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The majority of patients with resected stage II‐IIIA non‐small cell lung cancer (NSCLC) are treated with platinum‐based adjuvant chemotherapy (ACT) in a one‐size‐fits‐all approach. However, a significant number of patients do not derive clinical benefit, and no predictive patient selection biomarker is currently available. Using mass spectrometry‐based proteomics, we have profiled tumour resection material of 2 independent, multi‐centre cohorts of in total 67 patients with NSCLC who underwent ACT. Unsupervised cluster analysis of both cohorts revealed a poor response/survival sub‐cluster composed of ~ 25% of the patients, that displayed a strong epithelial‐mesenchymal transition signature and stromal phenotype. Beyond this stromal sub‐population, we identified and validated platinum response prediction biomarker candidates involved in pathways relevant to the mechanism of action of platinum drugs, such as DNA damage repair, as well as less anticipated processes such as those related to the regulation of actin cytoskeleton. Integration with pre‐clinical proteomics data supported a role for several of these candidate proteins in platinum response prediction. Validation of one of the candidates (HMGB1) in a third independent patient cohort using immunohistochemistry highlights the potential of translating these proteomics results to clinical practice.

Abstract In this review, we cover the current understanding of BRAF mutations and associated clinical characteristics in patients with metastatic NSCLC, approved and emerging treatment options, BRAF sequencing approaches, and unmet needs. The BRAFV600E mutation confers constitutive activity of the MAPK pathway, leading to enhanced growth, proliferation, and survival of tumor cells. Testing for BRAF mutations enables patients to be treated with therapies that directly target BRAFV600E and the MAPK pathway, but BRAF testing lags behind other oncogene testing in metastatic NSCLC. Additional therapies targeting BRAFV600E mutations provide options for patients with metastatic NSCLC. Emerging therapies and combinations under investigation could potentially overcome issues of resistance and target non-V600E mutations. Therefore, because targeted therapies with enhanced efficacy are on the horizon, being able to identify BRAF mutations in metastatic NSCLC may become even more important.

Adoptive transfer of tumor infiltrating lymphocytes (TIL therapy) has proven highly effective for treating solid cancers, including non-small cell lung cancer (NSCLC). However, not all patients benefit from this therapy for yet unknown reasons. Defining markers that correlate with high tumor-reactivity of the autologous TIL products is thus key for achieving better tailored immunotherapies. We questioned whether the composition of immune cell infiltrates correlated with the tumor-reactivity of expanded TIL products. Unbiased flow cytometry analysis of immune cell infiltrates of 26 early-stage and 20 late-stage NSCLC tumor lesions was used for correlations with the T cell differentiation and activation status, and with the expansion rate and anti-tumor response of generated TIL products. The composition of tumor immune infiltrates was highly variable between patients. Spearman’s Rank Correlation revealed that high B cell infiltration negatively correlated with the tumor-reactivity of the patient’s expanded TIL products, as defined by cytokine production upon exposure to autologous tumor digest. In-depth analysis revealed that tumor lesions with high B cell infiltrates contained tertiary lymphoid structure (TLS)-related immune infiltrates, including BCL6+ antibody-secreting B cells, IgD+BCL6+ B cells and CXCR5+BLC6+ CD4+ T cells, and higher percentages of naïve CD8+ T cells. In conclusion, the composition of immune cell infiltrates in NSCLC tumors associates with the functionality of the expanded TIL product. Our findings may thus help improve patient selection for TIL therapy.

IntroductionResearch has confirmed the safety and comparable seroconversion rates following SARS-CoV-2 vaccination in patients with solid cancers. However, the impact of cancer treatment on vaccine-induced T cell responses remains poorly understood.MethodsIn this study, we expand on previous findings within the VOICE trial by evaluating the functional and phenotypic composition of mRNA-1273-induced T cell responses in patients with solid tumors undergoing immunotherapy, chemotherapy, or both, compared to individuals without cancer. We conducted an ELISpot analysis on 386 participants to assess spike-specific T cell responses 28 days after full vaccination. Further in-depth characterization of using flow cytometry was performed on a subset of 63 participants to analyze the functional phenotype and differentiation state of spike-specific T cell responses.ResultsELISpot analysis showed robust induction of spike-specific T cell responses across all treatment groups, with response rates ranging from 75% to 80%. Flow cytometry analysis revealed a distinctive cytokine production pattern across cohorts, with CD4 T cells producing IFNγ, TNF, and IL-2, and CD8 T cells producing IFNγ, TNF, and CCL4. Variations were observed in the proportion of monofunctional CD4 T cells producing TNF, particularly higher in individuals without cancer and patients treated with chemotherapy alone, while those treated with immunotherapy or chemoimmunotherapy predominantly produced IFNγ. Despite these differences, polyfunctional spike-specific memory CD4 and CD8 T cell responses were comparable across cohorts. Notably, immunotherapy-treated patients exhibited an expansion of spike-specific CD4 T cells with a terminally differentiated effector memory phenotype.DiscussionThese findings demonstrate that systemic treatment in patients with solid tumors does not compromise the quality of polyfunctional mRNA-1273-induced T cell responses. This underscores the importance of COVID-19 vaccination in patients with solid cancers undergoing systemic treatment.

Introduction: Brain metastases (BM) are common in patients with advanced EGFR-mutated (EGFRm+) NSCLC. Despite good BM-related outcomes of osimertinib, several patients still experience intracranial progression. A possible explanation is pharmacologic failure due to low plasma trough levels (Cmin,SS) and consequently limited intracranial osimertinib exposure. We investigated the relation between osimertinib Cmin,SS and BM development or progression. Methods: A prospective multicenter cohort study, including patients receiving osimertinib for advanced EGFRm+ NSCLC. At osimertinib start, patients were allocated to the BM or no or unknown BM cohort and were further divided into subgroups based on osimertinib Cmin,SS (low, middle, and high exposure). Cumulative incidence of BM progression or development and overall survival were determined for each group. Results: A total of 173 patients were included, with 49 (28.3%) had baseline BM. Of these patients, 36.7% experienced BM progression, of which 16.7% in the low (270.7 ng/mL) Cmin,SS subgroups. After 12 months, the cumulative incidence of BM progression for the BM cohort was 20% (95% confidence interval [CI] 2.6–49.0), 31% (95% CI:10.6–53.9), and 31% (95% CI:10.8–54.5) per Cmin,SS subgroup, respectively. After 20 months, this was 20% (95% CI:2.6–49.0), 52% (95% CI:23.8–74.2), and 57% (95% CI:24.9–79.7), respectively. For the no or unknown BM cohort, 4.0% developed BM without differences within Cmin,SS subgroups. Conclusions: No relation was found between osimertinib Cmin,SS and BM development or progression in patients with advanced EGFRm+ NSCLC. This suggests that systemic osimertinib exposure is not a surrogate marker for BM development or progression.

Summary: Background: The DRUG Access Protocol provides patients with cancer access to registered anti-cancer drugs that are awaiting reimbursement in the Netherlands and simultaneously collects prospective real-world data (RWD). Here, we present RWD from PD-1 blocker cemiplimab in patients with locally advanced or metastatic cutaneous squamous cell carcinoma (laCSCC; mCSCC). Methods: Patients with laCSCC or mCSCC received cemiplimab 350 mg fixed dose every three weeks. Primary endpoints were objective clinical benefit rate (CBR), defined as objective response (OR) or stable disease (SD) at 16 weeks, physician-assessed CBR, defined as clinician’s documentation of improved disease or SD based on evaluation of all available clinical parameters at 16 weeks, objective response rate (ORR), and safety, defined as grade ≥ 3 treatment related adverse events (TRAEs) occurring up to 30 days after last drug administration. Secondary endpoints included duration of response (DoR), progression-free survival (PFS), and overall survival (OS). Findings: Between February 2021 and December 2022, 151 patients started treatment. Objective and physician-assessed CBR were 54.3% (95% CI, 46.0–62.4) and 59.6% (95% CI, 51.3–67.5), respectively. ORR was 35.1% (95% CI, 27.5–43.3). After a median follow-up of 15.2 months, median DoR was not reached. Median PFS and OS were 12.2 (95% CI, 7.0-not reached) and 24.2 months (95% CI, 18.8-not reached), respectively. Sixty-eight TRAEs occurred in 29.8% of patients. Most commonly reported TRAE was a kidney transplant rejection (9.5%). Interpretation: Cemiplimab proved highly effective and safe in this real-world cohort of patients with laCSCC or mCSCC, confirming its therapeutic value in the treatment of advanced CSCC in daily clinical practice. Funding: The DRUG Access Protocol is supported by all participating pharmaceutical companies: Bayer, Janssen, Lilly, Merck, Novartis, Roche, and Sanofi.

IntroductionOsimertinib is recommended by major guidelines for use in the adjuvant setting in patients with EGFR mutation-positive NSCLC following the significant improvement in disease-free survival observed in the Phase III ADAURA trials. Due to limited real-world data in the adjuvant setting, little guidance exists on how to approach potential recurrences either during or after the completion of the treatment. This study aimed to reach a broad consensus on key treatment decision criteria in the events of recurrence.MethodsTo reach a broad consensus, a modified Delphi panel study was conducted consisting of two rounds of surveys, followed by two consensus meetings and a final offline review of key statements. An international panel of experts in the field of NSCLC (n=12) was used to provide clinical insights regarding patient management at various stages of NSCLC disease including patient monitoring, diagnostics, and treatment approach for specific recurrence scenarios. This study tested recurrences occurring 1) within or outside the central nervous system (CNS), 2) during or after the adjuvant-osimertinib regimen in NSCLC disease which is 3) amenable or not amenable to local consolidative therapy.ResultsPanellists agreed on various aspects of patient monitoring and diagnostics including the use of standard techniques (e.g., CT, MRI) and tumour biomarker assessment using tissue and liquid biopsies. Consensus was reached on 6 statements describing treatment considerations for the specific NSCLC recurrence scenarios. Panellists agreed on the value of osimertinib as a monotherapy or as part of the overall treatment strategy within the probed recurrence scenarios and acknowledged that more clinical evidence is required before precise recommendations for specific patient populations can be made.DiscussionThis study provides a qualitative expert opinion framework for clinicians to consider within their treatment decision-making when faced with recurrence during or after adjuvant-osimertinib treatment.

ABSTRACTBetter biomarkers for programmed death - (ligand) 1 (PD-(L)1) checkpoint blockade in non-small cell lung cancer (NSCLC) are needed. We explored the predictive value of early response evaluation using Fluor-18-deoxyglucose positron emission tomography and pre- and on-treatment flowcytometric T-cell profiling in peripheral blood and tumor-draining lymph nodes (TDLN). The on-treatment evaluation was performed 7–14 days after the start of PD-1 blockade in NSCLC patients. These data were related to (pathological) tumor response, progression-free survival, and overall survival (OS). We found that increases in total lesion glycolysis (TLG) had a strong reverse correlation with OS (r = −0.93, p = 0.022). Additionally, responders showed decreased and progressors increased Treg frequencies on-treatment. Frequencies of detectable PD-1-expressing CD8+ T cells decreased in responders but remained stable in progressors. This was especially found in the TDLN. Changes in activated Treg rates in TDLN were strongly but, due to low numbers of data points, non-significantly correlated with ΔTLG and reversely correlated with OS.

Abstract Background This study was designed to investigate the impact of age on the effectiveness and immune-related adverse events (irAEs) of programmed death-(ligand)1 [PD-(L)1] inhibitors in patients with non-small cell lung cancer (NSCLC) using a novel text-mining technique. Methods This retrospective study included patients with stage III/IV NSCLC treated with a PD-(L)1 inhibitor (nivolumab, pembrolizumab, atezolizumab and durvalumab) at Leiden University Medical Centre and Haga Teaching hospital, (both in The Netherlands) from September 2016 to May 2021. All the relevant data was extracted from the structured and unstructured fields of the Electronic Health Records using a novel text-mining tool. Effectiveness [progression-free survival (PFS) and overall survival (OS)] and safety (the incidence of nine potentially fatal irAEs and systemic corticosteroid requirement) outcomes were compared across age subgroups (young:

Summary: Background: Central nervous system (CNS) metastases are present in approximately 40% of patients with metastatic epidermal growth factor receptor-mutated (EGFRm+) non-small cell lung cancer (NSCLC). The EGFR-tyrosine kinase inhibitor osimertinib is a substrate of transporters ABCB1 and ABCG2 and metabolized by CYP3A4. We investigated relationships between single nucleotide polymorphisms (SNPs) ABCB1 3435C>T, ABCG2 421C>A and 34G>A, and CYP3A4∗22 and CNS treatment efficacy of osimertinib in EGFRm+ NSCLC patients. Methods: Patients who started treatment with osimertinib for EGFRm+ NSCLC between November 2014 and June 2021 were included in this retrospective observational multicentre cohort study. For patients with baseline CNS metastases, the primary endpoint was CNS progression-free survival (CNS-PFS; time from osimertinib start until CNS disease progression or death). For patients with no or unknown baseline CNS metastases, the primary endpoint was CNS disease-free survival (CNS-DFS; time from osimertinib start until occurrence of new CNS metastases). Relationships between SNPs and baseline characteristics with CNS-PFS and CNS-DFS were studied with competing-risks survival analysis. Secondary endpoints were relationships between SNPs and PFS, overall survival, severe toxicity, and osimertinib pharmacokinetics. Findings: From 572 included patients, 201 had baseline CNS metastases. No SNP was associated with CNS-PFS. Genotype ABCG2 34GA/AA and/or ABCB1 3435CC --present in 35% of patients-- was significantly associated with decreased CNS-DFS (hazard ratio 0.28; 95% CI 0.11–0.73; p = 0.009) in the multivariate analysis. This remained significant after applying a Bonferroni correction and internal validation through bootstrapping. ABCG2 421CA/AA was related to more severe toxicity (27.0% versus 16.5%; p = 0.010). Interpretation: ABCG2 34G>A and ABCB1 3435C>T are predictors for developing new CNS metastases during osimertinib treatment, probably because of diminished drug levels in the CNS. ABCG2 421C>A was significantly related with the incidence of severe toxicity. Pre-emptive genotyping for these SNPs could individualize osimertinib therapy. Addition of ABCG2 inhibitors for patients without ABCG2 34G>A should be studied further, to prevent new CNS metastases during osimertinib treatment. Funding: No funding was received for this trial.

BackgroundAlectinib is first-line therapy in patients with stage IV non-small cell lung carcinoma (NSCLC) and an anaplastic lymphoma kinase (ALK) fusion. A shorter median progression-free survival (mPFS) was observed when alectinib minimum plasma concentrations during steady state (Cmin,SS) were below 435 ng/mL. This may suggest that patients should have an alectinib Cmin,SS ≥ 435 ng/mL for a more favorable outcome. This potential target could be attained by using therapeutic drug monitoring (TDM), i.e. adjusting the dose based on measured plasma trough concentrations. Hypothetically, this will increase mPFS, but this has not yet been evaluated in a randomized controlled trial (RCT). Therefore, the ADAPT ALEC trial is designed, with the primary objective to prolong mPFS in NSCLC patients treated with alectinib by using TDM.MethodsADAPT ALEC is a multicenter, phase IV RCT, in which patients aged ≥ 18 years with advanced ALK positive (+) NSCLC eligible for alectinib in daily care are enrolled. Participants will be randomized (1:1 ratio) into intervention arm A (TDM) or B (control), stratified by brain metastases and prior ALK treatments. Starting dose in both arms is the approved flat fixed dose of alectinib 600 mg taken twice daily with food. In case of alectinib Cmin,SS < 435 ng/mL, arm A will receive increased doses of alectinib till Cmin,SS ≥ 435 ng/mL when considered tolerable. The primary outcome is mPFS, where progressive disease is defined according to RECIST v1.1 or all-cause death and assessed by CT-scans and MRI brain. Secondary endpoints are feasibility and tolerability of TDM, patient and physician adherence, overall response rate, median overall survival, intracranial PFS, quality of life, toxicity, alectinib-M4 concentrations and cost-effectiveness of TDM. Exploratory endpoints are circulating tumor DNA and body composition.DiscussionThe ADAPT ALEC will show whether treatment outcomes of patients with advanced ALK+ NSCLC improve when using TDM-guided dosing of alectinib instead of fixed dosing. The results will provide high quality evidence for deciding whether TDM should be implemented as standard of care and this will have important consequences for the prescribing of alectinib.Clinical trial registrationClinicalTrials.gov, identifier NCT05525338.

Introduction Previous studies have shown interference between epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors and chemotherapy in the cell cycle, thus reducing efficacy. In this randomised controlled trial we investigated whether intercalated erlotinib with chemotherapy was superior compared to erlotinib alone in untreated advanced EGFR-mutated nonsmall cell lung cancer (NSCLC). Materials and methods Treatment-naïve patients with an activating EGFR mutation, ECOG performance score of 0–3 and adequate organ function were randomly assigned 1:1 to either four cycles of cisplatin-pemetrexed with intercalated erlotinib (day 2–16 out of 21 days per cycle) followed by pemetrexed and erlotinib maintenance (CPE) or erlotinib monotherapy. The primary end-point was progression-free survival (PFS). Secondary end-points were overall survival, objective response rate (ORR) and toxicity. Results Between April 2014 and September 2016, 22 patients were randomised equally into both arms; the study was stopped due to slow accrual. Median follow-up was 64 months. Median PFS was 13.7 months (95% CI 5.2–18.8) for CPE and 10.3 months (95% CI 7.1–15.5; hazard ratio (HR) 0.62, 95% CI 0.25–1.57) for erlotinib monotherapy; when compensating for number of days receiving erlotinib, PFS of the CPE arm was superior (HR 0.24, 95% CI 0.07–0.83; p=0.02). ORR was 64% for CPE versus 55% for erlotinib monotherapy. Median overall survival was 31.7 months (95% CI 21.8–61.9 months) for CPE compared to 17.2 months (95% CI 11.5–45.5 months) for erlotinib monotherapy (HR 0.58, 95% CI 0.22–1.41 months). Patients treated with CPE had higher rates of treatment-related fatigue, anorexia, weight loss and renal toxicity. Conclusion Intercalating erlotinib with cisplatin-pemetrexed provides a longer PFS compared to erlotinib alone in EGFR-mutated NSCLC at the expense of more toxicity.

Introduction: We report brigatinib long-term efficacy and safety from phase 1/2 and phase 2 (ALTA) trials in ALK–rearrangement positive (ALK+) NSCLC. Methods: The phase 1/2 study evaluated brigatinib 30 to 300 mg/d in patients with advanced malignancies. ALTA randomized patients with crizotinib-refractory ALK+ NSCLC to brigatinib 90 mg once daily (arm A) or 180 mg once daily (7-d lead-in at 90 mg; arm B). Results: In the phase 1/2 study, 79 of 137 brigatinib-treated patients had ALK+ NSCLC; 71 were crizotinib pretreated. ALTA randomized 222 patients (n = 112 in arm A; n = 110 in arm B). Median follow-up at phase 1/2 study end (≈5.6 y after last patient enrolled) was 27.7 months; at ALTA study end (≈4.4 y after last patient enrolled), 19.6 months (A) and 28.3 months (B). Among patients with ALK+ NSCLC in the phase 1/2 study, median investigator-assessed progression-free survival (PFS) was 14.5 months (95% confidence interval [CI]: 10.8–21.2); median overall survival was 47.6 months (28.6–not reached). In ALTA, median investigator-assessed PFS was 9.2 months (7.4–11.1) in arm A and 15.6 months (11.1–18.5) in arm B; median independent review committee (IRC)-assessed PFS was 9.9 (7.4–12.8) and 16.7 (11.6–21.4) months, respectively; median overall survival was 25.9 (18.2–45.8) and 40.6 (32.5–not reached) months, respectively. Median intracranial PFS for patients with any brain metastases was 12.8 (9.2–18.4) months in arm A and 18.4 (12.6–23.9) months in arm B. No new safety signals were identified versus previous analyses. Conclusions: Brigatinib exhibited sustained long-term activity and PFS with manageable safety in patients with crizotinib-refractory ALK+ NSCLC.

Introduction: With the approval of first-line osimertinib treatment in stage IV EGFR-mutated NSCLC, detection of resistance mechanisms will become increasingly important—and complex. Clear guidelines for analyses of these resistance mechanisms are currently lacking. Here, we provide our recommendations for optimal molecular diagnostics in the post-EGFR tyrosine kinase inhibitor (TKI) resistance setting. Methods: We compared molecular workup strategies from three hospitals of 161 first- or second-generation EGFR TKI–treated cases and 159 osimertinib-treated cases. Laboratories used combinations of DNA next-generation sequencing (NGS), RNA NGS, in situ hybridization (ISH), and immunohistochemistry (IHC). Results: Resistance mechanisms were identified in 72 first-generation TKI cases (51%) and 85 osimertinib cases (57%). RNA NGS, when performed, revealed fusions or exon-skipping events in 4% of early TKI cases and 10% of osimertinib cases. Of the 30 MET and HER2 amplifications, 10 were exclusively detected by ISH or IHC, and not detected by DNA NGS, mostly owing to low tumor cell percentage (

Abstract Background Total metabolic active tumour volume (TMATV) and total tumour burden (TTB) are increasingly studied as prognostic and predictive factors in non-small cell lung cancer (NSCLC) patients. In this study, we investigated the repeatability of TMATV and TTB as function of uptake interval, positron emission tomography/computed tomography (PET/CT) image reconstruction settings, and lesion delineation method. We used six lesion delineation methods, four direct PET image-derived delineations and two based on a majority vote approach, i.e. intersection between two or more delineations (MV2) and between three or more delineations (MV3). To evaluate the accuracy of those methods, they were compared with a reference delineation obtained from the consensus of the segmentations performed by three experienced observers. Ten NSCLC patients underwent two baseline whole-body [18F]2-Fluoro-2-deoxy-2-D-glucose ([18F]FDG) PET/CT studies on separate days, within 3 days. Two scans were obtained on each day at 60 and 90 min post-injection to assess the influence of tracer uptake interval. PET/CT images were reconstructed following the European Association of Nuclear Medicine Research Ltd. (EARL) compliant settings and with point-spread-function (PSF) modelling. Repeatability between the measurements of each day was determined and the influence of uptake interval, reconstruction settings, and lesion delineation method was assessed using the generalized estimating equations model. Results Based on the Jaccard index with the reference delineation, the MV2 lesion delineation method was the most successful method for automated lesion segmentation. The best overall repeatability (lowest repeatability coefficient, RC) was found for TTB from 90 min of tracer uptake scans reconstructed with EARL compliant settings and delineated with 41% of lesion’s maximum SUV method (RC = 11%). In most cases, TMATV and TTB repeatability were not significantly affected by changes in tracer uptake time or reconstruction settings. However, some lesion delineation methods had significantly different repeatability when applied to the same images. Conclusions This study suggests that under some circumstances TMATV and TTB repeatability are significantly affected by the lesion delineation method used. Performing the delineation with a majority vote approach improves reliability and does not hamper repeatability, regardless of acquisition and reconstruction settings. It is therefore concluded that by using a majority vote based tumour segmentation approach, TMATV and TTB in NSCLC patients can be measured with high reliability and precision.

BackgroundCheckpoint inhibitors provided sustained clinical benefit to metastatic lung cancer patients. Nonetheless, prognostic markers in metastatic settings are still under research. Imaging offers distinctive advantages, providing whole-body information non-invasively, while routinely available in most clinics. We hypothesized that more prognostic information can be extracted by employing artificial intelligence (AI) for treatment monitoring, superior to 2D tumor growth criteria.MethodsA cohort of 152 stage-IV non-small-cell lung cancer patients (NSCLC) (73 discovery, 79 test, 903CTs), who received nivolumab were retrospectively collected. We trained a neural network to identify morphological changes on chest CT acquired during patients’ follow-ups. A classifier was employed to link imaging features learned by the network with overall survival.ResultsOur results showed significant performance in the independent test set to predict 1-year overall survival from the date of image acquisition, with an average area under the curve (AUC) of 0.69 (p < 0.01), up to AUC 0.75 (p < 0.01) in the first 3 to 5 months of treatment, and 0.67 AUC (p = 0.01) for durable clinical benefit (6 months progression-free survival). We found the AI-derived survival score to be independent of clinical, radiological, PDL1, and histopathological factors. Visual analysis of AI-generated prognostic heatmaps revealed relative prognostic importance of morphological nodal changes in the mediastinum, supraclavicular, and hilar regions, lung and bone metastases, as well as pleural effusions, atelectasis, and consolidations.ConclusionsOur results demonstrate that deep learning can quantify tumor- and non–tumor-related morphological changes important for prognostication on serial imaging. Further investigation should focus on the implementation of this technique beyond thoracic imaging.

Introduction: The TIGER-3 (NCT02322281) study was initiated to compare the efficacy and safety of rociletinib, a third-generation EGFR tyrosine kinase inhibitor (TKI) that targets EGFR T790M and common EGFR-activating mutations, versus chemotherapy in patients with NSCLC who progressed on first- or second-generation EGFR TKIs. Methods: Patients with advanced or metastatic EGFR-mutated NSCLC with disease progression on standard therapy (previous EGFR TKI and platinum-based chemotherapy) were randomized to oral rociletinib (500 or 625 mg twice daily) or single-agent chemotherapy (pemetrexed, gemcitabine, docetaxel, or paclitaxel). Results: Enrollment was halted when rociletinib development was discontinued in 2016. Of 149 enrolled patients, 75 were randomized to rociletinib (n = 53: 500 mg twice daily; n = 22: 625 mg twice daily) and 74 to chemotherapy. The median investigator-assessed progression-free survival (PFS) was 4.1 months (95% confidence interval [CI]: 2.6–5.4) in the rociletinib 500-mg group and 5.5 months (95% CI: 1.8–8.1) in the 625-mg group versus 2.5 months (95% CI: 1.4–2.9) in the chemotherapy group. An improved PFS was observed in patients with T790M-positive NSCLC treated with rociletinib (n = 25; 500 mg and 625 mg twice daily) versus chemotherapy (n = 20; 6.8 versus 2.7 mo; hazard ratio = 0.55, 95% CI: 0.28–1.07, p = 0.074). Grade 3 or higher hyperglycemia (24.0%), corrected QT prolongation (6.7%), diarrhea (2.7%), and vomiting (1.3%) were more frequent with rociletinib than chemotherapy (0%, 0%, 1.4%, and 0%, respectively). Conclusions: Rociletinib had a more favorable median PFS versus chemotherapy but had higher rates of hyperglycemia and corrected QT prolongation in patients with advanced EGFR-mutated NSCLC who progressed on previous EGFR TKI. Incomplete enrollment prevented evaluation of the primary efficacy end point.

Summary: Clinical implementation of tumor organoids for personalized medicine requires that pure tumor organoids can be reliably established. Here, we present our experience with organoid cultures from >70 non-small cell lung cancer (NSCLC) samples. We systematically evaluate several methods to identify tumor purity of organoids established from intrapulmonary tumors. Eighty percent of organoids from intrapulmonary lesions have a normal copy number profile, suggesting overgrowth by normal airway organoids (AOs). This is further supported by the failure to detect mutations found in the original tumor in organoids. Histomorphology alone is insufficient to determine tumor purity, but when combined with p63 immunostaining, tumor and normal AOs can be distinguished. Taking into account overgrowth by normal AOs, the establishment rate of pure NSCLC organoids is 17%. Therefore, current methods are insufficient to establish pure NSCLC organoids from intrapulmonary lesions. We discourage their use unless steps are taken to prevent overgrowth by normal AOs.

Acquired resistance to targeted drugs remains a major clinical challenge in lung adenocarcinoma patients. Here, the authors show how the acquired EGFR G724S mutation induces resistance to third-generation EGFR inhibitors and why the mutant kinase remains susceptible to second-generation inhibitors.

Background Immune checkpoint inhibitors have emerged as a standard of care treatment for non-small cell lung cancer (NSCLC). To get insight into variations in tumour growth kinetics and their potential predictive values for outcome, we evaluated tumour growth rate (TGR) in patients receiving programmed cell death 1 (PD-1) checkpoint inhibitors. Patients and methods Differences in TGR before and after the start of treatment were calculated by entering the sum of the longest diameters from computer tomography scans before and after the initiation of therapy into a formula that assumes volumetric exponential tumour growth. TGR variations, possible predictors for TGR changes and its relationship to overall survival (OS) were studied. For comparison, tumour response was assessed using Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1. Results Among the 58 evaluable patients, 37 patients (64%) showed deceleration of TGR and 16 patients (27%) showed an acceleration of TGR after initiation of therapy, with a significant difference in median OS of 18.0 months versus 6.0 months (hazard ratio 0.35, 95% CI 0.18–0.71) between these groups. Four patients (7%) were defined as having hyperprogressive disease. In five patients (9%), tumour growth remained stable. These TGR categories were not significantly different according to age, sex, histology, smoking or previous radiotherapy. Of the patients defined as having progressive disease by RECIST version 1.1 at first follow-up, 40% showed response to checkpoint inhibitors by a decrease in TGR. Conclusion Tumour growth kinetics can be used as a clinically relevant predictor for OS in anti-PD-1-treated patients with NSCLC, and may provide additional information to RECIST measurements.

Objectives Molecular profiling of tumours has become the mainstay of diagnostics for metastasised solid malignancies and guides personalised treatment, especially in nonsmall cell lung cancer (NSCLC). In current practice, it is often challenging to obtain sufficient tumour material for reliable molecular analysis. Cell-free DNA (cfDNA) in blood or other bio-sources could present an alternative approach to obtain genetic information from the tumour. In a retrospective cohort we analysed the added value of cfDNA analysis in pleural effusions for molecular profiling. Methods We retrospectively analysed both the supernatant and the cell pellet of 44 pleural effusions sampled from 39 stage IV patients with KRAS (n=23) or EGFR (n=16) mutated tumours to detect the original driver mutation as well as for EGFR T790M resistance mutations. Patients were diagnosed with either NSCLC (n=32), colon carcinoma (n=4), appendiceal carcinoma (n=2) or adenocarcinoma of unknown primary (n=1). Samples collected in the context of routine clinical care were stored at the Netherlands Cancer Institute biobank. We used droplet digital PCR for analysis. Results The driver mutation could be detected in 36 of the 44 pleural effusions by analysis of both the supernatant (35 out of 44 positive) and the cell pellet (31 out of 44 positive). In seven out of 20 pleural effusions from patients with EGFR mutation-positive tumours, a T790M mutation was detected. All seven supernatants and cell pellets were positive. Conclusions cfDNA in pleural effusion can be used to detect driver mutations as well as resistance mechanisms like EGFR T790M in pleural effusion with high accuracy and is therefore a valuable bio-source.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor, and no effective treatment is available to date. Mouse models of SCLC based on the inactivation of Rb1 and Trp53 show frequent amplifications of the Nfib and Mycl genes. Here, we report that, although overexpression of either transcription factor accelerates tumor growth, NFIB specifically promotes metastatic spread. High NFIB levels are associated with expansive growth of a poorly differentiated and almost exclusively E-cadherin (CDH1)-negative invasive tumor cell population. Consistent with the mouse data, we find that NFIB is overexpressed in almost all tested human metastatic high-grade neuroendocrine lung tumors, warranting further assessment of NFIB as a tumor progression marker in a clinical setting.

Extracellular vesicles (EVs) are cell-secreted membrane vesicles enclosed by a lipid bilayer derived from endosomes or from the plasma membrane. Since EVs are released into body fluids, and their cargo includes tissue-specific and disease-related molecules, they represent a rich source for disease biomarkers. However, standard ultracentrifugation methods for EV isolation are laborious, time-consuming, and require high inputs. Ghosh and co-workers recently described an isolation method utilizing Heat Shock Protein (HSP)-binding peptide Vn96 to aggregate HSP-decorated EVs, which can be performed at small ‘miniprep’ scale. Based on microscopic, immunoblot, and RNA sequencing analyses this method compared well with ultracentrifugation-mediated EV isolation, but a detailed proteomic comparison was lacking. Therefore, we compared both methods using label-free proteomics of replicate EV isolations from HT-29 cell-conditioned medium. Despite a 30-fold different scale (ultracentrifugation: 60 ml/Vn96-mediated aggregation: 2 ml) both methods yielded comparable numbers of identified proteins (3115/3085), with similar reproducibility of identification (72.5%/75.5%) and spectral count-based quantification (average CV: 31%/27%). EV fractions obtained with either method contained established EV markers and proteins linked to vesicle-related gene ontologies. Thus, Vn96 peptide-mediated aggregation is an advantageous, simple and rapid approach for EV isolation from small biological samples, enabling high-throughput analysis in a biomarker discovery setting.

Background: Extracellular vesicles (EVs) are small nanometre-sized vesicles that are circulating in blood. They are released by multiple cells, including tumour cells. We hypothesized that circulating EVs contain protein kinases that may be assessed as biomarkers during treatment with tyrosine kinase inhibitors. Methods: EVs released by U87 glioma cells, H3255 and H1650 non-small-cell lung cancer (NSCLC) cells were profiled by tandem mass spectrometry. Total AKT/protein kinase B and extracellular signal regulated kinase 1/2 (ERK1/2) levels as well as their relative phosphorylation were measured by western blot in isogenic U87 cells with or without mutant epidermal growth factor receptor (EGFRvIII) and their corresponding EVs. To assess biomarker potential, plasma samples from 24 healthy volunteers and 42 patients with cancer were used. Results: In total, 130 different protein kinases were found to be released in EVs including multiple drug targets, such as mammalian target of rapamycin (mTOR), AKT, ERK1/2, AXL and EGFR. Overexpression of EGFRvIII in U87 cells results in increased phosphorylation of EGFR, AKT and ERK1/2 in cells and EVs, whereas a decreased phosphorylation was noted upon treatment with the EGFR inhibitor erlotinib. EV samples derived from patients with cancer contained significantly more protein (p=0.0067) compared to healthy donors. Phosphorylation of AKT and ERK1/2 in plasma EVs from both healthy donors and patients with cancer was relatively low compared to levels in cancer cells. Preliminary analysis of total AKT and ERK1/2 levels in plasma EVs from patients with NSCLC before and after sorafenib/metformin treatment (n=12) shows a significant decrease in AKT levels among patients with a favourable treatment response (p

The treatment of advanced nonsmall cell lung cancer (NSCLC) with PD-1/PD-L1 immune checkpoint inhibitors has improved clinical outcome for a proportion of patients. The current challenge is to find better biomarkers than PD-L1 immunohistochemistry (IHC) that will identify patients likely to benefit from this therapy. In this exploratory study we assessed the differences in T-cell subsets and PD-1 expression levels on T-cells in tumour-draining lymph nodes (TDLNs) and peripheral blood mononuclear cells (PBMCs). To evaluate this, flow cytometric analyses were performed on endobronchial ultrasound-guided (EBUS) fine-needle aspirates (FNA) from TDLNs of patients with NSCLC, and the results were compared to paired PBMC samples. For a select number of patients, we were also able to obtain cells from a non-TDLN (NTDLN) sample. Our data show that the frequency of PD-1+ CD4+ and CD8+ T-cells, as well as the PD-1 expression level on activated regulatory T (aTreg) and CD4+ and CD8+ T-cells, are higher in TDLNs than in PBMCs and, in a small sub-analysis, NTDLNs. These elevated PD-1 expression levels in TDLNs may reflect tumour-specific T-cell priming and conditioning, and may serve as a predictive or early-response biomarker during PD-1 checkpoint blockade.

Lung adenocarcinoma is known for its high rate of somatic mutations and genomic rearrangements. The identification of epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements that sensitize tumors to specific drugs has changed the therapeutic approach and prognosis in these molecularly-defined subgroups. Several other key genetic alterations have been identified, of which BRAF mutations are found in 4% of non-small cell lung cancer (NSCLC) cases. Targeted drugs against BRAF and downstream MEK were recently approved for the treatment of BRAF-positive melanoma and have entered clinical evaluation in NSCLC. In this review we discuss the latest evidence on the treatment of BRAF-mutated NSCLC, including tumor biology, targeted treatment with BRAF and MEK inhibitors, therapeutic resistance and strategies to overcome resistance.

There are no effective therapies for the ∼30% of human malignancies with mutant RAS oncogenes. Using a kinome-centered synthetic lethality screen, we find that suppression of the ERBB3 receptor tyrosine kinase sensitizes KRAS mutant lung and colon cancer cells to MEK inhibitors. We show that MEK inhibition results in MYC-dependent transcriptional upregulation of ERBB3, which is responsible for intrinsic drug resistance. Drugs targeting both EGFR and ERBB2, each capable of forming heterodimers with ERBB3, can reverse unresponsiveness to MEK inhibition by decreasing inhibitory phosphorylation of the proapoptotic proteins BAD and BIM. Moreover, ERBB3 protein level is a biomarker of response to combinatorial treatment. These data suggest a combination strategy for treating KRAS mutant colon and lung cancers and a way to identify the tumors that are most likely to benefit from such combinatorial treatment.

Background: The finding of a renal mass on imaging is suggestive of metastatic non-small cell lung cancer in the presence of a lung tumor but can also have another origin. Case Report: We describe the case of a patient diagnosed with stage IV lung cancer based on a renal metastasis. A second opinion including review of histopathological data and additional imaging followed by lung surgery and cryoablation of the kidney lesion revealed two tumors of different origins, non-small cell lung cancer and a renal cell carcinoma. Discussion: The presence of a renal mass diagnosed on a CT scan in a patient with lung cancer is not always synonymous with metastatic disease. Confirmation of diagnosis by tissue sampling is mandatory, especially if a synchronous primary tumor is possible.

Polycomb group (PcG) genes are responsible for maintenance of cellular identity and contribute to regulation of the cell cycle. Recent studies have identified several PcG genes as oncogenes, and a role for PcG proteins in human oncogenesis is suspected. We investigated the expression of BMI-1 and EZH2 PcG oncogenes in human bronchial squamous cell carcinomas (SCCs) and bronchial premalignant precursor lesions (PLs). Whereas normal bronchial epithelium was associated with widespread expression of BMI-1 in resting EZH2-negative cells, neoplastic cells in lung carcinomas displayed altered expression of both BMI-1 and EZH2. Two patterns of abnormal PcG expression were observed: increased expression of BMI-1 in dividing neoplastic cells of PLs and SCCs, and enhanced expression of EZH2 and Ki-67 in BMI-1-positive cells according to severity of the histopathologic stage. We propose that altered expression of BMI1 and EZH2 is an early event that precedes high rates of proliferation in lung cancer. Because PcG complexes are normally involved in the maintenance of cell characteristics, abnormal PcG expression may contribute to loss of cell identity.

Background: Mediastinal lymphadenopathy in combination with lung cancer is suggestive for lymph node metastases but can also have other origins. Case report: We describe a patient diagnosed with stage IV lung cancer presenting with parenchymal lesions and enlarged mediastinal lymph nodes. A second opinion including FDG-PET scan review and a mediastinoscopy followed by surgery revealed tumor specimens originating from a single primary tumor with a sarcoid-like reaction in the mediastinal lymph nodes, changing the diagnosis from metastasized to resectable lung cancer. Discussion: PET positive lesions are not always synonymous with metastatic disease in the presence of a malignant tumor. Conscientious review of FDG-PET scans and tissue sampling are therefore mandatory to determine definitive staging and subsequent interventions.

A therapeutic plateau seems to have been reached with the standard treatment of cytotoxic chemotherapy alone for advanced stage non-small cell lung cancer (NSCLC) and new treatment options are urgently needed. Recent insight into the molecular biology of cancer has identified angiogenesis as one of the key biological processes. The major player in tumor angiogenesis is the vascular endothelial growth factor (VEGF) pathway. VEGF is expressed in the majority of NSCLC and overexpression is associated with a poor prognosis. The VEGF pathway can be inhibited in two main ways: targeting VEGF directly or inhibiting the VEGF receptors. The development of angiogenesis inhibitors has shown great promise in the treatment of NSCLC. Bevacizumab, an anti-VEGF antibody, has been approved for the treatment of advanced NSCLC and other drugs are undergoing phase III investigation. However, a number of unresolved issues remain. In this review, we discuss the main angiogenesis inhibitors in development for the treatment of NSCLC focusing on the VEGF pathway.