OveRcoming immunosupprEssion aNd rebAlancing the Immune reSponSe in ovAriaN CancEr Study

INTRODUCTION AND RATIONALE

Problem description/ unmet needs:

Ovarian cancer is the fifth leading cause of cancer death among women in the Western world. The high mortality rate is due to detection at an advanced stage of the disease and lack of curative therapies. Standard therapy consists of a combination of cytoreductive surgery and chemotherapy. Despite this aggressive treatment, recurrent disease almost invariably occurs resulting in a five-year survival rate of only 30%, which has not changed significantly over the last 30 years.

Recurrent ovarian cancer is generally treated with second/third line chemotherapy, and (costly) targeted therapies. Although targeted therapy options are rapidly expanding, response rates often fall short of expectations probably caused by heterogeneity of the disease. Therefore, new treatment options need to be explored. One such option is immunotherapy. However, the limited data on this topic show disappointing results.

Envisioned solution/research direction:

To improve the patients' outcomes and identify novel therapeutic targets, one needs a 'systems understanding' of the pathophysiology of tumors, which transcends beyond the process of carcinogenesis to encompass the complex interaction of the malignant cells with other cell types in the tumor and the tumor environment (TME), especially immune cells. The inflammatory TME has characteristics that promote and support the malignant phenotype, and inflammatory pathways have been proposed as new targets for cancer therapy. The TME conditions the local myeloid cells to become immunosuppressive. In this project we primarily focus on myeloid cells, in particular circulating monocytes and their progenitors, as well as the tumor-

associated macrophages (TAMs) which are pluripotent myeloid cells that represent the most abundant component of the TME in advanced OC arguing for their importance in the pathogenesis of OC.

In the present study, we aim to investigate the programming of myeloid cells in OC patients with advanced tumors in depth and to explore whether we can functionally reprogram myeloid cells from different bodily compartments (circulating monocytes, bone marrow (BM) and spleen progenitors) towards an anti-tumorigenic phenotype by trained immunity-modulating nanoparticles. If this hypothesis is proven to be correct, this mechanism could be explored in further studies including investigation of the interaction of myeloid cells with other immune cells and as a novel immunotherapy for OCs that are refractory to conventional treatment.

OBJECTIVES

To assess the transcriptional, epigenetic, and functional reprogramming of circulating monocytes and myeloid progenitor cells in OC by in vitro exposure to agents that modulate trained immunity.

To achieve this goal the study has a set of complementary key objectives:

To assess the transcriptional, epigenetic, and functional signature of i) circulating monocytes in patients with OC compared to controls in response to trained immunity inducers in vitro and ii) bone marrow and spleen myeloid progenitor cells in patients with OC compared to controls in response to trained immunity inducers in vitro.

STUDY DESIGN

This is an investigator-initiated, single-center cross-sectional explorative study that will be performed in the Catharina hospital Eindhoven at the Department of Obstetrics and Gynecology, Division of gynecological oncology in collaboration with the Eindhoven University of Technology and the department of internal medicine, Radboud UMC.

All OC patients and controls will donate blood samples during the operation. The OC patients will undergo surgery for removal of their tumor (as planned by their treating physician) and will be requested permission to use small samples of their removed tissue (malignant and benign samples), a BM sample from sternum or posterior iliac crest and spleen). The controls will undergo surgery for benign gynecological conditions (as planned by their treating physician) and will be requested permission to use a BM sample from sternum or posterior iliac crest.

The following set of measurements will be performed on peripheral blood samples:

Circulating and splenic monocytes will be purified and the following assessments will be performed before and after treatment with trained immunity inducers:

* Transcriptome analysis by RNA-sequencing * Epigenetic analysis of chromatin accessibility (ATACseq), whole-genome histone modification profiles, and DNA methylation * Immunological profiling of the monocytes by stimulation and trained immunity assays

The following set of measurements will be performed on bone marrow aspirates:

Bone marrow and splenic progenitor cells will be purified and the following assessments will be performed before and after treatment with trained immunity- inducing agents:

* Transcriptome analysis by RNA-sequencing * Epigenetic analysis of accessibility (ATACseq), whole-genome histone modification profiles, and DNA methylation * Composition, differentiation, and immunological profiling of the progenitors by colony formation, differentiation, stimulation, and trained immunity assays

All experiments and handling of samples will be performed according to Good Clinical Practice and the declaration of Helsinki.

The effect of exposure to trained immunity inducing agents on the functional phenotype of the monocytes and progenitors will be interrogated by pathway analysis of transcriptome and epigenome, to identify the major cellular processes in reprogramming circulating monocytes and progenitors.

STUDY POPULATION

The following patient groups will be included in the study:

Group 1. Patients with OC undergoing primary debulking surgery (N=30) Group 2. Patients with OC undergoing interval debulking surgery (N=30) Group 3. Controls as blood and bone marrow donors (N=30)

Rationale for the choice of study subjects:

Data from controls is crucial in order to provide a baseline, make useful comparisons, and to legitimately analyze immune cell status of OC patients and the effects of trained immunity-inducing agents. Both OC patients who are chemotherapy naïve (primary debulking) and who have had chemotherapy (carboplatin-paclitaxel) will be included in this study, to determine the effect of chemotherapy on the TAMs.

Subject recruitment: controls will be recruited from patients who undergo surgery for benign gynecological conditions. Patients will be invited to participate by their treating gynecologist during an outpatient clinic visit or via a letter. In addition, they will receive a letter with study-related information. Within one week they will be contacted to check if they require additional information and if they are willing to participate.

Patients with OC will be recruited if they undergo debulking surgery at our centers. Patients will be invited to participate by their treating gynecological oncologist during an outpatient clinic visit or via a letter; in addition, they will receive a letter with study-related information. Within one week they will be contacted to check if they require additional information and if they are willing to participate.

Approximately 150 patients undergo surgery for benign gynecological conditions at our department and around 100 patients undergo debulking surgery at our center annually. Therefore, inclusion of the limited number of subjects aimed for this study is feasible within three years.

Inclusion criteria:

In order to be eligible to participate in this study, a subject must meet all of the following criteria:

* Subjects should be at least 18 years old and mentally competent * Newly diagnosed patients with OC who go for primary debulking surgery or OC are scheduled for interval debulking * Controls: women who undergo abdominal surgery for benign gynecological conditions under general anesthesia

Exclusion criteria:

A potential subject who meets any of the following criteria will be excluded from participation in this study:

* Mentally incompetent * Pregnant or breastfeeding * Known inflammatory or infectious diseases or an immunosuppressive status; * Using medication interfering with the immune system * Severe comorbidities: other active malignancy (except for basal cell carcinoma and other in situ carcinomas) * Serious psychiatric pathology * A self-reported alcohol consumption of \>21 units per week

Sample size calculation:

The whole-genome analyses (transcriptome, epigenome) and systems biology models that will be performed during the current study will encompass a very large number of variables, each with different variations within the samples. Therefore, due to the nature of the methodological approach, the calculation of the precise number of samples is not possible. Based on the previous whole-genome transcriptional and epigenetic analyses of human macrophages, we will compare full transcriptional and epigenetic profiles of macrophages and TAMs differentiated from groups of 30 patients who received neoadjuvant chemotherapy and 30 patients who are chemo naïve and 30 patients with benign gynecological disease. The above-mentioned numbers of subjects are determined after careful consideration and shared consensus by members of the scientific team involved in the study. The exploratory nature of the study and, in case of invasive investigations, the risks and burden on participants were taken into account.

TREATMENT OF SUBJECTS Not applicable as the patients will not undergo other treatments within the study than those already planned by their treating physician corresponding to the standard of care.