bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential monitoring of androgen receptor expression and copy number variation in castration-resistant prostate cancer (CRPC).
Authors: Gross Mitchell E. , Agus David B. , Dorff Tanya B. , Pinski Jacek K. , Quinn David I. , Dago Angel E , Stepansky Asya , Carlsson Anders , Felch Natalie , Luttgen Madelyn , Kolatkar Anand , Hicks James B , Kuhn Peter
Published: March 2020

11047

Background: The high-definition circulating tumor cell (HD-CTC) assay provides for an enrichment-free approach to identify and characterize CTCs. Here, we utilized the HD-CTC assay to study androgen receptor (AR) expression combined with single-nucleus sequencing for genome-wide analysis of copy number variation (CNV) in sequential samples obtained from patients with CRPC treated with abiraterone acetate (AA). Methods: Patients were approached for participation in a study to provide peripheral blood at baseline, at 2-5 weeks, and at 9-12 weeks (or at progression). In each sample, 106cells (defined with a DAPI-intact nucleus) were quantitatively examined for the presence of cytokeratin and AR (CTCs) and CD45 (leukocytes). Initial results are available from 9 subjects treated with AA as standard of care. Results: At baseline, the median (range) CTC was 7.8 (1.1-57.2) cells/ml. Using a definition of AR positive (AR+) as >6 standard deviations over mean signal observed in leukocytes, the median (range) of AR+ and total CTCs observed at baseline were 3.1(0-33.8) and 7.8 (1.1-57.2) cells/ml, respectively. Detailed single-nucleus CNV analysis was performed in sequential samples in a single subject (Table). Complex genomic rearrangements were observed including AR amplification and 8p deletion in both AR+ and AR- negative (AR-) cells at baseline. During AA treatment, the frequency of AR+ CTCs decreased along with changes in the CNV pattern including loss of AR amplification. At 10 weeks, disease progression occurred coincident with re-emergence of an AR+ CTC population exhibiting AR amplification and a novel CNV pattern only distantly related to that of the baseline CTCs. While multiple complex abnormalities were noted, MYC amplification was observed at higher frequency in cells present at progression. Conclusions: Overall, our results demonstrate the feasibility of monitoring of CTCs for treatment emergent changes in protein which may be used to better monitoring and predict therapeutic responses in patients with metastatic cancer. [Table: see text]
Detection and genomic interrogation of invasive circulating tumor cells (iCTCs) derived from men with metastatic castration resistant prostate cancer (mCRPC).
Authors: Friedlander Terence W. , Weinberg Vivian K. , Roy Ritu , Ngo Vy , Dong Huang , Doty Shaun , Zhao Qiang , Premasekharan Gayatri , Gilbert Elizabeth , Ryan Charles J. , Chen Wen-Tien , Paris Pamela
Published: March 2020

11048

Background: Isolation, enumeration, and genomic profiling of CRPC CTCs offers the potential to discover genetic changes that occur in advanced disease. The Vitatex VitaCap platform captures CTCs based on their ability to invade a collagenous matrix (CAM), allows for capture of invasive CTCs (iCTCs) independent of EpCAM status, and yields viable cells suitable for comprehensive genomic study. Here we sought to compare CTC yields between the CAM and CellSearch platforms, to determine the utility of prostate-specific membrane antigen (PSMA) as an iCTC biomarker, to identify iCTC clusters and iCTCs expressing stem-like markers, and to explore the feasibility of iCTC epigenomic analysis. Methods: CTCs were isolated and enumerated simultaneously using the CellSearch and CAM platforms in 23 men with mCRPC. CAM-isolated iCTCs were defined as EpCAM+PSMA+ and were enumerated immunocytochemically (ICC) and by flow cytometry. iCTC clusters were enumerated by ICC. The Illumina Infinium HumanMethylation27 BeadChip was used to determine whole genome methylation status for CAM isolated cells. Results: 35 samples were collected for CAM analysis. A median of 27 (range 0-800) and 23 (range 2-390) iCTCs/ml were detected by ICC and flow respectively. In a subset of 20 samples, a median of 7 CTCs/ml (range 0-85) were detected by the CellSearch platform. CTCs were detectable by either CAM or CellSearch in >95% of samples. iCTC clusters were observed in 23% of samples with a median 7 clusters/ml (range 1-200). iCTCs expressing stem-like markers CD44 and Seprase were detected in 70% and 97% of samples by ICC and flow respectively, with a median of 9/ml (range 1-264) by flow. The iCTC methylation profile highly resembled mCRPC. Conclusions: The CAM and CellSearch platforms yield comparable CTC counts. iCTC clusters and iCTCs expressing stem-like markers are detectable using the CAM platform, and the iCTC methylome closely resembles that of mCRPC. Correlation with clinical data may yield further insight into the functional significance of these findings.
Clinical significance of BIM deletion polymorphism in non-small cell lung cancer with epidermal growth factor receptor mutation.
Authors: Isobe Kazutoshi , Hata Yoshinobu , Kaburaki Kyohei , Kobayashi Hiroshi , Sugino Keishi , Sakamoto Susumu , Tochigi Naobumi , Takai Yujiro , Shibuya Kazutoshi , Takagi Keigo , Homma Sakae
Published: March 2020

11052

Background: Germ line alterations in the proapoptotic protein BCL2-like 11 (BIM) can have a crucial role in how a tumor responds to treatment. To clarify the clinical usefulness of detecting BIM deletion polymorphism in non-small cell lung cancer (NSCLC) with positive epidermal growth factor receptor (EGFR) mutation we examined the prognosis in patients with or without the BIM changes. Methods: Seventy NSCLC patients with positive EGFR mutation treated with EGFR-tyrosine kinase inhibitor (EGFR-TKI) between January 2008 and January 2013 were enrolled in this study. BIM deletion polymorphism was analyzed by PCR in 58 peripheral blood samples, 24 formalin-fixed paraffin-embedded (FFPE) slides of surgical specimens (lung tissue in 20, brain tissue in 4), and 12 samples that included both blood and tissue specimens. We performed retrospective analyses on clinical characteristics, response rate and toxicity of EGFR-TKI, and estimated the prognosis in patients with or without BIM deletion polymorphism. Results: BIM deletion polymorphism was present in 13 of 70 patients (18.6%) with homozygous deletion in 1 and heterozygous deletion in 12. There was no discordance between the two types of samples among the 12 patients. There were no significant differences between patients with or without BIM deletion polymorphism on clinical characteristics, response rate to EGFR-TKI and incidence of EGFR-TKI toxicity. Patients with BIM deletion polymorphism showed significantly shorter PFS that in patients without BIM deletion polymorphism (median: 216 days vs. 430 days, p < 0.001). There was no significant difference in OS. In multivariate analyses using the Cox regression model, BIM deletion polymorphism (hazard ratio = 4.2, p< 0.001, 95% CI = 2.026-8.777) was identified as an independent factor for shorter PFS. Conclusions: BIM deletion polymorphism could be detected by PCR on peripheral blood samples and FFPE slides of surgical specimens. BIM deletion polymorphism has emerged as an independent prognostic factor for shorter PFS. Therefore, new treatment strategies should be established for patients with BIM deletion polymorphism.
Patient segmentation assay for MAPK pathway mutations.
Authors: Marton Matthew John , Chang Ken , Galuska Stefan , Samatar Ahmed , Zhang Ling , Weiner Russell
Published: March 2020

11059

Background: Mutational status of solid tumors is increasingly important for identifying the best treatment options. Tumors harboring activating mutations in RAS and RAF result in constitutive activation of the RAS/MAPK signal transduction pathway and do not respond well to MAPK pathway inhibitors. Scientific attention is mostly focused on the major mutational hotspots in these genes (e.g., KRAS codons 12, 13 and 61, BRAF codon 600). However, there is increasing evidence that other mutations can be tumorigenic. Thus, there is a need for highly sensitive and specific assays to detect these mutations. To address this need, we developed and analytically validated an assay that detects 33 mutations that activate the MAPK pathway. Methods: We constructed a set of multiplexed single nucleotide primer extension (SNPE) assays that detect 33 activating mutations in KRAS, NRAS, or BRAF. The assays were analytically validated using a set of 60 formalin fixed paraffin embedded (FFPE) tissue samples from various tumor types, cell lines, and oligos with specific mutations. Performance was compared to dideoxy sequencing. Discordant calls were resolved with next generation sequencing (NGS). The limit of detection was determined by serial dilution of mutant DNA into wildtype DNA. Results: The developed multiplexed assay requires only 15 ng genomic DNA, relies on established technology, is cost effective, is amenable to high throughput, and can yield a patient eligibility decision in a CLIA lab in 4 days, making it a practical alternative to a NGS-based assay. Since low allele frequency mutations may be critical to patient survival, we devised a replicate strategy to increase the specificity and sensitivity of the assay. We showed that analysis of technical triplicates relative to no replicates increased the sensitivity from 97 to 100% and the specificity from 91 to 100%. Limit of detection varied from 2 -12%, depending on the mutation. Conclusions: We developed a selective and sensitive SNPE assay capable of detecting 33 hotspots in the MAPK pathway. Our approach reduced false positive and false negative calls of low allele frequency samples. The assay has clinical applicability for the selection of patients for early phase clinical studies.
Exploration of a plucked hair gene signature as a potential pharmacodynamic marker for the dual PI3K/mTOR inhibitor VS-5584.
Authors: Weaver David T. , Paterson Daniel , Miele Gino
Published: March 2020

11064

Background: The PI 3-kinase/mTOR pathway plays a central role in cancer cell proliferation, survival, and cancer stem cell mechanisms. Depending on the mode of pathway activation, different PI3K isoforms and mTOR complexes have been shown to play essential roles in oncogenesis. VS-5584 inhibits PI3K/mTOR signaling with equipotency against all four human Class I PI3K isoforms and the mTOR kinase. VS-5584 has been shown to induce broad and robust antitumor activity with corresponding inhibition of PI3K/mTOR downstream targets in human xenograft models. Considering the inhibitory activity of VS-5584 against multiple targets within the PI3K/mTOR pathway, multiple clinical pharmacodynamic biomarkers need to be assessed. Methods: Due to its vascularization, ease of sampling and epithelial origin, anagen hair represents a practical surrogate tissue for monitoring drug effects in patients. Human anagen hair from volunteer donors were treated ex vivo with VS-5584 and another PI3K/mTOR dual inhibitor, BEZ235. Following 6, or 24 hour exposure to various drug concentrations, RNA was extracted, amplified and analyzed on Affymetrix PrimeView microarrays. Results: Transcriptomic profiles indicated a robust and dose-dependent response to VS-5584. A VS-5584-specific 91 gene signature was identified by 3-way ANOVA, and a Connectivity map evaluation indicated a strong positive correlation with other agents targeting the PI3K/mTOR pathway (wortmannin, LY-294002 and sirolimus). Likewise, VS-5584 exposure ex vivo in human anagen hair follicles resulted in a dose responsive suppression at both 6 and 24 hrs with an alternative PI3K/mTOR dual inhibitor 115 gene signature trained on BEZ235. A seven gene subset was consistently reduced by both VS-5584 and BEZ235 at 6 hrs and 24 hrs. Conclusions: Gene signatures from plucked anagen hair may provide a non-invasive approach to assess pharmacodynamic effects of VS-5584 in cancer patients.
Association of IGF2 DMR0 hypomethylation in esophageal squamous cell carcinoma with poor prognosis.
Authors: Baba Yoshifumi , Watanabe Masayuki , Murata Asuka , Shigaki Hironobu , Iwagami Shiro , Ishimoto Takatsugu , Baba Hideo
Published: March 2020

11065

Background: The insulin-like growth factor 2 gene (IGF2) is normally imprinted. Loss of imprinting (LOI) of IGF2 in humans is associated with an increased risk of cancer and is controlled by CpG-rich regions known as differentially methylated regions (DMRs). Specifically, the methylation level at IGF2 DMR0 is correlated with IGF2 LOI and is a suggested surrogate marker for IGF2 LOI. A relationship between IGF2 DMR0 hypomethylation and poor prognosis has been shown in colorectal cancer. However, no study has examined the relationships among the IGF2 DMR0 methylation level, LOI, and clinical outcome in esophageal squamous cell carcinoma (ESCC). Methods: The IGF2 imprinting status was screened using ApaI polymorphism, and IGF2 protein expression was evaluated by immunohistochemistry with 30 ESCC tissue specimens. For survival analysis, IGF2 DMR0 methylation was measured using a bisulfite-pyrosequencing assay with 216 ESCC tissue specimens. The Cox proportional hazards model was used to calculate mortality hazard ratios (HR) adjusted for clinical, epidemiologic, and pathological variables.The term “prognostic marker” is used throughout this article according to the REMARK Guidelines. Results: Twelve (40%) of 30 cases were informative (i.e., heterozygous for ApaI), and 5 (42%) of 12 informative cases displayed IGF2 LOI. IGF2 LOI cases exhibited lower DMR0 methylation levels (mean, 23%) than IGF2 non-LOI cases (37%). The IGF2 DMR0 methylation level was significantly associated with IGF2 protein expression. Among 202 patients eligible for survival analysis, IGF2 DMR0 hypomethylation was significantly associated with higher cancer-specific mortality (log-rank P=0.013; univariate HR=2.03, 95% confidence interval: 1.14–3.58, P=0.017; multivariate HR=2.34, 95% CI: 1.29-4.22, P=0.0054). Conclusions: The IGF2 DMR0 methylation level in ESCC was associated with IGF2 LOI and IGF2 protein expression. In addition, IGF2 DMR0 hypomethylation was associated with a shorter survival time, suggesting its potential role as a prognostic biomarker.
Analysis of the prognostic impact of Treg-related genes in tumor and stroma in resectable NSCLC.
Authors: Usó Marta , Jantus-Lewintre Eloisa , Sirera Rafael , Gallach Sandra , Blasco Ana , Martinez Nieves , Hernando Cristina , Guijarro Ricardo , Camps Carlos
Published: March 2020

11073

Background: Immunosuppressive regulatory T lymphocytes (Tregs) have been proved to play a critical role in immune tolerance to tumor. In this study we have analyzed the expression of 11 genes related to Tregs in both tumor and stroma samples of resectable NSCLC patients. Methods: Primary tumor tissues of FFPE samples from 125 early-stage NSCLC patients were used in this retrospective study. The most representative areas of tumor cells and tumor stroma of each sample were carefully micro-dissected. RTqPCR using hydrolysis probes (TaqMan, Applied Biosystems) was performed to assess the expression of Treg markers such as: CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNF-a as well as CD4 and CD8. Relative gene expression was assessed using GAPDH and CDKN1B as endogenous controls and results were normalized against a human cDNA (Clontech) as a reference. All statistical analyses were considered significant at p< 0.05. Results: In both tumor and stroma, we found over-expression of CD25 (5.40X and 7.95X, respectively) and down-expression of CD127 (0.28X and 0.37X, respectively). There was a tendency toward higher expression of FOXP3 (1.67X and 2.01X, respectively) and CTLA-4 (1.92X and 1.76X, respectively) as well. Paired Wilcoxon test showed significant gene expression differences between tumor and stroma in FOXP3 (p=0.006), CD25 (p<0.0001), CD4 (p<0.0001), CD8 (p=0.028), IL-10 (p<0.0001) and TGFB-1 (p<0.0001). Kruskal-Wallis test showed that FOXP3 expression was higher in adenocarcinoma than in SCC samples (2.56X vs 1.79X, p=0.002). Survival analyses revealed that patients with a “Treg profile” (↑CD25 and ↓CD127) had a reduced overall survival (OS) (median 29.90 vs 74.33 months, p=0.003). We also found that those patients with higher levels of the ratio FOXP3 stroma/tumor had worse time to progression (TTP) (median 32.50 vs NR, p=0.04). Conclusions: Treg markers in the tumor microenvironment seem to play an important prognostic role in early-stage NSCLC patients. Supported by grants PS09-01149 and RD06/0020/1024 from ISCIII.
Using 124I-PU-H71 PET imaging to predict intratumoral concentration in patients on a phase I trial of PU-H71.
Authors: Gerecitano John F. , Modi Shanu , Gajria Devika , Taldone Tony , Alpaugh Mary , Gomes DaGama Erica , Uddin Mohammad , Chiosis Gabriela , Lewis Jason Stuart , Larson Steven M. , Pillarsetty Naga Vara Kishore , Jhaveri Komal L. , Krichevsky Brian , Chen Mei Hsuan , Dixit Payal , Dunphy Mark
Published: March 2020

11076

Background: PU-H71 is a Heat Shock Protein 90 inhibitor that can be labeled with 124I without altering its biochemical properties. Intratumoral drug concentration can be calculated based on 124I-PU-H71 (*PU-H71) region of interest analysis and dilution principle. A microdose pilot study has shown uptake of *PU-H71 in a variety of tumors. *PU-H71 PET is currently being used to estimate intratumoral concentrations in subjects on our phase I study. Methods: Patients with previously treated solid tumors or lymphoma are eligible for this phase 1 trial. PU-H71 is given twice-weekly for 2 weeks each 21 days at escalating dose levels. A mix of *PU-H71 and unlabeled PU-H71 is given during cycle 2 followed by serial PET imaging. Patients on the pilot study are administered a microdose of *PU-H71 alone, followed by serial PET scans. Intratumoral PUH-71 concentration is measured directly in optional pre- and post- treatment core needle tumor biopsies (CNB). Results: To date, 13 patients have received PU-H71 on the phase I trial. Of these, 10 have undergone *PU-H71 PET imaging. 4 imaged patients also volunteered for CNBs, with results reported in the table. Of the 10 patients who underwent *PU-H71 imaging in the phase 1 study, 5 also underwent prior *PU-H71 imaging in the microdose pilot. Intratumoral concentrations as calculated in the pilot and phase I studies were in close concordance. Conclusions: *PU-H71 can be used to visualize PU-H71 uptake in a variety of solid tumors and lymphoma, and *PU-H71 PET scans can be used to estimate intratumoral concentrations of PU-H71. Direct intratumoral measurements of PU-H71 correlate reasonably closely with concentrations calculated from *PU-H71 PET imaging. Further refinement of this imaging tool will allow quantitative assessment of PU-H71 uptake in tumors during the ongoing phase I trial. Clinical trial information: NCT01393509. [Table: see text]
Target lesion selection as a source of variability of response classification by RECIST 1.1.
Authors: Kuhl Christiane K. , Barabasch Alexandra , Dirrichs Tim , Bruners Philipp , Kennes Lieven , Bruemmendorf Tim , Keil Sebastian
Published: March 2020

11077

Background: Response classification in RECIST is based on manual uni-dimensional quantification of changes of target lesion size. In RECIST 1.1, the maximum number of target lesions was reduced to 5, with a maximum of 2 per organ. We analyzed the importance of different factors (manual vs. automated size measurement, uni- vs. three-dimensional size assessment, and between-reader-variability of target lesion selection) on response categorization. Methods: 41 female patients (58.1±13.2 y) with metastatic breast cancer underwent contrast-enhanced thoraco-abdominal CT for initial staging and first follow-up after systemic chemotherapy. Data were independently and prospectively interpreted by three radiologists. In addition, response was evaluated by a CAD system that allowed automated uni- and three-dimensional assessment of target lesions. Results: Response classification differed between readers in 19/41 patients (46%). In 25/41 patients, readers chose the same target lesions. In 6 of these 25 patients (24%), readers disagreed with regards to response classification. In 16/41 patients, readers chose different target lesions. In 13 of these 16 patients, readers disagreed (81%) (p < 0.001). When dichotomizing response classification according to its therapeutic implication into progressive vs. non-progressive disease, readers disagreed in 11/41 patients (27%). In 9 of these 11 patients, readers had chosen different target lesions. Classification by manual vs. automated uni-dimensional measurement differed in 11/41 of patients (27%). Classification by uni-dimensional vs. volumetric measurements differed in 6/41 of patients (15%). Conclusions: Response classification by RECIST suffers from substantial between-reader variability. Major source of variability is not the manual or uni-dimensional measurement, but the variable choice of target lesions between readers. [Table: see text]
The CAP-IASLC-AMP molecular testing guideline for the selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors.
Authors: Ladanyi Marc , Cagle Phil T. , Beasley Mary Beth , Chitale Dhananjay , Dacic Sanja , Giaccone Giuseppe , Jenkins Robert B. , Kwiatkowski David J. , Saldivar Juan-Sebastian , Squire Jeremy , Thunnissen Erik , Lindeman Neal Ian
Published: March 2020

11085

Background: The College of American Pathologists (CAP), the International Association for the Study of Lung Cancer (IASLC), and the Association for Molecular Pathology (AMP) jointly initiated an effort to establish evidence-based recommendations for the molecular analysis of lung cancers required to guide EGFR- and ALK-directed therapies, addressing which patients and samples should be tested, and when and how testing should be performed. Methods: Three co-chairs without relevant conflicts of interest were selected, one from each of the sponsoring societies: CAP (P.T.C.), IASLC (M.L.), and AMP (N.I.L.). Writing and advisory panels were formed from additional experts from these societies. Unbiased literature searches were performed to capture articles up to February 2012, yielding 1,533 articles whose abstracts were screened to identify 521 pertinent articles that were then reviewed in detail for relevance. Evidence was formally graded for each of the recommendations first formulated by the co-chairs and panel members at a public meeting. Each guideline section was assigned to at least two panelists. Successive drafts were circulated for comments to the writing panel, the advisory panel, the public (online posting), and the three professional societies. Results: We generated 37 guideline items addressing 14 areas of EGFR and ALK testing. The major, evidence-based recommendations are to test for EGFR mutations and ALK fusions in all patients with advanced stage adenocarcinoma, regardless of sex, race, or smoking history, and to prioritize EGFR and ALK testing over other molecular predictive tests. Recommendations and expert consensus opinions were generated for all other key aspects of EGFR and ALK testing in lung cancer related to oncology and pathology practice and technical issues in molecular testing. Conclusions: As scientific discoveries and clinical practice outpace the completion of randomized clinical trials, evidence-based guidelines developed by expert practitioners are vital for communicating emerging clinical standards and thereby improving patient outcomes.
Tumor, skin, and plasma concentrations of protein kinase inhibitors (PKIs) in patients with advanced cancer.
Authors: Labots Mariette , Neerincx Maarten , Van der Mijn Johannes C. , Dekker Henk , Honeywell Richard , Rovithi Maria , Gotink Kristy , Voebel-De Jong Margreet , Van der Peet Donald L. , Meijerink Martijn R. , Meijer Gerrit A. , Jiménez Connie R. , Peters Godefridus J , Verheul Henk M.W.
Published: March 2020

11087

Background: PKIs are selective for target receptors at low concentrations, but they act promiscuously at higher concentrations (PMID 18183025). This lack of selectivity may be relevant for their antitumor activity and for the development of PKI treatment selection tools. To obtain more insight in their clinical mechanism of action, we designed a pilot study to determine PKI tumor, skin and plasma concentrations in patients (pts) after 2 weeks of treatment. Results are related to cell line sensitivity data. Methods: Prior to standard palliative systemic treatment, pts were allocated to standard-dose PKI treatment (N=5 per PKI) for 10-14 days. Plasma, tumor and skin biopsies were collected within 24 hours of last dose. Sample PKI concentrations were determined by liquid chromatography – tandem mass spectrometry (LC-MS/MS); tissue concentrations in pg/mg were converted to molarity for comparison with preclinical sensitivity data (PMID 21980135). Concentrations of sunitinib (SUN), sorafenib (SOR), erlotinib (ERL), dasatinib (DAS) and everolimus (EVE) that inhibit 50% of cell proliferation (IC50) were determined by MTT assay in 1 RCC (786-O) and 8 CRC cell lines (HCT 116, HT-29, RKO, SW480, SW1398, DLD-1, COLO 205, CaCo-2). Results: Since August 2011 samples were obtained from 27 pts; 5 received SUN, 4 SOR, 4 ERL, 5 DAS and 5 EVE. After 12 ± 1 days of treatment, median tumor concentration (TC) was 9.0 µM (2.3-50.0) (range) for SUN, 8.5 µM (3.7-22.0) for SOR, 5.3 µM (0.9-10.8) for ERL and 2.1 µM (0.2-64.0) for DAS. EVE was measurable in 2 of 5 tumors: 3.5 µM (3.4-3.6). On average, PKI skin concentrations were 2.4-fold lower than TCs. SOR and ERL plasma concentrations (PCs) were in the range of TCs while SUN and DAS PCs were at least 14-fold lower than in tumors. Mean IC50 of the cell line panel was 1.3 µM (0.8-1.4) for SUN, 2.2 µM (1.4-3) for SOR, 8.2 µM (4-11.5) for ERL, 0.06 µM (0.02-1.8) for DAS and 1.2 µM (0.05-11) for EVE. Conclusions: PKI tumor concentrations may vary considerably from plasma concentrations, but are in the IC50 range of cancer cells in vitro. These results are indicative for the inhibitory concentrations of PKIs in patient tumors and should be considered for the development of individualized treatment strategies. Clinical trial information: NCT01636908.
Objective measurement of breast density, a marker of breast cancer risk, using fully automated radiation- and compression-free MRI.
Authors: Wengert Georg , Helbich Thomas , Polanec Stephan , Dubsky Peter Christian , Bago-Horvath Zsuzsanna , Bartsch Rupert , Vogl Wolf-Dieter , Curda Michael , Pinker-Domenig Katja
Published: March 2020

11088

Background: Women may have contraindications to mammography (MG) or simply refuse the test due to subjective reservations usually concerning compression and/or radiation. Other than the detection of suspicious findings these women do not have standardized information concerning their breast density (BD), one of the strongest independent predictors of breast cancer (BC) risk. We developed a fully-automated quantitative magnetic resonance imaging (MRI) based BD measurement system, provided correlation to MG BD estimation and compared BD in BC patients and age-matched healthy controls. Methods: In this IRB-approved prospective study 35 healthy women and 19 BC patients age-matched to one of the healthy controls were included. BD for healthy women and BC patients was assessed using a) subjective qualitative radiologist´s review allocating 1 of 4 ACR BIRADS BD categories, b) Cumulus, a MG based semi-automatic method and c) a radiation- and compression-free MRI measurements system, which automatically calculates volume of breast (cm3), % of fatty tissue and % of glandular tissue. Descriptive statistics were used to define the typical range of quantitative MRI BD readings corresponding to the qualitative four ACR BIRADS BD categories. Áppropriate statistical tests were used to compare mean values of method b) and c) and BD readings of cancers and controls. Results: % MRI BD correlated well with % MG BD (r = 0.83; P < 0.0001). MRI BD measurements ranged from 1.7% to 61.9% (mean 29.05%). MG BD measurements ranged from 5.42% to 74.33% (mean 40.23%). Equivalent values were found with MRI BD readings of 0-7.2%, 7.3-26%, 26.1-39.6% and >39.7% respectively for each of the four ACR BIRADS BD categories. Mean BD (%) was higher in BC patients than in healthy controls: 24% (SD 16.9%) vs. 18.4% (16.6). Conclusions: MRI BD measurement strongly correlates with MG based BD readings. The data suggest that objective, radiation- and compression-free MRI BD measurement is a convenient alternative to MG for assessment of BD. MRI BD measurement confirms higher breast density in BC patients compared to healthy women. The data is entirely consistent with the fact that BD is a strong independent risk factor for BC.
Thymidylate synthase expression as predictive biomarker of pemetrexed sensitivity in advanced cancer patients.
Authors: Rojo Federico , Domine Manuel , Zazo Sandra , Chamizo Cristina , Serrano Gloria , Carames Cristina , Perez-Gonzalez Nuria , Hernandez Tatiana , Auz Carmen Laura , Carvajal Nerea , Moreno Irene , Martin-Valades Jose Ignacio , Izarzugaza Yann , Lendinez Alberto , Martinez-Ammoros Brezo , Arranz Juan Luis , Madoz Juan , Leon Ana , Lobo Francisco , Garcia-Foncillas Jesus
Published: March 2020

11089

Background: Although a high level of thymidylate synthase (TS) expression in malignant tumours has been suggested to be related to a reduced sensitivity to the antifolate drug pemetrexed, no direct evidence for such an association has been demonstrated in routine clinical samples from patients treated with this drug. The purpose of this study was to evaluate the impact of quantitative TS expression in tumor cells as predictor of the efficacy in patients with advanced non-small cell lung cancer, small cell lung cancer (SCLC) and mesothelioma treated with pemetrexed in our institution. Methods: 54 patients were included in this study: 40 stage IV NSCLC (26 adenocarcinomas, 11 large cell, and 3 squamous cell carcinoma), 3 SCLC and 11 mesothelioma. 21 patients received platins-pemetrexed as first line NSCLC, 20 pemetrexed in monotherapy as second and further lines and 3 carboplatin-pemetrexed fo extensive disease SCLC. Total RNA was isolated by RNeasy FFPE procedure (Qiagen). The expression of TS was analyzed by RT-qPCR using appropriate mRNA specific primers and probes in LightCycler 480II platform at 45 cycles. TS levels was calibrated to expression in normal tissue. Results: From 54 cases, TS expression data were available in 32 cases, detecting overexpression in 23 (71.8%) and low expression in 9 (28.2%) patients. The response rate for patients with low TS expression was 0.63 compared with 0.15 in patients with overexpression (p=0.015). A significant benefit in time to progression was observed in patients with low expression (median TTP 12 vs. 2 months respectively, p= 0.002), whereas did not impact on overall survival (median OS 20 vs. 19 months respectively, p= 0.595). Conclusions: TS overexpression in tumor cells correlated with a reduced response to pemetrexed-containing chemotherapy and might be used as a predictive biomarker in advanced lung and mesothelioma cancer patients.
Performance of next-generation sequencing for detection of clinically actionable genetic variants in cancer.
Authors: Hussaini Mohammed Omar , Hagemann Ian S. , Cox Teresa Mary , Lockwood Christina , Seibert Karen , Kulkarni Shashikant , Pfeifer John , Duncavage Eric James
Published: March 2020

11099

Background: Next-generation sequencing (NGS) allows for simultaneous detection of numerous actionable somatic variants in cancer. We have implemented a clinical NGS panel to detect genetic alterations in 25 genes with established roles in cancer and report here the frequency of clinically actionable genetic variants in a variety of cancer types. Methods: NGS testing was performed in a CAP-certified, CLIA-licensed environment on DNA extracted from FFPE tissue in 209 cases spanning 41 histologic tumor types. DNA was enriched by hybrid capture and sequenced to >1,000x average coverage on Illumina sequencers with 2x101bp or 2x150bp reads. Variants were called using clinically validated parameters using the Genome Analysis Toolkit, Pindel, and the custom-written Clinical Genomicist Workstation. Results: Non-small cell lung cancer (45%), pancreatic cancer (10%), and colorectal cancer (8%) were the most common tumors sent for NGS analysis. An average of 3 (range 1- 16) non-synonymous, non-SNP sequence variants per case (SNVs and indels) were detected in the 130kb exonic target. Variants were most commonly seen in TP53, KRAS, and EGFR. 27% of cases (56/209) had one or more variants with therapeutic implications for the tumor type tested (e.g., EGFR mutation in NSCLC). 15% of cases (32/209) showed actionable variants not generally associated with the malignancy tested (e.g., detection of an activating KITvariant in thymic carcinoma). 10% of cases (21/209) had variants that were prognostically significant but not directly targetable. Some cases (9%) had variants that were prognostic/diagnostic and targetable. In 117 cases (56% of total), no therapeutically or prognostically significant variants were identified. Overall, in 92 cases (44%), NGS testing yielded information with therapeutic (majority), prognostic, or diagnostic ramifications. Conclusions: We found that 44% of unselected cancer cases have clinically relevant sequence variants in a set of 25 commonly mutated cancer genes. Our data suggest that clinical NGS testing may serve as an integral tool in realizing the potential of precision medicine in oncology.
Application of MET pharmacodynamic assays to compare effectiveness of five MET inhibitors to engage target in tumor tissue.
Authors: Srivastava Apurva K. , Hollingshead Melinda G. , Weiner Jennifer , Covey Joseph M , Liston Dane , Peggins James O , Bottaro Donald P. , Wright John Joseph , Kinders Robert J. , Tomaszewski Joseph E. , Parchment Ralph E. , Doroshow James H.
Published: March 2020

11103

Background: Several MET inhibitors are currently being developed that block aberrant HGF/MET signaling in different cancers. We utilized validated MET pharmacodynamic (PD) assays to compare time course, magnitude, and reversal of MET suppression by 5 MET inhibitors in preclinical models. Methods: Immunoassays (total MET, pY1234/35MET, and pY1356MET) were developed and validated to measure modulation of MET by 5 MET inhibitors (crizotinib, tivantinib, cabozantinib, foretinib, and EMD1214063). The comparison was implemented in 3 sequential stages: 1) establish time course and magnitude of MET inhibition after single drug administration of 4 different doses; 2) determine dose(s) and schedule for sustained MET inhibition and downstream signaling at optimal levels; and 3) compare efficacy of MET inhibitors at MTD and equal MET inhibition. The preclinical models include an autophosphorylation gastric tumor (SNU5) model and a paracrine MET activation model in hHGF knock-in mice. Plasma and tumor exposures were measured using LC-MS/MS to correlate with PD effects. Results: We completed phase one in the SNU5 model and determined inhibition of pY1234/35MET and total MET in tumor tissues after single administration of MET inhibitors. Time course and magnitude of pY1234/35MET inhibition varied considerably among MET inhibitors, with the most rapid (>80% suppression in 30 min) and sustained inhibition (up to 48 h) observed with EMD1214063 at a dose of 30 mg/kg. The maximal inhibition of pY1234/35MET and time taken for biomarker recovery were wide-ranging among MET inhibitors. Tumor drug exposures were concomitantly higher than plasma for all drugs and correlated inversely with pY1234/35MET, except for tivantinib which, unlike other drugs, is not ATP competitive inhibitor. Conclusions: We applied validated PD assays to directly compare similarities and differences in extent and duration of MET inhibition by 5 MET inhibitors. Our results provide important foundation for head-to-head comparison of efficacies of MET inhibitors at MTD and equal MET inhibition. Funded by NCI Contract No HHSN261200800001E.
Plasma osteopontin and the prognosis of pleural mesothelioma.
Authors: Pass Harvey I. , Donington Jessica S. , Gadgeel Shirish M. , Chachoua Abraham , Wozniak Antoinette J. , Liu Geoffrey , Tsao Ming Sound , de Perrot Marc , Goparaju Chandra
Published: March 2020

11109

Background: Cytoreductive surgery for malignant pleural mesothelioma (MPM) should be reserved for patients with favorable tumor biology. Osteopontin (OPN) and the ratio of absolute neutrophil to absolute lymphocyte counts (NLR) have been reported as possible prognostic biomarkers. These were studied with other clinical/ laboratory variables in a mixed surgical/non-surgical MPM population to define independent predictors of survival (OS) and progression (TTP). Methods: Forty-four MPM patients (12 F, 32M; 26 cytoreduction, 18 no cytoreduction; 31 epithelial, 13 non-epithelial; 15 Stage I/II, 29 Stage III/IV) were examined with regard to pretreatment plasma OPN (ELISA, R&D, Minneapolis, MN), NLR age, gender, therapy, histology, stage, platelet count and WBC count. Cut points for age, OPN, NLR, platelets, and WBC were determined by X-tile Software (Yale, New Haven, CT) and univariate/multivariate Cox analyses performed. Results: Median OS were 11 m, 21m, and 8m for all 44 MPMs, cytoreduced and non-cytoreduced MPMs, respectively. Of platelet count, WBC, NLR, and OPN, only OPN was statistically significant between Stage I/II and Stage III/IV (80.3 ng/ml vs 148 ng/ml, p<0.018). The only independent covariate predictive of OS was plasma OPN. For TTP in cytoreduced patients, only age, stage, platelet count, and OPN were significant in univariate analysis, and multivariate modeling retained stage (p=0.04, HR=2.75, 95% CI=1.0517 to 7.1879) and OPN (p=0.0008, HR=17.471, 95% CI=3.3054 to 92.3461). Conclusions: Plasma OPN is promising for the stratification of tumors into good or bad risk categories and to help select potential candidates for cytoreduction and further postoperative therapy. [Table: see text]
The role of Pirh2 C-terminal residues and ubiquitin lysine chains in ubiquitination mechanism.
Authors: Abou Zeinab Rami Mahmoud , Leng Roger
Published: March 2020

11110

Background: Pirh2, a p53 inducible gene, is proposed to be a main regulator of p53 family proteins fine tuning the DNA damage response by acting as an E3 ligase. A negative feedback loop between Pirh2 and p53 exists where under unstressed conditions, Pirh2 induces p53 ubiquitination followed by proteosomal degradation. In case of cellular stress when the activity of tumor suppressors is essential, Pirh2 is self-ubiquitinated releasing p53 continuous repression. Interestingly among all E3 ligases, Pirh2 is the only one to be over-expressed in a wide range of human tumors. This over-expression indicates a disruption in the self-ubiquitination mechanism or a defect in the degradation mechanism post ubiquitination. Based on that, we aimed to analyze Pirh2 ubiquitination mechanism through mapping Pirh2 domains to reveal the essential residues for this process and the ubiquitin chains utilized. Methods: Pirh2 constructs deleting major residues in the three domains were designed, and also ubiquitin mutant constructs were designed through single/multiple mutations at specific positions where lysine residues are mutated to arginine. Using these constructs and in comparison to WT proteins, we tested Pirh2 in-vitro self and p53 ubiquitination activity. Results: Regarding Pirh2, we were able to reveal that residues 240-250 of the c-terminal along with the ring domain are essential for self-ubiquitination.K63R and K48R ubiquitin did not affect Pirh2 self-ubiquitination minimizing the impact of ubiquitin mutations on Pirh2 self-ubiquitination. However, KO, which had all lysine residues mutated to arginine, showed total inhibition of Pirh2 self-ubiquitination confirming the importance of lysine residues. Interestingly, Pirh2 self-ubiquitination reaction showed no difference in the presence or absence of p53 proteins. Concerning Pirh2-p53 ubiquitination, K48 was found to be critical for E3 ubiquitin ligase activity because K48R and not K36R showed defective ubiquitination. All results were confirmed by quantifying ubiquitin. Conclusions: Our data added knowledge to the Pirh2 self-ubiquitination mechanism that can resolve the constant overexpression of Pirh2 proteins hence maximizing p53 response to DNA damage.
Hormone receptor-dependent regulation of ABAT and beta-alanine metabolism in breast cancer.
Authors: Budczies Jan , Brockmoeller Scarlet F. , Mueller Berit , Dietel Manfred , Fiehn Oliver , Denkert Carsten
Published: March 2020

11112

Background: Recently, we identified beta-alanine as biomarker for breast cancer using GC-MS based metabolomics. Beta-alanine is increased in breast cancer compared to normal tissues and in the more aggressive ER- subtype compared to ER+ breast cancer. Beta-alanine is a substrate of 4-aminobutyrate aminotransferase (ABAT), can be catabolised to malonate semialdehyde and used for reduction of NAD and acetylation of coenzyme A. The aim of the current study is to analyze ABAT protein and RNA expression in a large cohort of breast cancers. Methods: The specificity of a polyclonal antibody against ABAT was validated using siRNA in MFC7 cells. A cohort of 164 paraffin-embedded breast cancer tissues from the METAcancer biobank was investgated by immunohistochemistry. Tumor cells were evaluated separately for staining intensity (low, moderate or high) and percentage of stained cells. A cohort of 156 METAcancer samples was investigated for gene expression using whole genome DASL. Results: ABAT protein intensity correlated strongly with estrogen receptor (ER) status (p < 0.001), but not with HER2 status. In particular, the ABAT protein was highly expressed in 41% of the ER+ tissues, but only in 2% of the ER- tissues. Further, ABAT intensity correlated strongly with tumor grade (p < 0.001). ABAT intensity did not correlate with tumor stage or nodal status. Explorative evaluation of ABAT protein in normal cells revealed weak expression in some of the ducts and negative expression in adipose cells. ABAT protein and RNA expression strongly correlated in the subcohort investigated by DASL. Analysis of a large external data set (publicly available at www.kmplot.com) showed that low ABAT expression is associated with an unfavorable prognosis of breast cancer. Both, ABAT protein and RNA expression, showed a strong negative correlation with beta-alanine abundance. Conclusions: We reported on changes in beta-alanine metabolism that occur between the molecular subtypes of breast cancer and normal breast tissue. High expression of ABAT in ER+ breast cancer is compatible with catabolic use of beta-alanine. Differently, beta-alanine might be preferable used anabolic in ER- breast cancer, possibly for the synthesis of carnosine.
Impact of lymph node ratio (LNR) on prognosis of early breast cancer.
Authors: Donnellan Eoin , O'Keefe Jo , O'Connor Derbrenn , O'Hanlon Deirdre , Murphy Conleth G. , Bird Brian
Published: March 2020

1115

Background: Breast cancer is currently staged according to the TNM (tumors, nodes, metastases) classification of the American Joint Committee on Cancer (AJCC) Staging System. Lymph node ratio (LNR, the ratio of positive axillary lymph nodes to the total number of nodes examined) may provide additional prognostic information to that provided by TNM scores. Furthermore, LNR may potentially identify subpopulations within the traditional AJCC stages that are at increased risk of adverse outcomes. Methods: We performed a single institution retrospective study of all patients diagnosed with early breast cancer between January 2000 and January 2011. Patients were divided into low- (≤0.14), intermediate- (0.15-0.39) and high-risk (≥0.4) LNR groups. We assessed the impact of LNR and conventional AJCC staging parameters on overall survival (OS) and disease-free survival (DFS). Results: 786 patients were included in the analsyis, 238 of whom were node positive. As expected nodal status according to pathologic nodal (pN) stage was prognostic for OS and DFS with OS decreasing from 88.3% in pN1 patients to 40.8% in those with pN3 disease (p<0.001). LNR was also significantly associated with prognosis. OS decreased from 94% in the low-risk LNR group to 64% in the high-risk group, while DFS decreased from 92% in the low-risk LNR group to 50% in the high-risk (p<0.001). When Stage III patients were divided into low- and high-risk LNR groups, OS decreased from 100% in the low LNR group to 63% in the high LNR group (p<0.05). Similarly, DFS decreased from 96% in the low LNR group to 56% in the high LNR group (p<0.05). A similar trend was not observed when Stage III patients were stratified according to pN status. LNR was also found to be prognostic when pN1 patients were divided into low- and high-risk LNR groups. Although both LNR and nodal status were significantly associated with OS and DFS on univariate analysis, LNR retained its significance on multivariate analysis, while nodal status did not. Conclusions: LNR can identify subpopulations within the traditional AJCC staging that are at higher risk of adverse outcomes. These findings should be examined in larger retrospective studies and, if validated, be considered as a stratification factor in future adjuvant trials.
Sentinel lymph node biopsy (SNB) in pregnancy-associated breast cancer (PABC).
Authors: Gropper Adrienne , Zabicki Calvillo Katherina , Troyan Susan , Dominici Laura Stewart , Rhei Esther , Economy Katherine E. , Schapira Lidia , Tung Nadine M. , Meisel Jane L. , Partridge Ann H. , Mayer Erica L.
Published: March 2020

1117

Background: SNB in PABC is not often pursued due to concerns for potential fetal harm. There are only limited data available regarding the safety and efficacy of SNB in patients (pts) with PABC. Methods: Pts with PABC who underwent SNB were identified from within an existing multi-institutional PABC cohort diagnosed 1996-2013. Factors evaluated included method and result of SNB evaluation, maternal disease outcome, and fetal outcomes. Results: Within a cohort of 78 PABC pts, 53 had breast surgery while pregnant; 23 (43%) underwent SNB, 27 (51%) underwent initial axillary node (AN) dissection, 18 of whom were clinically node negative, and 3 had no nodal evaluation. Of SNB pts, 21 (91%) had stage 1-2 disease; 14 (61%) had ER/PR+ disease and 7 (30%) HER2+. Eight (35%), 9 (39%), and 6 (26%) women had SNB in the first, second, and third trimesters, respectively. 99-Technetium-labelled sulfur colloid (99-Tc) alone was used for SNB in 14 pts; methylene blue (MB) dye alone was used in 7. SN was identified in 100% of pts; see Table. There were no SNB-associated complications. At a median of 2.4 years from diagnosis, there were no locoregional recurrences, 3 (13%) distant recurrences, and 1 (4%) death from breast cancer. Among pts who underwent SNB, there were 20 liveborn infants and 3 pregnancies ongoing. Of the 20 infants born, 18 were healthy, 1 unknown, and 1 had cleft palate (in setting of maternal risk factors including smoking and methadone). Conclusions: SNB in PABC appears to be a safe and accurate procedure using either 99-Tc or MB techniques. This is one of the largest experiences reported to date of SNB during PABC; however, numbers remain limited and rates of SNB in our cohort were lower than current rates in non-PABC patients. Additional research and monitoring for safety of this procedure is warranted in women with PABC. [Table: see text]