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Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive and accurate method for quantication of nucleic acid sequences. We used absolute quantication of mutated v-Ki-ras2 Kirsten rat sarcoma viral oncogene homology gene (KRAS) by ddPCR to investigate the prognostic role of mutated KRAS in patients with KRAS-mutated lung adenocarcinomas. Pre-treatment plasma samples from 60 patients with stages I–IV KRAS-mutated lung adenocarcinomas were analysed for KRAS mutations. The associations between survival, detectable KRAS mutations in plasma, and the plasma concentration of mutated KRAS were assessed. Overall, 23 of 60 (38%)patients had detectable KRAS mutation in plasma. The percentage of patients with detectable mutation was 8%in stage I, 30% in stage II, 71% in stage III, and 73% in stage IV. Estimated overall median progression-free survival (PFS) and overall survival (OS) were 26.2 months [95% condence interval (CI) 12.5–39.9] and50.8 months (95% CI 0–107.3), respectively. Patients with detectable mutations in plasma had signicantlyworse median PFS compared to patients with undetectable mutation (13.1 versus 70.1 months) and shorter median OS (20.7 versus not reached). High circulating tumour DNA (ctDNA) concentrations of mutated KRAS were signicantly associated with shorter PFS [hazard ratio (HR) 1.008, 95% CI 1.004–1.012] and OS(HR 1.007, 95% CI 1.003–1.011). All associations remained statistically signicant in multivariable analyses. Inconclusion, ddPCR is an accurate and easily feasible technique for quantication of KRAS mutations in ctDNA.The presence of detectable KRAS mutation in plasma at baseline was associated with worse PFS and OS. High concentration of mutated KRAS in ctDNA was an independent negative prognostic factor for both PFS and OS. © 2021 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland & John Wiley & Sons, Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Lung cancer is the leading cause of cancer death in both sexes worldwide and has a predicted 5-year survival rate of

Objectives: A prerequisite for utilizing the tumour, lymph-nodes, and metastases (TNM) for the staging of lung cancer patients is a high quality of the reported data on which the staging is based. The aim of this study was to investigate the concordance between the clinical, cTNM and the pathology, pTNM staging for lung cancer, version 8 as reported to the Cancer Registry of Norway (CRN). Materials and Methods: A total of 1284 patients who underwent surgery 2018–2019 with sufficient data regarding both clinical and pathology T and N descriptors were included. Results: The differences in tumour diameter reported in the clinical and the pathology notifications were ≤5 mm and ≤10 mm in 65.9 % and in 84.4 % of the cases, respectively. For the c- and pT categories, there was concordance in 53.4 % while 28.4 % were upstaged and 18.2 % were downstaged. For N categories there was concordance in 83.3 % while 13.7 % were upstaged and 3.0 % were downstaged. Unforeseen pN2 was found in 6.2 % of the cases. For TNM staging groups there was concordance in 48.1 % of the cases, while 33.4 % were upstaged and 18.5 % were downstaged. The calculated sensitivity and specificity for reported cTNM staging as diagnostic test for being eligible for adjuvant treatment (stage II–IIIA) were 0.65 and 0.91, respectively. Conclusions: These data on staging for lung cancer, as reported to the CRN, shows a disappointingly low precision and concordance in c- and pTNM staging. This urges a strategy for a marked improvement.

Background: due to emerging therapeutics targeting KRAS G12C and previous reports with conflicting results regarding the prognostic impact of KRAS and KRAS G12C in non-small cell lung cancer (NSCLC), we aimed to investigate the frequency of KRAS mutations and their associations with clinical characteristics and outcome. Since mutation subtypes have different preferences for downstream pathways, we also aimed to investigate whether there were differences in outcome according to mutation preference for the Raf, PI3K/Akt, or RalGDS/Ral pathways. Methods: retrospectively, clinicopathological data from 1233 stage I–IV non-squamous NSCLC patients with known KRAS status were reviewed. KRAS’ associations with clinical characteristics were analysed. Progression free survival (PFS) and overall survival (OS) were assessed for the following groups: KRAS wild type (wt) versus mutated, KRAS wt versus KRAS G12C versus KRAS non-G12C, among KRAS mutation subtypes and among mutation subtypes grouped according to preference for downstream pathways. Results: a total of 1117 patients were included, 38% had KRAS mutated tumours, 17% had G12C. Among KRAS mutated, G12C was the most frequent mutation in former/current smokers (45%) and G12D in never smokers (46%). There were no significant differences in survival according to KRAS status, G12C status, among KRAS mutation subtypes or mutation preference for downstream pathways. Conclusion: KRAS status or KRAS mutation subtype did not have any significant influence on PFS or OS.

Simple Summary Lung cancer is the leading cause of cancer-related death worldwide, accounting for nearly one-fifth of all cancer-related deaths. Immunotherapy with immune checkpoint inhibitors has become one of the most promising approaches in the treatment of advanced lung cancer, although beneficial responses are seen only in a proportion of patients. To improve immunotherapy treatment responses in lung cancer, we need to identify which immunosuppression mechanisms are activated in the tumor microenvironment. In this study, we investigated gene expression profiles in intra-tumoral immune cells in lung cancer, focusing on tumor-associated macrophages, and interactions with CD4+ and CD8+ T cells. Our data highlight two newly described immunosuppressive pathways, which may represent novel innate immune checkpoints dampening the anti-tumor T cell immune response in lung cancer. Our results substantiate the importance of tumor-associated macrophages as a mediator of immunosuppression and a promising target for immunotherapy. Abstract Non-small cell lung carcinoma (NSCLC) is one of the most commonly diagnosed cancers and a leading cause of cancer-related deaths. Immunotherapy with immune checkpoint inhibitors shows beneficial responses, but only in a proportion of patients. To improve immunotherapy in NSCLC, we need to map the immune checkpoints that contribute immunosuppression in NSCLC-associated immune cells and to identify novel pathways that regulate immunosuppression. Here, we investigated the gene expression profiles of intra-tumoral immune cells isolated from NSCLC patients and compared them to the expression profiles of their counterparts in adjacent healthy tissue. Transcriptome analysis was performed on macrophages, CD4+ and CD8+ T cells. The data was subjected to Gene Ontology (GO) term enrichment and weighted correlation network analysis in order to identify mediators of immunosuppression in the tumor microenvironment in NSCLC. Immune cells from NSCLC revealed a consistent differential expression of genes involved in interactions between myeloid cells and lymphocytes. We further identified several immunosuppressive molecules and pathways that may be activated in tumor-associated macrophages in NSCLC. Importantly, we report novel data on immune cell expression of the newly described CD200/CD200R1 pathway, and the leukocyte immunoglobulin-like receptors (LILRs), which may represent novel innate immune checkpoints, dampening the anti-tumor T cell immune response in NSCLC. Our study substantiates the importance of tumor-associated macrophages as a mediator of immunosuppression and a promising target for immunotherapy.

Background It is unclear if smoking-related DNA methylation represents a causal pathway between smoking and risk of lung cancer. We sought to identify novel smoking-related DNA methylation sites in blood, with repeated measurements, and to appraise the putative role of DNA methylation in the pathway between smoking and lung cancer development. Methods We derived a nested case-control study from the Trøndelag Health Study (HUNT), including 140 incident patients who developed lung cancer during 2009–13 and 140 controls. We profiled 850 K DNA methylation sites (Illumina Infinium EPIC array) in DNA extracted from blood that was collected in HUNT2 (1995–97) and HUNT3 (2006–08) for the same individuals. Epigenome-wide association studies (EWAS) were performed for a detailed smoking phenotype and for lung cancer. Two-step Mendelian randomization (MR) analyses were performed to assess the potential causal effect of smoking on DNA methylation as well as of DNA methylation (13 sites as putative mediators) on risk of lung cancer. Results The EWAS for smoking in HUNT2 identified associations at 76 DNA methylation sites (P Conclusions DNA methylation modifications in blood did not seem to represent a causal pathway linking smoking and the lung cancer risk.

Introduction Studies have indicated that detection of mutated KRAS or EGFR in circulating tumor DNA (ctDNA) from pre-treatment plasma samples is a negative prognostic factor for non-small cell lung cancer (NSCLC) patients. This study aims to investigate whether this is the case also for NSCLC patients with other tumor mutations. Methods Tumor tissue DNA from 107 NSCLC patients was sequenced and corresponding pre-treatment plasma samples were analyzed using a limited target next-generation sequencing approach validated in this study. Patients without detected mutations in tumor samples were excluded from further analyses. Results Mutations were detected in tumor samples from 71 patients. Median age was 68 years, 51% were female, and 88% were current/former smokers, 91% had adenocarcinoma, 4% had squamous cell carcinoma and 6% had other NSCLC. The distribution between stage I, II, III and IV was 33%, 8%, 30%, and 29%, respectively. Between one and three tumor mutation(s) were detected in ctDNA from corresponding plasma samples. Patients with detected ctDNA had shorter PFS (9.6 vs. 41.3 months, HR: 2.9, 95% CI: 1.6–5.2, p = 0.0003) and OS (13.6 vs. 115.0 months, HR: 4.0, 95% CI: 2.1–7.6, p = 0.00002) than patients without detected ctDNA. ctDNA remained a significant negative prognostic factor for OS (HR: 2.5, 95% CI: 1.1-5.7, p=0.0327), but not PFS, in the multivariable analyses adjusting for baseline patient and disease characteristics including stage of disease. Conclusions This study adds further evidence supporting that detectable tumor mutations in cfDNA is associated with a worse prognosis in NSCLC harboring a variety of tumor mutations.

Large prospective studies on asthma, especially asthma symptom control, as a potential risk factor for lung cancer are limited. We followed up 62,791 cancer-free Norwegian adults from 1995–1997 to 2017. Self-reported doctor-diagnosed asthma was categorized into active and non-active asthma. Levels of asthma symptom control were classified into controlled and partially controlled (including partly controlled and uncontrolled) according to the Global Initiative for Asthma guidelines. Incident lung cancer cases were ascertained from the Cancer Registry of Norway. Cox regression models were used to estimate hazard ratios (HRs) with 95% confidence intervals (CIs) for possible associations. Totally, 984 participants developed lung cancer during a median follow-up of 21.1 years. After adjustment for smoking and other potential confounders, an increased incidence of lung cancer was found for adults with partially controlled asthma (HR 1.39, 95% CI 1.00–1.92) compared with those without asthma at baseline. Adults with active asthma had a tendency of increased lung cancer incidence (HR 1.29, 95% CI 0.95–1.75). Sensitivity analyses indicated that the observed associations were less likely resulted from reverse causation or residual confounding by smoking. Our findings suggested that proper control of asthma symptoms might contribute to a reduced incidence of lung cancer. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Studies have indicated that detection of circulating tumor DNA (ctDNA) prior to treatment is a negative prognostic marker in non-small cell lung cancer (NSCLC). ctDNA is currently identified by detection of tumor mutations. Commercial next-generation sequencing (NGS) assays for mutation analysis of ctDNA for routine practice usually include small gene panels and are not suitable for general mutation analysis. In this study, we investigated whether mutation analysis of cfDNA could be performed using a commercially available comprehensive NGS gene panel and bioinformatics workflow. Tumor DNA, plasma DNA and peripheral blood leukocyte DNA from 30 NSCLC patients were sequenced. In two patients (7%), tumor mutations in cfDNA were immediately called by the bioinformatic workflow. In 13 patients (43%), tumor mutations were not called, but were present in ctDNA and were identified based on the known tumor mutation profile. In the remaining 15 patients (50%), no concordant mutations were detected. In conclusion, we were able to identify tumor mutations in ctDNA from 57% of NSCLC patients using a comprehensive gene panel. We demonstrated that sequencing paired tumor DNA was helpful to interpret data and confirm ctDNA, and thus increased the ratio of patients with detectable ctDNA. This approach might be feasible for mutation analysis of ctDNA in routine diagnostic practice, especially in case of suboptimal plasma quality and quantity.

We have studied the alterations in the use of curative treatment and the outcome for lung cancer patients in Norway 2001–2016. The Cancer Registry of Norway has a practically complete registration of all cancer diagnoses, treatments given and deaths. For the years 2001–2016, 43,137 patients were diagnosed with lung cancer. Stereotactic radiotherapy was established nationwide from 2008 and its use has increased, and in 2016, 8.8% were given this treatment. In addition 20.6% were operated and 8.5% were treated with conventional radiotherapy. Thus 37.9% of those diagnosed were treated with intention to cure, compared to 22.9% in 2001 (p

Purpose Circulating tumor DNA (ctDNA) can potentially be used to monitor cancer treatment response. In this study, we developed and used a sensitive and selective ultra-deep next-generation sequencing (NGS) approach for detection of ctDNA in lung cancer patients. Method Most studies use fixed gene panels for detection of ctDNA by NGS. Deep sequencing of large target regions is expensive and generates a myriad of variants, which are difficult to interpret. For the purpose of using ctDNA for monitoring, we try to overcome these challenges by sequencing only selected regions where mutations were previously found in the tumor. This method is more sensitive and cost-efficient. In this study, we included about 80 lung cancer patients. Tumor DNA was sequenced with a large gene panel and somatic mutations were identified in each tumor. We then defined the target region according to the tumor mutations and selected primers for constructing NGS libraries. Since the coverage region is small it enables ultra-deep sequencing of several samples simultaneously. We evaluated the lower limit of detection for this method by sequencing a series of artificially constructed samples with known mutant allele frequencies (MAFs). DNA from peripheral blood leucocytes was sequenced in parallel with plasma DNA to exclude variants from clonal hematopoiesis. Preliminary results Using 40 ng as input DNA, which contains approximately 12,000 haploid genomes, we detected mutations down to 0.02% MAF. This corresponds to about 6 mutated copies per mL plasma. Currently, we are applying this method on patient plasma DNA samples. Conclusion We have developed a sensitive method for ctDNA detection, and we will present the results of the lung cancer patient samples. Citation Format: Anine Larsen Ottestad, Elisabeth F. Emdal, Sissel G. Wahl, Bjørn Henning Grønberg, Hong Yan Dai. Ultra-deep next-generation sequencing of selected single genes for detecting circulating tumor DNA in lung cancer 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 3099.

Background Cancer of unknown primary site is still a demanding condition as it is per definition metastatic, with heterogeneous biological behavior, and it is often resistant to therapy. Cancer of unknown primary site accounts for approximately 1 to 5 % of all cancers, but is currently among the top six causes of cancer deaths in Western countries. To correctly identify the biological origin of the tumor, a large spectrum of differential diagnoses must be considered and scrutinized. At progression, re-biopsy might be necessary to reveal the true origin of the tumor or actionable targets. Case presentation A 62-year-old Norwegian woman, with a fast growing lump in her left groin, was primarily diagnosed as having undifferentiated carcinoma that was BRAF V600 positive. There was complete response with paclitaxel-carboplatin and she was recurrence-free for 18 months. She had recurrence in both lungs and subcutaneously in her left groin and thigh; a re-biopsy revealed transformation to a malignant melanoma. She was resistant to BRAF inhibitors, then treated with ipilimumab and is currently a long-term survivor of 4 years and 4 months since the first diagnosis, with no clinical or radiological evidence of recurrence. Conclusions A biopsy from patients with metastasis of unknown primary should be analyzed thoroughly to identify organ of origin, molecular make-up, and possible molecular targets. Re-biopsy of cancer of unknown primary site at progression can reveal the true cellular origin of the tumor as well as provide novel therapeutic opportunities, including immunotherapy. © The Author(s). 2017. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates and appears highly promising as a supplement to the tumor-node-metastasis (TNM) classification of various tumors. In colorectal cancer, an international task force has initiated prospective multicenter studies aiming to implement TNM-Immunoscore (TNM-I) in a routine clinical setting. In breast cancer, recommendations for the evaluation of tumor-infiltrating lymphocytes (TILs) have been proposed by an international working group. Regardless of promising results, there are potential obstacles related to implementing TNM-I into the clinic. Diverse methods may be needed for different malignancies and even within each cancer entity. Nevertheless, a uniform approach across malignancies would be advantageous. In nonsmall-cell lung cancer (NSCLC), there are several previous reports indicating an apparent prognostic importance of TILs, but studies on TILs in a TNM-I setting are sparse and no general recommendations are made. However, recently published data is promising, evoking a realistic hope of a clinical useful NSCLC TNM-I. This review will focus on the TNM-I potential in NSCLC and propose strategies for clinical implementation of a TNM-I in resected NSCLC.

Epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) are a relatively new class of drugs for treatment of non-small-cell lung cancer. The national professional group for lung cancer, The Norwegian Lung Cancer Group, recommends that patients with non-small-cell lung cancer are tested for mutations in the EGFR gene. Here, we report the experience collected after the introduction of such testing in Norway in 2010.Information on the number of patients tested, gender distribution, histopathological data and analysis results have been collected from the molecular-pathology laboratories at the university hospitals in Tromsø, Trondheim, Bergen and Oslo for the period from May 2010 to May 2011.During this period, altogether 1,058 patients with lung cancer were tested for mutations in the EGFR gene, equal to approximately half of all those who were diagnosed with non-small-cell lung cancer. A mutation was detected in 123 patients (11.6 per cent). There was a higher proportion of mutation-positive women than men (17.6 per cent, compared to 6.3 per cent, p0.001), and a lower proportion with squamous cell carcinoma than for other histopathological subtypes (3.0 per cent, compared to 12.9 per cent, p0.001). Of a total of 80 cytological tests, nine (11.3 per cent) were positive.In light of the relatively high mutation frequency and a considerable number of positives in the group with squamous cell carcinoma, we recommend to continue the practice of mutation-testing all patients with non-small-cell lung cancer.

BACKGROUND Epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) are a relatively new class of drugs for treatment of non-small-cell lung cancer. The national professional group for lung cancer, The Norwegian Lung Cancer Group, recommends that patients with non-small-cell lung cancer are tested for mutations in the EGFR gene. Here, we report the experience collected after the introduction of such testing in Norway in 2010. MATERIAL AND METHOD Information on the number of patients tested, gender distribution, histopathological data and analysis results have been collected from the molecular-pathology laboratories at the university hospitals in Tromso, Trondheim, Bergen and Oslo for the period from May 2010 to May 2011. RESULTS During this period, altogether 1,058 patients with lung cancer were tested for mutations in the EGFR gene, equal to approximately half of all those who were diagnosed with non-small-cell lung cancer. A mutation was detected in 123 patients (11.6 per cent). There was a higher proportion of mutation-positive women than men (17.6 per cent, compared to 6.3 per cent, p < 0.001), and a lower proportion with squamous cell carcinoma than for other histopathological subtypes (3.0 per cent, compared to 12.9 per cent, p < 0.001). Of a total of 80 cytological tests, nine (11.3 per cent) were positive. INTERPRETATION In light of the relatively high mutation frequency and a considerable number of positives in the group with squamous cell carcinoma, we recommend to continue the practice of mutation-testing all patients with non-small-cell lung cancer.