JC/in Pacho/ 1991;44:211-214
`
`211
`
`c-erbB-2 expression in different histological types
`
`
`
`of invasive breast carcinoma
`
`S Soomro, S Shousha, P Taylor, HM Shepard, M Feldmann
`
`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,s-12 although overexpression of the
`protein product can sometimes occur in the
`11 12
`absence of gene amplification.
`In breast carcinoma between 9-33% of
`invasive tumours overexpress the gene
`
`product,8911-2° and there is strong evidence
`
`that overexpression is associated with
`
`increased tumour aggressiveness.11 13 17 •9-25
`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.
`
`Abstract
`Sections of 149 breast carcinomas were
`examined for the over-expression of
`c-eTbB-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
`ductal cases had areas of comedo
`carcinoma. The intraductal elements in
`a further tumour were positively stained
`with 21N antibody only. None of the
`Methods
`Routinely processed paraffiin wax sections of
`second
`group of
`tumours, which
`149 invasive breast carcinomas were studied.
`included histological types known to
`have good prognosis, stained with 4D5,
`The cases were selected on the basis of their
`
`histological type and were divided into two
`although one mucinous carcinoma was
`groups. The first included 75 cases of invasive
`positively stained with 21N.
`
`ductal and classic invasive lobular carcinomas.
`These findings suggest that in invasive
`The second group included 74 cases with
`breast carcinoma immunostaining for
`c-erbB-2 is mainly seen in a subgroup of
`histological
`types known to have a good
`ductal tumours, and that almost all
`prognosis, including mucinous, medullary,
`
`
`tubular, cribriform, papillary and the alveolar
`other histological types, especially those
`variant of invasive lobular carcinoma (table
`associated with good prognosis, lack this
`1 ).21
`expression.
`Two specific antibodies were used: a poly­
`
`clonal antibody, 21N (kindly supplied by
`c-erbB-2 (also called HER2 and neu) is a
`Dr W J Gullick, ICRF Oncology Group,
`
`Hammersmith Hospital, London), raised to a
`
`proto-oncogene which encodes a 185-190
`
`kilodalton glycoprotein molecule that is
`
`
`synthetic peptide of the predicted sequence of
`
`the intracellular domain of c-erbB-2 gene
`
`closely related in structure to the epidermal
`
`product,28 and a monoclonal antibody, 4D5
`growth factor receptor.H It maps to human
`
`chromosome 17.5 Under experimental con­
`
`
`(Genentech, San Francisco, California, USA),
`
`ditions, c-erbB-2 becomes a potent oncogene
`
`raised to the extracellular domain of the gene
`product.29-31
`with transforming activity only when it is
`each 5 µm thick, were cut
`Four sections,
`
`overexpressed in the cells.6 In 1986 the
`
`from a representative paraffiin wax embedded
`oncogene was found to be amplified in a small
`tissue block of each case. Two of these sec­
`
`percentage of adenocarcinomas of various
`tions were intended for staining with the
`organs, including breast, but not in other
`
`specific antibodies, and two were used as
`types of tumours.7
`
`controls. All sections were incubated over­
`
`The gene product has been localised to the
`night at 37°C. On the following day they were
`
`cell membrane with extracellular, transmem­
`dewaxed in two changes of xylene for two
`
`brane, and intracellular domains.• Various
`minutes each and hydrated in graded alcohols.
`
`specific antibodies have been raised to the
`
`
`Endogenous peroxidase activity was blocked
`
`extra-and intracellular domains. Repeated
`
`Department of
`Histopathology,
`Charing Cross and
`Westminster Medical
`School, London,
`W68RF
`S Soomro
`S Shousha
`Charinr Cross Sunley
`Research Centre,
`London
`PTaylor
`MFeldmann
`Genentech Inc, San
`Francisco, California,
`USA
`HM Shepard
`Correspondence to:
`Or S Shousha
`Accepted for publication
`31October1990
`
`1 of 4
`
`BI Exhibit 1089
`
`

`

`212
`
`Soomro, Shousha, Taylor, Shepard, Feldmann
`
`Table I c-erbB-2 immunostaining of breast carcinoma according to histological type
`
`Histological type
`(all imJasivt)
`Ductal
`Lobular, classic
`Composite, ductal and lobular
`Mucinous
`Lobular, alveolar variant
`Medullary
`Tubular
`Cribriforrn
`Papillary
`
`Total
`
`Total No Casts positiw
`of casts
`with2IN (%)
`
`Cases positiw
`with 4DS (%)
`
`63
`II
`1
`23
`22
`13
`6
`6
`4
`149
`
`14(22%)
`0
`1
`I (4%)
`0
`0
`0
`0
`0
`
`16 (II%)
`
`14(22%)
`0
`I
`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 O· lM 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
`swine anti-rabbit
`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
`incubated with diaminobenzidine
`(DAB,
`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 so that ( - )
`( +)
`indicated absence of
`stained cells,
`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 I). Of these, 14 were invasive ductal
`(22 % of all ductal cases examined), I 0 of which
`had areas of intraductal comedo elements (fig I).
`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 imJasive ductal carcinoma
`( avidin-biotin cqmp/ex).
`
`....
`
`.. ..
`
`·�
`
`c-erbB-2 (21N) positive pure mudnous
`Figure 2
`carcinoma ( avidin-biotin cqmp/ex).
`
`Table 2 Comparison of semiquanciraiively assessed
`staining results of 21 N and 4D5 antibodies in invasive
`elements of 63 ductal carcinomas
`
`Antibody
`
`21N
`405
`
`Toca/
`
`63
`63
`
`+
`
`0
`2
`
`++ +++
`
`I
`6
`
`13
`6
`
`49
`49
`
`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
`
`2 of 4
`
`BI Exhibit 1089
`
`

`

`c-erbB-2 in breast carcinoma
`
`213
`
`especially so when the monoclonai antibody
`
`immunostaining results obtained with the two
`
`405, which recognises the extracellular
`
`antibodies used which were raised to different
`domain of the oncogene product, was used.
`domains of the oncogene product (intra-and
`The results obtained with the polyclonal
`
`
`extra-cellular). The polyclonal antibody raised
`
`antibody 21N were similar, except for one case
`
`to the intracellular domain (21N), however,
`of mucinous carcinoma which was positive
`tended to stain more cells in a given case,
`with this antibody but not with 405 (table 1).
`exclusively
`stained two (l ·4 % ) extra cases
`
`The findings provide indirect support for the
`(table 2), and in some tumours showed cyto­
`
`plasmic staining which is considered to be non­
`
`
`existence of an association between positive
`
`c-erbB-2 imrnunostaining and increased
`specific by most authors.
`
`
`aggressiveness of invasive tumours. As most of
`
`these special types of breast carcinoma, with
`We thank Dr W J Gullick of the ICRF Oncology Group,
`Hammersmith Hospital for supplying us with the 21 N antibody.
`the exception of the medullary type, are also
`Photography was carried out by Mr Ron Barnen.
`
`usually rich in oestrogen receptors,27 34 the
`This study was presented in the Summer meeting of the
`
`findings are in line with the presence, in general,
`Pathological Society of Great Britain and Ireland which was
`of an inverse relation between c-erbB-2 and
`held in Nottingham, England, between 10-13 July 1990.
`18 25 3s-39
`This work is pan of Dr Soomro's PhD thesis.
`
`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
`I Schechter AL, Stem OF, Vaidyanathen L, et al. The neu­
`
`
`are no published references about the c-erbB-2
`oncogene: an erbB related gene encoding a 185,000
`M tumour antigen. Nature 1984;312:513-16.
`
`expression of the alveolar variant of lobular
`2 Coussense L, Yang-Fang TL, Liao Y-C, tt al. Tyrosine
`
`carcinoma, but investigators who have
`kinase receptor with extensive homology to EGF receptor
`shares chromosomal location with neu oncogene. Science
`examined cases of the classic variant found
`1985;230:1132-9.
`
`them either all negative,12 17 35 40 or to have
`3 Yamamoto T, lkawa S, Akiyama T, et al. Similarity of
`
`included only an occasional positive case. 9 14 39 41
`protein encoded by the human c-erbB-2 gene to epidermal
`growth factor receptor. Nature 1986;319:230-4.
`
`In view of the recently reported absence of the
`4 Akiyama T, Sudo C, Ogawara H, Toyoshima K, Yamamoto
`T. The product of the human c-erbB-2 gene: A 185-
`
`
`oncoprotein in lobular carcinoma in situ4042 the
`kilodalton glycoprotcin with tyrosine kinase activity.
`
`findings suggest that overexpression of
`Science 1986;232:164<ki.
`5 Schechter AL, Hung M-C, Vaidyanathan L, et al. The neu
`
`c-erbB-2 may not have an important role in
`gene: An erb B-homologous gene distinct from and
`
`lobular neoplasia. The presence of c-erbB-2
`unlinked to the gene encoding the EGF receptor. Science
`l 985;229:976-8.
`imrnunostaining in the composite ductal/
`6 Di Fiore PP, Pierce JH, Kraus MH, Segatto 0, King CR,
`
`
`lobular tumour examined may indicate that the
`Aaronson SA. erb B-2 is a po1en1 oncogene when overex­
`pressed
`in NIH/3T3
`cells. Science
`1987;237:
`
`
`pathogenesis of the lobular-looking elements in
`178--82.
`this case is different from that of pure lobular
`7 Yokota}, YamamotoT, ToyoshimaK,era/. Amplification of
`c-erbB-2 in human adenocarcinomas in vivo. Lancet
`tumours.
`l 986;i:765-7.
`The only cases that were positively stained
`
`8 Venter DJ, Tuzi NL, Kumar S, Gullick WJ. Overexpression
`of the c-erb B-2 oncoprotein in human breast carcinomas:
`
`with the two antibodies used in this study were
`inununohistological assessment correlates with gene
`either purely or partly of ductal type, and most
`amplification. Lancet 1987;ii:69-72.
`9 Van de Vijver MD, Peterse JL, Mooi WJ, et al. Neu-protein
`of these cases also contained intraductal
`overexpression in breast cancer. Association with comedo­
`
`
`comedo elements. This is consistent with the
`type ductal carcinoma in situ and limited prognostic value
`in stage II breast cancer. N En.gt J Med 1988;319:1239-45.
`findings of most previous studies and strongly
`10 Walker RA, Senior PV, Jones JL, Critchley DR, Varley JM.
`An immunohistochcmical and in situ hybridisation study
`
`
`supports the suggestion that overexpression of
`
`c-erbB-2 oncoprotein in invasive breast carcin­
`of c-myc and c-erbB-2 expression in primary human
`breast carcinomas. J Pathol 1989;158:97-105.
`II Slamon DJ, Godolphin W, Jones LA, ti al. Studies of the
`
`oma is almost totally restricted to a subset of
`HER-2/neu proto-oncogcne in human breast and ovarian
`ductal tumours with specific morphological
`cancer. Science 1989;244: 707-12.
`42 It also seems that there are only
`features.91640
`12 Parkes HC, Lillycrop K, Howell A, Craig RK. C-erbB2
`mRNA expression in human breast tumours: comparison
`two specific types of in situ breast tumour which
`with c-erbB2 DNA amplification with prognosis. Br J
`Cancer I 990;61 :39-45.
`frequently overexpress the oncoprotein­
`13 Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A,
`
`
`namely, intraductal comedo carcinoma9 39 42 and
`McGuire WJ. Human breast cancer: correlation of relapse
`and survival with amplification of the HER-2/neu
`
`Paget's disease of the nipple.404344 It is tempt­
`oncogene. Science 1987;235:177-82.
`ing to suggest that a common thread may
`14 Barnes OM, Lammie GA, Millis RR, Gullick WL, Allen
`OS, Alunan DG. An immunohistochemical evaluation of
`
`connect these three lesions, one invasive and
`c-crbB-2 expression in human breast carcinoma. Br J
`
`two in situ, together. They are all characterised
`Cancer 1988;58:448-52.
`by large cell size, and although they may
`15 Gusterson BA, Machin LG, Gullick WJ, et al. C-erbB-2
`expression in benign and malignant breast disease. Br J
`
`
`occasionally occur separately, they are more
`Cancer 1988;58:453-7.
`
`commonly seen in a combination of two or
`16 Adnane J, Gaudray P, Simon M-P, Simony-Lafontaine J,
`Jcantcur P, Theillct C. Proto-oncogene amplification and
`
`three; and when they do, they almost all
`human breast cumor phenotype. Oncogene 1989;4:
`
`overexpress the c-erbB-2 oncoprotein.
`43 44 The
`1389-95.
`17 Walker RA, Gullick WJ, Varley JM. An evaluation of
`
`neoplastic changes are probably the same and
`inununorcactivicy for c-erbB-2 protein as a marker of poor
`shon-term prognosis in breast cancer. Br J Cancer
`
`
`involve specific cells at specific anatomical sites,
`1989;60:42&-9.
`and what determines what type of lesion(s)
`18 Zeillinger R, Kury F, Czcrwnka K, et al. HER-2 amplifica­
`
`develop(s) is the primary site of the target
`tion, steroid receptors and epidermal growth faccor recep­
`tor in primary breast cancer. Oncogene 1989;4:109-14.
`
`
`cell(s) involved in the neoplastic process and its
`19 Zhou D-J, Ahuja H, Cline MJ. Proto-oncogene abnor­
`
`
`
`original directional proliferation potential.
`malities in human breast cancer: c-ERBB-2 amplification
`docs not correlate with recurrence of disease. Oncogene
`Our study also shows that there is an
`1989;4:105-8.
`
`excellent correlation (98·7%) between the
`20 Paik S, Hazan R, Fisher ER, tt al. Pathologic findings from
`
`3 of 4
`
`BI Exhibit 1089
`
`

`

`214
`
`the National Surgical Adjuvant Breast and Bowel Proj«t:
`prognostic significan« of erb B-2 protein overexpression
`in primary breast can«r. JC/in C>Mo/ 1990;8:10'.>-12.
`21 Cline MJ, Battifora H, Yokota J. ProtcH>nCOJClle abnor­
`maliries
`in human breast can«r: correlations with
`anatomic fcarures and clinical course of disease. J Clin
`Oncol 1981;S:999-l006.
`22 Varley JM, Swallow JE, Brammer WJ, Whittalter JL,
`Walker RA. Alterations to either c-erbB-2 (neu) or c-myc
`proto-oncogcnes in brea.st carcinomas correlate with poor
`short-term prognosis. Oncoient 1987;1:42:>-30.
`23 Zhou D, Banifora H, Yokota J, Yamamoto T, Cline MJ.
`Association of multiple copies of the c-crbB-2 oncogcn
`with spread of breast carcinoma. Canur R<J 1987;
`47:612'.>-5.
`24 Tandon AK, Clark GM, Chamness GC, Ullrich A,
`McGuire WL. HER-2/neu oncogene protein and prog·
`nusis in breast cancer. J Clin Onco/ 1989;1:1120-8.
`25 De Pouer C, Beghin C, Makar AP, Vandekerckhove D,
`Roets HJ. The neu-oncogene protein as a predictive factor
`for haematogenous mewtases in breast cancer patients.
`Int J Cancer 1990;4S:5H.
`26 McDiviu RW, Stewart FW, Berg JW. Tumours of the
`breast. Fascicle 2. Jn: At/aJ of tumor patlw/ogy. Second
`series. Washington, DC: Armed Forces Instirute of
`Pathology, 1968:86.
`27 Shousha S, Backhous CM, Alagbband-Zadch J, Bum 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, Benneu PLP, Rothbard JB,
`Waterfield MD. Expression of the c-erbB-2 protein in
`normal and transformed cells. Int J Canur 1987;40:
`24(>-54.
`29 Hudziak RM, Scblessinger J, Ullrich A. Increased expres­
`sion of the putative growth factor receptor p l 85HER2
`cauSC1 transformation and tumorigcnesis of NIH 3T3
`�Its. Proc Natl Acad Sci USA 1987;84:715�3.
`30 Hudziak RM, Lewis GD, Shalaby MR, <t al. Amplified
`expression of the HER2/ERBB2 oncogene induces resis­
`tance to tumor necrosis factor alpha in NIH 3T3 �Its.
`Proc Natl Acad Sci USA 1988;85:5102-6.
`31 Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard
`HM, Ullrich A. p185HER2 monoclonal antibody has
`antiproliferative effects in vitro and sensitizes human
`breast tumor cells to rumor necrosis factor. Mo/ Cell Biol
`1989;9:1165-72.
`
`Soomro, Shousha, Taylor, Shepard, Feldmann
`
`32 Gusterson BA, Gullidt WJ, Veorer DJ, ti al. Immuoo­
`hisrocbcmical localizatfoo of c-erbB-2 in human breast
`carcinomas. Mo/ CAii Probu 1988;2:38>-91.
`33 De Potter CR, Quatadter J, Maenens G. The subc:cllular
`localiz.ation 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, Alaahband­
`Zadeh J. Oestrogen receptors in mucinous carcinoma of
`the breast: an immunohistological srudy using paraffin wax
`sections. J Clin Pa11w/ 1989;42:902-5.
`35 Garcia J, Dietrich P-Y, Aapro M, Vauthier G, Vadas L,
`Engel E. Genetic alterations of c-myc, c-crbB-2, and
`c-Ha-ras protoonoogcnes and clinical association in
`human breast carcinomas. Canur Ru 1989;49:6675-9.
`36 Roux-Dosseto M, Romain S, Dussault N, Martin PM.
`Correlation of erbB-2 gene amplification with low levels of
`estrogen and/or progesterone receptors in primary breast
`cancer: do crbB-2 productS delineate hormone-indepen­
`dent rumon? Bimmd Pharmaco1her 1989;43:641-9.
`37 Wright C, Angus B, Nicholson S, er al. Expression of
`c-erbB-2 oncoprotein: a prognostic indicator in human
`breast cancer. COJtUr Rts I 989;49:2087-90.
`38 Heintz NH, Leslie KO, Roaers LA, Howard PL. Amplifica­
`tion of the c-crbB-2 oncogene and pf'OIXIOliS of breast
`adenocarcinoma. Ards Patlwl Lab Mtd 1990;114:1�3.
`39 Marx D, Schauer A, Reiche Chr,t1 al. c-crbB2 expression in
`correlation t o other biological parameters of breast cancer.
`J Canar Rts Clin C>Mo/ 1990;116:15-20.
`40 Gustcrson BA, Machin LG, Gullick WJ, ti al. lmmuno­
`histochemical distribution of c-erbB-2 in infiltrating and in
`situ breast cancer. Int J Cancer 1988;42:842-5.
`41 Ro J, El-Na11ar A, Ro JY, er al. c-crbB-2 amplificarion in
`node neptive human breast cancer. Cancer Rts 1989;
`49:6941-4.
`42 Ramacbandra S, Machin L, Ashley S, Monaghan P, Gustcr­
`son BA. lmmunohistochemical distribution of c-crbB-2
`in in siru breast carcinoma-A detailed morphological
`analysis. J Patlto/ 1990;161:7-14.
`43 Lammie GA, Barnes OM, Millis RR, Gullick WJ. An
`immunohistochemical srudy of the presence of c-erbB-2
`protein in Paget's disease of the nipple. Histopatho/ogy
`1989;15:504-14.
`44 Kea tings L , Sinclair J, Wright C, ei al. c-crbB-2 oncoprotein
`expression in mammary and extramammary Paget's
`disease: an immunohistochemical srudy. HiJtopathology
`1990;17:24'.>-7.
`
`4 of 4
`
`BI Exhibit 1089
`
`