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Item does not contain fulltext, PURPOSE: Durable clinical benefit to PD-1 blockade in non-small cell lung cancer (NSCLC) is currently limited to a small fraction of patients, underlining the need for predictive biomarkers. We recently identified a tumor-reactive tumor-infiltrating T lymphocyte (TIL) pool, termed PD-1T TILs, with predictive potential in NSCLC. Here, we examined PD-1T TILs as biomarker in NSCLC. EXPERIMENTAL DESIGN: PD-1T TILs were digitally quantified in 120 baseline samples from advanced NSCLC patients treated with PD-1 blockade. Primary outcome was disease control (DC) at 6 months. Secondary outcomes were DC at 12 months and survival. Exploratory analyses addressed the impact of lesion-specific responses, tissue sample properties, and combination with other biomarkers on the predictive value of PD-1T TILs. RESULTS: PD-1T TILs as a biomarker reached 77% sensitivity and 67% specificity at 6 months, and 93% and 65% at 12 months, respectively. Particularly, a patient group without clinical benefit was reliably identified, indicated by a high negative predictive value (NPV) (88% at 6 months, 98% at 12 months). High PD-1T TILs related to significantly longer progression-free (HR 0.39, 95% CI, 0.24-0.63, P < 0.0001) and overall survival (HR 0.46, 95% CI, 0.28-0.76, P < 0.01). Predictive performance was increased when lesion-specific responses and samples obtained immediately before treatment were assessed. Notably, the predictive performance of PD-1T TILs was superior to PD-L1 and tertiary lymphoid structures in the same cohort. CONCLUSIONS: This study established PD-1T TILs as predictive biomarker for clinical benefit to PD-1 blockade in patients with advanced NSCLC. Most importantly, the high NPV demonstrates an accurate identification of a patient group without benefit. See related commentary by Anagnostou and Luke, p. 4835.

Contains fulltext : 252040.pdf (Publisher’s version ) (Open Access), INTRODUCTION: In 1-3% of non-small cell lung cancer (NSCLC) human epidermal growth factor 2 (HER2) mutations are identified as a genomic driver. Nevertheless, no HER2-targeted treatment is approved for NSCLC. In the Drug Rediscovery Protocol (DRUP), patients are treated with off-label drugs based on their molecular profile. Here, we present the results of the cohort 'trastuzumab/pertuzumab for HER2 exon20 mutation positive (HER2m+) NSCLC'. METHODS: Patients with treatment refractory, advanced HER2m+ NSCLC with measurable disease (RECISTv1.1) were eligible. Treatment with intravenous trastuzumab combined with pertuzumab every 3 weeks was administered. The primary end-point was clinical benefit (CB: either objective response or stable disease ≥ 16 weeks). Patients were enrolled using a Simon-like 2-stage design, with 8 patients in stage 1 and up to 24 patients in stage 2 if at least 1 patient had CB in stage 1. At baseline, a biopsy for biomarker analysis, including whole genome sequencing, was obtained. RESULTS: Twenty-four evaluable patients were enrolled and treated between May 2017 and August 2020. CB was observed in 9 patients (38%); including an objective response rate of 8.3% (2 patients had a partial response) and 7 patients with stable disease ≥ 16 weeks. The most frequently observed HER2 mutation was p.Y772_A775dup (71%, n = 20). Median follow-up was 13 months, median progression-free survival and overall survival 4 (95% CI 3-6) and 10 months (95% CI 4 - not reached), respectively. Whole genome sequencing data (available for 67% of patients) confirmed the inclusion mutation in all cases. No unexpected toxicity was observed. CONCLUSION: Despite the fact that the study did meet its primary end-point, trastuzumab/pertuzumab was only marginally active in a subset of patients with heavily pre-treated HER2m+ NSCLC.

Item does not contain fulltext, Stage II-IIIA nonsmall cell lung cancer (NSCLC) patients receive adjuvant chemotherapy after surgery as standard-of-care treatment, even though only approximately 5.8% of patients will benefit. Identifying patients with minimal residual disease (MRD) after surgery using tissue-informed testing of postoperative plasma circulating cell-free tumour DNA (ctDNA) may allow adjuvant therapy to be withheld from patients without MRD. However, the detection of MRD in the postoperative setting is challenging, and more sensitive methods are urgently needed. We developed a method that combines variant calling and a novel ctDNA fragment length analysis using hybrid capture sequencing data. Among 36 stage II-IIIA NSCLC patients, this method distinguished patients with and without recurrence of disease in a 20 times repeated 10-fold cross validation with 75% accuracy (P = 0.0029). In contrast, using only variant calling or only fragment length analysis, no signification distinction between patients was shown (P = 0.24 and P = 0.074 respectively). In addition, a variant-level fragmentation score was developed that was able to classify variants detected in plasma cfDNA into tumour-derived or white-blood-cell-derived variants with 84% accuracy. The findings in this study may help drive the integration of various types of information from the same data, eventually leading to cheaper and more sensitive techniques to be used in this challenging clinical setting.

Contains fulltext : 242594.pdf (Publisher’s version ) (Open Access), BACKGROUND: Bio-Rad droplet-digital PCR is a highly sensitive method that can be used to detect tumor mutations in circulating cell-free DNA (cfDNA) of patients with cancer. Correct interpretation of ddPCR results is important for optimal sensitivity and specificity. Despite its widespread use, no standardized method to interpret ddPCR data is available, nor have technical artifacts affecting ddPCR results been widely studied. METHODS: False positive rates were determined for 6 ddPCR assays at variable amounts of input DNA, revealing polymerase induced false positive events (PIFs) and other false positives. An in silico correction algorithm, known as the adaptive LoB and PIFs: an automated correction algorithm (ALPACA), was developed to remove PIFs and apply an adaptive limit of blank (LoB) to individual samples. Performance of ALPACA was compared to a standard strategy (no PIF correction and static LoB = 3) using data from commercial reference DNA, healthy volunteer cfDNA, and cfDNA from a real-life cohort of 209 patients with stage IV nonsmall cell lung cancer (NSCLC) whose tumor and cfDNA had been molecularly profiled. RESULTS: Applying ALPACA reduced false positive results in healthy cfDNA compared to the standard strategy (specificity 98 vs 88%, P = 10-5) and stage IV NSCLC patient cfDNA (99 vs 93%, P = 10-11), while not affecting sensitivity in commercial reference DNA (70 vs 68% P = 0.77) or patient cfDNA (82 vs 88%, P = 0.13). Overall accuracy in patient samples was improved (98 vs 92%, P = 10-7). CONCLUSIONS: Correction of PIFs and application of an adaptive LoB increases specificity without a loss of sensitivity in ddPCR, leading to a higher accuracy in a real-life cohort of patients with stage IV NSCLC.

Contains fulltext : 242641.pdf (Publisher’s version ) (Open Access), BACKGROUND: The widespread introduction of immunotherapy in patients with advanced non-small cell lung cancer (NSCLC) has led to durable responses but still many patients fail and are treated beyond progression. OBJECTIVE: This study investigated whether readily available blood-based tumor biomarkers allow accurate detection of early non-responsiveness, allowing a timely switch of therapy and cost reduction. METHODS: In a prospective, observational study in patients with NSCLC treated with nivolumab or pembrolizumab, five serum tumor markers were measured at baseline and every other week. Six months disease control as determined by RECIST was used as a measure of clinical response. Patients with a disease control < 6 months were deemed non-responsive. For every separate tumor marker a criterion for predicting of non-response was developed. Each marker test was defined as positive (predictive of non-response) if the value of that tumor marker increased at least 50% from the value at baseline and above a marker dependent minimum value to be determined. Also, tests based on combination of multiple markers were designed. Specificity and sensitivity for predicting non-response was calculated and results were validated in an independent cohort. The target specificity of the test for detecting non-response was set at > 95%, in order to allow its safe use for treatment decisions. RESULTS: A total of 376 patients (training cohort: 180, validation cohort: 196) were included in our analysis. Results for the specificity of the single marker tests in the validation set were CEA: 98·3% (95% CI: 90·9-100%), NSE: 96·5% (95% CI: 87·9-99·6%), SCC: 96·5% (95% CI: 88·1-99·6%), Cyfra21·1 : 91.8% (95% CI: 81·9-97·3%), and CA125 : 86·0% (95% CI: 74·2-93·7%). A test based on the combination of Cyfra21.1, CEA and NSE accurately predicted non-response in 32.3% (95% CI 22.6-43.1%) of patients 6 weeks after start of immunotherapy. Survival analysis showed a significant difference between pre

Item does not contain fulltext, Lactococcus lactis subsp. lactis KF147 as a non-dairy strain from lactic acid bacteria (LAB) can inhabit plant tissues. It can grow on complex carbohydrates derived from plant cell walls. Its genome size is one of the largest among the sequenced lactococcal strains, possessing many genes that do not have homologues in the published genome sequences of dairy-associated L. lactis strains. In silico analysis has identified a gene cluster encoding a hybrid NRPS-PKS system (composed of non-ribosomal peptide synthetases and polyketide synthases) in the L. lactis KF147 genome, as first example of a LAB possessing such hybrid mega-enzymes. Hybrid systems produce hybrid NRP-PK secondary metabolites (natural products) in a wide variety of bacteria, fungi, and plants. In the hybrid NRPS-PKS system of L. lactis KF147, a total of 21 NRPS and 8 PKS domains were identified that are arranged into 6 NRPS modules, 3 PKS modules, and two single functional domains (trans-acyl-transferase "transAT" and thioesterase). We found homologous hybrid systems having similar gene, module, and domain organization in six other L. lactis strains and 25 strains of the dental cariogenic Streptococcus mutans. This study mainly aimed to predict the structure and function of the hybrid NRP-PK product of L. lactis KF147 using comparative genomics techniques, and included a detailed analysis of the regulatory system. Various bioinformatical approaches were used to predict the substrate specificity of the six A domains and the iterative transAT domain. Functional conservation of the A domains within different-niche-associated strains supported the prediction of the primary core structure of the putative hybrid natural product to be Leu-DLeu-Asp-DAsn-Gly-MC-MC-MC-DAsp (MC = Malonyl-CoA). Oxidative stress resistance and biofilm formation are the most probable functions of this hybrid system. The need for such a system in two different niches is argued, as an adaptation of L. lactis and S. mutans to adhere to

Contains fulltext : 229836.pdf (Publisher’s version ) (Open Access), The ALSYMPCA study established a 3.6 month Overall Survival (OS) benefit in metastatic Castration Resistant Prostate Cancer (mCRPC) patients treated with Radium‐223 dichloride (Ra‐223) over placebo. Here we report clinical outcomes of Ra‐223 treatment in a nonstudy population. In this prospective registry, patients from 20 Dutch hospitals were included prior to Ra‐223 treatment. Clinical parameters collected included previous treatments and Adverse Events. Primary outcome was 6 months Symptomatic Skeletal Event (SSE)‐free survival, while secondary outcomes included Progression‐Free Survival (PFS) and Overall Survival (OS). Of the 305 patients included, 300 were evaluable. The mean age was 73.6 years, 90% had ≥6 bone metastases and 74.1% were pretreated with Docetaxel, 19.5% with Cabazitaxel and 80.5% with Abiraterone and/or Enzalutamide. Of all patients, 96.7% were treated with Ra‐223 and received a median of 5 cycles. After a median follow‐up of 13.2 months, 6 months SSE‐free survival rate was 83%, median PFS was 5.1 months and median OS was 15.2 months. Six months SSE‐free survival rate and OS were comparable with those reported in ALSYMPCA. “Previous Cabazitaxel treatment” and “bone‐only metastases” were independent predictors of a shorter and longer PFS, respectively, while above‐median LDH and “bone‐only metastases” were independent predictors of shorter and longer OS, respectively. Toxicity was similar as reported in the ALSYMPCA trial. These results suggest that in a nonstudy population, Ra‐223 treatment is well‐tolerated, equally effective as in the ALSYMPCA population and that patients not previously treated with Cabazitaxel benefit most from Ra‐223. Publisher: Abstract available from the publisher.

Contains fulltext : 215691.pdf (publisher's version ) (Closed access), BACKGROUND: Immune checkpoint inhibitors have improved survival outcome of advanced non-small-cell lung cancer (NSCLC). However, most patients do not benefit. Therefore, biomarkers are needed that accurately predict response. We hypothesized that molecular profiling of exhaled air may capture the inflammatory milieu related to the individual responsiveness to anti-programmed death ligand 1 (PD-1) therapy. This study aimed to determine the accuracy of exhaled breath analysis at baseline for assessing nonresponders versus responders to anti-PD-1 therapy in NSCLC patients. METHODS: This was a prospective observational study in patients receiving checkpoint inhibitor therapy using both a training and validation set of NSCLC patients. At baseline, breath profiles were collected in duplicate by a metal oxide semiconductor electronic nose (eNose) positioned at the rear end of a pneumotachograph. Patients received nivolumab or pembrolizumab of which the efficacy was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 at 3-month follow-up. Data analysis involved advanced signal-processing and statistics based on independent t-tests followed by linear discriminant and receiver operating characteristic (ROC) analysis. RESULTS: Exhaled breath data of 143 NSCLC patients (training: 92, validation: 51) were available at baseline. ENose sensors contributed significantly (P < 0.05) at baseline in differentiating between patients with different responses at 3 months of anti-PD-1 treatment. The eNose sensors were combined into a single biomarker with an ROC-area under the curve (AUC) of 0.89 [confidence interval (CI) 0.82-0.96]. This AUC was confirmed in the validation set: 0.85 (CI 0.75-0.96). CONCLUSION: ENose assessment was effective in the noninvasive prediction of individual patient responses to immunotherapy. The predictive accuracy and efficacy of the eNose for discrimination of immunotherapy responder types were replicated in an independent validation

Contains fulltext : 207015.pdf (publisher's version ) (Open Access), 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.

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