Most primary rectal cancers are adenocarcinomas, of which most are conventional gland-forming tumors (Fig. 4.3a, b). However, some special types need to be addressed as they exhibit different behavior and/or molecular phenotype. The special histopathologic sub-types of CRC recognized over the years include (a) mucinous adenocarcinoma: more than 50 % of the lesion is composed of pools of extracellular mucin (Fig. 4.3c); (b) signet-ring cell carcinoma: more than 50 % of tumor cells with prominent intracytoplasmic mucin, typically with displacement and molding of the nucleus (Fig. 4.3d); (c) medullary carcinoma: sheets of malignant cells with vesicular nuclei, prominent nucleoli and abundant cytoplasm exhibiting prominent infiltration by intraepithelial lymphocytes (Fig. 4.3e); (d) adenosquamous carcinoma: tumors show features of both squamous cell carcinoma and adenocarcinoma, either as separate areas within the tumor or admixed; (e) spindle cell carcinoma: a biphasic carcinoma with a spindle-cell sarcomatoid component in which the tumor cells are at least focally immunoreactive for keratins; and (f) undifferentiated carcinoma: tumors lack morphological, immunohistochemical, and molecular biological evidence of differentiation beyond that of an epithelial tumor and have variable histological features [29]. Micropapillary, serrated and cribriform comedo-type adenocarcinomas have been introduced as new distinct histological subtypes of CRC in the new WHO classification [29]. Micropapillary adenocarcinoma shows small clusters of tumor cells within retracted stromal spaces mimicking vascular channels (Fig. 4.3f). Micropapillary adenocarcinoma can be present in combination with other types in variable amounts. Although it is unclear how much of this component is significant, recognition of any micropapillary component, should be reported, as it imparts a significantly worse prognosis and a high incidence of LN metastasis [30]. Serrated adenocarcinoma shows architecture similar to serrated polyps and is believed to arise via serrated pathway of CRC (Fig. 4.3g). The tumors may show MSI-low, MSI-high or have K-RAS or B-RAF mutations, amongst other distinct molecular changes [31, 32]. Cribriform comedo-type adenocarcinoma shows extensive large cribriform glands with central necrosis analogous to breast adenocarcinomas (Fig. 4.3h), and is usually microsatellite stable with CpG island hypermethylation [33].

Other types of primary carcinoma such as clear cell carcinoma, choriocarcinoma-like carcinoma, large cell or small cell neuroendocrine carcinoma and hepatoid adenocarcinoma do occur in the rectum, but are uncommon. Squamous cell carcinomas are mostly seen as an extension from an anal primary, however, rarely these can be rectal primaries.

Adenocarcinomas are graded predominantly based on the extent of glandular appearance. Despite a significant degree of interobserver variability, histologic grade has repeatedly been shown to be an important stage-independent prognostic parameter. Specifically, it has been demonstrated that high tumor grade is an adverse prognostic factor. While traditionally 3- or 4-tiered grading systems: grade 1 (well-differentiated, lesions exhibit glandular structures in >95 % of the tumor) (Fig. 4.3a); grade 2 (moderately differentiated, adenocarcinoma has 50–95 % glands); grade 3 (poorly differentiated, adenocarcinoma has 5–50 % glands) and grade 4 (undifferentiated, < 5 % of tumor with glandular differentiation) have been used, the WHO classification now divides, these into low-grade (well and moderately differentiated adenocarcinomas) (Fig. 4.3b) and high-grade (poorly differentiated and undifferentiated adenocarcinomas) tumors [29]. Studies using the new 2-tiered grading stratification system suggest it is relatively simple, and more reproducibile, while maintaining its prognostic significance. Now CAP also recommends the 2-tiered grading system for grading CRC [34].

In practice, the weakness of the glandular-based grading system is that the estimation of the degree of gland formation is subjective that leads to significant interobserver variability, and ultimately limits the prognostic significance of the histological grade. Furthermore, grade should be established based upon the least differentiated component in heterogeneous tumors; however, the size of such a component has not been specified in any of the systems used in current practice. A proposal that takes into account the extent of poorly differentiated component, defined as a tumor area with no glandular formation, has been proposed recently [35]. Grade 3 was applied to tumors for which the poorly differentiated component fully occupied the microscopic field of an X40 objective lens. For tumors having a smaller component, cancer clusters composed of at least 5 cells, but not forming glands, were counted in the microscopic field of an X4 objective lens where the clusters were the most common. Tumors with less than 10 clusters were classified as grade 1 and those with more than 10 clusters were classified as grade 2. Grade 1 tumors demonstrated 99.3 % cancer-related 5-year survival; grade 2, 86.0 %; and grade 3, 68.9 %, independent of pT and pN stage [35]. Additional studies demonstrated that this tumor grading system based on counting poorly differentiated cell clusters provided significant prognostic information with regards to progression-free survival and was more reproducible than the conventional grading system [36].

Despite interobserver variability and absence of specific definition and diagnostic criteria, the nature of the advancing tumor margin and degree of lymphocytic infiltrate have been shown to be powerful prognostic indicators in rectal cancer. The majority of rectal cancers show a well- or moderately well-circumscribed (so-called “expanding”) margin. An infiltrative margin, however, is frequently associated with perineural and lymphovascular invasion and confers worse prognosis [37, 38]. As stated above, the recently recognized micropapillary pattern and/or tumor budding, which are often present at the invasive front, are also associated with poor prognosis [39].

Lymphocytic infiltration including tumor-infiltrating lymphocytes (TIL), peritumoral lymphocytes and peritumoral lymphoid aggregates in CRCs has long been considered as an indicator of host immune response to tumor cells, and some studies show a better prognosis in their presence irrespective of the MSI status. Recent studies have shown that lymphocytic infiltration, especially CD3+ T cells or FOXP3+ regulatory T cells along the tumor invasive border or within the tumor stroma, and tumor-infiltrating CD8+ lymphocytes in the cancer cell nests are related to longer survival, early tumor stage, expanding growth pattern and lower levels of LVI in patients with CRC [40, 41]. The peritumoral lymphoid aggregates have been named as Crohn’s-like reaction because it resembles inflammatory response in Crohn’s disease [42]. Recently, TILs and to a lesser extent Crohn’s-like reaction have gained attention because of their association with MSI-H status in most cases [43].

Identification of microsatellite instability-high (MSI-H) colorectal tumors is important, as DNA mismatch repair deficiency may serve as a prognostic marker of patient outcome, predict response to chemotherapy, and serve as a screening tool for Lynch syndrome. These tumors are commonly located in the right colon, however, they also can be seen in left side with approximately 8 % of rectosigmoid junctional and rectal cancers being MSI-H [44]. MSI-H left-sided and right-sided CRCs [45] have similar histologic features including TILs (Fig. 4.4a), Crohn’s-like lymphocytic reaction (Fig. 4.4b), mucinous/signet-ring differentiation, and/or a medullary growth pattern. These histologic features have a high predictive value for MSI-H; however, a significant portion of patients with Lynch Syndrome or sporadic MSI-H colorectal cancer will be missed if testing for MSI is solely based on tumor morphology or patient’s clinical/family history. Hence, recently universal testing for MSI has been advocated in all newly diagnosed CRCs regardless of patient’s clinical/family history and tumor morphology [46].

Tumor budding is described as the presence of detached single cells or clusters of up to 4 or 5 cells (Fig. 4.5a, b) along the invasive tumor front [47]. In contrast to the tumor border configuration (infiltrative or pushing pattern), tumor budding is best identified at high magnification, although one can suspect its presence by the typical cellular myofibroblastic response around the advancing edge of the tumor at low magnification that is typically seen in low grade tumors with pushing borders. This phenomenon has been suggested to represent epithelial–mesenchymal transition, thus increasing the migratory capacity, metalloproteinase expression, and resistance to apoptotic signals, corresponding to a more aggressive biological behavior [48, 49]. Tumor budding scores based on 10 high-power field evaluation show excellent inter-observer agreement and high-grade budding (>10 buds across 10 high-power fields) has been shown to be associated with a higher tumor grade, higher TNM stage, LVI, infiltrating tumor border and reduced survival [50]. Although tumor budding is independently associated with LN and distant metastases, and shorter disease-free and overall survival [51, 52], it is not yet universally reported by pathologists due to the absence of consensus criteria for assessment and cut-off values.

Vascular invasion is currently an independent prognostic factor in CRC influencing disease progression and survival [14]. The vascular system consists of arterial, venous and lymphatic vessels, however, it is not always possible to distinguish lymphatic channels from capillary-type vessels, because both are small, thin-walled structures. Theoretically, these two types of invasions should lead to different consequences: lymphatic invasion should be predictive of LN metastasis, whereas vascular invasion should be the source of systemic or hepatic metastases. Use of lymphatic endothelial markers, such as podoplanin (D2-40) or lymphatic endothelial hyaluronan receptor (LYVE-1) as well as capillary endothelial markers, such as CD31 or CD34 can distinguish between lymphatic and capillary vessels. However, these are not routinely used in practice. Thus, the presence of small vessel tumor invasion is best reported as lymphovascular invasion (LVI) or angiolymphatic invasion [53]. The AJCC staging manual (7th edition) combined lymphatic and venous invasion into LVI and recommends reporting its presence or absence in CRCs. Large vessel invasion, in particular of extramural venous invasion, has been shown to be an independent powerful indicator of unfavorable outcome and increased risk of synchronous or metachronous distant, especially liver metastasis; however, there are significant interobserver and interinstitutional variations in their recognition due to lack of standard guidelines [53–56].

Perineural invasion (PN) is an often under-reported high-risk pathologic feature in rectal cancer with a widely varying detection rate from 9 to 42 % [57]. It has a similar impact as LVI and should be reported as a prognostic (site-specific) factor, although it does not affect the tumor staging [5, 19]. A 3-tiered grading system (Pn0, no perineural invasion; Pn1a, intramural perineural invasion; Pn1b, extramural perineural invasion) proposed by the Japanese Society for Cancer of the Colon and Rectum (JSCCR) showed 5-year disease-free survival as 88 %, 70 %, and 48 %, respectively, independent of T or N stage [58]. However, most current systems just report the presence or absence of PN.

Tumor deposits (TD) are nodules of tumor present in the pericolonic/perirectal fat that lack recognizable lymphoid tissue. While we have been fixated with the notion that these likely represent nodal metastases with complete replacement of the nodal architecture by the tumor, studies suggest that these may also represent venous invasion, perineural invasion, discontinuous spread of tumor or the advancing edge of the tumor, each with different prognostic implication [59]. TDs have been shown to be associated with reduced disease-free and overall survival [60]. The interpretation of TDs which first appeared in the 5th edition AJCC staging system in 1997 has changed many times over the years. In the current 7th edition, TDs are considered as such recognizing their varied nature. Their number should be recorded in the pathologic report, and they are classified as pN1c in the absence of unequivocal LN metastases, regardless of the pT category; however, once nodal metastasis are identified, the final staging is performed as per the nodal counts (pN1-2). Equating them to LN metastasis likely underestimate their prognostic impact; we expect that this will be addressed in subsequent staging schemes [61].

With regards to rectal cancer it should be noted that neoadjuvant therapy may create residual tumor islands separate from the main tumor mass, which when located in perirectal fat are difficult to differentiate from true TDs. Since these islands are often the remains of advanced-stage tumors, their presence indicates that tumor regression has taken place, which can be linked to a better prognosis. Therefore, some advocate omitting TD terminology after neoadjuvant therapy, and simply to consider such residual islands in the ypT3 category [62].

Although the rectum is mostly extraperitoneal, the upper third is variably covered by peritoneum; as such, tumors occurring in upper rectum can potentially involve the serosa and be staged as pT4a. Serosal involvement is an independent prognostic factor in rectal cancer predicting a poor prognosis and should not be confused with CRM involvement [63]. Serosal involvement (T4a) implies a higher risk for intraperitoneal tumor spread and necessitates systemic chemotherapy, whereas positive circumferential margin often indicates increased risk of local recurrence and necessitates treatment modalities that can improve local control, including radiotherapy [64]. Visceral peritoneal involvement is often underestimated and can be easily missed without thorough sampling and/or sectioning and requires careful examination. Data suggest that presence of inflammatory reaction, mesothelial hyperplasia, and/or serosal erosion/ulceration when the tumor is present very close to the serosal surface (<1 mm) is sufficient to indicate serosal involvement and one need not wait to see free tumor cells on the serosal surface [11].

The colorectal serosa is formed by a mesothelial layer supported by a basement membrane containing elastic lamina. Use of elastin stain to demonstrate breach of the peritoneal elastic lamina has also been suggested to serve as evidence of possible serosal involvement [65, 66]. However, the lack of demonstrable peritoneal elastic lamina in all CRCs and the inconsistent sensitivity of the elastic staining reagents limit its wide acceptance in routine practice.

The CRM represents the adventitial soft tissue margin closest to the deepest penetration of tumor and is created surgically by blunt or sharp dissection of the retroperitoneum [11]. Multivariate analysis has suggested that CRM involvement is a critical factor in predicting cancer-specific survival, local recurrence, and distant metastasis in rectal cancer [4, 67, 68]. A positive CRM in rectal cancer increases the risk of recurrence by 3.5-fold and doubles the risk of death from disease [67]. The distance between the closest leading edge of the tumor and the CRM should be measured and recorded in mm in the report/staging form in all rectal carcinomas [19]. A positive CRM is defined as tumor ≤1 mm from the margin, because local recurrence rates are similar from 0 to 1 mm (Fig. 4.6). This assessment includes both tumor within a LN and direct tumor extension. A positive CRM secondary to LN metastasis in some studies has been associated with lower recurrence rates than that by direct extension [9, 69]. Thus, if CRM positivity is based solely on intranodal tumor, it should be stated in the pathologic report.

Involvement of the CRM in a patient after therapy with curative intent (e.g., surgical resection for cure) is designated under R classification [19]: R0 for complete tumor resection with all negative margins; R1 for microscopic positive margin and R2 for gross residual tumor. As mentioned above, grading of the completeness of the TME based on gross examination, however, is not assigned any category in the AJCC TNM system and the practice in various pathology laboratories is inconsistent. Of note, the CAP has now omitted the documentation of R category in pathologic reporting of CRCs.

Multimodality therapy has been successfully implemented in the treatment of locally advanced rectal cancers and increasing numbers of patients now receive pre-operative neoadjuvant therapy. The extent of tumor response to the neoadjuvant therapy has the strongest prognostic impact in the treated rectal cancers. The 7th edition AJCC Cancer Staging Manual [19], the CAP [11] and the NCCN rectal cancer guidelines [5] require comment on neoadjuvant treatment effect that should be reported as: 0 (complete response) – no viable cancer cells; 1 (moderate response) – single cells or small groups of cancer cells; 2 (minimal response) – residual cancer outgrown by fibrosis and finally 3 (poor response) – extensive residual cancer or minimal or no tumor kill. Of note, tumor regression should be assessed only in the primary tumor, LN metastases should not be included in the assessment.

Some tumors show little or no response to neoadjuvant therapy, however, microscopically a variety of morphological changes are often seen after neoadjuvant therapy and need to be recognized (Fig. 4.7a–d). The residual cells may show therapy induced nuclear pleomorphism, increased cytoplasmic eosinophilia and vacuoles, and degenerative changes. Some cases may show presence of neuroendocrine cells, admixed with or without an adenocarcinoma component, and it is speculated that these cells are chemoresistant and hence survive. Significance of residual endocrine cells post chemoradiation has been studied only in few studies which suggest they have no added adverse prognostic impact beyond that dictated by stage of residual tumor [70]. Sometimes mucin pools with or without associated tumor cells are seen. While mucin pools associated with viable appearing tumor cells are staged as per the deepest invasion of the tumor or mucin, evidence suggests that acellular mucin pools present in the bowel wall or LNs behave similar to when no residual tumor is identified [71]. Therefore, acellular mucin pools in specimens from patient receiving neoadjuvant therapy are considered as complete eradication of tumor. A variety of other secondary changes are seen that include stromal fibrosis, inflammatory cell infiltration, calcification and foreign body giant cell reaction which by themselves have no prognostic implication.

Pathologic tumor staging remains the fundamental guide for prognostication and treatment decision in managing rectal cancer. Significant improvements have been made in the staging system since the classical proposal introduced by Dukes in 1932 [72]. The original 1932 Dukes classification was based on the extent of disease, as evaluated by the degree of tumor infiltration through the bowel wall, and the presence or absence of LN involvement [72]. Dukes’ A, B, C staging system underwent several subsequent revisions and modifications by Dukes himself as well as other investigators, and it used to be the most popular CRC staging system (Table 4.1). Although Dukes’ staging was a simple, reproducible and widely recognized staging system, it did not take into account the extent of LN involvement, tumor grade, and other pathologic features of tumors. Also there was lack of incorporation of clinical information as well as difficulty in comparing one clinical trial to another due to various versions of Dukes’ classification. The Dukes’ staging system has largely been replaced by the more detailed AJCC/International Union Against Cancer (UICC) Tumor-Node-Metastasis (TNM) system.

The TNM staging system of the AJCC/UICC has gained popularity over the years and is nowadays the standard staging system for cancers including CRCs [73]. The TNM staging system describes the anatomic tumor extent. It is well known that the best estimation of prognosis in rectal cancer is related to the anatomic extent of disease determined by pathologic examination of the resection specimens. The TNM staging system has the ability to separately classify the individual tumor (T), lymph node (N), and metastatic (M) elements and then group them into different stages. Revisions of TNM staging are made periodically in response to newly acquired clinical data and improved understanding of cancer biology and factors affecting prognosis. Despite some criticisms, periodic update is one factor that makes the AJCC/UICC TNM staging system the most clinically useful staging system and accounts for its worldwide use [74].