Supplementary Table 1 from An Innovative Microarray Strategy Identities Informative Molecular Markers for the Detection of Micrometastatic Breast Cancer

BACKGROUND:To address the clinical relevance of molecular detection of occult breast cancer in sentinel lymph nodes and nonsentinel axillary lymph nodes (ALN), we initiated the Minimally Invasive Molecular Staging of Breast Cancer (MIMS) trial, a multi-institutional prospective cohort study. This trial represents the first prospective cohort study in which a multimarker, real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was applied to the detection of breast cancer micrometastases in ALN.MATERIALS AND METHODS:Sentinel and/or nonsentinel ALN from 501 breast cancer subjects with T1-T3 primary tumors were analyzed by standard histopathology and multimarker, real-time RT-PCR analysis. Seven breast cancer-associated genes (mam, mamB, PIP, CK19, muc1, PSE, and CEA) known to be overexpressed in metastatic breast cancer compared with control lymph nodes were used. Follow-up data were collected for 5 years.RESULTS:Of the 501 breast cancer subjects enrolled, 348 were node negative and completed the 5-year follow-up. Of these patients (n = 94), 27% demonstrated evidence of molecular overexpression. The 5-year relapse-free survival rate was 95.4% (95% confidence interval [95% CI], 92.4-97.2%). No single gene or combination of study genes was predictive of recurrence.CONCLUSIONS:The genes in this study panel failed to be predictive of clinical relapse. This may be a function of several factors: the low event rate at 5 years, the particular gene set, the methodology used for detection/analysis or that our original hypothesis was wrong and that the presence of positive marker signal by real-time RT-PCR is not associated with a worsened clinical outcome.

The epithelial to mesenchymal transition (EMT) is a developmental process enabling epithelial cells to gain a migratory mesenchymal phenotype. In cancer, this process contributes to metastases; however the regulatory signals and mechanistic details are not fully elucidated. Here, we sought to identify the subset of genes regulated in lung cancer by ZEB1, an E-box transcriptional repressor known to induce EMT. Using an Affymetrix-based expression database of 38 non-small cell lung cancer (NSCLC) cell lines, we identified 324 genes that correlated negatively with ZEB1 and 142 that were positively correlated. A mesenchymal gene pattern (low E-cadherin, high Vimentin or N-cadherin) was significantly associated with ZEB1 and ZEB2, but not with Snail, Slug, Twist1 or Twist2. Among eight genes selected for validation, seven were confirmed to correlate with ZEB1 by quantitative real-time RT-PCR in a series of 22 NSCLC cell lines, either negatively (CDS1, EpCAM, ESRP1, ESRP2, ST14) or positively (FGFR1, Vimentin). In addition, over-expression or knockdown of ZEB1 led to corresponding changes in gene expression, demonstrating that these genes are also regulated by ZEB1, either directly or indirectly. Of note, the combined knockdown of ZEB1 and ZEB2 led to apparent synergistic responses in gene expression. Furthermore, these responses were not restricted to artificial settings, since most genes were similarly regulated during a physiologic induction of EMT by TGF-β plus EGF. Finally, the absence of ST14 (matriptase) was linked to ZEB1 positivity in lung cancer tissue microarrays, implying that the regulation observed in vitro applies to the human disease. In summary, this study identifies a new set of ZEB-regulated genes in human lung cancer cells and supports the hypothesis that ZEB1 and ZEB2 are key regulators of the EMT process in this disease.

Coaxial connectors are used extensively in cables and microwave components. Any losses or reflections are detrimental to a microwave or RF system as they reduce the power transmitted and they become a source of noise thus reducing the overall system Signal to Noise ratio. In this paper a new type of connector is being introduced which avoids step discontinuities by incorporating smooth transitions. Measurements show that there is an improvement in the performance of such connectors.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Twenty-five to fifty per cent of early stage NSCLC patients who are treated by surgery alone will not survive beyond five years, suggesting a need for better prognostic factors. Using a novel bioinformatics approach, we identified 13 potentially prognostic NSCLC genes in which the expression levels of nine (69%) were highly inversely correlated in the NCI60 CGAP database with the epithelial-to-mesenchymal (EMT) inducers Zeb1 or Zeb2 (R values = 0.57-0.78; p-values 30K). To search for small molecules that might inhibit Zeb1, we constructed a CDH1 luciferase reporter containing a putative Zeb1 high-affinity binding site. Using transfected HEK293 cells, we screened 12,000 compounds from the ChemBridge library. One candidate stimulated endogenous Map7 expression 2.2-fold in HEK293 cells and 49-fold in lung cancer A549 cells. We are also screening the library for compounds that preferentially inhibit AGR2 expression with the goal of identifying novel agents for the treatment of lung cancer. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-264.

We have previously reported that a quantitative reverse transcription (QRT)-PCR assay accurately analyzes sentinel lymph nodes (SLNs) from breast cancer patients. The aim of this study was to assess a completely automated, cartridge-based version of the assay for accuracy, predictive value, and reproducibility. The triplex (two markers + control) QRT-PCR assay was incorporated into a single-use cartridge for point-of-care use on the GeneXpert system. Three academic centers participated equally. Twenty-nine positive lymph nodes and 30 negative lymph nodes were analyzed to establish classification rules. SLNs from 120 patients were subsequently analyzed by QRT-PCR and histology (including immunohistochemistry), and the predetermined decision rules were used to classify the SLNs; 112 SLN specimens produced an informative result by both QRT-PCR and histology. By histological analysis, 21 SLNs were positive and 91 SLNs were negative for metastasis. QRT-PCR characterization produced a classification with 100% sensitivity, 97.8% specificity, and 98.2% accuracy compared with histology (91.3% positive predictive value and 100% negative predictive value). Interlaboratory reproducibility analyses demonstrated that a 95% prediction interval for a new measurement (DeltaCt) ranged between 0.403 and 0.956. This fully automated QRT-PCR assay accurately characterizes breast cancer SLNs for the presence of metastasis. Furthermore, the assay is not dependent on subjective interpretation, is reproducible across three clinical environments, and is rapid enough to allow intraoperative decision making.

Colorectal cancer (CRC) is the second most frequent cause of cancer-related death in the United States. To determine whether certain molecular markers might be prognostic for survival, we measured by quantitative real-time RT-PCR the expression levels of 15 previously studied genes that are known to be up-regulated or down-regulated in the progression of epithelial cancers. The tumor samples were extracted from formalin-fixed paraffin-embedded primary tissues derived from patients with Stage II CRC who developed disease recurrence within two years (n=10), or were disease-free for at least 4 years (n=12). We were able to determine, by AUC curve analysis, that the ratio of microtubule associated protein 7 (Map7)/B2M was predictive of outcome in our sample set. Further, using Kaplan-Meier survival analysis, we observed significantly different curves as a function of marker positivity for the Map7/B2M (p=0.0001; HR=11) expression ratio. This suggests that the expression ratio of Map7/B2M may serve as a valuable prognostic marker in patients with Stage II colon cancer, and potentially guide therapeutic decision making.

BACKGROUND:We sought to examine the detection rate of cancer cells in peripheral blood (PBL) and in bone marrow (BM) using an established 7-gene marker panel and evaluated whether there were any definable associations of any individual gene with traditional predictors of prognosis.METHODS:Patients with T1-T3 primary breast cancer were enrolled into a prospective, multi-institutional cohort study. In this interim analysis 215 PBL and 177 BM samples were analyzed by multimarker, real-time RT-PCR analysis designed to detect circulating and disseminated breast cancer cells.RESULTS:At a threshold of three standard deviations from the mean expression level of normal controls, 63% (136/215) of PBL and 11% (19/177) of BM samples were positive for at least one cancer-associated marker. Marker positivity in PBL demonstrated a statistically significant association with grade II-III (vs. grade I; p = 0.0083). Overexpression of the mammaglobin (mam) gene alone had a statistically significant association with high tumor grade (p = 0.0315), and showed a trend towards ER-negative tumors and a high risk category. There was no association between marker positivity in PBL and the pathologic (H&E) and/or molecular (RT-PCR) status of the axillary lymph nodes (ALN).CONCLUSION:This study suggests that molecular detection of circulating cancer cells in PBL detected by RT-PCR is associated with high tumor grade and specifically that overexpression of the mam gene in PBL may be a poor prognostic indicator. There was no statistically significant association between overexpression of cancer-associated genes in PBL and ALN status, supporting the concept of two potentially separate metastatic pathways.

Colorectal cancer (CRC) is the fourth most common non-cutaneous malignancy in the United States and the second most frequent cause of cancer-related death. One of the most important determinants of CRC survival is lymph node metastasis. To determine whether molecular markers might be prognostic for lymph node metastases, we measured by quantitative real-time RT-PCR the expression levels of 15 cancer-associated genes in formalin-fixed paraffin-embedded primary tissues derived from stage I-IV CRC patients with (n = 20) and without (n = 18) nodal metastases. Using the mean of the 15 genes as an internal reference control, we observed that low expression of beta(2)microglobulin (B2M) was a strong prognostic indicator of lymph node metastases (area under the curve (AUC) = 0.85; 95% confidence interval (CI) 0.69-0.94). We also observed that the expression ratio of B2M/Spint2 had the highest prognostic accuracy (AUC = 0.87; 95% CI = 0.71-0.96) of all potential two-gene combinations. Expression values of Spint2 correlated with the mean of the entire gene set at an R-2 value of 0.97, providing evidence that Spint2 serves not as an independent prognostic gene, but rather as a reliable reference control gene. These studies are the first to demonstrate a prognostic role of B2M at the mRNA level and suggest that low B2M expression levels might be useful for identifying patients with lymph node metastasis and/or poor survival.

American Journal of HematologyVolume 83, Issue 1 p. 87-87 Letter to the American Journal of HematologyFree Access Detection of circulating tumor cells in the peripheral blood of patients with androgen-independent, advanced or metastatic prostate cancer Michael C. Mitas, Michael C. Mitas Department of Surgery, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this authorUzair B. Chaudhary, Uzair B. Chaudhary Department of Medicine, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this authorDavid T. Marshall, David T. Marshall Department of Radiation Oncology, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this authorSebastiano Gattoni-Celli, Sebastiano Gattoni-Celli Department of Radiation Oncology, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this author Michael C. Mitas, Michael C. Mitas Department of Surgery, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this authorUzair B. Chaudhary, Uzair B. Chaudhary Department of Medicine, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this authorDavid T. Marshall, David T. Marshall Department of Radiation Oncology, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this authorSebastiano Gattoni-Celli, Sebastiano Gattoni-Celli Department of Radiation Oncology, Medical University of South Carolina, Charleston, South CarolinaSearch for more papers by this author First published: 27 August 2007 https://doi.org/10.1002/ajh.21045Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume83, Issue1January 2008Pages 87-87 ReferencesRelatedInformation

A59 Treatment of colon cancer patients would benefit from the development of an accurate prognostic test that could also serve to guide therapeutic decisions. In an effort to meet this goal, we sought to i) develop a prognostic quantitative real-time RT-PCR assay using formalin fixed paraffin-embedded primary tumors, and ii), develop an assay that could be used for high throughput screening of drugs that would either abolish or restore functional activity of a viable target identified in the prognostic assay. To achieve these two objectives, we first measured the expression levels of 14 cancer-associated genes in Stage II colon cancer patients who developed disease recurrence within two years (n=9), or those who did not develop disease recurrence (n=12, mean follow-up time = 6.3 yrs). We observed that the expression ratio of (EpCAM1+Map7)/E-cadherin accurately predicted clinical outcome (Hazard ratio = 12; P=0.001 log-ranked test), such that good patient survival was associated with high expression of E-cadherin, a gene that has been shown to be downregulated by Zeb1 during colon cancer progression. Interestingly, we discovered that whereas the expression of E-cadherin was inversely correlated with Zeb1 (p=1.4E-7) in the CGAP NCI60 database, the expression of EpCAM1 was inversely correlated with Zeb2 (p=1.9E-10). We further discovered that the promoter region of EpCAM1 contained a 16 nucleotide match to the region including and surrounding the Zeb1 response element of the p73 gene (p=8.7E-7). Based on these findings, we concluded that E-cadherin represented the most viable therapeutic target of the three genes identified in the prognostic assay. To enable screening for drugs that might restore E-cadherin function, we developed a 20 microliter adhesion assay using an uncoated polystyrene surface in which it was observed that following 7 hours after cell trypsinization, the level of adhesion of highly metastatic SW620 colon cancer cell line was 15-fold (+/-4) lower compared to poorly metastatic HCT116, a cell line which we determined contains 100- and 8-fold higher levels of E-cadherin mRNA and protein, respectively, compared to SW620. This adhesion assay may allow for the identification of novel compounds that inhibit the epithelial-mesenchymal transition required for spread of metastatic disease.