Population-specific Mutation Patterns in Breast Tumors from African American, European American, and Kenyan Patients.

Women of African descent have the highest breast cancer mortality in the United States and are more likely than women from other population groups to develop an aggressive disease. It remains uncertain to what extent breast cancer in Africa is reminiscent of breast cancer in African American or European American patients. Here, we performed whole-exome sequencing of genomic DNA from 191 breast tumor and non-cancerous adjacent tissue pairs obtained from 97 African American, 69 European American, 2 Asian American, and 23 Kenyan patients. Our analysis of the sequencing data revealed an elevated tumor mutational burden in both Kenyan and African American patients, when compared with European American patients. TP53 mutations were most prevalent, particularly in African American patients, followed by PIK3CA mutations, which showed similar frequencies in European American, African American, and the Kenyan patients. Mutations targeting TBX3 were confined to European Americans and those targeting the FBXW7 tumor suppressor to African American patients whereas mutations in the ARID1A gene that are known to confer resistance to endocrine therapy were distinctively enriched among Kenyan patients. A Kyoto Encyclopedia of Genes and Genomes pathway analysis could link FBXW7 mutations to an increased mitochondrial oxidative phosphorylation capacity in tumors carrying these mutations. Finally, Catalogue of Somatic Mutations in Cancer (COSMIC) mutational signatures in tumors correlated with the occurrence of driver mutations, immune cell profiles, and neighborhood deprivation with associations ranging from being mostly modest to occasionally robust. To conclude, we found mutational profiles that were different between these patient groups. The differences concentrated among genes with low mutation frequencies in breast cancer. SIGNIFICANCE: The study describes differences in tumor mutational profiles between African American, European American, and Kenyan breast cancer patients. It also investigates how these profiles may relate to the tumor immune environment and the neighborhood environment in which the patients had residence. Finally, it describes an overrepresentation of ARID1A gene mutations in breast tumors of the Kenyan patients.

Diabetes-associated breast cancer is molecularly distinct and shows a DNA damage repair deficiency.

Diabetes commonly affects patients with cancer. We investigated the influence of diabetes on breast cancer biology using a 3-pronged approach that included analysis of orthotopic human tumor xenografts, patient tumors, and breast cancer cells exposed to diabetes/hyperglycemia-like conditions. We aimed to identify shared phenotypes and molecular signatures by investigating the metabolome, transcriptome, and tumor mutational burden. Diabetes and hyperglycemia did not enhance cell proliferation but induced mesenchymal and stem cell-like phenotypes linked to increased mobility and odds of metastasis. They also promoted oxyradical formation and both a transcriptome and mutational signatures of DNA repair deficiency. Moreover, food- and microbiome-derived metabolites tended to accumulate in breast tumors in the presence of diabetes, potentially affecting tumor biology. Breast cancer cells cultured under hyperglycemia-like conditions acquired increased DNA damage and sensitivity to DNA repair inhibitors. Based on these observations, we conclude that diabetes-associated breast tumors may show an increased drug response to DNA damage repair inhibitors.

Zinc finger protein 277 is an intestinal transit-amplifying cell marker and colon cancer oncogene.

Sustained proliferative signaling and resisting cell death are hallmarks of cancer. Zinc finger protein 277 (ZNF277; murine Zfp277), a transcription factor regulating cellular senescence, is overexpressed in colon cancer, but its actions in intestinal homeostasis and neoplasia are unclear. Using human and murine intestine, human colon cancer cells, and ApcMin/+ mice with dysregulated ß-catenin signaling and exuberant intestinal neoplasia, we explored the actions of ZNF277/Zfp277 and defined the underlying mechanisms. In normal human and murine intestine, ZNF277/Zfp277 was expressed uniquely in early stem cell progenitors, undifferentiated transit-amplifying cells (TACs). Zfp277 was overexpressed in the ApcMin/+ mouse colon, implicating ZNF277/Zfp277 as a transcriptional target of ß-catenin signaling. We confirmed this by showing ß-catenin knockdown reduced ZNF277 expression and, using chromatin IP, identified 2 ß-catenin binding sites in the ZNF277 promoter. Zfp277 deficiency attenuated intestinal epithelial cell proliferation and tumor formation, and it strikingly prolonged ApcMin/+ mouse survival. RNA-Seq and PCR analyses revealed that Zfp277 modulates expression of genes in key cancer pathways, including ß-catenin signaling, the HOXD family that regulates development, and p21WAF1, a cell cycle inhibitor and tumor suppressor. In both human colon cancer cells and the murine colon, ZNF277/Zfp277 deficiency induced p21WAF1 expression and promoted senescence. Our findings identify ZNF277/Zfp277 as both a TAC marker and colon cancer oncogene that regulates cellular proliferation and senescence, in part by repressing p21WAF1 expression.

Tangier Disease: An Unusual Cause of Chronic Diarrhea.

Tangier disease is a rare autosomal recessive disease resulting in cholesterol deposition in different organs. We report a case of a 52-year-old white man who presented for chronic diarrhea without significant findings on noninvasive testing. Subsequent colonoscopy revealed endoscopically normal mucosa, with random biopsies remarkable for foamy macrophages in the lamina propria. Genetic testing showed adenosine triphosphate-binding cassette transporter gene mutation with low high-density lipoprotein and low low-density lipoprotein. To the best of our knowledge, this is the first report of chronic diarrhea in a patient with Tangier disease without any other clear etiology.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

Iron pill induced gastritis causing severe anemia.

Iron supplementation is ubiquitously prescribed and considered a benign means of therapy. However, side effects such as iron pill gastritis can be life threatening prompting discontinuation. We describe a case of a 71-year-old man who presents with severe iron deficiency anemia on oral iron therapy. Esophagogastroduodenoscopy revealed mucosal injury in the fundus, including erythema and ulceration. Biopsy of the area was significant for pill debris. After switching to intravenous iron supplementation, his gastric mucosa healed and anemia improved. This case demonstrates the rare life-threatening side effect of iron pills causing corrosive mucosal damage and significant anemia from gastrointestinal bleeding.

NO• /RUNX3/kynurenine metabolic signaling enhances disease aggressiveness in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is refractory to available treatments. Delineating the regulatory mechanisms of metabolic reprogramming, a key event in pancreatic cancer progression, may identify candidate targets with potential therapeutic significance. We hypothesized that inflammatory signaling pathways regulate metabolic adaptations in pancreatic cancer. Metabolic profiling of tumors from PDAC patients with a high- (>median, n = 31) and low-NOS2 (inducible nitric oxide synthase;

Perianal Basal Cell Carcinoma Successfully Managed with Excisional Biopsy.

Basal cell carcinoma (BCC) is the most common cutaneous malignancy in the United States and is often nonaggressive. Its location in the perianal region is very rare and it is estimated that only 0.08% of all BCCs occur in this region. Herein, we present a case of perianal basal cell carcinoma, nodular type. The diagnosis was made using excisional biopsy of a skin lesion. Immunohistochemical staining confirmed the diagnosis: it showed diffuse and strong positivity for smooth muscle actin (SMA) and monoclonal antibody BER-Ep4 and was negative for carcinoembryonic antigen (CEA), pancytokeratin (AE1/AE3), and epithelial membrane antigen (EMA). The treatment of choice has traditionally been local excision to clear margins but the newest guidelines recommend Mohs Micrographic surgery (MMS) or standard 4mm surgical margins for this high-risk BCC. Our patient was successfully treated using excisional biopsy without recurrence. In select patients with lesions smaller than 1cm, excisional biopsy may be sufficient to treat the disease and may be better tolerated than MMS and wider surgical margins. Literature review suggests a predisposition for perianal BCC in individuals susceptible to cutaneous malignancies. Therefore, any history of cutaneous malignancy should further prompt clinicians to examine nonsun exposed areas on full body skin exams.

The role of purified Clostridium difficile glucosylating toxins in disease pathogenesis utilizing a murine cecum injection model.

Most pathogenic Clostridium difficile produce two major exotoxins TcdA and TcdB, in the absence of which the bacterium is non-pathogenic. While it is important to investigate the role of each toxin in the pathogenesis of C. difficile infection (CDI) using isogenic strains, it is impossible to precisely control the expression levels of individual toxins and exclude bacterial factors that may contribute to the toxins' effects during infection. In this study, we utilized an acute intestinal disease model by injecting purified toxins directly into mouse cecum after a midline laparotomy. We evaluated the physical condition of mice by clinical score and survival, and the intestinal tissue damage and inflammation by histology. Depending on the dose of the toxins, mice developed mild to severe colitis, experienced diarrhea or rapidly died. We found that both purified TcdA and TcdB were able to induce clinical disease, intestinal inflammation, and tissue damage that resembled CDI. TcdA was significantly faster in inducing intestinal inflammation and tissue damage, and was approximately five times more potent than TcdB in terms of inducing severe gut disease and death outcomes in mice. Moreover, we found that the two toxins had significant synergistic effects on disease induction. Comparison of the in vivo toxicity of TcdB from clinical strains revealed that TcdB from an epidemic RT 027 strain was more toxic than the others. Our study thus demonstrates that both TcdA and TcdB, independent of other factors from C. difficile bacterium, are able to cause disease that resembles CDI and highlights the importance of targeting both toxins for vaccines and therapeutics against the disease.

A prediction model for distinguishing lung squamous cell carcinoma from adenocarcinoma.

Accurate classification of squamous cell carcinoma (SCC) from adenocarcinoma (AC) of non-small cell lung cancer (NSCLC) can lead to personalized treatments of lung cancer. We aimed to develop a miRNA-based prediction model for differentiating SCC from AC in surgical resected tissues and bronchoalveolar lavage (BAL) samples. Expression levels of seven histological subtype-associated miRNAs were determined in 128 snap-frozen surgical lung tumor specimens by using reverse transcription-polymerase chain reaction (RT-PCR) to develop an optimal panel of miRNAs for acutely distinguishing SCC from AC. The biomarkers were validated in an independent cohort of 112 FFPE lung tumor tissues, and a cohort of 127 BAL specimens by using droplet digital PCR for differentiating SCC from AC. A prediction model with two miRNAs (miRs-205-5p and 944) was developed that had 0.988 area under the curve (AUC) with 96.55% sensitivity and 96.43% specificity for differentiating SCC from AC in frozen tissues, and 0.997 AUC with 96.43% sensitivity and 96.43% specificity in FFPE specimens. The diagnostic performance of the prediction model was reproducibly validated in BAL specimens for distinguishing SCC from AC with a higher accuracy compared with cytology (95.69 vs. 68.10%; P < 0.05). The prediction model might have a clinical value for accurately discriminating SCC from AC in both surgical lung tumor tissues and liquid cytological specimens.

Inducible nitric oxide synthase enhances disease aggressiveness in pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies and is refractory to the available treatments. Pancreatic ductal adenocarcinoma (PDAC) expresses high level of inducible nitric oxide synthase (NOS2), which causes sustained production of nitric oxide (NO). We tested the hypothesis that an aberrantly increased NO-release enhances the development and progression of PDAC. Enhanced NOS2 expression in tumors significantly associated with poor survival in PDAC patients (N = 107) with validation in independent cohorts. We then genetically targeted NOS2 in an autochthonous mouse model of PDAC to examine the effect of NOS2-deficiency on disease progression and survival. Genetic ablation of NOS2 significantly prolonged survival and reduced tumor severity in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice. Primary tumor cells isolated from NOS2-deficient KPC (NKPC) mice showed decreased proliferation and invasiveness as compared to those from KPC mice. Furthermore, NKPC tumors showed reduced expression of pERK, a diminished inactivation of Forkhead box transcription factor O (FOXO3), a tumor suppressor, and a decrease in the expression of oncomir-21, when compared with tumors in KPC mice. Taken together, these findings showed that NOS2 is a predictor of prognosis in early stage, resected PDAC patients, and provide proof-of-principle that targeting NOS2 may have potential therapeutic value in this lethal malignancy.

Endothelial Nitric Oxide Synthase Traffic Inducer (NOSTRIN) is a Negative Regulator of Disease Aggressiveness in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is refractory to available treatments. Delineating critical pathways, responsible for disease aggressiveness and therapeutic resistance, may identify effective therapeutic targets. We aimed to identify key pathways contributing to disease aggressiveness by comparing gene expression profiles of tumors from early-stage PDAC cases with extremely poor survival (≤7 months) and those surviving 2 years or more following surgical resection. EXPERIMENTAL DESIGN: Gene expression profiling was performed in tumors in a test cohort of PDAC (N = 50), which included short (≤7 months, N = 11) and long surviving (≥2 years, N = 14) patients, using affymetrix GeneChip Human 1.0 ST array. Key genes associated with disease aggressiveness were identified, using Cox regression, Kaplan-Meier, and pathway analyses with validations in independent cohorts for mechanistic and functional analyses. RESULTS: Gene expression profiling identified 1,820 differentially expressed genes between short and long survival groups with inflammatory gene network ranking first. Lower expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was associated with worst survival indicating its potential inhibitory role in disease progression. NOSTRIN overexpression suppressed migration and invasion of pancreatic cancer cells and enhanced sensitivity to chemotherapeutic drug gemcitabine. NOSTRIN inhibited production of nitric oxide (NO) by suppressing the activation of endothelial nitric oxide synthase (eNOS). Furthermore, miR-221, bound to the 3'UTR of NOSTRIN and suppressed its expression, and an increased miR-221 expression associated with poor survival in PDAC. CONCLUSIONS: Our findings showed that NOSTRIN is a potential negative regulator of disease aggressiveness, which may be targeted for designing improved treatment strategy in PDAC. Clin Cancer Res; 22(24); 5992-6001. ©2016 AACR.

Pilin Vaccination Stimulates Weak Antibody Responses and Provides No Protection in a C57Bl/6 Murine Model of Acute Clostridium difficile Infection.

Clostridium difficile is the leading cause of nosocomial infections in the United States, adding billions of dollars per year to health care costs. A vaccine targeted against the bacterium would be extremely beneficial in decreasing the morbidity and mortality caused by C. difficile-associated disease; a vaccine directed against a colonization factor would hinder the spread of the bacterium as well as prevent disease. Type IV pili (T4Ps) are extracellular appendages composed of protein monomers called pilins. They are involved in adhesion and colonization in a wide variety of bacteria and archaea, and are putative colonization factors in C. difficile. We hypothesized that vaccinating mice with pilins would lead to generation of anti-pilin antibodies, and would protect against C. difficile challenge. We found that immunizing C57Bl/6 mice with various pilins, whether combined or as individual proteins, led to low anti-pilin antibody titers and no protection upon C. difficile challenge. Passive transfer of anti-pilin antibodies led to high serum anti-pilin IgG titers, but to undetectable fecal anti-pilin IgG titers and did not protect against challenge. The low antibody titers observed in these experiments may be due to the particular strain of mice used. Further experiments, possibly with a different animal model of C. difficile infection, are needed to determine if an anti-T4P vaccine would be protective against C. difficile infection.

Urticarial Vasculitis-Associated Intestinal Ischemia.

Urticarial vasculitis (UV) is a rare small vessel vasculitis. UV is often idiopathic but can also present in the context of autoimmune disorders such as systemic lupus erythematosus, drug reactions, infections, or a paraneoplastic syndrome. Extracutaneous complications include intestinal ischemic injuries, in UV patients with nonspecific gastrointestinal symptoms such as abdominal pain and nausea. Prompt recognition and treatment can minimize morbidity and mortality. This paper describes a case of urticarial vasculitis-associated intestinal ischemia.

An Immune-Inflammation Gene Expression Signature in Prostate Tumors of Smokers.

Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer-prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers.

Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab.

Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibody-mediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fcγ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.

Integration of metabolomics and transcriptomics revealed a fatty acid network exerting growth inhibitory effects in human pancreatic cancer.

PURPOSE: To identify metabolic pathways that are perturbed in pancreatic ductal adenocarcinoma (PDAC), we investigated gene-metabolite networks with integration of metabolomics and transcriptomics. EXPERIMENTAL DESIGN: We conducted global metabolite profiling analysis on two independent cohorts of resected PDAC cases to identify critical metabolites alteration that may contribute to the progression of pancreatic cancer. We then searched for gene surrogates that were significantly correlated with the key metabolites, by integrating metabolite and gene expression profiles. RESULTS: Fifty-five metabolites were consistently altered in tumors as compared with adjacent nontumor tissues in a test cohort (N = 33) and an independent validation cohort (N = 31). Weighted network analysis revealed a unique set of free fatty acids (FFA) that were highly coregulated and decreased in PDAC. Pathway analysis of 157 differentially expressed gene surrogates revealed a significantly altered lipid metabolism network, including key lipolytic enzymes PNLIP, CLPS, PNLIPRP1, and PNLIPRP2. Gene expressions of these lipases were significantly decreased in pancreatic tumors as compared with nontumor tissues, leading to reduced FFAs. More importantly, a lower gene expression of PNLIP in tumors was associated with poorer survival in two independent cohorts. We further showed that two saturated FFAs, palmitate and stearate, significantly induced TRAIL expression, triggered apoptosis, and inhibited proliferation in pancreatic cancer cells. CONCLUSIONS: Our results suggest that impairment in a lipolytic pathway involving lipases, and a unique set of FFAs, may play an important role in the development and progression of pancreatic cancer and provide potential targets for therapeutic intervention.

Tumour antigen targeted monoclonal antibodies incorporating a novel multimerisation domain significantly enhance antibody dependent cellular cytotoxicity against colon cancer.

Tumour antigen targeted antibodies (mAbs) can induce natural killer (NK) cells to kill tumours through antibody dependent cellular cytotoxicity (ADCC) upon engagement of NK cell expressed FcγRIIIa. FcγRIIIa polymorphisms partially dictate the potency of the ADCC response. The high affinity FcγRIIIa-158-valine (V) polymorphism is associated with more potent ADCC response than the low affinity FcγRIIIa-158-phenylalanine (F) polymorphism. Because approximately 45% of patients are homozygous for the FcγRIIIa-158-F polymorphism (FF genotype), their ability to mount ADCC is impaired. We investigated whether a novel mAb capable of binding multiple antigen specific targets and engaging multiple low affinity FcγRIIIa receptors could further enhance ADCC against colon cancer in vitro. Specifically, we generated a novel anti-epidermal growth factor receptor (EGFR) antibody (termed a stradobody) consisting of an unmodified Fab sequence and two Immunoglobulin G, subclass 1 (IgG1) Fc domains separated by an isoleucine zipper domain and the 12 amino-acid IgG2 hinge. The stradobody framework induced multimerisation and was associated with increased binding to the EGFR and FcγRIIIa. From a functional perspective, when compared to an unmodified anti-EGFR mAb with a sequence identical to cetuximab (a commercially available anti-EGFR mAb), stradobodies significantly enhanced ADCC. These effects were observed using both KRAS wild type HT29 and KRAS mutant SW480 colon cancer cells as targets, and by NK cells obtained from healthy donors and a cohort of patients with colon cancer. These data suggest that high avidity cross-linking of multiple tumour surface antigens and multiple NK cell associated FcγRIIIa molecules can enhance ADCC and partially overcome impaired ADCC by FF genotype individuals in vitro.

FOXL1, a novel candidate tumor suppressor, inhibits tumor aggressiveness and predicts outcome in human pancreatic cancer.

The forkhead box L1 (FOXL1) transcription factor regulates epithelial proliferation and development of gastrointestinal tract and has been implicated in gastrointestinal tumorigenesis in mouse models. However, the role of FOXL1 in pancreatic cancer development and progression remains to be elucidated. Here, we report that higher expression of FOXL1 is significantly associated with better clinical outcome in human pancreatic ductal adenocarcinoma (PDAC). A lower FOXL1 expression is correlated with metastasis and advanced pathologic stage of pancreatic cancer. Mechanistic analyses showed that overexpression of FOXL1 induces apoptosis and inhibits proliferation and invasion in pancreatic cancer cells, whereas silencing of FOXL1 by siRNA inhibits apoptosis and enhances tumor cell growth and invasion. Furthermore, FOXL1 overexpression significantly suppressed the growth of tumor xenografts in nude mice. FOXL1 promoted apoptosis partly through the induction of TNF-related apoptosis-inducing ligand (TRAIL) in pancreatic cancer cells. In addition, FOXL1 suppressed the transcription of zinc finger E-box-binding homeobox 1 (ZEB1), an activator of epithelial-mesenchymal transition, and the negative regulation of ZEB1 contributed to the inhibitory effect of FOXL1 on tumor cell invasion. Taken together, our findings suggest that FOXL1 expression is a candidate predictor of clinical outcome in patients with resected PDAC and it plays an inhibitory role in pancreatic tumor progression.