JClin Pathol 1991;44:211-214
`
`211
`
`c-erbB-2 expression in different histological types
`of invasive breast carcinoma
`
`S Soomro, S Shousha, P Taylor, H M Shepard, M Feldmann
`
`Abstract
`Sections of 149 breast carcinomas were
`examined for the over-expression of
`c-erbB-2 oncoprotein using the avidin-
`biotin immunoperoxidase technique and
`two different specific antibodies. These
`included the polyclonal antibody 21N
`and the monoclonal antibody 4D5. The
`tumours were divided into two main
`groups. The first included 75 cases of
`invasive
`ductal and classic
`invasive
`lobular carcinomas. The second group
`consisted of 74 cases with histological
`types known to have a good prognosis,
`including mucinous, alveolar variant of
`invasive lobular, medullary, tubular,
`cribriform and papillary carcinomas.
`Fifteen (20%) tumours of the first group
`were positive with the two antibodies.
`Fourteen of these were of the ductal type
`and one was a mixed invasive ductal and
`lobular carcinoma. Ten of the pure
`of comedo
`ductal
`cases had areas
`carcinoma. The intraductal elements in
`a further tumour were positively stained
`with 21N antibody only. None of the
`of
`which
`second
`group
`tumours,
`included histological types known to
`have good prognosis, stained with 4D5,
`although one mucinous carcinoma was
`positively stained with 21N.
`These findings suggest that in invasive
`breast carcinoma immunostaining for
`c-erbB-2 is mainly seen in a subgroup of
`ductal tumours, and that almost all
`other histological types, especially those
`associated with good prognosis, lack this
`expression.
`
`c-erbB-2 (also called HER2 and neu) is a
`proto-oncogene which encodes a 185-190
`glycoprotein
`kilodalton
`molecule
`that
`is
`closely related in structure to the epidermal
`growth factor receptor."A It maps to human
`chromosome 17.5 Under experimental con-
`ditions, c-erbB-2 becomes a potent oncogene
`with transforming activity only when it
`is
`overexpressed in the cells.6 In 1986 the
`oncogene was found to be amplified in a small
`percentage of adenocarcinomas of various
`organs, including breast, but not in other
`types of tumours.7
`The gene product has been localised to the
`cell membrane with extracellular, transmem-
`brane, and intracellular domains.' Various
`specific antibodies have been raised to the
`extra- and intracellular domains. Repeated
`
`studies have shown a good correlation be-
`tween the amplification of the c-erbB-2 gene
`and positive immunostaining for its protein
`product in the cells using these specific
`antibodies,8-12 although overexpression of the
`protein product can sometimes occur in the
`absence of gene amplification." 12
`In breast carcinoma between 9-33% of
`invasive
`tumours
`gene
`overexpress
`the
`product,891120 and there is strong evidence
`that
`overexpression
`with
`is
`associated
`aggressiveness.11317 19-25
`increased
`tumour
`Most of these studies were carried out mainly
`on breast carcinomas of the ductal type. As
`there are other less common types of invasive
`breast carcinoma, some of which are known to
`have a relatively good prognosis,26 27 we inves-
`tigated the possibility that such tumours may
`have a lower incidence of c-erbB-2 protein
`overexpression. The study was carried out
`using two specific antibodies, one raised to the
`intracellular and the other to the extracellular
`domains of the c-erbB-2 gene product.
`
`Methods
`Routinely processed paraffiin wax sections of
`149 invasive breast carcinomas were studied.
`The cases were selected on the basis of their
`histological type and were divided into two
`groups. The first included 75 cases of invasive
`ductal and classic invasive lobular carcinomas.
`The second group included 74 cases with
`histological types known to have a good
`prognosis, including mucinous, medullary,
`tubular, cribriform, papillary and the alveolar
`variant of invasive lobular carcinoma (table
`1).27
`Two specific antibodies were used: a poly-
`clonal antibody, 21N (kindly supplied by
`Dr W J Gullick, ICRF Oncology Group,
`Hammersmith Hospital, London), raised to a
`synthetic peptide of the predicted sequence of
`the intracellular domain of c-erbB-2 gene
`product,28 and a monoclonal antibody, 4D5
`(Genentech, San Francisco, California, USA),
`raised to the extracellular domain of the gene
`product.293'
`Four sections, each 5 gm thick, were cut
`from a representative paraffiin wax embedded
`tissue block of each case. Two of these sec-
`tions were intended for staining with the
`specific antibodies, and two were used as
`controls. All sections were incubated over-
`night at 37°C. On the following day they were
`dewaxed in two changes of xylene for two
`minutes each and hydrated in graded alcohols.
`Endogenous peroxidase activity was blocked
`
`Department of
`Histopathology,
`Charing Cross and
`Westminster Medical
`School, London,
`W6 8RF
`S Soomro
`S Shousha
`Charing Cross Sunley
`Research Centre,
`London
`P Taylor
`M Feldmann
`Genentech Inc, San
`Francisco, California,
`USA
`H M Shepard
`Correspondence to:
`Dr S Shousha
`Accepted for publication
`31 October 1990
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1089 Page 1 of 4
`
`

`
`212
`
`Soomro, Shousha, Taylor, Shepard, Feldmann
`
`Table I
`
`c-erbB-2 immunostaining of breast carcinoma according to histological type
`
`Histological type
`(all invasive)
`
`Total No
`of cases
`
`Cases positive
`with 21N (%)
`
`Cases positive
`with 4D5 (%)
`
`Ductal
`Lobular, classic
`Composite, ductal and lobular
`Mucinous
`Lobular, alveolar variant
`Medullary
`Tubular
`Cribriforr
`Papillary
`Total
`
`63
`11
`1
`23
`22
`13
`6
`6
`4
`149
`
`14 (22%)
`0
`1
`1 (4%)
`0
`0
`0
`0
`0
`16 (11%)
`
`14 (22%)
`0
`1
`0
`0
`0
`0
`0
`0
`15 (10%)
`
`by 3% hydrogen peroxide in methanol for 30
`minutes at room temperature. This was foll-
`owed by rinsing three times in 0-iM TRIS-
`buffered-saline (TBS), pH 7-6.
`Sections intended for staining with the
`polyclonal antibody 21N, and their controls,
`were incubated with 10% normal swine serum
`in TBS. Sections intended for staining with
`the monoclonal antibody 4D5, and their con-
`trols, were incubated in 10% normal rabbit
`serum. After 30 minutes excess serum was
`removed and sections were incubated over-
`night at 4°C either with the specific primary
`antisera, or, for the negative controls, with
`TBS.
`On the following day sections were rinsed
`in TBS and then covered for 30 minutes with
`a 1 in 250 solution of the secondary antibodies
`in TBS (biotinylated
`anti-rabbit
`swine
`immunoglobulin for 21N, and biotinylated
`rabbit anti-mouse immunoglobulin for 4D5;
`both from Dakopatts, England).
`Sections
`were then rinsed three times with TBS and
`incubated for 60 minutes in avidin-biotin
`complex-horseradish peroxidase (Dakopatts,
`England). After rinsing in TBS sections were
`(DAB,
`diaminobenzidine
`incubated
`with
`Sigma, England) for six minutes and then
`counterstained with Harris's haematoxylin,
`dehydrated in graded alcohols, and mounted
`with Permount.
`The results were assessed
`semiquanti-
`tatively according to the percentage of cells
`showing membrane staining
`that (-)
`so
`cells,
`indicated
`of
`absence
`stained
`(+)
`indicated staining of less than 33% of tumour
`cells, (+ +) staining of 33-66% of tumour
`cells, and (+ + +) staining of more than 66%
`of the cells. Cells showing cytoplasmic stain-
`ing only were regarded as negative.32 33
`
`Results
`Only 15 tumours stained with both antibodies
`(table 1). Of these, 14 were invasive ductal
`(22% of all ductal cases examined), 10 of which
`had areas ofintraductal comedo elements (fig 1).
`The only other positive case was a composite
`tumour comprising two distinct zones, one
`invasive ductal and the other classic lobular;
`both showed strong positive staining (+ + +)
`with the two antibodies.
`Positive staining with 21N was seen in two
`other tumours. These included a moderately
`stained (+ +) pure mucinous carcinoma (fig 2)
`and the intraductal elements of an invasive
`
`Figure I
`c-erbB-2 positive invasive ductal carcinoma
`(avidin-biotin complex).
`
`-4,
`
`i
`
`:..
`
`A
`
`1
`
`1.
`
`%...", i-4 F
`
`1-
`
`A.
`
`I.
`
`t
`
`..j:1
`
`.I
`
`0
`
`UN... Is:4,
`
`Figure 2
`c-erbB-2 (21N) positive pure mucinous
`carcinoma (avidin-biotin complex).
`
`Table 2
`Comparison of semiquantitatively assessed
`staining results of 21N and 4D5 antibodies in invasive
`elements of 63 ductal carcinomas
`
`Antibody
`
`21N
`4D5
`
`Total
`
`63
`63
`
`-
`
`49
`49
`
`+
`
`0
`2
`
`+ +
`
`+ + +
`
`1
`6
`
`13
`6
`
`ductal tumour. These two tumours did not
`stain with 4D5.
`All remaining 133 tumours were negative
`with the two antibodies. These included all
`pure classic and alveolar lobular carcinomas,
`22 of the 23 mucinous carcinomas, and all
`medullary, tubular, cribriform and papillary
`tumours examined (table 1).
`Thus the two antibodies gave concordant
`results in 147 (98-7%) out of the 149 cases
`examined. In a given positive case, however,
`21N tended to stain more cells than 4D5 (table
`2). On the other hand, cytoplasmic staining,
`presumably non-specific, was often seen with
`21N, but was not encountered in cases stained
`with 4D5.
`
`Discussion
`The main finding of this study is the almost
`consistent absence of c-erbB-2 immunostaining
`in the uncommon histological varieties of
`invasive breast carcinoma, which are known to
`be associated with better prognosis than the
`common invasive ductal variety. This was
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1089 Page 2 of 4
`
`

`
`c-erbB-2 in breast carcinoma
`
`especially so when the monoclonal antibody
`4D5, which
`recognises
`the
`extracellular
`domain of the oncogene product, was used.
`The results obtained with the polyclonal
`antibody 21N were similar, except for one case
`of mucinous carcinoma which was positive
`with this antibody but not with 4D5 (table 1).
`The findings provide indirect support for the
`existence of an association between positive
`c-erbB-2
`immunostaining
`increased
`and
`aggressiveness of invasive tumours. As most of
`these special types of breast carcinoma, with
`the exception of the medullary type, are also
`usually rich in oestrogen receptors,27 34 the
`findings are in line with the presence, in general,
`of an inverse relation between c-erbB-2 and
`25 39
`oestrogen receptors.
`Almost all cases of invasive lobular carcin-
`oma examined, whether classic or alveolar in
`type, did not overexpress this oncoprotein. The
`only positive lobular elements were seen in a
`composite tumour which consisted of separate,
`but adjacent, lobular and ductal parts. There
`are no published references about the c-erbB-2
`expression of the alveolar variant of lobular
`carcinoma,
`but
`investigators who have
`examined cases of the classic variant found
`them either all negative,12 17 35 40 or to have
`included only an occasional positive case.9 14 39 41
`In view of the recently reported absence of the
`oncoprotein in lobular carcinoma in SitU40 42 the
`findings
`of
`that
`overexpression
`suggest
`c-erbB-2 may not have an important role in
`lobular neoplasia. The presence of c-erbB-2
`immunostaining in the composite ductal/
`lobular tumour examined may indicate that the
`pathogenesis of the lobular-looking elements in
`this case is different from that of pure lobular
`tumours.
`The only cases that were positively stained
`with the two antibodies used in this study were
`either purely or partly of ductal type, and most
`of these
`also contained intraductal
`cases
`comedo elements. This is consistent with the
`findings of most previous studies and strongly
`supports the suggestion that overexpression of
`c-erbB-2 oncoprotein in invasive breast carcin-
`oma is almost totally restricted to a subset of
`ductal tumours with specific morphological
`features.9 1640 42 It also seems that there are only
`two specific types ofin situ breast tumour which
`frequently
`the oncoprotein-
`overexpress
`namely, intraductal comedo carcinoma9 39 42 and
`Paget's disease of the nipple.4043
`It is tempt-
`ing to suggest that a common thread may
`connect these three lesions, one invasive and
`two in situ, together. They are all characterised
`by large cell size, and although they may
`occasionally occur separately, they are more
`commonly seen in a combination of two or
`three; and when they do, they almost all
`overexpress the c-erbB-2 oncoprotein.43 4 The
`neoplastic changes are probably the same and
`involve specific cells at specific anatomical sites,
`and what determines what type of lesion(s)
`develop(s) is the primary site of the target
`cell(s) involved in the neoplastic process and its
`original directional-proliferation potential.
`Our study also shows that there is an
`excellent correlation (98-7%) between the
`
`213
`
`immunostaining results obtained with the two
`antibodies used which were raised to different
`domains of the oncogene product (intra- and
`extra-cellular). The polyclonal antibody raised
`to the intracellular domain (21N), however,
`tended to stain more cells in a given case,
`exclusively stained two (1-4%) extra cases
`(table 2), and in some tumours showed cyto-
`plasmic staining which is considered to be non-
`specific by most authors.
`
`We thank Dr W J Gullick of the ICRF Oncology Group,
`Hammersmith Hospital for supplying us with the 21N antibody.
`Photography was carried out by Mr Ron Barnett.
`This study was presented in the Summer meeting of the
`Pathological Society of Great Britain and Ireland which was
`held in Nottingham, England, between 10-13 July 1990.
`This work is part of Dr Soomro's PhD thesis.
`
`1 Schechter AL, Stern DF, Vaidyanathen L, et al. The neu-
`oncogene: an erbB related gene encoding a 185,000
`M tumour antigen. Nature 1984;312:513-16.
`2 Coussense L, Yang-Fang TL, Liao Y-C, et al. Tyrosine
`kinase receptor with extensive homology to EGF receptor
`shares chromosomal location with neu oncogene. Science
`1985;230:1 132-9.
`3 Yamamoto T, Ikawa S, Akiyama T, et al. Similarity of
`protein encoded by the human c-erbB-2 gene to epidermal
`growth factor receptor. Nature 1986;319:230-4.
`4 Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto
`T. The product of the human c-erbB-2 gene: A 185-
`kilodalton glycoprotein with tyrosine kinase activity.
`Science 1986;232:1644-6.
`5 Schechter AL, Hung M-C, Vaidyanathan L, et al. The neu
`gene: An erb B-homologous gene distinct from and
`unlinked to the gene encoding the EGF receptor. Science
`1985;229:976-8.
`6 Di Fiore PP, Pierce JH, Kraus MH, Segatto 0, King CR,
`Aaronson SA. erb B-2 is a potent oncogene when overex-
`NIH/3T3
`pressed
`Science
`in
`cells.
`1987;237:
`178-82.
`7 Yokota J, Yamamoto T, Toyoshima K, et al. Amplification of
`c-erbB-2 in human adenocarcinomas in vivo. Lancet
`1986;i:765-7.
`8 Venter DJ, Tuzi NL, Kumar S, Gullick WJ. Overexpression
`of the c-erb B-2 oncoprotein in human breast carcinomas:
`immunohistological assessment correlates with gene
`amplification. Lancet 1987;ii:69-72.
`9 Van de Vijver MD, Peterse JL, Mooi WJ, et al. Neu-protein
`overexpression in breast cancer. Association with comedo-
`type ductal carcinoma in situ and limited prognostic value
`in stage II breast cancer. NEngl JMed 1988;319:1239-45.
`10 Walker RA, Senior PV, Jones JL, Critchley DR, Varley JM.
`An immunohistochemical and in situ hybridisation study
`of c-myc and c-erbB-2 expression in primary human
`breast carcinomas. JPathol 1989;158:97-105.
`11 Slamon DJ, Godolphin W, Jones LA, et al. Studies of the
`HER-2/neu proto-oncogene in human breast and ovarian
`cancer. Science 1989;244: 707-12.
`12 Parkes HC, Lillycrop K, Howell A, Craig RK. C-erbB2
`mRNA expression in human breast tumours: comparison
`with c-erbB2 DNA amplification with prognosis. Br J
`Cancer 1990;61:39-45.
`13 Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A,
`McGuire WJ. Human breast cancer: correlation of relapse
`and survival with amplification of the HER-2/neu
`oncogene. Science 1987;235: 177-82.
`14 Barnes DM, Lammie GA, Millis RR, Gullick WL, Allen
`DS, Altman DG. An immunohistochemical evaluation of
`c-erbB-2 expression in human breast carcinoma. Br J
`Cancer 1988;58:448-52.
`15 Gusterson BA, Machin LG, Gullick WJ, et al. C-erbB-2
`expression in benign and malignant breast disease. Br J
`Cancer 1988;58:453-7.
`16 Adnane J, Gaudray P, Simon M-P, Simony-Lafontaine J,
`Jeanteur P, Theillet C. Proto-oncogene amplification and
`human breast tumor phenotype.
`Oncogene
`1989;4:
`1389-95.
`17 Walker RA, Gullick WJ, Varley JM. An evaluation of
`immunoreactivity for c-erbB-2 protein as a marker ofpoor
`short-term prognosis in breast
`cancer. Br I Cancer
`1989;60:42619.
`18 Zeillinger R, Kury F, Czerwnka K, et al. HER-2 amplifica-
`tion, steroid receptors and epidermal growth factor recep-
`tor in primary breast cancer. Oncogene 1989;4:109-14.
`19 Zhou D-J, Ahuja H, Cline MJ. Proto-oncogene abnor-
`malities in human breast cancer: c-ERBB-2 amplification
`does not correlate with recurrence of disease. Oncogene
`1989;4:105-8.
`20 Paik S, Hazan R, Fisher ER, et al. Pathologic findings from
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1089 Page 3 of 4
`
`

`
`214
`
`the National Surgical Adjuvant Breast and Bowel Project:
`prognostic significance of erb B-2 protein overexpression
`in primary breast cancer. J Clin Oncol 1990;8:103-12.
`21 Cline MJ, Battifora H, Yokota J. Proto-oncogene abnor-
`in human breast cancer:
`malities
`correlations with
`anatomic features and clinical course of disease. J Clin
`Oncol 1987;5:999-1006.
`22 Varley JM, Swallow JE, Brammar WJ, Whittaker JL,
`Walker RA. Alterations to either c-erbB-2 (neu) or c-myc
`proto-oncogenes in breast carcinomas correlate with poor
`short-term prognosis. Oncogene 1987;1:423-30.
`23 Zhou D, Battifora H, Yokota J, Yamamoto T, Cline MJ.
`Association of multiple copies of the c-erbB-2 oncogen
`with spread of breast carcinoma. Cancer Res 1987;
`47:6123-5.
`24 Tandon AK, Clark GM, Chamness GC, Ullrich A,
`McGuire WL. HER-2/neu oncogene protein and prog-
`nosis in breast cancer. J Clin Oncol 1989;7:1120-8.
`25 De Potter C, Beghin C, Makar AP, Vandekerckhove D,
`Roels HJ. The neu-oncogene protein as a predictive factor
`for haematogenous metastases in breast cancer patients.
`Int J Cancer 1990;45:55-8.
`26 McDivitt RW, Stewart FW, Berg JW. Tumours of the
`breast. Fascicle 2. In: Atlas of tumor pathology. Second
`series. Washington, DC: Armed Forces Institute of
`Pathology, 1968:86.
`27 Shousha S, Backhous CM, Alaghband-Zadeh J, Burn I.
`Alveolar variant of invasive lobular carcinoma of the
`breast. A tumor rich in estrogen receptors. Am J Clin
`Pathol 1986;85:1-5.
`28 Gullick WJ, Berger MS, Bennett PLP, Rothbard JB,
`Waterfield MD. Expression of the c-erbB-2 protein in
`normal and transformed cells. Int J Cancer 1987;40:
`246-54.
`29 Hudziak RM, Schlessinger J, Ullrich A. Increased expres-
`sion of the putative growth factor receptor p185HER2
`causes transformation and tumorigenesis of NIH 3T3
`cells. Proc Natl Acad Sci USA 1987;84:7159-63.
`30 Hudziak RM, Lewis GD, Shalaby MR, et al. Amplified
`expression of the HER2/ERBB2 oncogene induces resis-
`tance to tumor necrosis factor alpha in NIH 3T3 cells.
`Proc Natl Acad Sci USA 1988;85:5102-6.
`31 Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard
`HM, Ullrich A. pl85HER2 monoclonal antibody has
`antiproliferative effects in vitro. and sensitizes human
`breast tumor cells to tumor necrosis factor. Mol Cell Biol
`1989;9:1 165-72.
`
`Soomro, Shousha, Taylor, Shepard, Feldmann
`
`32 Gusterson BA, Gullick WJ, Venter DJ, et al. Immuno-
`histochemical localization of c-erbB-2 in human breast
`carcinomas. Mol Cell Probes 1988;2:383-91.
`33 De Potter CR, Quatacker J, Maertens G. The subcellular
`localization of the neu protein in human normal and
`neoplastic cells. Int J Cancer 1989;44:969-74.
`34 Shousha S, Coady AT, Stamp T, James KR, Alaghband-
`Zadeh J. Oestrogen receptors in mucinous carcinoma of
`the breast: an immunohistological study using paraffin wax
`sections. J Clin Pathol 1989;42:902-5.
`35 Garcia I, Dietrich P-Y, Aapro M, Vauthier G, Vadas L,
`Engel E. Genetic alterations of c-myc, c-erbB-2, and
`c-Ha-ras protooncogenes and clinical association in
`human breast carcinomas. Cancer Res 1989;49:6675-9.
`36 Roux-Dosseto M, Romain S, Dussault N, Martin PM.
`Correlation oferbB-2 gene amplification with low levels of
`estrogen and/or progesterone receptors in primary breast
`cancer: do erbB-2 products delineate hormone-indepen-
`dent tumors? Biomed Pharmacother 1989;43:641-9.
`37 Wright C, Angus B, Nicholson S, et al. Expression of
`c-erbB-2 oncoprotein: a prognostic indicator in human
`breast cancer. Cancer Res 1989;49:2087-90.
`38 Heintz NH, Leslie KO, Rogers LA, Howard PL. Amplifica-
`tion of the c-erbB-2 oncogene and prognosis of breast
`adenocarcinoma. Arch Pathol Lab Med 1990;114:160-3.
`39 Marx D, Schauer A, Reiche Chr, et al. c-erbB2 expression in
`correlation to other biological parameters ofbreast cancer.
`J Cancer Res Clin Oncol 1990;116:15-20.
`40 Gusterson BA, Machin LG, Gullick WJ, et al. Immuno-
`histochemical distribution ofc-erbB-2in infiltratingand in
`situ breast cancer. Int J Cancer 1988;42:842-5.
`41 Ro J, El-Naggar A, Ro JY, et al. c-erbB-2 amplification in
`node negative human breast cancer. Cancer Res 1989;
`49:6941-4.
`42 Ramachandra S, Machin L, Ashley S, Monaghan P, Guster-
`son BA. Immunohistochemical distribution of c-erbB-2
`in in situ breast carcinoma-A detailed morphological
`analysis. JPathol 1990;161:7-14.
`43 Lammie GA, Barnes DM, Millis RR, Gullick WJ. An
`immunohistochemical study of the presence of c-erbB-2
`protein in Paget's disease of the nipple. Histopathology
`1989;15:504-14.
`44 Keatings L, Sinclair J, Wright C, et al. c-erbB-2 oncoprotein
`expression in mammary and extramammary Paget's
`disease: an immunohistochemical study. Histopathology
`1990;17:243-7.
`
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