A B S T R A C T PurposeContrary to the extensive data accumulated regarding pancreatic carcinogenesis, the clinical and molecular features characteristic of advanced stage (stage III and IV) disease are unknown. A comprehensive study of pancreatic cancers from patients who have succumbed to their disease has the potential to greatly expand our understanding of the most lethal stage of this disease and identify novel areas for intervention. Materials and MethodsRapid autopsies were performed on 76 patients with documented pancreatic cancer. The histologic features of end stage disease were determined and correlated to the stage at initial diagnosis, patterns of failure (locally destructive v metastatic disease) and the status of the KRAS2, TP53, and DPC4 genes. ResultsAt autopsy, 30% of patients died with locally destructive pancreatic cancer, and 70% died with widespread metastatic disease. These divergent patterns of failure found at autopsy (locally destructive v metastatic) were unrelated to clinical stage at initial presentation, treatment history, or histopathologic features. However, Dpc4 immunolabeling status of carcinoma tissues harvested at autopsy, a sensitive marker of DPC4 genetic status, was highly correlated with the presence of widespread metastasis but not with locally destructive tumors (P ϭ .007). ConclusionPancreatic cancers are represented by distinct genetic subtypes with significantly different patterns of failure. Determinations of DPC4 status at initial diagnosis may be of value in stratifying patients into treatment regimens related to local control versus systemic therapy.

Programmed ribosomal frameshifting is an essential mechanism used for the expression of orf1b in coronaviruses. Comparative analysis of the frameshift region reveals a universal shift site U_UUA_AAC, followed by a predicted downstream RNA structure in the form of either a pseudoknot or kissing stem loops. Frameshifting in SARS-CoV has been characterized in cultured mammalian cells using a dual luciferase reporter system and mass spectrometry. Mutagenic analysis of the SARS-CoV shift site and mass spectrometry of an affinity tagged frameshift product confirmed tandem tRNA slippage on the sequence U_UUA_AAC. Analysis of the downstream pseudoknot stimulator of frameshifting in SARS-CoV shows that a proposed RNA secondary structure in loop II and two unpaired nucleotides at the stem I-stem II junction in SARS-CoV are important for frameshift stimulation. These results demonstrate key sequences required for efficient frameshifting, and the utility of mass spectrometry to study ribosomal frameshifting.

Purpose: Aberrant activation of the Notch signaling pathway is commonly observed in human pancreatic cancer, although the mechanism(s) for this activation has not been elucidated. Experimental Design: A panel of 20 human pancreatic cancer cell lines was profiled for the expression of Notch pathway-related ligands, receptors, and target genes. Disruption of intracellular Notch signaling, either genetically by RNA interference targeting NOTCH1 or pharmacologically by means of the g-secretase inhibitor GSI-18, was used for assessing requirement of Notch signaling in pancreatic cancer initiation and maintenance. Results: Striking overexpression of Notch ligand transcripts was detectable in the vast majority of pancreatic cancer cell lines, most prominently JAGGED2 (18 of 20 cases, 90%) and DLL4 (10 of 20 cases, 50%). In two cell lines, genomic amplification of the DLL3 locus was observed, mirrored by overexpression of DLL3 transcripts. In contrast, coding region mutations of NOTCH1 or NOTCH2 were not observed. Genetic and pharmacologic inhibition of Notch signaling mitigated anchorage-independent growth in pancreatic cancer cells, confirming that sustained Notch activation is a requirement for pancreatic cancer maintenance. Further, transient pretreatment of pancreatic cancer cells with GSI-18 resulted in depletion in the proportion of tumor-initiating aldehyde dehydrogenase^expressing subpopulation and was associated with inhibition of colony formation in vitro and xenograft engraftment in vivo, underscoring a requirement for the Notch-dependent aldehyde dehydrogenase^expressing cells in pancreatic cancer initiation. Conclusions: Our studies confirm that Notch activation is almost always ligand dependent in pancreatic cancer, and inhibition of Notch signaling is a promising therapeutic strategy in this malignancy.Pancreatic cancer is an almost uniformly lethal disease with an overall 5-year survival of f5%, and this dire prognosis has not markedly improved over the last few decades (1). In the United States, f34,000 individuals succumb to this malignancy each year. To date, the only potentially curative therapeutic option is complete surgical resection, but unfortunately, the majority of patients are diagnosed at a locally advanced or distant metastatic stage, thus precluding surgical cure (2). Currently available treatment options for advanced pancreatic cancer, such as gemcitabine, have had minimal effect in ameliorating survival. Identification of aberrant signaling pathways that can also form the substrate for targeted therapies has thus become an area of foremost priority.The reactivation of embryonic signal transduction pathways, such as Notch and Hedgehog, have been reported in a variety of human cancers (3, 4); further, the availability of potent smallmolecule inhibitors has meant that these pathways can be targeted in these cancers, as we and others have recently shown

Background Vitamin D deficiency is associated with poor bone health and other adverse health outcomes. However, the associations are greatly attenuated in blacks compared with whites. One possible explanation for this attenuation could be different concentrations of bioavailable vitamin D metabolites in plasma, which are estimated using equations that include the total concentration of vitamin D binding globulin (VDBG) and haplotype-specific dissociation constants. Methods We developed a method to quantify VDBG using LC-MS/MS that could also identify the haplotypes/isoforms of VDBG present. We validated the method according to recent recommendations for publications of biomarker studies. We determined serum VDBG concentrations in samples from the Atherosclerosis Risk in Communities (ARIC) cohort and compared the results with a widely-used monoclonal immunoassay. Results An assay with a lower limit of quantification (<20% CV) of 71 µg/mL was developed using 10 µL of serum or plasma. The assay was linear from 64 to 434 µg/mL with total imprecision of 7.3–9.0% CV at ~250 µg/mL. Significant hemolysis interfered with quantification. The identification of isoforms was 97% concordant with genotyping (kappa coefficient). Method comparison with immunoassay revealed significant isoform-specific effects in the immunoassay. The concentration (SD) of VDBG by mass spectrometry was similar in whites and blacks [262 (25) vs. 266 (35) µg/mL, respectively; p=0.43]. Conclusions Validated mass spectrometric methods for the quantification of proteins in human samples are sensitive and specific and can provide additional information beyond immunoassay. Counter to prior observations by immunoassay, VDBG levels did not vary by race.