An expression signature of the angiogenic response in gastrointestinal neuroendocrine tumours: correlation with tumour phenotype and survival outcomes.

BACKGROUND: Gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are heterogeneous with respect to biological behaviour and prognosis. As angiogenesis is a renowned pathogenic hallmark as well as a therapeutic target, we aimed to investigate the prognostic and clinico-pathological role of tissue markers of hypoxia and angiogenesis in GEP-NETs. METHODS: Tissue microarray (TMA) blocks were constructed with 86 tumours diagnosed from 1988 to 2010. Tissue microarray sections were immunostained for hypoxia inducible factor 1α (Hif-1α), vascular endothelial growth factor-A (VEGF-A), carbonic anhydrase IX (Ca-IX) and somatostatin receptors (SSTR) 1–5, Ki-67 and CD31. Biomarker expression was correlated with clinico-pathological variables and tested for survival prediction using Kaplan–Meier and Cox regression methods. RESULTS: Eighty-six consecutive cases were included: 51% male, median age 51 (range 16–82), 68% presenting with a pancreatic primary, 95% well differentiated, 51% metastatic. Higher grading (P=0.03), advanced stage (P<0.001), high Hif-1α and low SSTR-2 expression (P=0.03) predicted for shorter overall survival (OS) on univariate analyses. Stage, SSTR-2 and Hif-1α expression were confirmed as multivariate predictors of OS. Median OS for patients with SSTR-2+/Hif-1α-tumours was not reached after median follow up of 8.8 years, whereas SSTR-2-/Hif-1α+ GEP-NETs had a median survival of only 4.2 years (P=0.006). CONCLUSION: We have identified a coherent expression signature by immunohistochemistry that can be used for patient stratification and to optimise treatment decisions in GEP-NETs independently from stage and grading. Tumours with preserved SSTR-2 and low Hif-1α expression have an indolent phenotype and may be offered less aggressive management and less stringent follow up

FOXP3 infiltrating lymphocyte density and PD-L1 expression in operable non-small cell lung carcinoma

Purpose/Aim: Regulatory FOXP3+ T-cells control the cytotoxic activity of effector cells and may have an essential role in the development of immune tolerance in cancer patients. Programed death ligand 1 PD-L1, expressed on cancer cell membranes also blocks the cytotoxic activity of PD1+ cytotoxic lymphocytes. Materials and Methods: We assessed the immunohistochemical detection of these immune-tolerance related markers in a series of 98 non-small cell lung carcinomas (NSCLC) treated with surgery. The Tumor Infiltration Lymphocyte TIL density (mean number per x400 optical field) and the percentage of FOXP3+ TILs were assessed. Results: PD-L1 expression was directly linked with the TIL density (p = 0.01) and with the extent of infiltration with FOXP3+ TILs, named as the FIL-score (p = 0.01). FIL-score was significantly higher in stage I disease (p = 0.04). IL6 expression was linked with high TIL-score. A low TIL-score, characterizing immune deficient tumors defined a significantly poorer prognosis subgroup of patients (p = 0.03). Stratification of these tumors according to the FIL-score showed that FOXP3 expression by TILs correlated with an even a poorer prognosis in univariate (p = 0.007; median survival 14 vs. 44 months, respectively) and in multivariate analysis (p = 0.01, hazard ratio 4.3). Conclusion: Tumor stroma infiltration by FOXP3+ Tregs is an early event in the progression of NSCLC. Low lymphocytic infiltration defines poor prognosis, which becomes worse when the small numbers of infiltrating lymphocytes characterizing these tumors contain FOXP3 + Tregs. Furthermore, the direct association of FOXP3+ Treg infiltration density with PD-L1 expression by cancer cells implies a co-ordinated immune-suppressive activity in NSCLC

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

Cancer stem cell markers in breast cancer: pathological, clinical and prognostic significance

INTRODUCTION: The cancer stem cell (CSC) hypothesis states that tumours consist of a cellular hierarchy with CSCs at the apex driving tumour recurrence and metastasis. Hence, CSCs are potentially of profound clinical importance. We set out to establish the clinical relevance of breast CSC markers by profiling a large cohort of breast tumours in tissue microarrays (TMAs) using immunohistochemistry (IHC). METHODS: We included 4, 125 patients enrolled in the SEARCH population-based study with tumours represented in TMAs and classified into molecular subtype according to a validated IHC-based five-marker scheme. IHC was used to detect CD44/CD24, ALDH1A1, aldehyde dehydrogenase family 1 member A3 (ALDH1A3) and integrin alpha-6 (ITGA6). A 'Total CSC' score representing expression of all four CSC markers was also investigated. Association with breast cancer specific survival (BCSS) at 10 years was assessed using a Cox proportional-hazards model. This study was complied with REMARK criteria. RESULTS: In ER negative cases, multivariate analysis showed that ITGA6 was an independent prognostic factor with a time-dependent effect restricted to the first two years of follow-up (hazard ratio (HR) for 0 to 2 years follow-up, 2.4; 95% confidence interval (95% CI), 1.2 to 4.8; P = 0.009). The composite 'Total CSC' score carried independent prognostic significance in ER negative cases for the first four years of follow-up (HR for 0 to 4 years follow-up, 1.3; 95% CI, 1.1 to 1.6; P = 0.006). CONCLUSIONS: Breast CSC markers do not identify identical subpopulations in primary tumours. Both ITGA6 and a composite Total CSC score show independent prognostic significance in ER negative disease. The use of multiple markers to identify tumours enriched for CSCs has the greatest prognostic value. In the absence of more specific markers, we propose that the effective translation of the CSC hypothesis into patient benefit will necessitate the use of a panel of markers to robustly identify tumours enriched for CSCs

A lactate shuttle system between tumour and stromal cells is associated with poor prognosis in prostate cancer

Background In a malignant tumour, cancer cells are embedded in stromal cells, namely cancer-associated fibroblasts (CAFs). These CAFs are now accepted as important players in cancer dynamics, being involved in tumour growth and progression. Although there are various reports on the interaction between tumour and stromal cells, the clinico-pathological significance of this cross-talk is still largely unknown. In this study, we aimed to characterise the expression of key metabolic proteins involved in glucose transport, pyruvate/lactate shuttle system, glycolytic metabolism and fatty acid oxidation in CAFs and tumour cells in different stages of malignant transformation. We further aimed to contextualise the clinico-pathological significance of these protein expression profiles with reference to known prognostic indicators, including biochemical recurrence in pT stage. Methods Prostate tissues were obtained from 480 patients with a median age of 64 years following radical prostatectomy with no previous hormonal therapy. Tissues were analysed for the expression of several key metabolism-related proteins in glands and surrounding fibroblasts by immunohistochemistry. Reliable markers of prognosis such as pT stage and biochemical recurrence were assessed for each case. Results We observed that prostate cancer cells did not rely mainly on glycolytic metabolism, while there was a high expression of MCT4 and CAIX - in CAFs. This corroborates the hypothesis of the "Reverse Warburg effect" in prostate cancer, in which fibroblasts are under oxidative stress and express CAIX, an established hypoxia marker. We found that alterations in the expression of metabolism-related proteins were already evident in the early stages of malignant transformation, suggesting the continuing alteration of CAFs from an early stage. Additionally, and for the first time, we show that cases showing high MCT4 expression in CAFs with concomitant strong MCT1 expression in prostate cancer (PCa) cells are associated with poor clinical outcome, namely pT3 stage of the tumour. Conclusions In summary, this work demonstrates for the first time the clinico-pathological significance of the lactate shuttle in prostate cancer. It also suggests that other alterations in CAFs may be useful prognostic factors, and further supports the use of MCT1/MCT4 as targets for PCa therapy.NPG received a fellowship from the Portuguese Foundation for Science and Technology (FCT), refs. SFRH/BD/61027/2009. This work was supported by the FCT grant ref. PTDC/SAUMET/113415/2009, under the scope of "Programa Operacional Tematico Factores de Competitividade" (COMPETE) of "Quadro Comunitario de Apoio III" and co-financed by Fundo Comunitario Europeu FEDER. JA was supported by a Boehringer Ingelheim Fonds fellowship

Autophagosome proteins LC3A, LC3B and LC3C have distinct subcellular distribution kinetics and expression in cancer cell lines

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies

Relationship of vascular maturation in breast cancer blood vessels to vascular density and metastasis, assessed by expression of a novel basement membrane component, LH39

Angiogenesis, the formation of new vessels, has been demonstrated to be an indicator of prognosis in breast cancer patients. The extent of differentiation of the tumour vessels may affect access of peripheral white cells and egress or invasion of tumour cells. This has not been assessed in relation to tumour microvessel density or other variables and may be a marker of vascular remodelling. LH39 is a monoclonal antibody recognizing an epitope located at the lamina lucida of mature small veins and capillaries but not in newly formed vessels. To study vascular differentiation in breast tumours, we examined the vascular maturation index (VMI) in 12 normal and 50 breast carcinomas and this was correlated with different clinicopathological variables including angiogenesis. Mature vessels were defined by staining with antibodies to both LH39 and to CD31, using double immunohistochemistry, whereas immature vessels stained only for CD31. VMI was defined as the % fraction of mature vessels (LH39-positive) / total number of vessels (CD31-positive). The VMI was significantly higher in normal (54–68.5%; median 66.5%) than in tumours (0–47%; median 8.8%) (P = 0.0005). There was a significant inverse correlation between the tumour VMI and nodal status (Fisher's exact test, P = 0.01) and between high VMI and low thymidine phosphorylase (TP) expression (Mann–Whitney U -test, P = 0.01). No significant association between VMI and tumour size, oestrogen receptor, epidermal growth factor receptor, grade, angiogenesis, patient age, or E-selectin was seen. There was a significant reduction in relapse-free survival (P = 0.01) with high angiogenesis. These findings show that the VMI gives new information on the mechanism of tumour angiogenesis independently from microvessel quantitation, there is a wide variation in the differentiation of tumour vasculature but the degree of capillary differentiation is not associated with quantitative angiogenesis. The VMI identifies a subset of patients who have a high chance of regional node involvement. © 2000 Cancer Research Campaig