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Hereditary Cancer Testing Panels. Jill Siegfried, RN, MS, CGC Certified Genetic Counselor. Elizabeth Chao, MD Director of Translational Medicine. Overview. Introduction to Ambry Current Testing and Next-generation (NGS) Methodologies Overview of NGS Panels and Indications Clinical Utility

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Hereditary cancer testing panels

Hereditary Cancer Testing Panels

Jill Siegfried, RN, MS, CGC

Certified Genetic Counselor

Elizabeth Chao, MDDirector of Translational Medicine


  • Introduction to Ambry

  • Current Testing and Next-generation (NGS) Methodologies

  • Overview of NGS Panels and Indications

  • Clinical Utility

  • Variants of Uncertain Significance

  • Insurance Coverage

Ambry s current major diagnostic methods
Ambry’s Current Major Diagnostic Methods

  • Applications:

    • Sanger Sequencing for single gene analysis

    • Pyrosequencing and NextGenSeq for targeted mutation analysis

    • Targeted enrichment and Next-gen sequencing for larger gene panels and exome analysis

    • MLPA for single gene deletion/duplication analysis

    • CMA and SNP-CGH array for genome wide deletion/duplication analysis and structural variant detection

Mission values
Mission & Values


Providing quality genetic and genomic answers and tools to our clients for the care and management of patients worldwide.




Client Care


On-Time Delivery

Ambry s cancer menu
Ambry’s Cancer Menu

Why cancer ngs panels
Why Cancer NGS Panels?

  • Efficient sequencing of targeted regions of cancer related genes

  • Many genes implicated in each cancer

    • Testing multiple genes simultaneously can be more time and cost effective

  • Aid in clinical diagnosis when clinical criteria are uncertain

  • There are different clinical implications for hereditary versus sporadic breast and colon cancer patients and their families. Important to understand genetic contribution for treatment and prevention

  • There are currently no dx breast cancer panels for intermediate risk genes

    • Need for expanded screening of breast cancer-related genes besides BRCA1 and BRCA2 as many patients are negative for BRCA1 and BRCA2 mutations

  • Scalability

Nextgen sequencing illumina gaiix hiseq2000 miseq
NextGen SequencingIllumina GAIIx, HiSeq2000, Miseq

Massive parallel sequencing---leap from capillary sequencing (96 well x 500 bp) to 250 GB of sequence in a run

Since 2007 at Ambry

Introduced a number of diagnostic panels (XLMR, Marfan, PCD panels, and today new cancer panels) on GAIIx, and Exome sequencing on Hiseq2000

Decreased per basepair cost allows for large panel design at reasonable cost and turn-around-time

Hereditary cancer testing panels

The Ambry RainDance Technologies NGS Panels

Alignment & NextGeneSequence Viewer

DNA iso from blood or saliva

TruSeq Library Prep,

IlluminaGAIIx Sequencing

RDT Enrichment

Sanger Verification

AVA: Ambry Variant Analysis


  • All identified Variants

  • All amplicons with low coverage

Deletion duplication analysis cancerarray design
Deletion/Duplication AnalysisCancerArray™ Design

Target gene implicated in cancer

Exon-level coverage




Targeted Regions

Backbone region

1 probe per 20kb

5.1 probes per exon

Next gen cancer panels hereditary breast ovarian and colorectal cancer
Next-gen Cancer PanelsHereditary breast, ovarian, and colorectal cancer

  • BreastNext

  • OvaNext

  • ColoNext

  • CancerNext

  • Comprehensive sequence and deletion/ duplication testing


  • Gene sequencing for all 14 genes

  • Deletion and Duplication Analysis

All breast cancer susceptibility genes are not created equal moderate to high penetrance alleles
All Breast Cancer Susceptibility Genes are Not Created EqualModerate to High Penetrance Alleles







Meindl et al 2011

In the context of family history atm brip1 chek2 palb2 and others with 2x increased lifetime risk
In the context of family historyATM, BRIP1, CHEK2, PALB2 and others with 2x increased lifetime risk

  • 70% of women have a lifetime risk below 10% (solid blue)

  • For women with a genetic mutation and FHx, 70% have a lifetime risk above 60% (dashed red)

Therefore, mutation testing of these genes for such women may be as clinically relevant as is mutation testing for BRCA1 and BRCA2. We argue that detection of mutations in these genes may be of considerable clinical consequence in terms of absolute breast cancer risk (that is, penetrance) for women with a strong family history

No Family History

Family History

Byrnes et al Br Cancer Res 2008

Moderate penetrance breast cancer genes
Moderate Penetrance Breast Cancer Genes

  • “Mutations in CHEK2, ATM, NBS1, RAD50, BRIP1, and PALB2 are associated with doubling of breast cancer risks”

    Walsh et al. Cancer Cell 2007

Hereditary cancer testing panels

Byrnes et al Breast Cancer Research 2008

Meta analysis of large c ase control s tudies of mild to moderate risks variants
Meta-analysis of large case-control studies of mild to moderate risks variants

  • ATM

    • O.R. 1.20- 4.56

  • CHEK2

    • O.R 1.52-3.10

  • NBN (NBS1)

    • O.R. 2.42

Zhang et al. Lancet Oncology 2011

Palb2 and breast cancer
PALB2 and Breast Cancer

  • Reported in 1-3% of BRCA1/2 negative families

  • Also in the FA-BRCA pathway

    • Estimate a 2-4 fold increase in breast cancer risk

    • Biallelic mutations result in Fanconi anemia type N (FANCN)

  • WECARE study (Tischkowitz et al. Hum Mut 2012)

    • O.R. : 5.3 (1.8-13.2)

    • n~500 cases and ~500 controls

  • Also at increased risk for:

    • Pancreatic Cancer (Jones et al Science 2009)

    • Ovarian Cancer (Walsh et al PNAS 2011)

  • Nbn nbs1 and breast cancer
    NBN(NBS1) and Breast Cancer

    • Founder mutation in Slavic populations of Central and Eastern Europe, c.657del5

      • Seen in 90% of NBS cases and 50% of heterozygotes with cancer

      • Frequency of this mutation is 1/100-1/200 but has been reported as high as 1/30

    • Truncating Mutations

    • Missense mutations may have decreased penetrance, such as p.R215W

    • Mutation prevalence is inversely correlated with age at diagnosis

    Bogdanova et al. Int J Cancer 2008

    Steffen et al. Int J Cancer 2006

    Mutyh and breast cancer review of conflicting data
    MUTYH and Breast CancerReview of conflicting data

    • Excess rate of extracolonic malignancies has been reported in individuals with MUTYH mutations (1,2)

      • Suggested elevated risk of CRC, gastric, ovarian, bladder, skin, breast and endometrial cancers

    • Follow-up showed no association of MUTYH and breast cancer risk (3,4)

    • Increased risk of breast cancer in familial cancer and polyposis families

      • 18% of female MAP patients with breast cancer (5)

      • 5-7% of familial colorectal and/or breast cancer families were heterozygotes (6)

        • 1.9% of controls

        • Rates were similar in predominantly CRC vs Breast families

    • Increased breast Cancer Risk in Sephardic Jews (7): O.R. 1.39-1.86

    • Trend towards association of MUTYH heterozygotes with sporadic breast cancer but insufficient power to detect O.R<2 (8)

    Win et al Fam Cancer 2010

    Vogt et al Gastroenterology 2009

    Zhang et al. CEPB 2006

    Beiner et al Br Can Res Treat 2009

    Nielsen et al. J Med Genet 2005

    Wasielewski et al. Br Can Res Treat 2010

    Rennert et al. Cancer 2011

    Out et al. Br Can Res Treat 2012


    • Gene sequencing for all 19 genes

    • Deletion and Duplication Analysis

    Atypical phenotypes
    Atypical Phenotypes

    Li-Fraumeni Syndrome

    “Mutations not associated with “typical phenotypes” are of particular interest. For example, the three TP53 mutations occurred in patients without a family history of Li–Fraumeni syndrome and the two MSH6 mutations occurred in patients without a family history of Lynch syndrome. As comprehensive genetic testing is undertaken for individuals not selected for established syndromic phenotypes, a wider range of expressivity associated with germ-line mutations of cancer susceptibility genes will become increasingly apparent.”

    Walsh et al. PNAS 2011

    Prevalence of hereditary ovarian cancer walsh et al pnas 2011
    Prevalence of Hereditary Ovarian Cancer (Walsh et al. PNAS 2011)

    • Previously reported by TCGA at 14%, only in BRCA1or BRCA2

    • Reported at 24% (62/282)

      • 7% of these are structural changes

    • 25% of hereditary ovarian cancer is related to a mutation in one of OvaNext genes

    Hereditary susceptibility to crc
    Hereditary Susceptibility to CRC


    Jasperson et al. Gastroenterology 2010


    • Gene Sequencing for all 14 genes

    • Deletion and Duplication Analysis

    Everybody in my family gets cancer
    Everybody in my family gets cancer.”

    Pancreatic ca dx 57

    Lung ca dx 80

    prostate dx 65

    breast dx 55

    Stomach ca dx 41

    64 y

    Colon ca dx 51

    d. 40 Accident

    2 polyps 39

    Lobular breast ca dx 32;

    2 polyps, 34y

    “GI” ca dx 45

    Leukemia dx 11

    Ductal breast ca dx 42


    • Gene Sequencing for 22 genes

    • Deletion and Duplication Analysis, plus additional 55 genes.

    Nccn guidelines
    NCCN Guidelines

    Lifetime breast cancer risk nccn
    Lifetime Breast Cancer Risk & NCCN

    • Lifetime Breast Cancer Risk of 20-25%

      • Threshold for designating women high risk for breast cancer, as stated in ACS Guidelines for breast screening with MRI as an adjunct to mammography (Saslowetal 2007)

        • “A high risk of breast cancer also occurs with mutations in the TP53 gene (Li-Fraumeni syndrome) and the PTEN gene (Cowden and Bannayan-Riley-Ruvalcaba syndromes).

        • “…In cases in which insufficient evidence to recommend for or against MRI screening, decisions should be made on a case-by-case basis…”

    Publications cited by NCCN guidelines for above statements:

    SaslowetalCa Cancer J Clin 2007;57:75-89.

    Murphy etalCancer 2008;113:3116-3120.

    Nccn example pten cowden syndrome genetic familial high risk assessment version1 2011
    NCCN Example – PTEN(Cowden Syndrome) Genetic/Familial High-Risk Assessment Version1.2011

    • Breast Cancer Screening Guidelines:

      • BSE beginning at age 18y

      • CBE every 6-12 mo, age 25y or 5-10y before the earliest known breast cancer

      • Annual mammography & breast MRI starting 30-35, or 5-10 y b/f earliest known ca in family (whichever comes first)

      • Discuss option of risk reducing mastectomy and hysterectomy on case-by-case basis….

    • Add’l recommendations for endometrial, thyroid, colon, and dermatologic screenings

    • Cited breast cancer risk:

    • An estimated breast cancer risk of 25-50% with average age of 38-46 years at diagnosis.

    Publications cited by NCCN Guidelines for above statements:

    StarinketalClin Genet 1986;29:222-233

    Brownstein etal. Cancer 1978;41:2393-2398

    Emerging data nccn guidelines
    Emerging Data & NCCN Guidelines

    • CDH1 - Discussed in:

      • Genetic/Familial High-Risk Assessment Version 1.2011

        • Cites a cumulative risk for female lobular breast cancer by age 75 as high as 52%, and that mutations may be associated with lobular breast cancer in the absence of diffuse gastric cancer.

      • Gastric Cancer Version 2.2011

        • “E-Cadherin mutations occur in approximately 25% of families with….[HDGC]”

        • “Consideration should be given to prophylactic gastrectomy in young asymptomatic carriers….”

    Publications cited by NCCN guidelines for above statements:

    KaurahetalJAMA.2007; 297:2360-2372.

    Schrader etalFam Cancer 2008;7:73-82.

    MasciarietalJ Med Genet 2007;44:726-731.

    Fitzgerald RC, etal. Gut 2004;53:775-778.

    Huntsman DG etal. N Engl J Med 2001;344:1904-1909.

    Emerging data nccn guidelines1
    Emerging Data & NCCN Guidelines

    • PALB2 - Discussed in:

      • Pancreatic Adenocarcinoma Version 2.2012

        • “… and particular mutations in PALB2 and MSH2 have been identified as possibly increasing pancreatic cancer susceptibility.”

        • “….Thus, gemcitabine plus cisplatin may be a good choice in selected patients with disease characterized by hereditary risk factors (egBRCA or PALB2 mutations).”

    Publications cited by NCCN guidelines for above statements:

    Canto etalClinGastroenterolHepatol 2006;4:766-781.

    Lowery etalOncologist 2011;16:1397-1402.

    Historic perspectives on hboc
    Historic Perspectives on HBOC

    Identification of Genetic Risk for Breast-Ovarian Cancer

    • 1866- Paul Broca provides a detailed scientific description of inherited breast-ovarian cancer.

    • Mid 1980s - Quest for genetic basis of hereditary breast and ovarian cancer

    • Dec 1990 - HBOC linked to chromsome 17q21 by Mary-Claire King & colleagues, UC Berkeley

      • Mar 1994 – Breast Cancer Linkage Consortium (BCLC) outlines risks for cancer beyond breast cancer for BRCA1

      • Oct 1994 – The cloning of BRCA1 described

    • Dec 1995 – The cloning/discovery of BRCA2

      • Aug 1999 – BCLC outlines risks for cancer beyond breast/ovarian cancer for BRCA2

    • Dec 1999 –Publication of evidence that BRCA1 and BRCA2 are involved in the DNA damage response. “BRCA1, BRCA2 and their possible function in DNA damage response.” Kate-Jarai Z etal. PMID: 10584867

    Historic perspectives on hboc1
    Historic Perspectives on HBOC

    Prophylactic surgical interventions for women with HBOC

    • 1995

      • Prophylactic Oopherectomyin inherited breast/ovarian cancer families.StruewingetalJNCI PMID: 8573450 :

        • Eg: StruewingetalJNCI PMID: 8573450 : Multi-center study to determine the incidence of post-oophorectomycarcinomatosis and to quantify the effectiveness of preventive surgery

    • Sept 1999

      • Studies demonstrate that prophylactic oophorectomy reduces ovarian cancer risk, and then specifically shows that prophylactic oophorectomy reduces both ovarian and breast cancer risk in BRCA carriers. RebbecketalJNCI PMID: 10469748

    • Jan 1999

      • “Efficacy of bilateral prophylactic mastectomy in women with a family history of breast cancer.” Hartman etalNEJM PMID: 9887158

        • Study supports the suggestion that prophylactic mastectomy reduces risk of breast cancer. However, it was retrospective and many women did not have genetic analysis performed

    • Mar 2004

      • Bilateral prophylactic mastectomy reduces breast cancer risk in BRCA1 and BRCA2 mutation carriers: the PROSE Study Group. Rebbecketal JCO PMID: 14981104

        • Demonstrates that bilateral prophylactic mastectomy reduces the risk of breast cancer in women with BRCA1/2 mutations (with or without prophylactic oopherectomy, by approximately 95%, and 90%, respectively).

    Historic perspectives on hboc2
    Historic Perspectives on HBOC


    • July/Sept 1998 –

      • Three papers describe three trials that investigate the role of Tamoxifen as a chemopreventive agent for breast cancer.

        • Fisher etal PMID: 9747868, PowlesetalPMID: 9672274,Veronesietal PMID: 9672273

        • Fisher B et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 study. J Natl Cancer Inst 1998;90:1371–88. [PubMed: 9747868]

    • June 2004

      • Analysis of 491 women with stage I or stage II breast cancer, for whom a BRCA1 or BRCA2 mutation had been identified in the family

      • Estimate risk of contralateral breast cancer in BRCA1 and BRCA2 carriers after diagnosis

      • Authors conclude, “The risk of contralateral breast cancer in women with a BRCA mutation is approximately 40% at 10 years, and is reduced in women who take tamoxifen or who undergo an oophorectomy.”

        • Metcalfe et al. Contralateral breast cancer in BRCA1 and BRCA2 mutation carriers. J ClinOncol2004;22:2328–35. PMID: 15197194

          MRI surveillance – similar trajectory

          Targeted chemotherapeutics - PARP inhibitors in Phase II clinical trials

    Hereditary cancer testing panels

    Variant Rates for Genes Previously Available for sequencing at Ambry

    ‘▪’ = sequencing available at Ambry for ~1-3 years

    Average chance of a variant by Gene ~5%

    Ambry s variant classification scheme
    Ambry’s at Ambry Variant Classification Scheme

    Always included in

    results report with interpretation of result and description of gene

    Always included in

    result report, with interpretation of result, description of gene, and supplementary data

    Reported only if requested


    Variant, suspected pathogenic

    Variant, unknown significance

    Variant, suspected benign


    Family studies program
    Family Studies Program at Ambry

    • Complimentary VUS analysis for informative relatives

    • Available to the families of probands with a VUS identified at Ambry

    • Ultimate goal is variant re-classification

      • Provide clinically-relevant information to our probands

    • Family VUS testing generates co-segregation data

      • Does the VUS track with disease?

      • Can provide powerful evidence to support benign or pathogenic role

      • Family studies data will be subsequently included in supplementary materials for that particular variant

    Enrolling in family studies program
    Enrolling in Family Studies Program at Ambry

    • Submit a detailed pedigree & application for family studies

      • Supplement to clinical information on Test Requisiton form



      • Reviewed by genetic counselor/medical director

    • Ambry GCs/MDs with clinician to select informative relatives for cosegregation analysis

      • Complimentary for approved relatives

    • Results reported back to ordering physician

    • Complementary for pre-approved relatives

    Vus reported data example palb2 p p864s c 2590c t
    VUS Reported Data at AmbryExample: PALB2 p.P864S c.2590C>T

    • First detected in 1/96 BRCA-negative high-risk breast cancer families and 0/96 controls

    • Allele frequency data

      • 1000genomes

        • 0.23% overall (5/2188)

        • 1.12% British sub-cohort (2/178)

      • NHLBI Exome Sequencing Project

        • 0.20% overall (21/10737)

        • 0.26% European American (EA) (18/7,020)

    • Genotype frequency data

      • NHLBI Exome Sequencing Project

        • T/C heterozygotes: 19/5359 (0.35%)

        • T/T homozygotes: 1/5359(0.02%)

    • Co-segregatation: not available

    • Co-occurrence: none

    Guenard F et al. Genet Test Mol Biomarkers. 2010 Aug;14(4):515-26.

    Insurance coverage pre verification
    Insurance Coverage/Pre-Verification at Ambry

    • Billing Options

      • Institution Billing

      • 3rd party payor (Insurance) Billing

        • Medicare and many state Medicaid plans are accepted

        • Extensive pre-verification department to assist

        • Pre-verification available before sample submission or at the time of sample submission

      • Patient Direct Payment

        • Payment Plan Options available

    Hereditary cancer testing panels

    Thank at Ambryyou!

    Any questions?