bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic Markers and Frequency Predict Functional Capacity of Natural Killer Cells Infiltrating Renal Cell Carcinoma
Authors: Schleypen Julia S. , Baur Nicole , Kammerer Robert , Nelson Peter J. , Rohrmann Karl , Gröne Elisabeth F. , Hohenfellner Markus , Haferkamp Axel , Pohla Heike , Schendel Dolores J. , Falk Christine S. , Noessner Elfriede
Published: March 2006

Purpose: Renal cell carcinoma harbors high numbers of infiltrating lymphocytes with apparent limited efficacy in tumor control. This study focused on the natural killer (NK) cells infiltrating renal cell carcinoma.

Experimental Design: Tumor-infiltrating lymphocytes (TIL) were isolated from renal cell carcinoma and analyzed for NK cell frequency and phenotype (n = 34). NK cells were enriched and tested for effector function.

Results: Two renal cell carcinoma subtypes were identified, one containing high (>20% of the lymphocyte population, n = 14), the other low (<20%, n = 20), NK cell numbers. NK cells of both groups were noncytolytic ex vivo but differed in CD16 and cytotoxic effector molecule expression as well as in their capacity to acquire cytotoxic activity: The majority of NK cells from tumors with high NK cell content (high NK-TIL) were CD16bright, whereas few CD16bright NK cells were found in tumors with low NK cell frequencies (low NK-TIL). The CD16 dichotomy correlated with different capacities to develop cytotoxicity after short-term activation with interleukin-2 ex vivo: Low NK-TIL remained noncytolytic against K562 and unresponsive to signals via the activating receptor NKp46 despite expression of receptor and adaptor molecules. In contrast, high NK-TIL acquired cytotoxic function. As described for peripheral CD16bright NK cells, NK cells from high-NK tumors showed high per cell expression of granzyme A, granzyme B, and perforin. NK cells from low NK-TIL resembled CD16neg/dim peripheral NK cells with few cytotoxin+ cells and lower expression of perforin.

Conclusion: The extent of NK cell infiltration and the expression of markers (CD16 and cytotoxins) predict the functional capacity of NK cells infiltrating renal cell carcinoma and can be used to characterize subgroups of renal cell carcinoma.
The Nuclear Transcription Factor κB/bcl-2 Pathway Correlates with Pathologic Complete Response to Doxorubicin-Based Neoadjuvant Chemotherapy in Human Breast Cancer
Authors: Buchholz Thomas A. , Garg Amit K. , Chakravarti Nitin , Aggarwal Bharat B. , Esteva Francisco J. , Kuerer Henry M. , Singletary S. Eva , Hortobagyi Gabriel N. , Pusztai Lajos , Cristofanilli Massimo , Sahin Aysegul A.
Published: March 2006

Purpose: Molecular factors involved in apoptosis may affect breast cancer response to chemotherapy. Herein, we studied the nuclear factor κB (NF-κB)/bcl-2 pathway to determine whether or not activation of this antiapoptotic pathway was associated with a poor response of human breast cancer to anthracycline-based neoadjuvant chemotherapy.

Experimental Design: We studied 82 human breast cancer samples from patients treated with neoadjuvant doxorubicin-based chemotherapy and studied whether or not nuclear location of the transcription factor NF-κB was associated with expression of bcl-2 and bax and whether or not expression of these proteins correlated with chemotherapy response. Protein expression was measured with immunohistochemical staining. A dedicated breast cancer pathologist who was unaware of the clinical outcome data dichotomized the slides as positive or negative based on the presence or absence of cytoplasmic staining for bcl-2 and bax or nuclear staining for NF-κB.

Results: Sixty-one percent of the tumors were positive for bcl-2, 85% were positive for bax, and 16% were positive for NF-κB. All bcl-2-positive tumors were also bax positive (P < 0.0001) and all NF-κB-positive tumors were both bcl-2 positive (P = 0.001) and bax positive (P = 0.113). Eleven of the 82 patients (13%) had a pathologic complete response (pCR) to chemotherapy. Patients with positive staining tumors for any of the markers less commonly achieved a pCR to chemotherapy than those with negative tumor staining. The pCR rates were NF-κB positive 0% (0 of 13) versus NF-κB negative 13% (11 of 69; P = 0.130); bcl-2 positive 4% (2 of 49) versus bcl-2 negative 27% (9 of 33; P = 0.004); and bax positive 6% (4 of 69) versus bax negative 58% (7 of 12; P < 0.001).

Conclusion: We conclude that nuclear localization of NF-κB correlates with bcl-2 and bax expression and that the NF-κB/bcl-2 pathway may be associated with a poor response to neoadjuvant doxorubicin-based chemotherapy.
A Phase I Study of the Dolastatin-15 Analogue Tasidotin (ILX651) Administered Intravenously Daily for 5 Consecutive Days Every 3 Weeks in Patients with Advanced Solid Tumors
Authors: Ebbinghaus Scot , Rubin Eric , Hersh Evan , Cranmer Lee D. , Bonate Peter L. , Fram Robert J. , Jekunen Antti , Weitman Steve , Hammond Lisa A.
Published: March 2006

Purpose: To determine the maximum tolerated dose, dose-limiting toxicity, and pharmacokinetics of the dolastatin-15 analogue, tasidotin (ILX651), when administered i.v. daily for 5 days every 3 weeks.

Experimental Design: Thirty-six patients with advanced solid tumors received a total of 114 courses through eight dose levels ranging from 2.3 to 36.3 mg/m2. Pharmacokinetic samples were collected in cycle 1.

Results: Neutropenia was the principal dose-limiting toxicity at 36.3 mg/m2/d along with grade 3 ileus and elevated aspartate amino transaminase/alanine amino transaminase (n = 1). At the maximum tolerated dose, 27.3 mg/m2, 4 of 14 patients experienced dose-limiting grade 4 neutropenia. The other principal toxicities consisted of mild-to-moderate elevated transaminases, alopecia, fatigue, and nausea. One patient with melanoma metastatic to liver and bone treated at 15.4 mg/m2/d experienced a complete response and received 20 courses of tasidotin. Two other patients with melanoma had mixed responses of cutaneous metastases at 27.3 mg/m2/d associated with either stable or progressive visceral disease. In addition, nine patients had stable disease. There was no accumulation of tasidotin following repeated daily dosing. Tasidotin decayed from plasma in a biphasic fashion with a half-life of <45 minutes in most cases.

Conclusion: The maximum tolerated dose and recommended phase II dose for tasidotin when administered on this schedule was 27.3 mg/m2/d. The favorable toxicity profile of tasidotin compared with other antitubulin agents (particularly the lack of severe cumulative neuropathy, peripheral edema, and fatigue), the observed antitumor activity of tasidotin, and its novel mechanism of action support further disease-directed evaluations of this agent on this 5-day schedule every 3 weeks.
Detection of Mononucleotide Repeat Sequence Alterations in a Large Background of Normal DNA for Screening High-Frequency Microsatellite Instability Cancers
Authors: Sun Xiyuan , Liu Yiding , Lutterbaugh Jim , Chen Wei-Dong , Markowitz Sanford D. , Guo Baochuan
Published: March 2006

Purpose: Mutations in mononucleotide repeat sequence (MRS) are good indicators of high-frequency microsatellite instability (MSI-H) cancers, but it has been a challenge to detect such mutations in a large background of wild-type DNA; as in this setting, PCR errors often generate false positive mutant alleles. In this study, we developed a general strategy, referred to as probe clamping primer extension-PCR (PCPE-PCR), to detect MRS alterations in a large background of wild-type DNA.

Experimental Design: In PCPE-PCR, genomic DNA is first subjected to PCPE, in which mutant single-strand DNA molecules are preferentially produced. Next, genomic DNA is removed to enrich for the mutant DNA fraction. Thereafter, PCR is carried out using the remaining single-strand DNA molecules as templates. Finally, the PCR products are analyzed to reveal the MSI-H status. In this study, the sensitivity of this new method was first examined by spiking mutant DNA into wild-type DNA at specific ratios followed by studying whether this method is applicable to fecal DNA testing.

Results: We showed that PCPE-PCR could detect both mutated BAT26 and transforming growth factor-β-RII (A)10 markers in the presence of 500-fold excess of normal DNA and that as few as three copies of mutated DNA could be detected. In addition, we showed that this technology could detect MSI-H colorectal cancer by fecal DNA analysis.

Conclusion: PCPE-PCR is sensitive. In addition, PCPE-PCR is simple and amendable to a cost-effective and high-throughput screening operation. This technology may be applicable to noninvasive screening of MSI-H cancer.
Sex-Specific Survival Advantage with Parathyroid Hormone–Related Protein in Non–Small Cell Lung Carcinoma Patients
Authors: Hastings Randolph H. , Laux Ann M.P. , Casillas Andrea , Xu Ronghui , Lukas Zenaida , Ernstrom Karin , Deftos Leonard J.
Published: March 2006

Purpose: Parathyroid hormone–related protein (PTHrP) is commonly expressed in non–small cell lung carcinomas (NSCLC). Expression of the protein could have implications for progression of the disease because it regulates cancer cell growth, apoptosis, and angiogenesis. However, its relationship with survival has not been evaluated in a large-scale investigation.

Experimental Design: PTHrP expression was assessed in paraffin-embedded tumor samples from 407 patients with NSCLC by immunohistochemistry. A pathologist unaware of the clinical history classified specimens as PTHrP positive or PTHrP negative. The log-rank test was used to compare survivals of PTHrP-positive and PTHrP-negative groups, and Cox regression was used to adjust for additional covariates.

Results: Median survival was 55 versus 22 months (P < 0.001) in female patients with and without tumor PTHrP, respectively. Male survival was 38 months independent of PTHrP status. Stage, histology, age, and smoking history were also associated with increased longevity. PTHrP remained a significant predictor of survival for female patients after controlling for stage, histology, and age.

Conclusions: In this study, PTHrP expression was associated with a survival advantage in female patients. Additional investigations must be done to ascertain whether the result is reproducible and independent of potential confounding covariates. Sex-dependent effects of PTHrP in lung cancer would open new avenues of research into the role of sex in cancer progression.
mdr-1Single Nucleotide Polymorphisms in Ovarian Cancer Tissue: G2677T/A Correlates with Response to Paclitaxel Chemotherapy
Authors: Gréen Henrik , Söderkvist Peter , Rosenberg Per , Horvath György , Peterson Curt
Published: March 2006

Purpose: P-glycoprotein, encoded by the mdr-1 gene, confers multidrug resistance to a variety of antineoplastic agents, e.g., paclitaxel. Recently, different polymorphisms in the mdr-1 gene have been identified and their consequences for the function of P-glycoprotein, as well as for the treatment response to P-glycoprotein substrates, are being clarified. We analyzed the allelic frequencies at polymorphic sites G2677T/A and C3435T in ovarian cancer patients with good or poor response to treatment with paclitaxel in combination with carboplatin in order to evaluate their predictive values.

Experimental Design: Fifty-three patients were included in the study; 28 of them had been relapse-free for at least 1 year and 25 had progressive disease or relapsed within 12 months. A reference material consisting of 200 individuals was also analyzed. The genotypes of each single nucleotide polymorphism (SNP) were determined using Pyrosequencing.

Results: The G2677T/A SNP was found to significantly correlate with treatment outcome. The probability of responding to paclitaxel treatment was higher in homozygously mutated patients (T/T or T/A; Fisher's exact test; P < 0.05). The frequency of the T or A alleles was also higher in the group of patients who had a good response (P < 0.05). There was also a dose-dependent influence of the number of mutated alleles on the response to paclitaxel treatment (χ2 test for linear-by-linear association; P = 0.03). However, the C3435T SNP was not found to correlate to treatment outcome.

Conclusions: The mdr-1 polymorphism G2677T/A in exon 21 correlates with the paclitaxel response in ovarian cancer and may be important for the function of P-glycoprotein and resistance to paclitaxel and provide useful information for individualized therapy.
Natural Killer Cells License Dendritic Cell Cross-Presentation of B Lymphoma Cell–Associated Antigens
Authors: Dao Tao , Gomez-Nunez Marta , Antczak Christophe , Kappel Barry , Jaggi Jaspreet Singh , Korontsvit Tatyana , Zakhaleva Victoriya , Scheinberg David A.
Published: March 2006

Purpose: Presentation of exogenous antigen by MHC class I molecules, or cross-presentation, is a property of dendritic cells, which is considered crucial for the priming of cytotoxic T-cell response to tumor antigens. However, the precise mechanisms of this process are not fully understood.

Experimental Design and Results: We show here in a human in vitro system, using B lymphoma cells as a tumor model, that the cross-presentation of cell-associated antigens to T cells by dendritic cells requires “help” from natural killer cells. When autologous dendritic cells that had taken up apoptotic B lymphoma cells and induced to a fully mature state were used to stimulate nonadherent cells of peripheral blood mononuclear cells from healthy donors, they induced strong cytotoxicity against B lymphoma cells in a HLA-A0201-restricted manner. The cells failed to induce cytotoxicity, however, when purified T cells were used as effector cells. Depletion of CD56+ cells, but not CD14+ or CD19+ cells, abrogated the cytotoxicity of nonadherent cells, showing that the help was provided by natural killer cells. Further, when natural killer cells were present in the cultures, a strong and persistent production of interleukin-18, but not interleukin-12 and interleukin-15, was observed. Blocking interleukin-18 significantly reduced the cytotoxicity of nonadherent cells against B lymphoma cells.

Conclusions: These results suggest that capture of tumor cells and a full maturation status of dendritic cells are not sufficient to cross-prime CD8 T cells. Effective cross-priming requires further activation of dendritic cells by natural killer cells and an abundant production of interleukin-18, which, along with other yet undefined mechanisms, contribute to the generation of CTL response against B-cell lymphoma.
Positive Correlation between Estradiol and Vascular Endothelial Growth Factor but not Fibroblast Growth Factor-2 in Normal Human Breast Tissue In vivo
Authors: Dabrosin Charlotta
Published: March 2006

Purpose: Angiogenesis is crucial in tumor development and progression. Ovarian hormones regulate angiogenesis in the reproductive tract but very little is known about its regulation in the normal breast. Sex steroids play an important role in breast cancer development by poorly understood mechanisms. Vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) are potent stimulators of angiogenesis. Both VEGF and FGF-2 function in autocrine/paracrine pathways and there is a major contribution of bioactive proteins by a posttranslational activation of sequestered molecules in the extracellular space. A direct measurement of these molecules in the extracellular compartment is, therefore, needed.

Experimental Design: In this study, microdialysis was used to measure extracellular VEGF and FGF-2 in normal human breast tissue in situ in 11 premenopausal and 5 postmenopausal women.

Results: Significantly higher level of VEGF in breast tissue of premenopausal women was found. Plasma as well as local estradiol and breast tissue VEGF exhibited significant correlations, whereas progesterone had no correlation with breast VEGF. FGF-2 did not correlate with either estradiol or progesterone.

Conclusion: The result suggests that estradiol is a more potent regulator of free VEGF levels than progesterone in the normal breast. The control of free FGF-2 seems to be independent of sex steroids in the breast. Estrogen induction of free extracellular VEGF may be one mechanism involved in sex steroid–dependent breast carcinogenesis.
Signal Pathway of Hypoxia-Inducible Factor-1α Phosphorylation and its Interaction with von Hippel-Lindau Tumor Suppressor Protein During Ischemia in MiaPaCa-2 Pancreatic Cancer Cells
Authors: Kwon Seok J. , Song Jae J. , Lee Yong J.
Published: March 2006

Purpose and Experimental Design: Previously, we observed that the activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK1) is mediated through the activation of apoptosis signal–regulating kinase 1 (ASK1) as a result of the reactive oxygen species–mediated dissociation of glutaredoxin and thioredoxin from ASK1. In this study, we examined whether p38 MAPK and JNK1 are involved in the accumulation of hypoxia-inducible factor-1α (HIF-1α) during ischemia. Human pancreatic cancer MiaPaCa-2 cells were exposed to low glucose (0.1 mmol/L) with hypoxia (0.1% O2).

Results and Conclusions: During ischemia, p38 MAPK and JNK1 were activated in MiaPaCa-2 pancreatic cancer cells. The activated p38 MAPK, but not JNK1, phosphorylated HIF-1α. Data from in vivo binding assay of von Hippel-Lindau tumor suppressor protein with HIF-1α suggests that the p38-mediated phosphorylation of HIF-1α contributed to the inhibition of HIF-1α and von Hippel-Lindau tumor suppressor protein interaction during ischemia. SB203580, a specific inhibitor of p38 MAPK, inhibited HIF-1α accumulation during ischemia, probably resulting from the ubiquitination and degradation of HIF-1α.
[131I]meta-Iodobenzylguanidine and Topotecan Combination Treatment of Tumors Expressing the Noradrenaline Transporter
Authors: McCluskey Anthony G. , Boyd Marie , Ross Susan C. , Cosimo Emilio , Clark Anne Marie , Angerson Wilson J. , Gaze Mark N. , Mairs Robert J.
Published: March 2006

Purpose: Both [131I]meta-iodobenzylguanidine ([131I]MIBG) and the topoisomerase I inhibitor topotecan are effective as single-agent treatments of neuroblastoma. The aim of this study was to investigate the efficacy of [131I]MIBG in combination with topotecan in vitro and in vivo.

Experimental Design: The cell lines used were SK-N-BE(2c) (human neuroblastoma) and UVW/NAT (glioma cell line transfected with the noradrenaline transporter gene). Three different treatment schedules were assessed: topotecan given before (schedule 1), after (schedule 2), or simultaneously (schedule 3) with [131I]MIBG. DNA strand breakage was evaluated by comet assay, and cytotoxicity was determined by clonogenic survival. Efficacy was also measured by growth delay of tumor xenografts in nude mice.

Results: Combination schedules 2 and 3 caused more cytotoxicity than schedule 1. Similarly, significant DNA damage was observed following treatment schedules 2 and 3 (P < 0.005) but not schedule 1. The mean number of days for a doubling in volume of SK-N-BE(2c) tumors and a 10-fold increase in volume of UVW/NAT tumors were 10.4 and 18.6 (untreated), 19.7 and 25.3 (topotecan alone), 22.8 and 31.9 ([131I]MIBG alone), 26.3 and 37.1 (combination schedule 1), 34.3 and 49.7 (combination schedule 2), and 53.2 and >71 (combination schedule 3), respectively. The highest rate of cure of both xenografts was observed following treatment with combination schedule 3.

Conclusions: The combination of topotecan and [131I]MIBG compared with either treatment alone gave rise to greater than additive DNA damage, clonogenic cell kill, and tumor growth delay. These effects were dependent on the scheduling of the two agents.
The Septin-Binding Protein Anillin Is Overexpressed in Diverse Human Tumors
Authors: Hall Peter A. , Todd Christopher B. , Hyland Paula L. , McDade Simon S. , Grabsch Heike , Dattani Mit , Hillan Kenneth J. , Russell S.E. Hilary
Published: March 2006

Anillin is an actin-binding protein that can bind septins and is a component of the cytokinetic ring. We assessed the anillin expression in 7,579 human tissue samples and cell lines by DNA microarray analysis. Anillin is expressed ubiquitously but with variable levels of expression, being highest in the central nervous system. The median level of anillin mRNA expression was higher in tumors than normal tissues (median fold increase 2.58; 95% confidence intervals, 2.19-5.68, P < 0.0001) except in the central nervous system where anillin mRNA levels were lower in tumors. We developed a sensitive reverse transcription-PCR strategy to show that anillin mRNA is expressed in cell lines and in cDNA panels derived from fetal and adult tissues, thus validating the microarray data. We compared anillin with Ki67 mRNA expression and found a significant linear relationship between anillin and Ki67 mRNA expression (Spearmann r ∼ 0.6, P < 0.0001). Anillin mRNA expression was analyzed during tumor progression in breast, ovarian, kidney, colorectal, hepatic, lung, endometrial, and pancreatic tumors and in all tissues there was progressive increase in anillin mRNA expression from normal to benign to malignant to metastatic disease. Finally, we used anti-anillin sera and found nuclear anillin immunoreactivity to be widespread in normal tissues, often not correlating with proliferative compartments. These data provide insight into the existence of nonproliferation-associated activities of anillin and roles in interphase nuclei. Thus, anillin is overexpressed in diverse common human tumors, but not simply as a consequence of being a proliferation marker. Anillin may have potential as a novel biomarker.
Tumor β-1,4-Galactosyltransferase IV Overexpression Is Closely Associated with Colorectal Cancer Metastasis and Poor Prognosis
Authors: Chen Wei-Shone , Chang Hong-Yi , Li Chung-Pin , Liu Jacqueline Ming , Huang Tze-Sing
Published: March 2006

Purpose: To elucidate the significance of β-1,4-galactosyltransferase IV (β-1,4-GT-IV) in the clinical presentation and prognostication of colorectal cancer.

Experimental Design: Tissue lysates from paired tumor and nontumor tissues of a colon cancer patient were labeled separately with fluorescent dyes Cy5 and Cy3 for two-dimensional difference in-gel electrophoresis. Subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and immunoblot analyses identified a down-regulated level of β-1,4-GT-IV in the tumor tissue. In the follow-up study, paired tissue lysates were obtained from 100 colorectal cancer patients with immunoblot analyses done to compare the levels of β-1,4-GT-IV expression in these patients.

Results: Of 100 colorectal patients studied, 48% had down-regulated expression of β-1,4-GT-IV in the tumor tissue but 28% of patients exhibited elevated β-1,4-GT-IV levels. Increased β-1,4-GT-IV in the tumor tissue was significantly coexistent with raised serum level of CA-199 and the presence of tumor metastasis (P = 0.006 and P < 0.001, respectively) but was independent of age and gender of patient, tumor site, tumor size, serum level of carcinoembryonic antigen, grade of tumor cell differentiation, and depth of tumor invasion. The results of logistic regression analyses suggested that tumor β-1,4-GT-IV overexpression and tumor invasion, but not other patient variables such as tumor size and serum levels of carcinoembryonic antigen and CA19-9, were significantly correlated with the occurrence of metastases (P < 0.05). In a multivariate regression analysis, the patient group with tumor β-1,4-GT-IV overexpression strongly predicted for tumor metastasis (odds ratio, 10.009; 95% confidence interval, 2.992-33.484; P < 0.001). Likewise, tumor β-1,4-GT-IV overexpression was significantly associated with poor overall survival (P < 0.01). By Cox regression analysis, this association remained significant even after adjustment for tumor metastasis (P = 0.048).

Conclusion: Increased β-1,4-GT-IV expression in tumor tissue was strongly associated with tumor metastases and poor prognosis in colorectal cancer.
Identification ofCCND3andBYSLas Candidate Targets for the 6p21 Amplification in Diffuse Large B-Cell Lymphoma
Authors: Kasugai Yumiko , Tagawa Hiroyuki , Kameoka Yoshihiro , Morishima Yasuo , Nakamura Shigeo , Seto Masao
Published: March 2006

Purpose: Increases in gene dosage through DNA amplification represents a common feature of many tumors and can result in the up-regulation of tumor-promoting genes. Our recent genome-wide, array-based comparative genomic hybridization analysis of 66 cases of diffuse large B-cell lymphoma found that genomic gain of 6p21 was observed in as many as 17 cases, including 14 cases with low-level copy number gain and three cases with high-level copy number gains (amplifications).

Experimental Design and Results: To identify the target gene(s) for 6p21 amplification, we constructed a detailed amplicon map at the region of genomic amplification with the aid of high-resolution contig array-based comparative genomic hybridization glass slides, consisting of contiguously ordered bacterial artificial chromosome/P1-derived artificial chromosome clones covering 3 Mb throughout the 6p21 amplification region. Alignment of the amplifications identified a minimally overlapping 800 kb segment containing 15 genes. Quantitative expression analysis of the genes from both patient samples and the SUDHL9 cell line revealed that CCND3 and BYSL (1.9 kb telomeric to the CCND3 gene locus) are the targets of 6p21 genomic gain/amplification.

Conclusions: Although it is known that t(6;14)(p21;q32) induces aberrant overexpression of CCND3 in B-cell malignancies, we were able to show that CCND3, which encodes the cyclin D family member protein that controls the G1-S phase of cell cycle regulation, can also be a target of genomic gain/amplification. Overexpression of CCND3 through genomic amplification is likely to lead to aberrant cell cycle control, although the precise biological role of BYSL with respect to tumorigenesis remains to be determined.
The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer.
Authors: Olivier Magali , Langer√∏d Anita , Carrieri Patrizia , Bergh Jonas , Klaar Sigrid , Eyfjord Jorunn , Theillet Charles , Rodriguez Carmen , Lidereau Rosette , Bi√®che Ivan , Varley Jennifer , Bignon Yves , Uhrhammer Nancy , Winqvist Robert , Jukkola-Vuorinen Arja , Niederacher Dieter , Kato Shunsuke , Ishioka Chikashi , Hainaut Pierre , B√∏rresen-Dale Anne-Lise
Published: March 2006

To investigate the clinical value of somatic TP53 mutations in breast cancer, we assembled clinical and molecular data on 1,794 women with primary breast cancer with long-term follow-up and whose tumor has been screened for mutation in exons 5 to 8 of TP53 by gene sequencing. TP53 mutations were more frequent in tumors of ductal and medullar types, aggressive phenotype (high grade, large size, node positive cases, and low hormone receptor content) and in women <60 years old. TP53 mutations within exons 5 to 8 conferred an elevated risk of breast cancer-specific death of 2.27 (relative risk >10 years; P < 0.0001) compared with patients with no such mutation. The prognostic value of TP53 mutation was independent of tumor size, node status, and hormone receptor content, confirming and reconciling previous findings in smaller series. Moreover, an interaction between TP53 mutation and progesterone receptor (PR) status was revealed, TP53 mutation combined with the absence of progesterone receptor being associated with the worst prognosis. Whereas previous studies have emphasized the fact that missense mutations in the DNA-binding motifs have a worse prognosis than missense mutations outside these motifs, we show that non-missense mutations have prognostic value similar to missense mutations in DNA-binding motifs. Nonetheless, specific missense mutants (codon 179 and R248W) seem to be associated with an even worse prognosis. These results, obtained on the largest series analyzed thus far, show that TP53 mutations identified by gene sequencing have an independent prognostic value in breast cancer and could have potential uses in clinical practice.
Tumor Necrosis Factor α and Adiponectin in Bone Marrow Interstitial Fluid from Patients with Acute Myeloid Leukemia Inhibit Normal Hematopoiesis
Authors: Iversen Per Ole , Wiig Helge
Published: March 2006

Purpose: Locally residing cytokines may inhibit bone marrow hematopoiesis in acute myeloid leukemia (AML). Using a novel method to isolate bone marrow interstitial fluid, we examined if this fluid from 10 adult AML patients could affect normal bone marrow hematopoiesis.

Experimental Design: Bone marrow interstitial fluid was isolated by centrifugation of bone marrow biopsies obtained at time of diagnosis and 2 to 4 weeks after start of induction therapy. The isolated fluid was added to normal bone marrow CD34 hematopoietic progenitor cells sampled from five healthy subjects.

Results: Unlike plasma, AML-derived bone marrow interstitial fluid clearly repressed hematopoietic progenitor cell growth as determined by an in vitro colony assay, an effect that was lost after successful induction treatment. Antibodies against tumor necrosis factor α (TNFα) and adiponectin abolished growth inhibition by bone marrow interstitial fluid, suggesting a mechanistic role of these cytokines in impairing normal hematopoiesis in AML. The plasma levels of adiponectin and TNFα were unaffected by therapy whereas bone marrow interstitial fluid levels of both cytokines fell significantly in patients entering remission. Transcripts for TNFα, but not for adiponectin, were found in AML blast cells. Neither the plasma levels nor the bone marrow interstitial fluid levels of the proangiogenic factors vascular endothelial growth factor or basic fibroblast growth factor were appreciably elevated in the patients nor did they change with treatment.

Conclusions: Specific analyses of bone marrow interstitial fluid may give novel information on normal and malignant hematopoietic activity and thus form the basis for mechanism-based therapy.
In vitro and In vivo Clinical Pharmacology of Dimethyl Benzoylphenylurea, a Novel Oral Tubulin-Interactive Agent
Authors: Rudek Michelle A. , Zhao Ming , Smith Nicola F. , Robey Robert W. , He Ping , Hallur Gurulingappa , Khan Saeed , Hidalgo Manuel , Jimeno Antonio , Colevas A. Dimitrios , Messersmith Wells A. , Wolff Antonio C. , Baker Sharyn D.
Published: March 2006

Dimethyl benzoylphenylurea (BPU) is a novel tubulin-interactive agent with poor and highly variable oral bioavailability. In a phase I clinical trial of BPU, higher plasma exposure to BPU and metabolites was observed in patients who experienced dose-limiting toxicity. The elucidation of the clinical pharmacology of BPU was sought. BPU, monomethylBPU, and aminoBPU were metabolized by human liver microsomes. Studies with cDNA-expressed human cytochrome P450 enzymes revealed that BPU was metabolized predominantly by CYP3A4 and CYP1A1 but was also a substrate for CYP2C8, CYP2D6, CYP3A5, and CYP3A7. BPU was not a substrate for the efflux transporter ABCG2. Using simultaneous high-performance liquid chromatography/diode array and tandem mass spectrometry detection, we identified six metabolites in human liver microsomes, plasma, or urine: monomethylBPU, aminoBPU, G280, G308, G322, and G373. In patient urine, aminoBPU, G280, G308, and G322 collectively represented <2% of the given BPU dose. G280, G308, G322, and G373 showed minimal cytotoxicity. When BPU was given p.o. to mice in the presence and absence of the CYP3A and ABCG2 inhibitor, ritonavir, there was an increase in BPU plasma exposure and decrease in metabolite exposure but no overall change in cumulative exposure to BPU and the cytotoxic metabolites. Thus, we conclude that (a) CYP3A4 and CYP1A1 are the predominant cytochrome P450 enzymes that catalyze BPU metabolism, (b) BPU is metabolized to two cytotoxic and four noncytotoxic metabolites, and (c) ritonavir inhibits BPU metabolism to improve the systemic exposure to BPU without altering cumulative exposure to BPU and the cytotoxic metabolites.
In vitro and In vivo Effects and Mechanisms of Celecoxib-Induced Growth Inhibition of Human Hepatocellular Carcinoma Cells
Authors: Cui Wei , Yu Chang-Hong , Hu Ke-Qin
Published: March 2006

Purpose: Cyclooxygenase-2 (COX-2) inhibitors cause growth inhibition of human hepatocellular carcinoma cells but it remains unclear whether this is both COX-2 dependent and independent. The related mechanisms remain to be determined. The present study was aimed to determine the effect of celecoxib on growth of hepatocellular carcinoma cells and xenografts and the related mechanisms.

Experimental Design: Both low COX-2 expressing PLC/PRF/5 and high COX-2 expressing HuH7 cells, and nude mice bearing hepatocellular carcinoma xenografts were used to study the effect and mechanisms of celecoxib on hepatocellular carcinoma cell growth.

Results: Celecoxib resulted in a comparable growth inhibition of both hepatocellular carcinoma cells that was associated with decreased production of prostaglandin E2 and increased peroxisome proliferator-activated receptor γ in both cells. Addition of prostaglandin E2 only partially counteracted the effect of celecoxib on both cells. Celecoxib resulted in a significant reduction of retinoblastoma phosphorylation and DP1/E2F1 complex in both cells. Celecoxib caused a significant increase of apoptosis and activation of caspase-3 and caspase-9 in both cells. In nude mice inoculated with HuH7 cells, celecoxib resulted in decreased frequency and mean weight of hepatocellular carcinoma xenografts.

Conclusion: The present study showed that celecoxib causes COX-2-dependent and COX-2-independent growth inhibition of hepatocellular carcinoma cells and xenografts by (a) decreased retinoblastoma phosphorylation and DP1/E2F1 complex; (b) increased activation of caspase-3 and caspase-9; and (c) increased expression of proliferator-activated receptor γ. The present study significantly extended our knowledge on the effect and mechanisms of celecoxib-induced inhibition of hepatocellular carcinoma cell growth.
A Simple and Fast Method for the Simultaneous Detection of Nine Fibroblast Growth Factor Receptor 3 Mutations in Bladder Cancer and Voided Urine
Authors: van Oers Johanna M.M. , Lurkin Irene , van Exsel Antonius J.A. , Nijsen Yvette , van Rhijn Bas W.G. , van der Aa Madelon N.M. , Zwarthoff Ellen C.
Published: March 2006

Purpose: Mutations in the fibroblast growth factor receptor 3 (FGFR3) occur in 50% of primary bladder tumors. An FGFR3 mutation is associated with good prognosis, illustrated by significantly lower percentage of patients with progression and disease-specific mortality. FGFR3 mutations are especially prevalent in low grade/stage tumors, with pTa tumors harboring mutations in 85% of the cases. These tumors recur in 70% of patients. Efficient FGFR3 mutation detection for prognostic purposes and for detection of recurrences in urine is an important clinical issue. In this paper, we describe a simple assay for the simultaneous detection of nine different FGFR3 mutations.

Experimental Design: The assay consists of one multiplex PCR, followed by extension of primers for each mutation with a labeled dideoxynucleotide. The extended primers are separated by capillary electrophoresis, and the identity of the incorporated nucleotide indicates the presence or absence of a mutation.

Results: The assay was found to be more sensitive than single-strand conformation polymorphism analysis. Mutations could still be detected with an input of only 1 ng of genomic DNA and in a 20-fold excess of wild-type DNA. Moreover, in urine samples from patients with a mutant tumor, the sensitivity of mutation detection was 62%.

Conclusions: We have developed a fast, easy to use assay for the simultaneous detection of FGFR3 mutations, which can be of assistance in clinical decision-making and as an alternative for the follow-up of patients by invasive cystoscopy for the detection of recurrences in urine.
In vivoTherapeutic Responses Contingent on Fanconi Anemia/BRCA2 Status of the Tumor
Authors: van der Heijden Michiel S. , Brody Jonathan R. , Dezentje David A. , Gallmeier Eike , Cunningham Steven C. , Swartz Michael J. , DeMarzo Angelo M. , Offerhaus G. Johan A. , Isacoff William H. , Hruban Ralph H. , Kern Scott E.
Published: March 2006

Purpose: BRCA2, FANCC, and FANCG gene mutations are present in a subset of pancreatic cancer. Defects in these genes could lead to hypersensitivity to interstrand cross-linkers in vivo and a more optimal treatment of pancreatic cancer patients based on the genetic profile of the tumor.

Experimental Design: Two retrovirally complemented pancreatic cancer cell lines having defects in the Fanconi anemia pathway, PL11 (FANCC-mutated) and Hs766T (FANCG-mutated), as well as several parental pancreatic cancer cell lines with or without mutations in the Fanconi anemia/BRCA2 pathway, were assayed for in vitro and in vivo sensitivities to various chemotherapeutic agents.

Results: A distinct dichotomy of drug responses was observed. Fanconi anemia–defective cancer cells were hypersensitive to the cross-linking agents mitomycin C (MMC), cisplatin, chlorambucil, and melphalan but not to 5-fluorouracil, gemcitabine, doxorubicin, etoposide, vinblastine, or paclitaxel. Hypersensitivity to cross-linking agents was confirmed in vivo; FANCC-deficient xenografts of PL11 and BRCA2-deficient xenografts of CAPAN1 regressed on treatment with two different regimens of MMC whereas Fanconi anemia–proficient xenografts did not. The MMC response comprised cell cycle arrest, apoptosis, and necrosis. Xenografts of PL11 also regressed after a single dose of cyclophosphamide whereas xenografts of genetically complemented PL11FANCC did not.

Conclusions: MMC or other cross-linking agents as a clinical therapy for pancreatic cancer patients with tumors harboring defects in the Fanconi anemia/BRCA2 pathway should be specifically investigated.
Mechanical Analysis of Tumor Growth Regression by the Cyclooxygenase-2 Inhibitor, DFU, in a Walker256 Rat Tumor Model: Importance of Monocyte Chemoattractant Protein-1 Modulation
Authors: Muta Mariko , Matsumoto Gaku , Nakashima Emi , Toi Masakazu
Published: March 2006

Cyclooxygenase (COX)-2 inhibition results in tumor regression; however, little is known about the mechanism. In the present study, using a Walker256 tumor model and a rat bone marrow–derived endothelial cell line TR-BME-2, we analyzed the effects of a new selective COX-2 inhibitor, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2-(5H)-furanone (DFU), on the production of chemokines and growth factors and on the neovascularization. The oral administration of DFU (5 mg/kg/d) significantly suppressed the tumor growth with decreasing microvessel density in vivo, although it showed no direct inhibition of Walker256 cell proliferation in vitro. It was newly found that the recruitment of systemically injected TR-BME-2 cells into the tumor site was significantly inhibited by DFU treatment. In addition, we found that DFU significantly reduced the production of monocyte chemoattractant protein-1 (MCP-1) both in tumor tissues and in the systemic circulation (P < 0.001 and P < 0.001, respectively). Such reduction was not observed in other chemotactic factors, vascular endothelial growth factor and stromal cell–derived factor-1. The induced chemotaxis of TR-BME-2 by serum of tumor-bearing rats was significantly reduced in DFU-treated rat serum, although DFU showed no direct inhibition for TR-BME-2 cells, either cell growth or chemotaxis. Treatment with neutralizing antibodies to soluble mediators, including MCP-1, significantly suppressed the chemotaxis. Regarding the down-regulation machinery of MCP-1 production in vivo, tumor-associated macrophages seem to play crucial roles, because DFU eliminated MCP-1 production in the activated macrophages remarkably but not in Walker256 tumor cells in vitro. In conclusion, COX-2 inhibitor DFU exerts tumor regression activity in a Walker256 tumor model by suppressing MCP-1 production in tumor tissues and in the circulation.
Myelomastocytic Leukemia: Evidence for the Origin of Mast Cells from the Leukemic Clone and Eradication by Allogeneic Stem Cell Transplantation
Authors: Sperr Wolfgang R. , Drach Johannes , Hauswirth Alexander W. , Ackermann Jutta , Mitterbauer Margit , Mitterbauer Gerlinde , Foedinger Manuela , Fonatsch Christa , Simonitsch-Klupp Ingrid , Kalhs Peter , Valent Peter
Published: March 2006

Purpose: Myelomastocytic leukemia is a term used for patients with advanced myeloid neoplasms, in whom elevated numbers of immature atypical mast cells are found, but criteria for a primary mast cell disease are not met. The origin of mast cells in these patients is presently unknown.

Patient and Methods: We have analyzed clonality of mast cells in an 18-year-old patient suffering from acute myeloid leukemia with a complex karyotype including a t(8;21) and mastocytic transformation with a huge increase in immature mast cells and elevated serum tryptase level, but no evidence for a primary mast cell disease/mastocytosis.

Results: As assessed by in situ fluorescence hybridization combined with tryptase staining, both the tryptase-negative blast cells and the tryptase-positive mast cells were found to contain the t(8;21)-specific AML1/ETO fusion gene. Myeloablative stem cell transplantation resulted in complete remission with consecutive disappearance of AML1/ETO transcripts, decrease of serum tryptase to normal range, and disappearance of neoplastic mast cells.

Conclusion: These data suggest that mast cells directly derive from the leukemic clone in patients with myelomastocytic leukemia.
Phase I Study of Cloretazine (VNP40101M), a Novel Sulfonylhydrazine Alkylating Agent, Combined with Cytarabine in Patients with Refractory Leukemia
Authors: Giles Francis , Verstovsek Srdan , Thomas Deborah , Gerson Stanton , Cortes Jorge , Faderl Stefan , Ferrajoli Alessandra , Ravandi Farhad , Kornblau Steven , Garcia-Manero Guillermo , Jabbour Elias , O'Brien Susan , Karsten Verena , Cahill Ann , Yee Karen , Albitar Maher , Sznol Mario , Kantarjian Hagop
Published: March 2006

Purpose: Cloretazine (VNP40101M) is a novel sulfonylhydrazine alkylating agent with significant antileukemia activity. A phase I study of cloretazine combined with cytarabine (1-β-d-arabinofuranosylcytosine, ara-C) was conducted in patients with refractory disease.

Design: Ara-C was given i.v. at a fixed dose of 1.5 gm/m2/d by continuous infusion for 4 days (patients ages <65 years at time of diagnosis) or 3 days (patients ages ≥65 years). Cloretazine was given i.v. over 15 to 60 minutes on day 2 at a starting dose of 200 mg/m2, with escalation in 100 mg/m2 increments in cohorts of three to six patients until a maximum tolerated dose was established. The DNA repair enzyme O6-alkylguanine DNA alkyltransferase (AGT) was measured at baseline.

Results: Forty patients, including 32 with acute myeloid leukemia, received 47 courses of treatment. Complete responses were seen at cloretazine dose levels of ≥400 mg/m2 in 10 of 37 (27%) evaluable patients, and in this patient subset, AGT activity was significantly lower in patients that responded to treatment than in patients who did not (P ≤ 0.027). Dose-limiting toxicities (gastrointestinal and myelosuppression) were seen with 500 and 600 mg/m2 of cloretazine combined with the 4-day ara-C schedule but not seen with the 3-day schedule.

Conclusion: The recommended cloretazine dose schedule for future studies is 600 mg/m2 combined with 1.5 gm/m2/d continuous infusion of ara-C for 3 days. The cloretazine and ara-C regimen has significant antileukemic activity. AGT activity may be a predictor of response to cloretazine.
Highly Sensitive Detection of Melanoma at an Early Stage Based on the Increased Serum Secreted Protein Acidic and Rich in Cysteine and Glypican-3 Levels
Authors: Ikuta Yoshiaki , Nakatsura Tetsuya , Kageshita Toshiro , Fukushima Satoshi , Ito Shosuke , Wakamatsu Kazumasa , Baba Hideo , Nishimura Yasuharu
Published: March 2006

Purpose: There are no available tumor markers detecting primary melanoma at an early stage. The identification of such serum markers would be of significant benefit for an early diagnosis of melanoma. We recently identified glypican-3 (GPC3) as a novel tumor marker but could diagnose only 40% of melanomas. Thereby, we focused out attention on secreted protein acidic and rich in cysteine (SPARC) overexpressed in melanoma as another candidate for tumor marker.

Experimental Design: Secreted SPARC protein was quantified using ELISA in the sera from 109 melanoma patients, five patients with large congenital melanocytic nevus, 61 age-matched healthy donors, and 13 disease-free patients after undergoing a surgical removal. We also quantified GPC3 and 5-S-cysteinyldopa in the same serum samples and compared these markers for their diagnostic value.

Results: The serum SPARC concentrations in melanoma patients were greater than those in healthy donors (P = 0.001). When we fixed a cutoff value at the mean concentration plus 2 SD of the healthy donors, the serum SPARC was found to have increased in the sera of 36 of the 109 (33%) melanoma patients, whereas there were three (4.9%) false-positive cases of 61 healthy donors. Surprisingly, 19 of 36 patients showing increased SPARC levels were in stages 0 to II. The serum SPARC level decreased under the cutoff level in 10 of 13 patients after surgical removal. Using SPARC and GPC3 in combination thus enabled us to diagnose 47 of 75 (66.2%) melanoma patients at an early stage (0-II).

Conclusions: SPARC or its combination with GPC3 is thus considered a potentially useful tumor marker, especially for melanoma at an early stage.
Plasmalemmal Vesicle Associated Protein-1 Is a Novel Marker Implicated in Brain Tumor Angiogenesis
Authors: Carson-Walter Eleanor B. , Hampton Jessica , Shue Eveline , Geynisman Daniel M. , Pillai Pramod Kumar , Sathanoori Ramasri , Madden Stephen L. , Hamilton Ronald L. , Walter Kevin A.
Published: March 2006

Purpose: Plasmalemmal vesicle associated protein-1 (PV-1) is up-regulated in the endothelium of human glioblastoma. We sought to further characterize the expression pattern of PV-1 in human brain tumors and interrogate its role in brain tumor angiogenesis.

Experimental Design: Quantitative reverse transcription-PCR and in situ hybridization were used to measure PV-1 expression in a panel of 46 human brain tumors and related pathologic states. Matrigel tubulogenesis assays and cell migration assays were used to show function of PV-1 in primary human endothelial cells (HMVEC) under gene knockdown conditions.

Results: PV-1 is selectively up-regulated in a variety of high-grade human brain tumors, including glioblastoma and metastatic carcinoma, as well as other cerebral disorders associated with blood-brain barrier disruption, such as acute ischemia. Expression levels were reduced in low-grade neoplasia; however, tumors associated with the ependyma and choroid plexus, known sites of PV-1 expression, also exhibited robust expression. Cerebral expression of PV-1 mRNA was confined to endothelial cells in all cases. PV-1 expression was induced in HMVEC cells in vitro by exposure to medium conditioned by U87MG and U251MG human brain tumor cell lines and by medium supplemented with exogenous vascular endothelial growth factor or scatter factor/hepatocyte growth factor. RNA interference–mediated inhibition of PV-1 induction in HMVEC cells blocked Matrigel-induced tubulogenesis and inhibited cell migration induced by conditioned medium or angiogenic growth factors.

Conclusions: Our results confirm that PV-1 is preferentially induced in the endothelium of high-grade human brain tumors. Inhibition of PV-1 expression is associated with failure of endothelial differentiation in vitro. PV-1 represents a novel marker of brain tumor angiogenesis and integrity of the blood-brain barrier and is a potential therapeutic target.