clinical dilemmas in genetic testing

Clinical Dilemmas in Genetic Testing

A Case-based Approach

Jeffrey A. Tice, MD

Division of General Internal MedicineUniversity of California, San Francisco I have no conflicts of interest

Definitions

• “Genetic Testing”

–Many types (full sequencing, single site, common mutations, chromosome analysis, etc.)

–Many tissues (tumors, blood, buccal mucosa)

–Not just a test, a process

–Specialty labs

• “Disease Predisposition”

–Risk is a complex issue, not “all or nothing”

–What to do with results?

4 possible cases

• Ivana Test: 24 year old, mother just diagnosed; aunt died of breast cancer “I just want the test”

• Cy Fibrosis: 28 year old with male infertility. His genetic testing for cystic fibrosis is prompting his wife to consider testing.

• Ima Clotter: Healthy 30 year old whose sister was found to carry “blood clotting genes” after several miscarriages. Not sure she wants to test, “How would it change things for me?”

• Dina Child: Healthy 36 year old woman who is pregnant and wants that new DNA test using her blood to tell her the sex of the fetus.

Ivana Test’s Family History

• Mother diagnosed last month with breast cancer

• Aunt with breast cancer was paternal, died at 45

• Eastern European

• Not close with paternal side

– Only one cousin

– Paternal grandmother died young

Three Generation Pedigree

85MI

45

22

47Breastca 65

2824

66

Breast ca 40, d45

d. 50 or so?StomachDiabetes

Breast cancer UCSF

Cancer Risk Program

d.71d.62

Three Generation Pedigree, Next Visit

85MI

45

22

47Breastca 65

2824

Prostate ca 55, Now 66

Breast ca 40, d45

d. 40OvarianDiabetes

Breast cancer

Prostate cancer UCSF

Cancer Risk Program

d.71d.82

Breast 33

Ovarian cancer

• “Cancer on the father’s side of the family doesn’t count.”

• “Ovarian cancer in the family history is not a factor in breast cancer risk.”

• “The most important thing in the family history is the number of women with breast cancer.”

Misconceptions About Family History

•Half of all women with hereditary risk inherited it from their father.•Ovarian cancer is an important indicator of hereditary risk, although it is not always present.•Age of onset of breast cancer is more important than the number of women with the disease.

© 2001 Myriad Genetic Laboratories

Genetic Counselor’s Family History

• Extensive pedigree, including cousins

• Verify cause of death, age of diagnosis and death

• Ovarian and “female” cancers often not discussed

• Ask about Jewish ancestry

• Next step is to test individual already affected with cancer

–“Genetics is a family business” ASCO

How Much Breast Cancer Is Hereditary?

Sporadic

Family clusters

Hereditary

Breast Cancer5%–10%

15%20%

Lifetime Risk of Breast Cancer by AgeBRCA1-2 Mutations Increase the Risk of

Cancer More Than Other Factors

Relative risk of breast cancer

Family history

BRCA1-2mutation

Early menarche

Late age at birth of 1st child

Benign breast

disease

Hormone replacement

therapy

Alcohol use

Cells have Two Copies of BRCA1

and BRCA2

BRCA2 BRCA1

Adapted from Tools for Understanding GeneticsNational Human Genome Research Institute

Office of Science Education and Outreachwww.nhgri.nih.gov/DIR/VIP

Each child has a 50% chance of inheriting an autosomal dominant disorder

Father withmutation on one chromosome

Autosomal Dominant Inheritance

BRCA 1 / 2 Associated Cancers:Lifetime Risk

Breast Cancer 12% 60-85%

Second Primary Breast <1% 40-50%

Ovarian Cancer 1.5%

BRCA1 - 20-40%

BRCA2 - 10-20%

General BRCA Population Population

Other cancers associated with BRCA2 mutations

Relative Risk*

• Melanoma 2.6

• Stomach 2.6

• Pancreatic 3.5

• Prostate 4.7

• Biliary tree 5.0

* Br Ca Linkage Consortium, JNCI 1999

ASCO

Features Indicating Increased Probability of BRCA Mutations

• Multiple cases of early onset breast cancer

• Ovarian cancer (with family history of breast or ovarian cancer)

• Breast and ovarian cancer in the same woman

• Bilateral breast cancer

• Male breast cancer

• Ashkenazi Jewish ancestry

Founder Mutations

General population

~ 1/500 carry BRCA mutations

Hundreds of different mutations identified

Ashkenazi Jewish population

1/40 carry one of 3 specific mutations

2 in BRCA 1 and 1 in BRCA 2

Other “founder” populations

French Canadians, Icelanders, Polish,…

Three Possible BRCA results

• Positive: Known deleterious mutation found

• Negative:

–True negative: Known mutation in family and patient doesn’t have it

–Uninformative negative: No mutation found, but family history is not explained

• Variant of Undetermined Significance: Change in DNA, but unsure whether it’s deleterious or benign

How many “uninformative negatives” are really positive?

• 300 very high risk families with “uninformative negative”results AND 4 family members with breast or ovarian cancer

• 12% had duplications (repeated words), deletions (missing word), or rearrangements (misplaced word) in BRCA1 or BRCA2

• Unclear how common these “false negatives” are in the larger population receiving BRCA testing

Walsh, JAMA 2006

What are the Options for Carriers of Mutations?

Breast Cancer

Increased and earlier surveillance

Chemoprevention

Prophylactic Mastectomy-- 90% risk reduction

Ovarian Cancer

Trans-vaginal ultrasound and CA-125 q 6 mos

Birth control pills—50% risk reduction in 5 yrs

Risk Reducing Salpingo Oophrectomy (RRSO)

Insurance Questions • Will health insurance cover testing?

– Most cover if medically indicated: Pre-authorization

• Can health insurance plans increase premiums with a positive test?

– It is illegal to change or drop coverage based on a test result; not a pre-existing condition (HIPAA)

• Are there any protections against life insurance discrimination?

– Currently, no federal laws include life insurance

Ivana Test, Conclusion

• Ivana’s father tested positive for a mutation common in the Jewish population

– Men can carry mutations in BRCA1/2

– Start with an affected individual if possible

• Ivana then tested using the Jewish panel and was negative

– A negative result is only a “true negative” when there is a positive result in the family

Cy Fibrosis’s History

• Infertility work-up showed azoospermia

• Congenital absence of vas defrens (1-2% of infertile men)

• Standard CF testing showed patient is a carrier of Delta F508

• He wants to use ICSI (intracytoplasmic sperm injection)

CF is Autosomal Recessive

Cystic Fibrosis Genetics

• CF is caused by mutations in a single large gene on chromosome 7 (codes CFTR protein)

• 250 kilobases, 1480 amino acid protein

• Wide phenotypic variation of disease

• 1998 consensus statement for screening– Family history of CF or partner’s family hx of CF

– Whites of European or AJ descent planning pregnancy or seeking prenatal care

Screen Cy’s wife? If so, how?

Ethnicity Incidence Carrier Frequency F508

White 1/3300 1/25 70%

Hispanic 1/8500 1/46 46%

AJ 1/29 30%

Black 1/15,300 1/65 48%

Native American

Zuni 1/3970 0%

Pueblo 1/1500 0%

Asian 1/32,100 1/90 30%

Cy’s wife has a “variant”

• “Variants of Undetermined Significance” (VUS) occur in over 5% of whites receiving full sequence testing, 20-40% of non-whites

• Fetus: 1/4 chance of

–Carrying VUS

–Carrying Delta F508 mutation

–No mutation/no VUS

–Mutation + VUS

Cy Fibrosis’s Messages

• In any testing process, plan for “next step”

• Use genetic counselors to counsel and provide “informed consent”

• VUS are becoming more common

–Full sequence testing becoming more common

–Testing is becoming more accepted and available in non-white populations

Ima Clotter’s Family History

• Ima is G1P1, on birth control pills, and healthy.

• After 3 miscarriages, Ima’s older sister was found to have a “double defect”

• A third sister is currently pregnant.

• Feels it’s “opened Pandora’s Box” and wonders “How will it change my care if I test?”

What is a “double defect?”

Two inherited thrombophilias

Factor V Leiden , nucleotide 1691 transition from guanine to adenine results in Arg506Gln protein

Prothrombin 20210, guanine to adenine, untranslated

MTHFR variant (C677T)

Protein C deficiency

Protein S deficiency

Risks of first venous thrombosis

Condition Relative Risk Annual Incidence

Normal 1.0 0.008

Hyperhomocysteinemia 2.5 0.02

(MTHFR C677T) 1.0

PT 20210 2.8 0.02

OCP’s 4.0 0.03

Factor V Leiden hetero 7.0 0.06

Plus OCPs 35 0.29

Factor V Leiden homo 80 0.5-1.0

Thromboembolism in Pregnant Women with Inherited Thrombophilias

Condition Probability per pregnancy

None 0.03%

Factor V Leiden 0.25%

PT 20210 0.5%

Factor V and PT 20210 4.6%

Anti-thrombin deficiency 0.4%

Gerhardt, NEJM 2000

Would testing change management?

• Indefinite anticoagulation recommended if

–2 or more spontaneous thromboses

–1 spontaneous thrombosis and-Antithrombin deficiency or antiphospholipid Ab -Life threatening or unusual site-”Double” or more defects

• Anticoagulate during pregnancy if

–Antithrombin deficiency or “double defect”

–Consider if personal or FH of thrombosis

Ima Clotter, Conclusion

• Ima is heterozygous for Factor V Leiden

– She stops OCPs

• Ima’s pregnant sister carries a “double defect”

– She is discussing anticoagulation with her OB

• Both defects are autosomal dominant

• Testing was fairly straightforward, as there were 2 genes with known mutations

Fetal Aneuploidy Detection by Maternal Plasma DNA Sampling

Cell-free fetal DNA testing

Background

Trisomy 21(Down’s)

Trisomy 18(Edwards)

Trisomy 13(Patau)

• Most common chromosome abnormality

• Prevalence increases with maternal advancing age

• 5-10% survive to 1 year

• Severe intellectual disability

• 80% die in the first year

• Severe intellectual disability in survivors

Prenatal Testing is widely used to test for chromosomal abnormalities

Prenatal Diagnostic Testing

Noninvasive tests (screening)

• Maternal serum markers interpreted in the context of maternal age

• Nuchal translucency• Tests often used in

combination

Invasive tests (diagnostic)

• Chorionic Villus Sampling (CVS)

• Amniocentesis

ACOG recommends that pregnant women be offered screening and that all should have the option of diagnostic testing

Prenatal Diagnostic Testing

• Noninvasive tests are “screen positive” or “screen negative”

- Screen negative: risk of a baby with Down syndrome is less than a predetermined level (eg <1/500)

Individualized risk scores provided

- Screen positive: Patient is offered option of CVS or amniocentesis

• Invasive tests are diagnostic

- Fetal karyotyping

- CVS performed earlier than amniocentesis

• Fetal blood found in the maternal circulation• Cell free nucleic acids more plentiful in maternal

circulation- Fetal DNA can be detected by 5th postmenstrual week- % increases with increasing gestational age

Cell-free Fetal DNA (cfDNA)

Massive Parallel Signature Sequencing (MPSS)

Directed Analysis(DANSR)

• Massive parallel sequencing random analysis of millions of cell free DNA fragments and assigns all free DNA fragments to specific chromosomes

• Number of chromosome counts compared to control values

• Directed DNA analysis selectively sequences relevant chromosomes

• Digital analysis of selected regions (DANSR)

• Uses fewer genetic fragments than MPSS

cfDNA: Potential Roles

Primary screening to replace current noninvasive tests

“Advanced”screening test

Potentially could reduce number of invasive tests and resulting loss of normal fetuses

Cannot replace diagnostic tests

Screening Test

Currently available cfDNA tests

Company LDT nameTrisomy identified Testing approach

T21(Down

Syndrome)

T18(Edwards

Syndrome)

T13(Patau

Syndrome)DANSR MPSS

AriosaDiagnostics

Harmony Prenatal Test

X X X X

Sequenom MaterniT21 X X X X

VerinataHealth

verifi prenataltest

X X X X

cfDNA: Evidence

• Eight studies evaluated cfDNa as screening for fetal aneuploidy

- 5 used MPSS

- 3 used DANSR

• Most studies were in high risk women

• Trisomies- All studies evaluated T21

- 6 studies evaluated T18

- 2 studies evaluated T13

• In all studies, true chromosome status was known

cfDNA: Evidence

Palomaki, 2011; Palomaki, 2012;

Number of fetuses with abnormal karyotypes ranged from 39 to 283 in the largest study

Bianchi Study Palomaki Study

Aneuploid fetuses 221 283

Trisomy 21 89 212

Trisomy 18 36 59

Trisomy 13 14 12Other aneuploidies 82 None

MELISSA: cfDNA Multicenter Study

• 2,882 high risk women undergoing prenatal diagnostic procedures at 60 sites

• Nested case-control- All singleton pregnancies with any abnormal karyotype- Balanced number of randomly selected euploid

pregnancies

Bianchi, 2012

522 samples (221 abnormal karyotypes*)# cases Sensitivity Specificity

Trisomy 21 89 100%

100%Trisomy 18 36 97.2%

Trisomy 13 14 78.6%

*139 other aneuploidies

cfDNA Multicenter Validation Study

• Nested case control study in cohort of 4,664 high risk pregnancies

• 212 Down Syndrome and 1,484 matched euploidpregnancies

• 98.6% sensitivity for Down Syndrome

• 0.2% false positive rate

• Testing failed in 0.8% of pregnancies

Palomaki, 2011

NICE: Prospective Cohort Study

Multi-center cohort study: 3,228 participants all undergoing prenatal diagnostic procedure for any

reason

Norton, 2012

N=3,228Number of aneuploidy

casesSensitivity Specificity

Trisomy 21 81 100% 99.97%

Trisomy 18 38 97.4% 99.93%

cfDNA: Comparative Studies

How does use of cfDNA compare with the established alternatives?

• Currently no direct evidence comparing cfDNA with established screening strategies for primary screening

• No studies have evaluated the potential role of cfDNAas a primary screening strategy

• To date studies have only evaluated high risk women using cfDNA as an advanced screening test

cfDNA: Clinical and Economic Model

• Used clinical data from MELISSA study

• Evaluated the impact of incorporating cfDNA into routine clinical care

- High risk women

- Advanced screening test

• 66% reduction in diagnostic induced miscarriages

• 38% more women receiving T21 diagnosis

• 1% overall reduction in prenatal screening and diagnostic costs

Garfield, 2012

Average risk women

• 2,049 women undergoing routine screening at 11-13 weeks gestation

• Fetal karyotyping in 86 cases

- Remainder considered phenotypically normal

• For all 8 cases of T21, the T21 Risk Score was >99%

• For 2 of the 3 cases of T18, the risk score was >99%

• cfDNA testing could not be performed on 4.9% of cases

cfDNA: Conclusions

• Promising new technology with high sensitivity and specificity for fetal aneuploidy when evaluated in high risk women

• To date, cfDNA has not been compared with current screening methods as an alternative to primary screening

• Currently no evidence to support use in average risk women

To summarize…

Why consider testing for predisposition genes?

• To identify patients not at risk despite family history - reassurance

• To identify patients at very high risk of disease

• To allow high risk patients to consider increased screening and prevention

• To assist with prenatal counseling

• To allow patient to enter screening/ prevention trials

• To provide important health info to extended family

ASCO

A Multi-Step Process: Pretest Genetic Counseling

AssessPersonal and family medical historyRisk perception and motivation for testing

EducateBasic genetics and inheritanceGenotype/phenotype disparities and risk

DiscussRisks, benefits, and limitations of testing Test procedure Alternatives to testingManagement options

ASCO

A Multi-Step Process: Post-test Genetic Counseling

ReviewEducational concepts and family historyRisk and prior probabilities

DiscloseTest results Interpretation of results

DiscussPlans for prevention and treatmentSharing results with family membersPotentially testing other family members

clinical dilemmas in genetic testing

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