Oncology and Genetics Who, What and Why

Dr Hilda High Genetic Oncologist

Why think about Genetics in Medical Oncology?

1. To provide best patient care

2. Because you’re a doctor and it’s in the papers and everyone expects you to know what it means

3. So you don’t look like an idiot in MDT’s

4. It’s fascinating !!

5. All of the above

Outline Germline (inherited) vs Somatic (acquired)

Overview of genetic testing and history Implications and limitations of Next Gen Seq (MPS)

Hereditary Cancer Services: What are they and What they do When, Why and How of referring

Cancer syndromes and genetics Familial bowel cancer (Lynch Syndrome) Haem cancer syndromes (Li Fraumeni (TP53))

Case study - EGFR

Somatic: occurred locally in an individual cell (eg breast, bowel, stem cell)

Random / spontaneous. Not inherited. This is most cancer

Germline: From germ cells (egg / sperm). Mutation in every cell

Somatic vs Germline Mutations

Types of Genetic Errors

Point mutations

Insertions / deletions (InDels)

Large deletions

Copy number variants CNV

Translocations and Rearrangements

Chromosomal

Infections eg HPV in tumour

Sequence

Sequence

MLPA

aCGH / FISH

aCGH / FISH

karyotype

Chromosomal Changes = Cytogenetics

Germline: Down Syndrome

Trisomy 21 Increased risk leukaemia

Somatic:

Philadelphia Chromosome Translocation: BRC-ABL EML4-ALK & lung cancer

Wikipedia

Polymerase Chain Reaction

DNA Two primers that are complementary to the DNA

(sense and antisense) DNA polymerase Nucleotides (dNTPs) Heat to denature DNA (single strand), cool to

anneal primers, warm to added nucleotides Repeat to get lots of short lengths of DNA

Genetic testing - Sanger Sequencing

Template DNA (from Human Genome Project)

PCR using dNTP and ddNTPs

Terminate when ddNTPs added

Each fluorescently labelled

Computer reads and shows nucleotide sequence

Forward and reverse to make sure is “real”

Sanger Sequence of part of BRCA2

Electropherogram of BRCA 2 mutation

Reference (wt)

Variant c.506A>G

Next Generation Sequencing aka Massively Parallel sequencing

Reagents cheaper and is faster

BUT Terabytes of data that needs to be interpreted

One person thousands of genes

whole exome sequencing (pt and tumour)

$1000 test but $1,000,000 analysis

Many people, thousands of genes (“bar code” the DNA!)

or thousand of people for a few genes

panel testing (multiple breast / bowel cancer genes)

MLPA

MRC Holland

How do you find something that is missing?

Gene Amplification

FISH or array

Frequently used in the tumour eg HER2 over-expression in breast cancer

Also used in germline eg Trisomy 21 (Down syndrome)

Array Comparative Genomic Hybridisation (aCGH) (eg GOLD)

Fluorescence measured for each spot (can you see the red one?)

Array CGH slide Plotted on chromosome map to reveal copy number variations

Genome Wide Association Study: GWAS

Stadler JCO Sept 2010

Finding Mutations is the Easy Part!

What amino acid substitution means: SIFT – Sorting Intolerant From Tolerant

Polyphen – prediction of functional effect

Conservation across species

Software to Integrate Databases eg ALAMUT

Functional studies = gold standard but expensive

In silico – guessing from experience

CIRCOS plots Colon Cancer from ring outer to inner Chromosomes

Insertions and deletions = Small rectangles

Heterozygous and homozygous substitutions

Coding substitutions, coloured according to type Silent/missense/nonsense /splice site

Copy number

Loss of heterozygousity

Intra chromosomal rearrangements

Inter chromosomal rearrangements

Landscape Plots

TP53

PIK3CA

Signalling Pathway diagrams

These, next and previous images from Primer of the Molecular Biology of Cancer. DeVita et al. Wolters Kluwer Health / Lippincott, Williams & Wilkins

Why Cancer Runs in Families?

Single Gene

Founder mutations

Gene/gene interactions

Gene/environment interactions

Diet / lifestyle

Chance / aging

Familial Cancer Service

Risk prediction Verification of history and pathology

Genetic counselling and testing Surveillance and Risk reduction strategies

Especially when genetic testing “uninformative”

Different treatment options Patient may be at risk of other cancers Other family members may be at risk Reproductive choices

When to refer 3:2:1 = 3 blood relatives, 2 generations, 1 <50yrs Patient characteristics

Cancer at young age Multiple cancers in patient or family Syndromal features or cancer clustering Ethnicity re founder mutations

Tumour characteristics Pathology:

loss of staining for MMR proteins on IHC

Rare tumour types Bilateral or multifocal tumours

eviQ Cancer Institute NSW

point of care

clinical information resource

current evidence based

peer maintained

best practice cancer treatment

www.eviQ.org.au

Risk of Contralateral Breast Cancer in BRCA1 mutation carrier <40 at diagnosis of first

breast cancer

1. 10%

2. 20%

3. 40%

4. 60%

5. >60%

Managing Cancer Risk in BRCA+ Breast:

Risk Reducing Surgery

Contralateral breast cancer risk:

BRCA1: 63% if pt <40 and 20% if pt >50 at diagnosis

Screening: Starting from age 30; Including MRI to age 50

Risk Reducing Medication eg Tamoxifen

Ovarian:

Risk Reducing Surgery

Screening with transvaginal US and Ca125 doesn’t work

Don’t forget the non-personalised (public health):

Diet / exercise / healthy body weight / lifestyle

Benefit of RRSO If around 40 / before menopause:

50% reduction in breast cancer risk

98% reduction in ovarian cancer risk

Domchek PROSE study 2010

all-cause mortality: 10% vs 3% HR = 0.40 [95% CI, 0.26-0.61]

HRT can be used to age of natural menopause

Polyps, Colon Cancer and Genes: Clues

Polyps: Number and age

Adenoma, hamartoma, juvenile, serrated, hyperplastic

Location (right sided vs left sided; duodenal)

Family history: including other cancers, rare tumours etc

Who didn’t get cancer and who lived to an older age

A “look” (phenotypic features)

Tumour testing (Lynch syndrome)

Average Risk

Bowel cancer: average age late 60’s Males 10% Females 6.6%

Screening:

Faecal occult blood test – 1 to 2 yearly from age 50

Daughter attends with mum 27

Mother Colon cancer at 66

Importance of Family History

Daughter attends with mum 27

Colon ca at 58 ? Cervical ca at 45

Mother Colon cancer at 66

Colon ca at 66

Moderate Risk

One close (1st) with colon cancer <55yr

or 2 relatives:

2 primary

or 1 primary and 1 secondary

Colonoscopy every 5 years from ? age 40.

Colon ca at 53 ? Cervical ca at 42

Mother Colon cancer at 66

Colon ca at 66

Diet / lifestyle

Healthy diet High fruit / vegetables; high fibre

Exercise 20 mins moderate exercise

Healthy body weight

=30% decrease cancer risk

(?aspirin to decrease polyp formation – CAPP3 study)

Need all cancers and pathology

Daughter attends with mum 27

Colon ca at 53 Uterine ca at 42

Mother Colon cancer at 66

Colon ca at 66

Colon ca at 30

Adenomatous Polyp

http://clinicfordigestivesurgery.com/

http://www.endoatlas.sk/

Lynch Syndrome

1895

in a single family

Dr Aldred Scott Warthin

Most died < 45

The History of Lynch Syndrome. CR Boland and HT Lynch. Familial Cancer 2013

Lynch Syndrome

Caused by a mutation in one of the mismatch repair genes

MLH1, MSH2, MSH6 or PMS2. (and EPCAM)

Proteins work in pairs MLH1 with PMS2 and MSH2 with MSH6

if the dominant protein is missing, partner missing also

MLH1 commonly somatic mutation

Do BRAF IHC (BRAF genetic test needed for therapy selection).

A BRAF mutation means NOT Lynch

MMR IHC

Slides provided by the pathology department at The Sydney Adventist Hospital, Wahroonga, Sydney

If gene not working, protein not made = loss of staining in tumour

What’s new?

MMR IHC in uterine and other cancers

can diagnosed in deceased relative due tissue stored

Penetrance lower than previously thought

can’t rely on FHx (identified <25%)

Universal testing

All CRC

? All uterine or all <60

Screening Works in Lynch Syndrome

Colonoscopy Removes polyps before can become cancer

Every 1 to 2 years

Start at age 25 or 5 years before earliest bowel cancer

Hereditary Cancer Registry

Screening not recommended rarer cancers Except gastroscopy from age 30 families with gastric cancers

Screening doesn’t work for ovarian cancer;

Screening not recommended for uterine cancer; hysterectomy + BSO at 40

www.eviQ.org.au

Familial Adenomatous Polyposis

http://www.endoatlas.sk/

Familial Adenomatous Polyposis 100 or 1000’s of adenomatous polyps in colon

(or multiple <30yr or > 20 after 30yr) Colon cancer almost 100% by age 40 without management

Caused by mutation in the APC gene 1/3 “de novo” Associated with:

Desmoids, especially abdominal in 12% Duodenal polyps (in 90%) or gastric polyps (in ~50%)

Ampula of vater cancers

Jaw osteomas (OPG Xray), extra teeth, CHPRE (ophthalmologist) Hepatoblastoma in children

Management of FAP

Can offer genetic testing to children

From age 12: flexible sigmoidoscopy. Switch to colonoscopy when polyps start

Colectomy when polyp load unmanageable Usually late teens / early 20’s

Upper endoscopy from age 25

Attenuated FAP – later, fewer polyps May not have non GI features

Autosomal Recessive Inheritance

Polyps > 30 by 29 yrs

MutYH associated polyposis: MAP 10s to 100s polyps Managed like AFAP

Adenoma: (Lynch, FAP, AFAP, MAP) but also common in older persons

Hamartoma: uncommon.

some with phenotype (Cowden; PJS)

Juvenile polyposis syndrome

Polyps

www.gastrointestinalatlas.com

www.endoatlas.sk http://www.casesjournal.com/content/1/1/68 Juvenile polyps

http://clinicfordigestivesurgery.com/

Syndromes with a phenotype and hamartomas

Peutz Jeghers syndrome (STK11)

Cowden syndrome (PTEN)

Juvenile Polyposis > 5 juvenile polyps of colon or multiple in GI tract

Genes: BMPR1A (20%) and SMAD4 (20%) genes

Cancer risk Colon = 7%-22% by age 35; gastric = 21% if polyps

Often bleeding and anaemia

Upper and lower endoscopy +/- resection

SMAD4 =hereditary hemorrhagic teleangectasis 15-22%

Serrated Hyperplastic

Pathological definition Epithelial lesion with unfolding of the crypt epithelium “serrated

appearance”

Polyps

http://www.endoatlas.sk/

Polyposis syndromes

Serrated Polyposis Syndrome (SPS): any of a) > 5 serrated polyps proximal to the sigmoid; 2 > 10 mm.

b) any number of serrated polyps proximal to sigmoid colon + a first-degree relative with SPS

c) > 20 serrated polyps, any size, anywhere in colon

Mixed Polyposis syndrome

Genes identified but only small % of families GREM1, POLE, PLOD1 etc...

Management for polyposis

Screening colonoscopy as directed by polyp load and family history

Colectomy if required due to polyp load Unclear if non GI cancer risks Unclear offspring risk in some families:

? Dominant or recessive inheritance ? Multiple genes ? Environmental interactions

Diet, Lifestyle and Health Body Weight

Who should see a Genetic Oncologist

Bowel cancer < 50 years or with loss of staining

Polyps Young age (eg 3 by age 30) or Lots of polyps ( >20 over time)

3 or more “special” polyps: hamartomatous or juvenile

Rare cancers eg ampula of vater

Woman with uterine cancer <50 (especially if not obese)

Family history: multiple, young onset, “syndromal”

Also: triple negative breast cancer, any age

High grade epithelial ovarian cancer, any age

Are Haematological Malignancies Heritable?

Li Fraumeni (TP53) and ALL

In 2001: RUNX1 (MDS/AML) In 2004: GATA2 (MDS/AML)

TERT/TERT2 (AML) CEBPA (MDS/AML) ETV6 (MDS/AML) DDX21 (MDS/AML) ANKRD26 (MDS/AML) PAX5 (ALL) SRP72 (MDS)

Hamish S. Scott, Centre for Cancer Biology,

SA Pathology, Adelaide, Australia

Australian Familial Haematological

Cancer Study (AFHCS)

Li Fraumeni syndrome

Mutation in TP53 (protein = p53)

Multiple cancers, young age

Sarcoma

Lung, leukaemia Chompret Criteria

Breast, Brain

TP53 commonly mutated in sporadic cancers

Li Fraumeni Cancer Risks Which is false

1. Risk of cancer is 15% by age 30

2. No screening except for breast in women

3. Breast cancers likely to be triple positive

4. Breast cancer screening, including MRI, starts at 20yrs

Li Fraumeni syndrome

Lifetime Cancer risks

Female

15% by 20 yrs 50% by 30 yrs >90% by 50 yrs

Male

15% by 20 yrs 20% by 30 yrs 60% by 50 yrs

Breast : 4.8% of breast cancer <30 (especially if triple positive: ER+/PR+/HER2+)

Risk Reducing Mastectomy or MRI from age 20

No evidence for screening for other cancers

Avoid smoking, UV and radiation

MDS/AML Families…Genetic Heterogeneity

MDS-AML GATA2

AML - Eosinophilia CEBPA

Familial Platelet

Disorder-AML

RUNX1

GATA2

Germline GATA2 mutations = predisposing, but require other factors for disease

Clinical utility is great for mutation negatives!

Unproven for mutation positives…

Selection of BMT donors from relatives

Prophylactic transplantation / identification of unrelated donor

Preimplantation diagnostics

Targeting Actionable Mutations

JK. 45, Asian, non smoker Adenocarcinoma of lung. Liver mets at Dx What to do? 1. Start chemo 2. Palliative care 3. Send tumour for EGFR mutation testing 4. I now do cancer genetics only, so not my problem

EGFR testing

EGFR mutation almost exclusively in Non Squamous

10% Western but 50% Asian

Higher in females and never smokers

NATA accredited lab: EGFR negative

EGFR-TKI use restricted to non squamous NSCLC that have mutations in the EGFR gene

Arrange for Massive Parallel Sequencing (genomic testing) of tumour at research lab

Research lab Identifies rare mutation in EGFR

States case study suggesting mutation “actionable”

You notify pt of result and state ~30% of pts with NSCLC have rare activating mutations

inform pt that EGFR-TKIs delay progression of cancer by 9 mths cf 6mths with chemotherapy

Mention clinical trials have demonstrated efficacy in common EGFR mutations

State effectiveness in pts with rare mutations unknown

On balance, recommend pt proceed with self funded EGFR-TKI

Commence TKI

Pt commences TKI

6 days later develops rapidly progressive interstitial lung disease

(is a rare side effect ~1.6% but 13% mortality)

Pt dies

Review of case instigated

Repeat molecular testing in a NATA lab fails to identify the mutation - ? Sample mix-up?

Are you going to be sued / disciplined ?

NHMRC: Principles for the translation of “omics”-based tests from discovery to health care

Sample transferred from NATA lab to research lab for the purpose of providing a test result for clinical use

Violates several domains:

Result not validated in NATA lab

No pt consent or ethics approval

Standards for data collection and transmission not followed

Pt not provided with “fair and balanced view”

Wasn’t told if EGFR wt

If treated with TKI PFS = 1.5mth vs 6mth with chemo (IPTASS study)

Interpretation of test result and risk benefit of treatment required MDT input

Pre-test and pre-treatment counselling not provided

Cancer Genetics and Malpractice Over 50 cases in 2011

Mainly physicians

Liable for failing to:

Are Liable if you:

1. Fail to take an adequate family history

2. Fail to recommend appropriate testing

3. Fail to refer to geneticist or genetics counsellor

4. Fail to interpret test result correctly or in timely manner

5. Fail to recommend risk mitigation strategies

6. Fail to disclose patient’s test results to at-risk family members

Cancer Genetics and Malpractice Failure to warn of genetic predisposition to cancer

FHx, not referred for testing. Developed breast cancer, BRCA1 found. Sued for failure to warn

Failure to utilise tumour genetic marker to tailor treatment Premature utilisation of genetic test to tailor treatment

Uses commercially available test to determine risk of breast cancer recurrence. Advises against chemo. Pt recurs. Sues for relying on non standard of care test

Failure to disclose genetic risk to patient’s family member Physician asks pt with BRCA mutation to inform daughter. Pt refuses. After pt

death, daughter develops breast cancer. Sues for failure to disclose

Breach of confidentiality: unauthorised disclosure of patient’s data Physician asks pt with BRCA mutation to inform daughter. Pt refuses. Dr

contacts daughter and discloses potential genetic risk. Pt sues

Who gets a Genetic Test? eviQ guidelines and risk calculators

Need a 10% “pretest” likelihood Clinical vs research testing

Person in the family most likely to have the mutation

Cost: Single gene (Lynch Syndrome) $850 BRCA1 and BRC2 (tested together)$1000 Predictive testing $200 to $400 Founder mutations (Ashkenazi heritage and 3 BRCA mutations) $350-400 Panel $3000 Tumour ~$5000

Why do you need counselling “Informed consent”

What to do if a mutation search is “positive”?

Risk management, guilt re children

What to do if a mutation search is “negative” (uninformative)?

Doesn’t exclude genetic cause

Risk management

What to do if a predictive test is negative? Population level risk, “survivour guilt”

How and when to tell children / other relatives?

What about insurance?

Insurance and Genetic Testing

Lots of media play but No effect medical or travel insurance No effect existing policies

May affect a new or changed life or disability policy Having had a cancer has greater effect Family history has to be disclosed May actually reduce premium

May affect a relative’s new life or disability insurance

Summary

All cancer is a genetic disease

Some families have inherited a mutation that significantly increases the risk of cancer(s)

In most cases, lots can be done to reduce that risk

Families can’t be seen if they’re not identified

Familial cancer services can’t see everyone

eviQ is a great resource

Distance isn’t an issue due to telehealth

Thank you