seqCNA: an R package for DNA copy number analysis in cancer using high-throughput sequencing.

BACKGROUND: Deviations in the amount of genomic content that arise during tumorigenesis, called copy number alterations, are structural rearrangements that can critically affect gene expression patterns. Additionally, copy number alteration profiles allow insight into cancer discrimination, progression and complexity. On data obtained from high-throughput sequencing, improving quality through GC bias correction and keeping false positives to a minimum help build reliable copy number alteration profiles. RESULTS: We introduce seqCNA, a parallelized R package for an integral copy number analysis of high-throughput sequencing cancer data. The package includes novel methodology on (i) filtering, reducing false positives, and (ii) GC content correction, improving copy number profile quality, especially under great read coverage and high correlation between GC content and copy number. Adequate analysis steps are automatically chosen based on availability of paired-end mapping, matched normal samples and genome annotation. CONCLUSIONS: seqCNA, available through Bioconductor, provides accurate copy number predictions in tumoural data, thanks to the extensive filtering and better GC bias correction, while providing an integrated and parallelized workflow.

Circulating tumor cells following first chemotherapy cycle: an early and strong predictor of outcome in patients with metastatic breast cancer.

We investigated the prognostic significance of circulating tumor cells (CTCs) determined immediately before the second cycle of chemotherapy in patients with metastatic breast cancer (MBC). The CTC counts were taken at baseline, before the first cycle of chemotherapy (CTC-0), and on day 21 before commencing the second cycle of chemotherapy (CTC-21) in consecutive MBC patients. The study's primary objectives were to analyze relationships between CTC-21 count and overall survival (OS). Based on the current literature, the CTC measurements were dichotomized as 0-4 versus ≥ 5 CTCs. Of 117 patients recruited, 99 were evaluable. Patients with 0-4 CTCs on day 21 had a significantly better OS than those with ≥ 5 CTCs (median OS: 38.5 months vs. 8.7 months). They also had a significantly better progression-free survival (PFS; median: 9.4 months vs. 3.0 months) and clinical benefit rate (77% vs. 44%). The OS of patients whose baseline CTCs were ≥ 5 but dropped to <5 on day 21 was apparently similar to those who had <5 CTCs at baseline. In a Cox regression analysis, CTC-21 was the only independent variable significantly predicting OS and PFS. Our data indicate that CTCs determined immediately before the second cycle of chemotherapy is an early and strong predictor of treatment outcome in MBC patients.

Vitamin D deficiency and vitamin D therapy in chronic hepatitis C.

BACKGROUND: Vitamin D has immunomodulatory properties, exerts an anti-hepatitis C virus (HCV) effect in vitro and improves response to interferon-based therapy in patients with chronic hepatitis C (CHC). Low serum levels of 25(OH) vitamin D [25(OH)D] are frequently found in CHC patients and seem to be related to more advanced stages of liver fibrosis. The study aims to establish the incidence of vitamin D deficiency in Spanish patients with CHC, its possible relation with features of liver damage and with the IL28B gene polymorphism, and the immediate effect of vitamin D therapy on CHC-related analytical variables. MATERIALS AND METHODS: Baseline serum 25(OH)D levels were measured in 108 consecutive CHC patients (60 men, age 54.3 ± 10.5 yrs). Results of transient elastography and of IL28B rs12979860C/T genotype were available in 89 and 95 patients, respectively. Forty one patients with insufficient levels of 25(OH)D received vitamin D supplements and were re-evaluated thereafter. RESULTS: Deficiency of vitamin D (< 20 µg/dL) and suboptimal levels (20-30 µg/mL) were detected in 36.1% and 40.9% of patients, respectively. No relationships were found between 25(OH)D levels and biochemical liver tests, fibrosis stage and IL28B genotype. Vitamin D therapy normalized 25(OH)D levels in all treated patients, but did not modify significantly HCV-RNA serum levels or biochemical tests. CONCLUSIONS: Vitamin D deficiency is common in Spanish patients with CHC but it is related neither to biochemical and virological variables nor with the fibrosis stage and IL28B polymorphism. Vitamin D therapy has no immediate effect on HCV-RNA serum levels.

Serum level of fibroblast growth factor 23 in maintenance renal transplant patients.

BACKGROUND: The discovery of fibroblast growth factor 23 (FGF23) provides a new conceptual framework that improves our understanding of the pathogenesis of post-transplant bone disease. Excess FGF23 is produced in the early post-transplant period; levels return to normal in the months following transplant. However, few manuscripts discuss FGF23 levels in stable long-term renal transplant recipients. METHODS: We performed a cross-sectional observational study of 279 maintenance kidney recipients with chronic kidney disease (CKD) Stages 1-4 and stable allograft function who had received their transplant at least 12 months previously. We calculated the estimated GFR (eGFR) using the MDRD4 equation. RESULTS: FGF23, parathyroid hormone (PTH) and phosphorus values were higher in more advanced stages, while the serum calcitriol levels and the phosphate reabsorption rate were lower. A significant inverse correlation was found between eGFR and FGF23 (r = -0.487; P < 0.001), PTH (r = -0.444; P < 0.001), serum phosphate levels (r = -0.315; P < 0.001) and fractional excretion of magnesium (r = -0.503; P < 0.001). Multivariable analysis showed that increased time on corticosteroids (P < 0.001), PTH (P < 0.001), serum phosphate (P = 0.003), decreased serum calcitriol (P = 0.049) and estimated glomerular filtration (P = 0.003) rate were associated with high FGF23 levels. In contrast with pre-transplant patients and first year post-transplant patients, higher FGF23 values were not correlated with increased phosphate excretion. An elevated phosphate reabsorption rate was associated with decreased PTH (P < 0.001) and calciuria (P = 0.028) and increased serum calcitriol (P = 0.009), plasma bicarbonate (P = 0.024) and estimated glomerular filtration (P = 0.003). CONCLUSIONS: Serum FGF23 concentrations remain increased in long-term kidney graft recipients, even in the early stages of CKD. It remains to be seen whether measures aimed at reducing serum levels of PTH and phosphate and/or corticosteroid doses might help to lower serum FGF23 and whether this will improve kidney recipient outcomes.

p16 gene methylation in colorectal cancer patients with long-term follow-up.

INTRODUCTION: p16 gene plays an important role in the cell cycle regulation and is considered an important tumor suppressor gene. Several mechanisms of gene inactivation have been described; in this study we have focused on p16 gene promoter methylation. In colorectal cancer p16 gene methylation is a frequent event. METHODS: 326 patients with sporadic colorectal cancer were included. DNA was extracted from tumor tissue samples obtained during the surgical procedure. Promoter methylation was analyzed using bisulfite modification and was detected by quantitative methylation-specific PCR. Frequency of p16 methylation was analyzed and compared with other clinicopathological variables. RESULTS: p16 gene methylation was detected in 24.8% of patients. Methylation was associated with differentiation grade and with tumor location: methylation was frequent in poorly differentiated tumors and had low frequency in distal colon. The p16 promoter methylation discriminated a subgroup of patients with better prognosis in poorly differentiated tumors. CONCLUSIONS: p16 methylation was a frequent event in our population and was able to induce differences in the overall survival of patients with poorly differentiated tumors.

Genetic alterations of IDH1 and Vegf in brain tumors.

BACKGROUND: This study evaluates the presence of R132H mutation in isocitrate dehydrogenase (IDH1) gene and the vascular endothelial growth factor (VEGF) +936 C/T polymorphism in brain tumors. The impact of these genetic alterations on overall survival (OS) and progression free survival (PFS) was evaluated. METHODS: A cohort of 80 patients surgically treated at Hospital Clínico San Carlos, Madrid, between March 2004 and November 2012, was analyzed. Tumors were distributed in 73 primary brain tumors (gliomas, meningiomas, hemangiopericytomas and hemangioblastomas) and seven secondary tumors evolved from a low grade glioma, thus providing a mixed sample. RESULTS: IDH1R132H gene mutation was found in 12 patients (15%) and appears more frequently in secondary tumors (5 (71.4%) whereas in 7 (9.7%) primary tumors (p < .001)). The mutation is related to WHO grade II in primary tumors and a supratentorial location in secondary tumors. The OS analysis for IDH1 showed a tendency towards a better prognosis of the tumors containing the mutation (p = .059).The IDH1R132H mutation confers a better PFS (p = .025) on primary tumors. The T allele of VEFG +936 C/T polymorphism was found in 16 patients (20%). No relation was found between this polymorphism and primary or secondary tumor, neither with OS or PFS. CONCLUSIONS: IDH1R132H gene mutation is exclusive in supratentorial tumors and more frequent in secondary ones, with a greater survival trend and better PFS in patients who carry it. The T allele of VEGF +936 C/T polymorphism is more common in primary tumors, although there is no statistical relation with survival.

Effect of ß-catenin alterations in the prognosis of patients with sporadic colorectal cancer.

CONTEXT: Wnt pathway activation represents a critical step in the etiology of most of colorectal cancer (CRC) and it is commonly due to mutations in the APC gene, which originates the loss of ß-catenin regulatory function. It has been suggested that APC inactivation or ß-catenin alteration have similar effects in tumor progression in CRC tumorigenesis. AIMS: The aim of this study was to analyze the frequency of ß-catenin gene mutation in patients with sporadic CRC and to determine its effect in prognosis. MATERIALS AND METHODS: This was a prospective cohort study, which included 345 patients with sporadic CRC. ß-Catenin gene mutations in exon 3 were detected by single strand conformation polymorphism (SSCP). Exon 3 deletion was studied by identifying differences in fragment length of specific amplification products. All the altered samples were confirmed by direct sequencing. RESULTS: In our population, point mutations were detected in 1.8% of the samples and 4.9% of the samples showed deletion. We observed association between exon 3 mutations and increased levels of Carcinoenbryonic Antigen (CEA). In these patients, clinically relevant improvement in overall survival was also observed. CONCLUSION: Frequency of point mutations in exon 3 ß-catenin gene is low in our population. It would be interesting to increase the population size to test the clinically relevant influence in the prognosis found, and to test the relation of these events with Microsatellite Instabillity (MSI) pathway. If these findings were confirmed, ß-catenin determination would help in the selection of patients with different prognosis.

Circulating tumor cells in solid tumor in metastatic and localized stages.

UNLABELLED: The aim of this study was the detection of circulating tumor cells (CTC) in three tumor types of epithelial origin. PATIENTS AND METHODS: Four hundred and thirty-eight patients with breast cancer (56.2% localized and 43.8% metastatic), 195 with colorectal tumors (84.1% localized and 15.9% metastatic) and 50 with prostate cancer (52% localized and 48% metastatic) took part in this study. CTC quantification was performed using the CellSpotter Analyzer (Veridex LLC). RESULTS: 31.5% of patients with cancer had > or =2 CTCs/7.5 mL but none of the healthy volunteers were above this level (p<0.001). Among patients with metastatic disease, 62.3% of them had > or =2 CTCs/7.5 mL but only 14.0% of those with localized disease were above this level (p<0.001). The presence of CTCs were correlated to stage in the three studied tumor types and no differences in the number of cells were found between them. CONCLUSION: The presence of more than 2 CTCs/7.5 ml is a frequent event in metastatic cases. In particular, patients with localized disease who have more than 2 CTCs/7.5 ml should be carefully studied to determine the possible prognostic and predictive value of this finding.

Circulating tumour cells in locally advanced breast cancer.

MATERIAL AND METHODS: A prospective study was conducted to determine the value of changes in circulating tumour cell (CTC) levels prior to and after the first cycle of neoadjuvant treatment in early prediction of pathologic response in locally advanced breast cancer (LABC). Two blood samples were obtained from 72 eligible LABC patients to isolate and enumerate CTCs before neoadjuvant chemotherapy started on day 1, and on day 21, immediately before second cycle administration. RESULTS: Sixty patients (83.3%) had <1 CTC in the first sample and response rates in this cohort were pathologic complete response (PCR) in 2 patients (5%), partial response (PR) in 35 (87.5%), stable disease (SD) in 2 (5%) and progressive disease (PD) in 1 (2.5%). Twelve patients (16.7%) had >2 CTCs in the first sample; these patients were more likely to have triple negative tumours. All 12 had fewer CTCs in the second sample. Response rates in this second cohort of 12 patients were PCR in 4 (34%), PR in 6 (50%), SD in 1 (8%) and PD in 1 (8%). PCR rate was markedly better in this second cohort (p<0.0042; OR 14.5, 95% CI 2.3-92). DISCUSSION: This study suggests that the presence of CTCs prior to neoadjuvant therapy might be a predictor of response to this therapy.

Circulating tumor cells in metastatic breast cancer: timing of blood extraction for analysis.

PURPOSE: Circulating tumor cells (CTCs) can be detected in the peripheral blood of around 50% of patients with metastatic breast cancer. Their numbers are an independent predictor of the patient's progression-free survival (PFS) and of overall survival (OS). However, to date, none of the studies carried out with the most commonly used system of CTC determination (the CellSearch System, approved by the US Food and Drug Administration) has examined the intra-patient variation in CTC numbers, a variation that could impact on prognosis assessment. EXPERIMENTAL DESIGN: To evaluate possible circadian variations in the number of CTCs in patients with breast cancer a pilot study was conducted in which these cells were quantified 12 h apart (at 8:00 a.m. and 8:00 p.m. of the same day) in a cohort of hospitalized patients with metastatic breast cancer. RESULTS: Out of the 58 patients included in the study, 51 were evaluable. No statistically significant differences between day-time and night-time CTC numbers were observed (p=0.8427, Wilcoxon matched pair test). Only two of the patients were classified in different prognostic categories in the morning and night determinations (5 or more CTCs=poor prognosis group; <5 CTCs=good prognosis group). The prognostic classification of the remaining 49 patients was the same at 8:00 a.m. and 8:00 p.m. CONCLUSION: The number of peripheral blood CTCs in metastatic breast cancer patients is not significantly different at 8:00 a.m. from that at 8:00 p.m. and, as such, indicates a lack of circadian rhythm with respect to CTC numbers in these patients.

Detección inmunomagnética de células tumorales circulantes en cáncer de mama metastásico: nuevas tecnologías / Inmunomagnetic detection of circulating tumor cells in metastatic breast cancer: new technologies

Las metástasis hematógenas son la mayor causa de mortalidad en el cáncer de mama. Está documentado que una vez las células tumorales se diseminan el resultado es, generalmente, letal. Las células tumorales circulantes han sido consideradas por largo tiempo un reflejo de la agresividad de los tumores, y entre ellos uno de los más agresivos es el cáncer de mama metastásico. Los primeros resultados clínicos han permitido determinar una fuerte relación entre la detección y el número de las células tumorales circulantes, como un valor pronóstico y como marcador de la actividad antitumoral del tratamiento. El análisis inmunomagnético utilizando una nueva metodología permite determinar que un recuento de 5 células tumorales circulantes o más en 7,5 ml de sangre, en cualquier fase de la enfermedad, se asocia a un mal pronóstico, y es predictivo de una supervivencia global más corta.

Hematogenous metastasis is the major cause of mortality in breast cancer. Evidence indicates that tumor cells escape from the primary tumor mass into the blood stream and that these disseminated cells are the source of increased lethality. Circulating or metastatic tumour cells have been considered as useful indicators of the aggressiveness of breast cancer tumours. The first clinical results obtained with such assays strongly suggest that in metastatic breast cancer, circulating tumour cells detection and enumeration can be used to estimate prognosis and may serve as an early marker to assess anti-tumour activity of a treatment. Immunomagnetic analysis using a new methodology, determine that a circulating tumour cells count of 5 or more per 7,5 ml of blood, at any time during the course of the disease is associated with a poor prognosis and is predictive of shorter progression and overall survival.