microRNA and lung cancer

Gabriella Sozzi

Lung Cancer Screening by LDCT : critical issues

Recent clinical trials results (DANTE, 2009, DLCST , 2012, MILD 2012; ~10.000 subjects overall; NLST 2011,53.454 persons), indicate that LDCT screening in high risk subjects may reduce lung cancer mortality.

The high false positive rates of LDCT, leading to multiple screening rounds, over-diagnosis, unnecessary (harmful?)diagnostic follow –up and costs underscore the need for non-invasive complementary biomarkers for standardized use.

Host

Tumor microenvironment

Cancer cells

Peripheral circulation

Biomarkersof risk

Biomarkers of risk of

aggressive disease

Diagnosticbiomarkers

Prognostic biomarkers

Sources of blood-based biomarkers. Novel promising biomarkers are generated by cancer cells, tumor

microenvironment, the host response and their dynamic interaction.

Microsatellite changes, KRAS and p53 mutation in case-control series (Sozzi G. Cancer Research 1999; Andriani F. IJC 2004)

Methylation markers plasma samples in case-control series (Bearzatto A. Clin Cancer Res. 2002 )

Circulating plasma DNA (hTERT) quantification in case-control series and in pilot INT-IEO and MILD screening trial (Sozzi G., Can Res 2001; Sozzi G., J Clin Oncol. 2003; Sozzi G., AJRCCM 2009; Roz L. Lung Cancer 2009)

Highthroughput microRNAs expression profiles in plasma samples in independent screening trials (INT-IEO, MILD) (Boeri M. et al., PNAS 2011; Sozzi G. Cell Cycle 2011; Boeri M. et al., Cancer J 2012)

Plasma biomarkers studies at INT (Milan)

mRNA

One miRNA

mRNA

mRNA

mRNA

mRNA

…

small noncoding RNAs that regulate gene expression by binding complementary sequences of target mRNAs and inducing their degradation or translational repression

Evolutionary conserved

One miRNA has multiple targets

microRNA: a new class of biomarkers

     

microRNA : plasma/serum-based biomarkers for cancer detection?

• Blood-based miRNA studies are in their infancy

• miRNA remain rather intact and stable in plasma/serum

• Simple universally applicable assay for quantification (i.e.

qRT-PCR)

In lung cancer plasma/serum levels of miRNAs might have

diagnostic (Silva J, ERJ. 2010; Shen J, Lab Invest. 2010; Foss KM, J TO 2011; Hennessey PT,

PLoS One 2012) and prognostic value (Hu et al.,

JCO, 2010).

miRNAs have been found packaged in exosomes derived from multivesicular bodies

(7) or be exported in the presence of RNA-binding proteins (i.e. Ago-2)(8) or might be

exported microvesicles shed during membrane blebbing (9). Once in the

extracellular space, these miRNAs could be taken up by other cells, degraded by RNases,

or excreted(10).

microRNA in LUNG TISSUE SAMPLES

TUMOUR TISSUES

mir-128amir-129mir-369mir-193mir-339mir-185mir-346mir-340mir-133b

NORMAL TISSUES

miR-126*mir-126let-7cmir-222mir-30emir-16-2mir-29b-1mir-030dmir-15alet-7a-2

microRNA in PLASMA SAMPLES

1196ALIVE

40 samples (19 patients)

DEAD 824

At disease1y before2y beforeenrollment

TAC- TAC- TAC+

CONTROLS

5 POOLS

(28 individuals)

CONTROLS

5 POOLS

(28 individuals)

Training Set (INT- IEO trial)

CONTROLS

10 POOLS

(54 individuals)

CONTROLS

10 POOLS

(54 individuals)1377ALIVE

34 samples (22 patients)

DEAD 322

At disease1y before2y beforeenrollment

TAC- TAC- TAC+

Validation Set (MILD trial)

ANALYSIS ON PLASMA SAMPLES

NORMALIZATION ISSUE

TaqMan® Array Human MicroRNA Cards Megaplex™ :378 microRNAs per sample

NORMALIZATION ON THE MEAN OF EACH CARD

TRAINING SET

TECHNICAL VALIDATION (training set)

& VALIDATION SET

TaqMan® MicroRNA Assays Multiplex™ Only on miRNA

of interest

TO BYPASS NORMALIZATION ISSUE (no plasma housekeeping miRNA):

analysis of miRNAs RATIOSRECIPROCAL RATIOS AMONG miRNAs

Risk

10 Diagnosis

94

11

RiskAggressive

disease (RAD)

DiagnosisAggressive

disease (PAD)

22

66

miRNA and miRNA ratios distribution and discrimination power in the analyses

miRNA RATIOS

miRNA

Risk

Diagnosis

RiskAggressive

disease (RAD)Diagnosis

Aggressive disease (PAD)

32

2

2

1

1

1

12

1 3

2

ANALYSIS OF miRNA RATIOS in plasma

Technical ValidationMultiplex™

Validation SetMultiplex™

Filter for Ratios with a minimal intra-pool

variability

Training SetTaqMan® Array Human

MicroRNA CardsMegaplex™

24 microRNAs

RISK OF DISEASE (1-2 yrs before disease)

RISK OF AGGRESSIVE DISEASE (1-2 yrs before disease)

DIAGNOSIS(at disease)

DIAGNOSIS OF AGGRESSIVE DISEASE (OUTCOME)

ANALYSIS OF miRNA RATIOS

AUC=0.85 p<0.0001

RISK OF LUNG CANCER 1-2 YEARS BEFORE CT

RISK OF AGGRESSIVE LUNG CANCER (RAD)

Time (months)

p=0.0006

Sample size 25

Patients 15

Controls 10

Sample size 37

RAD+ 17

RAD- 20

DIAGNOSIS OF LUNG CANCER

AUC=0.88 p<0.0001Sample size 26

Patients 16

Controls 10

p=0.0001

Time (months)

Sample size 31

PAD+ 13

PAD- 18

DIAGNOSIS OF AGGRESSIVE LUNG CANCER (PAD)

CONTROLS

100 disease free individuals

CONTROLS

100 disease free individuals

40 19 4Alive

51 PATIENTS

Dead 871

At disease1-2y before>2y beforeenrollment

TAC- TAC- TAC+

24 microRNA signatures: extended Validation Set (MILD trial) using custom-made microfluidic card

mir-16, mir-17, mir-21, mir-101, mir-126, mir-145, mir-197, mir-221, mir-320, mir-451, mir-660, mir-106a, mir-133a, mir-140-3p, mir-140-5p, mir-142-3p, mir-148a, mir-15b, mir-19b, mir-28-3p, mir-30b, mir-30c, mir-486-5p and mir-92a.

AUC = 0.89

RISK

AUC = 0.97

RISK of AGGRESSIVE

DISEASE

AUC = 0.92

AUC = 0.93

PRESENCE of AGGRESSIVE

DISEASE

PRESENCE of DISEASE

Performance of the miRNA signatures in plasma samples collected pre-disease, at the time of disease detection, and

after surgery (disease-free) from 19 patients.

Pre At Post

High

Low

No

1000 controls (no disease)

130 samples (13%): haemolyzed

872 controls suitable for analyses:

594 LDCT arm; 286 observational arm

85 lung cancer

patients

9 patients (11%) : samples not

available

7 patients (9%): haemolyzed

samples

69 Lung Cancer patients: 44 at cancer diagnosis, 54 before diagnosis, 19 post-

surgery

Study Design &

Aims

Diagnostic performance of miRNA test for lung cancer detection across LDCT and observational arms

Combination of LDCT and plasma miRNA test

Prognostic value of the miRNA assay

bioMILD prospective trial

4,000 smokers ≥ 50 anni Grants from AIRC and MoH

bioMILD - 2011biomarker-based prospective study

validate miRNA profile

combination of CT e miRNA

CT dose according to risk

Biology-based therapy

Pharmacologic prevention

ci servono 4000 volontari

www.biomild.org

TUMOR GENOMICS UNIT

Mattia Boeri

Carla Verri

Davide Conte

Mavis Mensah

Luca Roz

Federica Facchinetti

Francesca Andriani

Ohio State University Cancer Center, Columbus, OH, USA

Carlo M. Croce

UNIT of THORACIC SURGERY

Paola Suatoni

Ugo Pastorino

Special Program “Innovative Tools for Cancer Risk Assessment and early Diagnosis”, 5X1000/5 per mille Supported by Italian Ministry of

Heatlh Ricerca Finalizzata 20120