International consensus for a dissection room quality system (DRQS): A Delphi panel study.

Dissection Rooms (DRs) are key facilities that allow teaching and research on human anatomy, where students and researchers work with human bodies to acquire, increase, or create new knowledge. Usually, DRs work with a Body Donation Program (BDP), where living donors bequeath their bodies for use in teaching and research after they expire. Despite DRs being part of universities worldwide, no common guidelines, regulations, or quality management systems (QMS) exist that could be applied to different countries. With that purpose in mind, we aimed to develop a QMS that could be applied to DRs globally, using a Delphi panel to achieve consensus about the items that should constitute the QMS. The panel was constituted by 20 anatomy professors from 20 different countries, and the 167 standards to create the rules or guidelines that constitute the QMS were divided in five categories: direction, body donation, students, instructors, and research. After two rounds of revisions, 150 standards were considered "essential" or "important" by more than 70% of the participants, thus being incorporated to the Dissection Room Quality System (DRQS). The results of this panel represent a minimum list of items of the DRQS for improving the functioning of DRs globally.

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.

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.

Single-agent paclitaxel by 3-hour infusion in the treatment of non-small cell lung cancer: links between p53 and K-ras gene status and chemosensitivity.

Currently available cytotoxic drugs are only moderately active in non-small cell lung cancer (NSCLC) and prolong survival only slightly. In two published trials, single-agent paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) was reported to have significant activity in NSCLC, with response rates of 21% and 24%. Treatment-limiting hypersensitivity reactions, however, were noted in a phase I trial of paclitaxel given as a 3-hour infusion at doses > or = 190 mg/m2. We report the results of a phase II trial of paclitaxel given by 3-hour intravenous infusion at 210 mg/m2 every 3 weeks in an outpatient setting. The study was conducted simultaneously at three centers and included chemotherapy-naive patients with unresectable locoregional or metastatic NSCLC. The study objectives were to evaluate response rate, the potential link between p53 and K-ras gene mutations and increased paclitaxel resistance, and toxicity. Sixty-two patients were eligible for this study. All patients were premedicated with dexamethasone 20 mg given orally or intravenously 12 and 6 hours before paclitaxel infusion and cimetidine 300 mg and diphenhydramine 50 mg, both given 60 minutes prior to initiation of paclitaxel infusion. Of the 62 patients who were initially enrolled, 50 (44 men and six women) were evaluable for toxicity at interim analysis; 47 of these patients were evaluable for response. Twenty-four had squamous cell carcinoma, 20 had adenocarcinoma, and six had undifferentiated large cell carcinoma. The median age was 61 years (age range, 36 to 75 years). The median Zubrod performance status was 1 (range, 0 to 2). Seventeen (36%) patients achieved either partial or complete response. Among 24 patients with squamous cell carcinoma, eight (33%; 95% confidence interval, 15% to 61%) had a partial response. Seven (41%; 95% confidence interval, 18% to 64%) of 17 patients with adenocarcinoma had a partial or complete response. Tissue blocks were obtained for analysis of K-ras and p53 gene mutations by means of polymerase chain reaction followed by single-strand conformation polymorphism assay. Our findings indicate that mutations are associated with a poor clinical course and may be prognostic of paclitaxel resistance. Paclitaxel was well tolerated. None of the patients experienced allergic reactions. Granulocytopenia was generally mild. Therapy was interrupted in only two patients because of the development of grade 3 neuropathy. In our experience, paclitaxel is one of the most active cytotoxic drugs targeting NSCLC.

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.

Effect of ascitic liquid on growth in vitro of embryoid bodies derived from teratocarcinoma.

Embryoid bodies (EB) derived from teratocarcinoma (TC) OTT6050 were cultured with ascitic liquids (AL) from animals carrying 16-, 22- and 35-day evolved EB. At the same time the presence of fibronectin (FN) in AL were analyzed by immunoblotting. Results indicate the probable existence of growth-stimulatory factors for EB, as well as the presence of FN in the 22-day AL.

Alpha-fetoprotein in tumours derived from cystic and simple embryoid bodies.

Cellular aggregates called embryoid bodies (EB) have been obtained from the experimental teratocarcinoma (TC) 0TT6050. Two morphological types of EB can be differentiated, which are injected subcutaneously into isogenic 129/Sv mice. The tumors are collected 20 and 30 days after EB injection and processed histologically, and immunohistochemically with anti-alpha-fetoprotein (alpha-FP) antibodies. Our results indicate that the histological pattern of the tumors is related to the degree of morphological organization of the EB used.

Laminin and fibronectin expression during in vivo growth of embryoid bodies derived from teratocarcinoma.

Embryoid bodies (EB) derived from teratocarcinoma OTT6050 are a useful model for the study of early embryogenesis and tumorigenesis and are found in two forms: cystic and simple. After injection of cystic EB intraperitoneally into isogenic 129/Sv mice, their growth and the expression of laminin (LAM) and fibronectin (FN) were studied at 5, 7 and 9 days post-injection. LAM was highly expressed on the internal face of endodermal and on some internal cells in cystic type EB and in both endodermal cell faces in simple EB. Only slight FN expression was observed in cystic and simple EB. Extracellular matrix proteins can be studied in this model.

Growth control of embryonic stem cells injected into mouse uterus on fifth day of pregnancy.

Previous studies have shown that embryonic stem cells (ES) may participate in normal embryonic development when they are injected into the blastocyst. In contrast, ES cells develop into tumors if injected in ectopic sites in adult mice. In this study we injected ES-D3 cells, with the LacZ gene incorporated, into 5-day pregnant mouse uteri, into pregnant unilaterally salpingectomized uteri, into pseudopregnant uteri and into non-pregnant uteri. X-gal staining enabled us to identify injected ES cells on the 7th, 9th, 10th, 12th and 15th days post-injection. In pregnant decidua, the ES cells were located initially in the mesometrial decidua and later distributed in the basal and capsular decidua and in the endodermic layer of the visceral yolk sac. In pregnant, unilaterally salpingectomized mouse uteri, ES cells were mainly located in the uterine lumen and tumors were not observed in either case. In contrast, ES cells injected into pseudopregnant uteri often developed into tumors and those injected into non-pregnant uteri always developed into teratocarcinomas. We conclude that the pregnant-uterine microenvironment may participate in the control of ES cell growth.

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 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.