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Introduction Indolent systemic mastocytosis (ISM) is a rare clonal mast cell disease driven by the KIT D816V mutation in nearly all patients. Patients with ISM can present with debilitating acute and chronic cutaneous, gastrointestinal, neurocognitive, and systemic symptoms caused by the release of inflammatory mediators from abnormal mast cells. In some patients, symptoms are not adequately controlled with best supportive care (BSC) medications. Elenestinib (BLU-263) is a novel, investigational, oral, next-generation tyrosine kinase inhibitor that potently and selectively inhibits KIT D816V with limited central nervous system penetration and pharmacokinetics (PK) that support once daily dosing. HARBOR (NCT04910685) is a randomized, double-blind, placebo-controlled, phase 2/3 study assessing efficacy and safety of elenestinib in patients with ISM. Methods Eligible adult patients with ISM, confirmed following central review of bone marrow (BM) pathology, clinical, and laboratory findings per World Health Organization criteria and a moderate-to-severe symptom score based on minimum mean total symptom score (TSS) of the ISM-Symptom Assessment Form (ISM-SAF©2018), were stratified by baseline serum tryptase (<20 ng/mL vs ≥20 ng/mL) and randomly assigned 3:1 to elenestinib 25 mg, 50 mg, or 100 mg once daily + BSC (“elenestinib”) or placebo + BSC (“placebo”). The primary objective of Part 1 was to determine the recommended dose based on safety, PK, and pharmacodynamics. Secondary endpoints included changes in biomarkers of disease burden (serum tryptase, KIT D816V variant allele fraction [VAF], and BM mast cells) and ISM-SAF TSS. Three additional open-label PK groups enrolled patients at 50 mg, 75 mg, and 100 mg to further characterize PK and safety. Results As of data cutoff on July 3, 2023, a total of 122 patients with ISM have received their assigned treatment in Part 1 of the study; 39 were blinded and randomized to elenestinib or placebo, and 83 were treated with open-label elenestinib in the PK groups (21 patients at 50 mg, 34 patients at 75 mg, and 28 patients at 100 mg). Baseline demographics were similar to those reported for the general ISM population. After 12 weeks of therapy, symptom improvement was observed for all dose cohorts, and symptom reduction by TSS was greater for patients on elenestinib versus placebo in the blinded portion of Part 1. Patients receiving elenestinib at 25 mg, 50 mg, and 100 mg doses showed reduction (mean percent) from baseline for tryptase (-15.4%, -50.9%, and -68.4% vs 3.3%, respectively) and KIT D816VVAF (-37.5%, -70.3%, and -77.0% vs -2.5%, respectively) as compared to placebo. Similar reductions in TSS and disease-related biomarkers were observed in the open-label PK cohorts. After a median treatment duration of 35.3 weeks, elenestinib was well-tolerated at all dose levels. There were no treatment-related serious adverse events and no treatment-related adverse events that led to drug discontinuation. Conclusion Elenestinib at all tested doses demonstrated beneficial effects on disease-related symptoms and biomarkers of mast cell burden in this large and maturing cohort of patients with ISM with a moderate-to-severe symptom burden. Elenestinib was well tolerated across all dose levels with a promising early benefit-risk profile in ISM. These preliminary findings will inform dosing for the planned Part 2 of HARBOR, which will examine elenestinib versus placebo in a randomized, blinded, placebo-controlled setting.

Introduction Golcadomide (GOLCA, CC-99282) is an orally available cereblon E3 ligase modulator (CELMoD ®) that potently degrades target proteins Ikaros and Aiolos, enhancing antiproliferative, apoptotic, and immunomodulatory activity and preferentially distributing to lymphoid organs. In a phase 1 study, GOLCA monotherapy previously demonstrated a manageable safety profile with promising clinical activity in relapsed/refractory non-Hodgkin lymphoma (Michot Blood 2021). In the ongoing CC-220-DLBCL-001 study (NCT04884035), GOLCA dose finding is underway in patients with previously untreated aggressive B-cell lymphoma (BCL), in combination with R-CHOP in 21-day (D) cycles (C), at a variety of GOLCA dose levels (DLs) and schedules: DL-1 = 0.2 mg D1-7, DL1 = 0.4 mg D1-7, and DL2 = 0.4 mg D1-10. Here, we report Ikaros degradation, immune modulation, circulating tumor DNA (ctDNA) assessment, and baseline tumor genomics. Methods Peripheral blood was collected to analyze Ikaros and Aiolos levels in T cells by flow cytometry (C1D1, C1D7, and C2D1). Immune phenotyping of T and natural killer (NK) cells was performed by flow cytometry (C1D1, C1D15, C2D1, and C3D1). ctDNA levels were analyzed by the PhasED-Seq assay (timepoints C1D1, C2D1, C3D1, and end of treatment [EOT]; Kurtz Nat Biotechnol 2021); somatic variants were filtered by performing PhasED-seq on buffy coats. Pretreatment biopsies were analyzed by whole genome sequencing and whole transcriptome sequencing. DL1 (0.4 mg D1-7) and DL2 (0.4 mg D1-10) data were pooled. Results Ikaros levels in T cells at 5 hours after the first dose had a 1.1% median increase with 0.2 mg and 44% median decrease with 0.4 mg; by C1D7 pretreatment, Ikaros substantially decreased with both DLs (median: 0.2 mg, 87% decrease; 0.4 mg, 83% decrease). Ikaros differences between 0.2 mg and 0.4 mg were not substantial. By C2D1, Ikaros levels recovered to baseline. Based on preclinical benchmarks, steady-state Ikaros degradation in T cells suggested that direct tumor cell killing by GOLCA was achieved. Changes in T and NK cell phenotypes in peripheral blood included activation, increased proliferation, increased T effector memory and regulatory cell levels, and decrease in naive T cells. Changes were seen by C1D15 and maintained at C2D1 and C3D1. Overall, immune changes between 0.2 mg and 0.4 mg dose levels were not substantially different. Phased variant data from ctDNA and buffy coats were evaluable for 34 patients (0.2 mg, n =15; 0.4 mg, n = 19). Baseline ctDNA levels were similar between the 0.2 mg and 0.4 mg cohorts and, consistent with prior studies, correlated with International Prognostic Index and cell of origin. Previously reported landmarks such as early molecular response (EMR; ≥ 2 log decrease) at C2D1 and major molecular response (MMR; ≥ 2.5 log decrease) at C3D1 were assessed (Kurtz J Clin Oncol 2018) . A high fraction of patients designated high risk by pretreatment ctDNA (≥ 2.5 log haploid genome equivalents [HGE]/mL; Kurtz J Clin Oncol 2018) were clinical responders. Patients who achieved minimal residual disease (MRD) negativity on treatment generally remained MRD negative at subsequent timepoints. At C2D1, the EMR and MRD negativity rates for 0.2 mg were 73% (11/15) and 20% (3/15), respectively; for 0.4 mg these rates were 83% (15/18) and 44% (8/18). At C3D1, the MMR and MRD negativity rates for 0.2 mg were 83% (10/12) and 42% (5/12), respectively; for 0.4 mg these were 83% (10/12) and 58% (7/12). Although ctDNA differences between 0.2 mg and 0.4 mg were not substantial, numerically greater ctDNA changes were seen with 0.4 mg GOLCA + R-CHOP vs 0.2 mg GOLCA + R-CHOP and vs R-CHOP and R-CHOP-like regimens from other cohorts ( Figure). Insufficient samples were available to draw conclusions about EOT, but additional ctDNA data will be presented. Whole genome and transcriptome sequencing data from GOLCA + R-CHOP-treated patients will be shown. Conclusions Pharmacodynamic biomarkers and ctDNA levels indicate that both tumor-intrinsic and immune-modulating mechanisms of action of GOLCA remain relevant in combination with R-CHOP in previously untreated aggressive BCL. ctDNA data during treatment are consistent with the efficacy noted on clinical restaging. Translational data demonstrate the potency of GOLCA and will support future frontline clinical trial designs in patients with untreated aggressive BCL. Study support: Celgene, a Bristol-Myers Squibb Company

Background Diffuse large B cell lymphoma (DLBCL) is the most common aggressive lymphoid malignancy in adults. Though standard immunochemotherapy regimens can result in clinical remission and cure in a majority of patients, approximately 30% of patients are primary treatment resistant or eventually relapse (rrDLBCL), and their prognosis is very poor. Recent molecular classification by large scale genomic and transcriptomic profiling of newly diagnosed DLBCL tumors has resulted in a deeper understanding of disease drivers, however, the molecular heterogeneity of rrDLBCL is yet to be fully explored. Therefore, in this study, we used an unsupervised approach to better define transcriptional and genetic programs in rrDLBCL samples hypothesizing that molecular stratification within this high-risk group may lead to a better understanding of this disease, which remains an unmet need, and a more personalized therapeutic approach. Methods Data used in this study included RNA (n=143) and whole exome (n=126) sequencing data from available FFPE tumor samples at the time of a relapse (any line of treatment, r1-r10 relapse timepoints included in analysis, one per patient), consented to the Molecular Epidemiology Resource (n=61), banked in the Mayo Lymphoma Biobank (n=50), or consented to the CC-122-ST-001 clinical trial (n=32, NCT01421524). Unsupervised clustering was performed on protein coding gene expression values using the non-negative matrix factorization (NMF) approach. NMF was carried out by performing 200 runs on cluster sizes from 2 to 6. Cell of origin (COO) was determined using the method described by Reddy et al. The tumor microenvironment was analyzed using CIBERSORTx and Lymphoma Microenvironment Classification (LME). Genetic classification was done using LymphGen. Results In order to identify unique transcriptional programs in rrDLBCL, we implemented NMF and consensus clustering to define an ideal quantity of stable patient clusters. We identified 4 rrDLBCL patient clusters (rrC1-rrC4) as the most stable solution, based on consensus clustering selection criteria of iterative NMF runs. While relapse timepoint was not associated with individual clusters, we did observe significant differences in tumor characteristics between the clusters. COO classification found that rrC1 and rrC4 were enriched for GCB-DLBCL while rrC3 was enriched for ABC-DLBCL classification (P < .001). To explore the biologic programs in each cluster, we used pathFindR, which leverages protein-protein interaction networks to identify disease related pathways, rrC1 and rrC4 were defined by pathways involved in cell cycle and mismatch repair; oxidative phosphorylation in rrC2; and MAPK and NF-kB in rrC3. Next, LME classification was done to define patterns in the tumor microenvironment and we found a significant difference between groups (P < .001), with rrC2 and rrC4 tumors enriched for a depleted TME, while rrC1 and rrC3 had a more immune rich or inflammatory TME. This was supported by CIBERSORTx analysis identifying significant differences (P > .001) in abundance of CD8 T cells, memory CD4 T cells, and M1 macrophages between clusters, with rrC4 having the lowest abundance. We then explored genetic patterns in each cluster. Gene level mutation enrichment analysis showed BCL7A mutation exclusively in C4 (6/6, P = .03). The clusters were not associated with LymphGen classification. Lastly, our recently published high risk signature classification that is associated with early clinical failure and is characterized by metabolic dysregulation, a depleted TME, T53 alterations, and poor outcome, was significantly different between the groups (P = .03) with enrichment for high-risk cases in rrC4. In summary, we show for the first time that rrDLBCL patients can be classified into four gene expression clusters that are associated with distinct pathway, TME, and genetic programs. These clusters should now be tested to learn if they can help select patients for newer therapies for rrDLBCL such as CAR-T and bispecific antibodies.

Presence of deletion17p (del17p) in frontline (1L) and relapsed/refractory (R/R) CLL has been associated with shorter time to treatment initiation, early relapse after therapy and inferior overall survival (OS) (Mato et.al., Haemotologica 2022). However, there exists notable clinical heterogeneity in terms of outcome within this group (Tam et.al., Blood 2009, Stephens et.al; AJH 2015). Previous reports in 1L and R/R CLL have identified percentage of del17p nuclei by fluorescence in situ hybridization (FISH), having TP53 mutations on the second allele, copy number alterations and complex karyotype (CK) as additional risk factors within the del17p population (Yu et al., CCR 2017; Baliakas et al., Blood 2019). Here we profile a cohort of wait and watch (W&W, n=14), 1L treated (n=36) and R/R CLL (n=3) patients by whole genome sequencing (WGS) and RNA sequencing (RNAseq) to gain insights into genomic factors and gene expression pathways contributing to the high-risk nature of del17p across the spectrum of CLL patients. Using the Mayo Clinic CLL Database, we identified 53 patients with del17p (identified by FISH testing in routine clinical practice at diagnosis) who had stored peripheral blood mononuclear cells (PBMCs) available. RNA and DNA from matched peripheral blood (tumor) and DNA from CD19- (germline) samples at diagnosis (n=50) and relapse (n=3) was extracted using Qiagen RNeasy (RNA) and Purene (DNA) kits and subjected to RNAseq and WGS respectively. Additional FISH assays (del11q, trisomy 12, del13q) and CK by chromosomal banding analysis (CBA) were performed. Baseline clinical characteristics, time to first treatment, types of treatment, and survival were abstracted from the CLL Database. The Mayo Clinic IRB approved this study. Copy number analysis identified deletions in 3p, 4p, 8p, 9p, 9q and gains in 2p, 8q, 15q and 17q chromosomal lesions occurring in patients. We explored the association of clinical and genetic factors, and genomic instability signatures with survival to identify high-risk features in del17p patients. Both total number of deletions by WGS and number of chromosomal aberrations by CBA as a continuous variable were significantly associated with unfavorable OS in the entire cohort (n=53) in a univariate analysis (Hazard ratio, HR= 2.29, p= 0.00054 and HR= 2.33, p=0.0054 respectively). However, there was no significant association of number of gained genes by WGS or aneuploidy scores with survival (HR=0.74, p=0.37). In patients with CK data (n=41), deletion burden, but not gains, were highly correlated to chromosomal aberrations by CBA. Using cutpointr package for identifying deletion threshold corresponding to CK≥ 3, we defined patients with deletion burden ≥ 514 as enhanced-high risk (EHR; median OS = 4.83 years) compared to the group with high-risk (HR; median OS=9.94 years) (p=0.025). There was no significant difference in number of deleted genes between WW and treated patients. The EHR group is enriched in patients with high clonal cell fraction of del17p by FISH, unmutated IGHV and CK≥ 3. The EHR group remains significant in a multi-variable analysis with IGHV-mutation status, ZAP70, and age (HR: 5.16, 95% CI: 1.04 - 25.6, p=0.04). Mutational analysis identified 35 known CLL driver mutations with TP53 (86%), ATM (15%), NOTCH1(13%), MGA (13%), CHD2 and MED12(11%) as the top mutated genes. Total number of driver genes mutated was not significantly different between EHR and HR subgroups, but the EHR subset was significantly enriched with mutations in NOTCH1 (21%), and MGA (21%) as compared to no mutations in the HR group in these genes. Deleted genes (≥ 514) was prognostic in bi-allelic TP53 aberration patients as well (p=0.021) Differential expression gene-set enrichment analysis indicated a significant enrichment of DNA repair, cellular response to DNA damage and downregulation of inflammatory responses in the EHR group. CK is a known prognostic marker which includes both chromosomal losses and gains. In this small cohort of del17p we were able to show the prognostic value of deletions only, but not gains, as captured by WGS. Using genome wide sequencing, we identify increasing genomic deletions as a feature of enhanced-high risk del17p. While deletion burden cut-offs identified here are specific to our research method and require further validation in additional independent cohorts, EHR subgroup remained significant after adjusting for other known prognostic variables .

Background: Tumor mutational burden (TMB), defined as the number of nonsynonymous mutations per megabase (Mb) in the coding regions of a tumor genome, has been associated with improved response to immune checkpoint blockade treatments and more broadly with overall survival (OS) in many cancers. Limited prior studies have found that TMB is higher in diffuse large B-cell lymphoma (DLBCL) compared to many other cancers. However, only two studies have evaluated TMB and prognosis in newly diagnosed DLBCL patients. The prognostic role of TMB in diffuse large B-cell lymphoma (DLBCL) remains unclear as prior studies have been limited to panel sequencing of cancer genes and/or lacked matched germline-tumor samples for filtering. Methods: We characterized TMB from whole exome sequencing (WES) in newly diagnosed DLBCL patients with matched diagnostic tumor-normal samples prior to treatment with standard immunochemotherapy (IC). Tumor DNA was extracted from formalin-fixed, paraffin embedded tissue sections and germline DNA from peripheral blood. We evaluated the association between TMB and event-free (EFS) and OS using Cox regression adjusted for the international prognostic index (IPI) and other factors including cell of origin (COO), MYC double hit (DH) with BLC2 or BCL6 by FISH, and TP53 mutation status. EFS and OS were defined as time from diagnosis to earliest of treatment failure (EFS only), death, or last follow-up; EFS at 24 months (EFS24) was also modeled as a marker of early aggressive DLBCL. An optimal high/low TMB cutpoint was determined based on EFS using maximally selected rank statistics; sensitivity analyses used an 20/80 percentile to define the threshold. We also evaluated the association of driver tumor mutational load (driverTML) with EFS and OS, defined as the total number of mutations in 292 DLBCL driver genes. Results: In the analysis cohort of 394 DLBCL patients, the median age at diagnosis was 65 years (interquartile range: 56-73), 43% were female, 62.2% had IPI 0-2, and 57.4% germinal center B-cell COO. MYC was DH in 7.5% and TP53 was mutated in 16.2% of patients. Through a median follow-up of 7.5 years, there were 141 events and 122 deaths. EFS24 was achieved in 75.1% of the cohort; 5-year EFS was 69% (95% CI 64-74%) and 5-year OS was 75% (95% CI 71-80%). Median TMB in our cohort of DLBCL patients was 3.6 mutations/Mb, which was the fifth highest compared with 32 cancers in The Cancer Genome Atlas. As a continuous variable, increasing TMB (mutation/Mb) was associated with superior EFS (HR=0.89, 95% CI 0.81-0.98) and OS (HR=0.89, 95% CI 0.81-0.99) after adjustment for IPI. Spline analyses confirmed linear associations of TMB with EFS and OS. The optimal modeled cutpoint based on EFS was 4.5 mutations/Mb, with 31% classified as high TMB (>4.5). No significant ( P >.05) differences were observed between high and low TMB patients with clinical (i.e., age, sex, IPI category) or molecular (cell of origin, MYC DH, TP53 mutated) factors. In Cox regression, high-TMB was significantly protective for EFS and for OS compared to low-TMB after adjustment for each clinical factor ( Figure 1), with simultaneous adjustment for all these factors associated with superior EFS (HR=0.52, 95% CI 0.33-0.84) and OS (HR=0.59, 95% CI 0.36-1.00). TMB (high/low) combined with IPI (0-2, 3-5) was significantly ( P =.001) associated with EFS in Kaplan-Meier analysis ( Figure 2). Among patients with IPI 3-5, high TMB was significantly associated with superior EFS (HR=0.47, 95% CI 0.27-0.81) and OS (HR=0.57, 95% CI 0.33-0.99). Among patients with IPI 0-2, we also observed longer EFS (HR=0.62, 95% CI 0.34-1.11) and OS (HR=0.62, 95% CI 0.32-1.20) in high TMB patients, although the associations were not statistically significant ( P >.05). In sensitivity analyses, a 20/80 percentile TMB threshold found similar results for EFS (HR=0.64, 95% CI 0.41-1.03) and OS (HR=0.69, 95% CI 0.42-1.12) as in our main analyses. TMB and driverTML were significantly correlated ( R=0.65, P <10 -16) and we found similar effect sizes for EFS (HR=0.45, 95% CI 0.22-0.91) and OS (HR=0.55, 95% 0.27-1.13) as in our main analyses. Conclusions: In a large and well-defined cohort of IC treated DLBCL patients, we found DLBCL patients with higher TMB had superior EFS and OS after adjustment for other clinical and molecular prognostic factors, and the prognostic association for TMB was robust to threshold and gene set.

Background: Proprotein convertase subtisilin/kexin 9 (PCSK9) raises low-density lipoprotein cholesterol (LDL-C) levels and is associated with inflammation, which is elevated in HIV and hepatitis C virus (HCV) infection. We compared PCSK9 levels in people with co-occurring HIV and HCV (HIV/HCV) vs. HIV alone, and evaluated the impact of HCV direct-acting antiviral (DAA) therapy on PCSK9. Design: A prospective, observational cohort study. Methods: Thirty-five adults with HIV/HCV and 37 with HIV alone were evaluated, all with HIV virologic suppression and without documented cardiovascular disease. Circulating PCSK9 and inflammatory biomarkers were measured at baseline and following HCV treatment or at week 52 (for HIV alone) and compared using Wilcoxon tests and Spearman correlations. Results: At baseline, PCSK9 trended higher in HIV/HCV vs. HIV alone (307 vs. 284 ng/ml, P = 0.06). Twenty-nine participants with HIV/HCV completed DAA therapy with sustained virologic response. PCSK9 declined from baseline to posttreatment 1 (median 7.3 weeks after end of therapy [EOT]) and posttreatment 2 (median 43.5 weeks after EOT), reaching levels similar to HIV alone; median within-person reduction was -60.5 ng/ml (P = 0.003) and -55.6 ng/ml (P = 0.02), respectively. Decline in PCSK9 correlated with decline in soluble (s)E-selectin and sCD163 (r = 0.64, P = 0.002; r = 0.58, P = 0.008, respectively), but not with changes in LDL-C or other biomarkers. No significant change in PCSK9 occurred in the HIV alone group over 52 weeks. Conclusion: PCSK9 declined with DAA therapy in participants with HIV/HCV, correlating with declines in several inflammatory biomarkers but not LDL-C. Elevated PCSK9 with HCV may be linked to particular HCV-associated inflammatory pathways more so than cholesterol homeostasis.

ABSTRACTObjectivesWe aimed to investigate the gender-based differences in idiopathic inflammatory myopathies (IIMs), with a particular focus on patient-reported outcomes, utilizing the data obtained through the international COVID-19 vaccination in autoimmune disease e-survey.MethodsPatient-reported outcomes including fatigue, pain, and physical function were extracted from the COVID-19 vaccination in autoimmune disease database and compared between genders, adjusting for demographics and IIM subgroups by multivariable analysis. Inclusion body myositis (IBM) was analysed separately because of the substantial differences in outcomes.ResultsA total of 1197 complete responses from patients with IIMs as of 31 August 2021 were analysed. Seventy percent were women. Women were younger (58 [48–68] vs. 69 [58–75] years old, median [interquartile range], P < .001) and were more likely to suffer from autoimmune multimorbidity, defined as three or more autoimmune diseases in an individual patient (11.4% vs. 2.8%, P < .001). In non-IBM IIMs, fatigue visual analogue scale scores were higher in women (5 [3–7] vs. 4 [2–6], median [interquartile range], P = .004), whereas no significant gender-based differences were noted in IBM. Multivariable analysis in non-IBM IIMs revealed that women, residence in high-income countries, overlap myositis, and autoimmune multimorbidity were independently associated with increased fatigue.ConclusionsWomen with IIMs suffer from autoimmune multimorbidity and experience increased fatigue compared to men.