Immunocytochemical Staining For the C-Erbb-2 Gene Product In Breast Aspirates: A Preliminary Report

The results of the determination of c-erbB-2 gene expression by immunocytochemical staining of cytological aspirates, prepared by cytocentrifugation, have been compared with paraffin-embedded tissue sections from the same tumour. Our results show equivalent staining in 20/22 cases, with six cases being both scored positive and fourteen cases being both negative. Two samples gave conflicting results. One case was scored as being positive on the cytological aspirate, whereas in the tissue sections taken from the same tumour positive staining was only seen in areas of non-invasive intraduct carcinoma. This sample was scored as being negative. In another case, cytoplasmic staining with less than 50% of the cells showing any positivity was observed in the cytospin sample, with negative staining in the corresponding tissue section. We conclude that expression of c-erbB-2 immunostaining is detectable on cytological preparations prepared by cytocentrifugation but must be interpreted with caution in tumours which may have a large intraduct component or which give predominant cytoplasmic staining.     

p53 Protein expression and proliferative activity in ductal breast carcinomas

The immunocytochemical expression of p53 protein and Ki-67 labelling index in tumour cells of 100 ductal breast carcinomas of different histological grade and stage was evaluated in cytological material. In order to investigate p53 expression and Ki-67 expression an avidin-extravidin immunocytochemical technique was applied to imprints. Monoclonal antibody (MoAb) DO-p53 and proliferating cell monoclonal antibody were used as primary antibodies. A statistically significant difference was observed between p53 protein expression and grade of malignancy and clinical stage (P = 0.001, P < 0.001, respectively). A statistically significant difference was also observed between Ki-67 LI and histological grade and stage of the tumours (P < 0.001, P < 0.001 correspondingly). A correlation was observed between p53 protein expression and Ki-67 LI (P < 0.001). The immunocytochemical study of p53 protein and Ki-67 expression in cytological material represents a simple method which can be applied in routine cytological laboratories for the investigation of potential malignancy of ductal breast cancer.     

Assessment of cell proliferation by Ki-67 staining and flow cytometry in fine needle aspirates (FNAs) of reactive lymphadenitis and non-Hodgkin's lymphomas

This study was undertaken to assess cell proliferation in FNAs from a series of 57 non-Hodgkin's lymphomas (NHL) and 11 cases of reactive lymphadenitis using Ki-67 staining and flow cytometry (FCM). The results were compared and correlated to the cytomorphological subgrouping according to Kiel classification. The mean percentages of Ki-67 positivity were 16.6% and 61.1% for low and high grade lymphomas, respectively (P < 0.001). The mean S-phase fraction (SPF) determined by FCM was 4.61% for low grade and 12.9% for high grade lymphomas (P < 0.001). The figures for Ki-67 positivity and S-phase fraction in reactive lymphadenitis were 16.8% and 40%, respectively. We observed a strong correlation in low grade lymphomas between Ki-67 and SPF. A good correlation was also found in reactive lymphadenitis. In high grade lymphomas, however, with highly scattered Ki-67 and S-phase values, this correlation was lost. In some cases this discrepancy can be explained by a rich admixture of non-neoplastic, non-proliferating cells in aspirates from diploid tumours. In addition, the existence of a minor aneuploid tumour cell population of high proliferation such as that in Ki-1 lymphomas will not be accurately analysed by FCM but is easily assessed by Ki-67 staining. However, the main reason seems to be a high variability between the fraction of cells in S-phase and the total number of cells in G1, S and G2 in individual tumours.     

Cell proliferation in human tumour xenografts: measurement using antibody labelling against bromodeoxyuridine and Ki-67

Cell proliferation was investigated in human tumour xenografts using bromodeoxyuridine (BrdUrd) labelling, evaluated either by flow cytometry or in tissue sections, and also using the proliferation marker Ki-67. BrdUrd labelling was found to increase when cryostat tumour sections were digested with an enzymic solution. This yielded a labelling index up to four times higher than that obtained using the flow cytometer. Ki-67 indices were found to be higher than those reported for human tumour biopsies, as may be expected due to the enhanced growth rate of the xenografts. Significant heterogeneity was observed in the results for cervix, breast and bladder tumours, and the results of the three methods were poorly correlated. However, three of the four tumour types showed that the tumour with the lowest Ki-67 index also had the longest potential doubling time. Since the measurement of Ki-67 index was found technically easier to perform, and also adequately reflects relative tumour cell proliferation, it is preferred over the other techniques.     

Preliminary study on oncogene product immunohistochemistry (c-erbB-2, c-myc, ras p21, EGFR) in breast pathology.

The expression of oncogene products related to cell growth (c-erbB-2, c-myc, ras p21, EGFR) was investigated in benign (15 cases) and malignant breast lesions (20 cases) by means of immunohistochemistry using the avidin-biotin-peroxidase technique with polyclonal and monoclonal antibodies. The aim of this study was to evaluate the relationship between the staining positivity and various morphological and biological features, such as tumour type, grading, hormone receptor status and cell kinetic parameters. In benign breast lesions, as expected, the kinetic parameters were low, both for Ki-67 and LI. All the specimens showed a diploid condition (the DI being equal to 1) and we found a limited degree of immunoreactivity for all the growth factors and oncogene products. In breast cancer we studied the distribution of immunohistochemical positivity for EGFR, c-erbB-2, c-myc, ras p21 and Ki-67, which was related to age, nodal status, ER and PgR receptor status, LI, DI and histopathological grading. A significant positive correlation was found both between ras p21 expression and nodal status and ER-ICA positivity. We observed a strong correlation between LI and Ki-67 and an inverse relation between Ki-67 and ER expression. These findings suggest the importance of studying the relationship between prognostic factors which may provide preoperative prediction in the biological behaviour of breast cancer, not only on biopsy specimens, but also on fine needle aspirates.     

Necrotic Debris In Thyroid Aspirates: A Feature of Follicular Carcinoma of the Thyroid

Fine needle aspirates from 44 follicular thyroid tumours (30 adenomas, 14 carcinomas) have been studied. All aspirates contained neoplastic cells in follicular and trabecular arrangements. The individual tumour cells showed varying degrees of anisonucleosis and nuclear pleomorphism. Colloid was scanty or absent from all smears. Granular or filamentous necrotic material was observed in both biopsies and smears from one moderately and two poorly differentiated follicular carcinomas, but in none of the adenomas. This suggests that necrotic debris may be a feature of follicular carcinoma of the thyroid.     

Prediction of aggressiveness of gastrointestinal stromal tumours based on immunostaining with bcl-2, Ki-67 and p53

OBJECTIVE: While the use of fine needle aspiration (FNA) in the diagnosis of gastrointestinal stromal tumours (GISTs) is well-established, it can be difficult to predict the prognosis of GIST based on morphology alone. The objective of the current study was to determine if expression of bcl-2, Ki-67 and p53 correlated with the outcome of GISTs based on cytological material. METHODS: Cell-blocks from 14 GISTs diagnosed by FNA were retrieved. Immunostaining was performed with antibodies against bcl-2, Ki-67 and p53. All cytological diagnoses were confirmed by positive immunostaining with c-kit and/or subsequent histological evaluation. Positivity for bcl-2, Ki-67 and p53 was defined as the presence of > or =10% cytoplasmic staining, > or =5% nuclear staining and > or =5% nuclear staining respectively. RESULTS: The 14 patients consisted of seven males and seven females with a mean age of 58 years. The average follow-up interval was 46 months. Six had a benign course and eight developed recurrences/metastases. Thirteen (93%) cases showed positive staining for bcl-2. Positive Ki-67 and p53 staining was noted in one (7%) and seven (50%) cases respectively. The difference in staining for p53 between aggressive and non-aggressive GISTs was statistically significant. No statistically significant difference was noted for bcl-2 staining or Ki-67 labelling index between the two groups. CONCLUSIONS: According to our observations, p53 immunostaining may be useful in predicting the outcome of GIST diagnosed by FNA; Ki-67 and bcl-2 are not useful as prognostic markers for GIST in FNA specimens.     

Analysis of proliferating cell fraction determined by monoclonal antibody to M1-subunit ribonucleotide reductase and Ki-67 in relation to p53 protein expression in fine-needle aspirates from non-Hodgkin's lymphomas

The purpose of this study was to analyse the proliferative fraction with the monoclonal antibody M1-R-R to M1-subunit ribonucleotide reductase and with MIB-1 to Ki-67 antigen in relation to p53 protein expression in fine needle aspirates from B-cell non-Hodgkin's lymphomas. One hundred and thirty-seven cases, previously diagnosed and sub-typed according to the Kiel classification and characterized by immunophenotyping, were included in the study. The M-1 subunit ribonucleotide reductase (M1-R-R), Ki-67 and p53 antigens were detected using monoclonal antibodies on stored cytospin preparations. There was a good correlation (r = 0.72) between Ki-67 and M1-R-R positive cell fraction in both high and low grade lymphomas. High-grade lymphomas had a median percentage of M1-R-R/MIB-1 positive cells of 53.0/73.0 for lymphoblastic, 61.0/52.0 for immunoblastic and 33.5/41.0 for centroblastic lymphomas, respectively. In low grade lymphomas figures of median percentage of M1-R-R/MIB-1 were 9.0/15.0 for centroblastic/centrocytic, 11.0/9.5 for chronic lymphocytic leukaemia, 16.0/27.0 for centrocytic and 12.0/9.0 for immunocytomas, respectively. The median percentages of M1-R-R/MIB-1 for high and low grade lymphomas were 37.0/50.5 and 11.0/12.0, respectively. In the p53 positive cases the proliferation rate as measured by staining for M1-R-R and MIB-1 was higher than in p53 negative cases, but the difference was not statistically significant. The results show that cytospin material obtained by fine needle aspiration and stored at -70 degrees C for years can be used reliably for both peroxidase-avidin-biotin and three-step alkaline phosphatase immunocytochemical staining. In addition, proliferation fraction determined by M1-R-R monoclonal antibody staining correlates well with that measured by an established marker for cell proliferation, the Ki-67 antibody. However, the proliferation fraction as measured by the two antibodies differs in the various subtypes of non-Hodgkin's lymphoma which indicates that they may contribute different prognostic information.     

Ki-67 and B72.3 expression in breast cancer: an immunohistochemical study

The present study was performed to evaluate Ki-67 and B72.3 immunostaining in 20 selected cases of breast cancer. In particular, we have examined the intracellular localization of TAG 72 and the tumour growth fraction, identified by Ki-67 antibody, on frozen sections of mammary carcinoma, by immunohistochemical technique (ABC method sec.Hsu). Immunostaining of TAG 72 and Ki-67 antigen was related to histologic subtype, diameter, nodal involvement, and number of positive axillary nodes. The preliminary results suggest that: (a) the presence of Ki-67 nuclear staining appeared to be associated with a poorer degree of differentiation, but no direct relationships were observed with diameter and nodal involvement; (b) no correlation between Ki-67 labelling rates and B72.3 intracytoplasmic immunostaining was observed; (c) myoepithelial cells show weak intracytoplasmic positivities.     

Immunocytochemical detection of prognostic markers in breast cancer; technical considerations

The purpose of this study was to establish a good technical procedure for immunocytochemical (IC) staining of prognostic markers in breast cancer specimens. The influence of various preparation, fixation and storage methods on ER, P53 and Ki-67 IC staining was assessed, using cells of two breast cancer cell lines T47D (ER/P53+) and ZR-75-ER (ER+, P53-). In addition we searched for a suitable transport medium. Depending on the technical procedure, great variations in expression of the tested antigens were found. Cytospins fixed and stored according to the Abbott method gave the best results. Histocon appeared to be the medium of choice. A good concordance of IC and immunohistochemical (IH) results was found when the adopted method was tested on material of 10 breast cancers. This study underlines the importance of quality controlled standardization of cell processing, fixation and storage of fine needle aspiration (FNA) aspirates in order to obtain reproducible and consistent IC results.     

Comparison of monoclonal antibodies PC 10 and MIB 1 on microwave-processed paraffin sections

Abstract. Antibodies against the proliferation-associated nuclear antigen (PCNA) and against the Ki-67 protein are widely used as operational proliferation markers in human tumour diagnostics. The original Ki-67 antibody had the inherent drawback in that it could only be used when fresh-frozen material was available. The antibody PClO was supposed to offer the advantage that it could be applied on formalin-fixed and paraffin-embedded tissues. However, in cases in which the formalin fixation exceeded 4 h, PC10 staining proved to be inconsistent and often failed.
The aim of this study was to compare a recently prepared Ki-67 equivalent monoclonal antibody (MIB 1) and PC10 in routinely fixed histopathological material using antigen retrieval by microwave processing.
Antibody MIB 1 stained the nuclei of cells known to belong to the proliferative compartments in microwave-processing paraffin sections of formalin-fixed tissues. Quiescent cells were consistently negative for MIB 1 staining. In contrast, PC10 was positive in almost all nuclei of different tissues in microwave-treated paraffin sections. Thus, antigen retrieval by microwave processing is beneficial for the detection of the Ki-67 protein in paraffin sections, whereas it is not needed for the detection of the PCNA.     

The hTERT-protein and Ki-67 labelling index in recurrent and non-recurrent meningiomas

Meningiomas are considered as benign neoplasms affecting the coverings of the central nervous system and compromise approximately 20% of all intracranial tumours. However, a number of these tumours recur even after total resection. The aim of this study is to evaluate the prognostic significance for recurrence of the human telomerase catalytic subunit (hTERT) in the cells of meningiomas. The expression of hTERT-protein can be evaluated by immunohistochemical staining using a monoclonal antibody against hTERT (clone 44F42, NCL-L-hTERT). The interdependence between tumour recurrence and cell proliferation in this study is analysed by Ki-67 immunoreactivity (clone MIB-1). Archival material from 29 non-recurrent and 32 recurrent tumours has been evaluated, including specimens from World Health Organization (WHO) stages I (n = 73), II (n = 2) and III (n = 12). Although the tumours were categorized as benign meningiomas following the WHO classification, recurrence in 22 of 50 cases did not correlate with the tumour stage. For hTERT staining, the following results were found for nucleolar and total nuclear staining, respectively: non-recurrent meningiomas, 2.9% (+/- 7.7) and 3.0% (+/- 8.0); recurrent meningiomas at first resection, 16.8% (+/- 19.7) and 31.6% (+/- 30.2). Concerning the Ki-67 labelling index (LI): for the group of non-recurrent meningiomas, results were 2.1% (+/- 1.7) and for the recurrent group at first resection, 1.7% (+/- 2.0). A significant difference was seen for the hTERT staining (P < 0.001) between the non-recurrent and recurrent meningiomas, whereas no statistical significance was found for Ki-67. In conclusion hTERT-positive meningiomas had a high incidence for recurrence. Ki-67 was a good marker of cell proliferation status of the tumours, but did not correlate with recurrence; thus, hTERT alone seemed to be a potential predictor for recurrence.     

飞燕草素通过Wnt/β-catenin信号通路抑制乳腺癌的机制研究

为研究飞燕草素对乳腺癌MDA-MB-231细胞Wnt/β-catenin信号通路的影响。免疫组化检测裸鼠乳腺肿瘤组织和肺组织转移瘤Ki-67及乳腺肿瘤组织蛋白水解酶超家族基质金属蛋白酶-7(matrix metallopeptidase 7,MMP-7)的表达水平;Western blot检测移植瘤Wnt/β-catenin通路β-联蛋白(β-catenin)、磷酸糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)及通路下游细胞周期相关蛋白cyclinD1、原癌基因c-myc和MMP-7的蛋白水平表达,体内外实验发现飞燕草素不仅能抑制裸鼠异种移植瘤生长及乳腺癌肿瘤组织和肺组织转移瘤Ki-67表达还可以明显降低乳腺癌MDA-MB-231细胞Wnt/β-catenin信号通路β-catenin和p-GSK-3β下游靶基因c-myc、cyclin D1和MMP-7蛋白的表达。本研究证实飞燕草素能通过抑制Wnt/β-catenin信号通路,发挥抑制乳腺癌的作用。     

Tumor Cell Identification in Ki-67 Images on Deep Learning

The proportion of cells staining for the nuclear antigen Ki-67 is an important predictive indicator for assessment of tumor cell proliferation and growth in routine pathological investigation. Instead of traditional scoring methods based on the experience of a trained laboratory scientist, deep learning approach can be automatically used to analyze the expression of Ki-67 as well. Deep learning based on convolutional neural networks (CNN) for image classification and single shot multibox detector (SSD) for object detection are used to investigate the expression of Ki-67 for assessment of biopsies from patients with breast cancer in this study. The results focus on estimating the probability heatmap of tumor cells using CNN with accuracy of 98% and detecting the tumor cells using SSD with accuracy of 90%. This deep learning framework will provide an objective basis for the malignant degree of breast tumors and be beneficial to the pathologists for fast and efficiently Ki-67 scoring.     

Comparison of Four Immunochemical Methods For the Measurement of Oestrogen Receptor Levels In Breast Cancer

Four methods of assessing oestrogen receptor (ER) status were compared in 33 patients with operable primary breast cancer. The methods used to assess the ER status were immunocytochemical assay (ER-ICA) of frozen sections, fine needle aspirates and imprint material and enzyme immunoassay (ER-EIA) of tumour tissue. A mean overall ER positivity of 45% (15 out of 33), 41% (13 out of 32) and 21% (six out of 29) was obtained by immunocytochemical (ER-ICA) staining of frozen sections, fine needle aspirates and tumour imprints, respectively, and a mean overall ER positivity of 42% (14 out of 33) was obtained by ER-EIA. The concordance of ER positivity in pairs of data obtained from different method combinations was found to range between 72 and 91%. However, statistically there was no significant difference between the four methods on the basis of the data obtained. Good comparability has been shown between the three tissue analyses and therefore the method of choice is technically not immediately apparent.     

Fine needle aspiration cytology (FNAC) of mammary granular cell tumour: a report of three cases

This report describes the FNAC findings in three cases of granular cell tumour of the breast. The patients comprised two females aged 59 and 62 years and one male aged 28 years. All patients presented with a breast lump which was clinically and radiologically suspicious of malignancy. FNAs yielded moderately cellular specimens which on cytologic examipation consisted of groups of cells and single cells with small regular nuclei and abundant granular cytoplasm. Bare nuclei were also present but these did not have the characteristic bipolar appearance of myoepithelial cells. In two cases there was a granularity to the background. The aspirates were reported as equivocal or atypical, probably benign, and surgical biopsy was performed. Histological examination showed typical benign granular cell tumours with strong positive staining for S-100 protein. Pathologists should be aware that granular cell tumour may occur in or around the breast and should consider this diagnosis in aspirates containing a population of cells with regular nuclei and abundant granular cytoplasm. The main cytologic differential diagnoses are likely to be apocrine cells and histiocytes. The suspicion of a granular cell tumour should be heightened when these features are present in an aspirate from a clinically and radiologically suspicious mass. These cases highlight the role of the triple approach encompassing clinical, radiological and cytological features in the assessment of a breast lesion.     

Elevated Ki-67 expression is correlated with TNFalpha- and IFNgamma-induced apoptosis in tumour cells

Abstract. The expression of Ki-67 in tumour cells induced to apoptosis by tumour-necrosis-factor α (TNFα) and interferon γ (IFNγ) was studied. Ki-67 is known as a proliferation marker which is expressed in cycling cells, but not in resting quiescent or G_o cells. In numerous studies, the proportion of tumours expressing Ki-67 was determined and related to tumour grade or prognosis. A high percentage of Ki-67 expressing cells and a low apoptotic index were regarded as an indication of a progressive tumour. This implied that Ki-67 expression and apoptosis were contrary traits. In this study, the level of Ki-67 expression in human tumour cells in culture was measured after induction of apoptosis. The Ki-67 level was determined by flow cytometry and apoptosis was measured by various methods including PARP degradation (western blot) in detached and floating cells. While the floating cells were all apoptotic, more than 80% of the attached cells showed no apoptotic signs. The Ki-67 level of apoptotic cells was elevated about 3-fold compared to viable attached control cells. However, the cytokine-treated attached cells also expressed Ki-67 at similar high levels to the apoptotic floating cells, depending on sensitivity. The plot of Ki-67 level vs. remaining cells after treatment revealed a strong correlation between the level of Ki-67 expression and the sensitivity to cytokine-induced apoptosis. This implies that proliferation pathways and apoptotic signal transduction are connected.     

Immunocytochemical Detection of the Androgen Receptor In Fine Needle Aspirates From Benign and Malignant Human Prostate

A monoclonal antibody to the androgen receptor was applied to fine needle aspirates from patients with benign and malignant prostatic disease. The series includes six patients with benign hyperplasia and 24 patients with prostatic carcinomas. The androgen receptor was detected in most nuclei of both benign and malignant epithelial cells. The intensity of immunostaining varied. No obvious relation was observed between the intensity of the staining in benign versus malignant cells. In addition no clear differences were found in the proportion of androgen receptor positive cells in benign aspirates as compared with aspirates from well differentiated or moderately well differentiated prostatic carcinomas. The relative number of androgen receptor positive cells was highest in smears from poorly differentiated prostatic carcinomas.     

Ki-67 antigen detection in unstained and destained cytologic samples

OBJECTIVE: To evaluate the effect of different tissue preservation, fixation and staining procedures on the expression of proliferation-associated Ki-67 antigen in cytologic samples to establish an easy and uniform way to handle cytologic specimens with an automated staining technique. STUDY DESIGN: Multiple touch imprints were made from eight breast tumors. The specimens were treated according to different protocols, and Ki-67 nuclear expression was compared to that in the corresponding histologic sections. RESULTS: In the unstained specimens, air drying at room temperature for up to four months or ethanol spray fixation preserved the material and offered excellent results. Processing effectively removed previous stain without additional chemical destaining. Antigen retrieval was not achieved in the previously Giemsa stained imprints and was suboptimal in those stained according to Papanicolaou. CONCLUSION: Immunocytochemical detection of Ki-67 is recommended for previously unstained cytologic specimens.     

Prognostic evaluation of breast cancer in cytologic specimens

OBJECTIVE: To evaluate prognostic factors in breast cancer using cytologic samples and to determine the correlation between those factors and ploidy. STUDY DESIGN: Two hundred sixteen fine needle aspirates from patients with primary breast cancer were analyzed for expression of estrogen receptors (ERs), progesterone receptors (PRs), Ki-67 antigen, expression of p53 tumor suppressor gene and overexpression of c-erbB-2 using a standard immunochemical method. Not all subjects had all biomarker information because of the study design (c-erbB2 added later). The specimens were analyzed also for ploidy. We used the SAMBA 4000 image analysis system for quantification of the percent of cells stained positively by the different immunocytochemical stains andfor ploidy. RESULTS: A significant correlation wasfound between ER and PR and between Ki-67 and positive p53. Steroid receptor content was not significantly related to p53, Ki-67 or c-erbB2. No correlation was found between c-erbB2 and the other biomarkers. Ploidy had a significant correlation with all the biomarkers used. CONCLUSION: A reliable and rapid evaluation of markers for breast cancer can be achieved by measuring cells stained positively by immunocytochemical stains, as well as ploidy, by means of an image analysis system. ER, PR Ki-67, p53 and c-erbB2 had a significant correlation with ploidy and overall prognostic value in breast cancer.