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.     

Phosphorylated VEGFR2/KDR receptors are widely expressed in B-cell non-Hodgkin's lymphomas and correlate with hypoxia inducible factor activation

The expression of hypoxia inducible factors (HIF) 1alpha and 2alpha, of vascular endothelial growth factor (VEGF) and of the phosphorylated form of its receptor VEGFR2 (pVEGFR2/KDR) were investigated immunohistochemically in a series of 146 B-cell non-Hodgkin's lymphomas and 48 normal lymph nodes. These proteins were significantly up-regulated in neoplastic lymphocytes and a significantly higher expression of HIF1alpha (p = 0.05), VEGF (p = 0.02) and pVEGFR2/KDR (p = 0.007) was recorded in diffuse large B-cell lymphomas(DLBCL) compared to follicular lymphomas (FL). A strong statistical association of pVEGFR2/KDR expression with high HIF1alpha, HIF2alpha and VEGF was noted in both DLBCL and FL. HIF1alpha andHIF2alpha were linked with VEGF expression but no association between HIF1alpha and HIF2alpha was noted. Vascular density was significantly higher in the lymphoma compared to normal tissue, but there was no association with any of the examined parameters. It is concluded that the VEGF/receptor pathway is active in more than half of NHLs and particularly in DLBCL. The intimate correlation of VEGF production with the presence of phosphorylated VEGF-receptors strongly supports the concept of an autocrine pathway. The strong association of HIFalphas with the expression of VEGF and pVEGFR2/KDR found in the study provide strong evidence on the role of HIFalphas inthe activation of angiogenic and VEGF-autocrine pathways that may be critical therapeutic targets for HIF-inhibitors or other anti-angiogenic agents.     

Predicting Distant Failure in Nasopharyngeal Cancer

In a prospective study of 78 patients with nasopharyngeal cancer, we examined the prognostic significance of T stage, histology, parapharyngeal involvement, and lymph node dimensions, size, and level concerning distant metastasis. All patients were treated with radical radiotherapy alone and completed 3 to 7 years of follow-up. In univariate analysis of time to metastasis, there was a significant difference stratifying for T stage (T1 and 2 versus T3 and 4), node dimensions (less than 6 versus more than or equal to 6 cm), neck level (above versus below the thyroid notch), and parapharyngeal involvement, but not for bilaterality of lymphadenopathy. Histology was an important prognostic factor related to distant metastasis since none of the 24 World Health Organization class I cases showed distant metastasis versus 14 (26%) of 54 patients with World Health Organization class II/III carcinoma. A multivariate duration model of time to metastasis within the later histologic group suggested that lymph node dimensions, node level, and T stage were the most important factors related to distant metastases, with the hazard ratios being 3.98, 3.23, and 1.76, respectively. Multivariate analysis within the T3- to T4-stage group showed that node dimension was the only significant variable, with an associated hazard ratio of 4.09. Cases with upper-neck lymphadenopathy and node dimensions of less than 6 cm had a distant metastasis rate of <5%. We conclude that adjuvant chemotherapy for nasopharyngeal cancer is justified in T3- and T4-staged cases with nonkeratinizing or undifferentiated histology and with lymph nodes larger than 6 cm and/or located below the thyroid notch.     

BNIP3 expression is linked with hypoxia-regulated protein expression and with poor prognosis in non-small cell lung cancer.

BNIP3 is a proapoptotic protein regulated by hypoxia-inducible factor 1. We analyzed BNIP3 expression in 105 tumor samples from early operable, non-small lung cancer and the relationship of expression to hypoxia-inducible factor 1alpha, other hypoxia-regulated pathways, and prognosis. There was strong cytoplasmic expression in >10% of cells in 40 of 105 cases. BNIP3 expression was associated significantly with high hypoxia-inducible factor 1alpha (P = 0.003), carbonic anhydrase 9 (P = 0.04), and was inversely associated with bcl-2 expression (P = 0.009). High BNIP3 expression was a major independent factor for overall survival. Thus, high expression of a hypoxia regulated proapoptotic pathway was associated with a selection of an aggressive phenotype in vivo.     

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.     

Angiogenesis vs. response after combined chemoradiotherapy of squamous cell head and neck cancer

Oxygen/drug supply to cancer cells is an important factor defining response to radiotherapy and chemotherapy. Although tumor angiogenesis is considered an important prognostic marker, its role in the outcome of chemotherapy and radiotherapy is unknown. In the present study we examined the possible correlation of the degree of angiogenesis with response to cytotoxic therapy in locally advanced squamous cell head and neck cancer (HNC). Vascular grade (VG) was assessed immunohistochemically using the JC70 monoclonal antibody (MAb) in tumor specimens from 76 patients treated with platinum/5-fluorouracil (with or without methotrexate) induction chemotherapy (ICT) (n = 37) or concurrent chemoradiotherapy (CCRT) with cisplatin or carboplatin (n = 39). Seventeen of 76 analyzed patients had an overall microvessel score of < 11 (VGI), 25/76 of 11-30 (VG2), 16/76 of 31-50 (VG3) and 18/76 of > 50 (VG4). Complete response rate after ICT or after CCRT was higher in cases with an intermediate vascularization (VG2,3). Both local relapse-free and overall survival were significantly better in the VG2 group. Patients treated with CCRT had a better survival compared to those treated with ICT. This was mainly observed in VG1 tumors. Multivariate analysis showed that VG and treatment modality were independent prognostic factors for local relapse and survival. Intratumoral angiogenesis correlation with the cytotoxic therapy outcome is likely to follow a bell-shaped relation, the response being better in cases with an intermediate VG. This may be the consequence of 2 vasculature-dependent factors, i.e., the drug/oxygen availability and the ability of cancer cells to undergo rapid repopulation in optimally oxygenated conditions. Our pilot study stresses the importance of individualization of therapy according to VG.     

Comparison of angiogenic activities after urethral reconstruction using free grafts in rabbits

OBJECTIVE: To determine the most suitable type of graft-free penile skin grafts or mucosal grafts from bladder or buccal regions - for urethral reconstruction in an animal model, as evaluated on the basis of angiogenic activity. METHODS: Twenty-two male White New Zealand rabbits were randomly divided into four groups. In the control group (group O, n=4) a simple urethrotomy and closure was performed, whereas a ventral urethral defect was created in groups A, B, and C and then bridged using the following onlay patches: free penile skin (group A, n=6), buccal mucosal graft (group B, n=6), and bladder mucosal graft (group C, n=6). On the 21st postoperative day, the animals were sacrificed and the retrieved implants were subjected to macroscopic and microscopic analysis. The angiogenic activity was assessed with immunohistochemistry, using the anti-CD31 MoAb and the phosphatase antialkaline phosphatase procedure. The native vascularity of penile skin as well as buccal and bladder mucosa was assessed in rabbits from group O (n=3). Statistical analysis was performed using the one-way ANOVA. RESULTS: The angiogenesis in a magnification of x200 in groups O, A, B, and C was 34.1+/-4.1 (mean+/-SD), 61.7+/-6.4, 94.3+/-6.4, and 91.5+/-7.2 vessels per optical field, respectively. There were, statistically significant differences (p<0.001) between groups A and B and between groups A and C, but not (p>0.05) between groups B and C. The native vascularity of penile skin, buccal mucosa and bladder mucosa was 23.3+/-3.0, 24.6+/-3.7 and 17.0+/-2.6 vessels per optical field, respectively. CONCLUSION: The viability of mucosal grafts from bladder or buccal regions is better than that of a free penile graft because of higher angiogenic activity. Although the mucosal grafts showed the same angiogenic activity, the buccal mucosa graft is preferable because of its easier harvesting.     

Thymidine phosphorylase expression in patients with chronic viral hepatitis

BACKGROUND: The role of thymidine phosphorylase (TP), an angiogenic factor, in chronic viral hepatitis is unclear. In the present study we investigated TP expression in chronic viral hepatitis due to hepatitis B virus (HBV) and hepatitis C virus (HCV) infection. METHODOLOGY: Fifty-eight patients suffering from chronic viral hepatitis and 13 cirrhotic patients due to either HBV or HCV were studied. TP expression was investigated using a standard immunohistochemical technique and the specific antibody P-GF.44C. RESULTS: The pattern of TP staining was predominantly nuclear, or mixed nuclear/cytoplasmic, but no significant difference in such expression was noted between chronic hepatitis B and C: nuclear immunostaining, t=0.0616, p=0.9511; cytoplasmic immunostaining, t=0.5188, p=0.6060. There was, however, a marked difference in TP expression between patients with chronic hepatitis B/C and cirrhosis. Notably, this finding applied to both nuclear (p=0.00754, p=0.0143, respectively) and cytoplasmic (p=0.000091, p=0.000031, respectively) expression. CONCLUSION: These observations indicate that TP expression, although linked to liver cirrhosis, may not be virus-related.