Detección mediante análisis de ADN, falsificación en documento público (Tasa de alcoholemia) / Breath alive drivers alcohol in blood valuation. Subsequent DNA analysis to compare switched samples blood

Muestras de sangre analizadas previamente para la determinación del grado de alcoholemia, fueron contrastadas mediante el estudio de polimorfismos del ADN con el fin de esclarecer un supuesto delito de falsificación en documento público y poder así confirmar si pertenecían a la misma persona. En los resultados obtenidos no se detectó la presencia de alcohol etílico en ninguna de las muestras. Los perfiles genéticos obtenidos en las muestras analizadas no eran coincidentes con el perfil genético obtenido de las muestras indubitadas (AU)

Concordancia entre perfil genético en zona externa de preservativo y víctima / Genetic profile matching between victims and outer surface of preservatives

Se analizó la fracción no espermática de la zona externa de tres preservativos procedentes de tres asuntos no relacionados entre sí, de supuesta agresión sexual. Los perfiles genéticos obtenidos fueron posteriormente cotejados con el perfil genético de la víctima correspondiente a cada caso, con el fin de comprobar la compatibilidad existente. El estudio de los perfiles genéticos dió, en dos de los asuntos investigados, como resultado la ausencia de concordancia entre las zonas externas de los preservativos y sus víctimas. Se concluye que la recogida y conservación correcta de las muestras así como la aportación de una información más completa de las circunstancias de los hechos son cruciales para obtener resultados satisfactorios (AU)

Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders

The detection of 14-3-3 protein in cerebrospinal fluid by immunoblotting is useful for the diagnosis of Creutzfeldt-Jakob disease (CJD). We found 14-3-3 protein in 10 of 80 (12.5%) patients with paraneoplastic neurological disorders (PNDs), whose presenting symptoms may mimic those of CJD. In 47 of 48 CJD patients, the 14-3-3 protein was detected as a single band, and it was detected as a double band in 1 patient. The double-band pattern was observed in 9 of the 10 14-3-3 proteinpositive patients with PNDs. The 14-3-3 protein assay may be positive in PND patients, but the immunoblotting pattern distinguishes most PND samples from those of CJD.

Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders.

The detection of 14-3-3 protein in cerebrospinal fluid by immunoblotting is useful for the diagnosis of Creutzfeldt-Jakob disease (CJD). We found 14-3-3 protein in 10 of 80 (12.5%) patients with paraneoplastic neurological disorders (PNDs), whose presenting symptoms may mimic those of CJD. In 47 of 48 CJD patients, the 14-3-3 protein was detected as a single band, and it was detected as a double band in 1 patient. The double-band pattern was observed in 9 of 10 14-3-3 protein-positive patients with PNDs. The 14-3-3 protein assay may be positive in PND patients, but the immunoblotting pattern distinguishes most PND samples from those of CJD.

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.

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.

Microsatellite alterations at 5q21, 11p13, and 11p15.5 do not predict survival in non-small cell lung cancer.

We investigated the clinical implications of allelic deletions at three common sites of loss of heterozygosity (LOH) in regions 5q21, 11p15.5, and 11p13 in 86 patients with non-small cell lung cancer (NSCLC). We performed a PCR-based microsatellite polymorphism assay for detection of LOH. The microsatellite markers used were D5S82 (proximal to the APC gene), MCC (within the MCC gene), D11S904 (11p13), HRAS (within the H-ras gene), and D11S860 (11p15.5). Of the 68 informative cases at 5q21 loci, LOH was found in 14 cases (20%), whereas LOH frequency in 11p15.5 and 11p13 was 31% (19 of 61 informative cases) and 19% (12 of 63 informative cases), respectively. There was a significant correlation between 5q21 LOH and mediastinal lymph node involvement (P = 0.03). However, no differences were observed in median survival times (26 months in patients with 5q21 LOH versus 37 months in the remainder; P = 0.33) nor in patients with 11p LOH (38 months versus 32 months, respectively; P = 0.72). Cox's proportional hazards model predicted that stage was the only independent poor prognostic marker in the entire cohort of NSCLC patients. Thus, the present study revealed two important abnormalities, LOH at chromosome 5q21 and LOH at chromosome 11p, both implied in NSCLC development.

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.

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.

Molecular staging of non-small cell lung cancer according to K-ras genotypes.

We have previously demonstrated a strong association between K-ras gene mutations, as determined by PCR followed by allele-specific oligonucleotide hybridization (ASO-h), and survival in non-small cell lung cancer patients. The purpose of this study was to determine the relationship between tumor aggressiveness and specific-type K-ras point mutations in non-small cell lung cancer. We developed procedures to examine the status of the K-ras gene by ASO-h and by single-strand conformation polymorphism assay of DNA obtained from formalin-fixed paraffin-embedded tumors. K-ras point mutations at codons 12 and 61 were assessed in 275 consecutively treated stage I-IV non-small cell lung cancers. Among patients with stage I disease, median survival time was 41.5 months in those whose tumors had no evidence of K-ras mutations and 27 months in those with K-ras 12 mutations; among patients with stage IIIA disease, median survival time was 7 months in those with K-ras codon 12 aspartic and serine mutations and 15 months for those with other K-ras mutations (P = 0.01). In a multivariate analysis, specific-type K-ras codon 12 point mutation remained a strong predictive factor (hazard ratio for death, 2.06; 95% confidence interval, 1.11-3.81; P = 0.02) after adjustment for other evaluated factors, including TNM stage and histology. Thus, we concluded that in patients with non-small cell lung cancer, specific K-ras 12 point mutations detected by DNA amplification and either ASO-h or single-strand conformation polymorphism methods predicted a significantly increased risk of recurrence and death, independently of stage and histology.

The role of induction (neoadjuvant) chemotherapy in stage IIIA NSCLC.

Induction (neoadjuvant) chemotherapy has become an accepted treatment for stage IIIA (T1-3N2M0) non-small cell lung cancer. In two recent randomized trials, neoadjuvant chemotherapy plus surgery gave an increase in median survival at least fivefold greater than surgery alone. The Spanish Lung Cancer Group trial of preoperative chemotherapy, in which the cisplatin dose was randomized to either 50 mg/m2 or 100 mg/m2 plus 3 g/m2 ifosfamide and 6 mg/m2 mitomycin, examines the effect of K-ras gene mutations on tumor response and survival. Patients whose tumors contain K-ras gene mutations are more likely to develop distant metastases and have lower median survival than patients without such mutations. Microsatellite instability seems to be a frequent mechanism of genetic aberrations. Knowledge about these genetic alterations could have prognostic importance and may identify the patients who should receive the most aggressive additional treatment.

Mutated K-ras gene analysis in a randomized trial of preoperative chemotherapy plus surgery versus surgery in stage IIIA non-small cell lung cancer.

The observation that the proteins encoded by ras genes play a central role in the signalling pathways used by cells to respond to growth factors and the fact that mutated ras proteins are constantly promoting cell division have led to a PCR-based hunt for additional clinical information. In the present study, K-ras analysis draws the following conclusions: (1) K-ras point mutation frequency was higher in the surgery group (10 of 24 patients) than in the chemotherapy-surgery group (3 of 20 patients). (2) Mutated K-ras was predominantly observed at codon 12 but five mutations appeared at codon 61. (3) Mutations were identified in the squamous cell carcinoma histological NSCLC subtype except in four cases corresponding to adenocarcinoma. (4) A multifarious pattern of substitutions, especially at codon 12, were noted with aspartic K 12 substitutions more prone to develop bone metastases. (5) Although a genotypic K-ras classification of NSCLC may not yet be formulated, our accumulated data (unpublished) suggest a trend toward it. (6) Patients with mutated K-ras tumors in the surgery group had no different survival than those with normal K-ras. However our pooled data as well as other authors' results assert that mutated K-ras constitute an additional prognostic datum that deserves to be included together with TNM classification. In the design of new preoperative (neoadjuvant) chemotherapy trials, stratification of tumors by K-ras status deserves to be further investigated in order to correlate with response, relapse and survival. Mutated K-ras genotype merits further research. Finally, the paradigm of uneven histological distribution and mutated K-ras spectra among researchers should serve as a stimulus to search for further contributions in this field.

K-ras genotypes and prognosis in non-small-cell lung cancer.

BACKGROUND: Despite major advances in the treatment of many kinds of cancer over the past 25 years, the overall 5-year survival of non-small-cell lung cancer patients has scarcely improved. Even in stage I which has the best outcome long-term survival still falls below 70%. Since intriguing data suggest that the identification of genetic markers might allow prognosis to be assessed case by case. We were prompted to evaluate K-ras gene mutations as a putative prognostic marker in this neoplasm. MATERIALS AND METHODS: We used the polymerase chain reaction (PCR) followed by allele specific oligonucleotide (ASO) hybridization or single-strand conformation polymorphism (SSCP) assays, to detect K-ras mutations in DNA from formalin-fixed, paraffin-embedded tumor samples. K-ras mutations were examined in 192 stage I to IV non-small-cell lung cancer patients. RESULTS: K-ras mutations were detected in 51 of 192 of the cases studied (27%). All K-ras mutations detected by PCR/ASO hybridization were also identified by SSCP. In stage I disease, the median survival was 46 months in those patients whose tumors had no K-ras mutations and 21 months in those with aspartic acid and serine mutations at K-ras codon 12; in patients with stage IIIA disease, median survival time was 16 months in the K-ras negative group and 7 months in the aspartic acid and serine mutation group. No significant differences were observed for the remaining amino acid substitutions of K-ras, nor were they observed at all in more advanced disease. CONCLUSIONS: K-ras gene status has strong prognostic value in patients with stage IIIA non-small-cell lung cancer. The survival curve for patients with stage I and K-ras codon 12 aspartic or serine mutations is close to that of patients with stage IIIA without K-ras mutations. However, a non-small-cell lung cancer K-ras genotypic classification should be validated in larger studies.

Prognostic value of k-ras 12 genotypes in patients with advanced nonsmall cell lung-cancer receiving Carboplatin with either intravenous or chronic oral dose Etoposide.

Despite the knowledge of strong prognostic factors such as stage and performance status, the outcome of individual patients with non-small cell lung cancer is not yet predictable, although it has been observed that patients whose tumors contain K-ras gene mutations at codon 12 have a shortened survival. We compared response rate, toxicity and survival of patients with non-small cell lung cancer receiving carboplatin 325 mg/m2 on day 1 with either intravenous etoposide (100 mg/m2 days 1-3) or oral etoposide (50 mg/m2/day) for 21 consecutive days. Secondly, we sought to find whether K-ras mutations or their genotypes could help to distinguish tumor types with clinical implications on prognosis. 180 patients were entered in this randomized study. Tumor specimens obtained at the time of bronchoscopy were available in 71 cases. 167 patients were assessable for response. We obtained 24 objective responses out of 88 patients (27%) in the intravenous etoposide plus carboplatin arm and 14 out of 79 patients (18%) in the oral etoposide plus carboplatin arm. Neither the objective response rate nor the survival time showed a significant difference between the two groups. Toxicity consisted mainly of myelosuppression. We detected 20 ras gene mutations in the 71 (28%) tumors analyzed. None of the 7 patients with aspartic and serine ras mutations had objective response as compared with 2 of 13 patients (15%) whose tumors contained cysteine, valine and arginine mutations and 16 of the 51 patients (31%) who had no ras mutations. Patients whose tumors had aspartic and serine mutations had a median survival time of 18 weeks in contrast with 36 weeks for the remainder (P=0.04). This study highlights the fact that K-ras genotypes may be an important prognostic variable in patients with advanced non-small cell lung cancer.

Study of carbendazin residue accumulation on greenhouse and field-grown strawberries, after successive treatments with benomyl.

Benomyl, a systemic fungicide used in preharvest treatments to prevent Botrytis and other fungal diseases, metabolizes to carbendazim (MBC). A study was undertaken to determine if the total residues of MBC of greenhouse and field-grown Douglas strawberries accumulate in fruits after successive fortnightly treatments with a commercial preparation of benomyl. Statistical analysis of the data obtained indicates that residues of MBC do not accumulate in fruits; on the contrary, they decrease over time.

Monitoring residues of carbendazim (applied as benomyl) and thiabendazole in Wellspur apples.

Residues of benomyl (determined as carbendazim, widely accepted as MBC) and of thiabendazole in Wellspur apples were monitored after the apples were treated postharvest by immersion in a bath with each of the fungicides separately and stored under refrigeration. Whole fruit and pulp analyses were conducted over the period beginning 1 day after treatment and lasting up to 200 days afterwards. Under the conditions described, both benomyl (expressed as parts per million of carbendazim) and thiabendazole were found in the fruits in the following average amounts: 0.44 +/- 0.03 and 0.11 +/- 0.03 ppm benomyl (as carbendazim) in whole fruit and in pulp, respectively; 0.98 +/- 0.10 and 0.39 +/- 0.10 ppm thiabendazole in whole fruit and in pulp, respectively.