Eli Rosenbaum

Quantitative Methylation-Specific Polymerase Chain Reaction Gene Patterns in Urine Sediment Distinguish Prostate Cancer Patients From Control Subjects

PURPOSE Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of prostate cancers and is a promising marker for cancer detection. We sought to develop a test for prostate cancer based on a quantitative methylation-specific polymerase chain reaction (QMSP) of multiple genes in urine sediment DNA. PATIENTS AND METHODS We tested urine sediment DNA for aberrant methylation of nine gene promoters (p16INK4a, p14(ARF), MGMT, GSTP1, RARbeta2, CDH1 [E-cadherin], TIMP3, Rassf1A, and APC) from 52 patients with prostate cancer and 21 matched primary tumors by quantitative fluorogenic real-time polymerase chain reaction. We also analyzed urine sediments from 91 age-matched individuals without any history of genitourinary malignancy as controls. RESULTS Promoter hypermethylation of at least one of the genes studied was detected in urine samples from all 52 prostate cancer patients. Urine samples from the 91 controls without evidence of genitourinary cancer revealed no methylation of the p16, ARF, MGMT, and GSTP1 gene promoters, whereas methylation of RARbeta2, TIMP3, CDH1, Rassf1A, and APC was detected at low levels. CONCLUSION Overall, methylation found in urine samples matched the methylation status in the primary tumor. A combination of only four genes (p16, ARF, MGMT, and GSTP1) would theoretically allow us to detect 87% of prostate cancers with 100% specificity. Our data support further development of the noninvasive QMSP assay in urine DNA for early detection and surveillance of prostate cancer.

Mutational analysis of BRAF in Fine needle aspiration biopsies of the thyroid: A potential application for the preoperative assessment of thyroid nodules

Background: Fine needle aspiration (FNA) is routinely used in the preoperative evaluation of thyroid nodules, but subsequent patient management is often complicated by the inability to decisively recognize malignancy on cytologic grounds alone. Activating mutations of the BRAF oncogene commonly occur in papillary thyroid carcinomas (PTCs) but not in other types of benign and malignant thyroid lesions. Mutational analysis of FNAs could enhance selection of thyroid nodules for surgical removal. Methods: Ninety-five excised PTCs along with 49 corresponding FNAs were evaluated for BRAF mutations by a newly developed assay that uses a novel primer extension method (MutectorR assay) and by direct sequencing. An additional 42 FNAs from thyroid nodules that were excised based on a suspicion of malignancy were also evaluated. Results:BRAF mutations were identified in 36 of the 95 (38%) excised PTCs. By histological subtype, BRAF mutations were more common in conventional PTCs than in the follicular variant (67% versus 12%; P < 0.0001; χ2). Analysis of the preoperative FNAs accurately reflected BRAF status of the resected PTC in 46 of the 49 paired samples (94% concordance). In FNA samples grouped according to the preoperative cytologic findings (malignant, n = 25; benign, n = 11; and indeterminate, n = 55), a BRAF mutation confirmed the diagnosis of PTC in 72% of carcinomas within the malignant group, and it established the diagnosis of PTC in 16% of carcinomas within the indeterminate group. BRAF mutations were not detected in FNAs from 32 benign thyroid lesions. Direct sequencing and the MutectorR assay yielded completely concordant results. Conclusions:BRAF mutations are common in conventional PTCs, and they are specific for PTC. A BRAF mutation can be reliably detected in cells aspirated from a thyroid nodule suggesting a role for this marker in the preoperative evaluation of thyroid nodules.

Detection of aberrant methylation of four genes in plasma DNA for the detection of breast cancer.

PURPOSE Novel approaches to breast cancer screening are necessary, especially in the developing world where mammography is not feasible. In this study, we explored the hypothesis that blood-based biomarkers have potential for biomarkers for breast cancer. PATIENTS AND METHODS We first determined the frequency of aberrant methylation of four candidate genes (APC, GSTP1, Rassf1A, and RARbeta2) in primary breast cancer tissues from West African women with predominantly advanced cancers. We used a high-throughput DNA methylation assay (quantitative methylation-specific polymerase chain reaction) to examine plasma from 93 women with breast cancer and 76 controls for the presence of four methylated genes. Samples were randomly divided evenly into training and validation data sets. Cutoff values for gene positivity of the plasma-based assay and the gene panel were determined by receiver operating characteristic curves in the training data set and subsequently evaluated as a screening tool in the validation data set. RESULTS Methylation of at least one gene resulted in a sensitivity of 62% and a specificity of 87%. Moreover, the assay successfully detected 33% (eight of 24) of early-stage tumors. CONCLUSION These data suggest that epigenetic markers in plasma may be of interest for detection of breast cancer. Identification of additional breast cancer specific methylated genes with higher prevalence in early stage cancers would improve this approach.

Association of aberrant methylation of tumor suppressor genes with tumor aggressiveness and BRAF mutation in papillary thyroid cancer

The role of aberrant tumor suppressor gene methylation in the aggressiveness of papillary thyroid cancer (PTC) has not been documented. By showing promoter methylation‐induced gene silencing in PTC‐derived cell lines, we first demonstrated the functional consequence of methylation of several recently identified tumor suppressor genes, including those for tissue inhibitor of metalloproteinase‐3 (TIMP3), SLC5A8, death‐associated protein kinase (DAPK) and retinoic acid receptor β2 (RARβ2). We then investigated the role of methylation of these genes in the aggressiveness of PTC by examining the relationship of their aberrant methylation to clinicopathological characteristics and BRAF mutation in 231 primary PTC tumors. Methylation of TIMP3, SLC5A8 and DAPK was significantly associated with several aggressive features of PTC, including extrathyroidal invasion, lymph node metastasis, multifocality and advanced tumor stages. Methylation of these genes was also significantly associated with BRAF mutation in PTC, either individually or collectively in various combinations. Methylation of these genes, either individually or collectively, occurred more frequently in more aggressive classical and tall‐cell PTC subtypes than in less aggressive follicular‐variant PTC, with the latter known to infrequently harbor BRAF mutation. Several other tumor suppressor genes investigated were not methylated. These results suggest that aberrant methylation and hence silencing of TIMP3, SLC5A8, DAPK and RARβ2, in association with BRAF mutation, may be an important step in PTC tumorigenesis and progression.

BRAF mutations in anaplastic thyroid carcinoma: implications for tumor origin, diagnosis and treatment

Anaplastic thyroid carcinoma is a highly aggressive neoplasm. Affected patients typically present with advanced disease where there is little hope for cure using conventional therapeutic modalities. Understanding the genetic alterations underlying the development of anaplastic thyroid carcinoma, such as mutational activation of BRAF, could help clarify its relationship with well-differentiated forms of thyroid carcinoma (ie follicular and papillary carcinoma) and could help select patients most likely to benefit from novel therapeutic strategies targeting BRAF. We tested 16 anaplastic thyroid carcinomas for the thymine (T) → adenine (A) missense mutation at nucleotide 1796 in the BRAF gene using a newly developed assay that employs a novel primer extension method (Mutector® assay). Seven of these anaplastic thyroid carcinomas arose in association with a well-differentiated thyroid carcinoma, and these were also evaluated. The 1796T → A mutation was detected in eight (50%) of the anaplastic thyroid carcinomas, in four of five (80%) associated papillary thyroid carcinomas, and in zero of two (0%) associated follicular carcinomas. In all seven cases where anaplastic thyroid carcinoma arose in association with a well-differentiated thyroid carcinoma, BRAF status in the two components was concordant. Like papillary thyroid carcinoma, a significant percentage of anaplastic thyroid carcinomas also harbor BRAF mutations. Indeed, when papillary thyroid carcinoma and anaplastic thyroid carcinoma occur together, they consistently share the same BRAF profile, supporting the notion that many anaplastic thyroid carcinomas actually represent progressive malignant degeneration of a pre-existing well-differentiated thyroid carcinoma. The high frequency of BRAF mutations in a tumor that is generally regarded as uniformly fatal justifies evaluation of the potential benefits of anti-BRAF therapy for patients with anaplastic thyroid carcinoma.

Promoter Hypermethylation as an Independent Prognostic Factor for Relapse in Patients with Prostate Cancer Following Radical Prostatectomy

Purpose: To analyze the prognostic significance of six epigenetic biomarkers (APC, Cyclin D2, GSTP1, TIG1, Rassf1A, and RARβ2 promoter hypermethylation) in a homogeneous group of prostate cancer patients, following radical prostatectomy alone. Patients and Methods: Biomarker analyses were done retrospectively on tumors from 74 prostate cancer patients all with a Gleason score of 3 + 4 = 7 and minimum follow-up period of 7 years. Using quantitative methylation-specific PCR, we analyzed six gene promoters in primary prostate tumor tissues. Time to any progression was the primary end point, and development of metastatic disease and/or death from prostate cancer was a secondary point. The association of clinicopathologic and biomolecular risk factors to recurrence was done using the log-rank test and Cox proportional hazards model for multivariate analysis. To identify independent prognostic factors, a stepwise selection method was used. Results: At a median follow-up time of 9 years, 37 patients (50%) had evidence of recurrence: biochemical/prostate-specific antigen relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression (TTP), the significant factors were age > 60 [hazard ratio (HR), 0.4; 95% confidence interval (95% CI), 0.2-0.8; P = 0.01], hypermethylation of GSTP1 (HR, 0.23; 95% CI; 0.09-0.64; P = 0.004), and hypermethylation of APC (HR, 3.0; 95% CI, 1.42-6.32; P = 0.004). In another multivariate analysis, a profile of hypermethylation of APC and cyclin D2 hypermethylation was significant as well: if either any one was hypermethylated (HR, 1.84; 95% CI, 0.92-3.72; P = 0.09) or if both were hypermethylated (HR, 4.3; 95% CI, 1.52-12.33; P = 0.01). Conclusions: Methylation status of selected genes in the prostate cancer specimen may predict for time to recurrence in Gleason 3 + 4 = 7 patients undergoing prostatectomy. These results should be validated in a larger and unselected cohort.

Exon 15 BRAF Mutations Are Uncommon in Melanomas Arising in Nonsun-Exposed Sites

Purpose: An activating point mutation of the BRAF oncogene has been identified in a high proportion of cutaneous nevi and cutaneous melanomas, but its frequency in melanomas arising from the mucosa of head and neck is unknown. Experimental Design: We tested 17 malignant mucosal melanomas of the head and neck for the thymine (T)→adenine (A) missense mutation at nucleotide 1796 in the BRAF gene using direct sequencing and a newly developed assay that uses a novel primer extension method (Mutector assay). We also tested 21 cutaneous melanomas, including 13 arising from sun-exposed sites and 8 from a nonsun-exposed site, the vulvar skin. Results: The 1796T→A mutation was detected in only 1 (6%) of the sinonasal melanomas. As for cutaneous melanomas, a BRAF mutation was detected in 8 (62%) of the tumors arising in sun-exposed sites but in none (0%) of vulvar melanomas. Conclusions: In contrast to cutaneous melanomas arising in sun-exposed sites, mucosal melanomas of the head and neck do not frequently harbor an activating mutation of BRAF. This finding additionally supports the view that the various subtypes of melanoma are not equivalent and that distinct genetic alterations may underlie well recognized differences in risk factors and behavioral patterns. Accordingly, patients with melanomas should not be collectively regarded as a uniform group as new strategies are developed that target specific genetic alterations.

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, Pearsons 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.

Feasibility of quantitative PCR-based saliva rinse screening of HPV for head and neck cancer

Human papilloma virus (HPV) 16 is likely to be an etiologic factor in a subset of head and neck squamous cell carcinomas (HNSC). We investigated the ability of a quantitative PCR‐based assay to detect HPV 16 in salivary rinses as a screening method for HNSC. Real time quantitative PCR was applied to detect HPV16 E6 and E7 DNA level in 92 primary tumors and salivary rinses from patients with HNSC and 604 control subjects without HNSC. A total of 45.6% (42/92) of primary HNSC and 32.6% (30/92) of saliva rinse samples from HNSC patients had any detectable HPV 16 DNA, 30.4% (28/92) of tumors had HPV 16 levels >0.1 copies/cell. HPV16 was detected in 50% (confidence interval [CI] = 34.2–65.8%) (21/42) of the saliva rinse samples from HNSC patients with detectable HPV16 level in their tissue, 18% (CI = 8.4–30.9%) (9/50) of saliva rinse samples from patients with HPV 16 negative primary HNSC, in 2.8% (CI = 1.7–4.5%) (17/604) of the normal controls (p < 0.001). Using a cutoff of HPV 16 >0.001 copies/cell in saliva rinse yielded a sensitivity of 30.4% and a specificity of 98.3%. Nonsmokers had significant higher HPV 16 level than smokers in the cohort of cancer patients. HPV 16 DNA in saliva rinses can reflect HPV 16 status of primary HNSC. Quantitative analysis of HPV 16 DNA in salivary rinses allows for detection of HPV‐related HNSC, however, specific limitations exist that prevent the application of this as a screening technique for a broad population.

Pretreatment Neutrophil-to-Lymphocyte Ratio in Metastatic Castration-Resistant Prostate Cancer Patients Treated with Ketoconazole: Association with Outcome and Predictive Nomogram

BACKGROUND The neutrophil-to-lymphocyte ratio (NLR), an inflammation marker, is prognostic in several cancers. We assessed the association between the pretreatment NLR and outcome of patients with metastatic castration-resistant prostate cancer (mCRPC) treated with the CYP17 inhibitor ketoconazole. METHODS This was an international, retrospective study of 156 mCRPC patients treated with ketoconazole. The independent effect of the pretreatment NLR and factors associated with treatment outcome were determined by multivariate analysis. RESULTS Seventy-eight patients (50%) had a ≥50% decline in prostate-specific antigen (PSA). The median progression-free survival (PFS) time was 8 months. Excluded from the analysis were 23 patients without available data on their NLR and those with a recent health event or treatment associated with a blood count change. Sixty-two patients (47%) had a pretreatment NLR >3. Risk factors associated with the PFS outcome were a pretreatment NLR >3 and PSA doubling time (PSADT) <3 months and a prior response to a gonadotropin-releasing hormone agonist of <24 months or to an antiandrogen of <6 months. The number of risk factors was used to form a predictive nomogram by patient categorization into favorable (zero or one factor), intermediate (two factors), and poor (three or four factors) risk groups. CONCLUSIONS In mCRPC patients treated with ketoconazole, the pretreatment NLR and PSADT, and prior response to androgen-deprivation therapy, may be associated with the PFS time and used to form a risk stratification predictive nomogram.