Association Between Clonal Hematopoiesis of Indeterminate Potential and Brain ß-Amyloid Deposition in Korean Patients With Cognitive Impairment.

Few studies have focused on the association between clonal hematopoiesis of indeterminate potential (CHIP) and ß-amyloid (Aß) deposition in the brain, which causes Alzheimer's disease. We aimed to investigate the potential role of CHIP in brain Aß deposition in Korean patients. We enrolled 58 Korean patients over 50 yrs of age with cognitive impairment who underwent brain Aß positron emission tomography. We explored CHIP in their peripheral blood using deep-targeted next-generation sequencing. Irrespective of the presence or absence of brain Aß deposition, mutations in DNMT3A and the C:G>T:A single-nucleotide variants were identified as the primary characteristics, which reflect aged hematopoiesis in the study population. Multivariate logistic regression revealed that the presence of CHIP was not associated with brain Aß deposition. As both CHIP and brain Aß deposition are associated with aging, further research is required to elucidate their possible interplay.

Multiple Primary Cancers With Hematologic Malignancies and Germline Predisposition: A Case Series.

The term "multiple primary (MP) cancers" refers to the existence of more than one cancer in the same patient. The combination of MP cancers with hematological malignancies is relatively uncommon. In this study, we present five patients diagnosed with MP cancers concomitant with hematological malignancies. We comprehensively analyzed their clinical characteristics, cytogenetic profiles, and germline and somatic variants. As first primaries, two patients had solid cancer not followed by cytotoxic therapy and three had hematologic cancer, followed by cytotoxic therapy. The second primaries were all hematologic malignancies that did not meet the criteria for therapy-related myeloid neoplasm. Notably, two (40%) out of the five patients harbored pathogenic potential/presumed germline variants in cancer predisposition genes. Therefore, germline variant testing should be considered when MP cancers with hematological malignancies require consideration for related donor stem cell transplantation.

Cryptic KMT2A/MLLT10 fusion detected by next-generation sequencing in a case of pediatric acute megakaryoblastic leukemia.

KMT2A (11q23.3) gene rearrangements are found in acute leukemia and are associated with a poor or intermediate prognosis. MLLT10 is the fourth most common gene fusion partner for KMT2A. A reciprocal translocation t(10;11) is insufficient to produce an in-frame KMT2A/MLLT10 fusion, because the genes involved in the rearrangement have opposite transcriptional orientations. In order to bring KMT2A and MLLT10 into juxtaposition, complex rearrangements are required. Until now, conventional chromosome, fluorescence in situ hybridization (FISH), and reverse transcriptase-polymerase chain reaction (RT-PCR) studies have been used to detect KMT2A/MLLT10 fusions. However, conventional studies have limitations, such as poor and inconsistent resolution, when compared to next-generation sequencing (NGS). In this study, we report a pediatric patient with acute megakaryoblastic leukemia, in whom the cryptic KMT2A/MLLT10 fusion was not detected by KMT2A break-apart probe FISH and chromosome analysis, but detected by NGS. In this patient, NGS showed cryptic insertion of MLLT10 exons 9-24 into intron 9 of KMT2A, resulting in a KMT2A/MLLT10 fusion. Therefore, NGS is a valuable complementary option for the evaluation of structural aberrations, especially those with a cryptic size.

Distinct mutational pattern of T-cell large granular lymphocyte leukemia combined with pure red cell aplasia: low mutational burden of STAT3.

T-cell large granular lymphocyte leukemia (T-LGL) is often accompanied by pure red cell aplasia (PRCA). A high depth of next generation sequencing (NGS) was used for detection of the mutational profiles in T-LGL alone (n = 25) and T-LGL combined with PRCA (n = 16). Beside STAT3 mutation (41.5%), the frequently mutated genes included KMT2D (17.1%), TERT (12.2%), SUZ12 (9.8%), BCOR (7.3%), DNMT3A (7.3%), and RUNX1 (7.3%). Mutations of the TERT promoter showed a good response to treatment. 3 of 41 (7.3%) T-LGL patients with diverse gene mutations were revealed as T-LGL combined with myelodysplastic syndrome (MDS) after review of bone marrow slide. T-LGL combined with PRCA showed unique features (low VAF level of STAT3 mutation, low lymphocyte count, old age). Low ANC was detected in a STAT3 mutant with a low level of VAF, suggesting that even the low mutational burden of STAT3 is sufficient for reduction of ANC. In retrospective analysis of 591 patients without T-LGL, one MDS patient with STAT3 mutation was revealed to have subclinical T-LGL. T-LGL combined with PRCA may be classified as unique subtype of T-LGL. High depth NGS can enable sensitive detection of concomitant MDS in T-LGL. Mutation of the TERT promoter may indicate good response to treatment of T-LGL, thus, its addition to an NGS panel may be recommended.

Analysis of clinical and genomic profiles of therapy-related myeloid neoplasm in Korea.

BACKGROUND: Therapy-related myeloid neoplasm (T-MN) rarely occurs among cancer survivors, and was characterized by poor prognosis. T-MN has germline predisposition in a considerable proportion. Here, clinical characteristics and germline/somatic variant profiles in T-MN patients were investigated, and the findings were compared with those of previous studies. METHODS: A review of medical records, cytogenetic study, targeted sequencing by next-generation sequencing, and survival analysis were performed on 53 patients with T-MN at a single institution in Korea. RESULTS: The patients were relatively younger compared to T-MN patients in other studies. Our T-MN patients showed a high frequency of complex karyotypes, -5/del(5q), and -7/del(7q), which was similar to the Japanese study group but higher than the Australian study group. The most common primary disease was non-Hodgkin lymphoma, followed by breast cancer. The detailed distributions of primary diseases were different across study groups. Seven patients (13.2%) harbored deleterious presumed/potential germline variants in cancer predisposition genes (CPG) such as BRIP1, CEBPA, DDX41, FANCM, NBN, NF1, and RUNX1. In the somatic variant profile, TP53 was the most frequently mutated gene, which was consistent with the previous studies about T-MN. However, the somatic variant frequency in our study group was lower than in other studies. Adverse factors for overall survival were male sex, older age, history of previous radiotherapy, previous longer cytotoxic therapy, and -5/del(5q). CONCLUSION: The findings of our study corroborate important information about T-MN patients. As well as a considerable predisposition to CPG, the clinical characteristics and somatic variant profile showed distinctive patterns. Germline variant testing should be recommended for T-MN patients. If the T-MN patients harbor pathogenic germline variants, the family members for stem cell donation should be screened for carrier status through germline variant testing to avoid donor-derived myeloid neoplasm. For the prediction of the prognosis in T-MN patients, sex, age, past treatment history, and cytogenetic findings can be considered.

Novel THPO variant in hereditary thrombocytopenia: A potential candidate variant for predisposition to myeloid neoplasm.

Hereditary thrombocytopenia is a heterogeneous group of congenital disorders with a wide range of symptoms depending on the severity of platelet dysfunction or thrombocytopenia. Because of its clinical phenotypes and the bone marrow morphology associated with this condition, hereditary thrombocytopenia can be misdiagnosed as primary immune thrombocytopenia and myelodysplastic syndrome. Therefore, genetic evidence is necessary for the accurate diagnosis of hereditary thrombocytopenia. Refractory cytopenia of childhood is a subgroup of myelodysplastic syndrome that was added to the World Health Organization classification in 2008. To investigate the germline and somatic variants associated with refractory cytopenia of childhood, we performed targeted multigene sequencing in three patients with refractory cytopenia of childhood. Of the three patients, one progressed from megakaryocytic hypoplasia with thrombocytopenia, and targeted multigene sequencing revealed THPO variants in this patient and his sister. We propose that the monoallelic deletion of THPO is a potential candidate for germline predisposition to myeloid malignancy.

Laboratory Perspective of Concurrent Acute Myeloid Leukemia and COVID-19: a Case Report and Review of Literature.

BACKGROUND: Whether the diagnosis and treatment of coronavirus disease 2019 (COVID-19) affect the clinical course of patients with hematologic malignancies has been of interest during the COVID-19 pandemic. METHODS: We describe a 47-year-old female who was concurrently diagnosed with COVID-19 and acute myeloid leukemia (AML). RESULTS: She developed COVID-19 pneumonia which required treatment with remdesivir and dexamethasone, and induction therapy for t-AML was delayed. After her COVID-19 was resolved and isolation ended, follow-up bone marrow examination showed decreased leukemic burden. CONCLUSIONS: This case describes possible effects of COVID-19 treatment on the clinical course of patients with AML from a laboratory perspective.

Heterogeneous genetic landscape of congenital neutropenia in Korean patients revealed by whole exome sequencing: genetic, phenotypic and histologic correlations.

Congenital neutropenia (CN) is a hematological disease heterogeneous in its genetic, phenotypic and histologic aspects. We aimed to identify the genetic etiology of Korean CN patients in the context of bone marrow (BM) histology and clinical phenotype. Whole-exome sequencing (WES) or targeted sequencing was performed on the BM or peripheral blood specimens of 16 patients diagnosed with CN based on BM exam from 2009 to 2018. Absolute count of myeloperoxidase (MPO)-positive cells was calculated using ImageJ software. Semi-quantitation of MPO-positive cells in BM sections was performed by MPO grading (grades 0-3). Comprehensive retrospective review on real-world data of 345 pediatric patients with neutropenia including 16 patients in this study during the same period was performed. Seven disease-causing variants were identified in ELANE, G6PC3 and CXCR4 in 7 patients. A novel homozygous G6PC3 variant (K72fs) of which the mechanism was copy-neutral loss of heterozygosity was detected in two brothers. A low myeloid-to-erythroid ratio (0.5-1.5) was consistently observed in patients with ELANE mutations, while MPO-positive cells (40%-50%) with MPO grade 1 or 2 were detected in myelokathexis caused by G6PC3 and CXCR4 mutations. Meanwhile, disease-causing variants were detected in ELANE, TAZ and SLC37A4 in 5 patients by retrospective review of medical records. Our results suggest that following the immunological study and BM exam, WES or an expanded next generation sequencing panel that covers genes related to immunodeficiency and other inherited bone marrow failures as well as CN is recommended for neutropenia patient diagnosis.

Clinical and Genomic Profiles of Korean Patients with MECOM Rearrangement and the t(3;21)(q26.2;q22.1) Translocation.

The translocation (3;21)(q26.2;q22.1) is a unique cytogenetic aberration that characterizes acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) in patients with AML and myelodysplastic syndrome (MDS) or a therapy-related myeloid neoplasm. Using multigene target sequencing and FISH, we investigated the clinical and genomic profiles of patients with t(3;21) over the past 10 years. The frequency of t(3;21) among myeloid malignancies was very low (0.2%). Half of the patients had a history of cancer treatment and the remaining patients had de novo MDS. Twenty-one somatic variants were detected in patients with t(3;21), including in CBL, GATA2, and SF3B1. Recurrent variants in RUNX1 (c.1184A>C, p.Glu395Ala) at the same site were detected in two patients. None of the patients with t(3;21) harbored germline predisposition mutations for myeloid neoplasms. MECOM rearrangement was detected at a higher rate using FISH than using G-banding, suggesting that FISH is preferable for monitoring. Although survival of patients with t(3;21) is reportedly poor, the survival of patients with t(3;21) in this study was not poor when compared with that of other AML patients in Korea.

Genetic profile of primary plasma cell leukemia in Korea: comparison with plasma cell myeloma.

Plasma cell leukemia (PCL) is clinically and genetically distinct from multiple myeloma (MM), despite controversies regarding the disease definition. To determine the distinct features of PCL, the genetic property of primary PCL (pPCL) was compared with that of secondary PCL (sPCL) and MM. In patients with pPCL, Eighty-nine non-synonymous mutations were observed in 68 genes. The most frequently mutated genes were TP53, TSC2, and TYK2. In comparison with genetic abnormalities of sPCL and MM, 45 genes were present only in pPCL while 28 genes were only in sPCL and 22 genes only in MM. Among the common genes between pPCL and MM, a higher prevalence of TP53 was observed in pPCL, compared to MM (p < 0.05), while similar, compared to sPCL (p = 0.64). In summary, pPCL patients showed a higher level of genetic heterogeneity and distinctive genetic signature in their mutational profile compared to patients with MM and sPCL.

Evaluation of Three Multiplex Real-time Reverse Transcription PCR Assays for Simultaneous Detection of SARS-CoV-2, Influenza A/B, and Respiratory Syncytial Virus in Nasopharyngeal Swabs.

BACKGROUND: In the coronavirus disease 2019 (COVID-19) pandemic era, the simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (Flu), and respiratory syncytial virus (RSV) is important in the rapid differential diagnosis in patients with respiratory symptoms. Three multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been recently developed commercially in Korea: PowerChek™ SARS-CoV-2, Influenza A&B Multiplex Real-time PCR Kit (PowerChek; KogeneBiotech); STANDARD™ M Flu/SARS-CoV-2 Real-time Detection Kit (STANDARD M; SD BioSensor); and Allplex™ SARS-CoV-2/FluA/FluB/RSV Assay (Allplex; Seegene). We evaluated the analytical and clinical performances of these kits. METHODS: A limit of detection tests were performed and cross-reactivity analysis was executed using clinical respiratory samples. Ninety-seven SARS-CoV-2-positive, 201 SARS-CoV-2-negative, 71 influenza A-positive, 50 influenza B-positive, 78 RSV-positive, and 207 other respiratory virus-positive nasopharyngeal swabs were tested using the three assays. The AdvanSure™ respiratory viruses rRT-PCR assay (AdvanSure; LG Life Sciences) was used as a comparator assay for RSV. RESULTS: Except in influenza B, in SARS-CoV-2 and influenza A, there were no significant differences in detecting specific genes of the viruses among the three assays. All three kits did not cross-react with common respiratory viruses. All three kits had greater than 92% positive percent agreement and negative percent agreement and ≥ 0.95 kappa value in the detection of SARS-CoV-2 and flu A/B. Allplex detected RSV more sensitively than AdvanSure. CONCLUSION: The overall performance of three multiplex rRT-PCR assays for the concurrent detection of SARS-CoV-2, influenza A/B, and RSV was comparable. These kits will promote prompt differential diagnosis of COVID-19, influenza, and RSV infection in the COVID-19 pandemic era.

Double positivity of anti-ß2-glycoprotein I domain I and anti-phosphatidylserine/prothrombin antibodies enhances both thrombosis and positivity of anti-ADAMTS13 antibody.

Although a few antiphospholipid syndrome (APS) occurs with acquired thrombotic thrombocytopenic purpura (TTP), the relationship between antiphospholipid antibodies (aPL) and anti-ADAMTS13 (anti-a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) antibody remains uncertain. We investigated the relationship between high-risk thrombotic aPL and anti-ADAMTS13 antibody. Two hundred and thirty-seven patients with positive lupus anticoagulant and/or anticardiolipin antibody were included. Anti-ß2GPI (anti-ß2-glycoprotein I), anti-ß2GPIdI (anti-ß2-glycoprotein I domain I), anti-PS/PT (anti-phosphatidylserine and prothrombin), ADAMTS13 activity, and anti-ADAMTS13 antibody were measured. Double positivity of anti-ß2GPI and anti-PS/PT increased thrombotic risk more than three-fold and showed increased positivity of anti-ADAMTS13 antibody in comparison with the double negative group. Double positivity of anti-ß2GPIdI and anti-PS/PT presented both effects even more. In the linear regression analysis, double positivity of anti-ß2GPI and anti-PS/PT independently affected the anti-ADAMTS13 antibody level (ß = 1.982, P = 0.042). Our results revealed that double positivity of anti-ß2GPI or anti-ß2GPIdI and anti-PS/PT increased not only thrombotic risk but also the positivity of anti-ADAMTS13 antibody, especially indicating anti-ß2GPIdI showed a higher synergistic effect with anti-PS/PT. We suggest a possible association of anti-ADAMTS13 antibody with a high thrombotic risk of APS. Double positivity of anti-ß2GPI (anti-ß2-glycoprotein I) and anti-PS/PT (anti-phosphatidylserine and prothrombin) antibodies enhanced not only thrombotic risk but also positivity of anti-ADAMTS13 (anti-a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) antibody. Furthermore, double positivity of anti-ß2GPIdI (anti-ß2-glycoprotein I domain I) combined with anti-PS/PT even more elevated both thrombosis and positivity of anti-ADAMTS13 antibody. Double positivity of ß2GPI and anti-PS/PT was found as an independently significant contributing factor to anti-ADAMTS13 antibody level. We suggest the association between anti-ADAMTS13 antibody and the pathophysiology of antiphospholipid syndrome, which should be further evaluated.

Prognostic value of integrated cytogenetic, somatic variation, and copy number variation analyses in Korean patients with newly diagnosed multiple myeloma.

BACKGROUND: To investigate the prognostic value of gene variants and copy number variations (CNVs) in patients with newly diagnosed multiple myeloma (NDMM), an integrative genomic analysis was performed. METHODS: Sixty-seven patients with NDMM exhibiting more than 60% plasma cells in the bone marrow aspirate were enrolled in the study. Whole-exome sequencing was conducted on bone marrow nucleated cells. Mutation and CNV analyses were performed using the CNVkit and Nexus Copy Number software. In addition, karyotype and fluorescent in situ hybridization were utilized for the integrated analysis. RESULTS: Eighty-three driver gene mutations were detected in 63 patients with NDMM. The median number of mutations per patient was 2.0 (95% confidence interval [CI] = 2.0-3.0, range = 0-8). MAML2 and BHLHE41 mutations were associated with decreased survival. CNVs were detected in 56 patients (72.7%; 56/67). The median number of CNVs per patient was 6.0 (95% CI = 5.7-7.0; range = 0-16). Among the CNVs, 1q gain, 6p gain, 6q loss, 8p loss, and 13q loss were associated with decreased survival. Additionally, 1q gain and 6p gain were independent adverse prognostic factors. Increased numbers of CNVs and driver gene mutations were associated with poor clinical outcomes. Cluster analysis revealed that patients with the highest number of driver mutations along with 1q gain, 6p gain, and 13q loss exhibited the poorest prognosis. CONCLUSIONS: In addition to the known prognostic factors, the integrated analysis of genetic variations and CNVs could contribute to prognostic stratification of patients with NDMM.

Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones.

OBJECTIVES: We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. METHODS: We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens. RESULTS: A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation. CONCLUSIONS: Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.

Algorithmic decomposition for efficient multiple nuclear spin detection in diamond.

Efficiently detecting and characterizing individual spins in solid-state hosts is an essential step to expand the fields of quantum sensing and quantum information processing. While selective detection and control of a few 13C nuclear spins in diamond have been demonstrated using the electron spin of nitrogen-vacancy (NV) centers, a reliable, efficient, and automatic characterization method is desired. Here, we develop an automated algorithmic method for decomposing spectral data to identify and characterize multiple nuclear spins in diamond. We demonstrate efficient nuclear spin identification and accurate reproduction of hyperfine interaction components for both virtual and experimental nuclear spectroscopy data. We conduct a systematic analysis of this methodology and discuss the range of hyperfine interaction components of each nuclear spin that the method can efficiently detect. The result demonstrates a systematic approach that automatically detects nuclear spins with the aid of computational methods, facilitating the future scalability of devices.

Minimal residual disease negativity by next-generation flow in non-CR myeloma patients.

Next-generation flow (NGF) has detected minimal residual disease (MRD) in numerous myeloma patients who achieve a complete response (CR). However, when MRD is not detected via NGF in non-CR patients, its clinical meaning is uncertain. Here, we investigated the correlation between MRD findings on NGF and the response criteria, paying special attention to patients with discrepant results. We performed NGS analysis of IgH rearrangements on bone marrow samples from the non-CR patients with negative MRD on NGF. NGS detected residual abnormal clones in those patients, suggesting that NGF and NGS should be used in a complementary manner for MRD investigation.