Autologous hematopoietic stem cell transplantation (AHCT) is an accepted strategy for various hematologic malignancies that can lead to functional impairment, fatigue, muscle wasting, and reduced quality of life (QOL). In cancer cachexia, these symptoms are associated with inflammation, hypermetabolism, and decreased anabolic hormones. The relative significance of these factors soon after AHCT setting is unclear. The purpose of this study was to characterize the acute effects of AHCT on physical function, body composition, QOL, energy expenditure, cytokines, and testosterone. Outcomes were assessed before (PRE) and 30 ± 10 days after (FU) AHCT in patients with multiple myeloma (n = 15) and non-Hodgkin lymphoma (n = 6). Six-minute walk test (6MWT; p = 0.014), lean mass (p = 0.002), and fat mass (p = 0.02) decreased; nausea and fatigue increased at FU (both p = 0.039). Recent weight change and steroid exposure were predictors of reduced aerobic capacity (p < 0.001). There were no significant changes in interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF), energy expenditure, or bioavailable testosterone. Alterations in cytokines, energy expenditure, and testosterone were not associated with functional impairment acutely following AHCT. Recent history of weight loss and steroid exposure were predictors of worse physical function after AHCT, suggesting that targeting nutritional status and myopathy may be viable strategies to mitigate these effects.

Sensitive and specific detection of low frequency mutations in acute myeloid leukemia (AML) is critical in research of minimal residual disease (MRD). At variant allele frequencies (VAF) below approximately 1%, PCR and sequencing errors result in prohibitive signal-to-noise ratios with next generation sequencing (NGS). Duplex Sequencing (DS) relates the original top and bottom DNA strands to make double stranded consensus sequences to greatly reduce errors. In two experiments we show further improvement of DS of AML-related genes by the use of enzymatic fragmentation (EF) and an updated gene panel that incorporates 2022 European LeukemiaNet (ELN) recommendations. In the first DS study, 29 AML-related genes (59 kb) were targeted in hybrid capture. Mutant cell line DNA was mixed into DNA from a healthy young donor to simulate MRD. Expected VAFs in mixtures were 1.0-0.003%. Samples comprised a mix of 4 insertions and deletions (indel), a mix of 15 single nucleotide variants (SNV), and 4 serial dilutions of a FLT3 ITD plus an NPM1 insertion. Pure diluent DNA was used as a negative control. Each sample was prepared in quadruplicate with mechanical fragmentation (MF) vs EF. DNA input mass was 1,500 ng per replicate, except for 50-250 ng for the FLT3/NPM1 mixes with expected VAF of 1% and 0.1%, respectively. Input masses were set to ensure &gt;95% probability of detection of all mutations in the combined data. All targeted variants down to 0.003% VAF were detected in spike-in mixtures, with expected vs observed VAF highly correlated whether using MF or EF (R2 &gt;0.98 for all mixtures). Duplex molecular depth was 1.2-2.0x higher with EF vs MF across input masses. Panel-wide mean duplex depth per 1,500 ng replicate was 30,376x for MF and 48,036x for EF, for combined mean depths of 121,508x for MF and 192,144x for EF. In the negative control, background mutational calls at spike-in positions were 4/2,993,429 duplex bases using MF and 0/4,780,491 duplex bases with EF, reflecting increased specificity with EF. Next, a revised panel targeting 36 AML-related genes (80 kb) was used for DS with EF. Here differing mutant cell line DNA was mixed into the same diluent from above (the negative control). This mixture harbors 27 variants with expected VAFs of 0.125-0.006% (4 indels, 21 SNVs, a FLT3 ITD and an NPM1 insertion), 17 of which (2 indels, 14 SNVs and the NPM1 insertion) span VAFs of 0.011-0.009% to establish a limit of detection of 0.01%. With 2,000 ng (+/- 10%) input, for 14 samples, panel-wide mean duplex depth was 56,528x. For variants with expected VAF ≥ 0.009%, the assay had &gt;98% sensitivity, &gt;96% specificity, and &gt;98% accuracy at genomic positions of known true positive variants. In summary, DS with EF yields more data per nanogram of DNA than MF, maximizing the use of precious samples. This method exhibits extremely low background mutation signal and a low limit of detection across targets that are valuable in AML MRD research. Citation Format: Elizabeth Schmidt, Devon M. Fitzgerald, Camila Zanette, Gavin D. Meredith, Mark A. McElwain, Raul Burciaga, Kevin C. Vavra, Thomas H. Smith, Jesse J. Salk, Jake Higgins. Accurate detection of low frequency AML-associated mutations in vitro using Duplex Sequencing with enzymatic fragmentation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB254.

Background: Hematopoietic stem cell transplantation (HCT) is a potentially curative treatment for hematologic malignancies, but is associated with negative side effects, including reduced sexual function. It was recently reported that estradiol may exert an effect on sexual desire & erectile function, independent to that of testosterone, in healthy men. We hypothesized that changes in estrogen would be directly associated with changes in sexual function in men undergoing HCT. Methods: Nineteen men planning autologous HCT completed the International Index of Erectile Function (IIEF) & provided a morning, fasted blood sample at Baseline (before initiating preparatory chemotherapy) & 30±10 days post-HCT (FU). IIEF contains sub-categories of Erectile Function (EF), Orgasmic Function (OF), Sexual Desire (SD), Intercourse Satisfaction (IS), & Overall Satisfaction (OS). Plasma samples were analyzed for total testosterone (TT), dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), sex-hormone binding globulin (SHBG), 17-OHprogesterone, 17-OHpregnenelone, androstenedione, androsterone, pregnenolone, progesterone, estrone (E1), & estradiol (E2) via LCMS; bioavailable (BT) & free T (FT) were calculated using local laboratory-derived albumin levels. Follicle-stimulating hormone (FSH) was analyzed via ELISA. Non-parametric, paired t-tests compared Baseline to FU & multiple regression identified significant predictors of sexual function changes using forced variables (age, HCT-comorbidity index (CI), cumulative steroid exposure, post-HCT testing day, diagnosis) & conditional variables (change scores for each hormone & relative fat mass change). Data are median ± SEM. Results: Men were 68±3 years of age (multiple myeloma, N=15; leukemia, N=6). Significant changes were observed at FU for E2 (-5.2±1.9 pg/mL, p=0.048), E1 (-6.6±2.2 pg/mL, p=0.002), FSH (4.6±0.9 IUL, p=0.002), TT (1.3±0.4 ng/mL, p=0.02), SHBG (41.2±5.5 nmol/L, p=0.001), DHT (0.06±0.03 ng/mL, p=0.03), androsterone (0.02±0.02 ng/mL, p=0.045), androstenedione (0.54±0.16 ng/mL, p=0.001), progesterone (0.02±0.01 ng/mL, p=0.01), & 17-OHprogesterone (0.27±0.08 ng/mL, p=0.001). Sexual function significantly decreased at FU for EF (p=0.003), OF (p=0.01), SD (p=0.009), IS (p=0.03), & IIEF Total (p=0.004). There were no significant predictors of OF, SD, or OS change scores. Diagnosis, HCT-CI, & change scores for E2 & 17-OHpregnenolone were significant predictors of EF change (F=13.3, p=0.005). Age, diagnosis, HCT-CI, & change scores for E2 androstenedione were significant predictors of IS change (F=14.5, p=0.004). Age, diagnosis, HCT-CI, & change scores for E2 & DHEA were significant predictors of Total IIEF changes (F=20.1, p=0.002). Conclusion: Acute changes in sexual function after HCT are likely influenced by HCT-induced reductions in estradiol, independent of changes in testosterone.