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