Ninke Leffers

The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis.

Background:Tumour-infiltrating lymphocytes (TILs) are often found in tumours, presumably reflecting an immune response against the tumour. We carried out a systematic review and meta-analysis, aiming to establish pooled estimates for survival outcomes based on the presence of TILs in cancer.Methods:A Pubmed and Embase literature search was designed. Studies were included, in which the prognostic significance of intratumoural CD3+, CD4+, CD8+, and FoxP3+ lymphocytes, as well as ratios between these subsets, were determined in solid tumours.Results:In pooled analysis, CD3+ TILs had a positive effect on survival with a hazard ratio (HR) of 0.58 (95% confidence interval (CI) 0.43–0.78) for death, as did CD8+ TILs with a HR of 0.71 (95% CI 0.62–0.82). FoxP3+ regulatory TILs were not linked to overall survival, with a HR of 1.19 (95% CI 0.84–1.67). The CD8/FoxP3 ratio produced a more impressive HR (risk of death: HR 0.48, 95% CI 0.34–0.68), but was used in relatively few studies. Sample size and follow-up time seemed to influence study outcomes.Conclusion:Any future studies should be carefully designed, to prevent overestimating the effect of TILs on prognosis. In this context, ratios between TIL subsets may be more informative.

Prognostic significance of tumor-infiltrating T-lymphocytes in primary and metastatic lesions of advanced stage ovarian cancer

PurposeOvarian cancer patients with intra-tumoral CD3+ T-lymphocytes in primary tumor tissue have a better prognosis. This study aims to analyze the presence and relative influence of three important T-lymphocyte subsets, tumor-infiltrating CD8+ cytotoxic T-lymphocytes (CTL), CD45R0+ memory T-lymphocytes, and FoxP3+ regulatory T-lymphocytes (Treg), in primary tumor tissue and omental metastases of patients with ovarian cancer.Experimental designThe number of CD8+, CD45R0+, and FoxP3+ T-lymphocytes was determined by immunohistochemistry on a tissue micro array containing ovarian tumor tissue and/or omental metastases obtained at primary debulking surgery from 306 FIGO stage I–IV ovarian cancer patients. Immunohistochemistry data were correlated to clinicopathological parameters and survival data.ResultsHigh number of CD8+ CTL and a high CD8+/FoxP3+ ratio in ovarian-derived tumor tissue were associated with increased disease-specific survival and proved to be independent prognostic factors in multivariate analyses. In advanced stage patients, the presence of CD8+ CTL, CD45R0+ memory T-lymphocytes, FoxP3+ Treg or a high CD8+/FoxP3+ ratio in ovarian-derived tumor tissue was associated with an increased disease specific survival in univariate analysis, as was the presence of CD45R0+ memory T-lymphocytes and FoxP3+ Treg in omental metastases. Furthermore, in advanced stage patients CD8+ cytotoxic and FoxP3+ regulatory T-lymphocytes infiltrating ovarian-derived tumor tissue were independent predictors of increased prognosis.ConclusionsT-lymphocytes infiltrating primary and metastatic ovarian cancer sites are associated with improved prognosis. These associations are especially distinct in advanced stage patients, underlining the potential for immunotherapy as a broadly applicable therapeutic strategy.

Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial

The prognosis of ovarian cancer, the primary cause of death from gynecological malignancies, has only modestly improved over the last decades. Immunotherapy is one of the new treatment modalities explored for this disease. To investigate safety, tolerability, immunogenicity and obtain an impression of clinical activity of a p53 synthetic long peptide (p53‐SLP) vaccine, twenty patients with recurrent elevation of CA‐125 were included, eighteen of whom were immunized 4 times with 10 overlapping p53‐SLP in Montanide ISA51. The first 5 patients were extensively monitored for toxicity, but showed no ≥ grade 3 toxicity, thus accrual was continued. Overall, toxicity was limited to grade 1 and 2, mostly locoregional, inflammatory reactions. IFN‐γ producing p53‐specific T‐cell responses were induced in all patients who received all 4 immunizations as measured by IFN‐γ ELISPOT. An IFN‐γ secretion assay showed that vaccine‐induced p53‐specific T‐cells were CD4+, produced both Th1 and Th2 cytokines as analyzed by cytokine bead array. Notably, Th2 cytokines dominated the p53‐specific response. P53‐specific T‐cells were present in a biopsy of the last immunization site of at least 9/17 (53%) patients, reflecting the migratory capacity of p53‐specific T‐cells. As best clinical response, stable disease evaluated by CA‐125 levels and CT‐scans, was observed in 2/20 (10%) patients, but no relationship was found with vaccine‐induced immunity. This study shows that the p53‐SLP vaccine is safe, well tolerated and induces p53‐specific T‐cell responses in ovarian cancer patients. Upcoming trials will focus on improving T helper‐1 polarization and clinical efficacy.

Presence of tumor-infiltrating lymphocytes is an independent prognostic factor in type I and II endometrial cancer

OBJECTIVE Presence of tumor-infiltrating lymphocytes (TIL) is of prognostic importance in a variety of malignancies. This study aims to determine the prognostic value of CD8(+) cytotoxic T-lymphocytes (CTL), FoxP3(+) regulatory T-lymphocytes (Treg) and CD45R0(+) memory T-lymphocytes in endometrial cancer. METHODS The number of tumor-infiltrating CD8(+), FoxP3(+), and CD45R0(+) T-lymphocytes was determined by immunohistochemistry on tissue microarrays containing tumor material from 368 FIGO stage I-IV endometrial cancer patients. Results from immunohistochemistry were correlated with clinicopathological parameters and survival. RESULTS High numbers of intra-tumoral CD8(+) T-lymphocytes, a high CD8(+)/FoxP3(+) ratio and the presence of CD45R0(+) T-lymphocytes were strongly associated with well-known favorable prognostic factors in endometrial cancer. Furthermore, high numbers of CD8(+) T-lymphocytes and a high CD8(+)/FoxP3(+) ratio were associated with a better disease free survival (DFS). High numbers of CD8(+) T-lymphocytes and the presence of CD45R0(+) T-lymphocytes were associated with a prolonged overall survival (OS). In multivariate analysis, high numbers of CD8(+) T-lymphocytes had an independent prognostic impact for overall survival in the entire cohort (HR 0.48, 95% C.I. 0.26-0.89, p=0.019) and in type II endometrial cancer (HR 0.17, 95% C.I. 0.08-0.36, p<0.001). A high CD8(+)/FoxP3(+) ratio was independently associated with improved survival in type I endometrial cancer (HR 0.44, 95% C.I. 0.23-0.84, p=0.013). CD45R0(+) lymphocytes were an independent factor for improved OS (HR 0.42, 95% C.I. 0.19-0.93, p=0.033). CONCLUSION This study shows that the presence of TIL is an independent prognostic factor in endometrial cancer and indicates an important role for the immune system in endometrial cancer.

Serum Cytokine Profiling as a Diagnostic and Prognostic Tool in Ovarian Cancer: A Potential Role for Interleukin 7

Purpose: To evaluate if serum cytokine levels could be used as diagnostic or prognostic markers in ovarian cancer. Experimental Design: A cytokine bead array was done to simultaneously analyze 14 cytokines in the sera of 187 ovarian cancer patients with complete clinicopathologic data and follow-up, 45 patients with benign ovarian tumors, and 50 healthy controls. Serum levels of the well-known serum tumor marker CA-125 were routinely measured in all patients. Results: Serum levels of CA-125, interleukin 6 (IL-6), IL-7, and IL-10 were elevated in ovarian cancer patients compared with patients with benign ovarian tumors. Analyzing the cytokines in combination with CA-125 showed that a combination of IL-7 and CA-125 serum levels could accurately predict 69% of the ovarian cancer patients, without falsely classifying patients with benign pelvic mass. The cytokines IL-6, IL-7, IL-8, IL-10, monocyte chemotactic protein-1 (MCP-1), and IP-10 and CA-125 were associated with disease-free and overall survival in univariate analysis. In multivariate analysis, IL-7 and IP-10 were independent predictors of overall survival, although after inclusion of the clininopathologic parameters, only stage and residual disease remained as independent predictors of survival. Conclusions: IL-7 levels were found to be strongly associated with ovarian cancer and could be used in combination with CA-125 to distinguish between malignant and benign ovarian tumors.

Potentiation of a p53‐SLP vaccine by cyclophosphamide in ovarian cancer: A single‐arm phase II study

The purpose of the current phase II single‐arm clinical trial was to evaluate whether pretreatment with low‐dose cyclophosphamide improves immunogenicity of a p53‐synthetic long peptide (SLP) vaccine in patients with recurrent ovarian cancer. Patients with ovarian cancer with elevated serum levels of CA‐125 after primary treatment were immunized four times with the p53‐SLP vaccine. Each immunization was preceded by administration of 300 mg/m2 intravenous cyclophosphamide as a means to affect regulatory T cells (Tregs). Vaccine‐induced p53‐specific interferon‐gamma (IFN‐γ)‐producing T cells evaluated by IFN‐γ ELISPOT were observed in 90% (9/10) and 87.5% (7/8) of evaluable patients after two and four immunizations, respectively. Proliferative p53‐specific T cells, observed in 80.0% (8/10) and 62.5% (5/8) of patients, produced both T‐helper 1 and T‐helper‐2 cytokines. Cyclophosphamide induced neither a quantitative reduction of Tregs determined by CD4+FoxP3+ T cell levels nor a demonstrable qualitative difference in Treg function tested in vitro. Nonetheless, the number of vaccine‐induced p53‐specific IFN‐γ‐producing T cells was higher in our study compared to a study in which a similar patient group was treated with p53‐SLP monotherapy (p ≤ 0.012). Furthermore, the strong reduction in the number of circulating p53‐specific T cells observed previously after four immunizations was currently absent. Stable disease was observed in 20.0% (2/10) of patients, and the remainder of patients (80.0%) showed clinical, biochemical and/or radiographic evidence of progressive disease. The outcome of this phase II trial warrants new studies on the use of low‐dose cyclophosphamide to potentiate the immunogenicity of the p53‐SLP vaccine or other antitumor vaccines.

The prognostic role of classical and nonclassical MHC class I expression in endometrial cancer

The aim of this study was to investigate classical MHC class I and nonclassical MHC (human leukocyte antigen‐G [HLA‐G]) expression in a large cohort of patients with endometrial cancer, to determine the prognostic value of these cell surface markers and their relation with clinicopathological variables. Tissue microarrays containing epithelial endometrial carcinoma tissue from 554 patients were stained for classical and nonclassical MHC class I using the following monoclonal antibodies: 4H84 (anti‐HLA‐G), β2‐m (anti‐beta‐2‐microglobulin) and HC‐10 (MHC class I antigen heavy chain). Expression data were linked to known clinicopathological characteristics and survival. HLA‐G upregulation and MHC class I downregulation in neoplastic cells was observed in 40% and 48%, respectively. Nonendometrioid tumor type, advanced stage disease (FIGO stage ≥II) and poorly or undifferentiated tumors were associated with MHC class I downregulation. Absence of HLA‐G expression was independently associated with MHC class I downregulation. In univariate analysis, MHC class I downregulation was a predictor of worse disease‐specific survival. Prognostic unfavorable tumor characteristics were correlated with downregulation of MHC class I expression in endometrial cancer cells. Furthermore, downregulated MHC class I has a negative impact on disease‐specific survival, observed in a large cohort of patients with endometrial cancer. As there seems to be a relation between classical and nonclassical MHC class I molecules (HLA‐G), further research is warranted to unravel this regulatory mechanism.

Long‐term clinical and immunological effects of p53‐SLP® vaccine in patients with ovarian cancer

Vaccine‐induced p53‐specific immune responses were previously reported to be associated with improved response to secondary chemotherapy in patients with small cell lung cancer. We investigated long‐term clinical and immunological effects of the p53‐synthetic long peptide (p53‐SLP®) vaccine in patients with recurrent ovarian cancer. Twenty patients were immunized with the p53‐SLP® vaccine between July 2006 and August 2007. Follow‐up information on patients was obtained. Clinical responses to secondary chemotherapy after p53‐SLP® immunizations were determined by computerized tomography and/or tumor marker levels (CA125). Disease‐specific survival was compared to a matched historical control group. Immune responses were analyzed by flow cytometry, proliferation assay, interferon gamma (IFN‐γ) ELISPOT and/or cytokine bead array. Lymphocytes cultured from skin biopsy were analyzed by flow cytometry and proliferation assay. Of 20 patients treated with the p53‐SLP® vaccine, 17 were subsequently treated with chemotherapy. Eight of these patients volunteered another blood sample. No differences in clinical response rates to secondary chemotherapy or disease‐specific survival were observed between immunized patients and historical controls (p = 0.925, resp. p = 0.601). p53‐specific proliferative responses were observed in 5/8 patients and IFN‐γ production in 2/7 patients. Lymphocytes cultured from a prior injection site showing inflammation during chemotherapy did not recognize p53‐SLP®. Thus, treatment with the p53‐SLP® vaccine does not affect responses to secondary chemotherapy or survival, although p53‐specific T‐cells do survive chemotherapy.

P53-specific T cell responses in patients with malignant and benign ovarian tumors : Implications for p53 based immunotherapy

Despite intensive treatment, 70% of the ovarian cancer patients will develop recurrent disease, emphasizing the need for new approaches such as immunotherapy. A promising antigenic target for immunotherapy in ovarian cancer is the frequently overexpressed p53 protein. The aim of the study was to evaluate the nature and magnitude of the baseline anti‐p53 immune response in ovarian cancer patients. P53‐specific T cell responses were detected in both half of the ovarian cancer patients as in the group of control subjects, consisting of women with benign ovarian tumors and healthy controls. Importantly, while in the control group p53‐specific immunity was detected among the CD45RA+ naïve subset of T cells only, the p53‐specific T‐cell responses in ovarian cancer patients were also present in the CD45RO+ memory T‐cell subset, suggesting that in the cancer patients sufficient amounts of cancer‐derived p53 was presented to induce the formation of a p53‐specific memory T‐cell response. Further characterization of the p53‐specific memory T‐cell responses revealed that in addition to the type 1 cytokine IFN‐γ also the type 2 cytokines IL‐4 and IL‐5, as well as the immunosuppressive cytokine IL‐10 were produced. Notably, p53‐specific T cells were not only detected in the peripheral blood, but also among tumor infiltrating lymphocytes and in tumor‐draining lymph nodes. In conclusion, the existence of a weak mixed T‐helper type 1 and 2 p53‐specific T‐cell repertoire supports the rationale of using p53 long peptides in vaccination strategies aiming at the induction of p53‐specific Th1/CTL immunity.

Identification of genes and pathways associated with cytotoxic T lymphocyte infiltration of serous ovarian cancer

Background:Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer.Methods:For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated.Results:The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment.Conclusion:Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.