Intertumoral heterogeneity of bifocal breast cancer: a morphological and molecular study.

PURPOSE: To analyze concordance rates between individual foci of bifocal BC for histological grade, type and intrinsic subtype based on immunohistochemical (IHC) and mRNA-testing using MammaTyper. METHODS: We evaluated histological grade and type as well as intrinsic subtype based on IHC status for estrogen and progesterone receptors, HER2 and the mitotic activity index in 158 individual foci of 79 bifocal BC. A subgroup of 31 cases additionally underwent mRNA-based subtyping using the MammaTyper (MT) test. We calculated concordance rates between individual foci, as well as Cohen's Kappa (K). RESULTS: For 79 bifocal BC, concordance rates between individual foci for grade, histological type, and IHC-based subtype were 69.6% (K=0.53), 92.4% (K=0.81), and 74.7% (K=0.62), respectively. In the MT subgroup of 31 bifocal BC, concordance rates between individual foci for grade, histological type, IHC-based and mRNA-based intrinsic subtype were 87.1% (K=0.78), 90.3% (K=0.73), 87.1% (K=0.82), and 87.1% (K=0.7), respectively. Overall concordance between IHC- and mRNA-based subtype in the MT subgroup was 79% (K=0.7). In 6/79 cases (7.6%), testing of the smaller focus added clinically relevant information either on IHC- or mRNA-level: four cases showed high hormonal receptor expression while the expression in the larger focus was negative or low, warranting additional endocrine treatment; two cases presented with higher proliferative activity in the smaller focus, warranting additional chemotherapy. CONCLUSION: In bifocal BC, intertumoral heterogeneity on the morphological, immunohistochemical and molecular level is common, with discordant intrinsic subtype in up to 25% between individual foci, with about 8% clinically relevant discordances.

Systematically higher Ki67 scores on core biopsy samples compared to corresponding resection specimen in breast cancer: a multi-operator and multi-institutional study.

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error (p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections (p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

Luminal A versus luminal B breast cancer: MammaTyper mRNA versus immunohistochemical subtyping with an emphasis on standardised Ki67 labelling-based or mitotic activity index-based proliferation assessment.

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.

Analytical validation of a standardised scoring protocol for Ki67 immunohistochemistry on breast cancer excision whole sections: an international multicentre collaboration.

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.

Performance of 4 Immunohistochemical Phosphohistone H3 Antibodies for Marking Mitotic Figures in Breast Cancer.

BACKGROUND: Phosphohistone H3 (PHH3) has been suggested to facilitate and improve mitotic activity assessment in breast cancer and other tumor entities, but the reliability of respective immunohistochemical antibodies has not yet been compared for routine purposes. Our aim was to test the performance of 4 different PHH3 antibodies on a series of highly proliferating breast cancers with good preservation of morphology. METHODS: Four commercially available PHH3 antibodies were tested on 9 grade 3 invasive breast cancers processed in the same batch. We analyzed the number of antibody stained and nonstained mitotic figures as well as the total of cells observed in 10 high power fields per tumor to calculate sensitivity, specificity, and accuracy of the respective antibodies for staining mitotic figures, taking morphologically defined mitotic figures as gold standard. RESULTS: Sensitivity, specificity, and accuracy of the respective PHH3 antibodies for staining mitotic figures were 54.51%, 99.98%, and 98.79% for Cell Marque, 87.48%, 67.62%, and 67.47% for Epitomics, 98.62%, 99.73%, and 99.49% for Merck 06-570, and 99.74%, 99.52%, and 99.51% for Merck 09-797, respectively. Sensitivity was lowest for telophase. In statistical analysis, the Cell Marque antibody demonstrated significantly lower sensitivity and Epitomics substantially lower sensitivity and specificity than Merck 06-570 and Merck 09-797 antibodies (P<0.0001, respectively). CONCLUSIONS: Performance and reliability varied significantly between the 4 tested antibodies. For faster identification of mitotic hot spots and as potential marker in digital image analysis, the Merck antibodies seem to be most suitable.

Interlaboratory variability of Ki67 staining in breast cancer.

BACKGROUND: Postanalytic issues of Ki67 assessment in breast cancers like counting method standardisation and interrater bias have been subject of various studies, but little is known about analytic variability of Ki67 staining between pathology labs. Our aim was to study interlaboratory variability of Ki67 staining in breast cancer using tissue microarrays (TMAs) and central assessment to minimise preanalytic and postanalytic influences. METHODS: Thirty European pathology labs stained serial slides of a TMA set of breast cancer tissues with Ki67 according to their routine in-house protocol. The Ki67-labelling index (Ki67-LI) of 70 matched samples was centrally assessed by one observer who counted all cancer cells per sample. We then tested for differences between the labs in Ki67-LI medians by analysing variance on ranks and in proportions of tumours classified as luminal A after dichotomising oestrogen receptor-positive cancers into cancers showing low (<14%, luminal A) and high (≥14%, luminal B HER2 negative) Ki67-LI using Cochran's Q. RESULTS: Substantial differences between the 30 labs were indicated for median Ki67-LI (0.65%-33.0%, p < 0.0001) and proportion of cancers classified as luminal A (17%-57%, p < 0.0001). The differences remained significant when labs using the same antibody (MIB-1, SP6, or 30-9) were analysed separately or labs without prior participation in external quality assurance programs were excluded (p < 0.0001, respectively). CONCLUSION: Substantial variability in Ki67 staining of breast cancer tissue was found between 30 routine pathology labs. Clinical use of the Ki67-LI for therapeutic decisions should be considered only fully aware of lab-specific reference values.

St Gallen 2015 subtyping of luminal breast cancers: impact of different Ki67-based proliferation assessment methods.

Ki67 has been proposed as prognostic proliferation marker in luminal breast cancer (BC), but little is known on the influence of Ki67 assessment methods on subtyping into luminal A- and B-like tumors. Our aim was to study the influence of different Ki67-labeling index (Ki67-LI) assessment methods on the proportion of BCs classified as luminal A-like. 280 early BCs were subtyped according to the St Gallen 2015 definitions into 71 % luminal (HER2 negative), 6 % luminal B-like (HER2 positive), 13 % triple negative, 1 % HER2 positive (nonluminal), and 9 % special type. Digitized whole slides were counted manually on the screen. We used nine defined counting methods to assess the Ki67-LI (including the International Ki67 in Breast Cancer Working Group recommendations), and compared the resulting medians and the proportions of cancers classified as luminal A-like according to the formerly used cut-off <20 %. Methods assessing hot spots and tumor periphery resulted in significantly higher Ki67-LI medians than those measuring an average proliferation (27.45 % vs 16.96 %, p < 0.0001). Substantially lower median Ki67-LI were found when assessing 1020 compared to counting 100, 200, 300 cells (17.65 vs 33 %, vs 28 %, vs 24.33 %, respectively; p < 0.0001), or 510 cells (20.59 %, p = 0.019). Applying a standard Ki67-LI cut-off <20 % to define low proliferation for all methods, the proportion of luminal A-like cancers varied between 13 and 44 %. The proportion of BCs classified as luminal A-like is highly influenced by the Ki67-LI assessment method. As a consequence, the selection of a specific Ki67-LI assessment method may have a direct effect on the proportion of patients considered having low-risk disease and thus influence therapeutic decision making. This calls for a standardized assessment method.

The reliability of histological grade in breast cancer core needle biopsies depends on biopsy size: a comparative study with subsequent surgical excisions.

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.

Intratumoral heterogeneity of Ki67 expression in early breast cancers exceeds variability between individual tumours.

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.

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.