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1.  The evolution of treatment strategies for patients with chronic myeloid leukemia relapsing after allogeneic bone marrow transplantation: Can tyrosine kinase inhibitors replace donor lymphocyte infusions? 
Leukemia & lymphoma  2014;56(1):128-134.
The optimal treatment for chronic myeloid leukemia (CML) relapsing following allogeneic bone marrow transplantation (alloBMT) is unknown. We performed a single-center retrospective analysis of 71 consecutive patients undergoing alloBMT for CML from 1995–2008. A multi-state model was used to quantify the cumulative incidences of complete molecular response (CMR) and death following alloBMT. The primary analysis was the comparison of three treatment interventions (tyrosine kinase inhibitor: TKI, donor lymphocyte infusion: DLI, and TKI+DLI) for relapsed disease post-alloBMT. Forty-five (63%) patients relapsed post-alloBMT (molecular relapse: n=16, cytogenetic relapse: n=20, hematologic relapse: n=2, advanced phase relapse: n=7) and 40 patients underwent one of three treatments: TKI-only (n=13), DLI-only (n=11), or TKI+DLI (n=16). Although not statistically significant, the TKI-only group had the highest cumulative incidence of CMR and the lowest cumulative incidence of death compared to DLI and TKI+DLI. These data support the finding that TKI therapy is active in the post-alloBMT setting.
PMCID: PMC4268088  PMID: 24712979
chronic myeloid leukemia; relapse; allogeneic transplantation
2.  Combining the pan-aurora kinase inhibitor AMG 900 with histone deacetylase inhibitors enhances antitumor activity in prostate cancer 
Cancer Medicine  2014;3(5):1322-1335.
Histone deacetylase inhibitors (HDACIs) are being tested in clinical trials for the treatment of solid tumors. While most studies have focused on the reexpression of silenced tumor suppressor genes, a number of genes/pathways are downregulated by HDACIs. This provides opportunities for combination therapy: agents that further disable these pathways through inhibition of residual gene function are speculated to enhance cell death in combination with HDACIs. A previous study from our group indicated that mitotic checkpoint kinases such as PLK1 and Aurora A are downregulated by HDACIs. We used in vitro and in vivo xenograft models of prostate cancer (PCA) to test whether combination of HDACIs with the pan-aurora kinase inhibitor AMG 900 can synergistically or additively kill PCA cells. AMG 900 and HDACIs synergistically decreased cell proliferation activity and clonogenic survival in DU-145, LNCaP, and PC3 PCA cell lines compared to single-agent treatment. Cellular senescence, polyploidy, and apoptosis was significantly increased in all cell lines after combination treatment. In vivo xenograft studies indicated decreased tumor growth and decreased aurora B kinase activity in mice treated with low-dose AMG 900 and vorinostat compared to either agent alone. Pharmacodynamics was assessed by scoring for phosphorylated histone H3 through immunofluorescence. Our results indicate that combination treatment with low doses of AMG 900 and HDACIs could be a promising therapy for future clinical trials against PCA.
PMCID: PMC4302682  PMID: 24989836
AMG 900; aurora kinase inhibitor; histone deacetylase inhibitors; prostate cancer; synergy; valproic acid; vorinostat
3.  Aldehyde Dehydrogenase Expression Drives Human Regulatory T Cell Resistance to Posttransplantation Cyclophosphamide 
Science translational medicine  2013;5(211):211ra157.
High-dose, posttransplantation cyclophosphamide (PTCy) is an effective strategy for preventing graft-versus-host disease (GVHD) after allogeneic blood or marrow transplantation (alloBMT). However, the mechanisms by which PTCy modulates alloimmune responses are not well understood. We studied early T cell reconstitution in patients undergoing alloBMT with PTCy and the effects of mafosfamide, a cyclophosphamide (Cy) analog, on CD4+ T cells in allogeneic mixed lymphocyte reactions (MLRs) in vitro. Patients exhibited reductions in naïve, potentially alloreactive conventional CD4+ T cells with relative preservation of memory CD4+Foxp3+ T cells. In particular, CD4+CD45RA−Foxp3+hi effector regulatory T cells (Tregs) recovered rapidly after alloBMT and, unexpectedly, were present at higher levels in patients with GVHD. CD4+Foxp3+ T cells from patients and from allogeneic MLRs expressed relatively high levels of aldehyde dehydrogenase (ALDH), the major in vivo mechanism of Cy resistance. Treatment of MLR cultures with the ALDH inhibitor diethylaminobenzaldehyde reduced the activation and proliferation of CD4+ T cells and sensitized Tregs to mafosfamide. Finally, removing Tregs from peripheral blood lymphocyte grafts obviated PTCy's GVHD-protective effect in a xenogeneic transplant model. Together, these findings suggest that Treg resistance to Cy through expression of ALDH may contribute to the clinical activity of PTCy in preventing GVHD.
PMCID: PMC4155575  PMID: 24225944
4.  Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances the clinical responses to Interferon-α (IFN) in newly diagnosed chronic myeloid leukemia (CML) 
Leukemia research  2014;38(8):886-890.
The majority of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs) remain with residual disease. In contrast to TKIs, interferon (IFN) is directly toxic to CML progenitor cells, and myeloid growth factors such as GM-CSF may enhance IFN’s cytotoxicity. We performed a phase 2 study of IFN+GM-CSF in 58 newly diagnosed CML patients before imatinib approval. Short-term clinical responses included: 60% major cytogenetic response, 28% complete cytogenetic response and 19% complete molecular response. Six patients remain off all therapy for CML (range: 15 months–12 years) after IFN+GM-CSF treatment. IFN+GM-CSF shows promise as an adjunctive therapy for CML.
PMCID: PMC4135379  PMID: 25012565
chronic myeloid leukemia; tyrosine kinase inhibitor; cure; discontinuation of therapy; interferon: growth factors
5.  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;31(5):581-588.
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.
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.
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.
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.
PMCID: PMC3227793  PMID: 21816640
6.  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.
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.
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.
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).
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.
PMCID: PMC3715048  PMID: 22290566
The Prostate  2011;72(10):1133-1139.
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.
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).
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.
PMCID: PMC3360823  PMID: 22127895
8.  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.
PMCID: PMC3414391  PMID: 22694820
BRAF; RARβ2; RASSF1A; TIMP3; biomarkers; hypermethylation; thyroid cancer; thyroid tissue
9.  Lenalidomide Modulates IL-8 and Anti-Prostate Antibody Levels in Men with Biochemically Recurrent Prostate Cancer 
The Prostate  2011;72(5):487-498.
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.
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.
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.
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.
PMCID: PMC3248613  PMID: 21748755
prostate cancer; antibody; cytokine; IL-8; lenalidomide
10.  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.
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).
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.
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.
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.
PMCID: PMC3252493  PMID: 21960118
11.  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.
PMCID: PMC3483826  PMID: 23118755
12.  Lenalidomide in Nonmetastatic Biochemically Relapsed Prostate Cancer: Results of a Phase I/II Double-Blinded, Randomized Study 
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.
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.
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.
PMCID: PMC3444815  PMID: 20978144
13.  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.
PMCID: PMC3390263  PMID: 21756035
Cyclophosphamide; NHL; Mantle Cell Lymphoma
14.  An epigenetic marker panel for detection of lung cancer using cell-free serum DNA 
We investigated the feasibility of detecting aberrant DNA methylation of some novel and known genes in the serum of lung cancer patients.
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.
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%).
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.
PMCID: PMC3131425  PMID: 21610147
DNA methylation/epigenetics; serum; lung cancer
15.  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.
PMCID: PMC3388433  PMID: 22778748
16.  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.
PMCID: PMC3010878  PMID: 21132730
chronic myeloid leukemia; CML; leukemia stem cell; WT1; PRAME
17.  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.
PMCID: PMC2848533  PMID: 19961944
18.  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.
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.
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.
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.
PMCID: PMC2799055  PMID: 19720908
19.  Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors 
PLoS ONE  2010;5(5):e10828.
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.
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.
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.
PMCID: PMC2877085  PMID: 20520817
20.  A Multiple Loop, Double Cube Microarray Design Applied to Prostate Cancer Cell Lines with Variable Sensitivity to Histone Deacetylase Inhibitors 
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.
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.
A multiple loop, double cube microarray design can be used to identify HDACI induced changes in gene expression, possibly related to drug resistance.
PMCID: PMC2603330  PMID: 18980983
21.  TIMP3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer 
The Journal of urology  2007;179(2):743-747.
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.
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).
These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.
PMCID: PMC2674621  PMID: 18082200
22.  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.
PMCID: PMC2633246  PMID: 18489989
Bone marrow transplantation; Cyclophosphamide; Histocompatibility antigens; Conditioning regimens; Leukemia; Lymphoma
23.  Alterations of GPI transamidase subunits in head and neck squamous carcinoma 
Molecular Cancer  2007;6:74.
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).
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).
An increased expression level and elevated copy number for GAA1 suggest a role for this GPI anchor subunit in HNSCC.
PMCID: PMC2204038  PMID: 18028549
24.  Pre-processing Agilent microarray data 
BMC Bioinformatics  2007;8:142.
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.
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.
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.
PMCID: PMC1876252  PMID: 17472750

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