Digital image analysis of Ki67 in hot spots is the marker of choice for routine analysis of proliferation in breast cancer.

BackgroundTranscriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics.MethodsIn this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications.ResultsMolecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications.ConclusionsOur results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.Electronic supplementary materialThe online version of this article (10.1186/s12885-017-3815-2) contains supplementary material, which is available to authorized users.

A 24-year-old 0-para presented to our department with a history of severe dysmenorrhea and pelvic pain refractory to intensified analgesia and continuous hormonal contraceptive treatment. She underwent expert 2D-and 3D-ultrasound assessment. We used transvaginal (TVU) and transrectal ultrasound (TRU). We found a thick-walled cystic lesion with hypoechoic to ground-glass content, protruding from the right lateral uterine wall below the insertion of the round ligament. There were no signs of adenomyosis or deep invasive endometriosis (DIE). No other pathologies or anatomical anomalies were found on ultrasound or magnetic resonance imaging (MRI). The mass was highly suggestive of ACUM given its morphology, the normally shaped uterine cavity and the absence of adenomyotic features. Due to failure of conservative treatment and ongoing severe interval pain the patient was scheduled for uterine-sparing robotic assisted laparoscopic surgery. The postoperative recovery was uneventful and the woman reported substantial symptom relief at the 3-months follow-up visit. Pathologic analysis suggested ACUM and no histological features of adenomyosis were found. We believe that ACUM is a diagnosis that is not commonly known. TVU or TRU are non-invasive and costeffective examination techniques to evaluate congenital uterine malformations. Robotic assisted laparoscopic surgery is a safe and feasible way to treat this condition.

INTRODUCTION Proliferative activity is one of the most important prognostic parameters in cancer. During the pathological examination of breast tumors, it is routinely evaluated by a count of the number of mitoses. Adding immunohistochemical stains of the nuclear protein Ki67 provides extra prognostic and predictive information. However, the currently used methods for both of these evaluations battle imperfections, primarily in reproducibility. In this study, we make an equally broad and detailed evaluation of mitoses, Ki67 and the more recently described Phosphohistone H3 (PHH3) in primary breast cancer using digital image analysis (DIA). Furthermore, we aim to investigate the prognostic and predictive value of proliferation-associated biomarkers in breast cancer stromal cells in relation to patient outcome. MATERIALS AND METHODS Two cohorts of primary breast cancer specimens (total n=297) with clinicopathological data including >10 years survival data, were sectioned and stained for Ki67, PHH3 and pancytokeratin (CKMNF116) and all glass slides were digitally scanned at x20. The DIA software used was the Visiopharm Integrator System (VIS) by Visiopharm A/S, Hoersholm, Denmark. VIS operates by a 'digital fusion' method that automatically excludes non-epithelial tissue restricting the analysis of the biomarkers (Ki67 and PHH3) to CKMNF116 positive cells. Both manual and DIA scores were compared for sensitivity and specificity for the gene expression based Luminal B versus A subtype, for high versus low transcriptomic grade as well as for their prognostic value in terms of Cox regression hazard ratios and breast cancer specific and overall survival. Further, we investigated whether the expression of Ki67 in the tumors' hot spots, invasive edges or as an average across all regions should be assessed for maximum power in relation to these outcomes. In addition, by inverting the DIA algorithm run by the VIS on the same cohorts, the expression of Ki67 and PHH3 was evaluated in the tumor stromal compartment. RESULTS Regardless of tumor region, DIA of Ki67 outperformed the other markers in sensitivity and specificity for gene expression subtypes and transcriptomic grade. In contrast to mitotic counts, tumors with high expression of Ki67 as defined by DIA, had significantly increased hazard ratio for all-cause mortality within 10 years from diagnosis. DIA of Ki67 was superior to manual Ki67 and PHH3 evaluations as well as to mitotic counts in terms of separation of patients with poor versus relatively good survival. Finally, we replaced the manual mitotic counts with DIA of Ki67 in hot spots as the marker for proliferation when determining histological grade. This increased the differences in estimated mean overall survival between the highest and lowest grades and added significantly more prognostic information to the classic Nottingham histological grade. CONCLUSIONS We conclude that digital image analysis of Ki67 in hot spots should be suggested as the marker of choice for proliferative activity in breast cancer. Citation Format: Robertson S, Stålhammar G, Wedlund L, Gholizadeh S, Lippert M, Rantaleinen M, Bergh J, Hartman J. Digital image analysis of Ki67 in hot spots is superior to alternative proliferation associated markers in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-07.