Carlo-Federico Zambon

Decreased total lymphocyte counts in pancreatic cancer: an index of adverse outcome.

Objectives: An impaired host immunity might concur in determining the dismal prognosis of patients with pancreatic cancer (PC). Our aim was to ascertain whether the immunophenotype pattern of blood lymphocytes in PC correlates with tumor stage, grade, or survival. Methods: We studied 115 patients with PC, 44 with chronic pancreatitis (CP), 23 with tumors of the pancreatico-biliary tract, and 34 healthy controls (CS). Survival data were available for 77 patients with PC. Lymphocyte subsets were determined by fluorescent activated cell sorter (FACS) analysis. Results: In patients with PC, total lymphocyte counts were lower than in CP or CS, and CD8+ lymphocyte subset levels were higher with respect to CS. Lower circulating lymphocytes were found in advanced PC stages (IIB-IV; χ2 = 11.55, P < 0.05) compared with stages 0 to IIA. Cox regression analysis, made considering total lymphocyte counts and tumor stage as covariates, was found to be significant for both tumor stage (P < 0.001) and total lymphocyte counts (P < 0.05). Conclusions: The reduction of total lymphocytes in blood is the main immunologic change in advanced PC. The survival of these patients depends mainly on tumor stage, but it is also affected by the number of circulating lymphocytes, suggesting that the immune system plays an important role in pancreatic adenocarcinoma immunosurveillance and immunoediting.

Antibodies against Synthetic Deamidated Gliadin Peptides for Celiac Disease Diagnosis and Follow-Up in Children

BACKGROUND AGA IgA II and AGA IgG II have recently been suggested as reliable tools for celiac disease (CD) diagnosis. We compared their utility for diagnosis and monitoring CD in children with that of tTG IgA, an established CD marker. METHODS We studied a cohort of 161 CD and 129 control children in whom CD was histologically confirmed or ruled out. We followed 37 children with CD on a gluten-free diet for 12-84 months. In fasting sera, we measured AGA IgA II, AGA IgG II, and tTG IgA using ELISAs. RESULTS The best sensitivity (92.5%), specificity (97.6%), positive predictive value (98%), and negative predictive value (91.2%) were obtained using tTG IgA. AGA IgG II correctly identified 3 of 3 children with CD with total IgA deficiency who had negative AGA IgA II and tTG IgA results. In children <2 years old without total IgA deficiency, AGA IgG II and tTG IgA performed equally well (sensitivity 96.4% and specificity 100%). AGA IgA II, AGA IgG II, and tTG IgA concentrations diminished significantly (P < 0.0001) after 1 year of a gluten-free diet, reaching values below the cutoff in 87%, 70%, and 51% of cases, respectively. CONCLUSIONS The best available index for diagnosing CD in children was tTG IgA. In infants <2 years old, AGA IgG II performed as well as tTG IgA in cases without total IgA deficiency and allowed detection of CD when total IgA was <0.06 g/L. Gluten-free diet monitoring can be achieved using any of the studied serum markers.

Mitochondrial DNA D-loop in pancreatic cancer: somatic mutations are epiphenomena while the germline 16519 T variant worsens metabolism and outcome.

We ascertained the frequency of mitochondrial DNA (mtDNA) D-loop region somatic mutations in pancreatic cancer (PC) and verified whether polymorphisms were linked to diagnosis, prognosis, and PC-associated diabetes mellitus (DM) in 99 PC cases, 42 chronic pancreatitis (CP) cases, 18 pancreatobiliary tract tumors, and 87 healthy control subjects (CSs). Tissue samples were obtained from 19 patients with PC and 5 with CP. The D-loop region was sequenced from all tissue samples and from blood DNA of the same patients and 12 CSs. D-loop somatic mutations were found in 3 PC tissue samples (16%). Four single nucleotide polymorphisms (SNPs; T152C, T16189C, T16519C, A73G), more frequently found in PC than in CS, were analyzed by denaturing high-performance liquid chromatography-restriction fragment length polymorphism using blood DNA as the starting template in all cases. The T allele of 16519 SNP correlated with DM. The survival of patients with PC correlated with tumor stage and grade and with DM at diagnosis. When survival analysis was performed considering only patients with locally advanced disease, the T allele of mtDNA 16519 SNP correlated with shorter life expectancy. mtDNA D-loop somatic mutations, rarely found in PC, cannot be considered causative events for this tumor type and probably are epiphenomena; the mtDNA D-loop 16519 variant, which worsens PC prognosis, seems to be a predisposing genetic factor for DM.

CEA mRNA identification in peripheral blood is feasible for colorectal, but not for gastric or pancreatic cancer staging.

Objective: It has been suggested that the molecular identification of cancer cells in the circulation may be useful in predicting the presence of micrometastasis in several cancer types. The aim of the present study was therefore to assess the feasibility of CEA mRNA identification in blood for diagnosing and staging colorectal, gastric and pancreatic cancer. Methods: We studied 16 control subjects, 69 patients with colorectal (CRC), 30 with gastric (GC), 27 with pancreatic cancer (PC) and 8 with benign diseases of the pancreatobiliary tree. At diagnosis CEA mRNA was identified in peripheral blood by means of a RT-PCR procedure. Results: The specificity of this test in control subjects was 94%, and its sensitivity in identifying CRC, GC and PC were 34, 37 and 41%, respectively. False-positive findings were recorded in 25% patients with benign diseases. No association was found between CEA mRNA and stage in patients with GC or PC. In CRC patients, positive CEA mRNA findings were correlated with local spread (χ2 = 14.6, p < 0.01), lymph node (χ2 = 18.95, p < 0.001) and distant metastasis (χ2 = 11.3, p < 0.001). In these cases, CEA mRNA, but not CEA, was entered in stepwise discriminant analysis to classify the presence of lymph node metastasis. Conclusions: The molecular detection of micrometastasis in the blood by means of CEA mRNA identification is feasible for colorectal, but not for gastric or pancreatic cancer staging. Further studies are needed in order to define the clinical utility of this marker also in follow-up protocols.

VKORC1, CYP2C9 and CYP4F2 genetic-based algorithm for warfarin dosing: an Italian retrospective study.

AIM A total of 371 patients under stable warfarin therapy were retrospectively selected to develop a pharmacogenetic algorithm to identify the individual maintenance dose. MATERIALS & METHODS The variables that were entered into the algorithm were: VKORC1, CYP2C9 and CYP4F2 polymorphisms, body surface area and age. RESULTS The percentage of cases whose predicted mean weekly warfarin dose was within 20% of the actual maintenance dose was 51.8% considering patients overall, and were 36.2, 66.2 and 55.4%, respectively, taking into account patients requiring low (≤25 mg/week), intermediate (25-45 mg/week) and high (≥45 mg/week) doses. The algorithm could correctly assign 73.8 and 63.2% of patients to the low- and high-dose regimens, respectively. We developed and validated a pharmacogenetic algorithm in a series of Italian patients, we then tested, in the same series of italian patients, the formulas of three published algorithms. These three algorithms were developed and validated by their authors in a series of patients different from our own. The performance of our algorithm in our patients series was slightly higher than that achieved when using the three other algorithms in our patients series. CONCLUSION The high predictive accuracy of low and high warfarin requirements of our algorithm warrants its application in prospective studies for clinical validation.

Helicobacter pylori Infection in Children and Adults: A Single Pathogen But a Different Pathology

Background. The aims of this retrospective study were to ascertain in large series of children and adults: the relationship of the infecting strain to gastric mucosal lesions; and the relationship of the infecting strain to its duodenal localization.

Altered glucose metabolism and proteolysis in pancreatic cancer cell conditioned myoblasts: searching for a gene expression pattern with a microarray analysis of 5000 skeletal muscle genes

Background and aims: We verified whether conditioned media (CM) from pancreatic cancer cell lines (MIAPaCa2, CAPAN-1, PANC-1, BxPC3) alter glucose metabolism and gene expression profiles (microarray experiment with a platform of 5000 skeletal muscle cDNA) in mice myoblasts. Methods: Myoblasts were incubated with control or pancreatic cancer CM for 24 and 48 hours. Results: Lactate significantly increased in CM compared with non-conditioned myoblasts. No variations in expression levels of the main genes involved in glycolysis were found in CM myoblasts. Propionyl coenzyme A carboxylase and isocitrate dehydrogenase 3 beta genes, which encode enzymes of the tricarboxylic acid cycle, were overexpressed, while IGFIIR and VAMP5 genes were underexpressed in CM myoblasts. PAFAH1B1 and BCL-2 genes (intracellular signal transduction) and the serine protease cathepsin G (proteolysis), were overexpressed in CM myoblasts. Tyrosine accumulation in CM myoblasts suggested that proteolysis overcomes protein synthesis. Sorcin, actin alpha, troponin T1, and filamin A were underexpressed in CM myoblasts. Conclusions: Our findings demonstrate that pancreatic cancer cell conditioned media enhanced lactate production and induced proteolysis, possibly by altering expression levels of a large number of genes, not only those involved in protein biosynthesis and degradation or glucose metabolism, but also those involved in the tricarboxylic acid cycle and in vesicle traffic.

Interleukin 12 gene polymorphisms enhance gastric cancer risk in H pylori infected individuals

Bacterial, environmental, population related, and individual host factors are major determinants of the outcome of H pylori infection.1,2 Many bacterial virulence genes—including the pathogenicity island cagA , the s1m1 vacA alleles, babA2 , sabA , and oipA —have been associated with a higher degree of gastric mucosal inflammation, intestinal metaplasia, gastric or duodenal ulcer, gastric adenocarcinoma, and MALToma.1,3–7 H pylori triggers and maintains gastric mucosal inflammation by different mechanisms, which are partly strain dependent and partly strain independent.1 T and B lymphocyte activation and infiltration of the gastric mucosa depend on H pylori antigen processing. The number of infiltrating polymorphonuclear cells varies depending on the virulence of the infecting strain, being much greater when infections are caused by cagA positive strains.3,5,7–9 The inflammatory cells infiltrating H pylori infected gastric mucosa produce a pattern of proinflammatory cytokines.10,11 High mucosal levels of mononuclear cytokines (IL8, IL6, IL1β, tumour necrosis factor α (TNFα), and interferon γ (IFNγ)) and lymphocytic derived cytokines (IL2, IL2R) have been described in H pylori infected patients.10–13 H pylori infection also induces the production of IL12,14–16 a heterodimeric proinflammatory protein that triggers the production of IFNγ and favours the differentiation of T helper 1 (Th1) cells,17,18 which, in H pylori infected mucosa, prevail over Th2 cells.15,16,19 The ability of IL12 to induce Th1 is one of the biological bases of the importance of this cytokine in resisting most bacteria, including H pylori , and also intracellular protozoa and fungal pathogens.18,20,21 Cellular sources of IL12 in response to infections are mainly dendritic cells and phagocytes.16–21 The two subunits of IL12—p35 and p40—are encoded by different genes, named IL12A and IL12B respectively, which are unrelated …

Pancreatic cancer biomarkers discovery by surface-enhanced laser desorption and ionization time-of-flight mass spectrometry.

Abstract Background: Surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF/MS), a laboratory-friendly technique, is used to identify biomarkers for cancer. The aim of the present study was to explore the application of SELDI proteomic patterns in serum for distinguishing between cases of pancreatic cancer, chronic pancreatitis, type 2 diabetes mellitus and healthy controls. Methods: Sera from 12 healthy controls, 24 patients with type 2 diabetes mellitus, 126 with pancreatic cancer, including 84 with diabetes, and 61 with chronic pancreatitis, 32 of which were diabetics, were analyzed using SELDI-TOF/MS. Spectra (IMAC-30) were clustered and classified using Biomarker Wizard and Biomarker Pattern software. Results: Two decision tree classification algorithms, one with and one without CA 19-9, were constructed. In the absence of CA 19-9, the splitting protein peaks were: m/z 1526, 1211, and 3519; when CA 19-9 was used in the analysis, it replaced the m/z 3519 splitter. The two algorithms performed equally for classifying patients. A classification tree that considered diabetic patients only was constructed; the main splitters were: 1211, CA 19-9, 7903, 3359, 1802. With this algorithm, 100% of patients with type 2 diabetes mellitus, 97% with chronic pancreatitis and 77% of patients with pancreatic cancer were correctly classified. SELDI-TOF/MS features improved the diagnostic accuracy of CA 19-9 (AUC=0.883 for CA 19-9; AUC=0.935 for CA 19-9 and SELDI-TOF/MS features combined). Conclusions: SELDI-TOF/MS allows identification of new peptides which, in addition to CA 19-9, enable the correct classification of the vast majority of patients with pancreatic cancer, which can be distinguished from patients with chronic pancreatitis or type 2 diabetes mellitus. Clin Chem Lab Med 2009;47:713–23.

Inflammatory bowel diseases: from pathogenesis to laboratory testing

Abstract Inflammatory bowel diseases (IBDs), which comprise the two major clinical subtypes, Crohn’s disease and ulcerative colitis, incur high morbidity and potential mortality. The present study reviews data on the pathogenesis and diagnosis of IBDs. The pathogenesis depends on complex interactions between susceptibility genes, environmental factors, and innate and adaptive immunity, the understanding of which is crucial to discovering novel laboratory biomarkers. Traditional laboratory tests for the diagnosis, prognosis and assessment of disease activity of IBDs are reported on, and the biochemical properties, pre-analytical and analytical aspects and clinical utility of the fecal markers lactoferrin and calprotectin are described. DNA testing and established (ASCA and pANCA) and emerging (ACCA, ALCA, AMCA, OmpC) serum markers are described; a further aspect to be addressed is the clinical use of pharmacogenetics for the treatment of IBDs.