| ACCURATE
DIAGNOSIS AND STAGING OF PROSTATE CANCER USING RT-PCR IDENTIFICATION
OF SENSITIVE SPECIFIC MARKERS
Dr Christiane Fenske, et al below St George's Hospital Medical School, London |
Background
The accuracy of current diagnostic methods for prostate cancer, including
serum PSA measurements and biopsy, has been questioned and has been
the subject of much recent debate.
On the basis of these tests, men have been recommended to undergo often
unnecessary surgery (radical prostatectomy), which itself carries side
effects and reduces quality of life.
There is a need for the development of new, non-invasive and sensitive
diagnostic and prognostic CaP tests for the accurate diagnosis of stage
of development of prostate cancer and monitoring response to therapy
or surgery.
Research aims
Our aim is to develop a molecular test for early diagnosis of prostate
cancer and the accurate identification of the stage of development,
thus enabling accurate tailor made therapy, avoiding unnecessary treatment
and side effects. In addition, the aim is make this test non-invasive,
thereby encouraging men to seek medical attention early on, and enable
early treatment.
Molecular research
The reverse transcriptase-polymerase chain reaction (RT-PCR) is a powerful
and sensitive technique that is able to detect prostate cells in patients’ blood
samples. The method is so sensitive that one prostate cell in 100 million
blood cells can be picked up.
The method uses markers or genes - molecular flags - that
identify the cells as being from the prostate and no other organ. The
more specific and sensitive the marker is the more accurate the diagnosis
is and the more likely it is to pick up prostate cells in the blood.
We have developed relative quantitative RT-PCR whereby we can measure
the actual levels of the cancer markers in the blood. Markers may be
turned up or down with the progression of prostate disease, and different
levels are demonstrated at different stages of the disease. These levels
may be used in diagnosis and to monitor a patient’s response to
therapy.
We have correlated our qRT-PCR results with known patient diagnosis and
known stage of disease development. This has enabled us to determine
the accuracy and strength of the marker in potential diagnosis of prostate
cancer.
Research Developments
The patients: 400 patients attending the
Uro-oncology out patients clinic at St George’s and consenting
to take part in our study, were grouped according to their clinical diagnosis.
This relied exclusively on a prostate cancer database set up at St George’s
Hospital in 1995. This database includes information on the method of
diagnosis, grade, and stage of any tumour present together with serum
PSA values. The database is vital for our work- the diagnosis and clinical
details of each patient whose blood we use in our analyses must be accurate
to enable correlation of our results with the clinical diagnosis.
Markers: The markers we decided to include in our study were identified through literature searches and prioritised according to their characteristics. Our experiments showed that each marker has its own peculiar expression profile; different levels at different stages of the disease. Some markers may have high levels early on in the development of the disease (possibly playing a part in the establishment of the tumour). At later stages, these may be turned down or off and other markers increase in levels.
We concluded that the principles of a diagnostic test able to distinguish between all stages of prostate cancer development are :
- It must involve several markers - each specifically regulated and sensitive to the different stages.
- That no single stand alone marker would be able to be used to give all information necessary for accurate diagnosis.
- That a combination of markers may be used to increase the accuracy of diagnosis of any one stage - specific markers with low sensitivity may be combined with sensitive markers with low specificity.
The enormous amount of data which resulted from our relative quantitative
RT-PCR on the patient samples, then underwent statistical analysis.
Statistical analysis
Statistical analysis identified extremely specific and sensitive markers
which are excellent in the differential diagnosis between cancer and
non-cancer patients. Even greater accuracy and sensitivity could be
achieved by combining the results of some markers.
Having diagnosed a patient as having or not having the disease, the second
phase of the test when it is present would be to differentiate between
the individual developmental stages of the disease.
Analysis identified several excellent markers which give accurate diagnosis
at all stages of disease development and others which are excellent in
diagnosing the more specific stages. Again, the strength of these markers
could be increased by combining their use.
Additional interesting results for potential inclusion in test
Response to therapy
Specific markers were shown to discriminate accurately between metastatic
and benign disease. This could be potentially useful in monitoring
a patient’s response to therapy.
If the levels of these markers were shown to increase in a similar way
to metastatic disease levels, this may alert the clinician to the fact
that the therapy used may not be effective and that there is a continuing
risk of the cancer developing to the dangerous metastatic stage. This
would possibly indicate the need for alternative or more aggressive therapy.
However, if therapy is successful, marker levels may be seen to decrease
to those seen in patients with benign disease.
Inclusion of these markers then would make the test useful in determining
response to therapy.
Alternatively spliced markers
Alternative splicing is a phenomenon shown to be associated with tumour
development.
Mutations may result in additional material spliced into the mRNA, or
material may be spliced out, resulting in larger or smaller products
on RT-PCR than those expected or calculated. These alterations in the
mRNA may occur before the tumour forms and as such, it has the potential
of diagnosing risk of tumour development. Identification of this phenomenon
is very easily achieved alongside the main test. If seen in benign patient
samples, then this may indicate risk of cancer development.
We have identified several markers which show the possibility of alternative
splicing.
Markers showing an indication of aggressiveness of disease
One marker shows alternative splicing in a cell line which derives from
an aggressive form of prostate cancer. The fact that this alternative
spliceform does not appear in other cell lines, indicates that it may
be associated with an aggressive form of prostate cancer.
There are other markers that have been reported to be associated with
more aggressive forms of prostate cancer. If these were to be included
in the test marker panel, then not only would they add to the diagnostic
power of the test, but also be able to indicate speed of potential disease
progression. This in turn would enable appropriate choice of therapy.
Summary
We are well on the way to developing a non-invasive molecular test that
is able to diagnose prostate cancer accurately.
Strong markers have been identified that may be included in the test:
- in first line diagnostic approach i.e. diagnosing cancer from non-cancer
- in a subsequent stage, distinguishing the different developmental stages
- to monitor response to therapy
- to combine increasing sensitivity with accuracy of diagnosis
- to indicate the speed of disease development and therefore appropriate therapy
- to determine any risks of tumour development at a very early stage
A test that is able to give this accurate information will result in appropriate choice of effective therapy, avoiding unnecessary treatment, surgery and side effects.
Patent
A patent application has been filed covering this work. The filing was
completed in November 2005.
Further work is being undertaken, together with WestFocus, regarding
the next stages in the development of the product towards a format where
it may be used in routine diagnosis.
The research team
Dr Christiane Fenske
Christodoulos Pipinikas (PhD student)
Sabarinath Nair (Clinical Research Fellow-/ MD student)
Professor Nick Carter (Biochemist)
Dr Cathy Corbishley (Histopathologist/ CaP database)
Professor Roger Kirby (Consultant Urologist)
Research summary dated 05 December 2005
Project 2003/09