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Arch. Dermatol. Res. 264, 319-326 (1979)

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9 Springer-Verlag 1979

Monocytopoiesis in Malignant Melanoma: Untreated, During Immunotherapy and Chemoimmunotherapy* Eva Schmitt, Gerhard Meuret, Franz Waldermann, and Manfred Hagedorn University Clinics of Internal Medicine and Dermatology, University of Freiburg, Hauptstral3e 7, D-7800 Freiburg i.Br., Federal Republic of Germany

Summary.Monocytopoiesis and blood monocytes were investigated in patients with superficial spreading melanoma stages I and II. Monocyte production was moderately increased in 4 of 9untreated patients. Postoperative prophylactic BCG-vaccination gave rise to increased proliferation activity in 3 of 4patients with previously normal monocytopoiesis. However, monocyte production returned to normal between the 4th and 6th month of BCG immunotherapy. Monocytopoietic hyperproliferation did not occur if DTIC was administered simultaneously with BCG. These results indicate that BCGvaccination increases monocytopoiesis during the first months of treatment only. This effect is abrogated by concomitant chemotherapy. Key words: Malignant melanoma - BCG vaccination immunotherapy - Monocytopoiesis - Blood monocytes

-

Chemo-

Zusammenfassung. Bei Patienten mit malignem Melanom yon oberfl~chlich spreitendem Typ im klinischen Stadium I oder II wurden die Blutmonocyten und die Proliferationsaktivit/it der Monocytopoese im Knochenmark bestimmt. Die Monocytopoese war in 4 yon 9 unbehandelten Patienten stimuliert. Bei der postoperativ durchgeftihrten prophylaktischen BCG-Behandlung konnte bei 3 yon 4Patienten eine Zunahme der vorher normalen Proliferationsaktivit/it der Monocytopoese beobachtet werden, die allerdings nach dem 4. bis 6. Monat der Immunotherapie wieder in den Normalbereieh abfiel. Bei Patienten im klinischen Stadium II, die eine zus~itzliche cytostatische Therapie mit DTIC erhielten, konnte keine Zunahme der monocytopoetischen Proliferationsaktivitfitfestgestellt werden. Die Ergebnisse machen deutlich, dab die unspezifische Immunstimulation mit BCG in der von uns durchgefiihrten Weise die Proliferationsaktivitgt der Monocytopoese steigert, dab dieser Effekt * This investigation was supported by the Deutsche Forschungsgemeinschaft Offprint requests to." Priv.-Doz. Dr. M. Hagedorn (address see above)

0340-3696/79/0264/0319/$1.60

320

E. Schmitt et al. aber passager ist. Eine Zunahme der monocytopoetischen Proliferationsaktivit/it tritt nicht auf, wenn BCG gleichzeitig mit einer cytostatischen Therapie appliziert wird. Schliisselw6rter: Malignes Melanom -

BCG-Impfung therapie - Monocytopoese - Blut-Monocyten

Chemoimmun-

it is well established that malignant tumors frequently give rise to inflammatory reactions within the vicinity of the tumor and in draining lymph nodes. These reactions are associated with the accumulation of blood-born macrophages. In malignant melanoma the macrophage content of the tumor ranged from 8 % to 30 % in local disease and from 0 % to 8 % in metastatic disease [1]. This result as well as pathohistological observations [2, 3] indicate that inflammatory reactions in malignant melanomas tend to correlate with a favorite prognosis. Alexander et al. [4] using transplanted rat sarcoma demonstrated that macrophage sequestration by tumors increases with the immunogenicity of the tumor. Part of this process is mediated by Tqymphocytes [5], thus representing a delayed type of hypersensitivity reaction. Mitotic figures are absent in tumor-associated macrophages. Thus, formation and maintenance of the macrophage pools within and around the tumors seem to be completely dependent on macrophage recruitment of blood monocytes. Monocyte recruitment may be facilitated by monocytosis which frequently occurs in malignant diseases [6]. However, monocyte recruitment should be compromised by tumor-induced [7, 8] defective monocyte chemotaxis [9 - 11]. This defect of blood monocytes has been described in patients with malignant melanoma. It disappeared during vaccination with BCG [11]. Moreover, immunopotentiating manipulations were shown to render macrophages tumoricidal [12] and to give rise to a transitory increase of monocytopoiesis in mice [13]. The present study seeks to get some insight into the variations of monocyte production in patients with superficial spreading melanoma before and after tumor excision, during BCG-vaccination, and during chemoimmunotherapy. Monocyte production was estimated by determination of the fraction of mononuclear phagocytes in bone marrow (MNP) and the tritiated thymidine labeling index in vitro (3H-TDR L.L). The product of both quantities is approximately equivalent with monocyte production. Monocyte blood counts and naphthol-AS-D-chloroacetate esterase activity of blood monocytes being additionally determined, generally increase in stimulated monocytopoiesis [14, 15]. Patients and Treatment

A total of 36 patients with superficial spreading melanoma were investigated after giving their informal consent. They were hematologically normal and did not display clinical signs of inflammation or infection. The depth of skin invasion ranged from Clark's level [16] II to level IV. The behaviour of the count and naphthol-AS-D-chloroacetate reaction intensity of blood monocytes was investigated in 27 patients, 22 of whom had local disease (stage I) and five showed

Monocytopoiesis in Malignant Melanoma

321

clinical involvement of regional draining l y m p h nodes (stages II). Monocytopoiesis in the bone m a r r o w was analyzed in 9 patients in stage I. Surgery was restricted to the excision o f the primary lesion in stage I. Additional l y m p h node dissection was done in stage II. One m o n t h after the operation stage I patients were submitted to adjuvant i m m u n o t h e r a p y ; stage I[ patients received adjuvant c h e m o i m m u n o t h e r a p y . F o r i m m u n o t h e r a p y the patients received a b o u t 6 x 108 viable B C G germs of the Pasteur strain monthly. B C G was applied intradermally using a heaf gun. The site o f administration alternated between the anterior thigh and the dorsal surface of the upper arm. Stage II patients were treated with D T I C [5-(3.3-dimethyl-ltriazeno) imidiazole-4-carboxamide] 250 m g / m 2 i.v. for 5 consecutive days. B C G scarification was carried out at the 5th day o f D T I C treatment. Cycles were repeated monthly, i.e., every 28 days.

Methods The investigation of blood monocytes and of monocytopoietic proliferation activity was carried out before surgery and just before each application of BCG or DTIC-BCG. The methods used in this study were described in detail in previous publications [17, 18]. The number of blood monocytes was obtained from the leukocyte count and by differentiation of 400 leukocytes. Activity indices of naphthol-AS-D-chloroacetate esterase reaction in blood monocytes were determined as follows : 500 blood monocytes were classified into 4 groups, i.e., 0, 1, 2, 3 according to its cytochemical reaction intensity. The cell number within a group was multiplied with the corresponding group number, and the sum of the products was related to 100monocytes. Proliferation activity of monocytopoiesis was assessed by determination of the fraction of mononuclear phagocytes (MNP) in bone marrow and 3H-thymidine labeling index in vitro (3H-TDR L.I.) of these cells. The term MNP includes all medullar cells belonging to the monocyte-macrophage system; these cells comprise:monoblasts, promonocytes, monocytes, and macrophages. MNP were rendered identifiable on smears of bone marrow spicules by cytochemical reactions which simultaneously demonstrated NaF-resistant and NaF-sensitive naphthol-AS-D-acetate esterase. The latter is specific for MNP. Following autoradiographic processing and staining of the nuclei, 3,000 nucleated bone marrow cells were evaluated in order to determine the fraction of MNP. One thousend MNP were inspected individually for evaluation of their 3H-TDR L.I. The quantity fraction x 3H-TDR L.I, of MNP is approximately proportional with the monocyte birth rate in bone marrow.

Results Figure 1 illustrates the time course o f the count and n a p h t h o l - A S - D - c h l o r o a c e t a t e esterase reaction intensity o f b l o o d m o n o c y t e s during i m m u n o - and chemoimm u n o t h e r a p y . In patients with stage I superficial spreading m e l a n o m a who underwent m o n t h l y BCG-vaccination, the average b l o o d m o n o c y t e count lay at the upper limit of the normal variation during the first 6 m o n t h s o f treatment. Thereafter, mean blood m o n o c y t e counts fell into the lower part o f normal variation. N a p h t h o l - A S - D - c h l o r o a c e t a t e esterase activity of b l o o d monocytes showed a similar behavior. The mean values exceeded normal variation during the first 7 m o n t h s o f BCG-scarification; thereafter they gradually fell. Different results were obtained in stage II patients who, after excision o f the primary t u m o r and l y m p h node dissection, were submitted to c h e m o i m m u n o therapy. In this group, mean b l o o d m o n o c y t e counts definitely decreased already after the first course o f D T I C - B C G treatment (Fig. 1, right upper diagram). M e a n

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Fig. 1. Time course of blood monocyte counts and naphthol-AS-D-chloroacetate esterase activity of blood monocytes in patients with superficial spreading melanoma. Patients with localized disease (stage I) received prophylactic monthly BCG-scarifications after removal of the primary tumor. Patients with regional lymph node involvement (stage I1) received prophylactic immunochemotherapy using DTIC and BCG

activity indices ofnaphthol-AS-D-chloroacetate esterase, however, remained at the upper limit of normal variation (Fig. 1, right diagram at the bottom). Results of the investigation of monocytopoiesis in patients with superficial spreading melanoma stage I are given in Table 1 and Fig. 2. The fraction of 3HTDR labeled mononuclear phagocytes (MNP) in bone marrow exceeded the normal range in 5 of 9 untreated patients. After surgery and 2 months of BCG treatment, this parameter was increased in all 4patients examined. However, normal values were obtained after the 4th month of BCG-scarification. The fraction of medullar MNP lay in the normal range in most patients examined. The product of fraction of MNP and 3H-TDR L.I. which reflect the monocyte birth rate exceeded the normal range in half of the patients with untreated, superficial spreading melanoma. The values of the remaining 5 patients fell into the upper part of the normal range. After surgical removal of the primary tumor and after the second course of monthly BCG-scarification, a moderate rise of the product fraction times 3H-TDR L.I. of MNP was observed in 3 of 4patients. In

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contrast, no change or even a fall of initially increased values into the normal range was observed after the 4th course of BCG-scarification. Discussion

Monocyte production was moderately increased in about half of the patients with untreated superficial spreading melanoma in stage I. Normal skin reactivity in this early disease [21] indicated that blood monocytes enter sites of inflammation. Thus, monocyte chemotaxis seems not be substantially compromised. The combination of enhanced monocyte production and intact chemotaxis would allow adequate monocyte recruitment of the tumor. This is reflected by the relatively high macrophage content of localized malignant melanoma [1]. In contrast to localized melanoma, few numbers of macrophages were detected in the primary tumor and metastases of disseminated melanoma [4]. This finding as well as impaired monocyte chemotaxis in vitro [11] indicate defect monocyte chemotaxis which appears and increases with progressing tumor cell load. Postoperative BCG-vaccination was able to increase monocyte production. However, this effect was transitory; it disappeared after the 4th to 6th month of BCG treatment, and was abrogated by concomitant chemotherapy using DTIC. A similar phenomenon was observed in patients with operable breast cancer who after surgery were first submitted to a 6 months lasting chemotherapy using

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cyclophosphamide, methotrexate, and 5-fluorouracil. There after, therapy was continued by monthly BCG-scarifications. In these patients, being pretreated by chemotherapy, BCG-scarifications were unable to increase the proliferation activity of monocytopoiesis (Fig. 3). Equally, maximal doses of corynebacterium parvum were unable to induce monocytopoiesis in patients with metastasizing t e r a t o c a r c i n o m a of the testis who were pretreated by c h e m o t h e r a p y (Fig. 4). It is i m p o r t a n t to note that these in latter p a t i e n t c h e m o t h e r a p y was stopped at least one m o n t h before a p p l i c a t i o n of c o r y n e b a c t e r i u m p a r v u m due to t u m o r progression and that they were hematologically n o r m a l at the time of the b e g i n n i n g of immunotherapy. A t present, this c h e m o t h e r a p y - r e l a t e d a b r o g a t i o n of the monocytopoiesise n h a n c i n g action of B C G is n o t clearly u n d e r s t o o d . It might arise from a c h e m o t h e r a p y - i n d u c e d long-lasting reduction of h e m a t o p o i e t i c stem cells,

References

I. Gauci, C. L., Alexander, P. : The macrophage content of some human tumors. Cancer Letters 1, 29- 32 (1975) 2. Mehnert, J. H., Heard, J. L. : Staging of malignant melanoma by depth of invasion : a proposed index to prognosis. Am. J. Snrg. 110, 168-176 (1965) 3. Little, J. H.: Histology and prognosis in cutaneous malignant melanoma. Melanoma and skin cancer. Proceedings of the International Cancer Conference, Sidney 1972. McCarty, W. H. (ed.), pp. 107-120. Sidney: Blight 1972 4, Alexander, P., Eccles, S. A., Gauci, C. L. : The significances of macrophages in human and experimental tumors. Ann. N.Y. Acad. Sci. 276, 124-133 (1976) 5. Eccles,S. A., Alexander, P. : Macrophage content of tumours in relation to metastatic spread in host immune reaction. Nature 250, 667-669 (1974) 6. Eccles,S. A.,Bandlow, G.,Alexander, P.: Monocytosis associated with the growth oftransplanted syngeneic rat sarcoma differing in immunogenicity. Br. J. Cancer 34, 20-27 (1976)

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7. Snyderman, R., Pike, M. C. : Inhibitor ofmarcrophage chemotaxis produced by neoplasms. Science 192, 370-372 (1976) 8_ Norman, S. J., Sorkin, E. : Inhibition of macrophage chemotaxis by neoplastic and other rapidly proliferating cells in vitro. Cancer Res. 37, 7 0 5 - 7 / 1 (1977) 9. Boetcher, D. A., Leonard, E. J. : Abnormal monocyte chemotactic response in cancer patients. J. Nat. Cancer Inst. 52, 1091-1099 (1974) 10. Stevenson, M. M., Meltzer, M. S.: Depressed chemotactic responses in vitro of peritoneal macrophages from tumor-bearing mice. J. Nat. Cancer Inst. 57, 847-852 (1976) 11. Snyderman, R., Mergenhagen, S. E.: Chemotaxis of marcophages. In: Immunobiology of the Macrophage, Nelson, D_ S. (ed.), pp. 323-348. New York: Academic Press 1976 12. Alexander, P., Evans, R., Grant, C. K. : The interplay of lymphoid cells and macrophages in tumour immunity. Ann. Inst. Pasteur. 122, 645-658 (1972) 13. Fisher, B., Taylor, S., Levine, M., Saffer, E., Fisher, E. R. : Effect of mycobacterium bovis (strain Calmette-Gu6rin) on macrophage production by the bone marrow of tumor-bearing mice. Cancer Res. 34, 1668-1670 (1974) 14. Meuret, G., Schmitt, E., Hagedorn, M. : Monocytopoiesis in chronic eczematous diseases, psoriasis vulgaris, and mycosis fungoides, J. Invest, Dermatol. 66, 2 2 - 2 8 (1976) 15. Schmitt, E., Meuret, G., Stix, L.: Monocyte recruitment in tuberculosis and sarcoidosis. Br. J. Haemat. 35, 11 - 17 (1977) 16. Clark, W. H., From, L., Bernadino, E. H., Mihm, M. C. : The histogenesis and biologic behavior of primary human malignant melanomas of the skin. Cancer Res. 29, 705-727 (J 969) 17. Meuret, G., Batara, E., Fiirste, H. O. : Monocytopoiesis in normal man: pool size, proliferation activity and DNA synthesis time of promonocytes. Acta Haemat. (Basel) 54, 261-270 (1975) 18. Meuret, G., Detel, U., Kilz, H. P., Senn, H. J., Lessen, H., van: Human monocytopoiesis in acute and chronic inflammation. Acta Haemat. (Basel) 54, 328-335 (1975) 19. Volkman, A., Collins, F. M. : The cytokinetics of monocytes in acute salmonella infection in the rat. J. Exp. Med. 139, 264-277 (1974) 20. Meuret, G., Bammert, J., Hoffmann, G. : Kinetics of human monocytopoiesis. Blood 44, 801-816 (1974) 21. Eilber, F. R., Morton, D. L, : Impaired immunologic reactivity and recurrence following cancer surgery. Cancer 25, 362-367 (1970) 22. Lovereren, H., Otter, W. van den: Macrophages in solid tumors. I. Immunologically specific effector cells. J. Nat. Cancer Inst. 53, 1057-1060 (1974) 23. Meuret, G. : Monozytopoese beim Menschen. Blut, Suppl. 13, (1974) Received July 3, 1978