bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase I Trial of 17-Allylamino-17-Demethoxygeldanamycin in Patients with Advanced Cancer
Authors: Solit David B. , Ivy S. Percy , Kopil Catherine , Sikorski Rachel , Morris Michael J. , Slovin Susan F. , Kelly W. Kevin , DeLaCruz Anthony , Curley Tracy , Heller Glenn , Larson Steven , Schwartz Lawrence , Egorin Merrill J. , Rosen Neal , Scher Howard I.
Published: March 2007

Purpose: To define the maximum tolerated dose (MTD), toxicities, and pharmacokinetics of 17-allylamino-17-demethoxygeldanamycin (17-AAG) when administered using continuous and intermittent dosing schedules.

Experimental Design: Patients with progressive solid tumor malignancies were treated with 17-AAG using an accelerated titration dose escalation schema. The starting dose and schedule were 5 mg/m2 daily for 5 days with cycles repeated every 21 days. Dosing modifications based on safety, pharmacodynamic modeling, and clinical outcomes led to the evaluation of the following schedules: daily × 3 repeated every 14 days; twice weekly (days 1, 4, 8, and 11) for 2 weeks every 3 weeks; and twice weekly (days 1 and 4) without interruption. During cycle 1, blood was collected for pharmacokinetic and pharmacodynamic studies.

Results: Fifty-four eligible patients were treated. The MTD was schedule dependent: 56 mg/m2 on the daily × 5 schedule; 112 mg/m2 on the daily × 3 schedule; and 220 mg/m2 on the days 1, 4, 8, and 11 every-21-day schedule. Continuous twice-weekly dosing was deemed too toxic because of delayed hepatotoxicity. Hepatic toxicity was also dose limiting with the daily × 5 schedule. Other common toxicities encountered were fatigue, myalgias, and nausea. This latter adverse effect may have been attributable, in part, to the DMSO-based formulation. Concentrations of 17-AAG above those required for activity in preclinical models could be safely achieved in plasma. Induction of a heat shock response and down-regulation of Akt and Raf-1 were observed in biomarker studies.

Conclusion: The MTD and toxicity profile of 17-AAG were schedule dependent. Intermittent dosing schedules were less toxic and are recommended for future phase II studies.
Phase I Pharmacokinetic and Pharmacodynamic Study of 17-N-Allylamino-17-Demethoxygeldanamycin in Pediatric Patients with Recurrent or Refractory Solid Tumors: A Pediatric Oncology Experimental Therapeutics Investigators Consortium Study
Authors: Bagatell Rochelle , Gore Lia , Egorin Merrill J. , Ho Richard , Heller Glenn , Boucher Nichole , Zuhowski Eleanor G. , Whitlock James A. , Hunger Stephen P. , Narendran Aru , Katzenstein Howard M. , Arceci Robert J. , Boklan Jessica , Herzog Cynthia E. , Whitesell Luke , Ivy S. Percy , Trippett Tanya M.
Published: March 2007

Purpose: Heat shock protein 90 (Hsp90) is essential for the posttranslational control of many regulators of cell growth, differentiation, and apoptosis. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) binds to Hsp90 and alters levels of proteins regulated by Hsp90. We conducted a phase I trial of 17-AAG in pediatric patients with recurrent or refractory neuroblastoma, Ewing's sarcoma, osteosarcoma, and desmoplastic small round cell tumor to determine the maximum tolerated dose, define toxicity and pharmacokinetic profiles, and generate data about molecular target modulation.

Experimental Design: Escalating doses of 17-AAG were administered i.v. over 1 to 2 h twice weekly for 2 weeks every 21 days until patients experienced disease progression or toxicity. harmacokinetic and pharmacodynamic studies were done during cycle 1.

Results: Fifteen patients were enrolled onto dose levels between 150 and 360 mg/m2; 13 patients were evaluable for toxicity. The maximum tolerated dose was 270 mg/m2. DLTs were grade 3 transaminitis and hypoxia. Two patients with osteosarcoma and bulky pulmonary metastases died during cycle 1 and were not evaluable for toxicity. No objective responses were observed. 17-AAG pharmacokinetics in pediatric patients were linear; clearance and half-life were 21.6 ± 6.21 (mean ± SD) L/h/m2 and 2.6 ± 0.95 h, respectively. Posttherapy increases in levels of the inducible isoform of Hsp70, a marker of target modulation, were detected in peripheral blood mononuclear cells at all dose levels.

Conclusion: 17-AAG was well tolerated at a dose of 270 mg/m2 administered twice weekly for 2 of 3 weeks. Caution should be used in treatment of patients with bulky pulmonary disease.
Long-Lived Antitumor CD8+ Lymphocytes for Adoptive Therapy Generated Using an Artificial Antigen-Presenting Cell
Authors: Butler Marcus O. , Lee Jeng-Shin , Ansén Sascha , Neuberg Donna , Hodi F. Stephen , Murray Andrew P. , Drury Linda , Berezovskaya Alla , Mulligan Richard C. , Nadler Lee M. , Hirano Naoto
Published: March 2007

Purpose: Antitumor lymphocytes can be generated ex vivo unencumbered by immunoregulation found in vivo. Adoptive transfer of these cells is a promising therapeutic modality that could establish long-term antitumor immunity. However, the widespread use of adoptive therapy has been hampered by the difficulty of consistently generating potent antitumor lymphocytes in a timely manner for every patient. To overcome this, we sought to establish a clinical grade culture system that can reproducibly generate antigen-specific cytotoxic T lymphocytes (CTL).

Experimental Design: We created an off-the-shelf, standardized, and renewable artificial antigen-presenting cell (aAPC) line that coexpresses HLA class I, CD54, CD58, CD80, and the dendritic cell maturation marker CD83. We tested the ability of aAPC to generate tumor antigen-specific CTL under optimal culture conditions. The number, phenotype, effector function, and in vitro longevity of generated CTL were determined.

Results: Stimulation of CD8+ T cells with peptide-pulsed aAPC generated large numbers of functional CTL that recognized a variety of tumor antigens. These CTLs, which possess a phenotype consistent with in vivo persistence, survived ex vivo for prolonged periods of time. Clinical grade aAPC33, produced under current Good Manufacturing Practices guidelines, generated sufficient numbers of CTL within a short period of time. These CTL specifically lysed a variety of melanoma tumor lines naturally expressing a target melanoma antigen. Furthermore, antitumor CTL were easily generated in all melanoma patients examined.

Conclusions: With clinical grade aAPC33 in hand, we are now poised for clinical translation of ex vivo generated antitumor CTL for adoptive cell transfer.
Garlic-DerivedS-allylmercaptocysteine Is a NovelIn vivoAntimetastatic Agent for Androgen-Independent Prostate Cancer
Authors: Howard Edward W. , Ling Ming-Tat , Chua Chee Wai , Cheung Hiu Wing , Wang Xianghong , Wong Yong Chuan
Published: March 2007

Purpose: There is epidemiologic evidence that high garlic consumption decreases the incidence of prostate cancer, and compounds isolated from garlic have been shown to have cancer-preventive and tumor-suppressive effects. Recent in vitro studies in our laboratory have shown that garlic-derived organosulfur compound S-allylmercaptocysteine suppresses invasion and cell motility of androgen-independent prostate cancer cells via the up-regulation of cell-adhesion molecule E-cadherin. S-allylmercaptocysteine is therefore a potential antimetastatic drug with broad clinical applications that we tested in vivo for the first time in this study.

Experimental Design: We used a newly established fluorescent orthotopic androgen-independent prostate cancer mouse model to assess the ability of S-allylmercaptocysteine to inhibit tumor growth and dissemination.

Results: We showed that oral S-allylmercaptocysteine not only inhibited the growth of primary tumors by up to 71% (P < 0.001) but also reduced the number of lung and adrenal metastases by as much as 85.5% (P = 0.001) without causing notable toxicity. This metastatic suppression was accompanied by a 91% reduction of viable circulating tumor cells (P = 0.041), suggesting that S-allylmercaptocysteine prevents dissemination by decreasing tumor cell intravasation.

Conclusions: Our results provide in vivo evidence supporting the potential use of S-allylmercaptocysteine as an E-cadherin up-regulating antimetastatic agent for the treatment of androgen-independent prostate cancer. This is the first report of the in vivo antimetastatic properties of garlic, which may also apply to other cancer types.
DNA Methylation as a Therapeutic Target in Cancer
Authors: Issa Jean-Pierre J.
Published: March 2007

Targeting DNA methylation for cancer therapy has had a rocky history. The first reports on DNA methylation changes in cancer described global loss of methylation, which has been suggested to drive tumorigenesis through activation of oncogenic proteins or induction of chromosomal instability. In this context, reducing DNA methylation was viewed as a tumor-promoting event rather than a promising cancer therapy. The idea of inhibiting DNA methylation therapeutically emerged from subsequent studies showing that, in parallel to global decreases in methylation, several genes (including many critical to the tumor phenotype) displayed gains of methylation in their promoters during tumorigenesis, a process associated with epigenetic silencing of expression and loss of protein function. This led to revival of interest in drugs discovered decades ago to be potent inhibitors of DNA methyltransferases. These drugs have now been approved for clinical use in the United States in the treatment of myelodysplastic syndrome, thus opening the floodgate for a whole new approach to cancer therapy—epigenetic therapy.
Tumor Antigen–Specific T-Cell Expansion Is Greatly Facilitated byIn vivoPriming
Authors: Dang Yushe , Knutson Keith L. , Goodell Vivian , dela Rosa Corazon , Salazar Lupe G. , Higgins Doreen , Childs Jennifer , Disis Mary L.
Published: March 2007

Purpose: Adoptive T-cell therapy is a promising strategy for the treatment of patients with established tumors but is often limited to specific cancers where tumor-infiltrating lymphocytes, the source of T cells for ex vivo culture, can be obtained. In this study, we evaluated the feasibility of expanding HER-2/neu–specific T cells derived from peripheral blood ex vivo following in vivo priming with a HER-2/neu peptide vaccine.

Experimental Design: Peripheral blood mononuclear cells from cytomegalovirus (CMV)–seronegative and CMV-seropositive donors as well as HER-2/neu–positive cancer patients who had or had not been vaccinated with a HER-2/neu peptide–based vaccine was used as a source of T lymphocytes. Antigen-specific T-cell lines were generated by in vitro stimulation with antigen followed by nonspecific expansion on CD3/CD28 beads. The ability to expand antigen-specific T cells was assessed using IFN-γ and granzyme B enzyme-linked immunosorbent spot. The phenotype of the resultant T-cell lines was evaluated by flow cytometry, including the presence of FOXP3-expressing CD4+ T cells.

Results: The frequencies of CMV-specific T cells generated from CMV+ donors were >11-fold higher than the frequencies from CMV− donors (P = 0.001), with 22-fold increase of total number of CD3+ T cells. The frequencies of HER-2/neu–specific T cells generated from the primed patients were >25-fold higher than the frequencies from unvaccinated patients (P = 0.006), with an average of a 19-fold increase of total number of CD3+ T cells. Using peripheral blood as the source of T cells did not result in concurrent expansion of FOXP3+CD4+ regulatory T cells despite the use of interleukin-2 in in vitro culture. Both CD4+ and CD8+ HER-2/neu–specific T cells could be expanded. The extent of ex vivo expansion correlated with the magnitude of immunity achieved during immunization (P = 0.008).

Conclusion: Tumor-specific T cells can be efficiently expanded from the peripheral blood ex vivo following in vivo priming with a vaccine. This approach provides an effective method to generate tumor-specific polyclonal T cells for therapeutic use that could be applied to cancer patients with any tumor type.
Phase I and Pharmacodynamic Study of 17-(Allylamino)-17-Demethoxygeldanamycin in Adult Patients with Refractory Advanced Cancers
Authors: Ramanathan Ramesh K. , Egorin Merrill J. , Eiseman Julie L. , Ramalingam Suresh , Friedland David , Agarwala Sanjiv S. , Ivy S. Percy , Potter Douglas M. , Chatta Gurkamal , Zuhowski Eleanor G. , Stoller Ronald G. , Naret Cynthia , Guo Jianxia , Belani Chandra P.
Published: March 2007

Purpose: The primary objective was to establish the dose-limiting toxicity (DLT) and recommended phase II dose of 17-(allylamino)-17-demethoxygeldanamycin (17AAG) given twice a week.

Experimental Design: Escalating doses of 17AAG were given i.v. to cohorts of three to six patients. Dose levels for schedule A (twice weekly × 3 weeks, every 4 weeks) were 100, 125, 150, 175, and 200 mg/m2 and for schedule B (twice weekly × 2 weeks, every 3 weeks) were 150, 200, and 250 mg/m2. Peripheral blood mononuclear cells (PBMC) were collected for assessment of heat shock protein (HSP) 90 and HSP90 client proteins.

Results: Forty-four patients were enrolled, 32 on schedule A and 12 on schedule B. On schedule A at 200 mg/m2, DLTs were seen in two of six patients (one grade 3 thrombocytopenia and one grade 3 abdominal pain). On schedule B, both patients treated at 250 mg/m2 developed DLT (grade 3 headache with nausea/vomiting). Grade 3/4 toxicities seen in >5% of patients were reversible elevations of liver enzymes (47%), nausea (9%), vomiting (9%), and headache (5%). No objective tumor responses were observed. The only consistent change in PBMC proteins monitored was a 0.8- to 30-fold increase in HSP70 concentrations, but these were not dose dependent. The increase in PBMC HSP70 persisted throughout the entire cycle of treatment but returned to baseline between last 17AAG dose of cycle 1 and first 17AAG dose of cycle 2.

Conclusions: The recommended phase II doses of 17AAG are 175 to 200 mg/m2 when given twice a week and consistently cause elevations in PBMC HSP70.
A Phase I Study of 17-Allylaminogeldanamycin in Relapsed/Refractory Pediatric Patients with Solid Tumors: A Children's Oncology Group Study
Authors: Weigel Brenda J. , Blaney Susan M. , Reid Joel M. , Safgren Stephanie L. , Bagatell Rochelle , Kersey John , Neglia Joseph P. , Ivy S. Percy , Ingle Ashish M. , Whitesell Luke , Gilbertson Richard J. , Krailo Mark , Ames Matthew , Adamson Peter C.
Published: March 2007

Purpose: To determine the recommended phase 2 dose, dose-limiting toxicities (DLT), pharmacokinetic profile, and pharmacodynamics of the heat shock protein (Hsp) 90 inhibitor, 17-allylaminogeldanamycin (17-AAG).

Experimental Design: 17-AAG was administered as a 60-min infusion, on days 1, 4, 8, and 11 of a 21-day cycle at dose levels of 150, 200, 270, and 360 mg/m2/dose. Pharmacokinetic studies and evaluations for Hsp72 and Akt levels in peripheral blood mononuclear cells were done during the first course of therapy.

Results: Seventeen patients (7 males), median 7 years of age (range, 1-19 years), were enrolled using a standard dose escalation scheme. No DLTs were observed. Although there were no objective responses, three patients remain on therapy at 6+, 7+, and 9+ months with stable disease. One patient with hepatoblastoma had a reduction in α-fetoprotein and stable disease over three cycles. At 270 mg/m2/dose, the Cmax and areas under the plasma concentration-time curves of 17-AAG were 5,303 ± 1,591 ng/mL and 13,656 ± 4,757 ng/mL h, respectively, similar to the exposure in adults. The mean terminal half-life for 17-AAG was 3.24 ± 0.80 h. Induction of Hsp72, a surrogate marker for inhibition of Hsp90, was detected at the 270 mg/m2 dose level.

Conclusions: Drug exposures consistent with those required for anticancer activity in preclinical models were achieved without DLT. Evidence for drug-induced modulation of Hsp90 systemically was also detected. The recommended phase II dose of 17-AAG is 360 mg/m2/d. Non-DMSO–containing formulations may improve acceptance of this drug by children and their families.
A Pilot Trial of CTLA-4 Blockade with Human Anti-CTLA-4 in Patients with Hormone-Refractory Prostate Cancer
Authors: Small Eric J. , Tchekmedyian N. Simon , Rini Brian I. , Fong Lawrence , Lowy Israel , Allison James P.
Published: March 2007

Purpose: Blockade of the T-cell inhibitory receptor CTL-associated antigen-4 (CTLA-4) augments and prolongs T-cell responses and is a strategy to elicit antitumor immunity. The objectives of this pilot study were to establish the pharmacokinetic and safety profile for a single dose of 3 mg/kg of the anti-CTLA-4 antibody Ipilimumab (MDX-010, BMS-734016) and to assess if this therapy resulted in prostate-specific antigen (PSA) modulation and the development of polyclonal T-cell activation and/or clinical autoimmunity in patients with hormone-refractory prostate cancer treated with Ipilimumab.

Experimental Design: Patients with metastatic hormone-refractory prostate cancer received a single 3 mg/kg i.v. dose of Ipilimumab. Serologic measures of autoimmunity were obtained, and T-cell activation was evaluated by flow cytometry. Pharmacokinetic sampling of plasma for MDX-CTLA-4, PSA measurement, and diagnostic imaging were also undertaken.

Results: Fourteen patients were treated: 12 patients received a single dose of Ipilimumab, and 2 patients were re-treated with a second dose upon PSA progression. Two patients showed PSA declines of ≥50%. Treatment was well tolerated with clinical autoimmunity limited to one patient who developed grade 3 rash/pruritis requiring systemic corticosteroids. The mean ± SD Ipilimumab terminal elimination half-life was 12.5 ± 5.3 days.

Conclusions: A single dose of 3 mg/kg Ipilimumab, an anti-CTLA-4 antibody, given to patients with prostate cancer is safe and does not result in significant clinical autoimmunity. PSA-modulating effects observed warrant further investigation.
From mRNA Metabolism to Cancer Therapy: Chronic Myelogenous Leukemia Shows the Way
Authors: Perrotti Danilo , Neviani Paolo
Published: March 2007

Altered mRNA metabolism is a feature of many cancers including blast crisis chronic myelogenous leukemia. Indeed, loss of function of many tumor suppressors regulating cell proliferation, survival, and differentiation results from aberrant mRNA processing, nuclear export, and/or translation. Here, we summarize the effects of increased BCR/ABL oncogenic activity on the expression and function of RNA binding proteins (e.g., FUS, hnRNP A1, hnRNP E2, hnRNP K, and La/SSB) with posttranscriptional and translational regulatory activities and their importance for the phenotype of BCR/ABL-transformed hematopoietic progenitors. We also provide evidence that these studies not only advance our understanding on the molecular mechanisms contributing to tumor/leukemia emergence, maintenance, and/or progression but they also serve for the identification of novel molecular targets useful for the development of alternative therapies for imatinib-resistant and blast crisis chronic myelogenous leukemia and, perhaps, for other cancers characterized by similar alterations in the mRNA metabolism.
Identification and Prognostic Significance of an Epithelial-Mesenchymal Transition Expression Profile in Human Bladder Tumors
Authors: Baumgart Egbert , Cohen Michael S. , Neto Brasil Silva , Jacobs Micah A. , Wotkowicz Chad , Rieger-Christ Kimberly M. , Biolo Andreia , Zeheb Ron , Loda Massimo , Libertino John A. , Summerhayes Ian C.
Published: March 2007

Purpose: Epithelial to mesenchymal transition (EMT) is reportedly an important transition in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. In this study, we examined the expression pattern of EMT markers in vivo and determined the occurrence and clinical significance of these events in a series of bladder carcinomas.

Experimental Design: Eight hundred and twenty-five tumor samples from 572 bladder cancer patients were assembled in 10 tissue microarrays. Paraffin sections from each tissue microarray were subjected to antigen retrieval and processed by immunohistochemistry for the expression of E-cadherin, plakoglobin, β-catenin, N-cadherin, and vimentin.

Results: Pathologic expression of E-cadherin, β-catenin, plakoglobin, and vimentin were associated with the clinicopathologic variables of grade and stage with only the cytoplasmic localization of plakoglobin found associated with lymph node status. Associations between the aforementioned markers were found significant as determined by the Spearman correlation coefficient with N-cadherin showing no associations in this analysis. In univariate survival analysis involving patients who underwent cystectomy, the reduction or loss of plakoglobin significantly influenced overall survival (P = 0.02) in which the median time to death was 2 years compared with 4 years when a normal level of plakoglobin was recorded. When the analysis was done for cancer-specific survival, low levels of both plakoglobin (P = 0.02) and β-catenin (P = 0.02) significantly influenced survival.

Conclusion: The putative markers of EMT defined within a panel of bladder carcinoma cell lines were recorded in vivo, frequently associated with tumors of high grade and stage. Although multivariate analysis showed no significant influence of the EMT biomarkers on survival, alterations associated with plakoglobin were identified as significant prognostic features in these tumors.
Combination Therapy of an Orthotopic Renal Cell Carcinoma Model Using Intratumoral Vector-Mediated Costimulation and Systemic Interleukin-2
Authors: Kudo-Saito Chie , Wansley Elizabeth K. , Gruys M. Eilene , Wiltrout Robert , Schlom Jeffrey , Hodge James W.
Published: March 2007

Purpose: Interleukin (IL)-2 therapy is currently used for therapy of renal cell carcinoma (RCC). However, it is only effective in approximately 10% to 15% of patients, showing a need for additional therapies. We have previously described a replication-defective fowlpox vector encoding three costimulatory molecules (B7-1, ICAM-1, and LFA-3), designated rF-TRICOM. Here, we show that intratumoral administration of rF-TRICOM in an orthotopic RCC model effectively enhances tumor immunogenicity and reduces tumor burden in mice and the combination of rF-TRICOM and IL-2 is more effective than either therapy alone.

Experimental Design: RCC cells were implanted under the capsule of the kidney, and mice were given rF-TRICOM intratumorally 14 days later. We compared the effect of rF-TRICOM, rF-granulocyte macrophage colony-stimulating factor (GM-CSF), and two doses of IL-2 and combinations of the above on antitumor efficacy and survival. Host CD4+ and CD8+ T-cell responses were also evaluated.

Results: The results show that (a) systemic IL-2 therapy was moderately effective in the reduction of tumor burden in an orthotopic RCC model; (b) a single intratumoral injection of rF-TRICOM and rF-GM-CSF significantly reduced tumor burden; (c) the addition of systemic IL-2 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in further reduction of tumor burden, decrease in the incidence of metastasis, and extended survival in tumor-bearing mice above that seen with either treatment alone; and (d) CD8+ T cells played a critical role in the antitumor effect seen with rF-TRICOM/rF-GM-CSF + IL-2 therapy. Finally, the addition of systemic recombinant IL-15 or intratumoral vector-delivered IL-15 to intratumoral rF-TRICOM/rF-GM-CSF administration resulted in substantially more tumor-free mice than either therapy alone.

Conclusions: These studies show that intratumoral administration of rF-TRICOM admixed with rF-GM-CSF is effective at reducing tumor burden in mice and the addition of IL-2 further contributes to this effect. These studies thus form the rationale for combination immunotherapy clinical trials in patients with RCC.
Targeted Therapy for Brain Metastases: Improving the Therapeutic Ratio
Authors: Patel Rakesh R. , Mehta Minesh P.
Published: March 2007

Brain metastasis is the most common intracranial malignancy in adults. Improvements in modern imaging techniques are detecting previously occult brain metastases, and more effective therapies are extending the survival of patients with invasive cancer who have historically died from extracranial disease before developing brain metastasis. This combination of factors along with increased life expectancy has led to the increased diagnosis of brain metastases. Conventional treatment has been whole brain radiotherapy, which can improve symptoms, but potentially results in neurocognitive deficits. Several strategies to improve the therapeutic ratio are currently under investigation to either enhance the radiation effect, thereby preventing tumor recurrence or progression as well as reducing collateral treatment-related brain injury. In this review article, we discuss new directions in the management of brain metastases, including the role of chemical modifiers, novel systemic agents, and the management and prevention of neurocognitive deficits.
Relaxin Promotes Prostate Cancer Progression
Authors: Feng Shu , Agoulnik Irina U. , Bogatcheva Natalia V. , Kamat Aparna A. , Kwabi-Addo Bernard , Li Rile , Ayala Gustavo , Ittmann Michael M. , Agoulnik Alexander I.
Published: March 2007

Purpose: To understand the role of relaxin peptide in prostate cancer, we analyzed the expression of relaxin and its receptor in human prostate cancer samples, the effects of relaxin signaling on cancer cell phenotype in vitro, and the effects of increased serum relaxin concentrations on cancer progression in vivo.

Experimental Design: The relaxin and its receptor leucine-rich repeat containing G protein–coupled receptor 7 (LGR7) expression were studied by quantitative reverse transcription-PCR (11 benign and 44 cancer tissue samples) and by relaxin immunohistochemistry using tissue microarrays containing 10 normal and 69 cancer samples. The effects of relaxin treatment and endogenous relaxin/LGR7 suppression via short interfering RNA in PC-3 and LNCaP cells were analyzed in vitro. The effect of transgenic relaxin overexpression [Tg(Rln1)] on cancer growth and survival was evaluated in autochthonous transgenic adenocarcinoma of the mouse prostate (TRAMP).

Results: The relaxin mRNA expression was significantly higher in recurrent prostate cancer samples. In tissue microarrays of the 10 normal tissues, 8 had low staining in epithelial cells, whereas only 1 of 9 high-grade prostatic intraepithelial neoplasia lesions had low expression (P = 0.005) and only 29 of 65 cancers had low expression (P = 0.047). Stimulation with relaxin increased cell proliferation, invasiveness, and adhesion in vitro. The suppression of relaxin/LGR7 via short interfering RNAs decreased cell invasiveness by 90% to 95% and growth by 10% to 25% and increased cell apoptosis 0.6 to 2.2 times. The Tg(Rln1) TRAMP males had shorter median survival time, associated with the decreased apoptosis of tumor cells, compared with non-Tg(Rln1) TRAMP animals.

Conclusions: Relaxin signaling plays a role in prostate cancer progression.
130-nm Albumin–Bound Paclitaxel Enhances Tumor Radiocurability and Therapeutic Gain
Authors: Wiedenmann Nicole , Valdecanas David , Hunter Nancy , Hyde Stephen , Buchholz Thomas A. , Milas Luka , Mason Kathryn A.
Published: March 2007

Purpose: 130-nm albumin–bound paclitaxel (nab-paclitaxel) is a novel solvent-free albumin-bound paclitaxel, designed to avoid solvent-related toxicity. Nab-paclitaxel has been successfully introduced into the clinic but its radiation-enhancing potential has not yet been evaluated. We conducted a preclinical evaluation of the radiation-modulating effects of nab-paclitaxel in tumor and normal tissues.

Experimental Design: Mice bearing syngeneic ovarian or mammary carcinomas were treated with nab-paclitaxel, radiation, or combination of both. Nab-paclitaxel was administered at 90 mg/kg, 1.5 times the maximum tolerated dose for solvent-based paclitaxel. End points were antitumor efficacy (growth delay, radiocurability, and cellular effects) and normal tissue toxicity (gut and skin).

Results: Nab-paclitaxel showed single-agent antitumor efficacy against both tumor types and acted as a radiosensitizer. Combined with radiation, nab-paclitaxel produced supra-additive effects when given before radiation. Nab-paclitaxel significantly increased radiocurability by reducing the dose yielding 50% tumor cure (TCD50) from 54.3 to 35.2 Gy. Tumor histology following nab-paclitaxel treatment was characterized by pronounced necrotic and apoptotic cell death and mitotic arrest. Nab-paclitaxel did not increase normal tissue radioresponse.

Conclusions: Nab-paclitaxel exhibited strong antitumor efficacy against both tumors as a single agent and it improved radiotherapy in a supra-additive manner. These improved effects were achieved without increased normal tissue toxicity to either rapidly or slowly proliferating normal tissues although the drug dose was 1.5 times higher than the maximum tolerated dose of solvent-based paclitaxel. These preclinical findings show that combining nab-paclitaxel with radiotherapy would improve the outcome of taxane-based chemoradiotherapy. This novel taxane is thus a good candidate for testing in clinical chemoradiotherapy trials.
PD-1 Is Expressed by Tumor-Infiltrating Immune Cells and Is Associated with Poor Outcome for Patients with Renal Cell Carcinoma
Authors: Thompson R. Houston , Dong Haidong , Lohse Christine M. , Leibovich Bradley C. , Blute Michael L. , Cheville John C. , Kwon Eugene D.
Published: March 2007

Purpose: B7-H1 is expressed by clinically aggressive forms of renal cell carcinoma (RCC) and predicts adverse outcome. B7-H1 is known to impair host immunity via interaction with the Programmed Death-1 (PD-1) receptor, which is expressed by activated T cells. Levels of immune cells expressing PD-1 (PD-1+) in clinical RCC tumors have not been evaluated. Thus, we tested whether immune cell PD-1 expression is observed within aggressive RCC tumors.

Experimental Design: Between 2000 and 2003, 267 patients underwent nephrectomy at our institution for clear cell RCC and had fresh-frozen tissue available for review. These RCC specimens were immunostained using anti–PD-1 (clone MIH4) and outcome analyses were conducted.

Results: Mononuclear immune cell infiltration was observed in 136 (50.9%) specimens. PD-1+ immune cells were present in 77 of these 136 (56.6%) tumors. In contrast, RCC tumor cells did not express PD-1. Patients with PD-1+ immune cells were significantly more likely to harbor B7-H1+ tumor cells (P < 0.001), larger tumors (P = 0.001), and tumors of higher nuclear grade (P = 0.001). Likewise, intratumoral PD-1+ immune cells were associated with advanced tumor-node-metastasis stage (P = 0.005), coagulative tumor necrosis (P = 0.027), and sarcomatoid differentiation (P = 0.008). With a median follow-up of 2.9 years, 52 patients died from RCC. Univariately, patients with PD-1+ immune cells were at significant risk of cancer-specific death compared with PD-1− patients (risk ratio, 2.24; P = 0.004).

Conclusions: Levels of immune cells expressing PD-1 were increased in patients with high-risk RCC tumors. Interactions between immune cell PD-1 and B7-H1 may promote cancer progression by contributing to immune dysfunction in patients with RCC.
The Biology of Metastasis to a Sanctuary Site
Authors: Palmieri Diane , Chambers Ann F. , Felding-Habermann Brunhilde , Huang Suyun , Steeg Patricia S.
Published: March 2007

Metastasis to the brain is prevalent in solid tumors and lymphomas, and is associated with shortened survival. The brain is regarded as a sanctuary site for metastatic tumor cells where they exist partially protected from drugs by the blood-tumor barrier. Model systems for brain metastasis have been developed and are now yielding mechanistic insights into the roles of angiogenesis, energy metabolism, the Her-2 and Stat3 signaling pathways, and dormancy. Specific, new approaches to combat brain metastatic disease are needed.
The Blood-Brain Barrier and Cancer: Transporters, Treatment, and Trojan Horses
Authors: Deeken John F. , Löscher Wolfgang
Published: March 2007

Despite scientific advances in understanding the causes and treatment of human malignancy, a persistent challenge facing basic and clinical investigators is how to adequately treat primary and metastatic brain tumors. The blood-brain barrier is a physiologic obstruction to the delivery of systemic chemotherapy to the brain parenchyma and central nervous system (CNS). A number of physiologic properties make the endothelium in the CNS distinct from the vasculature found in the periphery. Recent evidence has shown that a critical aspect of this barrier is composed of xenobiotic transporters which extrude substrates from the brain into the cerebrospinal fluid and systemic circulation. These transporters also extrude drugs and toxins if they gain entry into the cytoplasm of brain endothelial cells before they enter the brain. This review highlights the properties of the blood-brain barrier, including the location, function, and relative importance of the drug transporters that maintain this barrier. Primary and metastatic brain malignancy can compromise this barrier, allowing some access of chemotherapy treatment to reach the tumor. The responsiveness of brain tumors to systemic treatment found in past clinical research is discussed, as are possible explanations as to why CNS tumors are nonetheless able to evade therapy. Finally, strategies to overcome this barrier and better deliver chemotherapy into CNS tumors are presented.
CNS Metastasis: An Old Problem in a New Guise
Authors: Aragon-Ching Jeanny B. , Zujewski Jo Anne
Published: March 2007

It is estimated that 10% to 30% of patients with solid tumors are diagnosed with central nervous system (CNS) metastasis. Common primary sites include lung, breast, melanoma, kidney, and colorectal. Brain metastases are increasing, due to the aging population, detection of subclinical disease, and control of systemic disease. CNS metastases are a major cause of morbidity and mortality affecting survival, neurocognition, speech, coordination, behavior, and quality of life. In pediatric acute lymphocytic leukemia event-free survival rates are >80% and the CNS is an important source of extramedullary relapse. CNS metastases are an increasing problem in solid tumors. In this CCR Focus series, four main topics are reviewed: (a) HER-2–positive breast cancer as a paradigm for the problem; (b) model systems for brain metastasis and mechanistic insights into the pathogenesis of brain metastasis; (c) the unique physiology of the blood brain barrier; (d) and the evolving role of radiotherapy in CNS disease and strategies to improve the therapeutic index. Areas for future research include the need for an understanding of site-specific metastasis, effective anticancer strategies for sanctuary sites, assays to detect drug accumulation in sanctuary sites, prevention of CNS metastasis, improving the therapeutic ratio of systemic and CNS-directed therapies, behavioral tools for anticipating/measuring long-term neurocognitive defects, and quality of life assessment of the long-term effect of systemic and CNS-directed therapies.
A fluorescence microplate cytotoxicity assay with a 4-log dynamic range that identifies synergistic drug combinations
Authors: Frgala Tomas , Kalous Ondrej , Proffitt Robert T. , Reynolds C. Patrick
Published: March 2007

Purpose: Cytotoxicity assays in 96-well tissue culture plates allow rapid sample handling for multicondition experiments but have a limited dynamic range. Using DIMSCAN, a fluorescence digital image system for quantifying relative cell numbers in tissue culture plates, we have developed a 96-well cytotoxicity assay with a >4-log dynamic range. Methods: To overcome background fluorescence that limits detection of viable cells with fluorescein diacetate, we used 2′4′5′6′-tetrabromofluorescein (eosin Y) to quench background fluorescence in the medium and in nonviable cells to enhance the reduction of background fluorescence achieved with digital image thresholding. The sensitivity and linearity of the new assay were tested with serial dilutions of neuroblastoma and leukemia cell lines. DIMSCAN was compared with other in vitro cytotoxicity assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, and trypan blue dye exclusion. Results: Without background fluorescence reduction, scans produced a nearly flat curve across various cell concentrations from 100 to 106 cells per well. Either digital image thresholding or eosin Y dramatically reduced background fluorescence, and combining them achieved a linear correlation (r > 0.9) of relative fluorescence to viable cell number over >4 logs of dynamic range, even in the presence of 4 × 104 nonviable cells per well. Cytotoxicity of deferoxamine for neuroblastoma cell lines measured by the DIMSCAN assay achieved dose-response curves similar to data obtained by manual trypan blue counts or colony formation in soft agar but with a wider dynamic range. Long-term cultures documented the clonogenic ability of viable cells detected by DIMSCAN over the entire dynamic range. The cytotoxicity of two drug combinations (buthionine sulfoximine + melphalan or fenretinide + safingol) was tested using both DIMSCAN and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and the wider dynamic range of DIMSCAN facilitated detection of synergistic interactions. Conclusion: DIMSCAN offers the ability to rapidly and efficiently conduct cytotoxicity assays in 96-well plates with a dynamic range of >4 logs. This assay enables rapid testing of anticancer drug combinations in microplates. [Mol Cancer Ther 2007;6(3):886–97]
Evaluation of locked nucleic acid–modified small interfering RNAin vitroandin vivo
Authors: Mook Olaf R. , Baas Frank , de Wissel Marit B. , Fluiter Kees
Published: March 2007

RNA interference has become widely used as an experimental tool to study gene function. In addition, small interfering RNA (siRNA) may have great potential for the treatment of diseases. Recently, it was shown that siRNA can be used to mediate gene silencing in mouse models. Locally administered siRNAs entered the first clinical trials, but strategies for successful systemic delivery of siRNA are still under development. Challenges still exist about the stability, delivery, and therapeutic efficacy of siRNA. In the present study, we compare the efficacy of two methods of systemic siRNA delivery and the effects of siRNA modifications using locked nucleic acids (LNA) in a xenograft cancer model. Low volume tail vein bolus injections and continuous s.c. delivery using osmotic minipumps yielded similar uptake levels of unmodified siRNA by tumor xenografts. Both routes of administration mediated sequence-specific inhibition of two unrelated targets inside tumor xenografts. Previous studies have shown that LNA can be incorporated into the sense strand of siRNA while the efficacy is retained. Modification of siRNA targeting green fluorescent protein with LNA results in a significant increase in serum stability and thus may be beneficial for clinical applications. We show that minimal 3′ end LNA modifications of siRNA are effective in stabilization of siRNA. Multiple LNA modifications in the accompanying strand further increase the stability but negate the efficacy in vitro and in vivo. In vivo, LNA-modified siRNA reduced off-target gene regulation compared with nonmodified siRNA. End-modified siRNA targeting green fluorescent protein provides a good trade-off between stability and efficacy in vivo using the two methods of systemic delivery in the nude mouse model. Therefore, LNA-modified siRNA should be preferred over unmodified siRNA. [Mol Cancer Ther 2007;6(3):833–43]
A novel class of pyranocoumarin anti–androgen receptor signaling compounds
Authors: Guo Junming , Jiang Cheng , Wang Zhe , Lee Hyo-Jeong , Hu Hongbo , Malewicz Barbara , Lee Hyo-Jung , Lee Jae-Ho , Baek Nam-In , Jeong Jin-Hyun , Kim Dae-Keun , Kang Kyung-Sun , Kim Sung-Hoon , Lu Junxuan
Published: March 2007

Androgen and the androgen receptor (AR)–mediated signaling are crucial for prostate cancer development. Novel agents that can inhibit AR signaling in ligand-dependent and ligand-independent manners are desirable for the chemoprevention of prostate carcinogenesis and for the treatment of advanced prostate cancer. We have shown recently that the pyranocoumarin compound decursin from the herb Angelica gigas possesses potent anti-AR activities distinct from the anti–androgen bicalutamide. Here, we compared the anti-AR activities and the cell cycle arrest and apoptotic effects of decursin and two natural analogues in the androgen-dependent LNCaP human prostate cancer cell culture model to identify structure-activity relationships and mechanisms. Decursin and its isomer decursinol angelate decreased prostate-specific antigen expression with IC50 of ∼1 μmol/L. Both inhibited the androgen-stimulated AR nuclear translocation and transactivation, decreased AR protein abundance through proteasomal degradation, and induced G0/1 arrest and morphologic differentiation. They also induced caspase-mediated apoptosis and reactive oxygen species at higher concentrations. Furthermore, they lacked the agonist activity of bicalutamide in the absence of androgen and were more potent than bicalutamide for suppressing androgen-stimulated cell growth. Decursinol, which does not contain a side chain, lacked the reactive oxygen species induction and apoptotic activities and exerted paradoxically an inhibitory and a stimulatory effect on AR signaling and cell growth. In conclusion, decursin and decursinol angelate are members of a novel class of nonsteroidal compounds that exert a long-lasting inhibition of both ligand-dependent and ligand-independent AR signaling. The side chain is critical for sustaining the anti-AR activities and the growth arrest and apoptotic effects. [Mol Cancer Ther 2007;6(3):907–17]
Macromolecular delivery of 5-aminolaevulinic acid for photodynamic therapy using dendrimer conjugates
Authors: Battah Sinan , Balaratnam Sherina , Casas Adriana , O'Neill Sophie , Edwards Christine , Batlle Alcira , Dobbin Paul , MacRobert Alexander J.
Published: March 2007

Intracellular porphyrin generation following administration of 5-aminolaevulinic acid (5-ALA) has been widely used in photodynamic therapy. However, cellular uptake of 5-ALA is limited by its hydrophilicity, and improved means of delivery are therefore being sought. Highly branched polymeric drug carriers known as dendrimers present a promising new approach to drug delivery because they have a well-defined structure capable of incorporating a high drug payload. In this work, a dendrimer conjugate was investigated, which incorporated 18 aminolaevulinic acid residues attached via ester linkages to a multipodent aromatic core. The ability of the dendrimer to deliver and release 5-ALA intracellularly for metabolism to the photosensitizer, protoporphyrin IX, was studied in the transformed PAM 212 murine keratinocyte and A431 human epidermoid carcinoma cell lines. Up to an optimum concentration of 0.1 mmol/L, the dendrimer was significantly more efficient compared with 5-ALA for porphyrin synthesis. The intracellular porphyrin fluorescence levels showed good correlation with cellular phototoxicity following light exposure, together with minimal dark toxicity. Cellular uptake of the dendrimer occurs through endocytic routes predominantly via a macropinocytosis pathway. In conclusion, macromolecular dendritic derivatives are capable of delivering 5-ALA efficiently to cells for sustained porphyrin synthesis. [Mol Cancer Ther 2007;6(3):876–85]
Dual mitogen-activated protein kinase and epidermal growth factor receptor inhibition in biliary and pancreatic cancer
Authors: Jimeno Antonio , Rubio-Viqueira Belen , Amador Maria L. , Grunwald Victor , Maitra Anirban , Iacobuzio-Donahue Christine , Hidalgo Manuel
Published: March 2007

This study aimed to develop rational combinations of targeted agents against biliary and pancreatic cancers. To this end, we compared the global gene expression profile of biliary cancer cell lines with different degrees of sensibility to the epidermal growth factor receptor tyrosine kinase inhibitors gefitinib and erlotinib using the Affymetrix U133A microarray platform. A set of 32 genes, including genes involved in signal transduction pathways, cell cycle regulation, and angiogenesis, was highly overexpressed in resistant cells. Five of these genes encoded proteins in the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway, a finding that was confirmed by Western blot and immunohistochemistry. Gefitinib failed to inhibit the MAPK pathway in resistant cell lines. Based on these data, we explored the activity of dual treatment with gefitinib in combination with CI-1040, a MAPK inhibitor. This strategy effectively resulted in inhibition of the MAPK signaling pathway and exerted antitumor effects in vitro and in vivo in tumors resistant to each of the agents alone. To further confirm these results, we tested the combined treatment in four tumor xenografts generated from patients with resected pancreatic cancer. Combined treatment was more effective than either single agent alone in this model. This study illustrates the value of global analysis of gene expression to rationally design combinations of mechanistic-based drugs. In addition, the data support the efficacy of combined epidermal growth factor receptor and MAPK inhibitors in biliary and pancreatic cancers, providing the basis to test this combination in the clinic. [Mol Cancer Ther 2007;6(3):1079–88]
Targeting BRAFV600E in thyroid carcinoma: therapeutic implications
Authors: Mitsiades Constantine S. , Negri Joseph , McMullan Ciaran , McMillin Douglas W. , Sozopoulos Elias , Fanourakis Galinos , Voutsinas Gerassimos , Tseleni-Balafouta Sophia , Poulaki Vassiliki , Batt David , Mitsiades Nicholas
Published: March 2007

B-Raf is an important mediator of cell proliferation and survival signals transduced via the Ras-Raf-MEK-ERK cascade. BRAF mutations have been detected in several tumors, including papillary thyroid carcinoma, but the precise role of B-Raf as a therapeutic target for thyroid carcinoma is still under investigation. We analyzed a panel of 93 specimens and 14 thyroid carcinoma cell lines for the presence of BRAF mutations and activation of the mitogen-activated protein/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. We also compared the effect of a B-Raf small inhibitory RNA construct and the B-Raf kinase inhibitor AAL881 on both B-Raf wild-type and mutant thyroid carcinoma cell lines. We found a high prevalence of the T1799A (V600E) mutation in papillary and anaplastic carcinoma specimens and cell lines. There was no difference in patient age, B-Raf expression, Ki67 immunostaining, or clinical stage at presentation between wild-type and BRAFV600E specimens. Immunodetection of phosphorylated and total forms of MEK and ERK revealed no difference in their phosphorylation between wild-type and BRAFV600E patient specimens or cell lines. Furthermore, a small inhibitory RNA construct targeting the expression of both wild-type B-Raf and B-RafV600E induced a comparable reduction of viability in both wild-type and BRAFV600E mutant cancer cells. Interestingly, AAL881 inhibited MEK and ERK phosphorylation and induced apoptosis preferentially in BRAFV600E-harboring cells than wild-type ones, possibly because of better inhibitory activity against B-RafV600E. We conclude that B-Raf is important for the pathophysiology of thyroid carcinomas irrespective of mutational status. Small molecule inhibitors that selectively target B-RafV600E may provide clinical benefit for patients with thyroid cancer. [Mol Cancer Ther 2007;6(3):1070–8]