Annechien Plat

Treatment regimen, surgical outcome and T cell differentiation influence prognostic benefit of tumor-infiltrating lymphocytes in high grade serous ovarian cancer

Purpose: Tumor-infiltrating lymphocytes (TIL) are associated with a better prognosis in high-grade serous ovarian cancer (HGSC). However, it is largely unknown how this prognostic benefit of TIL relates to current standard treatment of surgical resection and (neo-)adjuvant chemotherapy. To address this outstanding issue, we compared TIL infiltration in a unique cohort of patients with advanced-stage HGSC primarily treated with either surgery or neoadjuvant chemotherapy. Experimental Design: Tissue microarray slides containing samples of 171 patients were analyzed for CD8+ TIL by IHC. Freshly isolated CD8+ TIL subsets were characterized by flow cytometry based on differentiation, activation, and exhaustion markers. Relevant T-cell subsets (CD27+) were validated using IHC and immunofluorescence. Results: A prognostic benefit for patients with high intratumoral CD8+ TIL was observed if primary surgery had resulted in a complete cytoreduction (no residual tissue). By contrast, optimal (<1 cm of residual tumor) or incomplete cytoreduction fully abrogated the prognostic effect of CD8+ TIL. Subsequent analysis of primary TIL by flow cytometry and immunofluorescence identified CD27 as a key marker for a less-differentiated, yet antigen-experienced and potentially tumor-reactive CD8+ TIL subset. In line with this, CD27+ TIL were associated with an improved prognosis even in incompletely cytoreduced patients. Neither CD8+ nor CD27+ cell infiltration was of prognostic benefit in patients treated with neoadjuvant chemotherapy. Conclusions: Our findings indicate that treatment regimen, surgical result, and the differentiation of TIL should all be taken into account when studying immune factors in HGSC or, by extension, selecting patients for immunotherapy trials. Clin Cancer Res; 22(3); 714–24. ©2015 AACR.

CD103+tumor-infiltrating lymphocytes are tumor-reactive intraepithelial CD8+T cells associated with prognostic benefit and therapy response in cervical cancer

ABSTRACT Human papilloma virus (HPV)-induced cervical cancer constitutively expresses viral E6/E7 oncoproteins and is an excellent target for T cell-based immunotherapy. However, not all tumor-infiltrating T cells confer equal benefit to patients, with epithelial T cells being superior to stromal T cells. To assess whether the epithelial T cell biomarker CD103 could specifically discriminate the beneficial antitumor T cells, association of CD103 with clinicopathological variables and outcome was analyzed in the TCGA cervical cancer data set (n = 304) and by immunohistochemistry (IHC) in an independent cohort (n = 460). Localization of CD103+ cells in the tumor was assessed by immunofluorescence. Furthermore, use of CD103 as a response biomarker was assessed in an in vivo E6/E7+ tumor model. Our results show that CD103 gene expression was strongly correlated with cytotoxic T cell markers (e.g. CD8/GZMB/PD1) in the TCGA series. In line with this, CD103+ cells in the IHC series co-expressed CD8 and were preferentially located in cervical tumor epithelium. High CD103+ cell infiltration was strongly associated with an improved prognosis in both series, and appeared to be a better predictor of outcome than CD8. Interestingly, the prognostic benefit of CD103 in both series seemed limited to patients receiving radiotherapy. In a preclinical mouse model, HPV E6/E7-targeted therapeutic vaccination in combination with radiotherapy increased the intratumoral number of CD103+ CD8+ T cells, providing a potential mechanistic basis for our results. In conclusion, CD103 is a promising marker for rapid assessment of tumor-reactive T cell infiltration of cervical cancers and a promising response biomarker for E6/E7-targeted immunotherapy.

The Major Myelin-Resident Protein PLP Is Transported to Myelin Membranes via a Transcytotic Mechanism: Involvement of Sulfatide

ABSTRACT Myelin membranes are sheet-like extensions of oligodendrocytes that can be considered membrane domains distinct from the cells plasma membrane. Consistent with the polarized nature of oligodendrocytes, we demonstrate that transcytotic transport of the major myelin-resident protein proteolipid protein (PLP) is a key element in the mechanism of myelin assembly. Upon biosynthesis, PLP traffics to myelin membranes via syntaxin 3-mediated docking at the apical-surface-like cell body plasma membrane, which is followed by subsequent internalization and transport to the basolateral-surface-like myelin sheet. Pulse-chase experiments, in conjunction with surface biotinylation and organelle fractionation, reveal that following biosynthesis, PLP is transported to the cell body surface in Triton X-100 (TX-100)-resistant microdomains. At the plasma membrane, PLP transiently resides within these microdomains and its lateral dissipation is followed by segregation into 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS)-resistant domains, internalization, and subsequent transport toward the myelin membrane. Sulfatide triggers PLPs reallocation from TX-100- into CHAPS-resistant membrane domains, while inhibition of sulfatide biosynthesis inhibits transcytotic PLP transport. Taking these findings together, we propose a model in which PLP transport to the myelin membrane proceeds via a transcytotic mechanism mediated by sulfatide and characterized by a conformational alteration and dynamic, i.e., transient, partitioning of PLP into distinct membrane microdomains involved in biosynthetic and transcytotic transport.

CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαβ+ CD8αβ+ T cells that can be targeted for cancer immunotherapy

CD103+ tumor-infiltrating lymphocytes (TIL) have been linked to specific epithelial infiltration and a prolonged survival in high-grade serous epithelial ovarian cancer (HGSC). However, whether these cells are induced as part of an ongoing anti-HGSC immune response or represent non-specifically expanded resident or mucosal lymphocytes remains largely unknown. In this study, we first confirmed that CD103+ TIL from HGSC were predominantly localized in the cancer epithelium and were strongly correlated with an improved prognosis. We further demonstrate that CD103+ TIL were almost exclusively CD3+ TCRαβ+ CD8αβ+ CD4- T cells, but heterogeneously expressed T cell memory and differentiation markers. Activation of peripheral T cells in the presence of HGSC was sufficient to trigger induction of CD103 in over 90% of all CD8+ cells in a T cell receptor (TCR)- and TGFβR1-dependent manner. Finally, CD103+ TIL isolated from primary HGSC showed signs of recent activation and dominantly co-expressed key immunotherapeutic targets PD-1 and CD27. Taken together, our data indicate CD103+ TIL in HGSC are formed as the result of an adaptive anti-tumor immune response that might be reactivated by (dual) checkpoint inhibition.

Microsatellite instability derived JAK1 frameshift mutations are associated with tumor immune evasion in endometrioid endometrial cancer

JAK1 frameshift mutations may promote cancer cell immune evasion by impeding upregulation of the antigen presentation pathway in microsatellite unstable endometrial cancers (ECs). This study investigated the JAK1 mutation frequency, its functional implication in immune evasion and its prognostic significance in microsatellite unstable EC. Microsatellite instability and three microsatellite repeats within JAK1 were analyzed in 181 ECs. Sixty-two (34%) ECs showed microsatellite instability, of which 22 (35%) had a JAK1 mutation. LMP7, TAP1 and HLA class I protein expression and the presence of CD8-positive T-cells were analyzed in the microsatellite unstable ECs. JAK1 mutant microsatellite unstable ECs showed impaired upregulation of LMP7 (P=0.074) and HLA class I (P<0.001), validated using RNAseq data of the TCGA. TAP1 expression and presence of CD8-positive T-cells were not related to JAK1 mutations. In 198 additional microsatellite unstable ECs, the JAK1 mutation frequency was confirmed but no prognostic significance was found. For, JAK1 wildtype (n=135, 72%) and mutant (n=52, 28%) ECs, 10-year recurrence free rates were 84% and 77% (P=0.301). These observations show that JAK1 mutations are highly frequent in microsatellite unstable EC, not associated with survival, but are associated with impaired upregulation of LMP7 and HLA class I and may therefore facilitate immune escape.

Prediction model for regional or distant recurrence in endometrial cancer based on classical pathological and immunological parameters

Background:Adjuvant therapy increases disease-free survival in endometrial cancer (EC), but has no impact on overall survival and negatively influences the quality of life. We investigated the discriminatory power of classical and immunological predictors of recurrence in a cohort of EC patients and confirmed the findings in an independent validation cohort.Methods:We reanalysed the data from 355 EC patients and tested our findings in an independent validation cohort of 72 patients with EC. Predictors were selected and Harrell’s C-index for concordance was used to determine discriminatory power for disease-free survival in the total group and stratified for histological subtype.Results:Predictors for recurrence were FIGO stage, lymphovascular space invasion and numbers of cytotoxic and memory T-cells. For high risk cancer, cytotoxic or memory T-cells predicted recurrence as well as a combination of FIGO stage and lymphovascular space invasion (C-index 0.67 and 0.71 vs 0.70). Recurrence was best predicted when FIGO stage, lymphovascular space invasion and numbers of cytotoxic cells were used in combination (C-index 0.82). Findings were confirmed in the validation cohort.Conclusions:In high-risk EC, clinicopathological or immunological variables can predict regional or distant recurrence with equal accuracy, but the use of these variables in combination is more powerful.

TGF-β induced CXCL13 in CD8+ T cells is associated with tertiary lymphoid structures in cancer

Coordinated immune responses against human tumors are frequently characterized by tertiary lymphoid structures (TLS) which predict improved prognosis. The development of TLS is dependent on the chemokine CXCL13, reported to be secreted by dendritic cells and follicular helper T cells only. We report the unexpected finding that CXCL13 is also secreted by activated CD8+ T cells following stimulation by transforming growth factor beta (TGF-β). Using single cell RNA sequencing we found that expression of CXCL13 in CD8+ T cells was restricted to the intraepithelial CD103+ population. Accordingly, CD8+ T cells activated in the presence of TGF-β simultaneously upregulated CD103 and secreted CXCL13. CXCL13 expression was strongly correlated with neo-antigen burden and cytolytic gene signatures in bulk tumors. In line with this, TLS were abundant in neo-antigen-high, CD103+ T cell-enriched tumors. TGF-β thus appears to play a role in coordinating immune responses against human tumors through CD8-dependent CXCL13-associated formation of TLS.

Abstract 1687: Systemic immunological changes during first line chemotherapy in patients with high-grade serous ovarian cancer

Ovarian cancer remains the most lethal gynecological malignancy and new therapeutic strategies are urgently needed. High-grade serous ovarian cancer (HGSC), in particular, is associated with a five-year survival of only 40%. A promising new approach for treating HGSC is immunotherapy, which has resulted in complete and durable responses, albeit in a minority of patients. One potential approach for improving these response rates is by combining chemo- and immunotherapy. Indeed, carboplatin/taxol chemotherapy was shown to augment immune responses in cervical cancer patients by depleting circulating myeloid suppressor cells. As patients with HGSC are treated in first line with similar carboplatin/taxol chemotherapy, we explored the effects of chemotherapy on systemic immunity in HGSC patients. Within this prospective observational study, 75 patients with suspected ovarian cancer were included. Blood was collected at three different time points during first line chemotherapy treatment, namely: prior to chemotherapy, between the third and fourth cycle of chemotherapy and 4-6 weeks after the sixth cycle of chemotherapy. All patients received 6 cycles of carboplatin/taxol chemotherapy and cytoreductive surgery (either as primary debulking, or after 3 cycles of neo-adjuvant chemotherapy). Peripheral blood mononuclear cells were isolated and analyzed for a total of 36 immune markers using 9 flow cytometry panels, in total analyzing 49 immune cell subsets. Results were compared to an age-matched cohort consisting of women surgically treated for a benign disease, and a cohort of healthy young volunteers. 75 patients have been included so far from which 22 were diagnosed with benign disease, 3 were diagnosed with another malignancy, and 50 were diagnosed with OC. Of the 50 OC patients, 18 were diagnosed with HGSC and from 9 HGSC patients, multiple time points were available for analysis of chemotherapy-dependent changes in the immune cell subsets. All patients developed leukopenia as a result of chemotherapeutic treatment. Age-related changes in lymphocyte and myeloid cell subsets were observed in all HGSC patients and patients with benign disease. Chemotherapy-dependent depletion of myeloid cells was observed in a subset of patients. Depletion of myeloid subsets was equally distributed among monocytes, macrophages and dendritic cells. No HGSC- or chemotherapy-dependent changes in T cell subsets or migration and activation markers were observed. Taken together, we observed no major systemic changes in immune cell subsets during carboplatin/taxol chemotherapy treatment, suggesting that combined chemo-immunotherapeutic strategies could be feasible during first line treatment of HGSC. Citation Format: Fenne L. Komdeur, Florine A. Eggink, Ninke Leffers, Hagma H. Workel, Kim L. Brunekreeft, Annechien Plat, Marco de Bruyn, Hans W. Nijman. Systemic immunological changes during first line chemotherapy in patients with high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2017-1687

Abstract A108: CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαβ+ CD8αβ+ T cells that can be targeted for cancer immunotherapy

Introduction: CD103+ tumor-infiltrating lymphocytes (TIL) have been linked to specific epithelial infiltration and a prolonged survival in epithelial malignancies. However, whether these cells are induced as part of an ongoing anti-tumor immune response or represent non-specifically expanded resident or mucosal lymphocytes remains largely unknown. Methods: CD103+ TIL were quantified in a cohort of ovarian and endometrial cancer patients by immunohistochemistry. Localization of CD103+ cells and co-expression of CD103 with CD3, CD8, CD16 and FoxP3 was assessed by immunofluorescence. Further phenotyping and functional experiments of CD103+ cells was performed mainly by flow cytometry on primary tumor digests. Summary of the Data: In this study, we first confirmed that CD103+ TIL from ovarian and uterine cancer were predominantly localized in the cancer epithelium and were strongly correlated with an improved prognosis. We further demonstrate that CD103+ TIL were almost exclusively CD3+ TCRαβ+ CD8αβ+ CD4- T cells, but heterogeneously expressed T cell memory and differentiation markers. Activation of peripheral T cells in the presence of HGSC was sufficient to trigger induction of CD103 in over 90% of all CD8+ cells in a T cell receptor (TCR)- and TGFβR1-dependent manner. Finally, CD103+ TIL isolated from primary ovarian and uterine tumors showed signs of recent activation and dominantly co-expressed key immunotherapeutic targets PD-1 and CD27. Conclusion: Our data indicate CD103+ TIL are formed as the result of an adaptive anti-tumor immune response that might be reactivated by (dual) checkpoint inhibition. Citation Format: Hans Nijman, Fenne Komdeur, Maartje Wouters, Hagma Workel, Kim Brunekreeft, Annechien Plat, Florine Eggink, Bea Wisman, Toos Daemen, Evelien Duiker, Harry Hollema, Marco de Bruyn. CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαβ+ CD8αβ+ T cells that can be targeted for cancer immunotherapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A108.