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The contribution of DNA-methylation based gene silencing to carcinogenesis is well established. Increasingly, DNA-methylation is examined using genome-wide techniques, with recent public efforts yielding immense data sets of diverse malignancies representing the vast majority of human cancer related disease burden. Whereas mutation events may group preferentially or in high frequency with a given histology, mutations are poor classifiers of tumour type. Here we examine the hypothesis that cancer-specific DNA-methylation reflects the tissue of origin or carcinogenic risk factor, and these methylation abnormalities may be used to faithfully classify tumours according to histology. We present an analysis of 7427 tumours representing 19 human malignancies and 708 normal samples demonstrating that specific tumour changes in methylation can correctly determine site of origin and tumour histology with 86% overall accuracy. Examination of misclassified tumours reveals underlying shared biology as the source of misclassifications, including common cell of origin or risk factors.

Cancer recurrence after surgery remains an unresolved clinical problem1–3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4–6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy. In mouse models of pulmonary metastasis, adjuvant epigenetic therapy targeting myeloid-derived suppressor cells disrupts the premetastatic microenvironment after resection of primary tumours and inhibits the dissemination of residual tumour cells.

The development of the T- and natural killer (NK) cell growth factor IL-2 has been a sentinel force ushering in the era of immunotherapy in cancer. With the advent of clinical grade recombinant IL-2 in the mid-1980s, oncologists could for the first time directly manipulate lymphocyte populations with systemic therapy. By itself, recombinant IL-2 can induce clinical responses in up to 15% of patients with metastatic cancer or renal cell carcinoma. When administered with adoptively transferred tumor-reactive lymphocytes, IL-2 promotes T cell engraftment and response rates of up to 50% in metastatic melanoma patients. Importantly, these IL-2-driven responses can yield complete and durable responses in a subset of patients. However, the use of IL-2 is limited by toxicity and concern of the expansion of T regulatory cells. To overcome these limitations and improve response rates, other T cell growth factors, including IL-15 and modified forms of IL-2, are in clinical development. Administering T cell growth factors in combination with other agents, such as immune checkpoint pathway inhibitors, may also improve efficacy. In this study, we review the development of T- and NK cell growth factors and highlight current combinatorial approaches based on these reagents.

Colorectal cancer (CRC) remains the third most common malignancy and the second-leading cause of cancer-related deaths in the United States. Large multi-omic databases, such as The Cancer Genome Atlas and the International Colorectal Cancer Subtyping Consortium, have identified distinct molecular subtypes related to anatomy. The identification of genomic alterations in CRC is now critical because of the recent success and US Food and Drug Administration approval of pembrolizumab and nivolumab for microsatellite-instable tumors. In parallel, landmark studies have established the prognostic significance of the CRC tumor-infiltrating lymphocyte and the clinical impact of the tumor immune microenvironment. As a result, there is a growing appreciation for immunogenomics, the interconnected relation between tumor genomics and the immune microenvironment. The clinical implications of CRC immunogenomics continue to expand, and it will likely serve as a guide for next-generation immunotherapy strategies for improving outcomes for this disease. Cancer 2018;124:1650-9. © 2018 American Cancer Society.

2596 Background: There is currently a paucity of treatment options for checkpoint relapsed patients who have had an initial response but subsequently progress. N803, a novel IgG1 Fc-engineered IL-15-complexed protein may rescue checkpoint activity in a checkpoint independent manner via its selective enhancement of natural killer cell (NK) and CD8+ T cell number and function, without stimulation of T regs and MDSCs. Methods: QUILT 3.055, (NCT03228667) a phase 2b study of N803 plus investigator choice CPI in 11 tumor types: NSCLC, SCLC, Urothelial carcinoma, HNSCC, Merkel cell carcinoma, Melanoma (single PD-1/PD-L1 CPI or w/ ipilimumab), Renal cell carcinoma (RCC), Gastric cancer, Cervical cancer, Hepatocellular carcinoma, Microsatellite instability-high (MSI-H)/ mismatch repair deficient (dMMR) solid tumors, with a heterogeneous mix of prior therapies. We present interim data for 135 patients treated with CPI alone or in combination with chemotherapy as their most recent prior therapy. Trial inclusion required investigator assessed progression on last line of therapy, patients had either CR with relapse or partial response or stable disease for 6 months with progression as their most recent result of checkpoint therapy. Patients with hyperprogression or best initial response of progression were excluded. Subjects received N803 15mck/kg SC q 3 weeks in combination with the same checkpoint inhibitor on which they had their most recent progression. Results: Preliminary data from 135 patients (60% NSCLC) with treatment with checkpoint and N-803 following progression on the same checkpoint show CR 0%, PR 8%, Stable Disease 51%, Progression 29%, response unevaluable 12% to date. A PR or SD was seen in all subgroups. Median PFS 3.9 months (95% CI: 2.6,5.0). Median OS 13.8 months (95% CI: 11.8, 16.3) N-803 is well tolerated with grade 1-2 common N-803 treatment related adverse events (TRAE) were injection site reaction (68%), chills (32%) fatigue (26%), pyrexia (26%), flu-like illness (14%), nausea (12%) and no other individual AE > 10%. Grade 3 N-803 TRAE were 12% but no individual grade 3 AEs were greater than 5%. Conclusions: N803 demonstrates low toxicity in patients previously treated with CPI and promising efficacy of cessation of progression and induction of response and durable stable disease in patients who had previously progressed on a CPI containing regimen in multiple tumor types and different CPIs. Clinical trial information: NCT03228667.

9077 Background: NSCLC with EGFR ins20 represents a significant area of unmet need, with no approved targeted therapies. While several agents targeting EGFR ins20 are in development, wild-type (WT) EGFR-related adverse events (AEs) have been common and challenging to manage. CLN-081 is a novel oral EGFR TKI with broad activity against clinically relevant EGFR mutations, including ins20, and has attenuated activity against WT EGFR relative to EGFR ins20 in vitro, suggesting that CLN-081 may have a more favorable clinical therapeutic window. We present interim results of a multicenter, Phase (Ph) 1/2a trial evaluating CLN-081 in advanced, EGFR ins20 NSCLC (NCT04036682). Methods: Patients (pts) with EGFR ins20 previously treated with platinum-based therapy (tx) were eligible to enroll. Ph 1 dose escalation in this adaptive trial began with an accelerated titration (AT) design, and converted to a rolling six design based upon pre-specified safety criteria or at clinically active doses. Cohort expansion in Ph 1 occurred at any dose where responses were seen. Transition from Ph 1 to 2a was based on a Simon-Two Stage design. Prior tx with EGFR ins20-specific inhibitors was allowed in AT cohorts only. CLN-081 was dosed twice daily (BID) in 21-day cycles. Results: As of 10 November 2020, 37 pts [median age 64 years (44-82); median 2 (1-9) prior lines of tx] received CLN-081 at doses of 30 mg (n = 8), 45 mg (1), 65 mg (12), 100 mg (13), and 150 mg (3) BID. The most common all-grade (gr) treatment-related AEs (TRAEs) were rash (49%), diarrhea (24%), paronychia (16%), nausea (14%), stomatitis (14%), and dry skin (11%). Gr 3 TRAEs included anemia (5%), diarrhea (3%), and increased alkaline phosphatase (ALP) (3%). There was 1 DLT, gr 3 diarrhea at 150 mg BID. No gr ≥ 3 rash or gr 4/5 TRAEs were reported. Four pts (11%) required dose reductions for rash (2), diarrhea (1), and increased ALP (1). Two pts (5%) discontinued tx due to TRAEs of gr 2 hypersensitivity reaction (1) and gr 2 pneumonitis (1); the latter also experienced pneumonitis while receiving prior osimertinib. Among the 25 response evaluable pts (RECIST 1.1), 10 (40 %) had a partial response (PR) (6 confirmed, 2 pending confirmation, 2 unconfirmed), 14 (56%) had stable disease (SD), and 1 (4%) had progressive disease as best response. Of the 4 pts that received prior EGFR ins20 inhibitors, 2 had PR and 2 SD. Of pts with SD or PR as best response, 20/24 (83 %) experienced tumor regression [median regression: -18 % (-100 to +3)]. Enrollment is ongoing and updated data will be presented. Conclusions: CLN-081 has an acceptable safety profile, including diarrhea in < 25% of pts treated to date. CLN-081 has demonstrated encouraging preliminary anti-tumor activity across the full dose range tested, in multiple distinct EGFR ins20 variants, and in heavily pre-treated pts that are either naïve or refractory to other EGFR ins20 inhibitors. Since the time of the data cut, a Ph 2a expansion has been initiated at 100 mg BID. Clinical trial information: NCT04036682.

2552 Background: Nemvaleukin alfa (nemvaleukin, ALKS 4230) is a novel engineered cytokine that selectively binds the intermediate-affinity interleukin-2 receptor to preferentially activate CD8+ T and natural killer (NK) cells with minimal expansion of regulatory T cells (Tregs),designed for use as a cancer immunotherapy. ARTISTRY-2 (NCT03861793) is an ongoing phase 1/2 study evaluating the safety, efficacy, and pharmacokinetic and pharmacodynamic (PD) responses of subcutaneous (SC) nemvaleukin in combination with pembrolizumab in patients (pts) with advanced solid tumors. Methods: In phase 1, cohort-specific doses of SC nemvaleukin are administered on an every-7-day (q7d) or every-21-day (q21d) schedule during a 6-week monotherapy lead-in period, followed by combination with pembrolizumab 200 mg q21d. We present safety, PD effects, and preliminary clinical activity outcomes as of 12/02/2020. Results: 57 pts received nemvaleukin doses ranging from 0.3 mg to 6 mg q7d or 1 mg to 10 mg q21d. The most frequent tumor types (> 5 pts) were colorectal, pancreatic, ovarian, and lung; median number of prior therapies was 4. Treatment-related adverse events (TRAEs) in > 30% pts overall were pyrexia (43.9%), chills (38.6%), injection site erythema (33.3%), injection site reaction (33.3%), and fatigue (31.6%). 3 mg q7d (n = 7) had no drug-related dose reductions, discontinuations, or deaths during the monotherapy or combination periods. 6 mg was declared the maximum tolerated dose (MTD) for q7d dosing as 2 of 8 pts experienced dose-limiting toxicities (DLTs). For 6 mg q21d (n = 7), no drug-related dose reductions, discontinuations, or deaths have occurred during the monotherapy period; combination period data are not mature. 10 mg was declared the MTD for q21d dosing as 1 of 9 pts experienced DLTs and 3 had TRAEs leading to dose reductions. Systemic exposure to nemvaleukin increased with increasing dose. Increases in NK cells and CD8+ T cells of approximately 16-fold and 3-fold, respectively, at 3 mg q7d, and approximately 8-fold and 3-fold, respectively, at 6 mg q21d were observed, with minimal change in Tregs. 46 pts had at least 1 on-treatment scan as of the data cutoff date, and 30 (65%) had stable disease (SD) on the first scan. Of the 30 pts with ≥2 scans, 13 (43%) had 2+ consecutive scans of SD. 16 of 57 pts remain on therapy. Antitumor activity data for more recent cohorts are still maturing. Based on the totality of the safety, PD effects, and antitumor activity data, 3 mg q7d was selected as the RP2D for SC nemvaleukin. Conclusions: SC nemvaleukin 3 mg q7d was generally well tolerated as monotherapy and in combination with pembrolizumab, and demonstrated robust PD effects on NK cells and CD8+ T cells with minimal expansion of Tregs. These PD effects are similar to or greater than those observed with intravenous nemvaleukin. Thus, 3 mg q7d was selected as RP2D; phase 2 expansion cohorts for combination with pembrolizumab are enrolling. Clinical trial information: NCT03861793.

We identified unique molecular mechanisms of resistance to CDK4/6 inhibitors, an area of intense biomedical investigation., The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome–associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

e15008 Background: The oncotherapeutic promise of IL-15, a potent immune stimulant, is limited by short serum half-life. The fusion protein N-803 is an IL-15 superagonist complex that has > 20-fold longer half-life in vivo vs IL-15. This study characterized the pharmacokinetic (PK) profile, biological activity, and safety of N-803 after subcutaneous administration to healthy human volunteers. Methods: Volunteers were randomized 1:1 to receive 1 mg/mL or 2 mg/mL N-803. Each subject received 2 doses of N-803: 10 µg/kg followed 24 days later by 20 µg/kg. After each dose, PK and safety measures were assayed for 9 successive days. Primary endpoint was the PK profile of N-803; secondary was safety; and exploratory endpoints were cytokine levels, immune cell characterization, and immunogenicity. Results: N-803 resulted in no grade ≥3 or serious adverse events (AEs). Mild injection site reactions, chills, and pyrexia were the most common AEs. Serum N-803 concentrations peaked 10.3-15.4 hours after administration and declined with a half-life of 20.0-20.7 hours. Peak N-803 serum concentrations were dose-dependent, with a 1.5-fold increase in Cmax after administration of 20 µg/kg vs 10 µg/kg. In the peripheral blood, N-803 induced a transient decline, followed by a significant increase (3-fold) in NK cell number that persisted for ≥24 days. N-803 also caused a significant proliferation of NK (22-fold increase in Ki-67+ cells), CD8+ (27-fold) and CD4+ T (11-fold) cells; however, increased cell number occurred only in NK cells. N-803 administration also increased serum levels of interferon gamma, IL-10, and IL-6. One of 14 evaluable subjects had measureable anti-N-803 antibodies at the end-of-study visit. Conclusions: N-803 results in prolonged elevation of drug serum concentrations, contrasting with rapid clearance of recombinant human IL-15 (ie, half-life of ~20 vs < 1 hour). N-803 administration was well-tolerated in healthy volunteers, without evidence of adverse systemic inflammatory responses, and resulted in proliferation of NK cells and CD8+ T cells, as well as sustained increases in NK cell number. Findings in this study are consistent with published results from N-803 administration in treating liquid tumors and lung cancer. Our results demonstrate N-803 administration potentiates the proliferation and activity of lymphocytes with antitumor and antivirus properties, and suggest this investigational product holds promise in treatment of cancer as well as infectious disease such as HIV. Clinical trial information: NCT03381586 .

The emergence of immuno-oncology (IO) agents has led to unprecedented successes in the treatment of deadly cancers and sometimes even cures. These agents act by “releasing the brakes” and thereby unleashing the full potential of cells that fight tumors. Sadly, not every patient benefits from these new treatments. We need to know who will benefit and who may not. Here we approached the problem by designing a customized workflow by using high-dimensional single-cell mass cytometry (CyTOF) combined with machine-learning bioinformatics for the in-depth characterization of single immune cells in predicting and monitoring immune responses. The analysis is data driven, can be adapted to high-throughput approaches, and can model arbitrary trial designs such as batch effects and paired designs. We tested our workflow on two studies: a) Predict response to anti-PD-1 immunotherapy in melanoma. In our discovery cohort peripheral blood mononuclear cells (PBMCs) from 20 patients with stage IV melanoma before and after 12 weeks of anti-PD-1 therapy were analyzed. We observed a clear T-cell response on therapy. The most evident difference in responders before therapy was an enhanced frequency of CD14+ CD16+HLA-DRhi classical monocytes. We validated our results using conventional flow cytometry in an independent exploratory cohort of 31 patients before therapy. Finally, we correlated enhanced monocyte frequencies before therapy initiation with clinical response and could show association with lower hazard, extended progression-free and overall survival. b) Monitoring the immune response in anti-PD-1 immunotherapy-refractory non-small cell lung cancer to a novel combination immunotherapy of anti-PD-1 and an IL-15 superagonist. Twenty-one patients with non-small cell lung cancer who mostly were refractory to previous anti-PD-1 treatment received a novel combination therapy of IL-15 superagonist plus anti-PD-1. A response in the CD8+ T cell compartment was observed. Unexpectedly, our high-dimensional approach was able to detect and characterize a strong expansion of innate tumor-reactive effector NK cells starting around day 4 of therapy. Taken together, our unbiased artificial intelligence-driven immune workflow might support patient selection prior to therapy, select the right drug combination, and identify new druggable cell populations. Citation Format: Luis Cardenas, Silvia Guglietta, John Wrangle, Mark Rubinstein, Mark Robinson, Carsten Krieg. Using high-dimensional machine-assisted analysis for biomarkers detection and guidance of immunotherapy to cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA09.

Purpose: PD-1/L1 axis–directed therapies produce clinical responses in a subset of patients; therefore, biomarkers of response are needed. We hypothesized that quantifying key immunosuppression mechanisms within the tumor microenvironment by multiparameter algorithms would identify strong predictors of anti–PD-1 response. Experimental Design: Pretreatment tumor biopsies from 166 patients treated with anti–PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery (n = 24) and validation (n = 142) cohorts. Biomarker-positive cells and their colocalization were spatially profiled in pathologist-selected tumor regions using novel Automated Quantitative Analysis algorithms. Selected biomarker signatures, PD-1/PD-L1 interaction score, and IDO-1/HLA-DR coexpression were evaluated for anti–PD-1 treatment outcomes. Results: In the discovery cohort, PD-1/PD-L1 interaction score and/or IDO-1/HLA-DR coexpression was strongly associated with anti–PD-1 response (P = 0.0005). In contrast, individual biomarkers (PD-1, PD-L1, IDO-1, HLA-DR) were not associated with response or survival. This finding was replicated in an independent validation cohort: patients with high PD-1/PD-L1 and/or IDO-1/HLA-DR were more likely to respond (P = 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36; P = 0.0004) and overall survival (HR = 0.39; P = 0.0011). In the combined cohort, 80% of patients exhibiting higher levels of PD-1/PD-L1 interaction scores and IDO-1/HLA-DR responded to PD-1 blockers (P = 0.000004). In contrast, PD-L1 expression was not predictive of survival. Conclusions: Quantitative spatial profiling of key tumor-immune suppression pathways by novel digital pathology algorithms could help more reliably select melanoma patients for PD-1 monotherapy. Clin Cancer Res; 24(21); 5250–60. ©2018 AACR.

// Frank P. Vendetti 1 , Michael Topper 1 , Peng Huang 1 , Irina Dobromilskaya 1 , Hariharan Easwaran 1 , John Wrangle 1 , Stephen B. Baylin 1 , J. T. Poirier 2 , Charles M. Rudin 2 1 The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD 2 Memorial Sloan Kettering Cancer Center, NY 10065, New York Correspondence to: Charles M. Rudin, e-mail: rudinc@mskcc.org Keywords: epigenetic, azacitidine, entinostat, priming, chemosensitivity, non-small cell lung cancer (NSCLC) Received: July 25, 2014 Accepted: November 04, 2014 Published: November 25, 2014 ABSTRACT Recent clinical data in lung cancer suggests that epigenetically targeted therapy may selectively enhance chemotherapeutic sensitivity. There have been few if any studies rigorously evaluating this hypothesized priming effect. Here we describe a series of investigations testing whether epigenetic priming with azacitidine and entinostat increases sensitivity of NSCLC to cytotoxic agents. We noted no differences in chemosensitivity following treatment with epigenetic therapy in in vitro assays of viability and colony growth. Using cell line and patientderived xenograft (PDX) models, we also observed no change in responsiveness to cisplatin in vivo . In select models, we noted differential responses to irinotecan treatment in vivo . In vitro epigenetic therapy prior to tumor implantation abrogated response of H460 xenografts to irinotecan. Conversely, in vitro epigenetic therapy appeared to sensitize A549 xenografts (tumor growth inhibition 51%, vs. 22% in mock-pretreated control). In vivo epigenetic therapy enhanced the response of adenocarcinoma PDX to irinotecan. Taken together, these data do not support broadly applicable epigenetic priming in NSCLC. Priming effects may be context-specific, dependent on both tumor and host factors. Further preclinical study is necessary to determine whether, and in which contexts, priming with epigenetic therapy has potential to enhance chemotherapeutic efficacy in NSCLC patients.

Effector CD8+ T cells conditioned with IL12 during activation mediate enhanced antitumor efficacy after adoptive transfer into lymphodepleted hosts; this is due in part to improved IL7 responsiveness. Therefore, we hypothesized that increasing the intensity or type of lymphodepletion would deplete more IL7-consuming host cells and improve the persistence and antitumor activity of IL12-conditioned CD8+ T cells. Using cyclophosphamide, fludarabine, and total body irradiation (TBI, 6 Gy) either individually or in combination, we found that combined lymphodepletion best enhanced T-cell engraftment in mice. This improvement was strongly related to the extent of leukopenia, as posttransfer levels of donor T cells inversely correlated to host cell counts after lymphodepletion. Despite the improvement in engraftment seen with combination lymphodepletion, dual-agent lymphodepletion did not augment the antitumor efficacy of donor T cells compared with TBI alone. Similarly, IL7 supplementation after TBI and transfer of tumor-reactive T cells failed to improve persistence or antitumor immunity. However, IL15 or IL2 supplementation greatly augmented the persistence and antitumor efficacy of donor tumor-reactive T cells. Our results indicate that the amount of host IL7 induced after single agent lymphodepletion is sufficient to potentiate the expansion and antitumor activity of donor T cells, and that the efficacy of future regimens may be improved by providing posttransfer support with IL2 or IL15. Significance: The relationship between lymphodepletion and cytokine support plays a critical role in determining donor T-cell engraftment and antitumor efficacy. Cancer Res; 78(11); 3067–74. ©2018 AACR.

O1 Combinatorial CD8+ and PD-L1+ cell densities correlate with response and improved survival in non-small cell lung cancer (NSCLC) patients treated with durvalumab #### Sonja Althammer1, Keith Steele2, Marlon Rebelatto2, Tze Heng Tan1, Tobias Wiestler1, Guenter Schmidt1, Brandon Higgs2, Xia

Checkpoint inhibitors have significantly accelerated cancer treatment but still a majority of patients do not respond. Biomarker driven patient stratification early to the right immunotherapeutic might enhance response and patient survival. Here we used high-dimensional mass cytometry (CyTOF) combined with machine-learning bioinformatics for the in-depth characterization of immune responses before and during anti-PD-1 immunotherapy. CyTOF allows us to monitor protein expression of 34 markers on a single cell while running 20 samples simultaneously. The analysis is data driven, can be adapted to high throughput approaches and can model arbitrary trial designs such as batch effects and paired designs and is quantitative over millions of events. Using CyTOF as a precision medicine tool we could predict response to anti-PD-1 using liquid blood biopsies. Biobanked peripheral blood mononuclear cells (PBMCs) from 51 patients with stage IV melanoma before and after 12 weeks of anti-PD-1 therapy was analyzed. We observed a clear T cell response on therapy. The most evident difference in responders before therapy was an enhanced frequency of CD14+ CD16+HLA-DRhi classical monocytes. We validated our results using conventional flow and found a clear correlation of enhanced monocyte frequencies before therapy initiation with clinical response such as lower hazard and extended progression-free and overall survival. In a second study we used CyTOF to monitor immune response in 21 non small cell lung cancer (NSCLC) patients that initially responded and then progressed under anti-PD-1 to a novel combination immunotherapy of anti-PD-1 plus an IL-15 super-agonist (ALT-803). In this phase Ib clinical study a response in the CD8+ T cell compartment was observed. Unexpected our high dimensional unbiased analysis was able to detect and characterize a strong expansion of innate tumor-reactive effector NK cells starting around day 4 of therapy. Taken together, our unbiased artificial intelligence driven immune workflow might support patient selection prior to therapy, and serve as a novel tool for precision medicine to select the right drug combination and identify new drug-able cell populations. Citation Format: Carsten Krieg, Luis Cardenas, Silvia Guglietta, John Wrangle, Mark Rubinstein, Mark Robinson. Is biomarker-driven precision medicine possible by using high dimensional augmented intelligence assisted analysis of cancer immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4225.

8516 Background: Rovalpituzumab tesirine (Rova-T™) is an antibody-drug conjugate targeting DLL3, a Notch ligand expressed in SCLC but not normal tissue. Nivo ± Ipi has activity in 2L+ SCLC. Preliminary data suggest Rova-T may result in immunogenic cell death, complementing effects of Nivo ± Ipi. Methods: Eligibility: DLL3 expression (DLT phase only), progression after ≥1 line of therapy including a platinum-based regimen; ECOG 0-1; no prior immunotherapy. All pts received 0.3 mg/kg Rova-T IV on Day 1 of two 6-wk cycles. Cohort 1 (C1) also received two 3-wk cycles of 360 mg Nivo beginning on wk 4. Cohort 2 (C2) received four 3-wk cycles of 1 mg/kg Nivo and 1 mg/kg Ipi beginning on wk 4. Both cohorts then received 480 mg Nivo q4wks until PD. Primary objective: safety. Secondary: antitumor activity by RECISTv1.1, OS. Exploratory: PK. Results: As of Sep 7, 2018, 30 pts were dosed in C1 and 12 in C2. 55% were DLL3 high (≥75% DLL3 expression). 28 (67%) completed 2 planned cycles of Rova-T. 4 pts (1 in C1, 3 in C2) experienced DLTs including rash (3), pneumonitis (1) and colitis (1). C1 completed recruitment, and C2 enrollment was stopped after DLT evaluation phase. Preliminary PK showed Nivo±Ipi had no substantial effect on Rova-T exposure. Clinical trial information: NCT03026166. Conclusions: Despite activity in 2L+ ED-SCLC, Rova-T with Nivo/Ipi is not appropriate due to DLTs. Rova-T/Nivo demonstrated some durable responses; however, the safety data suggest that optimization of dose and schedule is warranted. NCT03026166.[Table: see text]

6055 Background: PD-1 inhibition therapy has revolutionized clinical medicine as it can mediate durable responses in a small cohort of patients. Yet, it remains incompletely understood why these patients respond. To address this question, we studied patients with oral cavity squamous cell carcinoma (OCSCC) to elucidate immune phenotypes associated with response to nivolumab. Methods: We defined the immune profile from the blood and tumor of patients on neoadjuvant nivolumab. We tested if tumor-infiltrating lymphocytes (TIL) could be preferentially expanded ex vivo from nivolumab-responsive patients versus those who were either non-responsive or had never received nivolumab. During the course of therapy, we comprehensively profiled a number of surface markers on patients’ T cells to define their activation status, cytotoxic capacity and memory phenotype. Moreover, the immune profile of the peripheral blood was assessed pre- and post-nivolumab using high dimensional mass cytometry. Results: Regardless of PD-1 therapy, TIL were successfully expanded from 11 of the 12 patients. TIL were comprised of both CD4+ and CD8+ T cells. Additional investigation revealed that the frequency of CD4+ T cells and effector memory T cells in TIL correlated with disease progression (CD4: p = 0.04, r = 0.74, effector memory: p = 0.046, r = 0.74). TILs from responders expressed higher CD26 (p = 0.007, r = -0.88) and Tim3 (p = 0.045, r = -0.74) while PD-1, Lag3, and Ox40 were not differentially expressed based on response. Spearman correlation and Mann Whitney U test were used to assess phenotypic differences. Conclusions: We demonstrate, for the first time, that TIL can be reliably expanded from OCSCC patients on neoadjuvant nivolumab. Moreover, individuals who were responsive to PD-1 blockade had TIL expressing high levels of CD26 and Tim3. Future studies will explore if these markers are predictive of responses and if they contribute to treatment outcome.

Innovative therapies are needed for advanced Non-Small Cell Lung Cancer (NSCLC). We have undertaken a genomics based, hypothesis driving, approach to query an emerging potential that epigenetic therapy may sensitize to immune checkpoint therapy targeting PD-L1/PD-1 interaction. NSCLC cell lines were treated with the DNA hypomethylating agent azacytidine (AZA – Vidaza) and genes and pathways altered were mapped by genome-wide expression and DNA methylation analyses. AZA-induced pathways were analyzed in The Cancer Genome Atlas (TCGA) project by mapping the derived gene signatures in hundreds of lung adeno (LUAD) and squamous cell carcinoma (LUSC) samples. AZA up-regulates genes and pathways related to both innate and adaptive immunity and genes related to immune evasion in a several NSCLC lines. DNA hypermethylation and low expression of IRF7, an interferon transcription factor, tracks with this signature particularly in LUSC. In concert with these events, AZA up-regulates PD-L1 transcripts and protein, a key ligand-mediator of immune tolerance. Analysis of TCGA samples demonstrates that a significant proportion of primary NSCLC have low expression of AZA-induced immune genes, including PD-L1. We hypothesize that epigenetic therapy combined with blockade of immune checkpoints – in particular the PD-1/PD-L1 pathway – may augment response of NSCLC by shifting the balance between immune activation and immune inhibition, particularly in a subset of NSCLC with low expression of these pathways. Our studies define a biomarker strategy for response in a recently initiated trial to examine the potential of epigenetic therapy to sensitize patients with NSCLC to PD-1 immune checkpoint blockade.

ICOS costimulation generates Th17 cells with durable memory responses to tumor. Herein, we found that ICOS induces PI3K/p110δ/Akt and Wnt/β-catenin pathways in Th17 cells. Coinhibiting PI3Kδ and β-catenin altered the biological fate of Th17 cells. Th17 cells inhibited of both pathways expressed less RORγt, which, in turn, reduced their ability to secrete IL-17. Unexpectedly, these cells were more effective (than uninhibited cells) at regressing tumor when infused into mice, leading to long-term curative responses. PI3Kδ inhibition expanded precursor Th17 cells with a central memory phenotype that expressed nominal regulatory properties (low FoxP3), while β-catenin inhibition enhanced Th17 multifunctionality in vivo. Remarkably, upon TCR restimulation, RORγt and IL-17 rebounded in Th17 cells treated with PI3Kδ and β-catenin inhibitors. Moreover, these cells regained β-catenin, Tcf7, and Akt expression, licensing them to secrete heightened IL-2, persist, and eradicate solid tumors without help from endogenous NK and CD8 T cells. This finding shines a light on ways to repurpose FDA-approved drugs to augment T cell-based cancer immunotherapies.

This abstract is being presented as a short talk in the scientific program. A full abstract is printed in the Proffered Abstracts section (PR05) of the Conference Proceedings. Citation Format: John Wrangle, Vamshidar Velchetti, Manish Patel, Samantha Suriano, Marzena Swiderska-syn, Kate Anderton, James Ravenel, Elisabeth Hill, Amy Rock, Hing Wong, Mark Rubinstein. Safety and activity of the IL-15/sIL-15Rα complex ALT-803 in combination with the anti-PD1 mAb nivolumab in metastatic non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B34.

Background: Despite the unprecedented efficacy of immunotherapy with PD-1/PD-L1 blockade in non-small cell lung cancer, treatment failure is common and an area of unmet clinical need. IL-2/IL-15Rbg agonists, which potently augment the activity of CD8+ T and NK cells, have shown the ability to induce complete and durable responses in select cancers. However, this class of drugs has been neither combined with PD-1 targeted immunotherapies nor examined extensively in NSCLC. We hypothesized that the addition of an IL-15 superagonist (ALT-803) would be safe and improve the efficacy of anti-PD-1 mAb (nivolumab) in metastatic NSCLC patients in second-line therapy. Methods: In this phase Ib trial, patients with metastatic NSCLC who were eligible to initiate or to continue receiving nivolumab (anti-PD-1 mAb) after first-line therapy were enrolled to trial, including patients with disease relapsed on or refractory to PD-1 blockade. Nivolumab was administered in combination with ALT-803 at four dose levels. The primary objective of the study was to define safety and tolerability and a recommended phase II dose of ALT-803 in combination with nivolumab, with secondary objectives including immune correlates and clinical parameters including response rate. Results: In a first-in-human combination, 21 NSCLC patients were treated at 4 dose levels of ALT-803 in combination with the approved dose of nivolumab. No dose-limiting toxicities were observed, and the most common adverse events were fever, flu-like symptoms, and injection site reactions. The recommended phase II dose (RP2D) of therapy is weekly subcutaneous ALT-803 at 20 mcg/kg in combination with intravenous nivolumab 240 mg every 14 days. Multiple objective responses were observed, including when ALT-803 was added to a failing PD-1 mAb regimen. Discussion: The recommended phase II dose of ALT-803 plus nivolumab was determined and the regimen can safely be administered in the outpatient setting. Responses among patients with PD-1 mAb relapsed disease with the addition of ALT-803 to their regimen demonstrate for the first time evidence of antitumor activity for a new class of agents for NSCLC and in patients with disease progression on PD-1/PD-L1 pathway blockade. This abstract is also being presented as Poster B34. Citation Format: John Wrangle, Vamshidar Velchetti, Manish Patel, Samantha Suriano, Marzena Swiderska-syn, Kate Anderton, James Ravenel, Elisabeth Hill, Amy Rock, Hing Wong, Mark Rubinstein. Safety and activity of the IL-15/sIL-15Rα complex ALT-803 in combination with the anti-PD1 mAb nivolumab in metastatic non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR05.

e24141Background: Current treatments for SCLC produce transient responses – 2-year survival is 5%. One reason is that the treatments apply stress upstream of p53 to upregulate it (cytotoxicity), bu...

e24134Background: Most advanced non-small cell lung cancer (NSCLC) cases do not respond to anti-PD1 therapy, because most NSCLC avoids immune-recognition in the 1st place, e.g, by epigenetic suppre...

Background: Phase I study with the antibody-drug conjugate anetumab ravtansine demonstrated promising safety and efficacy as monotherapy in mesothelin-expressing tumors, and in particular for patients with advanced metastatic malignant pleural mesothelioma. Pemetrexed in combination with cisplatin is standard for first-line treatment of patients with metastatic mesothelioma and NSCLC. We therefore conducted a phase Ib study to determine the safety, tolerability, and maximum tolerated dose (MTD) of anetumab ravtansine in combination with pemetrexed/cisplatin (Pem/Cis) in subjects with mesothelin-expressing predominantly epithelial mesothelioma or nonsquamous NSCLC. Methods: Patients with histologically confirmed, unresectable, locally advanced or metastatic, mesothelin-expressing predominantly epithelial pleural or peritoneal mesothelioma or nonsquamous NSCLC, who previously failed ≤3 prior lines of chemotherapy, were eligible. Patients were prescreened based on obligatory tumor staining for mesothelin as determined by the Ventana MSLN (SP74) immunohistochemistry assay. Anetumab ravtansine was administered by 1-hour IV infusion on day 1 of every 21-day treatment cycle (Q3W). Pem (500 mg/m2) and Cis (75 mg/m2) were administered as IV infusions Q3W. Serial plasma samples were collected and analyzed for anetumab ravtansine and its components, pemetrexed, total and free platinum. Response to therapy was assessed by RECIST 1.1 and modified RECIST criteria for mesothelioma. Results: As of June 12, 2017, 17 patients had been treated and had received ≥2 cycles of treatment, including 7 patients with pleural mesothelioma, 9 with peritoneal mesothelioma, and 1 with NSCLC. Three patients were treated with anetumab ravtansine 5.5 mg/kg in combination with Pem/Cis with no dose-limiting toxicity (DLT). Six patients were then treated with anetumab ravtansine 6.5 mg/kg in combination with Pem/Cis, again without DLTs. This dose in combination was deemed as the MTD. An additional 8 patients were enrolled in an MTD expansion cohort. Anetumab ravtansine-related adverse events were mainly grade (G)1 and G2. G1/G2 eye toxicities were possibly more frequent with the combination than in historical data. There was one G3 corneal microcystic event and one G3 hypertension. There was a 50% overall response rate (all partial responses) from 16 evaluable patients. Of 13 patients treated at the 6.5 mg/kg MTD dose of anetumab ravtansine in combination with Pem/Cis, the overall response rate was 46%. Based on comparisons to historical data and within-subject comparisons in small number of subjects, clinically relevant PK interaction was not observed. Conclusions: Anetumab ravtansine at 6.5 mg/kg dosed Q3W in combination with standard dosing of Pem/Cis had a manageable toxicity profile without evidence of PK interaction and with early signs of clinical efficacy. Anetumab ravtansine at 6.5 mg/kg in combination with Pem 500 mg/m2 and Cis 75 mg/m2, all administered in a Q3W regimen, is thus feasible and the recommended dose for future study. Citation Format: Raffit Hassan, Ding Wang, John Wrangle, Anish Thomas, Angus Byars, Lianne Asschert, Rolando Atienza, Prabhu Rajagopalan, Annette Walter, Jun Zhang, Nenad Sarapa, Hedy Kindler. Phase Ib study of anetumab ravtansine in combination with pemetrexed and cisplatin in patients with mesothelin-expressing epithelial mesothelioma or nonsquamous non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A095.

Purpose: Non–small cell lung cancer (NSCLC) is the leading cause of cancer mortality in the world. Novel diagnostic biomarkers may augment both existing NSCLC screening methods as well as molecular diagnostic tests of surgical specimens to more accurately stratify and stage candidates for adjuvant chemotherapy. Hypermethylation of CpG islands is a common and important alteration in the transition from normal tissue to cancer. Experimental Design: Following previously validated methods for the discovery of cancer-specific hypermethylation changes, we treated eight NSCLC cell lines with the hypomethylating agent deoxyazacitidine or trichostatin A. We validated the findings using a large publicly available database and two independent cohorts of primary samples. Results: We identified >300 candidate genes. Using The Cancer Genome Atlas (TCGA) and extensive filtering to refine our candidate genes for the greatest ability to distinguish tumor from normal, we define a three-gene panel, CDO1, HOXA9, and TAC1, which we subsequently validate in two independent cohorts of primary NSCLC samples. This three-gene panel is 100% specific, showing no methylation in 75 TCGA normal and seven primary normal samples and is 83% to 99% sensitive for NSCLC depending on the cohort. Conclusion: This degree of sensitivity and specificity may be of high value to diagnose the earliest stages of NSCLC. Addition of this three-gene panel to other previously validated methylation biomarkers holds great promise in both early diagnosis and molecular staging of NSCLC. Clin Cancer Res; 20(7); 1856–64. ©2014 AACR.

e14104Background: Ifosfamide is a moderate to highly emetogenic agent used in multiple chemotherapy regimens. Ifosfamide (Ifo) induced encephalopathy (IIE) is a neurotoxic adverse event with a repo...

The aim of this article is to summarize progress in understanding of the biology of squamous cell carcinoma of the head and neck and of trials to prevent malignant conversion of oral premalignant lesions and the development of second primary tumors in those already treated for squamous cell carcinoma of the head and neck.The understanding of squamous cell carcinoma of the head and neck biology is rapidly evolving. Clinical trials for chemoprevention are involving more diverse regimens, following disappointing results of retinoid monotherapy. In-vitro and animal studies form the rationale for the next generation of studies, employing combination, synergistic treatments.Based on trial data to date, no recommendation for intervention with a chemopreventive agent can be made. It is clear, however, that smoking cessation is an effective intervention for preventing oral premalignant lesions and second primary tumors. Promising trials are being conducted and designed currently. The future of this area of study will involve rational choice of multidrug regimens based on current understanding of the biology of squamous cell carcinoma of the head and neck.

Epigenetic treatment and immune checkpoint blockade are novel therapeutic approaches being investigated in NSCLC. Our previous studies showed that DNA methyltransferase inhibitor azacitidine (AZA) treatment in NSCLC cell lines led to up-regulation of genes related to the response to interferon and adaptive immune attack. We hypothesize that Interferon-γ(IFN-γ), an immune effecter made primarily by activated T cells and NK cells, has a direct tumor suppression effect in NSCLC cells, which can be enhanced by the DNA hypomethylation agent. To test this hypothesis, NSCLC cells (H838 and/or H1299) were first treated with or without 500nM AZA for 3 days, and then received IFN-γ. The samples were subsequently tested for cell viability, apoptosis, cell cycle, and gene expression assays. This preclinical study is part of the collective efforts to investigate the efficacy of combined epigenetic and immune therapy in NSCLC. IFN-γ causes moderate direct growth inhibition in H838 and H1299 cells, which was enhanced, using cell viability assays as a readout, by pretreatment with AZA. The AZA sensitizing effect lasted at least 3 weeks and was not detectable 8 weeks after stopping treatment. This timing is consistent with initial reprogramming of the cells and eventual waning of this effect. Flow cytometry studies showed that AZA enhances lung cell apoptosis induced by IFN-γ. However, no significant change in cell cycle was observed. To examine the mechanisms behind these effects on cell growth, we further determined how AZA altered IFN-γ induced gene expression in H1299 cells, first using Agilent expression arrays, and then validating specific changes in key IFN and cell death pathway genes with RT-PCR based methods. IFN-γ significantly up-regulated a group of known IFN targeted genes, including IFI27, IFITM1, ISG20, ICAM1, CCL5, CXCL10, MX1 and others, and this upregulation was further enhanced by AZA pretreatment. AZA also enhanced IFN-γ induced expression of CASP1, CASP4, TNFSF 10, and BCL2A. These AZA enhanced IFN-γ induced genes are important in tumor immune response and evasion, cell death, cytokine response and other critical cellular process. Our studies demonstrated that AZA enhanced lung cancer cell response to the immune effecter IFN-γ. This supports the clinical exploration of epigenetic “priming” and its combination with subsequent immune therapy in NSCLC treatment. It may also provide mechanistically derived biomarkers that can be used to monitor and predict epigenetic and immune response in NSCLC. Citation Format: Kai He, Xin Zhang, Ludmila Danilova, Xiaoyu Pan, Julie Brahmer, Drew Pardoll, Malcolm Brock, Stephen Baylin, James Herman, John Wrangle. Azacitidine pretreatment sensitizes NSCLC cells to interferon-γ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5033. doi:10.1158/1538-7445.AM2015-5033

76 Background: Preoperative chemoradiotherapy using a platinum plus either a taxane or 5FU results in a pathological complete response rate (pCR) of approximately 25% to 30% in patients with operable esophageal cancer. A predictive biomarker to personalize chemotherapy in an effort to increase pCR would be a significant advance. Checkpoint with forkhead and ring finger domains (CHFR) is an E3 ligase which plays a role in response to mitotic stress. In our retrospective data, hypermethylation and silencing of CHFR, correlates with improved response and prolonged survival following therapy with taxanes. The primary endpoint of this study is to determine if there is a higher pCR rate when CHFR methylation status (MS) is used to decide if patients receive a taxane or 5FU. Methods: All patients presenting to Johns Hopkins with operable T2-T4/N0-N3 esophageal cancer are eligibile. A methylation-specific PCR which demonstrates CHFR MS has been developed by our lab. Methylated (M) patients receive weekly cisplatin (30mg/m2)/paclitaxel (50mg/m2) and unmethylated (U) patients receive previously described regimens of folfox or cisplatin/5FU. All patients receive concurrent radiation. Results: From July 2011 to September 2013, 35 patients have been assessed, 18 U (51%), 14 M (40%) and 3 unknown (9%). Twenty-four patients (22 males and 2 females) have been treated on study (age 48 – 74 years old, median 63.8) with 14 tumors U (58%) and 10 tumors M (42%) respectively. To date, 2/13 U patients (15%) have had a pCR and 3/8 M patients (38%) have had a pCR (p=0.32). Toxicities of each regimen are consistent with previous reports. Conclusions: Trimodality therapy is standard of care for operable esophageal cancer in the United States. Different chemotherapies have been combined with radiation prior to surgery to improve pCR rates but no biomarkers to personalize chemotherapy exist at present. Epigenetic biomarkers such as CHFR MS potentially may be used to decide which patients are more sensitive to taxane based regimens. Accrual to this study is ongoing. Clinical trial information: NCT01372202.

Epigenetic alterations driving carcinogenesis and cancer progression can be specifically targeted by the demethylating agent azacitidine (Aza) and the histone deacetylase inhibitor entinostat. While this treatment combination has been effective in a limited number of patients (pts) with treatment-refractory non-small cell lung cancer (NSCLC), we observed clinical benefit in 5 of 5 patients who received immunotherapy with PD-1/PD-L1 pathway blockade immediately following epigenetic therapy. Three of 5 pts developed partial tumor regressions (RECIST criteria, duration 10+ to 20+ mo.) and 2 pts had stable disease ≥6 mo. This compares to the objective response + SD rates of NSCLC to monotherapy with anti-PD-1 (18% + 5%) or anti-PD-L1 (10% + 12%). To understand how epigenetic therapy may synergize with blockade of the immunosuppressive PD-1 pathway, we used genome wide methylation and expression profiling on 8 NSCLC cell lines treated with low dose Aza. We discovered complex immunomodulatory effects of Aza with up-regulation of diverse immune related pathways including Jun/Jnk, NFKB, viral defense, type I interferon signaling, the inflammasome, antigen processing and presentation and immune evasion including up-regulation of PD-L1 expression. Multiple cancer-testes antigens were also up-regulated, thereby conferring de novo antigenicity. Supporting the idea that Aza acts specifically through inhibition and degradation of DNA methyltransferase proteins, colon cancer cells genetically haplo-insufficient for DNMT1 and devoid of DNMT3b mirror the immunomodulatory effects of Aza. Upstream events potentially controlling these pathways were defined, and prominent among them was up-regulation of the transcription factor, interferon regulatory factor 7 (IRF7), a DNA hypermethylated gene. These data were used to query hundreds of primary NSCLC samples from the Cancer Genome Atlas project (TCGA). A low basal expression signature of interferon pathway related genes was significantly associated with low IRF7 expression and promoter methylation in squamous tumors. Another hypermethylated transcription factor, PITX1, which inhibits a subset of type I interferon signaling genes, tracked with non-squamous cancers. Together, these findings support a model in which epigenetic modulation activates innate and adaptive immune responses within the tumor microenvironment together with induction of counter-regulatory immune checkpoint ligands which can be therapeutically blocked with antibodies. Based on these findings, a clinical trial testing the efficacy of DNMT and HDAC inhibition combined with PD-1 pathway blockade is under development. This work will form the basis for an immune-classification of NSCLC, as well as biomarker discovery for a novel therapeutic paradigm combining epigenetic and immunotherapy with potentially synergistic activity against the world's most deadly malignancy. Supported by Stand Up to Cancer. Citation Format: John Wrangle, Wei Wang, Alexander Koch, Hariharan Easwaran, Helai Mohammad, Princy Parsana, Frank Vendetti, Kristen Rodgers, Xiaoyu Pan, Kirsten Harbom, Cynthia Zahnow, Janis Taube, Julie Brahmer, Peter Jones, Suzanne Topalian, Charles Rudin, Malcolm Brock, Drew Pardoll, Stephen Baylin. Epigenetic therapy and sensitization of lung cancer to immunotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4619. doi:10.1158/1538-7445.AM2013-4619

e18147 Background: A completed clinical trial has demonstrated activity of a combination of epigenetic therapies: entinostat, a histone deacetylase inhibitor, and azacytidine (AZA), a hypomethylating agent. A plasma-based biomarker assaying methylation status of key gene promoters from circulating DNA was developed. In order to test the relationship of activating mutations to sensitivity to epigenetic therapy, we treated non-small cell lung cancer (NSCLC) lines with AZA and assessed tumor growth in xenografts. Tumor from patients on trial was analyzed for mutational status. Methods: Nine NSCLC lines were treated in vitro for 72 hours with 500 nM AZA and injected to form flank tumors in NOG mice. Four lines contain no activating mutations. Of the five mutant lines, three were KRAS and two were EGFR mutations. Lines with no activating mutations are termed “mutation-negative” for known oncogenic drivers. Growth of mock versus treated tumors was compared over time to determine treatment phenotypes. Lines were grouped into four categories: augmented growth compared to mock, no treatment effect, mild inhibition of tumor growth, and marked inhibition. Seventeen tumors from the clinical trial were analyzed by SNaPshot multiplex PCR. Results: Among mutation negative lines three showed marked tumor growth inhibition and one showed mild inhibition. Among cell lines with activating mutations, one showed mild inhibition, three showed no effect, and one showed augmented tumor growth. Among 17 tumor samples from patients on study, four driver mutations were identified: 3 KRAS and one PTEN mutation. All four patients experienced progressive disease on study. Among thirteen patients with mutation negative tumors, three had stable disease and one had a complete response. Conclusions: The association between driver mutation negative status and treatment outcome in NSCLC cell lines in patients receiving AZA and entinostat merits further exploration. Future trials of epigenetic therapy in NSCLC may benefit from a biomarker enrichment strategy and the data presented here represents a potential way forward.

INTRODUCTION: Epigenetic therapy with a demethylating agent and an histone deacetylase inhibitor (HDACi) is now being pursued in hematological as well as solid tumor malignancies. Clinical responses have been varied with complete responses in some patients and no efficacy of the drugs in others. We investigated the effect of this therapy on two targets, DNA methyltransferase1 (DNMT1) and H3K4 modified histones, respectively. METHODS: Two human lung adenocarcinoma cell lines, A549 (Kras mutant) and H838 (Kras wild type) were pre-exposed for 72 hours with daily treatment of 500nM azacitidine alone or 72 hours of daily 500nM azacitidine followed subsequently with 10 nM, 100nM, 1000nM entinostat at 96 hours. DNMT1 and H3K4 protein levels were assayed by Western blot analysis at various time points after treatment. Cells were also re-plated after treatment to assess differential growth characteristics. RESULTS: In vitro growth of H838 cells is sensitive to azacitidine alone and in combination with entinostat whereas growth of the A549 cell line is only sensitive to azacitidine and entinostat in combination. Treatment with azacitidine, severely depleted DNMT1 protein expression in H838 cells both when given alone and in combination with most dose levels of entinostat. This decrease was transient and within 4 days, the protein was fully replenished. Azacitidine alone did not cause an appreciable decrease in DNMT1 protein expression of A549 cells but, when combined with increasing levels of entinostat, DNMT1 levels were significantly depleted and did not replenish for 7 days. Re-plating after treatment showed growth inhibition versus mock of all regimens containing azacitidine in the H838 cell line only whereas growth inhibition in the A549 cells was observed only in the combinatory therapy arms. Furthermore, in the A549 cell line, H3K4 protein levels (a marker of active gene expression) peaked at 7 days after treatment versus a peak in the first 24 hours in the H838 cell line. CONCLUSIONS: Differing sensitivities to epigenetic therapy in lung adenocarcinoma cell lines may reflect underlying differences in molecular phenotypes. Large decreases in DNMT1 expression are associated with sensitivity to azacitidine. In a cell line insensitive to azacidine alone, the addition of entinostat may accentuate DNMT1 protein depletion and delay its reexpression. This effect on DNMT1 may render this latter cancer cell line more sensitive to epigenetic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2618. doi:10.1158/1538-7445.AM2011-2618

Purpose: Within the HIV population, the incidence of lung cancer is estimated to be 2-4 times that of the general population. Of the non AIDS associated malignancies, lung cancer is the leading cause of death among HIV patients who develop malignancy because of its advanced stage of presentation. The National Lung Cancer Screening Trial recently reported a 20% reduction in mortality for patients undergoing low dose computed tomography (CT) screening, but individuals with HIV diagnosis were excluded. To report the results of an observational pilot study of lung cancer CT screening in a cohort of HIV heavy smokers. Materials and Methods: A prospective cohort study was performed with 185 asymptomatic individuals with confirmed HIV diagnosis who had smoked 20 pack-years or more. All participants had baseline (prevalence) chest CT scanning and 107 patients (58.2%) also received at least one subsequent annual (incidence) examination of the chest and upper abdomen. All patients underwent hand held spirometry testing, cytologic analysis of sputa, and detailed health status questioning. Results: Of the 185 individuals, there were 31.4% females, 68.6% males, 90.3% African-Americans, 8.7% Caucasian, and 1.1% Hispanic. The median age at enrolment was 48 years, and the median number of smoking pack-years was 34. Non-calcified nodules were detected in 4 participants (2.1%) by low-dose prevalent CT compared to no nodules on incident CT scans. Only one patient with malignancy was detected (0.54%) on prevalent scanning and none on incident scanning. Stage of malignancy was advanced. No biopsies were undertaken for benign disease. CT evidence of COPD and coronary artery disease were present in 24% and 27% of patients, respectively. Additional CT findings of clinical importance were noted in the chest in 132 (71.7%) patients and extrathoracically in 30 (16.3%) of patients, respectively. Conclusion: The prevalence results of this pilot study are in line with other observational studies of greater sample size for HIV indeterminate patients. Although the sample size is small, it suggests no elevated incidence of lung cancer in HIV heavy smokers. Although the rate of incident scanning is low, CT screening of HIV patients is feasible. This justifies accruing a larger sample of HIV patients perhaps from an already well defined, large, prospective cohort of HIV heavy smokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3679. doi:10.1158/1538-7445.AM2011-3679

Background: Epigenetic gene silencing is a key contributor to carcinogenesis. Pre-clinical studies suggest that combined inhibition of DNA methyltransferase and of histone deacetylase activity can synergistically re-express aberrantly silenced genes. Clinical studies combining 5-azacitidine (5AC) and entinostat have shown activity in leukemia. We hypothesized that a similar effect might be seen in patients with solid tumors. As a first test of this hypothesis, we conducted a phase II study in patients (pts) with advanced, recurrent non-small cell lung cancer (NSCLC). This was just completed under the aegis of a Stand up to Cancer (SU2C) project. Methods: Subjects included adults with recurrent metastatic NSCLC and progressive disease after ≥1 prior chemotherapy regimen. Pts were given 5AC 40 mg/m2 SQ days 1–6 and 8–10, and entinostat 7 mg days 3 and 10, on a 28 day cycle. A standard Simon two stage design was used. Results: 42 pts were enrolled on study, of whom, 32 completed 2 cycles and were evaluable for response. Mean # of previous therapies was 3. Treatment was well tolerated, with local injection site reactions being the most common toxicity. A complete response was observed in a pt with 3 prior therapies that also had the highest 5AC exposure by pharmacokinetic analysis. She remained on therapy for 14 months (m) and discontinued due to what proved to be a genetically distinct second primary lung cancer. This was resected, and she remained disease free off treatment for 12 m. A second pt with 3 prior therapies and metastases to the liver had a partial response for 8 m, including complete resolution of his liver disease. He also developed an independent primary, treated with chemoradiotherapy, and has been progression-free for over 18 m off epigenetic therapy. 9 pts had disease stabilization (SD) over multiple cycles, including 2 with SD for 14 m and 18 m, respectively, with marked symptomatic improvement. Median time to progression was 8 weeks and median overall survival was 8 m, comparing favorably with currently FDA approved drugs for this clinical context. Biomarker assessments included serial analysis of circulating tumor DNA methylation in plasma (loci: APC, RASSF1A, HCAD). 17 pts had ≥ 2 methylated loci detectable. 8 pts (47%) showed decreasing signal at all detectable loci of which 6 had SD or better, including both pts with RECIST criteria response. Conclusions: The combination of 5AC and entinostat is safe and well tolerated in advanced NSCLC pts. Durable patient benefit has been observed in this extensively pretreated population, including major objective responses. Correlative analyses are ongoing to identify characteristics of pts benefitting from this novel therapy. Based on these data, related studies are being initiated in colon, breast, and early stage NSCLC pts under the aegis of the SU2C. Supported by NCI, FAMRI, and AACR/SU2C. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-411. doi:10.1158/1538-7445.AM2011-LB-411