Electron microscopy evidence that cytoplasmic localization of the p16(INK4A) "nuclear" cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas.

It is well established that p16(INK4A) protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 (INK4A) usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16(INK4A) immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16(INK4) represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16(INK4A) staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16(INK4A) cytoplasmic expression. We observed p16 (INK4A) immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16(INK4A) immunoreactivity was detected only in the nuclei. We have demonstrated that p16(INK4A) cytoplasmic staining is specific and suggest that it represents a mechanism of p16(INK4A) inactivation similar to that observed in other tumor suppressor genes.

Interferon-a2b reduces neo-microvascular density in the 'normal' urothelium adjacent to the tumor after transurethral resection of superficial bladder carcinoma.

BACKGROUND: As angiogenesis represents one of the hallmarks of cancer we investigated whether intravesically administered interferon-a (IFN-a2b) reduces neo-angiogenesis in the 'normal' urothelium adjacent to the tumor in patients with superficial bladder carcinoma after complete transurethral resection (TUR) of the tumor. PATIENTS AND METHODS: In the present study 47 patients after TUR of the tumor were examined. 10 patients (group A) received no further treatment (control group); 37 patients (group B) received intravesical treatment with IFN-a2b. The instillations started within 7 days after TUR, were performed weekly for 2 months, twice a month for the next 4 months, and thereafter monthly for 6 more months. Cold cup biopsies were taken before TUR of the transitional cell carcinoma (TCC): from the tumor (T), near tumor (NT) and from normal epithelium (N). Cold cup biopsies 'near tumor', were also taken during follow-up cystoscopy (C1, C2, and C3) 2, 6, and 12 months after TUR, respectively. Angiogenesis was estimated by counting the microvessels detected with CD31 immunostaining. RESULTS: Significant differences of microvascular density (MVD) between patients of group A and B appear after TUR (p < 0.005, Kruskal-Wallis and Wilcoxon test). The MVD difference was maximal 6 months after TUR (C2(A)-C2(B), second cystoscopy) and measured at 12.17 microvessels/ mm(2) (26.2%). CONCLUSION: Our results show that the intravesical administration of IFN-a2b after TUR significantly decreases the angiogenic potential of the 'healthy' urothelium adjacent to the tumor in patients with TCC. This observation could possibly explain, to a certain extent, the mechanism by which IFN-a2b reduces the recurrence rate of primary TCC.

Expression of HLA-DR is reduced in tumor infiltrating immune cells (TIICs) and regional lymph nodes of non-small-cell lung carcinomas. A putative mechanism of tumor-induced immunosuppression?

BACKGROUND: Deregulation of MHC class II molecules consists of a favorable mechanism of tumor evasion from immune surveillance. Among these molecules, HLA-DR antigens are the predominant ones in cancer. In the present study we sought to investigate the ability of tumor infiltrating immune cells (TIICs) to express HLA-DR antigen in the primary tumor site and reactive regional lymph nodes (LNs) in non small cell lung cancer (NSCLC). MATERIALS AND METHODS: Material consisting of 60 NSCLCs with corresponding regional LNs was studied by immunohistochemistry for human leukocyte antigen D-region related (HLA-DR) expression. Control reactive LNs, regional to several different malignant and non-malignant disorders, were also included in the study. RESULTS: Primary tumor site investigation revealed positive HLA-DR cancer cells in 22% of cases, whereas TIICs rarely expressed HLA-DR antigens. The lack of HLA-DR expression in TIICs was gradually attenuated as the distance from the primary tumor site decreased. Regional LN investigation showed that all follicles (paracapsular and deep cortical ones) were HLA-DR-negative in 60% of the LNs; in the remaining 40%, the paracapsular follicles remained negative, while all deep cortical ones were positive. Interestingly, LNs possessing only HLA-DR-negative follicles were more proximal to the primary tumor site compared to those that had only the paracapsular follicles negative. All control reactive LNs, regional to several distinct malignant and non-malignant disorders, were found to be HLA-DR-positive. CONCLUSION: The impairment of HLA-DR expression, detected both in neoplastic and by-stander immune cells, may justify the immunosuppression observed in NSCLC. This phenomenon may be due to a putative soluble factor in the tumor environment secreted by cancer cells.

Low levels of p27 in association with deregulated p53-pRb protein status enhance tumor proliferation and chromosomal instability in non-small cell lung carcinomas.

BACKGROUND: Down-regulation or overexpression of the cyclin-dependent kinase inhibitor p27 have been observed in a range of malignancies, including lung cancer. To further elucidate the role of the molecule in tumor growth regulation, we evaluated p27 expression in a series of non-small cell lung carcinomas (NSCLCs), and examined its relation with histology, kinetic parameters, ploidy, and overall survival. We extended our investigation into the association of p27 levels with the presence of Ki-ras mutations, as well as with the expression status of p53 and pRb in tumor cells. MATERIAL AND METHODS: p27, p53, and pRb status were immunohistochemically evaluated in a total of 69 NSCLCs. In situ assays were employed to assess the kinetic parameters (Ki-67 immunohistochemistry for proliferation index, Tdt-mediated dUTP nick end labeling assay for apoptotic index). The ploidy status of the tumors was assessed after staining nuclei with the Feulgen procedure, and the presence of Ki-ras mutations was examined by restriction fragment length polymorphisms. All possible associations were assessed with a series of statistical methods. RESULTS: Immunoreactivity for p27 was observed in the entire series of specimens, with the mean percentage of positive cells being 33%. Adenocarcinomas (AdCs) exhibited higher p27 levels compared to squamous cell carcinomas (SqCCs) (p < 0.01). An inverse correlation was established between p27 expression and proliferation index (PI) (r = -0.834, p < 0.01) but not with apoptotic index (AI), whereas aneuploid tumors were characterized by lower p27 levels than diploid ones (p < 0.01). No difference in p27 immunostaining was observed with regard to the presence of Ki-ras mutations, whereas aberrant p53 and/or pRb expression patterns were associated with p27 underexpression (p < 0.01 for p53 status, p < 0.05 regarding pRb levels, and p < 0.01 for a combined deregulation of both proteins). Two or more alterations in the p27/p53/pRb protein network (i.e., p27 levels lower than the estimated mean value, overexpressed p53, and/or aberrant pRb) were associated with increased PI and aneuploidy (p < 0.001 and p < 0.01, respectively). A powerful trend was found between p27 expression and overall survival (p = 0.066). CONCLUSIONS: Our findings confirm the heterogeneity between AdCs and SqCCs, and are suggestive of an increased proliferative activity in NSCLCs underexpressing p27. Furthermore, our analysis supports the concept of p27 forming a functionally compact network with p53 and pRb, which is actively involved in the regulation of cellular proliferation and chromosomal stability.

Additional characterization of a hexanucleotide polymorphic site in the first intron of human H-ras gene: comparative study of its alterations in non-small cell lung carcinomas and sporadic invasive breast carcinomas.

Intron 1 of the human H-ras gene possesses a polymorphism consisting of repetitions of the GGGCCT consensus. Three alleles have been reported at this locus. We confirmed that two, P1 and P2, display four and two repeats, respectively, with their internal sequence structure similar to that previously described. The third, P3, previously assigned as a three-unit repetition allele according to its electrophoretic mobility and with no other information regarding its internal structure, was also found. Sequence analysis of the P3 allele revealed that it consists of three perfect repeats of the GGGCCT consensus. This polymorphism is present only in human c-H-ras gene, although single hexanucleotide repeats are found scattered within intron 1 of this gene in rodents. Analysis of this locus in matched tumor/distant normal samples from: (i) 38 patients with non-small-cell lung carcinoma (NSCLC), and (ii) 35 patients with sporadic invasive breast carcinoma, revealed: (1) 6.6% and 19% loss of heterozygosity (LOH) respectively, and (2) 10.5% and 2.9% hexanucleotide instability (HI) respectively, detected by the presence of shifted in length alleles. Shifted alleles exhibited altered internal sequence structure in comparison to normal ones, suggesting complex mutational events. The same pattern of alterations was also detected in tissues adjacent to lung adenocarcinomas and dysplasias adjacent to squamous cell carcinomas (7.7% LOH, 5.9% HI), implying that abnormalities at this locus may be early events in lung carcinogenesis. The frequency of alterations (LOH vs. HI) was significantly different among NSCLC and breast cancer (P=.005), probably due to the different tumor biology of each system. Finally, altered mRNA expression of H-ras gene was detected in all cases with HI, but this finding was also observed in samples without HI. In view of reports showing that elements in intron 1 of H-ras gene potentially influence its transcriptional regulation, from our results we cannot exclude that the hexanucleotide locus could be an element with possible involvement in expressional regulation of this gene.

A new rare allele at the CGG repeat polymorphism in the first intron of human c-H-ras gene.

We have examined a region in the first intron of the human c-H-ras gene containing a CGG repeat. This region was previously shown to be variable in length. The length variation was attributed to the presence of the CGG repeat after estimation of its electrophoretic mobility. In the present report we have characterized in detail this region by PCR-RFLP and automated sequencing, in a total of 102 histologically normal tissues from unrelated individuals affected by lung and breast cancer. Four alleles were detected and analysis of their internal sequence showed that the length alterations of this region were due to the presence of 5, 6, 8 and 9 CGG triplets respectively. The last three occur most often (44.1%, 34.8%, 20.6% respectively) and coincide with three previously reported alleles (Riggins et al, Hum Mol Genet 9: 775, 1992). The fourth allele consisting of 5 repeats is a rare one (0.5%), whilst alleles with 7, and a previously reported one suggested to comprise 11 repeats (1%) were not present in our cohort. This polymorphism coincides in position with an element that was previously shown to possess regulatory activity.

Diagnostic and prognostic significance of squamous cell carcinoma antigen in non-small cell lung cancer.

Squamous cell carcinoma antigen (SCC-Ag) is a glycoprotein secreted by non-small cell lung tumours (NSCLC). This study investigated the diagnostic and prognostic significance of SCC-Ag in NSCLC. Receiver operating characteristic (ROC) curve analysis was used to test the diagnostic performance of the SCC-Ag and determine the optimal threshold value in a group of 100 NSCLC patients undergoing surgery and 50 age matched healthy controls. This threshold was then prospectively validated in a group of 53 patients and 49 healthy controls. The prognostic significance of the preoperative SCC-Ag level and its postoperative decrease were tested using univariate and multivariate proportional hazard models. The area under the ROC curve was 0.71+/-0.04, and the best cutoff value was 1.4 ng/ml. This discriminated patients in the validation group, with a sensitivity of 0.55 and a specificity of 1.0. The hazard ratio was 0.144 (95% CI 0.074-0.281) for the postoperative decrease in the SCC Ag, and 5.823 (3.299-10.278) for the preoperative SCC Ag level. Multivariate analysis revealed that only disease stage and patients' age are strong prognostic factors for survival. In conclusion, the SCC-Ag serum level has moderate diagnostic role in NSCLC. Both the preoperative SCC-Ag level and its postoperative decrease have prognostic significance, yet inferior to the disease stage and the patient's age.

Deregulated expression of c-mos in non-small cell lung carcinomas: relationship with p53 status, genomic instability, and tumor kinetics.

Little is known about the status of the mitogen-activating protein kinase pathways in lung cancer. One of the key molecules taking part in these pathways is the product of the c-mos proto-oncogene, which plays an important role in oocyte maturation. In vitro investigations in somatic cells have shown that c-mos expression has opposing effects on the cell cycle, which suggests that this proto-oncogene may represent an important determinant of aberrant cell function (genomic instability and altered kinetics). A recent study suggests that these effects may be p53 dependent. In view of the apparent link between c-mos and p53, we investigated in a series of 56 non-small cell lung carcinomas: a) the status of c-mos; b) its relationship to genomic instability (aneuploidy) and two kinetic parameters of the tumors, proliferation and apoptotic indexes (AI); and c) its association with p53 alterations and their concomitant relationship with the above parameters. We found c-mos overexpression in 27% of the tumors. Expression was higher in stages II/III (34%) than in stage I (17%; P = 0.018). Complete concordance was observed between c-mos overexpression and elevated c-mos mRNA levels. Because c-mos gene amplification was not detected, its deregulated expression may be attributable to increased transcription. Of the c-mos positive [c-mos(P)] cases, 77% were associated with aneuploidy. Sequencing showed two silent mutations and one missense (R-->L) at codon 22, located in a region critical for c-mos stability. In contrast to the findings of some in vitro studies, c-mos(P) tumors had a lower mean AI score than the c-mos negative [c-mos(N)] tumors had, implying that induction of apoptosis may have been defective. Indeed, 86% of the tumors overexpressing c-mos showed p53 alterations. The carcinomas with concomitant alterations of c-mos and p53 [c-mos(P)/p53 positive] had significantly lower AI values (P < 0.001) and were more frequently associated with aneuploidy (P = 0.015) than the c-mos(N)/p53 negative tumors but not the c-mos(N)/p53 positive tumors, which suggests that p53 status is the main determinant of ploidy status and apoptosis in our series. This finding also strengthens the concept that wild-type p53 plays a "safeguard" role in preventing oncogene-mediated activation.

Association of allelic loss at the FHIT locus and p53 alterations with tumour kinetics and chromosomal instability in non-small cell lung carcinomas (NSCLCs).

The FHIT gene, located at the FRA3B fragile site of chromosome 3p14.2, encodes a 16.8 kD homologue of the yeast enzyme diadenosine tetraphosphate (Ap(4)A) hydrolase. Frequent allelic losses at this region in various malignancies, including non-small cell lung carcinomas (NSCLCs), imply that FHIT may represent a tumour suppressor gene (TSG). Increasing evidence suggests that multiple TSG impairment has a synergistic effect on tumour growth. The present study of 67 NSCLCs investigated the allelic imbalance (AIm) within the FHIT locus and its relationship with p53 abnormalities, kinetic parameters [proliferative activity or proliferation index (PI) and apoptotic index (AI)], and ploidy status of the carcinomas. Allelic imbalance at FHIT was observed in 35 out of 55 informative (heterozygous: H) cases (64%). Similar frequencies of loss of heterozygosity (LOH) were noticed among squamous cell lung carcinomas and adenocarcinomas. The high percentage of AIm in stage I tumours (71%) is indicative of its relatively early involvement in NSCL carcinogenesis. No association was found between LOH at FHIT, kinetic parameters, and ploidy status of the tumours. Concurrent loss at FHIT and p53 overexpression [FHIT(LOH)/p53(P)] was the most frequent pattern and was observed in 39% of the informative cases. The latter pattern was not associated with smoking, supporting the hypothesis that in patients with a history of tobacco exposure, FHIT allelic loss may not be a consequence of p53 checkpoint defects, but the outcome of tobacco-induced mutagenesis. Statistically significant differences in the presence of FHIT(LOH)/p53(P) and FHIT(LOH)/p53(N) patterns were noted at the proliferative and apoptotic level, whereas ploidy was similar amongst all groups, implying that wild-type (wt) p53 may play a safeguard role against altered FHIT function. However, the possibility of a masking effect from wt p53 cannot be excluded, since the FHIT(LOH)/p53(P) profile demonstrated a higher growth index (GI=PI/AI mean value ratio) than FHIT(H)/p53(P) (32 vs. 8), although this was not significant. Further studies are needed in order to elucidate the role of FHIT and its relationships with other cell-cycle regulatory molecules involved in NSCL carcinogenesis.

Relationship of the K-ras/c-mos expression patterns with angiogenesis in non-small cell lung carcinomas.

BACKGROUND: Neo-angiogenesis is an acquired capability vital for a tumor to grow and metastasize. Evidence has shown that the mitogen-activated protein (MAP) kinase pathway is involved in this process. Alterations of K-ras and c-mos, two pivotal components of this pathway, have been implicated in non-small cell lung carcinogenesis. In the present report, we examine, in a series of non-small cell lung carcinomas (NSCLCs), the status of K-ras and c-mos oncoproteins in correlation with the tumor neo-angiogenesis state and the major angiogenic factor, vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: c-mos and p-ERK1/2 status was evaluated immunohistochemically in a total of 65 NSCLCs, whereas the presence of K-ras mutations was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) in available matched normal tumor material from 56 cases. Microvessel density (MVD) was estimated by immunodetection of CD3, endothelial marker, and VEGF expression was assessed by immunohistochemistry. All possible associations were examined by a series of statistical methods. RESULTS: Expression of oncogenic activated K-ras and c-mos overexpression was observed in 12 of 49 (25%) and in 16 of 61 (26%) informative cases, respectively. Only 1 of the 25 deregulated for K-ras or c-mos cases exhibited both alterations, suggesting a mutually exclusive relationship between activated K-ras and c-mos overexpression (p = 0.074) in a subset of NSCLCs. In these cases, the MAPK kinase kinase/MEK/ERK pathway was found to be activated. MVD and VEGF expression were 36.9 +/- 10.6 mv/mm2 and 73.1 +/- 20.0%, respectively. The most intriguing finding was that the [K-ras(No)/c-mos(P)] profile was significantly associated with low MVD levels compared to normal cases (p = 0.004); by contrast, no correlation was found between the other K-ras/c-mos patterns and MVD. Furthermore, the former group exhibited the lowest VEGF levels. CONCLUSIONS: The mutually exclusive relationship between mutated K-ras and c-mos overexpression in a subset of NSCLCs implies a common signal transduction pathway in lung carcinogenesis. The effect of this pathway on NSCLC neo-angiogenesis seems to depend upon the status of c-mos, which acts as a molecular "switch," possibly exerting a negative selective pressure on tumor progression.

Association of allelic imbalance at locus D13S171 (BRCA2) and p53 alterations with tumor kinetics and chromosomal instability (aneuploidy) in nonsmall cell lung carcinoma.

BACKGROUND: BRCA2 gene, located at chromosome 13q12.3, frequently is altered in familial types of cancer in which a "double-hit" inactivation model seems to occur. In contrast, in sporadic forms of cancer there is frequent absence of a second event (point mutations) suggesting that allelic imbalance at the BRCA2 locus may be associated with a "gene dosage effect" of BRCA2 function. Little is known about BRCA2 allelic alterations in nonsmall cell lung carcinomas (NSCLCs). Furthermore, recent studies suggest that BRCA2 and p53 participate in a common pathway involved in DNA damage repair. In view of this putative link, the authors investigated in a series of 63 NSCLCs: 1) the allelic imbalance (AIm) at the D13S171 (BRCA2) locus, 2) the possible relation with tumor kinetics (proliferation [PI] and apoptotic indices [AI]) and chromosomal instability (aneuploidy) of the carcinomas, and 3) the mutual impact of D13S171 AIm and p53 altered status on the above-mentioned parameters. METHODS: Allelic status of the BRCA2 region was examined in a series of 63 NSCLCs, by using the polymorphic marker D13S171, which is located in the center of it. Most information regarding the status of p53 at the immunohistochemical and genetic levels was obtained from a previous analysis. Tumor kinetic parameters (proliferation and apoptotic indices) were determined using Ki-67 immunohistochemical analysis and Tdt-mediated dUTP nick end labeling assay, respectively. Chromosomal instability (aneuploidy) was assessed by measuring nuclear DNA ploidy with an image analysis system. RESULTS: Allelic imbalance at D13S171(BRCA2) was observed in 70% of the informative cases (H: heterozygous) with a rather high frequency of occurrence (50%) in Stage I disease, suggesting a possible early involvement in the development of NSCLCs. Although no association was found among loss of heterozygosity (LOH) at D13S171, kinetic parameters and ploidy status of the tumors and concurrent alterations in BRCA2 and p53 (BRCA2[LOH]/p53[P]), which was the most frequent profile (37.2%), had the highest growth index (PI/AI mean value ratio) that differed significantly only from the BRCA2(LOH)/p53(N) pattern (P = 0.027). This difference was attributed to the high AI of the BRCA2(LOH)/p53(N) pattern (P < 0.001), whereas PI was similar among all BRCA2/p53 profiles. Also the "full abnormal pattern" was associated with aneuploidy, whereas the BRCA2(LOH)/p53(N) profile was mainly diploid. When these indicators and conventional prognostic ones were examined for effect on patient survival, only stage and lymph node status showed a significant correlation, whereas LOH at D13S171 (BRCA2), p53 abnormalities, proliferative and apoptotic indices, ploidy status, smoking history, and histology and combinations of LOH and p53 abnormalities failed to show significant correlation with survival. CONCLUSIONS: These findings suggest that in BRCA2(LOH) NSCLCs the status of p53 (wild type or mutant) represents a decisive determinant of tumor growth and chromosomal instability. Nevertheless, a possible synergistic effect from loss of D13S171 region with p53 abnormalities cannot be excluded because the BRCA2(LOH)/p53(P) profile compared with the BRCA2(H)/p53(P) one had a higher PI/AI mean value ratio (31.05 vs. 22.97), although it was not statistically significant. However, we cannot exclude the possibility that LOH at D13S171 reflects deletion of other putative tumor suppressor gene(s) in the proximity of BRCA2. In this respect, more studies are needed to understand the involvement of BRCA2 region alterations in nonsmall cell lung carcinogenesis. (c) 2000 American Cancer Society.

Absence of mutations in the functional domains of the human MDM2 oncogene in non-small cell lung carcinomas.

Increasing evidence suggests that MDM2 oncoprotein participates in a complex array of interactions with a plethora of molecules, including cell-cycle and transcriptional regulators, as well as determinants of the cell differentiation and senescence. The tumorigenic potential of MDM2 is mainly determined by overexpression due to gene amplification, mRNA overexpression and possibly translational enhancement. Although artificially created mutations have been demonstrated to abolish normal MDM2 function, there is little information concerning its mutational status in human tissues. In this study, we screened all the functional domains of MDM2 for mutations in a series of 58 non-small cell lung carcinomas (NSCLCs), but none was found. Therefore, we report that MDM2 mutations are an extremely rare phenomenon of non-small cell lung carcinogenesis. A putative explanation for this observation may be the labyrinth of interactions necessary for cell viability, in which MDM2 takes part, a finding also supported by its stringent interspecies conservation.

c-mos immunoreactivity is an indicator of good prognosis in lung cancer.

AIMS: Reports concerning the expression of cytoplasmic components of the mitogen-activating protein kinase (MAPK) pathway in lung cancer are limited. One of the molecules participating in this pathway is the product of the c-mos proto-oncogene. In vitro investigations, in somatic cells, have shown that c-mos expression has opposing effects on cell cycle progression suggesting that it may represent an important determinant of aberrant cell function. In this study we analysed, by immunohistochemical means, its status in a series of lung carcinomas and correlated the findings with clinicopathological parameters and survival of the patients. METHODS AND RESULTS: Sixty cases of lung carcinomas were included in the study. These comprised 52 non-small (NSCLCs) and eight small cell lung carcinomas (SCLCs). Sections from the carcinomas were immunostained with the polyclonal anti-c-mos antibody P-19. Specificity was tested by using the appropriate control peptide and control cell lines. Expression was observed in 63% of the cases, with NSCLCs showing higher reactivity (67%) than SCLCs (37.5%). Staining was observed mainly to the cytoplasm and membranes of the cancerous cells, but some nuclei reacted as well. An intratumour heterogeneous immunoreactivity was noticed. The most interesting and unexpected finding was that c-mos positive staining was associated with better recurrence-free survival in our series, regardless of histological type (P = 0.035). Furthermore, favourable disease-related and recurrence-free survival was observed in the SqC group with c-mos immunoreactivity (P < 0. 001). CONCLUSIONS: c-mos proto-oncogene is expressed in a significant proportion of lung carcinomas and may play a role in its development. The fact that its expression is associated with a relatively good prognosis may be indicative of a negative impact on tumour growth.

Altered expression of the cell cycle regulatory molecules pRb, p53 and MDM2 exert a synergetic effect on tumor growth and chromosomal instability in non-small cell lung carcinomas (NSCLCs).

BACKGROUND: Recent in vitro studies provide evidence that the cell cycle molecules pRb, p53 and MDM2 form a tightly regulated protein network. In this study, we examined the relationship of this protein network in a series of non-small cell lung carcinomas (NSCLCs), with the kinetic parameters, including proliferative activity or proliferation index (PI) and apoptotic index (AI), and ploidy status of the tumors. MATERIAL AND METHODS: A total of 87 NSCLCs were examined using immunohistochemical and molecular methods in order to estimate the status of the pRb-p53-MDM2 network. The kinetic parameters and the ploidy status of the tumors were assessed by in situ assays. The possible associations between alterations of the network, kinetic parameters and ploidy status of the carcinomas were assessed with a series of statistical methods. RESULTS: Aberrant expression of pRb (Ab) and overexpression of p53 (P) and MDM2 (P) proteins were observed in 39%, 57%, and 68% of the carcinomas, respectively. The comprehensive analysis revealed that concurrent alterations in all three cell cycle regulatory molecules were the most frequent pattern, pRb(Ab)/p53(P)/MDM2(P); this "full abnormal" phenotype represented approximately 27% of the cases. This immunoprofile obtained the highest PI/AI value; whereas, the "normal" phenotype was the lowest one (p = 0.004). Furthermore, the pattern pRb(Ab)/p53(P)/MDM2(P) acquired the highest PI (p < 0.001) and lowest AI (p < 0.001) scores. Interestingly, the groups of carcinomas with impaired expression of one or two molecules attained PI/AI ratio values clustered in a narrow range placed in the middle of the scores exhibited by the "normal" and "full abnormal" phenotypes. These tumors had significantly lower AI, but similar PI values, compared with those noticed in the normal pattern. In addition, it was observed that the pRb(Ab)/p53(P)/MDM2(P) phenotype was also significantly associated with aneuploidy (p = 0.002) and a tendency was observed when the expression of two components was altered (p = 0.055). CONCLUSIONS: Our findings suggest that simultaneous deregulation of all members of the pRb-p53-MDM2 network confers an additive effect on tumor growth. The apoptotic pathway seems to be more susceptible to its defects than the cell proliferation machinery. The findings of the ploidy analysis, which are in parallel with those regarding the proliferative activity and the apoptotic rate study, further support the concept that these molecules constitute a tightly regulated network participating in cell cycle control and chromosomal stability.

Allelic imbalance at the 5q14 locus is associated with decreased apoptotic rate in non-small cell lung carcinomas (NSCLCs). Possible synergistic effect with p53 gene alterations on apoptosis.

Deletions in the 5q14 region have been described in a variety of neoplasms, such as testicular germ cell tumors, ovarian, gastric and lung cancer. The high frequency of allelic losses observed in this region implies the presence of putative tumor suppressor gene(s) (TSGs). In the present study, we investigated in a series of 56 non-small cell lung carcinomas (NSCLCs) the allelic imbalance (Alm) within the 5q14 region, employing the D5S644 marker, and its relationship with p53 abnormalities, the kinetic parameters [proliferation index (PI) and apoptotic index (AI)] and the ploidy status of the carcinomas. AI at D5S644 was found at a frequency of 51.2%. The rather high percentage of Alm in stage I tumors suggests an early involvement in NSCLC development. LOH at 5q14 was associated with decreased AR in lung tumors insinuating the presence of a putative TSG(s) (P=0.008). Simultaneous alterations of both p53 and D5S644 locus were the most frequent pattern observed (37.5%). Cases demonstrating this profile also exhibited a marked decrease in AI (P=0.001). These findings imply a synergistic mechanism of co-operation between different TSGs. However, proliferation activity was dependent only on p53 status, leading to the assumption that the putative TSG(s) present at 5q14 may probably be involved in normal apoptotic procedures. Further studies are needed to identify the candidate gene(s).

Sensitive differential detection of genetically related mycobacterial pathogens in archival material.

A polymerase chain reaction (PCR) assay targeted to the immunogenic protein MPB64 gene was used to detect members of the Mycobacterium tuberculosis complex, and an outward-primed PCR (OPPCR) designed on the IS6110 element allowed differentiation between Mycobacterium bovis and Mycobacterium tuberculosis. Additionally, the amplification of IS1110 and 16S ribosomal RNA sequences combined with a dot blotting assay were able to differentially detect Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium paratuberculosis. The validity of the experimental procedure was tested on reference material and formalin-fixed paraffin-embedded samples from patients with tuberculosis, sarcoidosis, or Crohn disease. We demonstrated mycobacterial DNA in 59 of 75 cases with histologic lesions typical of tuberculosis; we detected M tuberculosis and M paratuberculosis in 6 of 25 sarcoidosis cases and in 7 of 20 Crohn disease specimens, respectively. The proposed diagnostic procedure is directly applicable to archival material and allows differentiation of genetically related mycobacterial pathogens in more detail than other molecular methods. It provides a tool for the diagnostic study of tuberculosis, sarcoidosis, and Crohn disease.

Expression of p16(INK4A) and alterations of the 9p21-23 chromosome region in non-small-cell lung carcinomas: relationship with tumor growth parameters and ploidy status.

The 9p21-23 chromosome region harbors a number of known and putative tumor-suppressor genes (TSGs). The best characterized gene in this area is p16(INK4A) (CDKN2A). Alterations of its product have been observed in various malignancies, including non-small-cell lung carcinomas (NSCLCs). We earlier investigated the mechanisms underlying p16(INK4A) inactivation. In the present study, we examined, in a series of 87 NSCLCs, its relationship with the kinetic parameters [proliferation index (PI) and apoptotic index (Al)] and the ploidy status of the tumors. In addition, we extended our previous LOH analysis of the 9p21-23 region by examining flanking areas of p16(INK4A). Aberrant p16 expression was observed in 41.4% of the carcinomas. A significant association was found with increased PI (p = 0.037), but not with apoptosis. Aneuploid tumors were more frequently correlated with abnormal p16 staining (p = 0. 05). A high frequency of allelic imbalance (Alm) was noticed at the D9S161 (51.3%) and D9S157 (64.5%) loci, which lie approximately 4cM centromeric and 7cM telomeric, respectively, to CDKN2A. Abnormal p16(INK4A) expression was strongly correlated with Alm at D9S161 (p = 0.004). Allelic losses at D9S157 occurred more frequently in early stages (p = 0.018) and were significantly associated with deletions at D9S161 (p = 0.035). We conclude that, in a sub-set of NSCLCs, (i) abnormal p16 expression contributes to tumor growth mainly by increasing the proliferative activity in the initial stages of carcinogenesis; (ii) the association with aneuploidy merely reflects the impact of aberrant p16 on proliferative activity; and (iii) other putative TSGs possibly reside within the 9p21-23 region that possibly co-operate in certain cases with CDKN2A in the development of NSCLCs.

Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis.

The aims of the present study were: (1) to design a sensitive and specific polymerase chain reaction-based method that would allow detection of most common human typical and atypical mycobacterial strains and (2) to apply the method on formalin-fixed paraffin-embedded (FFPE) tissue and sputum samples from patients with clinicopathological evidence of tuberculosis and sarcoidosis. Three sets of primers were selected. The first detects specifically members of the Mycobacterium tuberculosis (M. tuberculosis) complex, amplifying a 243 bp fragment of the gene encoding the immunogenic protein MPB 64, whereas the second traces members of the Mycobacterium avium (M. avium) complex producing a 91 bp fragment of the IS1110 element. The third pair of primers is specific for slow-growing mycobacteria, amplifying a 383 bp region of the 65 kDa mycobacterial antigen gene. Our multiplex polymerase chain reaction assay identified mycobacterial DNA of 10(-3) colony-forming units (CFU)/mL from sputum samples, 10(-5) CFU/mL from FFPE tissue samples and 10(-6) CFU/mL from pure broth cultures. By performing the method on 75 FFPE tissue samples with histological and clinical evidence of tuberculosis and 300 sputum specimens from patients suspected of tuberculosis, we found 38 M. tuberculosis complex, 7 M. avium complex, and 14 slow-growing mycobacteria positive samples in the first case and in the second we found 95 M. tuberculosis complex, 21 M. avium complex, and 35 slow-growing mycobacteria positive samples. The sensitivity of the assay was significantly higher than that of Ziehl-Neelsen and in some cases higher than culture, especially when applied on atypical mycobacteria. In addition, 25 cases histologically and clinically characterized as sarcoidosis were investigated for mycobacterial DNA sequences and in nine of these, DNA corresponding to M. tuberculosis complex was detected. The method described can be applied directly on FFPE and sputum samples and allows not only the detection of mycobacterial DNA, but also an assessment concerning the species involved.

Aberrant p16 expression is correlated with hemizygous deletions at the 9p21-22 chromosome region in non-small cell lung carcinomas.

The p16 protein is encoded by the CDKN2 gene, and functions as an inhibitor of cyclin-dependent kinase 4 and 6 (CDK4/6). Phosphorylation of the retinoblastoma protein (pRb) by CDK4/6 represents a vital step in cell cycle progression. Alterations of p16INK4A are frequent events in human malignancies. In non-small cell lung carcinoma (NSCLC) the data concerning the mechanisms of p16INK4A inactivation suggest that point mutations and aberrant methylation of its promoter can only account for a proportion of the cases with abnormal p16 immunoexpression. The role of deletions in this procedure is not yet clarified. In order to gain more insight into the role of deletions in p16INK4A deregulated expression, we investigated the state of the chromosomal region 9p21-22 in a series of 57 NSCLCs, by performing a detailed mapping analysis, using a tight cluster of highly polymorphic microsatellite markers, and correlating the findings with p16 immunostaining. Abnormal p16 expression was observed in 46% of the NSCLCs examined. No relationship was observed between p16 abnormal staining and various clinicopathological parameters. Abnormal p16 protein staining was strongly associated with hemizygous deletions at the IFNA and D9S171 microsatellite loci, which demarcate the region encoding the p16INK4A gene (P = 0.002). These findings suggest that deregulated expression of p16 is involved in the multistage process of NSCL carcinogenesis and that deletions may represent a predominant mechanism of p16INK4A inactivation. A significant percentage also of LOH was noticed at the D9S162 (35%) and D9S126 (38%) loci which lie 6cM and 4cM, respectively, far from the area which encodes p16INK4A, implying that other tumor suppressor genes (TSGs) may reside in this region. Although the overall incidence of LOH at the examined region was high (58%), we did not observe any correlation with smoking habits, histology and lymph node status. Another noteworthy finding was the existence of microsatellite instability (MI) in 11% of the patients. MI provides a marker for replication error phenotype (RER+), a recently defined manifestation of genetic instability observed in a wide range of tumors. In conclusion, alterations (LOH + MI) at the 9p21-22 chromosome region are frequent events in NSCLCs and may affect directly or indirectly the expression of p16.

Modulation of wild-type p53 activity by mutant p53 R273H depends on the p53 responsive element (p53RE). A comparative study between the p53REs of the MDM2, WAFI/Cip1 and Bax genes in the lung cancer environment. WAFI/Cip1 = WAF1/Cip1.

Wild-type (wt) p53 is a tumor-suppressor protein which acts via transcriptional and transcriptional-independent mechanisms. The transcriptional function of p53 is mediated by specific responsive elements (REs). The MDM2, WAF1/Cip1 and Bax genes possess p53REs and their activation by wt p53 induces cell cycle progression, arrest and programmed cell death (apoptosis), respectively. Mutations of the p53 gene are detected in more than 50% of the human malignant tumors. p53 mutants seem to have a more stable conformation and are suggested to exert dominant-negative inhibition of wt p53 in cells containing both wt and mutant (mt) alleles. However, recent studies show that certain mt p53 proteins posses a "gain of function" phenotype. In the present study, we examined the effects of the second most frequent p53 mutant R273H on the p53REs of the MDM2, WAF1/Cip1 and Bax genes in the H1299 non-small cell lung carcinoma cell line. Although mt p53 R273H alone was unable to bind and transactivate the corresponding p53REs, it enhanced the MDM2-p53RE mediated gene transcription of wt p53 (positive-dominant effect) and prevented the wt p53 transactivation of the p53REs of WAF1/Cip1 and Bax genes (negative-dominant effect). Our data suggest that in the appropriate environment, differential transcription of critical p53 target genes by certain p53 mutant proteins may illustrate another mechanism implicated in tumor development.