(1) Seminar on "Retroviruses and Cancer: US-Japan Clinical Epidemiological Experiences"
This seminar was held at the Cloister on the NIH Campus on January 6-8, 1993. The organizers were Dr. William A. Blattner of the National Cancer Institute and Dr. Shigeo Ohino of Tottori University in Yonago, Japan.
Adult T-cell leukemia (ATL), recognized as a clinical entity in Japan in 1978 and in the Caribbean in 1982, is due to a retrovirus, human T-cell lymphotrophic virus type 1 (HTLV-I). In some carriers the virus causes lymphoma/leukemia; in others it causes neurologic, eye, joint and muscle disease; in children it causes skin disease. The purpose of the seminar was to exchange the most current information on HTLV-I/HTLV-II disease relationships and to identify areas for future research and cooperation.
Session I: Context and Perspectives
Dr. Sam Broder (Director, National Cancer Institute, Bethesda, MD)
Dr. Broder welcomed the participants. He expressed an appreciation to the group for their excellent collaborations over the years and encouraged the participants to continue to view their work in the global context. From his side, Dr. Broder said he would continue to endorse their efforts and give support whenever and wherever possible.
Dr. Robert W. Miller (National Cancer Institute, Bethesda, MD)
The first U.S.-Japan workshop on lymphocytic diseases, held in 1981, concerned reciprocal differences between Japan and the U.S. in lymphoproliferative disorders and autoimmune diseases. In comparison with the U.S., there was a marked deficiency of chronic lymphocytic leukemia in Japan, about half as much B-cell nodal lymphoma, and an absence of the peak in Hodgkin's disease at 20-29 years of age. In Japanese and other Asian people, systemic lupus erythematosus was about 2.8 times more common than in the U.S. Caucasians. Among patients treated surgically for thyroid disease 20.5% had Hashimoto's thyroiditis in Japan as compared with 8.3% in the U.S. and 2148 cases of Takayasu's aortitis were found in a national survey in Japan in 1975, whereas it is difficult to assemble even a small series of cases in the U.S. In addition, the Japanese had huge epidemics of Kawasaki's disease (mucocutaneous lymph node syndrome) before the disease was recognized in the U.S., and well as occasional cases of Takatsuki's disease, an unusual plasma cell dyscrasia with polyneuropathy; Kimura's disease, T-cell cervical lymphadenopathy with elevated IgE levels and eosinophilia; and Kikuchi's disease, a subacute necrotizing cervical lymphadenitis, especially in women 20-34 years old in Hokkaido. A second US-Japan workshop in 1982 expanded on this information (1). The explanation for the binational difference in the reciprocal relation between lymphoproliferative disorders and autoimmune diseases may be relevant to the diversity of findings due to HTLV-I.
1. Kadin ME, Berard CW, Nanba K, Wakasa H:
Lymphoproliferative diseases in Japan and western countries: proceedings of the United States-Japan seminar, September 6 and 7, 1982, in Seattle, Washington.
Human Pathol 14:745-772, 1983.
Dr. Shigeo Hino (Tottori University, Yonago, Japan)
Five years ago we started an intervention study, the ATL Prevention Program (APP) in Nagasaki to break the cycle of HTLV-I mother-to-infant transmission by discouraging breast feeding by HTLV-I carrier mothers. Among breast-fed infants, approximately 20% of the children were seropositive at 6 months of age and infection was usually confirmed by 12 months of age with occasional cases seroconverting later. As a result of intervention the incidence of maternal transmission of HTLV-I dropped from 20% to 3%. The results are consistent with the conjecture that milk-borne transmission is the major route of HTLV-I in Japan. Mothers with a seropositive child tend to infect successive children including, rarely, some who were bottle-fed. To determine predictors of intrauterine infection we performed nested PCR on cord blood of babies born to carrier mothers. There was no correlation between cord blood positivity and seroconversion in bottle fed babies. Additional studies are needed to further characterize the predictors of seroconversion in infants of HTLV-I positive mothers.
Dr. William A. Blattner (National Cancer Institute, Bethesda, MD)
A series of epidemiologic studies have been undertaken in HTLV-I and -H endemic areas outside of Japan to explore disease relationships and contrast these data with patterns observed in Japan. For example, adult T-cell leukemia/lymphoma (ATL) occurs at a rate similar to that in Japan but the peak occurrence is approximately 10 years younger, and the clinical presentations are more likely to be of the ATL-lymphoma type. Possible links to agricultural exposures are suggested in a case-control study analysis. Infective dermatitis has been confirmed as an HTLV-I associated entity in the West Indies, but no cases have been reported from Japan which raises the possibility of differences in susceptibility. A syndrome of persistent lymphadenopathy has been observed 'in children of HTLV-I seropositive mothers and it is hypothesized that these children may be manifesting an immune response to viral exposure, although they appear to be virus negative by polymerase chain reaction testing. The HTLV-II virus is endemic in Amerindian populations of the Western hemisphere and a number of possible disease associations are starting to be recognized including an HTLV-associated myelopathy syndrome and a form of T-cell leukemia involving natural killer cells. Contrasts in the patterns of disease and viral occurrence in different ethnic and racial groups afford new opportunities to define etiologic relationships.
Session II: Molecular Biology
Dr. Mitsuaki Yoshida (University of Tokyo, Tokyo, Japan)
MECHANISM OF TRANSCRIPTIONAL ACTIVATION BY ONCOGENIC PROTEIN OF HTLV-I
Persistent replication of HTLV-I and unusual proliferation of Infected cells seem to be associated with the initiation and/or progression of the diversity of clinical entities associated with HTLV-I infection, including lymphoproliferative malignancies and a spectrum of clinical conditions thought to result from HTLV-I induced immune perturbation.
Unlike other retroviruses, the HTLV subgroup has positive and negative regulatory genes for their replication. One of the regulatory genes, Tax, appeared to enhance expression of the viral genes and also of certain cellular genes. These cellular genes are of the growth factor receptor (IL-2R!
!!), cytokines (IL-2, GM-CSF, PTHrP, TNF and TGF!
!!), proto-oncogenes (c-fos, c-jun and c-egr) and some others (MHC class I and gp34). Through the activation of some of these cellular genes tax has been proposed to contribute to a) abnormal proliferation of infected T-cells and b) to the production of various cytokines that are frequently associated with disease phenotype. The element responsive to tax in the viral LTR is a 21 bp enhancer and that in the IL-2R!
!!is NF =kB; responsive elements in other genes have not been characterized. Tax protein thus appears to trans-activate at least two, likely more than two, enhancers that are structurally unrelated. However, it does not bind to these enhancer DNAs by this alone. As a consequence, participation of some cellular proteins has been proposed. Our previous observation using GAL-4 Tax fusion protein suggested that direct binding of the fusion protein to the DNA through GAL4 domain is required for activation. Thus, indirect association of Tax protein to the specific enhancers has been proposed. Such interaction might be mediated through enhancer binding proteins that bind to the Tax protein. To identify cellular proteins that contribute to trans-activation, we studied proteins that bind to the enhancer and those that bind to Tax protein. We found that leucine zipper proteins, CREB (CRE binding protein) and CREM (CRE modulator protein), do bind to Tax protein and then bind to the CRE sequence of DNA. Tax was also found to bind to a precursor p105 of the NF-Kb. These bindings were characterized and their involvements in the trans-activation mediated by Tax. Dr. Joseph Rosenblatt (UCLA School of Medicine, Los Angeles, CA)
HTLV: INTERACTIONS WITH VIRAL AND HOST GENES
HTLV-II has been reported in multiple cases of a T-cell lymphoproliferative disorder resembling, if not identical to, large granular cell leukemia (LGL) in which CD8 + cells contain integrated HTLV-II provirus, and in scattered cases of a chronic myelopathy resembling that induced by HTLV-I. In one such neurologic case, confected with HTLV-II and HIV-I, the cerebrospinal fluid contained oligoclonal antibodies, with specificity for at least one protein, known as rig., (the human homologue of the rat insulinoma gene), expressed in a brain CDNA library,.
The first complete HTLV-II provirus (designated NRA) was sequenced from a cell line obtained from a patient with LGL. This sequence is an HTLV-IIB subtype, that can be readily distinguished from the HTLV-IIA subtype based on numerous conserved differences within the GP46 and p21 transmembrane regions. Conservation was lowest in the LTR, and highest in protein coding regions. Isolates from U.S. and Italian IVDU and carriers can be classified by polymerase chain reaction into either HTLV-IIA (MO) or HTLV-IIB (NRA)-like viruses.
The downstream promoter for the hypercalcemic factor parathyroid hormone related peptide (PTHrP), is transactivated by HTLV-I/II tax, as well as by the AP-1/c-jun proto oncogene. A carboxy terminal HTLV-I Tax deletion mutant was deficient in transactivation of both IL-2R!
!!and PTHrP promoters, but not the HTLV-I LTR. Exogenous provision of NFKB rescued IL2R!!!expression but not the PTHrP promoter. This provides a possible explanation for the high incidence of hypercalcemia in ATL. The rex protein binds to specific RNA structural and sequence motifs present within the Rex response element (RxRE) within the LTR. The HTLV-II RxRE also contains cis-acting repressive sequences (CRS) that are distinct from the Rex binding domain. These CRS appear to impede nuclear-cytoplasmic transport of messenger RNA in the absence of Rex. Rex may therefore interact with cellular RNA binding proteins to regulate viral gene expression.
Dr. John Brady (National Cancer Institute, Bethesda, MD)
MOLECULAR PATHOGENESIS
Mutational activation of cellular proto-oncogenes or inactivation of tumor suppressor genes are common transforming events that may play a role in HTLV-I leukemogenesis. For example, our laboratory found that expression of the p53 suppressor gene is elevated in a number of HTLV-I transformed cell lines due to a prolonged half-life of p53 (2.5 to 9 hours compared to a normal 0.5 hour). Neither the PAb240 mutant-related p53 epitope nor hsc70, an indirect indicator of p53 mutation, was detected. DNA sequence analysis demonstrated that the p53 in each transformed cell line was wild-type. Stabilization of wild-type p53 in HTLV-I infected cells may represent its functional inactivation and contribute to lymphocyte transformation by HTLV-I.
HTLV-I-infected lymphocytes show increased expression of NF-Kb regulated cellular genes including IL2-R~, TNF-B and IL-6. The Tax~ gene product is associated with induction of the NF-KB transcription factor. When recombinant Tax1 protein is added to the tissue culture medium, it is taken up by uninfected cells and transported to cytoplasmic and nuclear compartments resulting in a rapid and transient increase in NF-Kb DNA binding. Since induction of NF-Kb did not require de novo protein synthesis it is possible that Tax1 induction of NF-kb occurs by a cytoplasmic activation mechanism which leads to the release of the IkB inhibitor from sequestered NF-Kb complexes. NF-Kb DNA binding is regulated in vitro by phosphorylation of IkB in NF-Kb complexes and alteration of the oxidation state of NF-Kb proteins. In recent experiments, Tax1 protein was found to associate with protein kinase C (PKC) by in vitro and the addition of Tax~ to the reaction leads to the phosphorylation of Tax~ and a 10-fold increase in the autophosphorylation of protein kinase C (PKC) in the absence of phosphatidylserine and diolein. Maximal Tax1-PKC activation occurred under reducing conditions. The role of the Tax1-PKC interaction and phosphorylation in the regulation of the NF-Kb transcription factor and Tax will provide important insight into the molecular mechanism by which the viral protein regulates viral and cellular transcription.
Dr. Genoveffa Franchini (National Cancer Institute, Bethesda, MD)
MOLECULAR MIMICRY: HTLV-I P12I PROTEIN EXHIBITS STRUCTURAL AND FUNCTIONAL SIMILARITY TO THE E5 ONCOPROTEIN OF BOVINE PAPILLOMAVIRUS
The transcriptional transactivator protein of HTLV-I, p40tax increases the transcription of the IL-2R and its ligand triggering an autocrine/paracrine mechanism which may represent the first step in the process of T-cell transformation. A protein of 12K (p12I), encoded by single and double-spliced mRNAs transcribed from the 3' end of the HTLV-I genome, localized in the perinuclear compartment and cellular endomembranes, exhibits significant amino acid sequence similarity to the E5 oncoprotein of bovine papillomavirus (BPV)-1. Both proteins are very hydrophobic, contain a glutamine residue in the middle of a potential transmembrane region(s) and are localized in similar cellular compartments. Because of these observations, we expressed the p12I protein to evaluate its ability to functionally cooperate with the BPV-1 E5 oncoprotein and to bind to a cellular target of the E5 protein, the 16K component of the vacuolar H + ATPase.
Cotransfection of the mouse C127 cell line with the p12I and E5 showed that although the P12I alone could not induce foci formation, it strongly potentiated the transforming activity of E5. In addition, the P12I protein bound to the 16K protein as efficiently as the E5 protein. These findings provide new insight for potential mechanisms of HTLV-I transformation and suggest that P12I and E5 may represent examples of convergent evolution between RNA and DNA viruses. The mechanism(s) of cell transformation by E5 in the C127 cell line is thought to involve activation of the platelet-derived growth factor receptor (PDGFR). Recent work suggests that 16K, E5, and the PDGFR form a three component complex which might lead to PDGFR activation and transformation of the C127 cells.
The finding of structural and functional similarities between proteins encoded by viruses with very different biological features suggests that these proteins may operate through common cellular mechanisms. Interestingly, the P12I alone does not bind to the PDGFR as has been reported for the E5 protein. These data indicate that binding to 16K is not sufficient for binding to the PDGRF, but that other parts of the E5 protein or other cellular proteins might be involved in the binding complex. However, preliminary data demonstrate that the HTLV-I P12I protein does bind specifically to the!
!!chain of the IL-2R. Although the functional and biological significance of this protein-protein interaction is not yet clear, the binding of a retroviral protein to the cell surface receptor has been shown to be biologically relevant in another model of transformation. In mouse erythroleukemia induced by the Friend spleen focus forming virus (F-SFFV), the binding of the carboxy-terminal 37 amino acids of the mutated F-SFFV gp55 envelope glycoprotein to the erythropoietin receptor is a crucial event for in vitro and in vivo transformation. Interestingly, an alignment of the amino acid sequences conserved between the E5 protein, the P12I protein, and the carboxy-terminal portion of the Mink-SFFV and Friend-SFFV envelope glycoproteins identifies a region which is redundant in Leucine and/or Isoleucine. Furthermore, other retroviruses, such as the feline leukemia virus and the simian sarcoma virus which cause leukemia in cats and sarcoma/leukemia in monkeys, respectively, share the same conserved region at the carboxsy-terminal region of the envelope glycoproteins. This signature sequence may define a particular class of viruses which use the binding to a relevant cellular receptor as a step in the mechanism of neoplastic transformation. The possible role of the 16K H + ATPase subunit in the HTLV-I and the BPV-1 transformation pathways remains to be clarified and the mechanism of cell transformation by P12I needs to be further investigated in human T-cells, the natural target in HTLV-I induced neoplasia.
The functional association of the HTLV-I P12I and BPV-I E5 raises the question of HTLV-I cooperation with the E5 protein of the human papillomaviruses. Basic research studies, combined with epidemiological studies, will be needed to determine whether HTLV-I can potentiate HPV in cervical cancer.
Session III: Epidemiology
Dr. Kazuo Tajima (Aichi Cancer Center Research Institute, Nagoya, Japan)
EPIDEMIOLOGY OF ATL
Although much is already known about the frequency of ATL in Japan, an ongoing series of surveys has continued to broaden our understanding of the distribution of these cases in Japan. Detailed knowledge is needed of the geographical distribution of this disease. There are four clinical sub-types of ATL: acute, chronic, lymphoma-type, and smoldering. These clinical types have some epidemiologic differences but all are ascertained by the following epidemiologic criteria:
1. Lymphoid malignancy such as in leukemia and lymphoma
2. Positive surface antigen for T-cells; T-cell lymphoma or leukemia
3. Positive antibody and/or antigen for HTLV-I.
Over a 2-year period all hospitals in Japan with 200 beds or more (60,000 hospitals) provided information on 1400 ATL cases. Geographically, ATL was concentrated primarily in Kyushu (60%), with 40% of cases from the northern part of Japan and the coastal area of Hokkaido. There is a clear correspondence between the distribution of patients with HTLV-I and patients with ATL. The Hokkaido area is heavily populated with the Ainu people who represent the aboriginal stock of the Japanese. The number of female HTLV-I carriers is higher than males but the number of males with clinical ATL is 50% greater than in females. The lifetime risk for the development of ATL in males was 5% and in woman 2%.
Only selected people progress to ATL after a long latency period and genetic and environmental factors may be involved. We do not know what these factors are but epidemiologically we can demonstrate that improved nutrition, especially an increase in the consumption of total protein over the past 25 years has significantly decreased the number of HTLV-I carriers that progressed to ATL. Since the end of WWII (1945) we see increases in the average height and weight of junior high school students, and infectious diseases have been substantially reduced.
Since the geographic distribution of malaria parallels ATL occurrence and malaria is a parasitic disease that attacks lymphatic tissue and stimulates T-cells, it is possible that infections which modulate immunity are associated with heightened progression of HTLV-I to ATL. There is a need to study such relationships since the decline in ATL in Japan may reflect the positive benefits of improved nutrition and infection control. Thus ATL might be prevented in carriers through such improvements in standard of living.
Another thing that seems to be an important factor in the manifestation of ATL and HTLV-associated myelopathy (HAM) is genetics. For example certain HLA haplotypes are specific to HAM and others specific to ATL.
On Tsushima Island, a high endemic area for HTLV-I, a prospective study of carriers at risk for ATL is underway. It shows a 0.1% risk for ATL in carriers, a figure that is the same in other parts of Japan. In the Tsushima Islands we cannot say that HTLV-I carriers are at a higher risk for other kinds of cancer.
Prevention trials to reduce HTLV-I transmission from mother to child are underway. Tsushima Island is a high endemic area, characterized by people who immigrated here over 100 years ago. We checked pregnant women in 113 villages and found a 5% to 8% positivity rate. This group was targeted for prevention of mother to child transmission through breast milk. The study is ongoing but the interim report on the breast-fed group shows that only 13% of the breast-fed babies are positive. If the babies were breast fed for less than 6 months, none were positive; over 6 months about 30% become infected. From this we conclude that shorter periods of breast feeding are not so dangerous for HTLV-I infection and have recommended this for carrier mothers in Tsushima Island.
HTLV-I infection rates by birth cohort show a declining prevalence which cannot be explained by changes in breast-feeding practices. Thus other factors must be involved in decreasing transmission. However, since Japan is a homogeneous country with similar lifestyles and social backgrounds it is difficult to do intersocial/interethnic comparisons between high endemic and low endemic areas. Thus comparative international studies from a global point of view for HTLV-I, ATL and other related diseases are needed.
Dr. Nancy Mueller (Harvard School of Public Health, Boston, MA)
EPIDEMIOLOGIC PERSPECTIVES ON HTLV-I
HTLV-I provides a useful model for understanding how oncogenic viruses cause diverse diseases such as ATL and HAM. Family studies provide a useful framework for understanding age at transmission and disease risk. For example in one study virtually all of the mothers of ATL cases were HTLV-I positive while for HAM, mothers were equally likely to be negative. Similarly in the Miyazaki Cohort Study sexual transmission has been documented primarily among older women married to seropositive men, a finding that is consistent with the cross-sectional age seroprevalence curve showing an excess of older seropositive women. Men who infect their wives are often older than age 60 years and have high antibody titer to HTLV-I and have antibody to the Tax protein of the virus. However, as demonstrated in this relatively large cohort, transmission events are rare and disease events even rarer. Thus as discussed below in the section outlining future research directions, large scale cooperative epidemiologic studies are needed to provide sufficient power to define the natural history of HTLV-I from infection to disease occurrence. This type of cooperation will also result in an opportunity to apply advanced biologic tests in order to define the mechanisms of disease pathogenesis.
Session IV: Clinical Pathogenesis
Dr. Kiyoshi Takatsuki (Kumamoto University Medical School, Kumamoto, Japan)
ETIOLOGIC INSIGHTS FROM CLINICAL AND CYTOGENETIC STUDIES OF ATL
Unusual Initial Manifestations in ATL.
In our recent experiences, the following patients were worthy of special mention to show the diversity of initial lesions in ATL.
Case 1.
A 77-year-old woman with a marked enlargement of the right tonsil was found to have lymphoma-type ATL. This case demonstrated that the Waldeyer ring can be the initial lesion in ATL.
Case 2.
A 32-year-old man complained of pain in the right forearm and was found to have a localized bone tumor in the right radius. Physical and laboratory examinations revealed no abnormality except for this bone lesion. Southern blot analysis demonstrated the monoclonal integration of HTLV-I proviral DNA in the bone tumor specimen. This case represents a rare example of bone lesion as an initial manifestation of ATL.
Case 3.
A 58-year-old patient with a small cell lung cancer (SCLC) was found to have high levels of soluble interleukin-2 receptors in the serum and in the supernatant of cultured SCLC cells obtained from the patient' s malignant pleural effusion. The expression of interleukin-2 receptors (Tac) on the SCLC cells was demonstrated by immunofluorescence, however, other lymphocytic markers, including CD2, CD4, CD8, CD19, and CD20 were not found on the cells with the exception of the natural killer cell marker, CD56. Southern blot analysis indicated the rearrangement of the T-cell receptor of the cancer cells. Moreover, monoclonal integration of HTLV-I provirus in DNA from the cancer cells was also demonstrated. These observations suggest that some SCLC in HTLV-I endemic areas are associated with HTLV-I.
Cases 4-7.
Four patients with adult T-cell leukemia (ATL) derived from a novel T-cell subset (CD4-, CD8- [double-negative, DN], T-cell receptor [TCR]!
!!+) have been identified. In the ATL cells of these patients, neither gene nor surface expression of CD4 and CD8 antigens were detected. In contrast to typical CD4+ ATL cells, DN-ATL cells were shown to express the protein and messenger RNA (MRNA) for S100!!!in immunocytochemical assay and the reverse-transcription polymerase chain reaction assay. The mean fluorescence intensity of the TCR/CD3 complex was extremely low in all four DN-ATL patients as well as in typical CD4 + ATL. All four patients had TCR!!!and!!!chain gene arrangements, with deletion of TCR!
!!chain gene and MRNA expression for TCR!!!,!!!, and CD3!!!but not for TCR!
!!and!!!chain genes. Thus, CD4-, CD8- TCR!!!T-cells are also a target for HTLVI-induced leukemogenesis. In addition, expression of the TCR!!!/CD3 complex on the DN-ATL cells was further diminished by the addition of anti-CD3 or anti-TCR!!!monoclonal antibody. These results suggest that the decreased TCR!!!/CD3 complex expression by ATL cells plays a key role in the development of ATL, irrespective of CD4 expression. Dr. Masanori Shimoyama, and the ATL Karyotype Review Committee recently published a detailed report on karyotypes of 107 cases with ATL (58 male, 49 female: 81 acute or lymphoma, 26 chronic or smoldering type). Clonal chromosome abnormalities were found 103 (96%) cases. The proportion of cases with aneuploid clones, the average numbers per case of both numerical and structural abnormalities, and marker chromosomes were larger in the aggressive acute or lymphoma type than in the nonaggressive chronic or smoldering type. The combination of rearrangement in 14q32 and monosomy X (seven cases) or deletion of 10p (six cases), and that of trisomy 3 and deletion in 6q21 (six cases), occurred only in the acute or lymphoma type and may be associated with the aggressiveness in adult T-cell leukemia/lymphoma.
Dr. Osao Sanada, a cytogeneticist in Kumamoto, studied chromosome abnormalities in ten cases of lymphoma-type ATL and six cases of HTLV-I negative non-Hodgkin lymphoma with peripheral T-cell type. The former group had a higher tendency for leukemic conversion and poorer prognosis than the latter. However, no definite difference on the numerical and structural chromosomal abnormalities between these two groups was found. The most frequent chromosome abnormalities: 14p+, 14q +, and chromosome 6 abnormalities were detected in both groups. These results support the conclusion that HTLV-I is not associated with specific chromosome abnormalities of non-Hodgkin lymphoma with peripheral T-cell type.
Dr. Sanada also performed a cytogenetic study on peripheral blood cells from a patient with smoldering ATL. Four types of primary abnormal clones were found upon examination of a large number of karyotypically analyzed cells cultured with and without phytohemagglutinin. However, HTLV-I proviral DNA was confirmed to be monoclonal. This discrepancy can be explained by the hypothesis that these four primary abnormal clones were all derived from a leukemic clone with a normal karyotype and the same integration site of HTLV-I proviral DNA.
Dr. Thomas Waldmann (National Cancer Institute, Bethesda, MD)
THE TREATMENT OF HTLV-I-ASSOCIATED ADULT T-CELL LEUKEMIA (ATL) WITH GENETICALLY ENGINEERED MONOCLONAL ANTIBODIES ARMED WITH RADIONUCLIDES
The leukemic cells of patients with HTLV-I-associated ATL express the IL-2 receptor!
!!,!!!and!!!subunits. In contrast, normal resting cells do not express the IL-2 receptor!!!-subunit identified by the anti-Tac monoclonal antibody. Patients with ATL were treated with different forms of IL-2 receptor-directed therapy to exploit the difference in IL-2 in receptor expression between normal and malignant cells. Using the unmodified anti-Tac monoclonal antibody, 1/3 of the 20 patients with ATL treated have undergone a remission, in two cases complete. There was no toxicity observed. However, unmodified murine monoclonal antibodies are limited by their immunogenicity and their poor effector functions. To address these issues we used genetic engineering to produce humanized anti-Tac as well as humanized MikB1 that is directed to IL-2RB. These humanized monoclonal antibodies contain the complementarity-determining regions from the mouse with the remainder of the antibody derived from human IgG1-k. These antibodies are dramatically less immunogenic than the murine versions, have improved pharmacokinetics and, in contrast to the parent antibodies, manifest antibody-dependent cellular cytotoxicity with human mononuclear cells. To enhance its effector function anti-Tac was armed with toxins or with!!!- and!!!-emitting radionuclides. In a clinical trial with 90Y-anti-Tac at the doses used (5, 10 and 15 mCi), 10 of the 15 patients with ATL underwent a partial or sustained complete remission. Thus, the clinical application of IL-2 receptor-directed therapy represents a new perspective for the treatment of certain neoplastic diseases including HTLV-I-associated ATL, autoimmune disorders, and for the prevention of allograft rejection. Dr. Toshiki Watanabe (The University of Tokyo, Tokyo, Japan)
PATHOGENETIC ROLE OF HTLV- I IN THE DEVELOPMENT OF UVEITIS
Based on seroepidemiologic, ophthalmologic and virologic data uveitis is another in a growing list of entities suspected to be caused by HTLV-I. In the highly HTLV-I endemic area, Miyakonojo in Miyazaki, the percentage of idiopathic uveitis (60% versus an expected prevalence of 40%). HTLV-I sero- prevalence is significantly high (37.9%, compared to 11.2% of general population in the area). In the younger age group of less than 50, the correlation was much more evident, with an odds ratio of HTLV-I for idiopathic uveitis at 11. O. Overall 20% of all uveitis in this area is HTLV-I associated.
HTLV-I uveitis is clinically characterized as follows: 1) average age of the onset in forties, 2) female predominance with the sex ratio of 1.82, 3) sudden onset of the symptoms such as floaters and/or blurred vision, 4) bilateral involvement in 45%, 5) intermediate uveitis with mild retinal vasculitis, 6) chronic and/or recurrent clinical course, 7) good prognosis of visual acuity in response to topical or systemic steroid therapy. The infiltrating cells in anterior chamber (AC) were shown to be mainly lymphocytes, of which about 90% was CD3 + T-cells in one case examined. Familial clustering of the uveitis was demonstrated. There was an association of Graves' disease to uveitis in 17% of the cases, where the onset of Graves' disease always preceded that of uveitis.
Virological studies revealed the presence of HTLV-I infected cells in the infiltrating cells of AC in all the cases examined, and the gene expression of Px but not env was demonstrated in two cases. These results provided support for the direct involvement of HTLV-I in the pathogenesis and/or the modulation of the inflammation. Compared to most asymptomatic patients there is an increase in the number of virus inflected cells in blood lymphocytes of uveitis patients and the level of virus resembled that reported in HAM/TSP by quantitative PCR method. Both of the major HTLV-I subtypes from Japan were found in these patients, indicating no disease specificity of the subtypes, while the so-called Pacific type clustered in Okinawa.
Session V: Immune Virus Interactions
Dr. Isao Miyoshi (Kochi Medical School, Kochi, Japan)
IMMUNOPROPHYLAXIS AGAINST HTLV-I AND HTLV-II
We have found that HTLV-I immune globulin is capable of blocking cell-to-cell infection of HTLV-I by blood transfusion and breast feeding in rabbits. In recent experiments our laboratory has investigated the protective effect of passive immunization against a Melanesian HTLV-I variant and HTLV-II.
HTLV-I IgG and HTLV-II IgG were prepared from pooled plasma from HTLV-I seropositive healthy Japanese and HTLV-II-seropositive healthy Americans, respectively. The neutralizing antibody titers of HTLV-I IgG and HTLV-II IgG were 1:3,900 and 1:1,900, respectively, when assayed by plaque inhibition of vesicular stomatitis virus bearing envelope antigens of HTLV-I or HTLV-II. Normal IgG was also prepared from pooled plasma from seronegative healthy Japanese.
To investigate cross neutralization against variant HTLV-I four groups of three rabbits (A, B, C and D) were studied. Groups A and B were first infused with 10 ml of normal IgG and HTLV-I IgG, respectively, and were immediately transfused with 5 ml of blood from a rabbit infected with a Melanesian variant, HTLV-IMEL5, which was isolated from a 58-year-old asymptomatic man from the Solomon Islands. Groups C and D were first transfused with 5 ml of blood from the HTLV-IMEL5-infected rabbit and infused with 10 ml of HTLV-I IgG after 24 and 48 hours, respectively. Rabbits receiving hyperimmune globulin remained seronegative during the observation period of 7-9 months while controls seroconverted. PCR analysis on lymphocyte DNA using HTLV-I Px primers confirmed the presence of HTLV-I sequences in all control rabbits and their absence in protected rabbits. These findings indicate that Japanese prototype and a Melanesian variant of HTLV-I share common neutralizing epitopes and are cross-neutralizable, although both are 8% divergent in the env region. It is emphasized that, for protective immunization, HTLV-I IgG must be given prior to or within 24 hours of transfusion exposure.
To evaluate immunoprophylaxis against HTLV-II, two groups of three rabbits were inoculated intravenously either with 3 x 107 RII cells (HTLV-II producing rabbit lymphocyte line) as controls or 24 hours after 10 ml of HTLV-II IgG was infused before challenge. All control rabbits seroconverted for HTLV-II after two weeks, while immunoglobulin infused rabbits showed a low level antibody response apparently to extracellular HTLV-II virions that were present in RII cultures. PCR analysis of lymphocyte DNA using HTLV-II-specific pol primers detected HTLV-II sequences in all controls but in none of immune globulin protected rabbits. These data indicate that HTLV-II hyperimmune globulin protects rabbits from HTLV-II infection.
To test cross-neutralization between HTLV-I and HTLV-II two groups of rabbits were either first infused with 10 ml of HTLV-II IgG or HTLV-I IgG. The HTLV-II IgG group was immediately transfused with 5 ml of blood from the HTLV-IMEL5-infected rabbit, and the HTLV-I IgG group was inoculated intravenously with 3 x 107 RII cells after 24 hours. Seroconversion occurred in both groups of rabbits after 24 weeks and HTLV-I and HTLV-II sequences were detected by PCR as well. These results suggest that there is no cross-neutralization between HTLV-I and HTLV-II, which is not unexpected in view of the 70% amino acid homology between these viruses.
Overall these data are important in the strategy for future vaccine development. Recent data indicate that the sequence divergence of other HTLV-I variants is confined to the variation level to HTLV-IMEL5. Therefore, two types of HTLV vaccines, one against cosmopolitan HTLV-I and the other against HTLV-II, would be sufficient for the prevention of HTLV infection worldwide.
Dr. Bernard Poiesz (SUNY Health Science Center, Syracuse, NY)
MOLECULAR BIOLOGIC CORRELATES
DNA from the peripheral blood mononuclear cells of 17 different individuals infected with the human T-cell lymphoma/leukemia virus type II (HTLV-II) amplified employing a pol gene primer pair by polymerase chain reaction (PCR). Subsequent cloning, sequencing and phylogenetic comparisons indicated that there are at least two distinct genetic strains of HTLV-II in the Western Hemisphere. HTLV-II isolates from Seminole, Guaymi, and Tobas Indians belong in the new substrain of HTLV-IIB. There was greater diversity among HTLV-II than HTLV-I New World strains with a level of heterogeneity similar to that observed in HTLV-I and STLV-I isolates from around the world, including Japan, Africa and Papua new Guinea. Given the above geographic and anthropological considerations and assuming similar mutation rates and selective forces among the PTLV (primate), these data suggest that either HTLV-II has existed for a long time in the indigenous Amerindian population and/or that HTLV-II isolates introduced into the New World were more heterogenous compared to HTLV-I strains.
The cell surface receptor for HTLV-I is unknown. A monoclonal antibody, Mab 34-23, inhibits the binding of HTLV-I to IL-2 and phytohemagglutinin-activation of peripheral blood mononuclear cells and HTLV-I entry into these cells. Analysis of a variety of target cells, including a human:mouse somatic hybrid which contains only human chromosome 17q, indicates that the binding of Mab 34-23 correlates with HTLV-I absorption and entry. SDS-PAGE and Western blot analysis shows that Mab 34-23 binds to 4 proteins of MW 31, 45, 55 and 70 kD. This binding can be inhibited by HTLV-I and not HIV proteins. HTLV-I virions bind to proteins of similar molecular weight and virus binding to these proteins can be inhibited by preincubation with Mab 34-23. These data suggest that Mab 34-23 may identify a specific cell surface receptor(s) for HTLV-I.
Classically, human retroviruses are believed to express only positive (+) sense messages. However, we have been able to detect both + and negative (-) strand HTLV-I transcripts in DNAse-treated, poly ARNA from two HTLV-I infected T-cell lines via reverse transcriptase-directed polymerase chain reaction (RTPCR) using the thermostable enzyme, Tth, and various primer oligonucleotides in sequential fashion. Both genomic and spliced + and - strand RNAs were detected. No RT-PCR products were observed if the target samples were first treated with RNAse, if neither primer was initially added during the reverse transcription step or if the Tth enzyme was used in solely the DNA-dependent mode. The presence of - strand HTLV-I messages was further confirmed by Northern and RNA dot blot analyses using a series of strand-specific probes. Interestingly, we find that HTLV-I infected T-cells have both + and - strand human!
!!-actin MRNA whereas in uninfected human T-cells we have been able to detect only + strand!!!-actin message. More importantly using RT-PCR we have been able, for the first time, to detect the presence of HTLV-I - strand RNAs in the fresh PBM's of three HTLV-I infected patients, thus ruling out the possibility of this phenomenon being a culture generated artifact. Further, our results suggest that the production of these - strand messages is via RNA dependent RNA synthesis. These observations include: (i) the absence of any template DNA copies corresponding to the spliced MRNA species detected in the infected cells; (ii) the similarity in size of + and - strand messages; and (iii) complementary sequence and similar splicing patterns for both + and - strand HTLV-I RNAs. We thus propose an alternative mechanism of RNA synthesis in the HTLV-I life cycle. Dr. Kazuo Sugamura (Tohoku University, Sendai, Japan)
VIRUS AND CELL INTERACTIONS
The interleukin 2 (IL-2)/IL-2 receptor (IL-2R) system has been considered to play a pivotal role in HTLV-I induced leukemogenesis. We have investigated the structure and function of the IL-2R subunit expressed on normal and HTLV-I infected T cells. So far, the functional IL-2R complex is known to contain the!
!!and!!!subunits. The!!!chain forms the low = affinity (Kd = ~ 10nM) receptor, and the!!!chain, which is essential for intracellular signal transduction, forms the intermediate-affinity receptor (Kd= ~ 10pM) and high-affinity receptor (Kd= ~ 10pM) in combination with the!!!in lymphoid cells. Unlike lymphoid cells, fibroblast cells transfected with IL-2R!!!, however, have no significant affinity to IL-2, suggesting existence of a lymphoid cell-specific third subunit,!!!chain, of IL-2R. We have recently succeeded in molecular cloning of the IL-2R!!!CDNA. Transfection experiments demonstrated that IL-2R!!!forms the high- or intermediate -affinity UK-2R along with Il-2R!!!and!!!, or with IL-2R!!!, respectively, in murine fibroblast L929 cells, and is involved in receptor-mediated Il-2 internalization. Although the cytoplasmic domain of IL-2R!!!is known to play a crucial role in intracellular signal transduction, it remains to be solved whether IL-2R!!!participates in the mechanisms of intracellular signal transduction as well as IL-2 binding abd IL-2 internalization. To answer this question, we examined the ability to transduce IL-2-mediated signals of murine fibroblast L929 cell lines transfected with cytoplasmic deletion mutants of the IL-2R!!!gene together with IL-2R!!!and!!!genes. We examined IL-2-induced tyrosine phosphorylation of IL-2R!!!and induction of the proto-oncogenes such as c-myc, c-fos, and c-jun in the L929 transfectants. Our results demonstrated that the cytoplasmic domain of IL-2R!!!is necessary for signal transduction that compromises at least two different signals.Our present study indicate that the functional IL-2R consists of the complexes of the!
!!,!!!and!!!chains. We also detected expression of these three receptor subunits on HTLV-I infected T cells as well as normal activated T cells. Dr. Shunro Sonoda (Kagoshima University, Kagoshima, Japan)
IMMUNOGENIC CORRELATIONS
Adult T-cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are caused by the same etiologic agent, HTLV-I but their clinical symptoms and pathogenesis are completely different.
Immunohematological features of ATL are characterized by leukemic outgrowth of CD4 T cells and decrease of CD8 T cells which leads to immunocompromised state in the immune response to HTLV-I. In contrast, HAM is characterized by a hyperimmune state in both humoral and cellular immunity to HTLV-I which is shown by an increased titer of anti-HTLV-I antibody and activated T cells in both the circulation and spinal fluid of patients.
Epidemiological surveillance revealed that most of the cases with ATL or HAM occur independently in a family or population of HTLV-I carriers, and cluster geographically in a certain ethnic group.
These clinico-epidemiological findings suggest that the etiology of ATL and HAM/TSP may be determined by an ethnically-defined host factor(s) whose immune response genes segregate a genetic group of HTLV-I carriers being susceptible to either ATL or HAM/TSP.
In order to characterize the immunogenetic host factors of ATL and HAM/TSP, we analyzed the HLA polymorphism and immune responsiveness to HTLV-I using peripheral blood lymphocytes (PBLs) and plasma collected from the patients and their relatives. When HLA haplotypes of ATL and HAM/TSP were analyzed by serological and PCR-RFLP DNA typings, two categories of HLA class II haplotypes were associated with Japanese ATL and HAM/TSP. Thus, ATL-associated HLA class II haplotypes were represented by DRB1*DQB1*0901-0303, 1501-0602, 0403-3-2 and 1302-0604. HAM/TSP-associated HLA class I haplotypes were represented by DRB1*DQB1*0101-0510, 1502-0601, 0405-0401, 0803-0601 and 1201-1301. The general population of Japanese showed an even distribution of the ATL- and HAM/TSP-associated haplotypes.
The similar genetic segregation of HLA class II haplotypes were confirmed by a pilot study on Caribbean and Colombian patients with ATL and HAM/TSP although they had another set of HLA haplotypes associating with the patients of African origin.
These results indicate that the HLA class II is one of the major genetic factors to segregate the patients with ATL and those with HAM/TSP.
In order to correlate the genetic polymorphism of HLA class II with immunopathology of ATL and HAM/TSP, we investigated the immune response of T-cells against HTLV-I using peripheral blood lymphocytes of ATL, HAM/TSP asymptomatic HTLV-I carriers (AC) and normal donors carrying HLA haplotypes associated with ATL and HAM/TSP.
HTLV-I specific immune response was measured by in vitro T-cell proliferation on the cultures of PBLs with or without purified HTLV-I virions. The spontaneous proliferation of T-cells without HTLV-I virion antigens was subtracted from total amount of T-cell response to get the specific T-cell response of HTLV-I. The HTLV-I specific T-cell response of HAM/TSP was about 10 times more than that of ATL. Asymptomatic carriers showed two patterns of T-cell response, one high and indistinguishable from HAM/TSP and the other low, resembling ATL. Immune response among normal donors was correlated with HLA class II haplotypes with those with the HAM/TSP type being high responders and those with the ATL haplotype being low responders. These results indicate that HLA class II genes of ATL and HAM/TSP are functionally segregated to be a low or high immune responders. The US-Japan collaborative study provides an opportunity to establish an international collaboration to investigate the genetic epidemiology of HTLV-I/II-associated diseases.
Dr. Steven Jacobson (National Institute of Neurological Disorders and Stroke, Bethesda, MD)
IMMUNE RESPONSE TO VIRAL INFECTION: IMPLICATION FOR DISEASE PATHOGENESIS
Cytotoxic T-Lymphocytes (CTL) have long been known to play an important role in the normal immunologically mediated recovery from infectious disease by recognition and subsequent elimination of foreign antigens. In general, two subsets of CTL have been identified. These CTL recognize foreign proteins, typically as short peptide fragments, in association with histocompatibility molecules (HLA). One population, expressing the CD8+ phenotype, recognize peptides through its antigen-specific T-cell receptor in the context of HLA class I molecules. A CD4 + population of CTL can also recognize peptide fragments but in association with HLA class II molecules. Both populations have been shown, in a number of systems, to be beneficial in eliminating infected cells and in recovery from a viral infection However, virus-specific CTL have also been shown to be immunopathologic and mediate certain diseases. If CTL destroy essential cellular structures, particularly if these CTL recognize viral determinants in the central nervous system, this can adversely affect the host. Therefore, in defining any novel virus-specific CTL response it is important to determine if these responses are beneficial or detrimental Obviously, this will dictate a strategy for potential immunotherapy either to augment the CTL response to help eliminate virus-infected cells or inhibit the CTL response to reduce the associated immunopathology.
Fresh PBL isolated on ficoll-hypaque gradients were used as effectors on a variety of 51CR-labelled targets with known HLA specificities. Targets that have been used are long term T-cell lines expressing HTLV-I proteins or EBV transformed lymphoblastoid cell lines (LCL) infected with HTLV-I vaccinia virus recombinants. These HTLV-I vaccinia recombinants have been a useful tool in expressing the different proteins of HTLV-I. Lysis occurs predominantly on targets infected with the HTLV-I p40x vaccinia constructs. This would indicate that a product of the HTLV-I Tax gene is being recognized by these CTL. Additional studies have demonstrated that these CTL responses are mediated by CD8+ cells and restricted by HLA class I molecules. These observations have been extended to a number of HAM/TSP patients. Other HTLV-I antigens, most notably the envelope region of HTLV-I can also be recognized by HTLV-I specific CTL from some individuals. However, these responses occur with a lower frequency or magnitude than the CTL responses to the Tax region of HTLV-I.
In contrast, fresh circulating PBL from HTLV-I seropositive, asymptomatic individuals had no detectable CTL or had significantly lower CTL responses than HAM/TSP patients. In all healthy HTLV-I seropositive carriers that we have tested as well as in seropositive individuals with other diseases not related to their HTLV-I infection or in two ATL patients, HTLV-I specific, CD8+, HLA class I restricted CTL from circulating PBL were not detected. This would imply that the presence of HTLV-I specific CTL in HAM/TSP patients may be a disease specific event, thereby contributing to the pathogenesis of this disorder.
Another interesting observation is the relative frequency of HTLV-I specific CTL in the peripheral blood of patients with HAM/TSP. By purifying CD8+ cells and plating these cells in limiting dilution an estimate of the precursor frequency of CTL can be obtained by enumerating the number of T-cell clones that are cytolytic. Results from such analysis demonstrate a precursor frequency for HTLV-I specific, CD8+ CTL in the peripheral blood as high as 1 in 86 cells. Since approximately 22% of PBL are CD8+, this would indicate that approximately 1 in 400 circulating PBL from this HAM/TSP patient is an HTLV-I specific CTL.
If CD8+, HTLV-I specific CTL contribute to the pathogenesis of HTLV-I associated neurologic disease then these cells would be expected to be present within affected tissues of the CNS. This is supported by earlier reports which demonstrate an infiltration of CD8+ cells within spinal cord lesions of HAM/TSP patients. In addition, the HTLV-I pl9 core protein, HTLV-I genomic sequences and HLA class I molecules were also present. Therefore, since all the requirements for CTL recognition were present in HAM/TSP lesion sites, it was of interest to speculate whether the CD8+ cells demonstrated within these lesions were HTLV-I specific CTL.
HTLV-I specific CTL were demonstrated from lymphocytes in the CSF of HAM/TSP patients. These CTL were HLA class I restricted and lysed targets expressing the p40x or env region of HTLV-I. Since HAM/TSP patients typically have intact blood/brain barriers this would indicate localized expansion of these CTL in the CNS.
Precursor frequency analysis of HTLV-I specific CTL from lymphocytes within the CNS showed a high frequency of these cells. As high as 1 in 60 CD8+ cells within the CSF was cytolytic and HTLV-I specific. Since CD8+ cells represented 48% of the total CSF lymphocytes, the precursor frequency of HTLV-I specific CTL within the total CSF was approximately 1 in 125. This frequency was comparable to that observed for HTLV-I specific CTL from the peripheral blood.
These results demonstrating CD8+, HTLV-I specific CTL in the CSF of patients with HTLV-I associated neurologic disease coupled with previous reports showing an infiltration of CD8+ cells in HAM/TSP lesions in the presence of HTLV-I genes, antigens and HLA class I molecules, strongly support a view that a component of these CD8 + cells may be HTLV-I specific CTL. If so, these lymphocytes may cause direct lysis of infected cells in the spinal cord, which may account for the pathological and clinical changes observed in this disease.
Ongoing experiments in our laboratory have begun to more precisely define the HTLV-I antigens that are recognized by these CTL. Our results indicate that small peptides (9 amino acids) of the HTLV-I tax region can be recognized by HTLV-I specific CTL in association with the HLA class allele, HLA-A2. HTLV-I Tax-specific CTL restricted by other HLA class I molecules have been shown, as expected, to recognize different peptides derived from the HTLV-I Tax protein. In this manner, mapping of predominant CTL epitopes as well as defining the T-cell receptor molecules used by these CTL for each patient is possible. This information could lead to tailored immunotherapeutic strategies which could specifically inhibit these CTL and potentially alter disease progression. This would be similar to the successful approaches that have been pioneered in the treatment and prevention of T-cell mediated experimental allergic encephalomyelitis. Moreover, an understanding of the involvement of HTLV-I specific CTL in the pathogenesis of HTLV-I associated neurologic disease could be extended to other neurologic disorders in which virus-specific CTL are thought to play a role or in diseases with suspected CTL involvement in which a viral agent has not yet been definitively identified (e. g. multiple sclerosis).
Session VI: Molecular Evolution
Dr. Raoul Benveniste (National Cancer Institute, Frederick, MD)
EVOLUTIONARY RELATIONSHIPS OF PRIMATE RETROVIRUSES: IMPLICATIONS FOR HUMAN RETROVIRUSES
The origins of the two groups of human retroviruses (HIV and HTLV) are unclear. Data were presented that show that mammalian retroviruses have in the past been transferred between vertebrate species that are only remotely related phylogenetically. Viral genes from one group of animals can give rise to infectious particles that can integrate into the DNA of another species, be incorporated into the germ line, and then be transmitted as cellular genes. There have been several documented examples of retrovirus transfer between species, including transfers from ancestors of primates to ancestors of carnivores (both in the New World and in Africa), from rodents to carnivores, from rodents to primates, from rodents to artiodactyla, and from primates to primates. After some of these transfers, the acquired retroviruses have remained infectious and still cause disease in the new host (for example, FeLV in cats, gibbon ape leukemia virus in gibbons, and SRV-type D retrovirus in macaques). Viewed against this backdrop, the presence in infected individuals of HIV-I/HIV-2 or HTLV-I/HTLV-II is but the most recently discovered example of interspecies transmission, with man this time as the unfortunate recipient.
Since HIV and HTLV-related retroviruses are found in Asian and African primate species, knowledge of the evolutionary relationships among primates and their geographic habitat will assist in the identification of possible non-human primate origins for these viruses, and help in determining the length of time that these viruses have been present in these species.
The SIV-macaque model is a useful animal model for human retroviral infection. For example, various vaccine immunization regimens and antiviral therapies have been tested. Recombinant and live-attenuated vaccines have already been developed that protect macaques against cell-associated, mucosal or intravenous SRV or SIV virus challenges. In addition, a maternal-fetal transmission model has been developed in macaques that closely approximates the frequency of transmission of HIV from mother to infant. A non-human primate model for HTLV-I, using either STLV-I infected macaques or baboons, is feasible in order to study the molecular determinants or pathogenicity, test various vaccine protocols, and develop a maternal-fetal transmission model. The apparent low rate of variability of the HTLV-I genome in vivo bodes well for the successful development of a recombinant HTLV-I-HTLV-II vaccine that would protect against all known variants of this virus. The more aggressive interventions that are possible in this kind of model will also assist in answering many questions concerning HTLV biology.
Dr. Richard Yanagihara (National Cancer Institute, Bethesda, MD)
MOLECULAR EPIDEMIOLOGY OF HTLV
The earlier conjecture, based on seroepidemiological data, that variants of HTLV-I are endemic in remote populations in the western Pacific region has been verified by the isolation of such variants among Melanesians of Papua New Guinea and Solomon Islands and among aboriginal people of central and northern Australia. Unlike cosmopolitan strains of HTLV-I from Japan, the Caribbean, the Americas and Africa which exhibit > 96.5% sequence similarity among themselves, these virus variants from Australasia and Melaneasia are markedly divergent at the nucleotide sequence level from cosmopolitan HTLV-I. Moreover, the env gene sequences of HTLV-I strains from a Solomon Islander with HAM/TSP and from Australian aboriginals with ATL are similarly divergent from cosmopolitan strains of HTLV-I, indicating that these variant viruses are capable of causing disease. These Australo-Melanesian variants of HTLV-I have multiple nucleotide substitutions not found in cosmopolitan strains of HTLV-I, supporting the concept of geographic-specific genotypes or topotypes of HTLV-I. Furthermore, when viewed within the context of sequence analysis of cosmopolitan strains of HTLV-I, as well as data from simian T lymphotropic virus type I (STLV-I) isolates from Africa and Asia, these Australo-Melanesian HTLV-I variants appear to have evolved from the common ancestor of primate T lymphotropic virus type I prior-to cosmopolitan strains of HTLV-I and African subtypes of STLV-I.
Although these data do not establish the origin of HTLV-I, they suggest that the common ancestor(s) of the Australo-Melanesian strains of HTLV-I evolved somewhere in the then Southeast Asian landmass (Sunda) or the offshore island in Wallacea. Two and possibly more of these ancestral virus variants were probably introduced by the Australoids who originally migrated from Sunda to settle the then single continent of Australia and New Guinea (called Sahul) and some of the Melanesian islands approximately 30,000 years ago.
As with the dissemination of other viruses, the spread of HTLV-I in the Pacific basin has been a dynamic process, and as in other parts of the world, HTLV-I endemicity in the Pacific is not uniform. On the one hand, studies conducted on sera collected during the 1960s to 1980s from indigenous populations in Micronesia (Mariana Islands, Caroline Islands), Polynesia (Cook Islands, French Polynesia, Marquesas, Anuta, Tikopia, American and Western Samoa) and certain regions in Melanesia (Fiji and New Caledonia) have failed to disclose evidence of HTLV-I infection. On the other hand, strains of HTLV-I isolated from inhabitants of Taiwan, the Marshall Islands, and the Polynesian Outlier Bellona are genetically more closely related to cosmopolitan strains of HTLV-I than to the sequence variants from Melanesia and Australia, suggesting a different source and subsequent evolution.
Disease-specific sequences have not been identified for HTLV-I strains isolated from patients with ATL or HAM/TSP, and virus strains from asymptomatic carriers are genetically indistinguishable from disease associated strains. Instead, the genomic variability of HTLV-I appears to depend on geographic origin, supporting the general concept of geographic-specific genotypes, or topotypes, of HTLV-I. Oligonucleotide primers derived from sequences unique to the gp46- and gp21-encoding regions of the env gene of the Australo-Melanesian variants of HTLV-I have now been successfully employed to discriminate HTLV-I strains from widely separated geographic regions into two major geographic-specific genotypes or topotypes: an Australo-Melanesian topotype and a cosmopolitan topotype.
Session VII: Perspectives on Vaccine Trials
Dr. Robert Gallo (National Cancer Institute, Bethesda, MD)
In the closing session, Dr. Robert Gallo posed a series of contextual questions for discussion of the research challenges indicated by the presentations and the potential responses for meeting these challenges. The heart of the matter, as he saw it, was whether there is a value to using HTLV-I as a model for other diseases and whether or not we should vaccinate people against it. If the direction is vaccination, then decisions must be made regarding when to do it, where to do it, to whom and why in that particular group.
He said it is important to do HTLV-I vaccine trials. Now, he said, is the time to select the place and the population. He said the vaccine will be available in 1993, that it will provide envelope protection. He suggested the place to do the trials will be those areas that have an epidemiological set-up, a case population and an existing supportive infrastructure. He also suggested this as an excellent area for US-Japan collaboration. The HTLV-I vaccine would be used as a prevention to infection rather than as a treatment.
After an extensive and productive discussion of these issues, the group agreed that the vaccine trials should be tried; a pilot project will be implemented in Japan and parallel trials will be done in other locations not yet determined.
Session VIII: Areas for specific Cooperation
Based on group discussions and Dr. Nancy Mueller's presentation, a series of issues were targeted for future research. Since many of these studies would benefit from large sample size or access to biologic specimens from patients with rare diseases, US-Japan bilateral collaborations might facilitate this process.
Disease-Oriented Studies. International studies have identified different patterns of disease and possibly different disease incidence in HTLV-inflected populations. Studies aimed at defining the incidence of specific HTLV-I associated diseases in different populations would be advantageous. For such studies standardized definitions of disease are needed and standardized protocols for data collection are required. The goals of this work would be to define the spectrum of disease in different ethnic and racial groups, to determine the incidence of disease in these populations and to discern differences in patterns of disease occurrence. As an outgrowth of these studies assessment of risk factors with attention to cases in younger and older age groups might provide useful insights. Definition of exposure source through testing of family members and immunogenetic and genetic analysis for host susceptibility factors are needed in comparative studies. Multi-disciplinary studies applying advanced laboratory techniques to patient populations in different geographic locales affords an opportunity to define common pathogenic mechanisms. Before these studies can be done it would be necessary to standardize laboratory approaches and exchange reagents, techniques, and personnel. And, since PCR is a standard tool for such research, it would be necessary to develop standardized panels of reagents for PCR. Perhaps international reference laboratories should be established to facilitate comparisons in the search for virus positive/Ab-negative persons.
Comparative treatment trials with standardized protocols are needed to evaluate new therapies especially since some subtypes of disease are extremely rare. Of particular interest are protocols to evaluate the effectiveness of combined AZT and interferon and anti IL-2Ra based approaches.
International studies to define the natural history of different HTLV-I-associated conditions are needed; the use of standardized sample and data collection approaches focused on studies of pathogenesis will enhance the value of such efforts. Similarly, the impact of HTLV-I infection on other cancers such as cervical carcinoma and lung cancer can be investigated; the impact of HTLV-I on incidence, stage and survival can be looked at as well.
Population Based Studies. Definition of the natural history of HTLV-I carriers requires large-scale studies because of the rarity of clinical outcomes. Pooling data from ongoing efforts is one approach to use in undertaking such studies; establishing collaborative cohorts of special populations would provide an extensive and powerful pool of information. One cohort could include persons of known source and age of infection for assessment of short-term morbidity. Again, there should be standardized protocols and inclusion of mutually-comparable assays of immune function and viral status. Another follow-up cohort could be made up of individuals at "high-risk for ATL" as defined by high viral load, abnormal lymphocytes and other consensus criteria based on standardized biomarker assays. This could potentially become an intervention cohort and may provide us with a consensual definition for "smoldering ATL". An additional cohort comprised of discordantly-infected couples could be used for clarification of risk factors for transmission. A collaborative re-examination of all the perinatal transmission studies including standardized assays of maternal sera for titer, anti-Tax, viral DNA, neutralizing antibody, etc. would create a database for reaching a consensus on population interventions.
This pooling effect would generate significant data with the potential to answer a number of significant questions: What is the spectrum of morbidity associated with HTLV-I infection? Does HTLV-I contribute to increased infant mortality? Are there identifiable risks among carriers and do these risks vary between populations? Do genetic factors, nutritional status, co-existing infections impact on this spectrum? Do risks vary by mode of and age at infection? Is the incidence of HTLV infection declining? Prospective cohort studies of large size in multiple locales could provide the information needed to answer these questions and many others.
Mathematical modeling provides another approach and a useful tool for investigating these questions; it would require the identification of model parameters and studies designed to measure them. The information provided by these studies would establish the bases for determining whether intervention trials are needed in some populations.
And, because of our limited knowledge of HTLV-II, there is a need to develop cohorts of HTLV-II-infected persons for collaborative studies of the spectrum of disease and transmission analyses
Intervention Research Experimental studies with animals provide ample evidence that an effective vaccine could be developed and administered but should the focus be on that approach; is that the place to invest limited resources? Is the incidence of new infections high enough to test efficacy? Is the morbidity and mortality associated with adult acquired infection high enough to warrant the expenditure of resources for such a vaccine? Where would the vaccine be given, and who would cover the costs? Do we have sufficient data to answer these questions? Should there be policy discussions between the US and Japan and other agencies such as WHO to address these questions? Should we consider a randomized trial of alternative interventions for mother-to-infant transmission such as targeting "high-risk" mothers vs all seropositives, and no breast-feeding vs short-term breast-feeding? What about sterilization of pumped breast milk, or immunizing mothers or infants with anti HTLV-I hyperimmune globulin? What of the characterization of non-breast-milk transmissions in terms of antibody pattern, etc to identify intervention strategies? Is the incidence of HTLV-I infection declining so that no intervention is required as suggested by some, or is just the opposite the case? All aspects of the debate need to be analyzed within the collaborative context.
Laboratory Studies The laboratory research agenda is consensually informed: What is the role of the CD-8 Class I restricted CTL in HTLV-I associated autoimmune, immune deficiency, and lymphoproliferation states? Can reagents be shared to enhance the spectrum of HLA types that can be mapped in different locales? Does HTLV-I infection cause cytokine imbalance leading to oncogenesis and immunosuppression? How can this best be studied in human populations? Based on current data there is no evidence that sequence variation of HTLV-I contributes to the pathogenesis of ATL and TSP/HAM. Is this question settled? Can data from multiple laboratories in the US and Japan be shared to fully address this issue? Is HTLV a model of human retrovirus oncogenesis? How can further molecular analysis of Tax and Rex function enhance the understanding of these regulatory proteins in cellular transformation? What is the function of new viral regulatory proteins pl2, pl3 and p30? What is the role of negative strand RNA expression in regulatory function?
The answers to these questions and others will be found in the context of bilateral agreements which foster multi-disciplinary studies linking biomarkers (e. g. cytokines, PCR, abnormal lymphocytes, anti-Tax, p53 assays) done in parallel among HTLV-I-endemic populations. The research designs should include comparative studies of assays (e. g. lymphocyte proliferation assays) to allow comparability of data and disease outcomes within cohorts and case-control studies based on specific HTLV-I-associated diseases.
(2) Seminar on "Biostatistics in Cancer Research"
This seminar, the fourth in a series dating back to 1978, was held at the Miyako Hotel in Tokyo, November 9-11, 1992. The organizers were Dr. David G. Hoel then of the National Institute of Environmental Health Sciences, and Dr. Takashi Yanagawa of Kyushu University.
A full meeting report has been prepared by Dr. Yanagawa in Japanese and the proceedings of the workshop may be published. Because Dr. Hoel moved from Research Triangle Park in February, he was able to provide only the following preliminary description of the workshop, dated April 13, 1993. "The meeting was outstanding and the best yet in the series. It had been decided to hold the meeting in Tokyo to improve the access by the many interested researchers in Japan and this was reflected by the audience of over 100 participants. The theme or emphasis of the meeting was to consider questions of the sources of error in the biostatistical analysis of epidemiological cancer data. There were several papers directed specifically to this topic.
"Besides the effects of errors in analysis, there were a number of interesting talks ranging from current techniques such as meta analysis in the study of multiple cancer epidemiology studies, so new techniques in the design of environmental health studies involving multiple exposures and multiple communities in the field of air pollution. There were several interesting papers on the modeling of lung cancer and cigarette smoking in Japan. What the speakers suggested in their presentations, is that the risk of lung cancer among smokers, which is generally considered lower among the Japanese, was shown instead to be similar to what was found by Doll and Peto in their studies of British physicians. The underestimation of effects of cigarette smoking in the Japanese evidently has much to do with the short length of smoking history. Using this information, a paper was presented concerning the projection of future lung cancer mortalities depending on various public health measures with regard to the control of cigarette smoking. This is an important step.... in cancer prevention for the Japanese in terms of trying to understand the health consequences of various control strategies.
"The meeting participants were given at registration time, an extended abstract book of about 140 pages containing materials provided by the speakers. This was quite useful for the participants [in] encouraging general participation. What was most impressive...was the length and quality of the discussion after each presentation. Both the Americans and Japanese asked very good and penetrating questions which helped make this an exciting and stimulating conference [as commented upon by several participants].
"All of the presentations are in hand and it is anticipated that they will be published in an Environmental Health Perspective monograph so the material will be available to the general public.
"In conclusion,...this series of conferences on biostatistics in cancer research has been very important to the development of the field in Japan and in fact goes beyond simply cancer epidemiology by influencing the general development of the field of biostatistics in human health research."
Dr. Hoel also provided the following partial summary of the meeting.
Epidemiologic Methodology
Dr. Greenland discussed the methods for the analysis of studies concerning multiple exposures. The usual approach involves the fitting of risk-regression models. Dr. Greenland compared these methods with two newer approaches of empirical-Bayes and "semi-Bayes" regression. These newer hierarchical methods are shown to be important in the analysis of multiple-exposure studies.
For traditional case-control studies, Dr. Langholz discussed matching in nested studies. Usually controls are matched to cases with respect to confounding variables. Dr. Langholz considered instead designs that select controls as dissimilar as possible to the cases. The design was considered for the situation where a surrogate measure of exposure is available for the entire cohort, and accurate data are available only for a nested case-control study. Dr. Sato reviewed recent developments in risk-ratio estimation procedures in case-control studies. In the crude analysis, maximum likelihood estimation procedures of crude risk-ratios was discussed.
Statistical Methods
Dr. Yanagawa reviewed a generalization of the Mantel-Haenszel procedure for 2xJ tables. He described tests and estimators for a common odds ratio and the generalized Breslow-Day test for homogeneity across strata. Dr. Nakamura reported on proportional hazard models where exact values of covariates are not observed but instead only surrogates with measurement errors are available. Maximum likelihood estimates based on partial likelihood with surrogate measures were shown using Monte-Carlo simulations.
Air Pollution and Cancer
In the air pollution and lung cancer area, Dr. Tango presented a Poisson regression model for time trends of mortality to the effects of air pollution levels in the Tokyo area. The analysis supported the existence of long-term effects of air pollution on lung cancer. Dr. Thomas described a hybrid epidemiological design which combines both analytic and ecologic approaches evaluating differences between individuals and groups. This was applied to the health effects of air pollution in 12 communities in Southern California and 3500 school children who will be followed for 10 years. The data will be used to develop a model for personal exposures in individual-level analyses and group-level analyses.
Meta-Analysis
Dr. Morris presented a review of the use of meta-analysis in cancer epidemiology, and described the use of the various methods in published studies. He discussed methods for assessing publication bias and techniques for combining dose-response data.
Diagnostic Testing
A general method of using regression to estimate smoothed ROC (receiver operating characteristic) curve for diagnostic testing was presented by Dr. Anna Tosteson. The methods were illustrated with multi-institutional data on magnetic resonance imaging with computed tomography in detecting liver metastases.
Validation Studies
A two-stage procedure for estimating sensitivity and specificity was described by Dr. Tor Tosteson. The study of risk factors for malignant melanoma was used to illustrate the method of two-stage validation. Using missing data methods for contingency tables, maximum likelihood estimators for the joint distribution of the test measure and the clinical evaluation were presented along with the estimators for sensitivity and specificity.
Questionnaires
Dr. Tokunaga examined the reproducibility of questionnaires after a one-year interval. The focus of the questionnaires was concerned with diet and "life-style." The conclusions were that these types of questionnaires are sufficiently reproducible to be useful in epidemiology studies.
Cigarette Smoking and Lung Cancer
Two important papers on lung cancer and cigarette smoking among the Japanese were presented. Dr. S. Akiba noted that while the proportion of female smokers has remained constant at 10- 15%, the proportion of male smokers has dropped from 80% in the 1960s to 60% currently. The important finding of Dr. Akiba's analysis was that the lung cancer mortality among male smokers smoking 20 cigarettes per day was similar to that observed by Doll and Peto in the British physicians ' study. Dr. Yamaguchi used a computerized simulation model to evaluate the potential impact of primary and secondary cancer prevention. He used smoking and lung cancer as his model and showed major lung rate changes through smoking cessation estimates.
SEMINAR AGENDA AND PARTICIPANTS
(1) Retroviruses and Cancer: US-Japan Clinical-Epidemiological Experiences
At The Cloisters on the NIH Campus
January 6-8, 1993
AGENDA