Cases Of Myeloid Leukemia Research Articles

Calreticulin Mutations in Myeloproliferative Neoplasms Patients Diagnosed in UKM Medical Centre

Introduction: Calreticulin (CALR) mutations are one of the molecular markers that has been incorporated for the diagnosis of myeloproliferative neoplasms (MPN) in the revised 2017 WHO Classification of Haematopoietic and Lymphoid Tumors. This study was performed to determine the prevalence of CALR mutations in patients with MPN diagnosed in UKMMC and to compare their demographics plus laboratory features with other MPN patients. Meth- ods: A total of 59 MPN patients who tested negative for JAK2V617Fmutation were selected and 21 MPN patients positive for JAK2V617F were included as controls. Screening for CALR exon 9 was done by multiplex polymerase chain reaction (PCR) followed by Sanger sequencing. Results: A total of six JAK2 V617F negative MPN samples were found to be positive for CALR mutations. Out of these six, three patients with CALR mutations were of type I mutation, two were type II while one was a mutation in the stretch III region. None of the twenty one JAK2 V617F positive MPN samples were positive for CALR mutation. Clinical phenotypes for those positive for CALR were restricted to Essential Thrombocythemia (ET), Primary Myelofibrosis (PMF) and one case of atypical Chronic Myeloid Leukaemia (CML). Conclusion: CALR mutations constituted 10.16% from the MPN patients who were negative for JAK2V617F mutation with no significant differences in platelet counts, hemoglobin (Hb), hematocrit and white cell counts as compared to MPN patients with JAK2 V617F mutations. Testing for CALR mutations among those who are negative for JAK2V617F within Malaysian population maybe worthwhile and require larger scale studies.

Case report: Sustained complete remission with ivosidenib in a patient with relapsed, IDH1-mutated acute leukemia

Older patients with acute lymphoblastic leukemia (ALL) have a poor prognosis, with a 5-year overall survival rate of only 10%–20%. This is attributable to patient comorbidities, poor performance status, and high-risk disease biology. The prognosis for patients with relapsed/refractory (R/R) disease remains poor, particularly for patients who are not candidates for therapies targeting CD19 or CD22. Additional treatment options are needed for these patient populations. The patient presented here is a 76-year-old man diagnosed with precursor B-cell ALL with aberrant expression of myeloid markers and lacking significant CD19 or CD22 expression. A 3-year remission was achieved with one cycle of CVP (cyclophosphamide, vincristine, and prednisone) followed by 22 months of maintenance DOMP (dexamethasone, vincristine, methotrexate, and 6-mercaptopurine) prior to relapse. He was then treated with one cycle of salvage CVP, which was complicated by a stroke resulting in hemiparesis. Next-generation sequencing (NGS) was performed on the relapsed bone marrow, which revealed the presence of an R132H mutation in the isocitrate dehydrogenase 1 (IDH1) gene. He was subsequently treated with the IDH1 inhibitor ivosidenib and remained in a second remission for nearly 1 year. IDH1 mutations are present in up to 14% of acute myeloid leukemia (AML) cases but are also seen more rarely in ALL, particularly in cases involving aberrant expression of myeloid markers. Ivosidenib has demonstrated efficacy in patients with IDH1-mutated AML but has not been extensively studied in other hematologic malignancies. This case demonstrates the role of NGS in revealing treatment options in patients with otherwise limited available therapies.

Comparison of DNA Sequencing and Multispecific Monoclonal Antibody (Msmab-1) based Immunohistochemical Detection of IDH1/2 Mutation in Acute Myeloid Leukemia Cases.

Background: The IDH1/2 mutation is an important epigenetic modifier involved in the pathogenesis of AML. It is associated with variable prognosis in AML cases. Lack of proper molecular diagnostic infrastructure and high cost limits the routine use of PCR with sequencing as routine diagnostic methodology. The aim of this study was to find the prevalence of IDH mutation in AML cases using both PCR with sequencing and Immunohistochemistry method.
 Methods: We evaluated 60 patients registered at KGMU, Lucknow for diagnosis and treatment of AML. PCR followed by sequencing was done. IHC staining of the IDH1/2 mutation was performed on all cases using bone biopsy or clot section (in cases of pediatric AML cases).
 Results: Out of the total 60 patients of AML 4(6.7%) patients had IDH1R312 mutation and 5(8.3%) Patients had IDH2R172 mutation. IDH2 R140 mutation was not detected in any sample. On immunohistochemistry analysis 10 cases showed positive staining against anti IDH1/2 mutant (R132/R172) antibody, clone MsMab-1 with a sensitivity of 77.8% and specificity of 94.1%.
 Conclusion: IHC could be an alternative method to direct Sanger sequencing for IDH1/2 mutation detection in AML cases. However, the antibody used in the study is not effective for individual assessment of IDH1 and IDH2 mutation.
 Keywords: Immunohistochemistry, Sanger sequencing, IDH1/2 Mutation, Acute Myeloid Leukemia

Unexpected diagnosis in not so usual clinical scenarios: Acute promyelocytic leukemia – A tertiary care hospital experience

Background: Acute promyelocytic leukemia (APL) is a distinct subtype of Acute myeloid leukemia (AML) with arrest in maturation of cells of myeloid series in bone marrow. PML-RARA fusion resulting from t(15;17) translocation forms the genetic basis of this haematological malignancy. Introduction of specific differentiating targeted therapy i.e. All-trans retinoic acid (ATRA) have remarkably improved the outcome of this previously dreaded AML. Aims and Objective: The current study presents five cases of Acute promyelocytic leukemia with unusual presentations followed by effective treatment to revert their adverse outcome. Materials and Methods: In this retrospective evaluation of Acute Myeloid leukemia cases, five cases were included from duration of November 2019 to November 2021. The diagnosis of AMLs was made according to EGIL 1998/WHO 2016 guidelines. Peripheral blood (PB) and bone marrow aspirate (BMA) were stained with Giemsa and cytochemistry included myeloperoxidase (MPO) and periodic acid-schiff (PAS). Results: Five color flowcytometric analyses were performed on BMA and PB samples. Chemotherapy was administered in the patients and follow-up was done. 03/05 patients faced mortality due to rapid disease progression. Conclusion: The fast track clinical course from presentation to the outcome of APL is dreadful for the treating clinician and the heamatopathologist. Cytomorphology and Cytochemistry are as important in rapid recognition of APL as Immunophenotyping and Cytogenetics for establishing accurate diagnosis.

Oncogenic FLT3 internal tandem duplication activates E2F1 to regulate purine metabolism in acute myeloid leukaemia

Oncogene FLT3 internal tandem duplication (FLT3-ITD) mutation accounts for 30 % of acute myeloid leukaemia (AML) cases and induces transformation. Previously, we found that E2F transcription factor 1 (E2F1) was involved in AML cell differentiation. Here, we reported that E2F1 expression was aberrantly upregulated in AML patients, especially in AML patients carrying FLT3-ITD. E2F1 knockdown inhibited cell proliferation and increased cell sensitivity to chemotherapy in cultured FLT3-ITD-positive AML cells. E2F1-depleted FLT3-ITD+ AML cells lost their malignancy as shown by the reduced leukaemia burden and prolonged survival in NOD-PrkdcscidIl2rgem1/Smoc mice receiving xenografts. Additionally, FLT3-ITD-driven transformation of human CD34+ hematopoietic stem and progenitor cells was counteracted by E2F1 knockdown. Mechanistically, FLT3-ITD enhanced the expression and nuclear accumulation of E2F1 in AML cells. Further study using chromatin immunoprecipitation-sequencing and metabolomics analyses revealed that ectopic FLT3-ITD promoted the recruitment of E2F1 on genes encoding key enzymatic regulators of purine metabolism and thus supported AML cell proliferation. Together, this study demonstrates that E2F1-activated purine metabolism is a critical downstream process of FLT3-ITD in AML and a potential target for FLT3-ITD+ AML patients.

Haematopoietic stem cell transplantation in RSUP Kariadi Semarang

In Indonesia, bone marrow transplantation is a treatment which not known yet to some people. Kariadi Hospital is the one hospital that do it first. On 1987, it was successful for acute myeloid leukemia case. Bone marrow transplantation is one of therapy used to treat patients with leukemia, severe aplastic anemia, Hodgkin's disease, non-Hodgkin's lymphoma, myeloma multiple, many blood disorder and solid tumor. This study described the patients characteristics who did hematopoietic stem cell transplantation during 2017-2021 at Kariadi General Hospital Semarang. This study is a quantitative research non-experiment with design retrospective analysis. Data was taken with a total sampling are 20 patients. Data analysis by describing the criteria. The results describe the sex of patients consist are 60% male and 40% female. Most range age at 35-44 years amount 30%. The diagnosis is 40% in the case of Myeloma Multiple. Most of transplantation doing an autologous transplant are 80%. It is indicated 90% of malignancy cases. All patient got 100% engraftment, all patients happen neutropenia, thrombocytopenia, oral mucositis, gut disorder, and electrolyte imbalance. Amount 15% are hematuria and 10% are urticaria, Veno Occlusive Disease, sepsis, brain infection and brain hemorrhage. The conclusion is patients who did hematopoietic stem cell transplantation have various adverse events and complications depending on many factors or comorbids. These are age, sex, diagnosis, stage of disease, state of remission, regiment conditioning. Health professionals should upgrade knowledge and skill for treatment of hematopoietic stem cell transplantation.

Classification of Acute Leukaemia based on Blast Morphology, Cytochemistry and Flow Cytometry

Introduction: Acute leukaemias are a heterogeneous group of malignancies due to the abnormal proliferation of immature cells. The classification of acute leukaemia has been transformed over a period. French American British’s (FAB) classification of acute leukaemia is based on morphology and cytochemistry. Later, the World Health Organization (WHO) classification stressed immunophenotyping to be done in the classification of acute leukaemia. Immunophenotyping is a powerful tool for classification and also for treatment and prognosis. Aim: To study the morphology of blasts in the peripheral smear and bone marrow cytochemistry and to compare with immunophenotyping in the classification of acute leukaemia. Materials and Methods: A cross-sectional study was undertaken in the Department of Transfusion Medicine and Immunohematology, at St. Johns National Academy of Health Sciences a Tertiary Care Hospital in Bengaluru over a period of one year from November 2018 to October 2019. The morphology of the blast was studied in peripheral blood and bone marrow aspirate stained by Leishman stain. Cytochemistry in peripheral smear and bone marrow with Sudan Black B and Periodic Acid-Schiff (PAS) stain was done in all acute leukaemia. The flow cytometry samples were processed within 24 hours of collection of the samples with a panel of markers including myeloid and lymphoid lineage and precursor markers. The results of morphology were confirmed with flow cytometry and final reports were released. Results: A total of 112 cases of acute leukaemia were studied and classified based on morphology, cytochemistry, and immunophenotyping of blasts. A total of 46 cases of Acute Myeloid Leukaemia (AML) and 63 cases of Acute Lymphoblastic Leukaemia (ALL). Two cases were found with Chronic Myeloid Leukaemia (CML) in blast crisis, one with myeloid and another with lymphoid blast crisis. One biphenotypic leukaemia was noted. Sudan Black was positive in 35 (76%) of AML and PAS was positive in 28 (44%) of ALL. The most common AML subtype was AML-M2 and the most common ALL subtype was B cell ALL. The most common symptom was fever and the sign was pallor. Conclusion: The present study showed that morphological diagnosis is important, based on which the cytochemistry and flow cytometry is performed for diagnosis. In cases of the diagnostic dilemma of acute leukaemia with morphology and cytochemistry, immunophenotyping supports diagnosing and classifying acute leukaemia.

Second-Line Therapy Tyrosine Kinase Inhibitor for Chronic Phase Chronic Myeloid Leukemia Resistant to Imatinib

Since the era of tyrosine kinase inhibitor (TKI) therapy, there has been an increase in the median survival of chronic myeloid leukemia. Nevertheless, nowadays there were several cases of chronic myeloid leukemia that are resistant to the first-line TKIs, so that several second-line TKIs were introduced. Assessment of the therapeutic response was performed using a complete peripheral blood examination, bone marrow aspiration cytogenetic examination, and quantitative BCR-ABL examination. The treatment failure criteria were applied based on the ELN criteria. In selecting the second line TKI should be based on the results of mutation analysis and consider the safety profile of the second line therapy. This paper will discuss the recommendations for second-line therapy TKI, adjusted to the resources in Indonesia.

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.

The impact of donor-to-recipient gender compatibility on outcomes of haploid hematopoietic stem cell transplantation in patients with hematological malignancies

Objective: To investigate how gender differences between the donor and the recipient affect the effectiveness of antithymocyte globulin (ATG) and pure peripheral blood stem cell (PBSC) hematopoietic stem cell transplantation (haplo-HSCT) in the treatment of malignant hematological diseases. Methods: From February 2015 to September 2020, 648 hematological malignancies patients underwent myeloablative condition regimen haplo-HSCT treatment at the Bone Marrow Transplant Center of the First Affiliated Hospital of Zhejiang University. The median age was 32 (14-62) years, with 363 males (56.0% ) and 285 females (44.0% ) present. 242 cases of acute lymphoblastic leukemia (ALL) (37.3% ) , 293 cases of acute myeloid leukemia (AML) (45.2% ) , 56 cases of myelodysplastic syndrome (MDS) (8.7% ) , 27 cases of non-Hodgkin's lymphoma (NHL) (4.2% ) , and 30 cases of other hematological malignancies (4.6% ) . Results: ① The 3-year overall survival (OS) , DFS, the incidence of Ⅱ-Ⅳ grade acute graft-versus-host disease (aGVHD) , the incidence of Ⅲ-Ⅳ grade aGVHD, the 3-year incidence of moderate & severe chronic GVHD (cGVHD) , severe cGVHD, the 3-year incidence of relapse, and NRM of the whole group were (73.10±1.90) % , (70.80±1.90) % , (33.96±1.87) % , (13.08±1.33) % , (35.10±2.14) % , (10.66±1.38) % , (19.43±1.67) % , and (9.80±1.24) % , respectively. ②There was no statistically significant difference between the donor-recipient gender match and donor-recipient gender mismatch groups in the 28-day cumulative neutrophil engraftment rate, 28-day cumulative platelet engraftment rate, the incidence of Ⅱ-Ⅳ grade aGVHD, the incidence of Ⅲ-Ⅳ grade aGVHD, 3-year OS, 3-year DFS, the cumulative incidence of relapse, NRM, and incidence of moderate & severe cGVHD, severe cGVHD. ③The 28-day cumulative neutrophil engraftment rate did not differ statistically between the male-female, female-female, male-male, and female-male groups (P=0.148) . The incidence of Ⅱ-Ⅳ grade aGVHD, the incidence of Ⅲ-Ⅳ grade aGVHD, 3-year OS, 3-year DFS, cumulative relapse rate, and NRM, and the incidence of cGVHD were not statistically different among the four groups (P>0.05) . The 28-day cumulative platelet engraftment rate of the female-male group was significantly lower than male-female group, and the female-female group [ (91.45±2.63) % vs. (94.77±1.75) % , P=0.004; (91.45±2.63) % vs. (95.54±2.05) % , P=0.005]. No significant difference existed in the 28-day cumulative platelet engraftment rate between the female-male group and the male-male group [ (91.45±2.63) % vs. (95.08±1.41) % , P=0.284]. ④Among patients ≤35 years old, the 3-year incidence of severe cGVHD patients receiving sister donors and sibling donors were (26.71±5.90) % and (10.33±4.43) % , respectively (P=0.054) . Patients accepting daughter donors and son donors had a 3-year incidence of moderate and severe cGVHD that was 40.07% vs. 27.41% , respectively, among those over 35 (40.07±6.65) % vs. (27.41±4.54) % (P=0.084) . ⑤Female donors to male recipients had a significantly lower 28-day cumulative platelet engraftment rate compared to the other groups [ (91.45±2.63) % vs. (95.08±0.95) % , P=0.037]. ⑥ Female donors to male recipients had a significantly lower 28-day cumulative platelet engraftment rate than the other groups in the ATG-Fresenius (ATG-F) 10 mg/kg group [ (89.29±4.29) % vs. (94.49±1.45) % , P=0.037]. But when compared to the other groups in the Rabbit Antihuman Thymocyte Immunoglobulin (rATG-T) 6 mg/kg group, the 28-day cumulative platelet implantation rate between female donors and male recipients was not significantly different [ (93.44±3.38) % vs. (95.62±1.26) % , P=0.404]. Conclusion: The main clinical outcomes of patients with malignant blood diseases following transplantation are unaffected by the gender combination of the donor and patient in the haplo-HSCT mode based on ATG and PBSC sources. Female donors to male recipients have a lower 28-day cumulative platelet engraftment rate and longer platelet engraftment times.

Clinical Analysis of Salvage Treatment of Glucocorticoid Resistant Graft-Versus-Host Disease with Vedolizumab in Children

To investigate the efficacy and safety of VDZ (Vedolizumab) in the salvage treatment of glucocorticoid resistance to gastrointestinal graft-versus-host disease (GR-GI GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in children. The clinical data of 5 patients with refractory GI GVHD who received allo-HSCT in Wuhan Children's Hospital from December 2020 to December 2021 were retrospectively analyzed with VDZ salvage therapy. Among the 5 children with refractory GI GVHD, there were 1 male and 4 female, including 2 cases of extremely severe aplastic anemia, 1 case of acute myeloid leukemia (M2, high-risk), 1 case of fanconi anemia and 1 case of myelodysplastic syndrome. The median age of transplant recipients was 54.4 (12-164) months. The median treatment time from transplantation to VDZ was 1.4 (0.6-6.8) months. On average, 3.5 (2-5) doses of VDZ were received. After receiving treatment, 2 patients achieved a complete response (CR), 2 patients achieved a very good partial response (VGPR), 1 patient was non-responsive (NR) after a short-term partial response (PR). Compared with that before VDZ treatment, the amount of diarrhea, stool color, blood and traits of the children after medication were effectively improved. The median follow-up time was 9.3 (7.23-12.83) months. No disseminated or severe bacterial/fungal infections occurred during VDZ treatment and follow-up, and 2 children died of leukemia recurrence and pulmonary bronchiolitis obliterans. VDZ salvage treatment of refractory GI GVHD in children has obvious short-term efficacy and good safety.

Розвиток лейкозу після перенесеної інфекції COVID-19

The purpose of the study was to review the literature on cases of leukemia in healthy people after the COVID-19 infection with a description of our own observation. Materials and methods. 26 literature sources were reviewed and analyzed using the PubMed/MEDLINE database regarding diagnosis of the leukemic process in patients after COVID-19. Analytical and bibliosemantic methods were used in the research. Results and discussion. Many aspects of the COVID-19 infection, especially its complications and long-term health consequences, are still unknown. Various reactive changes in blood tests during COVID-19 have been published. Leukocytosis, leukopenia, neutrophilia, lymphocytosis and lymphocytopenia, thrombocytopenia and rarely thrombocytosis were most often detected. The detected changes were usually not observable in patients. There are reports of leukemia after a recent COVID-19. Therefore, studying the features of the clinical picture and hematopoiesis in such patients during a viral infection, as well as at the initial manifestations of leukemia, is relevant. Costa B. and co-authors report cases of the development of the leukemic process in previously healthy young patients. Patients are aged 31-35 years with normal blood tests, in whom the manifestation of leukemia appeared approximately 2-3 months after COVID-19. The course of COVID-19 was not severe in all. Mild microcytaria was detected in one case of hypochromic anemia. The development of malignant hematological diseases in these patients with an unfavorable prognosis for the course and response to treatment attracts attention. These was T-cell acute lymphoblastic leukemia, myelodysplastic syndrome, acute myeloid leukemia with trigeminal dysplasia in the bone marrow, suggesting previous myelodysplastic syndrome. Nekooghadam S. M. and co-authors presented a case of acute myeloid leukemia in a man after recovery from COVID-19 after 1.5 months. The course of the infection was severe. The blood test showed slight leukocytosis. Manifestations of the described cases of leukemia included clinical gastroenteropathy and anemic syndrome. Two- or three-fold severe pancytopenia and dysplasia were more common in the analysis of bone marrow hematopoiesis. Low blastemia was a feature. Some authors suggest that SARS-Cov-2 plays a role in leukogenesis. The leading role in this is attributed to the imbalance of the renin-angiotensin system caused by the virus, which triggers leukogenesis in several mechanisms. An abnormal immune response to a viral infection can trigger secondary mutational events, contributing to the clinical development of leukemia. In addition to the effect of SARS-CoV-2 on renin-angiotensin system, other mechanisms of the potential development of cancer may underlie it. In particular, COVID-19 has been associated with T-cell exhaustion and activation of oncogenic pathways, including JAK-STAT, MAPK, and NF-kB. The coronavirus non-structural protein stabilizes factors by increasing RCHY1-mediated apoptosis-associated degradation of p53. The case of an acute leukemic process described by us was distinguished by a rapid atypical course, neurological symptoms, pancytopenia in peripheral blood during hospitalization, and a changed linear morphology of blast cells. Conclusion. According to the literature, the leukemic process after a sustained COVID-19 more often has an acute myeloid variant with hematopoietic dysplasia and a severe atypical course. Hematological monitoring of patients who have undergone COVID-19 in the presence of an oncology history or changes in the blood test during recovery from it is necessary. It is advisable to have a control of the hemogram after 2-3 months

Splicing Modulators Impair DNA Damage Response and Induce Killing of Cohesin-Mutant MDS/AML

Cohesin is a multi-subunit protein complex that is essential for sister chromatid cohesion, three-dimensional chromosome organization and gene regulation. Genes encoding components of the cohesin complex, most commonly STAG2, are frequently mutated in cancer, including 15-25% of cases of myelodysplastic syndromes MDS and secondary acute myeloid leukemia (sAML). Cohesin mutations are associated with poor overall survival and there are currently no targeted therapies available for patients with cohesin-mutant cancers. Modulation of RNA splicing with SF3B1-targeting drugs has shown efficacy in splicing-factor mutant MDS, but its use outside of this context is unknown. Prompted by our observations that SF3B1-mutations and STAG2-mutations tend not to co-occur in MDS patients and that that SF3B1-mutant cells depend on STAG2 for survival, we sought to determine the effects of SF3B1 splicing modulators on cohesin-mutant cells. First, we tested sensitivity of isogenic U937 cell lines engineered with different cohesin mutations to the SF3B1 modulators H3B-8800 and E-7107. STAG2- mutant cells showed increased sensitivity to both drugs and were out-competed by wild-type cells when treated with H3B-8800 both in vitro and in vivo. To understand the mechanism mediating the killing of cohesin-mutant cells, we quantified alternative splicing in cells treated with H3B-8800 relative to DMSO-treated controls. We identified widespread mis-splicing and corresponding reduced gene expression of DNA repair genes, including BRCA1 and BRCA2, whose mis-splicing led to complete loss of protein expression in cells. Cohesin mutant cells accumulate DNA damage and have increased reliance on proper DNA repair for survival. We therefore hypothesized that short-term treatment with SF3B1-targeting drugs could sensitize cohesin-mutant cells to DNA damage repair inhibitors, such as PARP inhibitors and chemotherapy. We tested this hypothesis using sequential treatment of isogenic cell lines and STAG2-mutant PDX models of AML with H3B-8800 and DNA repair inhibitors. Pre-treatment of cohesin-mutant cells with H3B-8800 for 3 days increased their sensitivity to the PARP inhibitor talazoparib 10-fold, while the sensitivity of wild-type cells to talazoparib was unaffected (Figure 1A). In addition, pre-treatment of mice with 5 days of E-7107 followed by talazoparib extended overall survival of multiple STAG2-mutant AML PDX models compared to talazoparib treatment alone (Figure 1B). In addition to talazoparib, we demonstrated significantly improved overall survival of STAG2-mutant AML PDX mice receiving sequential treatment of E-7107 and standard induction chemotherapy (doxorubicin and cytarabine), further expanding this strategy beyond PARP inhibitors. Finally, we validated the effects of SF3B1 splicing modulators on mis-splicing of DNA damage repair genes in a set of blood samples isolated from MDS/sAML patients enrolled in the Phase 1 trial with H3B-8800 (NCT02841540). We performed RNA-Seq immediately prior, and 2-4 hours post- treatment with H3B-8800 in three patients. We found a remarkable conservation at the event-level of the H3B-8800-induced mis-spliced targets as were observed in our cell line and PDX models, as well as the same trend for downregulation of DNA repair genes. This result suggests that H3B-8800 targets mis-splicing of DNA repair genes in primary MDS/sAML patients, and that sequential treatment with H3B-8800 and chemotherapeutic agents in vivo may provide additional therapeutic benefit to patients with cohesin-mutations. In summary, our study identifies a critical connection between cohesin mutations and splicing modulation in AML, creating a novel therapeutic strategy for these patients with very limited treatment options and poor outcomes. Our findings not only expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS/sAML but we also propose this as a broader strategy for therapeutic targeting of other DNA damage-repair deficient cancers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Clinical Laboratory-Oriented Targeted RNA-Seq System Accurately and Efficiently Detected Various Fusion Gene Mutations in Philadelphia Chromosome-like Acute Lymphoblastic Leukemia

Introduction: Genetic mutations that occur in blood cancer can be broadly classified into nucleotide sequence mutations including duplications or deletions, fusion gene mutations, and abnormal gene overexpression. Among these genetic mutations, gene fusion not only plays an important role in the development of blood cancers, such as leukemia, but is also important as an essential marker for the diagnosis, risk assessment, optimal treatment selection, and residual lesion detection. Most clinical laboratories use conventional cytogenetic testing, fluorescence in situ hybridization, and multiplex reverse transcriptase-polymerase chain reaction (PCR) to detect fusion gene mutations. However, the currently used methods have great limitations in detecting various gene fusions occurring in blood cancer. To overcome these shortcomings, RNA sequencing (RNA-seq) methods are being introduced in clinical laboratories. The authors developed a targeted RNA-seq panel, a bioinformatics pipeline used for targeted RNA-seq to detect gene fusions, sequence variants, and altered gene expression, which can be used more conveniently in clinical laboratories. Methods: The targeted RNA-seq panel and analysis pipeline developed based on information technology with an automated reporting system and commercialized by the authors was applied to a comparative evaluation with the commercialized targeted RNA-seq panel (Archer™ FusionPlex™ Heme Panel version 2; Archer DX, Boulder, CA, USA) using samples of acute leukemia patients. A laboratory-oriented targeted RNA-seq system consisting of 84 genes was developed in consideration of the test reporting time and operational efficiency in clinical laboratories (KBB-RNAseq NGS-Leukemia-PHB; KBlueBio Inc., Hwasun, Korea). For comparison with the existing analysis system, the Archer FusionPlex Heme v2 panel (ArcherDx) based on the cDNA library for specific genes using anchored multiplex PCR was used. To evaluate the test performance of these systems, bone marrow specimens were used while diagnosing 93 patients with leukemia (15 cases of acute myeloid leukemia, 35 cases of adult B-acute lymphoid leukemia, 30 cases of childhood B-acute leukemia, and 13 cases of T-acute lymphoid leukemia). Results: Among all 93 leukemia patients, tier 1 or tier 2 gene fusions were observed in 72 (77%) patients. Gene fusions were detected in 83% (25/30) of patients with pediatric B-acute lymphoid leukemia (B-ALL), and in 94% (33/35) of patients with adult B-ALL. In patients with acute myeloid leukemia (AML) and T-ALL, the proportions of fusion gene mutations were 53% (8/15) and 46% (6/13), respectively. For the comparative evaluation, 4 cases of B-ALL, 2 cases of AML, and 1 case of acute promyelocytic leukemia and T-ALL were used. The results of the two analysis systems were consistent in 7 of the 8 comparatively evaluated patients, but an IGH-CRLF2 fusion in a B-ALL case was detected only with the panel developed by the authors. It is known that the CRLF2 gene is a causative gene in half of the Philadelphia chromosome (Ph)-ALL cases, which account for 20-25% of all B-ALL cases and are classified as a new type of ALL showing a very poor prognosis. Conclusions: Our targeted RNA-seq system accurately and efficiently detected various fusion gene mutations that occur in acute leukemia. Importantly, our targeted RNAseq panel detected fusion gene mutations that cause Ph-like B-ALL more accurately than the c-ommercialized test system. This targeted RNA-seq system showed a reliable and stable performance in detecting various gene fusions occurring in blood cancers, such as leukemia, in clinical laboratories. Also, our targeted RNA-seq system was found to be very useful in diagnosing Ph-like B-ALL compared to the commercial RNA-seq panel. Keywords: Targeted RNA-seq, Clinical Laboratory, Ph-like B-ALL, Acute leukemia Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Rapid Etiological Detection Value of Metagenomic Next-Generation Sequencing for Bloodstream Infections in Patients with High-Risk Hematologic Malignancies

Background: Patients with hematological malignancies often suffer from infectious fever, rapid and accurate diagnosis of pathogens facilitates optimal and precise treatment of infectious diseases. In recent years, metagenomic next-generation sequencing (mNGS) technology has been widely used in the etiological detection of patients with respiratory tract infection, sepsis, etc, due to its advantages of rapid detection.However, at present, the clinical diagnosis of mNGS in patients with hematological malignancies in secondary bloodstream infections remains scarce. Objective: In this study, the application value of mNGS in the rapid etiological detection of bloodstream infection pathogens in patients with high-risk hematological malignancies was explored by comparing two etiological detection methods of mNGS and traditional pathogen culture. Methods: This study retrospectively analyzed 66 peripheral blood samples of patients with high-risk hematologic malignancies in our hospital from May 2020 to January 2022. SPSS 20.0 software was used to analyze the positive rate, species distribution and consistency of pathogens between mNGS and traditional pathogen culture method. The diagnostic efficiency of the two etiological detection methods was compared, and the risk factors affecting the positive rate of mNGS were investigated. Results: A total of 66 patients were enrolled in this study, including 38 males and 28 females, with an average age of (43.39±14.427) years. Among the 66 patients, 48 cases were acute myeloid leukemia (48/66, 72.7%), the rest included 2 cases of acute lymphoblastic leukemia (2/66, 3.0%), 3 cases of chronic myeloid leukemia (3/66, 4.5%), 3 cases of myelodysplastic syndrome (3/66, 4.5%), 4 cases of non-Hodgkin lymphoma (4/66, 6.0%), 4 cases of malignant tumor after autologous hematopoietic stem cell transplantation (4/66, 6.0%), and Hemophagocytic syndrome was found in 2 cases (2/66, 3.0%). Peripheral blood samples from 66 patients were both tested for mNGS and traditional pathogen culture. The positive rate of mNGS was 54.5% (36/66), while the traditional culture was 18.1% (12/66). There was a significant statistical difference between the two methods (P=0.000). Multivariate analysis was conducted on the influence of factors such as patient age, gender, disease diagnosis, and time from fever to the mNGS detection, and no statistical difference was found. Among the 36 mNGS-positive patients, a total of 64 pathogens were detected, including 19 bacteria (19/64, 29.7%), 12 fungus (12/64, 18.8%), 31 virus (31/64, 48.4%), and 2 special pathogens (2/64, 3.1%). Six drug-resistant bacteria were detected by mNGS in 5 patients .Among the 12 patients with positive traditional culture, 15 pathogens were detected, including 14 bacteria (14/15, 93.3%) and 1 fungus (1/12, 3.7%). In this study, 8 patients (8/66, 36.4%) were both positive for mNGS and traditional culture tests, and 5 of them (5/8, 62.5%) developed the same type of clinical infection with all or part identical detected pathogens. The time for positive and negative blood culture report was 72h and 7 days respectively, and 24h for mNGS. Among the 54 patients with negative results of traditional culture, 28 cases (28/54, 51.9%) were positive for mNGS, of which 15 cases (15/28, 53.6%) were made corresponding anti-infection regimens according to the positive results of mNGS, and the clinical efficacy was significantly improved. Seven of the 26 patients with both negative traditional culture and mNGS results were considered to have fungal infection based on chest imaging. After empirical antifungal treatment, the imaging results and clinical symptoms were improved; while only 4 of the remaining 19 patients (4/19, 21.5%) had clinical benefit from empirical anti-infective therapy. It demonstrated that pathogen-oriented therapy was significantly better than empirical anti-infective therapy (P=0.000). Conclusions: mNGS enhanced the positive rate of bloodstream pathogens detection and provided more accurate pathogen species in patients with high-risk hematologic malignancies.It could effectively detect multidrug-resistant bacteria and special pathogens,which provide an important reference for clinical antimicrobial treatment and improved their prognosis.

RIPK3 Deficiency Caused By DNA Hypermethylation Blocks R-2-Hydroxyglutarate Induced Necroptosis in AML with IDH Mutation

Background Mutations in isocitrate dehydrogenases (IDH) are frequently identified in 10% - 20% of acute myeloid leukemia (AML) cases. IDH mutations induce a neomorphic enzymatic function that catalyzes the conversion of α-ketoglutarate (α-KG) to the R enantiomer of 2-hydroxyglutarate (R-2HG). α-KG is also a co-substrate, together with oxygen and the co-factor iron, for α-KG-dependent dioxygenases. R-2HG is structurally close to α-KG and competitively inhibits the activity of α-KG-dependent dioxygenases. It has been reported that R-2HG inhibits the growth of IDH-wild-type AML cells by inhibiting the activity of fat mass and obesity-associated protein (FTO) and then reducing MYC and CEBPA expression. Recently, R-2HG was found to suppress aerobic glycolysis and proliferation in myeloid leukemia cells. However, why AML cells carrying IDH mutations tolerated high levels of R-2HG is not clear. Results In line with the previous study, we found that R-2HG markedly inhibited the viability of AML cells in a dose- and time-dependent manner. Interestingly, Necrostatin-1(Nec-1), a necroptosis inhibitor, could significantly prevent cell death induced by R-2HG in AML cell lines, but not Ferrostatin-1 (a ferroptosis inhibitor), Z-VAD (an apoptosis inhibitor), 3-MA (an autophagy inhibitor) and VX-765 (a pyroptosis inhibitor), suggesting that necroptosis contributed to the anti-leukemia activity of R-2HG. We evaluated the expression of genes involved in the necroptosis pathway and found the level of RIPK1 was significantly increased in R-2HG treated SKM-1 and THP-1 cells, while the expressions of RIPK3 and MLKL did not change. The knockdown of RIPK1 reduced the necroptosis caused by R-2HG in SKM-1 and THP-1 cells. Besides, a significant decrease in proliferation was observed when RIPK1 was overexpressed in AML cells which was significantly rescued by Nec-1. Among α-KG-dependent dioxygenases, the expression of lysine demethylase 2B (KDM2B) was negatively correlated with that of RIPK1. Moreover, KDM2B deficiency significantly increased RIPK1 expression and triggered necroptosis. ChIP followed by qPCR (ChIP-qPCR) showed that the RIPK1 promoter region was significantly enriched by H3K4me3, a main target of KDM2B. R-2HG treatment or knockdown of KDM2B activated H3K4me3 and increased RIPK1 expression. By comparing the expression of necroptosis-related genes between R-2HG-resistant and sensitive cell lines (GSE87187), we found that RIPK3, another key player of necroptosis, was under-expressed in R-2HG-resistant cell lines. Consistently, RIPK3 was lowly expressed in IDH-mutant primary AML cells compared to that of IDH wild-type primary AML cells. We found that enhanced RIPK3 levels increased the toxicity of R-2HG in R-2HG-resistant cell lines. Methylation-specific PCR (MS-PCR) revealed that methylation occurred in this region of genomic DNA near the TSS of RIPK3 in R-2HG-resistant cell lines. Primary cells from IDH-mutant AML patients were more sensitive to decitabine than those from IDH wild-type AML patients. Conclusion Herein, we reported that R-2HG triggered necroptosis in AML cell lines and IDH wild-type primary AML cells via KDM2B/RIPK1 signal pathway. The loss of RIPK3 expression resulting from RIPK3 promoter hypermethylation contributed to R-2HG tolerance in IDH mutated primary AML cells and R-2HG-resistant AML cell lines. IDH-mutant AML cells were more sensitive to DNMT inhibitor. These results implicate that restoring RIPK3 activity is a promising way to reverse R-2HG-resistant in AML cells with IDH mutation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunotherapy By Targeting CD45 in Mismatched HLA-DP to Treat Patients after Allogeneic Stem Cell Transplantation

Allogeneic stem cell transplantation (alloSCT) is a curative treatment for patients with hematological malignancies. The anti-tumor or Graft-versus-Leukemia (GvL) effect of alloSCT is mediated by donor T cells targeting patient hematopoietic cells. Donor T cells may, however, also target non-hematopoietic tissues of the patient, thereby inducing Graft-versus-Host Disease (GvHD). Patients without suitable related donors are transplanted with unrelated donors matched for HLA-A, -B, -C, -DRB1, and -DQB1 (10/10 match). HLA-DP alleles are mismatched in most patients transplanted with unrelated donors. HLA class II molecules are mainly expressed on hematopoietic cells, but under inflammatory conditions the expression of HLA class II on non-hematopoietic tissues is upregulated, thereby increasing the risk of GvHD. To reduce this risk and retain the anti-tumor effect, patients transplanted with unrelated donors may be treated with immunotherapy specifically targeting hematopoietic peptides presented by mismatched HLA-DP. CD45 is an attractive source protein for these peptides because of its broad expression in all hematopoietic lineages. Despite CD45 expression, healthy hematopoietic cells after alloSCT are of donor origin and will therefore be spared. To investigate whether CD45 peptides are presented by HLA-DP on hematopoietic cells, peptides were eluted from HLA-DP and measured by tandem mass spectrometry. HLA-DP peptidome data were generated from acute myeloid leukemia (AML) cases as well as hematopoietic (CD45pos) K562 cells separately transduced with 13 different HLA-DP alleles. Peptidome searching revealed one CD45 peptide on K562 cells transduced with HLA-DP2 (DPA1*01:03 & DPB1*02:01) or HLA-DP4 (DPA1*01:03 & DPB1*04:01 or DPB1*04:02) as well as AML cases expressing these HLA-DP alleles. Next, (CD45pos) K562 cells transduced with HLA-DP2 or HLA-DP4 and costimulatory molecules (CD40, CD80, CD86) were used to stimulate CD4 T cells from healthy individuals negative for these HLA-DP alleles. After 14 days of stimulation, CD4 T cells were restimulated and activated CD137+ and CD154+ CD4 T cells were single cell sorted. Growing T cell clones were screened for reactivity against CD45 peptide loaded on non-hematopoietic (CD45neg) HeLa cells transduced with HLA-DP2 or HLA-DP4. Three clones were able to specifically recognize CD45 in HLA-DP2 or HLA-DP4 (Figure 1A). T cell clones M6-5 and M6-6 were specific for CD45 in HLA-DP2, and clone M6-7 recognized the CD45 peptide in HLA-DP4. No cross-reactivity was observed against other HLA-DP alleles. In addition to HLA-DP transduced K562 cells, all T cell clones specifically reacted against HLA-DP2 or HLA-DP4 positive AML cases. We next investigated whether the T cell receptors (TCRs) of the T cell clones could be used to engineer and redirect CD4 T cells to CD45-derived peptides in mismatched HLA-DP. The HLA-DP2 restricted TCRs of M6-5 and M6-6 and HLA-DP4 restricted TCR of M6-7 were sequenced, cloned and transferred to CD4 T cells from three HLA-DP2 and HLA-DP4 negative healthy donors. TCR-T cells showed specific reactivity against K562 transduced with HLA-DP2 or HLA-DP4, whereas HeLa cells with these HLA-DP alleles were not recognized (Figure 1B, example of one donor). In conclusion, we identified three TCRs for a CD45 peptide presented by HLA-DP2 or HLA-DP4 that are able to target hematopoietic cells. The TCRs can be used for CD4 T cell immunotherapy to treat patients with hematological malignancies after HLA-DP mismatched alloSCT. Figure 1. CD45 as a target for specific CD4 T cells. (A) CD4 T cell clones recognizing CD45 in HLA-DP2 or HLA-DP4 on AML. (B) M6-5, M6-6 and M6-7-TCR transduced CD4 T cells recognize HLA-DP2 or -DP4 transduced K562 (CD45pos) but not HeLa (CD45neg) cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Liquid Biopsy-Based Next-Generation Sequencing Is an Alternative to Tissue Molecular Profiling of Lymphoid, Plasma-Cell, and Myeloid Neoplasms

Background: Liquid biopsy (LB)-based next-generation sequencing (NGS) of circulating tumor DNA (ctDNA) fragments can facilitate the molecular profiling of hematopoietic neoplasms (HNs), particularly in patients for whom conventional NGS is infeasible due to insufficient tumor in the blood, bone marrow, or tissue, or when tissue biopsy cannot be performed. Because HNs shed ctDNA into the plasma at levels comparable to those shed by solid tumors, LB assays initially designed for solid tumors can be adapted for use in hematology. We evaluated a LB-based ctDNA approach in patients with HNs. Methods: Retrospective study of LB samples from patients reported to have HNs tested using hybrid-capture NGS using the FoundationOne Liquid CDx (targeting 324 genes), FoundationOne Liquid (70 genes), or FoundationACT (62 genes) assays between 7/2016 and 3/2022. Paired buffy coat, bone marrow aspirate, or tissue samples undergoing conventional NGS with the FoundationOne Heme (406 genes), FoundationOne CDx (324 genes), or FoundationOne (322 genes) assays were sought where available, and genomic alterations baited across both the LB- and tissue-based assays were assessed for concordance. Results: Among 67,500 unique patient samples submitted for LB-based NGS, 271 were from HNs, including 89 non-Hodgkin lymphoma (NHL), 43 plasma-cell neoplasm (PCN), 41 histiocytosis, 27 myelodysplastic syndrome (MDS), 25 diffuse large B-cell lymphoma (DLBCL), 22 myeloproliferative neoplasm (MPN), 14 Hodgkin lymphoma (HL), and 10 acute myeloid leukemia (AML) cases. The median (interquartile range [IQR]) patient age was 68.0 (55.5-78.0) years, and 121 (44.6%) patients were female. Samples were sequenced to a median exon coverage of 8,564× and pathogenic alterations were detected in 73.4% (199/271) of cases. The median maximum somatic allele frequency (MSAF) among all cases was 7.3% (IQR, 1.1-42.7%), with higher median MSAFs in MPN (45.8%), AML (30.9%), and MDS (19.7%) than in NHL (8.4%), DLBCL (7.1%), histiocytosis (3.1%), HL (2.1%), and PCN (2.0%) cases (P = 0.02, Kruskal-Wallis test), not unexpected given that myeloid neoplasms naturally circulate (Figure 1). LB detected characteristic genomic alterations across HNs, including in TP53, KRAS, MYD88, and BTK in NHLs; TP53, KRAS, NRAS, and BRAF in PCNs; IGH in DLBCL; TP53, ATM, and PDCD1LG2 in HL; BRAF and MAP2K1 in histiocytoses; TP53, SF3B1, DNMT3A, TET2, and ASXL1 in MDS; JAK2 in MPNs; and FLT3, IDH2, and NPM1 in AML, among others (Figure 2). Paired buffy coat, bone marrow aspirate, or tissue specimens collected a median of 118 (IQR 36-346) days apart from the LB specimens sequenced to a median exon coverage of 810× were available for 42 patients. Of 95 variants in 20 HN-associated genes equally baited across the pairs, 31 (32.6%) were detected in both specimens. Of 42 variants detected on buffy coat, bone marrow aspirate, or tissue sequencing, 31 were also detected by LB (positive percent agreement = 73.8%). Eight pairs harbored variants detected in tissue only, all of which were explainable by either low ctDNA shed (e.g., a BRAF fusion was not detected in a LB sample with MSAF = 0.1%) or heterogeneity related to the disease course (e.g., a FLT3 tyrosine kinase domain variant identified by conventional tissue NGS was not detected on follow-up LB two years later, post-treatment). Conversely, 22 samples had ≥1 variant detected in the LB sample only, including in NF1 (n=4), KRAS (n=3), and BRAF (n=2), likely reflecting the enhanced sensitivity of LB for detecting low-level disease clones. Conclusions: LB detected relevant genomic alterations in the majority of patients with a variety of HNs. Given its analytical sensitivity, LB may also detect low-level residual or emerging therapy-resistant clones unable to be detected in a single tissue specimen and may play a role in the evaluation of the patient's disease course and in associated treatment decisions. This pilot study suggests the potential value of LB in the clinical management of patients; however, further prospective evidence would be required to quantify the utility of LB for patients with HNs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

105. Clinical whole-genome sequencing identifies NSD3 as the correct fusion partner of NUP98 in a patient with acute myeloid

<h3>Background</h3> Only rare cases of acute myeloid leukemia (AML) have been shown to harbor a t(8;11)(p11.2;p15.4). This translocation is believed to involve the fusion of NSD3 or FGFR1 with NUP98; however, apart from targeted mRNA quantitative PCR analysis, no molecular approaches have been utilized to define the chimeric fusions present in these rare cases. <h3>Case Presentation</h3> Here we present the case of a 51-year-old female with AML with myelodysplastic-related morphologic changes, 13q deletion and t(8;11), where initial fluorescence in situ hybridization (FISH) assays were consistent with the presence of NUP98 and FGFR1 rearrangements, and suggestive of NUP98/FGFR1 fusion. Using a streamlined clinical whole-genome sequencing approach, we resolved the breakpoints of this translocation to intron 4 of NSD3 and intron 12 of NUP98, indicating NUP98/NSD3 rearrangement as the underlying aberration. Furthermore, our approach identified small variants in WT1 and STAG2, as well as an interstitial deletion on the short arm of chromosome 12, which were cryptic in G-banded chromosomes. <h3>Conclusions</h3> NUP98 fusions in acute leukemia are predictive of poor prognosis. The associated fusion partner and the presence of co-occurring mutations, such as WT1, further refine this prognosis with potential clinical implications. Using a clinical whole-genome sequencing analysis, we resolved t(8;11) breakpoints to NSD3 and NUP98, ruling out the involvement of FGFR1 suggested by FISH while also identifying multiple chromosomal and sequence level aberrations.

Acute Erythroleukemia (AML-M6) in one-year-old boy: a case report

Background: Acute Erythroleukemia (AML-M6) is a rare case with approximately 3-4% of all Acute Myeloid Leukemia (AML) cases. AML-M6 is less common in the pediatric population, with an incidence of &lt;1% of all pediatric leukemia cases. This case study aims to evaluate Acute Erythroleukemia (AML-M6) in a one-year-old boy at Prof. dr. IGNG Ngoerah Denpasar. Case Presentation: A one-year-old boy with clinical manifestations of fluctuating fever for a month, paleness, bleeding gums, bruising all over his body, swelling of both eyelids and splenomegaly. Peripheral Blood Smear (PBS) examination revealed bicytopenia with moderate normochromic normocytic anemia, severe thrombocytopenia and leukocytosis. A hypercellular picture was obtained from the examination of Bone Marrow Aspiration (BMA) with an increase in erythroblast activity of 74%, proerythroblast at 50% and normal myeloblasts activity at 10%. The patient was diagnosed with Acute Erythroleukemia (AML-M6). The patient died on day 16 of treatment. Conclusion: Based on the examination results, it was concluded that the patient was diagnosed with AML-M6 according to the PBS and BMA evaluation.