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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- β 1 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- β 1 were all associated with poor prognosis but, in a multivariate analysis, the integrin- β 1 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- β 1 being the strongest and independent prognostic factor. Targeting CSC molecules with monoclonal antibodies or pharmaceutical agents may prove important for the treatment of HNSCC.

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 HIF1 α 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.

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.

Lactate dehydrogenase-5 (LDH-5) catalyses the reversible transformation of pyruvate to lactate, having a principal position in the anaerobic cellular metabolism. Induction of LDH-5 occurs during hypoxia and LDH-5 transcription is directly regulated by the hypoxia-inducible factor 1 (HIF1). Serum LDH levels have been correlated with poor prognosis and resistance to chemotherapy and radiotherapy in various neoplastic diseases. The expression, however, of LDH in tumours has never been investigated in the past. In the present study, we established an immunohistochemical method to evaluate the LDH-5 overexpression in tumours, using two novel antibodies raised against the rat muscle LDH-5 and the human LDH-5 (Abcam, UK). The subcellular patterns of expression in cancer cells were mixed nuclear and cytoplasmic. In direct contrast to cancer cells, stromal fibroblasts were reactive for LDH-5 only in a minority of cases. Serum LDH, although positively correlated with, does not reliably reflect the intratumoral LDH-5 status. Lactate dehydrogenase-5 overexpression was directly related to HIF1 α and 2 α , but not with the carbonic anhydrase 9 expression. Patients with tumours bearing high LDH-5 expression had a poor prognosis. Tumours with simultaneous LDH-5 and HIF1 α (or HIF2 α ) overexpression, indicative of a functional HIF pathway, had a particularly aggressive behaviour. It is concluded that overexpression of LDH-5 is a common event in non-small-cell lung cancer, can be easily assessed in paraffin-embedded material and provides important prognostic information, particularly when combined with other endogenous markers of hypoxia and acidity.

Hypoxia inducible factors HIF1α and HIF2α are important proteins involved in the regulation of the transcription of a variety of genes related to erythropoiesis, glycolysis and angiogenesis. Hypoxic stimulation results in rapid increase of the HIF1α and 2α protein levels, as a consequence of a redox-sensitive stabilization. The HIFαs enter the nucleus, heterodimerize with the HIF1β protein, and bind to DNA at the hypoxia response elements (HREs) of target genes. In this study we evaluated the immunohistochemical expression of these proteins in 108 tissue samples from non-small-cell lung cancer (NSCLC) and in normal lung tissues. Both proteins showed a mixed cytoplasmic/nuclear pattern of expression in cancer cells, tumoural vessels and tumour-infiltrating macrophages, as well as in areas of metaplasia, while normal lung components showed negative or very weak cytoplasmic staining. Positive HIF1α and HIF2α expression was noted in 68/108 (62%) and in 54/108 (50%) of cases respectively. Correlation analysis of HIF2α expression with HIF1α expression showed a significant association ( P < 0.0001, r = 0.44). A strong association of the expression of both proteins with the angiogenic factors VEGF ( P < 0.004), PD-ECGF ( P < 0.003) and bFGF ( P < 0.04) was noted. HIF1α correlated with the expression of bek-bFGF receptor expression ( P = 0.01), while HIF2α was associated with intense VEGF/KDR-activated vascularization ( P = 0.002). HIF2α protein was less frequently expressed in cases with a medium microvessel density (MVD); a high rate of expression was noted in cases with both low and high MVD ( P = 0.006). Analysis of overall survival showed that HIF2α expression was related to poor outcome ( P = 0.008), even in the group of patients with low MVD ( P = 0.009). HIF1α expression was marginally associated with poor prognosis ( P = 0.08). In multivariate analysis HIF2α expression was an independent prognostic indicator ( P = 0.006, t-ratio 2.7). We conclude that HIF1α and HIF2α overexpression is a common event in NSCLC, which is related to the up-regulation of various angiogenic factors and with poor prognosis. Targeting the HIF pathway may prove of importance in the treatment of NSCLC. © 2001 Cancer Research Campaign http://www.bjcancer.com

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.

Aims: Hypoxia inducible factors 1α and 2α (HIF1α and HIF2α) are hypoxia regulated transcriptional factors, which control the expression of a variety of genes responsible for angiogenesis, glycolysis, and the inhibition of apoptosis. Because angiogenesis and tissue regeneration are integral components of the inflammatory process, this study was designed to investigate the role of HIFα molecules in inflammatory bowel disease. Methods: Surgical specimens from patients with active ulcerative colitis (UC) and Crohn’s disease (CD) were assessed immunohistochemically for HIF1α and HIF2α reactivity, and the expression of these molecules was compared with the expression of the angiogenic factors thymidine phosphorylase (TP), vascular endothelial growth factor (VEGF), and VEGF–KDR activated vasculature. The vascular density of the lesions was also assessed using anti-CD31 immunostaining. Results: HIF1α was expressed focally (epithelial cells, stromal fibroblasts, and myocytes) in both UC and CD, whereas HIF2α was expressed focally in UC and diffusely in CD. TP expression was uniformly positive in both diseases. VEGF expression was absent in CD, and weakly positive in UC. The VEGF–KDR reactivity of the submucosal vasculature was only slightly increased in UC and CD compared with normal tissue. The inflammatory cells stained with HIF2α and TP in all cases, but the reactivity was generalised in CD and focal in UC. In both diseases, vascular density was significantly higher than that seen in normal tissue. Conclusions: The discordant expression of HIF2α and VEGF in CD suggests an inherent deficiency of the intestine to respond to various stresses by the induction of VEGF. This finding should be investigated further.

Aims: To investigate proliferation in disease free postmenopausal endometrium and that harbouring endometrial adenocarcinoma—is there a dynamic, yet lurking, potential for atrophic endometrium to give rise to endometrial adenocarcinoma? Material/methods: The study comprised 84 disease free endometria from asymptomatic postmenopausal women who had undergone hysterectomy for prolapse, and 50 endometrioid cell type endometrial adenocarcinomas with adjacent uninvolved postmenopausal endometrium. The non-neoplastic tissues were separated histologically into active, inactive, and mixed forms, although only the first two categories were studied immunohistochemically for oestrogen and progesterone receptors (ERs, PRs), epidermal growth factor receptor (EGFR), Ki-67, and angiogenic activity. Results: All postmenopausal endometria were atrophic, but only 42 were inactive; of the remaining samples, 22 were weakly proliferative and 20 were mixed active and inactive. In contrast, the non-neoplastic component of 43 of the 50 endometrial adenocarcinomas examined was of the active form; four specimens were of the pure and 39 of the mixed form. Interestingly, high ER and PR expression was seen in active and inactive endometria, but only the former were EGFR positive and had high proliferative (Ki-67) and angiogenic activity. A similar trend was also shown by the non-neoplastic atrophic endometrium adjacent to endometrial adenocarcinoma. Conclusions: At least half of the disease free postmenopausal atrophic endometria show a weak proliferative pattern, either diffuse or focal, probably as a response to continuous low level oestrogenic stimulation. These tissues have a latent, although very small, carcinogenic potential, as demonstrated by the immunohistochemical profile and their frequent association with adjacent endometrial adenocarcinoma.

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-1α; HIF1α) 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 HIF1α 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.

Aims:BNIP3 is a pro-apoptotic mitochondrial protein induced under hypoxic stress, with the BNIP3 gene being under direct regulation of the hypoxia-inducible HIF-1α transcription factor. Induction of BNIP3 leads to caspase-independent necrosis-like cell death and an aggressive tumour phenotype. The role of BNIP3 in endometrial cancer was examined.Methods:The immunohistochemical patterns of BNIP3 expression in 72 early endometrial adenocarcinomas of the endometrioid cell type were studied. Correlation of BNIP3 with the hypoxia-inducible factor HIF-1α pathway and with prognosis was also examined.Results:BNIP3 was strongly and extensively expressed in the cytoplasm of cancer cells in 23/72 (31.9%) cases. This high BNIP3 reactivity was not related to histological grade, depth of myometrial invasion or steroid hormone receptor expression. There was, however, a significant association of BNIP3 reactivity with HIF-1α (p = 0.04), VEGF (p = 0.04) and, particularly, LDH-5 expression (p = 0.0001). Furthermore, high BNIP3 was associated with poor survival in both univariate (p = 0.05) and multivariate (p = 0.03) models.Conclusion:BNIP3 seems to be an important hypoxia-regulated molecule involved in endometrial cancer pathology. Given that high BNIP3 reactivity, being linked with poor post-operative outcome, has been linked with a favourable response to cytotoxic therapy (as previously indicated in experimental studies), high BNIP3 expression may be an indicator for adjuvant chemoradiotherapy in stage I endometrial carcinomas.