Table of Contents  

West: Pathological assessment of colon cancer specimens

Introduction

There has been considerable improvement and standardization in the quality of the pathological assessment of rectal cancer specimens over the past 30 years following the recognition of the importance of total mesorectal excision and avoidance of circumferential resection margin involvement. Many of these principles can be easily translated into the pathological assessment of colon cancer specimens in order to provide maximum information to the multidisciplinary team to improve patient management.

Specimen receipt and photography

It is preferable that the fresh specimen is transferred to the pathology department as soon as possible after resection. The specimen should not be dissected by the surgical team in theatres without discussion with the pathologist in order to preserve the peritoneal surface and non-peritonealized resection margin around the tumour. Before any dissection takes place, it is preferable to gently wash the specimen and then take digital colour photographs of the front and back of the whole intact specimen alongside a metric scale (Figure 1). The mesentery should be laid out flat and the position of the tumour and high vascular tie indicated using labels. These pictures form a permanent record of the quality of the specimen and the height of the vascular ligation, and should be shown in multidisciplinary team meetings to provide feedback to the surgical team. If there are any mesocolic defects or perforations, close-up photographs of these should be taken.

FIGURE 1

Anterior (a) and posterior (b) photographs of a fresh unopened right hemicolectomy specimen taken to prospectively record the quality of surgery. A ruler is included as a metric scale to allow calibration. The position of the tumour and high vascular tie are clearly labelled.

HMJ-9-3-7-fig1a.jpgHMJ-9-3-7-fig1b.jpg

The anterior aspect of the specimen should then be opened along the teniae coli to a point approximately 2 cm above and below the tumour. This will allow the specimen to be cleaned and will aid fixation of the tumour. The tumour area should never be opened to facilitate cross-sectional slicing, including the intact peritoneal surface and non-peritonealized resection margin. If the specimen is opened through an area of involved peritoneum or resection margin, then these features may be damaged and therefore not reported. The opened specimen should then ideally be pinned to a cork board (to prevent shrinkage and distortion) and fixed in formalin for at least 48 hours. If the fixation time is less than this, the specimen is very difficult to slice at 3–4-mm intervals, as is required to adequately identify all of the high-risk features.

Assessing the plane of surgery

Assessing the plane of dissection in the mesorectum is now standard practice in many countries and has been shown to relate to the risk of local disease recurrence and, in some studies, survival.1 More recently, the plane of surgery for colon cancer resection has also been shown to be related to survival (Figure 2),2 and local recurrence.3 The recommended grading system is based on that used for mesorectal surgery and classifies the specimen as being in the mesocolic, intramesocolic or muscularis propria planes.

FIGURE 2

Kaplan–Meier survival curves and number at risk according to the plane of mesocolic dissection for colon cancers resected in Leeds for all cases (a) and stage III cases (b). Data from West NP, Morris EJ, Rotimi O, Cairns A, Finan PJ, Quirke P. Pathology grading of colon cancer surgical resection and its association with survival: a retrospective observational study. Lancet Oncol 2008; 9:857–65.2

HMJ-9-3-7-fig2a.jpgHMJ-9-3-7-fig2b.jpg

The assessment of the plane of surgery is best performed on the fresh specimen, and should be confirmed with findings on the formalin-fixed whole specimen and the serial cross-sectional slices. The grading should be performed in the ‘worst’ area within the region of lymphatic drainage of the tumour. For practical purposes, the whole specimen should be graded for a standard resection, although for extended resections (e.g. subtotal colectomy) only the area within the vascular arcades on either side of the tumour should be assessed.

The optimal plane of resection is the mesocolic plane, where the specimen should be resected with no significant defects into the bowel wall or mesentery. The peritonealized surface should be lined by non-interrupted peritoneum, and the non-peritonealized resection margins (where applicable) should be lined by fascia, except at the point of mesocolic transection from the root of the mesentery. Very occasional superficial defects are allowable, but these should be no greater than 5 mm in maximum dimension. The height of the vascular ligation is not assessed when determining the plane of surgery; therefore, mesocolic plane resections are applicable whether a small or large amount of mesentery has been resected, provided that it has been resected intact. An intermediate-quality specimen is classified in the intramesocolic plane, where significant defects will be present in the mesentery (i.e. more than 5  mm in size). None of these defects should expose the muscularis propria. The poorest plane of surgery is the muscularis propria plane, where significant defects expose the muscularis propria (Figure 3). Specimens should also be classified within this plane if the defects go beyond the muscularis propria into the submucosa or mucosa or are even full-thickness perforations (see below). Often defects can be noticed on the intact specimen, but it is not possible to judge how deep they go and whether they expose the muscularis. In this situation, it is advisable to put some coloured ink into the defect and examine its depth when performing the serial cross-sectional slices.

FIGURE 3

Grading the plane of colon cancer surgery including the mesocolic plane (a – intact specimen), intramesocolic plane (b – defect into the mesocolon but no exposure of the muscularis propria), and muscularis propria plane (c – major defects exposing the muscularis propria or perforations).

HMJ-9-3-7-fig3.jpg

The original study from Leeds demonstrated that for specimens resected between 1997 and 2002, the mesocolic plane rate was only 32%, with as many as 24% having major disruptions down to the muscularis propria.2 Patients with mesocolic plane surgery had a 15% greater overall survival at 5 years than those who underwent surgery in the muscularis propria plane. This difference was even more evident among patients with stage III disease, with the number alive at 5 years being twice the number of survivors of mesocolic plane surgery.

Grading the plane of the mesocolon is not an entirely objective measurement, and therefore there is some disagreement between observers.4 It is important that pathologists audit their grading rates to ensure consistency with colleagues, and that specimens are demonstrated by showing photographs at the multidisciplinary team meetings. Any disagreements in grading between the pathologists and the surgical team can then be discussed and a consensus reached.

Assessment of central radicality

In addition to determining the plane of surgery, it is also important that pathologists assess the degree of central radicality. Sticking to the mesocolic plane and undertaking a central vascular ligation are both essential components of complete mesocolic excision (CME), which has been shown to result in optimal patient outcomes when compared with non-CME surgery for colon cancer.5 It is possible to assess central radicality by measuring the length of the main supplying artery to the tumour. This should be done by measuring from the vessel ligation point to the nearest bowel wall and the position of the tumour. These measurements give an indication of the degree of central radicality, although they cannot accurately determine whether the vessel has been transected at its origin because of the significant population differences in vessel lengths that are known to exist. One possible way of auditing the radicality of the central tie is to look at the residual arterial stump lengths on postoperative computerised tomography imaging.6

In centres where CME is performed, the specimens have an average of 41 mm of additional tissue between the tumour and the vascular tie when compared with non-standardized conventional low-tie surgery.7 Japanese surgeons undertake a similar operation to European CME surgery termed D3 surgery. They similarly attempt to resect in the mesocolic plane and ligate the supplying artery at its origin, removing the central or D3 lymph nodes. The main difference is that Japanese surgeons resect a shorter length of colon, leading to a reduction in the overall area of mesentery and lymph nodes when compared with CME surgery.8

Assessing the plane of surgery and measuring the central radicality allows pathologists to monitor the effect of a change in practice following surgical education. One study, conducted in the Copenhagen region of Denmark, showed that surgeons at a hospital that had introduced CME as standard removed significantly more tissue between the tumour and the high tie and were more likely to resect in the mesocolic plane and with a higher lymph node yield than other hospitals in the region that undertook conventional low-tie surgery.9 These differences were observed for both right- and left-sided resections.

Assessing for perforations

Perforations in the specimen occur when a full-thickness defect is created, resulting in a communication between the lumen and the external surface (Figure 4). They can occur spontaneously prior to surgery or can be created at the time of surgery by the surgeon creating a hole in the specimen. Spontaneous perforations often occur directly through the muscle tube and peritoneal surface, although some may track through the mesenteric fat. There is usually evidence of serosal acute inflammatory exudate around the peritoneal defect. Surgical perforations will not usually be associated with inflammatory exudate and may occur when the plane of dissection cuts through a contained mesenteric perforation. Spontaneous perforations should not contribute to the assessment of the plane of surgery; however, surgical perforations should and therefore should be classified as muscularis propria plane resections.

FIGURE 4

Perforation in a sigmoid colon resection for sigmoid cancer resulting in communication between the lumen and the external surface of the specimen. Such perforations carry a higher rate of local recurrence and poorer survival than non-perforated specimens.

HMJ-9-3-7-fig4.jpg

It is important that perforations are accurately described in terms of their position, relationship to the tumour (i.e. within or outside) and size. Perforations within the tumour area that extend through the peritoneum should be staged as pT4, whether they are spontaneous or surgically created, because of the higher risk of disease recurrence. Whilst the TNM Classification of Malignant Tumours technically recognizes only tumour perforations through the peritoneum as pT4 disease, some countries also include perforations outside the peritonealized bowel (e.g. through the fascia at the non-peritonealized resection margin) because of the increased risk of local disease recurrence.10

Dissecting the specimen

The specimen should then be cross-sectionally sliced throughout the tumour segment at 3- to 4-mm intervals, and the slices laid out sequentially. Further digital colour photographs should be taken of the cross-sectional slices alongside a metric scale. It may be helpful to take additional close-up pictures of each of the tumour-bearing slices, or at least the ones demonstrating high-risk features. Slicing thinly is much easier if the specimen is well fixed and allows the pathologist to inspect a greater area of the tumour and identify the best blocks to select for microscopic examination. At least five blocks should be taken from the tumour demonstrating the area of maximal tumour extension beyond the muscularis propria, areas suspicious for peritoneal involvement, possible involved margins and anything that looks like extramural venous invasion (EMVI). EMVI is frequently missed by pathologists and is one of the strongest predictors of distant metastatic disease. It often appears as a serpiginous extension of tumour at right angles to the muscularis propria and can be several millimetres in width (Figure 5). Histologically, pathologists should look for rounded deposits of tumour in the subserosal fat that may lie next to an artery of a similar size. Residual components of the vessel wall can be identified through elastin stains or immunohistochemistry.11

FIGURE 5

Example of extramural venous invasion in an early pT1 cancer. The only extramural disease is contained within a large vein that extends for several millimetres into the mesocolic fat.

HMJ-9-3-7-fig5.jpg

All of the lymph nodes within the specimen should be identified and examined histologically. Whilst a minimum of 12 nodes is often quoted as an audit standard for pathologists, all of the nodes should be found. In many centres, average nodal yields for colon cancer specimens are now in excess of 20, even after preoperative chemotherapy.12 Lymph node yields are complex and influenced by a host of factors, including patient characteristics, tumour biology, surgical radicality and pathological diligence. One of the biggest influences on the ability to identify 12 or more lymph nodes is the pathologist.13 Pathologists should regularly audit their lymph node yields to ensure that they are sufficient. If low lymph node yields are a problem because of time constraints in the dissection room, ancillary techniques, such as methylene blue injection, can be used to improve the identification of small lymph nodes.14 For centres that undertake Dukes’ staging, the apical lymph node should be embedded separately.

The non-peritonealized resection margin

The colon has a variable sized and shaped non-peritonealized resection margin throughout its length depending on the site of the tumour and individual patient anatomy. This margin may be covered by either fascia (where appropriate) or bare fat at the point of mesocolic transection. The posterior retroperitonealized resection margin in the right colon is the largest colonic non-peritonealized margin and should be assessed in the same way as the mesorectal margin for rectal cancer. Ink should be applied to the external surface of the specimen and the minimum distance between the tumour and this margin measured microscopically. An involved margin has tumour sited ≤1 mm from the inked surface. Involvement of this margin occurs in approximately 8% of cases and has been shown to be a marker of advanced disease, with many cases also having other high-risk features (e.g. distant metastases, involved lymph nodes, extramural venous invasion and pT4 disease).15

Molecular pathology

Over recent years, molecular testing is increasingly being used to personalize the treatment of patients with cancer, including those with colon cancer. Around 15% of all colorectal cancers will show microsatellite instability. This is associated with a better prognosis and can be identified using immunohistochemistry for the mismatch repair proteins MLH1, MSH2, MSH6 and PMS2 (Figure 6). Microsatellite instability may be due to a germline defect in a mismatch repair gene (Lynch syndrome) or due to a sporadic event. Sporadic microsatellite instability is more common in the right colon and is associated with classical histopathological features, including poor differentiation, mucinous morphology and tumour-infiltrating lymphocytes. Older patients with high-risk features in the context of microsatellite instability are believed to derive less benefit from adjuvant treatment; therefore, testing should be undertaken for stratified treatment. Pathologists should have a robust process in place for the molecular testing of appropriate cases for microsatellite instability and subsequent clinical genetics referral where appropriate.

FIGURE 6

Immunohistochemistry for the mismatch repair protein MLH1, including normal expression in a microsatellite stable tumour (a), where the tumour cell nuclei retain expression of the protein, and abnormal expression in a microsatellite unstable tumour (b), where the tumour cell nuclei have lost the normal expression. Note that the stromal cell nuclei still express the protein in the unstable case providing an internal positive control to confirm that the stain has worked.

HMJ-9-3-7-fig6a.jpgHMJ-9-3-7-fig6b.jpg

Patients with metastatic disease may derive benefit from the addition of antiepidermal growth factor antibodies to standard chemotherapy. Recent research demonstrated that patients who are more likely to benefit are wild type for the downstream signalling proteins KRAS and NRAS. Mutations in the BRAF gene may also predict a poor response to such therapy and are associated with a poor prognosis independently of treatment. For this reason, patients with metastatic disease frequently undergo molecular testing of the mutational status of these proteins prior to the choice of therapy. A variety of techniques can be used but many centres are now opting for next-generation sequencing technologies to facilitate the simultaneous testing of multiple genes from multiple patients at minimal cost. A routine panel should now include the testing of KRAS (codons 12, 13, 59, 61, 117 and 146), NRAS (codons 12, 13, 59 and 61) and BRAF.16

Summary

The evaluation of colon cancer specimens by pathologists plays a vital role in improving the multidisciplinary treatment of patients with this disease. It is important that the specimens are sent fresh and photographed to capture a permanent record of the quality of surgery. The plane of surgery should be described, the presence or absence of perforations determined and the measures of central radicality assessed. Feeding these back to the surgical team via photographs during multidisciplinary team meetings should help to improve quality over time. The specimen should be meticulously dissected to identify the presence of high-risk features that should be sampled for histology, including the status of any non-peritonealized resection margins. All of the lymph nodes in the specimen should be identified and examined. Finally, molecular testing allows for the stratified treatment of patients with colon cancer. It is important that microsatellite instability testing and sequencing for KRAS, NRAS and BRAF is available in appropriate cases.

References

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Bosch SL, Nagtegaal ID. The importance of the pathologist’s role in assessment of the quality of the mesorectum. Curr Colorectal Cancer Rep 2012; 8:90–8. http://dx.doi.org/10.1007/s11888-012-0124-7

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West NP, Morris EJ, Rotimi O, Cairns A, Finan PJ, Quirke P. Pathology grading of colon cancer surgical resection and its association with survival: a retrospective observational study. Lancet Oncol 2008; 9:857–65. http://dx.doi.org/10.1016/S1470-2045(08)70181-5

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Quirke P, Thorpe H, Dewberry S, Brown J, Jayne D, Guillou P; on behalf of the MRC CLASICC trialists (2008). Prospective assessment of the quality of surgery in the MRC CLASICC trial evidence for variation in the plane of surgery in colon cancer, local recurrence and survival. Presented at the National Cancer Research Institute cancer conference, Birmingham, UK, 5–8 October, 2008.

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Munkedal DLE, Laurberg S, Hagemann-Madsen R, et al. Significant individual variation between pathologists in the evaluation of colon cancer specimens after complete mesocolic excision. Dis Colon Rectum 2016, in press.

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Kaye TL, West NP, Jayne DG, Tolan DJ. CT assessment of right colonic arterial anatomy pre and post cancer resection – a potential marker for quality and extent of surgery? Acta Radiol 2016; 57:394–400. http://dx.doi.org/10.1177/0284185115583033

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West NP, Hohenberger W, Weber K, Perrakis A, Finan PJ, Quirke P. Complete mesocolic excision with central vascular ligation produces an oncologically superior specimen compared with standard surgery for carcinoma of the colon. Clin Oncol 2010; 28:272–8. http://dx.doi.org/10.1200/JCO.2009.24.1448

8. 

West NP, Kobayashi H, Takahashi K, et al. Understanding optimal colonic cancer surgery: comparison of Japanese D3 resection and European complete mesocolic excision with central vascular ligation. J Clin Oncol 2012; 30:1763–9. http://dx.doi.org/10.1200/JCO.2011.38.3992

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West NP, Sutton KM, Ingeholm P, Hagemann-Madsen RH, Hohenberger W, Quirke P. Improving the quality of colon cancer surgery through a surgical education program. Dis Colon Rectum 2010; 53:1594–603. http://dx.doi.org/10.1007/DCR.0b013e3181f433e3

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Loughrey MB, Quirke P, Shepherd NA. Standards and datasets for reporting cancers. Dataset for colorectal cancer histopathology reports. The Royal College of Pathologists, London; 2014.

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Chand M, Palmer T, Blomqvist L, Nagtegaal I, West N, Brown G. Evidence for radiological and histopathological prognostic importance of detecting extramural venous invasion in rectal cancer: recommendations for radiology and histopathology reporting. Colorectal Dis 2015; 17:468–73. http://dx.doi.org/10.1111/codi.12920

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Foxtrot Collaborative Group. Feasibility of preoperative chemotherapy for locally advanced, operable colon cancer: the pilot phase of a randomised controlled trial. Lancet Oncol 2012; 13:1152–60. http://dx.doi.org/10.1016/S1470-2045(12)70348-0

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Morris EJ, Maughan NJ, Forman D, Quirke P. Identifying stage III colorectal cancer patients: the influence of the patient, surgeon, and pathologist. J Clin Oncol 2007; 25:2573–9. http://dx.doi.org/10.1200/JCO.2007.11.0445

14. 

Jepsen RK, Ingeholm P, Lund EL. Upstaging of early colorectal cancers following improved lymph node yield after methylene blue injection. Histopathology 2012; 61:788–94. http://dx.doi.org/10.1111/j.1365-2559.2012.04287.x

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Scott N, Jamali A, Verbeke C, Ambrose NS, Botterill ID, Jayne DG. Retroperitoneal margin involvement by adenocarcinoma of the caecum and ascending colon: what does it mean? Colorectal Dis 2008; 10:289–93. http://dx.doi.org/10.1111/j.1463-1318.2007.01365.x

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Wong NA, Gonzalez D, Salto-Tellez M, et al.; Association of Clinical Pathologists Molecular Pathology and Diagnostics Group. RAS testing of colorectal carcinoma – a guidance document from the Association of Clinical Pathologists Molecular Pathology and Diagnostics Group. J Clin Pathol 2014; 67:751–7. http://dx.doi.org/10.1136/jclinpath-2014-202467




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