{CANCER RESEARCH 53. 32l7~3220. July l5, l993l
`
`Advances in Brief
`
`Expression of Mutated Epidermal Growth Factor Receptor by Non-Small Cell
`
`Lung Carcinomas‘
`
`Irma E. Garcia dc Palazzof Gregory P. Adams, Padma Sundareshan, Albert J. Wong. Joseph R. Testa,
`Darell D. Bigner, and Louis M. Weiner’
`Department of Medical Oncology. Fox Chase Cancer Center, Philadelphia, Pennsylvania /91!) (I. E. 0.. G. F’. A..
`for 8min Tumor Research, Duke University Medical Camel: Durham, North Carolina 27710 [D. D. 8.]
`
`I’. S.. A. 1. 9V.. 1. R. 71. L M. W]. and the Preuss Laboratory
`
`Abstract
`
`The development of novel inununotherapy strategies for non-small cell
`lung cancer (NSCLC) will be facilitated by the identification of tumor-
`specific targets. Although the epidermal growth factor receptor (EGFR) is
`overexpressed in many cases of NSCLC, its wide distribution in normal
`tissue may limit its suitability as an imrnunotherapeutic target. However,
`mutations within the EGFR that are unique to malignanciu may provide
`specific targets for lmrnunotherapeutic intervention. For example, one
`mutant form, the type III deletion mutant of the EGFR, that has been
`identified in glloblastomas contains a novel peptide sequence In its extra-
`cellular domain which is detectable by anti-peptide antls-era. in this study,
`the prevalence of this type of mutation of the EGFR in NSCLC was
`determined. Thirty-two frozen sections of primary NSCLC were examined
`by lmmunocytochemistry to determine the presence of native and mutated
`EGFR. Native EGFR was overexpremed in 12 of the 32 sections and a
`diffuse cellular distribution of the EGFR type III deletion mutation was
`identified lnllve(l6%)ofthespecimens (2ofl3squamous,2of2mixed,
`0 of 10 adenocarclnoma, and I of 7 undifferentiated). This mutated EGFR
`was not detected in sections of normal breast, lung, sldn, ovary, colon,
`kidney, endometrlum, and placenta. The type III EGFR deletion mutant,
`expressed in some casw of NSCLC, may be a molecularly defined, tumor-
`specific antigen in lung cancer.
`
`Introduction
`
`NSCLC‘ is the most frequent cause of cancer death in the United
`States. Cure rates are directly related to clinical stage (70% for Stage
`I, decreasing to less than 5% for Stage IIIB) (1). Therapy using
`various permutations of surgery, radiotherapy, and chemotherapy has
`had limited effects on cure rates and median survival in advanced
`
`stages of NSCLC (l). Clearly, novel therapeutic targets and agents are
`needed for the management of this disease. One such approach in-
`volves immunothcrapy directed against antigens which are either se-
`lectively expressed or over expressed in malignant cells. A number of
`potential targets have been identified; the potential suitability of the
`EGFR as a target has been the subject of intensive study.
`Overexpression of EGFR has been associated with a number of
`neoplasms, including breast carcinoma (2. 3), adenocarcinoma and
`squamous cell carcinoma of the lung (3-7). large cell carcinoma of the
`lung (3, 5, 6), gliomas (8), and a variety of bladder (9, 10) and
`gynecological tumors (ll). In both bladder and breast cancer. a poor
`prognosis has been correlated with high expression of EGFR (2, 3, 9,
`
`Received 3/19/93; accepted 6/3/93.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`l8 USC. Section i734 solely to indicate this fact.
`' Supported by National Cancer institute Grants CA06927, CA50633, and CA5l880
`and the Bernard A. and Rebecca 8. Bernard Foundation.
`3 Present address: Department of Pathology and Labomwry Medicine. Temple Uni-
`versity. Philadelphia. PA l9l44.
`3 To whom requests for reprints should be addressed, at Fox Chase Cancer Center,
`Department of Medical Oncology. ‘/70! Burholme Avenue. Philadelphia. PA Will.
`‘ The abbreviations used are: NSCLC, non-small cell lung cancer; EGFR. epidermal
`growth factor receptor: PBS. phosphatebuffcred saline; iSCN, imemational System for
`Human Cytogenetic Nomenclature (i985).
`
`10). Due to its high expression by a number of neoplasms, the EGFR
`has been utilized as a tumor-associated target for tumor detection and
`therapy. In imaging studies performed in patients with squamous cell
`carcinoma of the lung, over 94% of primary lesions and sites of
`presumed metastases were detected when doses of 40 mg or more
`monoclonal antibody were utilized (i2, 13). In mice bearing a lethal
`inoculum of tumor cells expressing EGFR. treatment with transform-
`ing growth factor a-Pseudomonas exotoxin 40 fusion proteins more
`than doubled median survival. The fusion protein did not significantly
`prolong the survival of mice inoculated with EGFR-negative tumor
`cells (14, 15).
`Amplification of the EGFR gene has frequently been associated
`with EGFR overexpression in gliomas (8, 16, I7) and has been re-
`ported in squamous cell carcinomas (including those of the lung)
`(l8-20). breast carcinomas (21). and bladder tumors (9). Rearranged
`or truncated forms of EGFR have been identified and are often asso«
`
`ciated with gene amplification in gliomas (22-26). To date, three
`truncated forms of EGFR have been reported. The type I deletion
`mutant lacks the majority of the extracellular domain and is unable to
`bind epidermal growth factor (26). The type ll deletion mutant con-
`tains an in-frame deletion of 83 amino acids (520-603) in domain IV
`of its extracellular domain and is capable of binding epidermal growth
`factor and transforming growth factor a (23). The type Ill deletion
`mutant, reported to occur in 17% of the glioblastomas screened by
`Humphrey et al. (22), appears to be the most prevalent. This mutation
`generates a fusion junction sequence which is reported to be unique to
`malignancies. This sequence results from an in-frame deletion of 267
`amino acids (from amino acids 29-296) spanning the first and second
`extracellular domains of the receptor (22, 26, 27). A synthetic peptide
`spanning the deletion junction (lei-Leu~Glu~Glu-Lys-Lys—-Gly—Asn-
`Tyr-Val—Val-Thr—Asp—iiis—Oh) has been used to raise antisera spe-
`cific for this mutant protein. To determine if this mutated growth
`factor receptor merits consideration as a tumor-specific target
`in
`NSCLC, 32 sections of primary NSCLC were studied for the presence
`of the type lll EGFR deletion mutation.
`Materials and Methods
`
`Antibodies. Rabbit and goat antiserum specific for the EGFR type lll
`deletion sequence were prepared as described (22). Briefly. a l4-amino acid
`peptide spanning the predicted amino acid sequence of the fusion junction was
`synthesized. purified, and chemically conjugated to keyhole limpet hemocya~
`nin. Animals were immunized with a l:l emulsion of the conjugate in PBS and
`complete Freund's adjuvant. Boosts were performed on day 33 with con}ugate
`in a H emulsion with incomplete Freund’s adjuvant. The animals were bled
`and the anti-peptide antibody was purified from the antisera on a peptide-
`Sepharose affinity column.
`lmmunohlstocbemistry. Frozen sections of NSCLC and normal tissues
`were obtained from the Fox Chase Cancer Center Tumor Bank. The human
`
`glloblastoma multlforme xenograft (D-270 MG—X). expressing the type Ill
`EGFR deletion mutation, was utilized as a positive control. The sections were
`stained with hematoxylin-eosin to evaluate histology. Five-pm sections were
`fixed for 20 min in cold acetone and air-dried at room temperature. The
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`sections were then incubated in Tissue Conditioner Reagent (Biomeda Immu-
`nohistochernical Staining Kit. Foster City, CA) for 10 min at room tempera-
`ture. A blocking reagent (normal rabbit or goat serum) was then added and the
`sections were incubated at room temperature for 30 min. The blocking reagent
`was blotted and the samples were rinsed with 0.01 M PBS. pH 7.2. The sections
`were then incubated ovemiglit at 4°C with 5 p.g/ml of primary aritisera reactive
`with the type III deletion junction sequence, normal rabbit/goat serum. or 3
`pg/ml of affinity purified rabbit antibody to native EGFR (Ab-4; Oncogene
`Science, Inc, Manhasset. NY). After the slides were washed with PBS. 100 pl
`of a l:l dilution of biotinylated goat anti-rabbit lgG (Bioflenex Laboratories,
`San Ramon, CA) or rabbit anti-goat IgG (Biomeda) as appropriate were ap-
`plied and allowed to incubate at room temperature for 45 min. The sections
`then were washed twice with PBS and incubated with 100 ill of streptavidin
`alkaline phosphatase for 45 min at room temperature. They again were washed
`with PBS and incubated with 100 rd of Fast Red Chromogen (naphthol
`phosphate) (BioGenex Laboratories). The slides were washed in PBS and
`counterstained with Mayer's hematoxylin (Sigma Diagnostics, St. Louis, M0)
`for 5 min. The validity of immunohistochernical detection of cells expressing
`the type lll EGFR deletion mutation in gliomas, using these antiseia. previ-
`ously has been confimied by Western blot analysis (22).
`Karyotypic Analysis. In one case in which a fresh specimen was available,
`tissue was disaggregated by mechanical means (28). Actively growing cells
`were arrested in metaphase by exposure to 0.03 uglml colcernid (GIBCO
`Laboratories) for 16-20 h. Cells were then treated with a hypotonic solution of
`0.075 M KCl for 20 min at 37°C. They were fixed in a 3:l solution of
`methanoltglacial acetic acid. Chromosomes were analyzed using a G-banding
`technique (29). Chromosome identification and ltaryotypic designation were
`performed in accordance with the ISCN (30).
`
`Results
`
`lmmunoltlstochemlstry. Twelve of the 32 sections (38%) of
`NSCLC were stained by affinity purified rabbit antibody to native
`EGFR (Ab-4) by the irnrnunoperoxidase method (Table 1). Addition-
`ally, 5 of the 12 (16% overall) specimens with high levels of native
`EGFR also diffusely expressed the type III deletion mutant. Expres-
`sion of this deletion mutant was limited to malignant squamous epi-
`thelial cells and was not observed in cells with adenocarcinoma dif-
`
`ferentiation. ln the two positive cases of adenosquarnous carcinoma,
`both native EGFR and the deletion mutant were observed only in the
`squamous cell component (Fig. l). in the positive specimens, all of the
`squamous cells stained positive for the deletion mutant. All cells
`which were found to express the deletion mutant also reacted with the
`rabbit antiserum to native EGFR. The type III deletion mutant was
`found to be predominantly associated with the cellular membrane,
`although in some cases cytoplasmic staining was observed. Normal
`tissue samples were screened for the presence of both native EGFR
`and the deletion mutant. Native EGFR was found to be highly ex-
`pressed on skin (2) and endometriurn (2). diffusely expressed on
`scattered cells in the lungs (2) and the colon (3), and not observed on
`breast (3), ovary (3), kidney (2), and placenta (5). Antiserum directed
`against the type III deletion mutant did not bind to any of the above
`normal tissues.
`
`Table I Immunohistochemical detection of native EGFR and EGFR type III deletion
`mutant expressed in non-small cell lung carcinoma
`Expression of EGFR
`
`_~_ “_35_'_°‘38z >_‘M___>___mMm_“[‘{*jj’°_____’jf‘flf”‘
`Squamous
`13
`5
`2
`:fi::::‘}r";‘::";:
`1%
`g
`3:
`Undifferentiated
`7
`3
`1
`Total
`32
`l2
`5
`----—;l--16“-z~;-b;r—(<)~—f;I;a;;—---—---—-—-
`---—-—~
`b Pemmgc ofsampks positive for mum,‘
`“ Positive staining only observed on squamous component.
`
`% 0‘ mum”
`15
`mg
`M
`l6
`
`MUTATED EGFR lN NON-SMALL CELL LUNG CARCINOMA
`
`Karyotypic Analysis. One sample of squamous cell carcinoma,
`positive for expression of the type III deletion mutant, was available
`for ltaryotypic analysis. This specimen was found to be near triploid.
`Most cells examined had four copies of chromosome 7 plus two
`copies of a translocation derivative, der(2)t(2;7), containing an extra
`portion of most of the short arm of chromosome 7 (Fig. 2). The
`derivative included the region containing the EGFR gene, presumably
`resulting in six copies of the gene/cell.
`
`Discussion
`
`This study provides a preliminary assessment of the prevalence of
`EGFR type III deletion mutant expression in NSCLC. The mutant was
`present in l6% of all NSCLC specimens we tested and in 42% of the
`cases in which overexpression of the EGFR was detected. The prev-
`alence of the EGFR type III deletion mutant in NSCLC may actually
`be greater than reported here, because immunocytochemistry tech-
`niques are dependent upon both thesensitivity of the antibodies and
`detection systems which are used. For example, in this assay, we
`observed native EGFR expression in 38% of the NSCLC samples
`examined. However, EGFR expression in NSCLC, as detected by
`immunocytochemistry, has been observed in 52-83% of specimens
`studied using other reagents (9, 31).
`Expression of the deletion mutant was observed only in malignant
`squamous epithelial cells. In the cases of adenosquamous carcinoma
`studied, only the squamous component was positively stained. Normal
`breast, lung, skin, ovary, colon, and endometrium failed to react with
`antiserum against the deletion mutant fusion junction. The staining
`pattern of the positive cells revealed that the predominant expression
`of the EGFR type III deletion mutant was membrane associated.
`While cytoplasmic staining was observed in some cases, nuclear stain-
`ing was never observed. Expression of the type III deletion mutant
`was continued by Western blot analysis in the two cases where frozen
`tissue blocks were available (results not shown).
`Cytogenetic analysis of one squamous cell carcinoma revealed
`polysomy 7 and extra copies of most of 7p; the latter were contained
`within two identical translocation derivatives. der(2)t(2;7) (Fig. 2).
`The brealtpoint on chromosome 7 in the der(2)t(2;7) is at 7pl I .2-p12,
`which is near the location of the EGFR gene, but we do not know if
`this structural rearrangement has disrupted the EGFR locus.
`In a
`previous study, we observed polysomy for all or part of the short arm
`of chromosome 7 in 20 of 30 (67%) NSCLC specimens (32). The
`shortest region of overlap of partial gains of 7p was at region
`7pl l->pl3, where the EGFR gene is located (33). While an increase
`in the number of copies of EGFR might be expected to manifest itself
`in elevated expression of this gene, the correlation between polysomy
`of 7p and increased EGFR levels in NSCLC will require further study.
`The type III deletion mutant appears to be a promising target for
`therapy. Its fusion junction sequence is immunogenic. It is located
`near the amino-terminus of the extracellular domain, rendering it
`easily accessible to monoclonal antibodies. The detection of the
`EGFR type III deletion mutant by immunohistochemistry correlates
`extremely well with Western blot analysis and molecular detection of
`the deletion in tumor samples (22). Furthermore, antiserum directed
`against this sequence on EGFR type III mutant-expressing glioma
`cells (D4270 MG-X) has been demonstrated to be completely inter-
`nalized within 60 min (22), making this type III EGFR mutant po-
`tentially useful as a target for immunotoxin therapy.
`The frequency of expression of the EGFR type III deletion mutant
`in NSCLC (l6%) is very similar to that reported in glioblastomas
`(17%) (22) and suggests that this deletion mutant may occur in a
`.
`.
`.
`.
`.
`spectrum of EGFR-overexpressing malignancies. If this prevalence is
`verified by the study of more NSCLC samples, as many as 20,000
`individuals annually afflicted with NSCLC in the United States will
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`MUTATED EGFR IN NON-SMALL CELL LUNG CARCINOMA
`
`Fig. l. Adenosqnamous carcinoma of the lung.
`A, squamous component of the tumor. Tissue was
`stained as described in “Methods and Materials”
`using a biotinylated goat anti-rabbit IgG system
`following incubation with primary antibody to the
`type Ill deletion mutant. 8, adenocarcinoma com-
`ponent of the same tumor. Sections were treated as
`described above. Note that this component shows
`no reactivity for the type ll] deletion mutant anti—
`body. Frozen sections; Fast Red with hematoxylin
`counterstain (X 500).
`
`express this deletion mutant and may be candidates for treatments
`which exploit the presence of this antigen. Mutations of the EGFR
`would not be likely to occur in normal tissues and have not been
`detected in the limited panel which has been examined thus far. Thus,
`mutated growth factor receptors may be molecularly defined tumor-
`specific antigens. If this is indeed the case, the EGFR type III deletion
`mutant may provide a unique and powerful target for a number of
`forms of cancer detection and treatment, since the lack of antigenic
`targets on normal tissue should reduce the uptake of reagents and
`resultant toxicity to nontargeted tissues.
`
`These studies show that immunogenic mutations of the EGFR can
`be detected in at least two types of malignancies. Additional malig-
`nancies may be found to express the type III deletion mutation. Other
`mutations in the EGFR extracellular domain may be identified by
`systematically analyzing NSCLC and other tumor samples in which
`gene amplification of the EGFR is noted. Finally, it is possible that
`other growth factor receptors may undergo similar patterns of deletion
`mutations in the setting of malignant transformation. providing addi-
`tional examples of rnolecularly defined,
`turnonspecific antigens
`which can be exploited as new therapeutic targets.
`
`A
`
`
`
`‘oh
`
`2
`
`7
`
`Fig. 2. Chromosomes 2 and 7 from two different metaphase spreads (A and B) from in
`squamous cell lung carcinoma. Each metaphase has 3 or 4 copies of normal chromosome
`7 and two copies of a translocation derivative chromosome 2 containing most of the short
`arm of chromosome 7 translocated to the distal end of chromosome 2 (armws).
`
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`Cancer Research
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`Thejoumai of Cancer Research (1916»“'s93U} 3 The American journat of Cancer (19314 9&0}
`
`American Association
`for Cancer fiesearch ~.
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`
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`Expression of Mutated Epidermal Growth Factor Receptor by
`Non-Small Cell Lung Carcinomas
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`Irma E. Garcia de Palazzo, Gregory P. Adams, Padma Sundareshan, et al.
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`Cancer Res 1993;532:3217-3220.
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`Updated version
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`http://cancerres.aacrjourna|s.org/content/53/14/3217
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`Downloaded from cancerresaacrjournals.org on October 20, 2015. © 1993 American Association for Cancer
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`APOTEX EX. 1022-005