Background

Carcinoma with serrated features was originally described by Jass and Smith as carcinoma with features that resembled hyperplastic polyps.4 Serrated adenocarcinoma (SAC) as a distinctive morphologic category was first proposed in 2001, by Makinen and colleagues, to represent the malignant end-product of the serrated neoplasia pathway.5 As such, as an alternative to the conventional pathway of neoplasia,6 it has been suggested that SAC accounts for an entirely different category of CRC that are associated with serrated polyps, more commonly right sided, associated with deficient mismatch repair (dMMR) and CpG island methylator phenotype (CIMP) and associated with an aggressive phenotype.7–12 SAC is estimated to account for 8%–10% of all CRC.7 9 13

Clinical characteristics

The two largest studies on SAC reveal that there is no association with age (mean 697 and 69.1 (13)), no particular gender predilection, nor significant association with AJCC tumour stage.7 The largest study demonstrated that these tumours are more frequently right sided/proximal (p=0.003),7 while other studies failed to demonstrate such an association (p=0.73).13 Curiously, the original study on SAC revealed most to be found in the cecum (52%) and rectum (33%).9

Histologic and molecular features

The current edition of the WHO Classification of Digestive System Tumors14 recognises SAC as a distinct subset of CRC and defines SAC as tumours with morphological similarities to serrated polyps, which may have mucinous areas, and have cells that show low nuclear to cytoplasmic ratios (figure 1A, B).14 In a review on SAC in 2007, Makinen defined SAC as having epithelial serrations (resembling saw-tooth edges of a serrated bread knife), abundant clear or eosinophilic cytoplasm, vesicular nuclei, devoid of necrosis (<10% total volume), may demonstrate mucin production and have cell balls and papillary rods (figure 1A, B).9 He noted that these tumours are often associated with adjacent serrated polyps and may demonstrate a predominant pattern that is either mucinous or trabecular rather than serrated. As such, these tumours may have considerable overlap with tumours otherwise categorised as mucinous adenocarcinomas (MADs) or micropapillary adenocarcinomas (MPCs) (figure 1B).9

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

(A) Serrated adenocarcinoma, eosinophilic with prominent serrations. (B) High-grade serrated adenocarcinoma, cell balls and mucin.

The largest studies on SAC noted that serrated morphology was most specific for this phenotype relative to non-SAC CRC.7 ,13 Additional studies have demonstrated that this phenotype is frequently either not associated with a precursor polyp, or associated with an adenoma on the shoulder of the carcinoma.7 ,13 A study by our group evaluated SAC by percentage of epithelial serrations and found comparable outcome findings for tumours demonstrating some epithelial serrations (>5%) to tumours demonstrating more prominent serrations (>50%),13 highlighting again that serrations are the most specific finding,7 and that other patterns such as mucinous or trabecular can be the preeminent morphology in this subcategory of CRC.9

Prognosis and meaning

While SAC is an entity recognised by the WHO, its interpretation and meaning are controversial. Part of this stems from a limited threshold of definitions on what constitutes an SAC and its overlap with other entities such as MAD and micropapillary carcinoma. While some have proposed SAC to have a more aggressive prognosis,5 others have suggested no difference in survival13 or even improved survival.15 These differences may stem entirely from definitional criteria, locoregional variances and population cohorts, or interobserver variability, but do raise awareness regarding the thresholds of its histomorphologic criteria and overall clinical significance.

Recent publications, including one by our group, have argued that tumours with serrated morphology are associated with adenoma precursor polyps, are not associated with worse disease-free survival and have a similar incidence rate of dMMR, KRAS mutation and BRAF mutation as conventional carcinomas. Additionally, multiple studies have shown that carcinomas most frequently associated with serrated polyps do not exhibit a serrated phenotype, including carcinomas that arise in association with serrated polyposis syndrome.7 8 13 16 17 These findings raise the question of whether SAC represents the malignant end product of serrated neoplasia pathway. It is the opinion of the authors that additional studies need to be conducted that more precisely assess the significance of this pattern to establish whether SAC, as currently defined, represents a biologically and clinically relevant entity.

Background

MPC, originally described as a morphologic variant of invasive breast carcinoma,18 has subsequently been identified as a variant in a variety of organs, including bladder,19 lung,20 ovary,21 ampulla-pancreatobiliary22 and salivary gland,23 among others. It is currently recognised as a morphologic variant of CRC by the WHO.14 Indeed, its incidence as a variant of CRC is estimated to range from 5% to 19%, including the largest study performed by our group, which identified an incidence of 8.6%.24–29 A common thread for this tumour morphology is that they tend to be more commonly associated with high-risk features, including lymphovascular invasion, lymph node metastasis, poor response to adjuvant/neoadjuvant therapy and poor outcome measures.19 20 27 28 The metastatic component often displays MPC morphology, highlighting that the tumour morphology is at least in part driving the tumour biology.18 It is purported that the adverse features associated with MPC stem from its unique ‘inside-out’ tumour morphology (see below discussion in histology) as it may be indicative of an epithelial-mesenchymal transition, and in turn, a more infiltrative and aggressive phenotype. Indeed, ultrastructural studies have suggested that the cellular membrane reverses its polarity, with the apical end of the cell pointing towards the stroma, which likely contributes to the disruption and clefting of the malignant cells with the surrounding stroma and its more aggressive phenotype.30 Yet in this subsection of the review, we will highlight that the significance, diagnosis and role of this variant in the colon are both contentious and controversial.

Clinical characteristics

Age at diagnosis of MPC was younger in one examined study,31 and not significant in others25 28 29 with mean age ranging from 56.6 to 69 relative to non-MPC CRC. There is no apparent predilection in gender, tumour size or tumour sidedness, compared with non-MPC CRC, though multiple studies lacked power due to small sample size.22 32 A single study reported a greater rate of positive resection margins (R1 or R2) relative to conventional carcinoma.28 Another study demonstrated that MPC is more likely ulcerating (70%) than polypoid relative to conventional carcinoma.26

Histologic and molecular features

MPC is defined as nests of five or more tumour cells, devoid of fibrovascular cores and lacking a central lumen. The cellular nests are entirely surrounded by cleared-out/clefted spaces and are commonly mistaken for lymphatic invasion (figure 2A, B). Cytologically, the cells often have abundant eosinophilic cytoplasm and demonstrate prominent rounded nuclei with atypia and reverse nuclear polarity (figure 2B).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

(A) Micropapillary adenocarcinoma, numerous nests of cells with clefting. (B) Micropapillary adenocarcinoma, vesicular nuclei with reversed polarity.

Regarding CRC, the WHO requires at least 5% of the tumour morphology to be comprised of micropapillary architecture that can occur anywhere within the tumour.14 MPC is often comingled with other morphologies, including conventional adenocarcinoma, cribriform pattern, serrated pattern and mucinous variant.29 31 MPC is often only a minor component, with the largest study reporting a prevalence of MPC architecture of >50% in only 14.5% of cases,29 and to the authors’ knowledge, there is no reported case of a CRC with exclusive micropapillary architecture. Its overall morphology has been likened to overlap with high-grade SAC (figure 1B) and has commonly be described to be intermingled with more classic SAC morphology,9 29 raising the question of whether at least a subset of MPC is part of a biologic continuum with SAC.

While the definition of MPC seems straightforward, it could be (mis)interpreted as another tumour phenomenon. In particular, micropapillary clusters morphologically overlap with tumour budding and poorly differentiated clusters. Helpful clues in making this distinction are that tumour buds are, by definition, four cells or less in size and usually lack circumferential lacunae.33 The distinction with poorly differentiated clusters is more troublesome as the only apparent difference is that poorly differentiated clusters lack complete circumferential clefting. It has been suggested that these two processes represent the same biologic phenomenon34 35

MPC is a morphologic diagnosis, although immunohistochemistry has been used to demonstrate the inversion of the cell polarity seen in MPC. MUC1 is a glycoprotein present on the luminal apical surface of glands and may be a significant factor in gland formation. In MPC, MUC1 stains the inverted exterior of the cell clusters, which may be part of the mechanism disrupting its interaction with the stroma and leading to tumour clefting; epithelial membrane antigen and villin demonstrates a similar staining pattern.26 30 36 37 Immunostains for D2-40 may be of benefit to differentiate actual lymphatic invasion from the natural clefting seen in MPC, as it is quite prevalent in MPC.36

Regarding mismatch repair, the association is not clear; while some studies report no association,24 29 others report more frequent proficient mismatch repair.26 31 Two recent studies showed no association with KRAS or BRAFV600E mutation status.29 31

Prognosis and meaning

Intuitively, it would be expected that MPC of the colon would have similar high-risk associations as described for other organs. Indeed, multiple studies have demonstrated colonic MPC to be associated with higher tumour stage, more frequent lymphovascular invasion, lymph node metastasis and survival.24 26 38 Multiple studies have suggested that this more aggressive phenotype may be attributed to its differential expression of stem cell markers and mesenchymal markers, including SOX2 and NOTCH3, p53 and vimentin and loss of epithelial and junctional markers including AE1/AE3 and e-cadherin to indicate that the tumour is possibly undergoing an epithelial-mesenchymal transition.24 26 31 36 37 Additionally, our group identified that this group of tumours may be associated with an immune cold environment, including lower CD8 positive cells, to suggest a less robust tumor-host response; however, we note that multiple other immune biomarkers did not reach significance, and numerous other factors likely confound these findings.29

Recent studies, including the largest known study of colon MPC performed by our group, have found that when tumours are adjusted for grade and stage, the association with high-grade characteristics loses significance.28 29 31 Gonzalez and colleagues found that in CRC, lymph node metastases of colon cancers with a micropapillary component only demonstrated a micropapillary morphology 32% of the time, potentially suggesting that MPCs are inherently part of aggressive and high-grade tumours, and that the MPC component may not necessarily be the predisposing factor of a more aggressive phenotype.

Background

Medullary carcinoma (MC) of the colon is a rare variant of colon carcinoma that is often confused with poorly differentiated conventional adenocarcinoma, undifferentiated carcinoma and even large cell neuroendocrine carcinoma (NEC). Indeed, the evolution of MC stemmed from a necessity to identify ‘poorly differentiated’ tumours that had more favourable compared with other more aggressive phenotypes. Jessurun and colleagues performed one of the first studies to investigate this variant and argued that MC nomenclature, akin to the stomach,39 was more helpful as opposed to other more ominous terms like solid adenocarcinoma or large cell adenocarcinoma with minimal differentiation to highlight its better outcomes.40 41

Despite being a WHO entity, there appears to be a large variance in the reported incidence of MC. Indeed, MC is reported to represent a broad range of colon carcinomas (0.08% to over 4%), with a large meta-analysis finding an incidence of 2.2%.41–44 This may be due to a lack of a precise definition of the minimal histological, immunohistochemical and molecular features of MC.14 45 In this portion of the review, we not only seek to highlight the main distinguishing and potentially critical characteristics of MC but also underscore its controversy as a histologic entity.

Clinical characteristics

Most studies demonstrate an overwhelming predilection of MC for female sex, with one population-based analysis utilising the surveillance, epidemiology and end results dataed with a variebase (SEER) revealing a male-to-female ratio of 1:2.12,41 although we note that female sex did not reach statistical significance for one study that analysed 45 MC.42 Relative to other poorly differentiated carcinomas or non-MC, the vast majority of studies show that MC predominately occurs in older patients, with an average age of incidence ranging from approximately 76 to 79.43 45 46 The overwhelming majority of tumours are right sided (ranging from 72% to 94%),41 43 45 46 with tumours only rarely being seen in the rectum.43 MC appears to be larger than non-medullary or other poorly differentiated carcinomas with a mean size of approximately 60 mm–64 mm, including another study highlighting that two-thirds of examined cases were larger than 7 cm.43 45 46

Histologic and molecular features

Per WHO guidelines, MC is comprised of ‘malignant sheets with vesicular nuclei, prominent nucleoli and abundant eosinophilic cytoplasm’ (figure 3A, B).14 In MC, tumour cells are often uniform in appearance, mitotically active, contain intracytoplasmic Periodic acid-schiff (PAS) positive vacuoles, demonstrate a pushing rather than infiltrative border of invasion and lack obvious mucinous differentiation.40 45 MC has been described with a variety of names, including solid-type adenocarcinoma and solid-type carcinoma. The most relevant differential, therefore, are poorly differentiated adenocarcinoma and undifferentiated carcinoma. Unlike poorly differentiated adenocarcinomas, gland formation is vanishingly rare, and unlike undifferentiated carcinomas, they have more abundant cytoplasm and lack dense and highly pleomorphic nuclei. Another relevant differential is NEC, which is further complicated in that not all MCs are negative by immunohistochemical staining for neuroendocrine markers, including chromogranin, synaptophysin and INSM1.40 45

• Download figure
• Open in new tab
• Download powerpoint

Figure 3

(A) Medullary carcinoma, malignant sheets of cells and associated inflammation. (B) Medullary carcinoma, vesicular nuclei, prominent nucleoli, associated neutrophilic and lymphocytic inflammation.

It must be noted that there are shortcomings in the definition of MC that are not clarified. While the WHO sets percentage guidelines for mucinous and MPCs, it does not require a certain percentage for the percentage of tumour that must exhibit an MC morphology. In a study by Scott and colleagues, they found that over 20 years, only one-third of MC were correctly identified, often being mistaken for poorly differentiated conventional adenocarcinoma or NEC.45 Indeed, multiple studies have demonstrated different criteria, with some assessing MC predominant mixed morphology tumours, others MC only, while others do not clearly define this parameter.40–42 45 Another contentious element is that it lacks suitable criteria regarding allowable glandular definition. While most studies and the WHO elude to some permissible glandular differentiation, there is no clear-cut guideline as to what is suitable; in addition, some studies exclusively evaluated non-gland forming tumours.40–42 45

The WHO notes that MCs are characteristically associated with a brisk inflammatory infiltrate, including lymphocytes and neutrophils (figure 2B).14 Additional studies have demonstrated that this immune response is often associated with a prominent intratumoral component mimicking a lymphoepithelial carcinoma, as well as a prominent lymphoid response, or Crohn-like reaction.40 42 45 Friedman and colleagues interrogated the immune microenvironment of MC and found that the tumours more often demonstrated a hyperimmune tumour microenvironment that expresses immune regulatory proteins, to include PD-L1, PD-1 and IDO-1, and contain a more robust immune infiltrate comprised of CD8-positive lymphocytes and PD-L1-positive immune cells. This response is due to a high tumour mutational burden and may help explain the favourable outcomes of MC relative to other poorly differentiated adenocarcinomas.46

A natural segue from this discussion is MMR status. The WHO highlights a positive association with microsatellite instability, in particular, loss of MLH1 and BRAF mutation in MC.14 This again suggests that MC has a high mutational burden, and they are commonly sporadic though one study noted 11% of their MC cohort was comprised of Lynch patients.43 Indeed, some studies argue that MC requires dMMR status,43 while others include pMMR cases as well.44 It is not clear whether dMMR MC and pMMR MC have similar outcomes and characteristics due to limited cohort sizes. KRAS mutations appears infrequently associated with MC, while loss of ARID1A was more common.44

Due to the high-grade appearance of the tumour, immunostains are often employed to ensure that the tumours are indeed carcinomas that arise from colon. However, the MC immune profile often presents a non-conventional immunophenotype, including frequent loss of CDX-2, which is either diffuse or patchy in up to 87% of tumours.45 47 CK20 is similarly lost in up to 75% of MCs.45 48 49 Clues to support a diagnosis may hinge on evaluating the tumour cells for loss of mismatch repair proteins as well as staining for broader panel cytokeratins, epithelial membrane antigen,40 and SATB2.48 49

Prognosis and meaning

Many studies argue that MC is associated with more favourable outcomes relative to non-MC or other poorly differentiated carcinomas. In the only meta-analysis, MC had better overall and disease-specific survival relative to poorly differentiated and undifferentiated carcinomas, though there was no difference compared with conventional adenocarcinoma.44 Another study evaluating the SEER database revealed that relative to poorly differentiated carcinoma, which are most of stage III tumours, MCs are most often pT3 (48%) without lymph nodal involvement (60%); yet this study failed to demonstrate a significant difference in overall survival between these groups.41 Similarly, in a study by Knox and colleagues that used strict inclusion criteria of dMMR and non-gland forming tumours, there was no difference in survival between MC and non-MC. In fact, they found that MC has a higher 30-day mortality compared with conventional adenocarcinoma; though we note in this study that MC more frequently occurred in older patients with larger tumours arguing that these variables may have confounded the aforementioned outcome.43 In addition, relative to other dMMR colon carcinoma morphologies, MC was more likely to be stage 3 or 4 disease, with more frequent lymphovascular invasion and lymph node involvement.43

Given its complex morphology, molecular profile and immunohistochemical landscape, assessing the overall significance of MC is challenging. Notably, studies showing beneficial outcomes of MC often compare it to tumours with poorly or undifferentiated phenotypes. This suggests that MC’s unique characteristics may stem from its dMMR status, which is similar to carcinoma NOS. However, more studies with definitive diagnostic criteria are necessary to explore this further.

Background

NEC in CRC is critical yet challenging to identify. Unlike its counterpart in the lung, NECs are rare comprising less than 1% of all CRC primaries.50 Yet, NEC significantly influences outcomes and management strategies compared with stage-matched CRC.51 Additionally, some malignancies exhibit mixed phenotypes of NEC and mixed neuroendocrine non-neuroendocrine neoplasms (MiNEN), typically as mixed adenoneuroendocrine carcinoma. This section focuses on the diverse morphologies of NEC, their prognostic implications and the controversies and limitations of this classification. Importantly, this analysis excludes well-differentiated neuroendocrine tumours (NETs). Despite sharing neuroendocrine markers, NEC and NETs are essentially distinct, not part of the same biologic continuum.52

Clinical characteristics

Risk factors for NEC are less well established than its lung counterpart. The most recent edition of the WHO cites a large metanalysis that broadly evaluates neuroendocrine neoplasms (to include NET)53 and does not explicitly address NEC or mixed tumours. In turn, we identified relatively large studies from two separate institutions54 55 that more concisely evaluate this morphologic category. There appears to be a slight male predominance (~60%). Regarding disease distribution, one study showed nearly half the cases involve the rectum (42%)55 while the other showed a predisposition for the right colon (65%).54 The average age for NEC ranged from 55 to 65, while one study reported the average age of MiNEN to be slightly higher at 72.54–56 The majority of patients present with metastatic disease, though one study found this to be more frequently distant (67%),55 and the other regional lymph node metastasis (64%).54 Grossly NEC resembles conventional adenocarcinoma.14 A single study evaluating NEC in the entire luminal gut reported that small cell type NEC is less prevalent in the non-glandular gut (ie, anus and oesophagus), whereas mixed tumours are predominately found in the large bowel (82%).51 Other studies evaluating the colon and rectum found the overall incidence of NEC to be greater than MiNEN (27 vs 12), with 45% of the NECs demonstrating a small cell morphology.54

Histologic and molecular features

While this category of tumours are broadly categorised as NEC or mixed (ie, MiNEN), the morphologic spectrum is markedly heterogenous. NECs are conventionally categorised by the WHO as small cell or large cell type,14 though there are multiple additional descriptions in the literature to include a mixed or intermediate-type NEC with both large and small cell components.51 For the purposes of our discussion, however, we will focus on the entities recognised by the WHO.

Histomorphologically, similar to small cell carcinoma of the lung, small cell-type NEC demonstrates a diffuse/solid growth pattern of atypical cells with minimal cytoplasm, high nuclear to cytoplasmic ratio, hyperchromatic nuclei with inconspicuous nucleoli, pleomorphism, brisk mitoses, including atypical mitoses, crush artefact and prominent necrosis. In one study, the nuclei were regarded as being ‘more round’ compared with its pulmonary counterpart.51 It is also reported that up to 57% of patients with small cell-type NEC have colonic adenomas,56 though this study did not clarify whether these adenomas were directly associated with the malignancy. Additionally, immunohistochemistry is not diagnostically necessary when the appropriate classic morphology is present for this subcategory of NEC.14

Large cell-type NEC carcinoma demonstrates similar histologic characteristics to its lung counterpart. Briefly, this consists of solid nests of cells that frequently demonstrate organoid or trabeculae morphology, which may show rosette formation and cellular palisading. While glandular lumen and mucin are typically absent, it is suggested that these may be present in a ‘minor’ component of pure NEC.51 Cytologically, large cell carcinoma is comprised of a relatively monotonous-sized population, with a rounded shape, readily identifiable cytoplasm and vesicular nuclei with frequent prominent nucleoli. In addition, necrosis (often regional) and brisk mitotic activity (often much greater than 10 per/10 HPF) are usually present.14 An essential diagnostic criteria for large cell-type NEC are the demonstration by immunohistochemistry for at least one neuroendocrine biomarker including synaptophysin, chromogranin, or INSM1.

Mixed-type NECs are predominately mixed adenoneuroendocrine carcinoma though a subset is associated with squamous cell carcinoma. A single study reported adenocarcinoma more frequently associates with large cell-type NEC. While these morphologies are often spatially distinct, they can also be comingled.51 Both the NEC and non-NEC component need to comprise at least 30% of the tumour volume to qualify as a mixed tumour.14 ,51 54 A less defined area is the classification of tumours when either component is greater than 5% or less than 30% as was described in 11 of 39 cases in one study.54 While we agree that this is an area of controversy, we emphasise that at a minimum a tumour needs to demonstrate NEC morphology that is accompanied by expression of at least one neuroendocrine immunohistochemical biomarker to be considered as having neuroendocrine differentiation.14 51 We also note that NEC or MiNEN may be associated with a surface adenoma up to 31% of the time.51 54

High-risk histologic features are commonplace regardless of morphology. Lymphovascular invasion occurs in the vast majority of cases. Perineural invasion, while common, was less commonly seen in up to 50% of cases.51 54

Immunohistochemistry can be useful both in diagnosing a primary tumour, assessing a metastasis, and possibly in stratifying tumour aggressiveness. All/most tumours should stain at least partially for AE1/AE3, and the overwhelming majority should stain for at least one neuroendocrine marker most commonly including either synaptophysin, chromogranin and INSM1. There is no benchmark, per se, regarding how much the tumour needs to stain in order to be regarded neuroendocrine, but we endorse a benchmark of >20%.51 It is broadly reported that synaptophysin is the most sensitive neuroendocrine marker, though there are many occurrences where a tumour will stain only with chromogranin or CD56; indeed, a single study actually reported a higher sensitivity for chromogranin (18 of 19), relative to synaptophysin (10 of 15).14 50 54 57 58 Collectively, these findings highlight the necessity to perform a panel of stains to evaluate for neuroendocrine differentiation in the appropriate morphologic context. Immunostain for KI-67 has been purported to have a prognostic impact as a KI-67 of greater than 55% is suggested to have a worse outcome.59–61 A single study demonstrated that CD117 expression correlated with shorter patient survival on multivariate analysis.54

NECs and MiNENs demonstrate a rate of microsatellite instability (5 of 34; 15%) similar to conventional colon cancer, and these cases always revealed a CIMP (5 of 5) versus 64% of microsatellite stable cases.54 The same study also evaluated methylation profiles and found the methylation of three genes—ESR1, WT1 and GATA5—in >70% of cases to suggest a significant role of epigenetic alteration in NEC and MiNEN.54 NECs, particularly small cell carcinoma, harbour alterations in TP53, FHIT, MEN1 and RB1,62 63 which can consequently be used as diagnostic biomarkers in addition to highlighting the inherently high level of chromosomal instability found in this category of tumours. In particular, loss of RB1 and overexpression and loss of p53 can be used as valuable diagnostic immunohistochemical biomarkers.

Prognosis and meaning

NEC and MiNEN are significant morphologies that effect patient outcome and management decisions. A single study reported the absence of an adenocarcinoma component to be associated with a worse outcome,51 55 others reported no difference between NEC and MiNEN survival.50 54 While extent of disease (extensive vs local), vascular invasion, presence of synaptophysin staining and CD117 staining were all found to be independent prognostic factors in various studies, high stage seemed to be a common denominator as a poor prognosticator.50 51 54–56 Additionally, as discussed above, it is suggested that NECs and MiNENs with KI67 indices greater than 55% are associated both with a worse prognosis59 and a more favourable response to platinum-based chemotherapy.61 Given the impact of NEC and MiNEN on treatment and outcome, it is critical, therefore, to perform a panel of neuroendocrine immunohistochemical stains when NEC morphology is suspected.

NEC and mixed tumours frequently present as metastatic disease (up to 70%),50 with the most common site of metastasis being the liver.54 Of cases with metastatic disease, a single study reported that response to chemotherapy was the only factor associated with survival.55 The same study noted that relative to colonic primaries, anorectal primaries tended to more frequently present with local disease, likely because distal tumours are more often to be symptomatic at an earlier stage.55

NEC and MiNEN do worse than conventional adenocarcinomas, with a median survival of around 13 months and reported 3-year survival with metastatic disease being as low as 5%.55 The majority of patients receive first-line chemotherapy, with majority receiving platinum-based chemotherapy regardless of tumour stage.64 65 As in the lung, the role of surgery appears minimal. Even in cases of localised disease, surgery has been shown to have an overt survival benefit55; however there may nonetheless be a role for surgery in a minor subset of cases depending on the type of surgery and other clinicopathologic factors.55 For MiNENs, the use of 5-FU-based chemotherapy may also have utility as adjunctive therapy.55

Background

MAD is the most common histologic subtype of CRC, comprising up to 10%–20% of all CRCs.14 66 It was originally introduced and described by Parham in 1923 as colloid carcinoma named after the gelatinous nature of the tumour67 and is currently a formally recognised WHO CRC subtype.14

Clinical characteristics

Patients with MAD tend to present at a more advanced disease stage compared with their non-mucinous counterparts.68 It is associated with a higher proportion of nodal and peritoneal metastasis at presentation.68 69 MAD is also associated with higher rates of local and distant recurrence.70 The fact that these tumours are more commonly right sided and a softer tumour consistency seems to play a role in contributing to delayed symptomatic presentation until they progress into more-advanced disease.68

Histologic and molecular features

MADs are defined as tumours demonstrating greater than 50% of mucinous areas characterised by large pools of extracellular mucin (figure 4A) containing floating malignant epithelial strips, clusters and single cells (figure 4B). Tumours with less than 50% mucinous component are categorised as conventional adenocarcinoma with mucinous features.14 Associated signet-ring cells and micropapillary features may be seen as well, though they should only be present as a minor component.14 This 50% cut-off of mucinous component is somewhat an arbitrary criterion. In multiple studies,71 72 neither the proportion of mucin-producing malignant glands in a tumour nor the percentage of tumour volume composed of mucin had a significant impact on AJCC stage-matched disease-specific survival. Hence, the prognostic significance for this specific numerical cut-off remains unclear. In terms of molecular features, compared with conventional adenocarcinoma, MADs have higher rates MSI,73–75 MUC2 overexpression,76 CIMP,74 HATH1 expression,77 and BRAF mutations75 ; they are less frequently associated with TP53 mutations.75 Mucinous features have been reported to be associated with loss of HES1, which have been linked to a worse prognosis.78 Lastly, in one study, there was no difference between MAD and conventional adenocarcinoma regarding variables of the immune microenvironment, including CD8 and CD163.72

• Download figure
• Open in new tab
• Download powerpoint

Figure 4

(A) Mucinous adenocarcinoma, with large mucin pools. (B) Mucinous adenocarcinoma, with floating epithelial component.

Prognosis and meaning

The prognosis of MAD is overall comparable to conventional adenocarcinoma.14 66 71 72 79–81 Historically, patients with rectal disease tended to do worse when compared with those with colonic disease,66 80 however, this difference has narrowed with the introduction of total mesorectal excision and neoadjuvant therapy for rectal primaries.68 In advanced settings, however, metastatic MAD demonstrate worse prognosis compared with metastatic conventional adenocarcinoma owing to poor response to chemotherapy.82 While the clinical and prognostic significance of the histologic mucinous component itself is unclear, pathologists need to recognise and report this clinically aggressive histomorphologic feature regardless of percentage, to help guide subsequent molecular testing and therapeutic options.

Background

Signet-ring cell Carcinoma (SRC) was first described by Saphir and Laufman in 1951 as primary linitis plastica type carcinoma of the colon and rectum.83 It is a rare WHO-recognised histologic subtype accounting for up to 1% of all CRCs,14 66 84 and is associated with an aggressive clinical and biologic behaviour.

Clinical characteristics

SRC are generally associated with a more aggressive clinical course compared with conventional adenocarcinomas. They are more frequently right-sided and clinically present as higher stage (eg, AJCC Stage IIIC/IV) tumours compared with their conventional and mucinous counterparts.14 66 81 84 In addition, SRC is associated with higher and more rapid rates of metastatic disease to multiple sites (more commonly peritoneal), as well as higher rates of both locoregional and distant recurrences.84–86 This specific subtype is known for its linitis plastica-like growth pattern with somewhat elusive clinical appearance, and notoriously affects younger patients, all of which may in part contribute to delayed detection and more advanced disease at diagnosis.86 In one large populational study,66 more patients were diagnosed with SRC between ages 18–49, compared with their non-signet-ring counterparts (19% vs 10%). In another study,87 a large proportion (53%) of SRC were diagnosed under age 40, which is well below the recommended age for CRC screening in low- to average-risk individuals.

Histologic and molecular features

SRC is defined by adenocarcinoma with signet-ring cells comprising greater than 50% of the entire tumour, and thus, by definition, are poorly differentiated (ie, grade 3). A signet-ring cell is morphologically characterised by a large mucin-filled cytoplasmic vacuole that pushes its nucleus to the periphery, thereby creating a ‘signet-ring’-like appearance (figure 5A). Unlike conventional adenocarcinomas, SRCs tend not to form exophytic mass lesions, and instead are notable for their predominantly infiltrative concentric growth patterns leading to diffuse thickening of the colorectal wall.88 Histologically, the malignant cells infiltrate the colonic wall as discohesive sheets and clusters (figure 5B), and may be associated with mucin pools. Tumours with less than 50% signet-ring cell component are not technically SRCs, but should be reported as having signet-ring cell features14; signet-ring cells may be seen in association with adenocarcinoma with mucinous features. Compared with conventional counterparts, SRC are more frequently associated with BRAF mutations, CIMP status, and are MSI-H(19–48%).86 Furthermore, when SRC were grouped by methylation status, the hypermethylated group were associated with proximal location, high CD3-positive lymphoid infiltrate, and PDL1 expression, along with BRAF V600E mutation, CIMP, and MSI, whereas the hypomethylated tumours were commonly in the distal colon without significant molecular associations.89

• Download figure
• Open in new tab
• Download powerpoint

Figure 5

(A) Signet-ring cell carcinoma, signet-ring cells. (B) Signet-ring cell carcinoma, infiltrating sheets and clusters.

Prognosis and meaning

SRC are notoriously associated with worse clinical outcomes with poorer survival compared with conventional adenocarcinomas.66 81 However, proximal location have been shown to be an independently favourable prognostic factor, perhaps associated with the aforementioned hypermethylated genotype that may benefit from immune checkpoint inhibitor targeted therapy.89 90 Of note, studies have shown that signet-ring cells of any proportion, even existing as a minor component of MADs, are linked to poorer prognosis.91 92 This raises the question on the actual prognostic significance of the currently recommended 50% numerical cut-off for diagnosing SRC, and thus, pathologists should report any signet-ring cell component regardless of proportion to guide clinical management.

Background

Adenosquamous carcinoma (ASC) is an extremely rare subtype, reported to represent less than 0.1% of all CRCs.93 Recognition of ASC dates back to 1907 when Herxheimer94 first described this entity in the cecum.95 Previously classified as an independent entity, is now adopted as a formal subtype of CRC in the most recent WHO classification.14 96 Of note, pure colorectal squamous cell carcinoma (SCC) gained attention since its first introduction in 1919 by Schmidtmann.95 97 While their exact pathogenesis is unclear, some hypothesise colonic SCCs were at one point ASCs, and with progression only the squamous component remained.98 Specifically in the rectum however, primary rectal SCCs can be difficult to definitively distinguish from anal SCCs with rectal involvement. Nonetheless, colorectal SCC has been removed from the most recent WHO classification,14 96 likely due to extreme rarity and limited data to establish meaningful prognostic implications.

Clinical characteristics

ASC are more commonly right-sided lesions, are associated with poor tumour differentiation, and tend to present with higher rates of advanced disease.93 95 Human papilloma virus infection does not appear to play a role in the pathogenesis of ASC, unlike tumours with squamous differentiation of other sites (eg, oropharyngeal, cervical, anal).99 100

Histologic and molecular features

The histologic features of colorectal ASC are similar to ASC more frequently encountered in the oesophagus and stomach.14 This specific subtype is defined by having two distinct or admixed populations of glandular and squamous components resembling those of conventional adenocarcinoma and SCC, respectively. There is no specifically required percentage cut-off for each histologic component.14 96 IHC stains, though not necessary for diagnosis, may be useful in demonstrating the two components especially in the setting of non-keratinising squamous components. Cytokeratin 20 and CDX2 will highlight the glandular component whereas p40, p63, cytokeratin 34βE12, and cyclin D1 will highlight areas of squamous differentiation.101 In a molecular study including 22 cases of ASC, this subtype seems to harbour driver mutations similar to those we are already familiar with in conventional adenocarcinomas, including TP53, APC, KRAS, and BRAF.102 Only rare cases were dMMR.101 102 Of note, a rare but important histologic mimic of ASC is endometrioid adenocarcinoma with squamous differentiation involving the colorectum (ie, in the setting of endometriosis). While it is a potential diagnostic pitfall by morphology, its distinction from ASC becomes less challenging with the use IHCs as endometrioid adenocarcinomas are usually CK7-positive, CK20-negative, and immunoreactive for Mullerian markers (ie, PAX-8), whereas the glandular component in ASC are more commonly CK7-negative in addition to the aforementioned immunophenotype.103

Prognosis and meaning

Based on the currently available literature, the presence of squamous features in colorectal adenocarcinoma portends a worse prognosis. In one of the largest study93 to date including 99 cases of colorectal ASC, this subtype was associated with an approximately 3-times higher incidence of metastatic disease, significantly lower overall survival time, and increased overall and colorectal-specific mortality, compared with adenocarcinoma. Though extremely rare, it is important for pathologists to adequately sample resection specimens to identify and report any component of squamous differentiation, given its aggressive clinical behaviour. Additional molecular studies are warranted on larger cohorts to explore therapeutically targetable alterations.