Br. 1. Cancer (1993), 68, l62~l65
`
`© Macmillan Press Ltd», 1993
`
`The relationship of quantitative epidermal growth factor receptor
`expression in non-small cell lung cancer to long term survival
`
`D. Veale‘, N. Kerr‘, G.J. Gibson‘, R]. Kelly‘ & A.L. Harrisz
`
`‘Departments of Respiratory Medicine, Freeman Hospital, Newcastle upon Tyne, Medical Statistics, University of Newcastle upon
`Tyne and 2Imperial Cancer Research Fund Clinical Oncology Unit, Churchill Hospital, Headington, Oxford OX3 71.], UK.
`
`increased expression of epidermal growth factor receptor (EGFr) has been reported in non small
`Summary
`cell lung cancers (NSCLC) when compared to normal lung. We have examined post-operative survival in 19
`surgically treated patients with NSCLC who had full characterisation of EGFr on primary tumour membrane
`preparations from resection specimens. There were ten squamous, seven adeno and two large cell carcinomas.
`The median concentration of high atiinity sites was 31 fine] per mg of protein (44532) and the median
`dissociation constant (Kd) of these high affinity sites was 2.3 X l0"° per mol (l.2—30 x 10'”). Seven patients
`survived over 5 years. Twelve patients died between 8.5 and 55 months from the time of surgery. When >5
`year survivors were compared to non-survivors there was no difference as regards tumour size or stage, or as
`regards age or sex. The survivors had a median concentration of high affinity EGFr sites of 16.1 fmol mg”
`protein compared to a median concentration of 68.6 fmol mg“ protein in the nomsurvivors (P = 0.01
`Wilcoxon test). No long term survivor had >35 fmol mg” protein of receptor. Thus EGFr quantitation may
`give independent prognostic information in NSCLC and help to select patients for adjuvant therapy after
`surgery. These results need confinnation in a larger prospective study.
`
`(EGF) has been shown to be
`Epidermal growth factor
`(Cohen & Elliott,
`1963) and
`mitogenic to ectoderrnal
`endodermal (Konturek er al., l98l) cells in vivo. EGF binds
`to a receptor (EGFr) which is a transmembrane protein with
`an extracellular binding domain and an intracellular tyrosine
`kinase domain (Carpenter, 1983). Histological study has
`indicated that the EGFr may participate in EGF induced
`proliferation of the conducting airways of human foetal lung
`(Oliver, 1988). EGFr appears to play an important role in
`the development and proliferation of some human malignan-
`cies including those of neuroglia (Liberman et al., 1984),
`bladder (Neal et a1., 1985) and breast (Sainsbury et al., 1985).
`Increased expression of EGFr appears to be particularly
`common in squamous carcinomas (Hendler er al., 1988) and
`we have shown by immunoperoxidase studies using a mono~
`clonal antibody to EGFr that tumour cells in squamous lung
`cancers have stronger staining for EGFr then other non-
`small cell lung cancers (NSCLC) (Veale et al., 1987). In that
`study staining in stage three NSCLC where the tumour was
`locally invading or with mediastinal lymph node involvement
`was greater than in stage I and II tumours with no spread
`beyond the hilar nodes.
`We have,
`in addition, shown by ligand binding studies
`with 1”‘ radiolabelled EGF that there is increased concentra-
`tion of EGFr on NSCLC compared to normal
`lung. We
`failed to find any difference in EGFr concentration or affinity
`between NSCLC of different histological
`type or clinical
`stage in radioligand binding studies (Veale et al., 1989).
`Since NSCLC with a high proportion of cells expressing
`EGFr have a high rate of proliferation (Dazzi et al., 1990)
`and since the latter is associated with a poor prognosis, we
`have examined the prognostic significance of EGFr expres-
`sion in NSCLC measured directly by ligand binding studies.
`
`Patients and methods
`
`The study population comprised 19 patients who underwent
`surgical resection of bronchial carcinoma. Patients were of
`good performance status in order to be considered for oper-
`
`Correspondence: A.L. I-larris.
`Received 12 October 1992; and in revised form 24 February l993.
`
`ation. The tumours included ten squamous carcinomas, seven
`adenocarcinomas and two large cell carcinomas. Tumours
`were staged post operatively by the tumour, nodal involve-
`ment, metastasis (TNM) system on examination of resected
`material (pTNM) (Mountain et al.,
`l97-4). We have used this
`staging system as the tumours were resected between 1984
`and l986. By these criteriae T2 tumours are greater than
`3 cm in diameter or invading the visceral pleura or there is
`atalectasis of less
`than an entire lung. N defines nodal
`invasion with N1 signifying metastasis to ipsilateral hilar
`nodes and N2 means mediastinal or subcarinal lymph node
`involvement. EGFr binding was studied by multipoint bind-
`ing assay on tumour membrane preparations as previously
`reported (Veale et a1.,
`i989).
`Tumours were collected fresh at operation and stored in
`sucrose buffer at - 18°C. Membranes were prepared by
`homogenisation of finely cut
`tissue and differential centri~
`fugation. The homogenate was centrifuged at 300g at 4°C
`for 40min. The pellet obtained formed the membrane
`preparation which was confirmed by 5’ nucleotidase estima~
`tion (Gentry & Olsson, 1975). The protein concentration of
`the membrane preparation was measured by the Bradford
`method (Bradford,
`l976) and standardised to 1000 pg ml.
`The concentration of EGFr was measured by competitive
`ligand binding studies using radio~iodine labelled EGF in
`competition with ten to 14 varying concentrations of
`unlabelled ligand (Bonnet, 1978) as previously described.
`Briefly, membrane preparation (0.1 ml) was incubated at
`26°C with 0.l ml of 1”’ labelled EGF at a final concentration
`of 0.3 nM. To the incubation were added 12 to 14 varying
`concentrations of unlabelled EGF (from O to 200 mvt). The
`solution was incubated at 26°C for 2 h conditions which had
`been established as optimal in preliminary studies. Incuba~
`tion was terminated by the addition of 1 ml of ice-cold buffer
`and centrifugation at 14000 g. The binding reaction was
`linearly related to protein concentration up to 1.5 mg ml”.
`Post operatively the patients were seen 3 monthly for the
`first 6 months and thereafter annually. At each review the
`patient had a clinical examination and chest radiograph. On
`relapse patients were referred for radiotherapy if clinically
`indicated for symptom control. One patient with disease
`involving mediastinal nodes at surgery had post operative
`radiotherapy to the mediastinum. The minimum follow up
`period was 6 years. Patient’s general practitioners were con-
`tacted for details and all deceased patients had died from
`recurrent disease.
`
`APOTEX EX. 1024-001
`
`

`
`Statistical methods
`
`Table I Comparison of survivors > 5 years with patients who died
`
`INCREASE IN EGFr IN NSCLC AND LONG—TERM SURVIVAL
`
`I63
`
`The effect of age, staging, ceil type, operation performed and
`EGFr concentration on survival were assessed separateiy
`using Cox's regression model. Furthermore,
`the efl”ect of
`EGFr expression upon survival, adjusted for each of the
`other variables separately, was assessed by fitting each
`variable followed by EGFr concentration into the Cox
`regression modei as described by Altman (1991). The Cox
`regression models were implemented via the BMDP statistical
`package using program 2L. It was not possible to do any
`further multivariate analysis due to the small number of
`subjects in the study.
`
`Results
`
`Nineteen tumours were examined by 10~ 14 point Scatchard
`analysis (Scatchard, 1949) of EGFr binding and showed high
`and low aliinity binding sites. The median concentration of
`high affinity sites was 31 fmol per mg of protein (range
`4-1532) and the median dissociation constant (Kd) of these
`high aifinity sites was 2.3 X 10"” per mol (l.2~30 X 10'”).
`The median concentration of low atfinity sites was 255 fmol
`per mg of protein (53#3892) with a median binding Kd of
`1 X i0‘° (0.8 to 41).
`The clinical
`features of the patients and tumours are
`shown in Table I. The median age at operation was 60 years
`(39-74). Tumour stage by the TNM system showed a
`majority of patients to have large tumours T2-T3. Eleven
`patients had spread to the hilar lymph nodes only (Ni) and
`one had involvement of mediastinal nodes (N2). No patient
`showed evidence of systemic metastases at
`the time of
`surgery.
`The first patient died 7.5 months after surgery. Seven
`patients have survived over 5 years from the time of oper-
`ation with the iongest survival to the time of data analysis
`being 71 months. Twelve patients have died between 8.5 and
`55 months from the time of surgery. Ail patients died of
`metastatic disease.
`
`When 5 year survivors were compared to non-survivors
`
`Number
`Male
`Age
`Pneumonectomy
`Lobectomy
`Tumour size
`T3
`T2
`Tl
`Nodes positive
`Squamous
`Carcinoma
`Adenocarcinoma
`Large cell
`Differentiation
`Well
`Poor
`Median EGFr (fmol mg“)
`high affinity
`Range
`
`<5 yrs
`l2
`l0
`60 (S4~74)
`2
`10
`
`>5 yrs
`7
`5
`57 (39~67)
`4
`3
`
`3
`7
`2
`8
`8
`
`4
`0
`
`7
`5
`68.6
`
`2
`5
`O
`4
`2
`
`3
`2
`
`2
`5
`16.!
`
`l0.5— 1533
`
`4.3—34.4
`
`(Tabie I) there were no dilferences in tumour size, stage or
`type. The two large ceil cases were in the surviving group,
`and ten of the l2 patients who died had a lobectomy com-
`pared to three of the seven survivors. There was no difference
`between the groups as regards age or sex.
`The 5 year survivors had a median (range) concentration
`of high affinity EGFr sites of 16.1 (4.3~34.4) fmol mg“ pro-
`tein compared to a concentration of 68.6 (l0.5~l533)
`fmol mg“ protein in the non-survivors (P = 0.001 Wiicoxon
`test). All patients with high aflinity receptor concentrations
`greater than 35 fmol mg” had died within 5 years, whereas
`seven of ii patients with receptor concentrations less than
`this value were still alive after 5 years (P= 0.02 Log rank
`test) (Figure I). A univariate analysis of the influence of
`other prognostic factors in comparing patients with tumours
`having EGFr concentration <35>fmol mg“ protein showed
`
`Survival curve < 35 fmol >
`
`Survival and EGFR concentration
`72 —~
`0O
`-o
`-00
`
`b
`
` o
`
`10
`
`20
`
`30
`Survival (months)
`
`EL’!
`
`0I
`
`3HO
`
`°"
`
`DE)
`
`B
`
`<35 fmol mg“
`[EGFg]
`
`>35 fmol mg”
`
`as
`
`so
`
`Squamous carcinoma
`O
`Non-squamous carcinoma
`[:3
`Closed Pneurnoneetomy
`Open Lobectomy
`
`a, Log rank survival stratified by EGFr greater than 35 fmol mg" membrane protein or less than 35 fmol mg”'. Seven
`Figure I
`patients are alive after a minimum of 5 years follow up. There were eight patients in the group with > 35 fmol mg“ and 11 in the
`group with <35 fmol mg".
`I), Survival
`vs different histological
`subtypes and operations,
`stratified by EGFr> 35 or
`<35 fmolmg” membrane protein.
`
`APOTEX EX. 1024-002
`
`

`
`164
`
`D. VEALE et al.
`
`Table I! Univariate Cox regression results
`
`Hazard ratio
`
`95% C.1. for HR.
`
`P-valve
`
`1.06
`0.76
`1.08
`2.61
`2.95
`5.67
`
`0.98,
`0.2l,
`0.32,
`0.77,
`0.64,
`1.58,
`
`1.15
`2.83
`3.60
`8.81
`l3.50
`20.38
`
`0.12
`0.68
`091
`0.12
`0.18
`0.008
`
`Variable
`
`Age
`T
`N
`Cell type
`Op. type
`EGFR
`
`Table III Effect of EGFR adjusted for each of age, T, N, cell type and
`op. type separately
`
`EGFR
`(age adjusted)
`EGFR
`(T adjusted)
`EGFR
`(N adjusted)
`EGFR (cell type
`adjusted)
`EGFR (op. type
`adjusted)
`
`Hazard ratio
`4.75
`
`95% C.I. for HR.
`1.28,
`17.5
`
`P-valve
`0.02
`
`5.77
`
`8.59
`
`6.58
`
`4.73
`
`1.56,
`
`21.37
`
`1.83,
`
`40.26
`
`1.68,
`
`25.76
`
`1.25,
`
`17.97
`
`0.009
`
`0.006
`
`0.007
`
`0.02
`
`that no other variable significantly affected survival difference
`between the two groups (Table II). The unadjusted effect of
`having GFr > 35 frnol mg” is to have an approximate 5-fold
`increase in risk of death, as derived from a hazard ratio of
`5.67 for EGFr in Table II. Adjusting the effect of EGFr
`separately for each of age, T stage, N stage, cell type and
`operation type leads to similar conclusions (Table lll).
`
`Discussion
`
`lung
`We have shown that patients with non-small cell
`tumours which have a high concentration of EGFr have a
`shorter survival than those with tumours with a lower con-
`
`in
`expression of EGFr
`receptors. Over
`centration of
`squamous carcinoma of the head and neck has been found to
`be associated with poor survival (Hendler er al., 1988). In
`bladder cancer the level of EGFr is associated with the
`degree of invasion and with poor differentiation (Neal et al.,
`1985).
`The highest concentration of receptor in the survival group
`was 34.4 fmolmg“ of membrane protein. If we take this
`level as a cut—ofl' point and examine survival difference
`(Figure 1) there is a highly significant difference in survival
`(P= 0.02). Thus this cut-off point could be used to define
`prognostic groups.
`One possible mechanism by which EGFr might play a role
`in tumour progression is that subclones of tumour cells that
`express more EGFr may be selected for growth, invasion and
`metastasis. EGF: may be implicated in the growth and
`spread of tumours through an autocrine mechanism whereby
`tumour cells possessing receptors secrete the growth factor
`
`References
`
`which interacts with the receptor to stimulate further growth
`(Sporn & Todaro, 1980). The addition of EGF to culture
`medium has been shown to lead to increased growth in lung
`cancers of all types (Singletary et al., 1987). Infusion of EGF
`into athymic mice with implanted squamous tumours expres-
`sing a high concentration of EGFr led to increased growth of
`the tumours (Ozawa et al., 1987).
`The results presented here are comparable to those of
`Tateishi et al. who studied adenocarcinomas of the lung and
`showed by immunocytochemical staining that patients with
`EGFr positive tumours and strong staining for TGFa had a
`significantly reduced survival compared with those with
`positive EGFr and little TGFa staining (Tateishi et al., I990).
`In that study cases that demonstrated high expression of
`growth factors with co-expression of receptors were in
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`like us, a significantly higher concen-
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`
`Radioligand binding analysis may involve examination of
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`to immunohisto-
`chemistry. It is, however, quantitative and we ensured to cut
`tumour tissue from the centre of the tumour to prepare
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`antibody EGFR]
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`Although this study examines a small number of cases, no
`pre-selection was made to analyse these particular tumours.
`None of the other prognostic studies had quantitative data
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`then EGFr assay may be clinically useful in selecting patients
`for adjuvant therapy using either chemo or radiotherapy or
`new therapeutic approaches targeted at the receptor (Mul-
`shine er al., 1989).
`
`We acknowledge the helpful criticism of Dr D. Moro.
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`APOTEX EX. 1024-004