Steven A. Enkemann

Gene expression-based survival prediction in lung adenocarcinoma: A multi-site, blinded validation study

Although prognostic gene expression signatures for survival in early-stage lung cancer have been proposed, for clinical application, it is critical to establish their performance across different subject populations and in different laboratories. Here we report a large, training–testing, multi-site, blinded validation study to characterize the performance of several prognostic models based on gene expression for 442 lung adenocarcinomas. The hypotheses proposed examined whether microarray measurements of gene expression either alone or combined with basic clinical covariates (stage, age, sex) could be used to predict overall survival in lung cancer subjects. Several models examined produced risk scores that substantially correlated with actual subject outcome. Most methods performed better with clinical data, supporting the combined use of clinical and molecular information when building prognostic models for early-stage lung cancer. This study also provides the largest available set of microarray data with extensive pathological and clinical annotation for lung adenocarcinomas.

A sequence-based identification of the genes detected by probesets on the Affymetrix U133 plus 2.0 array

One of the biggest problems facing microarray experiments is the difficulty of translating results into other microarray formats or comparing microarray results to other biochemical methods. We believe that this is largely the result of poor gene identification. We re-identified the probesets on the Affymetrix U133 plus 2.0 GeneChip array. This identification was based on the sequence of the probes and the sequence of the human genome. Using the BLAST program, we matched probes with documented and postulated human transcripts. This resulted in the redefinition of approximately 37% of the probes on the U133 plus 2.0 array. This updated identification specifically points out where the identification is complicated by cross-hybridization from splice variants or closely related genes. More than 5000 probesets detect multiple transcripts and therefore the exact protein affected cannot be readily concluded from the performance of one probeset alone. This makes naming difficult and impacts any downstream analysis such as associating gene ontologies, mapping affected pathways or simply validating expression changes. We have now automated the sequence-based identification and can more appropriately annotate any array where the sequence on each spot is known.

Noninvasive Detection of Breast Cancer Lymph Node Metastasis Using Carbonic Anhydrases IX and XII Targeted Imaging Probes

Purpose: To develop targeted molecular imaging probes for the noninvasive detection of breast cancer lymph node metastasis. Experimental Design: Six cell surface or secreted markers were identified by expression profiling and from the literature as being highly expressed in breast cancer lymph node metastases. Two of these markers were cell surface carbonic anhydrase isozymes (CAIX and/or CAXII) and were validated for protein expression by immunohistochemistry of patient tissue samples on a breast cancer tissue microarray containing 47 normal breast tissue samples, 42 ductal carcinoma in situ, 43 invasive ductal carcinomas without metastasis, 46 invasive ductal carcinomas with metastasis, and 49 lymph node macrometastases of breast carcinoma. Targeted probes were developed by conjugation of CAIX- and CAXII-specific monoclonal antibodies to a near-infrared fluorescent dye. Results: Together, these two markers were expressed in 100% of the lymph node metastases surveyed. Selectivity of the imaging probes were confirmed by intravenous injection into nude mice-bearing mammary fat pad tumors of marker-expressing cells and nonexpressing cells or by preinjection of unlabeled antibody. Imaging of lymph node metastases showed that peritumorally injected probes detected nodes harboring metastatic tumor cells. As few as 1,000 cells were detected, as determined by implanting, under ultrasound guidance, a range in number of CAIX- and CAXII-expressing cells into the axillary lymph nodes. Conclusion: These imaging probes have potential for noninvasive staging of breast cancer in the clinic and elimination of unneeded surgery, which is costly and associated with morbidities. Clin Cancer Res; 18(1); 207–19. ©2011 AACR.

Dedifferentiation Precedes Invasion in the Progression from Barrett's Metaplasia to Esophageal Adenocarcinoma

Purpose: Adenocarcinoma arises in Barretts esophagus by progression from metaplasia to cancer through grades of dysplasia. Our aim in this exploratory study was to characterize the broad changes in gene expression that underlie this histologic progression to cancer and assess the potential for using these gene expression changes as a marker predictive of malignant progression in Barretts epithelium. Experimental Design: Microarray analysis was used to obtain individual gene expression profiles from endoscopic biopsies of nine esophageal adenocarcinomas and the Barretts epithelia from which three of the cancers had arisen. Pooled samples from the Barretts epithelia of six patients without cancer or dysplasia served as a reference. Results: Barretts epithelia from which cancer had arisen differed from the reference Barretts epithelia primarily by underexpression of genes, many of which function in governing cell differentiation. These changes in gene expression were found even in those specimens of Barretts epithelia from which cancer had arisen that lacked dysplasia. Each cancer differed from the Barretts epithelium from which it had arisen primarily by an overexpression of genes, many of which were associated with tissue remodeling and invasiveness. Cancers without identifiable Barretts epithelium differed from cancers that had arisen from a Barretts epithelium by having an even greater number of these overexpressed genes. Conclusions: Histologic progression from Barretts epithelium to cancer is associated with a gradient of increasing changes in gene expression characterized by an early loss of gene function governing differentiation that begins before histologic change; gain in function of genes related to remodeling and invasiveness follows later. This correlation of histologic progression with increasing changes in gene expression suggests that gene expression changes in biopsies taken from Barretts epithelium potentially could serve as a marker for neoplastic progression that could be used to predict risk for developing cancer.

Lung tumor NF-κB signaling promotes T cell–mediated immune surveillance

NF-κB is constitutively activated in many cancer types and is a potential key mediator of tumor-associated inflammation, tumor growth, and metastasis. We investigated the role of cancer cell NF-κB activity in T cell-mediated antitumor responses. In tumors rendered immunogenic by model antigen expression or following administration of antitumor vaccines, we found that high NF-κB activity leads to tumor rejection and/or growth suppression in mice. Using a global RNA expression microarray, we demonstrated that NF-κB enhanced expression of several T cell chemokines, including Ccl2, and decreased CCL2 expression was associated with enhanced tumor growth in a mouse lung cancer model. To investigate NF-κB function in human lung tumors, we identified a gene expression signature in human lung adenocarcinoma cell lines that was associated with NF-κB activity level. In patient tumor samples, overall lung tumor NF-κB activity was strongly associated with T cell infiltration but not with cancer cell proliferation. These results therefore indicate that NF-κB activity mediates immune surveillance and promotes antitumor T cell responses in both murine and human lung cancer.

Characterization of a R115777-Resistant Human Multiple Myeloma Cell Line with Cross-Resistance to PS-341

The farnesyl transferase inhibitor R115777 has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777-resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines, implying that farnesyl transferase (the drug target) has not been mutated. Whereas many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein, we found that P-glycoprotein expression is not increased in the 8226/R5 line and intracellular accumulation of R115777 was not reduced. In fact, 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS-341, and multidrug resistance did not correlate with the expression of heat shock proteins. Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Future experiments will attempt to identify genes that are directly linked to the resistant phenotype. Identification of molecules associated with R115777 and PS-341 resistance is clinically relevant because both compounds are being tested in solid tumors and hematopoietic malignancies.

A Mammaglobin-A Targeting Agent for Noninvasive Detection of Breast Cancer Metastasis in Lymph Nodes

Pathologic axillary lymph node (ALN) status is an important prognostic factor for staging breast cancer. Currently, status is determined by histopathology following surgical excision of sentinel lymph node(s), which is an invasive, time consuming, and costly procedure with potential morbidity to the patient. Here, we describe an imaging platform for noninvasive assessment of ALN status, eliminating the need for surgical examination of patients to rule out nodal involvement. A targeted imaging probe (MamAb-680) was developed by conjugation of a mammaglobin-A-specific monoclonal antibody to a near-infrared fluorescent dye. Using DNA and tissue microarray, mammaglobin-A was validated as a cell-surface target that is expressed in ALN-positive patient samples but is not expressed in normal lymph nodes. In vivo selectivity was determined by i.v. injection of MamAb-680 into mice with mammaglobin-A-positive and -negative mammary fat pad (MFP) tumors; and by peritumoral MFP injection of the targeted imaging probe in mice with spontaneous ALN metastases. Fluorescence imaging showed that probe was only retained in positive tumors and metastases. As few as 1,000 cells that endogenously express mammaglobin-A were detected in ALN, indicating high sensitivity of this method. Translation of this approach offers considerable potential as a noninvasive clinical strategy to stage breast cancer.

Identification of novel pancreatic adenocarcinoma cell-surface targets by gene expression profiling and tissue microarray

Pancreatic cancer has a high mortality rate, which is generally related to the initial diagnosis coming at late stage disease combined with a lack of effective treatment options. Novel agents that selectively detect pancreatic cancer have potential for use in the molecular imaging of cancer, allowing for non-invasive determination of tumor therapeutic response and molecular characterization of the disease. Such agents may also be used for the targeted delivery of therapy to tumor cells while decreasing systemic effects. Using complementary assays of mRNA expression profiling to determine elevated expression in pancreatic cancer tissues relative to normal pancreas tissues, and validation of protein expression by immunohistochemistry on tissue microarray, we have identified cell-surface targets with potential for imaging and therapeutic agent development. Expression profiles of 2177 cell-surface genes for 28 pancreatic tumor specimens and 4 normal pancreas tissue samples were evaluated. Expression in normal tissues was evaluated using array data from 103 samples representing 28 organ sites as well as mining published data. One-hundred seventy unique targets were highly expressed in 2 or more of the pancreatic tumor specimens and were not expressed in the normal pancreas samples. Two targets (TLR2 and ABCC3) were further validated for protein expression by tissue microarray (TMA) based immunohistochemistry. These validated targets have potential for the development of diagnostic imaging and therapeutic agents for pancreatic cancer.

The Impact of Genomics in Understanding Human Melanoma Progression and Metastasis

BACKGROUND Recent technological advances in the analysis of the human genome have opened the door to improving our primitive understanding of the gene expression patterns in cancer. For the first time, we have an overview of the complexities of tumorigenesis and metastatic progression of cancer. The examination of the phenotypic and (epi)genetic changes in cutaneous melanoma has identified several genes deemed central to the development and progression of melanoma. METHODS A review of the recent literature was performed to determine the role of array-based high-throughput gene expression analysis in understanding the specific genes involved as well as the pathways and the comparative gene expression patterns of primary and metastatic melanoma. RESULTS Most studies utilizing gene microarray analysis and other whole genome approaches reveal a wide array of genes and expression patterns in human melanoma. Furthermore, several of the same genes have been found in comparative studies, with some studies attempting correlation with clinical outcome. Several genes have been identified as potential prognostic markers of tumor progression and overall clinical outcome. CONCLUSIONS High-throughput gene expression analysis has had a major impact in melanoma research. Several gene expression platforms have provided insight into the gene expression patterns in melanoma. Such data will provide the foundations for the future development of prognostic markers and improved targeted therapies for patients with melanoma.

Transcriptional Response of the Sulfur Chemolithoautotroph Thiomicrospira crunogena to Dissolved Inorganic Carbon Limitation

The hydrothermal vent gammaproteobacterium Thiomicrospira crunogena inhabits an unstable environment and must endure dramatic changes in habitat chemistry. This sulfur chemolithoautotroph responds to changes in dissolved inorganic carbon (DIC) (DIC = CO(2) + HCO(3)(-) + CO(3)(-2)) availability with a carbon-concentrating mechanism (CCM) in which whole-cell affinity for DIC, as well as the intracellular DIC concentration, increases substantially under DIC limitation. To determine whether this CCM is regulated at the level of transcription, we resuspended cells that were cultivated under high-DIC conditions in chemostats in growth medium with low concentrations of DIC and tracked CCM development in the presence and absence of the RNA polymerase inhibitor rifampin. Induction of the CCM, as measured by silicone oil centrifugation, was hindered in the presence of rifampin. Similar results were observed for carboxysome gene transcription and assembly, as assayed by quantitative reverse transcription-PCR (qRT-PCR) and transmission electron microscopy, respectively. Genome-wide transcription patterns for cells grown under DIC limitation and those grown under ammonia limitation were assayed via microarrays and compared. In addition to carboxysome genes, two novel genes (Tcr_1019 and Tcr_1315) present in other organisms, including chemolithoautotrophs, but whose function(s) has not been elucidated in any organism were found to be upregulated under low-DIC conditions. Likewise, under ammonia limitation, in addition to the expected enhancement of ammonia transporter and P(II) gene transcription, the transcription of two novel genes (Tcr_0466 and Tcr_2018) was measurably enhanced. Upregulation of all four genes (Tcr_1019, 4-fold; Tcr_131, ∼7-fold; Tcr_0466, >200-fold; Tcr_2018, 7-fold), which suggests that novel components are part of the response to nutrient limitation by this organism, was verified via qRT-PCR.