Part of developing therapeutics is the need to identify patients who will respond to treatment. For HER2-targeted therapies, such as trastuzumab, the expression level of HER2 is used to identify ...patients likely to receive benefit from therapy. Currently, chromogenic immunohistochemistry on patient tumor tissue is one of the methodologies used to assess the expression level of HER2 to determine eligibility for trastuzumab. However, chromogenic staining is fraught with serious drawbacks that influence scoring, which is additionally flawed due to the subjective nature of human/pathologist bias. Thus, to advance drug development and precision medicine, there is a need to develop technologies that are more objective and quantitative through the collection and integration of larger data sets. In proof of concept experiments, we show multiplexed ion beam imaging (MIBI), a novel imaging technology, can quantitate HER2 expression on breast carcinoma tissue with known HER2 status and those values correlate with pathologist-determined IHC scores. The same type of quantitative analysis using the mean pixel value of five individual cells and total pixel count of the entire image was extended to a blinded study of breast carcinoma samples of unknown HER2 scores. Here, a strong correlation between quantitation of HER2 by MIBI analysis and pathologist-derived HER2 IHC score was identified. In addition, a comparison between MIBI analysis and immunofluorescence-based automated quantitative analysis (AQUA) technology, an industry-accepted quantitation system, showed strong correlation in the same blind study. Further comparison of the two systems determined MIBI was comparable to AQUA analysis when evaluated against pathologist-determined scores. Using HER2 as a model, these data show MIBI analysis can quantitate protein expression with greater sensitivity and objectivity compared to standard pathologist interpretation, demonstrating its potential as a technology capable of advancing cancer and patient diagnostics.
Antibodies are among the most frequently used tools in basic science research and in clinical assays, but there are no universally accepted guidelines or standardized methods for determining the ...validity of these reagents. Furthermore, for commercially available antibodies, it is clear that what is on the label does not necessarily correspond to what is in the tube. To validate an antibody, it must be shown to be specific, selective, and reproducible in the context for which it is to be used. In this review, we highlight the common pitfalls when working with antibodies, common practices for validating antibodies, and levels of commercial antibody validation for seven vendors. Finally, we share our algorithm for antibody validation for immunohistochemistry and quantitative immunofluorescence.
Quantification of mRNA has historically been done by reverse transcription polymerase chain reaction (RT-PCR). Recently, a robust method of detection of mRNA utilizing in situ hybridization has been ...described that is linear and shows high specificity with low background. Here we describe the use of the AQUA method of quantitative immunofluorescence (QIF) for measuring mRNA in situ using ESR1 (the estrogen receptor alpha gene) in breast cancer to determine its predictive value compared to Estrogen Receptor α (ER) protein.
Messenger RNA for ER (ESR1) and Ubiquitin C (UbC) were visualized using RNAscope probes and levels were quantified by quantitative in situ hybridization (qISH) on two Yale breast cancer cohorts on tissue microarrays. ESR1 levels were compared to ER protein levels measured by QIF using the SP1 antibody.
ESR1 mRNA is reproducibly and specifically measurable by qISH on tissue collected from 1993 or later. ESR1 levels were correlated to ER protein levels in a non-linear manner on two Yale cohorts. High levels of ESR1 were found to be predictive of response to tamoxifin.
Quantification of mRNA using qISH may allow assessment of large cohorts with minimal formalin fixed, paraffin embedded tissue. Exploratory data using this method suggests that measurement of ESR1 mRNA levels may be predictive of response to endocrine therapy in a manner that is different from the predictive value of ER.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Systematic and quantitative analysis of protein phosphorylation is revealing dynamic regulatory networks underlying cellular responses to environmental cues. However, matching these sites to the ...kinases that phosphorylate them and the phosphorylation-dependent binding domains that may subsequently bind to them remains a challenge. NetPhorest is an atlas of consensus sequence motifs that covers 179 kinases and 104 phosphorylation-dependent binding domains Src homology 2 (SH2), phosphotyrosine binding (PTB), BRCA1 C-terminal (BRCT), WW, and 14-3-3. The atlas reveals new aspects of signaling systems, including the observation that tyrosine kinases mutated in cancer have lower specificity than their non-oncogenic relatives. The resource is maintained by an automated pipeline, which uses phylogenetic trees to structure the currently available in vivo and in vitro data to derive probabilistic sequence models of linear motifs. The atlas is available as a community resource (http://netphorest.info).
BackgroundSpatial architecture within the tumor microenvironment (TME) can potentially impact disease pathology, progression and treatment response of DLBCL. Additionally, activation of host’s immune ...system greatly determines response to immunotherapy treatments. Multiplex imaging using fluorescence immunohistochemistry (FIHC) has found to enable characterization and quantification of tissue-specific proteins involved in immune activation, which in turn have enabled identification of distinct cell types, their functional status, and their organization in TME. We developed and validated two complementary quantitative multiplex fluorescence IHC (mFIHC) panels using hypothesis-driven spatial profiling algorithms (Automated Quantitative Analysis known as AQUA® Technology) to evaluate T and B cell populations and their functional status to generate detailed spatial information of tumor microenvironment of DLBCL. mFIHC assays when combined with the use of AQUA® Technology were able to predict immunotherapies in a systematic meta-analysis of over 8000 patients treated with PD1/L1 pathway blockers.1 Implementation of mFIHC assays coupled with robust image analysis may assist in risk stratification of patients or enable efficacy assessment of immunomodulating cancer therapies.MethodsWe designed two novel mFIHC assays to (1) characterize various T (CD2, CD3, CD8, CD28) and B cell populations (CD2, CD3, CD19, PAX5), and (2) evaluate T cell functional status (CD8, Granzyme B, PD1). We successfully validated clinical grade mFIHC assays using automated staining (Leica Bond RX), imaging (PhenoImager HT) and analysis (AQUA® Technology) workflow.ResultsSensitivity, accuracy and specificity were confirmed for all mFIHC assays on known positive and negative controls. Excellent reproducibility (less than 35% CV) and precision were observed across instruments, operators and independent experiments for all markers. Between these two panels, over 200 unique parameters were evaluated. The prevalence of CD2, CD3, CD19, and PAX5 ranged from 20% to >90% positive for the DLBCL samples tested and were overall highly concordant with each other.ConclusionsThe validated mFIHC assays examined B and T cell antigen expression in DLBCL and their interaction with PAX5+ and CD58+ cells within the TME. Implementation of mFIHC assays coupled with robust image analysis may assist in risk stratification of patients or enable efficacy assessment of immunomodulating cancer therapies. These assays may be used to further understand the complex immune cell and tumor cell spatial biology in the context of clinical trials.ReferenceLu, Steve, Julie E Stein, David L Rimm, Daphne W Wang, J Michael Bell, Douglas B Johnson, Jeffrey A Sosman, et al. ‘Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade: a systematic review and meta-analysis.’ JAMA oncology 5. 2019;8:1195–1204.
The RNA binding protein Lin28 and its paralog Lin28B are associated with advanced human malignancies. Blocking the biogenesis of let-7 miRNA, a tumor suppressor, by Lin28/Lin28B has been thought to ...underlie their roles in cancer. Here we report that the mRNA for the human epidermal growth factor receptor 2 (HER2), a HER-family receptor tyrosine kinase known to play a critical role in cell proliferation and survival and also a major therapeutic target in breast cancer, is among several targets of Lin28 regulation. We show that Lin28 stimulates HER2 expression at the posttranscriptional level, and that enforced Lin28 expression promotes cancer cell growth via multiple mechanisms. Consistent with its pleiotropic role in regulating gene expression, Lin28 overexpression in primary breast tumors is a powerful predictor of poor prognosis, representing the first report on the impact of Lin28 expression on clinical outcome in human cancer. While revealing another layer of regulation of HER2 expression in addition to gene amplification, our studies also suggest novel mechanistic insights linking Lin28 expression to disease outcome and imply that targeting multiple pathways is a common mechanistic theme of Lin28-mediated regulation in cancer.
Tumor survival is influenced by interactions between tumor cells and the stromal microenvironment. One example is Endosialin (Tumor Endothelial Marker-1 (TEM-1) or CD248), which is expressed ...primarily by cells of mesenchymal origin and some tumor cells. The expression, as a function of architectural masking, of TEM-1 and its pathway-associated proteins was quantified and examined for association with five-year disease-specific survival on a colorectal cancer (CRC) cohort divided into training (n=330) and validation (n=164) sets. Although stromal expression of TEM-1 had prognostic value, a more significant prognostic signature was obtained through linear combination of five compartment-specific expression scores (TEM-1 Stroma, TEM-1 Tumor Vessel, HIF2α Stromal Vessel, Collagen IV Tumor, and Fibronectin Stroma). This resulted in a single continuous risk score (TAPPS: TEM-1 Associated Pathway Prognostic Signature) which was significantly associated with decreased survival on both the training set HR=1.76 (95%CI: 1.44-2.15); p<0.001 and validation set HR=1.38 (95%CI: 1.02-1.88); p=0.04. Importantly, since prognosis is a critical clinical question in Stage II patients, the TAPPS score also significantly predicted survival in the Stage II patient (n=126) cohort HR=1.75 (95%CI: 1.22-2.52); p=0.002 suggesting the potential of using the TAPPS score to assess overall risk in CRC patients, and specifically in Stage II patients.
We sought to determine the predictive value of in situ mRNA measurement compared to traditional methods on a cohort of trastuzumab-treated metastatic breast cancer patients.
A tissue microarray ...composed of 149, classified as HER2-positive, metastatic breast cancers treated with various trastuzumab-containing chemotherapy regimens was constructed. HER2 intracellular domain(ICD), HER2 extracellular domain(ECD) and HER2 mRNA were assessed using AQUA. For HER2 protein evaluation, CB11 was used to measure ICD and SP3 to measure ECD of the HER2 receptor. In addition, HER2 mRNA status was assessed using RNAscope assay ERRB2 probe. Kaplan - Meier estimates were used for depicting time-to-event endpoints. Multivariate Cox regression models with backward elimination were used to assess the performance of markers as predictors of TTP and OS, after adjusting for important covariates.
HER2 mRNA was correlated with ICD HER2, as measured by CB11 HER2, with ECD HER2 as measured by SP3 (Pearson's Correlation Coefficient, r = 0.66 and 0.51 respectively) and with FISH HER2 (Spearman's Correlation Coefficient, r = 0.75). All markers, HER2 mRNA, ICD HER2 and ECD HER2, along with FISH HER2, were found prognostic for OS (Log-rank p = 0.007, 0.005, 0.009 and 0.043 respectively), and except for FISH HER2, they were also prognostic for TTP Log-rank p = 0.036, 0.068 and 0.066 respectively) in this trastuzumab- treated cohort. Multivariate analysis showed that in the presence of pre-specified set of prognostic factors, among all biomarkers only ECD HER2, as measured by SP3, is strong prognostic factor for both TTP (HR = 0.54, 95% CI: 0.31-0.93, p = 0.027) and OS (HR = 0.39, 95%CI: 0.22-0.70, p = 0.002).
The expression of HER2 ICD and ECD as well as HER2 mRNA levels was significantly associated with TTP and OS in this trastuzumab-treated metastatic cohort. In situ assessment of HER2 mRNA has the potential to identify breast cancer patients who derive benefit from Trastuzumab treatment.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
HER2/Neu (ErbB-2) overexpression, which occurs in 15-20% of breast cancer cases, is associated with better response to treatment with the drug trastuzumab. PhosphoHER2 (pHER2) has been evaluated for ...prediction of response to trastuzumab. Both markers are heterogeneously detected and are potentially subject to loss as a consequence of delayed time to fixation. Here, we quantitatively assess both markers in core needle biopsies (CNBs) and matched tumor resections to assess concordance between the core and the resection and between HER2 and pHER2.
A selected retrospective collection of archival breast cancer cases yielded 67 cases with both core and resection specimens. Both HER2 and pTyr(1248)HER2 were analyzed by the AQUA® method of quantitative immunofluorescence on each specimen pair.
Both HER2 immunoreactivity (P<0.0001) and pTyr(1248)HER2 immunoreactivity (P<0.0001) were lower in resections relative to CNB specimens. However, clinical implications of this change may not be evident since no case changed from 3+ (CNB) to negative (resection). Assessment of pTyr(1248)HER2 showed no direct correlation with HER2 in either CNB or resection specimens.
The data suggest that measurement of both HER2 and phospho- Tyr(1248)HER2, in formalin-fixed tissue by immunological methods is significantly affected by pre-analytic variables. The current study warrants the adequate handling of resected specimens for the reproducible evaluation of HER2 and pHER2. The level of pTyr(1248)HER2, was not correlated to total HER2 protein. Further studies are required to determine the significance of these observations with respect to response to HER2 directed therapies.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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