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IHC And Special Stains In Daily Practice
Dr Ian Brown
Envoi Pathology
Brisbane, Australia
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Tumour
• Classify disease
• Prognostication
• Predict response to therapy
• Identify an inherited syndrome
• Identify primary site of origin
Inflammatory pattern
• Look for infective cause
• Classify the immune reaction
• Exclude a haematological neoplasm
• Classify disease
Why do special stains?
• Never do a special stain without knowing what to do with the result
• I also like to get personal experience with special stains so that I know
how they will work when I really need them
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• IHCs• Gastrin• H.pylori• Use of p53• CD3/8 for RCD• MMR stains• MLH-1 in SSAD• Detection of enteroblastic differentiation• Vascular invasion markers• SATB2• DLBCL subtyping• Β catenin
• What I don’t do!!
• New and evolving concepts/general discussion
• Reference: An Update on the Role of Immunohistochemistry in the Evaluation of Gastrointestinal Tract Disorders. Adv Anat Pathol. 2018 Sep 18.
Overview
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• Features of AI gastritis
Gastrin for autoimmune gastritis
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• Some cases have• minimal or no intestinal metaplasia
• minimal or no history
• Just labelled as ‘gastric’
Gastrin for autoimmune gastritis
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Gastrin for autoimmune gastritis
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• Always do a H.pylori stain also• Co-existence
• mimic
• ? NE stains (synaptophysin) increase the diagnostic yield
Gastrin for autoimmune gastritis
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Atrophic pattern of collagenous gastritis
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Atrophic pattern of collagenous gastritis
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• Not routinely!
• Selective use and not if clearly evident on the H&E• active gastritis and gastric ulceration (unless clearly reactive gastropathy associated)
• moderate/florid chronic gastritis (? mild chronic gastritis)
• Autoimmune gastritis
• Lymphocytic gastritis (look very carefully!!)
• MALT lymphoma
• Previous H.pylori
• Positive urease
• Gastric intestinal metaplasia
• Gastric adenocarcinoma
• Duodenal ulceration
H.pylori
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H.pylori
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• Barrett’s dysplasia
• IBD dysplasia
Use of p53
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• British Society of Gastroenterology guidelines on the diagnosis and management of Barrett’s oesophagus Gut 2014;63:7–42.
“The addition of a p53 immunostain to the histopathological assessment may improve the diagnostic reproducibility of a diagnosis of dysplasia in Barrett’s oesophagus and should be considered as an adjunct to routine clinical diagnosis (Recommendation grade B)”
• I dabble!
• But I work in the ideal situation and the result of p53 seldom changes what I will call the atypia
p53 in Barrett’s dysplasia
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• However, there may be a role in the following situations
1) Isolated practice
2) Limited experience with BE dysplasia
3) Triage to know what is worth sending on
4) The stakes are high• Procedure will follow
• It might be adenocarcinoma versus just reactive
p53 in Barrett’s dysplasia
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• Our data on use of p53 in indefinite for dysplasia (retrospective)
p53 in Barrett’s dysplasia
125 indefinite cases with
follow up biopsies
Strong p53 in indefinite
focus
Weak p53 in indefinite
focusNo dysplasia 8 76
Persisting indefinite 1 18
No progression 9 94
LGD 1 7
HGD/IMCA 9 5
Dysplasia 10 12
~1/2 of our indefinite cases with strong p53 represented true dysplasia (usually HGD/IMC) while only ~ 10% represent no dysplasia/persisting indefinite
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• Patterns
p53 in Barrett’s dysplasia
Over-expression
Loss of expression
Courtesy of Priyanthi Kumarasinghe
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p53 in IBD dysplasia
AJG 2011
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p53 in IBD
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Disease not responding (or recurring) after 12 months of GFD or severe or deteriorating clinical symptoms
Need to considerInadequate GFD
Wrong diagnosis
Another condition causing the symptoms
Slow responding coeliac disease (RCD type 1)
Development of a clonal intraepithelial T cell process (RCD II)At high risk of progression to type 1 EATL
CD3/8 in refractory coeliac disease
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CD3/8 in RCD
Author Number Marsh 0 (%) Marsh 1/2 (%) Marsh 3 (%)
Bardella 114 18 20 62
Hutchinson 284 39 17 44
Martini 101 12 51 38
Ciacci 390 44 33 24
Lanzini 465 8 71 21
Wahab 158 39 51 10
Tuire 177 42 54 4
Haere 127 81 14 5
Range 8 – 81% 14 – 71% 4 – 62%
• Response to GFD
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Refractory coeliac disease
TCR rearrangement CD3/CD8
immunohistochemistry
Flow cytometry
Celiac disease Polyclonal Preserved No loss of surface T cell
markers
RCD1 Polyclonal (usual) Preserved No loss of surface T cell
markers
RCD 2 Monoclonal CD8 lost in > 50% of CD3
positive IEL
Loss of surface CD3,
CD7 and CD8 in > 20%
of IEL
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Refractory coeliac disease
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• 2 reasons
1) Detect Lynch syndrome
2) Indicate response for PD-1 inhibitors
? Universal testing or selective
MMR stains in CRC
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Universal MMR testing at Envoi
• 2,077 consecutive CRCs tested for MMRP status over 5 years
• From 2,016 patients with a median age at diagnosis of 70 years –females 45.5%
• MMR protein deficiency in 399 cases (19.2%)
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MMR IHC in adenomas
• Little benefit
• In known Lynch syndrome patients • Up to 70% of adenoma in LS show loss of corresponding MMR protein
• Higher rate in advanced adenomas: high grade dysplasia, villous component
• Normal MMR IHC expression does not exclude Lynch syndrome
• Best option: if known CRC in the family, test the cancer and test family members if a mutation is identified
• No indication for HP and SSA (but we have seen TSA’s in with MMR loss in Lynch syndrome)
Ferreira S et al. Dis Colon Rectum 2008;51:909–15.Walsh MD et al. Mod Pathol 2012;25:722–30.
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Significance of common MMR IHC patternsPattern of IHC expression Probability of LS Significance
All 4 MMR proteins (or PMS2/MSH6) normal Very unlikely Normal pattern. No further testing, unless strong clinical suspicion (MSI testing)
MLH1/PMS2 loss Sporadic or LS BRAF mutation testing▪ If present: Stop – sporadic CRC▪ If absent: MLH1, followed by PMS2 germline
testing
MSH2/MSH6 loss Likely MSH2, followed by MSH6 germline testing
MSH6 loss Likely MSH6, followed by MSH2 germline testing
PMS2 loss Likely PMS2, followed by MLH1 germline testing. Could a MLH-1 mutation with sufficient protein to be immunoreactive but non functional
MLH1/PMS2 loss and MSH6 loss (partial or complete)
Very unlikely MLH-1 hypermethylation with a secondary mutation in a coding region of MSH6
All 4 MMR proteins lost Likely Germline loss of MSH2 and hypermethylation of MLH-1
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• Can get biallelic sporadic loss of MMR protein vs constitutional biallelic loss of MMR protein
• Also somatic mosaicism (FAP)
• A side point:• Not all Lynch is the same
• PMS2 Lynch is commonly present but infrequently presents with clinical disease
MMR stains in CRC
Good review on MMR IHC patterns = Immunohistochemical Pitfalls: Common Mistakes in the Evaluation of Lynch Syndrome. Surgical Pathology Clinics 10 (2017) 977–1007
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• We do not do this routinely – but actually quite a bit because we garnering experience, particularly with ‘minimal deviation’ types
• Also, to get better at picking between early TSA arising in a SSA versus serrated pattern dysplasia, NOS (which is more biologically aggressive)
• Note – MLH-1 may be lost in occasional non dysplastic crypts in otherwise typical SSA (≠ SSA/D)
• (also isolated non dysplastic crypts adjacent to Lynch syndrome cancers may show loss of MMR protein) Mod Pathol. 2018 Oct;31(10):1608-1618
MLH1 in SSA/D
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SSA
SSAD MSI TSA
BRAF MSI CRC BRAF MSS CRC
Pathways to BRAF mutant colorectal carcinoma
MLH1 loss MLH1
preserved
P53, p16
SSAD MSS
Uncertain
?p53
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MLH1 in SSA/D – usual/NOS type
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MLH1 in SSA/D – minimal deviation
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MLH1 in SSA/D - serrated
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• αFP producing tumours of the upper GIT (often stomach)(1) hepatoid type
(2) yolk sac type
(3) enteroblastic type foetal or
• Enteroblastic type:(1) columnar carcinoma cells growing primarily in tubulopapillary and glandular patterns
(2) abundant glycogen, but no mucin production in the clear cytoplasm
(3) gut hormone-containing cells scattered among clear carcinoma cells
(4) carcinoma cells producing oncofoetal glycoproteins such as αFP, SALL 4, glypican 3 and CEA
(5) ultrastructurally, carcinoma cells showing well-developed microvilli with core filaments, whose rootlets formed occasional terminal webs, consistent with absorptive epithelium of foetal intestine or enteroblastic differentiation.
Detection of enteroblastic/foetal type differentiation
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Detection of enteroblastic/foetal type differentiation
αFP
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• Importance• Production/expression of αFP
• Poor prognosis
• ?under recognised (often a component of otherwise typical adenocarcinoma)
Detection of enteroblastic/foetal type differentiation
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Vascular invasion markers
• Lymphatic invasion (LI) and venous invasion (VI) are important Px markers in all GIT malignancies
• LI is and adverse factor in all luminal pT1 tumours → usually an indication for extended resection
• VI is a factor suggesting CT for stage II CRC
• VI is a quality marker for CRC reporting• RCP structured report Sept 2018 = “At the current time, individual units should closely
monitor venous invasion rates and, if they are consistently below the 30% threshold, then the adoption of elastic staining as standard is recommended”
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Vascular invasion markers
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Vascular invasion markers
• What to do:
• LI – D2/40 ( not of much benefit)
• VI• Orcein*
• Other elastin stains e.g. VVG
• Desmin
• Other?
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Vascular invasion markers
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SATB2
• Transcription factor• Osteoblasts, colorectum, appendix, some urothelial/renal
• Personally – usefulness has been limited (vs cdx2) but can be synergistic• Peritoneal disease of unknown primary especially if signet
ring morphology or goblet cell adenocarcinoma• Ovarian adenocarcinoma• Occasional liver metastases
• Often lost in MMR deficient CRC
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SATB2
IBD related neoplasia
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• Hepatocellular adenoma typing
• Solid cystic pseudopapillary tumour of pancreas
• Fibromatosis
• Conventional adenoma vs reactive (e.g. at ampulla)
• Foveolar dysplasia in FGPs
• ‘morules’
Β catenin
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Β catenin
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Β catenin
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• Covered in current WHO
• Hans classifier [CD10, bcl6, MUM1; 30% IHC positivity cut off for each]• Germinal centre cell like
• Better Px• CD10 +, bcl6 +, MUM1 –
• Activated B cell like• Worse Px• CD10 -, bcl6 +, MUM1 –• More aggressive therapy
• Also search for double/triple hit lymphoma (5-15%) = Ki67 >90%• C-myc IHC ± progress to FISH (also include bcl2 and bcl6)• Think of EBV association
DLBCL subtyping
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• PAS stain for candida (often)
• Alcian blue/other for detection of goblet cells in Barrett’s oesophagus
• Collagen stains in most cases of collagenous colitis
• Desmin stain for small colorectal leiomyoma
• Stains for ‘fibroblastic polyp’ of colon
• ‘Panels’ for bland mesenchymal tumours of GIT
What I don’t do (usually)
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• ?MMR testing in all adenocarcinomas• Detect MSI → predict response to PD1 inhibitors (also detects Lynch syndrome)
Evolving concepts
Tumour type Frequency, % (n)
Colorectal cancer 13% (1066)
Endometrial 22% (543), 33% (446)
Gastric 22% (295)
Hepatocellular carcinoma
16% (37)
Ampullary carcinoma 10% (144)
Thyroid 63% (30)
Skin (sebaceous tumors)
35% (20), 60% (25)
Skin (melanoma) 11% (56)
Tumor type Frequency, % (n)
Ovarian 10% (1234)
Cervical 8% (344)
Esophageal adenocarcinoma 7% (76)
Soft-tissue sarcoma 5% (40)
Head and neck SCC 3% (153)
Renal cell carcinoma 2% (152)
Ewing sarcoma 2% (55)
Clin Cancer Res; 22(4); 813–20.
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• MUC stains• Barrett’s dysplasia/gastric dysplasia classification
• Gastric type adenomas
• Other (EMA)
Evolving concepts
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• Anything else???• Her2 in CRC
Evolving concepts