Amanda Graham

Accurate discrimination of pancreatic ductal adenocarcinoma and chronic pancreatitis using multimarker expression data and samples obtained by minimally invasive fine needle aspiration

To augment cytological diagnosis of pancreatic ductal adenocarcinoma (PDAC) in tissue samples obtained by minimally invasive endoscopic ultrasound‐guided fine needle aspiration, we investigated whether a small set of molecular markers could accurately distinguish PDAC from chronic pancreatitis (CP). Expression levels of 29 genes were first determined by quantitative real‐time RT‐PCR in a training set of tissues in which the final diagnosis was PDAC (n = 20) or CP (n = 10). Using receiver operator characteristic curve analysis, we determined that the single gene with the highest diagnostic accuracy for discrimination of CP vs. PDAC in the training study was urokinase plasminogen activator receptor (UPAR; AUC value = 0.895, 95% CI = 0.728–0.976). In the set of test tissues (n = 14), the accuracy of UPAR decreased to 79%. However, we observed that the addition of 6 genes (EPCAM2, MAL2, CEA5, CEA6, MSLN and TRIM29; referred to as the 6‐gene classifier) to UPAR resulted in high accuracy in both training and testing sets. Excluding 3 samples (out of 44; 7%) for which results of the UPAR/6‐gene classifier were “undefined,” the accuracy of the UPAR/6‐gene classifier was 100% in training samples (n = 29), 92% in 12 test samples (p = 0.004 that results were randomly generated; p = 0.046 that the UPAR/6‐gene classifier was comparable to UPAR alone; χ2 test), 100% in 3 samples for which the initial cytological diagnosis was “suspicious” and 98% (40/41) overall. Our results provide evidence that molecular marker expression data can be used to augment cytological analysis.

Accurate discrimination of Barrett's esophagus and esophageal adenocarcinoma using a quantitative three-tiered algorithm and multimarker real-time reverse transcription-PCR.

Esophageal adenocarcinoma (EA) is increasing faster than any other cancer in the U.S. In this report, we first show that EA can be distinguished from normal esophagus (NE) and esophageal squamous cell carcinoma by plotting expression values for EpCam, TFF1, and SBEM in three-dimensional Euclidean space. For monitoring progression of Barretts esophagus (BE) to EA, we developed a highly sensitive assay for limited quantities of tissue whereby 50 ng of RNA are first converted to cDNA using 16 gene-specific primers. Using a set of training tissues, we developed a novel quantitative three-tiered algorithm that allows for accurate (overall accuracy = 61/63, 97%) discrimination of BE versus EA tissues using only three genes. The gene used in the first tier of the algorithm is TSPAN: samples not diagnosed as BE or EA by TSPAN in the first tier are then subjected to a second-tier analysis using ECGF1, followed by a third-tier analysis using SPARC. Addition of TFF1 and SBEM to the first tier (i.e., a five-gene marker panel) increases the overall accuracy of the assay to 98% (62/63) and results in mean molecular diagnostic scores (± SD) that are significantly different between EA and BE samples (3.19 ± 1.07 versus −2.74 ± 1.73, respectively). Our results suggest that relatively few genes can be used to monitor progression of BE to EA.

β2microglobulin mRNA expression levels are prognostic for lymph node metastasis in colorectal cancer patients

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 β2microglobulin (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 R2 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.

A simple two-gene prognostic model for adenocarcinoma of the lung

OBJECTIVE We hypothesized that clinical outcome of resected early-stage adenocarcinoma of the lung can be predicted by the expression of a few critically important genes as measured by quantitative real-time reverse-transcriptase polymerase chain reaction in formalin-fixed paraffin-embedded primary tumors. METHODS Twenty-two prognostic genes for the metastatic phenotype were identified through complementary DNA microarray analysis of 4 cancer cell lines and bioinformatics analysis. Expression levels of a subset of these genes (n = 13) were measured by real-time time reverse-transcriptase polymerase chain reaction in formalin-fixed paraffin-embedded primary adenocarcinoma from patients whose disease recurred within 2 years (n = 9) and in patients who did not have a recurrence (n = 11). Receiver operating characteristic curves were analyzed to establish prognostic values of single genes. The most informative gene was combined with the remaining genes to determine whether there was a particular pair(s) that yielded high diagnostic accuracy. A small validation study was performed. RESULTS Receiver operating characteristic curve analysis of the single genes revealed that high expression of CK19 was associated with nonrecurrence (area under the curve = 0.859, confidence interval = 0.651-0.970). The CK19/EpCAM2 gene ratio had the most reproducible prognostic accuracy, followed by the CK19/P-cadherin ratio. A Kaplan-Meier survival analysis generated from the CK19/EpCAM2 ratio resulted in highly significant curves as a function of marker positivity (P = .0007; hazard ratio = 10.7). Significance declined but was maintained in the validation study. CONCLUSIONS This preliminary study provides evidence that the CK19/EpCAM2 and/or CK19/P-cadherin ratio(s) may be a simple and accurate prognostic indicator of clinical outcome in early-stage adenocarcinoma of the lung. If further validation studies from large patient cohorts confirm the results, adjuvant therapy could be targeted to this high-risk group.

Abstract 4334: Tracking of leukemic stem cells from frozen and fresh samples in a mouse model of human AML using bioluminescent imaging

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The aim of this study was to develop and use in vivo bioluminescent imaging to track luciferase-labeled acute myelogenous leukemia (AML) stem cells in immunedeficient mice using AML blood samples collected from patients and subsequently frozen. The ability to utilize frozen pools as viable reservoirs for AML stem cells will allow the freezing back of samples from the clinic for later use as well as the use of previously frozen samples for engraftment and therapeutic studies. Using thawed, isolated cells, we assessed AML stem cell engraftment and quantitatively compared it to fresh AML stem cell engraftment in a xenogeneic transplant model using in vivo bioluminescent imaging. Frozen AML stem cell engraftment was also compared to both fresh and frozen normal human hematopoietic stem cell (HSC) engraftment. Human hematopoietic stem cells (CD34+CD38-) were isolated from both normal frozen cord blood and from peripheral blood of AML patients using modified Miltenyi Biotec (Auburn, CA) magnetic bead isolation kits. Isolated cells were transduced with a lentiviral vector (HIV-luc) pseudotyped with the VSV envelope able to express the firefly luciferase (fluc) gene in human HSC. Using a Caliper Life Sciences/Xenogen IVIS 200 optical imaging system, cells were assayed for fluc expression 48 hours after transduction. Transduced HSC and AML cells (6 × 104 each) were transplanted intravenously into sublethally irradiated adult NOD/SCID/IL2Rγnull mice and mice were imaged serially from day 1 up to day 160 post transplantation. Observed bioluminescent signal and patterns from thawed AML stem cell engraftment are similar to our results obtained using fresh AML stem cells in mice. Luminescent signal from peripheral blood and spleen engraftment of frozen AML stem cells was detectable within one day post-transplantation. Both fresh and frozen AML stem cell populations exhibited diffuse whole body signal, indicating luciferase-labeled stem cells in the peripheral blood, similar to a hallmark of the disease in humans. The presence of blast cells consistent with AML disease was observed by blood smears taken from mice engrafted with frozen AML stem cells. To our knowledge, we have demonstrated for the first time that frozen AML stem cells can be efficiently labeled with a luciferase enzyme for dynamic tracking in vivo. This work has resulted in a useful animal model that will enable us to elucidate the engraftment patterns of AML cells in living animals, learn the dynamics of the interplay of different stem cell populations, and provide information on the efficacy of new treatments in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4334.