Clinical Chemistry 49:10
`1579—1598
`(2003)
`
`Review
`
`
`
`Potential Clinical Utility of Serum HER-2/neu
`Oncoprotein Concentrations in Patients with
`Breast Cancer
`
`WALTER P. CARMEL"? RAINER NEUMANN,3 ALLAN LIPTON,4 KIM LEITZEL,4 SUHAIL ALI,“'5
`and CHRISTOPHER P. PRICE6’7
`
`
`Background: The HER-21mm oncogene and its p185
`receptor protein are indicators of a more aggressive form
`of breast cancer. HER-Zlneu status guides Herceptin
`therapy, specifically directed to the extracellular domain
`(ECD) of the HER-Zineu oncoprotein. The HER-Zlneu
`ECD is shed from cancer cells into the circulation and is
`measurable by immunoassay.
`Methods: We performed a systematic review of the
`peer-reviewed literature on circulating ECD with re-
`spect to prevalence, prognosis, prediction of response to
`therapy, and monitoring of breast cancer.
`Results: The prevalence of increased ECD in patients
`with primary breast cancer varied between 0% and 38%
`(mean, 18.5%), whereas in metastatic disease the range
`was from 23% to 80% (mean, 43%). Some women with
`HER-Zlneu-negative tumors by tissue testing develop
`increased ECD concentrations in metastatic disease.
`Increased ECD has been correlated with indicators of
`poor prognosis, e.g., overall survival and disease-free
`survival. Increased ECD predicts a poor response to
`hormone therapy and some chemotherapy regimens but
`can predict improved response to combinations of Her-
`
`' Oncogene Science, Bayer HealthCare, Cambridge, MA 02142.
`2 Tufts New England Medical School, Department of Pathology, Boston,
`MA 02111.
`3 Medizinische Fakultaet der Universitaet zu Koeln, D50931 Koeln, Ger-
`many, and Bayer Vital GmbH, D-51368 Leverkusen, Germany.
`4 Section of HematologyOncology, Pennsylvania State University/Her-
`shey Medical Center, Hershey. PA 17033.
`5 Department of Medicine, Veterans Administration Medical Center, Leb-
`anon, PA 17042.
`6 Bayer HealthCare, Diagnostics Division, Stoke Court, Stoke Poges,
`Slough, Berlehire SL2 4LY, United Kingdom.
`’Clinical Biochemistry, University of Oxford, Oxford 0x1 21D, United
`Kingdom.
`‘Address correspondence to this author at: Oncogene Science, Bayer
`HealthCare, 80 Rogers St., Cambridge, MA 02142, Fax 617-492-8438; e—mail
`walter.camey.b@oncogene.com.
`Received April 9, 2003; accepted July 2, 2003.
`
`ceptin and chemotherapy. Many studies support the
`value of monitoring ECD during breast cancer progres-
`sion because serial increases precede the appearance of
`metastases and longitudinal ECD changes parallel the
`clinical course of disease.
`
`Conclusions: The monitoring of circulating HER-Zlneu
`ECD provides a tool for assessing prognosis, for predict-
`ing response to therapy, and for earlier detection of
`disease progression and timely intervention with appro-
`priate therapy.
`li') 2003 American Association for Clinical Chemistry
`
`The activation and overexpression of cellular oncogenes
`has been considered to play an important role in the
`development of human cancer (1 ). An important member
`of the oncogene family is the growth factor receptor
`known as human epidermal growth factor receptor-2
`(HER-2)B (2), which is also referred to as HER-2/neu or
`c-erbB-Z. HER—Z/neu is structurally and functionally re—
`lated to the v-erbB retroviral oncogene (3) and is part of
`the HER family, which also includes HER-1, or epidermal
`growth factor receptor (EGFR), and HER-3 and HER-4 (4).
`The HER-Z/neu oncogene has been localized to chro-
`mosome 17q and encodes a transmembrane tyrosine
`kinase growth receptor that
`is expressed on cells of
`epithelial origin. The full-length glycoprotein has a mo-
`lecular mass of 185 000 Daltons (p185) and is composed of
`the internal tyrosine kinase domain, a short transmem—
`
`5 Nonstandard abbreviations: HER, human epidermal growth factor recep-
`tor; EGFR, epidermal growth factor receptor; ECD, extracellular domain:
`MMI’, matrix metalloproteinase; Mab, monoclonal antibody; MBC, metastatic
`breast cancer; 05, overall survival; lHC, immunohistochemistry; FISH, fluo-
`rescence in situ hybridization; FDA, Food and Drug Administration; PBC.
`primary breast cancer; CISH, chromogenic in situ hybridization; "ITP, time to
`progression; DFS, disease—free survival: HDCT, high-dose chemotherapy; ER,
`estrogen receptor; CMF, cyclophosphamide—metho-r. ., e—S-tluorouracil; and
`CEA, carcinoembryonic antigen.
`
`1579
`
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`Hospira v. Genentech
`IPR2017-00805
`(cid:44)(cid:51)(cid:53)(cid:21)(cid:19)(cid:20)(cid:26)(cid:16)(cid:19)(cid:19)(cid:27)(cid:19)(cid:24)(cid:3)
`Genentech Exhibit 2022
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`1580
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`Carney et al.: Clinical Utility of Serum HER-Zlneu in Breast Cancer
`
`brane portion, and an extracellular domain (ECD) that is
`similar to the three other members of the HER family
`(2, 5). The ECD portion of the receptor protein is heavily
`glycosylated, has a molecular mass in the 97- to 115-kDa
`range, and has been shown to be shed into culture fluids
`of SKBR—S cells (6) as well as plasma (7) and serum (8, 9)
`from healthy individuals and patients with breast cancer
`(7—9).
`The mechanism of activation of the HER-Z/neu path-
`way is not completely understood, but studies have
`shown that the ECDs of the HER family of receptor
`tyrosine kinases form homodimers and heterodimers and
`that receptor dimerization activates a cascade of events in
`the HER~2/neu signaling pathway (4). A ligand that binds
`to the HER-Z/neu receptor has not been identified, but a
`family of peptide ligands named neu differentiation fac-
`tors, or heregulins, has been identified. Heregulins are
`45-kDa growth factors, with homology to EGF, that bind
`directly to HER-3 and HER-4 receptors and cause the
`formation of heterodimeric receptor complexes. The
`dimers subsequently induce transphosphorylation and
`activate the HER—Z/neu receptor (Fig. 1).
`In the study by Aguilar and Salmon (10), the biological
`activity of the plasma membrane-anchored heregulin was
`evaluated in human breast cancer cells. It was reported
`that transmembrane heregulin binds to cells expressing
`HER-3 and induces HER-Z/neu phosphorylation and in—
`creases DNA synthesis in cells overexpressing HER-2/
`neu. The biological response to heregulin appears to
`depend directly on the degree of HER-Z/neu expression
`in breast cancer cells; therefore, the regulatory pathway
`for control of proliferation may depend on the concentra-
`tions of both the heregulins and HER-2/ neu ECD. Perhaps
`measuring growth factors such as heregulins and EGF
`will have a separate clinical utility or clinical utility in
`conjunction with measuring HER-2/neu ECD. In addi-
`tion, heterodimers can also form between the EGFR and
`HER-Z/neu on binding of EGF or transforming growth
`
`HER-2 Activation
`
`factor-a to the EGFR (11). Dimerization of the various
`HER receptors leads to tyrosine phosphorylation and
`activation of the HER-Z/neu kinase with subsequent acti-
`vation of downstream transduction pathways and signal—
`ing through ras, c-Src, phosphatidylinositol 3-kinase, and
`phospholipase Cy pathways. Formation of heterodirners
`increases the affinity of the partnering receptor for its
`ligand and leads to potentiation of the mitogenic signal
`(12).
`
`In a report by Codony—Servat et al. (13), it was sug—
`gested that cleavage of the HER-Z/neu ECD involves
`matrix metalloproteinase (MMP) activity and that the
`process of ECD cleavage was inhibited by the MMP
`inhibitor TIMP-l but not by TIMP-2. The MMPs have
`been strongly implicated in multiple stages of cancer
`progression, including invasion and metastasis (14), but it
`is not yet known whether the HER-2/neu ECD functions
`in the process of invasion and metastasis. Codony-Servat
`et al. (13) showed that HER—Z/neu ECD shedding was
`inhibited by broad-spectrum MMP inhibitors such as
`EDTA, TAPI-2, and Batimastat and confirmed the data
`from Christianson et al. (15) showing that HER-2/neu
`ECD cleavage leads to the release of a truncated phos-
`phorylated p95 fragment. Molina et al. (16) showed that
`the p95 fragment could be detected in only 14 of the 24 of
`the human breast tumors analyzed and that p95 band
`expression was highly variable. Studies continue to deter—
`mine both the biological role and the potential clinical
`value of the phosphorylated membrane fragment.
`In their report, Molina et al. (16) also showed that
`HER-Z/neu shedding was activated by 4-amino-phe-
`nylmercuric acetate (a well-known MMP activator) in
`HER-Z/neu—overexpressing breast cancer cells and that it
`could be blocked by the MMP inhibitor, Batimastat. The
`increase in ECD shedding also enhanced the production
`of the p95 fragment. In the same report (16), they dem-
`onstrated that Trastuzumab® [commonly referred to as
`Herceptin and manufactured by Genentech (San Fran-
`cisco, CA)], an anti-HER—Z/neu therapy, had a direct
`inhibitory effect on the basal and activated processes
`involved in HER-Z/neu cleavage from HER—Z/neu-over-
`expressing breast cancer cells. The HER—Z/neu ECD shed-
`ding that was activated by 4—arnino-pheny1mercuric ace—
`tate could be blocked with Herceptin, however, leading to
`a reduction in the release of the p95 fragment (16).
`Trastuzumab is a humanized monoclonal antibody
`(Mab) developed to target the HER—Z/neu receptor that is
`overexpressed in 25—30% of breast cancers cells. Herceptin
`binds with high affinity to the ECD of HER-2/neu and
`inhibits proliferation of tumor cells that overexpress the
`HER-Z/neu oncoprotein. The results of a large multicenter
`phase 3 clinical trial demonstrated that Herceptin, when
`added to conventional chemotherapy, can provide benefit
`to patients with metastatic breast cancers (MBCs) that
`overexpress HER-2/neu. Compared with the best avail-
`able standard chemotherapy, concurrent treatrnent with
`Herceptin and first-line chemotherapy was associated
`
` *Tymalna klnau
`Moment!"GrowthFactor: ”. .\J
`
`Reamer
`
`Receptor
`Dlmmutlcn
`
`Din-tuba"mg?
`
`
`amonhosphoryution
`
`“91-”HER-2
`
`MERJMER—l
`
`Gene Actlvatlon
`
`Fig. 1. HER-2 activation.
`HER-2/neu signaling pathways are activated by homo and heterodimerizations
`with £605 of the other HER family members. In addition. growth factors such as
`hereguiins can also induce dimerization and stimulate signal transduction along
`the HER-2/neu pathway by binding to the HER>3 and HERA receptors.
`
`

`

`Clinical Chemistry 49, No. 10, 2003
`
`1581
`
`with significantly longer times to disease progression, a
`higher rate of response, longer duration of response, and
`improved overall survival (OS) (17).
`Because Herceptin treatment [reviewed in Ref. (18)]
`has been in practice for only a few years, the mechanism
`of action by which Herceptin either alone or in combina-
`tion with chemotherapy enhances survival of MBC pa-
`tients still needs to be understood. According to a recent
`report (19), it is possible that Herceptin binds the ECD,
`causing internalization and degradation, and subse-
`quently inhibits signal transduction pathways. Another
`possible mechanism by which Herceptin is effective is the
`induction of cytolytic activity through cytotoxic lympho—
`cytes (19). However, many additional studies are neces-
`sary to understand the mechanism(s) by which Herceptin
`provides benefit to women with MBC.
`In this report, we will review HER-2/neu circulating
`ECD concentrations in relation to prevalence, prognosis,
`prediction of response to therapy, monitoring in MBC,
`and monitoring for early detection of recurrence.
`
`Methods of Determining her-2/neu Status
`The most widely accepted method for measuring HER-2/
`neu protein (p185) overexpression is immunohistochem-
`istry (IHC) (20, 21), whereas the number of HER-Z/neu
`gene copies or gene amplification is determined by a
`fluorescence in situ hybridization (FISH)
`test
`(20, 21).
`ELISAs have been used since 1991 to quantify either the
`full-length p185 in tumor tissue (7) or the soluble circu-
`lating HER-2/neu ECD in serum (8) or plasma (7).
`Since the earliest studies in 1987 (22) and 1989 (23),
`numerous tumor studies using formalin-fixed, paraffin-
`embedded tumor tissue have shown that HER-Z/neu
`DNA amplification and protein overexpression occur in
`~25—30°/o of women with primary breast tumors (20, 21).
`However, a few reports have described a wider range
`(IS—40%) of HER-Z/neu tissue expression, which appears
`to be a function of the composition of the sample being
`evaluated by the pathologist. For example, Latta et al. (24)
`reported that IHC staining was seen in 34% of women
`with pure ductal carcinoma in situ, 17% of women with
`invasive carcinomas with associated ductal carcinoma in
`
`situ, and 12.5% of pure invasive carcinomas. Sixty percent
`of cases of high-grade ductal carcinoma in situ showed
`increased IHC overexpression vs the 27% of high-grade
`invasive carcinomas with associated ductal carcinoma in
`
`situ and 22% of high—grade pure invasive ductal carcino-
`mas. In part the wide range of IHC staining of primary
`breast tumors could be attributable to a variety of factors.
`It could be the heterogeneity of the tumor specimen
`within the tissue section and/ or variations in methodol-
`ogies, antibodies, or operators. It could also be attribut—
`able to the time of diagnosis or the existence of the early
`detection programs at the various institutions.
`The current availability of commercial IHC reagent sets
`for HER-Z/neu detection has now led to greater standard—
`ization relative to past testing, when a variety of method—
`
`ologies and antibodies were used. For example, the US
`Food and Drug Administration (FDA) granted clearance
`for the use of two commercially available IHC assays to
`identify women who are candidates for Herceptin-based
`therapies. These include Herceptest, a rabbit polyclonal
`(DAKOCytomation, Copenhagen, Denmark), and Path-
`way (Ventana, Medical Systems, Tucson, AZ), which uses
`the Mab CBll. The first step in the IHC scoring system
`used to establish HER-Z/neu status is
`to determine
`whether >10% of the breast tumor cells are stained with
`
`a HER-Z/neu-specific antibody. The second step is to
`ascertain whether the membrane staining is partial or
`complete, and the third step is to determine the intensity
`of staining (3+, 2+, or 1+).
`The second method used to assess the HER-Z/neu
`status of a primary breast cancer (PBC) is FISH analysis.
`The advantages of this technique include the stability of
`chromosomal DNA and the ability to determine gene
`amplification or the number of HER-2/neu copies within
`tumor cells. The FISH technique is very reliable, provid-
`ing 95.5% sensitivity and 100% specificity for detection of
`the HER—2/neu gene amplification. Currently there are
`two commercially available FISH reagent sets cleared for
`use by the FDA. Patthsion (Vysis, Downers Grove, IL)
`detects the HER-2/neu gene with a directly labeled probe.
`The primary advantage of this test is that it includes an
`internal control probe for the assessment of polysomy.
`The control probe simultaneously detects the centromere
`of chromosome 17, the locus of the HER-Z/neu gene.
`Patthsion was approved originally to select node-posi-
`tive, stage II patients for anthracycline treatment and was
`recently expanded to include selection for Herceptin
`therapy. The second FISH test cleared by the FDA is called
`the Inform test (Ventana Medical Systems, Tucson, AZ)
`and uses a biotin-labeled oligonucleotide probe that is
`detected by an avidin-biotin FITC system for signal am-
`plification (21 ). This test was cleared for use as a prog-
`nostic factor in node-negative breast cancer patients.
`Overall,
`tissue testing with either IHC or FISH is
`performed on the primary tumor tissue, and these are the
`only methods cleared by the FDA to determine eligibility
`of women with MBC for Herceptin therapy (21). At
`present, the IHC and FISH methods are used in combina-
`tion to offer enhanced reliability for determining the
`HER-Z/neu status and eligibility of a woman for Hercep—
`tin—based therapies. Herceptin-based therapy is a term
`that is used to refer to any chemotherapy that is used in
`combination with Herceptin. Currently, a woman is con-
`sidered to have a positive HER-Z/neu tumor if she has
`>10% of the breast
`tumor cells staining with a 3+
`intensity. In contrast, women with <10% positive cells are
`considered not to have a HER—Z/neu—positive tumor and
`according to current guidelines would not be considered
`eligible for Herceptin therapy.
`Although tissue testing is very important in determin-
`ing the HER-Z/neu status of the primary tumor, there are
`limitations to tissue testing. The most important is that
`
`

`

`1 582
`
`Carney et al.: Clinical Utility of Serum HER-Z/neu in Breast Cancer
`
`they are one—time tests used to determine the HER-Z/neu
`status of the PBC, which in turn determines the eligibility
`for Herceptin. Herceptin is given primarily in the meta-
`static setting, but the HER-Z/neu status is determined
`from the original breast tumor, which may have been
`removed many years earlier.
`The tissue tests for protein overexpression by IHC and
`gene amplification by FISH are both subject to technical
`problems. For IHC these include, but are not limited to,
`differences in the methodology between laboratories and
`between operators, variability in operator interpretation,
`and variation in reagents. Although the FISH technology
`appears to be reproducible, there are several limitations to
`FISH testing. The FISH instrumentation is expensive and
`not widely available in diagnostic pathology laboratories.
`In addition,
`the standardization of fixative solutions,
`fixation times, and digestion conditions continue to be
`technical issues, suggesting that problems still exist with
`FISH as well as IHC.
`
`More recently, it has been proposed that chromogenic
`in situ hybridization (CISH) is an alternative to FISH. In
`contrast to FISH and fluorescence, CISH technology is
`based on a peroxidase reaction that can be visualized by
`light microscopy and eliminates the need for fluorescence
`equipment. CISH also provides a permanent record and is
`less expensive than FISH. Although CISH appears to be
`more practical and accurate, it had not been cleared by the
`FDA for use in the USA. Additional studies are also
`
`required to determine the exact concordance between
`FISH and CISH before CISH can be accepted as a method
`to select patients for Herceptin therapy (25, 26).
`A review of the recent literature comparing FISH and
`IHC methods on the same tumor specimens showed a
`considerable degree of concordance between the methods.
`Many reports in the past few years have shown a >90%
`concordance between the methods, which approached
`100% when comparing 3+ IHC readings with FISH
`(12,21). However, cases with a 2+ score are not very
`reproducible and therefore are no longer used alone to
`determine eligibility of a woman for Herceptin. Patients
`with a 2+ IHC score must have a confirmatory FISH test
`to be eligible for Herceptin therapy.
`In a retrospective analysis performed to explore the
`correlation between gene amplification by FISH and clin—
`ical outcomes of women treated with Herceptin-based
`therapies, it was concluded that FISH was the superior
`method for selecting patients likely to benefit from Her-
`ceptin therapy (27). Additional studies are warranted to
`substantiate this observation.
`
`Some studies have also used tissue testing to compare
`the HER-2/neu status of the primary tumor and the
`metastatic lesion. For examples, in a report by Gancberg et
`al. (28), the primary breast tumors and at least one distant
`metastatic lesion from 107 patients were analyzed by IHC
`and FISH. Similar degrees of amplification were observed
`in the primary (25%) and metastatic lesions (24%). How-
`ever, in the FISH analysis, performed on 68 paired pri-
`
`mary and metastatic tumors from breast cancer patients, 5
`of the 68 (7%) cases showed discordance; in 3 of the 5
`samples there was amplification in the metastasis but not
`in the primary tumor. In the case of IHC, there was 6% (6
`of 100) discordance, and in all 6 cases there was greater
`HER-2/ neu overexpression in the metastatic tissue than in
`the primary tumor.
`The third method used to determine the HER-2/neu
`status is the ELISA, and it can be used to quantify either
`the full—length p185 in tumor tissue or the circulating ECD
`in serum or plasma. Zabrecky et al.
`(6), using Mabs
`directed to the ECD (29), demonstrated that the ECD was
`shed into the culture supernatant of SK-BR—3 breast cancer
`cells. Studies using specific Mabs against the HER—2/neu
`protein combined with immunoprecipitation and Western
`blot techniques showed that the ECD was a glycoprotein
`with molecular mass between 97 and 115 kDa (6). Subse-
`
`quent studies illustrated that the ECD could be detected
`in the plasma of healthy individuals and was increased in
`women with primary and MBC (7). These observations
`were later confirmed by Leitzel et al. (8) and Pupa et al.
`(9).
`
`In the last few years many reports have described a
`variety of ELISA formats that have been used to quantify
`the ECD in serum or plasma of breast cancer patients and
`control groups. However, it has been difficult to compare
`results between publications because of a lack of stan-
`dardization between the ELISAs. For example, three pub—
`lications reporting ECD results with one particular com-
`mercial assay (Triton—Ciba Corning-Chiron) used at least
`three different cutoff values (3, 12, and 30 units/mL) to
`separate healthy and diseased populations (8, 30, 31). In
`some reports, antibody specificity or assay validation for
`HER-Z/neu has not been demonstrated (32), nor have
`adequate references been provided to demonstrate that
`the antibodies in the ELISAs specifically detect the HER-
`2 / neu ECD.
`In summary, IHC and FISH testing can be used to
`determine the HER-Z/neu status in primary tumor tissue
`but are not adequate for assessing the HER-Z/neu status
`of a woman after the tumor is removed. In contrast, the
`ELISA method for measuring the circulating HER-2/neu
`ECD is the only way to obtain the real-time status of
`HER-2/ neu and the only practical way to monitor changes
`in the HER-Z/neu ECD concentrations post surgery.
`
`Methodology of Literature Review
`An electronic search of the Medline database was per-
`formed using the following key words: human epidermal
`growth factor receptor-2, HER—Z/neu, and breast cancer.
`Only full articles in peer—reviewed journals were included
`in the search. After identifying the potentially relevant
`articles based on the inclusion criteria set out below, we
`included their reference lists. In addition, recent reviews
`were then searched for additional relevant articles.
`
`All of the titles generated by the search, and the
`abstracts when available, were reviewed for relevance by
`
`

`

`Clinical Chemistry 49, No. 10, 2003
`
`1583
`
`at least three of the authors, and the full articles were
`obtained. Each of the articles was then read by two of the
`authors, and those for subsequent inclusion in the data
`extraction stage were identified by the following criteria:
`(a) main objective of assessing the clinical utility of the
`HER-2/neu ECD measurement;
`(b) patient population
`defined,
`including age and pathology;
`(c) number of
`patients and any exclusion criteria identified; (d) timing of
`collection of blood samples identified; (e) analytical meth-
`ods defined; (f) cutoff values for HER-Z/neu ECD value
`defined; (3) data for assessing prognosis, predictive value,
`and clinical course available; and (h) indication whether
`the operators performing assays were blinded to the data
`from the assessment of the clinical status of the patient.
`Any discrepancy in the findings of the two primary
`reviewers was then assessed by a third reviewer, and a
`decision was made to include or exclude the data, the
`latter whenever the discrepancy could not be resolved.
`
`Results
`PREVALENCE or CIRCULATING CONCENTRATIONS HER-
`2/neu IN BREAST CANCER
`We found a total of 55 publications from which data on
`the prevalence (7, 8, 33—82) of increased concentrations of
`circulating HER-Z/neu could be extracted. The studies are
`summarized in Table 1 and represent circulating HER-Z/
`neu ECD measurements in >6500 patients with breast
`cancer. A review of 24 references used to evaluate ECD
`
`concentrations in PBC showed that in studies involving
`1923 patients, ~18.5% had circulating HER-Z/neu ECD
`concentrations that were above the control cutoff de—
`scribed in each publication.
`In contrast, a review of 45 references and 4622 patients
`with MBC showed that ("43% of the patients had circu-
`lating HER-2/neu ECD values above the cutoff for the
`control group presented in the publication. In 15 of the 45
`publications, ECD concentrations were increased above
`the control group in >50°/o of the patients studied. In a
`publication by Fehm et al. (49), 62% of the MBC patients
`were reported to have increased ECD. Fehm et al. also
`reported that 31% of the patients with PBC had increased
`ECD concentrations and in that population of patients,
`62% developed increased concentrations during the met-
`astatic disease.
`
`It is interesting to note that the data in Table 1 came
`from essentially six different assays. The automated Im-
`muno-l HER-Z/neu test (manufactured by Bayer Health-
`Care, Tarrytown, NY) and the Oncogene Science manual
`microtiter plate HER-Z/neu test (manufactured by Onco-
`gene Science, Cambridge, MA) are essentially the same
`assays in that both use anti-HER-Z/neu Mabs NB-3 and
`TA-l, (29), a soluble p97—115kDa calibrator, and a 15
`ug/ L cutoff (44, 74, 83). It should also be noted that there
`was a strong correlation between the automated and
`manual HER-Z/neu assays (44, 74, 83). Both the auto-
`mated and manual HER-Z/neu assays have been cleared
`by the FDA for use in the management and monitoring of
`
`women with MBC. These are the only two assays cur-
`rently cleared for the measurement of the circulating
`p97—115kDa HER-Z/neu ECD. There were 20 publications
`in Table 1 that used the two methods described above.
`There were 21 references to the Triton-Ciba-Chiron as-
`says, with 11 different cutoffs ranging from 5 to 30
`units /mL as well as a 120 or 450 fmol/mL cutoff. These
`assays are essentially the same and are no longer com-
`mercially available for studies. There were five references
`to the Nicherei assay, three references to the Calbiochem
`or ORP assay, three references to the Dianova assay, and
`two references to the Bender assay. We could not find any
`references that described the antibody specificities or
`standard materials used in the Calbiochem, Dianova, or
`Bender assays, nor could we find any references that
`validated biochemically that the assays clearly measured
`the circulating HER-Z/neu ECD. The Calbiochem, Di-
`anova, and Bender assays are available for research use,
`which means that their performance characteristics have
`not been determined. In fact, the Bender assay claims to
`measure the circulating soluble p185, but there has been
`never been a scientific report of a circulating full-length
`p185, nor does the manufacturer present data to support
`the claim. Several other reports, however, have reproduc-
`ibly demonstrated that
`the only HER—Z/neu fragment
`found circulating is the truncated p97—115kDa (6 —9 ) ECD,
`so it is unclear what the Bender assay actually measures.
`In their report, Andersen et al.
`(34) showed that
`increased HER-2/neu ECD concentrations were detected
`
`in the serum of 8% of preoperative breast cancer patients
`and in only % of postoperative sera from patients
`without recurrent breast cancer. In contrast, 59% (55 of 93)
`of patients with recurrent breast cancer developed in-
`creased HER-Z/neu ECD concentrations. They also re—
`ported that increased ECD concentrations were detected
`significantly more often in patients with distant metasta-
`ses than in patients with recurrent disease restricted to
`local metastasis (68% vs 19%). This observation was
`supported by Watanabe et al. (30), who concluded that
`the circulating HER-Z/neu ECD concentration was closely
`related to tumor mass because the HER-2/neu ECD con-
`
`centration in recurrent disease was found to be signifi-
`cantly higher than in nonrecurrent disease.
`Regardless of the ELISA used, studies show that there
`is great variation in shed ECD in patients with increased
`concentrations. The concentrations can range from thou-
`sands of micrograms per liter to slightly above the 15
`ug/ L cutoff in MBC patients. It is unclear what mecha—
`nisms account for such a wide variation in shedding and
`what is the biological or clinical significance of the shed-
`ding. However, as mentioned earlier, MMPs are involved
`in proteolytic cleavage of ECD, and the increased shed-
`ding may be indicative of up-regulated MMP activity.
`MMPs have been associated with tumor invasion and
`
`metastasis (14), and therefore, enhanced ECD shedding
`may be indicative of the more aggressive cancers.
`In their report, Andersen et al. (34) showed that 14 of
`
`

`

`1584
`
`Carney et al.: Clinical Utility of Serum HER-Z/neu in Breast Cancer
`
`
`
`Table 1. Prevalence 0! Increased HER-2/neu ECD concentrations In P36 and MBc.
`P86
`MES
`
`Studya
`H?
`
`comumcnbmm
`
`comwwwwmmmmmmmmMMMMHHHI—Ii—IHHHHH
`gggg3&33atc’33Sgggummbwnpoomumm>wwuommummnmmr—so
`
`55
`
`Amhom
`
`Nugent et al.
`Schwartz et at.
`Cheunget al.
`Cook et al.
`Dittadi et al.
`Lipton et al.
`Schoendorf et al.
`Koes’der et al.
`Ali et al.
`Hayes et al.
`Breuer et al.
`Breuer et al.
`Kasimir—Bauer et al.
`Anderson et al.
`Bewick et al.
`Yamauchi et al.
`Leitzel et al.
`Fontana et al.
`Krainer et al.
`Kandl et at.
`Wu et al.
`Wu et al.
`Kynast et al.
`Revilllon et al.
`Volas et al.
`Bewick et al.
`Isola et al.
`Narita et al.
`Harris et al.
`Molina et al.
`Molina et al.
`Molina et al.
`Molina et al.
`Lueftner et al.
`Fehm et al.
`Fehm et al.
`Colomer et al.
`lmoto et al.
`Hosono et al.
`Sugano et al.
`Sugano et al.
`Kath et al.
`Classen et al.
`Visco et al.
`Klein et al.
`Lipton et al.
`Chearskul et al.
`Fehm et al.
`Carney et al.
`Streckfus et al.
`Breuer et al.
`Harris et al.
`Wu et al.
`Dnistrian et al.
`
`Year
`1992
`2000
`2000
`2001
`2001
`2002
`2002
`2002
`2002
`2001
`1994
`1993
`2001
`1995
`2001
`1997
`1992
`1994
`1997
`1994
`1993
`1995
`1993
`1996
`1996
`1999
`1994
`1992
`2001
`1996
`1996
`1997
`1999
`1999
`1997
`1998
`2000
`1999
`1993
`1994
`2000
`1993
`2002
`2000
`1995
`2003
`2000
`2002
`1991
`2000
`1998
`1999
`1999
`2003
`
`56
`
`25
`
`16
`
`176
`
`36
`20
`128
`13
`
`125
`42
`
`25
`20
`
`000
`
`21
`14
`
`59
`81
`65
`
`3.4
`8.6
`22
`
`211
`
`12.3
`
`73
`
`38
`
`185
`158
`39
`
`28
`49
`
`94
`52
`33
`30
`67
`
`13
`22.8
`
`29
`
`13.8
`31
`15
`94
`4.50
`
`134
`
`17
`
`138
`30
`50
`55
`719
`19
`55
`566
`89
`
`93
`46
`94
`53
`
`62
`79
`16
`164
`35
`33
`300
`57
`
`51
`65
`29
`77
`106
`95
`35
`
`80
`58
`20
`21
`121
`
`61
`64
`43
`31
`562
`
`52
`105
`
`35
`92
`54
`
`50
`35.5
`57.1
`40
`51
`30.5
`63
`75
`30
`36
`
`59
`46
`34
`23
`
`44
`49
`75
`35
`26
`30
`19.3
`47.4
`50
`50.9
`29
`32.6
`45.4
`42
`28.4
`28.5
`
`38.8
`41
`80
`57
`51
`
`43
`40.6
`35
`65
`29.5
`
`62
`23
`
`28.5
`26
`67
`
`Assay
`Immuno 1
`lmmuno 1
`Immuno 1
`Immuno 1
`Immuno 1
`Immuno 1
`Immuno 1
`Immuno 1
`Immuno 1
`08c“1
`05c
`08C
`050
`08c
`050
`08c
`Triton
`Tn'ton
`Triton
`Triton
`Triton
`Triton
`Triton
`Triton
`Triton
`Triton
`Triton
`Triton
`Chiron
`Ciba
`Ciba
`Ciba
`Ciba
`Ciba
`Ciba
`Ciba
`Calbiochem
`Nichirei
`Nichirei
`Nichirei
`Nichirei
`Dianova
`Dianova
`Bender
`Bender
`Immuno 1
`Nichirei
`Dainova
`DuPont
`ORP
`OSc
`Chiron
`Calbiochem
`lmmuno 1
`
`(71)
`(74)
`(41)
`(44)
`(45)
`(63)
`(73)
`(107)
`(33)
`(53)
`(38)
`(37)
`(58)
`(34)
`(36)
`(82)
`(8)
`(50)
`(61)
`(57)
`(79)
`(80)
`(62)
`(72)
`(31)
`(35)
`(56)
`(70)
`(52)
`(66)
`(67)
`(68)
`(69)
`(65)
`(47)
`(48)
`(43)
`(55)
`(54)
`(76)
`(77)
`(59)
`(42)
`(78)
`(60)
`(64)
`(40)
`(49)
`(7)
`(75)
`(39)
`HNU/mL
`U/mL
`(51)
`(81)
`U/mL
`(46)
`pg/L
`" 05c, Oncogene Science: HNU. human Neu units; ORP. Oncogene Research-Calbiochem.
`
`Cute"
`
`Units
`
`Reference
`
`#g/L
`ug/L
`[Lg/L
`lug/L
`#g/L
`F-g/L
`rig/L
`rig/L
`rig/L
`U/mL
`U/mL
`lug/L
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`U/mL
`fmoI/mL
`fmoI/mL
`fmol/mL
`I-Lg/L
`U/mL
`#g/L
`ug/L
`HNU/mL
`U/mL
`
`#g/L
`[lg/L
`HNU/mL
`HNU/mL
`
`15
`20
`15
`13
`15
`20
`15
`15
`10.5
`3060
`3060
`15
`1600
`2500
`5000
`
`20
`10
`25
`24
`30
`27
`30
`21
`2O
`2O
`20
`20
`15
`15
`15
`15
`120
`120
`450
`5.4
`
`5.4
`5.4
`33
`40
`
`15
`5.4
`1900
`1900
`
`3271
`20
`40
`15
`
`

`

`Clinical Chemistry 49, No. 10, 2003
`
`1585
`
`24 patients who had IHC-positive breast tumors also had
`increased HER-Z/neu serum concentrations during the
`metastatic phase of the disease. In contrast, 28 of 82 (34%)
`patients who had IHC-negative primary breast tumors
`developed increased serum concentrations during the
`metastatic disease. Kandl et a1. (57) also reported that
`some patients with negative HER-2/neu tumor staining
`developed extremely high concentrations of circulating
`HER-Z/neu during MBC and that increasing concentra-
`tions correlated with progressive disease, which also
`correlated with extensive disease burden. Molina et al.
`
`(66) also reported that 23% of patients with recurrent
`breast cancer with no tissue overexpression had increased
`ECD concentrations, on