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Aims: Proliferation assessment by the use of Ki67 is a crucial component in intrinsic subtyping of luminal breast cancers (BCs), but suffers from variability between laboratories, observers, and methods. MammaTyper is a quantitative molecular tool that measures mRNA levels of ERBB2, ESR1, PGR and MKI67 in BC, and interprets the results according to the St Gallen 2013 consensus recommendations. We compared MammaTyper with immunohistochemistry (IHC)‐based subtypes, with a focus on standardised proliferation assessment. Methods and results: We analysed the agreement in assigning subtypes between MammaTyper and receptor IHC in 101 unifocal luminal HER2‐negative early BCs of no special type. Two Ki67 counting protocols, Ki67‐Global (Ki67‐G) and Ki67‐HotSpot (Ki67‐H), recommended by the International Ki67 in BC Working Group, and the mitotic activity index (MAI) were used for proliferation assessment. The proportions of BCs identified as luminal A and as luminal B were 55% and 45% for MammaTyper, 55% and 45% for IHC + Ki67‐G, 36% and 64% for IHC + Ki67‐H, and 56% and 44% for IHC + MAI. The levels of agreement between MammaTyper‐based and IHC‐based subtyping were 84% (κ = 0.679) for IHC + Ki67‐G, 72% (κ = 0.462) for IHC + Ki67‐H, and 89% (κ = 0.779) for IHC + MAI. Conclusions: High rates of agreement between mRNA‐based and IHC‐based intrinsic subtyping of luminal HER2‐negative BC can be achieved. However, the agreement between IHC‐based and MammaTyper‐based luminal subtypes depends on the proliferation assessment method, and was highest when the MAI was used. Further comparative clinical studies are needed to determine which method is to be preferred, including analysis of cost‐effectiveness. [ABSTRACT FROM AUTHOR]

Aims: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. Methods and results: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot‐spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799–0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot‐spot method (0.83; 95% CI = 0.74–0.90) did not. Visually, interobserver concordance in location of selected hot‐spots varies between cases. The median times for scoring were 9 and 6 min for global and hot‐spot methods, respectively. Conclusions: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further. [ABSTRACT FROM AUTHOR]

Aims In breast cancer patients undergoing neoadjuvant chemotherapy, histological grading needs to be performed on core needle biopsies ( CNBs) that may not be representative of the whole tumour when they are small. Our aim was to study the influence of biopsy size on agreement rates for histological grade between CNBs and subsequent surgical excision biopsies ( SEBs). Methods and results We calculated agreement and Cohen's κ between CNBs and SEBs of 300 early-stage breast cancers. The number of cores, total core length, total tumour length and tumour/tissue ratio were assessed for each CNB set. Agreement rates for grade were calculated for different classes of core number and tumour/tissue ratio, and for total core lengths and tumour lengths per CNB set in 5-15-mm intervals. Agreement on grade between CNBs and SEBs was 73% (κ = 0.59), with underestimation of grade in CNBs in 26% of cases and overestimation in 1% of cases. There was significantly higher concordance between CNBs and SEBs at a total core length of ≥50 mm than at a total core length of <50 mm (83% versus 68% agreement, P = 0.007), at a tumour length of ≥15 mm than at tumour length of <15 mm in CNBs (79% versus 67% agreement, P = 0.036), and at three or more cores than at fewer than three cores (75% versus 58% agreement, P = 0.048). The tumour/tissue ratio, pathological tumour size and radiological tumour size were not statistically different between concordant and discordant cases. Conclusions Agreement rates for histological grade in CNBs versus SEBs improve with increasing biopsy sample size. [ABSTRACT FROM AUTHOR]

Aims Regional differences in proliferative activity are commonly seen within breast cancers, but little is known on the extent of intratumoral heterogeneity of Ki67 expression. Our aim was to study the intratumoral heterogeneity of Ki67 expression in early breast cancers and its association with clinicopathological features, such as oestrogen receptor ( ER) status, grade and histological subtype. Methods and results The Ki67-labelling index (Ki67- LI) was assessed in hot, cold and intermediate spots of 233 invasive breast cancers by counting a total of 1020 cells, according to a protocol of the International Ki67 in Breast Cancer Working Group. Differences between the spots per tumour were analysed further for clinicopathological subgroups defined by ER status, grade and histological subtype. All clinicopathological subgroups showed significant differences in Ki67- LI between hot, intermediate and cold spots ( P < 0.0001). The coefficient of variance ( CV) between the spots was higher in ER-positive than in ER-negative cancers (72.6 versus 49.2%, P < 0.0001), and was highest in grade 3 (96.12%), grade 1 (87.27%) and invasive lobular tumours (83.59%) and lowest in medullary (26.48%) cancers. Nested analysis of variance indicated that in both ER-positive and ER-negative cancers, variance in Ki67- LI within tumours contributed more to the total variance (56% for ER-positive, 60% for ER-negative cancers) than the variance between tumours. Conclusion Intratumoral heterogeneity in Ki67- LI is a ubiquitous phenomenon across various pathological subgroups of breast cancer that may impact assessment of Ki67 levels for clinical decision-making, and sheds new light on recommended cut-offs. [ABSTRACT FROM AUTHOR]

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., Competing Interests: J.M.S.B. has consulted for Insight Genetics and BioNTech and received compensation. T.O.N. has consulted for Nanostring and received compensation. C.K.O. has consulted for Astra Zeneca, Genentech and NanoString and received compensation. The remaining authors declare no conflict of interest.