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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., Competing Interests: Conflicts of interest and sources of funding: none declared., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

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., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Rotman, Otter, Bleeker, Samuels, Heeren, Roemer, Kenter, Zijlmans, van Trommel, de Gruijl and Jordanova.)

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., (©2020 American Association for Cancer Research.)

Background: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression., Methods: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8)., Results: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8 + T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8 + FoxP3 + CD25 + T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8 + FoxP3 - counterparts., Conclusion: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8 + FoxP3 + CD25 + T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.

Purpose: Hybrid image-guided surgery technologies such as combined radio- and fluorescence-guidance are increasingly gaining interest, but their added value still needs to be proven. In order to evaluate if and how fluorescence-guidance can help realize improvements beyond the current state-of-the-art in sentinel node (SN) biopsy procedures, use of the hybrid tracer indocyanine green (ICG)- 99m Tc-nancolloid was evaluated in a large cohort of patients., Patients and Methods: A prospective trial was conducted (n = 501 procedures) in a heterogeneous cohort of 495 patients with different malignancies (skin malignancies, oral cavity cancer, penile cancer, prostate cancer and vulva cancer). After injection of ICG- 99m Tc-nanocolloid, SNs were preoperatively identified based on lymphoscintigraphy and SPECT/CT. Intraoperatively, SNs were pursued via gamma tracing, visual identification (blue dye) and/or near-infrared fluorescence imaging during either open surgical procedures (head and neck, penile, vulvar cancer and melanoma) or robot assisted laparoscopic surgery (prostate cancer). As the patients acted as their own control, use of hybrid guidance could be compared to conventional radioguidance and the use of blue dye (n = 300). This was based on reported surgical complications, overall survival, LN recurrence free survival, and false negative rates (FNR)., Results: A total of 1,327 SN-related hotspots were identified on 501 preoperative SPECT/CT scans. Intraoperatively, a total number of 1,643 SNs were identified based on the combination of gamma-tracing (>98%) and fluorescence-guidance (>95%). In patients wherein blue dye was used (n = 300) fluorescence-based SN detection was superior over visual blue dye-based detection (22-78%). No adverse effects related to the use of the hybrid tracer or the fluorescence-guidance procedure were found and outcome values were not negatively influenced., Conclusion: With ICG- 99m Tc-nanocolloid, the SN biopsy procedure has become more accurate and independent of the use of blue dye. With that, the procedure has evolved to be universal for different malignancies and anatomical locations.

Objective: Inguinofemoral lymphadenectomy for patients with vulvar squamous cell carcinoma is associated with a high incidence of postoperative wound complications, which may be influenced by inguinal drain management. The aim of this nationwide prospective study (MAMBO: Morbidity And Measurement of the BOdy) was to assess the feasibility and the incidence of complications after volume-controlled versus short drainage., Methods: The MAMBO study consisted of two observational studies in all eight oncology centers in the Netherlands, conducted between 2012 and 2016. In the first study, the drain was removed when the production was <30ml/24h, except in the first 48h, and after a maximum of 28days (MAMBO-IA). In the second study, the drain was removed five days postoperatively regardless of production (MAMBO-IB). We assessed the complications within eight weeks after surgery using logistic regression to compare the incidence of one or more complications between the two drainage protocols, adjusting for possible confounders., Results: We included 77 patients (139 groins) for volume-controlled drainage and 64 patients (112 groins) for short drainage. Volume-controlled drainage was associated with significant less lymphocele formation. Moreover, we found no difference in wound infection or primary wound breakdown. The estimated incidence of one or more complications was 46% per groin after volume-controlled drainage versus 75% after short drainage, (RD 29% (95% CI 8, 49) p=0.006)., Conclusions: This prospective study shows that volume-controlled drainage is associated with significantly less complications compared to short drainage. We therefore recommend volume-controlled drainage after inguinofemoral lymphadenectomy in patients with vulvar squamous cell carcinoma., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 MIP1β (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.