The intratumoural subsite and relation of CD8(+) and FOXP3(+) T lymphocytes in colorectal cancer provide important prognostic clues.

BACKGROUND: To find improved tools for prognostic evaluation in patients with colorectal cancer (CRC), we have analysed how infiltration of cytotoxic T lymphocytes (CD8(+)) and regulatory T lymphocytes (FoxP3(+)) correlates to prognosis, not only according to quantity and relation, but also to subsite within tumours of different molecular characteristics (microsatellite instability and CpG island methylator phenotype status). METHODS: CD8 and FOXP3 expression was evaluated by immunohistochemistry in 426 archival tumour tissue samples from patients surgically resected for CRC. The average infiltration of CD8(+) and FOXP3(+) cells was assessed along the tumour invasive front, in the tumour centre and within the tumour epithelium (intraepithelial). RESULTS: We found that infiltration of CD8(+) T lymphocytes within the tumour epithelium provided the strongest prognostic information (P<0.001). At the tumour invasive front and tumour centre, FOXP3 expression withheld the strongest association to prognosis (P<0.001), suggesting FOXP3(+) T-lymphocyte infiltration to be a better prognostic tool than CD8(+) T lymphocytes at these intratumoural subsites. We further analysed the possible prognostic impact of the relation between these T-cell subsets, finding that a high intraepithelial CD8 expression was associated with a better patient outcome, independent of FOXP3 infiltration. In groups of low intraepithelial CD8 expression, however, a high infiltration rate of FOXP3(+) cells at the tumour invasive front, significantly improved prognosis. CONCLUSIONS: Analyses of intraepithelial infiltration of CD8(+) T lymphocytes, infiltration of FOXP3(+) T lymphocytes at the tumour front or centre, and the relation between these subsets, may be a valuable tool for predicting prognosis in colon cancer.

The prognostic role of KRAS, BRAF, PIK3CA and PTEN in colorectal cancer.

BACKGROUND: Mutations in KRAS, BRAF, PIK3CA and PTEN expression have been in focus to predict the effect of epidermal growth factor receptor-blocking therapy in colorectal cancer (CRC). Here, information on these four aberrations was collected and combined to a Quadruple index and used to evaluate the prognostic role of these factors in CRC. PATIENTS: We analysed the mutation status in KRAS, BRAF and PIK3CA and PTEN expression in two separate CRC cohorts, Northern Sweden Health Disease Study (NSHDS; n=197) and Colorectal Cancer in Umeå Study (CRUMS; n=414). A Quadruple index was created, where Quadruple index positivity specifies cases with any aberration in KRAS, BRAF, PIK3CA or PTEN expression. RESULTS: Quadruple index positive tumours had a worse prognosis, significant in the NSHDS but not in the CRUMS cohort (NSHDS; P=0.003 and CRUMS; P=0.230) in univariate analyses but significance was lost in multivariate analyses. When analysing each gene separately, only BRAF was of prognostic significance in the NSHDS cohort (multivariate HR 2.00, 95% CI: 1.16-3.43) and KRAS was of prognostic significance in the CRUMS cohort (multivariate HR 1.48, 95% CI: 1.02-2.16). Aberrations in PIK3CA and PTEN did not add significant prognostic information. CONCLUSIONS: Our results suggest that establishment of molecular subgroups based on KRAS and BRAF mutation status is important and should be considered in future prognostic studies in CRC.

Calmodulin-dependent kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of IkappaB kinase.

Numerous fundamental biological processes involve the NFkappaB family of transcription factors. The mechanisms by which this family of proteins is regulated are therefore of widespread importance. In most cells, NFkappaB is bound to inhibitory IkappaB proteins and sequestered in the cytoplasm. NFkappaB-inducing signals result in activation of a large multisubunit kinase complex, IKK, which phosphorylates IkappaB. IkappaB is subsequently degraded, releasing NFkappaB, which translocates to the nucleus. We previously reported that inhibitors of the calcium-binding protein calmodulin (CaM) prevent phorbol ester-induced phosphorylation of IkappaB. Here we show that KN93, an inhibitor of CaM-dependent kinases (CaMKs), also inhibits the phosphorylation of IkappaB. The effect of both CaM and CaMK inhibitors on IkappaB phosphorylation is due to the inhibition of the activity of CaMK II because neither drug has any effect when a derivative of CaMK II that is insensitive to these inhibitors is expressed. When CaMK II is inhibited, phorbol ester is no longer able to activate IKK, placing CaMK II in the signaling pathway that leads to IKK activation. CaM and CaMK inhibitors also block T cell receptor/CD3-induced activation but have no effect on the ability of the cytokine tumor necrosis factor alpha or the phosphatase inhibitor calyculin A to induce degradation of IkappaB. Finally we show that expression of a constitutively active CaMK II results in the activation of NFkappaB. The results identify CaMK II as a mediator of IKK activation specifically in response to T cell receptor/CD3 and phorbol ester stimulation.