ColoNext

ColoNext is a next generation sequencing (NGS) panel that simultaneously analyzes 14 genes associated with increased risk for colorectal cancer.

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ColoNext is a next generation sequencing (NGS) panel that simultaneously analyzes 14 genes associated with increased risk for colorectal cancer.

Ambry utilizes next generation sequencing to offer a comprehensive panel for hereditary colorectal cancer.  Genes on this panel include APC, BMPR1A, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN, SMAD4, STK11, and TP53. Full gene sequencing is performed for 13 of the genes (excluding EPCAM). Gross deletion/duplication analysis is performed for all 14 genes. Specific-site analysis is available for individual gene mutations identified in a family.

Disease Name 
Colorectal cancer
Lynch Syndrome
Polyposis
Hereditary Cancer
Disease Information 

Colorectal cancer (CRC) affects about 1 in 20 (5%) men and women in their lifetime.[1]   The NCI estimates that approximately 136,830 new cases will be diagnosed and 50,310 CRC deaths will occur in the U.S. in 2014.[2]  The majority of CRC is sporadic, but approximately 30% are familial, a subset of which have a strong genetic cause.  Lynch syndrome is the most common form of hereditary CRC, but several other genes are associated with increased CRC risk as well.[3]

Hereditary cancer syndromes associated with genes on ColoNext include Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, PTEN hamartoma tumor syndrome, hereditary diffuse gastric cancer, Li-Fraumeni syndrome, Peutz-Jeghers syndrome, and juvenile polyposis syndrome.  Mutations in genes on this panel are associated with a 9% to nearly 100% lifetime risk for CRC and mutations in some genes can include increased risks for other cancers as well.

ColoNext Panel Genes:

APC germline mutations are well established as the primary cause of familial adenomatous polyposis (FAP) and attenuated familial adenomatous polyposis (AFAP). FAP and AFAP are autosomal dominant colon cancer predisposition syndromes characterized by hundreds to thousands of adenomatous polyps in the internal lining of the colon and the rectum. They affect 1 in 8,000 to 1 in 10,000 individuals and account for about 1% of all colorectal cancers.[4]  In individuals affected with classic FAP, colonic polyps generally begin developing at an average age of 16 years.[5]  In these families, colon cancer is inevitable without surgical intervention like colectomy, and the mean age of colon cancer diagnosis in untreated individuals is 35-40 years.[6]  Individuals with FAP or AFAP may also have increased risks to develop duodenal cancer, pancreatic cancer, papillary thyroid cancer, hepatoblastoma in childhood, and medulloblastoma.  Some individuals may also have non-malignant features such as osteomas, congenital hypertrophy of the retinal pigment epithelium (CHRPE), and/or desmoid tumors.[4] 

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 the development of polyps in the gastrointestinal tract.[7]  Malignant transformation can occur; risk of gastrointestinal cancer ranges from 40-50%. Juvenile polyposis of infancy, which is a rare presentation, involves the entire digestive tract and has the poorest prognosis.[8] Most 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 for those at risk. SMAD4 mutations may cause a combined syndrome of hereditary hemorrhagic telangiectasia (HHT) with JPS, as reported in 15-20% of JPS patients with SMAD4 mutations.[9]

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.[10-13]

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.[14]  HDGC patients 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%.[15]

MLH1, MSH2, MSH6, PMS2, and EPCAM germline mutations are associated with Lynch syndrome (previously known as Hereditary Nonpolyposis Colorectal Cancer, HNPCC). Lynch syndrome is an autosomal dominant condition estimated to cause 2-5% of all colon 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.[16-20]

MUTYH germline mutations are known to cause MUTYH-associated polyposis (MAP), an autosomal recessive condition predisposing to gastrointestinal polyposis and colorectal cancer. Individuals who carry two MUTYH mutations on different chromosomes (in trans) have an estimated lifetime colorectal cancer risk of up to 80%.[21]  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 endometrium (females),[22-24] 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.

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.[25, 26]  Recent studies noted increased risks for renal cell cancer, colorectal cancer, and other cancers.[27, 28]  One study quotes up to a 31-fold increase in RCC risk for PTEN mutation carriers as compared to the general population.[29]

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.[30, 31]  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%.[32]  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.[33, 34]  Studies have shown that a small percentage of women with early onset breast cancer who do not carry BRCA1 and BRCA2 mutations are identified to have mutations in TP53.[12, 35, 36]

 

Testing Benefits & Indication 

Indications for Testing

This panel may be appropriate in the following situations, combined with common red flags for hereditary cancer: 

  • Early-onset colorectal cancer (diagnosed < 50 years of age)
  • Multiple primary cancers in one person (e.g. two primary colorectal cancers or colon & uterine cancer)
  • Three or more family members with colorectal, uterine, ovarian, and/or stomach cancer*
  • 10 or more GI polyps during one’s lifetime (adenomatous, hyperplastic, hamartomatous, and/or other types of polyps)
  • The family history is suspicious for several different hereditary colorectal cancer syndromes
  • Previous genetic testing for hereditary colorectal cancer was uninformative

Common Red Flags for Hereditary Cancer

  • Cancer diagnosed at a younger age than expected for the general population (≤ 50 years, for most cancers)
  • Cancer diagnosed across generations, and in multiple generations within a family, especially if diagnosed younger than average
  • Individual with multiple primary cancers (either in paired organs or in different organs)
  • A pattern of cancer in the family that is typical of a known cancer predisposition syndrome (for example colon and uterine cancer in Lynch syndrome, or diffuse gastric cancer and lobular breast cancer with CDH1).

Benefits of Testing

 
Identifying patients with an inherited susceptibility for certain cancers can help with medical management. For example, this information can:
  • Modify colonoscopy and upper endoscopy screening frequency and age at initial screening.
  • Suggest specific risk-reduction measures (e.g. considering prophylactic oophorectomy, after childbearing is complete, for women with increased risk for uterine/ovarian cancer)
  • Clarify and stratify familial cancer risks, based on gene-specific cancer associations (e.g. risk for uterine, colon, and ovarian cancer with MLH1 mutations)
  • Offer treatment guidance (e.g. avoidance of radiation-based treatment methods for individuals with a TP53 mutation)
  • Identify other at-risk family members
  • Provide guidance with new gene-specific treatment options and risk reduction measures as they emerge  
Test Description 

ColoNext analyzes 13 of the 14 genes (APC, BMPR1A, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN, SMAD4, STK11, and TP53) by next generation sequencing 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). 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 targeted microarray identifies gross deletions and duplications in all 13 sequenced genes and EPCAM. Of note, the APC promoter 1B region is covered as part of this deletion/duplication analysis.  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.   

Mutation Detection Rate 

Analytical sensitivity for all genes is estimated to be greater than 99.97% of described mutations.

Specimen Requirements 

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 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.
Storage: -20°C.
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

 

Turnaround Time 
TEST CODE TEST NAME TURNAROUND TIME (Weeks)
8822 ColoNext (Ordered 11/3/14 or after) 2-4
  ColoNext (Ordered before 11/3/14) 8-12

 

 

 

Specialty 
References 

1. National Cancer Institute. Cancer Stat Fact Sheets.  Accessed October 22, 2014; Available from: http://seer.cancer.gov/.

2. National Cancer Institute.  Accessed October 22, 2014; Available from: http://www.cancer.gov/.

3. Hampel, H., Genetic testing for hereditary colorectal cancer. Surg Oncol Clin N Am, 2009. 18(4): p. 687-703.

4. Lipton, L. and I. Tomlinson, The genetics of FAP and FAP-like syndromes. Fam Cancer, 2006. 5(3): p. 221-6.

5. Petersen, G.M., J. Slack, and Y. Nakamura, Screening guidelines and premorbid diagnosis of familial adenomatous polyposis using linkage. Gastroenterology, 1991. 100(6): p. 1658-64.

6. Pedace, L., et al., Identification of a novel duplication in the APC gene using multiple ligation probe amplification in a patient with familial adenomatous polyposis. Cancer Genet Cytogenet, 2008. 182(2): p. 130-5.

7. van Hattem, W.A., et al., Large genomic deletions of SMAD4, BMPR1A and PTEN in juvenile polyposis. Gut, 2008. 57(5): p. 623-7.

8. Chow, E. and F. Macrae, A review of juvenile polyposis syndrome. J Gastroenterol Hepatol, 2005. 20(11): p. 1634-40.

9. Gallione, C.J., et al., A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet, 2004. 363(9412): p. 852-9.

10. Bahassi, E.M., et al., The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage. Oncogene, 2008. 27(28): p. 3977-85.

11. Cybulski, C., et al., CHEK2 is a multiorgan cancer susceptibility gene. Am J Hum Genet, 2004. 75(6): p. 1131-5.

12. Walsh, T., et al., Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. Jama, 2006. 295(12): p. 1379-88.

13. Walsh, T., et al., Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. Proc Natl Acad Sci U S A, 2011. 108(44): p. 18032-7.

14. Pharoah, P.D., et al., Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastroenterology, 2001. 121(6): p. 1348-53.

15. Guilford, P., B. Humar, and V. Blair, Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice. Gastric Cancer, 2010. 13(1): p. 1-10.

16. Hegde, M.R. and B.B. Roa, Genetic Testing for Hereditary Nonpolyposis Colorectal Cancer (HNPCC) Current Protocols in Human Genetics, 2009. 61(Unit 10.12): p. 10.12.1-10.12.28.

17. Capelle, L.G., et al., Risk and epidemiological time trends of gastric cancer in Lynch syndrome carriers in the Netherlands. Gastroenterology, 2010. 138(2): p. 487-92.

18. Bonadona, V., et al., Cancer risks associated with germline mutations in MLH1, MSH2, and MSH6 genes in Lynch syndrome. JAMA, 2011. 305(22): p. 2304-10.

19. Engel, C., et al., Risks of less common cancers in proven mutation carriers with lynch syndrome. J Clin Oncol, 2012. 30(35): p. 4409-15.

20. Win, A.K., et al., Colorectal and other cancer risks for carriers and noncarriers from families with a DNA mismatch repair gene mutation: a prospective cohort study. J Clin Oncol, 2012. 30(9): p. 958-64.

21. Jenkins, M.A., et al., Risk of colorectal cancer in monoallelic and biallelic carriers of MYH mutations: a population-based case-family study. Cancer Epidemiol Biomarkers Prev, 2006. 15(2): p. 312-4.

22. Win, A.K., et al., Cancer risks for monoallelic MUTYH mutation carriers with a family history of colorectal cancer. Int J Cancer, 2011. 129(9): p. 2256-62.

23. Vogt, S., et al., Expanded extracolonic tumor spectrum in MUTYH-associated polyposis. Gastroenterology, 2009. 137(6): p. 1976-85 e1-10.

24. Rennert, G., et al., MutYH mutation carriers have increased breast cancer risk. Cancer, 2012. 118(8): p. 1989-93.

25. Eng, C., Will the real Cowden syndrome please stand up: revised diagnostic criteria. J Med Genet, 2000. 37(11): p. 828-30.

26. Starink, T.M., et al., The Cowden syndrome: a clinical and genetic study in 21 patients. Clin Genet, 1986. 29(3): p. 222-33.

27. Heald, B., et al., Frequent gastrointestinal polyps and colorectal adenocarcinomas in a prospective series of PTEN mutation carriers. Gastroenterology, 2010. 139(6): p. 1927-33.

28. Tan, M.H., et al., Lifetime cancer risks in individuals with germline PTEN mutations. Clin Cancer Res, 2012. 18(2): p. 400-7.

29. Mester, J.L., et al., Papillary renal cell carcinoma is associated with PTEN hamartoma tumor syndrome. Urology, 2012. 79(5): p. 1187 e1-7.

30. Hearle, N., et al., Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clin Cancer Res, 2006. 12(10): p. 3209-15.

31. Lim, W., et al., Relative frequency and morphology of cancers in STK11 mutation carriers. Gastroenterology, 2004. 126(7): p. 1788-1794.

32. Hwang, S.J., et al., Germline p53 mutations in a cohort with childhood sarcoma: sex differences in cancer risk. Am J Hum Genet, 2003. 72(4): p. 975-83.

33. Birch, J.M., et al., Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Res, 1994. 54(5): p. 1298-304.

34. Olivier, M., et al., Li-Fraumeni and related syndromes: correlation between tumor type, family structure, and TP53 genotype. Cancer Res, 2003. 63(20): p. 6643-50.

35. Gonzalez, K.D., et al., Beyond Li Fraumeni Syndrome: clinical characteristics of families with p53 germline mutations. J Clin Oncol, 2009. 27(8): p. 1250-6.

36. McCuaig, J.M., et al., Routine TP53 testing for breast cancer under age 30: ready for prime time? Fam Cancer, 2012. 11(4): p. 607-13.