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Biomedical subjects

M Monzó

Publications and source records attributed to M Monzó.

At least 19 recordsLinked to original sources

Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues.

MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Although the number of verified human miRNA is still expanding, only few have been functionally described. However, emerging evidences suggest the potential involvement of altered regulation of miRNA in pathogenesis of cancers and these genes are thought to function as both tumours suppressor and oncogenes. In our study, we examined by Real-Time PCR the expression of 156 mature miRNA in colorectal cancer. The analysis by several bioinformatics algorithms of colorectal tumours and adjacent non-neoplastic tissues from patients and colorectal cancer cell lines allowed identifying a group of 13 miRNA whose expression is significantly altered in this tumor. The most significantly deregulated miRNA being miR-31, miR-96, miR-133b, miR-135b, miR-145, and miR-183. In addition, the expression level of miR-31 was correlated with the stage of CRC tumor. Our results suggest that miRNA expression profile could have relevance to the biological and clinical behavior of colorectal neoplasia.

Cluster Analysis↗

Prognostic value of K-ras mutations and allelic imbalance on chromosome 18q in patients with resected colorectal cancer.

PURPOSE: We designed this study to assess the frequency of K-ras mutations in patients with resected colorectal tumors and their association with survival. A second objective was to analyze the prognostic value of different K-ras genotypes. In a subgroup of patients we also investigated the presence of allelic imbalance on chromosome 18q and its relationship to clinical outcome. METHODS: One hundred fourteen colorectal tumors resected between 1983 and 1986 were analyzed to detect K-ras point mutations at codons 12, 13, and 61 by polymerase chain reaction followed by allele specific oligonucleotide hybridization. A subgroup of 77 tumors was further screened to detect loss of heterozygosity on chromosome 18q using three polymorphic microsatellite markers (D18S67, D18S474 and D18S58). RESULTS: K-ras mutations were detected in 29 percent (33/114) of patients. K-ras mutations correlated with age and preoperative carcinoembryonic antigen levels, and there was some indication that they may be linked to poor survival, especially in Stage II tumors, where a subgroup of patients with aspartic and serine mutations showed significantly reduced survival (P = 0.03) compared with K-ras-negative patients. 18q loss of heterozygosity was present in 39 percent (25/63) of tumors. A multivariate analysis of Stage II tumors showed that 18q loss of heterozygosity was significantly associated with a worse prognosis (P = 0.006). A significant decrease in survival was identified in ten patients harboring both genetic alterations (K-i mutations and 18q loss of heterozygosity; P = 0.02). CONCLUSIONS: In colorectal tumors, K-ras mutations and 18q loss of heterozygosity are two genetic markers which may identify patients with more aggressive behavior, mainly in Stage II tumors. These findings warrant further research, because they can be useful in customizing adjuvant chemotherapy.

Allelic Imbalance↗

Novel approaches in the treatment of non-small-cell lung cancer.

A wealth of data indicates that certain genetic abnormalities can target specific cytotoxic drugs and intervene at an early step as a mechanism of resistance in the treatment of non-small-cell lung cancer. Therefore prescribing certain combinations of cytotoxic anticancer agents to a vast majority of these patients is futile. Genetic abnormalities have been found to be useful surrogate markers for response, particularly in colorectal cancer: thymidylate synthase mRNA and ERCC1 mRNA levels. In addition, beta-tubulin mutations may also confer paclitaxel resistance in patients. An important target to be explored for gemcitabine resistance is the assessment of a particular region in chromosome 11p15.5 wherein lies the ribonucleotide reductase gene that could affect gemcitabine metabolism. Shedding light on this genetic framework, several proposed customized chemotherapy studies could help validate the relevance of these markers.

Antineoplastic Combined Chemotherapy Protocols↗

Failure of wild-type p53 gene therapy in human cancer cells expressing a mutant p53 protein.

The introduction of exogenous wild-type p53 into human cancer cells bearing p53 mutation does not necessarily result in inhibition of tumor growth. We have demonstrated this in MDA-MB468 breast cancer cells which are hemizygous for p53 mutation and also in KM12SM colorectal carcinoma cells which are heterozygous for p53 mutation. The wtp53 transfectants decreased three- to four-fold the number of colonies compared with controls. Most wtp53-expressing cells died by apoptosis at early passages, but some cells were able to form colonies and their proliferation rate was similar to control transfectants. This reversion was observed in three of the six MDA-MB-468 clones selected. When MDA-wtp53 transfectants were implanted orthotopically in nude mice only one clone showed prolonged tumor latency. No differences were found in either tumor proliferation or apoptosis in tumors. Integration and expression of exogenous wtp53 was assessed in early and late passages in vitro, and in tumors growing in vivo. Consistently, we found mutations in the exogenous wtp53 gene of MDA-MB468 transfectants. Excision of the exogenous gene was an alternative to abrogate the wtp53 function that was extremely efficient in KM12 cells, although they maintained resistance to geneticin. These results were corroborated by the functional assay in yeast. In conclusion, wtp53 is inactivated in these cancer cells by different mechanisms. The presence of mutated p53 may confer genome instability and mutator ability, which allows cells to escape the effects of the exogenous wtp53 and contributes to the failure of wtp53 gene therapy.

Animals↗

Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations.

PURPOSE: The mechanisms that cause chemoresistance in non-small-cell lung cancer (NSCLC) patients have yet to be clearly elucidated. Paclitaxel is a tubulin-disrupting agent that binds preferentially to beta-tubulin. Tubulins are guanosine triphosphate (GTP)-binding proteins. Beta-tubulin is a GTPase, whereas alpha-tubulin has no enzyme activity. We reasoned that polymerase chain reaction (PCR) and DNA sequencing of the beta-tubulin gene could reveal more information regarding the connection between beta-tubulin mutations and primary paclitaxel resistance. PATIENTS AND METHODS: Constitutional genomic DNA and paired tumor DNA were isolated from 49 biopsies from 43 Spanish and six American stage IIIB and IV NSCLC patients who had been treated with a 3-hour, 210 mg/m(2) paclitaxel infusion and a 24-hour, 200 mg/m(2) infusion, respectively. Oligonucleotides specific to beta-tubulin were designed for PCR amplification and sequencing of GTP- and paclitaxel-binding beta-tubulin domains. RESULTS: Of 49 patients with NSCLC, 16 (33%; 95% confidence interval [CI], 20.7% to 45.3%) had beta-tubulin mutations in exons 1 (one patient) or 4 (15 patients). None of the patients with beta-tubulin mutations had an objective response, whereas 13 of 33 (39.4%; 95% CI, 22.8% to 56%; P = 0.01) patients without beta-tubulin mutations had complete or partial responses. Median survival was 3 months for the 16 patients with beta-tubulin mutations and 10 months for the 33 patients without beta-tubulin mutations (P =.0001). CONCLUSION: We have identified beta-tubulin gene mutations as a strong predictor of response to the antitubulin drug paclitaxel; these mutations may represent a novel mechanism of resistance and should be examined prospectively in future trials of taxane-based therapy in NSCLC.

Adult↗

A novel anti-apoptosis gene: Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers.

PURPOSE: The survivin gene is a novel apoptosis inhibitor, related to the baculovirus gene, which is believed to play a pivotal role in fetal development and in cancer. We hypothesised that survivin would be expressed in tumors of patients with non-small-cell lung cancer (NSCLC), and we attempted to determine the influence of survivin re-expression on clinical outcome in patients with up to stage IIIA NSCLC who had undergone radical surgery. METHODS: We designed a reverse transcriptase polymerase chain reaction (RT-PCR) assay to study the expression of the survivin gene in 83 NSCLC tumor samples and compared the results with relevant clinical and pathologic data. RESULTS: The RT-PCR identified survivin gene transcript in 71 (85. 5%) of the tumor samples and in only 10 (12%) of the paired, histopathologically normal lung samples. There was no relationship between histologic subtype (squamous v nonsquamous) and survivin gene expression. The 12 patients without survivin expression had significantly better overall survival than the 71 patients with survivin expression (P =.01 by univariate analysis; relative risk, 2. 1). There was no significant correlation between survivin expression and age, sex, cigarette smoking, histologic subtype, tumor differentiation, tumor size, or the presence of mediastinal lymph node metastases in surgical specimens. CONCLUSION: The survivin gene was expressed in a vast majority of NSCLC tumors. We conclude that survivin transcript is a defining diagnostic marker for NSCLC that may also yield prognostic information and, as an apoptosis inhibitor, be an important target in cancer therapy.

Adult↗

Genetic susceptibility associated with rare HRAS1 variable number of tandem repeats alleles in Spanish non-small cell lung cancer patients.

The highly polymorphic HRAS1 variable number of tandem repeats (VNTR), which maps 1 kb downstream from the human H-ras1 gene, has been described as an inherited predisposing factor in many human cancers. Here, we investigated the association between the presence of rare HRAS1 minisatellite alleles and lung cancer in the population studied. Four hundred sixty-six HRAS1 VNTR alleles from 233 lung cancer patients and 892 alleles from 446 unaffected controls were typed using PCR-long agarose gel electrophoresis assay of peripheral blood lymphocyte DNA. Rare alleles were differentiated from common alleles (a1, a2, a3, and a4) by shifts in electrophoretic mobility. Odds ratio was calculated to evaluate increased risk of lung cancer associated to the presence of rare HRAS1 alleles. A higher percentage of rare HRAS1 VNTR alleles in lung cancer patients than in unaffected controls (32.7 versus 21.9%) was confirmed. The presence of rare alleles was associated with an increased risk of lung cancer (odds ratio = 1.68; P < or = 0.0001), indicating a genetic predisposition to lung cancer. No differences based on other clinicopathological variables were observed. Furthermore, a meta-analysis showed a higher distribution of rare alleles in our study of Caucasian Spaniards than was found in other studies of American and Northern European Caucasian populations. We conclude that the presence of rare HRAS1 VNTR alleles may be an inherited predisposing factor in lung cancer. This presence can be easily determined from peripheral blood samples by PCR-based methods. Furthermore, interracial variations in allele frequencies and variations between Caucasian subpopulations suggest that genetic variations may be involved in susceptibility to lung oncogenesis, especially in certain ethnic populations.

Alleles↗

[Analysis of telomeric length in epithelial carcinoma of the ovary].

BACKGROUND: As a consequence of the high cell division rate, telomeric repeat reduction in human tumor cells, giving rise to genetic instability, has recently been described. The aim of this study was to analyze by Southern blot telomeric length alterations in a retrospective group of patients with ovarian epithelial carcinoma. PATIENTS AND METHODS: Tumor and corresponding normal DNA were isolated from paraffin-embedded tissue of 16 patients with ovarian epithelial carcinoma. Telomeric Restriction Fragments (TRF) were studied by Southern blot and densitometric analysis. RESULTS: No telomere alterations were detected in 37.5% of patients (6/16). Of the remaining ten, 5 were found to have telomere reduction and five telomere elongation. No significant correlation was found between clinicopathological variables, response to chemotherapy, survival rate or time to progression, and telomere length alterations. CONCLUSIONS: In ovarian epithelial carcinoma telomere elongation may be a marker of the presence of immortal cells within the tumor, but telomere or the absence of telomeric alterations do not rule out the presence of these cells. Although TRF analysis can be performed in paraffin-embedded tissues, it is not the best indicator of telomerase activity and thus of tumor aggressiveness in early stages of this carcinoma.

Blotting, Southern↗

Allelic loss on chromosome 18q as a prognostic marker in stage II colorectal cancer.

BACKGROUND & AIMS: Loss of heterozygosity (LOH) on chromosome 18q is frequent in colorectal cancer (CRC) and has been associated with poor prognosis in stage II tumors. This study investigated the frequency of LOH in sporadic CRC and its effect on patient prognosis. METHODS: One hundred forty-four patients were screened for LOH at 18q by polymerase chain reaction using three polymorphic microsatellite markers. RESULTS: Nineteen patients were excluded because their tumors showed microsatellite instability in at least one marker. Of the remaining 125 patients, 121 were informative in at least one marker; 45% (54 of 121) showed 18q LOH. Five-year survival was 42% in those with 18q LOH and 73% in those without 18q LOH (P = 0.008). Multivariate analysis showed that tumor side (P = 0.0001) and 18q LOH (P = 0.01) were the only independent prognostic factors. Examining markers individually showed that only the lost of D18S474 had a significant influence on survival in patients with stage II CRC (P = 0.016). CONCLUSIONS: 18q LOH indicates an unfavorable outcome in patients with stage II CRC. Our results emphasize the importance of the 18q21.1 region, where several tumor-suppressor genes have been mapped. Microsatellite analysis may be useful in identifying high-risk patients who might benefit from adjuvant therapy.

Adult↗

Detection of chromosome 3p alterations in serum DNA of non-small-cell lung cancer patients.

BACKGROUND: The generally dismal outcome of non-small-cell lung cancer (NSCLC) is believed to be associated with the systemic nature of this disease. In current practice, the decision to begin adjuvant chemotherapy in completely resected early stages is based on empirical criteria and has not yet been influenced by the presence of individual risk factors. Nonetheless, recent studies indicate that soluble tumor DNA is found in the serum and plasma of cancer patients, and microsatellite alterations have been identified in small-cell lung cancer and in head and neck neoplasms. PATIENTS AND METHODS: We have investigated serum DNA from 22 completely resected stage I-IIIA NSCLC patients using a polymerase chain reaction microsatellite analysis with four microsatellite markers at chromosome 3p (D3S1038, D3S1611, D3S1067 and D3S1284). RESULTS: Our analyses showed serum tumor DNA in 6 of 22 (28%) cases, with microsatellite alterations, either as a shift (changes in the size of the microsatellite sequence in the autoradiograph) or as a loss of heterozygosity (LOH). LOH in both tumor and serum DNA at one or more microsatellite markers was found in four patients. Although it is still premature to look for prognostic implications, one patient with stage I serum DNA was identified prior to the development of distant metastases. CONCLUSIONS: The findings suggest that detection of free circulating DNA in sera of NSCLC patients is incidentally linked to the systemic nature of lung cancer even at the earliest stage. These observations provide the first hint that serum tumor DNA is present in NSCLC patients. The detection of DNA from cancer cells in the sera of NSCLC patients could be useful for monitoring relapse in a relatively non-invasive way, and the potential sensitivity of this test may help in selecting candidates for adjuvant chemotherapy.

Adult↗

TP53 mutational pattern in Spanish and Polish non-small cell lung cancer patients: null mutations are associated with poor prognosis.

Inactivation of TP53 tumor suppressor gene is the most frequent molecular alteration in NSCLC, involving up to 60% of cases. Furthermore, TP53 mutational spectrum is related to the type of mutagen exposure, as well as racial and/or diet differences. Nearly 95% of TP53 perturbations affect codons included within exons 5-8 which encode for almost the entire DNA-binding domain. In this study we addressed the possible prognostic value of the molecular alterations identified in exons 5-8 of the TP53 gene in DNAs from 151 paraffin-embedded NSCLC sections corresponding to 59 Spanish and 92 Polish stage I-IIIA resected patients. PCR/single-strand conformation polymorphism (SSCP) analysis revealed that the occurrence of TP53 exon 5-8 mutations was 17/59 (29%) in the Spanish cohort and 17/92 (18%) in the Polish group. However, when DNA sequencing analysis was performed, these frequencies were reduced because of the presence of SSCP-false positive, intronic and silent mutations and polymorphisms. Fifteen of the 59 Spanish NSCLC tumors (25%) harbored TP53 mutations affecting exons 5-8 coding sequences, whereas only 12 of 92 Polish neoplasms (13%) contained alterations in the central hydrophobic region of p53. Our results indicate that the occurrence of TP53 mutations affecting exon 5-8 coding sequences in some European NSCLC populations may be lower than previously reported, and that the TP53 mutational patterns of these cohorts differ somewhat. The Spanish NSCLC patients contained missense mutations (9/59, 15%) and a relatively high percentage of null mutations (5/59, 8%) while the Polish patients mostly harbored missense mutations (9/92, 10%) and only one tumor contained a null type (1/92, 1%). Moreover, most TP53 missense mutations in the Spanish group were located outside the conserved regions, whereas the same mutations in the Polish group affected conserved amino acids. Furthermore, the Polish patients harbored a high percentage of G-->A transitions (most of them at non-CpG sites), while G-->T transversions were predominant in the Spanish group. Our findings suggest that there may be different racial or exogenous factors in these two populations which may help to explain both the distinct TP53 mutational pattern and the lower frequency obtained in the Polish group. The presence of missense mutations did not confer a worse clinical outcome in these subsets of NSCLC patients. However, patients whose tumors contained null TP53 gene mutations had a 5 month median disease-free survival time in contrast with 42 months in those patients without mutations (P=0.008). These findings suggest that loss of p53 function may enhance tumor progression in NSCLC patients independently of whether dominant negative TP53 missense mutations are present.

Aged↗

Reduced survival in patients with stage-I non-small-cell lung cancer associated with DNA-replication errors.

To better understand whether replication-error-type instability (RER+) is a frequent genetic alteration event in surgical-pathologic stage-I non-small-cell lung cancer (NSCLC) and identify whether it constitutes an independent prognostic parameter, we examined 35 surgical-pathologic stage-I-NSCLC patients with complete follow-up in all cases for at least 49 months. The tumor samples and the paired histopathologically normal lung samples for each patient were analyzed for 8 microsatellite markers located at chromosomes 3p and 2p to investigate microsatellite alterations such as RER+ and loss of heterozygosity (LOH). Single-strand-conformation-polymorphism analysis for detection of p53 and k-ras gene mutations was also carried out. Genetic data were correlated with clinical outcome and histopathologically established prognostic factors. RER+ at one or both chromosomes was identified in 24 of the 35 patients; 9 patients showed LOH. A statistically significant correlation was found between RER+ and poor prognosis (p = 0.001). Furthermore, RER+ proved to be an independent factor that predicted decreased survival, ranking first, followed by visceral pleural invasion. A trend towards worse survival was strongest in the group of patients with tumor size greater than 3 cm (T2). Patients with other genetic abnormalities, such as K-ras mutations, p53 mutations or LOH, had prognoses similar to those of patients without such aberrations. The data suggest that RER+ is common in NSCLC, that it may provide important prognostic information in stage-I NSCLC and serve as a useful marker for relapse-risk assessment in operable NSCLC patients.

Adenocarcinoma↗

[Significance of heterozygosity loss in the long arm of chromosome 5 (5q21) in small cell carcinoma of the lung].

BACKGROUND: Loss of heterozygosity (LOH) at 5q21, where the APC/MCC genes reside, is one of the genetic alterations that characterizes lung cancer. The aim of this study was to analyse LOH frequency on chromosome 5q21 in patients with non-small cell lung cancer and its relationship with TNM staging and histological subtypes. PATIENTS AND METHODS: Tumor and corresponding normal DNA were isolated from 60 patients with non-small cell lung cancer and a subsequent polymerase chain reaction (PCR) with microsatellite markets within the APC/MCC region was performed. The PCR products were resolved by electrophoresis. The comparison between the normal and tumor DNA patterns allowed us to detect the samples harboring LOH. RESULTS: LOH at 5q21 was detected in 20% of the patients. LOH at 5q21 was found in 16% of stage I patients, 8% in stage II patients and 40% in stage IIIA patients. A trend towards worse survival was detected in stage IIIA patients with LOH at 5q21 (9 months median time survival) in comparison with patients with LOH (21 months), although the difference was not statistically significant (p = 0.11). No significant differences in LOH were observed among histological types. In addition, we found no correlation between LOH and higher frequency of mutations rate affecting K-ras and p53 genes. CONCLUSIONS: LOH at 5q21 is a frequent genetic alteration in non-small cell lung cancer and correlates with locally advanced disease (stage IIIA). Our results suggest that LOH at the 5q21 region could be a prognostic factor in non-small cell lung cancer.

Carcinoma, Non-Small-Cell Lung↗

Prognostic value of replication errors on chromosomes 2p and 3p in non-small-cell lung cancer.

As chromosomes 2p and 3p are frequent targets for genomic instability in lung cancer, we have addressed whether alterations of simple (CA)n DNA repeats occur in non-small-cell lung cancer (NSCLC) at early stages. We have analysed by polymerase chain reaction (PCR) assay replication errors (RER) and loss of heterozygosity (LOH) at microsatellites mapped on chromosomes 2p and 3p in 64 paired tumour-normal DNA samples from consecutively resected stage I, II or IIIA NSCLC. DNA samples were also examined for K-ras and p53 gene mutations by PCR-single-stranded conformational polymorphism (PCR-SSCP) analysis and cyclic sequencing, as well as their relationship with clinical outcome. Forty-two of the 64 (66%) NSCLC patients showed RER at single or multiple loci. LOH was detected in 23 tumours (36%). Among patients with stage I disease, the 5-year survival rate was 80% in those whose tumours had no evidence of RER and 26% in those with RER (P = 0.005). No correlation was established between RER phenotype and LOH, K-ras or p53 mutations. RER remained a strong predictive factor (hazard ratio for death, 2.89; 95% confidence interval, 2.23-3.79; P = 0.002) after adjustment for all other evaluated factors, including p53, K-ras, LOH, histological type, tumour differentiation and TNM stage, suggesting that microsatellite instability on chromosomes 2p and 3p may play a role in NSCLC progression through a different pathway from the traditional tumour mechanisms of oncogene activation and/or tumour-suppressor gene inactivation.

Adult↗

k-FGF protoncogene expression is associated with murine testicular teratogenesis, but is not involved during mouse testicular development.

The k-FGF gene, which belongs to the family of the fibroblast growth factor genes, is implicated in tumoral and developmental processes. It is expressed in embryonal carcinoma cells, in embryonic stem cells, during limb and tooth formation and in some germ cell tumors. However, the expression of this protooncogene during testicular development as well its relationship to spontaneous teratogenesis have not been determined. Here we investigate k-FGF expression during testicular development in mice, as well as in a spontaneous testicular teratoma (STT) and in the OTT6050 teratocarcinoma (TC) by Northern blotting, RT-PCR and it situ hybridization. Several data indicate that k-FGF gene contains downstream regulatory sequences which bind octamer factors. One of these transcription factors which binds to k-FGF enhancer is Oct-4. Although the k-FGF gene is activated by Oct-4 in embryonal carcinoma and embryonic stem cells and Oct-4 is expressed in the germ cells of the embryo, our results indicate that there is no detectable k-FGF expression in mouse testicular germ cells at any stage of development. This indicates that Oct-4 does not activate transcription of the k-FGF gene in mouse germ cells, and that k-FGF is not implicated during testicular development. We also show that there is a high k-FGF expression in the experimental OTT6050 TC, but only very low levels in a murine differentiated STT, suggesting that k-FGF activation may be responsible for the genesis and development of STT, behaving as a marker of malignancy in these neoplasms.

Animals↗