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Circulating tumor cells (CTCs) are becoming a scientifically recognized indicator of primary tumors and/or metastasis. These cells can now be accurately detected and characterized as the result of technological advances. We analyzed the presence of CTCs in the peripheral blood of patients with metastatic breast cancer by real-time reverse-transcription PCR (RT-qPCR) using a panel of selected genes. The analysis of a single marker, without an EpCAM based enrichment approach, allowed the positive identification of 35% of the metastatic breast cancer patients. The analysis of five genes (SCGB2, TFF1, TFF3, Muc1, KRT20) performed in all the samples increased the detection to 61%. We describe a sensitive, reproducible and easy to implement approach to characterize CTC in patients with metastasic breast cancer.

BRCA1 and BRCA2 genes are essential in preserving the integrity of genome, and it is not unambiguously clear whether the heterozygosity status may affect BRCA1 or BRCA2 functions. This may have implications for the clinical management of BRCA1 and BRCA2 mutation carriers both in breast cancer (BC) screening modality and in cancer treatment based on DNA-damaging or DNA-repair-inhibiting drugs. We investigated whether lymphocytes carrying BRCA1 or BRCA2 mutations displayed an increased sensitivity to radiation or mitomycin C (MMC) in vitro treatments. Peripheral blood from 21 BRCA1 mutation carriers (12 with BC and 9 healthy), 24 BRCA2 carriers (13 with BC and 11 healthy), 15 familial BC patients without detected mutation in BRCA1 or BRCA2 and 16 controls without familial history of cancer (5 with BC and 11 healthy) were irradiated or treated with MMC. Chromosomal damage was measured using the cytokinesis-block micronucleus assay. We evaluated micronuclei (MN) and nucleoplasmic bridges (NPBs). The BRCA2 mutation carriers and familial BC patients without detected mutation in BRCA1 or BRCA2 showed less basal NPB than BRCA1 carriers and controls. The BRCA1 (+/-) or BRCA2 (+/-) lymphocytes did not have increased frequencies of MN or NPB after irradiation. In contrast, BRCA2 (+/-) lymphocytes presented higher levels of MN after MMC exposure than BRCA1 carriers and controls. The monoallelic BRCA1 or BRCA2 pathogenic mutations seem not to be associated with an enhanced radiosensitivity. The mutation of one BRCA2 allele conferred an increased sensitivity to MMC, presumably because of the role of this gene in the repair of MMC-induced DNA damage. This finding indicates that the MMC-induced MN analysis could be useful in identifying functional deficiencies of BRCA2 or genes related to BRCA2. Since MMC can be used as an anti-cancer drug, these data may be relevant for the management and follow-up of BRCA2 mutation carriers.

SummaryLarge deletions of the factorVIII gene account for approximately 5% of severe haemophilia A patients. Although deletions are readily detectable in males, the identification of heterozygosity in possible carriers of these families still constitutes a challenge. In order to identify a deleted allele over the background of the normal allele in these carriers, we developed a rapid real-time quantitative PCR approach by means of LightCycler technology and SYBR green I for monitoring product formation. The method was applied to families with independent deletions (one in exon 14 and the other in exons 23-24) of the Factor VIII gene, thereby allowing a reliable determination of carrier or non-carrier status. The method is extremely versatile and can be adapted to other deletions within the factor VIII gene as well as to other diseases whose molecular pathology consists of deletions or duplications.

The modulation of disease severity in hemophilia A (HA) patients may be related to the co-inheritance of mutations in genes with a known thrombotic effect such as factor V Leiden (FVL) and prothrombin. In the Spanish population, the prothrombin 20210A (PT20210A) allele is the most prevalent genetic risk factor for venous thromboembolism.We investigated the presence of both mutations in a cohort of 265 hemophiliac patients divided into two groups: I) 140 unrelated patients with moderate and mild HA and II) 125 unrelated patients with severe HA (83 carrying an inversion of intron 22).In group I, 4 patients had the FVL (2.8% vs. 2.98% controls) and 5 had the PT20210A (3.6% vs. 6.46% controls). In group II, two patients with inversion had the FVL (1.6%) and PT20210A was found in 10 patients (8%), five of them with inversion of intron 22 without inhibitors. One of these patients had the FVL and PT20210A mutations concomitantly. In the subgroup of patients with inversion who were carriers of the PT20210A, three parameters i.e. spontaneous bleeding (p=0.008), factor VIII utilization (p=0.016) and number of hemophilic arthropathies (p0.0005) were significantly lower than in a subgroup of 11 age-matched non-PT20210A severe HA patients with inversion and without inhibitors.These results indicate that the inheritance of PT20210A could be a protective factor that mitigates the clinical severity of HA.