Somatostatin receptor type 2Aimmunohistochemistry in neuroendocrinetumors: a proposal of scoring systemcorrelated with somatostatin receptorscintigraphy

Marco Volante1, Maria Pia Brizzi1, Antongiulio Faggiano2, Stefano La Rosa3, Ida Rapa1,Anna Ferrero1, Gelsomina Mansueto4, Luisella Righi1, Silvana Garancini5, Carlo Capella3,Gaetano De Rosa4, Luigi Dogliotti1, Annamaria Colao2 and Mauro Papotti1

1Department of Clinical and Biological Sciences, San Luigi Hospital, University of Turin, Orbassano, Turin,Italy; 2Department of Molecular and Clinical Endocrinology and Oncology, ‘Federico II’ University, Naples,Italy; 3Section of Anatomic Pathology, Department of Human Morphology, University of Insubria andOspedale di Circolo, Varese, Italy; 4Department of General Pathology, Medicine, Human Pathology andClinical Pathology, University of Naples ‘Federico II’, Naples, Italy and 5Department of Nuclear Medicine,Ospedale di Circolo, Varese, Italy

Typing somatostatin receptor expression in neuroendocrine tumors is of relevance to target somatostatinanalogue-based diagnostic approach and treatment. The expanding use of immunohistochemistry to detectsomatostatin receptors is to date not paralleled by an accurate methodological setting and standardizedinterpretation of the results. A multicentric study was designed to compare somatostatin receptorimmunohistochemical expression with in vivo scintigraphic data and verify its usefulness in the clinicalmanagement of neuroendocrine tumors. After methodological setting by testing different somatostatin receptorantibodies, 107 cases of neuroendocrine tumors with available somatostatin receptor scintigraphy data andpathological material were retrospectively analyzed for somatostatin receptor types 2A, 3 and 5 immunohis-tochemical expression, and compared with scintigraphic images and, whenever available, with the clinicalresponse to somatostatin analogue treatment. Restricting ‘positive cases’ to the presence of a membranepattern of staining, an overall somatostatin receptor type 2A immunohistochemistry/somatostatin receptorscintigraphy agreement of 77% (v2 test Po0.0001) was reached. Lower concordance ratios were detected inpreoperative and metastatic tumor samples, possibly as a consequence of somatostatin receptor expressionheterogeneity. Pure somatostatin receptor type 2A cytoplasmic staining showed poor correlation withsomatostatin receptor scintigraphy (54% concordance rate). The immunohistochemical detection ofsomatostatin receptor types 3 and 5, which showed almost exclusively a cytoplasmic pattern, did not improvethe concordance with scintigraphic data. In a pilot series, somatostatin receptor type 2A immunohistochemistrycorrelated with clinical response in 75% of cases. In conclusion, we propose a scoring system for somatostatinreceptor type 2A immunohistochemistry in neuroendocrine tumors correlated with in vivo data, based onthe evidence that only membrane (rather that cytoplasmic) staining should be considered for a reliable,standardized and clinically relevant report.Modern Pathology (2007) 20, 1172–1182; doi:10.1038/modpathol.3800954; published online 14 September 2007

Keywords: somatostatin receptor; immunohistochemistry; neuroendocrine tumors; somatostatin receptorscintigraphy; scoring system

Neuroendocrine tumors belong to a spectrum oflesions with variable degree of differentiation andhave various locations and various disease stages:carcinoids (benign and malignant or typical andatypical) lie in the well differentiated extreme of thespectrum, while small and large cell neuroendo-crine carcinomas represent the poorly differentiatedvariants.1,2

Received 17 March 2007; revised 07 August 2007; accepted 09August 2007; published online 14 September 2007

Correspondence: Dr M Volante, MD, Department of Clinical andBiological Sciences, San Luigi Hospital, University of TurinMedical School, Regione Gonzole 10, Orbassano 10043, Torino,Italy.E-mail: marco.volante@unito.it

Modern Pathology (2007) 20, 1172–1182& 2007 USCAP, Inc All rights reserved 0893-3952/07 $30.00

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There are several therapeutic options for neuroen-docrine tumors. Apart from surgery, chemotherapyand biotherapy are generally the first line treat-ments. The latter includes in both functioning andnon-functioning neuroendocrine tumors somatosta-tin analogue administration, both in medical treat-ment and as radio-labeled agents, and interferon.3–5

Somatostatin is an acidic polypeptide that iswidely distributed throughout the central nervoussystem and different peripheral tissues and organs.Several biological functions related to somatostatinhave been described, and include potent inhibitionof basal and stimulated secretion from a widevariety of endocrine and exocrine cells, neuromo-dulatory actions in the central nervous system6–9 aswell as regulatory properties on cell proliferationand differentiation.10 The physiological actions ofsomatostatin are initiated by its interaction with afamily of specific membrane-bound receptors, thesomatostatin receptors family, which encompass todate five different subtypes, named somatostatinreceptors types 1–5, displaying a remarkable degreeof structural conservation across species (40–60%structural homologies), but mediating differentbiological actions of somatostatin, via the activationof different intracellular signaling pathways.11,12

The rationale for somatostatin analogue (eg octreo-tide) treatment is the presence of somatostatinreceptors on the surface of tumor cells. These bindoctreotide with different degrees of affinity, beingsomatostatin receptors types 2, 3 and 5 those mostlyinvolved in octreotide binding.

In the clinical practice, the presence of functionalsomatostatin receptors is documented by somatos-tatin receptor scintigraphy by 111In-pentetreotide: ifpositive, a binding of the radioligand with one ormore of the somatostatin receptors is demonstrated.Unfortunately, this technique does not identify thereceptor type and—most importantly—does notdetermine the cell population containing the bind-ing sites (tumor cells vs other intratumoral celltypes). Various methods to detect somatostatinreceptors in tumor specimens were reported, in-cluding autoradiography, RT-PCR, in situ hybridiza-tion, immunoblotting and immunohistochemistry.Several studies have documented that generallythese methods correlate each other in somatostatinreceptors positive cases, with only minor discre-pancies.13–22

The currently available somatostatin analoguesbind preferentially somatostatin receptors type 2(which is the most widely expressed subtype inneuroendocrine tumors) and to a lower extent types3 and 5, and somatostatin receptors profiling inindividual patients may be of relevance to bettertailor the somatostatin analogue-based treatment.This would hopefully allow to increase the responserate of each patient and reduce costs of biotherapy,which are generally high.

Immunohistochemistry is a cheap, reproducible,and easily accessible procedure that can be per-

formed in most pathology laboratories on fresh andalso archival tissue samples. Commercially availableantibodies specific for the different receptor sub-types are currently available, but they need to bevalidated in the clinical practice. No data exist infact on the quantification and interpretation ofsomatostatin receptor immunohistochemical find-ings, although scoring systems have been developedand applied for many years in the analysis of severalother receptors (eg estrogen receptor, c-erb-b2, etc).

Aim of the present study is therefore to define thesomatostatin receptors immunohistochemical pro-file in a large series of neuroendocrine tumors ofwhich surgical and/or biopsy material were avail-able, and compare the results with the in vivo dataon 111In-pentetreotide scintigraphy and response tosomatostatin analogue treatment.

Materials and methods

Case Selection

A series of 107 neuroendocrine tumors (from years1998 to 2006) treated in three different highlyspecialized Italian centers of the Universities ofTurin, Naples and Varese was selected according tothe following criteria: (i) revised histopathologicaldiagnosis; (ii) tissue material available for patholo-gical review and immunohistochemistry; (iii) 111In-pentetreotide scintigraphy data available.

For most cases, medical treatment administered,response to treatment (as progressive disease, partialremission or stable disease) and follow-up data werealso accessible.

Somatostatin receptor scintigraphy was per-formed preoperatively in all but seven patients,who had post-surgical somatostatin receptorscintigraphy in the presence of residual disease.111Indium-DTPA0-octreotide (135 MBq) was admi-nistered immediately after checking the specificradiochemical purity by chromatography, whichwas always higher than 95%. The scintigraphicprocedure included 4- and 24-h planar anterior andposterior images (matrix size 128� 128) of the chest,upper and lower abdomen and anterior views of thehead and proximal lower legs. At least 500 Kilo-counts were recorded over the chest and abdomenand 50–200 Kilocounts were obtained for the headand neck and lower leg views. SPECF of theabdomen was performed 24 h after injection. Sixty-four 40-s views (matrix size: 64� 64) were acquiredthrough a 3601 arc. Planar and SPECF images wereacquired using a large field of view g cameraequipped with a medium-energy, general-purposecollimator. Somatostatin receptor scintigraphyimages were scored in four groups according toKwekkeboom et al.23

The tissue material corresponded to 41 preopera-tive samples (fine-needle aspiration or core biop-sies) and 66 surgical samples. All surgical sampleswere fixed in buffered formalin; preoperative sam-

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ples were both fixed in formalin or in alcohol. Allcases have been reviewed and diagnosed accordingto years 2000 and 2004 WHO classifications ofEndocrine Tumors.1,2 They included 70 cases ofwell-differentiated neuroendocrine tumors (benigncarcinoids) or carcinomas (malignant/atypical carci-noids) (63 cases from lung, pancreas and gastro-intestinal tract, and 7 cases of liver or lymph nodemetastases in the absence of evident primarydisease), 18 cases of poorly differentiated neuroen-docrine carcinomas (mainly from lung), 9 cases ofmedullary thyroid carcinomas and 7 additionalcases of neuroendocrine tumors from skin, para-ganglia, thymus and pituitary, as well as 3 cases ofadenocarcinomas with neuroendocrine differentia-tion of the breast and prostate. In 31 cases, tissuematerial was obtained from metastatic deposits, andin 3 of these cases primary and metastatic lesionsfrom the same patient could be compared. The studywas approved by the Local Ethic Committee.

Immunohistochemistry

Serial 5-mm thick paraffin sections were collectedonto charged slides and processed by immuno-histochemistry in a single Institution (Orbassano,Turin, Italy). In a pilot series of 20 neuroendocrinetumors, as well as in control sections of normalpancreatic tissue, alternative polyclonal somatosta-tin receptor type 2A antibodies (BioTrend, Cologne,Germany, code SS-800; Santa Cruz Biotechnology,Santa Cruz, CA, USA, code sc-25676; LifeSpanBiosciences, Seattle WA, USA, code LS-A998) weretested. Although all three somatostatin receptor type2A antibodies were comparable in the majorityof normal and neoplastic specimens in terms ofstaining intensity, in our laboratory protocols Bio-Trend antibody appeared to be more sensitive ascompared to the others, in terms of both presence ofpositive internal reference controls (ie blood vessels)and demonstration of a membranous staining pat-tern in normal and neoplastic cell populations. Itwas therefore selected to be applied to the completeseries of cases and for comparison with in vivo data.Pre-absorption experiments, performed in controlsections by pre-incubation of the primary antibodywith a 10- to 50-fold excess of blocking peptide(BioTrend, code SS-801), could confirm the specifi-city of such antibody. Negative control experimentsby somatostatin receptor type 2A pre-absorptionwere therefore performed in each immunohisto-chemical run. In addition, polyclonal antibodies tosomatostatin receptor types 3 and 5 from threedifferent sources (BioTrend, codes SS-830 and SS-838, LifeSpan, codes LS-A2622 and LS-A2639, andAbcam, Cambridge, UK, codes ab28680 andab28618, for subtypes 3 and 5, respectively) werealso tested in the pilot series of 20 cases ofneuroendocrine tumors. According to the prelimin-ary immunohistochemical findings, Abcam antibo-

dies demonstrated the most intense and reliableresults and were therefore employed on the wholecase series. Moreover, pre-absorption experimentsfor BioTrend somatostatin receptor types 3 and 5antibodies, using the same conditions as for soma-tostatin receptor type 2A (blocking peptides fromBioTrend, codes SS-831 and SS-839-A, respectively)failed to confirm the specificity of these latterprimary antibodies, even applying different batches.

For somatostatin receptor immunohistochemistrythe following conditions were applied: antigenretrieval was performed in pH 6.0 citrate buffer ina microwave oven (three cycles 5 min each at 750 W)and the primary antibodies were incubated for 4 h at371C. Primary antibodies dilutions were 1/3000,1/1000 and 1/500 for somatostatin receptor types2A, 3 and 5. Immunoreactions were revealed by abiotin-free dextran-chain detection system (Envi-sion, DakoCytomation, Glostrup, Denmark), anddeveloped using diaminobenzidine as the chromo-gen. The specificity was also validated in parallelnegative control sections by omitting the primaryantibodies for each immunohistochemical run.

All somatostatin receptor immunohistochemicalfindings were analyzed independently by twoobservers (MV and IR) in one center (Orbassano,Turin, Italy).

In 14 selected cases, somatostatin receptor type2A immunohistochemistry was repeated in a differ-ent laboratory (Varese) following the same workingconditions, and the results checked to test inter-laboratory agreement.

Somatostatin Receptor ImmunohistochemistryScoring System

For somatostatin receptor type 2A a semiquantita-tive scoring system was designed, taking intoconsideration both the subcellular localization andthe extent of the staining, as follows: score 0:absence of immunoreactivity; score 1: pure cyto-plasmic immunoreactivity, either focal or diffuse;score 2: membranous reactivity in less than 50% oftumor cells, irrespective of the presence of cyto-plasmic staining; score 3: circumferential membra-nous reactivity in more than 50% of tumor cells,irrespective of the presence of cytoplasmic staining.

For somatostatin receptor types 3 and 5, acytoplasmic pattern of staining only was observed,and cases were scored as positive in the presence ofat least 10% of positive tumor cells.

Response to Treatment

Tumor responses was classified according to theRECIST criteria.24 Complete response was defined asthe absence of radiologically documented lesions.A partial response was defined as at least a 30%decrease in the sum of the longitudinal diameter oftarget lesions. Progressive disease was defined as at

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least a 20% increase in the sum of the longitudinaldiameter of target lesions or the appearance of one ormore new lesions. Stable disease was defined asneither a sufficient shrinkage to qualify for partialresponse, nor sufficient increase to qualify forprogression disease.

Statistical Analysis

Immunohistochemical results were compared tosomatostatin receptor scintigraphy status and avail-able clinico-pathological parameters by w2 test using1 degree of freedom (d.f.¼ 1), and a level ofstatistical significance of P¼ 0.05. For statisticalcomparison, somatostatin receptor type 2A immu-nohistochemical scores 0 and 1 were considerednegative, as opposed to scores 2 and 3 (see alsobelow).

Results

Somatostatin Receptors Distribution inNeuroendocrine Tumors

ImmunohistochemistrySomatostatin receptor types 2A, 3 and 5 antibodiesshowed the specific signal in internal referencecontrols (ie blood vessels, pancreatic islets) or incontrol sections of both formalin- and alcohol-fixedtissue samples. In the case of somatostatin receptortype 2A, pre-absorption experiments abolished the

immunoreactivity (both membranous and cytoplas-mic patterns) and confirmed the specificity of thereaction (Figure 1). Grouping somatostatin receptortype 2A immunohistochemical scores 2 and 3together as positive (see also below), somatostatinreceptor type 2A was detected in 79% of well-differentiated neuroendocrine tumors/carcinomasand in 44% of poorly differentiated neuroendocrinecarcinomas (Table 1). In somatostatin receptor type2A score 1 cases, the cytoplasmic reactivity wasgenerally weak to moderate; comparing scores 2 and3, apart from the percentage of positive tumor cellswhich determined the inclusion in one or the othergroup, a slightly weaker and often incompletemembrane staining was observed in score 2 cases,whereas a strong and circumferential membranouspattern was characteristic of score 3 cases (Figures 2and 3). Inter-observer cross validation between thetwo independent investigators (MV and IR) was upto 98%; for discrepant cases agreement was reachedby reviewing the slides at a multihead microscope.Inter-laboratory cross validation (between the slidesstained in Orbassano and in Varese) showed 100%scoring agreement in all 14 cases tested.

Somatostatin receptor types 3 and 5 showed acytoplasmic pattern only, with very occasionalmembrane reinforcement. They were positive in44 and 71% of well-differentiated neuroendocrinetumors/carcinomas and in 17 and 28% of poorlydifferentiated neuroendocrine carcinomas, respec-tively for types 3 and 5 (Table 1).

Somatostatin receptor scintigraphyIn vivo somatostatin receptor analysis by 111In-pentetreotide detected positive binding (includingscores 2–4 according to Kwekkeboom et al23 in 78/107 cases (73%), with a decreasing percentage ofpositive cases comparing well-differentiated lesions(79%), poorly differentiated neuroendocrine carci-nomas (44%) and the group miscellaneous neuroen-docrine tumors (21%).

Correlation of Immunohistochemistry with In VivoData

When checking the correlation of somatostatinreceptor type 2A antibody reactivity with in vivodata among the different immunohistochemicalgroups, scores 2 and 3 showed a high concordancewith somatostatin receptor scintigraphy, being cor-related to scintigraphic positivity in 87 and 94% of

Figure 1 Intense membranous pattern of staining with somatos-tatin receptor type 2A antibody (BioTrend, code SS-800) in apancreatic islet (a), completely abolished by pre-incubation of theprimary antibody with a 50-fold excess of blocking peptide(BioTrend, code SS-801) (b).

Table 1 Somatostatin receptors expression in 107 neuroendocrine tumors

Somatostatin receptortype 2A (scores 2 and 3)

Somatostatinreceptor type 3

Somatostatinreceptor type 5

WD NET/NEC (70 cases) 79% 44% 71%PD NEC (18 cases) 44% 17% 28%Others (19 cases) 21% 53% 74%

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cases, respectively, and were grouped together forfurther statistical comparison. By contrast, immu-nohistochemical scores 0 and 1 showed a poorcorrelation with somatostatin receptor scintigraphyresults, and therefore were grouped together asnegative for further statistical analysis (Figure 3).

An overall somatostatin receptor type 2A immuno-histochemistry/somatostatin receptor scintigra-phy agreement of 77% (82/107 cases, w2 Po0.0001)was reached (Table 2). Among the 82 concordantcases, 61 were both methods positive (Figure 4a–c)and 21 were both methods negative. Consistent

Figure 2 Representative illustration of somatostatin receptor type 2A immunohistochemical scores in surgical (left-sided pictures;(a) lymph node metastasis of medullary thyroid carcinoma; (c) atypical carcinoid of the lung, (e) well-differentiated neuroendocrinecarcinoma of the pancreas, not functioning) and preoperative cytological (right-sided pictures; (b) liver metastasis of well-differentiatedneuroendocrine carcinoma of the pancreas; (d) lymph node metastasis of well-differentiated neuroendocrine carcinoma, primaryunknown; (f) liver metastasis of well-differentiated neuroendocrine carcinoma of the small ileum) samples. The lesions representedincluded: (a, b) Score 1 (a and b) included pure cytoplasmic reactivity, either moderate/strong (a) or weak (b). Score 2 (c and d) wasconsidered in the presence of a membranous pattern of staining in less than 50% of tumors cells, either in scattered cells with completemembrane outlining (c) or with partial membrane staining in most tumor cells (d). Score 3 was clearly recognizable by a membranous,usually intense, staining in more than 50% of tumors cells (e and f).

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concordance rates were observed in the group ofwell-differentiated (79%, w2 P¼ 0.05) and poorlydifferentiated (94%, w2 Po0.0001) neoplasms. Con-versely, considering individual tumor locations, alow concordance rate was observed in the case ofmedullary thyroid carcinomas and occult primaryneuroendocrine carcinomas (67 and 54% immuno-histochemistry/somatostatin receptor scintigraphyagreement, respectively). The latter group sho-wed mainly an immunohistochemistry negative/somatostatin receptor scintigraphy positive profile(Figure 4d–f).

A rather poor correlation was observed in thegroup of preoperative biopsy samples (73% immuno-histochemistry/somatostatin receptor scintigraphyagreement, w2 P¼ 0.09) and in patients from whommetastatic tumor samples were available (65%immunohistochemistry/somatostatin receptor scin-tigraphy agreement, w2 P¼ 0.3).

When correlating scintigraphic data with all threesomatostatin receptor antibodies, the overall agree-

ment in the presence of at least one positivesomatostatin receptor type did not change (82/107cases, 77%), whereas an increase of immunohisto-chemistry positive/scintigraphy negative cases wasobserved (with a loss of ‘specificity’ if considering111In-pentetreotide scintigraphy the referencemethod; Table 3). In 28 patients, all bearing well-differentiated neuroendocrine carcinomas from pan-creas and gastrointestinal tract or unknown origin,homogeneous somatostatin analogue treatment wasadministered until progression, consisting in intra-muscular injection of the long-acting release octreo-tide (octreotide L-A-R) at a dose of 20 mg every 28days. Immunohistochemical somatostatin receptortype 2A status correlated to response to treatment in75% of cases (21/28); among these 21 concordantcases, 16 patients had a disease stabilization and2 cases had a partial response to treatment inthe presence of somatostatin receptor type 2Aimmunoreactivity, whereas the 3 remaining cases,negative to somatostatin receptor type 2A immuno-

Score 0 Score 1

SUBCELLULAR PATTERN

Purecytoplasmic

Membranoususually

incomplete

Membranouscircumferential

(Negative)

(Absent) 1-100%

CONCORDANCE WITH OCTREOSCAN DATA

EXTENSION OF POSITIVE TUMOR CELL POPULATION

<50% >50%

50% 54% 87% 94%

Score 2 Score 3

Figure 3 Schematic illustration of somatostatin receptor type 2A immunohistochemical scoring system and its concordance rate with111In-pentetreotide in vivo imaging.

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histochemistry, had disease progression. By con-trast, all seven discordant cases had disease pro-gression in spite of somatostatin receptor type 2protein immunohistochemical expression.

Discussion

The wide expression of somatostatin receptors inneuroendocrine tumors has been largely investi-gated and led nowadays to the development ofclinically relevant diagnostic and therapeutic stra-tegies.25 However, which methodology could betterdetermine the somatostatin receptor status of a giventumor and could predict its possible visualizationby somatostatin receptor scintigraphy and responseto somatostatin-analogue therapy is far from beingsettled in the clinical practice. At present for theclinicians, somatostatin receptor scintigraphy re-mains the golden standard method to detect soma-tostatin receptors. In fact, octreotide scintigraphyprovides information on the whole tumor massincluding metastases and its signal intensity is usedfor radiotherapeutic decisions.23 Among alternativemethods, immunohistochemistry seems to be areliable tool to detect the somatostatin receptorprofile in neuroendocrine tumors, due to thefollowing advantages: detection of the receptorprotein (instead of RNA, for example in PCR-basedmethods), possibility of detecting the cellular typeexpressing the receptor (neoplastic cells vs bloodvessels or reactive lymphocytes, etc), availability ofsubtype specific antibodies, applicability in archivalmaterial, low cost/benefit ratio which renders this

method applicable in most laboratories.13,15–17,19,21

On the contrary, major disadvantages are related tothe lack of standardization of the method (from bothtechnical and interpretation viewpoints) which is agreat limitation in diagnostic applications, the fail-ure of demonstrating ‘functional’ receptors (asopposed to autoradiography), and undeterminedsensitivity of the technique, since limited evidencehas been reported on the correlation betweenimmunohistochemistry and other in vivo techni-ques. In a recent paper, Korner et al15 tested differentsomatostatin receptor type 2A antibodies in differ-ent human tumors, and correlated the immunohisto-chemical pattern with previous autoradiographicdata on 37 cases, demonstrating a good correlationbetween the two methods applying the same anti-body selected in our study. Previous reports testingsomatostatin receptor immunohistochemistry inlung18 and gastrointestinal16 neuroendocrine tumorsand in pheochromocytomas17 in correlation tosomatostatin receptor scintigraphy are limited bythe relative low number of cases compared and by ageneral lack of standardization of the immunohisto-chemical interpretation (ie membranous vs cyto-plasmic pattern). Therefore, we designed the presentstudy on a large multicentric series of neuroendo-crine tumors to validate the reproducibility of theimmunohistochemical method and to compare theresults with somatostatin receptor scintigraphyimaging.

As a first aim, a practical immunohistochemicalscoring system for the most common somatostatinreceptor subtype—namely type 2A—based on thepercentage of positive tumor cells and the pattern of

Table 2 Concordance between immunohistochemistry and 111In-pentetreotide scintigraphy in detecting the presence of somatostatinreceptor type 2A in neuroendocrine tumors

Total (107) Location Concordant group Discordant group

No. (%) IHC/srs+ IHC/srs� No. (%) IHC+/srs� IHC�/srs+82 (77) 61 21 25 (23) 6 19

WD Lung (17) 13 (76) 11 2 4 (24) 1 3NET/NEC Pancreas (28) 24 (86) 23 1 4 (14) 2 2(70) GI tracta (18) 16 (89) 15 1 2 (11) 2 —

Primary unknown (7) 2 (29) 1 1 5 (71) — 5PD NEC Lung (13) 12 (92) 5 7 1 (8) 1 —(18) GI tract (1) 1 — 1 — — —

Primary unknown (4) 4 (100) 2 2 — — —Others Thyroid MTC (9) 6 (67) 1 5 3 (33) — 3(19) Merkel cell ca. (3) 2 2 — 1 — 1

Paraganglioma/Pheo (2) 1 1 — 1 — 1Thymic carcinoid (1) — — — 1 — 1ADCA with NE diff (3) 1 — 1 2 — 2Pituitary (1) — — — 1 — 1

Preoperative samples (41) 30 (73) 25 5 11 (27) 3 8Metastatic samples (31) 20 (65) 17 3 11 (35) 1 10

ADCA, adenocarcinoma; GI, gastrointestinal; IHC, immunohistochemistry; MTC, medullary thyroid carcinoma; NEC, neuroendocrine carcinoma;NET, neuroendocrine tumor; PD, poorly differentiated; srs, somatostatin receptor scintigraphy; WD, well differentiated.Bold values represent the total.Italic values represent the percentage.aIncluding one case of primary WD NEC of the liver and one of the gallbladder.

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staining was proposed and assessed. The correlationwith 111In-pentetreotide scintigraphy data indicatethat the higher concordance rates were achievedonly when a membranous pattern of staining wasobserved (corresponding to scores 2 and 3), whereas

pure cytoplasmic staining (score 1) presented arather poor and random concordance rate and wastherefore considered as negative for further analysis.Although ‘functional’ mechanisms for somatostatinreceptor type 2A cytoplasmic positivity (ie receptor

Figure 4 Fine-needle aspiration biopsy of a liver metastasis from a well-differentiated pancreatic neuroendocrine carcinoma (a) stronglyexpressing somatostatin receptor type 2A by immunohistochemistry (b, immunohistochemical score 3) and clearly visible by 111In-pentetreotide scintigraphy (c). By contrast, a case of fine-needle aspiration biopsy of a para-caval lymph node metastasis from a well-differentiated neuroendocrine carcinoma of primary unknown origin (d) showing discrepant results between negative immuno-histochemistry (e, immunohistochemical score 1) and positive scintigraphy (f).

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internalization) have been discussed in the litera-ture,26 it might be speculated that in the diagnosticpractice careful search for membranous pattern(associated or not with a faint cytoplasmic pattern)should be pursued to define a positive staining. Thesame consideration should be deserved for soma-tostatin receptor types 3 and 5 antibodies, whichshowed almost exclusively a cytoplasmic pattern ofstaining, and which did not improve significantlythe capability of somatostatin receptor type 2A aloneto correlate with octreotide scintigraphy.

Immunohistochemical somatostatin receptor type2A scores 2 and 3 were higher, as expected, inwell-differentiated neuroendocrine tumor/carcino-ma group as compared to poorly differentiatedcarcinomas (Table 1), without significant differencesconsidering the organs of origin, except for the casesof metastatic samples of unknown primary originwhich showed a lower rate of somatostatin receptortype 2A expression. When comparing the completesomatostatin receptor type 2A immunohistochem-ical data with somatostatin receptor scintigraphy, anoverall high agreement (77%, w2 test Po0.0001) wasobserved, irrespective of the degree of differentia-tion. However, when going through the major causesof discrepancy, some considerations might bedrawn. First of all, in most instances the source ofdiscrepancy was related to a negative somatostatinreceptor type 2A immunohistochemical result in thepresence of positive somatostatin receptor scintigra-phy. In this respect it is difficult, if not useless,to determine which method is more sensitive orspecific. It is well known, in fact, that falsesomatostatin receptor scintigraphy positivity maybe related to necrotic areas or inflammation whichmay be frequent within an otherwise somatostatinreceptor negative tumor,18 and therefore immuno-histochemistry represents, rather than an alterna-tive, a useful adjunct to somatostatin receptorscintigraphy. Moreover, the possible role of othersomatostatin receptor subtypes (namely types 3 and5), which possess high affinity for the currentlyavailable somatostatin analogues, was investigated,although their analysis did not improve the overallconcordance between scintigraphy and immunohis-tochemistry, and their application in the clinicalpractice still needs the availability of satisfactorycommercial reagents, in our opinion.

By contrast, it should be speculated that immu-nohistochemistry may present a lower sensitivitythan 111In-pentetreotide scintigraphy and the tissuematerial investigated may not represent the soma-tostatin receptor type 2A expression pattern of thewhole tumor. The frequent heterogeneity of soma-tostatin receptor distribution in tumor tissues maycause discrepant (either positive or negative) im-munohistochemical results as compared to soma-tostatin receptor scintigraphy, and this is moreevident in our study when analyzing separatelypreoperative (ie small tissue fragments) and meta-static samples, which showed lower concordancerates (73 and 65%, w2 P¼ 0.09 and P¼ 0.3, respec-tively). Technical artifacts related to tissue fixationand preservation are instead unlikely to explaindiscrepancies between the two methods considered,since comparable results were observed in bothformalin and alcohol-fixed tissue specimens, andirrespective of the length of tissue storage.

It is important to note that the correlation withsomatostatin receptor scintigraphy represents onlyone side of the matter, and the predictive value ofsomatostatin receptor immunohistochemistry indetermining the clinical response to somatostatinanalogues treatment would have comparable if notgreater clinical impact. Such a correlation waslimited in our study because of heterogeneousmedical treatments of the patients and differentintrinsic biological behavior of the diseases (ie fromwell- to poorly differentiated forms). However, in agroup of 28 cases with comparable disease andtherapeutic schedules, positive somatostatin recep-tor type 2A immunohistochemistry correlated withsomatostatin analogues response in up to 75% ofcases. Large clinical trials should be designed tovalidate the role of somatostatin receptor immuno-histochemical profile in the prediction of clinicalresponse.

In conclusion, our data indicate that (i) immuno-histochemistry represents a reliable and usefulmethod to characterize somatostatin receptor ex-pression in neuroendocrine tumors, with specialreference to type 2A, provided that accuratemethodological conditions and standardized inter-pretation of the immunohistochemical results(membranous vs cytoplasmic pattern) are obtained;(ii) a standardized scoring system for immunohis-

Table 3 Concordance between immunohistochemistry and 111In-pentetreotide scintigraphy, according to somatostatin receptor typeconsidered

Somatostatin receptortype 2A

Somatostatin receptortype 2A and/or 3

Somatostatin receptortype 2A and/or 5

At least one somatostatinreceptor type

Overall agreement 77% 80% 77% 77%Sensitivitya 80% 91% 89% 93%Specificitya 67% 60% 41% 35%

aSensitivity and specificity were calculated considering arbitrarily 111In-pentetreotide scintigraphy the reference method.

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tochemistry is proposed, with good clinical corre-lates; which support its role as a useful adjunct tosomatostatin receptor scintigraphy in the clinicalmanagement of these patients, providing additionalinformation on the tissue distribution of the recep-tors, heterogeneity among primary and metastaticlesions, and picking up false-positive scintigraphicimages; (iii) somatostatin receptor type 2A immuno-histochemistry may predict clinical response tosomatostatin analogue therapy, although furthervalidation on large series is needed.

Acknowledgement

The present paper has been supported by grantsfrom the Italian Ministry of University (ex 60%to MV and MP). Antibodies against somatostatinreceptors have been provided by an unrestrictedgrant to MV from Novartis Italia.

Disclosure/conflict of interest

The authors have no conflict of interest to declare.

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