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BACKGROUND: Circulating tumour cells (CTCs) can be used to monitor cancer longitudinally, but their use in non-small cell lung cancer (NSCLC) is limited due to low numbers in the peripheral blood. Through diagnostic leukapheresis (DLA) CTCs can be obtained from larger blood volumes.METHODS: Patients with all stages of NSCLC were selected. One total body blood volume was screened by DLA before and after treatment. Peripheral blood was drawn pre- and post DLA for CTC enumeration by CellSearch. CTCs were detected in the DLA product (volume equalling 2 × 108 leucocytes) and after leucocyte depletion (RosetteSep, 9 mL DLA product). Single-cell, whole-genome sequencing was performed on isolated CTCs.RESULTS: Fifty-six patients were included. Before treatment, CTCs were more often detected in DLA (32/55, 58%) than in the peripheral blood (pre-DLA: 18/55, 33%; post DLA: 13/55, 23%, both at p CONCLUSIONS: DLA detected nine times more CTCs than in the peripheral blood. The sustained presence of CTCs in DLA after treatment was associated with therapy failure and shortened PFS.TRIAL REGISTRATION: The study was approved by the Medical Ethical Committee (NL55754.042.15) and was registered in the Dutch trial register (NL5423).

BACKGROUND: The number of solitary pulmonary nodules to be evaluated is expected to increase and therefore we need to improve diagnostic and therapeutic tools to approach these nodules. To prevent patients from futile invasive procedures and receiving treatment without histological confirmation of cancer, we evaluated the value of virtual bronchoscopy navigation to obtain a diagnosis of the solitary pulmonary nodule in a real-world clinical setting.METHODS: In the NAVIGATOR single center, prospective, observational cohort study patients underwent a virtual bronchoscopy navigation procedure with or without guide sheet tunnelling to assess a solitary pulmonary nodule. Nodules were considered not accessible if a diagnosis could not be obtained by either by CT-guided transthoracic biopsy or conventional bronchoscopy.RESULTS: Between February 2021 and January 2022 35 patients underwent the virtual bronchoscopy navigation procedure. The overall diagnostic yield was 77% and was dependent on size of the nodule and chosen path, with highest yield in lesions with an airway path. Adverse events were few and manageable.CONCLUSION: Virtual bronchoscopy navigation with or without sheet tunnelling is a new technique with a good diagnostic yield, also in patients in whom previously performed procedures failed to establish a diagnosis and/or alternative procedures are considered not feasible based on expected yield and/or safety. Preventing futile or more invasive procedures like surgery or transthoracic punctures with a higher complication rate is beneficial for patients, and allowed treatment adaptation in two-third of the analyzed patient population.

Introduction: The phase 3 PACIFIC trial established consolidation therapy with durvalumab as standard of care for patients with unresectable, stage III NSCLC and no disease progression after definitive chemoradiotherapy (CRT). The observational PACIFIC-R study assesses the real-world effectiveness of durvalumab in patients from an early access program. Here, we report treatment characteristics and a preplanned analysis of real-world progression-free survival (rwPFS).Methods: PACIFIC-R (NCT03798535) is an ongoing, international, retrospective study of patients who started durvalumab (intravenously; 10 mg/kg every 2 wk) within an early access program between September 2017 and December 2018. The primary end points are investigator-assessed rwPFS and overall survival (analyzed by Kaplan–Meier method).Results: As of November 30, 2020, the full analysis set comprised 1399 patients from 11 countries (median follow-up duration, 23.5 mo). Patients received durvalumab for a median of 11.0 months. Median rwPFS was 21.7 months (95% confidence interval: 19.1–24.5). RwPFS was numerically longer among patients who received concurrent versus sequential CRT (median, 23.7 versus 19.3 mo) and among patients with programmed cell death-ligand 1 expression greater than or equal to 1% versus less than 1% (22.4 versus 15.6 mo). Overall, 16.5% of the patients had adverse events leading to treatment discontinuation; 9.5% of all patients discontinued because of pneumonitis or interstitial lung disease.Conclusions: Consolidation durvalumab after definitive CRT was well tolerated and effective in this large, real-world cohort study of patients with unresectable, stage III NSCLC. As expected, rwPFS was longer among patients who received concurrent versus sequential CRT and patients with higher programmed cell death-ligand 1 expression. Nevertheless, favorable rwPFS outcomes were observed regardless of these factors.

BACKGROUND: Patients with cancer have an increased risk of complications from SARS-CoV-2 infection. Vaccination to prevent COVID-19 is recommended, but data on the immunogenicity and safety of COVID-19 vaccines for patients with solid tumours receiving systemic cancer treatment are scarce. Therefore, we aimed to assess the impact of immunotherapy, chemotherapy, and chemoimmunotherapy on the immunogenicity and safety of the mRNA-1273 (Moderna Biotech, Madrid, Spain) COVID-19 vaccine as part of the Vaccination Against COVID in Cancer (VOICE) trial.METHODS: This prospective, multicentre, non-inferiority trial was done across three centres in the Netherlands. Individuals aged 18 years or older with a life expectancy of more than 12 months were enrolled into four cohorts: individuals without cancer (cohort A [control cohort]), and patients with solid tumours, regardless of stage and histology, treated with immunotherapy (cohort B), chemotherapy (cohort C), or chemoimmunotherapy (cohort D). Participants received two mRNA-1273 vaccinations of 100 μg in 0·5 mL intramuscularly, 28 days apart. The primary endpoint, analysed per protocol (excluding patients with a positive baseline sample [>10 binding antibody units (BAU)/mL], indicating previous SARS-CoV-2 infection), was defined as the SARS-CoV-2 spike S1-specific IgG serum antibody response (ie, SARS-CoV-2-binding antibody concentration of >10 BAU/mL) 28 days after the second vaccination. For the primary endpoint analysis, a non-inferiority design with a margin of 10% was used. We also assessed adverse events in all patients who received at least one vaccination, and recorded solicited adverse events in participants who received at least one vaccination but excluding those who already had seroconversion (>10 BAU/mL) at baseline. This study is ongoing and is registered with ClinicalTrials.gov, NCT04715438.FINDINGS: Between Feb 17 and March 12, 2021, 791 participants were enrolled and followed up for a median of 122 days (IQR 118 to 128). A SARS-CoV-2-binding antibody response was found in 240 (100%; 95% CI 98 to 100) of 240 evaluable participants in cohort A, 130 (99%; 96 to >99) of 131 evaluable patients in cohort B, 223 (97%; 94 to 99) of 229 evaluable patients in cohort C, and 143 (100%; 97 to 100) of 143 evaluable patients in cohort D. The SARS-CoV-2-binding antibody response in each patient cohort was non-inferior compared with cohort A. No new safety signals were observed. Grade 3 or worse serious adverse events occurred in no participants in cohort A, three (2%) of 137 patients in cohort B, six (2%) of 244 patients in cohort C, and one (1%) of 163 patients in cohort D, with four events (two of fever, and one each of diarrhoea and febrile neutropenia) potentially related to the vaccination. There were no vaccine-related deaths.INTERPRETATION: Most patients with cancer develop, while receiving chemotherapy, immunotherapy, or both for a solid tumour, an adequate antibody response to vaccination with the mRNA-1273 COVID-19 vaccine. The vaccine is also safe in these patients. The minority of patients with an inadequate response after two vaccinations might benefit from a third vaccination.FUNDING: ZonMw, The Netherlands Organisation for Health Research and Development.

Background Rheumatic and musculoskeletal immune-related adverse events (irAEs) are observed in about 10% of patients with cancer receiving checkpoint inhibitors (CPIs). Given the recent emergence of these events and the lack of guidance for rheumatologists addressing them, a European League Against Rheumatism task force was convened to harmonise expert opinion regarding their identification and management. Methods First, the group formulated research questions for a systematic literature review. Then, based on literature and using a consensus procedure, 4 overarching principles and 10 points to consider were developed. Results The overarching principles defined the role of rheumatologists in the management of irAEs, highlighting the shared decision-making process between patients, oncologists and rheumatologists. The points to consider inform rheumatologists on the wide spectrum of musculoskeletal irAEs, not fulfilling usual classification criteria of rheumatic diseases, and their differential diagnoses. Early referral and facilitated access to rheumatologist are recommended, to document the target organ inflammation. Regarding therapeutic, three treatment escalations were defined: (1) local/systemic glucocorticoids if symptoms are not controlled by symptomatic treatment, then tapered to the lowest efficient dose, (2) conventional synthetic disease-modifying antirheumatic drugs, in case of inadequate response to glucocorticoids or for steroid sparing and (3) biological disease-modifying antirheumatic drugs, for severe or refractory irAEs. A warning has been made on severe myositis, a life-threatening situation, requiring high dose of glucocorticoids and close monitoring. For patients with pre-existing rheumatic disease, baseline immunosuppressive regimen should be kept at the lowest efficient dose before starting immunotherapies. Conclusion These statements provide guidance on diagnosis and management of rheumatic irAEs and aim to support future international collaborations.

PURPOSE Immune checkpoint inhibitors (ICIs) are increasingly being used in non–small-cell lung cancer (NSCLC), yet biomarkers predicting their benefit are lacking. We evaluated if on-treatment changes of circulating tumor DNA (ctDNA) from ICI start (t0) to after two cycles (t1) assessed with a commercial panel could identify patients with NSCLC who would benefit from ICI. PATIENTS AND METHODS The molecular ctDNA response was evaluated as a predictor of radiographic tumor response and long-term survival benefit of ICI. To maximize the yield of ctDNA detection, de novo mutation calling was performed. Furthermore, the impact of clonal hematopoiesis (CH)–related variants as a source of biologic noise was investigated. RESULTS After correction for CH-related variants, which were detected in 75 patients (44.9%), ctDNA was detected in 152 of 167 (91.0%) patients. We observed only a fair agreement of the molecular and radiographic response, which was even more impaired by the inclusion of CH-related variants. After exclusion of those, a ≥ 50% molecular response improved progression-free survival (10 v 2 months; hazard ratio [HR], 0.55; 95% CI, 0.39 to 0.77; P = .0011) and overall survival (18.4 v 5.9 months; HR, 0.44; 95% CI, 0.31 to 0.62; P < .0001) compared with patients not achieving this end point. After adjusting for clinical variables, ctDNA response and STK11/ KEAP1 mutations (HR, 2.08; 95% CI, 1.4 to 3.0; P < .001) remained independent predictors for overall survival, irrespective of programmed death ligand-1 expression. A landmark survival analysis at 2 months (n = 129) provided similar results. CONCLUSION On-treatment changes of ctDNA in plasma reveal predictive information for long-term clinical benefit in ICI-treated patients with NSCLC. A broader NSCLC patient coverage through de novo mutation calling and the use of a variant call set excluding CH-related variants improved the classification of molecular responders, but had no significant impact on survival.

Contains fulltext : 288327.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Patients with life-threatening advanced non-small cell lung cancer (NSCLC) who harbor an exon 20 deletion and/or insertion mutation (EGFRex20 + ) have limited effective treatment options. The high dose 3rd generation tyrosine kinase inhibitor (TKI) osimertinib shows promising in vitro activity in EGFRex20 + NSCLC tumors. METHODS: The POSITION20 is a single arm phase II, multicenter study investigating 160 mg osimertinib in patients with EGFRex20+, T790M negative NSCLC. We allowed patients to be treatment naïve and to have asymptomatic brain metastases. The primary endpoint was overall response rate (ORR). Secondary outcomes were duration of response (DoR), progression free survival (PFS), overall survival (OS), and treatment related adverse events (trAEs). RESULTS: From June 2018 to October 2021, 25 patients were enrolled across five centers in the Netherlands. The median age was 70 years (range, 47-87), 20 patients (80%) were women, and the median number of previous lines of therapy was 1 (range, 0-3). The exon 20 mutations were clustered between A763 and L777. The most common exon 20 mutations were p.(N771_H773dup) (n = 3) and p.(A767_V769dup) (n = 3). The ORR was 28% (95% CI, 12-49%), including seven partial responses, with a median DoR of 5.3 months (range, 2.7-27.6). The median PFS was 6.8 months (95% CI, 4.6-9.1) and the median OS was 15.2 months (95% CI, 14.3-16.0). The most common trAEs were diarrhea (72%), dry skin (44%), and fatigue (44%). The primary reason for discontinuation was progressive disease in 14 patients (56%). CONCLUSION: The POSITION20 study showed modest antitumor activity in patients with EGFRex20 + NSCLC treated with 160 mg osimertinib, with a confirmed ORR of 28% and acceptable toxicity.

OBJECTIVES: Cancer stem cells (CSCs) have been implicated in disease progression of aggressive cancers including small cell lung carcinoma (SCLC). Here, we have examined the possible contribution of CSCs to SCLC progression and aggressiveness.MATERIALS AND METHODS: GLC-14, GLC-16 and GLC-19 SCLC cell lines derived from one patient, representing increasing progressive stages of disease were used. CSC marker expressions was determined by RT-qPCR and western blotting analyses, and heterogeneity was studied by CSC marker expression by immunofluorescence microscopy and flow cytometry. Colony formation assays were used to assess stem cell properties and therapy sensitivity.RESULTS: Increasing expression of stem cell markers MYC, SOX2 and particularly CD44 were found in association with advancing disease. Single and overlapping expression of these markers indicated the presence of different CSC populations. The accumulation of more homogeneous double- and triple-positive CSC populations evolved with disease progression. Functional characterization of CSC properties affirmed higher proficiency of colony forming ability and increased resistance to γ-irradiation in GLC-16 and GLC-19 compared to GLC-14. GLC-19 colony formation was significantly inhibited by a human anti-CD44 antibody.CONCLUSION: The progressive increase of MYC, SOX2 and particularly CD44 expression that was accompanied with enhanced colony forming capacity and resistance in the in vitro GLC disease progression model, supports the potential clinical relevance of CSC populations in malignancy and disease relapse of SCLC.

EGFR-mutated non-small cell lung cancer (NSCLC) patients can be effectively treated with tyrosine kinase inhibitors (TKI) but frequently present with an EGFR T790M resistance mutation at relapse. We aimed to screen for T790M in pre-treatment formalin-fixed and paraffin-embedded (FFPE) tissue samples of patients with a confirmed T790M mutation at progression. We analyzed 33 pre-treatment DNA samples of NSCLC patients who progressed upon TKI between 2013 to 2019. To establish storage-time dependent formalin fixation-induced background levels for C>T mutations, we analyzed DNA isolated from archival (stored >1 year, n = 22) and recently generated (stored

The number of genomic aberrations known to be relevant in making therapeutic decisions for non-small cell lung cancer patients has increased in the past decade. Multiple molecular tests are required to reliably establish the presence of these aberrations, which is challenging because available tissue specimens are generally small. To optimize diagnostic testing, we developed a transcriptome-based next-generation sequencing (NGS) assay based on single primed enrichment technology. We interrogated 11 cell lines, two patient-derived frozen biopsies, nine pleural effusion, and 29 formalin-fixed paraffin-embedded (FFPE) samples. All clinical samples were selected based on previously identified mutations at the DNA level in EGFR, KRAS, ALK, PIK3CA, BRAF, AKT1, MET, NRAS, or ROS1 at the DNA level, or fusion genes at the chromosome level, or by aberrant protein expression of ALK, ROS1, RET, and NTRK1. A successful analysis is dependent on the number of unique reads and the RNA quality, as indicated by the DV200 value. In 27 out of 51 samples with &gt, 50 K unique reads and a DV200 &gt, 30, all 19 single nucleotide variants (SNVs)/small insertions and deletions (INDELs), three MET exon 14 skipping events, and 13 fusion gene transcripts were detected at the RNA level, giving a test accuracy of 100%. In summary, this lung-cancer-specific all-in-one transcriptome-based assay for the simultaneous detection of mutations and fusion genes is highly sensitive.

Background: Circulating tumor cells (CTC) in non-small cell lung cancer (NSCLC) patients are a prognostic and possible therapeutic marker, but have a low frequency of appearance. Diagnostic leukapheresis (DLA) concentrates CTC and mononuclear cells from the blood. We evaluated a protocol using two VyCAP microsieves to filter DLA product of NSCLC patients and enumerate CTC, compared with CellSearch as a gold standard. Methods: DLA was performed in NSCLC patients before starting treatment. DLA product equaling 2×108 leukocytes was diluted to 9 mL with CellSearch dilution buffer in a Transfix CTC tube. Within 72 hours the sample was filtered with a 7 μm pore microsieve and subsequently over a 5μm pore microsieve. CTC were defined as nucleated cells which stained for cytokeratin, but lacked CD45 and CD16. CellSearch detected CTC in the same volume of DLA. Results: Of 29 patients a median of 1.4 mL DLA product (range, 0.5-4.1) was filtered (2% of total product) successfully in 93% and 45% of patients using 7 and 5 μm pores, respectively. Two DLA products were unevaluable for CTC detection. Clogging of the 5 μm but not 7 μm microsieves was positively correlated with fixation time (ρ=0.51, P

Introduction: Non-small-cell lung cancer exhibits a range of transcriptional and epigenetic patterns that not only define distinct phenotypes, but may also govern immune related genes, which have a major impact on survival.Methods: We used open-source RNA expression and DNA methylation data of the Cancer Genome Atlas with matched non-cancerous tissue to evaluate whether these pretreatment molecular patterns also influenced genes related to the immune system and overall survival.Results: The distinction between lung adenocarcinoma and squamous cell carcinoma are determined by 1083 conserved methylation loci and RNA expression of 203 genes which differ for > 80 % of patients between the two subtypes. Using the RNA expression profiles of 6 genes, more than 95 % of patients could be correctly classified as having either adeno or squamous cell lung cancer. Comparing tumor tissue with matched normal tissue, no differences in RNA expression were found for costimulatory and co-inhibitory genes, nor genes involved in cytokine release. However, genes involved in antigen presentation had a lower expression and a wider distribution in tumor tissue.Discussion: Only a small number of genes, influenced by DNA methylation, determine the lung cancer subtype. The antigen presentation of cancer cells is dysfunctional, while other T cell immune functions appear to remain intact.

BackgroundOlder patients with cancer often find it difficult to take part in shared decision making.AimTo assess the utility of the Outcome Prioritisation Tool (OPT), designed to aid discussion with a patient in regards to their treatment goals, to empower patients with cancer through structured conversations about generic treatment goals with GPs.Design and settingA randomised controlled trial of 114 Dutch participants recruited between November 2015 and January 2019, aged ≥60 years with non-curable cancer who had to make a treatment decision with an oncologist. The intervention group used the OPT while the control group received care as usual.MethodThe primary outcome was patient empowerment using the score on the decision self-efficacy (DSE) scale. Secondary outcomes were symptoms measures of fatigue, anxiety, and depression. The experiences of participants were also explored.ResultsNo effect was found on patient empowerment between the OPT group (n = 48; DSE 86.8; standard deviation [SD] = 18.2) and the control group (n = 58; DSE 84.2; SD = 17.6; P = 0.47). In the OPT group, although statistically non-significant, fewer patients had low empowerment (18.8%, n = 9 versus 24.1%, n = 14; P = 0.50), but they did have statistically significant lower mean anxiety scores (6.0, SD = 4.6 versus 7.6, SD = 4.4; Pn = 30 versus 77.2%, n = 44; P = 0.05). Overall, 44.8% (n = 13) of patients indicated that the OPT-facilitated conversation helped them make a treatment decision, and 31.1% (n = 14) of the GPs reported that they gained new insights from the conversation.ConclusionAn OPT-facilitated conversation about generic treatment goals between patients and their GPs is associated with less anxiety and fatigue, but did not show statistically significant improvements in patient empowerment. Adding the OPT to routine care might ensure more patient-tailored care.

PURPOSE Molecular tumor boards (MTBs) provide physicians with a treatment recommendation for complex tumor-specific genomic alterations. National and international consensus to reach a recommendation is lacking. In this article, we analyze the effectiveness of an MTB decision-making methodology for patients with non–small-cell lung cancer (NSCLC) with rare or complex mutational profiles as implemented in the University Medical Center Groningen (UMCG). METHODS The UMCG-MTB comprises (pulmonary) oncologists, pathologists, clinical scientists in molecular pathology, and structural biologists. Recommendations are based on reported actionability of variants and molecular interpretation of pathways affected by the variant and supported by molecular modeling. A retrospective analysis of 110 NSCLC cases (representing 106 patients) with suggested treatment of complex genomic alterations and corresponding treatment outcomes for targeted therapy was performed. RESULTS The MTB recommended targeted therapy for 59 of 110 NSCLC cases with complex molecular profiles: 24 within a clinical trial, 15 in accordance with guidelines (on label) and 20 off label. All but 16 recommendations involved patients with an EGFR or ALK mutation. Treatment outcome was analyzed for patients with available follow-up (10 on label and 16 off label). Adherence to the MTB recommendation (21 of 26; 81%) resulted in an objective response rate of 67% (14 of 21), with a median progression-free survival of 6.3 months (interquartile range, 3.2-10.6 months) and an overall survival of 10.4 months (interquartile range, 6.3-14.6 months). CONCLUSION Targeted therapy recommendations resulting from the UMCG-MTB workflow for complex molecular profiles were highly adhered to and resulted in a positive clinical response in the majority of patients with metastatic NSCLC.

The need for a liquid biopsy in non-small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM(high) circulating tumor cells (CTC), EpCAM(low) CTC, tumor-derived extracellular vesicles (tdEV) and cell-free circulating tumor DNA (ctDNA). EpCAM(high) CTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM(low) CTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had >= 2 EpCAM(high) CTC, 15% had >= 2 EpCAM(low) CTC, 27% had >= 18 tdEV and 19% had ctDNA with >= 10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM(high) CTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM(low) CTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.

Immunotherapy for metastasized non‐small‐cell lung cancer (NSCLC) can show long‐lasting clinical responses. Selection of patients based on programmed death‐ligand 1 (PD‐L1) expression shows limited predictive value for durable clinical benefit (DCB). We investigated whether early treatment effects as measured by a change in circulating tumor DNA (ctDNA) level is a proxy of early tumor response to immunotherapy according to response evaluation criteria in solid tumors v1.1 criteria, progression‐free survival (PFS), DCB, and overall survival (OS). To this aim, blood tubes were collected from advanced‐stage lung adenocarcinoma patients (n = 100) receiving immune checkpoint inhibitors (ICI) at baseline (t0) and prior to first treatment evaluation (4–6 weeks; t1). Nontargetable (driver) mutations detected in the pretreatment tumor biopsy were used to quantify tumor‐specific ctDNA levels using droplet digital PCR. We found that changes in ctDNA levels were strongly associated with tumor response. A > 30% decrease in ctDNA at t1 correlated with a longer PFS and OS. In total, 80% of patients with a DCB of ≥ 26 weeks displayed a > 30% decrease in ctDNA levels. For patients with a PD‐L1 tumor proportion score of ≥ 1%, decreasing ctDNA levels were associated with a higher frequency a DCB (80%) and a prolonged median PFS (85 weeks) and OS (101 weeks) compared with patients with no decrease in ctDNA (34%; 11 and 39 weeks, respectively). This study shows that monitoring of ctDNA dynamics is an easy‐to‐use and promising tool for assessing PFS, DCB, and OS for ICI‐treated NSCLC patients., Predictive markers to reliably monitor response in patients with lung adenocarcinoma treated with immune checkpoint inhibitors are currently lacking. Here, we show that tumor‐informed monitoring of a single bloodborne mutation using droplet digital PCR enabled the identification of early disease progression and durable treatment responses. The association between patient survival and changes in mutant circulating tumor DNA (ctDNA) levels was unrelated to programmed death‐ligand 1 expression. Altogether, our data show that ctDNA dynamics could offer a promising cost‐effective approach to monitor patients with metastasized lung adenocarcinoma.

INTRODUCTION: Non-small cell lung cancer (NSCLC) patients with Anaplastic Lymphoma Kinase (ALK) gene fusions respond well to ALK inhibitors but commonly develop on-target resistance mutations. The objective of this study is to collect clinical evidence for subsequent treatment with ALK inhibitors.PATIENTS AND METHODS: Local experience with on-target ALK resistance mutations and review of the literature identified 387 patients with ALK inhibitor resistance mutations. Clinical benefit of mutation-inhibitor combinations was assessed based on reported response, progression-free survival and duration of treatment. Furthermore, this clinical evidence was compared to previously reported in vitro sensitivity of mutations to the inhibitors.RESULTS: Of the pooled population of 387 patients in this analysis, 239 (62%) received at least 1 additional line of ALK inhibition after developing on-target resistance to ALK inhibitor therapy. Clinical benefit was reported for 177 (68%) patients, but differed for each mutation-inhibitor combination. Agreement between in vitro predicted sensitivity of 6 published models and observed clinical benefit ranged from 64% to 87%. The observed clinical evidence for highest probability of response in the context of specific on-target ALK inhibitor resistance mutations is presented.CONCLUSION: Molecular diagnostics performed on tissue samples that are refractive to ALK inhibitor therapy can reveal new options for targeted therapy for NSCLC patients. Our comprehensive overview of clinical evidence of drug actionability of ALK on-target resistance mechanisms may serve as a practical guide to select the most optimal drug for individual patients.

Background: Circulating tumour cell (CTC) number is an independent prognostic factor in patients with small cell lung cancer (SCLC) but there is no consensus on the CTC threshold for prognostic significance. We undertook a pooled analysis of individual patient data to clinically validate CTC enumeration and threshold for prognostication.Methods: Four European cancer centres, experienced in CellSearch CTC enumeration for SCLC provided pseudo anonymised data for patients who had undergone pre-treatment CTC count. Data was collated, and Cox regression models, stratified by centre, explored the relationship between CTC count and survival. The added value of incorporating CTCs into clinico-pathological models was investigated using likelihood ratio tests.Results: A total of 367 patient records were evaluated. A one-unit increase in log-transformed CTC counts corresponded to an estimated hazard ratio (HR) of 1.24 (95% CI: 1.19-1.29, PConclusions: Higher pre-treatment CTC counts are a negative independent prognostic factor in SCLC when considered as a continuous variable or dichotomised counts of ≥15 or ≥50. Incorporating CTC counts, as a continuous variable, improves clinic-pathological prognostic models.

Background The clinical relevance of epidermal growth factor receptor (EGFR) copy number gain in patients with EGFR mutated advanced non-small cell lung cancer on first-line tyrosine kinase inhibitor treatment has not been fully elucidated. Objective We aimed to estimate EGFR copy number gain using amplicon-based next generation sequencing data and explored its prognostic value. Patients and Methods Next generation sequencing data were obtained for 1566 patients with non-small cell lung cancer. EGFR copy number gain was defined based on an increase in EGFR read counts relative to internal reference amplicons and normal controls in combination with a modified z-score ≥ 3.5. Clinical follow-up data were available for 60 patients treated with first-line EGFR-tyrosine kinase inhibitors. Results Specificity and sensitivity of next generation sequencing-based EGFR copy number estimations were above 90%. EGFR copy number gain was observed in 27.9% of EGFR mutant cases and in 7.4% of EGFR wild-type cases. EGFR gain was not associated with progression-free survival but showed a significant effect on overall survival with an adjusted hazard ratio of 3.14 (95% confidence interval 1.46–6.78, p = 0.003). Besides EGFR copy number gain, osimertinib in second or subsequent lines of treatment and the presence of T790M at relapse revealed significant effects in a multivariate analysis with adjusted hazard ratio of 0.43 (95% confidence interval 0.20–0.91, p = 0.028) and 0.24 (95% confidence interval 0.1–0.59, p = 0.001), respectively. Conclusions Pre-treatment EGFR copy number gain determined by amplicon-based next generation sequencing data predicts worse overall survival in EGFR-mutated patients treated with first-line EGFR-tyrosine kinase inhibitors. T790M at relapse and subsequent treatment with osimertinib predict longer overall survival. Supplementary Information The online version contains supplementary material available at 10.1007/s11523-021-00798-2.

Background: The analysis of circulating tumor DNA (ctDNA) is a potential minimally invasive molecular tool to guide treatment decision making and disease monitoring. A suitable diagnostic-grade platform is required for detection of tumor-specific mutations with high sensitivity in circulating cell-free DNA (ccfDNA) of cancer patients. Methods: In an international multicenter study conducted by three laboratories participating in the CANCER-ID consortium, a cohort containing 177 cell-free plasma samples at baseline, follow-up or disease progression derived from 72 patients treated for advanced-stage non-small cell lung cancer (NSCLC), relevant mutations were tested using the UltraSEEK® Lung Panel on the MassARRAY® System covering 73 hotspot mutations in EGFR, KRAS, BRAF, ERBB2 and PIK3CA, and compared against mutation-specific droplet digital PCR (ddPCR) and tumor tissue next-generation sequencing (NGS) performed in routine diagnostics. Variant detection accuracy at primary diagnosis and during disease progression, and ctDNA dynamics as a marker of treatment efficacy were analyzed. Results: The UltraSEEK® Lung Panel revealed an overall detection rate of 87.2% irrespective of ccfDNA input, and a specificity of >99.5% using the reference material. When comparing the 131 variants identified in the patient-derived cell-free plasma using UltraSEEK® with mutation-specific ddPCR analyses, a concordance of 90% was found. The 77 diagnostically or clinically relevant variants identified in 66 pretreatment tumor tissue showed an overall concordance with UltraSEEK® of 73% at baseline with a false-negative rate of 3%. When restricting to therapeutically targetable mutations, the concordance elevated to 85%. A decrease in ctDNA levels at 4-6 weeks after start of treatment detected with UltraSEEK® correlated with a prolonged median PFS (46 vs 11 weeks; P Conclusion: Using plasma-derived ccfDNA, the UltraSEEK® Lung Panel with a mid-density set of the most common predictive markers for NSCLC is an alternative tool to detect both mutations at diagnosis and disease progression, and to monitor treatment response. Citation Format: Paul van der Leest, Melanie Janning, Naomi Rifaela, Maria L. Aguirre Azpurua, Jolanthe Kropidlowski, Sonja Loges, Nicholas Lozano, Alexander Sartori, Darryl Irwin, Pierre-Jean Lamy, T. Jeroen N. Hiltermann, Harry J. Groen, Klaus Pantel, Léon C. van Kempen, Harriet Wikman, Ed Schuuring. Detection and monitoring of tumor-derived mutations in ctDNA using the UltraSEEK Lung Panel on the MassARRAY System in metastatic NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3411.

Background: Salivary glands (SGs) can be damaged by immune checkpoint inhibitor (ICI) therapy. In patients with ICI-induced SG dysfunction, 60% progress to fulfill classification criteria for primary Sjogren's syndrome (pSS), owing to immune foci in SGs and/or anti-SSA autoantibody positivity. We report the SG tissue analysis of a patient with SG dysfunction after treatment with a programmed death ligand-1 (PD-L1) inhibitor, compared to that of a dry mouth ("sicca") control and pSS patient.Case presentation: The patient received the PD-L1 inhibitor durvalumab (10 mg/kg, every 2 weeks by intravenous infusion) as adjuvant treatment for stage 3 non-small cell lung carcinoma, following concurrent chemo radiotherapy. At 43 weeks after 21 cycles of Durvalumab, the patient was not capable of producing unstimulated or stimulated parotid gland saliva, and a biopsy was taken. Immunohistochemical analysis showed no classical AQP5(+) CK7(-) acinar cell clusters (CK7 marks intercalated ducts, IDs). In contrast, the parenchyma was dominated by hybrid epithelial "structures" with ID-like morphology, containing a mixture of AQP5(+)CK7(-), AQP5(-)CK7(+), and AQP5(+)CK7(+) cells (30 structures/mm(2)). These structures were present at lower frequencies in sicca control (2/mm(2)) and pSS (10/mm(2)) tissue. Hybrid structures contained proliferating (Ki67(+)) cells and senescent (p16(+)) cells. Striated ducts showed no abnormal morphology post PD-L1 treatment, in contrast to pSS tissue. PD-L1 expression was detected in the SG parenchyma following anti-PD-L1 therapy. The SG post-PD-L1 therapy further demonstrated focal lymphocytic sialadentitis, harboring disperse, and focal CD4(+) T cell-rich infiltrates. CD8(+) T cells were also present. In this patient, these CD4(+) and CD8(+) T cells were observed in-between and inside hybrid structures. CD20(+) B-cells were infrequently detected following PD-L1 blockade, in contrast to their preponderance in pSS SG tissue.Conclusion: This patient lacked conventional SG acinar cells following anti-PD-L1 therapy and demonstrated presence of hybrid intercalated duct-like structures. Understanding which mechanisms and dynamics underpinning this aberrant parenchyma may be crucial to understand how SG dysfunction post ICI therapy, and potentially other affected organs. Furthermore, although the patient treated with anti-PD-L1 antibody examined here fulfills the criteria for pSS and demonstrated focal lymphocytic sialadentitis, the further histopathological characteristics do not resemble pSS.

Circulating tumor cells (CTCs) detected by CellSearch are prognostic in non-small-cell lung cancer (NSCLC), but rarely found. CTCs can be extracted from the blood together with mononuclear cell populations by diagnostic leukapheresis (DLA), therefore concentrating them. However, CellSearch can only process limited DLA volumes (&asymp, 2 mL). Therefore, we established a protocol to enumerate CTCs in DLA products with Isolation by SizE of Tumor cells (ISET), and compared CTC counts between CellSearch&reg, and ISET. DLA was performed in NSCLC patients who started a new therapy. With an adapted protocol, ISET could process 10 mL of DLA. CellSearch detected CTCs in a volume equaling 2 &times, 108 leukocytes (mean 2 mL). CTC counts per mL were compared. Furthermore, the live cell protocol of ISET was tested in eight patients. ISET successfully processed all DLA products&mdash, 16 with the fixed cell protocol and 8 with the live cell protocol. In total, 10&ndash, 20 mL of DLA was processed. ISET detected CTCs in 88% (14/16), compared to 69% (11/16, p &lt, 0.05) with CellSearch. ISET also detected higher number of CTCs (ISET median CTC/mL = 4, interquartile range [IQR] = 2&ndash, 6, CellSearch median CTC/mL = 0.9, IQR = 0&ndash, 1.8, p &lt, 0.01). Cells positive for the epithelial cell adhesion molecule (EpCAM+) per mL were detected in similar counts by both methods. Eight patients were processed with the live cell protocol. All had EpCAM+, CD45&minus, CD235- cells isolated by fluorescence-activated cell sorting (FACS). Overall, ISET processed larger volumes and detected higher CTC counts compared to CellSearch. EpCAM+ CTCs were detected in comparable rates.

We read with great interest the article ‘Addressing immune-related adverse events of cancer immunotherapy: how prepared are rheumatologists?’ by Kostine et al .1 The introduction of immune checkpoint inhibitor (ICI) therapy has been a major breakthrough in the management of metastatic cancer. On the downside, ICI therapy may induce unwanted autoimmune effects, the so-called immune-related adverse effects (irAEs). Various irAEs have been described that resemble a regular rheumatic disease, including polymyalgia rheumatica (ICI-PMR).2 3 The authors report that rheumatologists may lack confidence in diagnosing irAEs. Therefore, recommendations for the diagnosis of rheumatic irAEs are needed. Based on our experience with ICI-PMR, we propose that imaging could be an important part of such recommendations. We investigated six consecutive patients with ICI-PMR by ultrasonography, and five of these patients also by [18F]-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG-PET/CT) scan. Five patients fulfilled the provisional American College of Rheumatology/European League Against Rheumatism classification criteria for PMR.4 A normal C-reactive protein level in the absence of an erythrocyte sedimentation rate (ESR) test precluded PMR classification in one patient. However, this patient fulfilled both the clinical and ultrasound criteria for PMR,4 and showed findings suggestive of PMR on the FDG-PET/CT scan.5 The median age was 73 years (range 59–83; online supplementary table 1). Patients received anti-programmed cell death protein 1 (PD-1) treatment, that …

Programmed cell death protein-1/ligand-1 (PD-1/PD-L1) blockade is effective in a subset of patients with several tumor types, but predicting patient benefit using approved diagnostics is inexact, as some patients with PD-L1-negative tumors also show clinical benefit1,2. Moreover, all biopsy-based tests are subject to the errors and limitations of invasive tissue collection3-11. Preclinical studies of positron-emission tomography (PET) imaging with antibodies to PD-L1 suggested that this imaging method might be an approach to selecting patients12,13. Such a technique, however, requires substantial clinical development and validation. Here we present the initial results from a first-in-human study to assess the feasibility of imaging with zirconium-89-labeled atezolizumab (anti-PD-L1), including biodistribution, and secondly test its potential to predict response to PD-L1 blockade (ClinicalTrials.gov identifiers NCT02453984 and NCT02478099). We imaged 22 patients across three tumor types before the start of atezolizumab therapy. The PET signal, a function of tracer exposure and target expression, was high in lymphoid tissues and at sites of inflammation. In tumors, uptake was generally high but heterogeneous, varying within and among lesions, patients, and tumor types. Intriguingly, clinical responses in our patients were better correlated with pretreatment PET signal than with immunohistochemistry- or RNA-sequencing-based predictive biomarkers, encouraging further development of molecular PET imaging for assessment of PD-L1 status and clinical response prediction.

Background: It is unknown whether the presence of circulating tumor cells (CTC), a known prognostic factor, influences treatment outcome. We investigated whether baseline CTC in non-small cell lung cancer (NSCLC) patients treated with tyrosine kinase inhibitors (TKI) or chemotherapy was associated with response to therapy. Methods: We included consecutive advanced NSCLC patients, stratified by therapy. Before treatment the number of CTC was measured by CellSearch. Tumor response rates, progression free survival (PFS) and overall survival (OS) in patients with and without CTC at baseline were compared. Results: We included 86 patients (34 treated by TKI). Response rates of patients with CTC were lower than in patients without CTC (OR =0.22, P

Purpose: Tumor cells from patients with lung cancer are expelled from the primary tumor into the blood, but difficult to detect in the peripheral circulation. We studied the release of circulating tumor cells (CTCs) during surgery to test the hypothesis that CTC counts are influenced by hemodynamic changes (caused by surgical approach) and manipulation. Experimental Design: Patients undergoing video-assisted thoracic surgery (VATS) or open surgery for (suspected) primary lung cancer were included. Blood samples were taken before surgery (T0) from the radial artery (RA), from both the RA and pulmonary vein (PV) when the PV was located (T1) and when either the pulmonary artery (T2 open) or the PV (T2 VATS) was dissected. The CTCs were enumerated using the CellSearch system. Single-cell whole-genome sequencing was performed on isolated CTCs for aneuploidy. Results: CTCs were detected in 58 of 138 samples (42%) of 31 patients. CTCs were more often detected in the PV (70%) compared with the RA (22%, P < 0.01) and in higher counts (P < 0.01). After surgery, the RA but not the PV showed less often CTCs (P = 0.02). Type of surgery did not influence CTC release. Only six of 496 isolated CTCs showed aneuploidy, despite matched primary tumor tissue being aneuploid. Euploid so-called CTCs had a different morphology than aneuploid. Conclusions: CTCs defined by CellSearch were identified more often and in higher numbers in the PV compared with the RA, suggesting central clearance. The majority of cells in the PV were normal epithelial cells and outnumbered CTCs. Release of CTCs was not influenced by surgical approach.

Background Non-small cell lung cancer (NSCLC) patients treated with checkpoint inhibitors show long lasting responses, but it is hard to predict which patients will profit from this treatment with the currently used marker, programmed death ligand 1 (PD-L1). We hypothesized that circulating tumor cells (CTC) or tumor derived extracellular vesicles (tdEV) are markers of treatment efficacy. Methods Patients with advanced NSCLC treated with checkpoint inhibitors were included. Blood was drawn at baseline (T0) and at 4 weeks of treatment (T1) for analysis of CTC and tdEV using CellSearch®. Tumor response was classified as partial or complete response based on the response evaluation criteria in solid tumors (RECISTv1.1) measured 4–6 weeks after start of treatment. Durable response was defined as stable disease, partial or complete response without disease progression at 6 months. Analyses were adjusted for covariables including PD-L1 expression. Results We included 104 patients (30 with a tumor response, 74 non-responders, 2 responses not evaluable due to early death); 63 patients provided T1 samples. All patients were treated with PD-L1 inhibitors. The majority of patients received second (85%) or third line (treatment with nivolumab monotherapy (89%). CTC were present in 33/104 patients at T0 (32%) and 17/63 at T1 (27%), 9/63 patients had CTC (14%) at both time points. The presence of CTC, both at T0 (OR = 0.28, p = 0.02,) and T1 (OR = 0.07, p

To explore the correspondence between primary and secondary healthcare providers about patients with lung, breast or colorectal cancer.Qualitative research.We collected the medical files of 50 patients with lung, breast or colorectal cancer by purposive sampling and selected the correspondence-related items from them. These concerned referral letters from primary to secondary caregivers and letters from specialists. A qualitative content analysis of these documents was performed. In addition, 4 general practitioners, 4 oncologists and 1 nurse specialist were interviewed.We analysed 50 referral letters and 369 letters from specialists. Content could be divided into 6 main themes in the referral letters, and it was noticeable that highly relevant information regarding the past medical history was often mixed with less relevant information. The same was true for the medication list and case history to a certain extent. We could distinguish 9 themes in the letters from specialists. All the letters from specialists did include information about the current treatment, but information about treatment intent (curative or palliative) or alternative treatment options was rarely available. Interviews with the healthcare providers confirmed these findings.The study findings indicate that referral letters and specialist correspondence are not sufficiently tailored to the needs of the recipient.

The prognostic value of markers of cancer stem cells and epithelial to mesenchymal transition in small cell lung cancer is not known. We retrospectively studied these markers in the biopsy tissue of patients with small cell lung cancer and correlated them with overall survival and the strongest known prognostic marker circulating tumor cells.Background: Small cell lung cancer (SCLC) has a poor prognosis, and even with localized (limited) disease, the 5-year survival has only been around 20%. Elevated levels of circulating tumor cells (CTCs) have been associated with a worse prognosis, and markers of cancer stem cells (CSCs) and epithelial to mesenchymal transition have been associated with increased chemoresistance and metastatic spread in SCLC. Patients and Methods: The biopsy specimens of 38 SCLC patients were used for marker evaluation by immunohistochemistry. The markers for CSCs were CD44 and SOX2. The markers for epithelial to mesenchymal transition were E-cadherin, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, vimentin, and c-MET. Staining was scored as low (weak) or high (strong) intensity for SOX2, epithelial cell adhesion molecule, cytokeratins 8, 18, and 19, and c-MET and using the immunoreactive score for CD44, E-cadherin, and vimentin, expressed as low or high expression. Results: High expression of c-MET (c-METH) and low expression of E-cadherin (E-cad(L)) showed a trend toward a better prognosis (P = .07 and P = .09, respectively). The combination of c-METH and E-cad(L) resulted in significantly better survival (P = .007). The tested markers were not associated with CTCs, although a trend was seen for c-(METE)-E-H-cad(L) (P = .09) with low CTCs. The CSC markers SOX2 and CD44 were not associated with overall survival in this patient cohort. Conclusion: SCLC with a mesenchymal-like phenotype (c-METHE-cad(L)) is associated with longer survival and showed a trend toward lower CTCs. (C) 2016 Elsevier Inc. All rights reserved.

Objective The relevance of EGFR gene amplification in patients with EGFR-mutated advanced non-small cell lung cancer (NSCLC) has not been fully elucidated. Moreover, a limited number of studies have been published on methods to estimate gene amplifications based on routinely performed targeted next generation sequencing (NGS). In this study we aimed to determine whether PCR-based targeted NGS data on tumor tissue can be used to estimate presence of gene amplifications and explored the prognostic value of EGFR gene amplification in EGFR mutated NSCLC patients. Materials and methods A total of 3,194 good quality targeted NGS data files were retrieved from 2014 to 2017. Among those, 1,729 NSCLC samples originated from 1,586 NSCLC patients of whom 134 had an EGFR mutation (8.2%). Clinical data were available for 66 of the patients. Raw sequencing data were re-analyzed using a custom designed pipeline. The presence of an amplification was based on the read depth of a given amplicon relative to a set of reference amplicons from the sample or relative to a set of normal control samples. Reference amplicons were selected based on low degree of variation in read depth amongst all tested samples. Amplifications were regarded significant when the ratio was ≥3 and the z score was ≥3.5. Technical validation was done by FISH and MLPA. Cox regression analysis on overall survival was done with each of the amplification parameters using SPSS. Results No amplifications were detected for the ALK, KIT, NRAS, PDGFRA, GNAQ and MAP2K1 gene loci, whereas amplifications for BRAF, ESR1, GNA11, HRAS, KRAS, MET and PIK3CA were observed at a low frequency. Depending on the amplification analysis strategy (within sample or relative to normal controls), 19% and 13% of the EGFR mutated group had an EGFR amplification, respectively. In EGFR wild type patients, amplifications were detected in 5% and 4% of the patients using the two methods, respectively. The sensitivity and specificity of the NGS based estimation of amplifications for EGFR was 94% (14/15) and 97% (34/35) respectively for both data analysis approaches. Patients with concurrent EGFR mutations and amplifications (estimated within sample) treated with EGFR-TKI had a significantly worse overall survival compared with those without concurrent EGFR amplifications (ratio, p=0.047 and z score, p=0.015). Cox regression analysis indicated a borderline significant interaction between ratio and z score (p=0.052). Conclusion Routinely obtained amplicon-based NGS data can be used to identify gene amplifications. The presence of EGFR amplifications in EGFR mutant patients is predictive of a worse overall survival. Keywords: Lung adenocarcinoma, EGFR, survival, tyrosine kinase inhibitor Citation Format: Pei Meng, Jiacong Wei, Miente Martijn Terpstra, Anke van Rijk, Menno Tamminga, Frank Scherpen, Arja ter Elst, Mohamed Z. Alimohamed, Lennart F. Johansson, T. Jeroen N. Hiltermann, Harry J. Groen, Klaas Kok, Anthonie J. van der Wekken, Anke van den Berg. Clinical value of EGFR gene amplifications detected using amplicon based targeted next generation sequencing data in lung adenocarcinoma patients [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 4335.

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

Crizotinib is an effective drug for patients with anaplastic lymphoma kinase (ALK)-positive non-small-cell lung cancer (NSCLC), but upon treatment, the tumors inevitably become crizotinib resistant in time. The resistance mechanisms are only partly understood. In this study, we aim to identify gene mutations associated with resistance in ALK positive advanced non-squamous NSCLC treated with crizotinib. Four ALK positive patients with progressive disease following crizotinib treatment were identified with paired pre- and post-crizotinib tumor tissue from our previously published cohort. Somatic variants in these samples were detected by whole exome sequencing. In one of the four patients, an ALK-resistance associated mutation was identified. In the other three patients, no ALK-resistance associated mutations were present. In these patients we identified 89 relevant somatic mutations in 74 genes that were specific to the resistant tumors. These genes were enriched in 15 pathways. Four pathways, were related to epithelial-mesenchymal transition (EMT): proteoglycans in cancer, HIF-1 signaling, FoxO signaling pathway, and ECM-receptor interaction. Analysis of other EMT-related pathways revealed three additional genes with mutations specific to the crizotinib-resistant tumor samples. The enrichment of mutations in genes associated with EMT-related pathways indicates that loss of epithelial differentiation may represent a relevant resistance mechanism for crizotinib.

Cytokine interleukin-1β (IL-1β) has multiple pro-tumorogenic effects on tumor microenvironment, thereby promoting carcinogenesis, tumor invasiveness, and immunosuppression. CAN is a selective IL-1β inhibitor that aims to target tumor-promoting inflammation to reduce immune suppression, thereby potentiating effects of immunotherapy with PD-1 inhibitors such as PEM. Results of phase 3 CANTOS study have shown that IL-1β inhibition with CAN was associated with reduced incidence of lung cancer and lung cancer mortality, thus providing a rationale to investigate therapeutic role of CAN in lung cancer. CANOPY-1 (NCT03631199) is a placebo-controlled, double-blind, randomized, phase 3 trial designed to evaluate efficacy and safety of PEM + Ctx ± CAN in previously untreated pts with stage IIIB/IIIC (not eligible for definitive chemo-radiation curative tx) or stage IV squamous and nonsquamous NSCLC. The study was divided into 2 parts: part 1 is non-randomized, safety run-in part where pts received CAN 200 mg s.c Q3W + PEM 200 mg i.v Q3W + platinum-based Ctx [Cohort A (non-squamous), carboplatin + pemetrexed; Cohort B (non-squamous), cisplatin + pemetrexed; Cohort C (squamous or non-squamous), carboplatin + paclitaxel]. Part 2 of the study randomizes pts to evaluate efficacy and safety of CAN combination regimen vs placebo combination regimen. Primary objective of safety run-in part: RP3R for CAN in combination with PEM + Ctx. Secondary objectives: ORR, DCR, DOR, safety, PK, and immunogenicity. As of 14 May 2019 (follow-up of ≥42 days from C1D1 unless pt discontinued earlier), 10 pts in cohort A (A), 11 pts in cohort B (B), and 9 pts in cohort C (C) were treated, of which 73% were male, median age was 63 yrs. In total, 24/30 (80%) pts enrolled were still receiving tx; primary reason for tx discontinuation was progressive disease (5 pts; 3 pts in A and 1 pt each in B and C) and 1 patient died due to study indication. Dose-limiting toxicity (DLT) occurring during first 42 days of study tx was reported only in 1 pt (cohort C: grade 3 hepatitis, not related to CAN). Recommended phase 3 regimen (RP3R) of CAN in combination with standard dose of PEM + Ctx was confirmed as 200 mg SC Q3W based on Bayesian logistic regression model (BLRM). Serious AEs regardless of study drug relationship were reported in 8 (27%) pts (2 pts in A and 3 pts each in B and C), none of which considered to be related to CAN. Most common AEs (≥20%, any grade) across all cohorts (n=30) were nausea (37%), vomiting (30%), constipation and fatigue (each 23%), and neutrophil count decrease (20%). Overall, 14 pts (47%) experienced grade 3 AEs and 1 pt experienced grade 4 AE (cardiac tamponade unrelated to study drugs). No fatal serious AEs were reported. AEs leading to discontinuation of one of the study drugs were reported in 3 (10%) pts (hepatitis, peripheral neuropathy, and polyneuropathy) but none were CAN related. AEs leading to dose reduction and dose interruption of one of study drugs were reported in 3 (10%) pts and 5 (17%) pts, respectively. Only 1 DLT was reported with this triplet combination of CAN + PEM + Ctx. Based on BLRM and all relevant data, the RP3R of CAN as 200 mg SC Q3W combination was considered safe and well tolerated. Enrolment is ongoing in randomized phase 3 part of study to evaluate efficacy and safety. Citation Format: Bruce E. Johnson, Tae Min Kim, T. Jeroen N. Hiltermann, Fabrice Barlesi, Christian Grohe, Yasushi Goto, Orvar Gunnarsson, Tobias Overbeck, Noemi Reguart, Martin Wermke, Gilberto Castro, Enriqueta Felip, Alastair Greystoke, Benjamin J. Solomon, Noelia Nebot, Stephanie Deudon, Anne-Laure Louveau, Vanessa Q. Passos, Daniel SW Tan. Safety run-in results from phase 3 study of canakinumab (CAN) or placebo in combination with pembrolizumab (PEM) plus platinum-based doublet chemotherapy (Ctx) as 1st line therapy in patients (pts) with advanced or metastatic NSCLC (CANOPY-1) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C100. doi:10.1158/1535-7163.TARG-19-C100

Introduction: Immunotherapy is a breakthrough in non-small cell lung cancer (NSCLC), showing long lasting responses in metastasized disease. However, only a limited number of patients responds to treatment. Selection of patients by known biomarkers (eg PD-L1, TMB) and combining PD-(L)1 inhibitors with other treatments (eg. chemotherapy or CTLA4 inhibitors) enriches the population for responders, however still around 60% do not respond with a durable response. We hypothesized that in KRAS mutant positive advanced NSCLC the detection of changes of ctDNA levels will be a relevant predictive marker in immunotherapy ( and stronger than eg PD-L1 expression in tumor tissue).. Purpose: To test ctDNA as an early predictive marker for durable response in KRAS mutated lung cancer, in patients treated with PD-1 inhibitors after progression on platinum containing chemotherapy. Methods: Patients with advanced NSCLC treated with nivolumab with a known KRAS or BRAF mutation from primary biopsy, with disease progression after platinum based chemotherapy are eligible. ctDNA obtained from EDTA plasma at baseline, 1, 2, 4, 6 weeks and thereafter every 12 weeks compared to durable response as primary outcome (ie. benefit from treatment> 6month). Secondary outcomes were tumor response according to RECISTv1.1, progression-free and overall survival. ctDNA was determined and analyzed using a digital droplet PCR assay (Biorad) that was confirmed by a second technique (Idylla). ctDNA was categorized according to its response pattern in time: increasing, decreasing and undetectable KRAS mutant allele frequencies over time. Results: In this ongoing study 31 patients with a known KRAS and 2 patients with a BRAF mutation participated. 24 (72%) had detectable levels of KRAS (n=22) or BRAF (n=2) in plasma. Concordance between Idylla and ddPCR was good and correlation was high (R2 =0,52). Of first 27 patients ctDNA increasing n=11, decreasing n=10, no change n=6. Data on all included patients will be presented. Conclusion: Early detection of decreasing KRAS/BRAF mutant alleles in ctDNA predicted durable tumor responses, and increasing levels were associated with early disease progression. Citation Format: T.Jeroen.N. Hiltermann, Lucie B.M. Hijmering-Kappelle, Maria L. Aguirre Azpurua, Anthonie.J. vd Wekken, Birgitta.I. Hiddinga, Wim Timens, Harry.J.M. Groen, Ed Schuuring. ctDNA a promising predictive marker for treatment with PD-1 inhibitors in KRAS mutated NSCLC after platinum based chemotherapy [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 399.

Mutations in epithelial growth factor receptor (EGFR), as well as in the EGFR downstream target KRAS are frequently observed in non-small cell lung cancer (NSCLC). Chronic obstructive pulmonary disease (COPD), an independent risk factor for developing NSCLC, is associated with an increased activation of EGFR. In this study we determined presence of EGFR and KRAS hotspot mutations in 325 consecutive NSCLC patients subjected to EGFR and KRAS mutation analysis in the diagnostic setting and for whom the pulmonary function has been determined at time of NSCLC diagnosis. Information about age at diagnosis, sex, smoking status, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) was collected. Chronic obstructive pulmonary disease(COPD) was defined according to 2013 GOLD criteria. Chi-Square, student t-test and multivariate logistic regression were used to analyze the data. A total of 325 NSCLC patients were included, 193 with COPD and 132 without COPD. COPD was not associated with presence of KRAS hotspot mutations, while EGFR mutations were significantly higher in non-COPD NSCLC patients. Both female gender (HR 2.61; 95% CI: 1.56-4.39; pT and G>C transversions were significantly more frequent in females (86.2%) than in males (61.5%) (p = 0.008). The exon 19del mutation was more frequent in non-smokers (90%) compared to current or past smokers (36.8%). In conclusion, KRAS mutations are more common in females and smokers, but are not associated with COPD-status in NSCLC patients. EGFR mutations are more common in non-smoking NSCLC patients.

In the CellSearch&reg, system, blood is immunomagnetically enriched for epithelial cell adhesion molecule (EpCAM) expression and cells are stained with the nucleic acid dye 4&prime, 6-diamidino-2-phenylindole (DAPI), Cytokeratin-PE (CK), and CD45-APC. Only DAPI+/CK+ objects are presented to the operator to identify circulating tumor cells (CTC) and the identity of all other cells and potential undetected CTC remains unrevealed. Here, we used the open source imaging program Automatic CTC Classification, Enumeration and PhenoTyping (ACCEPT) to analyze all DAPI+ nuclei in EpCAM-enriched blood samples obtained from 192 metastatic non-small cell lung cancer (NSCLC) patients and 162 controls. Significantly larger numbers of nuclei were detected in 300 patient samples with an average and standard deviation of 73,570 &plusmn, 74,948, as compared to 359 control samples with an average and standard deviation of 4191 &plusmn, 4463 (p &lt, 0.001). In patients, only 18% &plusmn, 21% and in controls 23% &plusmn, 15% of the nuclei were identified as leukocytes or CTC. Adding CD16-PerCP for granulocyte staining, the use of an LED as the light source for CD45-APC excitation and plasma membrane staining obtained with wheat germ agglutinin significantly improved the classification of EpCAM-enriched cells, resulting in the identification of 94% &plusmn, 5% of the cells. However, especially in patients, the origin of the unidentified cells remains unknown. Further studies are needed to determine if undetected EpCAM+/DAPI+/CK-/CD45- CTC is present among these cells.