11 14 2006 Oncology Virus Cancer

Virus and Leukemia/Lymphoma * Leukemia/lymphoma: ATL (Adult T-cell leukemia/lymphoma) Burkitt's lymphoma, B-cell, T-cell, T/NK-cell, Hodgkin’s disease * Virus: HTLV-1, EBV, (HIV/AIDS),………… * Carcinogenesis (transformation) * Virus and cancer behaviors? Drug resistance Anti-apoptosis Metastasis/invasion Viral genes Cellular genes

To become a cancer ( 身體內的罪 犯 ) A. Mutations (for transformation, invasion, metastasis…) 性本善 HTLV-1 Tax and EBV LMP-1 ( 結交損友 ) B. Survival (anti-apoptosis): Not to commit suicide. 喪失良心 BHRF-1 and LMP-1 of EBV C. Survival (evade immunity): “Hide” themselves. MHC↓ D. Survival (attack immunity): IL-10, TGF-β, Fas ligand …

Viral Oncogenesis * 1898: Transmission of warts ( 疣 ) by cell-free extracts in dogs. (HPV) * 1907: Transmission of warts by cell-free extracts in humans. (HPV) * 1911 (Peyton Rous): Cell-free extracts of tumor  chicken sarcoma. (Rous Sarcoma Virus, a retrovirus) (1966 年諾貝爾生理醫學獎 ) * 1950s (Ludwik Gross): Cell-free extracts of tumor  murine leukemia. (Gross murine leukemia virus, GMuLV, a retrovirus) * 1960s : SV40, adenovirus …… * 1958 (Dennis Burkitt): Burkitt’s lymphoma. * 1964 (Epstein, Achong, and Barr of Bristol): Herpesvirus-like particles/EM. * 1970s~1980s : HBV and HCC; HPV and cervical cancer; HTLV-1 and ATL (adult T-cell leukemia/lymphoma) Anti-HBV vaccine against HCC: 1984/1985( 台灣 ); Anti-HPV vaccine against CC: 2005/2006( 全球 )

15% worldwide cancer burden is caused by viruses. (Taiwan: HCC/CC/NPC…~30%)

Difficulties in demonstrating a causal involvement of virus in human cancers * Suspected viruses are ubiquitous. ONLY a small % infected individuals develops cancers. * Decades often required between primary infection and cancer onset. * Monoclonal nature of cancer (inconsistent with a systemic infection). * Other chemical and physical carcinogens are related to the same cancer types.

Koch’s postulates ( 柯霍氏法則 / 病原 菌 ): •Bacteria can be found in all people with the disease. •Bacteria can be isolated from the infected sites. •The pure culture inoculated into a susceptible individual produces the symptom of the disease. •The same bacterium can be re-isolated from the intentionally infected animal or human.  Seroepidemiological evidence as an additional proof of causation.

Mechanisms of retroviral oncogenesis A. Acutely transforming retroviruses (e.g., RSV) incorporate and exert control over cellular growth-related genes (protooncogene capture) and subsequently transfer these deregulated genes into new cells. (v-src) B. Slowly transforming viruses (e.g., ALV) alter cellular gene expression by chance insertion of cis-acting viral regulatory sequences adjacent to these genes (insertional mutagenesis). (LTR) C. Trans-acting retroviruses (e.g., HTLV-1) alter cellular gene expression and function through viral regulatory proteins that act in trans. (Tax)

Mutation rate of retrovirus: 106-fold of normal cellular DNA (10-4 vs. 10-10) Somatic mutation rates in cells latently infected by HTLV-1: also elevated 106-fold (partly due to Tax) !!!

ORF II ( p30I & p13I )

(ORF IV)

ORF I ( p12I )

( ORF III )

Proviral HTLV-1 DNA = 9032 bp

HTLV-1 (Human T-cell Leukemia/Lymphoma Virus Type I) * Isolated in 1978 from ATL (adult T-cell leukemia/lymphoma, CD4+). * Southwestern Japan, Caribbean islands, Central Africa, Papua New Guinea. * 100% HTLV-1 seropositivity in ATL. 5%~15% in adults of endemic areas. * Breast feeding, blood transfusion, sexual contacts ( M  F ). MF horizontal transmission; MotherKids vertical transmission. * Unlike HIV, not transmitted by cell-free blood products. HTLV-1 keeps the virus burden low but promote amplification of cells harboring proviral DNA. * All ATL cells contain HTLV-1 provirus. * HTLV-1 immortalizes primary T cells (IL-2 dependent).  Transformation (IL-2 independent, acute ATL). (Other events required) * Primary HTLV-1 infection  asymptomatic carrier state  ATL (lifetime 4%). Average age of onset 58 years (24 ~ 85) (40~60 yr latency for ATL). * ATL: Multiple chromosomal aberrations with little common abnormalities. * ATL is monoclonal or oligoclonal (Inverse PCR). * Acute ATL infiltrates multiple organs (lymph nodes, liver, spleen, skin, and lung). Aggressive, poor prognosis, treatment resistant. Survival is within months. * Proviral insertions are random in ATL patient  Insertional mutagenesis does not play a major role, but more likely, the transactivating Tax.

Host #1 Host #2

Inverse PCR Host DNA

Proviral DNA

Restriction enzyme cut and self re-ligation

Host DNA

PCR

Causal association of HTLV-1 with ATL * Identical geographic distributions. * Almost 100% HTLV-1 infection in ATL patients. * All leukemia cells are HTLV-1 positive and contain monoclonal proviral DNA. * HTLV-1 immortalizes CD4-positive T cells.

Interesting observations * HTLV-1 infection through transfusion  HAM/TSP (but not ATL). HAM/TSP (HTLV-1-Associated Myelopathy / Tropical Spastic Paraparesis) ( 神經性脊髓疾病 / 熱帶痙攣性下肢癱瘓 ) HTLV-1-infected T cells infiltrate into cerebrospinal fluid and spinal cord. * HTLV-1 transmitted sexually in adulthood does NOT result in ATL. *Age of HTLV-1 infection is important determinant for ATL!? WHY??

HTLV-1 Tax and Rex genes * Both required for viral replication and gene expression. * Rex: through interaction with a human nuclear export receptor, CRM-1, acts as an adaptor for the nuclear export of unspliced retroviral mRNA. Promotes the cytoplasmic accumulation of singly spliced (env) and unspliced (genomic) mRNAs, inhibits splicings into gag-pol and pX mRNA. (early↓, late↑) * Tax: nuclear transactivator acting on LTR (21-bp repeat U3) and cellular genes. * Tax does not bind DNA but interacts with transcription factors: IKK, NF-κB/c-Rel, SRF (serum response factor), TBP (TATA-Binding Protein), ATF/CREB, CBP/p300 (CREB-binding protein)… * Tax interacts with cell-cycle proteins: p16 (INK4A), MAD1, p53, Chk1, Chk2… * Cellular genes transactivated by Tax: IL-2R (all)  IL-2 (?) (when this autocrine loop established  IL-2 independent) IL-2 signaling through JAK-STAT pathwayIL-2 independent when constitutive. Constitutive activation of the pathway in IL-2 independency. GM-CSF, c-fos, c-jun, c-sis, vimentin, TNF, iNOS, MDR-1, IL-13, IL-15 … * Cellular genes down-regulated by Tax: β-DNA polymerase(repair)  accelerated mutations!!! * Tax gene can immortalize CD4-positive cells (IL2-dependent) and transform rodent fibroblastic cell lines. Induce mesenchymal tumors in Tax-transgenic mice.

HTLV-1 Tax: Potential transforming functions * Activation of cellular transcription factors (through respective enhancers). Lymphokines (IL-2, IL-3, IL-5, IL-10, IL-13, IL-15, GM-CSF, TNF-β, TGF-β, PTHrP) Lymphokine receptors (IL-2Rα, IL-2Rγ) Nuclear oncogenes (c-fos, c-jun, c-egr) Tax uncouples activations and regulations of transcription factors. (CREB, CBP, NF-κB, SRF, and TBP …) (Bridging between Enhancer and TATA box for initiation complex formation) * Suppression of transcriptional inhibitory proteins. -----| IκBα, the major IκB in human T cells. (through IKK activation) Stimulus  NF-κB  IκBα -----| NF-κB (inhibitory feedback loop) NF-κB activation AND IκBα inhibition by Tax (Constitutive activation) * Suppression of cell cycle inhibitors. -----| p16(INK4A) -----| CDK-4/6 -----| Rb -----| E2F  Cell cycling  cyclin D2 and D3 

TBP TATA box Initiation Complex

Figure 3. Diagram of the CREB-dimer bound to the core CRE of the HTLV-1 21 bp-repeat. Tax interacts with CREB as a dimer and independently recruits the cellular coactivators, p300/CBP and P/CAF, which activate transcription by histone-acetylation/chromatinremodeling.

Proviral

( ORF III )

Cellular factors ( ORF IV )

Cellular factors

Cellular

ORF I

p12I

In ER, interferes with the assembly and trafficking of MHC class I molecules on cell surface. ↓ Escape immune detection

Enhancers

Genes

p12I reduces the MHC I expression on the plasma membrane

Expression of HTLV-1 genes in ATL cells * 90% leukemic cells express no or little HTLV-1 mRNA. In vivo infected non-leukemic cells also express little HTLV-1 mRNA. Even low level Tax expression is sufficient to maintain ATL phenotypes. (OR, a Hit-and-Run model?) (Once switched ON by Tax, e.g. NF-κB & IL-2R  Maintained ON ever) * When such cells are cultured in vitro with FCS  HTLV-1 expression Presence of suppressive factor(s) in the blood (other than antibody). * ATL cells do not replicate in peripheral blood but in lymph nodes, spleen, skin.

Models of disease progression: pathways to ATL

( Decades )

Models of disease progression: pathways to HAM/TSP

APCs strong

Control of Infection and Clinical Management: * Primary prevention: avoid viral infection. (a) Breast feeding (major) (Mother  Child: Vertical transmission) 30% childhood infection  Just a few% (by avoiding BF) (b) Sexual activities (Husband  Wife : Horizontal transmission) (c) Blood transfusion (2/3 chance of infection from a HTLV-1+ blood ) HTLV-1 screening in blood banks Transmission reduced ( 日本 ) * Secondary prevention: inhibition of HTLV-1 replication and/or clonal expansion of infected cells in asymptomatic or carrier patients. As2O3 , in combination with IFN-α or Retinoic acid ? * Tertiary prevention: treatment of ATL cancer patients. Chemotherapy, As2O3 , in combination with IFN-α or Retinoic acid ?

HTLV-1 Tax: centrosome amplification and cancer. Pumfery et al., 2006 Aug 9;3:50. Involvement of HTLV-1 Tax and CREB in aneuploidy: a bioinformatics approach. de la Fuente et al., Retrovirology. 2006 Jul 5;3:43. Requirement of the HTLV-1 tax-stimulated HIAP-1 gene for the survival of transformed lymphocytes. Waldele et al., Blood. 2006 Jun 1;107(11):4491-9. HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP). Krueger et al., Blood. 2006, 107(10):3933-9. HTLV-1 oncoprotein tax inhibits Fas-mediated apoptosis by inducing cellular FLIP through activation of NF-kappaB. Okamoto et al., Genes Cells. 2006 Feb;11(2):177-91. HTLV-1 Tax Protein Down-regulates the Pre-TCR-α Gene Transcription in Human Immature Thymocytes. Wencker et al., J Virol. 2006 Oct 18; [Epub] Correlation of MHC class I downregulation with resistance of HTLV-1-infected T cells to cytotoxic Tlymphocyte killing in a rat model. Ohashi et al., J Virol. 2002 Jul;76(14):7010-9. IL-10 gene expression and adult T-cell leukemia (ATL). Mori et al., Blood. 1996 Aug 1;88(3):1035-45. Mori et al., Leuk Lymphoma. 1998 Apr;29(3-4):239-48. Overexpression of TGF-β in transgenic mice carrying the HTLV-1 tax gene. Kim et al., Mol Cell Biol. 1991 Oct;11(10):5222-8. Transactivation of the TGF-β1 gene by HTLV-1 tax: a potential mechanism for the increased production of TGF-β1 in adult T cell leukemia. Kim et al., J Exp Med. 1990 Jul 1;172(1):121-9. Involvement of NF-AT in HTLV-1 Tax-mediated Fas ligand promoter transactivation. Rivera et al., J Biol Chem. 1998 Aug 28;273(35):22382-8.

Epstein-Barr Virus (EBV): * 1958 (Dennis Burkitt): Burkitt lymphoma. * 1964 (Epstein, Achong, and Barr of Bristol): Herpesvirus-like particles/EM. * Readily immortalize normal B-cells and establish latency, (remain fully differentiated with phenotypes similar to activated normal B cells) * Gene expression in latent infection: 6 EBNAs, 3 LMPs, 2 EBERs. * Lytic infection (induction) in cell cultures by TPA (PKC activation). * Early proteins: viral DNA synthesis Late proteins: structural components of virion. * BK cells (latent EBV): EBNAs (and/or LMPs) BK cells + TPA: Early and late (structural) proteins BK cells + TPA + Acyclovir: Viral early proteins only (Acyclovir : viral DNA synthesis inhibitor) * Ab to EBV is prevalent in all human populations. High titers in BK lymphoma, NPC, and Hodgkin’s disease. * Host range of EBV, in vitro, is limited to primate B lymphocytes. CD21: EBV receptor, surface glycoprotein.

Human herpesvirus and subfamily

Common name

Associated disorders

HHV-1 (Alpha)

Herpes simplex virus 1 (HSV-1)

Orofacial herpes (cold sores), ocular lesions, erythema multiforme, genital herpes

HHV-2 (Alpha)

Herpes simplex virus 2 (HSV-2)

Genital herpes, orofacial herpes

HHV-3 (Alpha)

Varicella-zoster virus (VZV)

Chickenpox, herpes zoster (shingles), Ramsay-Hunt syndrome

HHV-4 (Gamma)

Epstein Barr virus (EBV)

Infectious mononucleosis (glandular fever), Burkitt’s lymphoma, nasopharyngeal carcinoma

HHV-5 (Beta)

Cytomegalovirus (CMV)

Usually asymptomatic in immune-competent individuals. Neonatal CMV infection resulting in mental retardation and hearing loss. Interstitial pneumonitis, retinitis and colitis in the immunocompromised patients

HHV-6 (Beta)

Human herpesvirus 6

Exanthema subitum (roseola), infantilefebrile illness, hepatitis, interstitial pneumonitis, more frequent and severe graft vs host disease, multiple sclerosis

HHV-7 (Beta)

Human herpesvirus 7

Pityriasis rosea, exanthema subitum(roseola), infantile febrile illness

HHV-8 (Gamma)

Human herpesvirus 8

Kaposi’s sarcoma (AIDS), primary effusion lymphomas

Basics of EBV: * Enveloped ds DNA (172-kb, ~100 genes) virus. * Transmitted through saliva, blood transfusion, transplantation. * > 90% of adults worldwide are infected. 1. Early childhood infections: subclinical or undistinguishable from other respiratory illnesses.  latency (episomal EBV DNA) 2. Older childhood or adult infections: 25~75% develops infectious mononucleosis (IM)  CD8+ T-cell response  Latency ↘ May be fatal if immune compromised. * Targets initially B cells located in pharyngeal tissues. * Readily immortalize B-cells after primary infection. * Life-long latent infection: 1 in 105~106 circulating WBC. * EBV infection increases expression of NF-κB, c-myc, Ig, RAG... * EBV infection prevents cell apoptosis.

Varied Copies of Terminal Tandem Repeats (500 bp)

Copies of Tandem Repeats Indicate Clonality

(Latency)

Immunocompromised

BL ↗ Further c-myc Activation

TPA

(Zta/Zebra) (Bcl-2 family)

EBV-associated malignancies: Long after primary infection, many malignancies may occur: (EBV association)

Burkitts’s lymphoma (100%, 15~88%, 30~40%) Hodgkin’s disease (lymphoma) (40~60%) Nasal T/NK-lymphoma (>90%) Asians, Peruvians Post-transplantation lymphoma (>95%) NPC (>95%) … Elevated anti-EBV titers precedes diagnosis of cancers.

◆

◆

◆

◆

Burkitt’s lymphoma * Endemic: 10/105, ~100% EBV+ (Central Africa, Papua New Guinea) Sporadic: 0.1/105, 15~88% EBV+ AIDS-related: 608/105, 30~40% EBV+ * All contains translocations between immunoglobulin gene and c-myc. t(8:14) ---- Ig heavy chain gene + c-myc t(2:8) ---- Ig light chain gene κ + c-myc t(8:22) ---- Ig light chain gene λ + c-myc

Endemic BL: * All BL cells are EBV positive. EBV-negative BL cell lines are difficult to infect with the virus. * Uniclonality of EBV in tumors. * Holoendemic malaria infection Malaria infection stimulates B-cell proliferation and depresses cytotoxic T-cells. * Familial aggregation (HLA types/Genetics, Environments) * Euphorbia plants. ( 綠珊瑚 )

Endemic BL Sporadic BL

? 鹿角草 ?

綠珊瑚 (Euphorbia tirucalli) 又名青珊瑚、鐵羅。散生在台灣南部、小琉球及澎湖群島之海濱砂 礫地。乳白色汁液含有 diterpene (phorbol) esters 。又稱「未婚青年 不能摸的樹」──光棍樹（ Euphorbia tirucalli Linn ）

Euphorbia plants and EBV-associated cancers: (Burkitt’s lymphoma, NPC) * Euphorbia plants in central Africa (BL) and southern China (NPC). * Used in herbal medicines in Africa for headaches, sore throats, diarrhea, toothaches. (Used in Taiwan for bone spur?) * Phorbol esters detected in vegetables, water, soil, everywhere in endemic areas. * Euphorbia extracts induce chromosome translocations and deletions involving chromosome 8 (c-myc) in B cells in vitro. * Euphorbia extracts reduce EBV-specific cellular immunity by 70%. * Euphorbia extracts enhance EBV-induced outgrowth of primary B cells. EBV genomes latently present in B cells were reactivated in 5~15% of the cells.

Hodgkin’s disease (lymphoma) * One of the most frequent malignancies affecting young adults. * Rare malignant Hodgkin and Reed-Sternberg cells (2% tumor mass) amidst the majority of mixed nonmalignant inflammatory infiltrating cells. * Loss of lymph-node architecture. * EBV+ at 40~50% * 3~5 fold risk in persons with history of infectious mononucleosis (IM). * Age-specific rates of HD:Bimodal peaking in young adults and those >60 yr.

Nasal T/NK- lymphoma * EBV+ at ~90% * Common in Asia, very rare in Western countries.

Post-transplant lmphoproliferative disorder lymphoma * EBV+ at >95% * Due to immuno suppression * Maybe monoclonal, oligoclonal, or polyclonal.

Epstein-Barr Latent Genes: * EBNA1: Ori-P binding protein, episome maintenance and transcriptional activation. Totally Non-immunogenic!! (escaping surveillance) * EBNA2: Acidic type transcriptional activator that interacts with cell proteins. * LMP1: Transforms rodent fibroblasts. In B cells: Induces adhesion markers, NFκB, Bcl-2, A20 Essential for B-cell transformation by EBV. In epithelial cells: Induces NFκB, EGFR, A20, inhibits differentiation (NPC). Acts like a constitutively active TNFα receptor and CD40: TNF signaling is critical in normal lymphoid development B-lymphocytic CD40 : activates NFκB and promotes growth * LMP2: Colocalizes with LMP1 in the cytoplasmic membrane. Maintaining latency by preventing lytic infection in response to lymphocyte activation signals. * BHRF1: a homologue of Bcl-2. * EBER1 and EBER2: 170-bases RNA exist (105~106/cell) as ribonuleoprotein. * In acute infection, as many as 10% circulating B cells may be EBV-infected.  Cytotoxic CD8+ T-cell response  Infected B cells down to <1% EBNA1-only (Type I) latency: Undetectable by T-cell surveillance. In normal people, high-level CD8+ T-cells persists for life!

Life-long battles between EBV and immune cells!

Prevention and Control: Malignancies are associated with latent EBV which is maintained by the Host DNA polymerase.  NOT susceptible to most antiviral therapies targeting viral enzymes. * Vaccination and Immunotherapy Need to neutralize EBV at mucosal surfaces (IgA induction). (a) With a transformation-defective EBV that replicate in mucosa. (b) Subunit vaccine using the most abundant viral glycoprotein. (c) Use synthetic peptides to induce CTL. (d) Enhance CTL by inducing expression of EBV viral proteins. (e) …

Does expression of viral genes affect drug resistance or apoptosis of cancer cells? * Tax gene of HTLV-1 * LMP1 of EBV

MDR-CAT plus Tax+ or Tax-

The interaction of EBV viral gene (LMP1) and cellular drug-resistance associated genes.

Since LMP1 may participate in the development of epithelial transformation via a mechanism different from that in lymphoid malignancy, we examine the effect of the LMP1 on the epithelial and lymphoid cells.

Transfection of the LMP1

RHEK1 Epithelial

H9 or

BJAB

T or B lymphoid

The drug sensitivity of LMP1-transfected stable clones. Epithelial cell

T lymphoid cell

Cell viability in Doxorubicin-treated RHEK1 Cell Viabilities (%)

Cell Viabilities (%)

Mock pLMP1-low pLMP1-high

60 40 20

Cells Viabilities (%)

100

80

80 60 40

Mock

20

pLMP1

0

0 0

0.01

0.1

Dosage (uM)

1

Cell viability in Doxorubicin-treated BJAB 100

Cell viability in Doxorubicin-treated H9

120 100

B lymphoid cell

80 60 Mock pLMP1

40 20 0

0

0.01

0.1

Dosage (uM)

1

0

0.01

0.1

1

Dosage (uM)

Ectopic expression of LMP1 sensitized epithelial RHEK1 cells but not T or B lymphoid cells to chemotherapeutic drugs. (The protein tyrosine kinase AXL is up-regulated by LMP1 in lymphoid cells but not in RHEK1)

EBV LMP-1↑micronucleus formation, ↓DNA repair Genomic instability (oncogenesis)

Liu MT et al., Oncogene. 2004; 23(14):2531-2539 ( 國衛院陳振陽老師 )

Liu MT et al., Oncogene. 2004; 23(14):2531-2539 ( 國衛院陳振陽老師 )

Recombinant adeno-associated virus mediated RNA interference inhibits metastasis of NPC cells in vivo and in vitro by suppression of EBV encoded LMP-1. Li et al., Int J Oncol. 2006 Sep;29(3):595-603. Suppression of EBV-encoded LMP-1 by RNA interference inhibits the metastatic potential of NPC cells. Li et al., Biochem Biophys Res Commun. 2004 Feb 27;315(1):212-8. Promotion of metastasis in NPC by EBV LMP-1. Yoshizaki, Histol Histopathol. 2002;17(3):845-50. Review. MMP-9 expression is induced by EBV LMP-1 C-terminal activation regions 1 and 2. Takeshita et al., J Virol. 1999 Jul;73(7):5548-55. EBV induces invasion and metastasis factors. Wakisaka et al., Anticancer Res. 2003 May-Jun;23(3A):2133-8. Review. Prognostic role of EBV LMP-1 and IL-10 expression in patients with NPC. Ozyar et al., Cancer Invest. 2004;22(4):483-91. (immune suppression) EBV LMP-1 induces IL-8 through the NF-kB signaling pathway in EBV-infected NPC cell line. Ren et al., Laryngoscope. 2004 May;114(5):855-9. Induction of IL-8 by EBV LMP-1 and its correlation to angiogenesis in NPC. Yoshizaki et al., Clin Cancer Res. 2001 Jul;7(7):1946-51.

Many WHYs: * HTLV-1 prevalence F=1.6M, but ATL M=1.4F why?. Routes of HTLV-1 infection: blood transfusion, sex, milk. HTLV-1 transmission: Why M  F, but not F  M? * Male predominance in Virus-associated cancers? (BL: 3~4x, HCC:3~4x, NPC: ~3x)… * Why c-myc translocation breakage points different between Endemic BL and sporadic BL? * Based on existing clues, make a story for Burkitt’s Lymphoma EBV, Euphorbia plants, c-myc translocations, Two age peaks in endemic Africa: 5 yr and 8 yr. BL Site: jaws (abdomen in sporadic BL) Holoendemic malaria

Endemic Burkitt’s Lymphoma

75

Hong Kong Chinese 25

Male Female

30

California Chinese 7

1.8

Whites 0.7

B-cells in Pharyngeal ?

Type II Type III Type II

Type III

Type I Type II

Type I

Type I

Type II

Type I Immune normal

Immune suppression

Type III

EBNA-1 and EBERs are expressed in all types

Enhanced transforming potential?

Table 52.1-6: Risks of Transfusion-Transmitted Disease Organism

Estimated Risk per Unit Transfused in the United States per Transfusion

Pretransfusion Testing

Hepatitis B virus

1:205,000

HBsAg, anti-HBc

Hepatitis C virus (HCV)

1:1,935,000 (postnucleotide testing)

Anti-HCV, nucleotide testing

Human immunodeficiency virus (HIV) types 1 and 2

1:2,135,000 (postnucleotide testing)

Anti-HIV-1/2, (p24 antigen), nucleotide testing

Human T-cell lymphotropic virus (HTLV) I and II

1:2,993,000

Anti-HTLV-1/2

Cytomegalovirus (CMV)

1:10 to 1:20 (see text)

Some units tested for antiCMV antibodies

Parvovirus B19

Unknown

None

Bacterial contamination

1:1500 to 1:2500

None

Treponema pallidum

Rare

Rapid plasma reagin

Parasites (Plasmodium sp., Ehrlichia sp., Babesia microti)

Rare

None

vCJD prion

Rare (see text)

Deferral based on history

HBsAg, hepatitis B surface antigen; anti-HBc, hepatitis B core antibody; vCJD, variant Creutzfeldt-Jacob disease.

Table 7.2-1: Human Viruses with Oncogenic Properties Virus Family

Type

Human Tumor

Cofactors

Adenoviruses

Types 2, 5, 12

None

—

Flaviviruses

HCV

Hepatocellular carcinoma

—

Hepadnavirus

HBV

Hepatocellular carcinoma

Aflatoxin, alcohol, smoking

Herpesviruses

EBV

Burkitt's lymphoma

Malaria

Immunoblastic lymphoma

Immunodeficiency

Nasopharyngeal carcinoma

Nitrosamines, HLA genotype

Hodgkin's disease

—

Leiomyosarcomas

—

Gastric cancers

—

Kaposi's sarcoma

HIV infection

Body cavity–based lymphoma

HIV infection

Castleman's disease

HIV infection

HPV-16, -18, -33, -39

Anogenital cancers and some upper airway cancers

Smoking, ? other factors

HPV-5, -8, -17

Skin cancer

EV, sunlight, immune suppression

SV40, JC, BK

? Brain tumors

—

? Insulinomas

—

? Mesotheliomas

—

HTLV-1

Adult T-cell leukemia/lymphoma

Uncertain

HTLV-1I

Hairy cell leukemia

Unknown

HHV-8

Papillomaviruses

Polyomaviruses

Retroviruses

EBV, Epstein-Barr virus; EV, epidermodysplasia verruciformis; HBV, hepatitis B virus; HCV, hepatitis C virus; HHV, human herpesvirus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HTLV, human T-cell leukemia virus; SV40, simian vacuolating virus 40.

Table 41.2-4: Revised European-American Lymphoma/World Health Organization Classification of Lymphoid Neoplasms B-cell neoplasms Precursor B-cell neoplasm Precursor B-lymphoblastic leukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) Mature (peripheral) B-cell neoplasmsa Chronic lymphocytic leukemia/B-cell small lymphocytic lymphoma B-cell prolymphocytic leukemia Lymphoplasmacytic lymphoma Splenic marginal zone B-cell lymphoma (splenic lymphoma with villous lymphocytes) Hairy cell leukemia Plasma cell myeloma/plasmacytoma Extranodal marginal zone B-cell lymphoma (MALT lymphoma) Nodal marginal zone B-cell lymphoma Follicular lymphoma Mantle cell lymphoma Diffuse large B-cell lymphomas

Burkitt's lymphoma/leukemia (EBV) T- and NK-cell neoplasms Precursor T-cell neoplasm Precursor T-lymphoblastic leukemia/lymphoma (precursor T-cell acute lymphoblastic leukemia) Blastoid NK cell lymphoma Mature (peripheral) T-cell neoplasms T-cell prolymphocytic leukemia T-cell large granular lymphocytic leukemia Aggressive NK cell leukemia

Adult T-cell lymphoma/leukemia (HTLV-1+) Extranodal NK/T-cell lymphoma, nasal type Enteropathy-type T-cell lymphoma Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Mycosis fungoides/Sézary syndrome Primary cutaneous anaplastic large cell lymphoma Peripheral T-cell lymphoma, not otherwise specified Angioimmunoblastic T-cell lymphoma Primary systemic anaplastic large cell lymphoma HTLV, human T-cell lymphotropic virus; MALT, mucosa-associated lymphoid tissue; NK, natural killer. a B- and T/NK-cell neoplasms are grouped according to major clinical presentations (predominantly disseminated/leukemic, primary extranodal, predominantly nodal).

Multiple mechanisms were identified that may contribute to its constitutive activation. Among them, mutations (inactivation) of the IkBa gene (in EBV-negative HD) and expression of EBV-encoded latent gene products appear to give feasible explanations for this phenotype.  Both leads to NFkB activation.

Involvement of HTLV-1 Tax and CREB in aneuploidy: a bioinformatics approach. Retrovirology. 2006, 3:43 de la Fuente C, Gupta MV, Klase Z, Strouss K, Cahan P, McCaffery T, Galante A, Soteropoulos P, Pumfery A, Fujii M, Kashanchi F.

Background Adult T-cell leukemia (ATL) is a complex and multifaceted disease associated with human T-cell leukemia virus type 1 (HTLV-1) infection. Tax, the viral oncoprotein, is considered a major contributor to cell cycle deregulation in HTLV-1 transformed cells by either directly disrupting cellular factors (protein-protein interactions) or altering their transcription profile. Tax transactivates these cellular promoters by interacting with transcription factors such as CREB/ATF, NF-κB, and SRF. Therefore by examining which factors upregulate a particular set of promoters we may begin to understand how Tax orchestrates leukemia development. Results We observed that CTLL cells stably expressing wild-type Tax (CTLL/WT) exhibited aneuploidy as compared to a Tax clone deficient for CREB transactivation (CTLL/703). To better understand the contribution of Tax transactivation through the CREB/ATF pathway to the aneuploid phenotype, we performed microarray analysis comparing CTLL/WT to CTLL/703 cells. Promoter analysis of altered genes revealed that a subset of these genes contain CREB/ATF consensus sequences. While these genes had diverse functions, smaller subsets of genes were found to be involved in G2/M phase regulation, in particular kinetochore assembly. Furthermore, we confirmed the presence of CREB, Tax and RNA Polymerase II at the p97Vcp and Sgt1 promoters in vivo through chromatin immunoprecipitation in CTLL/WT cells. Conclusion These results indicate that the development of aneuploidy in Tax-expressing cells may occur in response to an alteration in the transcription profile, in addition to direct protein interactions.

Oncogene. 2005 Sep 5;24(39):5976-85. Molecular mechanisms of cellular transformation by HTLV-1 Tax. Grassmann R, Aboud M, Jeang KT. Institut fur Klinische und Molekulare Virologie, Universitat Erlangen-Nurnberg, Schlossgarten 4, Germany. [email protected] The HTLV Tax protein is crucial for viral replication and for initiating malignant transformation leading to the development of adult T-cell leukemia. Tax has been shown to be oncogenic, since it transforms and immortalizes rodent fibroblasts and human T-lymphocytes. Through CREB, NF-kappaB and SRF pathways Tax transactivates cellular promoters including those of cytokines (IL-13, IL-15), cytokine receptors (IL-2Ralpha) and costimulatory surface receptors (OX40/OX40L) leading to upregulated protein expression and activated signaling cascades (e.g. Jak/STAT, PI3Kinase, JNK). Tax also stimulates cell growth by direct binding to cyclindependent kinase holenzymes and/or inactivating tumor suppressors (e.g. p53, DLG). Moreover, Tax silences cellular checkpoints, which guard against DNA structural damage and chromosomal missegregation, thereby favoring the manifestation of a mutator phenotype in cells.

Lancet Oncol. 2004 Dec;5(12):738-46. Is endemic Burkitt's lymphoma an alliance between three infections and a tumour promoter? van den Bosch CA. Communicable Disease Control, Surrey Health Protection Unit, Wolfson Institute of Preventive Medicine, Barts and the London Queen Mary's School of Medicine and Dentistry, London, UK. [email protected] Malaria and Epstein-Barr virus (EBV), recognised cofactors for endemic Burkitt's lymphoma, are ubiquitous within the lymphoma belt of Africa, and, unless other cofactors are involved, the tumour should be much more common than it is. Malaria and EBV alone cannot account for the occasional shifting foci and space-time case clusters of endemic Burkitt's lymphoma. Arboviruses and plant tumour promoters are other possible local cofactors that could explain such characteristics. The geographical and age distributions of endemic Burkitt's lymphoma parallel those of potentially oncogenic, mosquito-borne arboviruses. Arboviruses seem to be associated with case clusters of endemic Burkitt's lymphoma, and symptoms compatible with arbovirus infection have been seen immediately before the onset of the tumour. RNA and DNA viruses, including EBV, are promoted by extracts of a commonly used plant, Euphorbia tirucalli, the distribution of which coincides with the boundaries of the lymphoma belt. Extracts of E tirucalli are tumour promoters and can induce the characteristic 8;14 translocation of endemic Burkitt's lymphoma in EBV-infected cell-lines. They also activate latent EBV in infected cells, enhance EBV-mediated cell transformation, and modulate EBV-specific immunity.

Nat Rev Cancer. 2004 Oct;4(10):757-68. Epstein-Barr virus: 40 years on. Young LS, Rickinson AB. Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TT, UK. [email protected] Epstein-Barr virus (EBV) was discovered 40 years ago (1964) from examining electron micrographs of cells cultured from Burkitt's lymphoma, a childhood tumour that is common in sub-Saharan Africa, where its unusual geographical distribution - which matches that of holoendemic malaria -indicated a viral aetiology. However, far from showing a restricted distribution, EBV - a gamma-herpesvirus - was found to be widespread in all human populations and to persist in the vast majority of individuals as a lifelong, asymptomatic infection of the Blymphocyte pool. Despite such ubiquity, the link between EBV and 'endemic' Burkitt's lymphoma proved consistent and became the first of an unexpectedly wide range of associations discovered between this virus and tumours.

Semin Cancer Biol. 2004 Dec;14(6):453-71.

Infectious agents and cancer: criteria for a causal relation. Pagano JS, Blaser M, Buendia MA, Damania B, Khalili K, Raab-Traub N, Roizman B. Lineberger Comprehensive Cancer Center and Departments of Medicine and Microbiology, University of North Carolina at Chapel Hill, Campus Box 7295, Mason Farm Road, Chapel Hill, NC 27599-7295, USA. [email protected] Infectious agents, mainly viruses, are among the few known causes of cancer and contribute to a variety of malignancies worldwide. The agents and cancers considered here are human papillomaviruses (cervical carcinoma); human polyomaviruses (mesotheliomas, brain tumors); Epstein-Barr virus (B-cell lymphoproliferative diseases and nasopharyngeal carcinoma); Kaposi's Sarcoma Herpesvirus (Kaposi's Sarcoma and primary effusion lymphomas); hepatitis B and hepatitis C viruses (hepatocellular carcinoma); Human T-cell Leukemia Virus-1 (T-cell leukemias); and helicobacter pylori (gastric carcinoma), which account for up to 20% of malignancies around the globe. The criteria most often used in determining causality are consistency of the association, either epidemiologic or on the molecular level, and oncogenicity of the agent in animal models or cell cultures. However use of these generally applied criteria in deciding on causality is selective, and the criteria may be weighted differently. Whereas for most of the tumor viruses the viral genome persists in an integrated or episomal form with a subset of viral genes expressed in the tumor cells, some agents (HBV, HCV, helicobacter) are not inherently oncogenic, but infection leads to transformation of cells by indirect means. For some malignancies the viral agent appears to serve as a cofactor (Burkitt's lymphoma-EBV; mesothelioma - SV(40)). For others the association is inconsistent (Hodgkin's Disease, gastric carcinomas, breast cancer-EBV) and may either define subsets of these malignancies, or the virus may act to modify phenotype of an established tumor, contributing to tumor progression rather than causing the tumor. In these cases and for the human polyomaviruses the association with malignancy is less consistent or still emerging. In contrast despite the potent oncogenic properties of some strains of human adenovirus in tissue culture and animals the virus has not been linked with any human cancers. Finally it is likely that more agents, most likely viruses, both known and unidentified, have yet to be implicated in human cancer. In the meantime study of tumorigenic infectious agents will continue to illuminate molecular oncogenic processes.

Br J Haematol. 2004 May;125(3):267-81. Epstein-Barr virus-associated Hodgkin's lymphoma. Gandhi MK, Tellam JT, Khanna R. Department of Tumour Immunology, Division of Infectious Diseases and Immunology, Queensland Institute of Medical Research, Herston, Brisbane, Queensland, Australia. [email protected] Survivors of Hodgkin's lymphoma (HL) frequently have many years to experience the long-term toxicities of combined modality therapies. Also, a significant proportion of HL patients will relapse or have refractory disease, and less than half of these patients will respond to current salvage strategies. 30-50% of HL cases are Epstein-Barr virus associated (EBV-positive HL). The virus is localized to the malignant cells and is clonal. EBV-positive HL is more frequent in childhood, in older adults (>45 years) and in mixed cellularity cases. The survival of EBV-positive HL in the elderly and the immunosuppressed is particularly poor. Despite improvements in our understanding of EBV-positive HL, the true contribution of EBV to the pathogenesis of HL remains unknown. Increased knowledge of the virus' role in the basic biology of HL may generate novel therapeutic strategies for EBV-positive HL and the presence of EBV-latent antigens in the malignant HL cells may represent a target for cellular immunotherapy.

Clin Cancer Res. 2004 Feb 1;10(3):803-21. Epstein-Barr virus and cancer. Thompson MP, Kurzrock R. Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. EBV was the first human virus to be directly implicated in carcinogenesis. It infects >90% of the world's population. Although most humans coexist with the virus without serious sequelae, a small proportion will develop tumors. Normal host populations can have vastly different susceptibility to EBV-related tumors as demonstrated by geographical and immunological variations in the prevalence of these cancers. EBV has been implicated in the pathogenesis of Burkitt's lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, and lymphomas, as well as leiomyosarcomas arising in immunocompromised individuals. The presence of this virus has also been associated with epithelial malignancies arising in the gastric region and the breast, although some of this work remains in dispute. EBV uses its viral proteins, the actions of which mimic several growth factors, transcription factors, and antiapoptotic factors, to usurp control of the cellular pathways that regulate diverse homeostatic cellular functions. Recent advances in antiviral therapeutics, application of monoclonal antibodies, and generation of EBV-specific CTLs are beginning to show promise in the treatment of EBV-related disorders.

Oncogene. 2005 Sep 5;24(39):6047-57. Natural history of adult T-cell leukemia/lymphoma and approaches to therapy. Taylor GP, Matsuoka M. Department of GU Medicine & Communicable Diseases, Faculty of Medicine, Imperial College, Norfolk Place, London W2 1PG, UK. After cell-to-cell transmission, HTLV-1 increases its viral genome by de novo infection and proliferation of infected cells. Proliferation of infected cells is clonal and persistent in vivo. During the carrier state, infected cells are selected in vivo by the host's immune system, the genetic and epigenetic environment of proviral integration sites, and other factors. In leukemic cells, tax gene expression is frequently impaired by genetic and epigenetic mechanisms. Such loss of Tax expression enables ATL cells to escape the host immune system. On the other hand, ATL cells acquire the ability to proliferate without Tax by intracellular genetic and epigenetic changes. Despite advances in support and the development of novel treatment agents, the prognosis for ATLL remains poor. A number of therapies, however, do appear to improve prognosis compared to CHOP (VEPA). These include interferon-alpha plus zidovudine (probably after 1-2 cycles of CHOP), intensive chemotherapy as in LSG-15 with G-CSF support and Allo-SCT (which includes the potential for cure). Emerging novel approaches include HDAC inhibitors, monoclonal antibodies, and proteasome inhibitors. Comparison between different therapeutic approaches is complicated by the range of natural history of ATLL, different recruitments of naive-to-therapy, refractory or relapsed patients, and variations in the reporting of outcome that frequently excludes difficult-to-evaluate patients. Moreover, results from relatively small proof-of-principle studies have not been extended with randomized, controlled trials. As a result, currently, there is no clear evidence to support the value of any particular treatment approach over others. To avoid further unnecessary patient suffering and to identify optimal therapy as rapidly as possible, large randomized, controlled trials encompassing multicenter, international collaborations will be necessary.

In the early 1950s, Ludwik Gross found that a virus could transmit leukaemia: ground-up, filtered leukaemia cells induced a malignancy when injected into infant mice (Gross, 1951). This finding was anathema to the biological establishment—Gross commented that some oncologists "even doubted my integrity; one of the well-known pathologists [...] refused to shake my hand when I greeted him before one of my lectures" (Kevles, 1997). Gross, who later won the Lasker prize, might today have found himself hauled up on a charge of scientific misconduct or even fraud for announcing such a controversial discovery (Kevles, 1997). In the 1960s, Howard Temin faced resistance—even ridicule— when he suggested that viral RNA could generate complementary DNA. "I'll give Howard's idea the amount of time it's worth—none," said a leading virologist during a meeting at the time. About a decade later, Temin was awarded the 1975 Nobel Prize in Physiology or Medicine for his discovery. In the 1970s, J. Michael Bishop and Harold Varmus faced similar problems when they proposed the oncogene theory of carcinogenesis, which won them the Nobel Prize in 1989 (Kevles, 1997).

More than 50 years ago, a young woman named Henrietta Lacks was diagnosed with cervical cancer. Despite surgery and aggressive radiation therapy, the cancer soon spread throughout her body, and on October 4, 1951, she died. It was a cruel death for the 31-year-old mother of five, but Lacks’ story didn’t end there. George O. Gey, M.D., head of tissue culture at Johns Hopkins University, where Lacks was treated, had been searching, for research purposes, for a line of human cells that could live indefinitely outside the body. He got his wish when cells from Lacks’ cancerous tumor were cultured. Just as they had done in her body, the cells multiplied ferociously in the lab, crawling up the sides of test tubes and consuming the medium around them. An entire generation of the cells reproduced every 24 hours. Referring to Lacks’ cells, Gey declared at the time, “It is possible that, from a fundamental study such as this, we will be able to learn a way by which cancer can be completely wiped out.” To this day, Lacks’ cells, known as the HeLa cell line, are some of the most robust and rapidly growing cells known to science. They are still used by thousands of researchers around the world to decipher the complexities of cell biology, particularly as they apply to cancer. At Yale, scientists are using the HeLa cell line to study, among other things, the human papillomavirus (HPV) that causes the cervical cancer that killed Lacks. “Her legacy,” says Daniel C. DiMaio, M.D., Ph.D., the Waldemar Von Zedtwitz Professor of Genetics and professor of therapeutic radiology, “is that her cells are helping us unravel the pathogenesis of cervical cancer, so that some day we might be able to prevent and treat it. It’s rather remarkable.” More recently, DiMaio’s lab demonstrated that cervical cancer cells need the viral proteins to grow, thus raising the possibility that the cancers can be treated with antiviral drugs. DiMaio, Janet L. Brandsma, Ph.D. ’81, and others are currently working on a vaccine to treat patients with cervical cancer. HPV is the best-understood example of how a virus leads to cancer. Two things have to happen: First, viral gene products cause the cells to become genetically unstable and accumulate mutations that render cells unresponsive to aspects of growth control and the immune response. Second, the viral oncogenes provide a sustained stimulus to cell growth.

Tight corsets and HPV Early thinking on cervical cancer and what causes it would hardly suggest such a rosy scenario. In 1842 an Italian physician in Florence observed that married women in the city were getting cervical cancer, but nuns in nearby convents weren’t. Although this observation would seem to point to a link between sexual activity and cervical cancer, the physician did not make this connection. He also observed that nuns had higher rates of breast cancer, and suggested that the nuns’ corsets were too tight. “Clearly they had no clue,” DiMaio says, “but the observation was significant.” Beginning in 1975, the virologist Harald zur Hausen, M.D., D.Sc., figured out what had eluded the Florentine physician. Zur Hausen, who for 20 years headed the German Cancer Research Center in Heidelberg, showed that HPV, a common infection spread through skinto-skin contact and sex, could lead to cervical cancer. He and his research team successfully isolated several genotypes of the virus, some of which they linked to genital warts and others to cervical cancer. Today, cervical cancer is responsible for 250,000 deaths each year worldwide, according to Charles J. Lockwood, M.D., the Anita O’Keefe Young Professor of Women’s Health and chair of the Department of Obstetrics, Gynecology and Reproductive Sciences. In the United States, where early screening has greatly reduced the mortality rate due to cervical cancer, about 5,000 women a year still die of the disease.

HTLV-1 Tax: centrosome amplification and cancer. Pumfery et al., 2006 Aug 9;3:50. Involvement of HTLV-I Tax and CREB in aneuploidy: a bioinformatics approach. de la Fuente et al., Retrovirology. 2006 Jul 5;3:43. Requirement of the human T-cell leukemia virus (HTLV-1) tax-stimulated HIAP-1 gene for the survival of transformed lymphocytes. Waldele et al., Blood. 2006 Jun 1;107(11):4491-9. HTLV-1 Tax Protein Down-regulates the Pre-TCR-α Gene Transcription in Human Immature Thymocytes. Wencker et al., J Virol. 2006 Oct 18; [Epub] HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICEinhibitory protein (c-FLIP). Krueger et al., Blood. 2006, 107(10):3933-9. Activation of the anaphase promoting complex by HTLV-1 tax leads to senescence. Kuo et al., EMBO J. 2006 Apr 19;25(8):1741-52. HTLV-1 Tax transgenic mice develop spontaneous osteolytic bone metastases prevented by osteoclast inhibition. Gao et al., Blood. 2005, 106:4294-302.