Formalin-fixed Paraffin-embedded Patient Tissue Research Articles

The role of miRNA in colorectal cancer diagnosis: A pilot study.

Despite recent advances in diagnosis and treatment, colorectal cancer (CRC) remains the third most common cancer worldwide, and has both a poor prognosis and a high recurrence rate, thus indicating the need for new, sensitive and specific biomarkers. MicroRNAs (miRNAs/miRs) are important regulators of gene expression, which are involved in numerous biological processes implicated in tumorigenesis. The objective of the present study was to investigate the expression of miRNAs in plasma and tissue samples from patients with CRC, and to examine their potential as CRC biomarkers. Using reverse transcription-quantitative PCR, it was revealed that miR-29a, miR-101, miR-125b, miR-146a and miR-155 were dysregulated in the formalin-fixed paraffin-embedded tissues of patients with CRC, compared with the surrounding healthy tissue, and these miRNAs were associated with several pathological features of the tumor. Bioinformatics analysis of overlapping target genes identified AGE-RAGE signaling as a putative joint regulatory pathway. miR-146a was also upregulated in the plasma of patients with CRC, compared with the healthy control group, and had a fair discriminatory power (area under the curve, 0.7006), with 66.7% sensitivity and 77.8% specificity. To the best of our knowledge, this distinct five-miRNA deregulation pattern in tumor tissue, and upregulation of plasma miR-146a, were shown for the first time in patients with CRC; however, studies on larger patient cohorts are warranted to confirm their potential to be used as CRC diagnostic biomarkers.

Comparison of 3 PCR-based assays for microsatellite instability detection in formalin-fixed paraffin-embedded tissues of patients with colorectal cancer.

e15042 Background: Routine assessment of microsatellite instability (MSI) status in colorectal cancer (CRC) patients can help with initial screening for Lynch syndrome, prediction of tumor prognosis and selection of patients for immunotherapy. Immunohistochemistry (IHC) and PCR-based approaches represent the current clinical laboratory standard for the evaluation of MSI status. Both Bethesda and Promega marker panels have been used widely in the world and the Bethesda panel and the six mononucleotides panel (NR21/BAT25/NR24/NR27/MONO27/BAT26) have been approved by the National Medical Products Administration (NMPA) in China. The Idylla MSI Test, a fully automated system based on real-time PCR, which incorporates 7 novel MSI loci (ACVR2A/BTBD7/DIDO1/MRE11/RYR3/SEC31A/SULF2) can perform MSI analysis within 150min using 1-3 tissue slides. The aim of this study was to evaluate the performance of the three 3 PCR-based assays for MSI detection including Idylla MSI Test, Bethesda and that six mononucleotides panel. Methods: One hundred and fifty-three formalin-fixed paraffin-embedded (FFPE) tumor tissues from patients with CRC were collected and their MSI status were analyzed using 3 PCR-based assays, respectively, i.e. the Idylla MSI, “2B3D” NCI panel and the six mononucleotides panel. All samples were previously evaluated by IHC method. Results: In the all 153 patients, IHC identified 27 cases to be MMR-deficient (dMMR), and 126 to be MMR-proficient (pMMR). Using IHC as a reference, the Idylla MSI had a higher consistency that showed overall agreement, sensitivity and specificity as 93.46%, 92.89% and 93.65%, respectively, while the corresponding values of the six mononucleotides panel were 84.31%, 92.59% and 82.54% respectively, and the “2B3D” NCI panel showed a sensitivity of 92.16%, a specificity of 88.89% and an overall concordance of 92.86%. The PCR-based approaches identified 11 more MSI-H cases than IHC while 22 dMMR cases identified by IHC were detected by all the 3 assays, 2 dMMR by 2 assays, 1 dMMR by one assay only and 1 dMMR by neither assays above. Moreover, the Idylla MSI showed advantages in ease of use, turnaround time (TAT) and small dose of tissue input. Conclusions: Our findings support that the Idylla MSI assay provides a rapid, reliable, and ease-to-use solution to MSI detection in Chinese CRC patients with high sensitivity and specificity. The inconsistent cases between IHC and PCR-based approaches need a further analysis.

Comparison detection methods for EGFR in formalin-fixed paraffin-embedded tissues of patients with NSCLC

Epidermal growth factor receptor (EGFR) is an important gene in the development of lung cancer. Non-small cell lung cancer (NSCLC) is the most common lung cancer. In the present study, the expression of EGFR in 717 patients with NSCLC was detected by Ventana automatic immunohistochemical technique, and the samples was verified by Real-time PCR, and then the results were compared with the data acquired by next-generation sequencing technology (NGS), which is the high throughput, multiple sites for EGFR gene mutation testing. The expression of Ventana EGFR in 717 cases of NSCLC was detected by immunohistochemistry, and the positive rate was 60.70 % (435 / 717). The mutation rate of EGFR was 57.60 % (413/717). The coincidence rate of Ventana EGFR immunohistochemical assay and Real-time PCR assay reached 94.94 %, and the two had high consistency. The coincidence rate of Ventana EGFR immunohistochemical assay and NGS were high correlation. Based on these results, Ventana EGFR automatic immunohistochemical detection has high accuracy, simple operation process, low price and easy interpretation. It can be used as the preferred method for EGFR detection.

EGFR T790M detection in formalin-fixed paraffin-embedded tissues of patients with lung cancer using RNA-based in situ hybridization: A preliminary feasibility study.

BackgroundFollowing drug resistance in patients with lung cancer treated by EGFR TKIs, a biopsy is required to obtain sufficient cancer tissue for T790M detection in order to select potential beneficiaries suitable for third‐generation EGFR TKIs, such as osimertinib. The purpose of this study was to explore the feasibility of using a new in situ analysis technique based on RNA target sequences to detect EGFR T790M in lung cancer.MethodsA total of 28 formalin‐fixed paraffin‐embedded (FFPE) samples from 24 lung adenocarcinoma patients archived in Peking Union Medical College Hospital from 2015 to 2016 were collected. The BaseScope T790M detection technique by in situ hybridization on FFPE slides was used to analyze the mutation of EGFR T790M, and then the results were compared with the data acquired by Scorpions ARMS assay, which is the so‐called gold standard for EGFR gene mutation testing. The sensitivity and specificity of the BaseScope T790M detection technique were preliminarily evaluated.ResultsOf the 28 FFPE specimens, the average proportion of T790M‐positive cells was 35.78% ± 17.68% in 18 samples with EGFR T790M, confirmed by Scorpions ARMS assay, Compared with real‐time PCR assay, the sensitivity and specificity of BaseScope T790M were all 100% in our cohort.ConclusionBaseScope T790M assay could be completed on only one FFPE slide and the visualized molecular result overplayed with histomorphological information perfectly, so it may be the alternative method for EGFR T790M evaluation. BaseScope assay has potential clinical utility, and it will be necessary to carry out validation with a large number of cases.

Identifying Mcl-1 protein dependencies using dimerization-specific antibody biomarker for predicting response to targeted apoptosis inducing therapies.

7038 Background: The anti-apoptotic Bcl-2 family proteins facilitate pro-survival and resistance to anti-cancer therapies. Measuring the function of these proteins has shown utility in predicting response to therapy. A method that expands the principle of BH3 profiling to solid tumors is presented. Measuring the occurrence of heterodimers of Myeloid Leukemia Cell Differentiation Protein (Mcl-1), and pro-apoptotic binding protein Bim is an indicator of cancer cell apoptotic priming state. The readout of Mcl-1 containing complex-specific biomarkers can identify survival dependencies in cancer cells potentially providing clinical utility in guiding cancer treatments. Methods: Engineered immunogens that recapitulate conformation-specific epitopes induced during binding of the Mcl-1/Bim protein complex were used to generate. monoclonal antibodies. One Heterodimer Specific Mcl-1 Bim (HsMcB) was chosen. The selective binding was confirmed using ELISA, fluorescence polarization, immunofluorescence microscopy and flow cytometry in Bim or Mcl-1 knockdown cells, and by immunohistochemistry in formalin-fixed paraffin-embedded patient tissue. Correlation of HsMcB measurements to BH3 profiling readouts from the Mcl-1 restricted Noxa peptide was explored. Results: HsMcB signal depends on both Mcl-1 and Bim protein levels. The ratio of the HsMcB to unbound protein (Mcl-1) signal ([HsMcB]/[Mcl-1]) was measured. Disruption of the complexes by BH3 mimetics targeted to Mcl-1 and depletion of Mcl-1 level using CDK9 inhibitors diminished the [HsMcB]/[Mcl-1] readout. We see correlations between these readouts and BH3 mimetic peptides readouts from the Mcl-1 specific Noxa and MS1 BH3 mimetic peptides in AML patient samples that have been treated with Mcl-1 inhibitors. Conclusions: Mcl-1 dependence is a predictive biomarker for venetoclax resistance and for response to Mcl-1 targeted therapies. Flow cytometric and IHC based measurements of a heterodimer complex offer a direct and simpler approach that harbors potential for use in clinical settings. Additional antibodies targeting Mcl-1/Bak and Mcl-1 Noxa complexes are being tested.

ERBB2 Expression as Potential Risk-Stratification for Early Cystectomy in Patients with pT1 Bladder Cancer and Concomitant Carcinoma in situ

Background/Aims/Objectives: It is difficult to identify patients with a non-muscle-invasive bladder cancer (NMIBC) at stage pT1 with concomitant carcinoma in situ (Cis) who will benefit from an early cystectomy. Methods: We retrospectively analyzed clinical data and formalin-fixed paraffin-embedded tissues of patients with NMIBC. Messenger ribonucleic acid (mRNA) expression of progesterone receptor (PGR), estrogen receptor (ESR1), ERBB2, and marker of proliferation Ki-67 (MKI67) was measured by single-step reverse transcription quantitative real-time polymerase chain reaction using RNA-specific TaqMan assays. Relative gene expression was determined by the normalization of 2 reference genes (CALM2, B2M) using the 40 ΔΔCT method and relative gene expression was correlated to the histopathological stage and oncological outcome. Results: Of 302 patients with pT1 NMIBC in the initial transurethral resection of the bladder, 65 had a concomitant Cis. Elevated ERBB2 expression (>40.1) significantly correlated with progress in patients with and without concomitant Cis (p = 0.020 and p = 0.049, respectively). For the subgroup of pT1 with concomitant Cis, elevated ERBB2 expression significantly discriminated between a high-risk group of 55% progression-free survival (PFS) and a low-risk group of 90% PFS after a 5-year follow-up (p = 0.020). Cox-regression analysis revealed ERBB2 expression as the only independent prognostic factor for PFS (p = 0.0037). Conclusions: High mRNA expression of ERBB2 can identify patients with pT1 NMIBC with concomitant Cis, who have a high risk of progression and might benefit from an early cystectomy.

Machine Learning-based Classification of Diffuse Large B-cell Lymphoma Patients by Their Protein Expression Profiles

Characterization of tumors at the molecular level has improved our knowledge of cancer causation and progression. Proteomic analysis of their signaling pathways promises to enhance our understanding of cancer aberrations at the functional level, but this requires accurate and robust tools. Here, we develop a state of the art quantitative mass spectrometric pipeline to characterize formalin-fixed paraffin-embedded tissues of patients with closely related subtypes of diffuse large B-cell lymphoma. We combined a super-SILAC approach with label-free quantification (hybrid LFQ) to address situations where the protein is absent in the super-SILAC standard but present in the patient samples. Shotgun proteomic analysis on a quadrupole Orbitrap quantified almost 9,000 tumor proteins in 20 patients. The quantitative accuracy of our approach allowed the segregation of diffuse large B-cell lymphoma patients according to their cell of origin using both their global protein expression patterns and the 55-protein signature obtained previously from patient-derived cell lines (Deeb, S. J., D'Souza, R. C., Cox, J., Schmidt-Supprian, M., and Mann, M. (2012) Mol. Cell. Proteomics 11, 77–89). Expression levels of individual segregation-driving proteins as well as categories such as extracellular matrix proteins behaved consistently with known trends between the subtypes. We used machine learning (support vector machines) to extract candidate proteins with the highest segregating power. A panel of four proteins (PALD1, MME, TNFAIP8, and TBC1D4) is predicted to classify patients with low error rates. Highly ranked proteins from the support vector analysis revealed differential expression of core signaling molecules between the subtypes, elucidating aspects of their pathobiology.

Abstract 721: Multiplexed systems pathology for in-depth analysis of the tumor microenvironment: a strong correlation between pAkt and androgen receptor in the epithelial component of prostate cancer.

Abstract It is important to understand the heterogeneity across the cancer tissue as well as the spatial interactions and signaling between the different components that constitute the tumor microenvironment; epithelium, stroma, inflammatory cells, etc. The state of the tumor microenvironment and its variability within and in between tumors may give important insights on cancer initiation and progression, diagnostics, prognostics and therapeutic assessment. Thus, there is a major need to assess multiple molecular targets simultaneously, quantitatively, and within the specific tumor sub-compartments. Here, we demonstrate a systems pathology approach for quantitative detection of multiple proteins in formalin-fixed paraffin-embedded patient tissue samples using a combination of immunohistochemistry and high-throughput fluorescence imaging. The study cohort consisted of 50 prostate cancer tissue samples from 25 patients exhibiting different disease stages in a tissue microarray (TMA) format. We developed a multiplexed assay for detecting pAkt, AR, and cytokeratin 8 (CK8) simultaneously using fluorescent nanoparticle labeling and high-throughput quantitative imaging. Fluorescent signals of pAkt and AR were automatically quantified within the segmented CK8-positive prostate epithelium. The stromal compartment and the non-malignant prostate tissue were analysed separately. Our results showed that the expression of pAkt and AR correlated strongly in the prostate cancer epithelium (Pearson correlation coefficient 0.89, p < 0.001). This suggests that the androgen-dependent signaling is strongly associated with the pAkt signaling in cancer, with potential ramifications for administrating and monitoring anti-androgens and/or Akt-inhibitor therapy. The systems pathology approach enables quantitative in situ multianalyte detection for any key cancer signaling proteins. The analyses can be readily expanded to TMAs of large prospective or clinical trial cohorts with automated imaging. The ability to quantitatively study the spatial arrangement of the tumor microenvironment could have decisive advantages and improve the diagnostic and therapeutic predictions. Citation Format: Sami Blom, Petra Mäki-Teeri, Tuomas Mirtti, Antti Rannikko, Jenni Säilä, Teijo Pellinen, Olli Kallioniemi. Multiplexed systems pathology for in-depth analysis of the tumor microenvironment: a strong correlation between pAkt and androgen receptor in the epithelial component of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 721. doi:10.1158/1538-7445.AM2013-721

Abstract C153: Quantitative analysis of IGF-1R expression in FFPE human rhabdomyosarcoma tumor tissue by mass spectrometry.

Abstract The IGF-1R pathway is activated in many cancer types which has led to clinical development of many IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing's sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify patients most likely to benefit from anti-IGF1R therapies. We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform which enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. The IGF1R-specific SRM assay was preclinically validated on cell lines expressing a range of IGF1R protein. The expression levels of IGF1R in seven formalin fixed rhabdomyosarcoma (RMS) cell lines were measured by SRM mass spectrometry. We detected a range of IGF1R expression from 68 to 333 amol/ug for these cell lines, values that showed extremely high correlation with antibody based techniques. Analysis of FFPE human rhabdomyosarcoma xenograft explants demonstrates that variable levels of IGF1R expression can be measured accurately. In xenografts where treatment with a therapeutic IGF1R antibody caused tumor regression there was a concomitant loss of IGF1R expression. We have extended these studies by characterizing the expression of IGF1R in a clinical cohort of 25 RMS tumors. IGF1R was detected in 13/14 embryonal RMS tumors and all 11 syncytial RMS tumors. The range of expression in these tissues was 50–250 amol/ug of protein tissue. However, there was one tumor which expressed ∼4500 amol/ug of IGF1R and may represent a stable amplification, or mutation resulting in this extremely high level of IGF1R expression. Additional studies are underway to continue characterization of this tumor. These novel techniques for quantifying IGF1R protein expression in FFPE RMS tumor tissue may enhance our ability to select patients for IGF1R targeted therapy. We are extending this work in two ways. First we are initiating retrospective analysis of IGF1R levels in a sarcoma trial of an IGFR targeted therapy, and second, we are also preparing to use this assay for prospective analysis of FFPE sarcoma tissue in a future IGF1R targeted therapy trial. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C153.

Quantitative analysis of IGF-1R expression in FFPE human rhabdomyosarcoma tumor tissue by mass spectrometry.

10069 Background: The IGF-1R pathway is activated in many cancer types which has led to clinical development of many IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing’s sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify patients most likely to benefit from anti-IGF1R therapies. Methods: We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue-SRM technology platform which enables r...

Abstract 2211: Quantitative analysis of IGF-1R expression and signaling pathway activation in FFPE xenograft and human tumor tissues

Abstract The IGF-1R signal pathway is activated in many cancer types which has led to active clinical development of both antibody and small molecule IGF1R targeted therapeutics. Multiple clinical trials targeting IGF1R are currently underway, however after initial optimism in NSCLC and Ewing's sarcoma, several trials have failed to show efficacy or had severe toxicity. Pretreatment quantitation of tumor IGF1R protein expression remains a challenge due to high homology between IGF1R and the insulin receptor. Thus, there is a need for a specific and quantitative clinical assay for IGF-1R protein expression. The ability to quantify IGF1R expression and determine signaling pathway activation status directly in formalin-fixed paraffin-embedded (FFPE) patient tissue biopsies should help identify and select patients most likely to benefit from anti-IGF1R therapies. We have developed a quantitative IGF1R assay which can be performed directly in FFPE patient tissue. This approach is based on the Liquid Tissue®-SRM technology platform which enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed tissue. The IGF1R- specific SRM assay was developed and preclinically validated on cell lines expressing a range of IGF1R protein. The final assay has a LOD of <20 attomol/ug and an LOQ of ∼35 attomol/ug, values that are well in line with expected levels of expression in primary tumor sourced cell lines and xenograft explants. The expression levels of IGF1R in seven formalin fixed rhabdomyosarcoma (RMS) cell lines were measured by SRM mass spectrometry. We detected a range of IGF1R expression from 68 to 333 amol/ug for these cell lines, values that showed extremely high correlation with antibody based techniques (Cao et al, Cancer Res. 68:8039). We extended this quantitation study by measuring expression of IGF-1R in human rhabdomyosarcoma xenograft explants and a cohort of ∼40 embryonal and alveolar rhabdomyosarcoma tumors. Interestingly, in xenografts treated with a therapeutic IGF1R antibody, tumor regression was seen, and a loss of IGF1R expression was noted by SRM analysis. Our goal is to define expression levels of IGF1R in these tumors, and to validate this assay for use as a companion diagnostic in follow on clinical trials of IGF1R inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2211. doi:10.1158/1538-7445.AM2011-2211