According to Cancer Research UK, the incidence of ovarian cancer in the UK is around 7500 new cases per year, accounting for 4% of all new cancer cases in females. The overall 5-year survival rate is around 46%, dropping to 35% at the 10-year mark, a relatively poor prognosis, but almost double that of 40 years ago. These aggregated statistics hide a more complicated story and, as is often the case, diagnosis at an early stage leads to markedly better survival figures (90% survival for patients diagnosed at stage 1). However, one of the reasons for the poor prognosis is that ovarian cancer is often diagnosed at stage 3 or 4 (22% survival), which is likely to be the result of the rather vague and non-specific nature of symptoms in the early stages. Recent screening trials based on the detection of sudden increases in CA-125 level and ultrasound have shown slight improvements in diagnosis at an earlier stage but no significant reduction in mortality. A recent paper by Russell et al.*, however, demonstrated that use of a panel of four biomarkers results in the potential to diagnose ovarian cancer up to 2 years earlier than current tests can. The four biomarkers included in the panel were identified from serum samples collected in a time span up to 7 years before diagnosis (obtained from one of the screening trials mentioned earlier), which is likely to be the window in which early, treatable, asymptomatic disease occurs. The biomarkers used were ovarian cancer-related tumour marker CA125 (CA-125), vitamin K-dependent protein Z (PROZ), phosphatidylcholine-sterol acyltransferase (LCAT) and C-reactive protein (CRP) and dysregulation against baseline was calculated, with specificity thresholds for dysregulation set from control data. The authors, however, outline and discuss a number of limitations and caveats that mean that they are not suggesting that this biomarker panel is ready to be rolled out in a screening programme, but that the potential is there to warrant a larger scale study. This paper not only demonstrates a real step forward in the early detection of ovarian cancer but also very eruditely describes what is needed from biomarkers and a screening programme that is intended to identify early disease and is worth reading for that alone.

* Russel MR, et al. Diagnosis of epithelial ovarian cancer using a combined protein biomarker panel. Br J Cancer 2019; doi: 10.1038/s41416-019-0544-0.

Plasma cell disorders are detected in the clinical lab by finding the monoclonal immunoglobulin (M-protein) they produce. Serum protein electrophoresis methods have been employed widely to detect and isotype M-proteins. Increasing demands to detect residual disease and new therapeutic monoclonal immunoglobulin treatments have stretched electrophoretic methods to their limits. Newer techniques based on mass spectrometry are emerging which have improved clinical and analytical performance. These techniques are beginning to gain traction within routine clinical lab testing.

by Dr David L. Murray

Background
In a healthy immune system, the terminally differentiated white blood B-cells (i.e. plasma cells) each produce a unique immunoglobulin (Ig, or antibody) which was selected for by its fitness to bind to foreign invaders (antigens). This legion of plasma cells resides within our bone marrow and serves as a protective library manufacturing a diverse protective cacophony of Ig proteins whose aim is to protect us from recurrent infections. The total production of Igs in a healthy individual is remarkably highly regulated in the non-infected state with no particular plasma cell out-producing other plasma cells. As a result, the electrophoretic separation of healthy human serum results in a cathodically broad distribution of Ig proteins, which is labelled the gamma region (Fig. 1a).

In contrast, plasma cell proliferative disorders (PCDs) consist of a group of diseases stemming from clonal proliferation of a dysregulated plasma cell clone. PCDs range from relatively common benign conditions, such as monoclonal gammopathy of undetermined significance (MGUS), to frank malignant conditions, such as multiple myeloma (MM) [1]. Central to the detection of PCDs in serum is the detection of the over-produced monoclonal Ig by the dysregulated plasma clone (termed M‑protein or paraprotein). M‑proteins are a relatively common laboratory finding occurring in approximately 3 % of adults over the age of 50 [2]. The majority of these patients will live unaffected by the presence of the M‑protein while some patients will progress to more serious disease, such as MM, at a rate of 1 % per year. Currently, it is not possible to know which patient is going to progress and patients with an M‑protein undergo surveillance for M‑protein concentration changes yearly.

Electrophoresis-based assays
By nature, M‑proteins are heterogeneous and thus diverse methodologies are currently used to detect, characterize and quantitate serum M‑proteins in the clinical laboratory. Serum protein electrophoresis (PEL) was the first method available to detect and quantitate M‑proteins. To increase the specificity and sensitivity, a second technique known as immunofixation electrophoresis (IFE) enables establishment of M‑protein isotype (IgG, IgA, IgM, IgD, IgE or free light chain kappa or lambda) by examining multiple electrophoretic gel lanes in which the serum proteins were ‘fixed’ to the gel using reagents specific for human immunoglobulin components
(Fig. 1). A third assay, the serum free light chain (sFLC) assay, uses specific antibodies for quantitation of circulating free kappa and lambda light chains. This assay has demonstrated superior detection of PCDs, such as amyloid light chain (AL) amyloidosis, which can result from low levels of circulating monoclonal free light chains [3]. Currently, the International Myeloma Working Group recommends a panel of serum tests that include PEL, IFE and a sFLC assay quantitation to maximize the sensitivity of PCD screening [4].

Need for improved detection sensitivity
At our institution, agarose gel electrophoresis methods (PEL and IFE) have been used for detecting M‑proteins since 1967. While the utility of the electrophoretic methods to screen and monitor PCDs has been well established, several changes in the treatment of PCDs are pushing these methods to their analytical limits. Dramatic improvement in the treatment response of MM patients to new chemotherapies and immunotherapies is challenging long-held assumptions about this ominous disease. In particular, there is renewed hope that MM may be curable and perhaps it is time to start treating MM patients until all signs of the disease are eradicated. The long-standing routine serum electrophoretic methods are not capable of providing the analytical sensitivity needed to assess minimal residual disease (MRD). A few laboratorians have turned to using bone marrow biopsies to hunt for traces of the malignant plasma cells by high sensitivity flow cytometry and next-generation sequencing [5, 6]. In addition, new monoclonal therapeutic antibodies (t‑mAbs) designed to eradicate malignant plasma cells are producing interferences making it difficult to distinguish between a patient’s M‑protein and the t‑mAb drug. A search for a more convenient serum-based test to complement bone marrow MRD detection and aid in resolving t‑mAb interferences was sought to address limitations in traditional testing. Mass spectrometry (MS) is aptly suited for this task as the improvements in MS instrumentation and techniques have resulted in increased resolution and mass accuracy that have outpaced improvements in electrophoresis.
MS-based methodsFor Igs, both the overall charge of the protein (the basis of electrophoretic separation) and the mass of the protein (the basis of MS separation) are diverse among Igs owing to Ig gene rearrangement in which the adaptive immune system optimizes the affinity of the antigen binding region of the Ig to its target antigen. The unique amino acid sequence of the antigen binding domain results in a unique molecular mass (and peptide sequence) which is the basis of the mass spectrometric detection. Efforts to optimize M‑protein detection by MS have resulted in two methods differing in the analytical target used to detect the M‑protein. One method based on a tryptic digest of Igs and using selective reaction monitoring (SRM) MS to detect unique peptides from the Ig antigen binding region (also termed the ‘clonotypic’ peptide approach) [7] and a second method based on disassembling Igs by chemical reduction and measuring the mass distribution of Ig light chain [termed monoclonal immunoglobulin Rapid Accurate Mass Measurement (miRAMM)] [8]. Of these two approaches, the miRAMM method was suitable for adaptation to our high volume reference laboratory. The adaptation of the miRAMM method to MALDI-TOF mass spectrometers [9] eliminated the need for chromatography and allowed for throughputs suitable for PCD screening. The simplicity of MALDI-TOF data files also allowed our lab to build software capable of rapidly displaying multiple spectra which can be automatically analysed for an M‑protein. The current clinically validated version of the assay consists of five separate immune-enrichments for IgG, IgA, IgM, kappa and lambda which are separately analysed and the light chain mass distributions are examined for a ‘spike’ in a similar fashion to gel electrophoretic densitometry (Mass-Fix; Fig. 2). Mass-Fix has demonstrated overall superior analytical and clinical sensitivity to serum IFE [9, 10]. Mass-Fix has been automated and validated as a laboratory developed test and our one-year experience has confirmed that the assay is robust, sensitive and more labour efficient than our traditional gel IFE assay.
One of the benefits of using Mas-Fix over electrophoresis is the ability to determine a fundamental feature of the M‑protein, its light chain mass. Reporting the light chain mass allows for a more specific description of M‑protein than is currently available by electrophoresis. Current reporting of serum electrophoresis allows for placing an M‑protein within a region of the electropherorgram (alpha, beta or gamma) which is less specific than reporting an IgG kappa M‑protein with a light chain mass of 23 425 Da. Using the mass of the M‑protein light chain could allow other clinical labs using MS to assess the same patient for over-expressed clones of the same light chain mass increasing the confidence of M‑protein identity. By measuring the mass of the light chain of a t‑mAb, the lab will be able to determining if the detected over-expressed clone is due to the presence of a t‑mAb (such as daratumumab) or the patient’s M‑protein [11]. Additionally, the mass of the M‑protein light chain detected in other body fluids, such as urine, was found to be the same as in serum. This again affords more specificity than is currently available by electrophoresis.

The Mass-Fix assay has also shed light on M‑protein structural features that were not previously appreciated using electrophoretic techniques. In particular, the presence of monoclonal Ig light chains with masses outside the expected mass range were encountered in a small subset of patients. These light chains also had broader mass ranges than typically encountered with M‑proteins. Additional work revealed these light chains contained N-linked glycosylation [12]. Furthermore, patients with light chain glycosylated M‑proteins were found to be more likely to have a rarer form of a PCD (AL amyloidosis) than patients without light chain glycosylation.

Challenges and future perspectives
Challenges remain for these new assays to gain broad acceptance in the medical field. One feature that facilitates acceptance is Conformité Européene (CE) or U.S. Food and Drug Administration (FDA) approval in a format that is scalable and generalizable to a majority of clinical labs. Electrophoretic methods were employed prior to the FDA 510K process and thus have been grandfathered into the FDA approval system. This will not be the case for newer MS assays and thus time will be needed to get FDA approval. With increasing sensitivity, hematologists have also expressed concern over the potential increase in the detection of pre-malignant benign condition MGUS, as this would increase the number of consults.
These challenges need to be assessed in light of the numerous clinical advantages. The addition of the mass measurement allows for simpler conformation of peak as to its origin: disease or t‑mAb, the discovery of new risk factors for the formation of AL amyloidosis, and the ability to standard the detection from lab to lab.

Figure 1. Traditional detection of M-protein by immunofixation electrophoresis. (a) Healthy human serum demonstrating the albumin, alpha 1, alpha 2, beta and the broad gamma region which results from the diverse repertoire of Igs with slightly differing amino acid sequences and hence overall charge. (b) A patient with a plasma cell disorder demonstrating a relatively restricted band in the gamma region with immunofixation with anti-IgG (G) and anti (K) consistent with an IgG kappa M-protein.
Figure 2. Comparison of traditional immunofixation results and the new Mass-Fix spectra. (a) Healthy human serum demonstrating broad gamma region of IFE (left) and normal Gaussian [LC+2] m/z distribution for all immune-enrichments (IgG (black top), IgA (black middle), IgM (black, lower), kappa (orange, all spectra) and lambda (blue, all spectra). (b) A patient with a plasma cell disorder demonstrating a relative restricted band in the gamma region consistent with IgG kappa (left) and a non-Gaussian distribution of light chains with a peak in the IgG light mass distribution (black top) along with same peak in the total kappa light chain mass distribution (orange).

References
1. Willrich MAV, et al. Clin Biochem 2018; 51: 38–47.
2. Kyle RA, et al. N Eng J Med 2002; 346(8): 564–569.
3. Katzmann JA, et al. Clin Chem 2009; 55(8): 1517–1522.
4. Dimopoulos M, et al. Blood 2011; 117(18): 4701–4705.
5. Martinez-Lopez J, et al. Blood 2014; 123(20): 3073–3079.
6. Rawstron AC, et al. J Clin Oncol 2013; 31(20): 2540–2547.
7. Barnidge DR, et al. J Proteome Res 2014; 13(4): 1905–1910.
8. Barnidge DR, et al. J Proteome Re 2014; 13(3): 1419–1427.
9. Mills JR, et al. Clin Chem 2016; 62(10): 1334–1344.
10. Milani P, et al. Am J Hematol 2017; 92(8): 772–779.
11. Mills JR, et al. Blood 2018; 132(6): 670–672.
12. Kumar S, et al. Leukemia 2019; 33(1): 254–257.

The author
David L. Murray MD, PhD
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55906, USA
E-mail: Murray.David@mayo.edu

Drugs of abuse testing is performed to identify drug abuse, to monitor someone with a substance abuse problem, or to detect drug intoxication and overdose. The identification of drug abuse in biological samples can be used in court as scientific evidence, and it can help to improve the quality of clinical management during emergencies. One of the most common screening methods used for the detection of drugs in urine and other matrices are immunoassays. They are convenient, but they have their limitations: results are often class-specific and cannot be attributed to a specific drug or drug metabolite. Antibodies are also susceptible to cross-reactivity with structurally related and unrelated compounds, which increases the risk of false-positive results. This is why data gathered by immunoassays are considered as presumptive.

GC-MS vs LC-MS/MS
An immunoassay requires a second analytical procedure to confirm the quantitative determination, and this is usually performed by either GC-MS or LC-MS. The mass spectra obtained from the GC-MS can be compared with large databases, enabling the unknown abusive drugs to be identified – this is one of the reasons why GC-MS has been the gold standard in drugs of abuse testing for many years. However, most compounds of interest need to undergo a chemical derivatization to make them more volatile and compatible with GC analysis – without derivatization, GC-MS generally offers poor peak shapes, lower resolutions and reduced sensitivities. However, undertaking more sample preparation steps also increases the risk of errors, and acidic derivatization can be prone to uncertainties, such as the reagent quality, the presence of interferences, and variable lab conditions.
In contrast, LC-MS is ideal for polar and non-volatile molecules such as those analysed in drugs of abuse testing. An efficient separation and ion generation can be achieved without derivatization and LC-MS generally requires less sample preparation than GC-MS. Among the different mass spectrometry platforms, triple quadrupole mass spectrometry with multiple reaction monitoring (MRM) is the most commonly adapted technique.
LC-MS/MS – from theory to practice
A laboratory tested a commercial LC-MS/MS assay (MassTox® Drugs of Abuse Testing, Chromsystems) and compared it with GC-MS, with a focus on routine analysis [1]. The sample prep for the amount they routinely deal with usually takes 6 hours (excluding hydrolysis), but by using the commercial assay, the lab was able to reduce the time down to 2 hours. The switch from GC-MS to LC-MS reduced the resources required for the sample prep, and the sample volume required for the sample preparation was also significantly lower (see Table 1),
The lab also conducted comparative analysis between the commercial assay and an in-house LC-MS assay used by an external accredited laboratory. The values obtained correlate very well with each other across a range of concentrations demonstrating a high accuracy, as showcased by the linear results. Therefore, the commercial assay (Chromsystems) is suitable for replacing LC-MS/MS in-house methods and allows for the target screening and/or quantitative confirmation of 106 drugs in a single run (Fig. 1). Proficiency testing schemes from GTFCh and RfB, in which the assay has been used, also confirmed its accuracy [1].
100% hydrolysis, 0% doubt
In the human body, many drugs undergo glucuronidation, which requires an enzymatic or acidic hydrolysis prior to the analysis – a challenge for many assays. Enzymatic hydrolysis varies in its effectiveness depending on the drug and the enzyme [2]. Erratic quality assurance results for codeine – one of the more difficult to hydrolyse compounds – are considered to be based on an incomplete hydrolysis [3]. Consequently, some papers recommend the use of an acidic hydrolysis, however, this can degrade both opioids and other substances [4]. Furthermore, this approach can convert oxycodone to oxymorphone, and codeine to morphine by demethylation, which increases the risk of false-negative or false-positive results. To overcome this drawback, our lab has developed an enzymatic hydrolysis process that is effective for hydrolysing all glucuronides within 2 hours. This has been achieved by using a carefully selected enzyme that ensures a complete and selective hydrolysis of all 106 drugs that are covered in the assay, including codeine. The effectiveness has been demonstrated by measuring the hydrolysis of several substances over time: Easy-to-hydrolyse glucuronides become fully hydrolysed quickly, while others, such as codeine, require longer. After 2 hours, the hydrolysis is complete for all the compounds (Fig. 3).

Target screening and confirmation in one run
Immunoassays often require an alternative method to confirm the results, and this is how many organisations have laid out their drug abuse testing schemes. However, LC-MS/MS has an accuracy and selectivity that is a sufficient to do both in one step. This is why commercial assays, such as those from Chromsystems, enable the target screening and quantitative confirmation of more than 100 drugs in a single run (Fig.2), including benzodiazepines, opioids, booster, and Z-drugs. In the case of a positive result, the quantification can be evaluated straight away from the same peak. Labs might find this option in drug of abuse testing appealing, as it reduces the resources required without compromising on the accuracy.
[1] Geffert et al., Validation of a New LC-MS/MS Assay for the Analysis of Drugs in Urine and Comparison with Established Analytical Methods (GC-MS and LC-MS/MS):
Advantages for daily Laboratory Routine. GTFCh Symposium 2019.
[2] Wang P et al., Incomplete Recovery of Prescription Opioids in Urine using Enzymatic Hydrolysis of Glucuronide Metabolites. J. Analytical Toxicology, (2019), 571-575.
[3] Hackett LP et al., Optimizing the hydrolysis of codeine and morphine glucuronides in urine. Ther Drug Monit., (2002), 652-657.
[4] Opiate & Benzodiazepine Confirmations: To Hydrolyze or Not to Hydrolyze is the Question. J. of Appl. Lab Med., (2018), 1-9.

The author
Marc Egelhofer, PhD, Head of Marketing Communications, egelhofer@chromsystems.de