**1. Introduction**

100 Prostate Cancer – Diagnostic and Therapeutic Advances

Yao, V., Berkman, C.E., Choi, J.K., O'Keefe, D.S. & Bacich, D.J. (2009). Expression of prostate-

Zhong, X.S., Matsushita, M., Plotkin, J., Riviere, I. & Sadelain, M. (2010). Chimeric antigen

Zhou, J., Neale, J.H., Pomper, M.G. & Kozikowski, A.P. (2005). NAAG peptidase inhibitors

folic acid. *Prostate,* Vol. 70, No. 3, pp. 305-316.

*Mol Ther,* Vol. 18, No. 2, pp. 413-420.

pp. 1015-1026.

specific membrane antigen (PSMA), increases cell folate uptake and proliferation and suggests a novel role for PSMA in the uptake of the non-polyglutamated folate,

receptors combining 4-1BB and CD28 signaling domains augment PI3kinase/AKT/Bcl-XL activation and CD8+ T cell-mediated tumor eradication.

and their potential for diagnosis and therapy. *Nat Rev Drug Discov,* Vol. 4, No. 12,

Prostate biopsy is a motor, driving force and entrance ticket for dealing with parade discipline in urology of recent years – prostate cancer. Therefore it receives a lot of attention and standard techniques are constantly challenged by new developments. Enormous amount of literature seem to have addressed all possible aspects regarding prostate biopsy. This chapter focuses less on historical overview but mainly on recent developments, controversies and questions.

Prostate cancer has already become leading cancer among males in developed world (Jemal et al., 2011), second reason for death due to cancer among men in US (Jemal et al., 2010) and third in Europe (Malvezzi et al., 2011). Future is looking even more serious – numbers are expected to rise much further, as recently predicted in Canadian forecast analysis (Quon et al, 2011). The authors claim expected estimates of increase in prostate cancer cases should not be limited only to aging of population, which is huge itself and is expected to cause 39% increase in prostate cancer cases. At least three further factors should be taken into account. First and most important is lowering of PSA threshold for biopsy. It seems the move of decreasing PSA cutoff from 4 to 2.5 is getting from university centers to every urologist's and generalist's office (this is especially important as they are the ones who pick and refer patients to urologists for biopsy). Increase in number of people, referred to biopsy for this reason is estimated to be much greater compared to aging of population and may increase prostate cancer incidence by 200%. If it will increase prostate cancer incidence by 200%, increase in number of biopsies should be disproportionally higher, as biopsies have lower yield for this new target population with PSA values between 2.5 and 4. PSA screening is creating at present a lot of debate, it is a very hot topic and there are very strong opponents and supporters. Introduction of formal screening would of course increase burden of cancer and burden of biopsy. It is at present unlikely to happen, probably because people who decide on health policies and their advisers do not meet, treat and care for people with advanced prostate cancer. But, call it case finding or however one prefers, a "non-formal" screening programs are actually already available in many health systems, not only in selected first adopters, like Tyrol in Austria (Oberaigner et al., 2006), but also for example in Slovenia, where every general practitioner has available extra funds for PSA measurement on all of his male patients, every two years. Extent of "nonformal" screening programs can be seen from well-known PLCO trial, where control,

Future of Prostate Biopsy: Who Will Get It and How? 103

are not catalytically active. Active PSA in part undergoes further degradation/internal cleavage by different proteases, thereby also forming inactive PSA. In seminal plasma approximately 30% of PSA is in active form. Remaining PSA represents different forms of inactive PSA. Some PSA (all forms) leaks to circulation and can be measured in serum. Active form of PSA in serum binds (complexes) to protease inhibitors, mostly to alpha1 antichymotrypsin (ACT). It represents main part of total serum PSA (70-90%) and can be measured as cPSA. Catalytically inactive PSA forms circulate freely in serum as they are not complexed to ACT or other inhibitors. This fraction represents main result of free PSA assay. Antibodies were also developed for measuring serum concentrations of specific PSA isoforms, for example [-5]pro PSA and [-2]proPSA. Assays which measure total PSA aim at

As prostate cancer characteristic is disruption of basal cell layer and basement membrane, this allows increased amount of PSA and its isoforms to enter circulation (Balk et al, 2003). Free PSA fraction is decreased in serum of cancer patients. It is hypothesized there is relatively less free PSA in serum in cancer patients because more PSA enters circulation directly and complexes immediately to protease inhibitors. As there is less exposure of PSA to luminal and seminal fluid proteases, there is less chance for inactivating and producing inactive, free PSA. ProPSA isoforms are increased in serum of prostate cancer patients. Two hypotheses aim to explain this. One believes there is decreased cleavage of proPSA by hK2 in prostate cancer tissue (Balk et al., 2003). Other hypothesis believes proPSA isoforms are increased in cancer as a result of increased proPSA production from benign looking cancer

Higher serum PSA values were related to prostate cancer in 1980-ties. Hybritech, first commercial PSA measurement kit manufacturer, identified in a small study in 1986 in their sample 99% of tested men (different ages) had PSA below 4 and suggested this value as a cut-off. With this in hand, Catalona conducted a trial and published in 1991: 8% of tested men (mean age 68) had PSA values above 4, 22% of those with PSA between 4 and 10 had prostate cancer detected on sextant biopsy (and 67% among those with PSA above 10). Biopsy was performed using 18G needle. PSA was independent prognostic factor and positive DRE increased chance of positive biopsy (Catalona et al., 1991). 20 years later – we made a progress – now we do 12 core biopsies, use PSA cut-off 2.5 and use some other PSA derivatives to help in decisions. This modifies our outcomes a few percentages here and there, but main achievement, decrease in mortality due to prostate cancer, which was, after two decades of doubt, finally recently confirmed by a few large independent trials from both US and EU (see Introduction), was a result of a twenty years old approach, which is, with all its drawbacks and deficiencies, still valid and widely practiced around the world. Inadequacy of PSA cut-off value of 4 was in depth explained by publication of results of control biopsies in prostate cancer prevention trial (Thompson et al., 2004). They found, among men in age range 62 to 91, median 69, 6.6% of cancers among those with PSA 0.5 or less and 12.5% among them with Gleason 7 or more (=0.8% of all men with median age 69 with PSA below 0.6 had important cancer). Among men with PSA of 1.1 to 2.0, cancer was found in 10% and 10% of those had Gleason 7 or more (=1% of all men with PSA between 1 and 2, median age 69, had Gleason 7 or higher prostate cancer). For PSA 2-3, the percentage of Gleason 7 or more among all tested was 4.6% and for PSA 3-4 it was 6.7%. Those numbers were found in a prescreened population and with sextant biopsy. Now it is believed those numbers are still accurate for overall cancer presence, but for Gleason 7 or higher cancers in general referral population, real numbers are about twice those estimates. Several thoughts

detecting all isoforms, active (complexed) and inactive PSA.

associated areas in prostate gland (Makarov et al., 2009).

supposedly non-screened arm had 50% screening in comparison to intervention arm, supposedly "screened", where the rate was approximately 85% (Andriole et al., 2009). Some do not believe into usefulness of screening (Miettinen, 2010) and results of studies, which clearly show improved survival and necessity for screening, for example of European Randomized Trial of Screening for Prostate Cancer (Schröder et al., 2009). Despite those doubts, screening in one way or another is in fact increasingly taking place in every-day life. Further increase in screening is expected because recent analyzes of prostate screening studies seem to greatly reduce predicted numbers needed to screen and numbers needed to treat to save a life in prostate cancer and dispel previous doubts about usefulness of screening (Crawford et al., 2011) and treatment (Bill-Axelson et al., 2011).

Last reason for estimation of huge increase in prostate cancer cases expected in the next decade is stated (Quon et al., 2011) as "improved sensitivity of prostate biopsy". Will this really happen? This review will summarize some of the present developments in this field. If this is really the case, it may be at least one good sign, sign of relief: with improved biopsy specificity, number of repeat biopsies, which at present already represent significant burden and may also represent group of patients with higher risk for biopsy-related complications, may decrease.

Prostate biopsy aims to detect prostate cancer with as little problems and sequela as possible. Further, it aims to predict prognosis of detected cancer, therefore sampling must not only take any cancer, but representative cancer tissue samples from possibly all cancer focuses in the prostate. Third goal of modern prostate biopsy is to guide targeted therapy, which is expected to rise with increased number of lower stage cancers detected.

### **2. Selection of biopsy candidates**

Only main criteria, which are in clinical use or are available and may be used in Europe in 2011 are included here and they are: 3 PSA's (total, free and -2proPSA), urine marker (PCA3), race, family history, previous biopsy technique and result, age and life expectancy.

There are markers, which were proposed and also used in the past, but are now outdated, like cPSA and other PSA's, not mentioned above. There are markers, which may become very useful in the future, but are at present not ready yet (for example SNP's and other genetic data or new urine markers, which may use advantage of being excreted and therefore no aggressive rectal prostate massage would be necessary (Pandha et al., 2011)).

Should we really biopsy all men with PSA above 2.5? Obviously we do not want to do that. It remains unresolved task, how should we select between those who would benefit from biopsy even earlier, at PSA 1, from those who do not need biopsy at PSA 10.

#### **2.1 PSA's**

Prostate specific antigen, during history of its discovery named with other names, for example semenogelase, is an androgen-regulated serine protease (EC 3.4.21.77), human tissue kallikrein 3 (Balk et al, 2003). Gene is located on 19q13.4. Mainly it is produced in secretory epithelial cells in prostate acini and ducts and secreted into lumen. Its function is in ejaculate liquefaction.

PSA is expressed in preproPSA form (17 aminoacid leading peptide), excreted into prostate lumen as proPSA (7 aminoacid propeptide on N-terminal) and activated by trypsine-like human kallikrein 2 (hK2), which is expressed on prostate secretory epithelial cells (Balk et.al, 2003). ProPSA can undergo cleavage at position -7, removing 7 aminoacid propeptide to form active PSA. ProPSA cleavage can also occur at positions -5 or -2. Those PSA isoforms

supposedly non-screened arm had 50% screening in comparison to intervention arm, supposedly "screened", where the rate was approximately 85% (Andriole et al., 2009). Some do not believe into usefulness of screening (Miettinen, 2010) and results of studies, which clearly show improved survival and necessity for screening, for example of European Randomized Trial of Screening for Prostate Cancer (Schröder et al., 2009). Despite those doubts, screening in one way or another is in fact increasingly taking place in every-day life. Further increase in screening is expected because recent analyzes of prostate screening studies seem to greatly reduce predicted numbers needed to screen and numbers needed to treat to save a life in prostate cancer and dispel previous doubts about usefulness of screening

Last reason for estimation of huge increase in prostate cancer cases expected in the next decade is stated (Quon et al., 2011) as "improved sensitivity of prostate biopsy". Will this really happen? This review will summarize some of the present developments in this field. If this is really the case, it may be at least one good sign, sign of relief: with improved biopsy specificity, number of repeat biopsies, which at present already represent significant burden and may also represent group of patients with higher risk for biopsy-related complications, may decrease. Prostate biopsy aims to detect prostate cancer with as little problems and sequela as possible. Further, it aims to predict prognosis of detected cancer, therefore sampling must not only take any cancer, but representative cancer tissue samples from possibly all cancer focuses in the prostate. Third goal of modern prostate biopsy is to guide targeted therapy,

Only main criteria, which are in clinical use or are available and may be used in Europe in 2011 are included here and they are: 3 PSA's (total, free and -2proPSA), urine marker (PCA3), race,

There are markers, which were proposed and also used in the past, but are now outdated, like cPSA and other PSA's, not mentioned above. There are markers, which may become very useful in the future, but are at present not ready yet (for example SNP's and other genetic data or new urine markers, which may use advantage of being excreted and therefore no aggressive rectal prostate massage would be necessary (Pandha et al., 2011)). Should we really biopsy all men with PSA above 2.5? Obviously we do not want to do that. It remains unresolved task, how should we select between those who would benefit from

Prostate specific antigen, during history of its discovery named with other names, for example semenogelase, is an androgen-regulated serine protease (EC 3.4.21.77), human tissue kallikrein 3 (Balk et al, 2003). Gene is located on 19q13.4. Mainly it is produced in secretory epithelial cells in prostate acini and ducts and secreted into lumen. Its function

PSA is expressed in preproPSA form (17 aminoacid leading peptide), excreted into prostate lumen as proPSA (7 aminoacid propeptide on N-terminal) and activated by trypsine-like human kallikrein 2 (hK2), which is expressed on prostate secretory epithelial cells (Balk et.al, 2003). ProPSA can undergo cleavage at position -7, removing 7 aminoacid propeptide to form active PSA. ProPSA cleavage can also occur at positions -5 or -2. Those PSA isoforms

which is expected to rise with increased number of lower stage cancers detected.

family history, previous biopsy technique and result, age and life expectancy.

biopsy even earlier, at PSA 1, from those who do not need biopsy at PSA 10.

(Crawford et al., 2011) and treatment (Bill-Axelson et al., 2011).

**2. Selection of biopsy candidates** 

**2.1 PSA's** 

is in ejaculate liquefaction.

are not catalytically active. Active PSA in part undergoes further degradation/internal cleavage by different proteases, thereby also forming inactive PSA. In seminal plasma approximately 30% of PSA is in active form. Remaining PSA represents different forms of inactive PSA. Some PSA (all forms) leaks to circulation and can be measured in serum. Active form of PSA in serum binds (complexes) to protease inhibitors, mostly to alpha1 antichymotrypsin (ACT). It represents main part of total serum PSA (70-90%) and can be measured as cPSA. Catalytically inactive PSA forms circulate freely in serum as they are not complexed to ACT or other inhibitors. This fraction represents main result of free PSA assay. Antibodies were also developed for measuring serum concentrations of specific PSA isoforms, for example [-5]pro PSA and [-2]proPSA. Assays which measure total PSA aim at detecting all isoforms, active (complexed) and inactive PSA.

As prostate cancer characteristic is disruption of basal cell layer and basement membrane, this allows increased amount of PSA and its isoforms to enter circulation (Balk et al, 2003). Free PSA fraction is decreased in serum of cancer patients. It is hypothesized there is relatively less free PSA in serum in cancer patients because more PSA enters circulation directly and complexes immediately to protease inhibitors. As there is less exposure of PSA to luminal and seminal fluid proteases, there is less chance for inactivating and producing inactive, free PSA. ProPSA isoforms are increased in serum of prostate cancer patients. Two hypotheses aim to explain this. One believes there is decreased cleavage of proPSA by hK2 in prostate cancer tissue (Balk et al., 2003). Other hypothesis believes proPSA isoforms are increased in cancer as a result of increased proPSA production from benign looking cancer associated areas in prostate gland (Makarov et al., 2009).

Higher serum PSA values were related to prostate cancer in 1980-ties. Hybritech, first commercial PSA measurement kit manufacturer, identified in a small study in 1986 in their sample 99% of tested men (different ages) had PSA below 4 and suggested this value as a cut-off. With this in hand, Catalona conducted a trial and published in 1991: 8% of tested men (mean age 68) had PSA values above 4, 22% of those with PSA between 4 and 10 had prostate cancer detected on sextant biopsy (and 67% among those with PSA above 10). Biopsy was performed using 18G needle. PSA was independent prognostic factor and positive DRE increased chance of positive biopsy (Catalona et al., 1991). 20 years later – we made a progress – now we do 12 core biopsies, use PSA cut-off 2.5 and use some other PSA derivatives to help in decisions. This modifies our outcomes a few percentages here and there, but main achievement, decrease in mortality due to prostate cancer, which was, after two decades of doubt, finally recently confirmed by a few large independent trials from both US and EU (see Introduction), was a result of a twenty years old approach, which is, with all its drawbacks and deficiencies, still valid and widely practiced around the world.

Inadequacy of PSA cut-off value of 4 was in depth explained by publication of results of control biopsies in prostate cancer prevention trial (Thompson et al., 2004). They found, among men in age range 62 to 91, median 69, 6.6% of cancers among those with PSA 0.5 or less and 12.5% among them with Gleason 7 or more (=0.8% of all men with median age 69 with PSA below 0.6 had important cancer). Among men with PSA of 1.1 to 2.0, cancer was found in 10% and 10% of those had Gleason 7 or more (=1% of all men with PSA between 1 and 2, median age 69, had Gleason 7 or higher prostate cancer). For PSA 2-3, the percentage of Gleason 7 or more among all tested was 4.6% and for PSA 3-4 it was 6.7%. Those numbers were found in a prescreened population and with sextant biopsy. Now it is believed those numbers are still accurate for overall cancer presence, but for Gleason 7 or higher cancers in general referral population, real numbers are about twice those estimates. Several thoughts

Future of Prostate Biopsy: Who Will Get It and How? 105

High increase of PSA with infection or any irritation of prostate is well known and sometimes antibiotic or anti-inflammatory treatment is suggested and repeat measurement. PSA decreases in high body-mass index-correction and decreased of cutoff values may be needed (Pater et al., 2011). Diseases (liver cirrhosis) and medications (NSAID's, thiazides and statins) may also decrease PSA values (Nieder et al., 2011). If using free PSA values as trigger for biopsy decisions, it may be important to note that use of herbal products, like

Risk of prostate cancer significantly increases with age. Age is significant independent predictor of high risk prostate cancer. Although we need age as a criterion (we do not start to think about prostate cancer before age of 40 or 35), it is clear age could not be the only factor which would preclude decisions about PSA measurement, digital rectal exam and biopsy. Age is only one parameter in estimation of life expectancy. Life expectancy, not age, is crucial factor in decisions regarding prostate cancer screening interventions. Age does increase probability of cancer and cancer related death and therefore higher age – for example 75 years or more (with additional criterion of more than 10 years life expectancy) means strong indication in-favor of PSA screening and prostate biopsy. It is true that not all men at age 75 or more have more than 10 years of life expectancy, but significant number have and for them, prostate cancer screening is most useful and fruit bearing (much higher yield compared to younger men). This thinking is in strong contrast to past belief (and even now supported by some outdated recommendations of non-urological organizations), when

Digital rectal exam remains important part of prostate evaluation (Gosselaar et al., 2008). Although still on occasion declined by a patient and despite hopes PSA or any other method would completely replace it (Schroder et al., 1998), one still finds from time to time a case, where PSA is low and DRE is suspicious. For such a patient, next year, when PSA has increased, may be (and cases were confirmed it was) too late. Although after many suspicious DRE's biopsy comes negative, we are more and more aware of the fact that prostate cancer may progress without PSA increase. DRE can estimate sphincter tone, in a way prepare patient for biopsy, if needed, on occasion register some other, non-urological pathology and appreciate prostate size, which may give different dimension compared to ultrasound impression. DRE is necessary for sampling prostate cells in post-massage urine – at present for PCA3 only, but in the future maybe also for other markers. DRE is necessary for clinical staging of prostate cancer. DRE is limited, it can not palpate whole gland, but at

Comparison of mRNA expression patterns of prostate cancer and benign tissues identified significantly different expression of non-coding mRNA sequence, first called DD3 (Bussemakers et al., 1999) and later renamed PCA3. Technology (a variant of quantitative nucleic acid amplification test) was developed to identify very minute amounts of PCA3 mRNA from prostate cells shed in prostate urethra after prostatic massage and washed out immediately in first voided urine portion. Its estimated amount (number of copies) is

Serenoa repens, may (artifically?) increase free PSA values.

PSA testing was not recommended generally for all older men.

present it is here to stay (Yossepowitch, 2008).

**2.2 Age and life expectancy** 

**2.3 Digital rectal exam** 

**2.4 PCA3** 

follow from this data. One is related to high percentage of high grade cancers in population with median age 69 and relatively low PSA values. Once upon a time, it was suggested we should not measure PSA in men above 70. Should we now turn this completely around and biopsy all men with life expectancy of 10 yeas or more at the age of 70 irrespectively of their PSA? Or should we use other tests, like PSA isoforms derivatives?

Free PSA is measured to calculate fraction towards total PSA value, which is expressed as percentage. In PSA ranges between 4 and 10, with % free PSA below 10%, probability for biopsy detected prostate cancer in DRE negative patients was 56% and with % free PSA above 25% probability of cancer was only 8%. In PSA range between 2.5 and 4, with % free PSA below 10%, probability of cancer was 46% and with % free PSA above 20% probability of cancer was 8% (Catalona et al., 1998).

[-2]proPSA or p2PSA is new kid on the block, which is not yet universally available or accepted. It seems it will become next widely used PSA derivative, which is also used together with free PSA value (p2PSA/freePSA) or together with total and free PSA as value, calculated as p2PSA/freePSA times square root of total PSA and proprietary named by Beckman Coulter (Brea, California, USA) as PHI – prostate health index. Cut-off values for biopsy decisions using %p2PSA (p2PSA/free PSA) and PHI are not yet universally accepted and differ with regard to free PSA and total PSA calibration method (Hybritech or WHO), but it seems PHI values above 40 or 45 indicate high and below 21 low risk of prostate cancer. PHI of more than 48.5 was reported as 43% specific at 90% sensitive for detecting prostate cancer at initial prostate biopsy (Guazzoni et al., 2011). Another study reported PHI values above 34.2 to show increased probability for high risk disease (Isharwal et al., 2011). PHI was shown to indicate development of prostate cancer years before biopsy and correlated well with grade of future prostate cancer (Bektic et al., 2010). On negative side, using p2PSA compared to free PSA using ROC curves at area of high sensitivity (if we are to find cancer), increase in performance may seem marginal.

PSA density and different variants (PSA density of transitional zone etc), calculated from total PSA value and measurement of prostate volume using transrectal ultrasound, were in the past extensively evaluated. Drawback is variability of prostate volume measurements, need for this additional investigation and low sensitivity (Pepe et al., 2009). One example of cutoff value was density above 0.15 ng/ml PSA /ml prostate volume indicating higher risk of cancer. It may represent additional useful piece of information, also for example regarding prediction of disease course for patients on watchful waiting (Kotb et. al, 2011).

PSA velocity was in recent years proposed as another potentially helpful tool in picking cancers or high-risk cancers. Cutoff value of 0.35 ng/ml/year was proposed when total PSA values are less than 4. This may need adjustment for race (Tang et al., 2011). Although some doubts about value of PSA velocity in biopsy decisions were posed from analysis of PCPT trial data (Vickers et al., 2011), there are suggestions a lot of clinically important cancers in younger men may be detected by regularly and meticulously following and analyzing sequential PSA values (Bektic et al., 2011).

PSA values increase with age due to BPH. This became known early after PSA test introduction and "age-specific" PSA values were suggested. For Caucasian Americans, median, 75th and 95th percentiles of PSA were for age group 40-49 – 0.7, 1.0 and 2.1, for 50-59 age group 1.0, 1.6 and 3.6, for 60-69 age group 1.4, 2.5 and 4.3, for 70-79 decade 1.8, 3.5 and 5.8 (Morgan et al., 1996). One approach of integrating age specific PSA values in prostate biopsy decisions may be using as cutoff half of age-specific 95th percentile PSA value together with %free PSA below 18%, which was in part used in Innsbruck (Tyrol, Austria). High increase of PSA with infection or any irritation of prostate is well known and sometimes antibiotic or anti-inflammatory treatment is suggested and repeat measurement. PSA decreases in high body-mass index-correction and decreased of cutoff values may be needed (Pater et al., 2011). Diseases (liver cirrhosis) and medications (NSAID's, thiazides and statins) may also decrease PSA values (Nieder et al., 2011). If using free PSA values as trigger for biopsy decisions, it may be important to note that use of herbal products, like Serenoa repens, may (artifically?) increase free PSA values.
