Cathepsin B (CB) is involved in degradation of extracellular matrix proteins 
during tumor progression in human solid organ tumors (such as colorectal, 
bladder, and breast cancers), including human prostate cancer. Its activities 
are regulated by endogenous inhibitors (such as stefins or cystatins). Increased 
expression of cathepsin B message, protein, and membrane association have been 
linked to malignancy, but there are very few studies of their mRNA expression in 
prostate cancer using in situ hybridization techniques. Our objective was to 
determine the relationship of CB and stefin A (cystatin A) mRNA localization to 
the Gleason grading system for histologic scores in the hope of distinguishing 
aggressive and less aggressive variants of prostate cancer. We used a 25-base 
biotinylated oligonucleotide CB cDNA antisense probe to localize CB message and 
a 27-base biotinylated oligonucleotide stefin A cDNA antisense probe to localize 
stefin A message. Prostate samples from 41 prostatectomy patients were collected 
along with their pre-surgery serum PSA levels and clinical stage of the disease. 
Sections prepared from frozen prostate tissue samples were hybridized with the 
CB and stefin A and control pBR 322 probes using techniques reported by Sinha et 
al. [1] and their distribution quantitated by an image analysis system. Prostate 
sections treated with RNAse before hybridization or incubated with the pBR 322 
control probe showed little or no reaction products, confirming that 
localization of CB and stefin A probes was specific. In prostate cancer, the 
reaction products were found in neoplastic and invasive cells and occasionally 
in stromal cells. The ratios of CB to stefin A were similar in normal prostate 
and benign prostatic hyperplasia (BPH) whereas they varied consistently within 
and between Gleason histologic scores for prostate cancer. These variations 
showed three localization patterns; namely, prostate cancers with higher levels 
of CB than stefin A, lower levels of CB than stefin A, and similar levels of CB 
and stefin A. All three patterns and ratios for CB and stefin A were found in 
prostate samples (22/41) represented by the Gleason histologic score 6 tumors. 
In these tumors, serum PSA levels ranged from 1 to 78 ng/ml and prostate cancers 
showed B, C, and D clinical stages. There was no correlation of CB/stefin A 
ratio and serum PSA values or clinical stage in a limited number of prostate 
cancer cases. Our data showed that there were prostate cancer cases within 
Gleason histologic scores which expressed high, similar, and low levels of CB 
when compared to stefin A. We postulate that prostate cancer cases showing 
higher levels of CB compared to stefin A probably represent an aggressive 
variant of this cancer within any one Gleason histologic score. If this is the 
case, aggressive variants of prostate cancer would occur within Gleason scores 3 
to 10 even though higher scores are usually considered more aggressive forms of 
prostate cancers. Since our study is based upon a very limited number of frozen 
prostate samples, we emphasize that a larger series of archival prostate cancer 
samples along with their survival data should be analyzed to establish any 
relationship of CB/stefin A ratio and aggressive variants of this cancer. 
Therefore, our conclusion is tentative. Our study provides a partial explanation 
for differences in the clinical course of prostate cancer in patients. This is 
the first study to show that determination of CB and stefin A mRNA ratios may 
lead to identification of aggressive and less aggressive variants of prostate 
cancer within a Gleason histologic score.