IASG IASG Romanian Society of SurgeryRomanian Society of Surgery

RomtransplantRomtransplant

The V-th Symposium andThe V-th Symposium and PPostostgraduate Course ofgraduate Course of IASG IASG

Bucharest 9-11 April 2003Bucharest 9-11 April 2003

““DOMINO” LIVER DOMINO” LIVER TRANSPLANTATIONTRANSPLANTATION

WITH PARTICULAR INDICATIONWITH PARTICULAR INDICATION(FAMILIAL HYPERCHOLESTEROLEMIA)(FAMILIAL HYPERCHOLESTEROLEMIA)

““DOMINO” LIVER DOMINO” LIVER TRANSPLANTATIONTRANSPLANTATION

WITH PARTICULAR INDICATIONWITH PARTICULAR INDICATION(FAMILIAL HYPERCHOLESTEROLEMIA)(FAMILIAL HYPERCHOLESTEROLEMIA)

I POPESCU, M SIMIONESCU, I POPESCU, M SIMIONESCU, D TULBURE, A SIMA, C CATANA, D TULBURE, A SIMA, C CATANA,

L NICULESCU, N HANCU, L GHEORGHE, L NICULESCU, N HANCU, L GHEORGHE, M MIHAILA, S CIUREA, V VIDU, M MIHAILA, S CIUREA, V VIDU,

D HREHORETD HREHORET

I POPESCU, M SIMIONESCU, I POPESCU, M SIMIONESCU, D TULBURE, A SIMA, C CATANA, D TULBURE, A SIMA, C CATANA,

L NICULESCU, N HANCU, L GHEORGHE, L NICULESCU, N HANCU, L GHEORGHE, M MIHAILA, S CIUREA, V VIDU, M MIHAILA, S CIUREA, V VIDU,

D HREHORETD HREHORET

CENTER OF GENERAL SURGERY AND LIVER TRANSPLANTATIONCENTER OF GENERAL SURGERY AND LIVER TRANSPLANTATIONDEPARTMENT OF INTENSIVE CARE AND ANESTHESIOLOGYDEPARTMENT OF INTENSIVE CARE AND ANESTHESIOLOGY

CENTER OF GASTROENTEROLOGYCENTER OF GASTROENTEROLOGYFUFUNNDENIDENI CLINICAL INSTITUTE CLINICAL INSTITUTE

INSTITUTE OF CELLULAR BIOLOGY AND PATHOLOGY "NICOLAE SIMIONESCU“INSTITUTE OF CELLULAR BIOLOGY AND PATHOLOGY "NICOLAE SIMIONESCU“

INSTITUTE OF DIABETES AND NUTRITIONAL DISEASES CLUJ-NAPOCA INSTITUTE OF DIABETES AND NUTRITIONAL DISEASES CLUJ-NAPOCA

INTRODUCTIONINTRODUCTION

number of donors in the worldnumber of donors in the world

0.25-32 / 1,000,000 inhabitants0.25-32 / 1,000,000 inhabitants status in Romaniastatus in Romania

1 / 1,000,000 inhabitants1 / 1,000,000 inhabitants death on the waiting listdeath on the waiting list alternatives ? alternatives ?

ALTERNATIVESALTERNATIVES

Marginal liversMarginal livers• Marginal cadaver donorsMarginal cadaver donors

• Old donorsOld donors• Non-heart beating donorsNon-heart beating donors• B or C positive donorsB or C positive donors• Split liversSplit livers• Metabolically defective liversMetabolically defective livers

Ethical problems of allocationEthical problems of allocation

• Marginal recipientsMarginal recipients• Cancer patients- short life expectancyCancer patients- short life expectancy

““DOMINO” LIVER DOMINO” LIVER TRANSPLANTATION:TRANSPLANTATION:

REASONS ?REASONS ? low number of donorslow number of donors patients in which transplantation patients in which transplantation

is a marginal solutionis a marginal solution it had only been performed for:it had only been performed for:

familial amyloidosis familial amyloidosis (polyneuropathy)(polyneuropathy)

primary hyperoxaluria primary hyperoxaluria

Homozygous Familial Homozygous Familial HypercholesterolemiaHypercholesterolemia

DefinitionDefinition• disorder of the lipoprotein metabolism, disorder of the lipoprotein metabolism,

autosomally dominant, determined by the autosomally dominant, determined by the absence or dysfunction of low density absence or dysfunction of low density lipoproteins receptors (LDL-R), which causes an lipoproteins receptors (LDL-R), which causes an increase in the plasmatic level of LDL-increase in the plasmatic level of LDL-cholesterol, and consequently premature cholesterol, and consequently premature atherosclerosisatherosclerosis

The liver plays a major role in cholesterol The liver plays a major role in cholesterol synthesis and catabolismsynthesis and catabolism

• 50-75% of LDL-R are in the liver50-75% of LDL-R are in the liver• 25-50% are in extrahepatic tissues such as 25-50% are in extrahepatic tissues such as

muscles, adipocytes, endotheliummuscles, adipocytes, endothelium frequency frequency

• homozygous type 1/1,000,000 inhabitantshomozygous type 1/1,000,000 inhabitants• heterozygous type 1/500 inhabitantsheterozygous type 1/500 inhabitants

PATHOGENESISPATHOGENESIS

genetic defficiency of LDL receptorsgenetic defficiency of LDL receptors RRLDLLDL 50-75% hepatic; 25-50% 50-75% hepatic; 25-50%

extrahepaticextrahepatic responsible gene (allele 150-500); responsible gene (allele 150-500);

chromosome 19chromosome 19 Genetic categoriesGenetic categories

1. R - negative1. R - negative

2. R - defective (R2. R - defective (RLDLLDL: 5-25%) : 5-25%) 3. R - deficit of internalization3. R - deficit of internalization

HOMOZYGOUS TYPEHOMOZYGOUS TYPE

familiy history familiy history early onset < 10 yearsearly onset < 10 years xanthomas, xanthelasmas xanthomas, xanthelasmas severe early systemic severe early systemic

atheromatosisatheromatosis RRLDLLDL < 20% < 20%

HETEROZYGOUS TYPEHETEROZYGOUS TYPE

late onset > 40 yearslate onset > 40 years moderate systemic moderate systemic

atheromatosisatheromatosis RRLDLLDL 20-70% 20-70%

TREATMENTTREATMENT

1984 - Th.E. STARZL 1984 - Th.E. STARZL - LIVER - LIVER TRANSPLANTATION TRANSPLANTATION in the homozygous in the homozygous typetype

STARZL TE, BILHEIMER DW, BAHNSON HT, SHAW BW, Jr., HARDESTY RL, GRIFFITH BP, IWATSUKI S, ZITELLI BJ, GARTNER JC,

Jr., MALATACK JJ. - Heart-liver transplantation in a patient with familial hypercholesterolaemia. Lancet, 1984, 1(8391):1382-1383

CASE PRESENTATIONCASE PRESENTATION

DOMINODOMINOLIVER TRANSPLANTATIONLIVER TRANSPLANTATION

- THE DONOR -- THE DONOR -

HISTORY OF THE DISEASEHISTORY OF THE DISEASE 22 year-old female 22 year-old female homozygous type FHChomozygous type FHC –– onset onset at the age of at the age of

1212 family historyfamily history

mothermother heterozygous Rheterozygous RLDLLDL = = 29.229.2 dyslipidemia ( cholesterol - 372mg/dL)dyslipidemia ( cholesterol - 372mg/dL) systemic atheromatosissystemic atheromatosis arterial hypertensionarterial hypertension

fatherfather heterozygous Rheterozygous RLDLLDL = = 31,431,4 dyslipidemia (cholesteroldyslipidemia (cholesterol - 400 mg/dl)- 400 mg/dl) systemic atheromatosissystemic atheromatosis

Genealogical tree of the patient with homozygous familial hypercholesterolemia

SYMPTOMATOLOGYSYMPTOMATOLOGY

cutaneouscutaneous• xanthomasxanthomas• xanthelasmasxanthelasmas

cardiovascularcardiovascular• low threshold anginalow threshold angina• dyspnea after minimal dyspnea after minimal

efforteffort

BIOLOGYBIOLOGY

DYSLIPIDEMIADYSLIPIDEMIA total cholesteroltotal cholesterol

800 mg/dl800 mg/dl500 mg/dl under Simvastatin (NV 150-200 500 mg/dl under Simvastatin (NV 150-200

mg/dl)mg/dl) LDL cholesterol 286 mg/dl (NV<120 LDL cholesterol 286 mg/dl (NV<120

mg/dl)mg/dl) triglycerides – 84 mg/dl (NV< 100 triglycerides – 84 mg/dl (NV< 100

mg/dl)mg/dl)

other liver tests within normal limitsother liver tests within normal limits

INDICATION FOR LIVER INDICATION FOR LIVER TRANSPLANTATIONTRANSPLANTATION

Severe hypercholesterolemia, non-Severe hypercholesterolemia, non-responsive to medical treatmentresponsive to medical treatment

Severe cardio-vascular diseaseSevere cardio-vascular disease

The donor liverThe donor liver

normal histology on biopsy

DOMINODOMINOLIVER TRANSPLANTATIONLIVER TRANSPLANTATION

- THE RECIPIENT -- THE RECIPIENT -

HISTORY OF THE DISEASEHISTORY OF THE DISEASE

42 year-old female42 year-old female HEPATOCELLULAR CARCINOMA HEPATOCELLULAR CARCINOMA

developed on Child B HBV LIVER developed on Child B HBV LIVER CIRRHOSIS CIRRHOSIS

HBV chronic hepatitisHBV chronic hepatitis – diagnosed at 18 – diagnosed at 18 years (no antiviral therapy)years (no antiviral therapy)

liver cirrhosisliver cirrhosis – at 41 years – at 41 years Treatment with Lamivudine startedTreatment with Lamivudine started Unfavorable course towards Child B cirrhosis, with Unfavorable course towards Child B cirrhosis, with

frequent decompensationsfrequent decompensations HCCHCC – at 42 years ( AFP- 500 ng/dl, 5 cm – at 42 years ( AFP- 500 ng/dl, 5 cm

tumoral nodule – segment VIII)tumoral nodule – segment VIII)

intraarterial chemoembolizationintraarterial chemoembolization(Doxorubicin 50 mg - 2 sessions )(Doxorubicin 50 mg - 2 sessions )

INDICATION FOR LIVER INDICATION FOR LIVER TRANSPLANTATIONTRANSPLANTATION

CHILD BLIVER CIRRHOSIS

HEPATOMA DEVELOPEDON CIRRHOSIS

MARGINALCANDIDATE

MACROSCOPIC ASPECT OF RESECTED LIVER

5 cm tumoral nodule in segment VIII

MICROSCOPIC ASPECTMICROSCOPIC ASPECT Micronodular cirrhosisMicronodular cirrhosis Poorly-differentiated, microtrabecular Poorly-differentiated, microtrabecular

and acinary hepatocellular carcinoma and acinary hepatocellular carcinoma (Edmon(Edmonssoonn III-IV) III-IV)

SURGICAL TECHNIQUESURGICAL TECHNIQUE

P2 – FHCP2 – FHCP2 – FHCP2 – FHC

P3 P3 P3 P3

P4 – HEPATOMAP4 – HEPATOMAP4 – HEPATOMAP4 – HEPATOMA

SPLIT

SPLIT

DOMINO

P1 - CADAVER

Patient withfamilial

hypercholesterolemia

Child with glycogenosis

Patient withhepatoma developed

on cirrhosis

domino

split

split

BIOCHEMICAL ASSAYSBIOCHEMICAL ASSAYSSerum total cholesterol and triglycerides were measured Serum total cholesterol and triglycerides were measured using enzymatic commercial kits from Bio-Merieux, France using enzymatic commercial kits from Bio-Merieux, France

•Isolation of mononuclear cellsIsolation of mononuclear cells From each subject, blood was collected both on EDTA (for From each subject, blood was collected both on EDTA (for monocytes separation) or without EDTA (for serum monocytes separation) or without EDTA (for serum isolation). isolation). Mononuclear cells were isolated on Histopaque® (Sigma Mononuclear cells were isolated on Histopaque® (Sigma Chem. Co., USA), by centrifugation on a density gradient (30 Chem. Co., USA), by centrifugation on a density gradient (30 min, 400xg), followed by sequential washing with PBS (10 min, 400xg), followed by sequential washing with PBS (10 min, 300xg)min, 300xg)

•Quantification of LDL receptors on monocytesQuantification of LDL receptors on monocytes was was performed with a LDL-R kit from ORPEGEN - performed with a LDL-R kit from ORPEGEN - PHARMA (HEIDELBERG, GERMANY)PHARMA (HEIDELBERG, GERMANY)•Flow cytometry analysisFlow cytometry analysis was performed with a was performed with a Beckman Coulter apparatus on isolated Beckman Coulter apparatus on isolated mononuclear cellsmononuclear cells

ASSAY FOR EXTRAHEPATIC LDL ASSAY FOR EXTRAHEPATIC LDL RECEPTORS BY RECEPTORS BY

FLOW-CYTOMETRY FLOW-CYTOMETRY

Cells: Cells: • Monocyte-derived macrophages (MDM) cultureMonocyte-derived macrophages (MDM) culture• Fibroblast cell culture Fibroblast cell culture - Skin biopsies obtained from both - Skin biopsies obtained from both

patientspatients ExtractExtraction ofion of ttotal RNA from cultured cells using a high pure otal RNA from cultured cells using a high pure

RNA isolation kit (Roche)RNA isolation kit (Roche) RT-PCR RT-PCR -- performed in a single step (with a kit from Roche), performed in a single step (with a kit from Roche),

employing an Applied Biosystems thermocycler employing an Applied Biosystems thermocycler • expression of mRNA coding for LDL-R expression of mRNA coding for LDL-R -- evaluated with the evaluated with the

appropriate primers, normalized against the expression of appropriate primers, normalized against the expression of glyceraldehyde-phosphate-dehydrogenase (GAPDH) glyceraldehyde-phosphate-dehydrogenase (GAPDH)

• Agarose gel bands stained with ethidium bromide Agarose gel bands stained with ethidium bromide -- used for the used for the semi-quantitative evaluation of LDL-R expression; densitometric semi-quantitative evaluation of LDL-R expression; densitometric analysis analysis -- performed with a GeneAmp PCR System 2700 (Applied performed with a GeneAmp PCR System 2700 (Applied Biosystems)Biosystems)

Flow cytometry and reverse-transcription coupled with Flow cytometry and reverse-transcription coupled with polymerase chain reaction (RT-PCR) methods polymerase chain reaction (RT-PCR) methods -- used to used to assess the expression of LDL receptor on monocytes, MDM assess the expression of LDL receptor on monocytes, MDM and cultured fibroblast obtained from the two patientsand cultured fibroblast obtained from the two patients• The protocol allowed the sorting of CD14-positive cells The protocol allowed the sorting of CD14-positive cells

(monocytes) and, out of them, the LDL receptor-expressing cells(monocytes) and, out of them, the LDL receptor-expressing cells

QUANTIFICATION AND GENE QUANTIFICATION AND GENE EXPRESSION OF LDL RECEPTORS EXPRESSION OF LDL RECEPTORS

RESULTSRESULTS

POST-TRANSPLANTATION POST-TRANSPLANTATION EVOLUTION IN THE EVOLUTION IN THE DONORDONOR

normalization of cholesterolemianormalization of cholesterolemia disappearance of cubital xanthomasdisappearance of cubital xanthomas regression of xanthelasmasregression of xanthelasmas

BEFORE BEFORE TRANSPLANTATIONTRANSPLANTATION

AFTER AFTER TRANSPLANTATIONTRANSPLANTATION

cardio-vascular disease – cardio-vascular disease – unchangedunchanged coronarography 12 months after coronarography 12 months after

transplantation showed the same image transplantation showed the same image of ostial stenosis on the right coronary of ostial stenosis on the right coronary artery artery

PTCA was performed (stent in the right PTCA was performed (stent in the right coronary artery)coronary artery)

elevated levels of serum elevated levels of serum creatinin – 2creatinin – 2 mg/dlmg/dl

Current therapyCurrent therapy immunosuppressor – TACROLIMUSimmunosuppressor – TACROLIMUS antianginal agentsantianginal agents

• Beta-Adrenergic AntagonistBeta-Adrenergic Antagonist• Calcium Channel BlockerCalcium Channel Blocker• Antiplatelet AgentAntiplatelet Agent

POST-TRANSPLANTATION POST-TRANSPLANTATION EVOLUTION IN THE EVOLUTION IN THE RECIPIENTRECIPIENT

normal liver functionnormal liver function AFP - 0.72 ng/dl ( NV < 10 ng/dl)AFP - 0.72 ng/dl ( NV < 10 ng/dl) cholesterolemia – 271 mg/dl - cholesterolemia – 271 mg/dl -

under cholesterol lowering under cholesterol lowering therapy – Simvastatin (Zocor)therapy – Simvastatin (Zocor)

HDL – 37 mg/dlHDL – 37 mg/dl triglycerides – 266 mg/dltriglycerides – 266 mg/dl

Current therapyCurrent therapy immunosuppressorimmunosuppressor

TACROLIMUSTACROLIMUS antiviralantiviral

LAMIVUDINE + HBIG (HB hyperimmune LAMIVUDINE + HBIG (HB hyperimmune globulin)globulin)

cholesterol lowering agentcholesterol lowering agent SIMVASTATINSIMVASTATIN

Quantification of the expression of LDL-R on monocytes by flow-cytometry

analysis, one year after the transplant operations

MonocytesMean fluorescence

(arbitrary units)LDL-R

(%)

Control*130 - 277

(mean 216)100.0

Patient A (FHC) 14.4 6.7

Patient B (Cirrhosis) 153.5 71

*mean value given by LDL receptor kit

Histogram showing quantification of LDL receptors on monocytes isolated from patient A (familial

hypercholesterolemia) and B (cirrhosis) by flow cytometry, using an anti-LDL receptor monoclonal

antibody

Gene expression of LDL-R of monocyte-derived macrophages isolated from patient A (familial hypercholesterolemia) and B (cirrhosis); agarose gel electrophoresis of RT-PCR products.

Note the presence of LDL receptor (LDL-R) expression in both cases, although apparently reduced in the case of patient B due

to the reduced activation of monocytes. GAPDH and LDL-R gene expression are identified by comparing

with a ladder DNA (lane 1)

Gene expression of LDL-R in fibroblasts obtained from patient A (familial hypercholesterolemia) and B (cirrhosis).

Agarose gel electrophoresis of RT-PCR products of RNA isolated from primary culture fibroblasts from the two

subjects. Expression of mRNA coding for the LDL receptor (LDL-R) as compared with the GAPDH expression.

Lane 1: ladder DNA

DISCUSSIONDISCUSSION

the synthesis of the LDL receptors in extrahepatic the synthesis of the LDL receptors in extrahepatic cells (monocytes and fibroblasts) appears to be cells (monocytes and fibroblasts) appears to be normalnormal

hhowever, their functioning seemed to be impeded owever, their functioning seemed to be impeded because, as demonstrated by flow cytometry (the because, as demonstrated by flow cytometry (the test with monoclonal anti-LDL-R antibody), there is test with monoclonal anti-LDL-R antibody), there is only a very low level (6.7%) of functional LDL-R on only a very low level (6.7%) of functional LDL-R on the surface of circulating monocytes of FHC patientthe surface of circulating monocytes of FHC patient

tthese data suggest the existence of a defect in the hese data suggest the existence of a defect in the pathway of LDL-R that occurs after their synthesis pathway of LDL-R that occurs after their synthesis and before they become expressed on the cell and before they become expressed on the cell surface surface

immediately after transplantation, the FHC patient immediately after transplantation, the FHC patient exhibited a decrease in serum cholesterol level of exhibited a decrease in serum cholesterol level of 50-80%, as expected50-80%, as expected

tthe normalization of serum cholesterol and he normalization of serum cholesterol and triglycerides levels is due to the presence of the new triglycerides levels is due to the presence of the new normal liver that was transplanted normal liver that was transplanted

TThehe FHC FHC patientpatient

showed after surgery an increase in the cholesterol and triglycerides showed after surgery an increase in the cholesterol and triglycerides values but, under treatment with HMG-CoA inhibitors (Simvastatin), values but, under treatment with HMG-CoA inhibitors (Simvastatin), those values decreasedthose values decreased

ccurrently, more than one year after the operation, the serum urrently, more than one year after the operation, the serum cholesterol level is slightly higher than normal (249 mg/dl), with no cholesterol level is slightly higher than normal (249 mg/dl), with no clinical expression and no cholesterol deposits detected by clinical expression and no cholesterol deposits detected by ultrasonography.ultrasonography.

Considering that the quantity of LDL receptors from monocytes, Considering that the quantity of LDL receptors from monocytes, lymphocytes and fibroblasts is a marker of hepatic LDL receptors lymphocytes and fibroblasts is a marker of hepatic LDL receptors (16), the liver grafted from the FHC patient has diminished (16), the liver grafted from the FHC patient has diminished functionality (the level of functional LDL-R detected in the FHC functionality (the level of functional LDL-R detected in the FHC patient circulating monocytes was 6.7% of the normal value)patient circulating monocytes was 6.7% of the normal value)

It is possible that the patient who received the FHC liver to develop It is possible that the patient who received the FHC liver to develop with time “acquired hypercholesterolemia”, but we consider this risk with time “acquired hypercholesterolemia”, but we consider this risk acceptable, given the rate of tumoral recurrence after acceptable, given the rate of tumoral recurrence after transplantation (43 - 60% over a 5 year period)transplantation (43 - 60% over a 5 year period)

after transplantation after transplantation the patientthe patient regained menstruation (the regained menstruation (the protective role of estrogens against the cholesterol effects is well protective role of estrogens against the cholesterol effects is well known)known)

the lipidogenic effect of immunosuppressive drugs seems to be the lipidogenic effect of immunosuppressive drugs seems to be similar for Cyclosporine® and Prograf®similar for Cyclosporine® and Prograf®

if the patient lives beyond the expected survival and if the if the patient lives beyond the expected survival and if the hypercholesterolemia becomes clinically significant, a further hypercholesterolemia becomes clinically significant, a further solution could be the hepatocyte transplantationsolution could be the hepatocyte transplantation,, gene therapy gene therapy or or retransplantationretransplantation

The The domino – recipientdomino – recipient patient patient

CONCLUSIONS (1)CONCLUSIONS (1)

Liver transplantation from a FHC patient to Liver transplantation from a FHC patient to another patient with normal LDL-R may be a another patient with normal LDL-R may be a justified therapeutical optionjustified therapeutical option

The capacity to maintain serum cholesterol levels The capacity to maintain serum cholesterol levels within an almost normal range in the domino within an almost normal range in the domino recipient can be explained by the extrahepatic LDL recipient can be explained by the extrahepatic LDL receptors, such as those on monocytes and receptors, such as those on monocytes and fibroblasts; their efficient functioning combined fibroblasts; their efficient functioning combined with adjuvant treatment can adjust the excess with adjuvant treatment can adjust the excess cholesterol in the plasma and allow the patient to cholesterol in the plasma and allow the patient to have a normal lifehave a normal life

These are preliminary results (1 year These are preliminary results (1 year and a half post-transplantation): at and a half post-transplantation): at least a 5-year follow-up period is least a 5-year follow-up period is necessarynecessary

CONCLUSIONS (CONCLUSIONS (22))

ENDEND