MUC5B expression in gastric carcinoma: relationship with clinico-pathological parameters and with expression of mucins MUC1, MUC2, MUC5AC and MUC6

The tissue microarray technology is a high-throughput technique that allows studies of multiple markers in large tumor materials. We performed immunohistochemical profiling using tissue microarray and immunostaining for Ki-67, p53, bcl-2, CD44, cyclin A and Pgp in a series of 211 malignant fibrous histiocytomas (MFHs) with correlation to prognosis. Tissue from 50 local recurrences and 20 metastases was available for comparison with the primary tumors. In univariate analysis, Ki-67 was the only immunohistochemical marker significantly correlated with metastasis with a hazard ratio of 1.9. Multivariate analysis, with tumor size, depth, necrosis, vascular invasion, mitotic rate and Ki-67 expression, revealed an independent prognostic value of tumor size and Ki-67. Local recurrences did not differ from the corresponding primary tumors, whereas metastases showed a trend for upregulation of cyclin A and Pgp. In this large series of MFHs, a tumor size greater than 8 cm and a Ki-67 index of more than 20% were strong and independent prognostic factors for metastasis. In contrast, p53, bcl-2, CD44, cyclin A and Pgp, which have previously been suggested as prognostic factors in soft tissue sarcomas, did not show such correlations. Hence, we suggest that proliferation, as measured by Ki-67 index, should be considered as a prognostic marker in clinical management of pleomorphic soft tissue sarcomas.     

细胞周期蛋白D1、Ki-67和bcl-2的表达与CD117阳性的胃肠道间质瘤生物学行为的关系

目的 检测细胞周期蛋白D1（cyclin D1）、Ki-67和bcl-2在胃肠道间质瘤（GIST）中的表达,探讨它们在GIST发生、发展中的作用及临床病理意义。方法 59例手术切除GIST标本进行CD117、CD34、平滑肌肌动蛋白（SMA）、结蛋白、S-100、cyclin D1、bcl-2和Ki-67免疫组织化学染色,同时进行病理形态学观察包括形态学类型、肿瘤大小、坏死和核分裂象。所有病例随访2～9年。所有数据进行单因素、多因素和相关分析。结果 随访40例患者一直健在,15例患者死于GIST,4例患者死于其他原因。统计学分析显示肿瘤直径〉5cm、有坏死、核分裂象在每50个高倍视野〉5个、Ki-67增殖指数（LI）〉5％、cyclin D1和bcl-2免疫组织化学染色强阳性都可以作GIST患者手术后的预测指标,且具有统计学意义;核分裂象和Ki-67增殖指数是独立的预测指标;Ki-67 LI≥5％和核分裂象≥5／50 HPF呈正相关（r=0．532,P〈0．01）;cyclin D1与bcl-2强阳性表达呈正相关（r=0.273,P〈0．05）。结论 肿瘤大小、坏死、核分裂象、cyclin D1、Ki-67增殖指数和bcl-2可作为GIST患者临床预测指标;核分裂象和Ki-67增殖指数可作为独立的预测指标;cyclinD1与bcl-2呈明显相关性,Ki-67免疫组织化学染色可以代替核分裂象作为一项很有用的预测指标。     

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.     

The Impact of Phosphohistone-H3-Assisted Mitotic Count and Ki67 Score in the Determination of Tumor Grade and Prediction of Distant Metastasis in Well-Differentiated Pancreatic Neuroendocrine Tumors

This study investigated the impact of phosphohistone-H3 (PHH3)-assisted mitotic count by comparing its performance with conventional mitotic count and Ki67 score as well as the status of distant metastasis. A total of 43 surgically resected pancreatic neuroendocrine tumors (panNET) with complete follow-up information has been subjected to a standardized assessment with respect to mitotic count (both conventional and PHH3-assisted) and Ki67 score. Five participants assessed mitotic count and the time spent was recorded in both methods. All tumors were assigned to a G1 category of mitotic rate on conventional mitotic count that failed to identify three tumors with a G2 category of mitotic rate on PHH3. Near-perfect and fair agreements were achieved among observers when using PHH3 and conventional method, respectively. The mean time spent to determine mitotic count on PHH3-stained slides was significantly shorter (p?<?0.001). The performance of PHH3-assisted mitotic grade category was significant as the three cases with a G2 mitotic category were associated with distant metastasis (p?=?0.01). Despite its performance, the PHH3-assisted mitotic count downgraded 17 cases that were classified as G2 based on Ki67 scores in this series. The Ki67 grade category was either the same or higher than the mitotic grade category. Ten patients developed distant metastasis. Eleven tumors exhibited vascular invasion characterized by intravascular tumor cells admixed with thrombus. Our results indicate that PHH3-assisted mitotic count facilitates an accurate mitotic count with a perfect agreement among observers. The small size of this cohort is an important limitation of the current study, a G2 mitotic grade category based on PHH3 immunohistochemistry was one of the correlates of panNETs with distant metastasis. While the prognostic impact of PHH3-assisted mitotic count needs to be clarified in larger cohorts, Ki67 scores designated higher grade category in all cases; thus, it was the best determinant of the tumor grade. More importantly, the presence of vascular invasion along with the Ki67 grade category was found to be independent predictors of distant metastasis.     

Prognostic impact of histological grade and vascular invasion compared with tumour cell proliferation in endometrial carcinoma of endometrioid type

AIMS: The relative impact of different prognostic factors is important for endometrial carcinoma patients. The aim of our study was to examine the combined value of histological grade [International Federation of Gynaecology and Obstetrics (FIGO)] and vascular invasion in comparison with tumour cell proliferation assessed by mitotic count and Ki67. The recently proposed binary architectural grade was also evaluated, in addition to age, depth of myometrial infiltration and FIGO stage in our population-based series of 237 endometrioid carcinomas. METHODS AND RESULTS: The tumours were studied for several histological features, including FIGO grade, binary grade, vascular invasion, mitotic count, myometrial invasion and expression of Ki67. FIGO grade was significantly associated with all investigated histological features, including Ki67 expression. Vascular invasion was significantly more frequent in FIGO grade 3 tumours, and was associated with a diffusely infiltrative growth pattern, solid growth, necrosis and deep myometrial invasion. All variables showed a highly significant relationship with patient survival in univariate analysis. In multivariate models, FIGO grade, vascular invasion, and proliferation assessed by Ki67 expression all had independent prognostic influence in this population-based study. Comparing tumour cell proliferation (Ki67) with vascular invasion as a marker of metastatic spread, the latter had a stronger survival impact. CONCLUSIONS: Vascular invasion and tumour cell proliferation measured by Ki67 both had independent prognostic influence, and should be considered to identify aggressive tumours of the endometrioid subtype.     

Prognostic indicators for gastrointestinal stromal tumours: a clinicopathological and immunohistochemical study of 108 resected cases of the stomach

AIMS: Whether immunohistochemical markers increase accuracy in predicting prognosis for gastrointestinal stromal tumours (GISTs) remains uncertain. However, past studies have used only small, heterogeneous patient groups. Our aim was to test previously studied and more novel morphological features as well as four immunohistochemical markers as prognostic indicators amongst a large cohort of surgically resected, gastric GISTs. METHODS AND RESULTS: Tissues from 127 gastric mesenchymal tumours were collected retrospectively and subjected to repeat histological assessment and immunophenotyping. Further immunohistochemistry was performed for Ki67, p53, Bcl-2 and cyclin D1. Complete follow-up data were collected for 108 patients with immunophenotyped diagnoses of GIST (i.e. c-kit+ tumours). At the census point, 52 patients were alive, 24 had died from their GISTs and the remainder of other causes. Univariate analysis showed the following predicted for shorter disease-specific survival: size > or =50 mm; necrosis, no intratumoral lymphocytes; mitotic count > or =5/50 high power fields; Ki67 labelling index > or =5%; p53 immunopositivity. Of these variables, multivariate analyses showed only mitotic count and, to a lesser extent, Ki67 labelling to be independent prognostic indicators. CONCLUSIONS: Mitotic count remains the best predictor of outcome following surgical resection of gastric GISTs. Ki67 immunohistochemistry does not provide better prognostication and p53, Bcl-2 and cyclin D1 immunohistochemistry provide no additional prognostication.     

Image analysis is an excellent tool for quantifying Ki‐67 to predict the prognosis of gastrointestinal stromal tumor patients

We investigated the quantification of Ki‐67 staining using digital image analysis (IA) as a complementary prognostic factor to the modified National Institutes of Health (NIH) classification in patients with gastrointestinal stromal tumor (GIST). We examined 92 patients, focusing on the correlation between age, sex, primary tumor site, tumor size, predominant histologic type, mitotic index, modified NIH classification (low/intermediate vs high), Ki‐67 quantitation, and recurrence‐free survival (RFS). We compared two IA processes for whole slide imaging (WSI) and manually captured image (MCI) methods. A Ki‐67 quantitation cutoff was determined by receiver operator characteristics curve analysis. In the survival analysis, the high‐risk group of a modified NIH classification, a mitotic count >5 per 20 high‐powered fields, and Ki‐67 cutoffs of ≥6% and ≥8% obtained by IA of the WSI and MCI methods, respectively, had an adverse impact on RFS. On multivariate analysis, each Ki‐67 quantitation method strongly predicted prognosis, more strongly than the modified NIH classification. In addition, Ki‐67 quantitation using IA of the MCI method could stratify low or intermediate risk and high risk GIST patients. Thus, IA is an excellent tool for quantifying Ki‐67 to predict the prognosis of GIST patients, and this semiautomated approach may be preferable for patient care.     

Gastroenteropancreatic neuroendocrine neoplasms: Historical context and current issues

The digestive organs contain a large number of neuroendocrine cells as part of the diffuse neuroendocrine system. Neuroendocrine tumors can occur in every digestive organ. It has long been recognized that this is a diverse group of tumors with very different clinical outcomes; however, well-recognized prognostic parameters had been elusive until recently. Over the years, there have been several different classification schemes, each with different strengths and weaknesses. In an effort to standardize the classification and grading criteria for gastroenteropancreatic neuroendocrine tumors, the current World Health Organization classification includes a histologic grade based on proliferative rate (mitotic rate and Ki67 index) and a TNM stage that varies from organ to organ. The prognostic value of both the grade and stage has been validated in multiple studies. However, several issues remain, including the lack of standardized methods to assess proliferative rate, potential discrepancies between the mitotic count and the Ki67 index; intratumoral heterogeneity in proliferative rate; and the need for refinement in proliferative cut-points to define the grades. More studies are needed to further improve the classification of neuroendocrine tumors, thus guiding optimal treatment for these tumors.     

Apoptosis and proliferation in relation to histopathological variables and prognosis in hepatocellular carcinoma

The prognosis of patients with resectable hepatocellular carcinoma depends mainly on the anatomical extent of the tumour and on the general condition of the patient. Given the growing evidence that proliferation indices may be of prognostic significance in hepatocellular carcinomas and that parameters of cell loss (usually, but not exclusively, due to programmed cell death) are biologically relevant, the identification and quantitation of proliferative capacity and apoptosis may be of prognostic importance. In this study four different methods have been used to assess proliferation in a series of 193 curatively (R0) resected hepatocellular carcinomas: mitotic count, immunohistochemical assessment of MIB-1 (Ki-67), proliferating cell nuclear antigen (PCNA), and silver-stained nucleolar organizer regions (AgNORs). Apoptosis was assessed using the in situ-end labelling (ISEL) technique in combination with morphological criteria. Patients who received liver transplantation were excluded. The results obtained were compared with histopathological stage (according to UICC), Edmondson grade, several other histopathological factors, and survival rate. Significant statistical correlations were seen between the mitotic index, the rate of nuclear positivity for MIB-1 and PCNA, and the number of AgNOR dots. In univariate survival analysis, tumour stage and Edmondson grade, mitotic index, MIB-1 and PCNA index, and mean AgNOR number were significant factors influencing patients' survival. On multivariate Cox survival analysis, mitotix index, concomitant cirrhosis, Edmondson grade, and patient age were the only significant independent prognostic factors. Apoptosis was not related to prognosis or to other parameters examined. These results indicated that mitotic index is an additional prognostic parameter which could provide auxiliary information for patients' outcome. MIB-1 and PCNA immunostaining and AgNORs showed a good correlation among themselves. Apoptosis did not predict prognosis in hepatocellular carcinoma. Copyright © 1999 John Wiley & Sons, Ltd.     

Correlation between grade and prognosis in metastatic gastroenteropancreatic neuroendocrine tumors

Three-tiered grading systems (low, intermediate, and high grade) have been proposed for neuroendocrine tumors. These classifications have not been rigorously evaluated in neuroendocrine malignancies of the digestive tract. We performed a retrospective chart analysis of 83 patients with metastatic gastroenteropancreatic neuroendocrine tumors, correlating tumor grade with overall survival. We also analyzed available biopsy specimens (on 40 patients), examining hematoxylin and eosin stains for mitotic rate and immunostaining for measurement of the Ki-67 index. Tumor grades were assigned based on the mitotic rate and the Ki-67 index, and the prognostic validity of each grading method was assessed. A highly significant correlation existed between the reported tumor grade and overall survival. Five-year survival rates for patients with low-, intermediate-, and high-grade tumors were 87%, 38%, and 0%, respectively. On biopsy specimen analysis, both mitotic rates and Ki-67 indexes correlated strongly with overall survival. We conclude that a 3-tiered grading classification for gastroenteropancreatic neuroendocrine tumors correlates with survival in the metastatic setting. Both mitotic rates and Ki-67 indexes are inversely associated with survival and can be analyzed independently for assignment of grade.     

Proliferation, ploidy and prognosis in uterine smooth muscle tumours

DNA ploidy, mitotic rate (per 10 high power fields), mitotic index (per 1000 tumour nuclei), Ki-67 labelling index and S phase fraction were measured in 23 uterine leiomyosarcomas and 10 tumours of uncertain malignant potential. Correlations were calculated by Spearmann rank correlation. Univariate survival analysis was performed by log rank analysis and multivariate analysis performed by the Cox linear regression method. Ki-67 index and S phase fraction were significantly higher in leiomyosarcomas than in tumours of uncertain malignant potential. There was significant correlation between mitotic rate, mitotic index, Ki-67 index and S phase fraction in cases of leiomyosarcoma. Fifteen of 22 leiomyosarcomas and one of 10 tumours of uncertain malignant potential were DNA aneuploid. On univariate analysis of all the smooth muscle tumours, DNA ploidy, presence of significant nuclear atypia and presence of coagulative tumour cell necrosis were associated with outcome. Only DNA ploidy was associated with outcome in the group of leiomyosarcomas. On multivariate analysis of all of the smooth muscle tumours. DNA ploidy, age and grade of atypia were independently associated with outcome. No single factor was independently predicitive of outcome in the group of leiomyosarcomas. Alternative indices of cell proliferation correlate with mitotic rate in uterine leiomyosarcoma and do not provide additional useful prognostic information. DNA ploidy, age and grade of atypia are independently associated with outcome in uterine smooth muscle tumours and measurement of DNA ploidy may be useful in identification of cases with an adverse prognosis.     

Proliferative activity of urothelial neoplasms: comparison of BrdU incorporation, Ki67 expression, and nucleolar organiser regions.

AIMS: To evaluate the proliferative activity of urothelial neoplasms, compare it with that of the normal urinary tract epithelium, and determine its relation to morphological grade and presence of invasion. METHODS: Multiple biopsy specimens from 53 individuals--eight normal controls, five patients with severe urothelial atypia, and 40 with transitional cell carcinomas (TCCs)--were studied using in vitro bromodeoxyuridine (BrdU) incorporation, Ki67 antigen expression, and quantitation of the nucleolar organiser regions (NORs). RESULTS: The percentage of nuclei labelled by BrdU (BrdU index) correlated well with the percentage of nuclei expressing the Ki67 antigen (Ki67 index). These proliferation indices were very low (less than 0.1% in 60% of samples) in the urothelium of normal controls and the morphologically unremarkable epithelium of patients with TCCs. Non-invasive TCCs had increased proliferation (BrdU index 6.32 (SD 0.8)%, Ki67 index 5.04 (0.6)% but lagged behind the invasive tumours (BrdU 20.9 (3.2)%, Ki67 18.6 (2.8)%). The average NOR count was 1.57 (0.03) in morphological normal epithelium, which increased progressively with grade in non-invasive TCCs, but varied greatly in invasive tumours and did not correlate with the proliferation indices. The spectrum of values for both proliferation indices and NORs was particularly wide in grade 2 TCCs. Severe atypias without exophytic growth had an increase in BrdU and Ki67 indices comparable with that found in grade 3-4 invasive TCCs; these also had the highest NORs per nucleus. CONCLUSIONS: The growth potential of urothelial neoplasms is an important indicator of their aggressive course. In particular, growth indices over 10% are strongly associated with the presence of invasion. Papillary grade 2 TCCs show heterogeneity in their growth characteristics which may relate to their diverse clinical course. The mitotic count underestimates the growth potential of papillary TCCs and the addition of proliferation indices such as BrdU incorporation or the Ki67 index may enhance the prognostic accuracy of conventional morphological grading.     

Soft tissue sarcomas. Classifying and/or grading?

Grading of soft tissue sarcomas cannot substitute for their accurate classification. The latter, according to the historical typing of soft tissue tumors, as proposed by WHO (1969), has to be considered as presumptive for morphological grading. The parameters given for grading are subject to individual bias and artefacts. They are also influenced by the "histogenetic types" of tumors. To achieve some correlations between morphological grading and prognosis, grading parameters (atypia and mitotic activity) have to be weighted differently, according to tumor type. Immunohistochemical methods are helpful in accurate typing of tumors where classification is difficult. The future search will be concerned with more objective methods for grading, including immunohistochemical determination of proliferative markers (similar to Ki67) or flow-through cytophotometry. All efforts are oriented to achieving an optimal correlation between grading parameters on the one hand, and prognostic predictions on the other.     

PCNA and Ki67 expression in breast carcinoma: correlations with clinical and biological variables.

AIMS: To investigate the expression of two cell cycle related antigens (proliferating cell nuclear antigen (PCNA) and Ki67 related antigen) in a series of breast cancers; and the possible correlations between the PCNA and Ki67 labelling indexes (PCNA-LI and Ki67-LI) and their associations with other biological and clinicopathological variables. METHODS: Ninety six ductal and 10 lobular carcinoma specimens were investigated. Samples were fixed in formalin and in Methacarnoy for localisation of PCNA. Ki67 was immunostained on frozen sections. The PCNA-LI and Ki67-LI were evaluated in relation to tumour size, mitotic count, histological grade, nodal state as well as receptor content and altered expression of the p53 gene. RESULTS: PCNA-LI did not correlate with Ki67-LI, nor was it associated with any other variable examined. A high KI67-LI (above the median value of 13.5) was associated with high grade and mitotic count, negative receptor content, and altered expression of the p53 gene, but not with other variables. CONCLUSIONS: The PCNA-LI does not seem to be a substitute for the Ki67-LI in evaluating the growth fraction in breast cancer.     

Altered expression of cell cycle regulators in myxofibrosarcoma, with special emphasis on their prognostic implications

Myxofibrosarcoma/myxoid malignant fibrous histiocytoma (MFH) has continued to be considered a distinct entity even after recently published reassessments of pleomorphic sarcomas and MFH. Several cell cycle-regulated proteins have already been screened by immunohistochemistry with the aim of finding the reliable prognostic indicator of soft tissue sarcomas; however, it is still unknown whether their altered expression affects patient survival in myxofibrosarcoma. In this study, we evaluated the expression of p53, MDM2, MIB-1 (Ki-67), p21, p27, p16, cyclin A, cyclin D1, and cyclin E by immunohistochemistry in 45 cases of myxofibrosarcoma. First, we searched for possible clinicopathologic prognostic factors in 61 cases of myxofibrosarcoma for which follow-up data were available. In univariate analysis, large tumor size (> or =5 cm), deeply situated tumor, and high histological grade (grade 2 or 3) significantly decreased survival (log-rank test, P <0.05). Among 43 cases of myxofibrosarcoma for which immunohistochemical findings were available, high MIB-1 labeling index (LI) (cutoffs of 10 and 22.5 on average), high cyclin A LI (cutoffs 10% and 13.8% on average), low p21 LI (cutoffs 10 and 20.7 on average), and reduced abnormal expression of p16 were adverse prognostic factors. In multivariate analysis (Cox proportional hazards model), high mitotic rate (>15/10 high-power fields), p53 immunoreactivity (cutoff 10%), high MIB-1 LI (>22.5), low p21 LI (<20.7), and low p27 LI (<47.8 on average) were independent poor prognostic factors. Our results suggest that reduced expression of p21 could be considered a new parameter to be evaluated, along with classical clinicopathologic prognostic factors, for identifying those at high risk for myxofibrosarcoma.     

Prognostic value of Ki67 index in anaplastic oligodendroglial tumours--a translational study of the European Organization for Research and Treatment of Cancer Brain Tumor Group

Preusser M, Hoeftberger R, Woehrer A, Gelpi E, Kouwenhoven M, Kros J M, Sanson M, Idbaih A, Brandes A A, Heinzl H, Gorlia T, Hainfellner J A & van den Bent M
(2012) Histopathology  60, 885–894
Prognostic value of Ki67 index in anaplastic oligodendroglial tumours – a translational study of the European Organization for Research and Treatment of Cancer Brain Tumor Group Aims: To evaluate the prognostic value and clinical utility of Ki67 tumour cell proliferation index in anaplastic oligodendroglial tumours (AOT). Methods and results: We performed anti‐Ki67 immunostaining (MIB‐1 antibody) of formalin‐fixed and paraffin‐embedded tumour tissue specimens of 128 patients with newly diagnosed AOT that were treated in a randomized Phase III trial. Ki67 index was assessed by three independent observers and was correlated to clinical, histopathological and molecular features (including 1p/19q co‐deletion, epithelial growth factor receptor gene (EGFR) amplification, isocitrate dehydrogenase (IDH1) mutations, O6‐methylguanine‐DNA methyltransferase gene (MGMT) promoter methylation, and patient survival times. Intra‐ and inter‐observer agreement of Ki67 index assessment was excellent. Univariable analysis (n = 79) showed that patients with a low Ki67 index had significantly more favourable progression‐free survival (PFS) (P‐value = 0.004, log‐rank test) and overall survival (OS) (P‐value = 0.003, log‐rank test) than patients with a high Ki67 index, respectively. On multivariable analysis (n = 43), Ki67 index showed no independent association with PFS or OS. Conclusions: In AOT the Ki67 index has a strong prognostic impact on univariable analysis, but no independent influence on multivariable analysis. However, further prospective studies including larger numbers of cases and standardized evaluation of Ki67 index in conjunction with other relevant prognostic parameters are needed to draw definitive conclusions.     

Current status of the histopathological assessment,diagnosis, and reporting of colorectal neuroendocrine tumors: A Web survey from the Japanese Society for Cancer of Colon and Rectum

Although new classifications for neuroendocrine tumors were established by the World Health Organization, the current procedures and terms used in pathology laboratories are not known. A Web‐based questionnaire was distributed to 491 institutions affiliated with the Japanese Society for Cancer of the Colon and Rectum, and 150 participated. The questionnaires included questions regarding routine pathological reporting, staining, and assessment of neuroendocrine tumors. Next, the time taken to assess Ki‐67 index and mitotic count according to recommendation was evaluated to determine its feasibility. Most laboratories recorded diagnostic term, depth of invasion, size, lymph‐vascular invasion, Ki‐67 index, and mitotic count. However, only 32.2% reported tumor stage. Chromogranin A and synaptophysin were common neuroendocrine markers. D2‐40 and elastica stain were frequently used to confirm lymph‐vascular invasion. Only 62.1% counted more than 500 cells for the Ki‐67 index, and only 17.0% counted more than 50 fields for the mitotic count, as suggested by the recommendations. Median time of 7 cases was 18.0 and 27.3 min to assess mitotic count in 50 fields with Ki‐67 index in 500 and 2000 cells, respectively. For more standardized pathological reporting, education about standardized staging systems are needed in Japan. Practical and standardized procedures for mitotic index and Ki‐67 index are also required.     

Tumor cell growth fractions in human malignant melanomas and the correlation to histopathologic tumor grading.

The growth fraction (GF) of 72 human malignant melanomas was determined by immunostaining with monoclonal antibody Ki-67. A positive correlation of GF and histopathologically-assessed prognostic variables, such as tumor thickness, mitotic rate, and prognostic index, was found. Individual Ki-67 values were considerably scattered in all histologically defined groups of malignancy. Thus, GF as determined by Ki-67 was used to calculate a modified prognostic index. In contrast to the histologically defined prognostic index, the Ki-67 based prognostic index allows further subdivision of thin malignant melanomas with no or few mitotic figures. This may be of help in identifying tumors with a high recurrence potential.     

Proliferation markers and DNA content analysis in urinary bladder TaT1 urothelial cell carcinomas: identification of subgroups with low and high stage progression risks

AIMS: To evaluate whether in situ biomarkers Ki67, mitotic activity index (MAI), p53, mean area of the 10 largest nuclei (MNA10), and whole genome DNA ploidy by flow and image cytometry (FCM and ICM, respectively) have independent prognostic value in urinary bladder urothelial cell carcinomas (UCs). METHODS: Ki67 and p53 immunoquantitation was performed in TaT1 consensus diagnosis UCs. MAI and MNA10 were also determined. Single cell suspensions were stained (DAPI for FCM; Feulgen for ICM). There was enough material for all measurements in 171 cases. Kaplan-Meier curves and multivariate survival analysis (Cox) were used to assess the prognostic value of all features (including classic clinicopathological risk factors, such as stage, grade, multicentricity, carcinoma in situ). RESULTS: Thirteen (7.6%) patients progressed. Of the classic factors, grade was strongly prognostic in univariate analysis, as were all the biomarkers. In multivariate analysis, the strongest independent combinations for progression were MNA10 (threshold (T) = 170.0 micro m(2)) plus MAI (T = 30), or MNA10 (T = 170.0 micro m(2)) plus Ki67(T = 25.0%). p53 (T = 35.2%) plus Ki67 (T = 25.0%) also predicted progression well, with high hazard ratios, but p53 measurements were not as reproducible as the other features. The prognostic value of the quantitative biomarkers exceeded that of the classic risk factors and DNA ploidy. The sensitivity, specificity, positive, and negative predictive values of MNA10/MAI or MNA10/Ki67 at the thresholds mentioned were 100%, 79%, 57%, and 100%, respectively. These feature combinations were also strongest prognostically in the high risk treatment subgroup. CONCLUSIONS: The combined biomarkers MNA10/Ki67 or MNA10/MAI are more accurate and reproducible predictors of stage progression in TaT1 UCs than classic prognostic risk factors and DNA ploidy.     

Phosphohistone‐H3 (PHH3) is prognostic relevant in Merkel cell carcinomas but Merkel cell polyomavirus is a more powerful prognostic factor than AJCC clinical stage,PHH3, Ki‐67 or mitotic indices

Merkel cell carcinomas (MCCs) associated with Merkel cell polyomavirus (MCPyV) have better prognosis than those without MCPyV. The relationship between mitotic index (MI) and MCC outcome has remained elusive because of the difficulty in differentiating mitotic cells from apoptotic ones. We evaluated the role of phosphohistone‐H3 (PHH3) (Ser10), a new mitotic count biomarker, in MCPyV‐positive or ‐negative MCC patients, and assessed its prognostic value in comparison to Ki‐67 labeling index or MI using hematoxylin and eosin (HE) staining. We compared the prognostic value of PHH3 mitotic index with that of MI by HE in 19 MCPyV‐positive and 9 MCPyV‐negative MCC patients. PHH3‐positive immunoreactivity was mostly observed in mitotic figures. Multivariate analysis significantly showed that MCPyV status (HR, 0.004; 95% CI 0.0003–0.058) and the American Joint Committee of Cancer (AJCC) stage (HR, 5.02; 95% CI 1.23–20.51) were observed as significantly independent prognostic factors for OS. PHH3‐positive cell counts/10 HPF was a slightly significant independent prognostic factor for OS (HR, 4.96; 95% CI 0.93–26.55). PHH3‐positive MI and MCPyV status in MCC patients are useful in prognostication, although MCPyV‐infection is a more powerful prognostic factor in MCCs than the AJCC scheme on proliferation or mitotic indices.