David A. Levison

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

SummaryImmunohistochemical 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.

Immunohistochemical detection of p53 protein in mammary carcinoma: An important new independent indicator of prognosis?

In an immunohistochemical pilot study of 195 primary breast cancer patients with a 10-year median follow-up we found that patients with carcinomas who express p53 protein in the majority of their tumor cells (19% of the cases) have a considerably worse prognosis than those who do not. The effect of the presence of the protein is seen on disease-free interval (chi-square, 11.69; P < .001), overall survival (chi-square, 19.68; P < .001), and survival after relapse (chi-square, 4.93; P < .02), and is seen in node-negative (chi-square, 6.99; P < .009) and node-positive (chi-square, 13.05; P < .001) patients. Furthermore, the effect is most apparent in patients with infiltrating lobular and grade II infiltrating ductal carcinomas (chi-square, 27.97; P < .001) that have a rather heterogeneous clinical behaviour and are difficult to subdivide on the basis of currently available markers. Cox multivariate analysis shows that p53 majority staining is second only to node status in significance of effect on overall survival.

The assessment of proliferating cell nuclear antigen (PCNA) immunostaining in primary gastrointestinal lymphomas and its relationship to histological grade, S + G2 + M phase fraction (flow cytometric analysis) and prognosis

PCNA is a nuclear protein that is synthesized in late G1 and S phases of the cell cycle and is, therefore, correlated with the cell proliferative state. A new monoclonal antibody (PC10) to genetically engineered PCNA has been shown to label proliferating cells in formalin‐fixed paraffin‐embedded normal human tissues. Previous studies in lymphomas, using various markers of cell proliferation, have shown a strong correlation between indices of cell proliferation and histological grade. These studies have shown that within each histological subtype there is often a wide range of prolifeative indices and that these may be of some prognostic significance. Thirty‐one gastrointestinal lymphomas were studied. Our results show that there is a good correlation between PC10 index and histological grade of tumour (0.01 > P>0.001) and also a significant relationship between PC10 index and S + G2 + M phase fraction as measured by flow cytometric analysis (r2= 0.62; P≤0.01). Twenty‐three cases were available for survival analysis. In these cases a high PC10 score correlated with poor survival (P=0.04). Based on this series, it appears that there is a significant relationship between PC10 index and histological grade, and between PC10 index and S+G2+M phase as measurred by flow cytometric analysis. In addition, our results suggest that a high PC10 index is an adverse prognostic factor primary gastrointestinal lymphoma.

Prognostic value of proliferating cell nuclear antigen in gastric carcinoma.

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.

Haemangiopericytomas: the prognostic value of immunohistochemical staining with a monoclonal antibody to proliferating cell nuclear antigen (PCNA)

Forty‐tow cases of haemangiopericytoma were studied retrospectively using immunohistochemical staining with PC10, a monoclonal antibody to PCNA. The percentage of tumour cells with positive staining for PCNA was found to correlated well with histological grading. Clinical follow‐up data were available in 25 adults and showed no known deaths in 11 cases with a low proportion (<14%) of positive cells. Out of 14 cases with a high number (≥14%) of positive cells, seven patients are known to have died, two had metastases, and in a further two there have been multiple recurrences of tumour. DNA flow cytometry was performed on 26 cases but this showed no correlation with PC10 staining or clinical outcome. Staining with PC10 may be of particular value in the identification of patients at greatest risk of rapid tumour metastasis and early death.

The prognostic value of individual histologic grading parameters in small lingual squamous cell carcinomas. The importance of the pattern of invasion

Background. The histologic grading of the deep invasive margin of oral squamous cell carcinoma recently has been shown to have prognostic value, but previous series have not been homogeneous enough to allow grading parameters to be assessed individually.

Aberrant expression of p53 tumour‐suppressor protein in non‐melanoma skin cancer

Expression of the cellular p53 tumour‐suppressor protein was examined in 78 epidermal tumours, including basal and squamous cell carcinomas, keratoacanthomas, solar keratoses, Bowens disease and viral warts. An immunohistochemical study was employed using the antibody CM‐1, raised against recombinant human p55 protein. Positive staining for p53, not detectable in normal cells because wild‐type p53 is rapidly degraded, reflects abnormal stabilization of p53 protein, and in many cases suggests p53 gene mutation.

p53 immunoreactivity in human malignant melanoma and dysplastic naevi.

Expression of the tumour suppressor protein, p53, was determined in 77 cutaneous melanocytic lesions, and in five lymph node metastases from malignant melanoma, in an immunohistochemical study employing CM‐1, an antiserum raised against recombinant human p53 protein. Because wildtype p53 protein is rapidly degraded in normal cells, p53 immunoreactivity suggests the presence of an abnormally stable p53 protein. This may occur through either post‐translational mechanisms or gene mutation.

Posttransplant cutaneous lymphoma

BACKGROUND Posttransplant lymphoma is closely associated with Epstein-Barr virus (EBV) infection and appears to have a predilection for extranodal sites. We describe four cases of primary cutaneous posttransplant lymphoma. OBJECTIVE Our aim was to determine cell lineage and any possible association with EBV in each case of cutaneous lymphoma. METHODS Tumor tissue was examined by light microscopy, immunohistochemistry, nonisotopic in situ hybridization and polymerase chain reaction. RESULTS The data were consistent with a diagnosis of EBV-associated cutaneous B-cell lymphoma in three cases and cutaneous T-cell lymphoma not associated with EBV in one case. No patient with B-cell lymphoma had extracutaneous involvement during a mean follow-up of 3.9 years. The patient with cutaneous T-cell lymphoma died of cerebral involvement 9 months after initial presentation. CONCLUSION These data suggest a possible role for EBV infection in the origin of cutaneous B-cell lymphoma in immunosuppressed patients.

Exogenous pigment in Peyer's patches

Dark brown granular pigment was found consistently in macrophages in the deep aspect of adult Peyers patches. Tissue sections from intestinal resections of 35 patients with a variety of pathologic diagnoses and of seven postmortem cases with no evidence of gastrointestinal disease were examined for the presence of this pigment. It was found in all patients over the age of 6 years (34 cases) but was not found in any children below that age (eight cases). Scanning electron microscopy with secondary and backscattered electron imaging and x-ray energy spectroscopy were performed on routine histologic sections. The pigmented macrophages contained aluminum and silicon, diffusely present throughout the cytoplasm, and numerous discrete foci of titanium. Pigment containing these same elements has also been found around dilated submucosal lymphatics, in mesenteric lymph nodes, and in some transmural inflammatory aggregates of Crohns disease. The pigment probably is derived from the diet and actively taken up by Peyers patches, which are able to incorporate inert particulate matter.