Heleen van Beekhuizen

Epigenetic and Genomic Hallmarks of PARP-Inhibitor Resistance in Ovarian Cancer Patients

Background: Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after surgery and chemotherapy. Unfortunately, many patients experience disease progression because of acquired therapy resistance. This study aims to characterize epigenetic and genomic changes in cell-free DNA (cfDNA) associated with PARPi resistance. Materials and Methods: Blood was taken from 31 EOC patients receiving PARPi therapy before treatment and at disease progression during/after treatment. Resistance was defined as disease progression within 6 months after starting PARPi and was seen in fifteen patients, while sixteen patients responded for 6 to 42 months. Blood cfDNA was evaluated via Modified Fast Aneuploidy Screening Test-Sequencing System (mFast-SeqS to detect aneuploidy, via Methylated DNA Sequencing (MeD-seq) to find differentially methylated regions (DMRs), and via shallow whole-genome and -exome sequencing (shWGS, exome-seq) to define tumor fractions and mutational signatures. Results: Aneuploid cfDNA was undetectable pre-treatment but observed in six patients post-treatment, in five resistant and one responding patient. Post-treatment ichorCNA analyses demonstrated in shWGS and exome-seq higher median tumor fractions in resistant (7% and 9%) than in sensitive patients (7% and 5%). SigMiner analyses detected predominantly mutational signatures linked to mismatch repair and chemotherapy. DeSeq2 analyses of MeD-seq data revealed three methylation signatures and more tumor-specific DMRs in resistant than in responding patients in both pre- and post-treatment samples (274 vs. 30 DMRs, 190 vs. 57 DMRs, Χ2-test p < 0.001). Conclusion: Our genome-wide Next-Generation Sequencing (NGS) analyses in PARPi-resistant patients identified epigenetic differences in blood before treatment, whereas genomic alterations were more frequently observed after progression. The epigenetic differences at baseline are especially interesting for further exploration as putative predictive biomarkers for PARPi resistance.

Serous endometrial intra-epithelial carcinoma: an observational study

Serous endometrial intra-epithelial carcinoma is described as a malignant, superficial spreading lesion with risk of extra-uterine spread at time of diagnosis, and poor outcome. To evaluate the surgical management of patients with serous endometrial intra-epithelial carcinoma and its impact on oncologic outcomes and complications. This Dutch observational retrospective cohort study evaluated all patients diagnosed with pure serous endometrial intra-epithelial carcinoma in the Netherlands, between January 2012 and July 2020. The pathological examination was reviewed by two pathologists with expertise in gynecological oncology. Clinical data were obtained when the diagnosis was confirmed. Primary outcome is progression-free survival, secondary outcomes are duration of follow-up, adverse events related to surgery, and overall survival. A total of 23 patients from 13 medical centers were included, of whom 15 (65.2%) presented with post-menopausal blood loss. In 17 patients (73.9%) the intra-epithelial lesion was present in an endometrial polyp. All patients underwent hysterectomy, of whom 12 patients (52.2%) were surgically staged. None of the staged patients showed extra-uterine disease. Two patients received adjuvant brachytherapy. There were no recurrences of disease (median follow-up duration of 35.6 months (range 1.0-108.6) and no disease-related deaths in this cohort. In patients with serous endometrial intra-epithelial carcinoma, median progression-free survival reached nearly 3 years and no recurrences have been reported. Our results do not endorse World Health Organization 2014 advice to treat serous endometrial intra-epithelial carcinoma as high-grade, high-risk endometrial carcinoma. Full surgical staging might possibly lead to 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.

Improving imiquimod use in cervical high‐grade squamous intraepithelial lesions: A qualitative study

AbstractIntroductionTo investigate patient experiences and satisfaction with imiquimod for cervical high‐grade squamous intraepithelial lesions and its side effects, possibly influencing the (dis)continuation of treatment. Secondary objectives include evaluating patient counseling and guidance before and during imiquimod treatment for cervical high‐grade squamous intraepithelial lesions by gynecologists and nurse practitioners to improve patient guidance.Material and MethodsSemi‐structured interviews were conducted with patients treated with imiquimod for cervical high‐grade squamous intraepithelial lesions and with healthcare professionals from hospitals in the Netherlands. Interviews were transcribed and transcripts were analyzed using thematic analysis.ResultsA total of 15 patients, 11 of whom finished imiquimod treatment and four who discontinued imiquimod treatment, and six healthcare professionals were interviewed. Three main themes were identified: counseling and side effects, use of imiquimod, and contact moments during treatment. Many patients believed that sharing patient experiences would help in making treatment decisions. Patients discontinued treatment early due to side effects and the mode of application. Detailed information on application methods and side effect severity during counseling is desirable. Healthcare professionals agreed on the factors that need to be addressed during counseling but held different opinions on imiquimod's side effect severity and application methods.ConclusionsAdequate patient counseling on imiquimod treatment, including the possible application methods and potential side effects, along with optimal guidance during treatment, contributes to a better patient experience and reduces the likelihood of early discontinuation of treatment.

Poor accuracy of endometrial sampling in patients with uterine carcinosarcomas: a nationwide analysis

To determine the accuracy of aspiration biopsy (AB), hysteroscopic biopsy (HB), and dilatation & curettage (D&C) in detecting uterine carcinosarcoma (UCS). Pathology reports were retrieved from the Dutch Nationwide Pathology Databank PALGA for patients with a certain or suggested diagnosis of UCS in pre- and/or postoperative histology between 2001 and 2021. Patients without available pre- or postoperative pathology reports were excluded. The accuracy measures sensitivity, positive predictive value (PPV), accuracy, and concordance using Cohen's kappa were calculated for AB, D&C, and HB, using postoperative histology as the reference. This was analyzed for 2 scenarios: Analysis A compared samples with a certain or suggested diagnosis of UCS vs. no mention of UCS. Analysis B compared samples with a certain diagnosis of UCS vs those without UCS. The study included 1,481 patients, totaling 1,685 samples. Sensitivity was similar for AB and HB (52.4% and 50.5%, respectively, for analysis A; 45.1% and 42.2% for analysis B). D&C showed the highest sensitivity (70.8% and 64.9% for analysis A and B, respectively). AB had the highest PPV (85.3% and 90.9% for analysis A and B, respectively), HB had the lowest PPV (79.7% and 80.9%, respectively). Accuracy was highest for D&C (44.4%) compared to AB (32.8%) and HB (29.5%). All Cohen's kappa values were below 0.20, indicating poor correlation between preoperative and postoperative diagnoses. The study reveals low accuracy measures across all conventional endometrial sampling techniques, highlighting the need for research to identify markers or tools to diagnose UCS.

Unraveling Differences in Molecular Mechanisms and Immunological Contrasts between Squamous Cell Carcinoma and Adenocarcinoma of the Cervix

This study aims to refine our understanding of the inherent heterogeneity in cervical cancer by exploring differential gene expression profiles, immune cell infiltration dynamics, and implicated signaling pathways in the two predominant histological types of cervix carcinoma, Squamous Cell Carcinoma (SCC) and Adenocarcinoma (ADC). Targeted gene expression data that were previously generated from samples of primary cervical cancer were re-analyzed. The samples were grouped based on their histopathology, comparing SCC to ADC. Each tumor in the study was confirmed to be high risk human papilloma virus (hrHPV) positive. A total of 21 cervical cancer samples were included, with 11 cases of SCC and 10 of ADC. Data analysis revealed a total of 26 differentially expressed genes, with 19 genes being overexpressed in SCC compared to ADC (Benjamini–Hochberg (BH)-adjusted p-value < 0.05). Importantly, the immune checkpoint markers CD274 and CTLA4 demonstrated significantly higher expression in SCC compared to ADC. In addition, SCC showed a higher infiltration of immune cells, including B and T cells, and cytotoxic cells. Higher activation of a variety of pathways was found in SCC samples including cytotoxicity, interferon signaling, metabolic stress, lymphoid compartment, hypoxia, PI3k-AKT, hedgehog signaling and Notch signaling pathways. Our findings show distinctive gene expression patterns, signaling pathway activations, and trends in immune cell infiltration between SCC and ADC in cervical cancer. This study underscores the heterogeneity within primary cervical cancer, emphasizing the potential benefits of subdividing these tumours based on histological and molecular differences.

Topical imiquimod treatment of residual or recurrent cervical intraepithelial neoplasia lesions (TOPIC‐2): A randomised controlled trial

AbstractObjectiveTo investigate the efficacy of imiquimod in women with residual or recurrent cervical intraepithelial neoplasia (rrCIN), compared with large loop excision of the transformation zone (LLETZ).DesignRandomised controlled non‐inferiority trial.SettingOne academic and one regional hospital in the Netherlands.PopulationThirty‐five women with rrCIN were included in the study between May 2016 and May 2021.MethodsWomen were randomised to receive treatment with 5% imiquimod cream (12.5 mg) intravaginally (three times a week for a duration of 16 weeks) or a LLETZ procedure (standard treatment).Main outcome measuresThe primary outcome was reduction to normal cytology at 6 months after starting treatment. Secondary outcomes were clearance of high‐risk human papilloma virus (hr‐HPV) in both groups and reduction to ≤CIN1 in the imiquimod group. Side effects were monitored.ResultsTreatment success was 33% (6/18) in the imiquimod group versus 100% (16/16) in the LLETZ group (P < 0.001), whereas HPV clearance was 22% (4/18) in the imiquimod group versus 88% (14/16) in the LLETZ group (P < 0.001). After the randomisation of 35 women, the futility of treatment with imiquimod was proven and the trial was prematurely finished. In the follow‐up period, three patients remained without additional treatment, whereas all other patients underwent LLETZ, conisation or hysterectomy. In the LLETZ group none of the patients received additional treatment during 2 years of follow‐up.ConclusionsThis is the first randomised controlled trial to show that topical imiquimod has a significantly lower success rate in terms of reduction to normal cytology and hr‐HPV clearance, compared with LLETZ, in women with rrCIN. Additionally, imiquimod has numerous side effects and after using imiquimod most women with rrCIN still required additional surgical treatment.

Clinical validation and comparison of the Comprehensive Complication Index and Clavien-Dindo classification in predicting post-operative outcomes after cytoreductive surgery in advanced ovarian cancer

The Comprehensive Complication Index (CCI) is an instrument used to measure cumulative post-operative complications. Our study aimed to validate the CCI after cytoreductive surgery for primary advanced-stage epithelial ovarian cancer, and to compare its diagnostic performance with the Clavien-Dindo classification. This prospective cohort study classified post-operative complications according to the Clavien-Dindo classification and the CCI. Logistic regression was used to determine the association between both classifications with intensive care unit admission, prolonged length of hospital stay (defined as stays longer than the 75th percentile of all stays in this study), 30-day readmission, and time to initiating chemotherapy after surgery >42 days. Area under the receiver operating characteristic curves (AUC) were used to assess the discriminative performance of each classification. A total of 300 patients were included in the analysis. Most patients (n=255, 85%) underwent interval cytoreductive surgery. Complete cytoreduction was achieved in 235 (78%) patients. Overall, 30-day post-operative complications classified by the Clavien-Dindo classification occurred in 147 (49%) patients. Severe complications (grade ≥3a) occurred in 51 (17%) patients. Approximately 30% (n=82) had multiple complications. The CCI showed an excellent correlation with the Clavien-Dindo classification ( Both the Clavien-Dindo classification and CCI showed significant associations with all surgical outcomes. However, the cumulative complications score of the CCI demonstrated a more superior discriminative performance than the Clavien-Dindo classification for prolonged length of hospital stay in advanced-stage epithelial ovarian cancer.

Cost Study of the PlasmaJet Surgical Device Versus Conventional Cytoreductive Surgery in Patients With Advanced-Stage Ovarian Cancer

PURPOSEAdjuvant use of Neutral Argon Plasma (PlasmaJet Surgical Device) during cytoreductive surgery (CRS) for advanced-stage epithelial ovarian cancer improves surgical outcomes. The aim of this study is to examine the costs of adjuvant use of the PlasmaJet during surgery compared with conventional CRS in advanced-stage epithelial ovarian cancer.MATERIALS AND METHODSThe patients were randomly assigned to surgery with or without the PlasmaJet. Analysis of the intra- and extramural health care costs was performed. Costs were divided into three categories: costs of the diagnostic phase (T1), inpatient care up to discharge including costs of surgery (T2), and outpatient care including chemotherapy until 6 weeks after the last cycle of chemotherapy (T3).RESULTSOverall, 327 patients underwent CRS (surgery with PlasmaJet: n = 157; conventional surgery: n = 170). The mean total health costs were significantly higher for CRS with adjuvant use of PlasmaJet compared with conventional CRS (€19,414 v €18,165, P = .017). Costs are divided into costs of the diagnostic phase (€2,034 v €1,974, P = .890), costs of inpatient care (€10,956 v €9,556, P = .003), and costs of outpatient care (€6,417 v €6,628, P = .147).CONCLUSIONMean total health care costs of the use of PlasmaJet in CRS were significantly higher than those for conventional CRS. This difference is fully explained by the additional surgery costs of the use of PlasmaJet. However, surgery with the use of the PlasmaJet leads to a significantly higher percentage of complete CRS and a halving of stomas. A cost-effectiveness analysis will be performed once survival data are available (funded by ZonMw, Trial Register NL62035.078.17).

Factors predicting postoperative morbidity after cytoreductive surgery for ovarian cancer: a systematic review and meta-analysis

Advances in ovarian cancer cytoreductive surgery have enabled more extensive procedures to achieve maximal cytoreduction but with a consequent increase in postoperative morbidity and mortality. The aim of this study was to evaluate factors for postoperative morbidity after extensive cytoreductive surgery for primary epithelial ovarian cancer (EOC), particularly those which may be modifiable. Electronic databases were searched. Meta-analysis was conducted using random-effects models. Fifteen relevant studies, involving 15,325 ovarian cancer patients, were included in this review. Severe 30-day postoperative complications occurred in 2,357 (15.4%) patients. The postoperative mortality rate was 1.92%. Meta-analysis demonstrated that patient with following risk factors; age (p0 (p=0.001), albumin level <3.5 g/dL (p<0.001), presence of ascites on CT scan (p=0.013), stage IV disease (p<0.001) and extensive surgical procedure (p<0.001) has a significantly increase risk of developing postoperative complications. Surgical procedures including peritonectomy (p=0.012), splenectomy (p<0.001) and colon surgery (p<0.001) were significant predictors for postoperative complications. Moreover, we found that patients who received neoadjuvant chemotherapy followed by interval debulking surgery (NACT-IDS) had a lower risk of developing severe complications compared to those who underwent primary debulking surgery (PDS) (p<0.001). Our study demonstrated that patient performance status and hypoalbuminemia were the only significant adjustable preoperative risk factors associated with postoperative complications. Patients who underwent NACT-IDS had a lower risk of developing severe complications compared to PDS. International Prospective Register of Systematic Reviews (PROSPERO) Identifier: CRD42021282770.

Relevance of routine pathology review in cervical carcinoma

To determine the impact of pathology review on the management of patients with cervical carcinoma, 264 reports of pathology review from 230 patients referred to Erasmus MC (2010-2012) were studied retrospectively. Discrepancies between pathologic diagnoses were classified as 'major' if they led to changes in treatment, and as 'minor' where there was no change. Patient and tumor characteristics were analyzed to identify the factors influencing these discrepancies. Fifty-eight (25.2%) discrepancies were identified; 28 (12.2%) were major, these resulted frequently from missing essential information, or discordant assessment of tumor invasion. Pathology review prevented under-treatment of 3.5%, over-treatment of 1.3%, treatment for incorrect malignancy of 1.3%, and enabled definitive treatment of 6.1% of patients. This highlights the importance of pathology review for appropriate management. Major discrepancies were rare (1%) for patients with macroscopic tumor and histologic diagnosis of squamous cell carcinoma (n = 100). For these patients, yield of pathology review may be limited.

Quantitative Proteomic Analysis of MCM3 in ThinPrep Samples of Patients with Cervical Preinvasive Cancer

Triage methods for cervical cancer detection show moderate accuracy and present considerable false-negative and false-positive result rates. A complementary diagnostic parameter could help improve the accuracy of identifying patients who need treatment. A pilot study was performed using a targeted proteomics approach with opportunistic ThinPrep samples obtained from women collected at the hospital’s outpatient clinic to determine the concentration levels of minichromosome maintenance-3 (MCM3) and envoplakin (EVPL) proteins. Forty samples with ‘negative for intraepithelial lesion or malignancy’ (NILM), 21 samples with ‘atypical squamous cells of undetermined significance’ (ASC-US), and 33 samples with ‘low-grade squamous intraepithelial lesion and worse’ (≥LSIL) were analyzed, using cytology and the patients’ histology reports. Highly accurate concordance was obtained for gold-standard-confirmed samples, demonstrating that the MCM3/EVPL ratio can discriminate between non-dysplastic and dysplastic samples. On that account, we propose that MCM3 and EVPL are promising candidate protein biomarkers for population-based cervical cancer screening.

High acceptability of point‐of‐care HIV testing at the colposcopy outpatient clinic in hospitals in the Netherlands

AbstractObjectiveCervical cancer and its precursor are labeled as HIV indicator conditions, justifying the recommendation of HIV testing for all patients. This study aimed to assess the acceptance and patients' and gynecologists' perceptions on HIV testing in patients with cervical dysplasia at the colposcopy outpatient clinic in hospitals in the Netherlands.MethodsA cross‐sectional study was conducted between May 2021 and February 2023 to implement point‐of‐care HIV testing in five hospitals in the Rotterdam region, the Netherlands. We included patients aged 18 years and older, without documented HIV, presenting with cervical dysplasia. The primary outcome was the HIV test acceptance rate. Secondary outcomes were the HIV positivity rate and the association between dysplasia severity and test acceptance. We also assessed patients' and gynecologists' perspectives on this testing strategy with a questionnaire.ResultsOf 563 patients, 523 accepted HIV testing, resulting in a test acceptance rate of 92.9% (95% confidence interval, 90.45%–94.88%). Testing rates were consistent among participating hospitals (91.6%–100.0%) None of the patients tested positive for HIV. Severity of dysplasia was not associated with test acceptance (P = 0.768). Most patients (n = 500, 96.0%) reported good or higher experience with this testing strategy. The main barriers for gynecologists to offer HIV testing were lack of time (n = 22, 33.9%) and fear to offend a patient (n = 7, 12.5%). Less than half (n = 19, 39.3%) of gynecologists believed an HIV test should be offered to all patients with cervical dysplasia.ConclusionPoint‐of‐care HIV testing at colposcopy outpatient clinics was well accepted by patients. The data indicate that the primary barrier lies with the physicians to offer such testing.

Serous Endometrial Intraepithelial Carcinoma (SEIC): Prospective Registration Study

Background: Serous endometrial intra-epithelial carcinoma (SEIC) is a rare condition affecting the lining of the uterus. It's a pre-cancerous change that might lead to cancer if not treated. Doctors aren't sure of the best way to manage it because it's so uncommon. A previous study looked at past cases in the Netherlands from 2012 to 2020. This study found that none of the 23 women with SEIC had their cancer spread during surgery, and none had a recurrence in the following three years. This suggests that a simpler surgery might be safe for patients with SEIC. Methods: Information on disease details from patients who agree to participate in our study will be collected. All participants will fill out online forms and health questionnaires at several points: when they join the study, and again at six months, two years, and five years after their diagnosis. During the years after diagnosis, various details like health status, test results, treatments will be checked in the electronic medical record. Results: This study is just starting, with the first patients expected to join on January 1, 2025. Conclusion: A large international study is created to track SEIC patients over time. This will help us learn more about the condition and improve treatment strategies.