Novel method for highly multiplexed gene expression profiling of circulating tumor cells (CTCs) captured from the blood of women with metastatic breast cancer.

BACKGROUND: Enumeration of circulating tumor cells (CTCs) has proven clinical significance for monitoring patients with metastatic cancers. Multiplexed gene expression profiling of CTCs is a potential tool for assessing disease status and monitoring treatment response. The Parsortix® technology enables the capture and harvest of CTCs from blood based on cell size and deformability. The HyCEAD™ (Hybrid Capture Enrichment Amplification and Detection) assay enables simultaneous amplification of short amplicons for up to 100 mRNA targets, and the Ziplex™ instrument quantifies the amplicons for highly sensitive gene expression profiling down to single cell levels. The aim of the study was to functionally assess this system. METHODS: The HyCEAD/Ziplex platform was used to quantify the expression levels for 72 genes using as little as 20 pg of total RNA or a single cultured tumor cell. Assay performance was evaluated using cells or total RNA spiked into Parsortix harvests of healthy donor blood. The assay was also evaluated using total RNA obtained from Parsortix harvests of blood from metastatic breast cancer (MBC) patients or healthy volunteers (HVs). RESULTS: Using genes with low expression in WBC RNA and/or in unspiked Parsortix harvests from HVs, the assay distinguished between the different breast cancer and ovarian cancer cell lines with as little as 20 pg of total RNA (equivalent to a single cell) in the presence of 1 ng of WBC RNA. Single cultured cells spiked into Parsortix harvests from 10 mL of HV blood were also detected and distinguished from each other. CVs from repeatability experiments were less than 20%. Hierarchical clustering of clinical samples differentiated most MBC patients from HVs. CONCLUSION: HyCEAD/Ziplex provided sensitive quantification of expression of 72 genes from 20 pg of total RNA from cultured tumor cell lines or from single cultured tumor cells spiked into lysates from Parsortix harvests of HV blood. The HyCEAD/Ziplex platform enables the quantification of selected genes in the presence of residual nucleated blood cells in Parsortix harvests. The HyCEAD/Ziplex platform is an effective tool for multiplexed molecular characterization of mRNA in small numbers of tumor cells harvested from blood.

Transcriptome Profiling of Circulating Tumor Cells to Predict Clinical Outcomes in Metastatic Castration-Resistant Prostate Cancer.

The clinical utility of circulating tumor cells (CTC) as a non-invasive multipurpose biomarker is broadly recognized. The earliest methods for enriching CTCs from whole blood rely on antibody-based positive selection. The prognostic utility of CTC enumeration using positive selection with the FDA-approved CellSearchTM system has been demonstrated in numerous studies. The capture of cells with specific protein phenotypes does not fully represent cancer heterogeneity and therefore does not realize the prognostic potential of CTC liquid biopsies. To avoid this selection bias, CTC enrichment based on size and deformability may provide better fidelity, i.e., facilitate the characterization of CTCs with any phenotype. In this study, the recently FDA-approved Parsortix® technology was used to enrich CTCs from prostate cancer (PCa) patients for transcriptome analysis using HyCEADTM technology. A tailored PCa gene panel allowed us to stratify metastatic castration-resistant prostate cancer (mCRPC) patients with clinical outcomes. In addition, our findings suggest that targeted CTC transcriptome profiling may be predictive of therapy response.

Malignancy Assessment Using Gene Identification in Captured Cells Algorithm for the Prediction of Malignancy in Women With a Pelvic Mass.

OBJECTIVE: To evaluate the detection of malignancy in women with a pelvic mass by using multiplexed gene expression analysis of cells captured from peripheral blood. METHODS: This was an IRB-approved, prospective clinical study. Eligible patients had a pelvic mass and were scheduled for surgery or biopsy. Rare cells were captured from peripheral blood obtained preoperatively by using a microfluidic cell capture device. Isolated mRNA from the captured cells was analyzed for expression of 72 different gene transcripts. Serum levels for several commonly assayed biomarkers were measured. All patients had a tissue diagnosis. Univariate and multivariate logistic regression analyses for the prediction of malignancy using gene expression and serum biomarker levels were performed, and receiver operating characteristic curves were constructed and compared. RESULTS: A total of 183 evaluable patients were enrolled (average age 56 years, range 19-91 years). There were 104 benign tumors, 17 low malignant potential tumors, and 62 malignant tumors. Comparison of the area under the receiver operating characteristic curve for individual genes and various combinations of genes with or without serum biomarkers to differentiate between benign conditions (excluding low malignant potential tumors) and malignant tumors showed that a multivariate model combining the expression levels of eight genes and four serum biomarkers achieved the highest area under the curve (AUC) (95.1%, 95% CI 92.0-98.2%). The MAGIC (Malignancy Assessment using Gene Identification in Captured Cells) algorithm significantly outperformed all individual genes (AUC 50.2-65.2%; all P <.001) and a multivariate model combining 14 different genes (AUC 88.0%, 95% CI 82.9-93.0%; P =.005). Further, the MAGIC algorithm achieved an AUC of 89.5% (95% CI 81.3-97.8%) for stage I-II and 98.9% (95% CI 96.7-100%) for stage III-IV patients with epithelial ovarian cancer. CONCLUSION: Multiplexed gene expression evaluation of cells captured from blood, with or without serum biomarker levels, accurately detects malignancy in women with a pelvic mass. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT02781272. FUNDING SOURCE: This study was funded by ANGLE Europe Limited (Surrey Research Park, Guildford, Surrey, United Kingdom).

Derivation and Validation of a Serum Biomarker Panel to Identify Infants With Acute Intracranial Hemorrhage.

Importance: Abusive head trauma is the leading cause of death from physical abuse. Missing the diagnosis of abusive head trauma, particularly in its mild form, is common and contributes to increased morbidity and mortality. Serum biomarkers may have potential as quantitative point-of-care screening tools to alert physicians to the possibility of intracranial hemorrhage. Objective: To identify and validate a set of biomarkers that could be the basis of a multivariable model to identify intracranial hemorrhage in well-appearing infants using the Ziplex System. Design, Setting, and Participants: Binary logistic regression was used to develop a multivariable model incorporating 3 serum biomarkers (matrix metallopeptidase-9, neuron-specific enolase, and vascular cellular adhesion molecule-1) and 1 clinical variable (total hemoglobin). The model was then prospectively validated. Multiplex biomarker measurements were performed using Flow-Thru microarray technology on the Ziplex System, which has potential as a point-of-care system. The model was tested at 3 pediatric emergency departments in level I pediatric trauma centers (Children's Hospital of Pittsburgh of University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania; Primary Children's Hospital, Salt Lake City, Utah; and Lurie Children's Hospital, Chicago, Illinois) among well-appearing infants who presented for care owing to symptoms that placed them at increased risk of abusive head trauma. The study took place from November 2006 to April 2014 at Children's Hospital of Pittsburgh, June 2010 to August 2013 at Primary Children's Hospital, and January 2011 to August 2013 at Lurie Children's Hospital. Main Outcomes and Measures: A mathematical model that can predict acute intracranial hemorrhage in infants at increased risk of abusive head trauma. Results: The multivariable model, Biomarkers for Infant Brain Injury Score, was applied prospectively to 599 patients. The mean (SD) age was 4.7 (3.1) months. Fifty-two percent were boys, 78% were white, and 8% were Hispanic. At a cutoff of 0.182, the model was 89.3% sensitive (95% CI, 87.7-90.4) and 48.0% specific (95% CI, 47.3-48.9) for acute intracranial hemorrhage. Positive and negative predictive values were 21.3% and 95.6%, respectively. The model was neither sensitive nor specific for atraumatic brain abnormalities, isolated skull fractures, or chronic intracranial hemorrhage. Conclusion and Relevance: The Biomarkers for Infant Brain Injury Score, a multivariable model using 3 serum biomarker concentrations and serum hemoglobin, can identify infants with acute intracranial hemorrhage. Accurate and timely identification of intracranial hemorrhage in infants without a history of trauma in whom trauma may not be part of the differential diagnosis has the potential to decrease morbidity and mortality from abusive head trauma.

Marital status, life stressor precipitants, and communications of distress and suicide intent in a sample of United States Air Force suicide decedents.

Life stressor precipitants and communications of distress and suicide intent were examined among a sample of United States Air Force (USAF) married versus unmarried suicide decedents. A total of 100 death investigations conducted by the Office of Special Investigations on active duty USAF suicides occurring between 1996 and 2006 were retrospectively reviewed. Married decedents were twice as likely 1) to have documented interpersonal conflict 24 hours prior to suicide and 2) to have communicated suicide intent to peers or professionals. Themes of distress communication for all decedents were intrapersonal (perceived stress, depression, psychological pain) and interpersonal (thwarted belongingness, rejection, loneliness). Suicide prevention programs and policies are encouraged to adapt efforts to the unique needs of married and unmarried individuals.

Suicide in the United States Air Force: Risk factors communicated before and at death.

BACKGROUND: Over the last decade, suicide rates in the U.S. military have steadily increased, resulting in a call for suicide-related research with military populations. The present project aimed to describe and evaluate the communications (i.e., verbally and in suicide notes) of 13 suicide risk factors in the suicide death investigation files of 98 active duty U.S. Air Force (USAF) members. METHODS: Two-hundred thirty-seven suicide death investigation files were coded. Ninety-eight decedents left suicide notes and were included in the current analyses. Descriptive statistics were computed to evaluate the types of risk factors most commonly communicated prior to and at the time of death as well as the medium for their communication. Specifically, verbal and note communications were compared to evaluate which medium decedents most often used to communicate risk factors. Also, the frequency that interpersonal compared to intrapsychic risk factors were communicated was evaluated. RESULTS: Hopelessness (35.7% of cases) and perceived burdensomeness (31.6% of cases) were the risk factors most often communicated in suicide notes but not verbally. Thwarted belongingness (29.6% of cases) was the risk factor most often communicated verbally and in the suicide note. Further, evaluated risk factors were more frequently communicated in suicide notes than verbally. Finally, interpersonal risk factors were more often communicated than intrapsychic risk factors. LIMITATIONS: The validity of the data relies on interviews of decedents' acquaintances and various medical/military records. CONCLUSIONS: Our findings support emphasizing certain risk factors over others in USAF suicide prevention efforts. Further, interpersonal risk factors appeared to be more salient than intrapsychic risk factors in the minds of decedents.

The chemiluminescence based Ziplex automated workstation focus array reproduces ovarian cancer Affymetrix GeneChip expression profiles.

BACKGROUND: As gene expression signatures may serve as biomarkers, there is a need to develop technologies based on mRNA expression patterns that are adaptable for translational research. Xceed Molecular has recently developed a Ziplex technology, that can assay for gene expression of a discrete number of genes as a focused array. The present study has evaluated the reproducibility of the Ziplex system as applied to ovarian cancer research of genes shown to exhibit distinct expression profiles initially assessed by Affymetrix GeneChip analyses. METHODS: The new chemiluminescence-based Ziplex gene expression array technology was evaluated for the expression of 93 genes selected based on their Affymetrix GeneChip profiles as applied to ovarian cancer research. Probe design was based on the Affymetrix target sequence that favors the 3' UTR of transcripts in order to maximize reproducibility across platforms. Gene expression analysis was performed using the Ziplex Automated Workstation. Statistical analyses were performed to evaluate reproducibility of both the magnitude of expression and differences between normal and tumor samples by correlation analyses, fold change differences and statistical significance testing. RESULTS: Expressions of 82 of 93 (88.2%) genes were highly correlated (p < 0.01) in a comparison of the two platforms. Overall, 75 of 93 (80.6%) genes exhibited consistent results in normal versus tumor tissue comparisons for both platforms (p < 0.001). The fold change differences were concordant for 87 of 93 (94%) genes, where there was agreement between the platforms regarding statistical significance for 71 (76%) of 87 genes. There was a strong agreement between the two platforms as shown by comparisons of log2 fold differences of gene expression between tumor versus normal samples (R = 0.93) and by Bland-Altman analysis, where greater than 90% of expression values fell within the 95% limits of agreement. CONCLUSION: Overall concordance of gene expression patterns based on correlations, statistical significance between tumor and normal ovary data, and fold changes was consistent between the Ziplex and Affymetrix platforms. The reproducibility and ease-of-use of the technology suggests that the Ziplex array is a suitable platform for translational research.

Mental health evaluations of U.S. Air Force basic military training and technical training students.

A 2000 report by Staal, Cigrang, and Fiedler and a 1998 report by Cigrang, Carbone, Todd, and Fiedler described the attrition of U. S. Air Force basic military trainees due to mental health disorders for the year 1997. This article looks at the population of Air Force basic military trainees and technical training school students located at the same base during the year 2001. In addition, we look at the effect of allowing basic trainees and those in technical school to refer themselves for mental health evaluations as opposed to only evaluating those referred by secondary sources. Primary results of the data analysis suggest that mental health-related separation rates for calendar year 2001 basic military trainees are consistent with past years at 4.2%. For both basic trainees and technical school training students, adjustment disorders and depressive disorders are the top diagnostic categories related to recommendation for separation.

Psychological intervention with mortuary workers after the September 11 attack: the Dover Behavioral Health Consultant model.

Numerous descriptive as well as prospective studies have indicated that mortuary workers, body handlers, and those tasked to work with human remains are at risk for symptoms related to post-traumatic stress disorder and other psychological sequelae. There have been no controlled studies outlining psychological interventions to prevent or minimize the impact of being assigned to perform such duties. In this article, we describe a method of psychological intervention with mortuary workers mobilized as a result of the September 11 attack on the Pentagon. The intervention included the use of Critical Incident Stress Teams and was based on a behavioral health consultant model that has been demonstrated to be effective for behavioral interventions in primary care settings. The model incorporated knowledge from previous studies on mortuary workers and individuals tasked to work with human remains. Qualitative analysis indicated that the behavioral health consultants were well received, recommendations were implemented, and few personnel were removed from duty as a result of psychological factors leading to impaired performance. Implications related to popular models of critical incident intervention and directions for future controlled research are discussed.