ColoNextTM is a next generation sequencing panel that simultaneously analyzes 17 genes associated with increased risk for colorectal cancer.


ColoNextTM is a next generation sequencing panel that simultaneously analyzes 17 genes associated with increased risk for colorectal cancer.

Ambry utilizes next generation sequencing (NGS) to offer a comprehensive panel for hereditary colorectal cancer.  Genes on this panel include: APC, BMPR1A, CDH1, CHEK2, EPCAM, GREM1, MLH1, MSH2, MSH6, MUTYH, PMS2, POLD1, POLE, PTEN, SMAD4, STK11, and TP53. Full gene sequencing is performed for 15 of the genes (excluding EPCAM and GREM1). Gross deletion/duplication analysis is performed for all 17 genes. Specific Site Analysis is available for individual gene mutations identified in a family.

Disease Name 
Colorectal cancer
Hereditary cancer
Lynch syndrome
Disease Information 

Colorectal cancer (CRC) affects about 1 in 20 (5%) men and women in their lifetime.1 The National Cancer Institute estimates that approximately 132,700 new cases will be diagnosed and 49,700 CRC deaths will occur in the U.S. in 2015.2 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.

gene specific lifetime colorectal cancer risk

ColoNext Panel Genes:

APC germline mutations are 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 rare, 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  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%.15

GREM1 A large duplication upstream of GREM1 has been identified families of Ashkenazi Jewish descent with hereditary mixed polyposis. To date, only this founder mutation has been reported, but the possibility of GREM1 mutations in individuals of other ethnicities has not been excluded.16  Manifestations of the GREM1 duplication appear to be limited to the intestinal tract, and include mixed morphology colon polyps and cancer; however, lifetime risk estimates for mutation carriers are not currently available.16,17

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.18-22

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%.23  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 endometrium24-26; 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.

POLD1 and POLE mutations are implicated in an emerging syndrome of colorectal cancer and polyposis called polymerase proofreading-associated polyposis (PPAP) by some.27  Exact cancer risks for mutation carriers have not yet been determined; however, published studies support POLD1 and POLE mutations as highly penetrant, conferring increased risk for early-onset colorectal cancer and/or multiple adenomas.28,29  Associations between POLD1 and POLE mutations and elevated incidence of extra-intestinal tumors have been suggested, although data is currently limited.

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.30,31  Recent studies noted increased risks for renal cell cancer, colorectal cancer, and other cancers.32,33  One study quotes up to a 31-fold increase in RCC risk for PTEN mutation carriers as compared to the general population.34

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.35,36  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%.37  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.38,39  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 inTP53.12,40,41


Testing Benefits & Indication 


Indications for Testing
ColoNext 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 colorectal and uterine cancer)
  • ≥3 family members with colorectal, uterine, ovarian, and/or stomach cancer*
  • ≥10 GI polyps during one’s lifetime (adenomatous, hyperplastic, hamartomatous, and/or other types of polyps)
  • A family history suspicious for several different hereditary colorectal cancer syndromes
  • Previous uninformative genetic testing for hereditary colorectal cancer
    *On the same side of the family

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.42 Establishing a molecular diagnosis can help guide preventive measures, direct surgical options and estimate personal and familial cancer risk.

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 (e.g.  colorectal and uterine cancer in Lynch syndrome, or diffuse gastric cancer and lobular breast cancer with CDH1 mutations)

Benefits of Testing
Identifying patients with an inherited susceptibility for certain cancers can help with medical management and risk assessment. For example, this information can:

  • Modify cancer surveillance options and age of 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 17 genes (listed above). 15 genes (excluding EPCAM and GREM1) are evaluated by next generation sequencing (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). For POLD1 and POLE, missense variants located outside of the exonuclease domains (codons 311-541 and 269-485, respectively) are not routinely reported. The inversion of coding exons 1-7 of the MSH2 gene is 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. Reportable small insertions and deletions, potentially homozygous variants, variants in regions complicated by pseudogene interference, and single nucleotide variant calls not satisfying 100x depth of coverage and 40% het ratio thresholds are verified by Sanger sequencing.43  Gross deletion/duplication analysis is performed for the covered exons and untranslated regions of all 17 genes using read-depth from NGS data with confirmatory multiplex ligation-dependent probe amplification (MLPA) and/or targeted chromosomal microarray. For GREM1, only the status of the 40kb 5’ UTR gross duplication is analyzed and reported.  For APC, all promoter 1B gross deletions as well as single nucleotide substitutions within the promoter 1B YY1 binding motif are analyzed and reported. 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 

ColoNext can detect >99.9% of described mutations in the included genes listed above, when present (analytic sensitivity).

Specimen Requirements 

Complete specimen requirements are available here or by downloading the PDF found above on this page.

Turnaround Time 
8822 ColoNext 14 - 21


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