Thymostimulin therapy in melanoma patients: Correlation of immunologic effects with clinical course

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Thymostimulin Therapy Immunologic

28, 311-324 (1983)

in Melanoma Patients: Correlation Effects with Clinical Course

of

MARIA GRAZIA BERNENGO, PAOLA FRA, FRANCESCOLISA, MASSIMO MEREGALLI, AND GIUSEPPEZINA Dermatologic

Clinic University of Torino, Via Cherasco 23, 10126 Torino, Italy

Thirty-two nonmetastatic melanoma patients with low T-lymphocyte values were treated with a thymic extract, thymostimulin (TS) (8 patients), DTIC (8 patients), or surgery alone (16 patients). In the 8 patients receiving TS, active E-rosette (T-Ea) and total E-rosette (T-Et) counts rose to normal levels and there was a significant rise in IgM and IgD receptors. Six out of eight patients treated with TS showed no evidence of metastases after 34 months, while 7/8 patients on DTIC and 13/16 patients on surgery alone developed metastases. Twenty metastatic patients with low T-lymphocyte values received either DTIC plus TS or DTIC alone. Total lymphocyte, T-Ea, and T-Et counts did not increase in either group nor was there a significant difference between the group on DTIC plus TS and the group on DTIC alone. The survival rate of patients on DTIC plus TS did not differ significantly from that on DTIC alone.

INTRODUCTION

Thymic hormones have proved effective in the treatment of primary immunodeficiencies (1 - 3) and viral infections (4-6) because of their ability to stimulate cell-mediated immunity. The close relationship between cancer and cellular immunity has been widely demonstrated in recent years. Studies on patients with lung cancer on immunosuppression indicate that thymic hormones may have a positive effect on defective T-lymphocyte production (7, 8). Active E rosettes were significantly increased in patients with carcinoma of the pancreas, colon, and stomach with decreased cellular immunity due to drug therapy (9). A significant correlation between prognosis and cellular immunity was noted in patients with malignant melanoma: survival expectancies were shorter in patients with low T-lymphocyte counts than in those with normal values, and a fall in T lymphocytes occurred concomitantly with or immediately prior to the development of metastases (10, 11). A recent study demonstrated that, in patients with malignant melanoma, a reduction in T lymphocytes, particularly in the early stages, is linked to an increase in cells able to mature and acquire surface markers when incubated with thymus extracts (12). The above findings and the in viva response obtained in a preliminary report (13) indicated that a study was necessary to determine the behavior of certain immunological parameters in patients with both metastatic and nonmetastatic melanoma during treatment with a thymus extract (thymostimulin) over a period of 27 months. This was done for the following reasons: (1) to run an in vivo check on immunological responses obtained in vitro; (2) to assess whether these responses could in some way prevent the develop311 0090-1229183 $1 SO Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved

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ET AL.

ment of metastases in high-risk patients in the years immediately following surgery; (3) to determine whether thymic hormones can augment cellular immunity in patients with progressively decreasing T-lymphocyte values, thereby encouraging a better response to chemotherapy. MATERIAL Thymostimulin

AND METHODS

(73)

TS was prepared by the method of Falchetti et al. (14) and was kindly provided by the Istituto Farmacologico Serono, Rome. Fifty-two patients (25 women and 27 men, aged 20-77 years, mean 45) with histologically proven malignant melanoma were included in the study. The M.D. Anderson Hospital classification (15) was used to assign 32 patients to stage I, 1 to stage IIIA, 2 to stage IIIB, 4 to stage IIIAB, and 13 to stage IV. A previous long-term study indicated that a sudden drop in T-cell values frequently predicts the subsequent development of metastatic or recurrent disease, although it is impossible to say whether the reduction in T cells is a cause or a result of metastatic development (11). In addition, we found that the patients who died during the first 2 years after excision of primary melanoma, in spite of their favorable Clark and Breslow levels, had total T-cell values constantly below 1000/mm3. We therefore considered stage I patients with total T-cell counts below 1000/mm3 as high-risk patients, and chose those with values below 1000/mm3 for treatment. In our previous experience, we found that 15% only of untreated stage I patients displayed total T-cell values constantly lower than 1000/mm3 in the first year after excision of the primary melanoma. Of 211 patients studied with serial immunologic tests every 3 months between l/1977 and lo/1981 36 developed, in the first 2 years after excision, a reduction of T lymphocytes that remained unchanged over at least four controls performed every 15 days. Four were excluded from the study: 2 because they displayed hyperthyroidism and 2 because they were pregnant. The stage I patients with T-cell depression were too few for randomization. Their consecutive assignment to each of the three treatment groups was thus determined by the prognostic factors illustrated in Table 1 in an effort to obtain uniform groups. At the beginning of the study, the 32 stage I patients were tumor free as judged by clinical evaluation, blood chemistry, computed tomography, and liver scan. They were subdivided into three groups: 8 received TS, 25 mg/week; 8 DTIC, 200 mg/m2 for 5 days repeated every month over a period of 6 months; and 16 did not receive any treatment. In 15 patients with low T cells (normal values total E rosettes 59.2 + 8.5%), we tested the in vitro effects of TS, at different concentrations, on the percentage of E-RFC. The results are reported in Table 2. In 10 cases, the in vitro response was better with the lowest dose (5 pg/106 cells). Since immunomodulating agents may not only be inactive at high doses but may even become immunosuppressive and enhance tumor growth (16-20), we used

THYMOSTIMULIN

IN

IMMUNOLOGlC

WVO:

TABLE COMPARISON

OF PROGNOSTIC

Male/female Age (years) mean 2 SD Range Anatomic location Extremity Trunk Head and neck Histologic type SSM” SSM + lV’ NM’ Level of invasion II III IV Tumor thickness (mm) 0.7-2 2.1-3 >3 Mean * SD Range

FACTORS

EFFECTS

313

1

IN THE THREE

TREATMENT

STAGE I GROUPS

Surgery plus TS

Surgery plus DTIC

Surgery alone

513 44.2 2 16.9 22-75

6l2 49.5 k 15.0 20-72

8/8 44.0 f 12.8 26-71

5 2 1

5 2

11 4 1

3 2 3

3 2 3

6 4 6

2 5 1

1 6 1

3 12 1

3 1 4 3.37 ” 2.1 0.8-8

3 1 4 4.4 2 3.2 0.8-11

7 3 6 3.17 + 2.3 0.8-9

d SSM, superficial spreading melanoma. B SSM + N, superficial spreading melanoma with vertical growth phase. c NM, nodular melanoma. TABLE IN VITRO

Patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

EFFECT

OF TS ON PERCENTAGE

2

OF E-RFC

AT DIFFERENT

CONCENTRATIONS

TS pg/106 cells

Baseline % E-RFC

5

10

50

250

39 47.5 29.5 45.5 31.5 35.5 44.5 50 49 51.5 40 53 47.5 42.5 32.5

48 45 47 47.5 58 63.5 49 63 67.5 59 36 69 49.5 52.5 40

41 ND” 48 45.5 58 61.5 ND ND 59 ND ND ND ND ND 40

47 ND 31 45 58 52.5 ND ND 59 ND ND ND ND ND 53.5

47 39 41 41.5 65 68 43.5 65.5 74.5 47.5 28 38 39 51 55

Note. Patients 1-6 entered the study under TS treatment. a ND, not done.

314

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ET AL.

TABLE IN VIVO EFFECT

OF TS (25 mg/week)

3

ON PERCENTAGE

AND

E-RFC

ABSOLUTE

IN FOUR

PATIENTS

15

21

DW’ Patient

1

1

2

% E-RFC E-RFC/mm3 % E-RFC E-RFC/mm3 % E-RFC E-RFC/mm3 % E-RFC E-RFC/mm3

39 390 46 644 52 1040 43.5 794

n TS was administered

on Days

2 3 4

31 381 46 644 42.5 850 47.5 902

3 35 432 -

5

7

10

61 833 -

20.5 254 52.5 630 78 1560 50.5 1552

54.5 1277 76 1003

-

14 66 950 52 1327 37 901

-

67.5 1170 63 950 -

62 2109 43.5 1583

1, 8, 15, and 22.

the least E-RFC-increasing TS dose. In the first in viva study, this was half the 1-mg/kg dose effective in the treatment of primary immunodeficiencies (1, 4, 21). The body weight for our first four patients was about 50 kg and they thus received 25 mg. During the first week of treatment and after only one administration, an increase began on the third day with a fall on the seventh day (Table 3). A higher dose or a shorter administration interval failed to raise T-lymphocyte values, and caused a reduction in four other cases (Table 4). Therefore we treated 16 patients with 25 mg/week and determined E-RFC values every week for 3 weeks. In four (Nos. 1, 2, 4, 5) patients, body weight ranged from 60 to 70 kg. Even though the dose administered was thus lower than 0.5 mg/kg, after 1 week an increase in E-RFC was observed and no change in dose was necessary. Weekly monitoring of E-RFC confirmed the increase and the normalization of T cells. Percentage and absolute E-RFC values before and during TS treatment are reported in Table 5. Twenty patients with metastases were randomized to receive either DTIC (200 mg/m2 for 5 days repeated every month) plus TS (25 mg/week) or DTIC alone. Therapy continued for as long as possible until death occurred. Immunological

Studies

Full blood and differential counts, T-lymphocyte E-rosette (T-Ea) and total E-rosette (T-Et) estimation TABLE IN VIVO EFFECT

Patient F.R. M.C. V.E. C.G.

OF TS AT Two

TS dose 25 25 50 50

mg twice mg twice mg/week mgiweek

a week a week

DIFFERENT

60.5 51.5 47.5 47.5

4 DOSES

Baseline (692) (772) (756) (953)

assays, including “active” with and without addition of

ON E-RFC

OF FOUR

Percentage

(mm?

After

1 week

63 (567) 62.5 (594) 44.5 (691) 55.5 (1282)

PATIENTS

E-RFC After 62 59.5 29.5 52.5

3 weeks (697) (863) (472) (539)

THYMOSTIMULIN

IN VIVO EFFECT

OF TS (25 mg/week)

IN VIVO:

IMMUNOLOGIC

315

EFFECTS

TABLE 5 ON PERCENTAGE AND ABSOLUTE AND METASTATK PATIENTS*

E-RFC

IN NONMETASTATIC?

Day Patient

Baseline

1 S.G.G. 2 C.C. 3 B.P. 4 F.P.P. 5 A.S. 6 S.P. 7 C.A. 8 B.C. 9 C.E. 10 N.S. 11 A.S. 12 M. 13 M.G. 14 P.L. 15 G.A. 16 CA. n Patients * Patients

43 41 30 40 43.5 28 41 29.5 43.5 44.5 35 38.5 44.5 31.5 39 34.5

(853) (750) (792) (560) (794) (444) (787) (756) (674) (583) (454) (700) (982) (451) (785) (584)

7 54.5 45 41.5 58 50.5 33.5 42 60 51.5 59.5 53 48 57 41 44.5 45

(971) (777) (1096) (1054) (1552) (687) (684) (1469) (894) (756) (1102) (636) (1267) (597) (981) (787)

l-8 entered the present study under TS treatment; 13- 16 are the responders in the present study.

14 53.5 (959) 40 (873) 63.5 (1361) 49.5 (594) 44 (752) 64 (1792) 54.5 (1318) 61.5 (783) 59.5 (1416) 51.5 (964) 58.5 (1800) 63 (1260) 56 (909) 36 (739) 50 (850) patients

9- 12 were

21 66 50 58 55 43.5 58 51 47.5

(1176) (1235) (1775) (1004) (1583) (901) (1552) (1102) 48.5 (1018) 60

(1200) -

49.5 (1275) 50 (850) treated

thereafter.

TS, and B-lymphocyte assays including EAC rosettes and detection of surface membrane immunoglobulins (Sm-Ig) were performed at least three times during the month prior to the treatment, and monthly thereafter for the first year, every 2 months for the second year and every 3 months thereafter on the surviving patients. Lymphocyte stimulation assays with mitogens were performed on the day prior to treatment and at 3 monthly intervals. T-lymphocyte assays. Peripheral blood samples were collected in heparin (20 pg/ml) and Plasmagel (4 vol blood and I vol plasma gel) and allowed to stand at 37°C for 30 min. The supernatant was layered onto a Lymphoprep gradient (Nyegaard and Co-As-Oslo) and centrifuged at 4008 for 30 min. The interface was removed, washed three times with Hanks’ balanced salt solution (HBSS) and resuspended in McCoy’s 5A modified medium without calf serum. Cell viability was determined by the trypan blue exclusion test. The lymphocyte suspension (0.25 ml) was mixed with 0.25 ml of 0.5% washed sheep red blood cells (SRBC) (SRBC:lymphocyte ratio 20: l), centrifuged, and incubated at 37°C for 5 min. For determination of T-Ea, the pellet was resuspended immediately and at least 300 lymphocytes were counted under a light microscope at x 1000. Lymphocytes binding three or more SRBC were considered positive. For the determination of T-Et, the cells were incubated in ice for 8 hr prior to counting. T-cell assays were also performed after incubation with TS according to the method of Wara et al. (3). The lymphocyte suspension (0.25 ml) was mixed with 0.25 ml of 0.5% SRBC and 5 pg/ml of TS and incubated at 37°C for 5 min. The suspension was centrifuged at 200g for 5 min and then incubated at 4°C for 18 hr.

316

BERNENGO

ET

AL.

Supernatant was removed (0.25 ml), the pellet was resuspended, and the rosettes were counted. B-lymphocyte assays. EAC rosettes were estimated according to the method: 5.0 ml of washed SRBC was incubated at 37°C for 30 min with 5.0 ml of a 1 in 7000 dilution of rabbit anti-sheep erythrocyte antibody (A). After washing three times, the antibody-coated sheep erythrocytes (EA) were resuspended in 5.0 ml HBSS, 5.0 ml of fresh human serum complement (C) diluted 1 in 20 with the medium was added to the 5.0 ml of EA cells in HBSS. and incubated for 30 min at 37°C. The complement-antibody-coated cells (EAC) were washed three times and adjusted to a 1% solution; 0.20 ml of EAC cells was mixed with 0.20 ml of IO6 lymphocytes, centrifuged for 5 min at 2OOg, and incubated at 37°C for 20 min. The pellet was resuspended by vigorous mixing. At least 300 cells were counted under a sealed cover slip (22). The detection of surface membrane immunoglobulins (SmIg) by immunofluorescence was carried out according to the method of Pemis et al. (21) using polyspecific and monospecific fluorescein-conjugated goat anti-human immunoglobulin (anti-IgG, JgA, IgM, IgD, and IgE) (Cappel Labs, Downington) after overnight incubation at 37°C. Mitogen stimulation. Lymphocytes were incubated with phytohemagglutinin (PHA) (Wellcome), pokeweed mitogen (PWM) (Grand Island Biological Co.), and concanavalin-A (conA) (Pharmacia, Uppsala. Sweden) for 48 and 72 hr. Cells (2.0 x 105) in 0.1 ml RPM1 1640 (GIBCO) plus 10% heat-inactivated fetal calf serum (FCS), L-glutamine, and antibiotics were incubated in triplicate with 10 &ml PHA, PWM, and ConA at 37°C in an atmosphere containing 5% CO,. In addition, controls were set up without mitogens. Cultures were pulsed with [3H]thymidine (1.0 Ci/ml), harvested 16 hr Iater with a Skatron multiple automatic sample harvester (Lieben, Norway), and counted in a Packard liquid scintillation counter. Results were expressed as counts per minute (cpm). The stimulation index (SI) was assessed by deducting the counts per minute of the controls from the counts per minute of the stimulated samples and dividing the result by the former. LABORATORY

Nonmetastatic

RESULTS

patients

Baseline absolute T-lymphocyte values were not significantly different in the three groups (TS 717 -+ 131; DTIC 727 + 235; surgery alone 712 + 111/mm3) and all values were significantly lower than normal controls (mean 1340 +_ 426/mm3). The in vitro addition of TS was effective in raising the percentage of T lymphocytes in all eight patients receiving TS (P < 0.001) (Table 6). Mean absolute T-Et values during a 27-month follow-up in the three groups are shown in Fig. 1. Absolute values were more significant than percentage values. Patients receiving TS showed a significant increase (P < 0.01) in T-Et values to reach the normal levels, which were maintained for the whole observation period, while patients on DTIC had transient increases with frequent falls to the pretreatment values. Constantly low T-Et values were observed in the patients with surgery alone. A significant (P < 0.01) normalization of T-Et was observed in this group at the 24-month follow-up after excision of metastases in the survivors.

THYMOSTIMULIN

IN VITRO

IN

EFFECT OF TS ON BASELINE E-RFC

1 2 3 4 5 6 I 8 Mean A SD Student’s

IMMUNOLOGIC

TABLE 6 IN EIGHT

t test:

317

EFFECTS

NONMETASTATIC

MELANOMA

Percentage E-RFC without TS (4

Patient

Note.

VIVO:

PATIENTS

Percentage E-RFC with TS (b)

43 41 30 40 43.5 28 41 29.5 37 f 6.2

56.5 63 56 44 52.5 48 64 59.5 55.4 -c 6.5

a vs h, P < 0.001.

Baseline T-Ea values were significantly reduced (P < 0.01) in patients receiving TS only (327 ? 20Umm3) compared with normal controls (691 + 296/mm3) whereas in the other two groups T-Ea was within the normal range. In six out of eight patients on TS, increased T-Ea levels were noted during the first week of treat-

1 OCCURIENGE

OF MTASTASES

+ MATH

o-0

0.5

’

3

I

6

.

9

12

0-a

SURGERY

+ TS

m-s-=

WY

+DTJC~

I----A

%RGRIY+ALONE

15

I

16

r 21

24 MONTHS

FIG.

1. Follow-up

analysis

of T-Et/mm3

values

in stage I patients

during

therapy.

d 27

318

BERNENGO

ET AL.

ment. After 6 months of treatment, all patients on TS showed a significant increase (P < 0.05) in the levels, and values remained within the normal range for the following study period. Patients on DTIC showed increases that were not statistically significant. In patients with surgery alone, T-Ea remained unchanged. In the group on TS, baseline EAC rosettes (mean 23 1 2 50/mm3) increased (P < 0.05) at the fourth month (373 k 168/mm”), whereas in both the other groups EAC rosettes did not change. There was also a significant increase in surface immunoglobulins M and D in this group (baseline values IgM 4.4 k 2.7%, 93 2 61/mm3: sixth month 7.4 t 3.2%. 173 k 81/mm3, P < 0.05; baseline values IgD 3 t 1.4%, 65 ? 32/mm3; ninth month 4.8 k 0.8%, 107 t 17, P < 0.01). When we compared the in vitro and in vivo results, we observed that TS became ineffective in vitro when total T cells in vivo reached the mean value of healthy controls. Metastatic Patient,5 Pretreatment T-lymphocyte values in the group on DTIC plus TS (mean 771 L 264/mm3) was not different from the group on DTIC alone (mean 885 + 252/mm3) and both were significantly below the normal range (mean 1340 _+ 426/mm3). In vitro incubation with TS induced a greater than 10% increase in T lymphocytes in four patients only (Table 7). Mean T-Ea, T-Et, and total lymphocyte values during therapy are shown in Fig. 2. T-Ea, T-Et, and total lymphocyte values were similar in both groups, although a more pronounced decrease in all parameters was observed after three cycles of treatment in the group on DTIC alone. There was no change in EAC rosettes and SmIg levels in either group after therapy. Inconsistent results were obtained with mitogen stimulation in both nonmetastatic and metastatic patients. IN VITRO EFFECT

OF TS ON BASELINE

48 31.5

2

3 4 5 6 7 8 9 10 Note.

Student’s

7 IN 10 METASTATIC

Percentage E-RFC without TS (a)

Patient

Mean

TABLE E-RFC

42.5 44.5 46.5 40 32 34.5 39 40 39.8 _t 5.7

t SD t test: a vs b,

P < 0.001.

MELANOMA

PATIENX

Percentage E-RFC with TS (b)

46 53 52 60 43 46.5 56.5 44 54 47 50.2 -t 5.4

THYMOSTIMULIN

IN

VIVO:

IMMUNOLOGIC

EFFECTS

0-e .-.-.

300

2

4 01 1760~~.:.:.:.:.:.:.:.:.:.:.:.;.:.:.:.:.:.~:,:...:.:.:.:.:.~:.:.~;.:.~:.:.~:.~

1000 4 O-h-!-

3

9

12

319

DTc*T!s m

15

MONTHS

FIG. 2. Mean absolute T-Ea, T-Et, and total lymphocyte values in metastatic patients during

CLINICAL DATA Nonmetastatic Patients

Observation time ran from the documentation of low T-cell numbers to the stopping date of 30 April 1982. In most cases (TS 4/8, DTIC 6/8, surgery alone

320

BERNENGO

ET AL.

10116) low T-cell numbers were noted for the first time the day prior to excision of the primary melanoma. In 6 patients (1 TS, 2 DTIC, 3 surgery alone), reductions appeared after 6 to 12 months; in 6 patients, more than 19 months elapsed before reductions appeared, 3 in the TS group (19,21, and 25 months) and 3 in the surgery alone group (19, 22, and 28 months). The mean time between completion of surgery and documentation of low T-cell numbers was 10.4 2 9.8, 3.5 ? 2.1. and 7.6 2 8.5 months in the TS-treated, DTIC-treated, and surgery alone groups, respectively. The metastasis-free interval was defined as the time between the documentation of low T-cell values (i.e., commencement of treatment in the TS- and DTICtreated groups) and appearance of the first metastasis. Two out of eight patients receiving TS developed metastases 25 and 28 months, respectively, after the beginning of therapy. In these patients we stopped the therapy after 12 months. In one patient, 6 months after the suspension was observed a drop in T-Et values (827/mm3) which lasted for 6 months. During this period a chest X-ray revealed lung metastases. The other patient developed 10 cutaneous metastases in the form of hemorrhagic lesions, 2-4 mm in diameter. Two of these were excised and histologically confirmed. The patient was treated with CCNU 126 mg/m2 orally every 6 weeks and TS 25 mgweek. After 10 months. his metastases were flatter, and his blood chemistry tests, computed tomography of the brain, and radionuclide scan of the liver and bones were normal. The remaining six patients were metastasis-free after 30.6 + 2.9 months. Metastases developed in 718 patients receiving DTIC within 2- 10 months (mean 1I ? 7.5) and in 13/16 patients who received surgery alone within 2- 11 months (mean 7 + 2.9) (Fig. 3). Since 3 patients were metastasis free after 30, 43, and 48 months, the metastasis-free interval for the latter group was 13.5 t 13.5. Using the log-rank test the metastasis-free interval was significantly longer (P < 0.005) in the group on TS than in the other groups. With the Mann-Whitney U test, the metastasis free-interval (or observation time in patients that did not develop metastases) in the group on TS was significantly higher than in that on DTIC (P = 0.0005) and the surgery alone group (P = 0.0038). The differences between TS and the other two groups were also significant in function of the interval between excision of the primary melanoma and the appearance of metastasis. Survival curves generated by the product limit estimate of Kaplan and Meier in the three groups did not differ significantly (Fig. 4). Metastatic

Putients

The survival rate of patients on DTIC plus TS did not differ significantly from those on DTIC alone. A better response to therapy was observed during the first 3 months in the group on DTIC plus TS. DISCUSSION

In this study T-Ea and T-Et levels increased to normal in all nonmetastatic patients treated with TS for 25-34 months and in vitro incubation with TS revealed a statistically significant increase in TS-responsive cells. However,

THYMOSTIMULIN

[3

IN VIVO:

IMMUNOLOGIC ----.-

1000

SURGERY SURGERY

+ TS + DTIC

SURGERY

ALONE

2

34

90.

8

60.

r-‘-.‘-.-.,....-mm...

%

321

EFFECTS

,..... I

. . . . ..w

-1I

r.i /

70=

60=

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F--j

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20. !y lo-

0

F=‘. lj !i I, 7

14

, 26

21 MONTHS

3. Occurrence of metastases in stage I melanoma patients with “low”

pretreatment

T-cell

metastatic patients showed an increase in T-Ea and T-Et levels during chemotherapy only. Subsequently, the appearance of further metastases coincided with a fall in T-Ea and T-Et values against which TS therapy had no effect. There was also no significant difference in T-Et and T-Ea levels between the two groups (DTIC and DTIC plus TS), and the response to in vitro incubation with TS was much lower in metastatic patients with a tendency to decrease as the disease advanced. These results indicate the ineffectiveness of TS in the latter stages of melanoma, and are substantiated by an earlier study of 113 patients followed immunologically at 3-month intervals for 5 years, where a negative correlation was shown between the duration of observation and mean null cell values in patients with advanced metastases (11). In contrast, untreated nonmetastatic patients showed a positive correlation between mean null cell values and the duration of observation over the 5-year period, with a significant in vitro increase in TS-responsive cells. These results, together with those obtained in the present study, indicate a progressive decrease in the number of cells capable of maturation as the disease advances, but

322

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ET AL.

I: ---------;

L .-.-.-.

La.

-

SURGERY+TS

-.-

suwjERY+m

----

SURGERY

ALONE

Ii I ‘i-------7 i-.,.

:-,

1

i

a

L,,,,,,

---B-

---------

i.-.-.-.--.

-.-.

1

i

i 30

20

//.

10

180

,

.

360

540 TIME

FIG.

4.

Survival

of stage

I melanoma

patients

with

.

.

f

720

900

1080

FROM FIRST TREATMENT “low”

pretreatment

T-cell

(DAYS)

counts.

point to the positive role of thymic extracts in transforming immature lymphoid cells into mature T cells, especially during the early stages of the disease. The present study does not indicate whether helper or suppressor T-cell populations are stimulated, although the significant rise in IgM and IgD membrane receptors in nonmetastatic TS-treated patients only points to an increase in the helper subpopulation. Preliminary results with monoclonal antibodies OKT,, OKT,, OKT8 indicate that OKTJOKT, ratio was maintained within the normal range during a 1 year follow-up with TS therapy. Thymic extracts have been shown to directly stimulate B-cell differentiation (12). Previous in vitro studies have not demonstrated a significant effect of TS on patients with normal T-cell values, as is found in normal subjects. In nonmetastatic patients a steady state was obtained once immunological values returned to normal on TS therapy. We used the dose of 25 mg/week in the light of our first experiments in vitro and in vivo, and continued the follow-up with this dose in order to evaluate the immunological effect over a long period of time. Since in 5/H patients we had obtained a better in vitro response with greater doses, it is possible that the

THYMOSTIMULIN

IN

VIVO:

IMMUNOLOGIC

EFFECTS

323

increase of the in vivo dose produces similar or better effects on the patients responding in vitro. Clinical data suggest that, in nonmetastatic patients with low T-cell levels treated with TS, the in vivo immunological response is linked to a significantly longer metastasis-free interval in comparison with untreated controls. The time between the fall in T-cell values and the appearance of metastases was treated as the metastasis-free interval. Even if the time between excision and the appearance of metastases is taken as the metastasis-free interval, this was significantly longer in TS-treated patients, especially when compared with the surgery alone group. Several studies have reported that T-cell values were normal in stage I patients (23, 24) while T-cell depression occurs in the advanced stages of the disease. On the other hand Wybran and Fudenberg (25) found that in cancer patients (including melanoma) a fall in T-Ea preceded the appearance of metastasis. Since in one long-term study we observed that patients with a decrease in T-Ea and T-Et displayed metastases usually within 2- 10 months, we considered T-cell depression a high-risk prognostic factor (11). The number of patients is small because only 15520% of stage I patients display a constant reduction in T lymphocytes. It is clear that the size of the study is too small and that the duration of follow-up is not long enough to draw any specific conclusions. However, the absence of side effects, the fact that we did not observe tumor enhancement, and the fact that after surgery of primary melanoma no adjuvant chemotherapy can prevent the development of metastases suggest that a controlled study with a larger number of patients should be undertaken in view of these preliminary results. ACKNOWLEDGMENT The authors thank Mr. Giampaolo Berruto of Istituto di Ricerche Biomediche “A. Marxer” for statistical analysis.

di Ivrea

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13. Bernengo, M. G., Barbera, C., De Matteis, A., Meregalli, M., and Zina, G., In “Thymus, Thymic Hormones and T-Lymphocytes” (F. Aiuti and H. Wigzell, Eds.), pp. 313-322, Academic Press, New York, 1980. 14. Falchetti, R., Bergesi, G., Eshkol, A., Cafiero, C.. Adorini, L.. and Caprino, L.. Drugs hp. Ch. Res. 3, 39. 1977. 15. Sugarbaker, E. V., and MC Bride, C. M., Cancer 37, 188, 1976. 16. Shoham, J., Eshel, I., Aboud, M., and Salzberg, S., J. Zmmunol. 125, 54, 1980. 17. Small, M., and Trainin, N.. Inr. J. Cancer 15, 962, 1975. 18. Stutman, O., Advan. Cuncer Res. 22, 261, 1975. 19. Trainin, N., Linker-Israeli, M., and Boiato-Chen, L., Int. J. Cancer 2, 326, 1967. 20. Mikulski, S. M., and Muggia, F., Cancer Treat. Rep. 4, 103, 1977. 21. Aiuti, F., Fiorilli, M., Ammirati, P., D’Amelio, R., and Businco, L., “IV. International Symposium, Japan Medical Research Foundation,” p. 331, 1978. 22. Pernis, B., Forni, L.. and Amante, L., J. Exp. Med. 132, 1001, 1971. 23. Koziner, B.. Cosimi. A. B., and Bloch, K. .I., Nat. Cancer Inst. 55, 1295. 1975. 24. Silverman, N. A., Alexander, J. C., Jr., Potvin. C., and Chretien, P. B., Surgery. 79, 332, 1976. 25. Wybran, J., and Fudenberg, H. H., J. C/in. Znvesr. 52, 1026. 1973. Received

October

14, 1982: accepted

with

revisions

January

19, 1983.