European Journal of Endocrinology (2008) 159 275–282 ISSN 0804-4643

CLINICAL STUDY Tachykinins in endocrine tumors and the carcinoid syndrome Janet L Cunningham1, Eva T Janson1, Smriti Agarwal1, Lars Grimelius2 and Mats Stridsberg1 Departments of 1Medical Sciences and 2Genetics and Pathology, University Hospital, SE 751 85 Uppsala, Sweden (Correspondence should be addressed to J Cunningham who is now at Section of Endocrine Oncology, Department of Medical Sciences, Lab 14, Research Department 2, Uppsala University Hospital, Uppsala University, SE 751 85 Uppsala, Sweden; Email: [email protected])

Abstract Objective: A new antibody, active against the common tachykinin (TK) C-terminal, was used to study TK expression in patients with endocrine tumors and a possible association between plasma-TK levels and symptoms of diarrhea and flush in patients with metastasizing ileocecal serotonin-producing carcinoid tumors (MSPCs). Method: TK, serotonin and chromogranin A (CgA) immunoreactivity (IR) was studied by immunohistochemistry in tissue samples from 33 midgut carcinoids and 72 other endocrine tumors. Circulating TK (P-TK) and urinary-5 hydroxyindoleacetic acid (U-5HIAA) concentrations were measured in 42 patients with MSPCs before treatment and related to symptoms in patients with the carcinoid syndrome. Circulating CgA concentrations were also measured in 39 out of the 42 patients. Results: All MSPCs displayed serotonin and strong TK expression. TK-IR was also seen in all serotonin- producing lung and appendix carcinoids. None of the other tumors examined contained TK-IR cells. Concentrations of P-TK, P-CgA, and U-5HIAA were elevated in patients experiencing daily episodes of either flush or diarrhea, when compared with patients experiencing occasional or none of these symptoms. In a Spearman partial rank test, the correlation of P-TK with daily diarrhea was independent of both U-5HIAA and CgA levels. Conclusion: We found that TK synthesis occurs in serotonin-IR tumors and that P-TK levels are significantly correlated with symptoms of flush and diarrhea in patients with MSPCs. This is, to our knowledge, the first report demonstrating an independent correlation of P-TKs with carcinoid diarrhea, a symptom that is customarily regarded as serotonin mediated. Further investigations may present opportunities for new therapeutic possibilities.

European Journal of Endocrinology 159 275–282

Introduction -Phe-Xaa-Gly-Leu-Met-NH2, where Xaa is hydrophobic and either an aromatic or a branched-chain aliphatic Patients with metastasizing ileocecal serotonin-produ- . The C-terminal sequence is important for cing carcinoid tumors (MSPCs; also called midgut the activation of each of the three known receptors, carcinoids and EC-cell carcinoids in the literature) often NK1, NK2, and NK3. The N-terminal sequence differs suffer from the carcinoid syndrome that includes considerably in both length and amino acid compo- hormone associated symptoms such as diarrhea and sition. The activation of these receptors has been flush, carcinoid heart disease, and bronchial constriction. correlated with a range of biological activities, e.g., Tachykinins (TKs) are present in normal enterochro- smooth-muscle contraction, vasodilatation, pain trans- maffin cells in the gastrointestinal (GI) tract and in mission, activation of the immune system, and MSPCs (1–4). TKs have also been found in other stimulation of endocrine gland secretion (15). endocrine tumors, such as appendix carcinoids (5), The biological action of TKs suggests a role in the serotonin-producing lung carcinoids (6), ovarian carci- development of both flush and diarrhea but few studies noids (7), medullary carcinoma of the thyroid (8, 9), correlate TKs with specific symptoms in patients with and in pheochromocytomas (10). the carcinoid syndrome. A correlation between TK Among the members of the TK family are levels and flush has been described (16, 17), but not (SP), (NKA), K (NPK), between plasma SP concentrations and pentagastrin- (NKB), and hemokinin-1; these hormones induced flush (18). It has also been shown that are coded on three genes: TAC1, TAC3, and TAC4 intraluminal TK concentrations were higher in patients (11). Recent reviews have provided more details with MSPCs than in healthy control subjects (19).A (12–14). TKs are biologically active neuropeptide study in rats showed that serotonin but not SP increased hormones that share a conserved C-terminal sequence, gastric emptying and upper gastrointestinal transit

q 2008 European Society of Endocrinology DOI: 10.1530/EJE-08-0196 Online version via www.eje-online.org

Downloaded from Bioscientifica.com at 09/28/2021 10:30:21PM via free access 276 J L Cunningham and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2008) 159 times (20). No positive association between carcinoid differentiated endocrine carcinoid (rectum) (nZ2), diarrhea and TKs has been reported, as far as we know. appendix carcinoid (nZ4), goblet cell carcinoid In this study, a new polyclonal antibody to the (nZ2), L-cell rectal carcinoid (nZ1) pheochromocy- common C-terminal of the TK family was used in a RIA toma (nZ5), neuroblastoma (nZ2), adrenal cortical and in immunohistochemical analysis (IHC) to study adenoma (nZ2), adrenal cortical carcinoma (nZ7), P-TK association with clinical symptoms in MSPCs and follicular thyroid adenoma (nZ5), follicular thyroid to screen other endocrine tumor tissues for the carcinoma (nZ2), papillary thyroid carcinoma (nZ2), expression of members of the TK family. medullary thyroid carcinoma (nZ5), parathyroid adenoma (nZ4), and parathyroid carcinoma (nZ1), simple goiter (nZ2) see Table 1. The tumors were diagnosed histopathologically at the Laboratory for Materials and methods Pathology and Cytology and patients with MSPCs were treated at the Department of Endocrine Oncology at The Sample composition University Hospital, Uppsala. All MSPCs displayed In this retrospective study, a total of 137 patients were chromogranin A (CgA) immunoreactive (IR) and/or included, 65 of whom were diagnosed with MSPCs argyrophil (Grimelius) reaction (21); and also showed (tumor tissue available from 33 patients) and 72 with serotonin and vesicular monoamine transporter 1 IR other types of endocrine tumors. Plasma samples from and/or argentaffin (Masson) reaction (22). At the time 42 consecutive patients with MSPCs were collected for of the study, 48 MSPC patients had metastases and the RIA analysis before treatment was initiated. In the IHC rest of these patients developed metastases during the analysis tumor tissues were studied from 33 patients course of the disease. Tumor diagnosis was based on with MSPCs where 10 patients were included in both recommendations in the World Health Organization the RIA and IHC analysis. The IHC analysis also entailed classification of endocrine tumors (23). 72 patients with various types of endocrine tumors Forty-four of the patients with MSPCs had symptoms elsewhere in the GI tract and other organs including: of either flush and/or diarrhea. Medical records were ECLoma type 1 (nZ4), ECLoma type 3 (nZ2), typical reviewed in order to establish the symptoms evident at lung carcinoid (nZ3), atypical lung carcinoid (nZ3), diagnosis. All patients were asked specifically about endocrine pancreatic tumor (nZ11), poorly differen- symptoms of diarrhea and flush. When symptoms tiated endocrine carcinoid (stomach) (nZ3), poorly were absent, a negative entry was correspondingly

Table 1 Immunohistochemical detection of tachykinins, serotonin, and chromogranin A in endocrine tumors.

Tumor type N Tachykinin Serotonin Chromogranin A

Foregut ECLoma type 1 4 0 0a 4 ECLoma type 3 2 0 0 2 Typical lung carcinoid 3 1 1 3 Atypical lung carcinoid 3 1 1 3 Endocrine pancreatic tumor 11 0 0 11 Poorly differentiated endocrine carcinoid (stomach) 3 0 0 3b Midgut Ileocecal serotonin-producing carcinoids 33 33 33 33 Appendix carcinoid 4 4 4 4 Goblet cell carcinoid 2 1 1 2 Hindgut L-cell rectal carcinoid 1 0 0 1 Poorly differentiated endocrine carcinoid (rectum) 2 0 0 2b Other endocrine tumors Pheochromocytoma 5 0c 05 Neuroblastoma 2 0 0 2 Adrenal cortical adenoma 2 0 0 0 Adrenal cortical carcinoma 7 0 0 0 Follicular thyroid adenoma 5 0 0 0 Follicular thyroid carcinoma 2 0 0 0 Papillary thyroid carcinoma 2 0 0 0 Medullary thyroid carcinoma 5 0 0 5 Parathyroid adenoma 4 0 0 2 Parathyroid carcinoma 1 0 0 1 Simple goiter 2 0 0 0 aFewer than 1% of tumor cells were serotonin immunoreactive. bChromogranin A expression was seen focally in 20–80% of tumor cells. cTwo out of five pheochromocytomas contained tachykinin positive nerve cells.

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Downloaded from Bioscientifica.com at 09/28/2021 10:30:21PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2008) 159 Tachykinins and neuroendocrine tumors 277 documented. Patients were grouped according to The coupled peptide was then purified on a PD-10 symptom frequency into three groups; no symptoms, column (Amersham Biosciences, Freiburg, Germany) occasional symptoms (at least once in a week), and daily with PBS as the moving phase. Aliquots of 200 mg symptoms of either flush or diarrhea. Eight patients coupled peptide were frozen and stored at K20 8C until included in the RIA analysis had symptoms of both immunization. The peptide complex was injected into diarrhea and flush, 16 only diarrhea, 8 only flush, and New Zealand white rabbits, using the intradermal 10 did not experience symptoms. injection technique to produce polyclonal antibodies.

Antibody construction Development of the RIA Based on the amino acid sequence of human TKs, a All chemicals used were of pro analysis grade (Merck). polypeptide was synthesized by a solid-phase system Dilutions in the RIA were performed in the assay buffer, using Fmoc chemistry. The peptide was purified by a 0.05 M sodium phosphate buffer at pH 7.4, with reverse-phase chromatography and analyzed by plasma 0.15 M sodium chloride, 0.02% sodium azide, 0.2% desorption mass spectrometry. BSA, and 0.5% Tween 20. The antibodies and the The sequence was selected to be specific for the synthesized peptide were used to develop a specific RIA. conserved c-terminal of human TKs (Fig. 1). A cysteine For preparation of the tracer, the peptide was labeled residue was added to the N-terminal to facilitate with 125I (Amersham International plc, Amersham) coupling to the carrier protein. To facilitate iodine- using the chloramine-T method as previously described labeling, a tyrosine residue was added in the position (24). The assay was devised as follows: standards and between the cysteine residue and the N-terminal of unknown samples were incubated with a tracer the peptide. (30 000 c.p.m./tube) and primary antibodies, at a Before immunization, the peptide was coupled to final dilution of 1/45 000, for 3 days at C4 8C. All a carrier protein. One milligram peptide was dis- standards and samples were assayed in duplicate. solved in 100 ml DMSO after which, 1 mg Imject Antibody-bound radioactivity was separated from free maleimide-activated keyhole limpet hemocyanin tracer by adding a second antibody, goat anti-rabbit IgG (Pierce Biotechnology, Rockford, IL, USA) was coupled to a solid phase (Decanting suspension 3, added. This mixture was allowed to react for 2 h at Pharmacia Biotech). The antibody-bound radioactivity room temperature. was then measured in a g-counter (Autogamma, Wallac, Pharmacia Biotech) and the data were calcu- lated with a logit-log transformation program (Multi- calc, PerkinElmer, Massachusetts, USA). The cross-reactivity and specificity of the antibodies were tested in the RIA by serial dilutions of the commercial TK : SP1–11, Tyr-SP1–11, SP1–9, SP7–11, SP4–11, NPK, NKA and NKB, and the synthesized peptide (Nova Biochem Darmstadt, Germany and Sigma Chemical Co., LabKemi).

Collection of plasma samples for TK analysis Plasma samples from the 42 consecutive patients were collected after an overnight fast. TK-related IR in plasma samples was measured without prior extraction. Plasma samples were collected in heparinized tubes, stored on ice, and centrifuged within 30 min. The plasma was frozen immediately and stored at K20 8C until analysis.

Measurement of urinary-5 hydroxyindoleace- tic acid (U-5HIAA) and P-CgA Figure 1 The TK antibodies are specific for the TK family common C-terminal, as shown in a competition study where labeled Thirty-seven of the above-mentioned plasma samples, synthesized peptide and unlabeled TK peptides, listed in table collected after an overnight fast before treatment was below, compete to bind the antibody in a RIA. All peptides with intact C-terminal bound to the antibodies, while the peptide missing two initiated, were also analyzed for P-CgA concentrations amino acids, Substance P (1–9), did not bind. Thus, an intact using a previously described method (25). In all 42 C-terminal was mandatory for antibody binding. patients, U-5HIAA before treatment start was measured

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Downloaded from Bioscientifica.com at 09/28/2021 10:30:21PM via free access 278 J L Cunningham and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2008) 159 using HPLC and calculated as the mean amount (mmol) bound the antibodies competitively in the RIA setting, of two 24 h urine collections (26). whereas the peptide lacking two C-terminal amino acids did not (see Fig. 1). IHC TK expression in endocrine tumors Tumor biopsies were fixed in buffered formalin, dehydrated, and embedded in paraffin wax. Sections, All 33 MSPC specimens included in this study showed w4 mm thick, were attached to positively charged glass intense TK-IR. No difference in staining intensity was slides, deparaffinized in xylene, and rehydrated using noted in patients with when compared to those without decreasing concentrations of ethanol. Before immunos- the carcinoid syndrome. Furthermore, TK-IR was taining, the sections were treated in a microwave oven detected in 2 out of 5 lung carcinoids, 5 out of 6 at 700 W for 15 min and 350 W for 10 min in 50 mM appendix carcinoids; all TK-IR tumors also displayed Tris buffer (pH 8.0). DAKO EnVisionCSystem-RP was serotonin IR. TK expression could not be detected in the used as a staining technique with diaminobenzidine as other endocrine tumors studied (see Table 1 and Fig. 2). chromogen substance. The sections were counter- TKs were, as reported earlier (13), expressed in stained with hematoxylin. neuronal tissue. Slender cells surrounding the Brun- A dilution series of the TK antibody was tested on ner’s glands showed TK-IR possibly explained by the fact tumor sections from a patient with MSPC known to that these glands are innervated by TK signaling have elevated plasma levels of NPK. A distinct staining neurons (27). TK-IR cells were rarely seen in mucosa of tumor cells was noted at a dilution of 1:5000. IHC from the distal duodenum but were abundant the without antigen retrieval and with microwave pre- jejunal/ileal mucosa. treatment in citrate buffer pH6 was also tested but gave weaker staining. As a negative control, an absorption test was also performed with the synthesized peptide, Hormone markers in relation to the carcinoid using the concentrations 10, 1, and 0.1 nmol/ml. syndrome A Spearman correlation test showed that P-TK, Statistical analysis U-5HIAA, and P-CgA concentrations were significantly correlated with each other in the 42 consecutive A possible correlation between the biochemical patients diagnosed with MSPCs. The correlation markers and the presence of daily, occasional or coefficient was the highest for P-CgA and U-5HIAA the absence of symptoms at diagnosis and before the levels (0.93), 0.60 for P-TKs and U-5HIAA levels, and initiation of treatment, was tested using a Spearman 0.49 for P-TK and CgA. Ten patients, all with high rank test. A P value less than 0.05 was considered a TK-IR in the IHC analysis, displayed P-TK levels ranging statistically significant result. In order to analyze the from 30 to 186 pmol/l (median 49.5). independent effects of the three hormones on the The median P-TK level in patients with liver severity of diarrhea, each marker was tested while metastases (nZ22) was 65.5 pmol/l (range 30–570) the others were held constant in a Spearman partial in comparison to those without 46 pmol/l (range 30– rank test. 96). Patients with lymph node metastases had a median of 62 pmol/l (30–570) vs 43 pmol/l (43–186) for those without. In patients where neither liver nor lymph node Ethics metastases had yet developed (10) the median was The study was reviewed and approved by the local 44 pmol/l (range 30–87). Patients with metastases Medical Ethics Board at Uppsala University Hospital. tended to have higher concentrations of P-TK but this did not reach significance in this study. Results from a Spearman rank test, performed to determine the association of the hormone levels with the Results grade of clinical symptoms, are shown in Table 2. All Antibody specificity tumor marker concentrations were elevated in patients with daily episodes of flushing. However, the hormone Clear IHC staining of tumor cells in a patient with effects were not significant in the partial Spearman Rank elevated plasma levels of NPK was noted. In the IHC test and therefore not independent effects. In patients specificity test of anti-TK antibody, a complete elimina- with daily episodes of flushing at diagnosis (nZ8), tion of IR was seen when the TK antibody was pre- the median P-TK level was 86.5 pmol/l (range 30–493), incubated in a solution containing either 10 or for those with occasional flushes (nZ8), 53 pmol/l 1 nmol/ml of the synthesized peptide (see inset, (range 30–186), whereas for patients without flushes Fig. 2B). The specificity of the anti-TK antibody was (nZ26), 44 pmol/l (range 30–570) (see Fig. 3). also tested using TK-peptides in a competitive-binding All three hormone marker concentrations were assay. All TK-peptides with an intact TK C-terminal significantly elevated in patients with daily episodes of

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Figure 2 TK-IR occurs in serotonin-immunoreactive tumors. (A) TK-IR in ileum mucosa. Inset shows a TK-immunoreactive cell. (B) Intense TK-IR in a MSPC metastasis. Inset shows absorption test blocking IHC staining with 1 nmol/ml peptide. (C) TK-IR in the enterochromaffin tumor cell component of a goblet cell appendix carcinoid. (D) TK-IR in a serotonin-producing lung carcinoid showing diffuse intermediate staining intensity. diarrhea, though the association between P-TK and the medullary thyroid carcinoma and pheochromocytomas severity of diarrhea was independent of both CgA and (8–10, 30). In our study, TK-IR was not found in non- U-5HIAA concentrations (correlation coefficient: 0.4, serotonin-producing endocrine tumors; however, our P!0.01). In patients with daily episodes of diarrhea at sample population is too small to rule out the existence diagnosis (nZ11), the median P-TK level was of other TK-IR endocrine tumors. 105 pmol/l (range 30–570), for those with occasional In contrast to homogenous TK-IR in tumor tissue diarrhea (nZ12), 49.5 pmol/l (range 30–96), whereas variation in P-TK was noted in patients with MSPCs. for patients free from diarrhea (nZ19), 30 pmol/l (range 30–129) (see Fig. 3). Table 2 Biochemical markers associated with flush and diarrhea in patients with metastasizing ileocecal serotonin-producing carcinoid tumors before treatment start.

Discussion Grade Grade Grade of Grade of Marker of flusha of flushb diarrheaa diarrheab

The new antibody to the common C-terminal of Plasma-tachykinins 0.3 0.18 0.6 0.4 members of the TK protein family has been designed PZ0.03 PZ0.3 P!0.001 P!0.01 and raised in our laboratory and is shown to bind U-5 HIAA 0.4 0 0.4 K0.1 specifically to members of the TK family and useful in PZ0.01 PZ0.8 P!0.05 PZ0.5 both RIA and IHC analyses. In the RIA setting, we could Chromogranin A 0.4 0.2 0.4 0.2 show that an intact C-terminal was mandatory for P!0.01 PZ0.2 P!0.01 PZ0.2 antibody binding. TK expression was seen only in All markers are elevated in patients with daily episodes of flush and diarrhea tumors that simultaneously expressed serotonin. All when compared with those experiencing occasional or no symptoms. MSPCs studied displayed strong TK-IR, which may be of Plasma-tachykinin association with the severity of diarrhea is independent from urinary-5 hydroxyindoleacetic acid (U-5HIAA) and plasma-chromo- diagnostic value. Our results corroborate earlier granin A effects. findings indicating that serotonin and TK are often aSpearman’s rank correlation (r) between measure levels and severity of diarrhea. expressed in the same cell (1, 28, 29). Previously, focal bPartial Spearman’s, correlation coefficients adjusted for the other two SP expression had been described in a minor subset of biochemical markers.

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Figure 3 Plasma tachykinins (P-TK), urinary-5 hydroxyindoleacetic acid (U-5HIAA), and plasma chromogranin A (P-CgA) in relation to flush and diarrhea severity in patients with MSPCs.

This may be due to several factors. Patients with diarrhea (31, 35). Serotonin receptor antagonists metastases had higher levels when compared with those alleviate gastrointestinal symptoms in some but not all without. Notably, all patients with P-TK over patients (18, 35). A more recent study found that wide 100 pmol/l had extensive liver metastases. Other variation in, rather than the absolute value of, diurnal factors, such as the secretory activity of the tumor, the U-5HIAA concentrations was correlated with the short half-life of TK peptides in circulation, and severity of the diarrhea (36). In our study, all variation in liver function may also be important. biochemical marker concentrations were elevated in MSPCs produce many biologically active substances patients with daily episodes of diarrhea, though the with partially overlapping biological functions. The association between increased P-TK and the severity of biological processes underlying the specific symptoms of diarrhea was independent of both CgA and U-5HIAA the carcinoid syndrome are probably multifactorial. In concentrations. the present study, we could confirm results from earlier There is the evidence of a biological mechanism that studies showing that TK and U-5HIAA levels are could explain this association between TK and carcinoid elevated in patients with daily episodes of flushing (16, diarrhea. P-TKs are involved in other diarrheic 31). The hormone effects were not mutually indepen- conditions such as the irritable bowel syndrome (37, dent in this study. It is possible that the development of 38) and ulcerative colitis (39). In the normal gut, SP flushes is the result of multi-hormonal stimulation. and NKA, via TK receptors on muscle cells and Other biologically active substances, such as kallikrein, secretomotor neurons, have stimulatory effects on gut (32, 33) and prostaglandins (34), may also contribute. motility (37, 40). There is also evidence that TK can Secretory diarrhea in patients with MSPCs is induce serotonin release from colonic mucosa via NK2 currently thought to be related to serotonin production and NK3 receptors (41). P-SP levels have been shown to based on the functional role of serotonin in the normal correlate with the severity of diarrhea in cryptospor- gut. However, studies have not shown any significant idiosis (42). Earlier studies have not shown any correlation between U-5HIAA levels and the severity of significant correlation between individual members in

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Downloaded from Bioscientifica.com at 09/28/2021 10:30:21PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2008) 159 Tachykinins and neuroendocrine tumors 281 the TK family and carcinoid diarrhea (31, 35, 43). Here, References total P-TK concentrations are independently associated with diarrhea in agreement with results from the 1 Sundler F, Alumets J & Ha˚kanson R. 5-Hydroxytryptamine- studies mentioned above, that demonstrate overlapping containing enterochromaffin cells: storage site of substance P. Acta Physiologica Scandinavica 1977 452 121–123. effects of the members of the TK family that may have a 2 Wilander E, Portela-Gomes G, Grimelius L & Westermark P. combined effect on intestinal motility. Argentaffin and argyrophil reactions of human gastrointestinal P-TK may be important in treatment response carcinoids. Gastroenterology 1977 73 733–736. evaluation. 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