Development and validation of an integrative pan-solid tumor predictor of PD-1/PD-L1 blockade benefit.

BACKGROUND: Anti-PD-1 and PD-L1 (collectively PD-[L]1) therapies are approved for many advanced solid tumors. Biomarkers beyond PD-L1 immunohistochemistry, microsatellite instability, and tumor mutation burden (TMB) may improve benefit prediction. METHODS: Using treatment data and genomic and transcriptomic tumor tissue profiling from an observational trial (NCT03061305), we developed Immunotherapy Response Score (IRS), a pan-tumor predictive model of PD-(L)1 benefit. IRS real-world progression free survival (rwPFS) and overall survival (OS) prediction was validated in an independent cohort of trial patients. RESULTS: Here, by Cox modeling, we develop IRS-which combines TMB with CD274, PDCD1, ADAM12 and TOP2A quantitative expression-to predict pembrolizumab rwPFS (648 patients; 26 tumor types; IRS-High or -Low groups). In the 248 patient validation cohort (248 patients; 24 tumor types; non-pembrolizumab PD-[L]1 monotherapy treatment), median rwPFS and OS are significantly longer in IRS-High vs. IRS-Low patients (rwPFS adjusted hazard ratio [aHR] 0.52, p = 0.003; OS aHR 0.49, p = 0.005); TMB alone does not significantly predict PD-(L)1 rwPFS nor OS. In 146 patients treated with systemic therapy prior to pembrolizumab monotherapy, pembrolizumab rwPFS is only significantly longer than immediately preceding therapy rwPFS in IRS-High patients (interaction test p = 0.001). In propensity matched lung cancer patients treated with first-line pembrolizumab monotherapy or pembrolizumab+chemotherapy, monotherapy rwPFS is significantly shorter in IRS-Low patients, but is not significantly different in IRS-High patients. Across 24,463 molecularly-evaluable trial patients, 7.6% of patients outside of monotherapy PD-(L)1 approved tumor types are IRS-High/TMB-Low. CONCLUSIONS: The validated, predictive, pan-tumor IRS model can expand PD-(L)1 monotherapy benefit outside currently approved indications.

Therapies activating the immune system (checkpoint inhibitors) have revolutionized the treatment of patients with advanced cancer, however new molecular tests may better identify patients who could benefit. Using treatment data and clinical molecular test results, we report the development and validation of Immunotherapy Response Score (IRS) to predict checkpoint inhibitor benefit. Across patients with more than 20 advanced cancer types, IRS better predicted checkpoint inhibitor benefit than currently available tests. Data from >20,000 patients showed that IRS identifies ~8% of patients with advanced cancer who may dramatically benefit from checkpoint inhibitors but would not receive them today based on currently available tests. Our approach may help clinicians to decide which patients should receive checkpoint inhibitors to treat their disease.

Prevalence of asymptomatic cytomegalovirus (CMV) infection in newborns in northeast Florida.

Background: Congenital cytomegalovirus (CMV) infection is the leading cause of hearing loss and neurocognitive delay among children. Affected infants may be asymptomatic at birth and even pass their universal hearing screen. Early identification of CMV-infected infants will allow earlier detection, evaluation and management. The prevalence of congenital CMV infection in the developed world varies geographically from 0.6% to 0.7% of all deliveries and certain regions are at higher risk. The prevalence of congenital CMV is unknown for our region. Aim: The purpose of this study was to determine the prevalence of CMV infection among the neonatal population at an urban, tertiary hospital in northeast Florida which serves a large population of patients with low socioeconomic status to assess if universal screening program for congenital asymptomatic CMV infection can be determined. Methods: The study was submitted and approved by our Institutional Review Board. We tested the urine for CMV infection in 100 asymptomatic newborns (>32 weeks gestational age and >1,750 g weight at the time of delivery) delivered between June 2016 and July 2017. Results: Urine CMV was tested on 100 infants. One infant had a positive urine NAAT for CMV, making the prevalence of congenital CMV infection among asymptomatic newborns in our hospitals' population 1%. Conclusion: CMV prevalence in our setting of an urban, tertiary hospital is relatively consistent with the national average of all congenital CMV infections. A policy of universal screening for congenital CMV may be necessary.

Deep oncopanel sequencing reveals within block position-dependent quality degradation in FFPE processed samples.

BACKGROUND: Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized diploid cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified. RESULTS: Tissue sections that fail more frequently show low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces are more likely to show FFPE-specific errors, akin to "edge effects" seen in histology, while the inner samples display no quality degradation related to fixation time. CONCLUSIONS: To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.

Hepatic kinome atlas: An in-depth identification of kinase pathways in liver fibrosis of humans and rodents.

BACKGROUND AND AIMS: Resolution of pathways that converge to induce deleterious effects in hepatic diseases, such as in the later stages, have potential antifibrotic effects that may improve outcomes. We aimed to explore whether humans and rodents display similar fibrotic signaling networks. APPROACH AND RESULTS: We assiduously mapped kinase pathways using 340 substrate targets, upstream bioinformatic analysis of kinase pathways, and over 2000 random sampling iterations using the PamGene PamStation kinome microarray chip technology. Using this technology, we characterized a large number of kinases with altered activity in liver fibrosis of both species. Gene expression and immunostaining analyses validated many of these kinases as bona fide signaling events. Surprisingly, the insulin receptor emerged as a considerable protein tyrosine kinase that is hyperactive in fibrotic liver disease in humans and rodents. Discoidin domain receptor tyrosine kinase, activated by collagen that increases during fibrosis, was another hyperactive protein tyrosine kinase in humans and rodents with fibrosis. The serine/threonine kinases found to be the most active in fibrosis were dystrophy type 1 protein kinase and members of the protein kinase family of kinases. We compared the fibrotic events over four models: humans with cirrhosis and three murine models with differing levels of fibrosis, including two models of fatty liver disease with emerging fibrosis. The data demonstrate a high concordance between human and rodent hepatic kinome signaling that focalizes, as shown by our network analysis of detrimental pathways. CONCLUSIONS: Our findings establish a comprehensive kinase atlas for liver fibrosis, which identifies analogous signaling events conserved among humans and rodents.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Real-World Performance of a Comprehensive Genomic Profiling Test Optimized for Small Tumor Samples.

PURPOSE: Tissue-based comprehensive genomic profiling (CGP) is increasingly used for treatment selection in patients with advanced cancer; however, tissue availability may limit widespread implementation. Here, we established real-world CGP tissue availability and assessed CGP performance on consecutively received samples. MATERIALS AND METHODS: We conducted a post hoc, nonprespecified analysis of 32,048 consecutive tumor tissue samples received for StrataNGS, a multiplex polymerase chain reaction (PCR)-based comprehensive genomic profiling (PCR-CGP) test, as part of an ongoing observational trial (NCT03061305). Sample characteristics and PCR-CGP performance were assessed across all tested samples, including exception samples not meeting minimum input quality control (QC) requirements (< 20% tumor content [TC], < 2 mm2 tumor surface area [TSA], DNA or RNA yield < 1 ng/µL, or specimen age > 5 years). Tests reporting ≥ 1 prioritized alteration or meeting TC and sequencing QC were considered successful. For prostate carcinoma and lung adenocarcinoma, tests reporting ≥ 1 actionable or informative alteration or meeting TC and sequencing QC were considered actionable. RESULTS: Among 31,165 (97.2%) samples where PCR-CGP was attempted, 10.7% had < 20% TC and 59.2% were small (< 25 mm2 tumor surface area). Of 31,101 samples evaluable for input requirements, 8,089 (26.0%) were exceptions not meeting requirements. However, 94.2% of the 31,101 tested samples were successfully reported, including 80.5% of exception samples. Positive predictive value of PCR-CGP for ERBB2 amplification in exceptions and/or sequencing QC-failure breast cancer samples was 96.7%. Importantly, 84.0% of tested prostate carcinomas and 87.9% of lung adenocarcinomas yielded results informing treatment selection. CONCLUSION: Most real-world tissue samples from patients with advanced cancer desiring CGP are limited, requiring optimized CGP approaches to produce meaningful results. An optimized PCR-CGP test, coupled with an inclusive exception testing policy, delivered reportable results for > 94% of samples, potentially expanding the proportion of CGP-testable patients and impact of biomarker-guided therapies.

Development and Validation of StrataNGS, a Multiplex PCR, Semiconductor Sequencing-Based Comprehensive Genomic Profiling Test.

Despite widespread use in targeted tumor testing, multiplex PCR/semiconductor (Ion Torrent) sequencing-based assessment of all comprehensive genomic profiling (CGP) variant classes has been limited. Herein, we describe the development and validation of StrataNGS, a 429-gene, multiplex PCR/semiconductor sequencing-based CGP laboratory-developed test performed on co-isolated DNA and RNA from formalin-fixed, paraffin-embedded tumor specimens with ≥2 mm2 tumor surface area. Validation was performed in accordance with MolDX CGP validation guidelines using 1986 clinical formalin-fixed, paraffin-embedded samples and an in-house developed optimized bioinformatics pipeline. Across CGP variant classes, accuracy ranged from 0.945 for tumor mutational burden (TMB) status to >0.999 for mutations and gene fusions, positive predictive value ranged from 0.915 for TMB status to 1.00 for gene fusions, and reproducibility ranged from 0.998 for copy number alterations to 1.00 for splice variants and insertions/deletions. StrataNGS TMB estimates were highly correlated to those from whole exome- or FoundationOne CDx-determined TMB (Pearson r = 0.998 and 0.960, respectively); TMB reproducibility was 0.996 (concordance correlation coefficient). Limit of detection for all variant classes was <20% tumor content. Together, we demonstrate that multiplex PCR/semiconductor sequencing-based tumor tissue CGP is feasible using optimized bioinformatic approaches described herein.

Suitability of Placental Blood Samples of Newborns for Pre-Transfusion Testing.

Objective: To show concordance between heel stick and placental blood sample pairs for newborns' pre-transfusion testing and to validate placental blood's tube and gel methodology. Methods: Placental samples were collected for pre-transfusion testing at birth from 78 singleton and twin newborns admitted to our Mother-Baby Unit to compare with the results of heel stick samples taken from same newborns. Gestational age ≥35 weeks, weight ≥2,000 g. The study was approved by the Institutional Review Board (IRB). Informed consent was obtained from newborn parents. ABO blood group, Rhesus factor (Rh), direct antiglobulin test (DAT), and antibody screen were performed. Ortho ProVue Analyzer was used for tube and gel methods. McNemar's test for paired categorical data was performed. Results: One hundred percent concordance in 78 pairs for ABO and Rh. Seventy-four pairs were tested for antibodies, 72 were both negative, 1 was both positive, and 1 gave discordant result. Ninety-nine percent concordance, p = 0.999. Sixty-five pairs were both DAT negative, seven were both DAT positive, and six gave discordant results. Ninety-two percent concordance, p = 0.68. Placental blood gave identical results comparing tube with gel methods. Conclusions: Placental blood is suitable for pre-transfusion testing and can replace heel sticks. Placental blood tube and gel methods are validated.

Saving Time for Patient Care by Optimizing Physician Note Templates: A Pilot Study.

Background: At times, electronic medical records (EMRs) have proven to be less than optimal, causing longer hours behind computers, shorter time with patients, suboptimal patient safety, provider dissatisfaction, and physician burnout. These concerning healthcare issues can be positively affected by optimizing EMR usability, which in turn would lead to substantial benefits to healthcare professionals such as increased healthcare professional productivity, efficiency, quality, and accuracy. Documentation issues, such as non-standardization of physician note templates and tedious, time-consuming notes in our mother-baby unit (MBU), were discussed during meetings with stakeholders in the MBU and our hospital's EMR analysts. Objective: The objective of this study was to assess physician note optimization on saving time for patient care and improving provider satisfaction. Methods: This quality improvement pilot investigation was conducted in our MBU where four note templates were optimized: History and Physical (H and P), Progress Note (PN), Discharge Summary (DCS), and Hand-Off List (HOL). Free text elements documented elsewhere in the EMR (e.g., delivery information, maternal data, lab result, etc.) were identified and replaced with dynamic links that automatically populate the note with these data. Discrete data pick lists replaced necessary elements that were previously free texts. The new note templates were given new names for ease of accessibility. Ten randomly chosen pediatric residents completed both the old and new note templates for the same control newborn encounter during a period of one year. Time spent and number of actions taken (clicks, keystrokes, transitions, and mouse-keyboard switches) to complete these notes were recorded. Surveys were sent to MBU providers regarding overall satisfaction with the new note templates. Results: The ten residents' average time saved was 23 min per infant. Reflecting this saved time on the number of infants admitted to our MBU between January 2016 and September, 2019 which was 9373 infants; resulted in 2.6 hours saved per day, knowing that every infant averages two days length of stay. The new note templates required 69 fewer actions taken than the old ones (H and P: 11, PN: 8, DCS: 18, HOL: 32). The provider surveys were consistent with improved provider satisfaction. Conclusion: Optimizing physician notes saved time for patient care and improved physician satisfaction.

Loss of hepatic PPARα promotes inflammation and serum hyperlipidemia in diet-induced obesity.

Agonists for PPARα are used clinically to reduce triglycerides and improve high-density lipoprotein (HDL) cholesterol levels in patients with hyperlipidemia. Whether the mechanism of PPARα activation to lower serum lipids occurs in the liver or other tissues is unknown. To determine the function of hepatic PPARα on lipid profiles in diet-induced obese mice, we placed hepatocyte-specific peroxisome proliferator-activated receptor-α (PPARα) knockout (PparaHepKO) and wild-type (Pparafl/fl) mice on high-fat diet (HFD) or normal fat diet (NFD) for 12 wk. There was no significant difference in weight gain, percent body fat mass, or percent body lean mass between the groups of mice in response to HFD or NFD. Interestingly, the PparaHepKO mice on HFD had worsened hepatic inflammation and a significant shift in the proinflammatory M1 macrophage population. These changes were associated with higher hepatic fat mass and decreased hepatic lean mass in the PparαHepKO on HFD but not in NFD as measured by Oil Red O and noninvasive EchoMRI analysis (31.1 ± 2.8 vs. 20.2 ± 1.5, 66.6 ± 2.5 vs. 76.4 ± 1.5%, P < 0.05). We did find that this was related to significantly reduced peroxisomal gene function and lower plasma ß-hydroxybutyrate in the PparaHepKO on HFD, indicative of reduced metabolism of fats in the liver. Together, these provoked higher plasma triglyceride and apolipoprotein B100 levels in the PparaHepKO mice compared with Pparafl/fl on HFD. These data indicate that hepatic PPARα functions to control inflammation and liver triglyceride accumulation that prevent hyperlipidemia.

Myeloperoxidase deficiency attenuates systemic and dietary iron-induced adverse effects.

Iron deficiency is routinely treated with oral or systemic iron supplements, which are highly reactive and could induce oxidative stress via augmenting the activity of proinflammatory enzyme myeloperoxidase (MPO). To investigate the extent to which MPO is involved in iron-induced toxicity, acute (24 h) iron toxicity was induced by intraperitoneal administration of FeSO4 (25 mg/kg body weight) to MPO-deficient (MpoKO) mice and their wild-type (WT) littermates. Acute iron toxicity was also assessed in WT mice pretreated with an MPO inhibitor, 4-aminobenzoic acid hydrazide. Systemic iron administration up-regulated circulating MPO and neutrophil elastase and elevated systemic inflammatory and organ damage markers in WT mice. However, genetic deletion of MPO or its inhibition significantly reduced iron-induced organ damage and systemic inflammatory responses. In contrast to the acute model, 8 weeks of 2% carbonyl iron diet feeding to WT mice did not change the levels of circulating MPO and neutrophil elastase but promoted their accumulation in the liver. Even though both MpoKO and WT mice displayed similar levels of diet-induced hyperferremia, MpoKO mice showed significantly reduced inflammatory response and oxidative stress than the WT mice. In addition, WT bone-marrow-derived neutrophils (BMDN) generated more reactive oxygen species than MPO-deficient BMDN upon iron stimulation. Altogether, genetic deficiency or pharmacologic inhibition of MPO substantially attenuated acute and chronic iron-induced toxicity. Our results suggest that targeting MPO during iron supplementation is a promising approach to reduce iron-induced toxicity/side effects in vulnerable population.

Cytomorphologic characteristics and differential diagnoses of lymphoepithelial carcinoma of the parotid.

INTRODUCTION: Lymphoepithelial carcinoma of the salivary gland is an extremely rare neoplasm and is challenging to diagnose by fine needle aspiration (FNA). There are rare reports on the cytopathologic features of lymphoepithelial carcinoma, which may be mistaken for other high-grade salivary gland neoplasm or a metastasis to the salivary gland. MATERIALS AND METHODS: A retrospective review was undertaken of 7 cases of lymphoepithelial carcinoma of the parotid diagnosed on FNA with histologic confirmation from 4 major medical centers. RESULTS: Cytomorphologic features of lymphoepithelial carcinoma include smears with moderate cellularity displaying a rich nonneoplastic population of lymphoplasmacytic cells admixed with tissue fragments of high grade, malignant undifferentiated epithelial cells with high nuclear to cytoplasm ratio, hyperchromasia, prominent nucleoli, and scant to abundant, indistinct cytoplasm. DISCUSSION: Diagnostic pitfalls of lymphoepithelial carcinoma include metastatic squamous cell carcinoma, metastatic nasopharyngeal carcinoma, and other high grade primary salivary gland neoplasms. Recognizing this lesion may help guide clinicians to perform additional imaging studies to exclude a primary from other sites.

Diagnostic yield of ThinPrep preparation in the assessment of fine-needle aspiration biopsy of salivary gland neoplasms.

BACKGROUND: Fine-needle aspiration (FNA) has been widely recognized as an important modality in assessment of salivary gland neoplasms, and specimens are often processed as conventional smears. We conducted the current study to evaluate the diagnostic utility of ThinPrep preparation as an alternative method for assessment of salivary gland neoplasms. METHODS: A computer SNOMED search from the pathology database at our institution between July 1999 and June 2012 was conducted to identify FNA cytology specimens of salivary gland lesions for which follow-up surgical specimens revealed neoplasms. The FNA specimens were divided into two cohorts: one cohort consisted solely of the specimens in which all needle passes were collected into CytoLyt solution and only ThinPrep slides were prepared; and the other cohort included the specimens prepared with conventional smears. Diagnostic performance of the two cohorts was compared. RESULTS: Nondiagnostic rate of ThinPrep preparation was significantly higher than that of conventional smears (40% vs.18%; P <0.001). Among the diagnostic specimens, although more indeterminate diagnoses were generated in ThinPrep preparation compared to conventional smears (40% vs. 26%; P = 0.024), absolute cytohistologic concordant rate for the positive cases (type of neoplasms specified) is similar between the two preparations (80% vs. 86%; P = 0.354). Furthermore, there is no significant difference in rate of accurate diagnosis (correct typing of benign versus malignant neoplasm) between the two preparations (70% vs. 81%; P = 0.057). CONCLUSIONS: ThinPrep may be considered as another practical method of specimen preparation in the assessment of salivary gland neoplasms, particularly when FNA is performed without immediate assistance from cytology.

Prospective evaluation of impact of using the Bethesda System for Reporting Thyroid Cytopathology: an institutional experience.

INTRODUCTION: The Bethesda System for Reporting Thyroid Cytology (BSRTC) refines the definition of and provides specific diagnostic criteria for fine-needle aspiration (FNA) assessment of thyroid lesions. This study was conducted to prospectively evaluate the diagnostic and clinical impact of using BSRTC for management of thyroid lesions and to compare diagnostic performance of post-BSRTC period with that of pre-BSRTC period. MATERIALS AND METHODS: The study included FNA specimens obtained in our institution 2.5 years prior to and 2.5 years after implementing BSRTC. Nondiagnostic rate, distribution of the diagnostic categories, rate of surgical follow-up, cytohistologic concordant rate, and risk of malignancy were calculated and compared between pre- and post-BSRTC periods. RESULTS: In comparison to the pre-BSRTC period, the post-BSRTC period generated a lower nondiagnostic rate (19.9% versus 15.8%), a greater proportion of benign (65.3% versus 69.2%) and atypia of undetermined significance or follicular lesion of undetermined significance (4.4% versus 7.4%) in contrast with a decreased proportion of follicular neoplasm or suspicious for follicular neoplasm categories (5.6% versus 2.2%). Rate of surgical follow-up decreased for benign (13.8% versus 7.6%) and atypia of undetermined significance or follicular lesion of undetermined significance (61.5% versus 42.1%) categories, and overall surgical rate reduced (24.2% versus 18.1%). Implementation of BSRTC did not affect overall rate of cytohistologic concordance (78.4% versus 80.5%) or the overall rate of histologically proven malignancy (30.6 versus 36.9%), whereas the individual cytohistologic concordant rate and the malignant rate for each of the diagnostic categories did not differ between pre- and post-BSRTC. CONCLUSIONS: The implementation of BSRTC resulted in a decreased overall surgical rate, particularly for benign and follicular lesion of undetermined significance categories, without affecting overall cytohistologic concordance and rate of malignancy.

Image microarrays derived from tissue microarrays (IMA-TMA): New resource for computer-aided diagnostic algorithm development.

BACKGROUND: Conventional tissue microarrays (TMAs) consist of cores of tissue inserted into a recipient paraffin block such that a tissue section on a single glass slide can contain numerous patient samples in a spatially structured pattern. Scanning TMAs into digital slides for subsequent analysis by computer-aided diagnostic (CAD) algorithms all offers the possibility of evaluating candidate algorithms against a near-complete repertoire of variable disease morphologies. This parallel interrogation approach simplifies the evaluation, validation, and comparison of such candidate algorithms. A recently developed digital tool, digital core (dCORE), and image microarray maker (iMAM) enables the capture of uniformly sized and resolution-matched images, with these representing key morphologic features and fields of view, aggregated into a single monolithic digital image file in an array format, which we define as an image microarray (IMA). We further define the TMA-IMA construct as IMA-based images derived from whole slide images of TMAs themselves. METHODS: Here we describe the first combined use of the previously described dCORE and iMAM tools, toward the goal of generating a higher-order image construct, with multiple TMA cores from multiple distinct conventional TMAs assembled as a single digital image montage. This image construct served as the basis of the carrying out of a massively parallel image analysis exercise, based on the use of the previously described spatially invariant vector quantization (SIVQ) algorithm. RESULTS: Multicase, multifield TMA-IMAs of follicular lymphoma and follicular hyperplasia were separately rendered, using the aforementioned tools. Each of these two IMAs contained a distinct spectrum of morphologic heterogeneity with respect to both tingible body macrophage (TBM) appearance and apoptotic body morphology. SIVQ-based pattern matching, with ring vectors selected to screen for either tingible body macrophages or apoptotic bodies, was subsequently carried out on the differing TMA-IMAs, with attainment of excellent discriminant classification between the two diagnostic classes. CONCLUSION: The TMA-IMA construct enables and accelerates high-throughput multicase, multifield based image feature discovery and classification, thus simplifying the development, validation, and comparison of CAD algorithms in settings where the heterogeneity of diagnostic feature morphologic is a significant factor.

SIVQ-aided laser capture microdissection: A tool for high-throughput expression profiling.

INTRODUCTION: Laser capture microdissection (LCM) facilitates procurement of defined cell populations for study in the context of histopathology. The morphologic assessment step in the LCM procedure is time consuming and tedious, thus restricting the utility of the technology for large applications. RESULTS: Here, we describe the use of Spatially Invariant Vector Quantization (SIVQ) for histological analysis and LCM. Using SIVQ, we selected vectors as morphologic predicates that were representative of normal epithelial or cancer cells and then searched for phenotypically similar cells across entire tissue sections. The selected cells were subsequently auto-microdissected and the recovered RNA was analyzed by expression microarray. Gene expression profiles from SIVQ-LCM and standard LCM-derived samples demonstrated highly congruous signatures, confirming the equivalence of the differing microdissection methods. CONCLUSION: SIVQ-LCM improves the work-flow of microdissection in two significant ways. First, the process is transformative in that it shifts the pathologist's role from technical execution of the entire microdissection to a limited-contact supervisory role, enabling large-scale extraction of tissue by expediting subsequent semi-autonomous identification of target cell populations. Second, this work-flow model provides an opportunity to systematically identify highly constrained cell populations and morphologically consistent regions within tissue sections. Integrating SIVQ with LCM in a single environment provides advanced capabilities for efficient and high-throughput histological-based molecular studies.

Ethanol alters the osteogenic differentiation of amniotic fluid-derived stem cells.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is a set of developmental defects caused by prenatal alcohol exposure. Clinical manifestations of FASD are highly variable and include mental retardation and developmental defects of the heart, kidney, muscle, skeleton, and craniofacial structures. Specific effects of ethanol on fetal cells include induction of apoptosis as well as inhibition of proliferation, differentiation, and migration. This complex set of responses suggests that a bioinformatics approach could clarify some of the pathways involved in these responses. METHODS: In this study, the responses of fetal stem cells derived from the amniotic fluid (AFSCs) to treatment with ethanol have been examined. Large-scale transcriptome analysis of ethanol-treated AFSCs indicates that genes involved in skeletal development and ossification are up-regulated in these cells. Therefore, the effect of ethanol on osteogenic differentiation of AFSCs was studied. RESULTS: Exposure to ethanol during the first 48 hours of an osteogenic differentiation protocol increased in vitro calcium deposition by AFSCs and increased alkaline phosphatase activity. In contrast, ethanol treatment later in the differentiation protocol (day 8) had no significant effect on the activity of alkaline phosphatase. CONCLUSIONS: These results suggest that transient exposure of AFSCs to ethanol during early differentiation enhances osteogenic differentiation of the cells.

Functional genomics: new insights into the 'function' of low levels of gene expression in stem cells.

Understanding the global gene expression profile of stem cells and their multilineage differentiation will be essential for their ultimate therapeutic application. Efforts to characterize stem cells have relied on analyzing the genome-wide expression profiles that are biased towards the identification of genes that display the most pronounced differential expression. Rather than being viewed as a "blank" state, recent studies suggest that stem cells express low levels of multiple lineage specific genes prior to differentiation, a phenomenon known as "lineage priming." It is not likely that low levels of lineage-specific genes produce sufficient amounts of differentiation factors, but rather to provide rapid transcription to a wide range of lineage programs prior to differentiation. Thus, stem cell differentiation may involve the elimination of other potential pathways and the activation of a specific lineage program.