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IRCCS Istituto Tumori Giovanni Paolo II, Bari, Italy
S. TommasiS. Tommasi
Omica e nanotecnologie negli ESSERI VIVENTI Il Progetto ONEV
Biomarkers for therapeutic Biomarkers for therapeutic response in metastatic melanomaresponse in metastatic melanoma
Bari, 1 Dicembre 2014
APPROVED AGENTS USED FOR THE TREATMENT OF METASTATIC MELANOMAAPPROVED AGENTS USED FOR THE TREATMENT OF METASTATIC MELANOMA
Treatment type Approved drugs Mechanism of action Mechanism of resistance
Chemotherapy DTIC, TMZ DNA methylation High expression MGMTMMR deficiencyBER deficiency
Targeted therapy
VemurafenibDabrafenib
Inhibition of BRAFV600 mutant
MAPK pathway reactivationPI3K/AKT/mTOR reactivation
Trametinib Blockade of MEK in presence of BRAFV600 mut
MAPK pathway reactivation
Immunotherapy IL2 Stimulation of T and NK cell-mediated acitivities
Tumor escape from immune surveillance mechanisms
IpilimumabTremelimumab
CTLA4 blockade with enhacement of effector T cell function and inhibition of Treg activity
Low melanoma antigen expression
PembrolizumabNivolumab
Binding to PD-1 resulting in enhancement of the immune response against tumor
-
Liu L , and Gerson S L Clin Cancer Res 2006;12:328-331
Base excision repair (BER) key factors: Base excision repair (BER) key factors: predictive biomarkers - new promising targets for therapiespredictive biomarkers - new promising targets for therapies
in detail
Tuominen et al. 2014
PROGNOSTIC AND PREDICTIVE ROLE OF MGMT METHYLATIONPROGNOSTIC AND PREDICTIVE ROLE OF MGMT METHYLATION
Liu L , and Gerson S L Clin Cancer Res 2006;12:328-331
Base excision repair (BER) key factors: new promising targets for therapiesBase excision repair (BER) key factors: new promising targets for therapies
in detail
Prognostic role in pts BRAF V600 untreated by VemurafenibPrognostic role in pts BRAF V600 untreated by Vemurafenib
Abbotts R – Madhusudan 2014
APE1 inhibition induces a synthetic lethal response in PTEN-deficient cells by causing accumulation of abasic sites and subsequent strand breaks, and ultimately the induction of apoptosis
BRAFBRAF
NRAS & MEK mutations
Stromal secretion of HGF-like
Disrupted feedback regulation
Alternative splicing of BRAF mRNA↓ BIM
expression via PTEN loss
COT or EGFR activation
NF1 lossBRAF amplification
CAUSES of RAF kinase INHIBITORS RESITANCECAUSES of RAF kinase INHIBITORS RESITANCE
PI3K pathway:PIK3CA, PTEN, PIK3R1
HOXD8
RAC1
MAPK pathway
Downstreem effectors:NRAS, BRAF, MAP2K1, MAP2K2, MITF, NF1
44%
Van Allen EM 2014
NRAS as predictive biomarkerNRAS as predictive biomarker
IL2
Ipilimumab
MEK i
PI3K i
Next Generation Sequencing and Custom Ampliseq Panels: a new opportunity to predict response to chemotherapy,
targeted therapy, immunotherapy……….
SENSITIVITY TO ……….
Project PON01_01297 “Virtualab”
Next Generation Sequencing and Custom Ampliseq Panels: Next Generation Sequencing and Custom Ampliseq Panels: a new opportunity to predict response to targeted therapya new opportunity to predict response to targeted therapy
The aim was to identify mutations able to predict therapy response in a quick, accurate and cost effective method. The panel was developed to analyze the coding region of 11 genes with a coverage of 93.85% . The melanoma custom panel size was 39.08Kb, contains 303 amplicons and for the analysis is required an input of 20ng of FFPE DNA (2 pools).
Pinto confidential 2014
Patients in which at least one gene results mutatedPatients in which at least one gene results mutated
Pinto R confidential 2014
ION PGM Sanger Method ARMS Method
BRAFV600 mut n.11 1 false negative
BRAF V600 wt n.6 2 false positive
NRASGlu61 mut n.2 1 false negative
ION PGM, ARMS AND SANGER METHODSION PGM, ARMS AND SANGER METHODS
Pinto R confidential 2014
USING cfDNA ADVANTAGESUSING cfDNA ADVANTAGES
Analysis in the lack of tumor material in patients whose tumors are difficult to biopsy
The small amount of archival tumor tissue mixed with normal stroma tissue Degraded DNA by formalin fixation – cfDNA presented less non specific
amplifications Potentially mutant clones could be missed in biopsy from small part of tumor Shorten turnaround timefor testing
PRE-ANALYTICAL AND ANALYTICAL PHASES
SERUM PLASMASensitivity Specificity Sensitivity Specificity
44% 96% 52% 96%95%CI 35%-
53%95%CI 90%-
99%95%CI 43%-
61%95%CI 90%-
99%
Aung 2014
More BRAF mutations were detected in plasma than in serum although differences in assay sensitivity and specificity are not statistical differentPlasma contains less wt DNA than serum but higher mutation fraction
Discordance between tumor and cfDNA could depend:on time of samplingArchival tumor biopsy did not fully represent the tumor heterogeneityDifferent mutation status in primary and metastatic sitesSensitivity of assays: ARMS 2%
CONCORDANCE
SERUM PLASMA
64% 70%
Ascierto JCO 2013Biomarker analysis in Phase II Dabrafenib studyBiomarker analysis in Phase II Dabrafenib study
CORRELATION tumor tissue - cfDNA
SPECIFICITY SENSITIVITY
V600E 84% 100% 79%
V600K 97% 99% 89%
Significant association between baseline cfDNA mut fraction and OS
(HR:0,83; 95%CI, 0,72-0,96)
P=0.0134P=0.0006
Significant association between baseline cfDNA mut fraction and PFS
(HR:1,09; n=46)
39 patients - serumcharge range1,5 – 50 D IMAC 30
19 BRAF V600E/K Vemurafenib
17 BRAF wt Temozolomide/FM3 BRAF V600E
STUDY DESIGNAIM: to individuate novel candidate peptides useful as biomarkers to predict the
response or resistance to treatment in two sets of patients treated with TMZ/FM and vemurafenib
Garrisi, Strippoli Plos One 2014
M/Z P RegulationROC
Area
Intensity
BRAF mut
Intensity
BRAF wt
9446 0,0148 down 0,715 6,182 8,43
9295 0,0217 up 0,715 16,709 8,296
1883 0,023 up 0,296 14,284 7,596
9176 0,025 down 0,704 6,9 8,22
4652 0,027 down 0,696 7,4 11,351
BRAF mut vs BRAF wt
Acrostic name Description
Sequence
coverage
(%)
Score
SLAIN1 SLAIN motif-containing protein 1 20 38
GLIS2 Zinc finger protein 21 36
ABCC12 Multidrug resistance-associated protein 9 6 27
ATF6Cyclic AMP-dependent transcription
factor12 18
Invasiveness
Resistance
Garrisi, Strippoli Plos One 2014
Overexpressed
Basal Level
P=0.0011PFS
M/Z p-valueRegulation in
Shorter Resp
5900 0.019 up
12544 0.033 up
49124 0.048 up
11724 0.022 up
VEMURAFENIB RESPONDERS VS NON RESPONDERS
Acrostic
nameDescription
Sequence
coverage (%)Score
KLF17 Krueppel-like factor 31 40
RBM10 RNA-binding protein 12 26
TOX3 TOX high mobility group box
family member 320 22
Regulatory activity of gene transcription
RNA alternative splicing
Garrisi, Strippoli Plos One 2014
M/Z p-valueRegulation in
Resp
6411 0.022 up
4075 0.020 up
Overexpressed
Basal level
P=0.0024PFS
BRAF wild-type patients
Acrostic name Description
Sequence coverage
(%)Score
NQO1 NAD(P)H dehydrogenase [quinone] 1 21 28
COMD5COMM domain-containing protein - Hypertension-
Related Calcium-Regulated Gene Protein25 28
CA4 Carbonic anhydrase 4 18 26
VATL V-type proton ATPase 16 kDa proteolipid subunit 41 26
TM50A Transmembrane protein 50A 37 26
TMZ / FM RESPONDERS VS NON RESPONDERS
Pathway of detoxification
Cell acidification
Garrisi, Strippoli Plos One 2014
miRNAsmiRNAs
NEW PREDICTIVE BIOMARKERS?NEW PREDICTIVE BIOMARKERS?
Greemberg 2014
miRNA expression in Metastatic Melanoma
* Our cohort included 43 patients (treatment naïve and with histologically confirmed stage IV of metastatic melanoma), 30 cases were BRAF mutated at the codon 600, while 13 were wild type; * We have selected 15 miRNAs that scientific reports and informatics tools have established to target the crucial genes involved in melanoma biology.; * We analyzed miRNAs expression and the expression of the correspondent target genes by TaqMan probes; * We correlated miRNAs and gene expression data to time to progression of 20 patients treated with Vemurafenib.
Pinto R confidential 2014
Tumor miRNA biogenesis
miRNA release
Microvescicles
Proteic complexes
Apoptotic bodies
Passive delivery into bodily fluidsmiRNA extraction
(blood)
Quantification
Clinical biomarkers
qRT-PCR
Clinical decision
Prognosis
Tailored therapy
EXPERIMENTAL WORKFLOW
Non-invasive biomarkers: circulating miRNASNon-invasive biomarkers: circulating miRNAS
BLOOD BASED miRNA STUDY AND ADVANTAGESBLOOD BASED miRNA STUDY AND ADVANTAGES
Non invasive technique Possibility to multiple time point sampling
Tissue specific dysregulationStability to RNAase digestion, harsh conditions. Extended storage and multiple freeze-thaw cycles.
DNA repair and damage prevention
Cellular senescence
TP53
Circulating mir125b expression in response to TMZ/FE treatment: preliminary resultsCirculating mir125b expression in response to TMZ/FE treatment: preliminary results
De Summa confidential 2014
CONCLUSIONS
Only integrated studies on genetics and epigenetics are needed to evidence a complete pattern of pharmacological
sensitivity
Deregulated miRNAs and their respective targets may serve as molecular targets/tools for future platform
of molecular directed therapy or as predictive biomarkers in melanoma patients.
Pinto R.De Summa S.Garrisi V.M.
Guida M.Strippoli S.
Guida GMaida I.Cocco T.Grieco C.
Azzariti A.Porcelli L.Quatrale A.E.
Molecular Biology LaboratoryMedical Oncology Unit
In vitro Pharmacology Laboratory
Anatomopathology Unit
Simone G.Popescu O.
Biology & Biochemistry Lab
Univ Bari
THANKS to:THANKS to:
PON01_01297 VirtualabPONa_00034 ONEV