the value of immunocytochemistry - icc & ish · •light chain restriction ... • quality...
TRANSCRIPT
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
• Needs appropriate tissue preparation • Fixation • Slide preparation
• 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
• Needs appropriate tissue preparation • Fixation • Slide preparation
• 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
Factors affecting immunocytochemistry
• Slide preparation • Temperature and duration of slide drying
• Duration of slide storage has no influence
on quality of staining
Factors affecting immunocytochemistry
• 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
Grogan et al., (2006) Interpretation Guide for Ventana INFORM® HPV Probes In Situ Hybridization (ISH) Staining of Cervical Tissue
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
Grogan et al., (2006) Interpretation Guide for Ventana INFORM® HPV Probes In Situ Hybridization (ISH) Staining of Cervical Tissue
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.”
Efficacy and validation of HPV RNA-ISH
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)
Efficacy and validation of HPV RNA-ISH
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
Images: Prof M Novelli
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
Image: Prof M Novelli
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
Images: Prof M Novelli
CMV inclusions are often
rare, and present in a very
patchy distribution
Carefully examine all
levels on IHC, ISH may be
useful
Images: Prof M Novelli
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.
Image: Prof M Novelli
H pylori IHC
Membranous colonisation pattern Low-level infection (residual)
Images: Prof M Novelli
H pylori IHC
Minimal chronic gastritis post-Rx
Images: Prof M Novelli
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