Assessment Of Ki67 Research Articles

An international multicenter study to evaluate reproducibility of automated scoring for assessment of Ki67 in breast cancer

The nuclear proliferation biomarker Ki67 has potential prognostic, predictive, and monitoring roles in breast cancer. Unacceptable between-laboratory variability has limited its clinical value. The International Ki67 in Breast Cancer Working Group investigated whether Ki67 immunohistochemistry can be analytically validated and standardized across laboratories using automated machine-based scoring. Sets of pre-stained core-cut biopsy sections of 30 breast tumors were circulated to 14 laboratories for scanning and automated assessment of the average and maximum percentage of tumor cells positive for Ki67. Seven unique scanners and 10 software platforms were involved in this study. Pre-specified analyses included evaluation of reproducibility between all laboratories (primary) as well as among those using scanners from a single vendor (secondary). The primary reproducibility metric was intraclass correlation coefficient between laboratories, with success considered to be intraclass correlation coefficient >0.80. Intraclass correlation coefficient for automated average scores across 16 operators was 0.83 (95% credible interval: 0.73–0.91) and intraclass correlation coefficient for maximum scores across 10 operators was 0.63 (95% credible interval: 0.44–0.80). For the laboratories using scanners from a single vendor (8 score sets), intraclass correlation coefficient for average automated scores was 0.89 (95% credible interval: 0.81–0.96), which was similar to the intraclass correlation coefficient of 0.87 (95% credible interval: 0.81–0.93) achieved using these same slides in a prior visual-reading reproducibility study. Automated machine assessment of average Ki67 has the potential to achieve between-laboratory reproducibility similar to that for a rigorously standardized pathologist-based visual assessment of Ki67. The observed intraclass correlation coefficient was worse for maximum compared to average scoring methods, suggesting that maximum score methods may be suboptimal for consistent measurement of proliferation. Automated average scoring methods show promise for assessment of Ki67 scoring, but requires further standardization and subsequent clinical validation.

Ki-67 and presence of liver metastases identify different progression-risk classes in pancreatic neuroendocrine neoplasms (pNEN) undergoing resection

In pancreatic neuroendocrine neoplasms (pNEN), size ≤2 cm and Ki-67 < 3% suggest indolent behavior, but no factor alone predicts prognosis. We investigated factors predictive of tumor progression in 80 pNENs surgically resected in a single Institution from 1995 to 2015. At multivariable analysis the only two independent variables related to PFS were Ki-67 (HR 2.97; 95%CI 1.26–7.02) and presence of synchronous liver metastases (HR 3.60; 95%CI 1.70–7.61). Using Ki-67 < 3% and M0 as reference, the HR for tumor progression was 3.21 (95%CI 1.18–8.74) for M0 patients with Ki-67 3–20%, 5.06 (2.29–11.2) for M1 patients with Ki-67 ≤ 20% and 24.3 (6.64–89.2) for those with Ki-67 > 20%. Tumor size (≤2 vs. >2 cm) was not a predictive factor at any analysis. Intra-class correlation of Ki-67 values on pre-surgical biopsies vs. surgical specimens was 0.99 and Ki-67 classes were correctly identified in 97% of biopsies.Ki-67 and presence of liver metastases are the major prognostic factors in pNEN and identify different progression risks regardless of tumor size. Pre-surgical pNEN biopsy for Ki-67 assessment should be included in the evaluation of patients with 1–2 cm tumors to help in the decision on whether to perform surgical resection.

Ki 67 assessment in breast cancer in an Egyptian population: a comparative study between manual assessment on optical microscopy and digital quantitative assessment

BackgroundBreast cancer is by far the most frequent cancer among women. The proliferative index, Ki-67, is more and more taken into consideration for treatment decisions. However, the reliability of the established Ki-67 scoring is limited. Digital pathology is currently suggested to be a potential solution to Ki 67 assessment problems.MethodsThis is a retrospective and prospective study including 100 patients diagnosed with invasive breast cancer. Three senior pathologists have been asked to estimate the Ki-67 proliferative index for each of the 100 cases by examining the whole glass slides on optical microscope and providing a continuous score then a categorical score (‘high’ and ‘low’ Ki 67 index) using once 14%, once 20% as threshold indicative of high Ki67 status. Finally, a digital quantitative assessment of Ki67 was performed.ResultsA high inter-observer agreement was found when using optical microscopy for Ki 67 assessment, with correlation coefficient (CC) estimated at 0.878 (p value < 0.01). The overall agreement between manual and automated evaluation of Ki 67 was only substantial (CC estimated at 0.745 (p value < 0.01)). When using categorical scores, the inter-observers concordance was substantial using both cutoff points with kappa value estimated at 0.796 ([0.696–0.925] while using 14% as a cut off point and at 0.766 ([0.672–0.938] while using 20% as a cutoff point (p value < 0). The inter-observers agreement was better while using 14% as cutoff point. Agreement between manual and automated assessment of Ki 67 indices using both cutoff points was only substantial (Kappa estimated at 0.623, p value < 0.01). In comparison to automated assessment of Ki 67 index, while using 14% as a cutoff point, the overall tendency of all observers was to overestimate the Ki 67 values but to underestimate the proliferation index while using 20% as a cutoff point.ConclusionAutomated assessment of Ki 67 value would appear to be comparable to visual Ki 67 assessment on optical microscopy. Such study would help define the role of digital pathology as a potential easy-to use tool for a robust and standardized fully automated Ki 67 scoring.

Abstract B025: The proteome of prostate cancer bone metastases

Abstract Bone is the most predominant site of distant metastasis in prostate cancer. Patients who develop bone metastases have limited therapeutic options and poor prognosis. Understanding the cellular and molecular changes occurring at this stage is critical to find new treatments and to stratify patients for personalized therapies. In the current study, we used a system-wide quantitative proteomic approach in order to identify (i) cellular alterations occurring during the transition from localized to metastasized prostate cancer and (ii) phenotypic subgroups and potential novel targets for treatment of metastatic prostate cancer. Mass spectrometry-based quantitative proteomic profiling was applied to paraffin-embedded, formalin-fixed bone metastasis tissue from prostate cancer patients (n=22) operated to relieve spinal cord compression. At the time of surgery, 5 patients were previously untreated, 16 had relapsed after androgen-deprivation therapy (ADT), and 1 was under early ADT. Mechanisms behind disease progression were identified through comparative analysis with proteomic profiles of localized prostate cancer (n=26) and adjacent normal tissue (n=8) obtained from radical prostatectomies. Expression levels of selected proteins were verified by immunohistochemical analysis of metastases from our extended bone-metastasis cohort. A mean of 5,067 proteins were identified and quantified per tumor. Compared to primary prostate tumors (1) bone metastases showed increased levels of proteins involved in cell cycle progression, DNA damage response, RNA processing, lipid transport, and fatty acid β-oxidation as well as reduced levels of proteins involved in cell adhesion and carbohydrate metabolism. Based on their proteomes, prostate cancer metastases were more heterogeneous (ρ:0.64±0.077) than localized tumors (ρ:0.8±0.037). We identified two phenotypically different subgroups of metastatic tumors: (i) those expressing higher levels of androgen receptor canonical targets together with elevated levels of mitochondrial and Golgi apparatus resident proteins and (ii) those with increased expression of proliferation and DNA repair-related proteins. Importantly, the two subgroups were seen among both treatment-naïve and castration-resistant cases and thus unrelated to previous treatment. The inverse correlation between tumor cell proliferation and metabolically/secretory active tumor cells was verified by assessment of Ki67 and PSA immunoreactivity in 78 bone metastases (Rs=-0.36, P=0.001). Identification of novel therapeutic targets/strategies for the two subgroups is ongoing. In conclusion, this work describes the first system-wide quantitative characterization of the proteome in prostate cancer bone metastases. By deep proteomic analysis we revealed the existence of at least two different subtypes of prostate cancer bone metastasis according to proliferative and metabolic features, suggesting that personalized therapies could be considered based on molecular signatures in individual cases. Reference: 1. Iglesias-Gato D, et al. The proteome of primary prostate cancer. Eur Urol 2016:69:942-52. Citation Format: Diego Iglesias-Gato, Elin Thysell, Sead Crnalic, Matthias Mann, Anders Widmark, Anders Bergh, Amilcar Flores-Morales, Pernilla Wikström. The proteome of prostate cancer bone metastases [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B025.

GDF9-β promotes folliculogenesis in sheep ovarian transplantation onto the chick embryo chorioallantoic membrane (CAM) in cryopreservation programs

Ovarian tissue (OT) cryopreservation is a treatment option for fertility preservation among young cancer patients. However, the procedure may involve a reduction in the GDF9-β expression and a delay in follicular growth after thawing and transplantation. The aim of this study was to evaluate whether supplementation of GDF9-β can compensate the reduction of this factor during the cryopresevation process and promote folliculogenesis after transplantation of thawed sheep ovarian tissue. Sheep OT was cryopreserved using two methods of vitrification and slow freezing. Fresh and thawed OTs were then transplanted onto chick embryo chorioallantoic membrane (CAM) and then divided into two groups based on the addition of GDF9-β to the grafted tissue. After 5days of culture, both histological and immunohistological (Ki-67) assessments were performed to evaluate follicular structure, development, and proliferation. The fibrotic and necrotic areas were measured using MICROVISIBLE software. Folliculogenesis took place in all culture groups, but was significantly improved only in the +GDF9-β cultured group. Also, better follicular structure was preserved in the aforementioned group (p < 0.05). When GDF9-β was supplemented to the culture medium, more neovascularization (p < 0.05) and better transplantation (p > 0.05) was observed. Furthermore, the areas of fibrosis and necrosis were lower in this group rather than the controls. Follicular proliferative activity was significantly higher only in the slow freezing +GDF9-β cultured group. GDF9-β, as a stimulatory factor, not only promoted the folliculogenesis in the fresh ovarian transplant, but also compensated for its reduction during the cryopreservation process.

Ki-67 expression in mature B-cell neoplasms: a flow cytometry study.

Ki-67 is a nuclear protein associated with cellular proliferation in normal or leukemic conditions that can help identify more aggressive diseases and is usually evaluated with immunohistochemistry. The aim of this was to assess Ki-67 expression on mature B-cell neoplasms samples with flow cytometry immunophenotyping. After surface staining with CD19 and CD45, intracellular staining for Ki-67 was performed in leukemic mature B-cells. Ki-67 expression was evaluated with flow cytometry. Ki-67 expression was higher in mantle cell lymphoma, Burkitt lymphoma, and diffuse large B-cell lymphoma cases. It was also associated with CD38 mean fluorescence intensity. Ki-67 expression evaluated by flow cytometry can be a useful tool in the diagnosis of mature B-cell neoplasms. More studies are needed to validate Ki-67 assessment with flow cytometry immunophenotyping.

Assessment of Ki-67 for Predicting Effective Prognosis in Breast Cancer Subtypes

Assessment of Ki-67 for Predicting Effective Prognosis in Breast Cancer Subtypes

Assessment of Ki-67 as a potential biomarker in patients with breast cancer.

Breast cancer is the most common cancer in females, it accounts for one third of all malignancies affecting women. Appropriate biomarkers play significant role in predicting the prognosis and decide the specific therapy to each patient. In this study we aimed at evaluating the value of Ki-67 as a prognostic marker in breast cancer patients and to analyze the associations between Ki-67 and their clinicopathological parameters. This study included 92 patients with developed non metastatic breast cancer and 10 women had benign breast tumor served as controls. We measured the serum level by ELISA technique and tissue expression of Ki-67 by immunohistochemical technique. Our results showed that there were no statistically significant differences in serum Ki-67 levels between the two studied groups. As for Ki-67expression in breast cancer cells, the score increases with increase of tumor size, grade, premenopausal, Ki-67 expression in estrogen and progesterone receptor positive tumors showed lower values than estrogen and progesterone negative tumors, while higher Ki-67 expression was more frequently associated with HER2-positive. In conclusion; our study supports the finding that tissue Ki-67 expression may add prognostic information to that obtained from classical prognostic factors and can provide data of significant value to other important prognostic indicators such as pathological grading, and axillary lymph node involvement.

Assessment of Ki67 in relation to radiotherapy (RT) fractionation and prognosis in the CHHiP (CRUK/06/016) trial.

98 Background: A uniform fractionation schedule is used to deliver external beam RT for localised prostate tumours Individualising fractionation according to tumour biology may improve outcomes. In addition recurrence rates after RT vary considerably and better prognostic markers are needed to guide treatment choices. This study aims to identify if the cell proliferation marker Ki67 predicts response to RT fractionation in CHHiP, a randomised trial of 3 RT fractionation schedules. It also aims to identify if Ki67 predicts prognosis. Methods: A matched case:control study design was used, patients with biochemical or clinical failure &gt; 2 years after RT (BCR) were matched to patients without recurrence according to established prognostic factors (Gleason score, PSA, Tumour-stage) and fractionation schedule. Immunohistochemical (IHC) staining of diagnostic biopsy sections was carried out using the MIB1 Ki67 antibody. Two independent investigators scored Ki67 using the unweighted global method (1) to derive a mean and maximum percentage of cells staining positive (mean Ki67 and maximum Ki67 respectively). Conditional logistic regression models were fitted with interaction terms between the biomarker and the fractionation schedules to determine whether Ki67 predicted BCR according to fractionation. Secondly models were fitted using the entire case:control study sample to estimate the prognostic value of Ki67 on risk of BCR. Results: Ki67 results were available for 173 matched pairs. The interaction terms between Ki67 and the fractionation schedules were not significant. However mean and maximum Ki67 were significant prognostic markers for BCR in a model adjusted for established prognostic factors. Conditioning on matching variables and age, the odds of BCR is estimated to increase by 9% per 1 point increase in mean Ki67 (OR = 1.09, 95%CI:1.04–1.15, p = 0.001). Conclusions: Ki67 did not predict BCR according to fractionation schedule in CHHiP, however it did predict prognosis independently of established prognostic factors. Additional IHC biomarkers are under evaluation.

Abstract P1-06-11: Prediction of oncotype DX® results based on local gene expression measurements by MammaTyper®

Abstract Background: Oncotype DX® recurrence score (RS) has emerged as a recommended risk classifier for patients with ER+/HER2- early-stage breast cancer. While RS is one of the most rigorously studied risk scores, it is also one of the most expensive tests, thus remaining beyond reach for a many patients. The necessity for an affordable method for estimating risk of recurrence has motivated investigations on the correlation between RS and traditional parameters such as IHC for ER, PR and Ki67. However, semi-quantitative IHC lacks standardization across different laboratories especially for Ki67. In this study we therefore investigated whether the standardized assessment of HER2, ER, PR, and Ki67 on mRNA level could better serve for prediction of low risk RS cases. Methods: ERBB2, ESR1, PGR and MKI67 mRNA expression was measured by RT-qPCR in extracts from FFPE breast cancer samples using the MammaTyper® test. Complete data for RS, IHC, grading and mRNA measurement was available for 198 samples. Tumor subtypes according to St Gallen surrogate definition from 2013 were assigned based on binary mRNA marker classification (pos/neg) according to pre-defined cut-offs. Subtype results were compared to RS risk classes based on commercial and TAILORx-trial cut-offs. RS low risk classification (RS ≤25) based on four IHC markers and grading was estimated using the online tool breastrecurrenceestimator.onc.jhmi.edu and compared to observed RS classes. Finally, the prediction of continuous RS values by mRNA or semi-quantitative IHC measurement was compared by linear regression and subsequent ROC analysis of prediction models. Results: The distribution of RS risk classes in the set of samples with full data was 21% RS 0-10, 39% RS 11-17, 27% RS 18-25, 7% RS 26-30 and 7% RS &amp;gt;30. MammaTyper® called 38% (76) of the samples as Luminal A-like. From these samples 70% and 99% had RS values below 18 and 25 respectively. Only 1 MammaTyper® Luminal A-like sample had an RS &amp;gt;30. Estimation of RS according to the online tool resulted in classification of 61% (121) of the samples as low risk (RS ≤25). Of these 74% and 98% of samples had observed RS values below 18 and between 18 and 25 respectively. 2 and 1 samples called as low risk by the online tool had an RS of 26-30 and &amp;gt;30 and, respectively. In linear regression analysis of IHC against RS only PR and Ki67 were significant predictors (p-values &amp;lt;0.0001 and 0.0128) while when using mRNA values ESR1, PGR and MKI67 were found as predictors of RS in the multivariate model (all p-values &amp;lt;0.0001). On a training set (67% of samples) the IHC based prediction model was correlated to the observed RS with an R2 of 0.305 whereas the mRNA based model achieved an R2 of 0.489. When the models were applied to training and validation dataset (33% of samples) for prediction of an RS &amp;gt;25 result, the IHC based model had AUCs of 0.887 and 0.836, respectively, while the mRNA based model achieved AUCs of 0.909 and 0.899, respectively. Conclusion: mRNA based prediction of RS was considerably better than prediction based on IHC. As Ki67 IHC standardization is reaching its limits, local gene expression measurements with their high degree of standardization could serve as a safer way for prediction of Oncotype low risk results. Citation Format: Lehr H-A, Aulmann S, Laible M, Etzrodt A, Hartmann K, Gürtler C, Sahin U, Varga Z. Prediction of oncotype DX® results based on local gene expression measurements by MammaTyper® [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-06-11.

Abstract PD5-04: Ki67 changes and PEPI score in the LORELEI trial: A phase II randomized, double-blind study of neoadjuvant letrozole plus taselisib versus letrozole plus placebo in postmenopausal women with ER-positive/HER2-negative early-stage breast cancer

Abstract Background: Taselisib is an oral, potent, selective inhibitor of Class I PI3-kinase (PI3K) alpha, gamma, and delta isoforms with enhanced activity against PIK3CA mutant cancer cells. LORELEI trial demonstrated a significant improvement in ORR (objective response rate) centrally assessed by MRI with neoadjuvant taselisib plus letrozole compared to letrozole plus placebo in all randomized patients as well as in the PIK3CA mutant cohort (Saura et al, ESMO 2017). Methods: 334 postmenopausal women with newly diagnosed ER+/HER2-, untreated, Stage I-III operable breast cancer and evaluable tumor tissue for PIK3CA genotyping were randomized (1:1) to receive daily letrozole (2.5 mg) with either taselisib (4 mg on a 5 days on/ 2 days off schedule) or placebo for 16 weeks, followed by surgery. Tumor tissue collection was performed at baseline, week 3 (W3) and at surgery. Secondary objectives included, but were not restricted to, ORR assessed by MRI in patients with PIK3CA wild type (WT) tumors, ORR using alternative methods of tumor assessment (ultrasound, mammogram and clinical breast exam) in all patients and patients with PIK3CA mutant and WT tumors, central assessment of Ki67 at different timepoints (baseline, W3 and surgery), and the centrally derived PEPI score. Central Ki67 was assessed by two independent readers blinded to treatment arms and PIK3CA status (Vall D'Hebron Institute of Oncology, Barcelona). Results: ORR by centrally assessed MRI was similar in the two treatment arms in patients with PIK3CA WT tumors (45.7 vs 40.4% for taselisib and placebo, respectively). ORR assessed by breast US was also significantly higher with taselisib compared to placebo in all randomized patients and in the PIK3CA mutant cohort. The highest concordance rate between MRI and other imaging modalities was found with breast ultrasound (53.7%). Centrally assessed Ki67 changes are reported in Table 1. Ki67 values decreased from baseline to W3 and from baseline to surgery in both treatment arms. No significant differences in the decrease of Ki67 values between treatment arms were detected. Unplanned analysis of Complete Cell Cycle Arrest (CCCA) at W3 was numerically higher with taselisib than with placebo in all randomized patients (49.6% vs 38.5%) and in the PIK3CA mutant cohort (60.9% vs 47.5%). Due to the variability in timing between the last dose of taselisib (median time 11 days; interquartile range 6-16 days) and tissue collection at surgery, considering the half-life of taselisib of approximately 40 hours, centrally derived PEPI score is not interpretable. Ki67 proportional changes, %Taselisib + letrozolePlacebo + letrozoleBaseline to W3All patients-83.8-80.4PIK3CA mutant-84.5-79.1PIK3CA WT-82.8-81.1Baseline to surgeryAll patients-75.6-80.5PIK3CA mutant-71.9-79.9PIK3CA WT-78.2-81.2 Conclusion: Among the investigated alternative methods for assessing ORR, breast ultrasound performed similar to MRI. Decrease in the Ki67 values from baseline to W3 and to surgery were observed in both treatment arms. The time interval between taselisib cessation and tissue collection at surgery are being further investigated. Clinical trial information: NCT02273973 Citation Format: de Azambuja E, Saura C, Nuciforo P, Frantal S, Oliveira M, Zardavas D, Jallitsch-Halper A, de la Pena L, Dubsky P, Lombard JM, Vuylsteke P, Castaneda Altamirano C, Sanchez C, Ballestrero A, Colleoni M, Santos Borges G, Ciruelos E, Bardia A, Fornier M, Boer K, Wilson TR, Stout TJ, Hsu JY, Shi Y, Piccart M, Baselga J, Gnant M. Ki67 changes and PEPI score in the LORELEI trial: A phase II randomized, double-blind study of neoadjuvant letrozole plus taselisib versus letrozole plus placebo in postmenopausal women with ER-positive/HER2-negative early-stage breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD5-04.

The use of automated Ki67 analysis to predict Oncotype DX risk-of-recurrence categories in early-stage breast cancer.

Ki67 is a commonly used marker of cancer cell proliferation, and has significant prognostic value in breast cancer. In spite of its clinical importance, assessment of Ki67 remains a challenge, as current manual scoring methods have high inter- and intra-user variability. A major reason for this variability is selection bias, in that different observers will score different regions of the same tumor. Here, we developed an automated Ki67 scoring method that eliminates selection bias, by using whole-slide analysis to identify and score the tumor regions with the highest proliferative rates. The Ki67 indices calculated using this method were highly concordant with manual scoring by a pathologist (Pearson’s r = 0.909) and between users (Pearson’s r = 0.984). We assessed the clinical validity of this method by scoring Ki67 from 328 whole-slide sections of resected early-stage, hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer. All patients had Oncotype DX testing performed (Genomic Health) and available Recurrence Scores. High Ki67 indices correlated significantly with several clinico-pathological correlates, including higher tumor grade (1 versus 3, P<0.001), higher mitotic score (1 versus 3, P<0.001), and lower Allred scores for estrogen and progesterone receptors (P = 0.002, 0.008). High Ki67 indices were also significantly correlated with higher Oncotype DX risk-of-recurrence group (low versus high, P<0.001). Ki67 index was the major contributor to a machine learning model which, when trained solely on clinico-pathological data and Ki67 scores, identified Oncotype DX high- and low-risk patients with 97% accuracy, 98% sensitivity and 80% specificity. Automated scoring of Ki67 can thus successfully address issues of consistency, reproducibility and accuracy, in a manner that integrates readily into the workflow of a pathology laboratory. Furthermore, automated Ki67 scores contribute significantly to models that predict risk of recurrence in breast cancer.

Expression of chosen cell cycle and proliferation markers in pancreatic intraepithelial neoplasia.

IntroductionPancreatic ductal adenocarcinoma is one of the most aggressive tumours that develops from precursor lesions, most frequently including pancreatic intraepithelial neoplasia (PanIN). Deregulation of the cell cycle, responsible for uncontrolled cell proliferation, is an important phenomenon in the development of this cancer.AimTo evaluate the cell cycle and the expression of proliferation markers, namely Ki67, PCNA, and cyclin D1 in pancreatic intraepithelial neoplasia at its different stages of progression.Material and methodsThe study group consisted of 70 patients with different pancreatic diseases (pancreatic ductal adenocarcinoma, pancreatitis, and pancreatic cysts), who also had pancreatic intraepithelial neoplasia. Expression of Ki67, PCNA, and Cyclin D1 was analysed immunohistochemically using appropriate antibodies.ResultsStatistically significant differences were demonstrated in Ki67, PCNA, and Cyclin D1 expression between normal pancreatic ducts and various stages of PanIN (p < 0.001). Expression of these proteins increased from normal pancreas to PanIN 1, 2, and 3. Expression of these proteins was higher in stages PanIN 1, 2, and 3 compared to normal pancreas. The expression of Ki67, PCNA, and cyclin D1 was associated with age (p < 0.001), Ki67 and PCNA with sex (p < 0.001), and PCNA with the type of primary disease (p = 0.031). Simultaneously, a directly proportional relationship was established between the expression of all proteins examined (p < 0.001).ConclusionsAn increase in the expression of Ki67, PCNA, and cyclin D1 suggests that these proteins may enhance epithelial cell proliferation and may influence the development of pancreatic intraepithelial neoplasia. Moreover, immunohistochemical assessment of Ki67, PCNA, and cyclin D1 expression may be helpful in the differential diagnosis of PanIN.

Comparison of digital and visual methods for Ki-67 assessment in invasive breast carcinomas

to compare two methods for quantitative assessment of the proliferative activity index (PAI): a visual estimation method by several investigators and digital image analysis (DIA). The use of the Ki-67 index in the daily clinical practice of a Morbid Anatomy Department is associated with the problem of reproducibility of quantitative assessment of the Ki-67 PAI. Due to the development of digital imaging techniques in morphology, new methods for PAI evaluation using the DIA are proposed. The Ki-67 PAI data obtained during visual assessment and digital image analysis were compared in 104 cases of grades 2-3 breast carcinoma. The histological sections were scanned using a Panoramic III scanner (3D Histech, Hungary) and digital images were obtained. DIA was carried out using the software 3D Histech QuantCenter (3D Histech, Hungary), by marking 3-10 zones. Evaluation of the obtained sections was done independently by two investigators engaged in cancer pathology. The level of agreement between visual and digital methods did not differ significantly (p>0.001). The authors selected a gray area in the range of 10-35% IPA, where the Ki-67 index showed a weak relationship between the analyzed groups (ICC, 0.47). The Ki67 index below 10% and above 35% showed a sufficient reproducibility in the same laboratory. The authors consider that the scanned digital form of a histological section, which can be evaluated using automated software analysis modules, is an independent and objective method to assess proliferative activity for Ki-67 index validation.

The reliability of assessment of Ki-67 and HER-2/neu expression on breast carcinoma agarose cell blocks

Background Assessment of multiple prognostic and/or predictive biomarkers in patients with breast cancer with good reliability and reproducibility using samples obtained by fine-needle aspiration (FNA) can be of significant clinical value. Objective The objective of this study was to determine the reliability of assessment of breast carcinoma markers, Ki-67 and HER-2/neu, on agarose cell blocks (CBs). Patients and methods FNA samples were obtained from 29 female patients presenting with primary breast carcinoma. To prepare CBs (cytoblocks), cells were fixed in 10% neutral formalin for 6–12 h, embedded in 2% agarose gel, processed using standard laboratory protocol for biopsy tissue, and paraffin-embedded. The expression of Ki-67 and HER-2/neu was evaluated by immunohistochemistry (IHC) on cytoblocks and their corresponding formalin-fixed, paraffin-embedded histological resection specimens. Results Tissue sections exhibited high Ki-67 expression in 27/29 (93.1%) invasive breast carcinoma cases, whereas 2/29 (6.9%) tumors revealed low Ki-67 immunoreactivity. MIB-1-stained CBs showed high Ki-67 expression in 24/29 (82.7%) breast carcinoma cases, whereas 5/29 (17.3%) tumors revealed low expression. MIB-1 IHC staining on agarose CBs showed 26/29 (89.6%) tumors correlation with tissue results. IHC analysis of HER-2/neu protein expression on tissue sections revealed that 23/29 (79.3%) breast carcinoma cases were negative for overexpression (score 0 or 1+) and 6/29 (20.7%) were positive for overexpression (3+). On agarose CBs, 21/29 (72.4%) invasive breast carcinoma tumors were negative (0 or 1+) for HER-2/neu overexpression, 2/29 (6.9%) showed equivocal IHC staining (2+), and 6/29 (20.7%) tumors were positive (3+). IHC staining for HER-2/neu on agarose CBs showed 93.1% correlation with tissue results. Conclusion In invasive breast carcinoma, Ki-67 and HER-2/neu (negative/positive) expression could be determined with good reliability on agar CBs prepared from FNA using standard IHC procedures.

Anti-tumor Effect of Rhaponticum uniflorum Ethyl Acetate Extract by Regulation of Peroxiredoxin1 and Epithelial-to-Mesenchymal Transition in Oral Cancer.

Objective: To explore whether Rhaponticum uniflorum (R. uniflorum) had anti-tumor effects in oral cancer and investigate the molecular mechanisms involved in these anti-tumor effects.Methods: Chemical compositions of R. uniflorum ethyl acetate (RUEA) extracts were detected by ultra-performance liquid chromatography-Q/time-of-flight mass spectrometry (UPLC-Q/TOF-MS), followed by pharmacology-based network prediction analysis. The effects of RUEA extracts on proliferation, apoptosis, migration, and invasion ability of human oral squamous cell carcinoma (OSCC) cell line SCC15 were evaluated by CCK8 assay, Annexin V- fluorescein isothiocyanate/propidium iodide staining, wound healing assay, and Matrigel invasion assay, respectively. The mRNA and protein expression of peroxiredoxin1 (Prx1), the epithelial-to-mesenchymal transition (EMT) marker E-cadherin, vimentin, and Snail were determined by quantitative real-time reverse transcription polymerase chain reaction and western blotting. A mouse xenograft model of SCC15 cells was established to further evaluate the effect of RUEA extracts in vivo. Immunohistochemical assessment of Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling staining of apoptotic cells were performed on the tumor tissues to assess the effects of RUEA extracts on proliferation and apoptosis.Results: Fourteen compounds were identified from RUEA extracts by UPLC-Q/TOF-MS. The pharmacology-based network prediction analysis showed that Prx1 could be a potential binder of RUEA extracts. In SCC15 cells, RUEA extracts inhibited cell viability, induced apoptosis, and suppressed cell invasion and migration in a concentration-dependent manner. After treatment with RUEA extracts, the mRNA and protein expression of E-cadherin increased, whereas those of Prx1, vimentin, and Snail decreased. RUEA extracts also affected the EMT program and suppressed cell invasion and migration in Prx1 knockdown SCC15 cells. In an OSCC mouse xenograft model, RUEA extracts (25 and 250 mg/kg) significantly inhibited the growth of tumors. Compared with the control group, Ki67 expression was reduced and apoptosis rates were elevated in the transplanted tumors treated with RUEA extracts. RUEA extracts increased the expression of E-cadherin and decreased the expression of Prx1, vimentin, and Snail in vivo.Conclusion: RUEA extracts inhibited tumor growth and invasion by reducing Prx1 expression and suppressing the EMT process in OSCC. RUEA extracts may be a potential candidate for OSCC treatment.

The Reliability of Core-Needle Biopsy in Assessment of Hormone Receptor, HER2, and Ki-67 in Breast Carcinoma

Objective: The purpose of this study was to compare the estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2) status and Ki-67 index by immunohistochemical (IHC) analysis in breast carcinoma to determine the level of concordance between core-needle biopsy (CNB) and surgical specimens. Summary of Background: Accurate preoperative diagnosis of a breast lesion has recently been considered essential to the treatment strategy to achieve optimal treatment without delay. However, the reliability of using CNB specimens for IHC assessment is in relatively small number of cases and differing results between previous studies. Methods: The patients included in this study were 255 patients with primary breast carcinoma who had CNB and subsequent surgical resection at the Hospital of Dokkyo Medical University between 2010 and 2016. We compare the ER, PgR, HER2 status, and Ki-67 index by IHC analysis in breast carcinoma between CNB and surgical specimens. Results: There was a concordance rate between the ER, PgR, HER2, and Ki-67 IHC assessment of CNB and surgical specimens in 99.0%, 92.1%, 86.3%, and 91.5%, respectively. We also found small numbers of discordant cases in the estimation for which a discrepancy in determination led to a change in treatment. Conclusions: Our results do not entirely invalidate the use of CNB for assessment if they are the only source of tumor tissue available, but suggest a more cautious approach in their interpretation when clinical decisions are being made.

Assessment of Ki67 and uPA/PAI-1 expression in intermediate-risk early stage breast cancers

BackgroundThe objective of this study was to compare the efficacy of biomarkers in assessing the risk of breast cancer recurrence in patients with node-negative or micrometastatic grade II breast cancer. Specifically, we compared risk assessments based on the St. Gallen clinicopathological criteria, Ki67 expression and urokinase plasminogen activator (uPA)/plasminogen activator inhibitor-1 (PAI-1) expression.MethodsThis retrospective study included 347 patients with breast cancer followed at Limoges University Hospital. The optimal cut-off for high Ki67 expression (Ki67hi) was established as 20%. The threshold for uPA and PAI-1 positivity was 3 ng/mg and 14 ng/mg, respectively.ResultsKi67 expression was lower in uPA/PAI-1-negative than in uPA/PAI-1-positive tumours (227 tumours; P = 0.04). The addition of Ki67 status to the St. Gallen criteria resulted in a 28% increase in the rate of identification of high-risk tumours with a potential indication for chemotherapy (P < 0.001). When considering uPA/PAI-1 levels together with the St Gallen criteria (including Ki67 expression), the number of cases identified as having a high recurrence risk with a potential indication for adjuvant chemotherapy increased by 20% (P < 0.001). Adjuvant chemotherapy was 9% less likely to be recommended by a multidisciplinary board when using the current criteria compared with using a combination of the St. Gallen criteria and Ki67 and uPA/PAI-1 status (P = 0.03).ConclusionsTaken together, our data show discordance among markers in identifying the risk of recurrence, even though each marker may prove to be independently valid.

Cyto-histology in NET: what is necessary today and what is the future?

The carcinoid as originally described is part of the relatively large family of neuroendocrine neoplasia found in almost every organ. Historical reasons back their current definitions. Neuroendocrine cancer is most frequently observed in the lung and the digestive tract. In the lung is defined as carcinoid (typical and atypical) for well differentiated, low to intermediate grade, and small cell and large cell neuroendocrine carcinoma for poorly differentiated, high grade. In the digestive system are respectively defined as neuroendocrine tumor (NET) and neuroendocrine carcinoma (NEC) of small and large cell types. Grading and staging are developed for their clinical classification by the World Health Organization (WHO) and the American Joint Committee on Cancer (AJCC). In both anatomical sites the morphological features are overlapping, with bland histology for carcinoid and NET, and aggressive features with extensive necrosis, severe atypia and abundant, atypical mitoses for high grade cancer types. Such features are also essential diagnostic clues in cytological preparations. The confirmation of the neuroendocrine signature by immunohistochemistry is mandatory for the diagnosis; a minimum panel comprising chromogranin A and synaptophysin is recommended in the digestive system. In addition, the application of grading requires the mitotic count and or spotty necrosis assessment for lung, or the mitotic count and the Ki67 assessment in the digestive system.

Interlaboratory variability of Ki67 staining in breast cancer

BackgroundPostanalytic issues of Ki67 assessment in breast cancers like counting method standardisation and interrater bias have been subject of various studies, but little is known about analytic variability of Ki67 staining between pathology labs. Our aim was to study interlaboratory variability of Ki67 staining in breast cancer using tissue microarrays (TMAs) and central assessment to minimise preanalytic and postanalytic influences. MethodsThirty European pathology labs stained serial slides of a TMA set of breast cancer tissues with Ki67 according to their routine in-house protocol. The Ki67-labelling index (Ki67-LI) of 70 matched samples was centrally assessed by one observer who counted all cancer cells per sample. We then tested for differences between the labs in Ki67-LI medians by analysing variance on ranks and in proportions of tumours classified as luminal A after dichotomising oestrogen receptor–positive cancers into cancers showing low (<14%, luminal A) and high (≥14%, luminal B HER2 negative) Ki67-LI using Cochran's Q. ResultsSubstantial differences between the 30 labs were indicated for median Ki67-LI (0.65%–33.0%, p < 0.0001) and proportion of cancers classified as luminal A (17%–57%, p < 0.0001). The differences remained significant when labs using the same antibody (MIB-1, SP6, or 30-9) were analysed separately or labs without prior participation in external quality assurance programs were excluded (p < 0.0001, respectively). ConclusionSubstantial variability in Ki67 staining of breast cancer tissue was found between 30 routine pathology labs. Clinical use of the Ki67-LI for therapeutic decisions should be considered only fully aware of lab-specific reference values.