Xpert(®) MTB/RIF detection of Mycobacterium tuberculosis from sputum collected in molecular transport medium.

BACKGROUND: The Xpert(®) MTB/RIF assay is widely used for Mycobacterium tuberculosis detection. However, specimen transport remains a challenge. PrimeStore Molecular Transport Medium(®) (PS-MTM) inactivates specimens and stabilizes DNA/RNA at ambient temperature for subsequent molecular detection. OBJECTIVE: To compare the detection of M. tuberculosis concentrations in PS-MTM using Xpert and real-time polymerase chain reaction (RT-PCR), and smear-positive sputum specimens collected using a flocked swab. METHODS: Dilutions of M. tuberculosis in PS-MTM and phosphate buffered saline (PBS) were analyzed using the Xpert assay and commercial RT-PCR. Smear-positive (1+ to 3+) sputum specimens (n = 17) were transferred by flocked swab into PS-MTM and PBS, and were compared to standard 1.0 ml sputum Xpert analysis. RESULTS: Using the Xpert assay, cycle threshold values from high M. tuberculosis concentrations in PS-MTM (>10(3) colony forming units [cfu]/ml) were increased compared to control. In contrast, M. tuberculosis samples containing <10(3) cfu/ml, i.e., low concentrations, suspended in PS-MTM resulted in detection down to 10 cfu/ml. Xpert detection efficiency in PS-MTM treated samples (63.2%) was improved compared to PBS controls (34.9%). Xpert detected M. tuberculosis in all sputum specimens collected by flocked swabs in PS-MTM, and correlated with routine Xpert detection. CONCLUSIONS: PS-MTM enhances M. tuberculosis detection at low concentrations of M. tuberculosis, and provides a simplified and efficient collection method for Xpert detection.

Short- and long-term effects of a training session on pathologists' performance: the INQAT experience for histological grading in breast cancer.

Although the introduction of the Nottingham/Tenovus classification has improved the criteria to assess the histological grading in breast cancer, several quality control studies have shown that good results in terms of reproducibility are hard to obtain. This paper reports the results obtained during an implemented quality control programme for histological grading aimed at evaluating the short- and long-term effects of a training session on pathologists' performance. The interobserver reproducibility for grading score along with its components was assessed. The reproducibility between each pathologist and the reference values was also evaluated, as well as the contribution of each grading category to the observed reproducibility. Results show the weakness of a single training course in improving the long-term performance of the participating pathologists, suggesting the need to continuously monitor the quality of the grading determination by planning quality control studies and training sessions sequentially repeated in short time intervals.

Fifteen-year results of a randomized phase III trial of fenretinide to prevent second breast cancer.

PURPOSE: The synthetic retinoid fenretinide administered for 5 years for prevention of second breast cancer showed no difference after a median of 8 years, but a possible reduction in premenopausal women. We conducted a long-term analysis in a subgroup of women who were regularly followed up in a single center. PATIENTS AND METHODS: We analyzed data after a median follow-up of 14.6 years (IQ range, 12.3-16.3 years) from 1739 women aged 30-70 (872 in the fenretinide arm and 867 in the observation arm), representing 60% of the initial cohort of 2867 women. The main efficacy endpoint was second primary breast cancer (contralateral or ipsilateral). RESULTS: The number of second breast cancers was 168 in the fenretinide arm and 190 in the control arm (hazard ratio = 0.83, 95% CI, 0.67-1.03). There were 83 events in the fenretinide arm and 126 in the observation arm in premenopausal women (HR = 0.62, 95% CI, 0.46-0.83), and 85 and 64 events in postmenopausal women (HR = 1.23, 95% CI, 0.63-2.40). The younger were the women, the greater was the risk reduction associated with fenretinide, which attained 50% in women aged 40 years or younger and disappeared after age 55 (P-age*treatment interaction = 0.023). There was no difference in cancers in other organs, distant metastases or survival. CONCLUSIONS: Fenretinide induces a significant risk reduction of second breast cancer in premenopausal women, which is remarkable at younger ages, and persists several years after treatment cessation. Since adverse events are limited, a trial in young women at high-risk is warranted.

Statistical diagnostics emerging from external quality control of real-time PCR.

Besides the application of conventional qualitative PCR as a valuable tool to enrich or identify specific sequences of nucleic acids, a new revolutionary technique for quantitative PCR determination has been introduced recently. It is based on real-time detection of PCR products revealed as a homogeneous accumulating signal generated by specific dyes. However, as far as we know, the influence of the variability of this technique on the reliability of the quantitative assay has not been thoroughly investigated. A national program of external quality assurance (EQA) for real-time PCR determination involving 42 Italian laboratories has been developed to assess the analytical performance of real-time PCR procedures. Participants were asked to perform a conventional experiment based on the use of an external reference curve (standard curve) for real-time detection of three cDNA samples with different concentrations of a specific target. In this paper the main analytical features of the standard curve have been investigated in an attempt to produce statistical diagnostics emerging from external quality control. Specific control charts were drawn to help biochemists take technical decisions aimed at improving the performance of their laboratories. Overall, our results indicated a subset of seven laboratories whose performance appeared to be markedly outside the limits for at least one of the standard curve features investigated. Our findings suggest the usefulness of the approach presented here for monitoring the heterogeneity of results produced by different laboratories and for selecting those laboratories that need technical advice on their performance.

Prognosis in node-negative primary breast cancer: a neural network analysis of risk profiles using routinely assessed factors.

BACKGROUND: The present study investigated complex time-dependent effects of routinely assessed factors on the risk of breast cancer recurrence over follow-up time, with a partial logistic artificial neural network (PLANN) model. PATIENTS AND METHODS: PLANN was applied to data from 1793 patients with node-negative breast cancer, not submitted to any adjuvant treatment and with a minimal potential follow-up of 10 years. RESULTS: The shape of the hazard function changed according to histology, which showed a time-dependent effect, partly modulated by estrogen receptors (ERs). Age and progesterone receptors (PgR) showed protective effects; the latter was more evident for short follow-up and high ER values. Tumour size and ER content showed time-dependent unfavourable effects at early and long follow-up times, respectively. Predicted values of disease recurrence probability at 2 years of follow-up showed that low steroid-receptor content, young age and large tumour size were associated with the highest risk of relapse. Although the oldest patients with high ER content seem to be those most protected overall, high risk predictions tend to spread also to higher steroid-receptor contents, intermediate ages and small tumour size, with an increase in follow-up time. CONCLUSION: PLANN with suitable visualisation techniques provided thorough insights into the dynamics of breast cancer recurrence for improving individual risk staging of node-negative breast cancer patients.

Vascular endothelial growth factor in node-positive breast cancer patients treated with adjuvant tamoxifen.

In 212 postmenopausal women with node-positive oestrogen receptor-positive (ER(LBA)) breast cancer subjected to radical surgery and adjuvant tamoxifen, the risk of 6-year relapse increased with increasing values of intratumoral vascular endothelial growth factor (VEGF) in patients whose tumours had a low/intermediate ER(LBA) content compared to patients with high-ER(LBA) tumours. These findings indicate that tumour progression, activated or sustained by high VEGF levels, may be counteracted in high-ER(LBA) cancers by tamoxifen, which in contrast fails to contrast the metastatic potential in low-ER(LBA) tumours.

Quality control for biomarker determination in oncology: the experience of the Italian Network for Quality Assessment of Tumor Biomarkers (INQAT).

Biomarker analysis and evaluation in oncology is the product of a number of processes (including managerial, technical and interpretation steps) which need to be monitored and controlled to prevent and correct errors and guarantee a satisfactory level of quality. Several biomarkers have recently moved to clinical validation studies and successively to clinical practice without any definition of standard procedures and/or quality control (QC) schemes necessary to guarantee the reproducibility of the laboratory information. In Italy several national scientific societies and single researchers have activated -- often on a pilot level -- specific external quality assessment protocols, thereby potentially jeopardizing the clinical reality even further. In view of the seriousness of the problem, in 1998 the Italian Ministry of Health sponsored a National Survey Project to coordinate and standardize the procedures and to develop QC programs for the analysis of cancer biomarkers of potential clinical relevance. Twelve QC programs focused on biomarkers and concerning morphological, immunohistochemical, biochemical, molecular, and immunoenzymatic assays were coordinated and implemented. Specifically, external QC programs for the analytical phase of immunohistochemical p53, Bcl-2, c-erb-2/neu/HER2, and microvessel density determination, of morphological evaluation of tumor differentiation grade, and of molecular p53 analysis were activated for the first time within the project. Several hundreds of Italian laboratories took part in these QC programs, the results of which are available on the web site of the Network (www.cqlaboncologico.it). Financial support from the Italian Government and the National Research Council (CNR) will guarantee the pursuit of activities that will be extended to new biomarkers, to preanalytical phases of the assays, and to revision of the criteria of clinical usefulness for evaluating the cost/benefit ratio.

Prognostic role of serum CA15.3 in 362 node-negative breast cancers. An old player for a new game.

The aims of the present investigation were to evaluate the association between serum CA15.3 levels and other biological and clinical variables and its prognostic role in patients with node-negative breast cancer. We evaluated 362 patients operated upon primary breast cancer from 1982 to 1992 (median follow-up 69 months). Serum CA15.3 was measured by an immunoradiometric assay. The association between variables was investigated by a Principal Component Analysis (PCA) and the prognostic role of CA15.3 on relapse-free survival (RFS) was investigated by Cox regression models adjusting for age, oestrogen receptor (ER), tumour stage, and ER x age interaction, with both the likelihood ratio test and Harrell's c statistic. The prognostic contribution of CA 15.3 was highly significant. Log relative hazard of relapse was constant until approximately 10 (U/ml) of CA15.3 and increased thereafter with increasing marker levels. CA15.3 showed a significant contribution using as a cut-off point a value of 31 U/ml. However, the contribution to the model of the marker as a continuous variable is much greater. From these findings, we can conclude that: (i) CA15.3 is a prognostic marker in node-negative breast cancer; (ii) its relationship with prognosis is continuous, with the risk of relapse increasing progressively from approximately 10 U/ml.

Radiotherapy after breast-conserving surgery in small breast carcinoma: long-term results of a randomized trial.

BACKGROUND: Breast-conserving surgery followed by radiotherapy is a widely accepted form of treatment in patients with breast cancer of limited extent. Many attempts have been made to identify subgroups of patients who might avoid radiotherapy. PATIENTS AND METHODS: Between 1987 and 1989, 579 women with carcinoma of the breast were randomly assigned to quadrantectomy, axillary dissection and radiotherapy (299) and to quadrantectomy with axillary dissection without radiotherapy (280). Eligible patients were women with a breast carcinoma less than 2.5 cm in maximum diameter up to 70 years of age. Primary endpoints were intra-breast tumour reappearance (IBTR) and all-cause mortality. RESULTS: The number of IBTRs was significantly higher in patients treated with surgery alone (59 cases out of 273; 10-year crude cumulative incidence of 23.5%) than in patients treated with surgery plus radiotherapy (16 cases out of 294; 10-year crude cumulative incidence of 5.8%). The difference in IBTR frequency between the two treatments appeared to be particularly high in women up to 45 years of age, tending to decrease with increasing age up to no apparent difference in women older than 65 years. Overall survival curves for the two groups, did not differ significantly (P = 0.326). However, a limited survival advantage was evident after radiotherapy for node-positive women. CONCLUSIONS: After breast-conserving surgery radiotherapy appears indicated in all patients up to 55 years of age, in patients with positive axillary nodes, and in patients with extensive intraductal component at histology. The data suggest that radiotherapy may be avoided in patients older than 65, and may be optional in women aged 56-65 years with negative nodes.

Ticlopidine versus aspirin after myocardial infarction (STAMI) trial.

OBJECTIVES: We sought to compare the efficacy of aspirin and ticlopidine in survivors of acute myocardial infarction (AMI) treated with thrombolysis. BACKGROUND: The role of ticlopidine in secondary prevention after AMI has not yet been explored. METHODS: Of 4,696 patients with AMI treated with thrombolysis who were screened, 261 died in the hospital (5.6%) and 1,470 were enrolled in this randomized, double-blind, multicenter trial and allocated to treatment with either aspirin (160 mg/day) or ticlopidine (500 mg/day). The most frequent reasons for exclusion were refusal to give informed consent, planned myocardial revascularization, risk of noncompliance with study procedures, need for anticoagulant therapy and contraindications to the study treatments. The primary end point was the first occurrence of any of the following events during the six-month follow-up: fatal and nonfatal AMI, fatal and nonfatal stroke, angina with objective evidence of myocardial ischemia, vascular death or death due to any other cause. RESULTS: The primary end point was recorded in 59 (8.0%) of the 736 aspirin-treated and 59 (8.0%) of the 734 ticlopidine-treated patients (p = 0.966). Vascular death was the first event in five patients taking aspirin and in six patients taking ticlopidine (0.7% vs. 0.8%; p = NS); nonfatal AMI in 18 and 8 (2.4% vs. 1.1%; p = 0.049); nonfatal stroke in 3 and 4 (0.4% vs. 0.5%; p = NS); and angina in 33 and 40 (4.5% vs. 5.4%; p = NS), respectively. The frequency of adverse reactions was not significantly different between the two groups. CONCLUSIONS: No difference was found between the ticlopidine and aspirin groups in the rate of the primary combined end point of death, recurrent AMI, stroke and angina.

Safety of the synthetic retinoid fenretinide: long-term results from a controlled clinical trial for the prevention of contralateral breast cancer.

PURPOSE: To describe the pattern of occurrence of adverse events commonly arising during treatment with fenretinide, a synthetic retinoid under investigation for cancer prevention. PATIENTS AND METHODS: The series includes 2,867 women accrued in a trial aimed at assessing the effect of fenretinide on the prevention of second breast malignancy. Women were randomly assigned to receive no treatment (1,435 patients) or 5-year fenretinide treatment (1,432 patients). In terms of disease recurrence in the breast, the trial showed a possible beneficial effect of the compound in premenopausal women, and an opposite trend in postmenopausal women. End points considered for safety assessment were the occurrence of diminished dark adaptation, dermatologic disorders, gastrointestinal symptoms, disorders of the ocular surface, and abnormal laboratory values. RESULTS: The most common adverse events were diminished dark adaptation (cumulative incidence, 19.0%) and dermatologic disorders (18.6%). Less common events were gastrointestinal symptoms (13.0%) and disorders of the ocular surface (10.9%). In comparison, incidence figures in the control arm were 2.9% for diminished dark adaptation, 2.9% for dermatologic disorders, 5.4% for gastrointestinal symptoms, and 3.2% for disorders of the ocular surface. Symptoms occurring during fenretinide treatment tended to recover with time. No between-group difference was observed for the occurrence of laboratory data abnormalities. Overall, 63 (4.4%) treatment discontinuations were caused by adverse events. CONCLUSION: Given the number of patients involved in the study and the prolonged intake of the drug, the experience on fenretinide tolerability can be considered sufficiently reassuring to justify further testing of the retinoid.