Unlocking the therapeutic potential of primary tumor-draining lymph nodes

Lymph nodes draining the primary tumor are essential for the initiation of an effective anti-tumor T-cell immune response. However, cancer-derived immune suppressive factors render the tumor-draining lymph nodes (TDLN) immune compromised, enabling tumors to invade and metastasize. Unraveling the different mechanisms underlying this immune escape will inform therapeutic intervention strategies to halt tumor spread in early clinical stages. Here, we review our findings from translational studies in melanoma, breast, and cervical cancer and discuss clinical opportunities for local immune modulation of TDLN in each of these indications

Immunotherapeutic Approaches for the Treatment of HPV-Associated (Pre-)Cancer of the Cervix, Vulva and Penis

Human papillomavirus (HPV) infection drives tumorigenesis in almost all cervical cancers and a fraction of vulvar and penile cancers. Due to increasing incidence and low vaccination rates, many will still have to face HPV-related morbidity and mortality in the upcoming years. Current treatment options (i.e., surgery and/or chemoradiation) for urogenital (pre-)malignancies can have profound psychosocial and psychosexual effects on patients. Moreover, in the setting of advanced disease, responses to current therapies remain poor and nondurable, highlighting the unmet need for novel therapies that prevent recurrent disease and improve clinical outcome. Immunotherapy can be a useful addition to the current therapeutic strategies in various settings of disease, offering relatively fewer adverse effects and potential improvement in survival. This review discusses immune evasion mechanisms accompanying HPV infection and HPV-related tumorigenesis and summarizes current immunotherapeutic approaches for the treatment of HPV-related (pre-)malignant lesions of the uterine cervix, vulva, and penis

Immunotherapeutic Approaches for the Treatment of HPV-Associated (Pre-)Cancer of the Cervix, Vulva and Penis

Human papillomavirus (HPV) infection drives tumorigenesis in almost all cervical cancers and a fraction of vulvar and penile cancers. Due to increasing incidence and low vaccination rates, many will still have to face HPV-related morbidity and mortality in the upcoming years. Current treatment options (i.e., surgery and/or chemoradiation) for urogenital (pre-)malignancies can have profound psychosocial and psychosexual effects on patients. Moreover, in the setting of advanced disease, responses to current therapies remain poor and nondurable, highlighting the unmet need for novel therapies that prevent recurrent disease and improve clinical outcome. Immunotherapy can be a useful addition to the current therapeutic strategies in various settings of disease, offering relatively fewer adverse effects and potential improvement in survival. This review discusses immune evasion mechanisms accompanying HPV infection and HPV-related tumorigenesis and summarizes current immunotherapeutic approaches for the treatment of HPV-related (pre-)malignant lesions of the uterine cervix, vulva, and penis

Immune landscape in vulvar cancer-draining lymph nodes indicates distinct immune escape mechanisms in support of metastatic spread and growth

Background Therapeutic immune intervention is highly dependent on the T-cell priming and boosting capacity of tumor-draining lymph nodes (TDLN). In vulvar cancer, in-depth studies on the immune status of (pre)metastatic TDLN is lacking. Methods We have phenotyped and enumerated various T-cell and myeloid subsets in tumor-free (LN-, n=27) and metastatic TDLN (LN+, n=11) using flow cytometry. Additionally, we studied chemokine and cytokine release profiles and assessed expression of indoleamine 2,3-dioxygenase (IDO) in relation to plasmacytoid dendritic cell (pDC) or myeloid subsets. Results Metastatic involvement of TDLN was accompanied by an inflamed microenvironment with immune suppressive features, marked by hampered activation of migratory DC, increased cytokine/chemokine release, and closely correlated elevations of pDC and LN-resident conventional DC (LNR-cDC) activation state and frequencies, as well as of terminal CD8 + effector-memory T-cell (TemRA) differentiation, regulatory T-cell (Treg) rates, T-cell activation, and expression of cytotoxic T-lymphocyte protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1) immune checkpoints. In addition, high indoleamine 2,3-dioxygenase (IDO) expression and increased frequencies of monocytic myeloid-derived suppressor cells (mMDSC) were observed. Correlation analyses with primary and metastatic tumor burden suggested respective roles for Tregs and suppression of inducible T cell costimulator (ICOS) + T helper cells in early metastatic niche formation and for CD14 + LNR-cDC and terminal T-cell differentiation in later stages of metastatic growth. Conclusions Metastatic spread in vulvar TDLN is marked by an inflamed microenvironment with activated effector T cells, which are likely kept in check by an interplay of suppressive feedback mechanisms. Our data support (neoadjuvant) TDLN-targeted therapeutic interventions based on CTLA-4 and PD-1 blockade, to reinvigorate memory T cells and curb early metastatic spread and growth

Immune landscape in vulvar cancer-draining lymph nodes indicates distinct immune escape mechanisms in support of metastatic spread and growth

Background Therapeutic immune intervention is highly dependent on the T-cell priming and boosting capacity of tumor-draining lymph nodes (TDLN). In vulvar cancer, in-depth studies on the immune status of (pre)metastatic TDLN is lacking. Methods We have phenotyped and enumerated various T-cell and myeloid subsets in tumor-free (LN-, n=27) and metastatic TDLN (LN+, n=11) using flow cytometry. Additionally, we studied chemokine and cytokine release profiles and assessed expression of indoleamine 2,3-dioxygenase (IDO) in relation to plasmacytoid dendritic cell (pDC) or myeloid subsets. Results Metastatic involvement of TDLN was accompanied by an inflamed microenvironment with immune suppressive features, marked by hampered activation of migratory DC, increased cytokine/chemokine release, and closely correlated elevations of pDC and LN-resident conventional DC (LNR-cDC) activation state and frequencies, as well as of terminal CD8(+) effector-memory T-cell (TemRA) differentiation, regulatory T-cell (Treg) rates, T-cell activation, and expression of cytotoxic T-lymphocyte protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1) immune checkpoints. In addition, high indoleamine 2,3-dioxygenase (IDO) expression and increased frequencies of monocytic myeloid-derived suppressor cells (mMDSC) were observed. Correlation analyses with primary and metastatic tumor burden suggested respective roles for Tregs and suppression of inducible T cell costimulator (ICOS)(+) T helper cells in early metastatic niche formation and for CD14(+) LNR-cDC and terminal T-cell differentiation in later stages of metastatic growth. Conclusions Metastatic spread in vulvar TDLN is marked by an inflamed microenvironment with activated effector T cells, which are likely kept in check by an interplay of suppressive feedback mechanisms. Our data support (neoadjuvant) TDLN-targeted therapeutic interventions based on CTLA-4 and PD-1 blockade, to reinvigorate memory T cells and curb early metastatic spread and growth

PD-L1 and PD-L2 Expression in Cervical Cancer: Regulation and Biomarker Potential

PD-1/PD-L1 immune checkpoint inhibitors show potential for cervical cancer treatment. However, low response rates suggest that patient selection based on PD-L1 protein expression is not optimal. Here, we evaluated different PD-L1 detection methods and studied transcriptional regulation of PD-L1/PD-L2 expression by The Cancer Genome Atlas (TCGA) mRNAseq analysis. First, we determined the copy number of the PD-L1/PD-L2 locus by fluorescence in situ hybridization (FISH), PD-L1 mRNA expression by RNA in situ hybridization (RNAish), and PD-L1/PD-L2 protein expression by immunohistochemistry (IHC) on tissue microarrays containing a cohort of 60 patients. Additionally, distribution of PD-L1/PD-L2 was visualized based on flow cytometry analysis of single-cell suspensions (n = 10). PD-L1/PD-L2 locus amplification was rare (2%). PD-L1 mRNA expression in tumor cells was detected in 56% of cases, while 41% expressed PD-L1 protein. Discordant scores for PD-L1 protein expression on tumor cells between cores from one patient were observed in 27% of cases. Interestingly, with RNAish, PD-L1 heterogeneity was observed in only 11% of the cases. PD-L2 protein expression was found in 53%. PD-L1 mRNA and protein expression on tumor cells were strongly correlated (p < 0.001). PD-L1 and PD-L2 protein expression showed no correlation on tumor cells (p = 0.837), but a strong correlation on cells in stromal fields (p < 0.001). Co-expression of PD-L1 and PD-L2 on macrophage-like populations was also observed with flow cytometry analysis. Both PD-L1 and PD-L2 TCGA transcript levels strongly correlated in the TCGA data, and both PD-L1 and PD-L2 strongly correlated with interferon gamma (IFNG) expression/transcript levels (p < 0.0001). Importantly, patients with high PD-L1/PD-L2/IFNG transcript levels had a survival advantage over patients with high PD-L1/PD-L2 and low IFNG expression. Based on these findings, we conclude that PD-L1/PD-L2 expression in cervical cancer is mainly associated with interferon induction and not gene amplification, which makes FISH unsuitable as biomarker. The heterogeneous PD-L1 and PD-L2 expression patterns suggest IHC unreliable for patient selection. RNAish, in conjunction with interferon signaling evaluation, seems a promising technique for immune checkpoint detection. These results warrant further investigation into their prognostic and predictive potential

Immune landscape in vulvar cancer-draining lymph nodes indicates distinct immune escape mechanisms in support of metastatic spread and growth

Background Therapeutic immune intervention is highly dependent on the T-cell priming and boosting capacity of tumor-draining lymph nodes (TDLN). In vulvar cancer, in-depth studies on the immune status of (pre)metastatic TDLN is lacking. Methods We have phenotyped and enumerated various T-cell and myeloid subsets in tumor-free (LN-, n=27) and metastatic TDLN (LN+, n=11) using flow cytometry. Additionally, we studied chemokine and cytokine release profiles and assessed expression of indoleamine 2,3-dioxygenase (IDO) in relation to plasmacytoid dendritic cell (pDC) or myeloid subsets. Results Metastatic involvement of TDLN was accompanied by an inflamed microenvironment with immune suppressive features, marked by hampered activation of migratory DC, increased cytokine/chemokine release, and closely correlated elevations of pDC and LN-resident conventional DC (LNR-cDC) activation state and frequencies, as well as of terminal CD8 + effector-memory T-cell (TemRA) differentiation, regulatory T-cell (Treg) rates, T-cell activation, and expression of cytotoxic T-lymphocyte protein-4 (CTLA-4) and programmed cell death protein-1 (PD-1) immune checkpoints. In addition, high indoleamine 2,3-dioxygenase (IDO) expression and increased frequencies of monocytic myeloid-derived suppressor cells (mMDSC) were observed. Correlation analyses with primary and metastatic tumor burden suggested respective roles for Tregs and suppression of inducible T cell costimulator (ICOS) + T helper cells in early metastatic niche formation and for CD14 + LNR-cDC and terminal T-cell differentiation in later stages of metastatic growth. Conclusions Metastatic spread in vulvar TDLN is marked by an inflamed microenvironment with activated effector T cells, which are likely kept in check by an interplay of suppressive feedback mechanisms. Our data support (neoadjuvant) TDLN-targeted therapeutic interventions based on CTLA-4 and PD-1 blockade, to reinvigorate memory T cells and curb early metastatic spread and growth

PD-L1 and PD-L2 Expression in Cervical Cancer: Regulation and Biomarker Potential

PD-1/PD-L1 immune checkpoint inhibitors show potential for cervical cancer treatment. However, low response rates suggest that patient selection based on PD-L1 protein expression is not optimal. Here, we evaluated different PD-L1 detection methods and studied transcriptional regulation of PD-L1/PD-L2 expression by The Cancer Genome Atlas (TCGA) mRNAseq analysis. First, we determined the copy number of the PD-L1/PD-L2 locus by fluorescence in situ hybridization (FISH), PD-L1 mRNA expression by RNA in situ hybridization (RNAish), and PD-L1/PD-L2 protein expression by immunohistochemistry (IHC) on tissue microarrays containing a cohort of 60 patients. Additionally, distribution of PD-L1/PD-L2 was visualized based on flow cytometry analysis of single-cell suspensions (n = 10). PD-L1/PD-L2 locus amplification was rare (2%). PD-L1 mRNA expression in tumor cells was detected in 56% of cases, while 41% expressed PD-L1 protein. Discordant scores for PD-L1 protein expression on tumor cells between cores from one patient were observed in 27% of cases. Interestingly, with RNAish, PD-L1 heterogeneity was observed in only 11% of the cases. PD-L2 protein expression was found in 53%. PD-L1 mRNA and protein expression on tumor cells were strongly correlated (p < 0.001). PD-L1 and PD-L2 protein expression showed no correlation on tumor cells (p = 0.837), but a strong correlation on cells in stromal fields (p < 0.001). Co-expression of PD-L1 and PD-L2 on macrophage-like populations was also observed with flow cytometry analysis. Both PD-L1 and PD-L2 TCGA transcript levels strongly correlated in the TCGA data, and both PD-L1 and PD-L2 strongly correlated with interferon gamma (IFNG) expression/transcript levels (p < 0.0001). Importantly, patients with high PD-L1/PD-L2/IFNG transcript levels had a survival advantage over patients with high PD-L1/PD-L2 and low IFNG expression. Based on these findings, we conclude that PD-L1/PD-L2 expression in cervical cancer is mainly associated with interferon induction and not gene amplification, which makes FISH unsuitable as biomarker. The heterogeneous PD-L1 and PD-L2 expression patterns suggest IHC unreliable for patient selection. RNAish, in conjunction with interferon signaling evaluation, seems a promising technique for immune checkpoint detection. These results warrant further investigation into their prognostic and predictive potential

Delta-like ligand-notch1 signaling is selectively modulated by HPV16 e6 to promote squamous cell proliferation and correlates with cervical cancer prognosis

Human papillomavirus (HPV) drives high-grade intraepithelial neoplasia and cancer; for unknown reasons, this occurs most often in the cervical transformation zone. Either mutation or HPV E6- driven inhibition of Notch1 can drive neoplastic development in stratified squamous epithelia. However, the contribution of Notch1 and its Delta-like ligands (DLL) to site susceptibility remains poorly understood. Here, we map DLL1/DLL4 expression in cell populations present in normal cervical biopsies by immunofluorescence. In vitro keratinocyte 2D monolayer models, growth assays, and organotypic raft cultures were used to assess the functional role of DLL-Notch signaling in uninfected cells and its modulation by HPV16 in neoplasia. AnRNAsequencing-based gene signature was used to suggest the cell of origin of 279 HPV-positive cervical carcinomas from The Cancer Genome Atlas and to relate this to disease prognosis. Finally, the prognostic impact of DLL4 expression was investigated in three independent cervical cancer patient cohorts. Three molecular cervical carcinoma subtypes were identified, with reserve cell tumors the most common and linked to relatively good prognosis. Reserve cells were characterized as DLL1-/DLL4+, a proliferative phenotype that is temporarily observed during squamous metaplasia and wound healing but appears to be sustained by HPV16 E6 in raft models of lowgrade and, more prominently, high-grade neoplasia. High expression of DLL4 was associated with an increased likelihood of cervical cancer-associated death and recurrence. Taken together, DLL4- Notch1 signaling reflects a proliferative cellular state transiently present during physiologic processes but inherent to cervical reserve cells, making them strongly resemble neoplastic tissue even before HPV infection has occurred

Adenocarcinoma of the Uterine Cervix Shows Impaired Recruitment of cDC1 and CD8+ T Cells and Elevated β-Catenin Activation Compared with Squamous Cell Carcinoma

PURPOSE: Adenocarcinoma of the uterine cervix is the second most common type of cervical cancer after squamous cell carcinoma (SCC). Although both subtypes are treated similarly, patients with adenocarcinoma have a worse prognosis. In this study, immunologic features of the tumor microenvironment in these two subsets were pursued with potential therapeutic implications. EXPERIMENTAL DESIGN: The immune microenvironment of primary tumors and nonmetastatic tumor-draining lymph nodes (TDLN) was compared between patients with cervical adenocarcinoma (n = 16) and SCC (n = 20) by polychromatic flow cytometry and by transcriptional profiling of the primary tumors (n = 299) using publicly available data from The Cancer Genome Atlas (TCGA). RESULTS: Flow cytometric analyses revealed intact T-cell differentiation in TDLNs, but hampered effector T-cell trafficking to the primary tumors in adenocarcinoma, as compared with SCC. TCGA analysis demonstrated higher expression of chemokines involved in effector T-cell homing (CXCL9/10/11) in SCC primary tumors as compared with adenocarcinoma primary tumors, which was highly correlated to a transcriptional signature for type I conventional dendritic cells (cDC1). This was consistent with elevated frequencies of CD141/BDCA3+cDC1 in primary tumor SCC samples relative to adenocarcinoma and correspondingly elevated levels of CXCL9 and CXCL10 in 24-hour ex vivo cultures. Hampered cDC1 recruitment in adenocarcinoma was in turn related to lower transcript levels of cDC1-recruiting chemokines and an elevated β-catenin activation score and was associated with poor overall survival. CONCLUSIONS: Our data have identified an opportunity for the investigation of potentially novel therapeutic interventions in adenocarcinoma of the cervix, that is, β-catenin inhibition and cDC1 mobilization