Lactate dehydrogenase 5 isoenzyme overexpression defines resistance of prostate cancer to radiotherapy.

BACKGROUND: Radiotherapy provides high-cure rates in prostate cancer. Despite its overall slow clinical growth, high proliferation rates documented in a subset of tumours relate to poor radiotherapy outcome. This study examines the role of anaerobic metabolism in prostate cancer growth and resistance to radiotherapy. METHODS: Biopsy samples from 83 patients with prostate cancer undergoing radical hypofractionated and accelerated radiotherapy were analysed for MIB1 proliferation index and for lactate dehydrogenase isoenzyme LDH5, a marker of tumour anaerobic metabolism. Ninety-five surgical samples were in parallel analysed. Correlation with histopathological variables, PSA and radiotherapy outcome was assessed. Dose-response experiments were performed in PC3 and DU145 cancer cell lines. RESULTS: High MIB1 index (noted in 25% of cases) was directly related to Gleason score (P<0.0001), T3-stage (P=0.0008) and PSA levels (P=0.03). High LDH5 (noted in 65% of cases) was directly related to MIB1 index (P<0.0001), Gleason score (P=0.02) and T3-stage (P=0.001). High Gleason score, MIB1, LDH5 and PSA levels were significantly related to poor BRFS (P=0.007, 0.01, 0.03 and 0.01, respectively). High Gleason score (P=0.04), LDH5 (P=0.01) and PSA levels (P=0.003) were significantly related to local recurrence. MIB1 and T-stage did not affect local control. Silencing of LDHA gene in both prostate cancer cell lines resulted in significant radiosensitisation. CONCLUSIONS: LDH5 overexpression is significantly linked to highly proliferating prostate carcinomas and with biochemical failure and local relapse following radiotherapy. Hypoxia and LDHA targeting agents may prove useful to overcome radioresistance in a subgroup of prostate carcinomas with anaerobic metabolic predilection.

Prospective neoadjuvant analysis of PET imaging and mechanisms of resistance to Trastuzumab shows role of HIF1 and autophagy.

BACKGROUND: Although Trastuzumab has improved survival of HER2+ breast cancer patients, resistance to the agent pre-exists or develops through the course of therapy. Here we show that a specific metabolism and autophagy-related cancer cell phenotype relates to resistance of HER2+ breast cancer to Trastuzumab and chemotherapy. METHODS: Twenty-eight patients with locally advanced primary breast cancer were prospectively scheduled to received one cycle of Trastuzumab followed by a new biopsy on day 21, followed by taxol/Trastuzumab chemotherapy for four cycles before surgery. FDG PET/CT scan was used to monitor tumour response. Tissue samples were immunohistochemically analysed for metabolism and autophagy markers. RESULTS: In pre-Trastuzumab biopsies, the LC3A+/HER2+ cell population was correlated with HIF1α expression (P=0.01), while GLUT1 and LC3B expression were correlated with Ki67 proliferation index (P=0.01 and P=0.01, respectively). FDG PET tumour dimensions before therapy were correlated with LC3B expression (P=0.005). Administration of Trastuzumab significantly reduced clinical and PET-detected tumour dimensions (P<0.01). An inverse association of tumour response with the percentage of cells expressing HIF1α at baseline was documented (P=0.01). Administration of Trastuzumab resulted in a decrease of the proliferation index (P=0.004), GLUT1 (P=0.04) and HER2 (P=0.01) expression. In contrast, the percentage of LC3A+/HER2+ cells was increased (P=0.01). High baseline HIF1α expression was the only parameter associated with poorer pathological response to preoperative chemotherapy (P=0.001). CONCLUSIONS: As the HER2+/LC3A+ phenotype, which often overexpresses HIF1α, is a major subpopulation increasing after therapy with Trastuzumab, LC3A- and HIF1α-targeting therapies should be investigated for the augmentation of anti-HER2 therapy efficacy.

Autophagy and hypoxia in colonic adenomas related to aggressive features.

AIM: The study investigated whether autophagic activity and hypoxia parallel the adenoma-carcinoma sequence. METHOD: The study comprised 120 tubular adenomas with high-grade dysplasia, including 22 with questionable evidence of invasion, 37 with definite stromal invasion and 29 with severely dysplastic adenoma, 10 traditional serrated adenomas and 22 classical tubular adenomas lacking aggressive features. The samples were stained immunohistochemically for autophagy (LC3A and Beclin-1) and hypoxia-inducible factor1-alpha (HIF1α) markers. RESULTS: LC3A was detected as diffuse cytoplasmic staining and as dense "stone-like" structures (SLS) within cytoplasmic vacuoles. Beclin-1 reactivity was purely cytoplasmic, whereas that of HIF1α was both cytoplasmic and nuclear. SLS counts in noninvasive, nontransformed areas of tubular adenomas were consistently low (median SLS = 0.5; 200× magnification), whereas a progressive increase was noted from areas of equivocal invasion (median SLS = 1.3; 200× magnification) and intramucosal carcinoma (median SLS = 1.4; 200× magnification) to unequivocal invasive foci (median SLS = 2.1; 200× magnification) (P < 0.0001). A similar association was shown for Beclin-1 and HIF1α expression (P < 0.05). Traditional serrated adenomas yielded low SLS counts and weak HIF1α reactivity, but high cytoplasmic LC3A and Beclin-1 expression (P < 0.01). CONCLUSION: A hypoxia-driven autophagy in adenomatous polyps, when particularly intense and localized, is commonly associated with early invasion or severely dysplastic adenoma.

Cancer stem cell phenotype relates to radio-chemotherapy outcome in locally advanced squamous cell head-neck cancer.

BACKGROUND: Cancer stem cells (CSCs) tend to repopulate malignant tumours during radiotherapy and, therefore, prolongation of the overall treatment time may result in radiotherapy failure. Thus, an estimate of the number of CSCs in tumour biopsies may prove most useful in predicting resistance to radiotherapy and a guide for development therapies aimed to eradicate a cancer cell population with effects on radiotherapy-related cancer regrowth. METHODS: The CSC population was investigated semi-quantitatively in 74 locally advanced squamous cell head-neck cancers (HNSCC) from an equal number of patients, treated with accelerated platinum-based radiotherapy. A standard immunohistochemical technique and the CSC markers CD44, CD24, Oct4, integrin-ß1 and aldehyde dehydrogenase isoform 1A1 (ALDHA1) was used, in parallel with the proliferation marker MIB-1. The results were correlated with the site of the tumour, the MIB-1 index, the tumour grade and stage, and prognosis. RESULTS: The expression of CD44, CD24 and Oct4 were significantly associated with the MIB-1 proliferation index. In addition, the CD44 was linked with the better differentiated HNSCC. The CD44, Oct4 and integrin-ß1 were all associated with poor prognosis but, in a multivariate analysis, the integrin-ß1 had an independent statistical significance in terms of local relapse, distant metastases and overall survival. Interestingly, ALDH1 was associated with favourable prognosis. CONCLUSION: CSC markers are linked with poor radiotherapy outcome in HNSCC, with integrin-ß1 being the strongest and independent prognostic factor. Targeting CSC molecules with monoclonal antibodies or pharmaceutical agents may prove important for the treatment of HNSCC.

Vascular density analysis in colorectal cancer patients treated with vatalanib (PTK787/ZK222584) in the randomised CONFIRM trials.

BACKGROUND: Pharmacological inhibitors of vascular endothelial growth factor (VEGF) receptors, like vatalanib, have been tested in randomised trials (CONFIRM (Colorectal Oral Novel therapy For the Inhibition of Angiogenesis and Retarding of Metastases) 1 and 2) in colorectal cancer showing activity in a subgroup of patients with high serum LDH expression. In the current study, we assessed the predictive role of vascular density (VD) in patients treated in the above trials. METHODS: Paraffin-embedded materials from 141 patients were analysed with immunohistochemistry for the expression of the CD31 (pan-endothelial cell marker) and of phosphorylated pVEGFR2/KDR on endothelial cells. The VD was correlated with response to therapy and with progression-free (PFS) and overall survival (OS). RESULTS: A significant association of pVEGFR2/KDR+ VD with poor response in the placebo group was noted (response rates (RRs) 15% (3/20) when high VD vs 52% (26/50) when low VD; P=0.006). The RR increased from 15 (3/20) to 50% (11/22) in tumours with high VD when vatalanib was added to chemotherapy (P=0.02). A significantly improved PFS was noted in patients with high pVEGFR2/KDR+ VD when treated with vatalanib (P=0.002). A similar effect was also noted in patients with high CD31+ VD (P=0.07). Overall survival was marginally improved (P=0.07). CONCLUSION: Assessment of the activated vessel density may allow the stratification of patients recruited in randomised trials with VEGFR-targeting anti-angiogenic agents, unmasking their therapeutic potential and enabling their introduction in the clinical practice for the benefit of specific patient subgroups, at the same time reducing the cost of therapy.

Targeting the epigenome in malignant melanoma: Facts, challenges and therapeutic promises.

Malignant melanoma is the most lethal type of skin cancer with high rates of mortality. Although current treatment options provide a short-clinical benefit, acquired-drug resistance highlights the low 5-year survival rate among patients with advanced stage of the disease. In parallel, the involvement of an aberrant epigenetic landscape, (e.g., alterations in DNA methylation patterns, histone modifications marks and expression of non-coding RNAs), in addition to the genetic background, has been also associated with the onset and progression of melanoma. In this review article, we report on current therapeutic options in melanoma treatment with a focus on distinct epigenetic alterations and how their reversal, by specific drug compounds, can restore a normal phenotype. In particular, we concentrate on how single and/or combinatorial therapeutic approaches have utilized epigenetic drug compounds in being effective against malignant melanoma. Finally, the role of deregulated epigenetic mechanisms in promoting drug resistance to targeted therapies and immune checkpoint inhibitors is presented leading to the development of newly synthesized and/or improved drug compounds capable of targeting the epigenome of malignant melanoma.

Beclin 1 over- and underexpression in colorectal cancer: distinct patterns relate to prognosis and tumour hypoxia.

INTRODUCTION: Autophagy enables cells to recycle long-lived proteins or damaged organelles. Beclin 1, the mammalian orthologue of the yeast Apg6/Vps30 gene, functions as a scaffold for the formation of autophagosomes. MATERIALS AND METHOD: The immunohistochemical patterns of Beclin 1 expression and their prognostic relevance were studied in formalin-fixed tissues from 155 patients with colorectal adenocarcinoma treated with surgery alone. RESULTS: Using the weak homogeneous expression of Beclin 1 in normal colonic tissues as a basis for assessing tumours, the following grouping/staining patterns were recognised in colorectal carcinomas: a normal-like pattern in 62 of 155 (40%) cases, an underexpression pattern in 24 of 155 (15.5%) cases, extensive overexpression of Beclin 1 in 33 of 155 (21.3%) tumours and limited overexpression of the protein in 36 of 155 (23.2%) tumours. Extensive overexpression of Beclin 1 was significantly linked with overexpression of HIF1α and LDH5, as well as with high histological grade, vascular invasion and nodal involvement. Furthermore, patients with extensive over- or underexpression of Beclin 1 had a significantly poorer overall survival compared with the other two groups (P<0.0001). Beclin 1 had an independent prognostic relevance in multivariate analysis. CONCLUSIONS: Beclin 1 has an important role in growth and metastasis of colorectal cancer. Loss of Beclin 1 expression (allelic loss or microRNA regulatory activity, as suggested in the literature) defines poor prognosis presumably by promoting anti-apoptotic pathways, while overexpression of the protein, being linked with tumour hypoxia and acidity, also defines subgroups of tumours with aggressive clinical behaviour.

Angiogenesis and angiogenic factor expression in thyroid cancer.

PURPOSE: Increased expression of angiogenic factors and high vascular density characterize tumors with increased invasive and metastatic capability. Anti-vascular endothelial growth factor (VEGF) therapies have shown an important potentiation of chemotherapy and radiotherapy in experimental and clinical studies. The purpose of this study was to investigate whether it could be possible to identify a subgroup of thyroid cancer patients with high angiogenic activity. METHODS: Formalin-fixed paraffin-embedded tissues from 25 papillary and 18 follicular thyroid carcinomas were assessed immunohistochemically for angiogenic activity, i.e. vascular density (VD) and expression of VEGF and basic fibroblast growth factor (bFGF). RESULTS: VD was significantly higher in follicular tumors (p=0.05). Tumors > 4 cm had a significantly higher VD (p=0.001). High VEGF expression was significantly related to high VD (p=0.05). There was no association of bFGF with histological characteristics. CONCLUSIONS: Increased angiogenic activity is a common feature of thyroid carcinomas, particularly in follicular tumors and larger carcinomas. These results support the testing of anti-VEGF therapies in combination with radiotherapy and chemotherapy in advanced thyroid tumors.

Tumor microenvironment, immune response and post-radiotherapy tumor clearance.

Radiotherapy is the treatment of choice for many cancer patients. Residual tumor leads to local recurrence after a period of an equilibrium created between proliferating, quiescent and dying cancer cells. The tumor microenvironment is a main obstacle for the efficacy of radiotherapy, as impaired blood flow leads to hypoxia, acidity and reduced accessibility of radiosensitizers. Eradication of remnant disease is an intractable clinical quest. After more than a century of research, anti-tumor immunity has gained a dominant position in oncology research and therapy. Immune cells play a significant role in the eradication of tumors during and after the completion of radiotherapy. The tumor equilibrium reached in the irradiated tumor may shift towards cancer cell eradication if the immune response is appropriately modulated. In the modern immunotherapy era, clinical trials are urged to standardize immunotherapy schemes that could be safely applied to improve clearance of the post-radiotherapy remnant disease.

Characterization of the "Autophagic Flux" in Prostate Cancer Tissue Biopsies by LC3A/LAMP2a Immunofluorescence and Confocal Microscopy.

A method is described for the immunostaining of human prostate cancer biopsies for the autophagic marker LC3 and the lysosomal protein LAMP2A. Using this combination we can provide some information on autophagic flux, and specific patterns of staining have been recognized for normal prostate tissue and prostatic carcinomas.

Human papillomavirus in endometrial adenocarcinomas: infectious agent or a mere "passenger"?

AIMS: To investigate the possible association of human papillomavirus (HPV) with endometrial hyperplasias and neoplasia. Does HPV play any role in the initiation or prognosis of endometrial adenocarcinomas? METHODS: Twenty-five endometrial adenocarcinomas of the endometrioid cell type, with and without squamous differentiation, and twenty-four endometrial hyperplasias of various forms (simple, complex, and atypical) were analyzed for the presence of type 16 and 18 HPV by the polymerase chain reaction (PCR). The results were related to histopathological features of the tumour, and the patients' age, and prognosis. RESULTS: Six of 25 endometrial adenocarcinomas were HPV 16-positive (24%), and 5 of 25 (20%) were HPV 18-positive. Simple endometrial hyperplasias was associated somewhat more commonly with HPV 16 and 18 (2/8 and 1/8 cases, resp.) than hyperplasias progressing to endometrial adenocarcinomas, namely, atypical endometrial hyperplasia (1/8 and 0/8 cases, resp.). None of the positive cases in the series, whether hyperplastic or neoplastic, demonstrated cytological evidence of HPV infection. There was no relation between HPV-positive cases and squamous differentiation, depth of myometrial invasion, lymphatic involvement, lymphocytic response, patients' age, or prognosis. CONCLUSION: It appears that the presence of HPV in the endometrium, as detected by PCR, does not play any role in the initiation or prognosis of endometrial adenocarcinoma.

Squamous cell nose and a synchronous in-situ vocal cord carcinoma: a case report.

Reports indicate that the incidence of multiple primary tumours in head and neck cancers is high. However, most of these tumours are either metachronous primary or secondary tumours of the same histopathological type. The development of a synchronous primary squamous cell skin cancer of the nose and an in-situ vocal cord carcinoma is something unusual. We present the case of a patient with a primary neoplasm along the lateral side of the nose up to the bone of the pyramid, including the skin of the inner side of the nose and an infiltration of the inferior nasal concha on the right side, together with a small synchronous primary lesion of the left vocal cord. To the best of our knowledge the case described is the first in the English medical literature and we discuss the complete management of synchronous head and neck malignancies, emphasising the importance of panendoscopy in the prevention of pitfalls in diagnosis and the therapeutic procedure.

Hypoxia inducible factor (HIF-1a and HIF-2a) expression in early esophageal cancer and response to photodynamic therapy and radiotherapy.

Hypoxia inducible factor 1a and 2a (HIF-1a and HIF-2a) are key proteins regulating cellular response to hypoxia. Because the efficacy of photodynamic therapy (PDT) is dependent on the presence of oxygen, the assessment of HIF-1a and HIF-2a expression may be of value in predicting clinical response to PDT. Using recently produced MoAbs, we examined the expression of HIF1a and HIF2a in a series of 37 early-stage esophageal cancers treated with PDT and with additional radiotherapy in case of incomplete response after PDT. Strong expression of the HIF1a and of HIF2a proteins in all optical fields examined was noted in 51% and in 13% of cases, respectively. High expression was associated with a low complete response (CR) rate and with the absence of bcl-2 protein expression. On the contrary, bcl-2 expression was associated with a high CR rate. Combined analysis of HIF1a and bcl-2 protein expression revealed that of 16 cases with high HIF1a expression and the absence of bcl-2 reactivity, only 1 (7%) responded completely to PDT (P = 0.007). Bivariate analysis showed that HIF1a expression was independently related to response to PDT (P = 0.04; t ratio = 2.8), whereas bcl-2 approached significance (P = 0.07; t-ratio = 1.8). The final response to radiotherapy was high (70%) and independent of the HIF and bcl-2 status, which may be a result of reoxygenation after cellular depletion mediated by PDT. The present study suggests that assessment of HIF and of bcl-2 expression are important predictors of in vivo sensitivity to PDT. Modulation of PDT response with bioreductive drugs and/or drugs targeting bcl-2 (i.e., taxanes) may prove of significant therapeutic importance in a subgroup of patients with high HIF expression.

Expression of hypoxia-inducible carbonic anhydrase-9 relates to angiogenic pathways and independently to poor outcome in non-small cell lung cancer.

Carbonic anhydrase-9 (CA9), a transmembrane enzyme with an extracellular active site, is involved in the reversible metabolism of the carbon dioxide to carbonic acid. Up-regulation of CA by hypoxia and the hypoxia-inducible factor (HIF) pathway has been recently postulated (Wykoff et al. Cancer Res., 60: 7075-7083, 2000). In the present study we examined the expression of this enzyme in non-small cell lung cancer. Of 107 cases analyzed, 39 (36.4%) had strong membrane/cytoplasmic expression of CA9 and were grouped as positive. The staining was confined around areas of necrosis, and a significant association of CA9 expression with the extent of necrosis was noted (P = 0.004). Nevertheless, 38 of 74 cases with focal or extensive necrosis did not express CA9. CA9 expression was more frequent in the squamous cell histology (P = 0.001) and with advanced T stage (P = 0.009). A significant coexpression of CA9 with platelet-derived endothelial cell growth factor and basic fibroblast growth factor receptor expression was noted. Double staining of CA9 with anti-CD31 monoclonal antibody revealed an overall higher microvessel density in the areas expressing CA9 than in negative areas (P = 0.0005). Thirty-one of 38 CA9-positive cases were positive for HIF1a/HIF2a, but HIF positivity was a more common event (68 of 107) and their patterns of expression were diffuse (not confined in the necrotic areas). A direct association of CA9 expression with epidermal growth factor receptor, c-erbB-2, and MUC1 expression was also noted (P < 0.04). Survival analysis showed that CA9 expression is related to poor prognosis. CA9 expression in tumors with low vascularization defined a prognosis similar to the one of patients with highly angiogenic tumors. Multivariate analysis revealed that CA9 expression is a significant prognostic factor independent of angiogenesis. We conclude that CA9 is an important molecule in non-small cell lung cancer, the up-regulation of which occurs in highly hypoxic/necrotic regions of the tumors. The expression of CA9 is linked to the expression of a constellation of proteins involved in angiogenesis, apoptosis inhibition, and cell-cell adhesion disruption, which explains the strong association of CA9 with poor outcome.

Vascular endothelial growth factor/KDR activated microvessel density versus CD31 standard microvessel density in non-small cell lung cancer.

Vascular endothelial growth factor (VEGF) is an important angiogenic factor, linked to poor outcome in human malignancies including non-small cell lung carcinoma (NSCLC). We used the 11B5 monoclonal antibody recognizing the VEGF/KDR complex (R. A. Brekken et al., Cancer Res., 58: 1952-1959, 1998) to assess the VEGF expression in cancer cells and the VEGF/KDR activated microvessel density (aMVD) in early operable NSCLC. The JC70 anti-CD31 monoclonal antibody was used to assess the standard MVD (sMVD). The aMVD was significantly higher in the invading front of the tumors and in the normal lung adjacent to the tumors as compared with normal lung distant to the tumor or to inner tumor areas (P < 0.0002). The sMVD was higher in the normal lung and decreased from the invading front to inner tumor areas (P < 0.0001). However, the vascular activation (aMVD:sMVD) was 4-6 times higher in the tumor areas as compared with lung from normal individuals (36-58% versus 9%; P < 0.0001). Fibroblast 11B5 reactivity, noted in 25% of cases, correlated with high aMVD and sMVD in the inner tumor areas. Multivariate analysis showed that aMVD was the most potent and independent prognostic factor (P = 0.001; t-ratio, 3.28). It is concluded that intense VEGF/KDR angiogenic pathway activation is a tumor-specific feature in more than 50% of NSCLC cases and is associated with poor postoperative outcome. Clinical trials involving targeting of the VEGF/KDR-positive vasculature with specific antibodies, such as 11B5, are, therefore, encouraged.

Subcutaneous administration of amifostine during fractionated radiotherapy: a randomized phase II study.

PURPOSE: Amifostine (WR-2721) is an important cytoprotective agent. Although intravenous administration is the standard route, pharmacokinetic studies have shown acceptable plasma levels of the active metabolite of amifostine (WR-1605) after subcutaneous administration. The subcutaneous route, due to its simplicity, presents multiple advantages over the intravenous route when amifostine is used during fractionated radiotherapy. PATIENTS AND METHODS: Sixty patients with thoracic, 40 with head and neck, and 40 with pelvic tumors who were undergoing radical radiotherapy were enrolled onto a randomized phase II trial to assess the feasibility, tolerance, and cytoprotective efficacy of amifostine administered subcutaneously. A flat dose of amifostine 500 mg, diluted in 2.5 mL of normal saline, was injected subcutaneously 20 minutes before each radiotherapy fraction. RESULTS: The subcutaneous amifostine regimen was well tolerated by 85% of patients. In approximately 5% of patients, amifostine therapy was interrupted due to cumulative asthenia, and in 10%, due to a fever/rash reaction. Hypotension was never noted, whereas nausea was frequent. A significant reduction of pharyngeal, esophageal, and rectal mucositis was noted in the amifostine arm (P <.04). The delays in radiotherapy because of grade 3 mucositis were significantly longer in the group of patients treated with radiotherapy alone (P <.04). Amifostine significantly reduced the incidence of acute perineal skin and bladder toxicity (P <.0006). CONCLUSION: Subcutaneous administration of amifostine is well tolerated, effectively reduces radiotherapy's early toxicity, and prevents delays in radiotherapy. The subcutaneous route is much simpler and saves time compared with the intravenous route of administration and can be safely and effectively applied in the daily, busy radiotherapy practice.

Liposomal doxorubicin and conventionally fractionated radiotherapy in the treatment of locally advanced non-small-cell lung cancer and head and neck cancer.

PURPOSE: Stealth (ALZA Corporation, Palo Alto, CA) liposomal drug formulation allows a higher intratumoral accumulation and a prolonged plasma half-life of the encapsulated drugs. In the study presented here, we evaluated the feasibility of Stealth liposomal doxorubicin (Caelyx; ALZA Corporation) administered concurrently with conventionally fractionated radiotherapy in the treatment of non-small-cell lung cancer (NSCLC) and head and neck cancer (HNC). PATIENTS AND METHODS: Fifteen patients with NSCLC and 15 with squamous-cell HNC were recruited in two phase I dose-escalation trials. The starting dose of Caelyx was 10 mg/m(2) every 2 weeks (for three cycles during radiotherapy) and was increased by 5 mg/m(2) dose increments for every three patients. RESULTS: The maximum tolerated dose of Caelyx was 20 mg/m(2) for HNC and 25 mg/m(2) in NSCLC patients. Oral/pharyngeal mucositis was the dose-limiting toxicity for HNC patients. "In field" radiation skin toxicity was slightly increased. Hematologic toxicity was minimal. Single photon emission computed tomographic evaluation of Caelyx distribution, using technetium-99m-diethylenetriamine pentaacetic acid labeling, revealed a high intratumoral accumulation of the drug. The tumor to thoracic vessel area count ratio in the NSCLC cases ranged from 0.6 to 1.6 (mean +/- SD, 1.01 +/- 0.29), whereas this ratio was higher (0.8 to 1.85; mean +/- SD, 1.35 +/- 0.39) in HNC cases (P =.049). The complete response rate was 21% in the NSCLC cases and 75% in the HNC cases. NSCLC cases with higher Caelyx tumor accumulation responded better to the regimen. The tumor microvessel density assessed with the anti-CD31 monoclonal antibody directly correlated with the degree of the Caelyx accumulation (P =.007; r =. 92). CONCLUSION: We conclude that combination of radiotherapy with Stealth liposomal doxorubicin is feasible. The potential role of such a regimen in the treatment of highly angiogenic tumors requires further investigation.

Proliferating fibroblasts at the invading tumour edge of colorectal adenocarcinomas are associated with endogenous markers of hypoxia, acidity, and oxidative stress.

BACKGROUND: Stroma frequently forms at sites of active tumour invasion, and may be important for tumour growth and progression. The term "stromatogenesis" is used to describe this unique process that involves host peritumorous fibroblasts and is very different to reactive fibrosis. AIMS/METHODS: To investigate the activation status of host fibroblasts at the invading tumour edge, assessed as MIB1 proliferation index and thymidine phosphorylase (TP) expression. Results were related to vascular density and certain properties of invading cancer cells-MIB1 proliferation activity, TP expression, expression of endogenous markers of hypoxia (hypoxia inducible factor-1alpha; HIF1alpha) and acidity (lactate dehydrogenase-5; LDH5). Standard immunohistochemical techniques were applied to 150 colorectal adenocarcinomas. RESULTS: Normal fibroblasts at the tumour edge had a median MIB1 index of 2%-significantly higher than normal submucosal fibroblasts (0.3%) and significantly lower than cancer cells (40%). Normal peritumorous fibroblasts with a proliferation rate above the median strongly expressed TP and were supported by an increased vascular network. Cancer cells close to these fibroblasts had a high MIB1 proliferative index, high HIF1alpha and LDH5 reactivity, and a clear trend to extramural extension. All associations were significant. CONCLUSIONS: These results suggest that activated fibroblastic status at the invading tumour front sets the stage for stromatogenesis and new blood vessel formation, facilitating deep transmural invasion in colorectal adenocarcinomas. This complicity of peritumorous fibroblasts in the overall aggressiveness/invasive and metastatic ability of colorectal tumours, occurring within the framework of cancer-stromal cell interactions, is probably favoured by the altered microenvironmental conditions of hypoxia and acidity.

LYVE-1 immunohistochemical assessment of lymphangiogenesis in endometrial and lung cancer.

AIMS/METHODS: Normal and malignant pulmonary and endometrial tissues were analysed for lymphatic vessels to assess the process of lymphangiogenesis and its role at these sites, using specific immunostaining for LYVE-1 and the panendothelial marker CD31. RESULTS: Lymphatics were clearly demonstrated in some normal tissues (myometrium, bronchial submucosa, and intestinal submucosa), but not in others (endometrium and alveolar tissue). LYVE-1 positive lymphatic vessels were detected at the tumour periphery of endometrial and lung carcinomas, but not within the main tumour mass. Double staining for LYVE-1 and the MIB1 proliferation marker revealed a higher proliferation index in lymphatic endothelial cells at the invading front of endometrial carcinomas, compared with myometrial areas distal to the tumour. Lung and endometrial carcinomas did not have an intratumorous lymphatic network. CONCLUSIONS: Although lymphangiogenesis may occur at the invading tumour front, incorporated lymphatics do not survive. Therefore, the dissemination of cancer cells through the lymphatics may occur by invasion of peripheral cancer cells into the adjacent normal lymphatics, or through shunts eventually produced at the invading tumour front as a consequence of active angiogenesis and lymphangiogenesis.

Tumor angiogenesis is associated with MUC1 overexpression and loss of prostate-specific antigen expression in prostate cancer.

The biological potential of prostate cancer is highly variable and cannot be satisfactorily predicted by histopathological criteria alone. Therefore, additional and more precise information is desirable. Although angiogenesis has been suggested as being of prognostic importance in many human cancers, and MUC1, also known as episialin, was thought to be responsible for the development of metastasis, the role of these parameters in prostate cancer remains unclear. The aim of this study was to investigate whether angiogenesis, assessed as microvessel density (MVD), was correlated with the expression of prostate tumor MUC1 and prostate-specific antigen (PSA) or with histopathological grade at diagnosis, and to determine whether any of these factors might provide additional information with regard to prostate tumor biology. Paraffin-embedded material from 60 patients with prostate carcinoma was examined immunohistochemically, using the monoclonal antibody CD31 to determine MVD, and the monoclonal antibodies CCE831 and ER-PR8 to assess MUC1 and PSA expression, respectively. The tumors were categorized according to the Gleason grading system. MUC1 overexpression was significantly related to a high intratumoral angiogenesis (P = 0.02). By contrast, a high PSA expression by prostate cancer cells was associated with low MVD (P = 0.03). No correlation was found between MUC1 and PSA expression. Usually, high-grade tumors were not PSA-expressive and tended to display increased angiogenesis. These differences, however, were not of statistical significance. Similarly, there was no statistically significant association between histological grade and MUC1 expression or angiogenesis. It is suggested that PSA may have a direct suppressive effect on new blood vessel formation in prostate cancer, whereas the expression of MUC1 in this tumor may be connected with an angiogenic phenotype. Additional studies are obviously needed to clarify the precise role of these proteins in prostate cancer.