Clinical Endocrinology (2015) 83, 420–428

doi: 10.1111/cen.12775

ORIGINAL ARTICLE

Somatostatin and dopamine receptor expression in neuroendocrine neoplasms: correlation of immunohistochemical findings with somatostatin receptor scintigraphy visual scores Evanthia Diakatou*, Krystallenia I. Alexandraki†, Apostolos V. Tsolakis‡, George Kontogeorgos*, Eleftherios Chatzellis†, Anastasia Leonti§ and Gregory A. Kaltsas† *Department of Pathology, “G. Gennimatas” Athens General Hospital, †Department of Pathophysiology, University of Athens, Athens, Greece, ‡Department of Medical Sciences, Section of Endocrine Oncology, Uppsala University, Uppsala, Sweden, and §Department of Nuclear Medicine, Alexandra Hospital, Athens, Greece

Summary Context The expression of somatostatin (sstr1-5) and dopamine (DR) receptors in neuroendocrine neoplasms (NENs) facilitates diagnosis by tumour visualization with somatostatin receptor scintigraphy (SRS) and directs towards specific treatment with peptide receptor radionuclide therapy (PRRT) with radiolabelled somatostatin analogues. Objective To investigate the co-expression of sstrs, D2R in relation to pre-operative SRSs in NENs. Design Prospective two-centre study. Patients and measurements We analysed pre-operative SRS of 60 patients [44 with gastrointestinal (GI) NENs and 16 with lung NENs] and compared SRS results with immunohistochemical (IHC) reactivity for sstr2, sstr3, sstr5 in sample tissues from primary (n = 54) and metastatic (n = 27) lesions and IHC reactivity for D2R in 23 samples from primary GI-NENs lesions. Results Sstr2 was the commonest sstr expressed (654%) and was co-expressed with sstr3 and sstr5 in 321% and 247% of the specimens, respectively. In 67 of 81 specimens (827%), there was concordance of sstr2 immunohistochemistry with SRS findings (P < 0001). D2R was expressed in only 8 of 23 (348%) GI-NENs while was co-expressed with sstr2 in all cases. SRS grade, as per Krenning scale, was higher in metastatic foci, largesize (>2 cm) tumours and GI-NENs, whereas sstr2 intensity was greater in GI compared to lung NENs. SRS grade showed higher correlation with sstr2 (r = 06, P < 0001) and D2R (r = 05, P < 0001) IHC intensity scores than tumour size (r = 04, P < 0001) and sstr3 (r = 04, P < 0001) intensity score.

Correspondence: Krystallenia Alexandraki, Endocrine Unit, Department of Pathophysiology, National University of Athens, Laiko University Hospital, Mikras Asias 75, 115 27 Athens, Greece. Tel.: 0030 2107462657; Fax: 0030 2107462664; E-mail: [email protected] E.D. and K.I.A., contributed equally to this work.

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Conclusions Sstr2 IHC expression and SRS are useful tools for the diagnosis and management of NENs because they display a high concordance. IHC expression of DR2 seems to be of potential clinical significance in GI-NENs tumours. (Received 10 January 2015; returned for revision 5 February 2015; accepted 18 March 2015)

Introduction Somatostatin (sst) is a multifunctional oligopeptide capable of inhibiting cell secretion and proliferation acting through five G protein-coupled membrane somatostatin receptors (sstr1-5) encoded by five different genes.1,2 Somatostatin receptors have been detected and mapped in various neuroendocrine neoplasms (NENs), using reverse-transcriptase polymerase chain reaction (RT-PCR) m-RNA analysis, immunohistochemistry, autoradiography and in situ hybridization (ISH).2–4 Due to the inhibitory role of sst, long-acting synthetic sst analogues have been developed and shown to be effective in controlling both tumour growth and symptoms secondary to the secretion of bioactive substances from NENs.3–7 In addition, the expression of sstr permits visualization of NENs, known as sst scintigraphy (SRS), through labelling these receptors using radiolabelled sst analogues [111In-diethylene triamine pentaacetic acid (DTPA)-octreoscan]. SRS exerts high affinity for sstr types 2, 3 and 5 and provides information regarding the presence of at least one of these receptors in the tissue studied.8,9 The expression of sstr is utilized for the identification, staging, follow-up and therapy of NENs, using sst analogues and/or peptide receptor radionuclide therapy (PRRT) with radiolabelled sst analogues (SA).8,9 In addition, dopamine is an important neurotransmitter of the central nervous system (CNS) 10,11 that exerts its functions, after binding to five membranous dopamine receptors (DRs) D1R–D5R. DRs are G-coupled membrane receptors, similar to sstrs,10,11 and are divided further into two major groups D1R-like (D1R and D5R) and D2R-like (D2R, D3R and D4R). © 2015 John Wiley & Sons Ltd

Somatostatin and dopamine receptors in NENs 421 D2R has recently been shown to also be expressed in NENs using either immunohistochemical (IHC) or molecular techniques.10–12 Interestingly, sstrs and DRs crosstalk at the membrane level may trigger alternative intracellular pathways and this could modify their function.13 Sstrs can heterodimerize with other families of G-coupled membrane receptors, as DRs, producing heterodimers with properties that are different to those of the distinct receptors.13–17 Indeed, sstrs and DRs are co-localized in neuronal subgroups, where sst is involved in modulating dopamine-mediated control of motor activity and D2R and sstr5 interact physically through hetero-oligomerization to create a novel receptor with enhanced functional activity.17 However, the correlation of both DRs and sstrs with SRS imaging and its potential clinical significance have not been investigated as yet. Moreover, either SRS or IHC expression of sstrs and D2R has several limitations that could lead to false-negative results regarding tumour size or tissue heterogeneity.18 Regarding the immunohistochemistry, it is widely accepted that approximately 20–30% of tumour specimens’ exhibit only focal immunoreactivity and that in some cases, a negative result is not necessarily associated with absence of sstr expression. Alternatively, various factors may cause a false-positive SRS result, such as respiratory infections, surgical scars, concomitant granulomatous disease, adrenal uptake and accessory spleen.9 Hence, the aim of this study was to examine the IHC expression of sstr2, sstr3 and sstr5 in a series of gastrointestinal (GI) and lung NENs and for the first time, D2R in patients with GINENs, and to correlate their co-expression with pre-operative SRSs in order to identify any existing correlations.

Material and methods We analysed 55 specimens from 44 patients with GI-NENs (41 primary tumours and 14 metastases) and 26 specimens from 16 patients with lung NENs (16 primary tumours and 10 metastases) (Table 1). All neoplasmatic tissues were tested for

sstr2, sstr3, sstr5 and D2R IHC reactivity and graded for the radioligand uptake of the performed SRSs according to the Krenning scale.9,19,20 Neoplasmatic tissues were classified initially using conventional haematoxylin–eosin (H&E) sections, and their neuroendocrine nature was confirmed by immunohistochemistry using the neuroendocrine markers chromogranin A and synaptophysin. To grade all these neoplasmatic tissues, the proliferation label index Ki-67 was measured as percentage of approximately 2000 cells in areas with the strongest expression (‘hot spots’) and the greater result was considered.21 We also analysed the results according to the size of the neoplasmatic tissue, small (2 cm), as reported in the pathology report, metastatic foci versus primary sites and GI-NENs versus lung NENs. Sstr and D2R immunohistochemistry For sstr immunihistochemistry, polyclonal antisera were directed against sstr types 2, 3 and 5 (dilution 1:3000, Gramsch Laboratories, Schwabhausen, Germany) overnight at 4 °C and against D2R (dilution 1:300 in 02% bovine serum albumin, Novus Biologicals, USA) overnight at 4 °C; all antisera used have been repeatedly validated in previous studies.22,23 Sections of known sstr-positive pituitary adenomas were used as positive controls and sections where citrate buffer replaced the primary antisera served as negative controls. For evaluation of sstr immunopositivity, the three-scale scoring system standardized and proposed by Volante et al.24 was used. The receptor scoring was evaluated by two independent observers (ED and AVT), and the results obtained were compared to reach an agreement. Complete or incomplete membranous immunostaining (scores 3 and 2 respectively) was considered a positive result, whereas any cytoplasmic immunostaining was interpreted as negative result (score 1) (Fig. 1).24 D2R scoring was based on the intensity of the membranous immunostaining (0 = negative, 1 = weak positivity, 2 = moderate positivity and 3 = strong positivity).16.

Table 1. Patient and tumour characteristics of the 81 NENs specimens studied for sstr expression

Specimens/ parameters Age of patients (year old) Tumour size (mm) SRS grade Sstr2 IHC score Sstr3 IHC score Sstr5 IHC score Total

All

Primary site

Metastatic foci

Small-sized neoplasmatic tissue**

56  12

573  112

534  132

56  13

56  11

552  123

577  114

274 (18, 3–120)

233 (15, 3–75)

354 (25, 4–120)¶

109 (10, 3–18)

442 (40, 22–120)†

281 (18, 6–120)

258 (205, 3–70)

2 (2, 0–4) 19 (2, 0–3) 1 (1, 0–3) 08 (0, 0–3) 81

17 17 09 08 54

27 (3, 0–4)‡ 23 (3, 1–3)‡ 1 (1, 0–3) 07 (0, 0–3) 27

15 17 08 08 41

25 21 12 08 40

24 21 11 08 55

12 15 07 07 26

(15, 0–4) (2, 0–3) (1, 0–3) (0, 0–3)

(1, (2, (0, (0,

0–4) 0–3) 0–3) 0–3)

Large-sized neoplasmatic tissue**

GI-NENs

Lung NENs

(3, 0–4)† (2, 0–3)§ (1, 0–3)§ (05, 0–3)

(3, (2, (1, (0,

0–4) 0–3) 0–3) 0–3)

(1, (1, (1, (1,

0–4)* 0–3)* 0–3) 0–3)

GI-NENs, gastrointestinal tumours neuroendocrine neoplasms; IHC, immunohistochemical; SRS, somatostatin receptor scintigraphy; sstr, somatostatin receptor. P < 005: *vs GI-NENs; †vs small tumours; ‡vs primary site. P > 005 but

Somatostatin and dopamine receptor expression in neuroendocrine neoplasms: correlation of immunohistochemical findings with somatostatin receptor scintigraphy visual scores.

The expression of somatostatin (sstr1-5) and dopamine (DR) receptors in neuroendocrine neoplasms (NENs) facilitates diagnosis by tumour visualization ...
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