Hans W. Nijman

Response to Systemic Therapies in Patient-Derived Cell Lines from Primary and Recurrent Adult Granulosa Cell Tumors

Abstract In patients with the rare adult-type granulosa cell tumor (aGCT), surgery is the primary treatment for both primary and recurrent disease. In cases of inoperable disease, systematic therapy is administered, but variable response rates and drug resistance complicate predicting the most effective therapy. Drug screen testing on patient-derived cell lines may offer a solution. In a national prospective study on aGCT, fresh tissue was cultured into 2D cell lines, testing 27 clinical and experimental drugs. Dose–response curves and synergy were calculated using GraphPad Prism and CompuSyn software. We established 34 patient-derived cell lines from tissue of 20 patients with aGCT. Of these, seven patients had a primary diagnosis of aGCT and 13 patients had recurrent disease. In eight patients, multiple tumor locations were cultured. On each cell line, 10 monotherapies and 17 combinations of drugs were tested. Carboplatin/gemcitabine showed efficacy and synergy in almost all patient-derived cell lines. Synergy could not be detected in the regular carboplatin/paclitaxel and carboplatin/etoposide combinations. Experimental combinations alpelisib/fulvestrant and alpelisib/gemcitabine showed efficacy of more than 75%. Drug screens on patient-derived tumor cell lines reflect the reality of the variable response of systemic therapy in patients with aGCT. In future research, this technique may be used to personalize the systemic treatment of patients with aGCT in a clinical study. The good response to carboplatin/gemcitabine in our patient-derived cell lines can then be confirmed in a clinical setting.

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.

Prognostic refinement of NSMP high-risk endometrial cancers using oestrogen receptor immunohistochemistry

Abstract Background Risk-assessment of endometrial cancer (EC) is based on clinicopathological factors and molecular subgroup. It is unclear whether adding hormone receptor expression, L1CAM expression or CTNNB1 status yields prognostic refinement. Methods Paraffin-embedded tumour samples of women with high-risk EC (HR-EC) from the PORTEC-3 trial ( n  = 424), and a Dutch prospective clinical cohort called MST ( n  = 256), were used. All cases were molecularly classified. Expression of L1CAM, ER and PR were analysed by whole-slide immunohistochemistry and CTNNB1 mutations were assessed with a next-generation sequencing. Kaplan–Meier method, log-rank tests and Cox’s proportional hazard models were used for survival analysis. Results In total, 648 HR-EC were included. No independent prognostic value of ER, PR, L1CAM, and CTNNB1 was found, while age, stage, and adjuvant chemotherapy had an independent impact on risk of recurrence. Subgroup-analysis showed that only in NSMP HR-EC, ER-positivity was independently associated with a reduced risk of recurrence (HR 0.33, 95%CI 0.15–0.75). Conclusions We confirmed the prognostic impact of the molecular classification, age, stage, and adjuvant CTRT in a large cohort of high-risk EC. ER-positivity is a strong favourable prognostic factor in NSMP HR-EC and identifies a homogeneous subgroup of NSMP tumours. Assessment of ER status in high-risk NSMP EC is feasible in clinical practice and could improve risk stratification and treatment.

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.

Deep immune profiling of ovarian tumors identifies minimal MHC-I expression after neoadjuvant chemotherapy as negatively associated with T-cell-dependent outcome

Epithelial Ovarian cancer (EOC) is the most lethal gynecological malignancy and has limited curative therapeutic options. Immunotherapy for EOC is promising, but clinical efficacy remains restricted to a small percentage of patients. Several lines of evidence suggest that the low response rate might be improved by combining immunotherapy with carboplatin and paclitaxel, the standard-of-care chemotherapy for EOC. Here, we assessed the immune contexture of EOC tumors, draining lymph nodes, and peripheral blood mononuclear cells during carboplatin/paclitaxel chemotherapy. We observed that the immune contexture of EOC patients is defined by the tissue of origin, independent of exposure to chemotherapy. Summarized, draining lymph nodes were characterized by a quiescent microenvironment composed of mostly non-proliferating naïve CD4 + T cells. Circulating T cells shared phenotypic features of both lymph nodes and tumor-infiltrating immune cells. Immunologically 'hot' ovarian tumors were characterized by ICOS, GITR, and PD-1 expression on CD4 + and CD8 + cells, independent of chemotherapy. The presence of PD-1 + cells in tumors prior to, but not after, chemotherapy was associated with disease-specific survival (DSS). Accordingly, we observed high MHC-I expression in tumors prior to chemotherapy, but minimal MHC-I expression in tumors after neoadjuvant chemotherapy, even though there were no differences in the number of tumor-infiltrating lymphocytes (TIL) in both groups. We therefore speculate that the TIL influx into the chemotherapy tumor microenvironment may be a consequence of the general inflammatory nature of chemotherapy-experienced tumors. Strategies to upregulate MHC-I during or after neoadjuvant chemotherapy may thus improve treatment outcome in these patients.

Prognostic image-based quantification of CD8CD103 T cell subsets in high-grade serous ovarian cancer patients

CD103-positive tissue resident memory-like CD8

Combined STING levels and CD103+ T cell infiltration have significant prognostic implications for patients with cervical cancer

Activation of STimulator of INterferon Genes (STING) is important for induction of anti-tumor immunity. A dysfunctional STING pathway is observed in multiple cancer types and associates with poor prognosis and inferior response to immunotherapy. However, the association between STING and prognosis in virally induced cancers such as HPV-positive cervical cancer remains unknown. Here, we investigated the prognostic value of STING protein levels in cervical cancer using tumor tissue microarrays of two patient groups, primarily treated with surgery (n = 251) or radio(chemo)therapy (n = 255). We also studied CD103, an integrin that marks tumor-reactive cytotoxic T cells that reside in tumor epithelium and that is reported to associate with improved prognosis. Notably, we found that a high level of STING protein was an independent prognostic factor for improved survival in both the surgery and radio(chemo)therapy group. High infiltration of CD103+ T cells was associated with improved survival in the radio(chemo)therapy group. The combination of STING levels and CD103+ T cell infiltration is strongly associated with improved prognosis. We conclude that combining the prognostic values of STING and CD103 may improve the risk stratification of cervical cancer patients, independent from established clinical prognostic parameters.

Short‐term surgical complications after radical hysterectomy—A nationwide cohort study

AbstractIntroductionCentralization 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 methodsWomen 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.ResultsA 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.ConclusionsShort‐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.

Radical hysterectomy or chemoradiotherapy for clinically early-stage cervical cancer with suspicious lymph nodes on imaging: a retrospective cohort study

The optimal treatment of clinically early-stage cervical cancer with suspicious lymph nodes on pretreatment imaging is unclear. Therefore, we aimed to compare surgery (i.e., radical hysterectomy and pelvic lymphadenectomy±adjuvant therapy) with primary chemoradiotherapy as treatment strategies in this patient group regarding recurrence-free, overall survival and toxicity. Women diagnosed between 2009-2017 with the International Federation of Gynecology and Obstetrics (2009) stage IA-IIA and suspicious nodes based on radiologic assessment of pretreatment imaging were retrospectively selected from the Netherlands Cancer Registry. Cox proportional hazard was used to estimate survival and logistic regression for toxicity. Inverse probability weighting was used to correct for confounding. Grade ≥2 surgery-related (≤30 days) and grade ≥3 chemotherapy or radiotherapy-related (≤6 months) toxicity were collected. Missing data were imputed. Of 330 patients included, 131 (40%) received surgery (followed by adjuvant therapy in 54%) and 199 (60%) chemoradiotherapy. Pathological nodal status was known in 100% of the surgery group and 32% (n=63) of the chemoradiotherapy group, of whom 43% (56/131) and 89% (56/63), respectively, had metastases. After adjustment for confounders, the recurrence-free survival (hazard ratio [HR]=0.67; 95% confidence interval [CI]=0.34-1.31) and overall survival (HR=0.75; 95% CI=0.38-1.47) were not significantly different between both groups, while surgery was associated with more toxicity (odds ratio=2.82; 95% CI=1.42-5.60), mainly surgery-related. In patients with clinically early-stage cervical cancer and suspicious nodes on imaging, surgery and primary chemoradiotherapy yielded comparable results in terms of survival, whereas surgery might be associated with more (surgery-related) short-term toxicity.

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.