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UNIVERSITY CAMPUS BIO-MEDICO, ROME Bruno Vincenzi MD, PhD Medical Oncology University Campus Bio-Medico, Rome-Italy ROLE OF DICER.

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Presentation on theme: "UNIVERSITY CAMPUS BIO-MEDICO, ROME Bruno Vincenzi MD, PhD Medical Oncology University Campus Bio-Medico, Rome-Italy ROLE OF DICER."— Presentation transcript:

1 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

2 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

3 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

4 DROSHA: structure and function RNase III family, class II Tandem RNase III domain: dsRNA cleavage (endonuclease activity)

5 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)

6 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

7 microRNAs and angiogenesis miR-221/miR-222: anti-angiogenic let-7f/miR-27b: pro-angiogenic

8 microRNAs and cancer

9 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

10 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

11 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

12 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

13 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!

14

15 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

16 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.

17 DICER and DROSHA: ovarian cancer High Dicer expression and high Drosha expression are associated with increased median survival

18 ROLE OF DICER AND DROSHA AS PREDICTORS OF BEVACIVUMAB-BASED ANTICANCER TREATMENT EFFICACY IN ADVANCED COLORECTAL CANCER PATIENTS

19 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

20 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

21 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

22 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 RNA Isolation mRNA/miRNA Detection RNA Analysis

23 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).

24 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-specific primer (miScript Primer Assay); - mRNA detection: Gene-specific primer pairs will be used. mRNA/miRNA Detection Patients and methods Each sample was analysed in triplicate.

25 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.

26 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.

27 Thank you for the attention!


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