Vitamin D is derived from food, and human beings need exposure to sunlight for its synthesis. This is achieved through the absorption of ultraviolet B radiation by 7-dehydrocholesterol in the skin.  However, certain sections of the population cannot produce Vitamin D in sufficient quantities; such at-risk groups are the focus of screening.
The principal function of Vitamin D is to enable absorption of calcium and phosphorus and promote bone health. It is metabolized in the liver to 25-hydroxyvitamin D (25OHD). The kidneys activate 25OHD to 1,25-dihydroxyvitamin D, which in turn regulates calcium, phosphorus and bone metabolism.
25OHD is the key circulating form of vitamin D, and as a biomarker, is generally the target of screening.

 
The deforming condition known as rickets, caused by a deficiency of Vitamin D, was a scourge in children until the early 20th century. The vitamin was identified in the 1920s. Though the crucial role played by sunlight in avoiding rickets had been known since the 1820s, it was only endorsed officially after over a century. Accompanied by fortification of milk and infant food with Vitamin D, rickets in industrialized countries soon became a thing of the past.
Rickets has, however, recently returned. One reason is ironic: the excessive promotion of breastfeeding and an unawareness that human milk does not suffice for an infant’s Vitamin D needs. This is exacerbated by modern lifestyles which discourage exposure to the sun, along with an overuse of sun screens and other forms of protection.

Benefits likely for several diseases
More recently, researchers have found that Vitamin D may also play a role in controlling a host of other diseases. This has revived the debate about screening.
Diseases against which Vitamin D provides benefits role are believed to include cancer, cardiovascular and cerebrovascular disease, diabetes and metabolic disorders, multiple sclerosis, inflammatory bowel diseases and certain neurological conditions.

Ethnicity, age and gender
Since the mid-1980s, it is accepted that Vitamin D’s role in the context of certain major medical conditions depends on ethnicity. The re-emergence of rickets has been concentrated, for example, in African Americans in the US and Asians in the UK.
In 1988-1994, the third US National Health and Nutrition Examination Survey (NHANES III) found significant differences between different ethnic groups in terms of 25OHD levels, which in turn correlated to a predisposition to both high blood pressure and diabetes.
In 2002, researchers found that Vitamin D’s role in pediatric Crohn’s disease was particularly pronounced in African-American children. Likewise, in the case of coronary artery disease, one high risk group consists of African-Americans with HIV.

Age-specific risks too are an area of concern. The elderly have been specifically targeted to study Vitamin D’s role in hypertension and in cognitive decline. Older women, in particular, seem prone to suffer from its deficiency, and thereby to adverse effects on their musculoskeletal system.
On the other side of the age scale, Vitamin D deficiency in infants seems associated with a range of chronic diseases in later life, from multiple sclerosis to Type 1 diabetes and schizophrenia.

More research still needed
Nearly all the research efforts cited above add caveats to their findings, underlying the role of Vitamin D in avoiding several diseases, but also calling for further investigation. Two recent meta-studies, which assimilate the results of previous research in this area, illustrate the problem.
One meta-study, published at the end of 2012, focuses on Vitamin D and cardiovascular disease (CVD). Its scope extends to 19 previous studies covering 6,123 CVD cases in nearly 66,000 subjects. It notes that, in spite of evidence about a link between CVD and Vitamin D deficiency, “optimal” levels of 25OHD for cardiovascular health “remain unclear,” and underlines that there is a “generally linear, inverse association” between circulating 25OHD in the range of 20-60 nmol/L and a risk of CVD. However, it calls for “further research” on 25OHD levels higher than 60 nmol/L.
The second meta-study dates to the end of 2013 and covers 18 prospective studies on Type 2 diabetes and metabolic disorders, involving 210,107 participants. It begins by observing that though “several studies have assessed Vitamin D in relationship with metabolic outcomes”, “results remain inconsistent.” It concludes that Vitamin D-targeted interventions “may be a useful preventive measure for metabolic diseases”, but also warns that “reliable evidence from carefully designed intervention studies, particularly those based on healthy populations, is needed to confirm observational findings.”

Differences in definition of risk persist 
This bewildering array of qualifiers makes screening for Vitamin D problematic, for now.
Making things even more di ficult are continuing doubts about what level of 25OHD indicates a risk, and for who, when and where.

In the UK, the National Health Service (NHS) considers concentrations of less than 25 nmol/L to indicate “deficiency”, with “insufficiency” at 25-50 nmol/L, and “adequate” levels at more than 50 nmol/L. The American Journal of Clinical Nutrition (AJCN) specifies 25OHD below 50 nmol/L as an indicator of “deficiency”, while 51–74 nmol/L does so for “insufficiency”; the 74 nmol/L margin (as discussed below) has also been adopted in Poland. On its part, the National Institutes for Health (NIH) defines less than 30 nmol/L as “deficiency”, 30-50 nmol/L as “generally considered adequate” and more than 125 nmol/L to be associated with “potential adverse effects.”
Although the NIH says a 50 nmol/L concentration covers the needs of 97.5% of the population, it still leaves 7.5 million Americans in need of screening.

Seasonal factors confound the playing field further. In the UK, for example, the  “insufficiency” concentration of 25-50 nmol/L for 25OHD is found in as much as half the country’s population, during spring.
Last but not least are doubts about the relevance of 25OHD concentration itself. For instance, the European Food Safety Authority (EFSA) stated, as recently as 2012, that while it “is a good marker of vitamin D status”, 25OHD can only be used “as a biomarker of vitamin D intake in people with low exposure to sunlight.”

Recommendations for screening
In the face of this, there is little consensus about screening for Vitamin D.
Nevertheless, national recommendations to prevent Vitamin D deficiency were instituted in Poland in 2009, and three years later in Hungary and Germany. Underlying the ongoing lack of clarity on the issue,  the German Nutrition Society, which issued the recommendations, also evaluated Vitamin D as part of a separate investigation into vitamin availability, but this time was sanguine, except in the elderly. The elderly were also the subject of nutrition recommendations by the the International Osteoporosis Foundation in 2010.
In much of continental Europe, 50 nmol/L of 25OHD concentration is used to indicate Vitamin D sufficiency, in line with Britain’s NHS and the US NIH. However, most east and central Europeans tend to follow the 2009 Polish recommendations, which specified less than 50 nmol/L as deficiency, 50-75 nmol/L as ‘suboptimal’, with an ‘optimal’ target of 75–125 nmol/L; the latter straddles the lower and higher margins prescribed in the US by the AJCN and NIH, respectively (see above).
Iterations on thresholds, meanwhile, continue to proliferate, even with regard to at-risk populations. For example, while both Germany and the International Osteoporosis Foundation suggest 25OHD concentrations of 60 nmol/L in the elderly, in 2013 the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) recommended 50nmol/L for elderly women, and 75 nmol/L for “fragile” subjects.

The VITAL trial
It is to be hoped that many questions about the precise role of Vitamin D in battling some of the largest challenges facing modern medicine are answered in the years to come.
One such effort has, in fact, recently been launched. Known as VITAL (‘VITamin D and OmegA-3 TriaL), this interventional, randomized clinical trial is recruiting 20,000 men and women in the US to investigate whether daily dietary supplements of Vitamin D or omega-3 fatty acids reduces the risk of cancer, heart disease and stroke in people with no prior history of these illnesses.

Targeted supplementation most likely course
The above developments indicate that the argument about routine supplementation making “more sense” than screening is likely to yield place to “targeted” screening for at-risk populations, with an especially strong case for the elderly and infants.
Both groups were, in fact, highlighted in July 2011 Clinical Practice Guidelines on Vitamin D by the influential US-headquartered Endocrine Society. The Society also included “pregnant and lactating women,” “obese children and adults”, and patients on “anticonvulsant medications, glucocorticoids, antifungals such as ketoconazole, and medications for AIDS.”

Standardizing lab tests
In the meanwhile, one major concern for clinical laboratories with regard to Vitamin D tests seems to have been addressed. Until recently, serum 25OHD concentrations showed considerable variations, depending on the type of assays used (the most common is liquid chromatography). This led to differences among laboratories in their findings. The implication of such differences has been significant, especially given the differences in definitions of Vitamin D sufficiency and insufficiency.
In 2009, the US National Institute of Standards and Technology (NIST) released SRM 972, a reference material for 25OHD. SRM 972, updated by NIST in February 2013 to SRM 972a, is also used in Europe.

With research demonstrating the impact of vitamin D on a range of conditions, the importance of adequate levels of vitamin D is often in the spotlight. This has resulted in increased rates of testing from both GPs and self-referring individuals.

by Robyn Shea and Dr Jonathan Berg

Background
Vitamin D is an essential nutrient required for bone health and calcium homeostasis. It is also described as a pro-hormone as it is the biologically inactive precursor to the active secosteroid hormone 1,25-dihydroxyvitamin D [1,25(OH)2D, also known as calcitriol] [1].
Vitamin D is found in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Both forms are available as supplements or in a small number of foodstuffs, naturally occurring or fortified. However, the main source of vitamin D, around 90%, is through endogenous synthesis in the skin with the conversion of 7-dehydrocholesterol, via UVB radiation from the sun, into vitamin D3. Vitamin D2 or D3 are hydroxylated, first in the liver to form 25-hydroxyvitamin D2 [25(OH)D2] and  25-hydroxyvitamin D3 [25(OH)D3] respectively, and secondly in the kidney to form the active hormone 1,25(OH)2D. The first hydroxylation step is unregulated and 25(OH)D levels therefore depend on the availability of the vitamin D substrate. The second hydroxylation step is tightly controlled via parathyroid hormone and through a variety of negative feedback mechanisms including calcium and phosphate levels as well as 1,25(OH)2D itself [2]. Comprehensive discussion about sources and metabolism of vitamin D are covered elsewhere in the literature [3, 4].

How is vitamin D measured?
25(OH)D is the best marker of vitamin D status. It is a difficult analyte to measure – it is very hydrophobic, is present in two forms [25(OH)D3 and 25(OH)D2] and is bound to vitamin D binding protein or albumin. Despite the difficulty in measuring it, a number of analytical platforms are available for 25(OH)D measurement:

• enzyme immunoassay (EIA)
• high pressure liquid chromatography (HPLC)
• radioimmunoassay (RIA)
• enzyme-linked immunosorbent assay (ELISA)
• liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The agreement between the different methodologies is often poor and there can be quite large variation even within method groups, again highlighting how difficult it is to measure 25(OH)D. This has improved over time with the introduction of a NIST Standard Reference Material and the introduction of commercially available calibrators for chromatographic techniques [5].
Historically, manual techniques such as RIA and ELISA were used to measure 25(OH)D. Many laboratories have moved over to more automated random access immunoassays – either standalone automated analysers specifically for 25(OH)D analysis or as part of a large scale automated laboratory where 25(OH)D is just one test in a large repertoire – in order to cope with the ever increasing work load [6]. However, immunoassays, automated or not, have the disadvantages of performance changes over time, e.g. due to changes in kit reagents or antibody, and a large number of the assays are not able to detect both 25(OH)D3 and 25(OH)D2 and therefore may underestimate total 25(OH)D. Some of the immunoassays are also subject to interference from heterophilic antibodies and 24,25-dihydroxyvitamin D [5].
Direct detection methods such as HPLC and LC/MS-MS do not suffer from the disadvantages mentioned above; however, they can suffer from interference from the inactive 3-epi-25(OH)D isomer, which is often seen in high concentrations in infants. The equipment is expensive and specialist knowledge is required to set up and maintain a clinical service. However, the advantages of low consumables costs, potential for automation and high throughput, and the ability to detect both 25(OH)D2 and 25(OH)D3 explains its growing popularity with clinical laboratories [5].

Reasons for testing
Even though vitamin D can be made endogenously and is available through dietary sources, vitamin D deficiency is extremely common, verging on pandemic [7]. In inner-city Birmingham, 24% of the study population was found to be deficient [defined as having 25(OH)D levels <25 nmol/L], with 43% of Asian women deficient [8]. If vitamin D insufficiency is defined as having levels <75 nmol/L, then it has been estimated that 1 billion people worldwide are vitamin D deficient or insufficient [3]. Deficiency of vitamin D is most commonly linked with rickets in children and osteomalacia in adults, but in the last few years it has been associated with a range of non-musculoskeletal conditions such as diabetes, immune function, cardiovascular disease and cancer. As well as being deficient in vitamin D, it is possible to become intoxicated with vitamin D, often as the result of patients taking supraphysiological doses of vitamin D for a variety of reasons. Hypervitaminosis D can lead to hypercalcaemia as a result of increased intestinal calcium absorption and bone resorption, which can ultimately lead to kidney injury [9].
It is very hard to predict what a person’s vitamin D concentration is, even if you take into account factors such as age, supplement use, season, sun exposure, race and body mass intake [8]. However there are a range of risk factors and these can often help to identify people who are vitamin D deficient (Table 1) [7, 11–13].

Routine population screening is not advocated by most of the literature but there is still considerable debate as to when and who to test. The guidelines from the Endocrine Society suggest screening for vitamin D deficiency in individuals at risk for deficiency [7]. The National Osteoporosis Society guidelines suggest that vitamin D deficiency should be corrected before certain drug regimens begin, e.g. before starting treatment with a potent antiresorptive agent (zoledronate or denosumab) implying that 25(OH)D should be checked in these patients. They also recommend testing patients who have symptoms suggestive of osteomalacia or who have chronic widespread pain. They do not, however, recommend routinely testing 25(OH)D in asymptomatic individuals who may be at higher risk of vitamin D deficiency, but suggest that these individuals should have a higher intake of vitamin D [13]. The drawback of this approach is that the recommended daily allowance will not be enough to correct severe deficiency, and giving higher loading doses to those already replete may put them at risk of vitamin D intoxication.

Rate of testing
Many things in the area of vitamin D lack consensus, such as what is the optimal level of serum 25(OH)D; however, as far as the rate of testing goes the evidence is unequivocal: 25(OH)D testing is growing at a staggering rate. The author’s laboratory at City Hospital, Birmingham, UK, has seen a 75% increase in testing since 2012 and will measure around 80,000 serum samples this year for 25-hydroxy vitamin D (Fig. 1). This is even after demand management was introduced in 2010, whereby GPs could only have one 25(OH)D test per patient per year, unless there were strong clinical indications for measurement. Other laboratories have noticed an annual 80–90% growth in 25(OH)D test rates [14]. One report suggested that Quest laboratories in the USA were receiving 500,000 requests for 25(OH)D analysis per month [15].

Self-referral testing
Growth has come from testing in the GP population as well as hospital-based testing. In addition to this, our Dried Blood Spot (DBS) Vitamin D service, which is a direct-to-the-public service, has also grown steadily since its introduction in 2011. We have analysed close to 10,000 samples since the service inception and requests have been received from all over the world as well as the UK (Fig. 2).

The reasons why health professionals want to test 25(OH)D have been discussed but we have found that the reasons the general public want to test for 25(OH)D are very different. Many are knowledgeable about vitamin D and want to check their and their family’s levels are adequate. Many are taking supplements and they want to be sure that they are taking enough. Some have certain conditions, e.g. psoriasis, multiple sclerosis, cancer, and have read about connections between vitamin D and these conditions and want to make sure they have achieved recommended levels of 25(OH)D. Some are not able to get 25(OH)D tested through their GP. A small but significant proportion suspect that they have over-supplemented and they use the DBS service to check how high their levels have got [16].

The different reasons behind testing, as well as population differences such as ethnic distribution, have led to a significantly different distribution of 25(OH)D statuses in the two populations. During 2012, 54.7% of self-referred samples showerd adequate levels, compared to only 20.1% of GP samples were [25(OH)D >50 nmol/L, P<0.001, 95% confidence interval]. Only 0.1% of GP samples were high to toxic [25(OH)D >220 nmol/L] compared with 1% of the DBS samples (P<0.001, 95% confidence interval). For people who want to be pro-active about their health, the self-referral service is giving them this opportunity. It is also allowing a proportion of the population who may have been inadvertently over-supplementing to realise this and act before any long-term harm has occurred. Clinical laboratories are struggling to cope with the increase in demand for vitamin D testing, not just with physically performing the work but also because many are not paid appropriately for the analysis and this has led to financial difficulties for some laboratories. However, the demand from the public is not going to abate, especially as the media and public awareness of vitamin D grows. Self-referral vitamin D testing gives people a viable alternative when they are not able to access the testing through their health care system. Into the future…
Vitamin D testing is going to continue to rise and clinical laboratories are going to have to find strategies to cope. This may mean putting demand management strategies in place, negotiating prices with commissioners in order to be paid appropriately and looking at the methods used for measuring 25(OH)D to see if savings and efficiencies can be made.

As vitamin D remains in the media spotlight, and while vast numbers of papers relating to vitamin D continue to be published, hopefully the awareness of the importance of having adequate levels of vitamin D will continue to rise amongst healthcare professionals, and advice on what to do to achieve adequate levels will improve. One day we will have a national strategy on who to test, when and how often, but for now it seems that some of the population will continue to try to figure it out for themselves.

References
1. Pearce SHS, Cheetham TD. Diagnosis and management of vitamin D deficiency. BMJ 2010; 340: b5664.
2. Norman AW. From vitamin D to hormone D: fundamentals of the vitamin D endocrine system essential for good health. Am J Clin Nutr. 2008; 88(suppl): 491S-499S.
3. Holick MF. Vitamin D Deficiency. N Engl J Med. 2007; 357: 266-281.
4. Zerwekh JE. Blood biomarkers of vitamin D status. Am J Clin Nutr. 2008; 87(suppl): 1087S-1091S.
5. Carter GD. 25-Hydroxyvitamin D: a difficult analyte. Clin Chem. 2012; 58; 486-488.
6. Hollis BW. Measuring 25-hydroxyvitamin D in a clinical environment: challenges and needs. Am J Clin Nutr. 2008; 88(suppl): 507S-510S.
7. Holick MF, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an endocrine society clinical practice guideline. J Clin Endocrinol Metab. 2011; 96: 1911-1930.
8. Ford L, et al. Vitamin D concentrations in an UK inner-city multicultural outpatient population. Ann Clin Biochem. 2006; 43: 468-473.
9. Vogiatzi MG, et al. Vitamin D supplementation and risk of toxicity in pediatrics: a review of current literature [Epub ahead of print]. J Clin Endocrinol Metab. 2014; 99: doi 10.1210/jc.2013-3655.
10. Freedman DM, et al. Sunlight and other determinants of circulating 25-hydroxyvitamin D levels in black and white participants in a nationwide US study. Am J Epidemiol. 2013; 177: 180-192.
11. Harrison M, et al. The Royal College of Pathologists of Australia Position Statement on the use and interpretation of vitamin D testing. 2013.
12. Hull S, Anastasiadis T. Vitamin D guidance. Barts and the London Clinical Effectiveness Group. 2011.
13. Francis R, et al. Vitamin D and bone health: a practical clinical guideline for patient management. National Osteoporosis Society. 2013.
14. Singh RJ. Are clinical laboratories prepared for accurate testing of 25-hydroxy vitamin D? Clin Chem. 2008; 54: 221-223.
15. Carter GD. 25-Hydroxyvitamin D assays: the quest for accuracy. Clin Chem. 2009; 55: 1300-1302.
16. Shea RL, Berg JD. Self referral vitamin D testing: are we just testing the worried well or making an important contribution to healthcare? Ann Clin Biochem. 2013; 50(suppl): 34.

The authors
Robyn Shea* BSc, MSc, MSc, DipRCPath and Dr Jonathan Berg BSc, PhD, MCB, FRCPath, MBA
Department of Clinical Biochemistry, Sandwell and West Birmingham Trust Hospitals NHS Trust, Birmingham, UK

*Corresponding author
E-mail: robyn.shea@nhs.net

B-cell maturation antigen (BCMA) was originally identified as a cell surface receptor expressed on late-stage B cells, plasma cells, and B-cell malignancies including multiple myeloma (MM). We recently discovered that BCMA is shed into the blood of MM patients, and, therefore, serum BCMA may serve as a new prognostic marker to track disease status and response to treatment.

by Eric Sanchez, Suzie Vardanyan, Mingjie Li, Cathy Wang, Dr Haiming Chen, and Dr James R. Berenson

Background
B-cell maturation protein (BCMA, also referred to as TNFRSF17 or CD269) is a receptor shown to be expressed in B lymphocytes and at increasing levels as these cells mature [1]. BCMA binds the ligands BAFF (B-cell activating factor) and APRIL (a proliferation inducing ligand) [2, 3]. Membrane-bound expression of BCMA has been demonstrated in human CD138-expressing cells from multiple myeloma (MM) bone marrow (BM) and MM cell lines [4]. It has also been shown to be expressed on malignant cells from Hodgkin’s lymphoma and Waldenstrom’s macroglobulinemia (WM) patients [5, 6]. Although the response to treatment of MM patients is traditionally followed with measurement of their monoclonal immunoglobulin (Ig) levels, some patients do not produce this marker; and, moreover, in others, it is lost during the course of their disease.

Serum BCMA levels are elevated in MM patients
We measured serum BCMA by ELISA from patients with newly diagnosed MM, monoclonal gammopathy of undetermined significance (MGUS) and healthy control subjects. Using the International Staging System, 30, 12 and 7 patients with stages 1, 2 and 3, respectively, and one with unknown staging were analysed. We found that the serum levels of BCMA in MM patients (13.87 ng/ml) were elevated when compared to healthy controls (2.57 ng/ml; P <0.0001) and MGUS individuals (5.30 ng/ml; P = 0.0157; Fig.1A). We then determined that serum BCMA levels correlated with the MM patient’s response to therapy. Patients responding to their treatment regime had lower serum BCMA levels than patients with progressive disease (P = 0.0038; Fig. 1B). To confirm that the BCMA found in the blood of MM patients came from cells within the BM, BM aspirates were obtained from MM patients and BM mononuclear cells (MCs) were isolated and cultured for 48 hours. MM patients showed high levels of BCMA in culture medium whereas healthy subjects lacked significant amounts of BCMA.  Moreover, serum and supernatant BCMA levels from MM patients were compared and showed a strong correlation (r = 0.82) between the levels in the serum and supernatants from cultured MM BMMCs (Fig. 2). To exclude the possibility that the BCMA detected in MM patient serum and from cultured MM BMMCs may have been derived from non-malignant cells, we evaluated human BCMA levels in our human MM xenografts growing in severe combined immunodeficient (SCID) mice.  Animals were implanted with the human MM tumour LAGκ-2 and analysed for human serum BCMA levels. SCID mice dosed with bortezomib (0.5 mg/kg, twice weekly) had a reduction in tumour volume compared to untreated mice (P = 0.0067; Fig. 3A), and human serum BCMA levels from these bortezomib-treated animals were also markedly lower compared to untreated mice (P = 0.0006; Fig. 3B). Similar results were obtained when using our other MM xenograft models (LAGλ-1, LAGκ-1A). Human BCMA was not detected in the serum of non-tumour-bearing mice (data not shown). A rise in serum BCMA levels from the possible release of membrane-bound protein from dead MM cells was not observed in mice following drug treatment. Thus, it can be concluded that the serum levels of BCMA in the mice are derived from live MM cells. Soluble BCMA has been shown to inhibit normal B-cell development through interference with the binding of its ligands (BAFF and/or APRIL) to membrane-bound BCMA on normal B cells. One group demonstrated that administration of BCMA–Ig fusion protein to normal mice, which inhibits the binding of BAFF to B cells, resulted in a dramatic reduction in B-cell numbers in the blood and peripheral lymphoid organs [2]. Splenic B-cell reductions were shown to occur in an in vitro mouse splenocyte proliferation assay following in vivo administration of BCMA–Fc fusion protein to normal mice [7]. Other investigators have shown that injecting soluble BCMA–Fc fusion protein, which binds BAFF and APRIL both as free and membrane-bound ligands, into nude mice bearing human colon or lung carcinoma cell lines resulted in inhibition of tumour growth [3]. The authors suggested that BCMA bound its ligand APRIL, and prevented the known stimulatory effect of APRIL on these cell lines. Additionally, investigators have shown in vitro the existence of BCMA–BAFF complexes [3, 7, 8]. In the context of cancer growth, one group demonstrated that BCMA injected into mice reduced the growth of tumour cells from human colon or lung carcinoma cell lines in vivo by binding its ligand APRIL [3]. Thus, we are currently conducting studies to determine if such complexes exist in vivo and may block the immune function of myeloma patients.

Conclusions and future directions
Measurement of MM tumour mass is indirect, given the location of this BM based malignancy. Thus, assessment of tumour burden in response to therapy is difficult but essential to effectively monitor patients with MM. Traditionally, changes in Ig levels have been used to follow disease progression and response to treatment. In fact, for three decades Ig was a prognostic factor used in the Durie-Salmon staging system [9], which, until recently, was the most widely used staging system [10].  However, assessment of this protein does not always accurately reflect changes in MM tumour burden [11–13]. Additionally, small subsets of MM patients do not produce this marker. Thus, additional markers are needed to assess response to therapy in these non-secretory patients and in MM patients as a whole.

We have now shown that BCMA is present in the serum of MM patients; and, moreover, its levels correlate with the patient’s response to therapy [14]. We have also previously shown that supernatant from cultured BMMCs from MM patients with active disease contain much higher levels of this protein in their culture medium than in healthy subjects and those with MGUS or indolent MM [14]. SCID mice bearing human MM xenografts also showed high levels of BCMA in their sera, and these levels decreased in response to anti-MM therapy. We believe that that serum BCMA will eventually be used in the clinic as a diagnostic and prognostic marker for MM patients.

References
1. Laabi Y, Gras MP, Brouet JC, et al. The BCMA gene, preferentially expressed during lymphoid maturation, is bidirectionally transcribed. Nucleic Acids Res. 1994; 22(7): 1147–1154.
2. Thompson JS, Schneider P, Kalled SL, et al. BAFF binds to the tumor necrosis factor receptor-like molecule B cell maturation antigen and is important for maintaining the peripheral B cell population. J Exp Med. 2000; 192(1): 129–135.
3. Rennert P, Schneider P, Cachero TG, et al. A soluble form of B cell maturation antigen, a receptor for the tumor necrosis family member APRIL, inhibits tumor cell growth. J Exp Med. 2000; 192(11): 1677–1684.
4. Novak AJ, Darce JR, Arendt BK, et al. Expression of BCMA, TACI, and BAFF-R in multiple myeloma: a mechanism for growth and survival. Blood 2004; 103(2): 689–694.
5. Chiu A, Xu W, He B, et al. Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL. Blood 2007; 109(2): 729–739.
6. Elsawa SF, Novak AJ, Grote DM, et al. B-lymphocyte stimulator (BLyS) stimulates immunoglobulin production and malignant B-cell growth in Waldenstrom macroglobulinemia. Blood 2006; 107(7): 2882–2888.
7. Pelletier M, Thompson JS, Qian F, et al. Comparisons of soluble decoy IgG fusion proteins of BAFF-R and BCMA as Antagonist for BAFF. J Biol Chem. 2003; 278(35): 33127–33133.
8. Shu HB, Johnson H. B cell maturation protein is a receptor for the tumor necrosis factor family member TALL-1. Proc Natl Acad Sci U S A 2000; 97(16): 9156–9161.
9. Durie BG, Salmon SE. A clinical staging system for multiple myeloma: correlation of measured myeloma cell mass with presenting clinical features, response to treatment and survival. Cancer 1975; 36(3): 842–854.
10. Larson RS, Sukpanichnant S, Greer JP, et al. The spectrum of multiple myeloma: diagnostic and biological implications. Hum Pathol. 1997; 28(12): 1336–1347.
11. Sullivan PW, Salmon SE. Kinetics of tumor growth and regression in IgG multiple myeloma. J Clin Invest. 1972; 51(7): 1697–1708.
12. Kawano M, Huang N, Harada H, et al. Identification of immature and mature cells in the bone marrow of human myelomas. Blood 1993; 82(2): 564–570.
13. Zaanen HCT, Lokhorst HM, Aarden LA, et al. Chimaeric anti-interleukin 6 monoclonal antibodies in the treatment of advanced multiple myeloma: a phase I dose-escalating study. Br J Haematol. 1998; 102(3): 783–790.
14. Sanchez E, Li M, Kitto A, et al. Serum B-cell maturation is elevated in multiple myeloma and correlates with disease status and survival. Br J Haematol. 2012; 158(6): 727–738.

The authors
Eric Sanchez BA; Suzie Vardanyan BS; Mingjie Li BS; Cathy Wang BS; Haiming Chen MD, PhD; and James R. Berenson* MD
Institute for Myeloma & Bone Cancer Research, West Hollywood, CA, USA

*Corresponding author
E-mail: Jberenson@imbcr.org

 

Expressed prostatic secretion (EPS) is obtained after prostatic message by milking the urethra and collecting fluid directly from the urethral meatus. Clinicians have previously used this biospecimen primarily for the diagnosis of urinary infections. More recently it has been recognized as a rich source of biomarkers of prostate malignancy. Many of those biomarkers are accessible only in EPS, where it has shown a unique potential in both prostate cancer screening and active surveillance programmes.

by Dr J. Linehan, Dr J. Yamzon, Prof. T. Wilson and Prof. S. Smith

Current risk assessment tools
An estimated 238,590 new cases of prostate cancer will be diagnosed in the United States during 2013. A substantial proportion of these patients are considered ‘low-risk’ for progression and may be best served with conservative management. Yet many will undergo definitive treatment by means of radiotherapy or radical prostatectomy, both of which have risks of life-altering side effects. This huge – and ultimately unsustainable – pattern of over-treatment has incited a shift in practice patterns toward initial conservative management known as Active Surveillance (AS), the premise of which is to minimize unnecessary treatment by performing close monitoring. Should there be signs of tumour progression upon recurring reassessment, then treatment can be offered with curative intent. Currently, our ability to discern one’s risk for harbouring an indolent form of prostate cancer utilizes clinical parameters that lack predictive ability. Serum PSA level, clinical stage determined on digital rectal examination (DRE), and tumour grade determined upon biopsy, known as the Gleason Score (GS), are currently used to stratify the risk of tumour progression, although with only limited accuracy. This is evidenced by studies of men eligible for AS who are found to have more advanced disease upon treatment with radical prostatectomy [1] despite meeting AS criteria using the standard criteria. Tools based on the biology of prostate cancer offer a potential platform for improved risk stratification, and hence allow more appropriate levels of intervention.

Invasive and non-invasive testing
Current efforts in biomarker discovery and validation stand to complement the existing tools that gauge eligibility for AS. Several tools, such as Oncotype DX-Prostate® (Genomic Health) and Prolaris® (Myriad Genetics), are commercially available and generate risk scores for harbouring more advanced disease. These rely on extraction of RNA from tumour tissue, most commonly taken from the biopsy specimen that may not contain the sufficient amounts of cancer tissue needed to perform these proprietary tests. AS protocols require periodic repeat biopsies of the tumour, which may have potential complications of bleeding, infection, and pain. Thus, a less invasive method of assessment is ideal. Tools reliant on serum and urine specimens are attractive in that they are more readily obtained. Serum tests, such as free-to-total PSA ratios, the 4K Score (OPKO Health), and the Prostate Health Index (Beckman-Coulter), show promise in refining prostate cancer diagnosis, but these rely on proteins shed from the tumour into circulation. Urine based tests like PCA3 (Gen-Probe) or DNA based tests like ConfirmMDx (MDxHealth) rely on proteins shed into the urine after prostate massage, or DNA extracted from biopsy specimens. These latter tests are utilized in the clinical scenario where prostate cancer remains undiagnosed despite high suspicion by standard parameters.

The biological function of the prostate gland is the production of seminal fluid critical to reproduction. It contains DNA, RNA, proteins and metabolites that have the potential to serve as biomarkers useful in prostate cancer diagnosis and risk assessment. Its contents can be retrieved by two non-invasive methods. The first, variously called a post-DRE urine or post-massage urine, is obtained as a urine sample collected immediately after an attentive DRE. The second, termed prostatic fluid or expressed prostatic secretion (EPS) is obtained after prostatic massage by milking the urethra and collecting fluid directly from the urethral meatus. Neither specimen requires invasive techniques and both contain significant numbers of prostate cancer cells and prostate cancer biomarkers [2–5]. Each has advantages and disadvantages. Post-DRE urine is relatively straightforward to collect; however, the urine volume, and cell lysis that occurs in urine, can significantly dilute the informative cellular material and hinder the recovery of the informative components of the fluid. EPS specimens require more effort to collect but are undiluted and do not promote cell lysis or block activity measurements. Most clinicians would prefer to collect post-DRE urine samples, but the extra effort required to collect EPS could be justified because a number of useful biomarkers cannot be assessed in post-DRE urine. Patient acceptance of both biopsy and EPS collection are improved dramatically by the administration of valium prior to biopsy. Moreover, EPS collection prior to biopsy can be performed in a single clinical visit.

Both EPS and post-DRE urine specimens have proven successful in prostate cancer detection using gene expression biomarkers, where PCA3 and TMPRSS2:ERG fusions have proven effective. Here, sufficient quantities of RNA are present in either specimen, permitting roughly equivalent improvements in performance in the prediction of biopsy outcome (Table 1). The moderate improvement in test performance seen in EPS compared to post-DRE urine appears to be attributable to the need for normalization to PSA messenger RNA measured in the same specimen [6]. This requirement is unique to post-DRE urine as a specimen since urine volume collection is not constant, making it necessary to gauge the informative RNA signals against a fixed RNA signal. The choice of PSA messenger RNA in this application tends to suppress the informative signals since more of it is released into the urine from regions of prostate cancer where the basement membrane is compromised. Moreover it is not possible to measure the volume of the prostatic fluid after it is diluted by the variable urine catch.

DNA methylation is much more difficult to quantify, since DNA is present in much lower concentration in cells, and the required chemical treatments of the isolated DNA results in a severe reduction of the DNA available for quantitative PCR amplification. It has only been reported to be effective in the prediction of biopsy outcome in EPS [4], where the combination of hypermethylation at RARß, RASSF1 and GSTP1performed as well as PCA3 as a single marker.

The field defect and prostate function
Prostate functionality becomes an important diagnostic tool when one considers the extensive field defect that characterizes the disease [8]. Field defects are best defined as functional aberrations that extend beyond the morphologically recognizable tumour in otherwise histologically normal tissue. The concept dates to the early 1950s and has been shown to involve altered DNA methylation patterns extending several millimeters from the prostate tumour. In prostate tumorigenesis it has recently been documented that extensive chromosome rearrangements occur in single steps involving chromothripsis and chromoplexy [9, 10]. In this view the field defect represents clonal progeny of an earlier stage in tumour evolution from which additional chromothripsis and chromoplexy events generate the malignancy. Since most reports of hypermethyled loci fall at or extremely near fragile sites [11], DNA methylation alterations may be linked to chromoplexy and chromothripsis, making it possible to detect cancer by looking for early alterations DNA methylation patterning. Mitochondrial DNA aberrations also appear to track prostate tumour evolution and the field defect includes mitochondrial DNA deletions [12].

Each of these findings suggests that capacity of the prostate gland to produce functional prostatic fluid will be regionally compromised. Genome wide aberrations in DNA methylation coupled with the aberrant expression of transcription factors, such as ERG, could indicate wholesale aberrations in gene expression, while the mitochondrial deletions may underlie alterations in the unique metabolic pathways present in the normal gland. The field defect around larger or more highly evolved tumours could in fact affect the entire gland and can be expected to significantly alter not only the nature but also the amount of prostatic fluid that the gland can produce.

Anecdotally, surgeons collecting EPS by the non-invasive procedure prior to prostatectomy or by squeezing the excised gland just after prostatectomy note that a prostate with a larger tumour burden yields less fluid than a gland bearing small tumour foci, suggesting that the measured volume of the recoverable fluid should be an effective biomarker for prostate cancer. This was demonstrated in recent studies with patients who are eligible for active surveillance by various criteria but were treated with robot assisted radical prostatectomy instead.

For these studies, we used a biomarker set that measures the secretion capacity of the prostate gland. This set includes recovered EPS volume in microlitres and total recovered RNA in nanograms. It is measured along with pre-operative serum PSA value as baseline in multivariate logistic regression analysis (Fig. 1).

Future directions
Given the utility of EPS biomarkers in predicting the presence of occult extracapsular extension in an AS cohort, we believe that the advantages of EPS in assessing the functionality of the gland make it uniquely suited for initial risk stratification in determining eligibility for AS and also in monitoring the disease during surveillance. One of the most perplexing issues in this area is detecting misclassification. In preliminary work in this area (Wittig et al. in prepartion) we noted that more than 40% of patients initially scored as GS 6 in an NCCN cohort can be occult GS 7 comprising largely GS 3+4 patterns with relatively fewer pattern 4+3 GS 7s. Although GS 7 patients are referred for definitive treatment this data suggests that many have not progressed during AS. None of the available biomarkers that we have tested in EPS thus far (TMPRSS2:ERG, PCA3, Secretion Capacity, TXNRD1 or PSA-mRNA) reliably detect this relatively subtle change in the GS. Misclassification recognized at repeat biopsy is generally accepted as an error of initial biopsy undersampling. One can question the clinical significance of such a subtle nuance and its affect on the grander outcomes of disease metastasis and prostate cancer death. Biopsy misclassification is yet another example of the under-performance of standard parameters (like the GS) and the need for biomarker based determinations of disease aggressiveness.

Several new biomarkers are evaluable exclusively in EPS. One example is PSA activity. PSA is a proteolytic enzyme that cannot be assayed in urine specimens, although the assay may function in urine sediment. It is active in EPS and has been shown to be inversely proportional to tumour aggressiveness [13] . Another biomarker with potential in EPS is citrate, which has been shown to be an indicator of prostate cancer in seminal fluid. This marker [14] reflects the overall health of the gland, since the unique metabolism of the normal prostate is designed to secrete citrate into the prostatic fluid. A third biomarker approachable only in EPS is zinc [15]. This ion is taken up preferentially in normal prostate where it blocks the degradation of citrate by inhibiting aconitase in the citric acid cycle thus contributing to the normal function of the gland in citrate secretion.
In summary, the suboptimal performance of the current clinicopatholigic parameters utilized in prostate cancer diagnosis and risk stratification drive the need for additional tools. Biomarkers examined in EPS offer the additional dimension of prostate-function assessment through a non-invasive platform that has great potential to improve prostate cancer diagnosis, risk stratification, and AS.

Abbreviations
AS, active surveillance
AUC, area under curve
DRE, digital rectal examination
EPS, expressed prostatic secretion
GS, Gleason Score
PSA, prostate-specific antigen
ROC, receiver operating characteristic

References
1. Conti SL, Dall’era M, Fradet V, Cowan JE, Simko J, Carroll PR. Pathological outcomes of candidates for active surveillance of prostate cancer. J Urol. 2009; 181: 1628–1633 (Discussion pp. 1624–1633).
2. Groskopf J, Aubin SM, Deras IL, Blase A, Bodrug S, Clark C. APTIMA PCA3 molecular urine test: development of a method to aid in the diagnosis of prostate cancer. Clin Chem. 2006; 52: 1089–1095.
3. Crocitto LE, Korns D, Kretzner L, Shevchuk T, Blair SL, Wilson TG, Ramin, SA, Kawachi MH, Smith SS. Prostate cancer molecular markers GSTP1 and hTERT in expressed prostatic secretions as predictors of biopsy results. Urology 2004; 64: 821–825.
4. Clark JP, Munson KW, Gu JW, Lamparska-Kupsik K, Chan KG, Yoshida JS, Kawachi MH, Crocitto LE, Wilson TG, et al. Performance of a single assay for both type III and type VI TMPRSS2:ERG fusions in non-invasive prediction of prostate biopsy outcome. Clin Chem. 2008; 54: 2007–2017.
5. Laxman B, Tomlins SA, Mehra R, Morris DS, Wang L, Helgeson BE, Shah RB, Rubin MA, Wei JT, Chinnaiyan AM. Noninvasive detection of TMPRSS2:ERG fusion transcripts in the urine of men with prostate cancer. Neoplasia 2006; 8: 885–888.
6. Whelan C, Crocitto L, Kawachi M, Chan K, Smith D, Wilson T, Smith S. The influence of PSA-RNA yield on the analysis of expressed prostatic secretions (EPS) for prostate cancer diagnosis. Can J Urol. 2013; 20: 6597–6602.
7. Tomlins SA, Aubin SM, Siddiqui J, Lonigro RJ, Sefton-Miller L, Miick S, Williamsen S, Hodge P, Meinke J, et al. Urine TMPRSS2:ERG fusion transcript stratifies prostate cancer risk in men with elevated serum PSA. Sci Transl Med. 2011; 3: 94ra72.
8. Mehrotra J, Varde S, Wang H, Chiu H, Vargo J, Gray K, Nagle RB, Neri JR, Mazumder A. Quantitative, spatial resolution of the epigenetic field effect in prostate cancer. Prostate 2008; 68: 152–160.
9. Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y, Nezi L, Protopopov A, Chowdhury D, Pellman D. DNA breaks and chromosome pulverization from errors in mitosis. Nature 2012; 482: 53–58.
10. Baca SC, Prandi D, Lawrence MS, Mosquera JM, Romanel A, Drier Y, Park K, Kitabayashi N, MacDonald TY, et al. Punctuated evolution of prostate cancer genomes. Cell 2013; 153: 666–677.
11. Smith SS. Maintaining the unmethylated state. Clin Epigenetics 2013; 5: 17.
12. Maki J, Robinson K, Reguly B, Alexander J, Wittock R, Aguirre A, Diamandis EP, Escott N, Skehan A, et al. Mitochondrial genome deletion aids in the identification of false- and true-negative prostate needle core biopsy specimens. Am J Clin Pathol. 2008; 129: 57–66.
13. Ahrens MJ, Bertin PA, Vonesh EF, Meade TJ, Catalona WJ, Georganopoulou D. PSA enzymatic activity: A new biomarker for assessing prostate cancer aggressiveness. Prostate 2013; 73: 1731–1737.
14. Kline EE, Treat EG, Averna TA, Davis MS, Smith AY, Sillerud LO. Citrate concentrations in human seminal fluid and expressed prostatic fluid determined via 1H nuclear magnetic resonance spectroscopy outperform prostate specific antigen in prostate cancer detection. J Urol. 2006; 176: 2274–2279.
15. Zaichick V, Sviridova TV, Zaichick SV. Zinc in the human prostate gland: normal, hyperplastic and cancerous. Int Urol Nephrol. 1997; 29: 565–574.

The authors
Jennifer Linehan MD, Jonathan Yamzon MD, Timothy Wilson MD, and Steven Smith* PhD
NanoLab, Division of Urology, City of Hope, Duarte, California, USA

*Corresponding author
E-mail: ssmith@coh.org