Environmental epidemiology and risk assessment: Exploring a path to increased confidence in public health decision-making.

Throughout history, environmental epidemiology has proven crucial to identify certain threats to human health and to provide a basis for the development of life-saving public health policies. However, epidemiologists are facing challenges when studying tenuous threats such as environmental exposure to chemicals, whose association with adverse health effects may be difficult to characterize. As a result, epidemiological data can seldom be fully leveraged for quantitative risk assessment and decision-making. Despite two decades of efforts to improve a more systematic integration of human data to evaluate human health risks, assessors still heavily rely on animal data to do so, while epidemiology plays more of a secondary role. Although the need for more and better collaboration between risk assessors and epidemiologists is widely recognized, both fields tend to remain siloed. In 2017, the Health and Environmental Sciences Institute initiated a project engaging the epidemiology, exposure science, and regulatory communities with tripartite representation from regulators, industry, and academia in a dialogue on the use of environmental epidemiology for regulatory decision-making. Several focus groups attended by epidemiology, exposure science, and risk assessment experts were organized to explore incentives and barriers to collaboration, to ultimately bridge the gap between the various disciplines, and to realize the full potential of epidemiological data in risk assessment. Various ideas that have emerged from these meetings could help ensure the better integration of epidemiological data in quantitative risk assessment and contribute to building confidence in a robust and science-based regulatory decision-making process.

Menin immunoreactivity in secretory granules of human pancreatic islet cells.

The protein product of the Multiple Endocrine Neoplasia Type I (MEN1) gene is thought to be involved in predominantly nuclear functions; however, immunohistochemical (IHC) analysis data on cellular localization are conflicting. To further investigate menin expression, we analyzed human pancreas (an MEN1 target organ) using IHC analyses and 6 antibodies raised against full-length menin or its peptides. In 10 normal pancreas specimens, 2 independently raised antibodies showed unexpected cytoplasmic immunoreactivity in peripheral cells in each islet examined (over 100 total across all 10 patients). The staining exhibited a distinct punctate pattern and subsequent immunoelectron microscopy indicated the target antigen was in secretory granules. Exocrine pancreas and pancreatic stroma were not immunoreactive. In MEN1 patients, unaffected islets stained similar to those in normal samples but with a more peripheral location of positive cells, whereas hyperplastic islets and tumorlets showed increased and diffuse cytoplasmic staining, respectively. Endocrine tumors from MEN1 patients were negative for menin, consistent with a 2-hit loss of a tumor suppressor gene. Secretory granule localization of menin in a subset of islet cells suggests a function of the protein unique to a target organ of familial endocrine neoplasia, although the IHC data must be interpreted with some caution because of the possibility of antibody cross-reaction. The identity, cellular trafficking, and role of this putative secretory granule-form of menin warrant additional investigation.

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.

Three-dimensional mRNA measurements reveal minimal regional heterogeneity in esophageal squamous cell carcinoma.

The classic tumor clonal evolution theory postulates that cancers change over time to produce unique molecular subclones within a parent neoplasm, presumably including regional differences in gene expression. More recently, however, this notion has been challenged by studies showing that tumors maintain a relatively stable transcript profile. To examine these competing hypotheses, we microdissected discrete subregions containing approximately 3000 to 8000 cells (500 to 1500 µm in diameter) from ex vivo esophageal squamous cell carcinoma (ESCC) specimens and analyzed transcriptomes throughout three-dimensional tumor space. Overall mRNA profiles were highly similar in all 59 intratumor comparisons, in distinct contrast to the markedly different global expression patterns observed in other dissected cell populations. For example, normal esophageal basal cells contained 1918 and 624 differentially expressed genes at a greater than twofold level (95% confidence level of <5% false positives), compared with normal differentiated esophageal cells and ESCC, respectively. In contrast, intratumor regions had only zero to four gene changes at a greater than twofold level, with most tumor comparisons showing none. The present data indicate that, when analyzed using a standard array-based method at this level of histological resolution, ESCC contains little regional mRNA heterogeneity.

Kinase-impaired BRAF mutations in lung cancer confer sensitivity to dasatinib.

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 ((Y472C)BRAF); no inactivating BRAF mutations were found in the nonresponding tumors taken from other patients. Cells transfected with (Y472C)BRAF 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 (Y472C)BRAF 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.

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.

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

BACKGROUND: Esophageal 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. RESULTS: As 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. CONCLUSIONS: These 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.

Squamous Cell Carcinoma - Similarities and Differences among Anatomical Sites.

Squamous cell carcinoma (SCC) is an epithelial malignancy involving many anatomical sites and is the most common cancer capable of metastatic spread. Development of early diagnosis methods and novel therapeutics are important for prevention and mortality reduction. In this effort, numerous molecular alterations have been described in SCCs. SCCs share many phenotypic and molecular characteristics, but they have not been extensively compared. This article reviews SCC as a disease, including: epidemiology, pathology, risk factors, molecular characteristics, prognostic markers, targeted therapy, and a new approach to studying SCCs. Through this comparison, several themes are apparent. For example, HPV infection is a common risk factor among the four major SCCs (NMSC, HNSC, ESCC, and NSCLC) and molecular abnormalities in cell-cycle regulation and signal transduction predominate. These data reveal that the molecular insights, new markers, and drug targets discovered in individual SCCs may shed light on this type of cancer as a whole.

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.

MicroRNA analysis of microdissected normal squamous esophageal epithelium and tumor cells.

Previous studies have identified several dysregulated microRNAs in esophageal squamous cell carcinoma (ESCC); however, to date there are no ex vivo analyses comparing expression levels of these regulatory molecules in esophageal squamous cell tumors versus patient-matched normal epithelium. We describe here a technical strategy to evaluate microRNAs in normal esophageal basal cells (NB), normal esophageal differentiated cells (ND), and tumor cells (T). Laser capture microdissection was used to procure target populations from five cases and 18 ESCC-associated microRNAs were measured by RT-qPCR. Five microRNAs (miR-25, miR-106b, miR-21, miR-203, and miR-145) demonstrated consistent differential expression in at least one of the three comparisons: T vs. NB, T vs. ND, or NB vs. ND. The potential regulatory role of the microRNAs in ESCC was further evaluated by correlating their expression with a matched mRNA dataset, which included the same five cases and cell populations. In conclusion, the present work demonstrates the feasibility of studying microRNA levels in precisely dissected cell populations from clinical samples, and sheds light on the molecular mechanisms associated with ESCC.

Increased matrix metalloproteinase activation in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC. METHODS: Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RESULTS: Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment. CONCLUSIONS: Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.

Influence of hypoxia induced by minimally invasive prostatectomy on gene expression: implications for biomarker analysis.

Handling and processing of clinical specimens during and after surgical resection may significantly skew the molecular data obtained from analysis of those samples. Minimally invasive prostatectomy was used as a model to specifically study effects of surgical ischemia on gene expression in human clinical samples. Normal prostatic urethra cup biopsies were procured from 12 patients at three time points during laparoscopic radical prostatectomy. Homogeneous cells (stroma and epithelium) were microdissected. Transcript analysis of 3 oxygen-dependent, 3 oxygen-independent, and 3 control class genes was performed using quantitative RT-PCR. Data were analyzed by relative quantitation and two-sided t-test. Patient demographic and time covariates were fit by a linear mixed model. VEGF, an oxygen-dependent gene, showed significant expression alterations across three time points in epithelium (p=0.008), but not in stroma (p=0.66). Expression levels of VHL, STAT5B, and CYPA showed significant changes at the p<0.05 level in the stroma only. Effects of age, PSA, prostate size, Gleason score, surgery type, total surgery time, total ischemia time, and estimated blood loss on VEGF expression over time were not significant at the p<0.01 level. Therefore, surgical manipulation and tissue processing methods need to be taken into account when assessing prostatic biomarkers; however, resection does not dramatically alter mRNA profiles in prostate 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.

Identification of EpCAM as a molecular target of prostate cancer stroma.

To delineate the molecular changes that occur in the tumor microenvironment, we previously performed global transcript analysis of human prostate cancer specimens using tissue microdissection and expression microarrays. Epithelial and stromal compartments were individually studied in both tumor and normal fields. Tumor-associated stroma showed a distinctly different expression pattern compared with normal stroma, having 44 differentially expressed transcripts, the majority of which were up-regulated. In the present study, one of the up-regulated transcripts, epithelial cell adhesion activating molecule, was further evaluated at the protein level in 20 prostate cancer cases using immunohistochemistry and a histomathematical analysis strategy. The epithelial cell adhesion activating molecule showed a 76-fold expression increase in the tumor-associated stroma, as compared with matched normal stroma. Moreover, Gleason 4 or 5 tumor stroma was increased 170-fold relative to matched normal stroma, whereas the Gleason 3 tumor area showed only a 36-fold increase, indicating a positive correlation with Gleason tumor grade. Since the stromal compartment may be particularly accessible to vascular-delivered agents, epithelial cell adhesion activating molecule could become a valuable molecular target for imaging or treatment of prostate cancer.

Quantitative RT-PCR gene expression analysis of laser microdissected tissue samples.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a valuable tool for measuring gene expression in biological samples. However, unique challenges are encountered when studies are performed on cells microdissected from tissues derived from animal models or the clinic, including specimen-related issues, variability of RNA template quality and quantity, and normalization. qRT-PCR using small amounts of mRNA derived from dissected cell populations requires adaptation of standard methods to allow meaningful comparisons across sample sets. The protocol described here presents the rationale, technical steps, normalization strategy and data analysis necessary to generate reliable gene expression measurements of transcripts from dissected samples. The entire protocol from tissue microdissection through qRT-PCR analysis requires approximately 16 h.

Tissue microdissection.

Procurement of pure populations of cells from heterogeneous histological sections can be accomplished utilizing tissue microdissection. At present, a variety of different manual and laser-based dissection tools are available and each method has particular strengths and weaknesses. The types of biomolecular analyses that can be performed on microdissected cells depend not only on the method of cell procurement, but also on the effects of upstream tissue handling and processing. Tissue preparation protocols include two major approaches; snap-freezing, or, fixation and embedding. Snap-freezing generally provides the best quality tissue for subsequent study, including proteomic analyses such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Tissue fixatives include either precipitating reagents or biomolecular cross-linkers. The fixed samples are then further processed and embedded in a wax medium. In general, the biomolecules recovered from fixed and embedded tissue specimens are lower in both quantity and quality than those from snap-frozen specimens, although they are useful for certain types of analyses. The protocols provided here for tissue handling and processing, preparation of tissue sections, and microdissection are derived from our experience at the Pathogenetics Unit of the National Cancer Institute.

Global expression analysis of prostate cancer-associated stroma and epithelia.

Characterization of gene expression profiles in tumor cells and the tumor microenvironment is an important step in understanding neoplastic progression. To date, there are limited data available on expression changes that occur in the tumor-associated stroma as either a cause or consequence of cancer. In the present study, we employed a 54,000 target oligonucleotide microarray to compare expression profiles in the 4 major components of the microenvironment: tumor epithelium, tumor-associated stroma, normal epithelium, and normal stroma. Cells from 5 human, whole-mount prostatectomy specimens were microdissected and the extracted and amplified mRNA was hybridized to an Affymetrix Human Genome U133 Plus 2.0 GeneChip. Using the intersection of 2 analysis methods, we identified sets of differentially expressed genes among the 4 components. Forty-four genes were found to be consistently differentially expressed in the tumor-associated stroma; 35 were found in the tumor epithelium. Interestingly, the tumor-associated stroma showed a predominant up-regulation of transcripts compared with normal stroma, in sharp contrast to the overall down-regulation seen in the tumor epithelium relative to normal epithelium. These data provide insight into the molecular changes occurring in tumor-associated stromal cells and suggest new potential targets for future diagnostic, imaging, or therapeutic intervention.

Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples.

Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.

Layered peptide array for multiplex immunohistochemistry.

Layered peptide array is a new methodology for multiplex molecular measurements from two-dimensional life science platforms. The technology can be used in several different configurations depending on the needs of the investigator. Described here is an indirect layered peptide array (iLPA) that is capable of measuring proteins on a solid surface, such as target antigens on a tissue section. A prototype iLPA system was developed and subsequently examined for reproducibility and specificity and then compared with standard immunohistochemistry. Semiquantitative, multiplex proteomic analysis of histological sections was achieved with up to 20 membranes. The experimental variability was 18%. Overall, the data suggest that iLPA technology will be a relatively simple and inexpensive method for molecular measurements from tissue sections.

Tumor-associated endothelial cells display GSTP1 and RARbeta2 promoter methylation in human prostate cancer.

BACKGROUND: A functional blood supply is essential for tumor growth and proliferation. However, the mechanism of blood vessel recruitment to the tumor is still poorly understood. Ideally, a thorough molecular assessment of blood vessel cells would be critical in our comprehension of this process. Yet, to date, there is little known about the molecular makeup of the endothelial cells of tumor-associated blood vessels, due in part to the difficulty of isolating a pure population of endothelial cells from the heterogeneous tissue environment. METHODS: Here we describe the use of a recently developed technique, Expression Microdissection, to isolate endothelial cells from the tumor microenvironment. The methylation status of the dissected samples was evaluated for GSTP1 and RARbeta2 promoters via the QMS-PCR method. RESULTS: Comparing GSTP1 and RARbeta2 promoter methylation data, we show that 100% and 88% methylation is detected, respectively, in the tumor areas, both in epithelium and endothelium. Little to no methylation is observed in non-tumor tissue areas. CONCLUSION: We applied an accurate microdissection technique to isolate endothelial cells from tissues, enabling DNA analysis such as promoter methylation status. The observations suggest that epigenetic alterations may play a role in determining the phenotype of tumor-associated vasculature.