Epstein–Barr Virus–Related Gastric Adenocarcinoma: An Early Secondary Cancer Post Hemopoietic Stem Cell Transplantation
2005; Elsevier BV; Volume: 129; Issue: 6 Linguagem: Inglês
10.1053/j.gastro.2005.10.011
ISSN1528-0012
AutoresWing‐Yan Au, Annie Pang, Eunice C. Chan, Kent‐Man Chu, Tony W.H. Shek, Yok–Lam Kwong,
Tópico(s)Eosinophilic Disorders and Syndromes
ResumoBackground & Aims: Epstein–Barr virus (EBV) infection has been associated with some cases of gastric cancer. Methods: We studied a case of early onset gastric adenocarcinoma after nonmyeloablative hematopoietic stem cell transplantation for myeloma in a 56-year-old man. Results: The development of gastric adenocarcinoma was preceded by severe graft-versus-host disease (GVHD) necessitating strong immunosuppression, which resulted in an intense reactivation of EBV infection. Three sequential gastric biopsy examinations performed at 100, 130, and 150 days after hematopoietic stem cell transplantation showed gastritis, dysplasia, and adenocarcinoma, respectively. There was no evidence of Helicobacter pylori infection. Quantitative polymerase chain reaction for circulating EBV showed a surge of EBV DNA peaking at the time of gastritis, followed by a gradual decrease afterward with adequate control of GVHD and tailing of immunosuppression. In situ hybridization for EBV-encoded early small RNA showed absence of EBV in the gastritis specimen, but the presence of EBV in the dysplastic and carcinoma specimens. Aberrant promoter methylation of E-cadherin was observed only in the carcinoma specimens, showing that infection with EBV preceded E-cadherin methylation. Conclusions: Mucosal damage caused by GVHD, immunosuppression, and EBV reactivation combined to lead to EBV infection of the gastric cells and initiation of carcinogenesis, suggesting this case to be a genuine EBV-related opportunistic malignancy post-transplantation. An interesting proposition is that this case also might reflect a compacted timeline of events in EBV-related gastric cancers developing in immunocompetent patients. Background & Aims: Epstein–Barr virus (EBV) infection has been associated with some cases of gastric cancer. Methods: We studied a case of early onset gastric adenocarcinoma after nonmyeloablative hematopoietic stem cell transplantation for myeloma in a 56-year-old man. Results: The development of gastric adenocarcinoma was preceded by severe graft-versus-host disease (GVHD) necessitating strong immunosuppression, which resulted in an intense reactivation of EBV infection. Three sequential gastric biopsy examinations performed at 100, 130, and 150 days after hematopoietic stem cell transplantation showed gastritis, dysplasia, and adenocarcinoma, respectively. There was no evidence of Helicobacter pylori infection. Quantitative polymerase chain reaction for circulating EBV showed a surge of EBV DNA peaking at the time of gastritis, followed by a gradual decrease afterward with adequate control of GVHD and tailing of immunosuppression. In situ hybridization for EBV-encoded early small RNA showed absence of EBV in the gastritis specimen, but the presence of EBV in the dysplastic and carcinoma specimens. Aberrant promoter methylation of E-cadherin was observed only in the carcinoma specimens, showing that infection with EBV preceded E-cadherin methylation. Conclusions: Mucosal damage caused by GVHD, immunosuppression, and EBV reactivation combined to lead to EBV infection of the gastric cells and initiation of carcinogenesis, suggesting this case to be a genuine EBV-related opportunistic malignancy post-transplantation. An interesting proposition is that this case also might reflect a compacted timeline of events in EBV-related gastric cancers developing in immunocompetent patients. The Epstein–Barr virus (EBV) is linked pathogenetically with several malignancies, including lymphomas of diverse histologies1Timms J.M. Bell A. Flavell J.R. Murray P.G. Rickinson A.B. Travers-Glehen A. Berger F. Delecluse H.J. Target cells of Epstein-Barr-virus (EBV)–positive post-transplant lymphoproliferative disease similarities to EBV-positive Hodgkin's lymphoma.Lancet. 2003; 361: 217-223Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 2Siu L.L. Chan J.K. Kwong Y.L. Natural killer cell malignancies clinicopathologic and molecular features.Histol Histopathol. 2002; 17: 539-554PubMed Google Scholar, 3Horenstein M.G. Nador R.G. Chadburn A. Hyjek E.M. Inghirami G. Knowles D.M. Cesarman E. Epstein-Barr virus latent gene expression in primary effusion lymphomas containing Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8.Blood. 1997; 90: 1186-1191Crossref PubMed Google Scholar, 4Fukayama M. Ibuka T. Hayashi Y. Ooba T. Koike M. Mizutani S. Epstein-Barr virus in pyothorax-associated pleural lymphoma.Am J Pathol. 1993; 143: 1044-1049PubMed Google Scholar and nasopharyngeal carcinoma.5Hermann K. Niedobitek G. Epstein-Barr virus-associated carcinomas facts and fiction.J Pathol. 2003; 199: 140-145Crossref PubMed Scopus (77) Google Scholar In patients immunocompromised as a result of marrow or solid-organ allografting or infection with the human immunodeficiency virus, EBV is associated with another spectrum of malignancies. In organ allograft recipients, reactivation of EBV infection can lead to post-transplantation lymphoproliferative diseases.6Knowles D.M. Cesarman E. Chadburn A. Frizzera G. Chen J. Rose E.A. Michler R.E. Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders.Blood. 1995; 85: 552-565PubMed Google Scholar In human immunodeficiency virus–positive patients who develop lymphomas, EBV is found in about 30% of centroblastic and up to 90% of immunoblastic lymphomas.7Carbone A. Emerging pathways in the development of AIDS-related lymphomas.Lancet Oncol. 2003; 4: 22-29Abstract Full Text Full Text PDF PubMed Scopus (122) Google ScholarOccasionally, EBV infection might be associated with rare nonhematologic malignancies after organ allografting. Leiomyosarcomas developing after liver transplantation have been described, with shown clonal EBV infection in the tumor cells, suggesting that the virus plays a role in oncogenesis.8Lee E.S. Locker J. Nalesnik M. Reyes J. Jaffe R. Alashari M. Nour B. Tzakis A. Dickman P.S. The association of Epstein-Barr virus with smooth-muscle tumors occurring after organ transplantation.N Engl J Med. 1995; 332: 19-25Crossref PubMed Scopus (389) Google Scholar These tumors commonly develop after years of continuous immunosuppression.Interestingly, in immunocompetent patients, EBV infection also might be associated with gastric cancers. The association is strongest for gastric carcinomas of lymphoepithelioma-like histology, where EBV is detectable by in situ hybridization (ISH) or molecular techniques in 80%–100% of cases.9Osato T. Imai S. Epstein-Barr virus and gastric carcinoma.Semin Cancer Biol. 1996; 7: 175-182Crossref PubMed Scopus (86) Google Scholar, 10Wu M.S. Shun C.T. Wu C.C. Hsu T.Y. Lin M.T. Chang M.C. Wang H.P. Lin J.T. Epstein-Barr virus-associated gastric carcinomas relation to H. pylori infection and genetic alterations.Gastroenterology. 2000; 118: 1031-1038Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar These lymphoepithelioma-like carcinomas are similar pathologically to undifferentiated nasopharyngeal carcinoma, a tumor virtually always associated with EBV infection. In more conventional gastric adenocarcinomas of the diffuse and intestinal types, EBV is found in 7%–14% of cases.10Wu M.S. Shun C.T. Wu C.C. Hsu T.Y. Lin M.T. Chang M.C. Wang H.P. Lin J.T. Epstein-Barr virus-associated gastric carcinomas relation to H. pylori infection and genetic alterations.Gastroenterology. 2000; 118: 1031-1038Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 11van Beek J. zur Hausen A. Klein Kranenbarg E. van de Velde C.J. Middeldorp J.M. van den Brule A.J. Meijer C.J. Bloemena E. EBV-positive gastric adenocarcinomas a distinct clinicopathologic entity with a low frequency of lymph node involvement.J Clin Oncol. 2004; 22: 664-670Crossref PubMed Scopus (204) Google Scholar Interestingly, EBV-associated gastric adenocarcinomas have been reported to occur in younger patients, who have with fewer disease metastases and a better survival, when compared with EBV-negative cases.11van Beek J. zur Hausen A. Klein Kranenbarg E. van de Velde C.J. Middeldorp J.M. van den Brule A.J. Meijer C.J. Bloemena E. EBV-positive gastric adenocarcinomas a distinct clinicopathologic entity with a low frequency of lymph node involvement.J Clin Oncol. 2004; 22: 664-670Crossref PubMed Scopus (204) Google Scholar Apart from the different clinical behavior, the pathogenesis of EBV-associated gastric adenocarcinomas also appears to be discrete, in that there is less association with Helicobacter pylori infection,10Wu M.S. Shun C.T. Wu C.C. Hsu T.Y. Lin M.T. Chang M.C. Wang H.P. Lin J.T. Epstein-Barr virus-associated gastric carcinomas relation to H. pylori infection and genetic alterations.Gastroenterology. 2000; 118: 1031-1038Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar minimal microsatellite instability12Chang M.S. Lee H.S. Kim H.S. Kim S.H. Choi S.I. Lee B.L. Kim C.W. Kim Y.I. Yang M. Kim W.H. Epstein-Barr virus and microsatellite instability in gastric carcinogenesis.J Pathol. 2003; 199: 447-452Crossref PubMed Scopus (43) Google Scholar or loss of TP53,13van Rees B.P. Caspers E. zur Hausen A. van den Brule A. Drillenburg P. Weterman M.A. Offerhaus G.J. Different pattern of allelic loss in Epstein-Barr virus-positive gastric cancer with emphasis on the p53 tumor suppressor pathway.Am J Pathol. 2002; 161: 1207-1213Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar and a much higher frequency of aberrant gene promoter methylation14Kang G.H. Lee S. Kim W.H. Lee H.W. Kim J.C. Rhyu M.G. Ro J.Y. Epstein-Barr virus-positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma.Am J Pathol. 2002; 160: 787-794Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar when compared with EBV-negative gastric cancers. Furthermore, aberrant methylation of E-cadherin, a gene implicated in hereditary gastric cancer,15Fitzgerald R.C. Caldas C. Clinical implications of E-cadherin associated hereditary diffuse gastric cancer.Gut. 2004; 53: 775-778Crossref PubMed Scopus (83) Google Scholar occurs almost universally in EBV-positive gastric adenocarcinomas, but in only about half of EBV-negative cases.16Sudo M. Chong J.M. Sakuma K. Ushiku T. Uozaki H. Nagai H. Funata N. Matsumoto Y. Fukayama M. Promoter hypermethylation of E-cadherin and its abnormal expression in Epstein-Barr virus-associated gastric carcinoma.Int J Cancer. 2004; 109: 194-199Crossref PubMed Scopus (67) Google Scholar These findings strongly suggest that EBV plays a distinct role in gastric carcinogenesis.We report an exceptional case of early onset EBV-related gastric carcinoma 3 months after a nonmyeloablative hematopoietic stem cell transplantation (HSCT). This case allowed us the rare opportunity to examine the sequence of events leading to EBV infection, E-cadherin methylation, and gastric carcinogenesis.Materials and MethodsHistopathologic AnalysisPathology specimens were grossly examined, and representative tissue blocks were sectioned and stained with H&E. Classification of gastric cancer was performed according to the World Health Organization criteria.17Watanabe H. Jass J.R. Sobin L.H. Histologic typing of oesophageal and gastric tumours. Springer-Verlag, Berlin1990Crossref Google Scholar ISH for EBV encoded early small RNA (EBER) was performed as described.18Au W.Y. Shek W.H. Nicholls J. Tse K.M. Todd D. Kwong Y.L. T-cell intravascular lymphomatosis (angiotropic large cell lymphoma) association with Epstein-Barr viral infection.Histopathology. 1997; 31: 563-567Crossref PubMed Scopus (86) Google Scholar Fluorescence ISH for the X and Y chromosomes was performed with a commercial kit (CEP X spectrum orange/Y spectrum green; Vysis, Downers Grove, IL) as previously described.19Lau Y.L. Kwong Y.L. Lee A.C. Chiu E.K. Ha S.Y. Chan C.F. Chan V. Chan T.K. Mixed chimerism following bone marrow transplantation for severe combined immunodeficiency a study by DNA fingerprinting and simultaneous immunophenotyping and fluorescence in situ hybridisation.Bone Marrow Transplant. 1995; 15: 971-976PubMed Google ScholarMolecular AnalysisDNA was extracted from formalin-fixed paraffin-embedded, gastric biopsy specimens and the peripheral blood plasma with a commercial kit (QIAamp DNA mini kit; Qiagen GmbH, Hilden, Germany). Quantitative polymerase chain reaction (PCR) for EBV was performed on plasma DNA with real-time PCR as previously reported.20Au W.Y. Pang A. Choy C. Chim C.S. Kwong Y.L. Quantification of circulating Epstein-Barr virus (EBV) DNA in the diagnosis and monitoring of natural killer cell and EBV-positive lymphomas in immunocompetent patients.Blood. 2004; 104: 243-249Crossref PubMed Scopus (247) Google Scholar Briefly, plasma DNA was amplified in triplicates with a pair of primers and a TaqMan probe targeting the EBV nuclear antigen 1 (EBNA-1) gene. Quantification of the EBNA-1 gene copy number was performed against a standard plasmid where the targeted EBNA-1 gene fragment was cloned. A 300-bp type-specific polymorphic region in the EBV EBNA-2 gene was amplified by PCR from the plasma of the donor and recipient, followed by direct DNA sequencing as described21Chiang A.K. Wong K.Y. Liang A.C. Srivastava G. Comparative analysis of Epstein-Barr virus gene polymorphisms in nasal T/NK-cell lymphomas and normal nasal tissues implications on virus strain selection in malignancy.Int J Cancer. 1999; 80: 356-364Crossref PubMed Scopus (68) Google Scholar to determine if the EBV post-HSCT might be of donor or recipient origin. As a putative surrogate marker of the early stages of gastric carcinogenesis, aberrant promoter methylation of the E-cadherin gene was tested on the gastric biopsy specimens by methylation-specific PCR (MSP) as previously described.21Chiang A.K. Wong K.Y. Liang A.C. Srivastava G. Comparative analysis of Epstein-Barr virus gene polymorphisms in nasal T/NK-cell lymphomas and normal nasal tissues implications on virus strain selection in malignancy.Int J Cancer. 1999; 80: 356-364Crossref PubMed Scopus (68) Google Scholar Serial dilutions of methylated DNA in normal DNA showed that the MSP had a sensitivity of 10−3 for detecting the methylated allele.Serologic AnalysisAnalysis for antibodies against EBV viral capsid antigen and EBNA-1 (nuclear antigen-1) was performed by standard methods.23Reedman B.M. Hilgers J. Hilgers F. Klein G. Immunofluorescence and anti-complement immunofluorescence absorption tests for quantitation of Epstein-Barr virus-associated antigens.Int J Cancer. 1975; 15: 566-571Crossref PubMed Scopus (19) Google ScholarResultsCase ReportA 52-year-old man with multiple myeloma underwent a nonmyeloablative HSCT from his human leukocyte antigen–identical sister.24Au W.Y. Chan E.C. Pang A. Lie A.K.W. Liang R. Yuen A.P.W. Shek T.W.H. Kwong Y.L. Non-hematologic malignancies after allogeneic hematopoietic stem cell transplantation incidence and molecular monitoring.Bone Marrow Transplant. 2004; 34: 981-985Crossref PubMed Scopus (25) Google Scholar The source of HSC was unmanipulated bone marrow. He had no previous problems relating to his gastrointestinal tract. The condition regimen comprised fludarabine (30 mg/m2/day × 3) and total body irradiation (3 Gy). Prophylaxis for graft-versus-host disease (GVHD) consisted of cyclosporine (8 mg/kg/day) and mycophenolate mofetil (30 mg/kg/day). Engraftment occurred on day 24 without GVHD. A bone marrow biopsy examination showed no residual myeloma cells, and serum monoclonal paraprotein also became undetectable. Both cyclosporine and mycophenolate mofetil were tapered off by 3 months. However, this was followed shortly by GVHD affecting the skin and gut, necessitating treatment with cyclosporine (8 mg/kg/day) and methylprednisolone (1 mg/kg/day). Anemia prompted an upper endoscopy, which showed diffuse gastritis and a bleeding erosion. Helicobacter pylori was not detected by a rapid urease test,25Chu K.M. Poon R. Tuen H.H. Law S.Y. Branicki F.J. Wong J. A prospective comparison of locally made rapid urease test and histology for the diagnosis of Helicobacter pylori infection.Gastrointest Endosc. 1997; 46: 503-506Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar or on subsequent histologic examination. Retrospective analysis of a serum sample stored before HSCT by enzyme-linked immunosorbent assay also was negative (pylori DTect IgG test; Diagnostic Technology, Pymble, Australia). A sigmoidoscopy also showed inflamed mucosa that on biopsy examination showed GVHD. Concomitant cytomegalovirus reactivation occurred, which was treated with intravenous ganciclovir (5 mg/kg/day until cytomegalovirus PCR negative) and gammaglobulin (.4 g/kg/day for 3 doses). By day 130, the skin rashes and diarrhea had subsided. A repeat upper endoscopy showed a 3-cm gastric ulcer, which on biopsy examination showed epithelial dysplasia only. All corticosteroids were stopped, and the patient was treated with a proton-pump inhibitor. A repeat endoscopy 3 weeks later showed a nonhealing ulcer, which was confirmed to be malignant on biopsy examination. A computed tomographic scan showed no evidence of metastases. Distal radical gastrectomy was performed on day 160. At follow-up evaluation 2 years later, the patient remained well with mild chronic GVHD, and no evidence of recurrence of the carcinoma or the multiple myeloma.Histopathologic AnalysisThe first gastric biopsy examination (4 separate specimens) showed acute gastritis with markedly edematous lamina propria (Figure 1A). The second biopsy examination (2 separate specimens from the ulcer) showed severe epithelial dysplasia (Figure 1B). During the third endoscopy, 10 separate biopsy specimens of the ulcer all showed gastric adenocarcinoma. Two biopsy specimens taken from the antrum showed gastritis without dysplasia. The gastrectomy specimen showed a 4-cm superficial gastric ulcer. Histologic examination showed an invasive adenocarcinoma (Figure 1C) involving the mucosa and submucosa. There was no lymph node involvement. Random biopsy examinations of other parts of the gastrectomy specimen not involved by carcinoma showed gastritis with no evidence of dysplasia. Retrospective ISH study showed that the initial gastritis specimen was negative for EBER (Figure 1D). However, the gastric dysplasia specimen was EBER positive (Figure 1E). There also were numerous EBER-positive cancer cells in the carcinoma specimen (Figure 1F). Fluorescence ISH analysis of sections with predominantly cancer cells (>90%) showed that all analyzable cells possessed single X and Y hybridization signals (Figure 1G), suggesting that the cancer cells were recipient in origin.Molecular AnalysisSerial plasma EBV DNA monitoring showed a moderate increase after the conditioning regimen and at the time of engraftment (Figure 2). With the onset of severe GVHD, there was a rapid surge in the EBV-DNA level. Interestingly, the EBV-DNA level then started to decrease, coinciding with the development of gastric dysplasia and carcinoma. After surgical resection of the carcinoma, and together with the cessation of corticosteroids and improvement in the GVHD, the EBV-DNA level had decreased to undetectable levels (Figure 2). MSP for E-cadherin promoter methylation was unsuccessful for the gastritis specimen, probably because of DNA degradation. For the dysplastic specimen there was no E-cadherin methylation. However, E-cadherin methylation became positive in the carcinoma biopsy and gastrectomy specimens (Figure 3). Analysis of the EBNA-2 gene showed that the EBV from the donor and recipient collected before HSCT had identical sequences within the amplified polymorphic fragment (data not shown), meaning that it was not possible to use this to determine whether the EBV post-HSCT was donor or recipient in origin.Figure 2Quantification of circulating EBV-DNA level in a case of EBV-positive gastric cancer post-HSCT. An increase in EBV-DNA level occurred shortly after HSCT. Further increases ensued with the onset of GVHD and immunosuppression, peaking at the time of detection of gastritis. However, with adequate control of GVHD leading to a decrease in immunosuppression, the EBV-DNA level started to decrease. Cessation of immunosuppression and complete resection of the gastric cancer led to undetectable levels of EBV DNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3MSP for E-cadherin. The gastritis (gast) specimen did not give any PCR product. In the gastric specimen showing dysplasia (dys), amplification was positive for the unmethylated (U) E-cadherin gene, but negative for the methylated (M) gene. However, in the gastric cancer biopsy specimen (CA1) and the gastrectomy specimen (CA2), E-cadherin methylation was present. P, positive control with methylated DNA showing positive amplification in the M but not U reactions; N, normal control DNA showing positive amplification in the U but not M reactions; B, reagent blank; MW, molecular weight markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Serologic AnalysisAnalysis of serum samples from the donor, and from the patient collected before HSCT, showed positive antiviral capsid antigen and anti–EBNA-1 IgG (titer >1:10), indicating past infections with EBV in both of them.DiscussionWe report a case of EBV-associated gastric carcinoma after HSCT. Solid-organ allograft recipients are at an increased risk for secondary cancers. In one study of 1657 patients undergoing liver transplantation, gastric cancer as a secondary malignancy was diagnosed only in 2 patients.26Frezza E.E. Fung J.J. van Thiel D.H. Non-lymphoid cancer after liver transplantation.Hepatogastroenterology. 1997; 44: 1172-1181PubMed Google Scholar In another Korean study in which gastric cancer was the most common malignancy in the general population, 14 of 2000 renal allograft recipients subsequently developed gastric cancer.27Chen J. Cheong J.H. Hyung W.J. Kim J.U. Choi D.J. Kwon K.H. Kim S.I. Kim Y.S. Park K. Noh S.H. Gastric adenocarcinoma after renal transplantation.Hepatogastroenterology. 2004; 51: 895-899PubMed Google Scholar It therefore is uncertain if these cases of post-transplantation gastric cancer were merely coincidental. In 19,229 patients undergoing HSCT, not a single case of gastric cancer was recorded in 80 malignancies that developed during a 28-year study period.28Curtis R.E. Rowlings P.A. Deeg H.J. Shriner D.A. Socie G. Travis L.B. Horowitz M.M. Witherspoon R.P. Hoover R.N. Sobocinski K.A. Fraumeni Jr, J.F. Boice Jr, J.D. Solid cancers after bone marrow transplantation.N Engl J Med. 1997; 336: 897-904Crossref PubMed Scopus (769) Google Scholar Therefore, gastric cancer cannot be regarded as a common secondary malignancy after marrow and solid-organ allografting.In our patient, less than 2 months elapsed from the initial endoscopy during which no dysplasia was found to the third endoscopy during which gastric adenocarcinoma was detected. This time-frame of carcinogenesis is far shorter than that of years or decades as proposed in the classic gastritis-metaplasia-dysplasia-carcinoma paradigm, in which infection with H pylori plays a major role.29Correa P. The biological model of gastric carcinogenesis.IARC Sci Publ. 2004; 157: 301-310PubMed Google Scholar The antecedent presence of long-standing gastritis and dysplasia before HSCT also was unlikely. H pylori, the most common causative agent of gastritis-metaplasia-dysplasia, was not shown by different tests in our patient. Furthermore, multiple biopsy examinations at different time points in the nonulcerous areas did not show dysplastic features, suggesting that the dysplasia occurred only in the ulcer that finally progressed to adenocarcinoma.Therefore, we propose that a distinct sequence of events might have led to the development of the carcinoma in our patient. In the first stage, there was severe gastric mucosal damage caused by GVHD. The heavy immunosuppression needed to treat the GVHD led to an unchecked EBV replication and therefore a rapid surge of circulating EBV-DNA level. This most likely was caused by a reactivation of the latent EBV infection in the patient. However, the increase in EBV level also could represent an iatrogenic infection derived from the marrow of the donor who was an EBV carrier. Interestingly, although ganciclovir was used for cytomegalovirus infection, EBV replication did not seem to have been affected, suggesting that ganciclovir might not be totally effective in suppressing EBV.30Gao S.Z. Chaparro S.V. Perlroth M. Montoya J.G. Miller J.L. DiMiceli S. Hastie T. Oyer P.E. Schroeder J. Post-transplantation lymphoproliferative disease in heart and heart-lung transplant recipients 30-year experience at Stanford University.J Heart Lung Transplant. 2003; 22: 505-514Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar At this point, the gastric mucosa had remained free from latent EBV infection. In the second stage, there was morphologic dysplasia in the gastric mucosa. At this time, EBV infection had become detectable in the dysplastic gastric epithelial cells. However, E-cadherin methylation was not found. Because EBER ISH and MSP for E-cadherin were performed on the same biopsy specimen, and the high sensitivity of MSP meant it was unlikely that small foci of dysplastic cells with E-cadherin methylation had been missed; EBV infection clearly preceded E-cadherin methylation. Interestingly, the circulating EBV-DNA load had started to decrease, owing to the decreased immunosuppression and improvement in GVHD. In the final stage, frank carcinoma had developed. By this time, the GVHD and immunosuppression had abated, accounting for a low circulating EBV-DNA load.Therefore, somewhere between the first and second stage, a phase critical in the multistep carcinogenic process, EBV must have entered the gastric epithelial cells, perhaps opportunistically subsequent to the mucosal damage, immunosuppression, and the high EBV DNA load. This could have been an important initiating event of gastric carcinogenesis that preceded the methylation of E-cadherin. This also explains the observation, in other studies, of the consistent presence of E-cadherin methylation in gastric cancer positive for EBV.14Kang G.H. Lee S. Kim W.H. Lee H.W. Kim J.C. Rhyu M.G. Ro J.Y. Epstein-Barr virus-positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma.Am J Pathol. 2002; 160: 787-794Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 16Sudo M. Chong J.M. Sakuma K. Ushiku T. Uozaki H. Nagai H. Funata N. Matsumoto Y. Fukayama M. Promoter hypermethylation of E-cadherin and its abnormal expression in Epstein-Barr virus-associated gastric carcinoma.Int J Cancer. 2004; 109: 194-199Crossref PubMed Scopus (67) Google Scholar From this perspective, our case can be considered a genuine post-transplantation opportunistic malignancy, a consequence of the short-term heightened EBV reactivation and intense immunosuppression; which is similar to post-transplantation lymphoproliferative disease in both the time of onset and disease tempo. Finally, surgical eradication of the cancer together with subsidence of GVHD led to undetectable levels of circulating EBV DNA.Our observations provide important clues in the study of the pathogenesis of EBV-positive gastric cancers. Previous serologic studies suggested that inappropriate responses to EBV infection on a long-term basis might have existed years before the development of EBV-associated gastric cancer.31Levine P.H. Stemmermann G. Lennette E.T. Hildesheim A. Shibata D. Nomura A. Elevated antibody titers to Epstein-Barr virus prior to the diagnosis of Epstein-Barr-virus-associated gastric adenocarcinoma.Int J Cancer. 1995; 60: 642-644Crossref PubMed Scopus (120) Google Scholar The exact relationship between EBV infection and the initial steps of gastric carcinogenesis, however, has not been defined. In our patient, EBV infection occurred before E-cadherin methylation. Aberrant methylation of E-cadherin has been regarded as an alternative mechanism of silencing of E-cadherin expression, and an important early event in gastric carcinogenesis.22Chan A.O. Lam S.K. Wong B.C. Wong W.M. Yuen M.F. Yeung Y.H. Hui W.M. Rashid A. Kwong Y.L. Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer.Gut. 2003; 52: 502-506Crossref PubMed Scopus (260) Google Scholar Interestingly, our observations suggest EBV infection to be an even earlier and possibly more crucial event than E-cadherin methylation. Therefore, the molecular events subsequent to EBV infection of gastric epithelial cells should be a focus in the study of EBV-related gastric cancers. Whether our case represented an extraordinary sequence of events, or actually might reflect a compacted timeline of what normally happens in immunocompetent patients who develop EBV-positive gastric cancers, will need to be evaluated prospectively.Finally, recent evidence has suggested that hematopoietic stem cell might transdifferentiate into cells of other tissues, including the gastrointestinal tract.32Korbling M. Katz R.L. Khanna A. Ruifrok A.C. Rondon G. Albitar M. Champlin R.E. Estrov Z. Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells.N Engl J Med. 2002; 346: 738-746Crossref P
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