Tumor bed configuration and tumor boundaries are better observed after formalin fixation. Sectioning of the tumor with surrounding tumor is easy and does not produce disruptions or tissue curling after formalin fixation. Tumor dimensions, presence and dimension of other lesions, and status of nonneoplastic colorectal wall and adjoining organ should be assessed in the fixed state.

It is recommended that entire tumor bed or scar should be submitted for microscopic examination. In specimens with distinct grossly identifiable tumor, majority of gross tumor should be sampled for microscopic examination. There is more than one approach for sampling tumor for optimal assessment of residual tumor and status and distance of tumor from radial distal and other margins.

The approach of macroscopic examination being followed by majority of the pathology laboratories includes inking, examining, and opening the specimen in the fresh state followed by fixation and sampling in the fixed state. Tumor and adjacent nonneoplastic rectum are submitted by longitudinal or transverse sectioning of tumor to include tumor, rectal wall, mesorectum, and adherent organs. The tumor is serially sectioned in multiple slices, and each slice is divided into multiple sections to accommodate the tissue in a traditional paraffin block . A 5-micron thick section is procured and stained with H&E from each block. This sampling approach identifies discontinuous foci of tumor cells in tumor bed for appropriate pathologic tumor stage. In addition, this method is helpful in accurately measuring distance between tumor and radial or adherent organ margin under a microscope and status of peritumoral lymph nodes . In addition, due to each section derived from the paraffin block that has tissue thickness of <5 mm, the amount of tumor available for microscopic assessment is higher in the H&E-stained sections as compared to having >5 mm thick sections.

Approach for sampling distal margin depends upon the distance between tumor/scar and distal margin . When the distance between the distal edge of the tumor/scar and distal margin is ≤2 cm, perpendicular sections of tumor and corresponding margin should be submitted so that exact distance between tumor cells and/or acellular mucin and distal margin can be measured under the microscope. For specimens with broad distal margin, it is acceptable to submit part of distal margin which is in line with and closest to the tumor/scar in perpendicular sections and peripheral part of the margin as shaved sections with an en face margin. Highest vascular pedicle and proximal colonic margins should be submitted separately as shaved section for en face margins.

Major challenge for pathologic rectal cancer resection specimen evaluation of after neoadjuvant chemoradiation is finding adequate number of lymph nodes. Although processing by chemicals like Bouin’s solution has shown to increase yield of lymph nodes, there is no alternative to meticulous dissection of mesorectal and other soft tissue to identify as many lymph nodes as possible. Not infrequently, the lymph node loses the rounded contour and soft consistency due to radiation, so nodular firm area should be felt and visualized and if suspicious should be sampled. Any lymph node which grossly appears positive should be sampled in its entirety because not infrequently these lymph nodes show reaction to the therapy and/or acellular mucin and not tumor cells. Due to independent prognostic value, inferior mesenteric artery lymph nodes should be identified in advance and sampled separately [5]. It is also essential to make sure that lymph nodes suspected to be positive in the preoperative scans are sampled for microscopic assessment. A review of the preoperative scan by the pathologist is helpful in identifying need of search for specific positive lymph nodes.

Sampling of additional focal lesions such as polyps, ulcers, and diverticula along with random section of nonneoplastic rectum is routinely performed as for any other colorectal resection specimen. Table 17.1 lists the essential components to be included in the gross/macroscopic description of rectal resection specimens .

A different approach of sampling originally described by Quirke et al. [6] includes whole mount processing of the slices of the tumor and adjoining mesorectal and other soft tissue. In this method, the tumor is serially sliced in a transverse plane into 5–10 mm thick slices. The slice with maximal lateral spread of tumor is identified as “primary slice” and is blocked into multiple paraffin blocks with maintenance of orientation. A 5-micron thick section is procured and stained with H&E from each paraffin block. Remaining slices with tumor and mesorectal soft tissue are obtained, and the slices are whole mounted on paraffin blocks, and a 10-micron thick section is procured by sledge microtome followed by H&E staining . The strength of this approach is ability to consistently measure the distance between deepest extent of the tumor to radial margin and tumor extension beyond muscular layer and correlating the extent of the residual tumor with preoperative imaging findings. The limitations include training of the pathologist grossing these specimens and more demand on histology laboratory due to whole mount of the sections and less sampling of the tumor bed in the vicinity of the tumor epicenter as compared to the method described above, due to the fact that the amount of tissue remaining in the FFPE block and not assessed under microscope is higher than what is described in the first approach.

Optimal histopathologic examination includes all the histopathology findings which are assessed in any adenocarcinoma of colon or rectal resection specimen including pathologic tumor stage, nodal stage, distant metastases, presence (or absence) of lymphovascular and perineural invasion, and distance of tumor to the margins. In addition, histologic regression of the tumor characterized by reduction of tumor cells and replacement of the tumor bed by fibrosis with or without necrosis, granulation tissue, and inflammation is one of the essential parameters required to be reported in the pathology report.