Classical and non-classical HLA class I aberrations in primary cervical squamous- and adenocarcinomas and paired lymph node metastases.

BACKGROUND: Tumors avoid destruction by cytotoxic T cells (CTL) and natural killer (NK) cells by downregulation of classical human leukocyte antigens (HLA) and overexpression of non-classical HLA. This is the first study to investigate HLA expression in relation to histology (squamous cell carcinoma (SCC) vs. adenocarcinoma (AC)), clinicopathological parameters and survival in a large cervical cancer patient cohort. METHODS: Classical (HLA-A and HLA-B/C)- and non-classical HLA molecules (HLA-E and HLA-G) were studied on primary tumors and paired lymph node (LN) metastases from cervical cancer patients (n = 136) by immunohistochemistry. The Chi2 test was used for the comparison of clinicopathological characteristics between SCC and AC patients. The Related-Samples Wilcoxon Signed Rank test was used to compare HLA expression between the primary tumor and metastasis in LN. Patient survival rates were analyzed by Kaplan-Meier curves and Log Rank test. The Mann-Whitney U Test was used to compare the distribution of HLA class I expression between SCC and AC. RESULTS: Decreased expression of HLA-A (SCC P < 0.001), HLA-B/C (SCC P < 0.01; AC P < 0.01) and total classical HLA (SCC P < 0.001; AC P = 0.02) was apparent in metastatic tumor cells compared to the primary tumor. In primary SCC, there was a clear trend towards complete loss of HLA-A (P = 0.05). SCC metastases showed more complete loss of HLA-A, while AC metastases showed more complete loss of HLA-B/C (P = 0.04). In addition, tumor size and parametrium involvement were also related to aberrant HLA class I expression. No significant associations between HLA expression and disease-specific (DSS) or disease-free survival (DFS) were found in this advanced disease cohort. However, in the SCC group, samples showing loss of HLA-A or loss of total classical HLA but positive for HLA-G were linked to poor patient survival (DSS P = 0.001 and P = 0.01; DFS P = 0.003 and P = 0.01, for HLA-A and total classical HLA, respectively). CONCLUSION: These results strengthen the idea of tumor immune escape variants leading to metastasis. Moreover, SCC tumors showing downregulation of HLA-A or total classical HLA in combination with HLA-G expression had poor prognosis. Our findings warrant further analysis of HLA expression as a biomarker for patient selection for CTL- and NK- cell based immunotherapeutic intervention.

Indoleamine-2,3-dioxygenase (IDO) metabolic activity is detrimental for cervical cancer patient survival.

The expression of the immunomodulating enzyme indoleamine-2,3-dioxygenase (IDO) suppresses T-lymphocyte function, thus correlating with poor survival in a variety of cancer patients. IDO degrades the essential amino acid tryptophan leading to immunosuppressive kynurenines production. In the present study, concentrations of tryptophan, 3-hydroxykynurenine, and kynurenine were measured in pre-treatment serum samples of 251 cervical cancer patients by a mass-spectrometric method (XLC-MS/MS) and IDO activity determined by the kynurenine/tryptophan (Kyn/Trp) ratio. A low concentration of tryptophan was found to be significantly associated with tumors greater than 4 cm and lymph node metastatic spread. Furthermore, significant positive correlations were found between high concentrations of the tryptophan metabolites kynurenine and 3-hydroxykynurenine and advanced disease stage (FIGO >IIA) and lymph node metastases. High levels of kynurenine were further associated with parametrial invasion and tumor size. A high Kyn/Trp ratio was related to lymph node metastasis, FIGO stage, tumor size, parametrial invasion and poor disease-specific survival. These results suggest that IDO activation is linked to poor clinicopathological parameters and worse survival in cervical cancer, warranting the use of IDO inhibitors in future clinical trials.

High and interrelated rates of PD-L1+CD14+ antigen-presenting cells and regulatory T cells mark the microenvironment of metastatic lymph nodes from patients with cervical cancer.

A better understanding of the microenvironment in relation to lymph node metastasis is essential for the development of effective immunotherapeutic strategies against cervical cancer. In the present study, we investigated the microenvironment of tumor-draining lymph nodes of patients with cervical cancer by comprehensive flow cytometry-based phenotyping and enumeration of immune-cell subsets in tumor-negative (LN(-), n = 20) versus tumor-positive lymph nodes (LN(+), n = 8), and by the study of cytokine release profiles (n = 4 for both LN(-) and LN(+)). We found significantly lower CD4(+) and higher CD8(+) T-cell frequencies in LN(+) samples, accompanied by increased surface levels of activation markers (HLA-DR; ICOS; PD-1; CTLA-4) and the memory marker CD45RO. Furthermore, in LN(+), we found increased rates of a potentially regulatory antigen-presenting cell (APC) subset (CD11c(hi)CD14(+)PD-L1(+)) and of myeloid-derived suppressor cell subsets; the LN(+) APC subset correlated with significantly elevated frequencies of FoxP3(+) regulatory T cells (Treg). After in vitro stimulation with different Toll-like receptor (TLR) ligands (PGN; Poly-IC; R848), we observed higher production levels of IL6, IL10, and TNFα but lower levels of IFNγ in LN(+) samples. We conclude that, despite increased T-cell differentiation and activation, a switch to a profound immune-suppressive microenvironment in LN(+) of patients with cervical cancer will enable immune escape. Our data indicate that the CD14(+)PD-L1(+) APC/Treg axis is a particularly attractive and relevant therapeutic target to specifically tackle microenvironmental immune suppression and thus enhances the efficacy of immunotherapy in patients with metastasized cervical cancer.