UNIVERSITY CAMPUS BIO-MEDICO, ROME

Bruno Vincenzi MD, PhD

Medical Oncology

University Campus Bio-Medico, Rome-Italy

b.vincenzi@unicampus.it

ROLE OF DICER AND DROSHA AS PREDICTORS OF BEVACIVUMAB-BASED ANTICANCER TREATMENT

EFFICACY IN ADVANCED COLORECTAL CANCER PATIENTS

RNA interference: a breakthrough in modern biology

RNAi

Protects against viral infections

Secures genome stability

by keeping mobile elements silent

Repress protein synthesis and regulate the

development of organisms

Offers a new experimental tool to repress genes

specifically

Might be a useful approach

in future gene therapy

RNA interference: the role of DICER and DROSHA

Exogenous dsRNA

miRNA (19-21 nucletides)

Long-precursor microRNA

70 nucletides long fragments

Small interfering RNA

RNA induced silencing complex

DROSHA: structure and function

RNase III family, class II

Tandem RNase III domain: dsRNA cleavage

(endonuclease activity)

DICER: structure and function

RNase III family, class III

PAZ: dsRNA binding module

(3’ overhang)

Platform: guide dsRNA

(positively charged)

Tandem RNase III domain: dsRNA cleavage

(endonuclease activity)

DICER and angiogenesis

Dicer WT embryos

Dicer deficient embryos

Dicer WT yolk sacs

Dicer deficient yolk sacs

Yang WJ et al, J Biol Chem 2005

microRNAs and angiogenesis

miR-221/miR-222:

anti-angiogenic

let-7f/miR-27b:

pro-angiogenic

microRNAs and cancer

DICER and lung cancer

DICER IIC expression in

normal lung

DICER IIC overexpression in

precancerous lesions of

adenocarcinoma

(atypical adenomatous hyperplasia, A, bronchioloalveolar carcinoma, B e C

adenocarcinoma, D)

Chiosea S et al, Cancer Res. 2007 Mar 1;67

DICER and DROSHA: lung cancer

Low DICER expression is associated with a shortened post-operative

survival in NSCLC patients

(p= 0.0001)

Drosha status did not show a significant relationship with NSCLC

patients survival

(p= 0.06)

Karube Y et al, Cancer Sci. 2005 Feb;96

DICER and breast cancer

Low-intensity staining

IIC analysis of Dicer protein expression in normal and breast cancer tissues

High-intensity staining

Grelier G et al, Br J Cancer. 2009 Aug 18;101

DICER and resected breast cancer

Low mRNA DICER expression

1. Lymph node metastases (p= 0.02)

2. Lower metastatic-free survival(p= 0.003)

Grelier G et al, Br J Cancer. 2009 Aug 18;101

DICER and DROSHA: resected breast cancer

Dedes Kj et al, Eur J Cancer. 2011 Jan;47

No correlation

between DICER

and DROSHA

mRNA levels and

outcome!

DICER and DROSHA: ovarian cancer

• 111 ovarian-cancer specimens

• 11 benign epithelial ovarian specimens

Quantitative RT-PCR

for mRNA and IHC analysis

Mutational analysis

Transfection with Small Interfering

and Short Hairpin RNA

DICER and DROSHA: ovarian cancer

Low Dicer mRNA levels are significantly associated with advanced tumor stage;

low Drosha mRNA levels are significantly associated with suboptimal cytoreduction.

DICER and DROSHA: ovarian cancer

High Dicer expression and high Drosha expression are associated with

increased median survival

ROLE OF DICER AND DROSHA AS PREDICTORS OF BEVACIVUMAB-BASED ANTICANCER TREATMENT

EFFICACY IN ADVANCED COLORECTAL CANCER PATIENTS

Patients accrual

Inclusion criteria:

• Histologically proven diagnosis of colorectal cancer

• Not resectable metastatic colorectal cancer not previously treated with chemotherapy for metastatic disease;

• At least one measurable lesion according to RECIST criteria;

• Age 18-75 years;

• ECOG PS < 2 if age < 70 years, ECOG PS = 0 if age = 71-75 years;

• Adequate bone-marrow, liver and kidney function;

• Life expectancy of at least 3 months;

• Bevacizumab-based first line anticancer treatment for advanced colorectal cancer

Exclusion criteria:

• Radiotherapy to any site within 4 weeks before the study;

• Brain metastases;

• History or evidence upon physical examination of CNS disease;

• Serious, non-healing wound, ulcer, or bone fracture;

• Evidence of bleeding diathesis or coagulopathy;

• Uncontrolled hypertension;

• Clinically significant cardiovascular disease;

• Current or recent (within 10 days prior to study treatment start) ongoing treatment with anticoagulants for therapeutic purposes;

• Treatment with any investigational drug within 30 days prior to enrolment;

• Treatment with other immunomoulating agents or previous immunotherapy

Patients and methods

Follow up data:

• Data on clinical outcome were obtained from

patients' records.

• Responses to initial chemotherapy were recorded

as either sensitive or resistant according to

standard RECIST criteria. Moreover, a particular

attention was referred to those patients treated with

local treatment (thermoablation,

chemoembolization, surgical resection of mets).

Patients samples:

• This study include formalin-fixed paraffin

embedded (FFPE) 10 µm sections (not fixed on

glass) of:

• tumor specimens from 183 CRC patients

• normal colorectal tissues from 50 patients

Study end points

Primary end points:

• Evaluation of a potential association between Dicer

and Drosha modulation in specimens of advanced

colorectal cancer from patients treated with

bevacizumab and time to progression

Secondary end points :

• Evaluation of a potential association between Dicer

and Drosha modulation in specimens of advanced

colorectal cancer from patients treated with

bevacizumab and

• response rate

• rate of surgical resection of metastases

• overall survival

miScript Syber Green PCR kit (Qiagen)

Quantitec primer Assays (Qiagen) miScript primer Assays (Qiagen)

mRNAs (GAPDH, Dicer, Drosha)

miRNAs (miR-RNU6B, miR221, miR222, let-

7, miR27-b)

Isolation of total RNA: RNeasy FFPE Kit (Qiagen)

miScript Reverse Trascripion Kit (Qiagen)

cDNA

Patients and methods

FFPE sections

RN

A

Isola

tion

mR

NA

/miR

NA

D

ete

ction

RN

A

An

aly

si

s

Patients and methods

• Paraffin will be dissolved from two FFPE sections and then removed exposing samples for subsequent treatment with xylene and ethanol.

• The tissue will be dried and resuspended in Digestion Buffer and Proteinase K (Qiagen) to allow the lysis of the sample. The tissue will be digested during an o/n incubation at 56°C.

• Total RNA (including small RNA) will be purified from digested tissues.

• RNA is treated with DNase Buffer and DNase to avoid gDNA contamination and particularly to remove even trace amounts of small DNA fragments which can impair downstream assays such as Real Time PCR.

• RNA is purified using an RNeasy MinElute spin column (Qiagen), where RNA is bound, washed, and finally eluted in RNase-free water.

RNA isolation

• The RNA concentration will be quantified using NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific Inc).

• Total RNA will be reverse transcribed using oligo-dT primers and random primers allowed retrotrascription of mRNA and miRNA simultaneously.

• cDNA will be used as template for SYBR® Green based real-time PCR analysis:

- miRNA detection: Universal Primer serves to bind the Universal Tag sequence in combination with a miRNA-specificprimer (miScript Primer Assay);

- mRNA detection: Gene-specific primer pairs will be used.

mRNA/miRNA Detection

Patients and methods

• Each sample was analysed in triplicate.

Data Analysis

Patients and methods

• The expression of each miRNA and mRNA, relative to RNU-6b and GAPDH respectively, will be determined using the 2−ΔCT method.

• The relative mRNA/miRNA expression was also normalized against control tissue using the comparative 2−ΔΔCT.

Statistical analysis

Sample size calculation:

• Alpha error: 0.05

• Power: 0.80

• Median TTP in the best prognosis arm: 12

• Median TTP in the worse prognosis group: 8

• Further FUP time: 12 months

• Radio between arms: 1

Calculated sample size: 183 patients

Results analysis:

• Treatment activity: tumour control rate (% of

patients who had a best-response rate of complete

response, partial response, or stable disease

maintained for at least 28 days); TTP (period from

the beginning of treatment to the date of the first

observation of disease progression or death from

any cause within 60 days after the start of

treatment or the most recent tumour assessment).

• Stratified permutation tests will be carried out to

explore the association between tumour

response and Dicer and Drosha modulation.

Moreover, the differences in terms of TTP will be

evaluated by the log-rank test. The Cox

proportional hazards model was applied to the

multivariate survival analysis.

• SPSS software (version 17.00, SPSS, Chicago)

was used for statistical analysis. A P value of less

than 0.05 was considered to indicate statistical

significance.

Thank you for the attention!