Pathologist-initiated reflex testing for biomarkers in non-small-cell lung cancer: expert consensus on the rationale and considerations for implementation.

Biomarker tests in lung cancer have been traditionally ordered by the treating oncologist upon confirmation of an appropriate pathological diagnosis. The delay this introduces prolongs yet further what is already a complex, multi-stage, pre-treatment pathway and delays the start of first-line systemic treatment, which is crucially informed by the results of such analysis. Reflex testing, in which the responsibility for testing for an agreed range of biomarkers lies with the pathologist, has been shown to standardise and expedite the process. Twelve experts discussed the rationale and considerations for implementing reflex testing as standard clinical practice.

Large pulmonary masses containing varicose veins: a rare presentation of benign metastasising leiomyomas.

A 48-year-old woman presented with cough and chest pain. A chest radiograph and CT scans showed bilateral lung masses containing massive venous varices. A core biopsy specimen revealed benign metastasising leiomyoma with strong expression of progesterone receptors. A review of her medical history revealed a hysterectomy 11 years earlier. The lung masses showed significant reduction in size after induction of artificial menopause, although the pulmonary varices persisted.

EUELC project: a multi-centre, multipurpose study to investigate early stage NSCLC, and to establish a biobank for ongoing collaboration.

The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular-pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARbeta genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes.

Expression of p53, pRB, and p16 in lung tumours: a validation study on tissue microarrays.

Tissue microarrays have been created from 326 lung tumours, including 173 squamous cell carcinomas (SCCs) and 132 adenocarcinomas (ADs). In order to evaluate the usefulness of this microarray series, the expression of p53, p16, and Rb proteins was compared by immunohistochemistry on both the tissue microarrays and the corresponding whole sections for all 326 tumours. The presence of replicate punches improved both the yield and the concordance of data relative to the whole section results, so that the consensus score from the replicates agreed with the whole section result in more than 90% of informative tumours. The large number of tumours in this series also allowed significant differences in protein expression patterns to be detected between SCC and AD, the major subtypes of non-small cell lung carcinoma (NSCLC). SCC had higher levels of p53 staining (67% vs 52% in AD) and substantially increased p16 loss (SCC 75%, AD 53%) combined with greater retention of pRB expression (SCC 86% vs 67% in AD). The strong inverse correlation between p16 and pRB seen in SCC was essentially absent in AD. This study represents the largest single immunohistochemical survey of protein expression for p53, p16, and RB in NSCLCs.

Allelic imbalance at the DNA mismatch repair loci, hMSH2, hMLH1, hPMS1, hPMS2 and hMSH3, in squamous cell carcinoma of the head and neck.

BACKGROUND: Squamous cell carcinoma of the head and neck (SCCHN) is one of the 10 most frequently occurring cancers in the world. Defective mismatch repair, as exhibited by the phenomenon of microsatellite instability, has been observed in SCCHN although no reports of mismatch repair gene mutations or altered protein expression have been published. In a variety of microsatellite instability (MSI) positive cancers where mutations in the mismatch repair (MMR) genes were not observed, allelic imbalance at the loci of the MMR genes was prevalent. OBJECTIVE: To investigate whether allelic imbalance at the MMR genetic loci contributes to the development of SCCHN. MATERIALS AND METHODS: 35 matched normal/tumour SCCHN pairs were studied using 29 microsatellite markers located within and adjacent to six known DNA mismatch repair genes. In addition, mutational analysis and protein expression of hMSH2 and hMLH1 were investigated. RESULTS AND CONCLUSIONS: We demonstrated that 36 and 17% of the analysed SCCHN specimens exhibited allele imbalance at the hMLH1 and hMSH3 genetic loci, respectively. Allelic instability at these two loci was found to be correlated with the MSI status of the SCCHN tumours. Allelic instability was found to be uncommon at the other MMR gene loci analysed. One mutation was found in hMSH2 and none in hMLH1 in this series of tumours. 23 of 24 (96%) of the examined SCCHN tumours showed reduced expression of either hMSH2 or hMCH1 genes. Allelic instability in the MMR genes, hMLH1 and hMSH3, is proposed to be involved in the aetiology of SCCHN tumours.

Cancer-specific genomic instability in bronchial lavage: a molecular tool for lung cancer detection.

We examined genomic instability in DNA from 80 bronchial lavage samples from patients with lung cancer and individuals with no malignant lung disease. We used a multiplex assay of eight fluorescent-tagged microsatellite markers that have a very high incidence of allelic imbalance in lung tumors. When genomic instability at individual loci was analyzed statistically against diagnosis, markers D3S1289 (P = 0.033), D3S1300 (P = 0.001), D13S171 (P = 0.009), and D17S2179E (P = 0.017) demonstrated significantly higher frequency of instability in bronchial lavage specimens from lung cancer cases than those with nonmalignant conditions. In contrast, markers D9S157, D9S161, D13S153, and D5S644 demonstrated lower specificity (P > 0.05) for lung tumors. These results suggest that genomic instability in some loci may be related to high proliferation rates but not necessarily to cell commitment to malignancy. When genomic instability was scored with only the four cancer-specific markers, the assay produced a sensitivity of 73.9% and a specificity of 76.5%. On combining the results from the cytological examination and the molecular assay, the sensitivity reached 82.6%. These results indicate that in our efforts to investigate genomic instability as a potential marker for the early detection of lung cancer, we need to identify cancer-specific genomic instability markers. This paper has shown that these first four markers may be considered to form an individual set of cancer-specific genomic instability markers.

The evolution of loss of heterozygosity on chromosome 17 during the progression to barrett's adenocarcinoma involves a unique combination of target sites in individual specimens.

We have previously identified thirteen common minimally deleted regions (MRs) on chromosome 17 in twelve Barrett's esophageal adenocarcinoma (BOA) specimens using 41 precisely mapped microsatellite markers (Dunn et al., Oncogene, 17: 987-993, 1999). The aim of the present study has been to identify the earliest sites of loss on this chromosome that arise and persist during the progression to BOA. This has been undertaken by the analysis of multiple carefully microdissected tissue samples from each of five esophagectomy specimens, several of which contained identifiable premalignant tissue. Our data demonstrate a stepwise accumulation of loss in each analyzed specimen, consistent with a single clonal pathway in four specimens and several coexisting pathways in one specimen. Several clonal anomalies (loss preceding heterozygosity and variable intrasample degrees of loss at different markers) were also observed. Within extensively deleted regions of the tumor (seen in three specimens), small deletions were detected in premalignant tissue, predominantly at the site of our identified MRs, and these losses were seen to expand and merge during the progression to BOA. Clonal losses at MRs were first detected in tissue showing early changes histologically, including Barrett's intestinal metaplasia and intermediate-grade dysplasia. Our results provide further support for many of the MRs we have previously identified, thereby adding to evidence for the existence of multiple novel cancer-associated genes on chromosome 17 involved in the development of BOA.

hMLH1 and hMSH2 expression correlates with allelic imbalance on chromosome 3p in non-small cell lung carcinomas.

DNA mismatch repair genes have been implicated in the pathogenesis and predisposition of certain malignancies through a mutator phenotype. In this study, we investigated, in 150 non-small cell lung carcinomas, the expression levels of hMLH1 and hMSH2 proteins in relation to loss of heterozygosity on chromosomes 3p and 2p, the mutational status of these genes' promoters and the hot spot exons. We have demonstrated that 88 of 150 (58.6%) tumor specimens had reduced expression levels of the hMLH1 protein, whereas 85 of 147 (57.8%) specimens had reduced expression levels of the hMSH2 protein. Reduced expression levels of both proteins were observed in 51 of 150 (34%) specimens. In adenocarcinomas, the reduction of hMSH2 expression was more frequently observed than that of hMLH1 (P<0.003), whereas in squamous cell carcinoma of the lung hMLH1 expression was more frequently reduced than hMSH2 (P<0.006). Reduced expression of hMLH1correlated with allelic imbalance on loci D3S1289 (P<0.0002) and D2S391 (P<0.05). It is of note that an inverse correlation was found between hMSH2 reduced expression and loss of heterozygosity at locus D3S1300 (P = 0.016). In addition, hMLH1 reduced expression was more frequently associated with heavy smokers, assessed by daily tobacco uptake (P = 0.018) and total smoking exposure (pack-years; P<0.05). In addition, a correlation between hMLH1 reduced expression and nodal metastasis in squamous cell carcinoma of the lung was observed (P = 0.015). No mutations were identified in the promoters or exons examined in these two genes. These findings indicate that hMLH1 and hMSH2 gene inactivation is a common event in the development of non-small cell lung carcinoma and allelic loss seems to be a major genetic event involved in hMLH1 silencing. In addition, we propose that a putative negative regulator of hMSH2 gene may be located at the locus 3p14.

Fractional allele loss indicates distinct genetic populations in the development of squamous cell carcinoma of the head and neck (SCCHN).

Loss of heterozygosity (LOH) had been widely used to assess genetic instability in tumours and a high LOH on chromosome arms 3p, 9p and 17p has been considered to be a common event in squamous cell carcinoma of the head and neck (SCCHN). We have investigated LOH in 52 SCCHN using a range of microsatellite markers. LOH was observed in 69% of individuals on 17p using seven markers, in 64% of individuals on 3p using 17 markers and in 61% of individuals on 9p using 11 markers. Fractional allele loss (FAL) has been calculated for each tumour (FAL is the number of chromosomal arms showing LOH divided by the number of informative chromosomal arms) and a median FAL value of 0.25 was obtained in the 52 SCCHN studied. The LOH data were examined on the basis of FAL scores: low FAL (LFAL), 0.00-0.19; medium FAL (MFAL), 0.20-0.32; high FAL (HFAL), 0.33-0.88. HFAL tumours demonstrated a significantly higher LOH on chromosome arms 3p, 9p and 17p, with 94% LOH on 3p, 94% on 9p and 100% on 17p compared with LFAL tumours. Six of the 16 patients in the LFAL group were found to have no LOH on 3p, 9p or 17p and of these four had LOH at other sites, on chromosomes 2p25-p24, 5q21-22, 7pter-p22, 8q13-q22.1, 11q23.3, 13q32, 17q, 18p11.21, 18q21.31 and 19q12-q13.1. These results indicate that LFAL patients form a subset of SCCHN tumours with distinct molecular initiating events which may represent a discrete genetic population.

Telomerase activity in non-small cell lung carcinomas correlates with smoking status.

Human telomerase is a ribonucleoprotein DNA polymerase which maintains the telomeric region of human chromosomes and has been detected in all types of human cancer tested. We used the telomeric repeat amplification protocol (TRAP) assay to examine 71 non-small cell lung carcinomas (NSCLC) and their adjacent normal tissue. Telomerase activity was detected in 61 (86%) of the 71 NSCLC examined but not in any of the matched normal lung tissues. A significant correlation was found between the presence of telomerase activity and current smoking status at the time of diagnosis (p=0. 0076). In addition, a trend was found between telomerase activity and smoking exposure (p=0.06). Our findings demonstrate that telomerase activity is a common phenomenon in NSCLC cases but not in the normal lung. However, certain cases in former smokers may follow a telomerase independent pathway.

Pulmonary neuroendocrine cells in nitrofen-induced diaphragmatic hernia and the effect of prenatal glucocorticoids.

The high mortality associated with congenital diaphragmatic hernia (CDH) is due to pulmonary hypoplasia and hypertension, structural and functional abnormalities which can to some extent be ameliorated by prenatal administration of glucocorticoids. In the hypoplastic, hypertensive lungs of neonatal rats in which CDH has been induced by nitrofen, those pulmonary neuroendocrine cells (PNCs) containing calcitonin gene-related peptide (CGRP) increase in number, and it has been suggested that this might be due to inhibition of secretion of the peptide, the consequent decrease in its vasodilatory effects contributing to the hypertension. Whether this increase affects the entire population of PNCs, however, and how these cells are affected by administration of prenatal glucocorticoids, is unknown. As revealed by immunolabelling for protein gene product (PGP) 9.5, a general marker of NCs and expressed per cm2 tissue section, the total PNC population in rats with nitrofen-induced CDH was significantly greater than in controls receiving only olive oil (672 vs 375/cm2, P = 0.03) and was further increased (824 per cm2) in animals treated prenatally with dexamethasone (n = 8 in all groups). The increase in the total PNC population in rats with CDH is similar in magnitude to that described for the CGRP-containing subpopulation. Since the major role of the products of PNCs is now thought to be the regulation of development of pulmonary tissues and their response to injury, it is probable that the expansion of their population in the abnormal lungs associated with CDH is an adaptive response to pulmonary maldevelopment, a response possibly augmented by exogenous corticosteroids.

Sustentacular cells in pulmonary neuroendocrine tumours.

AIMS: To determine the prevalence of sustentacular cells across the range of pulmonary neuroendocrine tumours: typical and atypical carcinoid tumours and large cell and small cell neuroendocrine carcinomas. METHODS AND RESULTS: Sustentacular cells were sought in 80 pulmonary neuroendocrine tumours by immunolabelling for S100 protein, nerve growth factor receptor and glial fibrillary acidic protein. Intratumoural macrophages and Langerhans cells were identified with the KP 1 (CD68) and CD1A antibodies. S100-positive sustentacular cells were present in 25 of 30 typical carcinoids, 200 of 25 atypical tumours, six of 10 large cell carcinomas and six of 15 small cell lesions. They were most numerous in the typical carcinoids but very few in the small cell carcinomas, their prevalance being clearly related to grade of differentiation and, in particular, to the degree of architectural organization. CONCLUSIONS: Sustentacular cells are often found in pulmonary neuroendocrine tumours, especially better-differentiated lesions with a well-developed architecture. their prevalence clearly reflecting the degree of structural organization. Whether their prevalence is a useful prognostic indicator within a particular group of such tumours, such as the atypical carcinoids or the large cell carcinomas, as appears to be the case with paragangliomas, is unclear.

Multiple target sites of allelic imbalance on chromosome 17 in Barrett's oesophageal cancer.

Twelve Barrett's adenocarcinomas have been analysed for the occurrence of allelic imbalance (LOH) on chromosome 17 using 41 microsatellite markers. This study provides evidence for 13 minimal regions of LOH, six on 17p and seven on 17q. Four of these centre in the vicinity of the known tumour suppressor genes (TSGs) TP53 (17p13.1), NFI (17q11.2), BRCA1 (17q21.1), and a putative TSG (17p13.3). The tumours all displayed relatively small regions of LOH (1-10 cM), and in several tumours extensive regions of LOH were detected. One tumour displayed only two very small regions of LOH; 17p11.2 and 17p13.1. The frequency of allelic imbalance has been calculated based on the LOH encompassing only one minimal region, and based on all the LOH observations. By both evaluations the highest LOH frequencies were found for regions II (p53), III (17p13.1 centromeric to p53), IV (17p12), V (17p11.2) and VII (NF1, 17q11.2). Our data supports the existence of multiple TSGs on chromosome 17 and challenges the view that p53 is the sole target of LOH on 17p in Barrett's adenocarcinoma.

Effect of prenatal glucocorticoids on pulmonary vascular muscularisation in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Pulmonary hypertension (PH) contributes significantly to the mortality of congenital diaphragmatic hernia (CDH). Pulmonary vascular changes in CDH include a reduced vascular bed with increased arterial medial wall thickness and peripheral extension of muscle into intraacinar vessels. Antenatal steroids improve biochemical immaturity, lung compliance, and morphology in experimental CDH animals. The aim of this study was to examine the effects of prenatal glucocorticoid therapy on pulmonary artery muscularisation in CDH rats. METHODS: CDH was induced in fetal rats by the maternal administration of 100 mg of nitrofen by gavage on day 9.5 of gestation (term, day 22). Control animals received olive oil (OO). Dexamethasone (Dex, 0.25 mg/kg) or normal saline (NS) was given by intraperitoneal injection on days 18.5 and 19.5, and fetuses were delivered by caesarean section on day 21.5. Lung sections from five fetuses in each of four experimental groups were studied by a blinded investigator- OO-NS controls, CDH-NS, CDH-Dex, and non-CDH-NS. The external diameter (ED), medial wall thickness (MT), percent of medial wall thickness, and wall structure were evaluated from preacinar arteries accompanying conducting airways, and the intraacinar arterioles associated with the respiratory bronchi and saccules. RESULTS: In the preacinar arteries, CDH-NS animals had a significantly increased MT percentage compared with OO-NS controls (21.2+/-8.8 v 17.8+/-10.3, P = .0001). CDH-Dex rats had a lower MT percentage than CDH-NS rats (15.5+/-6.7 v 21.2+/-8.8, P = .0001). In the intraacinar region, CDH-Dex fetuses had a reduced percentage of muscularised intraacinar blood vessels compared with CDH-NS and OO-NS controls (10% v 24% and 28%, respectively, P = .01). Dexamethasone-treated CDH pups also displayed a significantly lower MT percentage of the intraacinar arteries compared with CDH-NS and OO-NS animals (15.7+/-13 v 23.4+/-9 and 25.4+/-12, P = .003). CONCLUSIONS: Medial hypertrophy is present in the preacinar but not the intraacinar blood vessels of CDH rats before birth. Dexamethasone inhibits medial hypertrophy and reduces the number of muscularised intraacinar vessels. Antenatal glucocorticoids may reduce the risk of PH developing in human newborns with antenatally diagnosed CDH.

Pulmonary neuroendocrine cell system in pediatric and adult lung disease.

In humans lungs affected by naturally occurring pulmonary disease, the pulmonary neuroendocrine cell system, which is normally arranged in a sparse but even distribution throughout the respiratory tract, increases in size. It is likely that the stimulus for this is pulmonary injury and that its purpose is the paracrine regulation of the restoration of pulmonary tissues to their normal state, an hypothesis supported by studies of animal lungs subjected to experimental injury as well as of the development of human and animal lungs in utero. Initially, this increase involves the development of interrupted rows of neuroendocrine cells. In the later stages, however, development of more disorderly intraepithelial aggregates can occur and the small, locally invasive neuroendocrine cell lesions known as tumourlets may occasionally result. Both of these latter structures often contain secretory products not found in the neuroendocrine cells of normal human lungs, probably indicating a derangement of what appears to be a fundamentally physiological response. It is likely that, in some circumstances, this disorderly change may contribute to pulmonary disease as well as being the result of it.

Pulmonary endocrine cells in anthracosilicotic lungs.

It has been suggested by some studies of human and animal lungs that the products of pulmonary endocrine cells, particularly gastrin-releasing peptide, might play a role in fibrogenesis, but more recent detailed studies of fibrotic human lungs have failed to confirm this. We have made a detailed quantitative examination of a series of fibrotic human lungs to see if we could determine whether there was any relationship between endocrine cells and fibrosis. Using immunocytochemistry, we investigated the morphology, content, distribution and number of pulmonary endocrine cells in 15 pairs of fibrotic lungs from coal miners, and compared their features with those of equivalent cells in age-matched controls. Proliferation of endocrine cells was seen in the lungs of just two miners, in which it was focal and associated with acute bronchitis and bronchopneumonia. There was no difference between the miners and controls in the appearance (mostly solitary cells), content (predominantly gastrin-releasing peptide and calcitonin), distribution (mainly in small bronchi and bronchioles), or number (4.5 vs 4.1 cells per 10,000 epithelial cells, respectively) of endocrine cells. It seems unlikely that the substances secreted by these cells play any role in stimulating fibrosis in human lungs, but rather that they have a function in the inflammatory response to pulmonary injury.