1. Hereditary Cancer Testing Panels
Jill Siegfried, RN, MS, CGC
Certified Genetic Counselor
Elizabeth Chao, MD Director of Translational Medicine

2. Overview Introduction to Ambry
Current Testing and Next-generation (NGS) Methodologies
Overview of NGS Panels and Indications
Clinical Utility
Variants of Uncertain Significance
Insurance Coverage

3. Ambry’s Current Major Diagnostic Methods
Applications:
Sanger Sequencing for single gene analysis
Pyrosequencing and NextGen Seq 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

4. Mission & Values Mission: Values:
Providing quality genetic and genomic answers and tools to our clients for the care and management of patients worldwide.
Values:
Partnership
Quality
Client Care
Flexibility
On-Time Delivery

5. Ambry’s Cancer Menu

6. Familial Adenomatous Polyposis (FAP), Gardner Syndrome, Turcot Syndrome, Attenuated FAP (AFAP)
APC Amplified
APC
PTEN-Related Disorders (including Autism Spectrum Disorder), Cowden Syndrome, PTEN HamartomaTUmor Syndrome (PHTS), Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, Autism / Autism spectrum disorder
PTEN-Related Disorders (including Autism Spectrum Disorder)
PTEN
PALB2-Related Cancer, Breast cancer, Familial, Fanconi Anemia, PALB2-Related, Pancreatic cancer, Familial
PALB2-Related Cancer
PALB2
CHEK2-Related Cancer
CHEK2
Malignant Melanoma, Cutaneous Malignant Melanoma Syndrome, Familial Atypical Mole-Malignant Melanoma Syndrome (FAMMM)
Malignant Melanoma (CDKN2A/p16)
CDKN2A
Pleuropulmonary Blastoma (PPB) Family Tumor and Dysplasia syndrome, DICER1 Syndrome
Pleuropulmonary Blastoma (PPB) Family Tumor and Dysplasia Syndrome, DICER1 Syndrome
DICER1
Multiple Endocrine Neoplasia Type 2 (MEN2), MEN2A (Sipple Syndrome), MEN2B (Mucosal Neuroma Syndrome), Familial Medullary Thyroid Carcinoma (FMTC)
Multiple Endocrine Neoplasia Type 2 (MEN2)
RET
Hereditary Diffuse Gastric Cancer
CDH1
Juvenile Polyposis Syndrome (JPS), HHT, SMAD4-Related
Juvenile Polyposis AMPLIFIED™
BMPR1A, SMAD4
HNPCC (Hereditary Non-Polyposis Colon Cancer), Lynch Syndrome, Muir-Torre Syndrome, Turcot Syndrome
HNPCC / Lynch syndrome DNA Analysis
EPCAM, MLH1, MSH2, MSH6, PMS2
HNPCC / Lynch Syndrome Tumor Testing
MLH1, MSH2, MSH6, PMS2, BRAF
Li-Fraumeni Syndrome
Li-Fraumeni Syndrome (TP53 AMPLIFIED)
TP53
Multiple Endocrine Neoplasia Type 1
Multiple Endocrine Neoplasia Type1 (MEN1)
MEN1
MUTYH-associated polyposis (MAP)
MUTYH-associated Polypsis (MAP)
MUTYH
Pancreatitis, CTRC-related
Pancreatitis, CTRC-Related
CTRC
Pancreatitis, PRSS1-Related
PRSS1
Pancreatitis, SPINK1-related
Pancreatitis, SPINK1-Related
Peutz-Jeghers Syndrome
Peutz-Jeghers AMPLIFIED™
STK11
Retinoblastoma
RB1
Von Hippel-Lindau Disease
VHL

7. 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

8. Next-generation technology

9. NextGen Sequencing Illumina 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

10. The Ambry RainDance Technologies NGS Panels
Alignment & NextGene Sequence Viewer
DNA iso from blood or saliva
TruSeq Library Prep,
Illumina GAIIx Sequencing
RDT Enrichment
Sanger Verification
AVA: Ambry Variant Analysis
Report
All identified Variants
All amplicons with low coverage

11. Deletion/Duplication Analysis CancerArray™ Design
Target gene implicated in cancer
Exon-level coverage
Exon
Targeted Regions
Backbone region
1 probe per 20kb
5.1 probes per exon

12. Panel Overviews and Indications

13. Very rare high-risk variants and rare moderate-risk variants
Hollestelle et al. 2010

14. Next-gen Cancer Panels Hereditary breast, ovarian, and colorectal cancer
BreastNext
OvaNext
ColoNext
CancerNext
Comprehensive sequence and deletion/ duplication testing

15. Other Associated Cancer
BreastNext
Gene sequencing for all 14 genes
Deletion and Duplication Analysis
Gene
Syndrome
Breast Cancer Risk
Other Associated Cancer
BARD1
HBOC
Increased
Ovarian
BRIP1
MRE11A
NBN
25-35%
RAD50
25-48%
RAD51C
ATM
Ataxia Telectangasia
~25-60%
PALB2
Hereditary Breast and Pancreatic
~25-40%
Breast Pancreas
STK11
Peutz-Jegher
30%
Colon Pancreas
CHEK2
Hereditary Breast and Colon
~25%
PTEN
Cowden
25-50%
Thyroid; endometrial; renal
TP53
Li-Fraumeni
50%
Sarcoma; brain; adrenocortical; leukemia
CDH1
Hereditary Diffuse Gastric Cancer
39-52%
Gastric colon
MUTYH
MUTYH-Associated Polyposis
20-25%
Colon

16. All Breast Cancer Susceptibility Genes are Not Created Equal Moderate to High Penetrance Alleles
CDH1
MRE11A
NBN
RAD50
BARD1
MUTYH
Meindl et al 2011

17. In the context of family history ATM, BRIP1, CHEK2, PALB2 and others with 2x increased lifetime risk“ ”
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
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)
Byrnes et al Br Cancer Res 2008

18. 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

19. Byrnes et al Breast Cancer Research 2008

20. Meta-analysis of large case-control studies of mild to moderate risks variants
ATM
O.R
CHEK2
O.R
NBN (NBS1)
O.R. 2.42
Zhang et al. Lancet Oncology 2011

21. PALB2 and Breast Cancer Also at increased risk for:
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 ( )
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)

22. 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

23. MUTYH and Breast Cancer Review 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
Trend towards association of MUTYH heterozygotes with sporadic breast cancer but insufficient power to detect O.R<2 (8)
Carrier of map recessive mtn
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

24. Other Associated Cancer
OvaNext
Gene sequencing for all 19 genes
Deletion and Duplication Analysis
Gene
Syndrome
Breast Cancer Risk
Ovarian Cancer Risk
Uterine Cancer Risk
Other Associated Cancer
BARD1
HBOC
increased
BRIP1
MRE11A
NBN
25-35%
RAD50
25-48%
RAD51C
ATM
Ataxia Telectangasia
~25-60%
PALB2
Hereditary Breast and Pancreatic
~25-40%
Pancreas
STK11
Peutz-Jegher
30%
Colon; Pancreas
CHEK2
Hereditary Breast and Colon
~25%
Colon
PTEN
Cowden
25-50%
5-10%
Thyroid; endometrial; renal
TP53
Li-Fraumeni
50%-70%
Sarcoma; brain; adrenocortical; leukemia
CDH1
Hereditary Diffuse Gastric Cancer
0.39
Gastric; colon
MUTYH
MUTYH-Associated Polyposis
20-25%
MLH1
Lynch
 ??
9-12%
20-60%
colon; stomach; other
MSH2
MSH6
PMS2
EPCAM

25. 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

26. 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

28. Hereditary Susceptibility to CRC?
Jasperson et al. Gastroenterology 2010

29. ColoNext Gene Sequencing for all 14 genes
Deletion and Duplication Analysis
Gene
Syndrome
Colon Cancer Risk
Polyposis
Other Associated Cancer
STK11
Peutz-Jegher
57-81%
Yes
breast, uterine; testicular; cervical; lung; pancreas
CDH1
Hereditary Diffuse Gastric Cancer
Increased
stomach; breast; colorectum
CHEK2
Hereditary Breast and Colon
increased (~10%)
breast; ovarian
PTEN
Cowden
increased
breast; thyroid, renal
TP53
Li-Fraumeni
breast; sarcoma; brain; adrenocortical; leukemia
MUTYH
MUTYH-Assoc Polyposis
35-53%
breast
APC
FAP
~99%
stomach; pancreas; thyroid; CNS; hepatoblastoma
MLH1
HNPCC
60-80%
uterine; ovarian; stomach; bladder; brain; ureter; other
MSH2
MSH6
PMS2
EPCAM
BMPR1A
JPS
9-50%
stomach; other
SMAD4

30. “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

31. Other Associated Cancer
CancerNext
Gene Sequencing for 22 genes
Deletion and Duplication Analysis, plus additional 55 genes.
Gene
Syndrome
Breast Cancer Risk
Ovarian Cancer Risk
Uterine Cancer Risk
Colon Cancer Risk
Polyposis
Other Associated Cancer
BARD1
HBOC
Increased
BRIP1
MRE11A
NBN
25-35%
RAD50
25-48%
RAD51C
ATM
Ataxia Telectangasia
~25-60%
biallelic: leukemia, lymphoma
PALB2
Her. Breast/Panc
~25-40%
pancreatic
CDH1
Her. Diffuse Gastric Ca
39%
gastric
STK11
Peutz-Jegher
0.3
57-81%
Yes
testicular, cervical, lung, pancreas
CHEK2
Her. Breast/Colon
~25%
increased (~10%)
PTEN
Cowden
25-50%
5-10%
thyroid; renal
TP53
Li-Fraumeni
50%-70%
sarcoma; brain; adrenocortical; leukemia
MUTYH
MUTYH-Assoc Polyposis
20-25%
35-53%
APC
FAP
~99%
stomach; pancreas; thyroid; CNS; hepatoblastoma
MLH1
HNPCC
9-12%
20-60%
60-80%
uterine; ovarian; stomach; bladder; brain; ureter; other
MSH2
MSH6
PMS2
EPCAM
BMPR1A
JPS
9-50%
Stomach; other
SMAD4

32. Clinical Utility Variable depending on gene.
Counseling patterns will change;
Discussion about potential VUSs up front
Discussion about varying clinical application of results, but hope for the future and evolving management options
Utility of testing unaffected relatives may vary

33. NCCN Guidelines
Gene
Syndrome
Discussed in NCCN Guidelines?
APC
Familial adenomatous polyposis Y
MUTYH
MUTYH-Associated polyposis
STK11
Peutz-Jegher syndrome
PTEN
Cowden syndrome
TP53
Li-Fraumeni syndrome
MLH1
Lynch Syndrome
MSH2
MSH6
PMS2
EPCAM
BMPR1A
Juvenile Polyposis syndrome
SMAD4
CDH1
Hereditary Diffuse Gastric Cancer
PALB2
Hereditary Breast/Pancreatic Cancer
ATM
Hereditary Breast Cancer
CHEK2
Hereditary Breast/Colon/Other Cancer
BARD1
Hereditary Breast and/or Ovarian Cancer
BRIP1
MRE11A
NBN
RAD50
RAD51C

34. 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 (Saslow etal 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…”
Clinical judgement
Breast can risk has been assessed by models based largely on family history has been used ins ome guidelines to identify a woman as being at high risk of breast ca. For eg, this risk threshold was used in recent updates to the ACS guidelines on breast screening which incorporates MRI
Screening MRI is recommended for women with an approximately 20-25% or greater lifetime risk of breast cancer, including women with a strong family history of breast or ovarian cancer and women who were treated for Hodgkin disease
“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). Accurate prevalence figures are not available, but these conditions appear to be very rare.”
Publications cited by NCCN guidelines for above statements:
Saslow etal Ca Cancer J Clin 2007;57:75-89.
Murphy etal Cancer 2008;113:

35. 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
Clinical Utility
Talk about younger age and MRI – reference NBN gene data presented by Dr. Chao. Mutation rate inversely proportionate to age at onset…
NBN OR 2.42
Cited breast cancer risk:
An estimated breast cancer risk of 25-50% with average age of years at diagnosis.
Publications cited by NCCN Guidelines for above statements:
Starink etal Clin Genet 1986;29:
Brownstein etal. Cancer 1978;41:

36. Emerging Data & NCCN Guidelines
CDH1 - Discussed in:
Genetic/Familial High-Risk Assessment Version
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
“E-Cadherin mutations occur in approximately 25% of families with….[HDGC]”
“Consideration should be given to prophylactic gastrectomy in young asymptomatic carriers….”
PTEN Cum Breast CA risk cited at 25-50
Carriers with germline CDH1 mutations in families with highly penetrant HDGC.
Publications cited by NCCN guidelines for above statements:
Kaurah etal JAMA.2007; 297:
Schrader etal Fam Cancer 2008;7:73-82.
Masciari etal J Med Genet 2007;44:
Fitzgerald RC, etal. Gut 2004;53:
Huntsman DG etal. N Engl J Med 2001;344:

37. Emerging Data & NCCN Guidelines
PALB2 - Discussed in:
Pancreatic Adenocarcinoma Version
“… 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 (eg BRCA or PALB2 mutations).”
Gemcitabine… based on MSK study of 5 of 6 patients with known BRCA mutations and metastatic pancreatic adenocarcinoma treated with platinum based regimen
Publications cited by NCCN guidelines for above statements:
Canto etal Clin Gastroenterol Hepatol 2006;4:
Lowery etal Oncologist 2011;16:

38. 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 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:
1866 – Paul Broca provides a detailed scientific description of inherited breast-ovarian cancer
Demonstrated the tranmission of an assumed underlying genetic defect, its expressions, the age-related and sex-limited penetrance, and the possibilities of modifying environmental and genetic factors.
Mid 1980s – global search for genetic basis of HBOC begins in ernest
Dec 1990 – MC King/UC Berkeley link HBOC to chromosome 17q21 “Linkage of early-onset familial breast cancer to chromosome 17q21” PMID: 
Oct M Stolink of Univ of Utah & Myriad genetics describe the cloning of the BRCA1 gene. “A strong candidate for the breast and ovarian cancer susceptibility gene, BRCA1. PMID:   ”
Rest of timeline – BRCA2 cloning, ionitial BRCA1/2 risk estimates based on linkage data, then mutation data.
seminal papers outlining the risks of other cancers, apart from breast cancer in BRCA1/2 carriers.
Ford D, Easton DF, Stratton MR, et al: Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families. Am J Hum Genet 1998, 62:676–689.
This paper describes the proportion of high-risk families due to BRCA1 and BRCA2 and their phenotypic characteristics and is used by cancer geneticists when quoting penetrance figures for the high-risk families.
31. Kote-Jarai Z, Eeles RA. BRCA1, BRCA2 and their possible function in DNA damage response. Br J Cancer 1999, 81:1099–1102.
This review summarises the evidence that BRCA1 and BRCA2 are involved in the DNA damage response
Lakhani S, Sloane JP, Gusterson BA, et al: A detailed analysis of the morphological features associated with breast cancer in patients harbouring mutations in BRCA1 and BRCA2 predisposition genes.
J Natl Cancer Inst 1999, 90:1138–1145.
This paper showed that the pathological features of breast cancers occurring in BRCA1 carriers are different from those in BRCA2 carriers and those without mutations in either of these genes.
40. Struewing JP, Watson P, Easton DF, Ponder BA, Lynch HT, Tucker MA: Prophylactic oophorectomy in inherited breast/ovarian cancer families. J Natl Cancer Inst Monogr 1995, 17:33–35.
This reference shows that prophylactic oophorectomy reduces ovarian cancer risk.
41. Rebbeck T, Levin AM, Eisen A, et al: Breast cancer risk after bilateral prophylactic oophorectomy in BRCA1 mutation carriers. J Natl Cancer Inst 1999, 91:1475–1479.
This reference shows that prophylactic oophorectomy reduces both ovarian and breast cancer risk in BRCA carriers.
1998 – studie sof tamoxifen for chemo prevention emerge
50. Hartmann LC, Schaid DJ, Woods JE, et al: Efficacy of bilateral prophylactic mastectomy in women with a family history of breast cancer. N Engl J Med 1999, 340:77–84.
This is the most commonly quoted reference to support the suggestion that prophylactic mastectomy reduces risk of breast cancer. The problem is that this study, of necessity, was retrospective and many women did not have genetic analysis performed.
2007 – ACS guidelines for MRI screening
– PARP inhibitors for BRCA1/2?
2010 – Parp inh may be effective in tumors with PALB LOH also
Nat Struct Mol Biol. 2010 Oct;17(10): Epub 2010 Sep 26.
Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.
Buisson R, Dion-Côté AM, Coulombe Y, Launay H, Cai H, Stasiak AZ, Stasiak A, Xia B, Masson JY.
– MC King on effective use of NGS for genetic testing
2012 – Lynch on underestimate of genetci risk

39. Historic Perspectives on HBOC
Prophylactic surgical interventions for women with HBOC
1995
Prophylactic Oopherectomy in inherited breast/ovarian cancer families. Struewing etal JNCI PMID: :
Eg: Struewing etal JNCI PMID: : Multi-center study to determine the incidence of post-oophorectomy carcinomatosis 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. Rebbeck etal JNCI PMID:
Jan 1999
“Efficacy of bilateral prophylactic mastectomy in women with a family history of breast cancer.” Hartman etal NEJM PMID:
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. Rebbeck etal JCO PMID:
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).
Struewing etal PMID: : On-going studies to determine the incidence of post-oophorectomy carcinomatosis and to quantify the effectiveness of preventive surgery, a multicenter study is ongoing between the National Cancer Institute (NCI), Creighton University, and the United Kingdom
Hartman etal PMID:
At the time, was the most commonly quoted reference to support the suggestion that prophylactic mastectomy reduces risk of breast cancer. However, it was retrospective and many women did not have genetic analysis performed
Breast Cancer Screening with MRI
Early 2000s
Studies of efficacy of MRI screening in BRCA1/2 carriers underway
2007
ACS guidelines published for MRI screening in women at high risk for breast cancer
Screening MRI is recommended for women with an approximately 20-25% or greater lifetime risk of breast cancer, including women with a strong family history of breast or ovarian cancer and women who were treated for Hodgkin disease.
Saslow etal. CA Cancer J Clin. 2007 Mar-Apr;57(2):75-89.
2008
ACS discusses importance of utilizing genetic testing in determining appropriate screening with Breast MRI
Authors conclude “The current results demonstrated a need for greater awareness of breast cancer risk factors in the screening mammography population, so that high-risk women can be identified and given access to genetic testing and counseling regarding all risk-reducing interventions
Murphy etal Cancer 2008;113:
Chemotherapeutic regimen selective for BRCA1 & BRCA2 mutation carriers
2007-present
Poly-ADP ribose polymerase (PARP) inhibitors emerge as new chemotherapy options for women with ovarian cancer.
Phase II clinical trials have shown great promise in treating women with hereditary breast and ovarian cancers associated with BRCA1/2 mutations, associated with only minimal adverse effects
Trials for PARP inhibitors in BRCA1 & 2 PARP inhibitors and epithelial ovarian cancer: an approach to targeted chemotherapy and personalised medicine. Mukhopadhyay A PMID:
2010-present
Studies demonstrates that PALB2-deficient cells are sensitive to PARP inhibitors. 
Continued biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair
“Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.” Buisson etal PMID:

40. Historic Perspectives on HBOC
Chemoprevention
July/Sept 1998 –
Three papers describe three trials that investigate the role of Tamoxifen as a chemopreventive agent for breast cancer.
Fisher etal PMID: , Powles etal PMID: , Veronesi etal PMID:
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: ]
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 Clin Oncol 2004;22:2328–35. PMID:
MRI surveillance – similar trajectory
Targeted chemotherapeutics - PARP inhibitors in Phase II clinical trials
Metcalfe etal Centre for Research on Women' s Health, Women's College Hospital, Toronto, Ontario, Canada.
Chemotherapeutic regimen selective for BRCA1 & BRCA2 mutation carriers
2007-present
Poly-ADP ribose polymerase (PARP) inhibitors emerge as new chemotherapy options for women with ovarian cancer.
Phase II clinical trials have shown great promise in treating women with hereditary breast and ovarian cancers associated with BRCA1/2 mutations, associated with only minimal adverse effects
Trials for PARP inhibitors in BRCA1 & 2 PARP inhibitors and epithelial ovarian cancer: an approach to targeted chemotherapy and personalised medicine. Mukhopadhyay A PMID:
2010-present
Studies demonstrates that PALB2-deficient cells are sensitive to PARP inhibitors. 
Continued biochemical insights into PALB2's function with piBRCA2 as a mediator of homologous recombination in DNA double-strand break repair
“Cooperation of breast cancer proteins PALB2 and piccolo BRCA2 in stimulating homologous recombination.” Buisson etal PMID:

41. Variants of Unknown Significance
Variant rates are expected to be higher than average for 2 reasons:
Shear number of genes – 22 genes on full panel
For the newer genes, not previously available for sequencing at Ambry, higher VUS rates are expected in the beginning, as is true with any genetic test
Example: XLMR next gen panel at Ambry – we have been offering this test for ~2 years.
In a review of our initial 30 cases, and then our most recent 30 cases the VUS rate has decreased dramatically’
Initial 30: Avg 2.5 VUS reported per case, with only 5 cases with no VUS’s report
Last 30: Ave 1 VUS reported per case, with ~half reported as variant suspect benign or susp pathogenic, and 13 cases with no VUSs reported

42. Variant Rates for Genes Previously Available for sequencing at Ambry
Panel Genes
PALB2 ▪
CDH1
STK11
CHEK2
PTEN
TP53
MUTYH
APC
MLH1
MSH2
MSH6
PMS2
EPCAM
BMPR1A
SMAD4
BARD1
BRIP1
MRE11A
NBN
RAD50
RAD51C
ATM
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%

43. Ambry’s Variant Classification Scheme
Always included in
results report with interpretation of result and description of gene
Mutation
Variant, suspected pathogenic
Variant, unknown significance
Variant, suspected benign
Polymorphisms
Always included in
result report, with interpretation of result, description of gene, and supplementary data
Reported only if requested

44. Family Studies Program
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

45. Enrolling in Family Studies Program
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
Informative relative selection based on family hx and relatives
Testing for known mutations - SMA fee…if VUSs mutations identified, still charge fee for mutation?!?

46. VUS Reported Data Example: 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
T/C heterozygotes: 19/5359 (0.35%)
T/T homozygotes: 1/5359 (0.02%)
Co-segregatation: not available
Co-occurrence: none
The p.P864S (also known as c.2590C>T) variant is located in exon 7 of the PALB2 gene. This alteration results from a C to T substitution at nucleotide position The proline at codon 864 is replaced by serine, an amino acid with similar properties. In one study, p.P864S was detected in (1/96) BRCA-negative high-risk breast cancer families and 0/96 controls (p-value 0.061) (Guenard F et al. Genet Test Mol Biomarkers Aug;14(4):515-26). This alteration was also previously reported in the SNPDatabase as rs with a T-allele frequency of approximately 0.2% in the general population (NCBI Database. Bethesda, MD: Single Nucleotide Polymorphism: Updated August 15, Accessed January 4, 2012). Based on data from the 1000 Genomes Project, the T-allele has an overall frequency of approximately 0.23% (5/2188). The highest observed frequency was 2 of 178 (1.12%) British chromosomes studied ( Released July Accessed January 4, 2012). To date, this alteration has been detected with an allele frequency of approximately 1.9% (>250 alleles tested) in our clinical cohort. Based on protein sequence alignment, this amino acid position is not conserved. In addition, this alteration is predicted to be benign and tolerated by Polyphen and SIFT in silico analyses, respectively. Based on the majority of available evidence to date, this variant is unlikely to be pathogenic; however, its clinical significance remains unclear.
Guenard F et al. Genet Test Mol Biomarkers Aug;14(4):

47. Insurance Coverage/Pre-Verification
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

48. Thank you!
Any questions?