ColoNext

ColoNextTM is a next generation (next-gen) sequencing panel that simultaneously analyzes 14 genes that contribute to increased risk for colon cancer.

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ColoNextTM is a next generation (next-gen) sequencing panel that simultaneously analyzes 14 genes that contribute to increased risk for colon cancer.

Ambry utilizes next generation sequencing to offer a genetic testing panel for hereditary colon cancer syndromes. Genes on this panel include APC, BMPR1A, CDH1, CHEK2,  EPCAM, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN, 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 13 of the 14 genes (excluding EPCAM). Gross deletion/duplication analysis is performed for all 14 genes. Specific-site analysis is available for individual gene mutations known to be in the family.  

Disease Name 
Cancer, Colon
Hereditary Colon Cancer
Disease Information 

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.CRC is the third leading cause of cancer-related deaths in the United States when men and women are considered separately, and second leading cause of cancer related deaths when combined.2

Colorectal cancer starts in the colon or the rectum. Colon cancer and rectal cancer have many features in common. CRCs starts in the lining of the bowel and if left untreated, can grow into the muscle layers underneath, and then through the bowel wall. Most CRCs begin with a small growth, a colorectal polyp or adenoma, on the bowel wall. These mushroom-shaped growths are usually benign, but some develop into cancer over time. Localized bowel cancer is usually diagnosed through colonoscopy. 

The majority of CRCs are sporadic; however, at least 25% are familial, a subset of which showing strong genetic etiology. As with most forms of cancer, early detection is crucial.  Studies demonstrate that mutations in genes on the ColoNext panel lead to a significant lifetime risk for colon cancer. Studies demonstrate that mutations genes on this panel confer a 10-99% lifetime risk for colon cancer and mutations in some genes can include increased risks for other cancers as well.

Hereditary cancer syndromes associated with genes on this panel include Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, PTEN-Related disorders, CHEK2-related cancers, hereditary diffuse gastric cancer, Li-Fraumeni syndrome, Peutz-Jegher syndrome, and juvenile polyposis syndrome.

ColoNext Panel Genes:

APC germline mutation are well established the primary cause of familial adenomatous polyposis (FAP) and attentuated familial adenomatous polyposis (AFAP).  Familial adenomatous polyposis (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).

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.6  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.7 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.8

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.9-13

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.14 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,15 with an estimated lifetime breast cancer risk of 39-52%.

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.16-18

Mutations in these genes can confer up to a 4-5% lifetime risk for upper urinary tract cancers, including the ureter and the renal pelvis, with mean age of diagnosis of 56 years. This represents a relative risk 22 times higher comparing to the general population for upper urinary tract cancers.30,31 The majority of urinary tract cancers are attributed to MSH2 mutations, with a lower incidence in MLH1 and MSH6.32 Recent studies also suggest an increased risk for pancreatic cancer in Lynch syndrome with up to a 8.5-fold increase in risk compared to the general population.35 Lynch syndrome-associated pancreatic cancers often have a distinctive medullary appearance.

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.19 Two common mutations, p.Y179C and p.G396D (originally designated as p.Y165C and p.G382D), have been reported as homozygous or compound heterozygous in about 70%-86% of MAP patients.20,21 Heterozygous mutations have also been associated with a 1.9 fold increased risk for breast cancer. In this series, characteristics of tumors and at of diagnosis in carriers with MUTYH variants were similar to those without MUTYH variants.22

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.23,24

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.25,26 Meta-analysis assessed the cancer risks of PJS and observed that STK11 mutation carriers ages 15-64 had a 36% risk of developing pancreatic cancer,31 while another study noted a risk of 11% for pancreatic cancer by 70 years.30

TP53 is a tumor suppressor gene that causes Li-Fraumeni syndrome (LFS)and Li-Fraumeni like (LFL) syndrome that can affect adults and children alike. An individual harboring a TP53 mutation has a 50% risk of developing cancer by age 30 and a lifetime cancer risk of up to 90%. The most common tumor types observed in LFS/LFL families include soft tissue and osteosarcomas, breast cancer, brain tumors (including astrocytomas, glioblastomas, medulloblastomas and choroid plexus carcinomas) and adrenocortical carcinoma (ACC); however, other cancers, including RCC, have been reported. Studies have shown that a small percentage of women who are negative for BRCA1 and BRCA2 negative are identified to have mutations in TP53.13,27,28 Multiple other cancer types have been observed in TP53 mutation carriers, including pancreatic cancer.34-36

Testing Benefits & Indication 

Indications for Testing

Some patterns of colon cancer, colon polyps and tumor characteristics in a family can clearly point towards a particular hereditary cancer syndrome. At other times, you may be considering a number of different genes associated with colon cancer in your differential diagnosis. These are the  cases where it would be appropriate to consider for the ColoNext panel. 

Common Red Flags for Hereditary Cancer

  • Cancer diagnosed at a younger age than expected for the general population (</= 50 for most cancers)
  • Cancer diagnosed across generations and in multiple generations within a family
  • 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 breast and pancreatic cancer with PALB2-related cancer)
     
Benefits of Testing
Knowing your patient has a genetic susceptibility for colon cancer 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. Consider prophylactic colectomy in individuals with a mutation in APC)
  • Clarify and stratify familial cancer risks, based on gene-specific cancer associations, such as risk for colon and breast cancer with mutations in STK11
  • Offer treatment guidance (e.g. Consider subtotal colectomy rather than resection for an early stage colon cancer in a person with a mutation in MLH1)
  • Identify other at-risk family members
  • Provide guidance with new gene-specific treatment options and risk reduction measures as they emerge  
Test Description 

The ColoNext Panel targets detection of mutations in 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 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 colon cancer gene coding exons followed by polymerase chain reaction (PCR) and next-generation sequencing. Additional Sanger sequencing 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 targeted microarray identifies gross deletions or duplications in all 14 genes (APC, BMPR1A, CDH1, CHEK2,  EPCAM, MLH1, MSH2, MSH6, MUTYH, PMS2, PTEN,  SMAD4, STK11, and TP53). If a deletion is detected in exons 13, 14, or 15 or 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% 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 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

Billing Codes 
Test Code Technique
8822 ColoNext Gene Analysis

 

Turnaround Time 
Technique weeks
ColoNext Gene Analysis
(Ordered 2/1/14 or after)
8 - 12
ColoNext Gene Analysis
(Ordered before 2/1/14)
12 - 16

 

Specialty 
References 

1. http://seer.cancer.gov

2. http://www.cancer.gov

3.  Lipton L et al., The genetics of FAP and FAP-like syndromes. Fam Cancer. 2006;5(3):221-226. [PMID: 16998667]

4.  Petersen GM et al. Screening guidelines and premorbid diagnosis of familial adenomatous polyposis using linkage. Gastroenterology. 1991; 100(6):1658-1664. [PMID: 1673441]

5.  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):130-135. [PMID: 18406876]

6. Van Hattem WA et al., Large genomic deletions of SMAD4, BMPR1A and PTEN in juvenile polyposis. Gut. 2008;57:623-627. [PMID: 18178612]

8. Chow E & Macrae F. J Gastroenterol Hepatol., A review of juvenile polyposis syndrome. 2005;20:1634-1640. [PMID: 16246179]

8. Gallione CJ et al., A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet. 2004;363:852-859 [PMID: 15031030]

9. Walsh et al., Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing. PNAS. 2011:108(44);10832-18037. [PMID: 22006311]

10. Bahassi, EM. et al., The checkpoint kinases Chk1 and Chk2 regulate the functional associations between hBRCA2 and Rad51 in response to DNA damage. Oncogene. 2008; 27, 3977–3985. [PMID: 18317453]

11. Orloff and Eng et al., Genetic and phenotypic heterogeneity in the PTEN hamartoma tumour syndrome. Oncogene. 2008; 27, 5387-5397. [PMID: 18794875]

12. Cybulski et al., CHEK2 is a multiorgan cancer susceptibility gene. Am J Hum Genet. 2004;75:1131–1135. [PMID: 15492928] 

13. Walsh et al., Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer. JAMA. 2006;295(12):1379-1388. [PMID: 16551709]

14. Narod SA., Testing for CHEK2 in the cancer genetics clinic: ready for prime time? Clin Genet. 2010;78:1-7. [PMID: 20597917]

15. Pharoah PD et al., Incidence of gastric cancer and breast cancer in CDH1 (E-cadherin) mutation carriers from hereditary diffuse gastric cancer families. Gastro. 2001;121:1348-1353. [PMID: 20597917]

16. Guilford P et al., Hereditary diffuse gastric cancer: translation of CDH1 germline mutations into clinical practice. Gastric Cancer. 2010;13:1-10 [PMID: 20373070]

17. Abdel-Rahman WM et al., The genetics of HNPCC: application to diagnosis and screening. Crit Rev Oncol Hematol. 2006;53:208-220. [PMID: 16434208]

18. Hedge MR & Roa BB. Genetic testing for hereditary nonpolyposis colorectal cancer (HNPCC). Curr Protoc Hum Genet. 2009;10.21(61):1-28. [PMID: 19360696]  

19. Filipe B et al., APC or MUTYH mutations account for the majority of clinically well-characterized families with FAP and AFAP phenotype and patients with more than 30 adenomas. Clin Genet. 2009;76:242-255. [PMID: 19793053]

20. Sampson JR & Jones N., MUTYH-associated polyposis. Best Pract Res Clin Gastroenterol. 2009;23(2):209-18. [PMID: 19414147]

21. Barnetson RA et al., Germline mutation prevalence in the base excision repair gene, MYH, in patients with endometrial cancer. Clin Genet. 2007; 72:551-555. [PMID: 17956577]

22. Rennert et al. MutYH mutation carriers have increased breast cancer risk. Cancer.  2012; 118(8):1989-93. [PMID: 21952991]

23. Eng C. J Med Genet. Will the real Cowden syndrome please stand up: revised diagnostic criteria. 2000;37:828-830. [PMID: 11073535]

24. Starink TM et al., The Cowden syndrome: a clinical and genetic study in 21 patients. Clin Genet. 1986;29:222–233. [PMID: 3698331]

25. Lim W et al., Relative frequency and morphology of cancers in STK11 mutation carriers. Gastroenterology. 2004;126:1788-1794. [PMID: 15188174]

26. Hearle et al., Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clin Cancer Res. 2006;12:3209-3215. [PMID: 16707622]

27. Birch JM et al., Prevalence and diversity of constitutional mutations in the p53 gene among 21 Li-Fraumeni families. Cancer Research. 1994;54: 1298-1304. [PMID: 8118819]

28. Olivier M et al., Li-Fraumeni and related syndromes: correlation between tumor type, family structure, and TP53 genotype. Cancer Research. 2003;63: 6643-6650. [PMID: 14583457]

29. Adapted from: http://www.nccn.org/professionals/physician_gls/f_guidelines.asp. Accessed Feb 28, 2012.   

30.Roupret, M., et al. (2008). Upper urinary tract urothelial cell carcinomas and other urological malignancies involved in the hereditary nonpolyposis colorectal cancer (Lynch syndrome) tumor spectrum. Eur Urol, 54:1226-1236.

31. Crockett, D., et al. (2011). Upper urinary tract carcinoma in Lynch syndrome cases. Jrn Urol, 185:1627-1630.

32. Van der Post, R., et al. (2010). Risk of urothelial bladder cancer in Lynch syndrome is increased, in particular among MSH2 mutation carriers. Jrn Med Genet, 47: 464-470.

33. Kastrinos, F., et al. Risk of pancreatic cancer in families with Lynch syndrome. JAMA, 302(16):1790-1795.

 

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

35. Hearle, et al. (2006). Frequency and spectrum of cancers in the Peutz-Jeghers syndrome. Clin Cancer Res, 12:3209-3215.

36. Giardiello, F., et al. (2000). Very high risk of cancer in familial Peutz-Jeghers syndrome. Gastroenterology, 119:1447-1453.