Lori A. Kelly

Optimization of the weck-Cel collection method for quantitation of cytokines in mucosal secretions.

ABSTRACT Measurement of immune components in mucosal secretions is important for the evaluation of local immunity at the mucosal surfaces. The Weck-Cel ophthalmic sponge provides a method for the collection of these secretions. The sponge absorbs a relatively large volume of material, therefore allowing for quantitation of multiple immune components. Additionally, it provides a method in which the same device may be used to collect specimens from different mucosal sites, such as the genital tract and oral cavity. This sampling technique has successfully been applied for collection and measurement of antibody in oral and genital tract secretions. The purpose of this work was to optimize the extraction of protein from the sponge matrix. Of particular interest was the recovery of cytokines from the sponge. Satisfactory recovery of the cytokines interleukin 1β (IL-1β), IL-2, IL-5, IL-12, IL-6, IL-8, IL-10, and granulocyte-macrophage colony-stimulating factor was obtained. However, IL-4 and gamma interferon recovery rates remained low. Using an alteration of the published extraction method, cytokine concentrations were measured in cervical secretions from women using oral contraceptives. The data revealed detectable concentrations of IL-6, IL-10, IL-8, and IL-12 on cycle days 9 and 20. The proposed technique provides an easy, practical, and consistent method for collection of nonconventional body fluids, such as cervicovaginal fluids and saliva, for the assay of immunoglobulins and several cytokines.

The degree of segmental aneuploidy measured by total copy number abnormalities predicts survival and recurrence in superficial gastroesophageal adenocarcinoma.

Background Prognostic biomarkers are needed for superficial gastroesophageal adenocarcinoma (EAC) to predict clinical outcomes and select therapy. Although recurrent mutations have been characterized in EAC, little is known about their clinical and prognostic significance. Aneuploidy is predictive of clinical outcome in many malignancies but has not been evaluated in superficial EAC. Methods We quantified copy number changes in 41 superficial EAC using Affymetrix SNP 6.0 arrays. We identified recurrent chromosomal gains and losses and calculated the total copy number abnormality (CNA) count for each tumor as a measure of aneuploidy. We correlated CNA count with overall survival and time to first recurrence in univariate and multivariate analyses. Results Recurrent segmental gains and losses involved multiple genes, including: HER2, EGFR, MET, CDK6, KRAS (recurrent gains); and FHIT, WWOX, CDKN2A/B, SMAD4, RUNX1 (recurrent losses). There was a 40-fold variation in CNA count across all cases. Tumors with the lowest and highest quartile CNA count had significantly better overall survival (p = 0.032) and time to first recurrence (p = 0.010) compared to those with intermediate CNA counts. These associations persisted when controlling for other prognostic variables. Significance SNP arrays facilitate the assessment of recurrent chromosomal gain and loss and allow high resolution, quantitative assessment of segmental aneuploidy (total CNA count). The non-monotonic association of segmental aneuploidy with survival has been described in other tumors. The degree of aneuploidy is a promising prognostic biomarker in a potentially curable form of EAC.

Renal Cell Neoplasms Contain Shared Tumor Type–Specific Copy Number Variations

Copy number variant (CNV) analysis was performed on renal cell carcinoma (RCC) specimens (chromophobe, clear cell, oncocytoma, papillary type 1, and papillary type 2) using high-resolution arrays (1.85 million probes). The RCC samples exhibited diverse genomic changes within and across tumor types, ranging from 106 to 2238 CNV segments in a clear-cell specimen and in a papillary type 2 specimen, respectively. Despite this heterogeneity, distinct CNV segments were common within each tumor classification: chromophobe (seven segments), clear cell (three segments), oncocytoma (nine segments), and papillary type 2 (two segments). Shared segments ranged from a 6.1-kb deletion (oncocytomas) to a 208.3-kb deletion (chromophobes). Among common tumor type-specific variations, chromophobes, clear-cell tumors, and oncocytomas were composed exclusively of noncoding DNA. No CNV regions were common to papillary type 1 specimens, although there were 12 amplifications and 12 deletions in five of six samples. Three microRNAs and 12 mRNA genes had a ≥98% coding region contained within CNV regions, including multiple gene families (chromophobe: amylases 1A, 1B, and 1C; oncocytoma: general transcription factors 2H2, 2B, 2C, and 2D). Gene deletions involved in histone modification and chromatin remodeling affected individual subtypes (clear cell: SFMBT and SETD2; papillary type 2: BAZ1A) and the collective RCC group (KDM4C). The genomic amplifications/deletions identified herein represent potential diagnostic and/or prognostic biomarkers.

Development of quantitative reverse transcriptase PCR assays for measuring gene expression.

Real-time, quantitative reverse transcriptase (RT)-PCR is a very useful and powerful technology for analysis of gene expression. At a first pass, real-time PCR appears to be a simple extension of regular PCR, and it should therefore be easy for an experienced PCR user to convert to quantitative assays. In practice, however, our experience would indicate that this is not usually the case, and most novice real-time PCR users run into problems even though they are very capable at regular PCR. One problem is that, unlike Northern blots, which are technically difficult but typically either work or do not, real-time PCR assays, even poorly designed ones, usually give data. Unfortunately, these data, or their interpretation, may be erroneous, since there are many potential pitfalls that need to be avoided when designing and using real-time PCR for measurement of gene expression. The purpose of this chapter is not to try to discuss the complexities of real-time PCR in detail (which would require a whole book), but, instead, to provide a simple outline for the development of real-time PCR assays. If followed, these guidelines should allow the reader to develop real-time PCR assays that avoid the most common pitfalls and that are capable of producing reliable and accurate gene expression data.