Mitotic activity: A systematic literature review of the assessment methodology and prognostic value in feline tumors.

Increased proliferation is a driver of tumorigenesis, and quantification of mitotic activity is a standard task for prognostication. This systematic review is an analysis of all available references on mitotic activity in feline tumors to provide an overview of the assessment methods and prognostic value. A systematic literature search in PubMed and Scopus and a nonsystematic search in Google Scholar were conducted. All articles on feline tumors that correlated mitotic activity with patient outcome were identified. Data analysis revealed that of the 42 eligible articles, mitotic count (MC, mitotic figures/tumor area) was evaluated in 39 studies, and mitotic index (MI, mitotic figures/tumor cells) in 3 studies. The risk of bias was considered high for most studies (26/42, 62%) based on small study populations, insufficient details of the MC/MI methods, and lack of statistical measures for diagnostic accuracy or effect on outcome. The MC/MI methods varied between studies. A significant association of MC with survival was determined in 20 of 28 (71%) studies (10 studies evaluated other outcome metrics or provided individual patient data), while 1 study found an inverse effect. Three tumor types had at least 4 studies, and a prognostic association with survival was found in 5 of 6 studies on mast cell tumors, 5 of 5 on mammary tumors, and 3 of 4 on soft-tissue sarcomas. MI was shown to correlate with survival for mammary tumors by 2 research groups; however, comparisons to MC were not conducted. Further studies with standardized mitotic activity methods and appropriate statistical analysis for discriminant ability of patient outcome are needed to infer the prognostic value of MC and MI.

Mitotic activity: A systematic literature review of the assessment methodology and prognostic value in canine tumors.

One of the most relevant prognostic indices for tumors is cellular proliferation, which is most commonly measured by the mitotic activity in routine tumor sections. The goal of this systematic review was to analyze the methods and prognostic relevance of histologically measuring mitotic activity that have been reported for canine tumors in the literature. A total of 137 articles that correlated the mitotic activity in canine tumors with patient outcome were identified through a systematic (PubMed and Scopus) and nonsystematic (Google Scholar) literature search and eligibility screening process. Mitotic activity methods encompassed the mitotic count (MC, number of mitotic figures per tumor area) in 126 studies, presumably the MC (method not specified) in 6 studies, and the mitotic index (MI, number of mitotic figures per number of tumor cells) in 5 studies. A particularly high risk of bias was identified based on the available details of the MC methods and statistical analyses, which often did not quantify the prognostic discriminative ability of the MC and only reported P values. A significant association of the MC with survival was found in 72 of 109 (66%) studies. However, survival was evaluated by at least 3 studies in only 7 tumor types/groups, of which a prognostic relevance is apparent for mast cell tumors of the skin, cutaneous melanoma, and soft tissue tumor of the skin and subcutis. None of the studies using the MI found a prognostic relevance. This review highlights the need for more studies with standardized methods and appropriate analysis of the discriminative ability to prove the prognostic value of the MC and MI in various tumor types. Future studies are needed to evaluate the influence of the performance of individual pathologists on the appropriateness of prognostic thresholds and investigate methods to improve interobserver reproducibility.

Assessment of cell cycle progression and mitotic slippage by videomicroscopy.

Senescence is a state of irreversible cell cycle arrest accompanied by the acquisition of the senescence-associated secretory phenotype (SASP), which is activated in response to a variety of damaging stimuli, including genotoxic therapy. Accumulating evidence indicates that mitotic stress also promotes entry into senescence. This occurs via a mechanism involving defective mitoses and mitotic arrest, followed by abortion of cell division and slippage in the G1 phase. In this process, mitotic slippage leads to the generation of senescent cells characterized by a large cell body and a multinucleated and/or enlarged nuclear size. Here, we provide a detailed protocol for the assessment of cell proliferation and mitotic slippage in colorectal cancer cells upon pharmacological inhibition of the mitotic kinesin KIF11, best known as EG5. This approach can be used for preliminary characterization of senescence induction by therapeutics, but requires validation with standard senescence assays.

Single-cell impedance cytometry of anticancer drug-treated tumor cells exhibiting mitotic arrest state to apoptosis using low-cost silver-PDMS microelectrodes.

Chemotherapeutic drugs such as paclitaxel and vinblastine interact with microtubules and thus induce complex cell states of mitosis arrest at the G2/M phase followed by apoptosis dependent on drug exposure time and concentration. Microfluidic impedance cytometry (MIC), as a label-free and high-throughput technology for single-cell analysis, has been applied for viability assay of cancer cells post drug exposure at fixed time and dosage, yet verification of this technique for varied tumor cell states after anticancer drug treatment remains a challenge. Here we present a novel MIC device and for the first time perform impedance cytometry on carcinoma cells exhibiting progressive states of G2/M arrest followed by apoptosis related to drug concentration and exposure time, after treatments with paclitaxel and vinblastine, respectively. Our results from impedance cytometry reveal increased amplitude and negative phase shift at low frequency as well as higher opacity for HeLa cells under G2/M mitotic arrest compared to untreated cells. The cells under apoptosis, on the other hand, exhibit opposite changes in these electrical parameters. Therefore, the impedance features differentiate the HeLa cells under progressive states post anticancer drug treatment. We also demonstrate that vinblastine poses a more potent drug effect than paclitaxel especially at low concentrations. Our device is fabricated using a unique sacrificial layer-free soft lithography process as compared to the existing MIC device, which gives rise to readily aligned parallel microelectrodes made of silver-PDMS embedded in PDMS channel sidewalls with one molding step. Our results uncover the potential of the MIC device, with a fairly simple and low-cost fabrication process, for cellular state screening in anticancer drug therapy.

SpindlesTracker: An Automatic and Low-Cost Labeled Workflow for Spindle Analysis.

Quantitative analysis of spindle dynamics in mitosis through fluorescence microscopy requires tracking spindle elongation in noisy image sequences. Deterministic methods, which use typical microtubule detection and tracking methods, perform poorly in the sophisticated background of spindles. In addition, the expensive data labeling cost also limits the application of machine learning in this field. Here we present a fully automatic and low-cost labeled workflow that efficiently analyzes the dynamic spindle mechanism of time-lapse images, called SpindlesTracker. In this workflow, we design a network named YOLOX-SP which can accurately detect the location and endpoint of each spindle under box-level data supervision. We then optimize the algorithm SORT and MCP for spindle's tracking and skeletonization. As there was no publicly available dataset, we annotated a S.pombe dataset that was entirely acquired from the real world for both training and evaluation. Extensive experiments demonstrate that SpindlesTracker achieves excellent performance in all aspects, while reducing label costs by 60%. Specifically, it achieves 84.1% mAP in spindle detection and over 90% accuracy in endpoint detection. Furthermore, the improved algorithm enhances tracking accuracy by 1.3% and tracking precision by 6.5%. Statistical results also indicate that the mean error of spindle length is within 1 µm. In summary, SpindlesTracker holds significant implications for the study of mitotic dynamic mechanisms and can be readily extended to the analysis of other filamentous objects. The code and the dataset are both released on GitHub.

Assessment of Spindle Shape Control by Spindle Poleward Flux Measurements and FRAP Bulk Analysis.

In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule subpopulations and interacting proteins. The majority of what we know about the assembly and shape control of the mitotic spindle arose from vertebrate model systems. The dynamic properties of the individual tubulin polymers are crucial for the accurate assembly of the spindle array and are modulated by microtubule-associated motor and non-motor proteins. The mitotic spindle relies on a phenomenon called poleward microtubule flux that is critical to establish spindle shape, chromosome alignment, and segregation. This flux is under control of the non-motor microtubule-associated proteins and force-generating motors. Despite the large number of (plant-specific) kinesin motor proteins expressed during mitosis, their mitotic roles remain largely elusive. Moreover, reports on mitotic spindle formation and shape control in higher plants are scarce. In this chapter, an overview of the basic principles and methods concerning live imaging of prometa- and metaphase spindles and the analysis of spindle microtubule flux using fluorescence recovery after photobleaching is provided.

Dose-dependent Transmissibility of Chromosome Aberrations at First Mitosis after Exposure to Gamma Rays. I. Modeling and Implications Related to Risk Assessment.

The relationship between certain chromosomal aberration (CA) types and cell lethality is well established. On that basis we used multi-fluor in situ hybridization (mFISH) to tally the number of mitotic human lymphocytes exposed to graded doses of gamma rays that carried either lethal or nonlethal CA types. Despite the fact that a number of nonlethal complex exchanges were observed, the cells containing them were seldom deemed viable, due to coincident lethal chromosome damage. We considered two model variants for describing the dose responses. The first assumes independent linear-quadratic (LQ) dose response shapes for the yields of both lethal and nonlethal CAs. The second (simplified) variant assumes that the mean number of nonlethal CAs per cell is proportional to the mean number of lethal CAs per cell, meaning that the shapes and magnitudes of both aberration types differ only by a multiplicative proportionality constant. Using these models allowed us to assemble dose response curves for the frequency of aberration-bearing cells that would be expected to survive. This took the form of a joint probability distribution for cells containing ≥1 nonlethal CAs but having zero lethal CAs. The simplified second model variant turned out to be marginally better supported than the first, and the joint probability distribution based on this model yielded a crescent-shaped dose response reminiscent of those observed for mutagenesis and transformation for cells "at risk" (i.e. not corrected for survival). Among the implications of these findings is the suggestion that similarly shaped curves form the basis for deriving metrics associated with radiation risk models.

Deep Reinforcement Learning-Based Progressive Sequence Saliency Discovery Network for Mitosis Detection In Time-Lapse Phase-Contrast Microscopy Images.

Mitosis detection plays an important role in the analysis of cell status and behavior and is therefore widely utilized in many biological research and medical applications. In this article, we propose a deep reinforcement learning-based progressive sequence saliency discovery network (PSSD)for mitosis detection in time-lapse phase contrast microscopy images. By discovering the salient frames when cell state changes in the sequence, PSSD can more effectively model the mitosis process for mitosis detection. We formulate the discovery of salient frames as a Markov Decision Process (MDP)that progressively adjusts the selection positions of salient frames in the sequence, and further leverage deep reinforcement learning to learn the policy in the salient frame discovery process. The proposed method consists of two parts: 1)the saliency discovery module that selects the salient frames from the input cell image sequence by progressively adjusting the selection positions of salient frames; 2)the mitosis identification module that takes a sequence of salient frames and performs temporal information fusion for mitotic sequence classification. Since the policy network of the saliency discovery module is trained under the guidance of the mitosis identification module, PSSD can comprehensively explore the salient frames that are beneficial for mitosis detection. To our knowledge, this is the first work to implement deep reinforcement learning to the mitosis detection problem. In the experiment, we evaluate the proposed method on the largest mitosis detection dataset, C2C12-16. Experiment results show that compared with the state-of-the-arts, the proposed method can achieve significant improvement for both mitosis identification and temporal localization on C2C12-16.

Assessment of mitotic activity in breast cancer: revisited in the digital pathology era.

The assessment of cell proliferation is a key morphological feature for diagnosing various pathological lesions and predicting their clinical behaviour. Visual assessment of mitotic figures in routine histological sections remains the gold-standard method to evaluate the proliferative activity and grading of cancer. Despite the apparent simplicity of such a well-established method, visual assessment of mitotic figures in breast cancer (BC) remains a challenging task with low concordance among pathologists which can lead to under or overestimation of tumour grade and hence affects management. Guideline recommendations for counting mitoses in BC have been published to standardise methodology and improve concordance; however, the results remain less satisfactory. Alternative approaches such as the use of the proliferation marker Ki67 have been recommended but these did not show better performance in terms of concordance or prognostic stratification. The advent of whole slide image technology has brought the issue of mitotic counting in BC into the light again with more challenges to develop objective criteria for identifying and scoring mitotic figures in digitalised images. Using reliable and reproducible morphological criteria can provide the highest degree of concordance among pathologists and could even benefit the further application of artificial intelligence (AI) in breast pathology, and this relies mainly on the explicit description of these figures. In this review, we highlight the morphology of mitotic figures and their mimickers, address the current caveats in counting mitoses in breast pathology and describe how to strictly apply the morphological criteria for accurate and reliable histological grade and AI models.

Visual assessment of mitotic figures in breast cancer: a comparative study between light microscopy and whole slide images.

BACKGROUND AND AIMS: Visual assessment of mitotic figures in breast cancer (BC) remains a challenge. This is expected to be more pronounced in the digital pathology era. This study aims to refine the criteria of mitotic figure recognition, particularly in whole slide images (WSI). METHOD AND RESULTS: Haematoxylin and eosin (H&E)-stained BC sections (n = 506) were examined using light microscopy (LM) and WSI. A set of features for identifying mitosis in WSI and to distinguish true figures from mimickers was developed. Changes in the mitotic count between the two platforms was explored. Morphological features of mitoses were recorded separately, including absence of nuclear membrane, chromatin hairy-like projections, shape, cytoplasmic features, mitotic cell size and relationship to surrounding cells. Each mitotic phase has its own mimickers. Fifty-eight per cent of mitoses showed absent hairy-like projection in WSI; however, 89% retained their ragged nuclear border, which distinguished them from mimickers including apoptotic cells, lymphocytes and dark elongated hyperchromatic structures. Mitosis in WSI showed loss of fine details, and there was a 20% average reduction rate of mitotic counts when compared to the same area on LM. Using refined mitosis recognition criteria in WSI resulted in a twofold improvement of interobserver concordance. However, when compared to LM, 19% of cases were underscored in WSIs. CONCLUSIONS: All morphological features of mitosis should be considered to enable recognition and differentiation from their mimickers, particularly in WSI, to ensure reliable BC grading. Refining mitotic cut-offs per specific area when using WSI, based on the degree of reduction and association with outcome, is warranted.

Assessment of proliferation in breast cancer: cell cycle or mitosis? An observational study.

BACKGROUND AND AIMS: Proliferation is an important indicator of breast cancer (BC) prognosis, but is assessed using different approaches. Not all cells in the cell cycle are committed to division. This study aimed to characterise quantitative differences between BC cells in the cell cycle and those in mitosis and assess their relationship with other pathological parameters. METHODS AND RESULTS: A cohort of BC sections (n = 621) was stained with haematoxylin and eosin and immunohistochemistry for Ki-67. The proportion of mitotic cells and Ki-67-positive cells was assessed in the same areas. The Cancer Genome Atlas (TCGA) BC cohort was used to assess MKI-67 transcriptome level and its association with the mitotic counts. The mean proportion of BC cells in the cell cycle was 24% (range = 1-90%), while the mean proportion of BC cells in mitosis was 5% (range = 0-73%). A low proportion of mitoses to whole cycling cells was associated with low histological grade tumours and the luminal A molecular subtype, while tumours with a high proportion of mitoses to the overall cycling cells were associated with triple-negative subtype, larger tumour size, grade 3 tumours and lymph node metastasis. The high mitosis/low Ki-67-positive cells tumours showed a significant association with variables of poor prognosis, including high-grade and triple-negative subtypes. CONCLUSION: The proportion of BC cells in the cell cycle and mitosis is variable. We show that not only the number of cells in the cell cycle or mitosis, but also the difference between them, provides valuable information on tumour aggressiveness.

A comprehensive assessment of a new series of 5',6'-difluorobenzotriazole-acrylonitrile derivatives as microtubule targeting agents (MTAs).

Microtubules (MTs) are the principal target for drugs acting against mitosis. These compounds, called microtubule targeting agents (MTAs), cause a mitotic arrest during G2/M phase, subsequently inducing cell apoptosis. MTAs could be classified in two groups: microtubule stabilising agents (MSAs) and microtubule destabilising agents (MDAs). In this paper we present a new series of (E) (Z)-2-(5,6-difluoro-(1H)2H-benzo[d] [1,2,3]triazol-1(2)-yl)-3-(R)acrylonitrile (9a-j, 10e, 11a,b) and (E)-2-(1H-benzo[d] [1,2,3]triazol-1-yl)-3-(R)acrylonitrile derivatives (13d,j), which were recognised to act as MTAs agents. They were rationally designed, synthesised, characterised and subjected to different biological assessments. Computational docking was carried out in order to investigate the potential binding to the colchicine-binding site on tubulin. From this first prediction, the di-fluoro substitution seemed to be beneficial for the binding affinity with tubulin. The new fluorine derivatives, here presented, showed an improved antiproliferative activity when compared to the previously reported compounds. The biological evaluation included a preliminary antiproliferative screening on NCI60 cancer cells panel (1-10 µM). Compound 9a was selected as lead compound of the new series of derivatives. The in vitro XTT assay, flow cytometry analysis and immunostaining performed on HeLa cells treated with 9a showed a considerable antiproliferative effect, (IC50 = 3.2 µM), an increased number of cells in G2/M-phase, followed by an enhancement in cell division defects. Moreover, ß-tubulin staining confirmed 9a as a MDA triggering tubulin disassembly, whereas colchicine-9a competition assay suggested that compound 9a compete with colchicine for the binding site on tubulin. Then, the co-administration of compound 9a and an extrusion pump inhibitor (EPI) was investigated: the association resulted beneficial for the antiproliferative activity and compound 9a showed to be client of extrusion pumps. Finally, structural superimposition of different colchicine binding site inhibitors (CBIs) in clinical trial and our MDA, provided an additional confirmation of the targeting to the predicted binding site. Physicochemical, pharmacokinetic and druglikeness predictions were also conducted and all the newly synthesised derivatives showed to be drug-like molecules.

Toxicity assessment of zinc sulfate: A commonly used compound.

Because zinc sulfate (ZnSO4) is widely used in many fields such as biomedicine, electronics, and chemistry, it is important to evaluate its toxic effects. In this study, the cyto-genotoxic effects of ZnSO4 on meristematic cells in the root tip of Allium cepa L. were investigated. After calculating the effective concentration (EC50 = 70 ppm) of ZnSO4, A. cepa root tip cells were suspended for 24, 48, 72, and 96 h in solutions of 35 ppm (EC50/2), 70 ppm (EC50), and 140 ppm (EC50 × 2) concentrations. Using the counts of dividing cells, the mitotic index (MI) was calculated. Chromosome aberration index (CAI) was determined from percentages of abnormal cells. When the obtained data were statistically evaluated, it was determined that all application concentrations caused a significant decrease in MI and an increase in CAI compared to the control group (distilled water). It was concluded that increased ZnSO4 dose concentrations and exposure times caused cytotoxicity and genotoxicity in the root cells of A. cepa L.

Toxicity assessment of palladium oxide nanoparticles derived from metallosurfactants using multi assay techniques in Allium sativum.

In today's world, nanotechnology is reaching practically every ground and entering the human lifestyle by becoming a part of it. Thus, it is vital to check the cytotoxic and genotoxic effects of nanosubstances on plants, as they are the base constituent of ecosystem. The present work deals with the toxicity evaluation of metallosurfactant derived palladium oxide nanoparticles towards Allium sativum (Garlic cloves). The nanoparticles were prepared using microemulsion quenching method (a softer approach) using palladium metallosurfactants as precursors. The three ligands used were cetyltrimethylammonium chloride (CTAC), dodecylamine (DDA) and hexadecylamine (HEXA). Further, their characterization was done using TEM, Size Distribution curve, FESEM, EDS, XRD and Zeta potential. Garlic (Allium sativum) cloves were used to investigate the cytotoxicity and genotoxicity of fabricated PdO NPs. To check the cytotoxicity, optical microscopy was employed and for the genotoxic assessment, different parameters such as chromosomal aberrations in the mitosis, circular dichroism, and gel electrophoresis were utilized. From mitosis study, chromosomes aberrations were confirmed such as chromosomes stickiness, breakage, C-Mitosis, delay in anaphase, spindle fibre abnormality, laggard, vagrant and condensed chromosomes. Morphology of A. sativum clove, rooting and shooting pattern in the presence of PdO nanosuspension was observed. From all the experiments, it was concluded that all the three PdO nanosuspension are toxic in nature to both the cells and to genome, although, bishexadecyltrimethyl ammonium palladium tetrachloride (PdCTAC) Ns was found to be the most cytotoxic and genotoxic. Gel electrophoresis also confirmed the complete degradation of DNA in the presence of PdCTAC Ns.

A large-scale dataset for mitotic figure assessment on whole slide images of canine cutaneous mast cell tumor.

We introduce a novel, large-scale dataset for microscopy cell annotations. The dataset includes 32 whole slide images (WSI) of canine cutaneous mast cell tumors, selected to include both low grade cases as well as high grade cases. The slides have been completely annotated for mitotic figures and we provide secondary annotations for neoplastic mast cells, inflammatory granulocytes, and mitotic figure look-alikes. Additionally to a blinded two-expert manual annotation with consensus, we provide an algorithm-aided dataset, where potentially missed mitotic figures were detected by a deep neural network and subsequently assessed by two human experts. We included 262,481 annotations in total, out of which 44,880 represent mitotic figures. For algorithmic validation, we used a customized RetinaNet approach, followed by a cell classification network. We find F1-Scores of 0.786 and 0.820 for the manually labelled and the algorithm-aided dataset, respectively. The dataset provides, for the first time, WSIs completely annotated for mitotic figures and thus enables assessment of mitosis detection algorithms on complete WSIs as well as region of interest detection algorithms.

Breast cancer in Tanzanian, black American, and white American women: An assessment of prognostic and predictive features, including tumor infiltrating lymphocytes.

INTRODUCTION: Breast cancer is a major cause of morbidity and mortality for women in Sub-Saharan Africa and for black American women. There is evidence that the pathologic characteristics of breast cancers in both African women and black American women may differ from their counterparts of European ancestry. However, despite the great burden of disease, data on pathologic features of breast carcinoma in Sub-Saharan Africa is limited and often contradictory. This lack of information makes it difficult to prioritize resource use in efforts to improve breast cancer outcomes in the region. METHODS: We examined consecutive cases of breast cancer in Tanzanian women (n = 83), black American women (n = 120), and white American women (n = 120). Each case was assessed for tumor type, grade, mitotic count, ER and HER2 status, and tumor infiltrating lymphocyte involvement. RESULTS: The Tanzanian subjects were younger and had higher stage tumors than the subjects in either American group. Breast cancers in the Tanzanian and black American groups were more likely to be high grade (p = 0.008), to have a high mitotic rate (p<0.0001), and to be ER-negative (p<0.001) than the tumors in the white American group. Higher levels of tumor infiltrating lymphocyte involvement were seen among Tanzanian and black American subjects compared to white American subjects (p = 0.0001). Among all subjects, tumor infiltrating lymphocyte levels were higher in tumors with a high mitotic rate. Among Tanzanian and black American subjects, tumor infiltrating lymphocyte levels were higher in ER-negative tumors. These findings have implications for treatment priorities for breast cancer in Tanzania and other Sub-Saharan African countries.

Targets and mechanisms of chemically induced aneuploidy. Part 1 of the report of the 2017 IWGT workgroup on assessing the risk of aneugens for carcinogenesis and hereditary diseases.

An aneuploidy workgroup was established as part of the 7th International Workshops on Genotoxicity Testing. The workgroup conducted a review of the scientific literature on the biological mechanisms of aneuploidy in mammalian cells and methods used to detect chemical aneugens. In addition, the current regulatory framework was discussed, with the objective to arrive at consensus statements on the ramifications of exposure to chemical aneugens for human health risk assessment. As part of these efforts, the workgroup explored the use of adverse outcome pathways (AOPs) to document mechanisms of chemically induced aneuploidy in mammalian somatic cells. The group worked on two molecular initiating events (MIEs), tubulin binding and binding to the catalytic domain of aurora kinase B, which result in several adverse outcomes, including aneuploidy. The workgroup agreed that the AOP framework provides a useful approach to link evidence for MIEs with aneuploidy on a cellular level. The evidence linking chemically induced aneuploidy with carcinogenicity and hereditary disease was also reviewed and is presented in two companion papers. In addition, the group came to the consensus that the current regulatory test batteries, while not ideal, are sufficient for the identification of aneugens and human risk assessment. While it is obvious that there are many different MIEs that could lead to the induction of aneuploidy, the most commonly observed mechanisms involving chemical aneugens are related to tubulin binding and, to a lesser extent, inhibition of mitotic kinases. The comprehensive review presented here should help with the identification and risk management of aneugenic agents.

Deep learning and manual assessment show that the absolute mitotic count does not contain prognostic information in triple negative breast cancer.

PURPOSE: The prognostic value of mitotic count for invasive breast cancer is firmly established. As yet, however, limited studies have been aimed at assessing mitotic counts as a prognostic factor for triple negative breast cancers (TNBC). Here, we assessed the prognostic value of absolute mitotic counts for TNBC, using both deep learning and manual procedures. METHODS: A retrospective TNBC cohort (n = 298) was used. The absolute manual mitotic count was assessed by averaging counts from three independent observers. Deep learning was performed using a convolutional neural network on digitized H&E slides. Multivariable Cox regression models for relapse-free survival and overall survival served as baseline models. These were expanded with dichotomized mitotic counts, attempting every possible cut-off value, and evaluated by means of the c-statistic. RESULTS: We found that per 2 mm2 averaged manual mitotic counts ranged from 1 to 187 (mean 37.6, SD 23.4), whereas automatic counts ranged from 1 to 269 (mean 57.6; SD 42.2). None of the cut-off values improved the models' baseline c-statistic, for both manual and automatic assessments. CONCLUSIONS: Based on our results we conclude that the level of proliferation, as reflected by mitotic count, does not serve as a prognostic factor for TNBC. Therefore, TNBC patient management based on mitotic count should be discouraged.