Dissertation committee:
Δούση-Αναγνωστοπούλου Υπατία, Αναπλ. Καθηγήτρια, Ιατρική, ΕΚΠΑ
Γοργούλης Βασίλειος, Καθηγητής, Ιατρική, ΕΚΠΑ
Κοτσίνας Αθανάσιος, Επικ. Καθηγητής, Ιατρική, ΕΚΠΑ
Κλέτσας Δημήτριος, Ερευνητής Ά, ΕΚΕΦΕ ΔΗΜΟΚΡΙΤΟΣ
Ευαγγέλου Κωνσταντίνος, Καθηγητής, Ιατρική, Πανεπιστήμιο Ιωαννίνων
Κουλούκουσα Μυσρίνη, Αναπλ. Καθηγήτρια, Ιατρική, ΕΚΠΑ
Χαβάκη Σοφία, Επικ. Καθηγήτρια, Ιατρική, ΕΚΠΑ
Summary:
The purpose of this thesis was to study the infiltration level and the spatial distribution of the immune cell subpopulations within the tumor, as well as the level of their correlation with clinicoopathological characteristics and the clinical outcome of patients with invasive, non-metastatic, ductal breast cancer (BCa). Furthermore, we studied the possible correlations between circulating factors at the preoperative stage and tumor infiltration status by immune cells, in order to create a combinational signature of prognostic significance.
The study is divided in three parts. The first part includes the total number of patients with breast cancer (BCa). The second part includes patients with available follow-up that were analyzed retrospectively. Finally, the third part is a prospective cohort of patients, with an additional blood sample collected one day before operation.
In the total number of patients, tests were carried out in tissue specimens to evaluate the presence, as well as the spatial-architectural distribution of immune cell subpopulations in the tumor center (TC) or in the invasive margin (IM), or/and in both. The results from differential tumor infiltration by CD8+ lymphocytes and CD163+ macrophages in a group of 162 patients were correlated with established BCa clinicopathological features, such as TNM stage, Grade, Hormone Receptors status, HER2/neu over-expression, proliferation marker Ki67% and patient age. From our results, it was demonstrated for the first time that the combined evaluation of CD8+ or/and CD163+ cell densities in TC and IM improves patients’ categorization and increases the prognostic value of the established TNM staging in BCa.
In the retrospective part of the study, 97 patients with available clinical follow-up were analyzed. Our data indicated that the combined assessment of the CD8+ and CD163+ cell densities in TC and IM could enhance the prognostic accuracy of disease-free survival-DFS and overall survival-OS. More specifically, two “signatures” were created, a favorable one –FCIS (Favorable Combined Immune Signatures) characterized by high CD8+ in TC and low in IM and/or low CD163+ in TC and high in IM and an unfavorable one -UCIS (Unfavorable Combined Immune Signatures) characterized by low CD8+ in TC and high in IM or high CD163+ in TC and low in IM (without being CD8+ HL or CD163+ LH). These two signatures cover approximately 50% of the patients with invasive, non-metastatic ductal BCa. The other patients remain at a “gray area” (REST).
At the prospective part of the study, six miRNAs levels, from serum and Peripheral Blood Mononuclear Cells-PBMCs, of 50 patients were evaluated. Furthermore, the expression levels of circulating cytokines/ chemokines were measured. Direct correlations, positive and negative, were revealed between circulating miRNAs and tumor infiltrated immune cell subpopulations, as well as with some cytokines/chemokines level. No correlation was observed between serum miRNAs of PBMCs miRNAs.
Using an algorithm, three distinguishable clusters of patients were created based on their serum-derived miRNA expression levels. There was a classification of patients with different clinicopathological characteristics, different levels of circulating cytokines/chemokines, different immune cell infiltration and a different HLA genotype.
Taking into account the limitations set in our study regarding the small sample size, we could argue that we have identified for the first time a "signature" that easily and with a minimally invasive method segregates patients with invasive, non-metastatic BCa into three different clusters with distinctive features. In addition, some of the clusters/groups, that have been created, seem to have a diagnostic role as well. These "signatures" remain to be confirmed in a larger sample of patients. Beside this, clinical follow-up over a period of time is essential to verify whether the factors measured in the peripheral blood, alone or in combination with the tumor microenvironment, could serve as a "liquid biopsy" and/or as a "signature" with the immune cell infiltration of the tumor giving at the time of the initial diagnosis an additional prognostic value to our "signature".
Keywords:
Immune signatures, Tumor infiltration, Breast cancer, Biomarkers, miRNAs
