Alfredo Hidalgo

Microarray comparative genomic hybridization detection of chromosomal imbalances in uterine cervix carcinoma

BackgroundChromosomal Comparative Genomic Hybridization (CGH) has been applied to all stages of cervical carcinoma progression, defining a specific pattern of chromosomal imbalances in this tumor. However, given its limited spatial resolution, chromosomal CGH has offered only general information regarding the possible genetic targets of DNA copy number changes.MethodsIn order to further define specific DNA copy number changes in cervical cancer, we analyzed 20 cervical samples (3 pre-malignant lesions, 10 invasive tumors, and 7 cell lines), using the GenoSensor microarray CGH system to define particular genetic targets that suffer copy number changes.ResultsThe most common DNA gains detected by array CGH in the invasive samples were located at the RBP1-RBP2 (3q21-q22) genes, the sub-telomeric clone C84C11/T3 (5ptel), D5S23 (5p15.2) and the DAB2 gene (5p13) in 58.8% of the samples. The most common losses were found at the FHIT gene (3p14.2) in 47% of the samples, followed by deletions at D8S504 (8p23.3), CTDP1-SHGC- 145820 (18qtel), KIT (4q11-q12), D1S427-FAF1 (1p32.3), D9S325 (9qtel), EIF4E (eukaryotic translation initiation factor 4E, 4q24), RB1 (13q14), and DXS7132 (Xq12) present in 5/17 (29.4%) of the samples.ConclusionOur results confirm the presence of a specific pattern of chromosomal imbalances in cervical carcinoma and define specific targets that are suffering DNA copy number changes in this neoplasm.

Chromosomal imbalances in brain metastases of solid tumors.

Metastases account for approximately 50% of the malignant tumors in the brain. In order to identify structural alterations that are associated with tumor dissemination into the central nervous system we used Comparative Genomic Hybridization (CGH) to investigate 42 brain metastases and 3 primary tumors of 40 patients. The metastases originated from lung cancer (14 cases), melanomas (7), carcinomas of breast (5), colon (5), kidney (5), adrenal gland (1) and thyroid (1). In addition, tumors of initially unknown primaries were assessed in 3 cases. The highest incidence of DNA gains were observed for the chromosomal regions 1q23, 8q24, 17q24‐q25, 20q13 (>80% of cases) followed by the gain on 7p12 (77%). DNA losses were slightly less frequent with 4q22, 4q26, 5q21, 9p21 being affected in at least 70% of the cases followed by deletions at 17p12, 4q32‐q34, 10q21, 10q23‐q24 and 18q21‐q22 in 67.5% of cases. Two unusual narrow regional peaks were observed for the gain on 17q24‐q25 and loss on 17p12.The incidence at individual loci can be viewed at our CGH online tumor database at http://www.amba.charite.de/cgh. The metastases of each tumor type showed a recurrent pattern of changes. In those cases with primary tumor and metastases available, the CGH pattern exhibited a high degree of conformity. In conclusion, our data suggests that specific genetic lesions are associated with tumor dissemination into the nervous system and that CGH analysis may be a useful supplementary tool for classification of metastases with unknown origin.

A simple method for the construction of small format tissue arrays

Tissue arrays can evaluate molecular targets in high numbers of samples in parallel. Array construction presents technical difficulties and tissue arrayers are expensive, particularly for small and medium sized laboratories. This report describes a method for the construction of 36 sample arrays using widely available materials. A blunted 16 gauge needle for bone marrow aspiration was used to extract paraffin wax cylinders and manually define a 6 × 6 matrix on a blank paraffin wax block. Tissue cores from 36 paraffin wax embedded premalignant lesions and invasive cervical carcinomas were injected into the matrix using a 14 gauge needle. This tissue array was sectioned using a standard microtome and used for the immunodetection of CD44 variant 9 and interleukin 18 with satisfactory results. This method can be applied in any laboratory, without the need of specialised equipment, offering a good alternative for the wider application of tissue arrays.

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines.

Human papilloma virus (HPV) infection is the crucial step in the initiation of cervical carcinomas. In addition, HPV18 has been implicated in tumour progression and adverse clinical outcome. We determined the HPV types in 12 primary cervical carcinomas and 12 cell lines and compared the findings with the comparative genetic hybridisation (CGH) pattern of chromosomal alterations. The most frequent alteration was the deletion at 3p14 followed by the loss of 2q34-q36 along with 3q gain. High risk HPV types were detected in all samples except one primary tumour. In contrast to the normal distribution, HPV18 was present in 75% of cases including all cell lines. The cell lines carried a higher number of genetic alterations and a different CGH pattern for several chromosomes than the primary tumours, despite microdissection. Purely HPV18 positive cases indicated a high incidence of imbalances at specific loci with peaks of the histogram coinciding with known HPV integration sites. The study suggests that HPV infection is associated with a recurrent pattern of chromosomal changes in cervical carcinomas and that the development and progression of these alterations is triggered by integration into the host genome.

Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines

BackgroundUterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV) infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH) experiments have detected a specific pattern of chromosomal imbalances during cervical cancer progression, indicating chromosomal regions that might contain genes that are important for cervical transformation.MethodsWe performed HPV detection and CGH analysis in order to initiate the genomic characterization of four recently established cervical carcinoma derived cell lines from Mexican patients.ResultsAll the cell lines were HPV18 positive. The most prevalent imbalances in the cell lines were gains in chromosomes 1q23-q32, 3q11.2-q13.1, 3q22-q26.1, 5p15.1-p11.2, this alteration present as a high copy number amplification in three of the cell lines, 7p15-p13, 7q21, 7q31, 11q21, and 12q12, and losses in 2q35-qter, 4p16, 6q26-qter, 9q34 and 19q13.2-qter.ConclusionsAnalysis of our present findings and previously reported data suggest that gains at 1q31-q32 and 7p13-p14, as well as losses at 6q26-q27 are alterations that might be unique for HPV18 positive cases. These chromosomal regions, as well as regions with high copy number amplifications, coincide with known fragile sites and known HPV integration sites. The general pattern of chromosomal imbalances detected in the cells resembled that found in invasive cervical tumors, suggesting that the cells represent good models for the study of cervical carcinoma.

Characterization of the global profile of genes expressed in cervical epithelium by Serial Analysis of Gene Expression (SAGE)

BackgroundSerial Analysis of Gene Expression (SAGE) is a new technique that allows a detailed and profound quantitative and qualitative knowledge of gene expression profile, without previous knowledge of sequence of analyzed genes. We carried out a modification of SAGE methodology (microSAGE), useful for the analysis of limited quantities of tissue samples, on normal human cervical tissue obtained from a donor without histopathological lesions. Cervical epithelium is constituted mainly by cervical keratinocytes which are the targets of human papilloma virus (HPV), where persistent HPV infection of cervical epithelium is associated with an increase risk for developing cervical carcinomas (CC).ResultsWe report here a transcriptome analysis of cervical tissue by SAGE, derived from 30,418 sequenced tags that provide a wealth of information about the gene products involved in normal cervical epithelium physiology, as well as genes not previously found in uterine cervix tissue involved in the process of epidermal differentiation.ConclusionThis first comprehensive and profound analysis of uterine cervix transcriptome, should be useful for the identification of genes involved in normal cervix uterine function, and candidate genes associated with cervical carcinoma.

Changes in retinoblastoma gene expression during cervical cancer progression

Summary. The role of tumour suppressor genes in the development of human cancers has been studied extensively. In viral carcinogenesis, the inactivation of suppressor proteins such as retinoblastoma (pRb) and p53, and cellular oncogenes overexpression, such as c‐myc, has been the subject of a number of investigations. In uterine‐cervix carcinomas, where high‐risk human papillomavirus (HPV) plays an important role, pRb and p53 are inactivated by E7 and E6 viral oncoproteins, respectively. However, little is known about the in situ expression of some of these proteins in pre‐malignant and malignant cervical tissues. On the other hand, it has also been demonstrated that c‐myc is involved in cervical carcinogenesis, and that pRb participates in the control of c‐myc gene expression. By using immunostaining techniques, we investigated pRb immunodetection pattern in normal tissues, squamous intraepithelial lesions (SILs) and invasive carcinomas from the uterine cervix. Our data show low pRb detection in both normal cervical tissue and invasive lesions, but a higher expression in SILs. C‐Myc protein was observed in most of the cellular nuclei of the invasive lesions, while in SILs was low. These findings indicate a heterogeneous pRb immunostaining during the different stages of cervical carcinogenesis, and suggest that this staining pattern could be a common feature implicated in the pathogenesis of uterine‐cervix carcinoma.

High prevalence of RET tyrosine kinase activation in Mexican patients with papillary thyroid carcinomas.

RET/PTC oncogene expression is restricted to papillary thyroid carcinomas (PTC). At least three forms of this oncogene have been described. These are generated by the rearrangement of the 5′-terminal region of different expressed genes with the tyrosine-kinase (TK) domain of the ret proto-oncogene. Several studies showing the correlation between the expression of this oncogene, clinical outcome, and histological subtypes have been published. Thirty-five paraffin-embedded PTC samples from patients without a history of radiation exposure were studied. Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to determine a possible correlation between RET activation, clinical outcome, and tumor subtype. Almost half of the studied cases presented with tumoral extension or metastases. Ret gene transcripts and protein were found in all PTC variants as well as in their corresponding metastases. In contrast, none of the follicular adenomas, goiters, or normal follicular cells from the thyroid gland showed evidence of ret activation. We observed a high frequency of ret expression in PTCs, suggesting that ret activation is a common event in nonradiation-related PTC from Mexican patients.