Absence of mutations in the VHL gene but frequent loss of heterozygosity at 3p25-26 in non-small cell lung carcinomas.

In this study we have examined 79 primary non-small cell lung tumours for the presence of mutations of the VHL gene as well as for allelic imbalance at the gene surrounding loci. While allelic imbalance was found in 83% of specimens, frequently affecting the whole 3p25-p26 region, no mutations were detected in the VHL coding region. The fractional regional loss (FRL) was significantly higher in squamous cell carcinomas (0.746) than adenocarcinomas (0.493) (Wilcoxon P=0.002). This is the first investigation of the VHL gene mutational status in primary lung tumours. Our results indicate that mutation is not a common means of VHL inactivation in NSCLC.

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.

K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification.

BACKGROUND: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMS(TM) allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. METHODS: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. RESULTS: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04-100%. CONCLUSIONS: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.

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.

Evaluation of telomerase activity in bronchial lavage as a potential diagnostic marker for malignant lung disease.

Telomerase is a ribonucleoprotein DNA polymerase that maintains the telomeric region of chromosomes lost during successive rounds of cell division. We used the telomeric repeat amplification protocol (TRAP) assay to examine telomerase activity in bronchial lavage (BL) samples from individuals undergoing diagnosis of lung cancer. Telomerase activity was detected in 17 (47%) of 36 samples examined. In particular, 16 (70%) of 23 BL specimens obtained from lung cancer patients showed detectable telomerase activity, while only 1 of 13 (8%) specimens obtained from patients without lung cancer demonstrated activity (P=0.00038). Moreover, 9 (90%) of 10 BL specimens, which were cytologically positive for lung cancer, were also positive for telomerase activity, while 7 (54%) of 13 cytologically negative BL specimens for lung cancer showed detectable telomerase activity. Detection of telomerase activity combined with cytology were able to identify 17 (74%) of 23 lung cancer cases whereas cytology alone identified 10 (43%) of 23 such cases (P=0.035). Our findings indicate that telomerase is a specific marker for malignant lung disease and a potential complementary tool to cytology in the diagnosis of certain lung cancer cases.

Sensitivity and limitations of high throughput fluorescent microsatellite analysis for the detection of allelic imbalance: application in lung tumors.

We have used two hexaplex fluorescent microsatellite assays and analysis on an automatic sequencer to determine allelic imbalance in lung tumors. The markers used are located close to tumor-suppressor genes, DNA repair genes and regions frequently lost in lung cancer. We present a reference interval and quantify the reproducibility of the assays as assessed by multiple repeat reactions for normal DNAs. The cut-off value was calculated to 0.77 (23% reduction of one allele intensity) which, to the best of our knowledge, is currently the lowest reported cut-off. Using these parameters we analysed 85 lung carcinomas. Eighty-three samples (97.6%) showed allelic imbalance in at least one locus. It is of note that by using a selection of only 6 markers, imbalance was detected in 81 (95.2%) of the samples. Loci 9p21 and 9p23 exhibited the greatest imbalance (77% and 75% respectively). The fractional allele loss (FAL) for the 3p markers examined was greater in squamous cell carcinomas than adenocarcinomas (t-test, p=0.0001) while no such difference was observed for 9p. The degree of imbalance of different markers within the same sample was divergent, indicating heterogeneity of genomic status (losses, amplifications, aneuploidy) in these tumors. In conclusion, we have established a robust experimental platform with high throughput, sensitivity and specificity for the detection of allelic imbalance in lung tumors. Such assays may be useful for the detection of allelic imbalance in clinical samples to trace genetically abnormal cells and thus assist in the identification of individuals at a high risk for developing lung cancer.

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.

Genetic alterations in bronchial lavage as a potential marker for individuals with a high risk of developing lung cancer.

Using 12 microsatellite markers, we have studied DNAs from the bronchial lavage of 90 individuals who were referred to an early-lung-cancer clinic in the Northwest of England with suspected lung cancer. Genetic alterations were detected in 15 (35%) of 43 patients with lung cancer but also in 11 (23%) of 47 patients with no cytological or radiological evidence of bronchial neoplasia. No significant differences were found between the referring symptoms in any of the second group of individuals with and without genetic alterations. No correlation was found between smoking exposure and loss of heterozygosity (LOH)/microsatellite alterations (MAs) in the microsatellite markers. On comparing LOH with MAs based on cytology review, we found that the prevalent type of alteration in specimens with cytological evidence of malignancy was LOH; in contrast, the individuals with no cytological evidence of malignancy showed a preponderance of MAs (P = 0.01). Our results indicate that a substantial proportion of cells in the bronchial lavage from suspected lung cancer patients carry identifiable genetic alterations. However, the presence of genetic alterations in the bronchial lavage of individuals with no clinical evidence of lung cancer raises the question whether instability is a phenomenon solely associated with cancer or represents a feature of nonneoplastic diseases. Our results suggest that microsatellite PCR-based assays can be developed as tools for the earlier identification of genetic changes in cells exfoliating in the bronchus.

The presence of herpesviruses in pterygium.

Pterygium is a chronic disease of unknown origin and pathogenesis. It is a vision threatening disease where surgical excision is effective. We examined surgically excised symptomatic pterygia for the presence of herpesviruses such as cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) DNA using the polymerase chain reaction (PCR) technique. Samples of normal conjuctival tissue from limpus at 12 or 6 hours were excised in some of the eyes treated; they were used as controls. HSV DNA was detected in 9 and CMV DNA in 8 out of the 20 examined samples. In 3 out of the 20 examined samples both HSV and CMV DNA were detected whereas EBV DNA was not found in any of the examined samples. These results suggest that HSV and CMV may contribute to the pathogenesis of pterygium.

Detection of human cytomegalovirus by the polymerase chain-reaction in immunosuppressed and immunocompromised patients.

Infections caused by Human Cytomegalovirus (HCMV) are very common in patients who undergo immunosuppression or immunocompromisation. The techniques used for routine HCMV detection are time-consuming and lack specificity and sensitivity. The ability of the Polymerase Chain Reaction (PCR) to amplify HCMV DNA from clinical samples of the patients is a valuable diagnostic tool for the detection of HCMV in the early stages of the infection. We used a pair of primers to amplify a 435 bp region of the immediate early-1 gene, to detect HCMV DNA in clinical samples from patients at high risk for HCMV infection. We found HCMV in the following type of patients: 6 out of 20 in immunosuppressed, 11 out of 31 in immunocompromised, 5 out of 8 in pregnant women, 4 out of 25 in patients with high anti-CMV IgM and IgG titres, 1 out of 2 in patients with kidney failure, and 6 out of 14 in patients with opthalmic disorders. Sixty-seven specimens, which were found to be negative for CMV by the PCR technique, were used to inoculate human fibroblast monolayer cultures and PCR was performed to the DNA extracted from the cultured cells. Only in 1 out of the 67 cases HCMV DNA was detected.

Detection of human cytomegalovirus and epstein-barr-virus by the polymerase chain-reaction in patients with Beta-thalassemia.

Infections caused by Human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) are common in multiple transfused patients, such as patients with beta-thalassaemia. The ability of the Polymerase Chain Reaction (PCR) to amplify HCMV and EBV DNA from blood and other samples makes this technique a valuable diagnostic tool for the detection of both viruses in the early stages of the infection. PCR was used for the amplification of a 435 bp region of the immediate early-1 (IE-1) gene of HCMV and a 375 bp sequence from the EcoRI B fragment of EBV genome. Blood samples from 80 patients with beta-thalassaemia were examined. HCMV was found in 14 and EBV in 12 patients. The results obtained confirm the implications of HCMV and EBV in the diagnosis of viral infections in multiple transfused patients as well as the importance of PCR technique as a valuable diagnostic tool.

Detection of hsv, CMV and ebv by the polymerase chain-reaction technique in patients with inflammatory eye-diseases.

Herpes simplex virus (HSV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) have been recognized as pathogenic agents of intraocular inflammatory conditions. The ability of the Polymerase Chain Reaction (PCR) technique to amplify HSV, CMV and EBV DNA from aqueous specimens makes this technique a valuable diagnostic tool for the detection of these viral pathogens in patients with ophthalmic lesions. We used PCR for the amplification of a 476 bp long sequence from the pol I gene of HSV genome, a 435 bp region of the immediate early-1 (IE-1) gene of CMV and a 375 bp sequence from the EcoRI B fragment of EBV genome. We examined 22 aqueous humour specimens from patients with uveitis and retinitis, inflammatory eye diseases, diagnosed clinically. We found HSV in 4 (18.2%), CMV in 6 (27.3%) and EBV in 1 (4.5%) out of the 22 examined patients. None of the 22 examined samples was found to be infected with more than one of the examined viral pathogens. These data confirm the implications of the members of Herpesvirus family in inflammatory inner eye diseases and the importance of PCR technique as a diagnostic tool in clinical virology.