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Background: Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL), and several studies suggest that hypogammaglobulinemia at the time of CLL diagnosis may predict shorter time to first therapy (TTFT; Parikh et al, 2015). However, it is unclear if hypogammaglobulinemia can predict TTFT independent of the CLL-International Prognostic Index (CLL-IPI) and the tumor mutational load (TML). We sought to determine if serum immunoglobulin (Ig) levels at diagnosis can independently predict outcomes in patients with CLL and its precursor, high-count monoclonal B-cell lymphocytosis (MBL). Methods: We used the Mayo Clinic CLL Database to identify newly diagnosed CLL/MBL consented within 2 years of diagnosis. Next generation sequencing (NGS) was performed using a 59 gene panel (SureSelect) to detect mutated CLL genes. Serum Ig levels were quantitated by radial immunodiffusion (Immunoplates). Serum Ig levels below the lower limit of normal were defined as hypogammaglobulinemia. We used Chi-squared or Fisher's exact tests to compare discrete variables and the Kruskal Wallis test for continuous variables. TTFT was calculated from date of diagnosis to date of first treatment or date last known to be untreated. We used Cox regression models to estimate hazard ratios (HRs) and 95% confidence intervals (CIs) for TTFT and overall survival (OS) associations. Results: We identified 895 newly diagnosed patients (588 CLL and 307 MBL) who had undergone NGS for recurrent somatic mutations and had serum Ig levels available at the time of diagnosis. The median absolute B-cell count was 6.7 x109/L; 613 (69%) were male, 375 (42%) had unmutated IGHV genes, and 81 (9%) had TP53 disruption. Based on CLL-IPI, 412 (46%) patients had low risk, 285 (32%) had intermediate risk, and 198 (22%) had high/very high risk. The most commonly mutated genes were NOTCH1 (14%), SF3B1 (11%), TP53 (10%), NFKBIE (8%), ATM (6%), and BIRC3 (6%). Based on the number of genes with high impact or hotspot mutations, we classified 40%, 29%, and 31% patients with TML scores of 0, 1, or 2+, respectively. At CLL/MBL diagnosis, the median serum IgG, IgA, and IgM was 891 mg/dL (111-2620), 121 mg/dL (1-1150), and 44 mg/dL (5-1320), respectively; 284 (32%) patients had low serum IgG, 128 (17%) had low serum IgA, and 322 (42%) had low serum IgM. Low serum IgA was associated with mutations in NOTCH1, SF3B1, ATM, BIRC3 and NFKBIE, low IgM was associated with SF3B1 and BIRC3 mutations (all comparisons, P < .05), and low IgG was associated with BIRC3 mutation (P = .05). The median follow-up for the cohort was 8.4 years. A total of 359 patients progressed requiring therapy (299 CLL and 60 MBL), and 265 patients died (177 CLL and 88 MBL). The TTFT was significantly shorter for patients with low serum IgG compared to normal serum IgG at diagnosis (median 4.4 vs 10.8 years) and similar findings were observed in individuals with low serum IgA (1.7 vs 10.8 years) and those with low serum IgM (4.6 vs 11.1 years). On univariable analysis, the following were associated with a shorter TTFT: low serum IgG (HR: 1.8, 95% CI 1.4-2.2), low serum IgA (HR: 3.5, 95% CI 2.7-4.4), low serum IgM (HR: 1.9, 95% CI 1.5-2.3), TML score of 1 (HR: 1.7, 95% CI 1.3-2.2), TML of 2+ (HR: 4.4, 95% CI 3.4-5.7), CLL-IPI intermediate risk (HR: 3.7, 95% CI 2.8-4.8), and CLL-IPI high/very high risk (HR: 7.6, 95% CI 5.8-10.1) (all comparisons, P < .0001). In multivariable analyses after adjusting for CLL-IPI and TML, low serum levels of IgG (HR: 1.8, 95% CI: 1.5-2.3), IgA (HR: 2.4, 95% CI: 1.8-3.0), and IgM (HR: 1.8, 95% CI: 1.5-2.3) were independently associated with a shorter TTFT (all comparisons, P < .0001). No significant differences in OS were detected between patients with low and normal serum IgG, IgM, and IgA on univariate analysis. Similar results for TTFT and OS were observed after adding clonal B-cell count to the Cox model. Conclusions: Our study shows that low serum Ig levels at the time of initial diagnosis is predictive of TTFT independently of the CLL-IPI and TML, which can further risk stratify individuals with newly diagnosed CLL/MBL. Additionally, low serum IgA, IgM, and IgG levels at diagnosis are significantly associated with specific somatic mutations in individuals with newly diagnosed CLL/MBL.

Chronic lymphocytic leukemia (CLL), one of the most common types of leukemia among adults in the US, is characterized by the accumulation of CD5+CD19+ monoclonal B cells. Current clinical treatment approaches for progressive CLL patients focus on novel agents targeting Bruton Tyrosine Kinase (BTK) and Bcl-2. Although clinical trials combining novel agent have shown significant efficacy with high overall response and, in some cases, achievement of high rates of undetectable measurable residual disease (MRD), CLL remains incurable with a majority of patients eventually experiencing relapse of their disease. Therefore, it remains of high interest to expand therapeutic options. Transcriptional chemical inducers of proximity (TCIPs) are small molecules designed and developed by Dr. Gerald R. Crabtree at Stanford University. TCIPs are a new class of anticancer drugs that recruit endogenous cancer drivers or a downstream transcription factor to the promoters of cell death genes and activate their expression, resulting in apoptosis of cancer cells. Current TCIPs are produced by covalently linking small molecules that bind B-cell lymphoma 6 (BCL6) to those that bind to transcriptional activators, such as Bromodomain-containing protein 4 (BRD4) or cyclin-dependent kinases 9 (CDK9). The Stanford team has demonstrated TCIPs generate remarkable killing in diffuse large B cell lymphoma (DLBCL) cell lines, including chemotherapy-resistant, TP53-mutant lines, with a half maximal effective concentration (EC50) of 1-10nM at 72hours (h) and exhibited cell-specific and tissue-specific effects (Gourisankar et al, 2023). We have initiated TCIPs cytotoxicity assessment in primary leukemic cells from untreated CLL patients. Testing two types of TCIPs with a flow cytometry-based apoptosis assay, we found time and dose-dependent drug-associated killing in primary CLL B cells collected from untreated CLL patients over a 24-72h interval. The IC50 for BRD4-BCL6 TCIP JWZ-7-133 at 72h was 560nM while CDK9-BCL6 TCIP BAK-04-232 at 72h was 10nM. After 24, 48, and 72h exposure of CLL B cells to TCIP JWZ-7-133, the IC50 was 1050, 820 and 560nM, respectively. We also found that TCIP JWZ-7-133 exhibited a similar ability to inhibit leukemic cell viability with an IC50 of 1070nM using a cell viability assay, compared to a flow cytometry-based cell death assay (24h IC50 1050nM). CDK9-BCL6 TCIPs are designed and produced by covalently linking BI-3812 (binding to BCL6) and SNS-032 (binding to CDK9) with a rigid linker. BI-3812 is a potent and efficacious BCL6 inhibitor, inhibiting the BTB domain of BCL6, and SNS-032 is a potent and selective inhibitor of CDK9. When CLL B cells were treated with only BI-3812 or SNS-032 or TCIP BAK-04-232 as single agents for 72h, we found that TCIP BAK-04-232 had superior and significant killing effects compared to the single agents, with an IC50 of 10nM. Specifically this killing activity was more than 10-fold compared to SNS-032 alone (IC50 over 100nM), and more than 1,000-fold compared to BI-3812 alone (IC50 over 10,000nM). There remains an urgent to identify new categories of potent drug, inhibitor, or small molecules to expand potential clinical treatment options for patients with CLL. The results presented here indicate that TCIPs targeting BCL6 and BRD4/CDK9 have a strong capacity to induce high levels of cell death in CLL B cells at nanomolar concentrations, and this remarkable killing effect may be due to its unique gain-of-function drug mechanism. TCIPs provide a completely new drug direction and strategy for the design of anticancer drugs: to rewire cancer drivers to activate apoptosis in cancer cells. This study is the first of a kind demonstration of TCIPs in robustly targeting CLL B cells. Further studies to fully validate the preclinical activity of TCIPs and investigate the molecular mechanism(s) for TCIP mediated killing of leukemic B cells will help to define the role of TCIPs as a novel therapeutic approach in CLL.

Introduction Venetoclax + rituximab resulted in a 2-year progression-free survival estimate of 85% in the mostly 2nd-line, post-chemotherapy setting of the MURANO clinical trial (Seymour NEJM 2018). Short remissions with venetoclax in patients (pts) with prior cBTKi treatment (tx) for CLL are reported. However, these cohorts have included heavily pre-treated pts. Here, we report outcomes with venetoclax-based regimens in a contemporary cohort with cBTKi-exposed CLL and no prior chemotherapy. Methods We conducted a retrospective study of pts who received venetoclax for relapsed CLL after prior cBTKi tx at Mayo Clinic and across 8 centers in France. Pts with any prior chemotherapy tx were excluded. Pts were grouped as cBTKi-resistant (CLL disease progression on cBTKi) and cBTKi-exposed (any prior cBTKi tx but without disease progression). Overall survival (OS) was analyzed as the time from initiation of venetoclax until date of death or last known to be alive. Time to next therapy or death (TTNTD) after venetoclax was defined as the time from venetoclax initiation until the earliest date of next tx or death. Cox proportional hazards regression models were used to estimate associations of factors with OS and TTNTD. Results A total of 85 pts received venetoclax, including: venetoclax monotherapy (n=13, 15%), venetoclax + rituximab (n=45, 53%), and venetoclax + obinutuzumab (n=27, 32%). Patient characteristics at the time of venetoclax start were as follows: median age 69 years (interquartile range [IQR] 62-77), 59 (69%) male, 45% (33/73 evaluated) with TP53 disruption (del(17p) or TP53 mutation), 82% (46/56 evaluated) with unmutated IGHV, and 41% (15/37 evaluated) with complex karyotype (CK; ≥3 clonal structural or numerical abnormalities on karyotype). The median prior lines of therapy was 1 (IQR 1, 2); 32 (38%) pts had received prior anti-CD20 monoclonal antibody and 8 (9%) pts had received 2 prior cBTKi. Prior cBTKi tx was stopped for a reason other than disease progression in 46 (54%) pts (cBTKi-exposed) and due to CLL progression in 39 (46%) pts (cBTKi-resistant). TP53 disruption, unmutated IGHV, and CK were more common in the cBTKi-resistant subgroup. Among pts with cBTKi-resistant CLL, 60% (12/20 evaluated) had a BTK and/or PLCG2 mutation. Median follow-up from start of venetoclax-based therapy was 20 months. The median TTNTD and OS for the entire cohort was 36 (95% CI 29-not evaluable [NE]) and 68 (95% CI 39-NE) months, respectively. Among the 46 pts with cBTKi-exposed CLL, the median duration of prior cBTKi tx was 6.7 months (IQR 1.8, 23.7). The median TTNTD and OS from venetoclax start were both 78 months in pts with cBTKi-exposed CLL. The 24-month TTNTD and OS estimates were 73% and 81%, respectively. Among the 39 pts with cBTKi-resistant CLL, the median duration of prior cBTKi tx was 42.3 months (IQR 23.4, 60.1). The median TTNTD from venetoclax start in pts with cBTKi-resistant CLL was 30 months. The 24-month TTNTD estimate was 65% in the cBTKi-resistant subgroup, 53% with venetoclax monotherapy (n=5), 62% with venetoclax + rituximab (n=30), and 100% venetoclax + obinutuzumab (n=4). The median OS from venetoclax initiation was 39 months overall in pts with cBTKi-resistant CLL. The 24-month OS estimate was 84% in the cBTKi-resistant subgroup overall, 53% with venetoclax monotherapy, 87% with venetoclax + rituximab, and 100% with venetoclax + obinutuzumab. On a univariate analysis in the whole cohort, only TP53 disruption was associated with shorter TTNTD (HR 2.23; 95% CI 1.02-4.87; P=0.04) and only older age was associated with shorter OS (HR 1.05; 95% CI 1.007-1.095, P=0.02). On a univariate analysis in the cBTKi-resistant subgroup, only TP53 disruption was associated with shorter TTNTD (HR 6.68; 95% CI 1.51-29.46; P=0.01) and OS (HR 7.95; 95% CI 1.02-61.97; P=0.04). Duration of prior cBTKi exposure (as a continuous variable) was not associated with survival outcomes in the cBTKi-resistant subgroup. Conclusions Pts with chemotherapy-naïve, cBTKi-treated CLL had worse than expected outcomes with venetoclax-based tx in a mostly 2nd-line tx cohort, particularly pts with cBTKi-resistant CLL. The frequency of high-risk disease features, shorter duration of disease control with their initial cBTKi tx, and small cohort size warrant caution in generalizing these results to all pts with cBTKi-resistant disease. Novel tx trial strategies in 1st- and 2nd-line settings remain a high priority for high-risk pts.

Background: Chronic lymphocytic leukemia (CLL) is one of the most common leukemias in the United States (US), with an estimated incidence of 20,700 cases in 2024. Population-based studies conducted in Olmsted County (OC), Minnesota, reported an increase in CLL incidence over a 55-year period (1935 through 1989), which was attributed to increased detection of early-stage cases in individuals over age 50. In an updated study from 2000 to 2010 in OC, we reported no further increase in the incidence of CLL using the 2008 International Workshop on CLL (IWCLL 2008) guidelines. Additionally, when we compared the 1996 National Cancer Institute Working Group (NCI-WG 96) criteria with the IWCLL 2008 guidelines incorporating the monoclonal B-cell lymphocytosis designation, we found that the use of the IWCLL 2008 guidelines reduced the overt CLL incidence, modified the distribution of Rai Stages at diagnosis, and shortened the median time to first treatment (TTFT). Over the last two decades, 5-year survival for CLL has increased due to new therapies and improved supportive care. Herein we provide updated data on the incidence, prevalence, and natural progression of CLL in OC and studied similar features in an expanded 6-county region around Mayo Clinic, Rochester. Methods: We utilized the Rochester Epidemiology Project (REP), a population-based medical records linkage system, to identify CLL cases from 2000 to 2020 using International Classification of Diseases codes. We included individuals residing in OC and the expanded 7-county region (REP-7, including OC), for which data became available starting in 2010. CLL cases were included if they met the IWCLL 2008 criteria. All CLL diagnoses were confirmed by manual review of medical records and data on CLL-related factors were collected, including treatment status, prior history of other cancers, and overall survival (OS). Age- and sex-specific incidence rates were derived from REP census data and adjusted to the 2020 US white population. OS analyses were conducted using the Kaplan-Meier method. TTFT was calculated with death as a competing risk. Cox regression was used to evaluate the effect of decade on TTFT and OS among patients in OC. Results: From 2010-2020, 242 patients were diagnosed with CLL in REP-7, and between 2000-2020, 174 patients were diagnosed in OC. Among the 242 CLL patients in REP-7, the median age at diagnosis was 71 years (range 30-96),158 (65%) were male, 236 (99%) were white, and 74 (31%) had a history of other cancer, with non-melanoma skin cancer being the most common. After age and sex adjustment to the 2020 US white population, the CLL incidence in REP-7 and OC from 2010 to 2020 was 7.8 and 8.1 per 100,000 individuals, respectively. In OC, the age- and sex-adjusted CLL incidence from 2000 to 2020 was 7.5 per 100,000 individuals, showing no significant trend changes over time. Among the 242 patients diagnosed with CLL in REP-7 between 2010 and 2020, 102 received treatment with a median TTFT of 9.8 years. In OC from 2000 to 2020, 79 out of 174 patients received treatment with a median TTFT of 9.4 years. When comparing across two decades in OC, the median TTFT was 12.8 years for the period from 2000 to 2009, decreasing to 5.9 years between 2010 and 2020, but was not significantly different after accounting for age and sex. The median OS for patients diagnosed with CLL in REP-7 from 2010 to 2020 was 11 years. The median OS for patients in OC between 2000 to 2020 was 12.6 years. When comparing across decades in OC, the median OS was 8.9 years for the period from 2000 to 2009, increasing to 13.6 years for those diagnosed between 2010 and 2020, but this trend was not significantly different after accounting for age and sex. Prevalence data for REP-7 and OC were analyzed as of 01/01/2020. Additionally, prevalence data for OC were available for 01/01/2010. In REP-7, the prevalence of CLL was 97 per 100,000 individuals on 01/01/2020. In OC, the CLL prevalence was 86 per 100,000 individuals on 01/01/2010, and 100 per 100,000 individuals on 01/01/2020. Conclusions: We observed no substantial increase in the incidence rates of CLL in REP-7 from 2010 to 2020, nor in the 20 years of available data from OC. However, the prevalence of CLL in OC increased by 16% from 2010 to 2020. Additionally, evidence indicated a longer OS among newly diagnosed CLL cases in OC compared to previous decades, suggesting a potential impact of improved therapeutic approaches on OS.

Introduction: Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant lymphocytes in the lymph nodes, spleen, blood, and bone marrow. Although current clinical treatment approaches targeting Bruton Tyrosine Kinase (BTK) and Bcl-2, either as monotherapy or in combination are highly effective with high overall response, complete responses, and achievement of undetectable measurable residual disease (MRD), most patients eventually experience disease relapse. In this study, we utilized our unique mesenchymal stromal cells (MSCs) system, which mimics the in vivo stromal environment in CLL, to explore the combination interaction effect of the first-in-class BTK inhibitor, ibrutinib, with novel tumor microenvironment (TME)-directed drugs in CLL. Methods: Since 1999, patients with CLL seen at Mayo Clinic in Rochester, MN have been offered participation in a longitudinal study of CLL B-cell Biology (CLL Tissue Bank). This Tissue Bank is seamlessly integrated into the Mayo Clinic CLL Database where the clinical characteristics of all patients are prospectively maintained. Using this resource, we identified previously untreated CLL patients (n=10) for our experiments. Individuals with high risk CLL (defined as those with both unmutated IGHV gene mutations and either del17p- or del11q- by FISH) (n=5) and with low risk CLL (defined as those with both mutated IGHV genes and no del17p- or del11q- by FISH) (n=5) were studied. Annexin-V/PI apoptotic cell death assays were employed to evaluate the effects of TME-directed drugs including everolimus (mTOR inhibitor), MI-2 (MALT1 inhibitor), ruxolitinib (JAK1/2 inhibitor), S63845 (MCL-1 inhibitor), and venetoclax (Bcl-2 inhibitor) either as single agent, or in combination with ibrutinib on CLL B cells in direct contact with MSCs. Dose response curves and IC50 levels were generated for single agent killing effect in CLL. The combination interaction effects were determined by Combination Index (CI) values generated by CalcuSyn software indicating synergistic (CI < 0.9), additive (CI between 0.9 - 1.1), or antagonistic (CI > 1.1) interactions. Among drug combinations with synergistic CI values, the following indicated different levels: below 0.1 very strong, 0.1 - 0.3 strong, 0.3 - 0.7 good, and 0.7 - 0.9 moderate synergism. Results: All tested drugs exhibited different IC50 levels in CLL cells co-cultured with MSCs: MI-2 IC50 of 3.5uM, ruxolitinib IC50 of 18.5uM, S63845 IC50 of 0.115uM, venetoclax IC50 of 0.006uM, and both everolimus and ibrutinib did not reach IC50 levels at maximum tested doses. Of interest we observed more killing in high versus low-risk cohort when treated with everolimus (19uM vs. undetectable), MI-2 (2.5 vs. 4uM) and S63845 (0.09 vs. 0.14uM) as single agents (IC50, high vs. low, respectively), while ruxolitinib and venetoclax showed no differences. Combination interaction effect results showed highly synergistic interactions for all patients (good to strong synergism) when ibrutinib was combined with S63845 or venetoclax, moderate to strong synergism for ibrutinib and everolimus, moderate synergism to additive levels for ibrutinib and MI-2, and moderate synergism to antagonism for ibrutinib and ruxolitinib. We also observed more synergism in the low-risk cohort when treated with ibrutinib and MI-2/ruxolitinib combinations, but no difference with ibrutinib and everolimus/S63845/venetoclax combinations between low and high-risk cohort. Conclusions: In summary, we evaluated the pre-clinical combination effects of ibrutinib when used together with other novel agents on CLL B cells in the presence of TME, where patients were assigned to low or high-risk cohorts. Our study showed that ibrutinib exhibits highly synergistic effects with venetoclax, S63845 and everolimus in both cohorts. The observed difference in effectiveness between high and low-risk cohorts when treated with ibrutinib plus MI-2 or ruxolitinib suggests these combinations would be beneficial for a subset of patients with high risk CLL. These preclinical data offer preliminary insights that support the evaluation of therapeutic combinations in patient with high-risk CLL: a population with significant unmet medical needs. Funding Statement: This study was funded and supported by Pharmacyclics LLC, an AbbVie company.

Abstract Purpose: Although the B-cell receptor (BCR) signal plays a critical role in chronic lymphocytic leukemia (CLL) cell survival and a target of current therapies (ibrutinib targets Bruton’s tyrosine kinase; idelalisib targets PI3Kδ), contribution of the cytokine-driven JAK2 pathway to the “CLL cell-survival signaling network” is largely undefined. Experimental Design: Patients with CLL were enrolled to investigate expression/activation of JAK2 and acylglycerol kinase (AGK), and their functional implication in primary CLL cell survival. A series of biochemical and molecular biology assays were employed to uncover the underlying mechanism. Results: We detected that compared with normal B cells, CLL cells aberrantly express constitutively active JAK2. Mechanistically, HSP90 forms a chaperoning complex with JAK2, resulting in its aberrant accumulation in CLL cells. We also discovered aberrant upregulation of a novel mitochondrial lipid kinase, AGK, which remains complexed with HSP90 in CLL cells activating JAK2. Although AGK is typically mitochondrial, we detected its nuclear localization in association with JAK2 in some CLL cells. Functionally, JAK2 phosphorylates its noncanonical substrate, histone H3(Y41), but not STAT3, activating transcription of diverse sets of genes in a patient-specific manner. Additionally, JAK2 activates the BCR signal in CLL cells via LYN/Bruton’s tyrosine kinase axis. Targeted inhibition of JAK2 as monotherapy, or in combination with the BCR inhibitors or venetoclax (a BCL2 inhibitor), induced apoptosis synergistically in CLL cells. Conclusions: These findings suggest that aberrantly expressed AGK activates JAK2, independent of cytokine, leading to activation of diverse sets of gene transcription in CLL cells. Combined targeting of JAK2 and BCR signals or BCL2 may be effective in some patients with CLL.

Abstract Chronic lymphocytic leukemia (CLL) patients are immunocompromised and highly vulnerable to serious recurrent infections. Conventional dendritic cells (cDCs) and plasmacytoid DCs (pDCs) are principal sensors of infection and are essential in orchestrating innate and adaptive immune responses to resolve infection. This study identified significant deficiencies in six functionally distinct DC subsets in blood of untreated CLL (UT-CLL) patients and selective normalization of pDCs in response to acalabrutinib (a Bruton tyrosine kinase inhibitor) therapy. DCs are continuously replenished from hematopoiesis in bone marrow (BM). Four BM developmental intermediates that give rise to cDCs and pDCs were examined and significant reductions of these were identified in UT-CLL patients supporting a precursor/progeny relationship. The deficiencies in blood DCs and BM DC progenitors were significantly associated with alterations in the Flt3/FL signaling pathway critical to DC development and function. Regarding clinical parameter, cDC subset deficiencies are associated with adverse prognostic indicators of disease progression, including IGHV mutation, CD49d, CD38, and ZAP-70 status. Importantly, UT-CLL patients with shared DC subset deficiencies had shorter time-to-first treatment (TTFT), uncovering a profound alteration in innate immunity with the potential to instruct therapeutic decision-making.

PURPOSE Chronic lymphocytic leukemia (CLL)–phenotype monoclonal B-cell lymphocytosis (MBL) is a premalignant condition that is roughly 500-fold more common than CLL. It is unknown whether the two-fold increased risk of developing melanoma associated with CLL extends to individuals with MBL. METHODS Using the Mayo Clinic Biobank, we identified participants who were 40 years or older with no previous hematological malignancies, who resided in the 27 counties around Mayo Clinic, and who had available biospecimens for screening. Eight-color flow cytometry was used to screen for MBL. Individuals with MBL were classified as low-count MBL (LC-MBL) or high-count MBL on the basis of clonal B-cell percent. Incident melanomas were identified using International Classification of Diseases codes and confirmed via medical records review. Cox regression models were used to estimate hazard ratios (HRs) and 95% CI. RESULTS Of the 7,334 participants screened, 1,151 were identified with a CD5-positive MBL, of whom 1,098 had LC-MBL. After a median follow-up of 3.2 years (range, 0-13.5), 131 participants developed melanoma, of whom 36 individuals were positive for MBL. The estimated 5-year cumulative incidence of melanoma was 3.4% and 2.0% among those with and without MBL, respectively. After adjusting for age, sex, and history of previous melanoma, individuals with MBL exhibited a 1.86-fold (95% CI, 1.25 to 2.78) risk of melanoma. This elevated risk persisted when analysis was restricted to those without a history of melanoma (HR, 2.05 [95% CI, 1.30 to 3.23]). Individuals with LC-MBL had a 1.92-fold (95% CI, 1.29 to 2.87) increased risk of developing melanoma overall and a 2.74-fold increased risk (95% CI, 1.50 to 5.03) of melanoma in situ compared with those without MBL. CONCLUSION LC-MBL is associated with an approximately two-fold increased risk of melanoma overall and a 2.74-fold increased risk of melanoma in situ.

Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNAs) and somatic mutations that are central to disease prognosis, risk stratification, and mechanisms of therapy resistance. Fluorescence in situ hybridization (FISH) panels are widely used in clinical applications as the gold standard for screening prognostic chromosomal abnormalities in CLL. DNA sequencing is an alternative approach to identifying CNAs but is not an established method for clinical CNA screening. We sequenced DNA from 509 individuals with CLL or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, using a targeted sequencing panel of 59 recurrently mutated genes in CLL and additional amplicons across regions affected by clinically relevant CNAs [i.e., del(17p), del(11q), del(13q), and trisomy 12]. We used the PatternCNV algorithm to call CNA and compared the concordance of calling clinically relevant CNAs by targeted sequencing to that of FISH. We found a high accuracy of calling CNAs via sequencing compared to FISH. With FISH as the gold standard, the specificity of targeted sequencing was >95%, sensitivity was >86%, positive predictive value was >90%, and negative predictive value was >84% across the clinically relevant CNAs. Using targeted sequencing, we were also able to identify other common CLL-associated CNAs, including del(6q), del(14q), and gain 8q, as well as complex karyotype, defined as the presence of 3 or more chromosomal abnormalities, in 26 patients. In a single and cost-effective assay that can be performed on stored DNA samples, targeted sequencing can simultaneously detect CNAs, somatic mutations, and complex karyotypes, which are all important prognostic features in CLL.

7050 Background: The management of Richter transformation (RT) of CLL represents an unmet clinical need. We previously reported modest efficacy of pembrolizumab monotherapy in an initial cohort of patients with RT (Ding, et al, Blood 2017). Here we report updated results of this trial, including an expansion cohort and a combination therapy cohort. Methods: Patients with biopsy proven RT to diffuse large B-cell lymphoma were enrolled and treated with single agent pembrolizumab (200 mg IV Q3W). Patients with progressive disease (PD) or stable disease (SD) after 3 months of pembrolizumab monotherapy were allowed to add a B-cell receptor (BCR) kinase inhibitor. The primary end point was overall response rate (ORR). Results: A total of 26 patients with RT were enrolled (9 in initial cohort and 17 in expansion cohort). The median age was 68.5 years. Ten (38%) patients had del(17p) or TP53 mutation. The median number of prior lines of therapy was 3 (range 1-10). Fifteen (58%) patients had received ibrutinib previously (14 of whom had PD on ibrutinib). With pembrolizumab monotherapy (n=26), the ORR was 23.1%, with 2 (7.7%) complete response (CR), 4 (15.4%) partial response (PR), and 7 (26.9%) SD. Of the 6 patients with confirmed CR or PR, responses occurred after a median of 2 cycles of monotherapy. The median duration of response (DOR) was 3.2 months (95% CI 2.8-NA). The median progression-free survival (PFS) was 2.6 months (95% CI 1.6-3.6). Sixteen patients went on to receive a BCR kinase inhibitor (ibrutinib [n=15], idelalisib [n=1]) in combination with pembrolizumab, after SD or PD on pembrolizumab monotherapy. The ORR to combination therapy was 62.5%, with 4 (25.0%) CR and 6 (37.5%) PR. Of the 10 patients with confirmed CR or PR to combination therapy, responses occurred after a median of 4 cycles of therapy. The median DOR was 4.5 months (95% CI 1.6-NA). The median PFS was 7.6 months (95% CI 2.3-13.3). After a median follow-up of 50.4 (95% CI 34.0-56.0) months, the median overall survival (OS) for all 26 patients with RT was 11.6 months (95% CI 7.5-20.1), and the 2-year OS rate was 27%. Treatment-related adverse events (TRAEs) of any grade occurred in 24 (92%) patients, including anemia (54%), thrombocytopenia (58%), neutropenia (54%), leukopenia (35%), lymphopenia (23%), dyspnea (23%), diarrhea (19%), and nausea (23%). Treatment-related grade 3 or above AEs occurred in 21 (81%) patients, including neutropenia (38%), leukopenia (31%), thrombocytopenia (27%), anemia (15%), febrile neutropenia (12%), and lung infection (12%). Conclusions: This expanded analysis confirmed that pembrolizumab has modest single agent activity in RT. Combination therapy with pembrolizumab and a BCR kinase inhibitor is associated with increased efficacy (ORR >60%), similar to other reports. These results support further investigation of immune checkpoint inhibitor-based combination therapy in RT. Clinical trial information: NCT02332980 .