Biomedical subjects
Publications and source records attributed to David Sidransky.
Alterations in gene sequences, expression levels and protein structure or function have been associated with every type of cancer. These 'molecular markers' can be useful in detecting cancer, determining prognosis and monitoring disease progression or therapeutic response. But what is the best way to identify molecular markers and can they be easily incorporated into the clinical setting?
OBJECTIVES: Routine histologic examination of resected lymph nodes in patients with stage I non-small cell lung cancer may underestimate the incidence of advanced disease. The presence of occult lymph node metastases may predict a higher risk of recurrence after intended curative resection. The purpose of this study was to determine the prognostic significance of TP53 and K-ras mutations in histologically determined negative lymph nodes from patients with stage I non-small cell lung cancer who underwent intended curative surgical resection. METHODS: Between July 1995 and March 1998, clinical data and tissue samples of primary tumors and lymph nodes were collected in a prospective fashion from 102 patients undergoing resection for non-small cell lung cancer (stage I, n = 55; stage II, n = 32; stage IIIA, n = 15). TP53 and K-ras mutations were detected by direct sequencing. If molecular alterations were found in the primary tumor, the corresponding lymph nodes were examined for these same TP53 (by oligonucleotide hybridization) and K-ras (by allele-specific ligation) mutations. RESULTS: TP53 mutations were found in 47 of 94 primary tumors (50%), and K-ras mutations were present in 26 of 55 adenocarcinomas (47%). A total of 134 lymph nodes from 32 patients with stage I disease were analyzed. In 9 cases (28%) the same TP53 or K-ras mutations were found in tumor and lymph node specimens, suggesting occult metastasis. On the basis of nodal location, 7 patients had their disease upstaged by a single stage and 2 patients by two stages. All 28 patients with stage II or III disease had pathologically determined positive nodes that were confirmed as positive by molecular analysis. Standard histopathologic assessment of regional lymph nodes failed to detect metastases at levels below 0.9% tumor-specific mutant TP53 clones per node. No statistically significant difference in disease-specific or overall survival was observed between patients with stage I disease with and without molecular lymph node metastases. CONCLUSIONS: Occult lymph node metastases are present in a significant percentage of patients with stage I non-small cell lung cancer. These data suggest that molecular analysis allows a more accurate assessment of staging. However, larger studies are needed to determine the clinical role of molecular staging.
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Human papillomaviruses (HPVs) play a central role in the development of cervical carcinoma. Plasma DNA from 232 patients taken at diagnosis or after treatment for invasive cervical cancer (n = 175) or carcinoma in situ (n = 57) and 60 normal controls were examined for HPV-16 or HPV-18 E7 DNA by conventional and real-time quantitative PCR assays. We found HPV-16 or HPV-18 E7 DNA in 6.9% (11 of 175) of invasive cervical cancer cases (18.1% of cases positive for HPV-16 or HPV-18 at the genital tract), 1.8% (1 of 57) of carcinoma in situ, and 1.7% (1 of 60) of normal controls by conventional PCR. Quantitative PCR identified the highest concentrations of HPV DNA (copy number of HPV/ml of plasma) in patients with invasive cervical cancer (mean, 11,163; median, 183.5), followed by a level of 8 in the single carcinoma in situ case and 0 copies in the normal control initially positive by conventional PCR. HPV DNA can be detected in the plasma of some patients with HPV-positive cervical tumors. It remains to be demonstrated whether quantitative PCR analysis of HPV DNA in plasma may have utility in patients at high risk of recurrent disease.
PURPOSE: Hepatocellular carcinoma (HCC) is a highly malignant tumor prone to multicentric occurrence. Differentiation between a true relapse of HCC and a second primary tumor is of clinical importance. We sought to identify mitochondrial mutations in HCC and test their use as clonal markers in this disease. EXPERIMENTAL DESIGN: Primary HCC tissue samples were obtained from 19 patients and analyzed for mutations within the mitochondrial displacement loop (D-loop). The discovered mutations were used to determine tumor clonality and provided the basis for detection of tumor DNA in corresponding plasma samples. RESULTS: Thirteen of 19 HCC cases (68%) were identified as having D-loop mitochondrial DNA (mtDNA) mutations in at least one tumor. In 3 of these 13 cases, the same mutation was observed in multiple tumors, indicating monoclonal origin. Remarkably, in 8 of 13 mutated cases, we detected deletion/insertion mutations in the C-tract, a recently reported hotspot and potential replication start site of the closed, circular mitochondrial genome. In addition, we detected mutant mtDNA in 8 of 10 tested paired plasma DNA samples using a highly sensitivity molecular assay. CONCLUSIONS: mtDNA mutations within the D-loop control region are a frequent event in HCC, providing a molecular tool for the determination of clonality. In addition, detection of tumor-specific mtDNA mutations in plasma DNA needs to be explored further for monitoring patients with primary HCC.
PURPOSE: Human papillomavirus (HPV) type-16 has been associated with invasive squamous cell carcinoma of the head and neck. This study examines the role of HPV-16 in the progression of oral head and neck cancer by determining the quantity of HPV-16 DNA in premalignant and malignant lesions, using real-time quantitative PCR, to more accurately determine the role of HPV-16 in oral head and neck squamous cell carcinogenesis. EXPERIMENTAL DESIGN: We examined 102 microdissected premalignant head and neck lesions (85 from the oral cavity), 34 invasive oral cavity squamous cell carcinomas, as well as 18 invasive tumors known to be HPV positive by traditional molecular technology for the presence of HPV-16 DNA using real-time quantitative PCR. RESULTS: HPV DNA was detected in 1 of 102 premalignant lesions (0.98%), 1 of 34 (2.9%) invasive oral cavity carcinomas, and 14 of 18 (78%) known HPV-positive tumors. CONCLUSIONS: HPV-16 infection and integration is seldom found in oral premalignant lesions and invasive carcinoma, and therefore rarely contributes to malignant progression in the oral cavity. Furthermore, quantitative PCR is a useful technique that reliably excludes contaminated samples and those with minimal HPV DNA content that is unlikely to be significant in carcinogenesis.
The GSTP1 gene encodes for an enzyme, glutathione S-transferase pi (GSTpi),involved in detoxification of carcinogens. An aminoacid substitution (I105V) in GSTP1 produces a variant enzyme with lower activity and less capability of effective detoxification. This variant GSTP*B allele has been associated with a propensity to develop several neoplasms. Because GSTP1 promoter hypermethylation and inactivation of GSTpi expression is a frequent alteration in prostate carcinoma, we hypothesized that this somatic epigenetic modification could obviate any reduced enzyme activity caused by the germ-line polymorphism. We tested for the GSTP1 genotype in a population of prostate cancer patients, and in a control group composed of patients with benign prostatic hyperplasia (BPH) and healthy blood donors. Tissue samples from the 105 prostate cancer cases (105 adenocarcinomas and 34 prostatic intraepithelial neoplasia lesions), and from 43 BPH patients were tested for GSTP1 hypermethylation by methylation-specific PCR. GSTpi protein expression was assessed by immunohistochemistry. No significant effect on prostate cancer risk was detectable for GSTP1 genotype compared with the control population (odds ratio, 1.02; 95% confidence interval, 0.59-1.75). Moreover, no association was found between this genotype and tumor or BPH methylation status. Patients with unmethylated carcinomas did not disclose significant differences in genotypic distribution compared with the control population. In adenocarcinoma, a strong association (P < 0.00001) between GSTP1 promoter hypermethylation and loss of GSTpi expression was observed; however, this trend was not retained in prostatic intraepithelial neoplasia or BPH lesions. Although the GSTP1 polymorphism is not associated with altered susceptibility to prostate cancer, somatic promoter hypermethylation is an effective, but not the only, cause of decreased GSTpi function.
PURPOSE: We evaluated the postoperative clinical course of patients with renal cancer identified preoperatively by microsatellite analysis to examine the correlation between microsatellite alterations and risk of disease recurrence and patient mortality 2 years after nephrectomy. EXPERIMENTAL DESIGN: A panel of 28 microsatellite markers was used previously to assess loss of heterozygosity and microsatellite instability in urine, serum, and tumor DNA of 30 patients with clinically organ-confined renal masses who underwent partial or radical nephrectomy. The clinical reports and imaging data in the medical records of patients with a minimum follow-up of 2 years were retrospectively reviewed to determine their postoperative course. RESULTS: Two-year follow-up was available for the 30 patients (100%) who entered the study. Mean age was 61.6 +/- 12.9 years (range, 21-77 years). Tumor stage was associated with patient mortality (P = 0.03). Tumor grade was associated with mortality (P = 0.03) and disease recurrence (P < 0.01). The frequency of microsatellite alterations (loss of heterozygosity) found in the preoperative serum of patients with renal masses served as a prognostic indicator for disease recurrence (P < 0.01). CONCLUSIONS: Analysis of microsatellite alterations found in preoperative blood samples is a promising method for the detection of renal cancer. The presence of frequent molecular changes in preoperative serum was associated with disease recurrence. These findings suggest a role for microsatellite analysis in future studies attempting to stratify patients with clinically organ-confined renal cancer into low- and high-risk prognostic groups. Larger prospective randomized trials are needed to validate the clinical utility of this observation.
PURPOSE: Although mitochondrial DNA mutations have been described recently in many different tumor types, the nature and timing of such alterations remain unclear. In an effort to further examine the role of mitochondrial DNA mutations in carcinogenesis, we examined 137 premalignant lesions of the head and neck from 93 patients for DNA alterations in the poly-cytosine tract (C-tract) of the displacement loop, discovered recently to be a hot spot of mitochondrial DNA alteration. EXPERIMENTAL DESING: All premalignant lesions were tested using a length-based PCR assay, which amplified the C-tract region of mitochondrial DNA. Somatic microsatellites at six loci were also tested on a subset of patients with metachronous or synchronous lesions found to possess a mitochondrial C-tract alteration. RESULTS: Thirty-four of 93 (37%) patients harbored lesions that displayed a C-tract alteration. There was a clear increase in incidence from histologically benign hyperplasia (22%) to squamous carcinoma in situ (62%: P < 0.01). We also tested synchronous dysplastic lesions, metachronous dysplastic lesions, and normal epithelium adjacent to dysplastic epithelium with this assay. In most cases, the mitochondrial C-tract status identified a clonal relationship between these lesions. Genomic microsatellites also confirmed that a clonal relationship was present in many of these cases. CONCLUSIONS: Mitochondrial DNA alterations in the head and neck occur in the earliest premalignant lesions and demonstrate a rising incidence that parallels histological severity. These alterations are valuable as additional markers of histopathological progression.
The ST7 gene was cloned and mapped to chromosome 7q31.1-q31.2, a region suspected of containing a tumor suppressor gene involved in a variety of human cancers. Subsequent investigation described the presence of ST7 mutations in human cell lines derived from breast tumors and primary colon carcinoma. Introduction of the ST7 cDNA into a prostate cancer-derived cell line abrogated in vivo tumorigenecity in nude mice. To clarify the role of the ST7 gene in cancer, we scrutinized primary head and neck squamous cell carcinomas, invasive ductal carcinomas of the breast, and adenocarcinomas of the colon. Loss of heterozygosity of D7S522/D7S677 was detected in 24% (4 of 17) of head and neck squamous cell carcinomas, 17% (2 of 12) of invasive ductal carcinomas of the breast, and 33% (8 of 24) of adenocarcinomas of the colon, but no somatic mutations were found in any of these specimens. We then searched for mutations in breast cancer cell lines and found a complete wild-type sequence in all, including cell lines previously reported to harbor mutations. We believe that the ST7 gene is not a primary target of inactivation in most human cancers with loss of heterozygosity at 7q31.1-q31.2.