Short Communication

IMMUNOCOMPETENT CELLS AND LYMPHOCYTE REACTIVITYTO MITOGENS IN LEVAMISOLE-TREATED BRAIN TUMOR CHILDREN

Tomislav F. Hajn Ïzic, MD h Department of Pediatrics, Hematology± Oncology,University Hospital Sestre milosrdnice, Zagreb, Croatia

Maja Ka Ïstelan, PhD, Josip Luka Ïc, PhD, and Tajana Hajn Ïzic, PhD h Departmentof Nuclear Medicine and Oncology, University Hospital Sestre milosrdnice,Zagreb, Croatia

h This study investigated the in¯ uence of levamisole therapy on immunocompetent cells and lym-phocyte reactivity to mitogens in 25 children with brain tumor. Eleven (11/25) patients were receivingchemotherapy and immunomodulating drug levamisole 3 months after neurosurgery, during main-tenance chemotherapy, 2.5 mg/kg of body weight per os, for three consecutive days every 2 weeks,for 6± 12 months. The proportion of lymphocytes, proportion and number of T- and B-lymphocytesand natural killer (NK) cells, as well as lymphocyte reactivity to mitogens were signi® cantly lowerin non-levamisole-treate d patients than in the healthy controls ( N = 18). Therapy with levamisolesigni® cantly augments the proportion of T lymphocytes, the number of T lymphocytes, NK cells, andthe lymphocyte reactivity to concanavalin A (Con A). Depression of the NK cells and the lympho-cyte reactivity to mitogens were much more pronounced in those patients who developed recurrences.Levamisole shortened the period of secondary immunode® ciency in immunocompromised childrenwith brain tumor.

Keywords. immunocompetent cells, levamisole, reactivity to mitogen, tumor cerebri

A signi® cant improvement of treatment effects in children with solid ma-lignant tumors is a result of multimodal therapy application with aggressivepolychemotherapy [1]. Together with such intensive therapy, the frequencyand intensity of many undesirable side effects have increased [2, 3]. Cyto-static chemotherapy by immunosuppression activity catches hold of variousmechanisms of immune defense [4, 5]. The aim of this research was to ® ndout about immunocompetence during chemotherapy in children with solidmalignant tumors, as well as to investigate whether it is possible to shortenthe period of secondary immunode® ciency with the immunomodulatingdrug levamisole. This paper presents the results of determining the propor-tion of all lymphocytes and the proportion and absolute number of T- andB-lymphocytes and natural killer (NK) cells, as well as lymphocyte reactivity

Received 23 June 1998; accepted 15 September 1998.This work was supported by Research Grant 3-01-207, Ministry of Science and Technology, Republic

of Croatia.Address correspondence to Tomislav Franjo HajnÏzi Âc, Department of Pediatrics Haematology±

Oncology, University Hospital Sestre milosrdnice, Vinogradska 29, 10000 Zagreb, Croatia.

Pediatric Hematology and Oncology, 16:335± 340, 1999Copyright C° 1999 Taylor & Francis

0888-0018/99 $12.00 + .00 335

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336 T. F. HajnÏziÂc et al.

to mitogens in levamisole-treated and non-levamisole-treated brain tumorpatients.

PATIENTS AND METHODS

Patients

Twenty-® ve patients (14 males, 11 females aged 3.4± 13.7 years, median5.6 years) treated at the Pediatric Oncology Department of the Sestre Milos-rdnice University Hospital during 1990± 1996 were studied. The number ofT- and B-lymphocytes and NK cells, the proportion of all lymphocytes, andlymphocyte reactivity to mitogens were determined in malignant brain tu-mor patients during maintenance therapy (vincristine, lomustin), 6 monthsafter neurosurgery, induction chemotherapy (GPO-SIOP MED 84, high-risk relapse protocol: procarbazine, methotrexate, vincristine, prednisolone)[6], and radiotherapy. The children had histologically veri® ed diagnosesof medulloblastoma (N = 14), anaplastic ependimoma (N = 6), glyoblas-toma (N = 1), and anaplastic astrocytoma (N = 4). The same parametersof cellular immunity were determined in 18 healthy children without ma-lignant or infectious disease, matched for age and gender (10 males, 8 fe-males aged 6.8± 14 years, median 9.2 years). The patients were divided intotwo subgroups: (A) fourteen (14/25) patients received only maintenancechemotherapy; (B) eleven (11/25) children with brain tumor were receiv-ing the chemotherapy and levamisole (Ergamisol, Decaris Janssen) 3 monthsafter neurosurgery, in doses of 2.5 mg/kg of body weight per os for 3 con-secutive days every 2 weeks for 6± 12 months.

Analysis of Lymphocyte Subpopulation

Puri® ed lymphocytes were analyzed by indirect immuno¯ uorescencetechnique for quantitation of subsets. The monoclonal antibodies used werefrom Ortho Diagnostics (Raritan, NJ) and identi® ed total T cells (CD3+ ) andNK cells (CD16+ ). B lymphocytes (slg+ ) were detected by direct immuno¯ uo-rescence using F(ab)2 portion antihuman immunoglobulin conjugated to¯ uorescein isothiocyanate. Lymphocyte subpopulations were expressed inproportion (P) and absolute number ( £ 109/L).

Blastogenic Response of Lymphocytes to Mitogens

The ability of lymphocytes to respond to optimal concentrations of mi-togens was assessed by standard techniques in which 1 £ 105 lymphocyteswere cultured in plastic microtiter plates with the appropriate mitogen for48 h [7]. The mitogens used were phytohemagglutinin (PHA; Wellcome Re-search Laboratories, Beckenham, England), concanavalin A (Con A; Sigma

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Immunocompetence in Brain Tumor Children 337

Chemical, Saint Louis, MO), and poke-weed mitogen (PWM; Grand IslandBiological, Grand Island, NY). The ® nal dilution of PHA and PWM was 1:100,and 10 l g of Con A per culture was added. Cultures were incubated at 37±Cin 5% CO2 atmosphere. For the last 18 h of the culture, cells were pulsed with1 l Ci of [3H]thymidine (Amersham, England), harvested, and counted on ascintillation counter. The results were expressed as relative response (RR%).

Statistical Analysis

A dependent (paired) t test was performed to determine differencesbetween the children with brain tumor and the control group. The resultsare expressed as mean values and standard errors. The level of statisticalsigni® cance was .05 (95%).

RESULTS

The proportion of all lymphocytes and the proportion and absolute num-ber of CD3+ , slg+ , and CD16+ immunocompetent cells are given in Tables 1and 2. The proportion of all lymphocytes and the proportion and abso-lute number of CD3+ , slg+ , and CD16+ were signi® cantly lower in the non-levamisole-treated patients than in the healthy controls. In Table 3, it canbe seen that lymphocyte reactivity to mitogens (PHA, Con A, PWM) in non-levamisole-treated children and reactivity to Con A and PWM in levamisole-treated patients with brain tumor were signi® cantly reduced in comparisonto control values. The difference was found between the levamisole-treatedpatients and those without levamisole therapy (A vs. B). The proportionof all and T lymphocytes, the absolute number of T lymphocytes and NKcells, as well as the lymphocyte reactivity to Con A were signi® cantly lower innon-levamisole-treated patients (A vs. B) (Tables 1± 3).

Depression of the absolute number of NK cells and the lymphocyte reac-tivity to mitogens were much more pronounced in seven (7/25) children with

TABLE 1 Total Lymphocytes and T-Lymphocytes in Non-Levamisole-Treated Patients(A), Levamisole-Treated Patients With Brain Tumor (B), and Controls (C)

CD3+ cells

Subjects NProportion of

ALL lymphocytes Proportion Number £ 109/L

Not treated (A) 14 0.36(0.021) 0.51(0.026) 1.38(0.106)Treated (B) 11 0.54(0.048) 0.63(0.042) 1.92(0.163)Control (C) 18 0.49(0.020) 0.78(0.045) 1.95(0.148)

p valueA vs. B .01 .05 .05A vs. C ¤ .001 .0001 .01B vs. C¤ NS .05 NS

Note. Results are the mean values(standard errors). NS, not signi® cant. ¤ p values arecalculated from a paired t test.

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TABLE 2 Number of B-Lymphocytes and NK Cells in Non-Levamisole-TreatedPatients (A), Levamisole-Treated Patients (B), and Controls (C)

sIg+ cells CD16+ cells

Number NumberSubjects N Proportion £ 109/L Proportion £ 109/L

Not treated (A) 14 0.09(0.010) 0.18(0.025) 0.05(0.007) 0.11(0.018)Treated (B) 11 0.12(0.015) 0.27(0.042) 0.08(0.014) 0.22(0.041)Control (C) 18 0.14(0.012) 0.31(0.036) 0.09(0.012) 0.24(0.029)

p valueA vs. B NS NS NS .05A vs. C¤ .01 .01 .05 .001B vs. C ¤ NS NS NS NS

Note. Results are the mean values(standard errors). NS, not signi® cant. ¤ p valuesare calculated from a paired t test.

brain tumor who developed recurrences and metastases during the period offollow-up (24 months). Five (5/14) non-levamisole-treated and two (2/11)levamisole-treated patients (35%/18%) with markedly decreased CD16+

cells and lymphocyte reactivity to mitogens, during antitumor chemother-apy period, developed recurrence. Five (5/7) of these patients died in theperiod of 24 months. At this therapeutic dose, levamisole was well toleratedand caused no complications. The only reaction observed was mild nauseain two children.

DISCUSSION

Almost all organic systems can be damaged in the treatment of patientswith malignant diseases. Children especially tend to undesirable side effectsat the growing and development stage. Hematopoetic and immune systemsare damaged very early during oncological treatment. Disorder of immunereactivity is observed immediately after surgery [8] and the signs of immuno-depression are most expressed at the time of radiotherapy and cytostatic

TABLE 3 Lymphocyte Reactivity to Mitogens in Non-Levamisole-Treated,Patients (A), Levamisole-Treated Patients With Brain Tumor (B), and Controls (C)

Subjects N PHA (RR%) Con A (RR%) PWM (RR%)

Not treated (A) 14 72(6.4) 52(5.8) 41(5.3)Treated (B) 11 76(8.1) 71(5.4) 44(6.6)Control (C) 18 92(5.4) 97(9.2) 85(9.6)

p valueA vs. B NS .05 NSA vs. C ¤ .05 .001 .001B vs. C¤ NS .05 .01

Note. Results are the mean values(standard errors). NS, not signi® cant. ¤ p valuesare calculated from a paired t test.

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Immunocompetence in Brain Tumor Children 339

chemotherapy [4, 5]. In these patients infections become the cause of mor-tality together with recurrence and metastases. This secondary immunode-® ciency can last for months. A further consequence is the appearance ofsecondary malignomas [9].

Because of the weakened immune status after oncologic treatment, thequestion is how to stop further weakening of immunocompetence and howto force convalescence of the immune system. Lower doses of cytostatics andradiation are hardly possible because successful treatment results would bejeopardized. For that reason, maximal efforts are directed to immunother-apy. Knowledge of protective antitumor systems and progress of technol-ogy will enable treatments by immunostimulating and immunomodulatingmeans. Bacteriological preparations have been tried in adult patients, but to-day, restorative immunotherapy with interferon and interleukin-2 [10] andadoptive cellular therapy [11] are more signi® cant.

Application of nonspeci® c immunostimulation in children with solid tu-mors was not successful, even with application of adjuvant therapy. Researchresults with ªtumor-in® ltrating lymphocytesº (TIL) and with interleukin-2are very promising [12]. Levamisole, a wide-spectrum antiparasitic drug at-tracted considerable interest when its stimulatory capacity was demonstrated[13]. It is being subjected to extensive clinical trials in a variety of disorders inwhich immunological impairment is postulated. Levamisole has prolongedremission and increased survival when given to patients with breast cancer[14], melanoma [15], lung cancer [16], and colon carcinoma [17, 18].

The results of our investigation so far in patients with soft tissue sarcoma[19] have shown that the immunomodulating drug levamisole improves im-munocompetence in patients during chemotherapy. The subjects of our in-vestigation were brain tumor patients during maintenance chemotherapy.The proportion of all lymphocytes, proportion and the absolute number ofT- and B-lymphocytes and NK cells, as well as the lymphocyte reactivity tomitogens were lower in non-levamisole-treated children than in the controls.Therapy with levamisole signi® cantly augments the proportion of all and Tlymphocytes, the absolute number of T lymphocytes and NK cells, as wellas the lymphocyte reactivity to Con A in secondary immunocompromisedpatients with brain tumor. Depression of the NK cells and the lymphocytereactivity to mitogens were much more pronounced in those children withmalignant brain tumor who developed recurrences during follow-up.

The data presented show that immunological reactivity during chemo-therapy is signi® cantly damaged in children with malignant brain tumor.Therapy with the immunomodulating drug levamisole during chemotherapystimulated immune response in immunocompromised patients with braintumor and shortened the period of secondary immunode® ciency.

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REFERENCES

1. Young JL, Reis LG, Silverberg E, et al. Cancer incidence, survival and mortality for children youngerthan age 15 years. Cancer (Suppl). 1986;58:598± 602.

2. HajnÏzi Âc TF, LovrenÏci Âc KM, JugoviÂc V. Effect of the chemotherapy and irradiation on the neuroen-docrinologic status in children with acute lymphoblastic leukemia. Eur J Cancer (Suppl). 1991;27:252.

3. Wright MJ, Halton JM, Martin RF, Bard RD. Long-term gross motor performance following treatmentof acute lymphoblastic leukemia. Med Pediatr Oncol. 1998;31:86± 90.

4. Abrahansson J, Marky I, Mellander L. Immunoglobulin levels and lymphocyte response to mito-genic stimulation in children with malignant disease during treatment and follow-up. Acta Paediatr.1995;84:177± 182.

5. Leikin S, Miller D, Sather H, et al. Immunologic evaluation in the prognosis of acute lymphoblasticleukemia: a report from children’s cancer study group. Blood. 1981;58:501± 507.

6. Neidhardt M, Bailey CC, Gnekow A, et al. Die Medulloblastom-Therapiestudien MBL 80 and MED84 der Gesellschaft fur Padiatrische Onkologie und Societe Internationale d’Oncologie Pediatrique.Klin Padiatr. 1987;199:188± 192.

7. KaÏstelan M, Rudolf M, HrÏsak M, StojkoviÂc R, Gamulin S. Lymphocyte reactivity to mitogens andtumor sex steroid receptors in breast cancer patients. Biomed Pharmacother. 1985;39:442± 444.

8. Cochran AJ, Spilg WGS, Mackie RM, Thomas CE. Postoperative depression of tumor-directed cell-mediated immunity in patients with malignant disease. Br Med J. 1972;4:67± 70.

9. Morris-Jones PH, Craft AW. Childhood cancer: cure at what cost? Arch Dis Child. 1990;65:638± 640.10. Toma S, Melioli P, Palumbo R, Rosso R. Recombinant interleukin 2 and alpha-2a-interferon in pre-

treated advanced soft tissue sarcomas. Int J Oncol. 1993;2:997± 1001.11. Topalian SL, Rosenberg SA. Adoptive cellular therapy: basic principles. In: De Vita VT, Hellman S,

Rosenberg SA, eds. Biologic Therapy of Cancer. Philadelphia: Lippincott; 1991:178± 196.12. Wexler L, Thiele C, McClure L, et al. Adoptive immunotherapy of refractory neuroblastoma with

tumor-in® ltrating lymphocytes (TIL), interferon-gamma and interleukin-2 (IL-2). Proc Am Soc ClinOncol. 1992;11:368.

13. Greespan EM, Erlich R. Levamisole and new era of chemoimmunotherapy. Cancer Invest. 1991;9:111± 124.

14. Treurniet-Donker AD, Meiscke deJongh ML, vanPuttens WIJ. Levamisole as adjuvant immunotherapyin breast cancer. Cancer. 1987;59:1590± 1593.

15. Quirt I, Shelley W, Bodwitha A, et al. Adjuvant levamisole improves SV and DFS in patients with poorprognosis in malignant melanoma. Proc Am Soc Clin Oncol. 1986;5:130± 131.

16. Herskovic A, Bauer M, Seydel HG, et al. Postoperative thoracic irradiation with or without levamisolein non-small cell lung cancer. Int J Radiat Oncol Biol Phys. 1988;14:37± 42.

17. Bjorge L, Matre R. Down-regulation of CD 59 (protectin) expression on human colorectal adenocar-cinoma cell lines by levamisole. Scand J Immunol. 1995;42:512± 516.

18. Moertel CG, Fleming TR, MacDonald JS, et al. Levamisole and ¯ uorouracil for adjuvant therapy ofresected colon carcinoma. N Engl J Med. 1990;322:352± 358.

19. HajnÏzi Âc TF, CvitanoviÂc L, LukaÏc J, JurÏci Âc Z. Phagocytic activity in levamisole treated children with softtissue sarcoma. Book of Abstract, 3. Int Soft Tissue Sarcoma Congress 1997, Stuttgart, Abstr.P-BS-15.

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