Sex-related differences in serum biomarker levels predict the activity and efficacy of immune checkpoint inhibitors in advanced melanoma and non-small cell lung cancer patients.

BACKGROUND: Immune Checkpoint Inhibitors (ICIs) lead to durable response and a significant increase in long-term survival in patients with advanced malignant melanoma (MM) and Non-Small Cell Lung Cancer (NSCLC). The identification of serum cytokines that can predict their activity and efficacy, and their sex interaction, could improve treatment personalization. METHODS: In this prospective study, we enrolled immunotherapy-naïve patients affected by advanced MM and NSCLC treated with ICIs. The primary endpoint was to dissect the potential sex correlations between serum cytokines (IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, MCP-1, TNF-ɑ, IP-10, VEGF, sPD-L1) and the objective response rate (ORR). Secondly, we analyzed biomarker changes during treatment related to ORR, disease control rate (DCR), progression free survival (PFS) and overall survival (OS). Blood samples, collected at baseline and during treatment until disease progression (PD) or up to 2 years, were analyzed using Luminex xMAP or ELLA technologies. RESULTS: Serum samples from 161 patients (98 males/63 females; 92 MM/69 NSCLC) were analyzed for treatment response. At baseline, IL-6 was significantly lower in females (F) versus males (M); lower levels of IL-4 in F and of IL-6 in both sexes significantly correlated with a better ORR, while higher IL-4 and TNF-ɑ values were predictive of a lower ORR in F versus M. One hundred and sixty-five patients were evaluable for survival analysis: at multiple Cox regression, an increased risk of PD was observed in F with higher baseline values of IL-4, sPD-L1 and IL-10, while higher IL-6 was a negative predictor in males. In males, higher levels of GM-CSF predict a longer survival, whereas higher IL-1ß predicts a shorter survival. Regardless of sex, high baseline IL-8 values were associated with an increased risk of both PD and death, and high IL-6 levels only with shorter OS. CONCLUSIONS: Serum IL-1ß, IL-4, IL-6, IL-10, GM-CSF, TNF-ɑ, and sPD-L1 had a significant sex-related predictive impact on ORR, PFS and OS in melanoma and NSCLC patients treated with ICIs. These results will potentially pave the way for new ICI combinations, designed according to baseline and early changes of these cytokines and stratified by sex.

Perfluoroalkyl substances and thyroid stimulating hormone levels in a highly exposed population in the Veneto Region.

BACKGROUND: Per- and poly-fluoroalkyl substances (PFAS) are persistent and widespread environmental pollutants. People living in Veneto Region (Italy) have been exposed from the late 1970s to 2013 to elevated concentrations of PFAS through drinking water. The effect of PFAS on thyroid function is still controversial and studies focusing on thyroid stimulating hormone (TSH) have shown inconsistent results. The aim of this study was to evaluate the association between serum PFAS and TSH levels and its dose-response relationship in a large population of highly exposed individuals. METHODS: A cross-sectional study was conducted on 21,424 individuals aged 14-39 living in the contaminated area. In the main analysis, participants with prevalent thyroid disease and pregnant women were excluded. Serum levels of perfluorooctanoic acid (PFOA), perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS) and perfluorononanoic acid (PFNA) were measured. Generalized Additive Models were used to evaluate the association between TSH levels and serum PFAS, using thin plate spline smooth terms to model the potential non-linear relationship. Models were stratified by sex and age group and adjusted for potential confounders. A secondary analysis was conducted to evaluate the association between PFAS with prevalent self-reported thyroid disorders. RESULTS: We found no association between TSH and any type of PFAS among adolescents or women. A decrease in TSH concentration was observed in association with an IQR increase in PFHxS and a mild decrease in TSH at low levels of PFOA, PFOS and PFHxS among male adults. Self-reported thyroid disease was more common among women with higher levels of PFNA concentrations, whereas all other PFAS were not associated with thyroid diseases regardless of sex or age. CONCLUSIONS: Overall there is no evidence of an association between TSH and PFAS. However, some results are suggestive of a possible inverse association of TSH with PFOA, PFOS and PFHxS among adult males.

Perfluoroalkyl substances are associated with elevated blood pressure and hypertension in highly exposed young adults.

BACKGROUND: Residents in a large area of North-Eastern Italy were exposed to perfluoroalkyl substances (PFAS) via drinking water. Studies on the association between PFAS and blood pressure levels are limited, and results are inconsistent. Using cross-sectional data from the Regional health surveillance program, we aimed to quantify the associations between PFAS serum concentrations and blood pressure and hypertension prevalence. METHODS: The study comprised 16,224 individuals aged 20-39 years. Pregnant women (n = 327), or individuals with missing information on the selected covariates (n = 111) were excluded, leaving 15,786 subjects for the analyses. Hypertension was defined as any self-reported diagnosis, use of antihypertensive drugs, or elevated systolic blood pressure (SBP ≥ 140 mmHg)/diastolic blood pressure (DBP ≥ 90 mmHg). Generalized additive models were used to investigate the relation between perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS), perfluorohexane sulfonic acid (PFHxS), and perfluorononanoic acid (PFNA)) natural log (ln) transformed and by decile, and SBP, DBP, hypertension, adjusted for potential confounders. RESULTS: Both SBP and DBP increased significantly with an increase in the ln-transformed serum PFAS concentrations in a monotonic way. The predicted increase in SBP and DBP were 1.54 mmHg (95%CI 0.61-2.47), 1.60 mmHg (95%CI 0.92-2.27) from lowest to highest decile of PFOA. The associations were stronger for SBP in men and for DBP in women. One unit increase in each In-transformed PFAS was positively associated with an increased odd of hypertension in men: PFOA OR = 1.06 (1.01-1.11), PFOS OR = 1.13 (1.03-1.23), PFHxS OR = 1.08 (1.02-1.15), PFNA OR = 1.20 (1.02-1.40). CONCLUSIONS: Our findings suggest that serum PFAS concentrations were associated with increased systolic and diastolic blood pressure in a large highly exposed young adult population. Although the magnitude of the observed effect was relatively small, if confirmed it would be of public health relevance since even small increases in blood pressure levels at the population level may be associated to a raised risk of adverse outcomes such as cardiovascular disease and target organ damage.

Insufficient uptake of systematic search methods in oncological clinical practice guideline: a systematic review.

BACKGROUND: The use of systematic review methods are widely recognized to be essential in the development of recommendations in clinical practice guidelines to prove their trustworthiness. The objective of this study was to assess the use of systematic search methods by authors of guidelines published in the oncology field. METHODS: We analyzed 590 guidance documents identified in PubMed, NGC, GIN and web sites for guidelines in 2009-2015 in oncology. The main outcome measure used was incidence of guidance documents supported by a systematic search of the literature. In addition to descriptive analyses, logistic regression was used to evaluate if adequate search methods were explained by guideline characteristics. RESULTS: Of 590 guidance documents included in the study, 305 (51.7%) declared the use of systematic search methods but only 168 (28.5%) applied methods meeting minimum standards for quality and provided sufficient details to allow classification. 164 (27.8%) guidance documents did not report any use of literature evaluation. Guidance documents produced by a Government Agency in North America (OR 2.16, 95% CI 1.16-4.17) and those with a focused scope (OR 2.35, 95% CI 0.97-5.56) were positively associated with the use of systematic search methods. We found no association between the year of publication and use of systematic search methods. CONCLUSIONS: A relatively small number of guidance documents was informed by scientific evidence identified through adequate systematic search methods. We observed substantial room for improvement of applied methods and reporting, especially in documents with a broad focus, or those produced by professional societies or independent expert panels in other continents than North America.

Preanalytical stability of [-2]proPSA in whole blood stored at room temperature before separation of serum and plasma: implications to Phi determination.

Background [-2]proPSA seems to outperform free/total prostate-specific antigen (PSA) ratio in prostate cancer diagnosis. However, [-2]proPSA stability remains an underestimated issue. We examined [-2]proPSA stability over time in whole blood before separation of serum and plasma and its implications for prostate health index (Phi) determination. Total PSA (tPSA) and free PSA (fPSA) stabilities were also assessed. Methods Blood was drawn from 26 patients and separated in two tubes for plasma (K2EDTA and K2EDTA plus protease inhibitors - P100) and one for serum (clot activator plus gel separator). Tubes were stored at room temperature before centrifugation 1, 3 and 5 h for serum and EDTA plasma or 1 and 5 h for P100 plasma. To investigate the influence of gel separator on markers' stability, blood was collected from 10 patients in three types of tubes to obtain serum: tubes with clot activator plus gel separator, with silica particles or glass tubes. Biomarkers were assayed with chemiluminescent immunoassays. Results [-2]proPSA and Phi levels significantly and progressively increased over time in serum (+4.81% and +8.2% at 3 h; +12.03% and +14.91% at 5 h, respectively, vs. 1 h; p<0.001). Conversely, [-2]proPSA levels did not change in plasma (EDTA or P100). tPSA levels did not change over time in serum or plasma, whereas fPSA decreased in serum. All markers were higher in plasma than in serum at any time point. This difference did not seem to be attributable to the use of gel for serum preparation. Conclusions EDTA prevented spurious in vitro modifications in PSA-related isoforms, confirming that a stabilized blood sample is a prerequisite for [-2]proPSA measurement and Phi determination.

Shed HER2 surrogacy evaluation in primary breast cancer patients: a study assessing tumor tissue HER2 expression at both extracellular and intracellular levels.

The aim of the present study was to investigate serum HER2 extracellular domain (ECD) as a putative surrogate marker of the shedding phenomenon of HER2 receptor from the tumor tissue of primary breast cancer (BC) patients. A pilot retrospective study was conducted on 100 matched serum and tissue samples from patients with node-positive primary BC, stage II/III. Analysis of association and concordance between serum HER2 ECD levels (measured by chemiluminescence immunoassay) and the expression in matched tumor tissue of HER2 ECD and intracellular receptor domain (ICD) (determined by immunohistochemistry) were performed. The median serum HER2 ECD level was 9.4 ng/ml and cutoff values were set at 15.2 ng/ml or 13.0 ng/ml. HER2 ICD and ECD were overexpressed in tumor tissue of 19.8% and 6.9% of patients, respectively. Statistically significant associations were found between serum HER2 ECD levels and tissue expression of both HER2 ICD and ECD (p < .001; Fisher analysis). Moreover, strong concordances were found between serum HER2 ECD levels and tissue expression of HER2 ICD or ECD (cutoff 15.2 ng/ml: 80 and 92.5%, respectively). Our findings support a role for serum HER2 ECD as a surrogate marker of tissue HER2 status in primary BC, both for HER2 ICD or ECD expression.

Human Chorionic Gonadotropin Assays for Testicular Tumors: Closing the Gap between Clinical and Laboratory Practice.

BACKGROUND: Clinical practice guidelines recommend the measurement of human chorionic gonadotropin (hCG) and/or hCGß in serum for management of testicular germ cell tumors (GCTs). These guidelines, however, disregard relevant biochemical information on hCG variants to be detected for oncological application. We set out to provide a critical review of the clinical evidence together with a characterization of the selectivity of currently marketed hCG immunoassays, identifying assays suitable for management of GCTs. CONTENT: Evidence sources in the available literature were critically appraised. Most instances of misdiagnosis and mismanagement of testicular GCTs have been associated with hCG results. According to the clinical evidence, 36% of patients with seminoma show an exclusive hCGß increase, and 71% of patients with nonseminomatous GCTs (NSGCTs) show an increase of intact hCG and/or hCG + hCGß, whereas the hCGß increase in NSGCTs is variable according to the tumor stage and histology. SUMMARY: hCG + hCGß assays that display an equimolar recognition of hCG and hCGß, or at least do not overtly underestimate hCGß, may be employed for management of testicular GCTs. Assays that underestimate hCGß are not recommended for oncological application. In addition to the hCG + hCGß assay in service, an additional assay with broader selectivity for other hCG variants should be considered when false-negative or false-positive results are suspected on the basis of clinical data.

Correction to: Observational study on the prognostic value of testosterone and adiposity in postmenopausal estrogen receptor positive breast cancer patients.

Following publication of the original article [1], the authors reported that the affiliation of author Annalisa Orenti was omitted.

Observational study on the prognostic value of testosterone and adiposity in postmenopausal estrogen receptor positive breast cancer patients.

BACKGROUND: Despite the clear endocrine-metabolic relationship between androgenic activity and adiposity, the role of androgens in breast cancer prognosis according to patient's adiposity is scarcely explored. Here, we aimed at investigating the prognostic value of circulating testosterone in association with patient's body mass index (BMI). METHODS: Circulating testosterone and BMI were evaluated at breast cancer diagnosis in 460 estrogen receptor (ER)-positive postmenopausal patients. Local relapse, distant metastasi(e)s and contralateral breast cancer were considered recurrence events. The Kruskal-Wallis test was performed to evaluate if testosterone levels differed within subgroups of categorical tumour characteristics. The Cox proportional hazard regression model was fitted to estimate the impact of standard prognostic factors on relapse-specific hazard ratio (HR). After backward selection, a model including continuous testosterone level, BMI categories (< 25, normal-weight; =25-30, overweight; ≥30 kg/m2, obese), tumour size and lymph nodes number was fitted. Furthermore, Cox models provided the relapse-specific HRs for median, third quartile and 95th percentile compared to the first quartile of testosterone levels, stratified by BMI categories. RESULTS: During a median follow up of 6.3 years, 45 patients relapsed. Testosterone levels significantly increased across BMI categories (p = 0.001). Both circulating testosterone and BMI were positively associated with disease free survival (p = 0.005 and p = 0.021, respectively). A significant interaction was found between testosterone and BMI (p = 0.006). For normal-weight women, testosterone concentration around median (0.403 ng/mL) or third quartile (0.532 ng/mL) showed a high significant HR of relapse (5.52; 95% CI:1.65-18.49 and 4.55; 95% CI:1.09-18.98, respectively). Overweight patients showed increased HR at increasing testosterone levels, reaching a significant high HR (4.68; 95% CI:1.39-15.70) for testosterone values of 0.782 ng/mL (95th percentile). For obese patients HR decreased (not significantly) at increased testosterone concentrations, explaining the interaction between testosterone levels and BMI categories. CONCLUSIONS: In ER-positive postmenopausal breast cancer patients, high testosterone levels are associated with worse prognosis in normal-weight and overweight women, whereas in obese seems to be associated with a better outcome. Although the results require further validation, they suggest that assessment of circulating testosterone and BMI could help to identify postmenopausal ER-positive patients at higher risk of relapse and potentially open new therapeutic strategies.

Phytosome complex of curcumin as complementary therapy of advanced pancreatic cancer improves safety and efficacy of gemcitabine: Results of a prospective phase II trial.

A large body of biomedical evidence indicates that activation of Nrf2 by curcumin increases the nucleophilic tone and damps inflammation cumulatively supporting the malignant phenotype. Conversely, genetic analyses suggest a possible oncogenic nature of constitutive Nrf2 activation since an increased nucleophilic tone is alleged increasing chemoresistance of cancer cells. Aiming to contribute to solve this paradox, this study addressed the issue of safety and efficacy of curcumin as complementary therapy of gemcitabine on pancreatic cancer. This was a single centre, single arm prospective phase II trial. Patients received gemcitabine and Meriva®, a patented preparation of curcumin complexed with phospholipids. Primary endpoint was response rate, secondary endpoints were progression free survival, overall survival, tolerability and quality of life. Analysis of inflammatory biomarkers was also carried out. Fifty-two consecutive patients were enrolled. Forty-four (13 locally advanced and 31 metastatic) were suitable for primary endpoint evaluation. Median age was 66 years (range 42-87); 42 patients had Eastern Cooperative Oncology Group performance status 0-1. The median number of treatment cycle was 4.5 (range 2-14). We observed 27.3% of response rate and 34.1% of cases with stable disease, totalizing a disease control rate of 61.4%. The median progression free survival and overall survival were 8.4 and 10.2 months, respectively. Higher IL-6 and sCD40L levels before treatment were associated to a worse overall survival (p < 0.01). Increases in sCD40L levels after 1 cycle of chemotherapy were associated with a reduced response to the therapy. Grade 3/4 toxicity was observed (neutropenia, 38.6%; anemia, 6.8%). There were no significant changes in quality of life during therapy. In conclusion, the complementary therapy to gemcitabine with phytosome complex of curcumin is not only safe but also efficiently translate in a good response rate in first line therapy of advanced pancreatic cancer.

HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) as diagnostic tools for ovarian cancer in patients with a pelvic mass: An Italian multicenter study.

OBJECTIVE: This multicenter study aims to evaluate HE4, CA125 and risk of ovarian malignancy algorithm (ROMA) performance in the differential diagnosis of epithelial ovarian cancer (EOC). METHODS: A total of 405 patients referred to gynecological oncologist with suspicious pelvic mass requiring a surgery for identification of EOC were consecutively enrolled; 387 patients satisfied inclusion criteria: 290 benign diseases; 15 borderline neoplasia and 82 tumors (73 EOC). RESULTS: Good diagnostic performance in discriminating benign from EOC patients was obtained for CA125, HE4 and ROMA when calculating optimal cut-off values: premenopause, specificity (SP) >86.6, sensitivity (SN) >82.6, area under the curves (AUC)≥0.894; postmenopause, SP>93.2, SN>82, AUC≥0.928. Fixing SP at 98%, performance indicators obtained for benign vs EOC patients were: premenopause, SN:65.2%, positive predictive value (+PV): 75%, positive likelihood ratio (+LR): 26.4 for CA125; SN:69.6%, +PV:76.2%, +LR:28.1 for HE4; SN:69.6%, +PV: 80%; +LR:35.1 for ROMA; postmenopause, SN:88%, +PV: 95.7%, +LR:38.7 for CA125; SN:78%, +PV:95.1%, +LR:34.3 for HE4; SN:88%, +PV:97.8%, +LR:77.4 for ROMA. When using routine cut-off thresholds, ROMA showed better well-balanced values of both SP and SN (premenopause, SN:87%, SP:86.1%; postmenopause, SN:90%; SP:94.3%). CONCLUSIONS: Overall, ROMA showed well balanced diagnostic performance to differentiate EOC from benign diseases. Meaningful differences of +PVs and +LRs between HE4 and CA125 suggest that the two markers may play at least in part different roles in EOC diagnosis, with HE4 seeming to be more efficient than CA125 in ruling in EOC patients in the disease group, also in early stages tumors, both in pre and postmenopause.

An epidemiology-based model as a tool to monitor the outbreak of inappropriateness in tumor marker requests: a national scale study.

BACKGROUND: Evaluation of appropriateness of laboratory tests on the basis of individual requests remains a serious problem as the clinical question is usually not reported with the test order. This study explored the comparison of the rate of tumor marker orders with cancer prevalence as a putative indicator of inappropriateness. METHODS: Tumor marker orders (2011 and 2012) were obtained from the Ministry of Health and cancer prevalence from the Italian Association of Cancer Registries. The rate of tumor marker orders was matched with demographic data and tumor prevalence and examined by using the confidence interval approach. Region-to-region and year-to-year variations were also examined. Focus was placed on CEA, CA125, CA19.9 and CA15.3. RESULTS: Tumor markers ordered in Italy were 13,207,289 in 2012 (221.3/1000 individuals). Given an estimated prevalence of 2,243,953 cancer cases, 7.04 tumor markers appear to be requested for each prevalent case of epithelial cancer per year. The rate of requests of CEA, CA125, CA19.9 and CA15.3 (in aggregate 5,834,167 requests in 2012, 44.2% of total) from the first and the last ranked region (96 and 244/1000 individuals) are significantly different (p<0.01). Region-to-region differences do not correspond to any known variation of prevalence in the different regions. CONCLUSIONS: The developed approach provides a proxy indicator of inappropriateness showing that tumor markers are overused in Italy and their ordering pattern is not related to tumor prevalence. The model is suitable to be validated in other laboratory tests used in diseases whose prevalence is known.

Sirtuin 1 stabilization by HuR represses TNF-α- and glucose-induced E-selectin release and endothelial cell adhesiveness in vitro: relevance to human metabolic syndrome.

Chronic inflammation and hyperglycaemia, typical features of metabolic diseases, trigger endothelial damage and release of E-selectin, a marker of endothelial activation. In the present study, we investigated molecular pathways involved in the regulation of endothelial cell activation induced by tumour necrosis factor-α (TNF-α) and high glucose. In cultured human umbilical vein endothelial cells (HUVECs), we studied the role of HuR, an ELAV (embryonic lethal, abnormal vision, Drosophila) family RNA-binding protein, and Sirtuin 1 (SIRT1) on E-selectin release and cell adhesion at different glucose concentrations. HuR expression and binding to SIRT1 were also analysed ex vivo in peripheral blood mononuclear cells (PBMCs) of subjects with and without the metabolic syndrome (MS), by immunoprecipitation (IP) of the ribonucleoprotein (RNP) complex. We found that SIRT1 overexpression prevented TNF-α- and high-glucose-dependent nuclear factor-κB (NF-κB)-p65 acetylation, E-selectin promoter activity, E-selectin release and adhesion of THP-1 cells to HUVECs. The same was mimicked by HuR overexpression, which binds and stabilizes SIRT1 mRNA. Importantly, in PBMCs of individuals with MS compared with those without, SIRT1 expression was lower, and the ability of HuR to bind SIRT1 mRNA was significantly reduced, while plasma E-selectin was increased. We conclude that post-transcriptional stabilization of SIRT1 by HuR represses inflammation- and hyperglycaemia-induced E-selectin release and endothelial cell activation. Therefore, increasing SIRT1 expression represents a strategy to counter the accelerated vascular disease in metabolic disorders.

An epidemiology-based model to estimate the rate of inappropriateness of tumor marker requests.

BACKGROUND: Appropriateness of tumor markers (TMs) has been retrospectively studied in limited patients' series, matching the requests to clinical records. Methods to monitor appropriateness suitable for use on a large scale are required. This study aims to establish and validate an innovative model to estimate appropriateness based on the comparison between the number of TMs requested and the expected requests inferred from epidemiological data. METHODS: The number of CA15.3, CA19.9 and CA125 requests theoretically expected according to the epidemiology of malignancies in a known geographic area (2 Italian regions) was compared with the number of TMs actually requested - the surveyed requests projected on a regional scale - during a given time span (1 year). The expected number of requests was calculated comparing TMs recommended by guidelines in different clinical scenarios with the prevalence or incidence figures of the examined diseases (carcinomas of breast, pancreas and biliary tract, ovary and endometrium). RESULTS: Suitability of the model was demonstrated with the analysis of 1,891,070 TM requests surveyed in 66 laboratories from Veneto and Tuscany regions. The percentage difference over the total of expected TMs (delta%) ranged from -6.9% for CA15.3 to +1022.6% for CA19.9 in Veneto and from +35.7% for CA15.3 to +1842.6% for CA19.9 in Tuscany. CONCLUSIONS: The presented model was effective in demonstrating higher than expected TM request rates, possibly associated with inappropriate use. Moreover, it can be applied on a large scale survey setting since it circumvents the unavailability of clinical information on test orders.

The role of HE4 in ovarian cancer follow-up: a review.

OBJECTIVE: The aim of this review was to analyze the state of the art about HE4 and follow-up in patients treated for ovarian cancer. METHODS: A literature search was conducted in the MEDLINE database using the key words "HE4" and "ovarian cancer" and "recurrence" or "relapse" or "follow up." RESULTS: Seven of 28 clinical studies were selected. Four studies were prospective, and all of them were based on a small number of patients (8-73 women). A failure of HE4 levels to normalize at completion of standard therapy may indicate a poor prognosis, thus suggesting the need of a closer follow-up. Moreover, HE4 showed better sensibility and specificity in the diagnosis of ovarian cancer recurrence with respect to CA-125, being also an earlier indicator of the relapse with a lead time of 5 to 8 months. HE4 showed a better performance in this setting if performed in association with other markers (CA-125, CA-72.4). HE4 seems to be an independent predictive factor for the surgical outcome at secondary cytoreductive surgery and to maintain its prognostic role even after the recurrence. CONCLUSIONS: These preliminary data start to suggest a superiority of HE4 over CA-125 in the detection of ovarian cancer recurrence. Moreover, the prognostic role of HE4 could help clinicians to personalize the follow-up program, whereas its predictive role could be useful to plan the treatment of the relapse. The role of HE4 in ovarian cancer follow-up deserves to be further investigated in prospective randomized multicentric studies.

Are E-test and Vitek2 good choices for tigecycline susceptibility testing when comparing broth microdilution for MDR and XDR Acinetobacter baumannii?

This study reports the results of antimicrobial susceptibility testing of 10 MDR and 74 XDR Acinetobacter bauman- nii clinical isolates from our hospital routine. We used three different methods: two automated systems (Sensititre and VITEK 2) and one standardized manual method (E-test). Since many published papers refer to in vitro tests performed by E-test, the aim of this study was to test if this method is reliable for testing tigecycline. The results obtained show that E-test significantly overestimates the MIC of the broth microdilution (reference test), thus ob- taining a significant number of major errors (resistant instead of sensitive). VITEK 2 also shows the same problem, but it is less critical. We therefore conclude that these methods do not seem to be very reliable in the performance of susceptibility testing of MDR and XDR Acinetobacter baumannii against tigecycline.

Cytokine and soluble programmed death-ligand 1 levels in serum and plasma of cancer patients treated with immunotherapy: Preanalytical and analytical considerations.

AIM: To evaluate cytokine and soluble programmed death ligand-1 (sPD-L1) levels in the serum and plasma of cancer patients treated with immunotherapy, and to test different assays. METHODS: Three Luminex xMAP assays and two ELLA microfluidic cartridges were used to screen 28 immune-related biomarkers in 38 paired serum and citrate-theophylline-adenosine-dipyridamole (CTAD) plasma samples collected from 10 advanced melanoma or non-small cell lung cancer (NSCLC) patients at different time points during immunotherapy. RESULTS: Twenty-three of 28 biomarkers were detected both in serum and plasma by at least one of the assays, including IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, GM-CSF, IFN-γ, TNF-α, VEGF, IP-10, MCP-1, eotaxin, fractalkine, G-CSF, IFN-α, IL-1RA, IL-13, IL-17A, MIP-1ß and sPD-L1. Conversely, FGF-2 and IL-1α were not detected in both matrices; GRO-α factor and EGF were detected only in serum and MIP-1α only in plasma. sPD-L1, MCP-1, IFN-γ, IL-8, MIP-1ß and VEGF were, respectively, 1.15-, 1.44-, 1.83-, 2.43-, 2.82-, 6.72-fold higher in serum, whereas IL-10, IL-4, IL-2 and IL-5 were 1.05-, 1.19-, 1.92- and 2.17-fold higher, respectively, in plasma. IP-10 levels were higher in plasma but, as well as for VEGF, the bias serum versus plasma varied depending on the assay used (IP-10: -5.7% to -145%; VEGF: 115% to 165%). No significant differences were found for the remaining nine analyzed cytokines. CONCLUSION: The cytokine and sPD-L1 levels may differ between serum and plasma samples collected from cancer patients treated with immunotherapy, and the results obtained can be influenced by the different characteristics of the tested assays. The standardization of pre-analytical and analytical procedures is therefore needed for the future implementation of these circulating biomarkers in clinical practice.

Design of tumor biomarker-monitoring trials: a proposal by the European Group on Tumor Markers.

A major application of tumor biomarkers is in serial monitoring of cancer patients, but there are no published guidelines on how to evaluate biomarkers for this purpose. The European Group on Tumor Markers has convened a multidisciplinary panel of scientists to develop guidance on the design of such monitoring trials. The panel proposes a 4-phase model for biomarker-monitoring trials analogous to that in use for the investigation of new drugs. In phase I, biomarker kinetics and correlation with tumor burden are assessed. Phase II evaluates the ability of the biomarker to identify, exclude, and/or predict a change in disease status. In phase III, the effectiveness of tumor biomarker-guided intervention is assessed by measuring patient outcome in randomized trials. Phase IV consists of an audit of the long-term effects after biomarker monitoring has been included into standard patient care. Systematic well-designed evaluations of biomarkers for monitoring may provide a stronger evidence base that might enable their earlier use in evaluating responses to cancer therapy.

Cancer antigen 125, human epididymis 4, kallikrein 6, osteopontin and soluble mesothelin-related peptide immunocomplexed with immunoglobulin M in epithelial ovarian cancer diagnosis.

BACKGROUND: Human epididymis protein 4 (HE4), kallikrein 6 (KLK6), osteopontin (OPN) and soluble mesothelin-related peptide (SMRP) are new promising biomarkers that could integrate CA125 in epithelial ovarian cancer (EOC) diagnosis. The autoantibody response to tumor antigens is a potential tool for improving the diagnostic performances of biomarkers. The aim of this study was to assess the diagnostic potential of these biomarkers in the form of free markers and immunocomplexed with immunoglobulin M (IgM). Moreover, we analyzed the association between these markers and clinico-pathological characteristics of EOC patients. METHODS: Serum and plasma samples of 60 healthy controls, 60 ovarian benign cysts, 60 endometriosis and 60 EOCs, collected before any treatment, were tested for CICs and free antigens by immunoassays. RESULTS: Immunocomplexes were characterized by poor sensitivity and specificity, since they allowed the detection only of a small number of EOC patients and were increased in patients with benign gynecological pathologies. However, the markers in the form of free antigens showed good diagnostic performances. Of note, CA125 and HE4 showed high sensitivity in the detection of the malignancy and HE4 emerged as a useful biomarker in differential diagnosis between EOC and endometriosis. Finally, elevated KLK6 and OPN, were associated with advanced FIGO stage, high grade disease, suboptimally debulked tumor and ascites. CONCLUSIONS: This study confirms the diagnostic role of CA125, HE4, KLK6, OPN and SMRP, and for the first time showed that CA125, HE4, KLK6, OPN and SMRP immunocomplexed with IgM are not a potential tool for EOC diagnosis.