Epigenomic, genomic, and transcriptomic landscape of schwannomatosis.

Schwannomatosis (SWNTS) is a genetic cancer predisposition syndrome that manifests as multiple and often painful neuronal tumors called schwannomas (SWNs). While germline mutations in SMARCB1 or LZTR1, plus somatic mutations in NF2 and loss of heterozygosity in chromosome 22q have been identified in a subset of patients, little is known about the epigenomic and genomic alterations that drive SWNTS-related SWNs (SWNTS-SWNs) in a majority of the cases. We performed multiplatform genomic analysis and established the molecular signature of SWNTS-SWNs. We show that SWNTS-SWNs harbor distinct genomic features relative to the histologically identical non-syndromic sporadic SWNs (NS-SWNS). We demonstrate the existence of four distinct DNA methylation subgroups of SWNTS-SWNs that are associated with specific transcriptional programs and tumor location. We show several novel recurrent non-22q deletions and structural rearrangements. We detected the SH3PXD2A-HTRA1 gene fusion in SWNTS-SWNs, with predominance in LZTR1-mutant tumors. In addition, we identified specific genetic, epigenetic, and actionable transcriptional programs associated with painful SWNTS-SWNs including PIGF, VEGF, MEK, and MTOR pathways, which may be harnessed for management of this syndrome.

A survey of methylated candidate tumor suppressor genes in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a rare malignancy with unique genetic, viral and environmental characteristic that distinguishes it from other head and neck carcinomas. The clinical management of NPC remains challenging largely due to the lack of early detection strategies for this tumor. In our study, we have sought to identify novel genes involved in the pathogenesis of NPC that might provide insight into this tumor's biology and could potentially be used as biomarkers. To identify these genes, we studied the epigenetics of NPC by characterizing a panel of methylation markers. Eighteen genes were evaluated by quantitative methylation-specific polymerase chain reaction (PCR) in cell lines as well as in tissue samples including 50 NPC tumors and 28 benign nasopharyngeal biopsies. Significance was evaluated using Fisher's exact test and quantitative values were optimized using cut off values derived from receiver-operator characteristic curves. The methylation status of AIM1, APC, CALCA, deleted in colorectal carcinomas (DCC), DLEC, deleted in liver cancer 1 (DLC1), estrogen receptor alpha (ESR), FHIT, KIF1A and PGP9.5 was significantly associated with NPC compared to controls. The sensitivity of the individual genes ranged from 26 to 66% and the specificity was above 92% for all genes except FHIT. The combination of PGP9.5, KIF1A and DLEC had a sensitivity of 84% and a specificity of 92%. Ectopic expression of DCC and DLC1 lead to decrease in colony formation and invasion properties. Our results indicate that methylation of novel biomarkers in NPC could be used to enhance early detection approaches. Additionally, our functional studies reveal previously unknown tumor suppressor roles in NPC.

KIF1A and EDNRB are differentially methylated in primary HNSCC and salivary rinses.

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. In this study, we aimed to evaluate to the utility of aberrant promoter hypermethylation for detection in a panel of 10 genes (KIF1A, EDNRB, CDH4, TERT, CD44, NISCH, PAK3, VGF, MAL and FKBP4) in head and neck squamous cell carcinoma (HNSCC) via a candidate gene approach. We investigated methylation of the gene promoters by bisulfite modification and quantitative methylation-specific PCR (Q-MSP) in a preliminary study of a limited cohort of salivary rinses from healthy subjects (n = 61) and patients with HNSCC (n = 33). The methylation status of 2 selected genes (EDNRB and KIF1A) were then analyzed in 15 normal mucosa samples from a healthy population, 101 HNSCC tumors and the corresponding salivary rinses from 71 out of the 101 HNSCC patients were collected before treatment. The promoter regions of CDH4, TERT, VGF, MAL, FKBP4, NISCH and PAK3 were methylated in normal salivary rinses while no methylation of CD44 was observed in either normal salivary rinses or tumor samples. However, KIF1A and EDNRB were methylated in 98 and 97% of primary HNSCC tissues respectively and were only methylated in 2 and 6.6% of normal salivary rinses. In addition, KIF1A and EDNRB were methylated in 38 and 67.6% of salivary rinses from HNSCC patients, respectively. Promoter hypermethylation of KIF1A and EDNRB is a frequent event in primary HNSCC, and these genes are preferentially methylated in salivary rinses from HNSCC patients. KIF1A and EDNRB are potential biomarkers for HNSCC detection.

Pharmacologic unmasking of epigenetically silenced genes in breast cancer.

PURPOSE: Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of various cancers including breast cancer. Many epigenetically inactivated genes involved in breast cancer development remain to be identified. Therefore, in this study we used a pharmacologic unmasking approach in breast cancer cell lines with 5-aza-2'-deoxycytidine (5-aza-dC) followed by microarray expression analysis to identify epigenetically inactivated genes in breast cancer. EXPERIMENTAL DESIGN: Breast cancer cell lines were treated with 5-aza-dC followed by microarray analysis to identify epigenetically inactivated genes in breast cancer. We then used bisulfite DNA sequencing, conventional methylation-specific PCR, and quantitative fluorogenic real-time methylation-specific PCR to confirm cancer-specific methylation in novel genes. RESULTS: Forty-nine genes were up-regulated in breast cancer cells lines after 5-aza-dC treatment, as determined by microarray analysis. Five genes (MAL, FKBP4, VGF, OGDHL, and KIF1A) showed cancer-specific methylation in breast tissues. Methylation of at least two was found at high frequency only in breast cancers (40 of 40) as compared with normal breast tissue (0 of 10; P<0.0001, Fisher's exact test). CONCLUSIONS: This study identified new cancer-specific methylated genes to help elucidate the biology of breast cancer and as candidate diagnostic markers for the disease.

ssDNA-binding protein 2 is frequently hypermethylated and suppresses cell growth in human prostate cancer.

PURPOSE: Prostate cancer is a major cause of cancer death among men and the development of new biomarkers is important to augment current detection approaches. EXPERIMENTAL DESIGN: We identified hypermethylation of the ssDNA-binding protein 2 (SSBP2) promoter as a potential DNA marker for human prostate cancer based on previous bioinformatics results and pharmacologic unmasking microarray. We then did quantitative methylation-specific PCR in primary prostate cancer tissues to confirm hypermethylation of the SSBP2 promoter, and analyzed its correlation with clinicopathologic data. We further examined SSBP2 expression in primary prostate cancer and studied its role in cell growth. RESULTS: Quantitative methylation-specific PCR results showed that the SSBP2 promoter was hypermethylated in 54 of 88 (61.4%) primary prostate cancers versus 0 of 23 (0%) in benign prostatic hyperplasia using a cutoff value of 120. Furthermore, we found that expression of SSBP2 was down-regulated in primary prostate cancers and cancer cell lines. Hypermethylation of the SSBP2 promoter and its expression were closely associated with higher stages of prostate cancer. Reactivation of SSBP2 expression by the demethylating agent 5-aza-2'-deoxycytidine in prostate cancer cell lines confirmed epigenetic inactivation as one major mechanism of SSBP2 regulation. Moreover, forced expression of SSBP2 inhibited prostate cancer cell proliferation in the colony formation assay and caused cell cycle arrest. CONCLUSION: SSBP2 inhibits prostate cancer cell proliferation and seems to represent a novel prostate cancer-specific DNA marker, especially in high stages of human prostate cancer.

The Secretomes of Painful Versus Nonpainful Human Schwannomatosis Tumor Cells Differentially Influence Sensory Neuron Gene Expression and Sensitivity.

Schwannomatosis is a multiple tumor syndrome in which patients develop benign tumors along peripheral nerves throughout the body. The first symptom with which schwannomatosis patients often present, prior to discovery of tumors, is pain. This pain can be debilitating and is often inadequately alleviated by pharmacological approaches. Schwannomatosis-associated pain can be localized to the area of a tumor, or widespread. Moreover, not all tumors are painful, and the occurrence of pain is often unrelated to tumor size or location. We speculate that some individual tumors, but not others, secrete factors that act on nearby nerves to augment nociception by producing neuronal sensitization or spontaneous neuronal firing. We created cell lines from human SWN tumors with varying degrees of pain. We have found that conditioned medium (CM) collected from painful SWN tumors, but not that from nonpainful SWN tumors, sensitized DRG neurons, causing increased sensitivity to depolarization by KCl, increased response to noxious TRPV1 and TRPA1 agonists and also upregulated the expression of pain-associated genes in DRG cultures. Multiple cytokines were also detected at higher levels in CM from painful tumors. Taken together our data demonstrate a differential ability of painful versus non-painful human schwannomatosis tumor cells to secrete factors that augment sensory neuron responsiveness, and thus identify a potential determinant of pain heterogeneity in schwannomatosis.

Epigenetic silencing of human T (brachyury homologue) gene in non-small-cell lung cancer.

Early detection of lung cancer is challenging due to a lack of adequate biomarkers. To discover novel tumor suppressor genes (TSGs) silenced by aberrant promoter methylation, we analyzed the gene expression profiles of two lung adenocarcinoma cell lines using pharmacologic-unmasking and subsequent microarray-analysis. Among 617 genes upregulated, we selected 30 genes and investigated the methylation status of their promoters by bisulfite sequencing analysis. Aberrant methylation was detected in four genes (CRABP2, NOEY2, T, MAP2K3) in at least one lung adenocarcinoma cell lines. Furthermore, the T promoter was methylated in 60% of primary lung adenocarcinomas versus 13% of non-malignant lung tissues. Conversely, RT-PCR analysis revealed T expression was low in lung tumors, while high in normal tissues. In addition, no non-synonymous mutations related to gene silencing were found. While further analysis is warranted, our results suggest that T has the potential to be a novel candidate TSG in lung cancer.

Tissue inhibitor of metalloproteinases-3 promoter methylation is an independent prognostic factor for bladder cancer.

PURPOSE: TIMP-3 (tissue inhibitor of metalloproteinases-3) is 1 of 4 members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases. We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from patients with bladder cancer with well characterized clinicopathological parameters, including patient outcome. MATERIALS AND METHODS: We examined urine sediment DNA for aberrant methylation of 9 genes, including TIMP-3, by quantitative fluorogenic real-time polymerase chain reaction. RESULTS: Using an optimal cutoff value by TaqMan(R) quantitation we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (HR 1.99, 95% CI 1.12 to 3.27, p = 0.01 and HR 1.66, 95% CI 1.00 to 2.76, p = 0.05, respectively) than in patients without/lower TIMP3 and ARF methylation in urine. A significant correlation was also seen between the risk of death and stage 3 tumor (HR 2.73, 95% CI 1.58 to 4.72, p = 0.003) and metastasis (HR 3.32, 95% CI 1.98 to 5.57, p = 0.0001). Multivariate analysis subsequently revealed that TIMP-3 methylation was an independent prognostic factor for bladder cancer survival with stage and metastasis (p = 0.001 and 0.02, respectively). CONCLUSIONS: These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.

Breast and ovarian cancer in relatives of cancer patients, with and without BRCA mutations.

BACKGROUND: First-degree relatives of patients with breast or ovarian cancer have increased risks for these cancers. Little is known about how their risks vary with the patient's cancer site, carrier status for predisposing genetic mutations, or age at cancer diagnosis. METHODS: We evaluated breast and ovarian cancer incidence in 2,935 female first-degree relatives of non-Hispanic White female patients with incident invasive cancers of the breast (n = 669) or ovary (n = 339) who were recruited from a population-based cancer registry in northern California. Breast cancer patients were tested for BRCA1 and BRCA2 mutations. Ovarian cancer patients were tested for BRCA1 mutations. We estimated standardized incidence ratios (SIR) and 95% confidence intervals (95% CI) for breast and ovarian cancer among the relatives according to the patient's mutation status, cancer site, and age at cancer diagnosis. RESULTS: In families of patients who were negative or untested for BRCA1 or BRCA2 mutations, risks were elevated only for the patient's cancer site. The breast cancer SIR was 1.5 (95% CI, 1.2-1.8) for relatives of breast cancer patients, compared with 1.1 (95% CI, 0.8-1.6) for relatives of ovarian cancer patients (P = 0.12 for difference by patient's cancer site). The ovarian cancer SIR was 0.9 (95% CI, 0.5-1.4) for relatives of breast cancer patients, compared with 1.9 (95% CI, 1.0-4.0) for relatives of ovarian cancer patients (P = 0.04 for difference by site). In families of BRCA1-positive patients, relatives' risks also correlated with the patient's cancer site. The breast cancer SIR was 10.6 (95% CI, 5.2-21.6) for relatives of breast cancer patients, compared with 3.3 (95% CI, 1.4-7.3) for relatives of ovarian cancer patients (two-sided P = 0.02 for difference by site). The ovarian cancer SIR was 7.9 (95% CI, 1.2-53.0) for relatives of breast cancer patients, compared with 11.3 (3.6-35.9) for relatives of ovarian cancer patients (two-sided P = 0.37 for difference by site). Relatives' risks were independent of patients' ages at diagnosis, with one exception: In families ascertained through a breast cancer patient without BRCA mutations, breast cancer risks were higher if the patient had been diagnosed before age 40 years. CONCLUSION: In families of patients with and without BRCA1 mutations, breast and ovarian cancer risks correlate with the patient's cancer site. Moreover, in families of breast cancer patients without BRCA mutations, breast cancer risk depends on the patient's age at diagnosis. These patterns support the presence of genes that modify risk specific to cancer site, in both carriers and noncarriers of BRCA1 and BRCA2 mutations.

Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients.

Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening.

Prevalence of BRCA1 mutation carriers among U.S. non-Hispanic Whites.

Data from several countries indicate that 1% to 2% of Ashkenazi Jews carry a pathogenic ancestral mutation of the tumor suppressor gene BRCA1. However, the prevalence of BRCA1 mutations among non-Ashkenazi Whites is uncertain. We estimated mutation carrier prevalence in U.S. non-Hispanic Whites, specific for Ashkenazi status, using data from two population-based series of San Francisco Bay Area patients with invasive cancers of the breast or ovary, and data on breast and ovarian cancer risks in Ashkenazi and non-Ashkenazi carriers. Assuming that 90% of the BRCA1 mutations were detected, we estimate a carrier prevalence of 0.24% (95% confidence interval, 0.15-0.39%) in non-Ashkenazi Whites, and 1.2% (95% confidence interval, 0.5-2.6%) in Ashkenazim. When combined with U.S. White census counts, these prevalence estimates suggest that approximately 550,513 U.S. Whites (506,206 non-Ashkenazim and 44,307 Ashkenazim) carry germ line BRCA1 mutations. These estimates may be useful in guiding resource allocation for genetic testing and genetic counseling and in planning preventive interventions.

The effects of BRCA1 missense variants V1804D and M1628T on transcriptional activity.

Many families with multiple cases of ovarian cancer, breast cancer, or both segregate inherited mutations in one allele of the tumor suppressor gene BRCA1. Genetic testing is used to assess cancer risk; however, testing can detect missense DNA alterations, called unclassified variants, of unknown functional and biological significance with uncertain risk implications. Some missense variants at the transcriptional activation domain of BRCA1 of cancer patients inactivate transcriptional activity of BRCA1, providing evidence that they are deleterious. We identified the variants V1804D and M1628T at the transcriptional activation domain of BRCA1 of two ovarian cancer patients without a family history of ovarian or breast cancer. To test if these residues are critical for transcriptional activation, we created V1804D and M1628T independently in BRCA1 cDNA via site-directed mutagenesis in a mammalian expression vector, pcDNA3.1. Wild-type, mutant, and empty vector constructs were tested in human kidney 293 cells using a p53-responsive luciferase reporter. M1628T had the same transcriptional activity as wild-type BRCA1 but V1804D and the empty vector control showed a 60% reduction. This indicates that V1804D is deleterious but M1628T is not.

cDNA sequence and in vitro folding of GsMTx4, a specific peptide inhibitor of mechanosensitive channels.

The peptide GsMTx4 from the tarantula venom (Grammostola spatulata) inhibits mechanosensitive ion channels. In this work, we report the cDNA sequence encoding GsMTx4. The gene is translated as a precursor protein of 80 amino acids. The first 21 amino acids are a predicted signal sequence and the C-terminal residues are a signal for amidation. An arginine residue adjacent to the N-terminal glycine of GsMTx4 is the cleavage site for release. The resulting peptide is 34 amino acids in length with a C-terminal phenylalanine and not a serine-alanine previously identified [J. Gen. Physiol. 115 (2000) 583]. We chemically synthesized this peptide and folded it in 0.1 M Tris, pH 7.9 with oxidized/reduced glutathione (1/10). Properties of the synthetic peptide were identical to the wild type for high performance liquid chromatography (HPLC), mass spectrometry, CD, and NMR. We also cloned GsMTx4 in a thioredoxin fusion protein system containing six histidines. Nickel affinity columns allowed rapid purification and folding occurred in conditions described above with 0.5 M guanidiniumHCl present. Thrombin cleavage liberated GsMTx4 with three extra amino acids at the N-terminus. The retention time in HPLC analysis and the CD spectrum was similar to wild type. Both the synthetic and cloned peptides were active in the patch clamp assay.

Differential promoter methylation of kinesin family member 1a in plasma is associated with breast cancer and DNA repair capacity.

Methylation alterations of CpG islands, CpG island shores and first exons are key events in the formation and progression of human cancer, and an increasing number of differentially methylated regions and genes have been identified in breast cancer. Recent studies of the breast cancer methylome using deep sequencing and microarray platforms are providing a novel insight on the different roles aberrant methylation plays in molecular subtypes of breast cancer. Accumulating evidence from a subset of studies suggests that promoter methylation of tumor-suppressor genes associated with breast cancer can be quantified in circulating DNA. However, there is a paucity of studies that examine the combined presence of genetic and epigenetic alterations associated with breast cancer using blood-based assays. Dysregulation of DNA repair capacity (DRC) is a genetic risk factor for breast cancer that has been measured in lymphocytes. We isolated plasma DNA from 340 participants in a breast cancer case control project to study promoter methylation levels of five genes previously shown to be associated with breast cancer in frozen tissue and in cell line DNA: MAL, KIF1A, FKBP4, VGF and OGDHL. Methylation of at least one gene was found in 49% of the cases compared to 20% of the controls. Three of the four genes had receiver characteristic operator curve values of ≥ 0.50: MAL (0.64), KIF1A (0.51) and OGDHL (0.53). KIF1A promoter methylation was associated with breast cancer and inversely associated with DRC. This is the first evidence of a significant association between genetic and epigenetic alterations in breast cancer using blood-based tests. The potential diagnostic utility of these biomarkers and their relevance for breast cancer risk prediction should be examined in larger cohorts.

Cigarette smoke induces methylation of the tumor suppressor gene NISCH.

We have previously identified a putative tumor suppressor gene, NISCH, whose promoter is methylated in lung tumor tissue as well as in plasma obtained from lung cancer patients. NISCH was observed to be more frequently methylated in smoker lung cancer patients than in non-smoker lung cancer patients. Here, we investigated the effect of tobacco smoke exposure on methylation of the NISCH gene. We tested methylation of NISCH after oral keratinocytes were exposed to mainstream and side stream cigarette smoke extract in culture. Methylation of the promoter region of the NISCH gene was also evaluated in plasma obtained from lifetime non-smokers and light smokers (<20 pack/year), with and without lung tumors, and heavy smokers (20+ pack/year) without disease. Promoter methylation of NISCH was tested by quantitative fluorogenic real-time PCR in all samples. Promoter methylation of NISCH occurred after exposure to mainstream tobacco smoke as well as to side stream tobacco smoke in normal oral keratinocyte cell lines. NISCH methylation was also detected in 68% of high-risk, heavy smokers without detectable tumors. Interestingly, in light smokers, NISCH methylation was present in 69% of patients with lung cancer and absent in those without disease. Our pilot study indicates that tobacco smoke induces methylation changes in the NISCH gene promoter before any detectable cancer. Methylation of the NISCH gene was also found in lung cancer patients' plasma samples. After confirming these findings in longitudinally collected plasma samples from high-risk populations (such as heavy smokers), examining patients for hypermethylation of the NISCH gene may aid in identifying those who should undergo additional screening for lung cancer.

Correlation between BRAF mutation and promoter methylation of TIMP3, RARß2 and RASSF1A in thyroid cancer.

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARß2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based "mutector assay" was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.

An epigenetic marker panel for detection of lung cancer using cell-free serum DNA.

PURPOSE: We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients. EXPERIMENTAL DESIGN: To determine the analytic sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of 15 gene promoters from 10 patients with primary lung tumors by using quantitative methylation-specific PCR. We then tested this 15-gene set to identify the more useful DNA methylation changes in the serum of a limited number of lung cancer patients and controls. In an independent set, we tested the six most promising genes (APC, CDH1, MGMT, DCC, RASSF1A, and AIM1) for further elucidation of the diagnostic application of this panel of markers. RESULTS: Promoter hypermethylation of at least one of the genes studied was detected in all 10 lung primary tumors. In majority of cases, aberrant methylation in serum DNA was accompanied by methylation in the matched tumor samples. In the independent set, using a single gene that had 100% specificity (DCC), 35.5% (95% CI: 25-47) of the 76 lung cancer patients were correctly identified. For patients without methylated DCC, addition of a logistic regression score that was based on the five remaining genes improved sensitivity from 35.5% to 75% (95% CI: 64-84) but decreased the specificity from 100% to 73% (95% CI: 54-88). CONCLUSION: This approach needs to be evaluated in a larger test set to determine the role of this gene set in early detection and surveillance of lung cancer.

Molecular analysis of plasma DNA for the early detection of lung cancer by quantitative methylation-specific PCR.

PURPOSE: Aberrant promoter hypermethylation of tumor suppressor genes is a promising marker for lung cancer detection. We investigated the likelihood of detecting aberrant DNA methylation of tumor suppressor genes in plasma samples of patients with abnormalities of the lung detected upon computed tomography (CT) scan. EXPERIMENTAL DESIGN: In a small evaluation cohort, four gene promoters (DCC, Kif1a, NISCH, and Rarb) were found to be methylated with increased frequency in samples from cancer patients specifically. We then examined DNA from 93 plasma samples from patients with abnormal findings in the lung detected upon CT scan for aberrant methylation of these four gene promoters by quantitative fluorogenic real-time PCR. The patients were divided into two groups, ground glass opacity (n = 23) and cancerous tumors (n = 70). Plasma DNA from age-matched nodule-free individuals were used as controls (n = 80). RESULTS: In plasma, 73% of patients with cancerous tumors showed methylation of at least one gene with a specificity of 71% (P = 0.0001). Only 22% patients with ground glass opacity exhibited methylation of at least one gene. When smoking history was taken into account, 72% of cancer patients with no smoking history or those who smoked <20 pack-years showed methylation of at least one gene with 100% specificity (P = 0.05) when compared with matched controls. Among heavy smokers with 20+ pack-years of smoking history, 30% of the control group and 73% of the patients with cancerous tumors showed methylation (P = 0.0001). CONCLUSIONS: These biomarkers can distinguish between cancerous and noncancerous abnormal CT findings.

Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.

BACKGROUND: Epigenetic alterations have been implicated in the pathogenesis of solid tumors, however, proto-oncogenes activated by promoter demethylation have been sporadically reported. We used an integrative method to analyze expression in primary head and neck squamous cell carcinoma (HNSCC) and pharmacologically demethylated cell lines to identify aberrantly demethylated and expressed candidate proto-oncogenes and cancer testes antigens in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We noted coordinated promoter demethylation and simultaneous transcriptional upregulation of proto-oncogene candidates with promoter homology, and phylogenetic footprinting of these promoters demonstrated potential recognition sites for the transcription factor BORIS. Aberrant BORIS expression correlated with upregulation of candidate proto-oncogenes in multiple human malignancies including primary non-small cell lung cancers and HNSCC, induced coordinated proto-oncogene specific promoter demethylation and expression in non-tumorigenic cells, and transformed NIH3T3 cells. CONCLUSIONS/SIGNIFICANCE: Coordinated, epigenetic unmasking of multiple genes with growth promoting activity occurs in aerodigestive cancers, and BORIS is implicated in the coordinated promoter demethylation and reactivation of epigenetically silenced genes in human cancers.