Page 296 – Clinical Laboratory int.

Biochemical investigation of monoclonal gammopathies

, 26 August 2020/in Featured Articles /by 3wmedia

Monoclonal gammopathy (MG) refers to the presence of monoclonal immunoglobulin produced by clonally expanded plasma cells or immunoglobulin-expressing lymphocytes. MG is a key feature of a wide spectrum of diseases ranging from the indolent MG of undetermined significance to the overt multiple myeloma. In this article, we discuss the utility and pitfalls of common biochemical techniques used to detect MG.

by Dr Michelle L. Parker and Dr Pak Cheung Chan

Introduction
The monoclonal immunoglobulins or ‘M-proteins’ detected in monoclonal gammopathy (MG) are produced by clonally expanded plasma cells, or less frequently by immunoglobulin-expressing lymphocytes at different stages of maturation. The prevalence of MG in the general population over 50 years of age is approximately 3 % and increases with age. M-proteins secreted by plasma cells (Fig. 1a) can be partial or intact immunoglobulins, with the latter consisting of two heavy chains and two light chains that together form a Y-shaped structure with constant and highly variable antigen-binding domains (Fig. 1b). M-proteins that are immunologically functional may cause disease by directly binding to self-antigens, e.g. in some peripheral neuropathy. Other unique chemical properties may cause the M-protein to transform into insoluble amyloids, to increase plasma viscosity, or even to block capillary blood flow by precipitating out at the low temperatures in the extremities. As the production of M-protein increases, the mass effect can be exerted through the expanded clonal plasma cells compressing neighbouring cell lineages in the bone marrow, resulting in reduced red blood cell production (anemia), pan-leukopenia (recurrent infections), thrombocytopenia (bleeding diathesis), suppressed non-involved plasma cells (immune paresis) and bone resorption (hypercalcemia and bone lesions). Large amounts of circulating M-protein could promote plasma hyperviscosity, thrombosis, and tissue and organ damage. For example, excess filtered free light chains in multiple myeloma can directly damage the kidney proximal tubules, form amyloids rupturing glomeruli and form obstructive casts in the distal tubules leading to cell death and nephritis. In general, measured M-protein concentration is taken to reflect the tumour burden and is prognostic for disease progression or survival, e.g. in monoclonal gammopathy of undetermined significance (MGUS), smouldering myeloma and multiple myeloma.

Conditions associated with MG cover a wide range of clinical presentations and severity, including MGUS, multiple myeloma, P.O.E.M.S., light chain deposition disease, plasmacytoma, Waldenstrom’s macroglobulinemia, non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. In some of these diseases, the severity of tissue or organ damage may not be related to the M-protein concentration. For example, in some amyloid light chain (AL)-amyloidosis, extensive kidney damage is reflected by massive proteinuria, yet the circulating monoclonal free light chain can be barely, or not at all, demonstrable by serum and/or urine testing [1]. Nevertheless, the presence of an M-protein can be a defining hallmark of many of these conditions and its detection provides a critical link to their final diagnosis.

Biochemical detection of monoclonal immunoglobulins
Five common biochemistry tests form the core of first-line MG investigations and will be discussed below: serum protein electrophoresis (SPE), serum immunofixation electrophoresis (IFE), urine protein electrophoresis (UPE), urine immunofixation electrophoresis (uIFE), and serum free light chain (sFLC) assays. Other techniques such as mass spectrometry-based assays and HevyliteTM analysis are increasingly available for specific circumstances but will not be discussed here.

SPE and UPE
SPE and UPE resolve serum and urine proteins respectively into five or six major fractions, viz. albumin, alpha-1, alpha-2, beta (total beta, or beta-1 and beta-2 depending on resolution), and gamma (Fig. 2). If a monoclonal antibody is present, an additional peak may be observed, most frequently in the gamma (hence the term gammopathy) (Fig. 2) but other regions such as beta and alpha-2 are also possible. Estimating the size of this extra peak gives the amount of M-protein present and is one of the recommended methods for monitoring disease activity. However, the detection of M-protein this way requires that it is readily distinguished from background polyclonal immunoglobulins or other co-migrating proteins, which not only limits the analytical sensitivity to around 0.5–2.0 g/L [2] and prevents its use to rule-out low abundance M-proteins [3, 11], but also limits the accuracy of quantification especially at low M-protein concentrations and/or high background in any electrophoretic regions.

Importantly, an ‘abnormal’ peak identified by SPE does not prove that it is an endogenous monoclonal immunoglobulin, as the peak may be due to a haptoglobin variant, iodinated contrast material, aminoglycoside, administered biologics, or increases in other proteins such as tumour markers, transferrin in severe iron deficiency, C-reactive protein in acute inflammation, and fibrinogen in plasma or incompletely clotted serum [4]. Similarly, a positive finding in UPE can only be regarded as presumptive and should be confirmed by techniques such as IFE.

Historically, qualitative deviations from the expected SPE pattern have been taken to imply clinical conditions such as bisalbuminemia, acute-phase inflammatory response, alpha-1-antitrypsin deficiency, nephrotic syndrome, cirrhosis, hypogammaglobulinemia, etc. However, not all of these conditions as predicted by SPE patterns have been validated, nor have their clinical utility in terms of MG investigation been established [5].

IFE and uIFE
For IFE, a combination of antisera against the heavy chains (IgG, IgA, IgM, IgD, IgE), the two light chains (total kappa and total lambda) and/or the free light chains (free kappa and free lambda) is selected and separately overlaid on the electrophoresed sample. Immuno-precipitation results in a blush of staining in the presence of polyclonal immunoglobulins, while a discrete band indicates the presence of an M-protein and its isotype is determined when discrete bands in the heavy and light chain lanes are aligned (Fig. 2 inset). This immunological detection not only characterizes the M-protein whose isotype provides prognostic information, but also improves the analytical sensitivity (typically 0.2 to 0.5 g/L) enabling detection of M-proteins even when the SPE pattern is visibly normal [2]. However, a notable short fall is that the interpretation is unavoidably subjective especially when bands are faint or not well defined.

In uIFE, the focus is to detect monoclonal free light chains or Bence Jones proteins that passed through the kidneys unabsorbed. In normal individuals, immunoglobulin light chains are produced in slight excess of the heavy chains and are secreted into the circulation. Because of their small sizes, free light chains are readily filtered through the glomeruli but are efficiently absorbed in the proximal tubules. Thus, in patients with MG, the detection of monoclonal free light chains in urine usually indicates an increased production exceeding renal reabsorbing capacity, compromised reabsorption, or both. Since the secretion of free light chains into the circulation is sporadic throughout the day, a ‘pooled’ sample such as a 24-h urine collection usually improves the sensitivity as well as the reliability of urine testing, although a first-morning urine has also been accepted for initial investigations.

sFLC assays
The fully automated sFLC measures polyclonal immunoglobulin free light chains individually with high analytical sensitivity (down to mg/L) and targets the light chain epitopes that are otherwise hidden when bound to heavy chains (Fig. 3)[2]. Patients with MG often have increased concentrations of the involved free light chains, resulting in a skewed free kappa/lambda ratio as the uninvolved free light chains remains normal or suppressed. A skewed ratio not only supports the diagnosis of MG but also provides prognostication information on malignant progression for MGUS, smouldering myeloma and multiple myeloma. A free kappa/lambda ratio >100 has even been taken as a defining feature for multiple myeloma [6].

Similar to many other immunoassays, the sFLC assay is subject to antigen excess and displays dilutional non-linearity, raising concern over the accuracy of results at both high concentrations (variation due to different dilution response) and low concentrations (high dose hook effect). Additionally, falsely abnormal free kappa/lambda ratios have been reported in individuals with polyclonal gammopathy, hospitalized patients and patients with renal dysfunction. In one study, the reported positive predictive value of an abnormal ratio amongst primary care patients was only 39 % [7], underscoring the high false-positive rate in unselected patients. Although there are sFLC assays reportedly less susceptible to these limitations [8], a general lack of standardization renders the results non-commutable and values cannot be interchanged between methods.

Diagnostic testing algorithms
Although the biochemical tests discussed above play an important role in the detection of M-proteins, the information that each test provides does overlap substantially, and different test combinations may be required for different monoclonal gammopathies. Moreover, these tests tend to be costly, labour intensive, and/or require expertise for result interpretation. There is ongoing debate on the optimal testing algorithm due to competing priorities such as maximizing clinical sensitivity or diagnostic efficiency, streamlining workflows, improving economic feasibility, and reducing unnecessary or redundant testing.

With a primary goal of maximizing clinical sensitivity, the International Myeloma Working Group (IMWG) recommends SPE, IFE and sFLC as first-line tests for confirming multiple myeloma and other plasma cell disorders, with the addition of 24-hour urine studies only if AL-amyloidosis is suspected [2, 8]. Although the recommendation falls short of indicating that these tests may be performed in tandem depending on findings, it does represent a welcomed change to previous versions as 24-h urine samples are inconvenient to collect and UPE and uIFE are expensive to perform. Although sFLC testing has largely obviated the need for first-line urine studies, no single serum test has adequate clinical sensitivity for screening all plasma cell disorders [8, 9]; in one large study, SPE, IFE and sFLC had clinical sensitivities of just 79, 87 and 74 % respectively [3].

The optimal combination of first-line and reflexed tests remains difficult to determine owing to the wide spectrum of MG diseases. There is substantial redundancy if SPE and IFE are performed simultaneously. IFE contains a protein lane that provides the same qualitative detection of M-proteins as SPE. A separate SPE only provides additional information regarding quantity of the M-protein, as there are no true positives that would be missed by IFE but identified by SPE. For economic and other reasons, SPE is often performed initially and is reflexed to IFE for confirmation if SPE presents with features suggestive of an M-protein, including the observation of restricted staining or a clearly discrete band, increased beta fraction [10], or decreased gamma fraction [11]. However, this approach has been shown to miss up to 20 % of cases [3, 10–12] as some M-proteins, especially free light chains and those existing in small concentrations, may not present with any abnormal features in SPE. Recently, it was argued that the increased sensitivity of IFE over SPE warrants its use as the first-line screening test, despite being more expensive and labour intensive. The use of modified IFE protocols such as combined light chain immunofixation (a mixture of anti kappa and anti-lambda antisera), or the penta-IFE using a mixture of five antisera (anti IgG, IgA, IgM, kappa, and lambda) seems to make this approach more feasible. The counterpoint to this approach, though, is that the detection of very low concentration M proteins by IFE may lead to unnecessary investigation of transient or low risk conditions [13]. On the other hand, without full characterization of the M-protein (both isotype and concentration), it may be premature to judge the significance of an M-protein based only on its low concentration.

Concluding Remarks
Clearly, further studies are needed to balance the competing priorities of various testing algorithms and provide evidence-based approaches to MG investigations suited to the diverse clinical environments, ranging from family practice to speciality hematology clinics. Irrespective of the algorithm used, it is good practice to interpret laboratory findings within the specific clinical context to mitigate the risk of false-positive or false-negative test results.

References
1. Truong D, Blasutig IM, Kulasingam V, Chan PC. A patient with monoclonal gammopathy-related nephrotic syndrome revealed no electrophoretic “nephrotic pattern” or skewed free light chain ratio. Clin Biochem 2018; 51: 110–111.
2. Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, et al. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2009; 23(2): 215–224.
3. Katzmann JA, Kyle RA, Benson J, Larson DR, Snyder MR, Lust JA, et al. Screening panels for detection of monoclonal gammopathies. Clin Chem 2009; 55(8): 1517–1522.
4. McCudden CR, Jacobs JFM, Keren D, Caillon H, Dejoie T, Andersen K. Recognition and management of common, rare, and novel serum protein electrophoresis and immunofixation interferences. Clin Biochem 2018; 51: 72–79.
5. Chan PC, Chen Y, Randell EW. On the path to evidence-based reporting of serum protein electrophoresis patterns in the absence of a discernible monoclonal protein – A critical review of literature and practice suggestions. Clin Biochem 2018; 51: 29–37.
6. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos MV, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol 2014; 15(12): e538–548.
7. Hill PG, Forsyth JM, Rai B, Mayne S. Serum free light chains: An alternative to the urine Bence Jones proteins screening test for monoclonal gammopathies. Clin Chem 2006; 52(9): 1743–1748.
8. Tate JR, Graziani MS, Mollee P, Merlini G. Protein electrophoresis and serum free light chains in the diagnosis and monitoring of plasma cell disorders: laboratory testing and current controversies. Clin Chem Lab Med 2016; 54(6): 899–905.
9. Willrich MAV, Murray DL, Kyle RA. Laboratory testing for monoclonal gammopathies: focus on monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. Clin Biochem 2018; 51: 38–47.
10. Chan PC, Lem-Ragosnig B, Chen J. Diagnostic implications of enumerating and reporting beta fraction(s) for the detection of beta-migrating monoclonal immunoglobulins in serum protein electrophoresis. Clin Biochem 2018; 53: 77–80.
11. Chan PC, Chen J. Value of reflex testing based on hypogammaglobulinemia as demonstrated in serum protein electrophoresis. Clin Biochem 2015; 48: 674–678.
12. Pretorius CJ. Screening immunofixation should replace protein electrophoresis as the initial investigation of monoclonal gammopathy: Point. Clin Chem Lab Med 2016; 54(6): 963–966.
13. Smith JD, Raines G, Schneider HG. Should routine laboratories stop doing screening serum protein electrophoresis and replace it with screening immune-fixation electrophoresis? No quick fixes: Counterpoint. Clin Chem Lab Med 2016; 54(6): 967–971.

The authors
Michelle L. Parker¹ PhD, Pak Cheung Chan*^1,2 PhD, DABCC, FCACB
¹Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
²Department of Laboratory Medicine & Molecular Diagnostics, Sunnybrook Health Sciences Centre, Toronto, ON, Canada

*Corresponding author
E-mail: pc.chan@sunnybrook.ca

The measurement of free 25-hydroxyvitamin D

, 26 August 2020/in Featured Articles /by 3wmedia

Vitamin D status is currently assessed by measurements of total 25-hydroxyvitamin D [25(OH)D]. However, over 99% of circulating 25(OH)D is bound to protein, vitamin D binding protein in particular. The free hormone hypothesis stipulates that only the free form crosses the cell membrane to exert biologic action. Measurement of free 25(OH)D is now available.

by Professor Daniel D Bikle

Introduction
Circulating levels of 25-hydroxyvitamin D [25(OH)D] are the most commonly used marker for the assessment of vitamin D nutritional status. This is because its concentration in blood is higher than all other vitamin D metabolites, making it easier to measure, and because its conversion from vitamin D is substrate dependent with minimal regulation. However, 25(OH)D is not the most biologically active metabolite of vitamin D. Instead 25(OH)D must be further metabolized to 1,25 dihydroxyvitamin D [1,25(OH)2D] for vitamin D to achieve its full biologic potential. 1,25(OH)2D is the ligand for a nuclear transcription factor, the vitamin D receptor (VDR), that mediates the genomic and at least some of the nongenomic actions of vitamin D within the cell. Nearly all, if not all, cells express the VDR at some stage in their development or activation. As the appreciation that vitamin D and its metabolites affect numerous physiologic processes and not just bone and mineral metabolism, and that these physiologic processes may have different requirements for these vitamin D metabolites, interest in determining optimal levels of the vitamin D metabolites to effect these different biologic processes has grown. Complicating this determination is the fact that all the vitamin D metabolites circulate in blood tightly bound to proteins, of which the vitamin D binding protein (DBP) plays the major role. For most cells, these binding proteins limit the flux of the vitamin D metabolites from blood into the cell where they exert their biologic activity. This raises the issue of what should we measure to determine vitamin D status: the total levels of these metabolites or their free levels?

The free hormone hypothesis: why measure free 25(OH)D
The free hormone hypothesis postulates that only the non-bound fraction (the free fraction) of hormones that otherwise circulate in blood bound to their carrier proteins is able to enter cells and exert their biologic effects. This hypothesis applies to steroid hormones, thyroid hormone and vitamin D. For the vitamin D metabolites this hypothesis needs to be qualified in that some tissues, kidney and parathyroid glands in particular, express a transport system, the megalin/cubilin complex, that enables 25(OH)D bound to DBP to be transported into these cells. However, for cells lacking this complex the free fraction is felt to be the fraction capable of entering these cells. In serum samples from normal individuals, ~85% of circulating vitamin D metabolites are bound to DBP, whereas albumin with its substantially lower binding affinity binds only ~15% of these metabolites despite its 10-fold higher concentration than DBP. Approximately 0.4% of total 1,25(OH)2D and 0.02–0.03% of total 25(OH)D is free in serum from normal non-pregnant individuals. The fraction of ‘bioavailable’ vitamin D metabolites is composed of the fraction of the free vitamin D and the fraction bound to albumin, thus measuring around 15% in normal individuals. At this point there is little evidence that the albumin fraction is truly bioavailable. A simple strategy might be to estimate the free concentration based on measurements of DBP and total 25(OH)D with known binding constants of DBP for 25(OH)D. This has in fact been done, but as subsequent research has documented, this relationship is affected by numerous clinical conditions and the different DBP variants with different affinities for 25(OH)D.

DBP
DBP is a 51–58 kDa multifunctional serum glycoprotein synthesized primarily in the liver. Initially, isoelectric focusing migration patterns identified phenotypic variants termed Group-Specific Component (Gc), the most common of which are Gc1f, Gcs and Gc2. Two common missense point mutations (SNPs) in exon 11 of the DBP gene, rs7041 (G/T single-nucleotide variation) and rs4588 (an A/C single-nucleotide variation), result in the three most common isoforms with amino acid changes at positions 416 and 420: Gc1f (Asp416, Thr420), Gc1s (Glu 416, Thr420), and Gc2 (Asp416, Lys420). Gc2 is the least abundant and Gc1f the most abundant. The distribution of the Gc alleles varies by race. Black and Asian populations are more likely to carry the Gc1f form, whereas the Gc2 form is rare, whereas Whites more frequently express the Gc1s and the Gc2 alleles. Although affinities of these DBP variants for 25(OH)D appear to vary, the rank order remains controversial, and their contribution of total 25(OH)D levels and the relationship between free and total 25(OH)D is modest in comparison to differences influenced by clinical condition. In the absence of disease or pregnancy, DBP levels are relatively constant over time in adults. That said, various substances in the blood such as polyunsaturated fatty acids may alter the affinity of DBP for the vitamin D metabolites, as can various clinical conditions. Liver disease leads to reduced levels of DBP, as do protein-losing nephropathies and acute illness (DBP is an acute phase reactant), whereas DBP levels are elevated during the latter stages of pregnancy. Moreover, various clinical conditions appear to shift the relationship between free and total 25(OH)D seemingly independent of DBP levels or DBP haplotypes. Thus, the measurement of total 25(OH)D may not provide the best assessment of vitamin D status. Calculation of free 25(OH)D from DBP and total 25(OH)D measurements using affinity constants obtained by measurements in normal sera may be inaccurate, at least in some clinical situations. Therefore, direct measurement of free 25(OH)D would appear to offer information about vitamin D nutritional status that at least complements that of total 25(OH)D.

The free 25(OH)D assay
The original free 25(OH)D assay employed centrifugal ultrafiltration. This was a labour- and reagent-intensive assay suitable only for a dedicated research laboratory. However, it sufficed to determine free 25(OH)D levels in a number of patient groups including cirrhotics and pregnant women, providing proof of concept that the free 25(OH)D measurement would add to the assessment of vitamin D nutritional status. This assay has subsequently been superseded by a much simpler method capable of high throughput.

A two-step ELISA that directly measures free 25(OH)D levels was recently developed by Future Diagnostics Solutions using monoclonal antibodies from DIAsource Immunoassays. In the first incubation step, an anti-25(OH)D monoclonal antibody immobilized on a microtitre plate binds the free 25(OH)D in the serum sample. The serum is removed and biotinylated 25(OH)D in a known amount is added to react with the unoccupied binding sites on the monoclonal antibody attached to the plate. The non-bound biotinylated 25(OH)D is then removed followed by the addition of streptavidin peroxidase conjugate and the substrate 3,3ʹ,5,5ʹ-Tetramethylbenzidine (TMB). The bound streptavidin peroxidase can be quantified by measuring the absorbance at 450 nm generated in the reaction. The intensity is inversely proportional to the level of free 25(OH)D. The limit of detection is 2.8 pg/mL. The antibody in the current assay does not recognize 25(OH)D₂ as well as 25(OH)D₃ (77% of the 25(OH)D₃ value), and so it underestimates the free 25(OH)D₂. However, under most situations where the predominant vitamin D metabolite is 25(OH)D₃ this issue is not a major concern. The data for both normal subjects and those with different DBP levels (cirrhotics, pregnant women) compare quite well to those obtained from similar populations using the centrifugal ultrafiltration assay.

Clinical implications
In a study currently under review for publication we compiled data from over 1600 individuals in whom free 25(OH)D had been measured by this ELISA. The samples included sera from both normal subjects and those with a variety of clinical conditions and a variety of DBP alleles. In the nearly 1000 normal and community dwelling outpatient subjects the normal range for free 25(OH)D was established at 4.3±1.9 pg/mL with a mean total 25(OH)D of 21.9±9.9 ng/mL, providing a percent free 25(OH)D of 0.02%. These results are essentially identical to those reported by the author using centrifugal ultrafiltration 30 years ago. As expected, clinical conditions affecting DBP values made a big difference. Liver disease resulted in lower DBP levels and higher percentage free 25(OH)D resulting in the population of cirrhotics studied having among the highest free 25(OH)D despite the lowest total 25(OH)D. Nursing home patients also had unexpectedly high free 25(OH)D, higher than that of the cirrhotics, with only modest reductions in DBP levels. Pregnancy (third trimester), however, resulted in increased DBP levels and the lowest free 25(OH)D levels, although the free fraction was not lower than that of the normal subjects. Overall, these results indicate that the free fraction is altered by the clinical situation not only in terms of altered DBP levels but in the relationship between total and free 25(OH)D for any given DBP level. Therefore, it is recommended that the free 25(OH)D level needs to be measured directly if the free level is thought to have particular relevance to the clinical situation that cannot be captured by measuring total 25(OH)D.

At this point it is not yet clear whether the determination of free 25(OH)D is a better marker of vitamin D nutritional status and biologic action than the determination of total 25(OH)D. Using a convenient marker such as parathyroid hormone (PTH), much as we use thyroid-stimulating hormone (TSH) as a marker of thyroid status, is problematic. First of all PTH levels are controlled by calcium as well as
vitamin D. Second, regulation of PTH secretion is mediated primarily by the 1,25(OH)₂D produced within the gland itself (much as TSH secretion is controlled by triiodothyronine (T3) produced within the pituitary). Third, the parathyroid gland has the megalin/cubilin transport system to enable 25(OH)D bound to DBP to enter the cells, obviating any advantage free 25(OH)D might have in cell uptake. However, several studies have demonstrated a stronger correlation between free 25(OH)D and bone markers than that observed with total 25(OH)D. But at this point, determining the role that free 25(OH)D measurements play in the assessment of vitamin D nutrition and action requires further investigation.

Bibliography
1. Bikle DD. Vitamin D Assays. Front Horm Res 2018; 50: 14–30.
2. Malstroem S, Rejmark L, et al. Current assays to determine free 25-hydroxyvitamin D in serum. J AOAC Internl 2017; 100: 1323–1327.
3. Bikle D, Bouillon R, et al. Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status? J Steroid Biochem Mol Biol 2017; 173: 1054–1116.
4. Bikle DD, Malmstroem S, Schwartz J. Current controversies: are free vitamin metabolite levels a more accurate assessment of vitamin D status than total levels? Endo Clinics NA 2017; 46: 901–918.
5. Lai JC, Bikle DD, et al. Total 25(OH) vitamin D, free 25(OH) vitamin D, and markers of bone turnover in cirrhotics with and without synthetic dysfunction. Liver Int 2015; 35: 2294–2300.
6. Schwartz JB, Lai J, et al. A comparison of direct and calculated free 25-OH vitamin D levels in clinical populations. J Clin Endocrinol Metab 2014; 99: 1631–1637.

The author
Daniel D Bikle MD, PhD
VA Medical Center and University of
California San Francisco, San Francisco,
CA 94158, USA

E-mail: Daniel.bikle@ucsf.edu

24,25-dihydroxyvitamin D: a new biomarker in non-parathyroid hypercalcemia diagnosis

, 26 August 2020/in Featured Articles /by 3wmedia

The 24,25-dihydroxyvitamin D [24,25(OH)₂D] is a catabolite of 25-hydroxyvitamin D [25(OH)D]. This transformation is performed by 1,25-hydroxyvitamin D 24-hydroxylase (or 24-hydroxylase, encoded by the CYP24A1 gene). Mutations in CYP24A1 can lead to severe diseases such as idiopathic infantile hypercalcemia (IIH). Explorations of hypercalcemia with suppressed parathyroid hormone levels and normal or high phosphatemia should now include 24,25(OH)2D determination to exclude CYP24A1 mutations. 24,25(OH)₂D and the vitamin D metabolite ratio (VMR) [i.e. 25(OH)D/24,25(OH)₂D] are now considered as new biomarkers for the assessment of functional vitamin D deficiency.

by L. Vranken, C. Fontaine, Prof. JC. Souberbielle and Prof. E. Cavalier

Vitamin D metabolism
Nowadays, there is an increased focus on the vitamin D and its benefits on health maintenance and disease prevention. Vitamin D is mainly produced following skin exposure to UVB rays. Additionally, it is found in several foods, such as oily fish, mushrooms and egg yolk. Vitamin D is considered as a pro-hormone owing to the fact that its production in the skin from 7-dehydrocholesterol could be sufficient when the sun exposure is adequate. Two forms of vitamin D coexist: vitamin D₂ produced by vegetables, and vitamin D₃ produced by animals and humans [2, 8]. After its synthesis in the skin or its intestinal absorption, this liposoluble vitamin is transported to the liver where it is hydroxylated by vitamin D 25-hydroxylase (or 25-hydroxylase, encoded by the CYP2R1 gene) to form 25-hydroxyvitamin D [25(OH)D]. This hydroxylation is very poorly regulated and, therefore, most of the circulating vitamin D will be metabolized into 25(OH)D. 25(OH)D is then transported to the kidney by a specific protein carrier [vitamin D binding protein (DBP)], and to a lesser extent by albumin, where it is hydroxylated by 25-hydroxyvitamin D-1 alpha hydroxylase (or 1α-hydroxylase, encoded by the CYP27B1 gene) on the carbon in position 1 to form the most active metabolite, 1,25-dihydroxy-vitamin D [1,25(OH)₂D]. This transformation is strictly regulated, notably by the parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23) and 1,25(OH)₂D itself (Fig. 1). The major role of vitamin D is the maintenance of calcium homeostasis, by acting on the vitamin D receptor (VDR). Calcium regulation is very complex and not fully understood yet. When ionized calcium decreases, the calcium sensing receptors (CaSR) located on the surface of the parathyroid glands stimulate PTH secretion.

PTH then acts on different targets to increase serum calcium concentration: it stimulates the release of calcium (and phosphate) from bones by acting on osteoclasts through osteoblasts and the RANK/RANKL system. It also decreases calcium excretion by the kidney and stimulates 1α-hydroxylase to produce 1,25(OH)₂D which, in turn, acts on the VDR of intestinal cells to produce calbindin 9k, TRPV6 and the NCX1 Ca/Na exchanger increasing intestinal absorption of calcium. The resulting increase of calcium levels inhibits CaSR-stimulated PTH production, but 1,25(OH)₂D also acts as a feedback loop to stop PTH synthesis. 1,25(OH)₂D finally acts on the VDR of the FGF23 gene to stimulate FGF23 production. In turn, FGF23, which is the most potent phosphaturic hormone (it inhibits Npt2a and Npt2c sodium-dependent phosphate co-transporters in the proximal renal tubule), blocks the activity of 1α-hydroxylase and stimulates 24-hydroxylase which leads to 25(OH)D and 1,25(OH)₂D catabolism (Fig. 2).

24-Hydroxylase is a key enzyme that catalyses the inactivation of svitamin D. It is expressed in most vitamin D target cells and is also stimulated by 1,25(OH)₂D, which hence regulates its own metabolism, therefore protecting against hypercalcemia and limiting the levels of 1,25(OH)₂D in cells [1]. Production of 1,24,25(OH)₃D and 24,25(OH)₂D is the first step of a five-step pathway that transforms vitamin D in a more hydrophilic compound, calcitroic acid, and allows its excretion in urine and in bile [2–6, 8]. 24,25(OH)₂D has a half-life of approximately 7 days and a concentration in the range of 1 to 10 ng/mL in healthy individuals.

CYP24A1 mutations
Loss-of-function mutations of the CYP24A1 gene have been identified in children presenting with idiopathic infantile hypercalcemia (IIH). These CYP24A1 gene product (24-hydroxylase) defects can be inherited as an autosomal recessive biallelic mutation. Infants present with severe hypercalcemia, suppressed PTH levels, hypercalciuria and medullary nephrocalcinosis owing to hypersensitivity to
vitamin D [4]. Indeed, there is no transformation of 25(OH)D and 1,25(OH)₂D to 24,25(OH)₂D and 1,24,25(OH)₃D leading to a prolonged and excessive elevation of 25(OH)D and 1,25(OH)₂D concentrations and an incapacity to clear them from plasma. By feedback, there will be a decrease of PTH and an increase in FGF23 concentrations (Fig. 2). These symptoms are similar to those met in vitamin D intoxication and it is important to make the distinction between these two diseases. In IIH, the vitamin D metabolite ratio (VMR), the ratio between 25(OH)D and 24,25(OH)₂D, allows the differential diagnosis of 24-hydroxylase defects from vitamin D intoxication. In IIH, the VMR will be high (>50–80); that is to say high 25(OH)D with low 24,25(OH)₂D, and is indicative of idiopathic hypercalcemia due to CYP24A1 gene mutations. In vitamin D intoxication, the VMR is normal because both 25(OH)D and 24,25(OH)₂D are increased. Moreover, the VMR may be more accurate for revealing this mutation than 24,25(OH)₂D alone because the ratio takes into consideration the circulating 25(OH)D and provides a clear distinction from a vitamin D deficiency, in which both 25(OH)D and 24,25(OH)₂D are low. Indeed, if the substrate decreases, in this case 25(OH)D, the activity of 24-hydroxylase is reduced, thus the production of 24,25(OH)₂D is low [4]. These genetic mutations indicate that vitamin D supplementation in children could be potentially deleterious. In these children, vitamin D supplementation must be eliminated. Indeed, they may have failure to thrive, vomiting, dehydratation, spikes of fever and nephrocalcinosis. Supplementation of mothers with 24-hydroxylase defects during pregnancy could lead to hypercalcemia associated with prematurity and intra-uterine growth retardation. Treatment of IIH encompasses the avoidance of sun and calcium- and vitamin D-rich foods. However, recently, it has been shown that isoniazid could induce the cytochrome P450 3A4, which is another vitamin D degradation pathway [9].

Thereafter, Molin et al. found that CYP24A1 gene mutations are frequently associated with renal complications including renal failure, nephrolithiasis and nephrocalcinosis. Also, they suggest that this loss-of-function of 24-hydroxylase is the most recently elucidated cause of hypercalcemia after parathyroid hypercalcemia, vitamin D intoxication and poorly regulated 1α-hydroxylation [3]. They have described patients with CYP24A1 heterozygous mutations, mostly asymptomatic, implying a hypothesis of an autosomal-dominant trait from which clinical consequences would vary throughout life and where hypercalcemia would appear only when vitamin D intakes are excessive.

Less severe mutations have been observed in patients with moderate hypercalcemia and inappropriately low PTH (<20 pg/mL). Those patients are likely to develop nephrolithiasis. 24,25(OH)₂D evaluation should be done on subjects with hypercalcemia and low PTH, especially as they suffer from nephrolithiasis. Not all the mutations have been discovered yet and further genetic studies are required. Moreover Ginsberg et al. found that lower 24,25(OH)₂D concentrations and lower VMR are associated with increased hip-fracture risk in community-living older men and women. They also noticed that higher 24,25(OH)₂D concentrations were associated with higher bone mineral density (BMD), whereas VMR was not. Additionally, 1,25(OH)₂D concentrations were not associated with BMD, consistent with previous studies in older adults [1]. In addition to catabolism, many studies tend to demonstrate that the 24,25(OH)₂D may have its own biological activity in vitro in calcium regulation [5, 6]. Finally, recent studies suggest that the assessment of 24,25(OH)₂D or the assessment of the VMR could better reflect the activity of the VDR and could be used as an index of vitamin D clearance [1, 3, 4]. The VMR may have the advantage of being uninfluenced by DBP concentrations, which affects both the numerator and denominator of the ratio.

Vitamin D metabolite evaluation
Quantitative evaluation of 24,25(OH)₂D is complicated by its presence at low concentrations. LC-MS/MS is currently the only alternative to evaluate 24,25(OH)₂D levels and has the great advantage to distinguish simultaneously the different metabolites and 25(OH)D in serum [6, 10]. The NIST (National Institute of Standards and Technology) has recently issued a new serum-matrix standard reference material [11] and Tai et al. published a reference measurement procedure for the determination of 24,25(OH)₂D in human serum using isotope-dilution LC-MS/MS [10].

Conclusion
In conclusion, the assessment of 25(OH)D alone is not always enough. 24,25(OH)₂D and VMR are other available tools to help for the diagnosis and the monitoring of abnormalities in phosphocalcic metabolism. The drawback is that it requires the determination of vitamin D metabolites by LC-MS/MS, and very few laboratories perform this determination [only 10 labs participate in the 24,25(OH)2D proficiency testing provided by the Vitamin D External Quality Assessment Scheme (DEQAS)]. Collaboration with a reference lab may be a good compromise. It is important to be aware of hypercalcemia caused by CYP24A1 mutants and their consequences on health. Further studies will be needed to explore the others mutations of CYP24A1 and the potential biological activity of 24,25(OH)₂D in vivo.

References
1. Ginsberg C, Katz R, de Boer IH, Kestenbaum BR, Chonchol M, Shlipak MG, Sarnak MJ, Hoofnagle AN, Rifkin DE, et al. The 24,25 to 25-hydroxyvitamin D ratio and fracture risk in older adults: the cardiovascular health study. Bone 2018; 107: 124–130.
2. Vranken L, Emonts P, Bruyère O, Cavalier E. Prévalence de l’hypovitaminose D chez la femme enceinte: quelle est la situation en région liégeoise? Revue Médicale de Liège 2018; 73 (1): 10–16 [in French].
3. Molin A, Baudoin R, Kaufmann M, Souberbielle JC, Ryckewaert A, Vantyghem MC, Eckart P, Bacchetta J, Deschenes G, et al. CYP24A1 mutations in a cohort of hypercalcemic patients: evidence for a recessive trait. J Clin Endocrinol Metab 2015; 100(10): E1343–E1352.
4. Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, Misselwitz J, Klaus G, Kuwertz-Bröking E, et al. Mutations in CYP24A1 and idiopathic infantile hypercalcemia. N Engl J Med 2011; 365(5): 410–421.
5. Van Leeuwen JPTM, an den Bemd GJCM, van Driel M, Buurman CJ, Pols HAP. 24,25-Dihydroxyvitamin D3 and bone metabolism. Steroids 2011; 66: 375–380.
6. Wagner D, Hanwell HE, Schnabl K, Yazdanpanah M, Kimball S, Fu L, Sidhom G, Rousseau D, Cole DEC, Vieth R. The ratio of serum 24,25-dihydroxyvitamin D3 to 25-hydroxyvitamin D3 is predictive of 25-hydroxyvitamin D3 response to vitamin D3 supplementation. J Steroid Biochem Mol Biol 2011; 126: 72–77.
7. Lu X, Chen Z, Mylarapu N, Watsky MA. Effects of 1,25 and 24,25 vitamin D on corneal epithelial proliferation, migration and vitamin D metabolizing and catabolizing enzymes. Sci Rep 2017; 16951: 1–12.
8. Bikle DD. Vitamin D and bone. Curr Osteoporos Rep 2012; 10(2): 151–159.
9. An inducible cytochrome P450 3A4-dependent vitamin D catabolic pathway. Wang Z, Lin YS, Zheng XE, Senn T, Hashizume T, Scian M, Dickmann LJ, Nelson SD, Baillie TA, et al. Mol Pharmacol 2012; 81(4): 498–509.
10. Tai SSC, Nelson MA. Candidate reference measurement procedure for the determination of (24R),25-dihydroxyvitamin D3 in human serum using isotope-dilution liquid chromatography-tandem mass spectrometry. Anal Chem 2015; 87: 7964–7970.
11. Tai SS, Nelson MA, Bedner M, Lang BE, Phinney KW, Sander LC, Yen JH, Betz JM, Sempos CT, Wise SA. Development of standard reference material (SRM) 2973 vitamin D metabolites in frozen human serum (high level). J AOAC Int 2017; 100(5): 1294–1303.

The authors
Laura Vranken¹, Corentin Fontaine¹, Jean-Claude Souberbielle² PhD, Etienne Cavalier¹ PhD
1Clinical Chemistry, University of Liège, CHU Sart-Tilman, Belgium
²Service des Explorations Fonctionnelles, Hôpital Necker-Enfants Malades, Paris, France

*Corresponding author
E-mail: Laura.vranken@chuliege.be

Scientific literature review: Prostate cancer

, 26 August 2020/in Featured Articles /by 3wmedia

CTC-derived AR-V7 detection as a prognostic and predictive biomarker in advanced prostate cancer
^{Bastos DA, Antonarakis ES. Expert Rev Mol Diagn 2018; 18(2): 155–163}

Prostate cancer is a highly heterogeneous disease, with remarkably different prognosis across all stages. Increased circulating tumour cell (CTC) count (≥5) using the CellSearch assay has been identified as one of the markers that can be used to predict survival, with added value beyond currently available prognostic factors. Recently, androgen receptor splice variant 7 (AR-V7) detection has been associated with worse outcomes for patients with castration-resistant prostate cancer (CRPC) treated with novel androgen receptor-signalling (ARS) inhibitors such as abiraterone and enzalutamide but not taxane chemotherapies. Areas covered: In this manuscript, the authors review the available biomarkers in CRPC and discuss emerging data on the value of CTC-derived AR-V7 status to assess prognosis and its potential role to guide treatment selection for patients with advanced prostate cancer. Expert commentary: Current evidence supports AR-V7 status as a prognostic biomarker and also as a potential predictive biomarker for patients with mCRPC. The authors expect that the incorporation of AR-V7 status and other biomarkers (e.g. AR mutations) in the sequential assessment of patients with advanced prostate cancer will lead to a more rational use of available and future therapies, with significant improvements in outcomes for our patients.

Defining a cohort of men who may not require repeat prostate biopsy based on PCA3 score and MRI: The dual negative effect
^{Perlis N, Al-Kasab T, Ahmad A, Goldberg E, Fadak K, Sayid R, et al. J Urol 2017; doi: 10.1016/j.juro.2017.11.07}

PURPOSE: Prostate cancer over diagnosis and overtreatment are concerns for clinicians and policy makers. Multiparametric magnetic resonance imaging and the PCA3 (prostate cancer antigen 3) urine test select for clinically significant cases. We explored how well the tests performed together in with previous biopsies.

MATERIALS AND METHODS: In accordance with ethics committee approval we collected clinicopathological data on all patients in whom a PCA3 test from was done 2011 to June 2016. This included patients on active surveillance for low-risk prostate cancer and those without prostate cancer who had previous negative biopsies and suspicion of occult disease. We explored whether age, prostate-specific antigen, PCA3 score, multiparametric magnetic resonance imaging, digital rectal examination, family history and prostate size would predict clinically significant prostate cancer on repeat biopsy. The negative predictive value of multiparametric magnetic resonance imaging and PCA3 score was calculated.

RESULTS: A total of 470 patients were included in study. The PCA3 score was abnormal at 35 or greater in 32.5 % of cases. In the multivariate model including 154 men only age (OR 1.08, 95 % CI 1.01–1.16), multiparametric magnetic resonance imaging PI-RADS™ (Prostate Imaging-Reporting and Data System) score 4 (OR 16.6, 95 % CI 3.9–70.0) or 5 (OR 28.3, 95 % CI 5.7–138) and PCA3 score (OR 2.9, 95 % CI 1.0–8.8) predicted clinically significant cancer on biopsy. No patient with negative multiparametric magnetic resonance imaging and a normal PCA3 score had clinically significant prostate cancer on biopsy for a negative predictive value of 100 % (p<0.0001).

CONCLUSIONS: In patients with dual negative tests (multiparametric magnetic resonance imaging and PCA3 score) clinically significant prostate cancer was never found on biopsy, which may be unnecessary in this group. This study was limited by its retrospective design, selection bias and lack of cost-effectiveness data.

Quantitative mass spectrometry-based proteomic profiling for precision medicine in prostate cancer
^{Flores-Morales A, Iglesias-Gato D. Front Oncol 2017; 7: 267}

Prostate cancer (PCa) is one of the most frequently diagnosed cancer among men in the western societies. Many PCa patients bear tumours that will not threat their lives if left untreated or if treatment is delayed. Our inability for early identification of these patients has resulted in massive overtreatment. Therefore, there is a great need of finding biomarkers for patient stratification according to prognostic risk; as well as there is a need for novel targets that can allow the development of effective treatments for patients that progress to castration-resistant PCa. Most biomarkers in cancer are proteins, including the widely-used prostate-specific antigen (PSA). Recent developments in mass spectrometry allow the identification and quantification of thousands of proteins and posttranslational modifications from small amounts of biological material, including formalin-fixed paraffin-embedded tissues, and biological fluids. Novel diagnostic and prognostic biomarkers have been identified in tissue, blood, urine, and seminal plasma of PCa patients, and new insights in the ethology and progression of this disease have been achieved using this technology. In this review, we summarize these findings and discuss the potential of this technology to pave the way toward the clinical implementation of precision medicine in PCa.

Biomarkers for prostate biopsy and risk stratification of newly diagnosed prostate cancer patients
^{Loeb S. Urol Pract 2017; 4(4): 315–321}

INTRODUCTION: Many new markers are now available as an aid for decisions about prostate biopsy for men without prostate cancer, and/or to improve risk stratification for men with newly diagnosed prostate cancer.

METHODS: A literature review was performed on currently available markers for use in decisions about prostate biopsy and initial prostate cancer treatment.

RESULTS: Although total prostate-specific antigen cutoffs were traditionally used for biopsy decisions, PSA elevations are not specific. Repeating the PSA test, and adjusting for factors like age, prostate volume and changes over time can increase specificity for biopsy decisions. The Prostate Health Index (phi) and 4K Score are new PSA-based markers that can be offered as second-line tests to decide on initial or repeat prostate biopsy. The PCA3 urine test and ConfirmMDx tissue test are additional options for repeat biopsy decisions. For men with newly diagnosed prostate cancer, genomic tests are available to refine risk classification and may influence treatment decisions.

CONCLUSIONS: Numerous secondary testing options are now available that can be offered to patients deciding whether to undergo prostate biopsy and those with newly diagnosed prostate cancer.

Bidirectional electrochemiluminescence color switch: an application in detecting multimarkers of prostate cancer
^{Wang YZ, Ji SY, Xu HY, Zhao W, Xu JJ, Chen HY. Anal Chem 2018; doi: 10.1021/acs.analchem.8b00014}

A selective excitation of [Ir(df-ppy)₂(pic)] and [Ru(bpy)3]²⁺ through tuning the electrode potential is reported in this work. Bidirectional colour change from blue-green to red could be observed along with increase and decrease of the potential, which was ascribed to the dual-potential excitation property of [Ir(df-ppy)₂(pic)]. Similar to the three-electrode system, selective excitation of ECL could be achieved at the anode of the bipolar electrode (BPE). Both increase and decrease of the faradic reactions at the cathode of the BPE could induce ECL reporting colour at the other pole switched from blue-green to red. We applied a closed BPE device for the bioanalysis of multicolour ECL since the organic solvent containing electrochemiluminophores could be separated from the bioanalytes. On the basis of BPE arrays coupled with the ECL switch, the detection of three biomarkers of prostate cancer, PSA, microRNA-141, and sarcosine were integrated in a same device. The cutoff values of the biomarkers could be recognized directly by the naked eye. Such a device holds great potential in the early diagnosis of prostate cancer.

Molecular biomarkers in the clinical management of prostate cancer
^{Udager AM, Tomlins SA. Cold Spring Harb Perspect Med 2018; doi: 10.1101/cshperspect.a030601}

Prostate cancer, one of the most common non-cutaneous malignancies in men, is a heterogeneous disease with variable clinical outcome. Although the majority of patients harbour indolent tumours that are essentially cured by local therapy, subsets of patients present with aggressive disease or recur/progress after primary treatment. With this in mind, modern clinical approaches to prostate cancer emphasize the need to reduce overdiagnosis and overtreatment via personalized medicine. Advances in our understanding of prostate cancer pathogenesis, coupled with recent technologic innovations, have facilitated the development and validation of numerous molecular biomarkers, representing a range of macromolecules assayed from a variety of patient sample types, to help guide the clinical management of prostate cancer, including early detection, diagnosis, prognostication, and targeted therapeutic selection. Herein, we review the current state of the art regarding prostate cancer molecular biomarkers, emphasizing those with demonstrated utility in clinical practice.

Genomic markers in prostate cancer decision making
^{Cucchiara V, Cooperberg MR, Dall’Era M, Lin DW, Montorsi F, Schalken JA, et al. Eur Urol. 2017; doi: 10.1016/j.eururo.2017.10.036}

CONTEXT: Although the widespread use of prostate-specific antigen (PSA) has led to an early detection of prostate cancer (PCa) and a reduction of metastatic disease at diagnosis, PSA remains one of the most controversial biomarkers due to its limited specificity. As part of emerging efforts to improve both detection and management decision making, a number of new genomic tools have recently been developed.

OBJECTIVE: This review summarizes the ability of genomic biomarkers to recognize men at high risk of developing PCa, discriminate clinically insignificant and aggressive tumours, and facilitate the selection of therapies in patients with advanced disease.

EVIDENCE ACQUISITION: A PubMed-based literature search was conducted up to May 2017. The most recent and relevant original articles and clinical trials that have provided indispensable information to guide treatment decisions were selected.

EVIDENCE SYNTHESIS: Genome-wide association studies have identified several genetic polymorphisms and inherited variants associated with PCa susceptibility. Moreover, the urine-based assays SelectMDx, Mi-Prostate Score, and ExoDx have provided new insights into the identification of patients who may benefit from prostate biopsy. In men with previous negative pathological findings, Prostate Cancer Antigen 3 and ConfirmMDx predicted the outcome of subsequent biopsy. Commercially available tools (Decipher, Oncotype DX, and Prolaris) improved PCa risk stratification, identifying men at the highest risk of adverse outcome. Furthermore, other biomarkers could assist in treatment selection in castration-resistant PCa. AR-V7 expression predicts resistance to abiraterone/enzalutamide, while poly(ADP-ribose) polymerase-1 inhibitor and platinum-based chemotherapy could be indicated in metastatic patients who are carriers of mutations in DNA mismatch repair genes.

CONCLUSIONS: Introduction of genomic biomarkers has dramatically improved the detection, prognosis, and risk evaluation of PCa. Despite the progress made in discovering suitable biomarker candidates, few have been used in a clinical setting. Large-scale and multi-institutional studies are required to validate the efficacy and cost utility of these new technologies.

PATIENT SUMMARY: Prostate cancer is a heterogeneous disease with a wide variability. Genomic biomarkers in combination with clinical and pathological variables are useful tools to reduce the number of unnecessary biopsies, stratify low-risk from high-risk tumours, and guide personalized treatment decisions.

The use of biomarkers in prostate cancer screening and treatment
^{Ashley VA, Joseph MB, Kamlesh KY, Shalini SY, Ashutosh KT, Joseph R. Rev Urol 2017; 19(4): 221–234}

Prostate cancer screening and diagnosis has been guided by prostate-specific antigen levels for the past 25 years, but with the most recent US Preventive Services Task Force screening recommendations, as well as concerns regarding overdiagnosis and overtreatment, a new wave of prostate cancer biomarkers has recently emerged. These assays allow the testing of urine, serum, or prostate tissue for molecular signs of prostate cancer, and provide information regarding both diagnosis and prognosis. In this review, we discuss 12 commercially available biomarker assays approved for the diagnosis and treatment of prostate cancer. The results of clinical validation studies and clinical decision-making studies are presented. This information is designed to assist urologists in making clinical decisions with respect to ordering and interpreting these tests for different patients. There are numerous fluid and biopsy-based genomic tests available for prostate cancer patients that provide the physician and patient with different information about risk of future disease and treatment outcomes. It is important that providers be able to recommend the appropriate test for each individual patient; this decision is based on tissue availability and prognostic information desired. Future studies will continue to emphasize the important role of genomic biomarkers in making individualized treatment decisions for prostate cancer patients.

A four-kallikrein panel and β-microseminoprotein in predicting high-grade prostate cancer on biopsy: an independent replication from the Finnish Section of the European Randomized Study of Screening for Prostate Cancer
^{Assel M, Sjöblom L, Murtola TJ, Talala K, Kujala P, Stenman UH, et al. Eur Urol Focus 2017; doi: 10.1016/j.euf.2017.11.002}

BACKGROUND: A panel of four kallikrein markers (total, free, and intact prostate-specific antigen [PSA] and human kallikrein-related peptidase 2 [hK2]) improves predictive accuracy for Gleason score ≥7 (high-grade) prostate cancer among men biopsied for elevated PSA. A four-kallikrein panel model was originally developed and validated by the Dutch centre of the European Randomized Study of Screening for Prostate Cancer (ERSPC). The kallikrein panel is now commercially available as 4Kscore™.

OBJECTIVE: To assess whether these findings could be replicated among participants in the Finnish section of ERSPC (FinRSPC) and whether β-microseminoprotein (MSP), a candidate prostate cancer biomarker, adds predictive value.

DESIGN, SETTING, AND PARTICIPANTS: Among 4861 biopsied screening-positive participants in the first three screening rounds of FinRSPC, a case-control subset was selected that included 1632 biopsy-positive cases matched by age at biopsy to biopsy-negative controls.

OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The predictive accuracy of prespecified prediction models was compared with biopsy outcomes.

RESULTS AND LIMITATIONS: Among men with PSA of 4.0–25 ng/ml, 1111 had prostate cancer, 318 of whom had high-grade disease. Total PSA and age predicted high-grade cancer with an area under the curve of 0.648 (95 % confidence interval [CI] 0.614–0.681) and the four-kallikrein panel increased discrimination to 0.746 (95 % CI 0.717–0.774). Adding MSP to the four-kallikrein panel led to a significant (Wald test; p=0.015) but small increase (0.003) in discrimination. Limitations include a risk of verification bias among men with PSA of 3.0–3.99 ng/ml and the absence of digital rectal examination results.
CONCLUSIONS: These findings provide additional evidence that kallikrein markers can be used to inform biopsy decision making. Further studies are needed to define the role of MSP.

PATIENT SUMMARY: Four kallikrein markers and β-microseminoprotein in blood improve discrimination of high-grade prostate cancer at biopsy in men with elevated prostate-specific antigen.

Combinations of elevated tissue miRNA-17-92 cluster expression and serum prostate-specific
antigen as potential diagnostic biomarkers for prostate cancer
^{Feng S, Qian X, Li H, Zhang X. Oncol Lett 2017; 14(6): 6943–6949}

The aim of the present study was to investigate the effectiveness of the miR-17-92 cluster as a disease progression marker in prostate cancer (PCa). Reverse transcription-quantitative polymerase chain reaction analysis was used to detect the microRNA (miR)-17-92 cluster expression levels in tissues from patients with PCa or benign prostatic hyperplasia (BPH), in addition to in PCa and BPH cell lines. Spearman correlation was used for comparison and estimation of correlations between miRNA expression levels and clinicopathological characteristics such as the Gleason score and prostate-specific antigen (PSA). Receiver operating curve (ROC) analysis was performed for evaluation of specificity and sensitivity of miR-17-92 cluster expression levels for discriminating patients with PCa from patients with BPH. Kaplan-Meier analysis was plotted to investigate the predictive potential of miR-17-92 cluster for PCa biochemical recurrence. Expression of the majority of miRNAs in the miR-17-92 cluster was identified to be significantly increased in PCa tissues and cell lines. Bivariate correlation analysis indicated that the high expression of unregulated miRNAs was positively correlated with Gleason grade, but had no significant association with PSA. ROC curves demonstrated that high expression of miR-17-92 cluster predicted a higher diagnostic accuracy compared with PSA. Improved discriminating quotients were observed when combinations of unregulated miRNAs with PSA were used. Survival analysis confirmed a high combined miRNA score of miR-17-92 cluster was associated with shorter biochemical recurrence interval. miR-17-92 cluster could be a potential diagnostic and prognostic biomarker for PCa, and the combination of the miR-17-92 cluster and serum PSA may enhance the accuracy for diagnosis of PCa.

Prostate-specific antigen screening impacts on biochemical recurrence in patients with clinically localized prostate cancer
^{Hashimoto T, Ohori M, Shimodaira K, Kaburaki N, Hirasawa Y, Satake N, Gondo T, Nakagami Y, Namiki K, Ohno Y. Int J Urol 2018; doi: 10.1111/iju.13563}

OBJECTIVE: To clarify the impact of prostate-specific antigen screening on surgical outcomes of prostate cancer.

METHODS: Patients who underwent radical prostatectomy were divided into two groups according to prostate-specific antigen testing opportunity (group 1, prostate-specific antigen screening; group 2, non-prostate-specific antigen screening). Perioperative clinical characteristics were compared using the Wilcoxon rank-sum and χ2 -tests. Cox proportional hazards models were used to identify independent predictors of postoperative biochemical recurrence-free survival.

RESULTS: In total, 798 patients (63.2 %) and 464 patients (36.8 %) were categorized into groups 1 and 2, respectively. Group 2 patients were more likely to have a higher prostate-specific antigen level and age at diagnosis and larger prostate volume. Clinical T stage, percentage of positive cores and pathological Gleason score did not differ between the groups. The 5-year biochemical recurrence-free survival rate was 83.9 % for group 1 and 71.0 % for group 2 (p<0.001). On multivariate analysis, prostate-specific antigen testing opportunity (hazard ratio 2.530; p<0.001) was an independent predictive factor for biochemical recurrence after surgery, as well as pathological T stage, pathological Gleason score, positive surgical margin and lymphovascular invasion. Additional analyses showed that prostate-specific antigen screening had a greater impact on biochemical recurrence in a younger patients, patients with a high prostate-specific antigen level, large prostate volume and D’Amico high risk, and patients meeting the exclusion criteria of the Prostate Cancer Research International Active Surveillance study.

CONCLUSIONS: Detection by screening results in favourable outcomes after surgery. Prostate-specific antigen screening might contribute to reducing biochemical recurrence in patients with localized prostate cancer.

bioLOGICAL ♦ Blood. A priceless good.

, 26 August 2020/in Featured Articles /by 3wmedia

The second edition of the Greiner Bio-One customer magazine bioLOGICAL is now available on their website. Interesting articles about capillary blood sampling are included in this issue. In the article by Jasna Lenicek Krleza, PhD, the reader will learn about which factors to pay special attention to in capillary blood collection to get high-quality samples. The neonatal station and small patients are also featured, together with the company’s MiniCollect^® capillary blood collection system. In addition, it includes tips to help users find the most suitable vein for venipuncture.
https://tinyurl.com/ycu2f3ne

Biochemical investigation of monoclonal gammopathies

The measurement of free 25-hydroxyvitamin D

24,25-dihydroxyvitamin D: a new biomarker in non-parathyroid hypercalcemia diagnosis

Scientific literature review: Prostate cancer

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Panasonic biomedical has become PHCbi

Make Needlestick Injuries History – VACUETTE Safety Products

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QDx Vit. D Rapid test for rapid results

RESIST – the new solution to detect carbapenem resistance in Acinetobacter spp.

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