Hepatitis C Virus Infection in Egyptian Children with Malignancy Thesis for fulfillment of master degree in pediatrics Presented by

Mona Samir Ali M.B.B.Ch Supervised by

Prof. Dr. Mona El-Said El-Raziky Professor of Pediatrics Faculty of Medicine Cairo University

Prof. Dr. Eman Fawzy Halawa Assistant Professor of Pediatrics Faculty of Medicine Cairo University

Dr. Eman Hassan Draz Lecturer of Pediatrics Faculty of Medicine Cairo University

Cairo University 2012

Acknowledgement I would like to thank ALLAH a lot for his kindness, patience and strength he gave me to achieve this work and made me able to finish it. I’m extremely grateful to Professor Dr/ Mona El-Said El-Raziky, Professor of Pediatrics, Faculty of Medicine, Cairo University for her meticulous supervision, her valuable comments and her kind support. I greatly appreciate her efforts to guide me to accomplish this work properly so to her I express my deepest gratitude. I would like to thank Professor Dr/ Eman Fawzy Halawa, Professor of Pediatrics, Faculty of Medicine, Cairo University, who guided me a lot in this work by encouragement and her great advices. My special thanks to Dr/ Eman Hassan Draz, Lecturer of Pediatrics, Faculty of medicine, Cairo University, for her encouragement and revision of this work; she gave me much of her time. I would also like to thank my dearest Dr/ Hanan Mina Fouad, fellow of Pediatrics, National Hepatology and Tropical Medicine Research Institute who helped me a lot in finishing this work and was always beside me.

A special dedication to my family for their never ending care. They were always supporting me and encouraging me to continue and to be successful especially my little children Habiba & Omar the best gift that God gave me.

Abstract

Abstract: Background: Children with cancer are at a high risk for hepatitis C virus infection due to immunosuppression secondry to chemotherapy and multiple transfusions of blood products during the course of the disease. Aim: The aim of this study is to evaluate the presence of HCV infection in children with malignant diseases, different risk factors, clinical course, laboratory, histopathological findings, natural history and type of response to HCV treatment. Methods: The medical records of

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patients recruited from the pediatric hepatology clinic at Cairo University pediatric hospital and presenting with post malignant virus C infection were reviewed retrospectively for data of medical history, physical examination and periodic evaluation clinically and laboratory during their follow up. Results: The mean age at diagnosis of HCV infection was 8+3.3 years, the period of follow up of the patients in the hepatology clinic ranged from 0.3 to 15 years with a mean of 2.6+ 2.3 years. Risk factors for HCV acquisition were chemotherapy in 93.5%, blood transfusions in 83.9%, operations in 64.5%. Out of the 31 cases, 51.6% were diagnosed as leukemia. At presentation serum ALT level was elevated in 83.9% and AST level was elevated in 80.6%. Liver biopsy were performed to 26 cases; 96.1% had mild to moderate activity, 32% had no fibrosis and 68% had mild to moderate fibrosis. Eighteen cases received HCV treatment. The response to HCV treatment was 27.7%. Conclusion: Although hepatitis C infection acquired by childhood cancer survivors was presented initially with high rate of elevated liver enzymes and PCR positivity, it seems to have a relatively benign clinical course with mild to moderate chronic hepatitis. Key words: HCV- Immunosuppression -Blood transfusions- EgyptMalignancy.

List of Contents List of Tables

………………………………………………………………...i

List of Figures ………………………………………………………………..iii List of Abbreviations …………………………………………………………iv Introduction

…………………………………………………………..…..1

Aim of Work

…………………………………………………….………...4

Review of Literature

…………………………………………………….….5

Chapter (I) Hepatitis C Virus infection in Children…………………….......5 Chapter (II) Neoplastic Diseases in Children

.................................……..34

Chapter (III) Hepatitis C Virus Infection in Children with Malignancy…....73 Patients and methods Results Discussion

……..……………………………………….……….85

……………………………………………………….…...90 ………………………………………………………………..110

Summary….……………………………….……………………………….....128 Conclusion ……………………………………………………………….…...130 Recommendations

....................………………………………….……..131

References …………………………………………………...........................132 ‫ اﻟﻤﻠﺨﺺ اﻟﻌﺮﺑﻰ‬.......................................................................................................150

List of Tables Table 1

Interpretation of HCV Assays.

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Table 2

Virological Responses during therapy and definitions.

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Table 3

International classification of childhood cancers.

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Table 4

Environmental causes of cancer.

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Table 5

Nonspecific signs and symptoms of childhood cancer

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The most common signs and symptoms of cancer in Table 6

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children. Clinical and laboratory features of rheumatologic

Table 7

and malignant conditions.

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Table 8

Signs and symptoms of brain tumors in children.

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Table 9

Presenting features of childhood Acute leukemia.

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Table 10

Presenting features of childhood lymphoma.

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Table 11

Sex distribution of the 31 studied cases.

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Table 12

Demographic data of the 31 studied cases.

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Table 13

Clinical presentation of 31 HCV infected children.

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Risk factors for HCV infection acquisition in 31 Table 14

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studied cases. Demographic data related to the history of

Table 15

malignancy in 31cases. i

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Table 16

Types of malignancies in 31 cases.

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Types of treatment for malignant diseases in 31 Table 17 Table 18

HCV infected children. Symptoms of HCV infection in 21 studied cases.

95 96

Examination at 1st presentation of HCV infected Table 19

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children.

Table 20

The ultrasonographic findings of 31 cases.

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Table 21

The 1st hematological results of 31 cases.

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Table 22

The 1st laboratory results of 31 cases.

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Table 23

HBsAg results of 31 HCV infected children.

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Table 24

The 1st liver histopathological findings of 26 cases.

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Table 25

The ALT course of the 31 cases.

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Table 26

The HCV PCR course of the 31 cases.

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Table 27

HCV treatment in 31 cases.

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Table 28

Table 29

Table 30

Histopathological data in relation to ALT in 25 cases. Histopathological data of 6 patients with multiple biopsies. The correlation between mean ALT & mean PCR in 10 HCV infected children.

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List of Figures Figure 1

The morphology of hepatitis C virus.

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Figure 2

The hepatitis C virus genome.

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Figure 3

Global prevalence of hepatitis C.

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Figure 4 Graphic display of virological responses.

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Lymphoblast on a peripheral blood smear, often seen at Figure 5

the time of diagnosis of acute lymphoblastic leukemia.

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Figure 6

Types of malignancies in 31 cases.

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Figure 7

Symptoms of HCV infection in 21 studied cases.

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The Correlation between basal ALT & basal PCR in 16 Figure 8

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HCV infected children. The response to different HCV treatment modalities of 18

Figure 9

105

cases.

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List of Abbreviations AAP

American Academy of Pediatrics

ALCL

Anaplastic Large Cell Lymphoma

ALL

Acute Lymphoblastic Leukemia

ALT

Alanin transaminase

AML

Acute Myeloid Leukemia

anti-HCV

Hepatitis C Virus antibody

AST

Aspartate transaminase

BL

Burkitt lymphoma

BMT

Bone Marrow Transplantation

CD4

cluster of differentiation 4

CRP

C Reactive Protein

CMV

cytomegalovirus

CNS

Central Nervous System

DLBCL

Diffuse Large B Cell Lymphoma

EBV

Epstein-Barr Virus

ESR

Erythrocyte Sedimentation Rate

ELISA

Enzyme Linked Immunosorbont Assay

ETR

End-of-treatment response

EVR

End Viral Response

FDA

Food and Drug Administration

GCTs

Germ Cell Tumors

GGT

Gamma-Glutamyltransferase

HAI

Histological Activity Index

HAV

Hepatitis A Virus

HBV

Hepatitis B Virus

HCV

Hepatitis C Virus

HCV-ab

Hepatitis C Virus antibody iv

HCV-PCR

Hepatitis C Virus- Polymerase Chain Reaction

HCC

Hepatocellular carcinoma Hepatic

HD

Hodgkin’s Disease

HDV

Hepatitis D Virus

HEV

Hepatitis E Virus

HHV-8

Human Herpes Virus 8

HIV

Human Immunodeficiency Virus

HLA

Human Leukocyte Antigen

HPV

Human Papilloma Viruse

HSCT

Hematopoietic Stem Cell Transplantation

HTLV-1

Human T-cell leukaemia virus type 1

IL-1b

Interleukin-1b

IRES

Internal ribosome entry site

INF

Interferon

INF-α

Interferon-α

IFN –γ

Interferon- γ

LL

lymphoblastic lymphoma

NHL

Non-Hodgkin’s lymphoma

NK

Natural killer

ORF

open reading frame

PCR

Polymerase Chain Reaction

PEG-IFNα-2b

Peginterferon -α-2b

RBV

Ribavirin

RIBA

Recombinant Immunoblot Assay

RVR

Rapid Virological Response

SLE

Systemic Lupus Erythematosus

SVR

Sustained Virological Response

TNF-α

Tumor Necrosis Factor-α

v

TSGs

Tumor Suppressor Genes

UTR

Untranslated Regions

WHO

World Health Organization

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Introduction

Introduction: Hepatitis C virus (HCV) was identified in 1989 and since then significant advances have been made in understanding the molecular biology, pathology, and treatment of HCV liver disease. HCV is an enveloped, single-stranded positive-sense RNA virus, belonging to the Hepacivirus genus within the flavivirus family. The HCV genome is approximately 10,000 kilobases with stereotypical genetic heterogeneity. Based on phylogenetic analysis of HCV sequences, 6 major HCV genotypes are recognized, designated 1 to 6, with multiple subtypes within each viral genotype (Mohan et al., 2010).

Egypt has the highest prevalence of adult HCV infection in the world, averaging 15%-25% in rural communities. The main (90%) HCV genotype is type 4. Egyptian studies documented that analysis of risk factors is significant for male circumcision by informal health care provider. Although blood transfusion, circumcision, vertical transmission, and living in a house with infected family member are the established risk factors for HCV transmission, approximately 70% of acquired infections are due to unidentified risk factors (El-Raziky et al., 2007).

Most chronically infected children are asymptomatic and have normal or only mildly abnormal alanine aminotransferase levels. Although the natural history of HCV infection acquired in childhood seems benign in the majority of instances, the infection takes an aggressive course in a proportion of cases leading to cirrhosis and end-stage liver disease during childhood; the factors responsible for a more aggressive course are 1

Introduction unidentified (Jonas, 2002). An Egyptian study reported that HCV infection is not always benign in the childhood period in children and Alanin transaminase (ALT) levels remain elevated in half of the children with histological abnormalities that are detected in three quarters of HCV RNApositive cases (El-Raziky et al., 2004).

Children who have had multiple transfusions as a result of chronic anemia, cancer or hemophilia are at a high risk for HCV and hepatitis B virus (HBV) infections. Patients who were treated for childhood cancer before HCV donor screening are a large population at a higher risk for transfusion acquired HCV infection. In countries with a high prevalence of HCV infection, exposure to such infection is a frequent problem for children with cancer (Ansari et al., 2008). A recent Egyptian study reported that a statistically significant correlation was found between HCV ribonucleic acid polymerase chain reaction (HCV-PCR) positivity (HCV viremia) and shorter inter-transfusion interval (Ragab et al., 2010).

Moreover, when compared with immunocompetent patients, the immunodepression caused by chemotherapy increases the chronicity rate of viral hepatitis. The successful cloning of HCV genome and the development of serologic markers of HCV infection showed that HCV was responsible for 85% to 90% of parenterally transmitted non-A, non-B hepatitis. The prognosis of chronic HCV is a matter of controversy. HCV could worsen the outcome of successfully treated pediatric oncology patients because a progression rate to cirrhosis of 20% has been documented in 20-year followup studies in HCV-infected adults with no other disease. Furthermore, recent 2

Introduction studies have shown that HCV infection is a risk factor for hepatocellular carcinoma (HCC). The prevalence of HCV infection did not show any significant change in the distribution between leukemia/ lymphoma and solid-tumor patients, even if the former group had a higher exposure to risk factors for HCV infection (Cesaro et al., 1997).

Patients with acute leukemia are immunosuppressed by nature of the disease and as a major side effect of the antineoplastic drugs. A recent study showed that immune reconstitution of T-cell, B-cell and Natural killer (NK) cell subsets is not gained for at least six months following therapy, and reconstitution is even more delayed in high-risk groups who receive more intensive chemotherapy (Torben et al., 2005). This immunosuppressed state increases risk of blood-borne viral infections (Sarper et al., 2008).

Older studies in pediatric cancer patients reported that, anti-HCV positive children had received significantly more blood products transfusion compared to seronegative patient (Fink et al., 1993). Also, a strong relation between the volume of blood infused and the risk of HCV infection, explains the higher prevalence of HCV infection among patients tested before anti-HCV blood screening was available (Strickland et al., 2000). Moreover, an Egyptian study has reported that evidences of Hepatitis B Virus (HBV) and HCV infection were present in around half of cases with childhood malignancies and there is a significant relation between HCV (antibody and RNA) and history of transfusions. Blood and platelets transfusions were the most identifiable risk factors for acquisition of HCV and HBV (Sharaf-Eldeen et al., 2007). 3

Aim of Work

Aim of work: The aim of this study is to evaluate the presence of HCV infection in children with malignant diseases, different risk factors, clinical course, laboratory, histopathological findings, natural history and type and response to HCV treatment.

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Chapter I

Hepatitis C Virus Infection in Children Hepatitis or inflammation of the liver can be due to a variety of causes of which viral infection is the most important, and leads to significant morbidity and mortality. Viral hepatitis is caused by infection with one of the five known viruses, which predominantly affect the liver the hepatitis A, B, C, D and E viruses (HAV, HBV, HCV, HDV and HEV) (Kumar et al., 2010). Many viruses in addition to the primary hepatotropic viruses (hepatitis A-E) should be considered in the etiology of hepatitis that occurs in children1. The nonhepatotropic viruses account for up to 10% of viral hepatitis and may cause severe liver disease especially in neonates and immunocompromised patients. Some of these relatively common non-hepatotropic viruses are Epstein-BarrVirus (EBV), cytomegalovirus (CMV), herpes simplex virus, enterovirus, adenovirus, rubella and parvovirus (Tezer et al., 2008). Hepatitis C virus (HCV) was identified in 1989 and since then significant advances have been made in understanding the molecular biology, pathology, and treatment of HCV liver disease (Mohan et al., 2010). HCV is a small, enveloped virus (Figure 1), a member of the Flaviviridiae family and the lone example of the genus Hepacivirus (Kumar et al., 2010). The HCV genome is approximately 10,000 kilobases with stereotypical genetic heterogeneity (Mohan et al., 2010).

Figure (1): The morphology of hepatitis C virus (Kumar et al., 2010) 5

Chapter I

Viral genome and replication: The genome consists of a single stranded positive sense RNA molecule approximately 9.6 kb long encoding a large open reading frame (ORF), which is flanked by highly structured 5′ and 3′ untranslated regions (UTR) (Figure 2). The 5′ UTR (341 nt) harbours the internal ribosome entry site (IRES) that mediates capindependent internal initiation of HCV RNA translation. The 3′ UTR varies between 200 and 235 nt, which includes a polyU/UC tract of variable length (~80 nt) and a conserved 3′ X tail region (98 nt) that helps in RNA replication (Kumar et al., 2010). Consistent with the known functions of most flavivirus proteins, the three Nterminal HCV proteins are probably structural (C, E1, and E2/NS2) and the four Cterminal proteins (NS2, NS3, NS4, and NS5) are believed to function in viral replication (Zein, 2000). The viral proteins are translated as a single polyprotein, which undergoes a series of co- and post-translational cleavage with the help of both host cell signal peptidases and viral proteases to generate the structural and non-structural proteins (Figure 2) (Kumar et al., 2010).

Figure (2): The hepatitis C virus genome. The boxed region represents the polyprotein coding segment. The 5′ and 3′ NCR contain highly structured internal ribosome entry site (IRES) and U/C-rich elements respectively. The polyprotein is processed into individual proteins by the viral NS2/3 cysteine protease and the NS3/4A serine protease, and a cellular signal peptidase as indicated. The functions of individual proteins are indicated

(Kumar et al., 2010) 6

Chapter I

Genotypes: Based on phylogenetic analysis of HCV sequences, 6 major HCV genotypes are recognized, designated 1 to 6, with multiple subtypes within each viral genotype. HCV genotypes are geographically clustered, with HCV genotypes 1 and 2 being prevalent worldwide, genotype 3 most common in Australia and the Indian subcontinent, and genotype 4 most common in Egypt, the Middle East, and central Africa, whereas genotypes 5 and 6 are seen in South Africa and Southeast Asia, respectively. Genotype 1 is the most common HCV genotype found in the United States (74%) and Europe (64%) (Mohan et al., 2010). Interestingly, genotype 4 accounts for over 90% of HCV infections in Egypt (Mostafa et al., 2004). More than 50 subtypes have also been described; the most common subtypes are 1a, 1b, 2a and 2b (Simmonds et al., 2005). These HCV genotypes differ in their pathogenicity, efficiency of translation/replication and responsiveness to antiviral therapy (Kumar et al., 2010). Typing of HCV isolates is of specific clinical interest, as the genotype is a marker of the likelihood of a response to pegylated interferon-ribavirin combination therapy and serves as a guideline for the duration of therapy. Determination of the HCV genotype is now part of medical practice for pre-treatment patient management. Genotyping is also useful for investigating outbreaks of infections and for understanding the epidemiology and virological features of this virus. Several methods targeting different regions of the HCV genome have been used for assessing genotypes. The most accurate method is to sequence an appropriate coding region that is divergent enough to allow the discrimination of types and subtypes. The three most studied regions are the core, E1, and NS5B (Murphy et al., 2006).

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Chapter I

Epidemiology: Infection with hepatitis C virus (HCV) is a major global health care problem. The World Health Organization (WHO) estimates that up to 3% of the world’s population has been infected with the virus. The infection rate ranges from as low as 0.1% in Canada to the extremely high rate of 18.1% in Egypt (Kalil et al., 2010). Egypt reports the highest prevalence of HCV worldwide, ranging from 6% to more than 40% with an average of 13.8%. In populations of blood transfusion recipients over the age of 30, this proportion has been reported to be as high as 73%, and in the general population aged 40–60 years it can be as high as 55% (Lehman and Wilson, 2009). The use of parenteral antischistosomal therapy in Egypt is thought to have contributed to a prevalence of antibodies against HCV in various regions ranging from 6 to 28 percent (mean, 22 percent) (Lauer and Walker, 2001). The overall worldwide prevalence of HCV varies geographically, but it is estimated at 3% with 150 million people with chronic infection. Data from the Centers for Disease Control and Prevention have shown that the seroprevalence of antibodies to HCV in the United States was 0.2% for children aged 6–11 years and 0.4% for those aged 12–19 years. Similar prevalence was found in Western Europe, North America, Mongolia, and Japan, whereas a higher prevalence, up to 10%–15%, was reported in Saudi Arabia and in parts of Africa (Indolfi et al., 2010).

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Chapter I

Figure (3): Global prevalence of hepatitis C (Lavanchy, 2009) The prevalence of HCV infection in children in developed countries ranges between 0.1% and 0.4% (Wirth et al., 2011). At the same time, the prevalence of HCV in Egyptian children is much lower, ranging between 2% and 10%. While the levels in children are much lower than those seen in the older population, they are still excessive by WHO standards, where prevalences greater than about 3–4% are considered high (Lehman and Wilson, 2009). In Egypt, the population-based HCV prevalence is about 18%. Communitybased data suggest that some 17.5% of HCV-infected Egyptians are children. Two studies from upper and lower Egypt revealed that the prevalence of HCV antibody (anti-HCV) in children is 3 and 9%, respectively (Habib et al., 2001; Medhat et al., 2002).

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