IHC And Special Stains In Daily Practice

Dr Ian Brown

Envoi Pathology

Brisbane, Australia

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

• 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

• Features of AI gastritis

Gastrin for autoimmune gastritis

• Some cases have• minimal or no intestinal metaplasia

• minimal or no history

• Just labelled as ‘gastric’

Gastrin for autoimmune gastritis

Gastrin for autoimmune gastritis

• Always do a H.pylori stain also• Co-existence

• mimic

• ? NE stains (synaptophysin) increase the diagnostic yield

Gastrin for autoimmune gastritis

Atrophic pattern of collagenous gastritis

Atrophic pattern of collagenous gastritis

• 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

H.pylori

• Barrett’s dysplasia

• IBD dysplasia

Use of p53

• 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

• 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

• 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

• Patterns

p53 in Barrett’s dysplasia

Over-expression

Loss of expression

Courtesy of Priyanthi Kumarasinghe

p53 in IBD dysplasia

AJG 2011

p53 in IBD

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

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

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

Refractory coeliac disease

• 2 reasons

1) Detect Lynch syndrome

2) Indicate response for PD-1 inhibitors

? Universal testing or selective

MMR stains in CRC

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%)

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.

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

• 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

• 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

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

MLH1 in SSA/D – usual/NOS type

MLH1 in SSA/D – minimal deviation

MLH1 in SSA/D - serrated

• 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

Detection of enteroblastic/foetal type differentiation

αFP

• Importance• Production/expression of αFP

• Poor prognosis

• ?under recognised (often a component of otherwise typical adenocarcinoma)

Detection of enteroblastic/foetal type differentiation

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”

Vascular invasion markers

Vascular invasion markers

• What to do:

• LI – D2/40 ( not of much benefit)

• VI• Orcein*

• Other elastin stains e.g. VVG

• Desmin

• Other?

Vascular invasion markers

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

SATB2

IBD related neoplasia

• 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

Β catenin

Β catenin

• 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

• 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)

• ?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.

• MUC stains• Barrett’s dysplasia/gastric dysplasia classification

• Gastric type adenomas

• Other (EMA)

Evolving concepts

• Anything else???• Her2 in CRC

Evolving concepts