Analytical and Clinical Validation of a Target-Enhanced Whole Genome Sequencing-Based Comprehensive Genomic Profiling Test.

Evaluation of the test performance of the Target enhanced whole-genome sequencing (TE-WGS) assay for comprehensive oncology genomic profiling. The analytical validation of the assay included sensitivity and specificity for single nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs), revealing a revealed a sensitivity of 99.8% for SNVs and 99.2% for indels. The positive predictive value (PPV) was 99.3% SNVs and 98.7% indels. Clinical validation was benchmarked against established orthogonal methods and demonstrated high concordance with reference methods. TE-WGS provides insights beyond targeted panels by comprehensive analysis of key biomarkers and the entire genome encompassing both germline and somatic findings.

Targeting FLT3-TAZ signaling to suppress drug resistance in blast phase chronic myeloid leukemia.

BACKGROUND: Although the development of BCR::ABL1 tyrosine kinase inhibitors (TKIs) rendered chronic myeloid leukemia (CML) a manageable condition, acquisition of drug resistance during blast phase (BP) progression remains a critical challenge. Here, we reposition FLT3, one of the most frequently mutated drivers of acute myeloid leukemia (AML), as a prognostic marker and therapeutic target of BP-CML. METHODS: We generated FLT3 expressing BCR::ABL1 TKI-resistant CML cells and enrolled phase-specific CML patient cohort to obtain unpaired and paired serial specimens and verify the role of FLT3 signaling in BP-CML patients. We performed multi-omics approaches in animal and patient studies to demonstrate the clinical feasibility of FLT3 as a viable target of BP-CML by establishing the (1) molecular mechanisms of FLT3-driven drug resistance, (2) diagnostic methods of FLT3 protein expression and localization, (3) association between FLT3 signaling and CML prognosis, and (4) therapeutic strategies to tackle FLT3+ CML patients. RESULTS: We reposition the significance of FLT3 in the acquisition of drug resistance in BP-CML, thereby, newly classify a FLT3+ BP-CML subgroup. Mechanistically, FLT3 expression in CML cells activated the FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway, which conferred resistance to a wide range of BCR::ABL1 TKIs that was independent of recurrent BCR::ABL1 mutations. Notably, FLT3+ BP-CML patients had significantly less favorable prognosis than FLT3- patients. Remarkably, we demonstrate that repurposing FLT3 inhibitors combined with BCR::ABL1 targeted therapies or the single treatment with ponatinib alone can overcome drug resistance and promote BP-CML cell death in patient-derived FLT3+ BCR::ABL1 cells and mouse xenograft models. CONCLUSION: Here, we reposition FLT3 as a critical determinant of CML progression via FLT3-JAK-STAT3-TAZ-TEAD-CD36 signaling pathway that promotes TKI resistance and predicts worse prognosis in BP-CML patients. Our findings open novel therapeutic opportunities that exploit the undescribed link between distinct types of malignancies.

Effectiveness of pegfilgrastim prophylaxis in preventing febrile neutropenia during R-FC chemoimmunotherapy for chronic lymphocytic leukemia: A multicenter prospective phase II study.

Background: A chemotherapy of rituximab, fludarabine and cyclophosphamide (R-FC) has been accepted as a promising frontline chemotherapy in selected patients with chronic lymphocytic leukemia (CLL). Although R-FC regimen is a relatively dose-dense regimen and neutropenia incidence is more than 50%, primary prophylactic pegfilgrastim was not fully recommended in the clinical field. Therefore, the study evaluated the prophylactic effectiveness of pegfilgrastim to reduce the incidence of febrile neutropenia associated with R-FC of patients with CLL. Patients and methods: A single-arm, multicenter, prospective phase II study was designed to assess the efficacy of prophylactic pegfilgrastim. Thirty-four CLL patients were enrolled and analyzed for neutropenia and other related factors, and comparative analysis was performed with historical cohort. Results: Compared with our historical cohort, incidence of grade 3-4 neutropenia and febrile neutropenia was remarkably reduced during any cycle of chemotherapy (14.7% vs. 48.2% of study cohort vs. historical cohort during C1, 5.9% vs. 65.8% during C2, 12.9% vs. 80.6% during C3, 10% vs. 84.6% during C4, 3.4% vs. 83.6% during C5, and 10.7% vs. 85.7% during C6, p <0.001). Also, cumulative incidence of disrupted chemotherapy was noticeably reduced in study cohort on any cycles of R-FC regimen (8.8% vs. 22.2% of study cohort vs. historical cohort on C2, 9.7% vs. 25.2% on C3, 13.4% vs. 26.9% on C4, 13.8% vs. 45.2% on C5, 17.9% vs. 47.3% on C6, p=0.007). In addition, treatment-related mortality was 5.9%, which significantly reduced compared to 9.6% of our historical cohort (HR 0.64, 95% CI 0.42-0.79, P = 0.032). Conclusion: Primary prophylactic pegfilgrastim is effective in the prevention of neutropenia/febrile neutropenia, and infection-related mortality during R-FC regimen in patients with CLL.

Exposure to Crude Oil-Related Volatile Organic Compounds Associated with Lung Function Decline in a Longitudinal Panel of Children.

BACKGROUND: Children in the affected area were exposed to large amounts of volatile organic compounds (VOCs) from the Hebei Spirit oil spill accident. OBJECTIVES: We investigated the lung function loss from the exposure to VOCs in a longitudinal panel of 224 children 1, 3, and 5 years after the VOC exposure event. METHODS: Atmospheric estimated concentration of total VOCs (TVOCs), benzene, toluene, ethylbenzene, and xylene for 4 days immediately after the accident were calculated for each village (n = 83) using a modeling technique. Forced expiratory volume in 1 s (FEV1) as an indicator of airway status was measured 1, 3, and 5 years after the exposure in 224 children 4~9 years of age at the exposure to the oil spill. Multiple linear regression and linear mixed models were used to evaluate the associations, with adjustment for smoking and second-hand smoke at home. RESULTS: Among the TVOCs (geometric mean: 1319.5 mg/m3·4 d), xylene (9.4), toluene (8.5), ethylbenzene (5.2), and benzene (2.0) were dominant in the order of air concentration level. In 224 children, percent predicted FEV1 (ppFEV1), adjusted for smoking and second-hand smoke at home, was 100.7% after 1 year, 96.2% after 3 years, and 94.6% after 5 years, and the loss over the period was significant (p < 0.0001). After 1 and 3 years, TVOCs, xylene, toluene, and ethylbenzene were significantly associated with ppFEV1. After 5 years, the associations were not significant. Throughout the 5 years' repeated measurements in the panel, TVOCs, xylene, toluene, and ethylbenzene were significantly associated with ppFEV1. CONCLUSIONS: Exposure to VOCs from the oil spill resulted in lung function loss among children, which remained significant up to 5 years after the exposure.

A novel bi-alleleic DDX41 mutations in B-cell lymphoblastic leukemia: case report.

BACKGROUND: The germline mutations of DDX41, also known as DEAD box RNA helicase 41, have been found in about 1.5% of myeloid neoplasms (MNs). Development of MDS/AML is relatively common in germline DDX41 mutations. However, a variety of hematological malignancies (HMs) have been reported. CASE PRESENTATION: We report a novel case of bi-alleleic DDX41 mutations in B-cell lymphoblastic leukemia (B-ALL), with unusual location of DDX41 mutations. The gene expression profile (GEP) of Ph + B-ALL with bi-alleleic DDX41 mutations showed heterogeneously transitional GEP and altered gene expression levels of genes involved in the process essential for red blood cells and myeloid cell differentiation were noted. CONCLUSIONS: We report that DDX41 mutations are unusual but can be an underlying event in Ph + B-ALL and screening DDX41 mutations can be also informative for patients awaiting for haploidentical stem cell transplantation and choosing the therapy.

Metabolite Profiling and Characterization of LW6, a Novel HIF-1α Inhibitor, as an Antitumor Drug Candidate in Mice.

A novel HIF (hypoxia-inducible factor)-1α inhibitor, the (aryloxyacetylamino)benzoic acid derivative LW6, is an anticancer agent that inhibits the accumulation of HIF-1α. The aim of this study was to characterize and determine the structures of the metabolites of LW6 in ICR mice. Metabolite identification was performed using a predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (pMRM-IDA-EPI) method in negative ion mode on a hybrid triple quadrupole-linear ion trap mass spectrometer (QTRAP). A total of 12 metabolites were characterized based on their MS/MS spectra, and the retention times were compared with those of the parent compound. The metabolites were divided into five structural classes based on biotransformation reactions: amide hydrolysis, ester hydrolysis, mono-oxidation, glucuronidation, and a combination of these reactions. From this study, 2-(4-((3r,5r,7r)-adamantan-1-yl)phenoxy)acetic acid (APA, M7), the metabolite produced via amide hydrolysis, was found to be a major circulating metabolite of LW6 in mice. The results of this study can be used to improve the pharmacokinetic profile by lowering the clearance and increasing the exposure relative to LW6.

Pharmacokinetic Characterization of LW6, a Novel Hypoxia-Inducible Factor-1α (HIF-1α) Inhibitor in Mice.

LW6, an (aryloxyacetylamino)benzoic acid derivative, was recently identified to be an inhibitor of hypoxia-inducible factor-1α (HIF-1α), which is an attractive target for cancer therapeutics. Although LW6 is known to act by inhibiting the accumulation of HIF-1α, pharmacokinetics needs to be evaluated to assess its potential as an anti-tumor agent. Here, we investigated the plasma pharmacokinetics and metabolism of LW6 in mice. LW6 exhibited a small volume of distribution (0.5 ± 0.1 L/kg), and a short terminal half-life (0.6 ± 0.1 h). Following intravenous or oral administration, LW6 was rapidly converted to its active metabolite, (4-adamantan-1-yl-phenoxy)acetic acid (APA). Although LW6 was rapidly absorbed, its oral bioavailability, estimated using AUClast values, was low (1.7 ± 1.8%). It was slowly degraded in mouse liver microsomes (t1/2 > 1 h) and serum (t1/2 > 6 h). About 54% or 44.8% of LW6 was available systemically as APA in the mouse after a single intravenous or oral administration, respectively. Thus, our results indicated the need to simultaneously consider the active metabolite as well as the parent compound for successful evaluation during lead optimization.

Heterogeneity of MYO15A variants significantly determine the feasibility of acoustic stimulation with hearing aid and cochlear implant.

Autosomal recessive nonsyndromic hearing loss 3 (DFNB3) mainly leads to congenital and severe-to-profound hearing impairment, which is caused by variants in MYO15A. However, audiological heterogeneity in patients with DFNB3 hinders precision medicine in hearing rehabilitation. Here, we aimed to elucidate the heterogeneity of the auditory phenotypes of MYO15A variants according to the affected domain and the feasibilities for acoustic stimulation. We conducted whole-exome sequencing for 10 unrelated individuals from seven multiplex families with DFNB3; 11 MYO15A variants, including the novel frameshift c.900delT (p.Pro301Argfs*143) and nonsense c.4879G > T (p.Glu1627*) variants, were identified. In seven probands, residual hearing at low frequencies was significantly higher in the groups with one or two N-terminal frameshift variants in trans conformation compared to that in the group without these variants. This is consistent with the 56 individuals from the previously published reports that carried a varying number of N-terminal truncating variants in MYO15A. In addition, patients with missense variants in the second FERM domain had better hearing at low frequencies than patients without these variants. Subsequently, acoustic stimulation provided by devices such as hearing aids or cochlear implants was feasible in patients with one or two N-terminal truncating variants or a second FERM missense variant. In conclusion, N-terminal or second FERM variants in MYO15A allow the practical use of acoustic stimulation through hearing aids or electroacoustic stimulation for aural rehabilitation.

Mutation of ten-eleven translocation-2 is associated with increased risk of autoimmune disease in patients with myelodysplastic syndrome.

BACKGROUND/AIMS: Myelodysplastic syndrome (MDS) is caused by genetic and epigenetic alteration of hematopoietic precursors and immune dysregulation. Approximately 20% of patients with MDS develop an autoimmune disease (AID). Here, we investigated whether particular genetic mutations are associated with AID in patients with MDS. METHODS: Eighty-eight genetic mutations associated with myeloid malignancy were sequenced in 73 MDS patients. The association between these mutations and AID was then analyzed. RESULTS: The median age of the 73 MDS patients was 70 years (interquartile range, 56 to 75), and 49 (67.1%) were male. AID was observed in 16 of 73 patients (21.9%). Mutations were detected in 57 (78.1%) patients. The percentage (68.8% vs. 80.7%, p = 0.32) and the mean number of mutations (1.8 ± 1.6 vs. 2.2 ± 1.8, p = 0.34) in MDS patients with or without AID were similar. However, the ten-eleven translocation- 2 (TET2) mutation rate was significantly higher in patients with AID than in those without (31.3% vs. 5.3%, respectively; p = 0.001). All TET2 mutations were variants of strong clinical significance. CONCLUSION: Mutation of TET2 in patients with MDS may be associated with increased risk of developing AID.

Telomere length and its correlation with gene mutations in chronic lymphocytic leukemia in a Korean population.

Telomere length (TL) is a prognostic indicator in Caucasian chronic lymphocytic leukemia (CLL), but its significance in Asian CLL remains unknown. To investigate the prognostic significance of TL and its correlation with cytogenetic aberrations and somatic mutations, we analyzed TL measurements at the cellular level by interphase fluorescence in situ hybridization in patients with CLL in Korea. The present study enrolled 110 patients (41 females and 69 males) diagnosed with CLL according to the World Health Organization criteria (2001-2017). TLs of bone marrow nucleated cells at the single-cell level were measured by quantitative fluorescence in situ hybridization (Q-FISH) in 71 patients. The correlations of TL with clinical characteristics, cytogenetic aberrations, genetic mutations, and overall survival were assessed. The median value of mean TL in CLL patients (T/C ratio 7.46 (range 1.19-18.14) was significantly shorter than that in the normal controls (T/C ratio 15.28 (range 8.59-24.93) (p < 0.001). Shorter TLs were associated with complex karyotypes (p = 0.030), del(11q22) (p = 0.023), presence of deletion and/or mutation in ATM and/or TP53 (p = 0.019), and SH2B3 mutation (p = 0.015). A shorter TL was correlated with lower hemoglobin levels and adverse survival (mean TL < 9.35, p = 0.021). When the proportion of cells with extremely short TLs (< 7.61) was greater than 90%, CLL patients showed poor survival (p = 0.002). Complex karyotypes, TP53 mutation, and the number of mutated genes were determined to be significant adverse variables by multivariable Cox analysis (p = 0.011, p = 0.002, and p = 0.002, respectively). TL was attrited in CLL, and attrited telomeres were correlated with adverse survival and other well-known adverse prognostic factors. We infer that TL is an independent adverse prognostic predictor in Korean CLL.

Characteristics of Waldenström Macroglobulinemia in Korean Patients According to Mutational Status of MYD88 and CXCR4: Analysis Using Ultra-Deep Sequencing.

BACKGROUND: Little is known about the mutational frequency of myeloid differentiation factor 88 (MYD88) and C-X-C chemokine receptor type 4 (CXCR4) and the corresponding characteristics in Asian individuals afflicted with Waldenström macroglobulinemia (WM). We investigated the characteristics of WM according to mutational status of MYD88/CXCR4, and attempted to determine the lineage commitment among hematopoietic cells by MYD88L265P single-cell sequencing on bone marrow (BM) smear slides. MATERIALS AND METHODS: CXCR4 mutations (muts) were detected using ultra-deep sequencing using target capture. Mutational burden of MYD88 was assessed using real-time polymerase chain reaction. Single-cell sequencing for MYD88 was performed on lymphocytes, plasmacytoid lymphocytes, plasma cells, and neutrophils using laser microdissection. RESULTS: Among 31 patients, the frequencies of MYD88/CXCR4 muts were as follows: MYD88 wild type (WT) CXCR4WT (6 patients, 19.4%), MYD88L265PCXCR4WT (19 patients, 61.4%), MYD88L265PCXCR4mut (6 patients, 19.4%; 1 frameshift and 5 nonsense muts). Immunoglobulin M levels of MYD88L265CXCR4WT patients were significantly higher than those of MYD88WTCXCR4WT patients (P = .024). Tumor burden in BM was highest in patients with MYD88L265PCXCR4mut (82.0%), followed by MYD88L265PCXCR4WT (52.8%) and MYD88WTCXCR4WT (14.2%) (P < .001). The quantity of MYD88-mutated DNA tended to correlate with tumor burden in BM (correlation coefficient 0.647; P = .009). MYD88L265P was detected in plasma cells, plasmacytoid lymphocytes, and lymphocytes but not neutrophils. CONCLUSION: The frequency of MYD88/CXCR4 muts in Korean and Caucasian patients with WM was similar, however 5 of the 6 CXCR4 muts were nonsense-a proportion higher than reported frequencies in Caucasian individuals. Ultra-deep sequencing was capable of detecting CXCR4 muts not detectable using Sanger sequencing, suggesting a possible replacement of the B-cell sorting.

Hebei Spirit oil spill and its long-term effect on children's asthma symptoms.

On December 7th, 2007, an estimated 12,547 kL of crude oil was spilled from the collision of Hebei Spirit near residential area. Our previous study demonstrated worsening of children's asthma symptoms one year after the accident. This study investigated long-term effect of the oil spill on children's asthma symptoms up to five years after the accident. All elementary and middle school students in the exposure area were surveyed on one year (n = 655), three years (664), and five years (611) after the accident. Oil spill exposure was estimated using two estimates including distance from the oil spill (A) and modeled estimates of benzene, toluene, ethylbenzene, and xylene (BTEX) compounds (B), and each was dichotomously categorized (A: high-exposure vs low-exposure; B: ≥20 mg/m3 vs < 20 mg/m3). Asthma symptoms were evaluated using a standard questionnaire. Oil spill exposure estimates were associated with asthma symptoms on one year (odds ratio (95% confidence interval) A: 1.9 (1.1-3.1); B: 1.6 (0.9-2.7)), three years (A: 1.9 (1.1-3.2); B: 1.3 (0.8-2.2)), and five years (A: 1.2 (0.7-1.9); B: 1.8 (1.1-2.8)) after the oil spill. Significant longitudinal relationship between oil spill exposure estimates and asthma symptoms was also observed (A: 1.6 (1.2-2.2); B: 1.6 (1.1-2.1)). Overall, the effect of oil spill exposure estimates was more severe on younger children. Oil spill exposure estimates were associated with asthma symptoms in children up to five years after the oil spill.

Monozygotic twins with shared de novo GATA2 mutation but dissimilar phenotypes due to differential promoter methylation.

A revised WHO classification of hematopoietic neoplasm introduced the new category 'Myeloid Neoplasms with Germline Predisposition', reflecting the growing importance of genetic testing for myeloid neoplasms. Here, we investigated monozygotic twins with the same de novo mutation in GATA2 but different phenotypes. The patient suffering a bleeding tendency was diagnosed with myelodysplastic syndrome (MDS), and her monozygotic twin showed dysmegakaryopoietic features in the bone marrow. Targeted sequencing revealed the same germline mutation in GATA2, c.1192C > T, in both sisters and different somatic mutations in 14 genes between the sisters. The GATA2 mutation was absent in both parents, and their hemograms were normal. The methylation profile of the GATA2 promoter region was different between the twins, showing denser promoter methylation in the patient, correlated with MDS. Thus, we concluded that the twins had acquired a de novo GATA2 mutation but showed different phenotypes, possibly due to the critical role of epigenetic changes.

ASXL1 is a molecular predictor in idiopathic cytopenia of undetermined significance.

We analyzed the mutational profile of idiopathic cytopenia of undetermined significance (ICUS) compared with that of myelodysplastic syndrome (MDS). Targeted sequencing of 88 genes associated with myeloid malignancies was performed using samples of bone marrow mononuclear cells from ICUS and MDS patients. Forty patients with ICUS and 128 patients with MDS were included in this study. The median mutational burden was 0.7 mutation/person in the ICUS group and 2.2 mutation/person in the MDS group. ASXL1 (seven patients) was the most frequently mutated gene. ASXL1 was an independent significant prognostic factor for event-free survival (EFS) and overall survival (OS) (hazard ratio (HR) = 10.07 and 30.63, p = .004 and .003, respectively). The ASXL1 mutation which is frequently detected in elderly patients is a molecular predictor for pancytopenia and survival in patients with ICUS. A larger prospective study is needed to validate the role of this genetic mutation in an ICUS prognosis.

Shifting of erythroleukemia to myelodysplastic syndrome according to the revised WHO classification: Biologic and cytogenetic features of shifted erythroleukemia.

The 2016 revision of the World Health Organization (WHO) classification of tumours of haematopoietic and lymphoid tissues was published. According to 2016 WHO criteria, diagnostic criteria of acute erythroid leukemia was revised. We reassessed 34 de novo acute erythroid leukemia (AEL) diagnosed by 2008 WHO criteria, according to 2016 WHO criteria. A total of 623 patients (excluding M3) with acute myeloid leukemia including 34 patients with AEL were enrolled. Among 34 patients diagnosed with AEL, diagnosis was shifted to MDS-EB in 28 patients (28/34, 82.3%) and MDS-U in 2 patients (2/34, 5.9%), while remained as AEL in 4 patients (4/34, 11.8%) according to 2016 WHO criteria. Interphase FISH for cytogenetic changes of MDS (-5/del(5q), -7/del(7q), del(20q), +8) revealed cytogenetic aberrations in 50.0% (17/34) of AEL 2008 group. AEL 2008 group showed higher frequency of complex cytogenetic abnormalities and higher MDS related cytogenetic abnormalities than AML excluding AEL group. Transformation to another AML subtype was noted in 10% in AEL shifted to MDS. Majority (88.2%) of AEL by 2008 WHO criteria was reclassified to MDS by 2016 WHO criteria. Clinical characteristics of shifted AEL were similar to those of MDS rather than de novo AML.