CD44 andOTPAre Strong PrognosticMarkers for PulmonaryCarcinoids
Dorian R.A. Swarts1, Mieke E.R. Henfling1, Leander Van Neste7, Robert-Jan van Suylen4,Anne-Marie C. Dingemans3, Winand N.M. Dinjens5, Annick Haesevoets1, Martina Rudelius8, Erik Thunnissen6,MarcoVolante9,WimVanCriekinge10,Manon vanEngeland2, FransC.S. Ramaekers1, andErnst-JanM.Speel2
AbstractPurpose: Pulmonary carcinoids are well-differentiated neuroendocrine tumors showing usually a
favorable prognosis. However, there is a risk for late recurrence and/or distantmetastasis. Because histologic
classification in typical and atypical carcinoids is difficult and its reliability to predict disease outcome varies,
we evaluated three genes as potential prognostic markers, that is, orthopedia homeobox (OTP), CD44, and
rearranged during transfection (RET).
Experimental Design: These genes were analyzed in 56 frozen carcinoids by quantitative real-time PCR
(qRT-PCR). RET was further studied by methylation and mutation analysis. Immunohistochemistry for
CD44 and OTP protein expression was conducted on 292 carcinoids.
Results: Low mRNA expression levels of CD44 (P ¼ 1.8e�5) andOTP (P ¼ 0.00054), and high levels of
RET (P ¼ 0.025), were strongly associated with a low 20-year survival of carcinoid patients. High RET
expression was not related to promoter hypomethylation or gene mutations. A direct link between gene
expression andprotein levels was confirmed forCD44 andOTPbut not forRET.Within all carcinoids aswell
as atypical carcinoids, absence of CD44 protein was significantly associated with low 20-year survival (P¼0.00014 and 0.00013, respectively). The absence of nuclear OTP followed by complete loss of expression
was also significantly associatedwithunfavorable disease outcome in all carcinoids (P¼5.2�6).Multivariate
analyses revealed that age at diagnosis, histopathology, stage, and cytoplasmic OTP immunoreactivity were
independent predictors of prognosis.
Conclusions: Our study indicates that CD44 and OTP are strong indicators of poor outcome. We
therefore argue for implementation of thesemarkers in routine diagnostics in addition to histopathology to
improve subclassification of pulmonary carcinoids into prognostically relevant categories. Clin Cancer Res;
19(8); 2197–207. �2013 AACR.
IntroductionPulmonary carcinoids comprise a group of well-differen-
tiated neuroendocrine tumors (NET) with little relation tocigarette smoking (1). In contrast to high-grade lung carci-
nomas, such as small-cell lung cancer (SCLC) and large-cellneuroendocrine carcinoma (LCNEC), carcinoids are char-acterized by a lowermetastatic rate and a relatively favorableprognosis. According to the World Health Organization(WHO) classification, lung carcinoids are subclassified astypical carcinoids or atypical carcinoids (2). Atypicalcarcinoids are more often characterized by malignantbehavior and have a lower 5-year survival rate as com-pared with typical carcinoids (61%–88% vs. 92%–100%,respectively; ref. 3). Metastases will develop in 4% to 64%of carcinoid patients (typical carcinoids, 4%–14%; atyp-ical carcinoids, 35%–64%), usually in regional lymphnodes, but also at distant sites including liver, bones,brain, subcutaneous tissue, and breast (3, 4). The onlycurative treatment of pulmonary carcinoids is surgicalresection, whereas the use of chemotherapy or radiother-apy for patients with metastatic disease has limited cura-tive potential (1). In the inherited multiple endocrineneoplasia type I (MEN1) syndrome, approximately 5% ofpatients develop bronchial carcinoids (5). The MEN1gene is mutated in approximately 18% of sporadic lungcarcinoids (6).
Authors' Affiliations: Departments of 1MolecularCell Biology, 2Pathology,and 3Pulmonology, GROW—School for Oncology & Developmental Biol-ogy, Maastricht University Medical Center, Maastricht; 4Department ofPathology, Jeroen Bosch Hospital, 's-Hertogenbosch; 5Department ofPathology, ErasmusMC—University Medical Center, Rotterdam; 6Depart-ment of Pathology, VU University Medical Center, Amsterdam, the Nether-lands; 7MDxHealth, Irvine, California; 8Institute for Pathology, TechnicalUniversity Munich, Munich, Germany; 9Department of Clinical and Biolog-ical Sciences, Division of Pathology, University of Turin at San LuigiHospital, Orbassano, Turin, Italy; and 10Laboratory of Bioinformatics andComputational Genomics, Ghent University, Ghent, Belgium
Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).
Corresponding Author:Dorian R.A. Swarts, Department of Molecular CellBiology, UNS50-17, Maastricht University Medical Center, P.O. Box 616,6200MDMaastricht, the Netherlands. Phone: 31-43-3882998; Fax: 31-43-3884151; E-mail: [email protected]
Histologic classification of lung carcinoids is difficult andits reliability to predict disease outcome is variable (com-pare refs. 7–9). Furthermore, although most patientsremain cancer-free within 5 years after surgery, there is arisk for late recurrence and/or distant metastasis, the occur-rence of which cannot be sufficiently predicted by thepresent WHO classification. Alternatives to subclassify car-cinoids into prognostically relevant categories are thereforedesired.
A few studies have reported clinical and molecular para-meters associated with a higher risk of developing metas-tases and a poor disease outcome of carcinoid patients, suchas size 3.5 cm or more, high Ki-67 and Bcl-2 expression,deletions of chromosome 11q22.3-q25, and in typicalcarcinoids low membranous expression of the standardsplice variant of CD44 (7, 10–14). To analyze novel andmore reliable prognosticmarkers for pulmonary carcinoids,we have selected 3 genes from a gene expression profilingscreen of pulmonary carcinoids, comparing cases with afavorable and a poor disease outcome. These genes includethe CD44 and orthopedia homeobox (OTP) genes, whichwere downregulated in tumors from patients with a poordisease outcome, and the rearranged during transfection(RET) proto-oncogene that showed upregulated expressionin these patients. We analyzed the value of these genes asprognostic indicators using quantitative real-time PCR(qRT-PCR) in a series of 72 pulmonary NETs, and assesseda potential link between mRNA and protein expressionusing immunohistochemistry. Protein expression was thenvalidated by immunohistochemistry on a series of 352cases, including 60 of 72 tumors analyzed by qRT-PCR.
Materials and MethodsSupplementary Materials and Methods
A detailed description of the usedmaterials andmethodsin this study can be found in the Supplementary Data.
Collection of tumor material, clinical data, and celllines
We collected frozen material of 56 carcinoid tumors and16 high-grade neuroendocrine carcinomas (Table 1A), aswell as cell lines derived from NETs and RNA from normaltissues. Furthermore, we acquired a large series of formalin-fixed paraffin-embedded (FFPE) tissue, including 227typical carcinoids, 64 atypical carcinoids, 1 not furthersubclassified carcinoid, 24 large cell neuroendocrine carci-nomas, 35 SCLCs, and 1 not further subclassified high-grade neuroendocrine carcinoma.
Clinicopathologic data from the majority of patientscould be collected, including survival data up to 20 years.
Selection of genes from gene expression profilingThree genes were selected from a gene expression profil-
ing screen of pulmonary carcinoids (our unpublished data;available at http://www.ebi.ac.uk/arrayexpress/ underaccession number E-MEXP-3790), comparing cases witha favorable and apoor disease outcome. In this comparison,the OTP gene, was most strongly downregulated in tumorsfrom patients with a poor disease outcome with a medianfold-change of 845.CD44, for which protein expression hasbeen reported earlier to be associated with a favorableprognosis in typical carcinoid tumors (11), was also amongthe 15 strongest downregulated genes with a median fold-change of 29. The RET proto-oncogene showed the highestupregulation in the cases with a poor disease outcome witha median fold-change of 60.
Quantitative real-time PCRRNA isolation from frozen tissuewas conductedusing the
RNeasy Mini Kit (Qiagen GmbH). After conversion of RNAinto cDNA using the iScript cDNA Synthesis Kit (BioRad),qRT-PCR was conducted to assess mRNA expression ofCD44, OTP, and RET, as well as of 4 housekeeping genes(ACTB, CYPA, GUSB, and HPRT) using primers listed inSupplementary Table S1A.
RET mutation and methylation analysisRET mutation analysis was conducted for exons 10, 11,
and 16, known hotspot regions of mutation, using primerslisted in Supplementary Table S1B. These primers wereM13-tailed to facilitate sequencing.
Promoter hypermethylation of the RET proto-oncogenewas assessed using nested methylation-specific PCR (MSP),as described previously (15). All primer sequences areprovided in Supplementary Table S1C. Methylation andunmethylation-specific primers were described previously(16).
ImmunohistochemistryImmunohistochemistry on FFPE tissue sections was con-
ducted using the following primary antibodies: (i) mouseanti-CD44 (standard variant) monoclonal antibody, cloneDF1485 (Dako); (ii) rabbit anti-OTP polyclonal antibody(HPA039365; Atlas Antibodies); and (iii) rabbit anti-RETpolyclonal antibody (HPA008356; Atlas Antibodies).
Translational RelevancePulmonary carcinoids are well-differentiated neuro-
endocrine tumors, which however show a risk for laterecurrence and/or distant metastasis. Histologic classifi-cation in typical and atypical carcinoids can be difficultand may complicate prediction of disease outcome.Alternatives to subdivide carcinoids into prognosticallyrelevant categories are therefore desired. The currentstudy examines the expression of 3 genes in relation topatient survival, that is, CD44, orthopedia homeobox(OTP), and rearranged during transfection (RET). Ourdata reveal that a combination of CD44 andOTP immu-nostaining with histopathology reliably predicts patientoutcome, evenwithin carcinoid subgroups.We thereforeargue for the implementation of thesemarkers in routinediagnostics to identify patients at risk for tumor relapse.These patientsmaybeoffered an intensified surveillance.
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Antibodies were detected by Bright Vision Poly-HRP-anti-mouse/rabbit/rat immunoglobulin G (IgG; Immunologic)followed by peroxidase-DAB (3,30-diaminobenzidine)visualization. All stained tumor sections were independent-ly scored by 2 of the authors (D.R.A. Swarts and R.-J. vanSuylen) who were blinded for patient outcome; in case ofdisagreement, a consensus was reached after analysis by athird observer (E.-J.M. Speel). Scoring results were catego-rized into 5 different groups (0–4; see SupplementaryData). Positive cases were defined as having a scoring index>1 and negative cases displayed scoring indices �1. ForOTP, a distinction was made between nuclear and cyto-plasmic staining, which was scored separately.
Statistical analysisPossible correlations between clinical data, qRT-PCR,
and immunohistochemistry results were determined using
the x2 test, the Fisher exact test, the Student t test, andPearson’s correlation, when appropriate. Survival curveswere created using the Kaplan–Meier method and thelog-rank test was used to test for differences between sub-groups. Cox-regression was used for multivariate analyses.
ResultsLung carcinoids with a poor disease outcome showdownregulation ofOTP and CD44 and upregulation ofRET mRNA transcripts
The expression levels of 3 genes selected from an expres-sion profiling study, that is, CD44, OTP, and RET, wereanalyzed by qRT-PCR on frozen material of 56 carcinoidtumors and16high-grade lungneuroendocrine carcinomas(see Table 1A), 4 normal tissues and 9 neuroendocrine celllines. In Fig. 1A–C, the qRT-PCR results are provided as
Table 1. Clinicopathologic charcateristics of theFFPEand frozen tissue series of pulmonary carcinoids andhigh-grade neuroendocrine carcinomas
scatterplots. The gene expression levels ofCD44 andOTP inthe different tumor subtypes were highly correlated (P ¼3.15e�7). The mean relative gene expression levels of CD44and OTP, normalized to the levels of the ACTB and CYPAhousekeeping genes, showed a large variation within thegroup of carcinoid tumors but were close to 0 in high-gradeneuroendocrine carcinomas (Fig. 1A, B, D, and E). RET
expression did not differ significantly between these 2groups, but was slightly lower in high-grade lung NETs ascompared with carcinoids (Fig. 1C and F). Relative expres-sion values of CD44 (Fig. 1D) and OTP (Fig. 1E) weresignificantly decreased for carcinoid patients with a poorprognosis (defined as having distant metastasis and/ordeceased within 20 years after initial diagnosis), whereas
Figure 1. qRT-PCR results for CD44, OTP, and RET. A–C, scatterplots showing the relative mRNA expression values of CD44 (A), OTP (B), and RET (C) fortypical carcinoids (TC), atypical carcinoids (AC), LCNEC, SCLC, neuroendocrine cell lines (NECL), and normal tissues (NT), normalized to the geometric meanof the expression levels of the housekeeping genes ACTB and CYPA. The mean relative expression levels for the respective subgroups are indicated.The only neuroendocrine cell line showingCD44 expressionwas the insulinomacell lineCM (relative expression level 0.157, A). Levels ofOTP expressionwerevirtually absent in all cell lines analyzed.Only thepancreatic endocrine cell lineQGPexhibited considerableRET expression (relative expression level 0.083,C).D–F, differences between themean values�SDof gene expression levels between the total group of carcinoids (CD) and carcinomas (CAR) forCD44 [D, 95%confidence interval (CI), �0.283/�0.152], OTP (E, 95% CI, �0.382/�0.224), and RET (F, not significantly different), and in carcinoids from patientswith a favorable (F) and a poor (P) disease outcome forCD44 (D, 95%CI, 0.166/0.349),OTP (E, 95%CI, 0.176/0.437) andRET (F, 95%CI,�0.233/0.012). G–I,time-dependent ROC curves depicting the trade-off between the sensitivity and specificity ofCD44 (G),OTP (H), and RET (I) expression in predicting diseaseoutcome. J–L, Kaplan-Meier analyses depicting the difference in 20-year overall survival for high (solid lines) or low (dotted lines) mRNA expressionlevels of CD44 (J, cutoff value 0.109), OTP (K, cutoff value 0.132), and RET (L, cutoff value 0.0256). Cutoff values were determined using the ROC curvesdepicted in (G)–(I). The period is indicated in months. Censored cases are indicated by an asterisk. AUC, area under the curve.
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the values forRET (Fig. 1F) were increased. Cutoff values forsurvival analyses were determined using time-dependentarea under the receiver operating characteristic (ROC) curveexamination and were chosen to maximize sensitivity andspecificity for adverse disease outcome (Fig. 1G–I). Down-regulationofCD44 andOTP, andupregulation ofREThad alarge negative impact on 20-year overall survival, bothwithin the complete group of lungNETs (CD44: P¼ 1.1e�7;OTP: P ¼ 6.7e�5; RET: P ¼ 0.011) and within the group ofcarcinoids (Fig. 1J–L). Within the qRT-PCR series, thedifferential expression levels for all 3 genes outperformedhistologic subclassification into typical carcinoids and atyp-ical carcinoids (P ¼ 0.029; not shown) as indicators ofpatient outcome (see P values in Fig. 1J–L).
RET is not activated by gene mutation orhypomethylation of its promoter region in lungcarcinoidsWe decided to further investigate the RET proto-onco-
gene, as this gene is mutated in the multiple endocrineneoplasia type II (MEN2) syndrome and in other sporadicneuroendocrine neoplasms (17). We investigated thepossibility that either a mutation or promoter hypo-methylation of the RET oncogene is responsible for theincrease in its gene expression levels. Therefore, we con-ducted mutation and methylation analyses of 5 carcinoidcases with high RET gene expression levels and a poorprognosis and 5 cases with low expression levels and afavorable disease outcome. However, we did not identifymutations in exons 10, 11, and 16, known to be thehotspot regions of RET mutations (18). Furthermore, we
were unable to detect methylation of the RET promoter inthese 10 tumors (data not shown).
Establishment of CD44 and OTP as strong prognosticindicators for pulmonary carcinoids usingimmunohistochemistry
To further validate CD44, OTP, and RET as prognosticmarkers for lung carcinoids, we conducted immunohis-tochemistry for their encoded proteins on a series of 292lung carcinoids and 60 high-grade pulmonary neuroendo-crine carcinomas (see Table 1B), including 60 of 72 casesincluded in the qRT-PCR analyses (Supplementary TableS2). A preselection wasmade for extra atypical carcinoids toenhance the number of carcinoids with an unfavorableprognosis to increase the power of the study. The resultsof the immunohistochemical studies are summarizedin Table 2. Representative images are provided for carcinoidtumors in Fig. 2, for normal (lung) tissue in SupplementaryFig. S1, and for high-grade lungneuroendocrine carcinomasin Supplementary Fig. S2.
The immunohistochemistry results were correlated withclinical follow-up data, the outcome of which is shownin Fig. 3. Also, histopathologic subclassification into typicalcarcinoids and atypical carcinoids was correlated to diseaseoutcome. Atypical carcinoid histology was strongly associ-ated with poor disease outcome (P ¼ 8.2e�7; Fig. 3A).
CD44. CD44 displayed a strong membranous stainingpattern formost positive carcinoid cases (Fig. 2A),whereas asmall number of the CD44-negative tumors (Fig. 2B)showed a weak nuclear staining in dispersed cells. Mostcases showed a homogenous staining pattern for CD44,
Table 2. Immunohistochemistry results of CD44 and OTP protein expression in neuroendocrine lungtumors
whereas a small minority of cases had somewhat moreheterogenous immunoreactivity pattern. Normal lung dis-played positivity of basal cells in the bronchial epitheliumand in seromucous glands (Supplementary Fig. S1A). Fromthedata shown inSupplementary Table S2, it is obvious thatthe gene and protein expression levels of CD44 are directlycorrelated (P¼ 4.3e�10). Of the carcinoid tumors, 102 cases(36.2%)werenegative forCD44,whereas 180 cases (63.8%)were scored CD44-positive (Table 2). Absence of CD44staining was strongly correlated with low 20-year overallsurvival in the complete group of carcinoid tumors (Fig. 3B),as well as within the subgroup of atypical carcinoids (Fig.3C). Within the total carcinoid tumor group, absence ofCD44was also strongly correlatedwith distantmetastasis (P¼ 7.0e�7). There was also a correlation between CD44-negativity and Ki-67 proliferative index (P ¼ 0.017, usinga Ki-67 cutoff point of 2%; our unpublished data).
The tumor cells of high-grade lung NETs were usuallyCD44-negative (50 cases, 94.3%), although strong mem-
branous staining in the proximity of necrotic areas wasregularly seen (Supplementary Fig. S2A). Also within thetotal group of NETs, absence of CD44 was significantlyassociated with a low 20-year overall survival rate (P ¼6.2e�8; 171 positive cases and 130 negative cases) anddistant metastasis (P ¼ 1.6e�10).
OTP. The large majority of cases had a homogenousOTP staining pattern. Three different OTP staining patternscould be discerned (Table 2). A strong nuclear staining,either with or without a cytoplasmic reactivity, was seen in207 carcinoid cases (71.6%; Fig. 2C), whereas exclusivelycytoplasmic staining was seen in 32 cases (11.1%; Fig. 2D),and 50 carcinoid cases (17.3%) were negative (Fig. 2E). Incase of nuclear reactivity, more than 90% of the tumor cellswere generally positive. OTP transcription levels and nucle-ar protein expression levels (Supplementary Table S2) werestrongly correlated (P ¼ 5.9e�11). The subset of cases thatlost nuclear staining, but retained cytoplasmic reactivity, aswell as the completely negative cases, showed lowOTP geneexpression levels.
In normal lung, cytoplasmic reactivity for OTP was evi-dent in bronchial epithelial cells, macrophages and inseromucous glands (Supplementary Fig. S1B).
Absence of nuclear OTP staining was correlated to a poorprognosis, within the complete group of carcinoid tumors(Fig. 3D) and within typical carcinoids (Fig. 3E). Thedifference in survival between cases with or without nuclearimmunoreactivity became particularly prominent after 5years following first diagnosis. Absence of nuclear OTPexpression was correlated to the occurrence of distantmetastasis (P¼ 0.00014). Similarly, absence of cytoplasmicOTP staining was associated with adverse disease outcomewithin the complete group of carcinoids (P ¼ 0.00038).Also, the subdivision of OTP expression according to the 3immunostaining patterns was strongly related to patientoutcome within the group of pulmonary carcinoids (Fig.3F), the patients with nuclear OTP reactivity having the bestoutcome, patients with cytoplasmic staining displayingintermediate survival and negative tumors having the worstdisease outcome.
High-grade neuroendocrine lung carcinomas most oftendid not exhibit nuclear OTP staining (Table 2). Only 5 cases(8.5%) exhibited nuclear reactivity (Supplementary Fig.S2B), 8 cases (13.6%) retained cytoplasmic expression, and46 cases (78.0%) were negative (Supplementary Fig. S2C).Interestingly, 2 OTP-positive, limited-stage SCLCs (Supple-mentary Fig. S2B) were disease-free 5 and 10 years aftersurgery, respectively. Also, within the combined group ofNETs, absenceofnuclear (P¼ 3.6e�12; 195positive cases and117negative cases) or cytoplasmic (P¼ 5.0e�11; 219 positivecases and 93 negative cases) OTP reactivity was associatedwith low 20-year survival. Absence of nuclear OTP immu-nostainingwasalso related todistantmetastasis (P¼1.8e�9).Again, the subdivision into 3 OTP staining categories (Sup-plementary Fig. S3) showed a strong relation to prognosis.
CD44 combined with OTP. Positive immunostainingfor membranous CD44 and nuclear OTP reactivity werefound to be highly correlated (P ¼ 3.3e�35). When
A B
C D
E F
Figure 2. CD44, OTP, and RET immunohistochemistry of pulmonarycarcinoids. Representative immunohistochemical staining patterns forCD44 (A and B), OTP (C–E) and RET (F). A, typical carcinoid of a patientwith no evidence of disease (NED) 7 years after diagnosis. A very strongmembranous CD44 staining is observed in all cells. B, atypical carcinoidof a patient that died from metastatic disease 12 years after initialdiagnosis. In this casemembranousCD44 staining is absent. However, ina subset of cells (5%–10%) faint nuclear staining is seen. C, typicalcarcinoid of a patient with NED 25 years after initial diagnosis. This tumorshows both strong nuclear and cytoplasmic OTP staining. When nuclearstaining was prominent cytoplasmic staining could be either strong as inthe depicted case, or faint to negative. D, atypical carcinoid of a patientalive 10 years after diagnosis. This case has lost nuclear OTP staining butretains strong cytoplasmic localization. E, typical carcinoid of a patientthat died 5 years after initial diagnosis, which lost OTP expressioncompletely. F, liver metastasis of an atypical carcinoid of a patient thatdied of the disease 13 years after initial diagnosis. Note the punctatecytoplasmic RET staining. Immunohistochemistry results for normal(lung) tissues are provided in Supplementary Fig. S1 and for high-gradelung NETs in Supplementary Fig. S2.
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combining OTP and CD44 results, no gain in survival ratewas seen when both were found positive, but in case of anegative reaction for 1 of 2, negativity for the other was ofstrong additive value in predicting poor outcomewithin thetotal group of carcinoids (Fig. 4A) as well as the total groupof pulmonary NETs (Fig. 4B).RET. In lung carcinoids, RET did not show the typical
membranous stainingpatternasdescribed for normal ileumandcolon(ref. 19; Supplementary Fig. S1C),butwas locatedcytoplasmically, often showing a punctate staining pattern
(Fig. 2F) and sometimes a weak nuclear staining in a subsetof cells. In normal lung, RET expression was found in thecytoplasm of the bronchial epithelial cells and was seen inmucous glands with amembranous staining with occasion-ally a punctate pattern (Supplementary Fig. S1D). Becausethe RET immunohistochemistry results could not be corre-lated to the qRT-PCR results (Supplementary Table S2), andbecause our initial immunostaining results could not beassociated with patient outcome (data not shown), we didnot proceed with the analysis of RET in the complete series.
Figure 3. Survival analyses ofpulmonary carcinoids usinghistopathology and proteinexpression of CD44 and OTP asprognostic indicators. Kaplan–Meieranalyses showing 20-year overallsurvival rates for (A, B, D, and F) thecomplete group of carcinoid tumors,(C) atypical carcinoids, or (E) typicalcarcinoids. The period is indicated inmonths. Censored cases areindicated by an asterisk. A,histopathologic classification for the281 cases that could besubclassified into typical carcinoidsand atypical carcinoids and forwhichfollow-up data were available. Thesolid line indicates typical carcinoidsand the dotted line refers to atypicalcarcinoids. B and C, correlationbetween survival and CD44immunostaining. Dotted linesindicate staining intensities � 1,whereas solid lines depict cases withstaining intensities > 1. Overallsurvival is depicted for the 264 of 282cases with available follow-up fromthe complete group of carcinoids (B)or for the 59 of 61 atypical carcinoidswith available follow-uponly (C). D–F,correlation between survival andOTP immunostaining. Overallsurvival for nuclear OTP reactivity forthe 269 of 289 cases with availablefollow-up from the complete group ofcarcinoids (D) or for the 207 of 225typical carcinoids with availablefollow-up only (E). Dotted linesindicate staining intensities � 1,whereas solid lines depict cases withstaining intensities > 1. F, gradient ofOTP immunostaining for the 269 of289 carcinoids with available follow-up. The solid lines indicate caseswithnuclear staining which may or maynot display cytoplasmicimmunoreactivity, the dashed linesindicate tumors which have lostnuclear staining but retaincytoplasmic positivity, and thedotted lines indicate negativetumors.
Prognostic Markers for Lung Carcinoids
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Univariate and multivariate analysis. Univariate analy-ses were conducted on tumor subgroups using clinicopath-ologic parameters (age at diagnosis, histopathologic dis-tinction between typical carcinoids and atypical carcinoids,sex, stage, and tumor diameter) and molecular parameters(CD44 and OTP immunohistochemistry results, Ki-67 pro-liferation index; Supplementary Table S3). In multivariateanalysis, age at diagnosis, histopathology, stage, andabsence of cytoplasmic reactivity for OTP were significantlyassociated with decreased 20-year overall survival withinthe group of carcinoids (Supplementary Table S3). Withinthe groupof atypical carcinoids, only stagewas significant inmultivariate analysis, although CD44 immunostainingshowed a trend toward significance (P ¼ 0.069). Withinthe group of typical carcinoids, age at diagnosis and nuclearOTP status were significantly correlated with disease out-come (Supplementary Table S3).
DiscussionSo far, only few prognostic parameters have been
described for pulmonary carcinoids, including histology(7, 10–14). In this report, we evaluated the value of CD44,OTP, and RET expression to improve the prediction ofpulmonary carcinoid prognosis. By analyzing a series ofalmost 300 carcinoids (227 typical carcinoids, 64 atypicalcarcinoids), we could show that loss of CD44 or OTPexpression is a strong indicator of adverse patient outcome.The immunohistochemical staining patterns are easy tointerpret and their detectability in paraffin-embeddedtissue sections makes them readily applicable in routinediagnostics.
With respect toCD44,weobserved a strong positive effecton patient outcome when membranous staining was pres-ent. This is the case for both typical carcinoids and atypical
carcinoids, the latter being largely neglected in the literature.Because the standard variant of CD44 was shown to be themost potent prognostic marker in the study by Granbergand colleagues (11), we chose to analyze only this variant inthe underlying study. Our results are in agreement with theliterature, where in smaller series of neuroendocrine lungtumors the presence of CD44 expression has been associ-ated with a favorable disease outcome. Carcinoids havebeen reported to be frequently positive for both the stan-dard and variant CD44 forms (11, 20–23). In the study byGranberg and colleagues (11), the presence of the standardvariant, as well as the CD44 variant 9, was associated with apositive outcome within 43 typical carcinoids. In a study ofSun and colleagues (23) on carcinoids, including 20 casesof pulmonary origin, CD44 negativity was associated withmetastasis. SCLCs are most often negative for CD44(20, 22), which is confirmed by our data. These combineddata point to a tumor suppressive role for CD44. Indeed,CD44-null fibroblasts are tumorigenic in nude mice, andCD44 has been suggested to promote apoptosis (24, 25).However, on the other hand, CD44 is involved in epithelial-to-mesenchymal transition and in promoting cell survival(24, 26). It is therefore not surprising that within humancancers CD44 expression has been described as anindicator of both a favorable (27–29) as well as a poorprognosis (30–32).
From the underlying study, using almost 300 cases ofcarcinoids with variable disease outcome, it is evident thatalso OTP downregulation is undeniably related to tumorprogression.Our data indicate a gradual loss ofOTP proteinexpression in correlation with prognosis. Nuclear OTPexpression was directly coupled to its mRNA expressionand strongly correlated to CD44 protein expression. OTPgene expression levels were close to 0 in neuroendocrine
Figure 4. Survival analyses of pulmonary NETs in relation to a combination of CD44 and OTP protein expression. Kaplan–Meier analyses showing 20-yearoverall survival rates for (A) the 261 of 279 carcinoidswith available follow-up and (B) the 298 of 331 cases of the combined group of carcinoids and high-gradeneuroendocrine carcinomas with available follow-up, using the combined CD44 and OTP immunohistochemistry results. The period is indicated in months.Censored cases are indicated by an asterisk. The solid lines indicate cases with both CD44 and OTP (nuclear and/or cytoplasmic) protein expression. Thedashed lines indicate tumors which have lost CD44 or OTP expression, and the dotted lines indicate tumors negative for both proteins.
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cancer cell lines and in most high-grade neuroendocrinecarcinomas, where the typical nuclear staining pattern wasvery rare. Because nuclear OTPwas also present in 2 limitedstage SCLC cases (both CD44-negative) with good out-come, OTP might also be a prognostic indicator in high-grade neuroendocrine lung carcinomas. For OTP, an evo-lutionary conserved homeodomain-containing transcrip-tion factor, currently only functions within the centralnervous system have been described (33–35). OTP hasimportant roles in the development of the neurosecretorysystem in the hypothalamus and in terminal differentiationof neuroblasts (33, 35). Especially differentiation, prolifer-ation, and migration of Otp-expressing cells were severelyabrogated in Otp�/� mice (33). Amir-Zilberstein and col-leagues (34) reported that Otp can be induced by stress,leading to transcriptional activation of, for example, corti-cotropin-releasing hormone. Only recently, Kim and col-leagues (36) have described OTP for the first time in thecontext of cancer, showing OTP methylation in breastcancer using CpG microarray analysis. Therefore, the needfor a study into the function of OTPwithin neuroendocrinetissues and neuroendocrine (lung) tumors is obvious.We noticed that the combination of CD44 and OTP
results allows an even better separation of tumors intoprognostic categories, because CD44 is a strong prognosticmarker within the group of atypical carcinoids, whereas theloss of nuclear OTP immunoreactivity has a strong prog-nostic value within the group of typical carcinoids. Inaddition, they partially compensate each other’s shortcom-ings from a methodologic point of view. CD44 showsoccasionally a heterogeneous staining pattern and the anti-body strongly reacts with near-necrotic cells, particularly inhigh-grade lung NETs. This might cause problems whenanalyzing small biopsy samples. For OTP no monoclonalantibody is currently available, which impedes standardi-zation of the staining procedure, and the cytoplasmic andnuclear immunoreactivity of OTP may make evaluationslightly more complicated.Besides CD44 and OTP immunostaining, also other
parameters such ashistopathologywere significantly relatedto disease outcome. Multivariate analysis showed that his-topathologic distinction between typical carcinoids andatypical carcinoids, stage and OTP immunostaining areindependent predictors of disease outcome within carci-noids. In addition, after carcinoid classification, OTP is themost optimal prognostic indicator in typical carcinoids. Inthe group of atypical carcinoids, CD44 showed a trendtoward significance, next to stage. Although histopathologyalone is a strong prognostic indicator in the underlyingstudy, classification of lung carcinoids using the WHOcriteriamaybedifficult and its ability to predict theoutcomeof especially atypical carcinoids varied in previous studies(7–9). Furthermore, counting mitoses is time consumingand may be difficult, because for example pyknotic apo-ptotic nuclei and mitoses are hard to distinguish (37, 38).Only 2 small studies showed interobserver variation to bepresent and more prominent in atypical carcinoids than intypical carcinoids (37, 39). Taken together, we argue for the
combined use of histopathology and CD44 and OTPimmunostaining.
Staining for bothCD44,which is usually negative inhigh-grade lung NETs, andOTP, which is negative or confined tothe cytoplasm in these tumors, may aid in the differentialdiagnosis between SCLC and carcinoid tumors, which isknown to be difficult in the case of small biopsies (40). Theabsence ofmembranous CD44 and nuclear OTP expressionin most high-grade lung NETs provides also further evi-dence for separate tumorigenesis pathways of these tumorsand carcinoids (1).
Expression of the RET proto-oncogene, a tyrosine kinasereceptor (17), was also correlated at the transcriptional levelwith patient outcome. Unfortunately, this could not beconfirmed at the protein level, and RET expression wastherefore not tested in the complete series. However, thetranscriptional upregulation of RET in aggressive carcinoidsmade it worthwhile to study the methylation status of itspromoter region. Methylation of the RET promoter has beenshown in colorectal cancer by Mokarram and colleagues(16), who did not study its effect on gene expression levels.We did not identify RET methylation in 10 pulmonarycarcinoid cases, andwe therefore concluded thathypomethy-lation of the promoter region does not underly its activationin pulmonary carcinoids, at least not in our studied cases.
Both gain and loss of function of RET is implicated indisease and can lead to MEN2 and Hirschprung disease,respectively (17). In contrast to the MEN1-syndrome,caused by mutations in the MEN1 gene, pulmonary carci-noids have not been associated with the MEN2-syndrome.In this study, we exclude mutation as the cause for RETupregulation in the ten cases that were studied for muta-tions in exons 10, 11, and 16. Few other studies haveassessedRETmutations in sporadic lungNETs. Komminothand colleagues (41) did not find mutations in 11 lungcarcinoids and 7 SCLCs. Futami and colleagues (42) iden-tified2 identicalmutations in 2of 6 SCLCcell lines and theirrespective primary tumors, but not in an additional 12 cases(43). No RETmutations were identified in a separate studyof 54 SCLC cell lines (44).
Alternatively, investigation of RET translocations hasbecome relevant because of the recent reports that suchtranslocations can take place in lung adenocarcinomas(45–47).
ConclusionUsing a series of almost 300 cases, we present CD44 and
OTP as powerful prognostic markers for pulmonary carci-noids. In lung NETs, CD44 and OTP gene expression levelsare directly correlated with their respective protein expres-sion levels. Furthermore, low transcriptional as well asprotein expression levels are strongly associated with a poorlong-term survival rate of pulmonary carcinoid patients.After independent validation, these markers may potential-ly be implemented in addition to the current histologicclassification of pulmonary carcinoid tumors to improveprediction of prognosis.
Prognostic Markers for Lung Carcinoids
www.aacrjournals.org Clin Cancer Res; 19(8) April 15, 2013 2205
Our resultsmay also have clinical implications, for exam-ple by increasing the frequency of follow-up when thesemarkers are negative, to detect metastatic disease as early aspossible.
Disclosure of Potential Conflicts of InterestW. Van Criekinge is employed as CSO in MdxHealth. E.-J.M. Speel has
honoraria from Speakers Bureau of Pfizer and Lilly and is a consultant/advisory board member of Pfizer. No potential conflicts of interest weredisclosed by the other authors.
Authors' ContributionsConception and design: D.R.A. Swarts, A.-M.C. Dingemans, W. Van Crie-kinge, M. van Engeland, F.C.S. Ramaekers, E.-J.M. SpeelDevelopment of methodology: D.R.A. Swarts, M.E.R. Henfling, L. VanNeste, M. van EngelandAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): D.R.A. Swarts, M.E.R. Henfling, R.-J. van Suylen,W.N.M. Dinjens, M. Rudelius, E. Thunnissen, W. Van CriekingeAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): D.R.A. Swarts, L. Van Neste, R.-J. vanSuylen, A.-M.C. Dingemans, W.N.M. Dinjens, E. Thunnissen, W. Van Crie-kinge, M. van Engeland, E.-J.M. SpeelWriting, review, and/or revision of themanuscript:D.R.A. Swarts, L. VanNeste, A.-M.C.Dingemans, E. Thunnissen,M. Volante,M. van Engeland, F.C.S. Ramaekers, E.-J.M. SpeelAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):M.E.R. Henfling, A. Haesevoets, M.RudeliusStudy supervision: M. Volante, F.C.S. Ramaekers, E.-J.M. Speel
AcknowledgmentsThe authors thank Ben van der Borne (Department of Pulmonology,
CatharinaHospital, Eindhoven, theNetherlands), Jos Broers (Department ofMolecular Cell Biology, Maastricht University Medical Center), Paul Klink-hamer (PAMM Laboratories, Eindhoven, the Netherlands); Marius Nap(Department of Pathology, Atrium Medical Center, Heerlen, the Nether-lands), Francien van Nederveen (Department of Pathology, Erasmus MC—University Medical Center), Aurel Perren (Department of Pathology, Uni-versity of Bern, Switzerland); Ren�e Schapers (Department of Pathology,Viecuri Medical Center, Venlo, the Netherlands), and Loes van Velthuysen(Divison of Pathology, Netherlands Cancer Institute, Amsterdam, the Neth-erlands) for providing tumor cases. The authors also thank Gerben Bootsma(Department of Lung Diseases, AtriumMedical Center, Heerlen, the Nether-lands), Sjaak Burgers (Department of Thoracic Oncology, NetherlandsCancer Institute, Amsterdam, theNetherlands), Harry Pouwels (Departmentof LungDiseases, Viecuri Medical Center, Venlo, the Netherlands), andMarcScheltinga (Department of Surgery, M�aximaMedical Center, Veldhoven, theNetherlands) for providing clinical data of the cases included in this studyand/or to allow collection of clinical data. Finally, the authors thank MurielDraht and Kim Wouters (Department of Pathology, Maastricht UniversityMedical Center) for providingMSPprimers forRETmethylation analysis andEdward Post (Department of Pathology, Erasmus MC—University MedicalCenter) for conducting RET mutation analysis.
Grant SupportThis study was sponsored by a grant from the Jan Dekkerstichting &
Ludgardine Bouwmanstichting (nr. 2010_010).The costs of publication of this articlewere defrayed in part by thepayment
of page charges. This articlemust therefore be herebymarked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received September 27, 2012; revised January 25, 2013; accepted February18, 2013; published OnlineFirst February 26, 2013.
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2013;19:2197-2207. Published OnlineFirst February 26, 2013.Clin Cancer Res Dorian R.A. Swarts, Mieke E.R. Henfling, Leander Van Neste, et al. CarcinoidsCD44 and OTP Are Strong Prognostic Markers for Pulmonary
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