CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA-D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p14(ARF) antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus.

Angiogenesis is an independent prognostic factor in malignant mesothelioma

Angiogenesis is essential for tumour growth beyond 1 to 2 mm in diameter. The clinical relevance of angiogenesis, as assessed by microvessel density (MVD), is unclear in malignant mesothelioma (MM). Immunohistochemistry was performed on 104 archival, paraffin-embedded, surgically resected MM samples with an anti-CD34 monoclonal antibody, using the Streptavidin-biotin complex immunoperoxidase technique. 93 cases were suitable for microvessel quantification. MVD was obtained from 3 intratumoural hotspots, using a Chalkley eyepiece graticule at × 250 power. MVD was correlated with survival by Kaplan-Meier and log-rank analysis. A stepwise, multivariate Cox model was used to compare MVD with known prognostic factors and the EORTC and CALGB prognostic scoring systems. Overall median survival from the date of diagnosis was 5.0 months. Increasing MVD was a poor prognostic factor in univariate analysis (P = 0.02). Independent indicators of poor prognosis in multivariate analysis were non-epithelial cell type (P = 0.002), performance status > 0 (P = 0.003) and increasing MVD (P = 0.01). In multivariate Cox analysis, MVD contributed independently to the EORTC (P = 0.006), but not to the CALGB (P = 0.1), prognostic groups. Angiogenesis, as assessed by MVD, is a poor prognostic factor in MM, independent of other clinicopathological variables and the EORTC prognostic scoring system. Further work is required to assess the prognostic importance of angiogenic regulatory factors in this disease. © 2001 Cancer Research Campaign.

Phase II clinical trial of first or second-line treatment with bortezomib in patients with malignant pleural mesothelioma

Based on promising preclinical efficacy of bortezomib in mesothelioma, a single-arm phase II trial (Ireland Cooperative Oncology Research Group 05-10 study), with Simon's two-stage design, was undertaken to assess efficacy of bortezomib monotherapy in the first-line (poor performance status) and second-line settings. The Bcl-2 homology domain 3-only protein Noxa has been implicated as a key inducer of apoptosis by bortezomib. Thus, in a biomarker research substudy, we hypothesized that deficiency in Noxa expression might correlate with resistance. In the second-line setting, 23 patients were enrolled. Partial response was confirmed in one patient (4.8%) who received four cycles of bortezomib. One patient had stable disease; however, progression occurred in the majority of patients within the first two cycles. Median progression-free survival and overall survival were 2.1 and 5.8 months, respectively. In the first-line setting, ten patients were accrued, and there was no evidence of objective response. In the tumor analysis, expression of Noxa was seen in all biopsies. Bortezomib monotherapy exhibits insufficient activity to warrant further investigation in unselected patients with mesothelioma. © 2012 by the International Association for the Study of Lung.

Prognostic value of angiogenesis in operable non-small cell lung cancer

Tumour angiogenesis is an important factor for tumour growth and metastasis. Although some recent reports suggest that microvessel counts in non-small cell lung cancer are related to a poor disease outcome, the results were not conclusive and were not compared with other molecular prognostic markers. In the present study, the vascular grade was assessed in 107 (T1,2-N0,1) operable non-small cell lung carcinomas, using the JC70 monoclonal antibody to CD31. Three vascular grades were defined with appraisal by eye and by Chalkley counting: high (Chalkley score 7-12), medium (5-6), and low (2-4). There was a significant correlation between eye appraisal and Chalkley counting (P < 0.0001). Vascular grade was not related to histology, grade, proliferation index (Ki67), or EGFR or p53 expression. Tumours from younger patients had a higher grade of angiogenesis (P = 0.05). Apart from the vascular grade, none of the other factors examined was statistically related to lymph node metastasis (P < 0.0001). A univariate analysis of survival showed that vascular grade was the most significant prognostic factor (P = 0.0004), followed by N-stage (P = 0.001). In a multivariate analysis, N-stage and vascular grade were not found to be independent prognostic factors, since they were strongly related to each other. Excluding N-stage, vascular grade was the only independent prognostic factor (P = 0.007). Kaplan-Meier survival curves showed a statistically significant worse prognosis for patients with high vascular grade, but no difference was observed between low and medium vascular grade. These data suggest that angiogenesis in operable non-small cell lung cancer is a major prognostic factor for survival and, among the parameters tested, is the only factor related to cancer cell migration to lymph nodes. The integration of vascular grading in clinical trials on adjuvant chemotherapy and/or radiotherapy could substantially contribute in defining groups of operable patients who might benefit from cytotoxic treatment.

Platelet-derived endothelial cell growth factor (thymidine phosphorylase) expression in lung cancer

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is an enzyme with angiogenic and cell motility properties. Moreover, it is involved in the transformation of fluoropyrimidines into active cytotoxic metabolites, In the present study, the expression of PD-ECGF in normal lung and lung cancer was immunohistochemically evaluated using the P-GF.44C monoclonal antibody. Alveolar and tumoural macrophages mere invariably stained and mere used as an internal control for assessment of the staining. Alveolar epithelium was always negative, whilst bronchiolar epithelium showed occasional positive reactivity. Normal lung and tumour endothelium was occasionally positive, Positive staining in more than 50 per cent of cells was observed in 23/71 squamous carcinomas (32 per cent), 16/38 (42 per cent) adenocarcinomas, and 2/6 (33 per cent) adenosquamous carcinomas. Differentiated areas and areas of squamous metaplasia mere more strongly positive than other tumour areas. All 22 small cell carcinomas and one carcinoid tumour were negative. The present study provides a baseline for future studies in non-small cell lung cancer to correlate PD-ECGF expression with tumour vascularization, prognosis, and response to chemotherapy.

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non- small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-NO, 1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P = 0.04) and both were inversely associated with microvessel density (P < 0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P < 0.01). A significant co-expression of c-erbB-2 with TP was noted (P = 0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P < 0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P = 0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c- erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c- erbB-2-related immune response remains to be further investigated.

Galectin-1 : a link between tumor hypoxia and tumor immune privilege

Purpose: To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. Methods: We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CDS (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. Results: SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O 2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CDS staining (P = .01). Expression of galectin-1 and CDS were significant predictors for overall survival on multivariate analysis. Conclusion: Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege. © 2005 by American Society of Clinical Oncology.

Regulation of microRNA-1288 in colorectal cancer : altered expression and its clinicopathological significance

We aim to examine the miR-1288 expression in cancer cell lines and a large cohort of patients with colorectal cancer. Two colon cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The miRNA expressions of miR-1288 were tested on these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). An exogenous miR-1288 (mimic) was used to detect cell proliferation and cell cycle changes in SW480 using MTT calorimetric assay and flow cytometry, respectively. In addition, tissues from 122 patients with surgical resection of colorectum (82 adenocarcinomas, 20 adenomas, and 20 non-neoplastic tissues) were tested for miR-1288 expression by qRT-PCR. The colon cancer cell lines showed reduced expression of miR-1288 compared to normal colonic epithelial cell line. Over expression of miR-1288 in SW480 cell line showed increased cell proliferation and increased G2-M phase cells. In tissues, reduced miR-1288 expression was noted in majority of colorectal adenocarcinoma compared to colorectal adenoma and non-neoplastic tissues. Reduced or absent expression of miR-1288 was noted in 76% (n = 62/82) of the cancers. The expression levels of miR-1288 were higher in distal colorectal adenocarcinomas (P = 0.013) and in cancers of lower T staging (P = 0.033). To conclude, alternation of miR-1288 expression is important in the progression of colorectal cancer. The differential regulation of miR-1288 was found to be related to cancer location and pathological staging in colorectal cancers.

Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis

Objective. The heritability of RA has been estimated to be ∼55%, of which the MHC contributes about one-third. HLA-DRB1 alleles are strongly associated with RA, but it is likely that significant non-DRB1 MHC genetic susceptibility factors are involved. Previously, we identified two three-marker haplotypes in a 106-kb region in the MHC class III region immediately centromeric to TNF, which are strongly associated with RA on HLA-DRB1*0404 haplotypes. In the present study, we aimed to refine these associations further using a combination of genotyping and gene expression studies. Methods. Thirty-nine nucleotide polymorphisms (SNPs) were genotyped in 95 DRB1*0404 carrying unrelated RA cases, 125 DRB1*0404 - carrying healthy controls and 87 parent-case trio RA families in which the affected child carried HLA-DRB1*04. Quantitative RT-PCR was used to assess the expression of the positional candidate MHC class III genes APOM, BAT2, BAT3, BAT4, BAT5, AIF1, C6orf47, CSNK2β and LY6G5C, and the housekeeper genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β2-microglobulin (B2M) in 31 RA cases and 21 ethnically, age- and sex-matched healthy controls. Synovial membrane specimens from RA, PsA and OA cases were stained by an indirect immunoperoxidase technique using a mouse-anti-human AIF1 monoclonal antibody. Results. Association was observed between RA and single markers or two marker haplotypes involving AIF1, BAT3 and CSNK. AIF1 was also significantly overexpressed in RA mononuclear cells (1.5- to 1.9-fold difference, P = 0.02 vs HPRT, P = 0.002 vs B2M). AIF1 protein was clearly expressed by synovial macrophages in all the inflammatory synovial samples in contrast to the non-inflammatory OA samples. Conclusions. The results of the genotyping and expression studies presented here suggest a role for AIF1 in both the aetiology and pathogenesis of RA.

Osteoclast formation from circulating precursors in osteoporosis

Objective: An imbalance between bone formation and bone resorption is thought to underlie the pathogenesis of reduced bone mass in osteoporosis. Bone resorption is carried out by osteoclasts, which are formed from marrow-derived cells that circulate in the monocyte fraction. Ihe aim of this study was to determine the role of osteoclast formation in the pathogenesis of bone loss in osteoporosis. Methods: The proportion of circulating osteoclast precursors and their relative sensitivity to the osteoclastogenic effects of M-CSF, 1,25(OH)2D3 and RANKL were assessed in primary osteoporosis patients and normal controls. Results: Although there was no difference in the number of circulating osteoclast precursors in osteoporosis patients and normal controls, osteoclasts formed from osteoporosis patients exhibited substantially increased resorptive activity relative to normal controls. Although no increased sensitivity to the osteoclastogenic effects of 1,25(OH)2D3 or M-CSF was noted, increased bone resorption was found in osteoporosis peripheral blood mononuclear cell (PBMC) cultures to which these factors were added. Conclusion: Our findings suggest that osteoclast functional activity rather than formation is increased in primary involutional osteoporosis and that dexamethasone acts to increase osteoclast formation.