Isolation and immune profiling of human B lymphocytes for the development of potential diagnostic and therapeutic antibodies

Uterine cancer (UC) is the most common gynecological malignancy in the Western
world. Standard treatment comprises surgery with or without
(chemo)radiotherapy. Despite an overall favorable prognosis, many women relapse
and succumb to the disease.
Approximately 30% of all uterine cancers are characterized by a high mutational
load resulting from: microsatellite instability (MSI-H), MisMatch Repair
deficiency (dMMR) or mutations in the exonuclease (proofreading) domain of DNA
polymerase epsilon (POLE-EDM).
MSI-H/dMMR and POLE-EDM occur in 20% and 12% of all uterine cancers (UCs),
respectively. These tumors are characterized by a high number of
mutation-associated neo-antigens (MANAs) and are therefore prime targets for
immunotherapy.
Evaluation of MANA-specific responses during clinical immunotherapy has focused
predominantly on CD8, and to a lesser extent CD4, T cell responses. However,
recent work has also identified a key role for B lymphocytes in immunotherapy.
In particular, MANA-rich tumors are characterized by extensive B cell
infiltration and so-called tertiary lymphoid structures (TLSs), de novo
lymphoid tissue associated with favorable prognosis and responses to immune
checkpoint inhibitors. The migration of circulating B cells to peripheral
organs has also been linked to potential immunotherapy-associated
autoimmune-related side-effects.
Based on our earlier work, we hypothesize that MANA-specific T cells recruit B
cells to tumors via release of the chemokine CXCL13. Infiltrating B cells could
subsequently contribute to antitumor responses through several mechanisms, most
notably through the formation of TLSs, production of anti-tumor antibodies, and
as potent antigen-presenting cells (APCs). Here, we propose a first step
towards addressing this hypothesis by analyzing de novo B cell antibody
responses towards endometrial cancer

We aim to study B cells derived from tumor and peripheral blood of uterine
cancer patients in order to evaluate B cell-produced (tumor binding) antibodies
for potential diagnostic and therapeutic interventions.

Prior to surgery (hysterectomy) PBMC*s (100mL) will be obtained. Tumor tissue
removed during surgery will be evaluated by pathological examination according
to standard-of-care procedures. The remaining *waste*-tumour tissue will be
used for study procedures. DMMR testing will be performed according to
standard-of-care by the pathologist. Additionally, tumor tissue sections from
the same block will be used for DNA isolation (POLE-EDM testing) and
immunohistochemical staining for at least CD3/CD20, CD4/CD8 and CD38/CD138 to
identify tertiary lymphoid structures and antibody-producing plasma cells.
Since a hysterectomy is performed as standard of care procedure, RNA and single
intratumoral B cells can be isolated and analyzed. B cells from different types
of uterine cancer (dMMR, POLE-EDM, TP53 mutant and NSMP) will be compared.
PBMC*s collected prior to surgery and during follow up (optional) are used to
assess the systemic B-cell responses. Single B-cells isolated from the surgical
specimen will be used for single cell B cell receptor (BCR) sequencing and for
antibody-screening. Whether patients will be approached for a second blood
sample to confirm findings of interest will depend on the primary results

Participants will be asked to donate one blood sample during their normal
diagnostic and therapeutic work-up. Some patients will be approached for a
second sample. The first sample will be obtained prior to standard-of-care
surgery (100 ml). Patients will be approached for a second blood sample to
validate notable findings.

Risk of participation is considered minimal as an ordinary venapunction will be
performed. Patients will not benefit from participation in the study.
This is a pilot study, if any potential diagnostic of therapeutic interventions
are found, further options can be explored in a large-scale research project to
contribute to the development of immunotherapy for uterine cancer.