Adjunctive use of p16 immunohistochemistry for optimizing management of CIN lesions in a high-risk human papillomavirus-positive population.

INTRODUCTION: Immunostaining with p16INK4a (p16), a tumor-suppressor surrogate protein biomarker for high-risk human papillomavirus (hrHPV) oncogenic activity, may complement standard hematoxylin and eosin (H&E) histology review, and provide more objective criteria to support the cervical intraepithelial neoplasia (CIN) diagnosis. With this study we assessed the impact of p16 immunohistochemistry on CIN grading in an hrHPV-based screening setting. MATERIAL AND METHODS: In this post-hoc analysis, 326 histology follow-up samples from a group of hrHPV-positive women were stained with p16 immunohistochemistry. All H&E samples were centrally revised. The pathologists reported their level of confidence in classifying the CIN lesion. RESULTS: Combining H&E and p16 staining resulted in a change of diagnosis in 27.3% (n = 89) of cases compared with the revised H&E samples, with a decrease of 34.5% (n = 18) in CIN1 and 22.7% (n = 15) in CIN2 classifications, and an increase of 18.3% (n = 19) in no CIN and 20.7% (n = 19) in CIN3 diagnoses. The level of confidence in CIN grading by the pathologist increased with adjunctive use of p16 immunohistochemistry to standard H&E. CONCLUSIONS: This study shows that adjunctive use of p16 immunohistochemistry to H&E morphology reduces the number of CIN1 and CIN2 classifications with a proportional increase in no CIN and CIN3 diagnoses, compared with standard H&E-based CIN diagnosis alone. The pathologists felt more confident in classifying the material with H&E and p16 immunohistochemistry than by using H&E alone, particularly during assessment of small biopsies. Adjunctive use of p16 immunohistochemistry to standard H&E assessment of CIN would be valuable for the diagnostic accuracy, thereby optimizing CIN management and possibly decreasing overtreatment.

Risk of Peritoneal Carcinomatosis After Risk-Reducing Salpingo-Oophorectomy: A Systematic Review and Individual Patient Data Meta-Analysis.

PURPOSE: After risk-reducing salpingo-oophorectomy (RRSO), BRCA1/2 pathogenic variant (PV) carriers have a residual risk to develop peritoneal carcinomatosis (PC). The etiology of PC is not yet clarified, but may be related to serous tubal intraepithelial carcinoma (STIC), the postulated origin for high-grade serous cancer. In this systematic review and individual patient data meta-analysis, we investigate the risk of PC in women with and without STIC at RRSO. METHODS: Unpublished data from three centers were supplemented by studies identified in a systematic review of EMBASE, MEDLINE, and the Cochrane library describing women with a BRCA-PV with and without STIC at RRSO until September 2020. Primary outcome was the hazard ratio for the risk of PC between BRCA-PV carriers with and without STIC at RRSO, and the corresponding 5- and 10-year risks. Primary analysis was based on a one-stage Cox proportional-hazards regression with a frailty term for study. RESULTS: From 17 studies, individual patient data were available for 3,121 women, of whom 115 had a STIC at RRSO. The estimated hazard ratio to develop PC during follow-up in women with STIC was 33.9 (95% CI, 15.6 to 73.9), P < .001) compared with women without STIC. For women with STIC, the five- and ten-year risks to develop PC were 10.5% (95% CI, 6.2 to 17.2) and 27.5% (95% CI, 15.6 to 43.9), respectively, whereas the corresponding risks were 0.3% (95% CI, 0.2 to 0.6) and 0.9% (95% CI, 0.6 to 1.4) for women without STIC at RRSO. CONCLUSION: BRCA-PV carriers with STIC at RRSO have a strongly increased risk to develop PC which increases over time, although current data are limited by small numbers of events.

Early prediction of post-molar gestational trophoblastic neoplasia and resistance to methotrexate, based on a single serum human chorionic gonadotropin measurement.

BACKGROUND: Clinicians are unable to provide individualized counseling regarding risk of progression for patients with a complete hydatidiform mole (CHM). We developed nomograms enabling early prediction of post-molar gestational trophoblastic neoplasia (GTN) and resistance to methotrexate (MTX) based on a single serum human chorion gonadotropin (hCG) measurement. METHODS: We generated two nomograms with logistic regression: to predict post-molar GTN, and MTX resistance. For patients with high probability to progress to post-molar GTN or MTX resistance, we determined hCG cut-offs at 97.5% specificity to select patients for additional- or adjustments in current treatment. RESULTS: The nomograms had a good to excellent ability to distinguish either between patients with uneventful hCG regression versus progression to post molar GTN, or between patients cured by MTX versus patients in whom resistance would occur. At 97.5% specificity, we identified 66% (95%CI 56-75) of the 149 patients who would progress to post-molar GTN, four weeks after initial curettage. For patients treated with MTX, we identified 55% (95%CI 23-83) of the 43 patients who would become resistant, preceding their third course at 97.5% specificity. CONCLUSION: The nomograms and cut-off levels can be used to assist in counseling for patients diagnosed with CHM.

Improving preoperative diagnosis in endometrial cancer using systematic morphological assessment and a small immunohistochemical panel.

Preoperative histopathological classification determines the primary surgical approach in endometrial carcinoma (EC) patients but has only moderate agreement between preoperative and postoperative diagnosis. The aim of the PIpelle Prospective ENDOmetrial carcinoma (PIPENDO) study is to determine whether histopathological assessment and a small panel of diagnostic biomarkers decreases discrepancies between preoperative and postoperative diagnosis in EC. Preoperative endometrial tissue of 378 included patients with EC was stained with 15 different antibodies. Clinically relevant discrepancies in grade or histological subtype between original preoperative and reviewed postoperative diagnosis were observed in 75 (20%) patients. Highest clinically relevant discrepancy was found in grade 2 ECs (20%), compared to 5% and 14% in respectively grade 1 and 3 endometrioid endometrial carcinomas (EECs). A practical two-biomarker panel with PR and p53 improved diagnostic accuracy (AUC = 0.92; 95%CI = 0.88-0.95) compared to solely morphological evaluation (AUC = 0.86). In preoperative high-grade EC, the diagnostic accuracy of histological subtype was improved by a three-immunohistochemical biomarker panel (PR, IMP3, and L1CAM) (AUC = 0.93; 95%CI = 0.88-0.98) compared to solely morphological evaluation (AUC = 0.81). In conclusion to improve correct preoperative diagnosis in EC, we recommend use of a panel of at least two easily accessible immunohistochemical biomarkers (PR and p53), only in grade 2 ECs. Overall, this will reduce clinically relevant discrepancies in tumor grade and subtype with postoperative diagnosis with 6% (from 20% to 14%). Addition of PR, IMP3, and L1CAM for histological subtyping in high-grade EECs resulted in a further decrease in discrepancies with 8% (from 20% to 12%).

The role of MMP-14 in ovarian cancer: a systematic review.

AIM: In order to evaluate the role of MMP-14 in ovarian cancer, a systematic review was conducted. METHODS: In March 2020, a search in Pubmed was performed with MMP-14 and ovarian cancer as search terms. After exclusion of the references not on MMP-14 or ovarian cancer or not in English, the studies found were classified into two categories: basic research and clinicopathological research. RESULTS: In total, 94 references were found of which 33 were excluded. Two additional articles were found in the reference lists of the included studies. Based on the full texts, another 4 were excluded. Eventually, 59 studies were included in the review, 32 on basic research and 19 on clinicopathological research. 8 studies fell in both categories. The basic research studies show that MMP-14 plays an important role in ovarian cancer in the processes of proliferation, invasion, angiogenesis and metastasis. In clinocopathological research, MMP-14 expression is found in most tumours with characteristics of poor prognosis but this immunohistochemical MMP-14 determination does not seem to be an independent predictor of prognosis. CONCLUSIONS: From this systematic review of the literature concerning MMP-14 in ovarian cancer it becomes clear that MMP-14 plays various important roles in the pathophysiology of ovarian cancer. The exact translation of these roles in the pathophysiology to the importance of MMP-14 in clinicopathological research in ovarian cancer and possible therapeutic role of anti-MMP-14 agents needs further elucidation.

Cervical intraepithelial neoplasia and the risk of spontaneous preterm birth: A Dutch population-based cohort study with 45,259 pregnancy outcomes.

BACKGROUND: Excisional procedures of cervical intraepithelial neoplasia (CIN) may increase the risk of preterm birth. It is unknown whether this increased risk is due to the excision procedure itself, to the underlying CIN, or to secondary risk factors that are associated with both preterm birth and CIN. The aim of this study is to assess the risk of spontaneous preterm birth in women with treated and untreated CIN and examine possible associations by making a distinction between the excised volume of cervical tissue and having cervical disease. METHODS AND FINDINGS: This Dutch population-based observational cohort study identified women aged 29 to 41 years with CIN between 2005 and 2015 from the Dutch pathology registry (PALGA) and frequency matched them with a control group without any cervical abnormality based on age at and year of pathology outcome (i.e., CIN or normal cytology) and urbanization (<100,000 inhabitants or ≥100,000 inhabitants). All their 45,259 subsequent singleton pregnancies with a gestational age ≥16 weeks between 2010 and 2017 were identified from the Dutch perinatal database (Perined). Nineteen potential confounders for preterm birth were identified. Adjusted odds ratios (ORs) were calculated for preterm birth comparing the 3 different groups of women: (1) women without CIN diagnosis; (2) women with untreated CIN; and (3) women with treated CIN prior to each childbirth. In total, 29,907, 5,940, and 9,412 pregnancies were included in the control, untreated CIN, and treated CIN group, respectively. The control group showed a 4.8% (1,002/20,969) proportion of spontaneous preterm birth, which increased to 6.9% (271/3,940) in the untreated CIN group, 9.5% (600/6,315) in the treated CIN group, and 15.6% (50/321) in the group with multiple treatments. Women with untreated CIN had a 1.38 times greater odds of preterm birth compared to women without CIN (95% confidence interval (CI) 1.19 to 1.60; P < 0.001). For women with treated CIN, these odds 2.07 times increased compared to the control group (95% CI 1.85 to 2.33; P < 0.001). Treated women had a 1.51 times increased odds of preterm birth compared to women with untreated CIN (95% CI 1.29 to 1.76; P < 0.001). Independent from cervical disease, a volume excised from the cervix of 0.5 to 0.9 cc increased the odds of preterm birth 2.20 times (37/379 versus 1,002/20,969; 95% CI 1.52 to 3.20; P < 0.001). These odds further increased 3.13 times and 5.93 times for women with an excised volume of 4 to 8.9 cc (90/724 versus 1,002/20,969; 95% CI 2.44 to 4.01; P < 0.001) and ≥9 cc (30/139 versus 1,002/20,969; 95% CI 3.86 to 9.13; P < 0.001), respectively. Limitations of the study include the retrospective nature, lack of sufficient information to calculate odds of preterm birth <24 weeks, and that the excised volume could only be calculated for a select group of women. CONCLUSIONS: In this study, we observed a strong correlation between preterm birth and a volume of ≥0.5 cc excised cervical tissue, regardless of the severity of CIN. Caution should be taken when performing excisional treatment in women of reproductive age as well as prudence in case of multiple biopsies. Fertile women with a history of performing multiple biopsies or excisional treatment for CIN may benefit from close surveillance during pregnancy.

Association of Salpingectomy With Delayed Oophorectomy Versus Salpingo-oophorectomy With Quality of Life in BRCA1/2 Pathogenic Variant Carriers: A Nonrandomized Controlled Trial.

IMPORTANCE: Most women with a BRCA1/2 pathogenic variant undergo premature menopause with potential short- and long-term morbidity due to the current method of ovarian carcinoma prevention: risk-reducing salpingo-oophorectomy (RRSO). Because the fallopian tubes play a key role in ovarian cancer pathogenesis, salpingectomy with delayed oophorectomy may be a novel risk-reducing strategy with benefits of delaying menopause. OBJECTIVE: To compare menopause-related quality of life after risk-reducing salpingectomy (RRS) with delayed oophorectomy with RRSO in carriers of the BRCA1/2 pathogenic variant. DESIGN, SETTING, AND PARTICIPANTS: A multicenter nonrandomized controlled preference trial (TUBA study), with patient recruitment between January 16, 2015, and November 7, 2019, and follow-up at 3 and 12 months after surgery was conducted in all Dutch university hospitals and a few large general hospitals. In the Netherlands, RRSO is predominantly performed in these hospitals. Patients at the clinical genetics or gynecology department between the ages of 25 and 40 years (BRCA1) or 25 to 45 years (BRCA2) who were premenopausal, had completed childbearing, and were undergoing no current treatment for cancer were eligible. INTERVENTIONS: Risk-reducing salpingo-oophorectomy at currently recommended age or RRS after completed childbearing with delayed oophorectomy. After RRSO was performed, hormone replacement therapy was recommended for women without contraindications. MAIN OUTCOMES AND MEASURES: Menopause-related quality of life as assessed by the Greene Climacteric Scale, with a higher scale sum (range, 0-63) representing more climacteric symptoms. Secondary outcomes were health-related quality of life, sexual functioning and distress, cancer worry, decisional regret, and surgical outcomes. RESULTS: A total of 577 women (mean [SD] age, 37.2 [3.5] years) were enrolled: 297 (51.5%) were pathogenic BRCA1 variant carriers and 280 (48.5%) were BRCA2 pathogenic variant carriers. At the time of analysis, 394 patients had undergone RRS and 154 had undergone RRSO. Without hormone replacement therapy, the adjusted mean increase from the baseline score on the Greene Climacteric Scale was 6.7 (95% CI, 5.0-8.4; P < .001) points higher during 1 year after RRSO than after RRS. After RRSO with hormone replacement therapy, the difference was 3.6 points (95% CI, 2.3-4.8; P < .001) compared with RRS. CONCLUSIONS AND RELEVANCE: Results of this nonrandomized controlled trial suggest that patients have better menopause-related quality of life after RRS than after RRSO, regardless of hormone replacement therapy. An international follow-up study is currently evaluating the oncologic safety of this therapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02321228.

Immune cell composition in the endometrium of patients with a complete molar pregnancy: Effects on outcome.

OBJECTIVE: In 15% of patients with complete hydatidiform mole (CHM), disease progresses to post-molar gestational trophoblastic neoplasia (GTN) after curettage. Tumor infiltrating lymphocytes (TILs) are essential in overcoming disease in many tumors. Infiltrating lymphocyte composition and density may influence trophoblast regression and development of post-molar GTN. We analyzed immune cell composition and density in curettaged endometrium of patients with CHM which spontaneously regressed, and of patients with CHM which progressed to post-molar GTN. METHODS: Sixteen patients with CHM and spontaneous regression, and 16 patients with CHM which progressed to post-molar GTN were selected. Immune cell composition and density of natural killer (NK) cells, natural killer T (NKT)-like cells, Cytotoxic T cells, T-Regulatory and T-Helper cells, were determined by multiplex immunohistochemistry (mIHC). RESULTS: Curettaged endometrium of patients with CHM and spontaneous regression contained a slightly higher number of immune cells compared to patients with CHM which progressed to post-molar GTN. NKT-like cell density was significantly higher in patients with spontaneous regression compared to patients with CHM which progressed to post-molar GTN (483 ± 296 vs.295 ± 143 (mean ± SD), p = 0.03) respectively. NKT-like cell density in the spontaneous regression group was split in 'high' and 'low' (i.e. above and below the median number of NKT-like cells). In patients with high NKT-like cell density, hCG normalized earlier than in patients with low NKT-like cell density (9.5 weeks, (range 3.7-14) vs. 12.9 weeks, (range 8.6-17.9), p = 0.05). CONCLUSION: A high number of NKT-like cells in the endometrium of CHMs may contribute to spontaneous regression of molar trophoblast cells.

TIGIT blockade enhances functionality of peritoneal NK cells with altered expression of DNAM-1/TIGIT/CD96 checkpoint molecules in ovarian cancer.

Advanced ovarian cancer (OC) patients have a poor 5-year survival of only 28%, emphasizing the medical need for improved therapies. Adjuvant immunotherapy could be an attractive approach since OC is an immunogenic disease and the presence of tumor-infiltrating lymphocytes has shown to positively correlate with patient survival. Among these infiltrating lymphocytes are natural killer (NK) cells, key players involved in tumor targeting, initiated by signaling via activating and inhibitory receptors. Here, we investigated the role of the DNAM-1/TIGIT/CD96 axis in the anti-tumor response of NK cells toward OC. Ascites-derived NK cells from advanced OC patients showed lower expression of activating receptor DNAM-1 compared to healthy donor peripheral blood NK cells, while inhibitory receptor TIGIT and CD96 expression was equal or higher, respectively. This shift to a more inhibitory phenotype could also be induced in vitro by co-culturing healthy donor NK cells with OC tumor spheroids, and in vivo on intraperitoneally infused NK cells in SKOV-3 OC bearing NOD/SCID-IL2Rγnull (NSG) mice. Interestingly, TIGIT blockade enhanced degranulation and interferon gamma (IFNγ) production of healthy donor CD56dim NK cells in response to OC tumor cells, especially when DNAM-1/CD155 interactions were in place. Importantly, TIGIT blockade boosted functional responsiveness of CD56dim NK cells of OC patients with a baseline reactivity against SKOV-3 cells. Overall, our data show for the first time that checkpoint molecules TIGIT/DNAM-1/CD96 play an important role in NK cell responsiveness against OC, and provides rationale for incorporating TIGIT interference in NK cell-based immunotherapy in OC patients.

EpCAM-Binding DARPins for Targeted Photodynamic Therapy of Ovarian Cancer.

Ovarian cancer is the most lethal gynecological malignancy due to late detection associated with dissemination throughout the abdominal cavity. Targeted photodynamic therapy (tPDT) aimed at epithelial cell adhesion molecule (EpCAM), overexpressed in over 90% of ovarian cancer metastatic lesions, is a promising novel therapeutic modality. Here, we tested the specificity and activity of conjugates of EpCAM-directed designed ankyrin repeat proteins (DARPins) with the photosensitizer IRDye 700DX in in vitro and in vivo ovarian cancer models. EpCAM-binding DARPins (Ec1: Kd = 68 pM; Ac2: Kd = 130 nM) and a control DARPin were site-specifically functionalized with fluorophores or IRDye 700DX. Conjugation of anti-EpCAM DARPins with fluorophores maintained EpCAM-specific binding in cell lines and patient-derived ovarian cancer explants. Penetration of DARPin Ec1 into tumor spheroids was slower than that of Ac2, indicative of a binding site barrier effect for Ec1. DARPin-IRDye 700DX conjugates killed EpCAM-expressing cells in a highly specific and illumination-dependent fashion in 2D and 3D cultures. Furthermore, they effectively homed to EpCAM-expressing subcutaneous OV90 xenografts in mice. In conclusion, the high activity and specificity observed in preclinical ovarian cancer models, combined with a high specificity in patient material, warrant a further investigation of EpCAM-targeted PDT for ovarian cancer.

Oestrogen receptor pathway activity is associated with outcome in endometrial cancer.

BACKGROUND: Oestrogen receptor (ER) expression is a prognostic biomarker in endometrial cancer (EC). However, expression does not provide information about the functional activity of the ER pathway. We evaluated a model to quantify ER pathway activity in EC, and determined the prognostic relevance of ER pathway activity. METHODS: ER pathway activity was measured in two publicly available datasets with endometrial and EC tissue, and one clinical cohort with 107 samples from proliferative and hyperplastic endometrium and endometrioid-type EC (EEC) and uterine serous cancer (USC). ER pathway activity scores were inferred from ER target gene mRNA levels from Affymetrix microarray data (public datasets), or measured by qPCR on formalin-fixed paraffin-embedded samples (clinical cohort) and related to ER expression and outcome. RESULTS: ER pathway activity scores differed significantly throughout the menstrual cycle supporting the validity of the pathway test. The highest ER pathway scores were found in proliferative and hyperplastic endometrium and stage I EEC, whereas stage II-IV EEC and USCs had significantly lower levels. Low ER pathway activity was associated with recurrent disease, and added prognostic value in patients with low ER expression. CONCLUSION: The ER pathway test reflects activity of the ER pathway, and may improve prediction of outcome in EC patients.

Preoperative risk stratification in endometrial cancer (ENDORISK) by a Bayesian network model: A development and validation study.

BACKGROUND: Bayesian networks (BNs) are machine-learning-based computational models that visualize causal relationships and provide insight into the processes underlying disease progression, closely resembling clinical decision-making. Preoperative identification of patients at risk for lymph node metastasis (LNM) is challenging in endometrial cancer, and although several biomarkers are related to LNM, none of them are incorporated in clinical practice. The aim of this study was to develop and externally validate a preoperative BN to predict LNM and outcome in endometrial cancer patients. METHODS AND FINDINGS: Within the European Network for Individualized Treatment of Endometrial Cancer (ENITEC), we performed a retrospective multicenter cohort study including 763 patients, median age 65 years (interquartile range [IQR] 58-71), surgically treated for endometrial cancer between February 1995 and August 2013 at one of the 10 participating European hospitals. A BN was developed using score-based machine learning in addition to expert knowledge. Our main outcome measures were LNM and 5-year disease-specific survival (DSS). Preoperative clinical, histopathological, and molecular biomarkers were included in the network. External validation was performed using 2 prospective study cohorts: the Molecular Markers in Treatment in Endometrial Cancer (MoMaTEC) study cohort, including 446 Norwegian patients, median age 64 years (IQR 59-74), treated between May 2001 and 2010; and the PIpelle Prospective ENDOmetrial carcinoma (PIPENDO) study cohort, including 384 Dutch patients, median age 66 years (IQR 60-73), treated between September 2011 and December 2013. A BN called ENDORISK (preoperative risk stratification in endometrial cancer) was developed including the following predictors: preoperative tumor grade; immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), p53, and L1 cell adhesion molecule (L1CAM); cancer antigen 125 serum level; thrombocyte count; imaging results on lymphadenopathy; and cervical cytology. In the MoMaTEC cohort, the area under the curve (AUC) was 0.82 (95% confidence interval [CI] 0.76-0.88) for LNM and 0.82 (95% CI 0.77-0.87) for 5-year DSS. In the PIPENDO cohort, the AUC for 5-year DSS was 0.84 (95% CI 0.78-0.90). The network was well-calibrated. In the MoMaTEC cohort, 249 patients (55.8%) were classified with <5% risk of LNM, with a false-negative rate of 1.6%. A limitation of the study is the use of imputation to correct for missing predictor variables in the development cohort and the retrospective study design. CONCLUSIONS: In this study, we illustrated how BNs can be used for individualizing clinical decision-making in oncology by incorporating easily accessible and multimodal biomarkers. The network shows the complex interactions underlying the carcinogenetic process of endometrial cancer by its graphical representation. A prospective feasibility study will be needed prior to implementation in the clinic.

Fallopian tube abnormalities in uterine serous carcinoma.

OBJECTIVE: Uterine serous carcinoma (USC) is presumed to arise from endometrial intra-epithelial carcinoma (EIC), whereas tubo-ovarian high-grade serous carcinomas have similar precursor lesions in the Fallopian tube, i.e. serous tubal intra-epithelial carcinoma (STIC). The presence of Fallopian tube abnormalities and their clonal relationship to the concurrent USC was investigated. METHODS: In this multicenter study, all patients treated for USC between 1992 and 2017 were retrospectively identified. Histopathological diagnosis of USC, EIC and STIC was revised by an expert pathologist. Additionally, all Fallopian tube sections were immunohistochemically stained (p53 and Ki-67). Fallopian tube abnormalities were classified as either p53 signature, serous tubal intra-epithelial lesion (STIL) or STIC. The USCs and Fallopian tube abnormalities were analyzed by targeted next-generation sequencing. RESULTS: In 168 included patients, Fallopian tube abnormalities were found in 27.4% (46/168): p53-signatures in 17.9% (30/168), STILs in 3.0% (5/168) and STICs in 6.5% (11/168). In subgroup analysis, STICs were found in 9.5% (11/115) of patients with at least one section of the fimbriated end embedded. Next-generation sequencing showed identical TP53-mutations in the STIC and corresponding USC. CONCLUSIONS: In conclusion, the presence of Fallopian tube abnormalities was shown in a high percentage of patients with USC, representing either true precursor lesions or metastasized disease.

Clonal Relationship Between Lichen Sclerosus, Differentiated Vulvar Intra-epithelial Neoplasia and Non HPV-related Vulvar Squamous Cell Carcinoma.

BACKGROUND/AIM: Differentiated vulvar intraepithelial neoplasia (dVIN) and lichen sclerosus (LS) can give rise to vulvar squamous cell carcinoma (VSCC), but genetic evidence is currently still limited. We aimed to determine genetic abnormalities in VSCC and backtrack these abnormalities in the dVIN and LS lesions. MATERIALS AND METHODS: DNA from VSCC and patient-matched dVIN and LS samples of twelve patients was collected. High-resolution genome-wide copy number analysis was performed and subsequently, we sequenced TP53. RESULTS: Copy number alterations were identified in all VSCC samples. One dVIN lesion presented with three copy number alterations that were preserved in the paired VSCC sample. Targeted sequencing of TP53 identified mutations in five VSCCs. All five mutations were traced back in the dVIN (n=5) or the LS (n=1) with frequencies ranging from 3-19%. CONCLUSION: Our data provide genetic evidence for a clonal relationship between VSCC and dVIN or LS.

The risk of cervical cancer after cervical intraepithelial neoplasia grade 3: A population-based cohort study with 80,442 women.

OBJECTIVE: To estimate the risk of cervical cancer in women with a history of cervical intraepithelial neoplasia (CIN) grade 3 and to review the compliance with post-treatment follow-up. METHODS: A population-based retrospective cohort study including 80,442 women with a median follow-up of 15.8 years, and 1,278,297 person years. Women with CIN3 between 1990 and 2010 were identified from the Dutch Pathology Registry (PALGA) and linked to the general female population from the Netherlands Cancer Registry. Cases of recurrent CIN3 and cervical cancer, defined as occurrence minimally two years post-treatment, were identified until 2016. Standardized incidence ratios (SIRs) were calculated for the risk of cervical cancer. RESULTS: 1554 women (1.9%) developed recurrent CIN3 and 397 women (0.5%) cervical cancer. Women with CIN3 were associated with a twofold increased risk of cervical cancer (SIR 2.29; 95%CI 2.07-2.52) compared with the general female population. Women aged ≥50 years during CIN3 diagnosis had a sevenfold and women with recurrent CIN3 a ninefold increased risk of developing cervical cancer. The increased risk up to 20 years of follow-up seems to be mostly attributable to ageing. 37.0% of women who developed cervical cancer after CIN3 did not complete the advised post-treatment follow-up. CONCLUSIONS: Women with CIN3 have a long-lasting twofold increased risk of developing cervical cancer, even when they complete the post-treatment follow-up and adhere to the regular screening program. This risk increases with CIN3 diagnosis at older age, further ageing during follow-up and in women with recurrent CIN3. Studies on optimizing follow-up strategies are warranted.

Short-term surgical complications after radical hysterectomy-A nationwide cohort study.

INTRODUCTION: Centralization has, among other aspects, been argued to have an impact on quality of care in terms of surgical morbidity. Next, monitoring quality of care is essential in identifying areas of improvement. This nationwide cohort study was conducted to determine the rate of short-term surgical complications and to evaluate its possible predictors in women with early-stage cervical cancer. MATERIAL AND METHODS: Women diagnosed with early-stage cervical cancer, 2009 FIGO stages IB1 and IIA1, between 2015 and 2017 who underwent radical hysterectomy with pelvic lymphadenectomy in 1 of the 9 specialized medical centers in the Netherlands, were identified from the Netherlands Cancer Registry. Women were excluded if primary treatment consisted of hysterectomy without parametrial dissection or radical trachelectomy. Women in whom radical hysterectomy was aborted during the procedure, were also excluded. Occurrence of intraoperative and postoperative complications and type of complications, developing within 30 days after surgery, were prospectively registered. Multivariable logistic regression analysis was used to identify predictors of surgical complications. RESULTS: A total of 472 women were selected, of whom 166 (35%) developed surgical complications within 30 days after radical hysterectomy. The most frequent complications were urinary retention with catheterization in 73 women (15%) and excessive perioperative blood loss >1000 mL in 50 women (11%). Open surgery (odds ratio [OR] 3.42; 95% CI 1.73-6.76), chronic pulmonary disease (OR 3.14; 95% CI 1.45-6.79), vascular disease (OR 1.90; 95% CI 1.07-3.38), and medical center (OR 2.83; 95% CI 1.18-6.77) emerged as independent predictors of the occurrence of complications. Body mass index (OR 0.94; 95% CI 0.89-1.00) was found as a negative predictor of urinary retention. Open surgery (OR 36.65; 95% CI 7.10-189.12) and body mass index (OR 1.15; 95% CI 1.08-1.22) were found to be independent predictors of excessive perioperative blood loss. CONCLUSIONS: Short-term surgical complications developed in 35% of the women after radical hysterectomy for early-stage cervical cancer in the Netherlands, a nation with centralized surgical care. Comorbidities predict surgical complications, and open surgery is associated with excessive perioperative blood loss.

Targeting Indoleamine 2,3-Dioxygenase in Cancer Models Using the Novel Small Molecule Inhibitor NTRC 3883-0.

Indoleamine 2,3-dioxygenase (IDO1) is a key regulator of immune suppression by catalyzing the oxidation of L-tryptophan. IDO1 expression has been related to poor prognosis in several cancers and to resistance to checkpoint immunotherapies. We describe the characterization of a novel small molecule IDO1 inhibitor, NTRC 3883-0, in a panel of biochemical and cell-based assays, and various cancer models. NTRC 3883-0 released the inhibitory effect of IDO1 on CD8-positive T cell proliferation in co-cultures of IDO1-overexpressing cells with healthy donor lymphocytes, demonstrating its immune modulatory activity. In a syngeneic mouse model using IDO1-overexpressing B16F10 melanoma cells, NTRC 3883-0 effectively counteracted the IDO1-induced modulation of L-tryptophan and L-kynurenine levels, demonstrating its in vivo target modulation. Finally, we studied the expression and activity of IDO1 in primary cell cultures established from the malignant ascites of ovarian cancer patients. In these cultures, IDO1 expression was induced upon stimulation with IFNγ, and its activity could be inhibited by NTRC 3883-0. Based on these results, we propose the use of ascites cell-based functional assays for future patient stratification. Our results are discussed in light of the recent discontinuation of clinical trials of more advanced IDO1 inhibitors and the reconsideration of IDO1 as a valid drug target.

RNA-based high-risk HPV genotyping and identification of high-risk HPV transcriptional activity in cervical tissues.

Nearly all cervical cancers are initiated by a persistent infection with one of the high-risk human papillomaviruses (high-risk HPV). High-risk HPV DNA testing is highly sensitive but cannot distinguish between active, productive infections and dormant infections or merely deposited virus. A solution for this shortcoming may be the detection of transcriptional activity of viral oncogenes instead of mere presence of high-risk HPVs. In this study, fresh-frozen cervical tissues (n = 22) were subjected to high-risk HPV DNA detection using the line probe assay and to targeted RNA next-generation sequencing using single-molecule molecular inversion probes. Targeted RNA sequencing was applied for (1) RNA-based genotyping of high-risk HPV, giving information on specific HPV-subtype (2) discrimination of E2, E6, and E7 transcripts and (3) discovery of possible non-HPV cancer biomarkers. Data were analyzed using computational biology. Targeted RNA sequencing enabled reliable genotyping of high-risk HPV subtypes and allowed quantitative detection of E2, E6, and E7 viral gene expression, thereby discriminating cervical lesions from normal cervical tissues. Moreover, targeted RNA sequencing identified possible cervical cancer biomarkers other than high-risk HPV. Interestingly, targeted RNA sequencing also provided high-quality transcription profiles from cervical scrape samples, even after 1 week of dry storage or storage in Preservcyt fixative. This proof of concept study shows that targeted RNA sequencing can be used for high-risk HPV genotyping and simultaneous detection of high-risk HPV gene activity. Future studies are warranted to investigate the potential of targeted RNA sequencing for risk assessment for the development of cervical lesions, based on molecular analysis of cervical scrapes.

Primary Ovarian Tumors With Lymphogenic and Hematogenic Metastasis Express High MMP-14, Which Colocalizes With Highly Sulfated Chondroitin Sulfate in the Stroma.

Lymphogenic and hematogenic metastases are uncommon in ovarian cancer, especially at presentation. We hypothesized that MMP-14 and MMP-2, CD44, and highly sulfated chondroitin sulfate (CS-E) may be overexpressed in tumors with these metastatic patterns. These molecules are all present in the ovarian tumor microenvironment, wherein they may interact. In an ovarian cancer cohort of 44 patients with metastases in lymph nodes, spleen, and/or liver, the presence of MMP-14, MMP-2, CD44, and CS-E in both the primary tumor and the metastases was determined with immunohistochemistry and related to clinical characteristics. Immunohistochemical expression was found for MMP-14 in all primary tumors as well as in all metastases and for MMP-2 expression in most of the samples. Most primary tumors with synchronous metastases were positive for CS-E, as well as most primary tumors with metachronous lymphogenic metastases. The expression of the MMPs and CS-E in the stroma seemed to colocalize. For CD44 immunohistochemical expression, this relationship was not found. Epithelial MMP-14 on the one hand and stromal CS-E on the other hand seem to be essential players in ovarian cancer with lymphogenic and hematogenic metastases. CD44 expression is not correlated with the other markers. More research on the interaction of these molecules and their role in the process of dissimination of disease is warranted.