| PLEIOTROPHIN
AND MIDKINE AS BIOMARKERS OF PROSTATE CANCER
Ms Emma Oldridge, Dr Martin Rumsby University of York |
 |
Introduction:
Worryingly for men, prostate cancer (PCa) has become the most common
cause of death in the UK and its incidence is increasing. This is
largely because of our longer life expectancy but also because there
is no effective treatment. However, as in all cancers, there is a
need to detect the disease as early as possible so treatment can
begin. In the UK a Prostate Cancer Risk Management has been introduced.
This includes advice about the prostate specific antigen (PSA) test.
PSA is a protein secreted by prostate cells: its level in blood can
rise in PCa but other prostate problems (infection, hypertrophy,
hyperplasia) may also lead to a rise in blood PSA. So an elevated
PSA level is not a definitive pointer of PCa and the PSA test is
subject to both false positive and false negative results. It would
be advantageous if other proteins which become elevated in PCa could
be identified to give a warning of early disease.
In cancer of the pancreas, colon, stomach, breast, brain, testes and lung researchers have identified two small proteins known as PTN (pleiotrophin) and MK (midkine) which increase in concentration and that become elevated in blood in the same disease. PTN and MK promote the cell survival, growth and migration: PTN also promotes tumour growth. In normal prostate PTN has been detected in stromal cells only; in PCa PTN is detected in epithelial cells as well. Clusters of epithelial cells in biopsies of invasive prostate carcinomas are PTN positive. MK is detectable in 80% of latent and 86% of clinical PCa cases but not in normal prostate. A positive correlation of MK protein expression with increasing severity has been reported in PCa.
Our current research has revealed that stromal cell preparations derived from PCa biopsy tissue secrete PTN. This summer project was to test an idea that this secreted PTN might be released to the blood where it could be assayed as a marker of PCa.
Aims of project:
i, to develop a method to measure levels of PTN in blood samples of
PCa patients
ii, to liaise with Dr D Ward at the Institute of Cancer Studies, University
of Birmingham, to compare PTN/MK assays.
Measuring PTN:
The project lasted 10 weeks and was undertaken by a second year biochemistry
undergraduate, Ms Emma Oldridge, to further her research career.
The interaction of an antibody with its target antigen is one of the most specific and sensitive reactions known. So we use this antibody-antigen reaction to measure the amount of PTN in blood samples. This is known as an immunoassay. Antibodies to PTN, bought commercially, are bound to special plastic in 5mm diameter wells set in a plate. Repeats are set up in triplicates for reproducibility. The antibody to PTN binds to the plastic surface, any uncoated sites being blocked with serum albumin. PTN is added and this binds to its antibody so is immobilised in the well. It is detected by adding another antibody to PTN which has a dye on it that gives a colour which can be measured. Thus the intensity of the blue colour reflects the amount of PTN initially bound. This sandwich arrangement is a common way of assaying proteins in solution. The reaction can be quantitated by adding known amounts of purified PTN to wells and assaying these along with the unknowns. Colour can be compared to give an estimate of the PTN in the unknown samples.
We developed the method and proved that the antibody to PTN did not recognise the closely related protein MK. This was important since MK could have interfered in the reaction by binding artifactually to the antibody to PTN. We showed that with this method we could detect PTN concentrations as low as 4ng/ml (0.004mg/ml) as shown in Fig. 1. However, this is not as sensitive as others have found in similar methods to detect PTN. In further experiments we found that adding blood serum to PTN samples resulted
Figure 1. Quantitisation of PTN in immunoassay from 0-300 ng/ml
in a lower detection sensitivity as if the serum was interfering in the reaction. We did not find this was so with plasma so we assayed 17 blood samples from PCa cases. Only 3 were positive for PTN above background with no correlation with severity of disease. We think that we have to increase the sensitivity of the method perhaps by testing other commercially-available antibodies to PTN in the sandwich. In addition we did not have proper control plasma samples from young healthy male donors. I have arranged to get these from the National Blood Service in the near future for further testing.
We visited Dr Ward in Birmingham to compare methods and results. He had assayed blood samples from liver and colon cancer for PTN. The PTN values in blood samples from liver patients (n=20) were elevated compared with controls (n=20) but were not in blood from colon cancer patients (n=17) compared with controls (n=23). He had also set up an immunoassay for MK. For lung cancer most samples (n=8) were below control levels (n=8) but some were as high as 10ng/ml though with no obvious link to the disease. For liver cancer MK values in control samples (n=20) were 0.4ng/ml while cancer samples (n=20) gave slightly higher values of 0.7ng/ml.
Conclusion:
Within the 10 week project we established an immunoassay for MK and
PTN which will now be refined to increase its sensitivity. We will
then re-assay blood samples from our bank of PCa samples using plasma
from healthy young males as controls.
Project summary, 18 January 2008.
Project 2006/51