Trisal, Preeti

Abstract Introduction: Deletion in the short arm of chromosome 17 (del17p) is a strong, negative prognostic biomarker found in about eight percent of patients with untreated chronic lymphocytic leukemia (CLL). CLL patients with del17p exhibit resistance to therapy, accelerated disease progression, and global immunodeficiency. It is unclear the extent to which global immunodeficiency impacts disease course and therapy outcomes. The goal of this study was to document the blood immune repertoire of patients with untreated CLL who have del17p and associate with clinical features, time to first treatment (TFT), and overall survival (OS). Methods: Biobanked blood samples from treatment-naïve del17p CLL patients (n = 58) and age-matched controls (n = 5) were obtained. B cells and T cells were analyzed with comprehensive, cell type-specific flow cytometry panels and visualized with unbiased dimensionality reduction. Lab values were log-transformed and compared using t-tests and one-way ANOVAs. TFT was calculated from date of sample collection to date of first CLL-directed therapy or last known untreated date; OS was calculated from date of sample collection to death or last known alive status. Cox proportional hazards regression analyses (results reported as hazard ratio [HR] and 95% confidence interval [CI]) were employed to identify associations of lab values with OS and TFT. Results: Patients with del17p CLL had increased proportions of T regulatory cells (Tregs) among total CD4+ T cells compared to controls (CLL 11.1%, control 5.7%, p = 0.05). A higher proportion of CD8+ T cells was associated with longer TFT (HR 0.57, 95%CI 0.36-0.89); in contrast, higher proportions of TIGIT+ (HR 1.83, 95%CI 1.03-3.28) and TIM3+ conventional (non-Treg) CD4+ T cells were associated with shorter TFT (HR 1.70, 95%CI 1.02-2.84). A higher proportion of PD1+ CD8+ T cells was associated with shorter OS (HR 1.79, 95%CI 1.05-3.06), in addition to PD1+ conventional CD4+ T cell proportion (HR 2.21, 95%CI 1.06-4.60). CLL B cells expressed PD-1 and inducible T cell costimulator (ICOS) at significantly lower median fluorescence intensities (MFIs) than control B cells (p < 0.0001 for both). The CXCR5+ MFI (HR 1.0002, 95%CI 1.0001-1.003) and CD23+ MFI (HR 1.88, 95%CI 1.01-3.50) of CLL B cells were associated with shorter TFT. Discussion: In this cohort of treatment-naïve del17p CLL patients, we determined that patients with higher proportions of exhausted CD4+ and CD8+ T cell phenotypes had significantly shorter OS. Cytotoxic T cell proportion positively associated with TFT, whereas proportions of CD4+ T cell exhaustion phenotypes negatively associated with TFT. Higher CXCR5+ and CD23+ MFIs of CLL B cells also associated with shorter TFT. The preliminary findings from our study suggest that the blood immune repertoire has strong associations with clinically relevant outcomes such as time to first treatment and overall survival in patients with untreated del17p CLL. Citation Format: Ethan J. Katcher, Kim A. Gwin, Sameer A. Parikh, Kari G. Rabe, Petra K. Lothert, Preeti Trisal, Esteban Braggio, Susan L. Slager, Nicholas Strong, Daniel L. Van Dyke, Zhiquan Wang, C. Chris Huang, Anita K. Gandhi, Kay L. Medina, Neil E. Kay. Correlating the blood immune repertoire and clinical features of del17p CLL patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB345.

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 .