Available online at www.sciencedirect.com

Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 295–301www.nanomedjournal.com

Original Article: Clinical

Mesothelin is a specific biomarker of invasive cancer in theBarrett-associated adenocarcinoma progression model:translational implications for diagnosis and therapy

Hector Alvarez, MD,a Pamela Leal Rojas, MD,a Ken-Tye Yong, PhD,b Hong Ding, PhD,b

Gaixia Xu, PhD,b Paras N. Prasad, PhD,b Jean Wang, MD,c Marcia Canto, MD,c

James R. Eshleman, MD, PhD,a,d Elizabeth A. Montgomery, MD,a Anirban Maitra, MBBSa,d,⁎aDepartment of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

bInstitute for Lasers, Photonics, and Biophotonics, Department of Chemistry, State University of New York, Buffalo, New York, USAcDivision of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

dDepartment of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Abstract Esophageal adenocarcinoma arises in the backdrop of Barrett metaplasia-dysplasia sequence, with

Received 13 JanuaThis work is suppo

P.N.P., and A.M. arR01CA119397.

⁎Corresponding aUniversity School of M

E-mail address: am

1549-9634/$ – see frodoi:10.1016/j.nano.20

Please cite this articmodel: translational

the vast majority of patients presenting with late-stage malignancy. Mesothelin, a glycopho-sphatidylinositol-anchored protein, is aberrantly overexpressed on the surface of many solid cancers.Mesothelin expression was assessed in esophageal tissue microarrays encompassing the entirehistological spectrum of Barrett-associated dysplasia and adenocarcinoma. Mesothelin expressionwas observed in 24/84 (29%) of invasive adenocarcinomas and in 5/34 (15%) lymph nodemetastases. In contrast, normal squamous and cardiac mucosa, as well as noninvasive Barrett lesions,failed to label with mesothelin. Mesothelin was expressed in the esophageal adenocarcinoma cell lineJH-EsoAd1 but not in primary human esophageal epithelial cells. Anti-mesothelin antibody–conjugated CdSe/CDS/ZnS quantum rods were synthesized, and confocal bioimaging confirmedrobust binding to JH-EsoAd1 cells. Anti-mesothelin antibody–conjugated nanoparticles can beuseful for the diagnosis and therapy of mesothelin-overexpressing esophageal adenocarcinomas.© 2008 Elsevier Inc. All rights reserved.

Key words: Barrett esophagus; Mesothelin; Immunohistochemistry; Quantum rods; Antibody-conjugated nanoparticles; Molecular imaging

Esophageal adenocarcinoma arises within the context ofprogressive histological alterations, originating with thedevelopment of a metaplastic epithelium (Barrett esopha-gus) and extending through low- and high-grade dysplasticstages to frank invasive cancer.1,2 The progression fromhigh-grade dysplasia to adenocarcinoma of the esophagus

ry 2008; accepted 25 June 2008.rted by the D'Amato Foundation. K.T.Y, H.D., G.X.,e supported by National Cancer Institute grant

uthor. Department of Pathology, Johns Hopkinsedicine, Baltimore, Maryland 21231, USA.aitra1@jhmi.edu (A. Maitra).

nt matter © 2008 Elsevier Inc. All rights reserved.08.06.006

le as: H. Alvarez, et al, Mesothelin is a specific biomarkerimplications for diagnosis and therapy, Nanomedicine: NB

occurs frequently, and approximately 50% to 60% ofpatients develop invasive cancer within 3 to 5 years of adiagnosis (on routine histological evaluation) of high-gradedysplasia.3,4 Esophageal carcinoma presently has one of themost rapidly increasing tumor incidences in the UnitedStates5; the American Cancer Society estimates that there willbe more than 15,500 new cases of esophageal carcinoma in2007.6 Unfortunately, despite an established precursor lesionand the availability of endoscopic screening programs, theoverwhelming majority of persons presenting with esopha-geal adenocarcinoma will do so at a late stage, and without apreexisting diagnosis of Barrett esophagus.6,7 As a conse-quence, of the 15,500 estimated new patients this year, almost14,000 (∼90%) are expected to succumb to their disease.8

of invasive cancer in the Barrett-associated adenocarcinoma progressionM 2008;4:295-301, doi:10.1016/j.nano.2008.06.006

296 H. Alvarez et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 295–301

Thus, there is an urgent need for developing betterbiomarkers for early diagnosis of esophageal cancer, aswell as potent molecularly targeted therapies that can be usedfor treatment of established, advanced disease.

Mesothelin is a 40-kDa glycophosphatidylinositol-anchored surface glycoprotein that is expressed on pleuraland peritoneal mesothelial cells, but not in other non-neoplastic tissues.9 Mesothelin is synthesized as a 69-kDaprecursor protein, which is subsequently cleaved into themembrane-bound mesothelin antigen, and a 31-kDa solublefragment known asmegkaryocyte-potentiating factor.10 Multi-ple lines of evidence have established that mesothelin isaberrantly overexpressed in certain cancers, including the vastmajority (80% to 100%) of pancreatic and nonmucinousovarian cancers, and in mesotheliomas.9,11-14 In addition, arecent broad survey byOrdonez of 471 archival human cancersby immunohistochemistry has found mesothelin expression inother neoplasms at a lower frequency, including in approxi-mately a third of pulmonary adenocarcinomas, cholangiocar-cinomas, and colorectal and gastric adenocarcinomas.15 In thisstudy four esophageal adenocarcinomas were also examined,and one (25%) labeled with mesothelin. However, there havebeen no comprehensive analyses of mesothelin expression inesophageal adenocarcinomas, specifically as it relates to themulti-step Barrett progression model. This is an importantquestion to address for multiple reasons with immediatetranslational impact. First, mesothelin expression is a usefulancillary diagnostic marker in clinical specimens15-22; second,surface mesothelin antigen has been used as a target forantibody-based cancer treatment approaches (e.g., usingimmunotoxins)10,23,24; third, mesothelin epitopes have beenidentified in the context of adoptive immunotherapy usinganticancer vaccines25-27; and fourth, elevated mesothelinlevels in circulation have emerged as a promising biomarkerin cancer.28-32 Therefore, we undertook this study to system-atically analyzemesothelin expression in the entire histologicalspectrum of Barrett esophagus, dysplasia, and adenocarci-noma. Our study indicates that mesothelin is a specific markerof invasive adenocarcinoma arising in the context of Barrettesophagus, and can potentially be used as a molecular target inthe diagnosis and treatment of esophageal cancer.

Methods

Esophageal tissue microarrays (TMAs)

TMAs were created representing the full histologicalspectrum of lesions observed in the Barrett esophagusprogression model.33 We have already used these TMAs forimmunohistochemical analysis of molecular alterations inesophageal adenocarcinoma.34,35 Briefly, the TMAs werecreated from 98 patients who underwent endoscopic biopsiesor surgical resection for Barrett esophagus and adenocarci-noma. Overall, 365 cores were evaluated for mesothelinlabeling, including 64 cores of squamous mucosa, 86 ofcardiac mucosa, 30 with nondysplastic Barrett esophagus,

23 with low-grade dysplasia, 41 with high-grade dysplasia,84 with adenocarcinoma, and 37 cores representing meta-static lesions (34 lymph node, 1 liver, and 2 lung metastases).

Immunohistochemistry and assessment of mesothelin labeling

Immunohistochemistry for mesothelin expression wasperformed on TMAs using an anti-mesothelin antibody(clone 5B2, dilution 1:20; Novocastra, Newcastle UponTyne, United Kingdom), as described.36,37 Briefly, unstained4-μm sections were cut from each esophageal TMA anddeparaffinized by routine techniques before placing in 200mLTarget Retrieval Solution, pH 6.0 (Envision Plus DetectionKit; Dako, Carpinteria, California) for 20 minutes at 100°C.After cooling for 20 minutes, slides were quenched with 3%H2O2 for 5 minutes, before incubating with anti-mesothelinantibody for 60minutes, using theDakoAutostainer. Labelingwas detected with the Dako Envision system as per themanufacturer's protocol. All sections were counterstainedwith Giles' hematoxylin. Two of the authors (H.A. and A.M.)evaluated the staining, using cytoplasmic labeling with apicalmembranous accentuation as the pattern of specificmesothelinlabeling.11 TMAcoreswere scored as negative (b5% labeling)or positive, based on a simple two-tier classification, andtabulated by histological subtype of the lesion.

Western blot analysis for mesothelin expression

Mesothelin expression was evaluated in the humanesophageal adenocarcinoma cell line, JH-Eso-Ad1, createdby one of the authors (J.R.E.) from a patient undergoingsurgical resection for esophageal adenocarcinoma at JohnsHopkins. The HeLa cell line was used as a positive control.In addition, we also obtained primary nonmalignant humanesophageal epithelial cells (HEEPICs; ScienCell Inc.,Carlsbad, California)38 as a negative control to excludemesothelin expression in the normal esophageal epithelium.A volume of 50 μg of protein was separated by sodiumdodecyl sulfate–polyacrylamide electrophoresis and trans-ferred to a nitrocellulose membrane. The membrane wasstained with mouse monoclonal anti-mesothelin antibody(clone K1, dilution 1:40; Invitrogen, Carlsbad, California),and incubated with a secondary horseradish peroxidase–conjugated antibody. The blots were then washed anddeveloped using the enhanced chemiluminescence detectionsystem (GE Healthcare, Piscataway, New Jersey). Actin wasused as loading control in these experiments. The westernblot was performed in triplicate, and densitometric compar-ison performed using ImageQuant software (GE Healthcare).

Synthesis of anti-mesothelin antibody–conjugatednanoparticles and confocal imaging

Our group has recently described the detailed synthesis ofcadmium selenide/cadmium sulfide/zinc sulfide (CdSe/CdS/ZnS) rod-shaped semiconductor nanocrystals, also known asquantum rods (QRs).39 The QRs have an average size of lessthan 15 nm, emit in the red spectrum, and are amenable to

Figure 1. Immunohistochemical labeling of mesothelin in Barrett esophagus and associated adenocarcinomas. No mesothelin labeling is observed in normalsquamous mucosa (A) or in nondysplastic Barrett esophagus (B). Absence of labeling is also observed in low-grade and high-grade dysplasia (not illustrated). Incontrast, robust mesothelin expression is seen in a representative example of esophageal adenocarcinoma (C). In (D), a high-power photomicrograph illustratesthe prominent apical membrane accentuation of mesothelin labeling.

Figure 2. Expression of mesothelin antigen in esophageal adenocarcinomacell line. Western blot analysis confirms robust mesothelin expression in theJHU-EsoAd1 esophageal adenocarcinoma cell line. The HeLa cell line isused as positive control, and primary nonmalignant human esophagealepithelial cells (HEEPICs) are used as negative control. Actin is used asloading control. Densitometric reading designated represents the average ofthree independent western blot experiments.

297H. Alvarez et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 295–301

surface functionalization (e.g., antibody conjugation), so asto ensure cell-specific targeting. We had earlier demonstratedthe efficacy of these QRs for confocal and two-photonfluorescence imaging of cancer cells in vitro.39 Briefly,CdSe/CdS/ZnS QRs were synthesized in organic media, andsubsequently dispersed in aqueous media using a mixture ofmercaptoundecanoic acid and lysine. Following aqueousdispersion, the QRs were conjugated with mouse mono-clonal anti-mesothelin antibody (clone K1; Invitrogen) usingcarbodiimide chemistry. Notably, K1 is the original antibodyclone that was used for identification of mesothelinantigen.40 For confocal imaging JH-Eso-Ad1 were seededovernight in 35-mm cell culture dishes and allowed to reach60% to 70% confluence. Thereafter, the cells were incubatedfor 2 hours in the presence of anti-mesothelin antibody–conjugated QRs or unconjugated lysine-coated QRs. Thecells were then washed three times with phosphate-bufferedsaline and directly imaged using a Leica TCS SP2 AOBSspectral confocal microscope (Leica Microsystems Semi-conductor GmbH, Wetzler, Germany) with laser excitationat 442 nm.

Results

Immunohistochemical analysis confirmed that mesothelinexpression is restricted to esophageal adenocarcinoma andrelatedmetastases,with 0/64 squamousmucosal samples, 0/86cardiac mucosal samples, 0/30 Barrett esophagus, 0/23 low-grade dysplasia, and 0/41 high-grade dysplasia labeling with

anti-mesothelin antibody. Cytoplasmic mesothelin labelingwith prominent apical membranous accentuation wasobserved in 24/84 (29%) primary adenocarcinomas (Figure 1).Mesothelin expressionwas usually intense and present inmorethan 50% of neoplastic cells, and not uncommonly, secretedprotein was observed within the lumens of neoplastic glands,as we have earlier reported in other mesothelin-positive cancertypes.11 A lower frequency of mesothelin labeling wasobserved in lymph node metastases (5/34, or 15%); the threeliver and lung metastases were negative.

Elevated mesothelin expression was observed in the JH-EsoAd1 cells, almost double that of levels seen in the HeLacell line by densitometric analysis, whereas no mesothelinexpression was observed in the nonmalignant HEEPICs(Figure 2). At 2 hours after incubation with anti-mesothelin

Figure 3. In vitro labeling of JH-EsoAd1 esophageal adenocarcinoma cell line using mesothelin-targeted nanoparticles. (A) and (B), Labeling of independentfields in the JH-EsoAd1 cell line with anti-mesothelin antibody–conjugated CdSe/CdS/ZnS quantum rods (QRs). (C), In contrast, no labeling is observed withunconjugated lysine-coated QRs. Each of the three figures includes a red channel, bright field, and “merge” panel.

298 H. Alvarez et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 295–301

Figure 3 (continued).

299H. Alvarez et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 295–301

antibody–conjugated QRs, optical imaging establishedrobust binding of the red-fluorescing QRs to JH-EsoAd1cells (Figure 3, A and B), whereas no binding was seen uponincubation with the unconjugated QRs (Figure 3, C),confirming that the cell surface mesothelin is amenable toactive targeting for diagnostic and/or therapeutic purposes.

Discussion

The vast majority of individuals with esophagealadenocarcinoma present with late-stage disease, and currentconventional therapies have not improved the prognosis ofthis neoplasm.1,2 We undertook this study to evaluate thepattern and frequency of mesothelin expression in the multi-step progression model of Barrett esophagus, becausemesothelin has garnered considerable promise recently asa diagnostic and therapeutic anticancer target. Our studydemonstrates that mesothelin expression is essentiallyrestricted to invasive adenocarcinoma, with precedingnoninvasive lesions, including high-grade dysplasia, notexpressing this antigen. The frequency of mesothelinlabeling in esophageal adenocarcinoma in our study(24 of 84 cases, or 29%) is comparable to that observedby Ordonez (1 of 4 cases, or 25%) in his broad survey ofhuman cancers for mesothelin expression.15 Notably, wehave already demonstrated that mesothelin expression isabsent in noninvasive precursor lesions of pancreatic andbiliary adenocarcinomas, but is upregulated during the

transition from carcinoma in situ (PanIN-3 in the pancreas)to invasive cancer.36,41 Thus, our study adds to the evidencethat mesothelin might have a function in the process ofinvasion during multi-step epithelial cancer progression. Onthe basis of our data, we propose that mesothelin expressionis a highly specific, albeit not particularly sensitive,biomarker of invasive adenocarcinoma in Barrett esopha-gus, amenable to application in routine clinical specimens.It is likely that mesothelin expression has to be used inconjunction with other molecular markers of advanceddisease, such as diffuse cdc2/CDK1 expression,42 so as toenhance diagnostic sensitivity.

Despite the restricted expression of mesothelin to aboutone third of esophageal adenocarcinomas, it is important tonote that at present approximately 90% of individuals withthis malignancy will succumb to their disease.6 Exploitingthe repertoire of mesothelin-targeted therapeutic approaches,therefore, provides an opportunity to improve prognosis inthe mesothelin-positive subset of esophageal adenocarcino-mas. Using novel anti-mesothelin antibody–conjugated QRnanocrystals, we have confirmed the feasibility of binding thesurface antigen expressed on esophageal adenocarcinomacell lines, and analogous strategies can be used for delivery ofimmunotoxins,10,23,24 immunoliposomes,43,44 or other nano-particle-encapsulated therapeutic payloads. The naturallyfluorescent anti-mesothelin QRs, or comparable mesothelin-targeted nanoimaging devices like quantum dots,45,46 canalso be used in the context of imaging of esophageal

300 H. Alvarez et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 295–301

adenocarcinoma in vivo, such as assessing for the presence ordepth of invasion in the primary lesion. Immunogenicmesothelin epitopes have been used as cancer vaccines inpreclinical models of ovarian cancer,25,26 and these could beextended to esophageal cancer as well. Most recently, phase Iclinical trials were conducted with a humanized monoclonalantibody targeting mesothelin (MORab-009; MorphotekInc., Downington, Pennsylvania) in persons with mesothe-lin-expressing solid cancers, and demonstrated a safe clinicalprofile. Antibody-based single-agent or combination thera-pies could be evaluated in the context of mesothelin-expressing advanced esophageal adenocarcinomas.47 Theavailability of a reliable immunohistochemical assay formesothelin expression in biopsy specimens would permitprior selection of patients most likely to benefit from suchmolecularly targeted therapies in clinical trials.

In summary, we have demonstrated mesothelin expres-sion in a subset (approximately one third) of esophagealadenocarcinomas, with absence of labeling in normalesophagus and noninvasive precursor lesions in the Barrettesophagus progression model. Future studies are warrantedexploiting mesothelin as a molecular target in esophagealadenocarcinoma for diagnostic and therapeutic purposes.

Acknowledgment

The authors thank Dr. Indrajit Roy for helpful discussions.

References

1. Paulson TG, Reid BJ. Focus on Barrett's esophagus and esophagealadenocarcinoma. Cancer Cell 2004;6:11-6.

2. Enzinger PC, Mayer RJ. Esophageal cancer. N Engl J Med 2003;349:2241-52.

3. Montgomery E, Goldblum JR, Greenson JK, Haber MM, Lamps LW,Lauwers GY, et al. Dysplasia as a predictive marker for invasivecarcinoma in Barrett esophagus: a follow-up study based on 138 casesfrom a diagnostic variability study. Hum Pathol 2001;32:379-88.

4. Weston AP, Sharma P, Topalovski M, Richards R, Cherian R, Dixon A.Long-term follow-up of Barrett's high-grade dysplasia. Am J Gastro-enterol 2000;95:1888-93.

5. Brown LM, Devesa SS. Epidemiologic trends in esophageal and gastriccancer in the United States. Surg Oncol Clin N Am 2002;11:235-56.

6. Sharma P, McQuaid K, Dent J, Fennerty MB, Sampliner R, Spechler S,et al. A critical review of the diagnosis and management of Barrett'sesophagus: the AGA Chicago Workshop. Gastroenterology 2004;127:310-30.

7. Spechler SJ. Clinical practice. Barrett's esophagus. N Engl J Med 2002;346:836-42.

8. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics,2007. CA Cancer J Clin 2007;57:43-66.

9. Chang K, Pastan I. Molecular cloning of mesothelin, a differentiationantigen present on mesothelium, mesotheliomas, and ovarian cancers.Proc Natl Acad Sci U S A 1996;93:136-40.

10. Hassan R, Bera T, Pastan I. Mesothelin: a new target for immunother-apy. Clin Cancer Res 2004;10:3937-42.

11. Argani P, Iacobuzio-Donahue C, Ryu B, Rosty C, Goggins M, WilentzRE, et al. Mesothelin is overexpressed in the vast majority of ductaladenocarcinomas of the pancreas: identification of a new pancreaticcancer marker by serial analysis of gene expression (SAGE). ClinCancer Res 2001;7:3862-8.

12. Iacobuzio-Donahue CA, Ashfaq R, Maitra A, Adsay NV, Shen-OngGL, Berg K, et al. Highly expressed genes in pancreatic ductaladenocarcinomas: a comprehensive characterization and comparison ofthe transcription profiles obtained from three major technologies.Cancer Res 2003;63:8614-22.

13. Hough CD, Sherman-Baust CA, Pizer ES, Montz FJ, Im DD,Rosenshein NB, et al. Large-scale serial analysis of gene expressionreveals genes differentially expressed in ovarian cancer. Cancer Res2000;60:6281-7.

14. Schaner ME, Ross DT, Ciaravino G, Sorlie T, Troyanskaya O, Diehn M,et al. Gene expression patterns in ovarian carcinomas. Mol Biol Cell2003;14:4376-86.

15. Ordonez NG. Application of mesothelin immunostaining in tumordiagnosis. Am J Surg Pathol 2003;27:1418-28.

16. McCarthy DM, Maitra A, Argani P, Rader AE, Faigel DO, Van HeekNT, et al. Novel markers of pancreatic adenocarcinoma in fine-needleaspiration: mesothelin and prostate stem cell antigen labeling increasesaccuracy in cytologically borderline cases. Appl ImmunohistochemMolMorphol 2003;11:238-43.

17. Chen Y, Zheng B, Robbins DH, Lewin DN, Mikhitarian K, Graham A,et al. Accurate discrimination of pancreatic ductal adenocarcinoma andchronic pancreatitis using multimarker expression data and samplesobtained by minimally invasive fine needle aspiration. Int J Cancer2007;120:1511-7.

18. Kachali C, Eltoum I, Horton D, Chhieng DC. Use of mesothelin as amarker for mesothelial cells in cytologic specimens. Semin DiagnPathol 2006;23:20-4.

19. Hassan R, Laszik ZG, Lerner M, Raffeld M, Postier R, Brackett D.Mesothelin is overexpressed in pancreaticobiliary adenocarcinomas butnot in normal pancreas and chronic pancreatitis. Am J Clin Pathol 2005;124:838-45.

20. Ordonez NG. The diagnostic utility of immunohistochemistry indistinguishing between epithelioid mesotheliomas and squamouscarcinomas of the lung: a comparative study. Mod Pathol 2006;19:417-28.

21. Hornick JL, Lauwers GY, Odze RD. Immunohistochemistry can helpdistinguish metastatic pancreatic adenocarcinomas from bile ductadenomas and hamartomas of the liver. Am J Surg Pathol 2005;29:381-9.

22. Jhala N, Jhala D, Vickers SM, Eltoum I, Batra SK, Manne U, et al.Biomarkers in Diagnosis of pancreatic carcinoma in fine-needleaspirates. Am J Clin Pathol 2006;126:572-9.

23. Hassan R, Viner JL, Wang QC, Margulies I, Kreitman RJ, Pastan I.Anti-tumor activity of K1-LysPE38QQR, an immunotoxin targetingmesothelin, a cell-surface antigen overexpressed in ovarian cancer andmalignant mesothelioma. J Immunother 2000;23:473-9.

24. Fan D, Yano S, Shinohara H, Solorzano C, Van Arsdall M, Bucana CD,et al. Targeted therapy against human lung cancer in nude mice by high-affinity recombinant antimesothelin single-chain Fv immunotoxin. MolCancer Ther 2002;1:595-600.

25. Hung CF, Calizo R, Tsai YC, He L, Wu TC. A DNAvaccine encoding asingle-chain trimer of HLA-A2 linked to human mesothelin peptidegenerates anti-tumor effects against human mesothelin-expressingtumors. Vaccine 2007;25:127-35.

26. Chang CL, Wu TC, Hung CF. Control of human mesothelin-expressingtumors by DNA vaccines. Gene Ther 2007;14:1189-98.

27. Thomas AM, Santarsiero LM, Lutz ER, Armstrong TD, Chen YC,Huang LQ, et al. Mesothelin-specific CD8(+) T cell responses provideevidence of in vivo cross-priming by antigen-presenting cells invaccinated pancreatic cancer patients. J Exp Med 2004;200:297-306.

28. Corso CD, Stubbs DD, Lee SH, Goggins M, Hruban RH, Hunt WD.Real-time detection of mesothelin in pancreatic cancer cell linesupernatant using an acoustic wave immunosensor. Cancer DetectPrev 2006;30:180-7.

29. Creaney J, van Bruggen I, Hof M, Segal A, Musk AW, de Klerk N, et al.Combined CA125 and mesothelin levels for the diagnosis of malignantmesothelioma. Chest 2007;132:1239-46.

301H. Alvarez et al. / Nanomedicine: Nanotechnology, Biology, and Medicine 4 (2008) 295–301

30. Badgwell D, Lu Z, Cole L, Fritsche H, Atkinson EN, Somers E, et al.Urinary mesothelin provides greater sensitivity for early stage ovariancancer than serum mesothelin, urinary hCG free beta subunit andurinary hCG beta core fragment. Gynecol Oncol 2007;106:490-7.

31. Huang CY, Cheng WF, Lee CN, Su YN, Chien SC, Tzeng YL, et al.Serum mesothelin in epithelial ovarian carcinoma: a new screeningmarker and prognostic factor. Anticancer Res 2006;26:4721-8.

32. Hellstrom I, Raycraft J, Kanan S, Sardesai NY, Verch T, Yang Y, et al.Mesothelin variant 1 is released from tumor cells as a diagnostic marker.Cancer Epidemiol Biomarkers Prev 2006;15:1014-20.

33. Antonioli DA, Wang HH. Morphology of Barrett's esophagus andBarrett's-associated dysplasia and adenocarcinoma. Gastroenterol ClinNorth Am 1997;26:495-506.

34. Montgomery E, Mamelak AJ, Gibson M, Maitra A, Sheikh S, Amr SS,et al. Overexpression of claudin proteins in esophageal adenocarcinomaand its precursor lesions. Appl Immunohistochem Mol Morphol 2006;14:24-30.

35. Powell EL, Leoni LM, Canto MI, Forastiere AA, Iocobuzio-DonahueCA, Wang JS, et al. Concordant loss of MTAP and p16/CDKN2Aexpression in gastroesophageal carcinogenesis: evidence of homozy-gous deletion in esophageal noninvasive precursor lesions andtherapeutic implications. Am J Surg Pathol 2005;29:1497-504.

36. Maitra A, Adsay NV, Argani P, Iacobuzio-Donahue C, De Marzo A,Cameron JL, et al. Multicomponent analysis of the pancreaticadenocarcinoma progression model using a pancreatic intraepithelialneoplasia tissue microarray. Mod Pathol 2003;16:902-12.

37. Cao D, Maitra A, Saavedra JA, Klimstra DS, Adsay NV, Hruban RH.Expression of novel markers of pancreatic ductal adenocarcinoma inpancreatic nonductal neoplasms: additional evidence of differentgenetic pathways. Mod Pathol 2005;18:752-61.

38. Ito T, Shimada Y, Kan T, David S, Cheng Y, Mori Y, et al. Pituitarytumor-transforming 1 increases cell motility and promotes lymph node

metastasis in esophageal squamous cell carcinoma. Cancer Res 2008;68:3214-24.

39. Yong KT, Qian J, Roy I, Lee HH, Bergey EJ, Tramposch KM, et al.Quantum rod bioconjugates as targeted probes for confocal and two-photon fluorescence imaging of cancer cells. Nano Lett 2007;7:761-5.

40. Chang K, Pastan I. Molecular cloning and expression of a cDNAencoding a protein detected by the K1 antibody from an ovariancarcinoma (OVCAR-3) cell line. Int J Cancer 1994;57:90-7.

41. Swierczynski SL, Maitra A, Abraham SC, Iacobuzio-Donahue CA,Ashfaq R, Cameron JL, et al. Analysis of novel tumor markers inpancreatic and biliary carcinomas using tissue microarrays. Hum Pathol2004;35:357-66.

42. Hansel DE, Dhara S, Huang RC, Ashfaq R, Deasel M, Shimada Y, et al.CDC2/CDK1 expression in esophageal adenocarcinoma and precursorlesions serves as a diagnostic and cancer progression marker andpotential novel drug target. Am J Surg Pathol 2005;29:390-9.

43. Pardridge WM. Gene targeting in vivo with pegylated immunolipo-somes. Methods Enzymol 2003;373:507-28.

44. Mamot C, Drummond DC, Noble CO, Kallab V, Guo Z, Hong K, et al.Epidermal growth factor receptor-targeted immunoliposomes signifi-cantly enhance the efficacy of multiple anticancer drugs in vivo. CancerRes 2005;65:11631-8.

45. Qian J, Yong KT, Roy I, Ohulchanskyy TY, Bergey EJ, Lee HH, et al.Imaging pancreatic cancer using surface-functionalized quantum dots.J Phys Chem B 2007;111:6969-72.

46. Gao X, Cui Y, Levenson RM, Chung LW, Nie S. In vivo cancertargeting and imaging with semiconductor quantum dots. NatBiotechnol 2004;22:969-76.

47. Armstrong DK, Laheru D, Ma WW, Cohen SJ, Phillips M, Brahmer J,et al. A phase 1 study of MORAb-009, a monoclonal antibody againstmesothelin in pancreatic cancer, mesothelioma and ovarian adenocarci-noma. J Clin Oncol 2007;25:14041.