Expression of Lewis-related antigen and prognosis in stage I non—small cell lung cancer

Expression of Lewis-Related Antigen and Prognosis in Stage I Non-Small Cell Lung Cancer Jun-ichi Ogawa, MD, Akemi Sano, Hiroshi Inoue, MD, and Shirosaku Koide, MD First Department of Surgery,, School of Medicine, Tokai University, Kanagawa, Japan

I m m u n o h i s t o c h e m i c a l e x p r e s s i o n of L e w i s y, sialyl Lewis x, and sialyl Lewis a were examined in relation to blood vessel invasion and prognosis in 133 patients with stage I n o n - s m a l l cell lung cancer w h o had a curative resection from 1980 to 1991. Expression of sialyl Lewis Xin adenocarcinomas was higher than in squamous cell and large cell carcinomas, and Lewis y immunoreactivity was the highest among the three antigens. The frequency of blood vessel invasion was significantly higher in tumors with expression of Lewis y or sialyl Lewis antigen (sialyl Lewis × or sialyl Lewisa), however, Lewis y expression was

even more significant. The postoperative survival was significantly shorter w h e n tumors expressed both the Lewis y and sialyl Lewis antigen. However, the survival of patients with either Lewis y or sialyl Lewis antigen expression was similar to that of patients w h o s e tumors did not express either the Lewis y or sialyl Lewis antigens. These results suggest that Lewis y and sialyl Lewis antigen may be of prognostic value for metastatic potential but have different functional roles in tumor cells.

b o u t 30% of patients with stage I lung cancer who u n d e r g o curative resection die of cancer due to m i n u t e metastatic foci, which p r o b a b l y are a l r e a d y p r e s e n t at the time of operation [1]. Although minute metastatic foci cannot be detected b y any currently available diagnostic or microscopic techniques, the ability to predict the likelihood of metastasis from the resected specimens could p r e s e n t an i m p o r t a n t contribution to t r e a t m e n t planning. There are several reports that the expression of Lewis-related c a r b o h y d r a t e antigens is associated with various cancers including the lung [2-4]; however, their functional roles for metastatic potential have not been fully established. W e therefore have exa m i n e d retrospectively the i m m u n o h i s t o c h e m i c a l expression of Lewis-related antigens in relation to prognosis in patients with stage I n o n - s m a l l cell lung cancer who h a d a curative resection.

sialyl Lewis ~ (CA19-9; Centocor, Malvern, PA) antigens was p e r f o r m e d on f o m a l d e h y d e - f i x e d tissue sections. Each diluted a n t i b o d y solution (BM-1, 1:100; FH6, 1:10; CA19-9, 1:1) was overlaid on thin-sliced sections a n d i n c u b a t e d at r o o m t e m p e r a t u r e overnight. Subsequently, the sections were i n c u b a t e d with a biotinylated sheep a n t i - m o u s e i m m u n o g l o b u l i n solution a n d then covered with a streptavidin-biotinyl peroxidase complex (LSAB kit; DAKO, Carpinteria, CA). The sections then were stained with 0.02% 3,3'-diaminobenzidine a n d 0.06% sod i u m azide (Wako Pure C h e m i c a l Osaka, Japan) a n d counterstained with 0.3% methyl green. I m m u n o h i s t o chemical staining was e x a m i n e d in at least 1000 cells over five h i g h - p o w e r fields (magnification, × 1,000). The m e a n percentage of the cells with Lewis y (LeY), sialyl Lewis x (SLe×), a n d sialyl Lewis ~ (SLe a) staining was 56%, 32%, a n d 24%, respectively. Of these antigens, positive incidence of Le y staining was significantly higher than SLe x a n d SLe a. Thus, the g r a d i n g of the Le y staining was defined as follows: (+), 50% or more of the cells h a d m e m b r a n e o u s staining; ( - ) , fewer than 50% h a d staining. The grading of the SLe × and SLe a staining were as follows: (+), 30% or m o r e h a d m e m b r a n e o u s staining; ( - ) , fewer than 30% h a d staining. Negative controls were done by omitting the p r i m a r y antibodies b u t using the same m e t h o d m e n t i o n e d earlier. The pathologic findings were r e v i e w e d separately by two pathologists who h a d no k n o w l e d g e of the clinical outcome, a n d the results were taken into account only if the two pathlogists r e a c h e d consensus. Blood vessel invasion (BVI) was exa m i n e d by victoria b l u e - h e m a t o x y l i n a n d eosin staining as previously r e p o r t e d [6] and their frequencies were evaluated by the X2 test. Survivals were calculated by the K a p l a n - M e i e r method, a n d statistical evaluation was done b y the log-rank test.

A

Patients and Methods One h u n d r e d thirty-three consecutive patients with stage I n o n - s m a l l cell lung cancer according to the TNM classification [5] who u n d e r w e n t a curative t u m o r resection a n d l y m p h node dissection from 1980 to 1991 were included in this study. The surgical protocols were the same for all the patients a n d none h a d received any adjuvant therapy. A streptavidin-biotinyl peroxidase complex m e t h o d using antibodies against Lewis y (BM-1; Japan I m m u n o r e search Lab, Takasaki, Japan), sialyl Lewis x (FH6; prov i d e d b y Otsuka Pharmaceutical, Tokushima, Japan), and Accepted for publication Sep 30, 1994. Address reprint requests to Dr Ogawa, First Department of Surge~,, School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa, 259-11, Japan. © 1995 by The Society of Thoracic Surgeons

(Ann Thorac Surg 1995;59:412-5)

0003-4975[95/$9.50 0003-4975(94)00866-6

Ann Thorac Surg 1995;59:412-5

OGAWAET AL LEWISANTIGENEXPRESSIONIN LUNGCANCER

Table 1. Patient Profile No. of Patients

Characteristic Total Age (y) Range Median Histology Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Tumor stage T1 NO T2 NO Blood vessel invasion ( ) (+) Recurrence Distant metastasis Lung Pleura Brain Bone Liver Skin Died of cancer

133 33-83 65 74 52 7 73 60 75 58 41 34 13 7 6 4 3 1 31

413

each SLe×(+), SLea(+), a n d LeY(+) tumors had significantly shorter survival than those with SLeK(-), SLea(-), a n d LeY(-) tumors (p < 0.02, p < 0.01, a n d p < 0.01, respectively). Because the expression of SLe x a n d SLe ~ are both significant factors for predicting the survival, and, as will be discussed in the c o m m e n t section, these antigens are likely to have similar function as adhesion molecules, probably due to the same structures with sialic acid conjugated in the teminal epitope, SLe×(+) or SLea(+) tumors are hereafter defined as sialyl Lewis antigen (SLe)(+) tumors.

Frequencies of Blood Vessel Invasion by Lewis y and Sialyl Lewis Antigen Expression For a distant metastasis to form, BVI is thought to be i m p o r t a n t as the first step. The relationship b e t w e e n BVI a n d the expression of Le y a n d SLe is shown in Table 3. The frequency of BVI was significantly higher in tumors expressing Le y as well as in tumors expressing SLe. However, the difference b e t w e e n the LeY(+) a n d LeY(-) tumors was even more significant than the difference b e t w e e n SLe(+) a n d S L e ( - ) tumors (p ( 0.01 a n d p ( 0.05, respectively).

Survival by Lewis y and Sialyl Lewis Antigen Expression

The age, histologic types, t u m o r stage, BVI, a n d recurrence in the patient population are s u m m a r i z e d in Table 1. Of the 41 patients with recurrent tumors, 34 (83%) had blood-borne metastases.

T h e 133 p a t i e n t s w e r e d i v i d e d i n t o t h o s e w i t h LeY(-)SLe(-) tumors (n = 33), LeY(-)SLe(+) tumors (n = 16), LeY(+)SLe(-) tumors (n = 35), a n d LeY(+)SLe(+) tumors (n = 49), a n d the postoperative survivals were calculated (Fig 2). Patients with LeY(+ )SLe(+) tumors had a significantly shorter survival than those with LeY(-)SLe(-) tumors (p < 0.01), LeY(-)SLe(+) tumors (p < 0.02), or LeY(+)SLe(-) tumors (p ( 0 . 0 5 ) . However, the survival of patients with LeY(-)SLe(+), LeY(+)SLe(-) a n d LeY(-)SLe(-) tumors were not significantly different.

Expression of Sialyl Lewis x Sialyl Lewis a, and Lewis y by Histologic Types

Comment

Results

Profiles of Patient Population

Sialyl Lewis × expression in adenocarcinomas was significantly higher than in s q u a m o u s cell carcinomas (p < 0.01). Expression of SLe x, SLe a a n d Le y in large-cell carcinomas were similar to those of s q u a m o u s cell carcinomas. The positive frequency of Le y antigen was highest a m o n g the three antigens (Table 2).

Survival by Expression of Sialyl Lewis x Sialyl Lewis ~ and Lewis y Postoperative survival curves according to the SLe ×, SLe a, a n d Le y expression are shown in Figure 1. Patients with

Our present study demonstrates that the expression of Le y, SLe ×, a n d SLe a may be of prognostic value for predicting survival. As shown in Table 4, Le y, SLe x, and SLe a have similar structures with sialic acid or fucose conjugated to their terminal epitope. Although these antigens accumulate in t u m o r tissues of patients with carcinoma [2-4], their functional roles have r e m a i n e d largely u n k n o w n . Recently, both SLe x a n d SLe a have b e e n shown to be ligands for the cell adhesion molecule called ELAM-1 (E-selectin, endothelial leukocyte adhesion molecule-I) [7-10], which is present on cytokine-

Table 2. Expression of Sialyl Lewis~, Sialyl Lewisa and Lewisy by Histologic Types Sialyl Lewis×

Sialyl Lewis"

Lewisy

Histology

N

(-)

(+)

(-)

(+)

(-)

(+)

Adenocarcinoma Squamous cell Large cell

74 52 7

36 47 6

38 (51%)~ 5 (13%)" 1 (14%)

37 32 5

37 (50%) 20 (38%) 2 (29%)

20 26 3

54 (73%) 26 (50%) 4 (57%)

ap < 0.01, comparisonof (+) with (-).

414

OGAWAET AL LEWISANTIGENEXPRESSIONIN LUNGCANCER

100

~

Ann Thorac Surg 1995;59:412-5

100

100

"N.,

•

"~". . . . . . "I - - .

".-......

P
t....-1 ........

P
. . . . ,. . . . . .

P
,

L..I •. . . . . . ] r.,o GI

SLex (--)(n=89)

rj

2

4

Patients at risk 89 84 ......44 36

0

69 27

Ley (--)(n--49) ...... Ley (+)(n=84)

SLea (--)(n=74) ...... SLea (+)(n=59)

...... SLex (+)(n=44)

6 Year~

0

2

4

6

8'

0

40 17 74 70 59 32 15 49 14 10 ...... 59 50 37 22 12 ...... 84 Fig 1. Survival by sialyl Lewis ~ (SLex), sialyl Lewis" (SLea), and Lewis y (Ley) expression.

activated vascular endothelial cells. Thus, SLe × a n d SLe ~ may contribute to the adhesion of t u m o r cells to the vascular beds a n d promote h e m a t o g e n o u s metastasis [11]. In addition, others have reported that cells expressing Le y repel each other [12], a n d may be correlated with cell motility [13]. Thus, Le y may influence the invasive potential of t u m o r cells and, consequently, prognosis [14]. To assess the contribution of these Lewis-related antigens, we selected patients with stage I n o n - s m a l l cell lung cancer and studied their immunohistochemical expression in relation to prognosis. Sialyl Lewis × immunoreactivity was more prevalent in the adenocarcinomas than in the s q u a m o u s a n d the large-cell carcinomas. In the developing lung of h u m a n embryos, SLe × appears in the terminal b u d cells for the future alveoli, not in the cells destined to produce the larger bronchi [15]. Adenocarcinoma mainly develops in the peripheral regions of the lung, a n d this may account for its higher frequency of SLe × expression. Lewis y immunoreactivity was higher than SLe x or SLe ~, which may be consistent with the findings that Le y antigen is observed from the most i m m a t u r e l u n g buds cells through

½

,~

6

47 73

39 57

26 28

11 16

the relatively mature cells appearing at a m u c h later stage [15]. For a distant metastasis to form, the following steps are thought to be important: individual t u m o r cells first detach from the primary lesion and then invade the blood vessels. Next, they attach to the vascular beds in the target organs after migration in the blood. They then penetrate the capillary walls a n d finally proliferate in the extracellular matrix. In fact, patients with BVI(+) tumors (n = 58) had a significantly shorter survival than those with BVI(-) tumors (n -- 75) (p < 0.01). In addition, we have reported that the disease-free survival of patients with BVI and SLe × expression was significantly worse [6]. In the present study, the BVI frequencies were significantly higher w h e n tumors expressed Le y or SLe a n d the survival was significantly shorter w h e n tumors expressed

10080-

!

~. ..... ~-.-.-,.xx,

"

: ..... ....... 1 ............... -I

:

60-

Table 3. Frequencies o f Blood Vessel I n v a s i o n by Lewis y a n d Sialyl Lewis A n t i g e n Expression ~

Blood Vessel Invasion Antigen Lewisy ( ) (+ ) Sialyl Lewis antigen ( ) (+) Sialyl Lewis antigen comparison w i t h ( ).

( )

(+)

49 84

38 37

11 (22%)b 47 (56%)b

68 65

46 29

22 (32%)~ 36 (55%)~ b p <

Ley(--)SLe(--)(n=33)

20"

N

= s i a l y l L e w i s x a n d s i a l y l L e w i s a. ~ p < 0.05, comparison w i t h ( ).

"~ 4 0 -

0.01,

Patien~ atrisk 33 •~16 -~ 35 ..... 49

.....

Ley()SLe(+)(n=16)

......

Ley(+)SLe(--)(n=35)

.......

Ley(+)SLe(+)(n=49)

Year 31 16 33 40

26 13 26 31

17 9 I3 15

7 4 6 10

Fig 2. Survival by Lewis y (Ley) and sialyl Lewis (SLe) antigen expression. (p < 0.01, LeY(-)SLe(-) versus LeY(+)SLe(+); p < 0.02, LeY(-)SLe(+) versus LeY(+)SLe(+); p < 0.05, LeY(+)SLe(-) versus Le~ ( + ) SLe( + ).)

Ann Thorac Surg

OGAWA ET AL LEWIS ANTIGEN EXPRESSION IN LUNG CANCER

1995;59:412-5

Table 4. Carbohydrate Structures of Sialyl Lewis ~, Sialyl Lewis a and Lewis y Antigen Sialyl Lewis × Sialyl Lewis ~ Lewis y

Structure Sia c~ 2--~3Gal/3 l ~ 4 [ F u c cr 1-+3]GlcNAc /3 1---~3R Sia c~ 2-~3Gal/3 1--~3[Fuc cr 1-+4]GlcNAc /3 1--~3R Fuc c~ 1--*2Gal 13 1--~[Fuc a 1--*3]GlcNAc /3 1--~3R

Fuc - fucose; Ga! = galactose; GIcNAc ~ N-acetylglucosamine, R ~ sugar residue; Sia = sialic acid.

b o t h Le y a n d SLe. H o w e v e r , Le y e x p r e s s i o n w a s a m o r e s i g n i f i c a n t f a c t o r for BVI t h a n SLe. M o r e o v e r , t h e s u r v i v a l of p a t i e n t s w h o s e t u m o r s e x p r e s s e d e i t h e r Le y or SLe w a s as l o n g as t h a t of t h e p a t i e n t s w h o s e t u m o r s d i d n o t e x p r e s s e i t h e r a n t i g e n , a l t h o u g h Le y a n d SLe w e r e i n d i v i d u a l l y s i g n i f i c a n t factors for t h e s u r v i v a l . T h e s e r e s u l t s s u g g e s t t h a t Le y a n d SLe h a v e a d i f f e r e n t f u n c t i o n a l role for d i s t a n t m e t a s t a s i s . B e c a u s e SLe is a l i g a n d for t h e E - s e l e c t i n r e c e p t o r , a n d Le y m a y b e i n v o l v e d w i t h cell motility, it is likely t h a t SLe p l a y s s o m e role i n t h e a d h e s i o n of c a n c e r cells to t h e v a s c u l a r b e d s , w h e r e a s Le y is i n v o l v e d in i n v a s i v e p o t e n t i a l , n a m e l y , b l o o d v e s s e l i n v a s i o n a n d t r a n s m i g r a t i o n f r o m t h e site of v a s c u l a r a r r e s t to t h e e x t r a c e l l u l a r m a t r i x . In c o n c l u s i o n , t h e e x p r e s s i o n of Le y SLe *, a n d SLe a a n t i g e n s m a y b e of p r o g n o s t i c v a l u e for p r e d i c t i n g p r o g n o s i s i n p a t i e n t s w i t h s t a g e I n o n - s m a l l cell l u n g c a n c e r , b u t h a v e d i f f e r e n t f u n c t i o n a l roles for m e t a s t a t i c p o t e n tial, p o s s i b l y in d e f i n i n g a d h e s i o n a n d t h e m o t i l i t y of c a n c e r cells.

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