and its prognostic value in non-small cell lung cancer
Ayşe YILMAZ1, İsmail SAVAŞ1, Serpil DİZBAY SAK2, Adem GÜNGÖR3, Akın KAYA1, Ebru SERİNSÖZ2, Berna SAVAŞ2
1 Ankara Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları Anabilim Dalı,
2 Ankara Üniversitesi Tıp Fakültesi, Patoloji Anabilim Dalı,
3 Ankara Üniversitesi Tıp Fakültesi, Göğüs Cerrahisi Anabilim Dalı, Ankara.
ÖZET
Küçük hücreli dışı akciğer kanserinde Bcl-2 gen ekspresyonunun dağılımı ve prognostik değeri
Bu çalışmada, küçük hücreli dışı akciğer kanseri tanısı almış hastaların parafin bloklarında Bcl-2 dağılımının saptanması, Bcl-2 ile tümör evresi arasındaki ilişkinin saptanması, Bcl-2 ekspresyonunun prognostik önemini ve yaşam süresiyle ilişki- sini ortaya koymak amaçlanmıştır. Bu çalışmada, Aralık 1996-Kasım 1999 tarihleri arasında kliniğimizde küçük hücreli dışı akciğer kanseri (KHDAK) tanısı alan 16 hasta ile Ekim 1996-Mayıs 1998 tarihleri arasında fakültemiz Göğüs Cerrahisi Kliniği’nde KHDAK tanısı almış 30 hasta olmak üzere toplam 46 hasta retrospektif olarak değerlendirilmiştir. Kliniğimizde takip edilen 16 (%34.8) hastanın tanısı fiberoptik bronkoskopla bronş mukoza biyopsisi ile, göğüs cerrahisinde takip edi- len 30 (%65.2) hastanın tanısı ise cerrahi rezeksiyon (lobektomi, pnömonektomi) materyallerinde konmuştur. Bu çalışma- da, 46 adet olgunun arşivinde mevcut olan %10’luk formalin fiksasyonu ve rutin doku takibi ile hazırlanmış parafin blok- larından elde edilen 4-6 µkalınlıktaki hemotoksilen-eozin (HE) ile boyanmış kesitleri kullanılmıştır. 4-6 µkalınlıktaki kesit- ler, polilizin ile kaplanmış lamlara alınmıştır. Kesitlere rutin deparafinizasyon işlemi uygulanmıştır. Tüm olgulara Bcl-2 an- tikorları ile immünhistokimyasal boyama yapılmıştır. Çalışmamızda 46 hastadan 9 (%19.6)’una ait olan kesitte pozitif bo- yanma gözlerken, 37 (%80.4)’sinde hiç boyanma gözlemedik. Yassı hücreli tümörlü 32 (%25) olgunun sekizinde boyanma gözlenirken, 24 (%75)’ünde boyanma olmamıştır. Hücre tipi adenoma olan 11 hastanın ve hücre tipi adenoskuamöz olan iki hastanın hiçbirinde (%100) boyanma olmamıştır. Büyük hücreli bir hastanın kesitinde ise pozitif boyanma (%100) sap- tanmıştır.
Anahtar Kelimeler:Bcl-2 geni, KHDAK.
Yazışma Adresi (Address for Correspondence):
Dr. Ayşe YILMAZ, Ankara Üniversitesi Tıp Fakültesi, Göğüs Hastalıkları Anabilim Dalı, Dikimevi, ANKARA - TURKEY
Lung cancer is the leading cause of death in both male and female cancers in United State of America (USA) (1). It also accounts for 28% of all deaths from cancer and 6% of all deaths (2).
In spite of sophisticated diagnostic procedures, mean five year survival after diagnosis is 10- 15%. Therefore, new methods are required for earlier diagnosis and treatment and prevention of its development. In the last decade, signifi- cant advances have been made in the unders- tanding of the molecular biology of lung cancer, leading to the earlier recognition of the disease at the molecular level and development of new treatments.
Apoptosis is one of the most important mecha- nism in the cell biology. Tumor cells, unlike nor- mal cells, have the ability to avoid programmed cell death (that is, apoptosis), which is a physi- ological response in cases of DNA damage or cellular injury. The most important components of normal apoptosis pathway are Bcl-2 protoon- cogen and products of p53 tumor suppressor gene. Bcl-2 protooncogene is a membrane pro-
tein increasing in the chromosomal 14; 18 trans- location, but not specific, discovered in B-cell malignities and is present predominantly in the cell, endoplasmic reticulum and mitochond- ria(3). The Bcl-2 gene prevents cells from apop- tosis (4). The expression of the Bcl-2 protein is normally found lymphoid cells and in several epithelial tissue (5). In addition, it is found lymphoproliferative disorders and in non-hema- topoietic malignancies incorporating breast car- cinoma, adenocarcinoma of the prostat and gastrointestinal tract, nasopharangeal carcino- ma, neuroblastoma and non-small cell lung can- cer (NSCLC) (6,7).
Investigations have demonstrated that Bcl-2 protein expression is more marked in small cell lung cancer than in NSCLC. In immunohistoche- mical investigations, Bcl-2 protein was expres- sed in 75-95% of small cell cancers (8). This is in contradiction with the susceptibility of small cell lung cancer, which is known to be much higher than that of NSCLC, to chemotherapy, which usually develops through apoptosis. In addition, SUMMARY
Distribution of Bcl-2 gene expression and its prognostic value in non-small cell lung cancer
Yilmaz A, Savas I, Dizbay Sak S, Gungor A, Kaya A, Serinsoz E, Savas B
Department of Chest Diseases, Faculty of Medicine, Ankara University, Ankara, Turkey.
The aim of this study was to determine the expression of Bcl-2 gene and prognostic importance of Bcl-2 expression in pa- raffin embedded blocks of patients diagnosed with non-small cell lung cancer (NSCLC). This study included the retrospec- tive analysis of overall 46 patients diagnosed with NSCLC in our clinic between 1996 and 1999. In 16 (34.8%) patients, the diagnosis was made on biopsy of bronchial mucosa and in 30 (65.2%) patients, on materials obtained by surgical resecti- on (lobectomy and pneumonectomy). We reviewed the sections 4-6 µin size and stained with Hemotoxylin-Eosine (HE) ob- tained from paraffin embedded blocks and fixed by 10% formalin. These sections are transferred on to slides covered with poly-L-lysine, then deparaffinization was made. In all cases, immunohistochemical staining with Bcl-2 antibodies was per- formed. Positive staining was observed in 9 (19.6%) patients, but not in 37 (80.4%) patients. Out of 32 cases with squamo- us cell tumor, 8 (25%) were observed to have positive staining in their sections, but 24 (75%) were not so. No staining was observed in 11 cases whose cell type was adenoma and two cases whose cell type was adenosquamos (100%). Staining was present in the section of one case with large cell (100%). Median survival time was 36.6 months in cases in which sta- ining was observed and 6.10 months in cases in which staining was not observed, with a significant difference (p< 0.05).
In conclusion, the rate of survival was higher in cases in which staining was present.
Key Words:Bcl-2 gene, NSCLC.
Bcl-2 positive NSCLC patients have been obser- ved to have higher survival rates (9).
The expression of Bcl-2 is much higher in squ- amous cell carcinoma (25%) than in adenocarci- noma (12%) (10). As Bcl-2 positive NSCLC show neuroendocrine markers, the expression of Bcl-2 may be related to neuroendocrine differen- tiation. In immunohistochemical analysis of re- sected NSCLC, Bcl-2 expression was found to be inversely proportional to the abnormal p53 exp- ression. These findings suggest the hypothesis that the increase in both Bcl-2 expression and the p53 mutation is not adequate to modify the apoptotic path in NSCLC. Bcl-2 positive tumors may exhibit less aggressive behavior (9).
Bax is a Bcl-2 related protein and it acts as a tu- mor suppressor gene (11). It is thought that Bax is step of p53 pathway. According to this model, the ratio of Bcl-2/Bax determine the apoptotic susceptibility of cells.
Therefore, the objective of this study was to de- termine the prognostic significance of Bcl-2 exp- ression encompassing 46 patients with NSCLC.
MATERIALS and METHODS Study Subjects
In the present study, it was attempted to deter- mine the relation of Bcl-2 protooncogen with tu- mor stage, and survival and its distribution in paraffin blocks of patients diagnosed with NSCLC, the prognostic significance of its exp- ression.
Staging procedure included physical and blood examination, chest radiography, and tomog- raphy of the chest, abdomen, and brain, bron- coscopy, abdominal ultrasonography or tomog- raphy, bone scanning and cranial tomography.
Thirty patients underwent pathological resection following surgical resection. Staging was carried out in accordance with the changes made by American Joint Committee on Cancer and Uni- on Internationale Contre Ie Cancer (12,13).
In Table 1, the characteristics of the patients inc- luded in the study are illustrated.
In the present study, sections of 46 cases that were stained with 4-6 µthick Hemotoxylin-Eosi-
ne obtained from paraffin blocks prepared with routine tissue follow up and 10% formalin fixati- on present in Pathology Department of our Uni- versity. Of 46 cases, 30 were surgical resection specimens and 16 small biopsy materials. Sec- tions at the thickness of 4-6 µ were placed on slides covered with poly-L-lysine. Routine depa- raffinization procedure was carried out on the sections. All cases were stained immunohistoc- hemically (85-9043 in 1/50 dilution, San Fran- cisco, California, USA). Lymph node sections showing lymphoma infiltration where this anti- body is positive were used as positive controls.
Table 1. The characteristics of the patients.
n %
Mean age (range) 55 (34-76)
Mean follow up (month) 23
Male 44 95.7
Female 2 4.3
Positive smoking history 43 93.5 Negative smoking history 2 4.3
Unknown smoking history 1 2.1
Histological subtypes
Squamous 32 69.6
Adeno 11 23.9
Adenosquamous 2 4.3
Large cell 1 2.1
Stage I 13 28.3
Stage II 3 6.5
Stage IIIA 13 28.3
Stage IIIB 9 19.6
Stage IV 8 17.4
Early stage (I, II, IIIA) 29 63 Advanced stage (IIIB, IV) 17 37
Liver alone -
Bone alone 1
Brain alone 3
Adrenal alone -
Liver and bone 2
Brain and surrenal 1
Skin, liver, renal ve surrenal 1
Died 31 67.4
Survived 15 32.6
As negative control, PBS was used instead of primary antibody and other steps were followed in the same way.
In tumor cells, brown-red cytoplasmic staining material was regarded as positive. Sections we- re evaluated as in Table 2 semiquantitatively.
Statistical Evaluation
In the analysis of survival according to factors, Kaplan-Meier analysis was used and Log-Rank test in comparisons. Chi-square analysis was employed in associations with fatality. In the analysis of the relation with staining, Fisher’s Exact test was used. Multivariate analysis was not possible as there were few cases.
In statistical evaluation, p< 0.05 was considered significant.
RESULTS
We observed positive Bcl-2 staining in sections of 9 (19.6%) patients. Staining was more than 10% (+2) in three patients and less than 10%
(+1) in six patients. No staining was observed in 37 (80.4%) patients. Sections that were stained positively are illustrated in Picture 1,2.
The association of staining with survival is shown in Table 3.
In the present study, it was determined that po- sitive staining was directly proportional to survi-
val rates, with a statistically significant differen- ce (p< 0.05). Mean survival time was 36.6 months in cases that were positively stained while it was 6.1 months in cases that were not positive. The relation between staining and sur- vival is shown in Graphic 1.
Of 16 patients diagnosed with biopsy, staining was found in 2 (12.5%) and was not present in
Table 2. Semiquantitative evaluation tissue secti- ons with lung cancer.
No staining in tumor cells 0
Staining less than 10% in tumor cells +1 Staining more than 10% in tumor cells +2
Table 3. The relation between Bcl-2 staining and survival in the tumor tissue of lung cancer patients.
Survivors Dying
Bcl-2 n % n % Total
Negative staining 9 24.3 28 75.7 37
Positive staining 6 66.7 3 33.3 9
Overall 15 32.6 31 67.4 46
Picture 1. Cytoplasmic marked staining in tumor tis- sue in squamous cell lung carcinoma case (x40).
Picture 2. Cytoplasmic marked staining in tumor tis- sue in squamous cell lung carcinoma case (x160).
14 (87.5%). As to 30 patients diagnosed with surgical resection, positive staining was obser- ved in 7 (23.3%), while no staining was seen in 23 (76.7%). No statistically significant differen- ce was found between patients diagnosed with biopsy and those diagnosed with surgical resec- tion in terms of staining (p> 0.05).
Of 32 squamous cell tumors, 8 (25%) were sta- ined positively while 24 (75%) did not. No sta- ining was observed in any of 11 patients with cell type adenoma and two patients with cell type adenosquamous tumors. In the present study, there was one patient with large cell lung cancer and his section was positively stained.
No statistically significant relation was found between cell type and positive staining (p>
0.05). Likewise, no significant relation was seen between cell type and survival (p> 0.05).
When the patients with squamous cell cancer were evaluated between themselves, median survival was found to be 34.95 months in those stained with Bcl-2 and 20.45 months in those that did not do so. Five years survival was found to be 62% in those stained and 17% in those that did not do so. In cases staining with Bcl-2 in squamous cell cancer, survival was found to be statistically longer compered to those that did not stain with Bcl-2 (p< 0.05).
While no staining was observed in 22 of 29 ca- ses in early stage (75.9%), positive staining was
seen in 7 (24.2%) cases. Of 17 cases at advan- ced stage, sections of 15 were not stained posi- tively, (88.2%), only two were stained positively (11.8%). No statistically significant relation was found between being at an early or advanced stage and positive staining (p> 0.05).
Of 38 cases without metastasis, sections of eight were stained positively, (21.1%), while no sta- ining was observed in the sections of 30 (78.9%). Of eight patients with metastasis, only 1 (12.5%) was found to be stained positively. No statistically significant relation between metas- tasis and positive staining was found.
Overall median survival time was 20.4 months.
The probability of 12 months survival time was 58.7% and that of 24 months 41.3%. Median survivals of patients diagnosed with biopsy and surgical resection were calculated separately. It was found to be 4.6 months in the former and 36.0 months in the latter. Survival period was shorter (p< 0.001) and survival rates were lo- wer (p< 0.001) in those diagnosed with biopsy, with a statistically significant difference.
In early stage tumors, median survival time 37.6 months and it was 5.8 months in late stage tu- mors. In advanced stages, survival period was shorter (p< 0.001) and survival rates were lower (p< 0.001), with a statistically significant diffe- rence.
Survival functions 1.0
0.8
0.6
0.4
0.2
0.0
Cum survival
Staining
Month
0 10 20 30 40 50 60
1.00
1.00-censored 0.00
0.00-censored
Graphic 1. The association between survival with the Bcl-2 staining in the tumor tissue of lung cancer patients.
Median survival time was 26.0 months in cases without metastasis and 6.1 months in those with metastasis. In cases with metastasis, survival period was shorter (p< 0.01) and survival rates were lower (p< 0.01), with a statistically signifi- cant difference.
DISCUSSION
In the present study, of 46 patients, staining was observed in the sections of 9 (19.6%) whilst no staining was observed in 37 (80.4%). Mean sur- vival of the patients whose sections were stained was 36.6 months while that of patients with no staining was 6.1 months, with a statistically sig- nificant difference (p< 0.05).
Pezzella et al have found, in their study including 122 patients that, paraffin blocks of 80 patients with squamous cell lung cancer, 20 (25%) were stained positive and of 42 adenocarcinoma pati- ents, 5 (12%) were stained positive and have stated that Bcl-2 expression may be of prognos- tic significance (10). Congruent with the fin- dings of the above study, we found Bcl-2 gene expression to be 25% in squamous cell lung cancers, yet our series had merely 11 patients with adenocarcinoma and none was stained po- sitive.
Anton and colleagues have published in 1997 a study including 427 cases, 252 of which had adenocarcinoma, 111 squamous cell and 64 lar- ge cell cancer. Immunopathological staining was scored in this study. However, in this large seri- es no relation was found between positive im- mune staining of Bcl-2 protooncogene and sur- vival and it was concluded that Bcl-2 staining had no prognostic value (14). We also scored positive immune staining, and discrepant with the findings of the above study, found that sta- ining and survival rates had statistically signifi- cant relation.
Another study by Pastorino et al, published in 1997, included 515 cases, 252 of whom had sta- ge I squamous cell carcinoma, 217 adenocarci- noma and 46 other cell types. Mean follow up period was 102 months. In the same sections, A blood group, laminin receptors, c-erbB1/epider- mal growth factor receptor (EGRF), c-erbB2/
neu, p53 and angiogenesis markers were inves-
tigated in addition to Bcl-2. None of the immuno- histochemical markers have been demonstrated to be a prognostic factor for survival. In this study, 83 of the cases (16%) were stained positi- ve with Bcl-2 and Bcl-2 positive tumors were shown to have better prognosis for recurrence (p= 0.03) (15).
In the investigation of Apolinario et al, published in 1997, p53, Bcl-2 and Bax markers were inves- tigated in 116 cases. 116 patients that had under- gone surgical resection and who were between stages I-III were evaluated. Bax oncogene was examined merely in 61 cases at stage I. Positive staining was also scored [negative (0), low (+), medium (++), high (+++)]. It has been determi- ned in the above study that Bcl-2 was expressed in higher degrees in squamous cell carcinoma (p< 0.001) and Bax expression was more mar- ked in males (p= 0.006) and adenocarcinoma (p= 0.0013). In addition, in stage I patients Bcl-2 expression was found to be associated with lon- ger survival (p= 0.0169). Prognosis was found to be worst in the group in which p53(+)/Bcl-2(-) was expressed (p= 0.034). In the evaluation of another combined group, prognosis was found to be more unfavorable in the patients with Bax(+)/Bcl-2(-) (p= 0.02) (16). In our study, we found that 25% of squamous cell tumors were sta- ined positive. In none of the sections of 11 patients with adenocarcinoma, could we find staining.
Brambilla et al have demonstrated that there was a inverse relation between immunohistoc- hemical staining of Bax and Bcl-2 in 121 neuro- endocrine lung cancer cases (29 large cell, 16 typical carcinoid, 71 small cell lung cancer and 5 atypical). They have shown that typical and atypical carcinoids express Bcl-2 at a low level and Bax at a high level and that it is just the contrary in many small cell lung cancer and lar- ge cell neuroendocrine tumors (17). This is con- sistent with the fact that carcinoids tend to ma- ke local invasion without metastasis whereas small cell cancers have usually display a more aggressive and metastatic clinical behavior. In the present study, there was only one large cell case. He was a forty seven year old patient at stage IIIA at presentation. Cytoplasmic focal sta- ining was present.
In another study by Laudansky et al published in 2001, 102 NSCLC cases were investigated for the association between clinicopathologic parame- ters and p53-M (mutations of p53 gene), p53Abs (antibodies to p53) and Bcl-2-Pe (protein ove- rexpression) and no significant realtion was fo- und. It was only establihed that the sole, indepen- dent unfavorable prognostic factor for survival was positive p53-m results and combination of p53 abnormalities with negative staining with Bcl- 2 reduced survival significantly (18-20).
In conclusion, Bcl-2 seems to be an important oncogene for prognosis in lung cancers. Eluci- dation of the molecular biological bases in lung cancer will help to the prevention and manage- ment of the disease.
REFERENCES
1. Newcomb PA, Carbone PP. The health consequences of smoking. Med Clin North Am 1992; 76: 305-31.
2. Beckett WS. Epidemiology and etiology of lung cancer.
Clin Chest Med 1993; 1-15.
3. Pezzella F, Tse AGD, Cordell JL, et al. Expression of the Bcl-2 oncogene protein is not specific for the 14; 18 chro- mosomal translocation. Am J Pathol 1990; 137: 225-32.
4. Hockenbery D, Nunez G, Milliman C, et al. Bcl-2 is an in- ner mitochondrial membrane protein that blocks prog- rammed cell death. Nature 1990; 348: 334-6.
5. Hockenbery DM, Zutter M, Hickey W, et al. Bcl-2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci USA 1991; 88:
6961-5.
6. Pezzella F, Gatter K. What is the value of Bcl-2 protein de- tection for histopathologists? Histopathology 1995; 26:
89-93.
7. Fleming MV, Guinee DG, et al. Correlates of Bcl-2 immu- nohistochemical staining in a surgical series of lung can- cer patients. Mod Pathol 1995; 8: 148A.
8. Kaiser U, Schilli M, et al. Expression of the Bcl-2 protein in small cell lung cancer. Lung Cancer 1996; 15: 31.
9. Fontanini G, Vignati S, Bigini D, et al. Bcl-2 protein: A prognostic factor inversely cor related to p53 in non- small cell lung carcer. Br J Cancer 1995; 71: 1003.
10. Pezzella F, Turley H, Kuzu I, et al. Bcl-2 protein in non- small cell lung carcinoma. N Engl J Med 1993; 329: 690-4.
11. Yin C, Knudson CM, Korsmeyer SJ, Van Dyke T. Bax suppresses tumorigenesis and stimulates apoptosis in vi- vo. Nature 1997; 385: 637.
12. Mountain CF. Revisions in the international system for staging lung cancer. Chest 1997; 111: 1710-7.
13. Lababede O, Moulay AM, Rice TW. TNM staging of lung cancer. A quick reference chart. Chest 1999; 115: 233-5.
14. Anton RC, Brown RW, Younes M, et al. Absence of prog- nostic significance of Bcl-2 immunopositivity in non- small cell lung cancer: Analysis of 427 cases. Hum Pat- hol 1997; 28: 1079-82.
15. Pastorino U, Andreola S, Tagliabue E, et al. Immunocy- tochemical markers in stage I lung cancer: Relevance to prognosis. J Clin Oncol 1997; 15: 2858-65.
16. Apolinario RM, Valk Paul, Jong JS, et al. Prognostic va- lue of the expression of p53, Bcl-2 and bax oncoprotein S and neovascularization in patients with radically resec- ted non-small-cell lung cancer. J Clin Oncol 1997; 15:
2456-66.
17. Brambilla E, Negoescu A, Ganzeri S, et al. Apoptosis-re- lated factors p53, Bcl-2 and Bax in neuroendocrine lung tumors. Am J Pathol 1996; 149: 1941-52.
18. Laudanski J, Niklinska W, Burzykowski T, et al. Prog- nostic significance of p53 and Bcl-2 abnormalities in ope- rable nonsmall cell lung cancer. Eur Respir J 2001; 17:
660-6.
19. Kaiser U, Schilli M, Haag U, et al. Expression of Bcl-2 pro- tein in small cell lung cancer. Lung Cancer 1996; 15-31.
20. Mountain CF, Dresler CM. Regional lymph node classifi- cation for lung cancer staging. Chest 1997; 111: 1718-23.
