Immune therapy has emerged as a powerful new approach to cancer therapy. Monoclonal antibodies are now front line agents in several oncology settings [1
]. Novel approaches to stimulating a patient's immune response to overcome a cancer are being assessed in multiple clinical trials [2
]. One question that is still subject to dispute concerns which molecules constitute appropriate targets for such therapies. Though it is rare to find a molecule that is expressed uniquely in the transformed target tissue, a number of candidates have been advanced. Understanding the tissue distribution of a given target molecule is essential for determining potential side-effects of the therapy, as well as for judging potential opportunities to exploit the therapy for new indications. Sipu-leucel-T is a United States Food and Drug Administration (FDA)-approved autologous cellular immunotherapy that targets prostatic acid phosphatase (PAP) as a treatment for advanced prostate cancer, and has demonstrated evidence of survival prolongation in men with asymptomatic or minimally symptomatic metas-tatic castrate resistant (hormone refractory) prostate cancer in randomized controlled Phase 3 clinical trials [3
]. It is of interest to know which other tissues express PAP in order to evaluate cross-reactivity and clinical safety.
In humans, PAP is one of the major proteins secreted by prostate columnar epithelium secretory cells following puberty. PAP protein has been determined to be approximately 0.5 mg/g wet weight of prostate tissue [5
] and approximately 1 mg/mL in seminal fluid [7
]. In healthy individuals, PAP serum levels are low, on the order of 1-3 ng/mL, whereas serum levels are elevated in many individuals with metastatic prostate cancer [8
]. Data from immunohistochemistry (IHC) studies indicate that > 95% of normal adult prostate tissue samples, including normal tissue adjacent to tumor, strongly express PAP [5
]. Analyses of PAP mRNA levels by RNA blotting methods [13
] and quantitative polymerase chain reaction (qPCR) using pooled tissue samples [14
] indicate high levels of PAP mRNA expression in normal prostate tissue. PAP has generally been considered a tissue-specific prostate antigen, highly expressed in both normal and malignant prostate cells.
Studies of PAP expression in non-prostate tissues have yielded conflicting results. The majority of these studies used IHC methodology. The studies have generally confirmed that the preponderance of PAP expression is in the prostate, though several studies have noted some extra-prostatic expression. PAP expression has been reported in liver cells, normal adult kidney [11
]; granulocytes, parietal cells of the stomach [15
]; urethral glands [16
]; anal gland non-mucosal epithelium, rectal tissue [16
]; salivary glands [18
]; and ectopic prostate tissue in both uterine cervix and vagina [19
In contrast, several studies failed to observe PAP staining in normal tissues, including bladder, breast, colon/rectum, epidermis, kidney, liver, lung, ovary, salivary glands, stomach, testes, and urethra [11
]. Using qPCR to measure relative levels of PAP mRNA transcripts, Cunha and colleagues [14
] found that, relative to prostate, PAP mRNA was expressed at low levels in several normal tissues, most notably in placenta, kidney, and testis. The kidney expression levels were 192-fold less (0.5%) than those seen in normal prostate tissue. Pancreas, small intestine, leukocytes, lung, ovary, colon, and spleen also exhibited low but detectable levels of PAP mRNA.
Several IHC studies provided evidence for PAP expression in normal pancreatic islet cells. Relative to prostate tissue extracts, normal pancreatic tissue had low but detectable PAP, as measured by electrophoresis [20
]. Li and co-workers observed weak occasional PAP IHC staining of pancreatic islets cells [15
]. Likewise, Cohen et al.
found weak diffuse PAP immunostaining in islets cells, but concluded the weak PAP signal was non-specific [21
]. Additional studies have found PAP expressed in normal pancreatic tissue [11
The correlation between increased PAP serum levels and metastatic prostate cancer has been well established [8
]. Detection of PAP in serum was the primary prostate cancer screening test used until the 1990s when it was supplanted by the more sensitive and specific assay for prostate-specific antigen (PSA). There is renewed interest in PAP as a prognostic tissue marker for T4 tumors [22
], as a serum marker in clinical staging of prostate cancer [23
], and for predicting recurrence after radical prostatectomy [24
]. PAP is expressed in > 95% of the primary prostate adenocarcinomas as determined by IHC [11
]. Goldstein observed that 80% (176 of 219) of PAP positive prostate adenocarcinomas from patients with Gleason scores of 6 to 10 expressed PAP in more than 25% of the malignant cells [25
]. Initial Phase 3 clinical protocols for the autologous cellular immunotherapy sipuleucel-T required subjects to have a tumor specimen submitted to a central pathology facility for IHC testing for PAP expression. Eligibility criteria required PAP expression in ≥ 25% of malignant cells by IHC. Of the 368 castrate resistant prostate cancer (CRPC) patients screened in two Phase 3 clinical trials for sipuleucel-T, 94% of the primary tumor specimens expressed PAP (unpublished results).
Other methods have been used to determine PAP expression in prostate cancer. PAP mRNA was present in a high percentage of prostate adenocarcinomas as determined by Northernblot hybridization methods [26
]. PAP peptide specific cytotoxic T lymphocytes (CTLs) were isolated from the peripheral blood mononuclear cells in 4 of 9 patients with prostate cancer but not from 3 healthy donors [27
Additional IHC studies have suggested PAP expression in non-prostate derived malignancies. PAP staining was observed in Warthan's tumors, mixed carcinomas, and pleomorphic adenomas of the salivary gland [18
One recent study [28
] questioned the specificity of PAP expression in the prostate. This study used RT-PCR to show widespread expression of PAP in mouse tissues, and IHC to demonstrate expression in human skeletal muscle. In addition, this study also suggested that PAP may exist in a form localized to the plasma membrane in addition to its well characterized secreted form.
Because of the disparate results regarding PAP expression reported in the literature, we undertook a series of studies on PAP expression using a variety of methodologies, including IHC, qPCR, in situ hybridization, and virtual Northern analysis. This comprehensive approach has confirmed that while the overwhelming majority of PAP expression is in the prostate, it is possible to observe low levels of PAP expression in several other normal tissues as well. This observation can be put to use in shaping clinical safety monitoring protocols in trials targeting PAP. We also observed that PAP may be expressed in some breast and colon cancers, but at levels considerably lower than that seen in prostate cancer.