OvaNext is a next generation sequencing panel that simultaneously analyzes 24 genes associated with increased risk for breast, ovarian, and/or uterine cancers.
OvaNext is a next generation sequencing panel that simultaneously analyzes 24 genes associated with increased risk for breast, ovarian, and/or uterine cancers.
Ambry utilizes next generation sequencing (NGS) to offer a comprehensive hereditary gynecologic cancer panel. Genes on this panel include: ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMARCA4, STK11, TP53. Full gene sequencing is performed for 23 of the genes (excluding EPCAM). Gross deletion/duplication analysis is performed for all 24 genes. Specific Site Analysis is available for individual gene mutations identified in a family.
Breast cancer is the most common cancer in women in developed countries, affecting about 1 in 8 (12.5%) women in their lifetime.1 The National Cancer Institute (NCI) estimates that approximately 231,840 new cases of female breast cancer and 2,350 new cases of male breast cancer will be diagnosed in the U.S. in 2015.2 The majority of breast cancers are sporadic, but 5-10% are due to inherited causes. Hereditary breast cancer tends to occur earlier in life than non-inherited sporadic cases, and is more likely to occur in both breasts. The highly penetrant genes, BRCA1 and BRCA2, appear to be responsible for around half of hereditary breast cancer.3-5 However, additional genes have been discovered that are associated with increased breast cancer risk as well.3-7 Mutations in the genes included in OvaNext can confer an estimated 20–87% lifetime risk for breast cancer. Some of these genes have also been associated with increased risks for other cancers, such as pancreatic cancer with PALB2, ovarian cancer with BRCA1, BRCA2, RAD51C, and others, and sarcoma with TP53.8-12
Ovarian cancer is the fifth most common cancer among women in developed countries, affecting approximately 1 in 71 (1.4%) women in their lifetime.1 The NCI estimates that approximately 21,290 new cases of ovarian cancer will be diagnosed and 14,180 ovarian cancer deaths will occur in the U.S. in 2015.2 It is the leading cause of death from gynecologic malignancy, usually characterized by advanced presentation with regional dissemination in the peritoneal cavity. Epithelial ovarian cancer is the most common form, and up to 25% of epithelial cases may be due to inherited gene mutations.13,14 BRCA1 and BRCA2 are the most common causes of hereditary ovarian cancer, but several other genes are associated with increased ovarian cancer risk as well.11,13,15,16
Uterine cancer affects about 1 in 38 (2.6%) women in their lifetime.1 The NCI estimates that approximately 54,870 new cases of uterine cancer will be diagnosed and 10,170 uterine cancer deaths will occur in the U.S. in 2015.2 Increased risk for uterine cancer has been identified in a number of hereditary cancer syndromes, including Lynch syndrome and Cowden syndrome.
Since the best approach to genetic testing for individuals with these cancer types is not always clear, OvaNext may be ideal.
OvaNext Panel Genes:
ATM is a gene associated with an autosomal recessive condition called ataxia-telangiectasia (AT). AT is characterized by progressive cerebellar ataxia with onset between ages 1 and 4, telangiectasia of the conjunctivae, oculomotor apraxia, immune defects, and a predisposition to malignancy, particularly leukemia and lymphoma. Women who carry ATM mutations also have an estimated 2-4 fold increased risk for breast cancer.17 Cancer risk estimates for male ATM mutation carriers are not currently available. Recent studies have also reported ATM germline mutations in individuals with familial pancreatic cancer. In one of these studies, ATM mutations were identified in 4/87 (4.6%) families with more than three affected members.18
BRCA1 and BRCA2 are tumor suppressor genes inherited in an autosomal dominant pattern. Mutations in these two highly penetrant genes increase the chance for cancer of the breast, ovaries (including primary peritoneal and fallopian tube), pancreas, and prostate. Studies suggest female BRCA1 mutation carriers have a 57-87% lifetime risk to develop breast cancer and a 39-40% lifetime risk to develop ovarian cancer by age 70.8-10,19-21 Male BRCA1 mutation carriers have a cumulative breast cancer lifetime risk of about 1.2% by age 70.(22, 23) Similar studies suggest female BRCA2 mutation carriers have a 45-84% lifetime risk to develop breast cancer and an 11-18% risk to develop ovarian cancer by age 70.8-10,24,25 Male BRCA2 mutation carriers have up a 15% lifetime prostate cancer risk and a cumulative lifetime breast cancer risk of 6.8% by ages 65 and 70 respectively.22,23,25,26 BRCA1/2 mutation carriers may also be at an increased risk for melanoma, pancreatic cancer, and potentially other cancers.27 BRCA2 is also known as FANCD1. Individuals who inherit a BRCA2/FANCD1 mutation from each parent may have a rare autosomal recessive condition called Fanconi anemia type D1 (FA-D1), which affects multiple body systems.
BARD1, BRIP1, MRE11A, NBN, RAD50, RAD51C, and RAD51D are genes involved in the Fanconi anemia (FA)-BRCA pathway, critical for DNA repair by homologous recombination, and interact in vivo with BRCA1 and/or BRCA2.4,15,28 Mutations in these genes are associated with an increased risk for female breast cancer. 28,29,30 The ovarian cancer risk associated with mutations in BRIP1, RAD51C and RAD51D has been estimated to be up to 9%, 5-9% and 10-12%, respectively.11,16,66, 67 It has been suggested that BARD1 is associated with an increased risk for ovarian cancer, and mutations in MRE11A, NBN, and RAD50.13,68 BRIP1, NBN, and RAD51C are each associated with a rare autosomal recessive disorder that affects multiple body systems.
CHEK2 is a gene that receives signals from damaged DNA, transmitted via ATM. CHEK2 interacts in vivo with BRCA1, BRCA2, and TP53, which have all been implicated in cellular processes responsible for the maintenance of genomic stability. Multiple studies indicate that mutations in CHEK2 confer an increased risk of developing many types of cancer including breast, colon, and other cancers. Mutations are more likely to be found among women with bilateral versus unilateral breast cancers. A female CHEK2 mutation carrier has approximately a two-fold increase in lifetime breast cancer risk, and has a 1% risk per year of developing a second breast primary cancer. Lifetime risks for other associated cancers are unknown. An increased risk for ovarian cancer has also been suggested.13,31-33
CDH1 germline mutations are associated with hereditary diffuse gastric cancer (HDGC) and lobular breast cancer in women. In one published study, the estimated cumulative risk of gastric cancer for CDH1 mutation carriers by age 80 years was 67% for men and 83% for women.34 Patients with HDGC typically present with diffuse-type gastric cancer, with signet ring cells diffusely infiltrating the wall of the stomach and, at advanced stages, linitis plastica. An elevated risk of lobular breast cancer in women is also associated with HDGC, with an estimated lifetime breast cancer risk of 39-52%.35
MLH1, MSH2, MSH6, PMS2, and EPCAM germline mutations are associated with Lynch syndrome (previously called hereditary non-polyposis colorectal cancer, HNPCC). Lynch syndrome is an autosomal dominant condition estimated to cause 2-5% of all colorectal cancer. It is associated with a significantly increased risk for colorectal cancer (up to 82% lifetime risk), uterine/endometrial cancer (25-60% lifetime risk in women), stomach cancer (6-13% lifetime risk), and ovarian cancer (4-12% lifetime risk in women). Risk for cancer of the small bowel, hepatobiliary tract, upper urinary tract (including transitional cell carcinoma of the renal pelvis), brain, and sebaceous glands may also be elevated.36-40
MUTYH germline mutations are known to cause MUTYH-associated polyposis (MAP), an autosomal recessive condition predisposing to gastrointestinal polyposis and colorectal cancer. Individuals that carry two MUTYH mutations on different chromosomes (in trans) have an estimated lifetime colorectal cancer risk of up to 80%.41 In addition, some studies suggest that MUTYH mutations confer an increased risk to develop female breast cancer; this is estimated to be a 1.5-fold lifetime increased risk within the North African Jewish population. MUTYH mutations in the carrier state may also increase lifetime risks for cancers of the duodenum, stomach, and endometrium42-44; however, these data are limited and risks may vary between populations. Two common mutations in the Caucasian population, p.Y179C and p.G396D (originally designated as p.Y165C and p.G382D), account for the majority of pathogenic MUTYH alterations reported to date. Breast cancer risk estimates for male MUTYH mutation carriers are not currently available.
NF1 mutations cause neurofibromatosis type 1 (NF1), an autosomal dominant disorder affecting multiple body systems. It is characterized by multiple café-au-lait spots, axillary and inguinal freckling, multiple cutaneous neurofibromas, and Lisch nodules. The most common neoplasms observed in individuals with NF1 include peripheral nerve sheath tumors, gastrointestinal stromal tumors (GIST), central nervous system gliomas, leukemias, paragangliomas (PGLs) and pheochromocytomas (PCCs), and breast cancer. Multiple population-based studies have demonstrated a 3 to 5-fold increase in lifetime breast cancer risk for women with NF1, with the highest risks for those less than 50 years of age. In addition, individuals with NF1 have an estimated lifetime risk for PGLs and PCCs of up to 7%.
PALB2 germline mutations have been associated with an increased lifetime risk for pancreatic cancer, breast cancer, and Fanconi anemia type N (FA-N). Familial pancreatic and/or breast cancer due to PALB2 mutations is inherited in an autosomal dominant pattern, while FA-N is a rare autosomal recessive condition affecting multiple body systems. Females with a PALB2 mutation have a 2- to 4-fold increase in risk for breast cancer.45,46 A 2014 article concluded that in the context of a strong family history, mutations in PALB2 may be associated with up to a 58% risk of female breast cancer. Without a family history, the risk for female breast cancer was estimated to be 33% (the difference attributed to genetic and/or environmental modifiers).47 Recent studies have identified PALB2 mutations in 1-3% of families with pancreatic cancer; however, the exact lifetime pancreatic cancer risk has not yet been established.48,49 Additionally, recent studies have shown an increased risk for ovarian cancer.13, 68
PTEN is a gene associated with Cowden syndrome (CS), PTEN hamartoma tumor syndrome (PHTS), Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome, and autism spectrum disorder. CS is a multiple hamartoma syndrome with a high risk of developing tumors of the thyroid, breast, and endometrium. Mucocutaneous lesions, thyroid abnormalities, fibrocystic disease, multiple uterine leiomyomata, and macrocephaly can also be seen. Affected individuals have a lifetime risk of up to 50% for breast cancer, 10% for thyroid cancer, and 5-10% for endometrial cancer. Over 90% of individuals with CS will express some clinical manifestations by their twenties.50,51 Recent studies noted increased risks for renal cell cancer, colorectal cancer, and other cancers.52,53 One study quotes up to a 31-fold increase in RCC risk for PTEN mutation carriers as compared to the general population.54
SMARCA4 truncating mutations cause rhabdoid tumor predisposition syndrome type 2. SMARCA4-associated tumors are highly aggressive and include atypical teratoid/rhabdoid tumors (AT/RT) of the central nervous system, malignant rhabdoid tumors of the kidney, and small cell carcinoma of the ovary, hypercalcemic type (SCCOHT). The lifetime cancer risks for SMARCA4 mutation carriers has yet to be defined; however, age of onset and penetrance are extremely variable, with some carriers presenting prenatally while others remain unaffected through adulthood.55-58
STK11 germline mutations are associated with Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by the development of gastrointestinal hamartomatous polyps, along with hyperpigmentation of the skin and mucous membranes. Overall, individuals affected with PJS have up to an 85% lifetime risk of developing cancer by the age of 70, with gastrointestinal and breast cancers being the most common.59,60 Individuals with PJS are also at elevated risk for tumors of the pancreas, lung, and, in females, ovarian tumors (specifically, sex cord tumors with annular tubules (SCTATs) and mucinous ovarian tumors).
TP53 is a tumor suppressor gene, and germline mutations within it are associated with Li-Fraumeni syndrome (LFS). An individual carrying a TP53 mutation has a 21-49% lifetime risk of developing cancer by age 30 and a lifetime cancer risk of 68-93%.61 The most common tumor types observed in LFS families include soft tissue and osteosarcomas, breast cancer, brain tumors (including astrocytomas, glioblastomas, medulloblastomas and choroid plexus carcinomas), and adrenocortical carcinoma (ACC); other cancers, including colorectal, gastric, ovarian, pancreatic, and renal, have also been reported.12,62 Studies have shown that a small percentage of women with early onset breast cancer that do not carry BRCA1 and BRCA2 mutations are identified to have mutations in TP53.33,63,64
Indications for Testing
Families with a combination of the cancers below and some common red flags for hereditary cancer in the family would be appropriate to consider for OvaNext.
Common Red Flags for Hereditary Cancer
If increased risk of a hereditary cancer syndrome is suspected, the American Congress (formerly College) of Obstetricians and Gynecologists (ACOG) recommends referral to a specialist in cancer genetics or a healthcare provider with expertise in genetics for complete hereditary cancer risk assessment, which may lead to genetic testing.65 Establishing a molecular diagnosis can help guide preventive measures, direct surgical options and estimate personal and familial cancer risk.
OvaNext analyzes 24 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMARCA4, STK11, TP53). 23 genes (excluding EPCAM) are evaluated by NGS or Sanger sequencing of all coding domains, and well into the flanking 5’ and 3’ ends of all the introns and untranslated regions. In addition, sequencing of the promoter region is performed for the following genes: PTEN (c.-1300 to c.-745), MLH1 (c.-337 to c.-194), and MSH2 (c.-318 to c.-65). The inversion of coding exons 1-7 of the MSH2 gene and the BRCA2 Portuguese founder mutation, c.156_157insAlu (also known as 384insAlu) are detected by NGS and confirmed by PCR and agarose gel electrophoresis. Clinically significant intronic findings beyond 5 base pairs are always reported. Intronic variants of unknown or unlikely clinical significance are not reported beyond 5 base pairs from the splice junction. Additional Sanger sequencing is performed for any regions missing or with insufficient read depth coverage for reliable heterozygous variant detection. Suspect variant calls are verified by Sanger sequencing. Gross deletion/duplication analysis is performed for the covered exons and untranslated regions of all 24 genes using read-depth from NGS data with confirmatory multiplex ligation-dependent probe amplification (MLPA) and/or targeted chromosomal microarray. If a deletion is detected in exons 13, 14, or 15 of PMS2, double stranded sequencing of the appropriate exon(s) of the pseudogene, PMS2CL, will be performed to determine if the deletion is located in the PMS2 gene or pseudogene.
OvaNext can detect >99.9% of described mutations in the included genes listed above, when present (analytic sensitivity).
|TEST CODE||TEST NAME||TURNAROUND TIME (days)|
|8830||OvaNext||14 - 21|