A COMPARISON OF THE BASAL CELL MARKERS LP34, p63 and MASPIN IN THE DIFFERENTIAL DIAGNOSIS OF PROSTATE CANCER IN NEEDLE BIOPSIES. Claire Krelle |
The diagnosis of prostate cancer from needle core biopsies is a challenging yet vitally important task facing a pathologist. Often the diagnosis cannot be made by light microscopy alone and so the use of an immunological technique is required. This technique, called immunohistochemistry uses commercially made antibodies that form a bond with certain tissue elements or antigens. Within the prostate it is the demonstration of the basal cell layer around the benign prostatic glands that excludes a diagnosis of cancer.
There are a number of antibodies which can be used to highlight basal cells. The most common are the high molecular weight (HMW) cytokeratins of which LP34 is one of the most widely used. This marker is found to be strongly positive in benign prostatic glands and negative in adenocarcinoma. There is, however, often weak staining in luminal cells (present in both) which causes problems with interpretation. For these reasons there is a need to find ways of improving the sensitivity and specificity of basal cell markers. The aim of this study was to make a comparison of three basal cell markers of the prostate: LP34, p63 and maspin, to determine the most effective marker for routine use in the histological diagnosis of prostate cancer.
P63 is expressed in the basal cells of many organs and irregularities in its expression have been associated with carcinogenesis. Many studies have looked at p63 expression in prostatic tissue and found it to be diagnostically useful in confirming the presence of adenocarcinoma in prostate specimens. P63 is strongly expressed in normal prostate, but negative in adenocarcinoma. Maspin is a serine protease inhibitor and has an integral role in many physiological and pathophysiological processes, including tumour invasion and apoptosis. To date most work carried out with maspin has been in human breast tissue. It has been shown to possess tumour suppressor activity for breast cancer progression and metastasis [ref]. There are relatively few studies directly linking maspin function to prostate cancer, however one study does confirm that maspin also has a tumour suppressive role in prostate cancer [ref]. Maspin is strongly expressed in benign prostatic tissue, with a loss of expression in adenocarcinoma.
This retrospective study used 212 formalin-fixed prostatic biopsy cases from Wycombe Hospital. 105 cases were benign and 106 had a diagnosis of adenocarcinoma. Three antibodies were used which required four slides for each case, one slide for each antibody and one for the negative control (no primary antibody applied). LP34 was already in routine use in the laboratory, however Maspin and p63 had not been used before and so suitable dilutions, pre-treatments and control tissue needed to be in place before the investigation could commence. For each of the antibodies a range of four dilutions were tried together with each of the pre-treatments; microwave, pressure cooker, chymotrypsin digestion and a combination of both microwave and chymotrypsin. Prostate chippings were used as the control tissue. Each case was stained with the three primary antibodies using a commercial kit on an automated staining machine. After staining was completed each slide was scored to reflect the efficiency of the antibody. The scoring was undertaken by a consultant Histopathologist specialising in Urology. The scoring system assessed the intensity of staining in each slide and each was allocated a grade between 0-3. The amount of background staining was also graded between 0-3.
Data was collated for benign and malignant cases, with the malignant
cases being categorized according to their Gleason score (tumour grade).
Positive staining in the malignant cases is found in benign tissue adjacent
to the tumour.
The analysis of the results was carried out using a two-tailed 95% confidence
test on a Microsoft excel spreadsheet. The analysis was carried out separately
on results for the benign cases and each Gleason score of the malignant
cases. The data was analysed with respect to intensity of staining, degree
of background staining and the differential between intensity and background.
In order to assess the staining value of LP34, p63 and maspin it was
necessary + to determine the mean score for staining intensity and the
mean score for background staining for each. The effectiveness of each
antibody in both benign and malignant biopsies is represented in the
graph below.
Figure 1. The mean efficiency of each antibody in benign and malignant
tissue
There is very little difference in the staining intensity between LP34 and p63, however the LP34 antibody produces considerably more background staining than does p63 in both benign and malignant biopsies. There is also more background staining in malignant biopsies with all three antibodies than there is in the benign biopsies. Overall the p63 antibody produces far less background staining than LP34.
The maspin antibody shows particularly weak staining intensity overall in both benign and malignant biopsies. There is also very little background staining as in many cases the antibody failed to stain any part of the biopsy. Maspin was therefore omitted from statistical analysis.
The statistical analysis of both the benign and malignant cases was carried out to ascertain any differences between LP34 and p63.
In the benign cases it was shown that there is no evidence at the 5% significance level of a difference between the intensity of staining between LP34 and p63. There was however extremely strong evidence at the 5% significance level of a difference in the efficiency of staining between LP34 and p63. p63 has significantly lower levels of background staining than LP34 as well as a significantly greater differential between staining intensity and levels of background than LP34.
The malignant biopsies were statistically assessed according to their Gleason scores. For each antibody in each Gleason group the mean background staining was subtracted from the mean staining intensity. This enabled the efficiency of each antibody in staining malignant biopsies to be ascertained. The statistical analysis showed at the 5% significance level there was strong evidence of a difference in the efficiency of staining between LP34 and p63 in each Gleason group. P63 has a significantly greater differential between staining intensity and levels of background than LP34.
Figure 2. Positive Staining with LP34
Figure 3. Positive Staining with p63
Figure 4. Positive Staining with maspin
In this study maspin proved to be unsuitable for use in the routine diagnosis of prostate biopsies. Suitable pretreatment and dilution proved to be problematic. When the standard protocol was used there was no positive staining seen where it was expected. It was believed this might be due to the fact that biopsy tissue, being small, could be subject to over-fixation by formalin, adversely affecting the staining ability of the maspin antibody, as the antigenic epitopes are not being unmasked. Varying the protocol did not overcome this problem to any great extent and despite achieving some positive staining in control biopsy slides the test cases were not staining as expected. Consequently, for the purposes of this study, maspin was not considered to be a suitable marker for the localization of basal cells in the prostate.
Since commencing this investigation, p63 has become the routine marker of choice for staining problematic prostate biopsy specimens within the histopathology department of Wycombe Hospital. By using LP34 in addition to p63 to stain particularly challenging cases has enhanced diagnostic ease.
Research summary dated 07 January 2006
Project 2003/14