Abstract: Introduction: Chronic lymphocytic leukemia (CLL) is characterized by multiple copy number alterations (CNA) and mutations that are central to disease pathogenesis, prognosis, risk-stratification, and identification of response or resistance to therapies. Fluorescence in situ hybridization (FISH) is gold standard in the clinical laboratory for detecting prognostic CNAs in CLL (e.g. deletion 17p13 (del(17p), deletion 11q23 (del(11q), deletion 13q14 (del(13q), and trisomy 12). Most clinical FISH assays have high specificity and sensitivity, but the technique can detect a limited number of alterations per assay. Importantly, next-generation sequencing (NGS) techniques have become more readily available for clinical applications and are increasingly being used for screening not only mutations, but also copy number abnormalities in multiple genes and chromosomal regions of interest in hematologic malignancies. Here, we evaluated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) using a custom targeted NGS assay for detecting common prognostic chromosomal alterations in CLL and high-count monoclonal B-cell lymphocytosis (MBL), the precursor to CLL. Methods : We designed a SureSelect DNA targeted sequencing panel, covering all exons of 59 recurrently CLL mutated genes and additional amplicons across regions affected by clinically relevant CNAs. All CLL (N=534) and MBL (N=162) patients had pre-treatment peripheral blood mononuclear cells (PBMC) collected within two years of diagnosis. DNA was extracted in cases with purity & gt;80% of CD5+/CD19+ cells. Clinical FISH data was available within 100 days of NGS in all untreated CLL and MBL cases. PatternCNV was used to detect clinically relevant CNAs in chromosomes 11, 12, 13 and 17. We performed a principal component analysis on the CNA calls, excluding chromosomes 11, 12, 13, and 17 to identify clusters of samples. Each cluster was then independently rerun with PatternCNV and the results from chromosomes 11, 12, 13, and 17 were extracted and further analyzed. We excluded samples with low tumor metrics identified by FISH (less than 20% of cells with del(17p), del(11q), trisomy 12 and del(13q)). Results: We sequenced a total of 696 patients of whom 162 were MBL and 534 were untreated CLL. The most commonly mutated genes were NOTCH1 (11.0%), TP53 (8.7%), SF3B1 (7.7%), ATM (4.1%), and CHD2 (3.8%). Based on CNA analyses from the NGS data, we identified 59 (9.1%) individuals with del(17p), 88 (13.4%) individuals with del(11q), 128 (20.0%) individuals with trisomy 12, and 329 (53.0%) individuals with del(13q). All 696 individuals had FISH panels conducted, with 39 (5.6%) individuals with del(17p), 68 (9.8%) individuals with (11q), 119 (17.1%) with trisomy 12, and 295 (42.4%) with del(13q). When we compared our CNA analyses with the FISH data, we found high concordance 95.0% for del(17p), 92.7% del(11p), 94.3% for trisomy 12, and 88.2% for del(13q). For del(17p) we found a sensitivity of 93.9%, specificity of 95.4%, PPV of 52.5%, and NPV of 99.7%. Del(11q) revealed a sensitivity of 88.1%, specificity of 94.0%, PPV of 59.1%, and NPV 98.8%. We found a sensitivity of 93.8%, specificity of 95.6%, PPV 82.0%, and NPV of 98.6% for trisomy 12 and for del(13q) we found a sensitivity of 92.6%, specificity of 90.9%, PPV of 91.7%, and NPV of 93.8%. We found lower PPVs in del(17p) and del(11q) likely due to lower prevalence of these chromosomal abnormalities. Conclusion: Here we show a high sensitivity, specificity, and NPV when comparing targeted sequencing with FISH. FISH panel testing is widely used in clinical practice to characterize highly prognostic chromosomal abnormalities in CLL. Comprehensive genetic profiling with NGS has become increasingly important in the work up of hematologic malignancies and provides additional prognostic and predictive information, including clinically relevant mutations such as TP53, SF3B1, and NOTCH1, tumor mutation load and mutations associated with resistance to chemo-immunotherapy and targeted therapies, such as BTK or BCL2 inhibitors, that FISH cannot offer. We show that NGS can infer clinically relevant CNA in cases without FISH testing while also providing additional clinically relevant information. Figure 1 Figure 1. Disclosures Cerhan: Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding; Genentech: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Genentech: Research Funding; MEI Pharma: Research Funding; Sunesis: Research Funding; Acerta Pharma: Research Funding; Abbvie: 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; AstraZeneca: 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; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; 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 Therapeutics: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Targeted 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; Behring: Membership on an entity's Board of Directors or advisory committees.

Abstract: Introduction: TP53 aberrations, including mutations and deletion of 17p (del17p), are important adverse prognostic markers in chronic lymphocytic leukemia (CLL). Prevalence of TP53 aberrations ranges from 7-11% in untreated CLL and increases with disease progression and treatment. Among CLL patients with TP53 aberrations, co-occurrence of TP53 mutations with del(17p) is common. CLL patients with TP53 mutations or del(17p) have significantly worse outcomes when compared to wild-type patients. Previous studies, focusing on CLL patients at time of treatment, are mixed as to whether a single or more than one TP53 aberration impacts outcomes. TP53 is less well studied in monoclonal B-cell lymphocytosis (MBL), an asymptomatic pre-malignant state of CLL. Del(17p) occurs in 3-4% of MBL individuals, and a study of 54 MBL individuals reported a 2% mutation frequency in TP53. Here we estimated prevalence and evaluated the impact of TP53 aberrations in a large cohort MBL or untreated CLL individuals. Methods : Patients with CLL or MBL diagnosed between 2000 and 2019 from the Mayo Clinic CLL Resource with pre-treatment peripheral blood mononuclear cells (PBMC) collected within two years of diagnosis were considered. DNA was extracted from PBMCs with purity & gt;80% or with sorted CD5+/CD19+ clonal cells. The entire coding regions of 59 CLL driver genes were paired end sequenced. Median coverage depth was & gt;1000x per nucleotide, allowing for detection of mutations with variant allelic fraction (VAF) as low as 1%. Somatic mutations were called using MuTect2 in tumor-only mode, and high impact mutations (frameshift, nonsense, and splicing variants) and missense mutations in CLL hot spots were selected. Somatic mutations and FISH del(17p) were used to define TP53 state for each patient: 1) wild-type [no TP53 mutations and normal del(17p)], 2) single-hit [one TP53 mutation or del( 17p)], or 3) multi-hit [multiple TP53 mutations or TP53 mutation and del(17p)] . Time to first treatment (TTFT) and overall survival (OS) were analyzed by TP53 state. TTFT and OS were defined as time from sample collection to first treatment or death, respectively, or last follow-up date. Median TTFT and OS was estimated by the Kaplan-Meier method. We used Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals for TTFT and OS associations. The models were adjusted for known adverse prognostic factors including clinical diagnosis (CLL or MBL), age at diagnosis, Rai stage, b2 microglobulin, and IGHV mutation status. Results: Individuals with CLL (N=597) or MBL (N=285) were analyzed for prevalence of TP53 mutations and del(17p). We found 58 CLL patients (9.7%) and 15 MBL individuals (5.3%) had TP53 mutations. The median VAF in CLL was 30.9% ( & lt;10% VAF, n=21) and in MBL was 13.5% ( & lt;10% VAF, n=7). Del(17p) was present in 37 patients with CLL (6.2%) and 7 individuals with MBL (2.7%). In total, 86 CLL/MBL patients had a TP53 mutation and/or del(17p): 56.8% (48 patients) were single-hit and 44.2% (38 patients) were multi-hit (Fig. 1a). Patients with any TP53 aberration had shorter TTFT than wild-type patients (median 2.3 vs 9.4 years). Among patients with TP53 aberrations, median TTFT was shorter in multi-hit patients (20 events, 1.8 years) compared to single-hit patients (24 events, 3.2 years) (Fig. 1b). In Cox regression, single-hit (HR = 1.7 [1.1-2.6]) and multi-hit (HR = 1.8 [1.1-2.9] ) patients had shorter TTFT compared to wild-type patients after adjusting for covariates (Fig. 1c). Multi-hit patients also had shorter OS compared to wild-type patients, while OS in single-hit patients did not significantly differ from wild-type patients (Fig. 1d). Median OS was 5.5 years in multi-hit patients (22 deaths) compared to 15.1 years in wild-type patients (196 deaths) and 14.3 years in single-hit patients (15 deaths). In the OS model adjusted for covariates, multi-hit patients had a significant increased risk (HR = 2.6 [1.6-4.1]), but single-hit patients did not (HR = 1.4 [0.8-2.5] ) compared with wild-type patients (Fig. 1e). OS HRs remained stable after censoring at time of treatment. Both TTFT and OS HRs were consistent when mutations with VAF & lt; 10% were excluded. Conclusions: This study suggests single versus multi-hit TP53 aberrations may be important for prognostic outcomes in untreated CLL and MBL patients. Prognostic metrics may need to consider single versus multi-hit TP53 aberrations and include TP53 mutations with low VAF. Figure 1 Figure 1. Disclosures Cerhan: Regeneron Genetics Center: Other: Research Collaboration; Celgene/BMS: Other: Connect Lymphoma Scientific Steering Committee, Research Funding; NanoString: Research Funding; Genentech: Research Funding. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: MEI Pharma: Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Research Funding; 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; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; 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; Behring: Membership on an entity's Board of Directors or advisory committees; Sunesis: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees.

Abstract: Chronic lymphocytic leukemia (CLL) is a neoplastic disease of mature B-cells with a highly heterogeneous clinical course. While European ancestry (EA) populations present an increased incidence of CLL, African ancestry (AA) populations have a younger median age of onset, higher frequency of adverse prognostic factors, and inferior clinical outcomes. Despite the considerable effort to characterize the genetic landscape of CLL, AA are overwhelmingly underrepresented. Our hypothesis is that the clinical differences observed between AA and EA populations are, in part, explained by underlying genetic features. To address this imbalance, we identified 90 AA patients diagnosed with CLL, 64% of which were untreated at sample collection. RNA and DNA were extracted from CD5+/CD19+ clonal B-cells. We performed mRNA-seq and targeted sequencing in 59 recurrently mutated somatic CLL driver genes. Differentially expressed genes were identified using edgeR. Data was compared to our previously analyzed EA CLL cohort (N=445). The median age at diagnosis was 59 years for AA and 66 for EA and 74% of AA and 50% of EA had unmutated IGHV (u-IGHV) status. When evaluating the entire AA and EA cohorts, there was a significant increased frequency of mutations in TP53, SF3B1, and NFKBIE, identified in 29%, 24%, and 20% of AA CLLs, compared to 5%, 9%, and 9%, respectively, in EA CLLs (p & lt;0.01). When exclusively evaluating the untreated and u-IGHV cases, AA CLLs showed greater proportion of TP53 (30% vs 12%; p=0.0145), NFKBIE (30% vs 15%; p=0.0492), BIRC3 (21% vs 10%; p=0.0697), and KRAS (15% vs 7%; p=0.1683). Furthermore, there was an increase in mutations targeting relevant molecular pathways, such as NF-κB (42% vs 15%) and MAPK (18% vs 8%). Upregulation MAPK pathway was also confirmed by mRNA-seq analysis in the AA u-IGHV CLLs. Because of the high prevalence of TP53 mutations in the AA cohort, we further evaluated differential gene expression in the DNA Damage/Telomere Stress-Induced Senescence pathway. AA CLLs presented a significant downregulation of multiple genes associated with genome stability and cellular DNA damage response - DDR (including TP53 and ATM), double strand break repair (H2AFX and RAD50), telomere maintenance (POT1 and ACD), and cell cycle regulation (RB1, CCNA1, and CCNE2) (FDR & lt;0.05). DDR is responsible for DNA repair or induction of apoptosis, with its deficiency resulting in the accumulation of chromosomal aberrations, negatively impacting clinical outcome in CLL. Disparities in cancer are influenced by numerous factors that affect disease risk, screening and diagnosis, access to treatment, and survival. We identified an increased number of genomic alterations in the AA CLL cohort, primarily inducing activation of NF-κB and MAPK pathways and DDR impairment, with the increased frequency of mutations, notably in TP53 and BIRC3, expected to negatively impact prognosis. Citation Format: Cecilia Bonolo de Campos, Daniel R. O'Brien, Chantal E. McCabe, Huihuang Yan, Geffen Kleinstern, Zhiquan Wang, Laura A. Bruins, Cristine Allmer, Nicholas J. Boddicker, Charla R. Secreto, Aaron D. Norman, Shulan Tian, Kari G. Rabe, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Richard Furman, J Brice Weinberg, James R. Cerhan, Celine M. Vachon, Neil E. Kay, Susan L. Slager, Esteban Braggio. Characterization of underlying genomic features among African ancestry populations diagnosed with chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2209.

Abstract: An improved understanding of childhood pneumonia etiology is required to inform prevention and treatment strategies. Lung aspiration is the gold standard specimen for pneumonia diagnostics. We report findings from analyses of lung and pleural aspirates collected in the Pneumonia Etiology Research for Child Health (PERCH) study. Methods The PERCH study enrolled children aged 1–59 months hospitalized with World Health Organization–defined severe or very severe pneumonia in 7 countries in Africa and Asia. Percutaneous transthoracic lung aspiration (LA) and pleural fluid (PF) aspiration was performed on a sample of pneumonia cases with radiological consolidation and/or PF in 4 countries. Venous blood and nasopharyngeal/oropharyngeal swabs were collected from all cases. Multiplex quantitative polymerase chain reaction (PCR) and routine microbiologic culture were applied to clinical specimens. Results Of 44 LAs performed within 3 days of admission on 622 eligible cases, 13 (30%) had a pathogen identified by either culture (5/44) or by PCR (11/29). A pathogen was identified in 12/14 (86%) PF specimens tested by either culture (9/14) or PCR (9/11). Bacterial pathogens were identified more frequently than viruses. All but 1 of the cases with a virus identified were coinfected with bacterial pathogens. Streptococcus pneumoniae (9/44 [20%]) and Staphylococcus aureus (7/14 [50%] ) were the predominant pathogens identified in LA and PF, respectively. Conclusions Bacterial pathogens predominated in this selected subgroup of PERCH participants drawn from those with radiological consolidation or PF, with S. pneumoniae and S. aureus the leading pathogens identified.

Abstract: Background: Upamostat is an orally available small molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2 and trypsin 3 (PRSS1/2/3) as well as urokinase-type plasminogen activator (uPA) which are expressed in many cancers and mediate cell migration, invasion and tissue remodeling. Opaganib (ABC294640), a novel, orally available small molecule is a specific inhibitor of sphingosine kinase 2 (SPHK2), which phosphorylates sphingosine to sphingosine-1-phosphate (S-1-P). While proliferation induced by S-1-P is regulated by both sphingosine kinase 1 (SPHK1) and SPHK2, SPHK2 appears to be more involved in cancer. We aimed to investigate the potential antitumor effect of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) patient derived xenografts (PDX) in nude mice. Methods: PAX165, a PDX from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2 and PRSS3. 4 groups of 18 mice were treated with either drug or both. Mouse weights and tumor volumes were measured. In addition, experiments were conducted using the chorioallantoic membrane (CAM) of chicken embryos. Results: Table 1 shows the average tumor size for the control, upamostat, opaganib, and upamostat+opaganib groups at the study end point (Day 42). Tumor volumes in the upamostat, opaganib, and upamostat+opagnib groups were significantly decreased compared to the control group. The CAM experiments are ongoing and will be presented at the AACR Annual Meeting. Change in tumor volumes (mean) of CCA PDX after opaganib, upamostat or combination treatmentControlOpaganibUpamostatOpaganib+UpamostatPre-treatment129.9128.7118.8126.8Day 42198.6102.093.3186.09Percent change Day 0-42+53%-21%-21%-32%P value vs. control0.00020.00100.0008 Conclusion: This preclinical study demonstrated that upamostat and opaganib resulted in tumor regression in mice. Body weights of the mice showed no significant inter- or intra- group differences. The combination of upamostat and opaganib treatment showed greater regression compared to either upamostat or opaganib alone. Studies are underway to identify the molecular mechanisms of their interaction. Citation Format: Faizal Z. Asumda, Mohamed A. Hassan, Yo Han Kim, Nellie A. Campbell, Xin Luo, Daniel R. O'Brien, Sarah A. Buhrow, Joel M. Reid, Michael J. Moore, Vered Katz Ben-Yair, Reza Fathi, Mark L. Levitt, Fabrice Lucien-Matteoni, Jennifer L. Leiting, Mark J. Truty, Lewis R. Roberts. Effects of upamostat and opaganib on cholangiocarcinoma patient derived xenografts [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 3078.

Abstract: Clinical practice guidelines recommend that individuals with HC MBL are evaluated annually for evidence of progression as it has been shown that they progress to develop CLL requiring therapy at a rate of 1-5%/year. Next-generation sequencing identified 59 genes recurrently mutated in CLL, and we previously showed that the tumor mutational load (TML), i.e., the total number of these genes mutated, is associated with a shorter time to first treatment (TTT) among 112 HC MBLs independent of the CLL international prognostic index (CLL-IPI), an established prognostic score. Herein, we continue to support our finding in additional HC MBLs and report the pooled results across the two cohorts. From the Mayo Clinic CLL Resource, we sequenced a total 167 HC MBL (112 from the initial round and 55 from additional sequencing). We extracted DNA from CD5+/CD19+ B-cells and sequenced the coding regions of 59 driver genes. Somatic mutations were called using MuTect2 in tumor-only mode. Germline variants were eliminated based on minor allele frequencies & gt;0.01% and by those identified in public databases, unless present in known CLL mutation hotspots. After filtering, we computed the TML based on the number of genes with high impact (nonsense, frameshift and splicing) and hotspot mutations. Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI), adjusted for sex and CLL-IPI. Stratified analyses were conducted by low/intermediate risk and high/very high CLL-IPI. Of the 167 HC MBL, low risk CLL-IPI comprised 60%, intermediate risk 28%, high risk 12%; and none were very-high risk. The most commonly mutated genes were SF3B1 (11%), NOTCH1 (9%), TP53 (7%), BIRC3 (7%), and MYD88 (6%); and 52 (31%) individuals had mutations in at least one gene. The median follow-up was 4.4 years, and 26 of the 167 individuals progressed to CLL requiring therapy. HC MBL with ≥2 mutated genes had a 28% 5-year risk of needing therapy compared to 6% among those with no mutated genes. Similar to our initial finding (HR=1.53, CI:1.12-2.07; P=0.007), we found a shorter TTT in the additional HC MBLs (HR=4.63, CI:1.57-13.7, P=0.006), and in the pooled cohort (HR=1.72; CI:1.29-2.28, P=0.0002). When we stratified by CLL-IPI risk, TML remained associated with TTT among individuals with low/intermediate CLL-IPI risk in the additional HC MBLs (HR=3.52, CI:1.11-11.2, P=0.03) and in the pooled cohort (HR=2.25, CI:1.34-3.77, P=0.002). A non-significant 2.8 fold association was observed with high risk CLL-IPI in the pooled cohort. In conclusion, the TML along with the CLL-IPI provide clinicians with a more accurate prognostic information regarding disease progression requiring therapy, especially for those MBLs with low to intermediate risk based on CLL-IPI. If validated, these findings may modify current practice guidelines for individuals with HC MBL. Citation Format: Geffen Kleinstern, Cecilia Bonolo de Campos, Nicholas J. Boddicker, Daniel R. O'Brien, Shulan Tian, Chantel Barney, Kari G. Rabe, Cristine Allmer, Laura Bruins, Aaron D. Norman, Timothy G. Call, Sameer A. Parikh, Jose F. Leis, Wei Ding, Huihuang Yan, James R. Cerhan, Neil E. Kay, Celine Vachon, Esteban Braggio, Susan L. Slager. Tumor mutational load derived from recurrently mutated genes in chronic lymphocytic leukemia (CLL) predicts time-to-first treatment in high-count monoclonal B-cell lymphocytosis (HC MBL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 884.