THIS IS AS YET JUST A THEORY.

By Jean Viviers M.B.,ch.b.
31 August 1996


General practitioner. PO Box 244, Sundowner, 2161, South Africa. (Correspondence should be addressed to: The South African Myeloma Foundation, PO Box 244, Sundowner, 2161, South Africa.
Tel: (+27) 1182 4599546,
Fax: (+27) 1182 131 4599546,
e-mail: viviers@icon.co.za


Abstract --- I propose, as an initiating cause for MM, MGUS and most likely PCL a retroviral infection. The different disease entities caused by retroviruses have a diverse clinical and pathophysiological expression, but a lot of similarities. A few of these I would like to refer to with reference to Multiple Myeloma.

The aetiology of multiple myeloma (MM) has eluded scientists for many years. Generally MM is believed to be caused by a multi step process. For an idiotypic immunoglobulin to be produced, it is believed that the malignant transforming event must occur in a late B cell precursor, some time after the immunoglobulin gene has been rearranged, and the idiotype determined (1-3) This would have to happen in the lymphoid tissue, before migration to the bone marrow. Genetic factors probably play an important role in determining whether the early B cells of an individual are susceptible to any of the malignant transforming events the person is exposed to.
I propose a retroviral etiology for the following reasons:

1. The incubation period of monoclonal gammopathy of determined significance (MGUS) development into MM is decades, and only 30% of these patients ever progress to MM (4).
HIV infection has a "incubation period" of about 10 years. TSP caused by HTLV-1 has a average age of onset of 42 years. The incubation period is unknown. There is an age dependent increase of sero prevalence to HTLV-1 and MM. Not all patients with HTLV-1 develop TSP or ATL.

2. MM has an altered CD4+ and CD8+ subpopulation. (5-8). CD4+ : CD8+ ratio is mainly related to the decrease, both in percentage and absolute number, of the CD4+ subset (8). CD8+ lymphocytes are monoclonally spontaneously activated (23) CD4+ are monoclonally activated.
HIV is well known to effect accessory immunoregulatory cells in the same way.
The majority of HTLV-1 induced leukemias and lymphomas involve CD4+. CD8+ cytotoxic T lymphocytes directed against HTLV-1 env, tax and rex proteins are activated and proliferate spontaneously (Harrisons edition 13). ATL is cause by this retrovirus. HTLV-1 has an affinity for CD4+ T helper cells causing reduction in functioning and syncytia formation. CD4+ T helper cells in ATL have at least 1 copy of the HTLV-1 provirus. The provirus seems to be clonal which suggests that the infection occurs before the first transformation. (9,10) HTLV-1 infected cells grow as multi nucleated giant cells in the absence or presence of IL-2. Growth can be stimulated indefinitely. Similarly HTLV-1 can transform normal T-cell, in vitro, to become immortal.( 11-13)

3. MM is thought to be prominently a B-cell malignancy. B cells are infected with HTLV-1 (14,15) In areas of the world where HTLV-1 is endemic, some B cell lymphoid malignancies and certain other cancers are associated with HTLV-1 infection more frequently than would be expected form the prevalence of the virus in the general population. In contrast to virus positive T cell leukemia, where the viral genes are integrated into the DNA of the leukemic cell, these B cell tumors have no HTLV-1 in their DNA. Instead, the virus is present in the normal T cells of these patients, and the role of the virus must be indirect, as in HIV-1 and B cell lymphomas and Karposi's sarcoma. Several of these B cell neoplasms have been shown to make specific antibody to HTLV-1; thus years of stimulation of some B cell populations by HTLV-1 proteins may be involved in their malignant transformation.

HIV patients show abnormal activation reflected by increased spontaneous proliferation and immunoglobulin secretion, and by increased spontaneous secretion of tumor necrosis factor alpha (TNF- ) and interleukin-6. ( Harrisons edition 13)

4. Transforming growth factor-B1 (TGF-B1) is produced by bone marrow stromal cells (BMSCs), after interaction with MM cells, and can regulate interleukin-6 secretion, (IL-6), ( A growth factor for MM) by BMSCs and MM cells. Moreover, MM cell growth may be enhanced by resistance of tumor cells to the inhibitory effects of TGF- B1 on normal B-cell proliferation and Ig secretion. Clinical immunodeficiency has also been attributed to increases in TGF- B1 in vivo (24) In patients infected with HIV and HTLV-1, high levels of TGF- B1 released by PBMCs lead to defects in both cellular and humoral immunity (16,17).

5. Decreased apoptosis is induced by Bcl-2. The Tax protein of HTLV-1 induces apoptosis, but the cells are immortalized by the protective effect of Bcl-2 (18). ADF (adult T-cell leukemia derived factor - a thioredoxin homologue) described by Matsuda et al. protects Fas expressing cells form anti-Fas mediated apoptosis.
This would explain how the initial infection in MM is not cleared by apoptosis.
Discussion

Although extensive work has been done to identify the etiology of MM it took 140 years to find the first virus which is important in the etiology of MM. It is the Karposi Sarcoma Herpes virus 8 which infects the dendritic cells in 100% of MM patients. By looking at the pathophysiology and clinical course of MM, it is likely that this disease is the direct cause of a retroviral infection, affecting mainly the T cells. It is activated by the KSHV 8. I postulate that the retro virus is an integral part of the human germ line and spread horisontally. The primary infection is CD4+ lymphocytes, monocytes and B cells.The infection is monoclonal and recognised as "own" by the immune system.

At this stage it is important to note that MM was first described in 1844 in England. It is probable that this is one of the older retro viruses infecting humans, and this may explain why MGUS has an incidence of 7-8% over the age of 55 years (22) and MM has an incidence of 30 per 100 000 over the age of 25 years. This increases with age and is remarkably similar throughout the world. The virus has established a degree of symbiosis. The increase with age is most likely because of progressive immunodeficiency developed in both T and NK cell subpopulations because of TGF- B1 inhibition of immune function and IL-6 stimulation of B cell replication. This may be stimulated by a retroviral gene similar to tat or tax in HIV and HTLV-1 respectively. We know now thanks to Dr Mathew Rettig et al. that KSHV infects the dendritic cells of all Multiple Myeloma patients tested so far. I is most likely very important in activating the endogenous retro virus, which would be dormant until activated by radiation, hormones or a DNA virus. I propose that KSHV activates the HERV. Secondly because of the immunodeficiency NK cell and T cell tumor control will be impaired, and escape from this mechanism is likely. The B cell 85-90% of MM patients show c-myc mRNA and protein comparable to those of actively proliferating cells, suggesting a possible role for c-myc in this neoplasm (19). Avian leukosis virus promote expression of cellular proto-oncogene myc, believed to be the first step in induction of B cell leukemia in chickens.
The amount of plasma cells replicating in MM are low 1-2%. This will make the detection of a retrovirus more difficult. The infection is monoclonal (as in other retro virusses) and the provirus is likely to rely on cell replication for multiplication. This is possible because the retrovirus immortalizes the cell. This retrovirus is not cytopathic as seen in HIV, but replicates by direct cell to cell spread as mediated by the various adhesion molecules, especially CD56. The same phenomena is seen in HTLV-1 infection. Somatic mutations without intraclonal variations have been detected in Ig genes of tumor plasma cells. (1-3). This finding indicates that the major oncogenic events yielding to a continuous proliferation of the tumor stem cell occur in a cell selected through antigen contact in lymphoid follicles.
A plasmacytoma of donor origin has been reported in a transplanted kidney, 14 years later. Non-Hodgkins lymphoma is also prevalent in this group of patients. The plasmacytomas described in this group illustrates the possibility of neoplasia arising from "passenger leucocytes" transferred with the graft but presenting at an unprecedented interval of 14 years (20). The potential for self-renewal and persistence of donor lymphocytes was demonstrated in the peripheral blood, lymph nodes and skin or renal transplant recipients > 25 years after transplantation (21).
Recent studies have shown that retroviruses containing myc and raf oncogenes induce plasmacytomas in pristane-injected wild type mice (18).

The cure:

The provirus incorporates into the host DNA for life. Thus the only way to get rid of an infection in a cell, is to get rid of the cell. CD4+ and dendritic cells are the infected cells, and this pool forms an important source of relaps. The human immune system has been working on this for a few million years. It will be egoistic to think we could improve on what has already been achieved by mother nature. For this reason it will be the easiest and the best to assist the immune system to get rid of these viruses. This can be done in a number of ways, but our best two possibilities I believe are anti-retro viral therapy and immunotherapy.

Anti retro viral therapy would consist of a Nuke like AZTof 3TC combined with a protease inhibitor. By this method we should be able to switch of viral replication and the immune system should have time to recover from the inhibitory effects of viral proteins. Hopefully the immune system will then have the ability to eliminate the infected clone and the infected clone may have the ability to undergo apoptosis. (More about this under anti-retro viral therapy!)

Immunotherapy is used to induce cell apoptosis. Allogenic "educated" leukocytes are primed to home in on cells displaying specific receptors. They recognize these cells as foreign and destroy them. CD95+ receptor which is expressed in late stage MM causes spontaneous apoptosis in MM cells after activation.
The reason the immune system does not recognize a neoplastic MM cell has eluded scientists for years. Looking at reports of graft-versus-leukemia, and graft-versus-myeloma effects (25), I postulated that it is reasonable to assume that the immune system has the ability to eradicate MM completely. An in vitro observation confirmed by Bianchi (26). The mechanisms employed by allo-immune cells are exactly the same as those employed by the MM patients immune system. Thus the immune cells are regulated in vivo by cell to cell messengers and a very intricate cytokine system. CD4+Th1 cells have been shown to be the important CD4+ anti-tumor effectors in vivo and specifically in B-cell malignancies, because they can specifically interact with HMC class II molecules highly expressed in B-cell malignancies. They can be suppressed by IL-6 produced by CD4+Th2 cells (27). Researchers from Stanford University working on HIV are expanding immune cells in vitro. The important step in the whole process is sequential activation of the T cell. (It won't help giving a soldier a gun before he knows how to shoot.) Thus we need naive T cells stimulated by a CD3/TCR followed by antigen stimulation, co-stimulated by CD28 and B7, then followed by cytokine addition. This I propose to be done by exposing MM PBMC to anti-fas antibody inducing apoptosis in the activated lines. The naive CD45RA+ cells will then be able become activated in the normal in vivo manner and recognize the mortally wounded MM plasmacells.
The second step would be to remove as many of the wrongful cytokine signals as possible in the MM patient. IL-6 is the cytokine with the most effect on MM. Treating the patient for at least a 6 day period with a super antagonist (28) will reduce the immunosuppression effect on CD4+Th1 lymphocytes and they will in turn be able to activate the more important CD8+ lymphocytes involved in the sustained anti-tumor response. As shown in a murine model by Ellenhorn et al (1990). Using a TGF- B1 monoclonal antibody to restore a normal cytokine milieu would do some good.

Lastly we need to make the plasma cells as visible as possible to the naive immune cells. By adenoviral vector transfer B7 can be expressed in B7- cells. ( Dr Rettig et al. work showed that dendritic cells are infected with the virus. This would partly explain why the immune system is not able to recognize MM myeloma cells.)(Dendritic cells are antigen presenting cells - APC) This can be done in vivo or in vitro. IFNg enhances the expression of HMC molecules and assists the immune system in this way.
These immune cells will now have the ability to eradicate the malignant clone and a normal immuno-surveillance should prevail.

Chemotherapy gives temporary remission in a most patients, most likely because of reduction of tumor mass. This method of treatment will not be successful in treating retroviruses because the infection is not eliminated.

Money for research is not directed in the MM direction because it is a disease affecting older people, and it has been thought largely incurable.
We are in the fortunate position that a lot of money and effort is being directed at our collegues, working on a cure for a more marketable retrovirus, being HIV. This virus is most likely more complex, and is definitely more variable than the virus causing MM. Most likely they have already come close to our answer or have found it!

REFERENCES

1. Billadeau D, Ahmann G, Greipp P, Van Ness B. The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell. J Exp Med 1993;78:1023@ 2. Ralph QM, Brisco MJ, Joshua DE, Brown R, Gibson J, Morley AA.
Advancement of multiple myeloma from diagnosis through plateau phase to progression does not involve a new B-cell clone: Evidence from the Ig heavy chain gene. Blood 1993; 82:202@
3. Bakkus M, Heirman C, Van Riet I, Van Camp B, Thielemans K. Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation. Blood 1992;80:2326@
4. Kyle RA @ .Monoclonal gammopathy of undetermined significance. Natural history of 241cases. Americal Journal of Medicine 1978; 64: 814-818.
5. Mellstedt H, Holm G, Peterson D et al @. T cells in monoclonal gammopathies. Scandinavian Journal of Haematology 1982; 29: 57-64.
6. Mills KH & Cawley JC. Abnormal monoclonal antibody-defined helper/suppressor T-cell subpopulations in multiple myeloma: relationship to treatment and clinical stage. British Journal of Haematology 1983; 53: 271-275.
7. Oken MM & Kay NE. T-cell subpopulations in multiple myeloma: correlation with clinical disease status. British Journal of Haematology 1981; 80: 305-309.@
8. San Miguel JF, Gonzalez M, Gascon A @. Lymphoid subsets and prognostic factors in multiple myeloma. British Journal of Haematology 1992; 80: 305-309.@
9. Sarin PS, Gallo RC. The involvement of human T-lymphotropic retrovirusses in T-cell leukemia and immune deficiency. Cancer Rev 1986;1:1-17.
10.Wong-Staal F, Gallo RC. Human lymphotropic retroviruss (HTLV). Nature 1985;317: 395-403.
11. Popovic M, Lange-Wantzin G, Sarin PS, et al@. Transformation of human umbilical cord blood T cells by human T cell leukemia /lymphoma virus. Proc Naatl Acad Sci USA 1983;80: 5402-5406.
12. Chen ISY, Quan SG, Golde DW. Human T cell leukemia virus Type II transforms normal human lymphocytes. Proc Natl Acad Sci USA 1983; 80: 7006-7009.
13. Miyoshi I, Kubonishi I, Yoshimoto S, et al@. Type C virus particles in a cord blood T cell line derived by cultivating normal human cord lymphocytes and human leukemic T cells. Nature 1981; 294: 770-771.
14. Gamamoto N, Matsumoto T, Koyanagi T, et al@. Unique cell line harboring both Epstein Barr virus and Adult T cell leukemia virus established from leukemia patients. Nature 1982; 249: 367-369.
15. Longo D, Gelmann EP, Cossman J, et al@. Isolation of HTLV-1 transformed B lymphocytes cloned form a patient with HTLV-1 associated adult T cell leukemia. Nature 1984; 300: 505-506.
16. Kekow J, Wachsman W, McCutchan A, Gross WL, Zachariah M, Carson DA, Lotz M. Transforming growth factor- and suppression of humoral immune responses in HIV infection. J Clin Immunol 1991; 87: 1010@
17. Niitsu Y, Urushizaki Y, Koshida Y, Terui K, Maraha K, Kohgo Y, Urushizaki I. Expression of the TGF- gene in adult T cell leukemia. Blood 1988;71:2633@
18. Yamada T, Yamaoka S, Goto T, Nakai M, Tsujimoto Y, Hatanaka M. The human T-cell leukemia virus type I Tax protein induces apoptosis which is blocked by the Bcl-2 protein. J Virol 1994; 68:3374@
19. Greil R, Fasching B, Loidl P, Huber H. Expression of the c-myc proto-oncogene in multiple myeloma and chronic lymphocytic leukemia: an in situ analysis. Blood 1991; 78:180-191.
20. Shustik C, Jamison B.M., Alfieri C, Scherer S, Loertscher R. A solitary plasmacytoma of donor origin arising 14 years after kidney allotransplantation. British Med Journ 1995;91:167@
21. Starzl T.E., Demetris A.J., Trucco M, Zeevi A, Ramos H, Terasaki P, Rudert W.A., Kocova M, Ricordi C, Ildstad S, Murase N. Chimerism and donor-spesific nonreactivity 27-29 years after kidney allotransplantation. Transplantation 1993; 55: 1272 @
22. Agussi F, Bergami MR, Gasparro C et al@ Occurence of monoclonal components in general practice: clinical implications. European Journal of Haematology 1992;48: 192-195
23. Moss P, Gillespie G, Frodsham P, Bell J, Reyburn H. Clonal populations of CD4 and CD8 cells in patients with multiple myeloma and paraproteinemia. Blood 1996; 87: 3297-3306.
24. Urashima M, Ogata A, Chauhan D, Hatziyanni M, Vidriales MB, Dedera DA, Schlossman RL, Anderson KC. Transforming growth factor- 1: Differential effects on multiple myleoma versus normal B cells. Blood 1996;87:1928-1938
25. Verdonck Leo F, Lokhorst Henk M, Dekker Adriaan W, Nieuwenhuis Karel H, Peterson Eefke J. Graft-versus-myeloma effect in two cases. Lancet 1996; 347: 800-01
26. Bianchi A, Montacchini L, Barral P, Borrione P, Attisano C, Orsini E, Boccarodo M, Pileri A, Massaia M. CD3-induced T-cell activation in the bone marrow of myeloma patients: major role of CD4+ cells. British Journal of Haematology 1995; 90: 625-632.
27. Romagnani, S. Lymphokine production by human T cells in disease states. Annual Reviews of Immunology 1994; 12: 227-257.
28. Sporeno E, Rocco S, Ciapponi L, Paonessa G, Cabibbo A, Lahm A, Pulkki K, Sun R, Toniatti C, Klein B, Ciliberto G. Human Interleukin-6 Super-antagonists with high potency and wide spectrum on Multiple Myeloma Cells. Blood 1996; 87: 4510-4519.

If you have anything to add please e-mail me at: viviers@icon.co.za