CancerNextTM is a next generation (next-gen) sequencing panel that simultaneously analyzes 28 genes that contribute to increased risk for breast, colon, ovarian, uterine and other cancers.
CancerNextTM is a next generation (next-gen) sequencing panel that simultaneously analyzes 28 genes that contribute to increased risk for breast, colon, ovarian, uterine and other cancers.
Please note, all samples received starting October 18, 2013 for CancerNext will automatically include CDK4, CDKN2A, NF1, and RAD51D gene sequencing and deletion/duplication analyses at no additional cost. Additionally, Ambry will contact clinicians to discuss any clinically-significant CDK4, CDKN2A, NF1, and RAD51D incidental findings on all in-house samples.
Ambry utilizes next generation sequencing to offer a genetic testing panel for hereditary cancers (cancer of the breast, colon, ovary, uterus and/or other), including BRCA1 and BRCA2. Genes on this panel include APC, ATM, BARD1, BRCA1, BRCA2, BRIP1, BMPR1A, CDH1, CDK4, CDKN2A, CHEK2, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMAD4, STK11, and TP53. Full gene sequencing and analysis of all coding domains plus at least 5 bases into the 5’ and 3’ ends of all the introns and untranslated regions (5’UTR and 3’UTR) is performed for 27 of the 28 genes (excluding EPCAM). Gross deletion/duplication analysis is performed for all 28 genes. Specific-site analysis is available for individual gene mutations known to be in the family.
Breast cancer is a disease in which cells in the breast become abnormal and multiply to form a malignant tumor. Breast cancer is the most common cancer in women in developed countries, affecting about 1 in 8 (~12.29%) women in her lifetime.1 The NCI estimates that approximately 227,000 new cases of female breast cancer and 2,200 new cases of male breast will be diagnosed in the U.S. in 2012.2
Ovarian cancer is the fifth most common cancer among women, affecting approximately 1 in 71 (1.4%) women in her lifetime.1 The NCI estimates that approximately 22,280 new cases of ovarian cancer will be diagnosed and 15,500 ovarian cancer deaths will occur in the U.S. in 2012.2 It is leading cause of death from gynecologic malignancy, characterized by advanced presentation with regional dissemination in the peritoneal cavity. Epithelial ovarian cancer is the most common form and arises as a result of genetic alterations sustained by the ovarian surface epithelium.
Hereditary breast cancers tend to occur earlier in life than non-inherited sporadic cases and are more likely to involve both breasts. Hereditary breast and ovarian cancers caused by mutations in the highly penetrant genes, BRCA1 and BRCA2, appear to be responsible for about 10-18% of total breast cancers and ovarian cancers.3,8,14 However, additional genes have been discovered that contribute to the incidence of breast and ovarian cancers as well.3-6,8,14
Colorectal (CRC) cancer affects about 1 in 20 (5.1%) of men and women in their lifetime.1 The NCI estimates that approximately 103,170 (colon) and 40,290 (rectal) new cases will be diagnosed and 51,690 CRC deaths will occur in the U.S. in 2012.2 CRC is the third leading cause of cancer-related deaths in the United States when men and women are considered separately, and the 2nd leading cause of cancer related deaths when combined.2 The majority of CRCs are sporadic, but approximately 25% are familial, a subset of which showing strong genetic etiology.
Uterine cancer affects about 1 in 38 (2.61%) women in her lifetime.1 The NCI estimates that approximately 47,130 new cases of uterine cancer will be diagnosed and 8,010 uterine cancer deaths will occur in the U.S. in 2012.2 Increased risk for uterine cancer has been identified in a number of hereditary cancer syndromes, including Lynch syndrome and Cowden syndrome.
CancerNext is a next-gen cancer panel that simultaneously analyzes selected genes associated with an increased risk for a wide range of cancers. While mutations in each gene on this panel may be individually rare, they collectively account for a significant amount of hereditary cancer susceptibility. This panel may be appropriate in a number of scenarios, particularly if the family history shares features of several different hereditary cancer syndromes.
Figure Legend: If there are multiple cancers in the family that could fit into different hereditary cancer syndromes, such as in the pedigree example above, consider CancerNext,
CancerNext Panel Genes
APC germline mutation are well established as the primary cause of familial adenomatous polyposis (FAP) and attentuated familial adenomatous polyposis (AFAP). FAP is an autosomal dominant colon cancer predisposition syndrome characterized by hundreds to thousands of adenomatous polyps in the internal lining of the colon and the rectum. It affects 1/8,000 to 1/10,000 individuals and accounts for about 1% of all colorectal cancers.7 In individuals affected with classic FAP, colonic polyps generally begin developing at an average age of 16 years.8 Colon cancer is inevitable without colectomy, and the mean age of colon cancer diagnosis in untreated individuals is age 35-40 years.9 Variants of FAP are Gardner syndrome, Turcot syndrome, and attenuated FAP (AFAP).7
ATM is a gene classically associated with an autosomal recessive condition called ataxia-telangiectasia (AT). AT is an autosomal recessive disorder characterized by progressive cerebellar ataxia with onset between ages one and four, telangiectases of the conjunctivae, oculomotor apraxia, immune defects, and a predisposition to malignancy, particularly leukemia and lymphoma. Heterozygous carrier females also have an estimated 2-4 fold increased risk for breast cancer.10
BRCA1 and BRCA2 are tumor suppressor genes. Mutations in these two highly penetrant genes increase the chance for cancer of the breast, ovaries and fallopian tubes, pancreas and prostate. Studies suggest female BRCA1 mutation carriers have between a 57-87% risk to develop breast cancer and between a 39-40% risk to develop ovarian cancer by age 70. Similarly male BRCA1 mutation carriers have a cumulative breast cancer risk of 1.2% by age 70.
BARD1, BRIP1, MRE11A, NBN, RAD50, and RAD51C are genes involved in the Fanconi anemia (FA)–BRCA pathway, which is critical for DNA repair by homologous recombination and interact in vivo with BRCA1 or BRCA2.3,11-13 Mutations in these genes are estimated to confer up to a 4 fold increase in breast cancer risk, and mutations in each have been reported in at least 1 identified case of ovarian cancer to date.14
BMPR1A and SMAD4 are genes implicated in juvenile polyposis syndrome (JPS), together accounting for 45-60% of JPS. JPS is an autosomal dominant disorder that predisposes to development of polyps in the gastrointestinal tract.15 Malignant transformation can occur with risk of gastrointestinal cancer ranging from 9% to 50%. Juvenile polyposis of infancy involves the entire digestive tract and has the poorest prognosis.16 Most other patients develop symptoms by age 20 though some are not diagnosed until the third decade of life. Common symptoms include gastrointestinal bleeding, anemia, diarrhea, and abdominal pain. Early detection of JPS allows for better treatment of polyps and surveillance of at-risk individuals. SMAD4 mutations may cause a combined syndrome of hereditary hemorrhagic telangiectasia (HHT) with JPS as reported in 22% of JPS patients with SMAD4 mutations.17
CHEK2 is a gene that receives signals from damaged DNA, transmitted to CHEK2 via ATM. Known substrates of CHEK2 include 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 the CHEK2 gene confer an increased risk of developing many types of cancer including breast, prostate, colon, thyroid, and kidney. Mutations are more likely to be found among women with bilateral versus those with unilateral breast cancers. A female carrier of a CHEK2 mutation has approximately a 2 fold increase in lifetime breast cancer risk and has a 1% risk per year of developing a second breast primary cancer. Ovarian cancer risk has also been suggested.14,18-21
CDH1 germline mutations have been 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 is 67% for men and 83% for women.22 HDGC patients typically present with diffuse-type gastric cancer with signet ring cells diffusely infiltrating the wall of the stomach and, at late stage, linitis plastica. An elevated risk of lobular breast cancer is also associated with HDGC,23 with an estimated lifetime breast cancer risk of 39-52%.
CDK4 is one of the genes implicated in cutaneous malignant melanoma (CMM) syndrome. Individuals with hereditary CMM have an increased genetic predisposition to develop hystologically dysplastic pigmented nevi and at an earlier age than the general population. Mutations in this gene are inherited in an autosomal dominant manner and contribute to increased risk for malignant melanoma. CDK4 mutations have been described in approximately 1% of hereditary form of malignant melanoma.
CDKN2A encodes two distinct proteins, p16 and p14ARF, which are both involved in cell cycle regulation. Germline p16/CKDN2A mutations are implicated in familial atypical multiple mole melanoma (FAMMM) syndrome. FAMMM is an autosomal dominant disorder characterized by an increased risk for atypical mole-malignant melanoma or skin cancer, often associated with dysplastic or atypical nevi. Studies show that germline CDKN2A mutations account for 10-39% of hereditary malignant melanoma cases. It is estimated that CDKN2A mutation carriers have a lifetime risk of 28-91% to develop malignant melanoma. CDKN2A mutations are also associated with an increased risk for pancreatic cancer. Studies note that kindreds with FAMMM have a 13- to 22-fold increased risk for pancreatic cancer.
EPCAM, MLH1, MSH2, MSH6, and PMS2 are mismatch repair genes that have been associated with Lynch syndrome (HNPCC). Lynch syndrome is estimated to cause 2-5% of all colon cancer. Lynch syndrome is associated with a significantly increased risk for colon cancer (60-80% lifetime risk), uterine/endometrial cancer (20-60% lifetime risk in women), stomach cancer (11-19% lifetime risk), and ovarian cancer (4-13% lifetime risk in women). Risk for cancer of the small intestine, hepatobiliary tract, upper urinary tract and brain are also elevated.24-26
MUTYH germline mutations are classically associated with an autosomal recessive form of hereditary polyposis. Clinical studies have shown that MUTYH mutations were detected in 33% and 57% of patients with clinical familial adenomatous polyposis (FAP)and attentuated familial adenomatous polyposis (AFAP), respectively, who are negative for mutations in the APC gene.27 Two common mutations, p.Y179C and p.G396D (originally designated as Y165C and G382D), have been reported as homozygous or compound heterozygous in about 70%-86% of MAP patients.28,29 Heterozygous mutations have also been associated with a 1.9 fold increased risk for breast cancer.30 In this series, characteristics of tumors and at of diagnosis in carriers with MUTYH variants were similar to those without MUTYH variants.27-30
NF1 mutations cause neurofibromatosis type 1 (NF1), an autosomal dominant disorder affecting multiple body systems. 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 of have demonstrated a 3-5 fold increase in breast cancer risk for women with NF1, with the highest risks for those under 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 increased risk for pancreatic cancer, breast cancer, and fanconi-anemia complementation group N (FA-N). Familial pancreatic and/or breast cancer due to PALB2 mutations is inherited in an autosomal dominant pattern, while FA-N is an autosomal recessive condition. Females with a PALB2 mutation have a 2 to 4 fold increase in risk for breast cancer,31-33 and a recent report has suggested an increased risk for ovarian cancer as well.14
PTEN is a gene that has been associated with Cowden syndrome, PTEN Hammartoma Tumor syndrome (PHTS), Bannayan-Riley-Ruvalcaba syndrome, Proteus syndrome and autism spectrum disorder. Cowden Syndrome is a multiple hamartoma syndrome with a high risk of developing tumors of the thyroid, breast, and endomentrium. Mucocutaneous lesions, thyroid abnormalities, fibrocystic disease, multiple uterine leiomyoma, 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 manifestation by their 20’s.34,35
RAD51D is a gene involved in the Fanconi anemia (FA)–BRCA pathway, which is critical for DNA repair by homologous recombination and interact in vivo with BRCA1 and/or BRCA2. RAD51D germline mutations confer an increased susceptibility for breast and ovarian cancer. Relative cancer risk is estimated at 1.32 and 6.3 respectively. This translates to nearly a 10% lifetime risk for ovarian cancer.
STK11 germline mutations have been associated with Peutz-Jegher syndrome (PJS), an autosomal dominant disorder characterized by the development of gastrointestinal hamartomatous polyps and melanin hyperpigmentation of the skin and mucous membranes. Overall, individuals affected with PJS have a 57-81% risk of developing cancer by age of 70, with gastrointestinal/colon and breast cancers being the most common.36,37
TP53 is a tumor suppressor gene that causes Li-Fraumeni and Li-Fraumeni like syndrome, which can affect adults and children. Individuals with TP53 mutations have a 50% risk of developing any of the associated cancers by age 30 and a lifetime risk up to 90%, including sarcomas, breast cancer, brain tumors (including astrocytomas, glioblastomas, medulloblastomas and choroid plexus carcinomas), and adrenocortical carcinoma (ACC). Studies have shown that a small percentage of women who are BRCA1 and BRCA2 negative are identified to have mutations in TP53.21,38,39
Indications for Testing
This panel may be appropriate in the following situations, combined with common red flags for hereditary cancer:
Common Red Flags for Hereditary Cancer
Selected Medical Management Guidelines* (based on NCCN guidelines).40
Breast Cancer (recommended for women with >/=20-25% lifetime risk)
*Medical management recommendations will depend on which gene is found to be mutated. Additional information regarding cancer risks and medical management options can be found in cited literature.
The CancerNext Panel targets detection of mutations in 27 of the 28 genes (APC, ATM, BARD1, BRCA1, BRCA2, BRIP1, BMPR1A, CDH1, CHEK2, CDK4, CDKN2A, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMAD4. STK11, and TP53) by next-generation sequencing or Sanger sequencing of all coding domains plus at least 5 bases into the 5’ and 3’ ends of all the introns and untranslated regions (5’UTR and 3’UTR). Genomic deoxyribonucleic acid (gDNA) is isolated from the patient’s specimen using a standardized kit and quantified by agarose gel electrophoresis. Sequence enrichment is carried out by incorporating the gDNA into microdroplets along with primer pairs designed to the target hereditary cancer gene coding exons followed by polymerase chain reaction (PCR) and next-generation sequencing. A secondary sequencing method is performed for any regions with insufficient read depth coverage for reliable heterozygous variant detection. Suspect variant calls other than those classified as "likely benign" or "benign" are verified by sanger sequencing in sense and antisense directions. Gene copy number analysis by a custom micrarray identifies gross deletions or duplications in all 28 genes (APC, ATM, BARD1, BRCA1, BRCA2, BMPR1A, BRIP1, CDH1, CHEK2, CDK4, CDKN2A, EPCAM, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, NF1, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, SMAD4, STK11, and TP53). 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.
Analytical sensitivity for all genes is estimated to be greater than 99% of described mutations.
Blood: Collect 6-10cc blood in purple top EDTA tube (preferred) or yellow top citric acetate tube.
Storage: 2-8°C. Do not freeze.
Shipment: Room temperature for two-day delivery.
For transfusion patients: Wait at least two weeks after a packed cell or platelet transfusion and at least four weeks after a whole blood transfusion prior to blood draw
DNA: Collect 20μg of of DNA in TE (10mM Tris-Cl pH 8.0, 1mM EDTA); preferred at 200 ng/μl.
Quality: Please provide DNA OD 260:280 ratio (preferred 1.7-1.9) and send agarose picture with high molecular weight genomic DNA, if available.
Shipment: Shipment frozen on dry ice is preferred, or ship on ice.
Saliva: Collect 2 tubes with 2cc per tube in Oragene Self Collection container
Storage: At room temperature in sterile bag.
Shipment: Ship room temperature for two-day deliver
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40. Adapted from: http://www.nccn.org/professionals/physician_gls/f_guidelines.asp. Accessed Feb 28, 2012.