美国外科病理学杂志09年4月 第18篇

Immunohistochemical Staining for CDX-2,PDX-1,NESP-55,and TTF-1Can Help Distinguish Gastrointestinal

Carcinoid Tumors From Pancreatic Endocrine and

Pulmonary Carcinoid Tumors

Amitabh Srivastava,MD*and Jason L.Hornick,MD,PhD w z

Abstract:Well-di?erentiated neuroendocrine tumors (WDNET)of the gastrointestinal tract,pancreas,and lung are histologi-cally similar.Thus,predicting the site of origin of a metastasis is not possible on morphologic grounds.Prior immunohisto-chemical studies of WDNET have yielded con?icting results,and pancreatic and duodenal homeobox factor-1(PDX-1)has not previously been evaluated in this context.We therefore analyzed the expression of CDX-2,PDX-1,TTF-1,and neuroendocrine secretory protein-55(NESP-55),a recently described member of the chromogranin family,in primary and metastatic WDNET.In total,64gastrointestinal carcinoids (5stomach;5duodenum;31ileum;11appendix;and 12rectum);39pancreatic endocrine tumors (PET);and 20pulmonary carcinoid tumors were studied.PET were positive for NESP-55(16/39)and PDX-1(11/39);3/31also showed heterogeneous positivity for CDX-2.Ileal carcinoids were exclusively positive for CDX-2(30/31)and negative for all other markers.Appendiceal carcinoids were uniformly positive for CDX-2(11/11).All rectal carcinoids were negative for CDX-2and TTF-1;2/12were positive for PDX-1,and 1/12for NESP-55.The gastric and duodenal carcinoids were only positive for PDX-1(7/10).TTF-1positivity was con?ned to pulmonary carcinoids (7/20);1/20was positive for NESP-55;and all were negative for CDX-2and PDX-1.NESP-55and PDX-1positivity,in the presence of negative CDX-2and TTF-1,was 97%speci?c for PET.The sensitivity and speci?city of CDX-2positivity for predicting an ileal primary,when PDX-1,NESP-55,and TTF-1were negative,was 97%and 91%,respectively.TTF-1positivity was con?ned to pulmonary carcinoids in our study but was present in only about a third of cases.A panel of these 4markers may be useful in predicting the primary site of metastatic WDNET.

Key Words:carcinoid tumor,endocrine,neuroendocrine,metastasis,immunohistochemistry

(Am J Surg Pathol 2009;33:626–632)

W

ell-di?erentiated neuroendocrine (carcinoid)tumors (WDNET)of pulmonary and gastrointestinal (GI)origin are morphologically similar to pancreatic endocrine tumors (PET).These tumors also share immunohistochem-ical reactivity for generic markers of neuroendocrine di?erentiation such as chromogranin and synaptophysin.Thus,unlike metastatic adenocarcinomas of unknown primary,where morphologic appearances and distinctive cytokeratin subsets can be used to suggest possible primary sites of origin,it is often not possible to predict the site of origin of a metastatic endocrine tumor when associated with a clinically occult primary.

Previous studies have attempted to evaluate cyto-keratin expression pro?les in endocrine tumors from various sites but have not proven to be useful in distinguishing endocrine tumors of pancreatic origin from those of GI or pulmonary primary sites.3The utility of immunohistochemical expression of transcription factors TTF-1and CDX-2for predicting a pulmonary or GI origin of carcinoid tumors,respectively,has also been studied previously,1,2,5–8,13,17,19but these markers are not widely used currently in this context in routine clinical practice.Islet-1was recently suggested as a useful marker for PET but 16%of pulmonary and 3%of ileal tumors were also positive for Islet-1in that study.20The pancreatic-duodenal homeobox 1(PDX-1)transcription factor is expressed in pancreatic progenitor cells in the developing embryo,and its expression is eventually restricted to a subset of endocrine and acinar cells.In adults,PDX-1expression can be detected in pancreatic polypeptide and insulin-secreting PP and b cells,respec-tively.4,12,22However,the utility of immunohistochemical expression of PDX-1in predicting a pancreatic primary endocrine tumor has not been evaluated thus far.Furthermore,neuroendocrine secretory protein-55(NESP-55)is a recently described member of the chromogranin family that seems to have a more restricted distribution in normal tissues as compared with chromo-granin A and has been proposed as a marker of cells di?erentiating toward an adrenal chroma?n cell or pancreatic islet cell phenotype.9,21

Copyright r 2009by Lippincott Williams &Wilkins

From the *Dartmouth Medical School,Dartmouth-Hitchcock Medical

Center Lebanon,NH;w Department of Pathology,Brigham and Women’s Hospital;and z Harvard Medical School,Boston,MA.Presented in part at the 97th annual meeting of the United States and

Canadian Academy of Pathology in Denver,CO,March 1to 7,2008.Correspondence:Jason L.Hornick,MD,PhD,Department of

Pathology,Brigham and Women’s Hospital,75Francis Street,Boston,MA 02115(e-mail:jhornick@https://www.360docs.net/doc/94281559.html,).O RIGINAL A RTICLE

The aim of our study was,therefore,to assess whether a panel of immunohistochemical markers includ-ing TTF-1,CDX-2,PDX-1,and NESP-55can be used to predict the site of origin of well-di?erentiated endocrine tumors of the gastro-enteric-pancreatic axis.

MATERIALS AND METHODS

A total of123well-di?erentiated endocrine tumors were retrieved from the surgical pathology archives of Brigham and Women’s Hospital.These included64 GI primary and metastatic carcinoid tumors(5stomach; 5duodenum;31ileum,including21primary and 10metastatic;11appendix;and12rectum);39PET (31primary,8metastatic);and20pulmonary carcinoid tumors.Hematoxylin and eosin and immunostained slides were available for review in all cases.A representa-tive para?n block with adequate tissue material for additional immunoperoxidase studies was selected from each case.

Immunoperoxidase staining for CDX-2,PDX-1, and TTF-1was performed using commercially available antibodies.A rabbit polyclonal antibody raised against the C-terminal octapeptide(GAIPIRRH)of the NESP-55 protein was obtained from Dr Fischer-Colbrie’s labora-tory and has been well-characterized previously.21The details of primary antibodies used in the study are summarized in Table1.Brie?y,5-m m-thick tissue sections were mounted onto charged slides,depara?nized,and rehydrated before immunostaining.All immunohisto-chemical staining was performed after antigen retrieval [using intermittent heating for4cycles of5min each in a 625-W microwave oven to maintain the temperature of the bu?er(0.01M citrate bu?er,pH6.0)at951C]and run in parallel with known positive and negative controls. After incubation with the primary antibody,the relevant secondary antibody was applied and the reaction product was developed using3,30-diaminobenzidine as the chro-mogen.Positive controls included sections of normal thyroid for TTF-1,normal colon for CDX-2,normal pancreas for PDX-1,and human adrenal medulla for NESP-55.Negative controls included those wherein the primary antibody was omitted and replaced with normal rabbit serum at equivalent concentration.

Positive staining for TTF-1,CDX-2,and PDX-1 was visualized as a dark brown reaction localized to the nucleus.A cytoplasmic staining pattern with prominent perinuclear accentuation was seen with NESP-55.The intensity of staining for each antibody was graded as weak,moderate,or strong and the extent of staining was scored from0to5(0=negative,1=<10%, 2=10%to25%,3=25%to50%,4=50%to75%, and5=>75%).The sensitivity and speci?city of each antibody for predicting a particular site of origin of endocrine tumors within the gastro-enteric-pancreatic axis was then calculated.

RESULTS

The staining pattern of the WDNETs included in this study is summarized in Table2.PET were positive for NESP-55(16/39)and PDX-1(11/39);7showed variable, heterogeneous reactivity for CDX-2(Fig.1),and none were positive for TTF-1.Uniformly strong immuno-reactivity was present in all PETs positive for NESP-55 and more than50%of the tumor cells were positive in 11/16of these cases.PDX-1,when present,was positive in>50%of tumor cells in all but one case,and the intensity of staining was moderate or strong in10/11 positive cases.In contrast to NESP-55and PDX-1,CDX-2 showed a variable intensity and heterogeneous distribution. Only3/31primary PETs were positive for CDX-2;the staining in these cases was weak or moderate and was present in<50%of the tumor cells in all cases.However, 4/8metastatic PETs showed CDX-2positivity that was of variable intensity but present in>50%of the tumor cells.

Ileal carcinoids were exclusively positive for CDX-2 (30/31)(Fig.2).The intensity of staining was moderate or strong in24/30cases and>50%of the tumor cells were positive in27/30positive cases.No staining was observed for PDX-1,TTF-1,or NESP-55in any of the primary or metastatic ileal carcinoids.The appendiceal carcinoids were also uniformly positive for CDX-2(11/11)and none for NESP-55or TTF-1.However,6/11appendiceal carcinoids showed PDX-1reactivity as well.All rectal carcinoids were negative for CDX-2and TTF-1;2/12 were positive for PDX-1,and1/12was positive for NESP-55.The gastric and duodenal carcinoids were only positive for PDX-1(7/10)and were negative for CDX-2, TTF-1,and NESP-55.All but one of7gastro-duodenal carcinoids that were positive for PDX-1showed staining in >50%of the tumor cells(Fig.3).

TTF-1positivity was con?ned to pulmonary carci-noids(7/20)in our study(Fig.4).The staining was weak

TABLE1.Details of Primary Antibodies Used in the Study

Primary

Antibody Clone Dilution Antigen Retrieval Vendor CDX-2CDX2-881:400Microwave;citrate bu?er Biogenex,San Ramon,CA

PDX-1Goat

polyclonal 1:100Microwave;citrate bu?er Santa Cruz Biotechnology,Santa Cruz,

CA

NESP-55Rabbit

polyclonal

1:4000Microwave;citrate bu?er Gift From Dr Fischer-Colbrie TTF-18G7G3/11:1000Microwave;citrate bu?er Dako,Carpenteria,CA

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in intensity in all positive cases and was present in >50%of the tumor cells in 5/7positive tumors.NESP-55positivity was present in 1/20pulmonary carcinoid tumor,and all cases were negative for CDX-2and PDX-1.

The speci?city of each antibody for predicting the site of origin of the primary tumor is summarized in Table 3.

Table 4illustrates the utility of a panel of immuno-histochemical markers to help predict site of origin of a WDNET.Thus,NESP-55and PDX-1positivity in the presence of negative CDX-2and TTF-1stains was highly speci?c (97%)for PETs in our study.Similarly,the sensitivity and speci?city of CDX-2positivity for predicting an ileal primary,when PDX-1,NESP-55,and TTF-1were negative,was 97%and 91%,respectively.TTF-1positivity was con?ned to pulmonary carcinoids in our study but was present in only about a third of cases.

DISCUSSION

Carcinoids are rare tumors that arise from neuro-endocrine cells of the GI or respiratory tract and comprise less than 1%of all cancers.Studies using the SEER database have suggested that the incidence of carcinoid tumors is increasing steadily.14,15Some recent studies have estimated that the incidence of carcinoid tumors has increased from 0.62to 5.17/100,000over

a

FIGURE 1.Pancreatic endocrine tumor (A;hematoxylin and eosin,?200)with diffuse NESP-55immunoreactivity (B),which shows the characteristic perinuclear accentuation.A subset of pancreatic endocrine tumors also stains with PDX-1(C)and shows weak,heterogeneous CDX-2positivity (D).

TABLE 2.Immunohistochemical Expression of CDX-2,PDX-1,NESP-55,and TTF-1in Well-differentiated Neuroendocrine Tumors

Site

CDX-2PDX-1NESP-55TTF-1Stomach (N =5)0(0%)3(60%)0(0%)0(0%)Duodenum (N =5)0(0%)4(80%)0(0%)0(0%)Ileum (N =31)30(97%)0(0%)0(0%)0(0%)Appendix (N =11)11(100%)6(55%)0(0%)0(0%)Rectum (N =12)0(0%)2(17%)1(8%)0(0%)Pancreas (N =39)7(18%)11(28%)16(41%)0(0%)Lung (N =20)

0(0%)

0(0%)

1(5%)

7(35%)

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20-year period from 1980to 2000.18Carcinoid tumors may present as metastatic tumors with an unknown primary,although the reported prevalence of this phenomenon is variable.Two of 101patients (2%)reported in the series of Zeitels et al,24and 50/8305cases (0.6%)reported by Modlin and Sandor 14were metastatic carcinoid tumors of unknown origin.In contrast,Kirshbom et al 10found 143of over 750cases (19%)to be metastatic tumors of unknown primary.Of note,in their study,15%of these patients with an unknown primary later on had a primary site identi?ed during follow-up.WDNET from other sites,such as the pancreas,share many clinical and pathologic character-istics with carcinoid tumors.These groups of tumors are histologically similar,with tumor cells arranged in solid,trabecular,nested,or acinar architecture,and the nuclei display a characteristic ?ne,granular (‘‘salt and pepper’’)chromatin pattern.Moreover,they also share reactivity for generic markers of neuroendocrine di?erentiation such as chromogranin and synaptophysin.Thus,it is morphologically di?cult to accurately predict the site of origin when confronted with a metastatic WDNET with an occult primary.

The results of our study suggest that a panel of immunohistochemical markers using the site-speci?c transcription factors CDX-2,PDX-1,and TTF-1,and NESP-55,a recently discovered member of the chromo-granin family,may be useful in predicting site of origin of metastatic WDNET that present with an unknown primary.For example,expression of TTF-1was con?ned to pulmonary carcinoids,whereas CDX-2expression,in the absence of NESP-55or PDX-1expression,was highly speci?c and sensitive for an ileal primary.CDX-2expression was also present in a minor subset of PET but was weak and heterogeneous,in contrast to the more uniform and strong staining for CDX-2seen in ileal carcinoids.When CDX-2was negative,the expression of PDX-1or NESP-55was highly speci?c for PET,and even greater speci?city was achieved when both markers were expressed together.PDX-1reactivity was seen in gastro-duodenal and PET,a subset of rectal carcinoids,and also in more than half of appendiceal carcinoids.However,all appendiceal carcinoids were also positive for CDX-2.

Several prior studies have evaluated the expression of the transcription factors TTF-1and CDX-2in pulmonary and GI carcinoids.2,6–8,11,13,17,19

However,

FIGURE 2.Primary ileal carcinoid tumor (A)and liver metastasis (C)showing diffuse strong CDX-2positivity (B and D;?200).

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Volume 33,Number 4,April 2009Well-differentiated Neuroendocrine

the clinically more important distinction between intestinal and PET has not been addressed in detail in published literature.Oliveira et al 17found TTF-1expression in 95%of pulmonary WDNET,in 80%of metastatic pulmonary WDNET,but none of the 50intestinal carcinoids.TTF-1positivity was also reported to be con?ned to pulmonary endocrine tumors by Saqi et al 19in a more recent study but in only 53%of pulmonary WDNET.Rare examples of TTF-1positivity in GI carcinoids have also been described previously.3In our study,the expression of TTF-1was restricted to pulmonary WDNET,but was present in only about a third of cases,which is a lower percentage than those reported previously.Nonetheless,the speci?city of TTF-1expression as a feature of pulmonary WDNET is rea?rmed by the ?ndings of our study.CDX-2expression was reported in 100%of midgut carcinoids and about a third of hindgut carcinoids,in a study by Ja?ee et al.8In this study,all foregut carcinoids (pulmonary and gastric)were negative for CDX-2,and expression of CDX-2in more than 50%of tumor cells was con?ned to midgut carcinoids.However,no PET were evaluated in that study.Werling et al 23described CDX-2expression in 42%of GI carcinoids but did not further elaborate on the speci?c tumor sites.Saqi et al 19reported CDX-2positivity,of variable intensity,in GI tumors from diverse sites,including foregut,midgut,and hindgut carcinoids,as well as in 26%of PET.CDX-2expression in 29%of PET was reported in a prior study by Moskaluk et al.16However,it must be emphasized that in both studies mentioned above,strong CDX-2positivity was also described in all pancreatic ductal epithelial

cells

FIGURE 4.Pulmonary carcinoid tumors (A;hematoxylin and eosin,?200)showed weak TTF-1positivity in only about a third of cases

(B).

FIGURE 3.Duodenal carcinoid tumor (A;hematoxylin and eosin,?200)with strong nuclear staining for PDX-1(B).

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and centroacinar cells in the surrounding non-neoplastic parenchyma.In our study,CDX-2positivity was present in7/39(18%)PET,but in a weak and heterogeneous pattern,and the pancreatic ductal epithelium was uniformly negative for CDX-2.A signi?cant di?erence in CDX-2positivity between nonfunctioning PET(2/31) and metastatic PET(9/19)was described by Barbareschi et al.2They also found CDX-2positivity in39%of all neuroendocrine carcinomas evaluated,irrespective of tumor site.Interestingly,in our study,we also found CDX-2expression in a higher percentage of metastatic PET(43%),as compared with localized primary PET (10%).Whether CDX-2expression in PET is an independent marker of aggressive behavior remains to be established.The role of Islet-1in separating PET from GI and pulmonary carcinoids was evaluated recently.20 Islet-1positivity was present in69%of PET,16%of pulmonary,3%of ileal,and20%of appendiceal endocrine tumors in that study.

NESP-55is a recently described member of the chromogranin family and is thought to be a marker of the constitutive secretory pathway.It has a more restricted pattern of expression,among endocrine tumors,than that of chromogranin A,and has been shown to be con?ned to PET and pheochromocytomas in previous studies.9,21Our results con?rm that di?use,strong expression of NESP-55 is highly speci?c for a pancreatic origin and only rare examples of rectal or pulmonary endocrine tumors show focal staining with this antibody.PDX-1expression,in adults,is restricted to pancreatic endocrine cells and acinar cells.In our study,PDX-1reactivity was present in 60%of gastric,80%of duodenal,and28%of pancreatic WDNET.PDX-1expression was not site-speci?c,as over half of appendiceal carcinoids and17%of rectal carcinoids were also positive with the PDX-1antibody used in our study.However,appendiceal carcinoids could be distinguished by their invariable positivity for CDX-2. Our?ndings suggest that the main utility of PDX-1is in the distinction between WDNET of pancreatic and ileal origin,as none of the31ileal WDNETs in our study were positive for PDX-1.Given the rarity of appendiceal or rectal carcinoids presenting with distant metastasis from an occult primary,the fact that some of these tumor types express PDX-1should not pose signi?cant problems.In view of the CDX-2reactivity seen in a minor subset of PET,the addition of PDX-1and NESP-55to the immunohistochemical panel can be expected to increase the speci?city of diagnosis.

In summary,the results of our study indicate that CDX-2is highly speci?c for midgut carcinoids while NESP-55and TTF-1show high speci?city for pancreatic and pulmonary WDNETs,respectively.PDX-1staining is absent in ileal and pulmonary carcinoids and positive staining may support a pancreatic or foregut WDNET.A panel of these four immunohistochemical markers may therefore help predict the primary site of metastatic WDNET and should be validated in future studies, including additional cases of metastatic tumors.

ACKNOWLEDGMENT

The authors are extremely grateful to Dr Reiner Fischer-Colbrie,Professor of Pharmacology,Innsbruck Medical University,Innsbruck,Austria,for a generous gift of the NESP-55antibody used in this study.

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TABLE4.Utility of a Panel of Immunohistochemical Markers Including CDX-2,PDX-1,NESP-55,and TTF-1in Predicting Site of Origin of Well-differentiated Neuroendocrine Tumors

Site N Pattern of Immunoreactivity Sensitivity

(%)

Speci?city

(%)

Pancreas39PDX-1or NESP-55positive

CDX-2and TTF-1negative

4682 Pancreas39PDX-1and NESP-55positive

CDX-2and TTF-1negative

4197

Ileum/ appendix 49CDX-2positive

PDX-1,NESP-55,and

TTF-1negative

9791

Lung20TTF-1positive

PDX-1,NESP-55,and CDX-2

negative 35100

TABLE3.Site-specific Immunohistochemical Expression of CDX-2,PDX-1,NESP-55,and TTF-1in Well-differentiated Neuroendocrine Tumors

Antibody Primary Tumor

Site N

Sensitivity

(%)

Speci?city

(%)

CDX-2Ileum/appendix429891

PDX-1Stomach/

duodenum/

pancreas

493689

NESP-55Pancreas393297

TTF-1Lung2035100

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