Test availability

Table 1 lists tests used to screen, diagnose, assess prognosis, select therapy, or monitor CRC.

Table 1. Tests Available for Screening, Diagnosis, and Management of Colorectal Cancer

Test code	Assaya	Method description		Clinical use
Screening
11290 (11293 for Medicare)	Fecal Globin by Immunochemistry (InSure®)	Fecal immunochemistry test targeting hemoglobin; colorimetric detection		Screen for lower GI bleeding associated with CRC, adenomas, polyps, and other lower GI conditions
Diagnosis and risk assessment
General pathology
14517	Tissue, Gastrointestinal Pathology Report	Hematoxylin and eosin stain; microscopy		Diagnose CRC and polyposis syndrome
Tumor testing for Lynch syndrome
16767	BRAF Mutation Analysisb	NGS analysis of exons 6, 8, 11, 12, 14, 15, and 17		Distinguish sporadic cancer from Lynch syndrome–associated cancer; determine suitability for EGFR-targeted therapy
92294	BRAF V600E Mutation, IHC With Interpretation	IHC
92295	BRAF V600E Mutation, IHC Without Interpretation
91332	Lynch Syndrome Tumor Panel, IHC With Interpretation Includes MLH1, MSH2, MSH6, and PMS2 proteins	IHC		Identify individuals with Lynch syndromec
91333	Lynch Syndrome Tumor Panel, IHC Without Interpretation Includes MLH1, MSH2, MSH6, and PMS2 proteins
14989	Microsatellite Instability (MSI)	Multiplex PCR amplification of 5 NCI-recommended microsatellites; fluorescent fragment analysis		Identify individuals with Lynch syndrome; determine suitability for checkpoint inhibitor immunotherapy in patients with advanced CRC; assess prognosis and predict response to fluoropyrimidine adjuvant therapy in patients with stage II CRC
39782	MLH1 Methylationb	Real-time PCR		Distinguish sporadic cancer from Lynch syndrome when MLH1 is absent on IHC
70196	MLH1, IHC With Interpretation	IHC		Identify individuals with Lynch syndromec
16967	MLH1, IHC Without Interpretation
70197	MSH2, IHC With Interpretation
16971	MSH2, IHC Without Interpretation
16938	MSH6, IHC With Interpretation
16252	MSH6, IHC Without Interpretation
16997	PMS2, IHC With Interpretation
16254	PMS2, IHC Without Interpretation
Blood tests for Lynch syndrome
91461	Lynch Syndrome Panelb Includes MLH1, MSH2, MSH6, PMS2, and EPCAM (dosage only) genes	NGS		Identify individuals with Lynch syndromec
91460	Lynch Syndrome, MLH1 Sequencing and Deletion/Duplicationb
91471	Lynch Syndrome, MSH2 Sequencing and Deletion/Duplication (Including EPCAM)b
91458	Lynch Syndrome, MSH6 Sequencing and Deletion/Duplicationb
91457	Lynch Syndrome, PMS2 Sequencing and Deletion/Duplicationb
Genetic testing for polyposis syndromes
93797	APC Sequencing and Deletion/Duplicationb	NGS		Identify individuals with FAP or AFAP
94053	Juvenile Polyposis Panel (BMPR1A and SMAD4)b			Identify individuals with JPS
93944	MUTYH Sequencing and Deletion/Duplicationb			Identify individuals with MAP
92566	PTEN Sequencing and Deletion/Duplicationb			Identify individuals with CS/PHTS
92565	STK11 Sequencing and Deletion/Duplicationb			Identify individuals with PJS
Multigene panels for hereditary cancer syndromes
38600	Comprehensive Hereditary Cancer Panelb Includes APC, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN1B, CDKN2A (p16, p14), CHEK2, DICER1, EGFR, EPCAM, FANCA, FANCC, FANCM, FH, FLCN, GALNT12, GREM1, HOXB13, MAX, MEN1, MET, MITF, MLH1, MRE11 (MRE11A), MSH2, MSH3, MSH6, MUTYH, NBN, NF1, NTHL1, PALB2, PMS2, POLD1, POLE, POT1, PTCH1, PTEN, RAD50, RAD51C, RAD51D, RECQL, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, SMARCA4, SMAD4, STK11, SUFU, TMEM127, TP53, TSC1, TSC2, VHL, and XRCC2	NGS		Identify individuals who may be at increased risk of hereditary cancers of the colon, rectum, adrenal glands, breast, endometrium, neuroendocrine system, ovary, pancreas, prostate, paraganglia, skin, stomach, thyroid, urinary tract, and other tissues
38611	Guideline Based Hereditary Cancer Panelb Includes APC, ATM, AXIN2, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p16, p14), CHEK2, EPCAM, GREM1, HOXB13, MLH1, MSH2, MSH3, MSH6, MUTYH, NF1, NTHL1, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53			Identify individuals who may be at increased risk of hereditary cancers of the colon, rectum, breast, endometrium, ovary, pancreas, prostate, stomach, urinary tract, and other tissues
38631	Hereditary Colorectal Cancer Panelb Includes APC, AXIN2, BMPR1A, CDH1, CHEK2, EPCAM, GREM1, MLH1, MSH2, MSH3, MSH6, MUTYH, NTHL1, PMS2, POLD1, POLE, PTEN, SMAD4, STK11, and TP53			Identify individuals who may be at increased risk of hereditary cancer of the colon, rectum, and other tissues
Therapy selection and prognosis
EGFR-targeted therapy
16510	KRAS Mutation Analysisb	NGS analysis of exons 2, 3, and 4		Determine suitability for EGFR-targeted therapy
16818	NRAS Mutation Analysisb	NGS analysis of exons 2, 3, and 4
16767	BRAF Mutation Analysisb	NGS analysis of exons 6, 8, 11, 12, 14, 15, and 17
92294	BRAF V600E Mutation, IHC With Interpretation	IHC
92295	BRAF V600E Mutation, IHC Without Interpretation
HER2-targeted therapy
30316	HER-2, IHC With Interpretation	IHC		Determine suitability for HER2-targeted therapy
19214	HER-2, IHC, Without Interpretation
15547	HER-2, IHC With Reflex to FISHd	IHC reflex to FISH
14620	FISH, HER-2/neu, Paraffin Blockd,e	FISH
19859	FISH, HER-2/neu With Reflex to IHCe	FISH reflex to IHC
Other variants
93234	Solid Tumor Core Panelb Includes AKT1, AKT2, ALK, AR, BAP1, BRAF, BRCA1, BRCA2, CDKN2A, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, FGFR1, FGFR2, FGFR3, FGFR4, GNA11, GNAQ, HRAS, IDH1, JAK2, KIT, KRAS, MAP2K1, MDM2, MET, MTOR, MYC, MYCN, NRAS, NTRK1, NTRK2, NTRK3, PDGFRA, PDGFRB, PIK3CA, PTCH1, PTEN, RET, ROS1, TERT, TMPRSS2, TP53, TSC1, and VHL. The genes tested for translocations include ALK, BRAF, EGFR, ERBB2, FGFR1, FGFR2, FGFR3, MET, NTRK1, NTRK2, NTRK3, RET, ROS1, and TMPRSS2. Includes TMB and MSI analysis.	NGS		Assess eligibility for clinical trial enrollment in patients with advanced CRC
93233	Solid Tumor Expanded Panelb Includes testing of 500+ genes (including the TERT promoter) for assessment of all DNA and RNA variant types, with testing of 55 genes for translocations. Includes TMB and MSI analysis. See appendix for the full list of genes.
Other tests
13682	Haystack MRD™ Baselineb	NGS		Predict recurrence risk; inform chemotherapy decisions; monitor treatment response and cancer recurrence
13151	Haystack MRD™ Monitoringb
14989	Microsatellite Instability (MSI)b	Multiplex PCR amplification of 5 NCI-recommended microsatellites; fluorescent fragment analysis		Identify individuals with Lynch syndrome; determine suitability for checkpoint inhibitor immunotherapy in patients with advanced CRC; assess prognosis and predict response to fluoropyrimidine adjuvant therapy in patients with stage II CRC
17813	UGT1A1 Gene Polymorphism (TA Repeat)b	PCR amplification; fluorescent detection		Predict irinotecan toxicity; assist in selecting initial dosage for patients with advanced or metastatic CRC
Monitor CRC
978	CEA	Immunoassay		Monitor therapeutic response; detect residual disease or recurrence; assess prognosis
13682	Haystack MRD™ Baselineb	NGS		Predict recurrence risk; inform chemotherapy decisions; monitor treatment response and cancer recurrence
13151	Haystack MRD™ Monitoringb

AFAP, attenuated familial adenomatous polyposis; CEA, carcinoembryonic antigen; CRC, colorectal cancer; CS/PHTS, Cowden syndrome/PTEN hamartoma tumor syndrome; FAP, familial adenomatous polyposis; FISH, fluorescence in situ hybridization; GI, gastrointestinal; IHC, immunohistochemistry; JPS, juvenile polyposis syndrome; MAP, MUTYH-associated polyposis; MSI, microsatellite instability; NCI, National Cancer Institute; NGS, next-generation sequencing; PCR, polymerase chain reaction; PJS, Peutz-Jeghers syndrome; TMB, tumor mutational burden.
a	Panel components may be ordered separately. Please note that Quest offers a variety of single-gene and gene panel testing. For genetic panels noted in this document, there may be single-gene tests or smaller panels that may be applicable for your patient. Refer to the Quest Diagnostics Test Directory for further information: TestDirectory.QuestDiagnostics.com/Test/Home.
b	This test was developed and its analytical performance characteristics have been determined by Quest Diagnostics. It has not been cleared or approved by the US Food and Drug Administration. This assay has been validated pursuant to the CLIA regulations and is used for clinical purposes.
c	Confirmatory Lynch syndrome tests are offered; assistance in test selection is available from our genomic science specialists at 1.866.GENE.INFO (1.866.436.3463).
d	Reflex tests are performed at an additional charge and are associated with an additional CPT code(s).
e	The analytical performance characteristics of this assay have been determined by Quest Diagnostics. The modifications have not been cleared or approved by the US Food and Drug Administration. This assay has been validated pursuant to the CLIA regulations and is used for clinical purposes.

 

Test selection and interpretation

Screening

Currently, colonoscopy is the most used method for screening CRC in the United States and is the gold standard for assessing other screening methods.3 Noninvasive methods are available as options to increase screening rates, such as fecal immunochemical tests (FITs).3

Fecal immunochemical tests (FITs)

Cancerous and precancerous colorectal lesions tend to cause low-level bleeding, making tests for occult blood in stool an important screening tool. Stool-based tests fall into 2 main categories: guaiac fecal occult blood tests (gFOBTs) and FITs. A drawback to gFOBTs is that they detect heme peroxidase activity and are not specific for human hemoglobin. In contrast, FITs do not react with non-human hemoglobin or peroxidase, eliminating the need for food restrictions. This advantage, along with relatively simple "brush" sample collection, may result in increased participation in stool-based screening.3,4

FITs are also specific for lower gastrointestinal bleeding because they target the globin portion of hemoglobin, which does not survive passage through the upper gastrointestinal tract. FITs have better sensitivity than gFOBTs for detecting CRC.3 InSure® ONE™ (test code 11290/11293) is a FIT that requires 2 samples collected from the toilet after a single bowel movement; it has been shown to exhibit acceptable overall agreement with InSure® FIT™.4

FITs are indicated for individuals at average risk of developing CRC.3 Positive FIT results generally reflect the presence of blood in the stool and may be associated with CRC. Positive results on FIT or other stool-based tests should be followed up with colonoscopy within 9 months.3 Negative FIT results do not rule out CRC; false-negative results can occur because of uneven distribution of blood in the feces or intermittent bleeding. Individuals with a negative FIT result should be rescreened with a FIT or another screening modality in a year.3

Diagnosis of CRC and associated syndromes

Tissue pathology

Most CRC cases are initially diagnosed through analysis of endoscopic biopsy or polypectomy specimens obtained during screening, follow-up, or diagnostic colonoscopy. Pathology review (test code 14517) assesses the state of neoplasia, histologic grade, the margin of the resected tissue, and the presence or absence of lymphovascular invasion.5 Follow-up depends on whether histologic features are favorable or unfavorable.5,6 Pathology results can also inform diagnosis of hereditary conditions associated with CRC (Figure).2

Testing for Lynch syndrome

Patients with newly diagnosed CRC should all be tested for mismatch repair (MMR) or microsatellite instability (MSI) to identify those with Lynch syndrome.2,5,6 Two types of tumor-based tests are available to screen for Lynch syndrome: (1) IHC analysis for the expression of MMR proteins and (2) analysis for MSI. The National Comprehensive Cancer Network® (NCCN®) recommends using one of the tests initially and using the other test when initial results are normal but Lynch syndrome is strongly suspected.2 Comprehensive tumor NGS panel and germline multi-gene testing may substitute MSI analysis and IHC but the relative sensitivity and specificity are not well defined.

IHC testing determines the expression of MMR proteins: MLH1, MSH2, MSH6, and PMS2 (Lynch Syndrome Tumor Panel, IHC With [test code 91332] and Without [test code 91333] Interpretation or as individual components [Table 1]). Loss of expression of 1 or more of these proteins may indicate defective DNA repair processes associated with Lynch syndrome. Compared to MSI testing, this approach has slightly lower sensitivity (93% for MSI, 89% to 92% for IHC); reported concordance between the methods is 99%.2 Abnormal IHC results alone do not rule out Lynch syndrome because the false-negative rate is 5% to 10%.2

Determining MSI status involves testing the tumor for short, repetitive DNA sequences called microsatellites (test code 14989), which indicate that MMR proteins are not functioning correctly. Results are reported as MSI- high (MSI-H) or microsatellite stable (MSS). An MSI-H result is reported if ≥2 of the 5 National Cancer Institute-recommended markers show instability; an MSS result is reported if 1 or no marker shows instability.7 MSS results alone do not rule out Lynch syndrome because the false-negative rate is 5% to 15%.2

When tumor tissue is insufficient for MSI or IHC testing, germline testing of the genes most commonly associated with Lynch syndrome (MLH1, MSH2, MSH6, PMS2, and EPCAM) using a blood specimen may be considered.2 These genes are preferably tested in a panel (test code 91461), especially for patients over 50 years old or with strong family history.2 If IHC or MSI results are abnormal, follow-up testing is recommended to differentiate somatic versus germline etiology because 10% to 15% of those abnormal results are caused by sporadic cancer.2 Appropriate follow-up testing depends on the abnormal protein (MLH1, MSH2, MSH6, PMS2). For example, when MLH1 expression is absent, testing for MLH1 hypermethylation (test code 39782), BRAF V600E pathogenic variant (test code 16767), or abnormal BRAF V600E protein (test code 92294 and 92295) is indicated before proceeding to germline testing.2 Refer to the most recent version of guidelines for follow-up testing.

Genetic testing for adenomatous polyposis

Individuals with 10 or more adenomas should be assessed for adenomatous polyposis, which includes FAP, AFAP, MAP, and rare genetic causes of multiple adenomatous polyps (Figure).2 These conditions are associated with pathogenic variants in several genes, such as APC, MUTYH, AXIN2, GREM1, NTHL1, POLE, POLD1, or MSH3. Whether to test a single gene or multiple genes in a panel depends on the individual's personal and family history as well as if there are known pathogenic variants. When no pathogenic variant can be identified, a diagnosis of colonic adenomatous polyposis of unknown etiology (CPUE) may be considered. Testing of these genes is also appropriate for assessing risk in family members of affected individuals.

Patients should be tested for adenomatous polyposis if they meet 1 of the following criteria2:

• The patient has ≥20 adenomas.
• A known pathogenic variant associated with adenomatous polyposis has been identified in the family.
• The patient has multifocal/bilateral congenital hypertrophy of retinal pigment epithelium (CHRPE).
• The patient has thyroid cancer (cribriform-morular variant).
• The patient has a family history of polyposis but the affected relative is unwilling/unable to have testing.

Patients may be considered for genetic testing if they meet 1 of the following criteria2:

• The patient has 10 to 20 cumulative adenomas.
• The patient has 1 of the following conditions: desmoid tumor, hepatoblastoma, or CHRPE.
• The patient has some adenomas and meets 1 of the following criteria for SPS:
• >20 serrated polyps or lesions distributed throughout the large bowel (5 or more are proximal to the rectum)
• ≥5 serrated polyps or lesions proximal to the rectum (all are at least 5 mm in size; 2 or more are at least 10 mm)

Quest offers single-gene tests for APC (test code 93797) or MUTYH (test code 93944) gene analysis that detect pathogenic variants. These genes, as well as genes associated with rare genetic causes of multiple adenomatous polyps, are also included as components of some panels (Table 1).

Interpretation of APC and MUTYH test results is as follows.2

• Presence of a pathogenic variant in APC confirms the diagnosis of FAP or AFAP in a symptomatic individual.
• Presence of a biallelic pathogenic variant in MUTYH confirms the diagnosis of MAP in a symptomatic individual.
• Presence of a monoallelic pathogenic variant (1% to 2% of the general population) in MUTYH does not indicate increased risk for colon cancer. These individuals (without family history of CRC or polyps) may be screened for CRC in the same manner as members of the general population.
• Absence of a bi- or monoallelic pathogenic variant does not rule out the diagnosis. Gene sequencing does not identify all pathogenic variants affecting APC or MUTYH mRNA splicing. Deletion/duplication analysis cannot detect deletions or duplications that affect regions of the APC or MUTYH gene not examined in the assay (eg, most of the intronic regions).
• Positive test results for the familial pathogenic variant for an asymptomatic, at-risk family member indicate that they are affected. Refer to the most recent version of guidelines for surveillance procedures.
• Negative test results for the familial pathogenic variant for an asymptomatic, at-risk family member indicate that they are unaffected and can be screened for CRC following guidelines for the general population.

Genetic testing for Peutz-Jeghers Syndrome, Juvenile Polyposis Syndrome, and Cowden Syndrome/PTEN Hamartoma Tumor Syndrome

Individuals with 2 or more hamartomatous polyps should be assessed for PJS, JPS, and CS/PHTS.2 Diagnosis of these syndromes is based on clinical criteria; distinguishing features include but are not limited to (see NCCN for full criteria6,8)

• Mucocutaneous hyperpigmentation for PJS
• Multiple juvenile polyps for JPS
• Macrocephaly and/or Lhermitte-Duclos disease and/or mucocutaneous lesions and/or breast, endometrial, or follicular thyroid cancer for CS/PHTS

Evaluation of the patient should also include genetic testing (Figure).2,9 Pathogenic variants in STK11 are present in up to 94% of PJS families; pathogenic variants in BMPR1A or SMAD4 are present in up to 64% of patients with JPS; pathogenic variants in PTEN are present in more than 80% of patients with CS/PHTS.2,8,9 Testing of these genes is also appropriate for patients with affected family members.2

Quest offers single-gene tests for STK11 (test code 92565) or PTEN (test code 92566) that detect pathogenic variants. Quest also offers a panel that includes testing both BMPR1A and SMAD4 genes (test code 94053). Testing for all 4 genes is also available in the context of larger panels (Table 1).

Interpretation of test results for these genes is as follows.2,9

• Presence of a pathogenic variant confirms the clinical diagnosis of the respective syndrome in a symptomatic individual.
• Absence does not rule out the diagnosis because gene sequencing does not identify all pathogenic variants affecting mRNA splicing. Deletion/duplication analysis cannot detect deletions or duplications that affect regions of the gene not examined in the assay (eg, most of the intronic regions). In addition, patients with these syndromes are diagnosed based on clinical criteria and may not have the pathogenic variants.
• Positive test results for the familial pathogenic variant for an asymptomatic, at-risk family member indicate that they are affected. Refer to the most recent version of guidelines for surveillance procedures.
• Negative test results for the familial pathogenic variant for an asymptomatic, at-risk family member indicate that they are unaffected and can be screened for CRC following guidelines for the general population.

Multigene testing

Testing for multiple genes simultaneously can improve the chances of identifying the cause of cancer.2 However, multigene testing can potentially introduce situations in which clinical management is uncertain (eg, if variants in >1 gene or variants of unknown clinical significance are identified). Different panels focus on different levels of coverage (eg, syndrome, cancer, comprehensive) and contain different numbers of genes that may have varying penetrance. Importantly, clinical context should be considered to identify the most appropriate panel and offer professional genetic expertise to the patient before and after testing.2

Guidelines do not recommend multigene testing if (1) a familial pathogenic variant has been identified and no other reasons for concern are present (eg, additional personal or family history suggestive of other pathogenic variants) or (2) family history strongly suggests a known syndrome.2

However, multigene panels may have an advantage in the following example scenarios (other scenarios may also be considered depending on clinical judgment)2:

• The patient's personal and family history can be explained by more than one gene.
• A syndrome-specific panel has negative results but the patient's personal and family history strongly suggest inherited syndromes.
• Syndrome-specific panel may miss pathogenic variants in multiple actionable genes that may inform the management of the patient and family members.

Refer to Table 1 for multigene panels relevant to CRC.

Selecting therapy and assessing prognosis

Testing CRC tumors for actionable variants influences clinical decisions regarding prognosis and therapy selection. The NCCN Guidelines® recommend tumor testing in all patients with metastatic CRC for pathogenic variants in KRAS and NRAS, HER2 overexpression, and MMR/MSI status.5,6 Refer to the most recent version of guidelines for detailed information.

KRAS, NRAS, and BRAF

The epidermal growth factor receptor (EGFR) is important for CRC initiation and progression.The KRAS, NRAS, and BRAF genes encode proteins that activate signaling pathways downstream of EGFR. Because such activation is independent of EGFR, pathogenic variants in these genes can render EGFR immunotherapies, such as cetuximab and panitumumab, ineffective. Quest offers tests for pathogenic variants in KRAS (test code 16510), NRAS (test code 16818), and BRAF (test code 16767) that can predict nonresponse.

Guidelines strongly recommend testing all metastatic CRC tumors for pathogenic variants in KRAS and NRAS to inform first-line treatments and to plan an early treatment continuum.5,6 They also recommend testing for the BRAF V600E pathogenic variant when stage IV CRC is diagnosed.5,6 The expression of BRAF V600E mutation can also be detected with IHC (test codes 92294 and 92295).5,6

The presence of a known pathogenic variant in KRAS or NRAS indicates that cetuximab and panitumumab should not be prescribed, alone or in combination with other therapies.5 However, in KRAS G12C mutation-positive patients, cetuximab and panitumumab may be prescribed in combination with KRAS G12C inhibitors (sotorasib or adagrasib).5 The presence of BRAF V600E indicates (1) that a response to cetuximab and panitumumab, alone or in combination with cytotoxic therapies (unless a component of a BRAF inhibitor regimen), is very unlikely; and (2) a poor prognosis.5

HER2 overexpression

The HER2 (also known as ERBB2) protooncogene encodes a tyrosine kinase receptor that is a member of the EGFR family. HER2 overexpression is detected in approximately 3% patients with CRC but up to in 14% patients who are negative for KRAS/NRAS/BRAF pathogenic variants.5 The NCCN Guidelines recommend HER2 testing for all patients with metastatic CRC to inform subsequent treatment decisions on HER2 overexpression.5,6

HER2 status can be assessed by IHC or fluorescence in situ hybridization (FISH). Quest offers HER2 tests using each method alone (test codes 30316 and 14620) or with reflex testing (test codes 15547 and 19859). If an initial IHC test yields a HER2 IHC score of 2+, then a reflex FISH test should be performed.5,6 Patients with tumors that are HER2 overexpressed but negative for KRAS/NRAS/BRAF pathogenic variants may be eligible for HER2-targeted therapies with signal transduction inhibitors.5,6 Patients with a HER2 IHC score of 3+ may be eligible for fam-trastuzumab deruxtecan-nxki monotherapy regardless of their KRAS/NRAS status.5,6

Other variants

In addition to the variants discussed above, Quest offers testing for other variants as part of large NGS panels for solid tumors spanning either 49 genes (test code 93234) or 522 genes and the TERT promotor (test code 93233). In these panels, common downstream acceptor genes are also sequenced from RNA to detect potential fusions and splice variants (Table 1 and Appendix). Reports from variant panel testing include the clinical significance, prognosis, and predicted response to therapy for the variant. The variants are classified into 4 tiers based on the strength of the current evidence for their clinical significance (Table 2).10 Some variants are detected only within targeted regions of the selected genes but not in the promoter and intronic variant regions (except for the TERT promoter, fusions, and splice site variants).

Table 2. Variant Classification Tiers

Tier10	Strength of significance	Type of evidence
1	Strong clinical significance	Actionability supported by large studies with expert consensus Included in professional guidelines to guide clinical decision-making for the given tumor type
2	Potential clinical significance	Actionability supported by multiple small or preclinical studies or case reports, with or without expert consensus Included in professional guidelines to guide therapy selection for a different tumor type Fulfills criteria for clinical trial inclusion
3	Uncertain clinical significance	No known actionability or significance in current literature Not found in the general population
4a	Benign or likely benign	No known actionability or significance in current literature Found in the general population

a Tier 4 variants are not reported.

Large NGS panels can also be used to simultaneously evaluate tumor mutational burden (TMB) and MSI. These are gene-agnostic measures of hypermutation and defective DNA repair mechanisms within tumor cells that can also be used to assess eligibility for some therapies.

Other tests

Mismatch repair or microsatellite instability

MMR or MSI testing (also see "Screening for Lynch syndrome") is recommended to inform decisions on immunotherapy for all patients with metastatic CRC and adjuvant chemotherapy for patients with stage II CRC, in addition to identifying individuals who may have Lynch syndrome. MSI status can inform options related to the immunotherapies, such as pembrolizumab, dostarlimab-gxly, nivolumab, and ipilimumab, in first-line or subsequent settings; in general, an MSI-H status indicates that these therapies may be options,5,6 but refer to the most current versions of guidelines for detailed information. MSI status can also inform prognosis and the use of fluoropyrimidine-based (eg, 5-fluorouracil [5-FU]) adjuvant therapy; stage II colon cancer patients with MSI-H have been found to have good prognosis but not to benefit from 5-FU adjuvant therapy.5

UGT1A1 polymorphism

Irinotecan (CAMPTOSAR®) therapy can cause dose-limiting toxicity that can manifest as neutropenia. The risk of neutropenia can be assessed by testing the gene UGT1A1, which encodes uridine diphosphate glucuronosyltransferase 1A1.5,11 This hepatic enzyme metabolizes the active form of irinotecan, SN-38, to an inactive state; variants that reduce enzyme activity are associated with drug toxicity.11 The irinotecan product insert suggests a reduced initial dose for patients with UGT1A1*28/*28, *6/*6, or *6/*28 genotypes.11 However, testing is not recommended for patients who already experience toxicity, because dose reduction is recommended regardless of the result.5

Quest offers a test for the polymorphic TA repeat (TA5, TA6, TA7, or TA8) in the promoter of UGT1A1 (test code 17813). Patients homozygous for UGT1A1*28 (TA7) are poor metabolizers of UGT1A1, which lead to increased likelihood of irinotecan toxicity. Consequently, the irinotecan product insert suggests a reduced initial dose and close monitoring for these patients.11 Patients heterozygous for UGT1A1*28 are intermediate metabolizers of UGT1A1, which can also lead to increased likelihood of irinotecan toxicity.11 Patients negative for the TA7 repeat may still suffer from dose-limiting toxicity because the assay does not detect other variants in UGT1A1 (eg, homozygous or heterozygous for UGT1A1*6 alleles) that may affect UGT1A1 enzyme activity.

Monitoring CRC

Carcinoembryonic antigen

A carcinoembryonic antigen (CEA) level (test code 978), measured preoperatively (to establish baseline level) and postoperatively, can be used for CRC surveillance.5,6,12 If tumor removal is complete, the CEA level should return to normal; persistently elevated levels suggest residual or metastatic disease. Serial CEA monitoring after surgery is useful for detecting recurrences.6,12 Preoperative CEA level is also a stage-independent marker for poor prognosis, although the cutoff level is not well-established.13

For patients with stage II/III/IV CRC who may be candidates for aggressive treatment, NCCN recommends CEA testing at baseline, every 3 to 6 months for 2 years, and then every 6 months for 3 more years.5,6 Stable or falling CEA levels suggest no disease progression. Elevated levels of serial CEA tests warrant reevaluation for recurrent and metastatic disease.5,6

Circulating tumor DNA (ctDNA)

Circulating tumor DNA (ctDNA; a subset of cell-free DNA [cfDNA]) has emerged as a prognostic marker to identify patients with elevated risk of recurrence and to potentially inform adjuvant treatment decisions. The detection of ctDNA after curative-intent treatment is associated with an increased risk of disease recurrence; conversely, when ctDNA is not detected, the risk of recurrence may be low.14–17 Clinical use of ctDNA in guiding adjuvant chemotherapy for stage II CRC has been demonstrated in a multicenter randomized controlled trial (DYNAMIC).18 The results indicated that postoperative ctDNA testing may be used to identify patients at low risk for recurrence who can forgo adjuvant chemotherapy without compromising recurrence-free survival.18 Although these data are promising, guidelines currently recommend further studies before ctDNA testing is routinely used.2,19

Quest offers Haystack MRD™ (test codes 13682 and 13151), a tumor-informed minimal residual disease (MRD) test, for patients with a previous or current diagnosis of CRC to aid in residual disease detection, treatment response assessment, and recurrence surveillance. Haystack MRD testing begins with tumor whole-exome sequencing to identify patient-specific somatic variants. Using this personalized assay, ctDNA is measured in an initial blood specimen and at subsequent timepoints to detect residual disease. Test results include a qualitative MRD status (detected or not detected) as well as the ctDNA levels if detected. An "MRD detected" result indicates the presence of residual cancer. An "MRD not detected" result reflects lack of ctDNA detection at the time of blood draw, which may indicate the absence but does not exclude the possibility of residual cancer.