Modulation of Peripheral Immune Cell Subpopulations After RapidArc/Moderate Hypofractionated Radiotherapy for Localized Prostate Cancer: Findings and Comparison With 3D Conformal/Conventional Fractionation Treatment.

Radiotherapy (RT) is an important therapeutic option in patients with localized prostate cancer (PC). Unfortunately, radiation treatment causes a decrease in peripheral lymphocytes and, consequently, influences the patients' immune status. Our aim was to study changes in peripheral blood immune cell subpopulations after RT and during 6 months' follow-up in 2 groups of PC patients irradiated with different techniques and dose fractions with curative intent. We also investigated the presence of correlation between immune cell modulation and genitourinary or gastrointestinal toxicity. We enrolled 44 patients treated with curative RT (RapidArc/hypofractionation regimen or 3D conformal/conventional fractionation) for localized PC. Total white blood cell (WBC), absolute lymphocyte counts (ALCs), and peripheral immune cell subpopulations were analyzed at baseline, at the end of RT, and 3 and 6 months after the end of RT. WBC and ALC greatly decreased at the end of RT with a trend to recover at 6 months' follow-up in the hypofractionation group but not in the conventional one. Furthermore, B, total T, T CD4+, T CD8+, and NK cell values dropped significantly in both groups at the end of RT, with a minor decrease detectable in the hypofractionation group for B, total T, and T CD4+ lymphocytes with respect to the other technique/fractionation group. Double-negative T (DNT), double-positive T (DPT), and NKT cells significantly decreased at the end of RT with a slight tendency to recover values during follow-up, particularly in the hypofractionation group. No correlation with genitourinary or gastrointestinal toxicity was found. In this study, we showed, for the first time, the effects of RapidArc/moderate hypofractionation RT on immune cell subsets in patients treated for localized PC. Due to the growing interest in minority T-cell subpopulations for immunotherapy, we also reported longitudinal monitoring of the effects of RT on DNT, DPT, and NKT, which was never studied before. Our preliminary data highlight the importance of considering the effects of different RT techniques/fractionation regimens on peripheral immune cells, in the era of RT and immunotherapy combination.

Circulating Regulatory T-Cell Number Does Not Predict Prognosis of Monoclonal Gammopathies of Uncertain Significance.

FOXP3-expressing regulatory T-cells (Tregs), which suppress aberrant immune response against self-antigens, also suppress anti-tumor immune response. It has been shown that there is an increased proportion of Tregs in several different human malignancies, although the actual mechanism remains unclear. The research aims to explore the relationship between the number of Tregs and a predict prognosis in particular hematological diseases as monoclonal gammopathies of uncertain significance (MGUS). Tregs were evaluated by means of flow cytometry (CD4+CD25high/+ CD127low/-) in whole peripheral blood of 56 patients with MGUS to predict progression to overt multiple myeloma (MM). In two groups of patients, MGUS versus MGUS evolved to MM, we found a significative difference for the number of white blood cells, but not in terms of clinical and laboratory features evaluated at diagnosis. The study demonstrated the absence of a prognostic relevance of Tregs in MGUS. Nevertheless, their role in these disorders is still to be defined.

Analysis of Amount, Size, Protein Phenotype and Molecular Content of Circulating Extracellular Vesicles Identifies New Biomarkers in Multiple Myeloma.

INTRODUCTION: Extracellular vesicles (EVs) are naturally secreted cellular lipid bilayer particles, which carry a selected molecular content. Owing to their systemic availability and their role in tumor pathogenesis, circulating EVs (cEVs) can be a valuable source of new biomarkers useful for tumor diagnosis, prognostication and monitoring. However, a precise approach for isolation and characterization of cEVs as tumor biomarkers, exportable in a clinical setting, has not been conclusively established. METHODS: We developed a novel and laboratory-made procedure based on a bench centrifuge step which allows the isolation of serum cEVs suitable for subsequent characterization of their size, amount and phenotype by nanoparticle tracking analysis, microscopy and flow cytometry, and for nucleic acid assessment by digital PCR. RESULTS: Applied to blood from healthy subjects (HSs) and tumor patients, our approach permitted from a small volume of serum (i) the isolation of a great amount of EVs enriched in small vesicles free from protein contaminants; (ii) a suitable and specific cell origin identification of EVs, and (iii) nucleic acid content assessment. In clonal plasma cell malignancy, like multiple myeloma (MM), our approach allowed us to identify specific MM EVs, and to characterize their size, concentration and microRNA content allowing significant discrimination between MM and HSs. Finally, EV associated biomarkers correlated with MM clinical parameters. CONCLUSION: Overall, our cEV based procedure can play an important role in malignancy biomarker discovery and then in real-time tumor monitoring using minimal invasive samples. From a practical point of view, it is smart (small sample volume), rapid (two hours), easy (no specific expertise required) and requirements are widely available in clinical laboratories.

MicroRNA-155 in serum-derived extracellular vesicles as a potential biomarker for hematologic malignancies - a short report.

PURPOSE: The use of extracellular vesicles (EVs) from body fluids as "liquid biopsies" is emerging as a promising approach for the diagnosis, prognosis and therapeutic monitoring of cancer patients. MicroRNA-155 (miR155), a non-coding transcript of the B-cell integration cluster (BIC) gene, has been reported to play a critical role in the pathogenesis of several types of hematologic malignancies (HMs) in which high miR155 levels have been found. At yet, however, the EV miR155 level and its putative clinical relevance in sera of HM patients have not been reported. METHODS: EVs from sera of representative patients with eight different HMs and healthy subjects (controls) were isolated using differential centrifugation. The identity and quality of the EVs were verified by atomic force and transmission electron microscopy. The EV miR155 levels were measured by quantitative RT-PCR. The sensitivity, specificity and area under the curve (AUC) of differences in EV miR155 levels were determined using ROC curve analyses. RESULTS: We found that the EV miR155 levels were significantly higher in chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and Waldenström's macroglobulinemia (WM) cases compared to controls. Conversely, we found that the EV miR155 levels were significantly lower in myelodysplastic syndrome (MDS) and multiple myeloma (MM) cases. No differences were found in follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL) or Hodgkin's Lymphoma (HL) cases compared to controls. EV miR155 ROC curve analyses revealed significantly different patterns in CLL and AML cases compared to controls, and in AML cases compared to MDS cases (p = 0.004, p = 0.01 and p = 0.04, respectively). In addition, we found that high EV miR155 levels correlated with high white blood cell counts in AML patients. CONCLUSION: Our data indicate that EV miR155 may serve as an attractive new, non-invasive diagnostic biomarker in human hematologic malignancies.

A Pyrazolo[3,4-d]pyrimidine compound inhibits Fyn phosphorylation and induces apoptosis in natural killer cell leukemia.

Natural killer (NK) cell neoplasms are characterized by clonal proliferation of cytotoxic NK cells. Since there is no standard treatment to date, new therapeutic options are needed, especially for NK aggressive tumors. Fyn tyrosine kinase has a key role in different biological processes, such as cell growth and differentiation, being also involved in the pathogenesis of hematologic malignancies. Our previous studies led us to identify 4c pyrazolo[3,4-d]pyrimidine compound capable of inhibiting Fyn activation and inducing apoptosis in different cancer cell lines. Here we investigated the presence of Fyn and the effect of its inhibitor in NK malignant cells. Firstly, we showed Fyn over-expression in NK leukemic cells compared to peripheral blood mononuclear cells from healthy donors. Subsequently, we demonstrated that 4c treatment reduced cell viability, induced caspase 3-mediate apoptosis and cell cycle arrest in NK cells. Moreover, by inhibiting Fyn phosphorylation, 4c compound reduced Akt and P70 S6 kinase activation and changed the expression of genes involved in cell death and survival in NK cells. Our study demonstrated that Fyn is involved in the pathogenesis of NK leukemia and that it could represent a potential target for this neoplasm. Moreover, we proved that Fyn inhibitor pyrazolo[3,4-d]pyrimidine compound, could be a started point to develop new therapeutic agents.

High serum levels of extracellular vesicles expressing malignancy-related markers are released in patients with various types of hematological neoplastic disorders.

Many cell types release extracellular vesicles (EVs), including exosomes, microvesicles (MVs), and apoptotic bodies, which play a role in physiology and diseases. Presence and phenotype of circulating EVs in hematological malignancies (HMs) remain largely unexplored.The aim of this study was to characterize EVs in peripheral blood of HM patients compared to healthy subjects (controls). We isolated serum EVs from patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), Waldenstrom's macroglobulinemia (WM), Hodgkin's lymphoma (HL), multiple myeloma (MM), acute myeloid leukemia (AML), myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDS), and controls. EVs were isolated from serum of peripheral blood by ultracentrifuge steps and analyzed by flow cytometry to define count, size, and immunophenotype. MV levels were significantly elevated in WM, HL, MM, AML, and some MPNs and, though at a lesser degree, in CLL and NHL as compared to healthy controls. HL, MM, and MPNs generated a population of MVs characterized by lower size (below 0.3 µm) when compared to controls. MVs from patients specifically expressed tumor-related antigens, such as CD19 in B cell neoplasms, CD38 in MM, CD13 in myeloid tumors, and CD30 in HL. Both total and antigen-specific count of MVs significantly correlated with different HM clinical features such as Rai stage in CLL, International Prognostic Scoring System in WM, International Staging System in MM, and clinical stage in HL. MVs may represent a novel biomarker in HMs.

Epidermoid cyst of the coronal sulcus mimicking penile cancer: a case report.

INTRODUCTION: Epidermoid cysts represent common benign tumors occurring anywhere in the body but very rarely in the penis. Only a few cases of penile localization have been reported in the literature so far, most of them being congenital and/or idiopathic, usually presenting in children as slow-growing, solitary, well-delimited cystic lesions. Here, we describe the case of a patient with a penile epidermoid cyst presenting as an ulcerated lesion of the coronal sulcus, thus mimicking penile cancer. CASE PRESENTATION: A 36-year-old Caucasian man presented with a three-month history of a rapidly growing asymptomatic ulcerated lesion in the ventral portion of the penile coronal sulcus. At surgical exploration, the area under the ulcerated lesion had a well-demarcated cystic shape; following its wide excision, an intraoperative histological examination revealed an epidermoid cyst. No recurrence had occurred at nine years of follow-up. CONCLUSIONS: Rare benign tumors of the penis, like the described epidermoid cyst, may mimic cancer. Nevertheless, penile ulcerated lesions should always be surgically explored as wide excision and intraoperative histological examination remain the only means of obtaining a precise disease definition and, consequently, administering the appropriate treatment.

Polymorphisms of the CYP1A1, CYP2E1 and XRCC1 genes and cancer risk in a Southern Italian population: a case-control study.

BACKGROUND: Polymorphisms in genes encoding enzymes involved in xenobiotic metabolism and/or in cellular defenses against carcinogen-induced DNA damage play an important role in determining individual cancer susceptibility. However, their distribution and association with cancer susceptibility can vary in different populations. MATERIALS AND METHODS: A case-control study including 290 cancer patients (cases) and 242 controls was performed to evaluate the relationship between polymorphisms of cytochrome P450 (CYP)1A1 and CYP2E1 and X-ray repair complementing defective repair in Chinese hamster cells (XRCC)1 genes and the risk of developing cancer in a Southern Italian (Basilicata) population. Genomic DNA was isolated from 5 ml whole blood and genotyping was performed using a PCR-RFLP technique. RESULTS: No significant differences were observed in the distribution of the CYP1A1, CYP2E1 and XRCC1 gene polymorphisms between the cases and controls in the population under study. CONCLUSION: The distribution of CYP1A1, CYP2E1 and XRCC1 gene polymorphisms in the Basilicata population is not different from that of other Italian regions or from that reported in the literature for Caucasian populations, and polymorphisms in these genes do not play an important role in determining cancer risk in the population under study.

High-intensity focused ultrasound (HIFU) in prostate cancer: a single centre experience in patients with low, intermediate or high-risk of progression.

OBJECTIVE: High-intensity focused ultrasound (HIFU) is a minimally invasive treatment based on thermal ablation of tissues which are warmed up to 85 degrees C in the focal area. Clinical studies have shown such treatment modality to be safe and effective in the management of localised prostate cancer as well as of local recurrences after radical prostatectomy or radiotherapy. MATERIAL AND METHODS: From May 2002 to June 2010, 171 patients with no previous treatment for prostate cancer, aged 44 to 86 years (mean 74.7) underwent 197 HIFU treatments; 22 patients needed a second treatment as the first was incomplete (4 patients) or because of recurrence (18 patients). The prognosis subgroups were defined as low-risk in 29 patients (clinical stage T1-T2a, PSA < or = 10 ng/mL and Gleason score lower than 7), intermediate-risk in 47 patients (clinical stage T2b or PSA 10 - 20 ng/mL or Gleason score of 7), and high-risk in 95 patients (clinical stage > or = T2c or PSA > 20 ng/mL or Gleason score higher than 7). RESULTS: At a mean follow-up of 67.9 months, biochemical success rate (PSA constantly < 0.5 ng/ml) was obtained in 84.2% of low and intermediate risk patients and in 43.1% of high risk patients; post-treatment biopsies (6 months after treatment) revealed no residual tumour in 93.4% of low or intermediate risk patients and in 63.1% of high risk patients. CONCLUSIONS: Radical prostatectomy remains the "gold standard" for localised prostate cancer. However, HIFU seems to be a promising alternative and less invasive treatment modality with an encouraging success rate, at least in the short-term, in patients with low and medium risk of progression, not candidates for radical surgery; in cancers with clinical stage > or = T2c, or PSA > 20 ng/mL, or Gleason score higher than 7 seems to get good results in about half of patients.

GSTM1 and NAT2 polymorphisms and colon, lung and bladder cancer risk: a case-control study.

BACKGROUND: Glutathione S-transferase M1 (GSTM1) and N-acetyltransferase-2 (NAT2) are phase II enzymes involved in the metabolism of xenobiotics and whose polymorphisms have been related to individual cancer risks. PATIENTS AND METHODS: A case-control study was performed including 92 colon, 75 lung and 23 bladder cancer patients and 121 corresponding controls to verify the existence of an association between the main genetic polymorphisms of GSTM1 and NAT2 and the risk to develop cancer. Genomic DNA, isolated from 5 mL whole blood, was used to study GSTM1 and NAT2 polymorphisms using multiplex PCR and a PCR-RFLP technique, respectively. RESULTS: GSTM1 homozygous null genotype was associated with an increased risk of colon cancer, especially in females and in younger patients. For NAT2 gene, the results suggest a role for the low acetylator phenotype in the development of colon and lung cancer, especially in females. In bladder cancer patients two rare NAT2 genotypes were found at a higher frequency compared with all the other groups. CONCLUSION: The results do not suggest a different distribution of GSTM1 and NAT2 polymorphisms in the studied population compared to those reported for other Caucasian populations and warrant further studies in order to evaluate their potential relationship with individual cancer risks.

Polymorphisms of the DNA repair genes XRCC1 and XRCC3 and risk of lung and colorectal cancer: a case-control study in a Southern Italian population.

BACKGROUND: Genetic polymorphisms in DNA repair genes may influence individual variation in DNA repair capacity, which may be associated with a higher risk of developing cancer. Studies on the association between DNA repair gene polymorphisms and lung and colorectal cancer risk appear to be very limited. This study was designed to examine the polymorphisms associated with two DNA repair genes, namely XRCC1 Arg194Trp, XRCC1 Arg399Gln and XRCC3 Thr241Met, and to investigate their role as susceptibility markers for lung and colorectal cancer. MATERIALS AND METHODS: A case-control study was conducted including 94 and 109 cases of lung and colorectal cancer, respectively, and 121 hospital-based age- and sex-matched healthy controls to examine the role of XRCC1 and XRCC3 genetic polymorphisms in the context of lung and colorectal cancer risk for a Southern Italian population. Genomic DNA isolated from 5 ml whole blood was used to genotype XRCC1 Arg194Trp, XRCC1 Arg399Gln and XRCC3 Thr241Met by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. RESULTS: No differences were observed among the studied groups with regard to the genotype distribution of XRCC1 codon 194 and 399, thus the risk for lung and colorectal cancer did not appear to be significantly influenced by polymorphisms of this gene. Significant differences were observed among the studied groups with regard to the genotype distribution of XRCC3 codon 241. In particular, the XRCC3 241Met allele was associated with an increased risk of lung and colorectal cancer. CONCLUSION: Our results showed no evidence of a relationship between the XRCC1 Arg194Trp and Arg399Gln polymorphisms and the risk of lung and colorectal cancer. On the other hand, they suggested an increased risk in individuals with the XRCC3 Thr241Met polymorphism thus warranting further study to definitively evaluate the role of DNA repair mechanisms in colorectal and lung cancer susceptibility.

Glutathione S-transferase (GST) polymorphisms as risk factors for cancer in a highly homogeneous population from southern Italy.

Glutathione S-Transferase (GST) is a phase II enzyme and catalyses reactions between glutathione and a variety of electrophilic compounds, including some environmental carcinogens. In man, at least 20 isoenzymatic forms of GST have been identified and many of them show genetically-based individual variability of enzyme activity. The GSTM1 and GSTT1 isoenzymes display several polymorphisms, including a homozygotic deletion, which have been associated with an increased risk for developing neoplastic diseases. There is geographical and ethnic variation in genotype frequencies for both genes. The available data suggest that cancer incidence varies amongst Italian regions, being higher in Northern that in Southern areas, though it is unknown whether this phenomenon is to be attributed to genetic and/or environmental factors. We performed a case-control study to evaluate the GSTM1 and GSTT1 polymorphisms in a series of cancer patients in Basilicata, a Southern Italian region, and in corresponding controls. The results obtained demonstrate that the occurrence of GST polymorphisms in the Basilicata population is not different from other Italian regions and suggest that the population attributable risk associated with these genotypes may be quite high. GSTM1 homozygous null genotype was associated with an increasing risk of cancer, especially in females. The strongest association was with colon and breast cancers. For the GSTT1 gene, the results obtained were suggestive of a decreased risk of cancer associated with the null genotype. Thus, similar studies on these and other susceptibility genes are warranted since they can help to identify susceptible subgroups of people who can be targeted for cancer prevention.