Joost Bart

Spatiotemporal Immune Landscape and Long-term Immune Memory in POLE-Mutant Endometrial Cancer at the Single-Cell Level

Abstract Polymerase epsilon–mutant (POLE-mut) endometrial cancers are characterized by a near 100% disease-specific survival rate, even when treated with surgery alone. This survival, combined with the ultramutated genome and high level of neoantigens in these tumors, indicates a substantial degree of immune control in preventing disease spread and recurrence. Although these features are intriguing, the analysis of immune infiltration in POLE-mut endometrial cancers has predominantly been confined to IHC studies. In this study, we used single-cell RNA and T-cell receptor sequencing to characterize the immune landscape of POLE-mut endometrial cancers. Moreover, we analyzed patient blood samples taken 2 to 8 years after curative treatment to assess the formation of long-term immune memory in circulation. We identified specialized tumor-infiltrating myeloid subsets at different stages of maturation, an array of lymphocytes ranging from immature to cytotoxic, and adaptive NK cells, as well as tumor-reactive exhausted and effector T cells, all contributing to a highly inflammatory antitumor response. Our analysis of blood samples taken years after curative treatment uncovered the presence of tumor-reactive T-cell clones that matched the primary tumor. This indicates the formation of systemic long-term memory immune responses in POLE-mut endometrial cancer survivors. Our study highlights the distinctive immunogenicity of POLE-mut endometrial cancer and identifies key features associated with persistent antitumor immunity that may contribute to prolonged, relapse-free survival.

High-Grade Serous Carcinoma at Risk-Reducing Salpingo-Oophorectomy in Asymptomatic Carriers of BRCA1/2 Pathogenic Variants: Prevalence and Clinical Factors

PURPOSE To investigate the prevalence of and clinical factors associated with high-grade serous carcinoma (HGSC) at risk-reducing salpingo-oophorectomy (RRSO) in asymptomatic BRCA1/2-pathogenic variant (PV) carriers. PATIENTS AND METHODS We included BRCA1/2-PV carriers who underwent RRSO between 1995 and 2018 from the Hereditary Breast and Ovarian cancer in the Netherlands study. All pathology reports were screened, and histopathology reviews were performed for RRSO specimens with epithelial abnormalities or where HGSC developed after normal RRSO. We then compared clinical characteristics, including parity and oral contraceptive pill (OCP) use, for women with and without HGSC at RRSO. RESULTS Of the 2,557 included women, 1,624 had BRCA1, 930 had BRCA2, and three had both BRCA1/2-PV. The median age at RRSO was 43.0 years (range: 25.3-73.8) for BRCA1-PV and 46.8 years (27.6-77.9) for BRCA2-PV carriers. Histopathologic review confirmed 28 of 29 HGSCs and two further HGSCs from among 20 apparently normal RRSO specimens. Thus, 24 (1.5%) BRCA1-PV and 6 (0.6%) BRCA2-PV carriers had HGSC at RRSO, with the fallopian tube identified as the primary site in 73%. The prevalence of HGSC in women who underwent RRSO at the recommended age was 0.4%. Among BRCA1/2-PV carriers, older age at RRSO increased the risk of HGSC and long-term OCP use was protective. CONCLUSION We detected HGSC in 1.5% ( BRCA1-PV) and 0.6% ( BRCA2-PV) of RRSO specimens from asymptomatic BRCA1/2-PV carriers. Consistent with the fallopian tube hypothesis, we found most lesions in the fallopian tube. Our results highlight the importance of timely RRSO with total removal and assessment of the fallopian tubes and show the protective effects of long-term OCP.

Vvax001, a Therapeutic Vaccine, for Patients with HPV16-Positive High-grade Cervical Intraepithelial Neoplasia: A Phase II Trial

Abstract Purpose: Human papillomavirus (HPV) infection is the major cause of (pre)malignant cervical lesions. We previously demonstrated that Vvax001, a replication-incompetent Semliki Forest virus vaccine encoding HPV type 16 (HPV16) E6 and E7, induced potent anti-E6 and -E7 cytotoxic T-cell responses. In this study, we investigated the clinical efficacy of Vvax001 in patients with HPV16-positive cervical intraepithelial neoplasia (CIN) grade 3 (CIN3). Patients and Methods: Patients with newly diagnosed HPV16-positive CIN3 were eligible for participation. Patients received three immunizations of Vvax001 (5 × 107 infectious particles) at a 3-week interval. Up to 19 weeks after the last immunization, patients were monitored for regression of CIN3 by colposcopy. A colposcopy-guided biopsy was taken at the last visit, and a standard-of-care loop excision was performed only in case of remaining CIN grade 2/CIN3. Histopathologic response rates, HPV16 clearance, treatment-related adverse events, and vaccine-induced immune responses were assessed. Results: A total of 18 patients were enrolled and fully immunized. Colposcopic examination revealed a reduction in CIN3 lesion sizes in 17/18 (94%) patients already evident from 3 weeks onward after the last immunization. A histopathologic complete response (regression to CIN grade 1 or no dysplasia) was observed in 9/18 patients (50%) and HPV16 clearance in 10/16 patients (63%). Vvax001 did not induce clearance of other HPV types. To date, no recurrences have been observed, with a median and longest disease-free survival of 20 and 30 months, respectively. No serious adverse events were observed. Conclusions: Treatment with Vvax001 is safe and feasible and shows preliminary clinical effectiveness in patients with HPV16-associated CIN3 lesions.

The risk of lymph node metastasis in the new FIGO 2018 stage IA cervical cancer with >7 mm diameter

In the 2018 FIGO staging system, cervical cancers with ≤5 mm depth of invasion (DOI) and a diameter of >7 mm, first classified as stage IB, are classified as stage IA. In this group, it is unclear what the risk of lymph node metastasis (LNM) is. This retrospective cohort study aims to determine the incidence of LNM and to study the association between disease-related characteristics and LNM. Women diagnosed with FIGO 2009 IB cervical cancer, with ≤5 mm DOI and a diameter >7 mm, treated with a radical hysterectomy and pelvic lymphadenectomy between 1985 and 2020 were selected from the databases of the Amsterdam University Medical Center and the University Medical Center Groningen. The specimens of patients with LNM were revised by expert pathologists. The incidence of LNM was calculated. The associations between LNM and DOI, diameter, histological type, clinical visibility and lymphovascular space invasion (LVSI) were evaluated by calculating odds ratios using logistic regression. Of the 389 patients included, 10 had pathologically confirmed LNM (2.6%, 95% confidence interval=1.3%-4.5%). In case of LVSI, univariate analysis showed an increased risk of LNM (p=0.003 and p=0.012, respectively). No difference in LNM was found between lesions diagnosed by microscopy and clinically visible lesions. No LNM were found in patients without LVSI and a DOI of ≤3 mm. For patients with stage IA cervical cancer with a diameter >7 mm, we recommend considering a pelvic lymph node assessment in case of DOI >3 mm and/or presence of LVSI.

High-grade serous carcinoma occurring after risk-reducing salpingo-oophorectomy in BRCA1/2 germline pathogenic variant carriers

Abstract Background Risk-reducing salpingo-oophorectomy (RRSO) effectively prevents high-grade serous carcinoma (HGSC) in BRCA1/2 germline pathogenic variant (GPV) carriers. Still, some women develop HGSC after RRSO without pathological findings. This study assessed long-term incidence and risk factors for developing HGSC after RRSO without pathological findings. Methods BRCA1/2 GPV carriers were selected from the Hereditary Breast and Ovarian cancer in the Netherlands (HEBON) cohort. Follow-up data for HGSC after RRSO were obtained from the Dutch Nationwide Pathology Databank (PALGA) and confirmed by histopathological review. Cumulative incidence rates of HGSC were calculated using Kaplan-Meier analyses. A Cox proportional hazards model was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for factors associated with an increased risk of HGSC after RRSO without pathological findings. Results A total of 2519 women were included, with a median follow-up of 13.4 years (range: 0.0-27.6 years). The 20-year cumulative incidence rate of HGSC was 1.5% (95% CI = 0.0 to 2.1) for BRCA1 and 0.2% (95% CI = 0.0 to 1.4) for BRCA2 GPV carriers. All women who developed HGSC underwent RRSO after the recommended age. Incomplete embedding of the RRSO specimen (HR = 4.2, 95% CI = 1.4 to 12.6), higher age at RRSO (HR per year = 1.1, 95% CI = 1.0 to 1.1), and carrying a BRCA1 GPV (HR = 12.1, 95% CI = 1.6 to 91.2) were associated with increased risk of HGSC. Conclusions In BRCA1/2 GPV carriers, long-term incidence of HGSC after RRSO without pathological findings was low. Strict adherence to guidelines regarding timely RRSO followed by complete specimen embedding can further reduce the risk of HGSC in the years after RRSO.

Use of p53 immunohistochemistry can improve diagnostic agreement for differentiated vulvar intraepithelial neoplasia ( dVIN ): an international reproducibility study

Aims Differentiated or HPV‐independent vulvar intraepithelial neoplasia (dVIN) can progress rapidly to invasive cancer and accurate pathological diagnosis is essential to facilitate appropriate interventions. Histological similarities of dVIN with non‐neoplastic lesions, however, often make the diagnosis less reproducible. We investigated among a diverse group of pathologists whether the diagnostic agreement improves with the use of p53 immunohistochemistry (IHC) interpreted using the pattern‐based schema. Methods and results Fifty haematoxylin–eosin (HE) stained archival slides (30 dVIN and 20 non‐dysplastic vulvar lesions) were selected and p53‐IHC was performed. Twenty‐four board‐certified pathologists from eight countries first assessed the HE slides alone, and after a washout period, re‐evaluated them alongside the p53‐IHC slides. During both rounds, slides were diagnosed as dVIN, favour dVIN, favour no‐VIN or no‐VIN. p53‐IHC was scored as wild‐type or mutant (diffuse, basal, cytoplasmic or null). Kappa ( κ ) statistics and McNemar's test were used for statistical analyses. Overall diagnostic agreement for dVIN saw a significant increase in the Kappa value ( κ  = 0.6 vs. κ  = 0.4, P  = 0.002) when HE and p53‐IHC slides were assessed together compared with histology assessment alone, although the level of agreement remained moderate. For p53‐IHC assessment, overall agreement was substantial ( κ  = 0.7). Diagnoses changing from no‐VIN/favour no‐VIN to dVIN correlated significantly with the identification of a p53‐mutant pattern ( P  < 0.001). Conclusions Our findings indicate that p53‐IHC is a robust ancillary tool that can be reproducibly interpreted by pathologists with varying experience levels and supports the routine use of p53‐IHC in cases where dVIN is considered in the differential diagnosis.

Evaluation of BRCA1/2 testing rates in epithelial ovarian cancer patients: lessons learned from real-world clinical data

Abstract Identification of somatic and germline BRCA1/2 pathogenic variants in epithelial ovarian cancer (EOC) patients is essential for determining poly-(ADP-ribose)-polymerase (PARP) inhibitor sensitivity and genetic predisposition. In the Netherlands, BRCA1/2 testing changed to a tumor-first approach to efficiently identify both somatic and germline pathogenic variants in all patients. Here, we performed an in-depth evaluation of the first four years of the tumor-first test-pathway. Data of consecutive series of patients diagnosed with EOC in two regions were obtained from the Netherlands Cancer Registry. Tumor and/or germline test data were retrieved from hospital databases. The primary outcome was the percentage of patients completing the BRCA1/2 test-pathway, defined as having a negative tumor test or a referral for a germline test in case of a positive tumor test or no tumor test. Factors associated with test-pathway completion were identified through multivariable logistic regression analysis. In total, 69.8% (757/1085) completed the test-pathway. This was 74.4% in the most recent year. Younger patients, patients diagnosed in year three or four, patients with high-grade serous/high-grade endometrioid carcinoma, advanced stage disease, middle or high socioeconomic status, and patients who underwent surgery or chemotherapy, were more likely to complete the test-pathway. We report inequalities in genetic testing access in EOC patients, which highlight the need for better guideline adherence, particularly in older patients, those with low socioeconomic status, low-grade histotypes, early-stage disease and those without surgery or chemotherapy. Additionally, timely testing of patients, and testing relatives if patients cannot be tested, are crucial to increase test uptake.

Neoadjuvant immune checkpoint blockade in women with mismatch repair deficient endometrial cancer: a phase I study

Neoadjuvant immune checkpoint blockade (ICB) has shown unprecedented activity in mismatch repair deficient (MMRd) colorectal cancers, but its effectiveness in MMRd endometrial cancer (EC) remains unknown. In this investigator-driven, phase I, feasibility study (NCT04262089), 10 women with MMRd EC of any grade, planned for primary surgery, received two cycles of neoadjuvant pembrolizumab (200 mg IV) every three weeks. A pathologic response (primary objective) was observed in 5/10 patients, with 2 patients showing a major pathologic response. No patient achieved a complete pathologic response. A partial radiologic response (secondary objective) was observed in 3/10 patients, 5/10 patients had stable disease and 2/10 patients were non-evaluable on magnetic resonance imaging. All patients completed treatment without severe toxicity (exploratory objective). At median duration of follow-up of 22.5 months, two non-responders experienced disease recurrence. In-depth analysis of the loco-regional and systemic immune response (predefined exploratory objective) showed that monoclonal T cell expansion significantly correlated with treatment response. Tumour-draining lymph nodes displayed clonal overlap with intra-tumoural T cell expansion. All pre-specified endpoints, efficacy in terms of pathologic response as primary endpoint, radiologic response as secondary outcome and safety and tolerability as exploratory endpoint, were reached. Neoadjuvant ICB with pembrolizumab proved safe and induced pathologic, radiologic, and immunologic responses in MMRd EC, warranting further exploration of extended neoadjuvant treatment.