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.

[Detection and quantification of circulating tumor cells in peripheral blood in patients with colon cancer]. / Detección y cuantificación de células tumorales en sangre periférica en pacientes con cáncer de colon.

BACKGROUND AND OBJECTIVE: The aim of this study is the detection and quantification of circulating tumor cells (CTC) in patients diagnosed with colon cancer and to establish whether they are related to the main clinicopathologic variables for this type of carcinoma. PATIENTS AND METHOD: Twenty-five colon cancer patients and 30 healthy volunteers were analysed. The quantification was performed using the CellSpotter Analyzer (Veridex LLC), that allows immunomagnetic isolation and immunospecific labelling of the cells for their enumeration. RESULTS: 72% of the colon cancer patients showed CTC and the mean number of cells found was 5 CTC/7.5 ml of peripheral blood. 52% of the samples contained 2 or more cells. Considering 2 cells as the cut-off point, a significant relationship with lactate dehydrogenase was found. CONCLUSIONS: This new technology which allows isolation and quantification of CTC in peripheral blood has proven to be valid for the detection of epithelial cells in colon cancer patients in every tumor stage. The results shown in this work confirm that cytokeratin 8, 18 and 19 are detected in CTC in this tumor type and will allow us to develop a protocol for the study of the relationship of quantification of theses cells and the clinical parameters involved in colon cancer.

[Microsatellite instability predicts better outcome in colorectal cancer patients]. / La inestabilidad de microsatélites predice un mejor pronóstico en los pacientes intervenidos de carcinoma colorrectal.

BACKGROUND AND OBJECTIVE: Two different pathways for the development of tumor have been described in colorectal carcinoma: the chromosomic instability, raised by suppressor genes and proto-oncogene alterations, and the microsatellite instability (MSI), caused by alterations in DNA repairing genes. PATIENTS AND METHOD: The frequency and the clinical meaning of the microsatellites instability pathway were determined in a consecutive prospective cohort of 106 patients who underwent surgical resection of colorectal carcinoma by a single surgeon. Microsatellite instability determination was established according to the criteria proposed by the National Cancer Institute in 1998. RESULTS: 9.4% of patients had a high instability and it was low in 11.3%; both groups displayed different clinico-pathological characteristics (age, sex, tumor site and histologic type). In the multivariant analysis of overall survival and disease free survival, high instability exhibited prognostic value independent of the rest of variables evaluated (p < 0.0001). CONCLUSIONS: The genetic alterations giving rise to microsatellite instability lead to a better prognosis in patients with colorectal cancer.

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.

A9 region in EPHB2 mutation is frequent in tumors with microsatellite instability. Analysis of prognosis.

AIM: The aim of the present study was to determine the relation of EPH tyrosine kinase receptor B2 (EPHB2) A9 region mutation and microsatellite instability (MSI); and to analyze their influence in prognosis of patients with sporadic colorectal cancer (CRC). PATIENTS AND METHODS: A total of 481 patients with CRC were examined. MSI (NCI criteria) and EPHB2 were analyzed using PCR and fragment analysis software. RESULTS: EPHB2 mutation was detected in 3.1% of patients. Mutation of EPHB2 was associated with location and with MSI status. We considered low instability (L-MSI) when only one marker showed instability, high instability (H-MSI) when two or more markers were positive and microsatelllite stable (MSS) when no instability was detected. The stratified analysis of overall survival (OS) and disease-free survival (DFS) in MSI according to EPHB2 status revealed no statistically significant differences. However, the risk of recurrence of H-MSI tumors with EPHB2 mutation carriers was 3.6-times higher than in non-mutation carriers. CONCLUSION: The frequency of EPHB2 mutation is higher in patients with H-MSI than MSS tumors. Promising results were found regarding the prognostic influence of EPHB2 in H-MSI.

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.

Genetic analysis of RET, GFR alpha 1 and GDNF genes in Spanish families with multiple endocrine neoplasia type 2A.

Multiple endocrine neoplasia type 2A (MEN 2A) is associated with specific germline missense mutations in the RET proto-oncogene. This locus encodes a receptor tyrosine kinase whose activation requires the formation of a multimeric receptor complex including GDNF as a ligand and GFR alpha 1 as a coreceptor. In order to explore the role of RET, GFR alpha 1 and GDNF genes in the variation of phenotypes observed in MEN2A families, we analysed germline mutations of these genes in 4 unrelated Spanish MEN2A families (23 cases studied). We found 2 novel variants corresponding to a single change in position + 47 (intron 12) of RET and position +22 (intron 7) of GFR alpha 1. Furthermore, we observed strong co-segregation between 2 polymorphisms of RET [G691S (exon 11) and S904S (TCC-TCG, exon 15) (100%, Fisher's exact test, p< 0.001)]. More interestingly, we found that these polymorphisms occurred at a significantly high frequency in patients with age at onset < 20 years old (Kruskal-Wallis's and Fisher's exact test, p = 0.007). These findings suggest that the G691S and S904S variants of RET may somehow play a role on the age of onset of MEN 2A.

Prognostic significance of p16INK4a alterations and 9p21 loss of heterozygosity in locally advanced laryngeal squamous cell carcinoma.

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin-dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous-cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico-pathological data and flow cytometric variables (DNA-ploidy and S-phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR-based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR-SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA-aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA-aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs.

Carcinoma invasor de la mama: estadificación molecular del ganglio centinela / Breast invasive carcinoma: the molecular stratification of sentinel node

Demostrar que existe un grupo de pacientes estadios patológicos N0 que presentan metástasis en el estudio del ganglio centinela no diagnosticados por métodos de rutina. El estudio del análisis molecular nos informa o no sobre el pronóstico, y podría considerarse como factor independiente. Se observó una relación agrupando los casos de mayor riesgo conocido, detectándose una población de bajo riesgo en la que se obviaría la quimioterapia. Observamos, que si agrupamos las pacientes con Ki-67 mayor de 5 por ciento, la mayoría de los casos en las que se observaron micro metástasis estaban en este grupo; aceptamos una relación entre los dos factores pronóstico. Algo similar sucede con pacientes grado II y III; la relación con la existencia de micrometástasis es evidente, pudiendo aceptarse que existe un grupo de carcinomas de mama de menor agresividad; se plantea que estas pacientes no reciban tratamiento adyuvante, y no planificar tratamientos quirúrgicos agresivos.

Demonstrated that exist a group of patients with pathological stage No who present metastases in the study of sentinel nodule no diagnostic for routine method. The study of molecular analysis inform or not about the prognosis and be considered a prognosis independent factor. We observed a relation between the mayor risk known cases and detected a low risk population in which obviated the chemotherapy. We observed that grouped patients with Ki-67 raised of 5 % the majority of the cases in which observed micro metastases were in these group; we accepted a relationship between this two prognosis factors. Some similar occur with patient’s grade II and grade III; the relation with the existence of micro metastases is evident, and we accepted that exist a group of breast carcinoma of less aggressive; for these reason we planted that these patient does not receive adjuvant treatment, and no planned surgical aggressive treatment.