university campus bio-medico, rome bruno vincenzi md, phd medical oncology university campus...
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UNIVERSITY CAMPUS BIO-MEDICO, ROME
Bruno Vincenzi MD, PhD
Medical Oncology
University Campus Bio-Medico, Rome-Italy
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!