Overexpression of heparin-binding EGF-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in both normal pancreatic islets and in pancreatic cancers, but its role in pancreatic physiology and disease is not known. This report examines the effects of HB-EGF overexpression in mouse pancreas. METHODS: Transgenic mice were established using a tissue-specific promoter to express an HB-EGF complementary DNA in pancreatic beta cells, effectively elevating HB-EGF protein 3-fold over endogenous levels. RESULTS: Mice overexpressing HB-EGF in pancreatic islets showed both endocrine and exocrine pancreatic defects. Initially, islets from transgenic mice failed to segregate alpha, beta, delta, and PP cells appropriately within islets, and had impaired separation from ducts and acini. Increased stroma was detected within transgenic islets, expanding with age to cause fibrosis of both endocrine and exocrine compartments. In addition to these structural abnormalities, subsets of transgenic mice developed profound hyperglycemia and/or proliferation of metaplastic ductal epithelium. Both conditions were associated with severe stromal expansion, suggesting a role for islet/stromal interaction in the onset of the pancreatic disease initiated by HB-EGF. Supporting this conclusion, primary mouse fibroblasts adhered to transgenic islets when the 2 tissues were cocultured in vitro, but did not interact with nontransgenic islets. CONCLUSIONS: An elevation in HB-EGF protein in pancreatic islets led to altered interactions among islet cells and among islets, stromal tissues, and ductal epithelium. Many of the observed phenotypes appeared to involve altered cell adhesion. These data support a role for islet factors in the development of both endocrine and exocrine disease.     

Non-islet origin of pancreatic islet cell tumors

The histogenesis of pancreatic islet cell tumors was investigated by morphological identification of putative precursor lesions in pancreatic tissue from patients with multiple endocrine neoplasia type 1 (MEN1), tissue microdissection, and genetic analysis. MEN1 mutation and absence of the MEN1 wild-type allele in different precursor lesions strongly suggest that pancreatic islet cell tumors are derived from the ductal/acinar system but not from pancreatic islet tissue. Pluripotent cells within the exocrine pancreas appear capable of formation into small atypical accumulations of MEN1-deficient cells with both exocrine and endocrine phenotype. The findings suggest presence of multiple developmental aberrations in MEN1 pancreas that potentially serve as precursor material for neuroendocrine tumors.     

Retinoid signaling controls mouse pancreatic exocrine lineage selection through epithelial-mesenchymal interactions

BACKGROUND & AIMS: The early embryonic pancreas gives rise to exocrine (ducts and acini) and endocrine lineages. Control of exocrine differentiation is poorly understood, but may be a critical avenue through which to manipulate pancreatic ductal carcinoma. Retinoids have been shown to change the character of pancreatic ductal cancer cells to a less malignant phenotype. We have shown that 9-cis retinoic acid (9cRA) inhibits acinar differentiation in the developing pancreas, in favor of ducts, and we wanted to determine the role of retinoids in duct versus acinar differentiation. METHODS: We used multiple culture systems for the 11-day embryonic mouse pancreas. RESULTS: Retinoic acid receptor (RAR)-selective agonists mimicked the acinar suppressive effect of 9cRA, suggesting that RAR-RXR heterodimers were critical to ductal differentiation. RARalpha was only expressed in mesenchyme, whereas RXRalpha was expressed in epithelium and mesenchyme. Retinaldehyde dehydrogenase 2, a critical enzyme in retinoid synthesis, was expressed only in pancreatic epithelium. 9cRA did not induce ductal differentiation in the absence of mesenchyme, implicating a requirement for mesenchyme in 9cRA effects. Mesenchymal laminin is necessary for duct differentiation, and retinoids are known to enhance laminin expression. In 9cRA-treated pancreas, immunohistochemistry for laminin showed a strong band of staining around ducts, and blockage of laminin signaling blocked all 9cRA effects. Western blot and RT-PCR of pancreatic mesenchyme showed laminin-beta1 protein and mRNA induction by 9cRA. CONCLUSIONS: Retinoids regulate exocrine lineage selection through epithelial-mesenchymal interactions, mediated through up-regulation of mesenchymal laminin-1.     

Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function

Platelets from a patient with a mild inherited bleeding disorder and abnormal platelet aggregation and secretion show reduced generation of inositol 1,4,5-trisphosphate, mobilization of intracellular Ca2+, and phosphorylation of pleckstrin in response to several G protein mediated agonists, suggesting a possible defect at the level of phospholipase C (PLC) activation (see accompanying report). A procedure was developed that allows quantitation of platelet PLC isozymes. After fractionation of platelet extracts by high-performance liquid chromatography, 7 out of 10 known PLC isoforms were detected by immunoblot analysis. The amount of these isoforms in normal platelets decreased in the order PLC- gamma 2 > PLC-beta 2 > PLC-beta 3 > PLC-beta 1 > PLC-gamma 1 > PLC- delta 1 > PLC-beta 4. Compared with normal platelets, platelets from the patient contained approximately one-third the amount of PLC-beta 2, whereas PLC-beta 4 was increased threefold. These results suggest that the impaired platelet function in the patient in response to multiple G protein mediated agonists is attributable to a deficiency of PLC-beta 2. They document for the first time a specific PLC isozyme deficiency in human platelets and provide an unique opportunity to understand the role of different PLC isozymes in normal platelet function.     

Increased expression and activity of phospholipase C in renal arterioles of young spontaneously hypertensive rats

BACKGROUND: The aims of this study were to document the presence of phospholipase C (PLC) isozymes beta(1), gamma(1), and delta(1) in freshly isolated renal glomeruli and resistance vessels, to compare their expression and activity to that in aorta, and to contrast values between 6-week-old Wistar-Kyoto (WKY) controls and 6-week-old spontaneously hypertensive rats (SHR) during the developmental phase of genetic hypertension. METHODS: Aorta, preglomerular arterioles, and glomeruli were isolated from 6-week-old rats using standard techniques. PLC isozyme protein level and activity were determined with Western blot analysis and by measuring inositol 1, 4, 5-trisphosphate (IP(3)) production, respectively. RESULTS: Immunoblots indicate that all three PLC isozymes examined are detectable in freshly isolated preglomerular arterioles, glomeruli, and aorta. Increased levels of PLC-beta(1), and -delta(1) were found in all tested vascular tissues of SHR v WKY. No strain difference was noted for PLC-gamma(1). The relative abundance for both groups was glomeruli > preglomerular arterioles = aorta. The strain difference in protein expression correlated with increased PLC activity in each vascular bed of SHR. CONCLUSIONS: Protein levels of PLC-beta(1) and -delta(1) and PLC activity are upregulated in the systemic and renal vasculature in 6-week-old SHR, suggesting a role in exaggerated vascular reactivity during the development of genetic hypertension. A more complete understanding of the physiologic roles of PLC isozymes and their contributions to specific aspects of cellular function should advance our understanding of vascular tone/reactivity and hypertrophy/remodeling in normal and hypertensive states.     

Peanut-like 1 (septin 5) gene expression in normal and neoplastic human endocrine pancreas

Peanut-like 1 (PNUTL1) is a septin gene which is expressed at high levels in human brain. There it plays a role in the process of membrane fusion during exocytosis by interacting with syntaxin and synaptophysin. As the secretory apparatus of pancreatic islet cells closely resembles that of neurons, we decided to study the expression of PNUTL1 in the human endocrine pancreas, both in normal islets and in pancreatic endocrine tumors (PETs). Normal pancreatic tissue, purified islets, 11 PETs and two cell lines were used to evaluate the presence of PNUTL1 by RT-PCR and Western blot. The expression of the PNUTL1 protein was also evaluated by immunohistochemistry on normal pancreas, additional 26 PETs, eight pancreatic adenocarcinomas, one mixed endocrine-exocrine pancreatic neoplasm, a specimen of solid papillary pseudomucinous tumor, an adult islet cell hyperplasia and a case of neonatal nesidioblastosis. In addition, a tissue array (LandMark High Density Cancer Tissue MicroArray) comprising 280 various tumor and matched normal specimens was utilized. In PETs, the expression of pancreatic hormones, chromogranin-A, synaptophysin and Ki-67 were also evaluated. In the normal pancreas PNUTL1 expression is almost exclusively confined to the islet cells, weak expression was occasionally seen in some acinar cells, while immunoreactivity was completely absent in the ductal epithelia. PNUTL1 expression is maintained at similar high levels in hyperplastic and neoplastic islet cells, but this did not correlate with any of the clinicopathological data nor with proliferation status in PETs. Weak immunoreactivity was also noted in a proportion of exocrine neoplasms. Our findings describe for the first time the high expression levels of PNUTL1 in human pancreatic endocrine cells that suggests a similar role of this protein in islet cells to that demonstrated in neuronal tissues, and warrants further functional studies of this protein.     

Immunocytochemical studies of pancreatic acinar cells from spontaneously diabetic BB Wistar rats

Amylase in pancreatic tissue from normal and spontaneously diabetic BB Wistar rats was assessed by immunocytochemistry and analyzed by biochemical approach. Amylase immunofluorescence, in pancreatic tissues from control "non-BB" Wistar rats, gave a positive reaction. By electron microscopy, it was detected in the rough endoplasmic reticulum, Golgi apparatus, immature, and mature secretory zymogen granules of the acinar cells. Quantitative evaluations of the intensity of labeling have demonstrated the presence of an increasing gradient which followed precisely the secretory pathway. In the spontaneously diabetic BB Wistar rats, concomitant with the disappearance of insulin containing cells in the islets of Langerhans, no reaction for amylase was found in the acinar cells. Labeling for amylase was markedly reduced in the cellular organelles and the gradient along the secretory pathway was altered. In insulin-treated diabetic rats, labeling for amylase was restored. These results were in agreement with those obtained by the biochemical approach and demonstrated that, in diabetic conditions, secretion of amylase by pancreatic acinar cells is selectively impaired. This alteration, found also in pancreatic tissue from streptozotocin-diabetic rats, demonstrates that the exocrine parenchyma is under the influence of islet hormones and that both the pancreatic exocrine and endocrine tissues are closely related forming an integrated organ.     

New advances in pancreatic cell physiology and pathophysiology

The mammalian pancreas originates from two developing buds on the dorsal and ventral side of the duodenum which fuse and convert into a single mixed gland, composed of exocrine and endocrine cells. In the adult organism, the exocrine pancreas consists of acinar and ductal cells which are organised in a lobular branched tissue architecture and secrete and transport digestive enzymes into the duodenum. Mature endocrine cells, which represent only 1-2% of the pancreatic organ volume, form aggregates of so called islets of Langerhans within the exocrine pancreatic tissue and control glucose homeostasis by secretion of glucagon, insulin and other hormones into the bloodstream. Pancreatitis is the most common and a potentially lethal disorder of the exocrine pancreas with limited therapeutic options. A major obstacle in the development of successful treatment strategies has, until today, been our limited knowledge of the disease pathophysiology. This review will summarise recent advances in our understanding of the physiological mechanisms involved in the early disease processes of the exocrine pancreas.     

Transdifferentiation of human islet cells in a long-term culture

It has been established that ductal cells or precursor cells within the ductal tree of the pancreas can differentiate into islet cells. Although islet cells can also form exocrine cells, it is unclear whether they arise from precursor (stem) cells or from mature endocrine cells by transdifferentiation. Using a defined culture medium and technique for islet purification, for the first time we were able to maintain human islets in culture for more than a year. Multilabeling immunohistochemical and immunoelectron microscopic examination of the islets at different days of culture using islet cell markers (antibodies to hormones, neuron-specific enolase, chromogranin A) and ductal cell markers (cytokeratins 7 and 19, carbonic anhydrase II, DU-PAN2, CA 19-9, and MUC1) revealed that endocrine cells gradually transdifferentiate to ductal, acinar, and intermediary cells. Although islet hormone secretion ceased after day 28 in culture, endocrine cells were still detectable at day 60. However, later, all endocrine and exocrine cells were replaced by undifferentiated cells that expressed neuron-specific enolase, chromogranin A, laminin, vimentin, cytokeratin 7 and 19, alpha-1-antitrypsin, transforming growth factor-alpha, and epidermal growth factor receptor. Our data thus show that, under proper conditions, human islets can be maintained in vitro over a long period and that, in the culture condition, islet cells seem to transdifferentiate to exocrine cells and undifferentiated cells, which may be considered pancreatic precursor (stem) cells.     

Chronic pancreatitis is associated with increased concentrations of epidermal growth factor receptor, transforming growth factor alpha, and phospholipase C gamma.

The epidermal growth factor (EGF) receptor is a transmembrane protein that binds EGF and transforming growth factor alpha (TGF alpha), and that stimulates phospholipase C gamma 1 (PLC gamma 1) activity. In this study the role of the EGF receptor in chronic pancreatitis was studied. By immunohistochemistry, the EGF receptor, TGF alpha, and PLC gamma 1 were found to be expressed at high concentrations in pancreatic ductal and acinar cells from chronic pancreatitis patients. Northern blot analysis showed that, by comparison with normal controls, 19 of 27 chronic pancreatitis tissues exhibited a 5.7-fold increase in EGF receptor mRNA concentrations, and 20 of 27 chronic pancreatitis tissues exhibited a sixfold increase in TGF alpha mRNA concentrations. In situ hybridisation confirmed that overexpression occurred in ductal and acinar cells, and showed that both mRNA moieties colocalised with their respective proteins. These findings suggest that TGF alpha may act through autocrine and paracrine mechanisms to excessively activate the overexpressed EGF receptor in the two major cell types of the exocrine pancreas, thereby contributing to the pathobiology of this disorder.     

Fuel and hormone regulation of phospholipase C beta 1 and delta 1 overexpressed in RINm5F pancreatic beta cells

The mechanism by which glucose and other fuels stimulate phosphoinositide-specific phospholipase C (PLC) in pancreatic islet beta cells is not known. Previous studies have suggested that glucose may couple to PLC beta 1 and PLC delta 1. To determine directly if fuels activate these PLC isozymes, clones stably overexpressing PLC beta 1 or PLC delta 1 were generated in the fuel-sensitive beta cell line RINm5F, and secretagogue regulation of these PLC isoforms was determined. Overexpression of PLC beta 1 or PLC delta 1 significantly increased PLC activity in isolated cell fractions, consistent with overexpression of active PLC isoforms in these clones. In paired experiments, stimulation of inositol phosphate (IP) accumulation by the fuel glyceraldehyde was enhanced in clones overexpressing PLC beta 1, in parallel with the G-protein alpha subunit activator, AlF(4)(-), suggesting a coupling between glyceraldehyde and this PLC isoform. In contrast, overexpression of PLC delta 1 had no effect on glyceraldehyde- or AlF(4)(-)-stimulated IP accumulation. Similarly, IP accumulation stimulated by ionomycin was enhanced in PLC beta 1, but not PLC delta 1 clones, indicating that increases in intracellular free calcium [Ca(2+)](i) can regulate PLC beta 1 but not PLC delta 1 overexpressed in this cell line. Interestingly, [Arg(8)] vasopressin-stimulated, but not carbachol-stimulated, IP accumulation was significantly increased in clones overexpressing either PLC beta 1 or PLC delta 1. These studies illustrate unique pathways coupling diverse secretagogues to specific PLC isoforms in islet beta cells, and demonstrate that glyceraldehyde can activate PLC beta 1 but not PLC delta 1; whereas, vasopressin, but not carbachol, can stimulate either isoform.     

Novel islet, duct, and acinar cell markers defined by monoclonal autoantibodies from prediabetic BB rats

Novel islet cell, duct cell, and acinar cell markers have been identified by monoclonal autoantibodies (Maab) derived from prediabetic BB rats. Spleen cells from two rats that both developed diabetes after splenectomy were fused with mouse myeloma cells. A cellular immunoradiometric assay for differential reactivity toward the surface of two closely related, insulin- and non-insulin-producing rat islet tumor cell lines was used to select and clone several IgM-producing hybridomas. The supernatants were finally characterized by two-color immunofluorescence with islet hormone antisera on frozen sections of human, monkey, and rat pancreas. Maab EB52 stained PP cells, but also few A cells on rat pancreas. Maab CA812 identified a subpopulation of islet D cells on rat, human and monkey pancreas. Although the CA812-reactive antigen and somatostatin were coexpressed in most D cells in adult rat pancreas, only a few islet D cells were stained in the newborn pancreas. The CA812-reactive antigen was not detected in somatostatin-producing cells in the duct epithelium. Maab H37 and IF5 selectively stained acinar cells in rat, human, and monkey pancreas, whereas Maab DA39 identified the rat ductal epithelium including the scattered endocrine cells of the ducts. In summary, B lymphocytes producing autoantibodies to pancreatic endocrine, exocrine, and ductal markers are present in prediabetic BB rats and can be detected by use of transformed pluripotent islet cells as target. Such B lymphocytes can be immortalized to produce monoclonal antibodies to study their role in insulin-dependent diabetes mellitus pathogenesis and to clarify the development of the pancreas.     

Diabetes and pancreatic tumours in transgenic mice expressing Pa × 6

Abstract Aims/hypothesis. Both endocrine and exocrine cells of the pancreas differentiate from epithelial cells of primitive pancreatic ducts, and four types of pancreatic islet cells (alpha, beta, delta, and PP cells) are derived from the common pluripotent precursor cells. Although Pa × 6 is expressed in all islet cells, Pa × 4 is detected only in beta cells. In homozygous Pa × 4-null mice, beta cells are absent, whereas the number of alpha cells is increased. Therefore, we hypothesized that the balance of Pa × 4 and 6 is one of the determinants by which the common progenitor cells differentiate into alpha or beta cells.?Methods. To change this balance, we generated transgenic mice overexpressing Pa × 6 driven by the insulin promoter or the PDX1 promoter.?Results. In both types of transgenic mice, normal development of beta cells was disturbed, resulting in apoptosis of beta cells and diabetes. In Insulin/Pa × 6-Tg mice, beta cells were specifically affected, whereas in PDX/Pa × 6-Tg mice, developmental abnormalities involved the whole pancreas including hypoplasia of the exocrine pancreas. Furthermore, PDX/Pa × 6-Tg mice experienced proliferation of both ductal epithelia and islet cells and subsequent cystic adenoma of the pancreas.?Conclusion/interpretation. These findings suggest that Pa × 6 promotes the growth of ductal epithelia and endocrine progenitor cells and that the suppression of Pa × 6 is necessary for the normal development of beta cells and the exocrine pancreas. [Diabetologia (2000) 43: 332–339] Received: 16 August 1999 and in final revised form: 3 December 1999     

Demonstration of epidermal growth factor binding sites in the adult rat pancreas by light microscopic autoradiography

The distribution of epidermal growth factor (EGF) receptors was studied in the pancreas using light microscopic autoradiography, which was performed at different time intervals (2-60 min) after injecting 125I-labeled EGF intravenously into the adult rat. In the exocrine pancreas, a labeling was found to occur over the pyramidal cells of the acini and cells lining the intercalated ducts. Moreover, substantial binding of EGF to cells of the islets of Langerhans was also revealed. At the 2-min time interval, most silver grains were found at the periphery of the target cells. The localization, as well as the diminution of silver grains over the cytoplasm of these cells, between 7 and 60 min, suggested the internalization and degradation of 125I-labeled EGF. Control experiments indicated that the autoradiography reaction was due to specific interaction of 125I-labeled EGF with its receptor. These results clearly indicate that EGF receptors are present in the acinar cells and the cells of intercalated ducts of the exocrine pancreas, as well as the cells of the endocrine pancreas. Finding that there are EGF binding sites in pancreatic acinar cells supports the physiological role of EGF in the regulation of pancreatic exocrine function. The presence of EGF receptors in cells of the islets of Langerhans suggests that EGF may play a role in the regulation of the endocrine pancreas.     

Betacellulin inhibits amylase and glucagon production and promotes beta cell differentiation in mouse embryonic pancreas

Aims/hypothesis Betacellulin, a member of the epidermal growth factor family, is expressed in the pancreas and is thought to regulate differentiation of beta cells during development. The aim of the present study was to investigate the effects of exogenous betacellulin on the development of the mouse embryonic pancreas. Materials and methods We used an in vitro culture model system based on the isolation and culture of the dorsal embryonic pancreas from day 11.5 embryos. Cultures were treated for up to 10 days with 10 ng/ml betacellulin and then analysed for changes in the expression of pancreatic exocrine, endocrine and ductal markers. Results Pancreases developed in culture and expressed the full complement of exocrine (both acinar and ductal) and endocrine cell types. Betacellulin enhanced branching morphogenesis and the proliferation of mesenchyme, increased Pdx1 and insulin production and inhibited the production of the exocrine cell marker amylase and the endocrine hormone glucagon. Conclusions/interpretation These results suggest betacellulin has distinct and separate effects on exocrine, endocrine and ductal differentiation. In the future, betacellulin could perhaps be utilised to increase the production of beta cells from embryonic pancreatic tissue for therapeutic transplantation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Pancreatic tumor pathogenesis reflects the causative genetic lesion.

Transgenic mice in which c-myc expression is targeted to pancreatic acinar cells develop mixed acinar/ductal pancreatic adenocarcinomas between 2 and 7 months of age. This contrasts with the effect on pancreas of the simian virus 40 tumor antigen or activated ras, which in adult mice causes lesions composed exclusively of acinar-like cells. Furthermore, during an early stage of myc-induced pathology, transformed acinar-derived cells appear within islets, suggesting that islet hormones may influence the progression of these exocrine pancreatic tumors. These findings demonstrate that the initial oncogenic alteration can influence the pattern of subsequent tumor pathogenesis and, given that human exocrine pancreatic tumors are predominantly ductal adenocarcinomas, support the suggestion that transformed acinar cells may contribute to the genesis of this serious disease in man.     

Topographical associations between islet endocrine cells and duct epithelial cells in the adult human pancreas

Objectives  The pancreatic ducts, endocrine islets and exocrine acini are three functionally related components. From birth to adulthood, the islets and ducts are regarded as independent entities. The objective of this study is to investigate the topographical associations between the islet endocrine cells and duct epithelial cells in the adult human pancreas.
Materials and methods  Panels of immunomarkers for the exocrine acinar cells (amylase), duct cells [cytokeratin 19 (CK19)], endocrine cells (chromogranin A, neurone specific enolase, synaptophysin) and islet hormones (glucagon, insulin, somatostatin, pancreatic polypeptide) were applied to sequential pancreatic tissue sections obtained from autopsy specimens of 10-nondiabetic human adults. Double immunofluorescent staining with CK19 and islet hormones was performed to confirm the islet to duct interrelationship.
Results  Sequential sectioning and immunostaining showed that 45% of the 172 islets examined appeared as single endocrine cell units or small clusters of < 10 endocrine cells on at least one plane of section. A topographical association was found between the islet endocrine cells and duct epithelial cells. Topographical associations with CK 19-stained duct cells occurred in 10·9% of the islet insulin-containing β-cells and in 8·9% of the islet glucagon-producing α-cells. The frequency of topographical associations increased toward the more distally located duct systems. The CK19-stained duct cells and amylase-labelled acinar cells were less frequently in association with other islet hormone-producing cells.
Conclusions  Topographical associations between islet endocrine cells and pancreatic duct cells are frequent in adult human pancreas. The islet–duct association suggests possible functional interactions between the two interrelated pancreatic compartments.     

Ductal and acinar differentiation in pancreatic endocrine tumors

Rare pancreatic endocrine tumors consisting of both exocrine and endocrine components have been reported sporadically. We investigated the ductal and acinar differentiation in pancreatic endocrine tumors. In immunohistochemical studies of 28 pancreatic endocrine tumors, staining with anti-carcinoembryonic antigen (CEA) or CA19-9 antibody indicated ductal differentiation, while staining with anti-amylase or anti-trypsin antibody indicated acinar differentiation. K-ras gene mutations and p53 gene alterations also were studied. Ten tumors were immunoreactive for CEA or CA19-9. Five tumors diffusely immunoreactive for CEA or CA19-9, in addition to endocrine markers, were diagnosed as duct–endocrine cell tumors of the pancreas. Two tumors diffusely immunoreactive for CEA or CA19-9 and also for pancreaticogut hormones as well as endocrine markers were diagnosed as duct–acinar–endocrine cell tumors. These tumors showed uniform histologic features and synchronous ductal, acinar, and endocrine differentiation, distinct from the coexisting different cellular populations seen in collision tumors. All tumors were malignant. These duct–endocrine cell tumors or duct–acinar–endocrine cell tumors of the pancreas may be derived from a stem cell that retains the capability of expressing either an exocrine or endocrine phenotype. Only one K-ras gene mutation and no p53 gene alterations were detected in these tumors, which suggests that they constitute an entity with a different origin than ductal carcinomas.