clones may pre-exist before therapy, which harbor or acquire mutations that confer selective advantage during chemotherapy, resulting in clonal expansion and eventually leading to relapse.

Nurse-Like Cells and Chronic Lymphocytic Leukemia B Cells: A Mutualistic Crosstalk inside Tissue Microenvironments

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is an example of hematological disease where cooperation between genetic defects and tumor microenvironmental interaction is involved in pathogenesis. CLL is a disease that is considered as “addicted to the host”; indeed, the crosstalk between leukemic cells and the tumor microenvironment is essential for leukemic clone maintenance supporting CLL cells’ survival, proliferation, and protection from drug-induced apoptosis. CLL cells are not innocent bystanders but actively model and manipulate the surrounding microenvironment to their own advantage. Besides the different players involved in this crosstalk, nurse-like cells (NLC) resemble features related to leukemia-associated macrophages with an important function in preserving CLL cell survival and supporting an immunosuppressive microenvironment. This review provides a comprehensive overview of the role played by NLC in creating a nurturing and permissive milieu for CLL cells, illustrating the therapeutic possibilities in order to specifically target and re-educate them.

Lack of Viral Load Within Chronic Lymphoproliferative Disorder of Natural Killer Cells: What Is Outside the Leukemic Clone?

Large granular lymphocyte leukemias (LGLL) are sustained by proliferating cytotoxic T cells or NK cells, as happens in Chronic Lymphoproliferative Disorder of Natural Killer cells (CLPD-NK), whose etiology is only partly understood. Different hypotheses have been proposed on the original events triggering NK cell hyperactivation and transformation, including a role of viral agents. In this perspective, we revise the lines of evidence that suggested a pathogenetic role in LGLL of the exposure to retroviruses and that identified Epstein Barr Virus (EBV) in other NK cell leukemias and lymphomas and focus on the contrasting data about the importance of viral agents in CLPD-NK. EBV was detected in aggressive NK leukemias but not in the indolent CLPD-NK, where seroreactivity against HTLV-1 retrovirus envelope BA21 protein antigens has been reported in patients, although lacking clear evidence of HTLV infection. We next present original results of whole exome sequencing data analysis that failed to identify viral sequences in CLPD-NK. We recently demonstrated that proliferating NK cells of patients harbor several somatic lesions likely contributing to sustain NK cell proliferation. Thus, we explore whether “neoantigens” similar to the BA21 antigen could be generated by aberrancies present in the leukemic clone. In light of the literature and new data, we evaluated the intriguing hypothesis that NK cell activation can be caused by retroviral agents located outside the hematopoietic compartment and on the possible mechanisms involved with the prospects of immunotherapy-based approaches to limit the growth of NK cells in CLPD-NK disease.

Comparison of Deep Whole Exome Versus Targeted Gene Sequencing for Assessment of Persistent Molecular Disease in Acute Myeloid Leukemia Samples

Background There is growing evidence that genomics-based assessment of persistent molecular disease (PMD) may be a useful risk stratification tool for patients with acute myeloid leukemia (AML). However, there is no consensus about the optimal approach for detection and monitoring of leukemia-associated mutations (LAMs) for PMD testing. One approach is whole exome sequencing (WES), which provides a comprehensive assessment of the clonal architecture by identifying LAMs across the exome at diagnosis, which can then be measured in follow-up samples. Another approach is to use highly sensitive targeted gene sequencing (TGS) to detect persistent LAMs even at very low levels, although detection is limited to genes interrogated by the panel. Although both of approaches have been shown to predict outcome in retrospective studies, there are substantial differences in the number of LAMs identified and the limit of detection, and a head-to-head comparison has not yet been reported. Here, we use a defined cohort enrolled in a prospective clinical trial to compare PMD results from deep WES to error-corrected TGS. Methods Cohort: Patients were age 18-60 with de novo AML classified as intermediate risk by European Leukemia Net criteria, who achieved a morphologic remission after undergoing standard induction therapy. All patients were enrolled in a prospective clinical trial (NCT02756962). Sequencing: Deep WES was performed using DNA from normal tissue (buccal swab or skin) and pre- and post-induction (~day 30) bone marrow (BM) samples to achieve an average coverage depth of ~600x. LAMs identified via paired tumor/normal analysis of the pre-induction sample were queried in the post-induction sample for assessment of PMD. PMD testing by TGS was performed using error-corrected sequencing of 40 genes recurrently mutated AML genes to an average error-corrected coverage depth of ~4500x. Definitions: Based on previously published work, we used two separate variant allele frequency (VAF) cutoffs to define PMD. For WES, PMD+ was defined as ≥1 LAM with a VAF >2.5% (Klco JAMA 2015). For TGS, PMD+ was defined as having ≥1 LAM with a VAF >0.5% (Duncavage NEJM 2018). LAMs were sub-classified per Table 1. TGS mutations not identified as LAMs by WES at diagnosis were excluded from the primary analysis. Results 31 patients were studied. LAMs are summarized in Table 1. 20 patients (65%) were PMD+ by WES after induction, and 22 patients (71%) were PMD+ by TGS. The concordance between WES and TGS was 81% (25/31) (Table 2). Two patients were PMD+ by WES only, due to the persistence of LAMs in the exome space, but not in the targeted panel. Four patients were PMD+ by TGS only, due to the presence of recurrent mutations at VAFs below the detection limit of WES (range: 0.59 - 1.90%). Two patients were PMD+ by both assays, but due to different mutations. All other patients who were PMD+ by both assays had at least one overlapping mutation. Analysis of the mutations that persisted after therapy in both assays showed that 26% of patients (8/31) were PMD+ by TGS because of mutations in DNMT3A, TET2, or ASXL1 (i.e., "DTA" mutations). All of these patients were also PMD+ by WES, with 7 of the 8 patients having ≥1 additional non-DTA mutation (median: 3, range: 1 - 7). In an exploratory analysis, 22 additional mutations in 13 patients were identified by TGS that were not detected by WES on the diagnostic sample. This included two patients who were PMD-, but who had new mutations in TET2 and DNMT3A, respectively, likely representing selection for ancestral clones that were unrelated to the AML founding clone. Conclusion Concordance between WES and TGS-based PMD assessment was high. Discordant results were generally driven by non-recurrent mutations detected by WES, and low-level mutations detected by the high coverage, error-corrected TGS. Although isolated DTA mutations were common on TGS, WES analysis showed additional LAMs accompanied these variants in most cases, indicating the persistence of an ancestral leukemic clone that may provide useful prognostic information. We also observed new, low-level mutations that emerged after therapy in 42% of patients, some of which were not part of the leukemic clone identified at diagnosis. This indicates that use of highly sensitive PMD approaches may be challenging without pre-induction mutation testing, which is required to understand the relevance of markers of persistent molecular disease. Disclosures Jacoby: AbbVie: Research Funding; Takeda: Consultancy; Jazz Pharmaceuticals: Research Funding.