Tammy Piper

An International Ki67 Reproducibility Study

BACKGROUND In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67s value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study. METHODS Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays-one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided. RESULTS Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation. CONCLUSIONS Substantial variability in Ki67 scoring was observed among some of the worlds most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited.

An international study to increase concordance in Ki67 scoring

Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 ‘training’ and ‘test’ web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90–0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.

Mutational Analysis of PI3K/AKT Signaling Pathway in Tamoxifen Exemestane Adjuvant Multinational Pathology Study

PURPOSE Deregulation of key PI3K/AKT pathway genes may contribute to endocrine resistance in breast cancer (BC). PIK3CA is the most frequently mutated gene in luminal BC (35%); however, the effect of mutations in helical versus kinase domains remains controversial. We hypothesize that improved outcomes occur in patients with estrogen receptor–positive (ER positive) BC receiving endocrine therapy and possessing PIK3CA mutations. MATERIALS AND METHODS DNA was extracted from 4,540 formalin-fixed paraffin-embedded BC samples from the Exemestane Versus Tamoxifen-Exemestane pathology study. Mutational analyses were performed for 25 mutations (PIK3CAx10, AKT1x1, KRASx5, HRASx3, NRASx2 and BRAFx4). RESULTS PIK3CA mutations were frequent (39.8%), whereas RAS/RAF mutations were rare (1%). In univariable analyses PIK3CA mutations were associated with significantly improved 5-year distant relapse-free survival (DRFS; HR, 0.76; 95% CI, 0.63 to 0.91; P = .003). However, a multivariable analysis correcting for known clinical and biologic prognostic factors failed to demonstrate that PIK3CA mutation status is an independent prognostic marker for DRFS (HR, 0.92; 95% CI, 0.75 to 1.12; P = .4012). PIK3CA mutations were more frequent in low-risk luminal BCs (e.g., grade 1 nodev 3, node-negative v -positive), confounding the relationship between mutations and outcome. CONCLUSION PIK3CA mutations are present in approximately 40% of luminal BCs but are not an independent predictor of outcome in the context of endocrine therapy, whereas RAS/RAF mutations are rare inluminal BC. A complex relationship between low-risk cancers and PIK3CA mutations was identified. Although the PI3K/AKT pathway remains a viable therapeutic target as the result of ahigh mutation frequency, PIK3CA mutations do not seem to affect residual risk following treatment with endocrine therapy.

Mammostrat As an Immunohistochemical Multigene Assay for Prediction of Early Relapse Risk in the Tamoxifen Versus Exemestane Adjuvant Multicenter Trial Pathology Study

PURPOSE Some postmenopausal patients with hormone-sensitive early breast cancer remain at high risk of relapse despite endocrine therapy and, in addition, might benefit from adjuvant chemotherapy. The challenge is to prospectively identify such patients. The Mammostrat test uses five immunohistochemical markers to stratify patients regarding recurrence risk and may inform treatment decisions. We tested the efficacy of this panel in the Tamoxifen versus Exemestane Adjuvant Multicenter (TEAM) trial. PATIENTS AND METHODS Pathology blocks from 4,598 TEAM patients were collected, and tissue microarrays (TMAs) were constructed. The cohort was 47% node-positive, and 36% of patients in the cohort were treated with adjuvant chemotherapy. Triplicate 0.6-mm(2) TMA cores were stained, and positivity for p53, HTF9C, CEACAM5, NDRG1, and SLC7A5 was assessed. Cases were assigned a Mammostrat risk score, and distant relapse-free survival (DRFS) and disease-free survival (DFS) were analyzed. RESULTS In multivariate regression analyses, which were corrected for conventional clinicopathologic markers, Mammostrat provided significant additional information on DRFS after endocrine therapy in estrogen receptor (ER) -positive node-negative patients (n = 1,226) who did not receive chemotherapy (P = .004). Additional analyses in all patients not exposed to chemotherapy, irrespective of nodal status (n = 2,559) and in the entire cohort (n = 3,837) showed Mammostrat scores provided additional information on DRFS in these groups (P = .001 and P < .001, respectively; multivariate analyses). No differences were seen between the two endocrine treatment regimens. CONCLUSION The Mammostrat score predicts DRFS for patients treated with exemestane and patients treated with tamoxifen followed by exemestane irrespective of nodal status and chemotherapy. The ability of this test to provide additional outcome data after treatment provides additional evidence of its use in risk stratification of ER-positive postmenopausal patients with breast cancer.

Analytical validation of a standardized scoring protocol for Ki67: phase 3 of an international multicenter collaboration

Pathological analysis of the nuclear proliferation biomarker Ki67 has multiple potential roles in breast and other cancers. However, clinical utility of the immunohistochemical (IHC) assay for Ki67 immunohistochemistry has been hampered by unacceptable between-laboratory analytical variability. The International Ki67 Working Group has conducted a series of studies aiming to decrease this variability and improve the evaluation of Ki67. This study tries to assess whether acceptable performance can be achieved on prestained core-cut biopsies using a standardized scoring method. Sections from 30 primary ER+ breast cancer core biopsies were centrally stained for Ki67 and circulated among 22 laboratories in 11 countries. Each laboratory scored Ki67 using three methods: (1) global (4 fields of 100 cells each); (2) weighted global (same as global but weighted by estimated percentages of total area); and (3) hot-spot (single field of 500 cells). The intraclass correlation coefficient (ICC), a measure of interlaboratory agreement, for the unweighted global method (0.87; 95% credible interval (CI): 0.81–0.93) met the prespecified success criterion for scoring reproducibility, whereas that for the weighted global (0.87; 95% CI: 0.7999–0.93) and hot-spot methods (0.84; 95% CI: 0.77–0.92) marginally failed to do so. The unweighted global assessment of Ki67 IHC analysis on core biopsies met the prespecified criterion of success for scoring reproducibility. A few cases still showed large scoring discrepancies. Establishment of external quality assessment schemes is likely to improve the agreement between laboratories further. Additional evaluations are needed to assess staining variability and clinical validity in appropriate cohorts of samples.

Time dependence of biomarkers: non-proportional effects of immunohistochemical panels predicting relapse risk in early breast cancer

Background:We investigated the impact of follow-up duration to determine whether two immunohistochemical prognostic panels, IHC4 and Mammostrat, provide information on the risk of early or late distant recurrence using the Edinburgh Breast Conservation Series and the Tamoxifen vs Exemestane Adjuvant Multinational (TEAM) trial.Methods:The multivariable fractional polynomial time (MFPT) algorithm was used to determine which variables had possible non-proportional effects. The performance of the scores was assessed at various lengths of follow-up and Cox regression modelling was performed over the intervals of 0–5 years and >5 years.Results:We observed a strong time dependence of both the IHC4 and Mammostrat scores, with their effects decreasing over time. In the first 5 years of follow-up only, the addition of both scores to clinical factors provided statistically significant information (P<0.05), with increases in R2 between 5 and 6% and increases in D-statistic between 0.16 and 0.21.Conclusions:Our analyses confirm that the IHC4 and Mammostrat scores are strong prognostic factors for time to distant recurrence but this is restricted to the first 5 years after diagnosis. This provides evidence for their combined use to predict early recurrence events in order to select those patients who may/will benefit from adjuvant chemotherapy.

Do type 1 receptor tyrosine kinases inform treatment choice? A prospectively planned analysis of the TEAM trial

Background:Epidermal growth factor receptors contribute to breast cancer relapse during endocrine therapy. Substitution of aromatase inhibitors (AIs) may improve outcomes in HER-positive cancers.Methods:Tissue microarrays were constructed. Quantitative analysis of HER1, HER2, and HER3 was performed. Data were analysed relative to disease-free survival and treatment using outcomes at 2.75 and 6.5 years.Results:Among 4541 eligible samples, 4225 (93%) had complete HER1–3 data. Overall, 5% were HER1-positive, 13% HER2-positive, and 21% HER3-positive; 32% (n=1351) overexpressed at least one HER receptor. In the HER1–3-negative subgroup, the hazard ratio (HR) for upfront exemestane vs tamoxifen at 2.75 years was 0.67 (95% confidence interval (CI), 0.52–0.87), in the HER1–3-positive subgroup, the HR was 1.15 (95% CI, 0.85–1.56). A prospectively planned treatment-by-marker analysis demonstrated a significant interaction between HER1–3 and treatment at 2.75 years (HR=0.58; 95% CI, 0.39–0.87; P=0.008), as confirmed by multivariate regression analysis adjusting for prognostic factors (HR=0.55; 95% CI, 0.36–0.85; P=0.005). This effect was time dependent.Conclusion:In the 2.75 years prior to switching patients initially treated with tamoxifen to exemestane, a significant treatment-by-marker effect exists between AI/tamoxifen treatment and HER1-3 expression, suggesting HER expression could be used to select appropriate endocrine treatment at diagnosis to prevent or delay early relapses.

Abstract S1-5: PIK3CA mutations are linked to PgR expression: a Tamoxifen Exemestane Adjuvant Multinational (TEAM) pathology study.

Background: PIK3CA is mutated in about 26% of breast cancers (BC) and is the most frequently mutated gene in (BC). Almost 95% of mutations occur in exons 9 (E9) or 20 (E20). PIK3CA mutations may be associated with increased survival in endocrine-treated patients; however the impact of mutations in E9 vs E20 is not clear. We assessed 10 common PIK3CA mutations (95% of all mutations), in ER-positive (+ve) samples from the TEAM pathology study (n = ∼4500), and determined the impact of PIK3CA mutations on survival. We report an interim analysis of 1969 TEAM cases. Methods: DNA was extracted from formalin-fixed paraffin embedded sections. Mutational analyses were performed on 25 mutations in 6 genes (PIK3CAx10, Akt1x1, K RAS x5, H RAS x3, N RAS x2 & B RAF x4), using Sequenom MassArray. Results: Mutations were found in PIK3CA: 37.5%; Akt1: 3.3%; K RAS: 0.3%; and BRAF : 0.1% of cases. No mutations were found in H RAS or N RAS (n = 1969). 90% of PIK3CA mutations were located in E9 and E20. Outcome data was available for 1958/1969 patients. Patients whose tumours contained any PIK3CA mutations (n = 739) were at lower risk of distant metastasis, Hazard ratio=0.86 (0.67–1.11), when compared to those without PIK3CA mutations (n = 1219); although this difference was not statistically significant (Cox Regression; p = 0.24). PIK3CA mutations were significantly more frequent in HER2-negative (−ve) (39%) than in HER2+ve samples (25%) (p = 0.001), without evidence that PIK3CA mutations differentially impacted outcome in HER2+ve vs HER2-ve patients. A positive correlation was demonstrated between PIK3CA mutations and PgR Allred score (p = 0.002) but not ER Allred score (p = 0.37). With increasing PgR Allred score increase there is an increased frequency of mutations in E20 but not E9 (Table 1). Discussion: This study indicates a higher percentage of PIK3CA mutations in ER+ve BC samples than previously demonstrated, either for BC as a whole or for ER+ve cases, suggesting that in ER+ve early BC, PIK3CA mutations are more common than previously reported. Furthermore, increased PIK3CA mutation frequency is significantly associated with increasing PgR Allred score and this appears solely due to increased numbers of patients with E20 mutations further complicating the analysis of the impact of PIK3CA mutations in BC. This may explain current uncertainty regarding the impact of PIK3CA mutations in E9 vs E20 with respect to clinical outcome. Whilst we were unable to show a significant impact on outcome in patients whose tumours contained PIK3CA mutations, we believe the complex relationship between PgR expression (good prognosis indicator) and PI3K mutations requires further exploration in the full dataset using interaction techniques adjusting for the impact of PgR on outcome. Mutational analysis and correlation with clinical outcome data for the remaining ∼2500 DNA samples, along with the existing data for 1969 patients, will be presented. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S1-5.

An international multicenter study to evaluate reproducibility of automated scoring for assessment of Ki67 in breast cancer

The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73–0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44–0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81–0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81–0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.

Abstract P5-09-01: Nottingham prognostic index plus (NPI+): Validation of the modern clinical decision making tool in breast cancer

Introduction Current management of breast cancer (BC) relies on risk stratification based on well-defined clinicopathologic factors. The Nottingham Prognostic Index Plus (NPI+) is based on the assessment of biological class combined with established clinicopathologic prognostic variables providing improved patient outcome stratification superior to the traditional NPI*. This study aimed to validate the NPI+ in an independent series of BC. Methods A Validation series of 469 primary early-stage BC cases treated in Edinburgh, UK were matched for size, stage and grade to cases from Nottingham, UK used to develop the NPI+ (Training series). Adjuvant therapy was similar in both series except that 143 Edinburgh cases received endocrine therapy whilst the matched Nottingham cases had no adjuvant therapy. However, there was no significant difference in 10 year BC specific survival (BCSS) between the Training and Validation series. Cases, prepared as TMAs, were immunohistochemically assessed for Cytokeratin (Ck)5/6, Ck18, EGFR, Estrogen Receptor (ER), Progesterone Receptor (PgR), HER2, HER3, HER4, Mucin 1 and p53 expression. NPI+ biological class based on the expression of the 10 biomarkers was determined. Subsequent NPI+ prognostic scores were assigned using individual algorithms for each biological class developed using the Training series incorporating clinicopathologic parameters: positive nodes (including nodal stage), tumour size, tumour grade (including mitotic index) and PgR. NPI+ biological classes, prognostic scores and prognostic groups were compared between the Validation and Training series and their role in prediction of patient outcome. A p-value of Results As anticipated, there was a comparable distribution of NPI+ biological classes between Training and Validation series: Luminal A, n=143 (31%) vs n=115 (25%); Luminal N, n=99 (21%) vs n=89 (19%); Luminal B, n=75 (16%) vs n=85 (18%); Basal p53 altered, n=54 (12%) vs n=72 (15%); Basal p53 normal, n=37 (8%) vs n=53 (11%); HER2+/ER+, n=31 (7%) vs 18 (4%); HER2+/ER-, n=30 (6%) vs n=37 (8%; X2=13.792, p=0.032). BCSS was analogous between the Validation and Training series in each of the NPI+ biological classes except Luminal B (p=0.042). Similar BCSS was observed in the NPI+ Biological classes of the Training versus Validation series when taking into consideration adjuvant treatment modalities. The mean NPI+ score was similar between the Validation and Training series (2.30 vs 1.89, Pearson’s Regression p=0.079). The NPI+ prognostic groups significantly predicted patient outcome in each molecular class (BCSS, p Conclusion This study validates the NPI+ in an independent series of primary BC confirming its’ reproducibility. The NPI+ provides improved individualised clinical decision making for breast cancer for both prediction of clinical outcome and relevant therapeutic options. Acknowledgements Funded by the MRC References *Rakha EA et al Br J Cancer. 2014 110:1688-97. Citation Format: Andrew R Green, Daniel Soria, Jacqueline Stephen, Desmond G Powe, Christopher C Nolan, Ian Kunkler, Jeremy Thomas, Gill Kerr, Wilma Jack, David Camreron, Tammy Piper, Graham R Ball, Jonathan M Garibaldi, Emad A Rakha, John MS Bartlett, Ian O Ellis. Nottingham prognostic index plus (NPI+): Validation of the modern clinical decision making tool in breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-09-01.