Epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2) are prognostic biomarkers for patients with resected colorectal cancer liver metastases.

BACKGROUND: Resection of colorectal cancer liver metastasis (CRCLM) with curative intent has long-term benefit in ~40% of cases. Prognostic biomarkers are needed to improve clinical management and reduce futile surgeries. Expression of epidermal growth factor receptor (EGFR) and prostaglandin-endoperoxide synthase 2 (PTGS2; also known as cyclooxygenase-2) has been associated with carcinogenesis and survival. We investigated the prognostic value of EGFR and PTGS2 expression in patients with resected CRCLM. METHODS: Formalin-fixed paraffin-embedded CRCLM tissue and corresponding primary tumour specimens from a multi-institutional cohort of patients who underwent liver resection between 1990 and 2010 were incorporated into tissue microarrays (TMAs). TMAs were stained for EGFR and PTGS2 by immunohistochemistry. The hazard rate ratio (HRR) for the association between expression in CRCLM and overall survival was calculated using a 500-fold cross-validation procedure. RESULTS: EGFR and PTGS2 expression could be evaluated in 323 and 351 patients, respectively. EGFR expression in CRCLM was associated with poor prognosis (HRR 1.54; P<0.01) with a cross-validated HRR of 1.47 (P=0.03). PTGS2 expression was also associated with poor prognosis (HRR 1.60; P<0.01) with a cross-validated HRR of 1.63 (P<0.01). Expression of EGFR and PTGS2 remained prognostic after multivariate analysis with standard clinicopathological variables (cross-validated HRR 1.51; P=0.02 and cross-validated HRR 1.59; P=0.01, respectively). Stratification for the commonly applied systemic therapy regimens demonstrated prognostic value for EGFR and PTGS2 only in the subgroup of patients who were not treated with systemic therapy (HRR 1.78; P<0.01 and HRR 1.64; P=0.04, respectively), with worst prognosis when both EGFR and PTGS2 were highly expressed (HRR 3.08; P<0.01). Expression of PTGS2 in CRCLM was correlated to expression in patient-matched primary tumours (P=0.02, 69.2% concordance). CONCLUSIONS: EGFR and PTGS2 expressions are prognostic molecular biomarkers with added value to standard clinicopathological variables for patients with resectable CRCLM.

Aurora kinase A (AURKA) expression in colorectal cancer liver metastasis is associated with poor prognosis.

BACKGROUND: Five-year survival after resection of colorectal cancer liver metastasis (CRLCM) is <30%. We recently found that aurora kinase A (AURKA) drives 20q gain-associated tumour progression and is associated with disease recurrence. This study evaluates the prognostic value of AURKA expression in CRCLM of patients who underwent liver resection. METHODS: Tissue microarrays (TMAs) were generated using formalin-fixed paraffin-embedded CRCLM and matched primary tumour from a multi-institutional cohort of patients with CRCLM who underwent liver resection between 1990 and 2010. Tissue microarrays were stained for AURKA using immunohistochemistry, and a hazard rate ratio (HRR) for the association between overall survival (OS) and nuclear AURKA expression in CRCLM was calculated. Results were validated by 500-fold cross-validation. RESULTS: The expression of AURKA was evaluated in CRCLM of 343 patients. High AURKA expression was associated with poor OS (HRR 1.55, P<0.01), with a cross-validated average HRR of 1.57 (P=0.02). Average HRR was adjusted for the established prognostic clinicopathological variables in a multivariate analysis (average HRR 1.66; P=0.02). The expression of AURKA in CRCLM was correlated to its expression in corresponding primary tumour (P<0.01). CONCLUSION: The expression of AURKA protein is a molecular biomarker with prognostic value for patients with CRCLM, independent of established clinicopathological variables.

A predictive role for noncancerous prostate cells: low connexin-26 expression in radical prostatectomy tissues predicts metastasis.

BACKGROUND: It is important to identify markers that predict whether prostate cancer will metastasise. The adjacent noncancerous cells (influenced by the tumour cells) may also express potential markers. The objective of this study was to determine the influence of cancer cells on noncancerous cells and to assess the value of the cell-communication protein connexin-26 (Cx26) as a marker to predict the development of metastasis. METHODS: The effect of conditioned medium (CM) from PrCa cells on in vitro noncancerous cell proliferation, migration and invasion and Cx26 expression was determined. Connexin-26 expression was investigated in prostatectomy tissues from 51 PrCa patients by immunohistochemistry and compared with various clinicopathological parameters. RESULTS: Proliferation, migration and invasion of noncancerous cells were influenced by CM from the PrCa cell lines. Importantly, a clear relation was found between low Cx26 expression in the noncancerous tissue in prostatectomy sections and the risk of development of metastasis (P<0.0002). Kaplan-Meier analysis showed a relation between low Cx26 expression in noncancerous tissues and time to biochemical recurrence (P=0.0002). CONCLUSION: Measuring Cx26 expression in the adjacent noncancerous tissues (rather than cancer tissues) of prostatectomy sections could help to identify high-risk patients who may benefit from adjuvant therapy to decrease the risk of metastasis.

New experimental markers for early detection of high-risk prostate cancer: role of cell-cell adhesion and cell migration.

Today's treatment and diagnosis of prostate cancer still exhibit major limitations. The search for new and additional prognostic markers is therefore still an actual field of interest. Potential markers involved in numerous biological processes in the tumor cell have been investigated intensively. For therapeutic interventions it is important to distinguish between harmless and aggressive disease in an early stage. Therefore the subject of this review is limited to markers associated with those functional processes, which discriminate early stage aggressive, metastatic cancer from harmless disease. Important processes in this respect are: altered cell adhesion and cellular migration. E-cadherin, N-cadherin, beta-catenin, integrins, focal adhesion kinase, connexins and matrix metalloproteinases all appear promising biological markers associated with the early stage metastatic process in prostate cancer. Here we discuss their potential to become valid biological markers based on literature data. Thus far, none of these markers proved to be a valid individual marker by itself due to prostate cancer heterogeneity and transient expression. Analyzing a combination of the potential markers discussed in this review is expected to be a better approach toward discriminating high- from low-risk tumors in an early stage of prostate cancer.

Evaluation of adenoviral oncolytic effect on glioma spheroids by 18F-DG positron-emission tomography.

Multicellular tumor spheroids are used as a model to assess the efficacy of replicating oncolytic adenoviruses. As most assays used to assess cellular viability are unsuitable for oncolytic viruses because of ongoing viral replication, we have used positron emission tomography (PET) to sequentially determine the incorporation of 18F-labeled deoxyglucose (18F-DG) as a measure of viability and compared the results to more commonly used assays for measuring the effect of oncolytic therapy. Glioma monolayer cultures and spheroids were infected with wild-type replicating adenovirus and viability was measured by 18F-DG incorporation, WST-1 assay, crystal violet assay, and spheroid volume 2 to 10 days following infection. Results show that volume measurements in adenovirus-infected spheroids are confounded by the cytopathic effect occurring in infected cells. 18F-DG PET provides a useful method to assess small differences in cell number and viability following oncolytic viral therapy in glioma monolayer cultures and spheroids without the need for disintegration of these cultures. Moreover, using 18F-DG PET, repeated sequential measurements of spheroid viability can be made, decreasing the required number of spheroids per experiment. This is a valuable feature when using spheroids derived from limited amounts of patient material.

The metastatic potential of rat prostate tumor variant R3327-MatLyLu is correlated with an increased activity of N-acetylglucosaminyl transferase III and V.

Enzyme activities of N-acetylglucosaminyltransferase (GlcNAc-Tase) I-V involved in N-linked complex-type carbohydrate synthesis were determined in a non-metastatic hormone-dependent rat prostate tumor (R3327-H) and a related, hormone-independent variant metastasizing to lymph nodes and lungs (R3327-MatLyLu). In the metastasizing variant a significantly increased activity of both GlcNAc-Tase III and GlcNAc-Tase V was observed, whereas the activities of GlcNAc-Tase I and II were essentially unchanged. The increase in activity of GlcNAc-Tase III is particularly noteworthy since it indicates that elevated expression of this enzyme cannot be considered as an exclusive marker of hepatic malignancy.

Radiotoxicity of 111indium.

Bone marrow cells were labelled with various concentrations of 111Indium-oxine and their capacity to form colonies (CFU-s) in an adoptive transfer was investigated. Labelling with more than 0.1 muCi/ml/107 cells impaired colony formation. It is concluded that the 111Indium-oxine complex is detrimental to cell proliferation.

Radiation sensitivity of Copenhagen rat prostatic carcinoma (R3327-AT and R3327-MATLyLu).

The radiosensitivity of two variants of the Dunning Copenhagen rat prostatic tumor R3327 was investigated. The R3327-AT variant, which is a poorly differentiated anaplastic, fast-growing tumor, was irradiated both in vivo and in vitro. Following irradiation, monodispersed cells were plated in vitro and colonies were counted after 7 days. The survival curve of R3327-AT cells irradiated in vivo showed an initial shoulder (Dq-value 0.97 Gy), followed by two exponential parts. The D0-value for the first part of the curve (0-10 Gy) was 2.76 Gy and for the second part of the curve (greater than 10 gy) 9.05 Gy. Extrapolation of the second part of the curve to the Y-axis indicated that the proportion of more radioresistant cells was about 10%. The survival curve for R3327-AT cells irradiated in vitro also suggested the presence of a radioresistant subpopulation, although the proportion was lower (about 3%). This difference might be due to the presence of an hypoxic fraction in the tumors irradiated in vivo, but not in vitro. Tumor cells from the R3327 tumor variant metastatic to lymph nodes and lungs (R3327-MATLyLu), were irradiated in vitro. The radiation effect was evaluated by in vitro colony formation in agar and by in vivo lung colony assay. The colony formation in agar yielded a D0-value of 1.09 Gy. No radioresistant subpopulation was identified in this variant. A similar radiosensitivity was observed by the in vivo lung colony assay (D0 1.39 Gy). The mean inactivation dose calculated for R3327-AT cells (3.45 Gy) was significantly higher than for the metastatic variant (2.00 Gy).

Combination 186Re-HEDP and cisplatin supra-additive treatment effects in prostate cancer cells.

UNLABELLED: Radionuclide therapy has proven to be an efficacious palliative treatment for metastatic prostate cancer. Its potential therapeutic possibilities may be substantially increased by combining it with effective radiosensitizing drugs. METHODS: This study explores the radiosensitizing properties of cisplatin when combined with 186Re-labeled hydroxyethylidene diphosphonate (HEDP) in the treatment of R3327-MATLyLu prostate cancer cells in vitro. A concomitant incubation during 4 d, combining various concentrations of cisplatin (0, 0.42, 0.83 and 1.67 micromol/L) and 186Re-HEDP (0, 1.84 and 3.69 MBq/mL [0, 50 and 100 microCi/mL, respectively]) was followed by the determination of the cell numbers surviving and the replating of these cells in semisolid agar. RESULTS: The surviving fraction of clonogenic tumor cells after combination treatment clearly showed synergism when analyzed by a panel of three different published analytical methods. In addition, analysis of variance demonstrated a significant interaction between radionuclide therapy and cisplatin-based chemotherapy (P < 0.001). Treatment with 186Re-HEDP and cisplatin by sequential incubation yielded similar, but never superior results. CONCLUSION: It is concluded that radionuclide therapy in combination with cisplatin is able, in principle, to improve therapeutic success rate in metastatic prostate cancer in a more than additive way.

Radiosensitizing effect of cisplatin in prostate cancer cell lines.

The radiosensitizing effect of platinum compounds has been demonstrated in a number of tumors. In prostate cancer, clinical and preclinical data concerning an eventual efficacy of the concept of radiosensitization are lacking. In the present study cisplatin and carboplatin have been used as a model to explore radiosensitization in in vitro prostate cancer cell lines. Human (DU-145) and rat (R3327-MATLyLu) prostate tumor cells were irradiated with doses ranging from 0 to 8 Gy in the presence of various concentrations of either cisplatin or carboplatin. For the evaluation of the combined effect of the two treatment modalities, a simple model is presented. Supra-additive treatment effects of combinations of platinum drugs with radiotherapy, both at clinically achievable doses, were shown on the basis of surviving fractions of tumor cells and proved to be significant. These data strongly suggest that radiotherapy may be effectively combined with radiosensitizers such as platinum drugs in prostate cancer therapy, to yield synergism in treatment efficacy.

Morphology, kinetics and secretory activity of antibody-forming cells.

Specific antibody-forming cells from spleen, bone marrow and popliteal lymph nodes were studied in mice after subcutaneous priming and intravenous boosting with horseradish peroxidase (HRP). Functional antibody-secreting capacity of these cells was correlated with their morphology at the cell population level. For this purpose, cells synthesizing anti-HRP antibody from the same cell suspensions were studied simultaneously by light and electron microscopy and by a plaque assay. It appeared that the population of cells responsible for antibody synthesis as well as antibody secretion was morphologically heterogeneous: besides plasma cells, considerable numbers of antibody-forming lymphocytes, antibody-forming plasmablasts and antibody-forming immature plasma cells were observed. Immature plasma cells constituted the majority of antibody-forming as well as antibody-secreting cells. Among the immature plasma cells in the popliteal lymph nodes proliferation occurred. Evidence is presented that the light-microscopically identified mature plasma cell is not the main antibody-forming cell. It does not show 3H-Thymidine incorporation and should be considered as a non-dividing end-cell.

Nerve-growth-factor-dependent neurite outgrowth assay; a research model for chemotherapy-induced neuropathy.

The rat clonal pheochromocytoma cell line PC12 can be induced to display neurite outgrowth upon induction with nerve growth factor (NGF). This NGF-dependent neurite outgrowth assay looks a promising model for research on toxic neuropathies. Using this assay we demonstrated that cisplatin caused a dose-dependent reduction of NGF-dependent neurite formation. Increasing doses of NGF, however, proved to exert protective activity against this cisplatin effect at an intermediate and clinically relevant cisplatin concentration of 1 micrograms/ml. Even at a high cisplatin concentration (10 micrograms/ml), the protective action of NGF, although less adequate, was observed. The value and strength of this model for screening neuropathogenic effects of anticancer agents at the cellular level and the possibly therapeutic action of neurotrophins are discussed and demonstrated. Furthermore, in the light of the urgent need for adequate models for neuropathy research, the PC12 neurite outgrowth protection assay may contribute to our knowledge of the mechanisms underlying the development of neuropathy.

Consideration of the use of 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5- alpha-androstan-3-one (4MA), a 5 alpha-reductase inhibitor, in prostate cancer therapy.

We investigated the effect of 17 beta-N,N-diethylcarbamoyl-4-methyl-4aza- 5 alpha-androstan-3-one (4MA), a 5 alpha-reductase inhibitor, on growth inhibition of androgen-sensitive rat prostatic tumour (R3327-H) and correlated it with changes in weight of normal androgen target tissues and with levels of androgens. Groups of male Copenhagen rats were treated for 28 days with a daily injection of various, increasing doses of 4MA (0.01-4.0 mg/day) and the results were compared with control (vehicle-treated) and with castrated animals. 4MA decreased tumour growth rate in a dose-dependent manner, which was reflected in a decreased incorporation of BrdUrd in DNA of glandular epithelial cells in the tumour. Normal prostate wet weight was also decreased after high-dose 4MA treatment while serum testosterone levels were not affected by 4MA treatment. Contrary to expectations, however, tissue levels of dihydrotestosterone in tumour and ventral prostate were still considerable in 4MA-treated animals. The tumour-inhibiting action of 4MA, therefore, has to be interpreted as not being purely due to 5 alpha-reductase inhibition. On the other hand, it was not possible to demonstrate any direct tumoricidal effect of 4MA in vitro. The relevance of these findings in terms of the endocrine mechanism of action of 4MA on tumour growth is discussed.

Nerve growth factor stimulates in vitro invasive capacity of DU145 human prostatic cancer cells.

The prevalence of nerve growth factor (NGF) production in different human prostatic tumor cell lines (DU145, PC-3, LNCaP-FGC) was investigated using a specific enzyme-linked immunosorbent assay (ELISA) and compared to that of different human and rat prostatic tissue samples. In addition, the biological effects of NGF beta addition to the human prostatic cancer cell cultures were investigated. The ELISA technique showed the DU145 cell line to secrete measurable levels of NGF in the culture medium. When neurite-outgrowth determination in a pheochromocytoma cell line was used as a bioassay, the NGF synthesized by DU145 cells was confirmed to exhibit functional biological activity. No effect of exogenously added NGF could be established on tumor cell proliferation, on the basis of either colorimetric tetrazolium-based staining assay or bromodeoxyuridine incorporation. Also the expression of prostate specific acid phosphatase was not influenced by NGF addition. However, the in vitro invasive capacity (Matrigel) of DU145 cells was significantly increased by inclusion of 50 ng or 100 ng NGF beta/ml culture medium. In view of the clinically well-known perineural invasion of prostate cancer cells, the possible involvement of NGF as a (paracrine) factor in prostatic cancer metastatic behavior should be investigated further.

Radionuclide therapy for prostate cancer lumbar metastasis prolongs symptom-free survival in a rat model.

OBJECTIVES: The present study was initiated to explore the effects of hydroxyethylidene diphosphonate labeled with rhenium 186(186Re-HEDP) treatment on the progression of lumbar skeletal metastasis in an animal model and to correlate the eventual treatment efficacy with the radioisotope tissue distribution. METHODS: The effect of 186Re-HEDP on the progression of lumbar metastasis from prostate cancer was investigated in the Copenhagen rat model. Metastatic prostate tumor deposits were induced in male rats by tail vein injection of R3327-MATLyLu prostate tumor cells under concomitant clamping of the inferior caval vein. The development of clinical symptoms such as onset of hind leg paralysis and urinary bladder swelling was monitored and related to the presence of tumor cells within histologic sections of L-5 and L-6 vertebrae. RESULTS: The 186Re-HEDP administration, given either 1 day or 8 days after surgical induction of lumbar metastasis, could significantly increase the symptom-free survival of the animals. These results were confirmed by a significant decrease in the presence of histologically detectable tumor tissue. Biodistribution studies demonstrated the uptake of the major part of the radioisotope within bone tissue. Uptake of radioactivity within the lumbar vertebrae on a microscopic scale, as shown by phosphor screen autoradiography, was concentrated in areas of bone formation and turnover. Signs of radiotoxicity, such as bone marrow replacement by fat cells and the absence of megakaryocytes, were observed. CONCLUSIONS: The results show that radionuclide treatment using 186Re-HEDP is a potentially efficacious treatment option in prostate cancer disseminated to the skeleton. The optimal treatment dose should be determined carefully and aimed at acceptable levels of myelotoxicity.

Cytotoxicity and neurocytotoxicity of new marine anticancer agents evaluated using in vitro assays.

PURPOSE: New classes of anticancer drugs, isolated from marine organisms, have been shown to possess cytotoxic activity against multiple tumor types. Aplidine, didemnin B, and isohomohalichondrin B (IHB), among the more promising antitumor candidates, have been evaluated in the present study on a comparative basis in terms of their antiproliferative activity and neurotoxic effects in vitro. METHODS: Using a panel of different human, prostatic cancer cell lines (DU 145, PC-3 and LNCaP-FGC) the effects of Aplidine, didemnin B, and IHB on tumor cell proliferation were tested in a colorimetric (XTT) assay and compared with the effects of vincristine, vinorelbine, and Taxol. Under analogous in vitro conditions these drugs were also monitored for neurocytotoxic effects using a PC 12 cell line based model. RESULTS: Didemnin B and - especially - Aplidine were more effective in the inhibition of prostate cancer cell proliferation than vincristine, vinorelbine or Taxol at concentration levels between 5 and 50 pmol/ml. At these same concentrations, however, Didemnin B and Aplidine were also most potent in the in vitro neurotoxicity assays. IHB was found to exert even more potent antiproliferative activity (at concentration levels between 0.05 and 0.1 pmol/ml). However, neurotoxic effects were also found to be present at these levels. After drug withdrawal, the neurotoxic damage, inflicted by aplidine or IHB appeared to be more long lasting than after vincristine or vinorelbine exposure. CONCLUSIONS: These results point to high antiproliferative activity of aplidine and IHB in prostate cancer. At the same time, the data urge some caution in the clinical use of these agents because of potential neurotoxic side-effects. The use of a newly formulated Aplidine may involve a more favorable therapeutic profile.

Clonally related but phenotypically divergent human cancer cell lines derived from a single follicular thyroid cancer recurrence (TT2609).

Starting from different regional samples taken from a heterogeneous follicular thyroid cancer recurrence in a male patient, a series of cell cultures was initiated. Three stable cancer cell lines were successfully established (TT2609-A02, TT2609-B02, and TT2609-C02) and kept in continuous culture for more than 3 years. The lines are each characterized by a unique set of biological parameters such as morphology, ploidy state, cell proliferation rate, ultrastructure, thyroid marker expression, p53 expression, karyogram, agar clonogenic capacity and tumorigenicity as xenografts in nude mice. These characterization studies point to a marked heterogeneity at the level of the clinical tumor recurrence. Karyotype analysis of the cell lines showed a pattern of aberrations indicating that the lines are clonally related and that the A02 and C02 lines are subsequently derived from the more "original" tumor cell type B02 after a tetraploidization event. It is concluded that the obtained cell lines represent an in vitro/in vivo model for human follicular thyroid cancer. The availability of a series of cell lines for human follicular thyroid cancer, mimicking the biological heterogeneity observed in patient tumors, enables both detailed fundamental investigation of thyroid cancer cell biology and the experimental exploration of new treatment approaches.

Models for cancer skeletal metastasis: a reappraisal of Batson's plexus.

While skeletal metastasis in prostate cancer is a major and frequent complication, literature data on the mechanisms involved are quite confusing. Recent efforts, however, to establish appropriate animal models for skeletal metastasis have finally yielded positive results which may provide clarity in this discussion. Models involving both human prostate cancer cell transplantation in nude mice as well as syngeneic rat models have contributed to the accumulated evidence in favor of the hypothesis of Batson. According to this hypothesis, prostate tumor cells reach the vertebral venous plexus of the spine especially under transient conditions of increased intraabdominal pressure and lead to metastatic tumor deposits in the vertebrae. Animal models displaying skeletal metastasis in conjunction with analysis of pathological findings have been instrumental in validating this concept. It is to be expected that such animal models will contribute to the development and optimization of new treatment approaches for prostate cancer bone metastasis.

Factors in prostate cancer metastasis.

Death related to prostate cancer is invariably due to the tumor metastasis (to lungs, skeleton and lymph nodes). Tumor cell metastatic behaviour is still poorly understood. The R3327 prostate tumor model in the male Copenhagen rat offers an opportunity to investigate the different aspects of metastatic processes in vivo and to evaluate effects of current and new treatment approaches. Lymphatic spread of cancer cells can be measured by determining volume of local load in successive draining lymph node stations. Surgical removal of primary tumor and inguinal lymph node shows that lymphatic metastasis in the R3327-MATLyLu tumor variant is a continuous progressive phenomenon, which starts already in early stages of tumor growth after subcutaneous transplantation. Spread to the lungs can be quantified by enumeration of macroscopically visible pleural lung colonies. Metastatic spread to the lungs can be mimicked by intravenous injection of monodispersed tumor cell suspension. Within 10 days pleural tumor colonies become visible. Effects of different agents and treatments like chemotherapeutic drugs, heparin, surgery and high energy shock wave (HESW) treatment have been described using these methods. Recently, metastasis to the vertebral column was induced by temporal occlusion of venous blood flow through the caval veins while injecting tumor cells in the lateral tail vein. The resulting osteoblastic metastatic lesions in the lumbar vertebrae and the concomitant spinal cord compression led in time to the loss of motoric and sensory function of the hind legs. These observations permit investigation of the mechanisms of bone metastasis based on biological functions, i.e. sensory and motoric function of the hind legs. Finally, it can be said that the various variants of the R3327 rat prostate tumor offer an appropriate model to study various aspects of prostate cancer and metastasis.

Doxorubicin treatment increases metastasis of prostate tumor (R3327-MatLyLu).

Doxorubicin (Dx) effects on metastatic spreading of prostate cancer were investigated using a rat metastatic tumor (R3327-MatLyLu). Intravenous injection of monodispersed tumor cells resulted in tumor nodule formation in the lungs. A simultaneous bolus injection of Dx (3.2 mg/Kg) caused a significant decrease in lung colony formation and growth. However, Dx administered during the course of spontaneous metastasis, or as adjuvant chemotherapy 24 hours before surgical removal of primary tumor, caused a significant increase in metastasis to the lungs. It is concluded that an intravenous doxorubicin bolus injection does not provide an antimetastatic effect, but increases metastatic spreading of prostate tumor.