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Peutz-Jeghers Syndrome: Quantitative Study on Enterochromaffin Cells In

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Peutz-Jeghers Syndrome: Quantitative Study on Enterochromaffin Cells In Srp Arh Celok Lek. 2013 Sep-Oct;141(9-10):602-607 DOI: 10.2298/SARH1310602K 602 ОРИГИНАЛНИ РАД / ORIGINAL ARTICLE UDC: 616.34-006.5:575.2 ; 536.5 Peutz-Jeghers Syndrome: Quantitative Study on Enterochromaffin Cells in Hamartomatous Intestine Polyps Miljan Krstić1, Vuka Katić1, Slavica Stojnev1, Dragan Mihailović , Marijola Mojsilović2, Zoran Radovanović3, Vladimir Živković4 1Department of Pathology, Faculty of Medicine, University of Niš, Niš, Serbia; 2Department of Histology, Faculty of Medicine, University of Niš, Niš, Serbia; 3Department of Radiology, Faculty of Medicine, University of Niš, Niš, Serbia; 4Department of Anatomy, Faculty of Medicine, University of Niš, Niš, Serbia SUMMARY Introduction Peutz-Jeghers (PJ) syndrome is a rare familial disorder with the autosomal transmission characterized by multiple intestinal polyps, mucocutaneous pigmentation and increased incidence of various malignancies. Some clinical manifestations of PJ syndrome may be associated with the serotonin secretion from the enterochromaffin cells (EC). Objective Since no data have been reported so far regarding EC cells in PJ polyps, the aim of our study was to quantitatively investigate EC population in hamartomatous intestinal polyps in patients with the PJ syndrome. Methods The samples of surgically removed PJ polyps from family members with the PJ syndrome were collected during 34-year follow-up period. Formalin-fixed paraffin-embedded specimens of twenty-one PJ polyps were stained with HE, AB-PAS, Van Gieson, Fontana-Masson, FIF and Grimelius. For immuno- histochemical analysis, the following antibodies were used: chromogranin A, serotonin, Ki-67, desmin, vimentin and cytokeratin in order to eliminate differential diagnostic possibilities and to confirm diag- nosis of PJ polyps. Results Strong EC cell hyperplasia was observed within the tissue of the investigated polyps. Statistical analysis demonstrated significantly higher content of EC cells in PJ polyps than in the normal ileal mucosa. Conclusion Marked hyperplasia of EC cells within the PJ polyps may be the most important contributor to functional disorders in patients with the PJ syndrome. Keywords: Peutz-Jeghers syndrome; hamartomatous polyp; enterochromaffin cells; chromogranin A INTRODUCTION Since some people with PJS are asymptomat- ic, genetic testing is suitable for confirmation of Peutz-Jeghers syndrome (PJS) is an inherited PJS. Nowadays, the only identifiable mutations cancer syndrome characterized by mucocutane- causing PJS affect the STK11 (serine/threonine ous melanin pigmentation and hamartomatous protein kinase 11 alias LKB1) gene, located gastrointestinal polyposis [1-5]. These patients on chromosome 19p13.3. Although the exact also have an increased risk of the extraintestinal mechanism of action of STK11 gene protein cancer, including tumours of the uterine cervix, products has not been elucidated completely, testis, breast and pancreas [1, 5, 6]. Polyps are these proteins are suggested to play significant the most common in the small intestine, but role in growth inhibition. Mutations in STK11 may occur anywhere in and outside the gas- may represent loss of heterozygosity at the tu- trointestinal tract. They are hamartomatous in mor suppressor gene locus [5, 11, 12]. Recent nature, with the bundles of smooth muscle cells study has strongly suggested that STK11 ge- extending out from the center of the lesion and netic alterations are disease-causing events in becoming progressively thinner as they reach the PJP [11]. polyp surface [5, 6, 7]. Cells of the normal small More than 30 peptide hormone genes ex- bowel mucosa, including goblet, absorptive, en- pressing more than 100 bioactive peptides, docrine and Paneth cells line the crypts and villi including the hormonal messengers, e.g. of the small intestinal PJS polyps. Columnar and monoamines and eicosanoids, have been rec- goblet cells predominate in the surface portion, ognized so far [9, 13, 14]. The abundance of whereas Paneth and endocrine cells lie at the multiple bioactive peptides secreted by the Correspondence to: crypt bases next to the muscularis framework cells interspersed in the gastrointestinal sys- Miljan KRSTIĆ [5-10]. The characteristic prominent muco- tem make the gut the largest endocrine organ Faculty of Medicine cutaneous pigmentation, if present, allows the in the body [15]. The enterochromaffin cells University of Niš diagnosis of asymptomatic patients in familial (EC) constitute the largest endocrine cell pop- Bul. Zorana Djindjića 81, 18000 Niš Serbia cases, although the hamartomatous PJS polyps ulation in the gastrointestinal tract, and were [email protected] constitute the main clinical hallmark [2, 4, 5]. the first gut endocrine cells to be identified [16, Srp Arh Celok Lek. 2013 Sep-Oct;141(9-10):602-607 603 17]. They have an almost exclusive intraepithelial location, and her son was prominently characterized with the secre- resting on the basal lamina and projecting into the gut tory diarrheas accompanied by the abdominal cramping, lumen with their apical portion. The cytoplasm of the EC reddish spots on the face and upper extremities, as well as cells is occupied by a large number of secretory granules, hypotension. which are the storage sites of the secretory products [15, 16, 17]. The main secretory product of EC cells is serot- onin and the EC cells account for more than 90% of all Specimen preparation, histochemical and serotonin synthesized in the body [15]. Minor amounts immunohistochemical staining of peptide hormones, e.g. tachykinins, enkephalins and motilin, may also be synthesized in subsets of EC cells. Selected samples of 21 surgically removed intestinal polyps Secreted serotonin influences adjacent cells by a paracrine of large diameters were fixed in 10% buffered formalde- action or reach target cells via the circulation. hyde, routinely processed and embedded in paraffin. Ten Clinical manifestations of PJS include abdominal dis- samples of normal ileal mucosa obtained during the en- comfort and pain, intestinal bleeding, anemia, diarrhea, doscopy served as control. Four µm thick tissue sections intussusception, obstruction and hypotension [1-5]. Some were stained with haematoxylin-eosin (HE) stain and Al- of these symptoms may be explained by the excessive se- cian blue pH 2.5 - Periodic acid Schiff (AB-PAS) methods. rotonin secretion. Following the histological confirmation of hamartomatous To the best of our knowledge, no data have been pub- polyps, the sections were further stained with histochemi- lished so far about the number and micromorphology cal argentaffin Fontana-Masson and formaldehyde induced of EC cells inside the polyps of PJS. In addition, domi- fluorescence (FIF) methods, detecting the serotonin, the nant symptoms of patients included in this study were main deposit within the EC cells. In addition, application abdominal cramping, flushing and secretory diarrhea af- of Grimelius technique localized neuroendocrine cells ter food intake, which may all be potentially associated through the impregnation of their cytoplasmic granules with the excessive serotonin discharge from EC cells. We with the silver salts. have hypothesized that hamartomatous polyps found in Immunohistochemical analysis was performed us- PJS patients may contain increased number of active EC ing the monoclonal antibodies against chromogranin A cells. (dilution 1:200), serotonin (1:50), Ki-67 (dilution 1:300), desmin (dilution 1:100), vimentin (dilution 1:100) and cy- tokeratin (dilution 1:50), all provided by Dako, Glostrup, OBJECTIVE Denmark) and a standard avidin–biotin immunoperoxi- dase complex detection system according to the manu- The aim of the present study was to investigate EC popula- facturer’s protocol (Dako LSAB2R system-HRP). Briefly, tion using the morphological and quantitative methodol- 4 μm tissue sections were deparaffinized and rehydrated. ogy in hamartomatous PJS associated polyps. Antigen retrieval was performed in 0.1 m citrate buffer (pH 6.0) in the microwave oven. Endogenous peroxidase activity was quenched with 0.3% hydrogen peroxide in METHODS methanol. After applying primary antibody, the slides were incubated for 60 minutes at room temperature, followed Patients by the extensive washes with the phosphate-buffered sa- line. Subsequently, the sections were incubated with the This is a report of a three-generation family with the PJS, secondary biotinylated antibody and with the streptavidin⁄ which has been followed up for 34-year period. The fa- avidin–biotin–peroxidase complex solution. Staining was ther presented solely with the mucocutaneous melanin developed using the liquid 3,3’-diaminobenzidine (DAB) pigmentation, but gastrointestinal polyposis had not been substrate kit and counterstained with Mayer’s haematoxy- observed. He died from consequences of the liver cirrhosis lin. Negative controls were carried out by omitting the at the age of 54. The older daughter had neither melanin primary antibodies. pigmentation nor intestinal polyposis, but she died from breast cancer at 34 years of age. The diagnosis of PJS has not been established in her two children. The younger Quantitative evaluation of EC cells and daughter and her son had typical facial pigmentations statistical analysis and PJS intestinal polyposis. She was 14 years old when she was operated on for removal of jejunal hamartoma- The number of argentaffine and serotonin positive EC cells tous polyp that caused intussusception. She died from non per 10 high-power fields (HPF), with
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