Cases Of Non-small Cell Lung Carcinoma Research Articles

Expression of Notch 1 and 3 is related to inhibition of lymph node metastasis and progression in non‐small cell lung carcinomas

ABSTRACT Background and aims: Notch signaling pathways are well known for their major role in stem cell differentiation. However, varied results have been reported in relation to their oncogenic role, depending on tumor type. We studied the expression of Notch receptors and other cell cycle proteins in human non‐small cell lung carcinomas (NSCLC) and analyzed their relationship to tumor progression. Methods: We examined the tissue microarrays of 158 cases of NSCLC for Notch1 and 3, and cell cycle regulatory proteins p53, p16INK4a, pRb and CDK2 by immunohistochemistry. Results: Notch1 and 3 receptors were noted in 81.6% and 79.7% of cases, respectively. Notch1 expression was negatively correlated with lymph node metastasis (P = 0.043) and Notch3 was negatively correlated with more advanced pathologic T stage (P = 0.024). However, expressions of Notch receptors didn't show any significant prognostic value for NSCLC. Of the cell cycle proteins, CDK2 and pRb were negatively correlated with Notch1 (P = 0.013) and Notch3 (P = 0.018). Conclusions: Notch receptors play an inhibitory role in metastasis to lymph node and in the activity of cell cycle proteins, which may lead to suppression of NSCLC.

Expression of glucose transporter in non-small cell lung carcinoma and its clinical significance

To investigate the expression of glucose transporter (Glut)1, Glut3, and hypoxia inducible factor (HIF)-1 alpha in human non-small cell lung carcinoma (NSCLC), and its clinical significance. Specimens of cancer tissues and paracancerous lung tissues from 34 cases of NSCLC and 17 specimens of benign lung lesions were collected. The expressions of Glut1, Glut3, and HIF-1 alpha were detected with immunohistochemical staining, RT-PCR, and Western blot. The relative mRNA expressions of Glut1 and HIF-1 alpha were 0.689 +/-0.245, 0.693 +/-0.248 in cancer tissues; and 0.338 +/-0.157, 0.351 +/-0.184 in paracancerous lung tissues (P <0.001); while those of Glut3 were 0.506 +/-0.246 in cancer tissues and 0.482 +/-0.238 in paracancerous tissues (P >0.05). The relative protein expressions of Glut1 and HIF-1 alpha were 0.582 +/-0.247, 0.525 +/-0.246 in cancer tissues and 0.288 +/-0.151, 0.261 +/-0.135 in paracancerous lung tissues (P<0.001), but the protein expressions of Glut3 were 0.551 +/-0.251 and 0.436 +/-0.224 respectively (P>0.05). Glut1 and HIF-1 alpha expressions were higher in poor differentiation group and in stage III group, than those in medium and well differentiation group and stage I and II group. Moreover, there was a significant correlation between the expression of Glut1 and HIF-1 alpha (r=0.854, P<0.01). Glut1 and HIF-1 alpha are highly expressed in NSCLC, and their expressions are associated with tumor differentiation and clinical stage.

Expression of Activated and Latent Signal Transducer and Activator of Transcription 3 in 303 Non–Small Cell Lung Carcinomas and 44 Malignant Mesotheliomas: Possible Role for Chemotherapeutic Intervention

Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in diverse human cancers and plays a critical role in tumor cell survival, proliferation, migration, invasion, angiogenesis, and inhibition of apoptosis. The phosphorylated active form of STAT3 (pSTAT3) mediates its effects via nuclear transcriptional activity. However, it was recently observed that the nonphosphorylated, cytoplasmic, inactive form of STAT3 is involved in cell motility and consequently tumor invasion. It appears that, although STAT3 is not absolutely required for tumor formation, tumors that develop in the presence of STAT3 become dependent on its expression for their survival, making it a potential therapeutic target. To investigate the possible utility of STAT3 as a future therapeutic target in non-small cell lung carcinoma (NSCLC) and malignant mesothelioma (MM). Immunohistochemical expression of MIB-1, STAT3, and pSTAT3 was assessed in 303 NSCLC and 44 MM archival cases. A more conspicuous expression of inactive STAT3 (91.44% in NSCLC and 79.5% in MM cases) was present compared with the nuclear activated form pSTAT3 (60.53% in NSCLC and 61.4% in MM cases). MIB-1 did not correlate with the expression of STAT3 or pSTAT3. The strong expression of cytoplasmic inactive STAT3 in NSCLC and MM cases implies a major role for STAT3 in tumor motility, invasion, and metastasis via a nontranscriptional pathway. We conclude that STAT3 and pSTAT3 are up-regulated in a high percentage of NSCLCs and MMs, regardless of tumor type, age, sex, smoking status, stage and grade of tumor, or survival, providing a basis for therapeutic intervention.

ErbB3 mRNA Expression Correlated with Specific Clinicopathologic Features of Japanese Lung Cancers

ErbB3 is a member of the epidermal growth factor receptor (EGFR) family of tyrosine kinase. A previous report has suggested that overexpression of ErbB3 protein was associated with lung cancer recurrence. Other reports suggested that high ErbB3 protein expression was associated with shorter survival in advanced non-small-cell lung carcinomas (NSCLC). The relationship between the erbB3 mRNA expression and EGFR mutation has not been reported. In this study, we have investigated the erbB3 mRNA levels in reference to clinicopathologic data and EGFR mutation status in Japanese lung cancer specimens. This study included 192 surgically removed NSCLC cases operated at Nagoya City University Hospital. The erbB3 mRNA levels were quantified by real-time polymerase chain reaction using LightCycler (Roche Molecular Biochemicals, Mannheim, Germany). The erbB3/glyceraldehydes-3-phosphatate dehydrogenase levels were significantly higher in female than in male (P < 0.0001), higher in nonsmoker than in smoker (P = 0.0007), higher in adenocarcinoma than in other types of lung cancers (P < 0.0001), and higher in well-differentiated than in less-differentiated subtypes (P = 0.0304). The erbB3/glyceraldehydes-3-phosphatate dehydrogenase levels were significantly higher in patients with EGFR gene mutation than in patients without mutation (P = 0.0124). The present study suggests that increased expression of erbB3 may play an additional role in NSCLC especially in female, nonsmoker, adenocarcinoma, and with EGFR gene mutation. Further studies on large sample sizes would help to determine if erbB3 expression is a clinically useful tool to further define patients who will benefit from EGFR TKIs.

An Alternative Approach to Determining Therapeutic Choices in Advanced Non-small Cell Lung Carcinoma (NSCLC): Maximizing the Diagnostic Procedure and the Use of Low-Volume Lung Biopsies

Accurate mutational analysis, especially epidermal growth factor receptor (EGFR) mutations, of diagnostic biopsies from all Asian NSCLC patients is crucial to their clinical management, but faces problems. Here, we explore, within usual hospital constraints, the practicalities of incorporating mutational analysis in every newly diagnosed case of NSCLC, namely, maximizing tissue acquisition during the diagnostic procedure and determining the maximum quantity and quality of DNA sequence data available from these biopsies. Sixty-eight Chinese patients were enrolled. Thirty-five underwent surgical resections for early-stage tumors. Thirty-three underwent diagnostic procedures, i.e., needle aspirates under bronchoscopic or computed tomographic/fluoroscopic guidance, or forceps biopsies via bronchoscopy. Separate samples for research purposes were obtained from these 33 patients during the diagnostic procedure. All samples were analyzed for mutations in EGFR exons 18 to 21, p53 exons 4 to 9, and Kras exon 2. No deaths occurred in this study. Success rates in obtaining sequence data from surgical samples versus low-volume samples for EGFR, p53, and Kras were 100% versus 85%, 100% versus 82%, and 100% versus 85%, respectively. Sequencing nine polymerase chain reaction products from each low-volume sample resulted in the exhaustion of all extracted DNA from three samples. Acquiring a separate low-volume lung biopsy sample for mutational analysis in lung cancer patients during the diagnostic procedure is feasible and may be a valuable complement to the usual diagnostic workflow in future.

MUC4 Expression in Non–Small Cell Lung Carcinomas: Relationship to Tumor Histology and Patient Survival

Abstract Context.—Mucin 4 (MUC4) is a high-molecular-weight membrane–bound glycoprotein that is expressed in the foregut before epithelial differentiation. It is found in normal adult airway epithelium, non–small cell lung carcinoma (NSCLC) and in other human malignancies independent of mucus secretion. Although its tissue distribution has been studied, its utility in predicting prognosis in NSCLC is unknown. Objective.—To evaluate the relationship between MUC4 overexpression and long-term survival in patients with NSCLC. Design.—Immunohistochemical staining for MUC4 was performed on formalin-fixed, paraffin-embedded tissue samples from 343 cases of NSCLC arranged in a high-density tissue microarray. Information about long-term survival and tumor stage was collected for all patients. Semiquantitative assessment of MUC4 staining was correlated with survival (Kaplan-Meier analysis). Results.—MUC4 was frequently expressed in adenocarcinomas (151/187 [81%]), squamous cell carcinomas (69/ 88 [78%]), adenosquam...

MUC4 Expression in Non–Small Cell Lung Carcinomas: Relationship to Tumor Histology and Patient Survival

Mucin 4 (MUC4) is a high-molecular-weight membrane-bound glycoprotein that is expressed in the foregut before epithelial differentiation. It is found in normal adult airway epithelium, non-small cell lung carcinoma (NSCLC) and in other human malignancies independent of mucus secretion. Although its tissue distribution has been studied, its utility in predicting prognosis in NSCLC is unknown. To evaluate the relationship between MUC4 overexpression and long-term survival in patients with NSCLC. Immunohistochemical staining for MUC4 was performed on formalin-fixed, paraffin-embedded tissue samples from 343 cases of NSCLC arranged in a high-density tissue microarray. Information about long-term survival and tumor stage was collected for all patients. Semiquantitative assessment of MUC4 staining was correlated with survival (Kaplan-Meier analysis). MUC4 was frequently expressed in adenocarcinomas (151/187 [81%]), squamous cell carcinomas (69/ 88 [78%]), adenosquamous carcinomas (6/8 [75%]), and large cell carcinomas (33/60 [55%]). High levels of expression (combined score, 2+/3+) for MUC4 were more characteristic of adenocarcinomas (126/187 [68%]) and adenosquamous carcinomas (6/8 [75%]) than of squamous cell carcinomas (46/88 [52%]) and large cell carcinomas (17/60 [28%]) (P < .001). In patients with stage I and II adenocarcinoma, there was a trend toward longer patient survival with higher levels of MUC4 immunoreactivity compared with lower levels (P = .11). MUC4 expression is common in pulmonary adenocarcinomas and may indicate a more favorable prognosis in early-stage adenocarcinomas.

Microsatellite Instability Is Uncommon in Lymphoepithelioma-like Carcinoma of the Lung

Primary lymphoepithelioma-like carcinoma of the lung (LELC) shares some morphologic and clinical characteristics with malignancies associated with microsatellite instability (MSI). The aims of our study were to determine the MSI status in LELC and compare these findings with stage I non-small cell lung carcinoma (NSCLC) with marked lymphocytic host response (MLHR). We assessed MSI by a DNA-based polymerase chain reaction assay using mononucleotide (BAT25 and BAT26) and dinucleotide (D2S123, D5S346, and D17S250) repeats. MSI was detected in 2 (29%) of 7 LELC cases with only 1 marker (D17S250), and in 3 (19%) of 16 NSCLC cases with MLHR with only 2 markers (1D2S123 and 2 D17S250). Loss of heterozygosity (LOH) was detected at 1 or 2 of 3 dinucleotide repeats in 11 NSCLC cases (69%) with MLHR and 3 LELC cases (43%) (P = .36). The overall frequencies of LOH in NSCLC with MLHR were 29% and 19% in LELC (P = .55). MSI is very uncommon in LELC, indicating that MSI is not an important event in carcinogenesis for this tumor subtype. The presence of LOH suggests a probable role of tumor suppressor genes in LELC carcinogenesis.

Expression of promyelocytic leukaemia protein in lung carcinomas and clinical significance thereof

To investigate the expression of promyelocytic leukaemia (PML) protein in lung carcinomas and the clinical significance thereof. A tumor tissue microarray with lung samples from 148 patients with lung carcinoma and 5 patients with pulmonary benign tumor, and 7 patients with other benign diseases resected during operation. The cases with at least triplicate 0.6-mm cores per tumor or tissue were analyzed. Immunohistochemistry (SP method) was used to detect the expression of PML protein. The association between PML expression and post-operational survival was evaluated. Four cases of lung carcinoma were excluded because their available cores were less than 3. The remaining 144 lung carcinoma cases included 45 cases with squamous cell carcinoma, 62 with adenocarcinoma, 23 with small cell lung carcinoma (SCLC), 7 with large cell carcinoma, 5 with pleomorphic carcinoma, 1 with carcinoid, and 1 with adenosquamous carcinoma. Forty of the 144 cases were PML protein positive in the nucleus, 26 were PML protein positive in the cytoplasm, and 12 were PML protein positive in both the nucleus and cytoplasm. Among the 5 cases with benign lung tumors, two cases with pulmonary leiomyomas had strong expression of PML. Among the 7 normal pulmonary tissues, one case with bronchial mucosa had a moderate expression of PML on the nuclei in the basilar part, and the 6 cases with normal alveolar epithelium did not express PML. The rates of PML expression on cell nuclei were 31.4% and 8.7% respectively in the non-small cell lung carcinoma (NSCLC) and SCLC samples (chi(2) = 4.968, P = 0.026). Farish, but not strong, expression on cytoplasm was found in 39.1% cases of SCLC and in 14% cases of NSCLC (chi(2) = 6.609, P = 0.010). PML protein was found in 9 patients with SCLC, but absent in the other 14 cases. The 5-year cumulative survival rate of the PML positive SCLC patients was 50%, significantly higher than that of the PML-negative SCLC patients (23%, chi(2) = 3.931, P = 0.047). Lack of PML protein (RR = 0.127, 95% CI = [0.027, 0.592]) and senior pathologic TNM stage (RR = 3.148, 95% CI = [1.029, 9.631]) were two hazardous factors that influenced long-term survival of postoperative SCLC patients. As an important suppressor of tumor, PML may be a useful indicator to predict postoperative prognosis in patients with SCLC.

Clinicopathological significance of BGP expression in non-small-cell lung carcinoma: relationship with histological type, microvessel density and patients’ survival

Brain-type glycogen phosphorylase (BGP) is the major isoform of glycogen phosphorylase found in fetal and neoplastic tissues, and is generally thought to induce glucose supply during an ischaemic period. This study was performed to investigate BGP expression in non-small-cell lung carcinoma (NSCLC). A total of 119 cases of NSCLC, including 63 squamous cell carcinomas (SqCCs) and 56 adenocarcinomas (ACs), were imunohistochemically evaluated for BGP expression, and its expression was correlated with clinicopathological parameters. In total, 76.5% were positive, while non-neoplastic bronchial epithelial cells were weakly positive and pneumocytes were negative. High BGP expression was noted in 78.6% of ACs and 36.5% of SqCCs (p=0.001). Microvessel density was higher in the low BGP expression tumours (29.6 +/- 16.9/mm(2)) than in the high expression tumours (22.8+/-13.8/mm(2)) (p=0.017). BGP expression did not correlate with patient age or tumour stage, but was more frequent in females than males. Kaplan-Meier analysis showed that high BGP expression was associated with poorer survival (p=0.032). BGP is expressed in NSCLC, particularly AC, and is an independent poor prognostic factor.

P21 as a prognostic factor in non-small cell lung carcinomas

Few studies have focused on the correlation between p21 expression and survival for patients with non-small cell lung carcinoma (NSCLC), and the results are not consistent. We investigated the expression of p21 in 90 cases of NSCLC to evaluate the correlation between the p21 expression level and the clinicopathologic characteristics with patient survival. p21 was expressed in the nuclei of all the NSCLCs. The percentage of immunoreactive cells varied from 1% to 70%. All the patients were subdivided into a high and a low p21 expression group on the basis of the median percentage (17.05). There was no significant correlation between the p21 expression level and age, gender, histologic type, histologic grade, or stage. Using uni- and multivariate analyses, survival was significantly associated with gender, stage, and the p21 expression level. The survival rate for the high p21 expression group was higher than that for the low p21 expression group in the entire patient group, and especially for stage II and III patients, males, adenocarcinomas, or p53-positive tumors. Our findings showed that high p21 expression was an independent prognostic factor for NSCLC. p21 may be useful for determining the candidates for adjuvant therapies and also for selecting the appropriate chemotherapeutic agents.

Co‐overexpression of fibroblast growth factor 3 and epidermal growth factor receptor is correlated with the development of nonsmall cell lung carcinoma

Lung cancer is a prevalent cancer with a poor prognosis. To develop a useful in vitro cell model, a cell line of lung squamous cell carcinoma (SCC-35) was established. The SCC-35 cell was characterized by comparative genomic hybridization (CGH) and spectral karyotyping (SKY). Chromosome microdissection, fluorescence in situ hybridization (FISH), and Southern and Northern blots analyses were used to study target genes. Two amplicons were found at chromosomes 7p12 and 11q13. Amplification and overexpression of epidermal growth factor receptor (EGFR) at 7p12 and fibroblast growth factor 3 (FGF3) at 11q13 were found. To understand the correlation between these two genes in nonsmall cell lung carcinoma (NSCLC) more comprehensively, overexpression of FGF3 and EGFR was investigated by immunohistochemistry with a tissue microarray containing 406 NSCLC samples. Cytoplasmic overexpression of FGF3 and EGFR was detected in 61% and 69% NSCLC cases, respectively. More interestingly, a significant correlation between overexpression of FGF3 and EGFR was found in NSCLC. These results suggest that co-overexpression of FGF3 and EGFR may play an important role in the pathogenesis of lung carcinoma.

Expression of Seminal Vesicle-Specific Antigen in Serum of Lung Tumor Patients

Protein markers are commonly used in forensic medicine to establish the origin of human fluids detected in crime scenes. Semenogelins, the major protein constituents of semen coagulum, are the most effective markers for semen detection. Recently, it has been demonstrated that semenogelins are also ectopically expressed in small cell lung carcinomas (SCLC) and in a minority of non-small cell lung carcinomas (NSCLC). This finding prompted us to look for semenogelin expression in the serum of lung cancer patients. A set of 13 serum samples (3 from SCLC and 10 from NSCLC patients) was screened by enzyme-linked immunosorbent assay (ELISA), using a commercially available kit. Four of the NSCLC cases showed positive results. Ectopic expression of marker proteins in individuals affected by cancer could represent a potential source of forensic pitfalls.

Prognostic Significance of Cyclooxygenase 2 Expression in 259 Cases of Non-Small Cell Lung Cancer

Abstract Context.—Previous studies report that increased expression of cyclooxygenase 2 (COX-2) correlates with poor clinical outcome in several malignancies, including non– small cell lung carcinoma (NSCLC). Cyclooxygenase 2 inhibitors have been reported to effectively inhibit carcinogenesis in colon cancer experimental models. Objective.—We examined COX-2 expression in 259 cases of NSCLC to evaluate its prognostic significance. Design.—Sections of NSCLC from patients with a median 5-year follow-up were immunostained with COX-2 monoclonal antibody (1:150) using the Dako mouse EnVision;pl system. Extent of COX-2 expression in neoplastic cells was recorded as follows: 0, 0% to 10% of cells positive; 1, 11% to 33% positive; 2, 34% to 66% positive; and 3, more than 66% positive. Intensity was scored as either increased (+) or not increased (−), compared to internal control smooth muscle and endothelial cells. Kaplan-Meier analysis was used to assess the relationship between survival and COX-2 expression, usi...

Prognostic Significance of Cyclooxygenase 2 Expression in 259 Cases of Non–Small Cell Lung Cancer

Previous studies report that increased expression of cyclooxygenase 2 (COX-2) correlates with poor clinical outcome in several malignancies, including non- small cell lung carcinoma (NSCLC). Cyclooxygenase 2 inhibitors have been reported to effectively inhibit carcinogenesis in colon cancer experimental models. We examined COX-2 expression in 259 cases of NSCLC to evaluate its prognostic significance. Sections of NSCLC from patients with a median 5-year follow-up were immunostained with COX-2 monoclonal antibody (1:150) using the Dako mouse EnVision;pl system. Extent of COX-2 expression in neoplastic cells was recorded as follows: 0, 0% to 10% of cells positive; 1, 11% to 33% positive; 2, 34% to 66% positive; and 3, more than 66% positive. Intensity was scored as either increased (+) or not increased (-), compared to internal control smooth muscle and endothelial cells. Kaplan-Meier analysis was used to assess the relationship between survival and COX-2 expression, using the log-rank test for statistical significance. No relationship was found between the extent and/or the intensity of COX-2 expression and patient survival when the entire cohort was considered. However, when separately analyzed according to disease stage and intensity of COX-2 expression, a significant relationship (P = .03) between increased COX-2 expression and shortened patient survival was found only in patients with stage I and II NSCLC. To our knowledge, this is the largest series of NSCLCs in which COX-2 has been investigated as a prognostic marker. The findings in this large series support previous studies of smaller cohorts that reported that increased COX-2 expression predicts poor outcome in patients with early-stage NSCLC.

Neuroendocrine differentiation in non‐small cell lung cancer and its relation to prognosis and therapy

Histopathologists report the presence of neuroendocrine (NE) differentiation in non-small cell lung carcinoma (NSCLC) in up to a third of cases and are often questioned about its clinical relevance. The conclusions of previous studies have been inconsistent. This paper aims to provide an answer by examining a large series together with a comprehensive critique of the literature. Four hundred and thirty-nine cases of NSCLC were examined, immunohistochemically, using antibodies to chromogranin A (CGA), synaptophysin (SYN) and CD56/neural cell adhesion molecule (NCAM). Three hundred and forty-one cases had been treated with surgical resection and the remainder with chemotherapy. The results were compared with clinical outcome. Thity-six percent of cases had positive staining for at least one NE marker. CGA was positive in 5.5% of cases, SYN in 16.5% and NCAM in 28%. There was no association between the presence of NE markers and survival in either the surgically treated group or the chemotherapy-treated group. There was also no association between NE markers and response to chemotherapy in the latter group. The presence of immunohistochemically detected NE differentiation in NSCLC is not of prognostic significance.

Relationship between Expression of XIAP Protein in Operable Non-small Cell Lung Carcinomas and Apoptosis Index and Postoperative Prognosis

Background : Dysregulation of apoptosis plays an important role in carcinogenesis, tumor progression, and resistance to chemotherapy. X-linked inhibitor of apoptosis (XIAP) is considered to be the most potent caspase inhibitor of all known IAP (inhibitor of apoptosis) family members. This study was designed to assess the pattern of expression and the prognostic value of XIAP in radically resected non-small cell lung carcinoma (NSCLC) patients. Method : The expression of XIAP and its relationship with clinicopathologic parameters (patient age, TNM stage, TNM-pT, TNM-pN, histologic type, VEGF expression, microvessel density, PCNA index) and overall survival were analysed with formalin-fixed, paraffin-embedded blocks from eighty cases of NSCLC. In addition, the apoptotic index (AI) was also assessed. Results : In a regard to histologic type, squamous cell carcinoma (SCC) showed XIAP expression in 91.3%(42/46) and adenocarcinoma (AC) in 61.8%(21/34). The difference was significant(p=0.001). There was no correlation between XIAP expression and other parameters. In the group of AC, XIAP expression showed the signifcant correlation with older age group and VEGF expression(p=0.028, p=0.014, respectively). The AI in the group with or without XIAP expression were and , respectively(p=0.001). Both groups just aforementioned showed no significant difference in median survival time (42.5 months, 29.8 months, respectively). Conclusion : This study suggests that the XIAP expression in NSCLCs could have relation to inhibition of apoptosis, and show differential expression according to histologic type. However, its prognostic role during the progression of NSCLC needs to be further defined.

Evaluation of immunohistochemical markers in non-small cell lung cancer by unsupervised hierarchical clustering analysis: a tissue microarray study of 284 cases and 18 markers.

This study has investigated a panel of immunomarkers in non-small cell lung carcinoma (NSCLC). Unsupervised hierarchical clustering analysis was used to investigate the possibility of identifying different subgroups in NSCLC based on their molecular expression profile rather than morphological features. A tissue microarray consisting of 284 cases of NSCLC was constructed. Immunohistochemistry was used to detect the presence of 18 biomarkers including synaptophysin, chromogranin, bombesin, NSE, GFI1, ASH-1, p53, p63, p21, p27, E2F-1, cyclin D1, Bcl-2, TTF-1, CEA, HER2/neu, cytokeratin 5/6, and pancytokeratin. Univariate analysis of all 18 markers for prognostic significance was performed. Immunohistochemical scoring data for NSCLC were analysed by unsupervised hierarchical clustering analysis. Kaplan-Meier survival curves were plotted for the different cluster groups of lung tumours identified by this method. Analysis of the three different World Health Organization (WHO) subtypes (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) of NSCLC individually showed that different markers were significant in different subtypes. For example, p53 and p63 were significant for squamous cell carcinoma (p = 0.007 and p = 0.03, respectively), whereas cyclin D1 and HER2/neu were significant prognostic markers for adenocarcinoma (p = 0.025 and p = 0.015, respectively). These markers were not significant prognostic predictors for NSCLC as a group. Hierarchical clustering analysis of NSCLC produced four separate cluster groups, although the vast majority of cases were found in two cluster groups, one dominated by squamous cell carcinoma and the other by adenocarcinoma. The clinical outcomes of cases from the four cluster groups were not significantly different. Prognostic indicators vary between different morphological subtypes of NSCLC. Unsupervised hierarchical clustering analysis, based on an extended immunoprofile, identifies two main cluster groups corresponding to adenocarcinoma and squamous cell carcinoma; cases of large cell carcinomas are assigned to one of these two groups based on their molecular phenotype.

Validating the prognostic value of marker genes derived from a non-small cell lung cancer microarray study

We previously reported that our cDNA microarray analysis of primary non-small cell lung carcinoma (NSCLC) could predict for patients at increased risk of cancer recurrence. From the result of this analysis, we selected 11 genes that were considered candidate prognostic marker genes and used the realtime reverse transcription polymerase chain reaction (RT-PCR) to investigate their expression in the same set of NSCLC cases used in the microarray study. Cluster analysis of the realtime RT-PCR data separated these patients into two groups with significantly different disease-free survivals (log-rank test, P<0.017). In contrast, cluster analysis failed to confirm the prognostic significance of the realtime RT-PCR results for these 11 genes in a validation series of 92 NSCLC cases. In univariate analysis, hypoxia inducible factor 1α, Rho-GDP dissociation inhibitor (GDI) α (RhoGDI) and Citron/rho-interacting serine–threonine kinase 21 (Citron K21) were significant prognostic factors for disease-free survival in the entire cohort of 130 NSCLC patients, but none were significant in multivariate analysis. The results demonstrate that the prognostic significance of microarray (SAM) results can be partially validated using realtime RT-PCR, but secondary validation using larger and independent series of tumors is necessary to identify true prognostic marker genes.

Hypermethylation of FHIT as a prognostic marker in nonsmall cell lung carcinoma.

Methylation of CpG islands in the promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of the current study was to determine the correlation between the aberrant promoter methylation of multiple genes and survival in patients with nonsmall cell lung carcinoma (NSCLC). The methylation status of nine genes was determined in 124 surgically resected NSCLC cases using methylation-specific polymerase chain reaction. The methylation frequencies of the genes tested in NSCLC specimens were 52% for E-cadherin (CDH1), 41% for RAS association domain family protein (RASSF1A), 38% for fragile histidine triad (FHIT) and adenomatous polyposis coli (APC), 27% for retinoic acid receptor beta (RARbeta) and H-cadherin (CDH13), 20% for p16INK4A, 0.8% for O6-methylguanine-DNA-methyltransferase (MGMT), and 0% for glutathione S-transferase P1 (GSTP1). The survival of the patients with FHIT methylation-positive tumors was found to be significantly shorter than that for those patients with methylation-negative tumors (P=0.03), even in those patients with International Union Against Cancer TNM Stage I or Stage II disease (P=0.007). In contrast, there were no significant survival differences noted between the methylation-positive and methylation-negative tumors for the other genes tested. In addition, based on multivariate analyses, FHIT methylation-positive status was found to be independently associated with poor survival (P=0.046) and disease stage (P<0.0001). The results of the current study suggest that methylation of FHIT is a useful biomarker of biologically aggressive disease in patients with NSCLC.