Dr. Regkli Areti, Kalargyroy K., Mallis Panayiotis., Gerontara G.,Kyriazi V., Matsis K. and Panagoula Kollia







Viral infections in hematological malignancies play significant role in the pathogenesis of  diseases and  have been of continued interest. An important number of molecular studies  have tried to establish a pathogenetic role for viral infections in lymphoid malignancies. Also there is strong correlation between HHV-6, EBV and HCV infections in childhood hematological malignancies but the results from molecular studies remain controversial. In particular, HHV-6 is increasingly recognized as an important opportunistic pathogen and its reactivation is common among recipients of allogenic stem cell transplantation, linked to various clinical manifestetions. Infections with EBV is widespread and affect at important grade patients with primary immunodeficiency diseases. Also hepatitis C virus infection plays a significant role in the etiology of liver disease in patients treated for childhood leukemia. The recent availability of serologic tests for detection of antibodies to HCV permits a reappraisal of the diagnosis of HCV infection in these patients. In this review we try to demonstrate the links between viral infections in development and during the treatment of childhood hematological and lymphoprliferative diseases.

Key words:Viral infections, childhood hematological malignancies, immunodefiency, EBV, HHV-6







Epstein-Barr Virus (EBV)


          In 1958 Dennis Burkitt described a lymphoma that represented the most common tumor affecting children in certain parts of East Africa. The geographical distribution of this malignancy suggested that the development of Burkitt’s lymphoma might be due to an infectious agent. In 1964 the successful establishment of cell lines from explants of BL enabled Epstein and Barr to identify herpesvirus-type particles by electron microscopy within a subpopulation of tumor cells in vitro.W and G Henle subsequently demonstrated that BL-derived cell lines expressed antigens that were recognized not only by sera from patients with BL but also by sera from patients with infectious mononucleosis (IM). Similar seoepidemiological studies also suggested a link between the EBV and undifferentiated nasopharyngeal carcinoma (NPC). The ability of this virus to efficiently immortalize B lymphocytes in vitro and to induce lymphomas in nonhuman primates established EBV as a putative oncogenic agent in humans. More recent studies have implicated EBV in a variety of other lymphoid and epithelial malignancies.

Immunodificiency patients and lymphomas

          Patients with primary immunodeficiency diseases such as X-linked lymphoproliferative syndrome (XLP) and Wiscott-Aldrich syndrome are at increased risk of developing EBV-associated B cell lymphomas1. Because these tumors are extremely rare little is known of their association with EBV infection. Mortality from XLP is high with around 50% of patients developing fatal IM after primary infection with EBV and an additional 30% of patients developing malignant lymphoma.                         Allograft recipients receiving immunosuppressive therapy and patients receiving immunosuppressive therapy with AIDS are also at increased risk for development lymphoproliferative-lymphomas. The incidence of B cell lymphomas in allograft recipients varies with the type of organ transplanted and with the type of immunosuppressive regimen used. Allogenic bone marrow transplantation into EBV seronegative children is particular risk factor for the development of virus associated B cells lymphomas1. The incidence of non-Hodgin lymphoma in AIDS patients is increased approxiamately 60 fold compared to the normal population. Around 60% of these tumors are large-cell lymphomas like those found in allograft recipients, 20% are primary brain lymphomas and 20% are of the BL type. Recent studies have demonstrated that 50% of AIDS lymphomas are EBV-positive and that this association varies with the histological tumor type. Thus, only 38% of the BL tumors are EBV-positive compared with 65% of the large-cell lymphomas.

T cell lymphomas

          Recent studies have demonstrated EBV infection in a considerable proportion of T cell non-Hodgkin’s lymphomas. Nasal T cell lymphomas, a tumor which is more common in the Far East, is inavariably EBV-positive whereas around 20% of T cell Lymphomas arising at other sites (gastrointestinal, lung, lymph nodes) are associated with EBV.    

Hodgkin’s disease

          Epidemiological studies originally suggested a possible role for EBV in the etiology of Hodgkin;s disease (HD). Thus, elevated antibody titers with HD and these increased antibody levels are present before the diagnosis of disease. Furthermore, there is an increased risk of HD following IM. EBV has been demonstrated in around 50% of HD cases with both viral nucleic acid (DNA/RNA) and virus latent antigens localized to the malignant component of  HD, the Reed-Sternberg cells and their variants2. The association of HD with EBV is age-related, pediatric and older adult cases are usually EBV-associated whereas HD in young  adults is less frequently virus-positive. The proportion of EBV-positive HD in developing countries is high consistent with a greater incidence of HD in children and more frequent prevalence of the mixed cellularity histiotype. Although the incidence of HD is relatively low (1-3/100000 per year) this tumor is not geographically restricted, making its association with EBV significant in world health terms.




Human Herpesvirus 6 (HHV-6)


          Human herpesvirus 6 was isolated in 1986 from the peripheral blood mononuclear cells of 6 patients affected with various lymphoproliferative disorders. This enveloped virion contains about 160 kb of  linear double-stranded DNA3, and is now classified as a member of the Roseolovirus genus in the Betaherpesvirinae subfamily of human herpesviruses. Type A and type B variants of HHV-6 have been identified, exhibiting different epidemiological and biological characteristics and disease associations4. HHV-6 is highly prevalent in the human population, infecting virtually all children with in the first few years of life5,6. Like the other herpesviruses HHV-6 is capable of persisting in the host after primary infection. Under conditions of immunosuppression, HHV-6 can reactive from latency. Both HHV-6A and HHV-6B replicate most efficiency in vitro in CD4+ T cells7. The host tissue range of HHV-6 in vivo is broad and includes peripheral blood mononuclear cells8, salivary glands, brain tissue, liver cells, lymph nodes and endothelial cells9. Candidate sites for latency are salivary glands10,11, brain tissue12,13, monocytes14 and early bone marrow progenitor cells15. To date, hunge numbers of investigations have examined the roles of HHV-6 in the development of hematological malignancies ( as an oncogene agent), and the significance of HHV-6 infection during the course of treatment ( as an opportunistic pathogen).

HHV-6 and Acute Leukemia

          Various hypotheses have been proposed concerning the involvement of infectious mechanism in the development of acute leukemia. The role of HHV-6 in acute leukemia, particularly childhood acute lymphoblastic leukemia (ALL), has been a matter of continuous interest, but remains controversial. Ablashi et al16 found high levels of HHV-6 antibodies in a small group of children with ALL compared with normal subjects, but a sequential study17 showed no significant differences in antibody titers between 50 patients with ALL and 50 patients sex aged mathed blood donors. The largest serological case control investigation showed a slight but significant association between HHV-6 antibody titers and acute myeloid leukemia (AML) patients, while no significant association was found between HHV-6 antibodies and ALL. In 2002, however , Salonen et al18 found the presence of IgM antibodies in 40% of children with leukemia (n=40) and high avidity of IgG compared with controls. These results again raise the possibility of the role for HHV-6 infection in childhood ALL. Bogdanovic et al19 analyzed HHV-6 and EBV DNA in Guthrie cards from children, but did not detect the DNA of these viruses in any samples from 54 subjects who later develop leukemia or 47 matched controls. These findings indicate that childhood ALL is unlikely to be associated with in utero infection byHHV-620.                                                                                                                                                                                          HHV-6 DNA was detected by PCR and in situ hybridization in bone marrow cells of children with T-ALL in 1991. However, Barozzi et al21 found that the presence of  HHV-6 DNA is not frequent in patients with ALL compared to normal subjects. Seror et al21 recently analyzed HHV-6 DNA copy number by real time PCR in bone marrow and peripheral blood from 36 children with ALL at diagnosis and during complete remission. Positive rates were 13,9% in leukemia samples and 34,1% in complete remission samples. Viral load was lower at diagnosis than at complete remission. Based on these findings, they concluded that HHV-6 may be unable to infect leukemia cells and reactivation may be observed during complete remission

HHV-6 and Chromosomal Integration

          The unique form of HHV-6 persistence is characterized by integration of the viral DNA sequences into chromosomes. The incidence of chromosomal integration (CI) for HHV-6 is about 2% in the population of the United Kingdom..Whether integrated HHV-6 is capable for replication or is associated with disease remains unclear. Daibata et al22 demonstrated integration of HHV-6 genome in Burkitt’s lymphoma cell line .Furthermore, they showed chromosomal transmission of HHV-6 DNA in ALL. These findings suggest the possibility of an association between chromosomally integrated HHV-6 and development of hematological malignancies. On the other hand, Hobacek et al23 recently reported the prevalence of HHV-6 Cl among children with ALL and AML. Among 339 patients, 5 patients (1,5%) were confirmed with HHV-6 CI. They concluded that the prevalence of HHV-6 CI in childhood leukemia does not differ from that published for other patients or healthy populations.




Hepatitis C Virus


Hepatitis C Virus (HCV) infection plays a significant role in the etiology of liver disease in patients treated for childhood leukemia24.The recent availability of serologic tests for the detection of antibodies to HCV (anti- HCV) permits a reappraisal of the diagnosis of HCV infection in these patients. However, only limited information, usually obtained by first-generation enzyme immunoassay (EIA l), is available on the overall prevalence of anti-HCV among patients cured of their leukemia25. Retrospective analysis of sera collected from on-therapy patients recently demonstrated a 19% prevalence of anti-HCV reactivity26. However, anti-HCV is not an indicator of active infection. Furthermore, patients receiving immunosuppressive therapy may develop anti-HCV reactivity only after treatment withdrawal Thus, studies of HCV prevalence among patients who undergo immunosuppressive therapy should include those with long-term follow-up, and study methods should be used that allow direct virus detection.

Maurizio Arico et al 27reported in their study that over 40% of patients who completed leukemia-directed treatment had evidence of exposure to HCV, and the majority of these showed ongoing HCV infection, as assessed by the presence of circulating HCV-RNA. This is in keeping with a recent study reporting anti-HCV prevalence of 35% in a similar population tested by EIA 1 at least 2 years after initiation of treatment28.Approximately one half of these patients were simultaneously seropositive for anti-HCV and HCV-RNA with evidence of liver enzyme elevation in 50% of the cases. This group showed biochemical and virologic features typically observed in children with transfusion-associated hepatitis C29.

About 10% of the patients were anti-HCV+ and HCVRNA- and had normal liver enzyme activity. These patients might have had recovered from HCV infection or might have had low fluctuating levels of viremia below the limits of detection of the RT-PCR assay. Approximately one third of HCV-infected patients did not have serologic evidence of infection. Specificity and reproducibility of the PCR results were confirmed by follow-up evaluation of nine patients, of whom four were repeatedly HCV-RNA+. It must be emphasized that HCV-RNA levels may fluctuate considerably30 as a function of time, thus providing an explanation for the five patients who were found to be PCR- upon follow-up evaluation. Indeed, two of them seroconverted to HCV by third generation ELISA. Locasciulli et al31have reported delayed appearance of anti-HCV in a population of viremic children who have been infected early in the course of antileukemic chemotherapy. All patients except one seroconverted after a median observation period of 10 months after treatment withdrawal. Indeed, the median follow-up period was not significantly different from that of our study population. However, in that study, patients were retrospectively selected for the presence of anti-HCV, and search for HCV-RNA was not systematically performed. The high number of seronegative infections observed in our patients is similar to that reported for HCV-infected organ transplant recipient or vertically infected newborns.I6 Although this phenomenon may be related to some extent to a long-term immunosuppressive effect of antileukemic therapy, it is not easily explainable because immunocompetence should have been fully restored after treatment withdrawal in the vast majority of our patients. Chronic hepatitis with defective serologic expression of HBV has been previously reported in leukemic children24, 32. Moreover, a pattern of seronegative HCV viremia has been occasionally found in immunocompetent blood donors,I as well as in nonleukemic children with chronic hepatitis, even though evaluated with first-generation assay33. All the viremic patients. except one who lacked anti-HCV, had normal transaminase activity, suggesting mild or absent liver damage. However, this does not exclude the presence of chronic liver disease in our patients, because it has been reported that viremic patients with normal transaminase levels may occasionally have chronic liver lesions." Our findings confirm that transaminase determination is inadequate to predict HCV infection, as has been previously reported in other risk categories. Analysis of HCV genotypes in our patients showed a relatively low prevalence of type I, whereas types I and II were equally represented. The proportion of the various genotypes was not significantly different from other anti-HCV patient groups. However, genotype 11 was more frequently associated with the absence of biochemical indicators of liver damage, as shown for other patient categories. The era of treatment was related to the risk of HCV infection in our series. Sixty percent of the patients treated up to 1984 were HCV+, and the infection rate decreased to about 29% after the introduction of donor screening for anti-HCV in 1990. However, two new HCV infections were found after blood transfusion since the introduction of EIA 2 as a donorscreening test. In view of the recently reported decline of the transfusion-associated risk of HCV infection,34 it is possible that the very high rate of HCV infection in these patients may not only depend on clearly documented parenteral exposure but also on other still unrecognized routes of transmission, possibly favored by associated conditions such as immunosuppressive therapy.







Viral infections play significant role in the pathogenesis of disease and especially affect patients with hematological diseases. HHV-6 is increasingly been recognized as an opportunistic pathogen rather than a casual pathogen among clinical hematologists. Particularly in the field of stem cell transplantation, HHV-6 is now considered as an important pathogen linked to life-threating encephalitis. On the other hand, the clinical syndrome of HHV-6 reactivation in patients with hematological malignancies who do not receive allogenic stem cell transplantation is not well defined. Hepatitis C Virus (HCV) infection plays a significant role in the etiology of liver disease in patients treated for childhood leukemia. Normal ALT levels did not exclude the presence of HCV infection in more than half of viremic patients. Thus, long term prospective studies of HCV infection in children, particularly in those cured of their leukemia, are mandatory to depict the current incidence and the natural history of liver disease in these subjects. The indications to perform liver biopsy in patients with subclinical HCV infection must be carefully considered, in accordance with current therapeutic options. Also EBV is a crusial pathogen for limphoproliferative diseases. Recent studies have demonstrated EBV infection in a considerable proportion of T cell non-Hodgkin’s lymphomas. Nasal T cell lymphomas, Hodgkin’s disease is inavariably EBV-positive whereas around 20% of T cell lymphomas arising at other sites (gastrointestinal, lung, lymph nodes) are associated with EBV.                                                Between the lines, pathogenetic roles of viral infections in hematological malignancies have been of continued interest. Many molecular studies have tried to establish a pathogenetic role of virus in development and during treatment of hematological malignancies. However whether viruses play role in these pathologies remains unclear, a positive polymerase chain reaction results may reflect latent infection or reactivation rather than presence of these viruses. In this way more studies need to be done for the extraction of sure conclusions.






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