bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiplexed mass spectometic quantitation of HER1-3, cMET, and IGF1R in FFPE tumor samples: Implications for targeted therapy and resistance.
Authors: Hembrough Todd A. , Liao Wei-Li , Thyparambil Sheeno , Darfler Marlene , Krizman David , Burrows Jon 
Published: March 2020

e21068 Background: The human EGF receptor family (HER’s) consists of two clinically validated drug targets (EGFR and HER2), and two receptors (HER3 and HER4) which are the subject of intensive preclinical and early clinical investigation. Although drugs inhibiting both EGFR and HER2 show significant antitumor activity in the clinic, the acquisition of resistance is a hallmark of these and other targeted therapies. In the case of both targets, one of the emerging resistance mechanisms is the co-expression of other receptor tyrosine kinases, including members of the EGFR superfamily, cMet and IGF1R. As an example, it was recently shown that HER2 co-expression mediates resistance in cetuximab treated head and neck cancer. Similarly, much attention has been paid to HER3 both as a bona fide drug target and a resistance mechanism. Methods: Using trypsin digestion mapping of recombinant proteins, we identified unique peptide sequences from each of these receptors, and built quantitative mass spectrometric (MS) assays which could be multiplexed into a single MS analysis of 1ug of tumor protein. Assays were preclinically validated on 10 formalin fixed cell lines, and FFPE human NSCLC primary tumor xenografts. Results: The validated multiplex assay was used to measure expression levels of HER1-3, cMET and IGF1R in two cohorts of clinical tumor tissue which had been treated with HER family antagonists. One, a set of gefitinib treated NSCLC tumors (N=15) , and a second, a cohort of advanced breast cancer tissues which had adjuvant treatment with trastuzumab(N=18). Here we present expression patterns for each of the RTKs studied, with the intent to begin to define the relationship between RTK expression and response to either gefitinib or trastuzumab treatment. Conclusions: It is important to not only understand primary mechanisms of tumor growth, but also mechanisms of resistance in patients undergoing targeted therapies. Our Liquid Tissue-SRM promises to be a platform which can deliver extremely high sensitivity, absolute specificity as well as multiplexing capabilities to assess critical oncology targets.
Panel of serum biomarkers to predict benefit from bevacizumab (BEV) in advanced NSCLC patients.
Authors: Braun Eduardo , Fidler Mary J. , Basu Sanjib , Gangaram Anjali , Walters Kelly Kaiser , Karmali Reem , Fhied Cristina , Lie Wen-rong , Borgia Jeffrey Allen , Bonomi Philip D. 
Published: March 2020

e21069 Background: BEV has produced modest benefits in patients (PTS) with advanced NSCLC. Identification of positive predictors for BEV would have important implications for individual PTS and health care costs. Methods: We performed a prospective exploratory analysis to identify serum biomarkers as predictors of improved outcomes with BEV. Pre treatment sera were collected from 93 pts prior to initiation of first line treatment for advanced NSCLC. Treatment drugs, including BEV, were prescribed according to treating physician’s discretion. Seventy two serum biomarkers, relevant to angiogenesis and tumor progression, were recorded using Luminex immunobead platform. Serum levels were correlated with progression free survival (PFS) and overall survival (OS) and compared between patient treated with or without BEV containing regimens, BEV + and BEV- groups respectively. Log-rank and interaction p value tests were used to identify markers associated with longer PFS and OS in the BEV+ group but not in BEV- group. Results: Characteristics for each group were: BEV+ (n=43, median age 65 y/o, 72% smokers, 60% females, 100% non-squamous). BEV– (n=50, median age 64 y/o, 84% smokers, 50% female, 70 % non-squamous). The BEV+ group had longer PFS (5.8 vs. 3.0 mos, log-rank p= 0.039) and OS (13.1 vs. 8.5 mos, log-rank p =0.11) when compared to the BEV- group. High serum levels of these markers resulted in a differential decreased hazard in the BEV+ group: PDGF-AB/BB (interaction p <0.01 for PFS, p=0.04 for OS), FGF (interaction p=0.15 for PFS, p<0.04 for OS), tenascin-c (interaction p=0.18 for PFS, p=0.04 for OS), RANTES (interaction p=0.04 for PFS, p=0.6 for OS), epiregulin (interaction p=0.31 for PFS, p=0.04 for OS) and anti-HGF (interaction p=0.18 for PFS, p=0.03 for OS). In the BEV+ group higher levels of PDGF-AB/BB were associated with a better outcome (log-rank p=0.05 and p=0.01 for PFS and OS respectively). We did not find significant correlations between serum levels of VEGF, anti-VEGF or VEGFR and benefit from BEV. Conclusions: This exploratory analysis suggests that these biomarkers may have predictive value for BEV in NSCLC PTS and should be considered for further studies.
Performance of the high-definition circulating tumor cells (HD-CTC) assay in patients with non-small cell lung cancer (NSCLC).
Authors: Bazhenova Lyudmila , Nieva Jorge J. , Kolatkar Anand , Luttgen Madelyn , Marinucci Dena , Bethel Kelly , Kuhn Peter 
Published: March 2020

e21074 Background: Technologies to isolate and study circulating tumor cells (CTCs) became available in recent years. To date, the most widely employed techniques to detect CTCs use EpCAM based enrichment protocols. However, those assays are only able to find CTCs in a minority of patients with NSCLC, thus limiting its potential applicability. We are reporting performance of a new enrichment-free HD-CTC assay in patients with NSCLC. Methods: This multi-institutional study recruited patients with all stages of NSCLC. Blood was collected at the time of the study entry, 3 weeks, 3m, 6m, 9m, and 12 m. CTCs were identified with enrichment free fluorescent labeling followed by automated digital microscopy. CTCs were physically and morphologically characterized by cytokeratin (CK) intensity, nuclear size, apoptotic changes, and aggregate formation. Cells were classified as HD-CTCs if they met strict morphologic and immunofluorescence characteristics. This assay has been previously validated in 15 normal controls. Results: 106 patients were recruited (I-10, II-3, III-34, IV-59), 78 were chemotherapy naïve. 85%, 79%, and 53% of patients with stages, I+II, III, and IV had detectable HD-CTCs. The chemotherapy naïve cohort had median HD-CTCs of 4.4/mL (range 0–515.6) and a mean of 44.1 (±95.2)/mL. No significant differences were detected in median HD-CTC count between tumor stages (p = 0.32). CK intensity, nuclear size, frequency of apoptosis and aggregate formation was also similar across stages. 28 patients with stage IV disease were analyzed with multiple draws over time. Mean CTC counts were averaged over multiple draws. In each individual patient there was a trend for increased CTC prevalence with advancement of the disease. Median survival was 244 days for patients with ≥5 CTC/mL vs. 304 days for patients with < 5 CTC/mL. HR 4.0, (p=0.0084). Conclusions: The HD-CTC assay can detect CTCs in a high percentage of patients with early and advanced NSCLC. Small subset analysis demonstrated the prognostic value for the enumeration of HD-CTCs. The high prevalence of HD-CTCs across stages could make this assay an ideal candidate for larger scale investigation into prognostic and predictive features of HD-CTCs.
Morphologic variation of circulating tumor cells in metastatic breast cancer patients visualized by immunofluorescent stain pattern.
Authors: Adams Daniel , Martin Stuart S , Charpentier Monica , Makarova Olga V , Zhu Peixuan , Li Shuhong , Amstutz Platte T , Tang Cha-Mei 
Published: March 2020

e21079 Background: Isolation of circulating tumor cells (CTCs) using microfiltration is a growing utility in the field of CTC detection. The microfiltration approach can be used on peripheral blood as a non-invasive liquid biopsy for cancer detection and subtyping. We present a utilization of the CellSieve microfilter to subtype CTCs based on immunofluorescent staining pattern of cytokeratin filamentation and EpCAM surface marker expression. Our initial study on CTCs in patient blood indicates that disseminated CTC populations have high rates of phenotypic heterogeneity. Further detailed molecular analysis and patient tracking of these phenotypes may lead to individualized patient assessment based on CTC characterization. Methods: 7.5 mL of whole blood collected from metastatic breast cancer patients were diluted in a fixative solution. An 8 µm CellSieve precision microfilter was placed into a filter holder and the samples were passed through the filter (~ 90 seconds). The cells captured on the filter were fixed, permeabilized, and stained with DAPI, cytokeratin (FITC), EpCAM (PE), and CD45 (Cy5). Cells without CD45 staining were classified by their morphology, nuclear integrity and the presence of cytokeratin and EpCAM staining. Results: In our initial assessment, patient samples were found to have a number of phenotypic CTC subtypes. Cytokeratin filamentation was clearly seen on a number of CTCs while other cells presented with spotted patterns, implying CTCs in various stages of apoptosis. Later stage apoptosis, with segmented nuclear signature, was also seen in various samples. Cell clusters and cells in division were DAPI positive, while EpCAM positivity was negligible, 0-3 cells/sample, correlating with established data from the CellSearch CTC assay. Conclusions: In addition to enumeration, phenotypic variation of CTCs may be a valuable tool for the personalized care of cancer patients. We have shown that individual breast cancer patients have overlapping phenotypes of CTCs circulating in their peripheral blood. We have begun categorizing patients based on these phenotypes and plan to correlate them with overall prognosis.
Value of circulating epithelial tumor cells and circulating endothelial cells (CTCs/CECs) in patients with HER2-negative recurrent or metastatic breast cancer treated with bevacizumab (B) in combination with paclitaxel (P) and gemcitabine (G) as first-line therapy (AVALUZ trial).
Authors: Codes Manuel , Manso Luis , Ciruelos Eva M. , De la Haba- Rodriguez Juan , Galan Antonio , Baena Jose Manuel , Jaen Ana , Gil Miquel , Murias Adolfo , Blancas Isabel , Gonzalez Encarnacion , Perez Diego , Bayo Juan L. , Mel Jose Ramón , Garcia-Martinez Elena , Cubedo Ricardo , Salvador Javier 
Published: March 2020

e21088 Background: CTCs in peripheral blood are an ideal source for the detection of tumor dissemination. Their prognosis significance has been demonstrated in metastasic breast carcinoma (Cristofanilli, NEJM 2004). Beacizumab in combination with CT, improves progression free survival (PFS) of first line treatments, may modify CTC and CEC levels. Aims of this study are the evaluation of the prevalence and kinetics of CTCs and CECs before and after treatment with B in pts with MBC Methods: Pts received B 10mg/kg/q2w combined with P 150mg/m2 and G 2000mg/m2 d1,15/q28d therapy until disease progression, unacceptable toxicity or withdrawal. CTC/CEC were measured in 7.5ml of blood at baseline and after the 1st cycle of treatment. Enumeration was performed by CellSearch System, Veridex Results: Data were available for 37 pts. Median of f-up was 16.2 m. The media of CTCs was 41 cells (min0-max845) in the 1st determination and 5 cells (min0-max99) in 2nd determination. Baseline CTCs>2 was associated with statistic lower PFS 10.8 m (95%CI:7.66-16.91) compared to those with CTCs<2, PFS 17.7m (95%CI:17.60-NA),p=0.046. Baseline CTCs>10 vs CTCs<10 was associated with statistic lower OS (14%vs45% of deaths), p=0.035. 92% (N=22) of pts that had stable disease/partial response, decreased or maintained CTCs value. CTC level was not correlated with CEC level, p=0.74. Media value of baseline CECs was 130 (min4-max1407) and 60.3 (min0-max349) in 2nd determination. Baseline CECs>200 was associated with lower PFS 8.2m (95%CI:0.6-10.8) compared to those with <200, PFS 16.9m (95%CI:8.78-NA),p=0.003. No difference was observed in OS. 74% of pts (N=14) that had stable disease/partial response, decreased or maintained CECs value. Baseline CTCs >5 was associated with a median PFS of 15.2 m (95%CI:7.6-16.9) Conclusions: Our study suggests significant correlations between levels of baseline CTCs and CECs and poor prognosis. Addiction of B to 1st-line CT was related with high reduction of CECs and CTCs count.
Molecular prognostic markers in colorectal cancer.
Authors: Ivanov Krasimir , Kolev Nikola , Tonev Anton , Tontchev Anton 
Published: March 2020

e21100 Background: Patient 5-year survival rate from colorectal cancer in the last few decades remains at 40 to 45 percent. Nearly one-half of all patients who undergo a potentially curative resection will relapse because of undetected metastasis. Overall survival rate remains poor which strongly suggests that the dissemination of these cells occurs early in the disease process there is a need of diagnostic methods which evaluate tumor aggressiveness. The p-53, MUC-2, Ki-67, VEGF, Bax, Bcl-2, Stat, MMP2 are markers, which could provide useful individual information about biological potential and prognosis. Methods: We evaluate the expression of those markers in group of 72 patients with colorectal carcinoma. We observed the relations between: 1) type of operation; 2) histological type; 3) clinical stage; 4) individual risk index (IRI), based on evaluated expression of tumor markers; 5) impact on survival rate. Results: Achieving 100% successful rate in our study, we statistically analyzed the data received from marker’s expression. We calculated an IRI for every patient, based on the data from imm3unohystochemical expression by already mentioned molecular markers and divided the patients in 3 groups. A Group A, formed by 28 (38%) patients, which are with low clinical stage and worse molecular prognosis. A Group B, formed by 20 (27%) of patients had low tumor aggression and III or IV clinical stage and Group C, formed by the other 26 (35%) of the patients where is a correlation between the molecular and clinical stage prognosis. We found significant differences in survival rate in Groups A and B, according to clinical stage and our proposed IRI. Conclusions: Multivariate analysis revealed that the IRI is independent prognostic factor for the tumor outcome. There were many evidences for the need of alteration of the surgical behavior in different stages according to biological nature of the tumor. Analysis of a combination of immunohistochemical molecular markers with the conventional diagnostic methods for colorectal cancer allows better prediction of the patients’ prognosis and more accurate and individualized therapeutic strategy. That could decrease the incidence of recurrence-rate and low the mortality rate of this society-significant disease.
Prognostic value of stem cell quantification in stage II colon cancer.
Authors: Vaz M. Angeles , Martinez Juan Carlos , Devesa Manuel , Die Trill Javier , Abraira Victor , Riquelme Alejandro , Carrato Alfredo 
Published: March 2020

e21104 Background: Cancer stem cells (CSCs) are a subset of tumor cells with capacity to self-renew and generate the diverse cells that comprise the tumor. They are considered cancer (Ca) initiating cells responsible for tumor recurrence and maintenance. These cells express pluripotency markers (CD133 and NANOG) and do not express markers of differentiation as cytokeratin 20 (CK20). The potential prognostic value of CSCs in colorectal cancer has been studied with conflicting results. However some of these series were based on heterogeneous situations, involving rectal and colon cancer (CC) and different tumor stages (I-IV). The aim of this study is to evaluate the prognostic value of CSCs in a highly homogeneous population of stage II CC. Methods: One hundred stage II CC patients (pts) treated by the same surgical team at Ramon y Cajal University Hospital between 1977 and 2005 were retrospectively analyzed. None of the pts received adjuvant chemotherapy. Inmunohistochemistry expression of CD133, NANOG and CK20 was scored on paraffin sections using four levels: <10%,11-25%, 26-50% and >50% positivity. Kaplan-Meier analysis and log rank test were used to compare survival. Results: Median age: 68 years (45-92). Median follow up: 5.8 years. Recurrent disease: 17 (17%). Expression of CD133 was shown in 60% of the tumors, in 95% for NANOG and 78% for CK20. No correlation was found among expression levels of CD133, NANOG or CK 20 and relapse-free survival (RFS) and overall survival (OS). However, a statistical significant correlation was found between established pathological prognostic factors and RFS and OS. Conclusions: Stem Cell quantification defined by CD133 and NANOG expression has no correlation with RFS or OS in this cohort of Stage II CC. However, a correlation with established prognostic pathological factors was found. Therefore, our results suggest that CSCs may not play a major role in early phases of CC.
Investigating cyclooxygenase-2 signaling in breast cancer-initiating cells.
Authors: Hall Carolyn S. , Walker Andrew , Laubacher Barbara A. , Singh Balraj , Lucci Anthony 
Published: March 2020

e21107 Investigating cyclooxygenase-2 signaling in breast cancer-initiating cells Background: Only a small fraction of breast tumor cells, breast cancer-initiating cells (CSC), have the ability to initiate tumor growth and metastasis. Some characteristics of breast CSCs have been described in vitro by employing non-adherent CSC-enriching culture conditions, however, little is known regarding prostanoid signaling or prostaglandin E2 (PgE2) production in CSCs. In this study we measured cyclooxygenase-2 (COX-2) and prostaglandin E2 receptor expression in CSCs and assessed the effects of COX-2 and Ep4 inhibition on in vitro CSC spheroid formation and PgE2 production. Methods: CSCs were cultured in serum-free medium using ultra low attachment plates. Cells were treated with vehicle, a COX-2 inhibitor (Celecoxib), or an EP4 inhibitor (GW627368X) at concentrations of 0.1, 1, 5, 10, 50, or 100uM for 10 days. Spheroids were quantified using a Gelcount machine. Non-linear regression was used to calculate IC50 values. Western blots were performed using anti-COX-2, anti- EP1, 2, 3 and 4 receptor antibodies. Prostaglandin E2 production was measured using a PgE2 immunoassay kit. Results: COX-2 protein expression was observed for MDA-MB-231 and SUM149 CSCs. EP2 and EP4 protein was detected for MCF-7, MDA-MB-231, and SUM149 CSCs. The Celecoxib IC50 values for MCF-7 and SUM149 CSC spheroid formation were 0.5 and 2.2 uM, respectively; GW627386X IC50 values for MCF-7 and SUM149 CSC spheroid formation were 1.2 and 0.3uM, respectively. No significant differences in PGE2 production were observed for MCF-7 CSCs compared to adherent MCF-7 cells (11.5 ± 4.4 vs. 8.7 ± 2.1 pg/mL/106 cells; p=0.88). However, PgE2 production in MDA-MB-231 CSCs was significantly higher than adherent MDA-MB-231 cells (1507.0 ± 329.0 vs. 13.8 ± 5.7 pg/mL/106 cells; p≤0.001) and SUM149 CSC PgE2 production was significantly higher than SUM149 adherent cells (4932.9 ± 501.7 vs. 194.5 ± 31.0 pg/mL/106 cells; p≤0.001). 10uM Celecoxib treatment inhibited PGE2 production in MDA-MB-231 and MDA-MB-231 CSCs. Conclusions: MDA-MB-231 and SUM149 CSCs express COX-2, EP2 and Ep4 and produce high levels of PGE2. CSC spheroid formation is significantly decreased with COX-2 and EP4 inhibition.
Immunologic changes in patients with metastatic melanoma on bevacizumab and chemotherapy versus chemotherapy alone.
Authors: Mansfield Aaron Scott , Nevala Wendy Kay , Markovic Svetomir 
Published: March 2020

e21112 Background: Metastatic malignant melanoma is difficult to treat and has a poor prognosis. Systemic therapy for metastatic melanoma has had limited success and novel drug combinations need to be tested. To that end, we have combined anti-angiogenic therapy (bevacizumab) with chemotherapy in clinical trials and have shown some clinical efficacy. Vascular endothelial growth factor (VEGF) is increasingly seen as an immunosuppressive factor that hampers dendritic cell maturation and reduces antigen presentation. We have recently shown that tumor-derived VEGF plays a role in Th-2 driven systemic chronic inflammation seen in advanced melanoma patients. Therefore, our data suggest that blocking tumor derived VEGF with the monoclonal antibody bevacizumab may impact immunity in such a way that augments anti-tumor immunity enhancing clinical efficacy. Methods: In order to understand the immune status in metastatic melanoma patients on chemotherapy (n=55) or chemotherapy plus bevacizumab (n=73), we measured levels of 42 cytokines, chemokines and growth factors in plasma by a mulit-plex bead assay. Matched pairs analysis was performed to determine if the average responses of the measured cytokines were different between the patients that received chemotherapy with bevacizumab or chemotherapy alone. The pre-treatment levels were compared with levels drawn four weeks after the first treatment. Results: There were significant differences in IL-1a, IL-1ra, IL-6, IL-8 and IP-10 between patients treated with chemotherapy alone relative to patients treated with chemotherapy plus bevacizumab. In all cases of patients treated with chemotherapy plus bevacizumab, the levels of these cytokines all decreased relative to pre-treatment samples. In patients treated with chemotherapy alone the levels of these cytokines increased relative to pre-treatment samples. Conclusions: Our data suggest that treating patients with the anti-angiogenic drug bevacizumab may affect the plasma cytokine milieu by reducing the level of many key inflammatory cytokines. Therefore, the use of bevacizumab in combination with chemotherapy may augment host immunity and elicit a more effective anti-tumor immune response.
Cellular responses to lactate in breast cancer.
Authors: Kennedy Kelly Marie , Dewhirst Mark W 
Published: March 2020

e21119 Background: Clinical studies across different tumor types have found that high lactate levels correlate with increased metastases and poor patient prognosis, overall and metastasis-free survival; however, mechanisms are unknown. There is increasing evidence of cancer stem cells (CSC). Lactate inducing a stem-like phenotype may be one explanation for the increased metastases seen in high-lactate solid tumors. We hypothesized that lactate induces EMT in human breast cancer cells and induces/selects for a stem-like phenotype. Methods: We examined lactate levels in locally advanced breast cancer (LABC) biopsies using bioluminescence. Breast cancer cell lines (MCF7, T-47D, MDA-MB-231) were treated acutely (hours) or chronically (weeks) with lactate (0-40mM) and tested for cell viability (Annexin V, clonogenics), EMT markers (Snail-1), cellular detachment, and anchorage-independent cell growth (mammosphere assays). There was one “no treatment” control group. Experimental groups were treated with lactate in the context of complete media or glucose-free conditions (to mimic the typical tumor microenvironment). For the mammosphere assays, cells were treated both prior to (attached as a monolayer) and during sphere formation. Results: Lactate concentrations in the LABC biopsies were between 0-7.5mM. Lactate levels relevant to solid tumors (<40mM) were not toxic to the cell lines tested. Cellular detachment after lactate treatment was not statistically significant; however, the percentage of viable detached cells was significantly increased, and they retained colony-forming abilities. Preliminary data shows a slight increase in Snail-1 levels after chronic lactate treatment. Chronic lactate treatment had a slight but statistically significant effect on secondary mammosphere formation in MCF7s, indicating the important self-renewal properties characteristic of CSCs. Conclusions: The effect of lactate treatment alone on human breast cancer cell lines is subtle but significant. Phenotypic changes elicited by lactate are dependent upon lactate concentration and duration of treatment. Results thus far support the hypothesis that lactate affects expression of stem-like properties in breast cancer cell lines. Supported by BC083154 and R01 CA40355