Dyke, Daniel L. Van

Introduction: Novel targeted therapies have revolutionized treatments for both upfront and relapsed CLL. However, effective therapies have not been established for progressive CLL after developing resistance to both BTK inhibitor and BCL-2 inhibitors. Similarly, Richter's transformation (RS) remains an area of unmet clinical need. Hypothesizing that progressive CLL and RS possess significant but differential molecular abnormalities, we aimed to dissect the initial genomic profiles in a clinical cohort of patients seen in our routine clinical practice using targeted DNA sequencing and transcriptome analysis. Methods: Twelve CLL patients who had lymph node biopsies for evaluation of clinical progression at Mayo Clinic were included in this study. RNA sequencing and targeted DNA sequencing on a focused oncology panel of 1,711 genes were performed on Illumina HiSeq through Tempus Labs, Inc. (Chicago, IL). Differentially expressed (DE) genes were identified using edgeR at the cutoff of FDR <=0.05 and fold-change >=2. Somatic mutations and copy number alternations (CNAs) were identified through Mutect2 and PatternCNV, respectively. CN loss was defined to have log2(ratio) <=-0.93 and CN gain to have log2(ratio) >= 0.585. Pathway analysis was done through the Enrichr package. Results: Among the 12 patients, 7 had confirmed RS (large cell lymphoma histology) and 5 had CLL based on pathological findings. The clinical and therapeutic history for each patient is illustrated in Fig 1A. FISH results from 11 of the patients showed chromosomal abnormalities including del(13q), del(11q), del(17p) and tri(12) (Fig 1A). Hierarchical clustering of expression profiles from the top 10,000 most variable genes in expression revealed two major clusters (Fig 1A). Cluster C1 contains five RS cases, of which case S207 did not receive ibrutinib, S256 was on high-dose steroid and anti-CD20 post-ibrutinib therapy for >1 year before biopsy, and case S025 was treated for < 6 months with ibrutinib. These three form a subgroup separate from the other two RS in C1 who had 8 and 42 months of continuous ibrutinib therapy pre-biopsy, respectively. C2 contains two RS and five CLL cases. Within C2, CLL and RS cases showed similar transcriptomic profiles. Over 90% of the DE genes showed an overall increase of expression in C1, particularly within the 3-sample subgroup (Fig 1B). These genes are enriched in pathways known to play key roles in CLL, including PI3K-AKT signaling, NOTCH signaling and RAS/MAPK signaling, while genes up-regulated in C2 are enriched in WNT signaling. The results suggest marked differences in gene expression profiles and indicate molecular heterogeneity among RS and CLL patients. In addition, targeted DNA sequencing data were analyzed to identify somatic mutations and CNAs. The analysis confirmed the FISH results of del(13q), del(11q), del(17p) or tri(12) in 8 of the cases (Fig 1A). The mutation profiles are similar between C1 and C2 for the recurrently mutated genes published in CLL. However, we identified recurrent CNAs that occurred preferentially in C1 (covering 9 genes) or C2 (48 genes) (Fig 1C). Manual inspections of the reads coverage revealed that 5 of the 9 genes are from chr1q21.1 to chr1q22 that showed clear CN gains largely in C1. Of the 48 genes, 6 are from chrXp11 that showed CN losses, and 27 are from chr17q12 to chr17q25 that showed CN gains. Therefore, the majority of the 57 genes are from large-scale CNAs. Notably, for the CNAs mostly occurring in C2, the affected genes are enriched in WNT signaling, Cell cycle/protein kinase activity and chromatin modifying enzymes. Collectively, the results support the variability of transcriptomic signatures and genetic lesions between RS and CLL. Conclusion: This study revealed a noticeable heterogeneity in gene expression and genetic variation between RS and CLL, albeit in a small cohort. The vast majority of the DE genes showed elevated expression in C1 containing only RS cases, and these genes are enriched in PI3K-AKT signaling, NOTCH signaling, and RAS/MAPK signaling. The pattern is more pronounced for a subgroup in C1 who were overall less exposed to ibrutinib. For somatic variation, there is a strong tendency of occurrence of recurrent CNAs towards C2 cohort, mainly involving gains of chr17q and deletions of chrXp11. Given these advances we are encouraged to further pursue the genetic abnormalities with defined roles in aggressive and transformed CLL. Disclosures Liu: Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Parikh:Genentech: Honoraria; Ascentage Pharma: Research Funding; AbbVie: Honoraria, Research Funding; Verastem Oncology: Honoraria; Pharmacyclics: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Merck: Research Funding; MorphoSys: Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Wang:Incyte: Research Funding; Innocare: Research Funding; Novartis: Research Funding. Kenderian:Gilead: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Mettaforge: Patents & Royalties; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; Juno: Research Funding; Kite: Research Funding. Kay:Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Acerta Pharma: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ding:Astra Zeneca: Research Funding; DTRM: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Introduction: PD-1 inhibits the cytotoxic T-cell functions via the interaction with its ligands (PDL1 and PDL2). Recently, our group showed a selective response (~40%) with PD-1 blocking antibody pembrolizumab in patients with Richter transformation (RT), particularly after prior exposure to ibrutinib (Wei Ding et al, Blood, 2017). Additionally, PD-1 showed an increased expression in tumor B-cells of patients with RT (Rong He et al, AJSP, 2018). Here we investigated the functional implications of the PD-1 signaling axis in lymphoma B-cell pathobiology. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN samples were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). Then, we checked PD-1 expression in 10 lymphoma cell lines and 1 CLL cell line (MEC-1) by both, flow cytometry and Western blot (WB) analysis. Over-expression of PD-1 using pLEX-lentiviral system (Thermo Scientific) was evaluated for in vitro cell cycle regulation and in vivo tumor growth. Overexpression of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Gene expression signatures in CLL and RT patients were also identified by Illumina-based RNA sequencing using Formalin-Fixed Paraffin-embedded (FFPE)-nodal tissue obtained by clinical biopsy (Tempus Labs; Chicago, IL). Results: The expression of PD-1 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) [Fig 1A]. 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 lymphoma B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable, but at very low-levels in lymphoma lines OCI-LY7 and OCI-LY19 (data not shown). 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 compared to original lines on day 4, respectively, Fig 1BI-II), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% and 24.53% in OCI-LY7 and OCI-LY19 cells (Fig 1B III), respectively. When the luciferase + PD-1 expressing OCI-LY7 cells were injected intravenously (1X106 cells) into NSG mice, increased tumor growth was observed at 22 days of follow up by bioluminescence imaging (p<0.01, Fig 1C). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and CHK2 (T68) (data not shown). This finding was further confirmed by WB analyses (Fig1DI). In addition, decreased of total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY19 and OCI-LY17 cell lines (Fig 1DI). Immunoprecipitation experiments with anti-PD1 antibody showed that PD-1 was in the same complex with SHP-1 and SHP-2 in OCI-LY7 cells but only with SHP-2 in OCI-LY19 cells (Fig 1DII). In parallel, mRNA sequencing was performed on 11 nodal tissues from either RT (n=8) or progressive CLL (n=3) after they developed clinical progression. The expression of PD-1 (PDCD1) was positively correlated with the expression of SHP-1 (PTPN6, r=0.56) and Cyclin E1 (CCNE1, r=0.62), implicating a direct role of PD-1 in regulating cellular proliferation and the induction in phosphatase expression in RT and CLL. Conclusion: Our data support the notion that PD-1 overexpression in lymphoma cells modifies cell proliferation in vitro and in vivo and regulates the function of phosphatase SHP and p53- pathways. These findings also indicate that aggressive lymphoma or CLL leukemic cells have the ability to hijack the PD-1 pathway and may result in downregulation of the p53 mediated DNA repair. This novel information provide for new strategies to further evaluate the interaction of checkpoint signals with DNA repair pathway, and possibly provide novel targets for Richter's transformation. Disclosures Ding: alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; MEI Pharma: Membership on an entity's Board of Directors or advisory committees. Sakemura:Humanigen: Patents & Royalties. Parikh:Janssen: Honoraria, Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria, Research Funding; Ascentage Pharma: Research Funding; GlaxoSmithKline: Honoraria; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Verastem Oncology: Honoraria; Genentech: Honoraria; AbbVie: Honoraria, Research Funding; TG Therapeutics: Research Funding. Wang:Novartis: Research Funding; Incyte: Research Funding; Innocare: Research Funding. Liu:Eisal: Research Funding; Genentech: Research Funding; GRAIL: Research Funding; Menarini Silicon Biosystems: Research Funding; Merck: Research Funding; Seattle Genetics: Research Funding; Tesaro: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Ansell:Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding; ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; Bristol Myers Squibb: Research Funding. Kenderian:Sunesis: Research Funding; Tolero: Research Funding; Torque: Consultancy; BMS: Research Funding; Gilead: Research Funding; Kite: Research Funding; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Novartis: Patents & Royalties, Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding; Juno: Research Funding. Kay:Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION Nearly 20% of patients (pts) are ≥ 80 years (yrs) of age at the time of CLL diagnosis. Increased medical comorbidities, as well as declining functional status and organ function, often accompany advanced age. However, clinical trials often enroll a disproportionally low number of people from this age demographic due to stringent eligibility parameters, which provides limited evidence to guide decisions. Here, we report clinical characteristics and outcomes of pts ≥ 80 yrs of age at the time of CLL diagnosis. METHODS Between 1/1995 and 3/2020, we identified previously untreated CLL pts from the Mayo Clinic CLL Database who were ≥ 80 yrs of age at the time of CLL diagnosis, and seen within 3 yrs of diagnosis. Baseline characteristics, treatments administered, and time to first treatment (TFT) were analyzed for all pts. Overall survival (OS) was measured from diagnosis date in all pts and separately from treatment date for treated pts. Time to next treatment (TTNT) was measured in treated pts from first treatment date to date of second treatment or last known treatment. TFT and TTNT were analyzed accounting for competing risk of death. Standardized mortality ratio (SMR) compared observed survival to survival in a Minnesota population. Cox multivariable regression models were used to determine which factors were associated with OS (treatment as a time-dependent covariate) and TTNT (accounting for competing risk of death). RESULTS Among the 213 pts identified, the median age was 83 yrs (range 80-97); baseline characteristics are summarized in Table 1. The median number of medical comorbidities among all pts was 4 (range 0-8). At least 1 comorbidity was present in 97% of pts, most commonly hypertension (57%), genitourinary (n=48%), rheumatological (46%), and dyslipidemia (n=43%). Median follow-up was 3.8 yrs. A total of 56 pts received treatment and the median TFT was 6.7 yrs. Over this 25 year interval, the treatment approaches included monoclonal antibody alone (n=17), chemotherapy alone (n=17), or chemoimmunotherapy (n=14), novel agents (n=3), and multi-agent Richter's transformation regimens (n=5). Chlorambucil-based treatments (chlorambucil n=13, chlorambucil, prednisone n=3) were the most frequently used chemotherapy regimens. The most common chemoimmunotherapy regimens included rituximab, cyclophosphamide, prednisone (RCP) with (n=3) or without vincristine (n=6), and chlorambucil with an anti-CD20 monoclonal antibody (n=4; obinutuzumab n=2, rituximab n=2). Among 17 pts who received monoclonal antibody alone, treatments included rituximab alone (n=11), rituximab plus methylprednisolone (n=2), obinutuzumab (n=2), and rituximab with alemtuzumab (n=2). Frontline novel agent treatments included ibrutinib alone (n=1), acalabrutinib alone (n=1), and ibrutinib, obinutuzumab, venetoclax (n=1). Five pts with Richter's transformation (diagnosed concurrent with CLL n=4) received multi-agent chemotherapy. Among the 56 treated pts, 22 pts received second line therapy after a median TTNT of 2.8 yrs from start of first therapy. The most common second line treatments were chlorambucil-based (n=6) and rituximab-based (n=6) regimens. Age, sex, absolute lymphocyte count, del17p, and unmutated IGHV were not predictive of TTNT. The median OS among all 213 pts was 4.6 yrs. SMR was 1.2 (p=0.008) when excluding pts with Richter's transformation. In univariable analyses, receipt of therapy (HR 3.92, 95%CI 2.11-7.28; p<0.001), age (per 5 year increase; HR 1.84, 95%CI 1.43-2.38; p<0.001) and unmutated IGHV (HR 2.03, 95%CI 1.12-3.67; p=0.02) were predictors of shorter OS. The OS among pts who received treatment (Figure 1) was similar when compared across time periods of treatment start year grouped as 1995-2001 (n=10), 2002-2013 (n=32), and 2014-2020 (n=14). Treatment categories by time period are shown in Table 2. CONCLUSIONS In this study of 213 pts ≥ 80 yrs of age at time of CLL diagnosis, the median time to first therapy was ~7 yrs. Although survival outcomes did not differ by era across the 25-year span of this study, pts treated with novel agents were underrepresented and are a patient population which warrants additional evaluation. The finding that pts with CLL in this cohort had a 20% higher risk of death compared to an age- and sex-matched population emphasize the need for ongoing efforts to improve clinical outcomes for CLL pts in this demographic category. Disclosures Kenderian: Torque: Consultancy; Mettaforge: Patents & Royalties; Humanigen: Consultancy, Patents & Royalties, Research Funding; Novartis: Patents & Royalties, Research Funding; Kite: Research Funding; Gilead: Research Funding; Juno: Research Funding; BMS: Research Funding; Tolero: Research Funding; Sunesis: Research Funding; MorphoSys: Research Funding; Lentigen: Research Funding. Wang:Incyte: Research Funding; Innocare: Research Funding; Novartis: Research Funding. Shanafelt:Genentech, Pharmacyclics LLC, an AbbVie Company, AbbVie, GlaxoSmithKline, and Merck: Research Funding; Mayo Clinic: Patents & Royalties: and other intellectual property. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; MEI Pharma: Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees. Ding:alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. Parikh:Merck: Research Funding; AstraZeneca: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; Verastem Oncology: Honoraria; GlaxoSmithKline: Honoraria; TG Therapeutics: Research Funding.

Background: Cytogenetic abnormalities detected by fluorescence in situ hybridization (FISH) are known to differentially impact prognosis in patients with chronic lymphocytic leukemia (CLL). Deletion6q23 (del6q23) has been reported in ~2-3% of CLL patients at diagnosis; however, its prognostic significance remains indeterminate. Methods: We identified previously untreated CLL patients from the Mayo Clinic CLL Database seen between 1/1995 - 2/2020 who had FISH performed. The clinical characteristics, concomitant cytogenetic abnormalities, time to first therapy (TTFT), and overall survival (OS) were compared between patients with del6q23 to those without this abnormality. TTFT was analyzed from FISH date until treatment or last known untreated date, accounting for competing risk of death. OS was analyzed using Kaplan-Meier methods from FISH date. In a matched (CLL-IPI risk score and sex) analysis, Cox regression analysis was used to determine the association between TTFT/OS and presence of del6q23. The Mayo Clinic IRB approved this study. Results: A total of 3101 CLL patients were identified; their median age at diagnosis was 64 years and 66% were male. Of these patients, 42 (1.3%) had evidence of del6q23 by FISH. Compared to patients without del6q23, patients with del6q23 carried a more adverse clinical and cytogenetic profile at the time of diagnosis including advanced Rai stage (22% Rai III/IV vs. 9% Rai III/IV; P = 0.002), unmutated IGHV genes (91% vs. 43%; P < 0.001), and high/very high risk CLL-IPI (50% vs. 27%; P < 0.001). Del6q23 was the sole abnormality in 13 patients (31%). Of the remaining patients, del6q23 was concomitantly present with del13q in 8 patients (19%), and with either del11q or del17p in 21 patients (50%). Of the 42 patients with del6q23, 29 received first line therapy: chemoimmunotherapy in 16 patients, BTK inhibitor in 7 patients, and an anti-CD20 monoclonal antibody alone in 6 patients. The median follow-up of the entire cohort was 7.3 years. The median TTFT in patients with del6q23 was 0.7 years while the 5-year OS was 76%. Patients with del6q23 demonstrated significantly shorter TTFT and reduced OS when compared to patients without del6q23 (Figures 1A and 1B). The shorter TTFT was also evident for patients with del6q23 compared to patients without del6q23 divided according to the Dohner risk category (Figure 2A), although there was no difference in OS between these groups of patients (Figure 2B). Because del6q23 patients were enriched for other unfavorable prognostic markers, we identified four control patients without del6q23, matched by CLL-IPI risk score and sex, to each treatment-naïve patient with del6q23. Patients with del6q23 showed a borderline statistically significant shorter TTFT (median 0.7 years versus 3.9 years; P = 0.07) and a nonsignificant association with OS (median 10.9 years versus 10.3 years; P = 0.28). Conclusion: Del6q23 by FISH in previously untreated CLL represents an uncommon abnormality. When compared to patients without del6q23, patients with del6q23 tend to have more adverse clinical and cytogenetic profiles at baseline, which likely confer a worse prognosis as reflected by shorter TTFT compared to patients without this abnormality. Figure Disclosures Ding: Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; DTRM: Research Funding; Beigene: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Astra Zeneca: Research Funding. Wang:Novartis: Research Funding; Innocare: Research Funding; Incyte: Research Funding. Braggio:DASA: Consultancy; Bayer: Other: Stock Owner; Acerta Pharma: Research Funding. Kay:Sunesis: Research Funding; Abbvie: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Acerta Pharma: Research Funding; MEI Pharma: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees. Kenderian:Juno: Research Funding; Gilead: Research Funding; Lentigen: Research Funding; MorphoSys: Research Funding; Sunesis: Research Funding; Kite: Research Funding; Novartis: Patents & Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents & Royalties, Research Funding; Mettaforge: Patents & Royalties; Tolero: Research Funding; BMS: Research Funding. Parikh:Ascentage Pharma: Research Funding; Merck: Research Funding; AbbVie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Honoraria, Research Funding; GlaxoSmithKline: Honoraria; Janssen: Honoraria, Research Funding; MorphoSys: Research Funding; Genentech: Honoraria; Verastem Oncology: Honoraria.

Abstract Background Approximately 25% of patients with chronic lymphocytic leukemia (CLL) experience a flare of disease following ibrutinib discontinuation. A critical question is whether this phenomenon may also occur when ibrutinib is temporarily held. This study aimed to determine the frequency and characteristics of disease flares in this setting and assess risk factors and clinical outcomes. Materials and Methods We identified all patients with CLL seen at Mayo Clinic between October 2012 and March 2019 who received ibrutinib. Temporary interruptions in treatment and associated clinical findings were ascertained. Results Among the 372 patients identified, 143 (38%) had at least one temporary interruption (median 1 hold, range 1–7 holds) in treatment. The median duration of interruption was 8 days (range 1–59 days) and the most common indication was periprocedural. Among the 143 patients with ≥1 hold, an associated disease flare was seen in 35 (25%) patients: mild (constitutional symptoms only) in 21 patients and severe (constitutional symptoms with exam/radiographic findings or laboratory changes) in 14 patients. Disease flare resolved with resuming ibrutinib in all patients. Predictive factors of disease flare included progressive disease at time of hold and ≥ 24 months of ibrutinib exposure. The occurrence of disease flare with an ibrutinib hold was associated with shorter event-free survival (hazard ratio 2.3; 95% confidence interval 1.3–4.1; p = .007) but not overall survival. Conclusion Temporary interruptions in ibrutinib treatment of patients with CLL are common, and one quarter of patients who held ibrutinib in this study experienced a disease flare. Resolution with resuming ibrutinib underscores the importance of awareness of this phenomenon for optimal management. Implications for Practice Ibrutinib is a very effective treatment for chronic lymphocytic leukemia (CLL) but needs to be taken continuously. Side effects, such as increased bleeding risk with procedures, require temporary interruptions in this continuous treatment. Rapid CLL progression following ibrutinib discontinuation has been increasingly recognized. This study demonstrates that similar flares in disease signs or symptoms may occur during ibrutinib holds as well. Importantly, management with restarting ibrutinib led to quick clinical improvement. Awareness of this phenomenon among clinicians is critical to avoid associated patient morbidity and premature cessation of effective treatment with ibrutinib if the flare is misidentified as true progression of disease.

Abstract Constitutive activation of B-cell receptor (BCR) signaling is a major driving mechanism for the proliferation and survival of various B-cell lineage non-Hodgkin lymphomas (NHL). Blocking BCR signaling using the first-in-class Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, has proved effective and is currently FDA approved for the treatment of several B-cell lymphomas. Unfortunately, significant subsets of patients possess either primary or acquired resistance to ibrutinib, often resulting in worse prognosis and poorer responses to subsequent therapies. Therefore, overcoming ibrutinib resistance is an urgent clinical need requiring new tools to both delineate the molecular mechanisms of resistance and develop viable therapeutics. To that end, we developed a mantle cell lymphoma (MCL) cell line, MCIR1, from a patient with clinically acquired ibrutinib resistance. MCIR1 is a bona fide MCL cell line featuring CD19+/CD5+/CD10-/CD23-/t(11;14)+/TP53+/-; and lacking the previously described mutations in BTK (C481S) and phospholipase C-gamma-2 (R665W, L845F). Importantly, MCIR1 cells possess hallmarks of ibrutinib resistance, including the insensitivity of ERK1/2 phosphorylation to ibrutinib in vitro, and the irresponsiveness of MCIR1 xenograft tumors to ibrutinib treatment in mice as compared with ibrutinib-sensitive Jeko-1 tumors. To delineate the molecular mechanism(s) of ibrutinib resistance, we identified by RNA-Seq analysis that MCIR1 cells possess robust activity of the non-canonical NFkB pathway and elevated expression of NFkB target genes, suggesting that this pathway likely drives their resistance to ibrutinib. Finally, we demonstrated the utility of MCIR1 as a tool for testing new drugs to combat ibrutinib resistance in vitro by showing its sensitivity to the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein inhibitor, venetoclax. To our knowledge, MCIR1 is the first patient-derived ibrutinib-resistant cell line to be established. Hence, MCIR1 is especially useful because uniquely possesses the relevant clinical and biological features of patients with ibrutinib-resistant MCL. We propose MCIR1 as a tool to further the understanding of molecular mechanisms conferring ibrutinib resistance by providing a clinically relevant system with the capacity for molecular and genetic manipulations. Further, these features make MCIR1 an ideal model to screen drugs for combating ibrutinib resistance through both in vivo and in vitro approaches. Citation Format: Kevin E. Nowakowski, Jithma P. Abeykoon, Mary J. Stenson, Michael M. Timm, Curtis A. Hanson, Daniel L. Van Dyke, Anne J. Novak, Xiaosheng Wu, Thomas E. Witzig. MCIR1: A patient-derived ibrutinib-resistant mantle cell lymphoma line for the study of ibrutinib resistance and drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 650.

e22002 Background: Mesenchymal stem cells (MSC) residing in the marrow support hematopoiesis and protect cancer cells from undergoing cell death induced by chemotherapy. Recent reports have described clonal cytogenetic abnormalities in the MSC of acute myeloid leukemia and myelodysplastic syndrome patients. To determine if cytogenetic abnormalities are present in MSC from CLL patients, we analyzed karyotypes of MSC from 13 CLL patients and 5 normal subjects. Methods: Stromal cells from marrow core biopsies of 13 CLL patients and 5 normal control subjects were isolated, cultured and confirmed as MSC based on their immunophenotype and capacity to differentiate into three lineages. After 3–4 non-stimulated cell culture passages, the karyotype was analyzed in 5–40 metaphase cells from each subject Abnormalities were considered clonal using the accepted convention of the same chromosomal gain or rearrangement in 2 or more cells or loss in at least 3 cells. For each CLL patient, interphase FISH analysis to detect the common CLL-associated abnormalities was performed on freshly isolated peripheral blood mononuclear cells (PBMC). Results: Clonal cytogenetic abnormalities of the cultured MSC were observed in 6 of 13 CLL patients (46%) and 3 of 5 control subjects (60%). The 6 CLL MSC had different clonal abnormalities than the PBMC CLL FISH studies. One CLL MSC exhibited trisomy 5, one exhibited trisomy 8, and one had monosomy X clone. One MSC had a balanced t(1;16). One MSC had a balanced t(3;12) and a del(3p), and one had a complex karyotype with 4 unbalanced rearrangements. The clonal aberrations observed in the 3 controls included a del(3p) in two cases, and in one case an inv(5) and a del(10q). There was no correlation of the chromosomal abnormalities in CLL MSC with clinical stage. Conclusions: Marrow MSC derived from CLL patients and normal subjects do show an array of cytogenetic abnormalities including clonal chromosomal abnormalities. However the genetic abnormalities found in both CLL and normal MSC could represent acquired genomic instability associated with advanced age, rather than oncogenesis associated with CLL. [Table: see text]

Context.— One goal of the joint College of American Pathologists/American College of Medical Genetics and Genomics Cytogenetics Committee is to ensure the accurate detection and description of chromosomal abnormalities in both constitutional and neoplastic specimens, including hematologic neoplasms. Objective.— To report a 20-year performance summary (1999–2018) of conventional chromosome challenges focusing on hematologic neoplasms. Design.— A retrospective review was performed from 1999 through 2018 to identify karyotype challenges specifically addressing hematologic neoplasms. The overall performance of participants was examined to identify potential recurring errors of clinical significance. Results.— Of 288 total conventional chromosome challenges from 1999–2018, 87 (30.2%) were presented in the context of a hematologic neoplasm, based on the provided clinical history, specimen type, and/or chromosomal abnormalities. For these 87 hematologic neoplasm challenges, 91 individual cases were provided and graded on the basis of abnormality recognition and karyotype nomenclature (ISCN, International System for Human Cytogenomic [previously Cytogenetic] Nomenclature). Of the 91 cases, 89 (97.8%) and 87 (95.6%) exceeded the required 80% consensus for grading of abnormality recognition and correct karyotype nomenclature, respectively. The 2 cases (2 of 91; 2.2%) that failed to meet the 80% consensus for abnormality recognition had complex karyotypes. The 4 cases (4 of 91; 4.4%) that failed to meet the 80% consensus for correct karyotype nomenclature were the result of incorrect abnormality recognition (2 cases), missing brackets in the karyotype (1 case), and incorrect breakpoint designation (1 case). Conclusions.— This 20-year review demonstrates clinical cytogenetics laboratories have been and continue to be highly proficient in the detection and description of chromosomal abnormalities associated with hematologic neoplasms.

Abstract Background: Results of conventional cytogenetic studies on specimens processed without B-cell mitogens are usually normal in B-CLL: this observation is often attributed to lack of mitotic B-CLL cells. Using metaphase FISH with D13S319, we now have evidence that these apparently normal mitotic B-CLL cells often exhibit submicroscopic interstitial deletions in 13q14. This observation indicates B-CLL cells do divide in unstimulated cultures, but the abnormal metaphases are not detected because the abnormalities are submicroscopic and represent a low percentage of proliferating cells. Methods: We studied 12 patients with B-CLL who had normal metaphases by conventional cytogenetics, but abnormal interphase nuclei by FISH with D13S319. This cohort consisted of 7 females and 5 males who ranged in age from 50 to 87 years. Clinical information was available for 11 patients: Rai stages were 0, I, II, III, or IV for 6, 1, 1, 2, and 1 patients, respectively; 8 patients received treatment and 3 have died. Conventional cytogenetic studies and metaphase FISH were done on bone marrow specimens that were processed by standard 24 and 48-hour unstimulated cultures with Chang Medium BMC. Interphase FISH studies were done on blood or bone marrow (collected within 3 days of specimens for conventional cytogenetic studies) using probes to detect anomalies of chromosomes 6, 11, 12, 13, 14 and 17. For each patient, metaphase FISH was done with commercial probes from Vysis Inc., Downers Grove, IL for D13S319/13q34 using leftover fixed cells from conventional cytogenetics studies. Results: Metaphase FISH results were normal for 3 bone marrow specimens: the remaining 9 specimens had 1.9 to 13.8% metaphases with interstitial deletions of 13q14. One patient had a cytogenetically visible chromosome 13 deletion in 2.2% of metaphases. In the remaining 8 specimens, the size of the abnormal 13 was similar to the normal 13 homolog and other acrocentric chromosomes. By comparison, interphase FISH revealed 13 to 93% nuclei with 13q-x1 or 13q-x2. Seven patients had additonal anomalies in their interphase nuclei; 3 had 11q-, 2 had 17p-, 1 had +12 and 1 had +IGH. Conclusions: This investigation indicates that metaphase FISH detects submicroscopic deletions in chromosome 13 as well as infrequent mitotic B-CLL cells in patients who have normal conventional cytogenetic results. Therefore, metaphase FISH is a novel and useful technique to detect abnormal mitotic cells in B-CLL in cultures without special B-cell mitogens. The prognostic significance of abnormal metaphase FISH results is still under investigation. We speculate that this method will allow for identification of more aggressive proliferating disease even in so-called “favorable” prognostic groups based on interphase FISH results.

Abstract Background: Loss of p53 is detected by fluorescence in situ hybridization (FISH) in interphase nuclei in 8–10% of patients with B-CLL and has been associated with aggressive disease and poor response to therapy. Results of chromosome studies suggest loss of p53 is often due to structural anomalies of chromosome 17 rather than monosomy 17. To characterize chromosome 17 anomalies in B-CLL, we used interphase and metaphase FISH to study a series of patients with this disorder. Methods: We studied 16 patients with B-CLL who had chromosome 17 anomalies detected by conventional chromosome studies or interphase FISH. The study cohort consisted of 8 females and 8 males (age 54 – 73 years). Clinical data was available for 15 patients: Rai stages were I, II, III and IV for 1, 2, 6 and 6 patients, respectively; 14 patients received treatment and 10 have died. Metaphase and interphase FISH studies were performed using left-over fixed bone marrow or peripheral blood cell pellets from conventional cytogenetic studies. To identify recurrent chromosome 17 breakpoint cluster regions, patients were first studied with p53(17p13.1) and D17Z1(centromere17), and then with FISH probes for SMS(17p11.2) and RARA(17q21). For patients retaining the SMS probe site, we did FISH for HNPP(17p11.2–12) and D17Z1(centromere17). A total of 200 interphase nuclei and up to 20 metaphases were analyzed by each probe set. Results: The karyotype was normal for 3 patients and complex involving various abnormalities of chromosome 17 for the remaining 13. All patients showed loss of one p53 probe site with retention of both chromosome 17 centromeres. The SMS locus was absent in 13 patients indicating a breakpoint cluster region between the centromere and SMS probe site. In the remaining 3 patients, HNPP was absent, but the SMS probe site was retained indicating chromosome breakage between the SMS and HNPP probe sites. Metaphase FISH results indicate loss of p53 was due to an isochromosome 17p11.2 in 4 patients and an unbalanced translocation involving 17p11.2 in 12 patients. Conclusions: This study shows that loss of p53 in B-CLL cells is due to isochromosome formation or unbalanced translocations that involve a ~6,400 kb breakpoint cluster region adjacent to the centromere on the short arm of chromosome 17. Importantly, our findings indicate that loss of p53 is not due to loss of chromosome 17, simple deletions of p53, or chromosome breakage near the p53 locus. The region between the centromere and HNPP on chromosome 17 is known to be rich in low copy repeats and homologous DNA sequences are dispersed throughout the genome. In somatic cells, these repeats can result in pairing of the same or different chromosomes; crossing over within these homologous regions can result in translocations or isochromosomes. Undoubtedly, this genetic mechanism plays an important role in the origin of structural anomalies of chromosome 17 in B-CLL.