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Abstract Background: Monoclonal B-cell lymphocytosis (MBL) is characterized by the presence of a circulating population of clonal B-cells where the total B-cell count is <5 × 109/L and criteria for the diagnosis of other lymphoproliferative disorders are not present. In most patients the immunophenotype is similar to that of chronic lymphocytic leukemia with co-expression of CD19 and CD5. The natural history of MBL is unclear. We used the Mayo Clinic hematopathology database to identify patients with MBL and abstracted clinical information on natural history and prognostic parameters from clinical and research records. Methods: After IRB review and approval, we reviewed the hematopathology records of 2292 patients seen at Mayo Clinic between August 1998 and May 2006 who had a clonal B-cell population on flow cytometry phenotypically consistent with CLL and fewer than <5 × 109/L B-cells. Clinical records were then reviewed to identify patients who fulfilled the criteria for MBL (no evidence of other lymphoproliferative disorders, no adenopathy on exam). Patient characteristics, prognostic parameters, and clinical outcomes were abstracted from the clinical and research records of these patients. Cox regression analysis was used to evaluate association between prognostic markers and time to treatment. Results: A total of 118 patients meeting the criteria for MBL were identified. Median age at the time of MBL diagnosis was 69.4 years (range 39.0 – 91.4). A slight male preponderance was observed (54% male). CD38 status was available for 106 (90%) patients of whom 20% were CD38 positive using the 30% threshold to classify CD38. Cytogenetic analysis by fluorescent in situ hybridization (FISH) was available in 58 (49%) patients. Using the hierarchical classification system of Dohner and colleagues, 26 (44.8%) were 13q-, 10 (17.2%) were trisomy 12, 2 (3.4%) were 11q-, 1 (1.7%) was 17p-, and 19 were (32.8%) no defect was detected. ZAP-70 status was available for 32 patients of whom 22% were ZAP-70+ positive. IgVH gene mutation status was only available for 15 (12.7%) patients of whom 40% were unmutated. Time from MBL diagnosis to treatment is shown in Figure 1. Survival free of treatment was 92.7% (95%CI: 86.8–99.1%) at 1 year, 90.9% (95%CI: 84.2–98.2%) at 2 years, and 87.5% (95% CI: 78.7–97.4%) at 5 years. While age (p=0.68) and gender (p=0.14) were not found to be associated with time to treatment (TTT), CD38 status showed a strong relationship with TTT (p<0.001, HR: 22.7, 95% CI: 3.4–239.6). Conclusion: The distribution of prognostic parameters (e.g. CD38, FISH) in patients with MBL appears similar to that of patients with early stage CLL. We observed a low rate of progression, with 5 year survival free of treatment greater than 87%. TTT among newly diagnosed MBL patients was associated with the CD38 status of clonal B-cells. Figure Figure

Background: The 2018 iwCLL guidelines recommend close observation of early stage CLL pts who do not meet indications for therapy ("watch and wait" strategy). Prior attempts at early therapeutic intervention in unselected early-stage CLL pts using alkylating agents such as chlorambucil and chemoimmunotherapy failed to show a significant benefit; and these therapies were associated with substantial toxicities. The introduction of Bruton tyrosine kinase (BTK) inhibitors has revolutionized the treatment landscape of CLL. The German CLL Study group recently presented results of the CLL12 study which demonstrated a significant improvement in event-free survival (EFS) among untreated early stage high risk CLL pts who received ibrutinib compared to placebo (median EFS not reached vs. 47.8 months, respectively; Langerbeins, EHA, 2019). Acalabrutinib is a more selective second-generation BTK inhibitor that has fewer adverse events in pts with CLL, and has shown equally impressive clinical activity for CLL pts. We are conducting an investigator initiated randomized phase 2 study to compare acalabrutinib with or without obinutuzumab (a glycoengineered anti-CD20 monoclonal antibody approved in the treatment of CLL) among high risk early stage CLL pts. Study Design: All pts with newly diagnosed (<2 years from registration) previously untreated early stage CLL who do not meet the 2018 iwCLL guidelines for initiation of therapy are eligible for enrollment in this study. Pts undergo risk stratification according to the CLL-International prognostic index (CLL-IPI) which consists of the following 5 variables: age >65 years (1 point), Rai stage I-IV (1 point), del17p and/or TP53 mutation (4 points), unmutated immunoglobulin heavy chain (IGHV) genes (2 points), and serum β2-microglobulin >3.5 g/dL (2 points). Pts with high (4-6) and very high (7-10) risk CLL-IPI are randomly (1:1) assigned to acalabrutinib 100 mg orally twice daily (Arm A) or acalabrutinib with obinutuzumab at a standard approved schedule (Arm B). Treatment with acalabrutinib is continued for a minimum of 2 years unless the patient has unacceptable side effects or withdraws consent. Pts with low (0-1) and intermediate (2-3) risk CLL-IPI score are assigned to an observation arm with follow-up once every 6 months for 2 years, and then according to routine clinical practice (Arm C). Endpoints: The primary endpoint of the intervention arms (Arms A and B) is the achievement of minimal residual disease (MRD)-negative complete remission in the bone marrow after 2 years of therapy. MRD is assessed using an 8-color flow cytometry with a detection limit of 0.01%. The primary endpoint of the observation arm (Arm C) is time to first therapy. Secondary endpoints for all arms are: progression-free survival, overall survival, and safety. Statistical design: A randomized trial comparing the experimental arm (acalabrutinib plus obinutuzumab) against the control arm (acalabrutinib alone) will be conducted as described by Rubinstein. A sample size of 36 pts per arm provides 80% power to detect an improvement in MRD-negative complete response rate from 10% to 30%, using a one-sided test at a significance level of 0.10 (EAST 6.4). We anticipate accruing an additional 8 high/very high risk pts (4 in each arm) to account for ineligibility, cancellation, or major treatment violation. The total enrollment for Arms A and B will be 80 pts. We will enroll 40 pts to Arm C; for a total enrollment of 120 pts. Key correlatives: Comprehensive profiling to assess the innate and adaptive immune system in paired peripheral blood and bone marrow will be conducted in all pts registered to Arms A and B at baseline, 12 and 24 months after enrollment. In addition, bone marrow hematopoietic function will be assessed in all pts at these time points. We will employ a CLL focused custom 61-gene panel to estimate tumor mutational burden as well as mutational profiles for each patient at baseline, 12, and 24 month time points, as well at the time of disease progression. Preliminary results: The trial is registered at clinicaltrials.gov NCT03516617. The trial opened to accrual at all three Mayo Clinic sites (Rochester, MN; Jacksonville, FL; and Scottsdale, AZ) in September 2018. Twenty-eight pts have been registered; the baseline characteristics are shown in Table 1. The trial is expected to complete enrollment in September 2020; and the primary analysis will be available in September 2022. Disclosures Parikh: Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Ding:DTRM Biopharma: Research Funding; Merck: Research Funding. Ailawadhi:Pharmacyclics: Research Funding; Cellectar: Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Chanan-Khan:Mayo Clinic: Employment; Ascentage: Research Funding; Millennium: Research Funding; Jansen: Research Funding; AbbVie: Research Funding; Xencor: Research Funding; Pharmacyclics: Research Funding; Merck: Research Funding. Kay:Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. OffLabel Disclosure: Drug: Acalabrutinib Purpose: treatment of CLL

INTRODUCTION Mutations in BTK and PLCG2 have been reported to occur in ~80% of CLL patients (pts) who have progression of disease on ibrutinib therapy (Woyach, JCO 2017; Ahn, Blood 2017). These mutations are described as appearing months before actual relapse and thus considered as a potential predictive biomarker for future relapse (Quinquenel, Blood 2019). However, the outcomes of these pts after disease progression are not well described. In this study, we seek to investigate time to next therapy and overall survival (OS) following progression among CLL pts on ibrutinib therapy with and without these resistance mutations. METHODS Between 10/2012 and 6/2019, we identified 34 pts in the Mayo Clinic clinical CLL resource who progressed while receiving ibrutinib therapy and also had testing for BTK and PLCG2 mutation performed as part of routine clinical practice at either NeoGenomics Laboratories or The Ohio State University. OS was calculated from time of ibrutinib progression to last known alive or death date; OS was plotted using Kaplan Meier methods and was compared using the log-rank test between various groups (e.g., mutation positive vs negative; CLL progression vs Richter's). Cumulative incidence of time to next treatment in those who had a treatment after progression was adjusted for the competing risk of death. RESULTS Of 34 pts who progressed while receiving ibrutinib, 26 pts experienced CLL progression and 8 pts had Richter's transformation; baseline characteristics in Table 1A. The presence of a BTK or PLCG2 mutation was found in 20/34 (59%) pts (specific mutations in Table 1B). BTK mutation alone was present in 9 pts, 7 pts had PLCG2 mutation alone, and 4 pts had both mutations. Median time between a positive test and start of next therapy was 4 months (range 1-19 months) and did not vary between BTK vs PLCG2 mutations. Among the 26 pts with CLL progression, 18 (69%) pts had a mutation present: BTK alone (n=8), PLCG2 alone (n=6), both (n=4). Therapy following progression on ibrutinib in these pts was as follows: venetoclax (n=16; 11 pts who continued ibrutinib in combination), idelalisib (n=4), investigational treatments (n=2), continued ibrutinib alone (n=2), dose-adjusted EPOCH-R (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab; n=1), and unknown (n=1). Twelve of the 26 pts with CLL progression on ibrutinib, including 8 pts with a prior resistance mutation detected, had subsequent progression of disease on the aforementioned next line therapy. Treatment of these patients consisted of the following: restarted ibrutinib in addition to current treatment of venetoclax (n=5), venetoclax (n=2), pembrolizumab (n=2; 1 pt with continued ibrutinib), obinutuzumab with continued ibrutinib (n=1), gemcitabine and vinorelbine with continued ibrutinib (n=1), and no further treatment (n=1). Among the 8 pts with Richter's transformation as the initial progression event on ibrutinib after mutation testing, 1 pt had a BTK mutation and 1 pt had a PLCG2 mutation. Treatments following progression on ibrutinib included multi-agent chemoimmunotherapy (n=3; 2 pts received rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone [R-CHOP] with continued ibrutinib and 1 pt received doxorubicin, bleomycin, vinblastine, dacarbazine [ABVD] alone), pembrolizumab (n=3; 1 pt in combination with continued ibrutinib), venetoclax in combination with continued ibrutinib (n=1), and venetoclax and obinutuzumab (n=1). The median time to next treatment (second line of treatment beyond ibrutinib) for the 31 pts who started another therapy following progression on ibrutinib was 16.7 months (95% CI 9.6-NE; Figure 1A) and was not significantly different for pts with or without a resistance mutation (p=0.57). Median OS for all 26 pts with CLL progression was 28.7 month and there was no difference according to presence or absence of a resistance mutation (median 28.7 months vs 18.2 months, p=0.53; Figure 1B). The 8 pts with Richter's transformation had a median OS of 7.1 months (95% CI 2.0-NE). CONCLUSION Approximately 60% of pts tested in this progression cohort had a BTK or PLCG2 mutation at time of or preceding progression on ibrutinib therapy. OS and time to next therapy did not differ statistically between pts with mutated vs non-mutated clones; however, caution should be applied with the conclusions given the limited sample size. Disclosures Ding: DTRM Biopharma: Research Funding; Merck: Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Parikh:Ascentage Pharma: Research Funding; Genentech: Honoraria; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding.

Background: The 2008 International Working Group for CLL (IWCLL) criteria define MBL as a clonal B-cell disorder with a peripheral blood B-cell count of <5 x 109/L, in the absence of cytopenias(s), lymphadenopathy and/or organomegaly on physical examination, and no other concomitant lymphoproliferative disorder. In the presence of palpable lymphadenopathy and/or organomegaly, and a B-cell count of < 5 x 109/L, a diagnosis of small lymphocytic lymphoma (SLL) is established. A study by Gentile et al (Am J Hematol. 2013) reported that 29 out of 62 individuals with clinically identified MBL (42%) had lymphadenopathy identified on CT imaging. Notably, however, after a median follow-up of 35 months, the rate of progression among cases of MBL with lymphadenopathy identified on CT imaging was only 6.9% (2/29 patients), which was no different than in cases of MBL without lymphadenopathy on CT imaging (rate of progression, 9%; 3/33 patients). Our study sought to 1) determine if lymphadenopathy and/or organomegaly detected by imaging studies performed for unrelated reasons in individuals with MBL can identify a subset with shorter time to first CLL therapy (TFT) and overall survival (OS); and 2) compare outcomes of individuals with MBL who have incidentally detected lymphadenopathy/splenomegaly and SLL patients. Methods: We used the Mayo Clinic CLL Database to identify individuals with high-count MBL (absolute B-cell count: 0.5 - 4.9 x 109/L) and divided them into the following three cohorts: a) Cohort A: no imaging studies available at MBL diagnosis; b) Cohort B: imaging studies done within one year of MBL with no evidence of lymphadenopathy and/or organomegaly; and c) Cohort C: imaging studies done within one year of MBL with lymphadenopathy and/or organomegaly, attributable to MBL. All individuals with MBL were first seen at Mayo Clinic after 1/1/2002; those with a concomitant lymphoproliferative disorder within 3 months of diagnosis were excluded. Patients with SLL seen during the same time interval were identified as a comparison cohort. We compared the baseline characteristics (Kruskal Wallis for continuous and Chi-square for categorical variables) and TFT and OS across the three MBL cohorts, and patients with SLL. Cumulative incidence of TFT was adjusted for the competing risk of death. Age- and sex-adjusted OS was analyzed using inverse weights methods. Results: 657 individuals with high-count MBL with a median follow-up of 6.7 years were identified: 415 (63%) individuals were in Cohort A, 142 (22%) in Cohort B, and 100 in (15%) Cohort C. Compared to Cohort B, individuals in Cohort C were more likely to have unmutated IGHV, high expression of CD38, and higher beta-2 microglobulin level (Table 1). SLL patients were younger, had higher rate of unmutated IGHV, and had high expression of CD49d compared to MBL individuals in Cohort C (Table 1). There was suggestive evidence of a difference in TFT between Cohorts B and C (estimated 10-year treatment rate 28.3% vs. 41.6%; p=0.16; HR 1.62 95% CI 0.84-3.15). TFT was significantly shorter in SLL patients compared to those with Cohort C (estimated 10-year treatment rate 71% vs. 41.6%, respectively, P<0.001; Figure 1A). There was a trend toward shorter OS in Cohort C compared to Cohort B (estimated 10-year OS rate 53.7% vs. 64.2%, respectively, P=0.09; Figure 1B). In contrast, there was no difference in OS among MBL individuals in Cohort C compared to those with SLL (estimated 10-year OS rate 56.9% vs. 62.6%, P=0.72). Summary/Conclusion: Our findings demonstrate that individuals with MBL who have incidentally identified lymphadenopathy/splenomegaly at diagnosis are more likely to have an unfavorable risk profile, shorter TFT and OS compared to patients who did not have lymphadenopathy/splenomegaly at MBL diagnosis. Additionally, these individuals have a longer TFT but similar OS compared to SLL patients seen during the same time interval. If externally validated, these findings have significant implications for individuals with newly diagnosed MBL. Disclosures Ding: Merck: Research Funding; DTRM Biopharma: Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Parikh:AstraZeneca: Honoraria, Research Funding; Janssen: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding.

Abstract Introduction: Richter transformation (RT) refers to transformation of chronic lymphocytic leukemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Most studies on the management of RT were either small retrospective series or early phase non-randomized trials before the era of novel agents. The natural history, prognostication and optimal treatment of patients with RT remain undefined. Here we report the clinical characteristics and outcomes of a large series of RT from a single center. Methods: Biopsy-confirmed RT (limited to non-Hodgkin lymphoma) diagnosed from 4/1993 to 4/2018 were identified from the Mayo Clinic CLL database. Clinical characteristics, treatment information and follow-up data were abstracted by chart review. Overall survival (OS) was defined as time from RT diagnosis to death from any cause and analyzed using the Kaplan-Meier method. Statistical analysis was done in SAS 9.4. Results: A total of 204 patients with CLL who developed RT were identified. The median age at CLL diagnosis was 62 years (range 22-85), and 148 (73%) were male. The median time to transformation was 4.7 years (range 0-34.5). Prior to RT, 68 (33.3%) patients received no treatment for CLL, 109 (53.4%) received chemoimmunotherapy (CIT) only, and 27 (13.2%) received at least one novel agent (idelalisib, ibrutinib, or venetoclax) for CLL. The median lines of CLL therapy prior to RT was 2 (range 0-13). The median age at RT diagnosis was 69 years (range 30-88). Pathology of RT was DLBCL and high grade B-cell lymphoma in 193 (94.6%) and 11 (5.4%) patients, respectively. The median LDH was 306 IU/L (range 99-9000). 62/125 (49.6%) patients had bulky disease (≥ 5 cm), and the median PET SUVmax was 13.9 (range 2.9-30.0). 45/131 (34.4%) patients had del(17p) or TP53 mutation, 12 (9.2%) had del(11q), 21 (16.0%) had trisomy 12, 27 (20.6%) had del(13q), and 25 (19.1%) had normal FISH. The CLL and RT were clonally related in 12/21 (57.1%) patients. For the transformed lymphoma, cell of origin by Han's algorithm was germinal center B cell-like (GCB) and non-GCB in 31/100 (31.0%) and 69/100 (69.0%) patients, respectively. EBV was positive in 14/52 (26.9%) patients. The median Ki-67 was 80% (range 10-100). Myc and Bcl-2 were positive by IHC in 31/43 (72.1%) and 83/103 (80.6%) patients, respectively; 27/56 (48.2%) were double-expressors. MYC, BCL2, and BCL6 rearrangement was positive by FISH in 18/68 (26.5%), 10/34 (29.4%), and 4/31 (12.9%) patients, respectively; 8/66 (12.1%) were double/triple-hit. The most common first-line treatment (Table 1 notes) of RT was R-CHOP-like regimen (n=114, 65.5%). Other treatments included R-EPOCH-like (n=6, 3.4%), high-intensity chemotherapy (n=15, 8.6%), novel agents (eg, ibrutinib, venetoclax, pembrolizumab; n=19, 10.9%), other chemotherapy (n=12, 6.9%), and palliative therapy (n=8, 4.6%). Response to first-line treatment was CR in 57 (38.0%), PR in 33 (22.0%), SD in 18 (12.0%), and PD in 42 (28.0%) patients. The median OS of the entire cohort after RT diagnosis was 12.0 months. The median OS for patients who received no prior CLL treatment, CIT only or at least one novel agent for CLL were 65.5, 7.3, and 12.0 months, respectively (P<0.0001; Figure 1). Of note, in patients who received CIT only for CLL, ~10% and 60% received high-intensity and R-CHOP/R-EPOCH-like chemotherapy, respectively, as first-line RT therapy. In contrast, in patients who had prior novel agents for CLL, 56% and 26% were treated with novel agents and R-CHOP/R-EPOCH-like chemotherapy, respectively, as first-line RT therapy. Patients with or without del(17p)/TP53 mutation had a median OS of 8.3 and 12.8 months, respectively (P=0.046). Patients who were treated with high intensity chemotherapy, R-CHOP/R-EPOCH-like regimens, novel agents, and other therapies for RT had a median OS of 35.1, 14.4, 10.9 and 6.1 months, respectively (P=0.02; Figure 2). OS comparisons by CLL/RT clonal relationship, double expressor or double/triple-hit status are shown in Table 1, with no significant differences noted. Conclusions: Over two thirds of RT were the non-GCB subtype, and about half were Myc/Bcl-2 double expressors. Patients who developed RT without prior CLL therapies had a significantly better OS. In contrast, patients who had received prior CLL therapies had poor outcomes. Myc/Bcl-2 double expressor and MYC/BCL2/BCL6 double/triple hit status had no impact on OS. Disclosures Kenderian: Humanigen: Research Funding; Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: 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; Janssen: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Cytomx Therapeutics: 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; Gilead: Membership on an entity's Board of Directors or advisory committees. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Gilead: Honoraria; Pharmacyclics: Honoraria, Research Funding. Ding:Merck: Research Funding.

Abstract Introduction: Given the established role of PD-1 in mediating immune suppression in chronic lymphocytic leukemia (CLL), we tested and reported the efficacy of PD-1 blocking antibody pembrolizumab in relapsed and transformed CLL patients. A selective response of pembrolizumab (~40%) in patients with RT, particularly after prior ibrutinib, was observed (Ding, Blood, 129:3419). Correlative analysis showed PD-1 expression in tumor B-cells of patients with RT and aggressive CLL after progression on ibrutinib. PD-1, an inhibitory receptor expressed on CLL T-cells, inhibits the immune synapse and cytotoxic T cell functions via the interactions with its ligands. However, the expression pattern and role of PD-1 in tumor B-cells is not well defined. In this study we investigated the functional implication of the PD-1 signaling axis in B-cell pathobiology in CLL and RT patients. Methods: 26 CLL-involved lymph node (LN) and 20 RT-involved LN were tested for PD-1 expression by immunohistochemistry (mouse clone NAT105, Abcam). For in vitro study, we checked PD-1 expression in 11 lymphoma cell lines and 1 CLL cell line by both flow cytometry and Western blot (WB) analysis. Effect of PD-1 knockdown using CRISPR/Cas9 system (Addgene) and over-expression of PD-1 using pLEX-lentiviral (Thermoscientific) or pRetro-retroviral (Clonetech) system were evaluated on pro-survival and apoptotic signaling pathways by Western blot analysis. Gene expression signatures in CLL and RT patients were also evaluated by Illumina-based RNA sequencing using 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). PD-1 expression was highest in patients with RT where the immediate prior CLL therapy was ibrutinib (Figure 1A). Among all cell lines tested for PD-1 expression, the expression of PD-1 by WB and flow was highest in Mino (mantle cell lymphoma line), followed by moderate expression in Jvm2 (B-PLL line) and Mec1 (CLL line), and very low-level expression in both Jeko-1 (B-NHL line) and lymphoma line 'Karpas299'. CRISPR/Cas9 mediated depletion of PD-1 in Mino cells inhibited constitutively active Akt, p70S6K and mTOR pathway, accompanied by significant downregulation of the anti-apoptotic proteins, Bcl-2, Mcl-1 and XIAP, but P-ERK1/2 was not affected. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in Jeko-1 and doxycycline regulated inducible retroviral (pRetro-PD-1) mediated overexpression of PD-1 in Karpas299 activated Akt, mTOR and p70S6K pathway. Overexpression of PD-1 in Jeko-1 significantly increased Bcl-2 and Mcl-1 and in Karpas299 increased Bcl-2, Mcl-1 and XIAP expression (Figure 1B). A parallel genetic analysis using RNA sequencing was performed on 5 nodal tissues involved by either RT or progressive CLL after these patients developed clinical progression after prior ibrutinib therapy. In all 5 patients, overexpression of PD-1 was associated with increased expression of Bcl-2 and mTOR regardless of the genetic mutations detected (including TP53, ATM, BTK, NOTCH1, XPO1, SF3B1, TET2 etc). However, in 2 patients who received prior chemoimmunotherapy, similar overexpression of gene signature was not observed by RNA sequencing analysis, alternative pathways including Met or NFkB overexpression was detected. Given these clinical and laboratory findings, we have treated 2 RT patients with a combination of BTK and Bcl-2 inhibitors (ibrutinib and venetoclax, respectively) whose CLL transformed after prior ibrutinib. Significant reduction of tumor burden was observed in both cases with one complete response and one mixed response. Conclusion: An increased expression of PD-1, Akt/mTOR and Bcl-2 gene signature was first observed in RT patients after prior ibrutinib therapy. PD-1 overexpression in the tumor B-cells of RT and progressive CLL patients likely regulate AKT/mtOR to upregulate Bcl-2. Targeting both BTK and Bcl-2 pathways in addition to PD-1 blockade appear to be a promising strategy to treat these aggressive diseases. Disclosures Parikh: Gilead: Honoraria; Janssen: Research Funding; Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding. Ansell:Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Celldex: Research Funding; Merck & Co: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Research Funding; Pfizer: Research Funding. Kay:Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: 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; Gilead: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: 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; Janssen: Membership on an entity's Board of Directors or advisory committees. Ding:Merck: Research Funding.

Abstract Background Predictors of outcomes after reduced intensity conditioning (RIC) stem cell transplantation (SCT) in chronic lymphocytic leukemia (CLL) are not well established. The Dana Farber (DF) investigators (Brown et al, leukemia 2013) developed a prognostic scoring model for overall survival (OS) and disease free survival (DFS) that assigned one point to each of comorbidity index, absolute lymphocyte count, LDH and disease status. The goals of the present study were to 1) validate the DF model 2) identify additional predictors of outcomes after SCT. Methods This study is a retrospective review of the Mayo Clinic CLL database and was approved by the Institutional Review Board. All CLL patients that underwent RIC-SCT were included. The prognostic significance of prognostic factors at time of RIC-SCT were analyzed, including the DF model. Survival was estimated and compared using the Kaplan Meier and Log Rank tests. Results From 2006 to 2012, 50 patients with CLL underwent RIC-SCT at the Mayo Clinic. Of these, 33 were male (66%) and the median age was 56 (29-69) years. The median time from diagnosis to RIC-SCT was 4.7 years (0.6-22.9). Indications for RIC-SCT were 17p deletion in 24 (48%) patients and purine nucleoside analogue (PNA) refractory disease in 26 (52%) patients. Transplant related mortality was 6% and non-relapse mortality at 5 years was 14%. Acute graft versus host disease (GVHD) developed in 30 (60%) patients and chronic (c) GVHD was noted in 32 patients (64%). The 5-year relapse rate was 38%. The DF prognostic score stratified three categories for OS (score of 0, 1-2, ≥ 3), but was not associated with DFS in this cohort. We next evaluated the impact of other patient, disease, and transplant characteristics on clinical outcome. Development of cGVHD post-transplant was the dominant predictor of both DFS (HR 0.26, 95% CI=0.10-0.69, P=0.006) and OS (HR 0.04, 95% CI=0.01-0.19, P<0.0001, figure 1). The presence of 17p deletion at time of RIC-SCT was associated with shorter DFS (HR 3.99, 95% CI=1.43-11.18, P=0.008), but did not impact OS. Developing cGVH but not DF score remained an independent predictor of OS on a multi-variate analysis including both factors. Given these findings, we evaluated what pre-treatment patient, disease, and transplant characteristics predicted for subsequent development of cGVHD. Patients with higher scores on DF prognostic model were less likely to develop cGVHD (score 0=66%, 1-2=76%, ≥3=31%; P=0.01). Three CLL characteristics (complete or partial remission at time of RIC-SCT, absence of 17p deletion, and absence of ZAP70 overexpression) were predictors for development of cGVHD while alemtuzumab exposure ≤3 months prior to RIC-SCT decreased likelihood of cGVHD. On multivariate modeling using these four predictors, only ZAP70 overexpression was an independent negative predictor of developing cGVHD (Table 1). Rates of cGVHD post RIC-SCT in ZAP 70 positive and ZAP70 negative patients were 50% and 92%, respectively (p=0.03). Conclusion This study found that while the DF prognostic score is an effective way to predict outcome prior to RIC-SCT, development of cGVHD post-transplant is ultimately the most significant predictor for both OS and DFS after transplant in patients with CLL. Pretransplant characteristics that predict subsequent cGVHD include DF prognostic score and ZAP70 status. This is the first report to identify biologic characteristics of CLL B-cells associated with development of cGVHD post-transplant. Disclosures: No relevant conflicts of interest to declare.

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is defined as somatic mutations in clonal blood cells of individuals without any other hematological abnormalities and have been identified using whole exome sequencing (WES) from pre-specified genes. CHIP is reportedly associated with aging, reduced overall survival, cardiovascular disease, and hematologic disease, mainly myeloid cancers. Additionally, whole genome sequencing and WES studies in CLL cases have identified putative CLL driver genes with recurrent somatic variants or copy number alterations. Here, we investigate the associations of CHIP and mutations in CLL somatic driver genes with risk of CLL and MBL, a precursor to CLL. Using WES, 571 individuals from 132 CLL families were sequenced from two different sites, Mayo Clinic (92 families) and National Cancer Institute (NCI, 40 families), including 251 CLLs, 74 MBLs, and 246 unaffected relatives. DNA was extracted from buccal cells (Mayo Clinic) and peripheral blood (NCI). Allele counts from gVCF files were extracted for specific genes and analyzed. Variants were annotated using the Variant Effect Predictor in Ensembl, genome build GRCh37 (http://useast.ensembl.org/index.html). Highly-sensitive flow cytometry of the blood was used to screen for MBL. CHIP was defined as in Genovese et al., 2014 as the presence of any of the following: A) disruptive mutations in ASXL1, TET2, or PPM1D; B) missense mutation p.617F in JAK2; C) disruptive or missense mutations in DNMT3Alocalized to exons 7 to 23D) any mutations reported at least 7 times in hematological malignancies in the Catalogue of Somatic Mutations in Cancer (COSMIC); and (E) No variant allelic fraction (VAF) threshold. CLL driver genes included disruptive or missense mutations in ATM, BIRC3, MYD88, NOTCH1, SF3B1, TP53, EGR2, POT1, NFKBIE, XPO1, or FBXW7with VAF greater than 0.1. Mutations from COSMIC and CLL driver genes were excluded if the minor allele frequency in 1000 Genomes was greater than 0.5%. Data was analyzed using logistic regression adjusting for age, sex, and site. Odds ratios (OR) and 95% confidence intervals (CI) were estimated. Median age for CLL, MBL, and unaffected was 63, 61.5, and 54, respectively. The unaffected relatives were significantly younger than both the CLL and MBL groups (p<0.0001), with no difference between CLL and MBL (p=0.46). A total of 70 (12%) individuals from 54 different families had CHIP. CHIP was characterized in the majority of samples as mutations in DNMT3A, ASXL1, and TET2with 43, 9 and 8 individuals, respectively. As expected, CHIP was associated with age (p<0.001) but not sex (p=0.87). CHIP was present in 42 (17%) CLLs, 9 (12%) MBLs, and 19 (8%) unaffected relatives. There was a significant association between the presence of CHIP and CLL risk compared to unaffected relatives (OR= 2.19, 95% CI: 1.20 - 4.13, p=0.013) but not with MBL risk (OR=1.34, 95% CI: 0.54 - 3.11, p=0.51). Using the CLL driver genes, we found mutations in 119 (47%) CLL, 20 (27%) MBL, and 70 (29%) unaffected relatives. Mutations in CLL driver genes were associated with CLL risk compared to unaffected relatives (OR = 2.51, 95% CI: 1.64 - 3.87, p<0.0001), but there was no evidence for increased MBL risk (OR = 1.00). These results held when we subset the analysis to the Mayo only data where the DNA was extracted from buccal cells. This study further supports the association between mutations in CHIP related genes and risk of hematologic malignancies, in particular CLL. This is the first report of the prevalence of CHIP in MBLs. Although MBL and CHIP represent clonal population of blood cells, our data suggest the clones are different. Mutations in CLL driver genes are also associated with CLL risk but show little to no evidence with increased MBL risk. Interestingly, our unaffected relatives of CLL cases also had a high frequency of CLL driver mutations. Future studies are needed to understand these acquired mutations (both in CHIP- and CLL driver- related genes) in the unaffected to MBL to CLL continuum. Disclosures Parikh: Abbvie: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria; Janssen: Research Funding. Ding:Merck: Research Funding. Cerhan:Jannsen: Other: Scientific Advisory Board; Celgene: Research Funding; Nanostring: Research Funding. Shanafelt:Jansen: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding; GlaxoSmithKline: Research Funding. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: 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; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Backgrounds B cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. Methods A phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. Results Nine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6). Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. Conclusion The Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. Acknowledgment This work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures: Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.