Determination of phosphorylated proteins in tissue specimens requires high‐quality samples collected under stringent conditions

Wolf, Corinna; Jarutat, Tiantom; Harring, Suzana Vega; Haupt, Kristin; Babitzki, Galina; Bader, Sabine; David, Kerstin A.; Juhl, Hartmut; Arbogast, Susanne

doi:10.1111/his.12268

Cited by 17 publications

(20 citation statements)

References 26 publications

(41 reference statements)

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“…Needle biopsies were collected under anesthesia and immediately flash frozen as controls. Tumors were excised and quarters transferred to sterile normal saline maintained at 25 C AE 3 C (cold ischemia) or 37 C (warm ischemia) for 1, 2, 3, 4, 5, 8, 10, 15, 30, or 60 minutes in temperature-controlled saline before being flash frozen. All flash-frozen samples were stored at À80 C, and lysates were processed within 2 weeks of collection.…”

Section: Xenograft Ischemia Studymentioning

confidence: 99%

“…Plates were incubated at ambient temperature (25 C AE 3 C) on an orbital shaker (600 rpm) for 1 hour to capture MET. After three washes with assay buffer, 100 mL/well 200 ng/mL biotin-conjugated mouse monoclonal MET antibody (clone L41G3) was added to the plates and incubated for 1 hour at 25 C AE 3 C, followed by a second wash and the addition of 100 mL/well 200 ng/mL streptavidin poly-HRP conjugate (Pierce). After 30-minute incubation with the HRP-conjugate, plates were washed 4 times and 100 mL SuperSignal ELISA Pico Chemiluminescent Substrate (Pierce) was added to each well.…”

Section: Met Immunoassay Proceduresmentioning

confidence: 99%

“…Preanalytic variables (specimen handling, shipping, and storage procedures) can have a significant impact on the reliability of biomarker measurements in the laboratory, and phosphoproteins involved in dynamic responses of signaling pathways are notoriously labile during specimen collection due to ischemia and other factors (23)(24)(25). The stability of full-length MET and its phosphospecies was characterized in biopsies of SNU5 xenografts subjected to increasing ischemia time, which was defined as the total time needed for core needle biopsy sampling, specimen handling, and flash freezing.…”

Section: Biopsy Handling To Control Preanalytic Variablesmentioning

confidence: 99%

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Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

Srivastava

Hollingshead

Weiner

et al. 2016

Clinical Cancer Research

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Purpose: Rational development of targeted MET inhibitors for cancer treatment requires a quantitative understanding of target pharmacodynamics, including molecular target engagement, mechanism of action, and duration of effect.Experimental Design: Sandwich immunoassays and specimen handling procedures were developed and validated for quantifying full-length MET and its key phosphospecies (pMET) in core tumor biopsies. MET was captured using an antibody to the extracellular domain and then probed using antibodies to its C-terminus (full-length) and epitopes containing pY1234/ 1235, pY1235, and pY1356. Using pMET:MET ratios as assay endpoints, MET inhibitor pharmacodynamics were characterized in MET-amplified and -compensated (VEGFR blockade) models.Results: By limiting cold ischemia time to less than two minutes, the pharmacodynamic effects of the MET inhibitors PHA665752 and PF02341066 (crizotinib) were quantifiable using core needle biopsies of human gastric carcinoma xenografts (GTL-16 and SNU5). One dose decreased pY1234/1235 MET: MET, pY1235-MET:MET, and pY1356-MET:MET ratios by 60% to 80% within 4 hours, but this effect was not fully sustained despite continued daily dosing. VEGFR blockade by pazopanib increased pY1235-MET:MET and pY1356-MET:MET ratios, which was reversed by tivantinib. Full-length MET was quantifiable in 5 of 5 core needle samples obtained from a resected hereditary papillary renal carcinoma, but the levels of pMET species were near the assay lower limit of quantitation.Conclusions: These validated immunoassays for pharmacodynamic biomarkers of MET signaling are suitable for studying MET responses in amplified cancers as well as compensatory responses to VEGFR blockade. Incorporating pharmacodynamic biomarker studies into clinical trials of MET inhibitors could provide critical proof of mechanism and proof of concept for the field.

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Section: Xenograft Ischemia Studymentioning

confidence: 99%

Section: Met Immunoassay Proceduresmentioning

confidence: 99%

Section: Biopsy Handling To Control Preanalytic Variablesmentioning

confidence: 99%

See 1 more Smart Citation

Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

Srivastava

Hollingshead

Weiner

et al. 2016

Clinical Cancer Research

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“…Their incorporation into systems modeling efforts will certainly allow for the complexity of the disease and provide the means to understand the process going forward. An additional consideration is the ability to effectively evaluate and quantify phosphorylated proteins in fixed/frozen tissue materials [34]. Careful consideration of appropriate biomarkers and tissue formats (needle biopsy vs resection specimens) must be considered in the overall utility of such markers for network/pathway analyses and model incorporation.…”

Section: Bladder Cancermentioning

confidence: 99%

Overcoming tumor heterogeneity in the molecular diagnosis of urological cancers

Donovan

Cordon‐Cardo

2014

Expert Review of Molecular Diagnostics

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Our understanding of tumor heterogeneity and impact on treatment response is still in its infancy, presenting significant challenges to the molecular pathologist, treating physician and ultimately for the patient. Given that tumor recurrence due to treatment resistance is the most common cause of cancer death, there remains a critical unmet need to change the current paradigm. The mechanisms which underlie tumor heterogeneity can be broadly divided into genomic instability and non-mutational processes, including stochastic variations in cellular responses, modulation by tumor microenvironment and or phenotypic/ functional plasticity relating to cancer stem cells. We believe that these biological mechanisms are not mutually exclusive and emphasize the need for more suitable methodologies to exploit the spatiotemporal patterns of intratumoral heterogeneity using novel approaches such as quantitative tissue-based biomarker assessment and systemic fluid analytics. Generating a comprehensive patient-centric phenotypic disease profile should generate a 'codex' which can be employed to change the current treatment decision process.

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“…The utility of phosphoproteins in clinical trials has been limited by the lability of the phosphoprotein network. Cold ischemia time, the time a tissue specimen sits ex vivo prior to fixation, is another preanalytical variable that has a demonstrated and profound effect on measurements of signaling proteins like phosphoproteins [11][12][13][14] . There is a clinical imperative to study and develop approaches that control and monitor the temperature and time that specimens experience prior to fixation.…”

mentioning

confidence: 99%

Effect of immediate cold formalin fixation on phosphoprotein IHC tumor biomarker signal in liver tumors using a cold transport device

Lerch

Kenerson

Chafin

et al. 2020

Sci Rep

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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. Cancer therapies often target specific proteins within pathways of biochemical networks. Precision medicine depends on the accurate diagnosis of disease using a variety of clinical assays to guide the selection of appropriate therapies 1. Diagnostic assays must be performed on high quality tissue where the biochemical networks are preserved and representative of the biological state of the highly dynamic signaling network. One example of a precision medicine test is immunohistochemistry (IHC), such as the test for BRAF V600E mutation using the VE1 clone IHC used in colon, thyroid, and melanoma for prognostic value to guide treatment decisions 2,3. BRAF V300E assays, like all IHC assays, are susceptible to preanalytical errors such as inadequate fixation or tissue processing, yet fixation and processing steps are often de-emphasized or taken for granted as an established part of the hospital workflow 1. Formalin fixation has been used for more than a century to preserve tissue for histopathology, our understanding of the biochemistry of formalin fixation is incomplete. There is evidence that cold formalin fixation improves the preservation of biochemical markers especially within signaling networks such as phosphoproteins 4,5 , and that cold formalin fixation has been shown to aid in the preservation of nucleic acids 6. The rapid two-temperature (2 + 2) protocol was developed to take advantage of the biochemistry of formaldehyde in solution, where methylene glycol exists in equilibrium with formaldehyde in a temperature dependent manner 4. At lower temperatures (4 °C), nonreactive methylene glycol is present in great excess o...

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Determination of phosphorylated proteins in tissue specimens requires high‐quality samples collected under stringent conditions

Cited by 17 publications

References 26 publications

Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

Pharmacodynamic Response of the MET/HGF Receptor to Small-Molecule Tyrosine Kinase Inhibitors Examined with Validated, Fit-for-Clinic Immunoassays

Overcoming tumor heterogeneity in the molecular diagnosis of urological cancers

Effect of immediate cold formalin fixation on phosphoprotein IHC tumor biomarker signal in liver tumors using a cold transport device

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