- Email: [email protected]
Magnetic resonance spectroscopy of bile in the detection of cholangiocarcinoma
Letters to the Editor J. Trojan received research funding and served on the advisory board for Bayer Health Care.
References [1] Bhoori S, Toffanin S, Sposito C, Germini A, Pellegrinelli A, Lampis A, et al. Personalized molecular targeted therapy in advanced, recurrent hepatocellular carcinoma after liver transplantation: a proof of principle. J Hepatol 2010;52:771–775. [2] Valdivieso A, Bustamante J, Gastaca M, Uriarte JG, Ventoso A, Ruiz P, et al. Management of hepatocellular carcinoma recurrence after liver transplantation. Transplant Proc 2010;42:660–662. [3] Graziadei I, Finkenstedt A, Nachbaur K, Zoller H, Mark W, Vogel W. Outcome of patients with recurrent hepatocellular carcinoma after liver transplantation. J Hepatol 2010;52:A214. [4] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378–390. [5] Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology 2008;135:1972–1983. [6] Toso C, Merani S, Bigam DL, Shapiro AM, Kneteman NM. Sirolimus-based immunosuppression is associated with increased survival after liver transplantation for hepatocellular carcinoma. Hepatology 2010;51:1237–1243.
[7] Schnitzbauer AA, Zuelke C, Graeb C, Rochon J, Bilbao I, Burra P, et al. A prospective randomised, open-labeled, trial comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing liver transplantation for hepatocellular carcinoma. BMC Cancer 2010;10:190. [8] Newell P, Toffanin S, Villanueva A, Chiang DY, Minguez B, Cabellos L, et al. Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. J Hepatol 2009;51:725–733. [9] Welker MW, Lubomierski N, Gog C, Herrmann E, Engels K, Vogl TJ, et al. Efficacy and safety of sorafenib in advanced hepatocellular carcinoma under daily practice conditions. J Chemother 2010;22:205–211. [10] Blanchet B, Billemont B, Barete S, Garrigue H, Cabanes L, Coriat R, et al. Toxicity of sorafenib: clinical and molecular aspects. Expert Opin Drug Saf 2010;9:275–287.
Oliver Waidmann Wolf-Peter Hofmann Stefan Zeuzem Joerg Trojan* Medizinische Klinik 1, Klinikum der Johann-Wolfgang, Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany * Tel.: +49 069 6301 7860; fax: +49 069 6301 83776 E-mail address: [email protected] (J. Troja)
Magnetic resonance spectroscopy of bile in the detection of cholangiocarcinoma To the Editor: We would like to draw your attention to some of the drawbacks in a recent article published by Wen et al. in your journal [1], on NMR-based metabolomics of bile samples in distinguishing cholangiocarcinoma from benign biliary diseases. The authors performed orthogonal partial least square discriminant analysis (OPLS-DA) of 1H NMR spectra of bile samples obtained from patients with cholangiocarcinoma and benign biliary diseases. They classified both groups with a sensitivity of 88% and a specificity of 81%. We are currently working on the utility of magnetic resonance spectroscopy in the study of hepatopancreatobiliary diseases [2–6] and have noticed some shortcomings in the above study. The authors have mentioned that their work was the first metabolomics approach reported in the diagnosis of human hepatobiliary diseases [1]. However, a similar metabolomic study using bile samples for the detection of cholangiocarcinoma was published by our group about two years ago [2]. In our study, we performed multivariate analysis of 1H NMR spectra of bile samples obtained from patients with cholangiocarcinoma and other benign biliary diseases (primary sclerosing cholangitis/choledocholithiasis) with a comparable sample size as that of Wen et al. [1]. We reported a sensitivity of 88.9% and a specificity of 87.1% in classifying cancer and control groups. Khan et al. [7] had also previously reported a study in which they analyzed bile samples from cholangiocarcinoma, pancreatic cancer, and other hepatobiliary diseases using 1H NMR spectroscopy. Although our study [2] and the one by Khan et al. [7] were undertaken with similar objectives as that of Wen et al., they were not cited.
398
We also have concerns regarding some of the metabolites quantified in the targeted metabolic profiling. Wen et al. [1] compared the levels of choline in both groups and the difference was not found to be statistically significant (p = 0.85). However, in our study, we quantified the predominant choline-containing phospholipid, phosphatidylcholine (PC) which was decreased in cancer patients compared to the benign group with the difference being statistically significant (p = 0.02). Khan et al. had also reported a reduced phosphatidylcholine signal in cancer patients (cholangiocarcinoma/pancreatic cancer) compared to the noncancer patients (p = 0.007) [7]. In a similar study, Nagana Gowda et al. observed decreased levels of phospholipids in cholangiocarcinoma patients compared to the non-liver disease control group (p = 0.001) [8]. Moreover, in a recent study, Sharif et al. made similar observations in comparing patients with cholangiocarcinoma and gallstone disease (p = 0.01) [9]. The above observations are consistent with the fact that phosphatidylcholine is an important component of bile protecting bile ducts from the harmful effects of bile acids and its depletion can lead to bile duct injury [3]. Second, the authors reported that citrate levels were elevated in cancer patients compared to the benign subjects with the difference being statistically significant [1]. Although citrate is a component of bile acting as a calcium chelator in gallbladder disease [10], its levels are considerably low (0–406 lM). As a result, detection of citrate in bile using 1H NMR spectroscopy would be difficult. Moreover, they have associated citrate with a signal resonating at 1.5 ppm (one of the signals found to be discriminatory in the OPLS-DA). This assignment is not correct as the –CH2– protons in citrate resonate at totally different chemical shift (in the region 2.55–2.75 ppm). Furthermore, the authors have misas-
Journal of Hepatology 2011 vol. 54 j 395–400
JOURNAL OF HEPATOLOGY signed a signal resonating at 3.70 ppm to a –CHn–OR moiety. We believe that this signal arises from the –CH2– protons of glycine conjugated to bile acids [6]. Finally, the bile samples in our study were analyzed without any sample pretreatment which reduced the experimental time, whereas in the study by Wen et al., the samples had undergone pretreatment. More specifically, the samples were freeze-dried and later dissolved in the solvent system, D2O–CD3OD (buffered with 10 mM sodium phosphate, pH 6). Their approach increased the procedure time considerably, which makes it less appealing to be used in the clinic. In a clinical setting, it is highly recommended that the minimum number of steps be used between sample collection and the generation of diagnostic output. Moreover, their methodology is expected to be more expensive as they used deuterated solvents to prepare the samples. Although bile is a complex biofluid, it is still possible to use the neat samples for the quantification of major biliary biochemicals, such as glycineconjugated bile acids, taurine-conjugated bile acids, and cholinecontaining phospholipids [6]. Although Wen et al. have presented some important NMRbased results in the diagnosis of cholangiocarcinoma and have done a thorough job in comparing their NMR data with other diagnostic markers (CEA, CA 19-9, and bile cytology), the errors we have noticed may mislead readers if left uncorrected. Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Wen H, Yoo SS, Kang J, Kim HG, Park JS, Jeong S, et al. A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer. J Hepatol 2010;52:228–233. [2] Albiin N, Smith ICP, Arnelo U, Lindberg B, Bergquist A, Dolenko B, et al. Detection of cholangiocarcinoma with magnetic resonance spectroscopy of
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
bile in patients with and without primary sclerosing cholangitis. Acta Radiol 2008;49:855–862. Ijare OB, Bezabeh T, Albiin N, Arnelo U, Bergquist A, Lindberg B, et al. Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of cholestasis: a 1H MR study. NMR Biomed 2009;22:471–479. Bezabeh T, Ijare OB, Albiin N, Arnelo U, Lindberg B, Smith ICP. Detection and quantification of D-glucuronic acid in human bile using 1H NMR spectroscopy: relevance to the diagnosis of pancreatic cancer. MAGMA 2009;22:267–275. Ijare OB, Smith ICP, Mohajeri S, Bezabeh T. Magnetic resonance spectroscopy of human bile in the detection of hepatopancreaticobiliary disease: past, present and future. In: Khetrapal CL, Kumar A, Ramanathan KV, editors. Future directions of NMR. 1st ed. New Delhi: Springer (India) Private Limited; 2010 [chapter 4]. Ijare OB, Bezabeh T, Albiin N, Bergquist A, Arnelo U, Smith ICP. Simultaneous quantification of glycine- and taurine-conjugated bile acids, total bile acids, and choline-containing compounds in human bile using 1H NMR spectroscopy. J Pharm Biomed Anal 2010;53:667–673. Khan SA, Cox IJ, Thillainayagam AV, Bansi DS, Thomas HC, Taylor-Robinson SD. Proton and phosphorus-31 nuclear magnetic resonance spectroscopy of human bile in hepatopancreaticobiliary cancer. Eur J Gastroenterol Hepatol 2005;17:733–738. Nagana Gowda GA, Shanaiah N, Cooper A, Maluccio M, Raftery D. Visualization of bile homeostasis using 1H-NMR spectroscopy as a route for assessing liver cancer. Lipids 2009;44:27–35. Sharif AW, Williams HRT, Lampejo T, Khan SA, Bansi DS, Westaby D. Metabolic profiling of bile in cholangiocarcinoma using in vitro magnetic resonance spectroscopy. HPB 2010;12:396–402. Vitetta L, Sali A. Citrate: a component of bile and calcium chelator in gallbladder disease. J Nutr Environ Med 1999;9:199–207.
Omkar B. Ijare1 Tedros Bezabeh1,* Nils Albiin2 Ian C.P. Smith1 1 National Research Council Institute for Biodiagnostics, Winnipeg, Manitoba, Canada 2 Karolinska University Hospital, Karolinska Institutet, Huddinge, Stockholm, Sweden * Address: NRC Institute for Biodiagnostics, 435 Ellice Avenue, Winnipeg, MB, Canada R3B 1Y6. Tel: +1 204 983 0994; fax: +1 204 984 7036 E-mail address: [email protected] (T. Bezabe)
A new NMR metabolomics-based diagnosis for hepatobiliary tract cancer Reply to Bezabeh et al.: Bezabeh and co-workers wrote a letter for their concerns about our recent article [1], and we would like to provide our reply as below. Bezabeh and co-workers did not recognize our article as ‘‘the first metabolomics approach reported in the diagnosis of human hepatobiliary diseases’’, providing their own [2] and another work [3] as similar studies. However, their argument seriously distorts our intent because they cut out the important last part of the original sentence that reads ‘‘. . .outperforming other conventional clinical criteria’’. The comparison with the conventional criteria obtained from the same patients is critical for the validation of the metabolomics approach, constituting one of the most important features of our article. This is because it is easy to
obtain a good diagnostic performance with a new approach if the benign and cancer samples have very different characteristics even with conventional criteria. In the two reports above, no quantitative comparison was provided. In addition, their work [2] leans more towards the subject of cholangiocarcinoma and primary sclerosing cholangitis (PSC), although they did provide the diagnosis for overall benign vs. cancer patients. This can be recognized from the title ‘‘Detection of Cholangiocarcinoma with Magnetic Resonance Spectroscopy of Bile in Patients with and without Primary Sclerosing Cholangitis’’. Furthermore, many (62%) of their benign samples had PSC, whereas a majority (75%) of the cancer samples were devoid of PSC. Therefore, each sample group has different characteristics with respect to the presence of PSC. The objective of Kahn et al.’s report [3] is also
Journal of Hepatology 2011 vol. 54 j 395–400
399
References [1] Bhoori S, Toffanin S, Sposito C, Germini A, Pellegrinelli A, Lampis A, et al. Personalized molecular targeted therapy in advanced, recurrent hepatocellular carcinoma after liver transplantation: a proof of principle. J Hepatol 2010;52:771–775. [2] Valdivieso A, Bustamante J, Gastaca M, Uriarte JG, Ventoso A, Ruiz P, et al. Management of hepatocellular carcinoma recurrence after liver transplantation. Transplant Proc 2010;42:660–662. [3] Graziadei I, Finkenstedt A, Nachbaur K, Zoller H, Mark W, Vogel W. Outcome of patients with recurrent hepatocellular carcinoma after liver transplantation. J Hepatol 2010;52:A214. [4] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378–390. [5] Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology 2008;135:1972–1983. [6] Toso C, Merani S, Bigam DL, Shapiro AM, Kneteman NM. Sirolimus-based immunosuppression is associated with increased survival after liver transplantation for hepatocellular carcinoma. Hepatology 2010;51:1237–1243.
[7] Schnitzbauer AA, Zuelke C, Graeb C, Rochon J, Bilbao I, Burra P, et al. A prospective randomised, open-labeled, trial comparing sirolimus-containing versus mTOR-inhibitor-free immunosuppression in patients undergoing liver transplantation for hepatocellular carcinoma. BMC Cancer 2010;10:190. [8] Newell P, Toffanin S, Villanueva A, Chiang DY, Minguez B, Cabellos L, et al. Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. J Hepatol 2009;51:725–733. [9] Welker MW, Lubomierski N, Gog C, Herrmann E, Engels K, Vogl TJ, et al. Efficacy and safety of sorafenib in advanced hepatocellular carcinoma under daily practice conditions. J Chemother 2010;22:205–211. [10] Blanchet B, Billemont B, Barete S, Garrigue H, Cabanes L, Coriat R, et al. Toxicity of sorafenib: clinical and molecular aspects. Expert Opin Drug Saf 2010;9:275–287.
Oliver Waidmann Wolf-Peter Hofmann Stefan Zeuzem Joerg Trojan* Medizinische Klinik 1, Klinikum der Johann-Wolfgang, Goethe-Universität, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany * Tel.: +49 069 6301 7860; fax: +49 069 6301 83776 E-mail address: [email protected] (J. Troja)
Magnetic resonance spectroscopy of bile in the detection of cholangiocarcinoma To the Editor: We would like to draw your attention to some of the drawbacks in a recent article published by Wen et al. in your journal [1], on NMR-based metabolomics of bile samples in distinguishing cholangiocarcinoma from benign biliary diseases. The authors performed orthogonal partial least square discriminant analysis (OPLS-DA) of 1H NMR spectra of bile samples obtained from patients with cholangiocarcinoma and benign biliary diseases. They classified both groups with a sensitivity of 88% and a specificity of 81%. We are currently working on the utility of magnetic resonance spectroscopy in the study of hepatopancreatobiliary diseases [2–6] and have noticed some shortcomings in the above study. The authors have mentioned that their work was the first metabolomics approach reported in the diagnosis of human hepatobiliary diseases [1]. However, a similar metabolomic study using bile samples for the detection of cholangiocarcinoma was published by our group about two years ago [2]. In our study, we performed multivariate analysis of 1H NMR spectra of bile samples obtained from patients with cholangiocarcinoma and other benign biliary diseases (primary sclerosing cholangitis/choledocholithiasis) with a comparable sample size as that of Wen et al. [1]. We reported a sensitivity of 88.9% and a specificity of 87.1% in classifying cancer and control groups. Khan et al. [7] had also previously reported a study in which they analyzed bile samples from cholangiocarcinoma, pancreatic cancer, and other hepatobiliary diseases using 1H NMR spectroscopy. Although our study [2] and the one by Khan et al. [7] were undertaken with similar objectives as that of Wen et al., they were not cited.
398
We also have concerns regarding some of the metabolites quantified in the targeted metabolic profiling. Wen et al. [1] compared the levels of choline in both groups and the difference was not found to be statistically significant (p = 0.85). However, in our study, we quantified the predominant choline-containing phospholipid, phosphatidylcholine (PC) which was decreased in cancer patients compared to the benign group with the difference being statistically significant (p = 0.02). Khan et al. had also reported a reduced phosphatidylcholine signal in cancer patients (cholangiocarcinoma/pancreatic cancer) compared to the noncancer patients (p = 0.007) [7]. In a similar study, Nagana Gowda et al. observed decreased levels of phospholipids in cholangiocarcinoma patients compared to the non-liver disease control group (p = 0.001) [8]. Moreover, in a recent study, Sharif et al. made similar observations in comparing patients with cholangiocarcinoma and gallstone disease (p = 0.01) [9]. The above observations are consistent with the fact that phosphatidylcholine is an important component of bile protecting bile ducts from the harmful effects of bile acids and its depletion can lead to bile duct injury [3]. Second, the authors reported that citrate levels were elevated in cancer patients compared to the benign subjects with the difference being statistically significant [1]. Although citrate is a component of bile acting as a calcium chelator in gallbladder disease [10], its levels are considerably low (0–406 lM). As a result, detection of citrate in bile using 1H NMR spectroscopy would be difficult. Moreover, they have associated citrate with a signal resonating at 1.5 ppm (one of the signals found to be discriminatory in the OPLS-DA). This assignment is not correct as the –CH2– protons in citrate resonate at totally different chemical shift (in the region 2.55–2.75 ppm). Furthermore, the authors have misas-
Journal of Hepatology 2011 vol. 54 j 395–400
JOURNAL OF HEPATOLOGY signed a signal resonating at 3.70 ppm to a –CHn–OR moiety. We believe that this signal arises from the –CH2– protons of glycine conjugated to bile acids [6]. Finally, the bile samples in our study were analyzed without any sample pretreatment which reduced the experimental time, whereas in the study by Wen et al., the samples had undergone pretreatment. More specifically, the samples were freeze-dried and later dissolved in the solvent system, D2O–CD3OD (buffered with 10 mM sodium phosphate, pH 6). Their approach increased the procedure time considerably, which makes it less appealing to be used in the clinic. In a clinical setting, it is highly recommended that the minimum number of steps be used between sample collection and the generation of diagnostic output. Moreover, their methodology is expected to be more expensive as they used deuterated solvents to prepare the samples. Although bile is a complex biofluid, it is still possible to use the neat samples for the quantification of major biliary biochemicals, such as glycineconjugated bile acids, taurine-conjugated bile acids, and cholinecontaining phospholipids [6]. Although Wen et al. have presented some important NMRbased results in the diagnosis of cholangiocarcinoma and have done a thorough job in comparing their NMR data with other diagnostic markers (CEA, CA 19-9, and bile cytology), the errors we have noticed may mislead readers if left uncorrected. Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Wen H, Yoo SS, Kang J, Kim HG, Park JS, Jeong S, et al. A new NMR-based metabolomics approach for the diagnosis of biliary tract cancer. J Hepatol 2010;52:228–233. [2] Albiin N, Smith ICP, Arnelo U, Lindberg B, Bergquist A, Dolenko B, et al. Detection of cholangiocarcinoma with magnetic resonance spectroscopy of
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
bile in patients with and without primary sclerosing cholangitis. Acta Radiol 2008;49:855–862. Ijare OB, Bezabeh T, Albiin N, Arnelo U, Bergquist A, Lindberg B, et al. Absence of glycochenodeoxycholic acid (GCDCA) in human bile is an indication of cholestasis: a 1H MR study. NMR Biomed 2009;22:471–479. Bezabeh T, Ijare OB, Albiin N, Arnelo U, Lindberg B, Smith ICP. Detection and quantification of D-glucuronic acid in human bile using 1H NMR spectroscopy: relevance to the diagnosis of pancreatic cancer. MAGMA 2009;22:267–275. Ijare OB, Smith ICP, Mohajeri S, Bezabeh T. Magnetic resonance spectroscopy of human bile in the detection of hepatopancreaticobiliary disease: past, present and future. In: Khetrapal CL, Kumar A, Ramanathan KV, editors. Future directions of NMR. 1st ed. New Delhi: Springer (India) Private Limited; 2010 [chapter 4]. Ijare OB, Bezabeh T, Albiin N, Bergquist A, Arnelo U, Smith ICP. Simultaneous quantification of glycine- and taurine-conjugated bile acids, total bile acids, and choline-containing compounds in human bile using 1H NMR spectroscopy. J Pharm Biomed Anal 2010;53:667–673. Khan SA, Cox IJ, Thillainayagam AV, Bansi DS, Thomas HC, Taylor-Robinson SD. Proton and phosphorus-31 nuclear magnetic resonance spectroscopy of human bile in hepatopancreaticobiliary cancer. Eur J Gastroenterol Hepatol 2005;17:733–738. Nagana Gowda GA, Shanaiah N, Cooper A, Maluccio M, Raftery D. Visualization of bile homeostasis using 1H-NMR spectroscopy as a route for assessing liver cancer. Lipids 2009;44:27–35. Sharif AW, Williams HRT, Lampejo T, Khan SA, Bansi DS, Westaby D. Metabolic profiling of bile in cholangiocarcinoma using in vitro magnetic resonance spectroscopy. HPB 2010;12:396–402. Vitetta L, Sali A. Citrate: a component of bile and calcium chelator in gallbladder disease. J Nutr Environ Med 1999;9:199–207.
Omkar B. Ijare1 Tedros Bezabeh1,* Nils Albiin2 Ian C.P. Smith1 1 National Research Council Institute for Biodiagnostics, Winnipeg, Manitoba, Canada 2 Karolinska University Hospital, Karolinska Institutet, Huddinge, Stockholm, Sweden * Address: NRC Institute for Biodiagnostics, 435 Ellice Avenue, Winnipeg, MB, Canada R3B 1Y6. Tel: +1 204 983 0994; fax: +1 204 984 7036 E-mail address: [email protected] (T. Bezabe)
A new NMR metabolomics-based diagnosis for hepatobiliary tract cancer Reply to Bezabeh et al.: Bezabeh and co-workers wrote a letter for their concerns about our recent article [1], and we would like to provide our reply as below. Bezabeh and co-workers did not recognize our article as ‘‘the first metabolomics approach reported in the diagnosis of human hepatobiliary diseases’’, providing their own [2] and another work [3] as similar studies. However, their argument seriously distorts our intent because they cut out the important last part of the original sentence that reads ‘‘. . .outperforming other conventional clinical criteria’’. The comparison with the conventional criteria obtained from the same patients is critical for the validation of the metabolomics approach, constituting one of the most important features of our article. This is because it is easy to
obtain a good diagnostic performance with a new approach if the benign and cancer samples have very different characteristics even with conventional criteria. In the two reports above, no quantitative comparison was provided. In addition, their work [2] leans more towards the subject of cholangiocarcinoma and primary sclerosing cholangitis (PSC), although they did provide the diagnosis for overall benign vs. cancer patients. This can be recognized from the title ‘‘Detection of Cholangiocarcinoma with Magnetic Resonance Spectroscopy of Bile in Patients with and without Primary Sclerosing Cholangitis’’. Furthermore, many (62%) of their benign samples had PSC, whereas a majority (75%) of the cancer samples were devoid of PSC. Therefore, each sample group has different characteristics with respect to the presence of PSC. The objective of Kahn et al.’s report [3] is also
Journal of Hepatology 2011 vol. 54 j 395–400
399