Ki67 index and mitotic count: Correlation and variables affecting the accuracy of the quantification in endocrine/neuroendocrine tumors.

Quantification of Ki67 and mitosis is time consuming and subject to inter-observer variabilities. Limited studies explored the impact of those variables on the results and the correlation between mitotic count and Ki67 index in endocrine/neuroendocrine tumors, particularly so since the advent of PHH3 antibody and digital pathology. Using Ki67 and mitosis as examples, this study is intended to reveal variables affecting accurate quantification of biomarkers, and to explore the relationship of Ki67 index and mitotic count/index in endocrine/neuroendocrine tumors. Using both manual and pathologist supervised digital image analysis (PSDIA) methods, we examined the impact of post-analytical variables on the quantification of mitosis and Ki67 index and studied the correlation between them in 41 cases of endocrine/neuroendocrine tumors of variable histological grades/proliferating rates. We found that the selection of hotspots, field size and especially threshold affected the outcome of quantification of mitosis and Ki67 index; that mitotic count/index strongly (p < 0.05) correlated with Ki67 index only in the tumors with peak Ki67 index less than 30% and the correlation was more monotonic (positive, non-linear) than linear. In the hotspots of these tumors, the ratio of mitotic count to proliferating cells defined by Ki67 detection averaged 0.04. We also found that the PHH3 antibody could markedly increase the efficiency and accuracy of mitotic quantification. A consensus among pathologists is needed for the selection of hotspots, field size and threshold for quantification of mitosis and Ki67 index.

Deep learning assisted mitotic counting for breast cancer.

As part of routine histological grading, for every invasive breast cancer the mitotic count is assessed by counting mitoses in the (visually selected) region with the highest proliferative activity. Because this procedure is prone to subjectivity, the present study compares visual mitotic counting with deep learning based automated mitotic counting and fully automated hotspot selection. Two cohorts were used in this study. Cohort A comprised 90 prospectively included tumors which were selected based on the mitotic frequency scores given during routine glass slide diagnostics. This pathologist additionally assessed the mitotic count in these tumors in whole slide images (WSI) within a preselected hotspot. A second observer performed the same procedures on this cohort. The preselected hotspot was generated by a convolutional neural network (CNN) trained to detect all mitotic figures in digitized hematoxylin and eosin (H&E) sections. The second cohort comprised a multicenter, retrospective TNBC cohort (n = 298), of which the mitotic count was assessed by three independent observers on glass slides. The same CNN was applied on this cohort and the absolute number of mitotic figures in the hotspot was compared to the averaged mitotic count of the observers. Baseline interobserver agreement for glass slide assessment in cohort A was good (kappa 0.689; 95% CI 0.580-0.799). Using the CNN generated hotspot in WSI, the agreement score increased to 0.814 (95% CI 0.719-0.909). Automated counting by the CNN in comparison with observers counting in the predefined hotspot region yielded an average kappa of 0.724. We conclude that manual mitotic counting is not affected by assessment modality (glass slides, WSI) and that counting mitotic figures in WSI is feasible. Using a predefined hotspot area considerably improves reproducibility. Also, fully automated assessment of mitotic score appears to be feasible without introducing additional bias or variability.

Reporting of mitotic rate in cutaneous melanoma: A study using the national cancer data base.

BACKGROUND: The seventh edition of the American Joint Commission on Cancer staging manual (AJCC7, published 2009), updated thin cutaneous melanoma staging protocols with the incorporation of mitotic rate (MR). In these patients, higher MR is associated with decreased survival. This study utilizes the National Cancer Data Base (NCDB) to evaluate MR reporting since AJCC7. METHODS: The NCDB was queried for patients with primary cutaneous melanoma from 1998 to 2013. Because MR reporting was infrequent prior to implementing AJCC7, records from 2010 to 2013 were analyzed. Categorical variables were compared with chi-square tests; univariate and multivariate logistic regression models were constructed to determine the effects of covariates on MR reporting. RESULTS: A total of 107,134 patients met inclusion criteria. From 2010 to 2013, MR reporting increased dramatically (64.3-80.9%). On multivariate analysis, factors significantly related to increased MR reporting include later diagnosis year, T-classification (T1a and b vs. T1), facility type (academic vs. other specified types of cancer programs), facility volume, patient income, level of education, and county population (metropolitan vs. urban and rural). CONCLUSIONS: MR reporting increased dramatically after the introduction of AJCC7; however, disparities in reporting remain across facility types. Further investigation of procedures performed in academic settings that may influence reporting of MR is warranted. J. Surg. Oncol. 2017;115:281-286. © 2017 Wiley Periodicals, Inc.

Genotoxicity of the food additive E171, titanium dioxide, in the plants Lens culinaris L. and Allium cepa L.

E171 (titanium dioxide, TiO2), an authorized foods and beverage additive, is also used in food packaging and in pharmaceutical and cosmetic preparations. E171 is considered to be an inert and non-digestible material, not storable in animal tissues, but the possible presence of TiO2 nanoparticles (NP) may present a risk to human health and the environment. We determined the presence of 15% TiO2 NP in a commercial E171 food additive product, by electron microscopy. The biological effects of E171 were assessed in Lens culinaris and Allium cepa for the following endpoints: percentage of germination, root elongation, mitotic index, presence of chromosomal abnormalities, and micronuclei. The results indicated low phytotoxicity but dose-dependent genotoxicity. We also observed internalization of TiO2 NP and ultrastructural alterations in the root systems.

Number of mitotic figures in microscopic picture of nephroblastoma histologic types--estimation of the value of the oldest known prognostic factor.

There are two main widely accepted prognostic factors in nephroblastoma group of tumors--histological type of tumor and clinical stage of disease. However nuclear anaplasia, one of the elements of Wilms' tumor microscopic picture was found as a new prognostic marker in this group of tumors. Estimation of nuclear anaplasia is an obligatory procedure in currently used therapeutic protocols. In our work we compared another element of nephroblastoma microscopic picture--number of mitoses as the oldest known indicator of activity of tumor cells with prognostic markers estimated in routine histologic examination according SIOP Protocols (histological risk, nephroblastoma type and the presence of diffuse anaplasia) and with CD44 expression--widely known marker of discussed prognostic value. We found statistically important correlation between number of mitoses and all the examined features. We believe that mitotic figure counting may become in future a helpful tool in the qualification of prognosis for individual patients in doubtful cases.

Evaluation of Mitotic Figures and Cellular and Nuclear Morphometry of Various Histopathological Grades of Oral Squamous Cell Carcinoma: Comparative study using crystal violet and Feulgen stains.

OBJECTIVES: The objectives of this study were to quantitatively estimate the number of mitotic figures (MFs) and evaluate the cellular and nuclear features of various histological grades of oral squamous cell carcinoma (OSCC) using Feulgen and 1% crystal violet stains. METHODS: This case-control study took place at the Dr D. Y. Patil Dental College & Hospital in Mumbai, Maharashtra, India, between June and December 2016. A total of 51 samples were retrieved from the hospital archives. Of these, 15 well-differentiated, 15 moderately-differentiated and six poorly-differentiated OSCC samples formed the case group while 15 samples of normal gingival mucosa constituted the control group. Each sample was dyed using Feulgen and 1% crystal violet stains and the mitotic count, nuclear area (NA), cellular area (CA), nuclear perimeter (NP), cellular perimeter (CP) and nuclear-to-cytoplasmic (N/C) ratio was calculated using computer-aided morphometry techniques. RESULTS: The number of MFs visible per field was significantly higher in Feulgen-stained sections as compared to those stained with crystal violet (P = 0.050). In addition, the NA, NP, CA and CP values and N/C ratios of samples in the experimental group increased significantly in accordance with an increase in OSCC grade (P <0.001). CONCLUSION: The Feulgen stain is more reliable than 1% crystal violet in terms of the selective staining of MFs. Moreover, the findings of this study indicate that computer-based morphometric analysis is an effective tool for differentiating between various grades of OSCC.

Mitotic rate in melanoma: a reexamination.

We reexamined the relationship between mitotic rate and overall survival in more than 1,200 cases of cutaneous melanoma with long-term follow-up. Like others, we found that mitotic rate was significantly associated with survival (P < 4 x 10(-8)) and more prognostic than tumor ulceration but was not an independent prognosticator because it was significantly associated with tumor thickness and ulceration. Thus, all 3 histologic variables are interrelated; among these, tumor thickness is the most important. Although mitotic rate can be effectively categorized in 3 groups (1/mm2, 1/mm2(-4)/mm2, and > 4/mm2), the optimal way to use mitotic rate remains unclear, and even this simplification requires determining the raw number per square millimeter. Because the collective information provided by tumor thickness, mitotic rate, ulceration, patient age, and site of tumor about hard outcomes such as 5-year fatality is limited and because measuring mitotic rate requires extra time, we recommend that mitotic rate need not be part of routine reports on cutaneous melanoma. Nevertheless, mitotic rate should continued to be measured in academic centers and other sites that maintain large prospective databases on melanoma, and it should be included in further studies of prognosis and adjuvant therapies for cutaneous melanoma.

Interobserver variability of mitotic index and utility of PHH3 for risk stratification in gastrointestinal stromal tumors.

OBJECTIVES: Accurate grading of gastrointestinal stromal tumors (GISTs), based on mitotic index, can be problematic. METHODS: In this study, we compared interobserver variability in detecting mitosis on H&E with PHH3 immunohistochemistry (IHC). In addition, we examined the correlation between H&E mitosis and Ki-67 and the association of PHH3 and Ki-67 with overall survival. Four pathologists independently reviewed 50 GIST cases. RESULTS: Intraclass correlation coefficients showed good interobserver variability for mitotic counts on both H&E (0.918; 95% confidence interval [CI], 0.874-0.950) and PHH3 IHC (0.923; 95% CI, 0.882-0.953). Nineteen (38%) cases were graded higher and five (10%) cases were downgraded by at least one observer using PHH3 compared with H&E. Using receiver operating characteristic curve analysis, a PHH3 cutoff of seven or more mitoses was associated with worse overall survival (P = .028). Ki-67 showed poor correlation with H&E mitotic counts and overall survival (P = .077). CONCLUSIONS: PHH3 may thus be a valuable adjunct for risk stratification in GISTs.

Cell cycle synchronization and BrdU incorporation as a tool to study the possible selective elimination of ErbB1 gene in the micronuclei in A549 cells.

Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1 could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells.

Prognostic significance of mitotic figures in metastatic mammary ductal carcinoma to the lymph nodes.

We previously reported that the number of mitotic figures in metastatic mammary carcinoma to the lymph nodes accurately predicted the outcome of patients with invasive ductal carcinoma with nodal metastasis. To confirm these previous findings, the present study investigated the number of mitotic figures and other histologic characteristics in metastatic mammary carcinoma to the lymph nodes and their associations with patient outcome according to nodal status and the histologic grade of primary invasive ductal carcinomas in a different series of 1039 patients with invasive ductal carcinoma. Multivariate analyses examining well-known clinicopathologic factors, the number of mitotic figures in the primary invasive ductal carcinomas, the grading system for lymph vessel tumor emboli, the p53 Allred score risk classes of tumor-stromal fibroblasts forming and not forming a fibrotic focus, and 9 histologic features of metastatic mammary carcinoma to the lymph nodes were performed. The presence of 6 or more mitotic figures in metastatic mammary carcinoma to the lymph nodes significantly increased the hazard ratios for tumor recurrence and tumor-related death among patients with invasive ductal carcinoma as a whole, those with nodal metastasis, and those with a histologic grade of 2 or 3. The presence of 6 or more mitotic figures in metastatic mammary carcinoma to the lymph nodes also significantly increased the hazard ratio for tumor recurrence among patients with histologic grade 1 invasive ductal carcinoma. In conclusion, this study clearly confirmed the excellent outcome predictive power of the number of mitotic figures in metastatic mammary carcinoma to the lymph nodes.

Ki67 immunohistochemistry: a valuable marker in prognostication but with a risk of misclassification: proliferation subgroups formed based on Ki67 immunoreactivity and standardized mitotic index.

AIMS: Counting mitotic figures is considered to be a reliable prognosticator, but evaluation of Ki67 immunohistochemistry has become more popular in evaluating proliferation. Our previous studies suggested an occasional discrepancy between mitotic figures and Ki67 fraction. The aim of this study was to investigate this more closely and also to study the associations between bcl-2 and p53 expression and proliferation. METHODS AND RESULTS: Two hundred and sixty-five infiltrating breast carcinomas were immunostained for Ki67, p53 and bcl-2. The standardized mitotic index (SMI) was determined. Four proliferation groups were based on Ki67 positivity fraction and SMI at optimal cut-off points. Cox's multivariate model was used to test the power of the prognosticators. SMI and nodal status were the most powerful individual prognosticators. Ki67 was an independent prognosticator if nodal status, tumour size, age and histological grade were included in the analysis but not if analysed with SMI. The group with low SMI and low Ki67 fraction had the best prognosis. Groups with high SMI had the poorest prognosis. The group with low SMI and high Ki67 fraction had a favourable prognosis. Bcl-2 negativity and p53 positivity correlated with proliferation. CONCLUSIONS: We have found a 'wrong positive' Ki67 group with favourable prognosis. SMI cannot be replaced by Ki67 because of the danger of misclassification of some patients.

Mitotic counting in surgical pathology: sampling bias, heterogeneity and statistical uncertainty.

Mitotic counting in surgical pathology: sampling bias, heterogeneity and statistical uncertainty Although several articles on the methodological aspects of mitotic counting have been published, the effects of macroscopic sampling and tumour heterogeneity have not been discussed in any detail. In this review the essential elements for a standardized mitotic counting protocol are described, including microscopic calibration, specific morphological criteria, macroscopic selection, counting procedure, effect of biological variation, threshold, and the setting of an area of uncertainty ('grey area'). We propose that the use of a standard area for mitotic quantification and of a grey area in mitotic counting protocols will facilitate the application of mitotic counting in diagnostic and prognostic pathology.