LETTER TO THE EDITORS

Intrahepatic Recurrence of HepatocellularCarcinoma 13 Years After Orthotopic LiverTransplantation for Hepatitis C–RelatedCirrhosis With Occult Hepatocellular

Carcinoma: A Case ReportReceived January 10, 2012; accepted February 4, 2012.

TO THE EDITORS:

Here we report the case of a patient who presentedwith well-differentiated, multifocal hepatocellular car-cinoma (HCC) limited to the allograft 13 years afterorthotopic liver transplantation (OLT) for hepatitis Cvirus (HCV)–related cirrhosis with occult HCC discov-ered in the explanted liver. Because of the extremelylong time interval from the transplant to the recur-rence of the carcinoma, molecular genetic techniqueswere used to determine whether the tumor was an in-trahepatic recurrence of the primary tumor or devel-oped de novo from the donor liver.

A 61-year-old male presented with a 3-month his-tory of progressively worsening nausea, vomiting, di-arrhea, and right upper quadrant pain associatedwith a 10-lb weight loss. His past medical history wassignificant for liver transplantation for end-stage cir-rhosis secondary to an HCV infection and alcoholabuse in 1998 (13 years before the current presenta-tion). An occult, well-differentiated HCC measuring4.5 cm in diameter with lymphovascular invasion wasfound during the pathological examination of the hep-atectomy specimen, but no metastasis was present inthe single hilar lymph node that was examined. Hismost recent liver biopsy (performed in 2006) showedrecurrent mild HCV (grade 1, stage 1). HCV serumantibody test results were positive at that time.

During the current presentation, the serum alpha-fetoprotein (AFP) levels were not increased. Computedtomography imaging of the abdomen and pelvisrevealed 3 large liver lesions between 4 and 9 cm indiameter as well as numerous other smaller lesions.No extrahepatic masses or abdominal lymphadenopa-thy was noted. Computed tomography–guided fine-needle aspiration biopsy and core-needle biopsy wereperformed for 1 of the hepatic lesions and revealed

well-differentiated HCC. The neoplasm appeared to bemorphologically very similar to the HCC diagnosed 13years previously in the patient’s hepatectomy speci-men. Because both tumors were well differentiated(Figs. 1A and 2A), the possibility that the currentHCC could represent a recurrence of the original neo-plasm in the hepatic allograft was considered, but denovo HCC arising because of recurrent chronic HCVcould not be entirely excluded. To address this ques-tion, we first performed immunostaining in parallel onthe HCC seen in the current biopsy specimen and onthe original HCC so that we could compare theirimmunophenotypes. Both the current tumor and theinitial tumor were weakly positive for cytokeratinAE1/AE3 and for cytokeratin 8/18. A b-catenin im-munostain showed a membrane-only pattern in bothtumors. AFP results were negative for both tumors,and this was consistent with the lack of a serum AFPelevation during the current presentation. CD34 deco-rated the endothelial cells rimming the expanded he-patocyte cords, and CD10 highlighted the biliarycanaliculi in both tumors (Fig. 2C,D). Interestingly,the hepatocyte paraffin 1 (HepPar1) immunostain wascompletely negative in the current biopsy specimen,whereas the initial tumor showed strong and diffusepositivity (Figs. 1B and 2B). We then performed a po-lymerase chain reaction (PCR)–based analysis ofhighly polymorphic DNA short tandem repeat (STR)loci to accurately discern the origin of the HCC afterOLT, as previously reported.1,2 A PCR-based STRanalysis (AmpFlSTR Profiler Plus PCR amplificationkit, Applied Biosystems, Foster City, CA) of the cur-rent HCC specimen was compared to analyses ofspecimens from the patient and the donor.

Testing demonstrated that the core-needle biopsysample consisted of recipient-derived tissue (94%)

Address reprint requests to Stefan E. Pambuccian, M.D., Department of Laboratory Medicine and Pathology, University of Minnesota MedicalSchool, 420 Delaware Street Southeast, C422 Mayo MMC 76, Minneapolis, MN 55455; E-mail: pambu001@umn.edu

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LIVER TRANSPLANTATION 18:612-614, 2012

VC 2012 American Association for the Study of Liver Diseases.

and donor-derived tissue (6%), which likely was dueto small amounts of nonneoplastic tissue obtainedfrom the liver allograft. All 9 STR loci and the amelo-genin locus were informative for distinguishing the or-igin of the HCC, with at least 1 disparate allelebetween the donor and recipient.

Two of the 9 loci suggested the presence of a smallquantity of donor tissue, whereas a third was equivo-cal because of a potential stutter peak. These resultsprovided conclusive evidence that the patient’s cur-rent HCC represented a multifocal recurrence of theoriginal tumor within the allograft 13 years after thetransplant was performed. Unfortunately, because ofthe multifocality of the tumor, the patient was not acandidate for surgery or chemoembolization. Thepatient elected to decline systemic chemotherapy, wasdischarged with home hospice care, and died soonthereafter.

Eighty percent of recurrent HCCs occur within thefirst 3 years of transplantation,3 and very late recur-rences more than 5 years after OLT are unusual. Toour knowledge, the 13-year interval between OLT andan intrahepatic HCC recurrence is the longestreported to date. A case of pulmonary HCC metastasisoccurring 143 months after liver transplantation hasbeen recently reported.4 This very long period betweentransplantation and recurrence in the liver allograftraises interesting questions about tumor cell dor-mancy and the role of the hepatic microenvironmentin the reactivation of such dormant cancer cells. It ishypothesized that single tumor cells, possibly havinga cancer stem cell phenotype, metastasize early in thedevelopment of HCC, reach the bone marrow hema-togenously, and survive there but are kept in adormant state by the influence of various factors,including the immune response, hormones, andangiogenesis inhibition,5 and, most importantly, bythe ectopic, unfavorable microenvironment. Because

most HCC recurrences after OLT occur in the hepaticallograft,3 the liver microenvironment appears to bevery important in the reactivation of cancer cells fromdormancy. Recent studies have suggested an impor-tant role for mechanical factors in the induction andmaintenance of tumor cell dormancy. Schrader et al.6

showed that a soft physiological environment (eg, thatencountered in the bone marrow niche) induces HCCcell dormancy, whereas increased matrix stiffnessmay be responsible for the reactivation of dormant tu-mor cells. This would explain the increased frequencyof HCC recurrence in fibrotic allografts and may helpto explain the very late recurrence seen in this patientafter a long evolution of chronic HCV in the allograftresulted in increased fibrosis. The process of reactiva-tion from dormancy, which may involve the mesen-chymal-to-epithelial transition, after the epithelial-to-mesenchymal transition undergone by the malignantcells to enter the dormancy state6 could explain theloss of HepPar1 staining in the recurrent HCC. Well-differentiated HCCs are almost universally positive forHepPar1, but this hepatocytic differentiation markerhas, to our knowledge, not been studied in the contextof intrahepatic recurrences of HCCs.

Molecular techniques previously used to determinethe donor-versus-recipient origin of HCC afterOLT have included fluorescent in situ hybridizationanalysis for X-Y chromosome discrimination, geneexpression array profiling, and genotyping by micro-satellite markers.2 STR-based microsatellite genotyp-ing provides a robust and effective method for identi-fying the source of an HCC occurring in an OLTpatient, as demonstrated by this case andothers.1,2,7,8 One of the advantages of STR genotypingover fluorescent in situ hybridization analysis is thatit does not require a sex mismatch between the donorand the recipient for discrimination. STR genotyping

Figure 1. Occult, well-differentiated HCC found in thehepatectomy specimen in 1998. (A) A hematoxylin and eosinstain shows the trabecular growth pattern with widenedtrabeculae and focally prominent nucleoli of hepatocytes. (B)A HepPar1 immunoperoxidase stain shows diffuse, intensestaining of the tumor cells (original magnification for bothimages �400).

Figure 2. Representative images of the 2011 needle biopsysample showing well-differentiated HCC. (A) A hematoxylinand eosin stain shows a trabecular growth pattern withwidened trabeculae. (B) A HepPar1 immunoperoxidase stainshows no staining of the tumor cells. (C) A CD34immunoperoxidase stain shows uniform staining of theendothelial cells rimming the hepatocyte trabeculae. (D) ACD10 immunoperoxidase stain highlights the bile canaliculi(original magnification for all images �200).

LETTER TO THE EDITORS 613

LIVER TRANSPLANTATION.DOI 10.1002/lt. Published on behalf of the American Association for the Study of Liver Diseases

is also more stable than gene expression profiling overlong periods of time and tumor evolution. Further-more, only small quantities of DNA are required, andin many transplant settings, specimens from the do-nor and the recipient are archived at the time oftransplantation and provide easily accessible andunequivocal material for comparison with the tumor.

Andrew C. Nelson, M.D., Ph.D.Jose Jessurun, M.D.

Randolph K. Peterson, M.D.Stefan E. Pambuccian, M.D.

Department of Laboratory Medicine and PathologyUniversity of Minnesota Medical School

Minneapolis, MN

REFERENCES

1. Altimari A, Gruppioni E, Fiorentino M, Petraroli R, PinnaAD, Petropulacos K, et al. Genomic allelotyping for dis-tinction of recurrent and de novo hepatocellular carci-noma after orthotopic liver transplantation. Diagn MolPathol 2005;14:34-38.

2. Mas VR, Maluf DG, Dumur CI, Archer KJ, Yanek K,Jackson-Cook C, Fisher RA. Molecular techniques foridentifying HCC origin and biology after orthotopic livertransplantation. Diagn Mol Pathol 2006;15:90-94.

3. Schlitt HJ, Neipp M, Weimann A, Oldhafer KJ, Schmoll E,Boeker K, et al. Recurrence patterns of hepatocellular andfibrolamellar carcinoma after liver transplantation. J ClinOncol 1999;17:324-331.

4. Viola C, Asselah T, Samuel D, Durand F, Boudjema H,Valla D, Marcellin P. Solitary pulmonary metastasis aris-ing thirteen years after liver transplantation for HBV-related hepatocellular carcinoma. World J Gastroenterol2006;12:4911-4913.

5. Naumov GN, Folkman J, Straume O. Tumor dormancydue to failure of angiogenesis: role of the microenviron-ment. Clin Exp Metastasis 2009;26:51-60.

6. Schrader J, Gordon-Walker TT, Aucott RL, van Deemter M,Quaas A, Walsh S, et al. Matrix stiffness modulates prolifer-ation, chemotherapeutic response, and dormancy in hepa-tocellular carcinoma cells. Hepatology 2011;53:1192-1205.

7. Flemming P, Tillmann HL, Barg-Hock H, Kleeberger W,Manns MP, Klempnauer J, Kreipe HH. Donor origin of denovo hepatocellular carcinoma in hepatic allografts.Transplantation 2003;76:1625-1627.

8. Vernadakis S, Poetsch M, Weber F, Treckmann J, MatheZ, Baba HA, et al. Donor origin de novo HCC in a noncir-rhotic liver allograft 3 years after liver transplantation.Transpl Int 2010;23:341-343.

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