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In recent years, there has been tremendous therapeutic progress for advanced lung cancer, leading to the identification of a multitude of therapeutic targets and significantly expanding the list of potential target genes to be tested. However, precision oncology requires knowledge of the exact biology of the tumor through sequencing of the cancer genome, which is hampered by limited tissue availability in thoracic malignancies. Liquid biopsy, namely plasma cell-free DNA (cfDNA) analysis, has expanded these opportunities and is now firmly established in the diagnostic algorithm of patients with lung cancer with metastatic disease. However, the full potential of this powerful tool has been largely underexplored. In this issue of Cancer Research, Nair and colleagues provide evidence of the clinical utility of bronchoalveolar lavage (BAL) cfDNA profiling through an ultra-deep next-generation sequencing approach. The study findings support further development of BAL cfDNA analysis for tumor genotyping, besides the current gold standard sources (tissue and plasma), as a complementary tool in patients undergoing diagnostic bronchoscopy for tumor diagnosis and/or rebiopsy, increasing the success rates of genomic analyses. In addition, BAL cfDNA profiling might represent an important diagnostic tool in early-stage lung cancer, outperforming plasma cfDNA in stage I–II and detecting field cancerization signs, potentially identifying tumors before their clinical appearance. Further studies should confirm the full potential of BAL cfDNA profiling in lung cancer and its place in the large family of liquid biopsies.See related article by Nair et al., p. 2838

Type-2 cytokines are hypothesized to promote an immune-permissive milieu for cancer to grow. Through scRNAseq and CITEseq on human non-small cell lung cancer (NSCLC) and the krasG12Dp53−/− lung cancer model we previously described a tumor-enriched dendritic cell program of concomitant immunosuppression and activation which we termed the “mregDC,” that was also notable for a strong Th2 immune signature. We subsequently blocked the canonical Th2 cytokine IL-4 in vivo in tumor-bearing mice, and found that this significantly decreased lung tumor burden, which was recapitulated in multiple other tumor models. Furthermore, this effect synergized with PD-L1 blockade. Based on this data, we designed a clinical trial to assess if the addition of dupilumab, an anti-IL-4Ra antibody widely used for treatment of atopic diseases, may rescue responses to checkpoint blockade (NCT05013450). In this Phase 1b/2 trial, up to 21 patients with NSCLC that have progressed on prior anti-PD-(L)1 therapy will be enrolled. Patients continue PD-(L)1 blockade while receiving three doses of dupilumab, administered every three weeks, with initial radiographic assessment of response at 9 weeks. Patients without progression of disease then continue anti-PD-(L)1 alone. The primary endpoint of Phase 1b is safety and tolerability, while the primary endpoint of Phase 2, inclusive of patients from Phase 1b, is efficacy as per RECIST. Patients undergo pre- and on-treatment biopsies, pre-treatment stool collection for microbiome analysis, as well as blood collection at 6 timepoints for PBMCs, plasma and ctDNA. Here we report the Phase 1b portion that has completed accrual in which 6 patients were enrolled in a Phase 1b run-in to confirm safety and tolerability. There were no adverse events attributable to study treatment during Phase 1b in any of the 6 patients treated. Serum proteomic analysis of this cohort revealed that dupilumab treatment induced a profound increase in proinflammatory/tumoricidal immune effector molecules, and reversed a systemic Th2 cytokine signature. Furthermore, mass cytometry of circulating immune populations showed an expansion of cytotoxic lymphocyte populations and a reduction in immunosuppressive myeloid cells. The fourth patient treated on trial who had progressive disease after 9 cycles of consolidation checkpoint blockade following concurrent chemoradiation for squamous NSCLC was enrolled, and had a partial response at 9 weeks, with deepening of the PR at 25 weeks. This patient’s on-treatment biopsy showed a major increase in CD8 T cell tumor infiltration. Histological analysis using spatial transcriptomics and multiplexed imaging from these patients is ongoing, and will be reported at the conference. Based on this promising signal in the initial lead-in, the trial will proceed through Simon’s Two Stage design and enroll a full 21 patients as part of the Phase 2 expansion cohort. This clinical trial is funded entirely through philanthropy support through the Tisch Cancer Institute. Citation Format: Thomas Urban Marron, Nelson M. LaMarche, Matthew D. Park, Samarth Hegde, Bailey Fitzgerald, Clotilde Hennequin, Raphael Mattiuz, Meriem Belabed, Jessica Le Berichel, Barbara Maier, Nicole Hall, Justin Miller, Deborah B. Doroshow, Nicholas Rohs, Rajwanth Veluswamy, Nicholas Venturini, Jorge E. Gomez, Christian Rolfo, David Yankelevitz, Udit Chaddha, Timothy Harkin, Mary B. Beasley, Seunghee Kim-schulze, Sacha Gnjatic, Fred R. Hirsch, Miriam Merad. Rescuing response to ICI by blocking the type-2 immune axis in patients with NSCLC progressing on immunotherapy: A phase 1b/2 trial of dupilumab administered with checkpoint blockade [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 CT199.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2–M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD. Significance: These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

Background: Malignant pleural mesothelioma (MPM) is usually fatal, though multimodality therapy— now including immunotherapy— has improved survival. Recurrence after surgery is close to 100%, even with adjuvant chemotherapy and radiation. Our collaborators have performed deep immunophenotyping of treatment-naïve MPM lesions using mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq) to define the tumor microenvironment. A population of rare CD141+ dendritic cells (DC1) is disproportionately represented in some MPM lesions analyzed. These DC1 cells— which express high levels of Toll-like receptor 3 (TLR3)— are among the most potent cross-presenters of antigen and are key to priming anti-tumor CD4+ and CD8+ T cell responses. Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC), is a double-stranded RNA host-targeted therapeutic viral-mimic. Poly-ICLC activates multiple innate immune receptors including TLR3 and melanoma differentiation-associated gene 5 (MDA5), leading to cross-presentation of antigen to T cells and induction of strong Th1 response. We hypothesize that injection of poly-ICLC prior to surgical resection may activate intratumoral (IT) DC1s, increase tumor antigen presentation to cytotoxic T cells, and induce tumor-specific immune surveillance. Methods: This is a phase I/Ib study to evaluate the safety of IT poly-ICLC prior to surgical resection for patients with MPM (NCT04525859). The primary endpoint is safety as assessed by frequency and severity of toxicities by CTCAE 5.0. Secondary endpoints are objective response as measured by RECIST 1.1 and recurrence free survival measured from the time of first poly-ICLC injection. Exploratory endpoints include evaluation of circulating immune cells (including regulatory T cells and NK cells), evaluation of immune cell infiltration in pre-injection tumor biopsy and surgically resected tissue, as well as characterization of immune parameters such as local B cell specificity. The protocol features a Simon’s two-stage design, with six patients enrolled in a phase I safety cohort, proceeding to a phase Ib expansion cohort (additional 13 patients) if no more than 1 dose limiting toxicity occurs. Eligible patients must have MPM deemed operable by the treating thoracic surgeon. Eligible subjects may not have uncontrolled immunocompromised states or autoimmune disorders. After enrollment, patients undergo biopsies at which time 2mg poly-ICLC is injected across two sites in the tumor. Patients then undergo resection of the tumor (pleurectomy/decortication or extra pleural pneumonectomy per standard of care) at day 21+/- 7 after poly-ICLC injection. Blood is drawn at three points (prior to poly-ICLC injection, at time of surgery, and at a post-operative visit) for immune profiling. At the time of submission six patients have been treated and phase Ib accrual is continuing as planned. Interim analysis of phase I safety and exploratory endpoints will be reported in late 2022. Citation Format: Bailey G. Fitzgerald, Thomas U. Marron, Robert Sweeney, Jorge Gomez, Nicole Hall, Daniel O'Grady, Christian Rolfo, Raj Veluswamy, Deborah Doroshow, John Mandeli, David Yankelevitz, Nina Bhardwaj, Sacha Gnjatic, Fred R. Hirsch, Miriam Merad, Alexander Tsankov, Raja Flores, Andrea Wolf. A phase I/Ib trial of intratumoral Poly-ICLC in resectable malignant pleural mesothelioma [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 CT205.

Background: Bintrafusp alfa is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. The global, phase 1b/2 INTR@PID LUNG 024 (NCT03840915) study evaluates bintrafusp alfa in combination with chemotherapy (CT) in patients with stage IV NSCLC. Here we report cumulative safety, which represents the first report of safety results from a clinical study with a bintrafusp alfa 2400-mg every-three-weeks (Q3W) regimen. Methods: Adults with stage IV nonsquamous or squamous NSCLC and an ECOG PS ≤1 are included. In cohorts A, B, and C, patients must not have received prior systemic therapy; in cohort D, patients must have had progression on previous anti-PD-(L)1 therapy. Patients received bintrafusp alfa 2400 mg Q3W intravenously in combination with CT for 4 cycles (A [nonsquamous only]: cisplatin or carboplatin + pemetrexed; B: carboplatin + nab-paclitaxel or paclitaxel; C: cisplatin or carboplatin + gemcitabine; D: docetaxel) followed by bintrafusp alfa maintenance (monotherapy or combination with pemetrexed in cohort A) for up to 31 cycles. The occurrence of dose-limiting toxicities (DLTs) was assessed during a 3-week observation period. The primary objective of this study was to evaluate the safety of bintrafusp alfa in combination with CT. Additionally, in cohort A, patients from the safety and expansion parts will be assessed for preliminary efficacy. Results: As of October 7, 2020, 64 patients received bintrafusp alfa in combination with CT. Of the 35 patients included in the DLT analysis, 4 experienced 1 DLT according to the safety monitoring committee (A: n=1/8; B: n=1/8; C: n=0/8; D: n=2/11). The cumulative safety is reported (Table). Conclusions: The combination of bintrafusp alfa 2400 Q3W in combination with CT was well tolerated. No new safety signals were identified. Cohort A (n=34)Cohort B (n=9)Cohort C (n=9)Cohort D (n=12)Treatment-emergent adverse events, n (%)Any*34 (100.0)9 (100.0)9 (100.0)12 (100.0)Grade ≥324 (70.6)6 (66.7)7 (77.8)12 (100.0)Grade ≥412 (35.3)3 (33.3)5 (55.6)6 (50.0)Serious22 (64.7)3 (33.3)6 (66.7)9 (75.0)Leading to death3 (8.8)1 (11.1)00Leading to dose reduction of CT9 (26.5)2 (22.2)5 (55.6)4 (33.3)Leading to permanent discontinuation of bintrafusp alfa12 (35.3)04 (44.4)4 (33.3)Leading to permanent discontinuation of CT13 (38.2)1 (11.1)4 (44.4)3 (25.0)Treatment-related adverse events, n (%)Any bintrafusp alfa-related adverse events28 (82.4)9 (100.0)9 (100.0)12 (100.0)Bintrafusp alfa-related serious adverse events9 (26.5)01 (11.1)3 (25.0)Bintrafusp alfa-related adverse events leading to death0000Any chemotherapy-related adverse events31 (91.2)8 (88.9)9 (100.0)12 (100.0)Chemotherapy-related serious adverse events10 (29.4)1 (11.1)4 (44.4)7 (58.3)Chemotherapy-related adverse events leading to death0000Adverse events of special interest, n (%)Skin lesions†2 (5.9)04 (44.4)1 (8.3)Immune-related adverse event15 (44.1)3 (33.3)5 (55.6)6 (50.0)Infusion-related reaction3 (8.8)1 (11.1)1 (11.1)1 (8.3)Anemia16 (47.1)5 (55.6)9 (100.0)9 (75.0)*Most common (≥50%) treatment-emergent adverse events in any cohort included anemia (cohort B: 55.6%; cohort C: 100.0%; cohort D: 75.0%), nausea (cohort A: 52.9%, cohort C: 66.7%), pruritus (cohort C: 66.7%), asthenia (cohort C: 55.6%; cohort D: 58.3%), neutropenia (cohort C: 55.6%), decreased appetite (cohort D: 50.0%), and diarrhea (cohort D: 50.0%).†Defined as actinic keratosis, basal cell carcinoma, Bowen's disease, hyperkeratosis, keratoacanthoma, lip squamous cell carcinoma, and squamous cell carcinoma of skin. Actinic keratosis, hyperkeratosis, and keratoacanthoma were reported in this study. Citation Format: Christian Rolfo, Laurent Greillier, Remi Veillon, Firas Badin, Francois Ghiringhelli, Nicolas Isambert, Astrid Paulus, Marc Lambrechts, Italia Grenga, Xiaoli You, Andre Koenig, Sandrine Hiret. Bintrafusp alfa in combination with chemotherapy in patients with stage IV NSCLC: safety results of the INTR@PID LUNG 024 study [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 CT104.

Background: The combinations of pembrolizumab plus chemotherapy (CT) and atezolizumab plus bevacizumab and CT have been approved for first-line therapy in patients with advanced non-small cell lung cancer (NSCLC) regardless of PD-L1 expression; however, there is still a significant unmet need for this patient population who may not respond to immune checkpoint treatment. Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor designed to function as a TGF-β “trap” fused to a human IgG1 mAb blocking PD-L1. Promising antitumor activity and a manageable safety profile were observed with bintrafusp alfa in multiple cohorts of a phase 1 study (NCT02517398) that enrolled patients with advanced, pretreated NSCLC who experienced disease progression either after platinum-based CT or anti-PD-(L)1 monotherapy. This study is evaluating the safety and efficacy of bintrafusp alfa in combination with CT in patients with stage IV NSCLC. Trial Design: This is a phase 1b/2, open-label, 4-cohort study (NCT03840915) evaluating bintrafusp alfa in combination with CT in patients with stage IV NSCLC. All patients will receive bintrafusp alfa 2400 mg Q3W intravenously and either cisplatin or carboplatin + pemetrexed (cohort A), carboplatin + nab-paclitaxel or paclitaxel (cohort B), cisplatin or carboplatin + gemcitabine (cohort C), or docetaxel (cohort D) Q3W for 4 cycles, followed by bintrafusp alfa maintenance (monotherapy or combination with pemetrexed [cohort A]) for up to 31 cycles or until disease progression, unacceptable toxicity, or death. Patients must be adults with histologically confirmed diagnosis of stage IV nonsquamous or squamous NSCLC (nonsquamous for cohort A), adequate organ function, ECOG PS ≤1, life expectancy ≥3 months, and measurable disease based on RECIST 1.1. Patients in cohorts A-C must not have received prior systemic therapy, and patients in cohort D must have disease progression on previous anti-PD-(L)1 therapy. Patients with tumors with actionable mutations (for which targeted therapy is locally approved), mixed SCLC and NSCLC, active CNS metastases, active autoimmune disease, known severe hypersensitivity, or interstitial lung disease are not eligible. The primary endpoint of this study is to assess the safety of bintrafusp alfa in combination with CT. Planned enrollment is 64 patients (32 patients among the 4 cohorts during the safety phase, and an additional 32 patients in cohort A for expansion phase). Citation Format: Fabrice Barlesi, Sandrine Hiret, Christian Rolfo, Italia Grenga, Andre Köenig, Yulia Vugmeyster, Leora Horn. A phase 1b/2, open-label study of bintrafusp alfa with chemotherapy in patients with stage IV non-small cell lung cancer [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 CT267.

Because of the refractory nature of mutant

Background Entrectinib is a potent inhibitor of ROS1 (in addition to TRKA/B/C), designed to effectively penetrate the central nervous system (CNS); brain metastases are common in patients with advanced ROS1 fusion-positive NSCLC. Entrectinib achieves therapeutic levels in the CNS with antitumor activity in multiple intracranial tumor models. We present an updated integrated safety and efficacy analysis from three Phase I/II studies of entrectinib (ALKA-372-001 [EudraCT 2012-000148-88], STARTRK-1 [NCT02097810], STARTRK-2 [NCT02568267]) in patients with locally advanced or metastatic ROS1 fusion-positive NSCLCs. Methods The analysis included patients with ROS1 inhibitor-naïve NSCLC harboring a ROS1 fusion identified via nucleic acid-based diagnostic platforms. The ROS1 safety-evaluable population included patients with ROS1 fusion-positive NSCLC who received ≥1 dose of entrectinib; the integrated efficacy analysis included patients with at least 6 months of follow-up. Tumor assessments were done at week 4 and every 8 weeks thereafter. Blinded independent central review (BICR), RECIST v1.1 was performed. Primary endpoints by BICR: overall response rate (ORR) and duration of response (DOR). Key secondary endpoints: progression-free survival (PFS), safety. Additional endpoints: intracranial ORR (complete/partial responses), DOR in patients with an intracranial response, PFS in patients with baseline CNS disease. Results In the ROS1 safety-evaluable population (n=134), at least one treatment-related AE (TRAE) of any grade was seen in 93% of patients. Patients with at least one TRAE by highest grade were: grade 1/2, 59%; grade 3, 31%; grade 4, 4%. There were no grade 5 TRAEs. TRAEs led to dose reduction or discontinuation in 34% and 5% of patients, respectively. In the efficacy-evaluable population (n=53 patients with treatment-naïve, ROS1 fusion-positive NSCLC; median age 53 years, 64% female, 59% never smokers), BICR-assessed ORR was 77% (95% CI 64-88), complete responses n=3 (6%). Median BICR-assessed DOR: 25 mo (95% CI 11-35). Median BICR-assessed PFS: 26 mo (95% CI 16-37) and 14 mo (95% CI 5-NR) for patients without (n=30) and with CNS disease (n=23) at baseline, respectively. In patients with baseline CNS disease (per BICR assessment, n=20), intracranial ORR was 55% (95% CI 32-77) and median intracranial DOR in patients with an intracranial response (n=11) was 13 mo (95% CI 6-not reached). Conclusion Entrectinib is highly active in patients with ROS1 fusion-positive NSCLC, including those with CNS disease. Entrectinib is well tolerated and has a manageable safety profile. Citation Format: Alexander Drilon, Fabrice Barlesi, Filippo De Braud, Byoung Chul Cho, Myung-Ju Ahn, Salvatore Siena, Matthew G. Krebs, Chia-Chi Lin, Tom John, Daniel SW Tan, Takashi Seto, Rafal Dziadziuszko, Hendrick-Tobias Arkenau, Christian Rolfo, Jürgen Wolf, Chenglin Ye, Todd Riehl, Susan Eng, Robert C. Doebele. Entrectinib in locally advanced or metastatic ROS1 fusion-positive non-small cell lung cancer (NSCLC): Integrated analysis of ALKA-372-001, STARTRK-1 and STARTRK-2 [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 CT192.

Background: Over the last decades, little progress has been made in the systemic treatment of patients with advanced or recurrent cervical cancer. We recently performed an silico analysis to identify potential driver pathways of cervical carcinogenesis and candidate targets for treatment.1 Expression2Kinases (E2K) analysis revealed a protein-protein interaction (PPI) network of 162 nodes (including 20 druggable kinases) consisting of 5 signaling modules associated with MYC signaling (module 1), cell cycle deregulation and cyclin signaling (module 2), TGFβ-signaling (module 3), a PI3K - MAPK signaling (module 4) and chromatine modeling (module 5). Methods: The cervical cancer cell lines included in the study were CaSki, SiHa and HeLa. Cells were incubated for 24 or 72 hours with the PI3K pathway inhibitor BYL719 (Novartis), the multiple pathway inhibitor INC280 (inhibits cMET-dependent PI3K and RAS signaling, Novartis), or the CDK4/6 inhibitor PD-0332991 (palbociclib, Selleck Chemicals) in monotherapy. Sensitivity to drug treatment (0 - 10 μM) was investigated using the colorimetric sulforhodamine B (SRB) assay. IC50 values were calculated using WinNonlin Software. Results: Incubation with BYL719 or INC280 for 24 or 72 hours in concentrations up to 10 μM was not able to establish a distinct cytotoxic effect in the three cervical cancer cell lines included in the study. Incubation with 10 μM BYL719 for 72h resulted in only 20% cell kill, while 10 μM INC280 for 72h caused around 30% cell kill in all three cell lines. However, incubation with palbociclib for 72 hours clearly induced a concentration-dependent cytotoxic effect, with IC50 values of 5.32 ± 0.03 μM, 7.69 ± 0.02 μM and 5.68 ± 0.12 μM for HeLa, SiHa and CaSki cells, respectively. Conclusion: CDK4/6 inhibition seems effective in cervical cancer cell lines and should be further evaluated alone and in combination with chemotherapy for the treatment of advanced and recurrent cervical cancer. References: Van Dam P, van Dam PJ, Rolfo C, Giallombardo M, Van Berckelaer C, Trin XB, Altintas S, Huizing M, Papadimitriou K, Tjalma W, Van Laere S. In silico pathway analysis in cervical carcinoma reveals potential new targets for treatment. Oncotarget 2016;7(3):2780-2795 Citation Format: Peter Van Dam, An Wouters, Luc Dirix, Christian Rolfo. Palbociclib monotherapy exhibits potent activity in cervical cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2317.

Introduction: Numerous studies have reported that tumor progression and invasiveness are determined not only by the malignant cancer cells themselves but also by the surrounding tumor microenvironment, including cancer-associated fibroblasts (CAFs). Although CAFs are implicated in tumor progression, their total depletion of CAFs has been demonstrated to induce more aggressive tumors, indicating that different CAF subpopulations have opposing tumor-promoting or tumor-inhibitory roles. Unfortunately, specific markers to target these subsets of CAFs are lacking. Expression of the immune checkpoint CD70 is normally tightly regulated and limited to cells of the lymphoid lineage only. Instead, tumors hijack CD70 to facilitate immune evasion by increasing the amount of suppressive regulatory T cells (Tregs), inducing T cell apoptosis and skewing T cells towards T cell exhaustion. Recently, a lot of clinical successes have been generated by the blockade of immune checkpoints. However, in colorectal cancer (CRC) the efficacy remains limited to a small subset of patients with mismatch repair-deficient (MSI) tumors which might be caused by the intense dialogue between stroma and malignant cells. Therefore, we have explored the expression patterns of the immune checkpoint molecule CD70 in CRC, with a particular focus on CAFs. Methods: The prognostic value of CD70 was analyzed by immunohistochemistry on 51 CRC specimens. In addition, the relationship with Tregs and microsatellite instability was explored. Furthermore, primary CAF cell lines were successfully cultured from 20 different primary resection specimens. These cell lines were used to study the effect of CD70 on the tumor microenvironment in vitro. Results: We revealed expression of CD70, not just on the malignant cells but on the majority of CAFs in invasive CRC specimens. Thereby, CD70-expression was significantly correlated with negative clinicopathological parameters such as metastasis (P=0.007), differentiation (P=0.053) and advanced stage (P=0.001). Moreover, CD70-positive CAFs proved to be a poor prognostic marker by univariate as well as multivariate analysis. We have also detected a significant association between elevated Treg amounts and CD70-expressing CAFs (P=0.012). In vitro data on the effects of CD70 on CRC behavior and immune escape are currently being analyzed. Conclusion: We have identified a new targetable CAF subpopulation, marked by the expression of CD70 and equipped with strong tumor-promoting properties. Thereby, we have found evidence of a potential cross talk between CD70+ CAFs and Treg, paving the way towards immune escape. The lack of association of CD70 expression and MSI-status, which highlights the potential of this target in CRC subsets that do not benefit from immune checkpoint blockade. We believe that targeting CD70 holds great potential in CRC, especially in light of the limited immunotherapeutic options available. Citation Format: Julie Jacobs, Vanessa Deschoolmeester, Karen Zwaenepoel, Christophe Hermans, Christian Rolfo, Marc Peeters, Filip Lardon, Vasiliki Siozopoulou, Evelien Smits, Patrick Pauwels. Blocking CD70+ cancer associated fibroblasts: Are we paving the way towards immunotherapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 958. doi:10.1158/1538-7445.AM2017-958

Introduction: In a previous work we found differences in immune gene sets enrichment in NSCLC between genders, regardless their smoking status, where women had higher expression of gene sets associated with immune processes. Based in this fact, we hypothesize fewer benefits from immune checkpoint inhibitors in women patients compared than men patients. Objectives: Evaluate the benefit (in terms of progression-free survival) of women patients from immune checkpoint inhibitors nivolumab and pembrolizumab in published randomized phase III trials. Methods: We evaluated by meta-analysis four randomized phase III studies, two of prembrolizumab (Herbst et al., 2015 and Reck et al., 2016) and two of nivolumab (Borghaei et al., 2015 and Brahmer et al., 2015). We analyzed TCGA data for lung adenocarcinoma to evaluate differences in the expression of PDCD1, CD274 genes (encoding for PD-1 and PD-L1, respectively). Results: For all studies, it was observed an overall HR=0.79 (CI95%:0.71-0.87;P Conclusion: Although there are not differences between genders in the expression of PDCD1 and CD274 genes , such as is previously described for expression for PD-1 and PDL-1 proteins, in our meta-analysis we shown that women have modest benefit from anti immune checkpoint inhibitors. Blockade of PD1 and PD-L1 is a promising therapy in lung cancer; however, a better stratification of patients should be done. Meta-analysis of four phase III randomized studiesStudyAll patientsMenWomenWeightHazard Ratio IV, Fixed, 95% CIWeightHazard Ratio IV, Fixed, 95% CIWeightIV, Fixed, 95% CIReck et al., 201610.6%0.50 [0.37, 0.68]10.4%0.39 [0.26, 0.581]8.0%0.71[0.40, 1.26Brahmer et al., 201513.7%0.63 [0.48, 0.8217.8%0.63 [0.46, 0.86]11.2%0.75[0.46, 1.22]Borghaei et al., 201530.3%0.91 [0.76, 1.0926.6%0.81 [0.63, 1.04]37.8%1.02[0.78, 1.33]Herbst et al., 201545.40.85 [0.73, 0.98]45.2%0.78 [0.64, 0.95]43.0%1.04[0.81, 1.33]Total (95% Cl)100.0%0.79 [0.71, 0.87]100.0%0.71 [0.62, 0.80100.0%0.97[0.82, 1.14]Heterogeneity: Chi2=14.73,df=3 (P=0.002); I2= 80% Test for overall effect: Z= 4.73 (P Citation Format: Joseph A. Pinto, Luis A. Mas, Carlos S. Vallejos, Jhajaira Araujo, Leny Bravo, Alfredo Aguilar, Zaida Morante, Denisse Bretel, Henry L. Gomez, Christian Rolfo. Decrease of benefit from immune checkpoint inhibitors in women with non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2614. doi:10.1158/1538-7445.AM2017-2614

Oncogenic drivers are often overexpressed in adenocarcinoma non-small cell lung cancer (NSCLC) patients, so this tumor type is often sensitive to targeted therapies. Sensitizing mutations in the epidermal growth factor receptor (EGFR) are good predictive biomarkers for response to EGFR-small molecule inhibitors, eg erlotinib. Amplification or exon 14 skipping of cMET are good biomarkers for cMET-small molecule inhibitors, eg crizotinib. In other subtypes of NSCLC, eg squamous, targeted therapy is not considered to be active. We tested the combination of erlotinib and crizotinib on a panel of squamous NSCLC cell lines that were wild-type for both EGFR and cMET. The effect of this drug combination was tested with different assays. The sulforhodamine B-assay was used to determine growth inhibition and combination indexes (CI) were calculated with the method of Chou&Talalay. A CI< 0.8 was synergistic, 0.81.2 was antagonistic. With flow cytometry the cell cycle was studied. With the wound-healing assay we determined the effect on cell migration. A 3D spheroid assay was performed to determine the effect of limited nutrient availability and hypoxia. Protein phosphorylation was studied with a pathscan array and phospho-specific western blotting. The LUDLU cell line gave a synergistic CI of 0.39±0.07, whereas the Calu1 (CI = 0.81±0.04), H520 (CI = 0.87±0.06) and SKMES1 (CI = 0.81±0.02) were additive and the H1703 cell line (CI = 1.20±0.15) was antagonistic. Cell cycle analysis revealed that erlotinib monotherapy caused a G1 phase arrest (38 to 76%) and crizotinib caused a G2/M (13 to 64%) phase arrest. In cells treated with the combination, the effect of crizotinib was dominant (13 to 52%). In the wound-healing assay the LUDLU cells were not migratory. The pathscan revealed a decrease in the phosphorylation of pERK1/2 (Thr202/204), pPRAS40 (Thr246) and pGSK3β (Ser9). Western blotting showed that the levels of pPRAS40 were decreased in the LUDLU cells after treatment with erlotinib or crizotinib, and almost gone when treated with the combination. In the SKMES1 cells only the combination decreased pPRAS40. H1703 cells showed no change in pPRAS40. These changes in phosphorylation make us hypothesize that Her3 and PI3K/Akt signaling may be important in the interaction of both drugs. Her3 is a promiscuous receptor that is able to heterodimerize with EGFR and cMET. When blocking one of these receptors, Her3 is still able to continue downstream PI3K/Akt signaling. When both EGFR and cMET are blocked, Her3 and PI3K/Akt signaling is inhibited, leading to synergy. The expression of total-Her3 was lowest in the antagonistic cell line. The combination of both inhibitors lead to a complete inhibition of pHer3 (Tyr1289). In conclusion: Blocking both EGFR and cMET signaling causes inhibition of Her3 downstream signaling through PI3K/Akt, leading to synergy in wild-type squamous NSCLC cells. Citation Format: Nele Van Der Steen, Christian Rolfo, Patrick Pauwels, Godefridus J. Peters, Elisa Giovannetti. Combining forces: Synergy of erlotinib and crizotinib in a wild-type squamous non-small cell lung cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2017-1074

The rise of liquid biopsy, including the molecular analysis of circulating cell-free tumor DNA (ctDNA), has enabled real-time follow up of cancer patients who receive targeted therapy. Here, we report the case of a lung cancer patient (adenocarcinoma), initially harboring the activating EGFR L858R mutation. Blood and tissue samples were collected during treatment with the EGFR tyrosine kinase inhibitor (TKI) gefitinib, and during the appearance of resistance. Analysis of the tissue biopsies consisted of EGFR mutational analysis by high-resolution melting analysis (HRMA) (all tissue samples), immunohistochemistry (IHC) of cMET (sample 2); FISH analysis of the cMET gene (sample 1 and 3); and next-generation sequencing (NGS) (sample 3). We performed digital droplet PCR (ddPCR) to detect EGFR mutations and cMET amplification in both tissue and plasma samples. To the best of our knowledge, this is the first time that ddPCR is used to screen for cMET amplification in ctDNA. The HRMA analysis of the biopsy during progression under EGFR TKI treatment revealed the EGFR T790M resistance mutation. Progression was also seen by radiological examination and this resulted in discontinuation of gefitinib treatment. As a third generation EGFR TKI was not available by that time, the patient was retreated with carboplatin plus pemetrexed, which resulted in a mixed response. In the second biopsy, only the EGFR activating L858R mutation was detected by HRMA. IHC analysis revealed a strong membrane positivity for cMET in 100% of the tumor cells. After progression upon rechallenging the patient with erlotinib, treatment with osimertinib (third generation EGFR TKI) was initiated. This resulted in only a partial response, with some strongly progressing lesions. NGS analysis of the third tissue biopsy indicated the presence of both the EGFR L858R and T790M mutations, while ddPCR analysis of the plasma sample only detected the EGFR L858R mutation. Due to the fast progression, it was opted to also screen for cMET amplification. DdPCR cMET analysis of the tissue and plasma resulted in a copy number variation of 10.0 and 3.7, respectively. FISH analysis confirmed the cMET amplification with a ratio of 3.1 and an average of 8.4 cMET-signals per nucleus. The results of the ddPCR EGFR and cMET analysis of both the plasma and tissue samples are now being processed to determine the course of the mutational load and the best course of treatment. As such, real-time follow-up of cancer patients is crucial to detect the underlying mechanisms of resistance to targeted therapies. Correlation of molecular analysis with the disease course is key. Our case study highlights the potential of ctDNA analysis for detection of both the EGFR T790M resistance mutation and cMET amplification. Hence, tissue biopsies can be supplemented by liquid biopsies in the screening for resistance mechanisms. Citation Format: Laure Sorber, Karen Zwaenepoel, An Wouters, Janssens Annelies, Birgitta Hiddinga, Jan Van Meerbeeck, Filip Lardon, Christian Rolfo, Patrick Pauwels. Detecting resistance mechanisms in patients on EGFR TKI treatment by liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2750. doi:10.1158/1538-7445.AM2017-2750

Introduction: In oncology, circulating cell-free DNA (cfDNA) has emerged as a new and sensitive biomarker. However, the low concentrations of cfDNA, especially in early stages of disease, make it a challenging analyte for extraction, consequently high isolation efficiency is essential. In this study we compared the isolation efficiency of one of the most commonly used DNA isolation kits, namely the QIAamp® circulating nucleic acid kit (QIA - Qiagen), with four newly launched cfDNA isolation kits. Material & Methods: The QIA kit was compared with the PME free-circulating DNA Extraction Kit (PME - Analytik Jenna), the Maxwell® RSC ccfDNA Plasma Kit (RSC - Promega), the NEXTprep-MagTM cfDNA Isolation Kit (NpM - BiooScientific), and the EpiQuickTM Circulating Cell-Free DNA Isolation Kit (EQ - Epigentek). CfDNA was extracted from 10 plasma samples of patients treated at the Antwerp University Hospital (UZA); including two patients with benign pancreatic cysts, three early stage pancreatic cancer patients, and four pancreatic cancer patients with metastasized disease, of which one had a second blood sample taken during follow up. Only the patients with metastasized disease were KRAS mutated. To determine the isolation efficiency of the different kits, the QX200TM Droplet DigitalTM PCR system (ddPCR - Bio Rad) was used. All samples were screened with the ddPCRTM KRAS Screening Multiplex kit (Bio Rad) for the presence of seven KRAS mutations and a specific wild type sequence, which results in the KRAS mutated cfDNA fraction and the quantification of the total cfDNA. Results: The QIA and the RSC kits displayed similar isolation efficiencies of both KRAS mutated and non-mutated cfDNA; with the detection of the KRAS mutated fraction ranging from approximately 1% to 40% and on average a yield of 76,7 and 83,1 copies/μl, respectively. On the other hand, the PME kit could not detect any KRAS mutations and in four cases there was not enough cfDNA isolated to be detected with ddPCR. DdPCR of cfDNA isolated with the NpM and EQ kits did not generate any positive signals. qPCR was performed on cfDNA isolated with both kits with control primers of the NpM kit. All samples of the NpM kit and three of the samples of the EQ kit were shown to contain cfDNA of at least 180 bp; which leads to the conclusion that ddPCR can't be used as a downstream procedure for the NpM and EQ kits. Conclusion: The RSC kit enables the isolation of a sufficient quantity of cfDNA similar to the yield generated with the QIA kit, and both kits have a higher isolation efficiency than the PME kit. This study presents two highly efficient isolation kits, of which the RSC kit has the advantage of a fully automated, magnetic beads based protocol over the labor intensive and time consuming QIA kit. We also concluded that ddPCR as a downstream procedure is not feasible with the NpM and EQ kits. However, an improved version of the NpM kit has recently been produced and will also be included in this comparison study. Citation Format: Laure Sorber, Karen Zwaenepoel, Vanessa Deschoolmeester, Geert Roeyen, Sofie Goethals, Christian Rolfo, Patrick Pauwels. A comparison of cfDNA isolation kits: isolation and quantification of cell-free DNA in plasma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 486.

Introduction - MErCuRIC is a Phase Ib/II clinical trial study using a combined MEK1/2 - cMET inhibition in RAS MT and RAS WT (with aberrant c-MET) colorectal cancer patients. As part of the discovery efforts on the cases enrolled in Phase I, we aimed to analyze the mutation status of 10 genes that could potentially be associated to the doublet MEK/MET inhibition. This study compared the MEK/MET-specific panel with the Ion Torrent 50 gene panel, aiming to compare: long-established, commercially available panels against this newly developed panel; the Ion Torrent PGM2 platform against Illumina MiSeq v.3 600 bp chemistry; hot-spot-based against full exomes-designed; and to compare the use of different bioinformatics reporter systems. The overlapping genes between the panels were: EGFR (n = 3); BRAF (n = 4); KRAS (n = 8); NRAS (n = 1); MET (n = 8); PIK3CA (n = 6); and ERBB2 (n = 5). Summary of Method NGS Design - The Multiplicom - MErCuRIC specific MASTR assay includes PTEN, MAP2K1 (MEK), EGFR, KRAS, NRAS, BRAF, PIK3CA, ERBB2, MET and PIK3R1, involving 257 amplicons in 4 plexes covering all coding exons of the 10 genes. Of the 257, 21 are control amplicons to allow for gene deletions/amplifications. The average length of the amplicons is 198 bp ranging from 124 bp to 255 bp. Validation - From a pool of 120 routine tumour samples characterised with a 50 gene mutation panel (Ion Torrent PGM2) and confirmed by Sanger sequencing and/or COBAS (Roche) QPCR, 24 FFPE cases were selected representing colorectal cancer and 4 other solid tumour types; all included a variety of DNA quality, and DNA concentration was standardized prior to library preparation. Pre-analytical handling was in accordance with established protocols in a laboratory, clinically-accredited in the UK for tissue-based, anatomical pathology testing. Results The evaluation of this MEK/MET-specific panel (Illumina MiSeq platform) resulted in 100% coverage of all targets, a higher than 97% on target reads and higher than 99% of all amplicons within 20% of mean coverage. The design minimized the areas of low coverage. A small part of exon 9 of ERBB2 was covered suboptimally: the low covered region is 30 bp in size located at the 5’ end of exon 9. It is unlikely that this low coverage led to false negative results since no mutations are reported in the COSMIC database for this DNA fragment. The results were concordant in relation to mutations involved in the genes stated above. Importantly, the percentages of allele frequency between both methods were similar, with variations ranging from 0.2% to 11.5% with an average variation of 5.2%. Insertion/deletion (Indel) detection however, required alternative bioinformatics pathways. Conclusion After combining well-established quality metrics to cover pre-analytical aspects with suitable technologies such as the MiSeq platform (Illumina) and appropriate bioinformatics, we recognize that this MEK/MET-specific NGS panel is fit for purpose. Citation Format: Perry Maxwell, Jurgen Del Favero, Marc-Aurel Fuchs, Josep Tabernero, Tim Maughan, Mark Middleton, Richard Addams, Christian Rolfo, Brian Hennessey, Pierre Laurent-Puig, Alberto Bardelli, Thierry Andre, Vlad Popovici, Patrick Johnstone, Richard Wilson, Mark Lawler, Sandra Van Schaeybroeck, Manuel Salto Tellez. Validation of a MEK/MET-specific NGS panel for early phase trial interrogation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1396.

Introduction: Upon interaction of CD70 with its receptor CD27, the NFκB pathway is activated, leading to proliferation and survival. Therefore, CD70 expression is tightly regulated and only transiently expressed on cells of the lymphoid lineage. Constitutive expression of CD70 by tumor cells can facilitate immune evasion by increasing the amount of suppressive regulatory T cells (Tregs), inducing T cell apoptosis and skewing T cells towards T cell exhaustion. Previously, we have detected CD70 positivity in non-small cell lung cancer (NSCLC) specimens, comprising patients that lack other targeted treatment options. This CD70 expression can be exploited by CD70-targeting antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies. Furthermore, anti-CD70 therapy has the exciting potential to enhance antitumor immune responses by blocking Treg proliferation. In this study, we have focused on the combination of anti-CD70 therapy with chemotherapeutic agents, to increase tumor cytotoxicity. Material and Methods: The cytotoxic effect of cisplatin was evaluated on a panel of five NSCLC cell lines (CRL-2868, CRL-5908, CRL-5883, LUDLU-1, A549), differing in their genetic aberrations, histological subtype and expression of CD70. Based on these cytotoxicity experiments 3.5μM, 7μM and 13μM were considered low, medium and high concentrations of cisplatin respectively. Using flow cytometry, we tested the effects of cisplatin on the expression of membranous CD70 at different time points (1h, 6h, 24h, 48h). Next, we evaluated whether an induction in CD70 expression could increase the ADCC potential of anti-CD70 therapy using the xCELLigence RTCA technology. Finally, we assessed the immune stimulatory potential of this combination strategy by analysis of Treg proliferation as well as cytokine production. Results: Flow cytometric analysis demonstrated the highest induction of CD70 expression, 24 hours after treatment with 7μM cisplatin, reaching significance in 4/5 cell lines. Next, we assessed whether this increase in CD70 expression could intensify the ADCC effect of anti-CD70 therapy. CRL-5883, a CD70- cell line bearing no response to anti-CD70 monotherapy, showed a significant inhibition of proliferation after sequential administration of cisplatin and anti-CD70 therapy. Also, in A549 (weak CD70+) and CRL-5908 (strong CD70+), the combination of anti-CD70 therapy and cisplatin resulted in a significant decrease in cell survival compared to monotherapy. Also our immune data point towards a favorable effect of the combination. Conclusion: Our preliminary data show that the combination of anti-CD70 therapy with medium doses of cisplatin significantly increases tumor specific cytotoxicity of the drug, compared to single treatment regimens. This combination strategy has the potential to reduce drug-related side effects and improve general outcome of NSCLC, still the most lethal type of cancer worldwide. Citation Format: Julie Jacobs, Vanessa Deschoolmeester, Karen Zwaenepoel, Christian Rolfo, Filip Lardon, Evelien Smits, Patrick Pauwels. Cisplatin and anti-CD70 therapy: Ideal partners in crime against NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4981.

A rapidly growing domain in present-day oncology is the development of targeted therapies, including several cMET inhibitors against non-small cell lung cancer (NSCLC). Nowadays, cMET amplification is used as a standard biomarker for patient selection, however there is no clear cut-off value to determine amplification, nor is there a real consensus about the way to test this. More recently, splicing variants of cMET, which skip exon 14, are gaining importance since it has been shown that patients harboring this mutation can respond to cMET directed targeted therapy. In this study, we explored the occurrence of cMET aberrations and their correlation with cMET expression in a population of 155 primary EGFR-TKI naive NSCLC tumors. Since cMET is considered as important in metastatic behavior, we also investigated the correlation of cMET expression and amplification between the primary tumor and metastases. Finally, given the link between the EGFR and cMET pathways, the EGFR status was also studied. Samples were collected at the Antwerp University Hospital and the Onze-Lieve-Vrouw Hospital Aalst. The expression of EGFR and cMET was determined by immunohistochemistry (Ventana, clones 3C6 and SP66 respectively), while cMET amplification was examined by in situ hybridization (Ventana, MET DNP and CEN7 DIG probes). EGFR mutations and cMET splice site mutations were detected by Sanger sequencing. Significant correlations were tested using the Chi2 and kappa test (SPSS 23). In 146/155 tumors cMET expression could be determined; 73/146 samples showed high (2+ or 3+ score) cMET expression and 4/118 samples showed amplification (ratio ≥ 2). No significant correlations could be determined between cMET expression and histological subtype of NSCLC (p = 0.065), differentiation degree (p = 0.468) and cMET amplification (p = 0.214). However, a significant correlation was found between cMET expression, EGFR expression (p = 0.015) and EGFR mutations (p = 0.029). Splice site mutation regions were sequenced in 87/155 samples, all of which were wild-type. When comparing cMET amplification between the primary tumor and the corresponding metastases (n = 40), only one sample showed amplification in the metastases and not in the primary tumor. Hence, cMET expression showed a significant correlation between primary tumors and their metastases (kappa = 0.003). The overall results of our study are in agreement with earlier data. Moreover, we showed that overall expression levels of cMET in primary tumors and metastases are very similar despite large intratumor heterogeneity. In conclusion, this study shows that high cMET expression in most NSCLC samples does not originate from presently known genetic cMET aberrations. High cMET expression is likely to be caused by temporary changes in transcription and translation, influenced by EGFR-signaling, miRNAs or other regulatory mechanisms. Citation Format: Nele Van Der Steen, Vanessa Deschoolmeester, Filip Lardon, Christian Rolfo, Elisa Giovannetti, Godefridus J. Peters, Patrick Pauwels. cMET in non-small cell lung cancer: pieces of the puzzle. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2242.

Intro: The use of targeted therapies confers great responses in non-small cell lung cancer patients, but ultimately results in acquired resistance, e.g. against epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) due to cMET amplification. Therefore, combination therapy with EGFR-TKI and cMET TKIs in cell lines with a different EGFR and cMET background was investigated. Materials & methods: All experiments were performed in 5 cell lines with a different EGFR and cMET background: the cMET amplified squamous lung cancer cell line EBC-1, the HGF-secreting myeloma cell line JJN3, the HCC827 and H1975 lung adenocarcinoma cell lines with an activating EGFR exon 19 deletion and an L858 as well as a T790M resistance mutation, respectively. Finally LUDLU-1 is a squamous lung cancer cell line, wild-type for both EGFR and cMET. The cytotoxicity of combination therapy with erlotinib (EGFR-TKI) and crizotinib (cMET-ALK-ROS1-TKI) or PHA665752 (cMET-TKI) was determined by the sulforhodamine B assay after 72h of simultaneous/sequential treatment (Table 1). Results: For all treatments, the JJN3, EBC-1 and HCC827 cell lines showed no synergistic effect. For the H1975, a decrease in resistance could be observed, albeit not significant. For LUDLU-1, a trend towards synergism was observed when erlotinib was administered simultaneously with crizotinib (p = 0.074) and PHA665752 (p = 0.055). Also a significantly synergistic effect was observed when treatment with erlotinib was followed by PHA665752 (p = 0.014). Discussion: Synergism between erlotinib and crizotinib/PHA665752 depends on the genetic background of the cells and the treatment schedule. This was most pronounced in the EGFR and cMET wild-type cell line. These results open up the discussion to include EGFR and cMET wild-type patients into future clinical trials. Cell lineErlotinib (μM)Crizotinib (μM)PHA665752 (μM)Erlo+CrizErlo = >CrizCriz = >ErloErlo+PHAErlo = >PHAPHA = >ErloEBC-10-100-0.025-0.050-0.1-0.150-0.025-0.050-0.1-0.20-0.3-0.40-0.1-0.2JJN30-100-2-30-2-30-2-30-5-70-5-70-5-6HCC8270-50-3-50-3-50-1-20-7-90-7-90-4-5H19750-100-2-30-2-30-1-20-6-70-6-70-2-3LUDLU-10-100-3-50-3-50-2-30-7-80-7-80-3.5-4 Table 1: Therapeutic concentrations per cell line & treatment schedule: control-IC20-IC40. Citation Format: Nele Van Der Steen, Vanessa Deschoolmeester, An Wouters, Filip Lardon, Christian Rolfo, Patrick Pauwels. Combination therapy with EGFR-TKI and cMET-TKIs in non-small cell lung cancer: the golden duo. [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 2593. doi:10.1158/1538-7445.AM2015-2593

Introduction: Tumors are often characterized by inactivating mutations in the TP53 gene. Mutations in this gene occur in about 50% of non-small cell lung cancers (NSCLC), and influence the response to common chemotherapeutics like CDDP (cisplatin). In addition, as hypoxic stress, which is often found in tumors, is able to induce p53 accumulation and p53 dependent apoptosis, the reactivation of mutant p53 might induce a strong cytotoxic effect in hypoxic tumor regions. Therefore, we treated NSCLC tumor cells with APR-246, a known activator of mutant p53, under normoxic and hypoxic conditions. In addition, we combined APR-246 with CDDP, to determine whether reactivation of mutant p53 could enhance the cytotoxic response of CDDP. Materials & Methods: The NSCLC cell line CRL-5908, harboring a R273H p53 mutation, was used. To determine the role of mutant p53 in response to APR-246, CRL-5908 was transduced with a p53 shRNA lentiviral vector to obtain a p53 deficient sub cell line. The SRB-assay was used to determine the cytotoxic effect of both mono-and combination therapies after which possible synergism was calculated by the Chou-Talalay method, both under normoxic and hypoxic conditions (0% O2, 5% CO2, 95% N2). In addition, we determined the p53 protein level as well as the mRNA and protein levels of its main transcription targets MDM2, p21, PUMA and BAX. The induction of apoptosis and cell cycle arrest were determined by flowcytometric analysis. Results: Our results show a clear response of CRL-5908 to APR-246, which was stronger under hypoxic conditions compared to normoxic conditions (IC50: 9.5 ± 2.5 vs. 16.3 ± 3.4). We observed that CRL-5908 was less sensitive to CDDP monotherapy than the p53 wild type cell line A549 (IC50: 15.2 ± 3.3 vs. 6.2 ± 1.6). We were able to overcome this resistance to CDDP by combining the drug with APR-246 (IC20, IC40, IC60), which resulted in CDDP IC50 values of respectively 11.5 ± 4.0; 5.0 ± 2.2 and 6.5 ± 2.0 under normoxic conditions. Discussion: Our initial results show that APR-246 monotherapy is promising for the treatment of p53 mutant NSCLC, particularly under hypoxic conditions. In addition, APR-246 seems to sensitize cells for CDDP treatment when combined simultaneously. We will further study the underlying mechanisms, like the activation of p53 and the transcription of its main transcription target MDM2, p21, PUMA and BAX as well as apoptosis and cell cycle arrest. Moreover, we will compare simultaneous treatment of CDDP and APR-246 with sequential treatment in which CDDP is followed by APR-246 treatment, and further sort out the response of this combination therapy under hypoxic conditions, as well as the role of mutant p53. Citation Format: Christophe Deben, Vanessa Deschoolmeester, An Wouters, Jolien Van den Bossche, Julie Jacobs, Filip Lardon, Christian Rolfo, Patrick Pauwels. APR-246 reactivates mutant p53 in non-small cell lung cancer cell lines and sensitizes cells for CDDP treatment under normoxic and hypoxic conditions. [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 3507. doi:10.1158/1538-7445.AM2015-3507

Introduction: Upon interaction of CD70, a type II transmembrane protein, with its receptor CD27, the NFκB pathway is activated, leading to proliferation and survival. Hence, CD70 expression is tightly regulated and only transient on cells of the lymphoid lineage. In contrast, constitutive overexpression of CD70 has been documented in malignancies, where it appears to mediate immune escape through recruitment of CD27+ regulatory cells. Soluble CD27 (sCD27), the extracellular domain of CD27, can be released after lymphocyte activation and has been detected at increased levels in serum samples of lymphoid malignancies. To our knowledge, expression of CD70 has never been systematically characterized in non-small cell lung cancer (NSCLC). Methods: Fourty-nine primary NSCLC formalin-fixed paraffin embedded and 9 metastatic biopsies were stained by immunohistochemistry (IHC) for expression of CD70 (Clone 301731, 1/40) and CD27 (Clone 137B4, 1/40), using the Dako autostainer Link and Ventana ultra, respectively. Additionally, sCD27 levels were analyzed in 19 serum samples, taken before of biopsy, with the PeliKine Compact™ human sCD27 ELISA kit. Finally, we tested the efficacy of ARGX-110, a CD70-blocking monoclonal IgG1 SIMPLE Antibody™ endowed with enhanced ADCC properties (POTELLIGENT®), using the xCELLigence RTCA technology in CD70+ and CD70- cell lines. Results: In tumor cells, CD70 positivity (>10%) was detected in 16% of cases with a higher percentage in squamous NSCLC (27%) than in adenocarcinoma (9%). Additionally, one neuro-endocrine NSCLC was CD70+. We demonstrated identical CD70 patterns in primary and metastatic tissue in 8/10 biopsies. Furthermore, CD70 was found in the micro-environment of the tumor (23/49). IHC revealed high levels of CD27 expressing tumor infiltrating lymphocytes (TIL) in all NSCLC biopsies, with increasing FOXP3 expression and higher CD4/CD8 ratios in stromal tissue adjacent to CD70+ tumor cells. High sCD27 levels (>500U/ml) were found to be significantly associated with poor overall survival and even though sCD27 levels did not show potential as a blood-based biomarker for CD70 overexpression on tumor cells, dual positivity of CD70 and sCD27 marked even worse prognosis. We also showed that a low concentration of ARGX-110 (0.5μg/ml) induces efficient NK cell based tumor lysis in CD70+ cell lines. Conclusion: Expression of CD70 was demonstrated in tumor cells and TILs of NSCLC. Moreover, all biopsies revealed infiltration of CD27+ TIL in the micro-environment of the tumor and a trend towards increased FOXP3 expression and higher CD4/CD8 ratios in CD70+ biopsies. In addition, serum sCD27 levels have potential as a prognostic marker for NSCLC. Lastly, our in vitro results showed a maximum ADCC of ARGX-110 in CD70+ target cells stained by IHC. Hence, our data suggest that CD70 might be an interesting therapeutic target in NSCLC. Citation Format: Julie Jacobs, Patrick Pauwels, Christian Rolfo, Filip Lardon, Vanessa Deschoolmeester, Christophe Deben, Jolien Van den Bossche, Karen Zwaenepoel, Christophe Hermans, Karen Silence, Alain Thibault. Unlocking the potential of CD70 as a therapeutic target in non-small cell lung cancer. [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 3563. doi:10.1158/1538-7445.AM2015-3563