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Prostate-specific antigen (PSA) is biochemically a 33-kDa serine protease and is the prototype of tumor marker most useful in investigating basic science and clinical application of prostate cancer. As an immunohistopathological marker, PSA is especially effective in the identification of distant metastatic prostate carcinoma and in the differential diagnosis of poorly differentiated transitional cell carcinomas of the bladder from prostate carcinoma. As a serologic marker, PSA is most useful in staging, monitoring and in early detection of recurrent disease. The greatest value of PSA is as a screening aid along with digital rectal examination for early detection of prostate cancer. PSA-based screening tests have been performed in various regions of the world and have yielded prostate cancer detection rates proportional to that of incidence rates in the countries. Most significantly, PSA detects early nonpalpable prostate cancer. Approximately 85% of the prostate tumors detected through PSA have the clinical features associated with medically important cancer. Further, a majority of the tumors, 70%, are confined to the organ. Although the issues of cost-benefit and increase in ultimate survival rate are still being evaluated in two large-scale randomized national trials, timely and definitive therapies of these potentially lethal tumors can result in high cure rate and improve the quality of patients' life.

Cellular protooncogenes, tumor suppressor genes (antioncogenes), and DNA mismatch repair mutators are generally the key molecular genetic biomarkers undergoing alterations during carcinogenesis, i.e., activation of oncogenes, inactivation of tumor suppressors, and DNA mismatch repair gene defects are essential events in cancer causation. In pancreas cancer, high incidence of oncogene K‐ras point mutations at the codon 12th is associated with premalignant and malignant transformation. Mutation in p53 tumor suppressor is also detected in pancreas adenocarcinoma. Concurrent loss of p53 and K‐ras function may contribute to the clinical aggressiveness of pancreas cancer. Microsatellite instability and DNA mismatch repair defects may represent new mutator phenotype for pancreas carcinogenesis. Mutation of cell cycle regulators, such as inhibitor of CDK4 or p16 tumor suppressor gene, is a new molecular event in pancreas cancer. Mutation of cyclin‐dependent kinases also may be involved in pancreas carcinogenesis. Loss or mutation of a new candidate tumor suppressor, DPC4 (deleted in pancreas carcinoma locus 4), is reported in pancreas cancer. The protein products of these gene mutations are potential tumor antigens, thus genotype expression can be detected by phenotype. Most of these emerging molecular genetic biomarkers are associated with regulation of cell growth and recognition, as well as gene expression, and may offer new insight into the cellular precursors to and genesis of pancreas cancer. J. Clin. Lab. Anal. 11:225–231, 1997. © 1997 Wiley‐Liss, Inc.

PROBLEM: Human seminal plasma is known to exhibit immunosuppressive activity. Transforming growth factor beta (TGF-beta) has been identified as an immunosuppressive factor in human seminal plasma. Biologically active TGF-beta represents a family of 25-kDa homodimeric proteins linked with disulfide bonds. TGF-beta associates with high molecular weight proteins noncovalently to form a type of latency that is biologically inactive. Quantitative distribution of active form of TGF-beta versus inactive latent form of of TGF-beta, and mechanism of the TGF-beta activation in human seminal plasma remain to be elucidated. PURPOSE: To characterize seminal plasma latent form of TGF-beta, including its concentration, and the mechanism underlying the activation of TGF-beta. METHOD: Gel filtrations on ACA-34 and Biogel P-60 were used to fractionate seminal plasma. TGF-beta was measured by enzyme immunoassay using antibodies specific for TGF-beta 1 and TGF-beta 2, respectively. Radioreceptor assay with recombinant human [125I]-TGF-beta 1 was applied to qualitatively identify TGF-beta 1. Kinetic experiments with various pH, temperature and time, along with protease inhibitors, were performed to delineate the activation mechanism of latent TGF-beta 1. RESULTS: Human seminal plasma contained both TGF-beta 1 and TGF-beta 2, predominantly in latent form. The total concentration of TGF-beta 1 averaged 238 ng/ml versus an average of 18 ng/ml for TGF-beta 2. The in vitro activation or release of TGF-beta 1 from latent TGF-beta 1 was achieved only at acidic pH of < 4.0, and was time and temperature dependent. At pH 3.7 and 37 degrees C, a significant activation of latent TGF-beta 1 was achieved after an incubation of only 15 min, reached the maximum at 120 min, and the activated TGF-beta 1 remained relatively stable for at least 24 h. The activation was not inhibitable by a series of protease inhibitors examined, alone or in combination (e.g., phenyl-methylsulfonyl fluoride, E-64, pepstatin, leupeptin, ethylenediamine tetraacetic acid). Competitive radioreceptor assay established the functional identity of TGF-beta 1 in human seminal plasma with recombinant human TGF-beta 1. CONCLUSION: Human seminal plasma TGF-beta is biologically activated from high molecular weight latent TGF-beta by acid pH. The acidic environment of female lower genital tract could represent an in vivo physiological condition for activation of seminal plasma TGF-beta that may immunologically protect the integrity of sperm.

Prostaglandin E2 (PGE2) is known to downregulate the generation of lymphokine-activated killer (LAK) cell activity. Indomethacin, an inhibitor of cyclooxygenase catalyzing the biosynthesis of PGE2, has been shown to augment LAK cell activities generated from peripheral blood mononuclear cells of normal healthy individuals. This study was undertaken to examine whether or not this augmentation is also a common phenomenon in cancer patients. LAK cell activities generated in the presence and the absence of indomethacin were examined in 15 normal healthy individuals and in 83 cancer patients. Paired data analysis revealed that indomethacin exhibited a significant augmentation of LAK activity generated from healthy individuals. Indomethacin enhanced LAK activity in patients with no distant metastases (TxNxM0); but depressed LAK activity in patients with distant metastases (TxNxM1). In patients without distant metastases, indomethacin showed an upregulating effect on LAK activity in those with an early T stage (T1-2NxM0), and no such effect was detected in those with a late T stage (T3-4NxM0). Indomethacin also significantly enhanced LAK cell generation in cancer patients with an ECOG performance status of 1, but significantly inhibited LAK cell generation in patients with a performance status of 4. These results indicated that indomethacin inhibited generation of LAK cell activity in cancer patients with a poor performance status or with distant metastatic disease, who normally would be the subjects of adoptive immunotherapy. Further, PGE2 production in cultured LAK cell medium was suppressed by indomethacin in all 20 cancer patients that were examined, suggesting that other yet to be identified factors or mechanisms may be responsible for the paradoxical effects of indomethacin on LAK cell activity.

Using viable adriamycin resistant human ovarian carcinoma cells 2780AD and colchicine resistant human oral epidermoid carcinoma cells KB-24 as the immunogen in primary and subsequent i.p. immunizations, followed by i.v. boostings with crude plasma membranes of 2780AD, KB-24, Chinese hamster lung cells resistant to vincristine DC-3F/VCRd-5L, and resistant to daunorubicin DC-3F/DMXX, we have generated a new murine monoclonal antibody (McAb), designated F4, of IgG1 isotype. McAb F4 reacted strongly with a cell surface epitope of drug resistant cells and insignificantly with their drug sensitive counterparts. Cell surface localization of F4 epitope was determined by immunofluorescence and laser scanning confocal imaging system. Results obtained from immunoprecipitation and immunoblot analyses using F4 and mdr1 P-glycoprotein specific McAb JSB-1 demonstrated the reactivity of P-glycoprotein with F4. These results along with those obtained from competitive binding-inhibition, chemical modification, and enzyme hydrolysis, revealed that McAb F4 detects an extracellular epitope of P-glycoprotein, and is different from other major McAbs directed against P-glycoprotein, e.g. C219, MRK16, JSB-1, HYB-241 and C494. Deduced from the putative structure of mdr1 protein and its orientation in cell membrane, it is proposed that F4 epitope is localized in or near the 3rd, and/or 6th extracellular transmembrane loops of P-glycoprotein.

PROBLEM: Human seminal plasma is known to exhibit immunosuppressive activity in seminal plasma. PURPOSE: The purpose was to characterize immunosuppressive proteins in seminal plasma. METHOD: Gel filtration fractions of 100 to >440 kDa were identified that inhibited DNA synthesis and killing activity of interleukin-2 stimulated lymphocytes. RESULTS: The fractions exhibiting immunosuppression also inhibited DNA synthesis in a mink lung cell bioassay commonly used to measure the activity for transforming growth factor β (TGF-β). The negative growth activity was diminished by aTGF-β neutralizing monoclonal antibody. TGF-β was further detected in the active fractions by Western immunoblot. CONCLUSIONS: These results identified TGF-β as an immunosuppressive protein in human seminal plasma and may provide insight into the role of immunosuppression played by seminal plasma, such as in reproduction and neoplasia.

To elucidate the inhibitory effect of prostaglandin E2 (PGE2) on the generation of recombinant interleukin-2 (IL-2)-induced lymphokine-activated killer (LAK) activity, we have ex

Cimetidine, an H2 histamine receptor antagonist, is a potent immunomodulating agent, which acts by inhibiting suppressor T lymphocyte function. The present work investigated the effect, if any, of cimetidine on interleukin-2 (IL-2)-induced natural killer (NK) and lymphokine-activated killer (LAK) cell activities, and on in vivo antitumor activity using syngeneic colon 26 adenocarcinoma as the model. Mimicking the clinical conditions, all in vitro experiments were evaluated with the splenocytes prepared from tumor-bearing BALB/c mice. Ten days after subcutaneous inoculation of tumor cells (5 x 10(5)), animals were treated intraperitoneally daily with phosphate-buffered saline (PBS), cimetidine (2 mg kg-1 day-1), IL-2 (300,000 IU/day), or cimetidine plus IL-2 for 7 consecutive days. The treatment of IL-2 plus cimetidine increased NK and LAK cell activities significantly and synergistically at the end of the treatment (i.e. on day 18) as well as 1 week after the treatment (i.e. on day 25), in comparison with those of the control groups (PBS, cimetidine alone, IL-2 alone). Also, in vivo antitumor activity, as analyzed by a Kaplan-Meier life table with the log-rank test, revealed a significantly prolonged survival in the group treated with IL-2 plus cimetidine compared to the control groups. Phenotyping performed on the murine splenocytes on day 18 indicated a significant reduction in Lyt2-positive cells in the cimetidine-treated group in comparison with the PBS group. A significant increase in asialo GM1-positive cells and IL-2-receptor-positive cells was detected in the group treated with IL-2 plus cimetidine in comparison with the PBS and IL-2 control groups. Therefore, this study indicates a synergistic enhancement of IL-2-induced NK and LAK cell activities in tumor-bearing hosts by cimetidine, a noncytotoxic inhibitor of suppressor T function, and a significantly prolonged survival of tumor-bearing animals treated by IL-2 plus cimetidine. It also suggests the clinical potential of combination therapy of IL-2 with cimetidine.

Prostate-specific antigen (PSA) is a well-characterized human prostate-specific glycoprotein. PSA has been shown to be the most effective immunohistologic marker for prostate cancer, as well as the most useful serologic test in staging and monitoring prostate cancer and in early detection of recurrent disease. The greatest clinical value of PSA is an aid for early detection of prostate cancer. Recent studies have indicated that PSA-based screening of the older population for organ-confined early-stage prostate cancer is an acceptable, practical, and reliable modality. The accuracy of PSA screening is within the same range as the mammogram. The cost-effectiveness of PSA is comparable to other cancer screening tests. Although the increase in the patient's survival due to PSA-based detection of early prostate cancer remains to be documented, it is generally agreed that the PSA test along with digital rectal examination (DRE) should be included in the annual physical examination for men 50 years of age or older. High-risk men are urged to commence at age 40. Asymptomatic men who have both a negative DRE and normal PSA blood test need only to continue an annual DRE and PSA check-up. Men who have a negative DRE and elevated PSA, and all those who have a suspicious DRE regardless of PSA results, should undergo further diagnostic workup, such as transrectal ultrasonography with biopsy of visible lesions. The cure rate is high with timely treatment, when prostate cancer is detected while still confined to the prostate.

The single most important development in clinical cancer markers since the early 1970s, when carcinoembryonic antigen (CEA) was approved by the Food and Drug Administration, is the approval of a prostate-specific antigen (PA or PSA) as a cancer-marker test for adenocarcinoma of the prostate in 1985. In fact, whatever CEA can do for gastrointestinal cancer, PSA can do, and do it better, for prostate cancer. The clinical application of PSA in diagnosis, prognosis, and monitoring of prostate cancer is the subject of this chapter. Basic characteristics of this marker of prostate cancer also will be reviewed. Reviews of similar topic are available elsewhere (Wang et al., 1982; Chu, 1985, 1990; Chu and Murphy, 1986; Brawer and Lange, 1989; Catalona et al., 1989; Killian and Chu, 1990). It should be noted that PSA is not a neoantigen of prostate cancer or a prostate cancer specific antigen; rather, it is expressed equally, but exclusively, by normal, benign hyperplastic and cancerous prostate glands (Wang et al., 1979; Papsidero et al., 1981).

In order to identify the endogenous phosphoprotein substrates for human prostatic acid phosphatase (PAP), cellular proteins of human normal, benign, and malignant prostatic tissues as well as carcinoma cell lines were phosphorylated by the cellular kinases in the presence of (gamma-32P)-ATP and then were subjected to dephosphorylation reaction by PAP. Of several endogenous phosphoproteins, PAP preferentially dephosphorylated a cytosolic protein of Mr 83 kDa. The dephosphorylation of the 83 kDa phosphoprotein (designated pp83) by PAP was uniformly observed in all cells/tissues of prostate origin, and was completely inhibited by L(+)-tartrate, the classic inhibitor of PAP. Phosphoamino acid analysis revealed that pp83 was a tyrosine-poor phosphoprotein and was mostly dephosphorylated by PAP at serine/threonine residues rather than tyrosine residues. Further comparison of dephosphorylation rate with that of an endogenous phosphotyrosine-containing phosphoprotein (pp53) revealed that PAP possessed both phosphoserine/threonine protein phosphatase and phosphotyrosine protein phosphatase activity. These results demonstrate that pp83 apparently is an endogenous substrate of PAP in human prostate, and that, instead of a phosphotyrosine protein specific phosphatase, PAP is a universal protein phosphatase hydrolyzing equally well the phosphotyrosine, serine, and threonine residues.

Acid phosphatase was purified to electrophoretic homogeneity from human normal lung and spleen and was characterized biochemically and immunologically in comparison with prostate acid phosphatase (PAP). The apparent MW of lung acid phosphatase (LAP) and spleen acid phosphatase (SAP) was 110,000 and 100,000, respectively, similar to that of PAP (100,000). All three enzymes exhibited similar electrophoretic mobility, optimal pH, substrate, and inhibitor specificity, except that PAP dephosphorylated profoundly the phosphate group from tyrosine phosphate in phosphoangiotensin (19,700 fmol/mg/min), whereas only marginal activities were detected for LAP and SAP (19 and 73 fmol/mg/min, respectively). Amino acid analysis revealed more similarity between SAP and LAP than PAP and LAP or PAP and SAP. An immunological cross-reactivity among these three acid phosphatases was detected by polyclonal and monoclonal antibodies raised against purified PAP, although unique epitopes were detected on the PAP molecule. This study provides data explaining why conventional biochemical methods are not specific for PAP measurement and why immunologic methods still detect other acid phosphatases, as observed in clinical laboratory assays. The data also suggest the possibility of using a new substrate or antibody reagent for a more specific assay for PAP.

With the use of five murine monoclonal antibodies (1A5, 2A4, 3F1, F5 and 3A12) and an antigen-affinity purified goat polyclonal IgG antibody, the presence of a prostate-specific antigenic domain in human prostate-specific antigen molecule was identified. The results were based upon a series of quantitative competitive inhibition assays of each 125I-labeled monoclonal antibody and polyclonal antibody binding to prostate-specific antigen by unlabeled monoclonal antibodies as inhibitors, and immunohistochemical examination of an extensive panel of human tissue specimens. A cluster of two epitopes that are spatially related or in close topographical proximity and represent a prostate-specific antigenic domain are defined by the monoclonal antibodies 1A5, 2A4, 3F1, and F5, 3A12, respectively.

Adrlamycin was conjugated to an IgG1 monoclonal antibody specific for human prostatic acid phosphatase via a dextran bridge. The adriamycin-monoclonal antibody conjugate retained substantially the original Immunological activity of the antibody. Antitumor effect of the conjugate in vitro was studied by Its Inhibition on RNA synthesis In human prostate tumor cells (LNCaP), which exhibited a higher inhibition than that by an identically prepared adrlamycin-normal mouse IgG conjugate but was less than that by free adrlamycin. The loss In pharmacological activity of the conjugate In vitro was proportional to the extent of dextran oxidation. Antitumor effect In vivo demonstrated that adriamycin-monoclonal antibody conjugate greatly Inhibited the growth of xenografted prostate tumor, as compared with control groups, only In the early phase of experiment. These results suggested that although adrlamycin conjugated to monoclonal antl-PAP antibody via a dextran bridge may be a potential reagent for experimental immunochemotherapy of prostate tumor, caution must be exercised at this stage of development.

A prospective trial from July 1976 to September 1980 by the National Prostatic Cancer Project randomized newly diagnosed metastatic prostate cancer patients to DES 1 mg orally three times daily or orchiectomy; or DES, 1 mg, three times daily, plus cyclophosphamide at 1 mg/m2 iv every three weeks, or cyclophosphamide 1 g/m2 iv every three weeks plus estramustine phosphate (Estracyt) at 600 mg/m2 orally daily in three divided doses. There were 246 patients evaluated for response of 301 entered. These consisted of 83 on the DES/orchiectomy arm, 77 on DES plus cyclophosphamide, and 86 on Estracyt plus cyclophosphamide. Objective response rates, initially evaluated at 12 weeks, were similar among the treatments. However, chemotherapy as used in this study early in the diagnosis of metastatic disease appears to exhibit an improved effect on overall survival compared to hormone therapy alone. Analysis within groups having pain versus no pain at entry revealed a marked advantage after 80 weeks for chemotherapy when pain was initially present and a slight advantage during treatment (throughout follow-up) when the pain was absent. Median survival times were 92, 91, and 94 weeks, respectively, for the three treatments. The progression-free interval for responders showed no difference between initial treatments, although nearly one half of the patients are still in remission; hence, further follow-up will be essential. Side effects were not excessive for the chemotherapy treatment arms and patient compliance was good. This national multicenter trial provides the basis for further testing of chemotherapy agents at an earlier phase for patients with newly diagnosed metastatic prostate cancer.

A sensitive and reproducible method for quantitation of polyamines in erythrocytes by high-performance liquid chromatography (HPLC) is described. Spermidine and spermine are first converted to fluorescent dansyl derivatives and are then separated in less than 15 minutes on a C18 reverse-phase cartridge using methanol in water mobile phase. Sensitivity of the method is 20 pmol with recoveries that average 96% and 95% for spermidine and spermine, respectively. Precision study revealed an average mean coefficient of variation of 5.42 ± 2.95 (mean ± S.D.) from four levels of the polyamines (8.2 to 354.9 nmol/g hemoglobin [Hgb]). The normal ranges for 30 apparently healthy men and women, aged 20–60 years, were 2.9–33.7 nmol/g Hgb (± 2 S.D. from mean) for spermidine and 0-20.9 nmol/g Hgb for spermine. The quantity of putrescine was negligible. Feasibility of this method was evaluated in serial specimens from an advanced-stage patient with prostate cancer who was receiving multiple-modality therapy. Results revealed that this HPLC method can be used in quantifying circulating polyamines in clinical specimens.

A long-term CEA follow-up was evaluated statistically for a series of 74 patients with primary colorectal carcinoma who underwent resections for cure. Thirty-three recurrences and 29 deaths were reported among this population after a median follow-up interval of 55 months. Preoperative CEA levels correlated with the Dukes' classification. However, the preoperative level adds significant information to Dukes' classification in the prediction of recurrence. Postoperative CEA assays taken later in the clinical evaluation process carry the most prognostic information for subsequent recurrence. Using matched-pairs techniques, the authors noted that CEA often rose in anticipation of recurrence, sometimes as early as one year before recurrence. The following four follow-up CEA events were evaluated: 1) three consecutive rising CEAs; 2) CEA rises 5.0 ng/ml; 3) CEA exceeds 5.0 ng/ml; 4) CEA exceeds 5.0 ng/ml two consecutive times. The follow-up CEA events examined were characterized by false positive rates ranging from 17% to 66% and true positive rates ranging from 43% to 56% in the prediction of recurrence. From follow-up CEA evaluations, further clinical investigation appeared most warranted when CEA exceeded 5.0 ng/ml at two consecutive follow-up evaluations, but the chances of finding any subsequent recurrent disease was near 50%. Among patients experiencing recurrences, although forewarned by CEA elevations, there was no subsequent time when patients were most likely to have recurrence of disease.

Ninety-four patients with carcinoma of the colon have been followed with serial determinations of plasma CEA (carcinoembryonic antigen) levels over a 3-year period using the Hansen assay. Nine hundred twelve CEA determinations have been made in these patients. Plasma CEA levels rose in 90% of the instances of clinical progression documented in these patients. In 30% of patients, this rise indicated progression 6 months or more before it was detected by standard clinical methods. Unfortunately, a few patients never developed elevated CEA levels even though disease clearly progressed. False positive results have also been encountered, with significant elevations occurring in patients who have since remained without evidence of disease for several months. Our data indicate that at least two sequential elevated CEA values, the second being higher, must be a minimal criterion for consideration of possible progression of disease. Even with this standard, we have encountered false positive results in 10% of our patients, indicating recurrence or progression where none has occurred clinically. CEA measurement is of limited usefulness for 30 days after curative surgery, because the elevation of CEA levels due to the original amount of tumor present as well as due to surgery per se may persist for this length of time in a significant number of patients. On the other hand, CEA levels have responded to chemotherapy in close correlation with observed clinical course in those patients with metastatic disease treated in this series. Initial pretherapy CEA values have so far proved to be good prognostic indicators of disease course following complete resection. With an initial CEA value of less than 2.5 ng/ml of plasma, recurrent has been rare (1/20). If the pretreatment CEA was greater than 7.0 ng/ml, it has been the rule (7/9).

There were 121 men with hormonally refractory metastatic cancer of the prostate who were randomized to receive estramustine phosphate or vincristine, or the combination of these 2 agents. All patients had received prior radiation therapy (greater than 2,000 rad). There were 90 patients who could be compared for response. The objective response rates (partial regression or stabilization of disease) for the 3 treatment groups were 26 per cent for estramustine phosphate, 24 per cent for estramustine phosphate plus vincristine and 15 per cent for vincristine. Subjective parameters varied little among the 3 regimens. The median duration of response for those responding to estramustine phosphate was similar (20 weeks) to that for vincristine (22 weeks) and greater than that for the combination (13 weeks). The probability of survival did not differ significantly for patients randomized to each of the 3 regimens. The addition of vincristine to estramustine phosphate did not enhance the response rate achieved by estramustine phosphate alone and vincristine alone produced the lowest response rate. Estramustine phosphate continues to be the most active agent in previously irradiated patients with hormonally refractory metastatic cancer of the prostate.

Hybridoma-derived monoclonal antibodies to human PAP have been produced by fusion of mouse myeloma cells [P3 x 63 Ag 8.653] with spleen cells from mice immunized with purified PAP. One hundred fifty-six out of 252 clones were found to produce antibodies against PAP. These monoclonal antibodies were classified into four different subclasses as IgM [3 clones], IgG2a [2 clones], IgG3 [1 clone], and IgG1 (150 clones] with k-chains. Four monoclonal antibodies, IgG1, IgG2 a, IgG3, and IgM, were selected from the 156 hybridoma clones for immunologic characterization. Results from a binding assay suggested that monoclonal anti-PAP antibodies IgG1 and IgM recognized two distinct antigenic determinants of the PAP molecule, while the hybridoma IgG2a and IgG3 antibodies recognized another antigenic determinant. The specificity of these four hybridoma anti-PAP antibodies has been evaluated by the immunohistochemical method and competitive-binding assay. Monoclonal antibodies IgG2a and IgG3 as well as polyclonal xenoantibodies were found to react with PAP as well as with nonprostatic acid phosphatases; and antibodies IgG1 and IgM reacted more specifically with PAP. These results indicated that monoclonal anti-PAP antibodies IgG1 and IgM possessed a higher specificity for human PAP in comparison with monoclonal antibodies IgG2a, IgG3, and xenoantibodies. The monoclonal anti-PAP IgG1 and IgM antibodies may be useful in delineating antigenic structure of the PAP molecule, as well as in the refinement of serologic determination and immunocytochemical study of PAP in human prostate cancer.

The clinical features of a new prostate tumor marker, prostate-specific antigen (prostate antigen, PA), has been reviewed. Although PA cannot be used in early detection of prostate cancer, simultaneous determination of PA and PAP yields an additive clinical value in immunodiagnosis of prostate cancer. At the present stage of development, PA is most useful as a prognostic marker for monitoring disease recurrence and treatment response. Also, PA is an effective immunohistologic marker for differential diagnosis of metastatic carcinomas with unknown primary, especially in the identification of metastatic prostate tumor in distant metastases and in the differentiation of primary prostate carcinoma from poorly differentiated transitional cell carcinoma of the bladder. Unequivocal evidence is not yet available on the role of circulating PA-binding globulin as an auto-antibody or an anti-tumor antibody as a result of patient's immune response. This observation is of clinical value for investigation of prostate cancer biology. The intriguing protease activity as detected in PA may provide new avenues for prostate cancer research.

A sensitive sandwich-type enzyme immunosorbent assay has been developed for quantitation of a new human pancreas-specific antigen (PaA). With this method, PaA at a concentration as low as 0.8 ng/ml can be detected. The assay was reproducible as shown by the coefficients of variation for within (4.8%) and between (6.1%) assays. Serum PaA levels from 51 healthy persons ranged from less than 4 to 34 ng/ml. Using 21.5 ng/ml (the upper 97.5 percentile of normal controls) as an upper limit, 42 of 60 pancreatic cancer (70%), 3 of 14 pancreatitis (21%) and 2 of 6 cholelithiasis (33%) had an elevated PaA. Very few patients with other cancers were shown to have an elevated PaA: 3/40 (8%) of lung cancer, 2/43 (5%) of colorectal cancer, 3/40 (8%) of prostate cancer and 1/39 (3%) of breast cancer. The sensitivity and specificity of the serum PaA test for the detection of pancreatic cancer were calculated to be 70% and 95%, respectively. These results indicate that PaA may be useful as an adjunctive tool in immunodiagnosis of pancreatic cancer.

Antiserum to a human prostate-specific antigen was raised in a rabbit and utilized by immunoperoxidase staining to evaluate its potential value as a diagnostic histologic marker for tumors of prostatic origin. All primary and metastatic prostatic malignancies reacted positively, whereas nonprostatic neoplasms did not stain with this procedure. This is the first immunohistochemical marker for prostate gland epithelium which does not represent prostatic acid phosphatase.

Large quantities of carcinoembryonic antigen (CEA) were detected by the radioimmunoassay method and confirmed by the column chromatography and immunodiffusion methods in the bowel contents from nine subjects: two healthy volunteers, two patients with colostomies and one with ileostomy free of disease, three patients with colostomies who had metastases outside the gastrointestinal tract, and one patient with carcinoma of the rectum (in place) before resection. The quantities of CEA in the intestinal contents did not correlate with the plasma levels. This may indicate that CEA could be present in large amounts in the normal epithelial cells of the gastrointestinal tract and does not appear conspicuously in blood until abnormal conditions develop.

An immunoglobulin M (IgM) antibody to a carcinoembryonic antigen (CEA)-like antigen was isolated from the ascites fluid of a patient with pancreatic cancer by ammonium sulfate precipitation (25–55% saturation) and subsequent purification by gel filtrations on Sephadex G-200 and Sepharose 6B followed by protein A-Sepharose CL-4B chromatography. The IgM antibody was found to be in complex with a CEA-like antigen as revealed by the multiple precipitin lines obtained on immunoelectrophoresis and double diffusion with a mixture of antihuman IgM and anti-CEA. Dissociation of the complex with 0.2M glycine HCl buffer pH 2.5 and further chromatography on Sepharose CL-6B. followed by Sepharose 6B separated the IgM antibody from the CEA-like antigen. The IgM antibody formed an immunoprecipitate upon double diffusion with a homologous CEA cross-reactive antigen isolated from the liver metastasis of another patient with pancreatic cancer. The antigen (molecular weight 70,000 ± 15,000 daltons) which reacted with the IgM antibody was purified by Con A Sepharose affinity chromatography followed by gel filtration on BioGel A 1.5M. The IgM antibody reacted with tumor antigens from pancreatic cancer only. These results suggest that the human IgM antibody response to antigens may be specific to the tumor type involved.

A mouse mammary tumor cell line, designated JC, has been established from a spontaneously developed primary adenocarcinoma of an aged virgin female BALB/c mouse. Isoenzyme analyses including glucose-6-phosphate dehydrogenase, lactate dehydrogenase, and peptidase proved that this cell line is of murine origin and devoid of contamination from other species. Karyotyping revealed that the number of chromosome ranged from 26 to 100, with a modal number of 40. Electron microscopic examination detected the presence of tonofilament and desmosomes confirming its epithelial nature. In addition, no type B or C virus particle was detected, although intracysternal A particle was observed occasionally. Tumorigenicity in immunocompetent syngeneic hosts was easily established by s.c., i.p., and i.v. injection of viable JC tumor cells. A very weak immunogenicity of the JC tumor was demonstrated through its immunization-challenging on syngeneic immunocompetent hosts. Although no rejection of JC tumor was noted, a significant prolongation for the incubation period before an obvious and palpable tumor growth was detected between the experimental and the control animals. Development of a concomitant immunity was also detected. The JC tumor represents a valuable murine mammary tumor model which is different from other available models because of its unique origin, absence of virus particles, very weak immunogenicity, and high tumorigenicity in syngeneic hosts. The cell line has been maintained for more than 5 yr and has been used for experimental immunotherapy in our laboratory.

Plasma carcinoembryonic antigen (CEA) assay was done in 30 patients with osteosarcoma. CEA was found positive (greater 2.5 ng/ml) in 17 of 21 patients who had active evidence of disease and negative (less than 2.5 ng/ml) in all 9 patients who were in complete remission resulting from previous amputation of chemotherapy. Serial CEA determinations demonstrated a fall to normal in 7 of 9 patients following successful surgery of chemotherapy and a ruse and fall (fluctuation) of levels in 8 patients who had tumor progression while on chemotherapy. Clinical recurrence of disease in two instances preceded or coincied with CEA elevation. The CEA assay in osteosarcoma although non-specific could be used as an inportant adjunct to experienced clinical judgment, periodic x-ray examination, and laboratory study to prognosticate the course of osteosarcoma during therapy. The interpretation of a rising or falling CEA titer alone, however, must be made with caution.

Clinical chemotherapy in the treatment of neuroblastoma is undergoing further evaluation. Chemotherapeutic agents, cyclophosphamide, vincristine, and hexamethylmelamine, were investigated in a murine neuroblastoma model that, in part, histologically and morphologically resembles the human tumor. The tumor was inoculated subcutaneously (SC), intramuscularly (IM), intraperitoneally (IP), intrarenally (IR). Growth of tumor was observed at all sites, as well as the appearance of metastases. Maximal survival of 32 +/- 1 days was seen (SC) and the minimum was 15 +/- 1 days (IR) (p less than 0.05). Corresponding survival rate for IM was 26 +/- 1 days and IP 21 +/- 1 days. For this reason SC was selected for drug testing. Cyclophosphamide 100 mg/kg/week showed the most favorable results, as determined by autopsy status with fewer tumors and a greater tumor weight decrease over time. This agent also showed a significantly longer survival in comparison to all other drugs (p less than 0.0001). The results with vincristine and hexamethylmelamine in the dose ranges employed differed little from the placebo, except for the finding of a slight decrease in body weight at autopsy.

Assays for serum placenta-like (Regan) isoenzyme of alkaline phosphatase were done on 157 samples from 96 patients with prostatic cancer entered into various National Prostatic Cancer Project protocols. Elevated placenta-like isoenzyme levels were found in 19 patients (20 per cent). Increased isoenzyme activity was seen in some patients with early clinical stages of the disease (B1 and B2), as well as in those with advanced disease. As in other reports results were not consistent in any individual patient. No correlations could be made between elevated isoenzyme activity and either stage of the disease or response to therapy. The placenta-like isoenzyme of alkaline phosphatase appears to be a nonspecific oncodevelopmental marker that is not helpful alone as a diagnostic indicator but that may be promising as part of a profile of markers for a patient with prostatic cancer.

The value of biological markers of bladder cancer was studied in 66 patients. The markers included serum and urine carcinoembryonic antigens, serum and urine fibrinogen degradation products, total lymphocyte counts, urine lymphocytes and urine cytology. A high degree of accuracy (90 per cent) was found in correlating cytology and urinary fibrinogen degradation products with the activity of the disease. Serum and urine carcinoembryonic antigens, serum fibrinogen degradation products, total lymphocyte counts and urine lymphocytes were found to have no value in screening bladder cancer patients. Urinary fibrinogen degradation products and cytology in combination are recommended for screening and followup of patients at high risk.

Data are presented suggesting the presence of pancreas tumor-associated antigens. Slow progress has been made during the past few years in the identification of pancreatic tumor antigens that may be of clinical usefulness and it seems unlikely that many of the practical problems now being faced in identification and isolation of these antigens and in development of a specific, sensitive assay will be solved by conventional immunochemical approaches. The study of antigen and/or antibody purified from immune complexes in the host and the application of leukocyte adherence inhibition techniques to immunodiagnosis of pancreatic cancer are among the new approaches that may provide effective alternatives in the study of pancreatic tumor antigens.

Bullous pemphigoid (BP) has been reported in association with malignant neoplasms of the urinary tract, lung, genitalia, breast, pancreas, gastrointestinal (GI) tract, skin, and lymphoreticular system. 1 The course of BP usually does not parallel the course of the malignant neoplasm. The malignant neoplasm may be evident at the onset of BP or present as an incidental finding at postmortem examination. 2 Carcinoembryonic antigen (CEA) levels greater than 2.5 ng/ml have been reported in serum samples from patients with pancreatic, colonic, rectal, gastric, mammary, and cervical malignant neoplasms, and in some leukemias. 3 The presence of CEA correlates with a high incidence of proved malignant neoplasms and is an early indicator of clinically unrecognized recurrence. 4 The purpose of this study was to determine the incidence of a malignant neoplasm in a large number of patients with BP using clinical as well as serologic evaluation. Subjects and Methods Thirty-three patients

We describe an episode of obstructive uropathy produced by xanthine precipitation in the tubules of the kidney of a patient with histiocytic lymphoma during intensive chemotherapy, despite allopurinol therapy. Urinary oxypurine-uric acid ratio suggested a subclinical deficiency of hypoxanthine-guanine phosphoribosyltransferase. Results of an assay of this enzyme confirmed the abnormality. Both parents and three brothers of the patient had normal enzyme activity. The continued importance of adequate hydration for patients who receive allopurinol during initial periods of cancer therapy is emphasized. ( Arch Intern Med 138:1017-1019, 1978)

According to the American Cancer Society, this year an estimated 57,000 new cases of prostate cancer (1979) will be diagnosed among American males (American Cancer Society, 1979). The Society also estimates that 20,000 lives will be lost to this form of cancer. One of the major reasons for this high mortality is that many cases are not detected until after metastases have occurred (Gittes and Chu, 1976). Therefore, markers that may serve as aids for early diagnosis of this disease and in monitoring the effectiveness of therapies have long been investigated. The purpose of this chapter is to review the prostate tumor markers that have been shown to be of significant value in diagnosis and treatment of this disease. It will include new information for old markers such as prostatic acid phosphatase, and data on new markers under investigation, such as prostate antigen. Other known markers that have been shown to be associated with cancer of the prostate are also described briefly.

By a specific immunochemical measurement, the activity of prostatic acid phosphatase (PAP) in prostate cancer was found to be about 25%, on average, based on micrograms DNA or per cell, of that in normal prostate or benign prostatic hypertrophy (BPH). The reduction of PAP in prostate cancer was further revealed by a decrease in PAP protein. The 125I-labeled anti-PAP IgG specifically bound to nascent peptides on PAP-synthesizing polysomes showed no qualitative differences among cancerous prostate, normal prostate and BPH. However, the quantitative binding of 125I-labeled anti-PAP IgG to polysomes of cancerous prostate was half that of normal prostate of BPH. These data suggest that a significant amount of PAP and its synthesizing polysomes was reduced in prostate cancer as a result of PAP gene suppression.

PCAA, a glycoprotein antigen fractionated from the ascites fluid of a patient with pancreatic cancer and sharing an identical immunogenicity with Gelder's POA, and PaA, a novel pancreatic tissue antigen, were studied immunohistologically. Serial paraffin sections were prepared from surgical specimens of 11 cases of pancreatic cancer, 15 of gastric cancer, 12 of colonic cancer, and 2 of gallbladder cancer; these were then subjected to immunofluorescence and immunoperoxidase stainings. In noncancerous tissues, PCAA was detected unexpectedly at goblet cells of the whole intestine, but was completely absent in pancreatic tissues, while PaA was not demonstrated in intestinal tissues, but was positive at acinar cells of the pancreas. In pancreatic cancer tissues, PCAA and PaA were detected at apical cytoplasm of cancer cells, although positive cells were in different proportions and had different distributions. PCAA-positive cells were mucin-producing (positive in PAS-alcian blue staining) and were well differentiated histologically, while PaA-positive cells were less differentiated and poor in mucin production. Among 11 cases of pancreatic cancer, both PCAA- and PaA-positive cells were demonstrated in 3 cases, PCAA-positive cells alone were found in 3 cases, and PaA-positive cells alone were seen in 4 cases. In 27 gastrointestinal cancer tissues, PCAA was detected in 7 cases of mucin-producing cancer, and PaA was demonstrated in 2 cases of poorly differentiated adenocarcinoma.

Tumor markers have become increasingly important in the management of genitourinary malignant neoplasms. For the purpose of this discussion, I will use a broad definition of tumor markers that is based on their potential clinical use. With this perspective, a tumor marker may be defined as any biochemical, hormonal, or immunologic assay which provides information regarding the diagnosis and/or prognosis of a malignant neoplasm. In addition to the obvious potential clinical importance of tumor markers, they may also prove to be useful tools for basic scientists in the study of the pathophysiology of human malignancy. Tumor markers may be highly specific for a given neoplasm or may be quite non-specific. However, their clinical relevance, which does not always correlate with specificity, will determine whether they will be incorporated into the urologic oncologist’s armamentarium or not. A number of factors will determine whether an individual tumor marker will be clinically accepted. Obvious relevant factors include the prevalence of the neoplasm; its actual or perceived impact on the population at risk; and the sensitivity and specificity of the tumor marker itself. Sensitivity refers to the accuracy of the tumor marker in detecting neoplasm when it is actually present. An insensitive test will produce false negative results, i.e., failure to detect neoplasm when it is actually present.

Serial levels of PAP and AcP activity were compared for their relative values in monitoring 57 early and 33 advanced prostate cancer patients. Several findings regarding the patients' disease status and the enzyme levels have been observed that may be beneficial to therapeutic management of these patients. They are: [1] an elevated PAP activity in disease recurrence and disease progression generally precedes an elevated AcP activity, and thus represents a more sensitive index for patients with early and advanced disease; [2] serial mean levels of PAP activity greater than the mean + 3 SD are more predictive for disease recurrence and progression than are those of AcP activity in both groups of patients; [3] PAP activity is a more sensitive monitor for changes in objective treatment response than is AcP activity; and [4] PAP is more specific than AcP for prostate, thus offering a more reliable marker to identify metastasis of unknown origin, or to confirm metastasis derived from a primary prostate tumor that may have been suggested by other non-prostate-specific marker[s]. In addition, data suggest a favorable prognosis for patients receiving therapy as inferred by a serial mean of PAP activity that is less than mean + 3 SD.

A human immunoglobulin that binds carcinoembryonic antigen (CEA) was isolated from four individual normal human sera by affinity chromatography with the use of a CEA-Sepharose solid adsorbent. The yield of isolated protein, termed human CEA-binding protein (HCBP), ranged from 1.8 to 10 microgram/ml serum. HCBP is a gamma-globulin of restricted electrophoretic heterogeneity as shown by immunoelectrophoresis. HCBP was shown to bind radioiodine-labeled CEA both by a radioimmune precipitation assay and by a radioimmunoelectrophoresis assay. This protein was of practical interest because of its potential usefulness as a carrier of radioactivity or therapeutic agents to a CEA-producing tumor for therapeutic or diagnostic purposes.

Doxorubicin (adriamycin) was conjugated via the dextran bridge method to a murine IgG3 monoclonal antibody, 1G3.10, directed against human bladder cancer. The drug-antibody conjugate, prepared from using 25% oxidized dextran as the linker, retained essentially the original immunological activity of the antibody using ELISA as tested against an antigen-positive target cell line (TSGH-8301), which has been shown to express an antigen recognized by the monoclonal antibody 1G3.10. Antitumor effect of the conjugate in vitro was evaluated by its inhibition on 3H-uridine incorporation into the established human bladder cancer cells. The conjugate exhibited a significantly higher cytotoxicity on target TSGH-8301 cells than that by a control antibody-doxorubicin conjugate prepared identically from an irrelevant mouse IgG3 monoclonal antibody. No apparently different cytotoxicity was detected on control antigen-negative bladder tumor cells of J82 between these two drug-antibody conjugates. Verapamil, a calcium channel blocker, enhanced the in vitro cytotoxicity of doxorubicin-1G3.10 monoclonal antibody conjugate. Results obtained from in vivo evaluation using xenografted target TSGH-8301 bladder tumor indicated that the 1G3.10 monoclonal antibody conjugate containing doxorubicin injected 4X, i.p., significantly inhibited TSGH-8301 bladder tumor growth in nude mice, whereas free monoclonal antibody, free drug and the mixture of both showed only moderate inhibition of tumor growth as compared to the untreated control. Verapamil also enhanced in vivo antitumor activity of the conjugate. There was no side effect (weight loss) detected on the conjugate-treated mice. Results obtained from in vivo evaluation using xenografted control J82 bladder tumor showed no specific antitumor activity as exhibited by doxorubicin-1G3.10 monoclonal antibody conjugate in comparison with free drug, mixture of drug and antibody without conjugation, or doxorubicin conjugated to the irrelevant antibody. These results suggested that doxorubicin conjugated with bladder tumor associated monoclonal antibody could be useful as a potentially cytotoxic agent in immunochemotherapy of human bladder cancer.

A pancreas-specific antigen was identified by immunologic techniques and purified from saline extract of human pancreas. The purified pancreas-specific antigen was shown to be homogeneous by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. It had a molecular weight of 44000 as estimated by gel filtration or sodium dodecyl sulfate-gel electrophoresis, and a sedimentation coefficient of 3.4 S as analyzed by sucrose gradient centrifugation. Pancreas-specific antigen possessed an isoelectric point of 4.9 and migrated to alpha-beta region upon immunoelectrophoresis. By colorimetric assay procedures, pancreas-specific antigen exhibited no enzyme activity, such as amylase, protease, esterase, lipase, acid phosphatase, alkaline phosphatase peroxidase, deoxyribonuclease or ribonuclease. Immunoreactivity of pancreas-specific antigen was sensitive to proteolytic enzymes, perchloric acid and high temperature (70 degrees C, 10 min); but insensitive to neuraminidase or beta-glucosidase. Immunohistochemical staining revealed that pancreas-specific antigen was located in acinar cells of human pancreas. In addition, a higher concentration of pancreas-specific antigen was detected in pancreatic juice than in the saline extract of pancreas. This newly identified pancreas-specific antigen, therefore, may be a useful marker protein in physiological studies of pancreas and pancreatic secretion.

Human prostate-specific antigen has been found to exhibit a mild activity of protease at neutral pH. This finding is based on two observations: a proteolytic activity was always associated with the antigen fractions during purification, and the proteolytic activity and the antigen were precipitated with specific antibody to the antigen. In comparison with physico-chemical and catalytic properties of known proteases, human prostate-specific antigen is a distinct neutral protease.

Hybridoma clones were established by fusing spleen cells from mice immunized with purified breast cancer-related immune complexes to drug-resistant nonproducing myelomas. Hybridoma cultures were evaluated for antibody production using immunofluorescence, radioimmunoassay and immunoperoxidase techniques. One antibody demonstrated strong reactivity against breast tumor cells (BT-20) in culture and also was found to bind ductal epithelial cells within sections of human mammary tissue. Antigen was detected within the soluble fraction of breast tumors with molecular weight greater than 500 K. The possible relationship between antigen detected in breast tumor cells and in immune-complexed form is discussed.