Background: The JAK2 V617F mutation is the most frequent recurring mutation noted in all three types of myeloproliferative disorders (polycythemia vera, essential thrombocytosis, myelofibrosis) and occurs less frequently in other myeloid malignancies. JAK2 V617F occurs in the JH2 pseudo kinase domain of JAK2 and results in hyperactivation of this kinase. The JH2 domain of JAK family kinase members are distinct whereas little difference exists between the JH1 kinase domain. Current available JAK2 inhibitor therapies such as ruxolitinib and fedratinib target the JH1 domain and provide symptomatic relief but are limited by non-selective JAK inhibition and disruption of WT JAK2 signaling. ZE74-2882 is a novel, small molecule designed to selectively inhibit pathologically activated JAK2—such as JAK2V617F—while sparing the wild-type (WT) JAK2 required for normal hematopoietic function described herein. Methods/Results: Utilizing both in-silico and artificial intelligence modeling derived from MolSoft LLC, we identified an optimal small molecule that binds preferentially to JH2 domain of JAK2 versus JH1 domain and to JH2 domain with V617F mutation versus wild type JH2 domain. ZE74-0282 demonstrated 500-fold selectivity to JAK2 V617F JH2 domain versus JAK2 JH2 WT protein in cytoxicity experiment in BaF3 JAK2 V617F cells versus BaF3 JAK2 WT cells. In vitro, ZE74-0282 inhibited BaF3 JAK2 WT cells pSTAT5 phosphorylation and growth at 50% (1456 nM and not reached at >10,000 nM) in contrast to ruxolitinib (30.3 nM and 444.3 nM). In contrast, ZE74-0282 inhibited BaF3 JAK2 V617F cell pSTAT5 phosphorylation and growth at 50% (52.1 nM and 40.6 nM) versus ruxolitinib (3.66 nM and 19.5 nM). SET-2 JAK2 V617F cell line demonstrated similar pSTAT5 inhibition between Ruxolitinib (4.97 nM) and ZE74-0282 (3.46 nM). In vitro, the CC50 of Ruxolitinib and ZE74-0282 was similar against SET-2 JAK2 V617F (10.9 and 24.3 nM) To evaluate the selectivity and functional activity of ZE74-282 we utilized human JAK2V617F+ -MPN whole blood where myeloid cells will have this mutation and lymphoid cells lack it along with murine JAK2V617F and WT cells for colony-forming assays. In JAK2V617F-positive human blood myeloid cells ZE74-282 reduced pSTAT5 levels with an EC50 of 25 nM. Importantly pSTAT5/3 levels were not affected in lymphoid cells, indicating the specificity. In contrast, pSTAT5/3levels were significantly reduced both in myeloid and lymphoid cells upon Ruxolitinib treatment. Viability was equally lost between ZE74-282 and ruxolitinib in human myeloid cells whereas only ruxolitinib resulted in loss of lymphoid cells. In murine cytokine containing colony forming assays, ZE74-282 selectively suppressed colony formation in JAK2ASXL1 mutant progenitors without affecting colonies from WT JAK2-expressing progenitors. In contrast to ruxolitinib, which reduced colony numbers in both genotypes. Pharmacology studies in mice demonstrated linear increases over time with a t1/2 of 7 hours at 28 days. Utilizing a subcutaneous xenograft model of the BaF3 JAK2 617F mouse model, treatment with vehicle, fedratinib (100 mg/kg BID) or ZE74-0282 (100 mg/kg BID) beginning day 14. Versus control, fedratinib modestly delayed growth (p=0.02) whereas growth was inhibited greater with ZE74-0282 (p=0.0001). Superior inhibition of pSTAT5 in vivo was observed with ZE74-0282 as compared to both control and fedratinib treatment. ADME properties of this molecule including mouse GLP toxicity have been favorable for clinical translation to date and will be presented Conclusion: ZE74-282 is a first-in-class, JH2 domain specific JAK2 inhibitor with potent and selective activity against mutant JAK2V617F while preserving normal JAK2-mediated function. Compared with JH1 directed JAK2 inhibitors ZE74-2882 offers a superior therapeutic index in preclinical models, pharmacology suggesting twice daily dosing, and exceptional “drug properties” with ADME studies completed to date. These findings support its further development as a potentially disease-modifying therapy for JAK2 V617F mutated diseases.

6524 Background: IRAK4, a kinase effector of MyD88 signaling downstream of Toll-Like Receptor and IL-1 receptor pathways, has recently been shown to function independently of MyD88 in Myelodysplastic Syndrome (MDS) and AML leukemic cells. Our previous research demonstrated that AML leukemic stem and progenitor cells (LSPC) rely on signaling through IRAK4. Notably, AML LSPCs express a hypermorphic long splice isoform of IRAK4 (IRAK4-L) resulting from the inclusion of exon 4. IRAK4 inhibitors, currently in clinical trials for the treatment of MDS and AML, show improved efficacy in spliceosome mutants, which preferentially express IRAK4-L. However, in patients without known spliceosome mutations, the extent of IRAK4-L expression remains unclear. Additionally, the impact of IRAK4-L levels in Venetoclax and Azacitidine treatment is unknown in refractory or unfit AML. Methods: Umbilical cord vein blood stem cells expressing MLL-AF9 and NRAS G12D (CD34+MA9.NRAS) were maintained in vitro using supplemented media. Venetoclax and Azacitidine doses were incrementally increased in combination until cells tolerated co-treatment with up to 1 µM of each compound with minimal cell death. Patient-derived xenograft (PDX) samples were obtained from the Cincinnati Children’s Biobank and included AML samples from diverse genetic backgrounds, relapsed/refractory cases, and pediatric populations (ages 1-20). Cell lysates were normalized to total protein and analyzed using a chemiluminescent capillary-based immunoblotting system, which requires minimal lysate quantities. An IRAK4 C-terminus antibody was used to detect both the shorter and longer IRAK4 protein isoform. RNA sequencing was performed, and transcripts were normalized per million counts. IRAK4 transcript ratios were calculated by dividing the total long isoform transcripts by the short isoform transcript for each condition in duplicate. Results: Analysis of PDX and human AML cell lines revealed that 8/9 PDX preferentially expressed the IRAK4-L isoform (>70% of IRAK4-L), with 4/9 PDX samples and 5/6 previously uncharacterized cell lines producing it almost exclusively (>95% IRAK4-L). MA9.NRAS cells predominantly express IRAK4-L, but transiently shifted to IRAK4-S during treatment with AZA/VEN (27% IRAK4-S), an effect reversed upon recovery from treatment. Conclusions: IRAK4-L is widely expressed in AML patient-derived samples, including those with complex karyotypes and TP53 mutations, suggesting that IRAK4 inhibitors may be useful beyond splicing factor-mutant MDS/AML. Our models demonstrate that changes in IRAK4-L expression during treatment with hypomethylating agents may be useful as a biomarker to guide therapy strategies. Further research is needed to understand how prolonged treatment and other standard of care therapies affect IRAK4-L expression and sensitivity to IRAK4 inhibitors.

Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in pre clinical studies and clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. In this study, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that cotargeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. Although IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complementary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via noncanonical MyD88-independent pathways. Genomic and proteomic analyses revealed that IRAK1 and IRAK4 preserve the undifferentiated state of MDS/AML LSPCs by coordinating a network of pathways, including ones that converge on the polycomb repressive complex 2 complex and JAK-STAT signaling. To translate these findings, we implemented a structure-based design of a potent and selective dual IRAK1 and IRAK4 inhibitor KME-2780. MDS/AML cell lines and patient-derived samples showed significant suppression of LSPCs in xenograft and in vitro studies when treated with KME-2780 as compared with selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for cotargeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.

Overexpression of immune-related genes is widely reported in acute myeloid leukemia (AML). Research from our lab and others has demonstrated that AML leukemic stem and progenitor cells (LSPC) rely on innate immune signaling through IRAK4 (reviewed in Bennett et al., Blood 2023). Moreover, AML LSPCs express a hypermorphic isoform of IRAK4 (IRAK4-L), due to U2AF1- and SF3B1-dependent mis-splicing. Building on this preclinical evidence, several IRAK4 inhibitors are now undergoing clinical trials in AML patients. In Phase-1 findings with a selective IRAK4 inhibitor (CA-4948; Curis Therapeutics), it was found that MDS and AML patients with splicing factor mutations responded best to monotherapy IRAK4 inhibition, although the overall response rate with monotherapy was modest. These findings suggest that IRAK4 is a relevant target in myeloid malignancies, but the identification of synthetic lethal dependencies may be crucial in revealing effective combination therapies. In this study, we sought to identify synthetic lethal interactions upon IRAK4 inhibition in AML. For this, we first generated isogenic AML cells that are proficient (WT THP1) or deficient for IRAK4 (IRAK4 KO THP1) using site-directed mutagenesis with CRISPR-Cas9. Deletion of IRAK4 resulted in a modest (~50%) reduction in leukemic colony formation and negligible differences in growth potential. We used the isogenic WT and IRAK4 KO AML cells to screen for synthetic lethal interactions by performing a 2400+ drug in vitro screen. Hits were prioritized for potency in IRAK4 KO cells vs WT AML cells, degree of fitness, and overall effect. Among these hits, the Cereblon E3 ligase modulator (CELMoD) CC-885 emerged as the top target. Independent validation confirmed that CC-885 significantly decreased proliferation and viability of isogenic IRAK4 KO MDS (MDSL) and AML (THP1) cells by approximately 3-4-fold compared to WT MDS or AML cells. Colony formation in methylcellulose after treatment with CC-885 was also significantly reduced in IRAK4 KO compared to WT MDS and AML cells. Based on Caspase-3 activation and AnnexinV staining, CC-885 induced significantly more apoptosis of IRAK4 KO cells as compared to WT AML cells. We extended our synthetic lethal analysis to a clinical-stage IRAK4 inhibitor (CA-4948). MDS and AML cells were treated with CA-4948 concurrently with CC-885 or pretreated for 7 days prior to treatment with CC-885. Unexpectedly, we found that only prior exposure of the AML cells to the IRAK4 inhibitors resulted in sensitivity to CC-885. These findings indicate that IRAK4 inhibition reprograms AML cells so that they acquire a synthetic lethal interaction with CC-885. CC-885 is known to target multiple substrates for degradation by the ubiquitin proteasome, including IKZF1, IKZF3, CK1a, and GSPT1. We compared the effect of CC-885 to CC-90009, a CELMoD with GSPT1 specific activity in the isogenic IRAK4 KO and WT AML cell lines. CC-90009 did not result in complete cell death up to concentrations of 10mM in both WT and IRAK4 KO, suggesting that the selective sensitivity of IRAK4 KO AML cells to CC-885 is not due to inhibition of GSPT1. In support of these interpretations, IRAK4 deficient AML cells exhibit increased protein expression of IKZF2 and IKZF3, but not GSPT1 or CK1a. Given the unique responses of IRAK4 KO cells to CELMoDs, we posited that IRAK4 inhibition alters the neosubstrate profile of CELMoDs in AML cells. Total proteome characterization of CC-885 treated WT and IRAK4 KO THP1 cells by mass spectrometry identified a significant decrease in 90 protein substrates in IRAK4 KO cells treated with CC-885 as compared to WT cells treated with CC-885. These findings suggest that IRAK4 inhibition alters the pool of neosubstrates in AML cells for certain CELMoDs. Overall, our study demonstrates that IRAK4 is a therapeutic target in AML, but that combination therapies, such as with certain CELMoDs, will be necessary to achieve better clinical responses.

Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators. This characteristic allows AML to thrive in a hostile and rapidly changing environment where it needs to adapt to stressors both extrinsic (chemotherapy) and intrinsic (replicative and oxidative stress) to survive. Our study is the first to demonstrate the mechanism of how AML uses epigenetic reprogramming to adjust to these stressors via lineage plasticity. Venetoclax and Azacitidine (Ven/Aza) co-treatment is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. However, most patients will relapse with a highly aggressive refractory AML. Some studies suggest that monocytic character of the AML is responsible for relapse, but more recent evidence demonstrates that Ven/Aza treatment itself can induce a lineage switch to monocytic character. A potent selection mechanism facilitated by epigenetic dysregulation generating plastic clones is likely driving resistance, though the mechanism remains unclear and represents a significant barrier for AML treatment. Using a combination of single cell transcriptomics with surface immunophenotype sequencing in multiple human xenograft and primary patient samples, we discovered that this plasticity is widespread and mediated by epigenetic reprogramming of the tumor in direct response to the therapy. Broadly, we found that AML responds to Ven/Aza by transitioning into a reversible state of cell cycle arrest with transcriptional character similar to hematopoietic stem cells. This transient state protects AML from Azacitidine through replication arrest and protects from Venetoclax by upregulation of anti-apoptotic transcriptional programs. These protections operate simultaneously with reprogramming events driven by a switch from MYC-driven transcriptional programs to a PU.1-driven program. Reprogramming is both facilitated by and dependent on repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation via its role as a nucleosome assembly factor. Repressing CAF1 facilitates reprogramming where the output is an altered cell population that is distinct from the initial tumor both transcriptionally and by ATACseq. AML can repeatedly return to this plastic state, regardless of the number of cycles of Ven/Aza, and introducing other cytotoxic compounds does not remove this plastic stem-like population but instead initiates the iterative process of reprogramming anew until a different resistant population emerges. To find therapeutic vulnerabilities, we generated several PDX models of Ven/Aza resistance so that we could track the stages of acquired resistance in real time. When single cell data from these experiments were projected onto a reference map of normal hematopoiesis, we found that Ven/Aza resistance stems from a plastic population of cells that actively samples the niche by generating short-lived multi-lineage hybrid differentiated products. While the predominating output of this process was monocytic, we found that the induced plastic stem-like population was durably retained even after Ven/Aza withdrawal. To undermine this process, we took advantage of the plastic state to drive differentiation through an external stimulus. We expected this strategy to be particularly effective because CAF1 was repressed (CAF1 repression facilitates differentiation in every metazoan model) and, based on our single cell data, these plastic populations of AML cells express pro-differentiation surface receptors. In particular, Ven/Aza induced the expression of CSF3R (the G-CSF receptor) which when stimulated, drives neutrophil maturation through the JAK/STAT pathway. While G-CSF as a single agent or in sequence with Ven/Aza was unable to achieve maturation, when used concurrently with Ven/Aza (or recapitulated by CAF1 depletion using shRNA) we were able to drive neutrophil differentiation in vitro and fully resolve primary mouse and PDX tumors in vivo, even when using Ven/Aza resistant tumors as starting material. Together, these data demonstrate the possibility that Ven/Aza resistance can be completely subverted by taking advantage of the plastic state induced by the therapy to stimulate differentiation and resolve the disease.

Inflammation is associated with the pathogenesis of myelodysplastic syndromes (MDS) and emerging evidence suggests that MDS hematopoietic stem and progenitor cells (HSPC) exhibit an altered response to inflammation. Deletion of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. Although this MDS subtype contains several haploinsufficient genes that impact innate immune signaling, the effects of inflammation on del(5q) MDS HSPC remains undefined. Utilizing a model of del(5q)-like MDS, inhibiting the IRAK1/4-TRAF6 axis improved cytopenias, suggesting that activation of innate immune pathways contributes to certain clinical features underlying the pathogenesis of low-risk MDS. However, low-grade inflammation in the del(5q)-like MDS model did not contribute to more severe disease but instead impaired the del(5q)-like HSPC as indicated by their diminished numbers, premature attrition and increased p53 expression. Del(5q)-like HSPC exposed to inflammation became less quiescent, but without affecting cell viability. Unexpectedly, the reduced cellular quiescence of del(5q) HSPC exposed to inflammation was restored by p53 deletion. These findings uncovered that inflammation confers a competitive advantage of functionally defective del(5q) HSPC upon loss of p53. Since TP53 mutations are enriched in del(5q) AML following an MDS diagnosis, increased p53 activation in del(5q) MDS HSPC due to inflammation may create a selective pressure for genetic inactivation of p53 or expansion of a pre-existing TP53-mutant clone.

In head and neck squamous cell carcinoma (HNSCC), anti-PD-1 inhibitors are approved for recurrent/metastatic (R/M) disease and anticipated to expand to other indications. The impact of p16 status and anatomical site on overall survival (OS) in immunotherapy-treated HNSCC patients remains unresolved. We performed a retrospective analysis of R/M HNSCC patients receiving anti-PD-1 immunotherapy at our academic medical center with an extensive community satellite network. Fifty-three R/M HNSCC patients were treated with anti-PD-1 immunotherapy and had a median OS of 6 months. Anatomical site was associated with distinct OS; oropharynx and larynx patients have superior OS compared to oral cavity patients. Analysis of the OPSCC subset showed p16+ status as a favorable, independent prognostic biomarker (HR 7.67 (1.23–47.8); p = 0.029). Further studies to assess the link between anatomical site, p16 status, and anti-PD-1 treatment outcomes in large cohorts of R/M HNSCC patients managed in real-world clinical practices and clinical trials should be prioritized.

e19086 Background: TNBC is a heterogeneous sub-type of breast cancer characterized by younger age of onset, more aggressive course, and higher incidence among African Americans who also experience disparities in access to health care including health insurance coverage and cancer screening. Methods: We performed a retrospective analysis of the NCDB to study the impact of race and insurance status on the stage at diagnosis and overall survival of people with TNBC. Chi-square tests analysis was used for univariate analysis. Cox models were used to test for survival differences between race and insurance status adjusted for other covariates such as age at diagnosis, gender. Results: Among 1,148,016 people with TNBC registered in the NCDB from 2010 to 2016, 87.7% were identified as white and 12.3% were identified as black. The majority (99%) were female. Mean age at diagnosis was 62.0 years for females and 65.9 years for males. Among white patients, 1.8% were uninsured while 3.6% of black patients were uninsured. Advanced stage at diagnosis was less common among white people (9.4%, Stage 3; 4.9%, Stage 4) than black people (13.3%, Stage 3; 7.6%, Stage 4). Uninsured patients had more advanced disease at time of diagnosis (16.2%, Stage 3; 14.1%, Stage 4) than insured patients (9.8%, Stage 3; 5.0%, Stage 4). Overall survival after adjustment for age at diagnosis, gender and insurance status was greater for white patients compared to black patients (harm ratio 0.60). Overall survival after adjustment for age at diagnosis, gender and race was lower for uninsured patients than insured patients (harm ratio 2.25). Conclusions: Racial disparities significantly affect TNBC patients, with black women having lower insurance coverage and worse overall survival. [Table: see text]

Background Immune checkpoint inhibitors (ICIs) are now approved for several cancer types due to superior outcomes compared to chemotherapy. PD-1/PD-L1 and CTLA-4 inhibitors reactivate T-cell mediated anti-tumor immunity but may also lead to immune-related adverse events (irAEs). Growing evidence suggests the onset of irAEs could be correlated with the efficacy of ICIs.1. 2 In this study, we investigated patterns and incidence of irAEs and their relationship to overall survival (OS) and progression-free survival (PFS) in multiple cancer types. Methods The electronic medical record was queried at the University of Cincinnati Medical Center for the administration of ICIs for the identification of irAEs. Data on new irAEs diagnosed after administration of at least one dose of ICI was collected along with relevant demographic and clinicopathologic variables including treatment type, cancer type, staging information, and the administration of immune suppression following the identification of an IRAE either inpatient or outpatient. Univariant and multivariant analysis were conducted and survival analysis was determined according to log-rank testing. Results Of our 210 initial patients, the median age was 64 (range 22–93), 37% were female, 72% had ECOG 0-1, and 79% were white. Cancer types included melanoma 24%, non-small cell lung cancer (NSCLC) 34%, small cell lung cancer 2%, renal cancer 12%, urothelial cancer 11%, head and neck cancers 12%, and 16% other primaries while 19% remained on ICI at the time of data entry. The most common ICIs were pembrolizumab, nivolumab, followed by ipilimumab-nivolumab, ipilimumab, and durvalumab. The overall incidence of irAEs was 22.6%. Overall survival and progression-free survival was improved for those who suffered an IRAE (median OS 8.3 vs. 3.5 years, HR 0.56, p=0.0092; median PFS 5.0 vs 2.5 years, HR 0.57, p=0.0052) (figure 1 and 2 respectively). ICI treatment in NSCLC was associated with decreased overall IRAE events by univariant analysis (Odds Ratio 0.39, 95% CI 0.17 - 0.86). Our multivariate analysis showed ICI treatment in hepatocellular carcinoma to be significantly associated with irAEs, however, this was likely due to low enrollment (n=4) and was not significant by univariant analysis. The most common IRAE overall was diarrhea/colitis (figure 3). Conclusions In our data set, irAEs were associated with increased OS and PFS regardless of disease site. ICI treatment of NSCLC was associated with significantly fewer irAEs compared to other malignancies. Further research is needed to determine irAE type-specific incidence, the incidence of multiple irAEs in a single patient, and response to corticosteroid therapy. Acknowledgements We would like to thank the Department of Hematology/Oncology of the University of Cincinnati, the University of Cincinnati Cancer Center, and the Department of Internal Medicine of the University of Cincinnati for their support. Ethics Approval Considered and Approved by the University IRB Approval #2019-0610. References Weber JS, Hodi FS, Wolchok JD, et al. Safety Profile of Nivolumab Monotherapy: A Pooled Analysis of Patients With Advanced Melanoma. J Clin Oncol 2017;35(7):785–792. Yamauchi I, Yasoda A, Matsumoto S, et al. Incidence, features, and prognosis of immune-related adverse events involving the thyroid gland induced by nivolumab. PLoS ONE. 2019;14(5):e0216954.

Background The use of immune checkpoint inhibitors (ICI) has increased significantly in the past five years, along with the number of available drugs and regimens. ICIs have dramatically increased survival in cancer patients, however, there has been data to show that they have reduced efficacy in the presence of antibiotics.1 Antibiotic use prior and during ICI therapy was associated with worsening clinical outcomes in patients with renal cell carcinoma,2 melanoma,3 and non-small-cell-lung-cancer.4 Specifically, exposure within 60 days of initiation of ICIs in NSCLC seemed most detrimental, however, some studies have suggested that antibiotics disrupt intestinal microflora for up to six months.5 6 Therefore, we hypothesized that the duration of the antibiotic effect on ICI efficacy may be present for a longer duration. Methods The electronic medical record was queried at the University of Cincinnati Medical Center for the administration of ICIs and antibiotics. Data was collected on the use, type, and duration of antibiotics before ICI use after administration with relevant demographic and clinicopathologic variables including treatment type, cancer type, staging information, and clinical course including subsequent antibiotic use. Univariant (Fischer’s Exact) and multivariant analysis (multiple logistic regression) were conducted and survival analysis was determined according to log-rank testing. Results 217 patients were examined. The median age was 64 (range 22-93), 38% were female, 73% had ECOG 0-1, and 77% were white. Cancer types included melanoma 24%, non-small cell lung cancer (NSCLC) 34%, small cell lung cancer 2%, renal cancer 11%, urothelial cancer 10%, head and neck cancers 11%, and 16% other primaries. 20% remained on ICI at the time of data entry. The most common ICIs were pembrolizumab, nivolumab, followed by ipilimumab-nivolumab, ipilimumab, and durvalumab. 81 patients of 218 received antibiotics within 6 months of receiving checkpoint inhibitors. Of antibiotics administered, the most common classes were cephalosporins (86%), fluoroquinolones (28%), and glycopeptides (23%) with substantial overlap. Overall survival and progression-free survival was improved for those who did not receive antibiotics prior to ICI therapy (median OS 6.5 vs. 2.3 years, HR 0.36, p<0.0001; median PFS 1.1 vs 0.5 years, HR 0.6, p=0.0027) (figure 1 and 2 respectively). Linear regression showed no significant association between antibiotic use prior to ICI use and age, sex, race, ICI type, or ECOG status. Conclusions This data adds to the growing body of knowledge that the use of antibiotics prior to ICI treatment leads to inferior overall and progression-free survival. Acknowledgements We would like to thank the Roman Jandarov, UC Cancer Center, the University of Cincinnati Division of Hematology and Oncology, Department of Internal Medicine of the University of Cincinnati, and the University of Cincinnati Medical Center for their continued support. Ethics Approval IRB 2019-0610 References Pinato DJ, Gramenitskaya D, Altmann DM, Boyton RJ, Mullish BH, Marchesi JR, et al. Antibiotic therapy and outcome from immune-checkpoint inhibitors. J Immunother Cancer 2019;7:287. Ueda K, Yonekura S, Ogasawara N, Matsunaga Y, Hoshino R, Kurose H, et al. The impact of antibiotics on prognosis of metastatic renal cell carcinoma in japanese patients treated with immune checkpoint inhibitors. Anticancer Res 2019;39:6265–71. Elkrief A, El Raichani L, Richard C, Messaoudene M, Belkaid W, Malo J, et al. Antibiotics are associated with decreased progression-free survival of advanced melanoma patients treated with immune checkpoint inhibitors. Oncoimmunology 2019;8:e1568812. Ruiz-Patiño A, Barrón F, Cardona AF, Corrales L, Mas L, Martín C, et al. Antibiotics impair immune checkpoint inhibitor effectiveness in Hispanic patients with non-small cell lung cancer (AB-CLICaP). Thorac Cancer 2020;(9):2552-2560. Pinato DJ, Howlett S, Ottaviani D, Urus H, Patel A, Mineo T, et al. Association of prior antibiotic treatment with survival and response to immune checkpoint inhibitor therapy in patients with cancer. JAMA Oncol 2019;5(12):1774-1778. Panda S, Khader IE, Casellas F, Vivancos JL, Cors MG, Santiago A, et al. Short-term effect of antibiotics on human gut microbiota. PLoS ONE 2014;9.

From the ∗Departments of Internal Medicine †Pathology ‡Pulmonology and Critical Care Medicine, University of Cincinnati Medical Center, Cincinnati, OH. The authors have no financial disclosures. It should be noted that this represents an off-label use of epoprostenol. Currently, it is only Food and Drug Administration approved in the United States for the treatment of pulmonary artery hypertension, and not as a treatment for Raynaud phenomenon. The authors have no conflicts of interest related to this topic. Correspondence: Eric Vick, MD, PhD, Department of Internal Medicine, University of Cincinnati Medical Center, Medical Sciences Building Room 6056, 231 Albert B. Sabin Way, Cincinnati, OH 45267-0557. E-mail: [email protected].

ATM mutations have been described in breast, lung, hematologic, gastrointestinal and genitourinary malignancies [1, 2]. Pritchard et al report an 11.8% incidence of pathogenic germline mutations in DNA-repair genes among metastatic prostate cancer patients [2]. Of 20 genes surveyed, all associated with hereditary cancer predisposition syndromes, ATM variants comprised 13% (11/84). Further, a “second “hit” somatic aberration” was found in 36/61 (59%) with available tumor sequencing data. We report a 70-year-old man diagnosed with new-onset acute myeloid leukemia (AML) following 14-year maintenance with imatinib for chronic-phase chronic myeloid leukemia (CML) with complete molecular remission and undetectable BCR/ABL transcripts. Cytogenetic analysis from initial bone marrow biopsy demonstrated karyotype 46,XY,del(7) (q22) with fluorescence in situ hybridization negative for BCR/ABL, t(8;21) and t(15;17). Next generation sequencing (NGS) (FoundationOne Heme panel, Foundation Medicine) described four pathogenic mutations: ATM (W579*), DNMT3A (R882H), IDH1 (R132C) and NPM1 (W288fs*10+). Variants of unknown significance (VUS) were found in six genes including ATM (G2863V), which in silico analysis utilizing PolyPhen-2 software (http://genetics.bwh.harvard.edu/pph2/) predicted as probably damaging (Table 1) [3]. Bone marrow biopsy following induction and salvage chemotherapy with cytarabine and idarubicin showed residual disease. During induction, gallbladder adenocarcinoma was diagnosed by biopsy. The patient subsequently elected for hospice care. During treatment, his family declined genetic counseling. One reality of NGS is the prospect of identifying pathogenic germline variants in cancer susceptibility genes [4]. Determining the significance of these mutations is difficult when NGS does not utilize matched-normal comparison DNA. Additionally, the clinical significance of VUSs identified by NGS remains elusive [5]. Following NGS, our CML patient was diagnosed with BCR/ABL-negative AML with concomitant single nucleotide variants in ATM: a pathogenic, non-sense mutation disrupting the protein kinase domain (W579*) and VUS point mutation (G2863V). Public databases including ClinVar and dbSNP supported the pathogenicity of W579*. A search for both variants on ExAc returned no results. Coupled with in silico analysis predicting G2863V as likely pathogenic, his AML harbored two presumed pathogenic mutations in ATM, although germline status was never established. This case illustrates both the tremendous value and unique challenge of interpreting genomic information supplied by NGS. Although rare, BCR/ABL-negative AML has been previously reported in CML patients following imatinib therapy [6-8], with one CML cohort identifying 3/1,701 patients with development of AML or high-risk myelodysplastic syndrome (MDS) with eventual AML transformation [6]. In our patient, we postulate the ATM lesions as contributing to susceptibility to AML following imatinib treatment. Additionally, the identification of coexistent mutations in ATM underscores a need for access to genetic counseling and clinical germline testing. Utilized in genetic counseling, we also propose in silico analysis as a clinical tool for oncologists to better characterize VUS supplied by NGS.

Radiation exposure for the average coronary stent placement varies based on a number of factors but typically amounts to 6-11 mSv per patient (compared to 3 mSv background). As with all procedures which utilize radiation, there is an inherent risk of genetic mutation and the possible development of malignancy. Here, we present the case of a 75-year-old male who presented with an exophytic mass on his back following prolonged coronary catheterization with a radiation burn seven years prior. Biopsy of the lesion revealed the mass was consistent with an undifferentiated pleomorphic sarcoma emanating from the site of the radiation burn. After staging studies demonstrated no evidence of metastatic disease, radical excision with negative margins was performed. This case demonstrates that despite the rarity of radiation injury, each incidence necessitates strict monitoring of radiation exposure and continual follow-up due to the risk of malignancy.

7551 Background: The prognosis, response to therapy and curability of FL3 is controversial. 5-year Overall Survival (OS) in the literature ranges from 35-72% (Ganti 2006). The aim of this study was to compare the OS for patients with early-stage FL3 managed with single- and multi-agent chemotherapy (CT) with and without radiotherapy (RT). Methods: We identified patients (pts) diagnosed with stage I & II FL3 between 2004 – 2012 from the NCDB and categorized into 3 groups based on therapy – pts given single agent CT with or without RT were combined due to small sample sizes (SA±RT), multi-agent CT without RT (MA-RT), and multi-agent CT with RT (MA+RT). We calculated OS for each group using Kaplan-Meier method and compared the results using Log Rank test. Cox regression model was used to identify factors which had significant impact on OS. Results: 1,563 pts were identified – 827 (53%) with stage I and 736 (47%) with stage II FL3. Median age was 61 yrs (range 18-90yrs); 750 (48%) males, 813 (52%) females; 1423 (91%) whites, 76 (5%) blacks. 112 (7%) received SA±RT, 886 (57%) MA-RT and 565 (37%) MA+RT. 5-year OS for MA+RT (95%) was significantly more than MA-RT (87%; HR 0.33, P<0.001) or SA±RT (88%; HR 0.38, P=0.007). Cox regression indicated that age (HR 1.05, P<0.001), sex (HR 0.66 for females, P=0.02), comorbidities (HR 1.60 for Charlson Deyo Score 1, P=0.04; HR 3.07 for Score 2, P=0.001), stage (HR 1.79, P=0.001), insurance status (HR 0.22 for insured, P<0.001) and increasing year of diagnosis (HR 0.92, P=0.03) also had significant impact on OS. Median radiation dose for the MA+RT was 36Gy (interquartile range 30.6 – 36Gy), and the proportion of patients who received greater than 36Gy decreased from 55% in 2004 to 38% in 2012 and at the same time, the proportion of patients who received intensity modulated RT increased from 5% in 2004 to 15% in 2012. Use of MA CT declined (2004 95% v 2012 89%, P=0.02) but there was no significant trend in use of RT (2004 39% v 2012 34%) during the periods studied. Conclusions: For pts with early-stage FL3, there was an association of improved survival with the use of MA+RT over other treatment strategies and appear to have outcomes superior to what has been previously reported.

e19022 Background: There has been some improvement in overall survival (OS) for patients with MCL over the past years [Gordon 2014]. However, side effects of treatment remain a major concern. With introduction of novel therapies like IT, it is imperative to optimize treatment regimens to improve survival while minimizing toxicity. Methods: MCL patients (pts) who were diagnosed in 2013 or later with information available about chemotherapy (CT) and IT and did not receive transplant were extracted from NCDB. Pts were assigned to age categories and were sorted into six groups based on different combinations of CT (none, single agent (SA), and multi-agent (MA)) with or without IT. Cox regression analysis was used to perform multivariate analysis that included age category, sex, race, clinical stage, Charlson/Deyo score, CT, radiation, and IT for each group. Multivariate p values (p) were used to analyze statistical significance. Kaplan Meier method was utilized to analyze OS. T-test was used to compare means (t-p). Results: 1438 total pts were identified with a mean age of 70 (range 24-90); 71% male; 93% white, 4% black, 3% others; 42% with stage III/IV disease. 667 pts did not receive CT or IT, and 40 received IT alone and both of these groups were excluded from further analysis. 52 pts received SA- (without) IT, 206 received SA+(with) IT and 260 pts received MA-IT and 213 MA+IT. Mean age was 72 and 66 for SA and MA groups, respectively (t-p<0.01). Mean OS for SA+IT was 27 months (m) vs SA-IT 16 m (p < 0.01). MA+IT v MA-IT showed no difference in mean OS (25 vs 26 m, respectively, p =0.49). Although there was a significant difference in OS between SA and MA groups without IT (16 vs 25 months, p < 0.01). SA + IT group showed comparable mean OS time to MA + IT (27 vs 26 m, p =0.145). Conclusions: For MCL pts, MA has superior OS to SA group. However, adding IT significantly improves OS for SA group and makes it comparable to MA. Adding IT to MA did not provide significant difference in OS. These results highlight the possibility of achieving same OS with less toxic regimens. Hence further evaluation in a prospective study to optimize treatment while reducing toxicity is warranted.

Background: In diffuse large B-cell lymphoma (DLBCL), chromosomal aberrations are known to increase with advancing age. Our study aims to determine if there are other genetic aberrations associated with DLBCL based on age. Methods: Using the Mitelman Database of Genetic Aberrations, we were able to find 749 cases of DLBCL with genomic aberrations with a median age of 62 years. Patients with DLBCL chromosomal aberration analysis results were divided into four groups based on age (0 - 30, 31- 50, 51- 70, > 71 years) and examined by chi-square analysis and Mantel-Cox for survival analysis. Results: Ten aberrations were found to be significant with a particular age range: t(2;3), trisomy 19p13, trisomy 18q21, trisomy 3, trisomy 7, trisomy 14, trisomy 16, trisomy 18, monosomy 3 and monosomy 11, and survival ranged from 7 to 25 months. Conclusion: This suggests that patients with DLBCL are likely to accumulate specific translocations depending on their age at the onset of DI,BCL.

Peripheral T-cell non-Hodgkin lymphoma (PTCL) are heterogeneous, rare, and aggressive diseases mostly incurable with current cell cycle therapies. Aurora kinases (AKs) are key regulators of mitosis that drive PTCL proliferation. Alisertib (AK inhibitor) has a response rate ∼30% in relapsed and refractory PTCL (SWOG1108). Since PTCL are derived from CD4+/CD8+ cells, we hypothesized that Program Death Ligand-1 (PD-L1) expression is essential for uncontrolled proliferation. Combination of alisertib with PI3Kα (MLN1117) or pan-PI3K inhibition (PF-04691502) or vincristine (VCR) was highly synergistic in PTCL cells. Expression of PD-L1 relative to PD-1 is high in PTCL biopsies (∼9-fold higher) and cell lines. Combination of alisertib with pan-PI3K inhibition or VCR significantly reduced PD-L1, NF-κB expression and inhibited phosphorylation of AKT, ERK1/2 and AK with enhanced apoptosis. In a SCID PTCL xenograft mouse model, alisertib displayed high synergism with MLN1117. In a syngeneic PTCL mouse xenograft model alisertib demonstrated tumor growth inhibition (TGI) ∼30%, whilst anti-PD-L1 therapy alone was ineffective. Alisertib + anti-PD-L1 resulted in TGI >90% indicative of a synthetic lethal interaction. PF-04691502 + alisertib + anti-PD-L1 + VCR resulted in TGI 100%. Overall, mice tolerated the treatments well. Co-targeting AK, PI3K and PD-L1 is a rational and novel therapeutic strategy for PTCL.

ABSTRACT Gardnerella vaginalis is a Gram-variable bacterium associated with bacterial vaginosis, a common vaginal inflammation in women of reproductive age. This study reports the whole-genome sequencing for the clinical isolate strain ATCC 49145. The draft genome is composed of 21 contigs containing 1,325 protein-coding sequences, 45 tRNAs and a single tmRNA (SsrA).

e23141 Background: Discovered as a novel aberration in congenital fibrosarcoma (CF), the ETV6-NTRK3 translocation (EN) confers oncogenic potential and is inhibited by crizotinib. The present study aims to survey the scope of neoplasms that harbor EN across tumor types. Methods: Utilizing the National Cancer Institute’s Mitelman Database (MD) of Chromosome Aberrations and Gene Fusions patients (pts) were identified with EN and categorized based on tumor type, subtype and incidence. Cancer pts who received tumor profiling with Caris were also surveyed for EN. Results: 47 pts with EN across 12 cancer types were extracted from the MD and had median age of 0.17 years (7 unreported); 38% male; 51% acquired malignancies, 49% congenital; 62% cases were pediatric, 23% adult and 15% unknown. 0/204 pts with Caris tumor profiling were found to have an EN. Cancers with the highest number of EN were: 15 (31.9% EN data set) congenital mesoblastic nephromas (CMN), 10 (21.3%) CF, 7 (14.9%) breast carcinoma (BC; 6 secretory ductal carcinoma (SD) and 1 invasive adenocarcinoma (IA)) and 3 (6.4%) colorectal carcinoma (CRC). EN were found in 8 other malignancies (Table 1). Cancer types with the highest incidence of EN+ cases in the MD were gastrointestinal stromal tumor (GIST; 100%), CMN (75%) and CF (23.3%). Conclusions: These results further our understanding of the distribution of ETV6-NTRK3 translocations in multiple tumor types across the age spectrum and suggest that pts with CMN, CF, BC and CRC requiring high order therapy should be considered for NTRK3-based treatment. [Table: see text]