Antiepileptic drugs (AEDs) are widely used and their number is steadily increasing. Therapeutic drug monitoring of AEDs, when performed correctly, can be a valuable tool for the treating physician. This article describes the indications, limitations and pitfalls that must be observed when measuring and interpreting AED serum concentrations.
by Dr Arne Reimers and Prof. Eylert Brodtkorb
Why measure antiepileptic drug serum concentrations?
Antiepileptic drugs (AEDs) are widely used, not only for epilepsy, but also for a range of non-epilepsy conditions, such as bipolar (manic-depressive) disorder, migraine and neuropathic pain [1]. Thus, the total number of AED users substantially exceeds the number of people with epilepsy. Therapeutic drug monitoring (TDM) has for many years been used to support AED treatment, as many of these drugs have unfavourable pharmacokinetic properties, a potential to problematic drug interactions as well as narrow therapeutic windows. TDM is a means of assisting clinical decision-making and should always be done with a specific question in mind. The general indications for TDM of AEDs are listed in Table 1.
Non-linear and linear pharmacokinetics
TDM of AEDs has a long clinical tradition. When the concept of TDM was introduced in the early 1970s, phenytoin was one of the first drugs to which it was applied [2]. This was mainly because phenytoin, then one of the most frequently used AEDs, has so-called non-linear pharmacokinetics. Linear kinetics means that the serum concentration is linearly correlated with dose – a doubling of the dose will double the serum concentration. This applies to almost all medicinal drugs. However, some drugs exhibit non-linear or saturation kinetics; phenytoin is one of them. Doubling the phenytoin dose may result in an unpredictable increase of the serum concentration. Thus, monitoring the phenytoin serum concentration was desirable and soon became available in large parts of the world.
Most other AEDs, however, exhibit linear kinetics. Why then is it important to measure their serum concentrations? One reason is the nature of epilepsy itself and the issue of prophylactic treatment. The only clinical marker for successful management is the extent of seizure control. However, epileptic seizures may occur in random patterns. The intervals between seizures may be minutes or months, and if a seizure occurs, it may have dramatic consequences, not only for the patient, but even for others. Thus, it can be very demanding to evaluate the therapeutic effect of AED treatment by clinical observation alone.
Absorption, distribution, metabolism and excretion
In addition, the pharmacokinetics of AEDs may be affected by changes in absorption, distribution, metabolism and excretion (ADME). Co-morbidity, pregnancy, drug interactions, pharmacogenetic polymorphisms, etc, all may considerably affect the ADME of AEDs (Fig. 1). Pregnancy may induce pronounced pharmacokinetic alterations, including increased volume of distribution, elevated renal clearance, and induction of hepatic metabolism. Breakthrough seizures in previously seizure-free patients may occur [3–5].
The serum concentration of carbamazepine may rise threefold and produce toxic symptoms when the patient is prescribed certain antibiotics which inhibit its metabolism, such as erythromycin. On the other hand, carbamazepine and other inducers of hepatic metabolism, may reduce serum concentrations of several other drugs, among them valproate, lamotrigine and hormonal contraceptives. Valproate is also a potent inhibitor of drug-metabolizing liver enzymes and may double lamotrigine concentrations. The clinically important induction of the metabolism of lamotrigine by combined oral contraceptives was detected by routine use of TDM [6]. Gabapentin is excreted almost exclusively by the kidneys; hence reduced kidney function will give increased serum concentrations.
Adherence
Poor adherence to prescribed treatment is one of the most important obstacles to the management of epilepsy [7, 8]. It has been documented that roughly half of all patients take their medicine more or less irregularly [9]. A recent study in patients admitted to hospital with acute epileptic seizures found that almost 40 % had less than 75 % of their usual trough AED serum concentration, indicating one or more missed doses [8] (Fig. 2). In such situations, it is crucial that the treating clinician receives the lab result as soon as possible to be able to decide on how to proceed with the management of the patient. Should the daily AED dose be increased or not? In the event that the seizure occurred because of a missed intake, it would not be appropriate; dose increase could even be harmful to the patient. If the serum concentration was adequate (according to prescribed dose), the occurrence of a seizure would suggest that the daily dose was too low and should be increased. This decision must be made quickly as the patient usually will be dismissed from hospital the next morning. It is essential to identify pseudo-refractory epilepsy. Clinically unrecognized non-adherence is often mistaken as drug-resistant epilepsy [10].
How it is normally done
The common convention is that blood samples for measuring the concentration of AEDs be taken drug-fasting in the morning (i.e. from 12 h to a maximum of 24 h after the last dose intake, and before the morning dose). Also, the patient must be in pharmacological steady state. This means that the amount of drug administered per unit time is in equilibrium with the amount of drug eliminated from the body during the same time. For all drugs, this state is reached after five times the drug’s plasma half-life. These rules apply after every dose change (Fig. 2E). The difficulties in complying with these rules are an important obstacle to TDM and is one major reason its routine use is discredited in many parts of the world. If a blood sample is taken before steady state is reached, or when the patient is not drug-fasting, the interpretation of the measured blood concentration is tricky and requires profound clinical-pharmacological experience.
Most commonly, the analyses are performed in a central lab using serum or plasma, either with immunologic or chromatographic methods. Usually, the total AED concentration (protein-bound plus unbound drug) is measured. In certain situations, e.g. in the elderly with hypoalbuminemia or in pregnant women, it is desirable to measure the unbound (free) proportion of an AED. This applies mainly to valproate and phenytoin which are >90 % protein bound. Hypoalbuminemia may cause signs of overdose despite only modest total AED concentration. However, unbound concentrations are rarely requested and not offered by all labs.
Reference ranges for antiepileptic drugs
It must be noted that reference ranges (RRs) for AEDs apply to the treatment of epilepsy. RRs for bipolar disorder have been suggested [11] but are not broadly established, whereas in the treatment of chronic pain states, treatment is usually guided by the clinical response alone. Unfortunately, with few exceptions, most RRs are not well documented. The exceptions are those AEDs that have been around for decades, e.g. phenytoin, carbamazepine and valproate. For them, broadly accepted RRs are supported by long clinical experience.
For the newer AEDs (introduced after 1990), there is a considerable lack of data. One of the reasons for the poor documentation is that drug manufacturers rarely publish serum concentrations obtained in clinical phase III or IV studies. Another reason is a lack of studies specifically aimed at examining the correlation between serum concentrations and effect. Thus, RRs for AEDs are often based on extrapolation of pharmacokinetic data obtained in preclinical studies, or on data from large routine databases, i.e. by applying some sort of population kinetics. Such data often lack clinical correlates owing to incomplete information provided on the request forms.
One consequence of the above is that the RRs used by different labs, and reported in the literature, are often incoherent. Another weakness of these population-based RRs is the fact that many patients achieve a satisfactory therapeutic effect with serum concentrations below the RR, while others need concentrations above the RR, yet without suffering symptoms of overdose. This is also the reason why the term ‘therapeutic range’ should not be used; it wrongly implies that any concentration outside that range is ‘non-therapeutic’.
The concept of individual RRs where each patient serves as his/her own reference [12] is an alternative approach. An obvious prerequisite for this concept is the availability of several consecutive serum concentration measurements (within reasonable time intervals) in the individual patient as well as close clinical follow-up, to correlate various serum concentrations with their corresponding clinical effect. It would also be desirable to have non-sufficient concentrations as well as toxic concentrations. Most of these individual therapeutic ranges would fall within the population-derived RRs. However, as mentioned above, some patients respond well to concentrations outside the common RR. For the sake of clarity, it has been suggested that such individual RRs be called individual therapeutic ranges [13]. Despite its advantages, neither the concept itself nor the term individual therapeutic range can be regarded as generally established.
Concluding remarks
TDM of AEDs is controversial, as it has been repeatedly emphasized that ‘treating patients is more important than treating blood levels’ [14]. Clinical evaluation and follow-up will continue to be the leading element in the management of epilepsy.
Nevertheless, when correctly applied, appropriately sampled and analysed, as well as correctly interpreted, TDM stands out as an important and relatively inexpensive tool for optimizing the drug treatment of epilepsy. Obviously, blinding for the actual serum concentrations may have severe untoward consequences in specific patient populations, such as pregnant women and patients with poor medication-taking behaviour.
References
1. Johannessen Landmark C. Antiepileptic drugs in non-epilepsy disorders: relations between mechanisms of action and clinical efficacy. CNS Drugs 2008; 22(1): 27–47.
2. Richens A. Drug estimation in the treatment of epilepsy. Proc R Soc Med 1974; 67(12 Pt 1): 1227–1229.
3. Cappellari AM, Cattaneo D, Clementi E, Kustermann A. Increased levetiracetam clearance and breakthrough seizure in a pregnant patient successfully handled by intensive therapeutic drug monitoring. Ther Drug Monit 2015; 37(3): 285–287.
4. Reimers A, Helde G, Becser Andersen N, Aurlien D, Surlien Navjord E, Haggag K, Christensen J, Lillestølen KM, Nakken KO, Brodtkorb E. Zonisamide serum concentrations during pregnancy. Epilepsy Res 2018; 144: 25–29.
5. Voinescu PE, Park S, Chen LQ, Stowe ZN, Newport DJ, Ritchie JC, Pennell PB. Antiepileptic drug clearances during pregnancy and clinical implications for women with epilepsy. Neurology 2018; 91(13): e1228–1236.
6. Sabers A, Buchholt JM, Uldall P, Hansen EL. Lamotrigine plasma levels reduced by oral contraceptives. Epilepsy Res 2001; 47(1–2): 151–154.
7. Faught E. Adherence to antiepilepsy drug therapy. Epilepsy Behav 2012; 25(3): 297–302.
8. Samsonsen C, Reimers A, Bråthen G, Helde G, Brodtkorb E. Nonadherence to treatment causing acute hospitalizations in people with epilepsy: an observational, prospective study. Epilepsia 2014; 55(11): e125–128.
9. Adherence to long-term therapies: evidence for action World Health Organization 2003; http://www.who.int/chp/knowledge/publications/adherence_report/en/.
10. Brodtkorb E, Samsonsen C, Sund JK, Bråthen G, Helde G, Reimers A. Treatment non-adherence in pseudo-refractory epilepsy. Epilepsy Res 2016; 122: 1–6.
11. Hiemke C, Bergemann N, Clement HW, Conca A, Deckert J, Domschke K, Eckermann G, Egberts K, Gerlach M, et al. Consensus guidelines for therapeutic drug monitoring in neuropsychopharmacology: update 2017. Pharmacopsychiatry 2018; 51(1–02): 9–62.
12. Landmark CJ, Johannessen SI, Tomson T. Dosing strategies for antiepileptic drugs: from a standard dose for all to individualised treatment by implementation of therapeutic drug monitoring. Epileptic Disord 2016; 18(4): 367–83.
13. Patsalos PN, Berry DJ, Bourgeois BF, Cloyd JC, Glauser TA, Johannessen SI, Leppik IE, Tomson T, Perucca E. Antiepileptic drugs – best practice guidelines for therapeutic drug monitoring: a position paper by the subcommission on therapeutic drug monitoring, ILAE Commission on Therapeutic Strategies. Epilepsia 2008; 49(7): 1239–1276.
14. Chadwick DW. Overuse of monitoring of blood concentrations of antiepileptic drugs. Br Med J (Clin Res Ed) 1987; 294(6574): 723–724.
The authors
Arne Reimers*1,2 MD PhD and Eylert Brodtkorb3,4 MD PhD
1Dept. of Clinical Chemistry and Pharmacology, Division of Laboratory Medicine, Skåne University Hospital, Lund, Sweden
2Department of Clinical Chemistry and Pharmacology, Lund University, Lund, Sweden
3Dept. of Neuromedicine and Movement Science, Norwegian University of Science and Technology (NTNU), Trondheim, Norway
4Dept. of Neurology and Clinical Neurophysiology, St. Olavs University Hospital, Trondheim, Norway
*Corresponding author
E-mail: arne.reimers@med.lu.se
Hereditary hemochromatosis type 1 is a disease of iron overload caused predominantly by a mutation in the homeostatic iron regulator (HFE) gene, p.Cyst282Tyr (p.C282Y). The incidence of the mutation is most common in people of northern European descent – with 1 in 8 people being carriers, making it the most common genetic condition in this population. Approximately 1 in 150 people are homozygotes, although a previous study suggested that only about 1% of homozygotes went on to develop “frank clinical hemochromatosis” involving liver disease. The overload of iron results in iron deposition in the liver, pancreas and joints, causing liver disease (cirrhosis and cancer), fatigue, diabetes and arthritis. Diagnosis if often missed or delayed because of the insidious onset of symptoms that often only become apparent later in life and which can easily be attributed to other causes. Currently, if hemochromatosis is suspected, diagnosis is made by testing for high blood iron levels. Genetic screening is limited only to close family members of hemochromatosis patients because of the suggestion of low general penetrance of the disease. The damaging effects of iron overload can be easily prevented if the disease is diagnosed early enough, largely by withdrawing blood on a regular basis. However, a recent study by Pilling et al. of nearly 500 000 UK Biobank volunteers is changing the way we think about the condition (Pilling LC, et al. Common conditions associated with hereditary haemochromatosis genetic variants: cohort study in UK Biobank. BMJ 2019; 364: k5222). This study involved a far larger number of people than previous studies, as well as involving older people – important for monitoring a disease where the effects are cumulative. The authors found a much higher prevalence of hemochromatosis and associated conditions than expected. Of the p.C282Y homozygous participants, 21.7% of men and 9.8% of women were eventually diagnosed with hemochromatosis. The results of this study have prompted the UK National Screening Committee to announce that it will review the evidence for hemochromatosis screening at its next routine review. However, in the meantime, we are actually in the fortunate position that this disease is easy to test for and easy to treat. No new methodology is needed, but simply a change in pathway, as advocated by Dr Ted Fitzsimons (consultant hematologist at Gartnavel Hospital, Glasgow, UK): if the results of a serum ferritin test are high and the patient is of northern European descent, the blood iron levels should automatically be tested. If this result is also high, then the patient should be screened for hemochromatosis. Many people have a lot to gain from this simple change.
For decades, attempts to biopsy or obtain fluid from eyes with retinoblastoma had been contraindicated, however recent changes in the management of retinoblastoma have allowed for safe sampling of the aqueous humour (AH) during therapy. Use of the AH as a liquid biopsy enables tumour biomarker analysis in these eyes; this has potential to dramatically alter the management of this pediatric cancer.
by Dr Benjamin K. Ghiam, Dr Liya Xu and Dr Jesse L. Berry
Introduction
Retinoblastoma (Rb) is the most common intraocular cancer in children, comprising 4 % of all pediatric malignancies [1, 2]. This potentially fatal malignancy often goes undiagnosed until the tumour is advanced and has damaged intraocular structures. Survival rates for Rb are in excess of 90 % in developed countries, though a critical, and often challenging, focus of Rb therapy is globe and vision preservation [3]. Throughout decades of ocular medicine and surgery, any attempt to biopsy these tumours, or even obtain fluid from Rb eyes had been fervently contraindicated for risk of tumour seeding and dissemination. Thus, much of the diagnosis and management of Rb is dependent on information gathered by the ophthalmologist through careful eye examination, and without histopathologic evidence.
In 2012, Munier et al. described a safety-enhanced protocol for intravitreal chemotherapy injections in the eyes of patients with Rb; this protocol requires an initial paracentesis [4]. As described by the authors of the study, a volume of 0.1 ml of aqueous fluid is aspirated to induce transient hypotony before the intravitreal injection as a safety measure to prevent reflux to the injection site. This protocol for intravitreal injection of chemotherapy has now been widely adopted worldwide and the risk of extraocular spread is considered extremely low (zero reported cases with the safety-enhanced procedure) [5]. This demonstrated safety record paved the way for aqueous humour (AH) extraction in eyes with Rb undergoing active therapy.
AH is the clear intraocular fluid produced by the ciliary processes that fills the front part of the eye (anterior chamber). The AH functions to maintain intraocular pressure, provide nutrients to the cornea, and remove waste products. It has also been shown to be a rich source of information for intraocular disease, including Rb [6]. Researchers have long sought to evaluate AH for the presence of biomarkers which may correlate with features of intraocular disease and provide diagnostic and prognostic value. However, before 2017, any evaluation of the AH was only done on eyes after enucleation. Now that the AH can be safely extracted during therapy, we hypothesized that previous evaluations of AH biomarkers (post-enucleation) may now be clinically applicable for the diagnosis, prognosis and/or management of Rb. This article excerpts our recently published systematic review, titled “Aqueous Humor Biomarkers for Retinoblastoma, a review” in the journal Translational Vision Science and Technology [7].
Lactate dehydrogenase
Lactate dehydrogenase (LDH) is an enzyme found in nearly all cells that acts as a regulator of metabolism; it has been used clinically as a non-specific marker found within body fluids in various pathological conditions, including malignant tumours.
In the early 1970s, Dias et al. examined LDH levels in the AH from enucleated Rb eyes [8]. Early reports demonstrated a significant increase in the levels of LDH within the AH of enucleated eyes with Rb when compared to patients without Rb, such that levels >1000 U/L strongly support the diagnosis of Rb (Table 1). Multiple studies on LDH levels in the AH from enucleated eyes were done between the years 1971 and 2008 which found that LDH levels were significantly elevated compared to controls, and more elevated in advanced eyes with delayed diagnosis; however, these levels did not correlate with other clinical features or outcomes. Elevation in AH LDH have been described in patients with other ocular conditions, including primary open angle glaucoma and Coats’ disease. Although LDH was the first described marker of tumour activity in the AH, the lack of specificity and correlation with patient or tumour features limits its use clinically. Owing to this lack of correlation this research was previously abandoned.
Enolase/neuron-specific enolase
Neuron-specific enolase (NSE) is an isoenzyme of the glycolytic enzyme enolase; it is highly specific for neurons and peripheral neuroendocrine cells. Increased body fluid levels of NSE occur with malignant proliferation and thus have been of value in the diagnosis and characterization of neuroendocrine tumours, including small cell lung cancer and retinoblastoma [9].
Evaluation of the isoenzyme patterns of enolase in the AH of enucleated Rb eyes demonstrated that NSE levels were elevated in AH Rb, whereas enolase was not detectable in the AH from controls (Table 1) [10–12]. Elevated levels of NSE significantly correlated with inflammation and tumour invasion into the anterior chamber [13]. NSE levels did not correlate with histological tumour parameters (tumour necrosis, calcification, optic nerve/choroidal invasion) as well as clinicopathological parameters (sex, enucleation age, presentation age, family history, previous treatment, and metastatic disease). Moreover, NSE levels were found to be within the control range in children more than 5 years after active therapy [14]. This suggests that NSE may be used clinically to indicate remission status. Although obtaining serial AH NSE measurements may have a significant role in determining tumour status in Rb patients in the future, additional evidence is required to further substantiate the use of this tumour marker clinically.
Surviving and transforming growth factor beta-1
Survivin is a protein that inhibits apoptosis. It has garnered significant interest as a diagnostic and prognostic factor in human neoplasms, including Rb. Elevated survivin levels are found in many human neoplasms, and it is used as a prognostic factor in several human neoplasms, including lung and colorectal cancers [15, 16]
Survivin expression in the AH from enucleated eyes of children with Rb was found to be significantly elevated, when compared to patients with non-malignant ophthalmic disease, such as congenital cataracts and glaucoma [17, 18]. AH survivin levels correlated with tumour stage and histopathologic post laminar optic nerve involvement.
Transforming growth factor beta-1 (TGF-β1) expression in the AH of enucleated Rb eyes was associated with poor differentiation of the tumour [17]. The authors demonstrated high sensitivity and specificity of these AH proteins which makes them promising markers for Rb, particularly of more aggressive pathologic features.
Uric acid and xanthine
During cell turnover, nucleic acids and nucleotides are degraded into xanthine and uric acid. Elevated levels of serum uric acid have been associated with many malignancies, as well as after rapid destruction such as after treatment with chemotherapy or radiation.
Elevated concentrations of uric acid and xanthines were found in the AH of children with Rb compared with control eyes (Table 1) [19]. Elevated levels of xanthine and uric acid in AH may support the diagnosis of Rb in children suspected of having the disease, however further studies are necessary to establish optimal cut-offs, explore clinicopathological correlations, and compare Rb levels to lesions simulating Rb (Coats’ disease and persistent fetal vasculature).
Protein content
Normally, the AH is virtually protein-free to ensure a clear optical media between the cornea and the lens. An increase in globulin content and an albumin/globulin ratio < 1 has been found in enucleated eyes with Rb [20]. Concentrations of interleukin (IL)-6, IL-7, IL-8, interferon gamma (IFN-γ), placental growth factor 1 (PlGF-1), vascular endothelial growth factor A (VEGF-A), beta-nerve growth factor (β-NGF), hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2) were significantly higher in the AH of patients with Rb than those in the control group [21]. Additionally, significantly decreased protein concentration was demonstrated in Rb eyes following treatment with selective intra-arterial chemotherapy (melphalan injection in the ophthalmic artery) that were subsequently enucleated after attempts at salvage, compared to primarily enucleated eyes [22].
Nucleic acids
Recent studies from Berry et al. demonstrated the presence of tumour-derived nucleic acids (DNA, RNA, miRNA) in the AH of Rb eyes [23]. Because of this, the authors suggest that the AH may be a rich source of tumour DNA and, thus, could be used as a liquid biopsy in children with Rb, without undergoing enucleation. A subsequent analysis by Berry et al. in 2018 showed that evaluation of the cell-free DNA (cfDNA) in the AH for chromosomal alterations has potential prognostic value as in indicator of aggressive disease [24]. Specifically, there was a significant increased odds of an eye failing therapy and requiring enucleation due to persistent and/or progressive cancer activity if gain of chromosome 6p was found in the AH cfDNA. Further research is required before this can be applied clinically, however this holds potential as a prognostic biomarker for Rb.
Conclusion
Despite significant investigation into tumour biomarkers for Rb spanning more than four decades, currently there are no active uses for the AH in a clinical setting. Diagnosis is made on the basis of examination and ancillary imaging findings without a biopsy, and molecular tumour markers are presently not used for diagnosis, prognosis, or to monitor therapeutic response. This is due in large part to the contraindication to biopsy in Rb; therefore, previously neither tumour nor AH or other ocular fluids were evaluated outside of specimens from enucleated eyes; clearly this limited the ability to correlate these markers with meaningful clinical outcomes. However, with recent advances in local therapy for Rb, paracentesis with extraction of the AH has now been shown to be safe in eyes being actively treated. This opens the door to for an AH liquid biopsy and thus there is renewed interest in these potential disease biomarkers.
Acknowledgement
This article excerpts our recently published systematic review, titled “Aqueous Humor Biomarkers for Retinoblastoma, a review” in the journal Translational Vision Science and Technology [7].
References
1. Shields, JA. Management and prognosis of retinoblastoma. In: Intraocular tumors: a text and atlas, pp377–391. WB Saunders 1992. ISBN 978-0721642680.
2. Shields JA, Shields CL. Intraocular tumors: an atlas and textbook, p574. Lippincott Williams & Wilkins 2008. ASIN B00XWR8WM6.
3. Pavan-Langston D. Manual of ocular diagnosis and therapy, p533. Lippincott Williams & Wilkins 2008. ISBN 978-0781765121.
4. Munier FL, Soliman S, Moulin AP, et al. Profiling safety of intravitreal injections for retinoblastoma using an anti-reflux procedure and sterilisation of the needle track. Br J Ophthalmol 2012; 96(8): 1084–1087.
5. Smith SJ, Smith BD, Mohney BG. Ocular side effects following intravitreal injection therapy for retinoblastoma: a systematic review. Br J Ophthalmol 2013; 98(3): 292–297.
6. Macknight AD, McLaughlin CW, Peart D, et al. Formation of the aqueous humor. Clin Exp Pharmacol Physiol 2000; 27(1-2): 100–106.
7. Ghiam BK, Xu L, Berry JL. Aqueous humor markers in retinoblastoma, a review. Transl Vis Sci Technol 2019; 8(2): 13.
8. Dias PL, Shanmuganathan SS, Rajaratnam M. Lactic dehydrogenase activity of aqueous humour in retinoblastoma. Br J Ophthalmol 1971; 55(2): 130–132.
9. Kivelä T. Neuron-specific enolase in retinoblastoma. Acta Ophthalmol 2009; 64(1): 19–25.
10. Wu Z, Yang H, Pan S, et al. Electrophoretic determination of aqueous and serum neuron-specific enolase in the diagnosis of retinoblastoma. Yan Ke Xue Bao 1997; 13(1): 12–16.
11. Shine BS, Hungerford J, Vaghela B, et al. Electrophoretic assessment of aqueous and serum neurone-specific enolase in retinoblastoma and ocular malignant melanoma. Br J Ophthalmol 1990; 74(7): 427–430.
12. Nakajima T, Kato K, Kaneko A, et al. High concentrations of enolase, alpha- and gamma-subunits, in the aqueous humor in cases of retinoblastoma. Am J Ophthalmol 1986; 101(1): 102–106.
13. Abramson DH, Greenfield DS, Ellsworth RM, et al. Neuron-specific enolase and retinoblastoma. Clinicopathologic correlations. Retina 1989; 9(2): 148–152.
14. Comoy E, Roussat B, Henry I, et al. Neuron-specific enolase in the aqueous humor. Its significance in the differential diagnosis of retinoblastoma Ophtalmologie 1990; 4(3): 233–235 [in French].
15. Andersen MH, Svane IM, Becker JC, et al. The universal character of the tumor-associated antigen survivin. Clin Cancer Res 2007; 13(20): 5991–5994.
16. Rohayem J, Diestelkoetter P, Weigle B, et al. Antibody response to the tumor-associated inhibitor of apoptosis protein survivin in cancer patients. Cancer Res 2000; 60(7): 1815–1817.
17. Shehata HH, Abou Ghalia AH, Elsayed EK, et al. Clinical significance of high levels of survivin and transforming growth factor beta-1 proteins in aqueous humor and serum of retinoblastoma patients. J AAPOS 2016; 20(5): 444.e1–444.e9.
18. Shehata HH, Abou Ghalia AH, Elsayed EK, Z et al. Detection of survivin protein in aqueous humor and serum of retinoblastoma patients and its clinical significance. Clin Biochem 2010; 43(4-5): 362–366.
19. Mendelsohn ME, Abramson DH, Senft S, et al. Uric acid in the aqueous humor and tears of retinoblastoma patients. J AAPOS 1998; 2(6): 369–371.
20. Dias PL. Postinflammatory and malignant protein patterns in aqueous humour. Br J Ophthalmol 1979; 63(3): 161–164.
21. Cheng Y, Zheng S, Pan C-T, et al. Analysis of aqueous humor concentrations of cytokines in retinoblastoma. PLoS One 2017; 12(5): e0177337.
22. Hadjistilianou T, Giglioni S, Micheli L, et al. Analysis of aqueous humour proteins in patients with retinoblastoma. Clin Experiment Ophthalmol 2012; 40(1): e8–15.
23. Berry JL, Xu L, Murphree AL, et al. Potential of aqueous humor as a surrogate tumor biopsy for retinoblastoma. JAMA Ophthalmol 2017; 135(11): 1221–1230.
24. Berry JL, Xu L, Kooi I, et al. Genomic analysis of aqueous humor cell-free dna in retinoblastoma predicts eye salvage: the surrogate tumor biopsy for retinoblastoma. Mol Cancer Res 2018; 16: 1701–1712.
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The authors
Benjamin K. Ghiam1 MD, Liya Xu2 PhD, Jesse L. Berry, MD*3,4 MD
1Oakland University, William Beaumont School of Medicine, Rochester, MI, USA
2Department of Biological Sciences, Dornsife College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, CA, USA
3The Vision Center at Children’s Hospital Los Angeles, Los Angeles, CA, USA
4USC Roski Eye Institute, Keck School of Medicine of USC, University of Southern California (USC), Los Angeles, CA, USA
*Corresponding author
E-mail: Jesse.Berry@med.usc.edu
Mesothelioma is a fatal cancer of mesothelial cells caused by previous asbestos exposure. Numerous biomarkers have been tested for their ability to diagnose or monitor pleural mesothelioma, but none are in routine clinical practice. This article aims to briefly outline the literature to date and future research directions.
by Dr David T. Arnold and Prof. Nick A. Maskell
Introduction
Mesothelioma is a cancer of mesothelial cells that carries a very poor prognosis. It is almost exclusively caused by previous inhalation of asbestos fibres, which is usually through industrial employments (ship building, lagging, railway work, etc) or from working on pre-existing asbestos products (plumbing, carpentry, etc). Given there is a 40-year mean latency from exposure to presentation, the European incidence of mesothelioma is expected to rise until around 2020, in keeping with the use and subsequent banning of asbestos in the 1980s [1]. However, given ongoing unregulated use of asbestos in China, India and Russia, cases of mesothelioma will continue to occur worldwide.
Mesothelioma can occur in the pleural cavity and peritoneum (with a 4 : 1 ratio) and more rarely in the pericardium and tunica vaginalis. Malignant pleural mesothelioma (MPM) is the most common with around 2500 new cases in the UK every year and will form the basis of the rest of this review [2].
Survival from MPM is dependent on histological subtype, of which there are four: epithelioid, sarcomatoid, biphasic and desmoplastic. Epithelioid accounts for 70 % of the overall cases and has the best prognosis, with a median survival of 13–14 months. Sarcomatoid has the worst prognosis, at 4 months, and is usually felt by clinicians to be not amenable to therapy.
Presentation
The main symptoms from MPM are shortness of breath, cough and chest pain. Given that it is a highly metabolically active tumour, patients can also develop systemic symptoms of fevers/sweats, fatigue and weight loss, indicating a more advanced stage. Around 90 % of individuals with MPM present with a pleural effusion (fluid collection around the lung), and any male with a history of asbestos exposure and a unilateral effusion has a 60 % of having malignancy [3]. MPM is highly locally invasive, which can cause chest pain, but rarely metastasises unless the pleura is disrupted by diagnostic or therapeutic procedures, which can cause tract metastases.
Diagnosis and imaging
Patients who present with a pleural effusion will invariably have cytological analysis of the fluid first. However, pleural fluid cytology alone is not usually sufficient to make a diagnosis of MPM [3]. If the patient is well enough then a biopsy is performed either radiologically, via medical thoracoscopy or surgically. These procedures are invasive, so there has been significant interest in additional diagnostic methods.
The mainstay of radiological investigations is computerised tomography (CT), with magnetic resonance imaging and positron emission tomography (PET) currently limited to the research setting. However, differentiating MPM from benign pleural thickening or other pleural malignancies is unreliable using CT alone [2]. Given the drawbacks in current cytopathological and radiological investigations for MPM there is a huge potential role for serum or pleural fluid biomarkers. Biomarkers would allow earlier detection of malignancy in at-risk groups (e.g. the asbestos exposed), reduce the need for invasive biopsies and speed the diagnostic pathway to treatment.
Treatment and monitoring
Sadly, due to the highly invasive nature of MPM, treatment is often palliative from diagnosis. The current standard of care is based on the results of a non-placebo-controlled trial from 2003. Vogelzang and colleagues used a combination of pemetrexed (an anti-folate) and cisplatin (platinum-based agent) chemotherapy [4]. This combination adds a modest 2 months to overall survival, with a response rate of only 30 %. Treatment for MPM had not significantly advanced until the publication of the Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS) trial in 2015, which showed a 2-month survival advantage when bevacizumab [an anti-vascular endothelial growth factor (VEGF) immunotherapy] was added to standard chemotherapy [5].
The role of surgery for MPM is highly controversial, with significant variation in operative rates internationally. This controversy exists because there are no randomised trials of radical surgical intervention against best medical therapy. Large case series of patients with positive surgical outcomes exist, but they are often highly selective of younger patients with good performance status.
Both chemotherapeutic and surgical management would benefit from a greater ability to prognosticate patients at baseline and assess response to treatment. Currently, serial CT scanning is the gold standard of disease monitoring in MPM. Similar to other malignancies an attempt to measure change in tumour is made using the RECIST criteria. However, unlike other malignancies, MPM grows as a rind around the chest wall so volume measurement is difficult. A modified RECIST criteria has been developed, but is time intensive, and, with the added complications of pleural fluid and plaques, is rarely used outside the research setting. Other radiological methods to monitor disease have been examined, with a recognition that biomarkers would be ideal as a method of monitoring disease in a non-invasive manner [6].
Mesothelin
Soluble mesothelin (SM) is a 40 kDa glycoprotein over-expressed by the epithelioid component of malignant mesothelial cells. Its exact biological role remains uncertain. Discovered in the serum of patients with ovarian cancer, it was subsequently found in serum, pleural fluid and urine of patients with MPM. See Figure 1 in the open access article by Hassan et al. for a schematic showing the maturation and structure of mesothelin [7].
There has been considerable research attention on its utility in diagnosis or monitoring MPM. The majority of these studies have used a commercial platform, the Mesomark® ELISA. Unfortunately, despite some positive initial signals, a meta-analysis by Cui and colleagues in 2014 demonstrated that the overall sensitivity for detecting MPM was 0.61 in serum and 0.79 in pleural fluid [8]. The level of SM rises with increased epithelioid disease bulk, and, therefore, can be low in early-stage disease and may never rise in sarcomatoid or desmoplastic subtypes. For a diagnosis with such profound consequences for the individual, as well as medico-legal implications, this inability to reliably detect MPM has limited its widespread diagnostic use.
More recent research has focused on its ability to monitor MPM during treatment or follow-up (Table 1).
Although there is considerable heterogeneity between these 10 studies in terms of primary outcome measures, each has demonstrated that a rising SM is correlated with clinical or radiological disease progression. A falling SM following chemotherapy or surgery was strongly indicative of treatment response. The future of SM in disease monitoring depends on the results of currently recruiting prospective trials.
Fibulin-3
Fibulin-3 is a glycoprotein that promotes tumour growth and invasion through the phosphorylation of epidermal growth factor. In 2012, the New England Journal of Medicine published the results of a landmark study which reported that serum fibulin-3 had a 100 % sensitivity for detecting early-stage MPM [9]. Unfortunately, several follow-up studies using the same commercial ELISA have been unable to replicate these results. Ren and colleagues published a meta-analysis of eight studies which found the sensitivity to be around 87 % with a specificity of 89 % [10].
Osteopontin
Osteopontin is over-expressed in several malignancies and several studies have focused on its prognostic abilities in MPM. Early studies used serum osteopontin, without appreciating the impact of its thrombin cleavage site on results. More recent studies have used more accurate plasma measurement and shown that osteopontin has no role in diagnosis or monitoring [11]. Interestingly, there is evidence that baseline osteopontin is a marker of poor prognosis, independent of histology, treatment modality or other biomarkers.
Vascular Endothelial Growth Factor (VEGF)
VEGF has been studied as a potential diagnostic or therapeutic target in MPM. Although baseline VEGF correlates with disease stage and survival it is not used in the clinical setting. However, following the publication of the MAPS trial (of the anti-VEGF immunotherapy bevacizumab) there has been a renewed focus on whether baseline VEGF can select responders from non-responders. These studies have been directed at pan-VEGF, as opposed to any specific isoform, and have not shown any definitive role to date.
Proteomic studies
A modern approach to biomarker discovery and validation is exemplified by the DIAPHRAGM study [12]. Tsim and colleagues took the results of a promising 13-protein diagnostic panel developed and internally validated by Ostroff [12]. The internal validation cohort had reported an area under curve (AUC) for detecting MPM of 0.95 (38 patients with MPM). A flaw of previous biomarker validation is that follow-up work is performed on small retrospective cohorts. It often takes several years or decades before the true utility of the biomarker is established. The DIAPHRAGM study aims to quickly and definitively validate or reject the SOMAscan assay, alongside fibulin-3, in a prospective, powered and clinically relevant manner. The final results of the research are awaited, but regardless, this approach has set a standard for biomarker discovery and validation in MPM.
Summary
MPM is a highly aggressive cancer that is difficult to diagnose and monitor. The potential scope for biomarkers is huge. Serum SM has shown the most promise in monitoring disease. A biomarker that can reliably diagnose early-stage MPM remains elusive.
References
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The authors
David T. Arnold* MBBCh, BSc, MRCP and Nick A. Maskell BMedSci, BM, BS, FRCP, DM, FCCP
Academic Respiratory Unit, Learning and Research Building, Southmead Hospital, Bristol, UK
*Corresponding author
E-mail: arnold.dta@gmail.com
November 2025
Prins Hendrikstraat 1
5611HH Eindhoven
The Netherlands
info@clinlabint.com
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