Proliferating cell nuclear antigen (PCNA) expression in Hodgkin's disease.

Previous studies of the proliferating cell fraction in Hodgkin's disease (HD) have been directed towards the classical Hodgkin and Reed-Sternberg cells (HRS) to the exclusion of the background population and have not included cases of nodular lymphocyte predominant Hodgkin's disease (NLPHD). Using an antibody to proliferating cell nuclear antigen (PCNA), we have determined the growth fraction of HRS cells and L&H cells in paraffin sections of 15 cases of classical HD [12 nodular sclerosis (NS), 3 mixed cellularity (MC)] and eight cases of NLPHD. By double staining with anti-PCNA and antibodies to B cells (CD20) and T cells (CD45RO), we also determined the growth fraction and immunophenotype of the background population in each case. In classical HD, 50.4 per cent of HRS cells were PCNA-positive and judged to be proliferating, which is comparable to previous studies, while in NLPHD 76.9 per cent of L&H cells were PCNA-positive. In both classical HD and NLPHD, the majority of PCNA-positive cells in the background were T cells, which showed a growth fraction of 57.8 and 68.5 per cent, respectively; in comparison, only 4 per cent of B cells were PCNA-positive in each type of HD. L&H cells are widely accepted to be B cells and there is growing evidence that HRS cells are also B cell-derived. Our results underline a relationship between classical HD and NLPHD and suggest that the characteristic histological features of both diseases may be caused by the production and release of cytokines from altered B cells.     

Proliferating cell nuclear antigen (PCNA) expression in formalin-fixed tissue of non-small cell lung carcinoma

An immunohistochemical study of non-small cell lung carcinoma using PC10, a monoclonal antibody against PCNA, was performed on tissues routinely processed with formalin fixation and paraffin embedding. The PCNA labelling index and mitotic index were determined from sections of these tissues. Tumours showed a high mean PCNA labelling index of 53.3%. The mean mitotic index was 10.3/1000 cells. Inter-examiner agreement of mitotic counting was good. A linear correlation between the PCNA labelling index and mitotic index was demonstrated (r = 0.71, P less than 0.00001). It is concluded that immunohistochemical nuclear labelling with anti-PCNA on routinely processed tissue is a simple technique for the assessment of proliferation in non-small cell lung carcinoma.     

Proliferating cell nuclear antigen (PCNA) and nucleolar organiser regions in Hodgkin's disease: correlation with morphology.

AIM--To define the distribution of proliferating cell nuclear antigen (PCNA) and silver staining nucleolar organiser regions (AgNORs) in Hodgkin's disease. METHODS--PCNA was shown in a series of 34 cases of Hodgkin's disease using immunohistochemical methods. In a second series of 46 cases the AgNOR technique for interphase nucleolar organiser regions was studied. Both series comprised routinely fixed and processed paraffin wax sections of three main Rye subtypes. RESULTS--In all cases, regardless of Rye subtype, most Sternberg-Reed cells and mononuclear Hodgkin cells showed nuclear PCNA immunoreactivity and such cells had 15 or more AgNOR sites. The Hodgkin cells had, in general, about half the number of AgNORs seen in Sternberg-Reed variants. CONCLUSIONS--These data support the notion that Hodgkin's disease can be regarded as a high grade lymphoma, the large Hodgkin's and Sternberg-Reed cells being the (PCNA positive and AgNOR rich) neoplastic elements with high proliferative capacity. A smaller proportion of the associated cells also showed evidence of proliferation.     

Proliferating cell nuclear antigen (PCNA) activity in hepatocellular carcinoma, benign peri-neoplastic and normal liver

Hepatocellular carcinoma (HCC) is among the ten most common cancers in Malaysian males. As cellular proliferation is an important feature of malignant transformation, we studied the proliferation pattern of normal and benign perineoplastic liver versus hepatocellular carcinoma in an attempt to further understand the tumour transformation process. 39 HCC (21 with accompanying and 18 without cirrhosis) histologically diagnosed at the Department of Pathology, University of Malaya Medical Centre between January 1992 and December 2003 were immunohistochemically studied using a monoclonal antibody to PCNA (Clone PC10: Dako). 20 livers from cases who had succumbed to traumatic injuries served as normal liver controls (NL). PCNA labeling index (PCNA-LI) was determined by counting the number of immunopositive cells in 1000 contiguous HCC, benign cirrhotic perineoplastic liver (BLC), benign perineoplastic non-cirrhotic (BLNC) and NL cells and conversion to a percentage. The PCNA-LI was also expressed as Ojanguren et al's grades. PCNA was expressed in 10% NL, 38.9% BLNC, 76.2% BLC and 71.8% HCC with BLNC, BLC and HCC showing significantly increased (p < 0.05) number of cases which expressed PCNA compared with NL. The number of BLC which expressed PCNA was also significantly increased compared with BLNC. PCNA-LI ranged from 0-2.0% (mean = 0.2%) in NL, 0-2.0% (mean = 0.3%) in BLNC, 0-3.6% (mean = 0.7%) in BLC and 0-53.8% (mean = 7.6%) in HCC with PCNA-LI significantly increased (p < 0.05) only in HCC compared with BLC, BLNC and NL. Accordingly, all NL, BLC and BLNC showed minimal (<5% cells being immunopositive) immunoreactivity on Ojanguren et al's grading system and only HCC demonstrated immunoreactivity which ranged up to grade 3 (75% of cells). From this study, there appears to be a generally increasing trend of proliferative activity from NL to BLNC to BLC and HCC. Nonetheless, BLNC and BLC, like NL, retained low PCNA-LI and only HCC had a significantly increased PCNA-LI compared with the benign categories. This is probably related to the malignant nature of HCC and may reflect the uncontrolled proliferation of the neoplastic hepatocytes.     

Proliferating cell nuclear antigen (PCNA) expression in tooth primordia in the field vole (Microtus agrestis,Rodentia)

Cell proliferation in developing tooth germs has been studied particularly using bromodeoxyuridine (BrdU) incorporation into growing tooth primordia and by counting and three-dimensional (3D) reconstruction of mitoses in serial sections of developing teeth. PCNA has been proposed as an alternative marker of proliferation activity. The aim of our study was to detect immunohistochemically locations of PCNA-positive cells in developing tooth germs of Microtus agrestis (Rodentia). PCNA expression could be distinguished in oral epithelium and mesenchyme before first signs of dental lamina elevation. During bud, cap, and bell stages, positive immunostaining could be observed at defined sites in enamel organ, tooth papilla, and dental follicle. Rudimental tooth germs of the upper diastema, enamel knots, and inner enamel epithelium at day of ontogeny 18 and 19 showed negative reaction. PCNA marks cycling and early G0 cells and can be used successfully as a proliferation marker even in collection material.     

Proliferating cell nuclear antigen in gallbladder carcinoma

Proliferating cell nuclear antigen (PCNA) expression was studied in 103 gallbladder carcinomas and 23 metastatic lesions as well as in 25 control non-neoplastic gallbladder specimens. Positive nuclear staining was observed in 88% of controls, in 92% of carcinomas and in 70% of metastases. The mean number of positive cells was 21.2% in controls, 44.1% in primary carcinomas and 32% in metastatic cancer cells. Differences which were significant were control v . primary tumour, P <0.000001; control v . metastasis, P <0.01 and primary tumour v . metastasis, P <0.006. In 57 (60%) of the primary tumours there was positive staining in over 40% of tumour cells. We were not able to demonstrate any relationship between macroscopic or microscopic features and PCNA expression. However, tumours confined to the mucosa expressed PCNA more frequently than did more advanced tumours.     

Proliferating cell nuclear antigen in neutrophil fate

The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack.     

Proliferating cell nuclear antigen in breast carcinomas

Proliferating cell nuclear antigen (PCNA), was examined by immunohistochemistry in 509 breast carcinomas. The immunoreactivity was found to be independent of the length of fixation when the tissue sections were microwaved before incubation with the primary antibody. The PCNA immunoreactivity was assessed by two semi-quantitative methods, which were correlated but not exchangeable. The comedo type of intraductal carcinomas and invasive ductal carcinomas had a higher PCNA score than other types. Lymph node metastases had a significantly higher PCNA score than primary carcinomas. High PCNA immunoreactivity was correlated with the presence of lymph node metastases, absence of tubule formation, numerous mitoses, severe nuclear pleomorphism, high histological grade and absence of progesterone receptors (PgR). PCNA in lymph node positive tumours was correlated with tumour type, especially with ductal carcinomas, absence of tubule formation, high histological grade and absence of PgR, whereas PCNA in lymph node negative tumours was correlated with large tumour size, numerous mitoses, severe nuclear pleomorphism and high histological grade. Number of mitoses and nuclear pleomorphism were the two most important factors in predicting the PCNA score; the absence of PgR and nuclear pleomorphism were important in lymph node negative and positive tumours, respectively. In a univariate analysis high PCNA score was found to be correlated with shorter relapse-free period and poorer over-all survival.     

Proliferating cell nuclear antigen expression in papillary thyroid carcinoma.

AIMS: To correlate the expression of proliferating cell nuclear antigen (PCNA) with various clinicopathological features of papillary thyroid carcinoma. METHODS: Sections from 131 formalin fixed, paraffin wax embedded papillary thyroid carcinomas were stained with a monoclonal antibody (PC10) directed against PCNA using the avidin-biotin immunoperoxidase (ABC) method. PCNA immunoreactivity was based on the PCNA labelling index (LI) following evaluation of at least 1000 tumour cells, and expressed as follows: grade A (LI < 10%), grade B (10% < or = LI < 25%), and grade C (LI > or = 25%). The relation between PCNA expression in these three groups and other clinicopathological factors, such as sex, age, tumour size, nodal metastases, and histological differentiation, were examined. RESULTS: Based on the labelling index, 57 (43.5%) cases were graded as A, 46 (35.1%) as B, and 28 (21.4%) as C. The female-:male ratios were 6.13:1 for group A, 2.83:1 for group B, and 2.11:1 for group C. The mean (SD) ages of the patients were 39.0 (16.1) in group A, 53.5 (14.4) in group B, and 55.8 (13.3) years in group C. The correlation between age and PCNA grade was strongest in women. CONCLUSIONS: PCNA immunoreactivity is correlated with sex and age in patients with papillary thyroid tumours.     

Proliferation in malignant mesothelioma as determined by mitosis counts and immunoreactivity for proliferating cell nuclear antigen (PCNA)

In order to assess its discriminating and prognostic value, we studied immunoreactivity for proliferating nuclear cell antigen (PCNA) in human malignant mesothelioma (31 cases) and in human non-neoplastic mesothelium (33 cases with reactive mesothelium and 20 cases of normal mesothellum) using the murine monoclonal antibody PC 10. We also compared it with mitosis counts expressed as the mitotic volume index (MV index). There were differences between malignant mesothelioma, reactive mesothelium, and normal mesothelium for percentage of PCNA immunoreactive cells (mean ± SD; 27 ± 9, 9·5 ± 5·1, and 3·6 ± 1·6, respectively) and for their MV index (20·3 ± 4·5, 9·4 ± 2·1, and 3·6 ± 0·6, respectively). The median actuarial survival was 10·1 months for patients with less than 25 per cent PCNA immunoreactive cells, 9·4 months for patients with less than 20 mitoses per mm² of tumoural tissue, 5·9 months for patients with more than 25 per cent PCNA immunoreactive cells, and 5·3 months for patients with more than 20 mitoses per mm² of tumoural tissue. Our results suggest that PCNA immunoreactivity is useful in discriminating between neoplastic and non-neoplastic mesothelium and that it may have prognostic value in malignant mesothelioma.     

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.     

Prognostic significance of proliferating cell nuclear antigen (PCNA) expression in gliomas

The relationship between proliferating cell nuclear antigen (PCNA) expression and various clinicopathological indices (age, sex, tumour location, histological type and grade and treatment) and post-operative survival were studied in patients with central nervous system gliomas using univariate and multivariate analysis. The expression of PCNA (PC10 score) was examined immunohistochemically using the monoclonal antibody PC10 on paraffin sections from 45 cases. Univariate analysis showed that a high PC10 score as well as older age, high histological grade and the histological type (astrocytoma) were associated with reduced survival. However, multivariate analysis revealed that only PC10 score and histological type had independent prognostic significance. The most important feature influencing PC10 score was the tumour grade. Regarding the patients who relapsed, the survival from the time of original diagnosis was related to the relapse-free period, while the PC10 score of the primary tumour emerged as the only independent predictor of survival following the first recurrence. These results indicate that PCNA expression is an independent prognostic indicator in CNS gliomas.     

Proliferating cell nuclear antigen immunostaining and survival in cerebral astrocytoma

In order to test the prognostic value of a proliferation index in cerebral astrocytoma, proliferating cell nuclear antigen immunostaining was performed on formalin-fixed and paraffin-embedded biopsies of 42 astrocytomas (21 serial stereotactic and 21 open surgical biopsies). Tumours were categorized as having a low (< 50%) or high (> 50%) labelling index. Tumour grading was also carried out. Several clinicotherapeutic factors were recorded. At least 40 months follow-up was available on all surviving patients. Survival estimated by the Kaplan and Meier method was significantly longer in tumours with a low proliferation index than in those with a high one (mean 20.4 months v. 10 months). According to Cox multiple regression analysis, the age of the patient and grading were significantly related to survival, whereas the proliferation index lost its significance.     

Proliferating cell nuclear antigen in normal and regenerating rat livers

Immunohistochemical analysis using proliferating cell nuclear antigen (PCNA) antibody shows a negligible number of cells stained in normal liver, but much higher numbers in regenerating liver 24 and 48 h after surgery. We also verified different results by biochemical analysis. Two forms of PCNA, L type (eluted at low concentrations of KCl from a phosphocellulose column) and H type (eluted at high KCl concentrations), were observed in the nucleoplasm of regenerating livers 24 and 48 h after surgery. Treatment of the H type fraction with nuclease caused the H type to disappear and the amount of L type to increase. PCNAs in the cytoplasm are P type (eluted in the pass through fraction) and L type. Surprisingly, the total amounts of P type and L type in cytoplasmic extracts are comparable to those of L type and H type in the nucleoplasm. These results suggest that newly synthesized PCNA is immediately converted into the P and L complex forms. The P type and some of the L type that lacks a nuclear localization signal remain in the cytoplasm; the rest of the L type with a nuclear localization signal is transferred into the nuclei. Then, some of the L type in the nucleoplasm forms the H type, which binds to DNA. These three types of PCNA are also found in significant amounts in the nucleoplasm and cytoplasm of normal rat liver despite its nonproliferating state.     

Expression of proliferative cell nuclear antigen (PCNA) in urinary bladder carcinoma. Evaluation of antigen retrieval methods.

Proliferative cell nuclear antigen (PCNA) expression was analyzed in formalin-fixed and paraffin-embedded specimens from patients with urinary bladder cancer using three different anti-PCNA monoclonal antibodies. In 20 recent cases a positive correlation was found between the extent and intensity of PCNA staining and grade of malignancy. In 95 specimens, three to six years old, extensive positive staining was detected in 15 and 23% of grade 2B and 3-4 tumors, respectively. No equivalent staining was found in the grade 1 and 2A tumors. In material more than six years old, a remarkably weak staining was observed regardless of grade. Similarly, in a test of archival material of tonsils a very weak immuno-reactivity was found as compared with fresh material. However, antigen retrieval by microwave heating of the tissue sections was possible in the majority of all cases, and the difference in extent and intensity of the staining between low and high grade tumors remained.     

Proliferating cell nuclear antigen. A marker for cell proliferation in autopsy tissues.

Antibodies to the proliferating cell nuclear antigen allow identification of proliferating cells in fresh tissue specimens using routine immunocytochemical methods. However, the use of such proliferation markers has not been verified for autopsy-derived tissue specimens, in which there is often a significant delay between the time of death and tissue specimen fixation. To assess the reliability of anti-proliferating cell nuclear antigen antibodies to identify proliferating cells in autopsy tissue specimens, an autopsy simulation was performed using fresh monkey and rat tissue specimens. These tissue specimens were kept at room temperature for predetermined numbers of hours before fixation. The proliferation specific staining was most reliable for tissue specimens obtained within 6 hours of death. There was reliable staining of proliferating regions up to 12 hours, although sensitivity was decreased. The only exception was skin, which was able to withstand much longer periods. Quantitative data from monkey spleen white-pulp regions showed 63% of the cells to stain for proliferating cell nuclear antigen when fixed immediately; this decreased to 29% of the cells after 12 hours and only 19% by 18 hours of postmortem simulation. Representative tissue specimens obtained from human autopsy material revealed similar postmortem staining patterns. Rapid procurement and fixation of tissue specimens and the use of control tissue specimens derived from the same autopsy material (eg, lymph node tissue) are recommended. These studies do suggest that anti-proliferating cell nuclear antigen antibodies can be used to identify proliferating cells in human autopsy tissue specimens obtained within approximately 12 hours of death, with some compromise in overall sensitivity.     

Proliferating cell nuclear antigen and S phase fraction in endometrial stromal sarcoma.

AIMS: To investigate the value of immunohistochemical staining for the cell cycle protein proliferating cell nuclear antigen (PCNA) and flow cytometric S phase fraction in determining prognosis in endometrial stromal sarcoma, graded according to mitotic count. METHODS: Seventeen endometrial stromal sarcomas from 13 patients treated at the Royal Marsden Hospital were analysed. Serial 5 microns sections were cut for haematoxylin and eosin and immunohistochemical staining for PCNA, performed using the murine monoclonal antibody PC10. PCNA positivity was expressed as a percentage of the total number of cells (PCNA index). Flow cytometric analysis was performed on nuclei extracted from paraffin wax sections. RESULTS: In the five patients who died of disease within five years, PCNA index varied between < 1% and 60% (mean 21%) and S phase fraction ranged from 11.3 and 20.1 (mean 13.8). Four patients who were apparently cured showed PCNA indices ranging from < 1% to 5% (mean 1.75%) and S phase fraction ranging from 1.4 to 3.5 (mean 2.3); and three patients alive with disease showed PCNA indices ranging from 1% to 15% (mean 8.6%) and S phase fraction ranging from 1.4 to 3.5 (mean 2.3). One patient who died from indolent local disease after nine years showed a PCNA of 1 or less and an S phase fraction of 0.9. CONCLUSIONS: PCNA staining was variable and therefore not a reliable prognostic indicator, but a high PCNA index was only found in those patients dying of disease within five years. A stronger association was seen between S phase fraction and prognosis; this also correlated well with histological grade determined by mitotic count. In individual borderline cases that are between low and high grade categories, these procedures may be useful.     

Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 in unicystic ameloblastoma

The expression of proliferating cell nuclear antigen (PCNA) and Ki-67 was studied in unicystic and solid ameloblastoma (follicular and plexiform types) using a biotin-streptavidin method on routinely processed paraffin sections. To determine percentage PCNA and Ki-67 labelling indices, positive tumour cells and total tumour cells were counted in areas of each unicystic ameloblastoma corresponding to cystic linings, intraluminal nodules and invading tumour islands, and in solid ameloblastomas. Positive cells in basal and suprabasal layers of cystic tumour lining were also counted with respect to the length of basement membrane determined by image analysis. In unicystic ameloblastoma the invading islands exhibited a significantly higher PCNA labelling index (29.2 ± 16.4%) than intraluminal nodules (13.6 ± 5.4%; P < 0.05). Cystic tumour lining had relatively few PCNA positive cells and a labelling index (5.5 ± 3.3%) significantly lower than invading islands (P < 0.001) or intraluminal nodules (P < 0.003). The labelling indices of solid ameloblastomas of follicular type (48.1 ± 12.9%) were significantly higher than those of cystic tumour lining (P < 0.0001), intraluminal nodules (P < 0.001) and invading islands (P < 0.04) in unicystic ameloblastoma. Similar relationships were found for Ki-67 expression except that comparisons involving invading islands and intraluminal nodules were not significant, a finding probably due to the smaller number of specimens available for quantitative analysis. These results indicate differences in proliferative potential between different areas of unicystic ameloblastoma and between unicystic and solid lesions. The fact that invading tumour islands within the fibrous tissue wall showed high labelling indices is in agreement with the clinical observation that their presence may be related to recurrence after conservative surgery. This provides a biological basis for indicating more radical surgical excision as the treatment of choice for this subgroup of lesions.