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D Sidransky

Publications and source records attributed to D Sidransky.

At least 73 records · Page 4Linked to original sources
PubMed2000

Genetic alterations in urinary bladder carcinosarcoma: evidence of a common clonal origin.

The cellular origin of carcinosarcoma of the bladder is unknown. We addressed this issue by using microsatellite analysis for loss of heterozygosity (LOH) in both the carcinomatous and sarcomatous components of 6 bladder tumors. We tested 40 microsatellite markers from 19 human chromosomes and compared the genetic alterations between the two separately isolated components. The potential relevance of the E-cadherin pathway was also evaluated by immunohistochemistry. All 6 cases revealed identical LOH on chromosomal arms 9p, 9q, 8p, and 8q, corresponding to relatively early events in bladder carcinogenesis. Discordant losses between two alleles in the remaining chromosomes, associated with progression, were seen in all tumors with a trend toward a higher incidence in the more advanced tumors (N1M1 and N1Mx). E-cadherin was strongly expressed in the carcinomatous components (5 of 6), whereas most of sarcomatous elements displayed absence of the protein product (4 of 6). These results indicate that both the carcinomatous and sarcomatous components of carcinosarcoma are derived from a common stem cell. Downregulation of E-cadherin may define one of the pathways responsible for conversion of epithelial cells to the sarcomatous phenotype.

Aged↗
PubMed2000

Genetic progression and clonal relationship of recurrent premalignant head and neck lesions.

We constructed a preliminary genetic progression model for head and neck squamous cell carcinoma (HNSC) based on the frequency of genetic alterations in preneoplastic and neoplastic lesions from single biopsy specimens. To firmly establish the temporal order of established genetic events in HNSC, we sampled serial biopsies from five patients with recurrent premalignant lesions at a single anatomic site over a period of time (1 month to 144 months). These lesions were examined by microsatellite analysis of the minimal regions of loss on the 10 most frequently lost chromosomal arms in HNSC. Each set of serial biopsies from all five patients demonstrated LOH (loss of heterozygosity) of identical alleles at multiple loci with identical boundaries between areas of LOH and retention of heterozygosity, indicating a common clonal origin for each set. Three patients demonstrated genetic progression (new regions of LOH) over time correlating with histopathological progression, one patient demonstrated lack of genetic progression associated with unchanged histopathological morphology, and one patient demonstrated histopathological progression without detection of a corresponding genetic progression event. For one of these patients with a laryngeal tumor, at least four separate steps in progression to malignancy could be determined, accompanied by spatial expansion of an increasingly altered clonal population from the ipsilateral to the contralateral side, ultimately resulting in a malignancy. Microsatellite-based genetic analysis of recurrent premalignant lesions indicates that these lesions arise from a common clonal progenitor, followed by outgrowth of clonal populations associated with progressive genetic alterations and phenotypic progression to malignancy.

Biopsy↗
PubMed2000

Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma.

The human papillomavirus (HPV) has been implicated as an etiological factor in a subset of head and neck squamous cell carcinoma (HNSCC). Because circulating tumor DNA has previously been detected in the sera of patients with advanced HNSCC (stage III or IV), we hypothesized that HPV DNA might be present in the sera of HPV-positive HNSCC patients. Serum DNA extracts from 70 patients with HNSCC were screened for HPV using conventional PCR and a real-time quantitative assay. All samples subjected to conventional PCR were further tested by dot blot hybridization, and positives were confirmed by Southern blotting. Paired tumor DNA from archived tissues was then similarly screened for HPV genomic material (n = 51) or tested by in situ hybridization (n = 19). HPV-16 DNA was detected with L1 primers in 0 of 65 sera and in 15 of 70 (21%) tumors. Conventional PCR with E7 primers and Southern blot hybridization detected HPV-16 DNA in four (6%) sera. Using real-time quantitative PCR, six samples were found to contain various levels of circulating HPV DNA (mean, 12 copies/ml; range, <1-35.) All six serum-positive patients had corresponding tumors positive for E7. Four of these patients with HPV-positive tumors later developed distant metastases, suggesting that HPV DNA in serum may represent occult hematogenous spread of cancer cells in this subset of patients. Although a much larger prospective trial is required, the presence of HPV genomic material in serum DNA of HPV-positive HNSCC patients may serve as a useful marker of early metastatic disease.

Adult↗
PubMed1999

Diagnosis of renal cancer by molecular urinalysis.

BACKGROUND: Organ-confined renal malignancies can be cured in the majority of patients, whereas more extensive lesions have a poor prognosis. We sought to develop a noninvasive test for renal cancer detection based on a novel molecular approach. METHODS: Matched urine and serum DNA samples were obtained before surgery from 30 patients with clinically organ-confined solid renal masses (25 with malignant tumors and five with tumors of low malignant potential) and were subjected to microsatellite analysis. Serum samples and urine samples obtained from 16 individuals without clinical evidence of genitourinary malignancy served as controls. RESULTS: Nineteen (76%) of the 25 patients with malignant tumors were found to have one or more microsatellite DNA alterations in their urine specimen, and 15 (60%) were found to have alterations in their serum DNA by microsatellite analysis. In every case, the microsatellite changes in urine or serum were identical to those found in the primary tumor. Three of five patients with tumors of low malignant potential were found to have DNA alterations in their urine, but none displayed alterations in their serum. Moreover, microsatellite alterations were not identified in either the urine or the serum samples from normal control subjects and patients with hematuria due to nephrolithiasis (renal stones). CONCLUSION: These data suggest that microsatellite DNA analysis of urine specimens provides a potentially valuable tool for the early detection of resectable kidney cancer. Furthermore, microsatellite analysis of serum samples reveals evidence of circulating tumor-specific DNA in approximately half of these patients and may reflect the propensity of these tumors to spread to distant sites at an early stage.

Aged↗
PubMed1999

Inactivation of the INK4A/ARF locus frequently coexists with TP53 mutations in non-small cell lung cancer.

Inactivation of the P16 (INK4A)/retinoblastoma (RB) or TP53 biochemical pathway is frequent event in most human cancers. Recent evidence has shown that P14ARF binds to MDM2 leading to an increased availability of wild type TP53 protein. Functional studies also support a putative tumor suppressor gene function for p14ARF suggesting that p14ARF or p53 inactivation may be functionally equivalent in tumorigenesis. To study the relative contribution of each pathway in tumorigenesis, we analysed and compared alterations of the p16, p14ARF and p53 genes in 38 primary non-small cell lung cancers (NSCLCs) (19 adenocarcinomas and 19 squamous carcinoma). The p16 tumor suppressor gene was inactivated in 22 of 38 (58%) tumors. Twelve of these samples (31%) had homozygous deletions by microsatellite analysis; eight of them (21%) had p16 promoter hypermethylation detected by Methylation Specific PCR (MSP) and the remaining two (5%) harbored a point mutation in exon 2 by sequence analysis. The absence of P16 protein in every case was confirmed by immunohistochemistry. Fourteen of the 22 tumors with p16 inactivation also inactivated the p14ARF gene (12 with homozygous deletions extending into INK4a/ARF and two with exon 2 mutations). Mutations of p53 were found in 18 (47%) of the tumors and nine of them (50%) harbored p14ARF inactivation. Thus, an inverse correlation was not found between p14ARF and p53 genetic alterations (P=0.18; Fisher Exact Test). Our data confirm that the p16 gene is frequently inactivated in NSCLC. Assuming that 9p deletion occurs first, the common occurrence of p53 and p14ARF alterations suggests that p14ARF inactivation is not functionally equivalent to abrogation of the TP53 pathway by p53 mutation.

Carcinoma, Non-Small-Cell Lung↗
PubMed1999

Allelotype of posterior uveal melanoma: implications for a bifurcated tumor progression pathway.

To further elucidate the somatic genetic alterations leading to acquired choroidal and ciliochoroidal melanoma, we screened every autosomal arm and the X chromosome of 50 primary posterior melanomas (31 choroidal tumors and 19 ciliochoroidal tumors). A minimum of two microsatellite markers were used to achieve at least 90% informativity (excluding X). Twenty-eight of 47 informative tumors (59%) showed allelic loss of all informative markers on chromosome 3, consistent with monosomy 3 (M3). Allelic imbalance of 8q was observed in 60% of tumors. A total of 28% of tumors displayed allelic loss of 6p. We then compared these genetic alterations with the status of chromosome 3 and found a relative absence of 6p alteration in tumors with M3 (P = 0.0005). Additionally, all observed 8q imbalance was associated with either M3 or alteration of 6p, suggesting that 8q alterations occur later in tumor progression. The mutual exclusivity of M3 and 6p alterations suggests a bifurcated tumor progression model. In this model, M3 or 6p loss identify distinct pathways, both followed by 8q loss in tumor progression.

Chromosome Mapping↗
PubMed1999

Rapid p53 sequence analysis in primary lung cancer using an oligonucleotide probe array.

The p53 gene was sequenced in 100 primary human lung cancers by using direct dideoxynucleotide cycle sequencing and compared with sequence analysis by using the p53 GeneChip assay. Differences in sequence analysis between the two techniques were further evaluated to determine the accuracy and limitations of each method. p53 mutations were either detected by using both techniques or, if only detected by one technique, were confirmed by using mutation-specific oligonucleotide hybridization. Dideoxynucleotide sequencing of the conserved regions of the p53 gene (exons 5-9) detected 76% of the mutations within this region of the gene. The GeneChip p53 assay detected 81% of all (exons 2-11) mutations, including 80% of the mutations within the conserved regions of the gene. The GeneChip assay detected 46 of 52 missense mutations (88%), but 0 of 5 frameshift mutations. The specificity of direct sequencing and of the p53 GeneChip assay at detecting p53 mutations were 100% and 98%, respectively. The GeneChip p53 assay is a rapid and reasonably accurate approach for detecting p53 mutations; however, neither direct sequencing nor the p53 GeneChip are infallible at p53 mutation detection.

Humans↗
PubMed1999

Cyclin D1 amplification is independent of p16 inactivation in head and neck squamous cell carcinoma.

Progression through the G1 phase of the cell cycle is mediated by phosphorylation of the retinoblastoma protein (pRb) resulting in the release of essential transcription factors such as E2F-1. The phosphorylation of pRb is regulated positively by cyclin D1/CDK4 and negatively by CDK inhibitors, such as p16 (CDKN2/MTS-1/INK4A). The p16/cyclin D1/Rb pathway plays a critical role in tumorigenesis and many tumor types display a high frequency of inactivation of at least one component of this pathway. In order to determine the overall contribution of these three components to progression of head and neck squamous cell carcinoma (HNSCC), we examined p16 inactivation, cyclin D1 amplification, and pRb expression in 23 primary HNSCC tumors and five cell lines. p16 inactivation was detected in 19/23 (83%) primary tumors by detailed genetic analysis and was confirmed by immunohistochemistry (IHC). Absence of Rb protein expression indicative of pRb inactivation was identified in 2/23 (9%) tumors. In this set of tumors, there was a perfect inverse correlation between p16 and pRb inactivation. Using fluorescence in situ hybridization (FISH) cyclin D1 amplification was identified in 4/5 (80%) cell lines and 4/11 (36%) primary tumors. However, 2/4 cell lines and all four primary tumors with cyclin D1 amplification contained a concomitant alteration of p16. Therefore 21/ 23 (91%) of primary HNSCC contained at least one alteration in the p16/cyclin D1/Rb pathway. Although p16 and Rb alteration are apparently exclusive, cyclin D1 amplification occurs concomitantly with the loss of p16 suggesting an additional role for this amplification in HNSCC.

Carcinoma, Squamous Cell↗
PubMed1999

Chromosome 9p21 loss and p16 inactivation in primary sclerosing cholangitis-associated cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma is a frequent complication of primary sclerosing cholangitis and is a leading cause of mortality in patients with this disease. The tumor suppressor gene p16 is commonly inactivated in many neoplasms; however, the role of p16 in the pathogenesis of cholangiocarcinoma is unclear. Therefore, we examined the role of p16 inactivation in the pathogenesis of cholangiocarcinoma associated with primary sclerosing cholangitis. MATERIALS AND METHODS: Paraffin-embedded sections from 10 patients who developed cholangiocarcinoma in the setting of primary sclerosing cholangitis were examined. Chromosomal loss at 9p21 was determined using microsatellite analysis. Methylation of a CpG island in the promoter region of the p16 gene was determined using methylation-specific polymerase chain reaction. p16 inactivation was also determined using immunohistochemistry. RESULTS: Allelic loss at chromosome 9p21 was present in 9 of 10 tumors (90%). Methylation of the p16 promoter was present in 2 of the 8 tumors examined (25%). Four of seven tumors (57%) analyzed by immunohistochemistry demonstrated an absence of p16 nuclear staining. CONCLUSIONS: Loss of chromosome 9p21 and inactivation of the p16 tumor suppressor gene are common events in primary sclerosing cholangitis-associated cholangiocarcinoma and may play a role in the high incidence of cholangiocarcinoma in patients with primary sclerosing cholangitis.

Adult↗
PubMed1999

Mutation analysis of hBUB1 in aneuploid HNSCC and lung cancer cell lines.

Aneuploidy is frequently observed in many types of human cancer cells, suggesting that mutations of genes required for chromosomal stability may occur in human tumors. The BUB gene is a component of the mitotic checkpoint in budding yeast that delays anaphase in the presence of spindle damage thus increasing the probability of successful delivery of a euploid genome to each daughter cell. Recently, human homologues of the BUB gene were identified and mutant alleles of hBUB1 were detected in two colorectal tumor cell lines. Transfection of one mutant allele led to dominant disruption of the mitotic checkpoint control in a euploid cell, suggesting that aneuploidy in some tumors could be due to defects in the mitotic checkpoint. We analyzed the entire coding sequence of hBUB1 for mutation in 31 head and neck squamous cell carcinoma (HNSCC) and lung cancer cell lines, most with severe aneuploidy. We found expression of the hBUB1 gene in all cell lines and only a single nucleotide substitution in one cell line without a resultant change in amino acid sequence. Our study demonstrates that hBUB1 is rarely a target for genetic alterations in tumors of the respiratory tract.

Aneuploidy↗
PubMed1999

Unknown primary head and neck squamous cell carcinoma: molecular identification of the site of origin.

BACKGROUND: Unknown primary head and neck squamous cell carcinoma (HNSCC) presents as a cervical lymph node metastasis without identification of the primary tumor, despite thorough diagnostic work-up that includes physical examination, computed tomography, esophagoscopy, laryngoscopy, bronchoscopy, and multiple surveillance biopsies. We investigated whether the site of origin of the primary tumor could be localized in the upper aerodigestive tract mucosa by detection of genetic alterations identical to those found in metastatic lesions. METHODS: Microsatellite analysis was performed on metastatic tumors obtained from 18 patients with unknown primary HNSCC. Histologically benign surveillance biopsy specimens were also analyzed. Patients were followed up to 13 years with continuing surveillance for primary mucosal tumors. Most patients were treated with neck dissection followed by radiation therapy to the affected neck and ipsilateral Waldeyer's ring. RESULTS: In 10 (55%) of the 18 patients, at least one histopathologically benign mucosal biopsy specimen from defined anatomic sites (i.e., most likely sites for an occult primary tumor) demonstrated a pattern of genetic alterations identical to that present in cervical lymph node metastases. One patient harboring genetic alterations in the base of the tongue and two patients harboring genetic alterations in a tonsillar fossa subsequently developed HNSCC in the identical or adjacent mucosal region; all three of the primary head and neck mucosal tumors that eventually appeared between 1 and 13 years later in these patients had genetic changes identical to those in the benign mucosal biopsy specimens and in the metastatic lymph nodes. CONCLUSIONS: These data support the hypothesis that histopathologically benign mucosa of the upper aerodigestive tract may harbor foci of clonal, preneoplastic cells that are genetically related to metastatic HNSCC and that such mucosal sites are the sites of origin of unknown primary HNSCC. Microsatellite analysis may represent a clinically useful tool for determining such sites.

Carcinoma, Squamous Cell↗
PubMed1999

Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer.

BACKGROUND: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage (BAL) fluid. METHODS: Tumor-specific oncogene mutations, CpG-island methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. RESULTS: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. CONCLUSION: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC.

Alleles↗
PubMed1999

Detection of aberrant promoter hypermethylation of tumor suppressor genes in serum DNA from non-small cell lung cancer patients.

Recent evidence suggests that tumor cells may release DNA into the circulation, which is enriched in the serum and plasma, allowing detection of ras and p53 mutations and microsatellite alterations in the serum DNA of cancer patients. We examined whether aberrant DNA methylation might also be found in the serum of patients with non-small cell lung cancer. We tested 22 patients with non-small cell lung cancer using methylation-specific PCR, searching for promoter hypermethylation of the tumor suppressor gene p16, the putative metastasis suppressor gene death-associated protein kinase, the detoxification gene glutathione S-transferase P1, and the DNA repair gene O6-methylguanine-DNA-methyltransferase. Aberrant methylation of at least one of these genes was detected in 15 of 22 (68%) NSCLC tumors but not in any paired normal lung tissue. In these primary tumors with methylation, 11 of 15 (73%) samples also had abnormal methylated DNA in the matched serum samples. Moreover, none of the sera from patients with tumors not demonstrating methylation was positive. Abnormal promoter methylation in serum DNA was found in all tumor stages. Although these results need to be confirmed in larger studies and in other tumor types, detection of aberrant promoter hypermethylation of cancer-related genes in serum may be useful for cancer diagnosis or the detection of recurrence.

Carcinoma, Non-Small-Cell Lung↗
PubMed1999

PGP9.5 as a candidate tumor marker for non-small-cell lung cancer.

PGP9.5 is a neurospecific peptide that functions to remove ubiquitin from ubiquitinated proteins and prevents them from targeted degradation by proteasomes. Using the serial analysis of gene expression method (SAGE), we observed that the PGP9.5 transcript was highly expressed in primary lung cancers and lung cancer cell lines but was not detectable in the normal lung. Here we examined the expression of PGP9.5 protein in normal lung epithelium, lung tumor cell lines, and 98 resected primary non-small-cell lung carcinomas (NSCLCs). We found PGP9.5 reactivity in normal lung in a pattern compatible with K-cells of the diffuse neuroendocrine system. However, the PGP9.5 was present in both small-cell lung cancer (SCLC) and NSCLC cell lines (22/24) independent of neuronal differentiation. In primary NSCLCs, 54% (53/98) of the cases had positive PGP9.5 staining, and the expression of protein was strongly associated with pathological stage of the cancer. It was present in 44% (29/66) of stage I NSCLCs and in 75% (24/32) of stage II and IIIA NSCLCs (p = 0.0032). These results suggest that the increased expression of PGP9.5 is specifically associated with lung cancer development and may serve as a potential marker for the detection of lung cancer.

Basic Helix-Loop-Helix Proteins↗
PubMed1999

Immunohistochemical detection of p53 protein accumulation in head and neck cancer: correlation with p53 gene alterations.

The genetic and functional status of the p53 gene may be an important factor in guiding therapeutic strategies for patients with cancer. The purpose of this study was to determine whether p53 immunohistochemistry (IHC) accurately reflects the mutational status of the p53 gene and to determine whether p53 IHC independently predicts tumor responsiveness to radiation therapy for patients with HNSCC. p53 IHC was performed using the monoclonal antibody DO7 on tumors from 85 patients with HNSCC treated with primary or adjuvant radiation. The p53 status in all of these tumors was previously assessed by direct sequence analysis of exons 5 through 9: 49 tumors were p53 wild-type, and 36 harbored p53 gene mutations. All patients were well characterized with respect to locoregional recurrence, distant spread, and survival. Positive p53 staining was observed in 53 of the 85 cases (62%). Only 27 (51%) of these 53 IHC-positive cases harbored gene mutations in exons 5 through 9; 23 (72%) of the 32 IHC-negative cases did not harbor mutations. The overall correlation rate between IHC and sequencing was 59% (P < .04, chi2). Discordant results were observed for 35 (41%) cases, including 26 IHC-positive cases and 9 IHC-negative cases. In 7 of 9 cases, false-negative staining was due to a nonsense or splice-site mutation. p53 IHC was not predictive of overall survival (P = .37) or disease-free survival (P = .95). In a sizable number of cases, p53 IHC does not reflect the mutational status of the p53 gene. Specific types of alterations (eg, truncating mutations) and other factors may contribute to this poor correlation. Moreover, p53 IHC does not appear to be an independent predictor of tumor responsiveness to radiation in patients with HNSCC.

Carcinoma, Squamous Cell↗
PubMed1999

Comparative microsatellite analysis in discerning origin of disseminated tumor: the case of a patient with malignant ascites and a history of multiple tumors.

The origin of metastatic carcinoma is now always easily resolved on the basis of conventional dinical and pathological parameters, particularly in patients with more than 1 primary tumor. When 1 of the tumors is a renal cell carcinoma, the clinical picture is further confounded by the tendency of these tumors to be locally silent, to metastasize to unusual sites, and to disseminate long after removal of the primary tumor. We compared tumors for loss (ie, deletion) of loci on chromosomal arms 3p, 5q, 11q, and 18q in a patient with a malignant ascites fluid, a remote history of renal and colonic neoplasms, and a strong clinical suspicion of disseminated gastrointestinal adenocarcinoma. DNA from microdissected tumors and normal tissues was subjected to polymerase chain reaction-based microsatellite analysis. Even though the clinical picture suggested a gastrointestinal origin, comparison of genetic alterations clearly showed that the malignant ascites represented recurrence of the renal cell carcinoma. The malignant ascites and the primary renal cell carcinoma showed identical patterns of allelic loss at all loci tested. In contrast, the malignant ascites and colonic adenoma showed discordant patterns of allelic loss. Comparative microsatellite analysis provides a rapid genetic approach for discerning the origin of metastatic tumor spread. This may be a useful diagnostic adjunct when tumor origin is not clear on clinical or morphological grounds. In some instances, it may even provide a reasonable alternative to an extensive and costly conventional work-up.

Adenoma↗