While pharmacogenetic testing has traditionally relied on array-based genotyping platforms, these methods are limited by incomplete variant coverage and inability to detect novel alleles. We hypothesize that the performance of genome sequencing (GS) is superior to that of an array-based genotyping method for gene-drug pairs that are clinically implemented at the NIH Clinical Center. DNA was collected from a cohort of 293 patients from a single-center prospective cohort study at the National Institutes of Health, Bethesda, MD. Post hoc analysis was conducted in probands with genetically inferred ancestry: European, African, African American, and Asian. The primary endpoint was to determine concordance between GS, exome sequencing (ES), and a commercial genotyping array while assessing accuracy of non-concordant alleles using orthogonal sequencing. In a cohort of 293 individuals genotyped with a commonly used array, GS (n = 120) and/or ES (n = 185) was conducted. GS demonstrated superior accuracy, resolving unknown or ambiguous array-based allele calls in 5% of cases, ultimately achieving 99% baseline accuracy. Moreover, we confirmed discordances between array-based calls and GS in favor of GS through validation by orthogonal sequencing methods such as long-read sequencing. We highlight clinically significant inaccuracies that include critical variants missed by arrays and ES. From a cost perspective, GS proved comparable or superior to array-based methods, with significantly greater clinical applicability and flexibility. Our findings underscore the feasibility and superiority of integrating GS-based pharmacogenetics testing into clinical settings, demonstrating robust analytical performance, improved patient management potential, and a pathway toward broader, lifetime utility of genomic data.

3027 Background: Extrapulmonary Small Cell Carcinoma (EPSCC) is a rare (0.1–0.4% of all cancers), aggressive neuroendocrine cancer with poor outcomes (Median PFS 2-4 months), no established second-line standard, and limited prospective data. We evaluated ATR inhibition with berzosertib combined with topoisomerase I (TOP1) inhibition with topotecan or sacituzumab govitecan (SG) and explored molecular correlates of response. Methods: Given the rare nature of EPSCC, patients were pooled from 3 prospective phase II trials evaluating topotecan or SG with berzosertib (NCT03896503, NCT02487095, NCT04826341) without intent for direct regimen comparison. Eligible patients had platinum-refractory EPSCC and received treatment at recommended phase II doses. Endpoints included efficacy and safety, with exploratory molecular correlates including somatic mutations, copy number alterations, circulating tumor DNA, tumor RNA sequencing, and pharmacogenomic analyses. Results: Of the 41 patients enrolled, 34 were evaluable (topotecan+berzosertib, n=20; SG+berzosertib, n=14). ORR based on RECIST was 10% (95% CI, 2.8–30.1) with topotecan+berzosertib and 21.4% (95% CI, 7.6–47.6) with SG+berzosertib; disease-control rates were 45% and 86%, respectively. Durable clinical benefit was observed in subsets of patients, with PFS ≥6.8 months and OS up to 19.7 months in the SG cohort, and OS >18 months in patients treated with topotecan+berzosertib. Patients with prolonged PFS represented multiple primary sites, including bladder, prostate and laryngeal. Grade 3–4 hematologic adverse events were more frequent with topotecan+berzosertib, whereas gastrointestinal toxicity and alopecia were more common with SG+berzosertib; no unexpected safety signals were observed. In the SG cohort, UGT1A1 intermediate and poor metabolizers experienced higher rates (90%) of grade 3–4 toxicities than normal metabolizers (10%). Early ctDNA declines correlated with radiographic response, while rising ctDNA preceded progression; baseline ctDNA correlated with tumor burden (r = 0.83; P = 0.006). Transcriptomic analyses demonstrated enrichment of E2F-driven and neuroendocrine lineage programs in responders (NES = 1.64–1.67; P < 0.001). Conclusions: In relapsed or refractory EPSCC targeting tumor replication stress through combined ATR and TOP1 pathway inhibition yielded clinically meaningful activity. Tumor-targeted TOP1 delivery with SG plus berzosertib achieved disease-control rate and a manageable safety profile. UGT1A1 phenotype was associated with toxicity in the SG cohort, supporting the relevance of pharmacogenomic-guided risk stratification. Correlative analyses identified E2F-driven transcriptional programs, neuroendocrine lineage signatures and early ctDNA suppression as promising biomarkers for future trials. Clinical trial information: NCT03896503 , NCT02487095 , NCT04826341 .

BACKGROUND:Hematopoietic cell transplantation (HCT) provides effective long-term management for some inborn errors of immunity. Genetic findings can inform donor selection, considerations in conditioning intensity and agents, and graft-versus-host disease prophylaxis. Exome/genome sequencing is increasingly accessible but of uncertain clinical utility. We aimed to evaluate the clinical utility of comprehensive genomic evaluations through review of HCT at our center. METHODS:We performed exome/genome sequencing on pre-HCT samples from participants between 2017 and 2023. We reported primary findings (PF) and secondary findings (SF). Post hoc, we analyzed medication and pharmacogenetic (PGx) data. RESULTS:We analyzed pre-HCT exome/genome sequencing (n = 84 exome, n = 63 genome, n = 32 with both) for 179 probands. Most (143/179; 79.9%) had a PF underlying the HCT indication, with GATA2 being most common (n = 59). Three percent of participants had an SF predisposing to cancer or cardiovascular disease. Most (n = 108/179; 60.3%) received ≥1 medication(s) that may have been further optimized with PGx. Using Kaplan-Meier survival analysis, we compared the survival rates of participants with 0, 1, and ≥2 genomic risk factors (GRF: absence of PF; presence of SF or PGx). Survival at 3 y was 94.8%, 84.8%, and 58.5% for those with 0, 1, and ≥2 GRF, respectively (log-rank: 16.10, df = 2, P  = 0.0003), indicating statistically significant survival differences by GRF. CONCLUSIONS:Comprehensive genomic evaluation is an emerging avenue for tailoring HCT approaches, and identification of HCT-relevant findings may be common. On multivariate analysis, GRF was associated with survival in this retrospective cohort. Prospective research is warranted to further integrate genomic data into precision treatment.

AbstractUnderrepresented populations’ participation in clinical trials remains limited, and the potential impact of genomic variants on drug metabolism remains elusive. This study aimed to assess the pharmacokinetics (PK) and pharmacogenomics (PGx) of ribociclib in self-identified Black women with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2) advanced breast cancer. LEANORA (NCT04657679) was a prospective, observational, multicenter cohort study involving 14 Black women. PK and PGx were evaluated using tandem mass spectrometry and PharmacoScan™ microarray (including CYP3A5*3, *6, and *7). CYP3A5 phenotypes varied among participants: 7 poor metabolizers (PM), 6 intermediate metabolizers (IM), and one normal metabolizer (NM). The area under the curve did not significantly differ between PMs (39,230 h*ng/mL) and IM/NMs (43,546 h*ng/mL; p = 0.38). The incidence of adverse events (AEs) was also similar. We found no association between CYP3A5 genotype and ribociclib exposure. Continued efforts are needed to include diverse populations in clinical trials to ensure equitable treatment outcomes.

IntroductionPost-transplant cyclophosphamide (PT CP) is given to deplete recipient alloreactive lymphocytes, particularly after haploidentical hematopoietic cell transplant (HCT). Several cytochrome P450s (CYPs), alcohol and aldehyde dehydrogenases (ADHs and ALDHs), and ABCB1 mediate the detoxification and clearance of cyclophosphamide (CP) and its metabolites. Furthermore, renal insufficiency, a common comorbidity in sickle cell disease, may affect CP pharmacokinetics (PK) and graft status. We hypothesize that interindividual variability affects CP PK and is associated with graft rejection in HCT patients.MethodsAdult patients with SCD transplanted on one of two nonmyeloablative haploidentical protocols (NCT00977691 and NCT03077542) were analyzed. Patients received one or two 50 mg/kg PT CP infusions; samples were drawn during the first dose and analyzed utilizing uHPLC-MS/MS to simultaneously detect CP and an active metabolite, 4OH-CP. Transplant outcomes and PK were compared with eGFR. The Pharmacoscan platform was used to genotype ABCB1, ADH1A, ADH1C, ADH4, ADH5, ADH7, ALDH1A1, ALDH3A1, CYP2B6, CYP2C9, CYP3A4, and CYP3A5. Genotypes were compared to PK parameters, CP toxicity, and transplant outcomes in both univariate and multivariate analyses.ResultsPK parameters were not associated with graft status (n=30; Table 1), and graft status did not depend on renal function. Pharmacogenomic (PG) analysis was conducted in a subset of patients (n=9). An intronic allele in ADH5 (rs2602836) was associated with prolonged graft survival (P<0.001; Figure 1). This association withstood multivariate analysis accounting for covariates (weight, age, race, sex, albumin, eGFR). CYP3A5*3 (rs776746) was associated with decreased CP clearance and higher AUC (P=0.024), and an ADH1A variant (rs1826909) was associated with shorter half-life and lower volume of distribution (P<0.048).ConclusionsADH5 detoxifies aldophosphamide by converting it into carboxyphosphamide, and we present the first evidence that a polymorphism in this enzyme is associated with graft status. These data need to be confirmed in a larger patient cohort. Although PG variants did impact PK, there was no association between PK parameters and graft status. Additionally, renal insufficiency did not impact PK or graft status.

1581 Background: Ribo is metabolized by CYP3A and used for the treatment of patients with hormone receptor-positive (HR+)/HER2- mBC. FDA recommends dose reduction if used with CYP3A inhibitors due to a 3.2x increase ribo area-under-the-curve (AUC). It is unknown if modifications are needed in patients who lack enzyme activity (e.g., genetic CYP3A5 poor metabolizers (PM)). CYP3A5varies by genetic ancestry, is known to affect dosing for other drugs (e.g., tacrolimus). CYP3A5, ~85% of people of European ancestry are PM, ~85% of African ancestry are normal or intermediate metabolizers (NM, IM), which may impact ribo exposure and response. 2 percent (41/2066) enrolled in MONALEESA 2, 3, and 7 were Black. Methods: This prospective, multicenter cohort study (NCT04657679) assessed the PK and PGx of ribo (600 mg daily + letrozole/fulvestrant) in self-identified Black women with HR+/HER2- mBC. PK (0.5, 1, 2, 4, and 6 hours after ribo) and PGx studies were performed during cycle 1 via liquid chromatography with tandem mass spectrometry and PharmacoScan (ThermoFisher) microarray, which tests 1,191 genes. Including variants in CYP3A5*3, *6,and *7. Phenotypes assigned: PM (2 variant alleles), intermediate metabolism (IM; 1 variant allele), NM (0 variant alleles). The area under the curve (AUCtau) was compared with the exact Wilcoxon rank-sum test; Fisher’s exact test assessed the AEs and grade 3+ AEs to day 28. Results: 14 completed the trial. CYP3A5 phenotypes were PM (7), IM (6), and NM (1). The primary endpoint, AUCtau, was similar between CYP3A5 PM (39,230 hr*ng/mL; interquartile range [IQR]: 18,745 to 57,566 hr*ng/mL) vs. IM/NM (43,546 hr*ng/mL; IQR: 35,298 to 46,647 hr*ng/mL; p = 0.38). Other PK properties were similar between groups (table). There was a non-statistically significant higher number of AEs and grade 3+ AEs in PMs, when compared to NM/IMs. Study was not powered to assess differences in AEs. Conclusions: This cohort study detected no association between CYP3A5 genotype and ribo exposure. However, PMs may have more AEs relative to IMs/NMs. Future steps include exploring the impact of rare variants, including ~70 variants in CYP3A 4 and 5, on ribo exposure in this population. We will explore the role of clinical and genetic factors on the interindividual variability of ribo. Diverse patient representation in clinical trials is critical to ensure research findings are applicable to all patients. Clinical trial information: NCT04657679 .[Table: see text]

S1. Expression of (A) doxycycline-inducible GFP-linked OATP1B3 was confirmed in the membrane and not cytosolic fractions of PC3 cells using a GFP-specific antibody Abcam goat antibody (#ab6673).

Supplementary Figure 5 - PDF file 60K, Changes in heart rate observed for patients in the NIH cohort during cycle 2 days 1, 8 and 15

Supplementary Figure 4 - PDF file 314K, Changes in mean heart rate during cycles 1 when patients taking betablockers were excluded

S3. t-SLCO1B3 expression was optimized using various concentrations of chetomin at different timepoints in (A) LNCaP and (B) 22Rv1 cell lines.

Supplementary Figure Legends - PDF file 25K, Legends describing supplementary figures

Supplemental Figure Legends 1-4

Supplementary Figure 6 - PDF file 60K, Changes in heart rate observed for each patient in the NIH cohort during cycle 2 days 1, 8 and 15, excluding the patients taking beta-blockers

Supplementary Figure 7 - PDF file 317K, Changes in mean heart rate occurring during cycle 1 days 1, 8 and 15 grouped by baseline heart rate

Genes Simultaneously Modulated in Cultured MES1 and MES7 Cells and Xenografts following Mithramycin Treatment.

Effect of p53 overexpression in SP1-depleted cells.

Effect of mithramycin on DNA damage.

S4. Both (A) lt-SLCO1B3 and (B) ct-SLCO1B3 were plotted as a function of t-SLCO1B3 in 384 tissue and tumor samples.

Supplementary Figure 1 - PDF file 69K, Changes in heart rate observed for patients in the NIH cohort during cycle 1 days 1, 8 and 15