The close relationship between growth factors and the nucleolar organizer regions in adenocarcinoma of the lung

EuropeanJournalof SurgicalOncoloyy 1995; 21:398--402

The close relationship between growth factors and the nucleolar organizer regions in adenocarcinoma of the lung Masahiro Tateishi, Satoshi Kaneko, Yasurou Fukuyama, Motoharu Hamatake, Satoshi Kohdono, Tetsuya Mitsudomi, Teruyoshi Ishida and Keizo Sugimachi Department of Surgery II, Faculty of Medicine, Kyushu University, Fukuoka, Japan

We examined immunohistochemically 111 cases of primary adenocarcinoma of the lung, for transforming growth factor at CI'GFat) or epidermal growth factor (EGF), and argyrophilic nucleolar organizer regions (AgNORs). The presence of more than 75% positive cells for both growth factors was designated as a high-GF, while all others were considered to be a Iow-GF. If AgNORs counts were more than 5.00, it was considered to be a high-AgNORs group, while less than 5.00 was designated as a Iow-AgNORs group. In our 111 examined specimens, there were 51 (46%) cases of high-GF, and 64 (58%) with high AgNORs. The 5-year survival rates of the patients with a high-GF and low-GF were 34% and 57% (P<0.05) respectively, while those with high-AgNORs and Iow-AgNORs were 21% and 81% (P<0.001), respectively. In the cases of high-AgNORs, the 5-year survival rates of the patients with high-GF and Iow-GF were 0% and 36% (P < 0.05), respectively. However, in the cases of Iow-AgNORs, the 5-year survival rates of the patients with high-GF and low-GF were 83% and 79%, respectively. These data suggest that growth factors might be related to the biological malignancy of turnouts with a high cell proliferation.

Key words: growth factor; lung cancer; nucleolar organizing regions.

Introduction It is known that the most reliable prognostic parameter in primary lung cancer is TNM staging) However, the pathological TNM staging can only be obtained after surgery. Many investigators have attempted to recognize alternative indicators from biopsy specimens. There have also been studies to identify the various prognostic factors in resected specimens of non-small cell lung cancer. We have previously reported on various prognostic parameters, identified as interstitial materials, 2-4 growth factors, s-7 cell proliferative activities,8-t° or oncogenesJ H'Among these, growth factors are considered to lead to tumour spread or metastasis. 5 Both TGFct and E G F are single polypeptides which activate the tyrosine kinase subunit after binding epidermal growth factor receptor (EGFR). On the other hand, the cell proliferative activity should be reflected in the biological malignancy of cancer cells. For example, AgNORs are loops of D N A which transcribe rRNA.~3 They are argyrophilic, acidic, non-histone proteins, and may be considered a marker of the D N A transcription activity. ~4 In this study, we attempted to clarify the relationship between growth factors and cell proliferative activities in adenocarcinoma of the lung.

Materials and methods

Specimens Tissue specimens examined were obtained at the time of surgery from 111 patients with primary adenocarcinoma of the lung. All patients had been diagnosed and treated in The Department of Surgery II, Faculty of Medicine, Kyushu University, between 1974 and 1986. Any patient who died within 1 month of surgery or who underwent an exploratory thoracotomy were excluded from the present analysis. The stage of the disease was classified according to the TNM classification of UICC. I There were 56 patients with stage I, 11 with stage II, 29 with stage IliA, nine with stage IIIB, and six with stage IV. Of these patients, 70 were men and 41 were women. The patient ages varied from 39 to 81 years (mean 62 years). The WHO classification of differentiation was used; 58 were well differentiated, 38 moderately differentiated, 15 poorly differentiated. For all patients, a curative resection was undertaken which consisted of a lobectomy with a complete hilar and mediastinum lymph node dissection. The.re was no evidence of residual tumour. The patients' records were reviewed and computerized in January 1994.

Immunostaining Correspondence to: Masahiro Tateishi Department of Surgery II, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan. 0748-7983/95/040398+ 05 $08.00/0

The resected specimens were fixed in 10% formalin and paraffin sections were prepared. For histological studies, the sections were stained with haematoxylin and eosin(H & E). © 1995W.B.SaundersCompany Limited

Gron'th factors and nucleolar organizer re.qions h~ lun9 cancer Immunostaining for TGF~ and EGF were carried out as follows. Primary anti-TGF~ goat serum was obtained from BIOTOP, s'6 while the anti-EGF rabbit serum came from Wakunaga Pharmaceutical Co. Ltd. s The avidin-biotin-peroxidase complex method was used. ts The deparaffinized sections were treated with 0.03% hydrogen peroxidase in methanol for 30 min at room temperature to inhibit the endogenous peroxidase. After washing in phosphate-buffered saline(PBS) and incubating with goat serum for TGFc~, and rabbit serum for EGF, each section was incubated at room temperature overnight with the primary antibody of TGF~ at a dilution of 100: I, EGF at a dilution of 50: 1. The sections were then exposed to a biotinylated secondary antibody and avidin with biotinylated horseradish peroxidase (Vector Laboratories). Peroxidase labelling was developed by the diaminobenzidinemethod, and the sections were counter-stained with methyl green. The staining reactions were evaluated as either positive or negative. A positive reaction was only considered when strong brown deposits were visible. Omission of the primary antibody resulted in negative staining. The proportion of positive cells was determined by counting 1000 cancer cells. We grouped the cases as follows: high TGF~ indicated that the proportion of TGF~ positive cells was greater than 75%; low TGFa indicated that the proportion was less than 75%. Similarly, a high EGF indicated that the proportion of EGF positive cells was greater than 75% of the tumour cells; while a low EGF indicated that the proportion was less than 75%.

(A)

(B)

AqNOR stahlhl9 ,m ~

The modified one-step silver colloid method was usedJ' The AgNOR solution was freshly prepared by dissolving gelatin at a concentration of 2g/dl in lg/dl aqueous formic acid to form the first solution. This solution was combined with a 50g/d I aqueous silver nitrate solution ( 1 : 2, v/v) to produce the final AgNOR solution. The AgNOR solution was then immediately poured over the deparaffinized sections, which were left in the dark at room temperature for 40 min. The silver colloid was washed from the sections with distilled deionized water. The evaluation for AgNOR was performed by careful focusing of the AgNORs in the nucleus which appeared as black dots. Using a × 1000 magnification and an oil immersion lens, the AgNORs were counted in 100 tumour cells. The mean number of AgNORs per nucleus was then calculated. We classified the cases into two groups: the high AgNORs group indicated that the number of AgNORs was greater than 5.00; and the low AgNORs group indicated that there were less than 5.00.

399

o

~

4 r

m

(C) Fig. 1. (A) lmmunostaining for TGF~ in adenocarcinoma of the lung. High TGF~ pattern ( x 760). (B) Immunostaining for EGF in adenocarcinoma of the lung. High EGF pattern (x 760). (C) AgNOR staining in adenocarcinoma of the lung. High AgNORs pattern ( x 650).

Statistical analysis The data were analysed by both the Student's t-test and the chi-square test. The survival rate was calculated by the Kaplan-Meier method, ~7 and comparisons with survival rates were made by the generalized Wilcoxon test) s

Results The immunoperoxidase reactivities for TGF~ or EGF were observed in the cytoplasm of the cancer cells (Fig. IA and

B), and AgNORs were evident in the nucleus (Fig. IC). A total of 74(67%) showed a high-TGF~, 56(50%) a highEGF, and 64(58%) a high-AgNORs. We placed the 111 cases into two groups according to the immunoreactivities of the growth factors as follows; a high-GF was considered to be both a high-TGF~ and a high-EGF, while all other cases were regarded as a low-GF. Accordingly, 51(46%) showed a high-GF and 60(54%) demonstrated a low-GF. The data assessed included sex, tumour status, node

400

M. Tateishi et al. Table 1. Relationship among various clinico-pathological factors according to the status

of growth factors or AgNORs in patients with adenocarcinoma of the lung GF

AgNORs

High

Low

High

Low

Male Female

30 21

40 20

35 29

35---a 12-*

1

18 19 8 6

26 26 5 3

19 26 11 8

25---~

2 3 4 0

30

39

25

44 ,

I

6

7

12

1-* *

2

15

14

27

2

0

48

57

58

47---1

1

3

3

6

0-*

I II IliA IIlB IV

22 5 15

34 6 14

14 10 26

6

3

8

3

3

6

42---a 1-** 3-** 1-** 0-**

well moderately poorly

25 21 5

33 17 10

30 25 9

28 13 6

curative non-curative

39 12

49 11

12 29

ll---a 12-**

Total

51

60

64

47

Sex

T 19 I 2-* * 1

N

i, I

M Stage

I

Differentiation

Curability

GF, growth factors. T, tumour status: N, node status; M, metastasis status. * The difference is statistically significant (P < 0.05). ** The differenceis statistically significant (P < 0.01).

status, metastasis status, stage, the pathological grade of differentiation, and the curability of operation according to the immunoreaetivities of G F and AgNORs counts (Table 1). No statistical difference was related to the G F status. However, a statistical significant difference was shown between factors of sex, T, N, M, Stage and Curability according to the AgNORs status. The 5-year survival rates of the patients with high-GF and low-GF were 34% and 57%, respectively, and the difference was statistically significant (P < 0.05). In addition, the 5year survival rates of the patients with high-AgNORs and low-AgNORs were 21% and 81%, respectively, and the difference was statistically significant (P < 0.001). When 111 eases were classified according to the status of G F and AgNORs, there was no statistical difference among the clinico-pathologieal factors (Table 2). In the highAgNORs cases, the 5-year survival rates of the patients with high-GF and low-GF were 0% and 36%, respectively, with a statistically significant difference (P < 0.05), as shown in Fig. 2A. However, in the low-AgNORs cases, the 5-year survival rates of the patients with high-GF and low-GF were 83% and 79%, respectively (Fig. 2B).

Discussion

It is widely accepted that the TNM classification is an independent prognostic factor.~ We previously reported that several prognostic factors exist for non-small cell lung cancer, however, we also noted that there were only a few independent indicators.2-~2 If a factor showed statistical difference in the univariate analysis, then several factors tended to show a relationship with TNM staging. However, TGF~ as a growth factor, and AgNORs as indicators of cell proliferation were shown to be independent prognostic parameters in non-small cell lung cancer. 6"~° TGFct binds to the epidermal growth factor receptor (EGFR) 19 and acts by self-stimulation to be recognized as to the autocrine growth mechanism.2° In addition, growth factors play an important role in cellular proliferation.2~We therefore hypothesize that the autocrine progressive system may be similar to the high proliferative activity of cancer cells. The status of AgNORs is closely related to survival.22We previously reported that the AgNOR count was statistically different among the factors of T, N and stage. 9 It was also

401

Growth factors and nucleolar organizer regions in lung cancer

100,

100 "--T--n ILl

u.I

Low-GF n---31 '--'%

,--I

> so

I

High-GF n=18 ,

it

L_ I

I

(~?, 9(

Low-GF n=29 ,

79~

.--I

X5o.

> tr

36% =:)

n=33

]•

0

I

0

YEARS AFTER OPERATION (A)

1

I

|

I

I

2 3 4 5 YEARS AFTER OPERATION (B)

Fig. 2. (A) Survival curves of the patients with high-AgNORs in adenocarcinoma of the lung according to the GF status. The difference is statistically significant between the two groups (P < 0.05). (B) Survival curves of the patients with Iow-AgNORs in adenocarcinoma of the lung according to the GF status.

shown to be an independent prognostic parameter of nonsmall cell lung cancer by multivariate analysis. ~° In this study, we demonstrate that growth factor status is a statistical prognostic parameter only when A g N O R counts are high. Our data demonstrate that growth factors show a close relationship with A g N O R s , and also possibly with cell Table 2. Relationship among the status of growth factors, AgNORs, and various clinicopathological factors in patients with adenocarcinoma of the lung

GF status

High-AgNORs

Low-AgNORs

High

Low

High

Low

17 16

18 13

13 5

22 7

Sex Male Female 1

8

2 3 4

12 8 5

11 14 3 3

10 7 0 I

15 12 2 0

0

13

12

17

27

I

6

6

0

I

2

14

13

1

1

0

30

28

18

29

1

3

3

0

0

proliferation. Therefore, even if a tumour has a high growth factor activity, it does not necessarily influence the prognosis when cell proliferative activity is low. Accordingly, the autocrine growth system may act under a high activity of cell proliferation. Based on recent experience, adjuvant chemotherapy provides no particular benefit in 'curatively resected' patients. 23 The merit of adjuvant chemotherapy remains questionable. The importance of this prospective study is that prognosis can be determined at the time of operation. Finally, the negative results for adjuvant chemotherapy may indicate a rather pessimistic future for the treatment of lung cancer. However, our investigation suggests some hope for the regulation of lung cancer when the growth factor status can be effectively controlled.

Acknowledgement

We thank Brian T. Quinn for critical comments.

N

M Stage I

5

9

II Ilia IIIB IV

5 15 5 3

5 I1 3 3

17 0 0 1 0

25 1 3 0 0

well moderately poorly

15 16 2

15 9 7

10 5 3

18 8 3

Differentiation

Curability curative non-curative

23 22 10 9 33(30) 31(28)

16 27 2 2 18(16) 29(26)

Total

64(58)

47(42)

Numbers in parentheses, percentages. GF, growth factors. T, tumor status; N, node status; M, metastasis status.

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