MARCH 1991 VOLMNO 3
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`
`URNAL OF
`I/CLINICAL
`PATHOLOGY
`
`BRITISH MEDICAL ASSOCIATION TAVlSTOCK SQUARE LONDON WClH 91R
`
`The journal of the Association of Clinical Pathologists
`
`in situ hybridisation in perspective A War/0rd, l Lauder
`Opportunistic protozoan infections in human immunodeficiency virus disease: Review highlighting
`diagnostic and therapeutic aspects A Curry, A J Turner. S Luca:
`.
`
`liistopathoioly and lmmunohistochemistry of the caecum in children with the Trich dysentery
`syndrome T T MacDonald. M-Y Chay. .I Spencer, 2: al
`
`lmmunohlstological characterisation of amyloid deposits
`C Hartley, J McClure, P Acktill
`
`renal biopsy specimens R J Pita-nitrite,
`
`Occult papillary mlcrocorcinoms of the thyroid — a potential pitfall of line needle aspiration
`cytology? H R Harach. E Saraw'a Day, S B Zunnan
`
`[Ki-67 staining in histological subtypes of breast carcinomn and fine needle aspiration smears
`V KWmamenn, Ti: H Van Der Kmart, H A J Van Laarhooen, S C Hem-Lopnam
`c-erbB-Z expression in different histological types of invasive breast carcinoma S Soomm, S Shouxlaa,
`P Taylor, H M Shepard. M Ftldmarnl
`DNA flow cytometry of follicular non-Hodgkin's lymphoma J C Macanney, R S Camplqohn, R Mom's.
`K Hollowaod. D Clarke, A Timothy
`
`Histometric studies on cellular infiltrates of tuberculin tests in patients with sareoidoais J G Lane,
`I H Gibbs, R C Putts. J L Stanford, J Swanson Beck
`
`Monocyte counting: Discrepancies in results obtained with diflerent automated instruments
`W Gama", L van Hove. R L Vn'wilghcn
`
`Measurement of acute phaae proteins for assessing severity of Plasmodium falciparum malaria
`S H Gillupit, C Bow, 1 G Rayner, R H Bohnm, P L Chabdml'. K P W} McAdam
`
`Plasma leucocyte elastaae concentrations in smokers C R K Hind, H Joyce, G A Tommi. M B Pepys.
`N B Pride
`
`Measurement of alkaline phosphatase of intestinal origin in plasma by P-bromotetramiaole
`inhibition T Karma, 8 B Rosalhi
`.
`
`Automated microparticie enzyme immunoassaya for 1:6 and 13M antibodies to Toxoplasma gondii‘
`J W Safimi, G 6 Abbott. M C Craim. R G MacDonald
`
`Plasma histamine in patients with chronic renal failure and nephrotic syndrome D S Gill,
`VA Fomeca. M A Barradal, R Balliod, J F Maorhtad, l’ Damiano
`
`Lack of specificity for antibodies to double stranded DNA found in four commercial kits
`M Kadinbomki, M Jackson, P L Yap, G Neill
`
`lmmnnocytologleal diagnosis of primary cerebral non-Hodgkin’s lymphoma A P Lax, A S Wurzbl'cb,
`P M Norman
`-
`
`Primary squamous cell carcinoma ofthe terminal ileum C C Plan. N Y Haboubu. P F Schofield
`
`Cells containing factor Xllla and pulmonary fibrosis induced by bleomyein M Taido. Y Charm],
`1‘ TM
`
`Audit of turnaround timoa in a microbiology laboratory S Rogers, M J Bywatn, D 5 Rune:
`Comparative yield of Salmonella typhi from blood and bone marrow cultures in patients with fever
`of unknown origin 8 J Far-Maui, M Khurshl'd, M K Alhqu. M Am KIran
`
`Recovery of CD3 + andCDS — lymphocyte subpopulatlon after autologous bone marrow
`transplantation and chunotherapy P J McKay, A McLaren. L M Andrew, N P Lucie
`
`202 Correspondence'Boohrevier'Noticea'
`
`PFIZER EX. 1089
`Page 1
`
`

`

`JOURNAL OF
`CLINICAL
`PATHOLOGY
`
`TM Journal of tht Association of Clinical Patlw/ogisu
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`BOARD
`
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`ASTM CODEN:JCPAAK 44(3) 177-264 (1991)
`ISSN 0021-9746
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`PFIZER EX. 1089
`Page 2
`
`

`

`1 Clin Patholl99l;44:2ll-2l4
`
`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(cid:173)
`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
`second group of
`tumours, which
`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.
`
`Department of
`Histopathology,
`Charing Cross and
`Westminster Medical
`School, London,
`W68RF
`S Soomro
`S Shousha
`Charing Cross Sunley
`Research Centre,
`London
`P Taylor
`MFeldmann
`Genentech Inc, San
`Francisco, California,
`USA
`H M Shepard
`Correspondence to:
`DrS Shousha
`Accepted for publication
`31 October 1990
`
`c-erbB-2 (also called HER2 and neu) is a
`proto-oncogene which encodes a 185-190
`that
`is
`kilodalton glycoprotein molecule
`closely related in structure to the epidermal
`growth factor receptor. H It maps to human
`chromosome 17.5 Under experimental con(cid:173)
`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 he amplified in a small
`percentage of adenocarcinomas of various
`organs, including breast, but not in other
`types oftumours.7
`The gene product has been localised to the
`cell membrane with extracellular, transmem(cid:173)
`brane, and intracellular domains. 1 Various
`specific antibodies have been raised to the
`extra- and intracellular domains. Repeated
`
`studies have shown a good correlation be(cid:173)
`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
`absence of gene amplification. 11 12
`In breast carcinoma between 9-33% of
`invasive
`tumours overexpress
`the gene
`product, 8 9 11
`20 and there is strong evidence
`-
`that overexpression
`is
`associated with
`increased
`tumour
`aggressiveness. 111 3171
`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/6 27 we inves(cid:173)
`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(cid:173)
`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/ 8 and a monoclonal antibody, 4D5
`(Genentech, San Francisco, California, USA),
`raised to the extracellular domain of the gene
`product. 2
`3 1
`'}-
`Four sections, each 5 Jlm thick, were cut
`from a representative paraffiin wax embedded
`tissue block of each case. Two of these sec(cid:173)
`tions were intended for staining with the
`specific antibodies, and two were used as
`controls. All sections were incubated over(cid:173)
`night at 3TC. 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
`
`PFIZER EX. 1089
`Page 3
`
`

`

`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 2/N (%)
`
`Cases p ositive
`with 4D5 ( % }
`
`Ductal
`Lobular, classic
`Composite, ductal and lobular
`Mucinous
`Lobular, alveolar variant
`Medullary
`T ubular
`Cribriform
`Papillary
`Total
`
`63
`11
`1
`23
`22
`13
`6
`6
`4
`149
`
`14 (22% )
`0
`1
`1 (4% )
`0
`0
`0
`0
`0
`16 (1 1% )
`
`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(cid:173)
`owed by rinsing three times in O·lM TRIS(cid:173)
`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(cid:173)
`trols, were incubated in 10% normal rabbit
`serum. After 30 minutes excess serum was
`removed and sections were incubated over(cid:173)
`night at 4oC 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 (cid:173)
`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(cid:173)
`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(cid:173)
`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 of intraductal 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
`
`c-erbB-2 positi'l!e in'l!asi'l!e ductal carcinoma
`Figure I
`(avidin-biotin complex).
`
`/
`
`..
`
`· '~ , -~
`Figure 2 c-erbB-2 ( 2I N ) positive pure mucinous
`carcinoma (avidin-biotin complex) .
`
`Table 2 Comparison of semiquanticatively assessed
`staining results of 21N and 4D5 antibodies in invasive
`elements of 63 ductal carcinomas
`
`Antibody
`
`Total
`
`+
`
`+ + + + +
`
`21N
`405
`
`63
`63
`
`49
`49
`
`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
`with4D5.
`
`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
`
`PFIZER EX. 1089
`Page 4
`
`

`

`c-erbB-2 in breast carcinoma
`
`213
`
`especially so when the monoclonai 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
`immunostammg
`and
`increased
`c-erbB-2
`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/7 34
`the
`findings are in line with the presence, in general,
`of an inverse relation between c-erbB-2 and
`oestrogen receptors. 18 25 3>-39
`Almost all cases of invasive lobular carcin(cid:173)
`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
`investigators who have
`carcinoma, but
`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
`that overexpression of
`findings
`suggest
`c-erbB-2 may not have an important role in
`lobular neoplasia. The presence of c-erbB-2
`immunostammg
`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 cases also contained
`intraductal
`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(cid:173)
`oma is almost totally restricted to a subset of
`ductal tumours with specific morphological
`features. 9 16 40 42 It also seems that there are only
`two specific types of in situ breast tumour which
`frequently overexpress
`the oncoprotein(cid:173)
`namely, intraductal comedo c-arcinoma9 39 42 and
`Paget's disease of the nipple.404344 It is tempt(cid:173)
`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.4344 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
`
`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(cid:173)
`plasmic staining which is considered to be non(cid:173)
`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.
`
`Schechter AL, Stern DF, Vaidyanathen L, eta/. The neu(cid:173)
`oncogene: an erbB related gene encoding a 185,000
`M tumour antigen. Nature 1984;312 :513- 16.
`Coussense L, Yang-Fang TL, Liao Y-C, eta/. Tyrosine
`kinase receptor with extensive homology to EGF receptor
`shares chromosomal location with neu oncogene . Science
`1985;230: 11 32-9.
`Yamamoto T , lkawa S, Akiyama T, et a/. Similarity of
`protein encoded by the human c-erbB-2 gene to epidermal
`growth factor receptor. Natu re 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.
`Schechter AL, Hung M-C, Vaidyanathan L, eta/. 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(cid:173)
`cells.
`1987;237:
`in NIH/3T3
`pressed
`Science
`178-82.
`Yokotaj, Yamamoto T, Toyoshima K, eta/. Amplification of
`c-erbB-2 in human adenocarcinomas in vivo. L ancet
`1986;i:765-7.
`8 Venter D J, 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 !987;i i:69-72.
`9 Van de Vijver MD , Peterse JL, Mooi WJ, eta/. Neu-protein
`overexpression in breast cancer. Association with comedo(cid:173)
`type ductal carcinoma in situ and limited prognostic value
`in stage II breast cancer. N Eng/J Med 1988;319:1239-45.
`10 Walker RA, Senior PV, j ones 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. J Patho/1989;158:97-105.
`II Slamon DJ, Godolphin W, j ones LA, eta/. Studies uf the
`HER-2/neu proto-oncogene in human breast and ovarian
`cancer. Sciena 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, U llrich 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 D G. An immunohistochemical evaluation of
`c-erbB-2 expression in human breast carcinoma. Br J
`Cancer 1988;58:448-52.
`IS Gusterson BA, Machin LG, Gullick WJ, et a/. 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 ],
`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 of poor
`short-term prognosis in breast cancer. Br J Cancer
`1989;60:426-9.
`18 Zeillinger R, Kury F, Czerwnka K , eta/. HER-2 amplifica(cid:173)
`tion, steroid receptors and epidermal growth factor recep(cid:173)
`tor in primary breast cancer. Oncogene 1989;4:109-14.
`19 Zhou D-J, Ahuja H , Cline MJ. Proto-oncogene abnor(cid:173)
`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 a/. Pathologic findings from
`
`PFIZER EX. 1089
`Page 5
`
`

`

`214
`
`Soomro, Shousha, Taylor, Shepard, Feldmann
`
`the National Surgical Adjuvant Breast and Bowel Project:
`prognostic significance of erb B-2 protein overexpression
`in primary breast cancer. J Clin Onco/1990;8:103--12.
`21 Cline MJ, Battifora H, Yokota J. Proto-oncogene abnor(cid:173)
`in human breast cancer: correlations with
`malities
`anatomic features and clinical course of disease. J Clin
`Onco/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(cid:173)
`nosis in breast cancer. J Clin Onco/1989;7:112~.
`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.
`lnt 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, Backhaus CM, Alaghband-Zadeh J, Burn I.
`Alveolar variant of invasive lobular carcinoma of the
`breast. A tumor rich in estrogen receptors. Am J Clin
`Patho/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. lnt J Cancer 1987;40:
`246-54.
`29 Hudziak RM, Schlessinger J, Ullrich A. Increased expres(cid:173)
`sion of the putative growth factor receptor p185HER2
`causes transformation and tumorigenesis of NIH 3T3
`cells. Proc Nat/ 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(cid:173)
`tance to tumor necrosis factor alpha in NIH 3T3 cells.
`Proc Nat/ A cad 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 tumor necrosis factor . Mol Cell Bioi
`1989;9: 1165-72.
`
`32 Gusterson BA, Gullick WJ, Venter DJ, et al. Immuno(cid:173)
`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. lnt J Cancer 1989;44:969-74.
`34 Shousha S, Coady AT, Stamp T, James KR, Alaghband(cid:173)
`Zadeh J. Oestrogen receptors in mucinous carcinoma of
`the breast: an immunohistological study using paraffin wax
`sections. J Clin Patho/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 of erbB-2 gene amplification with low levels of
`estrogen and/or progesterone receptors in primary breast
`cancer: do erbB-2 products delineate hormone-indepen(cid:173)
`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(cid:173)
`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 of breast cancer.
`J Cancer Res Clin Onco/1990;116: 15-20.
`40 Gusterson BA, Machin LG, Gullick WJ, et al. Immuno(cid:173)
`histochemical distributionofc-erbB-2 in infiltrating and in
`situ breast cancer. lnt J Cancer 1988;42:842-5.
`41 Ro J, EI-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(cid:173)
`son BA. Immunohistochemical distribution of c-erbB-2
`in in situ breast carcinoma-A detailed morphological
`analysis. J Patho/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.
`
`PFIZER EX. 1089
`Page 6
`
`