Abstract
Background
Conventional HER2-targeting therapies improve outcomes for patients with HER2-positive breast cancer (BC), defined as tumors showing HER2 protein overexpression by ...immunohistochemistry and/or ERBB2 gene amplification determined by in situ hybridization (ISH). Emerging HER2-targeting compounds show benefit in some patients with neither HER2 protein overexpression nor ERBB2 gene amplification, creating a need for new assays to select HER2-low tumors for treatment with these compounds. We evaluated the analytical performance of a targeted mass spectrometry-based assay for quantifying HER2 protein in formalin-fixed paraffin-embedded (FFPE) and frozen BC biopsies.
Methods
We used immunoaffinity-enrichment coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM-MS) to quantify HER2 protein (as peptide GLQSLPTHDPSPLQR) in 96 frozen and 119 FFPE BC biopsies. We characterized linearity, lower limit of quantification (LLOQ), and intra- and inter-day variation of the assay in frozen and FFPE tissue matrices. We determined concordance between HER2 immuno-MRM-MS and predicate immunohistochemistry and ISH assays and examined the benefit of multiplexing the assay to include proteins expressed in tumor subcompartments (e.g., stroma, adipose, lymphocytes, epithelium) to account for tissue heterogeneity.
Results
HER2 immuno-MRM-MS assay linearity was ≥103, assay coefficient of variation was 7.8% (FFPE) and 5.9% (frozen) for spiked-in analyte, and 7.7% (FFPE) and 7.9% (frozen) for endogenous measurements. Immuno-MRM-MS-based HER2 measurements strongly correlated with predicate assay HER2 determinations, and concordance was improved by normalizing to glyceraldehyde-3-phosphate dehydrogenase. HER2 was quantified above the LLOQ in all tumors.
Conclusions
Immuno-MRM-MS can be used to quantify HER2 in FFPE and frozen BC biopsies, even at low HER2 expression levels.
Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate ...protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues. Although the feasibility of using targeted, multiple reaction monitoring mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope-labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e., nine processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R 2 = 0.94) and immuno-MRM (R 2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens.
Phosphoproteins are the key indicators of signaling network pathway activation. Many disease treatment therapies are designed to inhibit these pathways and effective diagnostics are required to ...evaluate the efficacy of these treatments. Phosphoprotein IHC have been impractical for diagnostics due to inconsistent results occurring from technical limitations. We have designed and tested a novel cold transport device and rapid cold plus warm formalin fixation protocol using phosphoproteins IHC. We collected 50 liver tumors that were split into two experimental conditions: 2 + 2 rapid fixation (2 hours cold then 2 hour warm formalin) or 4 hour room-temperature formalin. We analyzed primary hepatocellular carcinoma (n = 10) and metastatic gastrointestinal tumors (n = 28) for phosphoprotein IHC markers pAKT, pERK, pSRC, pSTAT3, and pSMAD2 and compared them to slides obtained from the clinical blocks. Expression of pERK and pSRC, present in the metastatic colorectal carcinoma, were better preserved with the rapid processing protocol while pSTAT3 expression was detected in hepatocellular carcinoma. Differences in pSMAD2 expression were difficult to detect due to the ubiquitous nature of protein expression. There were only 3 cases expressing pAKT and all exhibited a dramatic loss of signal for the standard clinical workflow. The rapid cold preservation shows improvement in phosphoprotein preservation.
Abstract
Objectives
Standard implementations of amyloid typing by liquid chromatography–tandem mass spectrometry use capabilities unavailable to most clinical laboratories. To improve accessibility ...of this testing, we explored easier approaches to tissue sampling and data processing.
Methods
We validated a typing method using manual sampling in place of laser microdissection, pairing the technique with a semiquantitative measure of sampling adequacy. In addition, we created an open-source data processing workflow (Crux Pipeline) for clinical users.
Results
Cases of amyloidosis spanning the major types were distinguishable with 100% specificity using measurements of individual amyloidogenic proteins or in combination with the ratio of λ and κ constant regions. Crux Pipeline allowed for rapid, batched data processing, integrating the steps of peptide identification, statistical confidence estimation, and label-free protein quantification.
Conclusions
Accurate mass spectrometry–based amyloid typing is possible without laser microdissection. To facilitate entry into solid tissue proteomics, newcomers can leverage manual sampling approaches in combination with Crux Pipeline and related tools.
The development of precision testing for disease diagnosis has advanced medicine by specifically matching patients with drugs to treat specific diseases. High-quality diagnostics start with ...high-quality tissue specimens. The development and optimization of tissue handling and processing have lagged behind bioassay development. Ultrasound time-of-flight (TOF) technology has been successfully used to monitor the critical processing step of tissue fixation with formalin. In this study, we expand the use of this technology to monitor tissue dehydration and clearing by analyzing TOF signals from 270 different specimens, representing 13 different tissue types obtained through surgical resections. We determined the time constant τ
for each tissue type for the following tissue processing solvents: 70% ethanol, 90% ethanol, 100% ethanol, and xylene. The TOF signals were correlated with tissue morphology to ensure that high-quality tissue was produced. Tissues can be grouped into those exhibiting fast and slow reagent diffusion. We monitored incomplete dehydration of tissue by skipping a key processing step, dehydration in absolute ethanol, and then correlated the τ
with poor histomorphology, demonstrating that the technique can detect significant processing errors. Ultrasound TOF technology can therefore be used to monitor all phases of tissue processing cycle and yields an important preanalytical quality metric.
Personalized medicine promises diagnosis and treatment of disease at the individual level and relies heavily on clinical specimen integrity and diagnostic assay quality. Preanalytics, the collection ...and handling steps of a clinical specimen before immunohistochemistry or other clinical assay, are critically important to enable the correct diagnosis of disease. However, the effects of preanalytics are often overlooked due to a lack of standardization and limited assessment tools to quantify their variation. Here, we report a novel real-time ultrasound time-of-flight instrument that is capable of monitoring and imaging the critical step in formalin fixation, diffusion of the fixative into tissue, which provides a quantifiable quality metric for tissue fixation in the clinical laboratory ensuring consistent downstream molecular assay results. We analyzed hundreds of tissue specimens from 34 distinct human tissue types and 12 clinically relevant diseased tissues for diffusion and fixation metrics. Our measurements can be converted into tissue diffusivity constants that correlate with the apparent diffusion constant calculated using magnetic resonance imaging (R=0.83), despite the differences in the approaches, indicating that our approach is biophysically plausible. Using data collected from time-of-flight analysis of many tissues, we have therefore developed a novel rapid fixation program that could ensure high-quality downstream assay results for a broad range of human tissue types.
Five novel brominated polyacetylenic diols, diplynes A−E (2−6), and three sulfated analogues, diplyne A 1-sulfate (7), diplyne C 1-sulfate (8), and 2-deoxydiplyne D sulfate (9), were isolated from ...the Philippines sponge Diplastrella sp. by employing bioassay-guided fractionation using the HIV-1 integrase inhibition assay. The novel metabolites were characterized by interpretation of spectroscopic data.
Background
Numerous genome‐wide association studies (GWASs) have been performed for the determination of genes and variants contributing to Alzheimer’s disease (AD). We hypothesized that it is ...important to explore whether the previously‐reported AD‐associated genes in GWASs can have neuropathological effects in the brain and can be employed as potential biomarkers for neuropathological outcomes such as neuritic plaque development. Neuritic plaques (also known as amyloid plaques) are extracellular deposits of the amyloid beta protein primarily found in the grey matter of the brain and are the most discernible neuropathological alterations observed in individuals with dementia.
Method
Here, we report the genetic association study of neuropathology data such as neuritic plaque measurements in an AD cohort at the National Alzheimer’s Coordinating Center (NACC) using a sample of 1,283 individuals. We acquired the genetic data from the Alzheimer’s Disease Sequencing Project (ADSP) which generated whole genome sequencing data through the ADSP Quality Control pipeline. We employed 18 loci where the associations of these loci with AD risk were validated by the recent GWASs. Analysis was obtained using a linear mixed model after adjustment for covariates including age at death, gender, the number of the APOE e4 allele (i.e., 0, 1, and 2), ancestry representative principle components (i.e., population structure), and a genetic relatedness matrix (i.e., a polygenic effect due to genetic relatedness among individuals).
Result
From our analysis, we generalized to the NACC cohort significant associations after Bonferroni correction with neuritic plaque measurements and one previously identified locus such as BIN1 on chromosome 2 (p = 4.65×10−5). Furthermore, we detected, at the significance level after Bonferroni correction, a novel locus for neuritic plaque formation that has not previously been reported: PICALM (or nearby LINC02695 or nearby RNU6‐560P) on chromosome 11 (p = 1.02×10−3). PICALM is a strong candidate for playing a role in neuritic plaque formation because deficiency in PICALM results in diminished amyloid‐β clearance.
Conclusion
The results from our genetic association study of neuritic plaque formation in the AD cohort underline the potential of utilizing neuropathology data to discover additional support for relevant loci being associated with AD across diverse populations.
Two novel sulfated sterols, ibisterol sulfates B (
3) and C (
4), and an unprecedented non-sulfated sterol, 4β,5β-epoxy-2β,3α,12β,22
S-tetrahydroxy-14α-methylcholest-7,9(11)-dien-6,24-dione (
5) have ...been isolated from a
Xestospongia sp. collected in the Philippines. The structures were elucidated using spectroscopic data. These sterols were found to be inhibitors of HIV-1 integrase.
The structures of HIV-1 integrase inhibitors, ibisterol sulfates B (R=H) and C (R=Me) and 4β,5β-epoxy-2β,3α,12β,22
S-tetrahydroxy-14α-methylcholest-7,9(11)-dien-6,24-dione were elucidated using spectroscopic data.
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