Atezolizumab With or Without Radiotherapy for Advanced Squamous Cell Carcinoma of the Penis (The PERICLES Study): A Phase II Trial.

PURPOSE: Patients with advanced penile squamous cell carcinoma have a poor prognosis (21% 2-year overall survival [OS] from diagnosis). We assessed the activity of atezolizumab (anti-PD-L1) in patients with advanced penile cancer, with or without radiotherapy (RT). PATIENTS AND METHODS: A single-center, nonrandomized phase II study with two treatment arms was conducted in 32 patients with histologically confirmed advanced penile cancer. All patients received atezolizumab (1,200 mg) once every 3 weeks. Twenty patients, who were expected to benefit from RT for locoregional disease control, received additional irradiation. The primary end point was 1-year progression-free survival (PFS) for the complete cohort and was reached if the actual 1-year PFS was at least 35%. Secondary end points included OS, objective response rate (ORR), and tolerability. Exploratory biomarker analyses were conducted in pretreatment specimens. RESULTS: Median follow-up was 29.1 months (IQR, 18.1-33.5). Grade 3-4 adverse events related to atezolizumab or RT were observed in 3/32 (9.4%) and 13/20 (65%) patients, respectively. One-year PFS was 12.5% (95% CI, 5.0 to 31.3), which did not meet the study's primary end point. Median OS was 11.3 months (95% CI, 5.5 to 18.7). In the objective response-evaluable population (n = 30; 93.8%), the ORR was 16.7% (95% CI, 6 to 35), including 2 (6.7%) complete responders and 3 (10%) partial responders. Improved PFS was observed in patients with high-risk human papillomavirus (hrHPV)-positive tumors (P = .003) and those with high infiltration of intratumoral CD3+CD8+ T cells (P = .037). CONCLUSION: Although the primary end point of 1-year PFS was not met, durable antitumor activity to atezolizumab was observed in a subset of patients. Biomarkers, such as hrHPV and intratumoral CD3+CD8+ T-cell infiltration, may help to better select responders.

Cerebellar presence of immune cells in patients with neuro-coeliac disease.

Although various neurodegenerative disorders have been associated with coeliac disease (CD), the underlying neuropathological link between these brain and gut diseases remains unclear. We postulated that the neuronal damage sporadically observed in CD patients is immune-mediated. Our aim was to determine if the loss of neurons, especially Purkinje cells, coincides with microglia activation and T- and B-cell infiltration in the cerebellum of patients with CD and a concomitant idiopathic neurological disease affecting the cerebellum (NeuroCD). Post-mortem cerebellar tissue was collected of validated NeuroCD cases. Gender- and age-matched genetic spinocerebellar ataxia (SCA) controls and non-neurological controls (NNC) were selected based on clinical reports and pathological findings. Cerebellar tissue of seventeen patients was included (6 NeuroCD, 5 SCA, 6 NNC). In SCA cases we found that the Purkinje cell layer was 58.6% reduced in comparison with NNC. In NeuroCD cases this reduction was even more prominent with a median reduction of 81.3% compared to NNC. Marked increased numbers of both CD3+ and CD8+ cells were observed in the NeuroCD but not in SCA patients. This coincided with significantly more microglial reactivity in NeuroCD patients. These findings demonstrate that the massive loss of Purkinje cells in the cerebellum of neuro CD patients is accompanied by local innate and T-cell mediated immune responses.

Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy.

PURPOSE: Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. EXPERIMENTAL DESIGN: Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. RESULTS: It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. CONCLUSION: The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Increased Angiogenesis and Lymphangiogenesis in Adenomyosis Visualized by Multiplex Immunohistochemistry.

There is evidence for increased angiogenesis in the (ectopic) endometrium of adenomyosis patients under the influence of vascular endothelial growth factor (VEGF). VEGF stimulates both angiogenesis and lymph-angiogenesis. However, information on lymph vessels in the (ectopic) endometrium of adenomyosis patients is lacking. In this retrospective matched case-control study, multiplex immunohistochemistry was performed on thirty-eight paraffin embedded specimens from premenopausal women who had undergone a hysterectomy at the Amsterdam UMC between 2001 and 2018 to investigate the evidence for (lymph) angiogenesis in the (ectopic) endometrium or myometrium of patients with adenomyosis versus controls with unrelated pathologies. Baseline characteristics of both groups were comparable. In the proliferative phase, the blood and lymph vessel densities were, respectively, higher in the ectopic and eutopic endometrium of patients with adenomyosis than in the endometrium of controls. The relative number of blood vessels without α-smooth muscle actinin (α SMA) was higher in the eutopic and ectopic endometrium of adenomyosis patients versus controls. The level of VEGF staining intensity was highest in the myometrium but did not differ between patients with adenomyosis or controls. The results indicate increased angiogenesis and lymphangiogenesis in the (ectopic) endometrium affected by adenomyosis. The clinical relevance of our findings should be confirmed in prospective clinical studies.

Uterine Fibroids Causing Preterm Birth: A New Pathophysiological Hypothesis on the Role of Fibroid Necrosis and Inflammation.

According to recent studies and observations in clinical practice, uterine fibroids increase the risk of preterm birth. There are several theories on the pathogenesis of preterm birth in the presence of fibroids. One theory proclaims that fibroid necrosis leads to preterm birth, though pathophysiological mechanisms have not been described. Necrotic tissue secretes specific cytokines and proteins and we suggest these to be comparable to the inflammatory response leading to spontaneous preterm birth. We hypothesize that fibroid necrosis could induce preterm parturition through a similar inflammatory response. This new hypothesis generates novel perspectives for future research and the development of preventative strategies for preterm birth. Moreover, we emphasize the importance of the recognition of fibroids and especially fibroid necrosis by clinicians during pregnancy.

Low Transforming Growth Factor-ß Pathway Activity in Cervical Adenocarcinomas.

Cervical cancer is the fourth most common cancer in women worldwide. Squamous cell carcinoma (SCC) and adenocarcinoma (AC) are the most common histological types, with AC patients having worse prognosis. Over the last two decades, incidence rates of AC have increased, highlighting the importance of further understanding AC tumorigenesis, and the need to investigate new treatment options. The cytokine TGF-ß functions as a tumour suppressor in healthy tissue. However, in tumour cells this suppressive function can be overcome. Therefore there is an increasing interest in using TGF-ß inhibitors in the treatment of cancer. Here, we hypothesize that TGF-ß plays a different role in SCC and AC. Analysis of RNA-seq data from the TCGA, using a TGF-ß response signature, resulted in separate clustering of the two subtypes. We further investigated the expression of TGF-ß-signalling related proteins (TßR1/2, SMAD4, pSMAD2, PAI-1, αvß6 and MMP2/9) in a cohort of 62 AC patients. Low TßR2 and SMAD4 expression was associated with worse survival in AC patients and interestingly, high PAI-1 and αvß6 expression was also correlated with worse survival. Similar correlations of TßR2, PAI-1 and αvß6 with clinical parameters were found in previously reported SCC analyses. However, when comparing expression levels between SCC and AC patient samples, pSMAD2, SMAD4, PAI-1 and αvß6 showed lower expression in AC compared to SCC. Because of the low expression of core TßR1/2, (p-)SMAD2 and SMAD4 proteins and the correlation with worse prognosis, TGF-ß pathway most likely leads to tumour inhibitory effects in AC and therefore the use of TGF-ß inhibitors would not be recommended. However, given the correlation of PAI-1 and αvß6 with poor prognosis, the use of TGF- ß inhibitors might be of interest in SCC and in the subsets of AC patients with high expression of these TGF-ß associated proteins.

A Review of the Effects of Cervical Cancer Standard Treatment on Immune Parameters in Peripheral Blood, Tumor Draining Lymph Nodes, and Local Tumor Microenvironment.

Cervical cancer remains a public health concern despite all the efforts to implement vaccination and screening programs. Conventional treatment for locally advanced cervical cancer consists of surgery, radiotherapy (with concurrent brachytherapy), combined with chemotherapy, or hyperthermia. The response rate to combination approaches involving immunomodulatory agents and conventional treatment modalities have been explored but remain dismal in patients with locally advanced disease. Studies exploring the immunological effects exerted by combination treatment modalities at the different levels of the immune system (peripheral blood (PB), tumor-draining lymph nodes (TDLN), and the local tumor microenvironment (TME)) are scarce. In this systemic review, we aim to define immunomodulatory and immunosuppressive effects induced by conventional treatment in cervical cancer patients to identify the optimal time point for immunotherapy administration. Radiotherapy (RT) and chemoradiation (CRT) induce an immunosuppressive state characterized by a long-lasting reduction in peripheral CD3, CD4, CD8 T cells and NK cells. At the TDLN level, CRT induced a reduction in Nrp1+Treg stability and number, naïve CD4 and CD8 T cell numbers, and an accompanying increase in IFNγ-producing CD4 helper T cells, CD8 T cells, and NK cells. Potentiation of the T-cell anti-tumor response was particularly observed in patients receiving low irradiation dosage. At the level of the TME, CRT induced a rebound effect characterized by a reduction of the T-cell anti-tumor response followed by stable radioresistant OX40 and FoxP3 Treg cell numbers. However, the effects induced by CRT were very heterogeneous across studies. Neoadjuvant chemotherapy (NACT) containing both paclitaxel and cisplatin induced a reduction in stromal FoxP3 Treg numbers and an increase in stromal and intratumoral CD8 T cells. Both CRT and NACT induced an increase in PD-L1 expression. Although there was no association between pre-treatment PD-L1 expression and treatment outcome, the data hint at an association with pro-inflammatory immune signatures, overall and disease-specific survival (OS, DSS). When considering NACT, we propose that posterior immunotherapy might further reduce immunosuppression and chemoresistance. This review points at differential effects induced by conventional treatment modalities at different immune compartments, thus, the compartmentalization of the immune responses as well as individual patient's treatment plans should be carefully considered when designing immunotherapy treatment regimens.

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.

Epigenetic Modification of the von Willebrand Factor Promoter Drives Platelet Aggregation on the Pulmonary Endothelium in Chronic Thromboembolic Pulmonary Hypertension.

Rationale: von Willebrand factor (vWF) mediates platelet adhesion during thrombosis. While chronic thromboembolic pulmonary hypertension (CTEPH) is associated with increased plasma levels of vWF, the role of this protein in CTEPH has remained enigmatic. Objectives: To identify the role of vWF in CTEPH. Methods: CTEPH-specific patient plasma and pulmonary endarterectomy material from patients with CTEPH were used to study the relationship between inflammation, vWF expression, and pulmonary thrombosis. Cell culture findings were validated in human tissue, and proteomics and chromatin immunoprecipitation were used to investigate the underlying mechanism of CTEPH. Measurements and Main Results: vWF is increased in plasma and the pulmonary endothelium of CTEPH patients. In vitro, the increase in vWF gene expression and the higher release of vWF protein upon endothelial activation resulted in elevated platelet adhesion to CTEPH endothelium. Proteomic analysis revealed that nuclear factor (NF)-κB2 was significantly increased in CTEPH. We demonstrate reduced histone tri-methylation and increased histone acetylation of the vWF promoter in CTEPH endothelium, facilitating binding of NF-κB2 to the vWF promoter and driving vWF transcription. Genetic interference of NFκB2 normalized the high vWF RNA expression levels and reversed the prothrombotic phenotype observed in CTEPH-pulmonary artery endothelial cells. Conclusions: Epigenetic regulation of the vWF promoter contributes to the creation of a local environment that favors in situ thrombosis in the pulmonary arteries. It reveals a direct molecular link between inflammatory pathways and platelet adhesion in the pulmonary vascular wall, emphasizing a possible role of in situ thrombosis in the development or progression of CTEPH.

HPV-16 E6/E7 DNA tattoo vaccination using genetically optimized vaccines elicit clinical and immunological responses in patients with usual vulvar intraepithelial neoplasia (uVIN): a phase I/II clinical trial.

BACKGROUND: Usual vulvar intraepithelial neoplasia (uVIN) is a premalignancy caused by persistent infection with high-risk types of human papillomavirus (HPV), mainly type 16. Even though different treatment modalities are available (eg, surgical excision, laser evaporation or topical application of imiquimod), these treatments can be mutilating, patients often have recurrences and 2%-8% of patients develop vulvar carcinoma. Therefore, immunotherapeutic strategies targeting the pivotal oncogenic HPV proteins E6 and E7 are being explored to repress carcinogenesis. METHOD: In this phase I/II clinical trial, 14 patients with HPV16+ uVIN were treated with a genetically enhanced DNA vaccine targeting E6 and E7. Safety, clinical responses and immunogenicity were assessed. Patients received four intradermal HPV-16 E6/E7 DNA tattoo vaccinations, with a 2-week interval, alternating between both upper legs. Biopsies of the uVIN lesions were taken at screening and +3 months after last vaccination. Digital photography of the vulva was performed at every check-up until 12 months of follow-up for measurement of the lesions. HPV16-specific T-cell responses were measured in blood over time in ex vivo reactivity assays. RESULTS: Vaccinations were well tolerated, although one grade 3 suspected unexpected serious adverse reaction was observed. Clinical responses were observed in 6/14 (43%) patients, with 2 complete responses and 4 partial responses (PR). 5/14 patients showed HPV-specific T-cell responses in blood, measured in ex vivo reactivity assays. Notably, all five patients with HPV-specific T-cell responses had a clinical response. CONCLUSIONS: Our results indicate that HPV-16 E6/E7 DNA tattoo vaccination is a biologically active and safe treatment strategy in patients with uVIN, and suggest that T-cell reactivity against the HPV oncogenes is associated with clinical benefit. TRIAL REGISTRATION NUMBER: NTR4607.

Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer.

PURPOSE: Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. EXPERIMENTAL DESIGN: We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. RESULTS: HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2-15) from HPV ctDNA detection. CONCLUSIONS: HPV ctDNA detection is a useful marker to predict relapse in cervical cancer.See related commentary by Wentzensen and Clarke, p. 5733.

Distinct Patterns of Myeloid Cell Infiltration in Patients With hrHPV-Positive and hrHPV-Negative Penile Squamous Cell Carcinoma: The Importance of Assessing Myeloid Cell Densities Within the Spatial Context of the Tumor.

Comprehensive analysis of tumor infiltrating myeloid cells in the tumor microenvironment of penile squamous cell carcinoma (PSCC) is lacking. In this retrospective study, for the first time, PSCC resection specimens (N = 103) were annotated into the following compartments: intratumoral tumor (IT Tumor), intratumoral stroma (IT Stroma), peritumoral tumor (PT Tumor) and peritumoral stroma (PT Stroma) compartments. We then quantified CD14+, CD68+ and CD163+ myeloid cells within these compartments using an image analysis software and assessed their association with various clinical parameters, including high-risk human papillomavirus (hrHPV) status. In the total cohort, hrHPV status, grade of differentiation, age and tumor size were associated with myeloid cell densities. hrHPV+ tumors had higher infiltration rates of CD14+, CD68+ and CD163+ myeloid cells in the IT tumor compartment (p < 0.001, for all) compared to hrHPV- tumors. Furthermore, when examining the association between compartment-specific infiltration and differentiation grade, increased myeloid cell densities in the IT tumor compartment were associated with a more advanced histological grade (p < 0.001, for all). This association remained significant when the hrHPV- cohort (N = 60) was analyzed (CD14+ p = 0.001; CD68+ p < 0.001; CD163+ p = 0.004). Subgroup analysis in the hrHPV+ group (N = 43) showed that high infiltration rates of CD68+ and CD163+ cells in the PT tumor compartment were associated with lymph node (LN) metastasis (p = 0.031 and p = 0.026, respectively). Regarding the association between myeloid cell densities and disease-specific survival, the risk of death was found to decrease slightly as the number of myeloid cells in the IT tumor compartment increased (CD14+ p = 0.04; CD68+ p = 0.05; CD163+ p = 0.02). However, after adjusting for hrHPV, no independent association between myeloid densities and disease-specific survival were found. Altogether, these findings demonstrate the importance of assessing myeloid cell densities within the spatial context of the tumor. Further studies are needed to unravel the specific phenotype of myeloid cells residing in the different compartments, their effect on clinical parameters and the impact of hrHPV on the recruitment of myeloid cell populations in PSCC.

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. An RNA sequencing-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 low-grade 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. SIGNIFICANCE: This study investigates cervical cancer cell-of-origin populations and describes a DLL-Notch1 phenotype that is associated with disease prognosis and that might help identify cells that are susceptible to HPV-induced carcinogenesis.

Human papilloma virus (HPV) integration signature in Cervical Cancer: identification of MACROD2 gene as HPV hot spot integration site.

BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs. METHODS: Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study [NCT02428842]. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed. RESULTS: Episomal HPV was much less frequent in CC as compared to anal carcinoma (p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter- or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011). CONCLUSIONS: This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at MACROD2; involved in impaired PARP1 activity and chromosome instability.

Micro-environmental cross-talk in an organotypic human melanoma-in-skin model directs M2-like monocyte differentiation via IL-10.

Preclinical assessment of novel therapies to fight cancer requires models that reflect the human physiology and immune response. Here, we established an in vitro three-dimensional (3D) reconstructed organotypic human melanoma-in-skin (Mel-RhS) model to investigate cellular and molecular features of tumor formation over a period of 6 weeks. Tumor nests developed over time at the epidermal-dermal junction and spread towards the dermis, in places disrupting the basement membrane. This coincided with secretion of matrix metalloproteinase 9 (MMP-9) by melanoma cells. These features resemble the initial stages of invasive melanoma. Interestingly, while the SK-MEL-28 cell line did not secrete detectable levels of interleukin-10 (IL-10) in traditional two-dimensional monolayers, it did express IL-10 in the 3D Mel-RhS, as did the surrounding keratinocytes and fibroblasts. This cellular cross-talk-induced secretion of IL-10 in the Mel-RhS indicated the generation of an immune suppressive microenvironment. Culture supernatants from Mel-RhS interfered with monocyte-to-dendritic-cell differentiation, leading to the development of M2-like macrophages, which was in part prevented by antibody-mediated IL-10 blockade. Indeed, high-dimensional single-cell analysis revealed a shift within the monocyte population away from a CD163+PD-L1+ M2-like phenotype upon IL-10 blockade. Thus, the 3D configuration of the Mel-RhS model revealed a role for IL-10 in immune escape through misdirected myeloid differentiation, which would have been missed in classical monolayer cultures.

Adenocarcinoma of the Uterine Cervix Shows Impaired Recruitment of cDC1 and CD8+ T Cells and Elevated ß-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 ß-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, ß-catenin inhibition and cDC1 mobilization.

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.

Neoadjuvant cisplatin and paclitaxel modulate tumor-infiltrating T cells in patients with cervical cancer.

Resistance to chemotherapy is widely recognized as one of the major factors limiting therapeutic efficacy and influences clinical outcomes in patients with cancer. Many studies on various tumor types have focused on combining standard-of-care chemotherapy with immunotherapy. However, for cervical cancer, the role of neoadjuvant chemotherapy (NACT) on the local immune microenvironment is largely unexplored. We performed a pilot study on 13 primary cervical tumor samples, before and after NACT, to phenotype and enumerate tumor-infiltrating T-cell subpopulations using multiplex immunohistochemistry (CD3, CD8, FoxP3, Ki67, and Tbet) and automated co-expression analysis software. A significant decrease in proliferating (Ki67+) CD3+CD8- T cells and FoxP3+(CD3+CD8-) regulatory T cells was observed in the tumor stroma after cisplatin and paclitaxel treatment, with increased rates of cytotoxic CD8+ T cells, including activated and CD8+Tbet+ T cells. No effect was observed on the number of tumor-infiltrating T cells in the cervical tumor microenvironment after treatment with cisplatin only. Therefore, we conclude that patients treated with cisplatin and paclitaxel had more tumor-infiltrating T-cell modulation than patients treated with cisplatin monotherapy. These findings enhance our understanding of the immune-modulating effect of chemotherapy and warrant future combination of the standard-of-care therapy with immunotherapy to improve clinical outcome in patients with cervical cancer.