Journal Thoracic Oncology

Abstracts Journal of Thoracic Oncology • Volume 12 Issue S1 January 2017
controls. Synthesis of cDNA and qPCR were carried out using miRCURY LNA TM
Universal RT microRNA PCR with LNA TM enhanced PCR Primers (Exiqon).
Statistical calculations were executed on 11 samples as a Pre-eliminary data.
Results: Results are shown in the following table.
mir-19b and mir-320 was observed whereas mir-15b and mir-197 were upregulated
in the conversion of macrophages into M2 phenotype. Modulation
of miRNAs in immune and stromal cells was consistent with up-regulation of
the same miRNAs observed in plasma samples. Conclusion: Our findings on
the origin of circulating miRNAs support the conclusion that plasma miRNAs
are heterogeneous and secreted by different cellular components of lung
microenvironment rather than by tumor cells. In particular, we demonstrated
that a pro-tumorigenic and immunosuppressive microenvironment
contributes to the de-regulation of miRNAs observed in plasma of lung cancer
patients.
Keywords: lung cancer, microRNA, microenvironment
Conclusion: We assed level of 5 different miRNAs circulating in the blood of
NSCLC patients using qPCR. Our initial results show that different miRNA can
be used to stratify patients and miRNA. Expression of hsa-miR-451a is
decreasing in NSCLC versus negative control. Interestingly up-regulated
hsa-miR-660-5p was recently described as a prognostic marker in breast
cancer but our result preliminary results showed constant decrease in
hsa-miR-660-5p expression in all patients’ groups vs controls. The
examination on the bigger cohort of patients is necessary to receive a more
statistically significant and conclusive data.
Keywords: cell-free microRNA, biomarkers, miRNA expression profiling,
NSCLC.
POSTER SESSION 2 – P2.01: BIOLOGY/PATHOLOGY
ANALYSIS OF RNA –
TUESDAY, DECEMBER 6, 2016
P2.01-017 CIRCULATING MIRNAS IN LUNG CANCER ARE
ASSOCIATED TO PRO-TUMORIGENIC AND IMMUNOSUPPRESSIVE
MICROENVIRONMENT
Orazio Fortunato 1 , Cristina Borzi 1 , Giovanni Centonze 1 , Massimo Milione 2 ,
Davide Conte 1 , Mattia Boeri 1 , Carla Verri 1 , Linda Calzolari 1 , Francesca
Andriani 1 , Luca Roz 1 , Veronica Huber 3 , Agata Cova 3 , Chiara Camisaschi 3 , Chiara
Castelli 3 , Licia Rivoltini 3 , Claudio Tripodo 4 , Ugo Pastorino 5 , Gabriella Sozzi 1
1 Tumor Genomics Unit, Department of Experimental Oncology and Molecular
Medicine, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milano/Italy,
2 Anatomic Pathology Unit, Department of Pathology and Laboratory Medicine,
Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milan/Italy, 3 Unit of
Immunotherapy of Human Tumors, Department of Experimental Oncology and
Molecular Medicine, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milano/
Italy, 4 Tumor Immunology Unit Department of Health Science, Human Pathology
Section, University of Palermo School of Medicine, Palermo/Italy, 5 Thoracic
Surgery, Fondazione IRCCS Istituto Nazionale Dei Tumori Int, Milano/Italy
Background: We previously reported the identification of diagnostic miRNA
signatures in plasma samples of lung cancer patients detected by low dose
computed tomography (LDCT) screening. Circulating miRNAs are released
into the bloodstream by different mechanisms such as passive leakage from
damaged cells or active secretion through extracellular-vesicles or protein
complexes Methods: To evaluate the potential origin and the release of the
24 miRNAs of the diagnostic signature we analyzed their expression by realtime
or digital PCR in both cells and conditioned medium (CM) from cancer
cell and different cell types of the lung microenvironment. Lung tissues and
cell-blocks were analyzed by miRNAs in situ hybridization (ISH). Modulation
of miRNAs after in vitro treatments known to induce changes associated
with cancer progression, in different cell types was assessed and correlated
to changes observed in circulating miRNAs signatures. Results: 24-miRNAs
analysis showed higher abundance in specific cellular components such as mir-
145 in fibroblasts, mir-126 in endothelial cells, mir-133a in skeletal muscle cells
or mir-451 and 142-3p in hematopoietic cells. Generally, tumor cells showed
lower levels of miRNAs compared to bronchial epithelial cells. MiRNAs specific
localization in lung tissue was confirmed by ISH. We observed that mir-451
is specifically expressed in lung interstitial alveolar walls while mir-126 by
endothelial cells outside the tumor bulk; miR-145 is characteristic of fibroblast
and muscle cells and miR-142-3p of hematopoietic cells, fibroblast and muscle
whereas mir-21 is over-expressed in the tumor. The analysis of miRNAs in
CM showed that miRNAs secretion is correlated with cellular expression for
most cell types (Pearson correlation range: 0.41-0.80). Interestingly, platelets
and granulocytes were the components that mostly secreted miRNAs. In
vitro experiments showed that endothelial cells under hypoxic condition
up-regulate mir-126 and that mir-145 was up-regulated and secreted in lung
cancer-associated compared to normal fibroblasts. Interestingly, during
conversion of T lymphocytes into T regulatory cells up-regulation of mir-15b,
POSTER SESSION 2 – P2.01: BIOLOGY/PATHOLOGY
ANALYSIS OF RNA –
TUESDAY, DECEMBER 6, 2016
P2.01-018 DIFFERENTIAL MICRORNA EXPRESSION PROFILE
BETWEEN YOUNG AND OLD LUNG ADENOCARCINOMA PATIENTS
Mirella Giordano 1 , Laura Boldrini 1 , Adele Servadio 1 , Marco Lucchi 1 , Franca
Melfi 2 , Alfredo Mussi 3 , Gabriella Fontanini 1
1 Department of Surgical, Medical, Molecular Pathology and Critical Area, University
of Pisa, Pisa/Italy, 2 Unit of Thoracic Surgery, University Hospital of Pisa, Pisa/Italy,
3 Department of Surgical Medical Molecular Pathology and Critical Care, University
Hospital of Pisa, Pisa/Italy
Background: Lung cancer is the leading cause of cancer related mortality and
approximately 80% is represented by non-small cell lung cancer (NSCLC). In
the last decade, age of patients at diagnosis has decreased, with an incidence
of approximately 13.4% in patients under 50 years. Previous studies have
hypotesized that lung cancer in young patients could represent a separated
clinicalpathological entity, however it is still controversial whether younger
patients have a different outcome compared with their older counterparts.
MicroRNAs (miRNAs) have recently been defined to play a key role in cancer
pathogenesis and their aberrant expression has been suggested as a potential
biomarker of prognosis in lung adenocarcinoma. To understand the molecular
features of young and old adenocarcinoma patients, we investigated the
expression levels of a panel of miRNAs selected from recent literature.
Methods: Thirty-five lung ADC from patients under 50 years old were enrolled
as the younger group and thirty-five ADC older than 50 years were collected
as the older group. After miRNA isolation from formalin-fixed and paraffinembedded
tumor tissues, the expression levels of 30 miRNAs were analyzed
by NanoString technology and compared between the two groups. Survival
data were used to assess the prognostic impact of miRNAs. The software
miRgator v3.0 was used to predict the putative pathways targeted by
miRNAs. Results: 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