Crisis Chronic Myeloid Leukemia Research Articles

A Case of Central Venous Catheter-Related Candida parapsilosis Fungemia Evolved to Disseminated Infection in a Neutropenic Patient with Blast Crisis of Chronic Myeloid Leukemia.

Central venous catheter-related infections are of particular importance in onco-hematological patients. Candida parapsilosis is generally reported as a mild pathogen, however it is able to effectively colonize intravascular devices and potentially give rise to sustained fungemias. Here we report a case of invasive, potentially lethal C. parapsilosis disseminated infection in a neutropenic patient affected by chronic myeloid leukemia with blast crisis. We underline the importance of removing the central venous catheter as potential source of infection as soon as possible during the course of candidemia, and not replacing it with other polyurethan intravascular devices, which pose a risk for the maintenance of the fungemia despite the administration of the best antifungal therapy available.

Unusual presentation of extramedullary blast crisis in chronic myeloid leukemia: A case report

BACKGROUND Extramedullary blast crisis in chronic myeloid leukemia (CML) is an uncommon occurrence of leukemic blast infiltration in regions other than the bone marrow. Malignant infiltration of the serosal membranes should be considered in cases where CML presents with ascites or pleural effusion. CASE SUMMARY A 23-year-old female with CML presented with progressively worsening ascites and pleural effusion despite first-line tyrosine kinase inhibitor treatment. Her blood work indicated leukocytosis with myelocyte bulge and 2% blasts. Analysis of the patient’s bone marrow confirmed the chronic phase of CML. Abdominal ultrasound revealed hepatosplenomegaly with ascites. The fluid investigation of both ascites and pleural effusion revealed a predominance of neutrophils with exudate. However, no acid-fast bacilli or growth was observed after culturing. Although hydroxyurea reduced cell counts, there was no observed effect on ascites or pleural effusion. Repeat investigation of the ascitic and pleural fluid revealed a polymorphous myeloid cell population consisting of myelocytes, metamyelocytes, band forms, neutrophils and a few myeloblasts. Extramedullary blast crisis was suspected, and mutation analysis was performed. We switched the patient to dasatinib. The patient’s symptoms were relieved, and ascites and pleural effusion diminished. CONCLUSION Serosal membrane involvement in CML is extremely rare. In this case, the patient responded well to dasatinib treatment.

Secondary acute myeloid leukemia and de novo acute myeloid leukemia with myelodysplasia-related changes - close or complete strangers?

To compare the main features of patients with secondary acute myeloid leukemias (AMLs) after post-myelodysplastic syndrome (AML-post-MDS) or post-myeloproliferative neoplasms (AML-post-MPN) and myeloid blast crisis of chronic myeloid leukemia (CML-BC) vs. de novoAMLs with myelodysplastic characteristics (dn-AML-MDS).

Complete miRNA-15/16 loss in mice promotes hematopoietic progenitor expansion and a myeloid-biased hyperproliferative state

Dysregulated apoptosis and proliferation are fundamental properties of cancer, and microRNAs (miRNA) are critical regulators of these processes. Loss of miR-15a/16-1 at chromosome 13q14 is the most common genomic aberration in chronic lymphocytic leukemia (CLL). Correspondingly, the deletion of either murine miR-15a/16-1 or miR-15b/16-2 locus in mice is linked to B cell lymphoproliferative malignancies. However, unexpectedly, when both miR-15/16 clusters are eliminated, most double knockout (DKO) mice develop acute myeloid leukemia (AML). Moreover, in patients with CLL, significantly reduced expression of miR-15a, miR-15b, and miR-16 associates with progression of myelodysplastic syndrome to AML, as well as blast crisis in chronic myeloid leukemia. Thus, the miR-15/16 clusters have a biological relevance for myeloid neoplasms. Here, we demonstrate that the myeloproliferative phenotype in DKO mice correlates with an increase of hematopoietic stem and progenitor cells (HSPC) early in life. Using single-cell transcriptomic analyses, we presented the molecular underpinning of increased myeloid output in the HSPC of DKO mice with gene signatures suggestive of dysregulated hematopoiesis, metabolic activities, and cell cycle stages. Functionally, we found that multipotent progenitors (MPP) of DKO mice have increased self-renewing capacities and give rise to significantly more progeny in the granulocytic compartment. Moreover, a unique transcriptomic signature of DKO MPP correlates with poor outcome in patients with AML. Together, these data point to a unique regulatory role for miR-15/16 during the early stages of hematopoiesis and to a potentially useful biomarker for the pathogenesis of myeloid neoplasms.

Bilateral Massive Retinal Infiltrates: Sign of Blast Crisis in Chronic Myeloid Leukemia

Bilateral Massive Retinal Infiltrates: Sign of Blast Crisis in Chronic Myeloid Leukemia

Blast crisis in chronic myeloid leukemia: An immunophenotypic analysis

Background and Aim of Study: There are two different phases of untreated chronic myeloid leukemia – chronic phase, and blast crisis – according to the World Health Organization classification of Hematolymphoid tumors. The blast cells in the chronic myeloid leukemia blast crisis can express myeloid, lymphoid, bi-phenotypic, monocytic, megakaryocytic, and erythroid phenotypes. The immunophenotype of blast population determines how chronic myeloid leukemia – blast crisis patients respond to treatment, hence flowcytometric examination is required. The aim of the study: to assess immunophenotyping outcomes of flowcytometry performed on the chronic myeloid leukemia – blast crisis. Material and Methods: A five-year retrospective descriptive analysis was carried out in Pathology Department at King George’s Medical University Lucknow, India (2017-2021). The patient’s peripheral blood and bone marrow aspirate samples were analyzed. Clinical, hematological, and immunophenotypic data were retrieved. The flow cytometry samples were prepared using the standardized “lyse-stain-wash” method. Results: A total of 43 cases of chronic myeloid leukemia – blast crisis were retrieved from the departmental archive in 5 years. The mean age of study population was 39.62±14.86 years. There were 24 males and 19 females. 27 patients were diagnosed with myeloid blast crisis, 14 cases of B-lymphoid blast crisis and 2 cases of mixed phenotypic acute leukemia. Conclusions: Identification of the blast lineage of patients with chronic myeloid leukemia – blast crisis is crucial since the existence of atypical blast phenotypes influences the disease treatment and prognosis.

Multicenter Phase I Study of Post-Transplant Low-Dose Inotuzumab Ozogamicin to Prevent Relapse of Acute Lymphoblastic Leukemia

Background: The curative potential of allogeneic transplant (alloHCT) in patients with acute lymphoblastic leukemia (ALL) is hampered by relapse, the major cause of treatment failure. Risk factors for relapse include measurable residual disease (MRD) before or after alloHCT, transplantation in second complete remission (CR) or beyond, and reduced intensity conditioning (RIC) regimens. In ALL patients, relapse rates range from 30% to 50%, with most relapses occurring within the first year after alloHCT. After relapse, options for disease control are limited, and consequently overall survival (OS) is poor. Post-alloHCT maintenance may reduce MRD, provide time for a graft-versus-leukemia effect to develop, and ultimately decrease relapse rates and prolong progression-free-survival (PFS) and overall survival (OS). Inotuzumab ozogamicin (INO) is an anti-CD22 monoclonal antibody bound to calicheamicin and approved to treat relapsed ALL. With the hypothesis that low-dose INO would be safe and reduce relapse rates, we conducted a phase I study to define INO dose in this setting. Herein we present the results of this multicenter study. Methods: Eligibility included patients aged 16-75 who underwent alloHCT for CD22+ALL and were in CR after transplant, had a high risk for relapse after alloHCT (MRD before or after alloHCT, transplantation,in second CR or beyond, RIC regimen, lymphoid blast crisis of chronic myeloid leukemia, or Ph-like ALL), had adequate graft and organ function after transplant (platelets > 50 ANC > 1000), did not have active grade III/IV aGVHD or any active liver GVHD, and had no history of hepatic VOD. Patients were consented after transplant. The primary objective was to define a post-alloHCT maximum tolerated dose (MTD) of INO. Secondary objectives included describing the safety profile of INO, determining the rate of hepatic veno-occlusive disease (VOD) in this setting, evaluating non-relapse mortality (NRM), PFS and OS at 1-year post-alloHCT, and determining if INO at these doses is effective at eradicating MRD. Pre-treatment tests included a bone marrow biopsy and aspirate to assess disease state and MRD. The trial design was a 3+3 model. The study treatment consisted of 28-day cycles (initially planned for 4 cycles, increased up to 12 cycles) of INO starting at dose level 0 (0.3mg/m2; n= 4), then 0.4 mg/m2 (n= 3), 0.5 mg/m2 (n= 6) and 0.6 mg/m2 (n= 5) given on cycle day 1. The first cycle was initiated between day 40 and 100 post-alloHCT. Pre-defined dose limiting toxicities (DLT) included VOD, prolonged cytopenia (more than 28 days), severe toxicities ascribed to INO, and death. If there were no DLTs experienced in first two cycles of a cohort, subsequent patients were enrolled in the next highest dose level. Results: We have enrolled 19 patients (18 treated, Table 1) with a median follow up of 18.1 months and a median number of 3 doses (range, 1-9). Only treated patients are included in analyses. The median time to first INO dose was 84 days (range, 47-100) after alloHCT. There was one DLT (prolonged thrombocytopenia) and the study reached the final pre-defined dose of 0.6mg/m2. No VOD was observed. The most common toxicity was thrombocytopenia (42.1% of patients; 31.6% experienced grade 3 or 4 thrombocytopenia), neutropenia (31.6% of patients, 15.8% experienced grade 3 or 4 neutropenia), and nausea/vomiting (42% of patients, all grade 1 or 2). 68% of patients received reduced intensity or non-myeloablative conditioning. OS at 1-year post-alloHCT was 94.4% and PFS was 88.9%. One-year NRM was 5.5%. One patient died of aGVHD on 0.6mg/m2 and one patient relapsed on 0.5mg/m2 at 7 months post-alloHCT. Another patient relapsed from Ph-like ALL 23 months post-alloHCT. Seven of the patients enrolled either had MRD before (n = 4) or after alloHCT (n = 3) as measured by flow cytometry, PCR, or NGS. For patients with MRD before or after alloHCT, 1-year OS and PFS are 100% (Figure 1). The 1-year PFS and OS of recipients of RIC transplants was 83.3% and 91.7%. Conclusions: We have determined that INO at doses of 0.3, 0.4, 0.5 and 0.6mg/m2 was well tolerated and that 0.6 mg/m2 is the MTD. Thrombocytopenia may be the DLT. The low observed relapse rate and favorable safety profile justify investigating low-dose INO as maintenance therapy after alloHCT in a phase II trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Phase I Clinical Trial of Point-of-Care Manufactured Fresh Anti-CD19/20/22 Chimeric Antigen Receptor T Cells for Treatment of Relapsed or Refractory Lymphoid Malignancies (Non-Hodgkin Lymphoma, Acute Lymphoblastic Leukemia, Chronic Lymphocytic Leukemia, B Prolymphocytic Leukemia)

Background: CD19-targeted CAR-T cells have transformed the treatment of relapsed/refractory B-cell malignancies. However, relapse occurs in over 50% of patients treated with commercial CD19 directed CAR-T due to antigen escape from selective immune pressure either on the target antigen or due to exhaustion and lack of persistence of CAR-T cells. Hence there is an unmet need for next-generation CAR-Ts that have broad antigen recognition and novel costimulatory domains to promote survival and decrease exhaustion of CAR-Ts. Targeting three B-cell antigens simultaneously is a novel approach to prevent antigen-negative relapse while inclusion of the OX40 domain will promote CAR-Ts persistence and expansion. Whether targeting three commonly expressed antigens is feasible or safe and will result in durable remissions is not known. We hypothesize that this trispecific targeting of CD19/20/22 CAR-T construct will be safe and effective in patients with relapsed, refractory B-cell malignancies. Study Design and Methods: This parallel group phase 1 study follows a 3+3 phase 1 design to determine the safety of the infusion of point-of-care manufactured fresh CAR-T cells targeting CD19/20/22 and to find the recommended phase II dose for this cellular therapy. CAR-T cells will be manufactured over 6 days. Patients will be enrolled in one of the two cohorts: Cohort A (single infusion): Non-Hodgkin's Lymphoma (NHL) with lesions <= 5 cm; Indolent lymphomas; B-PLL (B-Pro lymphocytic leukemia); Chronic Lymphocytic Leukemia -CLL (without Richter's transformation), Cohort B (split infusion): B-ALL (B-Acute lymphoblastic leukemia); Lymphoid blast crisis of Chronic myeloid leukemia; CLL (with Richter's transformation); NHL with lesions > 5 cm and/or lymphoblastic lymphoma, or NHL with circulating lymphoma cells. Patients who are CAR-naïve will be enrolled in this study. The study involves infusion of anti-CD19/20/22 CAR-T cells administered after an immunosuppressive conditioning regimen of fludarabine and cyclophosphamide. Cyclophosphamide 60 mg/kg will be infused intravenously over 60 minutes on day -6. Fludarabine 25 mg/m2 will be infused intravenously daily over 30 minutes on days -5 to -3. In each group, patients will be enrolled sequentially to each dose level, starting with dose level 1. Three CAR-T dose-levels: 5x105, 1x106 & 2x106 cells/kg/dose will be explored to determine MTD. For cohort B patients, the total CAR-T cell dose infused, will be divided in two doses, dose 1 (day 0) is 40% of the total dose and dose 2 (day 7) is 60% of total dose. The minimum number of subjects enrolled in the complete study will be 18 (9 per cohort) and the maximum number in dose escalation will be 36 (18 per cohort). Patients in each cohort will be monitored for 30 days before enrollment of next patientTwo patients have been enrolled in the trial and received fresh infusion of Anti CD19/20/22 CAR-T cells and accrual is ongoing. Updates on the initial cohort of patients will be presented at the meeting. ClinicalTrials.gov NCT: NCT05418088 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Myeloid Sarcoma: Features in Cytological Preparations and the Utility of Rapid On-Site Evaluation (ROSE) in Triage

Introduction: Myeloid sarcoma (MS) is a rare extramedullary tumor consisting of blasts of granulocytic, monocytic, erythroid, or megakaryocytic lineage that disrupts the architecture of the involved tissue. MS shows vast clinical, morphologic, immunophenotypic, and genetic heterogeneity posing a diagnostic dilemma, especially in small biopsy specimens such as fine-needle aspiration (FNA) and core biopsy. The objective of this study is to highlight the morphologic features of MS in cytological preparations and investigate the efficacy of pathologist-performed rapid on-site evaluation (ROSE) in assuring accurate triage. Methods: A retrospective review was performed for cases of MS with concurrent cytology and ROSE results from 2006 to 2017. FNA smears and touch preparations were reviewed, and the results of ROSE, immunohistochemistry (IHC), flow cytometric immunophenotyping (FCI), cytogenetics/FISH, and histology were analyzed. Results: A total of 15 cases were found including 6 (40%) with monocytic morphology comprising promonocytes and monoblasts and 9 (60%) with conventional myeloblastic morphology. The most common genetic subgroup was KMT2A-rearranged MS (33.3%) followed by extramedullary blast crisis of chronic myeloid leukemia (26.6%). ROSE provided sufficient preliminary information and ensured the procurement of adequate tissue for histology, IHCs, FCI, and cytogenetics/FISH, leading to an accurate and complete diagnosis of MS in all cases. Discussion/Conclusion: MS is a rare malignancy that shows pronounced clinical, morphologic, immunophenotypic, and genetic heterogeneity that often overlaps with other neoplastic and non-neoplastic entities. Features including the presence of classic myeloblasts, promonocytes, monoblasts, nucleated red blood cells, left-shifted granulocytes, cytoplasmic granules, and pseudopods are helpful hints in cytological preparations. In the modern era where pathologists are increasingly expected to do extensive diagnostic, molecular, and therapeutic biomarker testing on tissue that is historically diminishing in size, ROSE is a highly effective tool to ensure effective triage of MS aiding in an accurate, timely, and complete diagnosis.

ALL-004 Philadelphia Chromosome-Positive T-Cell Acute Lymphoblastic Leukemia/Lymphoblastic Lymphoma: A Rare Case Report

T-acute lymphoblastic leukemia/lymphoblastic lymphoma (T-ALL/LBL) is a rare and aggressive leukemia. Philadelphia chromosome-positive (Ph+) T-ALL is exceedingly rare and has therapeutic and prognostic significance. The case study herein is a very rare presentation. A case report of a 52-year-old Egyptian female, who presented in 2012 with a 2-month history of fatigue, bone aches, and abnormal vaginal bleeding. Oncology Center Mansoura University. The patient appeared pale with significant splenomegaly. Complete blood count: white blood cell count 6.9 k/µL, microcytic hypochromic anemia with hemoglobin 10.4 g/dL, and platelet count 149 k/µL. A peripheral blood smear showed neutrophil toxic granulation. Bone marrow aspirate (BMA) was infiltrated with 45% blasts, and myeloid and erythroid series were suppressed. No significant dysplasia was noted. Flow cytometry analysis performed on BMA showed an abnormal population of blasts expressing TdT (20%), cytoplasmic CD3 (35%), CD7 (64%), CD2 (62%), CD5 (61%), CD1a (0.38%), CD4 (27%), CD8 (32%), CD10 (3.6%), CD117 (5.4%), CD 34 (2.41%), and CD79a (27%), revealing a case of ALL (T-cell lymphoblastic lymphoma/leukemia). The fluorescence in-situ hybridization (FISH) analysis performed on interphase nuclei obtained from a BMA sample revealed a dual-fusion hybridization signal pattern for BCR and ABL1 probes on chromosomes 9 and 22, indicating t(9:22) in 100%. Cytogenetic analysis revealed an abnormal complex karyotype, 46,XY,t(9;22),(q34;q11.2)[25], in all counted cells. BCR-ABL1 gene rearrangement by quantitative real-time PCR was 100% IS. The patient was diagnosed with a case of T-ALL/LBL with t(9;22);BCR-ABL1 (Ph+). The patient started the MRCUK protocol in combination with imatinib 400 mg/d. She finished maintenance therapy on 10/12/2016. The patient is still on imatinib 400 mg/d. The patient is still in complete remission and in deep molecular response with no detectable minimal residual disease. Ph+ T-ALL is rare. Differentiation between Ph+ T-ALL/LBL and T-LB crisis of chronic myeloid leukemia (CML) was difficult because it is diagnosed with no prior history of CML. The addition of tyrosine kinase inhibitors to intensive chemotherapy has greatly improved the outcomes of Ph+ ALL patients.

Minimal residual disease in BCR::ABL1-positive acute lymphoblastic leukemia: different significance in typical ALL and in CML-like disease.

Recently, we defined "CML-like" subtype of BCR::ABL1-positive acute lymphoblastic leukemia (ALL), resembling lymphoid blast crisis of chronic myeloid leukemia (CML). Here we retrospectively analyzed prognostic relevance of minimal residual disease (MRD)and other features in 147 children with BCR::ABL1-positive ALL (diagnosed I/2000-IV/2021, treated according to EsPhALL (n = 133) or other (n = 14) protocols), using DNA-based monitoring of BCR::ABL1 genomic breakpoint and clonal immunoglobulin/T-cell receptor gene rearrangements. Although overall prognosis of CML-like (n = 48) and typical ALL (n = 99) was similar (5-year-EFS 60% and 49%, respectively; 5-year-OS 75% and 73%, respectively), typical ALL presented more relapses while CML-like patients more often died in the first remission. Prognostic role of MRD was significant in the typical ALL (p = 0.0005 in multivariate analysis for EFS). In contrast, in CML-like patients MRD was not significant (p values > 0.2) and inapplicable for therapy adjustment. Moreover, in the typical ALL, risk-prediction could be further improved by considering initial hyperleukocytosis. Early distinguishing typical BCR::ABL1-positive ALL and CML-like patients is essential to enable optimal treatment approach in upcoming protocols. For the typical ALL, tyrosine-kinase inhibitors and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, no relevant prognostic feature applicable for therapy tailoring was found so far.

Metabolic plasticity in blast crisis-chronic myeloid leukaemia cells under hypoxia reduces the cytotoxic potency of drugs targeting mitochondria

Metabolic reprogramming (MR) influences progression of chronic myeloid leukaemia (CML) to blast crisis (BC), but metabolic programs may change transiently in a second dimension (metabolic plasticity, MP), driven by environments as hypoxia, affecting cytotoxic potency (CPot) of drugs targeting mitochondria or mitochondria-related cell stress responses (MRCSR) such as mitophagy and mitochondrial biogenesis. We assessed mitochondrial membrane potential (MMP), mitochondrial mass (MM), apoptosis, glucose uptake (GU), and CPot of arsenic trioxide (ATO), CCCP, valproic acid (VPA), vincristine (VCR), Mdivi1, and dichloroacetic acid (DCA) in CML BC cells K562 (BC-K562) under hypoxia through flow cytometry, and gene expression from GEO database. About 60% of untreated cells were killed after 72 h under hypoxia, but paradoxically, all drugs but ATO rescued cells and increased survival rates to almost 90%. Blocking mitophagy either with VCR or Mdivi1, or increasing mitochondrial biogenesis with VPA enhanced cell-survival with increased MM. DCA increased MM and rescued cells in spite of its role in activating pyruvate dehydrogenase and Krebs cycle. Cells rescued by DCA, VPA and CCCP showed decreased GU. ATO showed equal CPot in hypoxia and normoxia. MP was evidenced by differential expression of genes (DEG) under hypoxia related to Krebs cycle, lipid synthesis, cholesterol homeostasis, mitophagy, and mitochondrial biogenesis (GSE144527). A 25-gene MP-signature of BC-K562 cells under hypoxia identified BC cases among 113 transcriptomes from CML patients (GSE4170). We concluded that hypoxic environment drove a MP change evidenced by DEG that was reflected in a paradoxical pro-survival, instead of cytotoxic, effect of drugs targeting mitochondria and MRCSR.

S154: IMATINIB-RESISTANT CLONES ISOLATED FROM A MODEL OF BLAST CRISIS OF CHRONIC MYELOID LEUKAEMIA DIFFER IN MUTATIONS IN BCR::ABL1 AND OTHER CANCER RELATED GENES AND IN THEIR SENSITIVITY TO BH3-MIMETICS

Background: The treatment of blast crisis of chronic myeloid leukaemia (BC-CML) remains difficult and the outcome of patients is poor. A dysregulated apoptosis leads to survival of malignant cells. BH3-mimetics inhibit antiapoptotic proteins of BCL-2 family. These drugs have been recently introduced in the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. This work assumed that these drugs may have a treatment potential for myeloid and/or lymphoid BC-CML. Aims: The aim is to test the sensitivity of imatinib-resistant (IR) clones of KCL-22, model of BC-CML, and primary blast cells to BH3-mimetics and describe a mechanism of sensitivity/resistance using protein analysis of apoptotic pathways. Methods: Isolated IR-clones (n=10) of KCL-22 were characterized by NGS and cultivated with 4µM imatinib (IM) and dilution series of BH3-mimetics (venetoclax – anti-BCL-2, S63845 – anti-MCL-1, A-1155463 - anti-BCL-XL) for 72 hours. IC50 was determined based on proliferation. Protein expression of BCL-2 family (n=10) was conducted in 4 IR-clones cultivated with IM and with/without BH3-mimetics. For in vivo experiments NOD-SCID-gamma mice (n=16) were subcutaneously injected with 106 cells of BCR::ABL1-T315I clone and divided into control group and treated groups. Primary cells of patients with BC-CML (n=4) were exposed to IM and BH3-mimetics for 7 days and LC50 was estimated. Results: IR-clones of KCL-22 differ in their sensitivity to BH3-mimetics (Table 1). The majority of clones (8/10) including 4 clones with T315I were sensitive to MCL-1 inhibitor. A reduced sensitivity was observed in the T315I clone carrying mutated GATA2 and other clone with mutation Y253H and mutated BCOR. Moreover, two T315I clones were sensitive to venetoclax, but other T315I clones with mutations in other cancer related genes showed insensitivity (n=2) or decreased sensitivity (n=1). Interestingly, two T315I clones with mutations in other cancer related genes insensitive to venetoclax were sensitive to BCL-XL inhibitor and in general, it seems that clones insensitive to venetoclax were sensitive to BCL-XL inhibitor and vice versa. Protein analysis of parental KCL-22IR cells and 4 clones showed a high expression of BCL-2 and BCL-XL and undetectable MCL-1 expression. Except for the T315I clone (B8, Table 1), other analysed clones expressed MCL-1 after exposure to BH3-mimetics. The sensitivity of T315I clone to anti-MCL-1 can be explained by detection of cBAX (an active form of apoptotic effector) after exposure to the inhibitor. The parental cell line showed dissociation of antiapoptotic complex MCL-1/BIM after exposure to MCL-1 inhibitor. The released BIM accelerates the apoptosis as an apoptotic activator. In vivo analysis revealed a decreased tumour growth of xenografted T315I clone (B8) in IM, venetoclax or combined treatment compared to control. An additive effect of dual therapy against monotherapy was not observed. This can be explained by IM inhibition of non-mutated BCR::ABL1 in KCL-22IR carrying 2 Ph chromosomes. LC50 analysis of primary blasts (4 patients) showed anti-MCL-1 to be the most potent inhibitor. Mutations in other cancer related genes and/or changes in karyotype increased LC50 for venetoclax and BCL-XL inhibitor or venetoclax, respectively. Image:Summary/Conclusion: The preliminary data of this study showed that the MCL-1 inhibitor S63845 seems to be the most potent BH3-mimetic to induce apoptosis in BC-CML. The combined therapy of TKI and BH3-mimetics should reflect mutation status of BCR::ABL1 and other cancer related genes.

Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia.

Simple SummaryChronic myeloid leukemia is a type of blood cancer that is regarded as a success story in determining the exact biological origin, pathogenesis and development of a molecularly targeted (mutation-specific) therapy that has led to successful treatment of this fatal cancer. It is caused by the BCR-ABL fusion gene, which is formed from the translocation between chromosomes 9 and 22. Anti-BCR-ABL drugs, known as tyrosine kinase inhibitors (TKIs), have led to long-term remissions in more than 80% of CML patients and even cure in about one-third of patients. Nevertheless, many patients face drug resistance, and disease progression occurs in about 30% of CML patients, leading to morbidities and mortality. Unfortunately, no biomarkers of CML progression are available due to a poor understanding of the mechanism of progression. Therefore, finding reliable molecular biomarkers of CML progression is one of the most attractive research areas in 21st-century cancer research. In this study, we report novel genomic variants exclusively found in all our advanced-phase CML patients. This study will help in identifying CML patients at risk of disease progression and timely therapeutic interventions to avoid or at least delay fatal disease progression in this cancer.Background: Chronic myeloid leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(9;22) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKIs) has changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, the mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find novel biomarkers of CML progression by employing whole-exome sequencing (WES). Materials and Methods: WES of accelerated phase (AP) and blast crisis (BC) CML patients was carried out, with chronic-phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing were carried out using Illumina platforms. Statistical analysis was carried out using SAS/STAT software version 9.4, and R package was employed to find mutations shared exclusively by all AP-/BC-CML patients. Confirmation of mutations was carried out using Sanger sequencing and protein structure modeling using I-TASSER followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modeling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in humans. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in the biology and progression of CML.

DETECTION OF HEMATOPOIETIC NEOPLASMS IN CEREBROSPINAL FLUID BY IMMUNOPHENOTYPING: SINGLE-CENTER DATA ANALYSIS

Cerebrospinal fluid (CSF) infiltration can be identified at diagnosis and during the course of many hematopoietic malignancies, mainly acute leukemia and aggressive non-Hodgking lymphomas (NHL). The accurate diagnosis of central nervous system (CNS) infiltration allows patients with infiltrated CSF to be properly treated, avoiding the use of unnecessary CNS therapy for patients who do not have compromised CSF. Cytomorphology is the classic method for diagnosing CSF neoplastic infiltration, but it presents variability in sensitivity and specificity according to the experience of the morphologist and cell abnormalities resulting from sample preparation. Currently, multiparametric flow cytometry (MFC) has great sensitivity and specificity to detect cells of hematopoietic neoplasms in the CSF. This is a retrospective study to assess the frequency of CSF infiltration in a cohort of patients with hematologic malignancies treated at our institution. to evaluate the casuistry and frequency of CSF infiltration by hematologic neoplasms diagnosed by immunophenotyping in our institution. the results of CSF immunophenotyping were collected using the flow cytometry laboratory database. Assess period: from March 2008 to June 2021. Patients were grouped by type of disease and the frequency with which each one was represented in the cohort was verified. a total of 1572 CSF samples were analyzed by the laboratory during the study period. The frequency of diagnoses of patients whose samples were sent for evaluation were, in decreasing order: 956(60.8%), B-cell precursor acute lymphoblastic leukemia (BCP ALL) samples 238(15.1%) T-cell acute lymphoblastic leukemia (T-ALL) samples, 168(10.6%) AML samples, 120 (7.6%) B and T NHL, 42 (2.6%) mixed lineage acute leukemia (MPAL), 34(2.1%) blastic crisis of chronic myeloid leukemia (CML), 14(0.8%) other mature lymphoid neoplasms. Immunophenotyping showed 311(19.78%) positive results for CSF infiltration. The diagnoses of these patients and their respective frequency were: 156 (50.1%) BCP-ALL, 49(15.7%) AML, 41(31.1%) T-ALL, 37(11.8%) NHL, 13 (4.1%) blastic crisis of CML, 8 (2.5%) MPAL, 7(2.2%) other mature lymphoid neoplasms. The frequencies of NHL subtypes observed in this cohort were: 16 (1%) Large B Cell Lymphoma, 7(0.44%) Burkitt Lymphoma, 6 (0.39%) Mantle Cell Lymphoma, 5(0.31%) Adult T-Cell Leukemia-Lymphoma, 1 (0.06%) Marginal Zone Lymphoma, 1 (0.06%) Folicular Lymphoma and 1 (0.06%) Sezary Syndrome. It should be emphasized that among the samples from patients with other mature lymphoid neoplasms, 4 (0.25%) were from patients with Chronic Lymphocytic Leukemia (CLL), which is an unusual finding. this study observed that 91% of the samples with CSF infiltration were from patients diagnosed with acute leukemia and blastic crisis of CML. Mature lymphoid neoplasms have a lower frequency of CNS infiltration than acute leukemia, as also reflected in our series. These findings show the importance of an accurate assessment of the CSF for these diseases, because the results influence the choice of the therapeutic modality and intensity to be adopted in the different treatment protocols. we demonstrate the ability of immunophenotyping as a robust method for evaluating CSF samples from patients diagnosed with hematologic malignancies.

A Multicenter Retrospective Experience of Hematologic Neoplasms in Pregnancy

INTRODUCTION: Cancer is diagnosed in 0.07% to 0.1% of pregnancies and represent the second cause of maternal death after vascular complications. Hematologic malignancies account for 25% of all malignancies in pregnancy. It is always a challenge to confront a case of a pregnant woman with any type of cancer, requires a great multidisciplinary team and so far there is no a world-wide expert group in the treatment of this type of patients. The world's experience on approach and treatment of women with cancer is limited to cases series and recommendations with low scientific evidence. Latin America and particularly Mexico has a different clinical behavior to that reported in the developed countries and does not have a consensus of treatment for these patients.OBJETIVES: To report our multicenter experience in the setting of hematologic malignancies during the pregnancy.MATERIALS AND METHODS: This is a retrospective multicenter study that collects the information available in the databases of 9 hospitals in Mexico from January 1993 to June 2017 with diagnosis of pregnancy and hematologic cancer. Epidemiological data in relation to type of cancer, treatment as well as features of pregnancy, childbirth and newborn were colected.RESULTS: A total of 101 patients from 9 Mexican medical institutions were diagnosed with the following hematologic malignancies: 88 cases of leukemia (16% acute myeloid leukemia (AML), 34% acute lymphoblastic leukemia (ALL), 2 cases of T ALL, 3% Mixed lymphocytic leukemia, 35% chronic myeloid leukemia (CML), 7 cases of Hodgkin's lymphoma, 5 cases of non-Hodgkin's lymphoma, 1 case of myelofibrosis. The mean age at diagnosis was 23 years old (14-41), 46% were diagnosed before week 20 of gestation, 49% during week 21 to 35 and 6% after week 36. 35 patients received no treatment (34.6%). Of the women who received treatment, the drugs used according to the type of neoplasia were imatinib (28.7%), interferon alpha (10.6%), hydroxyurea (7.5%), cytarabine + doxorubicin (18.1%), vincristine + doxorubicin + prednisone + L-asparaginase (24.2%), Hyper CVAD (4.5%), ABVD (4.5%), R-CHOP (1.98%). 51 cases (50.4%) were diagnosed with hematological malignancies and under treatment, prior to pregnancy. Within the Acute Leukemia group (51%) there were 20 deaths; In those diagnosed with lymphoma no deaths were recorded and there was 1 death due to blast crisis in CML. In the group that received chemotherapeutic treatment (65.3%) there were 12 fetal deaths, treatment-related structural alterations in only 3 newborns, 45% healthy and the rest had mild reversible complications.CONCLUSIONS: We report the largest series of cases in malignant hemopathies in pregnancy. The results show the need to organize multidisciplinary work teams to improve the care and treatment of this type of patients. DisclosuresNo relevant conflicts of interest to declare.

Sequential High-Dose Chemotherapy Reinduction Followed By Haploidentical Transplantation in Acute Leukemias

Introduction: Outcomes after T-cell replete haploidentical stem cell transplantation (Haplo HSCT) with post- transplant Cyclophosphamide have been encouraging. As previously published (Schmid C. et al, JCO 2005; Ringdén O. et al, Br J Haematol. 2017), the sequential use of intensive chemotherapy and allogeneic transplantation represents a possible approach for the treatment of high-risk / relapsed-refractory Acute Myeloid Leukemia (AML).Patients and Methods: At our Center, 22 high-risk patients were transplanted during chemo-induced neutropenia after high-dose salvage chemotherapy. Median age at transplant was 49 years (range 21-62); 6/22 (27%) had received a previous allograft. All patients but 2, who presented with blast crisis of chronic myeloid leukemia, suffered from refractory (no. 12) or secondary AML (no. 8). All patients had active disease at the time of Sequential Therapy and median marrow blast count before reinduction was 25% (range 6-88%). All patients received high-dose Cytarabine (≥1g/sqm) containing regimens as reinduction therapy. Conditioning, myeloablative, in 14/22 (64%) and non-myeloablative in 8/22 (36%), was started at a median of 9 days (range 4-15) after the last day of chemotherapy. Graft-versus-host disease (GVHD) prophylaxis consisted of post-transplant Cyclophosphamide with calcineurine inhibitors and mycophenolic acid. Bone marrow was used as stem cell source in 18/22 (82%) patients.Results: One/22 patient (4%) experienced primary graft failure and was successfully rescued with a second haploidentical transplant from the same donor. Median day of neutrophil recovery was day +18 (range 14-24). Median follow-up of survivors was 4.1 years (range 5-69 months); 1-year Overall and Event-Free Survivals were 34.5% and 30.3%, while 1-year Relapse Incidence and Non Relapse Mortality were 41.9% and 28%, respectively. Overall cumulative incidences of acute and chronic GVHD were 41% and 33.6% at day +100 and + 400. Among patients who developed GVHD, 3 grade III-IV acute GVHD and 2 moderate-severe chronic GVHD were observed. At 4.1 years post Haplo-HSCT, 5/22 (23%) patients are alive and disease free.Conclusions: To our knowledge, we report the first experience of a sequential approach including Haplo HSCT. Though small the patient cohort, which also includes second transplants, our findings suggest that transplant-related toxicity was acceptable and early relapse was the major treatment-failure. However, long-term survival and disease-free rates in these very poor prognosis patients are rather encouraging. Only the prompt availability of a haploidentical donor allowed to implement this treatment modality. DisclosuresBoccadoro:Sanofi: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; AbbVie: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Mundipharma: Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Vitolo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Takeda: Honoraria; Mundipharma: Honoraria.

Detection of p53 Protein in Leukemic Cells by Flow Cytometry.

Although the alterations of TP53 gene are rare in malignant hematological disorders, these genetic alterations have strong correlations with unfavorable prognostic factors and are found in relapse acute lymphoid leukemia (ALL), relapse acute myeloid leukemia (AML), blastic crisis of chronic myeloid leukemia (CML) and evolution from chronic lymphoid leukemia to Richter's syndrome (CLL/SR). Most methods currently used to study TP53 gene employ molecular biology techniques such as single strand conformation polymorphism (PCR-SSCP) and direct DNA sequencing. Both techniques detect TP53 gene mutation but not p53 protein expression and are time-consuming and difficult to apply in the clinical setting. In the last years, p53 protein expression has emerged as a potential prognostic marker, which can be used to study TP53 gene alterations in malignant diseases. The aim of this work was to validate flow cytometry (FC) technique to detect p53 expression in different types of leukemias and analyze the relationship between p53 expression and status of disease. We studied p53 protein expression in 223 blood and bone marrow samples: 72 patients with CML, 54 in chronic phase (CML-CP), 7 in accelerated phase (CML-AP) and 11 in blastic crisis (CML-BC); 64 patients with CLL (34 at diagnosis and 9 with Richter's syndrome), 44 patients with ALL (36 at diagnosis and 8 in relapse) and 43 AML (27 de novo, 7 in relapse and 9 secondary AML). p53 protein expression was observed in 63 out of 223 patient's samples: 14/64 CLL (21.9%), 13/44 ALL (29.5%), 19/43 AML (44.2%) and 17/72 LMC (23.6%). The highest levels were detected in the advanced phases of CLL, ALL and CML. In addition, in AML cases, high levels of p53 expression were detected in secondary and relapse disease and also in de novo AML cases. Our results demonstrated that p53 expression levels are strongly associated with advanced disease. Based on these results flow cytometry can be a reliable approach to study p53 protein expression in leukemic patients. Further studies are needed to ascertain the role of p53 expression as a prognostic marker of disease progression.

Acquired hypofibrinogenemia in patients with leukemia during steroid therapy

Acquired hypofibrinogenemia is observed in patients with severe liver disease, disseminated intravascular coagulation, and high-volume perioperative fluid replacement. In lymphoblastic leukemia, hypofibrinogenemia is most frequently caused by the administration of L-asparaginase. Here we report the cases of two patients with acquired hypofibrinogenemia that occurred during steroid-containing chemotherapy treatment against lymphoblastic blast crisis of chronic myeloid leukemia in the first case and acute lymphoblastic leukemia in the second case. Administration of steroids repeatedly and promptly caused hypofibrinogenemia, irrespective of the products (prednisolone, dexamethasone, or methylprednisolone) or routes (oral or intravenous) that were used. Monitoring of the fibrinogen levels, especially during the first course of steroid therapy, would be useful for early diagnosis.

Analysis of Mechanisms of Blast Crisis in Chronic Myeloid Leukemia By Whole Genome Sequencing

Background: Thanks to tyrosine kinase inhibitors (TKIs) chronic myeloid leukemia (CML) has become a well manageable disease. This drastically changes once an individual progresses to blast crisis (BC), which carries a poor prognosis. Although progression to BC fortunately is a rare event, the mechanisms leading to transformation from chronic phase to BC are sparsely studied, which led us to perform in depth analyses of CML patients each at diagnosis (D), at molecular or hematological remission (REM) and at BC by whole genome sequencing (WGS). Aim: (1) Study chromosomal and mutational profiles at D, REM and BC (2) Identify chromosomal and molecular genetic mechanisms in progression to BC Patients and Methods: We performed in depth analyses of 11 CML patients with BC confirmed by cytomorphology. REM samples were available for 8 patients, in 1 patient 2 BC samples were sequenced. Median age at D was 59 years (range 31-70) and median time to BC was 2 years (range 0-6). Nine patients received only first line TKI and 2 patients had switched to second- or third line TKIs before or at time of BC. We sequenced DNA of bone marrow (n=17) or peripheral blood (n=14) by WGS at a median coverage of 106x and used Strelka2 for variant calling. Structural variations (SVs) were analyzed by Manta caller, copy number alterations (CNVs) were called using the GATK4 CNV calling pipeline. Results: None of the patients presented with known high risk additional chromosomal aberrations (ACA) at D (Hochhaus et al, Leukemia 2020). Using WGS at D, we found deletions in the breakpoint region of der(9)t(9;22) (n=2), der(22)t(9;22) (n=1) and a translocation involving 12p and der(22)t(9;22) (n=1). Mutations in known myeloid driver genes were found in 4 patients at D. In two patients (DNMT3A, ASXL1) mutations were present at D and BC, while in two patients three ASXL1 mutations were present at D (VAF 27%; 23% and 14%), but could not be detected at BC by WGS and more sensitive targeted sequencing. Both patients presented with complex ACAs detected both by chromosome banding analysis (CBA) and WGS at BC. Other known driver or resistance mutations were not detected in any other sample at D. We identified three mechanisms driving the transition from chronic phase to BC, the first being ABL1 resistance mutations which render one or several TKIs ineffective (n=6/11). All patients developed ABL1 mutations that conferred resistance to the TKI they were receiving. In BC we detected T315I, Y253H, F359V (n=2 each), E450K, Q252H and Q255K (n=1 each) mutations, one patient had T315I, Q255K and F259V mutation combined, all other patients had a single mutation. In one patient two BCs occurred which showed both an additional t(9;10) but differed in the ABL1 mutations: First Q252H was detected and after REM in second BC T315I was present whereas the Q252H was absent. Secondly, in 10/11 of all patients SVs or CNVs were identified in addition to t(9;22)(q34;q11) by WGS in BC. If CBA data was available it confirmed the WGS data. Patients showed an additional t(9;10) (n=1), t(10;11) (n=1), inv(16) (n=1) and CNVs involving virtually all chromosomes which were only detected in BC pointing towards a major role of chromosomal instability. Interestingly WGS detected de-novo leukemic driver mutations in BC that are described primarily in other myeloid malignancies, representing the third mechanism (n=7). Two patients gained truncating mutations in BCOR, one patient each gained a mutation in ASXL1 and CUX1, one patient 3 frameshift mutations in ETV6, another patient two mutations in WT1 and one case gained a CBFB-MYH11 rearrangement which is usually found in a subtype of AML. Conclusion: Using WGS we found three different contributing mechanisms to progression to BC in CML: 1) ABL1 resistance mutations, 2) gain of structural and copy number variations and 3) potentially rise of additional AML like mutations. Since none of these factors were identified at D and REM, a comprehensive screening is recommended to detect, at the earliest possible time point when molecular remission is lost, the drivers to BC and allow early clinical intervention such as allogeneic transplant. Disclosures No relevant conflicts of interest to declare.