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Background: RAF dimer inhibition can suppress RAF-dependent MEK reactivation leading to sustained MAPK pathway inhibition. RAF dimer inhibitor lifirafenib (L) synergized with MEK inhibitor mirdametinib (M) in RAS-mutated cancer models. In this ongoing Phase 1b study of L+M in patients (pts) with advanced/refractory solid tumors harboring MAPK pathway aberrations, we investigate preliminary safety, PK, and efficacy. Methods: Pts were enrolled by a 3+3 design and treated with L (15-20 mg QD) + M (2-4 mg QD or BID) across 9 dose levels (DLs). Primary objectives were to evaluate safety/tolerability, estimate MTD, and identify recommended Phase 2 dose (RP2D). Tumor responses were investigator assessed using RECIST v1.1. AEs were graded per NCI CTCAE v5.0. Results: Table 1 presents demographic, efficacy, and safety results as of 01 Sep 2022. Objective responses (1 CR, 14 PRs) were achieved in 15/54 (27.8%) efficacy-evaluable pts, including 10/17 low-grade serous ovarian cancer (LGSOC) (58.8%; median exposure ~23 mo), 2 NSCLC (1 NRAS Q61K, 1 BRAF-V600E), 2 endometrial cancer (1 BRAF ZC3HAv1 fusion, 1 KRAS G12A), and 1 LG serous adenocarcinoma of Mullerian origin (KRAS G12V). For L and M, plasma maximum drug concentration (Cmax) and exposure (AUC) were comparable to that of each compound at same DL in monotherapy studies, suggesting low likelihood of drug-drug interaction. L+M was generally well tolerated, with limited DLTs and discontinuations. There were 2 deaths due to TEAEs considered unrelated to L+M. The MTD/RP2D were not yet determined. Conclusions: L+M demonstrated a favorable safety profile and showed antitumor activity in pts with various KRAS, NRAS, and BRAF mutations across several solid tumor types, including LGSOC, NSCLC, and endometrial cancer. The combination warrants further clinical investigation. (Acknowledgements: We thank patients/their families, investigators, and site staff for participating in this study, which is sponsored by BeiGene. Support for abstract preparation was funded by BeiGene and provided by Traci Ginnona, inSeption Group, Lansdale, PA, USA) Table 1. Demographics (N=56) Age (y), median (range) 59.5 (29-78) ECOG PS 0/1, n (%) 56 (100.0) Prior lines of therapy, median (range) 1 (1-6) Efficacy Set (N=54), n (%) LGSOC (n=17) Other than LGSOC (n=37) All malignancies (n=54) ORR (CR+PR) 10 (58.8) 5 (13.5) 15 (27.8) CR 1 (5.9) 0 1 (1.9) PR 9 (52.9) 5 (13.5) 14 (25.9) SD 6 (35.3) 18 (48.6) 24 (44.4) DCR (CR+PR+SD) 16 (94.1) 23 (62.2) 39 (72.2) Safety Set (N=56), n (%) TEAEa 55 (98.2) SAE 23 (41.1) Grade 3 TEAE 24 (42.9) TEAE leading to treatment discontinuation 3 (5.4) DLT 6 (10.7) CR, complete response; DCR, disease control rate; DLT, dose-limiting toxicity; ECOG PS, Eastern Cooperative Oncology Group performance status; LGSOC, low-grade serous ovarian cancer; ORR, objective response rate; PR, partial response; SAE, serious adverse event; SD, stable disease; TEAE, treatment-emergent adverse event. a Commonly reported (>40%): fatigue (55.4%), dermatitis acneiform (46.4%), and diarrhea (44.6%). Citation Format: Benjamin Solomon, Bo Gao, Vivek Subbiah, Michael Millward, Lee Rosen, Jayesh Desai, Eric I. Sbar, Neal Collins, Thuy Hoang, Xi Song, Wenlin Shao, Jaspreet Jaggi, Badreddin Edris, Paraneedharan Ramachandran, Lusong Luo, Michael Friedlander. Safety, pharmacokinetics, and antitumor activity findings from a phase 1b, open-label, dose-escalation and expansion study investigating RAF dimer inhibitor lifirafenib in combination with MEK inhibitor mirdametinib in patients with advanced or refractory solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT033.

Therapeutic vaccines hold promise to broaden the potency of immune checkpoint blockade (ICB) therapy in tumors lacking immune reactivity. A heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based individualized neoantigen vaccine regimen in combination with nivolumab 480 mg IV and ipilimumab 30mg SC (NCT03639714) has previously demonstrated safety, durable immunogenicity, and clinical benefit in patients with previously treated metastatic disease. Genomic correlates of response were studied over time in 29 patients (13 MSS-CRC, 13 GEA, 3 NSCLC) to understand novel mechanisms of action. Exome and transcriptome sequencing from archival biopsies was used for neoantigen selection. Monthly circulating tumor DNA (ctDNA) samples were collected for monitoring using a comprehensive tumor-naïve and tumor-informed hybrid-capture based ctDNA assay. Paired pre- and post-vaccine tumor transcriptomes were analyzed for 10 patients with 6 having accompanying DNA T cell receptor Β CDR3 repertoire sequencing (TCRseq) in biopsies and longitudinal PBMCs. Prior to vaccination patient tumors were not enriched for immune infiltration or tumor mutation burden (TMB), median 4.3 mut/Mb (range: 2-17 mut/Mb). Minimal neoantigen and mutation drift was observed with a median of 92.5% of neoantigens (range: 45-100%) and a median of 84% (range: 24-99%) of individual mutations detected in biopsies and ctDNA. Notably, paired pre- and post-vaccine biopsy gene expression analyses show upregulation in gene signatures associated with immune infiltration aligning with evidence of T cell expansion measured by significantly expanding CDR3 clonotypes (p &lt0.01). Longitudinal TCRseq in PBMCs demonstrate vaccine induced TCR repertoire dynamics and expanding and contracting clones observed in tumor biopsies could be monitored throughout treatment. In 4 patients the most drastic TCR repertoire changes were observed at time points measured after a 2nd dose of ChAd68. Lastly, we observe evidence of acquired immune evasion through ctDNA monitoring in two patients each following a year of study treatment. One GEA patient acquired HLA-LOH after remaining stable on treatment and one MSS-CRC with a molecular response (MR) for &gt7 months acquired novel biallelic loss-of-function mutations in TAP1 following 1 year of study treatment. We demonstrate that our neoantigen-directed immunotherapy regimen drives durable immune pressure on the tumor in patients with advanced disease where CPI alone has provided minimal benefit. Further, the evidence of acquired resistance supports the induction of immune pressure on tumors following individualized neoantigen vaccination. Comprehensive ctDNA longitudinal monitoring enables real-time assessment of clinical response and acquired resistance. Citation Format: Desiree Schenk, Rita Zhou, Olivia Petrillo, Alexis Mantilla, Italo Faria do Valle, Steven Maron, Brian S. Henick, Chih-Yi Liao, Daniel V.T. Catenacci, Sameek Roychowdhury, Benjamin Solomon, Alexander I. Spira, Ankur Dhanik, Andrew R. Fergusson, Karin Jooss, Matthew Davis. Disease monitoring with comprehensive genomics provides evidence of mechanism of action and immune evasion in patients receiving an individualized neoantigen cancer vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1126.

Background: KRAS G12C oncogenic mutations occur in ~13% of non-small cell lung cancers (NSCLCs) and up to 4% of other solid tumors. JDQ443 is a selective, covalent, orally bioavailable, investigational KRASG12C inhibitor that irreversibly traps KRASG12C in the inactive, GDP-bound state. JDQ443 is structurally unique and forms novel interactions with KRAS in the switch II pocket. Methods: KontRASt-01 (NCT04699188) is a Phase Ib/II, open-label, multicenter, dose-escalation and dose-expansion trial of JDQ443 as monotherapy or in combination with TNO155 (SHP2 inhibitor) and/or tislelizumab (anti-PD-1 monoclonal antibody). Primary objectives of dose escalation are to assess safety and tolerability, and identify the maximum tolerated doses (MTDs) and/or recommended doses (RDs) and regimens for future studies. The primary objective of dose expansion is to assess efficacy. Key inclusion criteria: advanced, KRAS G12C-mutated solid tumors; previous standard-of-care treatment; age ≥18 yrs; ECOG PS 0-1. Key exclusion criteria for the JDQ443 monotherapy arm: active brain metastases, prior KRASG12C inhibitor treatment. Here, we present preliminary results for JDQ443 monotherapy dose escalation. Results: As of Nov 3, 2021, 39 pts were treated with JDQ443 PO continuously across 4 dose levels: 200 mg once daily (QD) (n=10), 400 mg QD (n=11), 200 mg twice daily (BID) (n=11), and 300 mg BID (n=7). Median age was 60 yrs (range 26-76), median prior lines of therapy was 3 (range 1-7), and indications included NSCLC (n=20) and colorectal cancer (CRC) (n=16). Median duration of exposure was 9.1 wks (range 0.9-21), with ongoing treatment in most pts (61.5%) at the time of cut-off. Treatment-related adverse events (TRAEs) occurred in 25 (64.1%) pts. Most TRAEs were Grade (Gr) 1-2. Four Gr 3 TRAEs occurred in 4 (10.3%) separate pts; there were no Gr 4-5 TRAEs. The most common TRAEs (occurring in ≥10% of pts) were fatigue (25.6%), nausea (15.4%), edema (12.8%), pruritus (10.3%), and vomiting (10.3%). There was one DLT (Gr 3 fatigue) and one treatment-related serious AE (Gr 3 photosensitivity reaction), each in separate pts treated at 300 mg BID. TRAEs led to dose reduction in 1 pt and discontinuation in 1 pt. A MTD was not reached. The RD was declared as 200 mg BID. At the RD, PK and PD modeling for JDQ443 predicted average KRASG12C target occupancy of >90% in >82% of pts. Using an efficacy cut-off date of Dec 13, 2021, for the 20 pts with NSCLC among the same 39 pts, the ORR (confirmed complete response or partial response) by RECIST 1.1 was 30.0% (6/20) across dose levels and 43.0% (3/7) at the RD. Additional data will be available at the time of presentation. Conclusions: JDQ443 demonstrates an acceptable safety and tolerability profile, with early signs of clinical activity in pts with NSCLC. Enrollment is ongoing to NSCLC and CRC dose-expansion groups for JDQ443 monotherapy at the RD, and to JDQ443 + TNO155 dose escalation. Citation Format: Daniel S. Tan, Toshio Shimizu, Benjamin Solomon, Rebecca S. Heist, Martin Schuler, Maria J. De Miguel Luken, Anas Gazzah, Martin Wermke, Christophe Dooms, Herbert H. Loong, Neeltje Steeghs, Enriqueta Felip, Conor E. Steuer, Eric van Cutsem, Ross A. Soo, Ashley C. Jaeger, Jaeyeon Kim, Kun Xu, Xueying Chen, Xiaoming Cui, Heather Burks, Anna Farago, Philippe A. Cassier. KontRASt-01: A phase Ib/II, dose-escalation study of JDQ443 in patients (pts) with advanced, KRAS G12C-mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT033.

Neoantigen directed immunotherapy holds promise to increase the likelihood of patients with solid tumor devoid of immune infiltration benefiting from immune checkpoint immunotherapy (CPI). A heterologous prime-boost vaccination approach consisting of Chimpanzee Adenovirus (ChAd) prime and multiple self-amplifying mRNA (SAM) boosts, delivering 20 neoantigens, has been evaluated in a Phase 1/2 clinical trial in late-stage solid tumor patients in combination with nivolumab and ipilimumab (NCT03639714). Neoantigen dynamics, tumor burden and genomic correlates of response were studied over time in 20 patients (8 GEA, 2 NSCLC, 10 MSS-CRC). Exome sequences from archival (sample used for neoantigen selection), baseline (start of immunization) and on-treatment biopsies were analyzed for 20, 16 and 10 patients respectively. Paired pre- and post-vaccine tumor transcriptomes were analyzed for 6 patients. Personalized capture baits were designed for all non-synonymous mutations detected in archival biopsies (mean 146; range: 67-402) for ctDNA monitoring. Longitudinal ctDNA samples were collected monthly on treatment (mean 7; range: 1-18). ctDNA duplex UMI libraries were captured and sequenced to a target mean raw depth >80,000x and reduced to 3x per strand consensus duplex reads. The majority of vaccine neoantigens were detected in ctDNA (87%; range 45%-100%) and mean neoantigen variant allele frequency (VAF) strongly correlated with all monitored mutations VAF (R2 = 0.90, p < 0.0001) through treatment. The percentage of vaccine neoantigens detected was higher than that for all monitored mutations in the same samples with a median of 80% (21%-98%) in ctDNA and 70% (44%-100%) in biopsies. Five of 9 MSS-CRC patients with measurable baseline ctDNA achieved molecular responses (MR, >50% reduction in ctDNA from baseline) that correlated with OS and PFS, and in some patients, was accompanied by radiologic tumor shrinkage. One MSS-CRC patient with MR for >7 months acquired novel biallelic loss-of-function mutations in TAP1 following 1 year of study treatment. Differential gene expression analysis from paired pre- and post-vaccine biopsies (including 2 MSS-CRC pairs with MR) demonstrated significant upregulation in gene signatures associated with immune-inflamed tumor microenvironments including interferon alpha and gamma responses. We demonstrate that tumor-informed neoantigen selection and vaccine manufacturing while patients receive chemotherapy is feasible, since the majority of neoantigens are retained in the tumor post-chemotherapy. Further, neoantigen-directed immunotherapy appears to drive clinical benefit in patients with advanced MSS-CRC tumors, where CPI alone has provided minimal benefit. Comprehensive ctDNA longitudinal monitoring enables real time assessment of clinical response and acquired resistance. Citation Format: Desiree Schenck, Rita Zhou, Alexis Mantilla, Oliver Spiro, Taylor Patch, Adrienne Johnson, Daniel Navarro Gomez, Brian S. Henick, Chih-Yi Liao, Sameek Roychowdhury, Steve Maron, Benjamin Solomon, Alexander I. Spira, Daniel V. Catenacci, Andrew R. Fergusson, Raphael F. Rousseau, Karin Jooss, Matthew J. Davis. Comprehensive ctDNA monitoring provides early signal of clinical benefit with a novel personalized neoantigen directed immunotherapy for late-stage cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1238.

Background: Lorlatinib improved progression-free survival (PFS) and demonstrated intracranial (IC) activity in patients (pts) with untreated advanced ALK+ NSCLC in the interim analysis of the randomized, Phase 3, CROWN study of lorlatinib vs crizotinib. We report updated 36-month follow-up data. Methods: 296 pts with previously untreated advanced ALK+ NSCLC were randomized 1:1 to oral lorlatinib (100 mg QD; n=149) or crizotinib (250 mg BID; n=147), stratified by presence of CNS metastases (mets) and ethnicity. Primary endpoint: PFS by blinded independent central review (BICR). Secondary endpoints included overall survival, PFS by investigator, and objective response (OR), IC-OR, IC time to progression (IC-TTP), duration of response (DR), IC-DR (all by BICR), and safety. Results: At data cutoff (Sep 20, 2021), median duration of follow-up for PFS was 36.7 months for lorlatinib and 29.3 months for crizotinib. Median PFS by BICR was NR (95% CI, NR-NR) for lorlatinib and 9.3 months (95% CI, 7.6-11.1) for crizotinib (HR, 0.27; 95% CI, 0.18-0.39). PFS by investigator results were similar (Table). For pts with brain mets at baseline (n=37 lorlatinib/n=39 crizotinib), the HR for IC-TTP for lorlatinib vs crizotinib was 0.10 (95% CI, 0.04-0.27), and for pts without brain mets (n=112/n=108) was 0.02 (95% CI, 0.002-0.14). OR, IC-OR, DR, and IC-DR were all improved with lorlatinib vs crizotinib (Table). All-cause grade 3-4 adverse events (AEs) and AEs leading to treatment discontinuation were reported in 76% and 7% of pts with lorlatinib and 57% and 10% of pts with crizotinib, respectively. No new safety signals emerged. Conclusions: These updated long-term data from CROWN confirm the efficacy of lorlatinib over crizotinib in pts with treatment-naïve ALK+ NSCLC, with no new safety signals detected, and support the use of lorlatinib in pts with untreated ALK+ NSCLC with and without brain mets. Summary of other efficacy resultsa Clinical trial information: NCT03052608 Funding: Pfizer Inc. ITT population Lorlatinib(n=149) Crizotinib(n=147) % alive without progression at:12 mo (95% CI) 78 (70–84) 38 (29–47) 24 68 (60–75) 22 (14–30) 36 64 (55–71) 19 (12–27) Median PFS by investigator, mo (95% CI) NR (NR–NR) 9.1 (7.4–10.9) HR (95% CI) 0.19 (0.13–0.27) Confirmed OR, n (%) [95% CI] 115 (77) [70–84] 86 (59) [50–67] Median DR,b mo (95% CI) NR (NR–NR) 9.6 (9.0–12.9) Patients with measurable or nonmeasurable brain metastases at baseline (n=37) (n=39) Median PFS, mo (95% CI) NR (18.2–NR) 7.2 (3.7–9.2) HR (95% CI) 0.21 (0.10–0.44) Confirmed IC-OR, n (%) [95% CI] 24 (65) [48–80] 7 (18) [8–34] Complete response, n (%) 22 (60) 5 (13) Median IC-DR,b mo (95% CI) NR (NR–NR) 9.4 (6.0–11.1) Patients without brain metastases at baseline (n=112) (n=108) Median PFS, mo (95% CI) NR (NR–NR) 11.0 (9.0–14.6) HR (95% CI) 0.29 (0.19–0.44) aBy blinded independent central review unless stated otherwise. bIn patients with a confirmed complete or partial response. CI, confidence interval; DR, duration of response; HR, hazard ratio; ITT, intention- to-treat; NR, not reached; IC, intracranial; OR, objective response; PFS, progression-free survival. Citation Format: Benjamin Solomon, Todd Bauer, Tony Mok, Geoffrey Liu, Julien Mazieres, Filippo de Marinis, Yasushi Goto, Dong-Wan Kim, Yi-Long Wu, Mikhail Dvorkin, Jacek Jassem, Froylán López-López, Ross Soo, Anna Polli, Elisa Dall'O, Laura Iadeluca, Francesca Toffalorio, Enriqueta Felip. Updated efficacy and safety from the phase 3 CROWN study of first-line lorlatinib vs crizotinib in advanced anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT223.

BACKGROUND: Lorlatinib, a 3rd generation ALK tyrosine kinase inhibitor, has shown overall and intracranial activity in ALK+ advanced NSCLC. In the randomized, multicenter, phase 3 study in pts with previously untreated ALK+ advanced NSCLC (CROWN; NCT03052608), lorlatinib resulted in a statistically significant and clinically meaningful improvement in progression-free survival (PFS) vs crizotinib.1 To identify molecular correlates of response, we performed molecular profiling of circulating free DNA (cfDNA) and tumor tissue. METHODS: Plasma and tumor tissue samples were available from 130 and 118 pts in the lorlatinib arm, and from 125 and 104 pts in the crizotinib arm, respectively. Plasma DNA was analyzed for ALK fusions and mutations by next-generation sequencing (NGS; Guardant360, Guardant Health, Inc., Redwood City, CA, USA); tumor tissue DNA was analyzed with an ALK-mutation focused NGS panel (MolecularMD, Portland, OR, USA). Objective response rate (ORR), duration of response (DOR), and PFS were evaluated by blinded independent central review according to EML4-ALK variant type and ALK resistance mutation status. RESULTS: At screening, 19 ALK missense mutations and 1 deletion were detected in plasma of 11 pts (5 and 6 in the lorlatinib and crizotinib arms, respectively). Most pts harbored 1 mutation, but 3 pts harbored ≥3 mutations. ALK fusions were detected in plasma of 48% of pts. EML4-ALK variants 1, 2, and 3 were detected in 14.6%, 5.4%, and 13.8% and in 20.0%, 1.6%, and 16.8% of pts, in the lorlatinib and crizotinib arms, respectively. Variants 4, 5, 7, and 8 were detected in 11.5% and 7.2% of pts, and less frequent fusion partners in 1.5% and 3.2% of pts, respectively. ORRs were generally higher in the lorlatinib arm vs the crizotinib arm, regardless of variant subtypes and/or mutational status, with no striking differences between EML4-ALK variants 1, 2, and 3 (range, 72% to 86% for lorlatinib and 50% to 76% for crizotinib). Median DOR and PFS were not reached for variants 1 and 3 in the lorlatinib arm, and ranged from 5.7 to 6.5 months, and from 7.4 to 7.6 months in the crizotinib arm, respectively. CONCLUSION: Pts with untreated ALK+ advanced NSCLC had higher ORRs and potentially longer DOR and PFS across predefined biomarker subgroups when treated with lorlatinib compared with crizotinib in a phase 3 CROWN study. Based on pretreatment cfDNA analysis, lorlatinib led to similar clinical benefit regardless of the type of EML4-ALK variant or presence of ALK kinase mutations. REFERENCE: 1. Shaw AT, et al. N Engl J Med. 2020;383:2018-2029. Citation Format: Alessandra Bearz, Jean-François Martini, Jacek Jassem, Sang-We Kim, Gee-Chen Chang, Alice Shaw, Deborah Shepard, Elisa Dall'O', Anna Polli, Holger Thurm, Gerard Zalcman, Maria Rosario Garcia Campelo, Konstantin Penkov, Hidetoshi Hayashi, Benjamin J. Solomon. Efficacy of Lorlatinib in Treatment-Naïve Patients (pts) With ALK-Positive Advanced Non-Small Cell Lung Cancer (NSCLC) in Relation to EML4-ALK Variant Type and ALK Mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB043.

BACKGROUND: Lorlatinib is a selective, potent, brain-penetrant, 3rd-generation (gen) ALK/ROS1 tyrosine kinase inhibitor (TKI) preclinically active against most known ALK resistance mutations. In pts with ALK+ advanced NSCLC with disease progression following 2nd-gen ALK TKIs, lorlatinib has shown robust clinical activity and we found that tumor genotyping for ALK mutations may identify such pts more likely to respond to lorlatinib treatment. Prior analyses have suggested that EML4-ALK variant type may influence ALK TKI treatment benefit. To further identify molecular correlates of response, we performed an exploratory subgroup analysis by EML4-ALK variant type and ALK resistance mutations in pts previously treated with 2nd-gen ALK TKIs and who received the recommended Ph 2 dose of lorlatinib (100 mg once daily). METHODS: Baseline plasma samples were collected from pts with ALK+ NSCLC with ≥1 prior 2nd-gen ALK TKI enrolled in the ongoing registrational Ph 2 study (NCT01970865). Circulating free DNA (cfDNA) was analyzed using Guardant360 (Guardant Health, Inc., CA, USA) to determine EML4-ALK variant and ALK kinase domain mutations. Objective response rate (ORR) and duration of response (DOR), by independent central review, were evaluated according to EML4-ALK variant type and ALK resistance mutation status. RESULTS: ALK fusions were detectable in 64 (41.0%) of 156 pt plasma samples. EML4-ALK variants 1, 2, and 3 were detected at frequency of 17.3%, 2.6% and 15.4%, respectively. Other EML4-ALK variants (including the variant types 4, 5, 7, and 8) were also detected in 3.2% of pts, as well as some other less frequent fusion partners (e.g., KIF5B) in 2.6% of pts. Based on cfDNA, ALK resistance mutations were detected in 40 pts; of whom, 6 pts harbored EML4-ALK variant 1, 1 pt had variant 2 and 18 had variant 3. The G1202R/Del mutation was detected in 23 pts samples; of which, 15 (65.2%) also harbored EML4-ALK variant 3. ORR was 33.3% (95% CI 16.5-54.0), 75.0% (95% CI 19.4-99.4) and 45.8% (95% CI 25.6-67.2) for variants 1, 2 and 3, respectively, while median DOR was similar for pts with variant 1 or 3 (both 6.9 months). Median DOR was not reached for variant 2. Of note, no responses were observed in the pts with other types of ALK rearrangements detected. Finally, ALK fusions were not detected in the cfDNA of 92 pts (59.0%), including 12 pts who had detectable ALK mutations. Among these 92 pts, ORR was 39.1% (95% CI 29.1-49.9) and median DOR was 7.1 months (95% C 5.5-not reached). Confirmation of these results in the tumor tissue is ongoing. CONCLUSION: In this heavily pretreated group of ALK+ NSCLC pts, the presence of an ALK resistance mutation might enrich for EML4-ALK variants 1 and 3. Lorlatinib exhibited antitumor activity irrespective of EML4-ALK variant and across a variety of ALK resistance mutations. Citation Format: Todd M. Bauer, Jean-François Martini, Benjamin Besse, Chia-Chi Lin, Ross A. Soo, Gregory J. Riely, Sai-Hong Ignatius Ou, Francesca Toffalorio, Antonello Abbattista, Holger Thurm, D. Ross Camidge, Steven Kao, Rita Chiari, Shirish Gadgeel, Enriqueta Felip, Alice T. Shaw, Benjamin J. Solomon. Impact of the EML4-ALK fusion variant on the efficacy of lorlatinib in patients (pts) with ALK-positive advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT025.

Cytokine interleukin-1β (IL-1β) has multiple pro-tumorogenic effects on tumor microenvironment, thereby promoting carcinogenesis, tumor invasiveness, and immunosuppression. CAN is a selective IL-1β inhibitor that aims to target tumor-promoting inflammation to reduce immune suppression, thereby potentiating effects of immunotherapy with PD-1 inhibitors such as PEM. Ph 3 CANTOS study has shown IL-1β inhibition with CAN was associated with reduced incidence of lung cancer (LC) and LC mortality in pts with atherosclerosis, providing a rationale to investigate therapeutic role of CAN in LC. CANOPY-1 (NCT03631199) is a PBO-controlled, double-blind, randomized, ph 3 trial designed to evaluate efficacy and safety of PEM + Ctx ± CAN in tx naive pts with stage IIIB/IIIC (not eligible for definitive chemo-radiation curative tx) or stage IV squamous and nonsquamous NSCLC. The study was divided into 2 parts: part 1 is open labelled, safety run-in part where pts received CAN 200 mg s.c Q3W + PEM 200 mg i.v Q3W + platinum-based Ctx (as induction during first 4 cycles only); Cohort A (A, non-squamous), carboplatin + pemetrexed; Cohort B (B, non-squamous), cisplatin + pemetrexed; Cohort C (C, squamous or non-squamous), carboplatin + paclitaxel. Part 2 is randomized and evaluates efficacy and safety of CAN comb regimen vs PBO comb regimen. Primary objective of safety run-in part: recommended ph 3 dose regimen (RP3R) of CAN comb. Secondary objectives: ORR, DCR, DOR, safety, PK, and immunogenicity. As of 14 May 2019 (follow-up of ≥42 days from C1D1 unless pt discontinued earlier), 10 pts in A, 11 pts in B, and 9 pts in C were treated, of which 73% were male, median age was 63 yrs. In total, 24/30 (80%) pts enrolled were still receiving tx; primary reason for tx discontinuation was progressive disease (3 pts in A and 1 pt each in B and C) and 1 pt died due to study indication. 1 pt reported DLT during first 42 days of study tx (C: grade 3 hepatitis, not related to CAN). RP3R of CAN in comb with standard dose PEM + Ctx was 200 mg SC Q3W based on Bayesian logistic regression model (BLRM). Serious AEs regardless of causality were reported in 8 (27%) pts (2 pts in A and 3 pts each in B and C), none of which were considered to be related to CAN. Most common AEs (≥20%, any grade) across all cohorts (n=30) were nausea (37%), vomiting (30%), constipation and fatigue (each 23%), and neutrophil count decrease (20%). 14 pts (47%) experienced grade 3 AEs and 1 pt experienced grade 4 AE (cardiac tamponade [unrelated]). No fatal serious AEs were reported. AEs leading to discontinuation of one of the study drugs were reported in 3 (10%) pts (hepatitis, peripheral neuropathy, and polyneuropathy) but none were CAN related. AEs leading to dose reduction and dose interruption of one of study drugs were reported in 3 (10%) pts and 5 (17%) pts, respectively. Only 1 DLT was reported with CAN + PEM + Ctx. Based on BLRM and all relevant clinical data, the RP3R of CAN as 200 mg SC Q3W comb was considered safe and well tolerated. Enrollment for the randomized part is completed. Citation Format: Bruce E. Johnson, Tae Min Kim, T. Jeroen N. Hiltermann, Fabrice Barlesi, Christian Grohe, Yasushi Goto, Orvar Gunnarsson, Tobias Overbeck, Noemi Reguart, Martin Wermke, Gilberto Castro Castro, Enriqueta Felip, Alastair Greystoke, Benjamin J. Solomon, Stephanie Deudon, Anne-Laure Louveau, Vanessa Passos, Daniel SW Tan. CANOPY-1: Safety run-in results from phase (ph) 3 study of canakinumab (CAN) or placebo (PBO) in combination (comb) with pembrolizumab (PEM) plus platinum-based doublet chemotherapy (Ctx) as 1st line therapy in patients (pts) with advanced or metastatic NSCLC [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT214.

Background: In the CANTOS study, treatment (tx) with Cana (selective IL-1β inhibitor) was associated with reduced incidence and mortality of NSCLC in stable post-myocardial infarction pts with elevated high-sensitivity C-reactive protein levels. The results provided a rationale to investigate the therapeutic role of Cana in NSCLC. Methods: The phase (ph) 2, open-label CANOPY-N study (NCT03968419) is evaluating Cana or pembrolizumab (pembro) alone or in combination as neoadjuvant tx in stage IB-IIIA, tx-naive NSCLC pts eligible for primary resection (except N2 and T4 tumors) with planned surgery in 4-6 weeks (wks) from the 1st dose of study tx. Pts (~110) are randomized 2:2:1 to Cana (2 doses 200 mg SC Q3W), Cana + pembro, or pembro (2 doses 200 mg iv Q3W) for 2 three-wk cycles. Randomization (R) stratification: histology (squamous [sq] vs non-sq). Primary endpoint: major pathological response rate at time of surgery.CANOPY-A (NCT03447769), CANOPY-1 (NCT03631199), and CANOPY-2 (NCT03626545) are ph 3, multicenter, double-blind studies. In CANOPY-A (Cana in adjuvant setting), pts (~1500) with stages IIA-IIIA and IIIB (T>5 cm N2), any histology, completely resected (R0) NSCLC post cisplatin-based chemotherapy (CTx) and radiation therapy (if applicable) are randomized 1:1 to Cana (200 mg SC Q3W)/placebo (PBO; SC Q3W) for 18 cycles. R stratification: AJCC/UICC v.8 stage (IIA vs IIB vs IIIA vs IIIB with T>5 cm, N2 disease), histology (sq vs non-sq), region (western Europe and North America vs eastern Asia vs rest of the world). Primary endpoint: disease-free survival. CANOPY-1 and CANOPY-2 consist of Part 1 (open-label, safety run-in; enrollment complete) and Part 2 (randomized 1:1, PBO-controlled, efficacy & safety; ongoing). CANOPY-1 eligibility: pts with previously untreated stages IIIB/IIIC or IV NSCLC and known PD-L1 status (for Part 2), without EGFR sensitizing mutations and/or ALK rearrangements. Part 1 (3 cohorts of ~9 pts each, based on different platinum-CTx): to confirm the recommended phase 3 regimen (RP3R) for Cana. In Part 2, pts (~600) are randomized to Cana (200 mg SC Q3W)/PBO + pembro + CTx for 4 cycles, followed by maintenance tx (Cana/PBO + pembro ± pemetrexed) until progressive disease (PD). R stratification: PD-L1 status (tumor proportion score Citation Format: Luis Paz-Ares, Edward B. Garon, Tony Mok, Andrea Ardizzoni, Fabrice Barlesi, Byoung Chul Cho, Gilberto de Castro, Pedro De Marchi, Enriqueta Felip, Yasushi Goto, Alastair Greystoke, Shun Lu, Darren Wan-Teck Lim, Martin Reck, Benjamin J. Solomon, David R. Spigel, Daniel SW Tan, Michael Thomas, James Chih-Hsin Yang, Jay M. Lee, Pilar Garrido, Edward Kim, Bruce E. Johnson. CANOPY program clinical trials: Canakinumab (Cana) in patients (pts) with non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT286.

Background: NSCLC is the leading cause of cancer death. Recently, much progress has been made to identify and target specific oncogenic drivers such as EGFR mutations and ALK translocations. Unfortunately, the majority of NSCLCs do not have a single driver, but instead harbor complex systems of molecular alterations. The advent of immunooncology has opened newer avenues for treating these patients, but still, many tumors are resistant. Therefore, selection of precise therapies targeting the appropriate pathways in these tumors is an important area of research. In previously published work, the Worldwide Innovative Network (WIN) Consortium has developed the Simplified Interventional Mapping System (SIMS) algorithm to match patients to a targeted drug triplet. The SIMS algorithm is based on the hypothesis that the use of both genomic as well as transcriptomic data is important in selecting an ideal tri-therapy regimen. Since the transcriptome varies widely between different types of normal tissue, the relative level of transcription of any given gene in a tumor must be normalized against the expected level of transcription of that gene in the analogous normal tissue. The SPRING trial is the first trial assessing a rational, SIMS-based tri-therapy regimen in advanced NSCLC. The drugs utilized for SPRING–avelumab (anti-PDL1), axitinib (VEGFR inhibitor) and palbociclib (CDK4/6 inhibitor)–were chosen because the SIMS genomic/transcriptomic analysis demonstrated that ~10% of NSCLCs have a combination of these targets. Methods: Patients with locally advanced or metastatic NSCLC without EGFR or ALK alterations are being enrolled and treated with avelumab, palbociclib, and axitinib after informed consent. During phase I, any prior treatment is allowed. SPRING will assess the safety of the tri-therapy combination and determine the recommended phase II dose (RP2D) (3+3 dose escalation). Once the RP2D is found, the study will proceed directly to the phase II portion, which will permit only previously untreated patients in the metastatic setting. A biopsy of both tumor and normal tissue (by endobronchial mucosal biopsy) is obtained on all patients, both of which are analysed in a central genomics and transcriptomics platform. The SIMS algorithm is being validated retrospectively by determining the degree of genomic and transcriptomic matching and correlating with outcome. Results: The phase I study is open in USA, Israel, Spain and Luxembourg and has enrolled 12 patients to date and has progressed to cohort 3. The phase II portion is expected to open later in 2019. This research is supported by the EMD Serono/Pfizer alliance and ARC Foundation for cancer research. Citation Format: Benjamin Solomon, Enriqueta Felip, Jair Bar, Guy Berchem, Lyudmila Bazhenova, Pierre Saintigny, Nicolas Girard, Raed Sulaiman, Catherine Bresson, Fanny Wunder, J Jack Lee, Jacques Raynaud, Eitan Rubin, Brandon Young, Vladimir Lazar, Amir Onn, Brian Leyland Jones, Razelle Kurzrock. Survival Prolongation by Rationale INnovative Genomics (SPRING): An international WIN Consortium Phase I/II proof-of-concept study to explore the safety and efficacy of a tri-therapy approach using avelumab, palbociclib and axitinib in advanced/metastatic non-small cell lung cancer (NSCLC) with integrated genomic and transcriptomic correlates [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 CT223.

Hypoxia is a prevalent feature of many tumors contributing to disease progression and treatment resistance, and therefore constitutes an attractive therapeutic target. Several hypoxia-activated prodrugs (HAP) have been developed, including the phase III candidate TH-302 (evofosfamide) and the preclinical agent SN30000, which is an optimized analogue of the well-studied HAP tirapazamine. Experience with this therapeutic class highlights an urgent need to identify biomarkers of HAP sensitivity, including enzymes responsible for prodrug activation during hypoxia. Using genome-scale shRNA screens and a high-representation library enriched for oxidoreductases, we identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in three different genetic backgrounds. No other genes consistently modified SN30000 sensitivity, even within a POR-negative background. Knockdown or genetic knockout of POR reduced SN30000 reductive metabolism and clonogenic cell death and similarly reduced sensitivity to TH-302 under hypoxia. A retrospective evaluation of head and neck squamous cell carcinomas showed heterogeneous POR expression and suggested a possible relationship between human papillomavirus status and HAP sensitivity. Taken together, our study identifies POR as a potential predictive biomarker of HAP sensitivity that should be explored during the clinical development of SN30000, TH-302, and other hypoxia-directed agents. Cancer Res; 75(19); 4211–23. ©2015 AACR.

Background: EGFR mutation testing is required to identify patients who may respond to TKI therapy. However, tumor biopsies from NSCLC patients can pose challenges for molecular analyses due to inadequate sample material, the inability to biopsy patients due to poor health status or inaccessible lesions, and tumor heterogeneity. We examined the detection of EGFR mutations in circulating cell-free DNA (cfDNA) from plasma and the concordance of EGFR mutation status with contemporaneously matched tumor tissue in TIGER-X, a Phase 1/2 clinical study of rociletinib (CO-1686) in previously treated advanced NSCLC patients harboring EGFR mutations in their tumors. Rociletinib is an oral, potent, small-molecule irreversible tyrosine kinase inhibitor that selectively targets mutant forms of EGFR, including T790M, L858R and Del(19), while sparing wild-type EGFR. Methods: Pretreatment matched tumor tissue and plasma from 139 Stage IIIB/IV NSCLC patients enrolled in TIGER-X were evaluated for EGFR status. Tumor tissue was processed as FFPE and tested by allele-specific PCR. Plasma was tested as a two mL aliquot using BEAMing, a quantitative and sensitive detection technology based on digital PCR. Results: Using tissue as the reference, the positive percent agreement between tumor and BEAMing plasma test results was 86% (102/119) for activating mutations and 77% (78/101) for T790M. Four plasma samples of the 23 tumor T790M+/plasma T790M- cases were also tested by three independent plasma testing platforms with claimed analytical sensitivities of Conclusions: The BEAMing plasma test identified EGFR mutations detected in tumor with high sensitivity. In addition, plasma testing identified T790M+ patients that were determined T790M- by the tumor test, which may be in part explained by tumor heterogeneity and/or inadequate biopsy. These findings demonstrate that BEAMing can be a useful tool for the non-invasive assessment of EGFR mutations in NSCLC. Citation Format: Jonathan W. Goldman, Chris Karlovich, Elaina Mann, Lindsey Rolfe, Shannon Matheny, Darrin Despain, Philipp Angenendt, Claudia Stamm, Heather A. Wakelee, Jean-Charles Soria, Benjamin Solomon, D. R. Camidge, Rafal Dziadziuszko, Leora Horn, Shirish Gadgeel, Mitch Raponi, Andrew R. Allen, Lecia V. Sequist. Pretreatment and serial plasma assessments of EGFR mutations in NSCLC patients treated with rociletinib (CO-1686). [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 927. doi:10.1158/1538-7445.AM2015-927

Background: We examined the detection of EGFR mutations in circulating free DNA from plasma and the concordance of EGFR mutation status between matched plasma and tumor tissue in a cohort of newly diagnosed or relapsed patients with advanced NSCLC. CO-1686 is an oral, potent, small-molecule irreversible tyrosine kinase inhibitor that selectively targets mutant forms of EGFR, including T790M, L858R and Del(19), while sparing wild-type EGFR. Promising clinical activity has recently been reported from an on-going Phase I/II trial. Methods: Matched tumor tissue and blood from 97 evaluable Stage IIIB/IV NSCLC patients, 40 treated with CO-1686, were tested using two allele-specific PCR assays, the cobas® EGFR FFPET and cobas® EGFR blood tests. Each test detects 41 mutations in EGFR, including the T790M resistance mutation, exon 19 deletions and L858R. In a subset of 30 patients, we compared the cobas plasma results to BEAMing, a highly quantitative and sensitive technology based on digital PCR. BEAMing was also used to serially monitor changes of the plasma EGFR mutation burden of several patients in response to CO-1686. Results: Using tissue as the reference, the positive percent agreement between tissue and plasma was 73% (57/78) for activating mutations and 62% (21/34) for T790M. The cobas® EGFR blood test identified two patients with T790M mutations in plasma that were not detected in the corresponding tumor biopsy_likely because of tumor heterogeneity. The M0/M1a/M1b status was known for 73 EGFR mutation-positive patients. Of the 49 with extrathoracic metastatic disease (M1b), 43 were found to have an activating mutation in plasma (88%). Conversely, only 50% (12/24) of EGFR mutation-positive patients with intrathoracic metastatic disease (M0/M1a) had detectable activating mutations in plasma (p Conclusions: Using the cobas® EGFR blood test, a high proportion of EGFR mutations identified in tissue were also detected in plasma. Mutations were more readily detectable in the plasma of patients with M1b rather than M1a/M0 disease. Serial monitoring identified quantitative changes in EGFR mutation levels over time in patients treated with CO-1686. These findings suggest that the cobas® EGFR blood test and BEAMing can be useful tools for the non-invasive assessment and monitoring of EGFR mutations in the plasma of NSCLC patients. Citation Format: Rafal Dziadziuszko, Chris Karlovich, Wei Wen, Jong-Mu Sun, Sean Chien, Elaina Mann, Patrick O'Donnell, Philipp Angenendt, Heather Wakelee, Leora Horn, David Spigel, Lecia Sequist, Jean-Charles Soria, Benjamin Solomon, D. Ross Camidge, Jonathan Goldman, Shirish Gadgeel, Mitch Raponi, Andrew Allen, Lin Wu, Keunchil Park. Serial monitoring of EGFR mutations in plasma and evaluation of EGFR mutation status in matched tissue and plasma from NSCLC patients treated with CO-1686. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5587. doi:10.1158/1538-7445.AM2014-5587

Lung neuroendocrine tumors (LNETs) comprise small-cell lung cancer (SCLC), large-cell neuroendocrine tumors (LCNEC), and pulmonary carcinoids (PCA), and account for 25% of all lung cancer cases. The low 5-year survival rate of the highly aggressive LNETs (SCLC and LCNEC) combined with the lack of an effective treatment, suggest that understanding how these tumors arise and identifying therapeutic targets are unmet needs. We performed genome/exome, and transcriptome sequencing of 29 SCLC (Ref.1), 60 LCNEC, and 45 PCA to better understand their molecular origin and identify altered genes that may offer therapeutic opportunities. In contrast to SCLC and LCNEC, we found that RB1 and TP53 mutations were rare events in PCA suggesting that PCA are not early progenitor lesions of SCLC or LCNEC, but arise through independent mechanisms. Moreover, GSEA analysis showed that genes of the RB1 pathway were downregulated in SCLC but not in PCA. Our data also show that inactivation of chromatin-remodeling genes, specifically genes involved in histone methylation and subunits of the SWI/SNF complex, is sufficient to drive transformation in PCA. In a preliminary analysis of 15 LCNEC (Ref.2) we observed a predominance of mutations typical of SCLC, such as RB1, TP53, and CREBBP/EP300. In this larger series, we additionally found samples with mutations frequent in adenocarcinoma (AD) or squamous (SQ) lung cancer. We could then distinguish two well-defined groups of LCNEC: a SCLC-like group, carrying MYCL1 amplifications and mutations in both RB1 and TP53 genes; and an AD/SQ-like group, harbouring CDKN2A deletions, TTF1 amplifications, and frequent mutations in KEAP1 and STK11. Interestingly, RB1, STK11, and KEAP1 mutations happened in an almost mutually exclusive way. These data suggest that LCNEC might represent an evolutionary trunk that can branch to SCLC or AD/SQ, and also that LNETs and non-LNETs are not completely different entities. This is already suggested by the fact that one of the resistance mechanisms of EGFR-mutant adenocarcinomas to tyrosine-kinase inhibitors is through trans-differentiation to SCLC (Ref.3). Finally, we also identified new targetable driver genes in SCLC and LCNEC: FGFR1 amplifications were observed in 6% and 18% of the cases respectively; PTEN mutations were identified in 10% of the SCLC cases; and interestingly, one of the LCNEC samples (belonging to the SCLC-like group) harboured an activating RFWD2-NTRK1 fusion gene suggesting that fusions affecting NTRK1 may not only be a targetable opportunity for AD (Ref.4) but also for LCNEC and, based on the molecular similarities, also SCLC. (1) Peifer and Fernandez-Cuesta et al. Nat Genetics 2012 (2) The Clinical Lung Cancer Genome Project (CLCGP) and Network Genomic Medicine (NGM) Sci Transl Med 2013 (3) Sequist et al., Sci Transl Med 2011 (4) Vaishnavi et al. Nat Medicine 2013 Citation Format: Lynnette Fernandez-Cuesta, Martin Peifer, Xin Lu, Danila Seidel, Thomas Zander, Frauke Leenders, Luka Ozretić, Odd-Terje Brustugun, John K. Field, Gavin Wright, Benjamin Solomon, Reinhard Buettner, Christian Brambilla, Elisabeth Brambilla, Roman K. Thomas. Cross-entity mutation analysis of lung neuroendocrine tumors sheds light into their molecular origin and identifies new therapeutic targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1531. doi:10.1158/1538-7445.AM2014-1531

Background. CDKN2A inactivation has been reported to be a frequent event in head and neck squamous cell carcinomas that correlates with worse patient outcome. The differential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas (OTSCC) have been poorly characterised. We sought to determine the frequency of potential mechanisms of CDKN2A inactivation in OTSCC and explore their impact on patient outcome. Material and methods. We investigated a cohort of 134 OTSCC patients treated between 2002 to 2008 with combinations of surgery, radiotherapy and chemotherapy, who have been comprehensively annotated for patient/tumour characteristics and outcome. 134 formalin fixed paraffin embedded blocks representative of primary tumours prior to treatment were collected. DNA was extracted from 115 patient blocks and a tissue microarray was made representing 123 patient samples. We assessed p16/CDKN2A expression by immunohistochemistry (IHC), promoter methylation status by methylation-sensitive high resolution melting, mutation status by Sanger sequencing, gene copy number variation by fluorescence in-situ hybridisation and correlated each of these with patient outcome. Results. The majority of OTSCC demonstrated loss of p16 expression (107/118, 90.7%), assessed by IHC. The frequency of CDKN2A inactivating mutations was 20.3% (21/103), homozygous CDKN2A loss was 4.1% (7/98), hemizygous CDKN2A loss 30.6% (30/98), and CDKN2A promoter methylation >10% was 17.7% (20/113). 34/107(31.8%) p16 IHC negative tumours had no evidence of CDKN2A mutation, copy number variation or promoter methylation identified. Of eleven p16 IHC positive patients, only two demonstrated evidence of HPV type 16 and 33 on PCR. No correlation was identified between any of the mechanisms of CDKN2A inactivation and clinicopathological features. Smoking status and age did not influence the frequency of any mechanism of CDKN2A inactivation or patient outcome. There was a non-significant trend for worse outcome for p16 IHC negative patients versus IHC positive patients (HR=0.65, 95% CI=0.23-1.79, p=0.40). No significant relationship between mechanisms of p16 inactivation and patient outcome were found. Conclusion. Loss of p16 is a frequent event in oral tongue squamous cell carcinomas that can occur through gene copy number variation, mutation, promoter methylation or through other unidentified mechanisms. We identified no correlation between mechanisms of p16 loss and clinicopathological characteristics or patient outcome. Citation Format: Annette M. Lim, Hongdo Do, Richard J. Young, Stephen Q. Wong, Christopher Angel, Marnie Collins, Elena Takano, June Corry, Stephen B. Fox, David Wiesenfeld, Stephen Kleid, Elizabeth Sigston, Bernard Lyon, Danny Rischin, Alexander Dobrovic, Benjamin Solomon. Characterization of the differential mechanisms of CDKN2A inactivation in oral tongue squamous cell carcinomas and correlation with patient outcome. [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 4693. doi:10.1158/1538-7445.AM2013-4693