Tissue-based Analyses Research Articles

Circulating tumor DNA as a biomarker for predicting progression-free survival and overall survival in patients with epithelial ovarian cancer: a systematic review and meta-analysis

ObjectivesCirculating tumor DNA (ctDNA) is emerging as a potential prognostic biomarker in multiple tumor types. However, despite the many studies available on small series of patients with ovarian cancer, a...

The Traumatic Inoculation Process Affects TSPO Radioligand Uptake in Experimental Orthotopic Glioblastoma.

The translocator protein (TSPO) has been proven to have great potential as a target for the positron emission tomography (PET) imaging of glioblastoma. However, there is an ongoing debate about the potential various sources of the TSPO PET signal. This work investigates the impact of the inoculation-driven immune response on the PET signal in experimental orthotopic glioblastoma. Serial [18F]GE-180 and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) PET scans were performed at day 7/8 and day 14/15 after the inoculation of GL261 mouse glioblastoma cells (n = 24) or saline (sham, n = 6) into the right striatum of immunocompetent C57BL/6 mice. An additional n = 25 sham mice underwent [18F]GE-180 PET and/or autoradiography (ARG) at days 7, 14, 21, 28, 35, 50 and 90 in order to monitor potential reactive processes that were solely related to the inoculation procedure. In vivo imaging results were directly compared to tissue-based analyses including ARG and immunohistochemistry. We found that the inoculation process represents an immunogenic event, which significantly contributes to TSPO radioligand uptake. [18F]GE-180 uptake in GL261-bearing mice surpassed [18F]FET uptake both in the extent and the intensity, e.g., mean target-to-background ratio (TBRmean) in PET at day 7/8: 1.22 for [18F]GE-180 vs. 1.04 for [18F]FET, p < 0.001. Sham mice showed increased [18F]GE-180 uptake at the inoculation channel, which, however, continuously decreased over time (e.g., TBRmean in PET: 1.20 at day 7 vs. 1.09 at day 35, p = 0.04). At the inoculation channel, the percentage of TSPO/IBA1 co-staining decreased, whereas TSPO/GFAP (glial fibrillary acidic protein) co-staining increased over time (p < 0.001). We identify the inoculation-driven immune response to be a relevant contributor to the PET signal and add a new aspect to consider for planning PET imaging studies in orthotopic glioblastoma models.

Impact of genomic alterations measured in circulating tumor DNA (ctDNA) on clinical response to telisotuzumab vedotin treatment in patients with non-small cell lung cancer (NSCLC).

9032 Background: The antibody-drug conjugate telisotuzumab vedotin (Teliso-V) is composed of the c-Met–targeting antibody telisotuzumab (ABT-700) linked to the microtubule inhibitor monomethyl auristatin E. In the LUMINOSITY study (NCT03539536), efficacy of Teliso-V was seen in patients (pts) with EGFR wildtype nonsquamous NSCLC and c-Met overexpression (≥25% tumor cells at 3+ intensity by IHC); overall response rate: 36.5%. Data are limited on whether specific driver oncogene states affect responses. We investigated genomic alterations in relation to response to Teliso-V in this population. Methods: Pts received 1.9 mg/kg Teliso-V monotherapy once every 2 weeks in LUMINOSITY. ctDNA was isolated from plasma collected at different timepoints. The PGDx elio plasma complete assay was used to identify genomic alterations in ctDNA samples. This assay included 521 genes for single nucleotide variants and insertion-deletion mutations, 38 for amplifications (amp), and 21 for translocations. Results: In total, 52 pts were included in the study; ctDNA from 48 pts was analyzed. The overall response rate among pts with ctDNA results was 37.5% (18 pts with partial response) compared with 36.5% for the ITT population of 52 pts. Genomic alterations are listed in the Table.Three out of 4 pts with MET amp at baseline responded, accounting for 17% of total responses. The observed MET amp frequency in this MET IHC preselected cohort was 8%, which is similar to the prevalence observed in tissue analysis by FISH. Of note, 1 nonresponder harbored a MET ex14del mutation at baseline, and a responder had a low-frequency mutation detected at the final visit. Mutations in KRAS were the most common genomic alteration and were detected in 13 (27%) pts at baseline. Three pts with a KRAS mutation were responders; among these, 2 out of 3 had a KRAS G12C mutation (seen in 3 pts total). Response rates were higher in pts with MET amp (75%; 95% CI: 0.30, 0.95) vs those without MET amp (34%; 0.22, 0.49), and higher in pts without KRAS mutations (43%; 0.28, 0.59) vs those with KRAS mutations (23%; 0.08, 0.50); however, confidence intervals were wide and larger sample sizes are needed. Conclusions: MET amp occurred more frequently in responders; however, Teliso-V activity was not restricted to these pts, as most responders were not MET amplified. Specific genomic alterations beyond MET may influence clinical response. The current analysis demonstrated numeric differences between pts with identified drivers who did or did not respond to Teliso-V. Additional research on this topic is needed in larger pt cohorts and/or with tissue-based NGS analyses. [Table: see text]

Detecting and analysing intraspecific genetic variation with eDNA: From population genetics to species abundance.

Advancements in environmental DNA (eDNA) approaches have allowed for rapid and efficient species detections in diverse environments. Although most eDNA research is focused on leveraging genetic diversity to identify taxa, some recent studies have explored the potential for these approaches to detect within-species genetic variation, allowing for population genetic assessments and abundance estimates from environmental samples. However, we currently lack a framework outlining the key considerations specific to generating, analysing and applying eDNA data for these two purposes. Here, we discuss how various genetic markers differ with regard to genetic information and detectability in environmental samples and how analysis of eDNA samples differs from common tissue-based analyses. We then outline how it may be possible to obtain species absolute abundance estimates from eDNA by detecting intraspecific genetic variation in mixtures of DNA under multiple scenarios. We also identify the major causes contributing to allele detection and frequency errors in eDNA data, discuss their consequences for population-level analyses and outline bioinformatic approaches to detect and remove erroneous sequences. This review summarizes the key advances required to harness the full potential of eDNA-based intraspecific genetic variation to inform population-level questions in ecology, evolutionary biology and conservation management.

U-shaped association between serum IGF2BP3 and T2DM: A cross-sectional study in Chinese population.

To clarify the expression of N6-methyladenosine (m6 A) modulators involved in the pathogenesis of type 2 diabetes mellitus (T2DM). We further explored the association of serum insulin-like growth factor 2 mRNA-binding proteins 3 (IGF2BP3) levels and odds of T2DM in a high-risk population. The gene expression data set GSE25724 was obtained from the Gene Expression Omnibus, and a cluster heatmap was generated by using the R package ComplexHeatmap. Differential expression analysis for 13 m6 A RNA methylation regulators between nondiabetic controls and T2DM subjects was performed using an unpaired t test. A cross-sectional design, including 393 subjects (131 patients with newly diagnosed T2DM, 131 age- and sex-matched subjects with prediabetes, and 131 healthy controls), was carried out. The associations between serum IGF2BP3 concentrations and T2DM were modeled by restricted cubic spline and logistic regression models. Two upregulated (IGF2BP2 and IGF2BP3) and 5 downregulated (methyltransferase-like 3 [METTL3], alkylation repair homolog protein 1 [ALKBH1], YTH domain family 2 [YTHDF2], YTHDF3, and heterogeneous nuclear ribonucleoprotein [HNRNPC]) m6 A-related genes were found in islet samples of T2DM patients. A U-shaped association existed between serum IGF2BP3 levels and odds of T2DM according to cubic natural spline analysis models, after adjustment for body mass index, waist circumference, diastolic blood pressure, total cholesterol, and triglyeride. Multivariate logistic regression showed that progressively higher odds of T2DM were observed when serum IGF2BP3 levels were below 0.62 ng/mL (odds ratio 3.03 [95% confidence interval 1.23-7.47]) in model 4. Seven significantly altered m6 A RNA methylation genes were identified in T2DM. There was a U-shaped association between serum IGF2BP3 levels and odds of T2DM in the general Chinese adult population. This study provides important evidence for further examination of the role of m6 A RNA methylation, especially serum IGF2BP3 in T2DM risk assessment.

EP05.01-014 Circulating Tumor DNA to Identify Genomic Biomarkers of Radiation Sensitivity in Locally Advanced Non-Small Cell Lung Cancer

Despite our increasing understanding of the genomic heterogeneity of non-small cell lung cancer (NSCLC), patients with locally advanced inoperable disease are treated with the same dose of radiation therapy (RT). Tissue-based analyses have identified likely biomarkers of RT response. We evaluated the feasibility of prospective molecular profiling with liquid biopsy ctDNA testing to identify genomic markers of RT sensitivity and resistance.

ME15 Current molecular treatment landscape of advanced colorectal cancer based on COLOMATE platform and collaboration with SCRUM-Japan GOZILA

Circulating tumor DNA (ctDNA)-based next-generation sequencing (NGS) assays offer meaningful advantages over tissue-based analyses given their improved representation of the totality of the cancer including real time and repeated sampling. Additional advantages include their low invasiveness and rapid turnaround time. Given the low incidence of most target gene alterations in colorectal cancer, large collaborative screening platforms are critically needed to develop rational genome-based molecularly targeted clinical trials.

Abstract IA21: Understanding responses to cancer therapy: The tissue is the issue, the scoop is in the poop, and you are what you eat

Abstract We have made major advances in cancer treatment using multi-modality therapy, with immunotherapy now playing a major role across cancer types. However, responses to treatment are not universal, and strategies to improve outcomes (and to ultimately prevent cancer altogether) are needed. Tissue-based analyses in neoadjuvant and other studies have yielded important insights into known and novel therapeutic targets, leading to rational combination strategies to improve outcomes to therapy. Additionally, next-generation sequencing approaches have allowed interrogation of the microbiome in the tumor, and also in the gut and other niches. Microbiota within the body have a profound impact on physiology, with emerging evidence that they may contribute to carcinogenesis and therapy response. Importantly, it is not only the microbes that matter, but also the substrate for energy use (i.e. our diet). All of these aspects will be discussed in this talk, and rational combination strategies to improve cancer treatment will be proposed. Citation Format: Jennifer A. Wargo. Understanding responses to cancer therapy: The tissue is the issue, the scoop is in the poop, and you are what you eat [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr IA21.

Comparing GOZILA and COLOMATE: Ongoing Umbrella/Basket Trials Examining Genetic Testing in Gastrointestinal Malignancies.

Circulating tumor DNA (ctDNA)-based next-generation sequencing (NGS) assays have advantages over classic tissue-based analyses because of their low invasiveness and availability of repeated sampling. Because of the low incidence of target gene alterations such as HER2 or BRAF V600E in gastrointestinal cancers, very large screening platforms are needed to develop genome-based clinical trials. For those reasons, ctDNA-based screening studies are being actively conducted; among them are the GOZILA (Guardant Originates in Zipangu Liquid biopsy Arrival) study in Japan and the COLOMATE (COlorectal Cancer Liquid BiOpsy Screening Protocol for Molecularly Assigned ThErapy) study in the United States. Although only patients with metastatic colorectal cancer (mCRC) who had previously received standard chemotherapies are eligible for the COLOMATE study, patients with various types of solid tumors at any line of treatment are eligible for GOZILA. This broad coverage of the eligible population allows a target of 5000 patients. By contrast, effective screening of selected candidate patients for companion trials using rapid turnaround time by ctDNA-based NGS assay is the key for COLOMATE. The companion trials of targeted therapies in mCRC are similar between GOZILA and COLOMATE. Both studies have identified patients eligible for studies by examining ERBB2 (HER2), BRAF V600E, BRAF non-V600E, and FGFR alterations, as well as MET amplification and rechallenge with anti-EGFR antibodies. The existence of various companion trials for common alterations that can be potential therapy targets on the 2 platforms can lead to future international collaboration.

Bifunctional molecules targeting SARS-CoV-2 spike and the polymeric Ig receptor display neutralization activity and mucosal enrichment

ABSTRACT The global health crisis and economic tolls of COVID-19 necessitate a panoply of strategies to treat SARS-CoV-2 infection. To date, few treatment options exist, although neutralizing antibodies against the spike glycoprotein have proven to be effective. Because infection is initiated at the mucosa and propagates mainly at this site throughout the course of the disease, blocking the virus at the mucosal milieu should be effective. However, administration of biologics to the mucosa presents a substantial challenge. Here, we describe bifunctional molecules combining single-domain variable regions that bind to the polymeric Ig receptor (pIgR) and to the SARS-CoV-2 spike protein via addition of the ACE2 extracellular domain (ECD). The hypothesis behind this design is that pIgR will transport the molecule from the circulation to the mucosal surface where the ACE ECD would act as a decoy receptor for the nCoV2. The bifunctional molecules bind SARS-Cov-2 spike glycoprotein in vitro and efficiently transcytose across the lung epithelium in human tissue-based analyses. Designs featuring ACE2 tethered to the C-terminus of the Fc do not induce antibody-dependent cytotoxicity against pIgR-expressing cells. These molecules thus represent a potential therapeutic modality for systemic administration of neutralizing anti-SARS-CoV-2 molecules to the mucosa.

Abstract 4969: Absolute quantification of immune cells in bulk DNA samples and liquid biopsies using digital PCR

Abstract The absolute quantification of adaptive immune cells in cancer is of clinical importance for diagnosis, prognosis and treatment. First, the cancer itself may be of lymphoproliferative origin, in which an accurate cell count facilitates the diagnosis. Alternatively, healthy T- and B-lymphocytes may be infiltrating a tumor, affecting tumor progression or representing a potential therapeutic target. However, sample quantity and quality do not always allow to perform cell- or tissue-based quantitative analyses. As an alternative, we identified generic DNA markers of a variety of adaptive immune cell types. These markers were translated into a digital PCR experimental setup, enabling the absolute quantification of these cell types in bulk DNA. The sensitivity and dynamic range of this approach were technically validated in control samples and calibration curves. Moreover, a biological validation showed a precision of our measurements comparable to flow cytometry (R2 &amp;gt; 0.9). Importantly, as our sample requirements are much lower (5-20 ng of DNA instead of intact cells), this approach provides a novel, sensitive way for the accurate quantification of adaptive immune cells independent of the cellular context. We evaluated our approach in the context of two clinical applications: diagnosis of vitreoretinal lymphoma by a liquid biopsy, and absolute deconvolution of the immune infiltrate in melanoma. Vitreoretinal lymphoma is a clinically challenging masquerade syndrome, frequently mimicking a benign uveitis. To confirm the diagnosis, a vitreous biopsy may be obtained and analyzed for malignant cells. However, analysis may be impeded by low cellularity or loss of cellular integrity. With our approach, we could acquire absolute immune cell quantifications from only 10 uL vitreous fluid, independent of cellular context. To analyze the immune infiltrate from bulk DNA or RNA samples in solid tumors, numerous deconvolution approaches are available. However, these methods typically rely on relative differences in gene expression, consequently lacking the precision to be compared with absolute measurements. Our DNA-based method allows to obtain absolute lymphocyte quantifications, comparable to (single) cell-based technologies. In both cutaneous and uveal melanoma, this facilitated an enhanced deconvolution of the bulk expression analysis. In conclusion, we developed and validated a digital PCR-based approach to quantify immune cells in bulk DNA samples. A similar precision could be obtained compared to flow cytometry, but with much lower requirements concerning sample quality and quantity. Therefore, this method can be broadly applied in a wide range of sample types, supporting cancer diagnosis, prognosis and treatment. Citation Format: Rogier J. Nell, Mieke Versluis, Willem H. Zoutman, Pieter A. Van der Velden. Absolute quantification of immune cells in bulk DNA samples and liquid biopsies using digital PCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4969.

Shallow Whole-Genome Sequencing from Plasma Identifies FGFR1 Amplified Breast Cancers and Predicts Overall Survival.

Background: Focal amplification of fibroblast growth factor receptor 1 (FGFR1) defines a subgroup of breast cancers with poor prognosis and high risk of recurrence. We sought to demonstrate the potential of circulating cell-free DNA (cfDNA) analysis to evaluate FGFR1 copy numbers from a cohort of 100 metastatic breast cancer (mBC) patients. Methods: Formalin-fixed paraffin-embedded (FFPE) tissue samples were screened for FGFR1 amplification by FISH, and positive cases were confirmed with a microarray platform (OncoscanTM). Subsequently, cfDNA was evaluated by two approaches, i.e., mFAST-SeqS and shallow whole-genome sequencing (sWGS), to estimate the circulating tumor DNA (ctDNA) allele fraction (AF) and to evaluate the FGFR1 status. Results: Tissue-based analyses identified FGFR1 amplifications in 20/100 tumors. All cases with a ctDNA AF above 3% (n = 12) showed concordance for FGFR1 status between tissue and cfDNA. In one case, we were able to detect a high-level FGFR1 amplification, although the ctDNA AF was below 1%. Furthermore, high levels of ctDNA indicated an association with unfavorable prognosis based on overall survival. Conclusions: Screening for FGFR1 amplification in ctDNA might represent a viable strategy to identify patients eligible for treatment by FGFR inhibition, and mBC ctDNA levels might be used for the evaluation of prognosis in clinical drug trials.

Toward a standard pathological and molecular characterization of recurrent glioma in adults: a Response Assessment in Neuro-Oncology effort

Regardless of subtype, diffuse gliomas of adulthood are characterized by inexorable progression through treatment. Cancer recurrence in the context of therapy is by no means unique to gliomas. For many tumors residing outside the central nervous system (CNS), tissue-based analyses are routinely employed to document the molecular and cellular features of disease recurrence. Such interventions are inconsistently applied for gliomas, however, and lack rigorous standardization when they are. While many of the reasons underlying these discrepancies reflect pragmatic realities inherent to CNS disease, the suboptimal employment of histological and molecular assessment at recurrence nevertheless represents a missed opportunity to proactively guide patient management and increase knowledge. Herein, we address this quandary by pairing a succinct description of the histological, biological, and molecular characteristics of recurrent glioma with recommendations for how to better standardize and implement quality pathological assessment into patient management. We hope this review will prompt thoughtful revision of standard operating procedures to maximize the utility of glioma re-biopsy.

Abstract 1392: Machine Learning Radiomic Biomarkers Non-invasively Assess Genetic Characteristics of Glioma Patients

Abstract Genetic heterogeneity of gliomas, both across and within patients, is a significant challenge in precision diagnostics and treatment planning. The emerging field of radiogenomics focuses on imaging signatures reflecting underlying genomic characteristics. In this study, we use advanced radiomic analysis of clinically acquired multi-parametric MRI (mpMRI) sequences to non-invasively detect clinically-relevant genetic alterations in gliomas, including isocitrate dehydrogenase (IDH1), epidermal growth factor receptor variant-III (EGFRvIII), and promoter methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene. Such non-invasive, in vivo determination of tumor genetic alterations could assist in assessing spatial heterogeneity (currently not captured via single-specimen analyses), as well as prognostic stratification, and treatment planning. We delineated all tumors, from a retrospective cohort of 202 glioma patients with available pre-operative mpMRI (T1, T1-Gd, T2, T2-FLAIR, DTI, DSC-MRI), into sub-regions of enhancement, non-enhancement, and peritumoral edema/invasion. We used CaPTk (www.cbica.upenn.edu/captk) to extract quantitative imaging phenomic (QIP) features across sub-regions from all mpMRI, describing size, morphology, texture, and intensity. Cross-validated (CV) sequential feature selection determined the most discriminative QIP features. The predicted classifications, following a 10-fold CV, were compared with the results of a next-generation sequencing panel performed on specimens from the patients in our cohort, as well as pyrosequencing for MGMT. The CV accuracy of the radiomic assessment was 85% (spec = 86%, sens = 83%, area under the curve [AUC] = 0.85), 87% (spec = 90%, sens = 79%, AUC = 0.86), and 83% (spec = 85%, sens = 83%, AUC = 0.84) for mutations in IDH1, EGFRvIII, and MGMT methylation, respectively. These signatures were consistent with EGFRvIII-mutated, IDH1-wildtype, and MGMT-methylated tumors having increased neovascularization and cell density, as well as a distinctive spatial pattern involving relatively more frontal and parietal regions in EGFRvIII-mutants, more frontal regions in IDH1-mutants, and relatively right spherical regions in MGMT-methylated tumors. These detections were independent of age, gender, and additional genetic changes. By non-invasively capturing the tumor in its entirety, our proposed QIP signatures can assist in evaluating the tumor’s spatial heterogeneity, hence overcoming common sampling limitations of tissue-based analyses. These signatures can non-invasively stratify patients for therapies targeting specific genes, and potentially monitor the dynamic mutational changes during treatment. Given the routine use of imaging in clinical practice, our QIP signatures may provide an unprecedented opportunity to improve decision-support in cancer treatment at low cost. Citation Format: saima rathore, Spyridon Bakas, Hamed Akbari, MacLean P. Nasrallah, Stephen Bagley, Christos Davatzikos. Machine Learning Radiomic Biomarkers Non-invasively Assess Genetic Characteristics of Glioma Patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1392.

A multicentre, prospective study of plasma circulating tumour DNA test for detecting RAS mutation in patients with metastatic colorectal cancer

BackgroundOncoBEAMTM RAS CRC kit using BEAMing technology is a circulating tumour DNA (ctDNA) test for detecting plasma RAS mutational status in metastatic colorectal cancer (mCRC). We conducted a multicentre, prospective study to investigate the concordance of the RAS mutational status between plasma ctDNA and tumour tissue DNA.MethodsmCRC patients without prior anti-EGFR antibodies or regorafenib treatment were enroled. Plasma- and tissue-based RAS mutational status were determined by BEAMing, respectively.ResultsA total of 280 patients from eight institutions were eligible. The overall agreement between plasma- and tissue-based analyses was 86.4%, with a positive percent agreement of 82.1% and negative percent agreement of 90.4%. From logistic regression analysis, lung metastasis alone indicated the most significant factor associated with discordance. The agreement between plasma- and tissue-based analyses was 64.5% in patients with lung metastasis alone (n = 31) indicating lower amount of ctDNA. Among the cases with lung metastasis alone, all plasma- and tissue-based analyses were perfectly concordant in cases with ≥20 mm of maximum lesion diameter or ≥10 lesions.ConclusionThe clinical validity of OncoBEAMTM RAS CRC kit was confirmed. Careful attention should be paid for mCRC patients with lung metastases alone having fewer metastases or smaller diameter lesions.

P3.02b-108 Assessment of Clinical Usability of a cfDNA-Based Assay Detecting EGFR T790M Mutation in EGFR-TKI Refractory NSCLC Patients: Topic: EGFR RES

Assessment of acquired resistant EGFR mutation T790M in circulating free DNA (cfDNA) in the plasma of EGFR-TKI treated NSCLC patients presents several challenges. Furthermore, the feasibility and required sensitivity of cfDNA-based detection methods in second-line therapy are not well elucidated. Here, we examined the cfDNA of patients for T790M and other activating mutations of EGFR to assess the clinical usability of such data for diagnosis purposes. cfDNAs were prepared from the plasma samples of 45 NSCLC patients who were confirmed as harboring activating EGFR mutations (exon19 deletion, n = 20; L858R, n = 23; and minor mutations, n = 2). EGFR mutations in cfDNA samples were detected using highly sensitive methods (NGS-utilizing ultra-deep sequencing, droplet digital PCR) and originally developed assays (BNA-clamped PCR/F-PHFA combined method, and BNA-clamped qPCR) and these results were compared to tissue-based definitive diagnoses. No significant change was observed in amounts of extracted cfDNA among EGFR-TKI naïve (n = 18) and refractory (n = 27) groups. There was a positive significant correlation between the amount of cfDNA and diameter in target regions, suggesting that tumor volume reflects the amount of cfDNA. Significant negative correlation was observed between cfDNA amounts and PFS following EGFR-TKI treatment in the TKI-naïve group. The overall percentage agreement between cfDNA and tissue-based analyses ranged from 89 to100 % in major activating mutations and was approximately 85% in T790M. Detected fragment number of each mutation in cfDNA samples by ultra-deep sequencing suggested that it caused the observed difference in the agreement rates between activating mutations and T790M. We confirmed the strong agreement between the high performance assays and definitive diagnosis when the same tissue samples were tested. Next, cfDNA genotyping results were compared to tissue-based definitive diagnosis. In the case of BNA-clamped qPCR, the positive percent agreement was 63% (26/41) in major activating mutations, whereas the negative percent agreement was 100% (45/45). T790M was detected in 46% (12/26) cfDNA samples derived from the EGFR-TKI refractory group. We performed re-biopsies in a proportion of enrolled patients and investigated the tissue-plasma results concordance in matched samples. The observed overall percent agreement was 63% in case of T790M and 88% regarding exon19 deletions. Due to heterogeneity or other biological features of drug-treated tumors, the cfDNA assay feasibility of detecting T790M was more limited than that of detecting activating mutations. To assess the T790M status in EGFR-TKI refractory patients, tissue-based assay and cfDNA-based assay should be performed complementarily.

MET amplification in metastatic colorectal cancer: an acquired response to EGFR inhibition, not a de novo phenomenon.

BackgroundMET amplification appears to be a predictive biomarker for MET inhibition. Prior studies reported a MET amplification rate of 9–18% in metastatic colorectal cancer (mCRC) but do not differentiate increased gene copy numbers due to chromosomal level aberrations from focal gene amplifications. Validation of MET amplification rate in mCRC is critical to this field.ResultsIn tumor tissue-based analyses, overall MET amplification rate was 1.7% (10/590). MET amplification was seen in 0/103 (0%), 4/208 (1.9%) and 6/279 (2.2%) cases, in cohorts 1, 2 and 3, respectively. Rate of MET amplification in cfDNA of cohort 4 patients refractory to anti-EGFR therapy (n = 53) was 22.6% (12/53) and was significantly higher compared to patients not exposed to anti-EGFR therapy (p < 0.001).Materials and MethodsWe analyzed MET amplification in mCRC (n = 795) using different methods across multiple cohorts. Cohort 1 (n = 103) and 2 (n = 208) included resected liver metastases and tumor biopsies, respectively, tested for MET amplification using fluorescence in-situ hybridization [amplification: MET/CEP7 ratio ≥ 2.0]. Using another tissue-based approach, cohort 3 (n = 279) included tumor biopsies sequenced with HiSeq (Illumina) with full exome coverage for MET [amplification: ≥ 4 copies identified by an in-house algorithm]. Using a blood-based approach by contrast, cohort 4 (n = 205) included patients in whom the full exome of MET in circulating-free DNA (cfDNA) was sequenced with HiSeq.ConclusionsContrary to prior reports, in this large cohort, MET amplification was a rare event in mCRC tissues. In plasma by stark contrast, MET amplification identified by cfDNA occurred in a sizable subset of patients that are refractory to anti-EGFR therapy.

Sunitinib-induced morpho-functional changes and drug effectiveness in malignant solitary fibrous tumours.

Sunitinib improves the outcomes of patients with solitary fibrous tumours (SFTs). The aim of this study was to investigate and contextualise sunitinib-induced morpho-functional changes in order to gain insights into the drug's mechanism of action.To this end, four surgical specimens obtained from two sunitinib-responsive patients with malignant SFT, and one primary cell culture obtained from fresh tumoral tissue and its stabilised cell line, were studied by means of immunohistochemistry, bright field in situ hybridisation, immunofluorescence/confocal microscopy, and biochemistry.The post-sunitinib surgical samples were characterised by two biologically relevant morpho-functional changes: clear areas and necrotic foci. The first were associated with the attenuation/loss of PDGFRB expression and decreased mTOR signalling, and corresponded to a pathological response. The second were associated with the over-expression of PDGFRB and VEGFA, strong mTOR signalling activation, and the appearance of HIF1α expression, hallmarks of pathological progression. The analysis clearly showed that sunitinib reduces the vascular supply network and inhibits tumoral cells. It also either induces autophagy, thus favouring drug response, or impairs autophagy as a result of lysosome sequestration, thus favouring disease progression. These distinct autophagic events were associated with different myeloid immune contextures. Finally, we also found that PDGFRB is one of the components of a complex that includes Beclin 1 and VPS34.The results of these tissue-based analyses provide new insights into sunitinib's mechanism of action in SFT patients.

Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics

Mass spectrometry (MS)-based proteomics is increasingly applied in a quantitative format, often based on labeling of samples with stable isotopes that are introduced chemically or metabolically. In the stable isotope labeling by amino acids in cell culture (SILAC) method, two cell populations are cultured in the presence of heavy or light amino acids (typically lysine and/or arginine), one of them is subjected to a perturbation, and then both are combined and processed together. In this study, we describe a different approach--the use of SILAC as an internal or 'spike-in' standard--wherein SILAC is only used to produce heavy labeled reference proteins or proteomes. These are added to the proteomes under investigation after cell lysis and before protein digestion. The actual experiment is therefore completely decoupled from the labeling procedure. Spike-in SILAC is very economical, robust and in principle applicable to all cell- or tissue-based proteomic analyses. Applications range from absolute quantification of single proteins to the quantification of whole proteomes. Spike-in SILAC is especially advantageous when analyzing the proteomes of whole tissues or organisms. The protocol describes the quantitative analysis of a tissue sample relative to super-SILAC spike-in, a mixture of five SILAC-labeled cell lines that accurately represents the tissue. It includes the selection and preparation of the spike-in SILAC standard, the sample preparation procedure, and analysis and evaluation of the results.

Abstract PR-05: Effects of presurgical administration of tea polyphenols in women with operable breast cancer

Abstract Background: Various epidemiologic studies have demonstrated an inverse association between green tea consumption and breast cancer risk. The primary polyphenol of green tea, epigallocatechin gallate (EGCG), is a potent antioxidant that acts on multiple stages of carcinogenesis, including proliferation and angiogenesis. The hepatocyte growth factor (HGF)/c-Met pathway is deregulated in breast cancer, with high levels of serum HGF and its tissue receptor c-Met being associated with a poorer prognosis in the clinical setting. Polyphenols have been shown to modulate these pathways in human breast cancer cell lines and mouse xenograft models, however, studies in humans are notably lacking. The purpose of this study was to determine the effects of short-term presurgical administration of an oral green tea extract, Polyphenon E (Poly E), on serum and tissue-based biomarkers in patients with operable breast cancer. Methods: This is a phase II single-arm open-label trial of oral Poly E 800 mg daily for 2-4 weeks in women with histologically confirmed breast cancer on core biopsy who were scheduled for surgical resection. Poly E is a purified tea fraction containing 800 mg of EGCG and lesser amounts of other green tea catechins. Safety was assessed by monitoring clinical and laboratory parameters at baseline and the end of treatment. Serum was collected at baseline and the day prior to surgery and analyzed for HGF and VEGF levels by ELISA. All pretreatment and posttreatment samples were run in the same batch and analyzed in a blinded fashion. Formalin-fixed paraffin-embedded tumor tissue from the diagnostic core biopsy (pretreatment) and surgical specimen (posttreatment) were analyzed for expression of the Ki-67 proliferation index by immunohistochemistry. Pre- and posttreatment values for each biomarker were compared using paired t-test or nonparametric analog. All statistical analyses were two-sided and performed using SAS version 9.1. Results: From February 2008 to September 2009, 25 women were enrolled of which 21 are evaluable. Median age: 49 (33-71); White/Hispanic/Black: 7/13/1; Stage 0/I/II/III: 3/11/4/2; Tumor hormone receptor +/−: 18/3. The study drug was well tolerated with no grade 2 or higher toxicities. Poly E administration was associated with a trend toward decreasing serum HGF levels (mean absolute change of −240 pg/ml, mean % change of −13.5%, p=0.077). No significant change in serum VEGF levels was observed. Tissue-based biomarker analyses are currently ongoing. Conclusions: Our results show a nonsignificant trend towards a decrease in serum HGF levels in women with newly diagnosed early stage breast cancer after short-term administration of Poly E. The treatment was well tolerated, with no significant elevation of liver or pancreatic enzymes. This presurgical study design is a useful and feasible model for testing the biologic effects of potential agents for breast cancer treatment and prevention. Citation Information: Cancer Prev Res 2010;3(12 Suppl):PR-05.