Germline investigation in male breast cancer of DNA repair genes by next-generation sequencing.

PURPOSE: In order to better define the breast cancer (BC) genetic risk factors in men, a germline investigation was carried out on 81 Male BC cases by screening the 24 genes involved in BC predisposition, genome stability maintenance and DNA repair mechanisms by next-generation sequencing. METHODS: Germline DNAs were tested in a custom multi-gene panel focused on all coding exons and exon-intron boundaries of 24 selected genes using two amplicon-based assays on PGM-Ion Torrent (ThermoFisher Scientific) and MiSeq (Illumina) platforms. All variants were recorded and classified by using a custom pipeline. RESULTS: Clinical pathological data and the family history of 81 Male BC cases were gathered and analysed, revealing the average age of onset to be 61.3 years old and that in 35 cases there was a family history of BC. Our genetic screening allowed us to identify a germline mutation in 22 patients (23%) in 4 genes: BRCA2, BRIP1, MUTYH and PMS2. Moreover, 12 variants of unknown clinical significance (VUS) in 9 genes (BARD1, BRCA1, BRIP1, CHEK2, ERCC1, NBN, PALB2, PMS1, RAD50) were predicted as potentially pathogenic by in silico analysis bringing the mutation detection rate up to 40%. CONCLUSION: As expected, a positive family history is a strong predictor of germline BRCA2 mutations in male BC. Understanding the potential pathogenicity of VUS represents an extremely urgent need for the management of BC risk in Male BC cases and their own families.

Evaluation of feline immunodeficiency virus ORF-A mutants as candidate attenuated vaccine.

Feline immunodeficiency virus (FIV) made defective in the accessory gene ORF-A were previously shown to be greatly attenuated in its ability to replicate in lymphocytes but to grow normally or near normally in other cell types. Here, we examined whether FIV thus mutated could protect specific pathogen-free cats against challenge with ex vivo fully virulent homologous virus. No reversion of the vaccinating infections to wild type ORF-A was noted over 22 months of in vivo infection. Following challenge, 6/6 unvaccinated control cats became readily and heavily infected. In contrast, 3/9 vaccinees showed no evidence of the challenge virus over a 15-month observation period. In the other vaccinees, the challenge virus was predominant for various periods of time, but pre-existing viral loads and CD4 lymphocyte counts were either unaffected or altered only marginally and transiently. These findings show that ORF-A-defective FIV should be further examined as a candidate live attenuated vaccine.

Identification of a novel HLA-DPB1 allele, DPB1*9701, by sequence-based typing.

This report describes the identification of a novel DPB1 allele, DPB *9701, found in an Italian Caucasian individual. The new allele was detected by human leukocyte antigen sequence-based typing carried out to investigate the role of genetic factors in determining the outcome of hepatitis C virus infection. DPB1*9701 was identical to DPB1*0501 except for a single-nucleotide substitution at codon 43 (GGG --> TGG). This nucleotide change is a non-synonymous mutation and results in the amino acid substitution glycine (G) --> tryptophan (W). The nucleotide sequence has been deposited in GenBank under the accession number AY033075, and denominated DPB1*9701 by the official World Health Organization Nomenclature Committee.

Development of feline immunodeficiency virus ORF-A (tat) mutants: in vitro and in vivo characterization.

A functional ORF-A is essential for efficient feline immunodeficiency virus replication in lymphocytes. We have characterized a series of mutants of the Petaluma strain, derived from p34TF10 and having different combinations of stop codons and increasingly long deletions in ORF-A. Six clones proved fully replicative in fibroblastoid Crandell feline kidney cells and monocyte-derived macrophage cultures but failed to replicate in T cell lines and primary lymphoblasts. Cats inoculated with three selected mutants had considerably milder infections than controls given intact ORF-A virus. In vivo, the mutants maintained growth properties similar to those in vitro for at least 7 months, except that replication in lymphoid cells was strongly reduced but not ablated. One mutant underwent extensive ORF-A changes without, however, reverting to wild-type. Antiviral immune responses were feeble in all cats, suggesting that viral loads were too low to represent a sufficiently powerful antigenic stimulus.

Impact of two different dose-intensity chemotherapy regimens on psychological distress in early breast cancer patients.

In order to improve outcome, new, often more toxic chemotherapy regimens are continuously investigated in early breast cancer patients. Because the expected survival improvement is small, the possible increase in the negative effects of the new treatments should be carefully evaluated. Negative effects are represented not only by acute and chronic toxicity, but also by the adverse psychological impact of chemotherapy. The aim of this study was to evaluate the effect on patient-reported psychological distress of an increase in the dose-intensity of adjuvant chemotherapy compared with a standard regimen. Psychological distress was evaluated at baseline, during chemotherapy and after 6 and 12 months in breast cancer patients enrolled in a phase III multicentre study comparing the standard adjuvant chemotherapy with cyclophosphamide, epirubicin and 5-fluorouracil every 21 days (CEF21) with the same chemotherapy given every 14 days (CEF14). 392 patients were randomised in participating centres, and 363 were evaluable for this study. Overall, 1095 out of 1446 expected questionnaires (75.7%) were collected and evaluable. At baseline, the mean scores of psychological distress were similar in the two arms. During chemotherapy, a significantly higher psychological distress was observed in the CEF14 compared with the CEF21 arm (32.3 +/- 1.3 versus 27.6 +/- 1.3; P=0.009), as well as a higher cumulative incidence of anaemia, mucositis, diarrhoea, alopecia, bone pain and fatigue was observed in the CEF14 arm. In multivariate analyses, mucositis (P=0.01), asthenia (P=0.059), and CEF14 treatment (P=0.054) were independently associated with a higher psychological distress. After 6 months, psychological distress was again similar in the two arms and significantly lower when compared with baseline within each arm. A dose-intensive adjuvant regimen induces a higher, although transient, psychological distress in early breast cancer patients. Final results of the randomised trial will indicate whether such higher adverse effects of the dose-intensive regimen are counterbalanced by a higher efficacy of the experimental treatment in terms of survival.

Predicting mood disorders in breast cancer patients.

Prediction of delayed psychiatric disorders in breast cancer patients by using a screening procedure was investigated. Two questionnaires, the Psychological Distress Inventory and the Hospital Anxiety and Depression Scale, were administered before and during chemotherapy, and at the first follow-up visit. A psychiatric diagnosis was assigned to 50 of the 132 patients (38%) evaluated at follow-up. Including a set of clinical and demographic variables in a logistic regression, increasing age (P=0.001) and psychiatric history (P<0.001) were associated with psychiatric morbidity at follow-up. The accuracy of the two questionnaires in predicting delayed psychiatric disorders increased from the evaluation before chemotherapy to the evaluation during chemotherapy. The most accurate prediction was observed for the concurrent evaluation at follow-up. The accuracy of three predictive models developed for each evaluation point by including age, psychiatric history and psychological distress measured with each of the two questionnaires was not significantly better than that observed using only the questionnaires' scores as predictors.

Immunogenicity of an anti-clade B feline immunodeficiency fixed-cell virus vaccine in field cats.

Attempts at vaccine development for feline immunodeficiency virus (FIV) have been extensive, both because this is a significant health problem for cats and because FIV may be a useful vaccine model for human immunodeficiency virus. To date, only modest success, producing only short-term protection, has been achieved for vaccine trials in controlled laboratory settings. It is unclear how relevant such experiments are to prevention of natural infection. The current study used a vaccine that employs cell-associated FIV-M2 strain fixed with paraformaldehyde. Subject cats were in a private shelter where FIV was endemic, a prevalence of 29 to 58% over an 8-year observation period. Cats roamed freely from the shelter through the surrounding countryside but returned for food and shelter. After ensuring that cats were FIV negative, they were immunized using six doses of vaccine over a 16-month period and observed for 28 months after the initiation of immunization. Twenty-six cats (12 immunized and 14 nonimmunized controls) were monitored for a minimum of 22 months. Immunized cats did not experience significant adverse effects from immunization and developed both antibodies and cellular immunity to FIV, although individual responses varied greatly. At the conclusion of the study, 0 of 12 immunized cats had evidence of FIV infection, while 5 of 14 control cats were infected. Thus, the vaccine was safe and immunogenic and did not transmit infection. Furthermore, vaccinated cats did not develop FIV infection in a limited clinical trial over an extended time period. Thus, the data suggest that a fixed, FIV-infected cell vaccine has potential for preventing natural FIV infection in free-roaming cats.

Detecting psychological distress in cancer patients: validity of the Italian version of the Hospital Anxiety and Depression Scale.

The psychometric properties of the Italian version of the Hospital Anxiety and Depression Scale and its utility as a screening instrument for anxiety and depression in a non-psychiatric setting were evaluated. The questionnaire was administered twice to 197 breast cancer patients randomised in a phase III adjuvant clinical trial: before the start of chemotherapy and at the first follow-up visit. The presence of psychiatric disorders was evaluated at the follow-up visit using the Structured Clinical Interview for DSM-III-R in 132 patients. Factor analyses identified two strictly correlated factors. Crohnbach's alpha for the anxiety and depression scales ranged between 0.80 and 0.85. At follow-up, 50 patients (38%) were assigned a current DSM-III-R diagnosis, in most cases adjustment disorders (24%) or major depressive disorder (10%). Receiver operating characteristics (ROC) analysis was used to test the discriminant validity for both anxiety and depressive disorders. The comparison of the areas under the curve (AUC) between the two scales did not show any difference in identifying either anxiety (P = 0.855) or depressive disorders (P = 0.357). The 14-item total scale showed a high internal consistency (alpha = 0.89 and 0.88) and a high discriminating power for all the psychiatric disorders (AUC = 0.89; 95% CI = 0.83-0.94). The cut-off point that maximised sensitivity (84%) and specificity (79%) was 10. These results suggest that the total score is a valid measure of emotional distress, so that the Italian version of HADS can be used as a screening questionnaire for psychiatric disorders. The use of the two subscales as a 'case identifier' or as an outcome measure should be considered with caution.