Ductal Precursor Research Articles

PanIN or IPMN? Redefining Lesion Size in 3 Dimensions.

Pancreatic ductal adenocarcinoma (PDAC) develops from 2 known precursor lesions: a majority (∼85%) develops from pancreatic intraepithelial neoplasia (PanIN), and a minority develops from intraductal papillary mucinous neoplasms (IPMNs). Clinical classification of PanIN and IPMN relies on a combination of low-resolution, 3-dimensional (D) imaging (computed tomography, CT), and high-resolution, 2D imaging (histology). The definitions of PanIN and IPMN currently rely heavily on size. IPMNs are defined as macroscopic: generally >1.0cm and visible in CT, and PanINs are defined as microscopic: generally <0.5cm and not identifiable in CT. As 2D evaluation fails to take into account 3D structures, we hypothesized that this classification would fail in evaluation of high-resolution, 3D images. To characterize the size and prevalence of PanINs in 3D, 47 thick slabs of pancreas were harvested from grossly normal areas of pancreatic resections, excluding samples from individuals with a diagnosis of an IPMN. All patients but one underwent preoperative CT scans. Through construction of cellular resolution 3D maps, we identified >1400 ductal precursor lesions that met the 2D histologic size criteria of PanINs. We show that, when 3D space is considered, 25 of these lesions can be digitally sectioned to meet the 2D histologic size criterion of IPMN. Re-evaluation of the preoperative CT images of individuals found to possess these large precursor lesions showed that nearly half are visible on imaging. These findings demonstrate that the clinical classification of PanIN and IPMN fails in evaluation of high-resolution, 3D images, emphasizing the need for re-evaluation of classification guidelines that place significant weight on 2D assessment of 3D structures.

Abstract I16: KRAS and environmental signals regulate MYC to drive lineage plasticity, pancreas cancer progression, and therapeutic resistance

Abstract Cellular plasticity in cancer is emerging as a biomarker of aggressive behavior and an important therapeutic resistance mechanism. The ability of cancer cells to switch lineage and differentiation states provides survival advantages in adverse tumor microenvironments and under therapeutic stress. Pancreatic ductal adenocarcinoma (PDAc) tumors display a dense immunosuppressive microenvironment and a high degree of cellular heterogeneity contributing to poor outcomes. Normally, pancreatic cellular plasticity allows for acinar-to-ductal metaplasia (ADM) in response to inflammation or injury as a reversible process and important mechanism for regeneration of functional tissue. However, mutant KRAS, which is a hallmark of over 90% of PDAc, drives ADM that progresses to ductal precursor lesions (PanINs) and PDAc. We have demonstrated that PDAc can further develop ductal to neuroendocrine lineage plasticity, and that this confers therapeutic resistance and is a biomarker of poor outcomes in patients. This discovery is consistent with other reports in prostate, lung, and breast cancer where neuroendocrine differentiation confers an aggressive, therapeutically resistant state. Mechanistically, we have demonstrated a role for the MYC oncoprotein in PDAc ductal – NE lineage plasticity. MYC is functionally activated by post-translational modifications to a conserved motif, Serine 62-Proline 63, in the MYC N-terminal transactivation domain. In PDAc, KRAS mutations and environmental signaling stimulate MYC Serine 62 phosphorylation and PIN1-mediated Proline 63 isomerization, which promote MYC DNA binding and regulation of pro-oncogenic target genes. We have also observed that cancer-associated fibroblasts (CAFs) can promote PDAc lineage plasticity and that the proline isomerase PIN1 involved in post-translational activation of MYC is also critical for fibroblast activation. Thus, PIN1 has a tumor cell intrinsic and stromal cell function, where loss of PIN1 in pancreatic stellate cells suppresses their activation to CAFs and their support of tumor growth. To understand crosstalk between the tumor microenvironment and cancer cell plasticity, and to develop effective therapeutic strategies that overcome this survival mechanism, we are using 3-D bioprinting to model tumor-stromal interactions and pursuing novel drugs that target post-translational regulation of MYC. Citation Format: Ellen Langer, Yulong Su, Meghan Joly, Brittany Allen-Petersen, Amy Farrell, Patrick Worth, Dan Liefwalker, Jason Link, Brett Sheppard, Rosalie Sears. KRAS and environmental signals regulate MYC to drive lineage plasticity, pancreas cancer progression, and therapeutic resistance [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I16.

Functional Maturation and In Vitro Differentiation of Neonatal Porcine Islet Grafts

There is a strong rationale to pursue the use of neonatal porcine islets (NPIs) as an unlimited source of islets for clinical xenotransplantation. Because NPIs are composed of immature insulin producing beta (ß) cells and ductal precursor cells, they provide an ideal model to examine culture conditions to enhance ß cell proliferation and/or ß cell neoformation from ductal cells. In an attempt to optimize the potential of NPIs as a source of ß cell grafts, we used an in vitro differentiation protocol and measured its effect on the functional maturation and differentiation of NPIs. Pancreata from 1- to 3-day-old neonatal pigs were digested and cultured in standard Ham's F10 media for 5 days. Each independent preparation was then further cultured in Dulbecco's modified Eagle medium nutrient mixture-F12 differentiation media containing growth factors added in a stepwise fashion, or cultured in control Ham's F10 media. After 20 days in culture, islets were assessed for insulin secretory capacity, cellular composition, gene expression, and metabolic activity after transplantation in immunodeficient mice with diabetes. Compared with control islets, differentiated islets exhibited a significantly higher proportion of endocrine cells, proliferating cell nuclear antigen double positive ß cells, and an enhanced glucose-stimulated insulin secretory activity. Mice transplanted with differentiated islets had significantly lower blood glucose values at weeks 18 and 20 compared with nondifferentiated controls and were shown to be more glucose tolerant. Culturing NPIs in a 20-day stepwise differentiation media increases the proportion of endocrine cells and augments both in vitro and in vivo function of the islets.

Abstract A40: Gene expression profiling of pancreatic cancer precursors directly from formalin fixed paraffin embedded (FFPE) tissue without nucleic acid extraction

Abstract Gene expression analysis of pancreatic intraepithelial neoplasia (PanINs), the pancreatic ductal adenocarcinoma precursor, has been challenging because extracting and purifying RNA from pancreatic tissue is difficult. Since the pancreas is rich in ribonucleases and PanINs are typically very small lesions, attempts to purify RNA of sufficient quality and quantity for microarray or RNAseq are usually unsuccessful. This is especially true if the starting material is formalin fixed paraffin embedded tissue, where the fixation process introduces further degradation of the RNA. We sought to solve this problem of PanIN gene expression profiling by using the HTG Edgeseq instrument platform. This instrument is designed to work with small FFPE specimens and perform RNA profiling even when RNA is partially degraded by employing a novel extraction free hybridization approach. In our pilot study, we show that the HTG Edgeseq can be used to profile ~2500 genes from a cohort of microdissected low and high grade PanIN from FFPE of human pancreatic adenocarcinoma resections. We speculate that most of these PanIN lesions would not have been amenable to other gene expression methodologies and that the PanIN data from the HTG Edgeseq will contribute to our understanding of the molecular pathways that underlie pancreatic cancer. Citation Format: Sheila Smiley, Celia Marginean, Bryan Lo.{Authors}. Gene expression profiling of pancreatic cancer precursors directly from formalin fixed paraffin embedded (FFPE) tissue without nucleic acid extraction. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A40.

Mutational spectrum of intraepithelial neoplasia in pancreatic heterotopia

Heterotopic pancreatic parenchyma recapitulates the normal pancreas in extrapancreatic locations and, on rare occasions, can even give rise to pancreatic adenocarcinoma. The genetic signatures of pancreatic adenocarcinoma and its precursor lesions are well characterized. We explored the genetic alterations in precursor lesions (intraductal papillary mucinous neoplasms [IPMN], pancreatic intraepithelial neoplasia [PanIN]) in patients with pancreatic heterotopias but without concomitant pancreatic ductal adenocarcinomas. This allowed us to determine whether the stereotypical dysplasia--infiltrating carcinoma sequence also occurs in these extrapancreatic foci. Seven cases of heterotopic pancreas with ductal precursor lesions were identified. These included 2 IPMNs with focal high-grade dysplasia and 5 PanINs with low- to moderate-grade dysplasia (PanIN grades 1-2). Neoplastic epithelium was microdissected and genomic DNA was extracted. Sequencing of commonly mutated hotspots (KRAS, TP53, CDKN2A, SMAD4, BRAF, and GNAS) in pancreatic ductal adenocarcinoma and its precursor lesions was performed. Both IPMNs were found to have KRAS codon 12 mutations. The identification of KRAS mutations suggests a genetic pathway shared with IPMN of the pancreas. No mutations were identified in our heterotopic PanINs. One of the possible mechanisms for the development of dysplasia in these lesions is field effect. At the time of these resections, there was no clinical or pathologic evidence of a prior or concomitant pancreatic lesion. However, a clinically undetectable lesion is theoretically possible. Therefore, although a field effect cannot be excluded, there was no evidence for it in this study.

Abstract PR01: Downregulation of PTF1A is a crucial and rate-limiting step in pancreatic cancer initiation

Abstract The goal of this study is to understand the endogenous mechanisms limiting the ability of oncogenic KRAS to initiate pancreatic tumorigenesis. Recently, our lab and others discovered that although pancreatic ductal adenocarcinoma (PDAC) shares phenotypic characteristics with normal pancreatic duct cells, it actually originates from mature exocrine acinar cells. In analyzing the initiating events of PDAC, we have identified the acinar cell transcription factor, PTF1A, as a critical factor inhibiting KRAS from reprogramming acinar cells to ductal tumor precursors. Binding sites for PTF1A are found upstream of essentially all acinar differentiation products and, importantly, PTF1A maintains its own expression through a positive autoregulatory loop. Given the central role of PTF1A in regulating acinar-specific gene expression, we wanted to determine if loss of this transcription factor plays a role in pancreatic intraepithelial neoplasia (PanIN) formation. Importantly, we find that PTF1A is downregulated in early (stage 1) PanINs of humans and mouse models, suggesting that loss of PTF1A expression could be a rate-limiting step in pancreatic cancer initiation. To test whether PTF1A loss is a functionally important step in PanIN development, we deleted the Ptf1a gene in mice using an acinar-specific inducible Cre (Ptf1a cKO). These studies confirm that PTF1A downregulation is an essential step in PanIN formation, as Cre-mediated deletion of acinar cell Ptf1a in the presence of oncogenic KRAS (KrasG12D) acutely and dramatically accelerates PanIN formation. Widespread PanINs are observed within two weeks of combined KRAS activation/Ptf1a deletion, by which time KRAS alone has induced few or no PanINs. At six weeks post-recombination, the combination of KRAS/Ptf1a cKO induces &amp;gt;15-fold more PanINs than KRAS alone. Additionally, we have found that Ptf1a heterozygosity sensitizes pancreatic cells to KRAS-mediated PanIN formation, indicating that transformation requires reduction of PTF1A activity below a critical autoregulatory threshold needed to maintain acinar identity. Based on these current studies, we hypothesize the existence of mutual antagonism between acinar differentiation, which is maintained through PTF1A, and transformation driven by KRAS. KRAS normally transforms acinar cells only weakly, but both endogenous and mutant KRAS activity can be increased by inflammation, such as that stimulated by caerulein-induced pancreatitis. As KRAS is potentiated by Ptf1a deletion, we asked whether inflammation would be sufficient to transform Ptf1a-deficient acinar cells even without mutant KRAS. In the absence of pancreatitis, Ptf1a deletion alone has relatively little short-term effect: Ptf1a-deficient acinar cells eventually lose their differentiated phenotype and express ductal markers, but they do not undergo hyperplasia or dysplasia. After subjecting Ptf1a cKO mice to caerulein-induced pancreatitis, however, we observed widespread acinar-to-ductal metaplasia, histological structures that resemble PanINs and stain positively with Alcian blue, and loss of amylase staining throughout the pancreas. Our current data indicate that PTF1A has a critical role in guarding against genetic (oncogenic KRAS) and environmental (pancreatitis) insults, and demonstrate that loss of Ptf1a expression is the critical event of acinar cell transformation. Going forward, we will use gene expression profiling and ChIP-seq analysis to identify PTF1A target genes responsible for inhibiting KRAS, and to characterize the epigenetic processes by which KRAS and inflammation reprogram acinar cells to a PanIN phenotype. This abstract is also presented as Poster A3. Citation Format: Nathan Michael Krah, Mary P. Bronner, Christopher V. Wright, L. Charles Murtaugh. Downregulation of PTF1A is a crucial and rate-limiting step in pancreatic cancer initiation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr PR01.

Periostin conditions the matrix to generate a niche for islet regeneration.

Islet replacement via the Edmonton Protocol offers proof of concept that transient insulin independence can be achieved in patients with diabetes (1–4). However, the need for life-long immunosuppression, an extreme shortage of human islets, along with graft failure due to rejection and continued autoimmunity, limits the widespread application of this approach. Although recent progress has been made in the derivation of “ -like” cells from human pluripotent stem cell sources (embryonic stem cells or induced pluripotent stem cells) (5–10), inducing the replication of surviving -cells in situ (11, 12) and/or stimulating new islet formation from progenitor cells within the pancreas (islet neogenesis) (13–16) represents the most direct way to increase -cell mass. Studies in both rodents and humans suggest endogenous islet repair mechanisms may remain intact during diabetes and can be activated by cellor pharmacologically-mediated stimuli. Evidence of ongoing -cell regeneration has been observed in type 1 diabetes (T1D) patients at early onset (17) or many years after diagnosis (18). Indeed, recent analysis of T1D patients with disease duration of more than 50 years (medalists) has uncovered sustained c-peptide and proliferating -cells within the pancreas in the face of ongoing autoimmunity (19). Moreover, the increase in -cell mass in response to pregnancy suggests that the human endocrine pancreas has the capacity to regenerate if we can understand how to “tip the balance” in favor of islet regeneration vs destruction during diabetes. Interestingly, increased -cell replication was first suggested as the mechanism for increased -cell mass during pregnancy, but a recent autopsy study of human pancreases during pregnancy showed an increased proportion of small islets and increased number of insulin cells in the ducts but no change in -cell replication or apoptosis frequency (20). Nonetheless, novel strategies to optimize islet regeneration, either by stimulating -cell replication or by harnessing endogenous islet neogenic processes within the diabetic pancreas, remain under intense investigation for the treatment of diabetes. In initial publications documenting endogenous islet regeneration by stem cells, transplantation of bone marrow-derived c-kit cells reduced hyperglycemia in mice with streptozotocin (STZ)-induced -cell deletion (21). Importantly, transplanted stem cells did not acquire insulin expression as previously thought. In contrast, donor cells surrounded regenerating islets or engrafted in ductal regions and stimulated proliferation within recipient islets via undetermined paracrine activities (21). Subsequently, other preclinical and early clinical reports have shown that both human hematopoietic (22–26) or multipotent stromal cell (25–28), also known as mesenchymal stem cell (MSC) lineages, can enhance islet regeneration. Furthermore, islet regeneration and immune protection may be achieved by coinfusion of murine hematopoietic cells with MSC in autoimmune non-obese diabetic mice (29). Although this concept termed “stem cell-stimulated islet regeneration” has emerged as a central process for pancreas repair, either by stimulating -cell proliferation (11, 24– 26) or by initiating new islet formation from ductal or islet-derived precursors (14–16, 25, 26), the molecular signaling mechanisms by which distinct stem and progenitor cell types mediate islet recovery are not well understood. More importantly, recent controversy of the role of betatrophin in islet regeneration (30, 31) has underscored

Evaluation of antidiabetic antihyperlipidemic and pancreatic regeneration, potential of aerial parts of Clitoria ternatea

The aim of the present study was to investigate the pancreatic regeneration potential of of diferent fractions of the ethanol extract Clitoria ternatea L., Fabaceae. The antidiabetic and antihyperlipidemic potential was evaluated in streptozotocin-induced diabetic rats and correlated with its in vivo and in vitro antioxidant activity. The extract and its fractions were initially screened for acute and sub-chronic antidiabetic activity in the dose range of 100–200mg/kg. The most potent extract and fractions were further evaluated for pancreatic β-cells regeneration activity along with antioxidant and antihyperlipidemic activity. The polyphenolic, flavonoid and flavanone contents were assessed and correlated with its antidiabetic activity. The most significant pancreatic regeneration activity, antidiabetic and antihyperlipidemic activity and was shown by ethanol extract and butanol soluble fraction at a dose level of 200mg/kg, while rutin was found to be least potent. In conclusion, pancreatic regeneration studies of ethanol extract treated rats show nesidioblastosis. It is also suggested that the factors causing regeneration are present within the pancreas. The newly generated islets may have formed from the ductal precursor cells and reduced oxidative stress helps in restoration of β-cell function.

Ready, Set, Go: The EGF Receptor at the Pancreatic Cancer Starting Line

Acinar-to-ductal metaplasia (ADM) results from pancreatic injury or KRAS activation, and is an early step in pancreatic cancer progression. In this issue of Cancer Cell, Ardito and colleagues and Navas and colleagues demonstrate that ADM- and KRAS-driven pancreatic cancer require EGFR signaling, revealing a mechanism for developmental reprogramming that primes tumorigenesis.

Severely fibrotic pancreases from young patients with chronic pancreatitis: evidence for a ductal origin of islet neogenesis

While it is known that islet cell mass increases considerably after birth, general uncertainty surrounds the source of new beta cells in humans. Chronic pancreatitis (CP) presents a natural injury model for studying postnatal beta-cell regeneration in the human pancreas. In this report, we present histological evidence from human CP pancreases to support the theory that islet neogenesis can occur from ductal precursor cells after birth. Three young patients (ages 16, 12, and 28years) underwent total pancreatectomy for the management of CP followed by islet isolation and autologous transplantation to prevent or minimize postsurgical diabetes. In all cases, the pancreases had extensive fibrosis, a rock-like consistency, and calcifications in the ducts. During islet isolations, we observed the unusual release of islets with many ductal fragments. In histopathological evaluation of these pancreases, solid cords of cells sometimes formed islet like structures intraductally or extending from ductal structures. Immunofluorescence staining for chromogranin, insulin, proinsulin, PDX1, glucagon, and cytokeratins confirmed these structures to be composed of chromogranin-positive endocrine cells which included both β-cells and α-cells. Labeling for Ki67 to demonstrate mitotic activity showed frequent labeling of duct epithelial cells and of some periductal cells. Using insulin and wide-spectrum cytokeratin double immunofluorescent labeling, we found insulin-positive cells to be present within the ductal lumens, among the cytokeratin-positive ductal epithelium, and extending from the ductal epithelium into surrounding connective tissues, providing evidence for a ductal origin of islet neogenesis.

Inactivation of Brca2 cooperates with Trp53R172H to induce invasive pancreatic ductal adenocarcinomas in mice

An inactivating germline mutation in BRCA2 is the most common known genetic basis for familial pancreatic cancer (FPC), accounting for 5-10% of inherited cases. A genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) arising on the backdrop of Brca2 deficiency is likely to elucidate valuable diagnostic and therapeutic insights for FPC. Both Brca2 alleles were conditionally deleted during development within the pancreatic epithelium by generating Pdx1-Cre; Brca2f/f (“CB”) mice; in addition, triple transgenic Pdx1-Cre; Brca2f/f; LSL-Trp53R172H (“CBP”) mice were generated, in order to determine the impact of p53 deregulation on Brca2-deficient carcinogenesis. Both “CB” and “CBP” mice developed non-invasive ductal precursor lesions (murine pancreatic intraepithelial neoplasia or mPanIN), although these were observed at an earlier time point (5 versus 8 months) and with higher prevalence in “CBP” mice. A minority of “CB” mice (15%) developed invasive and metastatic PDAC at a latency of 15 months or greater; in contrast, “CBP” mice of comparable age uniformly developed PDAC with variable histological features. Mortality in the absence of neoplasia in "CB" and "CBP" mice was associated with profound loss of pancreatic parenchyma, consistent with progressive elimination of Brca2-deficient cells. Widespread DNA damage, as evidenced by overexpression of the phosphorylated histone H2AXSer139, was observed in the non-neoplastic exocrine pancreas, as well as in the mPanIN and PDAC lesions of Brca2-deficient mice, independent of p53 status. Loss of Brca2 function predisposes the exocrine pancreas to profound DNA damage, and the frequency of invasive neoplasia is accentuated by the concomitant deregulation of p53.See commentary:Brca2 deficiency and Trp53 deregulation in pancreatic cancer: Implications for therapeutic targeting

Inactivation of BRCA2 cooperates with Trp53R172H to induce invasive pancreatic ductal adenocarcinomas in mice: A mouse model of familial pancreatic cancer.

4128 Background: Inactivating germline mutations in BRCA2 represent the most common known genetic basis for familial pancreatic cancer (FPC), accounting for approximately 5-10% of inherited cases. A genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC) arising on the backdrop of BRCA2 deficiency is likely to elucidate valuable diagnostic and therapeutic insights for FPC. Methods: Both BRCA2 alleles were conditionally deleted during development within the pancreatic epithelium by generating Pdx1-Cre; BRCA2f/f (“CB”) mice; in addition, triple transgenic Pdx1-Cre; BRCA2f/f; LSL-Trp53R172H (“CBP”) mice were generated, in order to determine the impact of p53 deregulation on BRCA2-deficient carcinogenesis. Results: Both “CB” and “CBP” mice developed non-invasive ductal precursor lesions (murine pancreatic intraepithelial neoplasia or mPanIN), although these were observed at an earlier time point (5 versus 8 months) and with higher prevalence in “CBP” mice. A minority of “CB” mice (15%) developed invasive and metastatic PDAC at a latency of 15 months or greater; in contrast, “CBP” mice of comparable age uniformly developed PDAC with variable histological features. Mortality in the absence of neoplasia in “CB” and “CBP” mice was associated with profound loss of pancreatic parenchyma, consistent with progressive elimination of BRCA2-deficient cells. Widespread DNA damage, as evidenced by overexpression of the phosphorylated histone H2AXSer139, was observed in the non-neoplastic exocrine pancreas, as well as in the mPanIN and PDAC lesions of BRCA2-deficient mice, independent of p53 status. Conclusions: Loss of BRCA2 function predisposes the exocrine pancreas to profound DNA damage and is sufficient to induce mPanIN precursor lesions as well as fully invasive cancers in a subset of cases. The frequency of invasive neoplasia is accentuated by the concomitant deregulation of p53.

Lineage Tracing Evidence for Transdifferentiation of Acinar to Duct Cells and Plasticity of Human Pancreas

Animal studies have indicated that pancreatic exocrine acinar cells have phenotypic plasticity. In rodents, acinar cells can differentiate into ductal precursors that can be converted to pancreatic ductal adenocarcinoma or insulin-producing endocrine cells. However, little is known about human acinar cell plasticity. We developed nongenetic and genetic lineage tracing methods to study the fate of human acinar cells in culture. Human exocrine tissue was obtained from organ donors, dissociated, and cultured. Cell proliferation and survival were measured, and cell phenotypes were analyzed by immunocytochemistry. Nongenetic tracing methods were developed based on selective binding and uptake by acinar cells of a labeled lectin (Ulex europaeus agglutinin 1). Genetic tracing methods were developed based on adenoviral introduction of a Cre-lox reporter system, controlled by the amylase promoter. Both tracing methods showed that human acinar cells can transdifferentiate into cells that express specific ductal markers, such as cytokeratin 19, hepatocyte nuclear factor 1β, SOX9, CD133, carbonic anhydrase II, and cystic fibrosis transmembrane conductance regulator. Within 1 week of culture, all surviving acinar cells had acquired a ductal phenotype. This transdifferentiation was decreased by inhibiting mitogen-activated protein kinase signaling. Human acinar cells have plasticity similar to that described in rodent cells. These results might be used to develop therapeutic strategies for patients with diabetes or pancreatic cancer.

6604 Exploring the role of CA 19–9 level as a predictor of survival in patients with advanced pancreatic adenocarcinoma

Heterotopic pancreatic parenchyma recapitulates the normal pancreas in extrapancreatic locations and, on rare occasions, can even give rise to pancreatic adenocarcinoma. The genetic signatures of pancreatic adenocarcinoma and its precursor lesions are well characterized. We explored the genetic alterations in precursor lesions (intraductal papillary mucinous neoplasms [IPMN], pancreatic intraepithelial neoplasia [PanIN]) in patients with pancreatic heterotopias but without concomitant pancreatic ductal adenocarcinomas. This allowed us to determine whether the stereotypical dysplasia--infiltrating carcinoma sequence also occurs in these extrapancreatic foci. Seven cases of heterotopic pancreas with ductal precursor lesions were identified. These included 2 IPMNs with focal high-grade dysplasia and 5 PanINs with low- to moderate-grade dysplasia (PanIN grades 1-2). Neoplastic epithelium was microdissected and genomic DNA was extracted. Sequencing of commonly mutated hotspots (KRAS, TP53, CDKN2A, SMAD4, BRAF, and GNAS) in pancreatic ductal adenocarcinoma and its precursor lesions was performed. Both IPMNs were found to have KRAS codon 12 mutations. The identification of KRAS mutations suggests a genetic pathway shared with IPMN of the pancreas. No mutations were identified in our heterotopic PanINs. One of the possible mechanisms for the development of dysplasia in these lesions is field effect. At the time of these resections, there was no clinical or pathologic evidence of a prior or concomitant pancreatic lesion. However, a clinically undetectable lesion is theoretically possible. Therefore, although a field effect cannot be excluded, there was no evidence for it in this study.

Heterogeneity in Mitotic Activity and Telomere Length Implies an Important Role of Young Islets in the Maintenance of Islet Mass in the Adult Pancreas

Understanding the mechanisms of beta-cell dynamics in postnatal animals is central to cure diabetes. A major obstacle in evaluating the status of pancreatic cells is the lack of surface markers. Here we performed quantitative measurements of two internal markers to follow the developmental history of islets. One marker, cell-cycle activity, was established by measuring expression of Ki67 and the incorporation of 5-bromodeoxyuridine. The other marker, the aging process, was delineated by the determination of telomere length. Moreover, islet neogenesis, possibly from ductal precursors, was monitored by pancreatic duct labeling with an enhanced green fluorescence protein (EGFP) transgene. We found that islets from younger animals, on average, expressed higher Ki67 transcripts, displayed elevated 5-bromodeoxyuridine incorporation, and had longer telomeres. However, significant heterogeneity of these parameters was observed among islets from the same mouse. In contrast, the levels of proinsulin-1 transcripts in islets of different ages did not change significantly. Moreover, mitotic activities correlated significantly with telomere lengths of individual islets. Lastly, after 5.5 d pancreatic duct labeling, a few EGFP-positive islets could be identified in neonatal but not from adult pancreases. Compared with unlabeled control islets, EGFP-positive islets had higher mitotic activities and longer telomeres. The results suggest that islets are born at different time points during the embryonic and neonatal stages and imply that young islets might play an important role in the maintenance of islet mass in the adult pancreas.

β-Cell Regeneration

It is now appreciated that both type 1 and type 2 diabetes are diseases of β-cell insufficiency. As such, there is a good deal of interest in mechanisms of β-cell regeneration that hold some therapeutic potential (1). This could apply to increasing numbers of remaining endogenous β-cells in vivo, or in vitro expansion of β-cells derived from isolated human islets, to be subsequently used for β-cell replacement therapy. Regeneration of β-cells may occur by several means, including replication of existing β-cells, generation of new β-cells from pancreatic ductal precursor cells (by a relatively undefined mechanism known as neogenesis), and perhaps transdifferentiation of pancreatic exocrine cells into endocrine β-cells. It has also been suggested that some pancreatic cell types, including β-cells, can undergo a reversible epithelial-mesenchymal transition (EMT) to enable a marked increase in cell numbers (2,3). However, in this issue of Diabetes , Chase et al. (4), by use of lineage-tracing, demonstrate that EMT is unlikely to be a major source of β-cell regeneration. What is EMT? Epithelial cells have a consistent and polarized morphology that are found in co-adherent organized multicellular assemblies, often in single-cell layers. In contrast, mesenchymal cells are of irregular shape, often with a spindle-like form characteristic of …

The impact of the mTOR inhibitor sirolimus on the proliferation and function of pancreatic islets and ductal cells

The Edmonton Protocol for islet transplantation has provided hope for type 1 diabetic patients. However, this protocol requires lifelong immunosuppression, specifically sirolimus, a cellular antiproliferate. The effect of sirolimus on human pancreatic ductal cells (HDCs) is not known. This may be important since HDCs are believed to be islet precursors. Since neonatal porcine islets (NPIs), which contain many ductal precursor cells, could be a potential clinical source of islets, we also tested the effects of sirolimus on this tissue. HDCs (n=4), NPIs (n=9) and human islets (n=5) were cultured with and without sirolimus (20 ng/ml) for 6 days. HDCs and NPIs cultured with sirolimus showed a 50 and 28% decrease, respectively, in cell number relative to control (p<0.05). Control cultures expanded 1.65- and 2.44-fold relative to time 0. Decreases in cell number of sirolimus-treated HDCs were not due to apoptosis as measured by TUNEL staining. No functional effects on human islets or NPIs were observed following static incubation with high glucose. Treatment of syngeneically transplanted and naïve BALC/c mice with sirolimus resulted in altered OGTT profiles with prolonged elevation of hyperglycaemia and weight gain. There was no difference in graft and organ insulin content between treatment groups. Our results indicate that sirolimus decreases ductal cell numbers in culture and alters glucose-stimulated insulin secretion in vivo. The administration of sirolimus to islet transplant recipients is likely to impair graft function as a result of decreasing ductal neogenesis and induction of insulin resistance.

Distinct progression pathways involving the dysfunction of DUSP6/MKP-3 in pancreatic intraepithelial neoplasia and intraductal papillary-mucinous neoplasms of the pancreas

DUSP6/MKP-3 is identified as a candidate tumor suppressor gene for pancreatic cancer. The aim of this study was to elucidate the roles of DUSP6 in the pancreatic carcinogenesis through the pancreatic intraepithelial neoplasia and/or intraductal papillary-mucinous neoplasms, both of which are considered to be precursor lesions of invasive carcinoma of the pancreas, by comparing with involvements of other major tumor suppressive pathways. Expressions of DUSP6, CDKN2A, TP53, and SMAD4 were investigated by immunohistochemistry in a total of 206 lesions of dysplastic ductal precursors and carcinomas retrieved from 52 pancreata with invasive ductal carcinomas and 51 of those with intraductal papillary-mucinous neoplasms. The intensity of staining was evaluated in lesions at different atypical grades and statistically compared among them. Mutations of KRAS2 were analyzed by methods of the allele-specific oligonucleotide hybridization and nucleotide sequencing. In pancreata with invasive ductal carcinomas, expressions of DUSP6 were abrogated exclusively in the invasive carcinoma cells in contrast to its fairly preserved expressions in pancreatic intraepithelial neoplasia. In pancreata with intraductal papillary-mucinous neoplasms, abrogated expressions of DUSP6 were observed in a relatively small fraction of intraductal adenoma/borderlines and intraductal carcinomas. Most of the intraductal adenoma/borderline lesions with abrogation of DUSP6 harbored mutations of KRAS2. None of the molecules was associated with each other in any grade of lesions. Morphological variations of papillae of the intraductal papillary-mucinous neoplasms were evaluated and analyzed for their associations with abrogations of the molecules, which resulted in finding of no significant associations. Our results suggest that the abrogation of DUSP6 is associated exclusively with progression from pancreatic intraepithelial neoplasia to the invasive ductal carcinoma while it is potentially associated with initiation of intraductal papillary-mucinous neoplasms with mutated KRAS2, which is independent of other major tumor suppressive pathways in both types of neoplasms.

Endocrine pancreatic tissue plasticity in obese humans is associated with cytoplasmic expression of PBX-1 in pancreatic ductal cells

In vivo lineage tracing experiments in mice have recently cast doubt on the potential islet neogenesis from ductal precursors in adult mammals. We examined, in human obesity, a model for pancreatic endocrine tissue plasticity, the gene and protein expression of PBX-1—a transcription factor expressed in regenerating rat ductules and potentially implicated in the pancreatic development, alone or in association with PDX-1. When comparing gene expression, by quantitative real-time RT-PCR, in pancreatic exocrine tissue from obese non-diabetic subjects with increased islet mass, we found that Pbx-1 and Pdx-1 were up-regulated (5.9 ± 1.2 and 2.4 ± 0.6 versus non-obese). Immunohistochemistry confirmed PBX-1 over-expression and its cytoplasmic sequestration in ductal cells of obese subjects, associated with pronounced islet neogenesis (cytokeratin 19/chromogranin A double labeling). cDNA microarray analysis also showed up-regulation of other genes implicated in islet regeneration, including betacellulin, laminin, TGFa, NeuroD1, Pax6, substantiating the role of the islet neogenesis pathway in human obesity.

The islet β-cell

The β-cell is one of four major types of cells present in the islets of Langerhans, which are islands of cells distributed throughout the endocrine pancreas in most mammals. The β-cell synthesizes and secretes the hormone insulin mainly in response to glucose but also in response to several nutrients, hormones and nervous stimuli. In adult rodents the β-cell has a slow mitotic rate. Recent studies provide novel insights into the functions of the β-cell. The presence of functional insulin-like growth factor-1 and insulin receptors and components of their signaling pathway indicate an important role for insulin/IGF-1 signaling in the regulation of β-cell function. Further, the recent discovery of glucokinase (GK) and the ATP-dependent potassium channels on insulin secretory granules, the detection of AMP-protein kinase in the β-cell and the identification of a new β-cell transcription factor, mMafA, are some exciting new areas of research currently underway to further understand the complex pathways that regulate the functions of β-cells. Cell facts • The insulin-secreting β-cell is one of four major types of cells that make up the mammalian pancreatic islet. • The β-cell secretes the hormone insulin, in response to nutrients, hormones and neuronal stimuli, and therefore plays a primary role in the maintenance of glucose homeostasis. • It is estimated that adult humans have approximately 2 million islets, which make up about 2% of the pancreas by weight. In rodents, each islet is composed of 2000–4000 cells of which 70–80% are β-cells, 5% are somatostatin-producing δ-cells and 15–20% are either glucagon-producing α-cells or pancreatic polypeptide-producing PP cells depending on whether the islets are located in the splenic portion of the pancreas (body and tail in humans), which is derived from a dorsal pancreatic bud, or in the duodenal pancreas (formed from the ventral pancreatic bud: the head or uncinate process of the pancreas), respectively [Diabetologia 28 (1985) 528]. A similar pattern may be observed in humans although precise data is lacking. • The replication rate and turnover of β-cells in humans is not precisely known. In rodents, new β-cells can form in adults either by slow replication of existing β-cells or by the formation of new cells from potential ductal precursors or β-cell “stem” cells. The half-life of mouse islet cells has been estimated to be approximately 47 days with the renewal rate of glucagon-producing cells being more rapid than insulin- or somatostatin-producing cells [Pancreas 24 (2002) 153].