Heidi S. Erickson

Molecular Alterations in Primary Prostate Cancer after Androgen Ablation Therapy

Purpose: After an initial response to androgen ablation, most prostate tumors recur, ultimately progressing to highly aggressive androgen-independent cancer. The molecular mechanisms underlying progression are not well known in part due to the rarity of androgen-independent samples from primary and metastatic sites. Experimental Design: We compared the gene expression profiles of 10 androgen-independent primary prostate tumor biopsies with 10 primary, untreated androgen-dependent tumors. Samples were laser capture microdissected, the RNA was amplified, and gene expression was assessed using Affymetrix Human Genome U133A GeneChip. Differential expression was examined with principal component analysis, hierarchical clustering, and Students t testing. Analysis of gene ontology was done with Expression Analysis Systematic Explorer and gene expression data were integrated with genomic alterations with Differential Gene Locus Mapping. Results: Unsupervised principal component analysis showed that the androgen-dependent and androgen-independent tumors segregated from one another. After filtering the data, 239 differentially expressed genes were identified. Two main gene ontologies were found discordant between androgen-independent and androgen-dependent tumors: macromolecule biosynthesis was down-regulated and cell adhesion was up-regulated in androgen-independent tumors. Other differentially expressed genes were related to interleukin-6 signaling as well as angiogenesis, cell adhesion, apoptosis, oxidative stress, and hormone response. The Differential Gene Locus Mapping analysis identified nine regions of potential chromosomal deletion in the androgen-independent tumors, including 1p36, 3p21, 6p21, 8p21, 11p15, 11q12, 12q23, 16q12, and 16q21. Conclusions: Taken together, these data identify several unique characteristics of androgen-independent prostate cancer that may hold potential for the development of targeted therapeutic intervention.

Robust gene expression signature from formalin-fixed paraffin-embedded samples predicts prognosis of non-small-cell lung cancer patients.

Purpose: The requirement of frozen tissues for microarray experiments limits the clinical usage of genome-wide expression profiling by using microarray technology. The goal of this study is to test the feasibility of developing lung cancer prognosis gene signatures by using genome-wide expression profiling of formalin-fixed paraffin-embedded (FFPE) samples, which are widely available and provide a valuable rich source for studying the association of molecular changes in cancer and associated clinical outcomes. Experimental Design: We randomly selected 100 Non–Small-Cell lung cancer (NSCLC) FFPE samples with annotated clinical information from the UT-Lung SPORE Tissue Bank. We microdissected tumor area from FFPE specimens and used Affymetrix U133 plus 2.0 arrays to attain gene expression data. After strict quality control and analysis procedures, a supervised principal component analysis was used to develop a robust prognosis signature for NSCLC. Three independent published microarray datasets were used to validate the prognosis model. Results: This study showed that the robust gene signature derived from genome-wide expression profiling of FFPE samples is strongly associated with lung cancer clinical outcomes and can be used to refine the prognosis for stage I lung cancer patients, and the prognostic signature is independent of clinical variables. This signature was validated in several independent studies and was refined to a 59-gene lung cancer prognosis signature. Conclusions: We conclude that genome-wide profiling of FFPE lung cancer samples can identify a set of genes whose expression level provides prognostic information across different platforms and studies, which will allow its application in clinical settings. Clin Cancer Res; 17(17); 5705–14. ©2011 AACR.

Kinase-Impaired BRAF Mutations in Lung Cancer Confer Sensitivity to Dasatinib

Induction of tumor cell senescence may explain the response of a patient with BRAF kinase–impaired lung cancer to the multikinase inhibitor dasatinib. A Lucky Break with BRAF The prognosis for those with metastatic non–small cell lung cancer (NSCLC) is bleak—the median survival time is measured in months. Therapeutic benefits have been achieved with targeted drugs in subpopulations of NSCLC patients with specific mutations, but the genetic changes responsible for this disease are undefined in most cases. Understanding why certain tumors respond to a given treatment might help determine useful therapeutic targets. Sen et al. now describe a striking case—the mutation responsible for the strong response of one patient with metastatic NSCLC to treatment with the tyrosine kinase inhibitor dasatinib. In a previous clinical trial of dasatinib treatment for metastatic NSCLC that lasted for 12 weeks, only a single patient responded to treatment; his tumor shrank and continued to shrink after treatment ended. Four years later, he appears free of active cancer. The researchers analyzed this patient’s tumor tissue and did not detect mutations that had been associated with NSCLC in other patients, but did find a mutation in the serine-threonine kinase BRAF that markedly impaired its kinase activity. (In contrast, another well-characterized oncogenic mutation in BRAF is kinase-activating.) Sen et al. found that in NSCLC cell lines with other kinase-inactivating BRAF mutations, dasatinib induced largely irreversible senescence—cell cycle arrest. Overexpression of kinase-active BRAF, however, increased dasatinib resistance in these cells, indicating that the inactive BRAF kinase was required for their dasatinib sensitivity. Furthermore, treatment of dasatinib-resistant cancer cells that express wild-type BRAF with a BRAF inhibitor increased their sensitivity to dasatinib. Exactly how dasatinib induces senescence in NSCLC cells with kinase-impaired BRAF is not yet clear, but the finding opens new possibilities for treatment. Cancers in which BRAF is impaired may respond well to dasatinib; more broadly, dasatinib in combination with BRAF inhibitors may be useful for treating tumors that express wild-type BRAF. During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non–small cell lung cancer (NSCLC), one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase-inactivating BRAF mutation (Y472CBRAF); no inactivating BRAF mutations were found in the nonresponding tumors taken from other patients. Cells transfected with Y472CBRAF exhibited CRAF, MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), and ERK (extracellular signal–regulated kinase) activation—characteristics identical to signaling changes that occur with previously known kinase-inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells’ dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by Y472CBRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization, resulting in ERK activation in NSCLC cells with kinase-inactivating BRAF mutations. The sensitivity of NSCLC with kinase-impaired BRAF to dasatinib suggested synthetic lethality of BRAF and an unknown dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells’ dasatinib sensitivity. Thus, the patient’s BRAF mutation was likely responsible for his tumor’s marked response to dasatinib, suggesting that tumors bearing kinase-impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF.

Uncommon epidermal growth factor receptor mutations in non-small cell lung cancer and their mechanisms of EGFR tyrosine kinase inhibitors sensitivity and resistance.

Therapy targeted against the epidermal growth factor receptor (EGFR) has demonstrated dramatic tumor responses and favorable clinical outcomes in a select group of non-small cell lung cancer (NSCLC) patients whose tumors harbor EGFR activating mutations. The best characterized of the mutations conferring sensitivity to EGFR tyrosine kinase inhibitors (TKIs) are deletions in exon 19 and a point mutation in exon 21 (L858R). Likewise, the most common mutation that confers resistance is the T790M point mutation. However several other mutations have been reported and several have been characterized as regards their role in sensitivity or resistance to EGFR TKIs. Resistance to the EGFR TKIs erlotinib and gefitinib, and the newer irreversible EGFR TKIs is a problem of fundamental importance. Recognition of the presence and significance of specific EGFR mutations is important for appropriate therapeutic implementation of EGFR TKIs and research and development of mutation-specific inhibitors. We summarize the literature and present an overview of the subject of less common EGFR mutations and their clinical significance, with an emphasis on EGFR TKI sensitivity or resistance.

Identification of unique expression signatures and therapeutic targets in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is characterized by high mortality. Previous work identified important mRNA expression differences between normal and tumor cells; however, to date there are limited ex vivo studies examining expression changes occurring during normal esophageal squamous cell differentiation versus those associated with tumorigenesis. In this study, we used a unique tissue microdissection strategy and microarrays to measure gene expression profiles associated with cell differentiation versus tumorigenesis in twelve cases of patient-matched normal basal squamous epithelial cells (NB), normal differentiated squamous epithelium (ND), and squamous cell cancer. Class comparison and pathway analysis were used to compare NB versus tumor in a search for unique therapeutic targets.ResultsAs a first step towards this goal, gene expression profiles and pathways were evaluated. Overall, ND expression patterns were markedly different from NB and tumor; whereas, tumor and NB were more closely related. Tumor showed a general decrease in differentially expressed genes relative to NB as opposed to ND that exhibited the opposite trend. FSH and IgG networks were most highly dysregulated in normal differentiation and tumorigenesis, respectively. DNA repair pathways were generally elevated in NB and tumor relative to ND indicating involvement in both normal and pathological growth. PDGF signaling pathway and 12 individual genes unique to the tumor/NB comparison were identified as therapeutic targets, and 10 associated ESCC gene-drug pairs were identified. We further examined the protein expression level and the distribution patterns of four genes: ODC1, POSTN, ASPA and IGF2BP3. Ultimately, three genes (ODC1, POSTN, ASPA) were verified to be dysregulated in the same pattern at both the mRNA and protein levels.ConclusionsThese data reveal insight into genes and molecular pathways mediating ESCC development and provide information potentially useful in designing novel therapeutic interventions for this tumor type.

Measuring molecular biomarkers in epidemiologic studies: laboratory techniques and biospecimen considerations

The future of personalized medicine depends on the ability to efficiently and rapidly elucidate a reliable set of disease-specific molecular biomarkers. High-throughput molecular biomarker analysis methods have been developed to identify disease risk, diagnostic, prognostic, and therapeutic targets in human clinical samples. Currently, high throughput screening allows us to analyze thousands of markers from one sample or one marker from thousands of samples and will eventually allow us to analyze thousands of markers from thousands of samples. Unfortunately, the inherent nature of current high throughput methodologies, clinical specimens, and cost of analysis is often prohibitive for extensive high throughput biomarker analysis. This review summarizes the current state of high throughput biomarker screening of clinical specimens applicable to genetic epidemiology and longitudinal population-based studies with a focus on considerations related to biospecimens, laboratory techniques, and sample pooling.

2D-PCR: a method of mapping DNA in tissue sections

A novel approach was developed for mapping the location of target DNA in tissue sections. The method combines a high-density, multi-well plate with an innovative single-tube procedure to directly extract, amplify, and detect the DNA in parallel while maintaining the two-dimensional (2D) architecture of the tissue. A 2D map of the gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was created from a tissue section and shown to correlate with the spatial area of the sample. It is anticipated that this approach may be easily adapted to assess the status of multiple genes within tissue sections, yielding a molecular map that directly correlates with the histology of the sample. This will provide investigators with a new tool to interrogate the molecular heterogeneity of tissue specimens.

High Throughput Screening of Normal and Neoplastic Tissue Samples

The capacity to rapidly and efficiently elucidate a reliable set of disease specific biomarkers is paramount to enable a future of personalized medicine. High throughput screening methods applied to human clinical samples for the discovery of diagnostic, prognostic, and therapeutic targets address this need. Although the ability to analyze either thousands of markers from one sample or one marker from thousands of samples is the current state of high throughput screening, it would be ideal to have the ability to analyze thousands of markers from thousands of samples to expedite the early discovery phase of biomarkers and their validation. This review summarizes the current state of high throughput screening of tissue specimens and discusses its applications. In addition, the rationale, difficulties, strategies, and development of new technologies to address the need for improved high throughput capabilities are discussed.

Abstract 272: Nanoscale high-throughput quantitative RT-PCR for the characterization of targeted-therapy related molecular biomarkers from recurrent and non-recurrent NSCLC tissues

Currently, there are no good biomarkers to predict targeted therapy outcomes for patients whose cancers recur vs. those that do not. Technology limitations have prevented extensive qRT-PCR biomarker analysis on small tumor tissue samples obtained in our unique lung cancer MD Anderson BATTLE clinical trials. Clinical sample size limitations are inherent due to the extremely small core needle biopsies (CNB) and fine needle aspirates (FNA). Identifying technology that can reliably analyze, in a quantitative manner, molecular biomarker signatures of 200-300 genes from CNB and FNA, will allow for comparative analysis of patients’ untreated tumor with treated follow-up tissue samples. We hypothesize RNA amplification coupled with nanoscale high-throughput (HT) qRT-PCR, will allow analysis of 320 gene expression patterns from 10ng RNA from frozen NSCLC tissues. We believe primary NSCLC tumors that recur have a different pattern of molecular abnormalities than NSCLCs from non-recurrent tumors ( Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 272. doi:1538-7445.AM2012-272

Interrogation of Chromosome 13q12-14 in Esophageal Squamous Cell Carcinoma

Previous studies of esophageal squamous cell carcinoma (ESCC) suggested chromosome region 13q12-14 harbors a familial ESCC gene. DNA sequencing of the BRCA2 gene, located on 13q12, showed evidence of both germline and tumor specific alterations but the frequency of changes was low and did not fit the classic Knudsen two-hit gene inactivation model. To further investigate chromosome 13q12-14 in ESCC, quantitative expression measurements were performed on BRCA2 and 11 neighboring genes in matched normal epithelium and tumor from 17 cases. Transcript analysis showed normal levels of five genes, tumor down-regulation of two genes (TNFRS19 and TPT1), and tumor up- regulation of five genes, including BRCA2. No evidence of BRCA2 loss-of-function was detected based on reduced mRNA in tumor cells. Between 13q12.3 (KATNAL1) and 13q12.3-q13 (CCNA1) five adjacent genes showed increased mRNA expression raising the possibility of a DNA amplicon; however, qPCR analysis showed normal DNA amounts in this region. CCNA1 transcript was significantly up-regulated in tumors and was thus further interrogated at the protein level by immunohistochemistry. CCNA1 staining was restricted to normal basal epithelium and was not expressed in more superficial, differentiated regions. In contrast, the CCNA1 protein was ubiquitously and highly expressed throughout tumor foci. Overall, these data from a relatively small number of cases (17) suggest that TNFRS19 and TPT1 deserve further investigation as candidate tumor suppressor genes in esophageal cancer in a larger patient series; BRCA2 mRNA is increased in the tumors, likely as a compensatory response to the marked DNA damage that is present in these lesions; and, CCNA1 was identified as a novel up-regulated gene in ESCC.