Stepwise neoplastic transformation of a telomerase immortalized fibroblast cell line

We have described recently a human fibroblast cell line immortalized through ectopic telomerase expression (cen3tel), in which the extension of the life span was associated with the appearance of chromosomal aberrations and with the ability to grow in the absence of solid support. As reported in this article, on further propagation in culture, cen3tel cells became neoplastically transformed, being able to form tumors in nude mice. The analysis of the cells, during the gradual transition toward the tumorigenic phenotype, allowed us to trace cellular and molecular changes associated with different phases of transformation. At the stage in which they were able to grow in agar, cen3tel cells had lost contact growth inhibition but still retained the requirement of serum to proliferate and were not tumorigenic in immunocompromised mice. Moreover, they showed a down-regulation of the INK4A locus and were resistant to oncogenic Ras-induced senescence but still retained a functional p53. Subsequently, cen3tel cells became tumorigenic, lost p53 function because of a mutation in the DNA-binding motif, and overexpressed c-myc. Interestingly, tumorigenic cells did not carry activating mutations either in the ras proto-oncogenes (H-ras, N-ras, and K-ras) or in B-raf. Cen3tel cells gradually became hyperdiploid but did not display centrosome abnormalities. To our knowledge, cen3tel is the first telomerase immortalized fibroblast line, which became neoplastically transformed. In this system, we could associate a down-regulation of the INK4A locus with anchorage-independent growth and with resistance to Ras-induced senescence and link p53 mutations and c-myc overexpression with tumorigenicity.     

The predictive value of clinical evaluation of response to neoadjuvant chemoradiation therapy for rectal cancer

INTRODUCTION: Multimodality therapy has become the standard treatment for patients with locally advanced (T3 and T4) rectal carcinoma. Accurate preoperative staging of the patients with rectal cancer has increased in importance because the selection of patients with transmural rectal cancer (T3 or T4) or node-positive disease leads to a previous nonsurgical neoadjuvant treatment. The purpose of this study was to evaluate the predictive value of the clinical response to neoadjuvant therapy on the basis of pathological results obtained on rectal cancer patients treated by chemoradiotherapy and surgery. METHODS: From 1994 to 2003, 58 patients with a primary diagnosis of rectal cancer were studied at our department and enrolled in a neoadjuvant protocol of chemoradiotherapy followed by surgery. All patients were treated by 30 days of chemoradiotherapy. At the end of the chemoradiotherapy, each patient underwent clinical examination, including digital rectal examination, proctoscopy and abdominal-pelvic computerized tomography to define the clinical response to the chemoradiotherapy. Surgical resection was performed in all patients three weeks after the end of chemoradiotherapy, and histological analysis was performed on all resected specimens. RESULTS: The clinical complete response rate corresponded to the pathological complete response rate, whereas the clinical evaluation overestimated partial response and stable disease. The pathologic examination revealed that 3.5% of clinical partial responses and 3.4% of clinical stable disease were really pathological progressive disease. Clinical partial response and clinical stable disease positive predictive values were 92.8% and 90.9%, respectively, whereas the clinical progressive disease negative predictive value was 20%. Then, 6.9% of patients believed to have responded to the therapy, or not to have responded or worsened, actually had worsened by the end of the chemoradiotherapy. CONCLUSIONS: Positive and negative predictive values, in particular for partial response and stable disease, of clinical evaluation of the response to chemoradiotherapy were not high enough to consider clinical evaluation accurate enough to make treatment decisions.     

Prevalence and correlates of abscesses among a cohort of injection drug users

Recent studies have indicated that injection-related infections such as abscesses and cellulitis account for the majority of emergency room visits and acute hospitalizations accrued by local injection drug users. The objective of this analysis was to examine the prevalence and correlates of developing an abscess among a cohort of injection drug users in Vancouver and to identify socio-demographic and drug use variables associated with abscesses at baseline. We examined abscesses among participants enrolled in a prospective cohort of injection drug users. Categorical variables were analyzed using the Pearson's chi-square test and continuous variables were analyzed using the Wilcoxon signed rank test. Among 1 585 baseline participants, 341 (21.5%) reported having an abscess in the last six months. In a logistic regression model that adjusted for all variables that were associated with having an abscess at p < 0.1 in univariate analyses, female gender [odds ratio (OR) = 1.7, [95%CI: 1.2 – 2.4]; p = 0.002), recent incarceration (OR = 1.7, [95%CI: 1.3 – 2.2]; p < 0.001), sex trade involvement (OR = 1.4 [95% CI: 1.0 – 2.0]; p = 0.03), frequent cocaine use (OR = 1.5 [95%CI: 1.2 – 2.0]; p = 0.002) and HIV serostatus (OR = 1.5, [95%CI: 1.2 – 2.0]; p = 0.003) were positively associated with having an abscess. Explanations for these associations require further study, and interventions are needed to address this highly prevalent concern.     

Bacterial contamination of human organ-cultured corneas

PURPOSE: This study was designed to define the risk of contamination of human corneas preserved by the organ-culture method. METHODS: We examined the microbial contaminations in 3,100 corneoscleral rims cultivated in our eye bank. Microbiologic tests were performed in the preservation medium 5 days after the beginning of cornea cultures and in the last day of culture (21.5 +/- 8.1 days), when the corneas were transferred to the deswelling medium. In 1,029 corneas a microbiologic test also was performed 1 day after the beginning of deswelling procedure. RESULTS: We found 206 microbial contaminations (6.65% of total) after 5 days and 17 (0.55%) at the end of the preservation period. The total number of contaminated samples during the cornea culture was 223 corresponding to 7.2% of the samples (95% confidence interval, 6.3-8.1). The 1,029 tests performed during the deswelling step disclosed 26 contaminated cornea cultures despite apparent sterility of the medium (2.5%; 95% confidence interval, 1.5-3.5). CONCLUSIONS: The observation of microbial contaminations in a time close to the transplant (i.e., at the end of the preservation period and in the deswelling step) showed that a fast microbial tests during the deswelling procedure may prevent the grafting of a contaminated cornea. The appearance of bacteria in the deswelling medium despite a negative culture medium suggests that bacteria penetrate the corneal tissues during the culture to be subsequently extruded when the internal fluids move outward.     

Lack of benefit of pelvic radiation in prostate cancer patients with a high risk of positive pelvic lymph nodes treated with high-dose radiation

PURPOSE: The use of pelvic radiation for patients with a high risk of lymph node (LN) metastasis (>15%) remains controversial. We reviewed the data at three institutions treating patients with a combination of external-beam radiation therapy and high-dose-rate brachytherapy to address the prognostic implications of the use of the Roach formula and the benefit of pelvic treatment. METHODS AND MATERIALS: From 1986 to 2003, 1,491 patients were treated with external-beam radiation therapy and high-dose-rate brachytherapy. The Roach formula [2/3 prostate-specific antigen + (Gleason score -6) x 10] could be calculated for 1,357 patients. Group I consisted of patients having a risk of positive LN < or = 15% (n = 761), Group II had a risk >15% and < or = 30% (n = 422), and Group III had a risk of LN disease >30% (n = 174). A >15% risk of having positive LN was found in 596 patients and was used to determine the benefit of pelvic radiation. The pelvis was treated at two of the cancer centers (n = 312), whereas at the third center (n = 284) radiation therapy was delivered to the prostate and seminal vesicles alone. Average biologic effective dose was > or = 100 Gy (alphabeta = 1.2). Biochemical failure was as per the American Society for Therapeutic Radiology and Oncology definition. Statistics included the log-rank test as well as Cox univariate and multivariate analysis. RESULTS: For all 596 patients with a positive LN risk >15%, median follow-up was 4.3 years, with a mean of 4.8 years. For all cases, median follow-up was 4 years and mean follow-up was 4.4 years. Five-year results for the three groups based on their risk of positive LN were significantly different in terms of biochemical failure (p < 0.001), clinical control (p < 0.001), disease-free survival excluding biochemical failure (p < 0.001), cause-specific survival (p < 0.001), and overall survival (p < 0.001). For all patients with a risk of positive LN >15% (n = 596), Group II (>15-30% risk), or Group III (>30% risk), no benefit was seen in the 5-year rates of clinical failure, cause-specific survival, or overall survival with pelvic radiation. In the Cox multivariate analysis for cause-specific survival, Gleason score (p = 0.009, hazard ratio [HR] 3.1), T stage (p = 0.03, HR 1.8), and year of treatment (p = 0.05, HR 1.1) were significant. A log-rank test for cause-specific survival for all patients (n = 577) by the use of pelvic radiation was not significant (p = 0.99) accounting for high-dose-rate brachytherapy dose, neoadjuvant hormones, Gleason score, prostate-specific antigen, T stage, and year of treatment as covariates. CONCLUSIONS: The use of the Roach formula to stratify patients for clinical and biochemical outcomes is excellent. Pelvic radiation added to high prostate radiation doses did not show a clinical benefit for patients at a high risk of pelvic LN disease (>15%) selected using the Roach formula.     

An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics

Many adverse drug reactions are caused by the cytochrome P450 (CYP) dependent activation of drugs into reactive metabolites. In order to reduce attrition due to metabolism-mediated toxicity and to improve safety of drug candidates, we developed two in vitro cell-based assays by combining an activating system (human CYP3A4) with target cells (HepG2 cells): in the first method we incubated microsomes containing cDNA-expressed CYP3A4 together with HepG2 cells; in the second approach HepG2 cells were transiently transfected with CYP3A4. In both assay systems, CYP3A4 catalyzed metabolism was found to be comparable to the high levels reported in hepatocytes. Both assay systems were used to study ten CYP3A4 substrates known for their potential to form metabolites that exhibit higher toxicity than the parent compounds. Several endpoints of toxicity were evaluated, and the measurement of MTT reduction and intracellular ATP levels were selected to assess cell viability. Results demonstrated that both assay systems are capable to metabolize the test compounds leading to increased toxicity, compared to their respective control systems. The co-incubation with the CYP3A4 inhibitor ketoconazole confirmed that the formation of reactive metabolites was CYP3A4 dependent. To further validate the functionality of the two assay systems, they were also used as a "detoxification system" using selected compounds that can be metabolized by CYP3A4 to metabolites less toxic than their parent compounds. These results show that both assay systems can be used to screen for metabolic activation, or de-activation, which may be useful as a rapid and relatively inexpensive in vitro assay for the prediction of CYP3A4 metabolism-mediated toxicity.     

Motor examination in children with Attention-deficit/hyperactivity Disorder and Asperger Syndrome

Aim: Evaluating whether motor skills could differentiate drug‐naive subjects with two neurodevelopmental disorders: Attention‐Deficit Hyperactivity Disorder (ADHD) and Asperger Syndrome (AS). Methods: Thirty‐six boys (12 with ADHD, 12 with AS and 12 with typical development) aged 8–12 were evaluated using the Physical and Neurological Examination for Subtle Signs. Three primary outcome variables were obtained as follows: (i) total speed of timed activities, (ii) total overflow and (iii) total dysrhythmia. Results: Children with AS performed more slowly than those with ADHD and healthy children independently of age and IQ. Total dysrhythmia differentiates ADHD and AS children from controls. Conclusion: Dysfunction of the fronto‐striatal–cerebellar networks related to motor control could be the physiopathological basis of the reported findings.