Bessonen, Kurt

Abstract Background: Clonal cytopenia of undetermined significance (CCUS) is defined as clonal hematopoiesis in the absence of a hematologic neoplasm, along with unexplained cytopenia. While several studies have identified risk factors and developed scores to predict progression to overt myeloid neoplasms, the clinical and molecular features during the progression process remain poorly understood. Aim: Evaluate the clinical and clonal characteristics of patients with CCUS, with a focus on the longitudinal evolution of clonal and clinical parameters over time. Methods: After IRB approval, we retrospectively reviewed charts of patients with unexplained cytopenia (2015-2024), recording data at the time of CCUS diagnosis (WHO 2022 diagnostic criteria) and at time of follow-up bone marrow biopsy after diagnosis. Follow-up CBC and karyotype data were recorded for patients who had repeat bone marrow biopsies (n= 111), while follow-up clonal parameters and risk scores were calculated for those who underwent repeat NGS (n= 74). A reduction or expansion in clone size was defined as a change exceeding 5% (CE5%), or change exceeding 10% (CE10%) from the baseline value. Overall survival (OS) was calculated from diagnosis to last follow-up. For statistical analysis, we used BlueSky Statistics V10.3.1. Results: 214 CCUS patients (median age 73, 68% males) were included. At baseline, 30% of patients had abnormal cytogenetics, with del (20q) being the most common one (8%; n= 18), followed by trisomy 8 (6%; n= 13), and loss of chromosome Y (5%; n= 10). The median number of mutations was 2 (range, 1-5), with the most common mutations TET2 (38%), SRSF2 (21.5%), ASXL1 (21%), ZRSR2 (18.5%), DNMT3A (9.5%), and U2AF1 (9%). Most patients had high risk CHRS (60%), followed by intermediate (30%), and low risk (10%). On the other hand, 41% had high risk CCRS, 29% had intermediate risk, and 30% had low risk. After a median follow-up of 46 months, 62 patients died (median OS not reached). A decrease in the white blood cell (WBC) count from the time of diagnosis to follow-up was more frequently seen among CCUS patients who progressed to MN (70% vs 50%, p= 0.04), however, changes in hemoglobin and platelets did not differ between those who progressed and those who did not (p> 0.05). Although the frequency of abnormal karyotypes at diagnosis was similar between patients who eventually progressed (28%) and those who did not (31%) (p= 0.6), 4 (2%) patients with initially normal karyotypes acquired chromosomal abnormalities during follow-up, and all progressed to MDS. Among the 74 patients who had follow-up NGS, 31% acquired new mutations, clonal expansion of pre-existing mutations were seen in 54% using CE5% and 19% per CE10%, and 16% developed new variants of uncertain significance (VUS). Acquiring new pathogenic mutations was similar in patients who progressed to MN compared to those who did not progress (34% vs 28%, p= 0.6). On the other hand, clonal expansion was significantly more observed in patients who progressed compared to those who did not, using both CE5% (73% vs 31%, p = <0.001) and CE10% (27% vs 9%, p= 0.03). At follow-up, ASXL1 was the most commonly acquired mutation among patients who did not progress (4 cases), followed by TET2 (2 cases), and U2AF1, GATA2, DNMT3A, SF3B1, ZRSR2, TP53, and CBL (1 case each). On the other hand, SRSF2, SETBP1, CBL, JAK2, TET2, and RUNX1 were more frequently acquired among those who progressed to myeloid neoplasms (each appeared in 2 patients). When recalculating CCRS, CHRS, IPSS-M, and IPSS-R at follow-up for patients who had repeat NGS, there was no significant difference in score changes - either upward or downward - between those who progressed and those who did not (p> 0.05). Conclusion: In CCUS patients who progressed to myeloid neoplasms, WBC decline, clonal expansion (more frequent), and evolving chromosomal aberrations (less frequent) were observed, supporting the value of longitudinal genomic and clinical monitoring in CCUS.

Abstract Prognosis in acute myeloid leukemia (AML) patients is influenced by various factors, most importantly, patient clinical status and cytogenetic/molecular abnormalities. Within those factors, elevated lactate dehydrogenase (LDH) is considered a poor prognostic indicator in AML patients. Our study aims at exploring the different factors associated with elevated LDH and their impact on overall survival (OS). After obtaining IRB approval, we reviewed the charts of 381 AML patients diagnosed at Mayo Clinic, Rochester, between the years of 2016 and 2023. All data was recorded at the time of AML diagnosis. Patients with no cytogenetic and molecular data were excluded from this study. LDH levels were recorded if measured within 10 days of diagnosis and treatment for AML was not yet given. Both risk stratification and response evaluation were done following the 2022 European LeukemiaNet (ELN) guidelines. OS was calculated from the date of AML diagnosis to the date of last follow-up or death. We used BlueSky Statistics V 10.3.4 for statistical analysis. We identified 299 patients (78.4%) who had elevated LDH levels (normal ≤ 222 U/L) at the time of diagnosis with a median of 417 U/L (range, 223-7989). The median age for those patients was 68 years (range, 18-94), with 78 (26.1%) of them having a prior myeloid disease (MDS, MPN, or MDS/MPN) before AML diagnosis. By risk stratification, more than half of the patients had an adverse risk profile (n=171, 57.2%), while 71 (23.7%) and 57 (19.1%) had an intermediate and favorable risk, respectively. Most of those patients had an abnormal karyotype (n=204, 68.7%), with complex karyotype (n=103, 34.7%) being the most common abnormality, followed by monosomy 7 (n=50, 16.8%) and trisomy 8 (n=46, 15.5%). Next-generation sequencing (NGS) was performed on 268 (89.6%) patients with elevated LDH, and the most common mutations were TP53 (n=60, 22.3%), FLT3-ITD (n=59, 21.1%), and NPM1 (n=51, 18.5%). TP53 mutations were more common in patients with normal/low white blood cell count (WBC) in contrast with FLT3 and NPM1, which were the most common mutations in patients with high WBC. After grouping the patients according to their LDH levels (group 1: 223-444 U/L, group 2: 444-666 U/L, group 3: > 666 U/L), we found that FLT3-ITD mutations were more likely to occur at higher LDH levels i.e. groups 2 and 3 (p= 0.002). The majority of patients with elevated LDH (n=262, 87.6%) received therapy following diagnosis, with the most common treatment regimens being a combination of a hypomethylating agent and venetoclax (HMA+VEN) (n=97, 37%) and a (7+3)-based induction therapy (n=77, 29.4%). When comparing patients with elevated LDH (n=299, 78.4%) to patients who had non-elevated LDH (n=82, 21.5%), no difference in both median age at diagnosis and risk stratification was found. We noticed an increase in WBC (median, 7.7 vs. 2.1) (p<0.001) along with an increase in peripheral blasts (median, 28% vs. 7%) (p<0.001) in elevated LDH patients. Among patients with non-elevated LDH, 21% (n=17) had a complex karyotype compared to 34.7% (n=103) in the elevated LDH group (p=0.022). The molecular profile was different between the 2 groups, where the most common mutations in patients with non-elevated LDH were IDH2 (n=19, 25.3%), RUNX1 (n=17, 23%), and DNMT3A (n=16, 21.6%). All patients (n=82, 100%) in the non-elevated LDH group received therapy after diagnosis. There was no difference in terms of treatment regimens used between the 2 groups. Moreover, no statistical difference in remission rates after one line of therapy was found between those groups where non-elevated LDH patients had a 65.9% remission rate compared to the 55.3% remission rate in patients with elevated LDH (p=0.098). Patients with elevated LDH had a shorter OS than patients with non-elevated LDH (9.5 vs. 26.4 months, p=0.0008). However, median OS was similar across the different groups of elevated LDH. Using univariate analysis in our cohort (n=381), age, HSCT, risk stratification, FLT3 mutation, and LDH elevation all had an impact on OS. However, when comparing those variables using multivariate analysis, age and FLT3 mutation lost their significance (p-value of 0.25 and 0.84, respectively).Complex karyotype and FLT3 mutations were more common in patients with elevated LDH. These patients tend to have higher WBC and peripheral blasts. Elevated LDH had a negative impact on patients' OS regardless of patients' risk classification.

Background: SETBP1 gene, located on 18q12.3, is a major oncogene in myeloid neoplasms. GATA2 gene, located on 3q21, is one of the six GATA transcription factors regulating gene expression via two conserved zinc finger domains (ZF). Previous data suggested that in patients with germline GATA2 mutation (m), acquisition of a somatic SETBP1 mutation (m) was associated with leukemic transformation among patients with excess blast MDS and CMML. Aim: To study the characteristics of patients with somatic SETBP1-GATA2 mutated myeloid neoplasms. Methods: This study is a retrospective study at Mayo Clinic. After obtaining IRB approval, we retrospectively reviewed the charts of patients who had myeloid NGS panel results between 2016-2023. All data were recorded at time of first NGS. Cohort 1 was defined as patients with SETBP1m and GATA2m, cohort 2; patients with SETBP1m and GATA2wt, and cohort 3; patients with SETBP1wt and GATA2m. Germline work-up was not performed in the majority of GATA2 cases. We stratified risk based on the European LeukemiaNet 2022 guidelines for AML, IPSS-M for MDS and MDS/MPN, and CPSS-M for CMML. Overall survival (OS) was calculated from date of NGS to date of last follow-up based on Kaplan-Meier estimates and Cox regression analysis. For statistical analysis, we used BlueSky Statistics V10.3.1. Results: General characteristics: 114 patients had SETBP1m (median VAF= 39%), and 63 patients had GATA2m (median VAF= 35%). Nine patients had co-occurring SETBP1m/GATA2m mutations (cohort 1). 4/9 patients in cohort 1 had MDS/MPN (44.4%), while only 13/105 in cohort 2 (12.4%) had MDS/MPN (P=0.02). AML was seen in 2 patients in cohort 1 (22.2%) compared to18 in cohort 2 (17.1%) (p=0.6). The median WBC count was 22.9 x 109 cells/L among cohort 1 compared to 9 x 109 cells/L among cohort 2 cases (p=0.09). BM blasts were higher in cohort 3 (7%) compared to cohort 1 (5%) and cohort 2 (3%) (p=0.09), while peripheral blasts tended to be lower in cohort 2 (1%) in comparison to cohort 1 (6%) and cohort 3 (4%) (p=0.48). Genetic characteristics: In cohort 1,7 GATA2m clustered in zinc finger 2 (ZF2) (77.8%), while the other two mutations were outside the ZF domains (22.2%). This was higher than the ZF2 mutated cases in cohort 3 (46.3%, p=0.1). Abnormal cytogenetics were seen in 66.7% of cohort 1 patients and 45% of cohort 2 (p= 0.3). Two cases with trisomy 8 were seen among patients in cohort 1 (22.2%), compared to only 4.9% in cohort 2 (p=0.09). Two incidences of isochromosome 17q [i(17q)] were seen in cohort 1 (22.2%), versus 5 cases in cohort 2 (4.8%) and 1 case in cohort 3 (1.9%) (p= 0.068). Co-mutations: The median number of co-mutationswas 3 in both cohort 1 and cohort 2 (p= 0.6). In cohort 1, ASXL1 and SRSF2 mutations were the most common (7/9, 77.8%) followed by NRAS (22.2%) and TET2 (22.2%). 77.8% of cohort 1 patients had SRSF2 co-mutation, compared to 44.8% in cohort 2 (p=0.08), and 27.8% in cohort 3 (p= 0.006). No isolated SETBP1m/GATA2m cases were found. Survival: Patients in cohort 1 had a numerically higher median OS compared to those in cohort 2 (19.4 vs 15.4 months, p= 0.3). When comparing with the cohort 3, patients in cohort 1 tended to have better outcomes (median OS= 19.4 vs 13.8 months, p=0.8). Among cohort 1, SRSF2 co-mutation was associated with worse survival outcomes compared to patients with SRSF2wt (median OS= 5.3 vs 19.4 months, p= 0.02); this association was not found in cohort 2 (12.5 vs 18.4 months, p= 0.3) and cohort 3 (13.2 vs 17.1 months, p= 0.4). Allogeneic hematopoietic cell transplantation was associated with favorable survival outcomes in all cohorts (cohort 1: 22.6 vs 7.8 months, p= 0.1; cohort 2: 20.9 vs 12.5 months, p= 0.04; cohort 3: 25.6 vs 13.1 months, p= 0.03). Conclusion: SETBP1m/GATA2m myeloid neoplasms included a significantly higher proportion of MDS/MPN overlap cases in comparison with the SETBP1m/GATA2wt group. The SETBP1m/GATA2m group had a higher proportion of ZF2 GATA2m and SRSF2 co-mutation compared to patients with SETBP1wt/GATA2m. Survival of SETBP1m/GATA2m patients was not worse compared to SETBP1wt/GATA2m or SETBP1m/GATA2wt.

Abstract Background In complex automated clinical laboratory Next Generation Sequencing (NGS) workflows, numerous opportunities exist for contamination events to occur. For oncology testing applications, detection of these events is crucial for accurate results reporting. A contamination detection module (MICon) using microhaplotype (MH) regions and an in-house designed analysis model was developed for use in a NGS assay for myeloid neoplasms (NGSHM) [1]. Variant detection for NGSHM has a validated analytical sensitivity of 2% variant allele frequency (VAF). Gross contamination events can potentially cause erroneous false positive variant detection whereas true low VAF mutations may be masked and evade detection. The following study was performed to evaluate MICon module performance on samples processed over the course of a year. Methods MH regions contain highly conserved tandem single nucleotide polymorphisms (SNPs) with high global heterogeneity occurring within a 300-nucleotide span. From the work of Kidd et al [2], 27 MH regions covering 92 SNPs and spanning 16 chromosomes were selected for inclusion in the 47 gene NGSHM target-capture panel. Sequencing was performed on a NovaSeq 6000 and analyzed through an internal bioinformatics pipeline, which incorporates the MICon module. MICon uses a binary classification model incorporating VAFs from MH regions, number of MH genotypes, and the contamination estimation score from verifyBamID to compute a single value on a 0-100 scale. Scores <50 are classified as non-contaminated and scores ≥50 require further investigation. Results The dataset was comprised of 10,990 samples. A total of 10,232 patient cases (93.1%) had non-contaminated MICon scores <50. Control replicates accounted for 261 samples (2.4%). There were 497 patient cases (4.5%) with MICon scores ≥50. Of the 497 patient cases, 137 (27.6%) had undergone hematopoietic stem cell transplantation (HSCT) and 114 (22.9%) contained gross chromosomal abnormalities, representing iatrogenic or tumor biological factors. An additional 135 (27.2%) of cases were contaminated from laboratory processing errors, 45 (9.1%) occurred due to chemistry failures during run processing, and 66 (13.2%) had an undetermined cause for a high score, representing laboratory-related and unknown factors. Overall, the 135 laboratory processing errors detected accounted for 1.3% of patient cases analyzed by the laboratory. These cases were repeated, and the non-contaminated results were accurately reported. Conclusions Herein it was shown the implementation of MICon has improved patient safety over the course of a year. The 1.3% of patient cases identified as arising from laboratory processing errors were successfully reprocessed. While the overall rate of laboratory processing errors appears low, the absolute number can be significant in a high-volume test setting, despite reliance on highly automated processes. Value is added by MICon through identifying errors that could result in the release of inaccurate patient results. By detecting previously unrealized errors, MICon has become an invaluable asset to the NGSHM assay by enhancing patient laboratory testing safety. References: [1] Balan J, et al. MICon Contamination Detection Workflow for Next-Generation Sequencing Laboratories Using Microhaplotype Loci and Supervised Learning. J Mol Diagn. 2023;25(8):602-610. doi:10.1016/j.jmoldx.2023.05.001. [2] Kidd KK, et al. Selecting microhaplotypes optimized for different purposes. Electrophoresis. 2018;39(21):2815-2823. doi:10.1002/elps.201800092.

Abstract The ZRSR2 gene is a member of the spliceosome gene family which are frequently mutated in myeloid neoplasms. ZRSR2 mutations (ZRSR2m) occur in less than 5% of MDS, CMML, and AML. Our study included 164 ZRSR2m patients (98.8% males) and 98 ZRSR2wt MDS control cohort. In the ZRSR2m group, there were 53 MDS (32%), 39 CCUS (24%), 33 MPN (20%), 23 MDS/MPN overlap (14%), 15 AML (9%), and 1 MPAL (0.6%). Most MDS patients were the low blast subtype (n=37, 70%). Twenty-seven MDS patients (51%), and 23 CCUS patients (59%) had absolute monocyte count ≥0.5 x109/L and 18 of 23 MDS/MPN overlap were CMML (78%). Mutations in ZRSR2 were spread across the entire gene. The median number of co-mutations was 2, with TET2 (51%) and ASXL1 (32%) being the most common. U2AF1 and SRSF2, previously described as mutually exclusive with ZRSR2, were found in 10.4% of patients. Median overall survival (OS) was 51 months, and significantly varied among MNs (p=0.004). ZRSR2m MDS patients had better mOS than the MDS control cohort with ZRSR2wt (35 vs 22 months, p=0.013). ZRSR2m patients with isolated TET2 co-mutation and higher hemoglobin showed improved survival, while patients with RUNX1m, higher WBC count showed worse OS.