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The College of American Pathologists offers these protocols to assist pathologists in providing clinically useful and relevant information when reporting results of surgical specimen examinations. The College regards the reporting elements in the “Surgical Pathology Cancer Case Summary (Checklist)” portion of the protocols as essential elements of the pathology report. However, the manner in which these elements are reported is at the discretion of each specific pathologist, taking into account clinician preferences, institutional policies, and individual practice.
The College developed these protocols as an educational tool to assist pathologists in the useful reporting of relevant information. It did not issue the protocols for use in litigation, reimbursement, or other contexts. Nevertheless, the College recognizes that the protocols might be used by hospitals, attorneys, payers, and others. Indeed, effective January 1, 2004, the Commission on Cancer of the American College of Surgeons mandated the use of the checklist elements of the protocols as part of its Cancer Program Standards for Approved Cancer Programs. Therefore, it becomes even more important for pathologists to familiarize themselves with these documents. At the same time, the College cautions that use of the protocols other than for their intended educational purpose may involve additional considerations that are beyond the scope of this document.
This protocol applies to all primary carcinomas of the colon and rectum. Well-differentiated neuroendocrine neoplasms (carcinoid tumors) are not included. The seventh edition TNM staging system for carcinoma of the colon and rectum of the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) is recommended.
* Data elements with asterisks are not required. However, these elements may be clinically important but are not yet validated or regularly used in patient management.
* Data elements with asterisks are not required. However, these elements may be clinically important but are not yet validated or regularly used in patient management.
* ____ BRAF V600E mutational analysis (specify testing method: _______________)
* ____ KRAS mutational analysis (specify testing method: _______________)
Other, specify: _______________
* ____ Not performed
The protocol applies to all carcinomas arising in the colon and rectum.1 It excludes carcinomas of the vermiform appendix and low-grade neuroendocrine neoplasms (carcinoid tumors).
The colon is divided as shown in Figure 1. The right colon is subdivided into the cecum and the ascending colon.2 The left colon is subdivided into the descending colon and sigmoid colon (see Table).1
The transition from sigmoid to rectum is marked by the fusion of the tenia coli of the sigmoid to form the circumferential longitudinal muscle of the rectal wall approximately 12 to 15 cm from the dentate line. The rectum is defined clinically as the distal large intestine commencing opposite the sacral promontory and ending at the anorectal ring, which corresponds to the proximal border of the puborectalis muscle palpable on digital rectal examination1 (Figure 2). When measuring below with a rigid sigmoidoscope, it extends 16 cm from the anal verge.
Tumors located at the border between 2 subsites of the colon (eg, cecum and ascending colon) are registered as tumors of the subsite that is more involved. If 2 subsites are involved to the same extent, the tumor is classified as an “overlapping” lesion.
A tumor is classified as rectal if its inferior margin lies less than 16 cm from the anal verge or if any part of the tumor is located at least partly within the supply of the superior rectal artery.3 A tumor is classified as rectosigmoid when differentiation between rectum and sigmoid according to the previously mentioned guidelines is not possible.4
For consistency in reporting, the histologic classification proposed by the World Health Organization (WHO) is recommended and follows.5
* By convention, signet-ring cell carcinomas, small cell carcinomas, and undifferentiated carcinomas are high grade (see note C). The only histologic types of colorectal carcinoma that have been shown to have adverse prognostic significance independent of stage are signet-ring cell carcinoma6 and small cell carcinoma (high-grade neuroendocrine carcinoma).7
† Medullary carcinoma is a distinctive histologic type strongly associated with high levels of microsatellite instability (MSI-H), indicative of defects in normal DNA repair gene function. Medullary carcinoma may occur either sporadically8 or in association with hereditary nonpolyposis colon cancer (HNPCC).9 This tumor type is characterized by solid growth in nested, organoid, or trabecular patterns, with no immunohistochemical evidence of neuroendocrine differentiation. Medullary carcinomas are also characterized by numerous tumor infiltrating lymphocytes (see note I).
‡ The term “carcinoma, NOS” (not otherwise specified) is not part of the WHO classification.
A number of grading systems for colorectal cancer have been suggested, but a single widely accepted and uniformly used standard for grading is lacking. Most systems stratify tumors into 3 or 4 grades as follows:
Despite a significant degree of interobserver variability,10 histologic grade has repeatedly been shown by multivariate analysis to be a stage-independent prognostic factor.11 Specifically, it has been demonstrated that high tumor grade is an adverse prognostic factor. It is noteworthy that in most studies documenting the prognostic power of tumor grade, the number of grades has been collapsed to produce a 2-tiered stratification for data analysis as follows:
The widest variations in grading concern the stratification of low-grade tumors into well- or moderately differentiated categories, while interobserver variability in diagnosing high-grade carcinoma is relatively small. Therefore, in light of its proven prognostic value, relative simplicity, and reproducibility, a 2-tiered grading system for colorectal carcinoma (ie, low grade and high grade) is recommended. The following criteria for grading based on gland formation alone are suggested12:
Colorectal adenomas containing invasive adenocarcinoma that extends through the muscularis mucosae into the submucosa have been defined as “malignant polyps.”13 This term encompasses cases in which the entire polyp head is replaced by carcinoma and adenomas with focal malignancy, but the definition excludes adenomas with high-grade dysplasia (intraepithelial carcinoma) or intramucosal carcinoma (invasive carcinoma limited to the lamina propria or invading no deeper than the muscularis mucosae) because these polyps possess negligible biologic potential for metastasis14 (see Tis in note M).
Malignant polyps removed by endoscopic polypectomy require evaluation of histologic factors related to the risk of adverse outcome (ie, lymph node metastasis or local recurrence from residual malignancy) following polypectomy.13,15 Factors shown to have independent prognostic significance and are important in determining the need for further surgical treatment include:
An increased risk of adverse outcome has been shown to be associated with:
Venous invasion has been demonstrated by multivariate analysis to be an independent adverse prognostic factor.11 Invasion of extramural veins, in particular, has been shown to be an independent indicator of unfavorable outcome and increased risk of occurrence of hepatic metastasis.16 The significance of intramural venous invasion is less clear, because data specific to this issue are lacking.
In several studies, both lymphatic invasion17 and perineural invasion18 have been shown by multivariate analysis to be independent indicators of poor prognosis. The prognostic significance, if any, of the anatomic location of these structures is not defined. Furthermore, it is not always possible to distinguish lymphatic vessels from post-capillary venules because both are small, thin-walled structures. Thus, the presence or absence of tumor invasion of small, thin-walled vessels should be reported in all cases.
Distinction should be made between traditional serrated adenomas, which exhibit cytologic features of adenomas, and the newly described sessile serrated adenomas.19 The sessile serrated adenoma may be the precursor lesion for colorectal carcinomas with MSI-H; they are more commonly found in the right colon and are characterized by serrated architecture with bulbous dilatation of deep crypts and lack of overt nuclear atypia, in most cases.
Tumor perforation is an uncommon complication of colorectal cancer, but one that is associated with a poor outcome, including high in-hospital mortality and morbidity.20 Perforation of the uninvolved colon proximal to an obstructing tumor is also associated with high mortality because of generalized peritonitis and sepsis. Reported perforation rates range from 2.6% to 9%. Perforation is more likely to occur in older patients.
The quality of the surgical technique is a key factor in the success of surgical treatment for rectal cancer, both in the prevention of local recurrence and in long-term survival. Numerous studies have demonstrated that total mesorectal excision (TME) improves local recurrence rates and the corresponding survival by as much as 20%. This surgical technique entails precise sharp dissection within the areolar plane outside (lateral to) the visceral mesorectal fascia to remove the rectum. This plane encases the rectum, its mesentery, and all regional nodes and constitutes Waldeyer fascia. High-quality TME surgery reduces local recurrence from 20% to 30%, to 8% to 10% or less, and increases 5-year survival from 48% to 68%.21,22 Adjuvant therapy in the presence of a high-quality TME may further reduce local recurrence (from 8% to 2.6%).22
Pathologic evaluation of the resection specimen has been shown to be a sensitive means of assessing the quality of rectal surgery. It is superior to indirect measures of surgical quality assessment, such as perioperative mortality, rates of complication, number of local recurrences, and 5-year survival. It has been shown that macroscopic pathologic assessment of the completeness of the mesorectum of the specimen, scored as complete, partially complete, or incomplete, accurately predicts both local recurrence and distant metastasis.22 Microscopic parameters, such as the status of the circumferential resection margin, the distance between the tumor and nearest circumferential margin (ie, “surgical clearance”), and the distance between the tumor and the closest distal margin, are all important predictors of local recurrence and may be affected by surgical technique. There is strong evidence that the status of the circumferential resection margin is a powerful predictor of local recurrence but is inconsistently evaluated and underreported.
The nonperitonealized surface of the fresh specimen is examined circumferentially, and the completeness of the mesorectum is scored as described in the following.22 The entire specimen is scored according to the worst area.
Identification of MSI-H colorectal tumors is important, as mismatch repair deficiency may serve as a prognostic marker of patient outcome, a predictive marker of response to chemotherapy, and as a screening tool for HNPCC (Lynch Syndrome). Revised Bethesda guidelines for HNPCC detection recommend testing colorectal tumors for MSI under the following circumstances23:
† MSI-H histologic features are defined as presence of tumor-infiltrating lymphocytes, Crohn-like lymphocytic reaction, mucinous/signet-ring cell differentiation, or medullary growth pattern.23
Tumor-infiltrating lymphocytes are closely associated with MSI and medullary architecture (see previous) and should be distinguished from Crohn-like peritumoral infiltrates (lymphoid aggregated or follicles are the tumor edge, not associated with preexisting lymph node).24 Although absolute cutoff values have not been established, only moderate- and high-density intratumoral lymphocytes (approximately 3 or more per high-power field using hematoxylin-eosin–stained sections) should be considered significant.25
Other pathologic features associated with MSI-H status in colorectal carcinomas include right-sided location, high-grade histology, and lack of dirty necrosis.25
It may be helpful to mark the margin(s) closest to the tumor with ink following close examination of the serosal surface for puckering and other signs of tumor involvement. Margins marked by ink should be designated in the macroscopic description of the surgical pathology report. The serosal surface (visceral peritoneum) does not constitute a surgical margin.
In addition to addressing the proximal and distal margins, the circumferential (radial) margin (Figure 3, A through C) must be assessed for any segment either unencased (Figure 3, C) or incompletely encased by peritoneum (Figure 3, B) (see note A). The circumferential (radial) margin represents the adventitial soft tissue margin closest to the deepest penetration of tumor and is created surgically by blunt or sharp dissection of the retroperitoneal or subperitoneal aspect, respectively. Multivariate analysis has suggested that tumor involvement of the circumferential (radial) margin is the most critical factor in predicting local recurrence in rectal cancer.26 A positive circumferential (radial) margin in rectal cancer increases the risk of recurrence by 3.5-fold and doubles the risk of death from disease. For this reason, the circumferential (radial) margin should be assessed in all rectal carcinomas as well as colonic segments with nonperitonealized surfaces. The distance between the tumor and circumferential (radial) margin should be reported (see note H). The circumferential (radial) margin is considered negative if the tumor is more than 1 mm from the inked nonperitonealized surface but should be recorded as positive if tumor is located 1 mm or less from the nonperitonealized surface because local recurrence rates are similar with clearances of 0 to 1 mm. This assessment includes tumor within a lymph node as well as direct tumor extension, but if circumferential margin positivity is based solely on intranodal tumor, this should be so stated.
The mesenteric resection margin is the only relevant circumferential margin in segments completely encased by peritoneum (eg, transverse colon). Involvement of this margin should be reported even if tumor does not penetrate the serosal surface.
Sections to evaluate the proximal and distal resection margins can be obtained either by longitudinal sections perpendicular to the margin or by en face sections parallel to the margin. The distance from the tumor edge to the closest resection margin(s) may also be important, particularly for low anterior resections. For these cases, a distal resection margin of 2 cm is considered adequate; for T1 and T2 tumors, 1 cm may be sufficient distal clearance. Anastomotic recurrences are rare when the distance to the closest transverse margin is 5 cm or greater.
In cases of carcinoma arising in a background of inflammatory bowel disease, proximal and distal resection margins should be evaluated for dysplasia and active inflammation.
Neoadjuvant chemoradiation therapy in rectal cancer is associated with significant tumor response and downstaging.27 Because eradication of the tumor, as detected by pathologic examination of the resected specimen, is associated with a significantly better prognosis,28 specimens from patients receiving neoadjuvant chemoradiation should be thoroughly sectioned, with careful examination of the tumor site. Minimal residual disease has been shown to have a better prognosis than gross residual disease.28 Although several grading systems for tumor response have been advocated, a 3-point tumor regression grade has been shown to provide good inter-observer reproducibility compared with 5-grade schemas, and to provide similar prognostic significance.29
Tumor regression should be assessed only in the primary tumor; lymph node metastases should not be included in the assessment.
Acellular pools of mucin in specimens from patient receiving neoadjuvant therapy are considered to represent completely eradicated tumor and are not used to assign pT stage or counted as positive lymph nodes.
Irregular discrete tumor deposits in pericolic or perirectal fat away from the leading edge of the tumor and showing no evidence of residual lymph node tissue, but within the lymphatic drainage of the primary carcinoma, are considered peritumoral deposits or satellite nodules1 and are not counted as lymph nodes replaced by tumor. Most examples are due to lymphovascular or, more rarely, perineural invasion. Because these tumor deposits are associated with reduced disease-free and overall survival,30,31 their number should be recorded in the surgical pathology report. If tumor deposits are observed in lesions that would otherwise be classified as pT1 (tumor confined to submucosa) or pT2 (tumor confined to muscularis propria), then the primary tumor classification is not changed, but the nodule is recorded in a separate N category as N1c1 (see note M).
Surgical resection remains the most effective therapy for colorectal carcinoma, and the best estimation of prognosis is derived from the pathologic findings on the resection specimen. The anatomic extent of disease is by far the most important prognostic factor in colorectal cancer.
The protocol recommends the TNM staging system of the AJCC and the UICC1 but does not preclude the use of other staging systems.
By AJCC/UICC convention, the designation “T” refers to a primary tumor that has not been previously treated. The symbol “p” refers to the pathologic classification of the TNM, as opposed to the clinical classification, and is based on gross and microscopic examination. pT entails a resection of the primary tumor or biopsy adequate to evaluate the highest pT category, pN entails removal or biopsy of nodes adequate to validate lymph node metastasis, and pM implies microscopic examination of distant lesions. Clinical classification (cTNM) is usually carried out by the referring physician before treatment during initial evaluation of the patient or when pathologic classification is not possible.
For identification of special cases of TNM or pTNM classifications, the “m” suffix and “y” and “r” prefixes are used. Although they do not affect the stage grouping, they indicate cases needing separate analysis.
The “m” suffix indicates the presence of multiple primary tumors in a single site and is recorded in parentheses: pT(m)NM.
The “y” prefix indicates those cases in which classification is performed during or following initial multimodality therapy (ie, neoadjuvant chemotherapy, radiation therapy, or both chemotherapy and radiation therapy). The cTNM or pTNM category is identified by a “y” prefix. The ycTNM or ypTNM categorizes the extent of tumor actually present at the time of that examination. The “y” categorization is not an estimate of tumor prior to multimodality therapy (ie, before initiation of neoadjuvant therapy).
The “r” prefix indicates a recurrent tumor when staged after a documented disease-free interval and is identified by the “r” prefix: rTNM.
For colorectal carcinomas, “carcinoma in situ” (pTis) as a staging term includes cancer cells confined within the glandular basement membrane (intraepithelial carcinoma, synonymous with high-grade dysplasia) or invasive into the mucosal lamina propria, up to but not through the muscularis mucosae (intramucosal carcinoma). Tumor extension through the muscularis mucosae into the submucosa is classified as T1.
Direct invasion of other organs or structures includes invasion of other segments of colorectum by way of the serosa or mesocolon (eg, invasion of the sigmoid colon by carcinoma of the cecum) is classified as pT4 (Figure 6). In such a case, both an adjacent organ and the visceral peritoneum are penetrated by tumor. Intramural extension of tumor from 1 subsite (segment) of the large intestine into an adjacent subsite or into the ileum (eg, for a cecal carcinoma) or anal canal (eg, for a rectal carcinoma) does not affect the pT classification.
Tumor that is adherent to other organs or structures macroscopically is classified as T4. However, if no tumor is found within the adhesion microscopically, the tumor should be assigned T3.1
For rectal tumors, invasion of the external sphincter is classified as T3, whereas invasion of the levator ani muscle(s) is classified as T4.
Tumor in veins or lymphatics does not affect the pT classification.
Serosal involvement by tumor cells (pT4a) has been demonstrated by multivariate analysis to have a negative impact on prognosis,32 as does direct invasion of adjacent organs (pT4b). Visceral peritoneal involvement can be missed without thorough sampling and/or sectioning, and malignant cells have been identified in serosal scrapings in as many as 26% of specimens categorized as pT3 by histologic examination alone.32 Although the absence of standard guidelines for assessing peritoneal involvement may contribute to underdiagnosis, the following findings are considered to represent serosal involvement by tumor:
Both types of peritoneal involvement are associated with decreased survival.
Although small studies suggested that serosal involvement was associated with worse outcome than invasion of adjacent organs, data from a large cohort of more than 100 000 colon cancer cases33 indicate that penetration of the visceral peritoneum carries a 10% to 20% better 5-year survival than locally invasive carcinomas for each category of N. Therefore, designation of the T4 subsets was changed in the seventh edition of the AJCC Cancer Staging Manual to reflect these new findings.
The regional lymph nodes for the anatomical subsites of the large intestine (Figure 7) are as follows:
Nodes along the sigmoid arteries are considered pericolic nodes, and their involvement is classified as N1 or N2 according to the number involved.
Perirectal lymph nodes include the mesorectal (paraproctal), lateral sacral, presacral, sacral promontory (Gerota), middle rectal (hemorrhoidal), and inferior rectal (hemorrhoidal) nodes. Metastasis in the external iliac or common iliac nodes is classified as distant metastasis.1
All grossly negative or equivocal lymph nodes are to be submitted entirely.12 Grossly positive lymph nodes may be partially submitted for microscopic confirmation of metastasis.
The accuracy and predictive value of stage II assignment are directly proportional to the thoroughness of the surgical technique in removing all regional nodes and the pathologic examination of the resection specimen in identifying and harvesting all regional lymph nodes for microscopic assessment. It has been suggested that 12 lymph nodes be considered the minimal acceptable harvest from a careful specimen dissection.12 In 2007, the National Quality Forum listed the presence of at least 12 lymph nodes in a surgical resection among the key quality measures for colon cancer care in the United States (see http://www.facs.org/cancer/qualitymeasures.html. Accessed June 2, 2009).
Increasingly, however, evidence indicates that this bar should be raised, as the greater the number of nodes examined, the greater the likelihood that metastasis will be found, suggesting that no minimum number of nodes accurately or reliably stages all patients.34,35
More importantly, it has been shown that clinical outcome is linked to lymph node harvest in stage II disease. Numerous studies have shown that conventional pathologic examination of increased numbers of lymph nodes is itself associated with an increased survival advantage in stage II disease,36 indicating a positive effect of optimal mesenteric resection by the surgeon, optimal lymph node harvest from the resection specimen by the pathologist, or both.
The number of lymph nodes recovered from resection specimen is dependent on several factors. Surgical technique, surgery volume, and patient factors (eg, age and anatomic variation) alter the actual number of nodes in a resection specimen, but the diligence and skill of the pathologist in identifying and harvesting lymph nodes in the resection specimen also are major factors. Lymph nodes may be more difficult to identify in specimens from patients who are obese37 or elderly, or after neoadjuvant therapy.38 Because it has been shown that nodal metastasis in colorectal cancer is often found in small lymph nodes (<5 mm in diameter), diligent search for lymph nodes is required on gross examination of resection specimens. If fewer than 12 lymph nodes are found, reexamining the specimen for additional lymph nodes, with or without visual enhancement techniques, should be considered.12 The pathology report should clearly state the total number of lymph nodes examined and the total number involved by metastases. Data are insufficient to recommend routine use of tissue levels or special/ancillary techniques.
For microscopic examination of lymph nodes in large resection specimens, lymph nodes must be designated as regional versus nonregional, according to the anatomic location of the tumor. Metastasis to nonregional lymph nodes is classified as distant metastasis and designated as M1.
A tumor nodule in the pericolonic/perirectal fat without histologic evidence of residual lymph node tissue is classified as a tumor deposit (peritumoral deposit or satellite nodule) and is not considered a positive lymph node. Such tumor deposits may represent discontinuous spread, lymph-vascular spread with extravascular extension, or totally replaced lymph nodes. In the absence of unequivocal lymph node metastases, tumor deposits are recorded as N1c.1
A micrometastasis is defined as tumor measuring greater than 0.2 mm but less than or equal to 2.0 mm in greatest dimension. Micrometastases are classified as N1(mic) or M1(mic) in lymph nodes or at distant sites, respectively. Isolated tumor cells (ITCs) are defined as single tumor cells or small clusters of tumor cells measuring 0.2 mm or less, usually found by special techniques such as immunohistochemical staining, and are classified as N0.4 Because the biologic significance of ITCs (either a single focus in a single node, multiple foci within a single node, or micrometastatic involvement of multiple nodes) remains unproven, N0 is considered justified. The number of lymph nodes involved by micrometastases or ITCs should be clearly stated.
Routine assessment of regional lymph nodes is limited to conventional pathologic techniques (gross assessment and histologic examination), and data are currently insufficient to recommend special measures to detect micrometastasis or ITCs. Thus, neither multiple levels of paraffin blocks nor the use of special/ancillary techniques such as immunohistochemistry are recommended for routine examination of regional lymph nodes. Specific denotation of the assigned N category is suggested as follows for cases in which ITCs are the only evidence of possible metastatic disease.1
Pathologic staging is usually performed after surgical resection of the primary tumor. Pathologic staging depends on pathologic documentation of the anatomic extent of disease, whether or not the primary tumor has been completely removed. If a biopsied tumor is not resected for any reason (eg, when technically unfeasible), and if the highest T and N categories or the M1 category of the tumor can be confirmed microscopically, the criteria for pathologic classification and staging have been satisfied without total removal of the primary cancer.
|Stage IVA||Any T||Any N||M1a|
|Stage IVB||Any T||Any N||M1b|
Detection of defects in mismatch repair in colorectal carcinomas is important for detection of Lynch syndrome (a subset of HNPCC accounting for approximately 2% of all colorectal carcinomas), and examination of the tissue for defective DNA mismatch repair is recommended if any of the criteria in the revised Bethesda guidelines23 (note I) are met. In addition, emerging data suggest that MSI-H in sporadic colon cancers is associated with better outcome and may serve as a predictor of response to 5-FU–based chemotherapy,39 although these latter indications for testing are not clearly established and have not been accepted as standard of care.
Most tumors from patients with HNPCC exhibit MSI-H due to defective DNA mismatch repair. Patients whose colorectal tumors do not exhibit an MSI-H phenotype are very unlikely to have HNPCC. MSI testing has high sensitivity but not necessarily high specificity for HNPCC, because an MSI-H phenotype can be observed in approximately 15% of sporadic colorectal cancer. The specificity of MSI testing can be increased by using it primarily on at-risk populations, such as colorectal cancer patients younger than 50 years or patients with a strong family history of HNPCC-associated tumors (eg, colorectal, endometrial, gastric, or upper urinary tract urothelial carcinoma).23
MSI testing can be used to cost-effectively screen at-risk colorectal cancer patients for possible HNPCC. Patients with an MSI-H phenotype may have a germline mutation in one of several DNA mismatch repair (MMR) genes (eg, MLH1, MSH2, MSH6, or PMS2) and after appropriate genetic counseling may want to consider having such testing. Follow-up germline testing for HNPCC may help in making a definitive diagnosis of the disorder and aid in the presymptomatic detection of carriers in at-risk individuals. Presymptomatic detection of carriers could lead to increased surveillance and potentially reduce morbidity and mortality.
MSI testing is generally performed with at least 5 microsatellite markers, generally mononucleotide or dinucleotide repeat markers. In 1998, a National Institutes of Health consensus panel proposed that laboratories use a 5-marker panel consisting of 3 dinucleotide and 3 mononucleotide repeats for MSI testing.40 Recent data suggest that dinucleotide repeats may have lower sensitivity and specificity for identifying tumors with an MSI-H phenotype. As a consequence, there has been a move toward including more mononucleotides and fewer dinucleotides in MSI testing panels. Many laboratories now use a commercially available kit for MSI testing that uses 5 mono-nucleotide markers.
The detection of MSI in a tumor by microsatellite analysis requires that the DNA used for the analysis be extracted from a portion of the tumor that contains approximately 40% or more tumor cells. Thus, pathologists should help identify areas of the tumor for DNA isolation that have at least this minimum content of tumors cells. MSI testing is frequently done in conjunction with immunohistochemical (IHC) testing for DNA MMR protein expression (ie, MLH1, MSH2, MSH6, PMS expression). If the results of DNA MMR IHC and MSI testing are discordant (eg, MSI-H phenotype with normal IHC or abnormal IHC with MSS phenotype), then the laboratory should make sure that the same sample was used for MSI and IHC testing and that there was no sample mix-up. External proficiency testing surveys are available through the College of American Pathologists Molecular Oncology resource committee and other organizations. These surveys are invaluable tools to ensure that the laboratory assays are working as expected.
Ideally, the results of DNA MMR IHC and MSI testing should be incorporated into the surgical pathology report for the colorectal cancer case and an interpretation of the clinical significance of these findings provided. If DNA MMR IHC has not been performed, this testing should be recommended for any cases that show an MSI-H phenotype because this information will help identify the gene that is most likely to have a germ-line mutation (eg, a patient whose tumor shows loss of MSH2 and MSH6 expression, but retention of MLH1 and PMS2 expression, is likely to have an MSH2 germline mutation).
Examination of expression of MLH1, MSH2, MSH6, and PMS2 is the most common IHC testing method used for suspected MSI-H cases; antibodies to these MMR proteins are commercially available. Any positive reaction in the nuclei of tumor cells is considered as intact expression (normal), and it is common for intact staining to be somewhat patchy. An interpretation of expression loss should be made only if positive reaction is seen in internal control cells, such as the nuclei of stromal, inflammatory, or non-neoplastic epithelial cells. Intact expression of all 4 proteins indicates that MMR enzymes tested are intact but does not entirely exclude Lynch syndrome, as approximately 5% of families may have a missense mutation (especially in MLH1) that can lead to a nonfunctional protein with retained antigenicity. Defects in lesser known MMR enzymes may also lead to a similar result, but this situation is rare. Loss of expression of MLH1 may be due to Lynch syndrome or methylation of the promoter region (as occurs in sporadic MSI colorectal carcinoma). Genetic testing is ultimately required for this distinction, although a specific BRAF mutation is present in many sporadic cases but not familial cancers. Loss of MSH2 expression essentially always implies Lynch syndrome. PMS2 loss is often associated with loss of MLH1 and is only independently meaningful if MLH1 is intact. MSH6 is similarly related to MSH2.
Analysis for somatic mutations in the V600E hot spot in BRAF may be indicated for tumors that show MSI-H, as this mutation has been found in sporadic MSI-H tumors but not in HNPCC-associated cancers.41 Use of BRAF mutational analysis as a step before germline genetic testing in patients with MSI-H tumors may be a cost-effective means of identifying patients with sporadic tumors for whom further testing is not indicated.42
The presence of the K-ras gene (KRAS) mutation has been shown to be associated with lack of clinical response to therapies targeted at the epidermal growth factor receptor (EGFR), such as cetuximab43 and panitumumab.44 While clinical guidelines for KRAS mutational analysis are evolving, current provisional recommendations from the American Society for Clinical Oncology are that all patients with stage IV colorectal carcinoma who are candidates for anti-EGFR antibody therapy should have their tumor tested for KRAS mutations (http://www.asco.org/. Accessed June 2, 2009). Anti-EGFR antibody therapy is not recommended for patients whose tumors show mutation in KRAS codon 12 or 13.
The authors have no relevant financial interest in the products or companies described in this article.