Volker Fendrich

Spontaneous induction of murine pancreatic intraepithelial neoplasia (mPanIN) by acinar cell targeting of oncogenic Kras in adult mice.

Pancreatic ductal adenocarcinoma (PDAC) is believed to arise through a multistep model comprised of putative precursor lesions known as pancreatic intraepithelial neoplasia (PanIN). Recent genetically engineered mouse models of PDAC demonstrate a comparable morphologic spectrum of murine PanIN (mPanIN) lesions. The histogenesis of PanIN and PDAC in both mice and men remains controversial. The most faithful genetic models activate an oncogenic KrasG12D knockin allele within the pdx1- or ptf1a/p48-expression domain of the entire pancreatic anlage during development, thus obscuring the putative cell(s)-of-origin from which subsequent mPanIN lesions arise. In our study, activation of this knockin KrasG12D allele in the Elastase- and Mist1-expressing mature acinar compartment of adult mice resulted in the spontaneous induction of mPanIN lesions of all histological grades, although invasive carcinomas per se were not seen. We observed no requirement for concomitant chronic exocrine injury in the induction of mPanIN lesions from the mature acinar cell compartment. The acinar cell derivation of the mPanINs was established through lineage tracing in reporter mice, and by microdissection of lesional tissue demonstrating Cre-mediated recombination events. In contrast to the uniformly penetrant mPanIN phenotype observed following developmental activation of KrasG12D in the Pdx1-expressing progenitor cells, the Pdx1-expressing population in the mature pancreas (predominantly islet β cells) appears to be relatively resistant to the effects of oncogenic Kras. We conclude that in the appropriate genetic context, the differentiated acinar cell compartment in adult mice retains its susceptibility for spontaneous transformation into mPanIN lesions, a finding with potential relevance vis-à-vis the origins of PDAC.

An orally bioavailable small-molecule inhibitor of Hedgehog signaling inhibits tumor initiation and metastasis in pancreatic cancer

Recent evidence suggests that blockade of aberrant Hedgehog signaling can be exploited as a therapeutic strategy for pancreatic cancer. Our previous studies using the prototype Hedgehog small-molecule antagonist cyclopamine had shown the striking inhibition of systemic metastases on Hedgehog blockade in spontaneously metastatic orthotopic xenograft models. Cyclopamine is a natural compound with suboptimal pharmacokinetics, which impedes clinical translation. In the present study, a novel, orally bioavailable small-molecule Hedgehog inhibitor, IPI-269609, was tested using in vitro and in vivo model systems. In vitro treatment of pancreatic cancer cell lines with IPI-269609 resembled effects observed using cyclopamine (i.e., Gli-responsive reporter knockdown, down-regulation of the Hedgehog target genes Gli1 and Ptch, as well as abrogation of cell migration and colony formation in soft agar). Single-agent IPI-269609 profoundly inhibited systemic metastases in orthotopic xenografts established from human pancreatic cancer cell lines, although Hedgehog blockade had minimal effect on primary tumor volume. The only discernible phenotype observed within the treated primary tumor was a significant reduction in the population of aldehyde dehydrogenase–bright cells, which we have previously identified as a clonogenic tumor-initiating population in pancreatic cancer. Selective ex vivo depletion of aldehyde dehydrogenase–bright cells with IPI-269609 was accompanied by significant reduction in tumor engraftment rates in athymic mice. Pharmacologic blockade of aberrant Hedgehog signaling might prove to be an effective therapeutic strategy for inhibition of systemic metastases in pancreatic cancer, likely through targeting subsets of cancer cells with tumor-initiating (“cancer stem cell”) properties. [Mol Cancer Ther 2008;7(9):2725–35]

Five years of prospective screening of high-risk individuals from families with familial pancreatic cancer

Objective: Familial pancreatic cancer (FPC) accounts for approximately 3% of all pancreatic cancer (PC) cases. It has been suggested that high-risk individuals (HRIs) should be offered a screening programme. Aim: To evaluate the diagnostic yield of a prospective screening programme in HRIs from families with FPC over a period of 5 years. Methods: HRIs of families with FPC of the National German Familial Pancreatic Cancer Registry (FaPaCa) were counselled and enrolled in a prospective, board-approved PC screening programme. Screening included clinical examination, laboratory tests, endoscopic ultrasound (EUS) and MRI with magnetic resonance cholangiopancreaticography (MRCP) and MR angiography. Results: Between June 2002 and December 2007, 76 HRIs of families with FPC took part in the screening programme with a total of 182 examination visits. Twenty-eight patients revealed abnormalities in EUS (n = 25) and/or MR/MRCP (n = 12). In 7 patients fine needle aspiration cytology was performed. Operative pancreatic explorations were performed in 7 individuals, resulting in limited resections in 6 cases. Histopathological examination of the resected specimens showed serous oligocystic adenomas (n = 3), pancreatic intraepithelial neoplasia 1 (PanIN1) lesions with lobular fibrosis (n = 1), PanIN2 lesions (n = 1) and PanIN1 lesion plus a gastric type intraductal papillary mucinous neoplasm (IPMN) (n = 1). Conclusions: In FPC an EUS/MR/MRCP-based screening programme leads to the detection of potential precursor lesions of PC. However, the yield of an extensive screening programme is low, especially since the tumourigenic value of low grade PanIN lesions is not yet defined. Taking into account the enormous psychological stress for the tested individual and the high costs, a general PC screening in HRIs is not justified.

Outcome of Duodenopancreatic Resections in Patients With Multiple Endocrine Neoplasia Type 1

Objective:To evaluate the outcome of an aggressive surgical approach for duodenopancreatic neuroendocrine tumors (PETs) associated with multiple endocrine neoplasia type 1 (MEN1). Summary Background Data:The management of PETs is still controversial in the setting of the autosomal dominant inherited MEN1 syndrome. Methods:MEN1 patients that had either biochemical evidence of functioning PETs or visualized nonfunctioning PETs larger than 1 cm in size on imaging were operated. Since 1997, patients were followed annually by biochemical testing and imaging studies. Results:Twenty-six genetically confirmed MEN1 patients underwent duodenopancreatic resection for functioning (n = 17) or nonfunctioning (n = 9) PETs. Ten (38%) patients had malignant PETs as characterized by the presence of lymph node (10 patients) and/or distant metastases (2 patients). The surgical approach was selected based on the type, location, and size of PETs. Four Zollinger-Ellison syndrome (ZES) patients required pylorus preserving pancreaticoduodenectomy (PPPD) as initial or redo procedure, 20 patients underwent other duodenopancreatic resections, and 2 patients had simple enucleations of PETs. After median 83 months (range, 5–241 months), 24 patients were alive and 2 patients died of an unrelated cause. All patients with insulinoma or vipoma and 7 of 11 patients with ZES were biochemically cured, including the ZES patients who underwent PPPD. However, 19 of 26 (73%) patients developed new small PETs (<1 cm) in the pancreatic remnant, but no patient had yet detectable metastases on imaging. Conclusions:Early and aggressive surgery of PETs in MEN1 patients prevents the development of liver metastases, which are the most life-threatening determinant. PPPD might be the procedure of choice for MEN1–ZES, which has to be proven in large scale studies.

Hedgehog inhibition prolongs survival in a genetically engineered mouse model of pancreatic cancer

Background and aims: Pancreatic cancer is among the most dismal of human malignancies. Current therapeutic strategies are virtually ineffective in controlling advanced, metastatic disease. Recent evidence suggests that the Hedgehog signalling pathway is aberrantly reactivated in the majority of pancreatic cancers, and that Hedgehog blockade has the potential to prevent disease progression and metastatic spread. Methods: Here it is shown that the Hedgehog pathway is activated in the Pdx1-Cre;LsL-KrasG12D;Ink4a/Arflox/lox transgenic mouse model of pancreatic cancer. The effect of Hedgehog pathway inhibition on survival was determined by continuous application of the small molecule cyclopamine, a smoothened antagonist. Microarray analysis was performed on non-malignant human pancreatic ductal cells overexpressing Gli1 in order to screen for downstream Hedgehog target genes likely to be involved in pancreatic cancer progression. Results: Hedgehog inhibition with cyclopamine significantly prolonged median survival in the transgenic mouse model used here (67 vs 61 days; p = 0.026). In vitro data indicated that Hedgehog activation might at least in part be ascribed to oncogenic Kras signalling. Microarray analysis identified 26 potential Hedgehog target genes that had previously been found to be overexpressed in pancreatic cancer. Five of them, BIRC3, COL11A1, NNMT, PLAU and TGM2, had been described as upregulated in more than one global gene expression analysis before. Conclusion: This study provides another line of evidence that Hedgehog signalling is a valid target for the development of novel therapeutics for pancreatic cancer that might be worth evaluating soon in a clinical setting.

Hedgehog Signaling Is Required for Effective Regeneration of Exocrine Pancreas

Although both endocrine and the exocrine pancreas display a significant capacity for tissue regeneration and renewal, the existence of progenitor cells in the adult pancreas remains uncertain. Using a model of cerulein-mediated injury and repair, we demonstrate that mature exocrine cells, defined by expression of an Elastase1 promoter, actively contribute to regenerating pancreatic epithelium through formation of metaplastic ductal intermediates. Acinar cell regeneration is associated with activation of Hedgehog (Hh) signaling, as assessed by up-regulated expression of multiple pathway components, as well as activation of a Ptch-lacZ reporter allele. Using both pharmacologic and genetic techniques, we also show that the ability of mature exocrine cells to accomplish pancreatic regeneration is impaired by blockade of Hh signaling. Specifically, attenuated regeneration in the absence of an intact Hh pathway is characterized by persistence of metaplastic epithelium expressing markers of pancreatic progenitor cells, suggesting an inhibition of redifferentiation into mature exocrine cells. Given the known role of Hh signaling in exocrine pancreatic cancer, these findings may provide a mechanistic link between injury-induced activation of pancreatic progenitors and subsequent pancreatic neoplasia.

An aggressive surgical approach leads to long-term survival in patients with pancreatic endocrine tumors.

Objective:To evaluate the outcome of reoperations in patients with duodenopancreatic neuroendocrine tumors (PETs) in a tertiary referral center. Summary Background Data:The management of reoperations in PETs is still controversial. Methods:A total of 125 patients with PETs that underwent surgery between 1987 and 2004 at our institution were retrospectively evaluated. The diagnosis of PETs was based on clinical symptoms, biochemical tests, and histopathology. Patients with at least one reoperation were analyzed regarding clinical characteristics, pathology, operations, and long-term follow-up. Results:A total of 33 patients with a median age of 42 years were identified for this study: 13 patients had gastrinomas, 12 patients had nonfunctional islet cell tumors, 6 patients had insulinomas, and 2 patients had vipomas; 24 patients had sporadic NETs, 9 patients had a MEN-1-syndrome; 27 patients had histologically verified malignant tumors; 33 initial operations and 50 reoperations were performed. The initial procedures comprised 27 resections of the primary tumor and 6 explorative laparotomies; 28 of all reoperations were resections of distant metastases, including 15 liver resections; 19 resections of the pancreas or duodenum were performed during reoperations. The overall morbidity and mortality was 45% and 4.8%, respectively. After a median follow-up of 124 months (range, 16–384 months), 27 of 33 patients are still alive, 12 without evidence of disease. All 6 patients with benign tumors are still alive. The 5-, 10-, and actuarial 25-year survival rate for patients with malignant tumors were 81%, 72%, and 36%, respectively. The survival rate was significantly related to the patients age at time of initial operation and better in patients younger than 50 years compared with patients older than 50 years (P = 0.0007), and the presence or development of metastases (none or lymph node metastases versus distant metastases: P = 0.01). Conclusion:We show that an aggressive surgical approach leads to long-term survival in patients with malignant PETs. Although long-term cure can only be achieved in a proportion of patients with malignant PETs, significant long-term palliation can be achieved.

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Significance We describe a blood test for detection of cell death in specific tissues based on two principles: (i) dying cells release fragmented DNA to the circulation, and (ii) each cell type has a unique DNA methylation pattern. We have identified tissue-specific DNA methylation markers and developed a method for sensitive detection of these markers in plasma or serum. We demonstrate the utility of the method for identification of pancreatic β-cell death in type 1 diabetes, oligodendrocyte death in relapsing multiple sclerosis, brain cell death in patients after traumatic or ischemic brain damage, and exocrine pancreas cell death in pancreatic cancer or pancreatitis. The approach allows minimally invasive monitoring of tissue dynamics in humans in multiple physiological and pathological conditions. Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

DYRK1B-dependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS

Synergism between the RAS and Hedgehog (HH) pathways has been suggested for carcinogenesis in the pancreas, lung and colon. We investigated the molecular cross-talk between RAS and HH signaling and found that, although mutant RAS induces or enhances SHH expression and favors paracrine HH signaling, it antagonizes autocrine HH signal transduction. Activated RAS can be found in primary cilia, the central organelle of HH signal transduction, but functions in a cilium-independent manner and interferes with Gli2 function and Gli3 processing. In addition, the cell-autonomous negative regulation of HH signal transduction involves the RAS effector molecule dual specificity tyrosine phosphorylated and regulated kinase 1B (DYRK1B). In line with a redirection of autocrine toward paracrine HH signaling by a KRAS-DYRK1B network, we find high levels of GLI1 expression restricted to the stromal compartment and not to SHH-expressing tumor cells in human pancreatic adenocarcinoma.

Prospective Evaluation of Imaging Procedures for the Detection of Pancreaticoduodenal Endocrine Tumors in Patients with Multiple Endocrine Neoplasia Type 1

Early identification of pancreaticoduodenal endocrine tumors (PETs) in multiple endocrine neoplasia type 1 (MEN-1) is mandatory, because these tumors represent the most common cause of death within the syndrome. The diagnostic value of imaging procedures has therefore been evaluated in a prospective observational study. Between December 1997 and June 2003 twenty-two MEN-1 patients with genetically confirmed disease were followed for PETs using a standardized screening program with serum hormone measurements, endoscopic ultrasonography (EUS), computed tomography (CT), and somatostatin-receptor scintigraphy (SRS). Results could be validated by surgery and histopathology in 13 patients during 18 operations. In 12 asymptomatic patients with tumors measuring 10 mm or less, who have not yet undergone operation, PETs were detected by EUS in 12/12, by CT in 1/12, and by SRS in 2/11 cases. In 13 patients who have undergone surgical exploration EUS, CT, and SRS were true positive in 12 of 16, 7 of 13, and 12 of 17 cases, respectively, although the number of tumors detected by each imaging procedure alone was lower than the number detected intraoperatively and histopathologically in almost every case. A solitary liver metastasis in one patient and a nonfunctioning PET recurrence in another were identified only by SRS. Endoscopic untrasonography is the most sensitive imaging procedure for the detection of small (≤ 10 mm) PETs in MEN-1, whereas SRS is the procedure of choice for the identification of metastases of MEN-1 PETs—i.e., for staging. Detection of PETs at an early stage by an aggressive screening program using EUS may lead to prompt surgical intervention and improved prognosis of MEN-1 PETs.