The evolution of non-small cell lung cancer metastases in TRACERx.

Metastatic disease is responsible for the majority of cancer-related deaths1. We report the longitudinal evolutionary analysis of 126 non-small cell lung cancer (NSCLC) tumours from 421 prospectively recruited patients in TRACERx who developed metastatic disease, compared with a control cohort of 144 non-metastatic tumours. In 25% of cases, metastases diverged early, before the last clonal sweep in the primary tumour, and early divergence was enriched for patients who were smokers at the time of initial diagnosis. Simulations suggested that early metastatic divergence more frequently occurred at smaller tumour diameters (less than 8 mm). Single-region primary tumour sampling resulted in 83% of late divergence cases being misclassified as early, highlighting the importance of extensive primary tumour sampling. Polyclonal dissemination, which was associated with extrathoracic disease recurrence, was found in 32% of cases. Primary lymph node disease contributed to metastatic relapse in less than 20% of cases, representing a hallmark of metastatic potential rather than a route to subsequent recurrences/disease progression. Metastasis-seeding subclones exhibited subclonal expansions within primary tumours, probably reflecting positive selection. Our findings highlight the importance of selection in metastatic clone evolution within untreated primary tumours, the distinction between monoclonal versus polyclonal seeding in dictating site of recurrence, the limitations of current radiological screening approaches for early diverging tumours and the need to develop strategies to target metastasis-seeding subclones before relapse.

The evolution of lung cancer and impact of subclonal selection in TRACERx.

Lung cancer is the leading cause of cancer-associated mortality worldwide1. Here we analysed 1,644 tumour regions sampled at surgery or during follow-up from the first 421 patients with non-small cell lung cancer prospectively enrolled into the TRACERx study. This project aims to decipher lung cancer evolution and address the primary study endpoint: determining the relationship between intratumour heterogeneity and clinical outcome. In lung adenocarcinoma, mutations in 22 out of 40 common cancer genes were under significant subclonal selection, including classical tumour initiators such as TP53 and KRAS. We defined evolutionary dependencies between drivers, mutational processes and whole genome doubling (WGD) events. Despite patients having a history of smoking, 8% of lung adenocarcinomas lacked evidence of tobacco-induced mutagenesis. These tumours also had similar detection rates for EGFR mutations and for RET, ROS1, ALK and MET oncogenic isoforms compared with tumours in never-smokers, which suggests that they have a similar aetiology and pathogenesis. Large subclonal expansions were associated with positive subclonal selection. Patients with tumours harbouring recent subclonal expansions, on the terminus of a phylogenetic branch, had significantly shorter disease-free survival. Subclonal WGD was detected in 19% of tumours, and 10% of tumours harboured multiple subclonal WGDs in parallel. Subclonal, but not truncal, WGD was associated with shorter disease-free survival. Copy number heterogeneity was associated with extrathoracic relapse within 1 year after surgery. These data demonstrate the importance of clonal expansion, WGD and copy number instability in determining the timing and patterns of relapse in non-small cell lung cancer and provide a comprehensive clinical cancer evolutionary data resource.

Neoadjuvant Nivolumab plus Chemotherapy in Resectable Lung Cancer.

BACKGROUND: Neoadjuvant or adjuvant chemotherapy confers a modest benefit over surgery alone for resectable non-small-cell lung cancer (NSCLC). In early-phase trials, nivolumab-based neoadjuvant regimens have shown promising clinical activity; however, data from phase 3 trials are needed to confirm these findings. METHODS: In this open-label, phase 3 trial, we randomly assigned patients with stage IB to IIIA resectable NSCLC to receive nivolumab plus platinum-based chemotherapy or platinum-based chemotherapy alone, followed by resection. The primary end points were event-free survival and pathological complete response (0% viable tumor in resected lung and lymph nodes), both evaluated by blinded independent review. Overall survival was a key secondary end point. Safety was assessed in all treated patients. RESULTS: The median event-free survival was 31.6 months (95% confidence interval [CI], 30.2 to not reached) with nivolumab plus chemotherapy and 20.8 months (95% CI, 14.0 to 26.7) with chemotherapy alone (hazard ratio for disease progression, disease recurrence, or death, 0.63; 97.38% CI, 0.43 to 0.91; P = 0.005). The percentage of patients with a pathological complete response was 24.0% (95% CI, 18.0 to 31.0) and 2.2% (95% CI, 0.6 to 5.6), respectively (odds ratio, 13.94; 99% CI, 3.49 to 55.75; P<0.001). Results for event-free survival and pathological complete response across most subgroups favored nivolumab plus chemotherapy over chemotherapy alone. At the first prespecified interim analysis, the hazard ratio for death was 0.57 (99.67% CI, 0.30 to 1.07) and did not meet the criterion for significance. Of the patients who underwent randomization, 83.2% of those in the nivolumab-plus-chemotherapy group and 75.4% of those in the chemotherapy-alone group underwent surgery. Grade 3 or 4 treatment-related adverse events occurred in 33.5% of the patients in the nivolumab-plus-chemotherapy group and in 36.9% of those in the chemotherapy-alone group. CONCLUSIONS: In patients with resectable NSCLC, neoadjuvant nivolumab plus chemotherapy resulted in significantly longer event-free survival and a higher percentage of patients with a pathological complete response than chemotherapy alone. The addition of nivolumab to neoadjuvant chemotherapy did not increase the incidence of adverse events or impede the feasibility of surgery. (Funded by Bristol Myers Squibb; CheckMate 816 ClinicalTrials.gov number, NCT02998528.).

Optimizing tissue stewardship in non-small cell lung cancer to support molecular characterization and treatment selection: statement from a working group of thoracic pathologists.

Many patients with non-small cell lung cancer do not receive guideline-recommended, biomarker-directed therapy, despite the potential for improved clinical outcomes. Access to timely, accurate, and comprehensive molecular profiling, including targetable protein overexpression, is essential to allow fully informed treatment decisions to be taken. In turn, this requires optimal tissue management to protect and maximize the use of this precious finite resource. Here, a group of leading thoracic pathologists recommend factors to consider for optimal tissue management. Starting from when lung cancer is first suspected, keeping predictive biomarker testing in the front of the mind should drive the development of practices and procedures that conserve tissue appropriately to support molecular characterization and treatment selection.

An endogenous inhibitor of angiogenesis downregulated by hypoxia in human aortic valve stenosis promotes disease pathogenesis.

Aortic valve stenosis is the most common valve disease in the western world. Central to the pathogenesis of this disease is the growth of new blood vessels (angiogenesis) within the aortic valve allowing infiltration of immune cells and development of intra-valve inflammation. Identifying the cellular mediators involved in this angiogenesis is important as this may reveal new therapeutic targets which could ultimately prevent the progression of aortic valve stenosis. Aortic valves from patients undergoing surgery for aortic valve replacement or dilation of the aortic arch were examined both ex vivo and in vitro. We now demonstrate that the anti-angiogenic protein, soluble fms-like tyrosine kinase 1 (sFlt1), a non-signalling soluble receptor for vascular endothelial growth factor, is constitutively expressed in non-diseased valves. sFlt-1 expression was, however, significantly reduced in aortic valve tissue from patients with aortic valve stenosis while protein markers of hypoxia were simultaneously increased. Exposure of primary-cultured valve interstitial cells to hypoxia resulted in a decrease in the expression of sFlt-1. We further reveal using a bioassay that siRNA knock-down of sFlt1 in valve interstitial cells directly results in a pro-angiogenic environment. Finally, incubation of aortic valves with sphingosine 1-phosphate, a bioactive lipid-mediator, increased sFlt-1 expression and inhibited angiogenesis within valve tissue. In conclusion, this study demonstrates that sFlt1 expression is directly correlated with angiogenesis in aortic valves and the observed decrease in sFlt-1 expression in aortic valve stenosis could increase valve inflammation, promoting disease progression. This could be a viable therapeutic target in treating this disease.

Plain language summary of the CheckMate 816 study results: nivolumab plus chemotherapy given before surgery for non-small-cell lung cancer.

WHAT IS THIS SUMMARY ABOUT?: In this article, we summarize results from the ongoing phase 3 CheckMate 816 clinical study that were published in The New England Journal of Medicine in 2022. The goal of CheckMate 816 was to find out if nivolumab, an immunotherapy that activates a person's immune system (the body's natural defense system) to fight cancer, plus chemotherapy works better than chemotherapy alone when given before surgery in people with non-small-cell lung cancer (NSCLC) that can be removed surgically (resectable NSCLC). WHAT HAPPENED IN THE STUDY?: Adults who had not previously taken medications to treat NSCLC and whose cancer could be removed with surgery were included in CheckMate 816. During this study, a computer randomly assigned the treatment each person would receive before surgery for NSCLC. In total, 179 people were randomly assigned to receive nivolumab plus chemotherapy, and 179 people were randomly assigned to receive chemotherapy alone. The researchers assessed whether people who received nivolumab plus chemotherapy lived longer without the cancer geting worse or coming back and whether there were any cancer cells left in the tumor and lymph nodes removed by surgery. The researchers also assessed how adding nivolumab to chemotherapy affected the timing and outcomes of surgery and whether the combination of these drugs was safe. WHAT WERE THE RESULTS?: Researchers found that people who took nivolumab plus chemotherapy lived longer without the cancer getting worse or coming back compared with those who took chemotherapy alone. More people in the nivolumab plus chemotherapy group had no cancer cells left in the tumor and lymph nodes removed by surgery. Most people went on to have surgery in both treatment groups; the people who took nivolumab plus chemotherapy instead of chemotherapy alone had less extensive surgeries and were more likely to have good outcomes after less extensive surgeries. Adding nivolumab to chemotherapy did not lead to an increase in the rate of side effects compared with chemotherapy alone, and side effects were generally mild and manageable. WHAT DO THE RESULTS OF THE STUDY MEAN?: Results from CheckMate 816 support the benefit of using nivolumab plus chemotherapy before surgery for people with resectable NSCLC. Clinical Trial Registration: NCT02998528 (ClinicalTrials.gov).

Strategies to Modulate MicroRNA Functions for the Treatment of Cancer or Organ Injury.

Cancer and organ injury-such as that occurring in the perioperative period, including acute lung injury, myocardial infarction, and acute gut injury-are among the leading causes of death in the United States and impose a significant impact on quality of life. MicroRNAs (miRNAs) have been studied extensively during the last two decades for their role as regulators of gene expression, their translational application as diagnostic markers, and their potential as therapeutic targets for disease treatment. Despite promising preclinical outcomes implicating miRNA targets in disease treatment, only a few miRNAs have reached clinical trials. This likely relates to difficulties in the delivery of miRNA drugs to their targets to achieve efficient inhibition or overexpression. Therefore, understanding how to efficiently deliver miRNAs into diseased tissues and specific cell types in patients is critical. This review summarizes current knowledge on various approaches to deliver therapeutic miRNAs or miRNA inhibitors and highlights current progress in miRNA-based disease therapy that has reached clinical trials. Based on ongoing advances in miRNA delivery, we believe that additional therapeutic approaches to modulate miRNA function will soon enter routine medical treatment of human disease, particularly for cancer or perioperative organ injury. SIGNIFICANCE STATEMENT: MicroRNAs have been studied extensively during the last two decades in cancer and organ injury, including acute lung injury, myocardial infarction, and acute gut injury, for their regulation of gene expression, application as diagnostic markers, and therapeutic potentials. In this review, we specifically emphasize the pros and cons of different delivery approaches to modulate microRNAs, as well as the most recent exciting progress in the field of therapeutic targeting of microRNAs for disease treatment in patients.

Large cell neuroendocrine lung carcinoma: consensus statement from The British Thoracic Oncology Group and the Association of Pulmonary Pathologists.

Over the past 10 years, lung cancer clinical and translational research has been characterised by exponential progress, exemplified by the introduction of molecularly targeted therapies, immunotherapy and chemo-immunotherapy combinations to stage III and IV non-small cell lung cancer. Along with squamous and small cell lung cancers, large cell neuroendocrine carcinoma (LCNEC) now represents an area of unmet need, particularly hampered by the lack of an encompassing pathological definition that can facilitate real-world and clinical trial progress. The steps we have proposed in this article represent an iterative and rational path forward towards clinical breakthroughs that can be modelled on success in other lung cancer pathologies.

Navigating Diagnostic and Treatment Decisions in Non-Small Cell Lung Cancer: Expert Commentary on the Multidisciplinary Team Approach.

Non-small cell lung cancer (NSCLC) accounts for approximately one in five cancer-related deaths, and management requires increasingly complex decision making by health care professionals. Many centers have therefore adopted a multidisciplinary approach to patient care, using the expertise of various specialists to provide the best evidence-based, personalized treatment. However, increasingly complex disease staging, as well as expanded biomarker testing and multimodality management algorithms with novel therapeutics, have driven the need for multifaceted, collaborative decision making to optimally guide the overall treatment process. To keep up with the rapidly evolving treatment landscape, national-level guidelines have been introduced to standardize patient pathways and ensure prompt diagnosis and treatment. Such strategies depend on efficient and effective communication between relevant multidisciplinary team members and have both improved adherence to treatment guidelines and extended patient survival. This article highlights the value of a multidisciplinary approach to diagnosis and staging, treatment decision making, and adverse event management in NSCLC. IMPLICATIONS FOR PRACTICE: This review highlights the value of a multidisciplinary approach to the diagnosis and staging of non-small cell lung cancer (NSCLC) and makes practical suggestions as to how multidisciplinary teams (MDTs) can be best deployed at individual stages of the disease to improve patient outcomes and effectively manage common adverse events. The authors discuss how a collaborative approach, appropriately leveraging the diverse expertise of NSCLC MDT members (including specialist radiation and medical oncologists, chest physicians, pathologists, pulmonologists, surgeons, and nursing staff) can continue to ensure optimal per-patient decision making as treatment options become ever more specialized in the era of biomarker-driven therapeutic strategies.

Tracking the Evolution of Non-Small-Cell Lung Cancer.

BACKGROUND: Among patients with non-small-cell lung cancer (NSCLC), data on intratumor heterogeneity and cancer genome evolution have been limited to small retrospective cohorts. We wanted to prospectively investigate intratumor heterogeneity in relation to clinical outcome and to determine the clonal nature of driver events and evolutionary processes in early-stage NSCLC. METHODS: In this prospective cohort study, we performed multiregion whole-exome sequencing on 100 early-stage NSCLC tumors that had been resected before systemic therapy. We sequenced and analyzed 327 tumor regions to define evolutionary histories, obtain a census of clonal and subclonal events, and assess the relationship between intratumor heterogeneity and recurrence-free survival. RESULTS: We observed widespread intratumor heterogeneity for both somatic copy-number alterations and mutations. Driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal. However, heterogeneous driver alterations that occurred later in evolution were found in more than 75% of the tumors and were common in PIK3CA and NF1 and in genes that are involved in chromatin modification and DNA damage response and repair. Genome doubling and ongoing dynamic chromosomal instability were associated with intratumor heterogeneity and resulted in parallel evolution of driver somatic copy-number alterations, including amplifications in CDK4, FOXA1, and BCL11A. Elevated copy-number heterogeneity was associated with an increased risk of recurrence or death (hazard ratio, 4.9; P=4.4×10-4), which remained significant in multivariate analysis. CONCLUSIONS: Intratumor heterogeneity mediated through chromosome instability was associated with an increased risk of recurrence or death, a finding that supports the potential value of chromosome instability as a prognostic predictor. (Funded by Cancer Research UK and others; TRACERx ClinicalTrials.gov number, NCT01888601 .).

Programmed death-ligand 1 expression influenced by tissue sample size. Scoring based on tissue microarrays' and cross-validation with resections, in patients with, stage I-III, non-small cell lung carcinoma of the European Thoracic Oncology Platform Lungscape cohort.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

Defining aggressive or early progressing nononcogene-addicted non-small-cell lung cancer: a separate disease entity?

A substantial proportion of patients with nononcogene-addicted non-small-cell lung cancer (NSCLC) has 'aggressive disease', as reflected in short time to progression or lack of disease control with initial platinum-based chemotherapy. Recently, clinical correlates of aggressive disease behavior during first-line therapy have been shown to predict greater benefit from addition of nintedanib to second-line docetaxel in adenocarcinoma NSCLC. Positive predictive effects of aggressive disease have since been reported with other anti-angiogenic agents (ramucirumab and bevacizumab), while such features may negatively impact on outcomes with nivolumab in nonsquamous NSCLC with low PD-L1 expression. Based on a review of the clinical data, we recommend aggressive nonsquamous NSCLC should be defined by progression within <6-9 months of first-line treatment initiation.

Whole Transcriptome Analysis of Pre-invasive and Invasive Early Squamous Lung Carcinoma in Archival Laser Microdissected Samples.

BACKGROUND: Preinvasive squamous cell cancer (PSCC) are local transformations of bronchial epithelia that are frequently observed in current or former smokers. Their different grades and sizes suggest a continuum of dysplastic change with increasing severity, which may culminate in invasive squamous cell carcinoma (ISCC). As a consequence of the difficulty in isolating cancerous cells from biopsies, the molecular pathology that underlies their histological variability remains largely unknown. METHOD: To address this issue, we have employed microdissection to isolate normal bronchial epithelia and cancerous cells from low- and high-grade PSCC and ISCC, from paraffin embedded (FFPE) biopsies and determined gene expression using Affymetric Human Exon 1.0 ST arrays. Tests for differential gene expression were performed using the Bioconductor package limma followed by functional analyses of differentially expressed genes in IPA. RESULTS: Examination of differential gene expression showed small differences between low- and high-grade PSCC but substantial changes between PSCC and ISCC samples (184 vs 1200 p-value <0.05, fc ±1.75). However, the majority of the differentially expressed PSCC genes (142 genes: 77%) were shared with those in ISCC samples. Pathway analysis showed that these shared genes are associated with DNA damage response, DNA/RNA metabolism and inflammation as major biological themes. Cluster analysis identified 12 distinct patterns of gene expression including progressive up or down-regulation across PSCC and ISCC. Pathway analysis of incrementally up-regulated genes revealed again significant enrichment of terms related to DNA damage response, DNA/RNA metabolism, inflammation, survival and proliferation. Altered expression of selected genes was confirmed using RT-PCR, as well as immunohistochemistry in an independent set of 45 ISCCs. CONCLUSIONS: Gene expression profiles in PSCC and ISCC differ greatly in terms of numbers of genes with altered transcriptional activity. However, altered gene expression in PSCC affects canonical pathways and cellular and biological processes, such as inflammation and DNA damage response, which are highly consistent with hallmarks of cancer.

Tracking genomic cancer evolution for precision medicine: the lung TRACERx study.

The importance of intratumour genetic and functional heterogeneity is increasingly recognised as a driver of cancer progression and survival outcome. Understanding how tumour clonal heterogeneity impacts upon therapeutic outcome, however, is still an area of unmet clinical and scientific need. TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy [Rx]), a prospective study of patients with primary non-small cell lung cancer (NSCLC), aims to define the evolutionary trajectories of lung cancer in both space and time through multiregion and longitudinal tumour sampling and genetic analysis. By following cancers from diagnosis to relapse, tracking the evolutionary trajectories of tumours in relation to therapeutic interventions, and determining the impact of clonal heterogeneity on clinical outcomes, TRACERx may help to identify novel therapeutic targets for NSCLC and may also serve as a model applicable to other cancer types.

Diagnostic procedures for non-small-cell lung cancer (NSCLC): recommendations of the European Expert Group.

BACKGROUND: There is currently no Europe-wide consensus on the appropriate preanalytical measures and workflow to optimise procedures for tissue-based molecular testing of non-small-cell lung cancer (NSCLC). To address this, a group of lung cancer experts (see list of authors) convened to discuss and propose standard operating procedures (SOPs) for NSCLC. METHODS: Based on earlier meetings and scientific expertise on lung cancer, a multidisciplinary group meeting was aligned. The aim was to include all relevant aspects concerning NSCLC diagnosis. After careful consideration, the following topics were selected and each was reviewed by the experts: surgical resection and sampling; biopsy procedures for analysis; preanalytical and other variables affecting quality of tissue; tissue conservation; testing procedures for epidermal growth factor receptor, anaplastic lymphoma kinase and ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) in lung tissue and cytological specimens; as well as standardised reporting and quality control (QC). Finally, an optimal workflow was described. RESULTS: Suggested optimal procedures and workflows are discussed in detail. The broad consensus was that the complex workflow presented can only be executed effectively by an interdisciplinary approach using a well-trained team. CONCLUSIONS: To optimise diagnosis and treatment of patients with NSCLC, it is essential to establish SOPs that are adaptable to the local situation. In addition, a continuous QC system and a local multidisciplinary tumour-type-oriented board are essential.

PD-L1 testing for lung cancer in the UK: recognizing the challenges for implementation.

A new approach to the management of non-small-cell lung cancer (NSCLC) has recently emerged that works by manipulating the immune checkpoint controlled by programmed death receptor 1 (PD-1) and its ligand programmed death ligand 1 (PD-L1). Several drugs targeting PD-1 (pembrolizumab and nivolumab) or PD-L1 (atezolizumab, durvalumab, and avelumab) have been approved or are in the late stages of development. Inevitably, the introduction of these drugs will put pressure on healthcare systems, and there is a need to stratify patients to identify those who are most likely to benefit from such treatment. There is evidence that responsiveness to PD-1 inhibitors may be predicted by expression of PD-L1 on neoplastic cells. Hence, there is considerable interest in using PD-L1 immunohistochemical staining to guide the use of PD-1-targeted treatments in patients with NSCLC. This article reviews the current knowledge about PD-L1 testing, and identifies current research requirements. Key factors to consider include the source and timing of sample collection, pre-analytical steps (sample tracking, fixation, tissue processing, sectioning, and tissue prioritization), analytical decisions (choice of biomarker assay/kit and automated staining platform, with verification of standardized assays or validation of laboratory-devised techniques, internal and external quality assurance, and audit), and reporting and interpretation of the results. This review addresses the need for integration of PD-L1 immunohistochemistry with other tests as part of locally agreed pathways and protocols. There remain areas of uncertainty, and guidance should be updated regularly as new information becomes available.

Immune Checkpoint Blockade: A New Era for Non-Small Cell Lung Cancer.

Despite better understanding of it's molecular biology, non-small cell lung cancer (NSCLC) remains a challenging disease to treat. Unfortunately, treatment options are still very limited and prognosis for advanced disease is poor. Immune surveillance plays a crucial role in a host's defence against tumour cells, and this is particular relevant for lung cancer due to it's high somatic mutational load, which increases the chances for the immune system to recognize cancer cells as 'non-self'. Novel immunotherapies are emerging as an effective treatment for this disease. In this review, we present the data on immune checkpoint inhibitors for NSCLC, describing their mechanism of action, data efficacy from recent clinical trials, and strategies to select patients more likely to benefit from these agents.

BAI3, CDX2 and VIL1: a panel of three antibodies to distinguish small cell from large cell neuroendocrine lung carcinomas.

AIMS: Discriminating small-cell lung carcinoma (SCLC) from large-cell neuroendocrine carcinoma (LCNEC) rests on morphological criteria, and reproducibility has been shown to be poor. We aimed to identify immunohistochemical markers to assist this diagnosis. METHODS AND RESULTS: Gene expression profiling on laser captured frozen tumour samples from eight SCLC and eight LCNEC tumours identified a total of 888 differentially expressed genes (DEGs), 23 of which were validated by qRT-PCR. Antibodies to four selected gene products were then evaluated as immunohistochemical markers on a cohort of 173 formalin-fixed paraffin-embedded (FFPE) SCLC/LCNEC tumour samples, including 26 indeterminate tumours without a consensus diagnosis. Three markers, CDX2, VIL1 and BAI3, gave significantly different results in the two tumour types (P < 0.0001): CDX2 and VIL1 in combination (either marker positive) showed sensitivity and specificity of 81% for LCNEC while BAI3 showed 89% sensitivity and 75% specificity for SCLC. Of the 26 indeterminate tumours 15 (58%) showed an immunophenotype suggesting either SCLC or LCNEC, eight (31%) showed staining of both tumour types, and three (11%) were negative for all markers. CONCLUSION: A panel of three markers, BAI3, CDX2 and VIL1, is a useful adjunct in the diagnosis of these tumour types.