Colorectal Cancer


Indications for Testing

  • Colorectal bleeding
  • Family history of colorectal cancer (CRC)
  • Clinical symptoms consistent with genetic/hereditary CRC (eg,  MUTYH-associated polyposis (MAP), familial adenomatous polyposis (FAP), Turcot syndrome, Gardner syndrome)


  • All polyps removed should have histologic examination
  • Tissue is gold standard for tumor classification and further testing (MSI, MMR, KRAS, NRAS, BRAF)
  • Immunohistochemistry – identifies mismatch repair (MMR) to guide MMR gene mutation testing
    • All tumors should be tested – ~15% of sporadic CRC will have microsatellite instability (MSI)
    • MLH1, MSH2, MSH6, PMS2 – genes to test based on IHC results

Genetic Testing

  • APC mutation testing
    • Confirm a clinical diagnosis of FAP or attenuated FAP
  • MUTYH testing
    • Confirm a clinical diagnosis of MAP
  • Mutation testing for other syndromes should be based on clinical presentation and family history
    • Peutz-Jeghers syndrome
    • Serrated polyposis syndrome


  • MMR-deficient tumors – generally more favorable prognosis
  • Chromosomal alterations in 8p, 17p, 18p – associated with poor prognosis
  • Serum markers
    • Carcinoembryonic antigen (CEA) – measure prior to surgery (ASCO, 2006)
    • Other potential but not validated serum markers – carbohydrate antigen CA19-9, CA242, CTC, and tissue inhibitor of metalloproteinase-1 (TIMP-1)
      • CTC – independent predictor of progression-free and overall survival
  • Mutation testing to predict the effectiveness of therapies that target the EGFR pathway

    Mutation Testing to Predict the Effectiveness of Therapies that Target the EGFR Pathway


    • 1 of 2 most common mutations associated with inhibited response to anti-EGFR therapy
    • Individuals who are candidates for anti-EGFR therapy should have testing for KRAS mutation (ASCO, NCCN)

    BRAF V600E

    • Other most common mutation associated with inhibited response to anti-EGFR therapy
    • Recommended in nonmutated KRAS cases (NCCN, 2012)


    • Rare mutation associated with relative resistance to anti-EGFR therapy


    • May be associated with relative resistance to anti-EGFR therapy


    • Exon 20 associated with relative resistance to anti-EGFR therapy
    • Exons 9 and 20 activating mutations indicate possible response to therapies targeting genes downstream of PI3K in the AKT/mTOR-signaling cascade
    ASCO = American Society of Clinical Oncology; NCCN = National Comprehensive Cancer Network

Differential Diagnosis


  • Direct evidence from clinical trials concludes that fecal occult blood testing and flexible sigmoidoscopy reduces mortality from colorectal cancer (CRC); however, National Comprehensive Cancer Network recommends colonoscopy as the preferred screening method (NCCN, 2014)

  • Fecal occult blood testing
    • 50% of documented colon cancers have a negative fecal occult blood test (FOBT)
      • If patient is at risk, consider sigmoidoscopy or colonoscopy even in the presence of a negative FOBT
    • Positive FOBT mandates further evaluation (eg, colonoscopy)
  • Flexible sigmoidoscopy/colonoscopy – negative result does not rule out CRC
    • 90% sensitivity for lesions ≥10 mm
  • Septin 9
    • Biomarker for presence of CRC – high negative predictive values
    • Indicated for individuals ≥50 years who have an average risk and no family history of CRC
    • Not recommended for individuals with
      • History of previous CRC
      • Above average risk (eg, family history of early onset CRC, hereditary CRC)
      • Previous polyp removal
    • Not intended as substitute for colonoscopy – may be useful as a complement to colonoscopy or for those who are unwilling or unable to have a colonoscopy
      • Patients with elevated level should undergo colonoscopy to rule out CRC
    • Has been shown to detect cancers in cecum, ascending colon, transverse colon, splenic flexure, descending colon, sigmoid, recto-sigmoid junction, and rectum
  • Colorectal screening for persons with average risk (beginning at 50 years)

    Colorectal Screening for Persons with Average Risk* (beginning at 50 years)



    USPSTF 2008; AAFP 2010

    50-75 years – one of the following screening regimens**

    • Annual screen for colorectal cancer (CRC) using FOBT
    • Sigmoidoscopy every 5 years combined with high-sensitivity FOBT every 3 years
    • Colonoscopy every 10 years

    76-85 years – recommend against routine screening for CRC; however, there may be considerations that support CRC screening in an individual patient

    >85 years – CRC screening not recommended

    ACS 2013

    One of the following regimens**

    • Flexible sigmoidoscopy every 5 years
    • Colonoscopy every 10 years
    • Double-contrast barium enema every 5 years
    • CT colonography every 5 years

    No upper age limit for screening

    *Average risk – age ≥50 years; no history of adenoma or inflammatory bowel disease (IBD); negative family history (not having one first-degree or two second-degree relatives with CRC, or a clustering of Lynch syndrome-related cancers in the family)

    **Patients with a family member diagnosed with colon cancer <60 years should begin colonoscopy screenings every 5-10 years starting at age 40 or 10 years before the age of the youngest family member diagnosed

    USPSTF = U.S. Preventive Services Task Force; AAFP = American Academy of Family Practice, ACS = American Cancer Society


  • Serum CEA
    • Elevated postoperative titer predicts tumor recurrence
    • Preoperative and postoperative monitoring for changes in concentration
      • Stage II or III tumors – measure every 3 months post operation, continuing for 3 years
    • Patient with metastatic disease – monitoring may help evaluate treatment response
  • Serum circulating tumor cell count (CTC) – in metastatic tumors, monitor disease progression and response to therapy
  • Others
    • Serum CA19-9
    • Deletion 18q – not enough data to recommend use
    • 9q22.2-31.2 – promising new marker for hereditary colorectal cancers

Pharmacogenetics and Therapeutic Drug Monitoring

  • UGT1A1 genotyping
    • Uridine diphosphate glucuronosyltransferase (UGT1A1) is responsible for clearance of irinotecan, a camptothecin analogue used in treatment of advanced colon cancer
    • Decreased gene expression may lead to drug toxicity (development of severe neutropenia)
      • Two gene variants responsible for 98-99% of genotypes in the Caucasian population – *1  and *28 (repeat TA sequence)
    • Routine reduction of dose in *28 homozygous is not recommended (Evaluation of Genomic Applications in Practice Working Group, 2009), but may identify patients at risk for adverse events and in need of closer monitoring
      • Selective genotyping based on patient preferences
        • Patients homozygous for *1 may tolerate aggressive treatment better than patients with the *28 variant
        • Patients homozygous for *28 may require a dose reduction to minimize dose-related adverse events
  • 5-fluorouracil (5-FU) sensitivity – genotyping of DYPD and TYMS
    • 5-FU is a fluoropyrimidine drug used in the treatment of colorectal cancer and other solid tumors
    • Pharmacogenetic variations in genes such as DPYD and TYMS may contribute to risk of toxicity or altered therapeutic benefits
      • DPYD or TYMS mutation detected – predictive of an increased sensitivity to 5-FU and may lead to increased risk for toxicity
        • Alternative chemotherapeutic agents, therapeutic drug monitoring, altered 5-FU doses, or increased surveillance for adverse drug reactions may be indicated

Clinical Background

Colorectal cancer (CRC) is the third most common form of cancer in the U.S. and can be roughly divided into sporadic, familial, and hereditary types.


  • Incidence – 43.7/100,000 (2011 U.S. SEER data)
    • Sporadic – most common form (~80%)
    • Hereditary
      • Hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome – accounts for 2-4% of colon cancer cases in the U.S. (NCCN, 2014)
      • Familial adenomatous polyposis (FAP) – occurs in 1/10,000 live births
        • Accounts for <1% of total colon cancers
      • MUTYH (formerly MYH)-associated polyposis (MAP) – rare
      • Others – rare
        • Cowden syndrome (multiple hamartoma syndrome)
        • Peutz-Jeghers syndrome (PJS) – 1/200,000
        • Juvenile polyposis syndrome (JPS) – 1/100,000
        • Hereditary diffuse gastric cancer
        • Serrated polyposis syndrome
  • Age 
    • Sporadic – median is 70 years
    • Hereditary – usually <60 years
      • FAP – average is 39 years
  • Sex – M>F


  • Sporadic  
    • TP53 (p53) – mutated in ~75% of sporadic tumors
    • DCC (deleted in CRCs) – mutated in ~70% of CRCs
    • DNA mismatch repair (MMR) genes – mutation or modification found in 15% of sporadic tumors
    • Hyperplastic polyposis syndrome (HPS) – rarely inherited
  • Hereditary
    • >10-15% of all CRC associated with familial clustering
    • Lynch syndrome – associated with microsatellite instability (MSI) and MMR gene mutation/modification (eg, MLH1, MSH2, MSH6, PMS2)
    • FAP
      • Mutations of APC gene – autosomal dominant inheritance, although 25% of cases are de novo
        • Classic FAP and attenuated FAP forms
    • MAP
      • Biallelic mutations in the MUTYH gene – autosomal recessive inheritance
      • Two MUTYH mutations, Y165C and G382D, account for 85% of MAP in Caucasians
    • PJS – mutations of STK11
    • JPS – mutations of SMAD4 or BMPR1A

Risk Factors

  • Diet high in animal fats (Western diet)
  • Patients with metabolic syndrome
  • Inflammatory bowel disease (eg, Crohn, ulcerative colitis)
  • Adenoma/sessile serrated polyp (SSP)
  • First-degree relative with colorectal adenoma or invasive CRC
  • Ureterosigmoidostomy – carcinoma can develop ≥15 years post procedure


  • Most CRCs arise from adenomatous polyps
    • Villous adenomas transform into adenocarcinomas more frequently than tubular adenomas
    • Subset of adenocarcinomas develop from hyperplastic-appearing polyps, especially large, right-sided polyps
    • Adenocarcinoma arising in a polyp is considered malignant when it penetrates into the submucosa 
  • Other less-common tumors can occur (lymphomas, endocrine, mesenchymal)

Clinical Presentation

  • Symptoms vary with tumor location – most are located in sigmoid colon and rectum
    • Cecum and ascending colon – tumors may be very large without causing obstruction
      • Anemia – a common presenting symptom
    • Descending and transverse colon – tumors tend to obstruct and cause annular lesions (apple core or napkin ring) with abdominal pain and bloating
    • Rectosigmoid – hematochezia, tenesmus, and narrowing of stool caliber
  • Sporadic tumors – usually single tumors
  • Inherited syndromes – tumors tend to develop at a younger age
    • Lynch syndrome – may have multiple tumors
      • Median age of 40-60 (10 years younger than sporadic colorectal carcinoma) 
      • For more information, see Lynch syndrome and Lynch syndrome testing algorithm
    • FAP (classic)
      • Affected persons develop hundreds to thousands of colon polyps and subsequent CRC; often present with multiple tumors; mean age of onset is 39 years
        • Presence of ≥100 polyps is sufficient for clinical diagnosis (or <100 polyps at younger ages in a family with identified FAP)
      • Increased risk for malignancy – thyroid, medulloblastoma, hepatoblastoma, pancreas, gastric/duodenal
      • Possible additional findings
        • Osteomas
        • Gastric polyps
        • Small intestinal polyps
        • Dental abnormalities
        • Polyps of the gastric fundus and duodenum
        • Congenital hypertrophy of the retinal pigment epithelium (CHRPE)
    • FAP (attenuated)
      • <10-100 adenomas (average is 30) – frequently right-sided distribution
      • Cancer appears at older age than classic FAP (mean age >50 years)
      • Risk of other cancers similar to classic FAP
      • Extraintestinal manifestations of classic FAP are rare
    • MAP
      • 10-100 polyps
      • Mean age of onset is >50 years
      • Often indistinguishable from attenuated FAP (may have similar extraintestinal manifestations)
    • PJS
    • JPS
      •  Juvenile polyps found mainly in colon
    • Serrated polyposis syndrome
      • Serrated polyps usually >10 mm

Indications for Laboratory Testing

  • Tests generally appear in the order most useful for common clinical situations
  • Click on number for test-specific information in the ARUP Laboratory Test Directory
Test Name and Number Recommended Use Limitations Follow Up
Occult Blood, Fecal by Immunoassay 2007190
Method: Quantitative Immunoassay

Screen for colorectal cancer (CRC)

Colonoscopy is the recommended test of choice for individuals >50 years

Septin 9 (SEPT9), Methylated DNA Detection by Real-Time PCR 2003243
Method: Polymerase Chain Reaction

Screen for CRC

Positive result indicates increased risk


  • All stages – 90%
  • Stages I-II – 87%
  • Decreased in adenomas

Specificity – 89%

Colonoscopy is the recommended test of choice for individuals ≥50 years

Do not use as screening in patients with previous history of CRC or in patients with family history of early onset colorectal or hereditary CRC

Individuals with positive results should have follow-up colonoscopy

Mismatch Repair by Immunohistochemistry 0049302
Method: Qualitative Immunohistochemistry

First-line screening test for Lynch syndrome (LS)

Directs additional molecular diagnostic testing for LS

Microsatellite Instability (MSI), HNPCC/Lynch Syndrome, by PCR 0051740
Method: Polymerase Chain Reaction/Fragment Analysis

First-line screening test for LS

Directs additional molecular diagnostic testing for LS

KRAS Mutation Detection with Reflex to BRAF Codon 600 Mutation Detection 2001932
Method: Polymerase Chain Reaction/Pyrosequencing

Evaluates two important genes in the EGFR pathway

Test detects mutations in codons 12, 13, and 61

If KRAS mutation is not detected, BRAF codon 600 mutation detection test will be performed

Clinical sensitivity – activating KRAS mutations found in ~40% of CRCs

Analytical sensitivity/specificity – 99%

Limit of detection – 10% mutant alleles

Oncogenic mutations outside of codons 12, 13, and 61 will not be detected

A substantial portion of individuals with wild-type KRAS still fail to respond to anti-EGFR agents, implicating downstream mutations

BRAF Codon 600 Mutation Detection by Pyrosequencing 2002498
Method: Polymerase Chain Reaction/Pyrosequencing

Detect activating BRAF mutations associated with anti-EGFR therapy resistance

For endometrial tissue evaluation, refer to MLH1 Promoter Methylation, Paraffin

Clinical sensitivity – activating BRAF found in ~10% of CRCs

Analytic sensitivity/specificity – 99% 

Limit of detection – 10% mutant alleles

Oncogenic mutations outside of codon 600 will not be detected

NRAS Mutation Detection by Pyrosequencing 2003123
Method: Polymerase Chain Reaction/Pyrosequencing

Detect rare NRAS mutations associated with relative resistance to anti-EGFR therapy

Clinical sensitivity – oncogenic NRAS mutation found in ~3% of CRCs

Analytic sensitivity/specificity – 99%

Limit of detection – 10% mutant alleles

Oncogenic mutations outside of codons 12, 13, and 61 are not detected

Presence or absence of mutations does not guarantee a positive response to anti-EGFR therapies

PTEN by Immunohistochemistry 2004115
Method: Immunohistochemistry

Detect loss of PTEN expression in tumor tissue

Possibly associated with relative resistance to anti-EGFR therapy

Clinical sensitivity/specificity – loss of expression found in up to 50% of CRCs

PIK3CA Mutation Detection 2004510
Method: Polymerase Chain Reaction/Pyrosequencing

Predicts response to anti-EGFR and AKT/mTOR pathway therapies in a variety of malignancies (eg, colorectal, ovarian, and breast cancer)

Detect activating PIK3CA mutations in exons 9 and 20

Clinical sensitivity – oncogenic PIK3CA mutation found in 10-20% of CRCs, mostly exons 9 (60-65%) or 20 (20-25%)

Analytic sensitivity/specificity – 99%

Limit of detection – 20% tumor cells, 10% mutant alleles

Oncogenic mutations outside of the hotspots in exons 9 and 20 will not be detected

Mutation presence or absence does not guarantee a response or lack of response to anti-EGFR therapy

Familial Mutation, Targeted Sequencing 2001961
Method: Polymerase Chain Reaction/Sequencing

Evaluate family members for a known family mutation

A copy of a relative's laboratory test report documenting the gene and specific mutation(s) for which testing is requested must be submitted with the test order

HNPCC/Lynch Syndrome Deletion/Duplication 2001728
Method: Polymerase Chain Reaction/Multiplex Ligation-dependent Probe Amplification
Order if sequencing studies have been performed at another laboratory and no mutations were detected    
Familial Adenomatous Polyposis Panel: (APC) Sequencing and Deletion/Duplication, (MUTYH) 2 Mutations 2004915
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Determine genetic basis for FAP

Test at-risk family members with family history but no known familial mutation

Do not use for identification of a known familial mutation

Familial Adenomatous Polyposis (APC) Sequencing 2004863
Method: Polymerase Chain Reaction/Sequencing

Detect gene mutations related to APC-associated FAP

Detects ~90% of APC gene mutations

Familial Adenomatous Polyposis (APC) Deletion/Duplication 2004920
Method: Polymerase Chain Reaction/Multiplex Ligation-dependent Probe Amplification

Detect APC deletions and duplications related to APC-associated FAP

Detects only APC gene mutations not identified by sequencing

MUTYH-Associated Polyposis (MUTYH) 2 Mutations 2004911
Method: Polymerase Chain Reaction/Sequencing

Diagnostic or presymptomatic testing for MAP and MUTYH-associated polyposis

Uncertain result means that a gene mutation was detected; it may be pathogenic or benign; clinician should rely on clinical findings and family history

Gastrointestinal Hereditary Cancer Panel, Sequencing and Deletion/Duplication, 15 Genes  2010198
Method: Massive Parallel Sequencing/Exonic Oligonucleotide-based CGH Microarray

Preferred test for individuals with suspected hereditary GI cancer syndrome


Analytical sensitivity/specificity – 99%

Not determined or evaluated

  • Mutations in genes not included on the panel
  • Deep intronic and regulatory region mutations
  • Breakpoints for large deletions/duplications
  • PMS2 gene (associated with Lynch syndrome) is not included on this panel
  • Sequence changes in EPCAM will not be evaluated
  • Deletions/duplications may not be detected in exon 1 in CDH1 and MSH2 genes; exons 4, 6, and 7 in STK11 gene; exon 8 in PTEN gene; exon 9 in BMPR1A gene

Diagnostic errors can occur due to rare sequence variations

Individuals with hematological malignancy and/or a previous allogenic bone marrow transplant should not undergo molecular genetic testing on peripheral blood specimen

  • Testing on cultured fibroblasts or buccal specimen is required for accurate interpretation of test results

Lack of a detectable gene mutation does not exclude a diagnosis of hereditary GI cancer syndrome

Not all predisposing genes are analyzed

Carcinoembryonic Antigen 0080080
Method: Quantitative Electrochemiluminescent Immunoassay

Monitor tumor recurrence

Not sensitive or specific enough for screening in the general population

Circulating Tumor Cell Count 0093399
Method: Immunomagnetic Separation/Immunofluorescent Stain/Computer Assisted Analysis

Use to determine prognosis, assess treatment efficacy, and aid in treatment decisions for patients with CRC

Cutoffs vary by tumor cell type

CTC test is not as accurate as imaging in assessing whether a patient has progressive disease

Doxorubicin therapy patients – allow at least 7 days following administration of dose before testing

Not detected – CTCs that do not express EpCAM; CTCs that express EpCAM but not cytokeratins 8, 18, and 19

Additional Tests Available
Click the plus sign to expand the table of additional tests.
Test Name and NumberComments
HNPCC/Lynch Syndrome (MLH1) Sequencing and Deletion/Duplication 0051650
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline MLH1 mutations

Use in mismatch repair (MMR)-deficient carcinoma with suggestive IHC (loss of MLH1 and PMS2 protein), absence of BRAF codon 600 mutation, and normal MLH1 methylation studies

HNPCC/Lynch Syndrome (MSH2) Sequencing and Deletion/Duplication 0051654
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline MSH2 mutations

Use in MMR-deficient carcinoma with suggestive IHC (loss of MSH2 and MSH6 protein)

Detects large MSH2 deletions and EPCAM 3 prime deletions

HNPCC/Lynch Syndrome (MSH6) Sequencing and Deletion/Duplication 0051656
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline MSH6 mutations

Use in MMR-deficient carcinoma with suggestive IHC (isolated loss of MSH6 protein)

HNPCC/Lynch Syndrome (PMS2) Sequencing and Deletion/Duplication 0051737
Method: Polymerase Chain Reaction/Sequencing/Multiplex Ligation-dependent Probe Amplification

Detect germline PMS2 mutations

Use in MMR-deficient carcinoma with suggestive IHC (isolated loss of PMS2 protein)

BRAF Codon 600 Mutation Detection with Reflex to MLH1 Promoter Methylation 0051750
Method: Polymerase Chain Reaction/Pyrosequencing

Recommended reflex test for differentiating between LS and sporadic CRC in tumors showing loss of MLH1

If no BRAF mutation is detected, MLH1 promoter methylation is evaluated

KRAS Mutation Detection 0040248
Method: Polymerase Chain Reaction/Pyrosequencing

Detect most activating KRAS mutations associated with anti-EGFR therapy resistance

5-Fluorouracil (5-FU) Toxicity and Chemotherapeutic Response, 7 Mutations 2007228
Method: Polymerase Chain Reaction/Single Nucleotide Extensions/Fragment Analysis

Predict toxicity and responsiveness of tumor to 5-FU therapy

Clinical sensitivity– estimated at 31% for the DPYD variants analyzed

Analytical sensitivity/specificity – 99%

UDP Glucuronosyltransferase 1A1 (UGT1A1) Genotyping 0051332
Method: Polymerase Chain Reaction/Fragment Analysis

Dosage planning for individuals

  • Who will receive high-dose irinotecan (>150 mg/m2)
  • With personal or family history of sensitivity to irinotecan
  • Who have experienced neutropenia while receiving irinotecan

Confirm suspected diagnosis of Gilbert syndrome

Solid Tumor Mutation Panel by Next Generation Sequencing 2007991
Method: Massively Parallel Sequencing

Detects hotspot mutations in PIK3CA, KRAS, BRAF, NRAS, and 44 other cancer-related genes