Counting mitoses: SI(ze) matters!

Mitoses are often assessed by pathologists to assist the diagnosis of cancer, and to grade malignancy, informing prognosis. Historically, this has been done by expressing the number of mitoses per n high power fields (HPFs), ignoring the fact that microscope fields may differ substantially, even at the same high power (×400) magnification. Despite a requirement to define HPF size in scientific papers, many authors fail to address this issue adequately. The problem is compounded by the switch to digital pathology systems, where ×400 equivalent fields are rectangular and also vary in the area displayed. The potential for error is considerable, and at times this may affect patient care. This is easily solved by the use of standardized international (SI) units. We, therefore, recommend that features such as mitoses are always counted per mm2, with an indication of the area to be counted and the method used (usually "hotspot" or "average") to obtain the results.

A new tool for technical standardization of the Ki67 immunohistochemical assay.

Ki67, a nuclear proliferation-related protein, is heavily used in anatomic pathology but has not become a companion diagnostic or a standard-of-care biomarker due to analytic variability in both assay protocols and interpretation. The International Ki67 Working Group in breast cancer has published and has ongoing efforts in the standardization of the interpretation of Ki67, but they have not yet assessed technical issues of assay production representing multiple sources of variation, including antibody clones, antibody formats, staining platforms, and operators. The goal of this work is to address these issues with a new standardization tool. We have developed a cell line microarray system in which mixes of human Karpas 299 or Jurkat cells (Ki67+) with Sf9 (Spodoptera frugiperda) (Ki67-) cells are present in incremental standardized ratios. To validate the tool, six different antibodies, including both ready-to-use and concentrate formats from six vendors, were used to measure Ki67 proliferation indices using IHC protocols for manual (bench-top) and automated platforms. The assays were performed by three different laboratories at Yale and analyzed using two image analysis software packages, including QuPath and Visiopharm. Results showed statistically significant differences in Ki67 reactivity between each antibody clone. However, subsets of Ki67 assays using three clones performed in three different labs show no significant differences. This work shows the need for analytic standardization of the Ki67 assay and provides a new tool to do so. We show here how a cell line standardization system can be used to normalize the staining variability in proliferation indices between different antibody clones in a triple negative breast cancer cohort. We believe that this cell line standardization array has the potential to improve reproducibility among Ki67 assays and laboratories, which is critical for establishing Ki67 as a standard-of-care assay.

Mitotic count: A need for standardization.

PURPOSE: The mitotic count (MC), number of mitosis per unit area, is a very important parameter frequently used for classification and grading of some tumors. Traditionally, the MC has been expressed in terms of number of mitoses per high power field. The size of the field of view can vary greatly among different microscopes. In order to avoid under or overestimation of mitotic count, a conversion needs to be made. METHODS: A simple formula based on a simple rule of three has been devised to standardize the mitotic count to the reference area by multiplying the number of mitotic figures by a correction factor which has been calculated for the most frequently used microscopes and various common tumors. RESULTS AND CONCLUSIONS: We propose this simple method, which involves only a single multiplication, to standardize the mitotic count to the reference area.

Standardized Method for Defining a 1-mm2 Region of Interest for Calculation of Mitotic Rate on Melanoma Whole Slide Images.

CONTEXT.­: Mitotic rate counting is essential in pathologic evaluations in melanoma. The American Joint Committee on Cancer recommends reporting the number of mitotic figures (MFs) in a 1-mm2 area encompassing the "hot spot." There is currently no standard procedure for delineating a 1-mm2 region of interest for MF counting on a digital whole slide image (WSI) of melanoma. OBJECTIVE.­: To establish a standardized method to enclose a 1-mm2 region of interest for MF counting in melanoma based on WSIs and assess the method's effectiveness. DESIGN.­: Whole slide images were visualized using the ImageScope viewer (Aperio). Different monitors and viewing magnifications were explored and the annotation tools provided by ImageScope were evaluated. For validation, we compared mitotic rates obtained from WSIs with our method and those from glass slides with traditional microscopy with 30 melanoma cases. RESULTS.­: Of the monitors we examined, a 32-inch monitor with 3840 × 2160 resolution was optimal for counting MFs within a 1-mm2 region of interest in melanoma. When WSIs were viewed in the ImageScope viewer, ×10 to ×20 magnification during screening could efficiently locate a hot spot and ×20 to ×40 magnification during counting could accurately identify MFs. Fixed-shape annotations with 500 × 500-µm squares or circles can precisely and efficiently enclose a 1-mm2 region of interest. Our method on WSIs was able to produce a higher mitotic rate than with glass slides. CONCLUSIONS.­: Whole slide images may be used to efficiently count MFs. We recommend fixed-shape annotation with 500 × 500-µm squares or circles for routine practice in counting MFs for melanoma.

Comparison of different anti-Ki67 antibody clones and hot-spot sizes for assessing proliferative index and grading in pancreatic neuroendocrine tumours using manual and image analysis.

AIMS: Ki67 proliferative index (PI) is essential for grading gastroenteric and pancreatic neuroendocrine tumours (GEP NETs). Analytical and preanalytical variables can affect Ki67 PI. In contrast to counting methodology, until now little attention has focused on the question of clone equivalence and the effect of hot-spot size on Ki67 PI in GEP NETs. Using manual counting and image analysis, this study compared the Ki67 PI achieved using MM1, K2 and 30-9 to MIB1, a clone which has been validated for, and is referenced in, guidelines relating to assessment of Ki67 PI in GEP NETs. METHODS AND RESULTS: Forty-two pancreatic NETs were each immunohistochemically stained for the anti-Ki67 clones MIB1, MM1, K2 and 30-9. Ki67 PI was calculated manually and by image analysis, the latter using three different hot-spot sizes. In manual comparisons using single hot-spot high-power fields, non-MIB1 clones overestimated Ki67 PI compared to MIB1, resulting in grading discordances. Image analysis shows good agreement with manual Ki67 PI but a tendency to overestimate absolute Ki67 PI. Increasing the size of tumour hot-spot from 500 to 2000 cells resulted in a decrease in Ki67 PI. CONCLUSION: Different anti-Ki67 clones do not produce equivalent PIs in GEP NETs, and clone selection may therefore affect patient care. Increasing the hot-spot size decreases the Ki67 PI. Greater standardisation in terms of antibody clone selection and hot-spot size is required for grading GEP NETs. Image analysis is an effective tool for assisting Ki67 assessment and allows easier standardisation of the size of the tumour hot-spot.

Interobserver Variability in Mitotic Count for Meningioma Grading: How Can We Reduce It?

AIM: To evaluate the interobserver variability in determining the number of mitoses in 10 high-power field (HPF) and thus the tumor grade, and to investigate how to reduce grade discordance between the observers and the most useful method to identify the patients who would receive an additional treatment. MATERIAL AND METHODS: Two hundred and seventy cases with meningioma were re-evaluated by three experienced pathologists and five senior residents. They determined the number of mitotic figures in 10 HPF in each slide. Re-evaluation of the cases, which were found of different grades from the reference observers was requested by full scan method. Statistical analysis was performed by using SPSS V23.0. RESULTS: A moderate agreement was found between the observers and the reference observer. After the evaluation of mitotic activity with the full scan method, the mean numbers of mitoses found by the observers in 10 HPF were increased. In the first evaluation, 4?6 cases were defined as Grade II by the observers. Whereas, 23?27 cases were defined as Grade II after the full scan method. CONCLUSION: If there are less than 16 mitotic figures throughout the slide, it is more difficult to find the 10 HPF including 4 or more mitosis. Interobserver variability in mitotic figure counting can be reduced by full scan method, and examining the hematoxylin and eosin stained slides by the full scan method helps us to determine the true histologic grade of meningiomas in most cases, who would receive an additional treatment.

Global and mitosis-specific interobserver variation in mitotic count scoring and implications for malignant melanoma staging.

AIMS: Staging is the gold standard for predicting malignant melanoma outcome but changes in its criteria over time indicate ongoing evolution. One notable recent change from the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual was removal of mitotic count. We explore the extent to which this feature is limited by interobserver error in order to find ways to improve its fitness for use should it be revisited in future staging versions. METHODS AND RESULTS: In a cohort of 476 patients with melanoma ≤1.0 mm, a mitotic count of 0 versus 1 was significant for metastasis-free survival, but not melanoma-specific or overall survival. In 10 melanomas that were 0.9-1.0 mm thick, the mitotic count intraclass correlation coefficient for histopathologists was 0.58 (moderate agreement). Uniquely, we also assessed agreement for specific putative mitotic figures, identifying precise reasons why specific mitotic figures qualified for scoring or elimination. A kappa score was 0.54 (moderate agreement). We also gathered data on other staging features. Breslow thickness had an intraclass correlation coefficient of 0.41 (moderate agreement) and there was a systematic difference between histopathologists among cases (P = 0.04). Every case had a range that crossed the AJCC8 0.8-mm pT1a/pT1b staging boundary. Ulceration was only identified in two of the 10 cases. For ulceration, kappa agreement score was 0.31 (fair). CONCLUSION: This study supports the removal of mitotic count from staging, but shows that its scoring is substantially affected by interobserver variation, suggesting that more prescriptive guidelines might have a beneficial impact on its prognostic value.

Evaluation of the proliferation marker Ki-67 in gliomas: Interobserver variability and digital quantification.

BACKGROUND: The Ki-67 Labelling Index (LI) is used as an ancillary tool in glioma diagnostics. Interobserver variability has been reported and no precise guidelines are available. Nor is it known whether novel digital approaches would be an advantage. Our aim was to evaluate the inter- and intraobserver variability of the Ki-67 LI between two pathologists and between pathologists and digital quantification both in whole tumour slides and in hot spots using narrow but diagnostically relevant intervals. METHODS: In samples of 235 low and high grade gliomas, two pathologists (A and B) estimated the Ki-67 LI (5-10% intervals) for whole tumour slides and for hot spots. In 20 of the cases intraobserver variability was evaluated. For digital quantification (C) slides were scanned with subsequent systematic random sampling of viable tumour areas. A software classifier trained to identify positive and negative nuclei calculated the Ki-67 LI. The interobserver agreements were evaluated using kappa (κ) statistics. RESULTS: The observed proportions of agreement and κ values for Ki-67 LI for whole tumour slides were: A/B: 46% (κ = 0.32); A/C: 37% (κ = 0.26); B/C: 37% (κ = 0.26). For hot spots equivalent values were: A/B: 14% (κ = 0.04); A/C: 18% (κ = 0.09); B/C: 31% (κ = 0.21). CONCLUSIONS: Interobserver variability was pronounced between pathologists and for pathologists versus digital quantification when attempting to estimate a precise value of the Ki-67 LI. Ki-67 LI should therefore be used with caution and should not be over interpreted in the grading of gliomas. Digital quantification of Ki-67 LI in gliomas was feasible, but intra- and interlaboratory robustness need to be determined.

Calculation of the Ki67 index in pancreatic neuroendocrine tumors: a comparative analysis of four counting methodologies.

Ki67 index is now an essential part of classification of pancreatic neuroendocrine tumors. However, its adaptation into daily practice has been fraught with challenges related to counting methodology. In this study, three reviewers used four counting methodologies to calculate Ki67 index in 68 well-differentiated pancreatic neuroendocrine tumors: (1) 'eye-ball' estimation, which has been advocated as reliable and is widely used; (2) automated counting by image analyzer; (3) manual eye-counting (eye under a microscope without a grid); and (4) manual count of camera-captured/printed image. Pearson's correlation (R) was used to measure pair-wise correlation among three reviewers using all four methodologies. Average level of agreement was calculated using mean of R values. The results showed that: (1) 'eye-balling' was least expensive and fastest (average time <1 min) but had poor reliability and reproducibility. (2) Automated count was the most expensive and least practical with major impact on turnaround time (limited by machine and personnel accessibility), and, more importantly, had inaccuracies in overcounting unwanted material. (3) Manual eye count had no additional cost, averaged 6 min, but proved impractical and poorly reproducible. (4) Camera-captured/printed image was most reliable, had highest reproducibility, but took longer than 'eye-balling'. In conclusion, based on its comparatively low cost/benefit ratio and reproducibility, camera-captured/printed image appears to be the most practical for calculating Ki67 index. Although automated counting is generally advertised as the gold standard for index calculation, in this study it was not as accurate or cost-effective as camera-captured/printed image and was highly operator-dependent. 'Eye-balling' produces highly inaccurate and unreliable results, and is not recommended for routine use.

Variability in mitotic figures in serial sections of thin melanomas.

BACKGROUND: T1 melanoma staging is significantly affected by tissue sampling approaches, which have not been well characterized. OBJECTIVE: We sought to characterize presence of mitotic figures across a minimum of 5 sequential sections of T1 melanomas. METHODS: A cohort of T1 melanomas with either 5 (single section per slide) or 10 (2 sections per slide) sequential sections (5-µm thickness) per case were prepared and examined for mitotic figures. RESULTS: In all, 44 of 82 T1 melanomas (54%) were classified as T1b. The number of sections with a mitotic figure present ranged from only 1 of 5 sections (n = 5 of 44 cases, 11.4%) to all 5 (n = 20 of 44 cases, 45.5%). A sequential approach versus a nonsequential approach did not appear to matter. LIMITATION: Cases were taken from a single pathology practice in the Pacific Northwest, which may not generalize to other populations in the United States. CONCLUSION: The variation in the presence of mitotic figures within sequential sections supports reviewing 3 to 5 sections to fulfill American Joint Committee on Cancer recommendations. The prognostic significance of a T1b melanoma with a rare mitotic figure on a single section versus a T1b melanoma with mitotic figures on multiple sections deserves more attention to see if further subclassification is possible or even necessary.

Are we counting mitoses correctly?

The number of mitotic figures in a predefined area is essential in pathologic evaluation for most tumors. This information sometimes provides clues in differentiating neoplastic lesions from nonneoplastic ones and sometimes in defining and grading of the tumors as well as prognosticating expected lifetime of the patient. As a generally accepted concept, scanning a certain number of consecutive nonoverlapping areas that are rich in viable tumor cells is required. Invasion fronts or the periphery of the tumors is preferred for counting mitosis. The target area to be counted for mitotic activity for various tumors is standardized as the number of mitosis in an established number of high-power fields. However, suggested mitotic counts, which constitute the basis of these studies, were obtained via the old microscopes, which usually had narrower visual fields than the state-of-the-art microscopes. Because the visual fields of the present microscopes provide larger areas compared with the older ones, corrections in mitosis counting are needed to make them compatible with the criteria, which had been put forward in the original reference studies.

Nevic mitoses: a review of 1041 cases.

The presence of dermal mitotic figures is helpful in identifying malignant melanocytic lesions but occasionally occur in benign nevi. We aim to determine a "benchmark" mitotic frequency for a wide variety of nevi. We prospectively collected 1041 cases of benign melanocytic nevi and reviewed them for the presence of mitotic figures. Specimens were collected from female (62%) and male (38%) participants, ages ranged from 1 to 90 years. Nevus types included compound melanocytic nevi (CMN), intradermal melanocytic nevi, junctional melanocytic nevi, lentiginous CMN (LCMN), lentiginous junctional melanocytic nevi, blue nevi, deep penetrating nevi, and pigmented spindle cell nevi. Nevi with congenital, mildly dysplastic, and Spitzoid features were included. A total of 82 of 1041 (7.9%) nevi contained one or more mitotic figures. Most (76.1%) mitoses were found in the papillary dermis. Single mitotic figures were more common (80.4%) than 2 (15.9%) or 3 (3.7%) within a single specimen. Of all cases containing mitotic figures, the most common nevus type was CMN. The nevus type most frequently demonstrating mitotic figures was CMN. The most common body site among cases with mitotic figures was trunk (53.7%), whereas the body site with the largest proportion of nevi demonstrating mitotic figures was special site (10.9%). The percentage of nevi containing mitotic figures was nearly the same among female (7.9%) and male (7.8%) participants. Results of this large review confirm that mitotic figures, even multiple ones, do not preclude benignity in a variety of melanocytic nevi.

Assessment of mitotic rate reporting in melanoma.

BACKGROUND: In patients with cutaneous melanoma, mitotic rate (MR) historically has been reported as the number of mitoses per high-power field (hpf) or per 10 hpf. The most recent revision of the American Joint Committee on Cancer melanoma staging system now incorporates MR and specifies that MR should be reported as mitoses per mm(2), with a conversion factor of 1 mm(2) equaling 4 hpf. However, because many pathologists continue to report MR in hpf units, we sought to compare the 2 conventions for reporting MR; this is important now that MR is used for staging and prognostic information. METHODS: A retrospective analysis was performed of a database that combined patients from a large multicenter study and our single-institution melanoma database. All patients with pathology reports that included MR were included. For patients with MR reported in hpf units, MR was converted to mitoses per 10 hpf. Statistical analysis was performed to test differences in Breslow thickness (BT), ulceration, sentinel lymph node (SLN) status, and overall survival (OS) (log-rank test) between the mitoses per mm(2) group versus the mitoses per 10-hpf group. RESULTS: A total of 1,148 patients were identified; of these, 759 were reported as per mm(2) and 389 were reported in hpf units. When patients were subdivided into categories of MR of 0, 1, or more than 1, there was no statistically significant difference in mean or median BT, ulceration, or SLN positivity within categories between patients with MR per mm(2) versus patients with MR reported per 10 hpf. There was also no difference in OS between groups. Subdividing into smaller categories of MR of 0, 1, 2, 3, 4, 5, or more than 5 did not yield different results. CONCLUSIONS: Although the American Joint Committee on Cancer staging system reports a conversion factor for MR of 1 mm(2) equals 4 hpf, no clinically meaningful differences in predictors of prognosis (BT, ulceration, SLN positivity) or OS were seen between groups when a conversion factor of 1 mm(2) equaling 10 hpf was used. Therefore, for practical purposes, MR reported per 10 hpf approximates MR per mm(2).

Prognostic differences of World Health Organization-assessed mitotic activity index and mitotic impression by quick scanning in invasive ductal breast cancer patients younger than 55 years.

The proliferation marker mitotic activity index is the strongest prognostic indicator in lymph node-negative breast cancer. The World Health Organization (WHO) 2003-defined procedure for determining WHO-mitotic activity index is often replaced by a quick scan mitotic impression. We evaluated the prognostic consequences of this practice in 433 T(1-3)N(0)M(0) lymph node-negative invasive ductal type breast cancers with long-term follow-up (median, 112 months; range, 12-187 months). Twenty-seven percent of the studied cases developed distant metastases, and 25% died of disease. Agreement between WHO-mitotic activity index (0-5 = 1, 6-10 = 2, >10 = 3) and mitotic impression (1, 2, 3) categories was 66% (kappa = 0.41), including 85% for category 1, 26% for category 2, and 52% for category 3. The WHO-mitotic activity index was a much stronger prognosticator than the mitotic impression, and the 10-year survival rates of the same categories (eg, mitotic activity index and mitotic impression category both 2) differed greatly. When grade was assessed by combining WHO-mitotic activity index or mitotic impression with the same values for tubular formation and nuclear atypia, grades disagreed in 18% of the cases. Deviation from the formal WHO-mitotic activity index assessment guidelines in breast cancer often results in erroneous prognosis estimations with therapeutic consequences and may explain why the prognostic value of proliferative activity in breast cancer is not always confirmed.

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.

An improved technique for mitosis counting.

Mitosis counting remains one of the most valuable prognostic indicators in tumor pathology; however, as currently carried out it is time consuming and not reproducible. In this study, 6 different pathologists, using different microscopes, arrived at widely different mitotic counts on the same slide, ranging from 4 to 16. These differences were mainly due to the different field areas of the various microscopes used and the method used for counting and recording. In evaluating the most active 10 HPF, the count ranged from 10 to 19. Instead, when an average of 40 fields was recorded, the range was 4-11. Using the mitosis/volume index, which expresses the number of mitotic figures per mm2 of viable tumor, the counts ranged from 8 to 10, a marked improvement. However, this method is complicated and not "user-friendly.'' We suggest a variation of the technique by which a 2 mm2 rectangle is drawn on a cover slip and mounted under the microscope, centered on the most mitotically active area of the tumor. The mitoses in that area are counted (=n) and the percent of viable tumor (=x%) is estimated under low magnification. The number of mitoses per mm2 of viable tumor (cs-MAI) is then calculated according to the formula Cs-MAI=100n/2x. Using this modified method, the range of mitoses counted by the different observers was very narrow (9 to 11), and the time required for the counting was only 5-10 minutes.

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.