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PROFILING FOR KSHV AND OTHER HERPESVIRUSES IN VIVO IN CLINICAL SPECIMENS

Kristen Marie Tamburro

A dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of

Philosophy in the Curriculum in Genetics and Molecular Biology.

Chapel Hill 2013

Approved by:

Dirk Dittmer, PhD

Charles van der Horst, MD

Blossom Damania, PhD

Mark Heise, PhD

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ABSTRACT

KRISTEN MARIE TAMBURRO: Profiling for KSHV and Other Herpesviruses In Vivo In Clinical Specimens

(Under the direction of Dirk Dittmer)

The 1994 discovery of Kaposi Sarcoma-associated herpesvirus (KSHV)

was an important finding, especially as the sharp rise in HIV infections in the

early 1990’s led to an explosive Kaposi Sarcoma (KS) epidemic in men who have

sex with men. This cancer-causing herpesvirus is the causative agent of three

diseases in addition to KS: Multicentric Castlemen Disease (MCD), Primary

Effusion Lymphoma (PEL), and the newly described condition KSHV

Inflammatory Cytokine Syndrome (KICS). By understanding viral gene

expression in human patients, we hope to better understand KSHV infection and

its diseases, with the hope to shed light on novel therapeutic techniques.

In order to quantify KSHV viral gene expression, I devised methods to

profile human clinical specimens using high-throughput qPCR. This technique

was used to profile microRNA expression and was published as a methods video

journal in the Journal of Visualized Experiments (Chugh, Tamburro, and Dittmer,

2010). The accompanying manuscript is found in the Appendix chapter. This

same high-throughput technique was employed in Chapter II to determine KSHV

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In Chapter II, we hypothesized that HIV patients with newly diagnosed KS

in Malawi could potentially benefit from treatment with the antiherpesviral drug

Ganciclovir (GCV). This was addressed through a pilot study with the AIDS

Malignancy Consortium (AMC). By using qPCR to assess gene expression of

two viral kinases critical to drug responsiveness, we found evidence that 78% of

patients express aviral kinase and would potentially benefit from this therapy.

This finding is important, as it indicates that patients can be stratified for

treatment based on kinase expression in a tumor biopsy, which will maximize

potential effectiveness and limit waste of drugs in a resource limited setting. This

work will lead to upcoming treatment studies in this population.

Next, we used whole-genome KSHV transcriptome profiling to understand

whether all KS patients from the Malawian cohort express similar gene

expression profiles or if subgroups can be distinguished. Our results indicate the

gene expression profiles vary significantly between patients. Specifically, two

subpopulations were discovered- those with and those without high levels of

genome-wide transcription. Patients with high transcription levels demonstrated

significantly higher CD4 counts and KSHV viremia in plasma, although HIV

viremia, number of tumors, or size of biopsied lesion were not significantly

associated with KSHV transcription. This work is described in Chapter III.

In Chapter IV, we employed high-throughput DNA sequencing as a

method to diagnose co-infections in an end-stage AIDS patient. This was

published in the Journal Virology (Tamburro et al. 2012). The patient had minimal

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the diagnosis of the newly described condition KSHV Inflammatory Cytokine

Syndrome (KICS). Additionally, sequencing uncovered a co-infection with

HHV6a, which may have accounted for Systemic Inflammatory Response

Syndrome (SIRS) that ultimately led to the patient’s death. Notable to the

research community, this work resulted in the first complete sequence of KSHV

using direct methods, rather than relying on pre-sequencing cloning or cell

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ACKNOWLEDGEMENTS

The work that is presented here would not have been possible without

help and support from several people whom I would like to acknowledge.

First, I would like to thank my mentor Dirk Dittmer for accepting me as a

student in his lab, and for his guidance over the years. Despite my initial lack of

knowledge in the field of Virology upon joining the lab, Dirk was patient and

supportive. He allowed me to find my niche in the lab, supporting my decision to

apply for the Translational Medicine Training Grant program, and helping to aim

my project to make the most out of this program.

Additionally, I would like to thank the rest of my thesis committee-

Blossom Damania, Mark Heise, Ronald Swanstrom, and Charles van der Horst- I

appreciate all of your help in preparing me over the years and getting me to this

point in my career. I would especially like to thank my clinical co-mentor Charlie

van der Horst for helping me achieve my certificate in translational medicine and

for exposing me to the clinical aspect of research.

I would like to thank my lab members and members of the Damania lab for

their support and helpful conversations throughout the years. I am grateful for all

of their helpful comments in lab meetings and on a day-to-day basis. In

particular I would like to thank my lab roommate and friend Pauline Chugh for

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I would like to thank my amazing friends from college and those that I met

along the way in graduate school. You know who you are- and I couldn’t have

gotten here without all of your support and friendship.

I would like to thank my incredible family for always supporting me and

helping me to succeed. Throughout my life, my parents have always been there

to support me and to celebrate every (even small) victory, and I am so grateful

for all that they have given me.

Lastly, I would like to thank my husband, Daniel Sweet. Dan has always

been in my corner and I am so grateful to have him to share my life with.

He is always there to listen and help me, both in life and in science. Dan is one

of the smartest and more optimistic person that I know, and I am lucky to have

his love and support.

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FIGURES AND TABLES

Figure 1.1 Kaposi Sarcoma lesions and histology ... 15

Figure 1.2 KS Belt in Africa ... 16

Figure 1.3 KSHV genome ... 17

Table 2.1 Cohort characteristics (N=70, unless noted) of Kaposi sarcoma patients evaluated at Lighthouse ART clinic ... 49

Figure 2.1 Lack of correlations between KSHV viral load, CD4 count and HIV viral load in ART naïve patients presenting with HIV-associated KS in an endemic KSHV area ... 50

Figure 2.2 Evidence for different types of KS ... 52

Table 3.1 Cohort demographics for 66 therapy-naïve KS HIV+ patients in Lilongwe, Malawi ... 75

Figure 3.1 Sample and data analysis work flow chart ... 76

Figure 3.2 KSHV array characteristics and its validation ... 78

Figure 3.3 Correlation of reference genes ... 79

Figure 3.4 Gene expression profiling of training subset of samples indicate two distinct clusters of samples exist ... 80

Table 3.2 Significant primers (p<0.1) were identified from training data set ... 81

Figure 3.5 Gene expression analysis of 33 additional samples confirm two clusters of biopsies are distinguishable ... 82

Figure 3.6 There are significant differences in CD4 count between the two groups, but no difference in HIV or KSHV viremia ... 83

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Figure 4.1 Patient was positive for Kaposi Sarcoma, as confirmed by autopsy, immunohistochemistry,

and PCR ... 117

Table 4.1 Comparison of viral loads for KSHV-associated

diseases, as compared to this case ... 118

Figure 4.2 Nextgen sequencing analysis reveals KSHV

and HHV6a coinfection ... 119

Table A1 Specific reagents and equipment ... 143

Figure A1 Pre-microRNA signatures emerge through

profiling using a novel qPCR-based array ... 144

Figure A2 Inclusion of internal controls in the

qPCR-based pre-microRNA array ... 146

Figure A3 Melting peak analysis of pre-microRNA primers

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LIST OF ABBREVIATIONS AND SYMBOLS

ACTG AIDS Clinical Trials Group

AIDS Acquired Immune Deficiency Syndrome

AMC AIDS Malignancy Consortium

BAC Bacterial Artificial Chromosome

BCBL Body Cavity-Based Lymphoma

BP base pair

CD Castleman Disease

cDNA Complementary DNA

Cp/mL copies per mL

DNA Deoxyribonucleic acid

DR direct repeat

EBV Epstein-Barr virus

ELISA Enzyme-Linked Immunofluorescence Antibody

FLICE FADD-like IL-1B-converting enzyme

FLIP FLICE-like inhibitory domain

GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase

HCMV Human Cytomegalovirus

HHV Human Herpesvirus

HIV Human Immunodeficiency Virus

HSV Herpes Simplex Virus

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KS Kaposi Sarcoma

KSHV Kaposi Sarcoma-associated Herpesvirus

LANA Latency Associated Nuclear Antigen

MCD Multicentric Castleman Disease

miRNA microRNA

NTC No Template Control

ORF Open Reading Frame

PBMC Peripheral Blood Mononuclear Cell

PCR Polymerase Chain Reaction

PEL Primary Effusion Lymphoma

Pre-miRNA Precursor microRNA

Pri-miRNA Primary microRNA

RNA Ribonucleic Acid

RRE RTA Responsive Element

RTA Replication Transcription Activator

SIRS Systemic Inflammatory Response Syndrome

SNP Single Nucleotide Polymorphism

TPA 12-O-tetradecanoyl phorbol-13-acetate

TR Terminal Repeat

vFLIP viral FLICE-like inhibitory domain

vGPCR viral G-protein Coupled Receptor

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CHAPTER I.

INTRODUCTION

Kaposi Sarcoma (KS)

The hungarian dermatologistMoritz Kaposi first described Kaposi

Sarcoma (KS) in 1872 as “an idiopathic multiple pigmented sarcoma of the

skin”(Kaposi, 1872). These red, vascular skin lesions were described in a

population of elderly men of Mediterranean descent, now known as the Classic

form of KS. Since the discovery of classic KS, three additional types of KS have

been designated, and named based on the population of patients that they

typically occur in: African endemic, transplant-associated iatrogenic, and

AIDS-associated (epidemic).

KS is a mesenchymal tumor that typically begins with multifocal patch

lesions on the skin or in the oral cavity (FIGURE 1.1 A, B). Lesions typically

either merge to form ulcerated plaques, or become hard solid nodular lesions

(Ganem, 2010). Edema surrounding the lesion and potentially involving the

entire foot, leg, or other regions is common. Aside from the skin, lesions of the

oral cavity and gastrointestinal tract can occur in approximately 10% of patients

(reviewed in (Iscovich et al., 2000)). Involvement of organs such as spleen, liver,

lung, and lymph node are less common in classic KS but observed in Acquired

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At the histological level, KS is very complex and is identified by long

spindle cells forming slit-like vessels, extravasated red blood cells, and

inflammatory infiltrate (Boshoff and Weiss, 2001) (FIGURE 1.1 C, D). The cell

type that spindle cells originate from is debated, as they express markers for

endothelial cells including lymphatic markers and vascular endothelial cell

markers, as well as that of dendritic cells, smooth muscle cells, and

macrophages (Beckstead, Wood, and Fletcher, 1985) (Dupin et al., 1999) (Wang

et al., 2004) (Sturzl et al., 1992). KS tumorigenesis has been described as

consisting of three concurrent processes- proliferation, inflammation, and

angiogenesis (Ganem, 2010).

Classic KS

Classic KS, also known as sporadic KS, is the initially described form of

the disease defined by Moritz Kaposi (Kaposi, 1872). Classic KS is benign and

slow progressing, and typically begins with patch lesions on the lower limbs. It

occurs most commonly in elderly men of Mediterranean and Eastern European

Ashkenazi descent (Ross et al., 1985; Rothman, 1962) (Franceschi and Geddes,

1995). Other risk factors include geographic location, being over 65 years of

age, and being of male gender. The highest rates have been observed in the

Italian islands of Sicily and Sardinia (Cottoni, De Marco, and Montesu, 1996)

(Geddes et al., 1994) as well as in Greece (Touloumi et al., 1997). Low rates of

classic KS are reported in England, Wales, and Denmark (Grulich, Beral, and

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caused by the herpesvirus Kaposi Sarcoma-associated herpesvirus (KSHV), and

it is likely that immune dysregulation plays a role in disease pathogenesis.

Endemic KS

While classic KS is a more moderate disease mostly limited to the

extremities, endemic of KS that occurs in Africa is highly aggressive. Both

adults and children suffer from KS (Ziegler and Katongole-Mbidde, 1996), and

the prognosis is poor if untreated. This is particularly true of the

lymphadenopathic form of the disease, which occurs in children and involves the

lymph nodes. It is typically fatal within 1 year of onset (reviewed in (Iscovich et

al., 2000)).

KS is the most common malignancy in East African men and the third

most common in women (Jemal et al., 2011). Importantly, high rates of KS

predated the AIDS epidemic in sub-Saharan Africa (Cook-Mozaffari et al., 1998).

For this reason, the region of Africa surrounding the equator is referred to as the

“KS Belt” (FIGURE 1.2).

Potential Cofactors in Classic and African Endemic KS

It is unclear whether geographic or genetic factors might be responsible

for the predisposition of KS in the HIV-negative population. Previous studies

have indicated that there is a higher incidence of KS in areas of Italy with more

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for example on the island of Sardinia which has one of the highest rates of KS in

Europe (Cottoni, De Marco, and Montesu, 1996). Depletion of insects due to

DDT and other vector-control acts resulted in a decrease in KS (Coluzzi et al.,

2003) although it is unclear if this is an effect of the decrease in malaria or in

insect bites directly.

Additionally, volcanic clay salts have been proposed as a potential

cofactor for KS (reviewed in (Simonart, 2006)). In Africa as well as in the

volcanic region of the Mediterranean, iron bi-products are found in wet clay and

can enter the skin through sweat glands, possibly from the feet (Price and Plant,

1990) (Montella et al., 1996; Simonart et al., 1999). Poverty-stricken peasants

tend to walk barefoot and are also more likely to have KS, particularly in the

lower extremities (Ziegler et al., 2003; Ziegler, 1993; Ziegler et al., 1997). Iron

also increases the risk for many cancers (Weinberg, 1996) (Huang, 2003), and it

is possible that consuming foods and water containing high levels of iron could

also be playing a role in the predisposition to KS in these regions (Simonart,

2006).

Iatrogenic KS

Iatrogenic KS occurs in transplant recipients on immunosuppressant

therapy, such as steroids (Penn, 1997; Trattner et al., 1993). Unlike classic KS,

males and females have approximately equal rates of iatrogenic KS (reviewed in

(Hardy et al., 1976); Penn, 1988). Transplant patients in the US have

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population (Penn, 1988). Additionally, iatrogenic KS is more common in regions

where KS is more prevalent, such as Jewish transplant recipients. In Saudi

Arabia, KS is the most common transplant-associated cancer (Qunibi et al.,

1988).

Iatrogenic KS is commonly seen in renal transplant patients, and is rare in

heart, liver, lung, or bone marrow transplant recipients (reviewed in (Iscovich et

al., 2000)) (Gentile et al., 2005). Lesions typically occur approximately 2 years

after pre-transplant therapy, and the average age of onset is 42 years (Penn,

1979) (Penn, 1988). Although both host and donor KSHV infection has been

proposed as the underlying cause for KS, it seems that reactivation of an

underlying host KSHV infection in the immune suppressed setting is likely

responsible (Becuwe et al., 2005). High KSHV viremia in renal transplant

patients is associated with a greater risk for developing KS (Weigert et al., 2004),

supporting the idea that host KSHV is an important factor in developing KS.

Tumors can regress with reduction of immune suppressants and

restoration of the immune system (Penn, 1988) (Trattner et al., 1993) (Frances,

1998). Additionally, treatment with an mTOR inhibitor Rapamycin (also known as

Sirolimus) has anti-angiogenic function (Guba et al., 2004) and has been

effective in regression of KS (Campistol, Gutierrez-Dalmau, and Torregrosa,

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AIDS-Associated KS

The last but possibly most important population of people with KS is in

AIDS patients. 15-20% of AIDS patients develop KS, and it is the most common

cancer in this population (Beral et al., 1990; Nguyen et al., 2008) as AIDS

patients have an approximate 3000 to 5000-fold increased risk for developing KS

over the general population (Grulich et al., 2007; Mesri, Cesarman, and Boshoff,

2010).

In the 1980’s, rare diseases including KS and Pneumocystis carinii

pneumonia (Gottlieb et al., 1981; Hymes et al., 1981), were diagnosed at an

increased frequency in men who have sex with men (MSM) in the metropolitan

areas of New York and San Francisco. This led to the discovery of Human

Immunodeficiency Virus (HIV) and AIDS. It is now understood that HIV causes a

progressive loss of CD4+ T cells resulting in a deficient immune system that is

unable to fight infections (Friedman-Kien, 1981; Gottlieb et al., 1981) (Masur et

al., 1981) (reviewed in (Mildvan et al., 1982)). KS was described as

AIDS-defining (Longo et al., 1984), and was commonly the first sign of an underlying

HIV infection or progression to AIDS (reviewed in (Martin et al., 1998)). Today,

KS is still observed more frequently in AIDS patients despite being rare in the

general population, and remains among the leading cause of death of AIDS

patients today. In up to a third of HIV+ patients that carry KSHV, KS may reoccur

despite successful antiretroviral therapy (ART) (Krown, Lee, and Dittmer, 2008;

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my work and justification for the continual search for novel methods of treating

KSHV-associated disease.

KSHV

The Discovery of an Infectious Cause of KS: KSHV

In 1994, Chang and Moore began the search for an infectious cause of

KS, on the premise that an infectious etiology would explain the epidemic in

immunocompromised patients, as a latent infection is able to flourish in this

setting (Chang et al., 1994). They used Representational Difference Analysis to

compare DNA from a KS biopsy and normal skin of the same AIDS patient, and

discovered the herpesvirus that they named Kaposi Sarcoma-associated

Herpesvirus (KSHV) (Chang et al., 1994) also known as Human Herpesvirus 8

(HHV8) (Levy, 1995).

Since its discovery, KSHV has been demonstrated to be in monocytes in

KS biopsies by immunohistochemistry (Blasig et al., 1997), and detected in

PBMCs, saliva, and plasma of KS patients (Carvalho et al., 2010; Humphrey et

al., 1996; Koelle et al., 1997; Miller et al., 2006; Vieira et al., 1997), as well as in

semen, albeit to a lesser extent (Pellett et al., 1999). Antibodies to KSHV have

been detected in blood via enzyme-linked immunofluorescence antibody (ELISA)

assays, western blots, and immunohistochemistry (Edelman, 2005; Tedeschi,

Dillner, and De Paoli, 2002). Because it is identifiable in every lesion (IARC,

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1999), and cause disease in healthy individuals (Moore et al., 1996; Whitby et al.,

1995), KSHV is widely accepted as the causative agent of KS (Koch, 1884).

KSHV Genes and Genome

In every case where it has been examined, 100% of KS tumors are

positive for Kaposi Sarcoma-associated Herpesvirus (KSHV, HHV8) (Chang et

al., 1994). The KSHV genome is a linear double-stranded DNA genome of

approximately 165kb in length. 140kb comprise approximately 87 open reading

frames (ORFs), while the additional sequence makes up the terminal repeat

regions that flank both ends of the viral genome (Ganem, 2010). KSHV-specific

ORFs have been named K1 through K15, based on their location in the viral

genome, while the remaining genes have homology to related alpha and beta

herpesviruses.

KSHV belongs to the subgroup gammaherpesvirus, with its closest

human-tropic relative being Epstein-Barr Virus (EBV). As a member of the

herpesviridae family of viruses, KSHV can undergo both lytic and latent life

cycles or infections. During latency, few viral genes are expressed (FIGURE

1.3). The latency genes include the Latency Associated Nuclear Antigen (LANA,

Orf73), viral cyclin homolog (v-cyclin, Orf72), viral FLICE inhibitory protein

(vFLIP, Orf71), kaposin (K12) (Dittmer et al., 1998), and the KSHV miRNA’s (Cai

et al., 2005; Marshall et al., 2007; Pfeffer et al., 2005). The LANA protein

maintains the viral episome in an inactive state, tethered to the host

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function to replicate the virus and modulate the host immune response (reviewed

in (West and Damania, 2010)). Reactivation begins with the transcription of

KSHV immediate early genes. Viral

Replication Transcriptional Activator gene

(RTA, Orf50) is known as the “lytic switch” and is critical in initiation of the

lytic cycle or viral replication. RTA and other early genes can in turn

control the expression of RTA Responsive Elements (RRE’s), either

through directly binding the promoter or through interactions with other

transcription factors. RREs include the immediate early and early genes

polyadenylated

nuclear RNA (PAN RNA), ORF6, ORF21, ORF57, ORF59,

ORF74, K1, K5, K8, K9, K12, K14, K15, and viral interleukin-6 (vIL-6)

(Hong et al., 2011). Expression of KSHV late genes includes viral

structural and capsid proteins necessary for the formation of new virions.

Transmission of KSHV

The mechanism of KSHV transmission is still debated. The discovery of

high levels of infectious viral particles in saliva (LaDuca et al., 1998; Lampinen et

al., 2000; Lucht et al., 1998; Vieira et al., 1997) suggests that oral shedding may

be important in person-to-person transmission. Related herpesviruses CMV,

EBV, and HSV-1 can be contracted through oral transmission. Although

mother-to-child vertical transmission may occur rarely (Brayfield et al., 2003;

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mothers as well as other children, particularly in regions where KSHV is endemic

or HIV is epidemic (Bourboulia et al., 1998; Malope et al., 2007; Minhas et al.,

2008). In Africa and regions of Europe where classic KS is observed, saliva is

likely an important method of transmission (Gessain et al., 1999; Kasolo,

Mpabalwani, and Gompels, 1997; Ziegler and Katongole-Mbidde, 1996).

Sexual transmission of KSHV is controversial. KSHV can be detected in

semen and in the female genital tract (Gupta et al., 1996; LaDuca et al., 1998;

Whitby et al., 1999). Studies have indicated sexual transmission is likely in MSM

(Beral et al., 1990; Martin et al., 1998; Smith et al., 1999), but possibly less likely

in heterosexuals (Engels et al., 2007; Smith et al., 1999) (de Sanjose et al.,

2009).

Transfusions and blood exchange as a result of drug use also serve as

potential means for transmission (Dodd, 2005) (Blackbourn et al., 1997) (Dollard

et al., 2005), although the low viremia and low prevalence in the healthy adult

population make this less of a threat than initially proposed (Engels et al., 1999;

Pica and Volpi, 2007). Similarly, although it is well known that transplant

recipients often develop KS lesions, it is unclear whether this is due to

reactivated host virus or virus from the donor organ, and both are likely (Becuwe

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Other KSHV-associated Diseases

Primary Effusion Lymphoma (PEL)

Primary Effusion Lymphoma (PEL) is a body cavity-based lymphoma

(BCBL) defined by effusion in the pleural, pericardial, or peritoneal cavities

(Cesarman et al., 1995; Cesarman et al., 1996). This lymphoproliferative

disease is rare, and occurs mostly in AIDS patients but can occur in HIV-negative

persons as well (Klepfish et al., 2001). PEL is caused by KSHV and can occur

with or without EBV co-infection (Arvanitakis et al., 1996) (Komanduri et al.,

1996). Typically, PEL in HIV-negative patients is also EBV negative (Nador et

al., 1996) (Hamoudi et al., 2004). PEL progresses rapidly, and prognosis is

typically poor, with median survival of 2-6 months (Komanduri et al., 1996).

Similar to iatrogenic KS, PEL can occur in transplant recipients either alongside

KS or alone (Dotti et al., 1999; Jones et al., 1998). Unlike KS, PEL is monoclonal

and derived from B cells (Reviewed in (Wen and Damania, 2010)).

PEL cell lines have been established, and are a useful tool in studying

KSHV in vitro (Arvanitakis et al., 1996; Boshoff et al., 1998; Drexler et al., 1998;

Renne et al., 1996). PEL cell lines have high copy numbers of latent KSHV,

typically around 40-80 copies per cell (Ablashi et al., 2002). KSHV can be

reactivated by treating cells with sodium butyrate (Yu et al., 1999), a protein

kinase C-activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) (Renne et al.,

1996), or the KSHV “lytic switch” gene RTA (Orf50) (Guito and Lukac, 2012;

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Multicentric Castleman Disease (MCD)

The plasma cell variant of Castleman Disease (CD), known as multicentric

Castleman Disease (MCD) due to its presence in several lymph nodes, is

associated with KSHV (Soulier et al., 1995). Notably, the idiopathic localized

hyaline vascular form of CD is more common and has not been associated with

KSHV infection (Ablashi et al., 2002; Cronin and Warnke, 2009). MCD is a rare

condition that arises mostly in HIV-positive immune suppressed patients, and

less commonly in HIV-negative persons who are immunocompromised

(Polizzotto et al., 2012b). The prognosis for MCD patients is poor, and median

survival is approximately 2 years (reviewed in (Stebbing et al., 2008))

(Oksenhendler et al., 2000). MCD is characterized by severe flares of symptoms

including fever, night sweats, malaise, splenomegaly, anemia, thrombocytopenia,

fatigue, edema, high KSHV viral loads and elevated inflammatory markers

(Polizzotto et al., 2012a) (Oksenhendler et al., 1996) (Menke, Camoriano, and

Banks, 1992).

Diagnosis of MCD is dependent on immunohistochemical staining of a

lymph node biopsy (Oksenhendler et al., 1996) (Du et al., 2001). MCD is also

called multicentric angiofollicular hyperplasia due to its characteristic

immunohistochemical appearance of germinal center vascular proliferation. The

lymph node architecture is generally maintained, however the germinal center is

typically hypocellular and the interfollicular region is proliferated (Chadburn et al.,

2008) (Schmidt et al., 2008). KSHV-positive polyclonal IgM lambda cells are

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viral interleukin-6 (vIL6) are expressed. Despite sharing molecular similarity of

approximately 25% (Neipel et al., 1997), vIL6 differs from its human homolog in

its ability to bind to the gp130 subunit directly without requiring the high affinity

IL6 receptor gp80 subunit (Molden et al., 1997). This results in an increased

activiy for vIL6, which may contribute to the high levels of inflammation

responsible for symptoms in MCD patients (reviewed in (Sin and Dittmer, 2012)).

KSHV Inflammatory Cytokine Syndrome (KICS)

KSHV Inflammatory Cytokine Syndrome (KICS) is a KSHV-associated

systemic inflammatory disease that has only been recently described (Uldrick et

al., 2010a). To date, only 4 publications exist on KICS- one of which is our case

study described in Chapter 4. The initial report describes a retrospective review

of six HIV-positive, KSHV-positive patients with a disease that symptomatically

mimics MCD, despite no pathological evidence of MCD (Uldrick et al., 2010b).

Symptoms included fevers, wasting, edema, anemia, thrombocytopenia, elevated

inflammatory markers, adenopathy, and splenomegaly, however MCD was

excluded after multiple attempts to confirm with lymph node biopsies. MCD was

not found in follow-up or autopsy, and symptoms were attributed to high levels of

IL6 and vIL6 production. Patients with KICS also had high KSHV load,

comparable to MCD patients, but significantly less than KS patients. Another

study found that KICS patients and MCD patients have more single nucleotide

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infection, although it is unknown whether this co-infection is correlated with KICS

or if this was unique to this patient (Tamburro et al., 2012).

A working definition of KICS diagnosis was characterized as having

indicators from at least 2 of these categories- Symptoms, Laboratory

abnormalities, and Radiographic abnormalities- along with elevated C-reactive

protein, elevated KSHV load in PBMCs, and the exclusion of MCD by

pathological evaluation (Polizzotto et al., 2012b).

As this disease is currently poorly understood, treatment options are

unclear, although it is possible that patients may respond to antiviral drugs such

as Ganciclovir alone or in combination with Zidovudine, or Rituximab and

liposomal doxorubicin as these drugs have had some success in treating MCD

and KS (Cianfrocca et al., 2010; Northfelt et al., 1998; Polizzotto et al., 2012a;

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TABLES AND FIGURES

FIGURE 1.1: Kaposi Sarcoma lesions and histology

(A) Gross lesions of KS on the palate of a patient with AIDS. (B) Nodular skin lesion seen in KS. (C) Histopathology of a nodular KS lesion, showing fascicles of elongated spindle cells and numerous slit-like neovascular spaces. (D) High-power image of KS spindle cells. All images reproduced from the CDC’s

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FIGURE 1.2: KS Belt in Africa

Cumulative incidence from birth to age 64 years of endemic (non-HIV-related)

Kaposi’ sarcoma among men in Africa. Estimates are from Cook-Mozaffari et al.

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FIGURE 1.3: KSHV Genome

Structure of the KSHV (HHV-8) genome. The central portion of the genome is

flanked by the terminal repeats, labeled TR. The KSHV genome contains close to

100 open reading frames. Many of these are conserved in most herpesviruses;

these are present in the conserved blocks (white boxes) and are not indicated.

Other open reading frames are unique to rhadinoviruses, gammaherpesviruses,

or KSHV and are present in more divergent areas of the genome (indicated by

gray boxes). ORFs that have homology with herpesvirus saimiri are assigned the

corresponding numbers, and ORFs without recognizable homologues were

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REFERENCES

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CHAPTER II:

ART NAÏVE KAPOSI SARCOMA (KS) PATIENTS EXPRESS KSHV

ORF21 AND ORF36 AND MAY BENEFIT FROM GANCICLOVIR

THERAPY– AN AIDS MALIGNANCY CONSORTIUM STUDY

(AMC-S001)

PREFACE

This work has been submitted to the Journal of Infectious Diseases and is

currently under review 1. Mina Hosseinipour designed the study along with Dirk

Dittmer, Jeanette Lee, and other members of the AIDS Malignancy Consortium

(AMC). I performed all experiments, analyzed the data, and assisted with

making the tables and figures and drafting the manuscript. Dirk performed much

of the statistical analysis, and wrote the manuscript together with Mina and

myself. Jie Xiong assisted with data analysis and statistics. Additional

collaborators on this study include Albert Mwafongo, Loreen Chiwoko, and

Deborah Kamwendo at the UNC Project in Lilongwe, Malawi, Dan Namarika and

Sam Phiri at the Kamuzu Central Hospital, and Michael Nyirenda at the

Lighthouse Trust. Irving Hoffman from UNC Center for Infectious Diseases

(cFAR) and Department of Medicine also assisted in the design and

implementation of this study.

_______________________________________

1 _{Hosseinipour, M.C., Tamburro, K.M., Xiong, J., Namarika, D., Nyirenda, M.,}