Letters to the Editor
SVR rate based on 12-week AUC median cumulative RBV exposure 100
P-value = 0.02 80
SVR (%)
1178
10. Stickel F, Helbling B, Heim M et al. Critical review of the use of erythropoietin in the treatment of anaemia during therapy for chronic hepatitis C. J Viral Hepat 2012;19:77–87. 11. Breadmore MC, Theurillat R, Thormann W. Determination of ribavirin in human serum and plasma by capillary electrophoresis. Electrophoresis 2004;25:1615–22.
60
40
20
0
Low-exposure group
High-exposure group
Figure 1. SVR rates in patients with low and high median cumulative RBV exposure (threshold set at ≥224.3 mg/dl/day). AUC, area under the curve; RBV, ribavirin; SVR, sustained virological response.
to the small number of patients (62.5 vs. 37.5%, P = 0.11). However, when comparing median cumulative exposure to RBV between groups as measured by the area under the drug exposure curve from week 0 to 12 based on biweekly measurements of RBV plasma levels rather than RBV dosage per se, cumulative exposure to RBV above ≥224.3 μg/dl/day was significantly associated with SVR (odds ratio 8.8; confidence interval 1.35–57.43, P = 0.02) (Figure 1). Anemia in group A was more severe than in group B (mean hemoglobin 99.6 vs. 106.3 g/l; P < 0.001), but well manageable with erythropoietin beta at doses between 9,000 and 30,000 IU per week according to a recently proposed consensus (10) Except for anemia, adverse events were similar in both groups. In conclusion—and in accordance with the study by Jin et al. (7)—optimal exposure to RBV guided by therapeutic drug monitoring significantly improves SVR in patients with CHC genotype 1. Therefore, regular RBV plasma level measurements at least for the first 12 weeks of therapy and RBV dose adjustment may be advocated. ACKNOWLEDGMENTS
The present investigator-initiated study (NCT00944684) has been funded, in part, by Roche Pharma (Switzerland). CONFLICT OF INTEREST
F.S. has received speaker honoraria from MSD, Roche, and Novartis and is a The American Journal of GASTROENTEROLOGY
member of the Advisory Board of MSD, Switzerland, and of Novartis, Switzerland. F.S. has received unrestricted research funding from MSD, Novartis and Roche unrelated to the present work. REFERENCES 1. Shiffman ML, Salvatore J, Hubbard S et al. Treatment of chronic hepatitis C virus genotype 1 with peginterferon, ribavirin, and epoetin alpha. Hepatology 2007;46:371–9. 2. Loustaud-Ratti V, Alain S, Rousseau A et al. Ribavirin exposure after the first dose is predicitve of sustained virological response in chronic hepatitis C. Hepatology 2008;47:1453–61. 3. Pearlman BL. Protease inhibitors for the treatment of chronic hepatitis C genotype-1 infection: the new standard of care. Lancet Infect Dis 2012;12:717–28. 4. Leroy V, Serfaty L, Bourlière M et al. Protease inhibitor-based triple therapy in chronic hepatitis C: guidelines by the French Association for the Study of the Liver. Liver Int 2012;32:1477–92. 5. Hézode C, Forestier N, Pol S et al. Telaprevir with or without ribavirin for chronic HCV infection. N Engl J Med 2009;360:1839–50. 6. Zeuzem S, Soriano V, Asselah T et al. SVR4 and SVR12 with an interferon-free regimen of BI201335 and BI207127, +/- ribavirin, in treatment-naïve patients with chronic genotype-1 HCV infection: interim results of SOUND-C2. J Hepatol 2012;56:S45–59. 7. Jin R, Cai L, McHutchison JG et al. Optimum ribavirin exposure overcomes racial disparity in efficacy of peginterferon and ribavirin treatment for hepatitis C genotype 1. Am J Gastroenterol 2012;107:1675–83. 8. Conjeevaram HS, Fried MW, Jeffers LJ et al. Peginterferon and ribavirin for the treatment of chronic hepatitis C in African American and Caucasian American patients with hepatitis C genotype 1. Gastroenterology 2006;131:470–7. 9. Lai M, Afdhal NH. Clinical utility of interleukin-28B testing in patients with genotype 1. Hepatology 2012;56:367–72.
1 Department of Visceral Surgery and Medicine, Inselspital, University of Berne, Berne, Switzerland; 2Division of Gastroenterology and Hepatology, CHUV, University of Lausanne, Lausanne, Switzerland; 3Department of Gastroenterology, Stadtspital Waid, Zürich, Switzerland; 4Department of Gastroenterology and Hepatology, Kantonsspital, St.Gallen, Switzerland. Correspondence: Felix Stickel, MD, Department of Visceral Surgery and Medicine, Inselspital, University of Bern, Murtenstr. 35, 3010 Bern, Switzerland. E-mail:
[email protected]
BAG3 Is a Novel Serum Biomarker for Pancreatic Adenocarcinomas Antonia Falco, PharmD, PhD1,2,10, Alessandra Rosati, PharmD, PhD1,2,10, Michelina Festa, MSc1,2, Anna Basile, PharmD, PhD1,2, Margot De Marco, MSc, PhD1,2, Morena d’Avenia, MSc, PhD1,2, Maria Pascale, MSc, PhD1,2, Fabrizio Dal Piaz, MSc, PhD1,2, Francesca Tavano, MSc, PhD3, Fabio Francesco Di Mola, MD, PhD3, Pierluigi di Sebastiano, MD3, Pasquale Bartolomeo Berloco, MD4, Francesco Nudo, MD4, Michele Caraglia, MD, PhD5, Antonio Febbraro, MSc6, Daniela Barcaroli, PhD7, Aldo Scarpa, MD, PhD8, Raffaele Pezzilli, MD9, Vincenzo De Laurenzi, MD, PhD1,2,7 and Maria Caterina Turco, MD, PhD1,2 SUPPLEMENTARY MATERIAL is linked to the online version of the paper at http://www.nature.com/ajg
doi:10.1038/ajg.2013.128
To the Editor: Pancreatic ductal adenocarcinoma (PDAC) is the only one of the five most lethal malignancies (lung, colorectal, breast, pancreatic, and prostate cancer) for which both the incidence rate and death rate have increased in recent VOLUME 108 | JULY 2013 www.amjgastro.com
Letters to the Editor
at e
b
C el ll ys
PDAC patients
rB AG 3
Healthy subjects
rB AG 3
a 75 kDa
75kDa
c
IB: PDAC patient Abs
C el ll ys
at e
theoretical MW 989.457 712.375 1819.894 984.499 2198.935 2311.077 1480.742 877.425 1277.673 1532.747 1412.715
AG 3
Experimental MW 989.47 712.38 1819.92 984.52 2198.95 2311.12 1480.76 877.43 1277.69 1532.77 1412.73
rB
Pept: BAG3 aa 46-53 54-60 107-121 124-133 174-196 231-249 250-261 268-276 283-294 432-445 461-473
75kDa IB: Healthy subjects Abs
BAG3 immunocomplexes (AU)
0.30 0.25 0.20 0.15 0.10 0.05 0.00
Median 95% CI for the median
Healthy subjects Pancreatitis N = 44 N = 17 0.062 0.062 0.058–0.075
0.047–0.078
Healthy subjectsand pancreatitis vs. neoplastic lesions*
Pancreatitis 0.397
e
PDAC N = 52 0.097
0.068–0.105
0.067–0.108
0.085–0.111
PDAC 1 × 10–5 0.0006 0.159 0.246
f
healthy subjects vs. PDAC
Pancreatitis vs. PDAC
100
100
80
80
80
60 40
Sensitivity
100
Sensitivity
Sensitivity
d
Ca in situ N = 19 0.084
Student′s t test: p value IPMN Ca in situ 0.009 0.025 0.011 0.027 0.986
VS Healthy subjects pancreatitis IPMN Ca in Situ
IPMN N = 28 0.088
60 40 20
20
60 40 20
AUC= 0.73 ± 0.048
AUC= 0.77 ± 0.049 0
0 0
20
40
60
80
100
100-Specificity Cutoff: 0.078 Sensitivity = 71% Specificity = 71% P < 0.0001 *IPMN, Ca in Situ and PDAC
AUC= 0.81 ± 0.059 0
0
20
40
60
80
100
100-Specificity Cutoff: 0.083 Sensitivity = 75% Specificity = 76% P < 0.0001
0
20
40
60
80
100
100-Specificity Cutoff: 0.078 Sensitivity = 81% Specificity = 77% P < 0.0001
Figure 1. (a) Proteins in 5 μl sera samples from four healthy donors and four pancreatic ductal adenocarcinoma (PDAC) patients were separated by 8% SDS-polyacrylamide gel electrophoresis and analyzed by western blotting with AC-2 anti-BAG3 monoclonal antibody (BIOUNIVERSA s.r.l., Fisciano, Italy) as described (7). The band recognized by AC-2 was obtained from a replica gel and analyzed by mass spectrometry (MASCOT), confirming the presence of BAG3- identical peptides (aa: amino acidic sequence; MW: molecular weight). (b) Antibodies obtained from PDAC patients’ (upper) or healthy donors’ (lower) serum samples by treatment with a low (2.5) pH buffer were used at a 1:200 dilution to detect either recombinant BAG3 or the protein in extracts from the human PDAC cell line PANC-1, by western blotting. (c) BAG3/antibody complexes were measured by enzyme-linked immunosorbent assay. Results are presented as arbitrary units (AU). Horizontal bars represent median AU value. (d–f) Receiver operating characteristic (ROC) curves obtained with results from the BAG3 IC enzyme-linked immunosorbent assay test. AUC, area under the curve.
years, and with a projected increase in the number of deaths. The lack of early symptoms results in late-stage detection © 2013 by the American College of Gastroenterology
and a near to 100% mortality rate, despite improvements in therapies (1–3). Therefore, there is an urgent need for novel
screening strategies to recognize early cancers or precursor lesions in the general population, and in individuals that are The American Journal of GASTROENTEROLOGY
1179
1180
Letters to the Editor
considered at highest risk on the basis of clinical or genetic characteristics. Owing to the vast genetic heterogeneity of this cancer, no single biomarker exists that is strongly correlated with its diagnosis. Efforts for detecting pancreatic adenocarcinoma at early stages are, therefore, focused on the identification of a large panel of appropriate sensitive and specific serum biomarkers, including some already characterized candidates and clearly novel ones (1,4). Recently, we reported that the intracellular anti-apoptotic protein BAG3 (5) was expressed in all 346 PDAC tumor samples examined, and not in the surrounding non-neoplastic tissue or in normal pancreas (6). Furthermore, in a cohort of 66 patients who underwent R0 resection, the level of BAG3 expression inversely correlated with patient survival (6). These results prompted us to investigate whether BAG3 protein was also present in patients’ sera and could potentially be used as a biomarker for PDAC. In a preliminary analysis by western blotting, we detected BAG3 protein in sera from four PDAC patients, but not in those from four healthy donors (Figure 1a). Furthermore, we found that BAG3 was complexed with anti-BAG3 antibodies: indeed, by dissociating such complexes and isolating antibodies, we demonstrated that these recognized BAG3 in western blotting (Figure 1b). We, therefore, decided to verify the presence and measure the levels of BAG3 and BAG3/antibody immune complexes (IC) in serum samples of a higher number of patients, by enzymelinked immunosorbent assay. We analyzed 52 pretreatment serum samples from PDAC patients (stage:T3; average age + s. e.:64.0 + 1.3; males:29, females:23), 44 samples from healthy subjects (average age + s. e.:58.7 + 1.6; males:31, females:13), 17 sera samples from patients having proven chronic pancreatitis (average age + s.e.:51.7 + 4.2; males:14, females:3), 28 sera from patients having main duct and/or branch duct intraductal papillary mucinous neoplasm (IPMN) (average age + s.e.:66 + 4.2; males:13, females:15) and 19 sera from resected IPMN patients with histologically demonstrated carcinoma in situ (average age + s.e.:72 + 2.1; males:8, The American Journal of GASTROENTEROLOGY
females:11). Levels of BAG3 (Supplementary Figure 1) and BAG3 IC (Figure 1c) were significantly higher in PDAC patients’ than in healthy donors’ or pancreatitis patients’ samples. Furthermore, also the levels detected in patients affected by IPMN or IPMN having carcinoma in situ were significantly (P < 0.05) higher than those measured in healthy donors’ or pancreatitis patients’ samples (Figure 1c). The test for BAG3 IC (Figure 1c) discriminated better in the different groups than that for BAG3 protein (Supplementary Figure 1). It is not clear at this stage why this is the case; it is possible that at the time of the diagnosis most of the protein is bound to antibodies reducing the discriminating potential of the protein detection test. The test discriminated between neoplastic lesions (PDAC, Ca in situ and IPMN) and healthy subjects or pancreatitis patients, with a sensitivity of 71% and a specificity of 71% at a cutoff value of 0.078 AU (P < 0.0001), corresponding to the criterion value (the maximum vertical distance between the ROC curve and the diagonal line) (Figure 1d). Particularly, the test appeared to discriminate PDAC patients from healthy donors with a sensitivity of 75% and a specificity of 75.6% at a cutoff value (criterion) of 0.083 AU (P < 0.0001) (Figure 1e), while chronic pancreatitis patients were discriminated from PDAC patients with a sensitivity of 80.8% and a specificity of 76.5% at a cutoff value of 0.078 AU (P < 0.0001) (Figure 1f). These findings demonstrate for the first time that BAG3/anti-BAG3 complexes are detected at higher levels in sera from PDAC patients as compared with sera from healthy donors or patients affected by chronic pancreatitis, and indicate that BAG3 IC could be a potential marker for screenings of high-risk groups or of the general population. Ideally one would have hoped that BAG3 IC levels could help discriminate between IPMN and carcinomas in situ, however, differences between these two groups were not significant in this relatively small set of patients. Clearly validation of these results in a larger cohort of patients with a broader disease spectrum is required to determine the clinical applications of this marker. Finally the fact that IPMN and in situ carcinomas are positive for this marker suggests that it could
be of great utility in early detection of PDACs and again indicates that additional studies in this direction should be undertaken. CONFLICT OF INTEREST Guarantor of the article: Maria Caterina Turco, MD, PhD. Specific author contributions: A.F., A.R., V.D.L., and M.C.T.: conception and experimental design; A.F., A.R., A.B., M.F., M.D.A., M.D.M., M.P., F.T., F.F.D., P.D.B., F.N., M.C., A.F., and D.B.: analysis and interpretation of data; V.D.L. and M.C.T.: drafting of the manuscript; R.P. and A.S.: revising it critically for important intellectual content; V.D.L. and M.C.T.: final approval of the manuscript submitted. Financial Support: This work was supported by Ministero dell’Università (FARB) to M.C.T., Associazione Italiana per la Ricerca sul Cancro (AIRC) IG 11450 and Ministero Sanità Ricerca Finalizzata to V.D.L. Potential competing interests: A.F., A.R., M.F., A.B., M.D.M., M.D.A., M.P., V.D.L., and M.C.T. are shareholders of BIOUNIVERSA s.r.l. that provided BAG3-specific antibodies and BAG3-specific enzymelinked immunosorbent assay tests free of charges for this work. All other authors have no industry relationship to disclose. REFERENCES 1. Brentnall TA. Pancreatic cancer surveillance: learning as we go. Am J Gastroenterol 2011;106:955–6. 2. van Lier MG, Wagner A, Mathus-Vliegen EM et al. High cancer risk in Peutz-Jeghers syndrome: a systematic review and surveillance recommendations. Am J Gastroenterol 2010;105:1258–64. 3. Greer JB, Whitcomb DC, Brand RE. Genetic predisposition to pancreatic cancer: a brief review. Am J Gastroenterol 2007;102:2564–69. 4. Poruk KE, Firpo MA, Adler DG et al. Screening for pancreatic cancer: why, how, and who? Ann Surg 2013;257:17–26. 5. Rosati A, Graziano V, De Laurenzi V et al. BAG3: a multifaceted protein that regulates major cell pathways. Cell Death Dis 2011;2:e141. 6. Rosati A, Bersani S, Tavano F et al. Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and overexpression is associated with poorer survival. Am J Pathol 2012;181:1524–29. 7. Ammirante M, Rosati A, Arra C et al. IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth. Proc Natl Acad Sci USA 2010;107:7497–502.
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/ licenses/by-nc-nd/3.0/ VOLUME 108 | JULY 2013 www.amjgastro.com
