The Value of Immunocytochemistry - ICC & ISH · •Light chain restriction ... • Quality control • Familiarity with staining patterns ... CMV, KSHV, HSV, EBV, VZV •Adenovirus

THE ROLE OF

IMMUNOHISTOCHEMISTRY

IN THE EVALUATION OF

LYMPHOID PROLIFERATIONS

Andrew C Wotherspoon

LYMPHOMA DIAGNOSIS

• Fixation • Processing • Sectioning

B-CELL NEOPLASMS Precursor B-cell neoplasms Precursor B lymphoblastic leukaemia / lymphoma Mature B-cell neoplasms Chronic lymphocytic leukaemia / small lymphocytic lymphoma B-prolymphocytic leukaemia Lymphoplasmacytic lymphoma Splenic marginal zone lymphoma Splenic B-cell lymphoma, unclassifiable Splenic diffuse red pulp small B-cell lymphoma Hairy cell leukaemia-variant Hairy cell leukaemia Plasma cell myeloma Solitary plasmacytoma of bone Extraosseous plasmacytoma Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) Nodal marginal zone B-cell lymphoma Follicular lymphoma Primary cutaneous follicle centre lymphoma Mantle cell lymphoma Diffuse large B-cell lymphoma, NOS T-cell/histiocyte-rich large B-cell lymphoma Primary DLBCL of CNS Primary cutaneous DLBCL, leg type DLBCL associated with chronic inflammation Lymphomatoid granulomatosis Primary mediastinal (thymic) large B-cell lymphoma Intravascular large B-cell lymphoma ALK-positive large B-cell lymphoma Plasmablastic lymphoma Large B-cell lymphoma arising in HHV-8 associated Castleman disease Primary effusion lymphoma Burkitt lymphoma B-cell lymphoma, unclassifiable, with features intermediate b/w DLBCL and BL B-cell lymphoma, unclassifiable, with features intermediate b/w DLBCL and cHL

T-CELL AND NK-CELL NEOPLASMS Precursor T-cell neoplasms Precursor T lymphoblastic leukaemia / lymphoma Blastic NK cell lymphoma Mature T-cell and NK cell neoplasms T-cell prolymphocytic leukaemia T-cell large granular lymphocytic leukaemia Chronic lymphoproliferative disorder of NK Aggressive NK cell leukaemia EBV-positive T-cell lymphoproliferative diseases of childhood Adult T-cell leukaemia/lymphoma Enteropathy-type T-cell lymphoma Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Mycosis fungoides Sezary syndrome Primary cutaneous CD30 positive lymphoproliferative disorders Primary cutaneous peripheral T-cell lymphomas, rare subtypes Peripheral T-cell lymphoma, NOS Angioimmunoblastic T-cell lymphoma Anaplastic large cell lymphoma, ALK positive Anaplastic large cell lymphoma, ALK negative

Genotypic alterations (DNA)

Transcription (mRNA)

Phenotype (protein expression)

Morphology (histology)

Immunocytochemistry

Role of immunocytochemistry

• Primary diagnosis

Immunocytochemistry

Uses • Identify cell type (lymphoid vs others)

Infarcted tumour in bone marrow

CD45

Focal sinus involvement by anaplastic large cell lymphoma

Focal sinus involvement by anaplastic large cell lymphoma

CD30

ALK-1

Immunocytochemistry

Uses • Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other)

Immunocytochemistry

Uses • Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other) • Determine clonality

• Light chain restriction • Aberrant/unusual expression (eg CD5 in B cell

NHL)

Lambda Kappa

Dense lymphoid infiltrate in stomach

CD5

CD20

Immunocytochemistry

Uses • Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other) • Determine clonality • Subclassify lymphoma

• Diffuse large B-cell lymphoma

Germinal centre B-like DLBCL Activated B-like DLBCL

B-cell neoplasms

Alizadeh et al 2000

Hans, C. P. et al. Blood 2004;103:275-282

“Activated B-cell” (non-GCB)

“Germinal centre B-cell”

CD10 GCB

BCL-6

Non -GCB

MUM-1

GCB

Non –GCB (ABC)

Neg Neg

Neg

pos pos pos

Diagnostic approach to HBCLs. Lymphomas that potentially fall into the HGBL categories can morphologically resemble B-lymphoblastic leukemia/lymphoma (B-LBL), BL, and DLBCL as

well as lymphomas that are intermediate between DLBCL and BL (DLBCL/BL).

Steven H. Swerdlow et al. Blood 2016;127:2375-2390

©2016 by American Society of Hematology

Immunocytochemistry

Uses • Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other) • Determine clonality • Subclassify lymphoma • Identify underlying structures and background

CD34

CD21

Immunocytochemistry

Uses • Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other) • Determine clonality • Subclassify lymphoma • Identify underlying structures and background • Identify abnormal/inappropriate protein

expression that characterizes disease type

ALK-1

Anaplastic large cell lymphoma

CyclinD1

Mantle cell lymphoma

Reactive lymph node

bcl-2

Follicular lymphoma

bcl-10 expression in MALT lymphoma

t(1;14) -ve

t(1;14) +ve

Immunocytochemistry Uses

• Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other) • Determine clonality • Subclassify lymphoma • Identify underlying structures and background • Identify abnormal/inappropriate protein

expression that characterizes disease type • Quantify cell attributes (eg proliferation)

Burkitt lymphoma

Immunocytochemistry

Role of immunocytochemistry

• Primary diagnosis

• Detection of residual disease

Hairy cell leukaemia Residual disease post therapy

CD20

CD20

CD5 CD3

CD23

Immunocytochemistry

• Needs appropriate tissue preparation • Fixation • Slide preparation

CD20 72hrs fixation

No treatment

Microwave

Factors affecting immunocytochemistry

• Fixative • Duration • pH • Type

• Processing • Temperature • Duration of dehydration and wax infiltration

Immunocytochemistry


• Quality control • Familiarity with staining patterns

B-CLL

Post-Rituximab

CD23

CD5

CD79a

CD20

Effect of microwave pre-treatment on poorly fixed/processed tissue

CD20

CD79a

Immunocytochemistry


• Quality control • Familiarity with staining patterns • Knowledge of possibilities of aberrant

antigen expression

CyclinD1 in myeloma

Immunocytochemistry

Aberrant expression • Epithelial markers in lymphoid tumours

• EMA • Plasmacytoma • Anaplastic large cell lymphoma

• Cytokeratin • Plasmacytoma

Cytokeratin in plasmacytoma

Immunocytochemistry

Aberrant expression • Epithelial markers in lymphoid tumours • Lymphoid markers in non-lymphoid

tumours • CD45 in undifferentiated and neuroendocrine

carcinomas • CD68 in melanoma, granular cell tumour

Immunocytochemistry

Aberrant expression • Epithelial markers in lymphoid tumours • Lymphoid markers in non-lymphoid

tumours • Non-lymphoid specific

• CD10 (CALLA) • CD56 (NCAM) • CD138

Immunocytochemistry

• While the results of immunocytochemical staining can be invaluable in the assessment of atypical cellular proliferations, it must be interpreted in context of • Morphology (H&E stained section) • Other special techniques • Clinical history

Immunocytochemistry

• One of many tools in the armamentarium of the Histopathologist trying to unravel the diagnosis of complicated cases.

• Widely available, but • needs to used in an organized fashion (panels) • needs to be interpreted with caution

Immunocytochemistry is a powerful tool in the investigation of lymphoid (and other) malignancies.

It maybe of vital help in unraveling difficult

diagnostic challenges but it may also mislead and confuse


• Slide preparation • Temperature and duration of slide drying

• Duration of slide storage has no influence

on quality of staining


• Different antibodies are affected by different variables

• Where weak staining is encountered antigen

retrieval/unmasking (eg microwave) can usually eliminate the problem

Immunocytochemistry in lymphoma diagnosis

• Use in panels • Be aware of staining profiles

• Tumour • Antibody

• Be aware of artefacts • Fixation/processing • Treatment related • Tumour related

A fool with a tool is still a fool

THE MOST

IMPORTANT STAIN

IN

HISTOPATHOLOGY

IS

H & E

Immunohistochemistry, ISH and the

Diagnosis of Infectious Agents

Dr Naomi Guppy, PhD FIBMS

UCL-Advanced Diagnostics

Picture credits: Roche, ACDBio, Prof M Novelli

Infectious Agents

• HPV – Human PapillomaVirus

• Herpesviruses – CMV, KSHV, HSV, EBV, VZV

• Adenovirus

• HIV – p24

• Bacteria – Treponema, Helicobacter, mycobacteria

• Parasites – Toxoplasma, Cryptosporidium

Human Papillomavirus (HPV)

• Many family

members

• Oncogenic and

non-oncogenic

members are α

papillomaviruses

• High risk and low

risk variants not

closely related Biology and life-cycle of human papillomaviruses. Doorbar et al

(2012) Vaccine 30(3): F55-70

HPV structure

Biology and life-cycle of human papillomaviruses. Doorbar et al

(2012) Vaccine 30(3): F55-70

Viral Proteins

• Early (E1-E7)

• Late (L1, L2)

E4

• Viral replication

• Accumulates in cells supporting viral

synthesis

E6

• Dysregulated in high risk

• Binds and degrades p53

• Promotes cell proliferation

E7

• Dysregulated in high risk

• Promotes cell proliferation via pRb

• Causes overexpression of p16

L1

• Abundant capsid protein, detectable

when viral particles about to be shed

HPV infection

• Wounding of stratified epithelia allows virus access to proliferating basal cells

• Viral episomes accumulate in normally proliferating basal cells

• Virus replicates as cells differentiate and rise towards the superficial layers

• Viral particles shed at the surface

Biology and life-cycle of human papillomaviruses. Doorbar et al (2012) Vaccine 30(3): F55-70

Productive vs. disordered HPV infection

Virus release

Virus assembly

Genome Amplification

Genome

maintenance/cell

proliferation

Genome maintenance

• Viral shedding

• E4 high, E6/E7 restricted

• L1 expression

• Episomal

• No viral shedding

• E4 low, E6/E7 widespread

• No L1 expression

• Integrated/transformative

Egawa, et al. (2015) Viruses 7: 3863-90

Productive vs. disordered HPV infection

Grogan et al., (2006) Interpretation Guide for Ventana INFORM® HPV Probes In Situ Hybridization (ISH) Staining of Cervical Tissue

Viral genome copy number increases with lesion growth until transformation

Copy number then declines following integration

HPV in the lower anogenital tract

• Warts (condylomata)

• HPV implicated in:

– cervical neoplasia (probably all)

– vulval, anal, penile squamous neoplasia

(most)

• Historical three-tier stratification (-IN1, 2, 3)

• Pathobiology favours two-tiers

– Low malignant potential, probable regression, productive infection

– High malignant potential, likely progression, disordered infection

Image: Prof M Novelli

HPV testing in lower anogenital lesions

Morphology

• koilocytes, dysplasia

IHC

• L1 - late capsid protein

expressed in productive

infection

• p16 - indicator of viral-

induced cell-cycle

dysregulation

• MIB-1 – indicator of

dysplasia

H&E HPV L1

MIB1 p16

Images: Prof M Novelli

HPV testing in lower anogenital lesions

• ISH – subtyping, episomal and integrated

• DNA-ISH for viral genome, active and ??latent (Roche INFORM)

• RNA-ISH for E6/E7 expression, active transcription (RNAScope)

• PCR – active and latent, with typing

• Does not differentiate active/established from transient infection

• Specificity lower than sensitivity

Episomal Integrated


Lower anogenital tract intra-epithelial lesions

LAST project (2012, CAP/NSCCP)

Darragh, et al. (2013) Int J Gyn Path 22: 76-115

All lower anogenital

tract squamous

lesions share

pathobiology

Standardise

terminology

LAST classification recommendations

• Important biological distinction between low and

high risk lesions

– LSIL (-IN1), productive viral infection, regress

• Low grade intra-epithelial lesion

– HSIL (-IN2, -IN3), disordered viral infection, progress

• High grade intra-epithelial lesion

• 30-50% progress, heterogeneous

Aims: reduce over-treatment of LSIL, improve

classification of –IN2 lesions and increase

reproducibility of diagnosis

LAST biomarker recommendations

• p16 IHC is recommended where:

– HSIL vs mimics

– CIN2 suspected

– Professional disagreement LSIL vs HSIL

– High risk but lesion morphologically <LSIL

?CIN2 Yes – “block positive” p16 IHC

LAST biomarker recommendations

• HPV testing/subtyping is NOT indicated – i.e. L1, HPV 16/18 mRNA, HPV genotyping

– Strong, block p16 positivity supports viral dysregulation and

diagnosis of HSIL

• MIB-1 may prove to be of value – More evidence required

• Although p16 costly, <20% of all cx. bx. would

require testing

Further developments

• Poor concordance grading CIN on morphology

• Transforming HPV infections fail to differentiate and produce E4

• Raised an antibody to E4 of 15 HR-HPV subtypes

• 104 cx samples positive for HR-HPV

• Graded by pathologists at 4 centres (morphology; κ ~ 0.4)

• IHC for panHPVE4, p16 and MDM2

• Scored for extent of all three markers and re-graded (κ ~ 0.9)

CIN1 CIN2 CIN3

E4 p16 MDM2 Van Baars et al (2015) Am J Surg Pathol

HPV in the oropharynx

• HPV-related SCC vs. others

– Distinct pathological entity

– “Basaloid” morphology,

lymphocytic infiltrate, non-

keratinising

• Lesions present at the CIS stage

• HPV typing predicts progression

• HPV subtyping indicated (2013

RCPath guidelines, NCCN, CAP)

Schache (2014) Meth. Mol. Biol. Ch. 13

HPV and oropharyngeal SCC

Current testing algorithm (Singh & Westra, 2010; Schache, 2014)

However…

• Discrepant cases do exist!

– p16+/HPV- ~5-20%

– p16-/HPV+ ~0-2% (contentious)

– Why?

– How do you manage these?

Efficacy of HPV DNA-ISH

Robinson, Schache, Sloan & Thavaraj (2012) Head and Neck Pathol. 6: S83-90

Issues with HPV typing via DNA-ISH

Ventana INFORM (subtypes 6/11; 16, 18, 31, 33, 35, 39, 45,

51, 52, 56, 58, and 66)

• Artefacts and non-specific staining

Precipitate – remove with acetone

Importance of slide choice – TOMO

Contingent on high contrast chromogen (BCIP/NBT)

• Low copy number integrated signal only visible at high

power

Leica Bond (subtypes 6/11; 16, 18, 31, 33, 51)

• DAB chromogen insufficient to reliably reveal low copy

number signal

Ventana INFORM interpretation pitfalls

Drying artefact Chromogen precipitate

Nucleolar staining Non-specific nuclear stain


Efficacy and validation of HPV RNA-ISH

Schache, et al., (2013) Br. J. Cancer

“…HR HPV RNAScope could

be developed to provide the

‘clinical standard’ for assigning

a diagnosis of HPV-related

OPSCC.”


Compared RNAScope probes for high risk E6/E7 with other RNA

and DNA ISH methods in p16+/HPV- cases (where HPV- was a

result of Roche INFORM HPV high testing)


Rooper et al, (2016) Oral Oncol. 55: 11-16

Ventana INFORM vs RNAScope (HPV hi)

Case 2

D-ISH -ive

Case 1

D-ISH +ive

A B

C D

Ventana INFORM RNAScope

Rooper et al, (2016) Oral Oncol. 55: 11-16

RNAScope

Single-molecule visualization

Novel probe design with a hybridization-based amplification system

Simultaneously amplifies signals and suppresses background

8000 labels for each target RNA with 20 probe pairs over 1kb

Wang, et al., (2012) J. Mol. Diag. 14: 22-29

RNAScope

PROS

• Novel, robust RNA-ISH technology

• Flexibility of probe design – detect more subtypes

• Sensitivity – can detect single mRNAs

• Specificity – “z-linker” oligo-probe pairs reduce non-specific binding

• Runs on automated platforms

– Ventana Ultra

– Leica Bond Rx

RNAScope

CONS

• Digestion times determined case-by-case – not needed in v3

• Expensive – but low volume

• May still have issues contingent on chromogen

– BCIP/NBT precipitate on Ventana

– But less likely to confuse due to high signal intensity

• Not yet available as CE-IVD – Developing IVD on Leica Bond III

for FDA approval in US

• ACDBio acquisition

Herpesviruses

– Cytomegalovirus (HHV-5)

– KSHV (HHV-8)

– Herpes simplex (Type 1 and Type 2)

– Epstein-Barr virus (HHV-4)

– Varicella (Herpes) zoster virus

• Typically:

– DNA genome (very large)

– Replication in cell nuclei, then cell death on release

– Latency and recurrence

HHV8

EBV


Cytomegalovirus (CMV)

• dsDNA virus (HHV-5)

• High prevalence - 60-90% population seropositive (USA)

• Vertical and horizontal transmission

• Asymptomatic infection – rarely, flu-like symptoms,

lymphadenopathy, splenomegaly (mono-like)

• Latency - reactivation/disease on immunosuppression

Images: M Novelli, XH Dai

Crough and Khanna (2009) Clin. Micro. Rev.

CMV Disease

• Neonatal/congenital – Prevalence 0.64%, due to primary infection

during pregnancy

– Deafness, mental impairment, cerebral palsy,

fatal in 10%

• Immunosuppression – Tx: Primary (with transplant) or recurrent (from

latency)

– Fever, pneumonia, hepatitis, encephalitis,

myelitis, retinitis, extensive colitis

– HIV: characteristic retinitis

– Dependent on viral load: viral activity

predicts outcome/severity


CMV Treatment

• Intravenous valganciclovir, ganciclovir - nucleoside

analogues that inhibit DNA synthesis

• Toxic – neutropaenia, thrombocytopaenia

• Prophylaxis - hampered by toxicity, drug interactions and antiviral

resistance

• Vaccines in development • Most failed at Phase I

• Phase II clinical trial results 2014

• gb/MF59 gave 50% efficacy in normal postpartum female recipients

• DNA vaccine TransVax limited viraemia in transplant patients

Rieder and Steininger (2014) Clin Microbiol Infect

CMV Diagnosis

• Always in the context of clinical suspicion

• Shell vial assay – tissue culture, lengthy

• Seropositivity - ↑IgM, ↑IgG if recent, false +ives from HHV-6/EBV

• Morphology – active disease, inclusion bodies on H&E

Inclusions

predominantly in

endothelial cells


CMV inclusions are often

rare, and present in a very

patchy distribution

Carefully examine all

levels on IHC, ISH may be

useful


But it’s not all biopsies…

Images: Prof M Novelli CMV appendicitis in HIV/AIDS patient

Also: CMV lymphadenitis can mimic Hodgkins in remission

CMV IHC More sensitive than

H&E

Nuclear or nuclear and

cytoplasmic depending

on clone

Antibodies to late

proteins (pp65) stain

nuclei and cytoplasm

Antibodies to late and

early proteins

(CCH2+DDG9, 8B1.2,

1G5.2 & 2D4.2) more

sensitive, additionally

stain nuclei of cells

with early infection Images: Prof M Novelli

Bacteria

• Helicobacter pylori

– Implicated in gastritis and gastric carcinoma

– CLO test for Dx but negative post-Rx

– Serology not useful post Rx

• Why not just H&E and special stains?

– Non-specific, background, debris

– Low level infection clinically significant

– Blanket prospective special stains

• Costly, low hit-rate

• IHC probably more efficient – Lash & Genta (2016) Helicobacter; Pitman et al.,

(2016) Human Pathol.; Yantis (2012) Am J Clin Pathol.


H pylori IHC

Membranous colonisation pattern Low-level infection (residual)


H pylori IHC

Minimal chronic gastritis post-Rx


Treponema pallidum

– Spirochaete

– Syphilis

• Congenital

• Primary, secondary, tertiary

• Painless sores

• Can be asymptomatic

– Huge rise in cases

• 20% rise 2014-15

• 76% rise since 2012

• 84% cases in young MSM

Public Health England (2016), Health Protection Report: Infection

Report 10: 22

Treponema pallidum

IHC

– Syphilis

– Intestinal spirochaetosis

– More sensitive than silver stains, PCR and serology

• 71% vs 41% for silver stains - Hoang, et al. (2004) J. Cutan. Pathol.

• 92% vs 72% for PCR - Buffet, et al. (2007) J. Invest. Dermatol.

• 90% vs 70% for serology - Phelps, et al. (2000) Int. J. Dermatol.

– Antibody specificities may be unknown

• May react with Borellia sp.(Lyme), T. pertenue (yaws),T.

carateum (pinta) and many other spirochaetes

– Organisms may be scanty (serial sections) - Putri, et al. (2013)

Int. J. Dermatol. Image: Prof M Novelli

Treponema pallidum

19yo ♂, lesion on lip

Syphilitic chancre

Further case showed

treponemes in tonsils

Acknowledgements

UKNEQAS ICC/ISH

UCL-Advanced Diagnostics

Pathological Society of Great Britain

and Ireland

UCL

Professor Marco Novelli

Dr Manuel Rodriguez-Justo

Dr Alex Freeman

Cambridge

Dr John Doorbar

Edinburgh

Professor Simon Herrington

The Value of Immunocytochemistry - ICC & ISH · •Light chain restriction ... • Quality control • Familiarity with staining patterns ... CMV, KSHV, HSV, EBV, VZV •Adenovirus

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