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.

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.

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.