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REVIEW ARTICLE
Year : 2020  |  Volume : 57  |  Issue : 3  |  Page : 239-252
 

Grossing and reporting of colorectal cancer resection specimens: An evidence-based approach


1 Department of Pathology, Columbia Asia Hospital Whitefield, Bangalore, Karnataka, India
2 Department of Pathology, Amrita Institute of Medical Sciences and Research Centre, Kochi, Kerala, India
3 Department of Medical Oncology, Columbia Asia Hospital Whitefield, Bangalore, Karnataka, India
4 Department of Radiology, Columbia Asia Hospital Whitefield, Bangalore, Karnataka, India

Date of Submission25-May-2020
Date of Decision20-Jun-2020
Date of Acceptance12-Jul-2020
Date of Web Publication10-Aug-2020

Correspondence Address:
Sanjeev Vasudev Katti
Department of Pathology, Columbia Asia Hospital Whitefield, Bangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijc.IJC_546_20

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 » Abstract 


Appropriate management of the patient with colorectal carcinoma depends on obtaining key prognostic and predictive information from the resection specimen. These include the quality of surgery, extent of lymph nodal clearance, presence of nodal disease, vascular invasion, residual disease post neoadjuvant treatment, and completeness of resection. A meticulous and structured approach to dissection of the resection specimen and subsequent histological examination by the pathologist is crucial in providing this information to the treating clinician. A good macroscopic examination also serves to audit the quality of other services including radiology, surgery, and oncology. This article attempts to review dissection and reporting guidelines with an evidence-based approach and hopes to guide pathologists to understand the basis behind the recommended protocols.


Keywords: Colorectal cancer, grossing, microscopy, total mesorectal excision


How to cite this article:
Katti SV, Paulose RR, Malipatil B, Verma NS. Grossing and reporting of colorectal cancer resection specimens: An evidence-based approach. Indian J Cancer 2020;57:239-52

How to cite this URL:
Katti SV, Paulose RR, Malipatil B, Verma NS. Grossing and reporting of colorectal cancer resection specimens: An evidence-based approach. Indian J Cancer [serial online] 2020 [cited 2020 Oct 1];57:239-52. Available from: http://www.indianjcancer.com/text.asp?2020/57/3/239/291780





 » Introduction Top


Colorectal cancer is the third most common cancer in men and women in developed countries and accounts for nearly 8% of all cancer-related deaths. The global incidence of colorectal cancer varies widely from 1.1 to 51.2 per 100,000 population.[1] In India, the annual incidence rates for colon cancer and rectal cancer are 4.4 and 4.1 per 100000, respectively. Colon cancer ranks 8th and rectal cancer ranks 9th among men. For women, rectal cancer does not figure in the top 10 cancers, whereas colon cancer ranks 9th.[2]

With the rapid advancement in therapeutic procedures, accurate pathology reporting of colorectal cancer resection specimens ensures appropriate patient management by providing confirmation of diagnosis and important prognostic information. Standardized reporting also helps in collection of relevant data for audit, research, and education, and in assessment of the quality of other services including radiology, surgery, and oncology.

The mainstay of treatment for colorectal carcinoma is surgery. Rectal magnetic resonance imaging (MRI) has evolved as the gold standard in the preoperative local staging of rectal carcinomas. Rectal MRI also predicts the presence of extramural vascular invasion. Neo adjuvant and adjuvant treatment is indicated in all stage II and stage III rectal cancers. In cancers of the colon, adjuvant chemotherapy is considered in patients with certain high-risk factors.[3] These factors are largely identified by meticulous gross and pathological examination of the resection specimen. The management flowcharts for colonic and rectal carcinomas are outlined in [Figure 1] and [Figure 2].
Figure 1: Management flowchart for colonic carcinoma. *High-risk features – poor differentiation, T4 disease, vascular or lymphatic invasion, inadequate nodal sampling (<12 nodes sampled). #Adjuvant chemotherapy includes capecitabine or 5-fluorouracil/leucovorin or 5-fluorouracil/leucovorin/oxaliplatin or capecitabine/oxaliplatin. ##In Stage II microsatellite instability-high cancers, chemotherapy is of doubtful benefit. Decision for chemotherapy is based on Multidisciplinary team (MDT) input

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Figure 2: Management flowchart for rectal carcinoma. *Neoadjuvant therapy – standard neoadjuvant therapy includes a combination of chemotherapy and radiation. Alternative approaches including short-course radiotherapy alone are considered in specific situations in a MDT setting. #Adjuvant chemotherapy includes capecitabine or 5-fluorouracil/leucovorin or 5-fluorouracil/leucovorin/oxaliplatin or capecitabine/oxaliplatin

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Evidence-based guidelines are already provided by the Royal College of Pathologists [4] and the College of American Pathologists [5] on the approach to, and reporting of, colorectal resections for cancer. The current challenges in reporting colorectal cancer are also discussed comprehensively in an excellent recent review.[6] The purpose of this article is to reinforce the standardized approach to the dissection of these specimens to obtain optimal information and to attempt to explain the how and why of the grossing techniques. A brief outline of histological assessment that is relevant to subsequent management of these cases is also provided.

Clinical information on the request form

It is the pathologist's responsibility to ensure that request forms are completed optimally as the information provided can decide the approach to dissection. It is good practice to mention the type of surgical procedure and the site of tumor as this provides confirmation to the pathologist that they are dealing with the right specimen. Knowledge of preoperative histology, history of familial cancer, or inflammatory bowel disease also may decide the extent of sampling, and these details should be included on the form. Details of preoperative radiological staging help in auditing radiology reporting. Nature of neoadjuvant therapy, if any should also be mentioned, as apart from assessment of treatment efficacy, this also assists the pathologist to select adequate blocks in case of significant tumor regression.

All clinical information provided on the request form should be transcribed on to the final histopathology report verbatim. This is useful not only for legal reasons but also because it serves as a documentation of the information that is received by the laboratory. It also gives important feedback to the clinicians that the time they took to write a clinical history was noted by the pathologist.[7]

Specimen handling

Specimens are ideally received as quickly as possible, fresh, and unopened. Fresh specimens allow for photography and optimal macroscopic assessment of the resection, crucial in rectal cancers, which otherwise may be affected by distortion resulting from formalin fixation. Tissue banking for research is also possible when the specimen is received fresh. However, it should be ensured that the diagnostic process is not affected before samples are retained for this purpose. It is important that a pathologist trained in proper dissection technique opens the specimen so as not to compromise the reporting of important prognostic parameters such as pathological stage and marginal status.

Although evidence is lacking and the recommendations are mainly anecdotal, molecular changes are said to set in after 1 hour of cold ischemia time.[8] If prompt transport to the laboratory is not possible, the specimen should be placed unopened in an adequate quantity of formalin fixative. Refrigeration should also be considered if a longer delay of a day or more is anticipated.[9]

Before the specimen is opened, a thorough inspection of the external features should be carried out. This includes an examination for possible serosal involvement and distant tumor deposits [Figure 3]. Macroscopic tumor perforation is also best assessed in an unopened specimen. The plane of surgical dissection for anterior resections and abdominoperineal resections is evaluated at this stage with photographic documentation of the findings (See section on macroscopic assessment).
Figure 3: Serosal deposit (black arrow) in a pT4a cecal carcinoma (blue arrow)

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A very critical step at this juncture is the inking of the non-peritonealized margin (NPM) or circumferential resection margin (CRM). These terms are used interchangeably to denote the bare area created by the plane of surgical excision which is not covered by peritoneum. Although described and studied quite extensively in the rectum, it is important to recognize that colonic resection specimens from all other sites also have an NPM; only its extent varies based on the tumor site. Surgical specimens of the lower rectum are completely surrounded by a CRM. In fact, this is the only site where the margin can be strictly called circumferential. Tumors in the upper rectum have a CRM posteriorly and laterally, the ascending and descending colons have a broad and jagged CRM posteriorly, and the transverse and proximal sigmoid colon only have a narrow CRM on the posterior aspect. The cecum is variable in its peritoneal covering, so some cases may have a large non-peritonealized area while others may have none. Care should be taken not to ink the serosa as this may result in misinterpretation of margin involvement. The ink can also obscure scanty tumor cells at the serosal surface during the histological examination.[10] India ink or a tissue marking dye of any color can be used for inking. Dabbing the CRM with 90% isopropyl alcohol before inking and then spraying the inked surface with 5% acetic acid, aid in fixing the ink to the surface. A handheld hairdryer can be used to dry the inked surface, and this can be very useful to prevent the ink from running onto serosal surfaces.

The specimen is then opened anteriorly with scissors from the proximal and distal ends to the level of the tumor, which is left unopened. For total mesorectal excisions, the entire length of the CRM is left unopened. A wick of paper or gauze soaked in formalin is then inserted into the unopened segment to facilitate fixation.

The specimen thus opened and cleaned is then allowed to fix in an adequate volume of formalin for at least 24 hours. An ideal period of fixation is 48 hours. Adequate fixation is shown to optimize lymph node yield and also permits thin transverse slices through the tumor.[9]

Macroscopic assessment

The adequately fixed specimen is then measured for its length. The overall length of the specimen has no prognostic significance, although the surgeons aim to achieve a 5 cm length of bowel in the sigmoid and a 1 cm length in the rectum between the neoplasm and the distal end of the specimen.[11] Shorter lengths at these sites are associated with a greater rate of recurrence. For low anterior resections and abdominoperineal resections, the length of the bowel below the anterior peritoneal reflection is measured.

The presence or absence of macroscopic tumor perforation is noted. As mentioned earlier, this is best assessed in the fresh state. Macroscopic tumor perforation is defined as a grossly identifiable defect through the tumor resulting in a communication of the bowel lumen with the external surface [Figure 4]. This is an adverse prognostic factor for colorectal cancers [12],[13] and is staged pT4a in the TNM8 classification, American Joint Committee on Cancer (AJCC) 8th edition [14] [Table 1] and [Table 2]. Macroscopic tumor perforation in the low rectum onto the CRM is also classified as pT4a. Not uncommonly, in obstructing tumors, perforation can be seen in the intestine proximal to the tumor as a result of a pressure effect. This does not constitute tumor perforation or pT4 disease.[4]
Figure 4: Macroscopic tumor perforation. The tumor is staged as pT4a in the TNM8 classification

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Table 1: Pathological tumor staging of colorectal carcinoma (pTNM, AJCC 8th edition)[14]

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Table 2: Pathological nodal staging of colorectal carcinoma (pTNM, AJCC 8th edition)[14]

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The site of the tumor, maximum tumor diameter, and the distance of the tumor from the proximal and distal resection margins are noted.

Tumor dimensions are difficult to measure accurately when the segment containing the tumor is left unopened and later when the tumor is sliced transversely. However, these dimensions have no independent prognostic value,[15],[16] and minor inaccuracies in measurements are a small price to pay for the important prognostic information obtained through this dissection technique.

For rectal tumors, the site of the tumor in relation to the level of the anterior peritoneal reflection should be recorded and classified as above, at or below the level of anterior peritoneal reflection. This is important because the rates of local recurrence for tumors situated below the level of the peritoneal reflection are significantly higher.[17]

In rectal cancers, the next step is to assess the quality of the surgery by evaluating the plane of mesorectal excision. Total mesorectal excision is the accepted standard of care for rectal cancers. Macroscopic assessment of the plane of resection of rectal cancers [Figure 5], [Figure 6], [Figure 7] by a trained pathologist and feedback about the quality of surgery has been shown to have a major impact on improving outcomes for the patient by reducing both likelihood of margin involvement and also local recurrence.[18],[19] Outcomes are best for surgeries done in the plane of the mesorectal fascia and worst for those in the plane of the muscularis propria. Descriptions of the three planes of excision are outlined in [Table 3].
Figure 5: Posterior mesorectum displaying good bulk and a smooth shiny surface with no defects

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Figure 6: Posterior mesorectum with a defect measuring more than 5 mm (black arrow). Muscularis is not exposed. Moderate amount of coning is seen distally (yellow arrows)

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Figure 7: Moderate degree of coning (yellow arrows) - (a) posterior view and (b) anterior view

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Table 3: Planes of mesorectal excision in anterior resection specimens[17]

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For abdominoperineal resections/excisions of the rectum (APER), in addition to the plane of mesorectal resection, the plane of excision of the levators/sphincters is also assessed [20] [Table 4]. Extralevator and sphincteric excisions are different surgical techniques and the coning seen at the level of the puborectalis muscle (surgical waist) in the latter, should not be interpreted as poor quality surgery. An intrasphincteric plane of excision however can lead to poorer outcomes.[21]
Table 4: Planes of sphincteric excision in abdominoperineal resections[17]

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The distance of the tumor from the dentate line in APER specimens is measured and this serves as a rough tool to audit the APER surgery rates.[4]

After optimal external macroscopic assessment, the entire tumor is sliced transversely at 3 to 4 mm intervals and the tumor slices are laid out sequentially and photographed [Figure 8]. A printout of the photograph may be used to mark the sites from where blocks are taken for histology.
Figure 8: Transverse slices from specimen in Figure 6 demonstrating mesorectal defect (red arc) measuring greater than 5 mm

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Block selection

The main aim of the pathological examination of colorectal cancer resection specimens, apart from confirmation of diagnosis, is to provide information on factors that decide the need for adjuvant therapy.

Adjuvant therapy will be considered in all cases with node-positive disease and for those node-negative tumors that have other adverse prognostic features including poor differentiation, vascular or perineural invasion, and positive marginal status.

Blocks for histological examination are selected to optimally assess these features. The standard blocks should include—

  1. The proximal and distal longitudinal margins, especially if these are close to the tumor, or the tumor is diffusely infiltrating on histology. The involvement of the longitudinal margins by invasive carcinoma is extremely rare and sampling is optional if the tumor is greater than 30 mm from the cut ends.[22] A similar approach can also be followed for proximal and distal donuts.
  2. A block from the tumor that demonstrates its deepest penetration through the wall.
  3. At least two blocks from the tumor where there is suspicion of serosal involvement. This can be usually identified adjacent to peritoneal reflections and in clefts formed by fat adjacent to the bowel wall. It can also be suspected in areas where the serosa appears dull, granular, or hemorrhagic.
  4. A block to demonstrate the closest approximation of the tumor to the CRM. This may either be the main tumor or an involved lymph node close to the CRM. This block can be omitted if the tumor is well clear of the inked margin and only the macroscopic distance is recorded in such cases.
  5. One or two blocks to demonstrate vascular invasion. Extramural vascular invasion can be identified on careful gross examination as pale white streaks radiating perpendicularly from the base of the tumor [Figure 9].
  6. Blocks to demonstrate invasion of adjacent organs if any.
  7. Rectal tumors treated with neoadjuvant therapy can demonstrate varying degrees of regression. If no tumor is visible grossly, the guidelines from the Royal College of Pathologists [4] recommend that five blocks from the original tumor bed should be examined in the first instance. If these show no microscopic evidence of tumor, then the entire tumor bed is sampled and examined at three levels. A complete pathological response is confirmed only if no tumor is identified after all these steps.
  8. In locally advanced rectal tumors, resection can be done sometimes en bloc with part of the sacrum. In such cases after inking the CRM, the bone is shaved off the main specimen. The exposed soft tissue at the site of the bone is inked a different color and sampled for histological examination. The bone is processed only if the soft tissue at the site of bony attachment shows a tumor.
  9. Sampling should be done of any background abnormalities including polyps and inflammatory bowel disease.
Figure 9: (a) Transverse slice demonstrating extramural venous invasion (EMVI) as a linear streak radiating from tumor base (arrowheads). The picture also demonstrates tumor extending close to a peritoneal crevice. (b) Rectal MRI T2W axial image demonstrating rectal wall thickening ( block white arrow) and serpiginous projections of hypointense signal intensity which follow the course of a perirectal vein (red arrow) suggestive of EMVI

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Identification and sampling of lymph nodes

There is a positive correlation between survival and the number of lymph nodes retrieved in resection specimens from patients with stage-II and stage-III cancer. The number of lymph nodes identified in colorectal cancer resection specimens are not only dissector dependent, but also depend on various clinicopathological factors [23],[24],[25] including the age of the patient, tumor site, and length of the resected segment.

It has been shown that the chance of finding a positive lymph node increases with the number of nodes found. The probability of detecting a positive lymph node, however, does not change after 12–15 nodes. It is recommended for audit purposes that an average of 12 lymph nodes be found.[9] However, it is important to identify and sample all regional lymph nodes that can be found in a specimen and not stop at 12. It is not required to examine all non-regional lymph nodes in case of extended or total colectomy specimens and a more pragmatic approach should be adopted in such cases.

Dissection should start at the high tie to first identify the apical node. The mesocolic/mesorectal fat is then sliced serially at 3 mm intervals to retrieve lymph nodes. In cases with neoadjuvant therapy, it may be difficult to identify lymph nodes.[24] Careful palpation of the fat with sampling of any firm focus increases yield. Additional techniques such as fat clearance protocols [26] or intra-arterial injection of methylene blue [27] may also be useful when the identification of lymph nodes is difficult. Fat clearance can be achieved by immersing the formalin-fixed mesocolic adipose tissue in 100% isopropyl alcohol for 24 hours. A modified Lillie alcohol-acetic acid-formalin fixative (GEWF solution) is also shown to be useful in fat clearance and increasing lymph node yield.[28]

Any lymph nodes that are close to the CRM should be sampled in continuity with that margin. If any of these lymph nodes are involved, the distance of the involved lymph node to the margin is more important than from the primary tumor.

There is still not enough evidence to justify the sampling of all lymph nodes, in their entirety. For grossly involved lymph nodes a single representative slice is adequate. All lymph nodes less than 4 mm in maximum dimension, should be entirely sampled. A single slice through the long axis, in larger grossly uninvolved nodes, should be processed for initial histological examination. The rest of the node is put through if isolated tumor cells are detected at histology.

Microscopy

A detailed discussion of the histological examination of a colorectal cancer resection specimen is beyond the scope of this article. But in brief, histology aims to answer key questions that decide prognosis and further management. These include—

  1. The type of tumor—Although the vast majority of colorectal carcinomas are conventional adenocarcinomas, histological typing of the tumor is important as this may decide prognosis and further management. Neuroendocrine tumors, particularly at the high-grade end of the spectrum and mixed neuroendocrine—non-neuroendocrine tumors (MINEN), can be misdiagnosed as poorly differentiated adenocarcinoma. It is important to recognize these histological types with appropriate markers as the adjuvant chemotherapy offered is different from conventional adenocarcinomas.


  2. Mucinous adenocarcinoma can be associated with microsatellite instability and may have a reduced response to conventional chemotherapy. A signet ring cell morphology is an independent adverse prognostic factor.[29] Variants of adenocarcinomas such as mucinous carcinoma and signet ring cell carcinoma are diagnosed when the variant type constitutes more than 50% of the tumor. Care should be taken before a mucinous adenocarcinoma is diagnosed in patients who have had neoadjuvant therapy, as this may induce a mucinous phenotype.[30]

  3. Differentiation or grade: As discussed earlier, poor differentiation is an adverse prognostic indicator and also a high-risk factor wherein adjuvant chemotherapy is considered. Assessment is based on architecture, specifically the extent of tubule formation, rather than on cytological features [Table 5]. The tumor is graded based on the predominant pattern of differentiation and not on the worst grade.[31]
  4. Local invasion (Depth of invasion): Assessment of the depth of invasion of the carcinoma is relatively easier when the tumor is appropriately sampled and the block properly oriented. However, certain important issues need to be considered when assessing the local staging—the presence of extramural vascular invasion or perineural invasion within the subserosal fat does not upstage a superficially invasive (pT1) or muscle-invasive (pT2) tumor. Identification of free lying tumor cells in the peritoneal cavity or tumor cells only separated by fibrin or inflammatory infiltrate from the serosal surface are sufficient to stage the tumor as pT4a [Figure 10]a and [Figure 10]b.
  5. Venous invasion: Invasion of veins outside the bowel wall (extramural vascular invasion—EMVI) has long been recognized as an independent poor prognostic indicator and when identified may be an indication for adjuvant chemotherapy. However, the significance of intramural venous invasion or lymphatic invasion is not clear in multivariate studies.[32],[33] A meta-analysis of early (pT1) cancers [34] has revealed submucosal lymphatic and venous invasion to be predictors of lymph node metastases in this group of cancers. It is therefore recommended that lymphatic and venous invasion should be assessed separately and the level of invasion—intramural or extramural—should be mentioned in the report.


  6. Venous invasion is sometimes difficult to identify on Hematoxylin and Eosin (HE)-stained sections. A useful clue to suspect venous invasion is the identification of an “orphan” artery adjacent to the tumor, without an accompanying vein [Figure 10]c. Immunohistochemistry (IHC) for endothelial cells or stains for elastin, such as a Van Geison stain or a Miller's elastic stain, are particularly useful in identifying the involved vein in such cases.

  7. Histological assessment of margins—The involvement of the longitudinal resection margins is extremely uncommon. The involvement of the CRM is associated with significantly higher rates of local recurrence and cancer-related mortality. Measurement of the distance of the tumor from the CRM should be done from the closest focus of tumor—this may be the main tumor, discontinuous tumor deposits, involved lymph nodes or tumor in veins or lymphatics [Figure 10]d. Poorer outcomes are seen in all forms of involvement.[35] The CRM is considered to be involved if the distance from the nearest focus of tumor is equal to or less than 1 mm.
  8. Lymph node status and presence of tumor deposits—Failure to retrieve at least 12 lymph nodes from a resection specimen is a high-risk factor and warrants consideration of adjuvant chemotherapy. However, if nodes are negative, even with a low yield, the nodal stage should still be categorized as pN0.


  9. Direct extension of a tumor into a lymph node is also considered nodal metastasis. Presence of isolated tumor cells (tumor clusters <0.2 mm) does not affect the prognosis adversely and cases where isolated tumor cells are the only form of nodal involvement, are classified as pN0 (i+).[14]

    The identification of tumor deposits confers a nodal stage of pN1c in the absence of nodal metastasis. Tumor deposits are defined as discontinuous or discrete foci of tumor in the pericolorectal adipose tissue and these should be—1) In the lymphatic drainage area of the primary tumor and 2) should not be associated with an identifiable residual lymph node, vascular embolus, or perineural invasion. Their presence or absence and number (less than 5 or more than 5) should be reported in all cases as tumor deposits are associated with a worse prognosis and confer an additional adverse outcome even in the presence of nodal metastasis.[36]

  10. Response to neoadjuvant therapy: Tumor response to neoadjuvant therapy is graded using the modified Ryan's system [37] [Table 6]. Gross residual disease has a worse prognosis compared to minimal residual disease. Grading of tumor regression should be done at the site of the primary tumor and not within lymph nodes. The presence of mucin pools at the site of the primary tumor and in lymph nodes without viable tumor cells indicates complete tumor response and should be staged ypT0 and ypN0 respectively. This is in contrast to cases without neoadjuvant treatment where the presence of mucin within lymph nodes, even when no tumor cells are identified, indicates nodal disease.
Figure 10: (a and b) Tumor involving peritoneum in fat crevices (H and E, ×20 and H and E, ×400), same case as in Figure 9a. (c) Orphan artery sign with tumor embolus obscuring accompanying vein (H and E, ×100). (d) Involved lymph node close to inked circumferential margin (H and E, ×100). Distance is measured from closest focus of tumor to circumferential resection margin

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Table 5: Grading of conventional adenocarcinoma[14]

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Table 6: Modified Ryan's scheme for grading tumor regression post-neoadjuvant therapy[35]

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Handling of local excision specimens

Local excision of colorectal cancer is performed for early (T1) cancers or as a palliative procedure in patients unfit for radical surgery. The different types of local excision specimens that pathologists can encounter are—polypectomies, endoscopic mucosal resection, endoscopic submucosal dissection, and endoscopic transanal microsurgical dissection.

Polypectomies with an intact stalk should be fixed intact. Mucosal resection specimens are ideally orientated and pinned out flat on a corkboard and then fixed. This allows for optimal assessment of the depth of invasion and the marginal status.

Pedunculated polyps are sliced longitudinally through the center of the stalk and the slices are embedded face down so that the stalk is visualized at histology. For larger polyps, additional lateral slices can be taken at 3 mm intervals and the entire sample embedded sequentially.

For mucosal resections, the base and the lateral mucosal margins are inked and the specimen is sliced serially at 3 mm intervals along the short axis. The slices are then processed for histology in sequentially labeled cassettes.

Histological assessment of local excision specimens

The most important question to be answered when local excision is performed for early cancer is whether the local excision is curative or a further, more radical surgery is required. An obvious factor that necessitates radical surgery is incomplete excision of the tumor. Other factors mainly predict the risk of nodal metastasis and these include the degree of differentiation, depth of submucosal invasion, and presence of lymphatic or vascular invasion.[34],[38]

  1. Degree of differentiation: Although the criteria to assess the degree of differentiation are the same, there is a key difference in the overall grading between local excision and resection specimens. In the latter, the predominant area of differentiation decides the final grade. However, in local excision specimens, there is no clarity from the literature if the worst area or predominant area should be used for grading. The Royal College of Pathologists recommends that grading should be based on the worst area of differentiation.[4] Poor differentiation is a potential indication for further surgery.
  2. Depth of submucosal invasion: The local extent of invasion can be assessed by the following two methods -


    1. The level of invasion by using the Haggitt classification for pedunculated polyps [Figure 11] and the Kikuchi classification [Figure 12] for sessile and flat lesions. In pedunculated lesions, invasion beyond the stalk into the submucosa,[39] and in sessile/flat lesions an sm3 level of invasion are adverse prognostic factors [40] with an increased risk of lymph node metastasis. However, since most local excision specimens do not include muscularis propria, using these classification systems is difficult, more so if the specimen is fragmented or poorly orientated.
    2. Measuring the absolute thickness of the invasive tumor beyond the muscularis mucosae. Surgical resection is recommended by the Japanese Society for Cancer of the Colon and Rectum (JSCCR) if the depth of invasion is greater than 1 mm.[41]
    Figure 11: Haggitt levels[39] – (a) Level 1 – invasion into head of polyp. (b) Level 2 – invasion into neck of polyp. (c) Level 3 – invasion into any part of stalk. (d) Level 4 – invasion beyond stalk

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    Figure 12: Kikuchi levels[40] – (a) sm1 – invasion into inner third of submucosa. (b) sm2 – invasion into mid-third of submucosa. (c) sm3 – invasion into outer third of submucosa

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    In the absence of firm evidence as to which system is better, it is recommended that both the level of invasion and the absolute thickness of invasion should be reported.

  3. Lymphovascular invasion: As discussed earlier, lymphatic and venous invasion should be reported separately. Lymphatic invasion in early cancers is a powerful predictor of nodal metastatic disease.[34] Sometimes, retraction artifact may mimic lymphatic invasion and the use of D2-40 IHC can be particularly helpful in such instances. Involved veins can be differentiated from lymphatics by the presence of red blood cells in the lumen and smooth muscle in their walls.
  4. Assessment of margins: Margins are measured in millimeters to one decimal point. Both the mucosal (lateral) margins and the deep margin are assessed. A distance of less than 1 mm from tumor to margin is considered as margin involvement. Most polyps are resected using diathermy and secondary changes including thermal and tissue retraction artifacts are produced, which make measurements difficult. Since thermal artifacts can be several millimeters thick, the outer edge of the diathermy zone should be considered as the true margin when measuring the distance from the tumor. However, infiltration of malignant glands into any part of the diathermy zone is regarded as margin involvement as it is not possible to confidently measure the true extent of invasion.[4]
  5. Tumor budding: Although there is as yet insufficient evidence, tumor budding, which is defined as the presence of single cells or clusters of less than five cells at the advancing edge of the tumor, is emerging as an independent factor predicting lymph node metastasis in local excision specimens.[38],[42],[43] It is recommended that tumor budding be assessed using the international tumor budding consensus conference (ITBCC) criteria [Table 7].[44] Although not a basis for altering management currently, reporting this parameter helps in the collection of data for future research.
Table 7: International tumor budding consensus conference (ITBCC) guidelines for assessment of tumor budding[42]

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Ancillary testing in colorectal cancer

(1) Testing for microsatellite instability (MSI): MSI reflects defective DNA Mismatch Repair (dMMR) system due to mutations in microsatellites, which may be germline or somatic or due to epigenetic inactivation (e.g., methylation of MLH-1 promoter gene), leading to loss function of the protein. Universal testing for microsatellite instability (MSI) in colorectal cancer (CRC) is recommended for prognostication as it confers favorable stage adjusted prognosis, for identification of Lynch syndrome and for therapeutic purposes, the latter most relevant in stage-II colorectal cancer where it has been shown that the patients with MSI-associated CRC are not benefitted with adjuvant 5-flurouracil (5-FU) chemotherapy.[45],[46],[47],[48] It is also performed in metastatic/refractory colorectal cancer to identify the subset of patients who would benefit from anti-programmed cell death-1 (PD-1)/programmed death ligand-1 (PDL-1) immune checkpoint inhibitors, for example, Pembrolizumab.[49]

Testing for MSI can be done by (i) IHC of MMR proteins (biomarkers MLH1, MSH2, MSH6, and PMS2) to show loss of nuclear expression of MMR proteins; and or (ii) molecular testing—polymerized chain reaction (PCR)-based test or next-generation sequencing (NGS), to reveal microsatellite alterations.[50]

IHC testing of MMR proteins: The interpretation of the biomarkers MLH1, MSH2, MSH6, and PMS2 evaluated on IHC is summarized in [Table 8].[51] It is recommended to use all four MMR antibodies either together or sequentially. IHC can be performed on the biopsy or surgical specimen. Normal colonic mucosa, lymphocytes, and stromal cells act as an internal control. The selection of tissue sections for IHC should include tumors with normal mucosa and or the other cells to ensure the presence of internal control. It is important to be aware that IHC expression of the MMR proteins can be adversely affected by pre-analytic factors such as poor tissue fixation, prolonged cold ischemia time resulting in false-negative staining or aberrant expression like cytoplasmic staining, dot-like/perinuclear positivity. Neoadjuvant therapy especially in rectal cancer can affect immunoexpression of the MMR proteins (especially MSH6) in the resection specimen and therefore pretreatment tissue biopsy is preferred for IHC studies.
Table 8: Immunohistochemical (IHC) interpretation of mismatch repair (MMR) protein(s) expression and consequent germ line testing for Lynch syndrome[51]

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Deletions of the epithelial cell adhesion (EpCam) gene located upstream of MSH2 has been described in a subset of families with Lynch syndrome.[52] This is evidenced by the loss of immunoexpression of MSH2, due to somatic hypermethylation of MSH2. Specific testing for EpCam (TACSTD1) can be performed if clinically indicated.

Molecular testing: Formalin-fixed, paraffin-embedded tissue is an acceptable specimen for molecular biomarker mutational testing in colorectal carcinoma. PCR-based testing is used to confirm MMR-IHC results or in cases of indeterminate IHC results (difficulty or discordant IHC results).[49] The PCR-based MSI testing of tumor DNA is performed using a panel of five microsatellite markers (mononucleotide and dinucleotide repeat markers) which can include two mononucleotides (BAT25, BAT 260) and three dinucleotides (D5S346, D2S123, and D17S250) repeats or 5 poly-A mononucleotide repeats (BAT-25, BAT-26, NR-21, NR-24, NR-27), the latter panel has higher sensitivity and specificity.[49],[53] MSI is defined by the loss of two or more of the five microsatellite markers. The usage of terms such as “MSI-High” and “MSI-Low” is discouraged; MSH-low is now categorized with microsatellite stable tumors (MSS). Approximately 5% of Lynch syndrome mutation carriers may be missed by PCR MSI testing; it is 86% sensitive in detecting MSI-associated CRC occurring in families with germline mutation in MSH6.[48]

Comparison of MSI testing by IHC and PCR based testing

PCR based molecular testing detects the changes in DNA caused by functional loss of MMR proteins as opposed to IHC which detects the presence or absence of the specific MMR proteins.

Results of both IHC or PCR based methods for MSI testing have been found to be largely concordant.[54] IHC method has the advantage that it is widely available, rapid, relatively inexpensive, and can help to direct the specific gene(s) to be tested for Lynch syndrome. IHC detection of isolated MSH6 deficiency is superior to PCR amplification. However, in rare cases, there may be functional loss of the MMR protein, due to missense mutations in any one of the MMR genes (e.g., MLH 1) but with retained antigenicity (maintain the ability to bind antibodies). So they are detected by IHC as proficient for MMR proteins despite being mutant primary proteins. In these cases, MSI by PCR will detect the presence of microsatellite instability.

A combination of IHC for MMR proteins and molecular testing will give the best rates for detection of dMMR, for consequent mutational analysis.

NGS can also be utilized as an alternative test for the determination of MSI status, the main advantage being that it can be coupled with an assessment of tumor mutational burden (TMB) and also can identify other treatment targets (e.g., KRAS, PD-1, PDL-1). NGS is expensive and should be carried out only in those centers with expertise in the field.[49]

MSI/MMR status assessment has predictive value in metastatic CRC where anti-PD1/PDL-1 inhibitors are considered for treatment. In these situations, it is recommended that tissue from the metastatic or recurrent colorectal cancer be used, if available, and sufficient. However, if not available, then the primary tumor tissue can be used.[48]

The algorithmic approach for MSI testing (IHC/PCR) in colorectal cancer for Lynch syndrome is depicted in [Figure 13].[47]
Figure 13: Algorithmic approach to microsatellite instability testing

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(2) Testing for B-Type RAF kinase (BRAF). BRAF V600E mutation occurs in 63.5% of CRCs with MLH1 promoter hypermethylation or MLH1/PMS protein loss. Testing for BRAF mutation has prognostic and predictive value as patients with advanced CRC and BRAF mutation have poorer progression free-survival, overall survival, and a decreased response to anti-EGFR therapy. BRAF mutation is not seen in Lynch syndrome-associated tumors.[55],[56] and its presence favors sporadic CRC. It is recommended to incorporate BRAF V600 mutational analysis in MSI-associated CRC with the loss of MLH1 to evaluate for Lynch syndrome risk.[48] [Figure 13]

(3) Testing for KRAS/NRAS: There is strong evidence that the biomarkers (KRAS and NRAS) are negative predictors of benefit from anti-EGFR therapies (Cetuximab or Panitumumab).

The molecular and IHC biomarker testing for colorectal cancer must follow validated protocols with sufficient performance characteristics for the intended clinical use and in accordance with the best laboratory practices.[48]


 » Conclusion Top


Accurate pathology reporting of colorectal cancer resection specimens is important in improving outcomes for patients and also the quality of radiological, surgical, and oncological services. A standardized and methodical approach to dissection and histological assessment is critical in obtaining this crucial information.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10], [Figure 11], [Figure 12], [Figure 13]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]



 

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