Biomarker development, from bench to bedside.

Critical Reviews in Clinical Laboratory Sciences

ISSN: 1040-8363 (Print) 1549-781X (Online) Journal homepage: http://www.tandfonline.com/loi/ilab20

Biomarker development, from bench to bedside Ulf-Håkan Stenman To cite this article: Ulf-Håkan Stenman (2016) Biomarker development, from bench to bedside, Critical Reviews in Clinical Laboratory Sciences, 53:2, 69-86, DOI: 10.3109/10408363.2015.1075468 To link to this article: https://doi.org/10.3109/10408363.2015.1075468

Published online: 18 Aug 2015.

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Date: 01 December 2017, At: 01:08

http://informahealthcare.com/lab ISSN: 1040-8363 (print), 1549-781X (electronic) Crit Rev Clin Lab Sci, 2016; 53(2): 69–86 ! 2015 Taylor & Francis. DOI: 10.3109/10408363.2015.1075468

REVIEW ARTICLE

Biomarker development, from bench to bedside Ulf-Ha˚kan Stenman

Downloaded by [RMIT University Library] at 01:08 01 December 2017

Department of Clinical Chemistry, Biomedicum, Helsinki University and Helsinki University Central Hospital (HUCH), Helsinki, Finland

Abstract

Keywords

This review describes studies performed by our group and other laboratories in the field aimed at development of biomarkers not only for cancer but also for other diseases. The markers covered include tumor-associated trypsin inhibitor (TATI), tumor-associated trypsin (TAT), human chorionic gonadotropin (hCG), prostate-specific antigen (PSA) and their various molecular forms, their biology and diagnostic use. The discovery of TATI was the result of a hypothesis-driven project aimed at finding new biomarkers for ovarian cancer among urinary peptides. TATI has since proved to be a useful prognostic marker for several cancers. Recently, it has been named Serine Peptidase Inhibitor Kazal Type 1 (SPINK1) after being rediscovered by several groups as a tumor-associated peptide by gene expression profiling and proteomic techniques and shown to promote tumor development by stimulating the EGF receptor. To explain why a trypsin inhibitor is strongly expressed in some cancers, research focused on the protease that it inhibited led to the finding of tumor-associated trypsin (TAT). Elevated serum concentrations of TAT-2 were found in some cancer types, but fairly high background levels of pancreatic trypsinogen-2 limited the use of TAT-2 for cancer diagnostics. However, trypsinogen-2 and its complex with a1-protease inhibitor proved to be very sensitive and specific markers for pancreatitis. Studies on hCG were initiated by the need to develop more rapid and sensitive pregnancy tests. These studies showed that serum from men and nonpregnant women contains measurable concentrations of hCG derived from the pituitary. Subsequent development of assays for the subunits of hCG showed that the b subunit of hCG (hCGb) is expressed at low concentrations by most cancers and that it is a strong prognostic marker. These studies led to the formation of a working group for standardization of hCG determinations and the development of new reference reagents for several molecular forms of hCG. The preparation of intact hCG has been adopted as the fifth international standard by WHO. Availability of several well-defined forms of hCG made it possible to characterize the epitopes of nearly 100 monoclonal antibodies. This will facilitate design of immunoassays with pre-defined specificity. Finally, the discovery of different forms of immunoreactive PSA in serum from a prostate cancer patient led to identification of the complex between PSA and a1-antichymotrypsin, and the use of assays for free and total PSA in serum for improved diagnosis of prostate cancer. Epitope mapping of PSA antibodies and establishment of PSA standards has facilitated establishment well-standardized assays for the various forms of PSA.

Epitope mapping, hCGb, human chorionic gonadotropin, immunoassay standardization, prostate-specific antigen, SPINK1, tumor markers, tumor-associated trypsin inhibitor, tumor-associated trypsin History Received 26 March 2014 Revised 19 May 2015 Accepted 7 June 2015 Published online 18 August 2015

Abbreviations: AAT: a1-antitrypsin; ACT: a1-antichymotrypsin; A2M: a2-macroglobulin; API: a1-protease inhibitor; BPH: benign prostatic hyperplasia; COPA: Cancer Outlier Profile Analysis; CEA: carcinoembryonic antigen; CRC: colorectal cancer; CRP: C-reactive protein; DELFIA: dissociation-enhanced lanthanide fluoroimmunoassay; DRE: digital rectal examination; ECM: extracellular matrix; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; ERCP: Endoscopic retrograde cholangiopancreatography; ELISA: enzyme-linked immunosorbent assay; ErbB1: epidermal growth factor receptor B1; ERK: extracellular signal-regulated kinase; ERSPC: European Randomized Screening for Prostate Cancer; fPSA: free PSA; %fPSA: proportion of free PSA; hCG: human chorionic gonadotropin; hCGa: a subunit of human chorionic gonadotropin; hCGb: b subunit of human chorionic gonadotropin; hCGbcf: core fragment of hCGb; hCG-h: hyperglycosylated hCG; hCGn: nicked form of human chorionic gonadotropin; hCGbn: nicked form of human chorionic gonadotropin b subunit; hK2: human kallikrein 2; HUVEC: human umbilical vein endothelial cells; HBV: hepatitis B virus; HCV: hepatitis C virus; Gln: glutamine; GnRH: gonadotropin releasing hormone; IFCC: International This article was originally published with errors. This version has been amended. Please see Erratum (http://dx.doi.org/10.3109/ 10408363.2016.1146497). Address for correspondence: Ulf-Ha˚kan Stenman, Department of Clinical Chemistry, Biomedicum, Helsinki University and Helsinki University Central Hospital (HUCH), PB 63, FIN-00014 Helsinki, Finland. E-mail: [email protected]

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Crit Rev Clin Lab Sci, 2016; 53(2): 69–86

Federation for Clinical Chemistry; IAPI: inter-a-protease inhibitor; ISOBM: International Society for Oncology and Biomarkers; IRR: international reference reagents; IS: international standard; KLK2: kallikrein-related peptidase 2; KLK3: kallikrein-related peptidase 3; LH: luteinizing hormone; LH/CG receptor: common receptor for LH and hCG; Lys: Lysine; MAPK: mitogenactivated protein kinase; MMP: matrix metalloproteinase; NSGCT: non-seminomatous germ cell tumors; PLCO: Prostate, Lung, Colorectal and Ovarian Cancer Screening; PSA: prostate-specific antigen; PSA-ACT: PSA–ACT complex; PSC: primary sclerosing cholangitis; PSTI: pancreatic secretory trypsin inhibitor; RIA: radioimmunoassay; RNAi: RNA interference; SPINK1: serine peptidase inhibitor Kazal type 1; TATI: tumor-associated trypsin inhibitor; TGFb: transforming growth factor b; tPSA: total PSA; uPA: urokinase-type plasminogen activator; USPSTF: US Preventive Services Task Force; WHO: World Health Organization


Introduction In recent years, research by our group and others has led to the discovery of tumor markers and the development and evaluation of assays for these. The development of new diagnostic markers may arise from hypothesis driven studies or the need to improve existing methods by making them more rapid, sensitive and specific. The use of these markers for diagnosis of cancer and other diseases will be described in this review.

Tumor-associated trypsin inhibitor and tumor-associated trypsin Since most tumor markers are proteins, it may be expected that tumor associated peptides also exist, which due to their small size, should be excreted into the urine. Research aimed at identification of new markers in urine from ovarian cancer patients was inspired by the way Gold and Friedman identified carcinoembryonic antigen (CEA)1. Our team used immunological techniques to identify and isolate a 6 kDa peptide from the urine of an ovarian cancer patient2. As the peptide inhibited trypsin, it was termed tumor-associated trypsin inhibitor or TATI. N-terminal sequencing showed that TATI was identical with the Kazal inhibitor, also called pancreatic secretory trypsin inhibitor (PSTI)3,4. Earlier studies had revealed an important role of PSTI in the pancreas5 and in pancreatitis6,7, but its role in cancer was a new finding. Later, characterization of the TATI/PSTI gene revealed close similarity with a family of protease inhibitors called serine peptidase inhibitor Kazal type (SPINK) and among these TATI/PSTI is SPINK1 (reviewed in Ref.8,9). Subsequent evaluation of TATI as a tumor marker by radioimmunoassay, showed that TATI proved to be a fairly good marker for ovarian cancer, and elevated serum and urine concentrations were also observed in several other cancers, including cervical, endometrial10, colorectal, bladder, liver, gastric, lung, breast, prostatic and renal cell carcinoma (reviewed in Ref.8). It is intriguing that TATI is useful for diagnosis of so many cancers, but, with the exception of hepatocellular and bladder cancer, TATI has so far not proved superior to existing tumor markers for other types of cancer. TATI is most useful as a prognostic marker, especially for ovarian11, bladder12,13 and hepatocellular cancer14. The function of PSTI in the pancreas is thought to inhibit trypsin that has been inadvertently activated within the pancreas, and thus, to protect against development of pancreatitis5. This function was confirmed when it was shown that mutations causing inactivation of PSTI increased

the risk of pancreatitis15,16. The fact that ovarian cyst fluid may contain very high concentrations of TATI17, combined with the confirmed co-expression of TATI and trypsin in the pancreas prompted the question of whether ovarian cysts also contain trypsinogen. In some mucinous cyst fluids, trypsinogen concentrations were found to exceed 20 mg/L, which is in the same range as in pancreatic fluid. As ovarian cyst fluid is available in large quantities, our laboratory has purified trypsinogen-1 and -218, and developed highly sensitive immunofluorometric assays for these19. Characterization of the isolated trypsinogens showed that they were otherwise identical to pancreatic trypsinogen-1 and -2, but the trypsinogens in ovarian cyst fluid had higher isoelectric points and slightly different enzymatic activity against chromogenic substrates18. Hence, they were termed tumor-associated trypsinogen-1 and -2 (TAT-1 and TAT-2). It was later shown that unlike the TATs, pancreatic trypsinogens contain a sulfate group at Tyr154, which explains the more acidic isoelectric points of pancreatic compared to tumor-associated trypsinogens20. Similar to the fact that TATI is produced by various types of cancers, several cancer cell lines were also found to secrete TATs, with TAT-2 being the major form. TAT-2 was produced in considerable amounts in the colorectal cancer (CRC) cell line COLO 205, the fibrosarcoma cell lines HT 1080 and 8387, and in K-562 erythroleukemia cells. Treatment with dexamethasone suppressed TAT-2 expression in HT 1080 but not in COLO 205 cells21, and treatment with doxycycline and chemically modified tetracycline, which downregulate matrix metalloproteinases (MMPs), also downregulated TAT-2 expression22. When grown on fibronectin-coated wells or on extracellular matrix (ECM), the cancer cell lines were shown to degrade ECM and release fibronectin fragments into the medium. This effect was enhanced by enteropeptidase and plasminogen, and the proteolysis was inhibited by addition of TATI and anticatalytic antibodies to trypsin-223,24. Further studies showed that TAT-2 activates type IV procollagenases, e.g. matrix metalloproteinases MMP2 and MMP925. In cyst fluids from ovarian tumors, the concentration of TAT-1 and -2 and their complexes with a1-protease inhibitor (API, also called a1-antitrypsin, AAT) correlated with the proportion of activated MMP-9 but inversely with that of MMP-226. In invasive squamous cell cancer, TAT-2 and MMP-9 co-localize in intracellular vesicles. Furthermore, these cells also express enterokinase (enteropeptidase), which is an efficient trypsinogen activator27. Together, these results indicate that TAT2 may form part of a proteolytic cascade that facilitates tumor invasion and metastasis.

Biomarker development, from bench to bedside

DOI: 10.3109/10408363.2015.1075468

Table 1. AUC values of various tumor markers for diagnosis of cholangiocarcinoma with and without concomitant primary sclerosing cholangitits (PSC).

Marker


Trypsinogen-2 hCGb CEA TATI Trypsinogen-1 CA 19-9 Trypsin-2-API

Cholangiocarcinoma no PSC

Cholangiocarcinoma and PSC

0.805 0.673 0.646 0.632 0.630 0.613 0.597

0.759 0.710 0.683 0.558 0.558 0.655 0.546

Earlier immunohistochemical studies had shown that trypsinogen is expressed by cholangiocarcinomas28,29 and that many patients with this disease have increased serum concentrations of trypsin-1-API30. Patients with primary sclerosing cholangitis (PSC) often develop cholangiocarcinoma. Liver transplantation is used to treat PSC in patients who do not have a concomitant cholangiocarcinoma. Markers indicating the presence of cholangiocarcinoma are therefore of value for selection of patients suitable for transplantation. Early studies showed that elevated serum concentrations of trypsinogen-2 and trypsin-2-API were common in patients with cancers of the biliary tract31. In a study comparing the ability of various serum markers to predict the presence of cholangiocarcinoma in patients with PSC, trypsinogen-2 had the largest area under the ROC curve (0.80), which was clearly larger than that for human chorionic gonadotropin beta (hCGb), CEA, TATI, trypsinogen-1, CA 19-9 and trypsin-2API (Table 1). Thus, trypsinogen-2 in serum is a useful marker for identification of PSC patients with cholangiocarcinoma32. Based on the finding of trypsinogen in normal prostatic tissue, investigation by our group has also shown that TAT-1 and TAT-2 are expressed in prostate cancer tissue and that expression increases with higher grade, while that of prostatespecific antigen (PSA) is decreased. Androgen stimulation of LNCaP cells, which increases PSA expression, suppressed that of TAT-1 and -2. This work shows that expression of TAT and PSA are regulated by different mechanisms. The results suggest that TAT may stimulate growth and invasion of prostate cancer in a paracrine way33. Recently, determination of trypsinogen in serum has also been found to be useful for differentiation between metastatic and localized breast cancer especially when combined with assay of TATI and epidermal growth factor receptor (EGFR) in serum34.

Trypsinogens in normal tissues Earlier studies have shown that trypsinogen immunoreactivity is found in the liver and especially in the peribiliary glands28. Bile was found to contain both TATI and trypsinogen-1 and -2 at highly variable concentrations. They could also be detected by immunohistochemistry in benign and malignant gall bladder epithelium35. Serum from pancreatectomized patients has been shown to contain trypsinogen-2 at concentrations that were about 15% of those in healthy controls. Trypsinogen-1 was detected only occasionally. Characterization of the immunoreactivity by ion exchange

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chromatography revealed the presence of isoforms resembling both TAT-2 and pancreatic trypsinogen-236. Studies on the expression of trypsinogens in the genitourinary tract showed that trypsinogen-1 and -2 occur in seminal fluid at median concentrations of 400 and 500 mg/L, respectively. Tissue expression was detected by immunohistochemistry and in situ hybridization in several tissues, i.e. prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands and testis37. Other research has shown that trypsinogen is expressed in Paneth cells of the ileum38. It co-localizes with defensin HD5 in secretory granules and cleaves the HD5 propeptide to active defensin. Trypsin acts as a prodefensin convertase and is thus involved in the regulation of innate immunity in the gastrointestinal tract. The isoenzyme pattern differs from that in the pancreas, trypsinogen-2 being the main form representing 86% and trypsinogen-3 (mesotrypsin) 14% at the mRNA level39. In broncoalveolar lavage fluid from patients with inflammatory lung disease, high concentrations of TAT-2-API were associated with increased activation of MMP-8 and MMP-9 but not of MMP-240. Increased concentrations of TAT-2 have also been observed in the lungs of infants with respiratory distress syndrome41. These results suggest that trypsin can be involved in inflammatory processes. Taken together, these results show that trypsinogen is expressed in many normal tissues and that it is involved in various physiological processes not associated with its role in food digestion. This also explains why trypsinogen occurs at low but measurable concentrations in serum from pancreatectomized patients36.

Trypsinogen in pancreatitis Recent studies of trypsinogen-1 and -2 in serum from healthy subjects confirmed earlier results, which showed that the concentrations of trypsinogen-1 were higher than those of trypsinogen-242. However, in pancreatitis patients, the concentration of trypsinogen-2 was much higher than that of trypsinogen-119. Thus, trypsinogen-2 is a more sensitive and specific marker of pancreatitis than total trypsin immunoreactivity or trypsinogen-18. As TATI serves as a first line of defense against premature activation of trypsinogen within the pancreas, a complex between trypsin and TATI would be expected to be the first sign of pancreatitis, but attempts to detect this complex have not been successful. This may be explained by rapid dissociation of TATI/PSTI from trypsin in circulation43. However, the trypsin-2-API complex can be detected in serum from healthy controls and patients with pancreatitis. Trypsin2-API has actually been found to be a better marker than trypsinogen-2 for pancreatitis with an area under the ROC curve of 1.0, while that for trypsinogen-2 was 0.996 and 0.929 for amylase44. Trypsin-2-API has also been demonstrated to be a better predictor of severe disease than trypsinogen-2 and the established markers amylase and C-reactive protein (CRP)45. Determination of amylase in urine is used for diagnosis of pancreatitis as an alternative to assay of serum, thus, there was some question as to whether determination of trypsinogen in

U.-H. Stenman 3500

Mild pancreatitis 3000

Serum amylase (IU/L)

urine is also of diagnostic utility. In healthy controls, the urine concentrations of both trypsinogen-1 and -2 were fairly low, but in pancreatitis, the concentration of urine trypsinogen-2 increased much more strongly than that in serum46. Therefore, urine trypsinogen-2 was equally useful for diagnosis of pancreatitis and, for prediction of disease severity, it was a better marker than amylase35. The urine concentrations of trypsinogen-1 were very low in comparison to those of trypsinogen-2. This is probably explained by the much more extensive breakdown of trypsinogen-1 during passage through the kidneys. This is surprising as the two isoenzymes are very similar in size and amino acid sequence. A rapid immunochromatographic test for urinary trypsinogen-2 has been developed based on the above findings. With a cut-off of 50 mg/L, the rapid test has a sensitivity and specificity of 94 and 95%, which is slightly better than that of serum trypsinogen and clearly better than those of serum and urine amylase47,48. Using a cut-off of 2000 mg/L, the rapid test is useful for predicting development of severe pancreatitis49. Serum trypsinogen-2 concentrations exceeding 1000 mg/L were found in 91% of the cases that developed necrotizing pancreatitis50. Endoscopic retrograde cholangiopancreatography (ERCP) is used for diagnosis and treatment of biliary and pancreatic disorders; however, this procedure is associated with potentially serious complications, especially pancreatitis. Assay of trypsinogen might therefore be useful to facilitate early detection of pancreatitis induced by ERCP. In patients developing pancreatitis, serum trypsinogen-2 increased on average 26-fold at 6 h after the procedure, while trypsin-2-API increased 11-fold after 24 h. A 3-fold increase in urine trypsinogen-2 at 6 h indicated pancreatitis with better than 90% sensitivity and specificity51. The rapid test could also be used to detect pancreatitis, but about 30% of the patients already had a positive test before the procedure, which limited its specificity51. The test is available from Medix Biochemica ([email protected]). The symptoms of pancreatitis are often non-specific and up to 40% of fatal cases are diagnosed at autopsy. Amylase is a very sensitive test at presentation, but if diagnosed several days after onset, amylase concentrations may have normalized. Analysis of the time course profiles of trypsinogen-2 and amylase in serum of pancreatitis patients revealed that in patients with mild pancreatitis, the concentrations of amylase normalized on average in 4 days, and in severe cases, within 6 days (Figure 1), while in contrast, the concentrations of trypsinogen-2 and trypsin-2-API remained elevated for 2 weeks in mild, and up to 4 weeks in severe cases (Figure 2). The prolonged elevation facilitates detection of pancreatitis in cases with atypical symptoms that may delay diagnosis. Taken together, these results show that trypsinogen-2 and trypsin-2-API are more reliable markers for pancreatitis than amylase and lipase52. The rapid trypsinogen-2 test is especially useful for ruling out pancreatitis in an outpatient setting53. Calibration of assays for trypsinogen-2 is complicated by the instability of the pure enzyme, which tends to autoactivate and degrade even when stored frozen. Active trypsin can be stabilized by inactivation with phenylmethylsulfonyl fluoride, but this affects immunoreactivity. Recombinant techniques


Severe pancreatitis

2500

2000

1500

1000

500

0

0

2

4

6

8

10 12 14 17 21

Time (d) Figure 1. Time course of serum of amylase in serum of patients with severe and mild acute pancreatitis. The dotted line indicates the upper reference limit. The amylase concentration normalizes in 3–6 days after presentation. Reproduced from Scandinavian Journal of Gastroenterology52 with permission. 3500

3000

Mild acute pancreatitis

Trypsinogen-2 in serum


72

Severe acute pancreatitis

2500

2000 1500

1000 500

0 0

2 4

6 8 10 12 14 16 18 21 24 27 29 34

Time (d) Figure 2. Time course of serum trypsinogen-2 concentrations (mean ± SD) in patients with severe and mild acute pancreatitis. The dotted line indicates the upper reference limit. The trypsinogen-2 concentrations remain elevated for the entire period of hospitalization. Reproduced from Scandinavian Journal of Gastroenterology52 with permission.

can be used to prepare mutated forms of trypsinogen-1 and -2 (Lys23Gln), which do not autoactivate and are thus stable during storage. With these standards, new reference values have been established for trypsinogen-1 and -254. Using monoclonal antibodies and recombinant trypsinogen-3 (also called mesotrypsinogen), a specific sandwich immunoassay has been developed55. Trypsin-3 may play a role in the development of pancreatitis because it is not inhibited by, but degrades, TATI/PSTI. Trypsin-3 may thus initiate activation

DOI: 10.3109/10408363.2015.1075468

of other enzymes within the pancreas5,56. The concentrations of trypsinogen-3 in serum are higher in patients with pancreatitis than in controls with upper abdominal pain of other causes55. It remains to be seen whether trypsinogen-3 provides additional information not obtained by measuring trypsinogen-2 and trypsin-2-API. Another reason to develop assays for trypsinogen-3 is its potential role in cancer.


Functions of TATI in cancer Studies on trypsinogens and TATI in cancer have suggested that the association between elevated TATI concentrations and cancer was dependent on co-expression with TAT, which was originally thought to be the main factor contributing to tumor aggressiveness8,57. However, studies on the expression of TATI and TAT in prostate cancer indicated that TATI by itself could play a role in cancer development. Expression of TATI was low in benign prostatic epithelium and moderate to strong in prostate cancer and high-grade prostatic intraepithelial neoplasia. Thus, strong TATI expression was associated with high Gleason grade. Interestingly, serum TATI was elevated in 44% of patients with advanced prostate cancer. TATI expression was observed in several prostate cancer cell lines and especially in 22Rv1 cells, in which its expression is suppressed by androgens58. Tomlins et al. used Cancer Outlier Profile Analysis (COPA) to identify candidate oncogenes from transcriptomic data based on high expression in a subset of prostate cancer cases (outlier expression). Several known oncogenes have been identified by this approach including ETS family members. Using COPA, it was shown that the TATI gene was overexpressed in ETS rearrangement-negative prostate cancers. Strong TATI expression was observed in about 10% of all cases and this was associated with adverse outcome in surgically treated patients. Knockdown of TATI in the aggressive prostate cancer cell line 22Rv1 reduced invasiveness. This suggests that TATI has a functional role in ETS rearrangement-negative prostate cancers59. Further studies showed that recombinant TATI/SPINK1 contributes to the aggressive phenotype of both benign as well as malignant prostatic cell lines60. Mechanisms by which SPINK1 could cause aggressive growth were studied using prostate cancer cell lines, in vitro invasion assays and xenografts in mice. Forced expression was found to enhance, whereas knockdown of SPINK1 reduced tumor growth and invasion. The effect of SPINK1 was at least partially mediated through the epidermal growth factor (EGF) receptor (EGFR). The effect of EGF and SPINK1 was additive, suggesting that different mechanisms were involved. Treatment of mice carrying SPINK1-expressing prostate cancer xenografts with antibodies to SPINK1 or EGFR reduced tumor growth by 60 and 40%, respectively, and by 75% when used together. This treatment had no effect on SPINK1-negative xenografts. The authors suggest that SPINK1 antibodies could be useful for targeted therapy of a subgroup of aggressive prostate cancers. Taken together, the results show that TATI may stimulate tumor invasion and this may at least partially be mediated through the EGFR60. The function of TATI as a growth factor was initially suggested based on the structural similarity between TATI


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and EGF, as well as the finding that TATI stimulates growth of human fibroblasts61 and endothelial cells62. Furthermore, overexpression of TATI in mice stabilizes intestinal mucosa and stimulates repair after injury of intestinal cells expressing functioning EGFR63. TATI has also been found to stimulate the growth of cells derived from rat pancreatic acinar cell tumors64 and from pancreatic65 and colorectal cancer66. Studies with recombinant EGFRs showed that TATI binds to the EGF receptor ErbB1 with an affinity that is half of that for EGF. TATI induces EGFR phosphorylation and stimulates growth of NIH 3T3 cells and several pancreatic cancer cell lines. These effects can be blocked by inhibitors of EGFR and MAPK/ERK kinase65. Marchbank et al. recently showed that TATI stimulates migration and invasion of bladder carcinoma cell lines. These effects were synergistic with that of EGF and were inhibited by an EGFR-blocking antibody. Knockdown of endogenous TATI also suppressed migration and invasion67. Taken together, these studies show that TATI acts as a growth factor that mediates its effect through the ErbB1 EGF receptor. TATI is secreted by many malignant tumors and cancer cell lines derived from various tissues, e.g. ovarian, endometrial, cervical10, prostatic59,60 pancreatic65, colon66, hepatocellular cancer68 and cholangiocarcinoma31,32,58,69. In all of these, and probably also in other cancers, TATI may act as an autocrine growth factor70. Tumor-associated trypsin inhibitor (TATI) has also been shown to suppress apoptosis induced by the serine protease granzyme A71–73. TATI binds granzyme A72 and the effect on the apoptotic machinery is dependent on the protease inhibitor activity as shown by lack of effect of K18Y mutated TATI73. Expression of TATI in the liver is upregulated by hepatitis B and C viruses (HBV and HCV)71 and it is increased in hepatocellular carcinoma (HCC) associated with hepatitis68. By inhibiting granzyme A, TATI attenuates the immune response and mediates evasion of apoptotic cell death. This is thought to facilitate persistent virus replication and eventual development of HCC71. Overexpression of TATI has been associated with adverse prognosis of HCC74. In hereditary hemochromatosis-related HCC, SPINK1 is the most strongly upregulated gene as demonstrated by global gene expression profiling and immunohistochemistry75. Thus, TATI is a potential marker for HCC75. Earlier studies have shown that the TATI concentrations in serum are elevated in HCC patients68. In these, TATI in serum was shown to be a stronger prognostic factor for adverse outcome than alpha fetoprotein14. It is intriguing that the tumor-association of TATI may be dependent on many different mechanisms in different tumors, i.e. inhibition of trypsin and granzyme A, as well as stimulation of EGFR. However, loss of TATI expression in tumor tissue is, on the other hand, associated with adverse prognosis in bladder76 and gastric cancer77. In these, TATI may act as a protective factor that may be dependent on its function as a protease inhibitor. The exact mechanisms by which TATI exerts its effect are only partially known. Further elucidation of these mechanisms may facilitate development of targeted therapies. In some tumors, TATI acts as an autocrine growth factor and blocking this activity with an antibody is feasible because TATI exerts its function in the extracellular space. It has been suggested that humanized

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anti-TATI antibodies could be used to treat cancer59,66. However, due to its protective function in the pancreas, this may be associated with increased risk of pancreatitis78. It is intriguing, that TATI was discovered more than 30 years ago as a tumor-associated peptide by an immunological approach and that it has been rediscovered by gene expression profiling of prostatic59, breast73, colorectal66, urothelial79 and liver cancers75. In many of these cancers, TATI is one of the most strongly upregulated genes. Thus, the TATI gene SPINK1 can be classified as an oncogene that may contribute to the development of cancer78.


Various forms of hCG in pregnancy diagnostics and as a tumor marker Diagnosis of pregnancy and trophoblastic cancer is based on determination of hCG. In the 1970s, biological assays taking several days were still used as pregnancy tests, however, rapid immunological methods, mainly based on agglutination or agglutination inhibition, were introduced around this time. The first assays were based on antisera that crossreacted with luteinizing hormone (LH), but this was not a problem because the detection limit of the assays was so high (100–1000 IU/L) that LH did not cause a positive result80. A lower detection limit was desirable for diagnosis of pregnancy-related disorders like ectopic pregnancy. The first sensitive and specific radioimmunoassay (RIA) for hCG was established using the SB6 antiserum prepared against the b subunit of hCG (hCGb) and the hCG standard CR-11981. The assay had a detection limit of 3 IU/L and took 24 h to perform. This was excellent for research work but the assay time was long when the aim was to detect ectopic pregnancy in an emergency setting82. The pregnancy tests available at that time had a detection limit of 300–500 IU/L, which was not sensitive enough for diagnosis of early pregnancy and gynecological emergencies83. Our group therefore modified the SB6 RIA by shortening the assay time to 2 h. The detection limit was 25 IU/L, which made it possible to detect a pregnancy at the time of the first missed period84. Studies with the SB6 RIA had shown that, in addition to patients with trophoblastic tumors, many with non-trophoblastic tumors had elevated serum concentrations of hCG85. While the rapid hCG RIA was useful for diagnosis of gynecological emergencies, it was not quantitative and sensitive enough for diagnosis of cancer. Furthermore, it detected not only hCG but also hCGb, and at this time, it was not clear which forms of hCG were produced by different tumors. Inspired by the studies of Miles and Hales86 and Engvall and Perlman87 on the use of labeled antibodies and sandwich assays, specific assays for the major forms of hCG were developed. Initially an antibody recognizing hCG and hCGb, but not LH, was used to replace the polyclonal antibody used in the rapid RIA88. Later improvements resulted in development of a very rapid and sensitive assay based on dissociation-enhanced lanthanide time resolved fluoroimmunoassays (DELFIA). The new DELFIA-based rapid and sensitive sandwich assay for hCG had an incubation time of 20 min and a detection limit of 1.7 IU/L. This assay was used to examine women with lower abdominal pain or vaginal bleeding.


Among 130 cases, 17 had apparently false positive results89. Initially, these results were confusing but later studies by Wilcox et al. showed that they were caused by early fetal loss. It is now well known that about 20% of all pregnancies end in early fetal loss90, and with modern pregnancy tests with detection limits of 10–25 IU/L, many of women with early fetal loss will test positive before the first missed period, which often is a few days delayed. This has caused much confusion and suspicion of ectopic pregnancy if examination of tissue obtained at surgery or curettage does not reveal trophoblastic tissue89. When combined with transvaginal ultrasound, the rapid and sensitive hCG assay was shown to be especially useful for early identification of ectopic pregnancies. This was valuable for screening of high-risk patients with a history of ectopic pregnancy. The change in serum hCG was found to facilitate identification of patients with ectopic pregnancy needing surgery91,92. Monitoring of the concentrations of hCG and hCGb after term pregnancy showed that hCGb disappeared more slowly than hCG, and thus, the ratio of hCGb to hCG increased from 0.8% at term to 27% 3 weeks later93. This means that the half-life of hCGb in plasma after a pregnancy is actually longer than that of hCG, which is contrary to results observed by injection of radiolabeled hCG and hCGb94. There has also been speculation as to whether monitoring of hCG, hCGb and hCGa in pregnancies achieved by in vitro fertilization could be used to predict outcome. Ectopic pregnancies were characterized by a 2-day delay in the increase in hCG concentrations, but thereafter the pattern was similar to that in successful pregnancies95. An hCG concentration below 76 IU/L 12 days after embryo transfer was found to predict high risk of early pregnancy loss96. For use in cancer diagnostics, a lower detection limit of the hCG assays was desirable, and with longer incubation times, the assay had a detection limit of 0.7 IU/L97. Further optimization of this assay improved the detection limit to 0.03 IU/L, allowing examination of whether hCG could be detected in serum of men and non-pregnant women. Earlier studies with an antiserum prepared against the unique Cterminal peptide had shown that hCG could be detected in urine98, and Borkowski and Muquardt had extracted hCC-like material from plasma of non-pregnant subjects99. Measurable concentrations of hCG could be detected in most men and women, in whom the concentrations increased with age. In women, the upper reference limit was 5 IU/L and 2 IU/L in men. Injection of gonadotropin releasing hormone (GnRH) caused an increase, while treatment with estrogen and progesterone resulted in suppression of hCG concentrations. This showed that most of the hCG was derived from the pituitary. The specificity of the assay, i.e. lack of crossreaction with LH, was confirmed by separation of hCG from LH by gel filtration100. Odell and Griffin showed that the secretion of hCG is pulsatile, confirming that hCG in nonpregnant women and men is secreted by the pituitary101. hCG was later isolated from the pituitary by Birken et al.102. Pregnancy serum was known to contain the free b subunit of hCG (hCGb) but results from different studies varied103,104. Monoclonal antibodies to hCGb and a sensitive and specific assay were developed, and the concentrations of hCG and hCGb in serum at various stages of pregnancy were


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determined. The results showed that the proportion of hCGb was highest (1–7%) in the first 4 weeks of pregnancy. In the second and third trimester, the median proportion was 51%. The specificity of the assay was confirmed by separating hCG and hCGb completely from each other by hydrophobic interaction chromatography105. With sensitive and specific assays for hCG, hCGb and for the core fragment of hCGb (hCGbcf) reference values could be established for serum and urine in men and non-pregnant women106. Most serum samples contained hCG and hCGb at measurable concentrations, but those of hCGb did not increase with age. While the concentrations of hCG could reach 15 pmol/L in post-menopausal women, the upper reference limit for hCGb, at 2 pmol/L, was much lower106. Early studies with RIA methods had shown that urine and serum from some cancer patients contain detectable concentrations of hCG immunoreactivity, which had not been wellcharacterized due to lack of sensitive and specific assays85,107. In most reports, it was assumed that the immunoreactivity consisted of hCG (reviewed in Ref.108). The concentrations of hCG, hCGb and hCGbcf in serum and urine of patients with pancreatic and biliary tract cancer were determined using the sensitive assays. The results showed that hCGb was the main form occurring in serum while hCGbcf was the main form in urine. hCGb in serum was the best marker because pituitary hCG contributed to the urine concentrations of hCGbcf. Thus, many non-trophoblastic cancers produce hCGb This was independently confirmed by Marillac et al.109. When excreted into urine, most of hCGb is degraded to hCGbcf110. Later studies have shown than about 20–50% of all non-trophoblastic cancers studied produce hCGb, i.e. colorectal111–114 squamous-cell115 ovarian and other gynecological adenocarcinomas11,116,117, breast118, bladder119,120, renal121,122, gastric123, fallopian tube cancer124 and cholangiocarcinoma32. As a diagnostic marker, hCGb is not superior to other tumor markers, but in most diseases, it is a strong prognostic marker that is independent of other markers and tumor histology108. The prognostic value of hCGb is especially strong in patients with ovarian cancer (Figure 3). Contrary to some earlier reports, our group has not found clearly elevated serum or urine concentrations of intact hCG in any patient with nontrophoblastic cancer108. Human chorionic gonadotropin (hCG) produced by choriocarcinoma has been shown to contain more complex and more extensively sialylated carbohydrates than pregnancy hCG125. This so-called hyperglycosylated hCG (hCG-h) could be detected with a monoclonal antibody, B152 produced by Birken et al.126, using an hCG-h preparation, C5, purified from urine of a choriocarcinoma patient by Cole et al.125. B152 recognizes a core 2 (i.e. biantennary) o-glycan on Ser132 and surrounding amino acids on the C-terminal peptide of hCGb127. This was confirmed by isolation of hCGh with a B152 affinity column and site-specific determination of the glycans on hCGb (Figure 4). This study further showed that hCG is highly heterogeneous mainly due to variation in carbohydrate composition128. The most striking difference in glycosylation is that pregnancy-derived hCG carries core 1 o-glycans at Ser127, Ser132 and Ser138 while core 2 o-glycans are more abundant in hCG-h from cancer patients128. hCG-h is also produced in early pregnancy,


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Figure 3. Survival curves for ovarian cancer patients presenting with pre-operative serum hCGb concentration below or above 2 pmol/L. Reproduced from International Journal of Cancer11 with permission.

when it is the major form of hCG129,130. There is no standard for hCG-h but it is presently defined as hCG reacting with B152. Our group is currently using hCG produced by the choriocarcinoma cell line JEG as a temporary standard, in which the content of hCG-h is determined on the basis of the hCG concentration measured by the Wallac assay for intact hCG131. In testicular cancer, hCG is an established marker and hCG-h has been found to be a major form of hCG in nonseminomatous germ cell tumors (NSGCT). hCG-h has been associated with aggressive forms of this disease132 but it does not add diagnostic or prognostic value additional to that of conventional hCG assays131. In 78% of patients with NSGCT, hCGb and hCG were simultaneously elevated but in seminoma, hCG was elevated in only 17%, while hCGb was elevated in 52% of the cases133. Thus, specific assay of hCGb is more useful than measurement of ‘‘total hCG’’ (i.e. intact hCG and hCGb) for monitoring of seminoma patients. Assays measuring ‘‘total hCG’’ will detect only part of the cases with elevated hCGb because the upper reference limit for hCG + hCGb is higher (18 pmol/L) than that for hCGb (2 pmol/L)133. Various forms of hCG can be detected by immunohistochemistry in testicular cancer tissue. Interestingly, staining for hCG-h is exclusively positive in NSGCT but not in seminomas. Staining for hCG-h is therefore of potential value for differential diagnosis in some cases that are difficult to classify134. Measurement of hCGb and its ratio to total hCG also appears to be useful for evaluation of patients with trophoblastic disease. During the more than 20 years that our group has used this approach, all patients with benign trophoblastic disease have had a proportion of hCGb less than 5%, while the proportion has been higher in patients with choriocarcinoma108. This finding needs to be confirmed as we see only about one case of choriocarcinoma each year. The proportion


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Figure 4. Structure of typical o-linked carbohydrate chains of hCG-h and mid-pregnancy hCG. The additional structures on hCG-h that are lacking on pregnancy hCG are indicated with red color in the online version. The core 2 o-glycan on Ser 132 is required for reactivity with B152. With the exception of the core 2 o-glyacn on Ser 121, the structures of the other glycans vary considerably. Modified from Ref.128.

Figure 5. Serum hCG and hCGb after successful therapy of a metastatic choriocarcinoma. The horizontal broken lines show the upper reference limits for HCG (thick line) and HCGb (thin line). As hCGb disappears from circulation more slowly than HCG, the ratio between these increases with time. After the initial decrease, serum HCG increases again to 8 pmol/l due to chemotherapyinduced gonadal suppression. Reproduced from Human Reproduction Update with permission210.

of hCGb must be determined before therapy because it disappears more slowly than hCG93, and thus the proportion increases when the concentrations decrease after successful therapy. Another important finding is that intense chemotherapy often suppresses gonadal function, which may cause a moderate increase in serum hCG after initial normalization of the concentration (Figure 5). This must not be interpreted as a relapse, and the condition can be identified by determination of serum LH, which is strongly elevated108,135. Human chorionic gonadotropin (hCG) has been shown to promote implantation and angiogenesis during placental development by stimulating the LH/CG receptor136,137. In addition, hCG-h has been claimed promote trophoblast invasion, and implantation independent of hCG138. Recently, hCG-h, but not ‘‘normal’’ hCG, was found to exert angiogenic activity, which was not mediated by the CG/LH receptor but by the transforming growth factor beta (TGFb) receptor139.

Interestingly, some studies have shown that hCGb, which does not bind to the classical CG/LH receptor, also promotes migration and invasion of prostate cancer cells140 and stimulates the growth of cancer cells141. The effect of various forms of hCG on invasion of JEG-3 choriocarcinoma cells has been studied, and intact hCG, hCG-h and hCGb all stimulated invasion to a similar degree in JEG-3 cells even when the CG/ LH receptor was down-regulated by RNAi142. Taken together, these results show that the proinvasive effect of different forms of hCG is not mediated by the LH/CG receptor. Increased invasion is associated with increased levels of matrix metalloproteinases (MMP)-2 and MMP-9 and active urokinase-type plasminogen activator (uPA)142. Some studies show that the effect of various forms of hCG is mediated by the TGFb receptor139, but results from different studies are contradictory, thus further research is needed to clarify the mechanism by which various forms of hCG exert their effects.

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Due to the ability of hCG-h to stimulate early trophoblast invasion during implantation, it is intriguing that pregnant women, who later developed pre-eclampsia, had a lower proportion of hCG-h in serum than healthy controls during the first143 but not the second trimester144. However, lower hCG-h concentrations have been observed in second trimester urine145.


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Table 2. Nomenclature of WHO International Reference Reagents (IRR) for various forms of hCG. So far no standard for hyperglycosylated hCG (hCG-h) is available. hCG: human chorionic gonadotropin hCGn: ‘‘nicked hCG’’, hCG with cuts in hCGb subunit between amino acids 42-48 hCGb: free b subunit hCGbn: ‘‘nicked hCGb’’, hCGb with cuts between amino acids 42-48 hCGbcf: ‘‘core fragment’’ of hCGb hCGa: free a subunit


Standardization of hCG determinations The relationship between various forms of hCG is relevant only if the concentrations are expressed in molar units. During a Bergmeier conference on immunoassay standardization organized by the International Federation for Clinical Chemistry (IFCC) in 1990, the participants were asked to suggest standardization projects. It was suggested that a project be developed for standardization of immunoassays for hCG, and a working group of major experts in the field was subsequently formed. The main goals of the project were: (A) establishment of a uniform nomenclature and abbreviations for the various molecular forms of hCG, (B) preparation of new calibrators for the major molecular forms of hCG and establishment of methods for determining the substance concentrations (mol/ L) of these, (C) improving quality assessment materials and procedures and (D) definition of methods for characterization of hCG measurement procedures146. In 1995, the IFCC appointed a working group (WG), whose first goal was to establish nomenclature and prepare pure standards and to establish methods for measurement of concentrations in mol/L for the various forms of hCG23,146. The expressions beta-hCG and hCGb have often erroneously been used to describe hCG assays using antibodies prepared against hCGb. The need for new standards was based on the fact that the third IS contained some partially degraded hCG, i.e. nicked hCG and some impurities, which caused problems when the preparations were used for immunoassay standardization. The decision to assign values to the standards in substance concentrations was based on the fact that immunoassays reflect molar concentrations of protein rather than bioactivity147. Financing of new hCG standards became possible through support from the IFCC and from 13 diagnostics companies that together provided 155 000 USD in exchange for early access to the hCG preparations. The six forms of hCG that were considered clinically important, i.e. hCG, hCGb, hCGa, hCGbcf and two partially degraded or nicked forms, i.e. hCGn and hCGbn were purified (Table 2)148. The nicked variants were needed for assay characterization because many antibodies specific for intact hCG underestimate hCGn, which may represent a substantial part of hCG in urine and also in serum128,149,150. A major form of hCG immunoreactivity in urine of pregnant women is hCGbcf, and it is of potential value as cancer marker in urine151,152. hCGbcf is also important for characterization of pregnancy tests and immunoassays used for detection of hCG immunoreactivity in urine. Urine often contains a large excess of hCGbcf, which may react with only one of the antibodies used in sandwich assays and thus it can cause false negative results in pregnancy tests153.

The new standards were assigned values in substance concentrations by amino acid analysis. In different assays, the mean bioactivity of the hCG preparation was 13 300 IU/mg148. Thus, its potency was about 40% higher than that of the identical third and fourth international standard (IS). The six preparations were approved by WHO as the First International Reference Reagents (IRR) for six hCG-related molecules154. The first IRRs have been used to characterize commonly used immunoassays. The results show that hCGn is differentially recognized by several assays. Recognition of hCGb by assays designed to measure hCGb together with hCG is variable and most assays overestimate this form. A few assays recognize hCGbcf but underestimate it155–157. These studies demonstrate the value of the new IRRs for assay standardization and characterization, which was a major goal of the hCG working group146. In 2013, WHO replaced the fourth IS for hCG with the first IRR, which is designated WHO International Standard, fifth WHO IS Chorionic Gonadotrophin, NIBSC Code: 07/364. This standard has been assigned a dual unitage by WHO. For calibration of immunoassays, each ampoule contains 179 IU corresponding to 0.39 nmol, while for use in bioassays the ampoule content is 162 IU. These values have been determined in a collaborative study organized by WHO, but the results have not yet been published (http://www.nibsc.org/ documents/ifu/07-364.pdf. Accessed 20 March 2014). One of the aims of the hCG Working Group was to establish reference methods for determination of hCG. This goal has not yet been achieved, but as a first step in this direction, the epitopes of a large number of monoclonal antibodies have been determined in two workshops organized in collaboration with the International Society for Oncology and Biomarkers (ISOBM). In the first workshop, 27 monoclonal antibodies were characterized158 while the second comprised 69 antibodies from research groups and diagnostic companies. The specificity of nearly all antibodies could be ascribed to 13 epitopes on hCGb, seven on hCGa and 4 present only on intact hCG. Knowledge of the epitope specificity will facilitate design of immunoassays with predefined specificity159. However, the final specificity of an immunoassay can only be determined by analysis of hCG standards (IRRs) and well-characterized samples from patients with various conditions.

Various forms of prostate-specific antigen in prostate cancer Studies on prostate-specific antigen (PSA) were initiated by the discovery of two samples with very high PSA


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concentrations that produced a hook effect, i.e. the concentrations increased when the samples were diluted. Characterization of PSA in these samples by gel filtration revealed two components, which could have been explained by complex formation or the occurrence of a fragment160. Based on earlier studies with complexes of trypsin and protease inhibitors, assays were established for putative PSA complexes using a catcher antibody to PSA and Eu-labeled antibodies for different plasma protease inhibitors. Fractions of prostate cancer serum were analyzed, and the larger component of PSA was shown to be a complex with a1antichymotrypsin (ACT), which was the main form of PSA in serum from prostate cancer patients161. Lilja et al. independently confirmed these results162 and furthermore showed that PSA reacts with ACT in vitro163. Interestingly, it was later shown that part of the PSA–ACT complex forms already within the prostate and that PSA–ACT levels are increased in moderately differentiated prostate cancer tissue164. Complexes with other inhibitors were also identified, i.e. a1-protease inhibitor (API) and inter-a-protease inhibitor (IAPI) and a2-macroglobulin (A2M) but their concentrations were low in comparison with those of PSA–ACT and free PSA. An important finding was that the proportion of PSA– ACT was higher, while that of free PSA was lower in prostate cancer patients than in men with benign prostatic hyperplasia (BPH). Thus, determination of the two major forms of PSA in serum improved the cancer specificity of the PSA test161,162. In order to reduce variation when measuring total PSA and complexed PSA with separate assays, a double label assay for these forms was developed. The assay used a capture antibody recognizing complexed and free PSA equally and separate detection antibodies to PSA and ACT, respectively. The antibody to PSA was labeled with Eu3+ and the ACT antibody with Sm3+. The two labels were measured separately at different wavelengths by time-resolved fluorometry. With this assay, it was confirmed that the proportion of PSA in complex with ACT was higher in patients with prostate cancer than in those with BPH. For diagnosis of prostate cancer, the area under the curve (AUC) value for total PSA was 0.64 while that for the proportion of PSA–ACT was 0.78. Assay of PSA– ACT was, however, hampered by non-specific absorption of ACT and ACT complexes to the solid phase. Due to this, the apparent proportion of PSA–ACT exceeded 100% in some samples165. To eliminate this problem, monoclonal antibodies were raised to the PSA–ACT complex and a dual label assay was developed with an antibody preferentially recognizing this complex. However, although the assay was improved, it was not superior to determination of free PSA166. Most clinical studies have used the assay for free (fPSA) and total PSA (tPSA) commercialized by LKB-Wallac167. Using this double label assay, reference values were established for free and total PSA in various age groups168. The first studies on PSA complexes showed that prostate cancer serum contained, in addition to ACT, complexes with other inhibitors. Assays were developed for PSA-A2M and PSA–API and examined for their ability to improve differentiation between prostate cancer and BPH. Using a specific sandwich assay, it was found that the proportion of PSA–API was lower in prostate cancer patients (0.9%) than in controls (1.6%). Like the PSA–ACT assay, this assay was also


hampered by a variable background caused by other API complexes. When the background was measured separately and subtracted, the proportion of PSA–API improved cancer specificity, but the complicated assay design precluded its routine use169. Later, an improved assay for PSA–API was developed based on the proximity-ligation principle. While this assay was sensitive enough and eliminated non-specific background enabling accurate measurement of PSA–API in serum specimens with moderately increased PSA concentrations, the complicated assay design prohibited its clinical use170. When added to serum, a major part of PSA binds to A2M, which engulfs PSA so that it can be detected only after denaturation of the complex. An assay was developed based on removal of PSA and PSA–ACT with antibody-coated magnetic beads, denaturation of serum at high pH and measurement of PSA released from A2M with a PSA assay for total PSA. Like with PSA–API, the proportion of PSA– A2M was higher in serum from BPH patients (11.6%) than in prostate cancer patients (8.2%). This may be explained by complex formation between partially nicked PSA and A2M occurring after sampling. Interestingly, the sum of PSA–A2M and fPSA improved detection of prostate cancer more efficiently than the proportion of free PSA (%fPSA)171,172. However, this assay was also too complicated for routine use. Studies on free and complexed PSA in serum showed that prostate cancer risk increased with decreasing %fPSA. In order to evaluate the risk of various combinations of tPSA and fPSA, logistic regression and multilayer perceptron networks were used to estimate the risk associated with these variables as well as the effect of heredity and a positive finding in digital rectal examination (DRE). All of these affected the risk and %fPSA had the strongest effect, i.e. at a tPSA concentration of 10 mg/L, the risk increased from 3% when %fPSA was 35% to nearly 40% when %fPSA was 10% (Figure 6)173. The algorithm was further developed by including prostate volume, which was also an independent variable174. When used to predict biopsy result in a screening program, the multivariable risk calculation model eliminated more false positive results than %fPSA alone175. Similar results have been obtained by logistic regression or with neural networks developed for other populations176–178. It is important to recognize that networks are assay-specific because various PSA assays often give slightly different results176. However, different risk calculation algorithms provide the same diagnostic accuracy if they are validated with the assay actually used179. Some of these algorithms are freely available (e.g. at http://personal.inet.fi/surf/finne/)175. In some hospitals, the probability of a positive biopsy is reported when a clinician orders a PSA test that includes free PSA. By this approach, it is possible to avoid prostate biopsy if cancer probability is low in spite of an elevated tPSA value. Use of risk calculation algorithms is a first step in order to reduce over-diagnosis and overtreatment of prostate cancer, which are the most important arguments against prostate cancer screening180.

Epitope mapping of PSA Early assays for PSA tended to give quite different results181. This was caused by preferential detection of free PSA and


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Figure 6. Calculation of the probability of a positive prostate biopsy based on logistic regression using PSA. The ratio of fPSA/tPSA in normal (A) or abnormal (B) DRE findings in men with PSA concentrations in the range 3–45 mg/. Reproduced from Ref.173.

complex formation of PSA with inhibitors when calibrators were prepared by adding purified PSA to a serum-based matrix. Much of the added PSA became undetectable when it reacted with A2M and some epitopes on PSA were blocked by complex formation with ACT and API182. To facilitate standardization of PSA assays, the TD-3 Workshop under the auspices of ISOBM was initiated. In this, 83 PSA antibodies from major assay manufacturers and research groups were characterized. Of the antibodies, 70 could be characterized for reactivity with 13 epitopes in six antigenic regions, some of which were specific for PSA while some also occurred on the related kallikrein, hK2 (presently KLK2). This information facilitated design of assays specific for fPSA and tPSA that recognized free and complexed PSA equally, so called equimolar assays. The study was performed by 11 research groups and the results described in 12 articles. The results were summarized in Ref.183. With well-characterized antibodies and standards for PSA and PSA-ACT, it became possible to standardize PSA assays in a satisfactory way184. Even if there is still need for improvement, PSA assays are probably now among the best standardized tumor marker determinations179. However, small differences in assay calibration have a considerable effect on the number of positive results in a screening setting. Therefore, diagnostic algorithms for prostate cancer have to be established separately for different assays176.

Prostate cancer screening Early studies on the use of the PSA-test for screening of prostate cancer were performed in 1991185; however, there were no prognostic studies showing whether a PSA-based screening would reduce mortality186. Our group had access to a serum bank comprising serum samples from 7125 men aged

Figure 7. Serum concentrations of PSA in relation to time to diagnosis of prostate cancer. Reproduced from the Lancet with permission187.

49–70 years. Among these, 44 developed prostate cancer within 12 years after blood sampling and their survival was recorded for 22 years. In most cases, PSA started to increase 6–10 years before diagnosis187 (Figure 7). The doubling time of PSA calculated by regression analysis was 3 years, which is in agreement with studies on patients under surveillance but not actively treated188. These results demonstrate that the development of prostate cancer is unusually slow. Long survival after the time of sampling further showed how slowly most prostate cancers progressed. Among prostate cancer cases with a PSA concentration below 4 mg/l, 53% survived for 15 and 43% for 20 years after sampling. Of those with higher PSA concentrations the survival rates at 15 and 20 years were 27% and 18%, respectively (Figure 8). Half of the


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Figure 8. Disease-specific survival after serum sampling of prostate cancer cases with serum PSA below (upper curve) and above 4 mg/l (lower curve) (cf. Figure 7). Reproduced from the Lancet with permission187.

false positive results in controls with PSA values in the range 2.5–25 mg/l could be eliminated by measuring the proportion of PSA–ACT. Based on these results, it was predicted that it is unlikely that screening of men with less than 10–15 years life expectancy will reduce mortality187. This prediction was confirmed in the Scandinavian SPCG-4 study. Radical prostatectomy reduced cancer-specific mortality of men less than 65 years of age but not in older men189. In later studies with long follow-up, mortality has been substantially lower. For instance, in the SPCG-4 study comparing watchful waiting versus radical prostatectomy, the cancer-specific mortality at 15 years was about 15% irrespective of treatment in men over 65 years but clearly higher (about 30%) in men under 65 years of age under active surveillance189. There are several possible reasons for the lower mortality in recent studies. In earlier studies, the cause of death was probably in many cases erroneously ascribed to prostate cancer once this diagnosis had been established. Furthermore, radical therapy was less common and endocrine therapy less effective. However, the main explanation is probably that most patients diagnosed based on an elevated PSA value have less aggressive diseases than those diagnosed based on the clinical symptoms. This explanation is supported by the even lower mortality in the European Randomized Screening for Prostate Cancer (ERSPC)190 than in SPCG-4189. Based on the promising results for PSA and PSA–ACT in our serum bank study, a randomized screening study was started in Finland in 1996187 and combined with ERSPC, in which the Finnish arm was the largest one. Now, after results for 11 years of follow-up have been reported, it is clear that screening reduces mortality by about 30%, when the effect of non-compliance is taken into account190. These results are far better than those obtained in the randomized Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial in the USA, in which the disease-specific survival was no better in the screening than in the control arm191. This can be explained by PSA testing in the control arm and noncompliance in the screening arm192.


The pros for and cons against prostate cancer screening have been extensively discussed in the US Preventive Services Task Force (USPSTF) recommendation statement based on the results of the PLCO and the ERSPC studies. The conclusion is that ‘‘PSA-based screening as currently implemented in the United States produces more harms than benefits in the screened population’’. The major problem is over-diagnosis and over-treatment. This is a readily accepted problem and therefore population screening is not recommended by either USPSTF or ERSPC. However, PSA-driven diagnostics can be recommended for well-informed men who wish to be screened190,191. It is obvious that further development of the screening algorithms is needed. Overtreatment can be reduced by active surveillance of men with well-differentiated tumors and it may even reduce mortality by eliminating treatment complications193. Screening markers are needed that identify men with aggressive tumors. Biopsy should be performed only on men with a clear risk of harboring life-threatening tumors based on serum (or urine) marker determinations. Promising results have been obtained with assay of PSA in combination with several mRNA-based urine markers194. Interestingly, preliminary studies suggest that TATI also is a promising prognostic marker for prostate cancer (Stenman et al. unpublished).

Development of peptides modulating the proteolytic activity of PSA and KLK2 The prostate produces a large number of proteases, many of which are members of the family of kallikrein-related peptidases (KLKs). These may affect the development and growth of prostate cancer195,196. Among the kallikreins, KLK2 and KLK3 (i.e. PSA) are most strongly expressed in the prostate and prostate cancer. Proteases have generally been associated with tumor invasion and aggressive disease, but they may also cause tumor suppression197. Kallikreins may thus affect development and growth of prostate cancer and are therefore potential targets for treatment. To explore this, peptides were developed that reacted with KLK3 and KLK2 using phage display198,199. Those reacting with KLK2 inhibited its enzymatic activity198,199 while those reacting with KLK3 stimulated its activity199. Interestingly, PSA has been shown to exert antiangiogenic activity200,201, and it was found that this activity was dependent on the enzymatic activity of PSA and could be inhibited by small molecule PSA-inhibitors202 and a monoclonal antibody to fPSA (KLK3). More importantly, the antiangiogenic activity of PSA was enhanced by peptides as studied in an in vitro model using human umbilical vein endothelial cells (HUVEC) grown on a basement membrane matrix (Matrigel)203,204. Studies have examined whether the peptides affect the growth of LNCaP xenografts in mice, but so far the effect has not been reproducible. This may be caused by the short halflife of the peptides caused by rapid renal excretion and breakdown in plasma by exopeptidases. Therefore, more stable peptide analogs and synthetic peptidomimetics are being developed, in which part of the peptide structure is replaced by building blocks that are resistant to proteolytic breakdown205,206. Recently, the first small drug-like molecule

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that stimulates PSA-activity using pharmacophore-based virtual screening has been identified207. Prostate-specific antigen (PSA)-binding peptides have also been used together with antibodies to develop assays recognizing only active free PSA208. Such assays have been hard to develop using antibodies only209. The ultimate goal is to develop compounds that stimulate the antiangiogenic activity of PSA, and thus slow down the growth of prostate cancers enough to prevent the tumor from surfacing clinically. In many prostate cancer patients, this might reduce the need for radical therapy.


Conclusions The common denominator for the research described in this review has been to improve diagnosis of cancer and certain other diseases. In many cases, markers intended for diagnosis of cancer have proved useful for other diseases, and research has strived to utilize these findings clinically. These studies have also elucidated the limitations of some cancer markers because their serum levels may also be elevated in benign diseases. An essential part of this work has concerned immunoassay standardization and development of better standards and mapping of epitopes recognized by the antibodies used in immunoassays.

Acknowledgements Throughout my career, I have been fortunate to collaborate with enthusiastic and skilled people. I have especially enjoyed tutoring doctoral students by helping them to experience the joy of discovering new phenomena. I have also had the luck to collaborate with many skilled clinicians, Markku Seppa¨la¨, Caj Haglund, Pekka Ylo¨stalo, Olavi Ylikorkala, Bruno Cacciatore, Dan Apter and Leo Dunkel, biochemists and biologists, Ursula Turpeinen, Jim Schro¨der, Marja-Liisa Huhtala, Hannu Koistinen, Leena Valmu. Thanks to these I have been able to develop clinically relevant methods. I have had the luck of having very skilled technicians working with me for decades, especially Taina Gro¨nholm, Maarit Leinimaa and, Marianne Niemela¨. The collaboration with Steven Birken, Peter Berger, Jean-Michel Bidart, Rob Norman, Catharine Sturgeon and Elisabeth Paus has been essential for the success of the hCG standardization and epitope mapping of hCG. Collaboration with other research groups has been essential for many projects and I would like to thank, Timo Lo¨vgren, Kim Pettersson, Hans Lilja, Anders Bjartell, Barry Dowell, Kjell Nustad and Olle Nilsson. Collaboration with diagnostics companies has been crucial by facilitating the use of cutting edge technology. I am grateful to Erkki Soini, who as the research director of LKBWallac gave me access to reagents and equipment for timeresolved fluorometry. Collaboration with PerkinElmerWallac, Orion Diagnostica, Medix Biochemica, and Abbott Diagnostics has been essential for the success of many of my projects. It has also been a necessity in order to make new assay commercially available.

Disclosures The author holds patents for TATI, tumor-associated trypsinogen, free and complexed PSA, PSA complexes with API


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and A2M and peptides modulating the activity of KLK2 and KLK3. He has served as a consultant for LKB-Wallac, Pharmacia-Wallac, PerkinElmer, Orion Diagnostica and Abbott Diagnostics.

Declaration of interest The author reports no conflicts of interest. The author alone is responsible for the content and writing of this article.

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