Protein expression profiles indicative for drug resistance of non-small cell lung cancer.

Data obtained from multiple sources indicate that no single mechanism can explain the resistance to chemotherapy exhibited by non-small cell lung carcinomas. The multi-factorial nature of drug resistance implies that the analysis of comprising expression profiles may predict drug resistance with higher accuracy than single gene or protein expression studies. Forty cellular parameters (drug resistance proteins, proliferative, apoptotic, and angiogenic factors, products of proto-oncogenes, and suppressor genes) were evaluated mainly by immunohistochemistry in specimens of primary non-small cell lung carcinoma of 94 patients and compared with the response of the tumours to doxorubicin in vitro. The protein expression profile of non-small cell lung carcinoma was determined by hierarchical cluster analysis and clustered image mapping. The cluster analysis revealed three different resistance profiles. The frequency of each profile was different (77, 14 and 9%, respectively). In the most frequent drug resistance profile, the resistance proteins P-glycoprotein/MDR1 (MDR1, ABCB1), thymidylate-synthetase, glutathione-S-transferase-pi, metallothionein, O6-methylguanine-DNA-methyltransferase and major vault protein/lung resistance-related protein were up-regulated. Microvessel density, the angiogenic factor vascular endothelial growth factor and its receptor FLT1, and ECGF1 as well were down-regulated. In addition, the proliferative factors proliferating cell nuclear antigen and cyclin A were reduced compared to the sensitive non-small cell lung carcinoma. In this resistance profile, FOS was up-regulated and NM23 down-regulated. In the second profile, only three resistance proteins were increased (glutathione-S-transferase-pi, O6-methylguanine-DNA-methyltransferase, major vault protein/lung resistance-related protein). The angiogenic factors were reduced. In the third profile, only five of the resistance factors were increased (MDR1, thymidylate-synthetase, glutathione-S-transferase-pi, O6-methylguanine-DNA-methyltransferase, major vault protein/lung resistance-related protein).

Protein expression profile of newly diagnosed acute lymphoblastic leukemia in children developing relapses.

The purpose of this investigation was to evaluate the expression profile of proteins involved in children with newly diagnosed acute lymphoblastic leukemia (ALL) children who are developing relapses. For this reason, the expressions of 10 proteins including proto-oncogene and tumor suppressor gene products, proliferative factors and resistance parameters in 104 initial cases of childhood ALL were analyzed and the proteins correlated with ALL patients who experienced relapses. Applying immunocytochemical assays, we found that 4 out of the 10 parameters revealed a relationship to developing relapses (Fisher's exact tests). These were the oncogene product Fos (p=0.002), the drug resistance proteins glutathione S-transferase (p=0.008) and P-glycoprotein (P-pg/MDR1) (p=0.07) and protein kinase C (p=0.01). By means of hierarchical cluster analysis, we were able to show that the patients could be separated according to their protein expression profile into clusters consisting of patients whose ALL relapsed later and of patients who did not show relapses in the future.

Vascular endothelial growth factor in newly diagnosed and recurrent childhood acute lymphoblastic leukemia as measured by real-time quantitative polymerase chain reaction.

PURPOSE: Overexpression of vascular endothelial growth factor (VEGF) is associated with increased angiogenesis, growth, and metastasis in solid tumors, but to date the significance of VEGF in leukemia has received only limited attention. Therefore, this study examined the cellular VEGF levels in 31 newly diagnosed and 22 recurrent cases of childhood acute lymphoblastic leukemia (ALL). EXPERIMENTAL DESIGN: VEGF was determined with real-time quantitative PCR methods. Kaplan-Meier statistical analyses were conducted for the relapse-free intervals and the overall survival times. The groups were compared by log-rank and rank-sum tests. RESULTS: The VEGF levels were significantly higher in recurrent ALL compared with newly diagnosed ALL (28.0 versus 3.1 units; P = 0.001). Kaplan-Meier estimates were conducted to analyze the prognostic value of VEGF levels in newly diagnosed ALL with regard to the relapse-free intervals and the overall survival times. In this analysis, the median relapse-free interval of patients with low VEGF levels was more than 10 years, whereas the relapse-free interval of patients with high VEGF expression was only 1.2 years. The median overall survival time for the collective with low VEGF levels was >10 years, whereas the survival of the group of patients with high VEGF levels was 3.9 years. This difference was not statistically significant. This may be attributable to the small number of patients involved. CONCLUSION: Our data suggest that VEGF may play an important role in the pathophysiology of ALL. The expression of VEGF raises the possibility of using angiogenesis inhibitors as a novel therapeutic strategy in childhood ALL.

UV-induced DNA damage and mutations in Hupki (human p53 knock-in) mice recapitulate p53 hotspot alterations in sun-exposed human skin.

The major etiological agent contributing to human nonmelanoma skin cancer is sunlight. The p53 tumor suppressor gene is usually mutated in these tumors, and the mutations are "UV signature" single or tandem transitions at dipyrimidine sequences in the DNA-binding domain (DBD). Cells that harbor these characteristic mutations are already present in sun-exposed skin areas of healthy individuals, and small epidermal patches that are immunoreactive to anti-p53 antibody accrue as exposure increases. To explore carcinogen-specific human p53 mutation patterns experimentally, we generated a knock-in (Hupki) mouse in which the murine DBD of the p53 gene has been replaced by the homologous human p53 DBD segment; thus, the precise base sequence context frequently targeted by mutagens or endogenous mutagenic processes in human carcinogenesis is present in this strain (J. L. Luo et al., Oncogene, 20: 320-328, 2001). Here we show that when epidermal cells of Hupki mice (p53(ki/ki)) are irradiated in vivo with a single acute dose of UVB light, they accumulate UV photoproducts at the same locations of the p53 gene as human cells. Chronic exposure of Hupki mice (4.5 kJ/m(2) 5x/week for 4 weeks) results in the appearance of cell patches that stain intensely with the anti-p53 antiserum CM1. DNA preparations from 2 cm(2) sections of chronically irradiated Hupki epidermis harbor C to T and CC to TT mutations at two mutation hotspots identified in human skin cancer, one at codons 278-279, and one at codons 247-248; the latter is the most frequent UVB-associated mutation site in humans but not in p53 wild-type mice. Thus, Hupki keratinocytes with these p53 mutations encode an aberrant DBD identical in amino acid sequence to the mutant p53 molecules in human UV-induced tumors. The Hupki mouse model offers a new experimental tool in molecular epidemiology and biomedical research.

Unusual profile and high prevalence of p53 mutations in esophageal squamous cell carcinomas from northern Iran.

Over 15,000 human tumor p53 mutations have been recorded in the scientific literature, including over 700 mutations in esophageal tumors. There are no data on p53 mutations in esophageal cancer patients from Iran yet; however, this country experiences one of the highest cancer mortality rates in the world for esophageal squamous cell carcinomas (ESCCs). The causes of this high cancer burden in Iran remain obscure and do not appear to be related to tobacco and alcohol consumption, the two major risk factors identified in Europe and North America. Because molecular analysis of tumors can provide clues to endogenous or environmental factors contributing to high cancer risk, we examined 74 Iranian ESCCs for the presence of mutations in exons 5-8 of the p53 gene by PCR and direct sequencing. Forty-eight of the 74 tumors (65%) had one or more p53 gene point mutations, including 5 patients with two or more mutations and one with a tandem mutation in codon 242. Surprisingly, over one-third of the 54 mutations we identified were transitions at CpG sites (20 of a total of 54 mutations, or 37%), a class of mutation that is significantly less common (16% of mutations) in the compilation of ESCC mutations from other countries (chi2 statistic, P < 0.0002), whereas transversions, which the literature shows to be common in ESCCs from non-Iranian patients, were infrequent in the tumors we examined here. Elevated levels of cyclooxygenase-2 and inducible nitric oxide synthase were observed in 74 and 91%, respectively, of tumors from Tehran as determined by immunohistochemistry, and high COX-2 expression correlated significantly with the presence of a p53 mutation in the tumor. Mediators of the inflammatory response in esophageal mucosa, perhaps in conjunction with specific dietary or cultural practices in Iran, may contribute importantly to the p53 mutation load in Iranian ESCC patients.

Angiostatin expression in non-small cell lung cancer.

Angiostatin, a potent inhibitor of angiogenesis, tumor growth, and metastasis, was examined in a panel of human lung cancer cell lines with Western blot analysis and in 143 primary non-small cell lung carcinomas with immunohistochemistry. Thirty-four of 143 cases (24%) stained positively. Patients with angiostatin-positive tumors survived longer (146 weeks) than patients with angiostatin-negative tumors (77 weeks; log-rank test: P = 0.07; rank-sum test: P = 0.02). To determine whether combining stimulating and inhibiting factors might improve the prognostic capability, both angiostatin and vascular endothelial growth factor (VEGF) were analyzed together with respect to patient survival. The median survival time of patients with angiostatin-positive/VEGF-negative carcinomas was 184 weeks, whereas the median survival time of patients with angiostatin-negative/VEGF-positive tumors was only 52 weeks. The angiostatin-positive tumors exhibited an increased incidence of apoptosis and a reduced capability to be transplanted into nude mice, but these differences did not reach or were only of borderline statistical significance.

Hypoxia-inducible factor (HIF-1) and its relationship to apoptosis and proliferation in lung cancer.

Hypoxia-inducible factor 1 (HIF-1) plays an important role in the pleiotropic response observed under hypoxia. In this study we examined whether a relationship exists between HIF-1 proteins and proliferation and apoptosis in lung cancer. To this purpose, we used immunohistochemistry to analyze HIF-1 alpha and HIF-1 beta in formalin-fixed, paraffin-embedded, non-small cell lung carcinomas (n = 96) and compared the HIF expression with cyclin A protein expression, cell cycle phases, the apoptotic index and the expression of caspase 3, Fas and Fas ligand. Additionally, we examined whether HIF-1 determinations can improve the prognostic information concerning a patient's overall survival. A relationship between HIF-1 alpha or HIF-1 beta and proliferation could not be observed. However, a significant correlation between HIF-1 expression, apoptosis and the pro-apoptotic factors caspase-3, Fas, and Fas ligand could be detected. Patients with HIF-positive carcinomas had significantly longer median survival times than patients with HIF-negative carcinomas (HIF-1 alpha: 191 vs. 60 weeks; P = 0.05; HIF-1 beta: 111 vs. 41 weeks; P = 0.003). Multivariate analyses demonstrated that the presence of HIF-1 at a given stage or extent of lymph node involvement is an independent prognostic factor for the survival of patients with non-small cell lung carcinomas.

Relevance of proliferative and pro-apoptotic factors in non-small-cell lung cancer for patient survival.

This investigation first set out to analyse which cellular proliferative and apoptotic factors, in addition to the clinical prognostic factors, are most predictive in patients with non-small-cell lung carcinomas (NSCLC). To this purpose, we related the proliferative factors proliferating cell nuclear antigen (PCNA), cyclin A, cyclin D1, cyclin-dependent kinase 2 (cdk2), cdk4 and the proportion of cell cycle phases in NSCLC to the survival times of 150 patients. Additionally, we associated the expressions of Fas, Fas ligand and caspase-3 in NSCLC to patient survival. Immunohistochemistry was used to determine the proteins and flow cytometry to assess the proportion of cell cycle phases. Patients with PCNA-positive carcinomas had significantly shorter survival times than patients with PCNA-negative carcinomas (median survival times: 51 vs 89 weeks). Corresponding results were obtained with the factor cyclin A (64 vs 92 weeks), with the factor cdk2 (76 vs 89 weeks), with the factor cdk4 (62 vs 102 weeks) and with the proportion of S phases (86 vs 121 weeks). Patients with an expression of the apoptotic factors had a more favourable prognosis than patients with negative carcinomas. The median survival times of cancer patients with Fas expression was 86 weeks and of those without Fas expression only 69 weeks. Corresponding results were obtained with the Fas ligand (87 vs 41 weeks) and caspase 3 (87 vs 34 weeks). In order to determine whether a combination of factors can yield improved prognostic information, we investigated all possible combinations of the proliferative and apoptotic factors. Patients with tumours having a high proliferative activity, but which did not express apoptotic factors had the shortest survival times while patients with a low proliferative activity and a high expression of apoptotic factors had the most favourable outcome. A multivariate analysis (Cox model) of the cellular and clinical prognostic factors indicated that stage, lymph node involvement, Fas, PCNA and cyclin A are the most important prognostic factors for the clinical outcome of patients with non-small-cell lung carcinomas.

Relationship between the expression of caspase-3 and the clinical outcome of patients with non-small cell lung cancer.

This study examined whether a relationship exists between the expression of caspase-3 in 135 non-small cell lung carcinomas and clinical outcome. Immunohistochemistry and Western blot analyses were used to analyze the expression of caspase-3 in solid tumors and cell lines. A significant correlation was observed between the expression of caspase-3, survival and metastasis. Caspase-3 expression correlated with a lower incidence of lymph node involvement (p = 0.0007). The median survival was longer for patients with caspase-3 positive carcinomas than for those with caspase-3-negative tumors (41 vs 87 weeks, p = 0.038).

Association of telomerase expression with successful heterotransplantation of lung cancer.

This study analyzed whether the expression of telomerase may serve as prognostic factor for the aggressiveness of human non-small cell lung carcinomas. To this purpose the expression of telomerase measured by immunohistochemistry was compared with the take rate of the primary tumors that were heterotransplanted into nude mice. Formalin-fixed, paraffin-embedded specimens of 97 non-small cell lung carcinomas from primarily untreated patients were analyzed for the expression of telomerase by a goat polyclonal antibody (clone C-20). Moderate or strong telomerase-staining was found in 78 (80%) cases. Age, gender, stage and histology had no influence on the telomerase expression. It was discovered that of the 19 telomerase-negative carcinomas only five (26%) exhibited growth in nude mice while of the 78 telomerase-positive cases 37 (47%) were successfully transplanted. To confirm these results, alcohol-fixed, paraffin-embedded cancer specimens from another group of patients (n=58) were analyzed for telomerase expression by a rabbit polyclonal antibody (clone H-231). Corresponding results were obtained. The take rate of telomerase-negative carcinomas was only 36%; the take rate of telomerase-positive carcinomas was 59%. These data suggest that high telomerase expression does indeed correlate with the aggressiveness of non-small cell lung carcinomas.

Prognostic relevance of c-Myc and caspase-3 for patients with non-small cell lung cancer.

This analysis attempted to ascertain whether combining the expression of c-Myc and caspase-3 can improve the available prognostic information for patients with non-small cell lung carcinomas. To this purpose, the expression of c-Myc and caspase-3 was determined in 128 cases of non-small cell lung carcinoma. The median survival time for patients with c-Myc-negative carcinomas was 89 weeks; it was only 43 weeks for patients with c-Myc-positive tumors (p=0.03). The estimated increased relative risk for patients with c-Myc-positive tumors was 1.6. The median survival time for patients with caspase-3-negative carcinomas was 41 weeks while patients with caspase-3-positive carcinomas survived for 79 weeks (p=0.06). The relative risk for patients with caspase-3-negative tumors was 1.5. A significant inverse relationship between the expression of c-Myc and caspase-3 was observed (p=0.04). To determine whether the combination of c-Myc and caspase-3 expression has a higher prognostic significance, patients were grouped based on their expressions of both variables. Patients with c-Myc-negative and caspase-3-positive tumors had the most favorable prognosis (102 weeks) while c-Myc-positive and caspase-3-negative carcinomas had the most unfavorable prognosis (22 weeks; p=0.01).

Inverse correlation between apoptotic (Fas ligand, caspase-3) and angiogenic factors (VEGF, microvessel density) in squamous cell lung carcinomas.

In order to explore whether apoptosis is associated with angiogenesis in lung cancer, immunohistochemistry was employed to determine the pro-apoptotic factors Fas ligand (FasL) and caspase-3 (Cas-3) in 70 squamous cell lung carcinomas. Furthermore, the vascular endothelial growth factor (VEGF) and the microvessel density (MVD) were analyzed. The comparison between MVD and the pro-apoptotic factors demonstrated that the apoptotic factors are inversely related to MVD (Cas-3: p = 0.011, FasL: not significant). In order to confirm this result, FasL and Cas-3 were also compared with the expression of VEGF. Again, an inverse correlation between VEGF and the pro-apoptotic factors was found (Cas-3: p = 0.019, FasL: p = 0.008). The inverse correlation between angiogenesis and apoptosis may be explained by the activation of pro-apoptotic and anti-angiogenic factors caused by hypoxia.

Xenotransplantability of human squamous cell lung cancer in nude mice is not affected by angiogenic factors.

Sixty-two human squamous cell lung carcinomas were analyzed for expression of various angiogenic growth factors and their receptors using immunohistochemistry. The data were correlated with xenotransplantability of these tumors in nude mice. None of the factors investigated did show an association with xenotransplantability. However, there was a trend that specimens lacking VEGF165 were established as xenografts at a higher incidence (52%) than those expressing VEGF165 (39%).

Association of cyclin D1 expression in lung cancer and the smoking habits of patients.

This study examined whether or not cyclin D1 expression is associated with the smoking habits of patients with non-small cell lung carcinomas (NSCLC). Immunohistochemistry was used to analyze 181 NSCLC samples for the expression of cyclin D1. Expression of cyclin D1 protein was found in 130 out of 181 cases (72%). A significant relationship between cyclin D1 expression and stage or histological classification was not observed. The carcinomas of smokers expressed cyclin D1 in 77% of the cases while carcinomas of non-smokers expressed this protein only 57% of the time (P < 0.01, Fisher's exact test). The correlation between smoking and cyclin D1 expression was maintained when the analysis was limited to squamous cell lung carcinomas. However, no correlation was found between cyclin D1 expression and the smoking habits of patients with adenocarcinomas. This can be explained by the fact that the development of adenocarcinomas--in contrast to squamous cell lung carcinomas--is not closely related to tobacco smoke.

Biological characterization of subgroups of squamous cell lung carcinomas.

Recently, Pezzella et al. (Am. J. Pathol., 1997, 151: 1417-1423, 1997) reported on a subgroup of non-small cell lung carcinomas that had no morphological evidence of neoangiogenesis but appeared to grow and were highly aggressive. In this investigation, we subdivided 87 squamous cell lung carcinomas into four subgroups according to angiogenesis (low and high vessel density) and tumor growth (low and high tumor cell proliferation). The aim was to find differences, if any, in the angiogenic status and clinical behavior between these subgroups. We identified a group of tumors with low angiogenesis and high tumor cell proliferation that was characterized by high expression of vascular endothelial growth factor, low expression of basic fibroblast growth factor, reduced apoptosis, increased incidence of metastases, and short survival times. These data show that even squamous cell lung carcinomas are a heterogeneous group of tumors that can be subdivided in tumors with different biological properties and different clinical behaviors.

Expression of Fas (CD95/APO-1) and Fas ligand in lung cancer, its prognostic and predictive relevance.

In order to examine whether or not the expression of the apoptosis-related receptor Fas (CD-95/APO-1) and its ligand (FasL) has relevance for patient survival, immunohistochemistry was used to analyze the proteins of both factors in 164 non-small cell lung carcinomas. Patients with Fas-positive tumors exhibited significantly longer survival times than patients with Fas-negative carcinomas. In contrast, FasL did not significantly influence patient survival time. A multivariate analysis of clinical and biological factors indicated that lymph node status and Fas expression were significant prognostic factors. Carcinoma patients who were negative for both Fas and FasL had a significantly higher incidence of lymph node involvement than did carcinoma patients who were positive for Fas and FasL. Carcinomas that were positive for Fas and FasL demonstrated a greater sensitivity to doxorubicin in vitro.

Angiogenesis and cigarette smoking in squamous cell lung carcinomas: an immunohistochemical study of 28 cases.

Angiogenesis in tumors is influenced by several factors which in turn are associated with chemoresistance or radioresistance. Moreover, the tumors of smokers are known to be relatively resistant to chemotherapy. This investigation attempts to determine whether or not a relationship exists between cigarette smoking and angiogenesis in lung cancer. Tumor samples from 14 non-smokers and 14 heavy cigarette smokers were selected for this study. The populations were matched for age, sex and tumor stage. Resistance to doxorubicin, microvessel density, the expression of vascular endothelial growth factor (VEGF) and thrombospondin (TSP) were analyzed in both populations. Tumors of smokers were more frequently resistant to doxorubicin in vitro, had lower vessel counts and a reduced expression of VEGF compared to tumors of nonsmokers. In contrast, TSP was significantly increased in the tumors of smokers. These data show that angiogenesis in lung tumors is linked to a patient's smoking habits.

PD-ECGF, bFGF, and VEGF expression in non-small cell lung carcinomas and their association with lymph node metastasis.

The angiogenic factors PD-ECGF, bFGF and VEGF were determined immunohistochemically in 168 non-small cell lung carcinomas to investigate whether the expression of these parameters is correlated with lymph node metastasis of patients. The expressions of the above mentioned factors was indeed associated with lymph node metastasis, but the results were not statistically significant. However, a combination of the factors PD-ECGF, bFGF and VEGF significantly improved the prognostic information. The number of tumors with lymph node involvement increased with the number of angiogenic factors. Only 43% of the patients had-lymph node involvement when all factors were negative whereas 77% showed metastasis when all factors were positive (one factor positive: 53%, two factors positive: 68). This result is statistically significant (p = 0.002, test for trend).

Relationship of urokinase and urokinase receptor in non-small cell lung cancer to proliferation, angiogenesis, metastasis and patient survival.

The purpose of the analysis was to ascertain whether the expression of components of the plasminogen activation system possesses an association with several biological parameters. The expression of urokinase (uPA) and urokinase receptor (uPAR) was analyzed in 137 non-small cell lung carcinomas by immunohistochemistry. No relationship could be observed between the proliferative activity of the carcinomas measured by flow cytometry and the expressions of uPA and uPAR. In addition, there was no association of the expressions of uPA or uPAR and vessel density (angiogenesis), neither any significant correlation between the expressions of uPA or uPAR and metastasis. The median survival was shorter for patients with uPA positive carcinomas than for those with uPA negative tumors (60 vs. 111 weeks; p=0.018). The relative risk estimate for patients with uPA positive tumors was increased by a factor of 1.8. In contrast, the expression of uPAR showed no significant correlation with overall survival.

The implications of proliferation and apoptosis for lung cancer metastasis.

The occurrence of metastatic spread depends on many factors both the condition of the patient and the properties of the tumor. In this investigation the association between proliferation and apoptosis and the incidence of lymph node involvement of patients with non-small cell lung carcinomas was analysed (n=215 patients). In order to analyse the relationship between lymph node metastasis and proliferative activity of the carcinomas, the distribution of cell cycle phases (flow cytometry), the expression of PCNA and cyclin A (immunohistochemistry) was determined. Fas, Fas-ligand, caspase-3 and Bcl-2 were determined by immunohistochemistry. In this retrospective analysis no association between proliferative activity of the tumors and lymph node status was found. In contrast, there existed a correlation between the apoptotic factors and lymph node metastasis. Higher expression of the pro-apoptic factors Fas, Fas-ligand and caspase-3 correlated with a lower incidence of lymph node involvement (Fas-ligand, p=0.004; caspase-3, p=0.007). The trend of an inverse correlation between the anti-apoptotic factor Bcl-2 and metastasis fits well into the present knowledge about the function of the bcl-2 gene. The results obtained from all the patients could be confirmed in patients with squamous cell lung carcinomas.