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Circulating tumor DNA (ctDNA) is emerging as a key potential biomarker for post-diagnosis surveillance but it may also play a crucial role in the detection of pre-clinical cancer. Small-cell lung cancer (SCLC) is an excellent candidate for early detection given there are no successful therapeutic options for late-stage disease, and it displays almost universal inactivation of TP53. We assessed the presence of TP53 mutations in the cell-free DNA (cfDNA) extracted from the plasma of 51 SCLC cases and 123 non-cancer controls. We identified mutations using a pipeline specifically designed to accurately detect variants at very low fractions. We detected TP53 mutations in the cfDNA of 49% SCLC patients and 11.4% of non-cancer controls. When stratifying the 51 initial SCLC cases by stage, TP53 mutations were detected in the cfDNA of 35.7% early-stage and 54.1% late-stage SCLC patients. The results in the controls were further replicated in 10.8% of an independent series of 102 non-cancer controls. The detection of TP53 mutations in 11% of the 225 non-cancer controls suggests that somatic mutations in cfDNA among individuals without any cancer diagnosis is a common occurrence, and poses serious challenges for the development of ctDNA screening tests., Highlights • CtDNA may play a crucial role in the detection of pre-clinical cancer. • TP53 mutations are detectable in the cfDNA of SCLC patients with early-stage tumors. • Detection of TP53 mutations in non-cancer controls poses serious challenges for the development of ctDNA screening tests. Cell-free DNA (cfDNA) has potential for monitoring response to treatment and relapse, but also for early detection. This is the first study reporting the detection of circulating-tumor DNA (ctDNA) in cases diagnosed with small-cell lung cancer (SCLC). Our results show that TP53 mutations are detectable in the cfDNA of SCLC patients including those with early-stage tumors. Importantly, we also provide evidence that cancer-like TP53 mutations are present in non-cancer controls, which poses serious challenges for the development of ctDNA screening tests.

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: “type I LCNECs” with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and “type II LCNECs” enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors., The molecular nature of large-cell neuroendocrine lung carcinomas (LCNEC) has remained unclear. Here, the authors show LCNECs represent a distinct transcriptional subgroup among lung cancers and comprise two molecular subgroups, type I (TP53 and STK11/KEAP1 alterations) and type II (TP53 and RB1 inactivation).

Plasma circulating cell-free (cf)DNA is of interest in oncology because it has been shown to contain tumour DNA and may thus be used as liquid biopsy. In nonsmall cell lung cancer (NSCLC), cfDNA quantification has been proposed for the monitoring and follow-up of patients. However, available studies are limited and need to be confirmed by studies with larger sample sizes and including patients who receive more homogenous treatments. Our objective was to assess the predictive and prognostic value of plasma cfDNA concentration in a large series of patients with NSCLC and treated with a standard chemotherapy regimen. We included samples from lung cancer patients recruited into the Pharmacogenoscan study. The cfDNA of 218 patients was extracted and quantified by fluorometry before and after two or three cycles of platinum-based chemotherapy. The association between baseline and post-chemotherapy concentrations and treatment response, assessed by RECIST (response evaluation criteria in solid tumours) or patient survival was analysed. Patients with high cfDNA concentrations (highest tertile) at baseline had a significantly worse disease-free and overall survival than those with lower concentrations (lowest and middle tertiles) (median overall survival 10 months (95% CI 10.7–13.9) versus 14.2 months (95% CI 12.6–15.8), respectively; p=0.001). In multivariate analysis, increased baseline concentration of cfDNA was an independent prognostic factor. However, we did not find any association between cfDNA concentration and response to treatment. cfDNA may be a biomarker for the assessment of prognosis in NSCLC. However, total concentration of cfDNA does not appear to predict chemotherapy response. Total cell-free DNA is not associated with chemotherapy response in advanced nonsmall cell lung cancer

Vascular endothelial growth factor-A (VEGF-A) is highly subjected to alternative pre-mRNA splicing that generates several splice variants. The VEGF

We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 377

Purpose Antiangiogenic therapies targeting the vascular endothelial growth factor (VEGF) pathway have yielded more modest clinical benefit to patients with non–small-cell lung cancer (NSCLC) than initially expected. Clinical data suggest a distinct biologic role of the VEGF pathway in the different histologic subtypes of lung cancer. To clarify the influence of histologic differentiation in the prognostic relevance of VEGF-mediated signaling in NSCLC, we performed a concomitant analysis of the expression of three key elements of the VEGF pathway in the earliest stages of the following two principal histologic subtypes: squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Patients and Methods We evaluated tumor cell expression of VEGF, VEGF receptor (VEGFR) 1, and VEGFR2 using automatic immunostaining in a series of 298 patients with early-stage NSCLC recruited as part of the multicenter European Early Lung Cancer Detection Group project. A score measuring the VEGF signaling pathway was calculated by adding the tumor cell expression value of VEGF and its two receptors. The results were validated in two additional independent cohorts of patients with NSCLC. Results The combination of high VEGF, VEGFR1, and VEGFR2 protein expression was associated with lower risk of disease progression in early SCC (univariate analysis, P = .008; multivariate analysis, hazard ratio, 0.62; 95% CI, 0.42 to 0.92; P = .02). The results were validated in two independent patient cohorts, confirming the favorable prognostic value of high VEGF signaling score in early lung SCC. Conclusion Our results clearly indicate that the combination of high expression of the three key elements in the VEGF pathway is associated with a good prognosis in patients with early SCC but not in patients with ADC.

The incidence of adenocarcinoma is increasing, particularly among females. We sought to assess the role of tobacco consumption in clinical presentation according to sex. In this retrospective study, 848 patients diagnosed between 1997 and 2006 at Grenoble University Hospital (Grenoble, France) were stratified into four groups according to smoking habits. Differences between sexes and two contrasting female profiles emerged. Female current smokers were younger than female never-smokers (median 51 versus 69 yrs; p < 0.001), more often had surgery (62.7% versus 39%; p = 0.01) and had a median (95% CI) estimated survival of 26.2 (18.1-49.2) versus 15.1 (12.8-22.2) months (p = 0.002). Both groups had similar survival when taking treatment into account. Among males, smoking did not influence presentation. Male current smokers were older than female current smokers (median 59 yrs; p < 0.001) and fewer had surgery (48.8%; p = 0.015), although the percentage of stage IIIb-IV disease was similar (53% and 46%; nonsignificant) and they had a poorer estimated survival of 14.3 (13.0-18.5) months (p = 0.0024). Males smoked more than females (median 41 versus 30 pack-yrs; p < 0.001). Quitting smoking delayed age at diagnosis by 11 yrs for females (p = 0.0035) and 8 yrs for males (p < 0.001). Our results support the hypothesis that carcinogenesis differs between males and females, and between female smokers and never-smokers.

Although genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations that drive squamous cell cancer (SCC) of the lung. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of lung SCCs and cell lines. Lung SCC cell lines harboring DDR2 mutations were selectively killed by knockdown of DDR2 by RNA interference or by treatment with the multitargeted kinase inhibitor dasatinib. Tumors established from a DDR2 mutant cell line were sensitive to dasatinib in xenograft models. Expression of mutated DDR2 led to cellular transformation that was blocked by dasatinib. A patient with lung SCC that responded to dasatinib and erlotinib treatment harbored a DDR2 kinase domain mutation. These data suggest that gain-of-function mutations in DDR2 are important oncogenic events and are amenable to therapy with dasatinib. Because dasatinib is already approved for use, these findings could be used to rapidly generate clinical trials. Significance: DDR2 mutations are present in 4% of lung SCCs, and DDR2 mutations are associated with sensitivity to dasatinib. These findings provide a rationale for designing clinical trials with the FDA-approved drug dasatinib in patients with lung SCCs. Cancer Discovery; 1(1); 78–89. ©2011 AACR. Read the Commentary on this article by Ohashi and Pao, p. 23 This article is highlighted in the In This Issue feature, p. 4

Senescence is a tumor suppression mechanism that is induced by several stimuli, including oncogenic signaling and telomere shortening, and controlled by the p53/p21WAF1 signaling pathway. Recently, a critical role for secreted factors has emerged, suggesting that extracellular signals are necessary for the onset and maintenance of senescence. Conversely, factors secreted by senescent cells may promote tumor growth. By using expression profiling techniques, we searched for secreted factors that were overexpressed in fibroblasts undergoing replicative senescence. We identified WNT16B, a member of the WNT family of secreted proteins. We found that WNT16B is overexpressed in cells undergoing stress-induced premature senescence and oncogene-induced senescence in both MRC5 cell line and the in vivo murine model of K-RasV12–induced senescence. By small interfering RNA experiments, we observed that both p53 and WNT16B are necessary for the onset of replicative senescence. WNT16B expression is required for the full transcriptional activation of p21WAF1. Moreover, WNT16B regulates activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Overall, we identified WNT16B as a new marker of senescence that regulates p53 activity and the PI3K/AKT pathway and is necessary for the onset of replicative senescence. [Cancer Res 2009;69(24):9183–91]

Inhibitor of growth 2 (ING2) is a candidate tumour suppressor gene the expression of which is frequently lost in tumours. Here, we identified a new function for ING2 in the control of DNA replication and in the maintenance of genome stability. Global replication rate was markedly reduced during normal S-phase in small interfering RNA (siRNA) ING2 cells, as seen in a DNA fibre spreading experiment. Accordingly, we found that ING2 interacts with proliferating cell nuclear antigen and regulates its amount to the chromatin fraction, allowing normal replication progression and normal cell proliferation. Deregulation of DNA replication has been previously associated with genome instability. Hence, a high proportion of siRNA ING2 cells presented endoreduplication of their genome as well as an increased frequency of sister chromatid exchange. Thus, we propose for the first time that ING2 might function as a tumour suppressor gene by directly maintaining DNA integrity.

The E-cadherin–β−catenin complex plays a pivotal role in epithelial cell–cell adhesion and in the maintenance of differentiated adult epithelia. Perturbation of its expression or function is widely involved in tumor progression and metastasis. Recent years have seen a rapid expansion in the understanding of the biology and the clinical relevance of the E-cadherin adhesion complex in human lung cancer. During human lung cancer progression genomic, transcriptionnal and post-transcriptionnal alterations of the E-cadherin–β-catenin adhesion system are implicated and comprise deletion of the chromosomic region 3p21 that comprise the locus of the gene encoding β-catenin, transcriptionnal downregulation of E-cadherin, cytoplasmic redistribution, phosphorylation of both proteins and proteolysis of E-cadherin. E-cadherin-inactivating mutations and oncogenic-activating mutation of β-catenin are not reported.

International audience; Amphiregulin (AR) and insulin-like growth factor-1 (IGF1) are growth factors known to promote non-small cell lung cancer (NSCLC) survival. We have previously published that 1) AR and IGF1, secreted by H358 NSCLC cells, cooperate to protect those cells and H322 NSCLC cells from serum-starved apoptosis; 2) H358 cells resist Bax-induced apoptosis through an inhibition of Bax conformational change. We show here that the antiapoptotic activity of the AR/IGF1 combination is specifically abolished by the PKC inhibitors calphostin C and staurosporine, but not by the MAPK and phosphatidylinositol 3-kinase inhibitors PD98059 and wortmannin, suggesting the involvement of a PKC-dependent and MAPK- and phosphatidylinositol 3-kinase-independent survival pathway. The PKCdelta inhibitor rottlerin restores apoptosis induced by serum deprivation. In addition, phosphorylation of PKCdelta and PKCzeta/lambda, but not of PKCalpha/beta(II), increases in serum-starved H358 cells and in H322 cells treated with an AR/IGF1 combination and is blocked by calphostin C. The combination of AR and IGF1 increases p90(rsk) and Bad phosphorylation as well as inhibiting the conformational change of Bax by a PKC-dependent mechanism. Finally, PKCdelta, PKCzeta, or p90(rsk) small interfering RNAs block the antiapoptotic activity of AR/IGF1 combination but have no effect on partial apoptosis inhibition observed with each factor used alone. Constitutively active PKC expression inhibits serum deprivation-induced apoptosis, whereas a catalytically inactive form of p90(rsk) restores it. Thus, AR and IGF1 cooperate to prevent apoptosis by activating a specific PKC-p90(rsk)-dependent pathway, which leads to Bad and Bax inactivation. This signaling pathway is different to that used by single factor.

Purpose: Telomerase, a ribonucleoprotein complex whose activity is related to the expression of its catalytic subunit human telomerase reverse transcriptase (hTERT), restores telomere length in tumor cells and enables immortality after p53/Rb inactivation has been achieved. To determine the timing of hTERT derepression during bronchial carcinogenesis and its relationship with telomere shortening and the p53/Rb pathway alterations, we did an immunohistochemical and in situ hybridization study in preinvasive and invasive bronchial lesions. Experimental Design: hTERT, P53, P16, cyclin D1, Bax-to-Bcl2 ratio, and Ki67 immunostainings were done in 106 preneoplastic lesions and in paired lung carcinoma and normal bronchial mucosae. Concomitantly, hTERT mRNA levels and qualitative telomere shortening were assessed by in situ hybridization and fluorescence in situ hybridization, respectively, in a subset of preneoplastic and neoplastic lesions. Results: Telomerase was increasingly expressed from normal epithelium to squamous metaplasia, dysplasia, and carcinoma in situ, and decreased in invasive carcinoma (P < 0.0001), with a direct correlation between protein and mRNA levels of expression (P < 0.0001). hTERT expression was directly correlated with P53, Ki67, and Bcl2-to-Bax ratio, suggesting a coupling between telomerase reactivation, proliferation, and resistance to apoptosis. Telomere signals significantly decreased as early as squamous metaplasia and progressively increased over the spectrum of preneoplastic lesions. Conclusions: Telomere shortening represents an early genetic abnormality in bronchial carcinogenesis, preceding telomerase expression and p53/Rb inactivation, which predominate in high-grade preinvasive lesions.

Pulmonary tumorlets and neuroendocrine (NE) cell hyperplasia are part of a continuous spectrum of NE-cell hyperplasia, going from NE hyperplasia to carcinoid. Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen that has been shown to be increased in hypoxic lung. We hypothesized that tumorlets and NE-cell hyperplasia, which occur frequently in this context, were partly responsible for VEGF secretion. Immunohistochemical analysis of VEGF and both VEGF-R1 and VEGF-R2 was performed on paraffin sections of 12 lung tissues containing tumorlets and NE-cell hyperplasia in parallel with a control group of 11 lung specimens. VEGF and its receptor expressions were compared in bronchial epithelial cells and endothelial cells in both groups. VEGF and its receptors were consistently expressed in tumorlets and in NE-cell hyperplasia. When compared with control group lungs, the staining score for VEGF in lung bearing tumorlets was significantly higher in endothelial cells, but was not different in bronchial epithelial cells. VEGF-R1 expression was significantly increased both on bronchial epithelial cells (P = 0.001) and endothelial cells (P = 0.006), and VEGF-R2 expression was significantly increased on endothelial cell (P = 0.044). There was a significant positive correlation between the level of expression of VEGF and VEGF-R1 (P = 0.04) in both control groups and lung bearing tumorlets, but there was no significant correlation between VEGF and VEGF-R2 expression (P = 0.1). We concluded that VEGF is highly expressed in localized NE cell proliferations without potential of malignancy and might participate in local development of fibrosis.

Pulmonary adenocarcinoma of the lung and its variants are well-defined entities, since the recent WHO classification of lung tumours. However, scant descriptions have been allocated to associated stromal changes, such as prominent inflammation and fibrosis, which can overshadow a tumoral proliferation and masquerade as a benign reactive process and this has not been recognised as a histopathological variant. The case of a 72-yr-old farmer who presented a multifocal well-differentiated adenocarcinoma that mimicked honeycomb lung with bronchiolectasis radiologically, on computed tomography scan and histologically at open lung biopsy, is reported. Histological pitfalls in the biopsy were represented by mild atypical cuboidal or columnar epithelial cells lining bronchiolar structures resembling florid bronchiolar metaplasia in a background of extensive fibrosis and inflammation, features that mimicked inflammatory honeycombing. However, histological analysis of the surgical resection of the main lesion, performed because of a clinical alteration of the patient, confirmed the diagnosis of multifocal adenocarcinoma of mixed subtype. A monomorphic proliferation of clear cells, lack of associated ciliated or squamous cells and presence of significant cytologic atypia gave a diagnosis of malignancy. This case illustrates how inflammatory and fibrotic changes may conceal a correct diagnosis of carcinoma and emphasises the importance of adequate sampling in such cases.

Human telomerase reverse transcriptase is a ribonucleoprotein that synthesises telomeric sequences, which decrease at each cell division. In cancer cells, its activity is linked to telomere maintenance leading to unlimited cellular proliferation and immortality. To evaluate the prognostic value of the catalytic subunit telomerase reverse transcriptase (hTERT), we analysed its expression by immunohistochemistry in 122 formalin-fixed lung tumours including 42 squamous cell carcinoma (SCC), 43 adenocarcinoma (ADC), 19 basaloid carcinoma (BC) and 18 small-cell lung carcinoma (SCLC) in comparison with detection of hTERT mRNA by in situ hybridisation and relative telomerase activity by TRAP assay in a subset of tumours. We observed a high concordance between hTERT protein expression and detection of hTERT mRNA and telomerase activity. Telomerase expression varied according to histology (P=0.0002) being significantly lower in ADC than in SCC, BC and SCLC (P

Fas (CD95) and its ligand FasL signal apoptosis and are involved in tissue homeostasis and the elimination of target cells by cytotoxic T cells. Corruption of this signalling pathway in tumour cells, for example by reduced Fas expression or increased FasL expression, can participate in tumour development and immune escape. The present study has analysed Fas/FasL expression and Fas death signalling function in vivo in lung tumour tissues [57 non-small cell lung carcinomas and 64 neuroendocrine lung tumours including small cell lung carcinoma (SCLC)] in comparison with normal lung tissue, and in vitro in neuroendocrine tumour cell lines in comparison with normal human bronchial epithelial cells. The Fas expression score was markedly decreased compared with normal lung tissue in 90% of the 121 lung tumours and was completely lost in 24%. The Fas staining pattern suggested cytoplasmic Fas expression in tumours, whereas membrane expression was observed in normal lung tissue. Loss of Fas at the cell surface was also shown in vitro by FACS analysis of neuroendocrine tumour cell lines and was concomitant with the resistance of tumour cells to FasL-mediated apoptosis according to in vitro cell viability. The lack of cell surface Fas expression in tumour cell lines resulted from the lack of intracellular Fas protein due to impaired Fas gene transcription. The FasL expression score was also decreased in most non-small cell lung carcinomas compared with normal bronchial cells, whereas 91% of SCLCs had higher expression than normal cells. FasL overexpression was related to advanced tumour stage as well as to a Fas/FasL ratio less than 1. It is concluded that a marked decrease in Fas expression may be part of lung tumourigenesis allowing tumour cells to escape from apoptosis. FasL overexpression in the context of Fas down-regulation in SCLC predicts the ability of SCLC cells to induce paracrine killing of Fas-expressing cytotoxic T cells. In lung tumours, Fas restoration may represent a key, although not unique, step in therapeutic strategies to reconstitute the ability of tumour cells to undergo apoptosis.

Study objectives Autofluorescence bronchoscopy (AFB), when used as an adjunct to standard white light bronchoscopy (WLB), enhances the bronchoscopist's ability to localize small neoplastic lesions, especially intraepithelial lesions. The current study was undertaken in order to define the population in which the rate of detection is higher using AFB. Design and patients Two hundred forty-four consecutive patients, who were symptomatic smokers or patients who previously had been treated for lung cancer or head and neck cancers, underwent WLB and AFB. All patients with endoscopic abnormalities underwent biopsies. Data concerning smoking history were prospectively registered. Results We report the prevalence of high-grade or invasive lesions at the time of examination. On a lesion-by-lesion analysis, 92 low-grade lesions, 42 high-grade lesions (ie, moderate dysplasia, severe dysplasia, and carcinoma in situ), and 39 invasive carcinomas were diagnosed. There was no effect of age, gender, and age at smoking initiation on the prevalence of preinvasive or invasive lesions. The 10 patients who previously had undergone surgery for lung cancer and exhibited high-grade preinvasive lesions had a history of carcinoma of the epidermoid histologic type (p = 0.01). These 10 patients displayed multiple lesions in the bronchial tree (mean No. of lesions, 1.8 per patient). In current smokers, the prevalence of high-grade or invasive lesions were both related to the number of pack-years smoking had occurred (p = 0.01) and to the duration of smoking (p = 0.01). In contrast, the prevalence of preinvasive lesions in former smokers was related to a history of epidermoid carcinoma. Conclusions AFB should be recommended in patients with a history of epidermoid carcinomas of the lung. Current smokers with a prolonged smoking history appear to comprise a population in which the rate of detection of preneoplastic lesions is high with AFB.

The World Health Organization's classification of lung tumors separately categorizes neuroendocrine (NE) lung tumors, small cell lung carcinoma (SCLC), and large cell neuroendocrine carcinoma (LCNEC) as high-grade NE malignancies and carcinoids (typical, [TC] and atypical [AC]) as low- and intermediate-grade malignancies. Although these NE tumors are considered with NE hyperplasia (NEH) and tumorlets as part of a spectrum of NE proliferations, their derivation from a common progenitor cell has not received full agreement. With the aim of refining their differential diagnosis and extending our understanding of their histogenesis, we studied the expression of thyroid transcription factor-1 (TTF-1), a transcription factor that regulates lung morphogenesis and differentiation, along the spectrum of NE lung tumors. Two hundred and twenty- seven NE proliferations and tumors were immunostained with TTF-1 antibody. Positive immunostaining for TTF-1 was detected in 47 of 55 (85.5%) pure SCLCs, in 31 of 64 (49%) pure LCNECs, but in none of 15 NEHs, 23 tumorlets, or 50 carcinoid tumors (27 TCs and 23 ACs). In 19 of 20 (95%) combined SCLCs and LCNECs, TTF-1 expression was identical in both NE and non-NE components. These results show that TTF-1 is not expressed in normal and hyperplastic NE cells or in carcinoids, but is expressed in high-grade NE proliferations and in lung adenocarcinomas. This challenges the concept of a spectrum of NE proliferations and tumors and lends credence to the alternative hypothesis of a common derivation for SCLC and non-SCLC including LCNEC, with carcinoids deriving from a different stem cell.

Purpose: The basaloid carcinoma (pure) and the (mixed) basaloid variant of lung squamous cell carcinoma (SCC) have a dismal prognosis but their underlying specific molecular characteristics remain obscure and no therapy has proven to be efficient. Experimental Design: To assess their molecular specificity among other lung SCCs we analyzed DNA copy number aberrations and mRNA expression pangenomic profiles of 93 SCCs, including 42 basaloid samples (24 pure, 18 mixed). Results: Supervised analyses reveal that pure basaloid tumors display a specific mRNA expression profile, encoding factors controlling the cell cycle, transcription, chromatin, and splicing, with prevalent expression in germline and stem cells, while genes related to squamous differentiation are underexpressed. From this signature, we derived a 2-genes (SOX4, IVL) immunohistochemistry-based predictor that discriminated basaloid tumors (pure and mixed) from non-basaloid tumors with 94% accuracy in an independent series. The pure basaloid tumors are also distinguished through unsupervised analyses. Using a centroid-based predictor, the corresponding molecular subtype was found in 8 independent public datasets (n = 58/533), and was shown to be associated with a very poor survival as compared with other SCCs (adjusted HR = 2.45; P = 0.000001). Conclusion: This study enlightens the heterogeneity of SCCs that can be subclassified in mRNA expression subtypes. This study demonstrates for the first time that basaloid SCCs constitute a distinct histomolecular entity, which justifies its recognition and distinction from non-basaloid SCCs. In addition, their characteristic molecular profile highlights their intrinsic resistance to cytotoxic chemotherapy and could serve as a guide for targeted therapies. Clin Cancer Res; 20(22); 5777–86. ©2014 AACR.

Basaloid carcinoma (BC) and large-cell neuroendocrine carcinoma (LCNEC) are 2 recently recognized variants of large-cell lung carcinomas that may overlap in their morphology, and are discriminated by expression of neuroendocrine markers in LCNEC. Because thyroid transcription factor 1 (TTF-1) is expressed in lung adenocarcinomas but not in squamous cell carcinomas (SCC), and 34betaE12 recognizes a set of high-molecular-weight cytokeratins characteristic of basal stem cells, we hypothesized that these 2 markers could help in distinguishing BC from LCNEC. Immunostaining for TTF-1 was detected in 40.9% of pure LCNEC but in no BC or basaloid variant of SCC. In contrast, immunoreactivity for 34betaE12 was shown in all BC and basaloid variant of SCC but in only 1 LCNEC. Bouin fixation was less efficient than formalin in the immunodetection of both markers for its well-known deleterious effect on antigen preservation. Specificity of TTF-1 for LCNEC (100%) and that of 34betaE12 for BC (98.3%) exceeded that of NE markers for distinction of these 2 entities. These data show that TTF-1 and 34betaE12, in association with specific neuroendocrine markers, represent a useful panel of antibodies in differentiating carcinomas presenting with a solid pattern, palisading, or pseudorosettes, the expression of TTF-1 excluding the diagnosis of BC, and staining with 34betaE12 excluding pure LCNEC.

Copious bronchorrhea can be related to bronchioloalveolar carcinoma, but reports of bronchorrhea related to lung metastasis are rare. We report the case of a woman presenting lung metastases of a cervical adenocarcinoma revealed by bronchorrhea, eventually identified as ectopic cervical mucus. Treatment included anticancer drugs and erythromycin, the latter in order to reduce the bronchorrhea, with eventually poor efficacy. This observation illustrates the importance of respiratory signs in the post-therapeutic follow up of cancer, especially cough and bronchorrhea in adenocarcinoma.

Erdheim-Chester disease (ECD) is a rare non-Langerhans' cell histiocytosis that may present with pulmonary symptoms. The condition seems to be nonfamilial and typically affects middle-aged adults. Radiographic and pathologic changes in the long bones are diagnostic, but patients often present with extraskeletal manifestations. Advanced pulmonary lesions are associated with extensive fibrosis that may lead to cardiorespiratory failure. The clinical, radiologic, and pathologic features of six patients with ECD with lung involvement are presented. The patients were three men and three women (mean age, 57). Five presented with progressive dyspnea, and one presented with diabetes insipidus. Open-lung biopsies showed histiocytic infiltrates in a lymphangitic pattern with associated fibrosis and lymphoplasmacytic inflammatory infiltrates. The histiocytes did not stain with periodic acid-Schiff. Immunoperoxidase studies performed on specimens from five of six patients showed that the histiocytes were positive for CD68 and Factor XIIIa and negative for CD1a. Specimens from two patients exhibited immunoreactivity for S-100 protein. Electron microscopy studies performed on specimens from two patients showed phagocytic lysosomes but no Birbeck granules. Clinical follow-up of up to 16 years was available. At the end of that time, five patients were dead of complications related to their disease; one patient remains alive 4 years after diagnosis but with severe respiratory compromise. ECD is a rare non-Langerhans' cell histiocytosis that may present as interstitial lung disease and resemble other pulmonary conditions, particularly usual interstitial pneumonitis and pulmonary Langerhans' cell histiocytosis. Recognition of this entity will allow better assessment of its true incidence, therapeutic options, and prognosis.

The formation of new vessels (angiogenesis) is essential for primary tumour growth and metastasis and is induced by several angiogenic factors, including vascular endothelial growth factor (VEGF). The microvascular density (MVD) in tumours was assessed and the expression of VEGF and its receptors VEGF-R1-Flt1 and VEGF-R2-KDR/Flk1 was investigated in the different cellular compartments in vivo, in order to establish their interrelationship and their prognostic influence. Immunohistochemical study of 69 stage I-II non-small cell lung carcinomas (NSCLCs) was performed on paraffin sections with CD34 antibody to estimate MVD, using a Chalkley eye-piece graticule and VEGF, VEGF-R1, and VEGF-R2 antibodies. There was strong expression of VEGF and its receptors in tumour cells, endothelial cells, and stromal fibroblasts. In tumour cells, the level of VEGF was correlated with that of VEGF-R1 ( p = 0. 018) but not that of VEGF-R2. In fibroblasts, high expression of VEGF was correlated with that of VEGF-R1 ( p = 0.0001) and VEGF-R2 ( p = 0.0001). In endothelial cells, expression of VEGF was correlated with that of VEGF-R1 ( p < 0.0001) and VEGF-R2 ( p = 0.04). The level of VEGF in fibroblasts was correlated with that of VEGF-R1 ( p = 0.0028) and VEGF-R2 ( p = 0.01) in endothelial cells. There was no correlation between the level of MVD and that of VEGF or VEGF-R1 or VEGF-R2. Neither the level of MVD, nor the level of expression of VEGF and VEGF receptors in any compartment influenced the patient's survival. In conclusion, although angiogenesis is essential for tumour growth, this study failed to demonstrate that MVD, VEGF, VEGF-R1, and VEGF-R2 are prognostic markers for stage I-II NSCLC. VEGF, however, might act as a direct autocrine growth factor for tumour cells via VEGF-R1 and angiogenesis could be promoted in a paracrine loop, where VEGF is produced by fibroblasts and tumour cells and then binds to endothelial cells via induced VEGF receptors. VEGF and its receptors thus appear as relevant therapeutic targets in NSCLC.

The aim of this study was to report our experience on the management of superior vena cava obstruction (SVCO) secondary to malignant disease, using endovascular procedures. Twenty-six patients with SVCO due to primary or secondary tumors of the lung or the mediastinum, or catheter inserted for treatment of an extra-thoracic neoplasm, had an endovascular therapy which consisted of stenting, angioplasty, thrombo-aspiration or local fibrinolysis. Immediately after the procedure, rapid relief of symptoms occurred in 24 (90 %) of the patients. The mean Kishi's score decreased from 5.5 to 0.96. Immediate complications included one death related to pericarditis bleeding following fibrinolysis. Three patients relapsed after 20 days, 4 months and 6 months, and needed a second stenting. At 6 months the primary patency rate was 83 % and the secondary patency rate was 89 %. Endovascular treatment of SVCOs is a simple and safe procedure to restore the patency of the superior vena cava in malignant SVCO. It should be indicated in most cases as first-line treatment and performed as early as possible.

Erdheim-Chester disease (ECD) is a rare focal or systemic infiltrative disorder resulting from xanthogranulomatous tissue deposition. Usually, bone marrow involvement affects long bone metaphyses symmetrically, but it spares the epiphyses. Retroperitoneal space, periaortic area, skin, and brain involvement have been described. Pulmonary involvement is frequent, occurring in 20% of cases. Reported histologic features in the lung include an infiltration of so-called lipid-laden macrophages and granulomatous lesions with fibrosis. Lung function outcome is unpredictable, but terminal respiratory failure is the most frequent cause of death. No effective treatment strategies have been described. We report a new case with lung and bone involvement occurring in a symptomatic woman. Histologic and electron microscopic analysis of the pulmonary infiltrate showed abnormal macrophages devoid of lipids forming nodular granulomas and rendering the previous hypothesis of this disease as a primary lipid storage disorder unlikely. These findings suggest that ECD histogenesis is instead based on a primary macrophage cell disease.

International audience; The retinoblastoma (RB) gene plays a key role in cell cycle control by regulation of G1 growth arrest. This gene is inactivated in some human cancers and in most small-cell lung carcinoma (SCLC) cell lines. The aim of this study was to analyze the mechanisms of RB silencing in freshly excised neuroendocrine (NE) tumors embracing the entire spectrum of NE lung neoplasms (typical and atypical carcinoids, large-cell neuroendocrine carcinomas [LCNECs], and SCLCs). To study the role and mechanism of RB inactivation in tumor differentiation and malignant potential, the status of the Rb protein was analyzed in 37 NE lung tumors, using immunohistochemistry with five Rb antibodies. Loss or altered expression of Rb protein was more frequently observed in high-grade NE lung carcinoma (23 of 28, 82%) than in typical and atypical carcinoids (1 of 9, 11%) (P < 0.001). Of 24 tumors with abnormal Rb staining, Southern blotting showed 1 to have undergone rearrangement, SSCP (single-strand conformation polymorphism) and sequencing showed that 6 (25%) exhibited mutations in exons 13-18 or 20-24 of the RB gene, and RT-PCR (reverse transcriptase-polymerase chain reaction) revealed that 14 (58%) showed a low level of or entirely absent RB mRNA (messenger RNA) expression, whereas hypermethylation of the CpG-rich island at the 5' end of the RB gene was not observed. Abnormal Rb protein expression was always associated with one of these three alternative mechanisms in the SCLCs analyzed, but in only 50% of LCNECs. These results indicate that inactivation of the RB gene is highly frequent in freshly excised high-grade NE lung tumors through distinct mechanisms including point mutations and frequent abnormal mRNA expression. Different modes of RB inactivation seem to be implicated along the spectrum of NE lung carcinomas, depending on differentiation state, phenotype, and malignancy grade.

Objectives Platinum-based chemotherapy regimens are the standard treatment of non-small cell lung cancer (NSCLC). In this study, our objective was to identify tumor tissue protein biomarkers that might predict a benefit from these treatments. Materials and methods The Pharmacogenoscan study prospectively included consecutive chemotherapy-naive NSCLC patients at any stage between 2005 and 2010 at six hospitals in the Rhone-Alpes-Auvergne region of France. Of the 537 patients in the full analysis set, 460 had a complete histological diagnosis. We used the tumor tissue samples for an immunohistochemical evaluation of eight biomarkers: ERCC1, BRCA1, p53, p27kip1, class III β-tubulin (TUBB3), Bax, Fas, and FasL. We looked for associations between these biomarkers and the disease control rate (DCR) after 2/3 cycles of platinum-based chemotherapy, progression-free survival (PFS), and overall survival (OS). Results A tissue sample adequate for testing at least one biomarker was available for 289 patients. We found no significant association between biomarker expression levels and clinical or pathological variables; TUBB3 showed a trend toward higher expression in adenocarcinomas ( P =0.005). For none of the biomarkers were significant associations found between expression level and DCR, PFS, or OS. TUBB3-negative and FasL-negative tumors showed associations of borderline significance with higher DCR. Conclusion In a large cohort of patients with predominantly advanced or metastatic NSCLC, none of eight tested immunohistochemical biomarkers predicted the chemotherapy response or survival. Our data indicate limited usefulness of protein biomarkers in metastatic NSCLC and a need for further research based on molecular signatures of greater complexity.

Purpose Characterization of the genomic changes that drive an individual patient's disease is critical in management of many cancers. In patients with non–small-cell lung cancer (NSCLC), obtaining tumor samples of sufficient size for genomic profiling on recurrence is often challenging. We undertook this study to compare genomic alterations identified in archived primary tumors from patients with NSCLC with those identified in metachronous or synchronous metastases. Patients and Methods Primary and matched metastatic tumor pairs from 15 patients were analyzed by using a targeted next-generation sequencing assay in a Clinical Laboratory Improvement Amendments laboratory. Genomic libraries were captured for 3,230 exons in 182 cancer-related genes plus 37 introns from 14 genes often rearranged in cancer and sequenced to high coverage. Results Among 30 tumors, 311 genomic alterations were identified of which 63 were known recurrent (32 in primary tumor, 31 in metastasis) and 248 were nonrecurrent (likely passenger). TP53 mutations were the most frequently observed recurrent alterations (12 patients). Tumors harbored two or more (maximum four) recurrent alterations in 10 patients. Comparative analysis of recurrent alterations between primary tumor and matched metastasis revealed a concordance rate of 94% compared with 63% for likely passenger alterations. Conclusion This high concordance suggests that for the purposes of genomic profiling, use of archived primary tumor can identify the key recurrent somatic alterations present in matched NSCLC metastases and may provide much of the relevant genomic information required to guide treatment on recurrence.

Study objective The cost and inconvenience of polysomnography make simplified techniques of screening desirable in the strategy of diagnosis of sleep apnea syndrome (SAS). We have evaluated, in a prospective study of 301 consecutive patients referred for suspected sleep disorders, an index (Δindex) that detects apneic events by quantifying arterial oxygen saturation (SaO 2 ) variability. Setting Regional sleep laboratory taking referrals from general practitioners and specialists. Design Classic polysomnography was the gold standard, with 15 apneas plus hypopneas per hour (RDI) being used as a threshold for definition of obstructive sleep apnea (OSA). Oximetry was recorded over the same night. Signal variability was quantified as a function of time, using digital processing of oximetric data. Sensitivity, specificity, and positive and negative predictive values of oximetry testing were calculated. A receiver operating characteristic (ROC) curve was constructed representing the comparative courses of sensitivity and 1-specificity at different thresholds of A index. Results Three hundred one patients were included (age, 56±12 years). Their RDI was 30±24. For a A threshold at 0.6, the sensitivity of oximetry for the diagnosis of OSA was 98% and the specificity was 46%. The positive and negative predictive values for diagnosing SAS were 77% and 94%, respectively. The three false-negative cases had a relatively high awake SaO 2 (97 vs 93.9±2.8%), a moderate RDI (23.3±1.6), and were less obese than the other patients (body mass index: 25±3 vs 33±8). The 58 false-positive cases had an RDI of 8±4, an awake SaO 2 of 93.1±3.6 vs 94.1±2.6 for the rest of the population (p=0.01). Finally, the false-positive cases had more airways obstruction (FEV 1 /VC=72±13 vs 77±15%; p=0.026). Using a A value of 0.8 leads to a sensitivity of 90% with 19 false-negative cases but with a higher specificity of 75%. Conclusions A nocturnal oximetry test with a A index below 0.6 is helpful in ruling out the diagnosis of SAS in patients being screened for this condition, as this yielded only three negative test results in 301 screening procedures.

Background. Mucinous cystadenoma is an unusual pulmonary tumor that must be distinguished from mucinous cystic carcinoma and mucinous cystic tumors of borderline malignancy. Methods. This study of two cases was performed to characterize mucinous cystadenoma clinically and immunohistochemically, using proliferation markers (proliferating cell nuclear antigen [PCNA], MIB1) and carcinoembryonic antigen expression. Results. Pathologic examination in each instance showed unilocular cysts containing abundant clear mucus. The cysts were lined by tall mucinous epithelium, with absence of cytologic atypia and invasive growth. Proliferation markers using immunohistochemical methods showed less than 10% and 5% of labeled nuclei, respectively. Carcinoembryonic antigen immunostaining in both cases was negative. Patients remained free from recurrence for at least 2 years after surgery. Conclusions. Mucinous cystadenoma of the lung appears to be a benign neoplasm because of its clinical course and immunohistochemical low expression of proliferation markers such as PCNA and MIB1. Cancer 1995; 76:1540–4.

CEA, SCC and CYFRA 21-1 were measured in samples of serum coming from 105 'Non small cell lung cancer' (NSCLC) patients. The present study has been carried out to compare these markers, to analyse their prognostic significance and to determine the best combination of tumor markers. The median value and interquartile range were: CYFRA 21-1: 2,3 ng/ml, CEA: 3,7 ng/ml, SCC: 1,2 ng/ml. CEA demonstrated higher values in adenocarcinomas (P = 0.04). SCC and CYFRA 21-1 were comparable in the different histologic groups. CYFRA 21-1 and CEA values were dependant on tumor stage. Advanced tumors (T3 and T4) demonstrated higher serum CYFRA 21-1 level (P = 0.0006). CYFRA 21-1 was higher than 3,3 ng/ml in 36% of patients. CEA was higher than 5 ng/ml in 38% of patients and SCC was higher than 2 ng/ml in 27% of patients. Patients with a high CEA and CYFRA21-1 serum level had a shorter survival than those with a normal serum level. In a Cox regression analysis four variables (TNM stage, age, CYFRA 21-1 and CEA level) were found to be significant in the prediction of survival; CYFRA 21-1 level had the lowest P value (P = 0.0002). The current study suggests the use of a combination of CEA and CYFRA 21-1 in the clinical care of NSCLC.

Clinically, our ability to predict disease outcome for patients with early stage lung cancer is currently poor. To address this issue, tumour specimens were collected at surgery from non-small cell lung cancer (NSCLC) patients as part of the European Early Lung Cancer (EUELC) consortium. The patients were followed-up for three years post-surgery and patients who suffered progressive disease (PD, tumour recurrence, metastasis or a second primary) or remained disease-free (DF) during follow-up were identified. RNA from both tumour and adjacent-normal lung tissue was extracted from patients and subjected to microarray expression profiling. These samples included 36 adenocarcinomas and 23 squamous cell carcinomas from both PD and DF patients. The microarray data was subject to a series of systematic bioinformatics analyses at gene, network and transcription factor levels. The focus of these analyses was 2-fold: firstly to determine whether there were specific biomarkers capable of differentiating between PD and DF patients, and secondly, to identify molecular networks which may contribute to the progressive tumour phenotype. The experimental design and analyses performed permitted the clear differentiation between PD and DF patients using a set of biomarkers implicated in neuroendocrine signalling and allowed the inference of a set of transcription factors whose activity may differ according to disease outcome. Potential links between the biomarkers, the transcription factors and the genes p21/CDKN1A and Myc, which have previously been implicated in NSCLC development, were revealed by a combination of pathway analysis and microarray meta-analysis. These findings suggest that neuroendocrine-related genes, potentially driven through p21/CDKN1A and Myc, are closely linked to whether or not a NSCLC patient will have poor clinical outcome.

International audience; Background: Data concerning the effects of prenatal exposures to phthalates and phenols on fetal growth are limited in humans. Previous findings suggest possible effects of some phenols on male birth weight.Objective: Our aim was to assess the relationships between prenatal exposures to phthalates and phenols and fetal growth among male newborns.Methods: We conducted a case-control study on male malformations of the genitalia nested in two French mother-child cohorts with recruitment between 2002 and 2006. We measured, in maternal urinary samples collected between 6 and 30 gestational weeks, the concentrations (micrograms per liter) of 9 phenol (n = 191 pregnant women) and 11 phthalate metabolites (n = 287). Weight, length, and head circumference at birth were collected from maternity records. Statistical analyses were corrected for the oversampling of malformation cases.Results: Adjusted birth weight decreased by 77 g [95% confidence interval (CI): -129, -25] and by 49 g (95% CI: -86, -13) in association with a 1-unit increase in ln-transformed 2,4-dichlorophenol (DCP) and 2,5-DCP urinary concentrations, respectively. Benzophenone-3 (BP3) ln-transformed concentrations were positively associated with weight (26 g; 95% CI: -2, 54) and head circumference at birth (0.1 cm; 95% CI: 0.0, 0.2). Head circumference increased by 0.3 cm (95% CI: 0.0, 0.7) in association with a 1-unit increase in ln-transformed BPA concentration. For phthalate metabolites there was no evidence of monotonic associations with birth weight.Conclusions: Consistent with findings of a previous study, we observed evidence of an inverse association of 2,5-DCP and a positive association of BP3 with male birth weight.

Standard treatment of small-cell lung cancer (SCLC) is a combination of etoposide and platinum for patients with extensive disease, associated with radiotherapy for patients with limited disease (LD). Therapeutic strategies for relapse, although well characterized, are disappointing. Between 1997 and 2009, 300 patients were treated for SCLC at Grenoble University Hospital. We analyzed patients' characteristics and outcomes at different treatment steps, to determine prognostic factors and propose subsequent treatment strategies according to "sensitive", "resistant" or "refractory" status established after first-line treatment (L1). The median patient age was 63.2 years, 46.3% had LD, and 23% were female. The objective response rate (ORR) to first-line chemotherapy was 73% [CI(95%): 67.6-77.9] and median survival was 13 months. After L1, comparison between "refractory" and "sensitive" groups showed more extensive disease (76.6% vs. 34.3%, p=0.003), poorer Performance Status (PS 0-1: 48.4% vs. 67.8%, p=0.008), more endocrine paraneoplastic syndrome (18.7% vs. 8.4%, p=0.03) and more composite histology (17.2% vs. 4.9%, p=0.004) in "refractory" patients. After second line (L2), ORR was 55.8% [CI(95%): 45.2-66.0] in "sensitive", 18.2% [CI(95%): 8.2-32.7] in "resistant", and 14.7% [CI(95%): 4.9-31.0] in "refractory" groups; with partial response only for the last two groups. After L3 and L4, ORR was 24.0% [CI(95%): 14.9-35.2] in "sensitive", 9.1% [CI(95%): 11.2-29.2] in "resistant" with partial response only. No response was observed for "refractory". After L1, the median survival was respectively 23, 10 and 6.4 months for "sensitive", "resistant" and "refractory" groups (p

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p

Background Environmental epidemiology and biomonitoring studies typically rely on biological samples to assay the concentration of non-persistent exposure biomarkers. Between-participant variations in sampling conditions of these biological samples constitute a potential source of exposure misclassification. Few studies attempted to correct biomarker levels for this error. We aimed to assess the influence of sampling conditions on concentrations of urinary biomarkers of select phenols and phthalates, two widely-produced families of chemicals, and to standardize biomarker concentrations on sampling conditions. Methods Urine samples were collected between 2002 and 2006 among 287 pregnant women from Eden and Pélagie cohorts, from which phthalates and phenols metabolites levels were assayed. We applied a 2-step standardization method based on regression residuals. First, the influence of sampling conditions (including sampling hour, duration of storage before freezing) and of creatinine levels on biomarker concentrations were characterized using adjusted linear regression models. In the second step, the model estimates were used to remove the variability in biomarker concentrations due to sampling conditions and to standardize concentrations as if all samples had been collected under the same conditions (e.g., same hour of urine collection). Results Sampling hour was associated with concentrations of several exposure biomarkers. After standardization for sampling conditions, median concentrations differed by ‒ 38 % for 2,5-dichlorophenol to +80 % for a metabolite of diisodecyl phthalate. However, at the individual level, standardized biomarker levels were strongly correlated (correlation coefficients above 0.80) with unstandardized measures. Conclusions Sampling conditions, such as sampling hour, should be systematically collected in biomarker-based studies, in particular when the biomarker half-life is short. The 2-step standardization method based on regression residuals that we proposed in order to limit the impact of heterogeneity in sampling conditions could be further tested in studies describing levels of biomarkers or their influence on health.

BACKGROUND: Basal cell carcinomas (BCCs) have been described in various locations, such as skin, anal canal, tongue, larynx, and recently, the lungs. These tumors seem to have a poor prognosis. METHODS: A series of 115 surgically resected lung tumors, previously classified as poorly or undifferentiated carcinoma, were reviewed retrospectively. From these, 37 cases were reclassified as BCCs and were compared in terms of clinical features and survival, with 40 cases reclassified as poorly differentiated squamous cell (PDSC) carcinoma of the lung. RESULTS: There was no difference between the groups regarding age, clinical presentation, pattern of relapse, and cause of death. Median and overall survival were different between the two groups, especially for Stage I and II patients: 5-year actuarial survival in the BCC group was 15% and in the PDSC group 47% (P = 0.004). CONCLUSIONS: This subset of non-small cell lung cancer (NSCLC) has a worse prognosis than other NSCLC, and this should be considered in survival studies and new treatment trials.

28 patients with primitive lung cancers were imaged by immunoscintigraphy (IS) with 111 indium-labelled F(ab′) 2 anti-carcinoembryonic antigen (CEA), to assess this technique for mediastinal staging. IS revealed primitive tumours in 21 cases in whom mediastinal extension was assessed. There was concordance between clinical staging and IS confirmed by surgery in 17 cases, and discordance in 4. After surgery, discordance was in favour of IS in 2 cases (1 true positive and 1 true negative) and in favour of clinical staging in 2 (false positive of immunoscintigraphy). Anti-CEA IS could be useful for improving mediastinal staging of lung cancer.

Sodium butyrate (NaBu) is a histone deacetylase inhibitor that exhibits numerous antiproliferative activities in various cancer cell lines, notably through the accumulation of the well-known cyclin-dependent kinase inhibitor p21(WAF1) . SRSF2 belongs to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Previous studies demonstrated that NaBu alters pre-mRNA splicing patterns through upregulation of SR proteins expression in non-tumor cells. In this study, we show that NaBu also induces the accumulation of SRSF2 in human lung carcinoma cell lines. We recently identified a signaling network involving the acetyltransferase TIP60, the deacetylase HDAC6 and the SRPK kinases that regulates SRSF2 protein turnover through phosphorylation/acetylation modifications in response to cisplatin. Here, we show that the same signaling pathway controls SRSF2 protein expression upon NaBu treatment. Importantly, we further demonstrate that SRSF2 is required for the accumulation of p21(WAF1) at both mRNA and protein levels in response to NaBu. Finally, we provide evidence that a long-term NaBu-treatment triggers senescence in our cellular models, a phenomenon that is prevented by the knockdown of SRSF2. Altogether, these results unravel a new function of SRSF2 in the process of cellular senescence and identify the cyclin-Cdk inhibitor p21(WAF1) as a key target of SRSF2 in this setting.

Introduction: Adenocarcinoma is the most common histologic type of lung cancer. To address advances in oncology, molecular biology, pathology, radiology, and surgery of lung adenocarcinoma, an international multidisciplinary classification was sponsored by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society. This new adenocarcinoma classification is needed to provide uniform terminology and diagnostic criteria, especially for bronchioloalveolar carcinoma (BAC), the overall approach to small nonresection cancer specimens, and for multidisciplinary strategic management of tissue for molecular and immunohistochemical studies. Methods: An international core panel of experts representing all three societies was formed with oncologists/pulmonologists, pathologists, radiologists, molecular biologists, and thoracic surgeons. A systematic review was performed under the guidance of the American Thoracic Society Documents Development and Implementation Committee. The search strategy identified 11,368 citations of which 312 articles met specified eligibility criteria and were retrieved for full text review. A series of meetings were held to discuss the development of the new classification, to develop the recommendations, and to write the current document. Recommendations for key questions were graded by strength and quality of the evidence according to the Grades of Recommendation, Assessment, Development, and Evaluation approach. Results: The classification addresses both resection specimens, and small biopsies and cytology. The terms BAC and mixed subtype adenocarcinoma are no longer used. For resection specimens, new concepts are introduced such as adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) for small solitary adenocarcinomas with either pure lepidic growth (AIS) or predominant lepidic growth with ≤5 mm invasion (MIA) to define patients who, if they undergo complete resection, will have 100% or near 100% disease-specific survival, respectively. AIS and MIA are usually nonmucinous but rarely may be mucinous. Invasive adenocarcinomas are classified by predominant pattern after using comprehensive histologic subtyping with lepidic (formerly most mixed subtype tumors with nonmucinous BAC), acinar, papillary, and solid patterns; micropapillary is added as a new histologic subtype. Variants include invasive mucinous adenocarcinoma (formerly mucinous BAC), colloid, fetal, and enteric adenocarcinoma. This classification provides guidance for small biopsies and cytology specimens, as approximately 70% of lung cancers are diagnosed in such samples. Non-small cell lung carcinomas (NSCLCs), in patients with advanced-stage disease, are to be classified into more specific types such as adenocarcinoma or squamous cell carcinoma, whenever possible for several reasons: (1) adenocarcinoma or NSCLC not otherwise specified should be tested for epidermal growth factor receptor (EGFR) mutations as the presence of these mutations is predictive of responsiveness to EGFR tyrosine kinase inhibitors, (2) adenocarcinoma histology is a strong predictor for improved outcome with pemetrexed therapy compared with squamous cell carcinoma, and (3) potential life-threatening hemorrhage may occur in patients with squamous cell carcinoma who receive bevacizumab. If the tumor cannot be classified based on light microscopy alone, special studies such as immunohistochemistry and/or mucin stains should be applied to classify the tumor further. Use of the term NSCLC not otherwise specified should be minimized. Conclusions: This new classification strategy is based on a multidisciplinary approach to diagnosis of lung adenocarcinoma that incorporates clinical, molecular, radiologic, and surgical issues, but it is primarily based on histology. This classification is intended to support clinical practice, and research investigation and clinical trials. As EGFR mutation is a validated predictive marker for response and progression-free survival with EGFR tyrosine kinase inhibitors in advanced lung adenocarcinoma, we recommend that patients with advanced adenocarcinomas be tested for EGFR mutation. This has implications for strategic management of tissue, particularly for small biopsies and cytology samples, to maximize high-quality tissue available for molecular studies. Potential impact for tumor, node, and metastasis staging include adjustment of the size T factor according to only the invasive component (1) pathologically in invasive tumors with lepidic areas or (2) radiologically by measuring the solid component of part-solid nodules.

International audience; SRSF2 is a serine/arginine-rich protein belonging to the family of SR proteins that are crucial regulators of constitutive and alternative pre-mRNA splicing. Although it is well known that phosphorylation inside RS domain controls activity of SR proteins, other post-translational modifications regulating SRSF2 functions have not been described to date. In this study, we provide the first evidence that the acetyltransferase Tip60 acetylates SRSF2 on its lysine 52 residue inside the RNA recognition motif, and promotes its proteasomal degradation. We also demonstrate that the deacetylase HDAC6 counters this acetylation and acts as a positive regulator of SRSF2 protein level. In addition, we show that Tip60 downregulates SRSF2 phosphorylation by inhibiting the nuclear translocation of both SRPK1 and SRPK2 kinases. Finally, we demonstrate that this acetylation/phosphorylation signalling network controls SRSF2 accumulation as well as caspase-8 pre-mRNA splicing in response to cisplatin and determines whether cells undergo apoptosis or G(2)/M cell cycle arrest. Taken together, these results unravel lysine acetylation as a crucial post-translational modification regulating SRSF2 protein level and activity in response to genotoxic stress.

International audience; ING2 (inhibitor of growth 2) is a candidate tumor-suppressor gene involved in cell cycle control, apoptosis and senescence. Although the functions of ING2 within the chromatin remodeling complex Sin3A/histone deacetylase (HDAC) and in the p53 pathway have been described, how ING2 itself is regulated remains unknown. In this study we report for the first time that ING2 can be sumoylated by small ubiquitin-like modifier 1 (SUMO1) on lysine 195 both in vitro and in vivo. Strikingly, ING2 sumoylation enhances its association with Sin3a. We provide evidences that ING2 can bind to the promoter of genes to mediate their expression and that sumoylation of ING2 is required for this binding to some of these genes. Among them, we identified the gene TMEM71 (transmembrane protein 71), whose expression is regulated by ING2 sumoylation. ING2 must be sumoylated to bind to the promoter of TMEM71 and to recruit the Sin3A chromatin-modifying complex to this promoter, in order to regulate TMEM71 transcription. Hence, sumoylation of ING2 enhances its binding to the Sin3A/HDAC complex and is required to regulate gene transcriptions.

International audience; ING2 (Inhibitor of Growth 2) is a candidate tumor suppressive protein frequently lost in human tumors. Recently, we have reported that ING2 downregulation impairs DNA replication forks progression and leads to genome instability. To better understand the tumor suppressive functions of ING2 and its role in the cell cycle, we downregulated its expression in cells and studied the consequences of this downregulation on the G(1)/S transition. We observed that the inhibition of ING2 expression accelerated the progression of cells from G(1) to S-phase, and was accompanied by a decrease of p21 expression. Moreover, we show that the regulation of p21 by ING2 is independent of the tumor suppressive protein p53. Interestingly, this function seems to be unique for ING2 since its closest homolog ING1 does not regulate the G(1)/S transition. It has been suggested previously that ING2 may modulate the trimethylation of H3K4 at the promoter of p21. Accordingly, our results suggest that there may be a link between the regulation of the G(1)/S transition by ING2 and the level of H3K4Me3. All together, these results bring new information concerning the role of ING2 in the regulation of the cell cycle and suggest that it may play important roles in controlling several S-phase checkpoints.

International audience; ING2 is a candidate tumor suppressor gene involved in cell cycle control, apoptosis and senescence. Furthermore, we have recently shown that loss of ING2 expression is associated with increased genome instability. We investigated its status in a series of 120 non-small cell lung cancer (NSCLC) by using immunohistochemistry (IHC). The results showed that ING2 protein expression is downregulated in more than 50% of NSCLC, with a higher frequency in adenocarcinoma (ADK) as compared to squamous cell carcinoma (SCC) (68% versus 45%, P=0.021). Loss of ING2 expression occurs in a high proportion of tumors from stage I and was not associated with patient's gender, age and 5-year survival. When investigating the possible mechanisms responsible for the decrease of ING2 expression, we did not observe any loss of heterozygosity or mutation in the ING2 gene. However, in 95% of the cases examined, we identified a silent single nucleotide polymorphism (SNP). By using quantitative RT-PCR, we found that ING2 loss of expression may be due to the decrease of its mRNA level. Analysis of CpG islands present in the promoter region of the ING2 gene did not allow for the detection of methylation. Mechanistically, although p53 can regulate ING2 transcription and ING2 enhances p53 activity, no correlation between ING2 and p53 IHC status was observed. Overall, these results indicate that loss of ING2 expression could contribute to lung tumorigenesis independently of p53.

The transcription factor E2F1 has a crucial role in the control of cell growth and has been shown to regulate neoangiogenesis in a p53-dependent manner through inhibition of activity of the VEGF-A (vascular endothelial growth factor) promoter. Besides being regulated by transcription, VEGF-A is also highly regulated by pre-mRNA alternative splicing, resulting in the expression of several VEGF isoforms with either pro-(VEGF(xxx)) or anti-(VEGF(xxx)b) angiogenic properties. Recently, we identified the SR (Ser-Rich/Arg) protein SC35, a splicing factor, as a new transcriptional target of E2F1. Here, we show that E2F1 downregulates the activity of the VEGF-A promoter in tumour cells independently of p53, leading to a strong decrease in VEGF(xxx) mRNA levels. We further show that, strikingly, E2F1 alters the ratio of pro-VEGF(xxx) versus anti-VEGF(xxx)b angiogenic isoforms, favouring the antiangiogenic isoforms, by a mechanism involving the induction of SC35 expression. Finally, using lung tumour xenografts in nude mice, we provide evidence that E2F1 and SC35 proteins increase the VEGF(165)b/VEGF ratio and decrease tumour neovascularization in vivo. Overall, these findings highlight E2F1 and SC35 as two regulators of the VEGF(xxx)/VEGF(xxx)b angiogenic switch in human cancer cells, a role that could be crucial during tumour progression, as well as in tumour response to antiangiogenic therapies.

Lung adenocarcinoma (AD) represents a predominant type of lung cancer demonstrating significant morphologic and molecular heterogeneity. We sought to understand this heterogeneity by utilizing gene expression analyses of 432 AD samples and examining associations between 27 known cancer-related pathways and the AD subtype, clinical characteristics and patient survival. Unsupervised clustering of AD and gene expression enrichment analysis reveals that cell proliferation is the most important pathway separating tumors into subgroups. Further, AD with increased cell proliferation demonstrate significantly poorer outcome and an increased solid AD subtype component. Additionally, we find that tumors with any solid component have decreased survival as compared to tumors without a solid component. These results lead to the potential to use a relatively simple pathological examination of a tumor in order to determine its aggressiveness and the patient's prognosis. Additional results suggest the ability to use a similar approach to determine a patient's sensitivity to targeted treatment. We then demonstrated the consistency of these findings using two independent AD cohorts from Asia (N = 87) and Europe (N = 89) using the identical analytic procedures.

International audience; Cancer cells frequently express genes normally active in male germ cells. ATAD2 is one of them encoding a conserved factor harbouring an AAA type ATPase domain and a bromodomain. We show here that ATAD2 is highly expressed in testis as well as in many cancers of different origins and that its high expression is a strong predictor of rapid mortality in lung and breast cancers. These observations suggest that ATAD2 acts on upstream and basic cellular processes to enhance oncogenesis in a variety of unrelated cell types. Accordingly, our functional studies show that ATAD2 controls chromatin dynamics, genome transcriptional activities and apoptotic cell response. We could also highlight some of the important intrinsic properties of its two regulatory domains, including a functional cross-talk between the AAA ATPase domain and the bromodomain. Altogether, these data indicate that ATAD2 overexpression in somatic cells, by acting on basic properties of chromatin, may contribute to malignant transformation.

Lung cancer remains one of the leading causes of cancer-related death in developed countries. Although lung adenocarcinomas with EGFR mutations or EML4-ALK fusions respond to treatment by epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) inhibition, respectively, squamous cell lung cancer currently lacks therapeutically exploitable genetic alterations. We conducted a systematic search in a set of 232 lung cancer specimens for genetic alterations that were therapeutically amenable and then performed high-resolution gene copy number analyses. We identified frequent and focal fibroblast growth factor receptor 1 (FGFR1) amplification in squamous cell lung cancer (n = 155), but not in other lung cancer subtypes, and, by fluorescence in situ hybridization, confirmed the presence of FGFR1 amplifications in an independent cohort of squamous cell lung cancer samples (22% of cases). Using cell-based screening with the FGFR inhibitor PD173074 in a large (n = 83) panel of lung cancer cell lines, we demonstrated that this compound inhibited growth and induced apoptosis specifically in those lung cancer cells carrying amplified FGFR1. We validated the FGFR1 dependence of FGFR1-amplified cell lines by FGFR1 knockdown and by ectopic expression of an FGFR1-resistant allele (FGFR1(V561M)), which rescued FGFR1-amplified cells from PD173074-mediated cytotoxicity. Finally, we showed that inhibition of FGFR1 with a small molecule led to significant tumor shrinkage in vivo. Thus, focal FGFR1 amplification is common in squamous cell lung cancer and associated with tumor growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option in this cohort of patients.

International audience; Lung cancer is the most important cause of cancer-related mortality. Resectability and eligibility for treatment with adjuvant chemotherapy is determined by staging according to the TNM classification. Other determinants of tumour behaviour that predict disease outcome, such as molecular markers, may improve decision-making. Activation of the gene encoding human telomerase reverse transcriptase (hTERT) is implicated in the pathogenesis of lung cancer, and consequently detection of hTERT mRNA might have prognostic value for patients with early stage lung cancer. A cohort of patients who underwent a complete resection for early stage lung cancer was recruited as part of the European Early Lung Cancer (EUELC) project. In 166 patients expression of hTERT mRNA was determined in tumour tissue by quantitative real-time RT-PCR and related to that of a house-keeping gene (PBGD). Of a subgroup of 130 patients tumour-distant normal tissue was additionally available for hTERT mRNA analysis. The correlation between hTERT levels of surgical samples and disease-free survival was determined using a Fine and Gray hazard model. Although hTERT mRNA positivity in tumour tissue was significantly associated with clinical stage (Fisher's exact test p=0.016), neither hTERT mRNA detectability nor hTERT mRNA levels in tumour tissue were associated with clinical outcome. Conversely, hTERT positivity in adjacent normal samples was associated with progressive disease, 28% of patients with progressive disease versus 7.5% of disease-free patients had detectable hTERT mRNA in normal tissue [adjusted HR: 3.60 (1.64-7.94), p=0.0015]. hTERT mRNA level in tumour tissue has no prognostic value for patients with early stage lung cancer. However, detection of hTERT mRNA expression in tumour-distant normal lung tissue may indicate an increased risk of progressive disease.