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To facilitate treatment and limit transmission of tuberculosis (TB), new methods are needed to enable rapid and affordable diagnosis of the disease in high-burden low-resource settings. We have developed a prototype integrated nucleic acid testing device to detect Mycobacterium tuberculosis (M.tb) in sputum. The device consists of a disposable cartridge and compact, inexpensive instrument that automates pathogen lysis, nucleic acid extraction, isothermal DNA amplification and lateral flow detection. A liquefied and disinfected sputum sample is manually injected into the cartridge, and all other steps are automated, with a result provided in

Next-generation sequencing (NGS) can identify and validate new biomarkers of cancer onset, progression and therapy resistance. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) cancer samples from patients represent a rich resource for linking molecular signatures to clinical data. However, performing NGS on FFPE samples is limited by poor RNA purification methods. To address this hurdle, we developed an improved methodology for extracting high-quality RNA from FFPE samples. By briefly integrating a newly-designed micro-homogenizing (mH) tool with commercially available FFPE RNA extraction protocols, RNA recovery is increased by approximately 3-fold while maintaining standard A260/A280 ratios and RNA quality index (RQI) values. Furthermore, we demonstrate that the mH-purified FFPE RNAs are longer and of higher integrity. Previous studies have suggested that pancreatic ductal adenocarcinoma (PDAC) gene expression signatures vary significantly under in vitro versus in vivo and in vivo subcutaneous versus orthotopic conditions. By using our improved mH-based method, we were able to preserve established expression patterns of KRas-dependency genes within these three unique microenvironments. Finally, expression analysis of novel biomarkers in KRas mutant PDAC samples revealed that PEAK1 decreases and MST1R increases by over 100-fold in orthotopic versus subcutaneous microenvironments. Interestingly, however, only PEAK1 levels remain elevated in orthotopically grown KRas wild-type PDAC cells. These results demonstrate the critical nature of the orthotopic tumor microenvironment when evaluating the clinical relevance of new biomarkers in cells or patient-derived samples. Furthermore, this new mH-based FFPE RNA extraction method has the potential to enhance and expand future FFPE-RNA-NGS cancer biomarker studies.

Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse® mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >104 fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse® method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 104 and 105 colony forming units (cfu)/mL M.tb. At 103 cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse® method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with "Equation missing"1 mL input volume (N = 41). The semi-automated PureLyse® method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths. Because there are virtually no biomarkers to aid in early detection or predicting therapy response, newly diagnosed patients have a dismal prognosis. Substantial archives of formalin-fixed, paraffin-embedded (FFPE) samples from PDAC tissue have been established and linked to clinical data. However, identifying biomarkers from these tumors is severely hindered because extracting high-quality nucleic acid material from FFPE samples is time-consuming and inefficient. Thus, developing improved methods for extracting high-quality RNA/DNA from FFPE samples represents an unmet need in the fields of cancer biology and Next-Generation Sequencing (NGS). We have tested Claremont BioSolutions’ microhomogenization (mH) tools in combination with commercial FFPE RNA extraction kits for their ability to improve high-quality RNA recovery from PDAC xenograft samples. Our results demonstrate that the addition of brief (< 5min) mH s...

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths. There are virtually no biomarkers to aid in early detection or predicting therapy response, newly diagnosed patients have less than a 7% 5-year survival rate and the median survival from the time of diagnosis is less than 12 months. Next-Generation Sequencing (NGS) together with in vitro/in vivo functional studies is likely to identify and validate new biomarkers of PDAC onset, progression and therapy resistance. Importantly, the substantial archives of formalin-fixed, paraffin-embedded (FFPE) samples from PDAC patients are likely to be a rich resource for linking molecular signatures to relevant clinical data. However, NGS methods on FFPE samples are severely hindered because extracting high-quality nucleic acid material from these samples is time-consuming and inefficient. We have sought to develop novel methods for improving the extraction of high-quality RNA from FFPE samples within the context of commercially-available FFPE RNA kits and protocols. Together with researchers and clinicians at Claremont BioSolutions, AntiCancer Inc. and UCSD our laboratory has developed a novel nucleic acid extraction method that significantly increases RNA yield and integrity from PDAC cell line and Patient-Derived Xenograft FFPE samples. By briefly ( Citation Format: Malachia Hoover, Yvess Adamian, Mark Brown, Ali A. Maawy, Robert Hoffman, Michael Bouvet, Robert Doebler, Jonathan A. Kelber. Analysis of microenvironment effects on pancreatic cancer biomarker expression using a novel method for FFPE RNA extraction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-263.