Discrimination of double primary lung cancer from intrapulmonary metastasis by p53 gene mutation

When multiple synchronous lung tumours are identified, discrimination of multicentric lung cancers from intrapulmonary metastases by clinical findings is often difficult. We used genetic alterations in p53 gene as a discrimination marker of double primary lung cancers from single lung cancer with intrapulmonary metastasis. Twenty of 861 patients with primary lung cancer who underwent lung resection were selected as subjects because they showed synchronous double solid tumours of the same histological type in the unilateral lung without distant metastases. In addition, they had been diagnosed as lung carcinoma with intrapulmonary metastasis by clinical and histological findings. DNAs were extracted from paraffin-embedded tissue of paired tumours from these 20 patients. Exons 5-9 of the p53 gene were examined for genetic alterations in the tumours by polymerase chain reaction, single-strand conformation polymorphism analysis and subsequent DNA sequencing analysis. Three different patterns in the distribution of p53 mutations in double lung tumours were observed: [A] mutation in only one of the tumours (four cases), [B] different mutations in the tumours (two cases), and [C] same mutation in both tumours (one case). The cases of [A] or [B] patterns could be classified as double primary lung cancers, while the case of the [C] pattern was suggested to be lung cancer with intrapulmonary metastasis. These results suggested that the multicentric cancers were more frequent than the intrapulmonary metastatic cancers in double cancer cases.     

Clonality and prognostic implications of p53 and epidermal growth factor receptor somatic aberrations in multiple primary lung cancers.

PURPOSE: For treatment decision and prognostic applications, we evaluated p53/epidermal growth factor receptor (EGFR) somatic aberrations in multiple primary lung cancers to differentiate multifocal tumors from intrapulmonary metastasis. EXPERIMENTAL DESIGN: Fifty-eight multiple primary lung cancers of 1,037 patients in a 10-year period were identified to investigate somatic mutations and altered expression of p53 and EGFR for clonality assessment. Genomic DNA was extracted from microdissected cells of paraffin-embedded multiple primary lung cancer tissues. Overexpression and somatic mutations in exons of p53 (exons 5-8) and tyrosine kinase domain of EGFR (exons 18-22) were examined by immunohistochemical staining and DNA sequencing, respectively. RESULTS: High frequency of somatic mutations in p53 (33 of 58, 56.9%) and/or EGFR (44 of 58, 75.9%) resulted in high discrimination rate of tumor clonality (50 of 58, 86.2%) of multiple primary lung cancers. Twenty-two cases (37.9%) were assessed as having the same clonality and 28 cases (48.3%) were determined as having different clonality, which further supported the carcinogenic theory of field cancerization. Notably, the occurrence of lymph node metastasis was more commonly observed in tumors with the same clonality (P = 0.045) and was associated with poor patient 5-year survival rate (P = 0.001). However, no correlation was found between tumor clonality and patient survival (P = 0.630). The EGFR somatic aberrations in 58 multiple primary lung cancers, including vascular invasion associated with EGFR overexpression (P = 0.012) and mutation (P = 0.025), further suggested the potential benefits of target therapy of inoperable multiple primary lung cancers. CONCLUSIONS: Our results suggest that analysis of somatic alterations in p53 and EGFR can significantly improve the clonality assessment and impact management of multiple primary lung cancer patients.     

Allelic loss on chromosome 17p and p53 mutations in human endometrial carcinoma of the uterus

To understand the involvement of allelic losses and inactivation of tumor suppressor genes for the development of endometrial carcinoma of the uterus (EC), 24 cases of EC were examined for loss of heterozygosity (LOH) using a total of 57 polymorphic DNA markers covering all 23 pairs of chromosomes. LOH was observed at 27 loci on 10 different chromosomes, i.e., chromosomes 1, 3, 6, 11, 13, 15, 17, 18, 20, and 21, but was not detected at loci on chromosomes 4, 5, 7, 9, 10, 12, 14, 16, and X. It was observed only in seven of 24 cases, and the other 19 cases did not show LOH at any loci examined, including five cases of tumors with a high proportion of adenomatous hyperplasia. Among seven tumors with LOH at one or more loci, five tumors showed LOH at loci on the short arm of chromosome 17. Furthermore, mutations of the p53 gene, which is located on the short arm of chromosome 17, were detected in three of these 24 tumors by a polymerase chain reaction-single strand conformation polymorphism analysis and subsequent DNA sequencing. In two of these three tumors, p53 mutations were accompanied by the loss of wild-type p53 alleles. These results suggest that inactivation of the p53 gene is involved in the development of EC as in the case of several other types of human cancers.     

p53 mutations in human lung tumors.

Mutation of one p53 allele and loss of the normal p53 allele [loss of heterozygosity (LOH)] occur in many tumors including lung cancers. These alterations apparently contribute to development of cancer by interfering with the tumor suppressor activity of p53. We directly sequenced amplified DNA in the mutational hot spots (exons 4-8) of p53 in DNA samples from 40 lung cancers. Most (31 of 40) samples were preselected for LOH in the region of p53. We detected 23 p53 mutations within these exons in 22 lung cancers; no p53 mutations were found in normal tissue of the patients. One-half of the mutations were G to T transversions on the nontranscribed strand, consistent with mutagenesis by tobacco smoke. Mutations of C to A on the nontranscribed strand, which would result from G to T mutations on the transcribed strand, were detected only in one sample. Three of 23 mutations were nonsense mutations; to date, nonsense mutations of p53 have not been reported in lung cancer. Mutation of this p53-coding region was detected in 20 of 27 small cell lung cancer samples, representing a 70% occurrence. Mutation of the p53 gene is apparently very frequent in small cell lung cancers. When LOH in the p53 region could be determined, complete concordance occurred between a sample having both a p53 mutation and LOH in the region of p53 (18 of 18 samples). Twelve samples of lung cancer had LOH in the region of p53, but the samples had no detectable p53 mutations, suggesting either alterations outside the known mutational hot spots of p53 or alterations of another unidentified tumor suppressor gene in the region of p53.     

Discrimination of multiple primary lung cancers from intrapulmonary metastasis based on the expression of four cancer‐related proteins

BACKGROUND:

Because distinguishing between multiple primary lung cancers and intrapulmonary metastasis is often difficult when the tumor histology is same, the feasibility of analyzing differential protein expression profiles to distinguish multiple primary lung cancers from intrapulmonary metastasis was evaluated.

METHODS:

This study enrolled 50 patients, with multiple primary lung cancers demonstrating the same histology, who underwent surgery between April 1994 and March 2006 and 20 patients who were diagnosed to have intrapulmonary metastasis during the same period. Thirty patients with lymph‐node metastasis were selected for comparison purposes. The sum value of the differences in the expression ratio of 4 proteins (p53, p16, p27, and c‐erbB2) was evaluated in immunohistochemically stained specimens among multiple primary lung cancers and intrapulmonary metastasis.

RESULTS:

None of the 30 patients with lymph‐node metastasis showed a sum value of the differences between primary tumor and lymph‐node metastasis in the 4 protein expression ratios >90. Therefore, when the difference between 2 tumors exceeded 90, the 2 tumors were considered to be different from each other, ie, multiple primary lung cancers. Forty‐one of 50 (82%) patients who were clinically diagnosed to have multiple primary lung cancers showed a sum value of the differences in their protein expression ratios >90, 4 of 20 (20%) patients who were clinically diagnosed to have intrapulmonary metastasis showed a sum value >90. Among the patients who were clinically diagnosed to have multiple primary lung cancers and intrapulmonary metastasis, the 5‐year survival of 70 patients who had a sum value of the differences in their 4 protein expression ratios, either >90 (newly classified multiple primary lung cancers) and ≤90 (newly classified intrapulmonary metastasis), were 81.1% and 40.2%, respectively (P = .002).

CONCLUSIONS:

The profile of protein expression in cancer‐related genes is, thus, considered to be a useful tool for distinguishing multiple primary lung cancers from intrapulmonary metastasis and for determining the appropriate biological staging of lung cancer. Cancer 2009. © 2009 American Cancer Society.     

Allelotype analysis of early colorectal cancers with lymph node metastasis

Several studies have indicated that frequent allelic losses in some specific chromosomal regions occur during colorectal cancer (CRC) progression. To clarify the correlation between such allelic losses and metastatic potential, the allelotype of lymph node-positive early CRCs, which are small but extremely malignant cancers consisting of metastatically competent cells, were investigated. Nineteen paraffin-embedded specimens of early CRC (pT1 tumors according to TNM classification) with positive lymph nodes were collected. The tumor tissues were examined for loss of heterozygosity (LOH), using microsatellite markers on chromosomes 1p34–36, 8p21–22, 14q32, 18q21 and 22q12–13. The relationship between p53 protein expression and the metastatic status was also investigated by immunohistochemical staining. A group of 20 early CRCs with negative lymph nodes having a similar distribution of macroscopic appearance were used as controls. Among the 19 node-positive tumors, LOH at 8p21–22 and 18q21 was detected in 11 cases (57.9%) and 17 cases (89.4%), respectively. Allelic losses within these 2 regions in node-positive tumors were significantly more frequent than that in node-negative ones (p< 0.01). No significant correlation was found between LOH at 1p34–36, 14q32 or 22q12–13 and lymph node metastasis. p53 protein expression was not significantly associated with lymph node metastasis. Our results suggest that putative tumor suppressor genes, which may be involved in the metastatic process of CRC, are located on chromosomes 8p21–22 and 18q21. Allelic losses in these regions are possible risk factors for lymph node metastasis of early CRC. Int. J. Cancer (Pred. Oncol.) 79:418–423, 1998. © 1998 Wiley-Liss, Inc.     

Correlation of allelic losses and clinicopathological factors in primary breast cancers

Human breast cancers frequently show allelic loss or loss of heterozygosity (LOH) at specific chromosomal regions. To understand the possible role of these genetic alterations in tumor development and progression, we examined LOH at loci on chromosomal arms lp, 3p, 11p, 13q, 16q, 18q, and 22q in 140 to 246 cases of primary breast cancers and compared it with lymph node metastasis, histological type, tumor stage, estrogen receptor (ER) and progesterone receptor (PgR) status. LOH at 1_p22-31 correlated with lymph node metastasis and a tumor size of greater than 2 cm. LOH at 13ql2-14 and 18q21 were most frequently observed in tumors of the solid-tubular type. LOH at 1p34-36 was more frequent in tumors of the scirrhous and solidtubular types than in other less aggressive histological types. Furthermore, a significant association was observed between LOH at 3pl4-21, 11p11-15 and 13ql2-14 and the absence of progesterone receptors. These results suggest that some clinical characteristics of breast cancers are determined by loss of tumor suppressor genes present at specific chromosomal regions.     

Tobacco smoke-induced DNA damage and an early age of smoking initiation induce chromosome loss at 3p21 in lung cancer

The short arm of chromosome 3 is thought to harbor a novel oncogenic locus that is important in the genesis of lung cancer. The region at 3p21 is believed to contain a distinct locus that is sensitive to loss from the action of tobacco smoke carcinogens and has been reported to be specifically targeted for deletion in lung cancer. To investigate whether 3p21 alteration in lung cancer is associated with carcinogen exposure, PCR-based analysis was performed to detect loss of heterozygosity (LOH) on chromosome 3 at 3p21 in non-small cell lung carcinoma (NSCLC). We also measured instability at the BAT-26 locus, because the mismatch DNA repair gene, hMLH1, is found at 3p21. LOH at 3p21 was analyzed for association with the clinical features of NSCLC, p53 mutation status, polynuclear aromatic hydrocarbon-DNA adduct levels (measured using 32P-postlabeling) and carcinogen exposure information including cigarette smoking and asbestos exposure. Of 219 lung cancers, 150 cases (68.5%) were informative at the D3S1478 locus, and 44.2% of squamous cell carcinoma cases and 30.2% of adenocarcinoma cases showed 3p21 LOH. None of the cancers showed BAT-26 instability. The prevalence of 3p21 LOH was higher in both current and former smokers compared with never smokers and was higher in p53 mutated cases. Among squamous cell carcinoma cases, there was a strong association of increased 3p21 LOH with increasing polynuclear aromatic hydrocarbon-DNA adducts levels (P = 0.03), as well as an increased prevalence LOH with earlier age of smoking initiation (P = 0.02). Our results confirm that 3p21 LOH is strongly associated with measures of biologically effective dose of exposure to tobacco carcinogens. Our results also suggest that alterations of hMLH1 are not related to any of the reported associations, because there was no evidence of microsatellite instability. Finally, LOH in 3p21 may be an early molecular event in NSCLC, because it is significantly associated with a tendency to start smoking at a young age.     

Molecular evidence supporting field effect in urothelial carcinogenesis.

PURPOSE: Human urothelial carcinoma is thought to arise from a field change that affects the entire urothelium. Multifocality of urothelial carcinoma is a common finding at endoscopy and surgery. Whether these coexisting tumors arise independently or are derived from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status in the cells from each coexisting tumor may further our understanding of urothelial carcinogenesis. EXPERIMENTAL DESIGN: We examined 58 tumors from 21 patients who underwent surgical excision for urothelial carcinoma. All patients had multiple separate foci of urothelial carcinoma (two to four) within the urinary tract. Genomic DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-capture microdissection. Loss of heterozygosity (LOH) assays for three microsatellite polymorphic markers on chromosome 9p21 (IFNA and D9S171), regions of putative tumor suppressor gene p16, and on chromosome 17p13 (TP53), the p53 tumor suppressor gene locus, were done. X-chromosome inactivation analysis was done on the urothelial tumors from 11 female patients. RESULTS: Seventeen of 21 (81%) cases showed allelic loss in one or more of the urothelial tumors in at least one of the three polymorphic markers analyzed. Concordant allelic loss patterns between each coexisting urothelial tumor were seen in only 3 of 21 (14%) cases. A concordant pattern of nonrandom X-chromosome inactivation in the multiple coexisting urothelial tumors was seen in only 3 of 11 female patients; of these 3 cases, only one displayed an identical allelic loss pattern in all of the tumors on LOH analysis. CONCLUSION: LOH and X-chromosome inactivation assays show that the coexisting tumors in many cases of multifocal urothelial carcinoma have a unique clonal origin and arise from independently transformed progenitor urothelial cells, supporting the "field effect" theory for urothelial carcinogenesis.     

Multiple high-grade bronchial dysplasia and squamous cell carcinoma: concordant and discordant mutations.

Loss of heterozygosity (LOH) involving chromosomes 3p, 5q, 9p, or 17p and aberrant expression or mutation of p53 are reported previously in selected bronchial dysplasias and squamous cell cancers (SCCs). Yet, comprehensive analyses of LOH patterns at these chromosomal sites and of p53 alterations are not reported for histologically normal bronchial epithelium, high-grade bronchial dysplasia, and SCC present in the same pulmonary resections. Whether concordant or discordant genetic changes are detected in these bronchial tissues, especially when multiple high-grade dysplastic bronchial lesions are present, was studied. Genomic DNA was microdissected from eight pulmonary SCCs and high-grade dysplastic lesions that were associated with SCC. In four cases, two independent high-grade dysplastic bronchial lesions were identified. When available, histologically normal bronchial epithelium was microdissected. Germ-line genomic DNA was isolated from normal lymph nodes. LOH was assessed for 15 microsatellite markers on chromosomes 3p, 5q, 9p, or 17p, sites frequently deleted in lung cancers. Immunohistochemical p53 expression was studied and correlated with p53 DNA sequence analyses. Progressive LOH for these markers was found when SCCs were compared with high-grade dysplasia and histologically normal bronchial epithelium present in the same resections. Histologically normal bronchial specimens had LOH in up to 27% of informative markers. High-grade dysplastic lesions exhibited LOH for 18-45% and SCC had LOH for 18-73% of the markers. Common regions of LOH were found in some dysplasias compared with SCCs. In other dysplasias, discordance was found relative to SCCs, especially for p53 mutations. In cases with a single or second high-grade dysplasia associated with SCC, heterogeneity in LOH markers was detected. These concordant and discordant changes were consistent with convergent and divergent clonal selection pathways in pulmonary squamous cell carcinogenesis. Some histologically normal bronchial epithelial tissues had genetic changes more similar to those in the SCCs than in dysplastic lesions. DNA loss or mutations accumulate in SCC, but discordant genetic changes can exist in the same carcinogen-exposed bronchial tissues. These findings have implications for lung cancer prevention trials.     

Molecular evidence for independent origin of multifocal neuroendocrine tumors of the enteropancreatic axis

Neuroendocrine tumors of the enteropancreatic axis are often multifocal. We have investigated whether multifocal intestinal carcinoid tumors and multifocal pancreatic endocrine tumors arise independently or whether they originate from a single clone with subsequent intramural or intrapancreatic spread. Twenty-four cases, including 16 multifocal intestinal carcinoid tumors and eight multifocal pancreatic endocrine tumors, were studied. Genomic DNA samples were prepared from 72 distinct tumor nodules using laser capture microdissection. Loss of heterozygosity (LOH) assays were done using markers for putative tumor suppressor genes located on chromosomes 9p21 (p16), 11q13 (MEN1), 11q23 (SDHD), 16q21, 18q21, and 18q22-23. In addition, X chromosome inactivation analysis was done on the tumors from eight female patients. Twenty-two of 24 (92%) cases showed allelic loss in at least one tumor focus, including 15 of 16 (94%) cases of multifocal carcinoid tumors and 7 of 8 (88%) cases of multifocal pancreatic endocrine tumors. Eleven of 24 (46%) cases exhibited a different LOH pattern for each tumor. Additionally, 9 of 24 (38%) cases showed different LOH patterns among some of the coexisting tumors, whereas other coexisting tumors displayed the same allelic loss pattern. Two of 24 (8%) cases showed the same LOH pattern in every individual tumor. X chromosome inactivation analysis showed a discordant pattern of nonrandom X chromosome inactivation in two of six informative cases and concordant pattern of nonrandom X chromosome inactivation in the four remaining informative cases. Our data suggest that some multifocal neuroendocrine tumors of the enteropancreatic axis arise independently, whereas others originate as a single clone with subsequent local and discontinuous metastasis.     

Loss of heterozygosity of chromosome 3p21 is associated with mutant TP53 and better patient survival in non-small-cell lung cancer

Allelic loss of chromosome region 3p21.3 occurs early and frequently in non-small-cell lung cancer, and numerous tumor suppressor genes at this locus may be targets of inactivation. Using an incident case series study of non-small-cell lung cancer, we sought to determine the prevalence of loss of heterozygosity (LOH) in the 3p21.3 region and to examine the associations between this alteration and patient outcome, exposure to tobacco smoke, occupational asbestos exposure, and additional molecular alterations in these tumors. We examined LOH at 7 microsatellite markers in the chromosome 3p21.3 region, and LOH was present in at least one of the loci examined in 60% (156 of 258) of the tumors, with the prevalence of LOH at individual loci ranging from 15 to 56%. Occupational asbestos exposure and TP53 mutation were significantly associated with more extensive 3p21 LOH. In squamous cell carcinomas, measures of cumulative smoking dose were significantly lower in patients with LOH at 3p21, particularly in TP53 mutant tumors. Examining patient outcome, we found that in squamous cell carcinomas, having any LOH in this region was associated with a better overall survival (log-rank test, P < 0.04). Together, these results indicate that allelic loss at 3p21 can affect patient outcome, and that this loss may initially be related to carcinogen exposure, but that extension of this loss is related to TP53 mutation status and occupational asbestos exposure.     

Correlation of abnormal RB, p16ink4a, and p53 expression with 3p loss of heterozygosity, other genetic abnormalities, and clinical features in 103 primary non-small cell lung cancers.

This study was performed to determine the frequency of inactivation and clinical correlates in non-small cell lung cancer (NSCLC) of three known tumor suppressor genes [TSGs; RB, MTS1/CDKN2 (p16), and p53] and various regions of 3p loss of heterozygosity (LOH) as other major potential TSG sites. Paraffin sections from 103 resected NSCLCs were analyzed for expression of pRB, p16, and p53 by immunohistochemistry, whereas DNA from tumor and normal tissue were tested for LOH at 3p25-26, 3p21, and 3p14. Previously published LOH data for 5q, 11p, 17q, and 18q were also available. Loss of pRB or p16 expression and overexpression of p53 were considered abnormal. The immunohistochemical and LOH data were correlated with a variety of clinical parameters including stage, age, sex, smoking history, and survival. With respect to pRB, p16, and p53, the tumors could be grouped into four categories: normal for all three proteins (21%); abnormal for pRB or p16 and normal for p53 (30%); normal for pRB and p16 and abnormal for p53 (20%); and abnormal in both pathways (28%). Aberrant expression of pRB, p16, p53, and 3p LOH, either individually or in combination, was not associated with survival differences or any other clinical parameters, with the exception that pRB/pl6 abnormalities were more common in older patients (P = 0.0005). pRB and p16 expression showed a strong inverse correlation (P = 0.002), whereas there was no correlation between expression of pRB, p16, and p53. Abnormal expression of any of the three genes inversely correlated with K-ras codon 12 mutations (P = 0.004), but not with 3p LOH or LOH at other TSG loci. We conclude that resectable NSCLCs show distinct patterns of TSG inactivation, but that no clear clinical correlates exist either alone or in combination for pRB, p16, p53, and 3p abnormalities.     

Clonal divergence and genetic heterogeneity in clear cell renal cell carcinomas with sarcomatoid transformation

BACKGROUND: Approximately 5% of clear cell renal cell carcinomas contain components with sarcomatoid differentiation. It has been suggested that the sarcomatoid elements arise from the clear cell tumors as a consequence of clonal expansions of neoplastic cells with progressively more genetic alterations. Analysis of the pattern of allelic loss and X-chromosome inactivation in both the clear cell and sarcomatoid components of the same tumor allows assessment of the genetic relationship of these tumor elements. METHODS: The authors of the current study examined the pattern of allelic loss in clear cell and sarcomatoid components of renal cell carcinomas from 22 patients who had tumors with both components. DNA samples were prepared from formalin-fixed, paraffin-embedded renal tissue sections using laser-capture microdissection. Five microsatellite polymorphic markers for putative tumor suppressor genes on 5 different chromosomes were analyzed. These included D3S1300 (3p14), D7S522 (7q31), D8S261 (8p21), D9S171 (9p21), and TP53 (17p13). In addition, X-chromosome inactivation analysis was performed in 14 tumors from female patients. RESULTS: The clear cell components showed loss of heterozygosity (LOH) at the D3S1300, D7S522, D8S261, D9S171, and TP53 loci in 18% (4/22), 18% (4/22), 50% (10/20), 15% (3/20), and 20% (4/20) of informative cases, respectively. LOH in the sarcomatoid components was seen at the D3S1300, D7S522, D8S261, D9S171, and TP53 loci in 18% (4/22), 41% (9/22), 70% (14/20), 35% (7/20), and 20% (4/20) of informative cases, respectively. Six cases demonstrated an LOH pattern in the clear cell component that was not seen in the sarcomatoid component. Different patterns of allelic loss were seen in the clear cell and sarcomatoid components in 15 cases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation in both clear cell and sarcomatoid components in 13 of the 14 cases studied. One case showed a random pattern of X-chromosome inactivation. CONCLUSION: X-chromosome inactivation analysis data suggest that both clear cell and sarcomatoid components of renal cell carcinomas are derived from the same progenitor cell. Different patterns of allelic loss in multiple chromosomal regions were observed in clear cell and sarcomatoid components from the same patient. This genetic heterogeneity indicates genetic divergence during the clonal evolution of renal cell carcinoma.     

Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines.

The tumor-suppressor genes p14(ARF), p16(INK4a) and Tp53 are commonly inactivated in many tumors. We investigated their role in the pathogenesis of 9 bile tract cancer cell lines and 21 primary sporadic extrahepatic bile duct carcinomas. p53 and p16 protein expression was examined by Western blot analysis and immunohistochemistry. Mutation screening of p53 was done by SSCP and direct sequencing. Inactivating mechanisms of p14 and p16 were addressed by screening for mutations, homozygous deletions, chromosomal loss of 9p21 (loss of heterozygosity [LOH] analysis) and promoter hypermethylation of the p14/p16 genes. p53 overexpression could be detected in 7 of 9 cell lines and 7 of 21 primary tumors, but mutations were found in 3 cell lines only. p16 expression was absent in all cell lines, due to homozygous deletion of the gene in 8 of 9 cell lines and hypermethylation of the p16 promoter in one cell line (CC-LP-1). p14 exon 1beta was homozygously deleted in 6 of 9 cell lines, while retained in CC-LP-1 and 2 additional lines. No p14 promoter hypermethylation could be detected. p16 expression was lost in 11 of 21 primary tumors. p16 promoter hypermethylation was present in 9 of 21 primary tumors, all with lost p16 expression. Allelic loss at 9p21 was detected in 13 of 21 primary tumors, 10 of 11 with lost p16 expression and 8 of 9 with methylated p16 promoter. No p14 promoter hypermethylation or p14/p16 mutations could be detected. Neither Tp53 nor p16 alterations showed obvious association with histopathologic or clinical characteristics. In conclusion, inactivation of the p16 gene is a frequent event in primary sporadic extrahepatic bile duct cancers, 9p21 LOH and promoter hypermethylation being the principal inactivating mechanisms. Therefore, p16, but not p14, seems to be the primary target of inactivation at the INK4a locus in bile duct cancers. Other mechanisms than Tp53 mutations seems to be predominantly responsible for stabilization of nuclear p53 protein in bile duct cancers.     

The origins of multiple squamous cell carcinomas in the aerodigestive tract

BACKGROUND: Chemoprevention and cessation of smoking and alcohol may prevent development of multiple tumors (MTs) in the aerodigestive tract if new MTs arise independently, but they are of no benefit if MTs are due to migration of an already transformed clone of tumor cells. This issue was addressed in this study by investigation of the clonality among MTs. METHODS: Mutation analysis of the entire coding region of p53 and loss of heterozygosity (LOH) pattern analysis of microsatellite markers on chromosome arms 3p, 9p, and 17p are promising for the investigation of clonality. In the first part of this study, the authors established the variability and stability of these clonal markers by comparing primary head and neck squamous cell carcinomas (HNSCCs) with their metastases. In the second part of this study, the authors evaluated nine patients with multiple HNSCCs using these markers. In the final part, the authors illustrate the use of these clonal markers in 11 patients for whom there was diagnostic uncertainty as to whether their second squamous cell carcinoma was either a new primary tumor, a metastasis, or a recurrence. RESULTS: Both p53 gene mutations and LOH patterns were stable during tumor progression. Furthermore, the variability of p53 gene mutations was high. More than 90% of the tumors contained a p53 mutation. A particular mutation never occurred more than twice in a total of 69 primary HNSCCs. Five of 69 cases presented a common mutation. In contrast, LOH patterns showed less variability; they were identical in 5 of 16 cases. The metachronous HNSCCs from nine patients all showed different p53 mutations, and in the three cases that were subjected to LOH analysis different patterns were observed. All 11 patients for whom there was diagnostic uncertainty about the origin of their second squamous cell carcinoma could be categorized as having multiple primary tumors, disseminated disease, or recurrent disease. CONCLUSIONS: Metachronous HNSCCs at different locations are not clonally related and thus have not developed from the migration of tumor cells.     

LOSS OF HETEROZYGOSITY AT 17p13 IN GASTRIC CANCER AND COLORECTAL CANCER

Bothgastriccancerandcolorectalcancerarecommonhumantumors.Althoughlittleisknownregardingthemolecularpathogenesisoftheneonlasms,itisnowgenerallyacceptedthattheactivationofoncogenesandinactivationoftumorsuppressorgenesmightbecriticalforthetumorcarcinogenesisandprogression.Inthepresentstudy,weemployedtheSouthernhybridizationbyprobesphP53BandPYNZ22,whichwereobtainedfromtheAmericanTypeCultureCollection,todetectthelossofheteroZygosity(LOH)at17pl3ingastriccancerandcolorectalcancer.Wefurtheranaly…     

Genetic changes in contralateral bronchioloalveolar carcinomas of the lung

OBJECTIVE: The pattern of metastases and recurrence of bronchioloalveolar carcinoma (BAC) differs from adenocarcinoma of the lung, occurring more frequently within the lung without extrapulmonary involvement. Analyses of genetic differences of contralateral BACs may help to explain these clinical differences. METHODS: We compared paired tumors from 5 patients with contralateral metachronous BACs for loss of heterozygosity (LOH) on 6 chromosomal arms (2q, 3p, 5q, 9p, 13q and 17p) and mutational analysis of p53 and K-ras. RESULTS: Two patients, patients 1 and 2, had discordant patterns of LOH on 2 and 3 of the chromosome arms, respectively. In addition, patient 2 had a detectable K-ras mutation in his initial tumor but not in his second. These results suggest that in patients 1 and 2, the contralateral tumors were clonally unrelated. Patient 3 had no mutations in the K-ras or p53 gene and no LOH on any of the 5 informative chromosome arms. Patient 4 had LOH of 9p and mutated K-ras in both the first and the second tumor, with a mutation in the p53 gene in the first but not in the second tumor. Patient 5 had LOH of 17p and the same p53 mutations in both the first and the second tumor, with a mutation of K-ras in the first tumor but not in the second. CONCLUSIONS: The preponderance of evidence suggests that in patients 3, 4 and 5, the paired tumors were clonally related. The different patterns of LOH and mutations in clinically similar contralateral metachronous BACs provide evidence of genetic heterogeneity in the tumors of this patient group.     

Loss of heterozygosity on the short arm of chromosome 3 in renal cancer

3% of human cancers are renal cell carcinomas (RCC). The most common chromosome abnormality found in this tumor is loss of heterozygosity (LOH) on the short arm of chromosome 3, which suggests that there must be one or more tumor suppressor genes between 3p14 and 3p21 near the VHL gene which play a relevant role in renal cancer development. DNA from normal and tumor tissue from 40 patients at various stages of RCC was analyzed for LOH at three microsatellites mapped to 3p (3p14.1-14.3; 3p21.2-21.3 and 3p25) by polymerase chain reaction). 42.5% of the tumors studied showed LOH on at least one locus. 30% showed LOH on only one locus; 5% on two loci and 7.5% on the three loci tested. LOH occurred only on nonpapillary tumors (p = 0.03). Interestingly, all the tumors with LOH on 3p21 were >/=25 mm (p = 0.04; relative risk 1.76, confidence interval: 1.3-2.3).     

Genetic changes in the spectrum of neuroendocrine lung tumors

BACKGROUND: Recent classifications identify four categories of neuroendocrine (NE) tumors of the lung: low grade typical carcinoid (TC), intermediate grade atypical carcinoid (AC), and high grade large cell neuroendocrine carcinoma (LC-NEC) and small cell lung carcinoma (SCLC). METHODS: The authors studied the molecular changes present in 59 archival NE tumors (10TCs, 11 ACs, 18 LNECs, and 20 SCLCs). Utilizing microdissection and polymerase chain reaction-based assays, the authors examined loss of heterozygosity (LOH) at ten chromosomal regions frequently deleted in lung tumors (3p, 5q, 11q, 13q, and 17p) and for mutations at the p53 and ras genes. RESULTS: With the exception of ras gene mutations, the majority of these changes frequently were present in carcinomas and were present at lower frequencies in carcinoids. LOH at one or more 3p regions was the most frequent change found in the carcinoids. A relatively high incidence of LOH at the MEN1 gene was common in all NE lung tumors. The incidence of LOH and p53 gene abnormalities progressively increased with increasing severity of tumor type. The patterns of p53 gene mutations were different between AC and high grade NE tumors. LOH at 5q21 was correlated with poor survival in the carcinoid group. CONCLUSIONS: Although NE lung tumors have varied etiologies, the results of the current study support the clinicopathologic concept that they represent a spectrum ranging from low grade TC to the highly malignant NE carcinomas.