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Frequency and reliability of oestrogen receptor I progesteronereceptor and HER2 in breast carcinoma determined byimmunohistochemistry in Australasia: results of the RCPAQuality Assurance ProgramGlenn D Francis, Margaret Dimech, leanne Giles, Alison Hopkins

J elin Patho/2007;60:1277-1283. doi: 10.1136/jcp.2006.044701

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Correspondence to:Dr Glenn D francis,Queensland HealthPathology Service, PrincessAlexandra Hospital, IpswichRd, Woolloongabba,Queensland, Australia4102; GlennJrancis@health.qld.gov.au

Accepted 8 January 2007Published Online First26 January 2007

Background and Aims: Immunohistochemistry (IHC) has replaced radioligand binding assay for thedetermination of oestrogen receptor (ER) status in breast carcinoma. IHC is also used for assessment ofprogesterone receptor (PRJ and HER2. The Royal College of Pathologists of Australasia (RCPA) QualityAssurance Program (QAP) introduced a breast markers module in 2003 to evaluate the performance oflaboratories with IHC for ER, PR and HER2.Methods: An audit of laboratories reporting breast carcinomas was performed in 2005 and 2006 to evaluatein-house results. Laboratories were asked to submit the hormone receptor and HER2 status on each invasivebreast carcinoma for the previous 6 month period up to a maximum of 100 cases. The time periods were 1July 2004 to 31 December 2004, and 1 July 2005 to 31 December 2005. A total of 55 laboratories returnedinformation for 2004 and 67 for 2005.Results: Complete data on 8128 patients was returned for both surveys, 3353 cases for 2004 and 4775 for2005. The results were simili;lr for both surveys. of the 8128 cases, 59.0% were ER+/PR+, 15.9% ER+/PR-,2.4% ER-/PR+ and 22.7% ER-/PR-. HER2 data were submitted for a total of 6512 patients (excludes 52patients with incomplete data sets); 17.1 %were reported as 3+ positive on IHC, 12.5% as 2+ and 70.4% asnegative.Conclusions: A laboratory audit was introduced into the RCPA QAP for breast markers due to concernsraised by participating laboratories about technical differences in supplied tissues for testing. This auditindicates that overall the results for ER, PR and HER2 fall inside established parameters. However, a numberof individual laboratories do not meet the target values and variation in results would impact on patienttreatment decisions.

In Australia there are approximately 12 000 new cases ofbreast carcinoma diagnosed each year. The dependence ofsome breast carcinomas on hormone receptors was identifled

more than 100 years ago with tumour regression in two of sixpatients after oophorectomy.' Assessment of hormone receptorstatus is utilised as a predictive marker to determine patienttreatment and is performed on all breast carcinomas. Currentassessment of hormone receptor status is based on immuno­histochemistry (IRC). This has supplanted the radioligandbinding assay that previously used frozen tissue.

The Royal College of Pathologists of Australasia (RCPA)Quality Assurance Program (QAP) introduced a breast markersmodule in 2003 because of the critical importance of hormonereceptor determination. It is a requirement of Australianlaboratories that they participate in a quality assuranceprogramme if this is available. While the majority of participat­ing laboratories are from Australia, there are a number oflaboratories from New Zealand and international locations.This module comprises two exercises each year consisting of anumber of patient samples in a ring trial format. The originalmaterial was a composite block, but this has been changed to atissue microarray construct for the past two years. Thespecimens consist of routine fonnalin fixed paraffin embeddedmaterial that has been supplied to the QAP by participants.Participating laboratories are sent these slides and asked toperform IHC for oestrogen receptor (ER), progesterone receptor(PR) and HERl on the sections, and return the slides forassessment as per their usual staining protocol. Homogeneity

slides are stained by H&E; IRC and stability testing has beenperformed on the test slides. The participant's slides arereviewed by a panel of scientists and pathologists and arescored individually from (1 to 5. Average scores are returned toparticipants. A score of 2 indicates that the result would affectpatient treatment or management. Scores of 3 and above areconsidered satisfactory. This evaluation of the laboratories'performance is included in the report as satisfactory, borderlineor unsatisfactory.

At anyone episode approximately 30% of participatinglaboratories will have an unsatisfactory result in at least oneof the three IRC markers. Using this system, a number ofparticipating laboratories that achieved unsatisfactory resultsraised concerns about the supplied material. This includeddifferences in fbmtion protocols, processing times and transittime to the laboratories. The laboratories indicated that theresults on in-house material were acceptable and therefore thering studies were not a tme reflection of the laboratory'sperformance.

To address these issues, laboratories were asked to performan audit in 2005 and 2006 for the previous 6 month period. Theaim was to truly indicate the reported results from eachlaboratory; this would overcome issues associated with external

Abbreviations: ER, oestrogen receptor; fiSH, Auorescence in-situhybridisotion; HR, hormone receptor; IHC, immunohistochemistry; ISH, in­situ hybridisation; PR, progesterone receptor; QAP, Quality AssuranceProgram; RCPA, Royal college of Pathologists of Australasia

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material and indicate if thein-house results were achievingacceptable results.

MATERIALS AND METHODSParticipants were requested to indicate each invasive breastcarcinoma case and return ER, PR and HERZ status for eachcase. This was retrieved from reports to a maximum of 100patients in each 6 month period. The time periods were 1 July2004 to 31 December 2004 and 1 July 2005 to 31 December2005. Participating laboratories used their own cut-off valuesfor hormone positivity; HERZ status was defined as for theHercepTest (Dako, CA., USA), scoring system. For this scoringsystem 3+ is positive, 2+ is equivocal and 1+ and 0 are scored asnegative. Duplicate cases were to be excluded.

The first exercise required the number of cases in eachcategory (hormone receptor and HERZ) to be returned. Thiswas modified for the second exercise to an Excel spreadsheet tofacilitate data collection. The results were analysed using SPSSV.14.0 for Windows (SPSS Inc, Chicago, lL, USA).

From the ring studies for ER and PR approximately 37% oflaboratories use a manual method, with the remaininglaboratories utilising various founs of automation. For ER,primary antibodies include 6Fll, IDS and SPl; for PR, PgR636,16, SP2 and 1A6; and for HERZ, A0485, SP3, CBll, 10A7, 4B5and TAB250 from a variety of suppliers. There are at least 20different retrieval systems and detection systems in use.

A number of participants reported that they used cur-offvalues for reporting positive results (table 1) and the cut-offsused are indicated in table 2.

RESULTSA total of 55 laboratories reurrned information for the timeperiods in 2004 and 67 for 2005. Por the 2005 series there were42 Australian sites, 9 New Zealand sites and 16 internationallaboratories. The results are very similar for both audits(table 3).

For the IHBR06 audit, data were returned for a total of 4807patients for ER/PR and 3980 patients for HERZ. However, 19patients were reported as ER+ with no PR status, 13 patientswere reported as ER- with no PR status (28 had HERZ data)and 20 patients had HERZ status reported with no ERIPR data.

Overall 59.0% of cases were ER+/PR+, 15.9% were ER+/PR-,2.4% were ER-/PR+ and 22.7% were ER-/PR- for patientswith complete data. Similarly there was little difference for thetwo years with respect to HER2 status and for the hormonereceptor subgroups within HERZ (tables 4-6).

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HERZ status was provided on 6512 patients (80.1%) withcomplete data sets. While it is recommended that laboratoriesperform HER2 testing at the tinle of diagnosis, this is not arequirement. Discussions with laboratories indicated that someuse a triage system and exclude patients based on clinicaldecisions (eg, patients who are ineligible for chemotherapy) orpathological decisions (eg, if the urmour is unlikely to bepositive, such as classic lobular carcinoma or tubular carci­noma). Overall 17.1% of patients were reponed as HERZpositive on mc with a rate of 16% for Australian cases.Collectively the results are similar to those reponed from otherstudies,' 3 however individual laboratories showed variation inthe percentages within each subgroup.

In the 2006 audit, ER positive rates varied from 26% to 100%,PR positive rates from 23% to 96%, and HER2 positive ratesfrom 0% to 66% (figs 1-3, table 7). .

Table 7 shows individual laboratory results for participatinglaboratories. There was still a large variation in positive rates,even in laboratories reporting 100 cases in the 6 month timeframe. Differences in rates were also observed across stateswithin Australia and between Ausualian, New Zealand andinternational laboratories (tables 8 and 9).

DISCUSSIONIn breast carcinomas, immunohistochemistry plays an essentialrole in determining patient treatment. The decision to useselective oestrogen receptor modulators or aromatase inhibitorsin adjuvant therapy is deternlined by the reported hormonereceptor starns. Similarly with the recen.! approval of trastuzu­mab for therapy in HERZ positive breast carcinomas inAustralia,IHC for HERZ has assumed equal inlportance.

In the current technical breast markers module for IHC thatuses supplied material in a ring stlJdy format, participants haveargued that the results do not truly reflect the in-house resultsand that the IHC performed by the laboratories is optimised forin-house material. This has been used as an argument forunsatisfactory results in the technical slides. The audit wasinuoduced in 2005 to assess the reported cases that shouldtherefore indicate the in-house results. Participants indicatedsome confusion for the requirements for this audit and as aresponse the reporting format was changed. The results for bothaudits are virtually identical, indicating little bias between thetwo time frames and data collection methods. For both audits,participants complained about the time taken to complete thesurvey. It should be remembered that for the audits these caseshave had reports generated and therefore action has already

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100 • 100

• • .. 90 00 090 • ••• '. 0 00 0 II 0 0 e• •• • • :. J 80 0 0080 • • • gx>o o~i5"< • • •• • • • i5"< 70 o~ • CD

70 • 0• • • •• 60 • tg' 0.l!l • *• •• OJ 0 CS'" 60 • • '" 0 o 0 •2 • .. c 50 00a • 0OJ 50 • • c.. 40 & • 0 t> '"~ OJ 0 • • .;.;;; 40 c< 30 ** t •0 • • .61'c.. 30 • 20 *. *,.4:.c< • •ill

20 10 *. * ~10

030 600 50

0 Total HER2 tests0 20 40 60 80

Total number of ER tests I. HER2 positive (%) *HER2 equivocal (%) 0 HER2 negative (%) IFigure 1 Positive oestrogen receptor (ER) rates for reporting laboratoriesplotted against number of reported cases for the 2006 audit.

been taken based on these results. This has the potential to

,iIrlpact significantly on patient treatment and outcome.'It 'was anticipated that the results of the audits would

indicate satisfactory performance for the majority of labora­tories. While a number of laboratories do achieve excellentrestilts, there are a number that have reported hormonereceptor and HER2 status positivity rates that fall outsideexpected results.

Arpino et al' reported on 54 865 patients from a database ofpatients diagnosed with breast cancer between 1970 and 1998.ERwas evaluated using the dextran-coated charcoal methodand PR was evaluated using sucrose density gradient (table 10).

Rhodes et al' reported on hormone status of 4053 breastcarcinomas using mc, Prancis et al' reported on 591 patients,Killeen et aI" reported 667 patients and Huang et af reported on1362 patients (table 10).

HER2 status in 669 patients was also assessed by Killeen et aI6

using image analysis; 69.5% were HERZ negative, 15.8% wereborderline and 14.6% were HER2 positive. Prancis et aI" reportedHERZ positivity in 15.4% of patients, HERZ was equivocal in18.4% and negative in 66.2%. Slamon et a[9 reported HERZoverexpression in 15-25% of breast carcinomas. Huang et af 10

reported an HER2 positive rate of 10.9% in 1362 patients asdefined by mc 3+ staining. Taucher et alII reported an HERZpositive rate of 17.3% in 923 patients, but this was defined as 2+or 3+ staining. Gancberg et aI IO reported an HER2 positive rate of23.1% in 160 tumours, and Yaziji et al lJ an HER2 positive rate of18.6% in 2913 tumours using fluorescence in-situ hybridisation(FISH). Lal et al I4 tested 561 tumours for HERZ using mc andFISH; 10.3% were positive with mc and 24.1 % showed gene

100 •90 • •80 •i5"< •• • • :..170 • • .~ • •.l!l • *••• •'"2 60 • • • ••OJ ~. • • •:£: 50 • • •• • ••.§ 40 .. .c..

30c< •c... •20100

0 20 40 60Total number of PR tests

Figure 2 Positive:rogesterone receptor (PR) rates for reportinglaboratories plotte against number of reported cases for the 2006 audit.

Figure 3 HER2 subgroup rates for reporting laboratories plotted againstnumber of reported cases for the 2006 audit.

amplification with FISH. Gusterson et ailS used mc to testHERZ status in 1506 patients; overall the positive rate was175% (16% for node negative patients and 19% for nodepositive patients), and a further 613 node negative patients hada 14.3% HER2 positive rate. I6 In a large study by Owens et aI,l73+ staining was seen in 10.9%, and 2+ staining in 9.1% of116 736 samples tested by HERZ mc and 22.7% of 6556specimens tested by FISH.

In the four hormone receptor (HR) me studies, the averageHR positive rate was 80.9% (SD 2.3%). The average HR negativerate was 19.0% (SD 2.2%). Therefore 95% of laboratories shouldhave an HR positive rate between 76.3% and 855%, and an HRnegative rate between 14.6% and 23.4%. Similarly ER+ ratesshould fall between 73% and 84.6% and PR+ rates between53.1% and 75.9%. For the 2006 audit, 22 laboratories had ERpositivity rates below 70%, and 8 laboratories had PR positivityrates below 50%. Of the laboratories reporting 100 patients inthe 6 month period for the 2006 audit, four had ER+ ratesbelow 70% and 2 had PR+ rates below 50%.

For the IHC studies with HERZ data: " 10 10 14 " 17 the averageHERZ 3+ positiveTaIe was 14.7% (SD 4.6%). In the 2006 audit,15 laboratories reported HER2 positive rates below 10%, and 17

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Francis, Dimech, Giles, et al

laboratories reported rates above 20%. Five laboratories report­ing 100 patients in the time period had HER2 positive ratesoutside the range of 10-20%.

The data from the 2006 audit were analysed in conjunctionwith the technical performance of each laboratory. The results

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were evaluated using the technical results obtained from 2003­05. An unsatisfactory perfOlTIlance was regarded if a laboratoryhad received this evaluation for any of the modules in that timeframe. There was no correlation between number of exercisesperformed or unsatisfactory perforrriance in the technical

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exercises and percentages of ER positive, PR positive or HER2positive cases by state (table 9). There was no correlationbetween the audit results and number of unsatisfactory resultsin the 2003-05 time frame (data not shown). There wasvariation in results in different states in Australia; interna­tional participants had lower rates of ER and PR positivity andhigher rates of HER2 positivity compared to other locations(table 9).

As 'already mentioned, each laboratory used their ownspecified cut-off value for ER and PR status, and while it ispossible. that changes to the cut points would have an impacton the percentage positivity rates for ER and PR, Layfield et al"showed no difference in concordance for ER when cut-pointswere· standardised. Only approximately 3% of breast carcino­mas have 1-4% of cells staining, and approximately 7% have1-19% of cells staining.~ This would be unlikely to account forthe wide variation of results in the two audits. It would also notaccount for differences in reporting of HER2 for which there arewell defined criteria. Since patients in these audits were non­selected and routinely reported, patient bias is unlikely to havea major impact for those laboratories reporting large numbersof cases. There was no correlation between methodology andpositivity rates; however, the wide variation in methods andreagents made statistical compalisons impossible (data notshown).

Rhodes et al" assessed the performance of 200 laboratories toperform ER by IHC. Their study showed a false negative rate forER status of 30-60%. The interlaboratory variation in resultspersisted with the laboratories' chosen threshold for determi­nation of ER status; however, a cut-off of 1% would have

resulted in a higher number of laboratories reporting ERpositivity. Considerable variability was also identified byLayfield et al'· with ER results in different laboratories; thiswould result in clinically significant differences in therapy.

In the technical component of the RepA QAP breast markersmodule similar results are observed, with an increasedproportion of laboratories achieving unsatisfactory results withlow ER expressing tumours.

The issues are similar for HER2. Press et apo evaluated HER2status in central and outside laboratories and concluded thatFISH .for assessment of HER2 status was superior whenperformed in a central laboratory.

Despite analysis of methodology, antibodies and detectionsystems, no single issue has been .identified as contributing tothe decreased performance; and conversely, no optimal methodhas been identified to achieve consistently good results.

CONCLUSiONSThe variation in results reported by laboratories has thepotential to impact significantly on patients. Despite the searchfor predictive and prognostic markers in breast carcinoma andthe description of hundreds of potential markers, only hormonereceptor and HER2 status have been translated into clinicalpractice." mc is at best a semiquantitative method, and whilecell lines and image analysis can be used to establish dermedstaining intensities and scoring systems, this does not enabletransition to solid tissues where the vagaries of fixation andprocessing impact significantly on staining quality. It isinteresting that HER2 gene amplification more accuratelyreflects response to trastuzumab, when logically it should beprotein expression.

IHC is limited by variability in tissue quality and methodol­ogy. While some of this can be overcome by meticulousattention to assay performance, correlation with patientoutcome and treatment response, the majority of laboratoriesdo not have me resources or access to patient information toclosely control or monitor these factors. Over me last decade,IHC has moved from a qualitative test (brown or not brown) toa quantitative test that determines selection of patient therapyand treatment outcome. With me development of new targetedtherapy, me imperative to identify predictive tests will increase.mc may be able to fulfil this role, but me data from this andother studies indicate it is fraught vvim difficulty. Thedifficulties with predicting response to epidermal growth factorreceptor by tyrosine kinase inhibitors or monoclonal antibodiesbased on THC graphically illustrates this point." 23

In the case of HER2, the American Society of ClinicalOncology/College of American Pathologists (ASCO/CAP) havereleased guideline recommendations for HER2 testing in breast

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cancer.24 These recommendations include testing on all invasivebreast cancers, validation of laboratory assays, use of standardisedoperating procedures and compliance with stringent proficiencyand accreditation requirements.

As from 1 October 2006 in Australia, in-situ hybridisation(ISH) is mandatory to be eligible for the government subsidiseddrug trastuzumab for adjuvant treatment in early breast cancer.It is the aim to test all patients with early breast cancer withISH for HERl, with a transition phase of 12 months forcontinued IHe testing. ISH testing for HERl has beenintroduced with stringent training and reporting requirementsto avoid similar issues identified with IRC testing.

While the guidelines for the introduction of ISH testing inAustralia have been developed independently to the ASCO/CAPguidelines, they are virtually identical. These guidelines includean (equivocal) borderline category for chromogenic ISH testingthat requires confirmatory FISH testing. They also includeminimum numbers to be performed by participating labora­tories and rep0l1ing pathologists. There are both training andaccreditation standards with mandatory performance ofongoing on-line training and evaluation. The audit will berepeated in 2007 and will include results for ISH testing on thepatient specimens.

However, for ER and PR, no alternative methodology to THCis available. Therefore it is essential that the clinician andlaboratory are at all times aware of the potential impactreporting of IHC has on patient treatment and outcome.Internal audits should be performed in the laboratories toensure results are as accurate as possible and referrallaboratories should be considered for laboratories perfomlingsmall numbers of cases.

ACKNOWLEDGEMENTSThe authors vlrish to acknowledge the support and assistance of the sraffand scientific and pathologist reviewers of RCPA QAP Pry Ltd.

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Authors' affiliationsGlenn D Francisr Queensland Health Pathology Service, PrincessAlexandra Hospital, Woolloongabba, Queensland, AustraliaMargaret Dimechr Leanne Gilesr Alison Hopkinsr RCPA Quality AssurancePrograms Pty Ltd, Anatomical Pathology, Burwood, Victoria, Australia

Competing interests: None declared.

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