Int. J. Cancer: 52,862-866 (1992) Q 1992 Wiley-Liss, Inc.

I

Publicationof the InternationalUnion Against Cancer Publicationde I'Union InternationaleContre le Cancer

EGF RECEPTOR EXPRESSION IN PRIMARY LARYNGEAL CANCER: CORRELATION WITH CLINICO-PATHOLOGICAL FEATURES AND PROGNOSTIC SIGNIFICANCE Maurizio M A U R I Z IGiovanni ~ ~ ~ , SCAMBIA', Pierluigi BENEDETTI PANICI?,Gabriella FERRANDINA~, Giovanni ALMADORI', Gaetano PALUDETTI~, Rosa DE VINCENZO?, Mariagrazia DISTEFANO~, Domenico BRINCHI?, Gabriella CADONI' and Salvatore MANCUSO~ Departments of 10tolaryngologyand 2Departmentof Obstetrics and Gynecology, Catholic University,L.go A . Gemelli, 8, 00168 Rome, Italy Epidermal-growth-factor-receptor(EGFR) expression was evaluated in 103 primary laryngeal tumors and in 42 normal laryngeal tissue specimens. Significantly higher EGFR levels were found in cancer specimens than in normal mucosa (p = 0.0053). EGFR expression did not correlate with age, tumor localization, T classification,cervical-lymph-nodeinvolvement or type of surgery, whereas it was higher in poorly differentiated tumors (G3) than in well/moderately differentiated (G 1 4 2 ) tumors (p < 0.05). Follow-up data were available for 74 patients. When EGFR status and the most important clinico-pathological characteristics were submitted to univariate analysis, tumor localization, type of surgery and EGFR status were found to be significantly correlated with disease-free survival. The 24-month disease-free survival rate was 58% for EGFR+ cancer patients and 82% for EGFR- ones. With multivariate analysis, only EGFR status and tumor localization were identified as significant independent prognostic parameters. Data reported here suggest that high EGFR levels may identify a sub-set of laryngeal-cancer patientswith a particularlyunfavorable prognosis. G 1992 Wiley-Liss,lnc.

Despite recent advances in surgery and radio-chemotherapy, the outcome of patients with squamous laryngeal cancer is still relatively variable. Tumor factors influencing prognosis, such as TNM classification and histopathological grading, provide only some useful prognostic information (Eiband et al., 1989), since they are semi-quantitative and are influenced by the examiner's evaluation. Host factors such as gender, age, performance status and lifestyle (smoking, alcohol, occupational and environmental factors) and treatment factors are less important predictors. Therefore efforts have been made to identify additional biological factors such as tumor ploidy, S-phase fraction, receptorial status (Rua et al., 1991; Reiner et al., 1988) which could provide more accurate understanding of tumor biology and useful information for predicting outcome and individualizing treatment. In this context, much attention has been focused on the role of peptidic growth factors in the onset and spread of human cancer. In particular, epidermal growth factor (EGF), a polypeptide acting through a specific trans-membrane receptor (EGFR), exerts mitogenic activity on several normal and neoplastic cell lines, including laryngeal cancer cells (Weber et al., 1988). E G F R has been described in many human neoplasms, and its expression might represent a parameter of poor prognosis in breast (Sainsbury et al., 1987; Battaglia et nl., 1988b),bladder (Neal et al., 1985), esophageal (Ozawa et al., 1989) and ovarian (Scambia et al., 1992) tumors. To date, only 2 studies (Scambia et al., 1991; Santini et al., 1991) have dealt with the characterization and quantification of E G F R expression in laryngeal tumors by using a ligandbinding technique. Significantly higher EGFR levels were found in laryngeal tumors than in normal mucosa, suggesting that E G F R may play a role in regulating the growth of laryngeal cancer cells. Moreover, poorly differentiated laryngeal tumors express higher EGFR levels than well-differentiated tumors. It has been suggested that EGFR could identify a class of more aggressive laryngeal tumors endowed with higher recurrence potential and an unfavorable prognosis.

In this study, EGFR expression has been prospectively analyzed in a large number of laryngeal cancer patients, in order to investigate the relationship with common histopathological parameters and to assess its prognostic value in relation to disease-free survival (DFS) in primary laryngeal cancer. MATERIAL AND METHODS

Between 1988 and 1991,103 consecutive untreated laryngeal carcinoma patients, 100 male and 3 female, aged between 37 and 83 (median, 62 years) were admitted to our study. Tumor site was classified as supraglottic or glottic, or was defined as transglottic when the extension of disease did not permit identification of the original site. Tumors were staged according to TNM classification and graded as well(Gl), moderately(G2) and poorly(G3) differentiated tumors. For evaluation of the results, G 1 and G2 laryngeal tumors were grouped and analyzed with respect to G 3 laryngeal tumors. All tumors were epidermoid squamous-cell carcinomas. The clinico-pathological features of the cases examined are shown in Table I. Fifty-seven patients underwent radical laryngectomy, while 46 had conservative surgery, i.e., 10 cordectomies, 31 horizontal supraglottic laryngectomies and 5 hemylaryngectomies. Patients treated with conservative laryngectomy (n = 46) included 8 patients at stage I, 20 patients at stage 11, 12 patients at stage 111, and 6 patients at stage IV. None underwent radioor chemotherapy. Patients treated with radical surgery (n = 57) included 6 patients at stage I, 11 patients at stage 11, 25 patients at stage 111, and 15 patients at stage IV. Radiotherapy was performed on 14 stage-IV patients (7 were T4NO and 7 were N2). None of the patients received chemotherapy. At the time of surgery, 29 patients underwent therapeutic neck dissection due to positive lymph-node involvement. Expression of E G F R was also analyzed in 42 normal laryngeal mucosa specimens obtained from the same patients in a corresponding non-tumor area of the larynx.

1251-EGF-bindit~g measurement Tissue specimens, collected during primary surgery, were frozen on dry ice shortly after surgical removal and stored at -80°C until processed. A representative section of specimens was retained for histopathological examination. The membrane fraction and cytosol were prepared as described elsewhere (Battaglia et al., 1988a,b). Briefly, tumor specimens were finely minced and homogenized in 5 volumes of ice-cold buffer consisting of 25 mM TRIS, 1.5 mM EDTA, 5 mM NaN3, 0.1% monothioglycerol and 20% glycerol (TENMG), by applying several intermittent bursts of an Ultra-Turrax homogenizer. The crude homogenate was centrifuged at 7,OOOg for 20

'To whom correspondence and reprint requests should be addressed. Fax: 06l3051343. Received: May 18.1992 and in revised form July 28. 1992.

863

CLINICAL ROLE OF EGFR EXPRESSION I N LARYNGEAL CANCER

TABLE I -CHARACTERISTICS OF PATIENTS Characteristic!,

Number of patients (9:)

Total Sex Male Female Age I 60 yrs > 60 yrs Tumor localization Supraglottic Glottic Transglottic T classification I I1

103

45 (44 58 (56)) 37 (36 32 (31{ 34 (33)

ing 300 to SO0 p.g protein) were incubated with '*'I-EGF (2.6 nM) in the presence or absence of unlabelled EGF (1 mM) for 16 hr at room temperature in a final volume of 400 p.1. Binding was stopped by the addition of 3 ml of 25 mM TRIS, 20% glycerol, 5 mM NaN3 and 0.1% BSA. Pellets were obtained by centrifugation at 2,OOOg for 20 min at 0°C and counted in a gamma counter for 1 min. Results were expressed as fmoles per mg of membrane protein (fmol/mg prot). Tissue-weight permitting, Scatchard analysis was carried out giving a range of constant dissociation from 0.8 nmolil to 3.2 nmol/l. Protein concentration was measured by the Bradford method (1976). Among the various cut-off values, the value of 8 fmol/rng prot, representing the median value of E G F R levels in laryngeal carcinomas, was demonstrated to be the best discriminating value, and was chosen as the cut-off to define EGFR status.

111

Statistical analysis The paired t-test was used to analyze E G F R expression in 69 67 normal and neoplastic laryngeal specimens from the same 34 {33] patient. The Wilcoxon rank-sum test was used to compare receptor levels and to analyze the relationship between EGFR NO 74 (72) expression and tumor characteristics. For disease-free survival N+' 29 (28) analysis, only patients (n = 74) with a follow-up of at least 12 Type of surgery months were considered. There was no difference in clinicoConservative? 46 (45 pathological parameters and in E G F R positivity between the Radical 57 (551 group of 103 patients and the group of 74 patients for whom 'Presence of metastatic involvement.-Tordectomy, hemilaryn- follow-up data were considered. All medians and life tables gectomy. supraglottic laryngectomies. were computed using the product-limit estimate by Kaplan and Meier (1958) and the curves were examined by means of the log-rank test (Mantel, 1966). The risk of recurrence was min at 0°C. The supernatants were then centrifuged at 105,OOOg estimated by Cox's proportional hazards model (Cox,1972). for 75 min at 0°C. Multivariate analysis was performed with BMDP statistical The membrane pellet was re-suspended in 25 mM TRIS, 1.5 software. A backward stepwise procedure was used to identify mM EDTA, 5 mM NaN3, 20% glycerol and 10 mM MgClz the major prognostic factors. Disease-free survival (DFS) was (TENG + MgCI2). Aliquots of the suspension (100 p.1 contain- calculated from the day of primary surgery to the date of IV Histopathological grading G1-G2 G3 Lymph-node involvement

60

50 u) Q)

-t 0

40

0

L P)

P

E 30 3

z

20

10

t

i

0 0-10

11-20

t

t 21-30

31-40

1y41-50

51-60

61-70

71-80

81-90

161 -170

EGFR (tMlmg protein) FIGURE 1 - Scattergram of the EGFR levels in the group of 103 primary-laryngeal-tumor patients consecutively admitted to the study.

864

MAURIZI ETAL. TABLE 11 - EGFK DISTRIBUTION IN LARYNGEAL CANCER AND NORMAL MUCOSA TISSUES

Number (%) of EGFR-positive

EGFR Numher of cases

Normal mucosa Laryngeal cancer

(tmolesimg protein)

42 103

median

range

cases

4.8 8.0

0-16.6 0-169.9

9 (21) 51 (50)

TABLE 111 - EGFR DISTRIBUTION IN LARYNGEAL CANCER ACCORDING T O CLINICO-PATHOLOGIC CHARACTERISTICS AND TYPE OF SURGERY IN THE GLOBAL POPULATION O F 103 CASES O F PRIMARY-LARYNGEAL-TUMOR PATIENTS CONSECUTIVELY ADMITTED TO THE STUDY

EGFR (fmoleqimg protein) Variables

Case5

Number

Age I60 yrs > 60 yrs Tumor localization Supraglottic Glottic Transglottic T classification

Median

Range

Number ("c ) of EGFR+

p value[

45 58

7.6 8.4

0-86.9 1.1-169.4

20 (44) 32 ( 5 5 )

ns

37 32 34

8.3 7.5 7.9

0-169.9 0-86.9 1.1-40.2

19 (51 15 (481 16 (47)

ns

59 44 Histopathological grading GIG2 69 G3 34 Lymph-node involvement 74 NO N+ 29 Type of surgery 46 Radical Conservative 57

7.2 8.7

0-1 69.9 0-115.2

7.0 11.9

0-86.9 0.3-169.9

28 40) 24 {70)

< 0.05

7.4 9.2

0-169.9 0-86.9

35 (47) 15 (52)

ns

7.2 8.2

0-169.9 1.1-115.2

1-11111-IV

ns

ns

'Significance by Wilcoxon rank-sum test.-ns, not significant. recurrence of disease and/or loco-regional lymph-node involvement. The relative risk corresponds to the number of adverse cvents in one category expressed as a proportion of the reference-category adverse events. The median follow-up in the group of 74 patients was 21 months. Analysis was as of December 1991.

100

\,

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--------

80 w Y

RESULTS

EGFR expressiori Figure 1 shows the scattergram of E G F R levels in 103 cases of primary laryngeal tumors, while Table I1 shows the distribution of EGFR in 103 laryngeal tumors with respect to 42 normal mucosa specimens. Significantly higher EGFR levels were found in laryngeal cancer (median, 8.0 fmolimg prot, range, 0 to 169.9) with respect to normal mucosa specimens (median, 4.8 fmolirng prot, range 0 to 16.6) (paired t-test: T = 2.95; D F = 41;p = 0.0053). When analyzing EGFR expression in laryngeal cancer according to the clinico-pathological parameters (Table III), we found no difference in EGFR content distribution in relation to age, tumor localization, T classification, lymphnode involvement or type of surgery. A statistically significant inverse correlation was demonstrated between grade of differentiation and E G F R expression, which is significantly higher in poorly differentiated (median, 11.9 fmolimg prot, range, 0.3 to 169.9) than in moderately and well differentiated tumors (median. 7.0 fmol/mg prot, range, 0 to 86.9) ( p < 0.05).

EGFR erprcssiori arid risk ofprogression For this analysis, 74 patients with a minimum of 12 months follow-up have been considered. This patient population did not differ from the overall study population (103 patients), according to the clinical parameters. During the follow-up period (Le., during the observation time) local recurrence and metastatic cervical-lymph-node involvement were observed in

d & a

60

Y

40

+

z

0

5 n

20

---

ENTERED EGFR

RELAPSE

+

41

13

EGFR-

33

6

8

16

24

32

40

MONTHS

FIGURE 2 - Disease-free survival rate according to EGFR status in 74 primary-laryngeal-cancer patients. Out of 19 cases who underwent recurrence of cancer, 13 have died, while 2 patients died from intercurrent diseases. At the end of the study, 59 patients were alive. EGFR-, 5 8 fMolesimg protein; EGFR+, > 8

fh4olesimg protein.

13 and 14 cases respectively in the 74 patients examined. Eight patients experienced both local recurrence and lymph-node involvement. In order to define the disease-free survival rate we took into consideration the occurrence of one or the other or of both events together ( i e . , 19 recurrences of disease). At the end of the study, 59 patients were alive, 13 had died from

865

C L I N I C A L R O L E OF E G F R E X P R E S S I O N IN L A R Y N G E A L C A N C E R

TABLE I\.- UNIVARIATE A N D MULTIVARIATE ANALYSIS O F PROGNOSTIC VARIABLES F O R DISEASE-FREE SURVIVAL (DFS) I N 74 PATIENTS WITH PRIMARY I ARYNGFAI CARCINOMA Prognostic variable

Age

< 60 yrs

560 yrs

Tumor localization Supraglottic Glottic Transglottic T classification

Lymph-node involvement NO N1 Type of surgery Conse rvat ive Radical EGFR status Negative Positive

Number of case5

RRl

p value

26 48

1* 3.1

0.056

25 23 26

7.6

0.0015

RR2

p balue

1*

2.7

42 32

1*

53 21

1* 1.8

0.24

53 21

I* 1.4

0.59

33 41

4.1

0.0039

33 41

I* 3.1

0.99

1.1

1*

0.02

The 74 patients included in this table had a minimum of 12 months follow-up. DFS, disease-free survival corresponds to the time from primary surgery to recurrence of disease.-RR, relative risk corresponds to the number of adverse events in one category, expressed as a proportion of reference-category events.-RR1, relative risk of relapse in the univariate analysis.-RR2, adjusted relative risk of relapse. taking into account all factors of the table.-ns, not significant.-*Reference category. cancer, and 2 had died from intercurrent disease. None of the patients underwent autopsy. The disease-free survival curves shown in Figure 2 demonstrated that E G F R + patients have a shorter DFS than EGFR- patients ( p = 0.02). The 24-month DFS was 58% [9S% confidence intervals (CI) 40% to 76%] for patients with E G F R + tumors and 82% (CI 70% to 94%) for EGFR- patients. The relative risk of recurrence of disease was estimated first of all in a univariate analysis and then in a multivariate analysis, using a backward stepwise procedure. When all parameters were considered separately, tumor localization, type of surgery and EGFR status were found to be significantly associated with a greater risk of recurrence, while in the multivariate analysis only E G F R status and tumor localization remained significantly associated with a high risk of progression (Table IV). DISCUSSION

The most commonly used criteria to determine the therapeutic approach to laryngeal cancer are tumor size and location and the nodal status (Eiband et a/., 1989). However, it is well-known that the outcome of patients with the same clinical stage and who underwent the same treatment may diverge considerably. Therefore efforts have been made over the years to identify additional factors which may be evaluated at the time of presentation and which could predict outcome and therefore help in choosing the best treatment. Previous studies have shown amplification of the E G F R gene in laryngeal-cancer cell lines (Weichselbaum et a/., 1989) and in primary laryngeal carcinomas (Hishitoya et a/., 1989). Over-expression of E G F R has also been found with different techniques (Scambia et a/., 1991; Santini et a/., 1991; Hishitoya et a/., 1989; Miyaguchi et a / . . 1990). Although immunohistochemistry allows a direct observation of E G F R at cellular level and avoids the diluting effects of stromal elements and nonmalignant cells, comparison of immunohistochemical studies is difficult owing to subjectivity in evaluating and categorizing the results. Moreover. the use of frozen sections instead of formalin-fixed paraffin-embedded sections may make it almost impossiblc to compare results. On the other hand, the ligandbinding technique we used provides quantification of E G F R

expression and characterization of binding parameters of the receptor. Moreover, there is general agreement regarding the substantially good correlation between immunohistochemical and radioreceptorial results. The present data confirm our previous observations (Scambia ef a/., 1991) that higher EGFR levels are present in laryngeal cancer than in normal tissues, and suggest that uncontrolled cell growth may be mediated by abnormal EGFR expression. The association of high EGFR levels with poor histopatological grading seems to confirm that EGFR expression may correlate with more aggressive tumor behavior. In our series, E G F R + laryngeal-tumor patients have a higher risk of recurrence and shorter DFS, suggesting that over-expression of EGFR may be associated with local aggressiveness and probably with metastatic capacity of the tumor. In the univariate analysis, age and histopathological grading did not significantly affect DFS, according to Eiband ef (11. (1989), while the primary tumor site, type of surgery and EGFR status were shown to condition DFS. We observed that only tumor location and EGFR status can improve the Cox model, thus showing independent prognostic significance. It can be hypothesized that the evaluation of EGFR expression may be clinically useful in identifying laryngeal-cancer patients with a high risk of relapse who should undergo more radical surgery, elective neck dissection and/or adjuvant chemo-radiotherapy. Our data support the hypothesis that EGFR assay could usefully integrate the clinically established prognostic parameters in laryngeal cancer. However, assessment of the negative prognostic significance of EGFR status in laryngeal cancer should be investigated. A large prospective clinical trial is now in progress in our Institute. Our results support the hypothesis that drugs and biologicalresponse modifiers which can interfere with the EGFRmediated pathways of cellular proliferation may be potentially useful in the treatment of laryngeal cancer. It is worth noting that monoclonal anti-EGFR antibodies (Masui et a/.. 1988; Hirota eta/., 1989) and synthetic EGF-like peptides (Eppstein et a/., 1989) have been shown to inhibit the growth of cancer cells.

866

MAURIZI E T A L .

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