Parikh, Sameer A.

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 .

Introduction: Chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) is a rare form of large granular lymphocytic leukemia. The clinical characteristics and outcomes of patients (pts) with CLPD-NK in the literature are limited. We aim to describe the patient characteristics, laboratory features, and clinical outcomes of CLPD-NK by reporting on, to our knowledge, the largest single-center cohort of CLPD-NK in the United States. Methods: We retrospectively identified Mayo Clinic pts with CLPD-NK, defined per the WHO 5 th edition and 2022 International Consensus Classification. We conducted chart review to collect clinical and laboratory data. Overall survival (OS) was plotted using Kaplan-Meier. Time to first treatment (TTFT) was displayed using cumulative incidence methodology accounting for competing risk of death. Univariable Cox regression models investigated associations between patient characteristics and outcomes (OS and TTFT). Results: We identified 45 pts who were diagnosed with CLPD-NK between 1997 and 2022. The median age at diagnosis was 69 years (range: 25-90) and 32 pts (71%) were male. At the time of diagnosis, 10 pts (22%) had a concomitant autoimmune disorder and 9 (20%) had laboratory evidence of a serologic abnormality (monoclonal gammopathy, rheumatoid factor, etc.). In addition, 10 pts (22%) had a prior diagnosis of a hematologic malignancy and 12 (27%) had a prior diagnosis of a solid tumor. Clinically, 4 pts (9%) had B symptoms, 15 (33%) had splenomegaly, and 13 (29%) were transfusion dependent. At diagnosis, median hemoglobin (hgb) was 10.2 g/dL, with 25 pts (56%) showing a hgb <11g/dL and 8 (18%) demonstrating a hgb <8 g/dL. The median platelet count was 202x10^9/L, with 12 pts (27%) demonstrating a platelet count <150x10^9/L. The median white blood cell count was 5.8x10^9/L. The median absolute neutrophil count (ANC) was 1.2x10^9/L, with 29 pts (64%) demonstrating an ANC <1.5x10^9/L. The median absolute lymphocyte count (ALC) was 3.8x10^9/L, with 20 pts (44%) demonstrating an ALC >4x10^9/L. The median absolute abnormal NK cell count per flow cytometry was 2.2x10^9/L. Flow cytometry revealed killer cell immunoglobulin-like receptor (KIR) restriction in all pts, with 27 (61%) lacking KIR expression and 17 (39%) demonstrating KIR restriction in CD158a, CD158b, and/or CD158e. CD7 expression was present in 42 pts (93%; 31% dim/partial), CD8 expression was present in 22 (49%; 42% d/p), CD56 expression was present in 17 (39%; 23% d/p), CD57 expression was present in 26 (59%; 46% d/p), CD94 expression was present in 33 (77%; 7% d/p), and NKG2A expression was present in 21 (50%; 5% d/p). Bone marrow examination (n=30) revealed that the median number of NK infiltrate was 11% (range: 5-40%) of cellularity, with 20 demonstrating an intrasinusoidal infiltrative pattern. The median follow-up was 41 months (mo), and 23 pts required at least one line of treatment. Treatment indications included anemia (39%), neutropenia (4%), bi/pancytopenia (39%), lymphocytosis (4%), or other (13%). The median TTFT was 32 mo (95% CI 2-93 mo). Hgb <11g/dL was associated with a shorter TTFT (9.7 vs 187 mo); however, neutropenia and thrombocytopenia were not associated with TTFT. For first-line treatment, in addition to frequent steroid use, 16 pts (70%) received a methotrexate-based regimen, 6 (26%) received a cyclophosphamide-based regimen, and 1 (4%) received a cyclosporine-based regimen. The median TTFT was not reached. Of those who required systemic treatment, 9 pts required a second-line therapy. The median OS from diagnosis was 162 mo (95% CI: 84-not estimable, NE), with 33 pts alive at last follow-up (Figure 1a). Increasing age (HR 1.2, 95% CI 1.1-1.3, p=0.002) and lack of CD94 expression (HR 5.6, 95% 1.3-25.0, p=0.02) were associated with worse OS. In the 23 pts who required treatment, the median OS from treatment initiation was 84 mo (95% CI: 52-NE) (Figure 1b). Conclusions: In this large cohort of 45 pts, the majority had neutropenia and anemia, and approximately half required systemic treatment. Estimated median OS from diagnosis exceeded a decade for the entire cohort and was seven years after treatment initiation. Anemia was associated with a shorter TTFT, while increasing age and lack of CD94 expression were associated with worse OS. This rare disease warrants additional investigations to understand optimal management and improve outcomes, especially for those requiring treatment.

Background: Understanding the symptoms and quality of life (QOL) over time of adults with follicular lymphoma (FL) is important for treatment decision-making and clinical management. However, there are limited population-level data on the long-term QOL of adults with FL. We aim to describe the real-world, long-term QOL of adults with newly diagnosed FL up to 5/6 years after diagnosis. Methods: We used the Mayo Clinic/Iowa Molecular Epidemiology Resource (MER) and the multi-institutional Lymphoma Epidemiology of Outcomes (LEO) to identify adults with grade 1-3A FL who completed QOL surveys at baseline and follow-up. Participants with lymphoma were prospectively enrolled within 9 months of diagnosis in the MER cohort from 2002-2015 and within 6 months of diagnosis in the multi-institutional LEO cohort study across 8 cancer centers from 2015-2020. The LEO/MER cohort studies systematically collected information on disease status, QOL, health behaviors, and functional assessment. Treatments, disease relapses, and deaths were verified by medical record review. Treating physicians determined clinical management. QOL was measured using the Functional Assessment of Cancer Therapy-General (FACT-G) at years 1, 2, 3, and 5/6 post-diagnosis. A higher FACT-G total score (range 0-108) indicated better QOL across 4 subscales (range): physical (0-28), social/family (0-28), emotional (0-24), and functional (0-28) well-being. We categorized participants based on their frontline management at time of diagnosis: “observation” (surveillance), “treatment” (systemic treatment with immunotherapy +/- chemotherapy), and “local” (radiation) groups. We employed a generalized linear mixed model to evaluate and compare the changes in QOL scores (as a continuous variable) from baseline for the observation, treatment, and local groups, adjusting for sex, race/ethnicity, age, FLIPI risk, and cohort (i.e., LEO or MER). We also evaluated QOL changes by FLIPI risk. Results: Our study included 1,544 participants with FL and QOL data. At the time of enrollment, median age was 61 years (range 19-91), 88% were non-Hispanic White, and 49% were female. Based on initial management, 529 (34%) were in the observation group, 880 (57%) were in the treatment group, and 135 (9%) were in the local group. FLIPI risk was high for 17% in the observation group, 29.3% in the treatment group, and 3% in the local group. The mean (standard deviation) baseline FACT-G total score was lowest in the treatment group with the following baseline scores: 86 (13) for the observation group, 83 (14) for the treatment group, and 88 (12) for the local group (Table 1). The observation and local groups reported a greater worsening of their baseline FACT-G total score vs the treatment group at both the 2-year (-1.2, -0.8 vs +1.8, respectively) and 5/6 year (-5.4, -6 vs. -1.6, respectively) timepoints (Figure 1). This appeared to be driven by worsened physical and functional well-being in the observation (-1.3, -2.2, respectively) and local (-0.9, -2.4, respectively) groups vs the treatment group (0, -0.3, respectively). These differences were statistically significant between the observation vs treatment groups. There were no statistically significant differences in the other subscales, i.e., social/family and emotional well-being, by frontline management. Social/family well-being decreased across all groups by 5/6 years (-2.3 in observation, -2.2 in treatment, and -3 in local, p<0.05). Regardless of FLIPI risk, the total FACT-G scores decreased within each FLIPI risk group at 5/6 years (-4.4 for low, -3.8 for intermediate, and -4.9 for high, p<0.05). This was primarily due to worsened social/family well-being (-2.4 for low, -2.2 for intermediate, and -3 for high, p<0.05) at 5/6 years. There was no statistically significant difference in QOL between FLIPI risk groups. Conclusion: This is one of the first and largest studies with real-world longitudinal QOL data for FL. Our study suggests that frontline systemic treatment initially improved QOL and resulted in a lower degree of QOL decline over time. Social/family well-being decreased over time regardless of frontline management or FLIPI risk. Further research is warranted to explore the impact of timing and specific systemic treatments on QOL, incorporate the patient/caregiver experience to address social/family well-being, and identify the clinical significance of our findings.

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.

INTRODUCTION Chronic lymphocytic leukemia (CLL) and SLL represent a single entity in current classifications per the WHO-HAEM5 and ICC. Both are characterized by malignant B cells with an identical immunophenotype, but they are distinguished by the predominant site of disease burden, such as peripheral blood for CLL and nodal compartments for SLL. Most clinical trials and retrospective studies involving CLL/SLL do not separate out SLL patients. In this study, we report reasons for treatment initiation and outcomes in a cohort of SLL patients seen at our institution. METHODS Using the Mayo Clinic CLL Database, we retrospectively identified patients with SLL who received first line therapy between 12/1995 and 2/2023. Patients were classified as having SLL if they met at least one of the following criteria for diagnosis: 1) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute clonal B cell count of < 5 x 109/L; or 2) presence of lymphadenopathy or splenomegaly (either by exam or scans) and an absolute lymphocyte count of < 11 x 109/L (if absolute clonal B cell count was not available). Although the presence of anemia (hemoglobin < 11 g/dL) and thrombocytopenia (platelets < 100 x 109/L) are considered as exclusion criteria for a diagnosis of SLL, patients were included if these cytopenia were due to autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP) with < 50% bone marrow involvement by SLL (bone marrow biopsy confirmed). Patients treated for SLL with extranodal involvement were also included. Indications for SLL treatment were abstracted by chart review and included one of the following: 1) symptomatic or progressive lymphadenopathy and/or splenomegaly, 2) autoimmune cytopenias, 3) extranodal/direct organ infiltration; and 4) other (e.g., cryoglobulinemia, paraneoplastic, non-infiltrative renal disease such as minimal change disease). Overall survival (OS) was defined as the time from treatment start until date of death or last known to be alive. Treatment-free survival (TFS) was defined as the time from treatment start until the earliest of date of next treatment, or death. Kaplan-Meier method was used to display OS and TFS. Cox proportional hazards regression models were used to estimate associations of factors with time-to-event outcomes. RESULTS We identified 182 patients with SLL who received first line treatment. The median age at treatment start was 66 years, and 132 (73%) were male. Unmutated IGHV was identified in 60/82 (73%) patients and peripheral blood fluorescent in situ hybridization (FISH) assessed pre-treatment start showed del13q in 10%, trisomy 12 in 43%, del11q in 7%, del17p in 8%, and no abnormalities in 33%. TP53 disruption (del17p and/or TP53 mutation) was present in 7/89 (8%) patients prior to first therapy. The median follow-up after diagnosis was 6.9 years. The indications for treatment were grouped as follows: symptomatic or progressive lymphadenopathy/splenomegaly (n=130), extranodal/direct organ infiltration (n=30), AIHA/ITP (n=10), and other (n=12). For the entire cohort, the median TFS was 2.9 years and the median OS was 12.3 years. The median TFS by the treatment indication groups were 3.3, 5.1, 1.6, and 5.3 years, respectively (Figure 1). The median OS by treatment indication groups were 11.1, 10.5, 8.8, and 19.9 years, respectively. Among the 160 patients with symptomatic or progressive lymphadenopathy/splenomegaly or extranodal disease, the treatments administered include: mAb alone (n=35), chemotherapy alone (n=33), chemoimmunotherapy (n=57), targeted agent (n=33; Bruton tyrosine kinase inhibitor [BTKi] based n=28; venetoclax-based n=3, BTKi + venetoclax n=2), and other (n=2). The median TFS by treatment groups were 2.4, 2.0, 3.8, and 6.0 years (Figure 2), respectively (excluding the small “other” subgroup of 2 patients). The median OS for these groups were 9.5, 9.7, 15.0, and not evaluable, respectively. Only older age at treatment start predicted for shorter TFS (HR 1.02; 95%CI 1.002-1.038; p=0.03) and shorter OS (HR 1.05; 95%CI 1.03-1.07; p<0.01). CONCLUSIONS In this study of patients with SLL who received therapy, the most frequent indications for treatment included symptomatic lymphadenopathy and/or splenomegaly, followed by extranodal disease. Trisomy 12 was the most frequently detected abnormality on peripheral blood FISH testing. Treatment with novel agents achieved durable TFS and OS.

Abstract In the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.