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Utilisation of MCM2 staining in assessment of pOLT liver biopsies assessment of pOLT liver biopsies
with HCV infection.
Dr S E DaviesAddenbrookes Hospital
CambridgeCambridge
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HCV infection post transplantHCV infection post transplant
� Universal but Unusual c.f. native
Spectrum of changes in the graft; mild and � Spectrum of changes in the graft; mild and stable or slowly progressive; rapidly progressive either to cirrhosis or cholestatic form (<10%)
� 10-30% cirrhosis at 5 years
� Apparent increase in rate of fibrosis
� Although short term survival for HCV negative and positive similar now decreased patient and graft survival
� Time from cirrhosis to decompensation ↓
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Factors implicated in rapid Factors implicated in rapid progressionprogression
� Donor age
Viral load pre-transplant� Viral load pre-transplant
� Warm ischaemia time
� Immunosupression
Number of episodes of rejection- treated� Number of episodes of rejection- treated
� Severity of hepatitis at recurrence
� Degree of fibrosis at 1 year
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Cell Cycle Markers Cell Cycle Markers --Minichromosome Minichromosome maintenance proteins maintenance proteins ––MCM2MCM2
� Best known of M2-7 – family, preserved in � Best known of M2-7 – family, preserved in eukaryotes cells Tye, Annu Rev Biochem 99
� Present in G1 phase – necessary for gene replication in S phase
� Down regulated in differentiation and quiescence; usually in restrictive proliferative quiescence; usually in restrictive proliferative compartments
� Antibodies raised against MCM2 protein; formalin fixed paraffin embedded tissues Freeman Clin Cancer Res 99
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Minichromosome maintenance proteinMinichromosome maintenance protein--2, MCM22, MCM2
Cyclin D1
p21
G0 Cyclin A
G1
SM
MCM2
p21
Cyclin B1
Phospho-Histone H3 G2
M
Marks entry into cell cycle
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Methodology 1Methodology 1
PositivePositive andand negativenegative cellscells countedcounted
MCM2
�� PositivePositive andand negativenegative cellscells countedcounted
�� HighestHighest andand 44 randomrandom highhigh powerpower fieldsfields countedcounted ––toto givegive percentagepercentage
�� HighestHighest andand predominantpredominant valuesvalues forfor MCMMCM22
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HCV InfectionHCV Infection
� In non-transplant HCV chronic infection, MCM2 expression was analysed in 87 patientsexpression was analysed in 87 patients
� Randomly chosen fields
� Compared with Ki67
� Compared with Stage of disease i.e. fibrosis
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MCMMCM--2 expression in chronic HCV 2 expression in chronic HCV ––according to Stage of fibrosisaccording to Stage of fibrosis (not Ishak)(not Ishak)
25*
Marshall Gastroenterology
5
10
15
20
MCM-2Ki 67
* *
*
*
Med
ian
Med
ian
labe
llin
g in
dex
labe
llin
g in
dex
Gastroenterology 2005;128:33-42
0
5
0 1 2 3 4 5
*p<0.01*p<0.01
Fibrosis stageFibrosis stage
Med
ian
Med
ian
labe
llin
g in
dex
labe
llin
g in
dex
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Neither of these had ACR treated, donors > 60yrs, nor different immunosupression….
8 monthsmonths
5 months
Case 1 Case 2
5 years
20 months
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Post Transplant HCVPost Transplant HCV
� 1990-1999 all HCV positive recipients 1st graft n=44
� Yearly biopsies + clinically indicated
� Exclusion− ‹ 5yrs follow up n=8
− Insufficient tissue n=11
− Chronic rejection n=2− Chronic rejection n=2
− Biliary disease n=1
− Autoimmune n=1 n=23
� Ishak score + steatosis + standard IHC
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Grouped into 3Grouped into 3
� Fibrosis stage at last biopsy divided by years post transplant = number of stages per yearpost transplant = number of stages per year
� No/Minimal Progression− ≤ stage 2 at 5yrs n=5
� Rapid Progression− Stage 6 by 2-3 yrs n=8 Stage 6 by 2-3 yrs n=8
� Intermediate Progression− > stage 2 at 3yrs <stage 6 at 5yrs n=8
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IHCIHC
� First biopsy available after biochemical recurrencerecurrence
� Labelling index of positive within the total
� Two observers, ~ 1000 cells; now semi-automated of whole biopsy
� Wide range of times; NP av 91d, RP 73d, IP 274274
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Histology of BiopsiesHistology of BiopsiesGroup NP IP RP p value
Fat (0-3) 0 (0-1) 0 (0-2) 0 (0-1) 0.78
Confluent (0-6)
0 (0) 0.5 (0-2) 0 (0-3) 0.13
Interface(0-4)
2 (0-2) 1 (0-2) 1.5 (0-3) 0.66
Lobular(0-4)
2 (1-2) 2 (1-4) 3 (2-4) 0.026
Portal (0- 2 (1-2) 1.5 (1-3) 2 (1-4) 0.85Portal (0-4)
2 (1-2) 1.5 (1-3) 2 (1-4) 0.85
HAI (0-18) 6 (2-6) 5 (2-11) 6.5 (3-13) 0.47
Fibrosis (0-6)
1 (0-2) 1 (0-3) 2 (1-5) 0.3
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MCM2 expression in early liver biopsies following MCM2 expression in early liver biopsies following OLT for HCV cirrhosisOLT for HCV cirrhosis
NonNon--progressorprogressor Intermediate Intermediate progressorprogressor
Rapid progressorRapid progressor
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NonNon--Progressor Progressor ––MCM2 stainingMCM2 staining
8 months
5 years
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Rapid Progressor Rapid Progressor –– MCM2 stainingMCM2 staining
5 months
20 months
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MCM2 labelling index in different MCM2 labelling index in different progression groupsprogression groups
Bars are the median
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ImmunosuppressionImmunosuppression
� All had initial triple therapy− Cyc or tacro + azathioprine + prednisolone− Cyc or tacro + azathioprine + prednisolone
� Prednisolone tapered to end at 3 months; Aza stopped at 6 months
� No difference between cyclosporin and tarcolimus in 3 groups
� 6 (29%) had x1 ACR; all Ŗ x3 iv methyl pred− All responded
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HCV versus/and/or ACRHCV versus/and/or ACR
� Immunostaining for Mcm-2 of portal tracts Unitt Liver Transpl 2009; 15: 306-312Liver Transpl 2009; 15: 306-312
� 99 liver biopsies − 31 patients with HCV graft infection,
− 22 patients with ACR
− 11 patients with HCV infection and unexplained graft dysfunction graft dysfunction
� Semi-automated counting per PT and per mm2 of PT
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Typical MCM2 staining in ACR Typical MCM2 staining in ACR ––non HCV patientnon HCV patient
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ImageJ software, NIH
I. Cut out of PT, subtract I. Cut out of PT, subtract background noise
II. Transfer into B&W – area of PT
III. Set threshold, identify positive cells
IV. and all nuclei determined
V. With set threshold mask and V. With set threshold mask and a watershed…ie operator dependant
VI. Nuclear count of positive and negative cells
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Cut off of 107 pos cells per portal tract, 1609 per mm 2, sensitivity of 81.8% and a specificity of 91.9%; positive predictive value of 66.67% and negative predictive value of 95.75%
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In difficult cases…In difficult cases…
� 11 HCV-infected patients with uncertain diagnosis, review of all clinical and histological diagnosis, review of all clinical and histological parameters, including improved LFTs post treatment for rejection
� 7 HCV thought graft infection alone, 4 superimposed corticosteroid-responsive ACR
MCM2 staining separated these; 85 v 235 per � MCM2 staining separated these; 85 v 235 per PT…but range (17-121) v (120-471)
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3 months in 33 months in 3rdrd graftgraft
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Possible focal rejection?Possible focal rejection?
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Use of MCM2?Use of MCM2?
� Possibility of identifying the rapid progressors early, alter immunosupression, consider other early, alter immunosupression, consider other anti-viral strategies.
� Help understand pathogenesis; implications for cell cycle arrest.
� Adjunct in complex histology of HCV and rejectionrejection
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AcknowledgementsAcknowledgements
� Aileen Marshall
Will Gelson� Will Gelson
� Esther Unitt
� Simon Rushbrook
Nick Coleman� Nick Coleman
� Graeme Alexander