A New Marker for Human Ductal Pancreatic Adenocarcinoma Gut: First Published As 10.1136/Gut.42.4.546 on 1 April 1998

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546 Gut 1998;42:546–550 Neurotensin receptors: a new marker for human ductal pancreatic adenocarcinoma Gut: first published as 10.1136/gut.42.4.546 on 1 April 1998. Downloaded from

J C Reubi, B Waser, H Friess, M Büchler, J Laissue

Abstract binding in vivo of the somatostatin analogue Background/Aims—New imaging possi- [111In]pentetreotide (Octreoscan) to sst2 soma- bilities for early diagnosis of the devastat- tostatin receptors expressed in high density in ing exocrine pancreatic adenocarcinomas neuroendocrine cancer.67 Unfortunately, this would be highly welcome. Recently, pan- method cannot be used for the diagnosis of creatic neuroendocrine tumours have exocrine pancreatic cancers since they do not been successfully visualised in vivo on the express suYcient amounts of these somatosta- basis of their high density of receptors for tin receptors.8–10 Therefore it is worth loooking the regulatory peptide somatostatin. Un- for receptors for other small regulatory pep- fortunately, exocrine pancreatic tumours tides that may be expressed in high density and do not express suYcient amounts of with high incidence in exocrine pancreatic somatostatin receptors. Therefore over- adenocarcinomas.10 One candidate peptide of expression of other regulatory peptide interest is neurotensin, a 13 amino acid peptide receptors in these tumours needs to be found in brain and gut. Neurotensin has, found. among other actions, a growth regulatory func- 11 12 Methods—Receptors for the regulatory tion in colon and pancreatic cell lines. In the peptide neurotensin were evaluated in present study, we evaluated neurotensin recep- vitro by receptor autoradiography in 24 tor expression in vitro in primary human pan- ductal pancreatic adenocarcinomas, 20 creatic adenocarcinomas, and compared the endocrine pancreatic cancers, 18 cases of results with its expression in normal human chronic pancreatitis, and 10 normal pan- pancreas and in chronic pancreatitis, as well as creatic glands. in endocrine pancreatic tumours, in order to Results—Some 75% of all ductal pancre- gauge the potential diagnostic value of in vivo atic adenocarcinomas, most of them dif- neurotensin receptor scintigraphy in pancreatic ferentiated, were neurotensin receptor adenocarcinomas. positive, whereas endocrine pancreatic http://gut.bmj.com/ cancers did not express neurotensin re- Methods ceptors. No neurotensin receptors were Aliquots of surgically resected tumours or of found in chronic pancreatitis or normal biopsy specimens submitted for diagnostic his- pancreatic tissues, including pancreatic topathology were collected. A total of 24 ductal acini, ducts, and islets. Conclusions—The selective and high ex- Differentiated tumours (grades I or II)

pression of neurotensin receptors in duc- Poorly differentiated tumours (grades II–III or III) on September 27, 2021 by guest. Protected copyright.

tal pancreatic adenocarcinomas could 4 form the molecular basis for potential 10 Division of Cell clinical applications, such as in vivo Biology and Experimental Cancer neurotensin receptor scintigraphy for early tumour diagnosis, radiotherapy with 3 Research, Institute of 10 Pathology, University radiolabelled neurotensin analogues, and of Berne, Berne, chemotherapy with neurotensin receptor Switzerland antagonists. J C Reubi (Gut 1998;42:546–550) 2 B Waser 10 J Laissue Keywords: neurotensin receptors; ductal pancreatic carcinomas; chronic pancreatitis; endocrine pancreatic Department of tumours; in vivo scintigraphy; regulatory peptides Visceral and 1 10 Transplantation Surgery H Friess Carcinoma of the exocrine pancreas is the ﬁfth

M Büchler largest cause of death from cancer in western Neurotensin receptor density (dpm/mg) 12 0 countries. It grows extremely rapidly, and A B C D Correspondence to: Professor J C Reubi, Division early diagnosis allowing therapeutic surgical 12 Figure 1 Density of neurotensin receptors in various of Cell Biology and resection is rarely possible. No alternative human pancreatic tissues. A, ductal pancreatic Experimental Cancer medical therapy is available. Therefore a adenocarcinomas (n = 24); 18 are diVerentiated tumours Research, Institute of non-invasive method of early diagnosis of this (grade I or II), and six are poorly diVerentiated tumours Pathology, University of (grade II–III or III). B, endocrine pancreatic cancers (n = Berne, Murtenstrasse 31, type of cancer would be extremely useful. 20). C, chronic pancreatitis (n = 18). D, normal pancreas CH-3010 Berne, In the last few years, a new in vivo diagnostic (n = 10). In A and B, the neurotensin receptor content of Switzerland. method has been developed and widely used each tumour (dpm/mg tissue) was measured by quantitative in vitro receptor autoradiography.13 In C and Accepted for publication with great success to localise neuroendocrine D, the pancreatic acini, ducts, and islets were evaluated for 2 December 1997 pancreatic tumours,3–5 based on the speciﬁc their neurotensin receptor content by the same method. Neurotensin receptors in pancreatic cancer 547

Neurotensin Acetyl neurotensin (8–13) Neurotensin (1–11)

100 Gut: first published as 10.1136/gut.42.4.546 on 1 April 1998. Downloaded from

I-Neurotensin specific binding (%) 0.01 0.1 1 10 100 1000

125 Peptides (nM) Figure 3 High aYnity and speciﬁcity of the 125I-[Tyr3]-neurotensin binding in a displacement experiment. Sections of a ductal pancreatic adenocarcinoma were incubated with 125I-[Tyr3]-neurotensin and increasing concentrations of unlabelled neurotensin, acetyl-neurotensin-(8–13), and neurotensin-(1–11). High aYnity displacement of the trace is found with neurotensin and acetyl-neurotensin-(8–13), whereas the biologically inactive neurotensin-(1–11) had no eVect. Non-speciﬁc binding was subtracted from all values. three days to improve adhesion of the tissue to the slide, as previously described.13 Each tissue underwent receptor autoradiographic processing, as described by Moyse et al,14 using 125I-[Tyr3]-neurotensin as radioligand. Neuro- tensin binding was achieved by incubating the sections with 40 000 dpm/100 µl monoiodo 125I-[Tyr3]-neurotensin (2000 Ci/mmol; Anawa, Wangen, Switzerland) in 50 mM Tris/

HCl buVer, pH 7.6, containing 5 mM MgCl2, 0.2% bovine serum albumin, and 5 × 10−5 M bacitracin at 4°C for one hour. Additional http://gut.bmj.com/ sections were incubated in the presence of increasing amounts of non-radioactive neurotensin, acetyl-neurotensin-(8–13), and neurotensin-(1–11), to generate competitive inhibi- tion curves. After incubation, the sections were washed for eight minutes at 4°C in four consecutive baths containing 50 mM Tris/HCl

buVer, pH 7.6. The sections were then dried on September 27, 2021 by guest. Protected copyright. Figure 2 Neurotensin receptors in two well diVerentiated ductal pancreatic under a stream of cold air at 4°C, apposed to adenocarcinomas. (A) and (B) show the haematoxylin-eosin stained sections representing 3H Hyperfilms (Amersham, Little Chalfont, tumour duct-like structures (arrowheads) surrounded by non-neoplastic pancreatic acini (asterisks). Bars = 1 mm. (C) and (D) show autoradiograms with total binding of Bucks, UK), and exposed for seven days to x 125I-[Tyr3]-neurotensin. The black areas (arrowheads) represent the neurotensin receptors in ray cassettes. the tumour ducts. The asterisks indicate the receptor-free pancreatic acini. (E) and (F) The autoradiograms were quantified using a show autoradiograms with non-specific binding of 125I-[Tyr3]-neurotensin (in the presence of 10−6 M neurotensin). computer assisted image processing system, as described.13 Tissue standards for iodinated pancreatic adenocarcinomas were included in compounds (Amersham) were used for this this study. They were obtained from 13 male purpose. A tumour was defined as receptor and 11 female patients, with an age range from positive when the optical density measured 49 to 89 years (mean age 68 years). Eighteen over a tumour area in the total binding section tumours were diVerentiated (grade I or II), and was at least twice that of the non-specific bind- six were poorly diVerentiated (grade II–III or ing section (in the presence of 10−6 M III). In addition, samples from 18 patients with neurotensin). In each experiment, rat and chronic pancreatitis, 10 normal pancreatic human brain tissues, rich in neurotensin recep- glands from organ donors, and 20 endocrine tors, were included as positive controls. pancreatic tumours (including gastrinomas, Somatostatin receptor autoradiography for insulinomas, vipomas, glucagonomas, and the sst2 subtype was performed on the non-secreting tumours) were collected. endocrine pancreatic cancers using 125I-[Tyr3]- All tissues were frozen in liquid nitrogen octreotide as radioligand, as described 613 immediately after surgical resection and stored previously. at −70°C. Receptor autoradiography was performed on 10 and 20 µm thick cryostat Results (Leitz 1720, Rockleigh, NJ, USA) sections of As shown in fig 1, 75% of all human ductal the tissue samples, mounted on microscope pancreatic adenocarcinomas expressed neuro- slides, and then stored at −20°C for at least tensin receptors. A considerable variability in 548 Reubi, Waser, Friess, et al Gut: first published as 10.1136/gut.42.4.546 on 1 April 1998. Downloaded from

Figure 4 Absence of neurotensin receptors in normal human pancreatic tissue (A–C) and in chronic pancreatitis (D–F).

(A), (D) Haematoxylin-eosin stained sections showing pancreatic acini (Ac), pancreatic ducts (arrowheads), and http://gut.bmj.com/ pancreatic islets (arrows). Bars = 1 mm. (B), (E) Autoradiograms showing total binding of 125I-[Tyr3]-neurotensin. (C), (F) Autoradiograms showing non-speciﬁc binding of 125I-[Tyr3]-neurotensin (in the presence of 10−6 M neurotensin).

receptor density is observed among individual The neurotensin receptors were also evalu- cases, ranging from a lack of neurotensin ated in large samples of normal pancreatic tis- receptors to very high amounts peaking at 8000 sue obtained from organ donors as well as in dpm/mg tissue, an observation that is compat- chronic pancreatitis: in these tissues, neither ible with the polyclonality and morphological the pancreatic acini and ducts nor the islets on September 27, 2021 by guest. Protected copyright. variability of neoplasms. We observed neuro- expressed these receptors in measurable tensin receptors more often in diVerentiated amounts (figs 1 and 4). Moreover, in a series of than in poorly diVerentiated tumours: indeed, endocrine pancreatic tumours, including in- as many as 83% of the diVerentiated adenocar- sulinomas, gastrinomas, glucagonomas, and cinomas were receptor positive; in particular, vipomas, neurotensin receptors were undetect- tumours with tubular diVerentiation were more able (fig 1), whereas somatostatin receptors often positive than solid tumours. Further, sev- were expressed at high density (fig 5), as eral cases displayed a heterogeneous distribu- reported previously.13 tion of the neurotensin receptors, even among histopathologically similar tumour areas. Discussion As shown in a characteristic example in fig 2, The results of the present study show the pres- the neurotensin receptors are exclusively lo- ence of neurotensin receptors in human ductal cated on tumour cells; the neoplastic duct-like pancreatic adenocarcinomas. These findings structures are clearly receptor positive, whereas extend the knowledge of the biology of these no receptors can be detected in adjacent adenocarcinomas, by identifying neurotensin normal pancreatic ducts, acini, and islets. The as an additional factor of their pathophysiology. binding characteristics of neurotensin recep- Exocrine pancreatic carcinomas not only syn- tors correspond to those established thesise and secrete several growth factors, such previously.11 14 As seen in the displacement as epidermal growth factor, and their respec- curve in fig 3, neurotensin receptors show high tive receptors,15 but may also be responsive to aYnity binding in the nanomolar range; hormones and regulatory peptides acting at moreover, the binding is specific, since the bio- specific membrane bound receptors, often logically active analogue acetyl-neurotensin- expressed in high density. Neurotensin may (8–13) has a similar aYnity to that of play a functional role, since it was shown in neurotensin, whereas the biologically inactive pancreatic cell lines to stimulate tumour cell neurotensin-(1–11) has no such aYnity. growth.12 The presence of a large number of Neurotensin receptors in pancreatic cancer 549

neurotensin receptors. However, the absence of neurotensin receptors from pancreatic tissue does not completely exclude the presence of

pancreatic cancer, since a minority of our exo- Gut: first published as 10.1136/gut.42.4.546 on 1 April 1998. Downloaded from crine tumours were found to be receptor nega- tive in vitro. The potential direct clinical consequences of the present data are considerable. In analogy to in vivo somatostatin receptor scintigraphy, it may be possible in the future to perform neurotensin receptor scintigraphy for early detection and diVerential diagnosis of exocrine pancreatic cancer. Three important prerequi- sites for the success of such a method are fulfilled. (1) High aYnity neurotensin receptors are specifically located on the tumour cells in three quarters of the cases. (2) An adequate radiotracer, a small neurotensin hexapeptide analogue linked to Ná-diethylenetriamine- pentaacetic acid (DTPA) which has the property to chelate 111In, a routinely used radioisotope in diagnostic nuclear medicine, and which retains high binding aYnity, is available.17 This neurotensin analogue, like DTPA-octreotide,4 is rapidly cleared through the kidneys and virtually not excreted through the biliary and gastrointestinal tracts,17 a significant advantage in terms of background activity in the pancreatic area. (3) The neurotensin receptors can be rapidly internal- ised, together with the bound radioligand, into target cells18: such a mechanism is likely to contribute to the high scintigraphic signal observed during in vivo somatostatin receptor 4 Figure 5 Endocrine pancreatic tumour lacking scanning of neuroendocrine tumours.

neurotensin receptors (B) but expressing a high density of Finally, neurotensin receptors may serve as a http://gut.bmj.com/ somatostatin receptors (C). (A) Haematoxylin-eosin highly specific therapeutic tool, especially in stained section showing the tumour. Bar = 1 mm. (B) Autoradiogram showing total binding of those tumours that express them in abundance. 125I-[Tyr3]-neurotensin. (C) Autoradiogram showing sst2 Radiotherapy of tumours with neurotensin somatostatin receptors identified by the binding of analogues linked to a â-emitting isotope may 125I-[Tyr3]-octreotide (OCT-R).In (B) and (C), non-specific binding was negligible. be envisaged, as shown in preliminary radio- therapeutic studies using radiolabelled somato- high aYnity neurotensin receptors in pancre- statin analogues in somatostatin receptor atic adenocarcinomas could therefore contrib- expressing tumours.19 20 Secondly, to counter- on September 27, 2021 by guest. Protected copyright. ute significantly to their aggressive growth and act the stimulatory eVect of neurotensin on the fatal outcome. tumour cells,12 the long-term eVect of neuro- The high selectivity of neurotensin receptor tensin antagonists, such as SR 48692,21 may be expression in ductal pancreatic adenocarcino- investigated. mas is underlined by the fact that no measurable levels of neurotensin receptors 1 Rosewicz S, Wiedenmann B. Pancreatic carcinoma. Lancet 1997;349:485–9. could be found in tissue samples from patients 2 Warshaw AL, Fernandes-Del Castillo C. Pancreatic carci- with chronic pancreatitis, a presumptive pre- noma. N Engl J Med 1992;326:455–64. 3 Krenning EP, Bakker WH, Breeman WAP, et al. Localisation malignant condition leading to pancreatic of endocrine-related tumours with radioiodinated analogue 16 adenocarcinomas. In contrast, epidermal of somatostatin. Lancet 1989;I:242–4. growth factor receptors are expressed in 4 Krenning EP, Kwekkeboom DJ, Pauwels S, et al. Somatosta- tin receptor scintigraphy. Nuclear Medicine Annual 1995; significant amounts in both chronic pancreati- 1995:1–50. 5 Gibril F, Reynolds JC, Doppman JL, et al. Somatostatin tis and the normal pancreas (J C Reubi, receptor scintigraphy: its sensitivity compared with that of unpublished work).15 Because of their overex- other imaging methods in detecting primary and metastatic gastrinomas. Ann Intern Med 1996;125:26–34. pression in ductal pancreatic adenocarcinomas 6 Reubi JC, Schaer JC, Waser B, Mengod G. Expression and and their absence in normal pancreas and localization of somatostatin receptor SSTR1, SSTR2 and chronic pancreatitis, neurotensin receptors can SSTR3 mRNAs in primary human tumors using in situ hybridization. Cancer Res 1994;54:3455–9. be considered as a marker for most ductal pan- 7 John M, Meyerhof W, Richter D, et al. Positive somatostatin receptor scintigraphy correlates with the presence of soma- creatic adenocarcinomas, an observation with tostatin receptor subtype 2. 1996; :33–9. 1 Gut 38 great clinical relevance. Indeed, simply the 8 Reubi JC, Horisberger U, Essed CE, et al. Absence of soma- demonstration of neurotensin receptors in the tostatin receptors in human exocrine pancreatic adenocarcinomas. Gastroenterology 1988;95:760–3. pancreas may indicate the presence of an 9 Buscail L, Saint-Laurent N, Chastre E, et al. Loss of sst2 adenocarcinoma in a patient. Neurotensin somatostatin receptor gene expression in human pancreatic and colorectal cancer. Cancer Res 1996;56:1823–7. receptors are also likely to discriminate be- 10 Reubi JC. Neuropeptide receptors in health and disease: the molecular basis for in vivo imaging. J Nucl Med tween malignant exocrine and endocrine pan- 1995;36:1825–35. creatic neoplasms, since the latter are lacking 11 Shulkes A. Neurotensin. New York: Raven Press, 1994. 550 Reubi, Waser, Friess, et al

12 Ishizuka J, Townsend CM, Thompson JC. Neurotensin 17 Tourwé D, Mertens J, Ceusters M, et al. The synthesis of regulates growth of human pancreatic cancer. Ann Surg metabolically stabilized peptides and their radiolabeling: 1993;217:439–46. new (111-In)- and (I)-neurotensin analogues. Tumor Target- 13 Reubi JC, Kvols LK, Waser B, et al. Detection of somatosta- ing (in press). tin receptors in surgical and percutaneous needle biopsy 18 Beaudet A, Mazella J, Nouel D, et al. Internalization and samples of carcinoids and islet cell carcinomas. Cancer Res intracellular mobilization of neurotensin in neuronal cells. Gut: first published as 10.1136/gut.42.4.546 on 1 April 1998. Downloaded from 1990;50:5969–77. Biochem Pharmacol 1994;47:43–52. 14 Moyse E, Rostène W, Vial M, et al. Distribution of 19 Krenning EP, Valkema R, Kooij PPM, et al. Radionuclide neurotensin binding sites in rat brain: a light microscopic therapy with (111-In-DTPA-D-Phe-1)-octreotide. Preliminary radioautographic study using monoiodo(125-I)Tyr3- data of a phase 1 study. Reston, VA: Society of Nuclear neurotensin. Neuroscience 1987;22:525–36. Medicine, 1996. 15 Korc M, Friess H, Kobrin MS, et al. Growth factors in 20 Stolz B, Smith-Jones P, Weckbecker G, et al. Radiotherapy human pancreatic cancer: update on the role of the epider- with Yttrium-90 labeled DOTA-Tyr3-octreotide in tumor mal growth factor receptor. Digestive Surgery 1994;11:147– bearing rodents. J Nucl Med 1997;38(suppl 5):18P. 9. 21 Gully D, Canton M, Goigegrain R, et al. Biochemical and 16 Löwenfels AB, Maisonneuve P, Cavallini G, et al. Pancreati- pharmacological proﬁle of a potent and selective nonpep- tis and the risk of pancreatic cancer. N Engl J Med tide antagonist of the neurotensin receptor. Proc Natl Acad 1993;328:1433–7. Sci USA 1993;90:65–9. http://gut.bmj.com/ on September 27, 2021 by guest. Protected copyright.