We have compiled from literature and other sources a list of 1261 proteins believed to be differentially expressed in human cancer. These proteins, only some of which have been detected in plasma to date, represent a population of candidate plasma biomarkers that could be useful in early cancer detection and monitoring given sufficiently sensitive specific assays. We have begun to prioritize these markers for future validation by frequency of literature citations, both total and as a function of time. The candidates include proteins involved in oncogenesis, angiogenesis, development, differentiation, proliferation, apoptosis, hematopoiesis, immune and hormonal responses, cell signaling, nucleotide function, hydrolysis, cellular homing, cell cycle and structure, the acute phase response and hormonal control. Many have been detected in studies of tissue or nuclear components; nevertheless we hypothesize that most if not all should be present in plasma at some level. Of the 1261 candidates only 9 have been approved as "tumor associated antigens" by the FDA. We propose that systematic collection and large-scale validation of candidate biomarkers would fill the gap currently existing between basic research and clinical use of advanced diagnostics.

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A list of candidate cancer biomarkers for targeted proteomics.

Tremendous efforts have been made over the past few decades to discover novel cancer biomarkers for use in clinical practice. However, a striking discrepancy exists between the effort directed toward biomarker discovery and the number of markers that make it into clinical practice. One of the confounding issues in translating a novel discovery into clinical practice is that quite often the scientists working on biomarker discovery have limited knowledge of the analytical, diagnostic, and regulatory requirements for a clinical assay. This review provides an introduction to such considerations with the aim of generating more extensive discussion for study design, assay performance, and regulatory approval in the process of translating new proteomic biomarkers from discovery into cancer diagnostics. We first describe the analytical requirements for a robust clinical biomarker assay, including concepts of precision, trueness, specificity and analytical interference, and carryover. We next introduce the clinical considerations of diagnostic accuracy, receiver operating characteristic analysis, positive and negative predictive values, and clinical utility. We finish the review by describing components of the FDA approval process for protein-based biomarkers, including classification of biomarker assays as medical devices, analytical and clinical performance requirements, and the approval process workflow. While we recognize that the road from biomarker discovery, validation, and regulatory approval to the translation into the clinical setting could be long and difficult, the reward for patients, clinicians and scientists could be rather significant.

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Translation of proteomic biomarkers into FDA approved cancer diagnostics: issues and challenges

The implantation process involves complex and synchronized molecular and cellular events between the uterus and the implanting embryo. These events are regulated by paracrine and autocrine factors. Trophoblast invasion and migration through the uterine wall is mediated by molecular and cellular interactions, controlled by the trophoblast and the maternal microenvironment. This review is focused on the molecular constituents of the human trophoblast, their actions and interactions, including interrelations with the uterine endometrium.

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Human trophoblast function during the implantation process

Human chorionic gonadotropin (hCG) is a glycoprotein hormone comprising 2 subunits, alpha and beta joined non covalently. While similar in structure to luteinizing hormone (LH), hCG exists in multiple hormonal and non-endocrine agents, rather than as a single molecule like LH and the other glycoprotein hormones. These are regular hCG, hyperglycosylated hCG and the free beta-subunit of hyperglycosylated hCG. For 88 years regular hCG has been known as a promoter of corpus luteal progesterone production, even though this function only explains 3 weeks of a full gestations production of regular hCG. Research in recent years has explained the full gestational production by demonstration of critical functions in trophoblast differentiation and in fetal nutrition through myometrial spiral artery angiogenesis. While regular hCG is made by fused villous syncytiotrophoblast cells, extravillous invasive cytotrophoblast cells make the variant hyperglycosylated hCG. This variant is an autocrine factor, acting on extravillous invasive cytotrophoblast cells to initiate and control invasion as occurs at implantation of pregnancy and the establishment of hemochorial placentation, and malignancy as occurs in invasive hydatidiform mole and choriocarcinoma. Hyperglycosylated hCG inhibits apoptosis in extravillous invasive cytotrophoblast cells promoting cell invasion, growth and malignancy. Other non-trophoblastic malignancies retro-differentiate and produce a hyperglycosylated free beta-subunit of hCG (hCG free beta). This has been shown to be an autocrine factor antagonizing apoptosis furthering cancer cell growth and malignancy. New applications have been demonstrated for total hCG measurements and detection of the 3 hCG variants in pregnancy detection, monitoring pregnancy outcome, determining risk for Down syndrome fetus, predicting preeclampsia, detecting pituitary hCG, detecting and managing gestational trophoblastic diseases, diagnosing quiescent gestational trophoblastic disease, diagnosing placental site trophoblastic tumor, managing testicular germ cell malignancies, and monitoring other human malignancies. There are very few molecules with such wide and varying functions as regular hCG and its variants, and very few tests with such a wide spectrum of clinical applications as total hCG.

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New discoveries on the biology and detection of human chorionic gonadotropin.

For centuries, physicians have attempted to use the urine for noninvasive assessment of disease. Today, urinalysis, in particular the measurement of proteinuria, underpins the routine assessment of patients with renal disease. More sophisticated methods for assessing specific urinary protein losses have emerged; however, albumin is still the principal urinary protein measured. Changes in the pattern of urinary protein excretion are not necessarily restricted to nephrourological disease; for instance, the appearance of β-human chorionic gonadotropin in the urine of pregnant women is the basis for all commercially available pregnancy kits. Similarly, microalbuminuria is a clinically important marker not only of early diabetic nephropathy but also of concomitant cardiovascular disease. With the emergence of newer technologies, in particular mass spectrometry, it has become possible to study urinary protein excretion in even more detail. A variety of techniques have been used both to characterize the normal complement of urinary proteins and also to identify proteins and peptides that may facilitate earlier detection of disease, improve assessment of prognosis and allow closer monitoring of response to therapy. Such proteomics-based approaches hold great promise as the basis for new diagnostic tests and as the means to better understand disease pathogenesis. In this review, we summarize the currently available methods for urinary protein analysis and describe the newer approaches being taken to identify urinary biomarkers.

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Urine proteomics: the present and future of measuring urinary protein components in disease

Background: Results on sera and calibrators vary 1.4- to 2.3-fold among commercial human chorionic gonadotropin (hCG) assays. The relative contributions of calibrators, standards, hCG charge isoforms, and major structural variants to this variation have not been quantified.

Methods: Purified hCG was separated by isoelectric focusing into four fractions with pI ranges of 3–4, 4–5, 5–6, and 6–7. These four fractions together with pure hCG, hyperglycosylated hCG, hCG β-subunit (hCGb), nicked hCG, and hCGb core fragment (hCGbcf) were tested in nine commonly used commercial serum assays for hCG. The compositions of pure hCG preparations, standards, and commercial hCG preparations were determined by immunoassay.

Results: The three pure hCG preparations and the four hCG charge isoforms each showed parallel responses in the nine commercial hCG assays. Although wide variations were found in the detection of hCG structural variants by the nine assays (range for hyperglycosylated hCG, 468-1544 IU/L; for hCGb, 3187-5535 IU/L; for nicked hCG, 2736-4240 IU/L; and for hCGbcf, <2–130 IU/L), this did not correlate with the between-method variation observed in results for the three pure hCG preparations. Commercial preparations of hCG and calibrators showed great variation in their content of hCG structural variants (from 34% to 100% intact hCG).

Conclusions: Intermethod differences in hCG results were not explained by changes in responses attributable to hCG charge isoforms or to hCG structural variants, but wide variation was observed in concentrations of hCG structural variants in calibrators and in detection of these structural variants. Differences in assay specificity and in composition of the calibrators are the most likely sources of between-method variation.

Between-Method Variation in Human Chorionic Gonadotropin Test Results

Hyperglycosylated hCG (invasive trophoblast antigen, ITA) a key antigen for early pregnancy detection.

Accuracy of home pregnancy tests at the time of missed menses

Human chorionic gonadotropin (hCG) is a glycoprotein composed of 2 dissimilar subunits, alpha and beta, joined non-covalently. This hormone is not only heterogeneous in peptide structure but also in combination of subunits and in the structure of carbohydrate side chains. Common hCG-related molecules in serum samples include regular hCG, hyperglycosylated hCG (ITA), nicked hCG, nicked ITA, hCG missing the beta-subunit C-terminal extension, free alpha-subunit, free beta-subunit, free beta-subunit missing the C-terminal extension, hyperglycosylatedfree beta-subunit and nickedfree beta-subunit. The same molecules plus beta-core fragment are present in urine samples. While ITA and regular hCG predominate in pregnancy samples, any one of these multiple hCG-related molecules may be the principal source of immunoreactivity in gestational trophoblastic disease, gestational trophoblastic neoplasm, choriocarcinoma and placental site tumor cases as well as in testicular cancer and germ cell tumor. As such it is critical to appropriately detect all these isoforms in the management of these diseases. Only 2 tests, the DPC Immulite (DPC, Inc., Los Angeles, California) and U.K. RIA (radioimmunoassay) (used at Charing Cross Hospital, London) appropriately detect all these hCG-related molecules. False positive hCG results are a major problem in the management of gestational trophoblastic disease and cancer. A particular problem is observed with the Abbott AxSym test. This test is flawed in design. It should be avoided in the management of gestational trophoblastic disease and cancer. As shown in a blind study, a proportion of false positive samples in the Abbott AxSym test (Abbott Laboratories, Inc., Chicago, Illinois) can also be false positive with the U.K. RIA; none are false positive with the DPC Immulite test. Results clearly show that the DPC Immulite/Immulite 2000 is the only appropriate assay for monitoring patients with gestational trophoblastic disease or cancer.

Selecting an appropriate hCG test for managing gestational trophoblastic disease and cancer.

Introduction to hCG and hCG-Related Molecules HCG is a glycoprotein composed of 2 dissimilar subunits, and -subunit, held together by charge interactions. HCG is an unusual glycoprotein in that as little as 65% of its molecular weight (MW) is due to amino acids or protein. The balance is due to large sugar side chains. HCG is sometimes considered a mucopolysaccharide, like collagen, because of the large carbohydrate component. There are 4 asparagine-linked (Nlinked) sugar side chains with 7 to 14 sugar residues attached to hCG, 2 on the -subunit and 2 on the -subunit; 4 serine-linked (Olinked) sugar side chains with 3 to 6 sugar residues, all on the -subunit. The combination of 2 subunits and 8 sugar side chains results in major variability in hCG structure. Free subunits, degraded hCG molecules, forms of hCG with irregular sugar side chains, and hCG and free subunit fragments are present in serum and/or urine samples during pregnancy and trophoblastic diseases. In addition to regular hCG (hCG), at least 5 key variants are present in serum samples: hyperglycosylated hCG, nicked hCG, hCG missing the -subunit C-terminal peptide, free -subunit, nicked free -subunit, and multiple combinations of these variations (ie, nicked hyperglycosylated hCG missing the -subunit C-terminal peptide). The same 6 molecule plus -core fragments are detected in urine samples. Table 1 summarizes the structure of the key Correspondence: Laurence A. Cole, PhD, USA hCG Reference Service, Department of Obstetrics and Gynecology, University of New Mexico Health Science Center, Albuquerque, New Mexico, USA, E-mail: larry@ hcglab.com Much of the research described in this paper was supported by NIH grant HD35654 and a research grant from Quest Diagnostics Inc. CLINICAL OBSTETRICS AND GYNECOLOGY Volume 46, Number 3, 523–540 © 2003, Lippincott Williams & Wilkins, Inc.

Jaime M. Sutton

Papers

Between-Method Variation in Human Chorionic Gonadotropin Test Results

Hyperglycosylated hCG (invasive trophoblast antigen, ITA) a key antigen for early pregnancy detection.

Accuracy of home pregnancy tests at the time of missed menses

Selecting an appropriate hCG test for managing gestational trophoblastic disease and cancer.

HCG tests in the management of gestational trophoblastic diseases.