1 Petrov Research Institute of Oncology, Leningrad, USSR.
2 The 1 st Leningrad Medical Institute, Leningrad, USSR.

Introduction

Multiple myeloma (MM) is a malignant clonal B-lymphoproliferative disease. Survival ranges from a few months to many years [7]. The determination of prognosis for the disease S course IS Important in therapeutic choice. At least two factors determine the course of MM: 1) the biology of myeloma cells and 2) the interrelation of malignant cells and host organism. That is why the morphology of myeloma cells is one of the most important prognostic factors of the disease [4]. There are also other prognostic factors associated with both malignant cell characteristics and the status of the host's immunocompetent system [7]. Taking into account the data on the decrease in colony-forming units -granulocyte/macrophage (CFU-GM) in MM [2, 11], and the antitumor effect of tumor necrosis factor alpha (TNFalfa) [8, 9] and interleukin-2 (IL-2) [10], we investigated myeloma cell morphology, the level of serum ß2-microglobulin (Sß2-M) and CFU-GM, TNF production by peripheral blood monocytes, and IL-2 production by peripheral blood mononuclear cells in different types ofMM course in order to determine their prognostic significance.

Materials and Methods

We investigated 101 patients with MM. Diagnosis was made according to previously described criteria [5].

Clinical Classification.

It is impossible to predict individual prognosis in MM according to the stage of the disease [6] at the first presentation. That is why we classified our patients according to their life duration, which determined the type of the disease. This classification is a result of prospective investigation. We divided the patients into three groups: first indolent myeloma, second -active myeloma, third -aggressive myeloma. The diagnosis was made on the following criteria:
1) Indolent myeloma. Patients who presented with Kyle's criteria [7], patients who had been in remission for more than 5 years without chemotherapy, and patients with monoclonal gammopathy of undetermined significance who had been under investigation from 2 to 5 years (Fig. 1).
2) Active myeloma. Patients in whom first line chemotherapy was effective m achieving remission or the plateau stage. Median survival was 96 months.
3) Aggressive myeloma. Patients who had primary or secondary resistance to chemotherapy. Median Survival was 12 months.

Fig.l. Survival of patients with various courses of multiple myeloma. The solid line shows the indolent course; the dash-dot line, the active course, the line of dashes, the aggressive course

Morphological Classification.

The degree of myeloma cell maturation was determined according to the following criteria of the morphological classification system for MM [4]: 1) mature -comprising more than 50 % mature plasma cells;
2) immature -comprising more than 50% proplasmacytes and mixed cell myeloma where none of the morphologically distinct plasma cell types exceed 50% (the proportion of lymphoplasmacytoid cells was always less than 50% of the bone marrow plasma cells);
3) plasmablastic -comprising more than 50% plasmablasts.

Serum ß2-Microglobulin.
Sß2-M was measured by 1251 radioimmunoassay.

Colony-Forming Unit-GranulocyteIMacrophage.
The cloning of hemopoietic cells was performed in a agar drop-liquid medium system [1].

Biological Activity of IL-2.
The biological activity of IL-2 in conditioned media of peripheral blood mononuclear cells was assayed according to the method reported by Bockman and Ropo [3].

TNF alfa Activity.
The spontaneous and pyrogen-stimulated (50 µl/ml) production of TNF alfa by peripheral blood mono cytes was assayed by exploring the immunoenzyme kits (performed by the Institute of High Purified Preparations (HPP) Leningrad, USSR), based on monoclonal and polyclonal antibodies to TNF alfa .In order to form the calibration curve in each case TNF alfa was used (made by NPO "Ferment", Tallin, USSR).

Results

It was revealed that mature plasma cells were the morphological substrate in indolent myeloma (Fig. 2) at the same time in active myeloma, mature cells predominated in 77% of the patients, whereby in the remaining 23% of the patients, the cells were immature. In aggressive myeloma, immature cells were the main morphological type in 56.3% of the patients, plasmablasts prevailed in 37.5 % of the patients, and mature cells were in 6.2% of the patients. We discovered that the level of Sß2-M correlated with the course ofMM in most of the patients (Fig. 3). There was a significant difference between the level of Sß2-M in patients with indolent myeloma (3.3 +- 0.4 mg/l) and in healthy controls (1.7 +- 0.4 mg/l; p < 0.05). We revealed a significant difference between the level of

Fig. 2. Morphology and course of MM. The white area shows the percentage of mature cells; the hatched area, the immature cells; and the black area, the plasmoblastic cells

Fig. 3. Serum ß2-microglobulin and types of courses in patients with multiple myeloma

Sß2-M in patients with indolent and active myeloma (4.9 I 0.4 mg/l; p < 0.05). There was also significant difference between the level of Sß2-M in patients with active and aggressive myeloma (8.6 I 1.2 mg/l; p < 0.05) and a significant difference between patients with aggressive and indolent courses (p < 0.01 ; Fig.4). Studies on the clonogenic ability of CFU-GM in patients with an indolent course gave unusual results. In most patients with an indolent course, the number of CFU -G M was higher than in healthy controls (Fig. 5). The number of colony-forming cells in agar culture ranged from 30 to 300 per 105 bone marrow cells, whereas in normal bone marrow cells it ranged from 40 to 60 per 105 bone marrow cells. High levels of CFU-GM were also found in some patients with active myeloma as compared with healthy controls. The number of colony-forming cells in agar culture in active myeloma ranged from 42 to 420 per 105 bone marrow cells. In patients with aggressive myeloma the number of colony-forming cells in agar culture ranged from 0 to 25 per 105 bone marrow cells, which was much less than in patients with indolent and active myeloma and less than in normal controls. There was an insignificant difference in IL-2 production between healthy con

Fig.4. Serum ß2-microglobulin and the course of multiple myeloma

trols (13.9 +-1.2 units of activity; p < 0.05) and patients with indolent (12.9+- 1.1 units of activity) (Figs.6, 7) and active courses (11.9+- 0.7 units of activity) and between the indolent and active courses. Then significant differences were found in the IL-2 production between patients with active and aggressive myeloma (8.6+- 0.6 units of activity; p < 0.01) and also between aggressive and indolent myeloma and healthy controls (p < 0.01). An insignificant difference between the pyrogen-stimulated production of TNF alfa in patients with an indolent course (1.5+- 0.3 ng/ml) and healthy controls (2.2 +- 0.38 ng/ml) was detected (p > 0.05; Fig. 8). A significant difference was found between the pyrogen-stimulated production of TNF alfa in patients with indolent and active courses (6.1 +- 1.1 ng/ml; p < 0.01 ). There was also a significant different in TNF alfa production between patients with active myeloma and healthy controls (p < 0.05), and also in TNFalfa production in patients with active and aggressive myeloma (2.0 +- 1.2 ng/ml; p < 0.05). There was an insignificant difference between TNF alfa production in patients with aggressive and indolent courses and healthy controls (p > 0.05; Fig. 9).

Fig. 5. Colony-forming unit granulocyte and macrophage of bone marrow and the multiple myeloma course

Fig.6. Production of interleukin-2 by peripheral blood mononuclear cells in patients with different types of courses in multiple myeloma

Fig.7. Interleukin-2 and the course of multiple myeloma

Conclusion

Our data suggest that: 1) Morphology of myeloma cells is the most important prognostic factor which, without a doubt, can be easily reproduced, and is widely used in clinical practice. 2) The level of Sß2-M may be used to determine the MM course type. 3) CFU-GM probably may also be used as a prognostic factor. An increased or normal level of CFU-GM confirms the indolent or active course of MM, while a decreased level of CFU-GM reflects the aggression of MM. 4) Decreased production of IL-2 by peripheral blood monocytes is characteristic of an aggressive MM course. 5) TNF alfa has a prognostic significance if it is used together with myeloma cell morphology as a) mature myeloma cells and normal production of TNF alfa by peripheral blood monocytes are typical of an indolent course; b) mature or immature morphology of myeloma cells and increased production of TNF alfa are typical of an active course; and c) immature or plasmablastic morphology and normal production of TNF alfa are typical of an aggressive course. The studies mentioned above were necessary in order to find out the role of these factors in MM pathogenesis.

Fig.8. Pyrogenal-stimulated tumor necrosis factor-alpha production by peripheral blood monocytes in patients with multiple myeloma

Fig. 9. Tumor necrosis factor-alpha and the course of multiple myeloma

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