First French Pilot Quality Assessment of the EndoPredict Test for Early Luminal Breast Carcinoma.

BACKGROUND/AIM: Genomic signatures are needed for the determination of prognosis in patients with early stage, estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancers. EndoPredict test is a RNA-based multigene assay that assesses the risk of 10-year relapse in this context. Quality assessment is a mandatory requirement for a laboratory to address the analytical quality of these molecular analyses. The aim of the study was to demonstrate the robustness of this prognostic test, its usefulness for the patient's treatment strategy, at the national level. MATERIALS AND METHODS: This study presents a pilot quality assessment (QA) of the EndoPredict test using composite design, including the follow-up of internal control values (qREF) of the 12 genes of the assay for 151 independent tests and one formalin-fixed paraffin embedded (FFPE) breast cancer sample. The evaluation of the test was performed by comparing the results of six independent medical laboratories. RESULTS: All measures were highly reproducible and quantification of the qREF showed a standard deviation of less than 0.50 and a coefficient of variation always of <2%. All laboratories found concordant results for the breast cancer samples. The mean EndoPredict (EP) score for the breast cancer sample was 4.97±0.24. The mean of EPclin score was 3.07±0.05. CONCLUSION: This first French independent reported QA assessed the robustness and reproducibility of the EndoPredict test. Such a simple composite design could represent an adapted QA for an expensive diagnostic test.

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment.

KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with anti-epidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allele-specific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

Urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 mRNA assessment in breast cancer by means of NASBA: correlation with protein expression.

Urokinase plasminogen activator (uPA) and its main inhibitor, plasminogen activator inhibitor type-1 (PAI-1) determined in tumor tissue by means of enzyme-linked immunosorbent assay (ELISA) can discriminate patients with primary breast cancer at high risk vs low risk for recurrence. The aim of this study was to analyze uPA and PAI-1 messenger RNA (mRNA) expression by means of quantitative nucleic acid sequence-based amplification (NASBA) on 77 primary breast tumor samples and to correlate this expression with the uPA and PAI-1 protein content. We observed that the 2 markers were significantly overexpressed (uPA, P < .0001; PAI-1, P = .0042) in mRNA in the ELISA+ group. The receiver operating characteristic (ROC) curves demonstrated high concordance between NASBA and ELISA (area under the ROC curve of 0.84 and 0.70 for uPA and PAI-1, respectively) and showed that uPA and PAI-1 status could be predicted by using the molecular assay with sensitivity and specificity values of 80.8% and 82.4% and sensitivity and specificity values of 66.7% and 74.0%, respectively.