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.

Telomere integrated scoring system of myelodysplastic syndrome.

INTRODUCTION: Recently, multigene target sequencing is widely performed for the purpose of prognostic prediction and application of targeted therapy. Here, we proposed a new scoring system that encompasses gene variations, telomere length, and Revised International Prognostic Scoring System (IPSS-R) together in Asian myelodysplastic syndrome. METHODS: We developed a new scoring model of these variables: age ≥ 65 years + IPSS-R score + ASXL1 mutation + TP53 mutation + Telomere length (<5.37). According to this new scoring system, patients were divided into four groups: very good score cutoff (≤3.0), good (3.0-4.5), poor (4.5-7.0), and very poor (>7.0). RESULTS: The median OS was 170.1, 100.4, 46.0, and 12.0 months for very good, good, poor, and very poor, retrospectively (p < 0.001). Meanwhile, according to the conventional IPSS-R scoring system, the median OS was 141.3, 50.2, 93.0, 36.0, and 16.2 months for very low, low, intermediate, high, and very high, retrospectively (p < 0.001). CONCLUSIONS: The newly developed model incorporating molecular variations and TL yielded more clear separations of the survival curves. By adding the presence of gene mutation and telomere length to the existing IPSS-R, its predictive ability can be further improved in myelodysplastic syndrome.

Application of Interphase Fluorescent in Situ Hybridization: a Screening Tool for the Diagnosis of Microdeletion Syndrome.

BACKGROUND: Most laboratories adopt the results of metaphase fluorescent in situ hybridization (FISH) for the diagnosis of microdeletion syndromes. To investigate the discrepancy between the results of interphase and metaphase, we compared the quantitative results of FISH for 5 kinds of microdeletion syndrome and gender determination disorders (SDD). METHODS: A total of 282 (135 for DiGeorge syndrome, 20 for Kalmann syndrome, 7 for Miller-Dieker syndrome, 38 for Prader Willi/Angelman syndrome, 62 for Williams syndrome, and 20 for SDD (SRY FISH)) were enrolled. For SRY FISH, we artificially mixed fresh blood of male and female with various ratios and then compared the results of metaphase and interphase SRY FISH. Using a bio-cell chip, we performed interphase FISH in 168 patients with microdeletion syndromes and compared the results with manual interphase. RESULTS: The concordance rate between the results of metaphase and interphase was 100% in microdeletion syndrome. In the disorders of gender development, SRY FISH showed 100% concordance between interphase and metaphase when we counted 50 metaphase cells and 100 interphase cells. Comparison with mixtures of male and female blood at various ratios also showed 100% concordance. The results of bio-cell chip showed 100% concordance between previous interphase FISH results. CONCLUSIONS: Considering the complete concordance between interphase and metaphase in microdeletion syndrome, the application of interphase FISH without performing metaphase FISH can be a screening test for microdeletion syndrome. Confirmation by metaphase FISH can be performed only in cases with abnormal results by interphase FISH.

Successful Treatment of Pediatric Epstein-Barr Virus-positive Aggressive Natural Killer-Cell Leukemia.

Epstein-Barr virus (EBV)-positive aggressive natural killer-cell leukemia (ANKL) is a rare malignancy of mature natural killer cells, with a very poor survival rate. Patients have a rapidly declining clinical course and a poor prognosis, with a median survival of only a few months. Herein, we describe a 16-year-old boy who was diagnosed with EBV-positive ANKL and successfully treated using combination chemotherapy and a subsequent allogeneic hematopoietic stem cell transplantation (alloHSCT). The patient is disease free 4 years and 9 months after alloHSCT. Thus, combination chemotherapy followed by alloHSCT seems to be a promising therapeutic option for EBV-positive ANKL.

The risk of second primary malignancy is increased in differentiated thyroid cancer patients with a cumulative (131)I dose over 37 GBq.

BACKGROUND: The aim of this study was to investigate the risk factors for second primary malignancy (SPM) diagnosed after differentiated thyroid cancer (DTC). METHODS: A total of 2468 DTC patients who underwent thyroidectomy were reviewed. SPM was defined as a non-thyroidal malignancy, diagnosed at least 1 year after the diagnosis of thyroid cancer. Patients were divided into five groups according to cumulative (131)I dose: very high-activity (≥ 37.0 GBq), high-activity (22.3-36.9 GBq), intermediate-activity (5.56-22.2 GBq), low-activity (1.1-5.55 GBq) and no RAI. RESULTS: Among the 2468 patients, 61 (2.5%) had SPMs during 7.0 (1.0-33.0) years of median follow-up. Age above 40 years, male sex and very high-activity RAI were independent risk factors for the development of SPM. SPM-related mortality was highest in the very high-activity group, while DTC-related mortality was highest in the high-activity group. The overall mortality both from SPM and DTC was highest in the high-activity group. CONCLUSION: A cumulative (131)I dose <37.0 GBq did not increase the risk of SPM. A cumulative (131) I dose ≥ 37.0 GBq increased the risk of SPM and SPM-related mortality and decreased the DTC-specific mortality, resulting in a similar all-cause mortality compared with the low-activity RAI group. Using repeated high-dose RAI for treating RAI-responsive but persistent DTC patients needs careful consideration of the individual benefits from RAI vs the risk of developing SPM.

Fluorescence in situ hybridization panel for monitoring of minimal residual disease in patients with double minute chromosomes.

A double minute chromosome (dmin) is a small fragment of extrachromosomal DNA bearing amplified genes observed in malignancies. We investigated the incidence and characteristics of dmins in hematologic malignancies, and the quantitative changes during the treatment follow-up. Once a dmin was observed in conventional G-banding, it was characterized using fluorescence in situ hybridization (FISH) with the panel of MYC, NMYC, and MLL probes. Quantitative changes of malignant cells were measured using G-banding and FISH during the follow up. Dmins were observed in 1.23% of patients (6/489) at the initial diagnosis including 4 with MYC amplification, 1 with MLL and 1 with NMYC. All 6 had complex karyotypes and showed short overall survival (7.7 months). In follow-up specimens, FISH detected dmins in 11 cases out of which G-banding detected dmins in 9 cases. The number of dmins detected by FISH and G-banding did not correlate well. Amplification of NMYC in dmins is reported for the first time. A FISH panel composed of frequently amplified oncogenes (MYC, NMYC, and MLL) in dmins is useful for characterization of dmins. FISH is a sensitive method in detecting dmins and will be useful in monitoring of the minimal residual disease.

Chromosome 13 deletion and hypodiploidy on conventional cytogenetics are robust prognostic factors in Korean multiple myeloma patients: web-based multicenter registry study.

This study was designed to evaluate the prevalence of chromosomal abnormalities and to identify the specific abnormalities associated with poor prognosis. A total of 2,474 patients whose conventional cytogenetics were available at the time of diagnosis were evaluated via a nationwide registry. Normal metaphase cytogenetics was observed in 2,012 patients (81.3%). Among the 462 patients with chromosomal abnormalities, there were 161 (34.8%) patients with hyperdiploidy, 197 (42.6%) with pseudodiploidy, 79 (17.1%) with hypodiploidy, and 25 (5.5%) with near-tetraploidy. Deletion 13 (Δ13) in metaphase was observed in 167 patients (6.8%). Fluorescent in situ hybridization (FISH) was carried out in 967 patients (39.1%), and 66 (13.7%) out of 482 and 63 (10.3%) out of 611 patients were positive for t(4;14) and del(17p), respectively. With a median follow-up duration of 25.1 months, the median overall survival (OS) was 51.2 months (95% confidence interval, 46.5-55.9 months). In univariate analysis, the following four chromosomal abnormalities were significantly associated with a poor survival outcome: Δ13, hypodiploidy, del(13q) in FISH, and del(17p) in FISH. In the subsequent multivariate analysis, in which del(13q) and del(17p) in FISH were excluded due to a relatively low number of patients, Δ13 and hypodiploid status were independently associated with a poor survival outcome after adjusting for important clinical factors, including age, sex, performance, beta2-microglobulin, albumin, and lactate dehydrogenase (LDH). Using conventional metaphase cytogenetics, we confirmed that both Δ13 and hypodiploid status were robust poor prognostic factors. The metaphase karyotyping should remain the primary cytogenetic tool and an essential investigation for risk stratification in newly diagnosed multiple myeloma patients.

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.

Quantitative comparison of CDKN2B methylation in pediatric and adult myelodysplastic syndromes.

BACKGROUND/AIMS: Transcriptional repression of tumor suppressor genes is determined by the quantity of promoter hypermethylation. We analyzed the methylation quantity of CDKN2B in pediatric myelodysplastic syndromes (MDS). METHODS: Quantitative measurement of CDKN2B methylation was performed in 25 pediatric MDS patients and 12 controls using pyrosequencing, and the result was compared with those from 74 adult MDS cases and 31 adult controls. The association between CDKN2B methylation quantity and factors related to prognosis including bone marrow blast percentage and karyotype was analyzed. RESULTS: Pediatric MDS patients showed a higher methylation level (MtL) of CDKN2B than pediatric controls (2.94 vs. 1.62; p = 0.031) but a lower level than adult MDS patients (8.76; p < 0.001). MtL was higher in pediatric MDS cases with >5% blasts than in pediatric controls (3.78 vs. 1.62; p = 0.052). Pediatric MDS cases with abnormal karyotype showed a higher MtL than pediatric controls (5.95 vs. 1.62; p = 0.045). CONCLUSIONS: We confirmed that methylation of CDKN2B is associated with the pathogenesis and prognosis in pediatric MDS. The difference in MtLs between pediatric and adult MDS might be related to the physiological hypermethylation of tumor suppressor genes in aging.

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.

Hema-seq reveals genomic aberrations in a rare simultaneous occurrence of hematological malignancies.

Co-occurrence of multiple myeloma and acute myelogenous leukemia is rare, with both malignancies often tracing back to multipotent hematopoietic stem cells. Cytogenetic techniques are the established baseline for diagnosis and characterization of complex hematological malignancies. In this study, we develop a workflow called Hema-seq to delineate clonal changes across various hematopoietic lineages through the integration of whole-genome sequencing, copy-number variations, cell morphology, and cytogenetic aberrations. In Hema-seq, cells are selected from Wright-stained slides and fluorescent probe-stained slides for sequencing. This technique therefore enables direct linking of whole-genome sequences to cytogenetic profiles. Through this method, we mapped sequential clonal alterations within the hematopoietic lineage, identifying critical shifts leading to myeloma and acute myeloid leukemia (AML) cell formations. By synthesizing data from each cell lineage, we provided insights into the hematopoietic tree's clonal evolution. Overall, this study highlights Hema-seq's capability in deciphering genomic heterogeneity in complex hematological malignancies, which can enable better diagnosis and treatment strategies.

Polymorphisms in genes that regulate cyclosporine metabolism affect cyclosporine blood levels and clinical outcomes in patients who receive allogeneic hematopoietic stem cell transplantation.

In patients who received allogeneic hematopoietic stem cell transplantation (HSCT), we investigated the correlations between single nucleotide polymorphisms (SNPs) in genes that regulate cyclosporine metabolism and clinical outcomes. All patients received sibling-matched HSCT. DNA samples of patients and donors were analyzed for 4 SNPs: MDR1 +1236C>T (rs1128503), +2677G>T>A (rs2032582), +3435C>T (rs1045642), and CYP3A5 +6986G>A (rs776746). A total of 156 patients (median age 40 years) were analyzed. Nineteen patients received HSCT for nonmalignant disease. The CYP3A5 +6986AA genotype was associated with a high cyclosporine blood level after transplantation. However, this genotype was not related to any particular clinical outcome. In contrast, the MDR1 +1236C>T SNP was correlated with specific clinical outcomes. When neither the donor nor the recipient had the CC genotype of MDR1 +1236, patients had lower creatinine levels (P < .001) and less transplantation-related mortality (TRM) (P = .012). These patients also showed longer overall survival (OS) in both univariate (P = .003) and multivariate (P = .003) analyses. Although the CYP3A5 +6986AA genotype was correlated with a high blood cyclosporine concentration, lack of the MDR1 +1236CC genotype in both the donor and recipient was correlated with less TRM and a longer OS in patients who received allogeneic HSCT.

Sequential vincristine, adriamycin, dexamethasone (VAD) followed by bortezomib, thalidomide, dexamethasone (VTD) as induction, followed by high-dose therapy with autologous stem cell transplant and consolidation therapy with bortezomib for newly diagnosed multiple myeloma: results of a phase II trial.

Incorporation of novel agents has resulted in an improved response rate and reduced side effects in multiple myeloma. This has prompted combining novel agents in induction chemotherapy in patients with newly diagnosed multiple myeloma. Our patients received 2 cycles of vincristine, adriamycin, dexamethasone (VAD) and then 2 cycles of bortezomib, thalidomide, dexamethasone (VTD) chemotherapy as an induction treatment. Subsequently, autologous stem cell transplantation was performed, and bortezomib was administered as a consolidation therapy. Seventy-one patients were enrolled, and 65 were evaluable for response. After 2 cycles of VAD, the overall response rate was 69%. After VTD, the response rate improved to 97% with a complete response (CR) and near CR rate of 27%. Importantly, patients with cytogenetics, having poor prognostic features, all responded after VTD. Autologous stem cells were successfully collected in all 58 patients with a median CD34+ cell count of 7.12 × 10(6)/kg (range, 1.94-44.7 × 10(6)/kg), except in 1 patient (2%). After ASCT, 36 patients completed bortezomib maintenance with a combined CR and near CR rate approaching 75%. Median time to response was rapid (1.6 months). With a median follow-up duration of 52.7 months, the median TTP was 29.4 months and median OS was not reached. Toxicities proved manageable. In conclusion, sequential VAD and VTD induction therapy in patients with newly diagnosed multiple myeloma was active with manageable toxicity and excellent stem cell yields. The incorporation of bortezomib as a consolidation therapy improved the clinical outcome with the expense of rather frequent development of peripheral neuropathy.

Impact of vitamin D receptor gene polymorphisms on clinical outcomes of HLA-matched sibling hematopoietic stem cell transplantation.

We hypothesized that polymorphisms of the vitamin D receptor (VDR) gene might affect clinical outcomes of allogeneic hematopoietic stem cell transplantation (HSCT). Three VDR gene polymorphisms (BsmI G>A, ApaI G>T, and TaqI T>C) were genotyped in 147 patients who underwent HLA-matched sibling allogeneic HSCT. Frequencies of infection, graft-vs.-host disease (GVHD), overall survival (OS), and disease-free survival (DFS) were compared according to genotypes and haplotypes. Infection and acute GVHD had trends to be less frequent in patients with ApaI TT genotype than non-TT genotypes (p = 0.061 and p = 0.059, respectively). For TaqI genotypes, there were no statistical differences in frequency of infection and acute GVHD (p = 0.84 and p = 0.30, respectively), but TC genotype was associated with longer OS and DFS than TT genotype (p = 0.022 and p = 0.038, respectively). In the ApaI-TaqI haplotype analysis, patients with TC haplotype had significantly longer OS and DFS than those without TC haplotype (p = 0.022 and p = 0.038, respectively). In multivariable analysis, TaqI genotype and ApaI-TaqI haplotype of recipients were independent prognostic factors for both OS and DFS. This study suggests that the genotype and haplotype of VDR in recipient might be associated with clinical outcome of sibling HLA-matched HSCT.

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.

Increased Apoptotic Activity in Low-Risk Myelodysplastic Syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous hematopoietic disorder associated with cellular proliferative and apoptotic activity. We retrospectively investigated these activities in bone marrow samples from 76 MDS patients using immunohistochemical staining for Ki-67 and cleaved caspase-3. We divided cleaved caspase-3 into two groups based on median value and compared the differences according to MDS risk scoring systems. We compared MDS patient indices with idiopathic cytopenia of undetermined significance (ICUS) and healthy control (HC) indices using our previously published data. Cleaved caspase-3 immunohistochemistry was highest in MDS patients, followed by ICUS patients and HCs. Similarly, the mean Ki-67 grade was also highest in MDS patients, followed by ICUS patients and HCs. Higher cleaved caspase-3 grade was significantly associated with lower IPSS-R score (p = 0.020), whereas Ki-67 was not associated with MDS. Interestingly, TET2 mutation was associated with decreased cleaved caspase-3 levels (p = 0.03). However, there was no significant association between proliferative/apoptotic activity and survival. Our results suggest that apoptotic activity gradually increases from healthy controls and ICUS patients to MDS patients. Furthermore, higher apoptotic activity was associated with better MDS patient prognostic scores. Further studies are needed to reveal the differences in apoptotic activity between lower- and higher-risk MDS.

The importance of critically short telomere in myelodysplastic syndrome.

A few critically short telomeres trigger genomic instability regardless of average telomere length (TL). Recently, the telomere shortest length assay (TeSLA) was developed to detect critically short telomeres and measure absolute telomeres. Using TeSLA with the internally labeled biotin probe, we measured the TL of bone marrow (BM) aspirates from 52 patients with myelodysplastic syndrome (MDS). A percentage of shortest telomeres (< 1.0 kb (ShTL1.0)) were calculated. ShTL1.0 was correlated to IPSS-R risk (spearman's rho = 0.35 and p = 0.0196), and ShTL1.0 and BM blast (2.61% in < 5% blast, 4.15% in 5-10% blast, and 6.80% in 10-20% blast, respectively, p = 0.0332). Interestingly, MDS patients with a shortest TL ≥ 0.787 kb at the time of diagnosis showed better overall survival (OS) and progression-free survival (PFS) than patients with a shortest TL < 0.787 kb in the multivariate analyses (HR = 0.13 and 0.30, p = 0.011 and 0.048 for OS and PFS, respectively). Our results clearly show the presence and abundance of critically short telomeres in MDS patients. These pathologic telomeres are associated with IPSS-R which is a validated prognostic scoring system in MDS. Furthermore, they are independent prognostic factors for OS in MDS patients. Future prospective studies are needed to validate our results.

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.

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.