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Immunohistochemistry and special stains in gastrointestinal pathology practice
MINI-SYMPOSIUM: GASTROINTESTINAL/HEPATO-PANCREATO-BILIARY PATHOLOGY
Immunohistochemistry and special stains in gastrointestinal pathology practice
This review will detail the ways in which IHC and special stains are useful in daily practice. It is beyond the scope of this review to detail every special stain, so we have chosen to concentrate on stains which are of particular importance to the gastrointestinal tract or require special attention in their interpretation. We will also consider stains that are potentially underutilized. Stains which are straightforward to interpret (e.g. CD117 in gastrointestinal stromal tumour, HepPar-1 in hepatocellular carcinoma) or serve a similar purpose across organ systems (e.g. haematolymphoid markers in lymphoma subtyping, chromogranin and synaptophysin in neuroendocrine tumours) will not be further discussed. There are two methods of presenting IHC and special stain information, entity-based or stain-based. Both have their advantages and disadvantages. Since an entity-based approach can be found in standard surgical pathology texts, a stain-based approach will be used in this review. We will further subdivide this into three broad categories, stains used in neoplastic processes, stains used in non-neoplastic processes, and stains used in tissue depositions.
Cheng Liu Masoumeh Ghayouri Ian S Brown
Abstract Immunohistochemistry and special stains play an increasingly important role in gastrointestinal pathology practice. In neoplastic disorders they are used to confirm the diagnosis, identify prognostic/predictive features, and screen for an underlying genetic syndrome. In nonneoplastic disorders they can identify an infectious organism, clarify the inflammatory infiltrate present, and confirm a tissue deposition. In this review we discuss the most important and topical of these stains, especially ones which require special care in interpretation. It should be emphasized that, in cases with equivocal or unexpected staining patterns, the results should be interpreted in the appropriate clinical, endoscopic and morphologic context.
Neoplastic processes Increasingly IHC and special stains are being applied to neoplastic processes, and virtually every gastrointestinal tract neoplasm is subject to some type of additional stain. Table 1 summarises the stains we find the most useful, and a selected number will be discussed in detail.
Keywords amyloid; cytomegalovirus; gastrointestinal pathology; Helicobacter pylori; HER2; immunohistochemistry; mismatch repair; mucin; PD-L1; SATB2; special stain
MLH1, PMS2, MSH2 and MSH6 Mismatch repair (MMR) is a mechanism of maintaining DNA integrity during cell division, where incorrectly matched base pairs are excised and repaired. This is performed by the MMR proteins MLH1, PMS2, MSH2 and MSH6. Loss of MMR protein function leads to progressive accumulation of errors throughout the genome, which are accentuated at microsatellites. Microsatellites are small repetitive DNA sequences most sensitive to replication error, and involvement of several predefined loci are referred to as microsatellite instability (MSI). Thus, detection of MMR deficiency can proceed via two ways, by detecting MMR protein loss via IHC, or MSI by DNA sequencing-based methods. Both methods have similar sensitivities and specificities.1 MMR IHC detects both inherited (Lynch syndrome-associated) and sporadic (MLH1-methylated) MSI colorectal carcinomas. Lynch syndrome accounts for 3e4% of all colorectal carcinomas.2 Although there is no universal agreement as to which colorectal carcinomas should be screened for Lynch syndrome, a growing number of international organisations have recommended to screen all tumours, or at least tumours in patients less than 70 years of age.3 In contrast, approximately 12% of colorectal carcinomas arise secondary to methylation-related silencing of MLH1. In both situations, microsatellite instability is associated with overall more favourable prognosis.4 Recently an additional benefit of MMR IHC has been the prediction of response to immune checkpoint inhibitor therapy. Carcinomas with MMR deficiency show improved response to PD-L1 blockade.5 With the approval of pembrolizumab for all MMRdeficient tumours by the United States Food and Drug
Introduction Immunohistochemistry (IHC) and special stains are indispensable adjuncts in the reporting of gastrointestinal tract specimens, whether these are procured by endoscopic biopsy or by surgical resection. In neoplastic disorders they may confirm a neoplastic process and/or provide clinically important information. The latter includes the identification of the site of origin of the neoplasm, prognostic parameters, prediction of response to therapy, or potentially identify an underlying genetic syndrome. Some stains serve more than one purpose, depending on clinical context. In non-neoplastic disorders IHC and special stains help to classify an inflammatory reaction pattern or identify an infectious aetiology. Both IHC and special stains may also help determine the nature of a tissue deposition.
Cheng Liu BMedSci MBBS FRCPA Envoi Specialist Pathologists, Brisbane, Faculty of Medicine, University of Queensland, Brisbane and The Conjoint Gastroenterology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. Conflicts of interest: none declared. Masoumeh Ghayouri MD Department of Pathology, H. Lee Moffitt Cancer Center, Tampa, FL, USA. Conflicts of interest: none declared. Ian S Brown BGEN MBBS FRCPA Envoi Specialist Pathologists, Brisbane and Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia. Conflicts of interest: none declared.
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Please cite this article as: Liu C et al., Immunohistochemistry and special stains in gastrointestinal pathology practice, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.10.010
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colorectal cancer cases demonstrating PMS2 loss (overwhelmingly due to MLH1 methylation),3 and it prevents falsely interpreting weak MSH6 expression as normal in patients with MSH2 germline mutation.9 The interpretation of MMR IHC is usually straightforward, where a tumour will show uniform retained staining or loss of staining of an MMR protein throughout, with retained staining in internal control cells. The terminology used in reporting MMR IHC must be precise, since a “positive” abnormal result is due to loss of staining, or “negative” staining. Thus, a statement such as “MLH1 IHC is negative” is ambiguous. Instead we recommend using “retained” or “loss of” staining, followed by an interpretation, such as “MLH1 shows loss of staining, which is an abnormal result”.3 Sometimes interpretation is challenging when there is weak/ heterogeneous staining or equivocal internal control staining. For example, in surgical resections where there may be areas of poor fixation or tumour hypoxia, up to 10% of the tumour cells may exhibit equivocal weak expression for MMR markers so long as other areas of the tumour show uniform strong expression.10 In tumours with MLH1 methylation, there may be variability in the intensity of expression of both MLH1 and PMS2, with areas of weak expression or loss of expression.11 The minimum cut off for an abnormal result is not well established. Even though it is sometimes recommended that any tumour staining which is at least as strong as background internal control be regarded as retained staining, we believe this scope is too narrow, and all cases with difficult-to-interpret or clearly heterogeneous staining should be flagged as possibly representing Lynch syndrome.12 Table 2 details the common patterns of staining identified with MMR markers and their clinical significance, and Figure 1 illustrates selected scenarios. Approximately 80% of colorectal carcinomas exhibiting MLH1 loss by IHC arise from sessile serrated lesions (SSLs). While SSLs are very common, SSLs with dysplasia are rare and capable of progressing to carcinoma rapidly. Dysplasia in SSLs is morphologically heterogeneous and can be very subtle. However, as 75% of SSLs with dysplasia demonstrate MLH1 loss, loss of staining is very helpful in confirming a diagnosis of dysplasia.13 In contrast, performing MMR IHC in conventional adenomas is of limited value. Lynch syndrome-associated conventional adenomas show loss of MMR protein staining in approximately 80% of cases (more likely if large, with a villous component and/or high-grade dysplasia).14 However, as non-syndromic conventional adenomas are much more common in practice, routine staining of all conventional adenomas is not an effective screening strategy.
Commonly used IHC and special stains in the gastrointestinal tract Examples Tumour subtyping within gastrointestinal tract
Neuroendocrine differentiation: chromogranin, synaptophysin, CD56 Adenocarcinoma vs. squamous cell carcinoma: CK7, CK20, p63, p40, mucin stains (e.g. PAS-D, mucicarmine) Subtyping of gastric adenocarcinoma: EBV (in situ hybridization), E-cadherin, MMR stains, p53 Prognostication and grading Neuroendocrine tumours: ki67 Colorectal carcinoma: MMR stains Vascular invasion/serosal involvement: elastic stains (e.g. Verhoeff-van Gieson, orcein, Movat) Predict response to therapy Gastro-oesophageal and gastric carcinoma: HER2 Colorectal carcinoma: MMR stains Multiple malignancies: PD-L1 Diagnosis of dysplasia IBD-related dysplasia: SATB2, p53, b-catenin Typing of dysplasia/ Pancreatic IPMN and associated adenocarcinoma carcinoma: intestinal (MUC2, CDX2), gastric (MUC5AC, MUC6), pancreaticobiliary (MUC1) Identifying an inherited tumour Lynch syndrome: MMR stains predisposition syndrome Carney-Stratakis syndrome: SDHA, SDHB Identifying primary site in poorly Multiple possibilities: CK7, CK20, differentiated malignancies TTF1, CDX2, SATB2, GATA3, PAX8, ER, calretinin, CK5/6, S100, SOX10, CD3, CD20 EBV, Epsten-Barr virus; MMR, mismatch repair; IBD, inflammatory bowel disease; IPMN, intraductal papillary mucinous neoplasm.
Table 1
Administration (FDA), it is increasingly likely that all gastrointestinal tract carcinomas will require routine MMR IHC in the future. IHC directed against the MMR proteins are widely available, and whether the stains are performed on endoscopic biopsy material or surgical resection material is a matter for individual laboratories. Our preference is for endoscopic biopsy material if available, because the stains are less likely to be affected by fixation artefact,6 a subsequent surgical resection may not be processed at the same laboratory, and neoadjuvant therapy may alter MMR IHC result.7 There is good evidence for strong concordance between the findings in biopsy material and resection tissue.8 Our preference is to stain for all four proteins upfront, as this avoids a second round of IHC in the 15e20% of
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SATB2 SATB2 is a protein with preferential expression in the lower gastrointestinal tract. Although its expression largely parallels CDX2, SATB2 is more site-specific and is less prone to methylation-related silencing than CDX2, making it more useful in mucinous, signet ring and undifferentiated colorectal carcinomas.15 SATB2 is highly sensitive and specific for lower gastrointestinal tract origin of a neuroendocrine tumour, particularly for well-differentiated neoplasms.16 Neuroendocrine carcinoma of the lower gastrointestinal tract demonstrates similar high expression, however metastasis from a non-gastrointestinal
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Please cite this article as: Liu C et al., Immunohistochemistry and special stains in gastrointestinal pathology practice, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.10.010
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Common MMR patterns and their significance Pattern of expression
Percentage
Probability of Lynch syndrome
Significance/further testing
MLH1, MSH2, MSH6 and PMS2 preserved
85%
Very unlikely
MLH1 and PMS2 loss
15%
Unlikely (sporadic in 80% of cases)
MSH2 and MSH6 loss MSH6 loss
<1% <1%
Likely Likely
PMS2 loss
<1%
Likely
Normal pattern; no further testing unless there is a strong clinical suspicious of Lynch syndrome BRAF mutation or MLH1 promoter methylation testing to confirm sporadic nature; if both absent, investigate for Lynch syndrome MSH2, followed by MSH6 germline testing MSH6, followed by MSH2 germline testing; may occur secondary to neoadjuvant therapy in rectal carcinoma PMS2, followed by MLH1 germline testing
Table 2
site needs to be excluded.16 SATB2 also allows for separation of primary small intestinal adenocarcinoma from colorectal carcinoma that has spread to the small intestine. While most primary colorectal carcinomas express this protein, less than half of primary small intestinal adenocarcinomas do. Furthermore, unlike the strong and diffuse staining pattern in CRC, the expression of SATB2 is weaker in small intestinal adenocarcinoma.17 The diagnostic uses of SATB2 continue to expand and there are recent reports of its potential role in diagnosing inflammatory bowel disease (IBD)-associated dysplasia and carcinoma. Loss of SATB2 staining is seen in 40% of cases of IBD related dysplasia, while it is retained in reactive atypia and conventional adenomas.18 This first allows for establishing a diagnosis of dysplasia versus reactive atypia, and secondly it separates IBDrelated dysplasia from a conventional adenoma occurring in a patient with IBD. Interestingly, SATB2 is also frequently lost in IBD-associated carcinomas, but is retained in most sporadic carcinomas.19 Figure 2 shows an example of IBD-related dysplasia with loss of SATB2 staining.
diagnosis of dysplasia where morphology is equivocal.20 In general, a dysplastic lesion should exhibit higher ki67 index, increased p53 staining towards the luminal aspect, and aberrant (cytoplasmic or nuclear) b-catenin expression, when compared with a reactive process. However, these stains are variably positive in reactive processes, especially when associated with inflammation or regeneration, and different studies have used different scoring systems and cut off thresholds for positivity. Also, the aberrant expression of b-catenin is restricted to lesions with WNT pathway activation, generally intestinal-type adenomas. The value of these stains is not an absolute cut off threshold but rather in comparing with background mucosa. If the lesion shows an abrupt difference in staining from the background, this is supportive of a dysplastic interpretation. b-catenin IHC has additional utility in unequivocally dysplastic lesions. Until recently it was used with p53 to separate IBD-related dysplasia from conventional adenoma, where IBDrelated dysplasia is usually negative for b-catenin and diffusely positive for p53, while conventional adenoma shows the opposite pattern.21 The potential of SATB2 in this setting has been discussed above. Since the management of dysplastic lesions in IBD is now dependent more on endoscopic appearance and resectability rather than aetiology, there is less need to separate IBD-related dysplasia from conventional adenoma.
ki67, p53 and b-catenin These three stains are discussed together due to their similar utility and interpretation. Within the tubular gastrointestinal tract, b-catenin, p53 and ki67 have all been used to support a
Figure 1 Mismatch repair protein immunohistochemistry. There is loss of expression of MLH1, PMS2 and MSH6 in this colorectal carcinoma. MSH2 is preserved (not shown). There is normal expression of the markers in the background lymphocytes and non-neoplastic epithelial cells. This abnormal pattern of expression is usually the result of sporadic methylation-induced silencing of MLH1 gene expression with secondary loss of PMS2 and MSH6.
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Figure 2 High-grade dysplasia and invasive adenocarcinoma developing in a colonic inflammatory polyp in a patient with long standing ulcerative colitis. There is strong nuclear expression of p53 in the dysplastic cells; in contrast, SATB2 expression is lost.
downregulates the immune response, and may lead to apoptosis of the T cell. In a similar vein, by expressing PD-L1, tumour cells mimic normal cells and escape destruction by cytotoxic T cells. The tumour cells are thus allowed to persist and proliferate. AntiPD-L1 therapy such as pembrolizumab “unmasks” tumour cells from the immune system and reverses the inhibitory effect of PDL1. IHC PD-L1 testing is most established in lung adenocarcinoma, and it is now being applied to an increasing number of tumours from diverse organ systems. However, accurate interpretation of PD-L1 IHC is complicated by several factors.26 Multiple antibody clones are available, and staining is usually heterogeneous. Different scoring systems are used for different tumours, with some also including tumour-infiltrating lymphocytes. As expected, PD-L1 positive tumours are associated with poor prognostic features,27 regardless of scoring system used, and response to PD-1/PD-L1 blockade correlates with degree of IHC PD-L1 positivity.28 In the gastrointestinal tract, PD-L1 IHC testing is driven by availability of pembrolizumab. The United States FDA data sheet for pembrolizumab includes oesophageal, gastric, hepatocellular and colorectal carcinomas, but other tumours are likely to become eligible in the future.
MUC1, MUC2, MUC5AC and MUC6 Mucins are high-molecular weight glycoproteins produced by epithelial cells, which have both gel-forming and signal transduction roles. Broadly the pattern of mucin expression corresponds to site-specific epithelial components of the gastrointestinal tract. For instance, pancreaticobiliary epithelium expresses MUC1, intestinal epithelium expresses MUC2, gastric foveolar epithelium expresses MUC5AC and gastric pyloric epithelium expresses MUC6. Mucin IHC can therefore be applied in the subclassification of pancreatic intraductal papillary mucinous neoplasm (IPMN)-derived carcinomas into pancreaticobiliary (MUC1), intestinal (MUC2) and gastric (MUC5AC and MUC6) phenotypes, which have differing prognoses.22 It should be emphasized, however, that the phenotype of these lesions can usually be determined via morphologic means and mucin stains have a supplementary role only. Table 3 summarises the morphologic and IHC features of these phenotypes. MUC1 and MUC2 staining may be useful in establishing a pancreaticobiliary or intestinal phenotype in neoplasms occurring at the duodenal ampulla. This is generally done in combination with CK20 and CDX2, and may guide further treatment of these tumours.23 Unfortunately, the stains sometimes do not establish a clear line of differentiation.24 Mucin stains may also be useful in separating gastric phenotype dysplasia from intestinal phenotype dysplasia of the upper gastrointestinal tract. Gastric pattern dysplasia is associated with different clinicopathological features.25 However, as with the pancreatic IPMN, the clinical utility of performing these stains routinely is yet to be firmly established. In addition, many examples of glandular dysplasia in the lower oesophagus display a hybrid pattern of mucin expression consistent with both gastric and intestinal differentiation.
HER2 HER2 is a member of the epidermal growth factor receptor family, where overexpression leads to activation of proliferation pathways in the absence of an extracellular ligand. It is overexpressed in approximately 20% of gastro-oesophageal junction and gastric adenocarcinomas, and treatment with trastuzumab increases survival.29 Although different antibody clones are available, the results are comparable between clones, and the interpretation methodology is standardized (see Table 4). Of note, interpretation differs from breast cancer in several respects. Because gastro-oesophageal junction and gastric cancers are commonly advanced (i.e. inoperable) at presentation, the only tissue available for testing may be an endoscopic biopsy specimen. To account for possible intratumoural heterogeneity in this setting, the cut off for an equivocal or positive result is much lower in biopsy compared with excision specimens. Complete circumferential membranous staining is also not required.
PD-1 and PD-L1 The PD-1/PD-L1 pathway allows cytotoxic T cells to selectively remove abnormal cells while leaving normal cells intact. To avoid damage, normal cells express the transmembrane protein PD-L1, which is detected by its receptor PD-1 on the cytotoxic T cell. When a cytotoxic T cell encounters a PD-L1-expressing normal cell, the PD-L1/PD-1 interaction inactivates the T cell,
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Morphologic features
MUC2
MUC5AC
MUC6
MUC1
Pancreaticobiliary
Columnar cells Round, uniform, basally located nuclei Abundant apical mucinous cytoplasm Columnar cells Hyperchromatic, pencillate, stratified nuclei Dense eosinophilic cytoplasm Cuboidal to columnar cells with pale clear to light eosinophilic cytoplasm Hyperchromatic round to oval nuclei Prominent nucleoli if high grade Closely packed tubules lined by cuboidal to columnar epithelium with pale to eosinophilic ground glass cytoplasm Round basal nuclei Nucleoli easily visible Cytological features intermediate between the above patterns, or an intimate admixture of both
e
e
e
þ
þ
e
e
e
þ
rare
e
e
þ (surface)
þ
e
Intestinal
Gastric foveolar
Gastric pyloric gland
Hybrid
Table 3
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Phenotype
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IPMN phenotypes as determined via mucin stains
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Interpretation of HER2 staining in the gastrointestinal tract HER2 score
Biopsy staining
Excision staining
Interpretation
0
No reactivity or no membranous reactivity in any cell 5 cells with a faint or barely perceptible membranous reactivity irrespective of percentage of cells positive 5 cells with a weak to moderate complete, basolateral, or lateral membranous reactivity irrespective of percentage of cells positive 5 cells with a strong complete basolateral, or lateral membranous reactivity irrespective of percentage of cells positive
No reactivity or membranous reactivity in <10% of cells Faint or barely perceptible membranous reactivity in 10% of cells; cells are reactive only in part of their membrane Weak to moderate complete, basolateral or lateral membranous reactivity in 10% of cells
Negative
Strong complete, basolateral or lateral membranous reactivity in 10% of cells
Positive
1þ
2þ
3þ
Negative
Equivocal
Table 4
of an SDHA mutation, but IHC is not available for SDHC and SDHD mutations. An example of a “positive” result (i.e. loss of staining) is shown in Figure 3.
SDHA and SDHB The succinate dehydrogenase (SDH) complex is a mitochondriaassociated enzyme complex involved in oxidative phosphorylation, comprising four subunits SDHA, SDHB, SDHC and SDHD. Mutation in the subunits result in Carney-Stratakis syndrome (gastric gastrointestinal stromal tumours [GISTs] and paragangliomas), and methylation-induced silencing of SDHC results in Carney triad (gastric GISTs, paragangliomas and pulmonary chondromas). Gastric GISTs that arise in the context of SDH mutation have distinct pathologic features.30 They grow as multinodular sheets which dissect the muscularis propria, display epithelioid cytology, and have a tendency towards lymph node metastases. However, they have an indolent course, so the prognostic algorithm used for usual GISTs does not apply. The interpretation of SDH IHC parallels that for MMR proteins. As mutations in any subunit results in loss of SDHB, SDHB IHC is an effective screening test.31 A normal result is granular cytoplasmic (i.e. mitochondrial) staining in tumour cells. Loss of staining is an abnormal result, which must be interpreted with an appropriately staining internal control such as endothelial cells. In the context of SDHB loss, SDHA IHC allows for identification
Elastic stains Venous invasion is an independent poor prognostic feature in malignant epithelial tumours of the gastrointestinal tract. However, its presence is underreported by pathologists. This is particularly problematic in colorectal carcinoma where it is expected that venous invasion should be present in at least 30% of resected tumours.32 Histological features suspicious for venous invasion include the “orphan (or unaccompanied) arteriole” sign, a well-circumscribed tumour nodule adjacent to a thickwalled artery without an accompanying vein; and the “protruding tongue” sign, a circumscribed protrusion of tumour into the pericolic fat. In these situations, at least, elastic tissue stains may demonstrate a residual vein wall, an example of which is shown in Figure 4. An elastin stain is also useful in confirming the presence of venous invasion in cases originally deemed equivocal on haematoxylin and eosin (H&E)-stained sections. Some have gone further and advocate routine use of elastin stains to increase venous invasion detection. It has been shown that use of
Figure 3 SDHB-deficient gastric gastrointestinal stromal tumour. Note loss of immunohistochemical SDHB expression in the tumour cells with preserved internal control staining of endothelial cells. Retained internal control expression is required to ensure the stain has worked correctly. (Photographs courtesy of Professor Anthony Gill, Royal North Shore Hospital, Sydney).
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Please cite this article as: Liu C et al., Immunohistochemistry and special stains in gastrointestinal pathology practice, Diagnostic Histopathology, https://doi.org/10.1016/j.mpdhp.2019.10.010
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Figure 4 Orcein stain demonstrating the residual elastic lamina of a vein that has been invaded by colorectal carcinoma. The "orphan artery" sign is apparent on the haematoxylin and eosin-stained section.
routine elastin stains is associated with a doubling of the venous invasion detection rate.33
There are however situations where stains should be performed if organisms are not identified in the H&E stained sections.35 These indications are listed in Box 1. H. pylori IHC may also be useful when there has been proton pump inhibitor therapy or incomplete antibiotic treatment,36 both of which lead to a reduction in the number of organisms. This is often accompanied by a loss or marked reduction in active inflammation. The organisms may be found only in the deep aspect of glands of the specialized mucosa, often closely applied to the canaliculi of parietal cells (see Figure 5).
Non-neoplastic processes Most stains performed in nonneoplastic processes are for characterizing an inflammatory infiltrate, or to identify an infectious organism. Table 5 summarises the stains we find the most useful, and a selected number will be discussed in detail. Helicobacter pylori stains The most common reason for performing a special stain in inflamed gastrointestinal tract tissue is to look for an infective cause, and Helicobacter pylori (H. pylori)-associated gastric inflammation represents the best example of this. While a variety of histochemical stains successfully highlight H. pylori (e.g. Wright-Giemsa, toluidine blue, Warthin-Starry), none of these have sensitivity or specificity that approaches the IHC, and this has become the gold standard test. While some laboratories advocate the routine use of stains to look for these organisms, there seems little point in doing this when the organism is readily identifiable on H&E in most cases.34
Cytomegalovirus immunohistochemistry Cytomegalovirus (CMV) is a herpesvirus which latently infects approximately 80% of the world’s population. In immunocompetent individuals the virus is dormant, but reactivation occurs in immune dysregulation, such as with steroid use, human immunodeficiency virus infection, bone marrow suppression, and old age.37 In the gastrointestinal tract any organ can be affected, manifesting as cytomegaly and characteristic “owl’s eye” nuclear inclusions. Infection preferentially affects stromal cells and
Common stains used in nonneoplastic processes Purpose
Example stains
Identifying infective organism
Bacteria: IHC or Giemsa for H. pylori, IHC or Warthin-Starry for T. pallidum Mycobacteria: Ziehl-Neelsen, Wade-Fite Fungi: PAS-D, Grocott Viral: IHC for HSV, IHC CMV, p16 for HPV Collagenous gastritis/sprue/colitis: trichrome, Verhoeff-van Gieson Lymphocytic gastritis/duodenitis/colitis: CD3 IgG4-related sclerosing disease: IgG, IgG4 Type 1: normal CD3 and CD8 in T cells Type 2: aberrant CD3 and CD8 in T cells
Confirm a specific inflammatory pattern
Plasma cell-rich fibrosing process Refractory coeliac disease
IHC, immunohistochemistry; HSV, herpes simplex virus; CMV, cytomegalovirus; HPV, human papillomavirus.
Table 5
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prominence, and a positive result in this case will always highlight more infected cells than appreciable on H&E. On the other hand, indiscriminate application of CMV IHC will detect latently infected cells, which may lead to an erroneous assignment of aetiology.39 Lastly, pigment-laden macrophages may simulate CMV-infected cells on IHC, due to their size and haemosiderin content.
Indications for H. pylori stains Acute gastritis and/or gastric ulceration (unless clearly reactive gastropathy associated) Active chronic gastritis Focally enhanced gastritis Moderate or severe chronic gastritis Autoimmune gastritis (H. pylori infection may be a precursor) Lymphocytic gastritis (organisms may be very sparse) Marginal zone lymphoma (MALT type) Previous H. pylori (to confirm eradication) Positive urease test without organisms seen in H&E stained sections Gastric intestinal metaplasia Gastric adenocarcinoma Duodenal ulceration
CD3 and CD8 Refractory coeliac disease is defined as symptoms not responding after 12 months on a gluten-free diet, or symptom recurrence while on a gluten-free diet.40 Most cases prove to represent an insufficient gluten-free diet or a slow healing response. Rarely, the persisting or recurring disease represents the development of a monoclonal T cell proliferation, which is initially confined to the epithelium but is at high risk of progression to enteropathy-associated T-cell lymphoma. Separating these two scenarios is clinically important, and CD3 and CD8 are helpful in this context.41 Normally at least 90% of CD3 þ intraepithelial T cells co-express CD8, and this is maintained in the first scenario (refractory coeliac disease type 1). Loss of CD3 and CD8 co-expression in greater than 50% of the intraepithelial T cells is a worrisome finding and corresponds to the second scenario (refractory coeliac disease type 2); an example of this is shown in Figure 6. Refractory coeliac disease type 2 is a difficult diagnosis and requires synthesis of clinical, H&E, IHC and flow cytometric findings; no feature should be considered diagnostic in isolation.
MALT, mucosa-associated lymphoid tissue; H&E, haematoxylin and eosin.
Box 1
endothelial cells, with epithelial cells being involved only in severe cases. This is typically associated with active chronic inflammation, and inflammation can be so pronounced as to obscure the diagnostic cytologic findings. CMV IHC aids in identifying the virus-infected cells in cases with a high index of suspicion.38 It is of greatest utility when the inclusions are equivocal and other entities enter the differential, chiefly inflammation/regeneration-associated nucleolar
IgG and IgG4 IgG4-related sclerosing disease has been recognized in almost all organ systems since its consensus diagnostic criteria were published in 2012.42 The three cardinal features are (1) IgG4 þ
Figure 5 Gastric biopsies in a patient taking a proton pump inhibitor. Mild active chronic inflammation is present in the gastric body. The gastric antrum is normal. H. pylori immunohistochemical stain demonstrates organisms restricted to the gastric body and closely applied to the parietal cell canaliculi.
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Figure 6 Refractory coeliac disease type 2 with loss of CD8 expression in >50% of CD3þ intraepithelial T cells.
plasma cells comprising at least 40% of all IgG þ plasma cells; (2) storiform fibrosis; and (3) obliterative phlebitis. At least two out of three features are required for diagnosis. In addition to the 40% minimum, an absolute number of IgG4 þ plasma cells is also required (expressed per high power field), which differs between organs. Recognition of the disorder is important as it respond well to steroid treatment.43 In the gastrointestinal tract the main organs involved are the pancreas and biliary tract. If not considered, the prominent fibroinflammatory reaction may be mistaken for chronic pancreatitis, primary sclerosing cholangitis, pancreatic ductal adenocarcinoma, or cholangiocarcinoma.
classification into either light chain (AL) or inflammationassociated (AA) amyloid, the two most common subtypes. IHC is available for a wider range of amyloid types, including AL, AA, transthyretin and b2-microglobulin.44 However, the efficacy of these stains is laboratory dependent, and nonspecific/equivocal staining is common; precise subtyping is better performed via mass spectrometry. Because gastrointestinal involvement by amyloidosis presents with nonspecific symptoms such as diarrhoea, constipation and abdominal pain,45 subtle deposits are easily missed. The deposits begin in the submucosal vessel walls, which can be mistaken for collagen or fibrin, and subsequently involve the lamina propria and submucosa proper. In our opinion, it is more important to recognize amyloidosis and recommend further investigation for a systemic cause, rather than to subtype the amyloid.
Assessment of tissue deposition Most tissue deposits are readily recognisable without IHC or special stains. Table 6 lists cases where stains may be of use.
Conclusions
Amyloid stains Amyloid represents abnormally aggregated protein which becomes deposited in extracellular sites as amorphous eosinophilic material. Histochemical stains such as Congo red and crystal violet can confirm the presence of amyloid, and pre-treatment of sections by potassium permanganate further allows sub-
It is sometimes challenging to keep abreast of available IHC and special stains in gastrointestinal pathology, and we have attempted to summarize the most topical and/or useful stains in this review. It should be emphasized, however, that stains should only be ordered to answer a specific clinical question, and the results interpreted along with the H&E findings. A REFERENCES 1 Lindor NM, Burgart LJ, Leontovich O, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 2002; 20: 1043e8. 2 Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer 2015; 15: 181e94. 3 Yozu M, Kumarasinghe MP, Brown IS, Gill AJ, Rosty C. Australasian Gastrointestinal Pathology Society (AGPS) consensus guidelines for universal defective mismatch repair testing in colorectal carcinoma. Pathology 2019; 51: 233e9. 4 Ward R, Meagher A, Tomlinson I, et al. Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut 2001; 48: 821e9. 5 Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017; 357: 409e13. 6 O’Brien O, Ryan E, Creavin B, et al. Correlation of immunohistochemical mismatch repair protein status between colorectal
Tissue deposition and their staining characteristics Tissue deposition
Example stains
Brown depositions
Iron and pseudomelanosis: Perls positive Melanosis and lanthanum: Perls negative Amyloid: Congo red, crystal violet Collagen: trichrome Elastin: Verhoeff-van Gieson, orcein, Movat Medication capsule: PAS Calcium/psammoma bodies, Osmoprep: von Kossa positive SIRT spheres: von Kossa negative
Eosinophilic depositions
Basophilic depositions
SIRT, selective internal radiation therapy.
Table 6
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carcinoma endoscopic biopsy and resection specimens. J Clin Pathol 2018; 71: 631e6. Bao F, Panarelli NC, Rennert H, Sherr DL, Yantiss RK. Neoadjuvant therapy induces loss of MSH6 expression in colorectal carcinoma. Am J Surg Pathol 2010; 34: 1798e804. Shia J, Stadler Z, Weiser MR, et al. Immunohistochemical staining for DNA mismatch repair proteins in intestinal tract carcinoma: how reliable are biopsy samples? Am J Surg Pathol 2011; 35: 447e54. Pearlman R, Markow M, Knight D, et al. Two-stain immunohistochemical screening for Lynch syndrome in colorectal cancer may fail to detect mismatch repair deficiency. Mod Pathol 2018; 31: 1891e900. Pai RK, Pai RK. A practical approach to the evaluation of gastrointestinal tract carcinomas for Lynch syndrome. Am J Surg Pathol 2016; 40: e17e34. Joost P, Veurink N, Holck S, et al. Heterogenous mismatch-repair status in colorectal cancer. Diagn Pathol 2014; 9: 126. Sarode VR, Robinson L. Screening for Lynch syndrome by immunohistochemistry of mismatch repair proteins: significance of indeterminate result and correlation with mutational studies. Arch Pathol Lab Med 2019; 143: 1225e33. Liu C, Walker NI, Leggett BA, Whitehall VL, Bettington ML, Rosty C. Sessile serrated adenomas with dysplasia: morphological patterns and correlations with MLH1 immunohistochemistry. Mod Pathol 2017; 30: 1728e38. Walsh MD, Buchanan DD, Pearson SA, et al. Immunohistochemical testing of conventional adenomas for loss of expression of mismatch repair proteins in Lynch syndrome mutation carriers: a case series from the Australasian site of the colon cancer family registry. Mod Pathol 2012; 25: 722e30. Ma C, Lowenthal BM, Pai RK. SATB2 is superior to CDX2 in distinguishing signet ring cell carcinoma of the upper gastrointestinal tract and lower gastrointestinal tract. Am J Surg Pathol 2018; 42: 1715e22. Bellizzi AM. SATB2 in neuroendocrine neoplasms: strong expression is restricted to well-differentiated tumors of lower gastrointestinal tract origin and is more frequent in merkel cell carcinoma among poorly differentiated carcinomas. Histopathology, 2019; https://doi.org/10.1111/his.13943 [Epub ahead of print]. Kim CJ, Baruch-Oren T, Lin F, Fan XS, Yang XJ, Wang HL. Value of SATB2 immunostaining in the distinction between small intestinal and colorectal adenocarcinomas. J Clin Pathol 2016; 69: 1046e50. Ma C, Henn P, Miller C, Herbst C, Hartman DJ, Pai RK. Loss of SATB2 expression is a biomarker of inflammatory bowel diseaseassociated colorectal dysplasia and adenocarcinoma. Am J Surg Pathol 2019; 43: 1314e22. Iwaya M, Ota H, Tateishi Y, Nakajima T, Riddell R, Conner JR. Colitis-associated colorectal adenocarcinomas are frequently associated with non-intestinal mucin profiles and loss of SATB2 expression. Mod Pathol 2019; 32: 884e92. Ma C, Pai RK. Predictive value of immunohistochemistry in premalignant lesions of the gastrointestinal tract. Semin Diagn Pathol 2015; 32: 334e43. Walsh SV, Loda M, Torres CM, Antonioli D, Odze RD. P53 and beta catenin expression in chronic ulcerative colitis–associated
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polypoid dysplasia and sporadic adenomas: an immunohistochemical study. Am J Surg Pathol 1999; 23: 963e9. Yonezawa S, Higashi M, Yamada N, et al. Mucins in human neoplasms: clinical pathology, gene expression and diagnostic application. Pathol Int 2011; 61: 697e716. Ang DC, Shia J, Tang LH, Katabi N, Klimstra DS. The utility of immunohistochemistry in subtyping adenocarcinoma of the ampulla of vater. Am J Surg Pathol 2014; 38: 1371e9. Reid MD, Balci S, Ohike N, et al. Ampullary carcinoma is often of mixed or hybrid histologic type: an analysis of reproducibility and clinical relevance of classification as pancreatobiliary versus intestinal in 232 cases. Mod Pathol 2016; 29: 1575e85. Park DY, Srivastava A, Kim GH, et al. Adenomatous and foveolar gastric dysplasia: distinct patterns of mucin expression and background intestinal metaplasia. Am J Surg Pathol 2008; 32: 524e33. Callea M, Pedica F, Doglioni C. Programmed death 1 (PD-1) and its ligand (PD-L1) as a new frontier in cancer Immunotherapy and challenges for the Pathologist: state of the art. Pathologica 2016; 108: 48e58. Wu P, Wu D, Li L, Chai Y, Huang J. PD-L1 and survival in solid tumors: a meta-analysis. PLoS One 2015; 10: e0131403. Herbst RS, Soria JC, Kowanetz M, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 2014; 515: 563e7. Bang YJ, Van Cutsem E, Feyereislova A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastrooesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010; 376: 687e97. Miettinen M, Wang ZF, Sarlomo-Rikala M, Osuch C, Rutkowski P, Lasota J. Succinate dehydrogenase-deficient GISTs: a clinicopathologic, immunohistochemical, and molecular genetic study of 66 gastric GISTs with predilection to young age. Am J Surg Pathol 2011; 35: 1712e21. Gill AJ. Succinate dehydrogenase (SDH)-deficient neoplasia. Histopathology 2018; 72: 106e16. Dawson H, Kirsch R, Driman DK, Messenger DE, Assarzadegan N, Riddell RH. Optimizing the detection of venous invasion in colorectal cancer: the ontario, Canada, experience and beyond. Front Oncol 2014; 4: 354. Kirsch R, Messenger DE, Riddell RH, et al. Venous invasion in colorectal cancer: impact of an elastin stain on detection and interobserver agreement among gastrointestinal and nongastrointestinal pathologists. Am J Surg Pathol 2013; 37: 200e10. Batts KP, Ketover S, Kakar S, et al. Appropriate use of special stains for identifying Helicobacter pylori: recommendations from the rodger C. Haggitt gastrointestinal pathology society. Am J Surg Pathol 2013; 37: e12e22. Genta RM, Lash RH. Helicobacter pylori-negative gastritis: seek, yet ye shall not always find. Am J Surg Pathol 2010; 34: e25e34. Panarelli NC, Ross DS, Bernheim OE, et al. Utility of ancillary stains for Helicobacter pylori in near-normal gastric biopsies. Hum Pathol 2015; 46: 397e403. Griffiths P, Baraniak I, Reeves M. The pathogenesis of human cytomegalovirus. J Pathol 2015; 235: 288e97.
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38 Ambelil M, Saulino DM, Ertan A, DuPont AW, Younes M. The significance of so-called equivocal immunohistochemical staining for cytomegalovirus in colorectal biopsies. Arch Pathol Lab Med 2019; 143: 985e9. 39 Solomon IH, Hornick JL, Laga AC. Immunohistochemistry is rarely justified for the diagnosis of viral infections. Am J Clin Pathol 2017; 147: 96e104. 40 Al-Toma A, Volta U, Auricchio R, et al. European Society for the Study of Coeliac Disease (ESsCD) guideline for coeliac disease and other gluten-related disorders. United Eur Gastroenterol J 2019; 7: 583e613. 41 Rubio-Tapia A, Murray JA. Classification and management of refractory coeliac disease. Gut 2010; 59: 547e57.
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42 Deshpande V, Zen Y, Chan JK, et al. Consensus statement on the pathology of IgG4-related disease. Mod Pathol 2012; 25: 1181e92. 43 Kamisawa T, Okazaki K. Diagnosis and treatment of IgG4-related disease. Curr Top Microbiol Immunol 2017; 401: 19e33. 44 Kebbel A, Rocken C. Immunohistochemical classification of amyloid in surgical pathology revisited. Am J Surg Pathol 2006; 30: 673e83. 45 Hokama A, Kishimoto K, Nakamoto M, et al. Endoscopic and histopathological features of gastrointestinal amyloidosis. World J Gastrointest Endosc 2011; 3: 157e61.
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Immunohistochemistry and special stains in gastrointestinal pathology practice
This review will detail the ways in which IHC and special stains are useful in daily practice. It is beyond the scope of this review to detail every special stain, so we have chosen to concentrate on stains which are of particular importance to the gastrointestinal tract or require special attention in their interpretation. We will also consider stains that are potentially underutilized. Stains which are straightforward to interpret (e.g. CD117 in gastrointestinal stromal tumour, HepPar-1 in hepatocellular carcinoma) or serve a similar purpose across organ systems (e.g. haematolymphoid markers in lymphoma subtyping, chromogranin and synaptophysin in neuroendocrine tumours) will not be further discussed. There are two methods of presenting IHC and special stain information, entity-based or stain-based. Both have their advantages and disadvantages. Since an entity-based approach can be found in standard surgical pathology texts, a stain-based approach will be used in this review. We will further subdivide this into three broad categories, stains used in neoplastic processes, stains used in non-neoplastic processes, and stains used in tissue depositions.
Cheng Liu Masoumeh Ghayouri Ian S Brown
Abstract Immunohistochemistry and special stains play an increasingly important role in gastrointestinal pathology practice. In neoplastic disorders they are used to confirm the diagnosis, identify prognostic/predictive features, and screen for an underlying genetic syndrome. In nonneoplastic disorders they can identify an infectious organism, clarify the inflammatory infiltrate present, and confirm a tissue deposition. In this review we discuss the most important and topical of these stains, especially ones which require special care in interpretation. It should be emphasized that, in cases with equivocal or unexpected staining patterns, the results should be interpreted in the appropriate clinical, endoscopic and morphologic context.
Neoplastic processes Increasingly IHC and special stains are being applied to neoplastic processes, and virtually every gastrointestinal tract neoplasm is subject to some type of additional stain. Table 1 summarises the stains we find the most useful, and a selected number will be discussed in detail.
Keywords amyloid; cytomegalovirus; gastrointestinal pathology; Helicobacter pylori; HER2; immunohistochemistry; mismatch repair; mucin; PD-L1; SATB2; special stain
MLH1, PMS2, MSH2 and MSH6 Mismatch repair (MMR) is a mechanism of maintaining DNA integrity during cell division, where incorrectly matched base pairs are excised and repaired. This is performed by the MMR proteins MLH1, PMS2, MSH2 and MSH6. Loss of MMR protein function leads to progressive accumulation of errors throughout the genome, which are accentuated at microsatellites. Microsatellites are small repetitive DNA sequences most sensitive to replication error, and involvement of several predefined loci are referred to as microsatellite instability (MSI). Thus, detection of MMR deficiency can proceed via two ways, by detecting MMR protein loss via IHC, or MSI by DNA sequencing-based methods. Both methods have similar sensitivities and specificities.1 MMR IHC detects both inherited (Lynch syndrome-associated) and sporadic (MLH1-methylated) MSI colorectal carcinomas. Lynch syndrome accounts for 3e4% of all colorectal carcinomas.2 Although there is no universal agreement as to which colorectal carcinomas should be screened for Lynch syndrome, a growing number of international organisations have recommended to screen all tumours, or at least tumours in patients less than 70 years of age.3 In contrast, approximately 12% of colorectal carcinomas arise secondary to methylation-related silencing of MLH1. In both situations, microsatellite instability is associated with overall more favourable prognosis.4 Recently an additional benefit of MMR IHC has been the prediction of response to immune checkpoint inhibitor therapy. Carcinomas with MMR deficiency show improved response to PD-L1 blockade.5 With the approval of pembrolizumab for all MMRdeficient tumours by the United States Food and Drug
Introduction Immunohistochemistry (IHC) and special stains are indispensable adjuncts in the reporting of gastrointestinal tract specimens, whether these are procured by endoscopic biopsy or by surgical resection. In neoplastic disorders they may confirm a neoplastic process and/or provide clinically important information. The latter includes the identification of the site of origin of the neoplasm, prognostic parameters, prediction of response to therapy, or potentially identify an underlying genetic syndrome. Some stains serve more than one purpose, depending on clinical context. In non-neoplastic disorders IHC and special stains help to classify an inflammatory reaction pattern or identify an infectious aetiology. Both IHC and special stains may also help determine the nature of a tissue deposition.
Cheng Liu BMedSci MBBS FRCPA Envoi Specialist Pathologists, Brisbane, Faculty of Medicine, University of Queensland, Brisbane and The Conjoint Gastroenterology Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. Conflicts of interest: none declared. Masoumeh Ghayouri MD Department of Pathology, H. Lee Moffitt Cancer Center, Tampa, FL, USA. Conflicts of interest: none declared. Ian S Brown BGEN MBBS FRCPA Envoi Specialist Pathologists, Brisbane and Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia. Conflicts of interest: none declared.
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colorectal cancer cases demonstrating PMS2 loss (overwhelmingly due to MLH1 methylation),3 and it prevents falsely interpreting weak MSH6 expression as normal in patients with MSH2 germline mutation.9 The interpretation of MMR IHC is usually straightforward, where a tumour will show uniform retained staining or loss of staining of an MMR protein throughout, with retained staining in internal control cells. The terminology used in reporting MMR IHC must be precise, since a “positive” abnormal result is due to loss of staining, or “negative” staining. Thus, a statement such as “MLH1 IHC is negative” is ambiguous. Instead we recommend using “retained” or “loss of” staining, followed by an interpretation, such as “MLH1 shows loss of staining, which is an abnormal result”.3 Sometimes interpretation is challenging when there is weak/ heterogeneous staining or equivocal internal control staining. For example, in surgical resections where there may be areas of poor fixation or tumour hypoxia, up to 10% of the tumour cells may exhibit equivocal weak expression for MMR markers so long as other areas of the tumour show uniform strong expression.10 In tumours with MLH1 methylation, there may be variability in the intensity of expression of both MLH1 and PMS2, with areas of weak expression or loss of expression.11 The minimum cut off for an abnormal result is not well established. Even though it is sometimes recommended that any tumour staining which is at least as strong as background internal control be regarded as retained staining, we believe this scope is too narrow, and all cases with difficult-to-interpret or clearly heterogeneous staining should be flagged as possibly representing Lynch syndrome.12 Table 2 details the common patterns of staining identified with MMR markers and their clinical significance, and Figure 1 illustrates selected scenarios. Approximately 80% of colorectal carcinomas exhibiting MLH1 loss by IHC arise from sessile serrated lesions (SSLs). While SSLs are very common, SSLs with dysplasia are rare and capable of progressing to carcinoma rapidly. Dysplasia in SSLs is morphologically heterogeneous and can be very subtle. However, as 75% of SSLs with dysplasia demonstrate MLH1 loss, loss of staining is very helpful in confirming a diagnosis of dysplasia.13 In contrast, performing MMR IHC in conventional adenomas is of limited value. Lynch syndrome-associated conventional adenomas show loss of MMR protein staining in approximately 80% of cases (more likely if large, with a villous component and/or high-grade dysplasia).14 However, as non-syndromic conventional adenomas are much more common in practice, routine staining of all conventional adenomas is not an effective screening strategy.
Commonly used IHC and special stains in the gastrointestinal tract Examples Tumour subtyping within gastrointestinal tract
Neuroendocrine differentiation: chromogranin, synaptophysin, CD56 Adenocarcinoma vs. squamous cell carcinoma: CK7, CK20, p63, p40, mucin stains (e.g. PAS-D, mucicarmine) Subtyping of gastric adenocarcinoma: EBV (in situ hybridization), E-cadherin, MMR stains, p53 Prognostication and grading Neuroendocrine tumours: ki67 Colorectal carcinoma: MMR stains Vascular invasion/serosal involvement: elastic stains (e.g. Verhoeff-van Gieson, orcein, Movat) Predict response to therapy Gastro-oesophageal and gastric carcinoma: HER2 Colorectal carcinoma: MMR stains Multiple malignancies: PD-L1 Diagnosis of dysplasia IBD-related dysplasia: SATB2, p53, b-catenin Typing of dysplasia/ Pancreatic IPMN and associated adenocarcinoma carcinoma: intestinal (MUC2, CDX2), gastric (MUC5AC, MUC6), pancreaticobiliary (MUC1) Identifying an inherited tumour Lynch syndrome: MMR stains predisposition syndrome Carney-Stratakis syndrome: SDHA, SDHB Identifying primary site in poorly Multiple possibilities: CK7, CK20, differentiated malignancies TTF1, CDX2, SATB2, GATA3, PAX8, ER, calretinin, CK5/6, S100, SOX10, CD3, CD20 EBV, Epsten-Barr virus; MMR, mismatch repair; IBD, inflammatory bowel disease; IPMN, intraductal papillary mucinous neoplasm.
Table 1
Administration (FDA), it is increasingly likely that all gastrointestinal tract carcinomas will require routine MMR IHC in the future. IHC directed against the MMR proteins are widely available, and whether the stains are performed on endoscopic biopsy material or surgical resection material is a matter for individual laboratories. Our preference is for endoscopic biopsy material if available, because the stains are less likely to be affected by fixation artefact,6 a subsequent surgical resection may not be processed at the same laboratory, and neoadjuvant therapy may alter MMR IHC result.7 There is good evidence for strong concordance between the findings in biopsy material and resection tissue.8 Our preference is to stain for all four proteins upfront, as this avoids a second round of IHC in the 15e20% of
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SATB2 SATB2 is a protein with preferential expression in the lower gastrointestinal tract. Although its expression largely parallels CDX2, SATB2 is more site-specific and is less prone to methylation-related silencing than CDX2, making it more useful in mucinous, signet ring and undifferentiated colorectal carcinomas.15 SATB2 is highly sensitive and specific for lower gastrointestinal tract origin of a neuroendocrine tumour, particularly for well-differentiated neoplasms.16 Neuroendocrine carcinoma of the lower gastrointestinal tract demonstrates similar high expression, however metastasis from a non-gastrointestinal
2
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Common MMR patterns and their significance Pattern of expression
Percentage
Probability of Lynch syndrome
Significance/further testing
MLH1, MSH2, MSH6 and PMS2 preserved
85%
Very unlikely
MLH1 and PMS2 loss
15%
Unlikely (sporadic in 80% of cases)
MSH2 and MSH6 loss MSH6 loss
<1% <1%
Likely Likely
PMS2 loss
<1%
Likely
Normal pattern; no further testing unless there is a strong clinical suspicious of Lynch syndrome BRAF mutation or MLH1 promoter methylation testing to confirm sporadic nature; if both absent, investigate for Lynch syndrome MSH2, followed by MSH6 germline testing MSH6, followed by MSH2 germline testing; may occur secondary to neoadjuvant therapy in rectal carcinoma PMS2, followed by MLH1 germline testing
Table 2
site needs to be excluded.16 SATB2 also allows for separation of primary small intestinal adenocarcinoma from colorectal carcinoma that has spread to the small intestine. While most primary colorectal carcinomas express this protein, less than half of primary small intestinal adenocarcinomas do. Furthermore, unlike the strong and diffuse staining pattern in CRC, the expression of SATB2 is weaker in small intestinal adenocarcinoma.17 The diagnostic uses of SATB2 continue to expand and there are recent reports of its potential role in diagnosing inflammatory bowel disease (IBD)-associated dysplasia and carcinoma. Loss of SATB2 staining is seen in 40% of cases of IBD related dysplasia, while it is retained in reactive atypia and conventional adenomas.18 This first allows for establishing a diagnosis of dysplasia versus reactive atypia, and secondly it separates IBDrelated dysplasia from a conventional adenoma occurring in a patient with IBD. Interestingly, SATB2 is also frequently lost in IBD-associated carcinomas, but is retained in most sporadic carcinomas.19 Figure 2 shows an example of IBD-related dysplasia with loss of SATB2 staining.
diagnosis of dysplasia where morphology is equivocal.20 In general, a dysplastic lesion should exhibit higher ki67 index, increased p53 staining towards the luminal aspect, and aberrant (cytoplasmic or nuclear) b-catenin expression, when compared with a reactive process. However, these stains are variably positive in reactive processes, especially when associated with inflammation or regeneration, and different studies have used different scoring systems and cut off thresholds for positivity. Also, the aberrant expression of b-catenin is restricted to lesions with WNT pathway activation, generally intestinal-type adenomas. The value of these stains is not an absolute cut off threshold but rather in comparing with background mucosa. If the lesion shows an abrupt difference in staining from the background, this is supportive of a dysplastic interpretation. b-catenin IHC has additional utility in unequivocally dysplastic lesions. Until recently it was used with p53 to separate IBD-related dysplasia from conventional adenoma, where IBDrelated dysplasia is usually negative for b-catenin and diffusely positive for p53, while conventional adenoma shows the opposite pattern.21 The potential of SATB2 in this setting has been discussed above. Since the management of dysplastic lesions in IBD is now dependent more on endoscopic appearance and resectability rather than aetiology, there is less need to separate IBD-related dysplasia from conventional adenoma.
ki67, p53 and b-catenin These three stains are discussed together due to their similar utility and interpretation. Within the tubular gastrointestinal tract, b-catenin, p53 and ki67 have all been used to support a
Figure 1 Mismatch repair protein immunohistochemistry. There is loss of expression of MLH1, PMS2 and MSH6 in this colorectal carcinoma. MSH2 is preserved (not shown). There is normal expression of the markers in the background lymphocytes and non-neoplastic epithelial cells. This abnormal pattern of expression is usually the result of sporadic methylation-induced silencing of MLH1 gene expression with secondary loss of PMS2 and MSH6.
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Figure 2 High-grade dysplasia and invasive adenocarcinoma developing in a colonic inflammatory polyp in a patient with long standing ulcerative colitis. There is strong nuclear expression of p53 in the dysplastic cells; in contrast, SATB2 expression is lost.
downregulates the immune response, and may lead to apoptosis of the T cell. In a similar vein, by expressing PD-L1, tumour cells mimic normal cells and escape destruction by cytotoxic T cells. The tumour cells are thus allowed to persist and proliferate. AntiPD-L1 therapy such as pembrolizumab “unmasks” tumour cells from the immune system and reverses the inhibitory effect of PDL1. IHC PD-L1 testing is most established in lung adenocarcinoma, and it is now being applied to an increasing number of tumours from diverse organ systems. However, accurate interpretation of PD-L1 IHC is complicated by several factors.26 Multiple antibody clones are available, and staining is usually heterogeneous. Different scoring systems are used for different tumours, with some also including tumour-infiltrating lymphocytes. As expected, PD-L1 positive tumours are associated with poor prognostic features,27 regardless of scoring system used, and response to PD-1/PD-L1 blockade correlates with degree of IHC PD-L1 positivity.28 In the gastrointestinal tract, PD-L1 IHC testing is driven by availability of pembrolizumab. The United States FDA data sheet for pembrolizumab includes oesophageal, gastric, hepatocellular and colorectal carcinomas, but other tumours are likely to become eligible in the future.
MUC1, MUC2, MUC5AC and MUC6 Mucins are high-molecular weight glycoproteins produced by epithelial cells, which have both gel-forming and signal transduction roles. Broadly the pattern of mucin expression corresponds to site-specific epithelial components of the gastrointestinal tract. For instance, pancreaticobiliary epithelium expresses MUC1, intestinal epithelium expresses MUC2, gastric foveolar epithelium expresses MUC5AC and gastric pyloric epithelium expresses MUC6. Mucin IHC can therefore be applied in the subclassification of pancreatic intraductal papillary mucinous neoplasm (IPMN)-derived carcinomas into pancreaticobiliary (MUC1), intestinal (MUC2) and gastric (MUC5AC and MUC6) phenotypes, which have differing prognoses.22 It should be emphasized, however, that the phenotype of these lesions can usually be determined via morphologic means and mucin stains have a supplementary role only. Table 3 summarises the morphologic and IHC features of these phenotypes. MUC1 and MUC2 staining may be useful in establishing a pancreaticobiliary or intestinal phenotype in neoplasms occurring at the duodenal ampulla. This is generally done in combination with CK20 and CDX2, and may guide further treatment of these tumours.23 Unfortunately, the stains sometimes do not establish a clear line of differentiation.24 Mucin stains may also be useful in separating gastric phenotype dysplasia from intestinal phenotype dysplasia of the upper gastrointestinal tract. Gastric pattern dysplasia is associated with different clinicopathological features.25 However, as with the pancreatic IPMN, the clinical utility of performing these stains routinely is yet to be firmly established. In addition, many examples of glandular dysplasia in the lower oesophagus display a hybrid pattern of mucin expression consistent with both gastric and intestinal differentiation.
HER2 HER2 is a member of the epidermal growth factor receptor family, where overexpression leads to activation of proliferation pathways in the absence of an extracellular ligand. It is overexpressed in approximately 20% of gastro-oesophageal junction and gastric adenocarcinomas, and treatment with trastuzumab increases survival.29 Although different antibody clones are available, the results are comparable between clones, and the interpretation methodology is standardized (see Table 4). Of note, interpretation differs from breast cancer in several respects. Because gastro-oesophageal junction and gastric cancers are commonly advanced (i.e. inoperable) at presentation, the only tissue available for testing may be an endoscopic biopsy specimen. To account for possible intratumoural heterogeneity in this setting, the cut off for an equivocal or positive result is much lower in biopsy compared with excision specimens. Complete circumferential membranous staining is also not required.
PD-1 and PD-L1 The PD-1/PD-L1 pathway allows cytotoxic T cells to selectively remove abnormal cells while leaving normal cells intact. To avoid damage, normal cells express the transmembrane protein PD-L1, which is detected by its receptor PD-1 on the cytotoxic T cell. When a cytotoxic T cell encounters a PD-L1-expressing normal cell, the PD-L1/PD-1 interaction inactivates the T cell,
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DIAGNOSTIC HISTOPATHOLOGY xxx:xxx
Morphologic features
MUC2
MUC5AC
MUC6
MUC1
Pancreaticobiliary
Columnar cells Round, uniform, basally located nuclei Abundant apical mucinous cytoplasm Columnar cells Hyperchromatic, pencillate, stratified nuclei Dense eosinophilic cytoplasm Cuboidal to columnar cells with pale clear to light eosinophilic cytoplasm Hyperchromatic round to oval nuclei Prominent nucleoli if high grade Closely packed tubules lined by cuboidal to columnar epithelium with pale to eosinophilic ground glass cytoplasm Round basal nuclei Nucleoli easily visible Cytological features intermediate between the above patterns, or an intimate admixture of both
e
e
e
þ
þ
e
e
e
þ
rare
e
e
þ (surface)
þ
e
Intestinal
Gastric foveolar
Gastric pyloric gland
Hybrid
Table 3
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Phenotype
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IPMN phenotypes as determined via mucin stains
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Interpretation of HER2 staining in the gastrointestinal tract HER2 score
Biopsy staining
Excision staining
Interpretation
0
No reactivity or no membranous reactivity in any cell 5 cells with a faint or barely perceptible membranous reactivity irrespective of percentage of cells positive 5 cells with a weak to moderate complete, basolateral, or lateral membranous reactivity irrespective of percentage of cells positive 5 cells with a strong complete basolateral, or lateral membranous reactivity irrespective of percentage of cells positive
No reactivity or membranous reactivity in <10% of cells Faint or barely perceptible membranous reactivity in 10% of cells; cells are reactive only in part of their membrane Weak to moderate complete, basolateral or lateral membranous reactivity in 10% of cells
Negative
Strong complete, basolateral or lateral membranous reactivity in 10% of cells
Positive
1þ
2þ
3þ
Negative
Equivocal
Table 4
of an SDHA mutation, but IHC is not available for SDHC and SDHD mutations. An example of a “positive” result (i.e. loss of staining) is shown in Figure 3.
SDHA and SDHB The succinate dehydrogenase (SDH) complex is a mitochondriaassociated enzyme complex involved in oxidative phosphorylation, comprising four subunits SDHA, SDHB, SDHC and SDHD. Mutation in the subunits result in Carney-Stratakis syndrome (gastric gastrointestinal stromal tumours [GISTs] and paragangliomas), and methylation-induced silencing of SDHC results in Carney triad (gastric GISTs, paragangliomas and pulmonary chondromas). Gastric GISTs that arise in the context of SDH mutation have distinct pathologic features.30 They grow as multinodular sheets which dissect the muscularis propria, display epithelioid cytology, and have a tendency towards lymph node metastases. However, they have an indolent course, so the prognostic algorithm used for usual GISTs does not apply. The interpretation of SDH IHC parallels that for MMR proteins. As mutations in any subunit results in loss of SDHB, SDHB IHC is an effective screening test.31 A normal result is granular cytoplasmic (i.e. mitochondrial) staining in tumour cells. Loss of staining is an abnormal result, which must be interpreted with an appropriately staining internal control such as endothelial cells. In the context of SDHB loss, SDHA IHC allows for identification
Elastic stains Venous invasion is an independent poor prognostic feature in malignant epithelial tumours of the gastrointestinal tract. However, its presence is underreported by pathologists. This is particularly problematic in colorectal carcinoma where it is expected that venous invasion should be present in at least 30% of resected tumours.32 Histological features suspicious for venous invasion include the “orphan (or unaccompanied) arteriole” sign, a well-circumscribed tumour nodule adjacent to a thickwalled artery without an accompanying vein; and the “protruding tongue” sign, a circumscribed protrusion of tumour into the pericolic fat. In these situations, at least, elastic tissue stains may demonstrate a residual vein wall, an example of which is shown in Figure 4. An elastin stain is also useful in confirming the presence of venous invasion in cases originally deemed equivocal on haematoxylin and eosin (H&E)-stained sections. Some have gone further and advocate routine use of elastin stains to increase venous invasion detection. It has been shown that use of
Figure 3 SDHB-deficient gastric gastrointestinal stromal tumour. Note loss of immunohistochemical SDHB expression in the tumour cells with preserved internal control staining of endothelial cells. Retained internal control expression is required to ensure the stain has worked correctly. (Photographs courtesy of Professor Anthony Gill, Royal North Shore Hospital, Sydney).
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Figure 4 Orcein stain demonstrating the residual elastic lamina of a vein that has been invaded by colorectal carcinoma. The "orphan artery" sign is apparent on the haematoxylin and eosin-stained section.
routine elastin stains is associated with a doubling of the venous invasion detection rate.33
There are however situations where stains should be performed if organisms are not identified in the H&E stained sections.35 These indications are listed in Box 1. H. pylori IHC may also be useful when there has been proton pump inhibitor therapy or incomplete antibiotic treatment,36 both of which lead to a reduction in the number of organisms. This is often accompanied by a loss or marked reduction in active inflammation. The organisms may be found only in the deep aspect of glands of the specialized mucosa, often closely applied to the canaliculi of parietal cells (see Figure 5).
Non-neoplastic processes Most stains performed in nonneoplastic processes are for characterizing an inflammatory infiltrate, or to identify an infectious organism. Table 5 summarises the stains we find the most useful, and a selected number will be discussed in detail. Helicobacter pylori stains The most common reason for performing a special stain in inflamed gastrointestinal tract tissue is to look for an infective cause, and Helicobacter pylori (H. pylori)-associated gastric inflammation represents the best example of this. While a variety of histochemical stains successfully highlight H. pylori (e.g. Wright-Giemsa, toluidine blue, Warthin-Starry), none of these have sensitivity or specificity that approaches the IHC, and this has become the gold standard test. While some laboratories advocate the routine use of stains to look for these organisms, there seems little point in doing this when the organism is readily identifiable on H&E in most cases.34
Cytomegalovirus immunohistochemistry Cytomegalovirus (CMV) is a herpesvirus which latently infects approximately 80% of the world’s population. In immunocompetent individuals the virus is dormant, but reactivation occurs in immune dysregulation, such as with steroid use, human immunodeficiency virus infection, bone marrow suppression, and old age.37 In the gastrointestinal tract any organ can be affected, manifesting as cytomegaly and characteristic “owl’s eye” nuclear inclusions. Infection preferentially affects stromal cells and
Common stains used in nonneoplastic processes Purpose
Example stains
Identifying infective organism
Bacteria: IHC or Giemsa for H. pylori, IHC or Warthin-Starry for T. pallidum Mycobacteria: Ziehl-Neelsen, Wade-Fite Fungi: PAS-D, Grocott Viral: IHC for HSV, IHC CMV, p16 for HPV Collagenous gastritis/sprue/colitis: trichrome, Verhoeff-van Gieson Lymphocytic gastritis/duodenitis/colitis: CD3 IgG4-related sclerosing disease: IgG, IgG4 Type 1: normal CD3 and CD8 in T cells Type 2: aberrant CD3 and CD8 in T cells
Confirm a specific inflammatory pattern
Plasma cell-rich fibrosing process Refractory coeliac disease
IHC, immunohistochemistry; HSV, herpes simplex virus; CMV, cytomegalovirus; HPV, human papillomavirus.
Table 5
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prominence, and a positive result in this case will always highlight more infected cells than appreciable on H&E. On the other hand, indiscriminate application of CMV IHC will detect latently infected cells, which may lead to an erroneous assignment of aetiology.39 Lastly, pigment-laden macrophages may simulate CMV-infected cells on IHC, due to their size and haemosiderin content.
Indications for H. pylori stains Acute gastritis and/or gastric ulceration (unless clearly reactive gastropathy associated) Active chronic gastritis Focally enhanced gastritis Moderate or severe chronic gastritis Autoimmune gastritis (H. pylori infection may be a precursor) Lymphocytic gastritis (organisms may be very sparse) Marginal zone lymphoma (MALT type) Previous H. pylori (to confirm eradication) Positive urease test without organisms seen in H&E stained sections Gastric intestinal metaplasia Gastric adenocarcinoma Duodenal ulceration
CD3 and CD8 Refractory coeliac disease is defined as symptoms not responding after 12 months on a gluten-free diet, or symptom recurrence while on a gluten-free diet.40 Most cases prove to represent an insufficient gluten-free diet or a slow healing response. Rarely, the persisting or recurring disease represents the development of a monoclonal T cell proliferation, which is initially confined to the epithelium but is at high risk of progression to enteropathy-associated T-cell lymphoma. Separating these two scenarios is clinically important, and CD3 and CD8 are helpful in this context.41 Normally at least 90% of CD3 þ intraepithelial T cells co-express CD8, and this is maintained in the first scenario (refractory coeliac disease type 1). Loss of CD3 and CD8 co-expression in greater than 50% of the intraepithelial T cells is a worrisome finding and corresponds to the second scenario (refractory coeliac disease type 2); an example of this is shown in Figure 6. Refractory coeliac disease type 2 is a difficult diagnosis and requires synthesis of clinical, H&E, IHC and flow cytometric findings; no feature should be considered diagnostic in isolation.
MALT, mucosa-associated lymphoid tissue; H&E, haematoxylin and eosin.
Box 1
endothelial cells, with epithelial cells being involved only in severe cases. This is typically associated with active chronic inflammation, and inflammation can be so pronounced as to obscure the diagnostic cytologic findings. CMV IHC aids in identifying the virus-infected cells in cases with a high index of suspicion.38 It is of greatest utility when the inclusions are equivocal and other entities enter the differential, chiefly inflammation/regeneration-associated nucleolar
IgG and IgG4 IgG4-related sclerosing disease has been recognized in almost all organ systems since its consensus diagnostic criteria were published in 2012.42 The three cardinal features are (1) IgG4 þ
Figure 5 Gastric biopsies in a patient taking a proton pump inhibitor. Mild active chronic inflammation is present in the gastric body. The gastric antrum is normal. H. pylori immunohistochemical stain demonstrates organisms restricted to the gastric body and closely applied to the parietal cell canaliculi.
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Figure 6 Refractory coeliac disease type 2 with loss of CD8 expression in >50% of CD3þ intraepithelial T cells.
plasma cells comprising at least 40% of all IgG þ plasma cells; (2) storiform fibrosis; and (3) obliterative phlebitis. At least two out of three features are required for diagnosis. In addition to the 40% minimum, an absolute number of IgG4 þ plasma cells is also required (expressed per high power field), which differs between organs. Recognition of the disorder is important as it respond well to steroid treatment.43 In the gastrointestinal tract the main organs involved are the pancreas and biliary tract. If not considered, the prominent fibroinflammatory reaction may be mistaken for chronic pancreatitis, primary sclerosing cholangitis, pancreatic ductal adenocarcinoma, or cholangiocarcinoma.
classification into either light chain (AL) or inflammationassociated (AA) amyloid, the two most common subtypes. IHC is available for a wider range of amyloid types, including AL, AA, transthyretin and b2-microglobulin.44 However, the efficacy of these stains is laboratory dependent, and nonspecific/equivocal staining is common; precise subtyping is better performed via mass spectrometry. Because gastrointestinal involvement by amyloidosis presents with nonspecific symptoms such as diarrhoea, constipation and abdominal pain,45 subtle deposits are easily missed. The deposits begin in the submucosal vessel walls, which can be mistaken for collagen or fibrin, and subsequently involve the lamina propria and submucosa proper. In our opinion, it is more important to recognize amyloidosis and recommend further investigation for a systemic cause, rather than to subtype the amyloid.
Assessment of tissue deposition Most tissue deposits are readily recognisable without IHC or special stains. Table 6 lists cases where stains may be of use.
Conclusions
Amyloid stains Amyloid represents abnormally aggregated protein which becomes deposited in extracellular sites as amorphous eosinophilic material. Histochemical stains such as Congo red and crystal violet can confirm the presence of amyloid, and pre-treatment of sections by potassium permanganate further allows sub-
It is sometimes challenging to keep abreast of available IHC and special stains in gastrointestinal pathology, and we have attempted to summarize the most topical and/or useful stains in this review. It should be emphasized, however, that stains should only be ordered to answer a specific clinical question, and the results interpreted along with the H&E findings. A REFERENCES 1 Lindor NM, Burgart LJ, Leontovich O, et al. Immunohistochemistry versus microsatellite instability testing in phenotyping colorectal tumors. J Clin Oncol 2002; 20: 1043e8. 2 Lynch HT, Snyder CL, Shaw TG, Heinen CD, Hitchins MP. Milestones of Lynch syndrome: 1895-2015. Nat Rev Cancer 2015; 15: 181e94. 3 Yozu M, Kumarasinghe MP, Brown IS, Gill AJ, Rosty C. Australasian Gastrointestinal Pathology Society (AGPS) consensus guidelines for universal defective mismatch repair testing in colorectal carcinoma. Pathology 2019; 51: 233e9. 4 Ward R, Meagher A, Tomlinson I, et al. Microsatellite instability and the clinicopathological features of sporadic colorectal cancer. Gut 2001; 48: 821e9. 5 Le DT, Durham JN, Smith KN, et al. Mismatch repair deficiency predicts response of solid tumors to PD-1 blockade. Science 2017; 357: 409e13. 6 O’Brien O, Ryan E, Creavin B, et al. Correlation of immunohistochemical mismatch repair protein status between colorectal
Tissue deposition and their staining characteristics Tissue deposition
Example stains
Brown depositions
Iron and pseudomelanosis: Perls positive Melanosis and lanthanum: Perls negative Amyloid: Congo red, crystal violet Collagen: trichrome Elastin: Verhoeff-van Gieson, orcein, Movat Medication capsule: PAS Calcium/psammoma bodies, Osmoprep: von Kossa positive SIRT spheres: von Kossa negative
Eosinophilic depositions
Basophilic depositions
SIRT, selective internal radiation therapy.
Table 6
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42 Deshpande V, Zen Y, Chan JK, et al. Consensus statement on the pathology of IgG4-related disease. Mod Pathol 2012; 25: 1181e92. 43 Kamisawa T, Okazaki K. Diagnosis and treatment of IgG4-related disease. Curr Top Microbiol Immunol 2017; 401: 19e33. 44 Kebbel A, Rocken C. Immunohistochemical classification of amyloid in surgical pathology revisited. Am J Surg Pathol 2006; 30: 673e83. 45 Hokama A, Kishimoto K, Nakamoto M, et al. Endoscopic and histopathological features of gastrointestinal amyloidosis. World J Gastrointest Endosc 2011; 3: 157e61.
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