Identification of a serum or urine paraprotein is a key element in the diagnosis of multiple myeloma. Traditionally, this has been achieved using a combination of serum and urine electrophoresis, but this can result in incomplete investigation. The use of serum free light chains as an alternative screening test has been advocated to overcome this.

by David Baulch and Beverley Harris

Multiple myeloma
Multiple myeloma (MM) accounts for 1% of all cancers, with nearly 5000 people in the UK being diagnosed each year. The average age of presentation is 70 with only 15% of patients presenting at less than 60 years of age [1]. Its prevalence has increased by 11% in the last decade, due mainly to increased survival rates in those diagnosed [2]. Despite this, MM still accounts for around 2700 deaths annually in the UK and over 70 000 worldwide with a median survival of only 3–4 years from diagnosis [3].

MM is characterized by the accumulation of clonal plasma cells, predominantly within the bone marrow, and subsequent clonal expansion of the plasma cell lineage [4]. It is almost always preceded by a premalignant, asymptomatic period of monoclonal gammopathy of undetermined significance (MGUS) [1]. The process of immunoglobulin (Ig) production by plasma cells is normally under a state of homeostasis, but random and non-random genetic aberrations, epigenetic changes and atypical interactions within the bone marrow microenvironment can cause uncontrolled proliferation of neoplastic plasma cells, leading to plasma cell disorders (PCDs) such as MM [4]. Clonal expansion of a plasma cell line under such circumstances can cause overproduction of intact monoclonal Ig (IgG, IgA, IgM, rarely IgD and IgE) or monoclonal free light chains (FLCs) kappa and lambda. Although the classification of PCDs is based on the immunoglobulin type secreted, 1–2% of MM cases are classified as non-secretory. This may be due to an absence of secreted monoclonal protein (M protein), or secretion at a concentration below the limits of the laboratory methods used for detection.

Compared with other cancers, diagnosis of MM is challenging. Patients present with a range of non-specific symptoms and as a result often have a string of primary care consultations resulting in diagnostic delay. Such delays significantly impact the clinical course of MM [5], for which a complete cure remains elusive.

Consequences of diagnostic delay
Studies have shown that over 50% of patients attending primary care institutions took 6 months (33% >12 months) from the onset of the first related symptoms to referral [5]. Another study showed the time to diagnosis of MM can be unacceptably prolonged [6] and the pathway to diagnosis in MM was more likely to include a string of repeated primary care consultations, infrequent use of urgent referral routes and increased emergency presentation [7]. In particular, patients whose referral was delayed by 6 months or more were more likely to suffer a greater number of more significant complications such as renal insufficiency which, if swift diagnosis had occurred, may have been reversible [5]. This highlights the need not only to raise awareness of disease symptoms, but to increase the sensitivity of laboratory detection.

Laboratory investigation of multiple myeloma
In addition to clinical and hematological investigations, screening for MM within the laboratory is based on the detection and classification of M proteins by serum protein electrophoresis (the separation of serum proteins according to molecular size, hydrophobicity and electric charge [8]), followed by immunofixation or immunotyping to identify and quantify the Ig isotypes. This method is less reliable for detecting disease when only FLCs are secreted, as these are rapidly cleared by the kidneys. Free light chains in the urine [known as Bence Jones protein (BJP)] can also be detected by electrophoresis followed by immunofixation. However, this methodology is time consuming and may not detect low concentration BJP in dilute urine samples [9]. Interpretation of the results can be difficult and should be performed by appropriately qualified and experienced laboratory staff. In addition, obtaining both urine and serum samples for screening can be problematic, with some laboratories reporting that both samples are received for only ~17% of MM screens.

There is growing evidence to support the direct measurement and quantitation of serum kappa and lambda FLCs in diagnosis, monitoring and prognosis of MM and related PCDs [4]. The serum FLC (sFLC) assay (The Binding Site™) was first developed in 2001 [10]. It is an immunoturbidimetric method using latex-enhanced polyclonal sheep antibodies targeted to epitopes on the light chains of Ig that are exposed when the light chain is ‘free’, i.e. not bound to heavy chain Ig. Results are expressed as a ratio of kappa : lambda light chains.

This sFLC assay can be used to replace traditional urine methods for the laboratory detection of FLCs. This practice has the obvious benefit of using a single serum sample and eliminating the need for a paired urine sample, which may not always be supplied. In addition to the reported increased diagnostic sensitivity of the sFLC assay, an unexpected finding by Dispenzieri et al. was that baseline sFLC results can be used in prognostication and risk stratification of MGUS [11]. Although the rationale for this is poorly understood, it is thought that a greater degree of abnormality in the sFLC ratio reflects an increasing tumour burden.

Studies such as these have informed changes to MM guidelines published in 2016 [12] to acknowledge that significantly abnormal FLC ratios, in the absence of clinical features of end organ damage, can be used in the diagnosis of MM [4]. This eliminates a traditional major challenge with MM diagnosis in that disease definition was clinicopathological. The use of the sFLC ratio in this way therefore marks a milestone in the early detection of MM and highlights a disease transition to being a laboratory-defined rather than a symptom-defined disease, allowing for earlier intervention.

There is, however, controversy as to whether the sFLC assay is indeed a robust candidate for inclusion in PCD screening strategies. There is currently only limited guidance on how it should be used in clinical practice [4] and there is ongoing debate regarding result interpretation, especially for those mildly abnormal ratios. There are, therefore, many considerations to be made before such screening could be implemented.

Study overview and results
Our real-time prospective study aimed to assess the clinical utility of three index laboratory investigations [serum and urine protein electrophoresis (sEP and uEP) and sFLC] to determine the most effective first-line testing strategy for detecting PCDs in primary care patients. These laboratory investigations were performed on 446 samples with no previous history of, or investigations for, MM. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and efficiency were calculated for our current screening tests (sEP and uEP) and the use of sEP with sFLC as an alternative strategy. Figures 1 and 2 outline the process for each of these screening strategies and a summary of the results is given in Table 1.

Conclusion
The purpose of a medical screening programme is to recognize a disease in its preclinical phase to allow intervention at an earlier stage. Such strategies have benefits, risks and costs and the final screening algorithm is often a compromise between these three. However, a proposed screening strategy should fulfil the criteria outlined by Wilson and Jungner in 1968 [13]. Of note, criterion 4 suggests there should be a detectable preclinical stage, in this case MGUS, and criterion 5 suggests there should be a suitable test for screening strategies. This real-time prospective study presents evidence of the clinical utility of the sFLC assay and its use in developing a more sensitive screening strategy for PCD detection.

Standard screening practice combining sEP and uEP increased the sensitivity of the constituent index tests (78% and 30% respectively) to 81%, meaning the addition of urinalysis to sEP increased the sensitivity by only 3%. This reinforces the need for a more sensitive method for detecting sFLC than sEP alone. This combination also displayed a good PPV without compromising efficiency (98%). Despite this, its use missed significant cases of PCDs including a light-chain multiple myeloma, a possible but unconfirmed (in the time frame of the study) case of MM and 10 cases of MGUS, highlighting its limitation as a first line screening investigation.

Combining sEP with sFLC analysis increased the sensitivity from sEP alone by 20% (data not shown), again suggesting singular sEP testing is not sensitive enough to detect minor abnormalities in FLC production. This proposed combination of screening tests increased sensitivity by 17% when compared with current protocols, indicating that the sFLC assay is more sensitive than urinalysis for detecting PCDs. The sFLC assay has been demonstrated to show a high sensitivity for light chain MM and non-secretory MM [14]. These often present with normal sEP and uEP, especially in low tumour burden stages when renal function remains adequate, which may explain the increased sensitivity of sFLC over uEP.

The results of this study confirm also those of others [15], which show that the addition of sFLC analysis to sEP increases the detection of MM and related PCDs. In our case, there was a 17% increase in patients with a PCD detected. However, a concurrent rise in false positive results (10%) was also seen when compared to traditional screening protocols. Investigation into this was beyond the scope of our study, though the false positive rate could potentially be reduced by employing screening strategies that apply renal reference intervals for the sFLC ratio for those with renal insufficiency.

Summary
On balance, there are several advantages to replacing urinalysis with the sFLC assay. These include increased clinical sensitivity for detection of early-stage disease, patient convenience in submitting a single serum sample rather than two separate specimens, increased use of automation and reduction in subjectivity in reporting of results. However, it is also important to consider the potential increased cost of performing sFLC on all samples submitted for myeloma screening, the importance of using appropriate reference ranges and the need to develop guidelines for interpretation of borderline results. This latter point is particularly important in order that unnecessary referrals are prevented, and should involve close liaison with local hematology teams to ensure that primary care clinicians are given clear guidance for further investigation and referral of their patients.

References
1. Bird JM, Owen RG, D’Sa S, Snowden JA, Pratt G, Ashcroft J, Yong K, Cook G, Feyler S, et al. Guidelines for the diagnosis and management of multiple myeloma 2011. Br J Haematol. 2011; 154(1): 32–75.
2. Brenner H, Gondos A, Pulte D. Expected long-term survival of patients diagnosed with multiple myeloma in 2006–2010. Haematologica 2009; 94(2): 270–275.
3. Rajkumar SV, Kyle RA, Therneau TM, Melton LJ, III, Bradwell AR, Clark RJ, Larson DR, Plevak MF, Dispenzieri A, Katzmann JA. Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance. Blood 2005; 106(3): 812–817.
4. Rajkumar SV, Dimopoulos MA, Palumbo A, Blade J, Merlini G, Mateos MV, Kumar S, Hillengass J, Kastritis E, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol. 2014; 15(12): e538–548.
5. Kariyawasan CC, Hughes DA, Jayatillake MM, Mehta AB. Multiple myeloma: causes and consequences of delay in diagnosis. QJM 2007; 100(10): 635–640.
6. Howell DA, Smith AG, Jack A, Patmore R, Macleod U, Mironska E, Roman E. Time-to-diagnosis and symptoms of myeloma, lymphomas and leukaemias: a report from the Haematological Malignancy Research Network. BMC Hematol. 2013; 13(1): 9.
7. Elliss-Brookes L, McPhail S, Ives A, Greenslade M, Shelton J, Hiom S, Richards M. Routes to diagnosis for cancer – determining the patient journey using multiple routine data sets. Br J Cancer 2012; 107(8): 1220–1226.
8. Bossuyt X. Separation of serum proteins by automated capillary zone electrophoresis. Clin Chem Lab Med. 2003; 41(6): 762–772.
9. Kaplan IV, Levinson SS. Misleading urinary protein pattern in a patient with hypogammaglobulinemia: effects of mechanical concentration of urine. Clin Chem. 1999; 45(3): 417–419.
10. Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, Drew R. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem. 2001; 47(4): 673–680.
11. Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, Palumbo A, Jagannath S, Blade J, 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.
12. Myeloma: diagnosis and monitoring. National Institute for Health and Care Excellence (NICE) 2016. (https://www.nice.org.uk/guidance/ng35)
13. Wilson JM, Jungner YG. [Principles and practice of mass screening for disease]. Bol Oficina Sanit Panam. 1968; 65(4): 281–393 (in Spanish).
14. Jagannath S. Value of serum free light chain testing for the diagnosis and monitoring of monoclonal gammopathies in hematology. Clin Lymphoma Myeloma 2007; 7(8): 518–523.
15. McTaggart MP, Lindsay J, Kearney EM. Replacing urine protein electrophoresis with serum free light chain analysis as a first-line test for detecting plasma cell disorders offers increased diagnostic accuracy and potential health benefit to patients. Am J Clin Pathol. 2013; 140(6): 890–897.

The authors
David Baulch* MSc, Beverley Harris MSc, FRCPath
Department of Clinical Biochemistry, Royal United Hospitals Bath NHS Foundation Trust, Bath, UK

*Corresponding author
E-mail: david.baulch@nhs.net

Prednisolone is an attractive once-daily option to treat adrenal insufficiency. Its prior association to osteoporosis and diabetes is possibly due to widespread over-replacement. With the availability of an ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) method to detect serum concentrations and guide treatment, we can assess the true effects of long-term low-dose prednisolone therapy.

by Dr Sirazum Choudhury and Dr Emma Williams

Introduction
Prednisolone is a pioneering synthetic corticosteroid synthesized by Arthur Nobile in 1950 as an anti-arthritic treatment [1, 2]. Sharing a similar structure to cortisol, prednisolone benefits from a longer half-life and increased potency compared to endogenous steroids, owing to a double bond found between C1 and C2 on the first carbocyclic ring (Fig. 1). Prednisolone has proven to be an indispensable anti-inflammatory drug and has long been used in the treatment of many conditions including asthma, inflammatory bowel disease and rheumatoid arthritis.

Use of prednisolone for adrenal insufficiency
More recently prednisolone is gaining traction as an option for glucocorticoid replacement therapy in adrenal insufficiency. There are an estimated 8400 individuals living with the condition in the UK, with an annual incidence of 4.4–6 cases per million in Europe [3]. The challenges of adrenal insufficiency are well characterized. In the era prior to the availability of effective treatment, the associated mortality was 85% in 2 years, and up to 100% in 5 years [4]. Over the last half-century, our increasing understanding of steroids has meant that patients are living longer, with a life expectancy approaching that of the normal population. However, a mortality gap does remain, which may in part be due to incorrect replacement of glucocorticoids, concurrently increasing the risk of diabetes, osteoporosis and cancer.

Oral hydrocortisone is the most commonly prescribed treatment for adrenal insufficiency, but is perhaps not the most ideal [5]. Due to the relatively short half-life, hydrocortisone must be administered three times daily, which can hinder compliance. For this reason it is our experience that some patients tend to omit the last dose of the day. Moreover, the price of hydrocortisone has been rising in the UK, costing £76 for a 1-month supply of 10 mg. This contrasts to a 1-month supply of 5 mg prednisolone tablets, which costs £0.88. With prednisolone offering a once-daily solution to adrenal insufficiency, it now features in the Endocrine Society clinical practice guidelines from earlier this year as an alternative to thrice-daily hydrocortisone therapy [6].

Prednisolone dose and adverse effects
The biggest obstacle to the widespread acceptance of prednisolone as a viable therapy has been its association with adverse metabolic effects such as osteoporosis. This is as a result of multiple studies purporting to show that ‘low dose’ prednisolone has a negative impact of the markers of bone turnover and bone absorptiometry [7]. Based on an assumed bioequivalence ratio of 4 : 1, 7.5 mg of prednisolone was judged to be equivalent to 30 mg of hydrocortisone and was considered ‘low dose’. The basis of this ratio is difficult to ascertain but was probably calculated from data on anti-inflammatory doses of prednisolone, which are significantly higher than the doses likely to be needed in steroid replacement therapy. More recently, a study comparing prednisolone to hydrocortisone in 44 children with congenital adrenal hyperplasia found that a lower dose of prednisolone than expected was required to control the condition [8]. Using objective biological markers, such as growth velocity, and hormonal markers such as androstenedione and 17-hyroxyprogesterone, the group discovered that prednisolone is 1.5 to 2 times more potent than previously thought, suggesting that a more appropriate prednisolone replacement dose is in fact 3 mg to 5 mg, and not as high as 7.5 mg.

To facilitate this shift towards the use of even lower doses of prednisolone, it is important to provide reassurance to both clinicians and patients that the lowest necessary dose of prednisolone is used to maintain an appropriate trough level towards the end of the day. This would be in keeping with the diurnal rhythm of cortisol. Our ability to more accurately and efficiently report serum prednisolone concentrations using an ultra-performance liquid-chromatography (UPLC) tandem mass spectrometry (MS/MS) technique provides this confidence.

Measurement of plasma prednisolone concentrations
Historically, the first assays to measure plasma prednisolone concentrations were competitive protein binding assays and radio-immunoassays [9]. The protein binding assays were designed to use cortisol binding globulin and were therefore non-specific to prednisolone. The early radio-immunoassays were prone to interference from other endogenous steroids and intermediaries, making them unreliable especially if patients continued to produce subclinical levels of cortisol. Specificity could be improved with the addition of a thin layer chromatography preparatory step; however, the lower limit of detection remained as high as 20 µg/L.

In the 1970s, high-performance liquid-chromatography (HPLC) methods gained popularity [10]. Offering greater specificity for prednisolone, the method involved a time-consuming liquid–liquid-extraction sample-preparation step. The extracted organic phase would be dried before being reconstituted with mobile phase and passed through a normal phase hydrophilic interaction chromatography HPLC column. Prednisolone concentrations were detected with ultraviolet absorbance spectrophotometry. Although this method could identify different corticosteroids, it proved to be cumbersome with retention times of up to 8 minutes for prednisolone, and 20 minutes for other steroids. With 76% recovery and a lower limit of detection of 25 µg/L, this technique is not suitable to assess trough levels of prednisolone, with a high likelihood of reporting undetectable results at the lower end, potentially facilitating over-replacement in patients.

Using a UPLC-MS/MS method, we are able to overcome the obstacles that have plagued prednisolone assays in the past (reference awaiting PubMed identifier). Serum samples are prepared using a protein precipitation method, involving zinc sulphate and the addition of deuterated (D6) prednisolone as internal standard. Following preparation, the extract is combined with both methanol and water based mobile phases before being passed through a C-18 chromatography column, which employs a reversed phase partition process. Prednisolone is eluted at approximately 1.0 minutes, before being detected by multiple reaction monitoring using electrospray ionization in positive ion mode. An example of the observed chromatograms can be found in Figure 2.

This method of measuring plasma prednisolone concentrations is linear to prednisolone concentrations of 1000 µg/L (Fig. 3), with an inter- and intra-assay co-efficient of variance at 50 µg/L of 4.1% and 2.5% respectively. The technique has proven more sensitive than HPLC with the lower limit of quantification at 10 µg/L without the HPLC recovery issues, and is equally as specific to prednisolone. By using a protein precipitation method, the preparation step is now significantly shorter. Additionally, with reduced prednisolone retention times, a prepared sample can now be analysed in 3.5 minutes before the next sample is immediately run. As a result, the UPLC-MS/MS technique is better suited to the modern clinical biochemistry laboratory being able to reliably cope with larger numbers of patient samples in shorter times than previously thought possible.

Measuring serum prednisolone concentrations has proven extremely valuable in monitoring glucocorticoid replacement therapy. There is observable variability in prednisolone metabolism between individuals, with terminal half-lives routinely varying between 1.75 and 3.75 hours. We currently measure a trough level at 8 hours post-prednisolone administration aiming for a concentration of 10–20 µg/L to ensure adequate replacement throughout the day and preserve an overnight corticosteroid nadir. The results are used clinically to inform the decision either to increase or decrease prednisolone doses as appropriate but also serve as objective proof to patients who are anxious about a reduction. The assay is also clinically useful in confirming patient compliance with their prescribed medication.

Future perspectives
Beyond the clinical utility in quantifying serum prednisolone levels, there is significant research potential for this assay. Addisonian crises are currently responsible for up to 15% of deaths in patients with adrenal insufficiency [11]. Our understanding of the disease process is limited by the urgency to provide treatment with either intravenous or intramuscular hydrocortisone, before a blood sample is taken. As this is detected by cortisol assays, it is difficult to interpret whether the pre-crisis hydrocortisone concentration was inadequate (suggesting non-compliance or reduced absorption) or appropriate (suggesting that the level was insufficient to match requirement). In patients treated with prednisolone who present with Addisonian crises, the assay will allow us to assess the pre-treatment serum prednisolone concentrations, even if the blood sample is taken after treatment with hydrocortisone.

More importantly in the immediate setting, it is anticipated that the previously accepted long-term effects of ‘low dose’ prednisolone can be explored. The availability of a reliable and specific assay will result in a greater number of patients on prednisolone who are appropriately treated and not over-replaced. In time, as more data becomes available, we will gain a clearer picture of the true effects of prednisolone.

References
1. Nobile A. The discovery of the delta 1,4-steroids, prednisone, and prednisolone at the Schering Corporation (USA). Steroids 1994; 59(3): 227–230.
2. Herzog HL, Nobile A, Tolksdorf S, Charney W, Hershberg EB, Perlman PL. New antiarthritic steroids. Science 1955; 121(3136): 176.
3. Charmandari E, Nicolaides NC, Chrousos GP. Adrenal insufficiency. Lancet 2014; 383(9935): 2152–2167.
4. Dunlop D. Eighty-six cases of Addison’s disease. Br Med J. 1963; 2(5362): 887–891.
5. Groves RW, Toms GC, Houghton BJ, Monson JP. Corticosteroid replacement therapy: twice or thrice daily? J R Soc Med. 1988; 81(9): 514–516.
6. Bornstein SR, Allolio B, Arlt W, Barthel A, Don-Wauchope A, Hammer GD, et al. Diagnosis and treatment of primary adrenal insufficiency: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2016; 101(2): 364–389.
7. Jodar E, Valdepenas MP, Martinez G, Jara A, Hawkins F. Long-term follow-up of bone mineral density in Addison’s disease. Clin Endocrinol. (Oxf) 2003; 58(5): 617–620.
8. Caldato MC, Fernandes VT, Kater CE. One-year clinical evaluation of single morning dose prednisolone therapy for 21-hydroxylase deficiency. Arq Bras Endocrinol Metabol. 2004; 48(5): 705–712.
9. Wilson CG, Ssendagire R, May CS, Paterson JW. Measurement of plasma prednisolone in man. Br J Clin Pharmacol. 1975; 2(4): 321–325.
10. Loo JC, Butterfield AG, Moffatt J, Jordan N. Analysis of prednisolone in plasma by high-performance liquid chromatography. J Chromatogr 1977; 143(3): 275–280.
11. Erichsen MM, Lovas K, Fougner KJ, Svartberg J, Hauge ER, Bollerslev J, et al. Normal overall mortality rate in Addison’s disease, but young patients are at risk of premature death. Eur J Endocrinol. 2009; 160(2): 233–237.

The authors
Sirazum Choudhury BSc, MBBS, MRCP
and Emma Williams* BSc, PhD, FRCPath
Charing Cross Hospital, Fulham Palace Road, London W6 8RF, UK

*Corresponding author
E-mail: emma.walker@imperial.nhs.uk

Cancer of the testicles, primarily the germ cells, is a highly treatable disease common to young men. This article describes how chemical biomarkers are central to the diagnosis, characterization, therapeutic monitoring, prognosis and long-term surveillance in patients with testicular cancer.

by Dr Angela Cooper and Dr Seán Costelloe

Incidence of testicular cancer
Testicular cancer (TC) is relatively rare, accounting for approximately 0.7% of all UK male cancers, with a worldwide incidence estimated as ~7 per 100 000 [1, 2]. Incidence of TC has noticeably increased in industrialized countries over the last few decades, particularly in white males of European descent, although the reasons for this remain unclear [2–5]. Amongst younger men aged between 15 and 49 years in the United Kingdom and the United States of America, TC is the most common type of cancer observed [2, 3, 6, 7].

Classification of TC
Approximately 95% of malignant TCs originate from primordial germ cells, also known as germ cell tumours (GCTs) [3, 7–9]. However, rarely these malignancies may arise from extragonadal primary sites such as the retroperitoneum, mediastinum or pineal gland [3–5, 8, 10]. Germ cell tumours classified as seminomas (~40%) are predominantly formed of uniform cell types, whereas non-seminomatous germ cell tumours (NSGCTs), also accounting for ~40% of GCTs, originate from multiple cell types such as embryonal carcinomas, teratomas, choriocarcinomas and yolk sac carcinomas. GCTs arising from mixed germ cells comprise the remaining 20%. The World Health Organization (WHO) classification system for testicular tumours (Table 1) define five basic GCT types based on histological examination:

• Seminomatous GCTs
• Non-seminomatous GCTs (NSGCTs)
• Embryonal cell carcinomas
• Yolk sac tumours
• Teratomas
• Choriocarcinoma

The vast majority of non-GCTs are sex cord-gonadal stromal tumours involving the Sertoli or Leydig cells of the testicles, and are often benign [8, 9, 11].

‘Burned-out’ GCTs, or spontaneous regression of a testicular GCT, is a very rare phenomenon occasionally observed in male patients presenting with metastatic malignancy with an absence of primary testicular tumour. Often, the only remaining evidence of malignancy are features such as homogeneous scarring, hemorrhage, intratubular calcification and testicular atrophy. This may be associated with choriocarcinomas or teratomas [5, 12].

Testicular GCTs exhibit very diverse histology and immunostaining profiles, and have varying clinical progression and prognosis outcomes as demonstrated by the numerous methods of GCT classification systems. It is outside the focus of this paper to consider histology or immunostaining used in the identification and differentiation of GCTs, as these topics has been extensively documented in other review articles.

Treatment and cure rates in TC
Advances in treatment strategies, such as the use of cisplatin therapies [13], careful staging at diagnosis, early intervention using multidisciplinary teams, rigorous surveillance follow-up, and salvage therapy, means that GCTs are highly curable. Currently, expected cure rates of 95% are observed in patients who receive a TC diagnosis, and cure rates of 80% in patients with a diagnosis of metastatic TC [3, 13].

Causes and presentation of TC
The causes of TC cancer are still unknown, although cryptochordism is the best-characterized risk factor associated with TC. Research has shown that timing of orchiopexy impacts on future risk of TC development, suggesting hormonal changes during puberty are strongly associated with TC etiology in males. However, prenatal risk factors, environmental exposures in adulthood, male infertility, certain genetic or congenital disorders such as Down’s syndrome, Klinefelter’s syndrome, human immunodeficiency virus infection and intersex patients have also been associated with an increased TC risk [3, 5, 7].

Presentation of TC is often a painless lump in the testis body, but due to a frequent lack of pain, medical opinion is frequently delayed. A testicular mass or swelling, or episodic diffuse pain may be observed. More rarely, metastatic symptoms such back pain arising from retroperitoneal lymph node involvement, or coughing, pain or hemoptysis due to lung metastasis may be reported [3, 7, 8].

Diagnosis and staging of TC
Clinical suspicion of TC, such as altered testicular shape or non-painful swelling, should prompt a full physical examination and patient history, imaging to include testicular and abdominal ultrasound, as well as chest X-ray [14]. If metastasis is suspected, chest, abdominal and brain computerized tomography (CT), and bone scintigraphy should be undertaken [9]. Final diagnosis and prognosis requires biopsy sampling for histology and immunostaining profiling as appropriate, and in the majority of cases, treatment options should be based on the histology results [10]. Biochemical analysis should include initial concentrations of serum tumour markers (STMs). Metabolic biochemistry, liver function tests and a full blood count should be undertaken to determine general organ function, and may demonstrate evidence of metastasis [9].

This collective information can be used to reference the Tumour-node-metastasis (TNM) Classification of Malignant Tumours staging system (Table 2). This cancer staging system is based on primary tumour site, nearby lymph node involvement, and presence of distal metastatic spread from initial primary tumour site [4, 15]. The use of STMs as a fourth staging system has added diagnostic and prognostic value, independent of the TNM system (Table 3) [9]. The decision for chemotherapy or radiotherapy treatment for non-surgical metastatic disease is based on CT and/or magnetic resonance imaging (MRI) results, and concentrations of STMs [4].

The majority of patients (~75%) presenting with a testicular mass are diagnosed at stage 1 [7, 8]. At this stage, treatment options are typically surgery with an excellent cure rate. For metastatic disease, combinations of surgery, chemotherapy or radiotherapy are required depending on cancer mass, location and distal lymph node involvement [13]. Greater than 80% of patients with metastatic GCTs are successfully treated and cured.

Treatment of TC
TC cells are extremely sensitive to chemotherapy [9, 10]. Specifically, the standard chemotherapy regime consists of 3 or 4 cycles of bleomycin, etoposide and cisplatin (BEP) chemotherapy, or etoposide and cisplatin (EP) chemotherapy every 21 days [8, 9]. Surgery may be considered to remove residual masses post-chemotherapy. Data suggests a higher relapse rate in patients with NSGCTs than seminomas following an initial chemotherapy regime. This relapse rate can be used to further classify patients into good, intermediate and poor prognostic groups, using a combination of STM concentrations and location of primary tumour or metastases. Around 50–99% of patients can still expect to survive [8].

Salvage therapy, often in combination with chemotherapy, is reserved for patients who have relapsed, or for patients where cancer progression continues after following a standard chemotherapy regime. High-dose chemotherapy with autologous bone marrow transplant is a controversial approach for patients with a poor prognosis, and where a standard chemotherapy regime and salvage therapy has been unsuccessful. Initial studies are encouraging but further trials are required. A small cohort of patients have been identified who suffer a late relapse, i.e. >2 years post-diagnosis but also potentially ≥10 years post-diagnosis. These patients are less responsive to chemotherapy, so are treated primarily with surgery. Unfortunately, less than half will remain disease-free following surgical intervention [8, 9]. Chemotherapy-induced side effects are governed by the dose and combination of drugs used. This has triggered more recent trials designed at maintaining a cure rate but with reduced associated chemotoxicity [8].

The use of serum tumour markers in TC
The discovery of serum and urine tumour markers and the advent of chemotherapy have significantly improved cancer staging, management and prognosis in patients with TC. The benefit of initial STMs is predominantly with regard to disease staging, whereas serial STMs are particularly useful for monitoring response to treatment after surgery, chemotherapy or radiation therapy. STMs are useful because they are often detectable well before clinical radiological detection in patients. Furthermore, concentrations can be helpful to differentiate GCT type. The detection of at least one elevated STM occurs in ~85% of NSGCTs, and the presence of elevated STMs occurs in significant numbers of pure seminoma cases [9, 10]. However, in rare cases where patients present with evidence of a testicular mass, radiographic evidence of metastatic disease, with significantly elevated alpha-fetoprotein (AFP) or human chorionic gonadotrophin (hCG) serum concentrations, it is advised that treatment is not delayed while awaiting histology results [10].

The American Society of Clinical Oncology recommend against using STMs as a screening test for GCTs in asymptomatic males. Given the low incidence and mortality of TC combined with the high cure rate, it is suggested a screening programme would be neither cost-effective nor decrease mortality [10]. Furthermore, although STMs can be helpful in combination with imaging techniques in the diagnosis of TC, normal STMs alone do not exclude TC and may also be raised in other conditions [3, 8–10]. Routine testicular examination via palpation is recommended in all males from puberty up to ~45 years. This is of particular importance for males with a past medical history that may suggest an increased GCT risk as detailed previously.

Commonly employed serum markers include: AFP and hCG as mentioned previously, hCG beta-subunit (hCGb), placental alkaline phosphatase (PLAP) and lactate dehydrogenase (LDH). Alpha-fetoprotein levels are elevated in teratocarcinoma or testicular embryonal carcinoma, while conversely, AFP is never elevated in pure seminomas. Human chorionic gonadotrophin elevations are associated with 10–15 % of pure seminomas. Lactate dehydrogenase is an enzyme found in all cell types, meaning it is less specific for TC, although it does have prognostic value in advanced stage GCTs [3, 9]. A decline in serial STM concentrations is useful to detect the presence of residual disease following surgery, or to assess response to chemotherapy. In both scenarios, the decline in STM concentrations should follow the half-lives of each marker [9].

There are detailed STM surveillance guidelines in place following surgery, which recommend a meticulous timetable of STM measurements and radiology imaging to detect disease recurrence depending on initial GCT type, thereby avoiding relapse and presentation at a later date with advanced stage disease [8, 9].

Future focus
While the majority of patients diagnosed with TC will survive, challenges still persist. Serum tumours markers have been pivotal to improved outcomes for patients with and without metastatic disease. Future research is focused on patients with an initial poorer prognosis, patients who have relapsed following first-line chemotherapy and patients who have a late relapse. Long-term health consequences for patients surviving TC, in particular side effects associated with chemotherapy and radiotherapy such as cardiovascular disease, impaired fertility and secondary cancers, continues to drive collaborative studies nationally and internationally to improve TC outcomes for the future.

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The authors
Angela Cooper* PhD, Seán Costelloe, PhD
Derriford Combined Laboratory, Plymouth Hospital NHS Trust, Plymouth, UK

*Corresponding author
E-mail: angelacooper5@nhs.net