| Abstract |
Lung cancer is the leading cause of cancer related deaths worldwide in both sexes, with 1.8
million deaths recorded in 2020. Five-year survival ranges from 10-20% of all lung cancer cases,
because most patients at the time of diagnosis already have advanced or metastatic disease,
which limits the patient's treatment options and significantly reduces the probability of survival
beyond five years.
Targeted molecular therapies and recently immunotherapy have significantly improved the
prognosis of lung cancer patients. Particularly, immunotherapy is an innovative therapeutic
approach for metastatic lung cancer, which aims to activate the immune system and its
components to suppress the tumor. However, despite the encouraging results from the
administration of immunotherapy, clinical benefits are limited to a relatively small percentage
of patients and therefore, the need to find new and more informative predictive biomarkers for
use in daily clinical practice remains imperative.
It is now known that tumors cause a disturbance in the peripheral immune response which
is associated with the progression of the disease and with an unfavorable prognosis of the
patients. Therefore, the analysis of biomolecules that have a key role in the regulation of the
immune response and anticancer immunity, in peripheral blood as "liquid biopsy" samples from
lung cancer patients, could reveal and highlight effective or easily accessible biomarkers.
In this direction, the study of small non-coding RNA molecules (miRNAs) consists an
important field of research as their expression has been found to be deregulated in a variety of
pathological conditions, including NSCLC, while at the same time more and more data are
converging that miRNAs can regulate the immune response against the tumor by modulating
the gene expression of immunoregulatory molecules, at the post-transcriptional level, in the
tumor and immune cells.
Due to the involvement of miRNAs in the different stages of tumor initiation and
progression, these molecules are being examined as potential prognostic and predictive
biomarkers in cancer. The effectiveness of miRNAs as biomarkers lies in their high specificity
and their expression pattern that differs between normal and pathological conditions. Their
significant advantages are that due to their small size (19-24 nucleotides), they are extremely
stable and can be easily determined in plasma samples with high reproducibility. Plasma
constitutes a pool of miRNAs secreted from different sites of the primary tumor or metastatic foci, thus reflecting tumor heterogeneity. As a result, changes in the expression of miRNAs
suggest a useful tool for early diagnosis and predicting the outcome of cancer patients.
The present research aimed to analyze the expression and clinical evaluation of circulating
miRNAs that regulate the immune response in the tumor microenvironment, in the plasma of
patients with advanced or metastatic NSCLC. The miRNAs examined are those that regulate
immune checkpoints (miR-34a, miR-200b, miR-200c), control the regulation of T regulatory cells
Tregs (miR-155, miR-146) and myeloid derived suppressor cells (MDSCs) (miR-223), and finally,
those that regulate macrophage differentiation towards M1 or M2 phenotype (let-7c, miR-26a,
miR-30d, miR-195, miR-202). Two independent groups of patients were used to implement the
objectives of this research. Group 1 included patients with advanced or metastatic NSCLC who
were treated with first-line chemotherapy based on platinum compounds and Group 2 included
patients with advanced or metastatic NSCLC who were treated with second-line
immunotherapy with PD-1/PD-L1 inhibitors.
Following that, quantification of the expression of the circulating miRNAs was performed
in the samples of the patients of both groups, through real-time quantitative PCR. Investigation
of the clinical significance of the studied miRNAs was performed through extensive statistical
analysis.
In the group of patients with advanced or metastatic NSCLC who were treated with firstline
chemotherapy based on platinum compounds, the results of the analysis initially showed
that miR-146a, miR-195, miR-200c and miR-223 exhibited differential expression between
patients and the control group. In addition, the expression levels of miRNAs were related to
various clinicopathological characteristics of the patients, such as age, performance status,
histological subtype, the number of metastatic foci, the presence of brain and liver metastases,
while they were also related to the objective response rate, with disease control rate and
prolonged duration disease control. Low expression of miR-146a and high expression of miR-
200c emerged as independent predictors of increased risk of developing disease progression as
an optimal response to treatment in the whole population, while in the subgroup of patients
with adenocarcinoma, low expression of miR-34a and high expression of miR-200c emerged as
independent predictors for the same factor.
High expression of miR-200c was associated with shorter overall survival in the whole
population and high expression of miR-202 was associated with shorter progression free
survival in the whole population, as well as with shorter overall survival in both the whole population and the subgroup of patients with adenocarcinoma. High expression of miR-26a was
associated with shorter overall survival, while high expression of miR-155 was associated with
shorter progression free survival and shorter overall survival, in the subgroup of patients with
squamous cell carcinoma. Finally, high miR-202 expression emerged as an independent
prognostic marker for shorter progression free survival in the whole population, while in the
subgroup of patients with adenocarcinoma it was found to be an independent prognostic
marker for shorter overall survival. Also, in the whole population, high expression of miR-200c
emerged as an independent prognostic indicator for shorter overall survival.
In the group of patients with advanced or metastatic NSCLC who had received second-line
immunotherapy with PD-1/PD-L1 inhibitors, the results of the analysis initially showed that miR-
26a, miR-30d, miR-34a, miR-146a, miR-155, miR-195, miR-200b, miR-200c, miR-202 and miR-
223 exhibited differential expression between patients and the control group. In addition, the
expression levels of miRNAs were related to various clinicopathological characteristics of the
patients, such as performance status, the stage of the disease, the number of metastatic foci,
the presence of liver and bone metastases, while at the same time they were also related to
the disease control rate and the prolonged duration disease control. Low expression of miR-34a
emerged as an independent predictor of increased risk of developing disease progression as an
optimal response to treatment in the subgroup of patients with adenocarcinoma.
Low miR-34a expression was associated with shorter progression free survival in the whole
population, while in the subgroup of patients with adenocarcinoma it was associated with both
shorter progression free survival and shorter overall survival. High expression of miR-200c was
associated with shorter overall survival in both the whole population and in the subgroup of
patients with adenocarcinoma, while in the subgroup of patients with adenocarcinoma, high
expression of let-7c was associated with progression free survival. Low expression of miR-26a
was associated with a shorter progression free survival in the subgroup of patients with
squamous cell carcinoma. Finally, high miR-200c expression emerged as an independent
predictor of shorter overall survival in both the whole population and the subgroup of patients
with adenocarcinoma. In addition, low miR-34a expression was identified as an independent
prognostic marker for shorter progression free survival and shorter overall survival in the
subgroup of patients with adenocarcinoma. Also, low expression of miR-26a emerged as an
independent predictor of shorter progression free survival in the subgroup of patients with
squamous cell carcinoma. The last part of the present thesis aimed to identify the origin of circulating miRNAs. From
the analysis of the results, the comparison of circulating miRNAs in matched samples of plasma
and mononuclear cells showed that the expression of miRNAs from these two sources are
independent and not related to each other, indicating that these miRNAs are possibly derived
by the tumor cells.
In conclusion, although the function of circulating miRNAs in the regulation of the immune
response remains unclear, the interpretation of the role of these molecules as potential
biomarkers is of great importance. The results of this research further support the hypothesis
that the expression pattern of circulating miRNAs involved in the regulation of immune
response and antitumor immunity through regulation of key elements of the immune system is
related to patient survival and these molecules should be further studied as potential
prognostic and predictive biomarkers in NSCLC.
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