Biotherapy 5: 107-112, 1992. O 1992 KtuwerAcademic Publishers. Printedin the Netherlands.

Immunological analysis and clinical effects of intraabdominal and intrapleural injection of lentinan for malignant ascites and pleural effusion Masaaki Oka, Shigefumi Yoshino, Shoichi Hazama, Kouji Shimoda & Takashi Suzuki The Second Department of Surgery, Yamaguchi University School of Medicine, Kogushi 1144, Ube City, Yamaguchi 755, Japan Received 9 November 199I; accepted 25 November 1991

Key words:

lentinan, malignant effusion, intracavitary injection

Abstract

Twenty effusions in sixteen patients with malignant peritoneal and/or pleural effusions were treated with intracavitary injection of lentinan. Lentinan was injected at a dosage of 4 rag/week for 4 weeks. In total, sixteen (80%) of twenty lesions demonstrated clinical responses. Performance status was improved in seven patients. The average survival time in responders was 129 days, while, in non-responders, it was 49 days. Serious toxidties were not observed. NK activity of PBMC significantly decreased after lentinan injection. NK activity of PEC in responders was augmented significantly. Anti-Daudi and lymphokine activated killer activity were also augmented or maintained after lentinan injection.

Introduction

Patients with end-stage cancer occasionally develop malignant ascites or pleural effusion which seriously impairs performance status. Therefore, successful management of these malignant effusions is required for avoiding such a detrimental effect on performance status. Recently, various Biological Response Modifiers (BRM) such as OK-432[1], interleukin-2 (IL-2)[2] and Corynebacterium parvum [3] were administered intraperitoneally or intrapleuraUy for the treatment of malignant effusion. However articles reported a high incidence of toxicity such as high fever, chill, gastrointestinal tract symptoms and abdominal pain. Lentinan is a beta [1-3] glucan extract from edible mushrooms, Lentinus edodes [4]. This non-toxic polysaccharide [5] has been found to augment immune responses to a variety of antigens and to increase host resistance against murine and human tumors [6]. Jeannin et al. reported that intraperitoneal injection of len-

tinan significantly increased the life span of carcinomatous rats and inhibited the growth of carcinomatoses [7]. The clinical efficacy of lentinan has been evaluated in patients with advanced or recurrent colorectal cancer in a randomized controlled trial [8]. However, effects of intracavitary injection of lentinan of malignant ascites or pleural effusion have not been reported yet. The aim of this study is to investigate the effect of intraabdominal or intrapleural injection of lentinan on malignant effusion and to evaluate changes in lymphocyte subsets and functions of peripheral blood mononuclear cells and peritoneal or pleural exudate cells.

Patients and method

Patients From 1988 to 1990, sixteen patients (Table 1) and their twenty lesions (nine malignant ascites,

108

Table t. Effect of instillations of lentinan in patients with malignant ascites or pteural effusion. case No

age

sex

primary lesion

effusion site

resection of primary lesion

disappearance of effusion

cytology pre post

PS pre

post

ascites ascites ascites ascites ascites pleural pleural pleural pleural ascites pleural ascites pleural pleural ascites pleural pleural ascites pleural pleural

+ + + + + + +

NC NC NC DE NC DA NC NC NC NC NC DA NC NC NC DA DA DA DA DE

V V V III V II V II V V III III V V V III IV V IV V

4 3 3 3 2 4 1 4 4

4 3 3 2 3 3 1 4 3

4

3

IM

1 4

3 4

2M 2M

3 4

2 3

2M 3M

4 2

2 1

4M 18M

1

36

F

2 3 4 5 6 7 8 9

76 53 65 66 68 56 68 82

F M M M F M M M

gastric ca. gastric ca. gastric ca. gastric ca. gastric ca. gastnc ca. gastric ca. gastric ca. gastric ca.

10

70

F

gastric ca.

11 12

72 64

M M

pancreatic ca. pancreatic ca.

13 14

53 45

M M

esophageal ca. anal ca.

15 16

77 78

M M

unknown unknown

+ + + + + -

V IV I IV V II IV I V IV II II V IV IV I IV I I III

survival 3M 6M 1M 9M 15 days 17 days 3M 2M 1M

DA: disappearance of effusion, DE: decrease of effusion, NC: no change, PS: performance status. Cytology was judged by Papanicolaou stain. e l e v e n p l e u r a l effusions) w e r e t r e a t e d with int r a p e r i t o n e a l o r i n t r a p l e u r a l i n j e c t i o n o f lent i n a n . I n f o r m e d c o n s e n t was o b t a i n e d . P r e t r e a t m e n t P a p a n i c o l a o u s m e a r r e v e a l e d V c y t o l o g y in t w e l v e l e s i o n s , I V in t w o , I I I in f o u r , II in two. F o u r o f s i x t e e n p a t i e n t s d i d n o t u n d e r g o resect i o n o f t h e i r p r i m a r y lesions w h i c h w e r e n o t d e t e c t e d in two o f t h e m . E l e v e n p a t i e n t s h a d l i v e r a n d / o r lung m e t a s t a s e s . F o u r p a t i e n t s with b o t h ascites a n d p l e u r a l effusion r e c e i v e d lent i n a n i n s t i l l a t i o n s to e a c h lesion. T h e s e p a t i e n t s did not receive diuretics and albumin agents but w e r e t r e a t e d with i n t r a v e n o u s h y p e r - a l i m e n t a tion. P e r f o r m a n c e status was also e v a l u a t e d acc o r d i n g to t h e c r i t e r i a o f W H O [9].

Protocol of lentinan injection Intraabdominal and/or intrapleural injection of l e n t i n a n (4 m g ) was d o n e w e e k l y for 4 weeks. I m m u n o l o g i c a l a n d cytological e x a m i n a t i o n s w e r e d o n e on the d a y p r i o r to the first i n j e c t i o n a n d o n e , t w o a n d f o u r w e e k s after the i n j e c t i o n . T e g a f u l ( 4 0 0 - 6 0 0 m g / d a y , p e r os) was also a d m i n i s t e r e d in t w e l v e p a t i e n t s with p r i m a r y lesions [2] a n d / o r o t h e r m e t a s t a t i c lesions [11]. E v e n if t h e effusion d i s a p p e a r e d , l e n t i n a n was i n j e c t e d in all p a t i e n t s , h o w e v e r , i m m u n o l o g i c a l

examinations were not done after the disappeara n c e o f effusion.

Criteria of response D i s a p p e a r a n c e o f ascites o r d e c r e a s e in a b d o m i nal girth b y m o r e t h a n 5 c m o r r e d u c t i o n in t h e size o f t h e p l e u r a l effusion a n d i m p r o v e m e n t o f c y t o l o g y class w e r e c o n s i d e r e d to be positive responses.

Isolation of mononuclear cells from peripheral blood or peritoneal and pleural effusion P e r i p h e r a l b l o o d m o n o n u c l e a r cells ( P B M C ) were isolated from heparinized venous blood, d i l u t e d 1 : 2 o r 3 in H B S S ( S i g m a , St. L o u i s , M O ) o r s a l i n e , a f t e r c e n t r i f u g a t i o n on a Hist o p a q u e ( S i g m a ) g r a d i e n t f o r 30 m i n u t e s at 4 0 0 g . T h e i n t e r p h a s e cells w e r e c o l l e c t e d , w a s h e d two t i m e s in R P M I - 1 6 4 0 ( S i g m a ) , a n d c o u n t e d using t r y p a n b l u e to assess viability. Peritoneal or pleural exudate mononuclear cells ( P E C ) w e r e i s o l a t e d as d e s c r i b e d [10]. I n b r i e f , s p e c i m e n s o f p e r i t o n e a l o r p l e u r a l effusions ( 5 0 - 1 0 0 cc) w e r e o b t a i n e d a n d i m m e d i a t e l y c e n t r i f u g e d for 5 m i n u t e s at 400 g. T h e cell p e l l e t was t h e n w a s h e d , a n d the cells w e r e a d j u s t e d to a c o n c e n t r a t i o n o f 1 × 106/ml in c o m p l e t e

109 medium. The cell suspension was layered on discontinuous gradients consisting of 10ml of 100% and 75% Ficoll-Hypaque (Sigma) in 50-ml plastic tubes and then centrifuged at 400 g for 30 minutes. Lymphocyte-rich mononuctear cells were collected from the 100% interphase, tumor cells and mesothelial cells from the 75% interphase, and erythrocytes, polymorphonuclear cells, and aggregated tumor cells from the bottom of the tube.

bation was performed for four hours at 37°C in a CO2-incubator. 51Cr release in each well was determined using a gamma-counter (Auto Well Gamma System ARC-202, Aloka, Japan). The percentage of cytotoxicity was calculated as follows:

% cytotoxicity = experimentalrelease- spontaneousrelease maximumrelease - spontaneousrelease × 100

Lymphocyte subsets Peripheral blood and peritoneal or pleural effusion were collected into tubes containing heparin. Specimens were reacted with saturating amounts of monoclonal antibodies for 30 minutes at 4°C. Single marker analysis was performed using the monoclonal antibodies (Ortho Diagnostics, Raritan, NJ), CD4 (helper/inducer T cell) and CD8 (suppressor/cytotoxic T cell). Double marker analysis was performed using the combination of the monoclonal antibodies (Becton, Dickinson Mountain View, CA) such as CD8 + C D l l - (cytotoxic T cell) and CD8 + C D l l - (suppressor T cell). Samples were analyzed by flow cytometry. These results were expressed as percentages.

Target cells without effector cells were mixed with 0.1 ml of the culture medium to obtain spontaneous release and with 0.1 ml of 0.1N hydrochloric acid to obtain maximum 51Cr release. When the maximum release was less than 1000 cpm or the spontaneous release from target cells exceeded 15% of maximum release, the data were excluded from the analysis.

Statistical analysis Statistical analysis was performed by Student's t test for paired mean and Fisher's exact method. A p value of less than 0.05 was considered to be significant. Results were given as the mean -+2 standard errors (S.E.).

Lymphokine-activated killer (LAK) cells For preparation of LAK cells, PBMC and PEC at a concentration of 1 × 106/ml were cultured for 4 days in complete medium containing 1 U/ ml of recombinant interleukin-2 (IL-2).

Results

Clinical response and prognosis Cytotoxic assay The cytotoxicity of PBMC, PEC and LAK were determined in a standard 4 hour 51Cr-release assy. The target cells consisted of K-562 cells for NK activity and Daudi cells for anti-Daudi and LAK activity. Target cells (1 × 106/ml) were labeled for 60 minutes at 37°C using 100/zCi/ml of radioactive sodium chromate (51Cr) (Amersham Japan, Tokyo) and washed four times in RPMI-1640. Labeled cells were resuspended in culture medium at a concentration of 1 x 106/ml. The effector cells were then suspended at 4 x 106/ml, and 0.1ml of effector cells were added to a microplate (Falcon) with 0.1 ml of target cells, to make the effector to target ratio 40 : 1. All tests were done in triplicate. The incu-

The effusion disappeared in six lesions (30%) and diminished in two (10%) (Table 1). In eight lesions in which the effusion did not change, there was an improvement in the cytologic class. In all sixteen lesions (80%) demonstrated clinical responses. The period from the first administration of lentinan to the disappearance of the effusion ranged from two to 28 days (mean 11.3 days). Pretreatment performance status (PS) was 1 in two patients, 2 in two, 3 in four, and 4 in eight. Therefore, baseline PS in 12 of 16 patients (75%) was greater than or equal to 3. After lentinan injection, an improvement in PS was observed in seven patients, while there was no change in seven and progression in two.

110 I00[,~

100

100

~ 8o

8O

~ 6O

_~

g. 4o

o 4o

6o

Q 2O 0 responder

0 w

I~

1

,

o

(mean: 129days) ,

,

~ so[

L

Fig. 1. Survival time of responders and non-responders. Average of survival time in responders was 129 days, while, in non-responders was 49 days. There was no statistical significant difference.

Changes of lymphocyte subsets of PBMC and PEC The percentages of CD4+ PBMC prior t o the lentinan injection and at weeks 1, 2, and 4 did not show any statistical difference (Figure 2). The percentages of CD4+ PEC, CD8+ PEC, CD8 + C D l l - and CD8 + C D l l + at each time point did not show any statistically significant variations (data not shown) and 19.0 -+ 10.3. The 10o

100

g 80

~ 8c

= 60

~ 6c

+ go ~t 121 2O

+ 40

0

0

1

2

4W

+

4W

/

~ 2o

0

1

2

4W

3o 2o

+

m

a o

to

~) 10

o' 1

2

4W

8o

Fig. 3. Change of lymphocyte subsets of peritoneal or pleural exudate mononuclear cells. There were no differences in the percentages of CD4+, CD8+, C D 8 + C D 1 1 - and CD8 + CD11+ cells during lentinan therapy.

percentages of CD8 + CD11+ cells were 12.04.0, 14.1 - 9.1, 11.2 _+4.5, and 10.4 - 4.7. These results were not statistically different (Figure 3).

Change of cytotoxicity of PBMC and PEC The NK activity of PBMC significantly decreased at 4 weeks compared with that prior to therapy ( p < 0 . 0 5 ) (Figure 4-a). In contrast, the NK activity of PEC in responders was augmented significantly after lentinan injection (p,~ > 0

•

•

Z

Non4~eSlDonders

Resp0nders

Patients Fig. 5. Comparison of pretherapeutic NK activity of peripheral blood mononuclear cells between responders and non-responders. NK activity in responders was significantly higher than that of non-responders (p = 0.045).

tients). The NK activities in seven of the nine responders were greater than 50%, while that in all non-responders were less than 50% ( p = 0.045) (Figure 5). Anti-Daudi activity of PEC was very low in 10 of 12 patients prior therapy and definitely increased in four patients after lentinan injection (Figure 6-a). LAK activity, measured in eight patients, was relatively high before therapy in all but one and was maintained during this therapy (Figure 6-b). Toxicity of lentinan injection There were no toxicities such as gastrointestinal symptoms or abdominal or thoracic pain, and (a)

(b)

(D I00 W O_

200

"5 8o

160

"a ~" so

120

40

•->

80