CP0106

ASESORAMIENTO GENÉTICO

Aurora Sánchez Díaz

Genética Clínica y Asesoramiento Genético. Servicio BGM. Hospital
Clínic de Barcelona
OBJETIVOS
» TO KNOW:

• CLINICAL SIGNS RELATED WITH HEREDITARY CANCER

• PENETRANCE VARIABILITY
• BREAST CANCER RELATED GENES
• MOLECULAR DIAGNOSIS METHODS
• THE NGS LIMITATIONS AND ADVANTAGEGENETIC CODE
• CLINICAL CRITERIA FOR BREAST CANCER GENETIC
STUDY
• GENETIC COUNSELING
BREAST CANCER
ØBreast cancer is the most common cancer in women: it
has been estimated that 10-12% of women develop
breast cancer over the course of their life
üMost of them appear sporadically
ØIn less than 25% of cases, a family susceptibility to
breast cancer is detected, and among these:
üOnly a small proportion are due to mutations in
genes that confer a susceptibility to cancer.
ü10% are hereditary following an autosomal
dominant pattern of transmission
Risk factors influencing breast
cancer development
Lifestyle risk factors
üDietary factors
üAlcohol consumption
üWeight-gain
üPhysical activity
üReduced BC mortality
üPregnancy
ü<29 years old risk
ü>35 years old risk
üPharmacotherapy
üPostmenopausal HRT
increases risks
üRadiation exposure
üThe BC risk correlates with
the dose of radiation
Genetic factors
ü Family history of BC
ü Number of cases
ü Relationship between
cases
ü Personal history of BC
ü Age of first BC
ü Contralateral BC risk
ü High breast tissue density
ü Menoactivity
ü Age at menopause
increases the risk by
3%/year
ü Decreased risk with late
menarche
ü Height
ü Tall women risk
ERRORS IN THE GENOME GIVING RISE
TO CANCER

In the tumor cells themselves the error can

appear on two levels :
Germline
DNA sequence changes:
• Loss related gene downregulates cell cycle
• Genetic mutations that can activate or
inactivate different proteins Hereditary
cancer
• Gene amplifications that lead to
overexpression of specific genes
•Epigenetic changes
• Gene silencing by hypermethylation of the
CpG islands in the promoter regions
HUMAN GENOME: variability
vMutation
A permanent alteration of nucleotide sequence in an organism.
Mutations may or may not produce discernible changes in the
observable characteristics (phenotype) of an organism
Somatic mutations Germline mutations

Ovum/sperm All cells

mutation affected

ØNot inheritable ØInheritable

ØAre tissue specific. NON germline ØTake place in germline
ØAcquired ØCongenital in offspring
Inheritance pattherns
Mitochondrial

X linked

Autosomal recesive

Autosomal dominant
Distribution of breast cancer according
to genetic risk

Zdenek Kleibl, Vessela N. Kristensen. The Breast 2016

Signs indicating the presence of a germline
mutation in a breast cancer-susceptibility gene

ØUnusual breast cancer appearance:

üEarly disease onset
üTumor recurrence
üBilateral tumor development
üMale breast cancer
üPresence of minor histopathological diagnosis (TNBC)
ØClustering of breast cancer in affected families
ØCancer multiplicity
üDevelopment breast cancer and other cancers
Breast cancer susceptibility genes

All breast cancer suceptibility genes have variable penetrance

The proportion of carriers who develop breast cancer over the

course of their lifetime

High Moderate low

The relative risk of breast cancer development in mutation carriers

the presence of mutation does not necessarily involve

disease development
 Genes related to hereditary breast cancer
Gen Phenotype Other neoplasias
BRCA1 HBOC Pancreas, ovary, uterus
BRCA2 HBOC Breast cancer in males, prostata, ovary,
melanoma
High PALB2 HBOC Anemia de Fanconi
penetrance TP53 Li-Fraumeni Sarcomas, adrenocortical, CNS
PTEN Cowden Hamartomas, non-medullary tyroid
STK11 Peutz-Jeghers Hamartomas, colorrectal, gastric,
CDH1 GCHD Diffuse gastric cancer
BARD1 BC
Moderate
CHEK2 BC Prostata, colorrectal, tyroid
penetrance
ATM BC Ataxia-telangiectasia (homozygosity)
C MRN BC ----
low
C BCDX2 BC Ovary
penetrance
FANCM BC Fanconi anemia (homozygosity)
HBOC: hereditary breast and ovary cancer; CNS: central nevous system; GCHD: gstric cancer
hereditary diffuse; BC: breast cancer
BRCA1 and BRCA2
They account for the development of 3 -6% of all breast cancers

BRCA1 and BRCA2 explain approximately 25 % of the familial

clustering of breast cancer

Responsible for 60 -80% of breast cancer and 65 -85% of hereditary

ovarian cancer

Frequency of carriers in general population 1/300 -1/500

BRCA1 BRCA2
Located in 17q21.31 Located in 13q13.1
Composed by 24 exons Composed by 27 exons
They are involved in the maintenance of genome integrity

They activate and regulate transcription and participate in DNA

repair
 Features of cancer associated with theBRCA1and BRCA2 genes
Phenotypic expresion BRCA1 BRCA2
Hormonal receptor Usually negative Mostly positive
HER2 Usually negative 10% amplified
Onset of disease 43 years old 47 years old
CR of BC a age 70 57% 49%
OC risk 40% 18%
Relative risk de PC 2.26 3.51
Risk of prostatic cancer low 20-fold increased
Melanoma risk - Increased
CR: cumulative risk; BC: breast cancer; OC: ovarian cancer; PC: pancreatic cancer
 BRCA1 BRCA2

20 higher than GP
Pancreatic cancer risk -----
82,5 in M/14 in F
Breast cancer in male 1,2% 8%
Contralateral breast
3%/year
cancer
Histopathological type Infiltrating ductal carcinoma
Ovarian cancer High-degree papillary serous and bilateral

GP: general population; M:male ;F: female

BRCA2 mutations are present in 15% of breast cancer in males

Mutations in BRCA2 confer a male a risk of breast cancer between

80-100 times higher than the general population
OTHER HIGH - PENETRANCE GENES INVOLVED IN
BREAST CÁNCER

Together they account for less than 1% of familial breast cancer

At age 30 At age 60
Breast cancer risk
50% 90%

TP53
Soft tissue sarcomas
Osteosarcomas
Síndrome de Li -Faumeni Brain tumors
Adrenocortical tumors
Colon and gastric cancer
Breast cancer

At premenopausal age Ductal carcinoma

Hormonal receptors + and HER2 -
Before 30 years old
 LI-FRAUMENI SYMDROME
Highly penetrating gene
100% of f women who carry such mutations
develop cancer

73% of men carrying mutation develop cancer

Attributable to breast cancer

Risk of cancer at age of 30 of 21%

Breast cancer 27%
The most common cancers
Sarcoma 25%
Childhood-onset rhabdomyosarcoma is also
associated with germline TP53mutations
The risk of second and subsequent malignancies is Brain tumours
Adrenocortical carcinoma
inversely correlated with younger age at diagnosis Leukaemia
of the first malignancy
There is an association between exposure to ionizing radiation and development
of cancer in individuals withTP53 mutations
 OTHER HIGH -PENETRANCE GENES INVOLVED IN BREAST
CANCER
STK11
PTEN CDH1
Peutz-Jeghers
Cowden syndrome
syndrome Involved in 35% of
Multiple hamartomas diffuse gastric cancer
Hamartomatuos
polyps en GI tract Usually it is lobular
Macrocephaly and
dysmorphia cancer
Oral and anal
hyperpigmented Breast cancer risk 52%
acral keratosis
macules at age 75
Breast cancer risk
Breast cancer risk
85%
45% at age 70
 MODERATE-PENETRANCE CANCER SUCEPTIBILITY
GENES
They are present in 1.1 to 9.4 % of individuals tested

The differentiation between high and moderate penetrance can

sometimes be arbitrary

There are some mutations in Certain mutations in high-

moderate-penetrance genes that penetrance genes might confer
confer a risk similar to high- more modest degrees of risk
penetrance genes

ATM c.7271T>G PALB2 c.3113G>A BRCA1 c.5096G>A

We need to individualize levels of cancer risk

 MODERATE-PENETRANCE GENES INVOLVED IN BREAST
CANCER
Overall risk of BC between 2-4 times greater than the general population

PALB2 CHEK2 ATM

Population-specific
Interact with BRCA’s
mutations Bialelic mutations :
Maintains genome Ataxia-telangiectasia
C.1100delC implies
Integrity
increase of BC of Frequency of carriers in
any type at an early GP 0,35-1%
Bialelic mutations:
age
Fanconi anemia
BC risk 2.4 times higher
Risk of BC 37% and than non-carriers
Risk of breast cancer
3.5 times more risk
47% at age 70
of contralateral BC
 LOW-PENETRANCE GENES IMPLICATED IN BREAST
CANCER
Increased risk of BC by almost twice the one of general population

Complex MRN Complex BCDX2 Other genes

Repair of DNA during implicated in
Genes that help DNA homologous Fanconi anemia
repair recombination
FANCM
MRE11 RAD51C
Increased risk for
RAD50 triple- BC
RAD51D

NBS1 Slight increase for triple

– BC
Slight increase for BC
Ovarian cancer
 MOLECULAR DIAGNOSIS
METHODS
ØIndividual gene sequencing by traditional Sanger method
ØIf the genetic testing results are negative, additional testing
may be indicated
ØThis method of diagnosis is indicated when the familiar
mutation is known
ØStudy of genomics rearrangements by multiplex ligation-dependent
probe amplification (MLPA)
ØDeletions and duplications are present in some cases
ØIt’s a sequencing testing complementary method
ØNext-generation testing/cancer panels
ØAllow for simultaneous analysis of multiple genes
Ø Cost and time effective
 NEXT-GENERATION SEQUENCING (NGS)

APC, ATM, BAP 1, BARD 1, BRCA 1, BRCA 2, BRIP1, BMPR 1A, CDH1,
CDK4, CDKN 2A, CHEK 2, EPCAM, FH, FLCN, GREM 1, MAX, MEN1,
MET, MITF, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB 2,
PMS2, POLD 1, POLE, PTEN, RAD 50, RAD 51C, RAD51D, RET, SDHA,
SDHAF 2, SDHB, SDHC, SDHD, SMAD 4, SMARCA 4, STK 11, TMEM127,
TP53, TSC1, TSC2, VHL.
TECHNIQUES TO DETECT MUTATIONS IN
BRCA1 & 2 GENES
TECHNIQUES TO DETECT MUTATIONS IN
BRCA1 & 2 GENES

MLPA: Multiplex ligation -dependent

-
probe amplification.

FAMILY MEMBERS
Sequencing (Sanger)
 BENEFITS AND LIMITATIONS OF NGS
Simultaneous analysis of multiple
genes
Allows to study in the same
analysis other genes than
BRCA’s
Increases detection rate by
30%
Greater sensitivity to assessing
cancer risk
Helpful when there are
overlapping phenotypes
Allows for testing patients who
not meet standard high-risk
criteria
Cost and time effective
Higher likelihood of identifying variants
of unknown significance
Difficulties for patients and
providers with respect to
counseling on true risk of
cancer
The panel tests includes genes that are
thought to be moderately penetrant
There is limited data about
cancer risks and penetrance
There are no clinical
management guidelines for the
clinical follow-up of the patients
More complexity for genetic
counseling
 NEXT -GENERATION SEQUENCING (NGS)
CLINICAL INTERPRETATION

To avoid maximum interpretation problems, only high-risk

individuals should be analyzed

Information about the risk of pathogenic variants is available for

high -penetrant genes
In BRCA1 and BRCA2 there are over 3500 distinct sequence
variants and more than half of hem are:

VUS Prediction of pathogenicity

Phylogenetic conservation
Impact on protein structure and function

Frequency in pathological population

Frequency in general population
HEREDITARY BREAST CANCER TESTING CRITERIA

• Breast cancer≤ 40 years old

• Triple negative breast cancer≤ 60 years old
• Breast cancer ≤ 50 years old in non-informative family
• Male breast cancer
• ≥ 3 first degree relatives with breast cancer, at least one of them diagnosis before
60 years old
• Two cases of breast cancer ≤ 50 years old
• Bilateral breast cancer ( the first diagnosed ≤ 50 years old)
• Bilateral breast cancer and another case with breast cancer, one of them
diagnosed ≤ 60 years old
• Metastatic HER2 negative breast cancer candidate for PARP inhibitors treatment
• High-grade ephitelial non-mucinous ovarian cancer. In low-grade tumors, an
individualized selection will made based on: age, family history and potential
benefit of family members
GENETIC DIAGNOSIS IN INHERITED BREAST CANCER

Affected breast cancer patient

We will choose for the study the individual of the family that meets more risk
criteria of hereditary cancer

+ TP53
MLPA NGS (panel)
PTEN
BRCA1 STK11
CDH1
BRCA2 No mutation PALB2
…..
First step
Second step according to clinical signs

Mutation

We extend the study at risk family members Gentic counseling process

MLPA or Sequencing (Sanger)

 GENETIC COUNSELING
A multi-step communication process dealing with complex and
often highly charged information which should be non-directive.
The objetives are:
Ø Provide explanations regarding cancer risk levels
to identify and counsel individuals at different risk of developing cancer

true high risk moderate risk Average risk

Ø Offer molecular diagnosis and gentic testing where appropiate

Ø Inform patients of the interpretation of the results
Ø Desing and propose individual risk reduction strategies
Ø Provide an emotional suport
Ø Coordinate heelthcare professionales to enable care delivery processes

should only be offered in the context of a comprehensive program of pre- and post-
test counseling, by professionals with appropriate knowledge and experience in
the field.
 GENETIC RESULTS ASSESSMENT

Informative test Non-informative test

True positive
Inconclusiveresult
Pathogenicmutation
No mutation in a healthy person.
BC patient presymptomatic We do not have a genetic study of
any family member with cancer

Diagnostic High risk of

confirmation cancer

True negative Variant of uncertain significance

No mutation
Genetic variant to which we
cannot attribute a possible
presymptomatic Cancer risk of pathogenic character
GP
 REPRODUCTIVE COUNSELING

Especially in healthy young people carrier of pathogenic mutation

The recurrence risks of this mutation Risk of cancer for offspring of

50% High risk

Depending on the experience of the disease within the family, there are
women who want to totally eradicate the possibility of transmitting the
mutation to their offspring

Reproductive options

Prenatal diagnosis IVF-PGD Gamete donation

mutation-free offspring
 CONCLUSIONS
Ø Cancer is caused by an accumulation of errors in the genome. When these
alterations occur in germline cells, they may be transmitted to the offspring,
generally following a pattern of autosomal dominant inheritance
ØAll breast cancer susceptibility genes have variable penetrance. The presence
of mutation implies more risk, but does not necessarily involve disease
development
ØThe highly penetrance genes, BRCA1 and BRCA2, are present in around 80% of
familial breast cancer. The rest of genes are implicated in less than 1% of
familial breast cancer
ØNext-generation sequencing panel testing has been integrated into clinical
practice. Benefits and limitations should be cautiously evaluated
Ø Due to the complexity and the high cost of the genetic study, it should be
initiated in the individual of the family who meets more clinical cancer risk
criteria
ØGenetic counseling allow individuals to learn how heredity contributes to
cancer risk, understand their personal risk, understand their options for
managing their cancer risk, choose a course of action that is appropriate for
them, and provide them reproductive options to have mutation-free offpring