Intraductal tubulopapillary neoplasm is a recently recognized distinct and rare entity of the pancreas, which may be unfamiliar to many physicians and laboratory personnel. However, recognizing this disease is critical for its proper clinical management and further study. Here, we discuss the clinical and pathological features of this neoplasm.

by Dr Shula Schechter and Dr Jiaqi Shi

Presentation and definition of intraductal tubulopapillary neoplasm of the pancreas
Intraductal tubulopapillary neoplasm (ITPN) was recently recognized as a distinct neoplastic entity of the pancreas in the 2010 edition of the World Health Organization (WHO) classification [1]. It was defined as an intraductal, grossly visible, tubule-forming epithelial neoplasm with high-grade dysplasia and ductal differentiation without overt production of mucin, although focal tubulopapillary growth is also acceptable [1].

ITPN is a rare entity. Although it was first described in the mid-1990s by Japanese investigators and has been termed ITPN since 2009 [2], its diagnostic criteria need to be refined and recognition of this disease needs to be improved. The differential diagnosis of ITPN can be complex because its features overlap with other more common intraductal neoplasms, such as intraductal papillary mucinous neoplasm (IPMN). A recently published literature review and a large study of 33 cases of ITPN have shed some light on the clinicopathologic and immunohistochemical features of this disease and further advance our knowledge of its diagnosis [3, 4].

The majority of the patients with ITPN present with abdominal pain, nausea, vomiting, weight loss and steatorrhea. A few patients have diabetes mellitus, acute pancreatitis, jaundice and fever. Incidental discovery of ITPN occurs in about one-third of patients. The risk factors for ITPN are not well defined, but there are reports of an association of ITPN with radiation exposure and with a family history of pancreatic cancer [4–6]. The incidence of ITPN in men and in women is comparable. Most ITPNs occur in the sixth decade with a range in age of 25 to 79 years. Nearly half of reported ITPNs are located in the head of the pancreas. In the remainder of cases, the location of the ITPN is divided between the body and tail with about one-quarter of lesions showing more extensive involvement of the entire pancreas [4]. ITPNs are often slow growing tumours and large at the time of discovery.

Imaging studies with dynamic contrast-enhanced computed tomography and magnetic resonance imaging are commonly used to assist with preoperative diagnosis. A helpful imaging clue for the diagnosis of ITPN is the two-tone duct sign, which is a reflection of tumour in the main pancreatic duct with ductal dilation upstream [7]. With magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography, ITPN also has a characteristic finding, the so called ‘cork-of-wine-bottle’ sign, which results from intraductal growth of the tumour [7].

Information on the prognosis of ITPNs is limited by the small number of reported cases, although data have suggested an excellent prognosis for patients without invasion (overall 5-year survival rate of 100%) and a significantly more favourable prognosis for ITPNs with a component of invasive carcinoma (overall 5-year survival rate of 71%) relative to the traditional invasive pancreatic ductal adenocarcinoma (overall 5-year survival rate <10%) [2, 8, 9]. However, the extent of invasion does not necessarily correlate with clinical outcome. Patients with minimal invasion can die of disease, whereas patients with a large volume of invasion can achieve long-term survival [4]. Unfortunately, invasive carcinoma is present in most (54–71%) ITPNs and may be more likely in men [3, 4, 10]. In addition, tumours that are large in size, or have increased mitosis and a high Ki-67 proliferation index may have an increased association with invasive carcinoma [2]. Despite the favourable prognosis, the possibility of invasive carcinoma, recurrence and metastasis has led to the general recommendation of surgery as treatment in most ITPN patients.

Diagnostic features of ITPN based on histology and immunohistochemistry
Macroscopically, the mean size of the tumour is 3.8 cm (range 0.5–15 cm). Most ITPNs are circumscribed solid or polypoid masses obstructing pancreatic ducts. They generally arise in the main pancreatic duct, but approximately 5% arise within the branch ducts [4, 10]. ITPNs may be cystic, this occurs in less than half of cases. However, ITPNs do not have grossly identifiable mucin.

Microscopically, ITPNs are characterized by back-to-back tubules forming complex cribriform structures (Fig. 1a, c) with focal areas of papillary architecture seen in 36% of ITPN cases [4]. Solid growth with necrotic foci can occur, occasionally with areas of comedo-like necrosis (Fig. 1b). Occasionally, there are apical apocrine snouts and intraluminal secretion; however, cytoplasmic and intraluminal mucin is scant to absent. The tubules are lined by cuboidal to low columnar epithelial cells with minimal to moderate amounts of eosinophilic or amphiphilic cytoplasm and round to oval nuclei with moderate to marked atypia (Fig. 1d). ITPNs classically have uniform high-grade dysplasia and increased mitotic figures. Uncommon clear cell morphology or stromal osseous and cartilaginous metaplasia has also been reported in an ITPN.

By immunohistochemistry, all ITPNs to date have stained positively with anti-cytokeratin (CK) 7 (Table 1) and CAM5.2 antibodies. CK19 is positive in 92% of the cases. Tumour markers CA19.9 and CEA (carcinoembryonic antigen) are expressed in 93% and 50% of the cases respectively. In contrast, CK20 and CDX2 (homeobox protein CDX-2) only stain rare cells in a minority of ITPNs. The mucin (MUC) family has a particular staining pattern in ITPNs (Table 1), which is sometimes helpful in its differential diagnosis. MUC1 and MUC6 are positive in the majority of cases (88% and 77% respectively) whereas MUC2 and MUC5AC are usually negative (only 2% and 6% ITPNs are positive respectively). Nuclear p53 and p16-INK4 (cyclin-dependent kinase inhibitor 2A) are expressed in 27% and 33% of the cases. Rare focal or scattered cells can be positive for HepPar-1 antigen, chromogranin or nuclear β-catenin. However, ITPNs do not express pancreatic enzymes, trypsin and chymotrypsin, or loss of E-cadherin or Smad4.

Recent molecular findings
Recent genetic studies have found evidence that ITPN is molecularly distinct from IPMN. The most commonly mutated genes in ITPN include PIK3CA, TP53 and CDKN2A, among others [8–18]. Other rare mutations in histone H3 methyltransferase genes, MLL2 and MLL3 (also known as KMT2A and KMT2C), and MCL1 amplification have also been identified in ITPN [19]. However, ITPNs have been shown to have no or rare mutations in KRAS, BRAF, or GNAS. In contrast, IPMNs have high mutation rates in multiple genes [20]. KRAS mutation is thought to be one of the driver genes during IPMN development and mutations in GNAS and RNF43 are also common.

Differential diagnosis
Despite its distinct molecular features, the histology of ITPN can resemble that of IPMN, especially the pancreatobiliary and oncocytic type, making it difficult to distinguish ITPNs from IPMNs by morphology alone. The key morphologic features that characterize ITPNs as compared to IPMNs are shown in Table 2 and Figure 2. Overall, cystic components are infrequent with ITPNs in contrast to IPMNs, which are predominantly cystic lesions. Mucin is another distinguishing feature, which is sparse or absent with ITPNs but abundant with IPMNs. IPMNs also have significantly more morphologic variation according to epithelial subtype, and their degree of cytologic and architectural atypia varies from low- to high-grade dysplasia, whereas ITPNs typically demonstrate uniform high-grade dysplasia. On the other hand, comedo-like necrosis is frequent with ITPNs but rare with IPMNs.

The cytologic and architectural distinctions between ITPNs and IPMNs are confounded by the wide spectrum of morphologies and degree of dysplasia that are seen with IPMNs. Among the four recognized epithelial subtypes of IPMN, the pancreatobiliary and oncocytic type IPMN are the types that are most easily confused with ITPN. Similar to ITPN, both pancreatobiliary and oncocytic type IPMNs have high-grade dysplasia and often complex architecture. Nevertheless, the architecture of these IPMN subtypes remains predominantly papillary in nature as compared to the tubular or tubulopapillary architecture of ITPNs. In addition, the oncocytic IPMN has intraepithelial lumens as well as cells with abundant granular eosinophilic cytoplasm. Subtypes of IPMN also differ from ITPN in their immunohistochemical profiles. Most ITPNs are MUC6 positive and MUC5AC negative, whereas the opposite is true for most IPMNs (MUC6 negative and MUC5AC positive). The immunohistochemical findings with the oncocytic subtype of IPMN are most similar to findings with ITPNs, although some studies found MUC5AC can be positive with oncocytic IPMNs. Use of a mitochondrial stain (e.g. phosphotungstic acid–hematoxylin, Novelli stain, anti-apoptin 111.3 antibody) may allow an oncocytic IPMN to be distinguished from an ITPN on the basis of abundant mitochondria in cytoplasm [12].

Intraductal acinar cell carcinoma can also be confused with ITPN due to its occasional intraductal growth pattern. However, intraductal acinar cell carcinoma will typically stain positively for pancreatic enzymes such as trypsin, chymotrypsin or Bcl-10 (B-cell lymphoma/leukemia 10), and negatively for CK7 and CK19 by immunohistochemistry [4, 21].

Conclusion
ITPN is a relatively new diagnostic entity that occurs infrequently, predominantly in older patients. One-third of patients can be asymptomatic. Although invasive carcinoma is present in most ITPNs, the prognosis of these tumours appears to be significantly more favourable than pancreatic ductal adenocarcinoma. ITPNs generally arise in and obstruct the main pancreatic duct with circumscribed, solid nodules that are grossly visible. Histologically, these tumours are characterized by back-to-back tubules forming complex cribriform structures and uniform high-grade dysplasia. Necrosis is frequent but cytoplasmic and intraluminal mucin is scant to absent, which is in contrast with IPMNs. Molecular studies support that ITPN is a distinct entity from other intraductal neoplasms of pancreas, such as IPMN. With increased recognition of ITPNs, we expect to learn more information about its pathological features and prognostic implications.

References
1. Adsay NV, Fukushima N, Furukawa T, Hruban RH, Klimstra DS, Klöppel G, et al. Intraductal neoplasms of the pancreas. In: Bosman FT, Carneiro F, Hruban RH, Theise ND, eds. World Health Organization Classification of Tumours of the Digestive System, pp. 304–313, 4th edn. International Agency for Research on Cancer 2010.
2. Yamaguchi H, Shimizu M, Ban S, Koyama I, Hatori T, Fujita I, Yamamoto M, Kawamura S, Kobayashi M et al. Intraductal tubulopapillary neoplasms of the pancreas distinct from pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms. Am J Surg Pathol 2009; 33(8): 1164–1172.
3. Rooney SL, Shi J. Intraductal tubulopapillary neoplasm of the pancreas: an update from a pathologist’s perspective. Arch Pathol Lab Med 2016; 140(10): 1068–1073.
4. Basturk O, Adsay V, Askan G, Dhall D, Zamboni G, Shimizu M, Cymes K, Carneiro F, Balci S et al. Intraductal tubulopapillary neoplasm of the pancreas: A clinicopathologic and immunohistochemical analysis of 33 cases. Am J Surg Pathol 2017; 41(3): 313–325.
5. Bhuva N, Wasan H, Spalding D, Stamp G, Harrison M. Intraductal tubulopapillary neoplasm of the pancreas as a radiation induced malignancy. BMJ Case Rep 2011; 2011.
6. Del Chiaro M, Mucelli RP, Blomberg J, Segersvard R, Verbeke C. Is intraductal tubulopapillary neoplasia a new entity in the spectrum of familial pancreatic cancer syndrome? Fam Cancer 2014; 13(2): 227–229.
7. Motosugi U, Yamaguchi H, Furukawa T, Ichikawa T, Hatori T, Fujita I, Yamamoto M, Motoi F, Kanno A et al. Imaging studies of intraductal tubulopapillary neoplasms of the pancreas: 2-tone duct sign and cork-of-wine-bottle sign as indicators of intraductal tumor growth. J Comput Assist Tomogr 2012; 36(6): 710–717.
8. Cooper CL, O’Toole SA, Kench JG. Classification, morphology and molecular pathology of premalignant lesions of the pancreas. Pathology 2013; 45(3): 286–304.
9. Kasugai H, Tajiri T, Takehara Y, Mukai S, Tanaka JI, Kudo SE. Intraductal tubulopapillary neoplasms of the pancreas: case report and review of the literature. J Nippon Medl Sch 2013; 80(3): 224–229.
10. Kolby D, Thilen J, Andersson R, Sasor A, Ansari D. Multifocal intraductal tubulopapillary neoplasm of the pancreas with total pancreatectomy: report of a case and review of literature. Int J Clin Exp Pathol 2015; 8(8): 9672–9680.
11. Amato E, Molin MD, Mafficini A, Yu J, Malleo G, Rusev B, et al. Targeted next-generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas. J Pathol 2014; 233(3): 217–227.
12. Bledsoe JR, Shinagare SA, Deshpande V. Difficult diagnostic problems in pancreatobiliary neoplasia. Arch Pathol Lab Med 2015; 139(7): 848–857.
13. Kloppel G, Basturk O, Schlitter AM, Konukiewitz B, Esposito I. Intraductal neoplasms of the pancreas. Semin Diagn Pathol 2014; 31(6): 452–466.
14. Reid MD, Saka B, Balci S, Goldblum AS, Adsay NV. Molecular genetics of pancreatic neoplasms and their morphologic correlates: an update on recent advances and potential diagnostic applications. Am J Clin Pathol 2014; 141(2): 168–180.
15. Schlitter AM, Jang KT, Kloppel G, Saka B, Hong SM, Choi H, Offerhaus GJ, Hruban RH, Zen Y et al. Intraductal tubulopapillary neoplasms of the bile ducts: clinicopathologic, immunohistochemical, and molecular analysis of 20 cases. Mod Pathol. 2015; 28(9): 1249–1264.
16. Urata T, Naito Y, Nagamine M, Izumi Y, Tonaki G, Iwasaki H, Sasaki A, Yamasaki A, Minami N et al. Intraductal tubulopapillary neoplasm of the pancreas with somatic BRAF mutation. Clin J Gastroenterol 2012; 5(6): 413–420.
17. Yamaguchi H, Kuboki Y, Hatori T, Yamamoto M, Shimizu K, Shiratori K, Shibata N, Shimizu M, Furukawa T. The discrete nature and distinguishing molecular features of pancreatic intraductal tubulopapillary neoplasms and intraductal papillary mucinous neoplasms of the gastric type, pyloric gland variant. J Pathol 2013; 231(3): 335–341.
18. Yamaguchi H, Kuboki Y, Hatori T, Yamamoto M, Shiratori K, Kawamura S, Kobayashi M, Shimizu M, Ban S et al. Somatic mutations in PIK3CA and activation of AKT in intraductal tubulopapillary neoplasms of the pancreas. Am J Surg Pathol 2011; 35(12): 1812–1817.
19. Bhanot U, Basturk O, Berger M, Shah R, Scott S, Adsay V, Offerhaus GJ, Hruban RH, Zen Y et al. Molecular characteristics of the pancreatic intraductal tubulopapillary neoplasm. Mod Pathol 2015; 28(2S): 440A.
20. Springer S, Wang Y, Dal Molin M, Masica DL, Jiao Y, Kinde I, Blackford A4, Raman SP5, Wolfgang CL et al. A combination of molecular markers and clinical features improve the classification of pancreatic cysts. Gastroenterology 2015; 149(6): 1501–1510.
21. Hosoda W, Sasaki E, Murakami Y, Yamao K, Shimizu Y, Yatabe Y. BCL10 as a useful marker for pancreatic acinar cell carcinoma, especially using endoscopic ultrasound cytology specimens. Pathol Int 2013; 63(3): 176–182.

The authors
Shula Schechter MD; Jiaqi Shi* MD, PhD
Department of Pathology, University of Michigan,
Ann Arbor, MI 48109 USA

*Corresponding author
E-mail: jiaqis@med.umich.edu

In recent decades liquid chromatography–tandem mass spectrometry (LC-MS/MS) has become more widespread in the clinical laboratory, bridging the analytical gap between high-throughput (but interference prone) immunoassays and the highly specific (but labour intensive) technique of gas chromatography–mass spectrometry (GC-MS). This article discusses serum steroid measurement by LC-MS/MS and describes a multiplexed LC-MS/MS steroid panel recently launched at Imperial College Healthcare NHS Trust.

by Dr Emma L. Williams

Introduction
Historically steroid hormones have been measured, primarily in urine, by GC-MS and in serum and plasma by radio-immunoassay. Both techniques require sample extraction prior to analysis and for the former there is a need for derivatization to form volatile derivatives. Thus the assays are laborious and time consuming and have been the preserve of research and specialist laboratories. More recently automated immunoassays have been used in routine clinical laboratories, but these are notorious for being highly prone to interference as a result of their inherent specificity problems [1]. In recent decades LC-MS/MS has come to the fore, offering a promising alternative to immunoassays for high-throughput, specific measurement of serum steroids and it is now the method of choice in many clinical laboratories. LC-MS/MS measurement of serum steroids is informative in the clinical investigation of conditions such as hirsutism, polycystic ovarian syndrome (PCOS) and infertility. In addition LC-MS/MS steroid measurement forms part of a diagnostic triad, along with urine steroid profiling by GC-MS and whole gene sequencing of genomic DNA, for inherited steroidogenic defects including the congenital adrenal hyperplasias (CAH) and disorders of sexual differentiation.

LC-MS/MS measurement
Significant advances in LC-MS/MS technology have enabled the development of high-throughput, sensitive and precise assays for steroid measurement. Figure 1 depicts the biosynthetic pathways of steroidogenesis. LC-MS/MS assays have now been published for all of the steroids in this pathway, using a variety of approaches for sample preparation prior to analysis. Protein precipitation, liquid–liquid extraction, solid phase extraction and supported liquid extraction have all been used for the preparation step. In my laboratory, semi-automated off-line solid phase extraction has been implemented in order to achieve higher throughput. This extraction approach is used to prepare samples prior to ultra-performance (UP)LC-MS/MS analysis using electrospray ionization with detection by multiple reaction monitoring (MRM). The majority of steroids are measured in positive ionization mode, although we use negative ionization mode for aldosterone and dehydroepiandrosterone sulphate (DHEAS).

For accurate LC-MS/MS quantitation, stable isotope internal standards (IS) are required. Addition of IS to all samples, calibrators and quality controls (QCs) is carried out prior to extraction and LC-MS/MS analysis. The ratios of analyte to IS signals are determined to correct for effects of the matrix upon signal intensity, which may be due to ion suppression or enhancement. Typically in LC-MS/MS assays the IS will have two or more hydrogens replaced by deuterium atoms. The IS has a different mass and ion transition to the analyte, while retaining its chemical and physical properties and thus behaves the same way as the analyte throughout the analytical procedure. Carbon-13 labelled IS are increasingly being used as they have become more available. These co-elute more completely with the non-labelled analyte and are, therefore, more effective at correcting for matrix effects compared to deuterium labelling, which alters polarity and increases the possibility of non-co-elution.

An important factor to consider in steroid LC-MS/MS assays is that of specificity, given the similarities in structures of the various steroid intermediates in the steroidogenic pathway.
There are several examples of steroids that have the same molecular weight and are, therefore, isobaric. It is vital that these isobaric steroids are chromatographically resolved as they will undergo the same ion transitions in the mass spectrometer. If not resolved, they would be measured as if they were the same steroid and, therefore, be a cause of positive interference. For example 11-deoxycortisol and 21-deoxycortisol have the same molecular weight (Fig. 2) and undergo the same ion transitions, but can be chromatographically resolved using the selectivity of the mobile phase. It can be seen in Figure 3 that these steroids are successfully resolved in our laboratory method, which uses reverse phase T3 chromatography.

LC-MS/MS steroid assays
In the clinical laboratory, testosterone is the serum steroid most frequently measured by LC-MS/MS analysis. In the external quality assessment scheme offered by the United Kingdom National External Quality Assessment Service (UK NEQAS), 43 (21%) participating labs use LC-MS/MS, with the remainder relying upon automated immunoassays. In my laboratory, both measurement techniques are used, whereby all female samples with elevated immunoassay testosterone results >2.0 nmol/L are reflexed for LC-MS/MS confirmation. In a recent audit of over 5000 female samples in which testosterone was measured we found that of over 800 elevated samples reflexed for confirmation, 23% of these are subsequently found to have normal LC-MS/MS results within the reference range. It is, therefore, essential that elevated female immunoassay results are confirmed by LC-MS/MS to avoid falsely elevated results being reported. Norethisterone, a synthetic form of progesterone used in hormonal contraceptives, is a commonly encountered cause of positive interference in immunoassays for testosterone in female samples [2].

Advantages of multiplexed assays
Testosterone is measured in the investigation of females presenting with clinical signs of hyperandrogenism, e.g. acne and hirsutism and in the investigation of infertility and PCOS. Following the introduction of LC-MS/MS assays into the clinical laboratory for the combined measurement of testosterone and androstenedione it became clear that androstenedione is the cause of hyperandrogenism in a subgroup of patients with PCOS [3]. These cases previously may have been undiagnosed when the testosterone measured in isolation was found to be normal. This observation highlights the benefits of being able to measure two or more steroids simultaneously, which is not possible with radio-immunoassays or in routine automated immunoassays.

17-Hydroxyprogesterone (17-OHP) measurement is used to screen for 21-hydroxylase deficiency; the most common cause of CAH, accounting for ~85% of cases. 17-OHP sits at a branch point for either cortisol or androgen synthesis (Fig. 1) and accumulates when 21-hydroxylase is deficient. However, it can also be raised in normal newborns, particularly in premature neonates, and is influenced by birth weight and stress. In 21-hydroxylase deficiency, 21-deoxycortisol is formed as a side product from the accumulated 17-OHP in a reaction catalysed by 11-beta hydroxylase. The LC-MS/MS measurement of 21-deoxycortisol for the diagnosis of CAH was first described by Cristoni et al. [4] and it allows accurate diagnosis of 21-hydroxylase deficiency in newborns independent of prematurity, birth weight and stress [5]. Shackleton has proposed that a second tier panel comprising 17-OHP, cortisol, 21-deoxycortisol and androstenedione is used in newborn screening for 21-hydroxylase deficiency with a third tier of urinary GC-MS analysis to clinch the final diagnosis [6]. The addition of 11-deoxycortisol to this panel permits the diagnosis of 11-beta-hydroxylase deficiency, the second most common form of CAH. Such a panel has been applied to second tier testing for CAH [7].

In my laboratory a semi-automated solid phase extraction (SPE) LC-MS/MS method for the simultaneous measurement of androstenedione, testosterone and 17-OHP has been in use since April 2016. The SPE uses Waters Oasis PRiME HLB, 96 well, μ-elution plates and is performed using a Tecan Freedom Evo automated Liquid Handler. One hundred microlitres of sample is mixed with IS and proteins are precipitated with methanol and water. Supernatants are applied to the wells of the SPE plate and drawn through under vacuum. Following washing with 0.1% formic acid in 35% methanol, steroids are eluted with methanol and water enabling direct LC-MS/MS analysis of the eluates.
Using a Waters Acquity UPLC system, samples are injected onto a Waters Acquity UPLC HSS T3 column (2.1 × 50 mm) and separated by water/methanol/ammonium acetate/formic acid gradient elution. The analysis is performed using a Waters Acquity-TQD mass spectrometer in electrospray positive ionization mode. The analytes and their co-eluting isotopic ISs are detected using MRM. Quantifier transitions (m/z) monitored are 287>97 for androstenedione, 289>97 for testosterone and 331>97 for 17-OHP.

The method underwent full validation prior to implementation according to Clinical and Laboratory Standards Institute (CLSI) guidelines and as recommended by Honour [8] and demonstrated excellent linearity over the analytical range, with all r2 values ≥0.99. Overall process efficiency was 100–108.3%, demonstrating excellent recovery and minimal ion suppression/enhancement. Intra-assay precision was 2.6–8.1% for all analytes across the measurement range, and inter-assay precision varied from 4.9 to 10.8%. Analysis of UK NEQAS samples revealed minimal negative bias and the high specificity of the assay was confirmed by spiking and interference studies. The newly developed assay compared favourably with the stand-alone LC-MS/MS methods in use previously in our laboratory, with no requirement to re-derive reference intervals. This supra-regional assay service (SAS) accredited steroid panel assay has been in routine use in our LC-MS/MS laboratory since April 2016, streamlining the analytical service. The assay is carried out two or three times a week, with each full plate accommodating around 80 patient samples, plus standards and controls, with automated sample extraction completed in ~ 90 minutes and the LC-MS/MS sample to sample injection time is 5 minutes.

We have recently evaluated a seven steroid LC-MS/MS assay with the addition of cortisol, DHEAS, 11-deoxycortisol and 21-deoxycortisol into the panel. Figure 3 shows the total ion chromatogram of the steroids quantified by this assay. Using a Waters Acquity-TQD mass spectrometer and a slightly modified experimental set-up, the lower limits of quantification obtained were 16.5 nmol/L for cortisol, 2nmol/L for DHEAS, 7nmol/L for 11-deoxycortisol and 2nmol/L for 21-deoxycortisol.
In conclusion, LC-MS/MS steroid panels are a valuable addition to the diagnostic work up of patients being investigated for hyperandrogenism and in the investigation of steroidogenic defects. The increased availability of semi-automated, high-throughput LC-MS/MS assays for multiplexed steroid measurement has opened the door for their future application in targeted metabolomic research. Finally, in the clinical laboratory setting the future continues to look bright for the role of accurate and robust measurement by LC-MS/MS in place of immunoassays as the method of choice for routine serum steroid measurement.

References
1. Jones AM, Honour JW. Unusual results from immunoassays and the role of the clinical endocrinologist. Clin Endocrinol Oxf 2006; 64: 234–244.
2. Jeffery J, MacKenzie F, Beckett G, Perry L, Ayling R. Norethisterone interference in testosterone assays. Ann Clin Biochem 2014; 51: 284–288.
3. Livadas S, Pappas C, Karachalios A, Marinakis E, Tolia N, Drakou M, Kaldrymides P, Panidis D, Diamanti-Kandarakis E. Prevalence and impact of hyperandrogenemia in 1218 women with polycystic ovarian syndrome. Endocrine 2014; 47: 631–638.
4. Cristoni S, Cuccato D, Sciannamblo M, Bernardi LR, Biunno I, Gerthoux P, Russo G, Weber G, Mora S. Analysis of 21-deoxycortisol, a marker of congenital adrenal hyperplasia, in blood by atmospheric pressure chemical ionization and electrospray ionization using multiple reaction monitoring. Rapid Commun Mass Spectrom 2004; 18: 77–82.
5. Janzen N, Peter M, Sander S, Steuerwald U, Terhardt M, Holtkamp U, Sander J. Newborn screening for congenital adrenal hyperplasia: additional steroid profile using liquid chromatography-tandem mass spectrometry. J Clin Endocrinol Metab 2007; 92: 2581–2589.
6. Shackleton C. Clinical steroid mass spectrometry: a 45-year history culminating in HPLC-MS/MS becoming an essential tool for patient diagnosis. J Steroid Biochem Mol Biol 2010; 121: 481–490.
7. Rossi C, Calton L, Hammond G, Brown HA, Wallace AM, Sacchetta P, Morris M. Serum steroid profiling for congenital adrenal hyperplasia using liquid chromatography-tandem mass spectrometry. Clin Chim Acta 2010; 411: 222–228.
8. Honour JW. Development and validation of a quantitative assay based on tandem mass spectrometry. Ann Clin Biochem 2011; 48: 97–111.

The author
Emma L. Williams PhD, FRCPath
North West London Pathology, Imperial College Healthcare NHS Trust, London
W6 8RF, UK

E-mail: emma.walker15@nhs.net

In the human body, steroid hormones are involved in a variety of regulatory processes, which makes them also important diagnostic markers for a range of diseases. However, due to their high chemical similarity, they can represent a challenge for many assays – immunoassays in particular suffer from cross-reactivities. In comparison, LC-MS/MS-based assays provide high specificity in combination with the ability to determine several steroids in one run.

by Dr Marc Egelhofer

Steroids have a common distinct chemical structure – they consist of a cholesterol backbone with 3 hexane rings and a pentane ring. The hormones are synthesized in the adrenal cortex (corticosteroids) as well as in the reproductive organs (androgens, estrogens). Several doping agents are also artificial derivatives of the male sexual hormone testosterone, called anabolics, and are used abusively to increase muscle and bone synthesis.

With a distinguished role in regulatory processes of the human body, dysfunctional steroid release can be responsible for many diseases with sometimes extremely unspecific symptoms (see Table 1). One example is aldosteronism, where the adrenal glands produce excessive amounts of the steroid hormone aldosterone. This leads to lowered levels of potassium in the blood (hypokalemia) and an increased excretion of hydrogen ions (alkalosis). Patients suffer from muscle spasms, fatigue, headaches, high blood pressure, and muscle weakness. However, these symptoms can be attributed to many diseases, and only the clinical evaluation of aldosterone plasma levels can ensure a correct diagnosis. 

Challenging targets
The chemical similarity of the steroid structure can be a challenge, in particular in a clinical setting where requirements in specificity and selectivity need to be met. This problem becomes evident when looking at epidemiological studies of major diseases, where many different assay methods with a varying performance are used, resulting in an inability to compare data [1]. The discrepancies in assay performance also limit investigations where comparisons of absolute steroid concentration values are used, rather than relative levels.  For example, absolute steroid hormone concentrations are needed when analysing effects of hormonal threshold concentrations to obtain a certain disease outcome – or not.

Steroid profiling
A lot of the published literature and of our knowledge about the physiology of steroid hormones is based on radioimmunoassays (RIA). One of the reasons for discrepancies in values, however, is that immunoassays suffer from various interferences due to antibody cross-reactions with other steroid hormones. In contrast, mass spectrometry has been recognized as the best available method for the accurate analysis of steroids in biological samples [2]. It overcomes limitations of immunoassays, while also simplifying the sample preparation in comparison to GC-MS/MS analysis that requires lengthy derivatization processes to obtain the analytes in the gaseous phase for separation.

We have developed a CE-IVD assay for mass spectrometry (MassChrom Steroids) for the determination of 15 steroid hormones. The subsequent analysis takes place in multi reaction monitoring mode (MRM). In this mode, the first and second mass spectrometers are set to a fixed certain mass. MS1 selects only the molecular ion, and ions with a different mass are disregarded. The molecular ion then fragments in the collision cell and MS2 detects the characteristic fragment. The MRM mode makes it possible to determine several steroids in a single run, thereby reducing the time for analysis and increasing the effectiveness of the method. The 15 hormones that can be analysed with this method are divided into two panels for a clear separation of each of the analytes (see Figure 1).

The chromatographic setup, including the analytical column, is identical for all analytes, thereby eliminating the need to change columns or mobile phases between separate runs. Depending on requirements and throughput, sample preparation can be performed in 96 SPE well plates or SPE columns.  The assay has been tested on a range of systems, such as the AB Sciex Triple Quad 4500 or the Waters Xevo TQS instruments.

Salivary sampling 
Plasma sampling can represent a problem, in particular for parameters that need to be collected several times a day or under stress-free conditions. Saliva consists of 99.5% water, electrolytes, mucus, white blood cells, epithelial cells, glycoproteins and enzymes, though saliva is also a carrier of steroid hormones.  The speed at which they are transferred from blood into saliva is controlled by passage through the lipophilic layers of the capillaries and glandular epithelial cells. Consequently, the more lipophilic the molecules the faster is the transfer through these barriers. Salivary concentrations are therefore dependent on the lipophilic properties of the molecule ­­— lipid-soluble steroids such as cortisol have higher concentrations, whereas more hydrophilic substances such as dehydroepiandrosterone-sulfate (DHEA-S) have much lower concentrations relative to the free plasma levels [3].

One of the common medical indications of cortisol testing in saliva is the screening for Cushing’s syndrome, a pathological increase of cortisol [4]. This hypercortisolism can be due to an endogenous overproduction or based on the intake of exogenous glucocorticoids. Symptoms may include obesity, hypertension, hyperglycemia, muscle weakness and osteoporosis. However, these symptoms are also not specific – the majority of individuals with some or all of the symptoms will not suffer from Cushing’s syndrome, therefore, the analysis of cortisol plays a significant role in the identification of the disease.

Cortisol levels do vary significantly over the course of the day (see Figure 2), making it a requirement to measure several times a day. Salivary sampling represents a simple, non-invasive and, for the patient, stress-free sampling method [5]. After a short introduction, patients can collect their sample by themselves at home, which results in a simple process to obtain samples at different stages of the circadian cycle.

The non-invasive nature of the collection procedure also enables samples to be obtained from patients afraid of venipuncture without provoking an unwanted adrenal stress response, especially in children and phobic patients. A disturbing influence of stress-induced adrenal activity is less likely, making salivary sampling more reliable compared with serum, in particular in stress research and pediatric applications [3].

We have developed a CE-IVD method for the determination of cortisol and cortisone in saliva with a sample prep procedure that is performed by filtration and in just a few steps (see Table 2).

The use of stable isotopically labelled internal standards for both analytes ensures reproducible and reliable quantification of the parameters. The performance data are 96-105% for the recovery of spiked samples, an intraassay variation of CV = 2-5%, and interassay variation of CV = 2-7 %, and the lower limit of quantification is 0.27 µg/l (see Figure 3).

Conclusions
Immunoassays are widely used for the measurement of steroids, though it is accepted that these methods suffer from various interferences due to antibody cross-reactions with other steroid hormones. In contrast, LC-MS/MS has been recognized as the best available method for the accurate analysis of steroids in biological samples. LC-MS/MS overcomes many limitations of immunoassays, enhances diagnostic utility of the testing, and expands diagnostic capabilities in endocrinology. In addition to the superior quality of the measurements, LC-MS/MS can help in the standardization and harmonization of steroid testing among clinical laboratories. Commercial suppliers offer complete solutions from sample to result that allow the determination of steroids with LC-MS/MS as the gold standard and without the need to go through the development of an in-house method.

References
1. Stanczyk F. et al. Standardization of Steroid Hormone Assays: Why, How and When? Cancer Epidemiol. Biomarkers Prev. 2017; 16(9): 1713-1719.
2. Rosner W. et al. Position statement: Utility, limitations, and pitfalls in measuring testosterone: an Endocrine Society position statement. J Clin Endocrinol Metab 2017; 92(2): 405-13.
3. Gröschl M. Current Status of Salivary Hormone Analysis Clin. Chem. 2008; 1759 54(11): 1759-69.
4. Nieman L.K. et al. The diagnosis of Cushing’s syndrome: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2008; 93(5):1526-40.
5. De Palo EF et al. Human saliva cortisone and cortisol simultaneous analysis using reverse phase HPLC technique. Clin Chim Acta. 2009; 405(1-2): 60-5.

The author
Marc Egelhofer PhD*
Chromsystems Instruments & Chemicals GmbH, Am Haag 12, 82166 Gräfelfing, Germany

*Corresponding author,
egelhofer@chromsystems.de

Mild traumatic brain injury (mTBI) and concussion from sporting/recreational activities are relatively common. However, assessment of mTBI is difficult and many incidents of mTBI and concussion are unrecognized and/or not reported. Levels of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) peptide, a product of the proteolytic degradation of AMPA receptors, have been found to be raised in mTBI and the development of point-of-care (POC) tests based on the recognition of the AMPAR peptide is underway. Such POC tests will be useful at the pitch sideline or in the combat field for aiding objective diagnosis and management of subtle brain injury.

by Prof. Svetlana A. Dambinova, Rozalyn Heath and Dr Galina A. Izykenova

Introduction
Mild traumatic brain injury (mTBI) including concussion is the most frequent form of injury in military and civilian settings with the highest prevalence among young adults aged 15 to 24 years. Each year sports and recreational activities contribute about 3.8 million cases of mTBI in the USA [1], whereas brain injuries caused by explosions are the most common combat wounds in the military arena.

Assessment of mTBI regardless of origin is complicated. Many primary mTBIs, and particularly concussions, go unrecognized or are not reported when there is no loss of consciousness. Additionally, without sufficient reports of previous incidents, soldiers and competitive athletes are often subjected to multiple concussions. There are several challenges to identify immediate (or primary acute and subacute within 24 hours and up to 2 weeks respectively) impact, secondary (beyond 14 days) and cumulative (brain-related seizures) consequences that might follow multiple concussions or mTBI.

Normally, acute subclinical concussions associated with micro-edema formation that is reversible and not visible on conventional computed tomography and magnetic resonance imaging (MRI) methods. Advanced neuroimaging techniques (diffusion tensor imaging, functional MRI, and positron emission tomography) that can register minor structural and microvascular changes are primarily used for research. These modalities are not available in emergency situations or for routine clinical evaluations and have a limited application in persons with metal implants [2] or claustrophobia [3].

Currently, there is an unmet diagnostic need to reveal brain micro-damage following concussion by use of a rapid and affordable assay detecting, for instance, neurotoxicity (immunoexcitotoxicity) biomarkers in the bloodstream [4]. Analogous to NR2 peptide as a biomarker for cortical lesions in transient ischemic attack (TIA)/strokes [5], we proposed that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) peptide marker is able to differentiate subtle brain injury in white matter associated with concussions.

Neurotoxicity biomarkers in mTBI
It is known that the family of ionotropic glutamate receptors (GluRs) implying N-methyl-D-aspartate (NMDAR), AMPAR and kainite receptors are involved in the regulation of synaptic connectivity in cortical/subcortical and brainstem areas [4,5]. Recently, it was shown that AMPAR represents a biomarker for the neurotoxicity cascade underlying subtle brain injury [6].

The family of location-specific GluRs is involved in more than 80% of cortical and subcortical neuronal communications underlying superior mental functions [7]. AMPAR is primarily distributed in the forebrain and subcortical pathways [8], and strategically located on surfaces of small cerebral arteries regulating blood circulations in white matter substructures [9]. Symptomatically the impact to brain may lead to executive brain dysfunctions associated with subcortical areas. Visual and cognitive deficits (including problems with memory, intellect, concentration and attention) might be an aftermath of subtle repetitive injuries to deep brain structures due to more severe cases of mTBI.

In acute and subacute phases of mTBI, a massive release of glutamate, which upregulates excitotoxic AMPARs has been detected [4]. The GluR1-subunit of N-terminal AMPAR fragments is rapidly cleaved by extracellular proteases and fragments carrying immune active epitopes released into the bloodstream through the compromised blood–brain barrier (BBB). This degradation product can be detected directly in the blood as AMPAR peptide fragments (molecular weight 5–7 kDa) [4]. The protective effects of a compromised BBB impacting neurotoxicity are exacerbated further when accompanied by a delayed immunological response generating peripheral anti-CNS antibodies [10].

Concussion assays development
To detect AMPAR peptide, magnetic-particle-based enzyme-linked immunosorbent assay (MP-ELISA) containing unique reagents has been developed. MP-ELISA involved a sandwich or ‘bridging’ assay where the suspended in solution microparticles coated by capture antibodies are binding to two epitopes of AMPAR peptide and reaction is revealed by probe-detection antibodies (Fig. 1). The probe is an enzyme that generates a colour reaction. The assay includes control samples (reference standards) produced synthetically or as a fusion human protein.

A feasibility study detecting the AMPAR peptide in a single blood draw taken from club sport athletes and professional football players in acute and subacute stage of concussions evaluated cut-offs of 0.4 and 1.0 ng/mL respectively for the assessment of single and recurrent concussions (Table 1) [11,12]. The predictive value of the test was assessed as 91% with a likelihood ratio of 11–12 for recognizing individuals with mTBI. If the test at a cut-off point of 0.4 ng/mL was negative, the post-test probability for a single concussion would be <4%.

AMPAR peptide concentrations in plasma for a military cohort suffering mTBI showed increased levels with an average concentration of 2.98 ng/mL [13]. In this study, the optimal cut-off value for recurrent mTBI was similar to professional players (Table 1), at which a positive predictive value of 93% was achieved. The trade-offs between true-positive and false-positive yielded in an area under the receiver operating characteristic (ROC) curve of 0.97.

Early experimental and clinical research of antibodies to AMPA receptors (AMPAR Ab) as an immunoexcitotoxicity biomarker has demonstrated their diagnostic value in detecting pathological brain-spiking activity and epileptic seizures [14,15] as a consequence of traumatic brain injury, thereby representing a prognostic risk factor [16]. Clinical studies of GluR1 antibodies in adult patients with different chronic neurological pathology (n=1866) performed in Russia, Germany, Ireland, Poland and the USA have demonstrated diagnostic potential (sensitivity of 86%-88% and specificity of 83–97%) in assessment of post-traumatic seizures.

In sport-related multiple concussions, AMPAR Ab values remained abnormally high in the blood of some athletes who had headaches and visual problems. It was suggested that this finding may reflect persistent changes in the subcortical areas of the brain. The diagnostic value of AMPAR Ab (sensitivity of 86–88%, specificity of 83–97% at 1.5 ng/ml cut-off) in assessment of seizures defined by electroencephalogram (EEG) with history of sustained single or multiple TBI have been demonstrated for children and adult patients indicating a development of ‘chronic’ conditions [13].

Sideline testing of AMPAR peptide and antibody
Recognizing the medical need for sideline testing for mTBI (largely for emergency care), a point-of-care (POC) testing platform has been recently undertaken for AMPAR peptide and antibody assays are being tested in clinical studies (www.drdbiotech.com).

A lateral flow sandwich assay to detect AMPAR Ab consists of a blood filtering sample pad, a pad containing gold nanoshells conjugated to protein A, a nitrocellulose strip with immobilized AMPAR peptide, a control line, and a cellulose absorbent. Applied to the blood filter, erythrocytes are removed from the sample, which passes to the conjugate pad where AMPAR IgGs are captured by protein A. When the complex reaches the peptide test line stripe on nitrocellulose, it binds to the test line yielding a visible signal with intensity proportional to the concentration of the AMPAR Ab presented in the sample (Fig. 2).

The control line then captures a portion of the remaining nanoshells regardless of the presence or absence of the peptide (Fig. 3).

The AMPAR peptide prototype test that works on a similar principle, employed gold nanoparticles as the signal-generating species covalently bound to specific antibodies against AMPAR peptide. This assay captures the AMPAR peptide from the blood sample between two different Abs, one immobilized on the nitrocellulose and the other on the gold nanoshells. This ‘bridging’ of the analyte leads to the immobilization of the particles at the test line producing a colour signal.

Conclusion
The laboratory assessment of concussion at the pitch sideline in competitive contact sports or on the field of combat is required to assist in the objective confirmation of when an injured athlete should return to play or a soldier may return to duty. Accurate diagnosis of concussion and appropriate management of subtle brain injury to prevent damage to the long-term health of athletes and others at risk of re-injury. Specific brain biomarkers detected by a rapid blood test would have important diagnostic/prognostic capabilities, particularly for concussion, to objectively evaluate early signs of further brain-function deterioration and assist in navigating personalized therapy when required. Clinical use of blood tests can reliably detect subtle brain impact, predict consequences and aid in triaging persons with persistent symptoms after a recurrent concussion for diffuse tensor or diffuse-weighted imaging modalities of MRI [17].

Early identification of concussion has the potential to become a key component of a successful treatment strategy and outcome monitoring. Advances in analytical assay technologies have made it possible to develop a rapid, cost-effective test that can be used to target populations and select a risk group for post-traumatic seizures to direct to the immediate attention of a specialist.

The opportunity of using ‘yes/no’ lateral flow tests without a need for an instrumental readout, being simply read by eye, would make a huge difference in supplying coaches with reliable, simple and rapid tests in return-to-play decisions. There is also a high demand for portable POC tests for mTBI diagnosis in military combat and civilian emergency settings.

References
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15. Dambinova SA, Granstrem OK, Tourov A, Salluzzo R, Castello F, Izykenova GA. Monitoring of brain spiking activity and autoantibodies to N-terminus domain of GluR1 subunit of AMPA receptors in blood serum of rats with cobalt-induced epilepsy. J Neurochem 1998; 71: 2088–2093.
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The authors
Svetlana A Dambinova*¹ DSc, PhD; Rozalyn Heath¹; Galina A Izykenova² PhD
¹Brain Biomarkers Research Laboratory, DeKalb Medical Center, Decatur, GA, USA
²GRACE Laboratories, LLC, Atlanta, GA, USA

*Corresponding author
E-mail: dambinova@aol.com