Growth fraction in lung tumours determined by Ki67 immunostaining and comparison with AgNOR scores

Estimation of the growth fraction with the use of monoclonal antibody Ki67, which recognizes a nuclear antigen in proliferating cells, was compared with the nucleolar organizer region staining in 95 lung tumours. There was nuclear staining in most tumours; 12 tumours were negative. Cytoplasmic staining was observed in another seven tumours. The small cell carcinoma group had the highest mean Ki67 index (23.75); squamous carcinomas had a mean value of 15.71, adenocarcinomas 10.99, and large cell anaplastic 20.76. Carcinoids had few stained cells. Nucleolar organizer regions were demonstrated by the argyrophilic method (AgNOR). No correlation was found between Ki67 indices and AgNOR scores. Kinetic data obtained by the AgNOR technique were less discriminating in view of the overlap between scores of various groups including carcinoids. We conclude that use of the monoclonal antibody Ki67 is a more reliable method of assessing proliferative activity in lung tumours. This antibody may be effective in identifying slowly proliferating tumours which are less sensitive to chemotherapeutic agents.     

New approach to assessing lung tumours in man

One hundred and four surgically resected lung tumours were labelled in either cryostat or freeze dried sections with a monoclonal antibody (Ki67), which reacts with a nuclear antigen expressed by proliferating cells. The tumours were categorised semiquantitatively into four proliferative grades, a classification that can be performed rapidly and reproducibly by the pathologist. In keeping with previous cell kinetic studies all small cell carcinomas had high proliferation rates, whereas the carcinoid tumours were in the lowest grade. In contrast, the adenocarcinomas (27 cases) and squamous cell carcinomas (63 cases) varied widely in their proliferative state, in keeping with their heterogeneous, morphological, and clinical behaviour. Immunocytochemical labelling of lung tumour biopsy specimens with antibody Ki67 is a simple technique within the scope of routine surgical pathology laboratories, which might enable these tumours to be classified according to their proliferative status and treatment to be selected accordingly.     

Expression of proliferation associated antigens and detection of numerical chromosome aberrations in primary human liver tumours: relevance to tumour characteristics and prognosis.

AIMS: To assess cell proliferation and the presence of numerical chromosome aberrations involving chromosomes 1 and 8 in benign and malignant liver tumours. METHODS: Cell proliferation was studied immunohistochemically in paraffin wax embedded material from 62 primary liver tumours (20 hepatocellular carcinomas, 16 cholangiocellular carcinomas, 15 liver cell adenomas, 11 focal nodular hyperplasias), and the results were compared with histological characteristics and clinical data. Copy numbers of chromosomes 1 and 8 were assessed by interphase fluorescence in situ hybridisation (FISH) with satellite probes in fresh tumour material. RESULTS: The expression of proliferation associated antigen Ki67, using the monoclonal antibody MIB-1, and proliferating cell nuclear antigen (PCNA), using the antibody PC10, was found to be significantly higher in malignant versus benign liver tumours. Neither Ki67 nor PCNA expression were independent prognostic parameters. However, there was a tendency for a worse outcome (survival < 12 months) for patients with a high MIB-1 labelling index (> 20%) compared with patients having the same tumour stage and a low MIB-1 index. Aneusomy for chromosomes 1 and 8 was demonstrated by FISH in malignant tumours (six of seven hepatocellular carcinomas, four of five cholangiocellular carcinomas) but not in benign tumours (none of nine) or non-neoplastic liver (none of nine). CONCLUSION: Both the determination of the proliferating cell fraction and FISH analysis are useful for distinguishing hepatocellular carcinoma from liver cell adenoma or focal nodular hyperplasia; high fractions of proliferating cells are predictive of an early relapse.     

A comparative study of cell proliferation markers in breast carcinomas

Aims—To investigate the tumour cell proliferative index obtained by immunostaining of paraffin wax sections of 30 cases of breast carcinoma with monoclonal antibodies MIB1, KiS1 and KiS5, and polyclonal Ki67 antisera to the Ki67 antigen and 19A2 and PC10 antibodies to proliferating cell nuclear antigen and the possible correlation between these indices and that of monoclonal Ki67 antibody in frozen sections of the same tumours.     

VS38: a new monoclonal antibody for detecting plasma cell differentiation in routine sections.

AIMS--To characterise a new mouse monoclonal antibody, VS38, which recognises an intracytoplasmic antigen of 64 kilodaltons present in normal and neoplastic plasma cells; and to establish its value as a diagnostic reagent for routine pathological practice. METHODS--A range of normal and neoplastic tissue sections, both frozen and routinely fixed, were immunostained, using the microwave method of antigen retrieval for routinely fixed specimens. The antibody was also tested on blood and bone marrow specimens and a range of human cell lines. The molecular weight of the antigen recognised by the antibody was obtained by western blot analysis. FACS analysis was used to demonstrate the cellular location of the antigen and its presence on tonsil cell suspensions and myeloma cases. RESULTS--VS38 recognised normal and neoplastic plasma cells in all of the tissues, including all routinely fixed plasma cell neoplasms tested. The antibody also weakly stained epithelial elements within the tissue but was absent from haemopoietic cells of other lineages. CONCLUSION--Antibody VS38 is of potential value in identifying myeloma or plasmacytoma in bone marrow or other tissues. It differentiates lymphoplasmacytoid lymphoma from lymphocytic and follicular lymphoma. It also subdivides large cell lymphomas into two groups which may be a more reliable method of separating these tumours than morphology alone.     

Detection of Ki67 antigen by a new sheep polyclonal antiserum.

This report describes the characterisation of a polyclonal sheep antiserum against the Ki67 antigen. On western blots, this antiserum recognises a pair of bands of high molecular weight identical with those seen with another polyclonal Ki67 antiserum and the MIB 1 monoclonal antibody. The new antiserum showed nuclear staining of a proportion of cells in paraffin wax embedded tissue sections following antigen retrieval using a microwave oven or pressure cooker. This staining pattern was blocked by incubating the serum with the peptide used as immunogen. The proportion and distribution of immunostained nuclei was identical with that seen with the alternative reagents that recognise the Ki67 antigen. The new reagent stained the same proportion of cells when used over a wide range of dilutions. There was no cross-reactivity with unrelated antigens sometimes detected by the monoclonal antibodies.     

Proliferation indexes--a comparison between cutaneous basal and squamous cell carcinomas.

AIMS: To compare differences in cell proliferation indexes and apoptotic indexes between cutaneous basal and squamous cell carcinomas, in an attempt to suggest an explanation for the differences in their biological behaviour. METHODS: Forty cases of cutaneous basal cell carcinoma (BCC) and 40 cases of moderately and well differentiated squamous cell carcinoma (SCC) were retrieved from the archives. Sections, 4 microns thick, were cut from formalin fixed, paraffin wax embedded tissue in each case and stained with haematoxylin and eosin. These were then examined for mitotic and apoptotic figures per 1000 cells. Sections from the same cases were also immunostained with the mouse monoclonal antibody Ki67 (MIB1); positive nuclear staining was counted per 1000 cells. RESULTS: No significant differences were found between the mitotic indexes and apoptotic indexes in these tumours. There was, however, a significant difference in Ki67 (MIB1) staining, with greater staining in the squamous cell carcinomas. CONCLUSION: Estimation of the mitotic and apoptotic indexes did not reveal any differences between these two tumour types. The proliferation indexes, assessed by Ki67 immunostaining, did differ. This may be one of the factors underlying the more aggressive behaviour of SCC.     

Comparison of proliferation rates assessed using "multiblock" and conventional tissue blocks of lung carcinoma.

AIMS: To compare the proliferative rates, assessed immunohistochemically, of human lung tumours using conventional paraffin wax blocks and the multitumour tissue block (MTTB) technique. METHODS: A multiblock containing 20 lung tumours (eight adenocarcinomas, five squamous cell, five small cell and two carcinoid tumours) was constructed. Sections were also cut from the original blocks of formalin fixed, paraffin wax embedded tissue used to construct the multiblock. Sections were stained with the monoclonal antibody PC10, which recognises a proliferating cell nuclear antigen, using the three stage immunoperoxidase technique. RESULTS: The proliferation rates of the lung tumours obtained using both techniques were, overall, significantly different (p = 0.05), although most cases showed good correlation. Some tumours displayed a high degree of intratumoral variation in PC10 staining. The degree of PC10 staining was in keeping with the known proliferative state of particular histological subtypes--that is, carcinoid tumours showed little staining and small cell carcinomas showed extensive positivity. CONCLUSION: The MTTB technique is a less suitable means of assessing proliferation rate in lung carcinomas than conventional tissue blocks. It should be restricted to qualitative antibody studies or quantitative studies using tumours with little intratumoral variation.     

Potential role of minichromosome maintenance protein 2 as a screening biomarker in esophageal cancer high-risk population in China

Minichromosome maintenance proteins are novel proliferative markers that have been proposed as diagnostic markers in many cancers. We evaluated the potential role of minichromosome maintenance protein 2 as a screening biomarker and compared it with proliferating cell nuclear antigen and Ki67 in a population survey of esophageal squamous cell carcinoma. A total of 299 esophageal samples from a high-risk region in China, including 171 from an endoscopy population survey, 30 from brushing cytology, and 98 from surgery and autopsy, underwent immunostaining with minichromosome maintenance protein 2, proliferating cell nuclear antigen, and Ki67 antibodies. Minichromosome maintenance protein 2 expression was confined to the proliferative compartment of normal and abnormal esophageal epithelium and particularly manifested in the surface layer of dysplasia and carcinoma in situ. The expression of proliferating cell nuclear antigen and Ki67 was positively correlated with that of minichromosome maintenance protein 2 (r(s) >0.39, P < .01); but their positive nuclei seldom reached the surface layer, and the labeling indices were significantly lower than those for minichromosome maintenance protein 2 in dysplasia (P < .05) and carcinoma in situ (P < .001). The sensitivity and specificity of minichromosome maintenance protein 2 in diagnosing dysplasia were 91.3% and 61.8%, respectively, higher than those for proliferating cell nuclear antigen (88.4% and 47.1%) and Ki67 (78.3% and 57.8%). Nine of 10 cancer and paracancerous surface-brushing samples expressed minichromosome maintenance protein 2, and the detection was higher than that for proliferating cell nuclear antigen (8/10 and 7/10) and Ki67 (7/10 and 7/10). However, none of 10 normal surface-brushing samples expressed the 3 markers. Minichromosome maintenance protein 2 is more sensitive and specific than proliferating cell nuclear antigen and Ki67 in indicating esophageal dysplasia. Minichromosome maintenance protein 2 immunostaining combined with surface brushing could be valuable in screening patients at high risk of cancer in mass surveys.     

Detection of growth fraction in tumors by Ki67 monoclonal antibody in cytologic smears: A prospective study of 40 cases

The monoclonal antibody (MAb) Ki67 detects a nuclear antigen in cycling tumor cells. Quantitation of proliferating cells is helpful in predicting the recurrence and metastatic potential of tumors as previously reported. We conducted a prospective study on 40 benign and malignant tumors by performing Ki67 immunocytochemical stains on cytologic smears and their corresponding frozen tissues. Quantitation of Ki67 positive cells was done by counting 300 cells in 5-7 high-power fields in cytologic smears and tissues. Only nuclear or nucleolar immunostaining was considered positive for MAb Ki67. The number of Ki67 positive tumor cells in cytologic smears correlated well with Ki67 positive cells from corresponding tissues. On the average, cytologic smears showed 1.9% higher Ki67 positivity in malignant tumors as compared to their corresponding frozen tissues (P < 0.001). The Ki67 positivity in malignant tumors was found to be significantly higher when compared with benign tumors (P < 0.001). We conclude that cytologic smears can be used for the determination of growth potential in tumors by MAb Ki67. Additionally, cytologic preparations can be used during intraoperative consultations when adequate tissue is not available for the above mentioned study.     

Which proliferation markers for routine immunohistology? A comparison of five antibodies.

AIMS--To determine the best of five antibodies for immunohistochemical assessment of growth fraction in formalin fixed, paraffin wax embedded tissues. METHODS--Sections from 100 recent, and 17 ten year old or over wax embedded blocks of normal and malignant tissues were immunostained with monoclonal Ki67, polyclonal Ki67, PC10, MIB1, and JC1. The antibodies were evaluated for specificity of nuclear versus cytoplasmic staining, cleanliness of background, and compared with the expected pattern of staining in normal tissues, defined immunohistochemically by monoclonal Ki67 antibody in frozen tissues or by tritiated thymidine uptake. RESULTS--No marker was ideal, but best results were obtained with MIB1 and polyclonal Ki67, followed by JC1, PC10, and monoclonal Ki67. CONCLUSIONS--For routine use, MIB1 or polyclonal Ki67 are the best proliferation markers in conventional histological preparations. The other markers tested cannot be recommended.     

DNA polymerase alpha. An immunohistochemical marker for proliferating cells in normal and neoplastic human tissues.

DNA polymerase alpha is a DNA replicating enzyme expressed in all proliferating cells. This nuclear antigen in paraformaldehyde-postfixed frozen sections of normal, benign, and malignant tissues was identified by the peroxidase-antiperoxidase technique with the use of a mouse monoclonal antibody against bovine/human DNA polymerase alpha. The nuclei of normal proliferating cells were positive. Malignant tumors (n = 95) showed a higher proportion of positive nuclei than did low-grade malignant tumors (n = 7) or benign lesions (n = 67). The number of positive nuclei in squamous cell carcinomas (n = 19) was higher than in adenocarcinomas (n = 45). Eight (18%) adenocarcinomas and all five renal cell carcinomas had less than 10% positive cells, whereas in benign tissues, such as pituitary adenomas, a thymoma, reactive lymphoid lesions, and some benign mammary nodules, more than 10% of nuclei were labeled. In addition, foci of proliferating cells were clearly recognized. DNA polymerase alpha is, therefore, an excellent marker of proliferative activity that provides an approach to analyzing tumor cell heterogeneity not only in fully developed neoplasms, but also in their precursor lesions.     

An assessment of MAC 387 antibody as a diagnostically useful marker of L1 epithelial antigen expression by lung tumours.

A recent report describing the distribution of L1 epithelial antigen in lung tumours in relation to the histological type claimed that this antigen was a highly reliable marker of squamous cell carcinoma. Our study was designed to test this claim and to examine the potential of this antigen in the typing of lung tumours in biopsy specimens. A total of 143 lung tumours were typed according to the WHO classification and examined immunohistochemically for L1 epithelial antigen expression using commercially available monoclonal mouse anti-human myeloid/histiocyte antigen (MAC 387). Positivity was found in 46 of 55 squamous cell carcinomas (84 per cent), 12 of 27 adenocarcinomas (44 per cent), 15 of 16 adenosquamous carcinomas (93 per cent), 10 of 15 large cell carcinomas (67 per cent), none of 20 small cell carcinomas, and none of 10 carcinoid tumours. Of those tumours expressing L1 epithelial antigen, most showed a patchy pattern of positivity. From this study it is clear that detection of L1 epithelial antigen by MAC 387 antibody is not specific for squamous cell carcinomas, but it may have a limited use in the diagnosis of small cell carcinomas and carcinoid tumours as these are consistently negative.     

IDENTIFICATION OF THE ANTIGEN RECOGNIZED BY THE MONOCLONAL ANTIBODY BU31 AS LAMINS A AND C

The murine monoclonal antibody BU31 binds to the nuclear membrane of many cell types. The expression of the BU31 antigen has previously been shown to have an inverse correlation with the proliferative index in lung tumours, defined by Ki67 staining. The distribution of BU31-positive cells is now shown to parallel the distribution of non-dividing cells in a range of normal human and rat tissues, although neuroendocrine cells and germ cells in the testis show no reactivity. Cells grown in culture and induced to undergo growth arrest show a higher level of labelling with BU31 than their proliferating counterparts. Confocal laser scanning microscopy reveals that the BU31 antigen is distributed predominantly along the nuclear lamina, with occasional internal foci. This distribution is very similar to that of the nuclear membrane proteins lamin A and lamin C, suggesting that the BU31 antigen and lamins A and C could be one and the same. Immunoblotting using recombinant lamin proteins confirmed this proposal. Moreover, a monoclonal antibody to the non-proliferation-associated antigen, statin, also recognizes lamins A and C. These data indicate that the demonstration of lamins A and C can be used to provide information on the proliferative activity of normal and neoplastic tissues. These data also suggest a role for nuclear lamins A and C during cellular quiescence, possibly through the reorganization and maintenance of nuclear structure, or more directly through interactions with the retinoblastoma gene product or related proteins.     

Expression of epithelial and neural antigens in small cell and non small cell lung carcinoma

Seventy-one lung carcinomas from 66 different patients were stained with a panel of monoclonal antibodies. Twenty-nine were small cell lung carcinoma (SCLC), 15 adenocarcinomas, 17 squamous carcinomas and 10 large cell carcinomas. Three of the monoclonal antibodies recognize different cytokeratins, three recognize other epithelial antigens and one recognizes a neural antigen. Both formalin-fixed and cryopreserved tumours were studied using an indirect immunoperoxidase method. 23/29 SCLC reacted with all but one of the antibodies which recognize epithelial antigens. This staining was similar to that seen in non small cell lung carcinomas (NSCLC) and provides further evidence that SCLC are true epithelial tumours. All but one of the SCLC stained with the antibody recognizing a neural antigen. This antibody did not stain squamous or adenocarcinomas. However, four of the large cell carcinomas stained well with this antibody, suggesting that SCLC and some large cell carcinomas share a common pathway of differentiation. There were variations of staining seen both within and between tumours. This has obvious implications if immunotargetting with monoclonal antibodies is to be used diagnostically or therapeutically.     

Immunohistochemical detection of proliferating cells in colorectal carcinomas and adenomas with the monoclonal antibody Ki-67. Preliminary data

Summary Twenty one cases of colorectal adenocarcinoma and six of adenoma have been studied using the monoclonal antibody Ki-67 which recognizes a nuclear antigen expressed by proliferating cells (PC). The quantitative evaluation of the stained nuclei showed that PC were more numerous in carcinomas than in adenomas although the difference did not reach a significant level. In each tumour, heterogeneity was noted. Furthermore, the superficial areas of both carcinomas and adenomas contained a greater number of PC than the deep. No difference was noticed in the various types and grades of differentiation for carcinomas. This preliminary report, compared with the only previous study of Shepherd et al. (1988) outlines the interest of the monoclonal antibody Ki-67 in the evaluation of growth fractions in colorectal tumours.     

Expression of the 67 kD laminin receptor in human cervical preneoplastic and neoplastic squamous epithelial lesions: an immunohistochemical study

Interactions of cancer cells with laminin play a critical role during the progression of solid malignant tumours. Increased expression of the 67 kD laminin receptor (67LR), one of the several laminin binding proteins, is associated with the invasive and metastatic capacity of various types of cancer, including breast, colon, ovary, lung, and endometrial carcinoma. In this study, 67LR expression was analysed in a series of cervical biopsy specimens including 16 normal cervical tissues, 36 low-grade squamous intraepithelial lesions (SILs), 24 high-grade SILs, and 11 invasive carcinomas. Detection of the 67LR was performed using immunoperoxidase staining and the monoclonal antibody MLuC5 which specifically recognizes the 67LR. Immunostaining of the 67LR was correlated with human papillomavirus (HPV) type detected by in situ hybridization and with proliferative activity of the lesion determined by immunohistochemistry with the MIB-1 monoclonal antibody, specific for the Ki67 antigen. Increased expression of the 67LR was correlated with the histological severity of the lesions, with the strongest immunoreactivity being found in invasive carcinomas. Significant differences in 67LR expression were found between normal cervical epithelium and high-grade SILs (P<0·05, non-parametric Mann-Whitney test) or invasive carcinomas (P<0·001), as well as between low- or high-grade SILs and invasive carcinoma (P<0·01 and P<0·05, respectively). Ki67 antigen expression also increased with the severity of the lesions. There was a positive correlation for each type of lesion between expression of the 67LR and of the Ki67 antigen. No specific relationship was found between 67LR or Ki67 antigen immunostaining and HPV type detected in SILs, segregated into low-grade and high-grade lesions. These data add weight to the evidence that increased expression of the 67LR is a consistent, but not sufficient feature of the invasive and metastatic phenotype and suggest that high expression of the 67LR might be associated with both more proliferative and more aggressive cervical (pre)neoplastic lesions. © 1997 John Wiley & Sons, Ltd.     

Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms

Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein associated with the cell cycle. A monoclonal antibody, PC10, that recognizes a fixation and processing resistant epitope has been used to investigate its tissue distribution. Nuclear PCNA immunoreactivity is found in the proliferative compartment of normal tissues. PCNA immunoreactivity is induced in lectin stimulated peripheral blood mononuclear cells in parallel with bromodeoxyuridine incorporation and the number of cells with PCNA immunoreactivity is reduced by induction of differentiation in HL60 cells. In non-Hodgkin's lymphomas a linear relation between Ki67 and PCNA staining was demonstrated. These data suggest that in normal tissues and lymphoid neoplasms, PCNA immunolocalization can be used as an index of cell proliferation. However, in some forms of neoplasia, including breast and gastric cancer and in vitro cell lines, the simple relation between PCNA expression and cell proliferation is lost. In some breast and pancreatic tumours there is apparent deregulation of PCNA with increased expression in tissues adjacent to the tumours. The over-expression in some tumours and in adjacent morphologically normal tissue may represent autocrine or paracrine growth factor influence on PCNA gene expression.     

Immunohistochemical Demonstration of Cell Proliferation and Estrogen Receptor Status in Human Breast Cancer

Cell proliferation and estrogen receptor (ER) status was investigated in 45 invasive ductal carcinomas of the breast by immunohistochemical methods using monoclonal antibodies Ki 67 (anti-human proliferating cell antibody) and ER ICA. The results were assessed on the basis of nuclear staining intensity and the percentage of positively stained tumor cell nuclei (index score). There was a significant inverse correlation between the Ki 67 and ERICA index scores, although 4 cases showed high index scores for both markers. We conclude that ER positive cells do not always have low proliferation activity, which may be one of the reasons why endocrine therapy is not effective against all ER positive breast cancers. Acta Pathol Jpn 40: 902 907, 1990.     

Transforming growth factor-beta 1 knockout mice. A mutation in one cytokine gene causes a dramatic inflammatory disease.

A monoclonal antibody (Ki-S1) has been raised that reacts with the nuclei of proliferating cells. The antigen recognized is resistant to formalin fixation and can be detected in frozen tissues as well as in routinely processed specimens. In immunohistochemistry, nuclear staining can be seen in those tissues and cellular compartments known to be actively proliferating. Peripheral blood lymphocytes are negative but show a strong increase in antigen expression after mitogen stimulation. Flow cytometric determination of DNA content and antigen expression revealed negativity of G0 cells and positivity of G1 to G2/M cells. A cytoplasmic co-reactivity, not associated with proliferation, was confined to Langerhans islands of the pancreas. The nuclear localized antigen has a molecular mass of 160 kd and therefore seems to be different from all other known immunohistochemical markers of proliferating cells. We conclude that the monoclonal antibody Ki-S1 might provide a useful tool for studying cell proliferation in situ under normal and pathological circumstances.