bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and Mutational Status of
c-kit
in Small-Cell Lung Cancer
Authors: Boldrini Laura , Ursino Silvia , Gisfredi Silvia , Faviana Pinuccia , Donati Valentina , Camacci Tiziano , Lucchi Marco , Mussi Alfredo , Basolo Fulvio , Pingitore Raffaele , Fontanini Gabriella
Published: September 2005

Purpose: The c-kit protein, also known as CD117, is a member of the type III receptor tyrosine kinase family. Kinase activity has been implicated in the pathophysiology of many tumors, including small-cell lung carcinoma (SCLC). Autocrine or paracrine activation of c-kit by its ligand has been postulated for lung cancer, but this receptor can also be activated by mutations of the c-kit gene. We examined c-kit expression and mutational status in SCLC to verify its putative expression and genetic alterations, as well as its eventual prognostic impact.

Experimental Design: We studied 60 SCLC samples to determine the mutations of the coding region of the gene; the exons 9 and 11 were analyzed by PCR-single-strand conformational polymorphism and automated sequencing. Moreover, c-kit expression was evaluated in 55 samples by immunohistochemical method.

Results: Expression of c-kit was demonstrated in about 40% of SCLC samples. Two mutations in exon 9 and three mutations in exon 11 were found. Kaplan-Meier analysis revealed no prognostic significance of c-kit expression for survival.

Conclusions: In our series, the expression of c-kit and its mutational status failed to appear relevant or to have a significant impact on survival; this makes the therapeutic approach with an inhibitor of tyrosine kinase more difficult in SCLC until a sure demonstration of c-kit implication is obtained for this tumor.
Prevention of Oxaliplatin-Related Neurotoxicity by Calcium and Magnesium Infusions
Authors: Gamelin Laurence , Boisdron-Celle Michele , Delva Remy , Guérin-Meyer Véronique , Ifrah Norbert , Morel Alain , Gamelin Erick
Published: September 2005

Purpose: Oxaliplatin is active in colorectal cancer. Sensory neurotoxicity is its dose-limiting toxicity. It may come from an effect on neuronal voltage-gated Na channels, via the liberation one its metabolite, oxalate. We decided to use Ca and Mg as oxalate chelators.

Experimental Design: A retrospective cohort of 161 patients treated with oxaliplatin + 5-fluorouracil and leucovorin for advanced colorectal cancer, with three regimens of oxaliplatin (85 mg/m2/2w, 100/2w, 130/3w) was identified. Ninety-six patients received infusions of Ca gluconate and Mg sulfate (1 g) before and after oxaliplatin (Ca/Mg group) and 65 did not.

Results: Only 4% of patients withdrew for neurotoxicity in the Ca/Mg group versus 31% in the control group (P = 0.000003). The tumor response rate was similar in both groups. The percentage of patients with grade 3 distal paresthesia was lower in Ca/Mg group (7 versus 26%, P = 0.001). Acute symptoms such as distal and lingual paresthesia were much less frequent and severe (P = 10-7), and pseudolaryngospasm was never reported in Ca/Mg group. At the end of the treatment, 20% of patients in Ca/Mg group had neuropathy versus 45% (P = 0.003). Patients with grade 2 and 3 at the end of the treatment in the 85 mg/m2 oxaliplatin group recovered significantly more rapidly from neuropathy than patients without Ca/Mg.

Conclusions: Ca/Mg infusions seem to reduce incidence and intensity of acute oxaliplatin-induced symptoms and might delay cumulative neuropathy, especially in 85 mg/m2 oxaliplatin dosage.
Confocal Microscopic Analysis of the Interaction between Cisplatin and the Copper Transporter ATP7B in Human Ovarian Carcinoma Cells
Authors: Katano Kuniyuki , Safaei Roohangiz , Samimi Goli , Holzer Alison , Tomioka Mika , Goodman Murray , Howell Stephen B.
Published: September 2005

Some cisplatin (DDP)-resistant cells overexpress the copper export transporter ATP7B, and cells molecularly engineered to overexpress ATP7B are resistant to DDP. The interaction of Cu with ATP7B normally triggers its relocalization from the perinuclear region to more peripheral vesicles. To investigate the interaction of DDP with ATP7B, we examined the effect of DDP on the subcellular localization of ATP7B using human ovarian carcinoma cells expressing a cyan fluorescent protein (ECFP)-tagged ATP7B (2008/ECFP-ATP7B). ATP7B expression was confirmed in 2008/ECFP-ATP7B cells by Western blotting, and its functionality was documented by showing that it rendered the cells 1.9-fold resistant to CuSO4 and 4.1-fold resistant to DDP and also reduced the accumulation of both drugs. There was greater sequestration of Pt into intracellular vesicles in the 2008/ECFP-ATP7B cells than in the 2008/ECFP cells. Confocal digital microscopy revealed that ECFP-ATP7B localized in the perinuclear region in absence of drug exposure and that both Cu and DDP triggered relocalization to more peripheral vesicular structures. A fluorescein-labeled form of DDP that retained cytotoxicity and was subject to the same mechanisms of resistance as DDP colocalized with ECFP-ATP7B in the 2008/ECFP-ATP7B cells, whereas the same fluorochrome lacking the DDP moiety did not. These results provide evidence that DDP directly interacts with ATP7B to trigger its relocalization and that ATP7B mediates resistance to DDP by sequestering it into vesicles of the secretory pathway for export from the cell.
Dendritic Cell Vaccination in Medullary Thyroid Carcinoma
Authors: Stift Anton , Sachet Monika , Yagubian Rubina , Bittermann Clemens , Dubsky Peter , Brostjan Christine , Pfragner Roswitha , Niederle Bruno , Jakesz Raimund , Gnant Michael , Friedl Josef
Published: September 2005

Purpose: Prognosis and treatment effectiveness for medullary thyroid carcinoma (MTC) are strictly related to tumor stage. Palliative treatment options show no significant benefit. A promising treatment approach for human cancer is based on the vaccination of autologous dendritic cells (DCs).

Experimental Design: The objective of this study was to evaluate the effectiveness of DC vaccines in MTC patients. Therefore, we generated autologous tumor lysate-pulsed DCs from 10 patients suffering from advanced MTC for repeated vaccination. Mature DCs were derived from peripheral blood monocytes by using CD14 magnetic bead selection and subsequent culture in the presence of granulocyte macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor α with or without addition of IFN-γ. DCs were loaded with tumor lysate and further injected into a groin lymph node. Toxicity, tumor marker profile, immune response, and clinical response were determined.

Results: Vaccination was well tolerated and induced a positive immunological response in all of the tested patients as evaluated by in vivo delayed-type hypersensitivity reactivity or in vitro intracytoplasmic IFN-γ detection assay. Three patients had a partial response, 1 patient presented a minor response, and 2 patients showed stable disease. The remaining 4 patients had progressive disease.

Conclusions: These data provide strong evidence that vaccination with tumor-lysate pulsed DCs results in the induction of a specific immune response in patients suffering from MTC. Objective clinical responses could be observed even for far-advanced disease. Therefore, we suggest that MTC is particularly suited for DC-based immunotherapy.
Gene Expression in Gastrointestinal Stromal Tumors Is Distinguished by
KIT
Genotype and Anatomic Site
Authors: Antonescu Cristina R. , Viale Agnes , Sarran Lisa , Tschernyavsky Sylvia J. , Gonen Mithat , Segal Neil H. , Maki Robert G. , Socci Nicholas D. , DeMatteo Ronald P. , Besmer Peter
Published: September 2005

Purpose: Gastrointestinal stromal tumors (GISTs) are specific KIT expressing and KIT-signaling driven mesenchymal tumors of the human digestive tract, many of which have KIT-activating mutations. Previous studies have found a relatively homogeneous gene expression profile in GIST, as compared with other histological types of sarcomas. Transcriptional heterogeneity within clinically or molecularly defined subsets of GISTs has not been previously reported. We tested the hypothesis that the gene expression profile in GISTs might be related to KIT genotype and possibly to other clinicopathological factors.

Experimental Design: An HG-U133A Affymetrix chip (22,000 genes) platform was used to determine the variability of gene expression in 28 KIT-expressing GIST samples from 24 patients. A control group of six intra-abdominal leiomyosarcomas was also included for comparison. Statistical analyses (t tests) were performed to identify discriminatory gene lists among various GIST subgroups. The levels of expression of various GIST subsets were also linked to a modified version of the growth factor/KIT signaling pathway to analyze differences at various steps in signal transduction.

Results: Genes involved in KIT signaling were differentially expressed among wild-type and mutant GISTs. High gene expression of potential drug targets, such as VEGF, MCSF, and BCL2 in the wild-type group, and Mesothelin in exon 9 GISTs were found. There was a striking difference in gene expression between stomach and small bowel GISTs. This finding was validated in four separate tumors, two gastric and two intestinal, from a patient with familial GIST with a germ-line KIT W557R substitution.

Conclusions: GISTs have heterogeneous gene expression depending on KIT genotype and tumor location, which is seen at both the genomic level and the KIT signaling pathway in particular. These findings may explain their variable clinical behavior and response to therapy.
Successful Engraftment After Reduced-Intensity Umbilical Cord Blood Transplantation for Adult Patients with Advanced Hematological Diseases
Authors: Miyakoshi Shigesaburo , Yuji Koichiro , Kami Masahiro , Kusumi Eiji , Kishi Yukiko , Kobayashi Kazuhiko , Murashige Naoko , Hamaki Tamae , Kim Sung-Won , Ueyama Jun-ichi , Mori Shin-ichiro , Morinaga Shin-ichi , Muto Yoshitomo , Masuo Shigeru , Kanemaru Mineo , Hayashi Tatsuyuki , Takaue Yoichi , Taniguchi Shuichi
Published: September 2005

Purpose: The purpose of this research was to evaluate the feasibility of reduced-intensity unrelated cord-blood transplantation (RI-UCBT) in adult patients with advanced hematological diseases.

Experimental Design: Thirty patients (median age, 58.5 years; range, 20–70 years) with advanced hematological diseases underwent RI-UCBT at Toranomon Hospital between September 2002 and August 2003. Preparative regimen composed of fludarabine 25 mg/m2 on days −7 to −3, melphalan 80 mg/m2 on day −2, and 4 Gy total body irradiation on day −1. Graft-versus-host disease prophylaxis was composed of cyclosporin alone.

Results: Twenty-six patients achieved primary neutrophil engraftment after a median of 17.5 days. Median infused total cell dose was 3.1 × 107/kg (range, 2.0–4.3 × 107/kg). Two transplant-related mortalities occurred within 28 days of transplant, and another 2 patients displayed primary graft failure. Cumulative incidence of complete donor chimerism at day 60 was 93%. Grade II-IV acute graft-versus-host disease occurred in 27% of patients, with median onset 36 days. Primary disease recurred in 3 patients, and transplant-related mortality within 100 days was 27%. Estimated 1-year overall survival was 32.7%. Excluding 7 patients with documented infection, 19 patients displayed noninfectious fever before engraftment (median onset, day 9). Manifestations included high-grade fever, eruption, and diarrhea. The symptoms responded well to corticosteroid treatments in 7 of 13 treated patients.

Conclusion: This study demonstrated the feasibility of RI-UCBT in adults.
Circulating Growth Factor Levels Are Associated with Tumorigenesis in Neurofibromatosis Type 1
Authors: Mashour George A. , Driever Pablo Hernáiz , Hartmann Melanie , Drissel Stephanie N. , Zhang Tingguo , Scharf Bianca , Felderhoff-Müser Ursula , Sakuma Sadatoshi , Friedrich Reinhard E. , Martuza Robert L. , Mautner Victor Felix , Kurtz Andreas
Published: September 2005

Purpose: Neurofibromatosis type 1 (NF1) is characterized by systemic development of neurofibromas. Early clinical diagnosis can be ambiguous, and genetic diagnosis can be prohibitively difficult. Dysregulation of a number of growth factors has been suggested to be a mechanism of pathogenesis. This study was performed to assess the contribution of circulating growth factors for diffuse tumorigenesis and the diagnostic value of circulating growth factor identification in serum.

Experimental Design: The growth stimulation of neurofibroma-derived cells by serum from NF1 patients was tested, and serum growth factor levels in a cohort of NF1 patients (n = 39) between the ages of 7 and 70 years were analyzed.

Results: Concentrations of midkine (MK) and stem cell factor, but not epidermal growth factor, were substantially increased in serum of NF1 patients when compared with healthy controls. Within the NF1 group, MK levels increased dramatically at puberty from an average of 0.79 ng/mL in patients <18 years to 1.18 ng/mL in patients >18 years old. Stem cell factor and MK concentrations above a defined threshold in serum of NF1 patients are of diagnostic benefit for 96% of patients in the cohort tested. Furthermore, serum from NF1 patients enhanced proliferation of human neurofibroma-derived primary Schwann cells and endothelial cells substantially better than normal serum.

Conclusions: Enhanced circulating growth factor levels contribute to diffuse tumorigenesis in NF1 and may provide the basis for molecular diagnosis.
Synergistic Effect of Histone Deacetylase Inhibitors FK228 and m-Carboxycinnamic Acid Bis-Hydroxamide with Proteasome Inhibitors PSI and PS-341 against Gastrointestinal Adenocarcinoma Cells
Authors: Adachi Masaaki , Zhang YuBin , Zhao Xudong , Minami Takae , Kawamura Rina , Hinoda Yuji , Imai Kohzoh
Published: September 2005

Purpose: We investigated whether the histone deacetylase inhibitors m-carboxycinnamic acid bis-hydroxamide (CBHA) and a bicyclic depsipeptide, FK228, can enhance the anticancer effect of the proteasome inhibitors PSI and PS-341 in gastrointestinal carcinoma cells.

Experimental Design: The anticancer effect of CBHA or FK228 and PSI or PS-341 was evaluated by cell death, caspase-3 activity, externalization of phosphatidylserine and DNA fragmentation, and colony formation assay. Expression of apoptosis-related molecules and cell cycle regulatory molecules, as well as phosphorylation of p38 were investigated by immunoblots. Generation of reactive oxygen species (ROS) was detected by intracellular oxidation of 5- (and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate.

Results: CBHA or FK228 plus PSI or PS-341 synergistically induced apoptosis in human colonic DLD-1 and gastric MKN45 carcinoma cell lines. CBHA or FK228, but not 5-fluorouracil, plus PS-341 strongly decreased plating efficiency of DLD-1 cells. FK228 elicited ROS generation, and the free radical scavenger l-N-acetylcysteine inhibited the synergistic anticancer effect of combined therapy. In addition, l-N-acetylcysteine inhibited the combined therapy-mediated elevation of a proapoptotic BH3-only protein Bim expression, phosphorylation of H2AX, and accumulation of 8-hydroxydeoxyguanosine.

Conclusions: FK228 or CBHA and PSI or PS-341 synergistically induce apoptosis in DLD-1 and MKN45 cells depending on ROS-mediated signals. Our data suggest that a combination of FK228 or CBHA with PSI or PS-341 may be a valuable therapy against gastrointestinal adenocarcinoma cells.
Plasma and Cerebrospinal Fluid Population Pharmacokinetics of Temozolomide in Malignant Glioma Patients
Authors: Ostermann Sandrine , Csajka Chantal , Buclin Thierry , Leyvraz Serge , Lejeune Ferdy , Decosterd Laurent A. , Stupp Roger
Published: September 2005

Purpose: Scarce information is available on the brain penetration of temozolomide (TMZ), although this novel methylating agent is mainly used for the treatment of malignant brain tumors. The purpose was to assess TMZ pharmacokinetics in plasma and cerebrospinal fluid (CSF) along with its inter-individual variability, to characterize covariates and to explore relationships between systemic or cerebral drug exposure and clinical outcomes.

Experimental Design: TMZ levels were measured by high-performance liquid chromatography in plasma and CSF samples from 35 patients with newly diagnosed or recurrent malignant gliomas. The population pharmacokinetic analysis was performed with nonlinear mixed-effect modeling software. Drug exposure, defined by the area under the concentration-time curve (AUC) in plasma and CSF, was estimated for each patient and correlated with toxicity, survival, and progression-free survival.

Results: A three-compartment model with first-order absorption and transfer rates between plasma and CSF described the data appropriately. Oral clearance was 10 liter/h; volume of distribution (VD), 30.3 liters; absorption constant rate, 5.8 h−1; elimination half-time, 2.1 h; transfer rate from plasma to CSF (Kplasma→CSF), 7.2 × 10−4h−1 and the backwards rate, 0.76 h−1. Body surface area significantly influenced both clearance and VD, and clearance was sex dependent. The AUCCSF corresponded to 20% of the AUCplasma. A trend toward an increased Kplasma→CSF of 15% was observed in case of concomitant radiochemotherapy. No significant correlations between AUC in plasma or CSF and toxicity, survival, or progression-free survival were apparent after deduction of dose-effect.

Conclusions: This is the first human pharmacokinetic study on TMZ to quantify CSF penetration. The AUCCSF/AUCplasma ratio was 20%. Systemic or cerebral exposures are not better predictors than the cumulative dose alone for both efficacy and safety.
A Potent Immunogenic General Cancer Vaccine That Targets Survivin, an Inhibitor of Apoptosis Proteins
Authors: Idenoue Satomi , Hirohashi Yoshihiko , Torigoe Toshihiko , Sato Yuriko , Tamura Yasuaki , Hariu Hiroyuki , Yamamoto Masaaki , Kurotaki Takehiro , Tsuruma Tetsuhiro , Asanuma Hiroko , Kanaseki Takayuki , Ikeda Hideyuki , Kashiwagi Kiyoteru , Okazaki Minoru , Sasaki Kazuaki , Sato Takashi , Ohmura Tousei , Hata Fumitake , Yamaguchi Koji , Hirata Koichi , Sato Noriyuki
Published: September 2005

We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8+ CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24+ cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24+ cancer patients.
Orthotopic Implantation of Human Hepatocellular Carcinoma in Mice
Authors: Armengol Carolina , Tarafa Gemma , Boix Loreto , Solé Manel , Queralt Rosa , Costa Dolors , Bachs Oriol , Bruix Jordi , Capellá Gabriel
Published: September 2005

Purpose: To allow the longitudinal investigation of molecular events associated with the progression of human hepatocellular carcinoma (HCC), we sought to develop a murine model by orthotopic implantation of tumor fragments obtained from patients diagnosed at early stage.

Experimental Design: Tumor pieces (2 × 2 mm) were implanted on the liver surface of nu/nu mice. After xenograft growing, subsequent passages were performed to achieve long-term implant viability. Isolation of tumoral hepatocytes was done to establish new cell lines. HCC characteristics, proliferation rate, apoptotic index (terminal deoxynucleotidyl transferase-mediated nick end labeling), and expression of cell-cycle regulators (cyclins E and A, p21Cip1, p27Kip1, p16INK4a, pRb, and p53) were assessed by Western Blot and immunohistochemistry, to correlate them with tumor progression.

Results: Five (50%) of the 10 primary HCCs resulted in small slow-growing liver implants. Three of them are viable after 48 months, whereas the remaining two survived for 15 and 13 months. Xenografts throughout passages exhibited a more aggressive phenotype with a poorer degree of differentiation, intense proliferation, moderate apoptosis, cell-cycle deregulation, p53 alterations, microvascular invasion, and dissemination. In one single passage, we observed critical growth delay, which was associated with significant p27kip1 overexpression. We established the anchor-free growing BCLC-9 cell line from one xenograft. This has gains of chromosomes 7, 5p, 6q, and 9q, is hepatitis B virus-DNA positive, does not secrete α-fetoprotein, and has TP53 missense mutations in codons 192 and 242.

Conclusions: The orthotopic implantation of early HCC fragments in nude mice provides a useful model to investigate the mechanisms of human HCC evolution and to establish new cell lines.
Competing Causes of Death and Second Primary Tumors in Patients with Locoregionally Advanced Head and Neck Cancer Treated with Chemoradiotherapy
Authors: Argiris Athanassios , Brockstein Bruce E. , Haraf Daniel J. , Stenson Kerstin M. , Mittal Bharat B. , Kies Merrill S. , Rosen Fred R. , Jovanovic Borko , Vokes Everett E.
Published: September 2005

Purpose: The purpose of this retrospective analysis was to evaluate the emergence of second primary malignancies and the contribution of different causes of death to the outcome of patients with locoregionally advanced head and cancer receiving primary chemoradiotherapy.

Experimental Design: We studied 324 patients with stage IV squamous cell head and neck cancer who were enrolled on five consecutive multicenter Phase II studies of concurrent chemoradiotherapy. All of the regimens included concurrent 5-fluorouracil and hydroxyurea on an alternate week schedule with radiotherapy, either alone (FHX) or with cisplatin (C-FHX) or paclitaxel (T-FHX). The cumulative incidence of second primary tumors or death from any cause was estimated using methods of competing risk analysis.

Results: Median follow-up of surviving patients was 5.2 years (2–10.6 years). The 5-year overall survival and progression-free survival of the cohort were 46% and 65%, respectively. Causes of death and median time of occurrence were as follows: disease (n = 88; 1.5 years), treatment-associated acute or late complications (n = 30; 4 months), second primary tumors (n = 18; 3.5 years), comorbidities (n = 41; 1.9 years), and unknown (n = 20; 5.1 years). Predominant causes of death from comorbidities were cardiac and respiratory illnesses. Twenty-six patients (8%) developed a second primary tumor at a median time of 2.8 years (4 months to 10 years). The cumulative incidence of second primary tumors was 5%, 7%, and 13% at 3, 5, and 10 years, respectively. The most frequent site of second primaries was the lung (n = 13), followed by the esophagus (n = 3) and head and neck (n = 2)

Conclusions: Patients with locoregionally advanced head and neck cancer treated with concurrent chemoradiotherapy are potentially curable but face significant risks of mortality from causes other than disease progression. Ameliorating toxicity, and implementing secondary screening and chemoprevention strategies are major goals in the management of head and neck cancer.
Neoadjuvant Endocrine Therapy as a Drug Development Strategy
Authors: Ellis Matthew J.
Published: September 2005

The aromatase inhibitors offer both toxicity and efficacy advantages over tamoxifen, but to date, their overall impact on breast cancer outcomes has been modest. Advanced breast cancer remains incurable, and for early stage disease, an improvement in survival with AI versus tamoxifen has yet to be demonstrated. Resistance to endocrine manipulation is at the core of the problem and must be overcome to make additional progress. A number of signal transduction inhibitors (STIs) are now under development as endocrine resistance modulators, including those targeting cyclooxygenase-2, HER1 and/or 2 kinase, mTOR, and farnesyl transferase. Developing STIs for this indication is a challenge, however, because we still do not have a clear understanding of the molecular basis of resistance. A complete understanding could translate into a series of endocrine therapy/STI combinations that would be tailored according to the biology of the individual tumor to achieve optimal efficacy and safety. The development of this strategy will require the ability to diagnose resistance mechanisms on a tumor-by-tumor basis, and this can only be attained through careful clinical investigation. Neoadjuvant endocrine therapy is an appealing context to conduct research in this area because clinical outcomes can be obtained within a few months of treatment, and repeated tumor sampling for biomarker analysis (pharmacodynamic tumor profiling) can be readily achieved. However, the optimal clinical investigative approaches, analytical techniques, and appropriate surrogate end points have yet to be identified and are the subject of several ongoing or planned clinical studies.
Cross-Talk between Estrogen Receptor and Growth Factor Pathways as a Molecular Target for Overcoming Endocrine Resistance
Authors: Schiff Rachel , Massarweh Suleiman A. , Shou Jiang , Bharwani Lavina , Mohsin Syed K. , Osborne C. Kent
Published: September 2005

Introduced more than 100 years ago, endocrine therapy is still the most important systemic therapy for all stages of estrogen receptor (ER) -positive breast tumors. A major clinical problem limiting the usefulness of this therapy is tumor resistance, either de novo or acquired during the course of the treatment. Relatively new discoveries emphasize the complexity of ER signaling and its multiple regulatory interactions with growth factor and other kinase signaling pathways. Both genomic (nuclear) and nongenomic (membrane and cytoplasmic) ER activities contribute to this intimate cross-talk, which is probably a fundamental factor in endocrine resistance. New targeted therapies, especially against the epidermal growth factor receptor/HER-2 pathway, should be carefully evaluated in more (bio)logical strategies to enable them to be exploited appropriately. A strategy of combining endocrine therapy (particularly tamoxifen) with these inhibitors, to circumvent de novo and acquired resistance, will be discussed. We will also emphasize open questions and future challenges in the dynamic research field of molecular ER biology from the endocrine therapy perspective.
The Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor ZD1839 (Iressa) Suppresses c-Src and Pak1 Pathways and Invasiveness of Human Cancer Cells
Authors: Yang Zhibo , Bagheri-Yarmand Rozita , Wang Rui-An , Adam Liana , Papadimitrakopoulou Vali Vassiliki , Clayman Gary L. , El-Naggar Adel , Lotan Reuben , Barnes Christopher J. , Hong Waun Ki , Kumar Rakesh
Published: September 2005

Purpose: Abnormalities in the expression and signaling pathways downstream of the epidermal growth factor receptor (EGFR) contribute to the progression, invasion, and maintenance of the malignant phenotype in human cancers, including those of the head and neck and breast. Accordingly, agents such as the EGFR tyrosine kinase inhibitor (EGFR-TKI) ZD1839 (Iressa) are promising, biologically based treatments that are in various stages of preclinical and clinical development. The process of tumor progression requires, among other steps, increased transformation, directional migration, and enhanced cell survival; this study explored the effect of ZD1839 on the stimulation of c-Src and p21-activated kinase 1 (Pak1), which are vital for transformation, directional motility, and cell survival of cancer cells.

Experimental Design: We examined the effect of ZD1839 on biochemical and functional assays indicative of directional motility and cell survival, using human head and neck squamous cancer cells and breast cancer cells.

Results: ZD1839 effectively inhibited c-Src activation and Pak1 activity in exponentially growing cancer cells. In addition, ZD1839 suppressed EGF-induced stimulation of EGFR autophosphorylation on Y1086 and Grb2-binding Y1068 sites, c-Src phosphorylation on Y215, and Pak1 activity. ZD1839 also blocked EGF-induced cytoskeleton remodeling, redistribution of activated EGFR, and in vitro invasiveness of cancer cells.

Conclusions: These studies suggest that the EGFR-TKI ZD1839 may cause potent inhibition of the Pak1 and c-Src pathways and, therefore, have potential to affect the invasiveness of human cancer cells deregulated in these growth factor receptor pathways.
Effect of Radiation and Ibuprofen on Normoxic Renal Carcinoma Cells Overexpressing Hypoxia-Inducible Factors by Loss of von Hippel–Lindau Tumor Suppressor Gene Function
Authors: Palayoor Sanjeewani T. , Burgos Melissa A. , Shoaibi Azadeh , Tofilon Philip J. , Coleman C. Norman
Published: September 2005

Purpose: Tumor hypoxia is a major limiting factor for radiation therapy. Hypoxia-inducible factors (HIFs) are overexpressed in several human cancers and are considered prognostic markers and potential targets for cancer therapy. The purpose of the present study was to investigate the impact of HIFs on radiosensitivity.

Experimental Design: Renal clear cell carcinoma (RCC) cell lines overexpressing HIFs under normoxic conditions because of inactivation of von Hippel–Lindau tumor suppressor gene function (VHL-ve) and their matched pairs in which overexpression of HIFs was abolished by expression of functional VHL (VHL+ve) were irradiated. Radiosensitivity was determined by clonogenic assay. HIF and VHL protein levels were evaluated by Western blot analysis. RCC cells were also treated with ibuprofen, a radiosensitizer and HIF inhibitor in prostate cancer cells. The effect of ibuprofen on radiosensitization and HIF and VHL proteins was compared in RCC matched-pair cell lines.

Results: The data showed only small differences in the radiosensitivity between the cells overexpressing HIFs and cells with basal HIF levels. The dose-modifying factors for C2, 786-0, and A498 RCC cells were 1.14, 1.14 and 1.15, respectively. Radiation did not alter HIF or VHL protein levels. Ibuprofen inhibited HIFs in VHL+ve cells expressing basal levels of HIFs. In VHL-ve cells overexpressing HIFs, the inhibition was very modest. Ibuprofen radiosensitized C2 RCC cells to the same extent irrespective of their HIF status.

Conclusions: Overexpression of HIFs in RCC cells harboring VHL mutations has only a modest effect on the radiosensitivity. Radiosensitization by ibuprofen appears to be independent of HIF status.
Phase I Trial of the Cyclin-Dependent Kinase Inhibitor Flavopiridol in Combination with Docetaxel in Patients with Metastatic Breast Cancer
Authors: Tan Antoinette R. , Yang Xiaowei , Berman Arlene , Zhai Suoping , Sparreboom Alex , Parr Allyson L. , Chow Catherine , Brahim Jaime S. , Steinberg Seth M. , Figg William D. , Swain Sandra M.
Published: September 2005

Purpose: The purpose of this study was to determine the toxicities and characterize the pharmacokinetics of docetaxel and flavopiridol in patients with metastatic breast cancer.

Experimental Design: Docetaxel was administered at an initial dose of 60 mg/m2 followed in 24 hours by a 72-hour infusion of flavopiridol at 50 mg/m2/d every 3 weeks. Because dose-limiting myelosuppression occurred, the schedule was amended to docetaxel, 50 mg/m2, followed by escalating doses of flavopiridol (starting dose, 26 mg/m2/d) as a 1-hour infusion daily for 3 days. Pharmacokinetic studies were performed. Ki67, p53, and phosphorylated retinoblastoma protein (phospho-Rb) in paired tumor and buccal mucosa biopsies (obtained pre- and posttreatment) were examined by immunohistochemistry.

Results: Eleven patients were enrolled. Five patients received docetaxel and 72-hour flavopiridol. Dose-limiting toxicity was grade 4 neutropenia. Six patients received docetaxel and 1-hour flavopiridol, and the dose-limiting toxicity was grade 3 hypotension. Pharmacokinetics of flavopiridol and docetaxel were consistent with historical data. Nuclear staining with p53 increased and phospho-Rb decreased in 10 pairs of buccal mucosa biopsies posttreatment (P = 0.002 and P = 0.04, respectively). No significant changes in Ki67, p53, or phospho-Rb were detected in six paired tumors. Two patients sustained stable disease for >3 months (72-hour flavopiridol), and one partial response was observed (1-hour flavopiridol).

Conclusions: Docetaxel combined with 72-hour flavopiridol was not feasible because of dose-limiting neutropenia. Dose escalation of a 1-hour infusion of flavopiridol with docetaxel was also not possible. The changes in p53 and phospho-Rb in buccal mucosa suggest that a biological effect with flavopiridol was achieved.
Optimal Use of a Panel of Methylation Markers with GSTP1 Hypermethylation in the Diagnosis of Prostate Adenocarcinoma
Authors: Tokumaru Yutaka , Harden Susan V. , Sun Dong-Il , Yamashita Keishi , Epstein Jonathan I. , Sidransky David
Published: September 2005

Purpose: In this study, we tested the ability of a panel of hypermethylation markers to improve the sensitivity of histologic prostate cancer detection in sextant needle biopsies.

Experimental Design: We obtained fresh-frozen sextant biopsies from 72 excised prostates and directly compared blinded histologic review and quantitative real-time methylation-specific PCR for hypermethylation of four genes, Tazarotene-induced gene 1 (TIG1), adenomatous polyposis coli (APC), retinoic acid receptor β2 (RARβ2), and glutathione S-transferase π (GSTP1) to detect the presence of prostate cancer. Results were compared with the final surgical pathological review of the resected prostates as the gold standard.

Results: Histologic review alone detected carcinoma with a sensitivity of 64% (39 of 61 cases) and 100% specificity. Quantitative real-time methylation-specific PCR for TIG1, APC, RARβ2, and GSTP1 detected carcinoma with a sensitivity of 70%, 79%, 89%, and 75%, respectively, with 100% specificity for all of the genes. Using this panel of methylation markers in combination with histology resulted in the detection of 59 of 61 (97%) cases of prostate with 100% specificity, a 33% improvement over histology alone.

Conclusion: The use of a panel of methylation markers as an adjunct to histologic review may substantially augment prostate cancer diagnosis from needle biopsies.
HER-2 Amplification, HER-1 Expression, and Tamoxifen Response in Estrogen Receptor-Positive Metastatic Breast Cancer
Authors: Arpino Grazia , Green Stephanie J. , Allred D. Craig , Lew Dannika , Martino Silvana , Osborne C. Kent , Elledge Richard M.
Published: September 2005

Purpose: Preclinical data indicate that expression of the ErbB family of receptors, such as HER-2 and HER-1 (EGFR) may be involved in endocrine resistance. Evidence of resistance from clinical studies has been inconsistent. The present study examined whether HER-2 gene amplification or HER-1 expression predicted response to tamoxifen.

Patients and Methods: Three hundred and forty nine patients had estrogen receptor (ER)-positive breast cancer and received daily tamoxifen as initial therapy for advanced disease. HER-2 gene amplification, detected by fluorescence in situ hybridization, and HER-1 expression, evaluated by immunohistochemistry, was determined on 136 and 204 patients, respectively.

Results: HER-2 amplification was correlated with lower ER (P = 0.02), HER-1 positivity (P = 0.004), and HER-2 protein overexpression (P < 0.00001). The response rate was 56% for HER-2 non-amplified versus 47% for HER-2 amplified tumors (P = 0.38), and 58% for HER-1–negative versus 36% for HER-1–positive (P = 0.05). Time to treatment failure (TTF) was 7 months for non-amplified HER-2 tumors and 5 months (P = 0.007) for amplified HER-2 tumors, and there was a trend toward a better overall survival (OS) in patients with non-amplified HER-2 tumors (median 31 versus 25 months, respectively, P = 0.07). For positive versus negative HER-1 tumors, TTF was 4 versus 8 months (P = 0.08) and median survival was 24 versus 31 months (P = 0.41). Combining HER-1 expression and HER-2 gene status, patients with both negative HER-1 expression and non-amplified HER-2 had longer TTF (P = 0.001) and OS (P = 0.03) than if either were positive. In multivariate analysis, HER-2 was not an independent factor for TTF and OS, although HER-1 was significant for TTF only (P ≤ 0.001).

Conclusion: Patients with HER-2 amplification and HER-1 expression had lower ER levels and were modestly less responsive to tamoxifen, suggesting that molecular events in addition to those involving the ErbB receptors are important in determining the endocrine-resistant phenotype.
Her-2/neu Expression in Prostate Cancer
Authors: Carles Joan , Lloreta Josep , Salido Marta , Font Albert , Suarez Marta , Baena Veronica , Nogue Miquel , Domenech Montserrat , Fabregat Xavier
Published: September 2005

The clinical effects of targeting Her-2/neu in prostate carcinoma are not known. This study explores the feasibility of molecular profiling to determine the correlation between Her-2/neu expression and hormonal sensitivity.

Patients with progressive androgen-independent prostate carcinoma were eligible to participate in the study. Her-2/neu expression was assessed on pretreatment tissue specimens and on bone marrow obtained in progressive androgen-independent disease. Her-2/neu expression was evaluated by immunohistochemistry and by fluorescence in situ hybridization in a consecutive series of 26 progressive androgen-independent prostate cancer patients.

Twenty four bone marrow biopsy specimens and 16 prostate biopsies from 26 patients were analyzed. These biopsies were categorized by androgen sensitivity at the time of the biopsy. In total, 90% of specimens from bone marrow were Her-2/neu positive, and 10% of the specimens were Her-2/neu negative. Of the prostate biopsies, all were from patients with androgen-dependent disease. Three of 13 androgen-dependent prostate biopsies (23%) overexpressed Her-2/neu. Of the 10 tumor samples analyzed by fluorescence in situ hybridization, genomic amplification of the Her-2/neu locus was not detected in any of the metastatic prostate tumors.

Her-2/neu expression varies with the clinical state of patients with prostate carcinoma: Accurate Her-2/neu profiling requires sampling metastatic tissue in patients with metastatic disease. Her-2/neu sampling from metastatic prostate carcinoma is not feasible until more reliable and practical methods can be developed.
Increased Expression of the Copper Efflux Transporter ATP7A Mediates Resistance to Cisplatin, Carboplatin, and Oxaliplatin in Ovarian Cancer Cells
Authors: Samimi Goli , Safaei Roohangiz , Katano Kuniyuki , Holzer Alison K. , Rochdi Myriam , Tomioka Mika , Goodman Murray , Howell Stephen B.
Published: September 2005

Purpose: The goal of this study was to determine the effect of small changes in ATP7A expression on the pharmacodynamics of cisplatin, carboplatin, and oxaliplatin in human ovarian carcinoma cells.

Experimental Design: Drug sensitivity and cellular pharmacology parameters were determined in human 2008 ovarian carcinoma cells and a subline transfected with an ATP7A-expression vector ATP7A (2008/MNK). Drug sensitivity was determined by clonogenic assay, platinum (Pt) levels were measured by inductively coupled plasma mass spectroscopy, copper (Cu) accumulation was quantified with 64Cu, and the subcellular distribution of ATP7A was assessed by confocal digital microscopy.

Results: The 1.5-fold higher expression of ATP7A in the 2008/MNK cells was sufficient to alter Cu cellular pharmacokinetics but not confer Cu resistance. In contrast, it was sufficient to render the 2008/MNK cells resistant to cisplatin, carboplatin, and oxaliplatin. Resistance was associated with increased rather than decreased whole-cell Pt drug accumulation and increased sequestration of Pt into the vesicular fraction. Cu triggered relocalization of ATP7A away from the perinuclear region, whereas at equitoxic concentrations the Pt drugs did not.

Conclusions: A small increase in ATP7A expression produced resistance to all three of the clinically available Pt drugs. Whereas increased expression of ATP7A reduced Cu accumulation, it did not reduce accumulation of the Pt drugs. Under conditions where Cu triggered ATP7A relocalization, the Pt drugs did not. Thus, although ATP7A is an important determinant of sensitivity to the Pt drugs, there are substantial differences between Cu and the Pt drugs with respect to how they interact with ATP7A and the mechanism by which ATP7A protects the cell.
A Novel Effective and Safe Consolidation for Patients Over 60 Years with Acute Myeloid Leukemia
Authors: Sperr Wolfgang R. , Piribauer Maria , Wimazal Friedrich , Fonatsch Christa , Thalhammer-Scherrer Renate , Schwarzinger Ilse , Geissler Klaus , Knöbl Paul , Jäger Ulrich , Lechner Klaus , Valent Peter
Published: September 2005

Purpose: High-dose intermittent cytarabine is an effective postremission treatment for patients with acute myeloid leukemia (AML). This regimen is a safe approach in patients < 60 years but produced severe neurotoxicity in the elderly.

Experimental Design: We have established a dose-reduced age-adapted consolidation using intermediate dose (IDAC; 2 × 1 g/m2 i.v., days 1, 3, and 5) for AML patients ≥ 60 years. Forty-seven de novo AML patients in complete remission (CR; median age, 70 years) were scheduled to receive four consolidation cycles of IDAC.

Results: In 25 of 47 patients (53%), all four cycles were administered: 9 (19%) received three cycles; 7 (15%) received two cycles; and 6 patients (12%) one cycle. Treatment was well tolerated without neurotoxicity. The median number of days with severe neutropenia (absolute neutrophil count < 500/μl) was 9. Neutropenic fever occurred in 22 of 47 patients (49%) during the first cycle, in 24 of 41 (60%) during the second, in 15 of 34 (44%) during the third, and in 18 of 25 (72%) during the fourth cycle. Only 1 patient died during consolidation (cardiac failure). The median overall survival, disease-free survival, and continuous CR were 10.6, 15.5, and 15.9 months, respectively. The probability of overall survival, disease-free survival, and continuous CR at 5 years were 18, 22, and 30%, respectively.

Conclusions: IDAC is a safe and effective postremission therapy for elderly patients with AML.
Antitumor Activity of Gefitinib in Malignant Rhabdoid Tumor Cells
In vitro
and
In vivo
Authors: Kuwahara Yasumichi , Hosoi Hajime , Osone Shinya , Kita Masakazu , Iehara Tomoko , Kuroda Hiroshi , Sugimoto Tohru
Published: September 2005

Purpose: Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. Current treatments have had only limited success. Epidermal growth factor receptor (EGFR) was found recently to be expressed on MRT cell lines. Gefitinib (trade name Iressa) is an oral and selective EGFR-tyrosine kinase inhibitor and has been demonstrated to be effective in inhibiting the proliferation of cancer cells in vivo as well as in clinical trials. This encouraged us to examine the antitumor effects of gefitinib on MRT cells in vitro and in vivo.

Experimental Design: The expression of EGFR in two MRT tumors and two MRT cell lines (MP-MRT-AN and KP-MRT-NS), established from these two tumor tissues, was examined by immunohistochemistry, immunofluorescence, and immunoblot. The effect of gefitinib on EGFR phosphorylation was examined by immunoblot. The effects of gefitinib on cell growth and apoptosis were examined by cell growth assay and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. The in vivo effect of gefitinib was assessed in athymic mice that had been xenografted with MRT cells.

Results: The expression of EGFR was detected in both tumor tissues and cell lines. Gefitinib inhibited EGFR-phosphorylation (IC50 < 0.1 μmol/L) and in vitro cell growth (IC50 = approximately 10–12 μmol/L), and a high concentration of gefitinib (20 μmol/L) induced apoptosis in vitro (MP-MRT-AN, 42.9% and KP-MRT-NS, 47.2%). Furthermore, gefitinib at 150 mg/kg had a cytostatic effect on established MRT xenografts (MP-MRT-AN, P = 0.039 and 0.0014; and KP-MRT-NS, P = 0.048 and 0.0086).

Conclusions: Our results demonstrate that gefitinib has antitumor effects in MRT cells in vitro and in vivo and, thus, has promise as a novel and therapeutic strategy for MRT.
Combinatorial Administration of Molecules That Simultaneously Inhibit Angiogenesis and Invasion Leads to Increased Therapeutic Efficacy in Mouse Models of Malignant Glioma
Authors: Bello Lorenzo , Lucini Valeria , Costa Francesco , Pluderi Mauro , Giussani Carlo , Acerbi Francesco , Carrabba Giorgio , Pannacci Marilou , Caronzolo Dario , Grosso Silvia , Shinkaruk Svetlana , Colleoni Federica , Canron Xavier , Tomei Giustino , Deleris Gerard , Bikfalvi Andreas
Published: September 2005

Purpose: We investigated the ability of the combinatorial administration of different inhibitors with activities on glioma angiogenesis, migration, and proliferation to produce a prolonged inhibition of glioma growth.

Experimental Design: We combined inhibitors affecting solely tumor angiogenesis (PF-4/CTF, cyclo-VEGI) or inhibitors affecting both angiogenesis and invasion together (PEX, PF-4/DLR).

Results: When administered in combination, these drugs produced a prolonged and increased inhibition of glioma growth independently from the type of inhibitor used. The combinatory administration was more effective than the administration of a single inhibitor alone, and a strong therapeutic response was reached with a significantly lower amount of protein. The strongest inhibition was observed when human PEX and PF-4/DLR, which affect both glioma angiogenesis and invasion by separate mechanisms, were combined.

Conclusions: This supports the concept that prolonged glioma growth inhibition can be achieved by simultaneous delivery of molecules that target both tumor and endothelial cells and acting by separate mechanisms.