Intra- and Interlaboratory Reproducibility of the Sensitivity to Endocrine Therapy Assay for Stage II/III Breast Cancer.

BACKGROUND: The sensitivity to endocrine therapy assay (SET2,3) predicts treatment outcomes in Stage II-III breast cancer. SET2,3 measures transcription related to estrogen and progesterone receptors (SETER/PR index) and the molecular subtype (RNA4: ESR1, PGR, ERBB2, AURKA) from formalin-fixed paraffin-embedded (FFPE) tissue sections. METHODS: We designed a nested study across 3 pathology laboratories, each testing 60 breast cancers twice in controlled batches. Laboratories macrodissected and directly homogenized the unstained FFPE tumor sections, then performed the QuantiGene Plex bead-based hybridization assay. SET2,3 was calculated centrally using predefined statistical R-scripts and applying pre-defined cutpoints. Concordance correlation coefficient (CCC) was calculated from continuous measurements and Kappa statistic from categorical results. A mixed-effects model estimated contributions to bias (fixed effects) and variance (random effects) from the replicated design. RESULTS: Intralaboratory (CCC 0.96-0.99) and interlaboratory (CCC 0.98-0.99) SET2,3 results were concordant, with rates of agreement for high/low categorization within (Kappa 0.83-0.93) and between laboratories (Kappa 0.87-0.88). The relative contributions to overall variance of SET2,3 measurements were 96.90% from biological differences between cancers, 0.67% from interlaboratory variability, and 2.44% from residual causes including intralaboratory replicates. Similar results were obtained with SETER/PR, the baseline prognostic index calculated using pathological or clinical tumor and nodal staging information, and the 4 individual genes (ESR1, PGR, ERBB2, and AURKA). CONCLUSION: Intra- and interpathology laboratory measurements of SET2,3 and its components were highly reproducible when tested from FFPE tumor sections.

Educating medical students in laboratory medicine: a proposed curriculum.

As the 100th anniversary of the Flexner report nears, medical student education is being reviewed at many levels. One area of concern, expressed in recent reports from some national health care organizations, is the adequacy of training in the discipline of laboratory medicine (also termed clinical pathology). The Academy of Clinical Laboratory Physicians and Scientists appointed an ad hoc committee to review this topic and to develop a suggested curriculum, which was subsequently forwarded to the entire membership for review. The proposed medical student laboratory medicine curriculum defines goals and objectives for training, provides guidelines for instructional methods, and gives examples of how outcomes can be assessed. This curriculum is presented as a potentially helpful outline for use by medical school faculty and curriculum committees.

Bacterial etiology for chronic villitis is not supported by polymerase chain reaction for 16S rRNA DNA.

Chronic villitis is characterized by chorionic villi infiltrated by lymphocytes, histiocytes, and sometimes plasma cells. In a small percentage of cases, an infectious agent can be demonstrated within areas of chronic villitis. However, the pathogenesis of most lesions is idiopathic. Chronic villitis may represent the direct spread of chronic endometrial infection by bacterial organisms that are particularly problematic for culture. To test this hypothesis, polymerase chain reaction (PCR) using primers for the universal bacterial 16S rRNA DNA was performed on DNA extracted from areas of chronic villitis selected from placentas in the Yale Pathology database. Specific areas of chronic villitis were first confirmed by examination of sections stained with hematoxylin and eosin and then removed from archived paraffin blocks. Control tissue spiked with known bacterial counts was also prepared to test the sensitivity of the experiment. All tissue was deparaffinized, dehydrated, and digested with proteinase K. DNA extraction was performed with the Gentra Puregene kit. PCR was done using primers p11 and p13 for the 16S rRNA DNA. The 233-bp amplified target product was identified by agarose gel electrophoresis. Nineteen specimens with multifocal chronic villitis without confinement to anchoring villi were studied. None of the chronic villitis specimens had a demonstrable product using the PCR primers for 16S rRNA DNA, despite adequate DNA in the samples and controls. The assay was sensitive down to approximately 1500 bacteria per specimen. In conclusion, these data do not support a bacterial etiology for chronic villitis.

Defective control of latent Epstein-Barr virus infection in systemic lupus erythematosus.

EBV infection is more common in patients with systemic lupus erythematosus (SLE) than in control subjects, suggesting that this virus plays an etiologic role in disease and/or that patients with lupus have impaired EBV-specific immune responses. In the current report we assessed immune responsiveness to EBV in patients with SLE and healthy controls, determining virus-specific T cell responses and EBV viral loads using whole blood recall assays, HLA-A2 tetramers, and real-time quantitative PCR. Patients with SLE had an approximately 40-fold increase in EBV viral loads compared with controls, a finding not explained by disease activity or immunosuppressive medications. The frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma was higher in patients with SLE than in controls. By contrast, the frequency of EBV-specific CD69+ CD8+ T cells producing IFN-gamma in patients with SLE appeared lower than that in healthy controls, although this difference was not statistically significant. These findings suggest a role for CD4+ T cells in controlling, and a possible defect in CD8+ T cells in regulating, increased viral loads in lupus. These ideas were supported by correlations between viral loads and EBV-specific T cell responses in lupus patients. EBV viral loads were inversely correlated with the frequency of EBV-specific CD69+ CD4+ T cells producing IFN-gamma and were positively correlated with the frequencies of CD69+ CD8+ T cells producing IFN-gamma and with EBV-specific, HLA-A2 tetramer-positive CD8+ T cells. These results demonstrate that patients with SLE have defective control of latent EBV infection that probably stems from altered T cell responses against EBV.

Real-time quantitative reverse transcription-PCR for cyclin D1 mRNA in blood, marrow, and tissue specimens for diagnosis of mantle cell lymphoma.

BACKGROUND: Overexpression of cyclin D1 mRNA, found in mantle cell lymphoma (MCL), is a critical diagnostic marker. We investigated the use of real-time reverse transcription-PCR (RT-PCR) for cyclin D1. METHODS: We studied 97 fresh specimens (50 blood, 30 bone marrow, 15 lymph node, and 2 other samples) from patients diagnosed with a variety of lymphoproliferative diseases, including 25 cases of MCL. We used real-time quantitative RT-PCR to evaluate cyclin D1 mRNA expression. Because blood and marrow specimens may contain only a minority of potentially malignant cells (as opposed to most lymph nodes) and to increase sensitivity, we normalized the cyclin D1 mRNA concentrations to mRNA of a B-cell-specific marker, CD19, as well as to previously characterized beta(2)-microglobulin mRNA. RESULTS: In 16 of 21 cases of MCL with overt disease, the ratio of cyclin D1 mRNA to beta(2)-microglobulin mRNA was increased, but all 21 cases showed increased ratios of cyclin D1 mRNA to CD19 mRNA. Cyclin D1 mRNA was low or undetectable in various lymphoproliferative diseases, including cases of ambiguous immunophenotype. The mRNA ratios were stable over 3-7 days of sample storage. CONCLUSION: Quantitative RT-PCR for cyclin D1 mRNA normalized to CD19 mRNA can be used in the diagnosis of MCL in blood, marrow, and tissue.