Immunohistochemical methods using antibodies to cell cycle-related antigens may be used as a means of assessing various aspects of proliferation in tissue, and have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Currently, the most widely available antibodies for this purpose are antibodies to bromodeoxyuridine (BrdU), Ki67 and antibodies to proliferating cell nuclear antigen (PCNA). BrdU is a thymidine analogue incorporated during the S phase of the cell cycle, which can be introduced by ‘in vivo’ administration or by ‘in vitro’ incubation, and monoclonal antibodies are available to display its localization. Ki67 demonstrates a nuclear antigen expressed in all phases of the cell cycle, except G0 and early G1, but can only be applied to frozen tissue. PCNA is a nuclear antigen which is essential for DNA synthesis, two commercially available antibodies to PCNA work in paraffin-embedded tissue, but may have different staining characteristics under different conditions of fixation. The main advantages and disadvantages of these different techniques are discussed, together with their main applications to date.

The assessment of cellular proliferation by immunohistochemistry: a review of currently available methods and their applications.

The standard method for assessment of cell proliferation in paraffin-embedded tissue sections is 5-bromodeoxyuridine (BrdU) immunohistochemistry (IHC). BrdU can be administered to laboratory animals via IP injections, is readily incorporated into nuclei during the DNA synthetic phase of the cell cycle, and is detected with an anti-BrdU antibody. This method has several disadvantages, and an accurate method for evaluation of proliferative activity that can substitute for BrdU IHC, when necessary, is of great interest to investigators. Alternative methods for detection of proliferating cells in tissue sections are proliferating cell nuclear antigen (PCNA) IHC, Ki-67 IHC, and in situ hybridization (ISH) for histone mRNA. To determine the optimal choice, we analyzed the correlation of anti-PCNA, anti-Ki-67(MIB-5), and histone mRNA labeling indices (LIs) with anti-BrdU LI in rat highly replicative (renewing) tissues. The correlation between anti-BrdU and histone mRNA LIs, as well as the correlation between anti-BrdU and anti-Ki-67 LIs, was statistically significant. There was no significant correlation between anti-BrdU and anti-PCNA LIs. These results suggest that both ISH for histone mRNA and IHC with MIB-5 are preferable techniques for assessment of cell proliferation in rat paraffin-embedded renewing tissues compared to PCNA IHC. They can substitute for BrdU IHC when necessary.

Evaluation of cell proliferation in rat tissues with BrdU, PCNA, Ki-67(MIB-5) immunohistochemistry and in situ hybridization for histone mRNA.

A major stimulus to study cell proliferation, particularly in rodent carcinogenicity assays and human tumors, has been the belief that the quantification of this fundamental biological process will provide the toxicologist and pathologist with objective data allowing a better understanding of the mechanisms involved in the toxicity and/or carcinogenicity of certain compounds as well as guiding more effective management of patients afflicted with neoplasia. Among the markers used for cell proliferation measurement, PCNA has recently gained much attention and holds much promise as it is intricately involved in the cell replication processes. It not only could allow measurement of the replication rates without necessitating pretreatment of the animal/tissue in prospective studies, but also would allow retrospective assessment of the proliferative rates in archival tissues due to the conservation of this marker in fixed and paraffin-embedded tissues. Finally, knowledge of the function of PCNA in the cell cycle and its regulation by other factors may help us understand the advantages and limitations of PCNA as a cell proliferation marker in its application in toxicology and as a prognostic marker in human tumors.

/pdf/toxicological-and-pathological-applications-of-proliferating-51erjofw0z.pdf

Toxicological and Pathological Applications of Proliferating Cell Nuclear Antigen (PCNA), A Novel Endogenous Marker for Cell Proliferation

Helicobacter pylori causes Hyperproliferation of the gastric Epithelium: Pre- and Post-Eradication indices of proliferating cell nuclear antigen

To investigate the contribution of various tumor components to tumor [{sup 3}H]FDG uptake, the size of proliferative cell and macrophage populations and the extent of necrosis, inflammatory infiltration and granulation tissue formation were evaluated in syngeneic rat mammary cancers (RMC) grown in immunocompetent rats, an animal tumor model that closely mimics human breast carcinoma. Tissue components of breast cancers grown in female Lewis rats (n=6) were identified histologically and immunohistochemically. Tracer uptake was studied by quantitative autoradiography 2 hr after an intravenous injection of 100 {mu}Ci [{sup 3}H]FDG. RMC tumors were glandular, with small foci of necrosis and were surrounded by a thin layer of granulation tissue. Tumors retained approximately 4% of the injected FDG dose (1.9 {plus_minus} 0.27 {mu}Ci/g). Macrophages numbered 0.5% of total cancer cells (1.2 {plus_minus} 1.0 of 246 {plus_minus} 77) and 18.0% {plus_minus} 3.9% of the nuclei of cancer cells were proliferating cell nuclear antigen (PCNA) positive (52 {plus_minus} 27 of 293 {plus_minus} 55). FDG uptake (in apparent disintegrations per minute per microgram of protein) in the cancer cell was 47.3 {plus_minus} 5.6, with the highest uptake in foci of high tumor cell density (82.1 {plus_minus} 6.3). Lower levels of FDG uptake were found in necroticmore » areas (19.8 {plus_minus} 22.9), granulation tissue (26.9 {plus_minus} 9.2) and areas of inflammatory infiltraton (20.5 {plus_minus} 15.5). These data suggest that FDG-PET imaging of untreated breast cancer mainly reflects tracer uptake in cancer cells. 45 refs., 4 figs., 2 tabs.« less

http://jnm.snmjournals.org/content/36/10/1854.full.pdf

Intratumoral distribution of tritiated fluorodeoxyglucose in breast carcinoma: I. Are inflammatory cells important?

A new monoclonal antibody to proliferating cell nuclear antigen (PCNA), PC10, which can be used on routinely processed tissue, was applied to 93 cases of gastric carcinoma. Significant intra-tumoural variation in staining occurred. In addition to a PCNA index (percentage of positive cells per 1000 tumour cells), a semiquantitative PCNA grading system was devised, based on estimates of less than or more than 50% of positive tumour cells in whole sections. Neither PCNA index nor PCNA grade showed any correlation with established histological variables, tumour stage, or the presence of lymph node metastases. No significant correlation was observed between PCNA index and S + G2M phase fraction measured by flow cytometric analysis. To analyse survival tumours with PCNA indices above and below the median level (41%) were compared. Those with a higher index tended to have a worse prognosis, but when PCNA grade was considered, it was found to have definite independent prognostic value, tumours of low grade surviving better than those of high grade. The ability of semiquantitative PCNA grading to allow for intra-tumoural variation suggests it may have advantages over absolute counting, which is prone to sampling error when tumour heterogeneity is a major factor. The prognostic value of PC10 staining in gastric carcinoma is therefore promising.

https://jcp.bmj.com/content/jclinpath/44/8/655.full.pdf

M I Filipe

Papers

Prognostic value of proliferating cell nuclear antigen in gastric carcinoma