Expression In Malignant Tumors Research Articles

Radiosynthesis of N5‐[18F]fluoroacetylornithine (N5‐[18F]FAO) for PET imaging of ornithine decarboxylase (ODC) in malignant tumors

AbstractPolyamines are naturally occurring polycations derived from amino acids via decarboxylation by ornithine decarboxylase (ODC). Ornithine is a substrate for ODC; decarboxylation of ornithine is inhibited by difluoromethylornithine (DFMO) and its derivatives. Polyamine contents are increased in many epithelial cancers, including breast cancer, melanoma, and prostate cancer. In order to image and measure the levels of ODC expression in malignant tumors, we have synthesized a derivative of ornithine, N5‐[18F]fluoroacetylornithine (N5‐[18F]FAO), for use in positron emission tomography. The precursor compound N2‐Boc‐N5‐bromoacetylornithine‐t‐butyl ester 2 was synthesized from 5‐amino‐2‐(tert‐butoxycarbonylamino)pentanoic acid, which was reacted with bromoacetyl chloride followed by esterification with tert‐butyl‐2,2,2‐trichloroacetamidate. Fluorination of the precursor produced a fluoro‐derivative, which was hydrolyzed in acid to obtain the desired compound, N5‐fluoroacetylornithine. The radiosynthesis of N5‐[18F]FAO was accomplished by radiofluorination of 2 with n‐Bu4N[18F], followed by high‐performance liquid chromatography (HPLC) purification and then by acid hydrolysis. The radiochemical yield was 6–10% (decay corrected) with an average of 8% (n=10) at the end of synthesis. The radiochemical purity was >99%, and specific activity was >1500 mCi/µmol. The synthesis time was 95–100 min from the end of bombardment. Copyright © 2010 John Wiley & Sons, Ltd.

Differential expression of hedgehog signaling components and Snail/E-cadherin in human brain tumors

The hedgehog (Hh) transcription factor Gli induces transformation of epithelial cells via induction of Snail, a repressor of E-cadherin. Epithelial-mesenchymal transition is also a determinant of the progression of tumorigenesis, following down-regulation of E-cadherin. However, the role of Hh signaling components and Snail/E-cadherin in brain tumors is not yet fully understood. We analyzed the expression of Hh signaling components and Snail/E-cadherin in 69 brain tumors by reverse transcription-polymerase chain reaction (RT-PCR). The data showed that overexpression of Smo (35/69), Ptch (50/69), Gli1 (56/69), Gli2 (29/69) and N-myc (39/69) might contribute to brain tumorigenesis. Our results also indicated that Snail and E-cadherin showed opposing expression in malignant tumors (high grade astrocytoma and metastasis). Snail and E-cadherin showed less correlation in benign brain tumors. We further investigated mutations of Gli2 and Snail by RT-PCR and direct sequencing. No mutation was observed on Gli2 but several sporadic mutations on Snail were found, including S96G, S111L, S111L/S119Y and one nonsense mutation at codon 158 (Y158*). An in vitro E-cadherin promoter assay showed that S96G, S111L, S111L/S119Y Snail mutants were decreased by 15, 25 and 50%, respectively, whereas Y158* was increased by 40% compared to wild-type. Furthermore, our data showed that wild-type Snail and S96G, S111L, S111L/S119Y translocated to the nucleus, while the Y158* mutant failed to translocate to the nucleus. Taken together, our results demonstrate that Hh signaling components, the expression and mutations of Snail and the expression of E-cadherin may play an important role in human brain tumorigenesis.

Kit formulation for 99mTc-labeling of recombinant anti-HER2 Affibody molecules with a C-terminally engineered cysteine

Molecular imaging of human epidermal growth factor receptor type 2 (HER2)-expression in malignant tumors provides potentially important information for patient management. Affibody molecules have shown to be suitable tracers for imaging applications using single photon emission computed tomography or positron emission tomography. Results from an earlier evaluation of the application of site-specific (99m)Tc-labeling of the Affibody molecule, Z(HER2:2395)-C, were favorable. As a preparation for clinical application of this tracer, we have developed and evaluated a robust single-vial freeze-dried kit, allowing labeling of the Affibody molecule, Z(HER2:2395)-C, with (99m)Tc. The composition of the kit [containing glucoheptonate, EDTA and tin(II)-chloride], as well as the protein amount and the pertechnetate volume were optimized for a high labeling yield (>90%) and minimal presence of reduced hydrolyzed technetium colloids (<1%). The specificity to HER2 receptors, the binding competence and the stability in phosphate-buffered saline and murine serum were verified in vitro. The shelf-life was also evaluated in vitro, showing no reduction in labeling yield or binding capacity to HER2-expressing cells after over 400 days of storage of the single-vial freeze-dried kit. Z(HER2:2395)-C labeled with (99m)Tc using the lyophilized kit was stable and resulted in a favorable biodistribution in an in vivo evaluation in normal Naval Medical Research Institute mice.

ScVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA conjugates: comparison of easy-to-label recombinant proteins for [68Ga]PET imaging of VEGF receptors in angiogenic vasculature

VEGF receptors play a key role in angiogenesis and are important targets for several approved and many experimental drugs. Imaging of VEGF receptor expression in malignant tumors would provide important information, which can influence patient management. The aim of this study was the development of an easy-to-label positron-emitting tracer for imaging VEGF receptors. The tracer is based on engineered single-chain VEGF (scVEGF), expressed with cysteine-containing fusion tag (Cys-tag) for site-specific conjugation of PEGylated bifunctional chelating agents, HBED-CC or NOTA, suitable for labeling with (68)Ga at ambient temperature. scVEGF-PEG-HBED-CC was synthesized by activating a single carboxyl group of the [Fe(HBED-CC)](-) complex with N-hydroxysuccinimide. Reaction of the activated complex with NH(2)-PEG-maleimide was followed by site-specific conjugation of PEGylated chelator to a thiol group in Cys-tag of scVEGF. The scVEGF-PEG-NOTA conjugate was synthesized using NHS-PEG-maleimide and p-NH(2)-Bn-NOTA. (68)Ga complexation was performed in HEPES buffer (pH 4.2) at room temperature. The functional activity after labeling was tested by radioligand cell binding assays. Biodistribution and PET studies in tumor-bearing mice were performed after 1, 2, 3 and 4 h postinjection. The radiolabeling of scVEGF-PEG-HBED-CC proved more efficient than scVEGF-PEG-NOTA allowing to stop the reaction after 4 min (>97% radiochemical yield). Radioligand cell binding assays performed on HEK-293 cells overexpressing VEGFR-2 revealed no change in the binding properties of (68)Ga-radiolabeled scVEGF relative to other scVEGF-based tracers. Both tracers showed comparable results in biodistribution, such as tumor accumulation and low liver uptake. The tracers were stable in 50% human serum for at least 72 h. The conjugates scVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA revealed comparable in vivo characteristics and allowed easy-to-perform labeling with high stability for fast [(68)Ga]PET imaging of VEGF receptors in angiogenic vasculature.

Targeting of HER2-Expressing Tumors with a Site-Specifically 99mTc-Labeled Recombinant Affibody Molecule, ZHER2:2395, with C-Terminally Engineered Cysteine

The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced (99m)Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. A C-terminal cysteine was introduced into the Affibody molecule Z(HER2:342) to enable site-specific labeling with (99m)Tc. Two recombinant variants, His(6)-Z(HER2:342)-Cys (dissociation constant [K(D)], 29 pM) and Z(HER2:2395)-Cys, lacking a His tag (K(D), 27 pM), were labeled with (99m)Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and gamma-camera imaging studies were performed in mice bearing HER2-expressing xenografts. (99m)Tc-His(6)-Z(HER2:342)-Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. (99m)Tc-Z(HER2:2395)-Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 +/- 2.5 (mean +/- SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 +/- 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 +/- 24 and 121 +/- 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical gamma-camera 1 h after injection of (99m)Tc-Z(HER2:2395)-Cys. The Affibody molecule (99m)Tc-Z(HER2:2395)-Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

On the Selection of a Tracer for PET Imaging of HER2-Expressing Tumors: Direct Comparison of a 124I-Labeled Affibody Molecule and Trastuzumab in a Murine Xenograft Model

Human epidermal growth factor receptor type 2 (HER2) is a tyrosine kinase, which is often overexpressed in many carcinomas. Imaging HER2 expression in malignant tumors can provide important prognostic and predictive diagnostic information. The use of anti-HER2 tracers labeled with positron-emitting radionuclides may increase the sensitivity of HER2 imaging. The goal of this study was to compare directly 2 approaches for developing anti-HER2 PET tracers: a (124)I-labeled monoclonal antibody and a small (7-kDa) scaffold protein, the Affibody molecule. The anti-HER2 Affibody Z(HER2:342) and humanized monoclonal antibody trastuzumab were labeled with (124/125)I using p-iodobenzoate (PIB) as a linker. Cellular processing of both tracers by HER2-expressing cells was investigated. The biodistributions of (124)I-PIB-Z(HER2:342) and (125)I-PIB-trastuzumab were compared in BALB/C nu/nu mice bearing HER2-expressing NCI-N87 xenografts using paired labels. Small-animal PET of (124)I-PIB-Z(HER2:342) and (124)I-PIB-trastuzumab in tumor-bearing mice was performed at 6, 24, and 72 h after injection. Both radioiodinated Z(HER2:342) and trastuzumab bound specifically to HER2-expressing cells in vitro and specifically targeted HER2-expressing xenografts in vivo. Radioiodinated trastuzumab was more rapidly internalized and degraded, which resulted in better retention of radioactivity delivered by Z(HER2:342). Total uptake of trastuzumab in tumors was higher than that of (124)I-PIB-Z(HER2:342). However, tumor-to-organ ratios were appreciably higher for (124)I-PIB-Z(HER2:342) due to the more rapid clearance of radioactivity from blood and normal organs. The ex vivo results were confirmed by small-animal PET. The use of the small scaffold targeting Affibody provides better contrast in HER2 imaging than does the monoclonal antibody.

Affibody Molecules for Epidermal Growth Factor Receptor Targeting In Vivo: Aspects of Dimerization and Labeling Chemistry

Noninvasive detection of epidermal growth factor receptor (EGFR) expression in malignant tumors by radionuclide molecular imaging may provide diagnostic information influencing patient management. The aim of this study was to evaluate a novel EGFR-targeting protein, the ZEGFR:1907 Affibody molecule, for radionuclide imaging of EGFR expression, to determine a suitable tracer format (dimer or monomer) and optimal label. An EGFR-specific Affibody molecule, ZEGFR:1907, and its dimeric form, (ZEGFR:1907)2, were labeled with 111In using benzyl-diethylenetriaminepentaacetic acid and with 125I using p-iodobenzoate. Affinity and cellular retention of conjugates were evaluated in vitro. Biodistribution of radiolabeled Affibody molecules was compared in mice bearing EGFR-expressing A431 xenografts. Specificity of EGFR targeting was confirmed by comparison with biodistribution of non-EGFR-specific counterparts. Head-to-tail dimerization of the Affibody molecule improved the dissociation rate. In vitro, dimeric forms demonstrated superior cellular retention of radioactivity. For both molecular set-ups, retention was better for the 111In-labeled tracer than for the radioiodinated counterpart. In vivo, all conjugates accumulated specifically in xenografts and in EGFR-expressing tissues. The retention of radioactivity in tumors was better in vivo for dimeric forms; however, the absolute uptake values were higher for monomeric tracers. The best tracer, 111In-labeled ZEGFR:1907, provided a tumor-to-blood ratio of 100 (24 h after injection). The radiometal-labeled monomeric Affibody molecule ZEGFR:1907 has a potential for radionuclide molecular imaging of EGFR expression in malignant tumors.

Galloyl cyclic-imide derivative CH1104I inhibits tumor invasion through suppressing matrix metalloproteinase activity

Matrix metalloproteinase (MMP)-2 and MMP-9 have been associated with the ability of tumor cells to metastasize because of their capacity to degrade type IV collagen, the main component of basement membrane, and to their elevated expression in malignant tumors. (S)-methyl 6-(benzyloxycarbonylamino)-2-(2-((S)-2,6-dioxo-3-(3,4,5-trimethoxybenzamido) piperidin-1-yl) acetamido) hexanoate (CH1104I) is a galloyl cyclic-imide derivative designed to fit and extend into the S1' active pocket of MMP-2 and MMP-9. We aimed to evaluate the efficacy of CH1104I as a candidate compound for antiinvasion and antimetastasis of tumor cells. CH1104I significantly blocked gelatinase activity as evidenced by a decrease in the degradation of succinylated gelatin. Gelatin zymography analysis showed that the compound (7-210 micromol/l) inhibited the activity of MMP-2 and MMP-9 produced by human ovarian carcinoma SKOV3 cells. Inhibition of MMP-2 and MMP-9 expression was also observed using the assays of immunocytochemical staining and western blot analysis. The results showed that CH1104I suppressed the expression of zymogens and active MMP-2 and MMP-9. The effects of CH1104I on the invasion and migration of SKOV3 cells were then measured. Both the trans-well motility assay and wound scratch assay indicated that CH1104I was very effective for the antiinvasion and antimigration of SKOV3 cells. Furthermore, the Lewis lung carcinoma model was used to evaluate the efficacy of CH1104I in vivo. A significant inhibition of pulmonary metastasis of carcinoma cells was observed in CH1104I-administrated mice (25-100 mg/kg). These results suggest that CH1104I is a potential MMP-2 and MMP-9 inhibitor that may effectively suppress tumor invasion and metastasis.

Native human autoantibodies targeting GIPC1 identify differential expression in malignant tumors of the breast and ovary

BackgroundWe have been studying the native humoral immune response to cancer and have isolated a library of fully human autoantibodies to a variety of malignancies. We previously described the isolation and characterization of two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1, an accessory PDZ-domain binding protein involved in regulation of G-protein signaling. Human monoclonal antibodies, 27.F7 and 27.B1, to GIPC1 demonstrate specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue.MethodsThe current study employs cELISA, flow cytometry, Western blot analysis as well as immunocytochemistry, and immunohistochemistry. Data is analyzed statistically with the Fisher one-tail and two-tail tests for two independent samples.ResultsBy screening several other cancer cell lines with 27.F7 and 27.B1 we found consistently strong staining of other human cancer cell lines including SKOV-3 (an ovarian cancer cell line). To further clarify the association of GIPC1 with breast and ovarian cancer we carefully studied 27.F7 and 27.B1 using immunocytochemical and immunohistochemical techniques. An immunohistochemical study of normal ovarian tissue, benign, borderline and malignant ovarian serous tumors, and different types of breast cancer revealed high expression of GIPC1 protein in neoplastic cells. Interestingly, antibodies 27.F7 and 27.B1 demonstrate differential staining of borderline ovarian tumors. Examination of different types of breast cancer demonstrates that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors.ConclusionThe present pilot study demonstrates that the GIPC1 protein is overexpressed in ovarian and breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role that this protein plays in malignant diseases. In addition, this study suggests that human monoclonal antibodies 27.F7 and 27.B1 should be further evaluated as potential diagnostic tools.

Increased Expression of CENP-H Gene in Human Salivary Gland Carcinomas

There have been very few studies on the expression of Centromere proteins in human salivary gland carcinomas. The purpose of this study was to clarify the correlation between Centromere protein H (CENP-H) expression and clinicopathologic factors in salivary gland carcinomas. The expression of CENP-H mRNA was investigated in 28 human salivary gland tumors (7 pleomorphic adenomas, 3 Warthin tumors, 6 mucoepidermoid carcinomas, 6 adenoid cystic carcinomas, 5 acinic cell carcinomas and 1 malignant myoepithelioma) and 8 normal submandibular glands using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The labeling index of PCNA and Ki-67 were also investigated immunohistochemically in 16 salivary gland carcinomas. The mean expression level of CENP-H mRNA was significantly higher in malignant tumors (0.55 ± 0.68) than normal submandibular glands (0.10 ± 0.029). A significant correlation between the PCNA labeling index and CENP-H mRNA expression was also found (Spearman's correlation coefficient by rank test, P=0.033). We also found a significant correlation between the Ki-67 labeling index and CENP-H mRNA expression in malignant tumors (Spearman's correlation coefficient by rank test, P=0.040). These results indicate that human CENP-H mRNA is closely linked to increased or abnormal cell proliferation in malignant salivary gland tumors.

Src kinase regulates metalloproteinase-9 secretion induced by type IV collagen in MCF-7 human breast cancer cells

Matrix metalloproteinases (MMPs) are a family of endopeptidases that collectively are capable to degrading all components of the extracellular matrix (ECM) and they have been implicated in several aspects of tumor progression, such as invasion through basement membrane (BM) and insterstitial matrices, angiogenesis and tumor cell growth. In particular, MMP-2 and MMP-9 have been associated with the ability of tumor cells to metastasize due to their capacity to degrade type IV collagen (Col-IV), the main component of BM, and to their elevated expression in malignant tumors. However, nothing is known about the regulation of MMP-9 secretion and expression in breast cancer cells stimulated with Col-IV. Our results demonstrate that stimulation of MCF-7 cells with Col-IV promoted the secretion of MMP-9, as revealed by gelatin zymography and Western blotting using specific antibodies that recognized MMP-9. In addition, inhibition of Src and FAK kinase activity prevented MMP-9 secretion. In contrast, MMP-9 expression was not up-regulated by treatment with Col-IV. These results demonstrate that Col-IV regulates the secretion of MMP-9 via a Src and FAK dependent pathway in MCF-7 cells.

Pre-clinical evaluation of [111In]-benzyl-DOTA-ZHER2:342, a potential agent for imaging of HER2 expression in malignant tumors

Imaging of expression of human epidermal growth factor receptor type 2 (HER2) in breast carcinomas may help to select patients eligible for trastuzumab therapy. The Affibody molecule Z(HER2:342) is a small (7-kDa) non-immunoglobulin affinity protein, which binds to HER2 with a picomolar affinity. Previously, a benzyl-DTPA conjugate of Z(HER2:342) was labeled with 111In and demonstrated good targeting in murine xenografts. We considered that the use of the macrocyclic chelator DOTA could increase the label stability and enhance a choice of nuclides, which could be used as a label for Z(HER2:342). The goal of this study was the preparation and pre-clinical evaluation of the indium-111- labeled DOTA-derivative of Z(HER2:342). Isothiocyanate-benzyl-DOTA was coupled to recombinant Z(HER2:342), and the conjugate was efficiently labeled with 111In at 60 degrees C. The specificity of 111In-benzyl-DOTA-Z(HER2:342) binding to HER2 was confirmed in vitro using HER2-expressing breast carcinoma BT474 and ovarian carcinoma SKOV-3 cell lines. Biodistribution of 111In-benzyl-DOTA-Z(HER2:342) was performed in nude mice bearing LS174T xenografts and compared directly with the biodistribution of 111In-benzyl-DTPA-Z(HER2:342). In vivo, 111In-benzyl-DOTA-Z(HER2:342) demonstrated quick clearance from blood and non-specific organs except the kidneys. Four hours post injection (pi), the tumor uptake of 111In-benzyl-DOTA-Z(HER2:342) (4.4+/-1.0% IA/g) was specific and the tumor-to-blood ratio was 23. The use of benzyl-DTPA provided higher tumor-to-blood and tumor-to-liver ratios. gamma-camera imaging showed clear visualization of HER2-expressing xenografts using 111In-benzyl-DOTA-Z(HER2:342). 111In-benzyl-DOTA-Z(HER2:342) has a potential for imaging of HER2 expression in malignant tumors.

Maspin Expression and Its Clinical Significance in Non-Small Cell Lung Cancer

Maspin is a member of the serpin (serine protease inhibitor) superfamily, and its exact function in the development and progression of malignant tumors remains controversial, though some experimental studies have revealed potential tumor-suppressor activities. In addition, there have been only a few clinical studies on maspin expression in malignant tumors including non-small cell lung cancer (NSCLC). The purpose of this study was to assess maspin expression and its clinical significance in NSCLC. A total of 210 consecutive patients with completely resected pathological (p-) stage I-IIIA NSCLC were retrospectively reviewed. Maspin expression along with intratumoral microvessel density, proliferative activity, and p53 status were evaluated immunohistochemically. The incidence of apoptotic cell death was also evaluated. The incidence of strong maspin expression was significantly higher in lung squamous cell carcinoma (56/76, 73.7%; P < .001) than in other histological types. The incidence of aberrant expression of p53 was significantly higher in maspin-strong than in maspin-weak tumors (56.2% and 35.8%, respectively; P = .005). There was no difference in prognosis according to maspin status for all patients. However, for squamous cell carcinoma patients, univariate analysis showed that enhanced maspin expression was a significant factor in predicting a favorable prognosis (5-year survival rates, 70.1% for maspin-strong tumors and 41.5% for maspin-weak tumors; P = .014), which was confirmed in a multivariate analysis (hazard ratio = .475, 95% confidence interval .241-.936; P = .032). Enhanced maspin expression was a significant and independent factor in predicting a favorable prognosis in lung squamous cell carcinoma.

Tumor Imaging Using a Picomolar Affinity HER2 Binding Affibody Molecule

The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z(HER2:342) showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging.

Importance of P-Cadherin, β-Catenin, and Wnt5a/Frizzled for Progression of Melanocytic Tumors and Prognosis in Cutaneous Melanoma

It has been proposed that melanoma cells shift from E-cadherin to N-cadherin expression during tumor development, and recent gene profiling has shown increased expression of Wnt5a/Frizzled in aggressive melanomas possibly by interactions with beta-catenin. We therefore wanted to investigate the role of cadherin subtypes, beta-catenin, and Wnt5a/Frizzled in melanocytic tumors, with focus on prognosis in nodular melanomas. The immunohistochemical expression of E-cadherin, N-cadherin, P-cadherin, beta-catenin, and Wnt5a/Frizzled was examined using tissue microarrays of 312 melanocytic tumors. Cytoplasmic expression of P-cadherin was associated with increasing tumor thickness (P=0.005) and level of invasion (P=0.019), whereas membranous staining was associated with thinner (P=0.012) and more superficial (P=0.018) tumors. Increased cytoplasmic P-cadherin was associated with reduced survival (P=0.047). Lack of nuclear beta-catenin expression was related to increased tumor thickness (P=0.002) and poor patient survival in univariate (P=0.0072) and multivariate (P=0.004) analyses. Membranous expression of N-cadherin was significantly increased from primary tumors to metastatic lesions, whereas E-cadherin staining tended to be decreased. Wnt5a and its receptor Frizzled were highly coexpressed, and nuclear expression of both markers was significantly reduced from benign nevi to melanomas, with a shift from nuclear to cytoplasmic expression in malignant tumors. In addition, Wnt5a expression was significantly associated with nuclear beta-catenin expression. Alterations in the expression and subcellular localization of cell adhesion markers are important in the development and progression of melanocytic tumors, and strong cytoplasmic P-cadherin expression and loss of nuclear beta-catenin staining were associated with aggressive melanoma behavior and reduced patient survival.

Correlation of CENP-F gene expression with tumor-proliferating activity in human salivary gland tumors

We examined the expression of Centromere protein F (CENP-F) mRNA in 26 human salivary gland tumors (seven pleomorphic adenomas, three Warthin tumors, seven mucoepidermoid carcinomas, four adenoid cystic carcinomas, four acinic cell carcinomas and one malignant myoepithelioma) and four normal submandibular glands using the real time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of CENP-F mRNA was higher in malignant tumors (1.05+/-0.32) than normal submandibular glands (0.11+/-0.05) and benign tumors (0.46+/-0.16). We found a significant association between the level of CENP-F mRNA expression and clinical stage in 16 malignant tumors (Mann-Whitney U test, p=0.027). We also found a significant correlation between the Ki-67 labeling index and CENP-F expression in malignant tumors (Spearmans correlation coefficient by rank test, p=0.029). These results indicate that human CENP-F mRNA is closely linked to the increased or abnormal cell proliferation in malignant conditions.

Silencing of Monocarboxylate Transporters via Small Interfering Ribonucleic Acid Inhibits Glycolysis and Induces Cell Death in Malignant Glioma: An in Vitro Study

Dependence on glycolysis is a hallmark of malignant tumors. As a consequence, these tumors generate more lactate, which is effluxed from cells by monocarboxylate transporters (MCTs). We hypothesized that 1) MCT expression in malignant tumors may differ from normal tissue in quantity, isoform, or both; and 2) silencing MCT expression would induce intracellular acidification, resulting in decreased proliferation and/or increased cell death. We quantified expression of MCT isoforms in human glioblastoma multiforme and glioma-derived cells lines by Western blot analysis. MCTs that were abundant or specific to glioma then were targeted in the model U-87 MG glioma cell line via small interfering ribonucleic acid-mediated gene silencing and tested for inhibition of lactate efflux, intracellular pH changes, reduced proliferation, and/or induction of cell death. MCT 1 and 2 were the primary isoforms expressed in human glioblastoma multiforme and glioma-derived cell lines. In contrast, MCT 3 was the predominantly expressed isoform in normal brain. Small interfering ribonucleic acid specific for MCT 1 and 2 reduced expression of these isoforms in U-87 MG cells to barely detectable levels and reduced lactate efflux by 30% individually and 85% in combination, with a concomitant decrease of intracellular pH by 0.6 units (a fourfold increase in intracellular H(+)). Prolonged silencing of both MCTs reduced viability by 75% individually and 92% in combination, as measured by both phenotypic and flow cytometric analyses. MCT targeting significantly reduced the viability of U-87 MG cells mediated by both apoptosis and necrosis. This indicates that the strategy may be a useful therapeutic avenue for treatment of patients with malignant glioma.

HER2/neu expression in bladder cancer: relationship to cell cycle kinetics

Background and objectives: The incidence of overexpression of HER2/neu in bladder cancer is one of the highest among all human malignancies tested; such overexpression is thought to play a role in the aberrant proliferation of cancer cells. This study was conducted to evaluate the quantitative assessment of HER2/neu expression by enzyme immunoassay (EIA) and its prognostic significance in differentiating between high and low proliferating tumors. Patients and methods: Tissue samples were collected from 35 patients with benign bladder lesions, 28 with bilharzial bladder cancer, and 25 with nonbilharzial bladder cancer. Twenty normal samples were obtained from normal safety margin areas in nonbilharzial bladder cancer patients. Out of the malignant samples, 22 were found to be squamous cell carcinoma and 31 were transitional cell carcinoma. All samples were examined for HER2/neu expression by EIA and Western blot (WB). Flow cytometric (FCM) analysis was also performed in all the samples provided. Results: HER2/neu was found to be significantly overexpressed in the malignant group compared to the benign and normal groups ( P < 0.001) and no significant difference was found between the bilharzial and nonbilharzial cancer groups or between the transitional and squamous cell carcinoma groups. HER2/neu was significantly correlated to ploidy ( P = 0.001), synthetic phase fraction, SPF ( P = 0.012), and DNA index ( P = 0.002). No significant correlation was found between HER2/neu and stage or grade while it was significantly associated with lymph node status of the tumour ( P = 0.02). Conclusion: HER2/neu can be measured reliably by the EIA method as confirmed by WB. The quantitative assessment of HER2/neu expression in malignant tumors aided by other proliferation markers such as SPF, DI, and ploidy could be useful in selecting patients for more aggressive treatment or for predicting outcome.

Cyclooxygenase-2 expression in stromal tumors of the gastrointestinal tract

Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostaglandins, prostacyclin, and thromboxane. COX-2 is expressed in many epithelial malignancies, particularly those of the gastrointestinal (GI) tract. COX-2 has been implicated in the pathogenesis of cancers and has a significant negative effect on survival. To date, little is known about the expression of COX-2 in nonepithelial tumors. The objective of this study was to evaluate the expression of COX-2 in GI stromal tumors (GISTs). We evaluated 15 GISTs using tissue microarray. Tissue blocks were retrieved and stained with hematoxylin and eosin to evaluate the histological tumor type. In addition, immunohistochemistry was performed for COX-2, the macrophage marker, CD68 (KP-1), and KIT (CD117). Two pathologists then evaluated the tissues to determine the extent and intensity of COX-2 expression. The location of CD68-positive cells, and whether these cells were COX-2 positive, was also evaluated. The results showed that 80% (12 of 15) of the tumors expressed COX-2. Expression was noted in the cytoplasm of the tumor cells, with variable intensity of staining among the tumors. COX-2 was expressed in both epithelial cell and spindle cell tumors, but appeared stronger in epithelial lesions. In mixed lesions, COX-2 was expressed to a greater extent in epithelial areas. There was a greater extent of COX-2 expression in malignant tumors and tumors located within the stomach. Tumor-infiltrating macrophages (CD68-positive cells) were identified in all of the lesions; in 80% of cases, those macrophages also expressed COX-2. This study is the first to demonstrate COX-2 expression in stromal lesions of the GI tract. The enzyme may play a role in the proliferation of these lesions, suggesting the potential use of nonsteroidal anti-inflammatory drugs in treatment.

Cyclooxygenase-2 expression in stromal tumors of the gastrointestinal tract

Cyclooxygenase (COX) is a key enzyme in the conversion of arachidonic acid to prostaglandins, prostacyclin, and thromboxane. COX-2 is expressed in many epithelial malignancies, particularly those of the gastrointestinal (GI) tract. COX-2 has been implicated in the pathogenesis of cancers and has a significant negative effect on survival. To date, little is known about the expression of COX-2 in nonepithelial tumors. The objective of this study was to evaluate the expression of COX-2 in GI stromal tumors (GISTs). We evaluated 15 GISTs using tissue microarray. Tissue blocks were retrieved and stained with hematoxylin and eosin to evaluate the histological tumor type. In addition, immunohistochemistry was performed for COX-2, the macrophage marker, CD68 (KP-1), and KIT (CD117). Two pathologists then evaluated the tissues to determine the extent and intensity of COX-2 expression. The location of CD68-positive cells, and whether these cells were COX-2 positive, was also evaluated. The results showed that 80% (12 of 15) of the tumors expressed COX-2. Expression was noted in the cytoplasm of the tumor cells, with variable intensity of staining among the tumors. COX-2 was expressed in both epithelial cell and spindle cell tumors, but appeared stronger in epithelial lesions. In mixed lesions, COX-2 was expressed to a greater extent in epithelial areas. There was a greater extent of COX-2 expression in malignant tumors and tumors located within the stomach. Tumor-infiltrating macrophages (CD68-positive cells) were identified in all of the lesions; in 80% of cases, those macrophages also expressed COX-2. This study is the first to demonstrate COX-2 expression in stromal lesions of the GI tract. The enzyme may play a role in the proliferation of these lesions, suggesting the potential use of nonsteroidal anti-inflammatory drugs in treatment. HUM PATHOL 34:1242-1246. © 2003 Elsevier Inc. All rights reserved.