Next-generation Sequencing Results Research Articles

Next-Generation Sequencing Testing and Triaging of Bone Marrow Samples: Indications, Pre-authorization and Medical Necessity for Patient Care

Abstract Introduction/Objective Bone marrow biopsy (BMBx) is an invasive procedure and samples are usually obtained for diagnosis, staging and prognosis of hematolymphoid disorders (HDs). Given the need for WHO and ICC classification of HDs; the Next Generation Sequencing (NGS) testing (in addition to other esoteric testing) has become relevant and medically necessary. It is important to triage BMBx in real-time based on clinical indications; however, pathologists are challenged by limited published guidelines as well as need for pre-authorization (PA) thus resulting in claim denials. Methods/Case Report Retrospective analysis was performed (2018 to 2022) on 1610 BMBx submitted for various clinical indications. These were categorized as follows: Cytopenias 220 (MDS); Cytosis 250 (MPN); Lymphomas work-up 350 (LY), acute leukemias 440 (AL) and plasma cell disorders 350 (PCD). In addition to esoteric testing; the decision was made to perform NGS testing based on preview of aspirate slides, clinical history and CBC. Results (if a Case Study enter NA) Providers initially ordered NGS in 10% of cases; however, NGS was added later by pathologists in 30% of BMBx samples. Abnormal NGS results were obtained as follows: MDS (70%); MPN (75%); LY (15%); AL (90%) and PCD (40%). NGS testing provided clinically useful data for MDS, MPN and AL groups. Even though LY and PCD groups also yielded positive results; the concurrent esoteric testing (Flow, FISH and Cytogenetics) was useful thus making indication of NGS in these two subset groups less indicated. Conclusion Appropriate triaging of BMBx for NGS testing is medically necessary for following reasons: 1) Enables precise diagnosis in a timely manner; 2) to avoid repeat BMBx if triaging guidelines are followed; and 3) decision to order NGS is made in real-time as BMBx are time sensitive samples thus eliminating the need for PA as this study emphasize that NGS has become standard of care in the work-up of HDs.

Feasibility and Relevance of Incorporating Genetic Testing By Next-Generation Sequencing for Acute Myeloid Leukemia Patients Treated in Low- and Middle-Income Countries

Background: The outcome of the treatment for acute myeloid leukemia (AML) in low- and middle-income countries (LMIC) is significantly worse than in high-income ones due to several factors, including higher non-relapse mortality from infections and limited access to hematopoietic stem-cell transplantation (HSCT). In this resource-constrained setting, AML-risk stratification is based exclusively on cytogenetics, which has longer turn-around time and higher failure rates compared to next-generation sequencing (NGS). Here, we report the benefit of adding a simplified NGS gene panel to improve risk stratification for 279 adult Brazilian patients with AML (male=146, female=136; mean age=50 years; range 18-65) included in International Consortium of Acute Leukemia (ICAL) 2015 Study. Material & Methods: Samples from 10 ICAL centers were shipped to the National Central Lab at the University of São Paulo at Ribeirão Preto Medical School, Brazil (located zero to 1420 kilometers from centers). All patient samples were assessed by conventional cytogenetics and molecular tests by RT-PCR for RUNX1::RUNX1T1 and CBFB::MYH11 , PCR and fragment analysis for NPM1 and FLT3-ITD allelic ratio, and Skyline AML profiler assay for CEBPA double mutant. We retrospectively searched for mutations in the ASXL1, CEBPA, FLT3, IDH1, IDH2, NPM1, RUNX1, TP53 genes using NGS in 53 out of the 279 samples. The tests were performed in the Illumina iSeq100 Sequencing System using reagents and methodology developed under the supervision of the Erasmus MC Cancer Institute (Valk Laboratory) as part of the activities of the ICAL Laboratory Subcommittee. Training, including bioinformatics analysis, was provided virtually and through a short-term visiting program (ASH Visitor Training Program) to Erasmus MC. Datasets with genetic testing results obtained locally were sent and reanalyzed at Erasmus MC and discussed in bi-monthly meetings. Risk stratification was determined either by cytogenetics only or by cytogenetic and molecular analysis according to the ELN 2017 proposal. Overall survival (OS) was defined from date of diagnosis to last follow up or death and compared by Kaplan-Meier and Log-Rank test followed by Benjamini-Hochberg p-value adjustment method, using R 4.3.1. Results: conventional cytogenetics was informative in 68% (190/279) of the cases, and based on those results 32 (16.8%), 116 (61%) and 42 (22.1%) patients were stratified into favorable, intermediate and adverse categories, respectively. However, no significant difference in the OS was detected according to cytogenetic risk. When genetic testing by NGS was added, 54 (27.3%), 98 (49.5%) and 46 (23.2%) patients were assigned into favorable, intermediate and adverse categories, respectively. Twenty-six (48%) patients had risk re-classified as a consequence of the NGS results. Moreover, two out of four patients whose risk stratification could not be assigned due to insufficient metaphasis were classified based on NGS results. Of note, the median OS of the favorable group was significantly longer (not reached) than that of intermediate and of the adverse groups (6 months), ( p<0.0001), but no difference was detected between the latter two. We also assessed whether genetic testing by NGS would change the risk category of AML patients previously classified based on conventional cytogenetics in association with the evaluation of gene rearrangements, NPM1, FLT3 and CEBPA mutational analysis by methods other than NGS. NGS results changed the category of 3 out of 54 patients. Our results indicate the feasibility of developing a simplified platform of genetic testing for AML based on NGS in LMIC, which was able to improve the identification of favorable-risk patients and may help to better allocate resources.

Only FLT3-ITD Mutation Did Not Have a Deleterious Effect on Acute Myeloid Leukemia Patients with NPM1 Mutation, but Concomitant with DNMT3A Mutation or a＜3log Reduction of MRD2 Predicted Poor Survival

Background Co-occurring mutations were observed in NPM1-mutated acute myeloid leukemia (AML) frequently, and the measurable residual disease (MRD) for NPM1 is effective for the prognosis of NPM1-mutated AML. However, the combined effects of co-occurring gene mutation at the baseline and MRD on survival are unclear. We investigated the impact of gene mutation status and NPM1 MRD on predictive value of AML patients with NPM1 mutation. Methord A single-center, retrospective cohort study was carried out in patients diagnosed as AML with NPM1 mutation in Peking University Peoples'hospital. Patients were treated by an anthracycline and cytarabine-based or homoharringtonine and cytarabine-based induction chemotherapy regimen and followed by high dose cytarabine as consolidation therapy. Patients who had the indications of allogeneic hematopoietic stem cell transplantation (allo-HSCT) underwent allo-HSCT. A small part of patients with FLT3-ITD mutation received FLT3 inhibitor. Targeted next-generation sequencing (NGS) were analyzed at diagnosis, and quantitative NPM1 mutation (A, B, and D type) were detected at diagnosis and each end of chemotherapy cycle. As MRD for NPM1 rare type (NPM1 RT) was not available, MRD, relapse free survival (RFS) or overall survival (OS) analysis were conducted in patients received chemotherapy only (NGS chemo), excluding harboring NPM1 RT. Results A total of 234 patients, with NGS results, diagnosed as NPM1-mutated AML were included. 68.4% (160/234) patients including 17 patients with NPM1 RT received chemotherapy only, and NGS chemo patients were 143. After chemotherapy, 31.6% (74/234) patients underwent allo-HSCT. 1.7% (4/234) patients died of infection or hemorrhage during induction therapy. 15.8% (37/234) patients received FLT3 inhibitor，and 92.2% (212/230) patients achieved complete remission rate (CR) or incomplete hematologic recovery (CRi). During median follow-up time of 24.9 [interquartile range (IQR) 11.6-47.8] months, 40.6% (95/234) patients died. Median transcription of NPM1 at diagnosis was 31.13 (IQR 17.23-56.91)%, and would take a median time of 4.1 (IQR 2.6-5.6) months to converted to negativity.Reduction of MRD at the end of second cycle of consolidation (MRD2) ≥3log associated with higher RFS (3-year RFS rate 66.3% vs. 11.1%, p＜0.001) and OS (3-year OS rate 78.4% vs. 32.4%, p＜0.001) than reduction＜3log, and consistent negativity of MRD also predicted higher RFS (3-year RFS rate, 98.1% vs. 42.3%, p＜0.001) and OS (3-year OS rate, 100% vs. 67.7%, p＜0.001) than those whose MRD turned from negativity to positivity. 9 genes were mutated in＞10% in 234 patients, and DNMT3A and FLT3-ITD were the most prevalence (Figure 1). Univariate analysis showed FLT3-ITD, DNMT3A and reduction of MRD2＜3log were associated with poorer RFS and OS in 143 NGS chemo patients. But In multivariate analysis, only DNMT3A mutation [RFS, hazard ratio (HR)=1.9, p =0.023; OS, HR=2.1, p =0.032] and reduction of MRD2 ≥3log（RFS, HR=0.1; OS, HR =1.9, both p＜0.001）were independent prognostic factors for survival. Four subgroups were divided by different status of DNMT3A and FLT3-ITD, that were both FLT3-ITD and DNMT3A negative, FLT3-ITD positive and DNMT3A negative, FLT3-ITD negative and DNMT3A positive, and both FLT3-ITD and DNMT3A positive. The concurrence of FLT3-ITD and DNMT3A was associated with lowest RFS and OS (3-year RFS rate 29.2%, p＜0.001; 3-year OS rate 53.5%, p＜0.001). However, patients with only FLT3-ITD mutation did not contribute to poor survival compared with patients without FLT3-ITD mutation (3-year RFS rate 71.4% vs. 74.4%, p＜0.843; 3-year OS rate 74.5% vs.75.8%, p = 0.759). Three risk group were re-stratified by the gene mutation and gene reduction of MRD2 (Figure2), and the RFS and OS of the poor risk group, defined by patients with ＜3log reduction of MRD2, or with both FLT3-ITD and DNMT3A mutation, could be improved by allo-HSCT(3-year RFS rate, 56.4% vs. 29.2%, p = 0.015; 3-year OS rate, 72.0% vs. 31.6%, p = 0.001). Conclusion In AML patients with NPM1 mutation, the deleterious effect of FLT3-ITD could be pronounced in patients concomitant with DNMT3A mutation. A＜3log reduction of MRD2 was also another independent poor prognostic factor for survival, which could be improved by allo-HSCT. Meanwhile, the conversion of MRD for NPM1 from negative to positive might be a poor prognostic factor, but it requires to be validated by multivariate analysis.

Harnessing Artificial Intelligence for Risk Stratification in Acute Myeloid Leukemia (AML): Evaluating the Utility of Longitudinal Electronic Health Record (EHR) Data Via Graph Neural Networks

Background: AML is a life-threatening disease, and to determine which patients need allogeneic stem cell transplantation, hematologists risk-stratify each case. However, standard risk stratification using the European LeukemiaNet (ELN) criteria is focused on baseline mutations and chromosomal aberrations, and the risk estimate is not updated during a patient's course. In other blood cancers, recalculating the risk with treatment response data can help guide the need for more intensive therapy (Kurtz, et al, Cell, 2019). Furthermore, deep learning graph neural networks (GNN) applied to EHR data have strong predictive power in a hematology context (Fouladvand, et al, J Biomed Inform, 2023). Thus, we evaluated the power of a GNN to predict survival in AML using longitudinal EHR data, specifically with labs and histological features that are not included in the ELN but may capture the treatment response. Methods: Patients who were seen at the Stanford Cancer Institute, had EHR data available within six months of diagnosis, and were diagnosed with AML between June 1998 and January 2021 were included in this retrospective analysis. The GNN was trained to predict survival at two years from diagnosis using the first six months of clinical data. Patients were excluded if they were lost to follow-up before two years or died before six months. Data were collected from structured databases associated with Stanford's EHR, except that diagnosis dates were from Stanford's Cancer Registry, and survival data was supplemented with other databases including the Social Security Death Index. Dysplasia, bone marrow cellularity, and bone marrow blast percentages from pathology reports (“pathology report data”) were extracted using text processing algorithms and weakly supervised machine learning (Ratner, et al, ArXiv, 2017). To represent time series information, we framed each patient's timeline as a network (or “graph”) of events. The primary GNN model was a heterogenous graph transformer classifier with two node types: complete blood count (CBC) data and pathology report data (Hu, et al, ArXiv, 2020). Data from the same week were assumed to be from the same timeframe and connected with bidirectional edges. Data separated by longer time periods were connected with unidirectional edges of a separate edge type. The independent test dataset consisted of patients whose ELN 2022 classification was available, and to train the model, the remaining data were divided into train/validation splits of 0.9/0.1. Results: Of the 2,535 patients with survival data, 1,029 met inclusion criteria. Table 1 summarizes the data available in the EHR for each variable, and nearly all patients had CBC and pathology report data. The area under the receiver operating characteristic (AUROC) using the ELN 2022 criteria for predicting survival in the test dataset was 0.79. The AUROC curve for the GNN model was comparable at 0.76, despite not using any variables from the ELN criteria, and the model effectively stratified patients' disease into high- and low-risk in the independent test dataset (hazard ratio [HR] 3.0, log-rank p = 0.0009). Interestingly, despite not having access to mutation or cytogenetic data, the high-risk cases were enriched in known high-risk mutations, like TP53 and RUNX1, and in high-risk chromosomal aberrations, like 5q deletion (Table 1). Although the model predictions correlated with the ELN criteria in some ways, they also stratified the ELN intermediate-risk AML cases into high and low risk (HR 6.1 for model-predicted high risk among ELN intermediate cases, p = 0.07). Conclusions: Risk stratification using artificial intelligence and longitudinal data from the EHR performed comparably to the ELN 2022 criteria and has the potential to further stratify the ELN categories. The model performed well despite only using histological features and lab values, which are more readily available and more frequently updated than next-generation sequencing results. In the future, this approach may further improve with a larger sample size and additional variables, such as measurable residual disease and treatment information. Given the heterogeneity and increasing complexity of AML classification, leveraging artificial intelligence to assist with classification will be crucial, and these results are a step towards a future where data are automatically extracted from the EHR and used for continuously updated risk stratification.

A Single Center Real World Study of Outcome and Resistance of Bruton Tyrosine Kinase Inhibitors (BTKi) in Chinese Patients with Chronic Lymphocytic Leukeima/Small Lymphocytic Lymphoma

Introduction Acquired BTK/PLCG2 mutations remain to be the driver of Bruton Tyrosine Kinase inhibitor (BTKi) resistance. But the underlying mechanisms need to be further explored. In this study, we retrospectively analyzed clinical and biological characteristics of BTKi-treated CLL patients and report the outcome and resistance in our center (the First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital). Method 262 patients treated with single BTKis continuously were collected and follow up the outcome in our center. Progression-free survival (PFS) was defined as from BTKi initiation to disease progression on BTKi. Next-generation sequencing (NGS) and High sensitivity droplet digital PCR (ddPCR) acquired at treatment initiation /or at the time of disease progression were used to describe mutational landscape. Kaplan-Meier method and the log-rank test were used for analysis. Results A total of 262 patients were enrolled from January 2014 to March 2023, and the median age was 63 (range 28-88) years. Ibrutinib, Zanubrutinib, Orelabrutinib were administered in 55.3%,18.3% and 17.9% of patients respectively, in addition to 6 patients with Acalabrutinib and one with Loxo-305, among them 47.7% (125/262) received BTKi as first line treatment. With a median follow up of 27.3 months, 208 patients were in durable remission while 54 patients relapsed, the median exposure of BTKi was 26.8 (range, 1-73.8) sand 27.7 (range, 3-69.8) months respectively. The median PFS was 5.8 years (5.0-NA, 95% CI) for the whole cohort. Clinical and biological characteristics of both cohorts at initiation of BTKi treatment were described and compared in Table 1. Higher proportion of bulky disease (50.0% vs 23.5%), IGHV unmutated status (84.3% vs 50.5%), del(17p) (44.7% vs 12.9%), TP53 mutation (37.8% vs 15.2%), TP53 aberrations (62.5% vs 24.3%) and complex karyotype (40.5% vs 21.1%) were shown in the BTKi relapsed cohort as compared to responsive cohort. Inferior PFS was observed in patients with TP53 aberration vs without TP53 aberration ( 45.6 mos vs not reached, p<0.0001), unmutated IGHV vs mutated IGHV (56.2 mos vs not reached, p=0.0005), with del(17p) vs without del(17p) (36.1 mos vs not reached, p<0.0001), with complex karyotype and without complex karyotype (69.8 mos vs not reached, p=0.0401), bulky disease vs no bulky disease (45.6 mos vs 69.8 mos, p=0.0006) and elevated LDH vs normal LDH (66.5 mos vs not reached, p=0.0083). Moreover, patients with del(11q) or BTKi given as second or greater line showed a tendency of inferior PFS ( p= 0.1963, p= 0.0735, respectively). Regarding the relapsed cohort, 43 patients displayed CLL progression while 11 patients displayed Richter transformation (RT, including 1 Hodgkin lymphoma [HL]) , the median time from initiation to progression were 33.7 months and 9.5 months, respectively（p<0.0001). BTK/PLCG2 mutations were the most common mutation at the timepoint of disease progression. By integrating NGS and ddPCR results, 54.9% (28/51) relapsed patients acquired BTK/PLCG2 mutation. BTKC481S mutation was the dominant hotspot mutation in all kinds of BTKi relapsed cases. Other mutations at the C481 site included C481R, C481Y, C481F. However, BTKT474 mutation was only detected in Orelabrutinib-resistant cases (Figure 1). 6 patients presented with multi-hit mutations. Treatment after disease progression on BTKis is still challenging. In our relapsed cohort, the median PFS was 5.7 months and poorer in RT as compared with CLL progression (3.0 months vs 6.7 months, p= 0.0874). Venetoclax-based regimen might be an effective salvage therapy which could showed a benefit trend in PFS as compared to other post BTKi therapy (10.1 months vs 3.1 months, p= 0.1818). Conclusion In our real world study, patients with IGHV unmutated status, TP53 aberrations, bulky disease and elevated LDH showed inferior PFS. Acquired BTK/PLCG2 mutations remained to be key drivers of BTKis resistance and BTKC481S mutation was the dominant mutation, while BTKT474 mutation was only detected in Orelabrutinib-resistant patients. The prognosis was rather poor for relapsed patients especially for RTs, treatment strategy after disease progression remains to be optimized.

Genetic Study of Patients with Severe Dense Granule Deficiency: Unraveling the Underlying Genetic Complexity of Hermansky-Pudlak Syndrome

Background: Severe platelet (plt) dense granule (DG) deficiency is a hallmark of Hermansky-Pudlak syndrome (HPS), a rare, heterogeneous group of genetic disorders characterized by oculocutaneous albinism, bleeding diathesis, and lysosomal storage defects. A subset of HPS patients (pt) who has HPS1, HPS2, and HPS4 may also experience pulmonary fibrosis, granulomatous colitis, and/or immunodeficiency (syndromic HPS). Platelet transmission electron microscopy (PTEM) has been considered a valuable tool to screen such patients, and molecular testing aids in confirming te diagnosis, subclassifying the HPS and providing information on prognosis in pt. To elucidate the effectiveness of PTEM and next-generation sequencing (NGS) to identify and subclassify HPS pt, we conducted a comprehensive study on a cohort of pt with severe plt DG deficiency. Methods: After IRB approval, consecutive samples submitted to our reference laboratory for PTEM testing were analyzed. Whole mount (WM) PTEM tests were performed on all pt. Targeted NGS of the ISTH tier 1 genes (including 11 HPS-related genes) was performed on a cohort of patients who had < 0.3 mean DG/plt (200 plt counted) by PTEM. Data collected included demographics, PTEM findings, and genetic variants classified based on the 2015 ACMG criteria. Results: A total of 38 pt met our study criteria, the median age at PTEM testing was 39 years (range, 5 months to 76 years), and 22 (58%) were female. Ethnic backgrounds included Caucasian, Albanian, Puerto Rican, and other (unavailable). The mean DG/plt was 0 (0-0.2). A variant in an HPS-related gene was found in 26/38 patients (68%). The spectrum of variants in HPS-related genes is shown in Table 1, encompassing single-nucleotide substitutions, insertions, deletions, and frameshift mutations involving HPS1, HPS3, HPS5, HPS6, and HPS7 ( DTNBP1) genes. In addition to the previously reported pathogenic variants, several novel/previously unreported variants were discovered (Table 1). Interestingly, some patients presented with multiple variants, suggesting potential compound heterozygosity. No variants were found for the remaining 12 patients (32%). Conclusion: Our NGS-based genetic study of 38 patients with severe DG deficiency provides valuable insights into the complexity of this rare disorder. The high yield of finding variants in HPS-related genes verified that WM-PTEM is a useful screening tool for identifying HPS patients. The identification of known variants in HPS-associated genes confirms the importance of genetic testing not only in the diagnosis of HPS but also provides information on prognosis in syndromic HPS. Pathogenicity of novel variants remains to be elucidated. Moreover, the observed 32% of patients who had negative NGS results suggest the existence of as yet undiscovered genetic mechanisms for the severe platelet DG deficiency.

Efficacy of Demethylated Drug Combine with Low Dose Chemotherapy in Juvenile Myelomonocytic Leukemia

Objection：Juvenile myelomonocytic leukemia (JMML) is a rare pediatric leukemia with a median survival time of less than 2 years. Currently, hematopoietic stem cell transplantation (HSCT) is the only possible cure of JMML. Most of the newly diagnosed pediatric patients with JMML have high tumor burden, and rapid disease progression, and may not tolerate HSCT. In the present study, we aimed to explore the effection of Demethylated drug in combination with low-dose chemotherapy on JMML patients before HSCT. Methods：A retrospective analysis of 16 JMML patients was performed from Jul 2017 to Nov 2021. All patients were given Demethylated drug in combination with low-dose chemotherapy, 10 patients decitabine at a dose of 20 mg/m2 for 5 days, supplemented with cytarabine (50-100 mg/m2×3~5 days), and /or etoposide (50 mg/m2×3~5 days), 6 patients Azacytidine at a dose of 75mg/m2 for 7 days combined with homoharringtonine 2mg/m2 for 5-7 days. Each treatment interval was 4 weeks, bridging to HSCT after three~four courses of treatment. Results：The median age of onset of the 16 JMML patients was 36 months (range 6-51 months), the male to female ratio was 14:2, and the median volume of the spleen at diagnosis was 357cm3 (range: 175-588) by Three-dimensional ultrasound of spleen. The median white blood cell (WBC) count was 39 (range 2-128) × 109/L, the median hemoglobin 84 (range 47-110) g/l, and the median platelet 36 (range 16-75) × 109/L，the median monocyte count was 8 (range 2-26) × 109/L at diagnosis, the median RDW-CV was 19 (15-30)%, the median RDW-SD 56 (45-77) fl, the median HbF 37(1-76)%. Chromosome was performed, of which 13 cases had normal karyotype. 2 cases had tirsomy 8, 1 case had structural abnormality in chrosome 9. Next-generation sequencing results showed that 11(68.8%) cases carried PTPN11 mutations, 6 (37.5%) cases NF1 mutation, 5 (31.3%) KRAS mutation, 4 (25%) NRAS mutation, 1 case without one of the Ras pathway genes. 8 cases (50%) had more than two types of canonical JMML mutations, among them 6 cases (75%) had PTPN11 and NF1 co-mutation. 10 cases had secondary gene mutations, 3 (3/10) cases JAK3 mutation, 2 (2/10) SETBP1 mutation, 2 (2/10) SH2B3 mutation, 2(2/10) ASXL1 mutation, 2 (2/10) ARID1A mutation, 1 (1/10) SRSF2 mutation. The median treatment course was three cycles (range: 1-6), and the response rate was 68.8% (11/16) after one cycle and 81.8% (9/11) after three cycles. One case achieved complete remission (CR) after four courses of therapy. The WBC and monocyte counts were significantly decreased after treatment (P<0.001), and the spleen volume showed a decreasing tendency. Eleven patients received HSCT, 9 cases were implanted successfully and 2 cases failed, of which 1 case was relieved by secondary transplantation and 2 patients died of grade IV graft-versus-host disease (GVHD) after transplantation. The median follow-up time was 31 months. The 2-year progression-free survival rate was 75.0±10.8%. Conclusions：Demethylated drug in combination with low-dose chemotherapy could reduce JMML patients' tumor burden, improve the general condition, and obtain a clinical response rate of 81.8% after 3 cycles therapy. Therefore, such a combination regimen could be used as a therapeutic option for JMML before HSCT.

NCOG-09. EXAMINING THE CLINICAL UTILITY OF NEXT GENERATION SEQUENCING IN ADULT GLIOBLASTOMA – A SINGLE CENTER RETROSPECTIVE STUDY

Abstract BACKGROUND Over the last 2 decades, the use of molecular targeted therapy has revolutionized the cancer treatment landscape and transformed the outcomes and disease prognoses of several cancer entities. The success of targeted therapy is closely tied to the advent of commercially available next generation sequencing (NGS) which has expanded our understanding and knowledge of the genetic drivers and mutations present in a variety of tumors. Unfortunately, in glioblastoma (GBM), the overall effectiveness of targeted therapies remains limited.1 Nonetheless, NGS for GBM patients has been integrated into the standard practice of many neuro-oncologists and is encouraged by the National Comprehensive Cancer Network (NCCN) Guidelines. The purpose of this retrospective chart review is to evaluate how NGS is incorporated into the management of glioblastoma at a single center academic institution. METHODS We identified 85 patients with pathologically confirmed GBM who had NGS done from January 2014-March 2021. The clinical records of all patients were reviewed to denote the frequency of clinical trial enrollment or off-label use of targeted therapy based on the results of NGS. We also denoted the time point in the disease course when NGS was ordered and the trend of frequency of ordering NGS from January 2014-December 2020. RESULTS 28.2% (95% CI 19 to 39.1%) of patients received treatment based on the results of NGS. 8% were enrolled in a clinical trial and 20% were treated with off-label use of targeted therapy at the time of disease progression. There was no statistically significant survival difference seen between patients who had treatment done based on results of NGS compared to those who did not. CONCLUSION NGS results are used to guide treatment or clinical trial enrollment in a significant number of glioblastoma patients, however, the clinical efficacy of this treatment is yet to be determined.

PATH-63. NEXT GENERATION SEQUENCING FOR CNS TUMORS IN CHILDREN; DOES IT ADD VALUE IN A MIDDLE-INCOME COUNTRY SETUP?

Abstract INTRODUCTION Advances in molecular diagnostics led to improved targeted interventions in the treatment of pediatric CNS tumors. However, the capacity to do these diagnostics is limited in LMICs, and consequently their value is questionable. METHODS We reviewed our experience of Next Generation Sequencing (NGS) testing (TruSight RNA Pan-Cancer seq panel) for pediatric CNS tumors at KHCC/Jordan (March/2022- April/2023). Paraffin blocks’ scrolls were shipped to Sickkids laboratory based on local multidisciplinary team recommendations. We reviewed patients’ clinical characteristics, tumor types and the impact on treatment. RESULTS There were 16 boys and 16 girls; median age at time of testing was 9.5 years (range, 0.9- 21.9 years). Twenty samples were sent at diagnosis and twelve upon tumor progression. Main diagnoses were Low-grade glioma (15), High-grade glioma (10), and other tumors (7). Reasons for NGS testing were: to find a molecular target (20), and to characterize more tumor/behavior (12). The median time from shipping the samples to receive the NGS result was 22 days (range, 15-49 days) at a cost of US$1000. Twenty (63%) tumors had targetable alterations (MAPK/PI3K inhibitors (16), NTRK inhibitors (2), check point inhibitors (2)). Two rare BRAF mutations were identified (BRAFp.G469A, BRAFp.K601E). One tumor diagnosed initially as undifferentiated round-cell sarcoma found to have NAB2::STAT6 fusion and re-diagnosed as aggressive metastatic solitary fibrous tumor. Eight patients received targeted therapy; Dabrafenib/Trametinib (5), Pembrolizumab (2), Entrectinib (1), while radio-chemotherapy was used in the others. Three patients died from disease progression (one used Entrectinib). CONCLUSIONS Sent abroad NGS testing was feasible, however local capacity building is necessary. In this highly selected tumor cohort, high percentage of targetable alterations were identified. NGS was helpful to characterize tumors more and to offer alternative therapies. Nevertheless, interpretation of the significance of NGS results and access to those “new” therapies continues to be a challenge in LMICs

Novel RICTOR amplification harbouring entities: FISH validation of RICTOR amplification in tumour tissue after next-generation sequencing

Alterations in mTOR signalling molecules, including RICTOR amplification, have been previously described in many cancers, particularly associated with poor prognosis. In this study, RICTOR copy number variation (CNV) results of diagnostic next-generation sequencing (NGS) were analysed in 420 various human malignant tissues. RICTOR amplification was tested by Droplet Digital PCR (ddPCR) and validated using the “gold standard” fluorescence in situ hybridisation (FISH). Additionally, the consequences of Rictor protein expression were also studied by immunohistochemistry. RICTOR amplification was presumed in 37 cases with CNV ≥ 3 by NGS, among these, 16 cases (16/420; 3.8%) could be validated by FISH, however, ddPCR confirmed only 11 RICTOR-amplified cases with lower sensitivity. Based on these, neither NGS nor ddPCR could replace traditional FISH in proof of RICTOR amplification. However, NGS could be beneficial to highlight potential RICTOR-amplified cases. The obtained results of the 14 different tumour types with FISH-validated RICTOR amplification demonstrate the importance of RICTOR amplification in a broad spectrum of tumours. The newly described RICTOR-amplified entities could initiate further collaborative studies with larger cohorts to analyse the prevalence of RICTOR amplification in rare diseases. Finally, our and further work could help to improve and expand future therapeutic opportunities for mTOR-targeted therapies.

INNV-06. SIGNIFICANCE OF MOLECULAR DIAGNOSTICS FOR THERAPEUTIC DECISION-MAKING IN RECURRENT GLIOMA

Abstract BACKGROUND Targeted therapies have substantially improved survival in cancer patients with malignancies outside the brain. Whether in-depth analysis for molecular alterations may also offer therapeutic avenues in primary brain tumors remains unclear. We herein present our institutional experience for glioma patients discussed in our interdisciplinary Molecular Tumor Board (MTB) implemented at the Comprehensive Cancer Center Munich (LMU). METHODS We retrospectively searched the database of the MTB for all recurrent glioma patients after previous therapy. Recommendations were based upon next-generation sequencing results of individual patient’s tumor tissue. Clinical and molecular information, previous therapy regimens and outcome parameters were collected. RESULTS Overall, 73 consecutive recurrent glioma patients were identified. In the median, advanced molecular testing was initiated with the third tumor recurrence. The median turnaround time between initiation of molecular profiling and MTB case discussion was 48 ± 75 days (range: 32 - 536 days). Targetable mutations were found for 50 recurrent glioma patients (68.5%). IDH1 mutation (27/73; 37%), EGFR amplification (19/73; 26%) and NF1 mutation (8/73; 11%) were the most detected alterations and a molecular-based treatment recommendation could be made for all of them. Therapeutic recommendations were implemented in 12 cases (24%) and one third of these heavily pretreated patients experienced clinical benefit with at least disease stabilization. CONCLUSION In-depth molecular analysis of tumor tissue may guide targeted therapy also in brain tumor patients and considerable anti-tumor effects might be observed in selected cases. However, future studies to corroborate our results are needed.

PATH-24. A CASE SERIES OF LONGER THAN EXPECTED SURVIVAL IN IDH-WILDTYPE, MGMT-UNMETHYLATED GLIOBLASTOMA PATIENTS

Abstract Despite interventions with surgery, radiation, and chemotherapy, Glioblastoma (GBM) remains a difficult cancer to treat. Only 6.9% of patients survive five years post-diagnosis1 and the median overall survival is reported to be in the range of 14-15 months.2Glioblastoma patients who harbor an IDH-mutation have a known improved survival rate over their IDH-wildtype counterparts and under the recent WHO 2021 Classification of CNS tumors, IDH-mutational status is now an established criteria for the classification of high-grade gliomas. MGMT methylation status is also a well-described prognostic factor in GBM. MGMT methylated patients have a more favorable prognosis and better response to conventional treatment with temozolomide.3 It is rare but there are some IDH-wildtype, MGMT-unmethylated GBM patients who survive longer than 4 years. Therefore, it is important to identify other potential favorable factors for longer survival in the unfavorable cohort of patients with IDH-wildtype and MGMT-unmethylated GBM. Case Series: Eighty-five GBM patients who had next generation sequencing completed at the University of California, Irvine were reviewed. Of 85 patients, 4 patients (4.7%) were IDH-wildtype, MGMT-unmethylated and lived longer than 4 years. We evaluated the pathology, clinical characteristics, treatment course, and molecular findings of these 4 long term GBM survivors with IDH-wildtype and MGMT-unmethylated status. Discussion: This case series of 4 cases of patients with IDH-wildtype, MGMT-unmethylated GBM with longer than expected survival did not reveal a ubiquitously prevalent molecular marker for long term survival based on review of next-generation sequencing results. 2 out of the 4 patients harbored an uncommon MTAP copy number loss mutation, which may warrant further investigation into the significance of this mutation on prognosis. 3 out of 4 patients received a vaccine-based intervention through clinical trial which may support ongoing investigation into the additional benefit this specific population of GBM patients may have from vaccine-based therapy.

Emerging Importance of Viral Transport Media in High-Throughput Sequencing Fidelity for Genomic Analysis

The impact of Viral Transport Media (VTM) components on the efficacy and accuracy of high-throughput Next-Generation Sequencing (NGS) techniques is a subject of paramount importance in the field of viral genomics and diagnostics. VTMs play a pivotal role in preserving viral samples, and their composition can profoundly influence the quality and reliability of NGS results. This study explores the intricate relationship between VTM components and NGS outcomes, with a focus on optimizing diagnostic precision and the efficiency of NGS in viral genomics. Through comprehensive investigation, this research sheds light on the critical implications of VTM composition for enhancing viral genomic analysis, streamlining diagnostic processes, and bolstering public health responses. The findings have far-reaching consequences, including the potential for personalized medicine, rapid detection of emerging viral threats, and the improvement of public health surveillance. Standardized protocols and ongoing technological advancements are essential in harnessing the full potential of NGS for genomics and metagenomics, marking a significant step toward more effective viral disease management and outbreak response.

Longitudinal Assessment of Minimal Residual Disease (MRD) in the ATLAS Randomized Phase 3 Trial of Post-Transplant Treatment with Carfilzomib, Lenalidomide, and Dexamethasone (KRd) Versus Lenalidomide (R) Alone in Patients with Newly Diagnosed Multiple Myeloma (NDMM)

Introduction The interim results of the ATLAS trial (Dytfeld, Lancet Oncology 2023) suggest a progression-free survival (PFS) benefit for NDMM patients after autologous hematopoietic stem cell transplantation (ASCT) treated with MRD-directed and risk-adapted maintenance using KRd compared to single-agent R. In order to gain a better understanding of the differences in response dynamics and depth achieved by the two regimens, we conducted a detailed analysis of MRD results assessed at multiple timepoints. Methods ATLAS is an ongoing phase 3 trial that randomized 180 patients with NDMM after ASCT to receive maintenance therapy with KRd or R. In the KRd arm, patients with standard-risk cytogenetics who achieved MRD-negativity after cycle 6 de-escalated therapy to receive R alone, starting from cycle 9. MRD assessments were performed using multicolor flow cytometry (MFC) with a limit of detection (LoD) of 10 -5 and next-generation sequencing (NGS) with LoD 10 -6 at screening, cycle 6 (C6), C12, C18, C24, and C36. Patients with MRD>10 -5 were considered MRD positive. When both MFC and NGS results were available, at least one positive result qualified a patient as MRD-positive. In line with the MRD reporting harmonization guidelines (Costa, Leukemia 2021), patients who have not reached a landmark timepoint for analysis were excluded from the denominator. MRD rates between the treatment arms were compared using the chi-square test. PFS and MRD-free survival (death, progression or MRD resurgence counted as an event) were analyzed using the Kaplan-Meier method to calculate survival rates, and the log-rank test was performed to compare the groups. Results We previously reported MRD rates at the end of 6 cycles (Dytfeld, Lancet Oncology2023). In this analysis, MRD results were evaluable for the following number of subjects at the respective timepoints: 180 at screening, 180 at C6, 180 at C12, 179 at C18, 175 at C24, and 160 at C36. All the reported data, including MRD results, response rates, and time to event, were obtained at the cut-off date 31-DEC-2021, as in the Lancet Oncology manuscript. In the landmark analysis, MRD-negativity after C6 was associated with improved PFS for the entire study patient population [HR=0.49 (95% CI: 0.27-0.90), p=0.01]. Although the proportion of MRD-negative patients at screening was higher in the R arm (46/87, 53%) compared to the KRd arm (36/93, 39%, p=0.06), MRD-negative rates were higher in the KRd arm in the subsequent landmark evaluations. The MRD-negativity rates for KRd and R were: 61/93 (66%) vs 41/87 (47%, p=0.01) at C6; 49/93 (53%) vs 41/87 (47%, p=0.46) at C12; 48/93 (52%) vs 35/86 (41%, p=0.14) at C18; 43/90 (48%) vs 26/85 (31%, p=0.02) at C24; 34/82 (41%) vs 16/78 (21%, p= 0.004) at C36. These findings were consistent with a higher number of patients in the KRd arm who converted from MRD-positivity at screening to MRD-negativity at any timepoint (Figure 1). Of the 50 patients with detectable MRD at screening in the KRd arm, 33 (66%) achieved MRD-negativity as their best response. Among them, 24 achieved MRD-negativity at the end of C6, with 2 patients converting as late as C24. The numbers were significantly lower for the R arm, with 14 out of 32 (44%) MRD-positive patients at screening achieving MRD-negativity (p=0.047). Sustained MRD-negativity for at least 12 months was reached in 51 out of 93 patients (55%) in the KRd arm and in 36 out of 87 (41%) in the R arm (p=0.06). Those who achieved sustained MRD-negativity have had significantly longer PFS (HR=0.35 (0.20-0.59), p<0.0001). Among patients with sustained MRD-negativity, patients in the KRd arm had superior PFS [HR=0.30 (0.12-0.79), p=0.01], and MRD-free survival [HR=0.42 (0.20-0.89), p=0.02, Figure 2]. In the KRd arm, sustained MRD-negativity was confirmed in 32 out of 41 (78%) patients who had de-escalated therapy from KRd to R. In this group there were 8 events (25%) of progressive disease or MRD resurgence, which compared favorably to 17/36 (47%, p=0.057) events observed in the R arm. Conclusions The improved PFS in patients with NDMM receiving post-ASCT KRd maintenance may be attributed to higher rates of MRD conversion and longer duration of MRD-negativity. Importantly, this benefit, compared to R alone, appears to be sustained even in the subset of patients who received only 8 cycles of MRD-directed, risk-adapted KRd and subsequently de-escalated to R alone.

How Induction Cycles before Remission Influence Long-Term Survival in Acute Myeloid Leukemia

Background ： Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by accumulation and expansion of immature myeloid cells in the bone marrow, peripheral blood and other tissues. Despite the progress made in mechanisms and approval of new therapeutics, the clinical outcome of AML remains suboptimal. The achievement of complete remission (CR) after induction chemotherapy is critical because the prognosis of refractory AML was especially unfavorable. However, little is known about the association between remission after first induction therapy and the overall survival (OS) in different subgroups of AML. Study Design and Methods: We retrospectively analyzed patients with newly diagnosed AML from January 2019 to August 2022. The diagnosis and treatment strategies were based on European Leukemia Net (ELN) criteria and National Comprehensive Cancer Network (NCCN) guideline. Composite complete remission (CRc) rate (CR+CR with incomplete recovery [CRi]) were assessed at the completion of induction or re-induction therapy. Minimal residual disease (MRD) was assessed using multiparameter flow cytometry with a minimum sensitivity of 0.1%. OS was measured from the day of diagnosis until death or the last follow-up date. Results : In total, 679 patients were included and the median age was 59.0 (range, 14.0-93.0) years old. There were 56.8% (386/679) of patients who achieved CRc after the induction therapy (group A), 18.1% (123/679) achieved CRc after the re-induction therapy (group B). The median follow-up period was 24.2 months. Even though there was no significant difference between group A and group B on OS, group A exhibited improved OS when censored at allogenic hematopoietic stem cell transplantation (HSCT) compared to that in group B (group A vs group B:3-year OS rates 65.7% vs 51.1%，p=0.004，Figure A). We further explored the effects of remission depth on survival. The results showed that, after induction therapy, patients achieved CR exhibited superior OS compared with that in CRi (CR vs. CRi, 3-year OS rates 69.4% vs 51.6%, p=0.006). Also, compared with these patients who failed to achieved MRD negativity within re-induction therapy, patients achieved MRD negativity within re-induction therapy showed superior OS when censored at HSCT (MRD- vs. MRD+, 3-year OS rates 64.7% vs 34.9%, p=0.040). After the first reduction therapy, patients achieved CRc but MRD positive benefit from HSCT (HSCT vs Non-HSCT, 3-year rates 83.0% vs 57.9%, p=0.017), but in patients with MRD negativity, HSCT did not improve the survival outcomes (HSCT vs Non-HSCT, 3-year rates 76.8% vs 65.7%, p=0.078). The next-generation sequencing (NGS) results were available in 520 patients. Based on the 2022 ELN risk stratification, the 3-year OS rates were 60.3%, 44.2% and 35.0% in favorable, intermediate and adverse group, respectively. Moreover, there were 43, 47, 60 patients in favorable, intermediate, and adverse subgroups underwent HSCT subsequently. In intermediate risk subgroup, patients who failed to achieve CRc after the first induction therapy, HSCT may serve as an effective intervention to prolong survival (3-year rates 100% vs 12.1%, P<0.001,). On the contrary, patients who achieved CRc in induction therapy, the overall survival were not influenced by HSCT status (3-year rates 81.5% vs 31.7%, p=0.153, Figure B). Conclusion: Our finding indicated that remission after the first cycle of induction therapy may be a prognostic factor for OS. Moreover, earlier and deeper remission may lead to improved survival. For those patients in intermediate risk group, who achieved CRc in induction therapy or achieved CRc with MRD negativity within re-induction therapy, HSCT may not influence the survival outcome.

A Universal CRISPR/Cas9n-Mediated Genome Editing Approach for ELANE-Related Severe Congenital Neutropenia

Severe congenital neutropenia (CN) is an inherited bone marrow failure syndrome characterized by impaired maturation of neutrophil granulocytes. Due to the lack or very low levels of neutrophils in the peripheral blood, patients experience severe, life-threatening bacterial infections as early as birth. Autosomal-dominant ELANE mutations are the most common cause of CN. Although most patients respond to daily treatment with recombinant human granulocyte colony-stimulating factor, about 15% of patients do not respond to this cytokine, and approximately 20% of patients develop myelodysplasia or acute myeloid leukemia. The only curative treatment available thus far is allogenic hematopoietic stem cell transplantation, which is associated with serious side effects. Autologous transplantation of gene-edited patients' HSPCs offers a novel curative therapy. Considering the wide range of mutations distributed throughout the ELANE gene, A universal, high-safety-profile approach could be advantageous for all ELANE-CN patients. Our group has already published a universal CRISPR/Cas9-mediated knockout approach for the ELANE gene; however, targeting the coding sequence region (CDS) of the ELANE gene could lead to the introduction of new disease-causing variants, so it may be harmful for patients. In order to address this concern, we reformatted our previously published knockout concept into a clinical-grade gene therapy strategy. We have screened cis-regulatory regions in the ELANE core promoter and, using CRISPR/Cas9n, introduced two nicks on opposing strands of the Goldberg-Hognes box region (TATA-box) to make the transcription starting process inefficient, as transcription factors and RNApol II cannot recognize the modified sequence. Theproposed strategy targets the non-coding region of the ELANE gene and thus does not generate new unwanted variants. Also, by replacing Cas9 with Cas9-ncikase, the off-target activity should decrease by up to 1000-fold. We have observed a markedly elevated neutrophil differentiation in gene-edited ELANE-CN patients HSPCs (n = 2), as assessed by the percentage of CD45 +CD11b +CD15 +, CD45 +CD15 +CD16 +, and CD45 +CD16 +CD66b + myeloid/granulocytic cells, compared to the mock electroporated ELANE-CN group, while no disruption of granulopoiesis in healthy donor HSPCs (n = 2) was detected. Results validated by the CFU assay. To have nucleotide-level resolution of on-target events, we performed targeted next-generation sequencing of edited loci on gene-edited healthy donors and ELANE-CN HSPCs. Next-generation sequencing results showed >90% on-target efficiency. CRISPR/Cas9n-mediated targeting of the TATA box rescued defective granulopoiesis in vivo, as gene-edited ELANE-CN HSPCs (n = 3) were transplanted in NSG mice and assessed by the percentage of neutrophils (hCD19 -hCD3 -hCD66b Int/lowhCD33 +hCD16 high) after 16 weeks. We have also observed efficient engraftment and preserved the multilineage potential of gene edited HSPCs in immunodeficient NSG mice. The strategy also depicted a safe profile upon applying gene editing to healthy donor CD34 + HSPCs and performing GUIDE-seq and CAST-seq. GUIDE-seq highlighted two potential off-target sites after >=6 mismatches, while no chromosomal translocations were detected by CAST-seq. RNA-sequencing of in vitro-generated neutrophils from gene-edited HSPCs of healthy donors (n = 2) confirmed a >9-fold reduction of ELANE gene expression level. Important to note, none of the other serine proteases or neighboring genes were down-regulated. Taken together, ex vivo CRISPR-Cas9n-based gene editing of ELANE's gene promoter in the setting of autologous stem cell transplantation could be a safe therapeutic approach for all ELANE-CN patients.

CORR Insights®: Next-generation Sequencing Results Require Higher Inoculum for Cutibacterium acnes Detection Than Conventional Anaerobic Culture.

CORR Insights®: Next-generation Sequencing Results Require Higher Inoculum for Cutibacterium acnes Detection Than Conventional Anaerobic Culture.

Expanding the genetic and clinical spectrum of osteogenesis imperfecta: identification of novel rare pathogenic variants in type I collagen-encoding genes.

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous skeletal disorder. The majority of affected cases are attributed to autosomal dominant pathogenic variants (PVs) found in the COL1A1 and COL1A2 genes, which encode type I collagen. However, PVs in other genes involved in collagen posttranslational modification, processing, crosslinking, osteoblast differentiation, and bone mineralization have also been associated with OI. In this study, we present the results of next-generation sequencing (NGS) analysis using a custom panel of 11 genes known to be associated with OI. This clinical study enrolled a total of 10 patients, comprising 7 male and 3 female patients from 7 families, all from the Puglia Region in South Italy, providing a detailed overview of their age, gender, family history, OI type, and non-skeletal features. The genetic analysis revealed 5 PVs in the COL1A1 gene and 2 PVs in the COL1A2 gene. Importantly, three of these PVs have not been previously reported in the literature. These include two novel heterozygous frameshift PVs in COL1A1 (c.2890_2893del and c.3887del) and one novel heterozygous missense PV in COL1A2 (c.596G>T). The identification of these previously unreported PVs expands the variant spectrum of the COL1A1 and COL1A2 genes and may have implications for accurate diagnosis, genetic counselling, and potential therapeutic interventions in affected individuals and their families.

Molecular oncology and the role of artificial intelligence in advancing cancer treatment

Artificial intelligence (AI) holds significant promise for advancing molecular oncology and improving personalized cancer care. This review highlights the numerous benefits of AI integration in various aspects of molecular oncology, from data analysis and interpretation to streamlining clinical trial matching. AI systems can aid clinical decision-making by rapidly analyzing complex molecular data, such as next-generation sequencing results, and suggesting treatment options based on the patient’s tumor profile. Furthermore, AI can facilitate collaboration among healthcare professionals, monitor treatment response, and serve as a valuable educational resource for oncologists. The incorporation of AI in electronic health records and pharmacogenomics can lead to improved clinical workflows and more personalized therapeutic approaches. In addition, AI can enhance precision oncology research by assisting in the identification of novel molecular targets and uncovering new therapeutic strategies. As AI technology continues to evolve, its role in molecular oncology is expected to expand, leading to better patient outcomes, and more personalized care. Nevertheless, ethical considerations and patient privacy remain crucial aspects that need to be addressed to ensure the responsible and effective use of AI in the field of molecular oncology.

B-253 Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing

Abstract Background High-resolution human leukocyte antigen (HLA) typing is a crucial process in transplant matching. Sequencing-based typing (SBT) has been the gold standard method used in HLA-based clinical applications. Recently, next-generation sequencing (NGS) has been adopted in clinical laboratories for HLA typing. NGS HLA typing assays cover whole or near-whole target HLA genes, allowing the detection of genetic variants outside of key exons. Multiplex amplicon-based NGS is an efficient high-resolution HLA typing method with minimal ambiguity and a high throughput. Here, we report the performance and validation of NGS for HLA typing and compare the results obtained using NGS with those of SBT to identify any discrepancies between the 2 methods. Methods A total of 237 samples from 2019 to July 2022 were collected from Chang Gung Memorial Hospital, including 42 samples previously subjected to SBT sequencing for 4-digit HLA analysis. NGS was performed for 5 whole genes (HLA-A/B/C/DQA1/DPA1) and 3 near-whole genes (HLA-DRB1/DQB1/DPB1; excluding exon 1 and part of intron 1) by using the AllType NGS kit (One Lambda), Illumina MiSeq platform, and Type Steam Visual NGS analysis software (One Lambda). Results Our validation process comprised 34 NGS runs and 237 samples. In total, 42 samples were analyzed with an overall accuracy and precision of 100% and 100% reproducibility. The overall ambiguity rate was 0% in HLA-A/B/C compared with 56.9% for SBT. Two discrepant alleles of HLA-A and DRB1 were noted in NGS and Sanger sequencing. In accordance with additional SBT exon analyses, one of the results was corrected according to the NGS results. Conclusion NGS HLA typing is a high-throughput approach with high accuracy and precision and a low ambiguity and is promising for use in clinical HLA laboratories.