Franke, Karl

Abstract Multiple myeloma (MM) is an incurable plasma cell neoplasm characterized by diverse and complex genetic abnormalities, including translocations of immunoglobulin (Ig) genes and a hyperdiploid (HRD) karyotype. Much of our current understanding of MM genetics, epigenetics, pathogenesis and response/resistance to myeloma directed therapies originated from studies on human MM-derived cell lines (HMCL). In this study, we conducted an integrated multi-omics analysis in five hyperdiploid cell lines established from serial samples from a single relapsed/refractory hyperdiploid MM patient, enabling detailed characterization of the genetic landscape and molecular alterations through disease progression.

Abstract Background: Minimal residual disease (MRD) is a critical prognostic and therapeutic response marker in multiple myeloma (MM). Two main technologies are used for MRD assessment in bone marrow: next-generation flow cytometry (NGF) and next-generation sequencing (NGS), from clonoSEQ. While both methods are FDA-cleared and clinically relevant, they differ in sensitivity, sample handling requirements, and technical differences. Comparative performance in real-world matched samples is understudied. Here we compared the detection rates and concordance between MRD by NGF (flow cytometry) and NGS-based clonoSEQ, using paired bone marrow aspirates collected at the same clinical timepoint. Methods We identified 246 paired bone marrow samples from MM patients, which were evaluated by both flow cytometry (Euroflow, typically sensitivity of 10-5) and clonoSEQ (sensitivity of up to 10-6) on the same collection date.MRD-negativity for clonoSEQ was defined as no detectable clones and below the assay's level of detection (LoD). Continuous variables (clonoSEQ and flow CPS) were analyzed using the Mann-Whitney U test. Categorical variables were assessed using chi-square tests with Yates continuity correction for 2x2. A cox proportional hazards model was used to evaluate the association between overall survival and cytogenetic abnormalities, MRD status, and other clinical covariates including age, sex, treatment type, and disease setting (newly diagnosed, relapsed/refractory). A p-value < 0.05 was considered significant. Results Out of 246 paired bone marrow samples, we identified 173 concordant samples, out of which 26 were concordant positive (clonoSEQ+/NGF+), 147 were concordant negative (clonoSEQ-/NGF-), and 73 were discordant (clonoSEQ+/NGF-). clonoSEQ detected MRD in all cases missed by NGF, including some with relatively high tumor burden, reflecting its higher sensitivity. Among discordant samples, 45 (61.6%) were following treatment of newly diagnosed MM (NDMM) and 28 (38.4%) treatment of relapsed/refractory (RRMM). Among the MRD-positive samples, the concordant group (n=26) showed a mean clonoSEQ count of 11923.5 cells per million (CPM) and a mean NGF CPM of 305.5 (p<0.01). In contrast, the 73 discordant samples had a lower mean clonoSEQ CPM of 156.1. For the 147 MRD-negative samples with concordant results, no residual disease was detected by either assay (CPM = 0 for both). When evaluating sex, race, ethnicity, chromosomal abnormalities, prior treatments to sample collection (immunomodulatory drugs, proteasome inhibitors, CD38 antibodies, CAR Ts/bispecific antibodies), and disease stage (NDMM, RRMM, untreated), patients with t(11;14) were significantly higher in the discordant group (17.9% concordant vs. 32.9% discordant; p = 0.016), while CAR T/bispecific antibody-treated patients were more likely concordant for the tests (27.2% concordant vs. 4.1% discordant; p<0.01). High-risk cytogenetics (t(4;14), t(14;16), gain 1q) were associated with worse survival (HR 6.22, 95% CI 2.02 – 19.2; HR 16.2, 95% CI 3.38-77.1; HR 4.0, 95% CI 1.28 – 12.5, respectively). Discordant MRD status did not impact survival, though only 13/246 patients were deceased (median follow-up approximately 50.6 months).Conclusion: ClonoSEQ demonstrates higher sensitivity compared to NGF for MRD detection in MM, identifying residual disease missed by NGF particularly in t(11;14) cases. Discordance did not affect survival, however, only 13 patients were deceased at the time of last follow up, suggesting longer follow up is needed.

Introduction: Sickle cell disease (SCD) is prevalent in sub-Saharan Africa. Phenotypic diversity in patients with similar SCD genotypes may result from genetic and environmental factors. Nigeria has the highest SCD prevalence, with homozygous SCD (HbSS) being the most severe form. Stroke affects about 11% of children under 20 years with SCD (Ohene-Frempong, Weiner, et al. 1998), primarily ischemic, involving medium to large intracranial arteries. Specific single nucleotide polymorphisms have been linked to ischemic stroke risk in some populations. (Earley, Kelly et al. 2023). This study aimed to identify genetic factors associated with early-age large-vessel stroke in SCD using whole-exome sequencing (WES) in a Nigerian cohort. Methods: The protocol was approved by the IRBs of TJU, Nemours, and 3 Nigerian sites. Subjects with SCD who experienced large vessel stroke early in life (16 years or less) were enrolled, along with controls (SCD patients without stroke). Subjects were recruited from 3SCD clinics in Kano Metropolis, Nigeria. Inclusion criteria were confirmed SCD diagnosis (SS and Sβ0 thalassemia genotypes), abnormal transcranial doppler (TCD) measurements, and clinical evidence of stroke confirmed by MRI by age 16. Controls included SCD patients (SS and Sβ0) aged 16-26 years without clinical stroke and at least one normal TCD. All cases underwent Magnetic Resonance Angiography (MRA) to establish arteriopathy. Immediate family members of subjects >16 years (siblings and parents) were used per best practice trio analysis to annotate variants with appropriate Mendelian inheritance patterns. WES was performed using SureSelect XT HS (Agilent) and Human All Exon V7 Rev B capture probes for library preparation on all samples. Libraries were sequenced on a NextSeq 550 to 50M paired-end 150bp reads. Variants were called using the GATK suite of algorithms following best practice guidelines and annotated using the VarSeq algorithm, including AlphaMissense annotations. Results: The control group had a median age of 21 years, and the stroke group had a median age of 9 years. The stroke group had a higher proportion of males (60%) compared to controls (30%) (p < 0.05). Fifty percent of the stroke group and 73% of the controls had a history of acute chest syndrome. History of avascular necrosis (7.5%), heart disease (2.5%), and splenic sequestration (1.3%) were present only in controls. Malaria (1.3%), sepsis (2.5%), and hand-foot syndrome (59%) affected the stroke group. History of priapism (6.3%) and leg ulcers (10%) had similar rates in both groups. Using AlphaMissense annotations, nine genes were identified to have more damaging mutations in the stroke cohort compared to the control cohort (odds ratio > 1 and p < 0.05). Pathway enrichment analysis utilizing genes prioritized with an odds ratio > 1 identified cadherin signaling, cellular junction/adhesion, and WNT signaling as significant (p < 0.05). The role of cellular adhesion has been previously described in SCD stroke subjects (Oni, Brito et al. 2024). The genes that we found that showed significance in our analysis include ADAMTSL2, ADCY9, OR3A1, OR3A2, XRRA1, ABTB2, CLDN18, CPNE7, EIF3A, SLC7A6, UBA7, PDE6A, GLOD5, and PCDHA1. Conclusion: Genes in the ADCY9 and ADAMSL2 families have been reported in previous studies to be associated with ischemic stroke. Though the small sample size limits this study, it offers preliminary insights into the genetic and clinical determinants of stroke in SCD patients in Northern Nigeria. The identified candidate genes potentially hold biological significance for the stroke phenotype; however, additional research with a larger sample size is required to validate these associations. These findings underscore the need for further whole-exome sequencing studies in Nigerian patients with the stroke/arteriopathy phenotype, which may augment our understanding of this severe complication. An in-depth examination of the genetic modifiers associated with stroke in SCD could lead to enhanced predictive models and targeted preventive interventions, reducing the burden of stroke in affected individuals. Our team has identified additional datasets in the United States to help validate molecular signatures of interest, inspiring us to continue our work in this important field.