Germline Pathogenic Variants in Patients With Pancreatic and Periampullary Cancers.

PURPOSE: Inherited cancer susceptibility is often not suspected in the absence of a significant cancer family history. Pathogenic germline variants in pancreatic cancer are well-studied, and routine genetic testing is recommended in the guidelines. However, data on rare periampullary cancers other than pancreatic cancer are insufficient. We compared the prevalence of germline susceptibility variants in patients with pancreatic cancer and nonpancreatic periampullary cancers. MATERIALS AND METHODS: Six hundred and eight patients who had undergone pancreaticoduodenal resection at a tertiary referral hospital were studied, including 213 with pancreatic ductal adenocarcinoma, 172 with ampullary cancer, 154 with distal common bile duct cancer, and 69 with duodenal adenocarcinoma. Twenty cancer susceptibility and candidate susceptibility genes were sequenced, and variant interpretation was assessed by interrogating ClinVar and PubMed. RESULTS: Pathogenic or likely pathogenic, moderate- to high-penetrant germline variants were identified in 46 patients (7.7%), including a similar percentage of patients with pancreatic (8.5%) and nonpancreatic periampullary cancer (7.1%). Low-penetrant variants were identified in an additional 11 patients (1.8%). Eighty-nine percent of the moderate- to high-penetrant variants involved the major cancer susceptibility genes BRCA2, ATM, BRCA1, CDKN2A, MSH2/MLH1, and PALB2; the remaining 11% involved other cancer susceptibility genes such as BRIP1, BAP1, and MSH6. Almost all pathogenic variant carriers had a family history of cancer. CONCLUSION: Patients with pancreatic and nonpancreatic periampullary cancer have a similar prevalence of pathogenic cancer susceptibility variants. Germline susceptibility testing should be considered for patients with any periampullary cancer.

Using Tumor Marker Gene Variants to Improve the Diagnostic Accuracy of DUPAN-2 and Carbohydrate Antigen 19-9 for Pancreatic Cancer.

PURPOSE: Circulating carbohydrate antigen 19-9 (CA19-9) levels reflect FUT3 and FUT2 fucosyltransferase activity. Measuring the related glycan, DUPAN-2, can be useful in individuals unable to synthesize CA19-9. We hypothesized that similar to CA19-9, FUT functional groups determined by variants in FUT3 and FUT2 influence DUPAN-2 levels, and having tumor marker reference ranges for each functional group would improve diagnostic performance. MATERIALS AND METHODS: Using a training/validation study design, FUT2/FUT3 genotypes were determined in 938 individuals from Johns Hopkins Hospital: 607 Cancer of the Pancreas Screening (CAPS) study subjects with unremarkable pancreata and 331 with pancreatic ductal adenocarcinoma (PDAC). Serum DUPAN-2 and CA19-9 levels were measured by immunoassay. RESULTS: In controls, three functional FUT groups were identified with significant differences in DUPAN-2 levels: FUT3-intact, FUT3-null/FUT2-intact, and FUT3-null/FUT2-null. DUPAN-2 training set diagnostic cutoffs for each FUT group yielded higher diagnostic sensitivity in the validation set for patients with stage I/II PDAC than uniform cutoffs (60.4% [95% CI, 50.2 to 70.0] v 39.8% [30.0 to 49.8]), at approximately 99% (96.7 to 99.6) specificity. Combining FUT/CA19-9 and FUT/DUPAN-2 tests yielded 78.4% (72.3 to 83.7) sensitivity for stage I/II PDAC, at 97.7% (95.3 to 99.1) specificity in the combined sets, with higher AUC (stage I/II: 0.960 v 0.935 for CA19-9 + DUPAN-2 without the FUT test; P < .001); for stage I PDAC, sensitivity was 62.0% (49.1 to 73.2; AUC, 0.919 v 0.883; P = .03). CA19-9 levels in FUT3-null/FUT2-null PDAC subjects were higher than in FUT3-null/FUT2-intact subjects (median/IQR; 24.9/57.4 v <1/2.3 U/mL; P = .0044). In a simulated CAPS cohort, AUC precision recall (AUCPR) scores were 0.51 for CA19-9 alone, 0.64 for FUT/CA19-9, 0.73 for CA19-9/DUPAN-2, and 0.84 for FUT/CA19-9/DUPAN-2. CONCLUSION: Using a tumor marker gene test to individualize CA19-9 and DUPAN-2 reference ranges achieves high diagnostic performance for stage I/II pancreatic cancer.

Using a CA19-9 Tumor Marker Gene Test to Assess Outcome After Pancreatic Cancer Surgery.

BACKGROUND: Cancer antigen 19-9 (CA19-9) is widely used as a marker of pancreatic cancer tumor burden and response to therapy. Synthesis of CA19-9 and its circulating levels are determined by variants encoding the fucosyltransferases, FUT2 and FUT3. Individuals can be grouped into one of four functional FUT groups (FUT3-null, FUT-low, FUT-intermediate, FUT-high), each with its own CA19-9 reference range based on its predicted capacity to produce CA19-9. The authors hypothesized that a FUT variant-based CA19-9 tumor marker gene test could improve the prognostic performance of CA19-9. METHODS: Preoperative and pre-treatment CA19-9 levels were measured, and FUT variants were determined in 449 patients who underwent surgery for pancreatic ductal adenocarcinoma (PDAC) at Johns Hopkins Hospital between 2010 and 2020, including 270 patients who underwent neoadjuvant therapy. Factors associated with recurrence-free and overall survival were determined in Cox proportional hazards models. RESULTS: Higher preoperative CA19-9 levels were associated with recurrence and mortality for patients in the higher-FUT groups (FUT-intermediate, FUT-high for mortality, with adjustment for other prognostic factors; hazard ratio [HR], 1.34 and 1.58, respectively; P < 0.001), but not for those in the lower-FUT groups (FUT3-null, FUT-low). As a tumor marker, CA19-9 levels of 100 U/ml or lower after neoadjuvant therapy and normalization of CA19-9 based on FUT group were more sensitive but less specific predictors of evidence for a major pathologic response to therapy (little/no residual tumor) and of early recurrence (within 6 months). CONCLUSION: Among patients undergoing pancreatic cancer resection, a CA19-9 tumor marker gene test modestly improved the prognostic performance of CA19-9.

Diagnostic Performance of a Tumor Marker Gene Test to Personalize Serum CA19-9 Reference Ranges.

PURPOSE: CA19-9 synthesis is influenced by common variants in the fucosyltransferase (FUT) enzymes FUT3 and FUT2. We developed a clinical test to detect FUT variants, and evaluated its diagnostic performance for pancreatic ductal adenocarcinoma (PDAC). EXPERIMENTAL DESIGN: A representative set of controls from the Cancer of the Pancreas Screening study was identified for each FUT functional group. Diagnostic sensitivity was determined first in a testing set of 234 PDAC cases, followed by a 134-case validation set, all of whom had undergone resection with curative intent without neoadjuvant therapy. Tumor marker gene testing was performed in the Johns Hopkins Molecular Diagnostics Laboratory. CA19-9 levels were measured in the Hopkins Clinical Chemistry lab. Receiver operating characteristic (ROC) curve analysis was used to evaluate the discriminative ability of CA19-9 alone versus with the gene test. RESULTS: Applying the CA19-9 standard cutoff (<36 U/mL) to all 716 subjects yielded a 68.8% sensitivity in the test set of cases, 67.2% in the validation set, at 91.4% specificity. Applying 99th percentile cutoffs according to each individual's FUT group (3, 34.9, 41.8, and 89.2, for the FUT3-null, FUT-low, FUT-intermediate, and FUT-high groups, respectively) yielded a diagnostic sensitivity for CA19-9 in the first set of cases of 66.7%, 65.7% in the validation set, at 98.9% specificity. ROC analysis for CA19-9 alone yielded an AUC of 0.84; with the tumor marker gene test, AUC improved to 0.92 (P < 0.001). CONCLUSIONS: Using a tumor marker gene test to personalize an individual's CA19-9 reference range significantly improves diagnostic accuracy.

Bone marrow-derived macrophages converted into cancer-associated fibroblast-like cells promote pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic reaction caused by cancer-associated fibroblasts (CAFs), which provokes treatment resistance. CAFs are newly proposed to be heterogeneous populations with different functions within the PDAC microenvironment. The most direct sources of CAFs are resident tissue fibroblasts and mesenchymal stem cells, however, the origins and functions of CAF subtypes remain unclear. Here, we established allogeneic bone marrow (BM) transplantation models using spontaneous PDAC mice, and then investigated what subtype cells derived from BM modulate the tumor microenvironment and affect the behavior of pancreatic cancer cells (PCCs). BM-derived multilineage hematopoietic cells were engrafted in recipient pancreas, and accumulated at the invasive front and central lesion of PDAC. We identified BM macrophages-derived CAFs in tumors. BM-derived macrophages treated with PCC-conditioned media expressed CAF markers. BM-derived macrophages led the local invasion of PCCs in vitro and enhanced the tumor invasive growth in vivo. Our data suggest that BM-derived cells are recruited to the pancreas during carcinogenesis and that the specific subpopulation of BM-derived macrophages partially converted into CAF-like cells, acted as leading cells, and facilitated pancreatic cancer progression. The control of the conversion of BM-derived macrophages into CAF-like cells may be a novel therapeutic strategy to suppress tumor growth.

Necroptosis in pancreatic cancer promotes cancer cell migration and invasion by release of CXCL5.

BACKGROUND: Necroptosis is a form of programmed cell death that is accompanied by release of intracellular contents, and reportedly contributes to various diseases. Here, we investigate the significance of necroptosis in pancreatic cancer. METHODS: We used immunohistochemistry and western blot analysis to evaluate expression of the key mediators of necroptosis-receptor-interacting serine/threonine protein kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL)-in human pancreatic cancer. We also tested the effects of conditioned media (CM) from necroptotic cells on pancreatic cancer cells in Transwell migration and Matrigel invasion assays. Protein array analysis was used to investigate possible mediators derived from necroptotic cells. RESULTS: RIP3 and MLKL are highly expressed in human pancreatic cancer tissues compared with normal pancreas. MLKL expression was particularly intense at the tumor invasion front. CM derived from necroptotic cells promoted cancer cell migration and invasion, but not CM derived from apoptotic cells. C-X-C motif chemokine 5 (CXCL5) was upregulated in CM derived from necroptotic cells compared with CM derived from control or apoptotic cells. Moreover, expression of the receptor for CXCL5, C-X-C-motif chemokine receptor-2 (CXCR2), was upregulated in pancreatic cancer cells. Inhibition of CXCR2 suppressed cancer cell migratory and invasive behavior enhanced by necroptosis. CONCLUSION: These findings indicate that necroptosis at the pancreatic cancer invasion front can promote cancer cell migration and invasion via the CXCL5-CXCR2 axis.

Neutrophil extracellular traps promote liver micrometastasis in pancreatic ductal adenocarcinoma via the activation of cancer­associated fibroblasts.

Cancer­associated fibroblasts (CAFs) promote the progression of pancreatic ductal adenocarcinoma (PDAC) via tumor­stromal interactions. Neutrophil extracellular traps (NETs) are extracellular DNA meshworks released from neutrophils together with proteolytic enzymes against foreign pathogens. Emerging studies suggest their contribution to liver metastasis in several types of cancer. Herein, in order to investigate the role of NETs in liver metastasis in PDAC, the effects of NET inhibitors on spontaneous PDAC mouse models were evaluated. It was demonstrated that DNase I, a NET inhibitor, suppressed liver metastasis. For further investigation, further attention was paid to liver micrometastasis and an experimental liver metastasis mouse model was used that was generated by intrasplenic tumor injection. Furthermore, DNase I also suppressed liver micrometastasis and notably, CAFs accumulated in metastatic foci were significantly decreased in number. In vitro experiments revealed that pancreatic cancer cells induced NET formation and consequently NETs enhanced the migration of hepatic stellate cells, which was the possible origin of CAFs in liver metastasis. On the whole, these results suggest that NETs promote liver micrometastasis in PDAC via the activation of CAFs.

S100P regulates the collective invasion of pancreatic cancer cells into the lymphatic endothelial monolayer.

Lymph node metastasis is an independent prognostic factor in pancreatic cancer. However, the mechanisms of lymph node colonization are unknown. As a mechanism of lymphatic metastasis, it has been reported for other types of cancer that spheroids from tumor cells cause circular chemorepellent­induced defects (CCIDs) in lymphatic endothelial monolayers. In pancreatic cancer, such mechanisms of metastasis have not been elucidated. The present study evaluated the involvement of this new mechanism of metastasis in pancreatic cancer and investigated the associated factors. In human pancreatic cancer tissue, it was observed that clusters of cancer cells penetrated the wall of lymphatic ducts around the primary tumor. An in vitro co­culture system was then used to analyze the mechanisms of tumor cell­mediated disruption of lymphatic vessels. Time­lapse microscopic imaging revealed that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. CCID formation ability differed depending on the cell line. Neither aggregation of spheroids nor adhesion to lymphatic endothelial cells (LECs) exhibited a significant correlation with this phenomenon. The addition of supernatant from cultured cancer cells enhanced CCID formation. Microarray analysis revealed that the expression of S100 calcium binding protein P (S100P) was significantly increased when LECs were treated with supernatant from cultured cancer cells. Addition of a S100P antagonist significantly suppressed the migration of LECs and CCID formation. The present findings demonstrated that spheroids from pancreatic cancer cells caused circular defects in lymphatic endothelial monolayers. These CCIDs in pancreatic cancer were partly regulated by S100P, suggesting that S100P may be a promising target to inhibit lymph node metastasis.

Inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer-stromal interaction and metastasis.

BACKGROUND: Extracellular signal-regulated kinases (ERKs) have been related to multiple cancers, including breast cancer, hepatocellular cancer, lung cancer and colorectal cancer. ERK1/2 inhibitor can suppress growth of KRAS-mutant pancreatic tumors by targeting cancer cell. However, no studies have shown the expression of ERK1/2 on pancreatic stromal and its effect on pancreatic cancer-stromal interaction. METHODS: Immunohistochemistry and western blotting were performed to detect the expression of p-ERK1/2 in pancreatic tissues and cells. Cell viability assay was used to study IC50 of ERK inhibitor on pancreatic cancer cells (PCCs) and primary cancer-associated pancreatic stellate cells (PSCs). Transwell migration, invasion, cell viability assay, senescence ß-galactosidase staining were performed to determine the effect of ERK inhibitor on PCCs and PSCs in vitro and in vivo. The expression of key factors involved in autophagy and epithelial-to-mesenchymal transition (EMT) process were evaluated by western blotting. The expression of key factors related to cell invasiveness and malignancy were confirmed by qRT-PCR. Co-transplantation of PCC Organoid and PSC using a splenic xenograft mouse model was used to evaluated combined treatment of ERK inhibitor and autophagy inhibitor. RESULTS: Immunohistochemical staining in pancreatic tumor samples and transgenetic mice detected p-ERK1/2 expression in both cancer cells and stromal cells. In pancreatic tissues, p-ERK1/2 was strongly expressed in cancer-associated PSCs compared with cancer cells and normal PSCs. PSCs were also significantly more sensitive to ERK1/2 inhibitor treatment. Inhibition of ERK1/2 suppressed EMT transition in HMPCCs, upregulated cellular senescence markers, activated autophagy in cancer-associated PSCs; and suppressed cancer-stromal interaction, which enhanced invasiveness and viability of cancer cells. We also found that chloroquine, an autophagy inhibitor, suppressed ERK inhibition-induced autophagy and promoted PSC cellular senescence, leading to significantly decreased cell proliferation. The combination of an ERK inhibitor and autophagy inhibitor suppressed liver metastasis in a splenic pancreatic cancer organoid xenograft mouse model. CONCLUSIONS: These data indicate that inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer-stromal interaction and metastasis.

Adipose tissue-derived stromal cells are sources of cancer-associated fibroblasts and enhance tumor progression by dense collagen matrix.

Although recent studies revealed that adipose tissue accelerates pancreatic tumor progression with excessive extracellular matrix, key players for desmoplasia in the adipose microenvironment remains unknown. Here, we investigated the roles of adipose tissue-derived stromal cells (ASCs) in desmoplastic lesions and tumor progression by in vitro and in vivo experiments. In a three-dimensional (3-D) organotypic fat invasion model using visceral fat from CAG-EGFP mice, GFP-positive fibroblastic cells infiltrated toward cancer cells. When tumor cells were inoculated into transplanted visceral fat pads in vivo, tumor weights and stromal components were enhanced compared to subcutaneous and orthotopic tumor cells inoculated without fat pads. Expression of αSMA in established human ASCs was lower compared to cancer associated fibroblasts, and the 3-D collagen matrices produced by ASCs cultured in cancer cell-conditioned medium changed from loose to dense structures that affected the motility of cancer cells. Microarray analyses revealed upregulation of S100A4 in ASCs, while S100A4-positive stromal cells were observed at extrapancreatic invasion sites of human pancreatic cancer. The present findings indicate that ASCs are recruited to extrapancreatic invasion sites and produce dense collagen matrices that lead to enhanced tumor progression. Both inhibition of ASCs recruitment and activation could lead to a novel antistromal therapy.

Cancer-associated acinar-to-ductal metaplasia within the invasive front of pancreatic cancer contributes to local invasion.

The pancreas is an organ prone to inflammation, fibrosis, and atrophy because of an abundance of acinar cells that produce digestive enzymes. A characteristic of pancreatic cancer is the presence of desmoplasia, inflammatory cell infiltration, and cancer-associated acinar atrophy (CAA) within the invasive front. CAA is characterized by a high frequency of small ducts and resembles acinar-to-ductal metaplasia (ADM). However, the clinical significance of changes in acinar morphology, such as ADM with acinar atrophy, within the tumor microenvironment remains unclear. Here, we find that ADM within the invasive front of tumors is associated with cell invasion and desmoplasia in an orthotopic mouse model of pancreatic cancer. An analysis of resected human tumors revealed that regions of cancer-associated ADM were positive for TGFα, and that this TGFα expression was associated with primary tumor size and shorter survival times. Gene expression analysis identified distinct phenotypic profiles for cancer-associated ADM, sporadic ADM and chronic pancreatitis ADM. These findings suggest that the mechanisms driving ADM differ according to the specific tissue microenvironment and that cancer-associated ADM and acinar atrophy contribute to tumor cell invasion of the local pancreatic parenchyma.

Basement membrane destruction by pancreatic stellate cells leads to local invasion in pancreatic ductal adenocarcinoma.

Stroma invasion is an important step in pancreatic cancer progression. However, how pancreatic ductal adenocarcinoma (PDAC) with ductal structure invades the surrounding stroma has not been clear. Here, we elucidated the mechanism of stromal invasion of PDAC, using organoids. From resected PDAC specimens, we established human PDAC organoids, which developed ductal and basement membrane (BM) structures. When the organoids were co-cultured with pancreatic stellate cells (PSCs) in a collagen matrix, organoids lost their BM and ductal structures, and invaded collagen matrix more frequently than did mono-cultured organoids. Interestingly, direct contact by PSCs to PDAC organoids was observed before BM destruction. Matrix metalloproteinase (MMP) 2 or membrane type-1 MMP (MT1MMP) knockdown in PSCs significantly attenuated BM destruction by PSCs, and retained the ductal structures in organoids. Our results imply that direct contact by PSCs induces BM destruction and stromal invasion of PDAC via MMP2 which binds to MT1MMP on PSCs.

Pancreatic stellate cells reorganize matrix components and lead pancreatic cancer invasion via the function of Endo180.

Specific cell populations leading the local invasion of cancer are called "leading cells". However, the underlying mechanisms are unclear. Here, we identified leading cells in pancreatic cancer and determined how these cells lead and promote cancer cell invasion in the extracellular matrix (ECM). Using three-dimensional matrix remodeling assay, we found that pancreatic stellate cells (PSCs) frequently invaded the collagen matrix with pancreatic cancer cells (PCCs), which invaded behind the invading PSCs. In addition, invading PSCs changed the alignment of collagen fibers, resulting in ECM remodeling and an increase in the parallel fibers along the direction of invading PSCs. Endo180 expression was higher in PSCs than in PCCs, Endo180 knockdown in PSCs attenuated the invasive abilities of PSCs and co-cultured PCCs, and decreased the expression level of phosphorylated myosin light chain 2 (MLC2). In mouse models, Endo180-knockdown PSCs suppressed tumor growth and changes in collagen fiber orientation in co-transplantation with PCCs. Our findings suggest that PSCs lead the local invasion of PCCs by physically remodeling the ECM, possibly via the function of Endo180, which reconstructs the actin cell skeleton by phosphorylation of MLC2.

Autophagy inhibition enhances antiproliferative effect of salinomycin in pancreatic cancer cells.

BACKGROUND: Salinomycin has cytotoxic effects on various types of malignancy and induces autophagy. However, it has not been clarified whether autophagy induced by salinomycin treatment has a protective or cytotoxic role. We investigated whether salinomycin affects autophagy in pancreatic cancer cells and whether autophagy induced by salinomycin treatment has a protective or cytotoxic role in these cells. METHODS: We investigated the effect of salinomycin using three pancreatic cancer cell lines. We investigated effect on proliferation and the CD133 positive fraction using flow cytometry. In addition, we monitored the change in autophagic activity after salinomycin treatment using fluorescent immunostaining, western blotting, and flow cytometry. Finally, knockdown of ATG5 or ATG7 by siRNA was used to investigate the impact of autophagy inhibition on sensitivity to salinomycin. RESULTS: Salinomycin suppressed the proliferation of pancreatic cancer cells in a concentration dependent manner, and reduced the CD133 positive fraction. Salinomycin enhanced autophagy activity in these cells in a concentration dependent manner. Autophagy inhibition made pancreatic cancer cells more sensitive to salinomycin. CONCLUSIONS: Our data provide the first evidence indicating that autophagy induced by salinomycin have a protective role in pancreatic cancer cells. A new therapeutic strategy of combining salinomycin, autophagy inhibitors, and anticancer drugs could hold promise for pancreatic cancer treatment.