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Digital archive of theses discussed at the University of Pisa


Thesis etd-04032017-113910

Thesis type
Tesi di dottorato di ricerca
Thesis title
Evaluation of a miRNA panel as biological marker for prognostic and predictive purposes in Non-small Cell Lung Cancer
Academic discipline
Course of study
tutor Mussi, Alfredo
  • microRNAs
  • Nanostring Technology
  • Non-small Cell Lung Cancer
  • prognosis
Graduation session start date
Release date
Lung cancer is the leading cause of cancer related mortality in both men and women, and approximately 80% are Non-small Cell Lung Cancer (NSCLC). Surgical resection represents the treatment of choice for NSCLC during early stages of the disease; however, the survival rates remain low, and the study of tumors and patient genetic profiles represents a priority for improving the knowledge of the molecular mechanisms involved in human tumorigenesis. Moreover, in the last decade, the incidence of NSCLC has been quickly increasing and patients’age at diagnosis keeps decreasing. The median age at diagnosis of NSCLC is 70 years and approximately 13% of patients are younger than 50 years. So far, it is still controversial whether younger patients have better or worse outcomes compared with the older counterparts with lung cancer.
microRNAs (miRNAs) are a class of small noncoding RNA that regulate the expression of many target gene via mRNA degradation or translation inhibition. Specific miRNA profiles could be useful in the diagnosis, subclassification, and prognosis of NSCLCs as well as the comprehension of mechanisms regulating the resistance or sensitivity of lung cancer to conventional chemotherapy. It is hypothesized that the integration of multidimensional molecular data may better capture the molecular heterogeneity of lung cancer patients, resulting in more robust biomarkers for patient classification.
The research project focused on the evaluation of miRNAs as biological markers with a prognostic and predictive role in two subset of NSCLC: early stage (T1N0) NSCLC patients and adenocarcinoma (ADC) patients younger than 50 years.
First, we evaluated the expression and the prognostic role of three miRNAs (let-7g, miR-21, and miR-205) in 105 early-stage (T1N0) NSCLC patients by quantitative retro-transcriptional PCR (qRT-PCR). A significant association between the low miR-205 expression and adenocarcinoma histotype (p < 0.0001) compared to squamous cell carcionoma was found; the validation of these findings on liquid biopsy could support the use of miR-205 as non-invasive biomarker for differential diagnosis. Moreover, survival analysis showed that tumors with high miR-205 expression had a significantly shorter mean progression free survival and overall survival compared to the patients with a low expression of this miRNA (p=0.02 and p=0.03, respectively). Regarding let-7g and miR-21 expression we did not observe any significant correlation with the clinico-pathological characteristics of the NSCLC patients.
Our results indicated that miR-205 could represent a useful marker in the diagnostic and prognostic management of the early-stage (T1N0) NSCLC patients.
Second, to understand the molecular features of young and old adenocarcinoma patients, we selected and investigated the expression level of a panel of 30 miRNAs togheter with some of their mRNA target (n=30) involved in different lung cancer related pathway such as angiogenesis, tumor immunity, cellular motility and proliferation. The expression analysis was performed by the nCounter System® (NanoString Technologies) directly on RNA, including small RNAs, that allows to detect simultaneously both miRNAs and mRNAs. The analysis revealed that 7 miRNAs (miR-25-3p, miR-29c-3p, miR-33a-5p, miR-144-3p, miR-153-3p, miR-342-5p and miR-485-3p) were differently expressed in the two groups (Mann-Whitney U test, p<0.05). All these miRNAs showed higher expression levels in young compared to old patients, and their predicted targets included EGFR, MET, VEGF-A, TP53 and PDGFRa. miR-144-3p had an opposite influence on overall survival, since its upregulation was associated with a worse prognosis in young patients (p=0.01) and a better outcome in the old group (p=0.03). We observed that lung cancer in young and old patients may be influenced by different regulatory mechanisms probably due to distinct age-related genetic and epigenetic alterations. Moreover, one of the deregulated miRNAs showed a different prognostic impact in the two groups thus confirming that young and old patients deserve a specific clinical approach.
Furthermore, comparing the expression levels of angiogenesis related genes (VEGF-A, FLT1, KDR, FLT4, PDGFRa and PDGFRb) with the different histological adenocarcinoma predominant patterns (lepidic, solid, acinar and papillary) regardless of age, we found a significant higher expression of VEGF-A in papillary than in other patterns (p=0.02). Four out of eight miRNAs that target VEGF-A were differentially expressed among ADC patterns and their expression profile was precisely opposite to the trend of VEGF-A. A distinct angiogenic miRNA-mRNA expression profile among the predominant patterns of ADC could represent a useful biomarker to stratify patients who can effectively treated with angiogenesis inhibitor. Moreover, the regulation of angiogenic mRNA factors by miRNAs could provide a novel therapeutic approach based on their expression pattern specific for distinct ADC patterns.
The idea of personalized medicine for cancer therapy is not novel and it is well-known the usefulness of biomarkers in treatment decision-making to improve patient outcome. However, the development of prognostic biomarkers is a multi-step process that requires optimization, for this reasons we believe that this study represents a starting point to better understand the role of miRNA in early stage NSCLCs and ADC younger than 50 years.