Circulating macrophage colony stimulating factor as a marker of tumour progression

Tumour expression of the macrophage colony stimulating factor (CSF-1 or MCSF) has been associated with an adverse prognosis in breast cancer, through an effect on the promotion of metastasis. The aim of the present study was to evaluate the clinical relevance of high circulating CSF-1 levels in patients with newly diagnosed breast tumours and correlate CSF-1 with clinico-pathological parameters. A secondary aim was to also measure CSF-1 in patients with other tumour types and at different stages of disease. Using a commercially available ELISA, pre-treatment plasma levels of CSF-1 were assessed, in 471 consecutive patients diagnosed with breast tumours, in 70 patients with newly diagnosed cancer of the head & neck, in 32 men with prostate cancer metastatic to bone and in 39 women with advanced metastatic breast cancer. Mean CSF-1 levels were significantly higher in patients with locally advanced (p <.015) or metastatic breast tumours (p <.048) and in a group of primary breast cancer patients (n = 26) selected for intensive chemotherapy because of multiple adverse tumour characteristics (p <.0002). Mean CSF-1 was also higher in patients younger than 35 years (p <.02) and in post-menopausal patients (p <.03). There was no significant association with tumour histologic type, grade, or other individual histopathologic parameters. No significant association was found between pre-treatment CSF-1 and overall/relapse free survival. Median CSF-1 levels were dramatically higher in patients with newly diagnosed tumours of the head & neck (604 pg/ml), in men with prostate cancer metastatic to bone (627 pg/ml) and women with advanced metastatic breast cancer (867 pg/ml) than those seen in patients with newly diagnosed breast tumours (334 pg/ml). Our data support the hypothesis that CSF-1 may play a functional role in tumour progression to metastasis as has previously been reported in animal models.     

Aberrant E-cadherin staining patterns in invasive mammary carcinoma

Background

Advances in our understanding of the molecular biology of colorectal cancer have fuelled the search for novel molecular prognostic markers to complement existing staging systems. Markers assessed in combination may perform better than those considered individually. Using high-throughput tissue microarray technology, we describe the prognostic value of combined p53 / Bcl-2 status in colorectal cancer.

Patients and methods

Tumour samples from 462 patients who underwent elective surgery to resect a primary colorectal cancer between 1994 and 2000 (mean follow-up of 75 months) were assembled in tissue microarray format. Clinico-pathological data including tumour grade, stage, vascular invasion status along with disease specific survival data has been collected prospectively. Immunohistochemical analysis of p53 and Bcl-2 expression was performed using antibodies DO-7 (p53) and 124 (Bcl-2), and results correlated with known clinico-pathological variables and outcomes.

Results

Abnormal nuclear p53 accumulation and Bcl-2 overexpression were detected in 221/445 (49.6%) and199/437 (45.5%) tumours respectively, with a significant inverse correlation between the two markers (p = 0.023). On univariate analysis no correlations were found between either marker and standard clinico-pathological variables, however nuclear p53 expression was associated with a significantly reduced survival (p = 0.024). Combined analysis of the two markers indicated that 112/432 (24.2%) cases displayed a p53(-)/Bcl-2(+) phenotype, this occurring more frequently in earlier stage tumours. Kaplan-Meier analysis revealed a significant survival advantage in these p53(-)/Bcl-2(+) tumours compared with the remaining cases (p = 0.0032). On multivariate analysis using the Cox proportional hazards model, neither p53 expression nor Bcl-2 expression alone were of independent prognostic significance, however the combined p53(-)/Bcl-2(+) phenotype was significantly associated with a good prognosis in this series (HR 0.659, 95%CI 0.452–0.959, p = 0.029).

Conclusion

Patient stratification by combined p53 / Bcl-2 phenotype provides stage-independent prognostic information in colorectal cancer. Specifically, that up to a quarter of patients display a good prognosis p53(-)/Bcl-2(+) phenotype. This may indicate a more clinically indolent phenotype and a subset of patients for whom less aggressive adjuvant treatment appropriate.     

Circular RNA MYLK promotes tumour growth and metastasis via modulating miR‐513a‐5p/VEGFC signalling in renal cell carcinoma

Growing evidence indicates that circular RNAs (circRNAs) are promising biomarkers, as they play significant roles in the development of various cancers. The circular RNA MYLK (circMYLK) has been reported to be involved in the development of malignant tumours, including liver, prostate and bladder cancers. Nevertheless, the biological function of circMYLK in renal cell carcinoma (RCC) remains unclear. In this study, we observed that circMYLK is notably up‐regulated in RCC. Increased circMYLK expression led to a larger tumour size, distant metastasis and poor prognosis of RCC patients. Moreover, circMYLK silencing repressed RCC growth and metastasis in vitro and in vivo. Mechanistically, circMYLK can capture miR‐513a‐5p to facilitate VEGFC expression and further promote the tumorigenesis of RCC cells. In summary, our findings demonstrate that circMYLK has an oncogenic role in RCC growth and metastasis by modulating miR‐513a‐5p/VEGFC signalling. Thus, circMYLK has potential as a diagnostic biomarker and therapeutic target in the treatment of RCC.     

Prognostic value of p53 protein expression and clinicopathological factors in infiltrating ductal carcinoma of the breast. A study of 192 patients

The p53 gene is located on the short arm of chromosome 17. It encodes a 53-kd nuclear protein (p53) found in scant amounts in normal tissue. Mutations of the p53 gene have been reported in different human tumours. In breast cancer, it has been noted that the overexpression of p53 protein in the nucleus is an indicator of poor prognosis, although there is a high degree of variability, which may be due to different immunohistochemical techniques, varying assessment of results and the type of monoclonal antibody used. This study is an immunohistochemical analysis of p53 expression in 192 cases of infiltrating ductal carcinoma of the breast, correlating it with clinicopathological factors and the clinical course of the disease. Of all the breast-cancer tissue analysed, stains for p53 antibody were found in 87 tumours (45.3%). The results of multivariate analysis show that the independent predictors related to recurrence are tumour size, lymph-node metastasis and p53, while those related to death are necrosis, lymph-node metastasis and p53. In summary, our series showed prognostic significance between the expression of p53 and shorter survival time and disease-free interval for all patients in general as well as those who presented lymph-node metastases at the time of diagnosis.     

Differential role of CD133 and CXCR4 in renal cell carcinoma

The chemokine receptor CXCR4 and CD133, putative stem cell markers, were previously described in renal cancer (RCC). To evaluate the biological and prognostic role of CD133 and CXCR4 in RCC the expression was evaluated through qPCR and immunoblotting in human renal cancer cell lines (786-O, A498, ACHN, CAKI-1, SN12C, TK10, UO31) and patients biopsies. Renal cancer cells and surgical biopsies expressed functional CXCR4 while CD133 was not detectable. CXCR4 and CD133 expression was then evaluated in 240 renal cancer patients through immunohistochemistry. CXCR4 and CD133 were low in 19.1% and 59.6%; intermediate in 20% and 17.9%; high in 60.8% and 22.5% of the cases, respectively. CXCR4 was overexpressed in tumours (p= 0.02), while CD133 was over expressed in healthy tissues (p= 0.04). Disease free survival Kaplan Meier plots suggest that prognosis is unfavourable for patients whose primary tumours express CXCR4 (p= 0.0199) but nor CD133 (p= 0.151) neither the concomitant CXCR4-CD133 (p=0.848) high expression affected prognosis. Analysis of prognostic factors suggests that age, clinical presentation, AJCC stage and CXCR4 had a significant prognostic value at the univariate analysis. The CXCR4 predictive ability was confirmed at the multivariate analysis while no prognostic role was identified for CD133. Thus concomitant CD133 and CXCR4 evaluation is not worth in RCC patient while the CXCR4 prognostic role encourage CXCR4 antagonists as promising therapeutic option.     

Molecular genetic investigations of the mechanism of tumourigenesis in von Hippel-Lindau disease: analysis of allele loss in VHL tumours

Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome characterised by the development of retinal and central nervous system haemangioblastomas, renal cell carcinoma (RCC), phaeochromocytoma and pancreatic tumours. The VHL disease gene maps to chromosome 3p25-p26. To investigate the mechanism of tumourigenesis in VHL disease, we analysed 24 paired blood/tumour DNA samples from 20 VHL patients for allele loss on chromosome 3p and in the region of tumour suppressor genes on chromosomes 5, 11, 13, 17 and 22. Nine out of 24 tumours showed loss of heterozygosity (LOH) at at least one locus on chromosome 3p and in each case the LOH included the region to which the VHL gene has been mapped. Chromosome 3p allele loss was found in four tumour types (RCC, haemangioblastoma, phaeochromocytoma and pancreatic tumour) suggesting a common mechanism of tumourigenesis in all types of tumour in VHL disease. The smallest region of overlap was between D3S1038 and D3S18, a region that corresponds to the target region for the VHL gene from genetic linkage studies. The parental origin of the chromosome 3p25-p26 allele loss could be determined in seven tumours from seven familial cases; in each tumour, the allele lost had been inherited from the unaffected parent. Our results suggest that the VHL disease gene functions as a recessive tumour suppressor gene and that inactivation of both alleles of the VHL gene is the critical event in the pathogenesis of VHL neoplasms. Four VHL tumours showed LOH on other chromosomes (5q21, 13q, 17q) indicating that homozygous VHL gene mutations may be required but may not be sufficient for tumourigenesis in VHL disease.     

SNAIL Induces EMT and Lung Metastasis of Tumours Secreting CXCL2 to Promote the Invasion of M2-Type Immunosuppressed Macrophages in Colorectal Cancer

Background: There is increasing evidence that tumour-associated macrophages (TAMs) are critical in the formation of lung metastases. However, the molecular mechanisms of tumour interactions with TAMs via EMT are largely unknown.Methods: The mechanism of lung metastasis was studied in patient tissues. The mechanism of SNAIL regulation of the interaction between mesenchymal cells and M2 macrophages was elucidated using coculture of M2 macrophages and Transwell assays in vitro and in vivo in nude mice and NOD-SCID mice.Results: We demonstrated for the first time that SNAIL and CXCL2 were abnormally overexpressed in colorectal cancer, especially lung metastasis, and were associated with poor prognosis in colorectal cancer patients. We demonstrated that SNAIL promoted the secretion of CXCL2 by mesenchymal cells and induced the activation of M2 macrophages. We found that CXCL2 attracted M2-type macrophages to infiltrate and promote tumour metastasis.Conclusion: These findings suggest that SNAIL promotes epithelial tumour transformation, and that transformed mesenchymal cells secrete CXCL2, which promotes M2 macrophage infiltration and tumour cell metastasis. These findings elucidate the tumour-TAM interaction in the metastatic microenvironment, which is mediated by tumour-derived CXCL2 and affects lung metastasis. This study also provides a theoretical basis for the occurrence of secondary lung cancer.     

p53 coordinates cranial neural crest cell growth and epithelial-mesenchymal transition/delamination processes

Neural crest development involves epithelial-mesenchymal transition (EMT), during which epithelial cells are converted into individual migratory cells. Notably, the same signaling pathways regulate EMT function during both development and tumor metastasis. p53 plays multiple roles in the prevention of tumor development; however, its precise roles during embryogenesis are less clear. We have investigated the role of p53 in early cranial neural crest (CNC) development in chick and mouse embryos. In the mouse, p53 knockout embryos displayed broad craniofacial defects in skeletal, neuronal and muscle tissues. In the chick, p53 is expressed in CNC progenitors and its expression decreases with their delamination from the neural tube. Stabilization of p53 protein using a pharmacological inhibitor of its negative regulator, MDM2, resulted in reduced SNAIL2 (SLUG) and ETS1 expression, fewer migrating CNC cells and in craniofacial defects. By contrast, electroporation of a dominant-negative p53 construct increased PAX7(+) SOX9(+) CNC progenitors and EMT/delamination of CNC from the neural tube, although the migration of these cells to the periphery was impaired. Investigating the underlying molecular mechanisms revealed that p53 coordinates CNC cell growth and EMT/delamination processes by affecting cell cycle gene expression and proliferation at discrete developmental stages; disruption of these processes can lead to craniofacial defects.     

Prognostic factors affecting disease-free survival rate following surgical resection of primary breast cancer.

In order to identify the prognostic factors that significantly influence the disease-free survival rate after surgical resection of primary breast cancers, we determined tumour and lymph node grades, and immunohistochemical staining for estrogen and progesterone receptors (ER and PR), c-erbB-2, p53, bcl-2, bax and PCNA in 76 patients. Univariate analysis showed that increased grade of tumour and lymph nodes, negative immunostaining for ER, positive immunostaining for c-erbB-2, and a high PCNA index (> or = 30%) negatively influenced the disease-free survival rate, but PR, p53, bcl-2 and bax had no predictive value. Although p53 was not an independent prognostic factor by itself, the combination of p53, bcl-2, and bax proved to correlate with the disease-free survival, with the best prognosis noted in tumours negative for p53 and positive for both bcl-2 and bax, intermediate prognosis in tumours negative for p53 and positive for either bcl-2 or bax and worst prognosis in tumors negative for p53 as well as bcl-2 and bax. Tumour grade correlated positively with PCNA index, while positive staining for ER correlated negatively with tumour grade as well as with PCNA index, although this was statistically insignificant. Immunostaining of breast cancers for bcl-2 correlated negatively with tumour grade and PCNA index. Immunostaining for c-erbB-2 correlated positively with PCNA but not with tumour grade. Immunostaining for p53 tended to correlate positively with PCNA, but not with tumour grade. Immunostaining for PR and bax did not correlate with tumour grade and PCNA index. These results suggest that in addition to tumour size and lymph node involvement, immunostaining for ER, c-erbB-2, and a high PCNA index are important prognostic factors in human breast cancer. Wild-type p53 with preserved bcl-2 and bax gene products is also a favorable prognostic factor indicating breast cancer at an early stage of cancer progression.     

肾癌预后分子生物标记物研究进展

肾癌发病率约占全身恶性肿瘤的3%。肾癌组织学行为多变,预后有不确定性。外科手术可以治疗局限性肾癌,但有将近20%的局限性肾癌患者原发肿瘤切除后出现转移,而且肾癌对化疗及放疗均不敏感。基于此临床上开展了许多辅助试验的研究,并建立了许多模型来研究肾癌术后的预后,而模型的精准度一般都需要依据肾癌的生物标记物监测。有很多分子生物标记物已经证实和肾癌预后相关,如VHL、P53、Ki-67等,本文综述了肾癌预后的分子生物标记物的最新进展。     

Computational Modelling of Metastasis Development in Renal Cell Carcinoma

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.     

Differential vascular expression and regulation of oncofetal tenascin-C and fibronectin variants in renal cell carcinoma (RCC): implications for an individualized angiogenesis-related targeted drug delivery

The study was aimed at determining the vascular expression of oncofetal fibronectin (oncfFn) and tenascin-C (oncfTn-C) isoforms in renal cell carcinoma (RCC) and its metastases which are well-known targets for antibody-based pharmacodelivery. Furthermore, the influence of tumour cells on endothelial mRNA expression of these molecules was investigated. Evaluation of vascular ED-A(+) and ED-B(+) Fn as well as A1(+) and C(+) Tn-C was performed after immunofluorescence double and triple staining using human recombinant antibodies on clear cell, papillary and chromophobe primary RCC and metastases. The influence of hypoxic RCC-conditioned medium on oncfFn and oncfTn-C mRNA expression was examined in human umbilical vein endothelial cells (HUVEC) by real time RT-PCR. There are RCC subtype specific expression profiles of vascular oncfFn and oncfTn-C and corresponding patterns when comparing primary tumours and metastases. Within one tumour, there are different vessel populations with regard to the incorporation of oncfTn-C and oncfFn into the vessel wall. In vitro tumour-derived soluble mediators induce an up regulation of oncfTn-C and oncfFn mRNA in HUVEC which can be blocked by Avastin(?). Vascular expression of oncFn and oncTn-C variants depends on RCC subtype and may reflect an individual tumour stroma interaction or different stages of vessel development. Therefore, oncFn or oncTn-C variants can be suggested as molecular targets for individualized antibody based therapy strategies in RCC. Tumour-derived VEGF could be shown to regulate target expression.     

Expression and localization of locally produced growth factors regulating lymphangiogenesis during different stages of the estrous cycle in corpus luteum of buffalo (Bubalus bubalis)

Recent experiments using expression, immunolocalization, and cell culture approaches have provided leading insights into regulation of luteal angiogenesis by different growth factor systems and its role in the function of corpus luteum (CL) in buffalo. On the contrary, lymphangiogenesis and its regulation in the CL are still poorly understood. The aim of this study was to evaluate the expression and localization of lymphangiogenic factors (vascular endothelial growth factor [VEGF]-C and VEGFD), their receptor (VEGFR3), and lymphatic endothelial marker (LYVE1) in bubaline CL during different stages of the estrous cycle and to investigate functional role of VEGFC and VEGFD in luteal lymphangeogenesis. The mRNA and protein expression of VEGFC, VEGFD, and VEGFR3 was significantly greater in mid and late luteal phases, which correlated well with the expression of LYVE1. The lymphangiogenic factors were localized in luteal cells, exclusively in the cytoplasm. Immunoreactivity of VEGFC was greater during midluteal phase and that of VEGFD was greater during the mid and late luteal phases. Luteal cells were cultured in vitro and treated for different time duration (24, 48, and 72 hours) with VEGFC and VEGFD each at 50, 100, and 150 ng/mL concentration and VEGFC with VEGFD at 100 ng/mL concentration. The temporal increase in LYVE1 mRNA expression was significant (P < 0.05) in VEGFC and VEGFC with VEGFD treatment and no significant change was seen in VEGFD treatment. Thus, it seems likely that VEGFD itself has little role in lymphangiogenesis but along with VEGFC it might have a synergistic effect on VEGFR3 receptors for inducing lymphangiogenesis. In summary, the present study provided evidence that VEGFC and VEGFD, and their receptor VEGFR3, are expressed in bubaline CL and are localized exclusively in the cell cytoplasm, suggesting that these factors have a functional role in lymphangiogenesis of CL in buffalo.