Novità nel campo della genetica Medica

Department of Oncology and Haematology Azienda Ospedaliera Policlinico di Modena.

Novità nel campo della Genetica Medica

Genetic Helps us to Identify Patients at High Risk of Developing Breast and Ovarian Cancer

Genetics

• Genetics is the study of heredity

While genetics influence genomics, genetics is responsible for only 5-10% of breast cancer

• Genetics focuses primarily on the likelihood of developing cancer

• Genetic tests find mutations, not disease

Source: Understanding Cancer Series: Gene Testing, National Cancer Institute

Key point: whole body has the mutation

Knudson A, PNAS 1971

Two Hits Hypothesis

Genetic Theory of Cancer

• Cancer is a genetic disease

• Most cancers have mutations in multiple genes

• The underlying defect in cancers is Genomic Instability

• Cancers require alterations in genes involved in

cellular proliferation, cell cycle, apoptosis, telomere maintenance and DNA repair

• Most inherited cancer syndromes are due to alterations in genes required for genomic stability.

Knudson A, PNAS 1971

As an autosomal dominant syndrome, a deleterious BRCA mutation can be transmitted through maternal or paternal lineages. As an incomplete

penetrance gene, interacts with environment to develop cancer

SYNDROME TUMORAL SPECTRUM TRANSMISSION GENES BREAST/OVARY Breast, ovary, uterus, prostate,

stomach, colo-rectum, pancreas, bilious tract, melanoma

Dominant autosomal BRCA 1 BRCA2 LI-FRAUMENI Soft tissues, breast, bone, leukemya,

brain, adrenal Dominant autosomal P53

COWDEN Breast, tyroid, endometrium (amartomes)

Dominant autosomal

PTEN Diffuse Gastric

Cancer Stomach, Lobular Breast Dominant autosomal CDH1 HNPCC

(Lynch Syndrome)

Colo-rectum, ovary, endometrium, bladder, urinary tract, pancreas,

stomach, bilious tract, breast

Dominant autosomal MLH1 MSH2 MSH6

PEUTZ-JEGHERS Colo-rectum, stomach, ovary, testis, cervix, pancreas, breast

Dominant autosomal

LKB1

72%

69%

Tumor sites in families with TP53 germline mutations

% of all tumors

0 5 10 15 20 25 30

Breast Brain GI Hematol Other

Bone Soft Tissue Gynecologic

Adrenal

.

Cowden Syndrome Cancer Risks

Tumor Site Risk

Pilarski R.

JGC.2009;18:13-27

Risk

Tan et al. Clin Can Res.

2012;18(2):400-7

Breast 25-50% 85%

Thyroid 3-10% 35%

Endometrial 5-10% 28%

Renal Cell Unknown 34%

Melanoma Unknown 6%

Colon Unknown 9%

Cancer Type Age Range Cancer Risk Risk for General Population

Gastric (male) To age 80 67-70% 0.6%

Gastric (female) To age 80 56-83% 0.6%

Female Breast To age 50 10% 1.9%

To age 80 39-52% 10.2%

Colorectal To age 80 Possible

Increased Risk

3.0%

Hereditary Diffuse Gastric Cancer

0 20 40 60 80 100

0 20 40 60 80

Colorectal Endometrial Stomach Biliary tract Urinary tract Ovarian

78 43 19 18 10 9

%

Age (years)

Peutz-Jeghers Syndrome



Pancreatic Cancer



Liver



Lung



Breast



Ovary



Uterine Cancer



Testis



Others

Website for BRCA classification

◦ BRCA exchange

◦ ClinVar

◦ BIC

◦ LOVD

◦ HCI

◦ A-GVGD

Website for functional analysis of BRCA

• BRCA Circos

• LOVD BRCA1

• LOVD BRCA2

Cortesi et al., Breast Can Res Treat 2012

BRCA1 p.His1673del is a pathogenic mutation associated with a predominant ovarian cancer

phenotype

Zuntini et al., Oncotarget 2017

Prevention of tumorigenesis

DNA repair Protein

Ubiquitination

Regulation of transcription

Regulation of cell cycle

Repair of double strand DNA

breaks / DNA adducts

Mediates E2- dependent ubiquitination

Modulates gene

expression in response to cell stress

P21 DNA damage

Induces cell cycle arrest at the G1/S, S and G2/M

checkpoints

Cellular function regulated by BrCa1

BRCA1 tumor suppressor protein

BRCA2

RAD51

DNA damaging RAD51 forms nucleoprotein filaments

RAD51 RAD51

RAD51

RAD51 RAD51

BRCA1

BRCA1 is a DNA damaging “detector”, blocks the transcription by cell cycle arrest at the G1/S, S and G2/M checkpoints and by ubiquitination of FANCD1

otherwise induces the DSB repair by chromatin remodelling

Chk2 ATR ATM

Toss & Cortesi, 2013 A

L

F

C E G

D1 Ub

B

M

I

Methods

Presented By Arielle Heeke at 2017 ASCO Annual Meeting

Results, Total HRD mutation frequency by lineage

Presented By Arielle Heeke at 2017 ASCO Annual Meeting

Results, HRD mutation landscape by lineage

Presented By Arielle Heeke at 2017 ASCO Annual Meeting

Niraparib Plus Carboplatin in Patients with Homologous Recombination Deficient Advanced Solid Tumor Malignancies

Presented By Arielle Heeke at 2017 ASCO Annual Meeting

Multi-Gene (NGS) Panels

• Genetic tests to look at dozens of genes related to cancer

• Similar cost and turn around time as gene specific testing

• Higher risk of uncertain results

ACCE FRAMEWORK

Parameter Definition

Analytic Validity

How well test measures property or chracteristics it is intended to measure Clinical

Validity

Accuracy of the test in diagnosing or

predicting risk for health condition (sensitivity, specificity, PPV, NPV)

ELSI Ethical, Legal and Social Implication

ACTIONABILITY

Summary of Clinical Validity

GENE BREAST OVARY OTHER

ATM Y N ?Pancreas

CHEK2 Y N ?Colon

PALB2 Y N

?Pancreas

NBN Y (657del5) N

NF1 Y N

BRIP1 N Y

RAD51C/D N Y

BARD1 N Y

Normal ATM protein Function

• A Serine-Protein Kinase as p53 or BRCA1

• Senses Double Stranded Breaks in DNA

• Activates cell cycle checkpoints

• Mutant ATM causes A-T, a genomic instability syndrome, which is lethal by age 20.

• Mutant ATM heterozygotes have increased risk of

breast, leukemia, lymphomas and stomach cancer.

Gene Variant Tumor type

CHEK2 pI157T Br, CCR,

Pr, Kidney, Bladder CHEK2 c1000delC Br, CCR, Pr

CHEK2 pS428F Br

CHEK2 Cdel5395 Br, Pr

CHEK2 c.IVS2 + 1G > A Br, Pr, Tyr

C

28 30 21 11

Br: 49 ER+ PgR+

50

Br: 45 Br: 50 Br: 50

D: 51

Br: 50 72

Br: 55 D: 55

PALB2 in BRCA1&2 negative pts

Cancer

Type Age Range Cancer

Risk Risk for General

Population

Breast To age 80 Up to 30% 10.2%

Prostate To age 80 Increased

Risk 10.9%

NBN Gene Mutation

46

NF1 Gene

 NF1 is a tumor suppressor gene

 NF1 is located on Chromosome 17 long arm

- NF2 on other hand is located on Chromosome 22

 NF1 gene encodes the protein

Neurofibromin

Relative Risk of cancers in NF1: UK

Overall risk of cancer was 2.7 times higher than in the general population

 Connective tissue SIR=122 (95% CI 7.8-24%)

 Brain SIR=22.6 (95% CI 3.9-16%)

 Breast SIR=1.87 (95% CI 0.61-4.37%)

Walker et al., 2006, Br J Can

The transfer of multigene panel testing for hereditary breast and ovarian cancer to

healthcare: What are the implications for the management of patients and families?

Eliade M et al., Oncotarget 2017

ASCO supports the

communication to patients of medically relevant incidental germline findings conducted in the clinical setting.

Oncology providers should communicate the potential for incidental germline

information to patients and should review the potential benefits, limitations, and risks before testing.

ASCO Policy Statement

NGS offers promise, but

poses significant challenges for oncologists who are ill

prepared to handle incidental findings that have clinical

implications for at-risk family members. This report

underscores the need for oncologists to develop a

framework for pre- and post- communication of risks to patients undergoing routine multi-gene panels testing

Ready for Surprises?

• All BRCA1 and BRCA2 pathogenic variants regardless of tumor type (NCCN guideline)

• Founder mutations (ie. MSH2 exon 1-6 deletion, TP53 R337H)

• Uncommonly mutated genes (ie. CHEK2, PALB2)

Germline Finding Report

N° of UV in different genes for patients

Kurian AW et al., JCO 2014

N° of UV per gene across 198 patients

Kurian AW et al., JCO 2014

Matsuzawa et al., Mol Cell 2014

BRCA1

BARD1

OLA1 ?

BRCA1

BARD1

OLA1 E168Q

?

OLA1

mutation BRCA1 mutation

BRCA1 I62V

BARD1

OLA1

?

BRCA1

BARD1 V695L

OLA1 ?

BARD1 mutation

Modified by Chiba N

H.Chen et al., Scient Rep 2015

Genome-wide association analysis of more than 120,000

individuals identifies 15 new susceptibility loci for breast cancer

Michailidou, Nat Gen 2015

CONCLUSIONS

• Oncogenetic counselling as prediction of treatment response and mutation carrier

• HRD analysis by NGS for PARP-inhibitor using

• Role for MGPs in individuals who are negative for hereditary syndrome but with suggestion for

susceptibility

• Refer results only for «actionable» genes

• A professional genetic expertise needs for MGPs

• GWA will be able to identify new susceptibility loci

for BC

BRCA1 Germline

8%

BRCA2 Germline

6%

BRCA1 Somatic

3% BRCA2 Somatic

3%

BRCA1 Methylation

11%

EMSY Amplification PTEN Loss 6%

Other HRD 5%

7%

CCNE1 Amplification

15%

MMR Germline

2%

Other 34%

HR deficiency No HR deficiency

Levine, D. The Cancer Genome Atlas, Molecular profiling of serous ovarian cancer. 2011

BRCA Mutation 31% (14%germline)

Others HR deficiency

18%

Gabai-Kapara E et al. ( 2014)

45 74 84 3% 50% 55%

45 74 84 1% 17% 65%

BRCA2

RAD51

DNA damaging RAD51 forms nucleoprotein filaments

RAD51 RAD51

RAD51

RAD51 RAD51

BRCA1

BRCA1 is a DNA damaging “detector”, blocks the transcription by cell cycle arrest at the G1/S, S and G2/M checkpoints and by ubiquitination of FANCD2

otherwise induces the DSB repair by chromatin remodelling

Chk2 ATR ATM

Toss & Cortesi, 2013

A L

F

C E G

D2 Ub

B

M

I

Santos-Pereira J. et al. Nat. Publ. Gr. (2015)

PARP

Inhibition of PARP-1

prevents recruitment of repair

factors to repair SSB

XRCC1

LigIII PNK 1

pol β

Replication (S-phase)

DNA DSB

DNA SSB

REPAIR

CELL SURVIVAL

CELL DEATH

BRCA deficient PARP

inhibitors

PARP inhibitors

Toss & Cortesi, 2013

Chemosensitivity/resistance

Tassone P. et al.; 2003:88; 1285-1291

N N O

N O

N O

F

IC50 on PARP-1 = 4.9 nM IC50 on PARP-2 ≈ 5nM IC50 on PARP-3 ≈ 50nM IC50 on Tankyrase >1M

• Olaparib (AZD2281; KU-0059436)

• Favorable PK

• Good bioavailability across species

• Tumor PK -Significant levels at 24 hrs following single oral dose

Menear et al. J Med Chem 2008

Olaparib: an Oral Inhibitor of

Poly (ADP-ribose) Polymerase (PARP)

BRCA² +/- BRCA2 -/- Wild type

Log

surviving fraction

- 4 0 - 3 - 2 - 1 0

PARP inhibitor concentration (M)

10^-9 10^-8 10^-7 10^-6 10^-5 10^-4

Increased levels of chromosomal aberrations in PARP inhibitor

treated BRCA2 ^-/- cells

WT BRCA^-/- 0

1 2 3 4

BRCA2^-/- + PARPi WT

+ PARPi

Mean number of chromatid aberrations per cell

Chromatid breaks Complex aberrations

Farmer et al. Nature 2005; 434:917-21

BRCA 1 & 2

ES Cells are Very Sensitive to

PARP Inhibition

Sono eleggibili al test BRCA tutte le pazienti con diagnosi di carcinoma epiteliale ovarico non mucinoso e non borderline, di carcinoma delle tube di Falloppio e di carcinoma peritoneale primitivo per completare la fase diagnostica molecolare in previsione di un eventuale utilizzo terapeutico e per favorire l’accesso ad una consulenza pre-test nell’ambito dei percorsi di prevenzione.

…Si sottolinea la necessità di definire percorsi aziendali in cui vengano indicate in modo chiaro per le pazienti ed i loro familiari, le funzioni e le responsabilità dell’equipe oncologica, del laboratorio e dell’equipe di genetica clinica oncologica nelle varie fasi del percorso individuato….

Breast Ovarian Cancer (BOC): Pazienti affette da BC e OC

Hereditary Ovarian Cancer (HOC): 2 o più pazienti affette da OC, con parentela di I grado e su 2 diverse generazioni

Sospetto ereditario per carcinoma mammario e/od ovarico (SHBOC):

3 o più pazienti affetti da BC e OC con parentela di I grado senza giovane età o bilateralità, oppure senza parentela di I grado e con giovane età o bilateralità

Hereditary Breast/Ovarian Cancer (HBC/HBOC): 3 o più pazienti affetti da BC (e OC), di cui uno con BC entro i 40 anni o bilaterale e parentela di I grado tra i 3 individui

Early Onset Breast Cancer (EOBC):

Pazienti affette da BC entro i 35 anni di età

Male Breast Cancer (MBC): Paziente affetto da BC maschile

Familiare per carcinoma mammario e/od ovarico (FBOC): 3 pazienti affetti da BC ed OC senza essere HBOC o SHBOC

Fortemente sospette per familiarità (SFBC+/SFBOC+/SFBOC): 2 parenti di I grado di cui 1 con età ≤ 40 anni o bilaterale oppure 1 paziente affetta da BC ≤ 40 anni o bilaterale e 1 da OC con familiarità di I

grado oppure 1 BC e 1 OC con parentela di I grado Triple Negative Breast Cancer (TNBC) < 60 anni Carcinoma Ovarico Non Mucinoso e Non Border-line

BRCAPRO o Tyrer-Cuzick > 40% in donna sana

CRITERI PER ACCEDERE AL TEST

Il test BRCA deve essere richiesto per la ricerca di varianti patogenetiche costituzionali e va valutata l’eventuale fattibilità del test BRCA somatico. Per un’adeguata esecuzione del test è necessaria per i laboratori una comprovata validazione ed un controllo di qualità esterno del test proposto

Mafficini A et al,. Oncotarget. 2016 Jan 12;7(2):1076-83

17%

6%

77%

Germline Mutation ( 9 )

Somatic mutation (3)

NON PATHOGENIC (40)

PROPORTION OF PATHOGENIC

VARIANTS, NON-PATHOGENIC VARIANTS IN TESTING SEROUS HIGH GRADE OC

BRCA1/2

Stili di vita e prevenzione

dei tumori

Biosintesi degli Estrogeni

20,22-Lyase

11b-Hydroxylase

18-Hydroxylase

17,20 Lyase

Pharmacological Target Cholesterol

Pregnenolone

Progesterone

11-Deoxycorticosterone

Corticosterone

17a-Hydroxylase

21a-Hydroxylase

11-Deoxycortisol

Testosterone Dehydroepiandrosterone

Androstenedione

Cortisol

Aldosterone

AROMATASI

Oestrone Oestradiol

(intermediate)

(intermediate) 17a-

Hydroxypregnenolone

17a-

Hydroxyprogesterone

Attività dell’ enzima aromatasi

ANDROGENS OESTROGENS

P-450 Aromatase

+ NADPH-cytochrome P-450 reductase

(Testosterone, androstenedione, 16-OH-testosterone)

(Oestradiol, oestrone)

tumour

growth

Breast Cancer Tumorigenesis

Situazione nutrizionale – pool di Asl 2009-12 (n=149.823)

Popolazione in eccesso ponderale

Sovrappeso* 31,4 %

Obeso** 10,5 %

Consigliato di perdere peso da un medico o operatore sanitario***

Sovrappeso 43,6%

Obesi 77,8 %

*sovrappeso = indice di massa corporea (Imc) compreso tra 25 e 29,9

**obeso = indice di massa corporea (Imc) ≥30

***tra coloro che sono stati dal medico negli ultimi 12 mesi

Attività fisica – pool di Asl 2009-2012 (n=147.020)

* lavoro pesante oppure adesione alle linee guida (30 minuti di attività moderata per almeno 5 giorni alla settimana, oppure attività intensa per più di 20 minuti per almeno 3 giorni)

** non fa lavoro pesante, ma fa qualche attività fisica nel tempo libero, senza però raggiungere i livelli raccomandati

*** non fa un lavoro pesante e non fa nessuna attività fisica nel tempo libero

Livello di attività fisica

Attivo* 33,1 %

parzialmente attivo** 35,7 %

Sedentario*** 31,1 %

The highest adherence score to an MD was significantly associated with

a lower risk of all-cause cancer mortality (RR: 0.87), colorectal cancer (RR: 0.83), breast cancer (RR: 0.93), gastric cancer (RR: 0.73), prostate cancer (RR: 0.96), liver cancer (RR: 0.58), head and neck cancer (RR: 0.40), pancreatic cancer (RR:

0.48),and respiratory cancer (RR: 0.10)

Cancer Med. 2015

1,784,404 subjects included in 56 studies

Toledo et al. JAMA Internal Medicine 2015

The multivariable-adjusted hazard ratios vs the control group were 0.32 (95%CI, 0.13-0.79) for the Mediterranean diet with extra-virgin olive oil group

and 0.59 (95% CI, 0.26-1.35) for the Mediterranean diet with nuts group

4282 women Follow up: 4,8 ys

Soy isoflavones consumption was inversely associated with risk of breast cancer incidence

(RR = 0.89, 95% CI: 0.79–0.99)

The protective effect of soy was only observed among studies conducted in Asian populations (RR = 0.76, 95% CI: 0.65–0.86) but not in Western

populations (RR = 0.97, 95% CI: 0.87–1.06)

Jia-Yi Dong • Li-Qiang Qin. Breast Cancer Res Treat (2011)

Soy intake consistent with a traditional Japanese diet (2-3 servings daily, containing 25-50 mg isoflavones) appears safe for breast

cancer survivors

While there is NO clear evidence of harm, better evidence confirming safety is required before use of high dose (≥ 100mg) isoflavones can

be recommended for breast cancer patients

MODERATE soy consumption appears to be safe and possibly beneficial for most women

Heidi Fritz. PLOS ONE. November 2013

Soy consumption may be associated with reduced risk of breast cancer incidence, recurrence, and mortality

Cancer 2017

a higher dietary intake of isoflavone was associated with reduced all-cause mortality

A 21% decrease was observed in all-cause mortality for women who had the highest versus lowest quartile of

dietary isoflavone intake

(>1.5 vs<0.3mg daily: HR, 0.79; 95% CI, 0.64-0.97; Ptrend 5.01)

6235 women with BC F-up approx. 9.4 ys1224 deaths documented

WCRF / AICR. CUP: Diet, Nutrition, Physical Activity and Breast Cancer Survivors. 2014

Linee guida sulla nutrizione

e l’attività fisica per la prevenzione dei

tumori.

American Cancer Society

© 2012 American Cancer Society

Studio su base di popolazione del Body mass index

Il RTM ha intrapreso uno studio relativo all’influenza del body mass index sul rischio di neoplasia mammaria.

Grazie alla collaborazione dei centri di screening è stato possibile effettuare misurazioni di altezza e peso delle donne invitate al programma di screening

mammografico.

Risultati su 14.255 donne:

N° Casi SIR Normopeso 6443 49 1.25 Sovrappeso 5334 60 1.85*

Obesità 2478 27 1.79*

* statisticamente significativo

Sebastiani F. J Breast Cancer 2016

Increased incidence and poor prognosis of breast cancer in postmenopausal women with high Body Mass Index

attending to the Mammography Screening Program in the province of Modena (Italy)

Normal weight Overweight Obesity

Normal weight Over weight Obesity

Sebastiani F. J Breast Cancer 2016

J Clin Oncol 2010

Obesity is an independent prognostic factor for

developing distant metastases and for death as a result of breast cancer

On a long-term basis, adjuvant therapy seemed to be

less effective for patients with breast cancer and obesity

After 10 years, both chemotherapy and endocrine therapy seemed to be less effective in patients with BMIs of 30 kg/m2 or greater

HR for death (all causes) in relation to follow-up time, BMI, and adjuvant treatment

J Clin Oncol 2010

J Clin Oncol 2010

ATAC study: 9366 postmenopausal women with early-stage breast cancer randomly assigned to oral daily anastrozole alone, tamoxifen alone, or

the combination in a double-blind fashion

There is a significantly greater risk of recurrence in overweight women receiving anastrozole

women with a high BMI at baseline had more recurrences

than those women with a low BMI (adjusted HR, 1.39) and

significantly more distant recurrences (adjusted HR, 1.46)

Is estrogen suppression with anastrozole complete in obese women?

J Clin Oncol 2010

LISTA DEGLI INVESTIGATORI

Sperimentatore principale Massimo Federico¹

Co-sperimentatore di riferimento Federica Sebastiani¹

Co-sperimentatori e collaboratori Nino Battistini¹, Silvia Toni¹

¹ Dipartimento di Medicina Diagnostica, Clinica e di Sanità Pubblica. Università di Modena e Reggio Emilia

Programma di educazione alimentare e al movimento rivolto alle donne operate per carcinoma della mammella

Obiettivi: valutare l’ efficacia di un programma di educazione alimentare e al movimento su:

 parametri antropometrici (BMI, peso corporeo)

 Livello di attività fisica

 Miglioramento della qualità di vita

OBIETTIVI

Analisi attività ambulatoriale Gen 2010-Mag 2016

Variabili considerate

BODY MASS INDEX (Kg/m²)

ATTIVITÀ FISICA PROGRAMMATA (ore/settim)

ANALISI STATISTICA Anova per misure ripetute

Campione

N°donne sottoposte alla prima visita: 442 N°donne con un follow-up di 24 mesi: 442

* differenza statistica con p<0.05

Campione: donne (n=442) con un f-up mediano di 24 mesi

Analisi per strato di BMI

103

170 169

103

150

130 103

150

125 103

135

115

Normopeso Sovrappeso Obese

P=0.74

P<0.001*

P<0.001*

103 donne

170 donne

169 donne

BMI (kg/m2 )

Base 6 M 12 M 24 M

0,00 0,50 1,00 1,50 2,00 2,50 3,00 3,50

BASELINE 6 MESI 12 MESI 24 MESI

* differenza statistica con p<0.05

ore/settim

P<0.001*

Attività fisica media

Campione: donne (n=442) con un f-up mediano di 24 mesi

Conclusioni

• L’analisi preliminare dei dati raccolti in 6 anni di attività dimostra come una corretta alimentazione ed un regolare esercizio fisico possono favorire una significativa riduzione del peso corporeo

• Il controllo del peso e del livello di attività fisica

attraverso strategie di intervento sullo stile di vita

dovrebbe rappresentare parte integrante del follow

up delle pazienti con tumore della mammella

Costi sanitari pubblici

Trattamenti

e cure Prevenzione

Ogni investimento in prevenzione produce, nel corso degli anni successivi, un risparmio triplo in trattamenti e cure

Prof. Cascinu Stefano Dr. Cortesi Laura

Dr. Toss Angela Dr Spaggiari Federica Dr. Razzaboni Elisabetta

Dr. Marchi Isabella Dr. Medici Veronica Dr. Venturelli Marta Inf. Bevini Paola

Prof. Pietro Torricelli Dr. Rachele Battista Dr. Barbara Canossi Dr. Annarita Pecchi Dr. Dal Molin Chiara Dr. Antonella Drago Dr. Giovanni Grandi

Programmi di screening di popolazione

 Gratuiti, ad invito

 Indicazioni dell’Unione Europea e del Ministero della Salute

 Donne fra i 45 ed i 74 anni

 Mammografia annuale in due

proiezioni dai 45 ai 50 anni poi

biennale

Learning Objectives

• To distinguish between role of Genetics and Genomics in clinical practice

• Focus on Hereditary Breast Cancer Syndromes and Multi-Gene Panels

• To explain the clinical meaning of Moderate Gene Risk and VUS

• To identify the patients for whom the Oncotype DX assay has been clinically validated

• To identify the patients for whom the Mammaprint assay could spare the chemotherapy

• To describe the most frequent mutated genes in

sub-type breast cancer and resistance mechanisms

by NGS

Examples of Genetic and Genomic Tests

Genetic Test

• BRCA1 and BRCA2

• The genetic make up of patients is tested for BRCA1 and BRCA2 mutations. Patients with those mutations have higher chances of developing breast cancer.

Genomic Test

• The expression level of 21 genes is measured in tumor tissue from patients that have already been diagnosed with breast cancer. This assay evaluates if a patient is going to recur (prognostic) and predicts benefit from chemotherapy and hormonal therapy (predictive)

• Mammaprint assay

• Breast Cancer Index

Moderate Risk Genes



Cancer risks may not be very high

◦ How high does risk need to be before we pursue surgery or medications?



Cancer risks may be unclear

◦ How do we make medical decisions when we don’t know the risks?



We’re still learning

◦ The recommendations you get today may be

different in 5 years

Results of Multi-Gene Panel Testing N=76574

Gene N % with mutations

CHEK2 641 0,84

ATM 503 0,66

PALB2 392 0,51

BRIP1 216 0,28

NBN 140 0,18

RAD51C/D 163 0,21

BARD1 47 0,14

Total 2164 2,54

Real World Classification can be Messy

[NOME CATEGORIA]

[NOME CATEGORIA]

[NOME CATEGORIA]

[NOME CATEGORIA]

RESULTS

32%

37%

5%

27%

First 599 of 1400 patients in the PROMPT registry-compared testing results to classification in CLINVAR

Development of Functional Assays

• Most variants of uncertain significance have only been observed in a small number of families

• These VUS will not be readily classified by epidemiological approaches

• Functional assays offer a useful option for evaluating these VUS

• Must use assay associated with risk associated protein activity

• Sensitivity and specificity are required

Br: 33 49

47 Stomach: 54

Oncotype DX® 21-Gene

Recurrence Score® (RS) Assay

PROLIFERATION Ki-67

STK15 Survivin Cyclin B1 MYBL2

ESTROGEN ER

PR Bcl2

SCUBE2 INVASION

Stromelysin 3 Cathepsin L2

HER2 GRB7 HER2

BAG1 GSTM1

REFERENCE Beta-actin GAPDH RPLPO GUS TFRC CD68

16 Cancer and 5 Reference Genes From 3 Studies

Oncotype DX ^® 21-Gene

Recurrence Score ^® (RS) Assay

RS = Coefficient x Expression Level + 0.47 x HER2 Group Score - 0.34 x ER Group Score

+ 1.04 x Proliferation Group Score + 0.10 x Invasion Group Score

+ 0.05 x CD68 - 0.08 x GSTM1 - 0.07 x BAG1

Calculation of the Recurrence Score Result

Category RS (0-100) Low risk RS <18

Int risk RS ≥18 and <31 High risk RS ≥31

The Trial Assigning Individualized Options for Treatment (TAILORx)

This trial was designed to further validate and refine the clinical usefulness of the 21-gene assay (Oncotype DX Recurrence Score, Genomic Health) in a specified low-risk cohort of women with hormone-receptor–positive, HER2-negative, axillary node–negative invasive breast cancer.

Node N-, ER+ Breast Cancer

RS <10 Hormone

Therapy Registry

RS 11-25 Randomize Hormone Rx

vs

Chemotherapy + Hormone Rx

RS >25

Chemotherapy +

Hormone Rx

Oncotype DX

Assay

Register Specimen banking

Primary study group

TAILORx Schema

Invasive Disease–free Survival

5-year event rate in cancers with recurrence scores 10 or less they did not receive chemotherapy

Freedom from Recurrence of Breast Cancer at Distant Site

Freedom from Recurrence

at Any Site Overall Survival

Among patients with hormone-receptor-positive, HER2- negative, axillary node-negative breast cancer who met established guidelines for the recommendation of

adjuvant chemotherapy on the basis of clinicopathologic

features, those with tumors that had a favorable gene-

expression profile had very low rates of recurrence at 5

years with endocrine therapy alone.

The Winners are…

1.

2.

3.

4.

5.

N+

T>2

GIII

HER2

ER

HR p

0.00 0.25 0.50 0.75 1.00

159 61 21 2

5/7

378 195 75 7

3-4

1549 914 343 32

0/2 Number at risk

2 5 8 10

0/2 3-4 5/7

A

0.00 0.25 0.50 0.75 1.00

88 42 8 2

5/7

210 108 26 1

3-4

675 372 113 8

0/2 Number at risk

2 5 8 10

0/2 3-4 5/7

B

Cumulative relapse probability

Follow-up, years

Tumor size, node status, grading, HER2 and

estrogen receptor status still retain a strong

value in patients with operable breast cancer

diagnosed in recent years

The MINDACT Study:Patient Enrollment

Enrolled N=6693

Clinical Risk:

Adjuvant Online

Genomic Risk:

Mammaprint

C low/G low

N=274⁵

Discordant

N=592 N=1550

C low/G high C high/G low C high/G high

N=1806 Random

No chemotherapy Chemotherapy

N=644

Clinical Outcome at 5-y Median Follow-up:

Concordant Risk Group

cL/gL 97.6% (96.9-98.1) cH/gH 90.6% (89.0-92.0)

92.8% (91.7-93.7) 85.3% (83.4-87.0)

98.4% (97.8-98.9) 94.7% (93.4-95.7)

The MINDACT Population: CT according Clinical or Genomic Strategy

Whole Population:

N=6693

N=2745 cLow/gLow

N=1806 cHigh/gHigh

Discordant

N=592 cLow/gHigh

N=1550 cHigh/gLow Clinical Strategy

CT=1550+1806=3356

Genomic Strategy CT=592+1806=2398 14% CT reduction

• MINDACT study provide a level 1A of evidence of the clinical utility of Mammaprint for assessing the lack of CT benefit in cHigh Risk population

• cHigh/gLow patients have a 5-y DMFS rate of excess of 94% regardless CT

• In the whole population genomic strategy leads to a 14% CT reduction compared with clinical strategy

• Among the cHigh Risk patients the clinical use of

Mammaprint is associated with a 46% CT reduction

HOXB13/IL17BR (H/I), is a prognostic biomarker in both untreated and tamoxifen-treated early-stage ER+ breast cancer patients.

Expression of H/I within primary tumors was determined by reverse-transcription polymerase chain reaction with a prespecified cutpoint. The predictive ability of H/I for

ascertaining benefit from letrozole was determined using multivariable conditional logistic regression including standard clinicopathological factors as covariates.

• Patients with High H/I had a 5yr absolute benefit of 16.5% from extended endocrine therapy with Letrozole (p=0.007)

• Patients with Low H/I had no significant benefit from extended endocrine therapy with Letrozole (p=0.35)

Prognostic ability of BCI, 21-gene recurrence score, and IHC4 for overall 10 year distant recurrence, for all patients and according to ATAC treatment group.

BCI-L (linear) was the only significant prognostic test for risk of both early and late distant recurrence. It could help to identify patients at high risk for late

distant recurrence who might benefit from extended endocrine or other therapy.

Sgroi DC et al. Lancet Oncol 2013

Tumor and normal interstitial fluid proteomic characterization in breast cancer patients receiving neoadjuvant chemotherapy

NGS meta-analysis: most frequently mutated

genes in each molecular subtype

Mechanisms of resistance to HER2- targeted therapy:

1. Impaired access to HER2 by expression of extracellular domain- truncated HER2 (p95 HER2) or overexpression of MUC4

2. Alternative signaling from IGF-1R and other HER family members or MET

3. Loss of downstream controllers (PTEN, p27)

4. Activation of downstream signaling pathways (PI3K-Akt, MAPK, mTOR)

Mechanisms of resistance to endocrine therapy:

1. Hyperactivation of the PI3K/AKT/mTOR pathway 2. Dysregulation of normal cell cycle control

3. Downregulation of ER through epigenetic aberrations (i.e. DNA methylation) 4. Amplification or overexpression of the ERBB2 proto-oncogene.

GENES PROTEINS

ERBB2 HER2

EGFR EGFR

HER3 HER3

AKT1 AKT2 AKT3

mTOR mTOR

PIK3CA PTEN

IGF1R ; FGFR1 ; MET IGF1R ; FGFR1 ; MET TP53 ; RB1

BRAF ; RAF1 KRAS ; NRAS

MEK1 (MAP2K1) ; MEK2 (MAP2K2)

ERK1 (MAPK3) ; ERK2 (MAPK1) ERK1 (MAPK3) ; ERK2 (MAPK1 MAP3K1

CCND1 CCND2 CCND3 CDK4 ; CDK6

ESR1