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
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Although controversy continues to surround the use of PSA testing as a screening aid, much of that actually relates to misconceptions about how to implement PSA and how best to follow-up on a suspicious test result. The dramatic spike in prostate cancer detection and decline in mortality due to prostate cancer that accompanied the introduction of PSA screening in the early 1990s, and the results of more recent long-term studies in large patient populations are evidence of the value of PSA testing when properly understood and applied.
Prostate cancer represents 27% of all cancers in men and is the second deadliest form of cancer in this population. In 2016, an estimated 26,000 men died of prostate cancer. The disease is especially prevalent among African-American men and men who have first-degree relatives with prostate cancer. More aggressive prostate cancer tends to occur more often in younger men…. Download white paper to continue reading
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, /in Featured Articles /by 3wmediaCharlie Gard, who recently died just before his first birthday, was admitted to Great Ormond Street Hospital (GOSH) when he was two months old. Founded 265 years ago, and classed as one of the top four pediatric hospitals globally, GOSH has a dedicated international workforce, including some of the world’s leading doctors, surgeons and pediatric nurses. As well as offering tireless and expert care for its young patients, GOSH is involved in over 800 research projects.
Charlie was provisionally diagnosed with mitochondrial DNA depletion syndrome (MDDS) soon after admission, and genetic tests confirmed the diagnosis. The baby had two mutations affecting his RRM2B gene, preventing the synthesis of ribonucleotide reductase, an enzyme that plays a key role in maintaining the mitochondrial deoxinucleotide triphosphate pool needed for mitochondrial DNA synthesis. Only 15 other patients have been described with RRM2B mutations in the medical literature: in all cases rapid encephalopathy and muscle breakdown caused death within a few months of onset. A team of GOSH experts did consider experimental nucleoside bypass therapy (NBT) when the baby was five months old (ethical approval was first necessary), but they concluded that his rapidly deteriorating condition did not warrant such an intervention.
One can sympathize with the child’s parents as they desperately searched for a possible cure for their terminally ill infant, waging a protracted litigation to have him treated with experimental NBT in the US by Columbia University’s Dr Michio Hirano. One cannot, however, condone the belligerent demands of around 200, 000 members of the medically ignorant public that Charlie be released from experts at GOSH, or the interventions of ill-informed public figures such as Donald Trump and even the Pope.
Dr Hirano claimed that NBT – he ‘retains a financial interest’ in the compounds he prescribes – could benefit the patient. His relevant work concerns the TK2 mutation, a more frequent cause of MDDS with a more varied prognosis. His team has recently published a paper showing that NBT is beneficial in TK2 deficient mice, and 18 patients, mostly in Italy and Spain, are undergoing experimental treatment. No peer-reviewed papers on patient prognosis have yet been published, and there are no ongoing clinical trials with NBT. Furthermore without seeing Charlie, the GOSH team or medical notes until the child was already 11 months old, Dr Hirano (and the popular press) blamed the legal procedure, declaring that it was now too late for treatment!
An evidence-based cure for MDDS is surely needed: hopefully that will be Charlie’s legacy.
Intraductal tubulopapillary neoplasm of the pancreas
, /in Featured Articles /by 3wmediaIntraductal 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
Urinary galactitol quantitation by gas chromatography–mass spectrometry for the diagnosis of galactosemia
, /in Featured Articles /by 3wmediaGalactosemia is an inborn error of metabolism caused by the deficiency of any of the three principal enzymes (GALT, GALK and GALE) involved in the Leloir pathway. The application of urinary galactitol as a diagnostic and monitoring marker for galactosemia has been extensively researched but the practice varies in different centres. The Willink Biochemical Genetic Laboratory has recently developed and evaluated a method to quantitate urinary galactitol by gas chromatography–mass spectrometry and revisited its use as a first-line diagnostic test for galactosemia. The analytical performance characteristics of the method, established age-related reference ranges, and the relationship between urinary galactitol excretion and hepatic dysfunctions will be discussed.
by Yuh Luan Choo, Teresa Hoi-Yee Wu, Jackie Till and Dr Mick Henderson
Galactosemia: an overview
Galactosemia is a group of three inborn errors of galactose metabolism each with an autosomal recessive inheritance pattern. The deficiency or absence of galactose-1-phosphate uridyltransferase (GALT), galactokinase (GALK) or galactose-4-epimerase (GALE) enzymes involved in the Leloir pathway leads to toxic accumulation of galactose, hence the term ‘galactosemia’. Classical galactosemia is the most common form of galactosemia caused by GALT deficiency. The prevalence of classical galactosemia varies greatly across different populations in the world, i.e. 1 : 10 000–1 : 20 000 live births in Ireland, 1 : 25 000-1 : 44 000 live births in the United Kingdom, 1 : 50 000 cases in the United States, 1 : 100 000 newborns in Japan, and relatively low frequency in Asian populations [1]. GALK deficiency has a high prevalence of 1 : 1600 in the Romani Gypsy population [2], but in other populations GALK and the GALE deficiency are more rare and can present with acute and life-threatening clinical signs and symptoms, typically manifested within the first few days to weeks of life after consumption of breast milk and galactose-containing formula. Clinical symptoms such as jaundice, vomiting, failure to thrive and poor feeding are commonly observed in galactosemic babies [3]. Signs and symptoms of abnormal carbohydrate metabolism, kidney and liver dysfunction including aminoaciduria, hepatomegaly, hypoglycemia and elevated blood galactose and urinary galactitol are characteristic of this disorder. Untreated galactosemia can potentially lead to neonatal death. Early diagnosis and treatment is critical and usually life-saving. However, there are long-term clinical complications, including cataracts, short stature, neurodevelopmental problems, premature ovarian failure, developmental delay and impaired cognitive functions [4].
Biochemical tests for galactosemia and their limitations
Newborn bloodspot screening (NBS) for galactosemia is not currently recommended by the United Kingdom Newborn Screening Committee because it fails to meet their strict criteria. Current tests have high false-positive rates and early treatment is only partially successful. However, galactosemia is frequently detected under the existing protocol owing to affected babies having elevated phenylalanine (≥200 µmol/L) and tyrosine (≥240 µmol/L) and so are investigated for probable liver diseases [5].
To date, a small number of laboratory tests are offered by specialist metabolic centres in the UK to aid the diagnosis and monitoring of galactosemia, including urinary sugar chromatography, the Beutler fluorescent spot test, urinary galactitol quantitation, quantitative assays of erythrocyte GALT, GALK and GALE enzymes, genetic analysis and galactose-1-phosphate (Gal-1-P) analysis (Table 1).
Urinary sugar chromatography
Increased urinary excretion of galactose, a feature of galactosemia, will give rise to a positive reducing substances result. The identification of the sugar is possible by a chromatography technique, as is the field method in the UK. These are useful first-line tests; however, false-negative results may be seen in patients who have already started a lactose-free diet.
Beutler test
Another commonly used first-line test that qualitatively detects the activity of GALT is the Beutler fluorescent spot test. This is a robust, technically simple test that works well in most situations. However, false-negative results could be expected following a blood transfusion. Also as the endogenous enzyme glucose-1-phosphate dehydrogenase (G6PD) is used as a linked enzyme in the Beutler method, G6PD deficiency will lead to a false-positive result.
GALT, GALK and GALE enzyme assay
The gold standard diagnostic tests are the quantitative assay for GALT, GALK and GALE to distinguish and confirm the three forms of galactosemia. However, blood transfusion will affect the validity of the enzyme results in the same manner as the Beutler test. Detection of the enzyme activities in lymphocytes may be helpful but all of these assays are laborious and time-consuming.
Galactose-1-phosphate (Gal-1-P) quantitation
The quantitative measurement of galactose-1-phosphate (Gal-1-P) is another technically complicated test that is useful to support the diagnosis in all forms of galactosemia. Gal-1-P has also been used as a biomarker to monitor dietary compliance in galactosemic patients; however, it is not a reliable marker for long-term monitoring because it reflects only the galactose ingestion in the past 24 hours and poorly correlates with long-term clinical outcome [6].
Urinary galactitol quantitation
Urinary galactitol, an end product of galactose formed by an alternative pathway, is invariably excreted in significant quantities in patients with all forms of galactosemia. As galactose is produced endogenously, the level of urinary galactitol is expected to be less affected by the dilutional effect of the blood transfusion or the exogenous/dietary source of galactose. In comparison to normal healthy controls, urinary galactitol excretion is significantly elevated at birth in all forms of galactosemia, including the milder phenotypes of GALT, i.e. S135L homozygosity [7] and in the Duarte variants [8]. The level of urinary galactitol decreases rapidly following commencement of dietary treatment but still remains above the reference ranges for normal healthy controls [7]. However, several studies have shown that galactitol is not correlated with dietary galactose intake or erythrocyte Gal-1-P concentration [8], nor with the development of long-term complications in patients with classical galactosemia [9]. In addition, the high intra-individual biological variability of urinary galactitol may limit its value in disease investigation and monitoring [10].
The practice in the diagnosis and monitoring of galactosemia varies widely, in particular on the use of urinary galactitol. The latest international guideline for classical galactosemia recommended that although urinary galactitol is unsuitable for disease monitoring, it could be used as a ‘supportive diagnostic test’ following blood transfusions [11], a treatment frequently used in neonatal care units. However, this test is not widely available and may be underused. Further research is necessary to evaluate the clinical usefulness of urinary galactitol in aiding the diagnosis and monitoring of galactosemia.
Measurement of urinary galactitol
Galactitol is the toxic metabolic by-product formed intracellularly following reduction of galactose by aldose reductase. Galactitol is subsequently excreted in the urine as it cannot be further oxidized by sorbitol dehydrogenase. This sugar alcohol has been extensively studied in urine, blood, amniotic fluid, liver, kidney, cardiac muscle, skeletal muscle, brain and the eye lens. Most clinically relevant data were derived from investigations on urinary galactitol. The analytical methods employed for identification and measurement of urinary galactitol have involved gas–liquid chromatography with trimethylsilyl (TMS) or methoxylamine-acetate derivatives, isotope dilution gas chromatography–mass spectrometry (GC-MS) with acetate derivative, reverse-phase high-performance liquid chromatography, thin-layer chromatography and proton magnetic resonance spectroscopy. Most research reported that GC-MS is particularly suitable for the quantitation of urinary polyols as it offers high resolution, great sensitivity and rapid analytical speed [12].
Urinary galactitol quantitation by gas chromatography–mass spectrometry
The Willink Biochemical Genetic Laboratory conducted a preliminary study on urinary galactitol quantitation by using a GC-MS method to evaluate the key analytical validation components, establish the age-related reference ranges, and to study the relationship between urinary galactitol excretion and hepatic dysfunctions. The study included plain urine samples from two known patients with galactosemia, random urine samples from eight unaffected patients with suspected hepatic dysfunction, and 120 individuals unaffected by galactosemia, received in the Willink Laboratory for a metabolic screen. The procedure was modified from the method described by Pettit et al. and Allen et al. based on the method principle of acetate derivatives formation followed by separation and detection using GC-MS [13, 14]. The method was linear from 2.5 µmol/L to 330 µmol/L. The lower limit of detection (LoD) and lower limit of quantification (LoQ) were 3 µmol/L and 9 µmol/L. Intra- and inter-assay precisions were 1.41–6.22% and 2.54–17.04% respectively at levels across the measuring range. We used a total of 27 samples from the ERNDIM (European Research Network for evaluation and improvement of screening, Diagnosis and treatment of Inherited Disorders of Metabolism) ‘Specialist Assays in Urine’ external quality assessment (EQA) scheme to test if our method was in agreement with those of other specialist laboratories. Figure 1 showed that the results from the GC-MS method were in good agreement with the method means (R2=0.944). We showed that samples for urinary galactitol measurement were stable up to 7 days under storage at −20 °C, 4 °C and room temperature. Our findings and other studies demonstrated that urinary galactitol excretion in both normal and galactosemic subjects are age-dependent, with the highest excretion at a younger age (Fig. 2). A minimal amount of galactitol can be found in urine samples of healthy individuals owing to the generation of galactose by endogenous metabolic reactions. Newborns are expected to excrete a greater amount of galactitol than older children as the neonatal liver is not yet fully developed and, thus, less effective in metabolizing the increased load of galactose after milk feeding. The age-related reference ranges were ≤85, ≤68, ≤29, ≤23, ≤9 and ≤4 µmol/mmol creatinine for the 0–3 months, 4–11 months, 1–2 years, 3–6 years, 7–15 years and >15 years age groups, respectively. In our study, galactosemic patients excreted 9-fold to ≥800-fold more urinary galactitol than the age-matched control group, whereas non-galactosemic patients with suspected hepatic dysfunction excreted 3-fold more. An elevated urinary galactitol result alone is does not identify whether galactosemia is caused by enzyme deficiency in the Leloir pathway or by other secondary causes. It is of utmost importance to consider further biochemical and radiological investigations for patients with hepatic dysfunctions and metabolic disorders in order to differentiate and confirm the diagnosis of hypergalactosemia.
Conclusion and future work
Further work is required for a comprehensive analytical and clinical validation of the test method, but our preliminary data are promising and demonstrate that the GC-MS quantitation of urinary galactitol would be acceptable for the diagnosis of galactosemia. Urinary galactitol is potentially very useful as a supportive diagnostic test following blood transfusions and its use should be encouraged. Its application as a first-line test for all forms of galactosemia is undisputable. A full evaluation of its clinical application will be possible following implementation of this assay into routine service in the Willink Biochemical Genetics Laboratory.
Acknowledgements
We would like to thank Graeme Smith and James Cooper for their technical expertise in helping to set up and validate the GC-MS assay for galactitol in our laboratory. We would also like to thank Ann Brown and the staff of the Clinical Chemistry Department at Southmead Hospital, Bristol, for sharing their in-house standard operating procedure for this method and demonstrating its use within their laboratory.
The Willink Laboratory acknowledges the use of data derived from ERNDIM EQA materials in this publication. The use of ERNDIM EQA materials does not imply that ERNDIM endorses the methods used or the scientific validity of the findings in this publication. ERNDIM (www.erndim.org) is an independent, not for profit foundation that provides EQA schemes in the field of inborn errors of metabolism with the aim of improving diagnosis, treatment and monitoring of inherited metabolic diseases.
References
1. Saleem U, Mahmood S, Kamran SH, Mutt MA, Ahmad B. Prevalence, epidemiology and clinical study of galactosemia. J App Pharm 2012; 4(1): 524–530.
2. Kalaydjieva L, Perez-Lezaun A, Angelicheva D, Onengut S, Dye D, Bosshard NU, Jordanova A, Savov A, Yanakiev P, et al. Founder mutation in the GK1 gene is responsible for galactokinase deficiency in Roma (gypsies). Am J Hum Genet 1999; 65(5): 1299–1307.
3. Waggoner DD, Buist NRM, Donnell GN. Long-term prognosis in galactosaemia: results of a survey of 350 cases. J Inherit Metab Dis 1990; 13(6): 802–818.
4. Walter JH, Collins JE, Leonard JV, Chiswick M and Marcovitch H. Recommendations for the management of galactosaemia commentary. Arch Dis Child 1999; 80(1): 93–96.
5. UK National Screening Committee. Screening for galactosaemia: external review against programme appraisal criteria for the UK National Screening Committee (UK NSC). Bazian Ltd. 2014. http://legacy.screening.nhs.uk/screening-recommendations.php.
6. Van Calcar SC, Bernstein LE, Rohr FJ, Scaman CH, Yannicelli S, Berry GT. A re-evaluation of life-long severe galactose restriction for the nutrition management of classic galactosemia. Mol Genet Metab 2014; 112(3): 191–197.
7. Palmieri M, Mazur A Berry GT Ning C, Wehrli S, Yager C, Reynolds R, Singh R, Muralidharan K, et al. Urine and plasma galactitol in patients with galactose-1-phosphate uridyltransferase deficiency galactosaemia. Metabolism 1999; 48: 1294–1302.
8. Ficicioglu C, Hussa C, Gallagher PR, Thomas N, Yager C. Monitoring of biochemical status in children with Duarte galactosemia: utility of galactose, galactitol, galactonate, and galactose 1-phosphate. Clin Chem 2010; 56(7): 1177–1182.
9. Cleary MA, Heptinstall LE, Wraith JE, Walter JH. Galactosaemia: relationshiop of IQ to biochemical control and genotype. J Inherit Metab Dis 1995; 18, 151–152.
10. Hutcheson ACJ, Murdoch-Davis C, Green A, Preece MA, Allen J, Holton JB, Rylance G. Biochemical monitoring of treatment for galactosaemia: biological variability in metabolite concentrations. J Inherit Metab Dis 1999; 22(2): 139–148.
11. Welling L Bernstein LE, Berry GT, Burlina AB, Eyskens F, Gautschi M, Grünewald S, Gubbels CS, Knerr I, et al. International clinical guideline for the management of classical galactosaemia: diagnosis, treatment, and follow-up. J Inherit Metab Dis 2017; 40(2): 171–176.
12. Haga H, Nakajima T. Determination of polyol profiles in human urine by capillary gas chromatography. Biomed Chromatogr 1989; 3(2): 68–71.
13. Pettit BR, King GS, Blau K. The analysis of hexitols in biological fluid by selected ion monitoring. Biomed Mass Spectrom 1980; 7(7): 309–313.
14. Allen JT, Holton JB, Lennox AC, Hodges IC. Early morning urine galactitol levels in relation to galactose intake: A possible method of monitoring the diet in galactokinase deficiency. J Inherit Metab Dis 1988; 11(S2): 243–245.
The authors
Yuh Luan Choo1 MSc; Teresa Hoi-Yee Wu2 MSc, FRCPath; Jackie Till2 BSc; Mick Henderson*2 PhD, FRCPath
1Faculty of Medical and Human Science, University of Manchester, Manchester
M13 9PL, UK
2Willink Biochemical Genetics Laboratory, Manchester, Manchester M13 9PL, UK
*Corresponding author
E-mail: Mick.henderson@nhs.net
LC-MS/MS measurement of serum steroids in the clinical laboratory
, /in Featured Articles /by 3wmediaIn 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
Making standardization simple
, /in Featured Articles /by 3wmediaMass spectrometry (MS) is a well-known and broadly used analytical technique, and one that is particularly effective when coupled with liquid chromatography (LC). LC-MS/MS operates by analyte separation, ionization, mass analysis and detection, and lends itself as an ideal technique to meet the needs of a range of laboratory types. Over the past decade, LC-MS/MS has been applied across several different fields of clinical diagnostics and has become commonplace for forensic and clinical toxicology. However, until now it has only been used across a limited number of specialities, including endocrinology and therapeutic drug monitoring.
By Professor Brian Keevil and Dr Sarah Robinson
Such a powerful technique has the potential to bring significant advantages to the clinical setting, and would enable clinicians to analyse multiple analytes at greater specificities than immunoassay-based methods. It has the potential to supersede alternative methods since it avoids the issues surrounding interferences and the subsequent generation of unreliable data. Even with such advantages, LC-MS/MS has not yet been further adopted by the clinical community. The lack of an automated system has limited its suitability to routine clinical use, while also presenting challenges to laboratories under pressures to standardize and harmonize their practices. Current LC-MS/MS systems involve multiple and complex manual stages that are open to human error while being both time- and labour-intensive. Furthermore, the lack of standardization of LC-MS/MS methods is deterring clinical labs from benefiting from their advantages.
Standardization is critical in clinical laboratories since it is necessary to ensure the correct results are obtained and they are in accordance with results from other labs, especially for therapeutic drug monitoring and endocrine applications.
The challenge of standardization
One of the barriers to more widespread LC-MS/MS use is the lack of properly standardized methods and different laboratories will often use a wide range of techniques, equipment and internal standards. Together, these factors may mean that different results are generated from the same sample.
This level of variation makes it challenging to obtain proper standardization of LC-MS/MS results and is highly problematic. Not only does it become difficult to control results within a lab and ensure they remain comparable year on year, but it can create discrepancies between labs. This could ultimately lead to incorrect patient diagnoses and clinicians recommending the wrong treatment programmes.
The drive for change
Until now, LC-MS/MS systems have been designed with the research laboratory in mind and, as such, are highly configurable making them great for developing methods. However, the needs of the clinical lab are different from those of the research community. The clinical setting requires a dedicated system that not only promotes, but also facilitates standardization. Studies have shown that, through careful use of the same instrument, column and methods, it is possible to generate consistent and reliable resulting data from LC-MS/MS systems based at different laboratories. There is currently a drive from organizations, such as the International Federation of Clinical Chemistry (IFCC), the Centers for Disease Control and Prevention (CDC), and the Endocrine Society, to harmonize assays across laboratories to improve levels of quality. The adoption of one dedicated system among an entire network of laboratories would not only satisfy this organizational drive, but also help clinicians be confident that the data across their entire network is standardized, and thus comparable and repeatable.
The availability of a dedicated system with standardized methods and procedures would make this process significantly easier and remove one of the primary barriers to uptake of this gold standard technique. A dedicated system would need to be optimized for the specific methods run by each laboratory, and available with columns, reagents, calibrators and controls that are consistent and designed specifically for the system. This would help to ensure all data generated is both reproducible and accurate – paramount to patient diagnosis and care. In addition, a clinical LC-MS/MS system would need to be automated and easy to use. Clinical labs are extremely busy so even the most junior members of the staff must be able to operate the instrument and walk away with the confidence that samples are being analysed without error or the need for manual intervention. A system such as this would help to ensure patients were properly diagnosed and appropriate treatment plans devised.
Breaking through the barrier
If a network of laboratories decided to start using a dedicated clinical analyser, it would be able to adopt common reference ranges and reagents, which would provide much greater confidence in the consistency of results. For example, if a patient was transferred to a different hospital mid-way through treatment then there would be a level of assurance that the test results would be the same from both facilities. The data would therefore be directly comparable as long as both labs were using the same dedicated LC-MS/MS system.
Proper standardization is extremely important, yet challenging, and is a key consideration when deciding on an analytical method for implementation. An automated, dedicated clinical LC-MS/MS system would enable inter-laboratory standardization, while allowing interference-prone immunoassay-based tests to be phased out and replaced by clinical LC-MS/MS analysers. The results obtained from one laboratory would then be consistent over many years, and match those results generated from the same patient samples in other labs using the same system. Furthermore, such a system could be operated by the entire laboratory team, removing the need for in-depth and specialist training. This ease of use would decrease the investment required in training, while freeing up more experienced team members to focus on their research.
Conclusion
Analytical techniques are a core component to clinical workflow to ensure accurate patient diagnosis and treatment. LC-MS/MS has clear advantages over alternative immunoassay-based methods, with the ability to analyse multiple analytes at greater specificities. However, its uptake across the clinical community has been slow. This is because LC-MS/MS systems to date have been developed for use in research laboratories, and although the data have been demonstrated to be of high quality, the technology does not simply translate to the needs of the clinical lab.
With analytical needs that directly correlate to patient treatment plans, analytical methods within the clinical lab need to be automated, standardized, reliable and provide walk-away capabilities. This clear need for a dedicated analytical technique has driven the development of the new Thermo Scientific™ Cascadion™ SM Clinical Analyzer*. This dedicated clinical LC-MS/MS system is accurate, easy to use, and has been designed specifically for the clinical laboratory, facilitating standardization both on an inter- and intra-laboratory level to enable clinicians to fully leverage the power of this technique. The impact of this system would help laboratories and laboratory networks to meet their clinical needs.
To find out more, visit www.thermofisher.com/cascadion
*This product is in development and not available for sale. This product is not CE marked or FDA 510(k) cleared.
The authors
Professor Brian Keevil1 and Dr Sarah Robinson2
1Consultant Clinical Scientist and Head of the Clinical Biochemistry Department, University Hospital of South Manchester
2Market Development Specialist, Thermo Fisher Scientific