Immunohistochemical study of P-glycoprotein distribution in lung cancer

LUNG CANCER

Lung Cancer IO (1993) I-12

Immunohistochemical study of P-glycoprotein distribution in lung cancer

Jean-Louis Pujol* a, Joslle Simony b, VCronique Gautiera, Charles Marty-AnCC, Henri Pujolb, FranCok-Bernard Michel”

‘Centre Hospitalier Universitaire, Hbpital Arnaud de Villeneuve. 34059 Montpellier Cedex, France ‘Centre Val d’Aurelle, Rue de la Croix Verte. 34094 Montpellier Cedex, France

‘Service de Chirurgie Thoracique, Centre Hospitalier Universitaire, H6pital Arnaud de Villeneuve, 34059 Montpellier Cedex, France

(Received 20 July 1992; revision received 4 December 1992; accepted 8 December 1992)

Abstract

Indirect immunoperoxidase was used to determine the reactivity of C219 (P-glycoCHEK C219TM, Centocor Diagnostics, Malvern, PA), a monoclonal antibody (Mab) with high affinity for an internal epitope of the P-glycoprotein encoded by the multidrug resistance (MDRl) gene, in 40 surgically resected primary lung tumours. C219 reactivity was qualitatively classified in seven small cell lung cancers (SCLC), 29 non small cell lung cancers (NSCLC), and four carcinoid tumours. Ploidy was analysed by means of static cytometry using a computer-assisted image processor following Feulgen staining of cytologic prints of 32/40 lung tumours. Indirect immunoperoxidase reactivities of Mabs S-L 11.14 and MOC-I were also studied to characterize the expression of cluster 1 lung cancer antigens and hence to determine among the NSCLC those which expressed the neural cell adhesion molecule (NCAM). Eighteen (45%) lung tumours strongly expressed P-glycoprotein as an immunostaining of many islets of malignant cells or almost all malignant cells. In addition, 8/40 tumours (20%) showed a weak reactivity (few immunostained cells) and 14140 (35%) no reactivity. There was no difference of reactivity when NSCLC were compared with SCLC. The expression of P- glycoprotein in NSCLC did not vary significantly when the stage of disease was considered. Among the 29 NSCLC, 10 (36%) expressed S-L 11.14 and MOC-1. The NCAM positive NSCLC did not show any difference of P-glycoprotein expression in comparison with NCAM negative ones. Finally, C219 immunoperoxidase reactivity did not significantly differ according to the ploidy status. In conclusion, the internal epitope of the P-glycoprotein encoded by the MDRl gene is frequently expressed by lung tumours of any histological type. This ex-

* Corresponding author.

0169-5002/931$06.00 0 1993 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0169-5002(93)00207-P

2 J.L. Pujol et al. /Lung Cancer IO (1993) I-12

pression is not higher in Stage III and IV lung cancers in comparison with Stage I and II ones, or in NSCLC in comparison with SCLC either. Thus, the C219 related epitope seems to have a weak implication in the lower chemosensitivity of both advanced stages and NSCLC.

Key words: Multidrug-resistance; Lung cancer; P-glycoprotein; Immunohistochemistry

1. Introduction

Chemotherapy is the main treatment for small cell lung cancer (SCLC) [15]. In contrast, non-small cell lung cancer (NSCLC) has a lower response rate following combination chemotherapy [ 141. Survival benefit in patients with unresectable NSCLC is still a subject of controversy [20,27]. Recent trials have demonstrated that chemotherapy in this setting leads to a significant improvement of median survival [27]; nevertheless this benefit remains poor. A meta-analysis recently showed that patients with unresectable NSCLC confined to one hemithorax (limited NSCLC) have a better response to chemotherapy when compared with patients with extensive (metastatic) NSCLC [9]. This finding is not surprising when one considers the fact that cytotoxic drugs kill a constant percentage of malignant cells instead of a fixed number of them [35]. Other investigators have studied the chemosensitivity of NSCLC which expressed heterotopic neuroendocrine antigens [ 131; they demonstrated these NSCLC respond better to chemotherapy than classic ones.

The P-glycoprotein (Mdrl) is encoded by the multidrug resistance (MDRZ) gene [3]. Its overexpression by lung cancer cell lines is associated with an increased eMux of many drugs from the cells [37]. However, recent studies have emphazised that P- glycoprotein expression is only one of the possible mecanisms of chemoresistance [6]. C219, a monoclonal antibody (Mab) with a high affinity for an internal epitope of P-glycoprotein, has been proposed to investigate the expression of Mdrl by normal tissues and tumours using immunohistochemistry [17]. If P-glycoprotein is an important mechanism of chemoresistance in lung cancer, the determination of its overexpression might be of clinical interest. In particular, it could be hypothezised that expression of P-glycoprotein is higher in NSCLC when compared with SCLC, in classic NSCLC when compared with NSCLC which express neuroendocrine antigens, and in Stage III and IV NSCLC when compared with Stage I and II ones. It has been demonstrated that bronchoscopically-obtained biopsy specimens are valid material for immunohistochemistry [2]. On the other hand, we reported previously an important phenotypic heterogeneity of lung cancer when large specimen are analysed [26]. Therefore, the present study aims to determine the P-glycoprotein distribution in large surgically resected specimens. This seems to us an important step before running through the analysis of smaller specimens.

Forty lung tumours were tested by immunohistochemistry using Mab C219. In addition, expression of neural cell adhesion molecule (NCAM) was determined by analysing the indirect immunoperoxidase reactivity of S-L Il. 14 and MOC-1 Mabs; in parallel, ploidy status was assessed by means of static cytometry using a computer-assisted image processor. This study was done to determine the P- glycoprotein distribution in lung cancers according to histology, stage of disease, NCAM expression, and ploidy.

J.L. Pujol et al. /Lung Cancer 10 (1993) l-12

2. Materials and methods

2.1. Patients

Forty patients were operated upon for a primary lung tumour. All surgical specimens were analysed according to the latest WHO classification [38] by light microscopy following hematoxylin-eosin staining. Characteristics of the patients are summarized in Table 1. Staging was performed according to the latest international TNM system [31,34]. For all patients staging procedure included clinical examina- tion, chest x-rays, computed tomographic scan (CT-scan) of chest and upper ab- domen, fiberoptic bronchoscopy, liver sonography and bone scanning. Patients for whom chest CT-scan disclosed enlarged mediastinal lymph nodes underwent a cervi- cal mediastinoscopy. Brain CT-scan was done in patients with central nervous system signs.

2.2. Immunohistochemistry

a. Tissue preparation The immunostaining was done on 5-pm thick frozen tissue sections obtained by

means of an automatic cryostat (Cryocut-Bright, Shandon, UK).

b. Antisera source C219 (P-glycoCHEK C21gTM, Centocor Diagnostics, Marvern, PA) is a murine

Mab (IgG2,) specific to the 170-kDa P-glycoprotein (Mdrl) [ 171.

Table 1 Patients’ characteristics

No. M/F Mean age (range) Surgery

Pneumectomies/lobectomies Diagnostic only

Histology Carcinoid tumour Small cell lung cancer Squamous cell carcinoma Adenocarcinoma

Stage of neuroendocrine tumours (SCLC and carcinoids) Stage I Stage II Stages IlIa and IlIb Stage IV

Stage of non-small cell lung cancers Stage I Stage II Stage IIIa Stage IV

40 3614 60 (41-83)

I5/20 5

4 7

26 3

4 I 5

I2 6

IO


Mabs MOC-I and S-L 11.14 defined the same neuronal-neuroendocrine antigen. Mab MOC-1 [8], an IgGl cluster 1 antibody, was kindly given By L. de Leij (State University, Groningen, Netherlands) and Mab S-L 11.14 [28] was kindly given by J.P. Laurent (SociCtC Civile Biocytex, Marseille, France). This IgG2, Mab was clustered in Group 1, as was the MOC-1, at the 1st and 2nd International Workshops On Lung Cancer Antigens [28]. Immunoprecipitation, using either Mab S-L 11.14 or Mab MOC-1, characterized the cluster 1 related antigen as a M, 145 000 membrane protein, referred to as one of the different isoforms of NCAM. This molecule is a sialoglycoprotein involved in cell-to-cell interactions of neural tissues. It has been demonstrated that both cell lines and specimens from almost all SCLC [28] and 20% of NSCLC [26] expressed NCAM.

c. Immunohistochemical reactions Frozen tissue sections were fixed with cold acetone. This led to cell permeabiliza-

tion and made the internal epitope of P-glycoprotein accessible to C219. Indirect immunoperoxidase tests were carried out using the biotin-streptavidin-peroxidase system (Amersham Les Ulis, France) following the three-stage procedure [16]. In stage 1, after rehydration in PBS and inhibition of endogen peroxidase, sections were incubated with 10 &ml of purified Mab for 40 min at room temperature; in stage 2, sections were then washed in PBS and incubated with sheep biotinylated anti- mouse IgG diluted 1:50 in PBS for 30 min at room temperature; in stage 3, sections were finally incubated with streptavidin-biotinylated horseradish peroxidase at 1:200 for 30 min at room temperature. Immunohistochemical reaction was then revealed in the dark with 3-amino-9 ethyl carbazol (Merck, Nogent/Marne, France) using hy- drogen peroxide as a substrate and counterstained with hematoxylin. Control slides were prepared with substitution of the primary Mab with similar dilution of an ir- relevant mouse antibody of the same isotype (DAKO, Glostrup, Denmark).

Indirect immunoperoxidase reactivity of cluster 1 Mabs was considered as positive when an homogeneous staining was observed. The reactivity of C219 was qualitatively analysed according to Table 2. Reactivity of this Mab was also tested in autologous normal lung tissue.

2.3. Cell DNA content analysis

a. Tissue preparation A cytological print from each specimen was air-dried. Slides were fixed in

Table 2 Qualitative analysis of C219 immunostaining

Score

- f + ++ +++

Feature

No staining Scattered cells Few stained cells but > 10% Numerous stained cells as islets Homogeneous staining of all cells

J.L. Pujol et al. /Lung Cancer 10 (1993) l-12 5

formaldehyde-alcohol solution for 10 min then washed three times in alcohol and stained by the para-rosaline Feulgen-Schiff technique [ 181.

b. Computer-assisted cytometry The stoichiometric reaction was analysed using the computer-assisted image pro-

cessor [29] (Systbme d’Analyse Micro-photomterique a Balayage Automatique; TITN; Grenoble, France). For each specimen, cell DNA content analysis’was carried out on 300 randomized malignant cells. The nuclear DNA values were com- puterized in order to produce histograms. The cell DNA content was expressed in c-units with 2c representing the mean value of normal diploid control cells (normal hepatic tissue). Ploidy was determined for each specimen using DI which represents the ratio of the cell DNA content of Go,, tumour cells to the diploid GoI1 peak (2~). Thus, a near diploid tumour referred to as a tumour with a DI = 1.

c. DNA histogram analysis The DNA histograms were classified into four types: type 1, Goil tumour cells in

the near diploid region (2c, DI = l), with few G2M tumour cells in the tetraploid region (4c); type 2, hyperdiploid Goi, tumour cells (DI 2 1.2); type 3, hypodiploid G,,, tumour cells (DI 5 0.8), with a hypodiploid peak containing at least 20% of cells; type 4, evidence of multiploid tumour cells with multiple aneuploid peaks, some of them in the octaploid region (SC). The percentage of cells in the hypodiploid modal DNA (percentage of hypodiploid cells, DI 5 0.8) was calculated for each histogram. In addition the percentage of cells in the modal DNA of Go,, phase was taken as an index of ploidy.

2.4. Study design

All patients had a complete staging. A surgically resected specimen from a non necrotizing area of the tumour was deep frozen and cytological prints were done. Neither chemotherapy nor radiotherapy were carried out prior to surgical resection. Evaluation of P-glycoprotein expression, NCAM expression and DNA content were done independently and without knowledge of the pTNM stage.

Statistical analyses were done by means of non-parametric tests. Chi square test was used to compare the distribution of P-glycoprotein expression according to histology, stage grouping, expression of NCAM, and DNA histogram types. Yates’ correction of the X2 test was used when appropriate. Differences in the percentage of cells in the modal DNA of G,i phase according to the P-glycoprotein expression were tested using the Kruskal Wallis test; a P level <0.05 was considered as significant.

3. Results

Respiratory epithelium autologous normal lung weakly expressed P-glycoprotein. The P-glycoprotein expression was increased in 18 (45%) lung tumours (Fig. 1). In these tumours the indirect immunoperoxidase C219 reactivity was observed either as many islets of positive malignant cells or as an homogeneously immunostaining


Fig. 1. C219 immunoperoxidase reactivity in a squamous cell carcinoma. Twenty to thirty percent of the cells demonstrated a strong membrane staning (magnification: x 400).

for all malignant cells. In addition, 8/40 tumours (20%) showed a weak reactivity and 14/40 (35%) no reactivity at all.

The reactivity of C219 did not significantly differ according to histology (X2 = 3.60; NS; Fig. 2), in particular the frequency of a strong expression of P- glycoprotein did not differed between SCLC and NSCLC. Among the 26 squamous cell carcinomas (SQC) 14 were well differentiated and 12 were poorly differentiated. The frequency of P-glycoprotein expression was 57% and 42%, respectively (X2 = 2.1; NS). The expression of P-glycoprotein in NSCLC did not vary significantly when the stage of disease was considered (X2 = 0.47; NS; Fig. 3). Clus- ter 1 neuroendocrine antigen expression was studied in 28/29 NSCLC. Among them 10 (36%) expressed S-L 11.14 and MOC- 1. These NCAM positive NSCLC did not show any difference of P-glycoprotein expression when compared with both NCAM negative NSCLC and SCLC (X2 = 0.79; NS; Fig. 4).

Among the 40 tumours, DNA content analysis has been possible in 32. Nine tumours were nearly diploid and 23 aneuploid (among them nine showed an aneuploid hypodiploid G,,, peak of malignant cells). The frequency of a strong P- glycoprotein expression was highest in hypodiploid tumours and lowest in diploid ones although this did not reach statistical significance (X2 = 1.52; NS). Moreover, the percentage of cells in the modal DNA of Go,, phase (taken as an index of diploidy) did not significantly varied according to the C219 reactivity (Kruskal Wallis KW = 156; NS).

J.L. Pujol et al. /Lung Cancer 10 (1993) I-12 7

+++

++

+

+

Ad SQCwd

0.0

0 0.0

SQCpd SCLC Car.

T

I- @ 0.0 1

0 I 0

Fig. 2. P-glycoportein distribution according to histology; Ad, adenocarcinoma; SQC, squamous cell carcinoma; pd, poorly differentiated; wd, well differentiated; SCLC, small cell lung cancer; Car., carcinoid

tumour; (X2 = 3.60; NS).

+++

++

+

+

I I I

??

??@a I I.0

I II llla Stage Grouping

IV

Fig. 3. P-glycoprotein distribution in non-small cell lung cancers according to stage grouping (X2 = 0.47; NS).

J.L. Pujol el al. /Lung Cancer IO (1993) 1-12

NE-NSCLC SCLC Fig. 4. P-glycoprotein distribution in non-small cell lung cancers with or without NCAM expression and

small cell lung cancers (X2 = 0.79; NS).

4. Discussion

In order to test the potential clinical interest of P-glycoprotein expression in SCLC and NSCLC we analysed the reactivity of C219, paying particular attention to tumour features known to affect chemosensitivity. There was no difference of P- glycoprotein expression according to histology, stage of disease, expression of neuroendocrine antigen and ploidy.

Response to chemotherapy in SCLC remains one of the most important factors with respect to long term survival [15]. In contrast, the use of chemotherapy in NSCLC is still a subject of controversy [20,27]. Sensitivity to chemotherapy is variable owing to heterogeneity of lung tumour phenotype and growth fraction [35]. The weak survival benefit obtained by chemotherapy of unresectable NSCLC might be explained by this heterogeneity. Thus, markers of chemosensitivity are needed. Recently, neoadjuvant chemotherapy was proposed in locally advanced NSCLC in an attempt to treat the microscopic metastatic disease [22,25]. It is noteworthy that a high response rate is observed in these selected Stage IIIa NSCLC. Cell kinetic par- ameters might be one of the reasons for the relatively lower chemoresistance of Stage III NSCLC in comparison with Stage IV ones. However, it could be questioned whether or not intrinsic cell drug resistance could also account for this phenomenon.

Heterogeneity of lung cancer phenotype has been widely investigated [5]. Im- munohistochemistry using Mabs directed against neuroendocrine antigens has demonstrated that NSCLC may express heterotopic antigens unrelated to cell

J.L. Pujol et al. /Lung Cancer 10 (1993) l-12 9

lineage [5,26]. Advanced clinical stage and aneuploidy are common features of NCAM positive NSCLC, suggesting a link between phenotypic heterogeneity and tumour progression [26]. Moreover, it has been suggested that NSCLC which express neuroendocrine antigens are less chemoresistant than are the classic NSCLC [ 131. Again, it could be of interest to determine the potential implication of Mdrl in the higher chemoresistance of classic NSCLC.

P-glycoproteins are membrane proteins detected in many normal tissues and tumours. In humans, they are encoded by two genes, MDRl and MDR2 [3,37]. The product of these genes are referred to as Mdrl and Mdr2. Only Mdrl, a 170-kDa glycoprotein is known to act as a membrane efflux transporter able to prevent the accumulation of intracellular chemotherapeutic agents such as doxorubicin, etoposide, vindesine, or vinblastine [19]. The P-glycoprotein (Mdrl) expression in normal tissue, tumours or cancer cell lines, can be analysed at different levels: measurement of MDRl gene amplification, measurement of MDRl mRNA, and detection of Mdrl using anti-Mdrl Mab. This latter method is known to have a lower specificity [37] and gives variable results according to the type of analysed specimens [4] and to the selectivity of the anti-P-glycoprotein Mab against Mdrl [32]. Moreover, the C219 Mab is known to recognize an epitope of the cytoplasmic region of the protein not absolutely specific to Mdrl [33]. The epitopes recognized by different anti-P-glycoprotein Mab have been analyzed by high resolution epitope map- ping [l I]. This study concluded that C219 bound strongly to an aminoacid sequence found in all P-glycoprotein isoforms. More recently, some samples of C219 and JSB- 1, two Mab raised against P-glycoporteins, have been tested in an indirect Coombs’- type-assay and immunostaining procedure of neutral glycolipids on thin-layer chro- matography [lo]. Both Mab demonstrated reaction with the carbohydrate structure of blood group A antigen referred to as Type 3 A structure. In the same study, the reactivity of these Mab in normal and malignant ovarian epithelium specimens has been tested using immunohistochemistry. All of the 13 C2 19-positive-specimens were from group A individuals [lo]. This might explain that discrepancies observed for ovarian cancer in which C219 reactivity is higher than overexpression of MDRl mRNA [24]. The relatively low specificity of anti-Mdrl Mab may be one explanation for this discrepancy; however, the sensitivity of northern blot analysis of MDRl mRNA could be questioned as the polymerase chain reaction is considered to be as the most accurate method of detecting MDRI mRNA [37]. The expression of MDRl mRNA in various histologies of lung cancer tissues and cell lines has been study by Goldstein [12] and Lai [21]. Slot blot and northern blot procedures showed that MDRl mRNA expression by both lung cancer tissues and lung cancer cell lines is low. The only positivity has been observed in the neuroendocrine subset of NSCLC: for this particular phenotype, one of two tumors was positive and 5/6 cell lines expressed intermediate or high level of MDRI RNA.

P-glycoprotein expression has been investigated in numerous cell lines whereas the analysis of its expression by tumour tissues is more recent. The in vitro implication of MDRl in the mechanism of chemoresistance against cytotoxic drugs depends on the cell lines studied. For example, the H69AR cell line derived from the H69 SCLC cell line, is resistant to doxorubicin and etoposide without any overexpression of MDRl; COR-L23, a subline of a large cell lung cancer cell line, also showed cross-

10 J.L. Pujol et al. /Lung Cancer 10 (1993) I-12

resistance to daunorubicin, vincristine and colchicine without overexpression of P- glycoprotein [7]. Conversely H69/VP, an etoposide resistant SCLC line, overexpress- ed MDRI mRNA, a finding which suggests that alteration of drug accumulation in the cell is mediated by P-glycoprotein [23]. A similar observation has been made with human squamous cell lung cancer cell lines selected from the SW-1573 line [ 11. In these sublines the selection of higher doxorubicin concentrations gave rise to vari- ants with a high transcriptional activation of the MDRZ gene although this phenomenon is not the unique explanation of resistance. Thus, in vitro overexpression of Mdrl seems to be very variable from one cell line to another and in many instances the cross-resistance to different drugs seems to belong to multiple mechanisms. Finally, the correlation between MDRI and aneuploidy has been analysed in kidney carcinomas but the relation seems to be very weak [36].

In our study P-glycoprotein encoded by the MDRI gene is frequently expressed by lung tumours of any histological type. We found out that hypodiploid tumours had a higher overexpression of Mdrl although this did not reach any statistical significance. However, this finding is in agreement with the observation that hypodiploid multiple myelomas share low chemosensitivity [30]. To establish the clinical implication of Mdr 1 overexpression in the chemoresistance phenomenon observed in previously untreated NSCLC, or in recurrences of SCLC, we started an ongoing study aimed at comparing C219 immunostaining of bronchoscopically- obtained biopsy specimens with clinical response. However, in the present study, reactivity of C219, a Mab directed against an internal epitope of P-glycoprotein, was not higher in Stage III and IV lung cancers in comparison with Stage I and II ones, or in NSCLC in comparison with SCLC either. Thus, this study weakens the implication of the Cl219 related epitope in the lower chemosensitivity of both advanced stages and NSCLC.

5. Acknowledgements

This work was supported by grants from the French League Against Cancer (Herault and Aude Committees), the GEFLUC, and Sarget Laboratories. The authors wish to thank Mrs Jo Baissus for help in preparing the manuscript and Mrs L. Ursule, Mrs M. Radal and Mrs N. Lequeu for technical assistance.

6. References

1 Baas F, Jongsma AP, Broxterman HJ et al. Non-P-glycoprotein expression during in vitro selection for doxorubicin resistance in a human lung cancer cell line. Cancer Res 1990; 50: 5392-5398.

2 Berendsen HH, De Leij LF, Poppema S, Postmus PE, Sluiter HJ, The TH. Simultaneous standard light microscopy and immunohistology on bronchoscopically procured lung cancer specimens. Eur J Cancer Clin Oncol 1988; 24: 915-922.

3 Bradley G, Juranka PF, Ling, V. Mechanism of multidrug resistance. Biochim Biophys Acta 1998; 948: 87-128.

4 Brambilla E, Moro D, Gazzeri S et al. Cytotoxic chemotherapy induces cell differentiation in small- cell lung carcinoma. J Clin Oncol 1991; 9: 50-61.

5 Broers JLV, Rot MK, Oostendorp T et al. Immunohistochemical detection of human lung heterogeneity using antibodies to epithelial, neuronal and neuroendocrine antigens. Cancer Res 1987; 47: 3225-3234.

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7

8

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14

15

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Cole SP, Chanda ER, Dicke FP, Gerlach JH, Mirski SE. Non-P-glycoprotein-mediated multidrug resistance in a small cell lung cancer cell line: evidence for decreased susceptibility to drug-induced DNA damage and reduced levels of topoisomerase II. Cancer Res 1991; 51: 3345-3352. Coley HM, Workman P, Twentyman PR. Retention of activity by selected anthracyclines in a multidrug resistant human large cell lung carcinoma line without P-glycoprotein hyperexpression. Br J Cancer 1991; 63: 351-357. De Leij LF, Poppema S, Nulend JK et al. Neuroendocrine differentiation antigen on human lung carcinoma and Kulchitski cells. Cancer Res 1985; 45: 2192-2200. Donnadieu N, Paesmans M, Sculier JP. Chimiothirapie des cancers bronchiques non a petites cellules. Meta-analyse de la litttratrue en fonction de I’extension de la maladie. Rev Malad Respir 1991; 8: 197-204. Finstad CL, Yin BWT, Gordon CM, Federici MG, Welt S, Lloyd KO. Some monoclonal antibody reagents (C219 and JSB-1) to P-glycoprotein contain antibodies to blood group A carbohydrate determinants: a problem of quality control for immunohistochemical analysis. J Histochem Cytochem 1991; 39: 1603-1610. Georges E, Bradley G, Gariepy J, Ling V. Detection of P-glycoprotein isoforms by gene-specific monoclonal antibodies. Proc Nat1 Acad Sci USA 1990; 87: 152-156. Goldstein LJ, Galski H, Fojo A et al. Expression of a multidrug resistance gene in human cancers. J Nat Cancer Inst 1989; 81: 116-124. Graziano SL, Mazid R, Newman N et al. the use of neuroendocrine immunoperoxidase markers to predict chemotherapy response in patients with non-small cell lung cancer. J Clin Oncol 1989; 7: 1398-1406. Gregor, A. Controversies in the treatment of non-small cell lung cancer. Eur J Cancer 1991; 27: 362-366. Hansen HH, Kristjansen, PEG. Chemotherapy of small cell lung cancer. Eur J Cancer 1991; 27: 342-349. Hsu SM, Raine L, Fanger, H. The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981; 29: 577-580. Kartner N, Evemde-Porelle D, Bradley G et al. Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. Nature 1985; 316: 820-823. Kenji M, Yoshikazu K, Sadamu N, Yoshihiro U. Automated Feulgen’s reaction in autosreening. J Jpn Sot Clin Cytol 1971; 10: 148-154. Konen PL, Currier SJ, Rutherford AV et al. The multidrug transporter: rapid modulation of emux activity monitored in single cells by morphologic effects of vinblastine and daunomycin. J Histochem Cytochem 1989; 37: 1141-1145. Lad TE, Nelson RB, Diekampu YX et al. Immediate versus postponed combination chemotherapy (CAMP) for unresectable non small cell lung cancer: A randomized study. Cancer Treat Rep 1981; 65: 973-8. Lai S, Goldstein LJ, Gottesman MM et al. MDR gene expression in lung cancer. J Nat Cancer lnst 1989; 81: 1144-1147. Martini N, Kris MG, Gralla RJ et al. The effects of preoperative chemotherapy on the resectability of non-small cell lung carcinoma with mediastinal lymph node metastases (N2MO). Ann Thorac Surg 1988; 45: 370-379. Minato K, Kanzawa F, Nishio K, Nakagawa K, Fujiwara Y, and Saijo, N. Characterization of an etoposide-resistant human small cell lung cancer cell line. Cancer Chemother Pharmacol 1990; 26: 313-317. Nooter K, Herweijer H. Multidrug resistance (mdr) genes in human. Br J Cancer 1991; 63: 663-669. Pujol JL, Rossi JF, Le Chevalier T et al. Phase II pilot study of neoadjvant ifosfamide, cisplatin, and etoposide in locally advanced non small cell lung cancer. Eur J Cancer 1990; 26: 798-801. Pujol JL, Simony J, Laurent JC et al. Phenotypic heterogeneity studied by immunohistochemistry and aneuploidy in non small cell lung cancers. Cancer Res 1989; 49: 2797-2802. Rapp E, Pater JL, Willian A et al. Chemotherapy can prolong survival in patients with advanced non-small cell lung cancer - Report of a Canadian Multicenter Randomized Trial. J Clin Oncol 1988; 6: 633-641.

12 J.L. Pujol et al. /Lung Cancer 10 (1993) I-12

28

29

30

31

32

33

34

35

36

37

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Rosier F, Richer G, Laurent JC. A group of antibodies having SCLC and neural reactivity define a restricted number of epitopes, as checked by competion experiments. Lung Cancer 1988; 4: 107-108. Simony J, Pujol JL, Radal M, Ursule E, Michel, FB, Pujol H. in situ evaluation of growth fraction determined by monoclonal antibody Ki-67 and ploidy in surgically resected non-small cell lung cancers. Cancer Res 1990; 50: 4382-4387. Smith L, Barlogie B, Alexanian R. Biclonal and hypodiploid multiple myeloma. Am J Med 1986; 80: 841-843. Sobin LH, Hermanek P, Hutter RVP. TNM classification of malignant tumours, 4th edn. UICC, Geneva 1987. Suguwara I, Kataoka I, Morishita Y et al. Tissue distribution of P-glycoprotein encoded by a multidrug-resistant gene as revealed by monoclonal antibody, MRK16. Cancer Res 1989; 48: 1926-1929. Thiebaut F, Tsuruo T, Hamada H et al. Immunohistochemical localization in normal tissues of different epitopes in a multidrug transport protein Pl70: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein. J Histochem Cytochem 1989; 37: 159-164. Tisi GM, Friedman PJ, Peters RM et al. American Thoracic Society: clinical staging of primary lung cancer. Am Rev Respir Dis 1982; 125: 659-64. Tubiana M. The growth and progression of human tumors: implications for management strategy. Radiother Oncol, 1986; 6: 167-184. Volm M, Efferth T. Relationship of DNA ploidy to chemoresistance of tumors as measured by in vitro tests. cytometry 1990; 1 I: 406-410. Weinstein RS, Kuszak JR, Kluskens LF, Coon JS. P-glycoproteins in pathology: the multidrug resistance gene family in humans. Human Pathol 1990; 21: 34-48. World Health Organization. The World Health Organization histological typing of the lung tumors, 2nd edn. Am J Clin Pathol 1982; 77: 123-36.

Immunohistochemical study of P-glycoprotein distribution in lung cancer

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