What is new in acute myeloid leukemia classification?

Recently, the International Consensus Classification (ICC) and the 5th edition of the World Health Organization classification (WHO2022) introduced diagnostically similar yet distinct approaches, which has resulted in practical confusion. This review compares these classification systems for acute myeloid leukemia (AML), building up on the revised 4th edition of WHO (WHO2016). Both classifications retain recurrent genetic abnormalities as a primary consideration. However, they differ in terms of blast threshold. The ICC mandates a minimum of 10% blasts in the bone marrow or peripheral blood, whereas the WHO2022 does not specify a blast cut-off. AML with BCR::ABL1 requires > 20% blast count in both classifications. In WHO2022, AML with CEBPA mutation requires > 20% blasts. TP53 mutation, a new entity is exclusive to ICC, diagnosed with > 20% blasts and variant allele frequency > 10%. AML with myelodysplasia-related changes is defined by cytogenetic or gene mutation-based criteria, not morphological dysplasia. Eight genes were common to both groups: ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2. An additional gene, RUNX1, was included in the ICC classification. AML cases defined by differentiation (WHO2022) and AML not otherwise specified (ICC) are categorized as lacking specific defining genetic abnormalities, WHO2022 labels this as a myeloid neoplasm post cytotoxic therapy (MN-pCT), described as an appendix after specific diagnosis. Similarly, in ICC, it can be described as "therapy-related", without a separate AML category.

The Discordance between G-Banding Karyotyping and Microarray in Structural Abnormality.

BACKGROUND: Cytomolecular genetic laboratory techniques have developed from conventional G-banding karyotyping to whole genome sequencing. Although resolution has greatly increased, various cytogenetic techniques have their advantages and limitations in detecting genomic variations. METHODS: We compared the chromosomal abnormalities detected by G-banding karyotyping and SNP-based microarray testing in 62 patients from July 2020 to December 2022. We analyzed their difference according to chromosomal abnormalities, including numerical and structural and others. RESULTS: Of the 62 patients, 28 patients showed chromosomal aberration detected in one or more of the two test methods. Aneuploidy was detected in both methods, while gain and loss less than 3 Mb were only detectable by the microarray. G-banding karyotyping is fundamental to detect structural chromosome rearrangement such as inversions, ring chromosomes, and translocations, but additional breakpoint or unknown origin materials informa-tion obtained from microarray. Loss of heterozygosity was only detectable in microarray, and mosaicism had limitations in both G-banding karyotyping and microarray. CONCLUSIONS: Various disease cause genomic structural variants, it is very important to detect this. We showed discordance between G-banding karyotyping and SNP based microarray in clinical laboratory. It can be helpful to clinical physicians to decide which diagnostic tool to use.

SARS-CoV-2 molecular diagnostic point-of-care testing based on loop-mediated isothermal amplification: A prospective, single-center validation study.

Objectives: Rapid and accurate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic tests are crucial for controlling the spread of infections in emergency settings. This study evaluated the diagnostic accuracy of a point-of-care (POC) test based on loop-mediated isothermal amplification (LAMP) that produces rapid results within 30 min. Methods: We prospectively included adult patients (age >19 years) who were diagnosed with SARS-CoV-2 infection within the last 3 days and symptomatic patients who had visited the emergency room. Posterior nasopharyngeal (PNP) swabs and throat swabs collected by physicians were used to test the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, and Cohen's Kappa coefficient (k) of the POC index and reference reverse transcription quantitative polymerase chain reaction (RT-qPCR) test devices. Results: Of the 352 participants, 102 (29.0%) tested positive via the RT-PCR-based reference test device; the RT-LAMP-based POC test had a sensitivity of 70.6% and specificity of 98.0%, with 93.5% PPV, 89.1% NPV, 35.5% PLR, and 3.4% NLR. Cohen's k correlation of results from the two devices was 0.74. The cycle threshold value between the positive and negative POC test results differed (17.6 vs. 24.6, p < 0.001). Conclusions: The RT-LAMP POC test in the emergency medical setting has a fair predictive value in high viral load cases in terms of infectivity.

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.

Usefulness of Genetic Aberration and Shorter Telomere Length in Myelodysplastic Syndrome: A Pilot Study.

OBJECTIVE: We aimed to evaluate the clinical usefulness of genetic aberration and shorter telomere length (TL) in individuals with myelodysplastic syndrome (MDS). METHODS: A targeted sequencing panel with 49 genes and TL measurement by quantitative real-time polymerase chain reaction were performed for 46 subjects. RESULTS: According to the revised International Prognostic Scoring System (IPSS-R) subtypes, the mutation frequency was 33.3%, 57.9%, and 100% in the very low/low, intermediate, and very high/high risk groups, respectively. A shorter telomere was detected in 43.5%. We defined group 1 as IPSS-R-high or -very high risk, group 2 as having 1 or more genetic aberrations, group 3 as having a shorter TL, and group 4 as having a longer TL than the age-matched reference. Group 1 and group 2 showed an adverse prognosis. The TL was not strongly correlated with MDS prognosis. However, it may be related to a poor long-term prognosis. CONCLUSION: Genetic variation and shorter TL may be helpful in reclassifying non-high-risk groups.

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.

The new diagnostic criteria for myelodysplasia-related acute myeloid leukemia is useful for predicting clinical outcome: comparison of the 4th and 5th World Health Organization classifications.

Mutations in myelodysplasia-related (MR) genes, rather than morphological features, have been included in the diagnostic criteria of the new 5th World Health Organization (WHO) classification for myelodysplastic syndrome (MDS)-associated acute myeloid leukemia (AML). This study compares the clinical relevance of the new criteria with those of the previous version. In a cohort of 135 patients with newly diagnosed AML, the MDS-related AML patients were classified according to the 5th and 4th edition of the WHO classification (AML, myelodysplasia-related [AML-MR5th] and AML with myelodysplasia-related changes [AML-MRC4th], respectively). The median age of the patients was 70.4 years. MR gene mutations were found in 48 patients (35.6%). Sixty-one patients (46.6%) were diagnosed with AML-MRC4th, while 71 patients (53.0%) were diagnosed with AML-MR5th. Patients with AML-MR5th were significantly older with significantly lower treatment response rate, higher recurrence rate, and shorter relapse-free survival after chemotherapy, whereas AML-MRC4th patients did not show any association with the treatment outcome. Overall, the following prognostic factors for survival were identified: age over 75 years, antecedent MDS or MDS/myeloproliferative neoplasm, chromosome 5 or 7 abnormalities, and KRAS and ZSZR2 mutations. The 5th WHO classification is more useful for predicting the treatment response of patients with AML-MR than the previous version. Among the MR genes, ZSZR2 mutations were found to be independent prognostic factors affecting survival.

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.

Korean clinical practice guidelines for the diagnosis of hereditary hemolytic anemia.

Although the prevalence of hereditary hemolytic anemia (HHA) is relatively low in Korea, it has been gradually increasing in recent decades due to increment in the proportions of hemoglobinopathies from immigrants of South East Asia, raising awareness of the disease among clinicians, and advances in diagnostic technology. As such, the red blood cell (RBC) Disorder Working Party (WP), previously called HHA WP, of the Korean Society of Hematology (KSH) developed the Korean Standard Operating Procedures (SOPs) for the diagnosis of HHA in 2007. These SOPs have been continuously revised and updated following advances in diagnostic technology [e.g., flow cytometric osmotic fragility test (FOFT) and eosin-5-maleimide (EMA) binding test], current methods for membrane protein or enzyme analysis [e.g., liquid chromatography-tandem mass spectrometry (LC-MS/MS), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), high-performance liquid chromatography (HPLC)], and molecular genetic tests using next-generation sequencing (NGS). However, the diagnosis and treatment of HHA remain challenging as they require considerable experience and understanding of the disease. Therefore, in this new Korean Clinical Practice Guidelines for the Diagnosis of HHA, on behalf of the RBC Disorder WP of KSH, updated guidelines to approach patients suspected of HHA are summarized. NGS is proposed to perform prior to membrane protein or enzyme analysis by LC-MS/MS, UPLC-MS/MS or HPLC techniques due to the availability of gene testing in more laboratories in Korea. We hope that this guideline will be helpful for clinicians in making diagnostic decisions for patients with HHA in Korea.

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.

Compound Heterozygous VPS13A Variants in a Patient with Neuroacanthocytosis: A Case Report and Review of the Literature.

Chorea-acanthocytosis (ChAc) is a rare autosomal recessive neurodegenerative disorder caused by pathogenic variants of the vacuolar protein sorting 13A (VPS13A). Only a few patients with ChAc have been reported to date, and the variant spectrum of VPS13A has not been completely elucidated. We describe the case of a 36-year-old woman who had been experiencing orofacial dyskinesia since age 30 years. In a genetic study using next-generation sequencing, 2 variants of VPS13A, the nonsense variant c.4411C>T (p.Arg1471Ter) and the splicing variant c.145-2A>T, were identified. The splicing variant c.145-2A>T was newly classified as a pathogenic variant through a literature review. Consequently, the patient was diagnosed with ChAc based on the typical clinical manifestations, laboratory findings, and imaging results.

A novel likely pathogenic PLCG2 variant in a patient with a recurrent skin blistering disease and B-cell lymphopenia.

Pathogenic variants of PLCG2 encoding phospholipase C gamma 2 (PLCγ2) were first reported in 2012 and their clinical manifestations vary widely. PLCG2-associated antibody deficiency and immune dysregulation (PLAID) and autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) are representative examples of PLCG2 pathogenic variants. In this report, we describe a 17-year-old male with recurrent blistering skin lesions, B-cell lymphopenia, and asthma. Distinct from the patients in previous reports, this patient had the heterozygous de novo c.2119T > C missense variant (NM_002661.4) resulting in a serine to proline amino acid substitution (p.Ser707Pro). The variant located to the PLCγ2 C-terminal Src homology 2 (cSH2) domain, which is a critical site for the restriction of intrinsic enzyme activity. This variant could be classified as "likely pathogenic" according to American College of Medical Genetics and Genomics guidelines. Laboratory results showed a reduction in circulating B cells without a decrease of serum IgG and IgA. Our findings expand the variety of clinical phenotypes for PLCG2 missense variants.

The Prognostic Ability of RAS Pathway-Related Gene Mutations in Patients with Myeloid Neoplasms Treated with Hypomethylating Agents.

INTRODUCTION: This study aimed to identify genetic predictors of treatment response and survival in patients with myeloid neoplasms treated with hypomethylating agents (HMAs). METHODS: We performed next-generation sequencing on bone marrow aspiration samples of 59 patients diagnosed with acute myeloid leukemia (AML), myelodysplastic syndrome with excess blasts-2, or chronic myelomonocytic leukemia and treated with decitabine or azacitidine as a frontline therapy. RESULTS: A single gene with the most common mutations was TP53 (14 of 59 patients), and mutations in RAS pathway-related genes including KRAS, NRAS, FLT3, PTPN11, CBL, and KIT were found in 28.8% of patients. The overall response rate to HMAs was 33.9%. Predictive factors for a poor response were an age >75 years (p = 0.007), 3 or more gene mutations (p = 0.004), mutations in RAS pathway-related genes (p = 0.033), and a mutated NRAS gene (p = 0.042). An age >75 years (hazard ratio 2.946), diagnosis of AML (hazard ratio 2.915), and mutations in NRAS (hazard ratio 4.440) were identified as poor prognostic factors for survival. CONCLUSION: In conclusion, mutations in RAS pathway-related genes were predictors of a poor response to HMAs. Particularly, mutated NRAS was associated with inferior survival rates.

A rapid quantitative on-site coronavirus disease 19 serological test.

On-site severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) serological assays allow for timely in-field decisions to be made regarding patient status, also enabling population-wide screening to assist in controlling the coronavirus disease 2019 (COVID-19) pandemic. Here we propose a rapid microfluidic serological assay with two unique functions of nanointerstice filling and digitized flow control, which enable the fast/robust filling of the sample fluid as well as precise regulation of duration and volume of immune reaction. Developed microfluidic assay showed enhanced limit of detection, and 91.67% sensitivity and 100% specificity (n = 152) for clinical samples of SARS CoV-2 patients. The assay enables daily monitoring of IgM/IgG titers and patterns, which could be crucial parameters for convalescence from COVID-19 and provide important insight into how the immune system responds to SARS CoV-2. The developed on-site microfluidic assay presented the mean time for IgM and IgG seroconversions, indicating that these titers plateaued days after seroconversion. The mean duration from day 0 to PCR negativity was 19.4 days (median 20 d, IQR 16-21 d), with higher IgM/IgG titres being observed when PCR positive turns into negative. Simple monitoring of these titres promotes rapid on-site detection and comprehensive understanding of the immune response of COVID-19 patients.

Identification of candidate gene variants of monogenic diabetes using targeted panel sequencing in early onset diabetes patients.

INTRODUCTION: Monogenic diabetes is attributed to genetic variations in a single gene. Maturity-onset diabetes of the young (MODY) is the most common phenotype associated with monogenic diabetes, but is frequently misdiagnosed as either type 1 or type 2 diabetes. Increasing our basic understanding of genetic variations in MODY may help to improve the accuracy of providing the correct diagnosis and personalize subsequent treatment regimens in different racial populations. For this reason, this study was designed to identify nucleotide variants in early onset diabetes patients with clinically suspected MODY in a Korean population. RESEARCH DESIGN AND METHODS: Among 2908 Korean patients diagnosed with diabetes, we selected 40 patients who were diagnosed before 30 years old and were clinically suspected of MODY. Genetic testing was performed using a targeted gene sequencing panel that included 30 known monogenic diabetes genes. The pathogenicity of the identified variants was assessed according to the American College of Medical Genetics and Genomics and Association for Molecular Pathology (ACMG-AMP) guidelines. RESULTS: A total of six rare missense variants (p.Ala544Thr in HNF1A, p.Val601Ile and p.His103Tyr in ABCC8, p.Pro33Ala in PDX1, p.Gly18Glu in INS, and p.Arg164Gln in PAX4) in five distinct MODY genes were identified in five patients. In addition, a variant was identified in mitochondrial DNA at 3243A>G in one patient. The identified variants were either absent or detected at a rare frequency in the 1000 Genomes Project. These variants were classified as uncertain significance using the ACMG-AMP guidelines. CONCLUSION: Using a targeted gene sequencing panel, we identified seven variants in either MODY genes or mitochondrial DNA using a Korean patient population with early onset diabetes who were clinically suspected of MODY. This genetic approach provides the ability to compare distinct populations of racial and ethnic groups to determine whether specific gene is involved in their diagnosis of MODY.

Differences in seroprevalence between epicenter and non-epicenter areas of the COVID-19 outbreak in South Korea.

To compare the standardized severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroprevalence of high epicenter region with non-epicenter region, serological studies were performed with a total of 3,268 sera from Daegu City and 3,981 sera from Chungbuk Province. Indirect immunofluorescence assay (IFA) for SARS-CoV-2 IgG results showed a high seroprevalence rate in the Daegu City (epicenter) compared with a non-epicenter area (Chungbuk Province) (1.27% vs. 0.91%, P = 0.0358). It is noteworthy that the highest seroprevalence in Daegu City was found in elderly patients (70's) whereas young adult patients (20's) in Chungbuk Province showed the highest seroprevalence. Neutralizing antibody (NAb) titers were found in three samples from Daegu City (3/3, 268, 0.09%) while none of the samples from Chungbuk Province were NAb positive. These results demonstrated that even following the large outbreak, the seropositive rate of SARS-CoV-2 in the general population remained low in South Korea.