Novel oncogene and tumor suppressor mutations in KIT and PDGFRA wild type gastrointestinal stromal tumors revealed by next generation sequencing

Among gastrointestinal stromal tumors (GISTs) of 10–15% are negative for KIT and PDGFRA, and most of these cases are SDH deficient. Recent studies have provided data on additional molecular alterations such as KRAS in KIT mutant GISTs. We aimed to assess the frequency and spectrum of somatic mutations in common oncogenes as well as copy number variations in GISTs negative for KIT and PDGFRA mutations. GISTs with wild type KIT/PDGFRA were tested via next generation sequencing for somatic mutations in 341 genes. SDHB immunohistochemistry to evaluate for SDH deficiency was also performed. Of 267 GISTs tested for KIT and PDGFRA mutations, 15 were wild type, of which eight cases had material available for further testing. All eight cases had loss of SDHB expression and had various molecular alterations involving ARID1A, TP53, and other genes. One case had a KRAS G12V (c.35G>T) mutation in both the primary gastric tumor and a post‐imatinib recurrence. This tumor had anaplastic features and was resistant to multiple tyrosine kinase inhibitors, ultimately resulting in cancer‐related mortality within 2 years of diagnosis. In conclusion, KRAS mutations occur in rare GISTs with wild type KIT and PDGFRA. These tumors may display immunohistochemical positivity for KIT and primary resistance to tyrosine kinase inhibitors. © 2014 Wiley Periodicals, Inc.     

High density DNA array analysis reveals distinct genomic profiles in a subset of gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or platelet‐derived growth facter receptor (PDGFRA). Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10–15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large‐scale genomic alterations, but little is known about the mutation‐negative tumors. Using genome‐wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/PDGFRA mutation‐negative tumors from nine adults and one pediatric case and 21 mutant tumors. Although all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, eight of the 10 KIT/PDGFRA mutation‐negative GISTs exhibited few or no genomic alterations. One KIT/PDGFRA mutation‐negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine‐threonine kinase BRAF. The only other mutation‐negative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome arm 9p. Similar findings in several KIT‐mutant GISTs identified a minimal overlapping region of deletion of ～0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell‐cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs. © 2009 Wiley‐Liss, Inc.     

Expression of KIT Receptor Tyrosine Kinase in Endothelial Cells of Juvenile Brain Tumors

KIT receptor tyrosine kinase is expressed in tumor endothelial cells of adult glioblastomas, but its expression in pediatric brain tumor endothelial cells is unknown. We assessed expression of KIT, phosphorylated KIT, stem cell factor (SCF) and vascular endothelial growth factor receptor-2 (VEGFR-2) in 35 juvenile pilocytic astrocytomas and 49 other pediatric brain tumors using immunohistochemistry, and KIT messenger RNA (mRNA) using in situ hybridization. KIT and phospho-KIT were moderately or strongly expressed in tumor endothelia of 37% and 35% of pilocytic astrocytomas, respectively, whereas marked SCF and VEGFR-2 expression was uncommon. KIT mRNA was detected in tumor endothelial cells. Tumor endothelial cell KIT expression was strongly (P < 0.01) associated with endothelial cell phospho-KIT and SCF expression, and with tumor KIT (P = 0.0011) and VEGFR-2 expression (P = 0.022). KIT and phospho-KIT were present in endothelia of other pediatric brain tumors, notably ependymomas. Endothelial cell KIT expression was associated with a young age at diagnosis of pilocytic astrocytoma or ependymoma, and it was occasionally present in histologically normal tissue of the fetus and children. We conclude that KIT is commonly present in endothelial cells of juvenile brain tumors and thus may play a role in angiogenesis in these neoplasms.     

Comparison of structural genetics of non-schistosoma-associated squamous cell carcinoma of the urinary bladder

Little is known about genetic changes in squamous differentiation of non-schistosomiasis-associated bladder cancer. Therefore, we investigated pure squamous cell carcinomas (SqCC), squamous parts of mixed urothelial carcinomas with squamous differentiation (MIX) and mere urothelial cancers (UC) for structural genetic differences. Tissue microarray slides (n = 29 SqCC, n = 35 MIX and n = 23 UC) were analyzed by ZytoLight SPEC p16/CEN3/7/17 Quadruple Color Probe fluorescence-in-situ-hybridization (FISH) and DNA was investigated by comparative genomic hybridization (CGH) (n = 35 SqCCs, n = 40 MIX and n = 36 UC). By FISH the mean number of polysomic cells was lowest in SqCC (CEN3 P = 0.0498, CEN17 P = 0.0009). A slight tendency of lower copy numbers of chromosomes 3, 7 and 17 and higher numbers of the p16-locus in SqCC (P = 0.45) indicated less aneuploid tumor cells in SqCC compared to MIX and UC. In CGH SqCC showed the lowest mean number of aberrations per tumor (SqCC 5.37 changes, MIX 6.75 and UC 7.64; P = 0.1754). Significant differences between the three groups were found for loss of chromosome 3p (P = 0.004), 6q (P = 0.028), 11p (P = 0.024) and gains of 5p (P = 0.020). Loss of 3p was more frequent in SqCC (51.4%) than in MIX (37.5%) or UC (13.9%). To conclude, SqCCs show less polysomy and genetic alterations than MIX and UC. Loss of 3p is more frequent in SqCC but there are no absolute specific alterations for each tumor group. Squamous parts of mixed tumors show similar alterations than UC and should be considered as further development of UC, while pure SqCC seem to be a separate tumor group.     

Alterations in cathepsin L expression in lung cancers

We herein investigated the potential role of cathepsin L in lung carcinogenesis. Lung cancer cell lines and surgically resected tumors were examined for the expression of the cathepsin L protein and copy number alterations in its gene locus. Cathepsin L was stably expressed in bronchiolar epithelial cells. Neoplastic cells expressed cathepsin L at various levels, whereas its expression was completely lost in most of the lung cancer cell lines (63.6%, 7/11) examined. Furthermore, expression levels were lower in a large fraction of lung tumors (69.5%, 139/200) than in bronchiolar epithelia. The expression of cathepsin L was lost in some tumors (16.0%, 32/200). In adenocarcinomas, expression levels were significantly lower in high‐grade tumors than in low‐grade tumors (one‐way ANOVA, P < 0.0500). Copy number alterations were found in 18.0% (36 [32 gain + 4 loss] /200) of lung tumors. No relationship existed between cathepsin L protein expression levels and the copy number of its gene locus (Spearman's rank‐order correlation, P = 0.3096). Collectively, these results suggest that the down‐regulated expression of cathepsin L, which is caused by an undefined mechanism other than copy number alterations, is involved in the progression of lung adenocarcinomas.     

Pan‐cancer analysis of copy number changes in programmed death‐ligand 1 (PD‐L1, CD274) – associations with gene expression,mutational load,and survival

Inhibition of the PD‐L1 (CD274) – PD‐1 axis has emerged as a powerful cancer therapy that prevents evasion of tumor cells from the immune system. While immunohistochemical detection of PD‐L1 was introduced as a predictive biomarker with variable power, much less is known about copy number alterations (CNA) affecting PD‐L1 and their associations with expression levels, mutational load, and survival. To gain insight, we employed The Cancer Genome Atlas (TCGA) datasets to comprehensively analyze 22 major cancer types for PD‐L1 CNAs. We observed a diverse landscape of PD‐L1 CNAs, which affected focal regions, chromosome 9p or the entire chromosome 9. Deletions of PD‐L1 were more frequent than gains (31% vs. 12%) with deletions being most prevalent in melanoma and non‐small cell lung cancer. Copy number gains most frequently occurred in ovarian cancer, head and neck cancer, bladder cancer, cervical and endocervical cancer, sarcomas, and colorectal cancers. Fine‐mapping of the genetic architecture revealed specific recurrently amplified and deleted core regions across cancers with putative biological and clinical consequences. PD‐L1 CNAs correlated significantly with PD‐L1 mRNA expression changes in many cancer types, and tumors with PD‐L1 gains harbored significantly higher mutational load compared to non‐amplified cases (median: 78 non‐synonymous mutations vs. 40, P = 7.1e‐69). Moreover, we observed that, in general, both PD‐L1 amplifications and deletions were associated with dismal prognosis. In conclusion, PD‐L1 CNAs, in particular PD‐L1 copy number gains, represent frequent genetic alterations across many cancers, which influence PD‐L1 expression levels, are associated with higher mutational loads, and may be exploitable as predictive biomarker for immunotherapy regimens. © 2016 Wiley Periodicals, Inc.     

Gain of FGF4 is a frequent event in KIT/PDGFRA/SDH/RAS‐P WT GIST

Gastrointestinal stromal tumors (GIST) lacking mutations in KIT/PDGFRA or RAS pathways and retaining an intact SDH complex are usually referred to as KIT/PDGFRA/SDH/RAS‐P WT GIST or more simply quadruple WT GIST (~5% of all GIST). Despite efforts made, no recurrent genetic event in quadruple WT GIST has been identified so far. To further investigate this disease, we performed high throughput copy number analysis on quadruple WT GIST specimens identifying a recurrent focal gain in band 11q13.3 (involving FGF3/FGF4) in 6/8 cases. This event was not found in the other molecular GIST subgroups. FGF3/FGF4 duplication was associated with high expression of FGF4, both at mRNA and protein level, a growth factor normally not expressed in adult tissues or in KIT/PDGFRA‐mutated GIST. FGFR1 was found to be the predominant FGF receptor expressed and phosphorylation of AKT was detected, suggesting that a FGF4‐FGFR1 autocrine loop could stimulate downstream signaling in quadruple WT GIST. Together with the recent reports of quadruple WT cases carrying FGFR1 activating alterations, these findings strengthen the hypothesis of a potential involvement of FGFR pathway deregulation in quadruple WT GIST, which may represent a rationale for novel therapeutic approaches.     

Significant high expression of cytokeratins 7, 8, 18, 19 in pulmonary large cell neuroendocrine carcinomas,compared to small cell lung carcinomas

The aim of the present study was to clarify protein profiling in small cell lung carcinoma (SCLC) and pulmonary large cell neuroendocrine carcinoma (LCNEC). The proteomic approach was used, and involved cell lysate from two cell lines (N231 derived from SCLC and LCN1 derived from LCNEC), with 2‐D gel electrophoresis (2‐DE). In the present study, 25 protein spots with greater than twofold quantitative differences between LCN1 and N231 cells on 2‐DE gels were confirmed. Within the 25 identified proteins, cytokeratins (CK) 7, 8, 18 and 19 were upregulated in LCN1 cells compared with N231 cells. The expression of CK7, 8, 18, and 19 was further studied on immunohistochemistry with 81 formalin‐fixed and paraffin‐embedded pulmonary carcinomas, which included 27 SCLC, 30 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas. Although the expression of CK7, 8, 18, and 19 was observed in all histological types, the mean immunostaining scores of CK7, 8, 18, and 19 were significantly higher in LCNEC than in SCLC (P < 0.001, P < 0.001, P < 0.01 and P < 0.001, respectively). These data suggest that the biological characteristics of LCNEC and SCLC may be different and the expression of CK may serve as differential diagnostic markers.     

Esophageal melanomas harbor frequent <Emphasis Type="Italic">NRAS</Emphasis> mutations unlike melanomas of other mucosal sites

Mucosal melanomas have genetic alterations distinct from those in cutaneous melanomas. For example, NRAS- and BRAF-activating mutations occur frequently in cutaneous melanomas, but not in mucosal melanomas. We examined 16 esophageal melanomas for genetic alterations in NRAS, BRAF, and KIT to determine whether they exhibit genetic features common to melanomas arising from other mucosal sites. A sequencing analysis identified NRAS mutations in six cases; notably, four of these mutations were located in exon 1, an uncommon mutation site in cutaneous and other mucosal melanomas. BRAF and KIT mutations were found in one case each. Immunohistochemistry showed KIT expression in four cases, including the tumor with a KIT mutation and two other intramucosal tumors. The low frequency of BRAF mutations and the presence of a KIT mutation-positive case are findings similar to those of mucosal melanomas of other sites, but the prevalence of NRAS mutations was even higher than that of cutaneous melanomas. The present study implies that esophageal melanomas have genetic alterations unique from those observed in other mucosal melanomas.     

Obesity related methylation changes in DNA of peripheral blood leukocytes

Background

Oncogenic point mutations in KIT or PDGFRA are recognized as the primary events responsible for the pathogenesis of most gastrointestinal stromal tumors (GIST), but additional genomic alterations are frequent and presumably required for tumor progression. The relative contribution of such alterations for the biology and clinical behavior of GIST, however, remains elusive.

Methods

In the present study, somatic mutations in KIT and PDGFRA were evaluated by direct sequencing analysis in a consecutive series of 80 GIST patients. For a subset of 29 tumors, comparative genomic hybridization was additionally used to screen for chromosome copy number aberrations. Genotype and genomic findings were cross-tabulated and compared with available clinical and follow-up data.

Results

We report an overall mutation frequency of 87.5%, with 76.25% of the tumors showing alterations in KIT and 11.25% in PDGFRA. Secondary KIT mutations were additionally found in two of four samples obtained after imatinib treatment. Chromosomal imbalances were detected in 25 out of 29 tumors (86%), namely losses at 14q (88% of abnormal cases), 22q (44%), 1p (44%), and 15q (36%), and gains at 1q (16%) and 12q (20%). In addition to clinico-pathological high-risk groups, patients with KIT mutations, genomic complexity, genomic gains and deletions at either 1p or 22q showed a significantly shorter disease-free survival. Furthermore, genomic complexity was the best predictor of disease progression in multivariate analysis.

Conclusions

In addition to KIT/PDGFRA mutational status, our findings indicate that secondary chromosomal changes contribute significantly to tumor development and progression of GIST and that genomic complexity carries independent prognostic value that complements clinico-pathological and genotype information.     

Recurrent somatic loss of TNFRSF14 in classical Hodgkin lymphoma

Investigation of the genetic lesions underlying classical Hodgkin lymphoma (CHL) has been challenging due to the rarity of Hodgkin and Reed‐Sternberg (HRS) cells, the pathognomonic neoplastic cells of CHL. In an effort to catalog more comprehensively recurrent copy number alterations occurring during oncogenesis, we investigated somatic alterations involved in CHL using whole‐genome sequencing‐mediated copy number analysis of purified HRS cells. We performed low‐coverage sequencing of small numbers of intact HRS cells and paired non‐neoplastic B lymphocytes isolated by flow cytometric cell sorting from 19 primary cases, as well as two commonly used HRS‐derived cell lines (KM‐H2 and L1236). We found that HRS cells contain strikingly fewer copy number abnormalities than CHL cell lines. A subset of cases displayed nonintegral chromosomal copy number states, suggesting internal heterogeneity within the HRS cell population. Recurrent somatic copy number alterations involving known factors in CHL pathogenesis were identified (REL, the PD‐1 pathway, and TNFAIP3). In eight cases (42%) we observed recurrent copy number loss of chr1:2,352,236‐4,574,271, a region containing the candidate tumor suppressor TNFRSF14. Using flow cytometry, we demonstrated reduced TNFRSF14 expression in HRS cells from 5 of 22 additional cases (23%) and in two of three CHL cell lines. These studies suggest that TNFRSF14 dysregulation may contribute to the pathobiology of CHL in a subset of cases. © 2015 Wiley Periodicals, Inc.     

CD274 (PDL1) and JAK2 genomic amplifications in pulmonary squamous‐cell and adenocarcinoma patients

Aims

CD274 (PDL1) and JAK2 (9p24.1) gene amplifications have been recently described in pulmonary carcinomas in association with programmed death‐ligand 1 (PD‐L1) expression. Furthermore, PTEN loss has been explored preclinically in relation to PD‐L1 expression. Our aim was to determine whether these genomic alterations affect PD‐L1 expression levels in non‐small‐cell lung cancer.

Methods and results

PD‐L1 and PTEN expression determined by immunohistochemistry (IHC), and CD274, JAK2 and PTEN copy number alterations (CNAs) determined by fluorescence in‐situ hybridisation, were studied in 171 pulmonary carcinoma specimens. PD‐L1 expression was positive in 40 cases (23.3%), and CD274 amplification was present in 14 tumours (8.8%). Concordance between both events was found in 12 of 14 amplified cases (P = 0.0001). We found nine JAK2‐amplified cases (5.7%), seven with PD‐L1 expression (P = 0.0006). Moreover, six of the seven cases had JAK2 and CD274 coamplification (9p24.1 genomic amplification). Remarkably, the average PD‐L1 IHC score was higher in these amplified cases (230 versus 80; P = 0.001). Non‐statistical associations were observed between PD‐L1 expression and PTEN loss and PTEN deletions.

Conclusions

We describe a subset of patients (8.2%) who had 9p24.1 amplifications resulting in high expression of PD‐L1. Our results provide evidence for genomic up‐regulation of PD‐L1 expression in non‐small‐cell lung cancer.     

Mechanisms of chromosomal instability in melanoma

A systems biology approach was applied to investigate the mechanisms of chromosomal instability in melanoma cell lines. Chromosomal instability was quantified using array comparative genomic hybridization to identify somatic copy number alterations (deletions and duplications). Primary human melanocytes displayed an average of 8.5 alterations per cell primarily representing known polymorphisms. Melanoma cell lines displayed 25 to 131 alterations per cell, with an average of 68, indicative of chromosomal instability. Copy number alterations included approximately equal numbers of deletions and duplications with greater numbers of hemizygous (?1,+1) alterations than homozygous (?2,+2). Melanoma oncogenes, such as BRAF and MITF, and tumor suppressor genes, such as CDKN2A/B and PTEN, were included in these alterations. Duplications and deletions were functional as there were significant correlations between DNA copy number and mRNA expression for these genes. Spectral karyotype analysis of three lines confirmed extensive chromosomal instability with polyploidy, aneuploidy, deletions, duplications, and chromosome rearrangements. Bioinformatic analysis identified a signature of gene expression that was correlated with chromosomal instability but this signature provided no clues to the mechanisms of instability. The signature failed to generate a significant (P = 0.105) prediction of melanoma progression in a separate dataset. Chromosomal instability was not correlated with elements of DNA damage response (DDR) such as radiosensitivity, nucleotide excision repair, expression of the DDR biomarkers γH2AX and P‐CHEK2, nor G1 or G2 checkpoint function. Chromosomal instability in melanoma cell lines appears to influence gene function but it is not simply explained by alterations in the system of DDR. Environ. Mol. Mutagen. 55:457–471, 2014. © 2014 Wiley Periodicals, Inc.     

Localization‐ and mutation‐dependent microRNA (miRNA) expression signatures in gastrointestinal stromal tumours (GISTs), with a cluster of co‐expressed miRNAs located at 14q32.31

The molecular biology and clinical behaviour of gastrointestinal stromal tumours (GISTs) are associated with their anatomical localization (stomach or intestine), and also with the mutation status of the receptor tyrosine kinases KIT and PDGFRA. Twelve GISTs were evaluated for differential miRNA expression signatures by use of microarrays representing 734 human miRNAs. Thirty‐two miRNAs were found to be differentially expressed according to localization and mutation status. Differential expression was further analysed and confirmed for four miRNAs (miR‐132, miR‐221, miR‐222, and miR‐504) by qRT‐PCR in 49 additional GISTs. Differentially expressed miRNAs were functionally mapped to KIT/PDGFRA signalling and G1/S‐phase transition of the cell cycle, revealing 22 predicted miRNA/mRNA interactions for ten gene targets from KIT/PDGFRA signalling, and 12 interactions for 12 gene targets of G1/S‐phase transition. Moreover, the expression of 44 miRNAs clustered in a genetically imprinted region at 14q32.31 was found to be strongly correlated in the microarray analysis. This was confirmed for two selected miRNAs (miR‐134 and miR‐370) from the 14q32.31 cluster by qRT‐PCR in 49 additional GISTs, and the expression of these two miRNAs was significantly lower in GISTs with 14q loss, and also in GISTs with tumour progress. miRNA profiling may prove to be a key determinant of the biology and clinical features of GISTs Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

High expression of the RNA-binding protein RBPMS2 in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n = 23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p < 0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy.     

Overexpression of EGFR and absence of C-KIT expression correlate with poor prognosis in salivary gland carcinomas

Aims: To evaluate the prognostic impact of expression of receptor tyrosine kinases epidermal growth factor receptor (EGFR), HER2, and C‐KIT in relation to established clinicopathological parameters in salivary gland carcinomas. Methods and results:  Immunohistochemistry for EGFR, HER2, C‐KIT and the proliferation marker Ki67 was performed in 101 cases of salivary gland carcinoma and related to long‐term clinical follow‐up. Immunopositivity of C‐KIT was common in adenoid cystic carcinoma (92%). Lack of C‐KIT expression occurred in salivary duct carcinoma (P < 0.001) and was associated with high‐grade tumours (P = 0.002), positive lymph nodes (P = 0.002) and high expression of Ki67 (P = 0.001). HER2 was typically expressed in salivary duct carcinomas (83%), but was not associated with any other parameter. EGFR overexpression occurred independently of histological type and clinical parameters. On univariate survival analysis, overexpression of EGFR (P = 0.011) and lack of C‐KIT (P = 0.014) were associated with worse prognosis, whereas HER2 was of no prognostic significance. On multivariate analysis, the strongest negative predictor of survival was high proliferative activity measured by Ki67 (P = 0.002), followed by presence of residual tumour (P = 0.006), overexpression of EGFR (P = 0.026) and advanced tumour stage (P = 0.041). Conclusions: The expression of receptor tyrosine kinases confers additional prognostic impact on disease‐specific survival. EGFR overexpression is an independent negative prognostic factor.     

Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability

den Bakker M A, Willemsen S, Grünberg K, Noorduijn L A, van Oosterhout M F M, van Suylen R J, Timens W, Vrugt B, Wiersma‐van Tilburg A & Thunnissen F B J M
(2010) Histopathology 56, 356–363 Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability Aims: To test the hypothesis that the published morphological criteria permit reliable segregation of small cell carcinoma of the lung (SCLC) and large cell neuroendocrine carcinoma (LCNEC) cases by determining the interobserver variation. Methods and results: One hundred and seventy cases of SCLC, LCNEC and cases diagnosed as neuroendocrine lung carcinoma before LCNEC had been established as a diagnostic category were retrieved from the archives of the assessor’s institutes. A representative haematoxylin and eosin section from each case was selected for review. Batches of cases were circulated among nine pathologists with a special interest in pulmonary pathology. Participants were asked to classify the cases histologically according to the 2004 World Health Organization (WHO) criteria. The diagnoses were collected and κ values calculated. Unanimity of diagnosis was achieved for only 20 cases; a majority diagnosis was reached for 115 cases. In 35 cases no consensus diagnosis could be reached. There was striking variability amongst assessors in diagnosing SCLC and LCNEC. The overall level of agreement for all cases included in this study was fair (κ = 0.40). Conclusions: Using non‐preselected cases, the morphological WHO criteria for diagnosing SCLC and LCNEC leave room for subjective pathological interpretation, which results in imprecise categorization of SCLC and LCNEC cases.