The Value of Immunocytochemistry - ICC & ISH · •Light chain restriction ... • Quality control...
Transcript of The Value of Immunocytochemistry - ICC & ISH · •Light chain restriction ... • Quality control...
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THE ROLE OF
IMMUNOHISTOCHEMISTRY
IN THE EVALUATION OF
LYMPHOID PROLIFERATIONS
Andrew C Wotherspoon
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LYMPHOMA DIAGNOSIS
• Fixation • Processing • Sectioning
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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
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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
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Genotypic alterations (DNA)
Transcription (mRNA)
Phenotype (protein expression)
Morphology (histology)
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Immunocytochemistry
Role of immunocytochemistry
• Primary diagnosis
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Immunocytochemistry
Uses • Identify cell type (lymphoid vs others)
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Infarcted tumour in bone marrow
CD45
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Focal sinus involvement by anaplastic large cell lymphoma
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Focal sinus involvement by anaplastic large cell lymphoma
CD30
ALK-1
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Immunocytochemistry
Uses • Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other)
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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)
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Lambda Kappa
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Dense lymphoid infiltrate in stomach
CD5
CD20
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Immunocytochemistry
Uses • Identify cell type (lymphoid vs others) • Determine cell lineage (B vs T vs other) • Determine clonality • Subclassify lymphoma
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• Diffuse large B-cell lymphoma
Germinal centre B-like DLBCL Activated B-like DLBCL
B-cell neoplasms
Alizadeh et al 2000
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Hans, C. P. et al. Blood 2004;103:275-282
“Activated B-cell” (non-GCB)
“Germinal centre B-cell”
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CD10 GCB
BCL-6
Non -GCB
MUM-1
GCB
Non –GCB (ABC)
Neg Neg
Neg
pos pos pos
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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
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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
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CD34
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CD21
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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
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ALK-1
Anaplastic large cell lymphoma
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CyclinD1
Mantle cell lymphoma
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Reactive lymph node
bcl-2
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Follicular lymphoma
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bcl-10 expression in MALT lymphoma
t(1;14) -ve
t(1;14) +ve
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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)
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Burkitt lymphoma
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Immunocytochemistry
Role of immunocytochemistry
• Primary diagnosis
• Detection of residual disease
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Hairy cell leukaemia Residual disease post therapy
CD20
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CD20
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CD5 CD3
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CD23
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Immunocytochemistry
• Needs appropriate tissue preparation • Fixation • Slide preparation
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CD20 72hrs fixation
No treatment
Microwave
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Factors affecting immunocytochemistry
• Fixative • Duration • pH • Type
• Processing • Temperature • Duration of dehydration and wax infiltration
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Immunocytochemistry
• Needs appropriate tissue preparation • Fixation • Slide preparation
• Quality control • Familiarity with staining patterns
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B-CLL
Post-Rituximab
CD23
CD5
CD79a
CD20
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Effect of microwave pre-treatment on poorly fixed/processed tissue
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CD20
CD79a
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Immunocytochemistry
• Needs appropriate tissue preparation • Fixation • Slide preparation
• Quality control • Familiarity with staining patterns • Knowledge of possibilities of aberrant
antigen expression
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CyclinD1 in myeloma
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Immunocytochemistry
Aberrant expression • Epithelial markers in lymphoid tumours
• EMA • Plasmacytoma • Anaplastic large cell lymphoma
• Cytokeratin • Plasmacytoma
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Cytokeratin in plasmacytoma
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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
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Immunocytochemistry
Aberrant expression • Epithelial markers in lymphoid tumours • Lymphoid markers in non-lymphoid
tumours • Non-lymphoid specific
• CD10 (CALLA) • CD56 (NCAM) • CD138
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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
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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
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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
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Factors affecting immunocytochemistry
• Slide preparation • Temperature and duration of slide drying
• Duration of slide storage has no influence
on quality of staining
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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
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Immunocytochemistry in lymphoma diagnosis
• Use in panels • Be aware of staining profiles
• Tumour • Antibody
• Be aware of artefacts • Fixation/processing • Treatment related • Tumour related
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A fool with a tool is still a fool
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THE MOST
IMPORTANT STAIN
IN
HISTOPATHOLOGY
IS
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H & E
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Immunohistochemistry, ISH and the
Diagnosis of Infectious Agents
Dr Naomi Guppy, PhD FIBMS
UCL-Advanced Diagnostics
Picture credits: Roche, ACDBio, Prof M Novelli
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Infectious Agents
• HPV – Human PapillomaVirus
• Herpesviruses – CMV, KSHV, HSV, EBV, VZV
• Adenovirus
• HIV – p24
• Bacteria – Treponema, Helicobacter, mycobacteria
• Parasites – Toxoplasma, Cryptosporidium
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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• 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)
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CIN1 CIN2 CIN3
E4 p16 MDM2 Van Baars et al (2015) Am J Surg Pathol
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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
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HPV and oropharyngeal SCC
Current testing algorithm (Singh & Westra, 2010; Schache, 2014)
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However…
• Discrepant cases do exist!
– p16+/HPV- ~5-20%
– p16-/HPV+ ~0-2% (contentious)
– Why?
– How do you manage these?
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Efficacy of HPV DNA-ISH
Robinson, Schache, Sloan & Thavaraj (2012) Head and Neck Pathol. 6: S83-90
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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
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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
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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.”
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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)
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Efficacy and validation of HPV RNA-ISH
Rooper et al, (2016) Oral Oncol. 55: 11-16
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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
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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
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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
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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
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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
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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.
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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
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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
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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
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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
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But it’s not all biopsies…
Images: Prof M Novelli CMV appendicitis in HIV/AIDS patient
Also: CMV lymphadenitis can mimic Hodgkins in remission
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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
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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
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H pylori IHC
Membranous colonisation pattern Low-level infection (residual)
Images: Prof M Novelli
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H pylori IHC
Minimal chronic gastritis post-Rx
Images: Prof M Novelli
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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
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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
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Treponema pallidum
19yo ♂, lesion on lip
Syphilitic chancre
Further case showed
treponemes in tonsils
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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