Br. J. Cancer (1986), 54, 265-269
`
`Expression of epidermal growth factor receptor (EGF-R) in
`human lung tumours
`
`T. Cerny1*, D.M. Barnesz, P. Hasleton3, P.V. Barber3, K. Healyz, W. Gullick“
`& N. Thatcherl
`
`‘CRC Department of Medical Oncology; 2Department of Clinical Research Christie Hospital, Manchester
`M20 9BX; 3Departments of Pathology & Medicine, Wythenshawe Hospital, Manchester M23 9LT; ‘Imperial
`Cancer Research Fund, Lincoln ’s Inn Fields, London WC2 3PX, UK.
`
`Summary Epidermal growth factor receptor (EGF-R) expression was assessed in 63 lung tumour samples
`with a monoclonal antibody (EGF—Rl) by indirect irnrnuuoperoxidase staining on cryostat sections. All 15
`small cell lung cancer samples were negative whereas over 80% of the 48 non small cell lung cancer stained
`positively.
`the cytoplasmic part of the EGF-R were
`In 30 bronchial biopsies two monoclonal antibodies against
`evaluated. These antibodies showed weaker staining than EGF-R1. No additional or enhanced staining as
`compared with EGF~Rl was observed, suggesting a lack of enhanced expression of a truncated EGF-R
`analogous to the v-erb~B oncogene product.
`Monoclonal antibodies against the EGF-R may be helpful diagnostically in differentiating small cell from
`non small cell lung cancer and may also be important in elucidating biological differences in primary lung
`0811061".
`
`During the last few years epidermal growth factor
`(EGF)
`and its
`receptor
`(EGF~R)
`have
`been
`intensively investigated in biological research (for
`review see Hunter & Cooper,
`i985). EGF-R is
`present in a wide range of normal epithelial tissues
`whereas EGF is found in normal human plasma
`and in almost all human body fluids (Gustcrson er
`al., 1984: Kasselberg er al., i985).
`The role of this particular growth factor and its
`receptor is still poorly understood both in normal
`and in disease states. As a result of EGF binding to
`its
`specific
`receptor
`there
`is
`increased DNA
`synthesis as well as other events
`such as cell
`proliferation, differentiation and repair of damaged
`epithelial
`tissue (Schlessinger et al., 1983; King er
`al.,
`1985). Conversely in cells possessing high
`numbers
`of EGF-R (A43l
`derived,
`vulval
`squamous carcinoma) a retardation of proliferation
`after incubation with EGF has been noted (Barnes,
`1982).
`A close similarity between the sequence of the
`v-erb-B oncogene of AEV-H (a strain of the avian
`erythroblastosis virus) and the cytoplasmic and
`transmembrane part of
`the EGF-R (truncated
`EGF-R) has been found (Downward et al., I984).
`It was hypothesised that an inappropriate activa-
`tion of the human erb~B gene either by truncation
`
`Correspondence: T. Cemy
`*Present
`address: Department of Medical Oncology,
`lnsehospital, University of Bern, CH-3010 Switzerland.
`Received l3 March i986; and in revised form 2 May
`1986.
`
`or overexpression plays a role in the development
`of malignancy (Newmark, 1984). This hypothesis is
`supported by preliminary studies which have shown
`an increased number of EGF-R in various malig-
`nant tumours (Hendler & Ozanne, 1984; Libermann
`er al.,
`i984; Neal et al., 1985; Gusterson er al.,
`i985; Gullick er al., 1986). In human breast cancer
`EGF-R was increased in metastases (Fitzpatrick et
`al., 1984) and was inversely related to the steroid
`receptor status (Sainsbury et al., 1985).
`In lung cancer there have been only a few reports
`using radioimmunoassay techniques involving small
`numbers of cases, probably because EGF-R assess-
`ment requires unfixed tissue (Hendler & Ozanne,
`1984; Sherwin et al., 1981). We have collected
`biopsies of 63 patients with lung cancer and
`assessed their EGF-R expression using an indirect
`immunoperoxidase method with a monoclonal
`antibody against
`the EGF-R (Waterfield et» al.,
`1982). In 30 of these 63 patients, two new mono-
`clonal antibodies against
`the cytoplasmic part of
`the EGF-R were evaluated. This was done in order
`to
`determine whether
`tumour
`samples
`ever
`expressed truncated EGF-R which would be a
`homologue of the v-erb-B oncogene product
`(see
`Figure 1).
`
`Materials and methods
`
`samples were obtained from 25
`Lung tumour
`patients undergoing thoracotomy and a further 42
`
`© The Macmillan Press Ltd., 1986
`
`APOTEX EX. 1018-001
`
`

`
`266
`
`T. CERNY er al.
`
`at bronchoscopy. Of the latter, 4 patients had
`pulmonary metastases
`from an extrapulmonary
`primary (1 breast cancer,
`1 ovarian cancer and 2
`non—Hodgkin lymphoma metastases). A further 20
`bronchoscopic tissue samples with no or very few
`tumour cells were also evaluated. Of the total 63
`patients with primary lung cancer the cell types are
`as follows: 15 small cell
`lung cancer (SCLC), 42
`squamous cell
`lung cancer, 4 adenocarcinoma,
`l
`pleomorphic adenoma and 1
`large cell carcinoma.
`There were 55 men and 8 women, with a mean age
`of 62 years. Placental
`tissue served as a positive
`control
`in EGF«R1 experiments and in addition
`cervical squamous epithelium was used for EGF-
`antibodies .
`RF4 and EGF-RDl0, monoclonal
`against a synthetic peptide from «the cytoplasmic
`domain of the EGF~R (residues 985~996). All three
`monoclonal antibodies against
`the EGF-R were
`also tested in A431 cells (kindly provided by Ana
`Schor). In each experiment a negative control using
`PBS instead of the primary antibody was used.
`In the cryostat, frozen sections (6 pm thick) were
`cut from lung biopsies obtained as described. All
`samples were snap frozen in liquid nitrogen and
`stored at ««l90°C. The sections were placed on
`slides
`treated with poly l~lysine
`(0.01%). Cut
`sections were kept
`in the cryostat cabinet until
`cutting was complete. One section from each
`sample was stained with haematoxylin and eosin.
`Sections were taken from cold cabinet and placed
`in acetone/chloroform (1:1)
`for 5min, and then
`washed in PBS for 2min. Endogenous peroxidase
`was blocked with 0.3% hydrogen peroxide in PBS
`for 10min, followed by 2x5min washes in PBS.
`The sections were then covered with normal rabbit
`serum/PBS 1:5 for 10 min and excess serum drained
`from and wiped off the slides but keeping the
`sections moist. The sections were thereafter covered
`with EGF-R antibody diluted l
`in 50 with diluted
`rabbit serum. They were left for 90min and washed
`in PBS for 2 x 5min after which they were covered
`in diluted rabbit serum for 10min. Excess serum
`was drained from the slides but the sections were
`again kept moist and covered with secondary
`antiserum (peroxidase conjugated rabbit anti~mouse
`antiserum) diluted 1/20 with diluted rabbit serum,
`left
`for 60min and then washed in PBS for
`2x 5min. Finally, peroxidase activity was demon-
`strated using diaminobenzidine solution (DAB),
`followed by counterstaining with haematoxylin.
`The histological sections were evaluated by two
`observers and the degree of staining scored as
`follows: 0=negative; + <25% positive
`stained
`tumour cells; + + x25-50% and + + + > 50%.
`In cases where observers disagreed the sections
`were studied together and agreement was reached.
`The chi~square test was used to analyse the
`results.
`
`External domain including
`the ligand binding site
`
` ”Truncated" EGF—R:homologous
`
`/
`mAb scan. //
`// /’
`
`
`to the v-erb B oncogene product
`(Dnca:d 1984)
`
`Figure 1 The transmembrane EGF-R and the three
`monoclonal antibodies (mAb) used in this study. EGF~
`RI mAb: (Waterfield et al.,
`i982).
`lgG2b subclass.
`Antiprotein mAb to the external domain of the EGF-
`R but does not compete for EGF-binding. EGF—RF4
`mAb and EGF-RD10 mAb:
`(Gulliek et al., 1986).
`lgGI
`subclass. Antipeptides mAb to a synthetic
`peptide from near the C-terminus of EGF~R, (residues
`98S~996).
`
`Results
`
`the staining with the EGF-Rl
`The results of
`monoclonal antibody are summarised in Table Ia.
`There is an obvious difference of EGF-R expression
`between the SCLC with no positive
`staining
`compared to the other lung primaries where 85% of
`cases were positively stained (p<0.0000l).
`Most squamous cell bronchial carcinoma were
`strongly positive in most of the tissues examined
`(Figure 2a). Often negative or weakly stained areas
`were adjacent
`to strongly positive areas. The 8
`negatively stained squamous cell carcinoma were
`all tiny biopsies. Two moderately well differentiated
`adenocarcinomas of the lung were strongly positive
`(Figure 2b);
`one
`poorly
`differentiated
`adeno~
`carcinoma was also positive whereas another was
`negative. In these samples the staining pattern was
`homogeneous. A pleomorphic adenoma of the lung
`and one large cell carcinoma were also positively
`stained.
`In the SCLC samples
`foci of slightly
`positively stained cells were often found. The cells
`of
`these
`groups
`in
`general
`contained more
`cytoplasm.
`Tissue of 4 lung metastases were also examined:
`two non Hodgkin lymphomas and a breast cancer
`metastasis were negative whereas a metastasis from
`an ovarian carcinoma was positive.
`In a further 20 lung biopsies no malignant cells,
`or only very few were found. In these samples, as
`well as in lung tissue adjacent
`to the tumour,
`squamous metaplasia
`and
`often macrophages
`stained positively. Also serous and mucinous glands
`often
`contained
`positive
`areas
`of EGF-R
`expression, as well as basal bronchiolar epithelial
`cells.
`the
`against
`antibodies
`two monoclonal
`The
`cytoplasmic part of the EGF—R (EGF-RF4 and
`
`APOTEX EX. 1018-002
`
`

`
`EPIDERMAL GROWTH FACTOR RECEPTOR
`
`267
`
`(a) Squamous cell carcinoma of the lung (Pt l6), moderately well differentiated, with strong
`Figure 2
`positive staining of the tumour cell membrane and less of the cytoplasm. In areas of early keiatinisation (left
`of the middle) the staining is less intensive (mamification: x160). (b) Adenomncinoma of the lung (Pt 19),
`positively stained. The staining pattern is slightly granular and more intense on the cell membrane (EGF-R1;
`magnification: x 320). (c) Small cell lung cancer (Pt 47), no positive staining (EGF-RI; magnifiation: x 320).
`(d) Squamous cell carcinoma of the lung (Pt 5), tiny bronchial biopsy which shows intensive staining of the
`cell membrane and only slight positivity of the cytoplasm (EGR-Rl; magnification: x 320).
`
`EGF-RDIO) were evaluated in 30 lung cancer
`biopsies and showed less intensive staining than
`with the EGF-R1 antibody (Table lb). EGF-RDIO
`was significantly weaker than EGF-RF4 when used
`at
`the
`same concentration. Positive EGF-RF4
`staining was demonstrated in l9 of 22 EGF—Rl
`positive squamous cell
`tumours,
`the pattern of
`staining being similar although weaker.
`In no
`instance was a positive result obtained with these
`two antibodies in an EGF-R1 negative tumour
`sample.
`
`n.
`
`.
`
`Small cell lung tancer is biologically different from
`other primary lung tumours since it often shows
`
`early widespread disease, a high growth fraction
`and a short cell doubling time. However,
`it does
`have a high response rate to chemotherapy. There:
`fore in oncological practice the classification of
`SCLC versus NSCLC is
`important
`in patient
`management.
`In our hands immunohistochemical analysis with
`monoclonal antibodies against the EGF-R showed
`no positive staining in any of the 15 tissue samples
`from SCLC cases wherms in all groups of NSCLC
`examined the majority of tumours were positive.
`Therefore
`for diagnostic purposes monoclonal
`antibodies against EGF~R may be important for
`the future. Moreover the lack of increased EGF-R
`expression in SCLC may reflect a more funda-
`mental difference from other primary lung tumours.
`Foci of faintly positive cells were seen in SCLC
`
`APOTEX EX. 1018-003
`
`

`
`268
`
`T. CERNY et al.
`
`Table la lndirect immunoperoxidase staining with EGF-Rl in lung tumour samples
`EGF-Rl
`Total
`+ +
`positive
`
`0
`
`+
`
`+ + +
`
`Tumour type
`
`6“
`0
`8
`28
`36
`Squamous cell lung cancer (n = 42)
`l
`0
`O
`3
`3
`Adenocarcinoma (n = 4)
`O
`0
`0
`I
`1
`Pleornorphic adenoma (n= 1)
`0
`0
`0
`1
`1
`Large cell lung cancer (:1: 1)
`00015" 0
`Small cell lung cancer (SCLC) (rt =15)
`
`
`
`
`
`
`
`
`
`Table lb Indirect immunoperoxidase staining with EGF—RF4 and EGF—_RI)l0
`EGF-RF4
`EGF—RDl0
`+
`+ + + + +
`+
`+ + + + +
`
`0 4
`
`0
`0
`5
`
`Tumour type
`
`Squamous cell lung cancer (1: =23)’
`Large cell lung cancer (7: =1)
`Adenocarcinorna (n = l)
`Small cell lungucancer (n = 5)
`
`0
`
`0
`0
`0
`0
`
`l4
`1
`l
`0
`
`5
`0
`0
`0
`
`l0
`l
`l
`5
`
`6
`0
`0
`0
`
`7
`0
`0
`O
`
`0
`O
`0
`O
`
`The histological sections were evaluated by two observers and the degree of staining scored as
`follows:
`
`0 -« negative;
`«I» = <25‘}{, positive stained tumour cells;
`+ + =25~50‘,’{,;
`+ + + m >50~l00%;
`nd = not done.
`
`‘All these samples were tiny biopsies.
`“Small foci of positive stained cells often seen.
`”22 of these samples were positive with EGF-R1.
`
`showed a decreased
`cells
`and these
`samples
`This may
`reflect
`ratio.
`nuclear/cytoplasmic
`squamous or adcnocarcinomatous differentiation in
`a few cell groups. It may be that all bronchogenic
`carcinomas have a common cell of origin and
`indeed on close inspection especially with mono-
`clonal antibodies one can find all
`the main cell
`types in selected tumours (Gatter et al., 1985).
`None of the SCLC samples showed detectable
`expression of the cytoplasmic part of the EGF—R
`(truncated EGF-R) as assessed by the two new
`monoclonal antibodies (EGF—RDl() and EGF-RF4).
`Therefore
`immunohistochemically
`no
`increased
`expression of a truncated EGF receptor analogous
`to the v-erb-B gene product could be found in
`this series.
`Forty-one of the 48 NSCLC samples showed
`positive staining with EGF-Rl. The group included
`squamous,
`adeno
`and
`large
`cell
`bronchial
`carcinoma as well as a pleomorphic adenoma. The
`conclusion of an earlier report {Hendler & Ozanne,
`1984) that adenocarcinomas may be distinguished
`from squamous cell carcinomas by their EGF-R
`expression could not be confirmed in our series.
`
`stain
`not
`did
`carcinoma
`cell
`Squamous
`homogeneously and negative areas were seen. This
`may
`reflect different
`cell
`clones or
`lack of
`expression of EGF-R_ in some cells. The six
`negative squamous cell
`tumour
`samples
`in our
`series were all tiny biopsies and may not have been
`representative of
`the overall
`staining pattern.
`However, none of the thoracotomy samples of the
`squamous
`cell carcinomas were negative. The
`inevitable crushing of the biopsy during bronchoscopy
`could have destroyed the cell membrane leading to
`negative staining in these very small samples.
`Interestingly
`one metastasis of
`an ovarian
`carcinoma was positive,
`in keeping with recent
`findings
`(Gullick
`et
`al.,
`1986) whereas
`one
`pulmonary breast carcinoma metastasis and two
`non Hodgkin lymphomas were negative. A further
`20 lung tissue samples with few or no malignant
`cells
`showed
`positive
`staining
`of
`squamous
`metaplasia. Serous and mucinous bronchial glands
`as well as macrophages were invariably positively
`stained. Recently a new v-erb-B related gene has
`been found in a human mammary carcinoma and
`other tumours and the immunological properties of
`
`APOTEX EX. 1018-004
`
`

`
`EHDERMAL ‘GROWTH FACTOR RECEPTOR
`
`269
`
`insight
`cancers.
`
`in biological differences of primary lung
`
`T. Cerny is a recipient of an EORTC Research
`Fellowship. This study was supported by the Cancer
`Research Campaign of Great Britain.
`
`(1985).
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`its oncogene product in comparison to the EGF-R
`has yet
`to be determined (King et al.,
`1985;
`Yamamoto et al., 1986; Bargmann et al., i986).
`We conclude that
`immunohistochemistry with
`monoclonal antibodies against EGF-R may play a
`role
`for diagnostic purpose by differentiating‘
`between SCLC and NSCLC and may provide new
`
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