Post on 29-Jul-2020
Matteo FASSANUNIVERSITÀ DEGLI STUDI DI PADOVA, ITALIA
Hot Issues in Molecular PathologyPopper H, Schaff Z, Tímár J
Technology Transfer in Diagnostic Pathology
7th Central European Regional Meeting
Siófok - Hungary, May 14-16 , 2012
MIRNAS expressionin oesophageal and gastric cancers
MIRNAS expressionin oesophageal and gastric cancers
MULTISTEP MODELOF SCIENTIFIC PARADIGMS
1761 1858 1953
Die ZellularpathologieDe sedibus et causis
morborum per anatomen indagatis
Molecular structure of nucleic acids; a structure
for deoxyribose nucleic acid
THE CODING-RNA PARADIGM
DNA
Gen
e
AA
CG
GC
TG
CG
CA
DNA strand
UUCC
GGCC
GGGG
GGUU
AACC
CCUU
mRNA
TRANSCRIPTION
protein
Cod
onTRANSLATION
The begin of a new story…
Holley RW et al - Science 1965
…the first non-coding RNA to be characterised was the alanine tRNA found in baker's yeast…
Cech TR et al - PNAS 1979
…ribosomal ribonucleic acid (rRNA) is the RNA component of the ribosome, the protein manufacturing machinery of all living cells…
NON-CODING RNAS’ WORLD
Bartel DP - Cell 2004
Small RNAS’ WORLD
THE INTERFERENCE ERA
Fire A et al - Nature 1998
…reported a potent gene silencing effect after injecting double stranded RNA into C. elegans…
ONCOMIRS: the new actors in the cancer scenario
Visone R & Croce CM - Am J Pathol 2009
Baffa R, Fassan M & Croce CM - Laborwelt 2009
Fassan M, Rugge M & Baffa R - Front Biosci 2011
… one-third of protein-coding human mRNAs are susceptible to this complex miRNA regulatory network…
let-7
RAS; C-MYC
Normal Cancer
miRNA as tumor suppressor gene
miR-21
PDCD4
miRNA as oncogene
PNASHuman microRNA genes are frequently located at fragile sites and genomic regions involved in cancers
Calin GA et al - PNAS 2003 Volinia S et al - PNAS 2005
A microRNA expression signature of human solid tumors defines cancer gene targets
Iorio MV & Croce CM – J Clin Oncol 2009
Mechanisms of miRNAs’ dysregulation in cancer
Baffa R, Fassan M, Gardiman M, Rugge M, Croce CM - J Pathol 2009
Fassan M, Palazzo JP, Rugge M, Croce CM - Breast Cancer Res 2009
The use ofFF-PE tissues
The pathologist’s point of view
PDCD4 nuclear loss inversely correlates with miR-21 levels in colon carcinogenesis
Fassan M, Pizzi M, Mescoli C, Ludwig K, Rugge M - Virchows Arch 2011
Fassan M, Pizzi M, Balistreri M, Rugge M - Dis Esophagus 2011
Programmed cell death 4 nuclear loss and miR-21 or activated Aktoverexpression in esophageal squamous cell carcinogenesis.
PRENEOPLASTIC LESIONS
Baffa R, Fassan M, Gardiman M, Rugge M, Croce CM - J Pathol 2009
Fassan M, Palazzo JP, Rugge M, Croce CM - Breast Cancer Res 2009
The pathologist’s point of view
The direct visualization in FF-PE tissues - ISH
MIRNAS IN SITU HYBRIDIZATION
Van Baal J, Fassan M, Rugge M, Siersema P – Gut 2012
Baffa R, Fassan M, Gardiman M, Rugge M, Croce CM - J Pathol 2009
Fassan M, Palazzo JP, Rugge M, Croce CM - Breast Cancer Res 2009
The pathologist’s point of view
The identification of the target genes - IHC
+3.00
0.00
-3.00
hsa-miR-205
N IM LG HG BAchsa-let-7c
hsa-mIR-605hsa-miR-100
hsa-miR-203
hsa-miR-99ahsa-miR-23ahsa-miR-147hsa-miR-192hsa-miR-215hsa-miR-560hsa-miR-326hsa-miR-615
Up
Down
13 MIRNAS DIFFERENTLY EXPRESSED IN BE-ONCOGENESIS:
hsa-miR-147 hsa-miR-192 hsa-miR-215 hsa-miR-326 hsa-miR-560 hsa-miR-615-3p
6 UP-REGULATED hsa-let-7chsa-miR-23a hsa-miR-99a hsa-miR-100 hsa-miR-203 hsa-miR-205 hsa-miR-605
7 DOWN-REGULATED
Fassan M, Croce CM, Rugge M – Int J Cancer 2011
hsa-let-7c TARGETS HMGA2/HMGI-C
� HMGA2/HMGI-C is a direct target of hsa-let-7c;
� HMGA2/HMGI-C is a small non-histone chromosomalprotein that modulates transcription;
� HMGA2/HMGI-C over-expression is a hallmark of variousbenign & malignant human tumors, including Gastric Cancer.
HMGA2/HMGI-C IHCRing chromosome
Differential Diagnosis: Liposarcoma lipoma-like versus lipoma.
N
20%
60%
100%
N IM LG HG BAc
BARRETT’S ONCOGENESIS: HMGA2 IHC
BAcHG
N
BAc
� Biopsy samples:
N=25, IM=25, LG=16, HG=11, BAc=10
� Score Values: 0= 0-5%; 1= 6-50%; 2= >50%
p < 0.001
Fassan M, Croce CM, Rugge M – Int J Cancer 2011
Fassan M, Croce CM & Rugge M – WJG 2011
The clinician’s point of view
miRNAs as new prognostic tools
1.0
1.0
0.5
Su
rviv
al p
rob
abil
ity
Time
1.0
0.5
Surv
ival
pro
babi
lity
Time
Kogo R et al – Clin Cancer Res 2011
…the miR-146a expression status is associated with patients’survival and the presence of lymph node metastases; miR-146ais an independent prognostic factor in GC…
high miR-146a
low miR-146a
MIR-27a POLYMORPHISM & GASTRIC ATROPHY
Arisawa T et al – Dig Dis Sci 2007
…the miR-27a genome region polymorphism may be an important definitive factor to develop the gastric mucosal atrophy in Japanese…
100
0
50
% o
f gas
tric
atr
oph
y
A/A G/G A/G
Fassan M, Croce CM & Rugge M – WJG 2011
The clinician’s point of view
miRNAs as non-invasive diagnostic
markers
MIRNAS AS PLASMA BIOMARKERS IN GC
Tsujiura M et al – BJC 2010
Plasma miRNAs are dysregulated in gastric cancer patients
7.0
3.0
1.0
5.0
.
.
.
.Rel
ativ
e qu
anti
ty
Normal Cancer
7.0
3.0
1.0
5.0
.
.
.
.Rel
ativ
e qu
anti
ty
Beforesurgery
Aftersurgery
miR-21 miR-21
Plasma miRNAs expression after 1 month from surgery
Fassan M, Croce CM & Rugge M – WJG 2011
The clinician’s point of view
miRNAs as biomarkers of therapeutic response
MIRNAS AND MULTI-DRUG RESISTANCE
Xia L et al – Int J Cancer 2008
miR-15b and miR-16 modulate MDR by targeting BCL2 in human gastric cancer cells
1
0
0.5
bcl2
rel
ativ
e ex
pre
ssio
n
miR15b miR16
1
0
0.5
bcl2
rel
ativ
e ex
pre
ssio
n
miR15b miR16
miR15b miR16ctrl
bcl2
β-actin
40
0
20A
pop
tosi
s %
3000
0
1500
Cas
pas
eac
tivi
ty
microarray qRT-PCR WB
Fassan M, Croce CM & Rugge M – WJG 2011
The clinician’s point of view
miRNAs as new therapeutic tools
MicroRNA Replacement Therapy for miR-145 and miR-33aIs Efficacious in a Model of Colon Carcinoma
polyethylenimine (PEI)-mediated delivery of unmodified miRNAs
miR-145 delivery reduced tumor proliferation and increased apoptosis, with concomitant repression of c-Myc and ERK5
Ibrahim AF et al – Cancer Res 2011
Beyond miRNAs…the UCRs(ultraconserved regions)
� noncoding RNA (ncRNA) transcripts (longer than 200 bp)
� a subset (n=481) of conserved sequences that are located in both intra- and intergenic regions
� conserved (100%) between orthologous regions of the human, rat, and mouse genomes
Fassan M & Rugge M – Unpublished data
2.0
0
BM
vs
N
uc.153 uc.472 uc.473
1.0
Human
2.0
0
1.0
BM
vs
N
uc.153 uc.472 uc.473
Mouse (model)2.0
0
BM
vs
N
uc.153 uc.472 uc.473
1.0
Rat (model)
Gastroduodenal anastomosis
CONCLUSIONS (1/2)
miRNAs expression patterns have a significant impact on human disease
miRNAs are dysregulated in GI diseases: from inflammation to cancer
miRNAs are fundamental in all the cellular processes
CONCLUSIONS (2/2)
miRNA expression profiling identifies cancer gene targets
Regulating miRNA expression might be a novel strategy for gastrointestinal tumors chemoprevention
miRNAs as novel diagnostic and prognostic tool
Laboratory of surgical pathology - Surgical pathology & cytopathology unit – University of Padua
EBRA registry (Barrett’s esophagus Veneto region registry; www.esofagodibarrett.org)
Ohio State University - Comprehensive Cancer Center
Istituto Oncologico del Veneto - Surgery & endoscopy units
Funding agencies
…a particular thank to…
Laboratory of molecular pathology - Surgical pathology & cytopathology unit - University of Padua