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1.  Comparison of Acute Toxicities in Two Primary Chemoradiation Regimens in the Treatment of Advanced Head and Neck Squamous Cell Carcinoma 
Annals of surgical oncology  2012;19(6):1980-1987.
Purpose
The optimal dosage and frequency of platinum-based chemoradiotherapy (CRT) regimen for treating advanced head and neck squamous cell carcinoma remains unresolved. This study aims to compare the toxicity and efficacy of weekly versus more dose-intensive cisplatin-based CRTs.
Methods
We reviewed 155 stage III/IV head and neck squamous cell carcinoma patients with no evidence of distant metastasis treated with one of two CRT regimens from 2000 to 2010 at Greater Baltimore Medical Center. Twice-daily radiation was provided as a split course over a 45-day period. Regimen A consisted of concomitant cisplatin (30 mg/m2/1 h) weekly for 6 cycles; regimen B consisted of concomitant cisplatin (12 mg/m2/1 h) and 5-fluorouracil (600 mg/m2/20 h) on days 1 through 5 and days 29 through 33. Main outcome measures included acute toxicities (myelosuppression, neurotoxicity, nephrotoxicity, gastrointestinal dysfunction), unplanned hospitalizations, and disease control at 12 months.
Results
Patients on regimen A were much less likely to experience ototoxicity due to their treatment (0% vs. 9.8%, P = 0.04). They were more likely to experience thrombocytopenia acutely (46% vs. 26%, P = 0.02), but the toxicity was not limiting (grade 1–2). No significant differences exist in the incidence of other toxicities or unplanned hospitalizations. At 1 year, 97% of patients on A vs. 86% of patients on regimen B were free of disease (P = 0.11).
Conclusions
With concurrent radiotherapy, low-dose, single-agent, weekly cisplatin is less likely than higher-dose daily cisplatin plus 5-fluorouracil provided at the beginning and end of treatment to be associated with ototoxicity. The preliminary data suggest at least equivalent efficacy, but longer follow-up is required.
doi:10.1245/s10434-012-2219-4
PMCID: PMC3715048  PMID: 22290566
2.  AIM1 PROMOTER HYPERMETHYLATION AS A PREDICTOR OF DECREASED RISK OF RECURRENCE FOLLOWING RADICAL PROSTATECTOMY 
The Prostate  2011;72(10):1133-1139.
Purpose
To evaluate the prognostic significance of six epigenetic biomarkers (AIM1, CDH1, KIF1A, MT1G, PAK3 and RBM6 promoter hypermethlation) in a homogeneous group of prostate cancer patients, following radical prostatectomy.
Patients and Methods
Biomarker analyses were performed retrospectively on tumors from 95 prostate cancer patients all with a Gleason score of 3+4=7 and a minimum follow up period of 8 years. Using Quantitative Methylation Specific PCR (QMSP), we analyzed the promoter region of six genes in primary prostate tumor tissues. Time to any progression was the primary endpoint and development of metastatic disease and/or death from prostate cancer was a secondary endpoint. The association of clinicopathological and biomolecular risk factors to recurrence was performed 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 10 years, 48 patients (50.5%) had evidence of recurrence: biochemical/PSA relapse, metastases, or death from prostate cancer. In the final multivariate analysis for time to progression, the significant factors were: older age, HR=0.95 (95% CI: 0.91, 1.0) (P=0.03), positive lymph nodes HR=2.11 (95%CI: 1.05, 4.26) (P=0.04) and decreased hypermethylation of AIM1 HR=0.45 (95%CI: 0.2, 1.0) (P=0.05).
Conclusions
Methylation status of AIM1 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.
doi:10.1002/pros.22461
PMCID: PMC3360823  PMID: 22127895
3.  Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer 
Epigenetics  2012;7(7):710-719.
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, RARβ2, 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.
doi:10.4161/epi.20524
PMCID: PMC3414391  PMID: 22694820
BRAF; RARβ2; RASSF1A; TIMP3; biomarkers; hypermethylation; thyroid cancer; thyroid tissue
4.  Lenalidomide Modulates IL-8 and Anti-Prostate Antibody Levels in Men with Biochemically Recurrent Prostate Cancer 
The Prostate  2011;72(5):487-498.
Background
We retrospectively explored changes in immunological parameters in men with biochemically recurrent prostate cancer treated with either 5mg or 25mg of lenalidomide in a randomized phase 2 trial, and determined whether those changes correlated with disease progression.
Methods
Cytokine levels were compared for each patient at baseline and after 6 months of treatment with lenalidomide. Regression models for correlated data were used to assess associations of cytokine levels with lenalidomide treatment effect. Estimates were obtained using generalized estimating equations (GEE). Changes in circulating anti-prostate antibodies were evaluated using a high-throughput immunoblot technique.
Results
Treatment with lenalidomide was associated with global changes in immune-reactivity to a number of prostate-associated antigens, as well as with changes in circulating levels of the TH2 cytokines IL-4, IL-5, IL-10 and IL-13. Disease progression in treated patients was associated with an increase in circulating IL-8 levels, while IL-8 levels decreased significantly in non-progressors.
Conclusions
Lenalidomide demonstrates immunomodulatory properties in patients with biochemically recurrent prostate cancer. The induction of novel anti-prostate antibodies is a potential mechanism for lenalidomide response. Changes in serum IL-8 levels may serve as a potential biomarker in treated patients. These hypotheses require formal testing in future prospective trials.
doi:10.1002/pros.21449
PMCID: PMC3248613  PMID: 21748755
prostate cancer; antibody; cytokine; IL-8; lenalidomide
5.  Changes in PSA Kinetics Predict Metastasis-Free Survival in Men with PSA-Recurrent Prostate Cancer Treated with Non-Hormonal Agents: Combined Analysis of 4 Phase II Trials 
Cancer  2012;118(6):1533-1542.
Background
Several phase II trials in men with non-castrate PSA-recurrent prostate cancer have assessed the impact of novel non-hormonal agents on PSA kinetics. However, it is unknown whether changes in PSA kinetics influence metastasis-free survival (MFS).
Methods
We performed a retrospective post hoc analysis of 146 men treated in four phase II trials examining the investigational agents marimastat (a matrix metalloproteinase inhibitor; n=39), imatinib (a tyrosine kinase inhibitor; n=25), ATN-224 (a copper/zinc-superoxide dismutase inhibitor; n=22), and lenalidomide (an antiangiogenic/immunomodulatory drug; n=60). We investigated factors influencing MFS, including within-subject changes in PSA kinetics (PSA slope, doubling time, and velocity) before and after treatment initiation.
Results
After a median follow-up of 16.8 months, 70 patients (47.9%) developed metastases. In multivariable Cox regression models, factors that were independently predictive of MFS after adjusting for age and other clinical prognostic variables were baseline PSA doubling time (PSADT) (P=.05), baseline PSA slope (P=.01), on-study change in PSADT (P=.02), and on-study change in PSA slope (P=.03). In a landmark Kaplan-Meier analysis, median MFS was 63.5 months (95% CI 34.6–not reached) and 28.9 months (95% CI 13.5–68.0) for men with or without any decrease in PSA slope by 6 months after treatment, respectively.
Conclusions
This hypothesis-generating analysis suggests that within-subject changes in PSADT and PSA slope after initiation of experimental therapy may correlate with MFS in men with biochemically-recurrent prostate cancer. If validated in prospective trials, changes in PSA kinetics may represent a reasonable intermediate endpoint for screening new agents in these patients.
doi:10.1002/cncr.26437
PMCID: PMC3252493  PMID: 21960118
6.  A non-comparative randomized phase II study of two doses of ATN-224, a copper/zinc superoxide dismutase inhibitor, in patients with biochemically recurrent hormone-naïve prostate cancer 
Urologic oncology  2011;null.
Objective
ATN-224 (choline tetrathiomolybdate) is an oral Cu2+/Zn2+-superoxide dismutase 1 (SOD1) inhibitor with preclinical antitumor activity. We hypothesized that ATN-224 may induce antitumor effects as an antiangiogenic agent at low dose-levels while possessing direct antitumor activity at higher dose-levels. The objective of this study was to screen its clinical activity in patients with biochemically recurrent hormone-naïve prostate cancer.
Methods
Biochemically-recurrent prostate cancer patients with prostate specific antigen doubling times (PSADT) <12 months, no radiographic evidence of metastasis, and no hormonal therapy within 6 months (with serum testosterone levels >150 ng/dL) were eligible. ATN-224 was administered at two dose-levels, 300 mg (n=23) or 30 mg (n=24) daily, by way of randomization. PSA progression was defined as a ≥50% increase (and >5 ng/mL) in PSA from baseline or post-treatment nadir. Endpoints included the proportion of patients who were free of PSA progression at 24 weeks, changes in PSA slope/PSADT, and safety. The study was not powered to detect differences between the two treatment groups.
Results
At 24 weeks, 59% (95% CI 33–82%) of men in the low-dose arm and 45% (95% CI 17–77%) in the high-dose arm were PSA progression-free. Median PSA progression-free survival was 30 weeks (95% CI 21–40+) and 26 weeks (95% CI 24–39+) in the low-dose and high-dose groups, respectively. Pre- and on-treatment PSA kinetics analyses showed a significant mean PSA slope decrease (p=0.006) and a significant mean PSADT increase (p=0.032) in the low-dose arm only. Serum ceruloplasmin levels, a biomarker for ATN-224 activity, were lowered in the high-dose group, but did not correlate with PSA changes.
Conclusions
Low-dose ATN-224 (30 mg daily) may have biologic activity in men with biochemically-recurrent prostate cancer, as suggested by an improvement in PSA kinetics. However, the clinical significance of PSA kinetics changes in this patient population remains uncertain. The absence of a dose-response effect also reduces enthusiasm, and there are currently no plans to further develop this agent in prostate cancer.
doi:10.1016/j.urolonc.2011.04.009
PMCID: PMC3227793  PMID: 21816640
7.  Multidisciplinary Service Utilization Pattern by Advanced Head and Neck Cancer Patients: A Single Institution Study 
Purpose. To analyze the patterns and associations of adjunctive service visits by head and neck cancer patients receiving primary, concurrent chemoradiation therapy. Methods. Retrospective chart review of patients receiving adjunctive support during a uniform chemoradiation regimen for stages III-IV head and neck squamous cell carcinoma. Univariate and multivariate models for each outcome were obtained from simple and multivariate linear regression analyses. Results. Fifty-two consecutive patients were assessed. Female gender, single marital status, and nonprivate insurance were factors associated with an increased number of social work visits. In a multivariate analysis, female gender and marital status were related to increased social work services. Female gender and stage IV disease were significant for increased nursing visits. In a multivariate analysis for nursing visits, living greater than 20 miles between home and hospital was a negative predictive factor. Conclusion. Treatment of advanced stage head and neck cancer with concurrent chemoradiation warrants a multidisciplinary approach. Female gender, single marital status, and stage IV disease were correlated with increased utilization of social work and nursing services. Distance over 20 miles from the center was a negative factor. This information may help guide the treatment team to allocate resources for the comprehensive care of patients.
doi:10.1155/2012/628578
PMCID: PMC3483826  PMID: 23118755
8.  Lenalidomide in Nonmetastatic Biochemically Relapsed Prostate Cancer: Results of a Phase I/II Double-Blinded, Randomized Study 
Purpose
To evaluate the safety and activity of 6 months of treatment with lenalidomide at 5 or 25 mg/d in nonmetastatic biochemically relapsed prostate cancer.
Experimental Design
Sixty men with non-castrate, nonmetastatic, biochemically relapsed prostate cancer were stratified by prostate-specific antigen (PSA) doubling time, surgery/radiation therapy, prior androgen deprivation therapy (ADT), and randomized to lenalidomide 5 mg (n = 26) or 25 mg/d (n = 34) for 3 weeks repeated monthly for 6 months or until dose-limiting toxicity or disease progression. Toxicity was evaluated monthly, and PSAs and X-rays/scans every 6 months. Study size was determined to detect a progression rate of 40% at 6 months in either arm with 85% power (compared with a rate of 80% in the population receiving no treatment). Changes in PSA slopes were calculated using the regression of the log PSA for each patient before and during the initial 6 months and compared by t test.
Results
Baseline variables were balanced between arms. Grade 3/4 toxicity rates were 12% (n = 3) with 5 mg and 29% (n = 10) with 25 mg (P = 0.1), most commonly neutropenia (five patients, all on 25 mg). Two patients per arm had thromboembolic events. The change in PSA slope was greater with 25 mg versus 5 mg [−0.172 (−0.24 to −0.11) versus −0.033 (−0.11 to 0.04); P = 0.005]. With a mean follow-up of 31.4 months (range 14–44), five patients on 25 mg and one patient on 5 mg remain on the study.
Conclusions
Lenalidomide has acceptable toxicity and is associated with long-term disease stabilization and PSA declines. Randomized studies evaluating conventional clinical disease end points in this patient population are planned.
doi:10.1158/1078-0432.CCR-10-1928
PMCID: PMC3444815  PMID: 20978144
9.  High-dose Cyclophosphamide and Rituximab without Stem Cell Transplantation: a Feasibility Study for Low Grade B cell, Transformed and Mantle Cell Lymphomas 
Leukemia & lymphoma  2011;52(11):2076-2081.
Relapse after autologous stem cell transplantation for low-grade B-cell lymphoma is common secondary to ineffective conditioning and/or tumor autograft contamination. We investigated high-dose cyclophosphamide and rituximab without stem cell rescue as first-line or salvage-therapy in lymphomas. After establishing safety, accrual was increased to evaluate event-free survival (EFS). 81 adults received rituximab [375mg/mm days 1, 4, 8, 11, 45, 52], cyclophosphamide [50mg/kg days 15-18] and pegfilgrastim (day 20). Forty-two patients had low-grade B-cell lymphoma [grade I/II follicular (69%), transformed lymphoma (17%), other (15%)]: 45% were treated without measurable disease. Thirty-nine patients had mantle cell lymphoma: 82% were treated without measurable disease. All achieved hematopoietic recovery; 46% required brief hospitalizations. The 5 year EFS and overall survival (OS) for low-grade B-cell and transformed patients was 40% and 72%, respectively. The 5 year EFS and OS for the MCL patients was 39% and 62%, respectively. This low-toxicity therapeutic approach obviates the need for stem cell products and establishes a platform for future therapies.
doi:10.3109/10428194.2011.594191
PMCID: PMC3390263  PMID: 21756035
Cyclophosphamide; NHL; Mantle Cell Lymphoma
10.  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 analytical sensitivity, we examined the tumor and the matched serum DNA for aberrant methylation of fifteen 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 AIM) 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.
doi:10.1158/1078-0432.CCR-10-3436
PMCID: PMC3131425  PMID: 21610147
DNA methylation/epigenetics; serum; lung cancer
11.  Hyperfractionated Radiotherapy with Concurrent Cisplatin/5-Fluorouracil for Locoregional Advanced Head and Neck Cancer: Analysis of 105 Consecutive Patients 
Objective. We reviewed a cohort of patients with previously untreated locoregional advanced head and neck squamous cell carcinoma (HNSCC) who received a uniform chemoradiotherapy regimen. Methods. Retrospective review was performed of 105 patients with stage III or IV HNSCC treated at Greater Baltimore Medical Center from 2000 to 2007. Radiation included 125 cGy twice daily for a total 70 Gy to the primary site. Chemotherapy consisted of cisplatin (12 mg/m2/h) daily for five days and 5-fluorouracil (600 mg/m2/20 h) daily for five days, given with weeks one and six of radiation. All but seven patients with N2 or greater disease received planned neck dissection after chemoradiotherapy. Primary outcomes were overall survival (OS), locoregional control (LRC), and disease-free survival (DFS). Results. Median followup of surviving patients was 57.6 months. Five-year OS was 60%, LRC was 68%, and DFS was 56%. Predictors of increased mortality included age ≥55, female gender, hypopharyngeal primary, and T3/T4 stage. Twelve patients developed locoregional recurrences, and 16 patients developed distant metastases. Eighteen second primary malignancies were diagnosed in 17 patients. Conclusions. The CRT regimen resulted in favorable outcomes. However, locoregional and distant recurrences cause significant mortality and highlight the need for more effective therapies to prevent and manage these events.
doi:10.1155/2012/754191
PMCID: PMC3388433  PMID: 22778748
12.  Characterization of chronic myeloid leukemia stem cells 
American journal of hematology  2011;86(1):31-37.
Though tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34+ stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34+CD38− CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34+CD38−ALDHhigh CML cells, similar to the normal CD34+CD38−ALDHhigh cells and less than in the total CML CD34+ cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34+ cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34+ subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34+ population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising.
doi:10.1002/ajh.21915
PMCID: PMC3010878  PMID: 21132730
chronic myeloid leukemia; CML; leukemia stem cell; WT1; PRAME
13.  Improved survival with inhibitory Killer Immunoglobulin Receptor (KIR) gene mismatches and KIR haplotype B donors after nonmyeloablative, HLA-haploidentical bone marrow transplantation 
Natural killer (NK) cell alloreactivity, which may contribute to the graft-versus-leukemia effect of allogeneic hematopoietic stem cell transplantation (HSCT), is influenced by the interaction of killer-cell immunoglobulin-like receptors (KIRs) on donor NK cells and their ligands, human leukocyte antigen (HLA) Class I molecules on recipient antigen-presenting cells. Distinct models to predict NK cell alloreactivity differ in their incorporation of information from typing of recipient and donor KIR and HLA gene loci, which exist on different autosomes and are inherited independently as haplotypes. Individuals may differ in the inheritance of the two KIR haplotypes, A and B, or in the expression of individual KIR genes. Here, we examined the effect of KIR and HLA genotype, in both the recipient and donor, on the outcome of 86 patients with advanced hematologic malignancies who received nonmyeloablative, HLA-haploidentical HSCT with high-dose, post-transplantation cyclophosphamide (Cy). Compared to recipients of marrow from donors with identical KIR gene content, recipients of inhibitory KIR (iKIR) gene-mismatched marrow had an improved overall survival (HR=0.37; CI: 0.21- 0.63; p=0.0003), event-free survival (HR=0.51; CI: 0.31- 0.84; p=0.01), and relapse rate (cause specific hazard ratio, SDHR=0.53; CI: 0.31-0.93; p=0.025). Patients homozygous for the KIR “A” haplotype, which encodes only one activating KIR, had an improved overall survival (HR=0.30; CI: 0.13-10.69; p=.004), event-free survival (HR=0.47; CI: 0.22-1.00; p=.05) and non-relapse mortality (NRM; cause specific HR=0.13; CI: 0.017-0.968; p=0.046) if their donor expressed at least one KIR B haplotype, which encodes several activating KIRs. Models that incorporated information from recipient HLA typing, with or without donor HLA typing, were not predictive of outcome in this patient cohort. Thus, nonmyeloablative conditioning and T cell-replete, HLA-haploidentical HSCTs involving iKIR gene mismatches between donor and recipient, or KIR haplotype AA recipients of marrow from KIR Bx donors, were associated with lower relapse and NRM and improved overall and event-free survival. These findings suggest that selection of donors based upon inhibitory KIR gene or haplotype incompatibility may be warranted.
doi:10.1016/j.bbmt.2009.11.022
PMCID: PMC2848533  PMID: 19961944
14.  Phase II, Randomized, Placebo-Controlled Trial of Neoadjuvant Celecoxib in Men With Clinically Localized Prostate Cancer: Evaluation of Drug-Specific Biomarkers 
Journal of Clinical Oncology  2009;27(30):4986-4993.
Purpose
Cyclooxygenase-2 (COX-2) is a potential pharmacologic target for the prevention of various malignancies, including prostate cancer. We conducted a randomized, double-blind trial to examine the effect of celecoxib on drug-specific biomarkers from prostate tissue obtained at prostatectomy.
Patients and Methods
Patients with localized prostate cancer and Gleason sum ≥ 7, prostate-specific antigen (PSA) ≥ 15 ng/mL, clinical stage T2b or greater, or any combination with greater than 45% risk of capsular penetration were randomly assigned to celecoxib 400 mg by mouth twice daily or placebo for 4 to 6 weeks before prostatectomy. The primary end point was the difference in prostatic prostaglandin levels between the two groups. Secondary end points were differences in COX-1 and -2 expressions; oxidized DNA bases; and markers of proliferation, apoptosis and angiogenesis. Tissue celecoxib concentrations also were measured. Tertiary end points were drug safety and compliance.
Results
Seventy-three patients consented, and 64 were randomly assigned and included in the intention-to-treat analysis. There were no treatment differences in any of the primary or secondary outcomes. Multivariable regression revealed that tumor tissue had significantly lower COX-2 expression than benign prostatic tissue (P = .01) and significantly higher levels of the proliferation marker Ki-67 (P < .0001). Celecoxib was measurable in prostate tissue of patients on treatment, demonstrating that celecoxib reached its target. Celecoxib was safe and resulted in only grade 1 toxicities.
Conclusion
Treatment with 4 to 6 weeks of celecoxib had no effect on intermediate biomarkers of prostate carcinogenesis, despite the achievement of measurable tissue levels. We caution against using celecoxib 400 mg twice daily as a preventive agent for prostate cancer in additional studies.
doi:10.1200/JCO.2009.21.9410
PMCID: PMC2799055  PMID: 19720908
15.  Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors 
PLoS ONE  2010;5(5):e10828.
Background
Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected.
Methodology
DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results.
Principal Findings
The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3.
Conclusions/Significance
Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors.
doi:10.1371/journal.pone.0010828
PMCID: PMC2877085  PMID: 20520817
16.  A Multiple Loop, Double Cube Microarray Design Applied to Prostate Cancer Cell Lines with Variable Sensitivity to Histone Deacetylase Inhibitors 
Purpose
Although microarray technology has been widely adopted by the scientific community, analysis of the ensuing data remains challenging. In this paper we present our experience with a complex design microarray experiment on resistance mechanisms of histone deacetylase inhibitors (HDACIs).
Experimental design
To improve our understanding of the underlying mechanism of HDACI resistance in prostate cancer (PCa) cells, we designed a novel “multiple loop, double cube” cDNA microarray experiment. In the experiment of 22 arrays, DU145 and PC3 cells were treated with two different HDACIs (vorinostat, and Valproic Acid, VPA) and incubation periods (48 and 96 hours). Pre-processing included exploratory analyses of the quality of the arrays and intensity-dependent within-array loess normalization. An ANOVA model was used for inference. The results were validated by western blot analysis of known treatment targets.
Results
Treatment of PC3 and DU145 cells with HDACIs caused 2.8–10% (p<.001) differential expression across conditions. 51–73% of these genes were up-regulated and 28–49% down-regulated. The extent of differential expression was associated with: cell line (DU145>PC3), HDACI (VPA≥vorinostat) and duration of treatment (96h>48h). We identified known and new treatment targets involved in cell cycle and apoptosis.
Conclusion
A multiple loop, double cube microarray design can be used to identify HDACI induced changes in gene expression, possibly related to drug resistance.
doi:10.1158/1078-0432.CCR-08-0119
PMCID: PMC2603330  PMID: 18980983
17.  TIMP3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer 
The Journal of urology  2007;179(2):743-747.
Purpose
Tissue inhibitor of metalloproteinases-3 (TIMP-3) is one of four members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases (MMP). We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from bladder cancer patients 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 PCR.
Results
Using an optimal cutoff value by Taqman quantitation, we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (hazard ratio [HR] =1.99, 95% confidence interval [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 risk of death and stage 3 tumor (HR=2.73, 95% CI=1.58 to 4.72; p=0.003 and the presence of 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).
Conclusion
These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.
doi:10.1016/j.juro.2007.09.019
PMCID: PMC2674621  PMID: 18082200
18.  HLA-Haploidentical Bone Marrow Transplantation for Hematologic Malignancies Using Nonmyeloablative Conditioning and High-Dose, Posttransplantation Cyclophosphamide 
We evaluated the safety and efficacy of high-dose, posttransplantation cyclophosphamide (Cy) to prevent graft rejection and graft-versus-host disease (GVHD) after outpatient nonmyeloablative conditioning and T cell-replete bone marrow transplantation from partially HLA-mismatched (haploidentical) related donors. Patients with advanced hematologic malignancies (n = 67) or paroxysmal nocturnal hemoglobinuria (n = 1) received Cy 50 mg/kg i.v. on day 3 (n = 28) or on days 3 and 4 (n 5 40) after transplantation. The median times to neutrophil (>500/µL) and platelet recovery (>20,000/µL) were 15 and 24 days, respectively. Graft failure occurred in 9 of 66 (13%) evaluable patients, and was fatal in 1. The cumulative incidences of grades II–IV and grades III–IV acute (aGVHD) by day 200 were 34% and 6%, respectively. There was a trend toward a lower risk of extensive chronic GVHD (cGVHD) among recipients of 2 versus 1 dose of posttransplantation Cy (P = .05), the only difference between these groups. The cumulative incidences of nonrelapse mortality (NRM) and relapse at 1 year were 15% and 51%, respectively. Actuarial overall survival (OS) and event-free survival (EFS) at 2 years after transplantation were 36% and 26%, respectively. Patients with lymphoid malignancies had an improved EFS compared to those with myelogenous malignancies (P = .02). Nonmyeloablative HLA-haploidentical BMT with posttransplantation Cy is associated with acceptable rates of fatal graft failure and severe aGVHD or cGVHD.
doi:10.1016/j.bbmt.2008.03.005
PMCID: PMC2633246  PMID: 18489989
Bone marrow transplantation; Cyclophosphamide; Histocompatibility antigens; Conditioning regimens; Leukemia; Lymphoma
19.  Alterations of GPI transamidase subunits in head and neck squamous carcinoma 
Molecular Cancer  2007;6:74.
Background
GPI anchor attachment is catalyzed by the GPI transamidase (GPIT) complex. GAA1, PIG-T and PIG-U are the three of five GPIT subunits. Previous studies demonstrated amplification and overexpression of GPIT subunits in bladder and breast cancer with oncogenic function. We performed an analysis of these subunits in head and neck squamous cell carcinoma (HNSCC).
Results
To evaluate GAA1, PIG-T and PIG-U in HNSCC, we used quantitative PCR (QPCR) and quantitative RT-PCR (QRT-PCR) to determine the copy number of those genes in primary tumors and the matching lymphocytes in 28 patients with HNSCC and quantified RNA expression of those genes in 16 primary HNSCC patients and 4 normal control tissue samples. GAA1 showed a significant increase in normalized mRNA expression, 2.11 (95% CI: 1.43, 2.79), in comparison to that of normal controls, 0.43 (95% CI: -0.76, 1.61), p = 0.014 (Mann-Whitney test). The mean genomic copy number of GAA1 was significantly increased in HNSCC, 0.59 (95% CI: 0.50, 0.79), in comparison to lymphocyte DNA, 0.35 (95% CI: 0.30, 0.50), p = 0.001 (paired t-test).
Conclusion
An increased expression level and elevated copy number for GAA1 suggest a role for this GPI anchor subunit in HNSCC.
doi:10.1186/1476-4598-6-74
PMCID: PMC2204038  PMID: 18028549
20.  Pre-processing Agilent microarray data 
BMC Bioinformatics  2007;8:142.
Background
Pre-processing methods for two-sample long oligonucleotide arrays, specifically the Agilent technology, have not been extensively studied. The goal of this study is to quantify some of the sources of error that affect measurement of expression using Agilent arrays and to compare Agilent's Feature Extraction software with pre-processing methods that have become the standard for normalization of cDNA arrays. These include log transformation followed by loess normalization with or without background subtraction and often a between array scale normalization procedure. The larger goal is to define best study design and pre-processing practices for Agilent arrays, and we offer some suggestions.
Results
Simple loess normalization without background subtraction produced the lowest variability. However, without background subtraction, fold changes were biased towards zero, particularly at low intensities. ROC analysis of a spike-in experiment showed that differentially expressed genes are most reliably detected when background is not subtracted. Loess normalization and no background subtraction yielded an AUC of 99.7% compared with 88.8% for Agilent processed fold changes. All methods performed well when error was taken into account by t- or z-statistics, AUCs ≥ 99.8%. A substantial proportion of genes showed dye effects, 43% (99%CI : 39%, 47%). However, these effects were generally small regardless of the pre-processing method.
Conclusion
Simple loess normalization without background subtraction resulted in low variance fold changes that more reliably ranked gene expression than the other methods. While t-statistics and other measures that take variation into account, including Agilent's z-statistic, can also be used to reliably select differentially expressed genes, fold changes are a standard measure of differential expression for exploratory work, cross platform comparison, and biological interpretation and can not be entirely replaced. Although dye effects are small for most genes, many array features are affected. Therefore, an experimental design that incorporates dye swaps or a common reference could be valuable.
doi:10.1186/1471-2105-8-142
PMCID: PMC1876252  PMID: 17472750

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