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Effect of continuous statistically standardized measures of estrogen and progesterone receptors on disease-free… Chapman, Judith-Anne W; Nielsen, Torsten O; Ellis, Matthew J; Bernard, Phillip; Chia, Stephen; Gelmon, Karen A; Pritchard, Kathleen I; Maitre, Aurelie L; Goss, Paul E; Leung, Samuel; Shepherd, Lois E; Bramwell, Vivien H C Aug 23, 2013

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RESEARCH ARTICLE Open AccessEffect of continuous statistically standardizedmeasures of estrogen and progesteronereceptors on disease-free survival in NCIC CTGMA.12 Trial and BC CohortJudith-Anne W Chapman1*, Torsten O Nielsen2, Matthew J Ellis3, Phillip Bernard4, Stephen Chia5, Karen A Gelmon5, Kathleen I Pritchard6, Aurelie Le Maitre1, Paul E Goss7, Samuel Leung2, Lois E Shepherd1 andVivien H C Bramwell8AbstractIntroduction: We hypothesized improved inter-laboratory comparability of estrogen receptor (ER) andprogesterone receptor (PgR) across different assay methodologies with adjunctive statistical standardization, akin tobone mineral density (BMD) z-scores. We examined statistical standardization in MA.12, a placebo-controlled pre-menopausal trial of adjuvant tamoxifen with locally assessed hormone receptor +/- tumours, and in a cohort ofpost-menopausal British Columbia (BC) tamoxifen-treated patients.Methods: ER and PgR were centrally assessed for both patient groups with real time quantitative reversetranscription polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Effects on disease-free survival(DFS) were investigated separately for 345 MA.12 and 673 BC patients who had both qPCR and IHC assessments.Comparisons utilized continuous laboratory units and statistically standardized z-scores. Univariate categorization ofER/PgR was by number of standard deviations (SD) above or below the mean (z-score ≥1.0 SD below mean; z-score <1.0 SD below mean; z-score ≤1.0 SD above mean; z-score >1.0 SD above mean). Exploratory multivariateexaminations utilized step-wise Cox regression.Results: Median follow-up for MA.12 was 9.7 years; for BC patients, 11.8 years. For MA.12, 101 of 345 (29%) patientswere IHC ER-PgR-. ER was not univariately associated with DFS (qPCR, P = 0.19; IHC, P = 0.08), while PgR was(qPCR, P = 0.09; IHC, P = 0.04). For BC patients, neither receptor was univariately associated with DFS: for ER, PCR, P= 0.36, IHC, P = 0.24; while for PgR, qPCR, P = 0.17, IHC, P = 0.31. Multivariately, MA.12 patients randomized totamoxifen had significantly better DFS (P = 0.002 to 0.005) than placebo. Meanwhile, jointly ER and PgR were notassociated with DFS whether assessed by qPCR or by IHC in all patients, or in the subgroup of patients with IHCpositive stain, for pooled or separate treatment arms. Different results by type of continuous unit supported theconcept of ER level being relevant for medical decision-making. For postmenopausal BC tamoxifen patients, higherqPCR PgR was weakly associated with better DFS (P = 0.06).Conclusions: MA.12 pre-menopausal patients in a placebo-controlled tamoxifen trial had similar multivariateprognostic effects with statistically standardized hormone receptors when tumours were assayed by qPCR or IHC,for hormone receptor +/- and + tumours. The BC post-menopausal tamoxifen cohort did not exhibit a significantprognostic association of ER or PgR with DFS. Adjunctive statistical standardization is currently under investigationin other NCIC CTG endocrine trials.* Correspondence: JChapman@ctg.queensu.ca1NCIC Clinical Trials Group, Queen’s University, Kingston, ON K7L3N6, CanadaFull list of author information is available at the end of the articleChapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71© 2013 Chapman et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.IntroductionThe growth of many breast cancers is hormone-dependent, with estrogen receptor (ER) and/or proges-terone receptor (PgR) expression a prerequisite forresponsiveness to endocrine therapy. Increased aware-ness about uncertainties in accurate assessment of thesepivotal breast cancer biomarkers has renewed interest instandardization; there is the potential that 20% ofcurrent immunohistochemical (IHC) assay results world-wide are either false negatives or false positives [1].Aspects affecting assays include tumor heterogeneity,acquisition and processing of specimens, antibodychoices, laboratory assessment protocols, reproducibilityof procedures, external assessment of process, profi-ciency of laboratory workers, sufficiency of scoring posi-tivity and cut-points for positivity [1]. The AmericanSociety of Clinical Oncology and the College ofAmerican Pathologists (ASCO/CAP) recently publishedguideline recommendations for IHC testing of ER andPgR in breast cancer [1]. The Panel recommended acut-off of a minimum of 1% of tumor cells positive forER/PgR for a specimen to be considered positive [1].Chia et al. [2] centrally assessed ER and PgR in theNCIC Clinical Trials Group Breast Committee Mammary(MA).12 (NCIC CTG MA.12) placebo-controlled trial oftamoxifen in premenopausal women; they utilized thenew 1% cut-off for IHC positivity to examine the prog-nostic and predictive associations of ER and PgR withrelapse-free and overall survival. Neither hormone recep-tor was found to be prognostic or predictive. However,intrinsic subtyping by PAM50 was prognostic and lumi-nal subtypes were predictive of benefit from tamoxifen.Welsh et al. focused standardization of ER assessmentson the determination of ER positivity using automatedquantitative immunofluorescence (QIF) [3] which has abroader range of detection than IHC, possibly minimizingfalse negative results. Cell lines with ER immunoreactivitywere analyzed with QIF for standardization reliant onthreshold intensity. Cut-offs at 10% or 1% did not greatlyalter the proportion of positive tumours [3]. Further, Iwa-moto et al. found that the small number of patients with1% to 9% positive tumours is molecularly similar to ER-positive patients [4].Bartlett et al. [5] investigated the role of continuousER and PgR with the Tamoxifen and Exemestane Adju-vant Multinational (TEAM) trial data. They found sig-nificant prognostic effects with increasing values ofcontinuous ER and PgR associated with higher disease-free survival (DFS) in the short (maximum 2.75 years)follow-up period before tamoxifen patients switched toexemestane [5].We hypothesized that the process of statistical stan-dardization originally envisaged to improve inter-labora-tory comparability of ER/PgR assay results might beuseful to improve comparability of results between assaymethods. We investigate here the association of contin-uous ER and PgR with DFS in patients randomized totamoxifen or placebo regardless of locally determinedER and PgR tumour status. Central review permittedinvestigation of statistical standardization for IHC andqPCR assessment modalities [6-10] across a broad rangeof hormone receptor values.MethodsPatientsNCIC CTG MA.12NCIC CTG MA.12 was a placebo-controlled trial oftamoxifen therapy following adjuvant chemotherapy inpremenopausal women with early breast cancer [11][see Additional file 1 CONSORT Diagram]. The studywas approved by local research Ethics Boards, andpatients provided written informed consent [11]. TheNCIC CTG MA.12 Study Chair (VHCB), PhysicianCoordinator (LS) and sources of qPCR and IHC hor-mone receptor data (TON, SC, PB, MJE) gave permis-sion to use MA.12 data in this work.Patients with pathological T1-4, N0-2, M0 tumourswere eligible. Local centre determination of levels of atleast one hormone receptor (ER and/or PgR), by bio-chemical (positive ≥10 fmol/mg protein) or immunohis-tochemical assay was required, but patients with anyreceptor status were eligible. The stratification factorswere type of chemotherapy (cyclophosphamide, metho-trexate and fluorouracil (CMF); cyclophosphamide, epir-ubicin, fluorouracil (CEF); doxorubicin (adriamycin)/cyclophosphamide (AC)), hormone receptor status (ERand/or PgR positive, ER and PgR negative) and nodalstatus (0, 1 to 3, 4 to 9, 10+). The primary endpoint wasoverall survival (OS). DFS was a secondary endpoint andwas defined as being the time from randomization tothe earliest date of recurrence or death; censoring wasthe last date the patient was known to be alive.A total of 672 women were accrued to MA.12, 338randomized to tamoxifen and 334 to placebo. Tumourhormone receptor status was positive in 505 (75%) ofwomen. At 9.7 years median follow-up, multivariateanalysis showed a DFS benefit for tamoxifen of border-line significance (P = 0.056) and a trend for improvedOS (P = 0.12). There was no evidence of greater efficacyfor tamoxifen in the hormone-receptor positive or ERreceptor-positive subgroups than in hormone-receptornegative or ER receptor-negative patients: interactiontest P -values were, respectively, 0.71 and 0.14.The process of statistical standardization requires con-tinuous assay assessments, assessed by the same assess-ment method, in the same laboratory, under similarcircumstances, for a sufficient number of patients tocharacterize the assay results with a normal distribution.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 2 of 13The 672 MA.12 patients were accrued at 44 Canadiancentres, with multiple different laboratories assayingtumours for hormone receptor status. Further, many ofthe patients entered MA.12 following biochemical assayof ER/PgR or with IHC results categorized as positive ornegative. Thus, the local hormone receptor data werenot suitable for our investigations. MA.12 patients withER and PgR centrally assessed by qPCR and IHC didnot apparently differ in baseline characteristics from allpatients randomized to the trial [2].British Columbia patient cohortAdjuvant endocrine therapy would currently be consideredfor patients assessed to have hormone receptor positivetumours, regardless of menopausal status. Our investiga-tions were augmented here with a cohort of 767 BritishColumbia breast cancer patients [12] who had centralassessment of ER and PgR in the same laboratory as theMA.12 patients. The BC patients were all women with newprimary breast cancer, who received adjuvant tamoxifen,without adjuvant chemotherapy. Only 22 of the patientswere pre-menopausal, and 11 had unknown menopausalstatus, so we restricted investigations to the post-menopau-sal patient group. We defined a MA.12 DFS endpoint forthe BC patients as time from randomization to the earliestdate of recurrence or death, censoring at the last date thepatient was known to be alive, or if alive, at June 30, 2004.Central reviewER and PgR were centrally assessed in the laboratory ofTN by real time quantitative RT-PCR (qRT-PCR) andby IHC. Following pathologist review of formalin-fixed,paraffin-embedded source blocks stored at the NCIC-CTG Pathology office, two 0.6 mm cores were removedfrom representative areas of viable invasive carcinomafor tissue microarray construction, and two 1.0 mmcores were removed for RNA purification and qPCRdetermination of ER (ESR1) and PR (PGR) using thePAM50 assay method [12]. IHC analyses were per-formed on 4-micron sections from the tissue microarray,with ER assessed using ASCO/CAP compatible methods[1] (MA.12 trial: SP 1 rabbit monoclonal antibody(ThermoFisher Scientific, Fremont, CA, USA), using1:50 dilution for 32 minutes with heat, and mild CC1on Ventana BenchMark. PgR was similarly assessed withrabbit monoclonal 1E2 (Ventana, Tuscon, AZ, USA),pre-diluted for eight minutes with heat, and standardCC1 antigen retrieval and incubation; BC cohort: 6F11mouse monoclonal antibody (Leica Biosystems Newcas-tle Ltd, UK), using 1:50 dilution for two hours with noheat, and standard CC1 on Ventana Dixcovery XT)). ERand PgR IHC were assessed by a pathologist as a visualscore from 0 to 100% based on the fraction of invasivecancer nuclei positive above background.Statistical methodsER and PgR qPCR data were log2 transformed; labora-tory ER and PR zeros were treated as missing. Mean-while, for ER and PgR IHC% positive stain, the Box-Coxloge transformation was indicated for variance stabiliza-tion, after addition of 0.1 to IHC ER and PgR zeros topermit the transformation. For each hormone receptorassessment method, the continuous logarithmic valueswere converted to statistically standardized z-scoresusing the assessment method mean and SD of logarith-mic values:z- score = ((log value - mean of log values)÷SD of logvalues), which has approximately a standard normal dis-tribution, N(0,1).For comparability, DFS investigations includedpatients who had both ER and PgR assays, by bothqPCR and IHC. With the MA.12 trial, we investigatedthe effects of ER and PgR for: 1) all women regardlessof ER and PgR status, referred to hereafter as allpatients; and 2) the subgroup of these patients with cen-trally confirmed positive IHC staining for ER and/orPgR tumours; for patients allocated to 1) placebo, 2)tamoxifen or 3) both arms together. All the BC postme-nopausal patients received tamoxifen and were assessedas a single group.DFS was the endpoint utilized here to investigate theassociation between ER and PgR and outcome. Univariatetests for MA.12 utilized the stratified log-rank statistic;for the BC cohort, we used the generalized Wilcoxon(Peto-Prentice) test statistic. Graphical description waswith Kaplan-Meier plots. For MA.12, we plotted theexperience for IHC ER/PR zero and for ER/PgR positivestain, while for the BC group all patients were IHC ERpositive. Analogous to bone mineral density (BMD), weused cut-points for positive stain categorization of num-ber of standard deviations (SD) above/below the mean(z-score ≥1.0 SD below mean; z-score <1.0 SD belowmean; z-score ≤1.0 SD above mean; z-score >1.0 SDabove mean).Exploratory multivariate examinations were withadjusted Cox regression, stratified for MA.12 by thestratification factors of nodal status and chemotherapytype. We investigated the effects of ER and PgR incontinuous laboratory and statistically standardizedz-scores. To permit comparison across assessmentmethods, ER and PgR had forced inclusion in all mod-els, while for MA.12 trial therapy and baseline patientcharacteristics (age, pathological stage, pathological Tstage, ECOG performance) were added in step-wisemode (P ≤0.05). Factors considered for the BC cohortwere age, MA.12 categories for number of positivenodes and clinical T stage; none of the patientsreceived adjuvant chemotherapy.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 3 of 13ResultsMA.12 patientsOf the 672 MA.12 patients, centrally assessed ER wasavailable by IHC for 392 (58%) and for PgR, for 376(56%) patients. There were 124 centrally reviewedpatients with no IHC staining for ER or PgR. Centrallyassessed ER was available by qPCR for 385 (57%)patients and for PgR for 389 (58%). Figure 1 shows theqPCR ER assay results for all patients; Figure 2, theqPCR ER results for central IHC positive hormonereceptor stain; Figure 3, the qPCR PgR results for allpatients; and Figure 4, the qPCR PgR results for centralIHC positive hormone receptor stain. Histograms ofqPCR ER and PgR values in Figures 1 and 3 covered thespectrum of negative and positive IHC stain and exhib-ited bimodality. Meanwhile, qPCR values for tumourswith only positive IHC stain in Figures 2 and 4 exhibitunimodal distributions. Corresponding IHC histogramsare provided [see Additional files 2 to 5, Figures S1 toS4]; the best Box-Cox transformation was a logarithm,although the resulting IHC histograms do not indicatethe same level of symmetry as those observed for qPCR.To have the same patients included in comparisonsacross assessment methods, all further examinationswere restricted to the group of 345 patients who hadboth ER and PgR assessed by both qPCR and IHC; 101(29%) of these patients had tumours with no IHC stainfor ER or PgR. The K-M plots (Figures 5 to 8) depictDFS experience for patients whose tumours under cen-tral review had no IHC ER or PgR staining, and DFSexperience for ER or PgR assay results categorized byFigure 1 Histogram of the NCIC CTG MA.12 qPCR (log2) ERassay results for all patients: N = 385. ER, estrogen receptor.Figure 2 Histogram of the NCIC CTG MA.12 qPCR (log2) ERresults for central IHC ER and/or PgR >0: N = 263. ER, estrogenreceptor; IHC, immunohistochemistry; PgR, progesterone receptor.Figure 3 Histogram of the NCIC CTG MA.12 qPCR (log2) PgRresults for all patients: N = 389. PgR, progesterone receptor.Figure 4 Histogram of the NCIC CTG MA.12 qPCR (log2) PgRresults for IHC ER and/or PgR >0: N = 262. ER, estrogen receptor;IHC, immunohistochemistry; PgR, progesterone receptor.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 4 of 13their Z-scores to be multiple SDs above or below themean: greater to or equal to 1 SD below mean, less than1SD below the mean, less than or equal to 1 SD abovethe mean, and greater than 1 SD above the mean. Uni-variately, qPCR ER was not associated with DFS (Figure5, P = 0.19), while both qPCR PgR (Figure 6, P = 0.09)and IHC ER (Figure 7, P = 0.08) had weak evidence ofassociation, and IHC PgR (Figure 8) achieved statisticalsignificance (P = 0.04). There was a general indicationthat patients with ER and PgR staining z-score values>1.0, that is, >1.0 SD above the standardized mean, hadbetter DFS, while those with no IHC ER and PgR stainhad worse DFS.Multivariate results are provided in Table 1. In allinstances, patients randomized to tamoxifen had signifi-cantly better DFS (P = 0.002 to 0.005) than those allo-cated to placebo. However, patients randomized to thetamoxifen arm did not have significantly different DFSby ER or PgR levels, in continuous or standardizedunits, whether assessed by qPCR or IHC, in all patients,or in the subgroup of patients with IHC positive stain.Likewise, with pooling of patients on both treatmentarms, there was no overall evidence for an associationfor ER or PgR with DFS. The single instance of standar-dized PgR being significant (P = 0.05) may easily be dueto chance with the number of tests performed.There is inconsistent evidence of a prognostic effectfor hormone receptors for patients randomized to theplacebo arm. The evidence was strongest for qPCRPgR (P = 0.01 to 0.04 in three of four scenarios). Theinconsistency is illustrated in two scenarios. For IHChormone receptor positive and negative patients,laboratory value qPCR assessment alone indicated sig-nificant association of PgR with DFS (P = 0.02) whileIHC alone indicated weak evidence for IHC PgR (P =0.08). However, the joint consideration of laboratoryIHC and qPCR assessments led to a qPCR PgR P-valueof 0.15 (changed from 0.02) and IHC PgR P-value ofFigure 5 NCIC CTG MA.12 Disease Free Survival by Standardized qPCR ER: Assay results are categorized by their z-scores (greater to orequal to 1 standard deviation (SD) below mean, less than 1 SD below the mean, less than or equal to 1 SD above the mean, and greater than1 SD above the mean) or whether tumors under central review had no IHC assay staining. ER, estrogen receptor; IHC, immunohistochemistry.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 5 of 130.41 (changed from 0.08), with significant associationfor continuous qPCR ER (P = 0.03, changed from P =0.12) and weak evidence for IHC ER (P = 0.08, chan-ged from P = 0.27). Thus, there is a reversed indicationof whether PgR or ER has the significant associationwith DFS.The second example occurs in the subgroup withpositive IHC hormone receptor stain. Laboratory qPCRassessment alone indicated PgR was significantly asso-ciated with DFS (P = 0.01), although in the joint consid-eration of IHC and qPCR, both qPCR ER (P = 0.04) andIHC ER (P = 0.01) were also significant. There was achange from only PgR being significant to both ER andPgR being significantly associated with DFS.Further, substantive differences were noted on the pla-cebo arm jointly considering both IHC and qPCRbetween ER assessed with or without standardization:for all patients regardless of IHC status, standardizedqPCR ER P = 0.12 versus laboratory units P = 0.03; withpositive IHC stain, standardized IHC ER, P = 0.21 ver-sus laboratory units P = 0.01; standardized qPCR ER, P= 0.11 versus laboratory units P = 0.04. There are differ-ences in results by type of continuous unit supportingthe concept that level of ER beyond a dichotomousnegative or positive stain could be relevant for medicaldecision-making.BC patientsER was centrally assessed by qPCR for 767 patients andIHC for 688 patients; PgR by qPCR for 767 patients andIHC for 717 patients. There were 673 of 767 (88%)patients who had central qPCR and IHC for ER and PgR.To have the same patients included in comparisonsacross assessment methods, all further examinations wererestricted to this group of 673 patients, all of whom hadIHC stain for ER and/or PgR. The K-M plots (Figures 9to 12) depict DFS experience for patients whose tumoursunder central review had ER or PgR assay resultsFigure 6 NCIC CTG MA.12 Disease Free Survival by Standardized qPCR PgR: Assay results are categorized by their z-scores (greater to orequal to 1 standard deviation (SD) below mean, less than 1 SD below the mean, less than or equal to 1 SD above the mean, and greater than 1 SDabove the mean) or whether tumors under central review had no IHC assay staining. IHC, immunohistochemistry; PgR, progesterone receptor.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 6 of 13categorized by their Z-scores to be multiple SDs above orbelow the mean: greater to or equal to 1 SD below mean,less than 1SD below the mean, less than or equal to 1SD above the mean, and greater than 1 SD above themean. Univariately, qPCR ER was not associated withDFS (Figure 9, P = 0.36), nor was qPCR PgR (Figure 10,P = 0.17), IHC ER (Figure 11, P = 0.24) or IHC PgR(Figure 12, P = 0.31). Similar to MA.12, there was ageneral indication that patients with ER and PgR stainingz-score values >0., that is, those above the standardizedmean had better DFS, while those with no IHC ER andPgR stain had worse DFS, although experience convergedto being similar by about 10 years.Multivariate results are provided in Table 2. All patientsreceived adjuvant tamoxifen, without adjuvant chemother-apy. There was no evidence that ER was associated withDFS, and only weak multivariate evidence (P = 0.06) thathigher PgR was associated with better DFS.DiscussionBreast cancer is a complex disease which displays bothinter-case and intra-tumour heterogeneity [13-16].Tumor ER and/or PgR positivity is a prerequisite forresponsiveness to targeted therapy with an endocrineagent. Yet, inter-laboratory comparability of hormonereceptor assay values is still problematic after decades ofroutine clinical assessment. Many laboratories do not par-ticipate in external quality assurance programs, and theuse of a uniform method of assessment is not assuredeven for those that do [1,3,5,16-19]. Tumour levels ofhormone receptors, ER a and PgR, and dynamic range ofassessment methodology [3,20,21] impact indications forthe presence of hormone receptors. Further, while mar-kers such as HER2 are quite homogeneously expressedacross a tumour, ER and, particularly, PgR [22] may bemore heterogeneous. Finally, the current multitude oflaboratory assessment methods, scoring and (prior to theFigure 7 NCIC CTG MA.12 Disease Free Survival by Standardized IHC ER: Assay results are categorized by their z-scores (greater to or equalto 1 standard deviation (SD) below mean, less than 1 SD below the mean, less than or equal to 1 SD above the mean, and greater than 1 SDabove the mean) or whether tumors under central review had no IHC assay staining. ER, estrogen receptor; IHC, immunohistochemistry.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 7 of 13recent ASCO/CAP Guideline recommendations1) dichot-omous cut-points for positivity from ‘any positivity’ to an‘H-score of 50’ [23] have been problematic [24-27].Part of the controversy about ER and PgR cut-pointsfor positivity has centred around the inability in mostendocrine clinical trials to assess the effects of therapyin patients with false negative ER and PgR. NCIC CTGMA.12 had the unusual feature of patients being rando-mized to tamoxifen or placebo regardless of their locallydetermined ER and PgR, permitting an examination ofthe effects of endocrine therapy for the spectrum of hor-mone receptor values.Central review of ER and PgR with both qPCR andIHC permitted a comparison of these two methods, aswell as a demonstration of benefit with higher levels ofER and PgR positivity.Lastly, statistical standardization within assessmentmethods provided a common set of z-scores whichwould be expected to improve inter-laboratorycomparability.The comparison in this work was across methodologicplatforms since patients may now have ER and PgRassessed clinically in a variety of ways, with differentintra-method variability as well as inter-laboratory varia-bility by method. Differences for IHC alone were the sub-ject of the ASCO/CAP guidelines1. PCR methods aremore quantitative, producing continuous assay levels;however, there is a need to establish validity by level. Werestricted investigations here, achieved in the samelaboratory, to be for the same patients for both methods.The methodology of categorizing ER- and PgR-posi-tive stain by cut-points corresponding to z-score stan-dard deviations (analogous to BMD studies) indicatedgeneral univariate support that high levels of hormonereceptors led to better DFS, and no receptors to a worseoutcome. IHC PgR was significantly (P = 0.04)Figure 8 NCIC CTG MA.12 Disease Free Survival by Standardized IHC PgR: Assay results are categorized by their z-scores (greater to orequal to 1 standard deviation (SD) below mean, less than 1 SD below the mean, less than or equal to 1 SD above the mean, and greater than1 SD above the mean) or whether tumors under central review had no IHC assay staining. IHC, immunohistochemistry; PgR, progesteronereceptor.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 8 of 13associated with DFS while qPCR PgR (P = 0.09), qPCRER (P = 0.19), and IHC ER (P = 0.08) were not.In the current study, IHC analyses for ER and PgRhad a stronger association with outcome than was seenwith single gene measurements for ESR1 and PGR.Tamoxifen acts against the ER protein rather than itsmRNA so perhaps this result is not surprising. Onestrength of qPCR over IHC is the ability to quantifymultiple genes simultaneously as a signature, allowing aquantitative association of multi-gene expression with aluminal centroid that is a stronger predictor of endo-crine therapy response than single gene measures (ChiaSK et al. [2]). Here, we confined our study to single bio-markers and focused particularly on IHC, the primarydiagnostic method used in current clinical practice.In MA.12, we found inconsistent multivariate indica-tions of prognostic effect for hormone receptors forpatients allocated to the placebo arm. High correlationsbetween ER and PgR likely influenced indications of sig-nificance; for example, for all patients, when qPCR andIHC were assessed separately, PgR significance was indi-cated, qPCR PgR (P = 0.02) and IHC PgR (0.08). Mean-while, in joint consideration of the two assessmentmodalities, only qPCR ER (P = 0.03) was significantlyprognostic.Likewise, for patients with positive IHC stain, qPCRPgR (P = 0.01) was significant, although in joint exami-nation we found qPCR PgR (P = 0.01) as well as qPCRER (P = 0.04) and IHC ER (P = 0.01) to be significantlyassociated.Previously, we saw indications that biochemical ER, orPgR, or both, were significantly associated with outcome[8]. The lack of consistent support for a single hormonereceptor, or for a single assessment method, precludesfocused application in clinical practice. Further, substan-tive differences were noted with or without statisticalstandardization of ER: respectively, qPCR ER, P = 0.12versus 0.03; IHC ER, P = 0.21 versus 0.01; qPCR ER, P= 0.11 versus 0.04. Differences in results by type of con-tinuous unit support the concept that level of ERbeyond a dichotomous negative or positive stain is rele-vant for medical decision-making. Further, we suggest itis prudent at this time to consider that the conservativeindications of significance with standardized units areappropriate. The literature is replete with transient indi-cations of biomarker significance, such that the require-ment for validation is now the norm.We found that patients allocated to tamoxifen did notexhibit significant multivariate ER or PgR effects on DFS,nor were there significant hormone receptor effects whenpatient data on both tamoxifen and placebo arms werepooled. These results held for all patients and for thosewith positive IHC ER and/or PgR stain, for qPCR andIHC assessments, and with or without standardized units.Table 1 NCIC CTG MAIHC ERP-value1IHCPgRP-value1qPCRERP-value1qPCRPgRP-value1TherapyP-value1I. Placebo ArmCox Model for AllPatients:All IHC valuesIHC 0.27 0.08 N/A N/A N/AStandardized IHC 0.96 0.07 N/A N/A N/AqPCR N/A N/A 0.12 0.02 N/AStandardized qPCR N/A N/A 0.12 0.02 N/AIHC and qPCR 0.08 0.41 0.03 0.15 N/AStandardized IHC andqPCR0.40 0.19 0.12 0.47 N/ACox Model for PatientswithPositive IHC ER and/orPgRIHC 0.25 0.29 N/A N/A N/AStandardized IHC 0.53 0.07 N/A N/A N/AqPCR N/A N/A 0.23 0.01 N/AStandardized qPCR N/A N/A 0.23 0.01 N/AIHC and qPCR 0.01 0.67 0.04 0.01 N/AStandardized IHC andqPCR0.21 0.50 0.11 0.04 N/AII. Tamoxifen ArmCox Model for AllPatients:All IHC valuesIHC 0.18 0.61 N/A N/A N/AStandardized IHC 0.44 0.89 N/A N/A N/AqPCR N/A N/A 0.46 0.83 N/AStandardized qPCR N/A N/A 0.46 0.83 N/AIHC and qPCR 0.26 0.69 0.79 0.95 N/AStandardized IHC andqPCR0.50 0.58 0.75 0.48 N/ACox Model for PatientswithPositive IHC ER and/orPgRIHC 0.30 0.57 N/A N/A N/AStandardized IHC 0.37 0.82 N/A N/A N/AqPCR N/A N/A 0.22 0.19 N/AStandardized qPCR N/A N/A 0.22 0.19 N/AIHC and qPCR 0.75 0.57 0.34 0.20 N/AStandardized IHC andqPCR0.67 0.14 0.22 0.07 N/AIII. Both ArmsTogetherCox Model for AllPatients:All IHC valuesIHC 0.20 0.45 N/A N/A 0.003Standardized IHC 0.63 0.21 N/A N/A 0.004Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 9 of 13Chia et al. [2] did not observe differences in baselinecharacteristics between the main MA.12 trial populationand those for whom there was central review of hormonereceptors. In the main trial, there was weak evidence(P = 0.056) that tamoxifen improved DFS [11]. However,trial therapy in the centrally reviewed population wasconsistently associated with significant multivariate DFSTable 1 NCIC CTG MA (Continued)qPCR N/A N/A 0.66 0.20 0.004Standardized qPCR N/A N/A 0.45 0.19 0.003IHC and qPCR 0.07 0.75 0.15 0.48 0.002Standardized IHC andqPCR0.28 0.22 0.20 0.99 0.003Cox Model for PatientswithPositive IHC ER and/orPgRIHC 0.18 0.43 N/A N/A 0.004Standardized IHC 0.47 0.05 N/A N/A 0.005qPCR N/A N/A 0.75 0.30 0.005Standardized qPCR N/A N/A 0.75 0.30 0.005IHC and qPCR 0.07 0.75 0.25 0.53 0.002Standardized IHC andqPCR0.34 0.10 0.55 0.97 0.0051P-value is for factor’s inclusion in stratified Cox model, with adjustment forstratification factors of nodal status and adjuvant chemotherapy.Figure 9 BC Cohort Disease Free Survival by Standardized qPCRER: Assay results are categorized by their z-scores (greater to orequal to 1 standard deviation (SD) below mean, less than 1 SD belowthe mean, less than or equal to 1 SD above the mean, and greaterthan 1 SD above the mean). P-value is that for the generalizedWilcoxon (Peto-Prentice) test statistic. ER, estrogen receptor.Figure 10 BC Cohort Disease Free Survival by Standardized qPCRPgR: Assay results are categorized by their z-scores (greater to orequal to 1 standard deviation (SD) below mean, less than 1 SD belowthe mean, less than or equal to 1 SD above the mean, and greaterthan 1 SD above the mean). P-value is that for the generalizedWilcoxon (Peto-Prentice) test statistic. PgR, progesterone receptor.Figure 11 BC Cohort Disease Free Survival by StandardizedIHC ER: Assay results are categorized by their z-scores (greater to orequal to 1 standard deviation (SD) below mean, less than 1 SDbelow the mean, less than or equal to 1 SD above the mean, andgreater than 1 SD above the mean). P-value is that for thegeneralized Wilcoxon (Peto-Prentice) test statistic. ER, estrogenreceptor; IHC, immunohistochemistry.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 10 of 13(P = 0.002 to 0.005), in all centrally reviewed patients, andin the subgroup of women whose tumours had positiveIHC stain for ER and/or PgR. The distribution of events inpatients with centrally reviewed tumors was not represen-tative of the full trial population.Our work was broadened here with a BC cohort of post-menopausal patients, all of whom received adjuvanttamoxofen, without adjuvant chemotherapy. The qPCRand IHC assessments of ER and PgR were performed inthe same laboratory (that of TON). As in the MA.12 trial,there was a general directional indication that univariateDFS was better with higher ER and PgR, although thereappeared to be no difference after 10 years follow-up, andoverall there was no significant effect of ER or PgR onDFS found by qPCR or IHC assay methods, with or with-out statistical standardization. ER and PgR did not exhibitsignificant multivariate effects on DFS, although there wasweak evidence (P = 0.06) that patients with higher qPCRPgR had better DFS. We recognize the limitations incohort data, that patient and tumour characteristics couldhave impacted clinical and patient decisions in treatmentchoice, affecting outcomes. We also recognize that thepatient spectrum was reduced when only hormone recep-tor positive patients are considered, and there was a deci-sion not to administer adjuvant chemotherapy.However, we note that there is some commonality forthis study as both MA.12 trial patients and the BCcohort had qPCR and IHC assay results assessed in thesame laboratory. The juxtaposition of the MA.12 pre-menopausal trial where patients with locally determinedhormone receptor positive and negative tumours wererandomized to receive tamoxifen or placebo, with theBC postmenopausal patients who, with locally deter-mined hormone receptor positive tumours, receivedtamoxifen extends the spectrum of patients, tumourcharacteristics, and experience. Both groups showedgeneral univariate directions that higher ER and PgRwere associated with better DFS. There was no multi-variate evidence that ER and PgR had a significant prog-nostic effect on DFS for either study population.Inter-laboratory comparability of ER assay results hasbeen problematic for decades. A proposal in the early1980s involved mathematical adjustment of laboratoryassay values utilizing reference laboratory values, likethe WHO mandated mathematical adjustment of pro-thrombin times. Meanwhile, a lack of inter-laboratorycomparability for bone mineral density (BMD) wasresolved for both research and clinical purposes by theWHO with mandated statistically standardized t-scoresand z-scores based on routine comparisons with refer-ence population values.Work on the proposal for adjunctive statistical stan-dardization of ER began with poor inter-laboratory com-parability in provincial quality control samples in thelate 1980s [6-10] with Ontario laboratories performingbiochemical ER assessments using the dextran-coatedcharcoal radioligand method, continued after labora-tories switched to the double monoclonal enzyme-immunoassay, ER-EIA [7], and eventually, to immuno-histochemical assays [8,9]. We hypothesized improvedinter-laboratory comparability of ER/PgR assay resultswith adjunctive statistical standardization, and weshowed improved comparability with provincial qualitycontrol samples, and examined the process in cohortstudies [6-10]. Continuous ER and PgR effects wereFigure 12 BC Cohort Disease Free Survival by StandardizedIHC PgR: Assay results are categorized by their z-scores (greater toor equal to 1 standard deviation (SD) below mean, less than 1 SDbelow the mean, less than or equal to 1 SD above the mean, andgreater than 1 SD above the mean). P-value is that for thegeneralized Wilcoxon (Peto-Prentice) test statistic. IHC,immunohistochemistry; PgR, progesterone receptor.Table 2 BC POST-MENOPAUSAL COHORT: MULTIVARIATEEFFECTS OF ER, PgR, ON DFSIHC ERP-value1IHC PgRP-value1qPCR ERP-value1qPCR PgRP-value1IHC 0.76 0.31 N/A N/AStandardized IHC 0.76 0.31 N/A N/AqPCR N/A N/A 0.17 0.06Standardized qPCR N/A N/A 0.17 0.06IHC and qPCR 0.97 0.58 0.17 0.06Standardized IHC and qPCR 0.97 0.58 0.17 0.061P-value is for factor’s inclusion in Cox model, with adjustment for significanteffects of age, number of positive nodes, and clinical T stage.Chapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 11 of 13indicated in time-to-event investigations with cohorts ofbreast cancer patients [8,9]. The routine clinical use oft-scores and z-scores for BMD demonstrates the feasi-bility of adjunctive statistical standardization of ER andPgR in breast cancer and suggests an approach that maybe clinically useful for delineating significant predictiveand prognostic effects of continuous ER and PgR atmultiple standard deviations below or above the mean.ConclusionsThe growth of many breast cancers is hormone-depen-dent, with estrogen receptor (ER) and/or progesteronereceptor (PgR) expression a prerequisite for responsive-ness to endocrine therapy. Increased awareness aboutuncertainties in accurate assessment of these pivotalbreast cancer biomarkers has renewed interest in stan-dardization; there is a potential that 20% of current IHCassay results worldwide are either false negatives or falsepositives [1].We hypothesized that the process of statistical stan-dardization, akin to bone mineral density (BMD) z-scores, and originally envisaged to improve inter-labora-tory comparability of ER/PgR assay results, might beuseful to improve comparability of results betweenqPCR and IHC assay methods. We demonstrated statis-tical standardization across assay methods in MA.12, aplacebo-controlled trial of adjuvant tamoxifen in preme-nopausal women, with locally assessed hormone recep-tor +/- tumours. We saw evidence suggestive of anunspecified continuous prognostic effect for hormonereceptors. This is the first clinical trial report about sta-tistical standardization in the unique MA.12 trial towhich premenopausal patients were accrued regardlessof their locally determined hormone receptor status.Further, there was also directional evidence that BCpostmenopausal patients receiving tamoxifen had betteroutcome in at least the first 10 years, when they havehigher hormone receptor assay values.A plethora of laboratory assessment methods are usedto assess hormone receptors. We showed here in MA.12that statistically standardized hormone receptors hadsimilar multivariate prognostic effects on DFS whentumours were assayed by qPCR or by IHC, across aspectrum of hormone receptor +/- tumours. The BCcohort did not exhibit significant prognostic effects onDFS for ER or PR, by qPCR or by IHC, with or withoutstatistical standardization. The process of statistical stan-dardization would need to be laboratory specific, estab-lished iteratively and cumulatively against externalquality assurance samples that cover the range of ERand PgR assay levels. We are examining statistical stan-dardization in other NCIC CTG endocrine trials.The process of statistical standardization is akin toBMD z-scores which are used in clinical practice, so itwould be feasible to consider statistically standardizinghormone receptor assays.Additional materialAdditional file 1: NCIC CTG MA.12 CONSORT Diagram.Additional file 2: Figure S1. Histogram of the NCIC CTG MA.12 IHC log(ER) assay results for all patients: N = 392.Additional file 3: Figure S2. Histogram of the NCIC CTG MA.12 IHC log(ER) results for central IHC ER and/or PgR>0: N = 266.Additional file 4: Figure S3. Histogram of the NCIC CTG MA.12 IHC log(PgR) results for all patients: N = 376.Additional file 5: Figure S4. Histogram of the NCIC CTG MA.12 IHC log(PgR) results for IHC ER and/or PgR>0: N = 262.AbbreviationsASCO/CAP, American Society of Clinical Oncology and the College ofAmerican Pathologists; BC, British Columbia; BMD, bone mineral density; DFS,disease-free survival; ER, estrogen receptor; IHC, immunohistochemistry;qPCR, quantitative reverse transcription polymerase chain reaction; PgR,progesterone receptor; SD, standard deviation; TEAM, Tamoxifen andExemestane Adjuvant Multinational; WHO, World Health Organization.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsJWC conceived the idea for the study, designed the study, oversaw analysesand drafted the manuscript. TON completed the qPCR and IHC assessments,suggested use of the data for this purpose, and worked on drafting themanuscript. MEJ, PB and SC participated in acquisition of the qPCR and IHCassessments. KAG, KIP, PEG and LES contributed to the exposition of thework and drafting the manuscript. ALeM performed the analyses andcontributed to the presentation of the work. VHCB participated in thedevelopment of the work in the context of the trial and worked on draftingthe manuscript. SL assisted in the integration of the BC cohort data. Allauthors read and approved the final manuscript.AcknowledgementsThis work was supported by the Canadian Cancer Society through a grantfrom the Canadian Cancer Society Research Institute to the NCIC ClinicalTrials Group, as well as by a Queen’s University start-up grant to J.W.Chapman. Paul Goss is supported by the Avon Foundation in New York. Wethank Christine Chow for technical assistance.Authors’ details1NCIC Clinical Trials Group, Queen’s University, Kingston, ON K7L3N6, Canada.2Pathology Department, Vancouver Hospital, University of British Columbia,Vancouver, BC V5Z1M9, Canada. 3Division of Medical Oncology, WashingtonUniversity School of Medicine, St. Louis, MO 63110, USA. 4Department ofPathology, University of Utah, Salt Lake City, UT 84112, USA. 5Division ofMedical Oncology, BCCA - Vancouver Centre, Vancouver, BC V5Z4E6, Canada.6Sunnybrook Odette Cancer Centre, University of Toronto, Toronto, ONM4N3M5, Canada. 7Massachusetts General Hospital, Boston, MA 02114, USA.8Department of Oncology, University of Calgary, Calgary, AB T2N4N2,Canada.Received: 11 September 2012 Revised: 29 April 2013Accepted: 23 August 2013 Published: 23 August 2013References1. 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Isaacs C, Stearns v, Hayes DF: New prognostic factors for breast cancerrecurrence. Sem Oncol 2001, 28:53-67.27. Cohen JL, Raam S, Gelman R: A blinded study of inter- and intra-laboratory variations in the performance of estrogen receptor (ER) assay.In Estrogen receptor Assays in Breast Cancer, Laboratory Discrepancies andQuality Assurance. Edited by: G.A. Sarfaty, A.R. Nash, D.D. Keightley. NewYork: Masson Publishing; 1981:43-56.doi:10.1186/bcr3465Cite this article as: Chapman et al.: Effect of continuous statisticallystandardized measures of estrogen and progesterone receptors ondisease-free survival in NCIC CTG MA.12 Trial and BC Cohort. BreastCancer Research 2013 15:R71.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitChapman et al. Breast Cancer Research 2013, 15:R71http://breast-cancer-research.com/content/15/4/R71Page 13 of 13

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