PD-L1 in squamous cell carcinoma of the oral tongue shows gender-specific association with prognosis.

OBJECTIVE: To use alternative quantitation approaches to clarify the clinical implication of programmed cell death ligand 1 (PD-L1) in squamous cell carcinoma of the oral tongue (SCCOT). MATERIALS AND METHODS: Ventana SP263 immunohistochemistry assay and a multiplicative QuickScore method were applied to quantify PD-L1 in tumor and surrounding immune cells from 101 patients with SCCOT. Tumor-infiltrating immune cells were estimated from bulk tissue transcriptional profiles of 25 patients. Circulating PD-L1 levels were measured in serum from 30 patients using an electrochemiluminescence assay platform. RESULTS: We found higher tumor cell PD-L1 levels in females than males (p = .019). For patients with low PD-L1 in tumor cells, better survival was seen in males than females (overall survival p = .021, disease-free survival p = .020). Tumor-infiltrating natural killer T cells, immature dendritic cells, and M1 macrophages were positively associated with tumor cell PD-L1 (p < .05). CONCLUSIONS: Our data confirmed the significance of gender on tumor cell PD-L1 expression and demonstrated combined effects of gender and PD-L1 levels on clinical outcome in patients with SCCOT. The data also indicated the involvement of specific immune cell types in PD-L1-regulated immune evasion.

Regional cyclin D1 overexpression or hypoxia correlate inversely with heterogeneous oestrogen receptor-alpha expression in human breast cancer.

The oestrogen receptor-alpha (ER alpha) differs in expression and regulation in breast cancer and, by studying the co-factor cyclin D1 as well as changes in the microenvironment, we here delineate two conditions that potentially cause regional down-regulation of the receptor. Heterogeneously expressed ER alpha was observed in 24 out of 134 of the studied breast cancer samples. In 6 out of 24 of the heterogeneous tumours, there was a clear inverse association between cyclin D1 protein/gene amplification and presence of ER alpha. In a subset of 120 tumours, analysed by Western blotting and ELISA, we further showed disparity in differentiation grade and proliferation between tumours with varied cyclin D1/ER alpha content. In another fraction (9 out of 24) of the heterogeneous tumours, there was an inverse association between the ER alpha and Hypoxia-Inducible Factor-1 alpha (HIF-1 alpha) suggesting that ER alpha was down-regulated during hypoxic conditions. These results were verified by culturing ER alpha breast cancer cell lines under hypoxic conditions showing a prominent down-regulation of the ER alpha. The two factors linked to ER alpha down-regulation can also be used to design new treatment approaches interfering with key proteins in these ER alpha regulatory pathways, thereby hopefully increasing the effect of anti-hormonal treatment.

C-erbB2, p27 and G1/S aberrations in human primary breast cancer.

BACKGROUND: C-erbB2 is overexpressed in approximately one-fourth of human breast cancers. In spite of numerous reports suggesting a connection of c-erbB2 overexpression with cell cycle regulation through p27, D cyclins and c-myc, the relationship between c-erbB2 and proliferation through de-regulation of the pRb pathway in primary breast cancer has not been fully clarified. MATERIALS AND METHODS: For this purpose we compared the expression of c-erbB2 in a total of 105 primary breast tumours with a variety of cell cycle proteins and clinical parameters. RESULTS: C-erbB2 was strongly correlated with down-regulation of p27 and overexpression of c-erbB2 was, as expected, associated with poor survival. However, there was no correlation with proliferation. There was, nevertheless, an association between c-erbB2 and proliferation in certain subtypes of breast cancer, as in oestrogen-receptor-positive tumours, tumours with high cyclin D1, and in tumours with an overall linear pRb pathway, i.e. tumours with a preserved linearity between cyclin D1, pRb phosphorylation and proliferation. CONCLUSION: Our results suggest that c-erbB2 may have alternative functions in different subtypes of breast cancer, and further stress that c-erbB2, in addition to promoting proliferation, also functions through other mechanisms in breast cancer.

Tumor specific VEGF-A and VEGFR2/KDR protein are co-expressed in breast cancer.

Angiogenesis is a prognostic indicator in primary breast cancer regulated by specific angiogenic factors and their receptors. Vascular endothelial growth factor-A (VEGF-A), so far considered the most important, acts through dimerization of the receptor VEGFR2/KDR within the receptor tyrosine kinase family of VEGF receptors. In order to study the interplay between VEGF-A and VEGFR2/KDR in breast cancer we evaluated their expression by immunohistochemistry in 102 breast cancers organized in a tumor tissue array system allowing semi-quantitative evaluation of cytoplasmatic staining intensity. In addition, VEGF-A165 was analyzed by an enzyme immuno assay (ELISA) in protein extracts prepared from frozen tissue from 98 of 102 tumors included in the array. Cytoplasmatic staining of VEGF of varying intensity was observed in all samples and correlated with the ELISA results of VEGF content (p = 0.007). Interestingly, VEGFR2/KDR expression correlated with VEGF expression using immunohistochemistry, indicating that VEGF and VEGFR2/KDR may be co-expressed in breast cancer. Furthermore, high levels of VEGF-A165 in the protein extracts was associated with impaired short time survival but not long term survival whereas immunohistochemically assessed VEGF and VEGFR2/KDR were not significantly associated with survival. In summary, immunohistochemically based analysis of VEGF using a tumor tissue array system seems to be a useful method for VEGF quantification in breast cancer here validated using an ELISA based method. The tumor tissue array system enables opportunities of simultaneous analysis of markers engaged in angiogenesis justifying further studies using larger series of tumors.

The cyclin D1 high and cyclin E high subgroups of breast cancer: separate pathways in tumorogenesis based on pattern of genetic aberrations and inactivation of the pRb node.

In an attempt to identify subtypes of breast cancer and pinpoint patterns of cell cycle regulatory defects associated with clinical behaviour, proliferation and other transformation associated events, a multitude of cell cycle regulatory proteins were analysed in a material of 113 primary breast cancers. Increased proliferation was observed in two different scenarios; (1) with high cyclin D1 and elevated retinoblastoma protein (pRb) phosphorylation, (cyclin D1(high) tumours) or (2) with high cyclin E protein but low cyclin D1 and lack of corresponding pRb phosphorylation (cyclin E(high) tumours) indicative of an interrupted pRb pathway. Characteristic for cyclin E(high) tumours were further defects in p53, p27 and bcl-2, while c-erbB2 overexpression and c-myc amplification was found in both cyclin D1(high) and E(high) tumours. Using transfected cell lines overexpressing cyclin E, cyclin E(high) and D1(high) tumours were mimicked and the cyclin D1(high) cell line normalized the cyclin E kinase activity by an induction and redirection of p21 and p27 to the cyclin E complex whereas cyclin E(high) cell lines obtained increased kinase activity without redirection of inhibitors. Based on differences in genetic aberrations as well as function of the pRb node we therefore propose a model in which cyclin D1(high) and cyclin E(high) tumours represent two alternative mechanisms to inactivate the pRb pathway and thereby achieve unrestrained growth in the tumorogenesis of breast cancer.