Flow cytometric analysis of B & T-lymphocyte phenotypes in breast cancer (CFS control group), 2020, Strachan (M.D. Thesis)

From: Dr. Marc-Alexander Fluks

Source: University of Leeds
Date: May 2020, online January 11, 2022
URL: https://etheses.whiterose.ac.uk/28168/

https://etheses.whiterose.ac.uk/28168/1/Final and corrected MD C.Strachan.pdf


A flow cytometric analysis of B and T-lymphocyte phenotypes in breast
cancer: implications for prognosis, treatment and recovery
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Caroline Louise Strachan
- Faculty of Medicine and Health, University of Leeds, U.K.


Abstract

Introduction:
It is widely understood that the T-lymphocyte is closely linked to
prognosis and treatment response in certain solid organ malignancies
including breast. B-Lymphocytes have, however, received comparatively
little attention in this field and it remains unclear what role, if any,
B-lymphocyte subtypes play in carcinogenesis or prognosis in breast
cancers. Likewise, it is unknown what effect systemic treatments such as
chemotherapy have on B-cell immunity, or the role these lymphocyte
subtypes may have in the side-effect profile of such therapies. Cancer
related fatigue (CRF) is one such side-effect of cancer and its
treatments, the pathogenesis of which has been linked to impaired
immunity including altered lymphocyte phenotypes.
My aim in this work was to perform detailed phenotypical analyses of B-
and Tlymphocytes in breast cancer, as well as phenotypical alterations
driven by cancer treatments, to provide insight into the function of
B-lymphocytes in this disease process, adding to the growing body of
knowledge driving immunotherapeutic development.

Methods
43 primary breast cancer patients, scheduled to receive adjuvant
chemotherapy, were recruited following resection surgery alongside 10
age- and sex-matched healthy controls. 27 Chronic fatigue syndrome (CFS)
patients were recruited at diagnosis, as a comparator group for
assessments relating to CRF. 15 further patients with primary breast
cancer were recruited prior to resection surgery for analyses of fresh
tumour tissue and peripheral blood. Phenotypes of circulating
lymphocytes were analysed using multicolour flow cytometry at various
time-points in the larger breast cancer cohort and its associated
comparator groups (CFS and healthy controls), along with general health
and well-being screening questionnaires as used in the diagnosis of CFS.
For the fresh tissue study, tumour tissue was biopsied and analyses of
tissue-resident B- and T-lymphocytes were performed, along with
circulating analyses as previously, focusing on regulatory B and T
subtypes. Results were analysed using paired T-tests, 2-way ANOVA, and
correlation analysis.

Results
Lymphocyte phenotype was vastly altered by chemotherapy. Within the
Tcell pool, the naïve subset is diminished with proportional increases
to the memory cell pool and the overall expansion of HLA-DR surface
expression on CD4+ T-cells. Regulatory T-cells were unchanged. Within
the B-cell pool, memory cells were 5 largely reduced by chemotherapy,
the resulting phenotype being naïve and transitional dominant.
Pro-inflammatory cytokine expression by regulatory B-cells was
diminished, yet IL-10 expression remains unchanged.
The CD20+CD27+ regulatory Memory B-cell subset is demonstrated as a
critical Bcell phenotype in breast cancer prognosis and in fatigue.
CD27+ regulatory memory B-cell dominates the phenotype in tumour tissue
where these subsets, in addition to naïve T-cells, correlated
positively with poor prognostic indicators in breast cancer both in
peripheral blood and tumour tissue. Additionally, expression of effector
regulatory cytokines IL-10 and TNF-alpha from the memory B-cell subsets
correlated with prognosis and fatigue.

Conclusions
Detailed B-lymphocyte phenotypes have been clearly demonstrated in
breast cancer and in fatigue, with the regulatory CD20+CD27+ memory
B-cell subset prevailing as the dominant cell type relating to breast
cancer prognosis, both in tumour and peripheral blood, as well as
fatigue scores. This first insight into the phenotype and function of
B-cells and B-regs in breast cancer highlights the memory B-cell subset
as a driver in the pathogenesis of this disease, and provides a target
for further research and potential novel lines of investigation in the
on-going search for immunotherapies.

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(c) 2020 University of Leeds

 

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