Andrea Padoan

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.

Pancreatic Tumors and Immature Immunosuppressive Myeloid Cells in Blood and Spleen: Role of Inhibitory Co-Stimulatory Molecules PDL1 and CTLA4. An In Vivo and In Vitro Study

Background Blood and spleen expansion of immature myeloid cells (IMCs) might compromise the immune response to cancer. We studied in vivo circulating and splenic T lymphocyte and IMC subsets in patients with benign and malignant pancreatic diseases. We ascertained in vitro whether pancreatic adenocarcinoma (PDAC)-associated IMC subsets are induced by tumor-derived soluble factors and whether they are immunosuppressive focusing on the inhibitory co-stimulatory molecules PDL1 and CTLA4. Methodology and Principal Findings 103 pancreatic and/or splenic surgical patients were enrolled including 52 PDAC, 10 borderline and 10 neuroendocrine tumors (NETs). Lymphocytes and IMCs were analysed by flow cytometry in blood, in spleen and in three PDAC cell conditioned (CM) or non conditioned PBMC. PDL1 and CTLA4 were studied in 30 splenic samples, in control and conditioned PBMC. IMCs were FACS sorted and co-coltured with allogenic T lymphocytes. In PDAC a reduction was found in circulating CD8+ lymphocytes (p = 0.004) and dendritic cells (p = 0.01), which were reduced in vitro by one PDAC CM (Capan1; p = 0.03). Blood myeloid derived suppressive cells (MDSCs) CD33+CD14−HLA-DR− were increased in PDAC (p = 0.022) and were induced in vitro by BxPC3 CM. Splenic dendritic cells had a higher PDL1 expression (p = 0.007), while CD33+CD14+HLA-DR− IMCs had a lower CTLA4 expression (p = 0.029) in PDAC patients. In vitro S100A8/A9 complex, one of the possible inflammatory mediators of immune suppression in PDAC, induced PDL1 (p = 0.018) and reduced CTLA4 expression (p = 0.028) among IMCs. IMCs not expressing CTLA4 were demonstrated to be immune suppressive. Conclusion In PDAC circulating dendritic and cytotoxic T cells are reduced, while MDSCs are increased and this might favour tumoral growth and progression. The reduced CTLA4 expression found among splenic IMCs of PDAC patients was demonstrated to characterize an immune suppressive phenotype and to be consequent to the direct exposure of myeloid cells to pancreatic cancer derived products, S100A8/A9 complex in particular.

An integrated system for monitoring the quality of sample transportation

OBJECTIVES Due to the consolidation of laboratory testing facilities, there is an increasing need for systems able to assure quality and safety in biological sample transportation, although little evidence on this aspect is available in literature. DESIGN AND METHODS An integrated system for sample transportation, implemented and monitored over a five-year period by our team, consists of secondary and tertiary containers, a device for temperature and time recording, and a system manager allowing the acceptance or rejection of biological samples through the immediate visualization and validation of registered data. RESULTS Data collected between 2009 and October 2011, after a preliminary phase for optimizing the temperature inside the containers, demonstrated the frequency of transportations at an acceptable temperature (<20 °C) had increased and that of transportations at an excessively high temperature (>25 °C) had decreased by ~80%. CONCLUSIONS The integrated system and related operating instructions allow improvement in the quality of sample transportation over time.

Effects of sample transportation on commonly requested laboratory tests

Abstract Background: Little evidence is available in literature on the effects of sample transportation. The aim of the present study was to evaluate the effects of an integrated system for sample transportation on the quality of six laboratory parameters considered representative of the quality of all tests requested and performed. Methods: The values of alanine aminotransferase (ALT), calcium (Ca), potassium (K), activated prothrombin time (APTT), prostate specific antigen (PSA), and hemoglobin (Hb) obtained in samples collected in peripheral centers in 2007 were compared with those obtained in 2011, following the introduction of the integrated transportation system entailing a tertiary and a secondary container, a data-logger for registering time and temperature, a mission starter and a system manager. Results: In 2007, for ALT, APTT and K, there were significant variations between findings for samples transported from long-term and those from short-term peripheral centers; following the introduction of the transportation system, no such variations were found. Conclusions: Improvement in the quality of sample transportation has been achieved, particularly for three of the six parameters evaluated, following the introduction of the integrated system described in the present study.

New screening tests enrich anti-transglutaminase results and support a highly sensitive two-test based strategy for celiac disease diagnosis.

BACKGROUND The identification of specific serological algorithms allowing the diagnosis of celiac disease (CD) is a new challenge for both the clinic and the laboratory. We compared the diagnostic accuracy of three new tests proposed for CD screening with that of the well established IgA tTG, and ascertained whether any combination of these tools might enhance accuracy in diagnosing CD. METHODS In sera from 329 CD and 374 control children, the following were assayed: IgA tTG; IgA/IgG, which identify tTG-gliadin complexes (Aeskulisa Celi Check and CeliCheck IgGA); IgA/IgG, which identify deamidated gliadin peptides and tTG (QUANTA Lite(TM) h-tTG/DGP Screen). RESULTS When specificity was set at 100%, the most sensitive index of CD was IgA tTG (75.7%, cut-off=100U), followed by QUANTA Lite(TM) h-tTG/DGP Screen (65.3%, cut-off 145U), Aeskulisa Celi Check (62.6%, cut-off 909U/mL) and CeliCheck IgGA (59.6%, cut-off 977U/mL). Three algorithms were obtained by combining IgA tTG with each of the new tests. The algorithm obtained by measuring IgA tTG and QUANTA Lite(TM) h-tTG/DGP Screen allowed the correct identification of CD in 78.7% of cases (negative predictive value=97.3%). CONCLUSIONS The two-test based strategy could be used for the cost effective diagnosis of CD.

PCA3 score of 20 could improve prostate cancer detection: Results obtained on 734 Italian individuals

BACKGROUND The role of PCa3 score in the diagnostics of prostate cancer (PCa) is still under debate, mainly due to the lack of a univocal cut-off useful alone or within nomograms proposed by Urologists. Aim of present study is to compare different PCA3 score cut-off values (20, 25, 35 and 50) observed in 734 patients with suspected PCa who were monitored for about three years with single or multiple biopsies. METHODS 734 patients who underwent first prostate biopsy for suspected PCa were enrolled. One month later the first biopsy result was obtained, both negative and positive PCa patients were investigated by means of PCA3 score, in order to establish risk of PCa presence on repeated biopsies. RESULTS PCA3 score was significantly higher (p<0.001) in PCa patients to the PCa negative ones, while tPSA did not significantly vary. The best negative predictive value (NPV 97.5%) and sensitivity (95.4%) result were obtained when a PCA3 score of 20 was used. At cut-off value of 50, the 75% of patients resulted as false positive. CONCLUSIONS PCA3 score of 20 could be safely introduced in the prostate cancer screening diagnostic flow chart, since it provides important information regarding the outcome of re-biopsy.

Usefulness of MALDI-TOF/MS identification of low-MW fragments in sera for the differential diagnosis of pancreatic cancer.

Objectives To identify new biomarkers of pancreatic cancer (PaCa), we performed MALDI-TOF/MS analysis of sera from 22 controls, 51 PaCa, 37 chronic pancreatitis, 24 type II diabetes mellitus (DM), 29 gastric cancer (GC), and 24 chronic gastritis (CG). Methods Sera were purified by Sep-Pak C18 before MALDI-TOF/MS Anchorchip analysis. Results Features present in at least 5% of all spectra were selected (n = 160, m/z range, 1200–5000). At univariate analysis, 2 features (m/z 2049 and 2305) correlated with PaCa, 3 (m/z 1449, 1605, and 2006) with DM. No feature characterized gastric cancer or chronic gastritis. Ten-fold cross-validation binary recursive partitioning trees were obtained for patients’ classification. The tree (CA 19-9, age, m/z 2006, 2599, 2753, and 4997), built considering only patients with diabetes, allowed a distinction between DM [area under the receiver operating characteristic curve (AUC), 0.997], chronic pancreatitis (AUC, 0.968), and PaCa (AUC, 0.980), with an overall correct classification rate of 89%. The tree including CA 19-9, 1550, and 2937 m/z features, achieved an AUC of 0.970 in distinguishing localized from advanced PaCa. MALDI-TOF-TOF analysis revealed the 1550 feature as a fragment of Apo-A1, which was determined as whole protein and demonstrated to be closely correlated with PaCa. Conclusions The findings made demonstrate a role for serum peptides identified using MALDI-TOF/MS for addressing PaCa diagnosis.

Pancreatic cancer alters human CD4+ T lymphocyte function: a piece in the immune evasion puzzle.

Objectives: To verify whether the dysregulation of CD4+ T cells concurs in worsening the outcome of pancreatic cancer, we compared the effects of pancreatic cancer and other gastrointestinal cancer cell-conditioned media on the (1) proliferation, migration, and differentiation of CD4+ T cells and (2) expansion of CD4+ memory (CD45RO), naive (CD45RA), activated (CD69), and regulatory (CD25) subsets. Methods: After culture of CD4+ T cells in control, pancreatic (BxPC3, Capan1, MiaPaCa2), or gastrointestinal cancer (AGS, HepG2, HT29) cell-conditioned media, we evaluated proliferation, migration, interferon &ggr; (IFN&ggr;) production, and CD45RA, CD45RO, CD69, and CD25 membrane expression in control and conditioned CD4+ T cells. Results: Only pancreatic cancer-conditioned media (1) inhibited CD4+ T-cell proliferation (P < 0.001) and migration under human stromal cell-derived factor-&agr; chemotaxis (P < 0.001) and (2) induced CD4+ T-cell IFN&ggr; production (P < 0.05) and the expansion of the CD69-positive subset (P < 0.001) with respect to the control, with no changes being found in the CD45RA, CD45RO, and CD25 subsets. Conclusions: The in vitro findings achieved in the present study demonstrate that pancreatic cancer cells inhibit CD4+ T-cell proliferation and migration, induce IFN&ggr; production, and favor a CD69+ subset expansion, suggesting that CD4+ T cells play an important role in pancreatic cancer immune evasion.

An approach for estimating measurement uncertainty in medical laboratories using data from long-term quality control and external quality assessment schemes

Abstract Background: The present study was prompted by the ISO 15189 requirements that medical laboratories should estimate measurement uncertainty (MU). Methods: The method used to estimate MU included the: a) identification of quantitative tests, b) classification of tests in relation to their clinical purpose, and c) identification of criteria to estimate the different MU components. Imprecision was estimated using long-term internal quality control (IQC) results of the year 2016, while external quality assessment schemes (EQAs) results obtained in the period 2015–2016 were used to estimate bias and bias uncertainty. Results: A total of 263 measurement procedures (MPs) were analyzed. On the basis of test purpose, in 51 MPs imprecision only was used to estimate MU; in the remaining MPs, the bias component was not estimable for 22 MPs because EQAs results did not provide reliable statistics. For a total of 28 MPs, two or more MU values were calculated on the basis of analyte concentration levels. Overall, results showed that uncertainty of bias is a minor factor contributing to MU, the bias component being the most relevant contributor to all the studied sample matrices. Conclusions: The model chosen for MU estimation allowed us to derive a standardized approach for bias calculation, with respect to the fitness-for-purpose of test results. Measurement uncertainty estimation could readily be implemented in medical laboratories as a useful tool in monitoring the analytical quality of test results since they are calculated using a combination of both the long-term imprecision IQC results and bias, on the basis of EQAs results.

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca2+]i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca2+] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca2+]i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca2+]i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca2+] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca2+]i oscillations and insulin release, probably by inducing Ca2+ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011.