RESUMO
BACKGROUND AND AIM: Resection of the distal part of the urethra is performed in 15% to 55% of women with vulvar cancer to achieve radicality of vulvectomy. Urinary reconstruction in these women may be complicated by urethral stenosis resulting from circular inset of the meatus. We report on our experience with 2 surgical techniques of noncircular inset to prevent such stenosis. METHODS: From January 2005 to January 2020, 42 urethral meatus reconstructions were performed in 41 women after vulvectomy for (pre)malignant skin disorders by a "limited" (n = 17) or "extended" (n = 25) anterior vaginal wall advancement technique, including V-Y insertion of part of the vaginal flap in a posterior longitudinal urethrotomy. Preoperative characteristics, procedural details, and surgical outcomes were reviewed. RESULTS: We observed 1 neomeatal stenosis and 1 case of partial vaginal wall flap necrosis as major complications following the "limited" technique and 1 circumferential neomeatal dehiscence and occlusion as major complication after the "extended" technique. Both the neomeatal stenosis and the dehiscence/occlusion are felt to have been preventable and not caused by a flaw of design of the advancement technique. CONCLUSIONS: We advocate applying these vaginal wall advancement techniques to prevent circular inset of the neomeatus. The "extended" technique offers a solution in cases where the periurethral vulvar defect cannot be closed by transpositioning of labial skin.
Assuntos
Procedimentos de Cirurgia Plástica , Vulvectomia , Constrição Patológica/cirurgia , Feminino , Humanos , Masculino , Procedimentos de Cirurgia Plástica/métodos , Retalhos Cirúrgicos/cirurgia , Uretra/cirurgiaRESUMO
BACKGROUND: Usual type vulvar intraepithelial neoplasia (uVIN) is caused by HPV, predominantly type 16. Several forms of HPV immunotherapy have been studied, however, clinical results could be improved. A novel intradermal administration route, termed DNA tattooing, is superior in animal models, and was tested for the first time in humans with a HPV16 E7 DNA vaccine (TTFC-E7SH). METHODS: The trial was designed to test safety, immunogenicity, and clinical response of TTFC-E7SH in twelve HPV16+ uVIN patients. Patients received six vaccinations via DNA tattooing. The first six patients received 0.2 mg TTFC-E7SH and the next six 2 mg TTFC-E7SH. Vaccine-specific T-cell immunity was evaluated by IFNγ-ELISPOT and multiparametric flow cytometry. RESULTS: Only grade I-II adverse events were observed upon TTFC-E7SH vaccination. The ELISPOT analysis showed in 4/12 patients a response to the peptide pool containing shuffled E7 peptides. Multiparametric flow cytometry showed low CD4+ and/or CD8+ T-cell responses as measured by increased expression of PD-1 (4/12 in both), CTLA-4 (2/12 and 3/12), CD107a (5/12 and 4/12), or the production of IFNγ (2/12 and 1/12), IL-2 (3/12 and 4/12), TNFα (2/12 and 1/12), and MIP1ß (3/12 and 6/12). At 3 months follow-up, no clinical response was observed in any of the twelve vaccinated patients. CONCLUSION: DNA tattoo vaccination was shown to be safe. A low vaccine-induced immune response and no clinical response were observed in uVIN patients after TTFC-E7SH DNA tattoo vaccination. Therefore, a new phase I/II trial with an improved DNA vaccine format is currently in development for patients with uVIN.
Assuntos
DNA/genética , Papillomavirus Humano 16/genética , Proteínas Oncogênicas Virais/imunologia , Vacinas de DNA/imunologia , Neoplasias Vulvares/genética , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Vulvares/terapiaRESUMO
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.
Assuntos
Linfonodos/patologia , Metástase Linfática/imunologia , Neoplasias Vulvares/imunologia , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Microambiente TumoralRESUMO
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.
Assuntos
Antígeno B7-H1/genética , Biomarcadores Tumorais , Expressão Gênica , Proteína 2 Ligante de Morte Celular Programada 1/genética , Neoplasias do Colo do Útero/genética , Adulto , Antígeno B7-H1/metabolismo , Variações do Número de Cópias de DNA , Feminino , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Imunofenotipagem , Hibridização in Situ Fluorescente , Pessoa de Meia-Idade , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma/imunologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Escamosas/imunologia , Células Dendríticas/imunologia , Neoplasias do Colo do Útero/imunologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Colo do Útero/imunologia , Colo do Útero/patologia , Conjuntos de Dados como Assunto , Células Dendríticas/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Ativação Linfocitária , Linfócitos do Interstício Tumoral/imunologia , Pessoa de Meia-Idade , Microambiente Tumoral/imunologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/patologia , beta Catenina/antagonistas & inibidores , beta Catenina/metabolismoRESUMO
Over-expression of matrilysin (MMP-7) is predominantly associated with epithelial (pre)malignant cells. In the present study MMP-7 expression is also found in endothelial cells in various human cancer types. Endothelial MMP-7 was associated with CD34 and/or CD105 expression. These immunohistochemical data were confirmed by RT-PCR on VEGF-stimulated endothelial cells. In addition, MMP-7 was also identified in sprouting endothelial cells in vitro. The potential clinical relevance of endothelial MMP-7 was assessed for cervical cancer patients by evaluating the association with overall survival. In contrast to MMP-7 in malignant epithelial cells, MMP-7 expression in endothelial cells showed a significant association with poor survival (LR 5.12, P=0.02, n=30). Our data suggest that MMP-7 is involved in tumor angiogenesis, thereby contributing to malignant growth and hence associated with decreased survival.
Assuntos
Carcinoma/enzimologia , Endotélio/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 7 da Matriz/biossíntese , Neoplasias do Colo do Útero/enzimologia , Linhagem Celular Tumoral , Feminino , Humanos , Invasividade Neoplásica , Neovascularização Patológica , Esferoides Celulares/patologia , Fatores de Tempo , Distribuição Tecidual , Veias Umbilicais/citologiaRESUMO
BACKGROUND: Proinflammatory cytokines are important in modifying the activity, differentiation, and migration of antigen-presenting cells and may influence the survival of cancer patients. The study assessed whether GM-CSF, TNF-alpha, and IL-12, produced by cervical cancer cells, are important for the activity, differentiation, and migration of antigen-presenting cells. METHODS: In 90 patients with cervical carcinoma the number of monocytes/tumor-associated macrophages (TAM), mature dendritic cells (DC), and Langerhans cells (LHC) was determined using immunohistochemistry. An RNA in situ hybridization technique was used to measure the expression level of GM-CSF, TNF-alpha, IL-12p35, and IL-12p40. RESULTS: TAM were detected intraepithelial as well as in the stroma of the tumor. LHC were only detected intraepithelial and mature DC only in the tumor stroma. The number of TAM correlated positively with the number of mature DC. The expression levels of GM-CSF and TNF-alpha correlated positively with the number of TAM and DC. TNF-alpha showed a negative correlation with the number of LHC. A significant correlation between the expression of functional IL-12 (IL-12p40) and stromal TAM was found. The expression of GM-CSF, TNF-alpha, and IL-12p40 did not correlate significantly with disease-free survival. However, high IL-12p40 expression was associated with a favorable cumulative overall survival. CONCLUSIONS: The results suggest that GM-CSF as well as TNF-alpha, produced by cervical carcinoma cells, may play a role in the differentiation of monocytes into mature DC. Furthermore, TNF-alpha may influence the migration of LHC from the tumor.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Diferenciação Celular , Movimento Celular , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Subunidade p40 da Interleucina-12/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias do Colo do Útero/imunologia , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adulto , Idoso , Carcinoma Adenoescamoso/imunologia , Carcinoma Adenoescamoso/metabolismo , Carcinoma Adenoescamoso/patologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Células Dendríticas/imunologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Hibridização In Situ , Células de Langerhans/imunologia , Macrófagos/imunologia , Pessoa de Meia-Idade , Monócitos/imunologia , Sondas RNA , Taxa de Sobrevida , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Tumor progression and recurrence of cervical cancer is associated with upregulation of matrix metalloproteinase 2 (MMP-2). We evaluated the location, origin and activity of MMP-2 in cervical squamous cell carcinomas in comparison with MT1-MMP (MMP-14), TIMP-2 and extracellular matrix metalloproteinase inducer (EMMPRIN). Positive immunostaining for MMP-2 in malignant cells was detected in 83% of the patients. Two patterns of tumor cell MMP-2 staining were observed: either homogenous in all tumor cells or confined to the cells neighboring the stroma (tumor-border staining pattern, TBS). Fluorescence in situ zymography showed active MMP-2 mainly around tumor nodules displaying TBS. The MMP-2 staining of TBS tumors correlated significantly with the presence of TIMP-2 and MT1-MMP, proteins involved in docking MMP-2 to the cell surface and essential for MMP-2 activation. In situ mRNA hybridization in TBS tumors demonstrated more abundant presence of MMP-2 mRNA in neighboring myofibroblasts than in the adjacent tumor cells. Moreover, the TBS MMP-2 pattern correlated with the presence of EMMPRIN (p = 0.023), suggesting that tumor cells induce MMP-2 production in nearby stromal cells. This pro-MMP-2 could subsequently be activated on tumor cells via the presence of MT1-MMP and TIMP-2. The biological relevance of this locally activated MMP-2 was underscored by the observation that only the TBS pattern of MMP-2 significantly correlated with decreased survival. In conclusion, the colocalization of EMMPRIN, MT1-MMP and TIMP-2 in human cervical carcinomas seems to be involved in a specific distribution pattern of tumor cell bound MMP-2, which is related with local proteolytic activity and therefore might be associated with worse prognosis of the patients.