Immunobiology of primary intracranial tumors Part 1: Studies of the cellular and humoral general immune competence of brain-tumor patients
M . STEPHEN M A H A L E Y , JR., M . D . , PH.D., WILLIAM H. BROOKS, M . D . , THOMAS L. ROSZMAN, PH.D., DARELL D. BIGNER, M . D . , PH.D., LYNN DUDKA, AND SHEILA RICHARDSON
Divisions of Neurosurgery and Department of Pathology, Duke University Medical Center, Durham, North Carolina, and University of Kentucky Medical Center, Lexington, Kentucky ~" Cellular and humoral immune parameters were evaluated in a series of braintumor patients, 42 with glioblastoma multiforme, 17 with other anaplastic gliomas, and 17 with meningiomas. A degree of anergy was found, which seems in the group as a whole to be proportional to the degree of anaplasia. In addition, serial bimonthly testing in individual cases revealed further reduction in certain immune responses coincident with clinical decline. KEY WORDS 9 brain neoplasm delayed hypersensitivity reaction
A
RATIONAL understanding of the immune capabilities of brain-tumor patients and, eventually, the institution of effective immunotherapy should be cultivated from an accurate comprehension of the general immune status of these patients? ,1~ This report describes our joint experience as regards serial, s y s t e m a t i c evaluation of several i m m u n e parameters, cellular and humoral, existing in patients with primary brain tumors at the time of diagnosis and thereafter. Clinical Material and Methods
Patients included in this study were on the neurosurgical services at Duke University J. Neurosurg. / Volume 46 / April, 1977
9 immune response 9 serial immune tests
9 anergy
9
Medical Center, the University of Kentucky Medical Center, the Lexington Veterans Administration Hospital, or Watts Hospital (Durham, N.C.). A total of 42 patients with the diagnosis of glioblastoma multiforme (the most anaplastic of gliomas), 17 with other anaplastic gliomas, and 17 with meningioma participated in this study. The histological slides on all cases underwent a c o m m o n pathological review. Initial studies were carried out at the time the patient was admitted with suspected brain t u m o r or soon after the diagnosis of a primary brain t u m o r was made by surgical resection. Patients with anaplastic gliomas or benign meningiomas were studied serially bimonthly in the postoperative period for as many occasions as 467
M . S. M a h a l e y ,
Jr., et al.
TABLE 1 Skin-test responders*
Skin Tests
Glioblastoma (42 Patients) ~o
SK-SD PPD trichophyton mumps responding to two or more
40 19 20 28
p p p p
Anaplastic Gliomas (17 Patients)
p value
7o
=.0002 = .0121 = .0719 = .0001
47 6 40 57
31 p = .00(02
p p p p
Meningioma (17 Patients)
p value
~o
=.0044 = .0034 = .7944 = .0013
53 24 31 69
50 p = .0019
Controls (20 Patients)
p value p p p p
= = = =
(7o)
.0114 .9020 .4136 .0081
90 50 45 100
59 p = .0077
95
*p values relate each group to the controls, using X 2 analysis of contingency tables. possible. Steroids were not administered during these periods for these patients. Two mechanisms for evaluation of cellular i m m u n e c o m p e t e n c e were used. The preoperative lymphocyte count (cells/cu m m ) of the peripheral blood was determined. Delayed hypersensitivity reactions ( D H R ' s ) at 48 hours following intradermal skin testing with 0.1 ml of each of four antigens were measured either preoperatively or 2 to 3 weeks postoperatively: streptokinase-streptodornase (SK-SD) (1:10 dilution), m u m p s antigen, trichophyton e x t r a c t (1:1000 dilution), and purified protein derivative (PPD). A positive D H R occurred when the diameter of skin induration was equal to or greater than 5 m m . Each of these evaluations was repeated bimonthly. Several studies were performed to assay humoral immune competence. Serum samples were collected preoperatively for immunoglobulin (Ig) IgG, l g M , and IgA levels determined by radial immunodiffusion and for tetanus and influenza antibody (AB) titer d e t e r m i n a t i o n s . Within 3 weeks postoperatively, intramuscular booster doses (0.5 ml) of tetanus toxoid and influenza virus (1200 C C A units) vaccine (U.S.P. bivalent types A and B), were given. These studies were repeated bimonthly, with baseline serum Ig levels and AB titers, followed by repeat booster injections. Controls for cellular i m m u n e evaluations consisted of n o n t u m o r patients of similar age on the neurosurgical services and included 45 peripheral blood lymphocyte counts and skin tests on 20 patients. S e r u m Ig controls for humoral i m m u n e responses were drawn from the hospital service laboratory where these 468
analyses are p e r f o r m e d regularly, but normal (control) values for serum tetanus and influenza antibody titers are not available. Serial, bimonthly data on normal or disease controls for either cellular or h u m o r a l responses using repeated skin tests and tetanus and influenza antigen injections are not available.
Results The results of the D H R ' s performed before or 2 to 3 weeks after surgery are shown in Table 1. These skin-test data are grouped together since the results of evaluations immediately preoperatively and those within 3 weeks after surgery were alike. Steroids were not being administered at the times of these skin tests. These results can be analyzed in terms of the percentage responding to two or more of the test antigens, as shown on the last line of this table. All brain-tumor patients had significantly (p = < .05, as determined by X 2 analysis of contingency tables) fewer responders to two or m o r e D H R ' s when compared to controls, the degree of anergy being directly related to the extent of anaplasia. The bimonthly data on these patients are shown in Table 2 and support in general this trend toward a smaller percentage of responders a m o n g patients with m o r e anaplastic t u m o r types. It is of interest that total removal of t u m o r in the m e n i n g i o m a patients did not cause an i n c r e a s e in the p e r c e n t a g e of responders to a normal level. Table 3 lists the preoperative baseline and serial bimonthly postoperative lymphocyte counts for all patients. The most significant lymphopenia was apparent in patients with
J. Neurosurg. / Volume 46 / April, 1977
Immunobiology of intracranial tumors TABLE 2 Responders to two or more of four skln-test antigens* Postoperative
At
Tumor Type glioblastoma other anaplastie gliomas meningioma
Surgery
2mos
4mos
6 mos
8 mos
31 (42) 50 (16) 59 (17)
41 (32) 60 (10) 43 (7)
25 (24) 29 (7) 80 (5)
35 (17) 33 (6) 40 (5)
40 (10) 40 (5) 75 (4)
10 mos 12 mos 43 (7) 60 (5) 75 (4)
0 (2) 75 (4) 50 (2)
*Numbers of those responding are percentages of the total cases, which are given in parentheses. Normal value is 95 7o.
g l i o b l a s t o m a m u l t i f o r m e , w h e r e a s patients with o t h e r t u m o r types t e n d e d to m a i n t a i n higher a l t h o u g h s u b n o r m a l c o u n t s . Preoperative and serial postoperative t e t a n u s a n d i n f l u e n z a A B titers are shown in T a b l e s 4 a n d 5, respectively. T h e s e A B titers were o b t a i n e d j u s t before the i n i t i a l a n t i g e n i c boost or the s u b s e q u e n t b i m o n t h l y boost. A n e l e v a t i o n o c c u r r e d in A B titers for m o s t p a t i e n t s at 2 to 4 m o n t h s , followed by s o m e w h a t lower titers later. B e y o n d 2 to 4 m o n t h s , p a t i e n t s with g l i o b l a s t o m a t e n d e d to show a progressive decline in i n f l u e n z a A B titers despite repeated b i m o n t h l y boosts.
Tables 6, 7, a n d 8 show p r e o p e r a t i v e a n d serial p o s t o p e r a t i v e d a t a on s e r u m I g G , I g A , and I g M levels, respectively. All a v e r a g e values were w i t h i n a n o r m a l range. H o w e v e r , patients with g l i o b l a s t o m a had the highest baseline values of I g M , with a n initial elevation at 2 m o n t h s followed by a progressive decline into the lower r a n g e of n o r m a l . The d e c l i n i n g n u m b e r s of p a t i e n t s s h o w n with serial t e s t i n g in T a b l e s 2 - 8 r e p r e s e n t deaths of several of the patients with a n a p l a s t i c g l i o m a s ( g l i o b l a s t o m a a n d others), and also p a t i e n t s e n t e r e d into the study who have n o t yet b e e n followed for longer periods.
TABLE 3 Preoperative and serial bimonthly postoperative lymphocyte counts* Tumor
Preop-
Type
erative
Postoperative 2 mos
4 mos
6 mos
glioblastoma range 4 6 8 - 4 0 3 5 408-2220 408-2494 392-1820 mean 1538 • 732 1299 -,- 517 1010 ~ 445 975 • 386 (P = < .05t) n o . cases 40 33 25 18
8 mos
10 mos
646-2025 540-1586 1059 • 553 930 • 431
389-1556 978 ~ 825
10
2
7
o t h e r anaplasficgliomas
450-1995 6 2 0 - 2 1 7 8 440-1760 500-2184 1268-4454 765-2050 2013 ~ 783 1256 ~ 480 1254 a= 496 1226 • 496 1077 • 542 1195 ~= 761 (p = n.s.) 9 7 5 5 no. easesl7 10
range mean
meningioma 980-1890 range 1116-3440 779-2184 mean 2160 • 759 1384 ~- 567 1471 • 448 (p -- n.s.) 5 no. cases 17 7
12 mos
500-1890 1265 a= 573 4
1312-2528 1155-1886 1776 • 464 1445 • 331
1254-1800 1565 • 288
1156-2295 1726 ~- 805
5
4
2
4
*Disease controls range = 429-5014 cells/cu ram; mean = 2181 =~ 1131. tP values relate mean values of each tumor type to controls, using X ~ analysis of contingency tables. J. Neurosurg. / Volume 46 / April, 1977
469
S. Mahaley, Jr.,
M,
et al.
TABLE 4
Serum tetanus antibody titers (serum dilutions) Tumor Type
Preoperative
glioblastoma range (ceUs/cu m m ) geometric m e a n no. cases
Postoperative 2 mos
4 mos
6 mos
8 mos
10 m o s
12 moS
0-19683 27 24
0-531442 582 24
0-531441 1756 17
81-19683 1409 11
81-6561 585 5
other anaplastic gliomas range (cells/cu m m ) geometric m e a n no. cases
0-531441 206 13
9-531441 13698 6
0-531441 1048 6
243-531441 3788 4
729-19683 3788 4
0-177147 1522 3
729 1
meningioma range (cells/cu m m ) geometric m e a n no. cases
0-6561 26 10
3-19683 113 6
3-19683 195 5
27-19683 195 5
243-531441 1756 4
243-6561 585 4
2187 2187 2
243-2187 729 3
6561 1
TABLE 5
Serum influenza antibody titers (serum dilutions) T u m o r Type
Preop-
Postoperative
erative
2 mos
4 mos
6 mos
8 mos
10 m o s
12 m o s
glioblastoma range (cells/cu m m ) geometric m e a n no. cases
10-160 20 26
10-640 78 22
10-640 101 15
10-640 85 12
10-80 50 6
10-80 50 3
20 1
other anaplastic gliomas range (cells/cu m m ) geometric m e a n no. cases
10-160 36 12
10-80 32 6
10-160 36 6
10-40 34 4
40-80 40 3
20-160 57 2
40-80 45 2
meningioma range (cells/cu m m ) geometric m e a n no. cases
10-80 20 9
10-300 57 6
10-320 46 5
20-320 70 5
20-160 95 4
20-80 57 4
40 40 2
TABLE 6
Serum immunoglobulin G measurements (rag 7o)* T u m o r Type
Preoperative
Postoperative 2mos
4mos
6mos
8 mos
10mos
12mos
glioblastoma range 550-1850 mean 925 • 314 no. cases 25
670-1560 1006 • 251 25
250-1275 890 • 270 17
670-1350 924 • 210 11
520-1330 915 • 314 5
770-1220 1190 -~ 401 3
1400 1
other anaplasfic g l i o m a s range 650-1750 mean 1003 ~= 280 no. cases 13
760-2300 1253 =L 546 6
840-1400 1083 • 233 6
880-1480 1122 • 270 4
760-1760 940 ~ 520 4
710-1700 1183 • 496 3
1880 1
meningioma range 440-1325 mean 866 • 250 no. cases 10
680-1410 1120 =~ 289 6
730-1250 1064 ~= 222 5
740-1100 956 • 153 5
1040-1190 1110 • 61 4
1100-1400 1231 • 124 4
970-1470 1220 • 353 2
*Normal range = 600 to 1600 mg~o. 470
J. Neurosurg. / Volume 46 / A p r i l , 1977
Immunobiology of intracranial tumors TABLE 7
Serum immuglobulin A measurements (mg To)* Tumor Type glioblastoma range mean no. eases
Postoperative
Preoperative
2mos
88-1120 439 • 220 25
24-1400 483 • 294 25
4mos
6mos
140-1520 19-1680 410 • 355 451 • 445 17 12
8 mos
10mos
97-540 290-520 351 • 188 380 • 122 5 3
12mos
580 1
other anaplastie gliomas range 275-1360 mean 560 • 295 no. cases 12
137-792 130-880 120-1020 185-830 462 • 193 467 • 204 461 • 275 509 ~ 328 6 4 4 6
84-1020 501 • 388 3
1032 1
meningioma range mean no. eases
137-450 306 • 117 6
145-480 153-480 307 • 120 279 • 128 5 5
305-420 351 • 39 4
280-370 325 • 63 2
120-840 350 ~ 207 10
254-345 312 ~ 40 4
*Normal range = 106 to 668 mgTo.
T h e d e c l i n e in n u m b e r s is not due to p a t i e n t s being lost to f o l l o w - u p a f t e r entry into the study. T h e r e l a t i v e l y b r i e f m e a n survival t i m e for p a t i e n t s w i t h g l i o b l a s t o m a u n d e r t r e a t m e n t (10 m o n t h s ) p r e c l u d e s serial f o l l o w i n g o f the m a j o r i t y o f t h e s e p a t i e n t s for l o n g e r periods. T h e l o w l y m p h o c y t e counts, declining s e r u m i n f l u e n z a A B titers, and d e c l i n i n g serum IgM o b s e r v e d in p a t i e n t s w i t h g l i o b l a s t o m a d u r i n g l a t e r f o l l o w - u p studies were n o t s i m p l y p e r s i s t e n t l y low i m m u n e p a r a m e t e r s in p a t i e n t s w h o lived longer t h a n others, b u t did r e p r e s e n t a c t u a l r e d u c t i o n s in p r e v i o u s l y h i g h e r v a l u e s in i n d i v i d u a l patients. T o i l l u s t r a t e the findings on in-
d i v i d u a l patients, Fig. 1 shows t h e d e c l i n i n g c o u r s e o f a p a t i e n t with a g l i o b l a s t o m a w h o lived t h e usual r e l a t i v e l y b r i e f t i m e f o l l o w i n g s u r g e r y a n d h a d a full c o u r s e o f c o b a l t t h e r a p y (5500 rads to the w h o l e head). F i g u r e s 2, 3, and 4 show the r e l a t i v e l y stable i m m u n e p a r a m e t e r s seen in t h r e e p a t i e n t s who h a d b e n i g n clinical c o u r s e s , o n e with g l i o b l a s t o m a , o n e with a n o t h e r t y p e o f a n a p l a s t i c g l i o m a , and o n e with m e n i n g i o m a . I n a s m u c h as p a t i e n t s with g l i o b l a s t o m a s e e m e d to h a v e t h e lowest D H R r e s p o n s e s and l y m p h o c y t e counts, as well as d e c l i n i n g i n f l u e n z a A B titers and s e r u m I g M levels, it was o f i n t e r e s t w h e t h e r any o n e o f t h e t h r e e
TABLE 8
Serum immunoglobulin M (mgTo)* Tumor Type glioblastoma range median no. eases
Preoperative 25-640 126 • 118 25
other anaplastie gliomas range 28-232 median 106 a= 65 no. eases 13 meningioma range
median no. eases
31-184 96 • 49 10
Postoperative 2 mos
4 mos
20-576 32-580 140 • 109 109 • 126 18 24
6 mos
8 mos
10 mos
31-530 108 • 141 12
34-124 59 • 37 5
44-124 79 • 41 3
69 1
40-57 46 • 9 3
50 1
34-110 68 • 26 6
27-78 62 • 19 6
31-95 70 a= 28 4
40-70 59 • 13 4
61-184 110 a= 47 6
49-200 114 • 55 5
64-244 118 • 76 5
78-135 76-120 104 • 50 93 • 55 4 4
12 mos
76-150 113 • 52 2
*Normal range = 37 t o 154 m g ~ .
J. Neurosurg. / Volume 46 / April, 1977
471
M. S. Mahaley, Jr., et al. TABLE 9
SKIN-TEST RESPONSES 0/4
T
0/4
1/4
Relationship o f therapy to immune responses (means) o f patients with glioblastoma*
0/4
~_ 2 0 0 0 U
u 0
1000
~,
0 1:300
Treatment
-la_
-5.z
:500000
1:2000
/
~ 1:200
i
1:1600 u_
~ 1:100 -
1:1200
Z < I--
0 ~"
~
I00
i
-
1:800
-
1:400
-
0
E
t~ I-I
5O 0 s
J c
I 2
I 4
I 6
u.op~ MOS. POSTOP. G
A L T
GLIOBLASTOMA--DECLINING CLINICAL COURSE
FIG. 1. Graph showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with a glioblastoma who died 8 months postoperatively.
modes of postoperative therapies (cobalt, chemotherapy, or both) influenced these results. Although the numbers of patients with glioblastoma in each treatment category who have been followed for at least 6 months is not great (eight to 10 cases each), Table 9 suggests that declining cell-mediated parameters ( D H R ' s and lymphocyte counts) were most prominent in patients receiving chemotherapy alone, and that these same patients showed an unusual elevation in serum influenza AB titers. The gradual decline in serum IgM levels and influenza AB titers, on the other hand, were accounted for primarily by the patients who received both cobalt and chemotherapy.
Discussion The occurrence of substandard immune capabilities has come to be associated with 472
Preoperativet
Postoperative 2 Mos 4 Mos 6 Mos
skin-test responses (of 4 tests) cobalt 1.4 1.2 chemotherapy 1 1.4 cobalt flchemotherapy .8 1.1 lymphocyte counts cobalt 1650 1261 chemotherapy 1595 1430 cobalt flchemotherapy 1293 1148 serum influenza antibody titers cobalt 1:41 1:151 chemotherapy 1:18 1:153 cobalt dchemotherapy 1:27 1:156 serum IgM cobalt 182 166 chemotherapy 72 110 cobalt dchemotherapy 116 87
1.2 .8
1.2 .3
1.1
1
1097 1007
1256 789
878
926
1 : 2 0 2 1:52 1 : 3 3 0 1:350 1:97
1:60
168 72
147 128
78
51
*The numbers of patients in each treatment group (8 to 10) at this time are not large enough to reveal any statistically significant difference with either Mann-Whitney U-test or X * analysis. ~Some skin tests were done soon postoperatively.
the presence, and usually the active progression, of neoplasia in a host. 15The cause versus effect relationships of immune responses to neoplasia remain to be defined precisely in humans, although the natural temptation is to regard i n a d e q u a t e i m m u n e reactions in cancer patients as an indication of the continued presence of the neoplasm, progressing in its course toward a worsening clinical state. In turn, one might equate adequate immune responses or any improvement in these functions as indicative of a favorable subsequent clinical course. Cell-mediated i m m u n e responses have classically been assayed by D H R ' s to skintest antigens and by blood-lymphocyte quantitation a n d / o r in vitro functional evaluation (that is, blast transformation). The first study of D H R in brain-tumor patients was reported by one of us 6 in 1972 and was expanded in 19747 A significant degree of anergy was J. Neurosurg. / Volume 46 / April, 1977
Immunobiology of intracranial tumors SKIN-TEST RESPONSE S
SKIN-TEST RESPONSES
2/4
T
~
Z
2/4
t
t
2/4
2/4
t
t
2/4
t
~" 2o00
t
:Z
o
1:800 ~_
~ 1:200 ~ 1:100
1:400
~
~S.~..~*A'~.,./~"
........
s ~
§ r
I
l
2
GLIOBLASTOMA
I
I
4 6 8 MOS. POSTOP.
G A L T
--
114
t
114
t
t
1000
o
1:300
..L ~ 1:200
/
1:2000
/
1:1600 :-~
t i
1:1200 u~
!
5O
tt
114
3
100
0
t
~ 1:100
0
H
O -r
i
......""
1/4
U
1000
/' .....,....,..,...'" .............. ...
t
T
2/4
2000
1:300
114
l
10
STABLE C L I N I C A L COURSE
FIG. 2. Graph
0
-
T
100
-
I-4
~
50
N
0
l
i
.9
Z
/
1:800
.:
1:400
1 l TT s C
U.Oo R B'X G. A L T
' ............... " , , 2 4
:E u~
0
I 6
MOS. P O S T O P
, 8
ANAPLASTIC GLIOMA- STABLE CLINICAL COURSE
FIG. 3. Graph
showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with a glioblastoma who is still living after the 10-month follow-up study.
showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with an anaplastic astrocytoma who remained stable neurologically after the 8-month follow-up study.
found preoperatively; it was most profound in patients with the most anaplastic gliomas where the responders to PPD were 5%, to SKSD, 50%, and to trichophyton, 4%. Only 35% of the patients with gliomas could be sensitized to dinitrochlorobenzene (DNCB), in contrast to 100% of controls. Patient's lymphocytes have an attenuated blastogenic response to PHA (phytohemagglutinin). 4'8'19 Lymphopenia in nervous system tumors was first reported by Bill and Morgan in 1970 s in children who succumbed early from neuroblastomas. Subsequent reports by Takakura, e t a l . ? ~ and Young, e t a l . , 23 have mentioned the existence of lymphopenia, reduced DHR's, and diminished lymphocyte blastogenesis with PHA stimulation. Our current report has evaluated DHR's and lymphocyte counts at the time of surgery and serially thereafter in 42 patients with glioblastoma multiforme, 17 with other
anaplastic gliomas, and 17 patients with meningiomas. At the time of surgery (Table 1), in the absence of steroids or other potentially immunosuppressive therapies, DHR's and the percentage of patients responding to two or more skin-test antigens were subnormal, and the magnitude of cellular anergy was proportional to the presence and extent of anaplasia of the three tumor categories studied. Preoperative lymphocyte counts (means) in the absence of steroids were most reduced in the patients with glioblastoma multiforme but were slightly reduced in the other patients as well. This lymphopenia is relevant to a reduction of certain subpopulations of lymphocytes, which is the subject of another report. 7 Serial bimonthly evaluations revealed a persistence of the lowest percent DHR responders and lymphocyte counts in patients with glioblastoma (Tables 2 and 3).
J. Neurosurg. / Volume 46 / April, 1977
473
M. S. M a h a l e y , Jr., et al. 3/4
SKIN-TEST RESPONSES 3/4 3/4 3/4 3/4
3/4
T
~ " 2000 o
1ooo
g o-
o
1:400 -~ Z
.............,.-"" ..............
1:300
o
~
~ 1:200 %,% "%
~ 1:100
%.%
0 ."T" 100 H
50
o
•
.s. u R G.
I 2
I 4
I I 6 8 MOS, POSTOR
I 10
MENINGIOMA-- STABLE CLINICAL COURSE
FIG. 4. Graph showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with a meningioma who received no postoperative adjunctive therapy and who is doing well more than 10 months postoperatively.
Studies of general humoral immunocompetence in brain-tumor patients are almost nonexistent, presumably due to the popular emphasis upon cell-mediated mechanisms of host response to neoplasia. Although reduced serum levels of IgA have been reported in epileptic patients receiving phenyltoin,~ no significant difference was found between our brain-tumor patients receiving phenyltoin (368 + 165 mg%) and those not on phenyltoin (454 4-221 mg%). Tokumaru and Catalano 21 have recently reported elevated IgM serum levels (up to 1700 mg%, mean of 420) in 15 patients with meningiomas, as compared with a range of 40 to 250 mg% (mean 145) for controls. Three of their patients with elevated levels reverted to normal 4 to 7 months after surgery. We have certainly not found any extraordinary abnormality of preoperative serum IgM, IgG, or IgA in any of our brain-tumor patients, including 17 meningioma patients, at the time of initial diagnosis (Tables 6-8), although 474
patients with glioblastoma had higher mean preoperative IgM levels than other patients, with further elevation at 2 months followed by a progressive decline afterward. Serum IgM is the serum Ig with the largest molecular weight and is over 70% intravascular in location. It is a strong binder of complement and may serve a recognition role in many immunologically specific reactions of both B and T lymphocytes,is The alterations in serum IgM concentrations in some patients with glioblastoma is intriguing and warrants further study. As regards serum tetanus and influenza AB titers, most patients showed postimmunization elevation of titers by 2 to 4 months, followed by lower titers later. After 2 to 4 months postoperatively, most patients with glioblastoma demonstrated a progressive decline in the bimonthly baseline influenza AB titers, despite repeated bimonthly immunizations. Several factors may influence the patterns of cellular and humoral immune parameters in patients before and after surgery for brain tumors. First, the histological nature of the brain tumor, as discussed earlier, seems relevant. In any studies of immune competence, it is important to control the use of steroids, as we have tried to do, since suppression of both serum Ig8 and cellular lymphocyte responses have been attributed to steroids. 18 Of particular pertinence is the postoperative therapeutic means (radiotherapy a n d / o r chemotherapy) and whether they, of themselves, are capable of producing immunosuppression? ,~6 In other tumor studies with drugs other than nitrosourea compounds, chemotherapy was not found to be necessarily suppressive for DHR's, and some patients undergoing therapy showed improved DHR's2 ,~~ It is interesting that essentially no studies have addressed themselves specifically to the issue of immunosuppression following either whole-head radiation in adults with brain tumors or administration of nitrosourea chemotherapeutic agents. In our own cases, transient increases in the number of skin-test responses occurred in 13 glioma patients and in lymphocyte counts in nine glioma patients while under treatment with cobalt therapy, chemotherapy, or both. Therefore, these therapies did not seem to preclude improved immune (although transient) responses in patients with brain J. Neurosurg. / Volume 46 / April, 1977
Immunobiology of intracranial tumors malignancies. Similar postoperative improvements in skin-test responses occurred in only two of the 17 meningioma patients treated with surgery only, none of whom showed any significant increases in lymphocyte counts. The data compiled in Table 9 suggest that patients with glioblastoma who received chemotherapy alone postoperatively had the most striking declines in cellular immune parameters, which could reflect either a direct relationship to drug administration or a group of patients who are declining imm u n o l o g i c a l l y because o f less effective t h e r a p e u t i c means. 12,22 W h y these same patients seemed to show more elevated postoperative influenza AB titers is unknown. The combination of cobalt plus chemotherapy seemed to relate to the declining serum IgM levels and influenza AB titers in these patients. The counterpart of immunosuppression would be possible i m m u n e stimulatory postoperative maneuvers. Surgical removal of a significant tumor-mass burden may play such a role, which is difficult to define in our studies partly because the relative amount of tumor removal in patients with gliomas is uncertain. Patients with benign meningiomas, usually totally resected at surgery and not otherwise treated postoperatively, did not show any striking elevation in skin-test responses or lymphocyte counts during serial tests, although their values all along were generally closest to normal. Future studies will c o n c e n t r a t e on a t t e m p t s to effect positively i m m u n e reactions in glioma patients. In a group of patients being treated with Protease I (Brinase), Thornes 2~ has reported one glioma patient who converted skin-test D H R ' s from negative to positive within 1 week, with a duration of conversion of 26 weeks. It is hoped that evaluations of the role of i m m u n o t h e r a p y in glioma models will promote the better understanding of its potential usefulness in treating patients with these malignant lesions. 1~
Summary Some but not all patients with brain tumors have, at the time of surgical diagnosis, a significant degree of anergy of the cellmediated immune system that is proportional to the degree of anaplasia of the neoplasm. During the postoperative period, while unJ. Neurosurg. / Volume 46 / April, 1977
dergoing whole head irradiation a n d / o r systemic chemotherapy ( B C N U or methylC C N U ) , a persistently lower level of cellular immunity was found in most patients with glioblastoma. Patients with glioblastoma have higher mean preoperative serum IgM levels than patients with other brain tumors, but most showed a progressive decline in serum IgM concentrations after surgery. Serum tetanus and influenza AB titers rose after an initial postoperative booster injection, but tended to decline later, with influenza AB titers showing a progressive decline in most patients with glioblastoma despite repeated bimonthly boosting. The relevance of these serial findings to biological progression of intracranial neoplasia may aid in the design of immunotherapy approaches to the problem of malignant brain tumors. Although not all patients with glioblastoma had subnormal general immune competence, many patients with this malignancy presented with low peripheral blood lymphocyte counts ( < 1000 cells/cm mm), elevated serum IgM levels ( > 150 mg%), and reduced numbers of D H R ' s . Serial evaluations frequently revealed declining blood lymphocyte counts, serum IgM levels, and serum influenza AB titers.
Acknowledgments The authors wish to thank the other members of the neurosurgical faculties at Duke University Medical Center and the University of Kentucky Medical Center and at the Durham Clinic for permission to include their patients in this study. Influenza antibody titer determinations were kindly performed by Mrs. Nelda Mold through the courtesy of Dr. Thomas Cate.
References 1. Aarli JA: Drug-induced IgA deficiency in epileptic patients. Arch Neurol 33:296-299, 1976 2. AI-Sarraf M, Sardesai S, Vaitkevicius VK: Clinical immunologic responsiveness in malignant disease. II. In vitro lymphocyte response to phytohemagglutinin and the effect of cytotoxic drugs. Ontology 26:357-368, 1972 3. Bill AH, Morgan A: Evidence for immune reactions to neuroblastoma and future possibilities for investigation. J Pediatr Surg 5:111-116, 1970 4. Brooks WH, Caldwell HD, Mortara RH: Immune responses in patients with gliomas. Snrg Neurol 2:419-423, 1974 475
M. S. Mahaley, Jr., et al. 5. Brooks WH, Netsky MG, Levine JE: Immunity and tumors of the nervous system. Surg Neurol 3:184-186, 1975 6. Brooks WH, Netsky MG, Normansell DE, et al: Depressed cell-mediated immunity in patients with primary intracranial tumors: characterization of a humoral immunosuppressive factor. J Exp Med 136:1631-1647, 1972 7. Brooks WH, Roszman TL, Mahaley MS, et al: Immunobiology of primary intracranial tumors. II. Analysis of lymphocyte subpopulations in patients with primary brain tumors. (In press) 8. Butler WT, Rossen RD: Effects of corticosteroids on immunity in man. II. Alterations in serum protein components after methylprednisolone. Transplant Proc 5: 1215-1219, 1973 9. Chang T-C, Stutzman L, Sokal JE: Correlation of delayed hypersensitivity responses with chemotherapeutic results in advanced Hodgkin's disease. Cancer 36:950-955, 1975 10. Hersh EM, Whitecar JP Jr, McCredie KB, et al: Chemotherapy, immunocompetence, immunosuppression and prognosis in acute leukemia. New Engl J Med 285:1211-1216, 1971 11. Mahaley MS Jr, Gentry RE, Bigner DD: Immunotherapy of primary intracranial tumors. IV. The evaluation of chemotherapy and immunotherapy protocols utilizing the Avian sarcoma virus (ASV) glioma model. Submitted for publication. 12. Mahaley MS Jr, Vogel FS, Burger P, et al: Neuropathological observations on tissues from patients treated by the Brain Tumor Study Group. J Natl Cancer Inst (In press) 13. Marcbalonis J J: Lymphocyte surface immunoglobulins. Science 190:20-29, 1975 14. Mavligit GM, Gutterman JU, Hersh EM: Primary brain tumors: tumor immunity and immunocompetence. Surg Neurol 1:261-263, 1973 15. Morton DL, Holmes EC, Eilber FR, et al: Immunological aspects of neoplasia: a rational basis for immunotherapy. Ann Intern Med 74:587-604, 1971
476
16. Stratton JA, Byfield PE, Byfield JE, et al: A comparison of the acute effects of radiation therapy, including or excluding the thymus, on the lymphocyte subpopulations of cancer patients. J Clin Invest 56:88-97, 1975 17. Takakura K, Miki Y, Kubo O, et al: Immunological treatment of malignant brain tumors. International Congress of Neurological Surgeons, T o k y o , Japan, October, 1973 (Abstract) 18. Tavadia HB, Fleming KA, Hume PD, et al: Circadian rhythmicity of human plasma cortisol and PHA-induced lymphocyte transformation. Clin Exp Immunol 22:190-193, 1975 19. Thomas DG, Lannigan CB, Beban PO: Impaired cell-mediated immunity in human brain tumours. Lancet 1:1389, 1975 (Letter) 20. Thornes RD: Unblocking or activation of the cellular immune mechanism by induced proteolysis in patients with cancer. Lancet 2:382-384, 1974 21. Tokumaru T, Catalano LW Jr: Elevation of serum immunoglobulin M (IgM) level in patients with brain tumors. Surg Neurol 4:17-21, 1975 22. Walker MD, Solowey M, Gehan EA: The treatment of malignant glioma in controlled, prospective and randomized trials. International Congress Series 293. Amsterdam: Excerpta Medica, 1973, p 66 23. Young HF, Sakalas R, Kaplan AM: Inhibition of cell-mediated immunity in patients with brain tumors. Surg Neurol 5:19-23, 1976
This study was supported in part by NCI Contract N01-CM-67010, American Cancer Society Grant IM-92, VA Project 596-1096-01, Public Health Service Research Grants CA-11898 and CA-14651, and 5P01 HD-00668-NICHD, and funds donated by the friends and relatives of patients with brain tumors. Address reprint requests to: M. Stephen Mahaley, Jr., M.D., Division of Neurosurgery, Box 3933, Duke Hospital, Durham, N o r t h Carolina 27710.
J. Neurosurg. / Volume 46 /April, 1977
M . STEPHEN M A H A L E Y , JR., M . D . , PH.D., WILLIAM H. BROOKS, M . D . , THOMAS L. ROSZMAN, PH.D., DARELL D. BIGNER, M . D . , PH.D., LYNN DUDKA, AND SHEILA RICHARDSON
Divisions of Neurosurgery and Department of Pathology, Duke University Medical Center, Durham, North Carolina, and University of Kentucky Medical Center, Lexington, Kentucky ~" Cellular and humoral immune parameters were evaluated in a series of braintumor patients, 42 with glioblastoma multiforme, 17 with other anaplastic gliomas, and 17 with meningiomas. A degree of anergy was found, which seems in the group as a whole to be proportional to the degree of anaplasia. In addition, serial bimonthly testing in individual cases revealed further reduction in certain immune responses coincident with clinical decline. KEY WORDS 9 brain neoplasm delayed hypersensitivity reaction
A
RATIONAL understanding of the immune capabilities of brain-tumor patients and, eventually, the institution of effective immunotherapy should be cultivated from an accurate comprehension of the general immune status of these patients? ,1~ This report describes our joint experience as regards serial, s y s t e m a t i c evaluation of several i m m u n e parameters, cellular and humoral, existing in patients with primary brain tumors at the time of diagnosis and thereafter. Clinical Material and Methods
Patients included in this study were on the neurosurgical services at Duke University J. Neurosurg. / Volume 46 / April, 1977
9 immune response 9 serial immune tests
9 anergy
9
Medical Center, the University of Kentucky Medical Center, the Lexington Veterans Administration Hospital, or Watts Hospital (Durham, N.C.). A total of 42 patients with the diagnosis of glioblastoma multiforme (the most anaplastic of gliomas), 17 with other anaplastic gliomas, and 17 with meningioma participated in this study. The histological slides on all cases underwent a c o m m o n pathological review. Initial studies were carried out at the time the patient was admitted with suspected brain t u m o r or soon after the diagnosis of a primary brain t u m o r was made by surgical resection. Patients with anaplastic gliomas or benign meningiomas were studied serially bimonthly in the postoperative period for as many occasions as 467
M . S. M a h a l e y ,
Jr., et al.
TABLE 1 Skin-test responders*
Skin Tests
Glioblastoma (42 Patients) ~o
SK-SD PPD trichophyton mumps responding to two or more
40 19 20 28
p p p p
Anaplastic Gliomas (17 Patients)
p value
7o
=.0002 = .0121 = .0719 = .0001
47 6 40 57
31 p = .00(02
p p p p
Meningioma (17 Patients)
p value
~o
=.0044 = .0034 = .7944 = .0013
53 24 31 69
50 p = .0019
Controls (20 Patients)
p value p p p p
= = = =
(7o)
.0114 .9020 .4136 .0081
90 50 45 100
59 p = .0077
95
*p values relate each group to the controls, using X 2 analysis of contingency tables. possible. Steroids were not administered during these periods for these patients. Two mechanisms for evaluation of cellular i m m u n e c o m p e t e n c e were used. The preoperative lymphocyte count (cells/cu m m ) of the peripheral blood was determined. Delayed hypersensitivity reactions ( D H R ' s ) at 48 hours following intradermal skin testing with 0.1 ml of each of four antigens were measured either preoperatively or 2 to 3 weeks postoperatively: streptokinase-streptodornase (SK-SD) (1:10 dilution), m u m p s antigen, trichophyton e x t r a c t (1:1000 dilution), and purified protein derivative (PPD). A positive D H R occurred when the diameter of skin induration was equal to or greater than 5 m m . Each of these evaluations was repeated bimonthly. Several studies were performed to assay humoral immune competence. Serum samples were collected preoperatively for immunoglobulin (Ig) IgG, l g M , and IgA levels determined by radial immunodiffusion and for tetanus and influenza antibody (AB) titer d e t e r m i n a t i o n s . Within 3 weeks postoperatively, intramuscular booster doses (0.5 ml) of tetanus toxoid and influenza virus (1200 C C A units) vaccine (U.S.P. bivalent types A and B), were given. These studies were repeated bimonthly, with baseline serum Ig levels and AB titers, followed by repeat booster injections. Controls for cellular i m m u n e evaluations consisted of n o n t u m o r patients of similar age on the neurosurgical services and included 45 peripheral blood lymphocyte counts and skin tests on 20 patients. S e r u m Ig controls for humoral i m m u n e responses were drawn from the hospital service laboratory where these 468
analyses are p e r f o r m e d regularly, but normal (control) values for serum tetanus and influenza antibody titers are not available. Serial, bimonthly data on normal or disease controls for either cellular or h u m o r a l responses using repeated skin tests and tetanus and influenza antigen injections are not available.
Results The results of the D H R ' s performed before or 2 to 3 weeks after surgery are shown in Table 1. These skin-test data are grouped together since the results of evaluations immediately preoperatively and those within 3 weeks after surgery were alike. Steroids were not being administered at the times of these skin tests. These results can be analyzed in terms of the percentage responding to two or more of the test antigens, as shown on the last line of this table. All brain-tumor patients had significantly (p = < .05, as determined by X 2 analysis of contingency tables) fewer responders to two or m o r e D H R ' s when compared to controls, the degree of anergy being directly related to the extent of anaplasia. The bimonthly data on these patients are shown in Table 2 and support in general this trend toward a smaller percentage of responders a m o n g patients with m o r e anaplastic t u m o r types. It is of interest that total removal of t u m o r in the m e n i n g i o m a patients did not cause an i n c r e a s e in the p e r c e n t a g e of responders to a normal level. Table 3 lists the preoperative baseline and serial bimonthly postoperative lymphocyte counts for all patients. The most significant lymphopenia was apparent in patients with
J. Neurosurg. / Volume 46 / April, 1977
Immunobiology of intracranial tumors TABLE 2 Responders to two or more of four skln-test antigens* Postoperative
At
Tumor Type glioblastoma other anaplastie gliomas meningioma
Surgery
2mos
4mos
6 mos
8 mos
31 (42) 50 (16) 59 (17)
41 (32) 60 (10) 43 (7)
25 (24) 29 (7) 80 (5)
35 (17) 33 (6) 40 (5)
40 (10) 40 (5) 75 (4)
10 mos 12 mos 43 (7) 60 (5) 75 (4)
0 (2) 75 (4) 50 (2)
*Numbers of those responding are percentages of the total cases, which are given in parentheses. Normal value is 95 7o.
g l i o b l a s t o m a m u l t i f o r m e , w h e r e a s patients with o t h e r t u m o r types t e n d e d to m a i n t a i n higher a l t h o u g h s u b n o r m a l c o u n t s . Preoperative and serial postoperative t e t a n u s a n d i n f l u e n z a A B titers are shown in T a b l e s 4 a n d 5, respectively. T h e s e A B titers were o b t a i n e d j u s t before the i n i t i a l a n t i g e n i c boost or the s u b s e q u e n t b i m o n t h l y boost. A n e l e v a t i o n o c c u r r e d in A B titers for m o s t p a t i e n t s at 2 to 4 m o n t h s , followed by s o m e w h a t lower titers later. B e y o n d 2 to 4 m o n t h s , p a t i e n t s with g l i o b l a s t o m a t e n d e d to show a progressive decline in i n f l u e n z a A B titers despite repeated b i m o n t h l y boosts.
Tables 6, 7, a n d 8 show p r e o p e r a t i v e a n d serial p o s t o p e r a t i v e d a t a on s e r u m I g G , I g A , and I g M levels, respectively. All a v e r a g e values were w i t h i n a n o r m a l range. H o w e v e r , patients with g l i o b l a s t o m a had the highest baseline values of I g M , with a n initial elevation at 2 m o n t h s followed by a progressive decline into the lower r a n g e of n o r m a l . The d e c l i n i n g n u m b e r s of p a t i e n t s s h o w n with serial t e s t i n g in T a b l e s 2 - 8 r e p r e s e n t deaths of several of the patients with a n a p l a s t i c g l i o m a s ( g l i o b l a s t o m a a n d others), and also p a t i e n t s e n t e r e d into the study who have n o t yet b e e n followed for longer periods.
TABLE 3 Preoperative and serial bimonthly postoperative lymphocyte counts* Tumor
Preop-
Type
erative
Postoperative 2 mos
4 mos
6 mos
glioblastoma range 4 6 8 - 4 0 3 5 408-2220 408-2494 392-1820 mean 1538 • 732 1299 -,- 517 1010 ~ 445 975 • 386 (P = < .05t) n o . cases 40 33 25 18
8 mos
10 mos
646-2025 540-1586 1059 • 553 930 • 431
389-1556 978 ~ 825
10
2
7
o t h e r anaplasficgliomas
450-1995 6 2 0 - 2 1 7 8 440-1760 500-2184 1268-4454 765-2050 2013 ~ 783 1256 ~ 480 1254 a= 496 1226 • 496 1077 • 542 1195 ~= 761 (p = n.s.) 9 7 5 5 no. easesl7 10
range mean
meningioma 980-1890 range 1116-3440 779-2184 mean 2160 • 759 1384 ~- 567 1471 • 448 (p -- n.s.) 5 no. cases 17 7
12 mos
500-1890 1265 a= 573 4
1312-2528 1155-1886 1776 • 464 1445 • 331
1254-1800 1565 • 288
1156-2295 1726 ~- 805
5
4
2
4
*Disease controls range = 429-5014 cells/cu ram; mean = 2181 =~ 1131. tP values relate mean values of each tumor type to controls, using X ~ analysis of contingency tables. J. Neurosurg. / Volume 46 / April, 1977
469
S. Mahaley, Jr.,
M,
et al.
TABLE 4
Serum tetanus antibody titers (serum dilutions) Tumor Type
Preoperative
glioblastoma range (ceUs/cu m m ) geometric m e a n no. cases
Postoperative 2 mos
4 mos
6 mos
8 mos
10 m o s
12 moS
0-19683 27 24
0-531442 582 24
0-531441 1756 17
81-19683 1409 11
81-6561 585 5
other anaplastic gliomas range (cells/cu m m ) geometric m e a n no. cases
0-531441 206 13
9-531441 13698 6
0-531441 1048 6
243-531441 3788 4
729-19683 3788 4
0-177147 1522 3
729 1
meningioma range (cells/cu m m ) geometric m e a n no. cases
0-6561 26 10
3-19683 113 6
3-19683 195 5
27-19683 195 5
243-531441 1756 4
243-6561 585 4
2187 2187 2
243-2187 729 3
6561 1
TABLE 5
Serum influenza antibody titers (serum dilutions) T u m o r Type
Preop-
Postoperative
erative
2 mos
4 mos
6 mos
8 mos
10 m o s
12 m o s
glioblastoma range (cells/cu m m ) geometric m e a n no. cases
10-160 20 26
10-640 78 22
10-640 101 15
10-640 85 12
10-80 50 6
10-80 50 3
20 1
other anaplastic gliomas range (cells/cu m m ) geometric m e a n no. cases
10-160 36 12
10-80 32 6
10-160 36 6
10-40 34 4
40-80 40 3
20-160 57 2
40-80 45 2
meningioma range (cells/cu m m ) geometric m e a n no. cases
10-80 20 9
10-300 57 6
10-320 46 5
20-320 70 5
20-160 95 4
20-80 57 4
40 40 2
TABLE 6
Serum immunoglobulin G measurements (rag 7o)* T u m o r Type
Preoperative
Postoperative 2mos
4mos
6mos
8 mos
10mos
12mos
glioblastoma range 550-1850 mean 925 • 314 no. cases 25
670-1560 1006 • 251 25
250-1275 890 • 270 17
670-1350 924 • 210 11
520-1330 915 • 314 5
770-1220 1190 -~ 401 3
1400 1
other anaplasfic g l i o m a s range 650-1750 mean 1003 ~= 280 no. cases 13
760-2300 1253 =L 546 6
840-1400 1083 • 233 6
880-1480 1122 • 270 4
760-1760 940 ~ 520 4
710-1700 1183 • 496 3
1880 1
meningioma range 440-1325 mean 866 • 250 no. cases 10
680-1410 1120 =~ 289 6
730-1250 1064 ~= 222 5
740-1100 956 • 153 5
1040-1190 1110 • 61 4
1100-1400 1231 • 124 4
970-1470 1220 • 353 2
*Normal range = 600 to 1600 mg~o. 470
J. Neurosurg. / Volume 46 / A p r i l , 1977
Immunobiology of intracranial tumors TABLE 7
Serum immuglobulin A measurements (mg To)* Tumor Type glioblastoma range mean no. eases
Postoperative
Preoperative
2mos
88-1120 439 • 220 25
24-1400 483 • 294 25
4mos
6mos
140-1520 19-1680 410 • 355 451 • 445 17 12
8 mos
10mos
97-540 290-520 351 • 188 380 • 122 5 3
12mos
580 1
other anaplastie gliomas range 275-1360 mean 560 • 295 no. cases 12
137-792 130-880 120-1020 185-830 462 • 193 467 • 204 461 • 275 509 ~ 328 6 4 4 6
84-1020 501 • 388 3
1032 1
meningioma range mean no. eases
137-450 306 • 117 6
145-480 153-480 307 • 120 279 • 128 5 5
305-420 351 • 39 4
280-370 325 • 63 2
120-840 350 ~ 207 10
254-345 312 ~ 40 4
*Normal range = 106 to 668 mgTo.
T h e d e c l i n e in n u m b e r s is not due to p a t i e n t s being lost to f o l l o w - u p a f t e r entry into the study. T h e r e l a t i v e l y b r i e f m e a n survival t i m e for p a t i e n t s w i t h g l i o b l a s t o m a u n d e r t r e a t m e n t (10 m o n t h s ) p r e c l u d e s serial f o l l o w i n g o f the m a j o r i t y o f t h e s e p a t i e n t s for l o n g e r periods. T h e l o w l y m p h o c y t e counts, declining s e r u m i n f l u e n z a A B titers, and d e c l i n i n g serum IgM o b s e r v e d in p a t i e n t s w i t h g l i o b l a s t o m a d u r i n g l a t e r f o l l o w - u p studies were n o t s i m p l y p e r s i s t e n t l y low i m m u n e p a r a m e t e r s in p a t i e n t s w h o lived longer t h a n others, b u t did r e p r e s e n t a c t u a l r e d u c t i o n s in p r e v i o u s l y h i g h e r v a l u e s in i n d i v i d u a l patients. T o i l l u s t r a t e the findings on in-
d i v i d u a l patients, Fig. 1 shows t h e d e c l i n i n g c o u r s e o f a p a t i e n t with a g l i o b l a s t o m a w h o lived t h e usual r e l a t i v e l y b r i e f t i m e f o l l o w i n g s u r g e r y a n d h a d a full c o u r s e o f c o b a l t t h e r a p y (5500 rads to the w h o l e head). F i g u r e s 2, 3, and 4 show the r e l a t i v e l y stable i m m u n e p a r a m e t e r s seen in t h r e e p a t i e n t s who h a d b e n i g n clinical c o u r s e s , o n e with g l i o b l a s t o m a , o n e with a n o t h e r t y p e o f a n a p l a s t i c g l i o m a , and o n e with m e n i n g i o m a . I n a s m u c h as p a t i e n t s with g l i o b l a s t o m a s e e m e d to h a v e t h e lowest D H R r e s p o n s e s and l y m p h o c y t e counts, as well as d e c l i n i n g i n f l u e n z a A B titers and s e r u m I g M levels, it was o f i n t e r e s t w h e t h e r any o n e o f t h e t h r e e
TABLE 8
Serum immunoglobulin M (mgTo)* Tumor Type glioblastoma range median no. eases
Preoperative 25-640 126 • 118 25
other anaplastie gliomas range 28-232 median 106 a= 65 no. eases 13 meningioma range
median no. eases
31-184 96 • 49 10
Postoperative 2 mos
4 mos
20-576 32-580 140 • 109 109 • 126 18 24
6 mos
8 mos
10 mos
31-530 108 • 141 12
34-124 59 • 37 5
44-124 79 • 41 3
69 1
40-57 46 • 9 3
50 1
34-110 68 • 26 6
27-78 62 • 19 6
31-95 70 a= 28 4
40-70 59 • 13 4
61-184 110 a= 47 6
49-200 114 • 55 5
64-244 118 • 76 5
78-135 76-120 104 • 50 93 • 55 4 4
12 mos
76-150 113 • 52 2
*Normal range = 37 t o 154 m g ~ .
J. Neurosurg. / Volume 46 / April, 1977
471
M. S. Mahaley, Jr., et al. TABLE 9
SKIN-TEST RESPONSES 0/4
T
0/4
1/4
Relationship o f therapy to immune responses (means) o f patients with glioblastoma*
0/4
~_ 2 0 0 0 U
u 0
1000
~,
0 1:300
Treatment
-la_
-5.z
:500000
1:2000
/
~ 1:200
i
1:1600 u_
~ 1:100 -
1:1200
Z < I--
0 ~"
~
I00
i
-
1:800
-
1:400
-
0
E
t~ I-I
5O 0 s
J c
I 2
I 4
I 6
u.op~ MOS. POSTOP. G
A L T
GLIOBLASTOMA--DECLINING CLINICAL COURSE
FIG. 1. Graph showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with a glioblastoma who died 8 months postoperatively.
modes of postoperative therapies (cobalt, chemotherapy, or both) influenced these results. Although the numbers of patients with glioblastoma in each treatment category who have been followed for at least 6 months is not great (eight to 10 cases each), Table 9 suggests that declining cell-mediated parameters ( D H R ' s and lymphocyte counts) were most prominent in patients receiving chemotherapy alone, and that these same patients showed an unusual elevation in serum influenza AB titers. The gradual decline in serum IgM levels and influenza AB titers, on the other hand, were accounted for primarily by the patients who received both cobalt and chemotherapy.
Discussion The occurrence of substandard immune capabilities has come to be associated with 472
Preoperativet
Postoperative 2 Mos 4 Mos 6 Mos
skin-test responses (of 4 tests) cobalt 1.4 1.2 chemotherapy 1 1.4 cobalt flchemotherapy .8 1.1 lymphocyte counts cobalt 1650 1261 chemotherapy 1595 1430 cobalt flchemotherapy 1293 1148 serum influenza antibody titers cobalt 1:41 1:151 chemotherapy 1:18 1:153 cobalt dchemotherapy 1:27 1:156 serum IgM cobalt 182 166 chemotherapy 72 110 cobalt dchemotherapy 116 87
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*The numbers of patients in each treatment group (8 to 10) at this time are not large enough to reveal any statistically significant difference with either Mann-Whitney U-test or X * analysis. ~Some skin tests were done soon postoperatively.
the presence, and usually the active progression, of neoplasia in a host. 15The cause versus effect relationships of immune responses to neoplasia remain to be defined precisely in humans, although the natural temptation is to regard i n a d e q u a t e i m m u n e reactions in cancer patients as an indication of the continued presence of the neoplasm, progressing in its course toward a worsening clinical state. In turn, one might equate adequate immune responses or any improvement in these functions as indicative of a favorable subsequent clinical course. Cell-mediated i m m u n e responses have classically been assayed by D H R ' s to skintest antigens and by blood-lymphocyte quantitation a n d / o r in vitro functional evaluation (that is, blast transformation). The first study of D H R in brain-tumor patients was reported by one of us 6 in 1972 and was expanded in 19747 A significant degree of anergy was J. Neurosurg. / Volume 46 / April, 1977
Immunobiology of intracranial tumors SKIN-TEST RESPONSE S
SKIN-TEST RESPONSES
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FIG. 3. Graph
showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with a glioblastoma who is still living after the 10-month follow-up study.
showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with an anaplastic astrocytoma who remained stable neurologically after the 8-month follow-up study.
found preoperatively; it was most profound in patients with the most anaplastic gliomas where the responders to PPD were 5%, to SKSD, 50%, and to trichophyton, 4%. Only 35% of the patients with gliomas could be sensitized to dinitrochlorobenzene (DNCB), in contrast to 100% of controls. Patient's lymphocytes have an attenuated blastogenic response to PHA (phytohemagglutinin). 4'8'19 Lymphopenia in nervous system tumors was first reported by Bill and Morgan in 1970 s in children who succumbed early from neuroblastomas. Subsequent reports by Takakura, e t a l . ? ~ and Young, e t a l . , 23 have mentioned the existence of lymphopenia, reduced DHR's, and diminished lymphocyte blastogenesis with PHA stimulation. Our current report has evaluated DHR's and lymphocyte counts at the time of surgery and serially thereafter in 42 patients with glioblastoma multiforme, 17 with other
anaplastic gliomas, and 17 patients with meningiomas. At the time of surgery (Table 1), in the absence of steroids or other potentially immunosuppressive therapies, DHR's and the percentage of patients responding to two or more skin-test antigens were subnormal, and the magnitude of cellular anergy was proportional to the presence and extent of anaplasia of the three tumor categories studied. Preoperative lymphocyte counts (means) in the absence of steroids were most reduced in the patients with glioblastoma multiforme but were slightly reduced in the other patients as well. This lymphopenia is relevant to a reduction of certain subpopulations of lymphocytes, which is the subject of another report. 7 Serial bimonthly evaluations revealed a persistence of the lowest percent DHR responders and lymphocyte counts in patients with glioblastoma (Tables 2 and 3).
J. Neurosurg. / Volume 46 / April, 1977
473
M. S. M a h a l e y , Jr., et al. 3/4
SKIN-TEST RESPONSES 3/4 3/4 3/4 3/4
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FIG. 4. Graph showing serial lymphocyte counts, serum influenza and tetanus AB titers, and serum IgM levels of a patient with a meningioma who received no postoperative adjunctive therapy and who is doing well more than 10 months postoperatively.
Studies of general humoral immunocompetence in brain-tumor patients are almost nonexistent, presumably due to the popular emphasis upon cell-mediated mechanisms of host response to neoplasia. Although reduced serum levels of IgA have been reported in epileptic patients receiving phenyltoin,~ no significant difference was found between our brain-tumor patients receiving phenyltoin (368 + 165 mg%) and those not on phenyltoin (454 4-221 mg%). Tokumaru and Catalano 21 have recently reported elevated IgM serum levels (up to 1700 mg%, mean of 420) in 15 patients with meningiomas, as compared with a range of 40 to 250 mg% (mean 145) for controls. Three of their patients with elevated levels reverted to normal 4 to 7 months after surgery. We have certainly not found any extraordinary abnormality of preoperative serum IgM, IgG, or IgA in any of our brain-tumor patients, including 17 meningioma patients, at the time of initial diagnosis (Tables 6-8), although 474
patients with glioblastoma had higher mean preoperative IgM levels than other patients, with further elevation at 2 months followed by a progressive decline afterward. Serum IgM is the serum Ig with the largest molecular weight and is over 70% intravascular in location. It is a strong binder of complement and may serve a recognition role in many immunologically specific reactions of both B and T lymphocytes,is The alterations in serum IgM concentrations in some patients with glioblastoma is intriguing and warrants further study. As regards serum tetanus and influenza AB titers, most patients showed postimmunization elevation of titers by 2 to 4 months, followed by lower titers later. After 2 to 4 months postoperatively, most patients with glioblastoma demonstrated a progressive decline in the bimonthly baseline influenza AB titers, despite repeated bimonthly immunizations. Several factors may influence the patterns of cellular and humoral immune parameters in patients before and after surgery for brain tumors. First, the histological nature of the brain tumor, as discussed earlier, seems relevant. In any studies of immune competence, it is important to control the use of steroids, as we have tried to do, since suppression of both serum Ig8 and cellular lymphocyte responses have been attributed to steroids. 18 Of particular pertinence is the postoperative therapeutic means (radiotherapy a n d / o r chemotherapy) and whether they, of themselves, are capable of producing immunosuppression? ,~6 In other tumor studies with drugs other than nitrosourea compounds, chemotherapy was not found to be necessarily suppressive for DHR's, and some patients undergoing therapy showed improved DHR's2 ,~~ It is interesting that essentially no studies have addressed themselves specifically to the issue of immunosuppression following either whole-head radiation in adults with brain tumors or administration of nitrosourea chemotherapeutic agents. In our own cases, transient increases in the number of skin-test responses occurred in 13 glioma patients and in lymphocyte counts in nine glioma patients while under treatment with cobalt therapy, chemotherapy, or both. Therefore, these therapies did not seem to preclude improved immune (although transient) responses in patients with brain J. Neurosurg. / Volume 46 / April, 1977
Immunobiology of intracranial tumors malignancies. Similar postoperative improvements in skin-test responses occurred in only two of the 17 meningioma patients treated with surgery only, none of whom showed any significant increases in lymphocyte counts. The data compiled in Table 9 suggest that patients with glioblastoma who received chemotherapy alone postoperatively had the most striking declines in cellular immune parameters, which could reflect either a direct relationship to drug administration or a group of patients who are declining imm u n o l o g i c a l l y because o f less effective t h e r a p e u t i c means. 12,22 W h y these same patients seemed to show more elevated postoperative influenza AB titers is unknown. The combination of cobalt plus chemotherapy seemed to relate to the declining serum IgM levels and influenza AB titers in these patients. The counterpart of immunosuppression would be possible i m m u n e stimulatory postoperative maneuvers. Surgical removal of a significant tumor-mass burden may play such a role, which is difficult to define in our studies partly because the relative amount of tumor removal in patients with gliomas is uncertain. Patients with benign meningiomas, usually totally resected at surgery and not otherwise treated postoperatively, did not show any striking elevation in skin-test responses or lymphocyte counts during serial tests, although their values all along were generally closest to normal. Future studies will c o n c e n t r a t e on a t t e m p t s to effect positively i m m u n e reactions in glioma patients. In a group of patients being treated with Protease I (Brinase), Thornes 2~ has reported one glioma patient who converted skin-test D H R ' s from negative to positive within 1 week, with a duration of conversion of 26 weeks. It is hoped that evaluations of the role of i m m u n o t h e r a p y in glioma models will promote the better understanding of its potential usefulness in treating patients with these malignant lesions. 1~
Summary Some but not all patients with brain tumors have, at the time of surgical diagnosis, a significant degree of anergy of the cellmediated immune system that is proportional to the degree of anaplasia of the neoplasm. During the postoperative period, while unJ. Neurosurg. / Volume 46 / April, 1977
dergoing whole head irradiation a n d / o r systemic chemotherapy ( B C N U or methylC C N U ) , a persistently lower level of cellular immunity was found in most patients with glioblastoma. Patients with glioblastoma have higher mean preoperative serum IgM levels than patients with other brain tumors, but most showed a progressive decline in serum IgM concentrations after surgery. Serum tetanus and influenza AB titers rose after an initial postoperative booster injection, but tended to decline later, with influenza AB titers showing a progressive decline in most patients with glioblastoma despite repeated bimonthly boosting. The relevance of these serial findings to biological progression of intracranial neoplasia may aid in the design of immunotherapy approaches to the problem of malignant brain tumors. Although not all patients with glioblastoma had subnormal general immune competence, many patients with this malignancy presented with low peripheral blood lymphocyte counts ( < 1000 cells/cm mm), elevated serum IgM levels ( > 150 mg%), and reduced numbers of D H R ' s . Serial evaluations frequently revealed declining blood lymphocyte counts, serum IgM levels, and serum influenza AB titers.
Acknowledgments The authors wish to thank the other members of the neurosurgical faculties at Duke University Medical Center and the University of Kentucky Medical Center and at the Durham Clinic for permission to include their patients in this study. Influenza antibody titer determinations were kindly performed by Mrs. Nelda Mold through the courtesy of Dr. Thomas Cate.
References 1. Aarli JA: Drug-induced IgA deficiency in epileptic patients. Arch Neurol 33:296-299, 1976 2. AI-Sarraf M, Sardesai S, Vaitkevicius VK: Clinical immunologic responsiveness in malignant disease. II. In vitro lymphocyte response to phytohemagglutinin and the effect of cytotoxic drugs. Ontology 26:357-368, 1972 3. Bill AH, Morgan A: Evidence for immune reactions to neuroblastoma and future possibilities for investigation. J Pediatr Surg 5:111-116, 1970 4. Brooks WH, Caldwell HD, Mortara RH: Immune responses in patients with gliomas. Snrg Neurol 2:419-423, 1974 475
M. S. Mahaley, Jr., et al. 5. Brooks WH, Netsky MG, Levine JE: Immunity and tumors of the nervous system. Surg Neurol 3:184-186, 1975 6. Brooks WH, Netsky MG, Normansell DE, et al: Depressed cell-mediated immunity in patients with primary intracranial tumors: characterization of a humoral immunosuppressive factor. J Exp Med 136:1631-1647, 1972 7. Brooks WH, Roszman TL, Mahaley MS, et al: Immunobiology of primary intracranial tumors. II. Analysis of lymphocyte subpopulations in patients with primary brain tumors. (In press) 8. Butler WT, Rossen RD: Effects of corticosteroids on immunity in man. II. Alterations in serum protein components after methylprednisolone. Transplant Proc 5: 1215-1219, 1973 9. Chang T-C, Stutzman L, Sokal JE: Correlation of delayed hypersensitivity responses with chemotherapeutic results in advanced Hodgkin's disease. Cancer 36:950-955, 1975 10. Hersh EM, Whitecar JP Jr, McCredie KB, et al: Chemotherapy, immunocompetence, immunosuppression and prognosis in acute leukemia. New Engl J Med 285:1211-1216, 1971 11. Mahaley MS Jr, Gentry RE, Bigner DD: Immunotherapy of primary intracranial tumors. IV. The evaluation of chemotherapy and immunotherapy protocols utilizing the Avian sarcoma virus (ASV) glioma model. Submitted for publication. 12. Mahaley MS Jr, Vogel FS, Burger P, et al: Neuropathological observations on tissues from patients treated by the Brain Tumor Study Group. J Natl Cancer Inst (In press) 13. Marcbalonis J J: Lymphocyte surface immunoglobulins. Science 190:20-29, 1975 14. Mavligit GM, Gutterman JU, Hersh EM: Primary brain tumors: tumor immunity and immunocompetence. Surg Neurol 1:261-263, 1973 15. Morton DL, Holmes EC, Eilber FR, et al: Immunological aspects of neoplasia: a rational basis for immunotherapy. Ann Intern Med 74:587-604, 1971
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16. Stratton JA, Byfield PE, Byfield JE, et al: A comparison of the acute effects of radiation therapy, including or excluding the thymus, on the lymphocyte subpopulations of cancer patients. J Clin Invest 56:88-97, 1975 17. Takakura K, Miki Y, Kubo O, et al: Immunological treatment of malignant brain tumors. International Congress of Neurological Surgeons, T o k y o , Japan, October, 1973 (Abstract) 18. Tavadia HB, Fleming KA, Hume PD, et al: Circadian rhythmicity of human plasma cortisol and PHA-induced lymphocyte transformation. Clin Exp Immunol 22:190-193, 1975 19. Thomas DG, Lannigan CB, Beban PO: Impaired cell-mediated immunity in human brain tumours. Lancet 1:1389, 1975 (Letter) 20. Thornes RD: Unblocking or activation of the cellular immune mechanism by induced proteolysis in patients with cancer. Lancet 2:382-384, 1974 21. Tokumaru T, Catalano LW Jr: Elevation of serum immunoglobulin M (IgM) level in patients with brain tumors. Surg Neurol 4:17-21, 1975 22. Walker MD, Solowey M, Gehan EA: The treatment of malignant glioma in controlled, prospective and randomized trials. International Congress Series 293. Amsterdam: Excerpta Medica, 1973, p 66 23. Young HF, Sakalas R, Kaplan AM: Inhibition of cell-mediated immunity in patients with brain tumors. Surg Neurol 5:19-23, 1976
This study was supported in part by NCI Contract N01-CM-67010, American Cancer Society Grant IM-92, VA Project 596-1096-01, Public Health Service Research Grants CA-11898 and CA-14651, and 5P01 HD-00668-NICHD, and funds donated by the friends and relatives of patients with brain tumors. Address reprint requests to: M. Stephen Mahaley, Jr., M.D., Division of Neurosurgery, Box 3933, Duke Hospital, Durham, N o r t h Carolina 27710.
J. Neurosurg. / Volume 46 /April, 1977
