Search

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F, and MPLW515L, or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK–mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2—Wilms' tumor 1 (WT1)–binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically.Significance:TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase–positive malignant hematopoietic cells to PARP inhibitors.

Supplementary Methods and Results

Objective To assess the safety and efficacy of convalescent plasma (CP) transfusion in elderly people with moderate to severe coronavirus disease 2019 (COVID-19) living in a long-term care facility (LTCF). Patients and Methods Twenty-two consecutive elderly patients with COVID-19 infection living in an LTCF in Lombardy, Italy, who were given CP during May 15 to July 31, 2020, were enrolled in a prospective cohort study. Their clinical, instrumental, and laboratory parameters were assessed following the CP treatment. The overall mortality rate in this group was compared with that recorded in other LTCFs in Lombardy during the 3-month period from March to May 2020. Results Of the 22 patients enrolled, 68.2% (n=15) received 1 CP unit, 27.3% (n=6) received 2 units, and 4.5% (n=1) received 3 units. Of the CP units transfused, 76.7% (23/30) had a neutralizing antibody titer of 1:160 or greater. No adverse reactions were recorded during or after CP administration. Improvements in clinical, functional, radiologic, and laboratory parameters during the 14 days after CP transfusion were observed in all 19 patients who survived. Viral clearance was achieved in all patients by the end of follow-up (median, 66 days; interquartile range, 48-80 days). The overall mortality rate was 13.6% (3/22), which compared favorably with that in the control group (38.3% [281/733]; P=.02) and corresponded to a 65% reduction in mortality risk. Conclusion Early administration of CP with an adequate anti–severe acute respiratory syndrome coronavirus 2 antibody titer to elderly symptomatic patients with COVID-19 infection in an LTCF was safe and effective in eliminating the virus, restoring patients’ immunity, and blocking the progression of COVID-19 infection, thereby improving patients’ survival. Trial Registration ClinicalTrials.gov : NCT04569188 .

Epstein–Barr Virus (EBV) is a human gamma-herpesvirus that is widespread worldwide. To this day, about 200,000 cancer cases per year are attributed to EBV infection. EBV is capable of infecting both B cells and epithelial cells. Upon entry, viral DNA reaches the nucleus and undergoes a process of circularization and chromatinization and establishes a latent lifelong infection in host cells. There are different types of latency all characterized by different expressions of latent viral genes correlated with a different three-dimensional architecture of the viral genome. There are multiple factors involved in the regulation and maintenance of this three-dimensional organization, such as CTCF, PARP1, MYC and Nuclear Lamina, emphasizing its central role in latency maintenance.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that affect the epigenetic regulation of DNA methylation. Mutations in both genes often co-occur with activating mutations in genes encoding oncogenic tyrosine kinases such as FLT3ITD, BCR-ABL1, JAK2V617F, and MPLW515L, or with mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Here, we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing these oncogenes. Malignant TET2-deficient cells displayed downregulation of BRCA1 and LIG4, resulting in reduced activity of BRCA1/2-mediated homologous recombination (HR) and DNA-PK–mediated non-homologous end-joining (D-NHEJ), respectively. TET2-deficient cells relied on PARP1-mediated alternative NHEJ (Alt-NHEJ) for protection from the toxic effects of spontaneous and drug-induced DNA double-strand breaks. Conversely, DNMT3A-deficient cells favored HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells were sensitive to PARP inhibitor (PARPi) treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity or TET2—Wilms' tumor 1 (WT1)–binding ability was responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Collectively, these findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Significance: TET2 and DNMT3A mutations affect distinct DNA repair mechanisms and govern the differential sensitivities of oncogenic tyrosine kinase–positive malignant hematopoietic cells to PARP inhibitors.

The Epstein Barr virus (EBV) infects almost 95% of the population worldwide. While typically asymptomatic, EBV latent infection is associated with several malignancies of epithelial and lymphoid origin in immunocompromised individuals. In latently infected cells, the EBV genome persists as a chromatinized episome that expresses a limited set of viral genes in different patterns, referred to as latency types, which coincide with varying stages of infection and various malignancies. We have previously demonstrated that latency types correlate with differences in the composition and structure of the EBV episome. Several cellular factors, including the nuclear lamina, regulate chromatin composition and architecture. While the interaction of the viral genome with the nuclear lamina has been studied in the context of EBV lytic reactivation, the role of the nuclear lamina in controlling EBV latency has not been investigated. Here, we report that the nuclear lamina is an essential epigenetic regulator of the EBV episome. We observed that in B cells, EBV infection affects the composition of the nuclear lamina by inducing the expression of lamin A/C, but only in EBV+ cells expressing the Type III latency program. Using ChIP-Seq, we determined that lamin B1 and lamin A/C bind the EBV genome, and their binding correlates with deposition of the histone repressive mark H3K9me2. By RNA-Seq, we observed that knock-out of lamin A/C in B cells alters EBV gene expression. Our data indicate that the interaction between lamins and the EBV episome contributes to the epigenetic control of viral gene expression during latency, suggesting a restrictive function of the nuclear lamina as part of the host response against viral DNA entry into the nucleus.

Summary: Synthetic lethality triggered by PARP inhibitor (PARPi) yields promising therapeutic results. Unfortunately, tumor cells acquire PARPi resistance, which is usually associated with the restoration of homologous recombination, loss of PARP1 expression, and/or loss of DNA double-strand break (DSB) end resection regulation. Here, we identify a constitutive mechanism of resistance to PARPi. We report that the bone marrow microenvironment (BMM) facilitates DSB repair activity in leukemia cells to protect them against PARPi-mediated synthetic lethality. This effect depends on the hypoxia-induced overexpression of transforming growth factor beta receptor (TGFβR) kinase on malignant cells, which is activated by bone marrow stromal cells-derived transforming growth factor beta 1 (TGF-β1). Genetic and/or pharmacological targeting of the TGF-β1-TGFβR kinase axis results in the restoration of the sensitivity of malignant cells to PARPi in BMM and prolongs the survival of leukemia-bearing mice. Our finding may lead to the therapeutic application of the TGFβR inhibitor in patients receiving PARPis.

The immunoassays methods need avoiding interferences that can influence result interpretation. Main sources of interference arise from either patient status, preparation and physiology or laboratory process and procedures. The aim of this non-systematic critical review is to highlight the preanalytical interferences on laboratory immunoassays. Blood hormone profile changes according with age and depending on sex: these are important variables, mainly in newborn, during both sexual maturation and childbearing. Gonadotropins FSH and LH show a sharp increase with age in females, whereas in males LH appears rather stable. With age both males and females show progressive decay of the hormone profile. Stress causes variations, as it influences GH, prolactin, Cortisol and the total/free ratio of thyroid hormone. Diurnal variations, day of cycle, influence by estrogens on thyroid hormone are relevant for result variability. Paraproteins and autoantibodies can interfere in some assays particularly drug, vitamin D and thyroid hormone. As regards the variables due to sample matrix, and to evacuated tubes components, some additives and anticoagulants have been reported to influence specific assays, e.g. thyroid hormone. Hemolysis, lipemia and bilirubin cause interferences on specific techniques/tests, e.g. ferritin, TSH, Vitamin B12, progesterone and folic acid. Nicotine and cocaine addictions interfere with some hormones. Thus, laboratory professionals should be aware of preanalytical problems particularly important when dealing with the immunoassays, by taking appropriate actions to avoid any relevant interferences.

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers.

Objectives: To evaluate associations of preoperative total prostate specific antigen (PSA) to free testosterone (FT), the PSA/FT index ratio, with features of pathology prostate cancer (PCA) and to investigate its prognostic potential in clustering the PCA population. Patients and methods: After excluding criteria, the records of 220 patients who underwent radical prostatectomy (RP) were retrospectively reviewed. Serum samples of PSA, total testosterone (TT) and FT were collected at 8.00 A.M., one month after biopsies and before RP. The PSA/FT ratio was computed in the population of patients who were clustered in groups according to ranking intervals of the PSA/FT ratio which identified at least 4 clusters which were coded as A, B, C, and D. The independent associations of the PSA/FT index ratio were assessed by statistical methods and a two-sided P < 0.05 was considered to indicate statistical significance. Results: TT correlated to FT which was a significant predictor of PSA in the population of patients who were subsequently clustered, according to increasing interval values of the PSA/FT index ratio, in groups that showed a stronger linear association of FT with PSA. The PSA/FT index ratio significantly associated with pathology features of prostate cancer such as pathology Gleason score (pGS), invasion of the seminal vesicles (pT3b), proportion of positive cores (P+) and proportion of cancer involving the volume of the prostate. In the population of patients, TT, PSA/FT index ratio and P+ independently associated with pGS ≥ 7 and pT3b; moreover, the odds ratio (OR) of the PSA/FT index ratio resulted 9.11 which was stronger than TT (OR = 1.11) and P+ (OR = 8.84). In the PCA population, TT, PSA/FT index ratio and P+ also independently associated with pT3b PCA; interestingly, the OR of PSA/FT index resulted 54.91 which was stronger than TT (OR = 1.31) and P+ (26.43). Conclusions: Preoperative PSA/FT index ratio is an independent strong factor which directly associates with aggressive features of pathology PCA; moreover, it might express prognostic potential for clustering the patient population in risk classes. Confirmatory studies are required.

Objective To investigate potential associations of preoperative total testosterone (TT) with tumor volume (TV) and grade of prostate cancer (PCa). Methods Patients who were under medications impacting on the hypothalamic-pituitary-adrenal-testis-prostate axis were excluded. TT was measured preoperatively at least 1 month after biopsies and TV was calculated on the removed prostate specimen. Other continuous variables included total prostate specific antigen (PSA), percentage of positive cores (P+) and weight (W) of the removed prostate. Patients were categorized according to the pathologic Gleason score (pGS) in 3 groups (pGS 6, 7 and > 7). Invasion of the seminal vesicles was coded as seminal vesicle invasion (SVI). Results The median levels of TT were significantly and increasingly higher from pGS 6 (14.7 nmol/L) to pGS 7 (15.0 nmol/L) and pGS > 7 (18.8 nmol/L). The median values of TV were also detected significantly and increasingly higher from pGS 6 (5.6 mL) to pGS 7 (8.1 mL) and pGS > 7 (14.8 mL). The median preoperative levels of PSA were also increasing from pGS 6 (5.9 μg/L) to pGS 7 (6.2 μg/L) and pGS > 7 (7.7 μg/L). There was a significant and positive correlation of TV to PSA, TT and P+. Multiple linear regression analysis showed that TV was significantly and independently predicted by TT, PSA and P+. High grade PCa (pGS > 7) independently associated with TV, TT, P+ and SVI. The median density values of TT relative to TV (quotient TT/TV) significantly decreased from pGS 6 (2.6 nmol/L/mL) to pGS 7 (1.9 nmol/L/mL) and pGS > 7 (1.4 nmol/L/mL). The median density values of PSA relative to TV (quotient PSA/TV) also significantly decreased from pGS (1.1 μg/L/mL) to pGS 7 (0.7 μg/L/mL) and pGS > 7 (0.6 μg/L/mL). Conclusion The investigation shows that TT relates to volume and grade of PCa; moreover, the density of TT relative to TV inversely associates with rate of increase of cancer that depends on the grade of the tumour.

Somatic variants in TET2 and DNMT3A are founding mutations in hematological malignancies that both affect the epigenetic regulation of DNA methylation. Although the proteins antagonistically regulate the epigenetic mark of 5-methylcytosine (5-mC), where DNMT3A catalyzes addition of 5-mC while TET2 oxidizes 5-mC as a first step in DNA demethylation, mutations in both genes appear in a similar spectrum of human hematopoietic malignancies. Mutations in both genes often co-occur with activating mutations in oncogenic tyrosine kinases (OTKs) such as FLT3ITD, BCR-ABL1, JAK2V617F, MPLW515L or mutations affecting related signaling pathways such as NRASG12D and CALRdel52. Moreover, while the mutations exert divergent effect on primitive hematopoietic progenitor cells, they lead to similar disease phenotypes, suggesting the roles of these mutations in hematopoietic malignancies may relate to mechanisms outside of DNA methylation. OTK-positive malignant cells accumulate high numbers of spontaneous and drug-induced DNA double-strand breaks (DSBs) in comparison to normal cells, but they manage to survive because of their enhanced/altered ability to repair these breaks. DSBs, the most lethal DNA lesions, are repaired by two major mechanisms, BRCA1/2-dependent homologous recombination (HR) and DNA-PK -mediated non-homologous end-joining (D-NHEJ). Both HR and D-NHEJ repair DSBs in proliferating cells, while D-NHEJ plays a major role in quiescent cells. PARP1 -dependent alternative NHEJ (Alt-NHEJ) serves as back-up in both proliferating and quiescent cells. The existence of these redundant pathways creates the opportunity to employ a phenomenon called "synthetic lethality", which was originally applied to eliminate cancer cells with mutations in BRCA1 and BRCA2 by PARP inhibitor (PARPi). Our previous report [M. Nieborowska-Skorska et al., Gene expression and mutation-guided synthetic lethality eradicates proliferating and quiescent leukemia cells. J Clin Invest 127, 2392-2406 (2017)] suggested that certain leukemias are sensitive to PARPi-triggered synthetic lethality. Here we show that TET2 and DNMT3A mutations exert divergent roles in regulating DNA repair activities in leukemia cells expressing OTKs. Malignant TET2-deficient cells display downregulation of BRCA1 and LIG4 resulting in reduced activity of HR and D-NHEJ, respectively, and rely on Alt-NHEJ to protect them from the toxic effects of replication stress and drug-induced DSBs. Conversely, DNMT3A-deficient cells favor HR/D-NHEJ owing to downregulation of PARP1 and reduction of Alt-NHEJ. Consequently, malignant TET2-deficient cells are sensitive to PARPi treatment in vitro and in vivo, whereas DNMT3A-deficient cells were resistant. Disruption of TET2 dioxygenase activity and/or TET2 - Wilms tumor 1 (WT1) binding ability were responsible for DNA repair defects and sensitivity to PARPi associated with TET2 deficiency. Moreover, mutation or deletion of WT1 mimicked the effect of TET2 mutation on DSB repair activity and sensitivity to PARPi. Our findings reveal that TET2 and WT1 mutations may serve as biomarkers of synthetic lethality triggered by PARPi, which should be explored therapeutically. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Tallman:Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Amgen: Research Funding; Bioline rx: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties. Martinelli:Roche: Consultancy; Pfizer: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy; Jazz: Consultancy; Janssen: Consultancy; Daichii Sankyo: Consultancy, Research Funding; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy; AbbVie: Consultancy, Research Funding. Vassiliou:Kymab Ltd - Monoclonal antibody company. Currently not working in myeloid cancers or clonal haematopoiesis.: Consultancy.

Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas.IMPORTANCE EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.

Aldosterone and renin measurement is a cornerstone for primary aldosteronism (PA) diagnosis, but different thresholds are used according to different assays. A fully automated chemiluminescence (CL) immunoassay for renin and aldosterone was recently proposed, showing good performance for PA screening by aldosterone to renin ratio (ARR). This study aimed to define the accuracy of this assay in the screening and in the most popular confirmatory test of autonomous aldosterone production, the intravenous saline loading test (ivSLT). We compared aldosterone results obtained by CL vs radioimmunoassay (RIA) in hypertensive patients investigated for PA (102 baseline and 85 after ivSLT). An excellent correlation was observed between RIA and CL in the entire population for aldosterone (r=0.922) and ARR (r=0.977). For ARR, Deming regression proved a good accordance between methods and, consistent with the fit model, our previous institutional ARR cut-off of 32 (pg ml−1)/(pg ml−1) corresponded to 20 pg ml−1 mU−1 l−1 in CL assay. However, the correlation was weaker in the low end of aldosterone concentrations (r=0.676 for aldosterone

Background: Growing evidence suggests that the protein galectin-3 may promote fibrosis and cardiac remodeling, thus being currently considered one of the most promising biomarkers for risk stratification of patients with acute myocardial infarction (AMI) and/or heart failure. Therefore, this study was planned to identify the main physiological determinants of serum galectin-3 in a reference population of middle-aged healthy subjects. Methods: The study population consisted of 44 healthy volunteers [14 males and 30 females; mean age, 47±11 years; body mass index (BMI), 23.6±3.6 kg/m 2 ], recruited from the personnel of the local laboratory. Demographical and biochemical measures were recorded early in the morning and potential associations with serum galectin-3 values were tested with both univariate and multivariate linear regression analyses. Results: In univariate analysis, the concentration of serum galectin-3 was found to be significantly associated with both BMI and estimated glomerular filtration rate (eGFR), but not with age, sex, serum albumin, serum cardiac troponin T, hemoglobin and red blood cell distribution width. In multivariate analysis, the concentration of serum galectin-3 remained inversely associated with eGFR, but not with BMI. The median value of galectin-3 was found to be significantly different across quartiles of eGFR (P=0.046). Results: The results of our study suggest that the serum concentration of galectin-3 in a population of middle-aged healthy subjects may be influenced by renal function, but not by age, sex, BMI, liver function and hemoglobin concentration.

The Epstein Barr virus (EBV) genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. Here, we demonstrate that binding of PARP1, an important cofactor of CTCF, at the BZLF1 lytic switch promoter restricts EBV reactivation. Knockdown of PARP1 in the Akata-EBV cell line significantly increases viral copy number and lytic protein expression. Interestingly, CTCF knockdown has no effect on viral reactivation, and CTCF binding across the EBV genome is largely unchanged following reactivation. Moreover, EBV reactivation attenuates PARP activity, and Zta expression alone is sufficient to decrease PARP activity. Here we demonstrate a restrictive function of PARP1 in EBV lytic reactivation.

Background and Objective: Prostate cancer is an endocrine-dependent tumor which is still under-investigated for physiopathology factors related to its natural history. The association of pretreatment total testosterone (TT) serum levels with prostate cancer is still a controversial topic. The objective of this study was to investigate potential associations and functional relationships of preoperative TT serum level and pathology-detected Gleason score (pGS). Materials and Methods: Pretreatment and pathological variables of 220 patients operated with radical prostatectomy were retrospectively reviewed. Age, prostate-specific antigen (PSA), percentage of positive biopsy cores (P+), biopsy Gleason score (bGS), pGS, TT and free testosterone were the continuous variables, while clinical stage (cT: cT1c, cT2/3), biopsy Gleason pattern (bGP: ≤3+3, 3+4, >3+4), pathology Gleason pattern (pGP: ≤3+3, 3+4, >3+4), pathology stage (pT: pT2, pT3a, pT3b), pathology nodal staging (pN: pN0, pN1, pNx) and surgical margin invasion by cancer (R-, R+) were the categorical variables. Statistical methods were computed for assessing associations of TT and pGS; moreover, simple and multiple linear regression analysis (SLRA and MLRA) were used for assessing functional relationships of TT and pGS. Results: High-grade tumors (pGS ≥8.0) were associated with bGS >6.0 (p < 0.0001), pGP ≥3+4 (p < 0.0001), P+ >0.31% (p = 0.006), cT2/3 (p = 0.01), TT >15.5 nmol/l (p = 0.0004) and, to a lesser extent, PSA >6.27 μg/l (p = 0.06). The odds ratio (OR) ranked as follows: 2.01 (PSA >6.27 μg/l), 2.88 (cT2/3), 3.23 (P+ >0.31%), 5.53 (TT >15.5 nmol/l) and 12.09 (pGP ≥3+4 and pGS ≥8.0). On SLRA, pGS variation was significantly predicted by bGS (p < 0.0001), P+ (p < 0.0001), PSA (p = 0.0005) and TT (p = 0.02); on MLRA, pGS variation was still significantly predicted by bGS (p < 0.0001), P+ (p = 0.04), PSA (p = 0.03) and TT (p = 0.002). When bGS, P+, PSA and TT were dichotomized to their median value, only bGS (p < 0.0001) and TT (p = 0.001) showed independence in predicting pGS variation. The best model for predicting pGS variations was by dichotomizing TT above its median (>15.5 nmol/l) because the predictive coefficient increased to 0.32, which means that patients with TT >15.5 have a significantly higher estimated risk for high-grade pGS than patients with TT ≤15.5 nmol/l (OR = 1.31). Conclusion: In a patient population undergoing radical prostatectomy, increased pretreatment serum measurements of TT are associated with and functionally related to high-grade pGS; moreover, baseline TT together with bGS and PSA are important factors for predicting pGS and assessing high-grade tumors. Baseline TT serum levels might have prognostic potential for assessing treatment response for continuous as well as intermittent androgen deprivation therapy.

Aim: To investigate the potential of preoperative serum total testosterone (TT) in contributing to the definition of separate prostatectomy Gleason score (pGS) groups of the prostate cancer (PCa) population. Materials and Methods: The data of 220 patients operated on for PCa were retrospectively reviewed. No patient had previously received 5α-reductase inhibitor, luteinizing hormone-releasing analogs or testosterone replacement treatment. The patient population was grouped according to the pGS as 6 = 3+3, 7 = 3+4, 7 = 4+3 and 8-10. Eight variables were simultaneously investigated in each group: prostate-specific antigen (PSA), TT, free testosterone, age, percentage of positive prostate biopsy cores (P+), biopsy Gleason score (bGS), overall cancer volume estimated as percentage of prostate volume (V+) and prostate weight (Wi). Univariate analysis of variance (ANOVA), multivariate analysis of variance (MANOVA) and multivariate discriminant analysis (MDA) were the statistical methods used for evaluating the data. Results: There were 89 patients in pGS 6 = 3+3, 84 in pGS 7 = 3+4, 24 in pGS 7 = 4+3 and 23 in pGS 8-10. ANOVA showed that bGS (p < 0.0001), P+ (p < 0.0001), V+ (p < 0.0001), PSA (p = 0.0001), Wi (p = 0.0002) and TT (p = 0.01) were significantly different in the four pGS groups. MANOVA tests showed that only bGS (p < 0.0001), V+ (p = 0.0003), TT (p = 0.001) and, to a lesser extent, PSA (p = 0.06) were the significant variables that individually and independently contributed a significant amount to separation of the four pGS groups of the PCa population. MDA showed that the independent variables ranked as bGS (p < 0.0001), TT (p = 0.001), V+ (p = 0.001) and PSA (p = 0.06). Conclusions: Serum TT is a significant preoperative variable that independently contributes to separating the PCa population into pGS score groups. Pretreatment baseline serum TT levels should be measured and their inclusion in neural networks predicting PCa natural history be considered in the patient population diagnosed with PCa.

Purpose: The study aims to investigate the potential associations between preoperative plasma levels of total testosterone (TT) and biopsy Gleason score (bGS) upgrading in prostate cancer (PCA) patients undergoing radical prostatectomy (RP). Materials and Methods: Exclusion criteria were treatment with 5α-reductase inhibitors, LH-releasing hormone analogues or testosterone replacement. Criteria of bGS upgrading were as follows: (i) bGS 6 to pathological Gleason score (pGS) >6, (ii) bGS 7 with pattern 3 + 4 to pGS 7 with pattern 4 + 3 or to pGS >7, (iii) bGS 7 with pattern 4 + 3 to pGS >7. Patients who showed bGS >7 were excluded from the cohort. Results: The study included 209 patients. Tumor upgrading was assessed in 76 (36.4%) cases of the entire cohort, in 51 out of 130 cases (39.2%) of the bGS 6 group and 25 out of 79 patients (31.6%) in the bGS 7 cluster. Logistic regression models showed that independent clinical covariates predicting the risk of bGS upgrading included TT (OR 1.058; p = 0.027) and prostate-specific antigen (PSA) density (OR 23.3; p = 0.008) as well as TT (OR 1.057; p = 0.029) with PSA (OR 1.061; p = 0.023). The model suggests that 1 unit increase in TT plasma levels increases the odds of bGS upgrading by 5.8 or 5.7%. Conclusions: In summary, we have determined that high TT preoperative plasma levels independently predict bGS upgrading in men with PCA undergoing RP. Preoperative plasma levels of TT might be included as a potential marker for assessing the risk bGS upgrading.

The value of red blood cell distribution width (RDW), a simple and inexpensive measure of anisocytosis, has been associated with the outcome of many human chronic disorders. Therefore, this retrospective study was aimed to investigate whether RDW may be associated with medium-term mortality and major adverse cardiac events (MACE) after an acute coronary syndrome (ACS).A total number of 979 patients diagnosed with ACS were enrolled from June 2014 to November 2014, and followed-up until June 2015.The RDW value in patients with 3-month MACE and in those who died was significantly higher than that of patients without 3-month MACE (13.3% vs. 14.0%; P0.001) and those who were still alive at the end of follow-up (13.4% vs. 14.4%; P0.001). In univariate analysis, RDW was found to be associated with 3-month MACE [odds ratio (OR), 1.70; 95% CI, 1.44-2.00, P0.001]. In multivariate analysis, RDW remained independently associated with 3-month MACE (adjusted OR, 1.36; 95% CI, 1.19-1.55; P0.001) and death (adjusted OR, 1.34; 95% CI, 1.05-1.71; P=0.020). The accuracy of RDW for predicting 3-month MACE was 0.67 (95% CI, 0.66-0.72; P0.001). The most efficient discriminatory RDW value was 14.8%, which was associated with 3.8 (95% CI, 2.6-5.7; P0.001) higher risk of 3-month MACE. Patients with RDW14.8% exhibited a significantly short survival than those with RDW ≤14.8% (331 vs. 465 days; P0.001).The results of this study confirm that RDW may be a valuable, easy and inexpensive parameter for stratifying the medium-term risk in patients with ACS.

Background The appropriate therapeutic management of patients with wasp-induced allergic reactions necessitates an accurate allergologic workup, entailing differentiation between sensitization against Vespula or Polistes venoms. Materials and methods We studied 52 consecutive adult subjects diagnosed with hypersensitivity to wasp venoms. Cap inhibition was performed using UniCAP Specific IgE. The concentration of serum IgE against two recombinant constituents of Antigen 5 (rVes v 5 and rPol d 5) was also measured using Immuno CAP 250. The ratio between values of specific IgE against recombinant allergens was calculated and a percentage difference > 50% was considered significant for specific immunization against one of the two venoms. Results The diagnostic agreement between recombinant allergens testing and CAP inhibition was 54% (kappa statistics, 0.34; 95% CI, 0.18–0.50) in the whole study population. In the 24 patients with recombinant allergens ratio > 50% and non dubious results of CAP inhibition assay the diagnostic agreement was perfect (100%; kappa of agreement, 1.00; 95% CI; 1.00–1.00). Discussion The results of this study show that the assessment of specific IgE against rVes v 5 and rPol d 5 may be regarded as a low-cost screening, providing valuable diagnostic information for differentiating the sensitization against Vespula or Polistes venoms. In patients with suggestive clinical history and ratio > 50% between specific IgE against rVes v 5 and specific IgE against and rPol d 5, the CAP inhibition assay may be safely withheld, thus allowing to achieve an early diagnosis at lower cost.

La necessita di promuovere l’appropriatezza delle procedure diagnostiche e terapeutiche e molto aumentata negli anni recenti a livello internazionale e nazionale. Le societa scientifiche sono state coinvolte dal principio nella definizione di modalita e strumenti per supportare i soci nella “traduzione” nella pratica clinica di tale necessita, per esempio nella definizione di liste delle procedure diagnostiche e terapeutiche a maggior rischio di inappropriatezza. Questo ha consentito non solo di condividere delle priorita nell’ambito delle singole discipline, ma anche di confrontarsi con gli altri specialisti e, aspetto sempre piu importante, con i pazienti e con i cittadini. I progetti che hanno avuto e continuano ad avere maggiore rilevanza a livello internazionale e nazionale sono, rispettivamente, Choosing Wisely e Slow Medicine. La SIPMeL ha aderito a Slow Medicine e, in occasione del 1° Congresso Nazionale, ha presentato la lista delle 5 procedure a maggiore rischio di inappropriatezza nell’ambito della Medicina di Laboratorio. Secondo i componenti del gruppo di studio Endocrinologia e Malattie del Metabolismo l’ambito di interesse del gruppo e di tale rilevanza diagnostica da suggerire l’utilita di proporre 5 procedure a rischio di inappropriatezza specifiche. A questo riguardo e stato compiuto nel 2015 un sondaggio che ha coinvolto la maggioranza dei componenti del gruppo, i quali hanno indicato le 5 procedure che consideravano maggiormente a rischio. Le procedure indicate dovevano essere corredate da motivazioni, punteggio (da 1 a 10) e note bibliografiche. Le 5 procedure che hanno ricevuto il punteggio piu alto e che sono state selezionate si riferiscono a: (1) diagnostica ottimale della funzionalita tiroidea; (2) diagnostica ottimale della Sindrome di Cushing; (3) diagnostica ottimale del feomocromocitoma; (4) diagnostica ottimale di tireopatia autoimmune; (5) determinazione della 25 (OH) vitamina D. Le 5 procedure sono state presentate in una sessione del 1° Congresso Nazionale della SIPMeL e diventeranno disponibili sul sito della Societa. Si propone che anche gli altri gruppi di studio di SIPMeL preparino una analoga lista e ne diano diffusione all’interno della Societa e della professione con modalita analoghe.

Aim: A preceding exploratory analysis has shown that follicle-stimulating hormone (FSH) was significantly correlated to and predicted by prostate-specific antigen (PSA) in a prostate cancer population. The aim of the study was to evaluate FSH physiopathology along the pituitary-testicular-prostate (PTP) axis at the time of initial diagnosis of prostate cancer in an operated population clustered according to the FSH/PSA ratio. Patients and Methods: The study included 93 patients who underwent standard radical prostatectomy. Age, percentages of positive cores at transrectal ultrasound scan biopsy (TRUSB) (P+), biopsy Gleason score (bGS), pathology Gleason score (pGS), luteinizing hormone (LH), FSH, prolactin hormone (PRL), total testosterone (TT), free testosterone (FT), estradiol (ESR) and PSA were the continuous variables. Category variables were pT and biopsy/pathology Gleason pattern I/II (b/pGPI/II). The population was clustered according to the FSH/PSA ratio which was computed from empirical data and then ranked for clustering the population as groups A (range 0.13 ≤ FSH/PSA ≤ 0.20), B (range 0.20 < FSH/PSA ≤ 0.50), C (range 0.50 < FSH/PSA ≤ 0.75), D (range 0.75 < FSH/PSA ≤ 1.00), E (range 1.00 < FSH/PSA ≤ 1.25), F (range 1.25 < FSH/PSA ≤ 2.00), G (range 2.00 < FSH/PSA ≤ 2.25), H (range 2.25 < FSH/PSA ≤ 6.40) and I (range 6.40 < FSH/ PSA ≤ 19.40). The model was assessed by simple linear regression analysis and differences between the groups were investigated by analysis of variance (ANOVA) for continuous variables and by contingency tables for category variables. Results: FSH was significantly correlated to and predicted by PSA in groups A (p = 0.04), B (p < 0.0001), C (p < 0.0001), D (p < 0.0001), E (p < 0.0001), F (p < 0.0001), G (p < 0.0001), H (p = 0.0001) and I (p = 0.001). Also, clusters (A–I) differed significantly for mean values of FSH (p < 0.0001), LH (p < 0.0001), TT (p = 0.04), PSA (p < 0.0001), bGS (p = 0.005), pGS (p = 0.01) and PSA/FT ratio (p < 0.0001); moreover, the nine groups showed significant different frequency distributions of pGPI (p = 0.02), pGPII (p = 0.0002) and bGPI (p = 0.04). Conclusion: The ranking FSH/PSA ratio significantly clustered, along the PTP axis, an operated population diagnosed with prostate cancer. Also, the ranking FSH/PSA ratio selected prostate cancer clusters expressing different levels of hormonal disorder along the PTP axis and prognostic potential with different risks of progression. As a theory, in the current advancing world, the ranking FSH/PSA model might be considered as an interesting and effective tool for prostate cancer study as well as individualized, risk-adapted approaches of the disease. However, confirmatory studies are needed.

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Poly(ADP-ribose) polymerase 1 (PARP1) regulates accessibility of chromatin, alters functions of transcriptional activators and repressors, and has been directly implicated in transcriptional activation. Previously we showed that LMP1 activates PARP1 and increases Poly(ADP-ribos)ylation (PARylation) through PARP1. Therefore, to identify targets of LMP1 that are regulated through PARP1, LMP1 was ectopically expressed in an EBV-negative Burkitt’s lymphoma cell line. These LMP1-expressing cells were then treated with the PARP inhibitor olaparib and prepared for RNA sequencing. The LMP1/PARP targets identified through this RNA-seq experiment are largely involved in metabolism and signaling. Interestingly, Ingenuity Pathway Analysis of RNA-seq data suggests that hypoxia-inducible factor 1-alpha (HIF-1α) is an LMP1 target mediated through PARP1. PARP1 is acting as a coactivator of HIF-1α-dependent gene expression in B cells, and this co-activation is enhanced by LMP1-mediated activation of PARP1. HIF-1α forms a PARylated complex with PARP1 and both HIF-1α and PARP1 are present at promoter regions of HIF-1α downstream targets, leading to accumulation of positive histone marks at these regions. Complex formation, PARylation and binding of PARP1 and HIF-1α at promoter regions of HIF-1α downstream targets can all be attenuated by PARP1 inhibition, subsequently leading to a buildup of repressive histone marks and loss of positive histone marks. In addition, LMP1 switches cells to a glycolytic ‘Warburg’ metabolism, preferentially using aerobic glycolysis over mitochondrial respiration. Finally, LMP1+ cells are more sensitive to PARP1 inhibition and, therefore, targeting PARP1 activity may be an effective treatment for LMP1+ EBV-associated malignancies., Author summary Epstein-Barr virus (EBV) is one of the most ubiquitous human viruses, with over 90% of adults worldwide harboring lifelong latent EBV infection in a small fraction of their B-lymphocytes. EBV is known to cause lymphoproliferative disorders and is associated with several other types of cancer, including Hodgkin's lymphoma, Burkitt's lymphoma and Nasopharyngeal carcinoma. However, in most cases, the approach to EBV-positive lymphomas does not differ from EBV-negative lymphomas of the same histology. Latent membrane protein 1 (LMP1) is the major transforming protein of EBV and is critical for EBV-induced B-cell transformation in vitro. LMP1 activates several epigenetic regulators to modify host gene expression, including the chromatin-modifying enzyme Poly(ADP-ribose) polymerase 1, or PARP1. In the current study we have determined that LMP1 can activate PARP1 to increase hypoxia-inducible factor 1-alpha (HIF-1α)-dependent gene expression, leading to a change in host cell metabolism indicative of a ‘Warburg effect’ (aerobic glycolysis). This subsequently provides a proliferative advantage to LMP1-expressing cells. The LMP1-induced increase in HIF-1α-dependent gene expression, alteration of cellular metabolism, and accelerated cellular proliferation, can be offset with the PARP inhibitor olaparib. Therefore, targeting PARP1 activity may be an effective treatment for LMP1+ EBV-associated malignancies.

Patients with Systemic Mastocytosis (SM) need a highly sensitive diagnostic test for D816V detection of the KIT receptor gene.Along with histology/cytology and flow cytometry evaluation, bone marrow (BM) from 110 consecutive adult patients referred with a suspicion of SM to Multidisciplinary Outpatient Clinic for Mastocytosis in Verona were tested both by Amplification Refractory Mutation System Reverse Transcriptase quantitative real time Polymerase Chain Reaction (ARMS-RT-qPCR) and RT-PCR. +. Restriction Fragment Length Polymorphism (RFLP) followed by Denaturing-High Performance Liquid Chromatography (D-HPLC) and Sanger sequencing.ARMS-RT-qPCR identified D816V mutation in 77 patients, corresponding to 100% of cases showing CD25+ mast cells (MCs) whereas RT-PCR+RFLP/D-HPLC+sequencing revealed D816V mutations in 47 patients.According to the 2008 WHO criteria 75 SM, 1 Cutaneous Mastocytosis (CM), 1 monoclonal MC activation syndrome (MMAS), and 1 SM Associated with Haematologic Non-Mast Cell Disorder (SM-AHNMD) were diagnosed. Seventeen out 75 SM patients (23%) would have not satisfied sufficient WHO criteria on the basis of the sole RT-PCR+RFLP: these patients had significantly lower serum tryptase levels and amount of CD25+ MCs.Therefore, ARMS-RT-qPCR might result particularly useful, in patients that do not fulfil major BM histological criterion, for the recognition of indolent SM with a very low MC burden.

Most of the immunoassays workload is processed in clinical laboratories within a time span comparable to the clinical chemistry and the haematology assays and the analytical work is usually completed between 1:00 and 2:00 p.m. In order to accelerate the auto-verification of the results of tumour markers (Total and free PSA, CEA, CA 125, CA 15-3, CA 19-9, TPA, AFP, NSE, S 100 protein), and hormones (TSH, FT4, FT3, Prolactin, total testosterone) and Procalcitonin (PCT) we used DNSevTM expert system implementing 13 rules based on decision levels/reference intervals and reference change values. The auto-verification procedure has been implemented after a 6-month trial in June 2004 and in 2005 the immunoassays section supervisor was able to verify about 500 results in about 30 min 5 days/week. We conclude that the auto-verification of immunoassays implemented in our laboratory is fast and consistent among different supervisors and leaves more time for an effective and timely interaction with clinicians and general practitioners.

Letter to the editor.-- et al.

Introduction Due to the budget limitations, laboratories mostly rely on the manufacturers' information about the influence of interfering substances on laboratory results. However, some manufacturers do not follow the recommended procedures for testing interferences (CLSI standard) and there is a great variability in the presentation of data regarding lipemia interference. Materials and methods We aimed to verify the manufacturers' specifications for lipemia interference for clinical chemistry reagents provided by Beckman Coulter, Roche and Siemens. Bias was determined using the Intralipid® simulated lipemic samples. Furthermore, we aimed to compare obtained data with the manufacturers' claims and desirable specification for imprecision derived from biological variation. Results i) Manufacturers' declarations were not confirmed for all three manufacturers; ii) the magnitude and direction of the effect of lipemia on laboratory results differ substantially between the three tested analytical systems; and iii) manufacturers are using arbitrary limits in declaring the expected effect of interference on laboratory results. Conclusions There is an urgent need to standardize the way manufacturers test and report their data on the lipemia interference. We propose that, instead of arbitrary limits, manufacturers use evidence based quality specifications for assessing the allowable biases. Moreover, laboratories should be aware of the possible lack of replicability of manufacturers' declarations.

Context: Metabolic inflexibility, ie, the impaired ability of the body to switch from fat to carbohydrate oxidation under insulin-stimulated conditions, is associated with insulin resistance. This alteration in metabolic plasticity can lead to organ dysfunction and is considered a key issue among the abnormalities of the metabolic syndrome. It is still unknown whether this phenomenon occurs in women with polycystic ovary syndrome (PCOS). Objective: Our objective was to examine whether metabolic inflexibility is a feature of PCOS women and whether hyperandrogenism may contribute to this phenomenon. Design and Patients: Eighty-nine Caucasian women with PCOS were submitted to hyperinsulinemic-euglycemic clamp. Respiratory exchange ratios were evaluated at baseline and during hyperinsulinemia by indirect calorimetry to quantify substrate oxidative metabolism. Total testosterone was measured by liquid chromatography mass spectrometry and free testosterone by equilibrium dialysis. Setting: Outpatients were seen in a tertiary care academic center. Main Outcome Measure: Metabolic flexibility was assessed by the change in respiratory quotient upon insulin stimulation. Results: Sixty-five of the 89 PCOS women (73%) had increased serum free testosterone, 68 (76%) were insulin resistant, and 62 (70%) had an impaired metabolic flexibility. Comparison of hyperandrogenemic and normoandrogenemic women showed that the 2 subgroups were of similar age but differed in terms of several anthropometric and metabolic features. In particular, hyperandrogenemic women had greater body mass index (32.9 ± 1.0 vs 24.7 ± 0.9 kg/m2, P < .001) and lower glucose utilization during the clamp (9.2 ± 0.4 vs 10.9 ± 0.7 mg/kg fat-free mass · min, P = .023) and metabolic flexibility (0.09 ± 0.06 vs 0.12 ± 0.01, P = .014). In univariate analysis, metabolic flexibility was associated with several anthropometric, endocrine, and metabolic features. In multivariate analysis, this feature was directly associated with baseline respiratory quotient and insulin sensitivity and inversely with free testosterone and free fatty acids concentrations under insulin suppression (R2 = 0.634, P < .001). Conclusions: Metabolic inflexibility is a feature of PCOS women. Both insulin resistance and androgen excess might contribute to this abnormality.

This retrospective study was planned to establish potential associations between circulating values of cardiac troponins and those of conventional blood lipids.The study population consisted of patients attending an inpatient clinic of the University Hospital of Verona during the year 2015 as part of routine cardiovascular risk assessment. No exclusion criteria were applied. Serum lipids including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) were measured using reference enzymatic techniques, whereas troponin T (TnT) was measured using a high-sensitivity (HS) immunoassay. A second analysis was also performed in the General Hospital of Verona, extracting data from the local laboratory database of all patients in whom troponin I (TnI) and blood lipids were simultaneously measured during the same year.In univariate analysis, HS-TnT was found to be associated with age, sex, TC, LDL-C, HDL-C, but not with TG. In multivariate linear regression analysis, age (positive correlation; P0.001) and HDL-C (negative correlation; P=0.032) remained significantly associated with HS-TnT. The frequency of HS-TnT values50 ng/L was higher in subjects with HDL-C1 mmol/L than in those with HDL-C ≥1 mmol/L [odds ratio (OR), 1.84; 95% confidence interval (CI), 1.03-3.32]. The frequency of HS-TnT values50 ng/L was also higher in elderly subjects than in younger ones (OR, 2.10; 95% CI, 1.15-3.84). The combination of age and HDL-C explained 35% of overall variability of TnT concentration. In the second analysis, HDL-C was also found to be an independent and negative predictor of TnI in multivariate linear regression analysis (P=0.010). The combination of age and HDL-C explained approximately 28% of the overall variability of TnI concentration.Our study suggests that HDL-C values inversely predict cardiac troponins concentration irrespective of age, sex and other blood lipids.

Background Systemic mastocytosis (SM) may be associated with hymenoptera allergy. In such cases, immunotherapy is a life-saving treatment, but a circumstantiated diagnosis is needed for its prescription. Patients with SM and previous reactions to stings, but with negative tests represent a diagnostic dilemma. The basophil activation test (BAT) may be helpful in refining the diagnosis. Objective We assessed the usefulness of BAT in subpopulations of mastocytosis patients, including those with negative tests for insect allergy. Methods Within a population of patients with mastocytosis and previous stings, we studied by BAT and augmented intradermal test (IDT) (10 µg/ml) two groups: (1) with reactions to stings and negative tests; (2) without reactions and negative tests. Basophil activation test was performed with different venoms, assessing at flow cytometry basophils’ activation. Results Sixty-three patients had mastocytosis and 52 had reactions to previous hymenoptera stings. Of them, seven proved negative to diagnostic tests. In six of seven of those patients, BAT was negative with all venoms, and in one, basophils resulted activated also with the negative control. In six patients without previous reactions and negative tests, BAT was totally negative in five of six patients and weakly positive to Hornet in one. Finally, the IDT at 10 µg/ml venom produced nonspecific positive results in most cases. Conclusion In patients with mastocytosis, the negative results of standard tests are reliable, because BAT and IDT at higher concentration do not add useful information.

To investigate, along the pituitary- testis- prostate axis, the potential of preoperative serum TT in contributing to defining separate prostatectomy Gleason score (pGS) groups of the prostate cancer (PC) population.The data of 126 patients operated on for PC were retrospectively reviewed. No patient had previously received 5α-reductase inhibitor, luteinizing hormone (LH)-releasing hormone analogs or testosterone replacement treatment. The patient population was grouped according to the prostatectomy Gleason score (pGS) as 6=3+3, 7=3+4, 7=4+3 and 8-10. Twelve variables were simultaneously investigated in each group: age, prolactin (PRL), follicle stimulating hormone (FSH), LH, total testosterone (TT), free testosterone (FT), estradiol (Er), prostate specific antigen (PSA), percentage of prostate biopsy positive cores (P+), biopsy Gleason score (bGS), overall cancer volume estimated as percentage of prostate volume (V+) and prostate weight (Wi). Univariate analysis of variance (ANOVA), multivariate analysis of variance (MANOVA) and multivariate discriminant analysis were the statistical methods used for evaluating the data.There were 38 patients in pGS 6=3+3, 57 in pGS 7=3+4, 15 in pGS 7=4+3 and 16 in pGS 8-10. ANOVA showed that bGS (p0.0001), P+ (p0.0001), V+ (p0.0001), PSA (p=0.02), Wi (p=0.001) and TT (p=0.04) were significantly different in the four pGS groups. MANOVA tests showed that only bGS (p0.0001) and TT (p=0.005) were the significant variables that individually and independently contributed a significant amount to separation of the four pGS groups of the PC population. Multivariate discriminant analysis confirmed that TT (p=0.005) and bGS (p0.0001) were the only variables that independently and significantly contributed to separating the pGS groups.along the pituitary- testis- prostate axis, serum TT is a significant preoperative variable that independently contributes to separating the prostate cancer population into pGS score groups. Pretreatment baseline serum TT levels should be measured for their inclusion in nomograms and future neural networks to be considered in the patient population diagnosed with PC.

We systematically assessed bone mineral density (BMD), bone turnover markers (BTM), and fractures in a large cohort of patients with Indolent Systemic Mastocytosis (ISM).Eighty-two patients (mean age 48 years, 37 women) with ISM were studied. BMD was measured by dual X-ray absorptiometry at the lumbar spine and proximal hip. The serum markers of bone turnover included bone-specific alkaline phosphatase, C-telopeptides of type I collagen, and serum osteocalcin. Previous clinical fractures were registered and spine X-ray was obtained from all patients.Three women were excluded for concomitant diseases associated to osteoporosis. Osteoporosis according with the WHO classification (T-score-2.5) was found in 16 patients (20.0%) (7 females and 9 men). Mastocytosis-related low BMD (Z-score at either the spine or the hip-2) was found in 3 women (9%) and 13 men (28%). The BMD was generally lower at the spine than at the hip. No significant correlation was observed between serum tryptase levels and T or Z-score BMD. One or more moderate or severe vertebral fractures were found in 17 patients (12 men); in 11 of them Z-score values were-2 or not valuable at the spine. No significant difference was found in the prevalence of mastocytosis-related low BMD and/or vertebral fractures between patients with or without skin involvement. Two patients had radiographic and densitometric osteosclerosis-like characteristics. In osteoporotic patients higher, normal or lower serum BTM were found, without correlations with serum tryptase levels, while in patients with osteosclerosis both BTM and serum tryptase values were particularly increased.Vertebral osteoporosis and fractures are frequent in patients with ISM. Spine X-ray and densitometric examination are warranted in all patients, also without skin involvement and particularly in males; Z-score other than T-score BMD must be evaluated. Patients with idiopathic osteoporosis should be evaluated for mast cell disease. Both high than low BTM can be observed in patients with osteoporosis while osteosclerosis is characterized by high bone turnover and serum tryptase levels.

Aim: To evaluate the physiopathology of follicle-stimulating hormone (FSH) along the pituitary-testicular-prostate axis at the time of initial diagnosis of prostate cancer in relation to the available clinical variables and to the subsequent cluster selection of the patient population. Patients and Methods: The study included 98 patients who were diagnosed with prostate cancer. Age, percentages of positive cores (P+) at transrectal ultrasound scan biopsy, biopsy Gleason score (bGS), luteinizing hormone (LH), FSH, total testosterone, free testosterone (FT) and prostate-specific antigen (PSA) were the continuous clinical variables. All patients had not previously received hormonal manipulations. FSH correlation and multiple linear analyses were computed in the population. The FSH/PSA ratio was computed and then ranked for clustering the population as groups A (0.13≤FSH/PSA≤0.57), B (0.57Results: In the patient population, FSH correlated to LH (p < 0.0001), FT (p = 0.007) and age (p = 0.004). FSH was independently predicted by both LH (p < 0.0001) and PSA (p = 0.04). PSA predicted FSH/PSA A (p < 0.0001), B (p < 0.0001) and C (p = 0.04). On multiple regression analysis, FSH/PSA A was predicted by PSA (p < 0.0001), P+ (p = 0.03) and bGS (p = 0.04); FSH/PSA B by LH (p = 0.002) and PSA (p < 0.0001); FSH/PSA C by LH (p < 0.0001) and PSA (p < 0.0001). Moreover, FSH/PSA A, B and C differed for mean values of FSH (p < 0.0001), LH (p < 0.0001), PSA (p < 0.0001) and PSA/FT ratio (p < 0.0001). FSH/PSA clusters showed features of decreasing aggressive disease as the FSH/PSA ratio progressed from A to C. Conclusion:At the diagnosis of prostate cancer and along the pituitary-testis-prostate axis in a patient population FSH significantly correlated to LH, FT and age, and FSH was independently and significantly predicted by both LH and PSA. Because of the independent prediction of PSA by FSH, the prostate cancer population at diagnosis was clustered and ranked according to the FSH/PSA ratio in groups A, B and C. Also, the predictive model of PSA on FSH for the different groups proved to be effective at selecting potential prognostic clusters in which the risk of progression might be assessed as low (group C), intermediate (group B) and high (group A). The FSH/PSA model might be considered as a tool for prostate cancer study and for use in individualized, risk-adapted approaches. However, confirmatory studies are needed.

Objectives: To explore, in operated prostate cancer patients, functional relationships of total testosterone (tt) predicting free testosterone (ft) and total PSA. Patients and Methods: 128 operated prostate cancer patients were simultaneously investigated for tt, ft and PSA before surgery. Patients were not receiving 5α-reductase inihibitors, LH-releasing hormone analogues and testosterone replacement treatment. Scatter plots including ft and PSA versus tt were computed in order to assess the functional relationship of the variables. Linear regression analysis of tt predicting ft and PSA was computed. Results: tt was a significant predictor of the response variable (ft) and different subsets of the patient population were assessed according to the ft to tt ratio. PSA was related to tt according to a nonlinear law. tt was a significant predictor of PSA according to an inversely nonlinear law and different significant clusters of the patient population were assessed according to the different constant of proportionality computed from experimental data. Conclusions: In our prostate cancer population, ft was significantly predicted by tt according to a linear law, and the ft/tt ratio was a significant parameter for assessing the different clusters. Also, tt was a significant variable predicting PSA by a nonlinear law and different clusters of the patient population were assessed by the different constants of proportionality. As a theory, we explain the nonlinear relation of tt in predicting PSA as follows: (a) the number of androgen-independent prostate cancer cells increases as tumor volume and PSA serum levels rise, (b) the prevalence of androgen-independent cells producing a substance which inhibits serum LH, and (c) as a result lower levels of serum tt are detected.

Summary Background: Cross-reactions between venoms may be responsible for multiple diagnostic positivities in hymenoptera allergy. There is limited data on the cross-reactivity between Vespula spp and Vespa crabro, which is an important cause of severe reactions in some parts of Europe. We studied by CAP-inhibition assays and immunoblotting the cross-reactivity between the two venoms. Methods: Sera from patients with non discriminative skin/CAP positivity to both Vespula and Vespa crabro were collected for the analyses. Inhibition assays were carried out with a CAP method, incubating the sera separately with both venoms and subsequently measuring the specific IgE to venoms themselves. Immunoblotting was performed on sera with ambiguous results at the CAP-inhibition. Results: Seventeen patients had a severe reaction after Vespa crabro sting and proved skin and CAP positive also to vespula. In 11/17 patients, Vespula venom completely inhibited IgE binding to VC venom, whereas VC venom inhibited binding to Vespula venom only partially (

Objectives: To explore the significance of the pretreatment total prostate-specific antigen (PSA) to free testosterone (FT) ratio (PSA/FT) as a marker for assessing the pathologic Gleason sum (pGS) and levels of tumor extension (pT) in prostatectomy specimens. Patients and Methods: 128 of 135 consecutive patients diagnosed with prostate cancer underwent radical prostatectomy. Simultaneous pretreatment serum samples were obtained to measure serum total testosterone, FT and total PSA levels. The statistical design of the study included 2 sections: the first part trying to explore the role of the PSA/FT ratio in clustering patients with different pathologic prognostic factors, and the second to investigate the PSA/FT ratio distribution in different groups of patients according to the pathologic stage and pGS of the specimen after radical prostatectomy. Results: The average age was 65.80 (range 51.21–77.26) years, mean PSA was 8.88 (range 1.22–44.27) µg/l, mean FT was 35.32 (range 13.70–69.30) pmol/l, and the mean PSA/FT ratio was 0.27 (range 0.04–1.48). The PSA/FT ratio significantly clustered both the pT and pGS groups. Analysis of variance for the distribution of the PSA/FT ratio was significant for the pT model groups. The mean PSA/FT ratio increased as the tumor extended and grew through the prostate gland (high-stage disease). Analysis of variance for the different distributions of the PSA/FT ratio was significant for all model pGS groups. In our investigation we also found (data not shown) that a PSA/FT ratio of ≧0.40 was strongly correlated with large extensive (pT3b+pT4) and high-grade cancers (pGS8+pGS9). Conclusions: Prostate cancer patients may be classified into 3 different pathologic prognostic groups according to the PSA/FT ratio: low risk (PSA/FT ≤0.20), intermediate risk (PSA/FT >0.20 and ≤0.40), and high risk (PSA/FT >0.40 and ≤1.5). The PSA/FT ratio may be considered as the marker expressing different biology groups of prostate cancer patients, and it is strongly associated with pT and pGS.