Seminal suPAR Levels as Marker of Abacterial Male Accessory Gland Inflammation in Hypogonadism.

BACKGROUND: Recent evidences suggest that hypogonadism is an important risk factor for lower urinary tract symptoms and benign prostatic hyperplasia. Several papers have discussed the role of chronic inflammation in the development of BPH, which may be modulated by the hypogonadal state. Soluble Urokinase-type Plasminogen Activator Receptor (suPAR), known protein marker of systemic inflammation, can be assayed in the seminal plasma and represents a reliable and sensitive marker of inflammation for the Male Accessory Gland Inflammation (MAGI). OBJECTIVE: The aim of this study has been to investigate if seminal suPAR is elevated in MAGI with hypogonadism and if suPAR represent a useful marker of abacterial inflammation in hypogonadism. METHODS: We included in the study twenty male patients aged between 25 and 55 year-old with secondary postsurgical hypogonadism. The same patients were also evaluated after a 3-month of Testosterone Replacement Therapy (TRT), to evaluate the effect of androgen replacement therapy on suPAR. Ten fertile men have been enrolled as a control group in the protocol. SuPAR concentrations were assayed on seminal plasma using an Enzyme-Linked Immunosorbent Assay (ELISA) kit. RESULTS: Hypogonadic patients presented significantly increased levels of seminal suPAR respect to controls (86.1±36.8 vs 55.2±20.0 ng/mL, p<0.05). TRT in hypogonadic patients has been associated with a significant reduction of suPAR levels as reported in the control group (50.9±22.91 vs 86.1±36.8 ng/ml p<0.05). CONCLUSIONS: These results confirm the role of suPAR as a protein marker of MAGI and support the hypothesis that hypogonadism induces a state of inflammation in male accessory glands which is involved in male infertility. Moreover demonstrated that testosterone treatment probably exerts a positive effect on MAGI and infertility as documented by reduction of suPAR levels in hypogonadic treated patients.

Enhanced liver fibrosis test as a reliable tool for assessing fibrosis in nonalcoholic fatty liver disease in a clinical setting.

BACKGROUND: Liver fibrosis is the main determinant and predictor of the clinical course of nonalcoholic fatty liver disease (NAFLD). To date, a liver biopsy is still considered the gold standard for staging fibrosis. The aim of this study was to investigate the diagnostic accuracy of the commercial enhanced liver fibrosis (ELF) test manufacturer's cutoff value (≥9.8) in identifying severe fibrosis for adult patients with histologically confirmed NAFLD. METHODS: We tested the ELF test in a clinical practice, prospective cohort of 82 consecutive patients who consecutively underwent percutaneous liver biopsy. RESULTS: All stages of liver fibrosis were represented in our cohort, and severe fibrosis was present in 15 of 82 patients (18.3%). The stage of fibrosis was significantly associated with ELF score (Spearman's rho = 0.483, p<0.001). The commercial ELF test manufacturer's cutoff identified severe fibrosis with good sensitivity (86.7%; 95% confidence interval [95% CI], 0.69-1.04) and high specificity (92.5%; 95% CI, 0.86-0.99), with a positive predictive value of 72% and negative predictive value of 97%. CONCLUSIONS: Our data could support the use of the ELF test in clinical practice.

Identification and Characterization of a New BRCA2 Rearrangement in an Italian Family with Hereditary Breast and Ovarian Cancer Syndrome.

INTRODUCTION: Many studies document the involvement of BRCA1/2 gene rearrangements in genetic predisposition to breast and ovarian cancer. Large genomic rearrangements (LGRs) of BRCA1 account for 0-27% of all disease-causing mutations in various populations, while LGRs in BRCA2 are rarer. Here, we describe a novel BRCA2 LGR, involving the duplication of exons 4-26, in an Italian family with hereditary breast and ovarian cancer (HBOC) syndrome. OBJECTIVE: Our purpose was to provide an effective characterization of this variant using a combination of different methods able to establish the exact breakpoints of the duplication. METHODS: A multiplex amplicon quantification (MAQ) assay was used as the primary screening method in the detection of LGRs. Array comparative genomic hybridization (CGH), reverse transcriptase polymerase chain reaction (RT-PCR) and long-range PCR were used for the careful characterization of the rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RESULTS: Array CGH and long-range PCR strategies revealed that the BRCA2 exons 4-26 duplication (g.12016_87170dup) involved exactly 75,154 bp nucleotides between intron 3 and intron 26 of the gene. Given that no Alu repeats were found at the junction sites, we support the hypothesis that the new duplication could be the result of a microhomology-mediated event (MH) involving very short homologous sequences at an upstream breakpoint. DISCUSSION: LGR investigation is mandatory in BRCA1/2 routine testing in order to provide a complete result for a targeted therapeutic decision. Nevertheless, the characterization and classification of novel BRCA1/2 variants represents a crucial step in the support of genetic counselling. Our results, including a comprehensive co-segregation analysis, indicate that the novel duplication identifed has a pathogenic role and would be considered a causing-disease variant in genetic and oncologic counselling.

Characterization of a new BRCA1 rearrangement in an Italian woman with hereditary breast and ovarian cancer syndrome.

BACKGROUND: We report a novel BRCA1 LGR, involving the complete duplication of exon 3, in an Italian patient with a strong family history of breast and ovarian cancer. Our purpose is to provide an effective characterization of this LGR using a combination of different methods able to establish the exact breakpoints of the duplication. METHODS: MAQ assay was used as primary screening method in LGRs detection. Array CGH, RT-PCR, and Long-PCR were used for a careful characterization of rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RESULTS: RNA analysis showed that this in tandem duplication of exon 3 causes an in frame insertion of 18 amino acids within the protein. Array CGH and Long-PCR strategies revealed that the duplication (g.100411_102863dup) involves exactly 2.452 nucleotides between intron 2 and intron 3 of the gene. In addition, while an Alu Sx sequence was identified at upstream breakpoint, no Alu repeats were found at downstream junction. This supports the hypothesis that the new duplication was the result of a non-homologous recombination event between Alu and Non-Alu sequences. CONCLUSION: Our strategy, which combines a comprehensive set of methodologies, has been able to characterize the new BRCA1 duplication confirming, as previously reported, that MAQ assay represents a reliable and effective method for a primary screening of BRCA rearrangements. We underline the relevance of incorporating quantitative methods for BRCA genes dosage testing into routine diagnostic practice.

Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: A new approach to assess quantitative status of BRCA1 gene in a reference laboratory.

AIM OF THE STUDY: Evaluation of copy number variation (CNV) in BRCA1/2 genes, due to large genomic rearrangements (LGRs), is a mandatory analysis in hereditary breast and ovarian cancers families, if no pathogenic variants are found by sequencing. LGRs cannot be detected by conventional methods and several alternative methods have been developed. Since these approaches are expensive and time consuming, identification of alternative screening methods for LGRs detection is needed in order to reduce and optimize the diagnostic procedure. The aim of this study was to investigate a Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) as molecular tool to detect recurrent BRCA1 LGRs. MATERIAL AND METHODS: C-PCR-HRMA was performed on exons 3, 14, 18, 19, 20 and 21 of the BRCA1 gene; exons 4, 6 and 7 of the ALB gene were used as reference fragments. RESULTS: This study showed that it is possible to identify recurrent BRCA1 LGRs, by melting peak height ratio between target (BRCA1) and reference (ALB) fragments. Furthermore, we underline that a peculiar amplicon-melting profile is associated to a specific BRCA1 LGR. All C-PCR-HRMA results were confirmed by Multiplex ligation-dependent probe amplification. CONCLUSIONS: C-PCR-HRMA has proved to be an innovative, efficient and fast method for BRCA1 LGRs detection. Given the sensitivity, specificity and ease of use, c-PCR-HRMA can be considered an attractive and powerful alternative to other methods for BRCA1 CNVs screening, improving molecular strategies for BRCA testing in the context of Massive Parallel Sequencing.

Fecal but not serum calprotectin is a potential marker of GVHD after stem cell transplantation.

Gastrointestinal graft-versus-host disease (GvHD) represents a life-threatening complication after stem cell transplantation. Differential diagnosis between gut GvHD and other causes of diarrhea after HSCT is still subjected to endoscopy and histological findings. The research for a reliable biomarker for gut GvHD might allow an early diagnosis of this condition and a consequent prompt treatment that could reduce unfavorable outcomes. Recently, fecal calprotectin was reported as reliable marker of gut involvement. We would evaluate if serum instead of fecal calprotectin could be considered a possible biomarker of gut GvHD. Serum calprotectin was measured in a cohort of 54 patients submitted to allogeneic stem cell transplantation using ELISA assay. For a subset of 21 patients, calprotectin serum levels were compared with fecal calprotectin detection. Contrary to fecal calprotectin, we found only a trend to high level of serum calprotectin for GvHD development and gut involvement, but statistical difference was not reached. Fecal but not serum calprotectin could be considered as possible biomarker for gut GvHD.

Follow-up of IgD-κ multiple myeloma by monitoring free light chains and total heavy chain IgD: A case report.

Immunoglobulin (Ig)D-κ multiple myeloma (MM) is a rare neoplastic disease characterized by an aggressive and rapidly progressing course, which constitutes only a very small proportion of all MM cases. In the present report, the clinical case of a 51-year-old Caucasian woman diagnosed with IgD-κ MM is described. The patient underwent different chemotherapeutic treatments subsequently to a single autologous stem cell transplantation. Despite the inherent difficulty of monitoring IgD levels and performing serum immunofixation electrophoresis, the clinical outcome of the patient was almost uniquely monitored by measuring the levels of κ and λ free light chains (FLCs) and total heavy chain IgD. The data suggest the non-invasive potential and usefulness of FLCs evaluation for early detection of stringent complete remission, follow-up and early detection of disease relapse. In addition, this diagnostic procedure has successfully been employed for the therapeutic monitoring of the present patient, and may represent a very helpful, non-invasive tool for the follow-up of IgD myeloma patients without the requirement of serial bone marrow aspirate.

Intraoperative high-dose calcium stimulation test in patients with sporadic medullary thyroid carcinoma is highly accurate in predicting lateral neck metastases.

BACKGROUND: Intraoperative measurement of calcitonin is not highly accurate in predicting the completeness of the operative resection after total thyroidectomy combined with central neck dissection (TT-CND) in patients with medullary thyroid carcinoma (MTC). We evaluated whether an intraoperative, high-dose calcium stimulation test (IO-CST) after TT-CND can predict lateral neck involvement. METHODS: Eleven patients who underwent primary operation for sporadic MTC were included. High-dose (25 mg/kg) calcium gluconate was administered after TT-CND with calcitonin measured at 2, 5, and 10 minutes after the calcium gluconate infusion. RESULTS: There were 2 males and 9 females (mean age, 51 years; range, 18-88). Three patients showed lateral neck metastases. At a mean follow-up of 7.0 months (range, 2-10), 1 patient showed distant metastases and 1 a slightly increased calcitonin level. After IO-CST, serum calcitonin increased in all the 3 patients with lateral neck metastases, and it remained unchanged or decreased in the other patients without lateral neck metastases. Percent variation of serum calcitonin after IO-CST was 92% in patients with lateral neck metastases and -3.1 ± 4.9% in patients without lateral neck metastases. CONCLUSION: Calcitonin measurement after IO-CST in patients with sporadic MTC can be highly accurate in predicting lateral neck nodes involvement. These results could represent a stimulus toward the development of a quick calcitonin assay.

Nasal lavage levels of granulocyte-macrophage colony-stimulating factor and chronic nasal hypereosinophilia.

BACKGROUND: The aim of the present study was to measure levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in nasal lavage of patients affected by chronic eosinophilic sinonasal inflammation to clarify the relationship with eosinophilic tissue infiltration and clinical features. METHODS: Between November 2012 and June 2013, we selected 70 patients with chronic eosinophilic inflammation (average age 41.8 years) who were classified into the following groups: persistent allergic rhinitis (group 1), noninfectious non-allergic rhinitis with eosinophilia syndrome (group 2) and chronic rhinosinusitis with polyps (group 3). Finally, we enrolled 20 healthy subjects as controls (group 4). All patients underwent symptoms score questionnaire based on a visual analogue scale, nasal endoscopy and/or computed tomography (CT) scan, and allergy testing. Nasal cytology by scraping of the mucosa and GM-CSF assays in nasal lavage were performed in all subjects. RESULTS: Detectable levels of GM-CSF were found in 34 of 70 (48.57%) patients, with an average concentration of 2.67 ± 0.8 pg/mL, whereas in controls only 1 of 20 individuals showed detectable GM-CSF levels. Eosinophil infiltration was significantly higher in patients with detectable GM-CSF compared to those with undetectable levels (49.4% vs 39.2%, respectively; p < 0.05). Furthermore, significant weakly-moderate correlation was found between GM-CSF levels and percentage of eosinophil infiltration in tissue (p < 0.05). Correlation between symptom scores and GM-CSF levels was significant only in group 2, which showed higher average concentrations of GM-CSF compared to groups 1 and 3 (2.9 pg/mL vs 1.6 pg/mL and 1.8 pg/mL, respectively; p < 0.05). CONCLUSION: Our data confirm that GM-CSF is more frequently detectable in nasal lavages of patients affected by chronic sinonasal eosinophilic inflammation than in controls. Statistical analyses revealed a significant weakly-moderate correlation between GM-CSF levels in nasal lavage of all patients and percentage of eosinophil infiltration of nasal mucosa.

A preliminary Quality Control (QC) for next generation sequencing (NGS) library evaluation turns out to be a very useful tool for a rapid detection of BRCA1/2 deleterious mutations.

BACKGROUND: Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the entire genomic region of BRCA1/2 genes and, to date, many studies report the effectiveness of these technologies. Here we show that Gene Scan (GS) labeling Quality Control (QC), performed before massive parallel pyrosequencing, coupled with Multiple Amplicon Quantification software (MAQ-S) analysis is a rapid and powerful tool in the detection of deleterious BRCA mutations carried by different patients. METHODS: GS labeling QC assay was performed according to the manufacturers' instructions and MAQ-S software was employed for analysis results. RESULTS: GS labeling QC was able to detect 14 different BRCA frameshift mutations in our patients. In addition, two novel BRCA mutations (c.1893_1894insTTAAGCCCACAAAT in BRCA1 gene and c.9413_9414insT in BRCA2 gene) were identified. CONCLUSION: We prove that a simple QC step may represent a valid and useful tool for a rapid detection of frameshift mutations in BRCA genes. For this reason, we recommend using this approach before massive parallel sequencing.

Nasal fluid release of eotaxin-3 and eotaxin-2 in persistent sinonasal eosinophilic inflammation.

BACKGROUND: The aim of the present study was to measure eotaxin-3 (CCL26) and eotaxin-2 (CCL24) in nasal lavage fluid of patients with different forms of chronic sinonasal eosinophilic inflammation to evaluate their role in the pathophysiology of nasal hypereosinophilia. METHODS: The study was an analytic cross-section study, level of evidence 3b. Patients (n = 80) with nasal hypereosinophilia were randomly recruited and grouped in the following categories: persistent allergic rhinitis (AR) (n = 25), nonallergic rhinitis with eosinophilia syndrome (NARES) (n = 30), and chronic rhinosinusitis with polyps (CRSwNP) (n = 25). Non-rhinitic volunteers (n = 20) were recruited as controls. CCL24 and CCL26 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) Quantikine Human Immunoassays (R&D Systems, Minneapolis, MN) in nasal lavage fluids. Differential cell counts were performed by microscopic cytological examination of nasal tissue scraped from the inferior turbinate. RESULTS: Mean CCL26 levels were significantly higher (p < 0.05) in AR and in NARES (132.0 pg/mL and 187.63 pg/mL, respectively) than in the control group (13.5 pg/mL); in patients with CRSwNP, CCL26 values were increased compared to controls even though the difference was not statistically significant (58.9 pg/mL vs 16.5 pg/mL). Mean CCL24 levels measured in AR, NARES, and CRSwNP were significantly increased (p < 0.05) compared to controls (96.7 pg/mL, 135.4 pg/mL, and 107.0 pg/mL, respectively, vs 32.2 pg/mL). Moreover, we observed a significant correlation between CCL24 and CCL26 levels, evaluating them intraindividually by Spearman's rank correlation test. Finally, a significant correlation was found between CCL24 and CCL26 levels and the percentage of eosinophilic infiltration of nasal mucosa. CONCLUSION: Our data suggest that CCL26 and CCL24 are likely involved in the pathogenesis of chronic nasal hypereosinophilia, with a complex cooperation and different involvement of the various members of eotaxin family. Further studies are necessary to better understand the actual physiopathologic mechanism, possible clinical relevance, and therapeutic implications.

DNA from buccal swab is suitable for rapid genotyping of angiotensin-converting enzyme insertion/deletion (I/D) polymorphism.

BACKGROUND: Plasma angiotensin-converting enzyme (ACE) variability between individuals is the results of an insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene. The I and D alleles differ for the presence or absence of a 288 bp Alu sequence DNA fragment. METHODS: The present paper regards the development of a single-tube High Resolution Melting Analysis (HRMA), applied to DNA extracted by buccal swabs, for determining three ACE I/I, I/D, D/D genotypes, in order to obtain a rapid and high throughput method. This method takes advantage of the presence of the 288 bp DNA fragment. Primer design was performed taking into account the possible different efficiency of allele I amplification compared to allele D, avoiding the misclassification of I/D with D/D genotypes. RESULTS: 50 samples previously genotyped by "conventional" PCR protocol already published in literature were 100% concordant with the HRMA results, showing high reproducibility, sensitivity and specificity. ACE genotypes were distinguished by normalized temperature melting curves and by derivate fluorescence plots. CONCLUSIONS: HRMA was confirmed as particularly suitable for the identification of ACE I/D polymorphism. Simple setup and rapidity of the analysis (about 1.5 h for 96 samples, including data interpretation) are other important advantages along with low-costs, making this technique useful in clinical research and diagnostics.

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

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

Is intraoperative calcitonin monitoring useful to modulate the extension of neck dissection in patients with medullary thyroid carcinoma?

BACKGROUND: The extension of the compartment-oriented neck dissection at primary surgery in medullary thyroid carcinoma (MTC) is controversial. Because a <50 % decrease in intraoperative calcitonin levels (IO-CT) after total thyroidectomy plus central neck dissection (TT-CND) has been associated with residual disease, IO-CT monitoring has been proposed to predict the completeness of surgery. The goal of the present prospective study was to verify the accuracy of IO-CT monitoring. METHODS: All patients scheduled for primary surgery for suspected or proven MTC between November 2010 and January 2013 were included. Calcitonin was measured pre-incision (basal level), after tumor manipulation, at the time TT-CND was accomplished (ablation level), 10 and 30 min after ablation. A decrease >50 % with respect to the highest IO-CT level 30 min after ablation was considered predictive of cure. RESULTS: Twenty-six patients were included, and IO-CT monitoring identified 18 of 23 cured patients (true negative results) and 2 of 3 patients with persistent disease (true positive result). In 5 patients with normal basal and stimulated postoperative calcitonin levels, a decrease <50 % was observed (false positive results). In one of three patients with persistent disease a >50 % decrease in IO-CT was observed (false negative results). Specificity, sensitivity, and accuracy of IO-CT were 78.2, 66.6, and 76.9 %, respectively. CONCLUSIONS: Intraoperative calcitonin monitoring is not highly accurate in predicting the completeness of surgical resection. In the present series, relying on IO-CT would result in limited resection in about one third of the patients with residual neck disease and in unnecessary lateral neck dissection in about 20 % of the cured patients.

Evaluation of the diagnostic and predictive power of PCA3 in the prostate cancer. A different best cut-off in each different scenario. Preliminary results.

INTRODUCTION: Aim of this study is to evaluate the diagnostic performance of PCA3 in patients with indication to perform a new biopsy, according to the histological doubt such as High Grade Prostatic Intraepithelial Neoplasia (HGPIN) or Atypical Small Gland Proliferation (ASAP) or the clinical suspicion. MATERIALS AND METHODS: One hundred men were enrolled. We used the PCA3 - PROGENSA™ procedure. After the PCA3 test a repeated prostate biopsy was proposed. The histological findings were correlated to the PCA3 scores. We calculated the positive predictive value (PPV), the sensibility, the specificity, the Youden's index, the ROC curves, the area under the curve (AUC) for each cut-off value of PCA3 score. RESULTS: These results are preliminary, because at present only 50 of the 100 enlisted men were subjected to rebiopsy. We calculated the best cut-off PCA3 score 20 at the first diagnosis; for patients with HGPIN or ASAP at first biopsy the best sensitivity cut-off is 45; the best cutoff is 45 when you already have a diagnosis of HGPIN, and 35 for ASAP. If we normalize the PCA3 score to the prostate volume, the best cut-off would be 20, with 100% sensitivity with a prostate volume of 65 ml. All results are statistically significant. The real problem, also present in literature, is the constant presence of not diagnosed prostate cancers, for any cut-off value. CONCLUSIONS: Our preliminary results suggest that, to get the best diagnostic performance, it would be wrong to maintain a single cut-off, but it should be chosen according to the scenario of the patients subgroup. It is to explore the possibility to search for the PCA3 in the serum to bridge the gap of the aggressive PCa missed by the urinary test.

Effects of lutein supplementation on biological antioxidant status in preterm infants: a randomized clinical trial.

OBJECTIVE: To test the hypothesis that lutein, compared to the placebo, would enhance the total antioxidant status (TAS) in the preterm infants. METHODS: Infants with gestational age (GA) ≤34 weeks were randomly assigned to receive a daily dose of lutein and zeaxanthin (0.5 mg + 0.02 mg/kg/d) or placebo from the 7th day of life until 40th week of postmenstrual age or until discharge. RESULTS: Seventy-seven preterm infants were randomized (38 in the Lutein group and 39 in the Placebo group) with mean GA of 30.4 (±2.3) weeks and the mean birth weight of 1415 (±457) grams. The TAS did not result statistically different between the two groups during all the study period, but a significant linear correlation was evidenced between plasma lutein concentration and TAS (r = 0.14, p = 0.012) and between plasma zeaxanthin concentration and TAS (r = 0.13, p = 0.02). CONCLUSIONS: Supplementation of preterm infants with orally lutein was ineffective in enhancing biological antioxidant capacity. Further studies need to better understand the bioavailability of lutein in the neonatal period in order to identify any best form of supplementation. TRIAL REGISTRATION NUMBER: UMIN000007041.

Circulating tumor cells in colorectal cancer patients.

The availability of sensitive methods has allowed the detailed study of circulating tumor cells only recently. Evolving evidence support the prognostic and predictive role of these cells in patients affected by several solid tumors, including colorectal cancer. Ongoing studies are aimed at confirming that the molecular characterization of circulating tumor cells in peripheral blood and in bone marrow of patients is a powerful tool to improve the patient risk-stratification, to monitor activity of the drugs, to develop more appropriate targeted therapies and tailored treatments. In parallel, results from these correlative studies promise to gain a better biological understanding of the metastatic process. The clinical utility of the detection of circulating tumor cells in patients affected by colorectal cancer is still hampered by a number of specific hurdles. Improvement in sensitivity and specificity of the available methods of detection, standardization of these methods and functional characterization of circulating tumor cells in well designed and statistically well powered studies are the key steps to reach these ambitious objectives in colorectal cancer patients as well.

Myocardial performance index and biochemical markers for early detection of doxorubicin-induced cardiotoxicity in children with acute lymphoblastic leukaemia.

BACKGROUND: Despite significant improvements in the prognosis of childhood acute lymphoblastic leukaemia (ALL), the risk of anthracycline-induced cardiovascular disease remains a major concern. This study was designed to investigate the role of the myocardial performance index (MPI) and serum concentrations of biomarkers (cTnT and NT-pro-BNP) in the early detection of subclinical anthracycline-induced functional alterations in children with ALL. METHODS: All children consecutively admitted to our Pediatric Oncologic Department from January 2009 to October 2010 with a diagnosis of ALL were enrolled in this study. cTnT and NT-pro-BNP were evaluated in all patients at diagnosis, before doxorubicin therapy and 2 and 24 h following each anthracycline administration. ECG and echocardiography were performed at diagnosis and 24 h after each anthracycline course. RESULTS: Nineteen children with standard-risk ALL were evaluated. The mean age was 6 years. The cumulative doxorubicin dosage was 240 mg/m(2) according to the AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) ALL 2000 protocol. None of the 19 patients developed congestive heart failure. With increasing cumulative dosages of anthracyclines a significant increase was observed in MPI. This increase was statistically significant starting from the cumulative dosage of 120 mg/m(2) compared to baseline, while the median NT-pro-BNP level did not change significantly during treatment and cTnT levels never exceeded the cut-off value for cardiac injury. CONCLUSION: MPI value is a sensitive and accurate parameter, allowing subclinical cardiac dysfunction to be detected in children receiving anthracyclines. Lifelong cardiac surveillance of these patients is warranted in order to determine the clinical implications of increased MPI on long-term cardiac status.

Small amplicons high resolution melting analysis (SA-HRMA) allows successful genotyping of acid phosphatase 1 (ACP1) polymorphisms in the Italian population.

BACKGROUND: The ACP1 gene, encoding a low-molecular-weight phosphotyrosine phosphatase (LMW-PTP), has been suggested as a common genetic factor of several human diseases, including inflammatory and autoimmune diseases, favism and tumors. For this reason, the ACP1 enzyme has been investigated by case-control studies for decades. Initially based on protein electrophoresis, the ACP1 phenotype is now determined by DNA-based techniques. METHODS: Here, we report a rapid optimized method which employs HRMA for ACP1 polymorphism identification, a molecular approach that we used to screen 80 healthy Italian subjects. RESULTS: HRMA proved particularly suitable for detecting ACP1 genotypes. In fact, HRMA results were 100% concordant with direct sequencing. In addition, ACP1 genotype frequency in the Italian population was in accordance with the literature [4% (*A/A), 36% (*A/B), 4% (*A/C), 50% (*B/B), 6% (*B/C)]. CONCLUSIONS: HRMA was found to be a simple, rapid, sensitive and low cost method potentially useful in research and diagnostic laboratories. Finally, use of small amplicons for the set-up allowed us a better optimization of HRMA. For this reason, we present such an approach as small amplicons high resolution melting analysis (SA-HRMA). Finally, ACP1 genotype frequency in the Italian population reported in this study may contribute to a better interpretation of ACP1 allelic frequency variation.

Potential usefulness of CTC detection in follow up of prostate cancer patients. A preliminary report obtained by using Adnagene platform.

OBJECTIVE: Prostate cancer (PCa) represents one of the most important medical problems for males, being the second major cause of cancer death. Routinely, PCa patients are followed up with both periodic evaluation of serum PSA levels and imaging. Recently, alternative laboratory methods were proposed for PCa patients' monitoring, with contrasting results. Aim of the present study was to evaluate the usefulness of a new commercially CE-IVD kit for detection of prostate circulating tumour cells. Our intention was to verify the Adnagene platform usefulness to identify patients with disease progression, whatever treatment ongoing, in order to modify the therapeutic process even before treatment failure is evident with imaging methods. MATERIALS AND METHODS: Twenty-one patients were enrolled and subdivided into three groups: n = 10 high risk tumor PCa patients; n = 6 low risk PCa patients; n = 5 sbjects without any signs of PCa. AdnaTest Prostate Cancer kit was used for enrichment and molecular characterization of prostate circulating tumour cells. RESULTS: Healthy subjects (with BPH) and patients without metastases resulted as negative, while 3 out of 10 high risk PCa patients were positive at least for one molecular marker like PSA, while only two showed positivity for PSMA mRNA. Our results indicate that the test specificity is 100% and the sensitivity is 100%; of course the sample is too small to give it statistical validity. In detail we verified that only the "not responder" patients resulted positive for AdnaTest. CONCLUSIONS: The present preliminary report provides evidence that isolation and detection of circulating tumour cells (CTCs) is feasible and it may be useful in the follow-up of patients with advanced prostate cancer. If the results of this preliminary study would be confirmed by a large prospective cohort study, it could be demonstrated that this test is a rapid diagnostic method, based on the analysis of a blood sample and useful to the clinician to decide when to change therapy for patients resistant to castration or able to confirm that, at that time, the therapy is effective.