bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of Lymph Node Micrometastases by Gene Promoter Hypermethylation in Samples Obtained by Endosonography- Guided Fine-Needle Aspiration Biopsy
Authors: Pellisé Maria , Castells Antoni , Ginès Àngels , Agrelo Rubén , Solé Manel , Castellví-Bel Sergi , Fernández-Esparrach Glòria , Llach Josep , Esteller Manel , Bordas Josep M. , Piqué Josep M.
Published: September 2005

Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has become a fundamental procedure for gastrointestinal and lung cancer staging. However, there is growing evidence that micrometastases are present in lymph nodes, which cannot be detected with standard pathological methods. The aim of this study was to evaluate whether hypermethylation gene promoter analysis was feasible on samples obtained by EUS-FNA from lymph nodes, as well as to establish the usefulness of this strategy for the detection of micrometastases in patients with gastrointestinal and non-small cell lung cancer.

Suspicious lymph nodes based on EUS findings from consecutive patients with esophageal, gastric, rectal, and non-small cell lung cancer were sampled by EUS-FNA. Hypermethylation analysis of the MGMT, p16INK4a, and p14ARF gene promoter CpG islands were performed by methylation-specific PCR. Effectiveness of conventional cytology, methylation analysis, and their combination were established with respect to the definitive diagnosis.

Twenty-seven patients were included, thus representing a total of 42 lymph nodes (esophageal cancer, n = 11; rectal cancer, n = 7; gastric cancer, n = 3; and lung cancer, n = 21). According to definitive diagnosis, 21 (50%) corresponded to metastatic lymph nodes. Sensitivity, specificity, and overall accuracy of conventional cytology were 76%, 100%, and 88%, respectively, whereas the corresponding values for the methylation analysis were 81%, 67%, and 74%, respectively. Combination of both techniques increased sensitivity (90%) but decreased specificity (67%) with respect to conventional cytology.

In conclusion, it is feasible to detect occult neoplastic cells in EUS-FNA samples by hypermethylation gene promoter analysis. Moreover, addition of methylation analysis to conventional cytology may increase its sensitivity at the expenses of a decrease in its specificity.
High Expression Levels of X-Linked Inhibitor of Apoptosis Protein and Survivin Correlate with Poor Overall Survival in Childhood
de Novo
Acute Myeloid Leukemia
Authors: Tamm Ingo , Richter Stephan , Oltersdorf Doreen , Creutzig Ursula , Harbott Jochen , Scholz Frank , Karawajew Leonid , Ludwig Wolf-Dieter , Wuchter Christian
Published: September 2005

Purpose: Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in adult acute myeloid leukemia (AML). However, data on the expression and prognostic impact of these molecules in childhood AML are rare.

Experimental Design: Using flow cytometry and Western blot analysis, we, therefore, investigated 45 leukemic cell samples from children with de novo AML enrolled and treated within the German AML-BFM93 study for the expression of apoptosis-regulating proteins [CD95, Bcl-2, Bax, Bcl-xL, procaspase-3, X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis protein–1 (cIAP-1), survivin].

Results: XIAP (P < 0.002) but no other apoptosis regulators showed maturation-dependent expression differences as determined by French-American-British (FAB) morphology with the highest expression levels observed within the immature M0/1 subtypes. XIAP (P < 0.01) and Bcl-xL (P < 0.01) expression was lower in patients with favorable rather than intermediate/poor cytogenetics. After a mean follow-up of 34 months, a shorter overall survival was associated with high expression levels of XIAP [30 (n = 10) versus 41 months (n = 34); P < 0.05] and survivin [27 (n = 10) versus 41 months (n = 34); P < 0.05].

Conclusions: We conclude that apoptosis-related molecules are associated with maturation stage, cytogenetic risk groups, and therapy outcome in childhood de novo AML. The observed association of XIAP with immature FAB types, intermediate/poor cytogenetics, and poor overall survival should be confirmed within prospective pediatric AML trials.
Treatment of Colon and Lung Cancer Patients withex VivoHeat Shock Protein 70-Peptide-Activated, Autologous Natural Killer Cells
Authors: Krause Stefan W. , Gastpar Robert , Andreesen Reinhard , Gross Catharina , Ullrich Heidrun , Thonigs Gerald , Pfister Karin , Multhoff Gabriele
Published: September 2005

Purpose: The 14 amino acid sequence (aa450–463) TKDNNLLGRFELSG (TKD) of heat shock protein 70 (Hsp70) was identified as a tumor-selective recognition structure for natural killer (NK) cells. Incubation of peripheral blood lymphocyte cells with TKD plus low-dose interleukin 2 (IL-2) enhances the cytolytic activity of NK cells against Hsp70 membrane-positive tumors, in vitro and in vivo. These data encouraged us to test tolerability, feasibility, and safety of TKD-activated NK cells in a clinical Phase I trial.

Experimental Design: Patients with metastatic colorectal cancer (n = 11) and non-small cell lung cancer (n = 1) who had failed standard therapies were enrolled. After ex vivo stimulation of autologous peripheral blood lymphocytes with Hsp70-peptide TKD (2 μg/ml) plus low-dose IL-2 (100 units/ml), TKD was removed by extensive washing, and activated cells were reinfused i.v. The procedure was repeated for up to six cycles, applying a dose escalation schedule in 4 patients.

Results: The percentage of activated NK cells in the reinfused leukapheresis products ranged between 8 and 20% of total lymphocytes, corresponding to total NK cell counts of 0.1 up to 1.5 × 109. Apart from restless feeling in 1 patient and itching in 2 patients, no negative side effects were observed. Concomitant with an enhanced CD94 cell surface density, the cytolytic activity of NK cells against Hsp70 membrane-positive colon carcinoma cells was enhanced after TKD/IL-2 stimulation in 10 of 12 patients. Concerning tumor response, 1 patient was in stable disease during therapy by formal staging criteria and another patient showed stable disease in one metastases and progression in another.

Conclusions: Reinfusion of Hsp70-activated autologous NK cells is safe. Immunological results warrant additional studies in patients with lower tumor burden.
A Phase I Pharmacokinetic and Biological Correlative Study of Oblimersen Sodium (Genasense, G3139), an Antisense Oligonucleotide to the Bcl-2 mRNA, and of Docetaxel in Patients with Hormone-Refractory Prostate Cancer
Authors: Tolcher Anthony W. , Kuhn John , Schwartz Garry , Patnaik Amita , Hammond Lisa A. , Thompson Ian , Fingert Howard , Bushnell David , Malik Shazli , Kreisberg Jeffrey , Izbicka Elzbieta , Smetzer Leslie , Rowinsky Eric K.
Published: September 2005

Purpose: To assess the feasibility of administering oblimersen sodium, a phosphorothioate antisense oligonucleotide directed to the Bcl-2 mRNA, with docetaxel to patients with hormone-refractory prostate cancer; to characterize the pertinent pharmacokinetic parameters, Bcl-2 protein inhibition in peripheral blood mononuclear cell(s) (PBMC) and tumor; and to seek preliminary evidence of antitumor activity.

Experimental Design: Patients were treated with increasing doses of oblimersen sodium administered by continuous i.v. infusion on days 1 to 6 and docetaxel administered i.v. over 1 h on day 6 every 3 weeks. Plasma was sampled to characterize the pharmacokinetic parameters of both oblimersen and docetaxel, and Bcl-2 protein expression was measured from paired collections of PBMCs pretreatment and post-treatment.

Results: Twenty patients received 124 courses of the oblimersen and docetaxel combination at doses ranging from 5 to 7 mg/kg/day oblimersen and 60 to 100 mg/m2 docetaxel. The rate of severe fatigue accompanied by severe neutropenia was unacceptably high at doses exceeding 7 mg/kg/day oblimersen and 75 mg/m2 docetaxel. Nausea, vomiting, and fever were common, but rarely severe. Oblimersen mean steady-state concentrations were 3.44 ± 1.31 and 5.32 ± 2.34 at the 5- and 7-mg/kg dose levels, respectively. Prostate-specific antigen responses were observed in 7 of 12 taxane-naïve patients, but in taxane-refractory patients no responses were observed. Preliminary evaluation of Bcl-2 expression in diagnostic tumor specimens was not predictive of response to this therapy.

Conclusions: The recommended Phase II doses for oblimersen and docetaxel on this schedule are 7 mg/kg/day continuous i.v. infusion days 1 to 6, and 75 mg/m2 i.v. day 6, respectively, once every 3 weeks. The absence of severe toxicities at this recommended dose, evidence of Bcl-2 protein inhibition in PBMC and tumor tissue, and encouraging antitumor activity in HPRC patients warrant further clinical evaluation of this combination.
Immunological Responses in Women with Human Papillomavirus Type 16 (HPV-16)-Associated Anogenital Intraepithelial Neoplasia Induced by Heterologous Prime-Boost HPV-16 Oncogene Vaccination
Authors: Smyth Lucy J. C. , van Poelgeest Mariëtte I. E. , Davidson Emma J. , Kwappenberg Kitty M. C. , Burt Debbie , Sehr Peter , Pawlita Michael , Man Stephen , Hickling Julian K. , Fiander Alison N. , Tristram Amanda , Kitchener Henry C. , Offringa Rienk , Stern Peter L. , van der Burg Sjoerd H.
Published: September 2005

Purpose: The purpose is to study the immunogenicity of heterologous prime-boost human papillomavirus (HPV) oncogene vaccination in patients with anogenital intraepithelial neoplasia (AGIN).

Experimental Design: Twenty-nine women with high-grade AGIN received three i.m. doses of TA-CIN (HPV-16 L2/E6/E7 protein) at four weekly intervals followed by a single dermal scarification of vaccinia HPV-16/18 E6/E7 and were followed up for 12 weeks. Immunity to HPV-16 was assessed by lymphoproliferation, IFN-γ enzyme-linked immunospot (ELISPOT), and ELISA.

Results: The patient group significantly responded to TA-CIN and not to the control antigen HPV-6 L2/E7 at all postvaccination time points when compared with baseline responses (P ≤ 0.05). Ten of the patients showed at least a 3-fold increase in TA-CIN-specific proliferation at one or more time points after vaccination. Comparison of stimulation with HPV-16 E6- or E7-GST fusion proteins showed that proliferative responses were biased to HPV-16 E6. This bias was also seen by IFN-γ ELISPOT using overlapping peptides, with HPV-16 E6- or E7-specific T cells being detected in 9 and 2 patients, respectively. In addition, vaccination resulted in the induction of antibodies against the HPV-16 oncoproteins. Of the 6 clinical responders, 2 patients showed both a proliferative TA-CIN-specific response and an E6-specific IFN-γ response, whereas 3 other patients displayed E6-specific reactivity only. Stable disease was recorded in 19 patients, 8 of whom showed a concomitant TA-CIN-specific proliferative and/or E6-specific T-cell response. Of the 4 progressors, 2 failed to make a T-cell response and 2 responded by either proliferation or E6 ELISPOT alone.

Conclusions: The prime-boost regimen is immunogenic in AGIN patients (humoral and cellular immunity), but there is no simple relationship between induction of systemic HPV-16-specific immunity and clinical outcome. Other factors that may play a role in the eradication of long-term established AGIN lesions need to be determined to identify the patient group that would benefit from immunotherapy with the vaccines used in this study.
Durable Carcinoembryonic Antigen (CEA)-Specific Humoral and Cellular Immune Responses in Colorectal Carcinoma Patients Vaccinated with Recombinant CEA and Granulocyte/Macrophage Colony-Stimulating Factor
Authors: Ullenhag Gustav J. , Frödin Jan-Erik , Jeddi-Tehrani Mahmood , Strigård Karin , Eriksson Emma , Samanci Ali , Choudhury Aniruddha , Nilsson Bo , Rossmann Eva D. , Mosolits Szilvia , Mellstedt Håkan
Published: September 2005

Purpose: Previous studies have indicated that carcinoembryonic antigen (CEA) might be a suitable immunotherapeutic target in colorectal carcinoma (CRC). The aim of the present study was to analyze the immunological and clinical effects of vaccination with CEA together with the adjuvant granulocyte/macrophage colony-stimulating factor (GM-CSF).

Experimental Design: Twenty-four resected CRC patients without macroscopic disease were immunized seven times with recombinant CEA at four different dose levels over a 12-month period. Half of the patients received GM-CSF (80 μg/day for 4 consecutive days) at each immunization. Patients were monitored immunologically for 36 months and clinically for 76 months. T-cell response was evaluated by a [3H]thymidine incorporation assay, and IgG response was determined by ELISA.

Results: Minor local side effects were common. All 12 patients (100%) in the GM-CSF group developed a CEA-specific T-cell as well as an IgG response. The corresponding figures in the CEA alone group were 9 of 12 (75%) and 8 of 12 (66%), respectively. GM-CSF significantly augmented the amplitude of the T-cell response and the IgG titers. No dose–response relationship was noted. The immune responses at 12 months persisted 24 months after the last vaccination. Anti-CEA IgG titers were associated with increased survival (P < 0.05), whereas standard prognostic factors had no relationship, with the exception of serum CEA value.

Conclusions: Vaccination with recombinant CEA and GM-CSF appears to be a nontoxic regimen inducing potent and durable antigen-specific IgG and T-cell response. The results of this study justify more extensive trials with recombinant CEA protein for immunotherapy of CRC.
Cell Cycle–Dependent Antagonistic Interactions between Paclitaxel and γ-Radiation in Combination Therapy
Authors: Sui Meihua , Dziadyk Jennifer M. , Zhu Xueming , Fan Weimin
Published: September 2005

Purpose: The promising clinical activity of paclitaxel, a naturally occurring antimicrotubule agent, has promoted considerable interest in combining this drug with radiation therapy, but it remains unclear whether such a combination would increase the therapeutic efficacy. This study is to assess the potential interactions between paclitaxel and γ-radiation against human tumor cells in vitro.

Experimental Design: Paclitaxel and γ-radiation were administered in three different sequences designated as pre-radiated, co-radiated, and post-radiated to BCap37 (human breast cancer cell line) and KB (human epidermoid carcinoma cell line) cells. The cytotoxic interactions between and mutual influences of these two agents on their antitumor activities were analyzed by a series of assays including cytotoxic, morphological, and biochemical examinations.

Results: The combination of paclitaxel and γ-radiation did not produce a synergistic or additive effect. Instead, the overall in vitro cytotoxicity of these combinations was much lower than that of paclitaxel treatment alone. DNA fragmentation and flow cytometric assays showed that the addition of γ-radiation interfered with paclitaxel-induced apoptosis. Further analyses indicated that the addition of γ-radiation resulted in a transient or prolonged cell cycle arrest at G2 phase, which likely prevented the cytotoxic effects of paclitaxel on both mitotic arrest and apoptosis. In addition, biochemical examinations revealed that γ-radiation inhibited paclitaxel-induced IκBα degradation and bcl-2 phosphorylation and increased the protein levels of cyclin B1 and inhibitory phosphorylation of p34cdc2.

Conclusions: Our results suggest that γ-radiation might specifically block the cell cycle at G2 phase, which in turn prevents the cytotoxic effects of paclitaxel on both mitotic arrest and apoptosis. Therefore, it eventually results in a cell cycle-dependent antagonistic effect on the antitumor activity of paclitaxel. This finding may be relevant to the clinical application of combination therapy with paclitaxel and radiation.
Class I Histone Deacetylase-Selective Novel Synthetic Inhibitors Potently Inhibit Human Tumor Proliferation
Authors: Park Jung-Hyun , Jung Yeonjoo , Kim Tai Young , Kim Sang Gyun , Jong Hyun-Soon , Lee Jung Weon , Kim Dae-Kee , Lee Jong-Soo , Kim Noe Kyeong , Kim Tae-You , Bang Yung-Jue
Published: September 2005

We have developed previously a class of synthetic hybrid histone deacetylase (HDAC) inhibitors, which were built from hydroxamic acid of trichostatin A and pyridyl ring of MS-275. In this study we evaluated the antitumor effects of these novel hybrid synthetic HDAC inhibitors, SK-7041 and SK-7068, on human cancer cells. Both SK-7041 and SK-7068 effectively inhibited cellular HDAC activity at nanomolar concentrations and induced the time-dependent hyperacetylation of histones H3 and H4. These HDAC inhibitors preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, as compared with the other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041 and SK-7068. We found that these compounds exhibited potent antiproliferative activity against various human cancer cells in vitro. Growth inhibition effect of SK-7041 and SK-7068 was related with the induction of aberrant mitosis and apoptosis in human gastric cancer cells. Both compounds induced the accumulation of cells at mitosis after 6 h of treatment, which was demonstrated by accumulation of tetraploid cells, lack of G2 cyclin/cyclin-dependent kinase inactivation, and higher mitotic index. After 12 h of treatment, apoptotic cells were increased through mitochondrial and caspase-mediated pathway. Finally, in vivo experiment showed that SK-7041 or SK-7068 was found to reduce the growth of implanted human tumors in nude mice. Therefore, based on isotype specificity and antitumor activity, SK-7041 and SK-7068 HDAC inhibitors are expected to be promising anticancer therapeutic agents and need additional clinical development.
The Potential Role of Neutrophils in Promoting the Metastatic Phenotype of Tumors Releasing Interleukin-8
Authors: De Larco Joseph E. , Wuertz Beverly R. K. , Furcht Leo T.
Published: September 2005

In the last decade, several groups have shown a direct correlation between the inappropriate or ectopic release of interleukin (IL)-8 by tumor cells in vitro and their growth and metastatic potential using in vivo models of tumor growth. IL-8 is a potent neutrophil chemoattractant. Neutrophils, as “early responders” to wounds and infections, release enzymes to remodel the extracellular matrix of the tissues through which they migrate to reach the site of the wound or infection. It is proposed that the host’s cellular response to IL-8 released by tumor cells enhances angiogenesis and contributes to tumor growth and progression. The activities released by the responding neutrophils could serve as enablers of tumor cell migration through the extracellular matrix, helping them enter the vasculature and journey to new, metastatic sites. The reactive oxygen species produced by neutrophilic oxidases to kill invading organisms have the potential to interact with tumor cells to attenuate their apoptotic cascade and increase their mutational rate. It is proposed that the increase in metastatic potential of tumors ectopically releasing IL-8 is, in part, attributable to their ability to attract neutrophils. Discussed here are possible mechanisms by which the neutrophils responding to ectopic IL-8 contribute to the in vivo growth, progression, and metastatic potential of tumor cells. Possible targets are also presented for the development of therapies to attenuate the effects of the ectopic IL-8 release by tumor cells.
Promoter Hypermethylation of Resected Bronchial Margins
Authors: Guo Mingzhou , House Michael G. , Hooker Craig , Han Yu , Heath Elizabeth , Gabrielson Edward , Yang Stephen C. , Baylin Stephen B. , Herman James G. , Brock Malcolm V.
Published: September 2005

Purpose: Histologically positive bronchial margins after resection for non-small cell lung cancer are associated with shortened patient survival due to local recurrence. We hypothesized that DNA promoter hypermethylation changes at bronchial margins could be detected in patients with no histological evidence of malignancy and that they would reflect epigenetic events in the primary tumor.

Experimental Design: Bronchial margins, primary tumor, bronchoalveolar fluid, and paired nonmalignant lung were obtained from 20 non-small cell lung cancer patients who underwent a lobectomy or greater resection. Disease-specific recurrence was the primary end point. The methylation status of p16, MGMT, DAPK, SOCS1, RASSF1A, COX2, and RARβ was examined using methylation-specific polymerase chain reaction.

Results: All malignancies had methylation in at least one locus. Concordance of one gene with an identical epigenetic change in the tumor or bronchial margin was observed in 85% of patients. Only one patient had methylation at the bronchial margin for a gene that was not methylated in the corresponding tumor. Median time to recurrence was 37 months (range, 5–71 months). There were two local recurrences and five metastases. There were no significant correlations between DNA methylation in tumor, margins, or bronchoalveolar fluid specimens and either regional recurrence or distant metastases.

Conclusions: Histologically negative bronchial margins of resected non-small cell lung cancer exhibit frequent hypermethylation changes in multiple genes. These hyper-methylation abnormalities are also present in the primary tumor and thus may represent a field defect of preneoplastic changes that occurs early in carcinogenesis.
Genomic Profiling of Viable and Proliferative Micrometastatic Cells from Early-Stage Breast Cancer Patients
Authors: Gangnus Rainer , Langer Sabine , Breit Elisabeth , Pantel Klaus , Speicher Michael R.
Published: September 2005

Purpose: Metastases in distant organs are the major cause of death for cancer patients, and bone marrow is a prominent homing organ for early disseminated cancer cells. However, it remains still unclear which of these cells evolve into overt metastases. We therefore established a new approach based on the analysis of viable and proliferating cancer cells by single-cell comparative genomic hybridization.

Experimental Design: The bone marrow of early-stage breast tumor patients (pN0M0) was screened for tumor cells by immunostaining. By applying special short-term culturing, we selected for viable and proliferative tumor cells. The short-term culturing allowed us to evaluate the proliferative potential of micrometastatic cells, which we had previously shown to represent an independent prognostic marker. We assessed genomic changes in single disseminated cancer cells by single-cell comparative genomic hybridization.

Results: We found that these viable disseminated cancer cells already had a plethora of copy number changes in their genome. All of these cells showed chromosomal copy number changes with a substantial intercellular heterogeneity and differences to the matching primary tumors.

Conclusions: The established experimental strategy might pave the way for the identification of metastatic stem cells in cancer patients. Our preliminary results support the new concept that early disseminated cancer cells evolve independently from their primary tumor.
A Concentrated Aglycone Isoflavone Preparation (GCP) That Demonstrates Potent Anti-Prostate Cancer ActivityIn vitroandIn vivo
Authors: Bemis Debra L. , Capodice Jillian L. , Desai Manisha , Buttyan Ralph , Katz Aaron E.
Published: September 2005

Purpose: Isoflavones have anticancer activities, but naturally occurring isoflavones are predominantly glycosylated and poorly absorbed. Genistein combined polysaccharide (GCP; Amino Up Chemical Co., Sapporo, Japan), is a fermentation product of soy extract and basidiomycetes mycillae that is enriched in biologically active aglycone isoflavones. This study analyzes GCP in vitro and in vivo for potential utility as a prostate cancer chemopreventative agent.

Experimental Design: Androgen-sensitive LNCaP and androgen-independent PC-3 cells were grown with various concentrations of GCP. In vitro cell growth was analyzed by the WST-1 assay, and apoptosis was assessed by fluorescence-activated cell sorting and detection of poly(ADP-ribose) polymerase cleavage using Western blot techniques. Effects of GCP on expression of cell cycle-regulatory proteins p53 (LNCaP only), p21, and p27 and the protein kinase Akt were considered using Western blot techniques. An in vivo LNCaP xenograft model was used to study the effects of a 2% GCP-supplemented diet on tumor growth in comparison with a control diet.

Results: GCP significantly suppressed LNCaP and PC-3 cell growth over 72 h (89% and 78% in LNCaP and PC-3, respectively, at 10 μg/ml; P < 0.0001). This reduction was associated with apoptosis in LNCaP cells, but not in PC-3 cells. GCP induced p27 and p53 (LNCaP only) protein expression within 6 h and suppressed phosphorylated Akt in both cell lines. The 2% GCP-supplemented diet significantly slowed LNCaP tumor growth, increasing apoptosis (P < 0.001), and decreasing proliferation (P < 0.001) over 4 weeks.

Conclusions: GCP has potent growth-inhibitory effects against prostate cancer cell lines in vitro and in vivo. These data suggest GCP has potential as an effective chemopreventive agent against prostate cancer cell growth.
A Randomized Phase II Feasibility Trial of BMS-275291 in Patients with Early Stage Breast Cancer
Authors: Miller Kathy D. , Saphner Thomas J. , Waterhouse David M. , Chen T.-T. , Rush-Taylor Anita , Sparano Joseph A. , Wolff Antonio C. , Cobleigh Melody A. , Galbraith Susan , Sledge George W.
Published: September 2005

Purpose: This pilot trial was performed to evaluate the safety, pharmacokinetics and feasibility of incorporating BMS-275291, a matrix metalloproteinase inhibitor (MMPI), into adjuvant breast cancer therapy.

Experimental Design: Patients with stage I (T1c)-IIIA breast cancer were eligible if planned adjuvant therapy consisted of either tamoxifen alone, doxorubicin + cyclophosphamide every 21 days for four cycles (AC), or AC followed by paclitaxel every 21 days for 4 cycles (AC>T). Patients were stratified by planned adjuvant therapy and randomized (2:1 ratio) to BMS-275291 (1200 mg/day) or matched placebo for 1 year.

Results: Seventy-two patients were recruited from March 2001 to July 2002. Grade ≥ 2 musculoskeletal toxicity, generally reversible arthralgia, was reported by 36.2% of patients receiving BMS-275291 compared with 16.7% of patients receiving placebo; difference = 19.5% (95% confidence interval: -0.06, 0.44; P = NS). Two patients receiving BMS-275291 developed palpable nodules along tendons. Grade ≥ 3 rash was reported by 8.5% of patients receiving BMS-275291 compared with 4.2% of patients receiving placebo; difference = 4.3% (95% confidence interval: -0.18, 0.3; P = NS). Overall, 33% of BMS-275291 patients and 21% of placebo patients discontinued treatment due to adverse events. BMS-275291 trough levels tended to decrease over time; 9 of 47 (19%) had ≥50% of trough concentrations > 124 ng/ml (IC90 for matrix metalloproteinase-9).

Conclusions: The pattern of arthralgia in BMS-275291-treated patients was consistent with matrix metalloproteinase inhibitor toxicity. Although the differential incidence of arthralgia did not reach statistical significance, the trial was terminated. An adjuvant trial in this patient population is not feasible.
Immunological Consequences of Interleukin 12 Administration after Autologous Stem Cell Transplantation
Authors: Pelloso David , Cyran Katherine , Timmons Lynette , Williams Brian T. , Robertson Michael J.
Published: September 2005

Purpose: The purpose is to determine the immunological effects of recombinant human interleukin (rhIL)-12 therapy after autologous stem cell transplantation.

Experimental Design: Twelve patients (8 non-Hodgkin’s lymphoma, 2 Hodgkin’s disease, and 2 plasma cell myeloma) were treated with rhIL-12 by bolus i.v. injection in doses of 30, 100, or 250 ng/kg starting at a median of 66 days posttransplant. Immunological assays were performed using serum and peripheral blood mononuclear cell (PBMC) samples obtained on study.

Results: Dose-dependent increases in the total lymphocyte count occurred during rhIL-12 therapy. The absolute number of peripheral blood CD4 T cells increased up to 16.3-fold, CD8 T cells up to 20.5-fold, B cells up to 11-fold, and natural killer (NK) cells up to 12.3-fold during rhIL-12 administration and returned to pretreatment baseline levels after discontinuation of rhIL-12. CD56bright NK cells expanded dramatically in the blood of a patient with baseline lymphopenia before rhIL-12 therapy. In vitro proliferation of patient PBMCs in response to IL-12 was indistinguishable from that of PBMCs obtained from healthy control sub-jects. Moreover, spontaneous in vitro proliferation of patient PBMCs increased significantly during rhIL-12 therapy. Increased levels of IFN-γ and IL-18 were detected in the serum of patients treated in the 100 and 250 ng/kg dose cohorts during the first multiple dose cycle.

Conclusions: Expansion of T, B, and NK cells occurs in vivo during rhIL-12 therapy after autologous stem cell transplantation for hematological malignancies. In contrast to their striking defect in IL-12-induced IFN-γ production, posttransplant patient PBMCs exhibit normal proliferative responses to IL-12 in vitro. Additional investigation of rhIL-12 for posttransplantation immunotherapy is warranted.
Malignant Mammary Cells Acquire Independence from Extracellular Context for Regulation of Estrogen Receptor α
Authors: Novaro Virginia , Radisky Derek C. , Castro Nancy E. Ramos , Weisz Alessandro , Bissell Mina J.
Published: September 2005

Interactions between luminal epithelial cells and their surrounding microenvironment govern the normal development and function of the mammary gland. Alterations of these interactions can induce abnormal intracellular signaling pathways that affect the development and progression of breast tumors. One critical component of mammary gland development, as well as breast cancer progression, is the expression of estrogen receptors. In a previous study using cultured nonmalignant mammary epithelial cells, we found that the basement membrane molecules, laminin-1 and collagen-IV, were involved in maintenance of estrogen receptor (ER) α expression, and that this response could be interfered with by disrupting cell-extracellular matrix adhesion. Here we use phenotypically normal mammary epithelial SCp2 cells to dissect the promoter region of the ERα that is involved in the selective response to basement membrane. We also analyze the alteration of this response in SCg6 cells, a malignant cell line that shares a common lineage with the SCp2 cells, to provide insight into the relative overexpression of ERα and the unresponsiveness to basement membrane regulation found in those malignant cells. Evidence is presented to show the relevance of the cross-talk between different signaling pathways in the constitution of a functional tissue organization and how this integration may be disrupted in the malignant phenotype.
Cardiovascular Safety Profile of Combretastatin A4 Phosphate in a Single-Dose Phase I Study in Patients with Advanced Cancer
Authors: Cooney Matthew M. , Radivoyevitch Tomas , Dowlati Afshin , Overmoyer Beth , Levitan Nathan , Robertson Kelly , Levine Sandra L. , DeCaro Kathleen , Buchter Carol , Taylor Anne , Stambler Bruce S. , Remick Scot C.
Published: September 2005

Purpose: The purpose of our study was to review and determine the cardiovascular safety profile of combretastatin A4 phosphate (CA4P) in a Phase I study in 25 patients with advanced solid tumors.

Experimental Design: CA4P was administered in a dose-escalating fashion starting at 18 mg/m2 i.v. every 21 days, and the maximal dosage was 90 mg/m2. Continuous evaluation included bedside blood pressure and pulse monitoring, 12-lead electrocardiogram (ECG) at fixed time points for measured QT interval determination, determination of the corrected QT interval (QTc) using Bazett’s formula QTc = QT/(R-R interval)1/2, and chart review. Pharmacodynamic correlations of CA4P dose, CA4P/CA4 area under the curve, and Cmax versus heart rate (HR), blood pressure, QT, and QTc intervals, over the first 4 h postdosing were analyzed.

Results: After CA4P administration, there were significant increases in QTc interval at the 3-h and 4-h time points [27.2 ms (P < 0.0001) and 30.8 ms (P < 0.0001), respectively] and HR at the 3- and 4-h time points [13.2 beats per minute (bpm; P < 0.01) and 15.1 bpm (P < 0.001), respectively]. Three of 25 patients had prolonged QTc intervals at baseline, whereas 15 (60%) of 25 and 18 (75%) of 24 patients had prolonged QTc intervals at 3 and 4 h. The slope of HR and QTc increasing as a function of time during the first 4 h was correlated to dose (in milligrams) of CA4P (P = 0.01 and r = 0.49 for HR, P = 0.005 and r = 0.55 for QTc) and to CA4 area under the curve (P = 0.04 and r = 0.41 for HR, P = 0.02 and r = 0.44 for QTc); blood pressure and uncorrected QTc interval dose-response correlations were not significant. Two patients had ECG changes consistent with an acute coronary syndrome within 24 h of CA4P infusion.

Conclusions: CA4P prolongs the QTc interval. There was a temporal relationship with the CA4P infusion and with ECG changes consistent with an acute coronary syndrome in two patients. It is advisable that future trials with CA4P have eligibility guidelines limiting patients with known coronary artery disease or those with multiple coronary artery disease risk factors until more experience is gained regarding potential cardiovascular toxicity with this agent.
Ubiquitous Aberrant RASSF1A Promoter Methylation in Childhood Neoplasia
Authors: Wong Ivy H. N. , Chan Jacqueline , Wong Joyce , Tam Paul K. H.
Published: September 2005

Purpose and Experimental Design: The role of RASSF1A has been elucidated recently in regulating apoptosis and cell cycle progression by inhibiting cyclin D1 accumulation. Aberrant RASSF1A promoter methylation has been found frequently in multiple adult cancer types. Using methylation-specific PCR and reverse transcription-PCR, we investigated epigenetic deregulation of RASSF1A in primary tumors, adjacent nontumor tissues, secondary metastases, peripheral blood cells, and plasma samples from children with 18 different cancer types, in association with their clinicopathologic features.

Results: Regardless of the tumor size, ubiquitous RASSF1A promoter methylation was found in 67% (16 of 24) of pediatric tumors, including neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, pancreatoblastoma, adrenocortical carcinoma, Wilms’ tumor, Burkitt’s lymphoma, and T-cell lymphoma. A majority (75%) of pediatric cancer patients with tumoral RASSF1A methylation was male. Methylated RASSF1A alleles were also detected in 4 of 13 adjacent nontumor tissues, suggesting that this epigenetic change is potentially an early and critical event in childhood neoplasia. RASSF1A promoter methylation found in 92% (11 of 12) of cell lines largely derived from pediatric cancer patients was significantly associated with transcriptional silencing/repression. After demethylation treatment with 5-aza-2′-deoxycytidine, transcriptional reactivation was shown in KELLY, RD, and Namalwa cell lines as analyzed by reverse transcription-PCR. For the first time, RASSF1A methylation was detected in 54% (7 of 13), 40% (4 of 10), and 9% (1 of 11) of buffy coat samples collected before, during, and after treatment, correspondingly, from pediatric patients with neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, rhabdomyosarcoma, Burkitt’s lymphoma, T-cell lymphoma, or acute lymphoblastic leukemia. Concordantly, RASSF1A methylation was found during treatment in plasma of the same patients, suggesting cell death and good response to chemotherapy.

Conclusions: RASSF1A methylation in tumor or buffy coat did not correlate strongly with age, tumor size, recurrence/metastasis, or overall survival in this cohort of pediatric cancer patients. Of importance, epigenetic inactivation of RASSF1A may potentially be crucial in pediatric tumor initiation.
Gene-Expression Profile Changes Correlated with Tumor Progression and Lymph Node Metastasis in Esophageal Cancer
Authors: Tamoto Eiji , Tada Mitsuhiro , Murakawa Katsuhiko , Takada Minoru , Shindo Gaku , Teramoto Ken-ichi , Matsunaga Akihiro , Komuro Kazuteru , Kanai Motoshi , Kawakami Akiko , Fujiwara Yoshie , Kobayashi Nozomi , Shirata Katsutoshi , Nishimura Norihiro , Okushiba Shun-ichi , Kondo Satoshi , Hamada Jun-ichi , Yoshiki Takashi , Moriuchi Tetsuya , Katoh Hiroyuki
Published: September 2005

Purpose: The purpose of this research was to identify molecular clues to tumor progression and lymph node metastasis in esophageal cancer and to test their value as predictive markers.

Experimental Design: We explored the gene expression profiles in cDNA array data of a 36-tissue training set of esophageal squamous cell carcinoma (ESCC) by using generalized linear model-based regression analysis and a feature subset selection algorithm. By applying the identified optimal feature sets (predictive gene sets), we trained and developed ensemble classifiers consisting of multiple probabilistic neural networks combined with AdaBoosting to predict tumor stages and lymph node metastasis. We validated the classifier abilities with 18 independent cases of ESCC.

Results: We identified 71 genes of 1289 cancer-related genes of which the expression correlated with tumor stages. Of the 71 genes, 47 significantly differed between the Tumor-Node-Metastasis pT1/2 and pT3/4 stages. Cell cycle regulators and transcriptional factors possibly promoting the growth of tumor cells were highly expressed in the early stages of ESCC, whereas adhesion molecules and extracellular matrix-related molecules possibly promoting invasiveness increased in the later stages. For lymph node metastasis, we identified 44 genes with predictive values, which included cell adhesion molecules and cell membrane receptors showing higher expression in node-positive cases and cell cycle regulators and intracellular signaling molecules showing higher expression in node-negative cases. The ensemble classifiers trained with the selected features predicted tumor stage and lymph node metastasis in the 18 validation cases with respective accuracies of 94.4% and 88.9%. This demonstrated the reproducibility and predictive value of the identified features.

Conclusion: We suggest that these characteristic genes will provide useful information for understanding the malignant nature of ESCC as well as information useful for personalizing the treatments.
Gene Expression Profiling of Differentiated Thyroid Neoplasms
Authors: Chevillard Sylvie , Ugolin Nicolas , Vielh Philippe , Ory Katherine , Levalois Céline , Elliott Danielle , Clayman Gary L. , El-Naggar Adel K.
Published: September 2005

Purpose: The purpose of this research was to identify novel genes that can be targeted as diagnostic and clinical markers of differentiated thyroid tumors.

Experimental Design: Gene expression analysis using microarray platform was performed on 6 pathologically normal thyroid samples and 12 primary follicular and papillary thyroid neoplasms. Microarrays containing probes for 5,760 human full-length cDNAs were used for hybridization with total RNA from normal and tumor thyroid samples labeled with Cy3-dUTP and Cy5-dUTP, respectively. Scanned array images were recorded, and data analysis was performed. Selected sets of differentially expressed genes were analyzed using quantitative real-time reverse transcription-PCR for verification.

Results: We identified 155 genes that differentiate histologically normal thyroid tissues from benign and malignant thyroid neoplasms. Of these 75 genes were differentiated between follicular neoplasms (adenoma and carcinoma) and the follicular variant of papillary carcinoma. Purely follicular neoplasms (adenomas and carcinomas) shared many genetic profiles, and only 43 genes were distinctly different between these tumors. Hierarchical cluster analysis also differentiated conventional papillary carcinoma from its follicular variant and follicular tumors. The differentially expressed genes were composed of members of cell differentiation, adhesion, immune response, and proliferation associated pathways. Quantitative real-time reverse transcription-PCR analysis of selected genes corroborated the microarray expression results.

Conclusions: Our study show the following: (1) differences in gene expression between tumor and nontumor bearing normal thyroid tissue can be identified, (2) a set of genes differentiate follicular neoplasm from follicular variant of papillary carcinoma, (3) follicular adenoma and carcinoma share many of the differentiated genes, and (4) gene expression differences identify conventional papillary carcinoma from the follicular variant.
Expression of the ELAV-Like Protein HuR Is Associated with Higher Tumor Grade and Increased Cyclooxygenase-2 Expression in Human Breast Carcinoma
Authors: Denkert Carsten , Weichert Wilko , Winzer Klaus-Jürgen , Müller Berit-Maria , Noske Aurelia , Niesporek Silvia , Kristiansen Glen , Guski Hans , Dietel Manfred , Hauptmann Steffen
Published: September 2005

Purpose: The human ELAV (embryonic lethal abnormal vision)-like protein HuR stabilizes a certain group of cellular mRNAs that contain AU-rich elements in their 3′-untranslated region. Cell culture studies have shown that the mRNA of cyclooxygenase (COX)-2 can be stabilized by HuR.

Experimental Design: To investigate a possible contribution of dysregulation of mRNA stability to the progression of cancer and to overexpression of COX-2, we studied expression of HuR in 208 primary breast carcinomas by immunohistochemistry.

Results: There were two different staining patterns of HuR in tumor tissue of breast carcinomas: nuclear expression was seen in 61% of cases; and an additional cytoplasmic expression was seen in 30% of cases. Expression of HuR was significantly associated with increased COX-2 expression; this association was particularly significant for cytoplasmic HuR expression (P < 0.0005). We further observed a significant association of cytoplasmic (P = 0.002) or nuclear HuR (P = 0.027) expression with increased tumor grade. Only 13% of the grade 1 carcinomas showed cytoplasmic expression of HuR, compared with 46% of the grade 3 carcinomas. There was no significant correlation between HuR expression and other clinicopathological parameters such as histological type, tumor size, or nodal status as well as patient survival.

Conclusions: Our results suggest that overexpression of HuR in tumor tissue may be part of a regulatory pathway that controls the mRNA stability of several important targets in tumor biology, such as COX-2. Based on our results, additional studies are necessary to investigate whether HuR might be a potential target for molecular tumor therapy.
Dose-Ranging Study of the Safety and Pharmacokinetics of Atrasentan in Patients with Refractory Malignancies
Authors: Ryan Christopher W. , Vogelzang Nicholas J. , Vokes Everett E. , Kindler Hedy L. , Undevia Samir D. , Humerickhouse Rod , André Amy K. , Wang Qiang , Carr Robert A. , Ratain Mark J.
Published: September 2005

Purpose: Atrasentan is an orally bioavailable selective antagonist of the endothelin receptor ETA. Due to the potential activity of this agent against prostate cancer, the majority of subjects enrolled in prior studies had been male. This Phase I study sought to determine the toxicity and pharmacokinetics of daily atrasentan in a population of both female and male subjects with advanced malignancies.

Experimental Design: Patients with refractory malignancies received atrasentan once daily at doses ranging from 5 mg to 75 mg. At least 3 subjects were treated at each dose level before enrollment began at the next higher dose level. Enrollment for specific dose levels was expanded if any subject experienced serious drug-related toxicity. Plasma concentration profiles for atrasentan were determined after dosing on days 1 and 28.

Results: Thirty-five patients received atrasentan at doses from 5 mg to 75 mg. The most frequent drug-related adverse events were headache (60%), rhinitis (49%), and peripheral edema (31%). These toxicities were mild to moderate in severity and reversible on cessation of treatment. Dose escalation was stopped at the 75-mg dose level due to the occurrence of three severe adverse events (2 hyponatremia and 1 hypotension). Atrasentan was rapidly absorbed after oral administration; mean time to maximum observed concentration ranged from 0.3 to 1.7 h. Terminal elimination half-life averaged 26 h. No significant difference between sexes was found in any atrasentan pharmacokinetic parameter tested, including maximum observed plasma concentration, time to maximum observed concentration, minimum observed plasma concentration, area under the plasma concentration-time curve, and elimination rate constant.

Conclusions: Atrasentan is well tolerated in both female and male cancer patients at doses of up to 60 mg/day with dose-limiting toxicity observed at 75 mg/day. The most frequently observed toxicities were headache, rhinitis, and edema. There was no statistically significant difference in atrasentan pharmacokinetics between sexes.
A Two-by-Two Factorial Trial Comparing Oral with Transdermal Estrogen Therapy and Fenretinide with Placebo on Breast Cancer Biomarkers
Authors: Decensi Andrea , Bonanni Bernardo , Baglietto Laura , Guerrieri-Gonzaga Aliana , Ramazzotto Francesca , Johansson Harriet , Robertson Chris , Marinucci Irene , Mariette Frederique , Sandri Maria Teresa , Daldoss Cristina , Bianco Vanda , Buttarelli Marco , Cazzaniga Massimiliano , Franchi Dorella , Cassano Enrico , Omodei Umberto
Published: September 2005

Purpose: Oral conjugated equine estrogen (CEE) and medroxyprogesterone acetate (MPA) increase breast cancer risk, whereas the effect of transdermal estradiol (E2) and MPA is less known. Fenretinide may decrease second breast malignancies in premenopausal women but not in postmenopausal women, suggesting a hormone-sensitizing effect. We compared the 6 and 12-month changes in insulin-like growth factor-I (IGF-I), IGF-binding protein-3 (IGFBP-3), IGF-I:IGFBP-3 ratio, sex-hormone binding-globulin, and computerized mammographic percent density during oral CEE or transdermal E2 with sequential MPA and fenretinide or placebo.

Experimental Design: A total of 226 recent postmenopausal healthy women were randomly assigned in a two-by-two factorial design to either oral CEE 0.625 mg/day (n = 111) or transdermal E2, 50 μg/day (n = 115) and to fenretinide 100 mg/twice a day (n = 112) or placebo (n = 114) for 12 months. Treatment effects were investigated by the Kruskall-Wallis test and analysis of covariance. P values were two-sided.

Results: After 12 months, oral CEE decreased IGF-I by 26% [95% confidence interval (CI), 22–30%] and increased sex-hormone binding-globulin by 96% (95% CI, 79–112%) relative to baseline, whereas no change occurred with transdermal E2 (P < 0.001 between groups). Fenretinide decreased IGFBP-3 relative to placebo (P = 0.04). Percentage of breast density showed an absolute increase of 3.5% (95% CI, 2.5–4.6%) during hormone therapy without differences between groups (P = 0.39).

Conclusions: Oral CEE has more favorable changes than transdermal E2 on circulating breast cancer risk biomarkers but gives similar effects on mammographic density. Fenretinide exerted little modulation on most biomarkers. The clinical implications of these findings require additional studies.