Correction: Novel Insight into Mutational Landscape of Head and Neck Squamous Cell Carcinoma.

[This corrects the article DOI: 10.1371/journal.pone.0093102.].

Overexpression of glycosylphosphatidylinositol (GPI) transamidase subunits phosphatidylinositol glycan class T and/or GPI anchor attachment 1 induces tumorigenesis and contributes to invasion in human breast cancer.

Based on the oncogenic role of phosphatidylinositol glycan (PIG) class U in human tumors, we explored the role of two additional subunits of the glycosylphosphatidylinositol (GPI) transamidase complex in human breast cancer. We found that PIG class T (PIG-T) and GPI anchor attachment 1 (GPAA1) were overexpressed in breast cancer cell lines and primary tumors. Forced expression of PIG-T and GPAA1 transformed NIH3T3 cells in vitro and increased tumorigenicity and invasion of these cells in vivo. Suppression of PIG-T expression in breast cancer cell lines led to inhibition of anchorage-independent growth. Moreover, we found that PIG-T and GPAA1 expression levels positively correlated with paxillin phosphorylation in invasive breast cancer cell lines. Furthermore, suppression of PIG-T and GPAA1 expression led to a decrease in paxillin phosphorylation with a concomitant decrease in invasion ability. These results suggest that the GPI transamidase complex is composed of a group of proto-oncogenes that individually or as a group contribute to breast cancer growth. This aberrant growth is mediated, at least partially, by phosphorylation of paxillin, contributing to invasion and progression of breast cancer.

The effect of p53-RNAi and p53 knockout on human 8-oxoguanine DNA glycosylase (hOgg1) activity.

Recent evidence indicates that in vitro p53 augments base excision repair (BER) activities in mammalian cells. To understand the role of p53 in BER, we analyzed the repair activity of hOgg1 in isogenic cell lines HCT116p53+/+ and HCT116p53-/-. We found that hOgg1 activity was significantly decreased in HCT116p53-/- cells as compared with HCT116p53+/+ cells, indicating a functional role for p53 in the regulation of hOGG1. Using gel-shift assays, we showed that p53 binds to its putative cis-elements within the hOGG1 promoter. In addition we demonstrated that supplementing p53 in HCT116p53-/- cells enhanced the transcription of hOGG1. To further strengthen our findings, we used p53-RNAi to study the effects of decreased p53 levels on hOgg1 activity. We observed that p53-RNAi resulted in decreased hOGG1 expression both at the mRNA and protein levels. This decrease in hOGG1 expression was associated with reduced cell viability upon oxidative damage and reduced hOgg1 activity as evidenced by the 8-oxoG incision assay. Taken together, our results indicate that loss of p53 function can lead to decreased hOgg1 repair activity.

Decreased mitochondrial DNA content in posttreatment salivary rinses from head and neck cancer patients.

PURPOSE AND EXPERIMENTAL DESIGN: Alterations in mitochondrial DNA (mtDNA) sequence and content have been described in human tissues and tumors in association with smoking exposure. We did quantitative PCR analysis of cytochrome c oxidase (Cox) I and Cox II genes to measure changes in mtDNA content in pretreatment and posttreatment salivary rinses obtained from 76 patients undergoing surgical resection for primary head and neck squamous cell carcinoma. We also examined the relationship between changes in mtDNA content and postoperative radiation therapy, smoking exposure, alcohol intake, and other clinical characteristics. RESULTS: Overall, mtDNA content in posttreatment saliva was significantly decreased. The mean change for Cox I was -0.21 [95% confidence interval (95% CI), -0.44 to 0.01, P = 0.06] and for Cox II was -0.31 (95% CI, -0.55 to -0.08, P = 0.01). Patients in the radiation therapy group exhibited a significant decrease compared with the nonradiated group (P = 0.03 for Cox I; P = 0.05 for Cox II). In addition, significant decreases in Cox I (-0.71; 95% CI, -1.17 to -0.25, P = 0.005) and Cox II (-0.65; 95% CI, -1.17 to -0.13, P = 0.02) were found in never-smoking patients but not in former or current smokers. CONCLUSION: Our data suggest that salivary mtDNA content is decreased in never smokers and in response to radiation therapy after primary surgical resection.

Oxidized guanine lesions and hOgg1 activity in lung cancer.

In humans, the oxidatively induced DNA lesion 8-hydroxyguanine (8-oxoG) is removed from DNA by hOgg1, a DNA glycosylase/AP lyase that specifically incises 8-oxoG opposite cytosine. We analysed the expression of hOGG1 mRNA in 18 lung cancer and three normal cell lines. Although hOGG1 was overexpressed in most cell lines, 2/18 (11.1%) showed a lower hOGG1 mRNA and protein expression (approximately 80% decrease) relative to normal cell lines. Liquid chromatography/mass spectrometry analysis showed increased levels of 8-oxoG in the two cell lines with the lowest hOGG1 mRNA expression. We examined the ability of nuclear and mitochondrial extracts to incise 8-oxoG lesion in cell lines H1650 and H226 expressing lower hOGG1 mRNA and H1915 and H1975 with higher than normal hOGG1 mRNA expression. Both nuclear and mitochondrial extracts from H1915 and H1975 cells were proficient in 8-oxoG removal. However, both cell lines with the lowest hOGG1 mRNA expression exhibited a severe reduction in 8-oxoG incision in both nuclear and mitochondrial extracts. Under-expression of hOGG1 mRNA and hOgg1 protein was associated with a decrease in mitochondrial DNA repair in response to oxidative damaging agents. These results provide evidence for defective incision of 8-oxoG in both nuclear and mitochondria of H1650 and H226 lung cancer cell lines. These results may implicate 8-oxoG repair defects in certain lung cancers.

Mitochondrial DNA content increase in response to cigarette smoking.

An increase in mitochondrial DNA (mtDNA) content and decline in mitochondrial function occurs with aging and in response to DNA-damaging agents, including tobacco smoke. We did a cross-sectional study and quantified changes in mtDNA content in a population of individuals with varied smoking and alcohol exposure. Age, smoking history, ethanol intake, and other demographic data were characterized for 604 individuals participating in a screening study for smoking-related upper aerodigestive malignancy. Total DNA was extracted from exfoliated cells in saliva. DNA from a nuclear gene, beta-actin, and two mitochondrial genes, cytochrome c oxidase I and II (Cox I and Cox II), were quantified by real-time PCR. mtDNA content was correlated with age, exposure history, and other variables using multivariate regression analyses. A significant increase (P<0.001) in mtDNA content was noted in smokers (31% and 29% increase for Cox I and Cox II, respectively) and former smokers (31% and 34%) when compared with never smokers. This association persisted after adjustment for other significant factors including age, alcohol drinking, and income (P<0.001). Increased mtDNA content was positively associated with pack-years of smoking (P=0.02). Despite an average smoking cessation interval of 21 years in former smokers, tobacco cessation interval was not statistically significantly associated with mtDNA content. Smoking is associated with increased mtDNA content in a dose-dependent fashion. Mitochondrial DNA alterations in response to smoking persist for several decades after smoking cessation, consistent with long-term, smoking-related damage.

Stunning and its effect on 3H-FDG uptake and key gene expression in breast cancer cells undergoing chemotherapy.

UNLABELLED: Shortly after chemotherapy, relatively little is known about the expression of key genes and proteins involved in glycolysis. Doxorubicin (DOX) and 5-fluorouracil (5FU) are two commonly used chemotherapy agents that work through differing pathways. Glucose transporter-1 (Glut-1) and hexokinase II (HKII) proteins are highly expressed in many breast carcinomas, but their status while undergoing DOX or 5FU chemotherapy has not been systematically evaluated. METHODS: We evaluated, in vitro, the messenger RNA (mRNA) and protein levels of Glut-1 and HKII in MCF-7, a breast adenocarcinoma cell line, and (3)H-FDG uptake, both in untreated conditions and during treatment with either DOX or 5FU for 24 h. Six time points were evaluated: untreated at time 0; treated for 1 h; treated for 24 h; and 1, 2, and 3 d after chemotherapy. We analyzed tumor cell Glut-1 and HKII mRNA expression with real-time polymerase chain reaction and Western blotting, (3)H-FDG uptake per cell, and cell viability with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. RESULTS: DOX was more effective than 5FU in killing the cancer cells under our study conditions. In untreated MCF-7 cells, the mRNA levels of HKII were typically higher than those of Glut-1, and (3)H-FDG uptake was strongly related to Glut-1 mRNA expression (R(2) = 0.85). Once treated with either drug, (3)H-FDG uptake declined initially, the mRNA ratio was reversed, and Glut-1 mRNA levels were higher than HKII levels. This was verified in the protein assay. With DOX treatment, the cells showed increased Glut-1 mRNA and decreased HKII mRNA for the duration of active treatment; these levels returned to those seen in the untreated cells once the treatment was stopped for 24 h. However, HKII protein levels remained somewhat low. No correlation was seen between (3)H-FDG uptake and HKII mRNA in DOX- and 5FU-treated cells (R(2) = 0.14 and 0.0038, respectively). CONCLUSION: After DOX or 5FU therapy, the relationship between (3)H-FDG uptake and viable cell number can become disjointed, with transient declines in (3)H-FDG uptake in excess of the decline in cell number despite increased Glut-1 mRNA levels. This transient "stunning" has potential implications for (3)H-FDG PET, especially soon after treatment is initiated. However, (3)H-FDG remains a generally valid marker of viable cell number after cancer chemotherapy.

Targeting of mutant hogg1 in mammalian mitochondria and nucleus: effect on cellular survival upon oxidative stress.

BACKGROUND: Oxidative damage to mitochondrial DNA has been implicated as a causative factor in a wide variety of degenerative diseases, aging and cancer. The modified guanine, 7,8-dihydro-8-oxoguanine (also known as 8-hydroxyguanine) is one of the major oxidized bases generated in DNA by reactive oxygen species and has gained most of the attention in recent years as a marker of oxidative DNA injury and its suspected role in the initiation of carcinogenesis. 8-hydroxyguanine is removed by hOgg1, a DNA glycosylase/AP lyase involved in the base excision repair pathway. METHODS: We over-expressed wild type and R229Q mutant hOGG1 in the nucleus and mitochondria of cells lacking mitochondrial hOGG1 expression through an expression vector containing nuclear and mitochondrial targeting sequence respectively. We used quantitative real time PCR to analyze mtDNA integrity after exposure to oxidative damaging agents, in cells transfected with or without mitochondrially-targeted mutant hogg1. RESULT: Over-expression of wild type hOgg1 in both nucleus and mitochondria resulted in increased cellular survival when compared to vector or mutant over-expression of hOGG1. Interestingly, mitochondrially-targeted mutant hogg1 resulted in more cell death than nuclear targeted mutant hogg1 upon exposure of cells to oxidative damage. Additional we examined mitochondrial DNA integrity after oxidative damage exposure using real-time quantitative PCR. The presence of mutant hogg1 in the mitochondria resulted in reduced mitochondrial DNA integrity when compared to the wild type. Our work indicates that the R229Q hOGG1 mutation failed to protect cells from oxidative damage and that such mutations in cancer may be more detrimental to cellular survival when present in the mitochondria than in the nucleus. CONCLUSION: These findings suggest that deficiencies in hOGG1, especially in the mitochondria may lead to reduced mitochondrial DNA integrity, consequently resulting in decreased cell viability.

Head and neck cancer cell lines exhibit differential mitochondrial repair deficiency in response to 4NQO.

Constituents of tobacco can cause DNA adduct formation and are implicated in head and neck squamous cell cancer (HNSC) development. We investigated the capacity of HNSC cell lines to repair mitochondrial DNA (mtDNA) damage induced by a DNA adduct-forming agent. HNSC cell lines underwent 4-nitroquinoline 1-oxide (4NQO) exposure with subsequent rescue with normal media. Real-time quantitative PCR for nuclear DNA (nDNA) and mtDNA was performed. mtDNA to nDNA ratios were calculated and standardized to mock-treated cells to assess mtDNA repair ability. Two of three tested cancer cell lines exposed to 4NQO exhibited consistent decreases in mtDNA/nDNA ratios throughout the different repair timepoints. At 24 h mtDNA/nDNA ratios of JHU-O19 and JHU-O22 decreased to 63% and 60% of controls, respectively. Conversely, a control keratinocyte cell line exhibited overall increases in mtDNA/nDNA ratios compared to baseline suggesting intact DNA repair mechanisms. By using a DNA adduct formation and repair model featuring 4NQO and HNSC cell lines, we have implicated faulty mtDNA repair as having a potential role in HNSC.

Increased mitochondrial DNA content in saliva associated with head and neck cancer.

Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure. We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II r = 0.33, P = 0.002, respectively, Pearson's correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

BRAF mutation in papillary thyroid carcinoma.

The BRAF gene has been found to be activated by mutation in human cancers, predominantly in malignant melanoma. We tested 476 primary tumors, including 214 lung, 126 head and neck, 54 thyroid, 27 bladder, 38 cervical, and 17 prostate cancers, for the BRAF T1796A mutation by polymerase chain reaction (PCR)-restriction enzyme analysis of BRAF exon 15. In 24 (69%) of the 35 papillary thyroid carcinomas examined, we found a missense thymine (T)-->adenine (A) transversion at nucleotide 1796 in the BRAF gene (T1796A). The T1796A mutation was detected in four lung cancers and in six head and neck cancers but not in bladder, cervical, or prostate cancers. Our data suggest that activating BRAF mutations may be an important event in the development of papillary thyroid cancer.

Defective repair of 8-hydroxyguanine in mitochondria of MCF-7 and MDA-MB-468 human breast cancer cell lines.

Breast cancer is one of the major causes of mortality among women in the United States. Although the causes of breast cancer remain unclear, it has been speculated that DNA base damage may lead to mutations that subsequently can be carcinogenic. Recently, defective oxidative DNA damage repair has been implicated in breast tumorigenesis. The major oxidative DNA lesion, 8-hydroxyguanine (8-oxoG), is increased in breast cancer, suggesting that this lesion may play a crucial role in the etiology of breast cancer. However, it is not known whether the repair of 8-oxoG or other oxidative base lesions is altered during breast carcinogenesis. We examined the ability of nuclear and mitochondrial extracts of two human breast cancer cell lines, MCF-7 and MDA-MB-468, to repair 8-oxoG lesion. We report that mitochondrial extracts from the two breast cancer cell lines are defective in the base excision repair of 8-oxoG relative to two noncancer cell lines. We also show that the incision activity of 8-oxoG was significantly lower in mitochondrial than in nuclear extracts in the breast cancer cell lines. The defective mitochondrial repair activity was not attributable to lower levels of human 8-hydroxyguanine DNA glycosylase, the base excision repair enzyme known to incise 8-oxoG in DNA. The repair of thymine glycol, another major oxidative DNA base lesion that blocks transcription and causes cell death, was similar in cancer and noncancer cells. Furthermore, nuclear extracts incised thymine glycol with a much higher efficiency than 8-oxoG. These data provide evidence for defective repair of 8-oxoG in mitochondria of MCF-7 and MDA-MB-468 breast cancer cell lines. These results may implicate 8-oxoG repair mechanisms in mitochondria of certain breast cancers.

Early occurrence of RASSF1A hypermethylation and its mutual exclusion with BRAF mutation in thyroid tumorigenesis.

Follicular epithelial cell-derived thyroid tumors are common neoplasms comprised mainly of benign thyroid adenomas, follicular thyroid cancers, and papillary thyroid cancers (PTCs). Hypermethylation of the tumor suppressor gene RASSF1A and activating mutation of BRAF gene have been reported recently in thyroid cancers. To additionally investigate the roles of these two epigenetic/genetic alterations in thyroid tumor progression, we examined their occurrences and relationship in both benign and malignant thyroid neoplasms. With real-time quantitative methylation-specific PCR, we found that 4 of 9 (44%) benign adenomas, 9 of 12 (75%) follicular thyroid cancers tumors, and 6 of 30 (20%) of PTC tumors harbored promoter methylation in > or = 25% of RASSF1A alleles. Additional quantitative analysis revealed RASSF1A methylation only in BRAF mutation-negative PTCs. A similar inverse correlation of RASSF1A methylation with BRAF mutation was seen in thyroid tumor cell lines. Our results, therefore, suggest that epigenetic inactivation of RASSF1A through aberrant methylation is an early step in thyroid tumorigenesis. Like the previously reported mutually exclusive relationship between BRAF mutation and other Ras pathway components such as RET/PTC rearrangement, a mutually exclusive relationship also exists between BRAF mutation and RASSF1A methylation in thyroid tumorigenesis.

Quantitative detection of promoter hypermethylation of multiple genes in the tumor, urine, and serum DNA of patients with renal cancer.

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of human cancers and is a promising marker for cancer detection. We investigated the feasibility of detecting aberrant DNA methylation in the urine and serum samples of renal cancer patients. We examined the tumor and the matched urine and serum DNA for aberrant methylation of nine gene promoters (CDH1, APC, MGMT, RASSF1A, GSTP1, p16, RAR-beta2, and ARF) from 17 patients with primary kidney cancer by quantitative fluorogenic real-time PCR. An additional 9 urine samples (total, 26) and 1 serum sample (total, 18) also were tested from renal cancer patients. Urine from 91 patients without genitourinary cancer and serum from 30 age-matched noncancer individuals were used as controls. Promoter hypermethylation of at least two of the genes studied was detected in 16 (94%) of 17 primary tumors. Aberrant methylation in urine and serum DNA generally was accompanied by methylation in the matched tumor samples. Urine samples from 91 control subjects without evidence of genitourinary cancer revealed no methylation of the MGMT, GSTP1, p16, and ARF genes, whereas methylation of RAR-beta2, RASSF1A, CDH1, APC, and TIMP3 was detected at low levels in a few control subjects. Overall, 23 (88%) of 26 urine samples and 12 (67%) of 18 serum samples from cancer patients were methylation positive for at least one of the genes tested. By combination of urine or serum analysis of renal cancer patients, hypermethylation was detected in 16 of 17 patients (94% sensitivity) with high specificity. Our findings suggest that promoter hypermethylation in urine or serum can be detected in the majority of renal cancer patients. This noninvasive high-throughput approach needs to be evaluated in large studies to assess its value in the early detection and surveillance of renal cancer.

Stage-Specific MicroRNAs and Their Role in the Anticancer Effects of Calorie Restriction in a Rat Model of ER-Positive Luminal Breast Cancer.

MicroRNAs have emerged as ubiquitous post-transcriptional regulators that coordinate many fundamental processes within cells, including those commonly linked to cancer when dysregulated. Profiling microRNAs across stages of cancer progression provides focus as to which microRNAs are key players in cancer development and are therefore important to manipulate with interventions to delay cancer onset and progression. Calorie restriction is one of the most effective preventive interventions across many types of cancer, although its effects on microRNAs have not been well characterized. We used the dimethylbenz[a]-anthracene-induced model of luminal mammary cancer in Sprague Dawley rats to elucidate which microRNAs are linked to progression in this type of cancer and, subsequently, to study how calorie restriction affects such microRNAs. We identified eight microRNAs (miR-10a, miR-10b, miR-21, miR-124, miR-125b, miR-126, miR-145 and miR-200a) to be associated with DMBA-induced mammary tumor progression. Calorie restriction, which greatly increased tumor-free survival and decreased the overall size of tumors that did develop, significantly decreased the expression of one microRNA, miR-200a, which was positively associated with tumor progression. We further showed that inhibition of miR-200a function, mimicking the effect of calorie restriction on this microRNA, inhibited proliferation in both rat (LA7) and human (MCF7) luminal mammary cancer cell lines. These findings present, for the first time, a stage-specific profile of microRNAs in a rodent model of luminal mammary cancer. Furthermore, we have identified the regulation of miR-200a, a microRNA that is positively associated with progression in this model, as a possible mechanism contributing to the anticancer effects of calorie restriction.

Uncommon mutation, but common amplifications, of the PIK3CA gene in thyroid tumors.

CONTEXT: As in many other human cancers, overactivation of the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway occurs frequently in thyroid cancer, but the mechanism is not completely clear. OBJECTIVE: Because activating mutations and genomic amplification of the PIK3CA gene, which encodes the p110a catalytic subunit of PI3K, are common in many cancers, we sought to investigate this phenomenon in thyroid tumors. DESIGN: To search for PIK3CA mutations, we isolated genomic DNA from primary thyroid tumors of various types and performed direct sequencing of the exons of PIK3CA gene that carry the most common mutations in other cancers. We used real-time quantitative PCR to investigate genomic amplification of the PIK3CA gene. RESULTS: We found no PIK3CA gene mutations in 37 benign thyroid adenomas, 52 papillary thyroid cancers, 25 follicular thyroid cancers, 13 anaplastic thyroid cancers, 13 medullary thyroid cancers, and seven thyroid tumor cell lines. We found a C3075T single-nucleotide polymorphism in exon 20 of this gene in two cases. With a copy number of 4 or more defined as amplification, we found PIK3CA gene amplification in four of 34 (12%) benign thyroid adenomas, three of 59 (5%) papillary thyroid cancer, five of 21 (24%) follicular thyroid cancer, none of 14 (0%) medullary thyroid cancer, and five of seven (71%) thyroid tumor cell lines. The PIK3CA gene amplification and consequent Akt activation were confirmed by fluorescence in situ hybridization and Western blotting studies using cell lines, respectively. CONCLUSION: These data suggest that mutation of the PIK3CA gene is not common, but its amplification is relatively common and may be a novel mechanism in activating the PI3K/Akt pathway in some thyroid tumors.

Somatic mutation and gain of copy number of PIK3CA in human breast cancer.

INTRODUCTION: Phosphatidylinositol 3-kinases (PI3Ks) are a group of lipid kinases that regulate signaling pathways involved in cell proliferation, adhesion, survival, and motility. Even though PIK3CA amplification and somatic mutation have been reported previously in various kinds of human cancers, the genetic change in PIK3CA in human breast cancer has not been clearly identified. METHODS: Fifteen breast cancer cell lines and 92 primary breast tumors (33 with matched normal tissue) were used to check somatic mutation and gene copy number of PIK3CA. For the somatic mutation study, we specifically checked exons 1, 9, and 20, which have been reported to be hot spots in colon cancer. For the analysis of the gene copy number, we used quantitative real-time PCR and fluorescence in situ hybridization. We also treated several breast cancer cells with the PIK3CA inhibitor LY294002 and compared the apoptosis status in cells with and without PIK3CA mutation. RESULTS: We identified a 20.6% (19 of 92) and 33.3% (5 of 15) PIK3CA somatic mutation frequency in primary breast tumors and cell lines, respectively. We also found that 8.7% (8 of 92) of the tumors harbored a gain of PIK3CA gene copy number. Only four cases in this study contained both an increase in the gene copy number and a somatic mutation. In addition, mutation of PIK3CA correlated with the status of Akt phosphorylation in some breast cancer cells and inhibition of PIK3CA-induced increased apoptosis in breast cancer cells with PIK3CA mutation. CONCLUSION: Somatic mutation rather than a gain of gene copy number of PIK3CA is the frequent genetic alteration that contributes to human breast cancer progression. The frequent and clustered mutations within PIK3CA make it an attractive molecular marker for early detection and a promising therapeutic target in breast cancer.

A quantitative promoter methylation profile of prostate cancer.

PURPOSE: Promoter hypermethylation is an alternative pathway for gene silencing in neoplastic cells and a promising cancer detection marker. Although quantitative methylation-specific PCR (QMSP) of the GSTP1 promoter has demonstrated near perfect specificity for cancer detection in prostate biopsies, we postulated that identification and characterization of additional methylation markers might further improve its high (80-90%) sensitivity. EXPERIMENTAL DESIGN: We surveyed nine gene promoters (GSTP1, MGMT, p14/ARF, p16/CDKN2A, RASSF1A, APC, TIMP3, S100A2, and CRBP1) by QMSP in tissue DNA from 118 prostate carcinomas, 38 paired high-grade prostatic intraepithelial neoplasias (HGPIN), and 30 benign prostatic hyperplasias (BPH). The methylation levels were calculated and were correlated with clinical and pathologic indicators. RESULTS: Only the methylation frequencies of GSTP1 and APC were significantly higher in prostate carcinoma compared with BPH (P < 0.001). Methylation levels of GSTP1, APC, RASSF1A, and CRBP1, differed significantly between prostate carcinoma and HGPIN, and/or HGPIN or BPH (P < 0.0001). With QMSP and empirically defined cutoff values, the combined use of GSTP1 and APC demonstrated a theoretical sensitivity of 98.3% for prostate carcinoma, with 100% specificity. Methylation levels were found to correlate with tumor grade (GSTP1 and APC) and stage (GSTP1, RASSF1A, and APC). CONCLUSIONS: Our data demonstrate the existence of a progressive increase of promoter methylation levels of several cancer-related genes in prostate carcinogenesis, providing additional markers to augment molecular detection of prostate carcinoma. Because methylation levels of GSTP1, APC, and RASSF1A are associated with advanced grade and stage, QMSP might augment the pathologic indicators currently used to predict tumor aggressiveness.

Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using a 51-gene enrichment panel.

BACKGROUND: Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures. METHODS: The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance. RESULTS: We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10(-16)), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CI = 56.3, 92.5) with 100% PPV (95% CI = 81.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CI = 66.4,97.2) while maintaining PPV. CONCLUSIONS: The results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens.

Clusterin is a gene-specific target of microRNA-21 in head and neck squamous cell carcinoma.

PURPOSE: MicroRNA-21 (miRNA-21) has proto-oncogenic properties, although no miRNA-21-specific targets have been found in head and neck squamous cell carcinoma (HNSCC). Further study of miRNA-21 and its specific targets is essential to understanding HNSCC biology. EXPERIMENTAL DESIGN: miRNA expression profiles of 10 HNSCCs and 10 normal mucosa samples were investigated using a custom miRNA microarray. Thirteen HNSCCs and five normal mucosa primary tissue specimens underwent mRNA expression microarray analysis. To identify miRNA-21 downstream targets, oral keratinocyte cells were subjected to microarray analysis after miRNA-21 transient transfection. miRNA and mRNA expression were validated by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of 16 HNSCCs and 15 normal mucosal samples. Microarray and bioinformatics analyses were integrated to identify potential gene targets. In vitro assays looked at the function and interaction of miRNA-21 and its specific gene targets. RESULTS: miRNA-21 was upregulated in HNSCCs and stimulated cell growth. Integrated analyses identified Clusterin (CLU) as a potential miRNA-21 gene target. CLU was downregulated after forced expression of miRNA-21 in normal and HNSCC cell lines. The activity of a luciferase construct containing the 3'-untranslated region (UTR) of CLU was repressed by the ectopic expression of miRNA-21. CLU was also downregulated in primary HNSCCs and correlated with miRNA-21 overexpression. CLU variant 1 (CLU-1) was the predominant splice variant in HNSCCs and showed growth suppression function that was reversed by miRNA-21 overexpression. CONCLUSIONS: CLU is a specific, functional target of oncogenic miRNA-21 in HNSCCs. CLU-1 isoform is the predominant growth-suppressive variant targeted by miRNA-21.