bluuebunny/crossref_metadata_2025 huggingface dataset:
The use of insulin and the effect on survival of non-small cell lung cancer patients
Authors: Farhan S. Y. , Jankowski M. , Hanbali A. , Wang D.
Published: August 2020

e22073

Background: Diabetes mellitus (DM) is one of the common morbidities in United States. Insulin therapy has been frequently used in its treatment. Some studies have postulated that activation of Insulin and Insulin-like growth factor (IGF) pathway may contribute to cancer proliferation, growth and resistance to anticancer therapies. The impact of insulin on diabetic patients with non-small cell lung cancer (NSCLC) has not been reported. Methods: 1,233 patients with NSCLC diagnosed between January 1999 and December 2004 were identified from the Tumor Registry at Henry Ford Health System. Based on inclusion criteria, data from 1,206 patients were extracted from the electronic medical records. Statistical analyses were performed between insulin and non insulin users. Results: Out of 1,206 patients, 193 patients had NSCLC and DM. Data was available from 192 patients. Sixty (31.3%) were on insulin at the time of NSCLC diagnosis and defined as insulin users, while 132 were not on insulin and defined as non- users (68.7%). Demographics, co-morbidities, disease stage at diagnosis, therapeutic interventions, and laboratory values were analyzed for overall and cancer-specific survival. More insulin users (81%) than non-users (64%) presented with advanced disease (stage ≥ 3) at diagnosis (P=0.02). Although no significant difference of Hemoglobin A1c was observed, the median survival from all causes of death was 6.5 months for insulin-users versus 9.9 months for non-users (P=0.08). When survival time was calculated from cancer-specific death, the median survival between insulin and non-insulin users was 7.2 months versus 38.7 months, respectively (P=0.002), with a hazard ratio (HR) for insulin use equals 1.91. Results from our univariable and multivariable analyses will be presented at the ASCO annual meeting in detail. Conclusions: This data indicates that the use of insulin may adversely affect the biology and clinical course of NSCLC with a tendency of advanced disease stage at diagnosis and a shorter cancer-specific survival time. Prospective study of DM with NSCLC or other cancer diagnosis will improve our understanding of this potential adverse effect on cancer biology and provide scientific rational to target IGF-pathway especially in diabetic patients.

No significant financial relationships to disclose.
A pilot study of biological markers (BM) as predictors for response and prognosis in patients (pts) with R0 resected gastric cancer (GC)
Authors: Li G. , Zhang Z. , Ma X. , Sheng W. , Zhu J. , Cai G. , Cai G. , Liu M. , Gu W. , Li J.
Published: August 2020

e22076

Background: To evaluate clinical value of biological markers and their prognostic significance in gastric cancer as well as prediction to clinic outcome; To select preference marker for targeted therapy in high risk patients hence to guide personalized treatment. Methods: Retrieved from total 394 patients treated during the period of Jan 2004 to June 2006 of Cancer Hospital, Fudan University with complete medical records and follow up data, 84 patients with pre- and post- operative biological markers examined were selected. All of these patients underwent R0 gastrectomy, 61 (72.6%) patients underwent D2 resection. Forty- one (48.8%) patients received ≥ 4 cycles adjuvant chemotherapy. Preoperative tumor markers include: CEA, AFP, CA199, CA50, CA72–4, CA24–2 and postoperative immunohistochemical examination include: Bax, Bcl-2, P27, CyclinD1, TOPO2, MDR, GST-π, Ki67, AgNOR, EGFR, P21, P53, PCNA, C-myc and Neu were analyzed. Univariate, multivariate and Logistic regression analysis were performed and Kapan-Meier estimates were calculated. Results: For the whole group, 2-year OS and PFS were 76% and 66% respectively. Logistic regression analysis showed bcl-2, ki67, c-myc2 and Neu were relative with more nodal involvement. Patients with lower level of CEA, CA72–4 and higher level of cyclin D1 suivived longer than the other patients. There were different clinical significance with those biomarkers. Patients with lower level of cyclinD1, CEA had more local regional recurrence (p=0.026) while patients with higher PCNA experienced more distant metastasis (p=0.005). Univariate analysis showed that clinical staging, level of cyclinD1 and CEA were correlated with OS (p<0.05). Conclusions: Biological markers may be helpful in predicting the clinical outcome and prognosis for R0 resected gastric cancer patients. Further study are needed to confirm their clinical significance.

No significant financial relationships to disclose.
Comparison of Lynch predictive models for identification of MLH1/MSH2 mutation carriers in a Spanish multicentre clinic- based cohort
Authors: Valenzuela I. , Balmaña J. , Rue M. , Blanco I. , Torres A. , Ramon y Cajal T. , Chirivella I. , Guillen C. , Brunet J. , Tejada I.
Published: August 2020

e22077

Background: Different predictive models for Lynch syndrome have recently been developed and their comparative performance in a clinic-based cohort has not been assessed. We aimed to analyze the accuracy of the MMRpro, Barnetson, and PREMM1,2 models to predict MLH1/MSH2 mutation carrier status in 564 unrelated probands with clinical suspicion of hereditary colorectal cancer and compare it with Wijnen model and clinical criteria. Methods: Overall, 538 individuals (95%) underwent mismatch repair (MMR) deficiency screening before germline genetic testing (sequencing with or without large rearrangement analysis) and 26 (5%) performed direct genetic testing. Prediction scores for all individuals were calculated by each model. Sensitivity, specificity, positive predictive value (PPV), and the areas under the receiver operating characteristics curves (AUC) for all models were calculated and compared with the Revised Bethesda Guidelines (RBG). Results: 114 individuals (20%) were mutation carriers (63 MLH1, 51 MSH2). The AUC was 0.95 (95% CI: 0.93–0.97) for MMRpro, 0.87 (95% CI 0.83–0.91) for the Barnetson model, 0.87 (95% CI 0.83–0.91) for PREMM1,2, and 0.75 (95% CI 0.69–0.80) for the Wijnen model (p<0.001). Testing thresholds and specificity at 100% and 90% sensitivity for each model were: 0.001/17% and 0.33/89% for MMRpro, 0.01/9% and 0.07/59% for Barnetson, 0.05/5% and 0.11/58% for PREMM1,2. Sensitivity and specificity of RBG were 86% and 14%, respectively. Calibration was 0.92, 1.05, 0.50, and 1.25 for PREMM1,2,Barnetson, Wijnen, and MMRpro, respectively. Conclusions: In a population of individuals at risk of Lynch syndrome, the MMRpro model has the largest AUC, although the Barnetson and PREMM1,2 model also show adequate discrimination. Any of the models perform better than the RBG and provide quantitative risk estimation of finding a MLH1/MSH2 mutation useful in genetic counselling.

No significant financial relationships to disclose.
A single genomic HER2 assay for the protein expression and the DNA amplification status
Authors: Carpentier S. , Bertucci F. , Tagett R. , Finetti P. , Jacquemier J. , Le Doussal J. M. , Hermitte F.
Published: August 2020

e22082

Background: Currently, up to 20% of HER2 tests in breast cancer may be inaccurate. To address this situation, the 2007 ASCO/CAP guideline for HER2 testing recommended using standardized IHC (for HER2 protein expression) and FISH (for HER2 DNA amplification) in a sequential testing algorithm. However, test standardization and equivocal results still remain an issue potentially leading to inappropriate treatment decisions. A new genomic HER2 (gHER2) assay was designed to address these issues. Methods: Frozen samples of 152 primary breast cancers with a positive or negative IHC result (126 IHC 0+ and 26 IHC 3+ determined by tissue microarray) were profiled using Affymetrix U133 Plus 2.0 gene chips. Recursive feature elimination algorithm by support vector machines (RFE-SVM) was used to select probesets correlated to IHC status; to compute a genomic HER2 (gHER2) expression index; and to set a threshold discriminating between IHC 0+ from IHC 3+ samples. This gHER2 assay was validated in 4 independent datasets (n=309) and tested for its ability to reclassify HER2 equivocal cases (IHC 2+, n=21). The assay was standardized using Ipsogen MapQuant Dx. Results: RFE-SVM identified 6 known and 1 unknown genes correlating with the IHC status, all located within the HER2 DNA amplicon. The gHER2 assay gave unequivoqual HER2 status in 96.2 % of samples. The genomic HER2 assay was concordant with IHC status in 94.5% of cases. 95 % of samples with equivocal IHC status (IHC 2+) could be re-classified in accordance with FISH status in 95 % of cases. Conclusions: We could identify a genomic signature of both HER2 DNA amplification and HER2 protein expression. A new standardized genomic HER2 assay was derived that matches the ASCO/CAP guideline requirements of 95 % concordance with validated techniques. It may contribute to reduce the number of equivocal results.

[Table: see text]
Intra tumoral CD8-mediated type 1 anti-tumor responses and differentiated triple negative (TN) and HER-2 advanced breast cancer under neoadjuvant chemotherapy
Authors: Marana H. C. , Benevides L. , Tiezzi D. G. , Andrade J. M. , Brito L. G. , Schiavon V. F. , da Silva J. S.
Published: August 2020

e22106

Background: To determine the predictive value of CD8 T cells tumor infiltrated lymphocytes (TIL) for treatment response in locally advanced breast cancer (LABC) primarily submitted to neoadjuvant chemotherapy. CD8 T cell-mediated immune responses fall into two distinct types based on effector cell-derived cytokine production. Type I CD8 T cells (Tc1) produce IFN-γ, whereas type 2 cells (Tc2) secrete IL-4, IL-5, IL-10, and GM-CSF. Methods: CD8 T cell, FoxP3 and stimulated anti-CD3 production of IFN-γ were quantitatively measured in the TIL sample from 22 consecutive patients with LABC using a FACS cell analyzing and enzyme immunoassay. Neoadjuvant treatment was given to all patients as Docetaxel 75 mg/m2 and Epirubicin 60 mg/m2 i.v. infusion, for one day, each 21/21 days. Results: Univariate analysis showed that CD8 T cell, FoxP3 and production of IFN-γ are more pronounced in Triple Negative (TN) tumor (39±4.1%, 44,15±11.2% and 17.66±9.18% respectivilly) than HER-2 tumor (30.2±3.4%; 31,7±2.62% and 9.69±6.07%) and this difference was not significantly changed by the neoadjuvant chemotherapy. Conclusions: The results suggest that mammary tumor growth and progression were dependent, in part, on effector cell-derived IFN-γ. Here we postulated that this condition was associated to response to neoadjuvante chemotherapy and was distinct expressed by different gene type of tumor breast. This suggested that CD8-mediated type 1 antitumor responses cannot only promote accumulation of distinct endogenous CD8 T cell subpopulations, but also facilitate and preferentially modulate their localization kinetics, persistence, states of activation/differentiation, and function within the primary tumor environment of tumor response/regression. The TN tumor was more chemo-sensitive and had high IFN-γ production. The action way could be done by CD44 high, FoxP3 and TGFβ1 and the activatioin of Treg in TIL that help in the tumor progression. The chemotherapy breaks this and facilitated the tumor regression. These studies offer insight into potential mechanisms for enhancing T cell-based immunotherapy in breast cancer.

[Table: see text]
Is age at the time of surgery a predictor of biochemical failure following radical prostatectomy?
Authors: Badalato G. , Barlow L. , Benson M. , McKiernan J.
Published: August 2020

e22110

Background: Preoperative PSA level, Gleason score, and tumor stage have all been shown to influence risk of recurrence after radical prostatectomy. Increasing age has been associated with more indolent behavior in some cancers. This study evaluates the effects of age at surgery on recurrence-free survival in prostate cancer patients at a single institution stratified by established preoperative risk factors. Methods: Using the Columbia Urologic Oncology Database, a retrospective analysis of 3,736 men treated with open or robotic-assisted laparoscopic radical prostatectomy for prostate cancer from 1988 to 2008 was conducted. Patients were divided into two groups by age at the time of surgery, and recurrence-free survival rates were analyzed using Kaplan-Meier survival curves. The subgroups were stratified by preoperative PSA level, biopsy Gleason score, and clinical stage; multivariate analyses with cox proportional hazards models were used to further identify independent predictors of recurrence. Recurrence was defined as a single PSA level of 0.2 ng/ml or greater at least 28 days after surgery. Results: 1,984 patients were divided into groups 1 (n=1,325 age 40–64) and 2 (n=659 age ≥65). Five-year recurrence-free survival rates were 80.6%(CI: 78.0–82.9%) and 75.6%(CI: 71.5–79.1%) for groups 1 and 2, respectively. In the univariate model, advanced age was significantly associated with an increased overall risk of recurrence (HR 1.30, p=0.012). However, in multivariate analyses accounting for PSA, Gleason score, and clinical stage, age was not shown to be an independent predictor of recurrence (HR 1.04, p=0.76). In a subset of patients with low-grade cancer (Gleason score 2- 6), advanced age was associated in a univariate analysis with an even greater relative risk of recurrence (HR 1.47, p=0.032). However, this was not significant in the multivariate model (HR 1.27, p=0.21). Conclusions: Older patients who undergo radical prostatectomy for prostate cancer appear to have an increased risk of recurrence, which is most notable in patients with low-grade disease. However, age is not an independent predictor of recurrence when accounting for PSA, grade, and stage.

No significant financial relationships to disclose.
MUC16/CA125 specific targeting of critical signaling kinases in ovarian cancer
Authors: Manson R. G. , Thapi D. , Ma X. , Rosales N. , Spriggs D.
Published: August 2020

e22112

Background: The serum marker CA125 is known to originate from the transmembrane protein, MUC16; however, the biological importance of MUC16 is not known. Our lab previously created fluorescent-labeled transfectants, containing truncated versions of the MUC16 molecule. MUC16 expression vectors were transfected into CA125-negative ovarian cancer cell lines. These cell lines have allowed for a unique means of studying MUC16 biology. In vivo data suggest that MUC16 drives a more aggressive phenotype in ovarian cancer, including increased growth, invasion and dissemination. MUC16 transfection is also associated with the upregulation of specific signaling kinases, including EGFR, Erk, Src and Akt. To validate these findings and explore the potential of MUC16 signal targeting through inhibition of downstream kinases, we are examining the effect of a kinase-directed siRNA library on the A2780 ovarian cancer cell line transfected with a carboxy-terminus MUC16-expression vector. With the identification of kinases important to the survival of CA125+ ovarian cancer tissue, we will identify molecular targets for novel, tumor-specific therapies. Methods: Pilot plates containing candidate kinase-directed, negative control and total kill siRNA (Qiagen) were applied to MUC16+ and MUC16- cells. Cells were grown in culture in parallel 96-well plates. Immunofluorescent readings and Alamar Blue viability staining were performed at 72 hours. Results: Transfection reagents and nonspecific siRNA did not substantially change the survival of fluorescent protein expression in MUC16± cells; while hrGFP and mCherry proteins were decreased by ∼75%. None of the final transfection conditions altered Alamar Blue survival. Preliminary studies suggest that siRNA against AKT2 and SRC had specific inhibition against MUC16+ cells compared to MUC16- controls. Studies of three additional AKT2 siRNAs and a full complement of 714 human kinases are now being tested. Kinases with MUC16-specifc effects will be confirmed with three additional siRNAs. Conclusions: Dual fluorescence isogenic cell lines can be used to explore the kinase profile of MUC16+ ovarian cancer cells. Identified target kinases will enhance our knowledge about MUC16/CA125 biology and provide future leads for the treatment of ovarian cancer.

No significant financial relationships to disclose.
p53 as a marker of prognosis in African American (AA) women with breast cancer
Authors: Dookeran K. A. , Dignam J. , Ferrer K. , Sekosan M. , McCaskill-Stevens W. , Gehlert S.
Published: August 2020

e22119

Background: Prior reports suggest that p53 may be of prognostic value in AA women with breast cancer. However, it remains to be determined whether p53 status would add prognostic value beyond the commonly used factors of stage and Intrinsic Subtype Classification (subtype). We evaluated p53 status as a prognostic factor among AA women treated at an urban community hospital. Methods: Cox proportional hazards regression models [results reported as hazard ratios (HR) with 95% confidence intervals (CI)] were used to select and evaluate prognostic factors [including stage, age, tumor grade of differentiation (grade), p53 status, subtype, & ER/PR status] for all-cause mortality in 331 consecutively treated AA women with breast cancer [42 months follow-up] and known subtype [luminal A = ER+, &/or PR+, & HER2-; luminal B = ER+, &/or PR+, & HER2+; HER2+ = ER-, PR-, & HER2+; basal = ER-, PR-, HER2-, cytokeratin (CK) 5/6+ &/or HER1+; & unclassified = negative for all 5 markers] and p53 [Pab1801 antibody] immunohistochemical status. Results: Tumors in 28% of women were p53+ and there were no chemotherapy and radiation treatment differences according to p53 status. However, 59% of p53+ women were ER/PR negative [Odds Ratio (OR), 0.37; 95% CI, 0.22–0.54; p=0.0003] and hence endocrine therapy was significantly less frequent in p53+ women [OR, 0.40; 95% CI, 0.23–0.69; p=0.0008]. p53+ tumors were also significantly more likely to be grade 3 [OR, 4.35; CI, 1.33–14.14; p=0.013]. Baseline prognostic factors were: stage [(II-IV/I) HR, 2.29; 95% CI, 1.86–2.81; p<0.0001]; age [HR, 1.003 per year; 95% CI, 0.99–1.02; p=0.697]; grade [(high/low) HR, 1.70; 95% CI, 1.22–2.37; p=0.0008]; p53 status [(±) HR, 1.76; 95% CI, 1.15–2.72; p=0.012]; subtype [(all other/luminal A) HR, 1.33; 95% CI, 1.14–1.55; p=0.0004]; ER/PR status [(±) HR, 0.47; 95% CI, 0.32–0.69; p=0.0001]. Cox multivariable models indicated that p53 status [HR, 1.59; 95% CI, 1.01–2.51; p=0.044] remained a significant prognostic factor when considered with stage [HR, 2.20; 95% CI, 1.71–2.84; p<0.001] and subtype [HR, 1.24; 95% CI, 1.04–1.49; p=0.016] and the other above-mentioned factors. Conclusions: Study results indicate that p53 status should be included with stage and subtype as markers to assess prognosis in AA women with breast cancer.

No significant financial relationships to disclose.
HMPS (2-hydroxy-4-methoxyphenylstilbene), a stilbene derivative of rhapontigenin, and cell death by mitochondrial apoptotic pathway in breast cancer cells
Authors: Jung J. , Park H. , Jung H. , Eun Y. , Kim J. , Chae Y. , Yang J. , Lee Y. , Aiyar S. E. , Santen R. J. , Kim S.
Published: August 2020

e22130

Background: Breast cancer with resistance to clinical therapy is a significant threat to live of recurrent breast cancer patients, and chemo-resistant breast cancer is increasing rapidly. During last several decades, natural stilbenoids have been studied on anticancer effects in vitro and in vivo, and resveratrol is the most famous stilbene as a leading compound in the studies of anticancer compounds derived from plants. HMPS (2-hydroxy-4-methoxyphenylstilbene) is an analogue derived from rhapontigenin (3,5,3'-trihydroxy-4'-methoxy-trans-stilbene), which is a stilbene of herbal plant Rheum undulatum. TMS (2,3',4,5'-tetramethoxystilbene), an another stilbene analogue from rhapontigenin, was reported potent anticancer effect on tamoxifen-resistant MCF-7 cells. In this study we investigated inhibitory effect of HMPS on proliferation of breast cancer and a potential for a new therapeutic candidate. Methods: We examined cell viability of MCF-7 and MDA-MB-231 by MTT assay after exposure to various concentrations of HMPS. Apoptotic cell death induced by HMPS was investigated by florescence microscopy, cell cycle analysis and western blotting. Results: Cell viability of breast cancer cells after 24 h exposure to HMPS decreased significantly, and both ER-positive and ER-negative breast cancer cells responded to HMPS. HMPS induced nucleus fragmentation and G2/M arrest followed by sub-G1 accumulation of apoptotic cells in time- and dose-dependent manner. During the process of cell death induced by HMPS, mitochondrial membrane potential was disturbed and caspase-3 and PARP cleavage were observed. Moreover, HMPS decreased cell number of LTED MCF-7 cells(Long term estradiol deprived cell) effectively. Conclusions: Our results demonstrates that proliferation inhibitory effect of HMPS is about 50-fold more potent than those of rhapontigenin and furthermore HMPS also inhibits cell growth of LTED cells which are difficult to treat therapeutic agents. Therefore, HMPS may be a potential therapeutic candidate to treat the recurrent breast cancer by alone or combination with other conventional anticancer agents.

No significant financial relationships to disclose.
Differential protein kinase activity in ER-positive and ER-negative breast cancer
Authors: Ruijtenbeek R. , Umar A. , van Houten L. , Hilhorst R. , Foekens J. A. , de Wijn R. , Martens J. W.
Published: August 2020

e22142

Background: Specific protein tyrosine kinases (PTKs) promote cancer progression and are potential drug targets. The presence of the estrogen receptor (ER) is an important criterion in deciding on treatment of patients with breast cancer. Since PTKs can affect the tumors' receptor function, we investigated in our first proof-of-principle study whether it is possible to discriminate ER-positive (ER+) from ER-negative (ER-) tumors based on PTK activity using microarrays containing many PTK peptide substrates. Methods: Cryosections of 12 ER+ and 12 ER- breast tumors were lysed in the presence of appropriate protease and phosphatase inhibitors. The protein kinase activity in the lysates was monitored in vitro using PamChip peptide microarrays, which comprise of 253 PTK peptide substrates derived from known human phosphorylation sites. Peptide phosphorylation through active kinases can be monitored in samples in real time, using a fluorescently labeled phospho-tyrosine specific antibody. Results: Phosphorylation activity profiles were determined in quadruplicate using multiple independent sample preparations. Using ANOVA analysis, out of 253 peptides, several peptides with a different phosphorylation signal in ER+ and ER- samples were found: 22 with p < 0.02 including VEGFR1, 2 and 3 and ADAM9. Multivariate unsupervised analysis using Principle Component Analysis showed a clustering of ER+ samples vs. ER- samples. Predictive analysis was performed using Partial-Least Squares Discriminant Analysis without explicit feature selection. The prediction error obtained from within experiment cross validation was typically in the range 10–20%. The error rate for between experiment prediction was 20%. An average predictive profile was constructed in which peptides with a relatively large weight were included, e.g. ADAM9 and BCAR1. Conclusions: Using PamChip peptide microarrays we have shown that differences in protein kinase activity exist between ER+ and ER- breast tumors. Our in vitro assay is a promising tool to investigate the interplay between kinase and nuclear receptor mediated signaling, with potential relevance to patient selection for targeted therapies.

No significant financial relationships to disclose.
Comparative proteomic analysis of radiation induced changes in non small cell lung cancer patients with or without radiation induced lung toxicity
Authors: Cai X. , Shedden K. , Ao X. , Lawrence T. S. , Lubman D. M. , Kong F.
Published: August 2020

e22146

Background: Radiation-induced lung toxicity (RILT) is dose limiting in the treatment of non small cell lung cancer (NSCLC). Early prediction of RILT would allow physicians to individualize treatment. This work aims to study if radiation induces differential changes in plasma proteomics in patients with and without RILT. Methods: 20 Patients with NSCLC in 3 stage- matched groups and treated with radiation therapy (RT) or chemoradiation were included in this analysis: 6 grade 0 (group 0), 8 grade 1 (group 1), and 6 grade ≥ 2 RILT (group 2). Platelet poor plasma was obtained pre-RT, at 2-, 4-, 6-week during-RT, and 1-, 3-month post-RT. The plasma proteomic characterizations from patients with and without RILT were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, reverse-phase high-performance liquid chromatography (RP-HPLC), and nano-LC electrospray tandem mass spectrometry (LC-ESI-MS/MS). ANOVA model was applied for significance test. Results: 23 common proteins were detected in all 20 patients, 9 of them, Ceruloplasmin, Prothrombin, Complement C3, Beta-2-glycoprotein (β2GP1) 1, Vitronectin, Complement factor I, Complement factor H, Complement C4-A, Complement C7, had significant difference among 3 groups. At baseline (Pre-RT), the levels of Vitronectin and C4-A in group 2 were both significantly higher than that in group 0 (p=0.008 and 0.021, respectively). At 2-week during RT, the levels of Ceruloplasmin, Prothrombin and β2GP1 in group 1 were significantly higher than that in group 0 (p=0.017, 0.011 and 0.013, respectively). At 4-week during RT, the levels of β2GP1 in group 1 and group 2 were significantly higher than that in group 0 (p=0.005 and 0.014). At 6-weeks during RT, Prothrombin, C3, Complement factor H, C7 and Complement factor I also had some significant changes among three groups. Conclusions: Plasma proteins related to inflammation, coagulation and fibrosis, such as β2GP1, change differently during RT in patients with and without RILT. This proteomic approach may identify new markers during treatment to predict RILT for adapting treatment to patient. Fundings: ASCO career developmental award, grant from Pardee foundation, and RTOG translational research program.

No significant financial relationships to disclose.
Enhancer of zeste homologue 2 ( EZH-2) expression in breast cancer: a novel marker and potential target
Authors: Bandyopadhyay S. , Nahleh Z. , Ali Fehmi R. , Arabi H. , Sakr W. , Munkarah A. , Kruger M.
Published: August 2020

e22154

Background: EZH-2 is a protein involved in cell cycle regulation; it belongs to the Polycomb group of proteins and has been suggested to be associated with aggressive breast cancer. In hormone receptor negative (HR-) breast carcinomas, novel therapeutic targets are needed. Few markers have achieved the success of Her-2/neu as targeted therapy making the search for novel treatment strategies imperative. In our study we investigate the expression of EZH2 in a cohort of hormone receptor negative breast carcinomas. Methods: We identified a consecutive cohort of 84 cases of HR- breast carcinoma in 2005–2006, from the Pathology department archives. Tumor grade, size, presence or absence of DCIS, lymph node status and Her2/neu expression were documented. The race of the patients was also noted. Immunohistochemical staining for EZH2 was performed on paraffin embedded sections. Nuclear expression of EZH2 was considered as positive and the percentage of cells staining positive was estimated. Using the Mann-Whitney U test, the expression of EZH2 was correlated with the tumor characteristics listed above. Results: The mean expression of EZH2 in HR- tumors was estimated at 74% with a median of 80% (0–90). Most of these cases (n= 61) were triple negative; 23 cases were HER-2/neu positive by established criteria. Increasing expression of EZH2 was correlated with increase in tumor size (>2 cm) and increased incidence of lymph node metastasis. No correlation was seen with tumor grade, the presence or absence of DCIS and Her2/neu expression. Race did not appear to have an impact on EZH2 expression. Conclusions: Our findings suggest that EZH2 is expressed in the majority of HR - breast cancer and is associated with aggressive breast carcinomas. EZH2 could be an important therapeutic target in this patient population. The study is ongoing to further characterize EZH2‘s role and its association with survival.

No significant financial relationships to disclose.
Prognostic role of insulin-like growth factor receptor-1 (IGFR-1) and insulin-like growth factor binding protein-3 (IGFBP-3) expression in small cell lung cancer
Authors: Chang M. , Won Y. , Han J. , Kim H. , Kwon O. , Lee J. , Park Y. , Ahn J. , Ahn M. , Park K.
Published: August 2020

e22155

Background: Insulin-like growth factor receptor-1 (IGFR-1) is a cellular membrane receptor overexpressed in many tumor cell lines and in some human tumors that seems to play a critical role in anti-apoptosis by enhancing cell survival. Also, insulin-like growth factor binding protein-3 (IGFBP-3) was reported to be a growth suppressor in variable pathways. Purpose of this study was to evaluate the state IGFR-1 and IGFBP-3 expression in patients with small cell lung cancer (SCLC) and its prognostic value. Methods: We analyzed IGFR-1 and IGFBP-3 expression in 194 SCLC tissues specimens by immunohistochemical stain. The relationship between IGFR-1 and IGFBP-3 expression and cliniopathological factors was evaluated. Univariate and multivariate analyses were performed to define its prognostic significance. Results: Median age was 63 years (range 38–85), 84% were men. One hundred-seventeen patients had extensive disease (60.3%), and 77 had limited disease (39.7%). With the median follow- up duration of 49.5 months (24–82), the median progression free survival (PFS) and overall survival (OS) were 8 months (95% CI: 7.3–8.7 months), and 14.4 months (95% CI: 12.7–16 months), respectively, The IGFR-1 expression was observed in 154 of 190 tumor tissues (79.4%), whereas there was no tissue stained by IGFBP-3. Multivariate analysis showed that stage (p<0.001), response to treatment (p<0.001), LDH level (p<0.001) were the independent prognostic factors for PFS, and age (p=0.014), LDH level (p<0.001), and stage (p<0.001) for OS. The IGFR-1 positivity was not associated with PFS or OS in the whole cohort. However, 84% of 115 extensive disease patients showed IGFR-1 positivity. The subgroup analysis revealed that OS was significantly longer for patients with IGFR-1 positive compared to those with IGFR-1 negative in extensive disease (11.3% vs 0% at 2year, p=0.034). Conclusions: These results suggest that IGFR-1 expression may be useful as a prognostic marker in patients with extensive disease of SCLC.

No significant financial relationships to disclose.
Prostacyclin synthase (PTGIS) expression and epigenetic regulation in non-small cell lung cancer (NSCLC)
Authors: Gray S. G. , Cathcart M. C. , Al-Sarraf N. , Pidgeon G. P. , O'Byrne K. J.
Published: August 2020

e22160

Background: Prostacyclin synthase (PTGIS) inhibits platelet aggregation and promotes vasodilation. Overexpression of this gene has been shown to inhibit lung cancer growth in a mouse model. Hypermethylation of the PTGIS promoter resulting in downregulation of PTGIS expression has been implicated in colorectal cancer. In this study we have examined both the expression patterns and epigenetic regulation of PTGIS in NSCLC. Methods: DNA/RNA and protein was extracted from matched tumour/normal samples (Thoracic Oncology Research Group BioBank, St James Hospital). PTGIS in these samples and a panel of retrospective resected lung samples was examined by immunohistochemistry and western blotting. A panel of NSCLC cell lines were treated with inhibitors to histone deacetylases (HDi) and DNA methyltransferases (DNMTi) and PTGIS expression examined by RT-PCR. Chromatin immunoprecipitation (ChIP) was used to examine changes to the PTGIS promoter in response to HDi. Results: PTGIS was found to have variable expression in both tumour samples and a panel of lung cancer cell lines. PTGIS expression was demonstrated in the vascular endothelial and bronchial epithelial cells of normal and cancerous sections. A striking variation in intratumoural PTGIS expression was observed. Methylation analysis of cell lines demonstrated hypermethylation of the PTGIS promoter. Treating cells with epigenetic inhibitors resulted in significant upregulation of PTGIS expression. Direct chromatin remodelling of the PTGIS promoter was confirmed. Conclusions: PTGIS is epigenetically regulated. The discrepancy between PTGIS mRNA and protein levels in tumor samples indicates that post-transcriptional and post-translational regulation of PTGIS is central to expression and requires further elucidation. Increased PTGIS expression is a potential therapeutic strategy for tumour prevention.

[Table: see text]
Development of a new micro-injection-system for adenoviral gene delivery (MAGD)
Authors: Bohlen G. , Raabe A. , Dahm-Daphi J. , Dikomey E. , Schild S. E. , Dunst J. , Rades D.
Published: August 2020

e22167

Background: Safe and effective delivery of viral vectors is important for gene therapy. Local application of such vectors is more efficient than systemic application. However, currently available injection systems are not optimal because placement of the injection needle and the vector application itself are generally not reproducibile. We developed a new injection system providing controlled injection of adenoviral vectors in tumors (xeno-transplants) in nude mice (NMRI nu/nu). Methods: The new MAGD consists of two fixation devices mounted on a 40x40cm plexi-glass plate, four injection units, and one pump unit with 4 infusion-pumps (Bee hive, Bioanalytical, Chesire, UK). Each injection unit is linked to a pump with commercially available plastic tube. Because the injection arms can be moved in x-,y- and z-direction, virus injection can easily performed for irregular tumor volumes. The entire injection volume is equally distributed to the 4 injection pumps allowing precise and steady injection rates between 0.1μl/min and 100μl/min. Success was confirmed with MRI-scans (Magnetom symphony, Siemens, Erlangen, Germany) 10 min after the injection. The efficacy of the MAGD was tested in 4 human squamous cell carcinoma cell lines from oro-/hypopharynx (FaDu, UD- SCC2, UD-SCC6, and UD-SCC7a). Virus transfection was performed with an adenovirus (serotype 5) expressing enhanced green fluorescent protein (eGFP). Transfection rates were quantified with flow cytometry. Successfully transfected cells expressed eGFP resulting in green fluorescence. In-vitro cell lines (concentration: 10 particles per cell, MOI 10) of FaDu, UD-SCC2, UD-SCC6 and UD-SCC7a served as controls. Results: The MAGD was easy to handle. Injection of 100μl (25μl per infusion pump) at an injection rate of 5μl/min took only 5 min. The in-vivo transfection rates achieved with the MAGD were 9±1% for FaDu, 39±2% for UD- SCC, 54±1% for UD-SCC6 and 54±9% for UD-SCC7a, respectively. The in-vitro transfection rates (controls) for these 4 cell lines were 2±1%, 23±4%, 29±4% and 3±2%, respectively. Conclusions: The new MAGD provided controlled injection of adenoviral vectors in tumors in nude mice. It proved to be effective, as it resulted in comparably high transfection rates.

No significant financial relationships to disclose.
Identifying the primary site using gene expression profiling in patients with carcinoma of an unknown primary (CUP): a feasibility study from the GEFCAPI
Authors: Loriot Y. , Gross-Goupil M. , Lesimple T. , Blot E. , Merrouche Y. , Mathieu M. C. , Fizazi K.
Published: August 2020

e22168

Background: CUP are an heterogeneous family of neoplasms with a dismal prognosis, with empiric chemotherapy as the recommended treatment. The aim of this study was to evaluate the feasibility of a 500-mRNA microarray to identify the tissue of origin in patients with CUP. Methods: Diagnostic biopsy formalin-fixed, paraffin-embedded (FFPE) specimens from 22 patients with CUP were prospectively collected. Gene expression profiling was performed using oligonucleotide microarray that contains 495 genes selected as highly differentially expressed between 49 tumor types (CupPrint). Results: The assay was successfully performed on specimens from 18 of the 22 patients (82%). It could not be performed because of a low RNA preservation in the remaining 4 cases. The median age was 57 years (range: 29–70 years). The median delay from tissue shipping to receipt of CupPrint result was 11 days (range: 1–26 days). The most common tissues of origin identified were lung cancer (22%) and colorectal cancer (17%). Of note, a primary cancer which would not be adequately treated by an empiric chemotherapy regimen currently recommended in CUP (like cisplatin-gemcitabine or carboplatin-paclitaxel) was identified in about half patients: kidney cancer (1), hepatocarcinoma (1), colorectal cancer (3), head and neck cancer (2) and cholangiocarcinoma (1). Conclusions: Gene expression profiling of FFPE biopsy specimens from patients with CUP is feasible in a reasonable delay, making it feasible in clinical practice. A phase III randomized trial is planned to compare therapy based on gene expression-suspected primary cancer versus empiric chemotherapy.

No significant financial relationships to disclose.
Predictive and prognostic functions of microtubule-associated protein-tau and topoisomerase IIα protein in early breast cancer
Authors: Choi M. , Won H. , Lee K. , Sung S. , Moon B. , Choi H. , Suh H. , Nam E. , Lee S.
Published: August 2020

e22169

Background: Topoisomerase IIα protein(topo II) is the molecular target of topo II inhibitors such as anthracyclines and Microtubule-Associated Protein(MAP)-tau protein is associated with taxane sensitivity. Anthracyclins and taxanes are major cytotoxic agents of breast cancer in the adjuvant setting. The aim of this study was to evaluate the predictive and prognostic functions of MAP-tau and topo II in early breast cancers. Methods: Representative breast tumor sections were constructed from paraffin embedded specimens from 78 node positive breast cancer patients. MAP-tau and topo II protein were assessed by immunochemistry using antibody clone 4F1(Affinity BioReagents,USA) and clone Ki-S1 antibody(Dakocytomation,USA). MAP-tau staining of tumor cells was semiquantatively scored as 0, 1+, 2+, 3+ and cases with 0 or 1+ staining intensity were considered MAP-tau negative. Topo II protein over- expression was defined as the detection of nuclear staining in more than median value of evaluated cells. Results: Thirty- four cases (43.6%) of 78 samples showed topo II overexpression and 35 cases(44.9%) showed MAP-tau overexpression in node positive breast cancers. HER2 overexpression was noted in 28 samples (35.9%) and 56 cases (71.8%) were compatible with the luminal type. In 43 patients (55.1%), anthracyclin and taxane were used as adjuvant therapy and in this group, both MAP-tau and topo II overexpression showed lower disease-free survival (DFS) than the others, but statistically not significant. In luminal type, MAP-tau overexpression was poor prognostic factor on DFS in Cox regression.(HR 5.644, 95% CI 1.14–28.07, p=0.034) Conclusions: Topo II overexpression and MAP-tau overexpression in node positive breast cancers were not significant predictive factors for anthracyclin and taxane therapies. As several investigators reported, MAP-tau is associated endocrine therapy sensitivity in patients without chemotherapy, but higher MAP-tau in luminal type was a strong poor prognostic factor in patients who were given chemotherapy and hormonal therapy.

No significant financial relationships to disclose.
TP53 codon 72 polymorphisms and breast carcinoma susceptibility: A meta-analysis
Authors: Zhu B. , Zhuo W. , Chen Z.
Published: August 2020

e22171

Background: Previously, TP53 codon 72 polymorphisms have been implicated as risk factors for various cancers. Several studies have conducted on the association of TP53 codon 72 polymorphisms with susceptibility to breast carcinoma and have yielded inconclusive results. The aim of the present study was to assess possible associations of breast cancer risk with TP53 codon 72 polymorphisms. Methods: We conducted a search in the Medline, EMBASE, OVID, Sciencedirect, and Chinese National Knowledge Infrastructure (CNKI) without a language limitation, covering all papers published up to Dec 2008. The associated literature was acquired through deliberate searching and selected based on the established inclusion criteria for publications. Results: Consequently, fifteen studies, including 3436 cases and 4394 controls, met the included criteria and thus were selected. Ultimately, the relevant data were extracted and further analyzed using systematic meta- analyses. The results showed that individuals carrying homozygote Arg/Arg genotype have a significant increased risk of breast cancer compared with those carrying Pro/Pro genotype (OR: 1.58, 95%CI:1.10–2.28). For Arg allele, no evidence indicated that individuals with Arg/Arg genotype have an increased risk of breast cancer compared with those with a combined Pro genotype (Arg/Pro+Pro/Pro) (OR: 1.68, 95%CI:1.24–2.29). For Pro allele, individuals with homozygote Pro/Pro genotype have a marked decreased susceptibility to breast cancer relative to those with a combined Arg genotype (Arg/Pro+Arg/Arg) (OR: 0.84, 95%CI:0.73–0.98). Conclusions: The results of the present study suggest that TP53 codon 72 polymorphisms might be a risk factor for breast cancer. Homozygote Arg allele genotype could significantly increase susceptibility to breast cancer, while Pro/Pro allele markedly decreases breast risk.

No significant financial relationships to disclose.
Malignant gastrointestinal stromal tumors treated with imatinib in France: Results in unselected patients
Authors: Mathieu-Boue A. , Bouché O. , Cioffi A. , Rios M. , Duffaud F. , Chaigneau L. , Ray-Coquard I. , Blay J. , Le Cesne A. , Deschaseaux C.
Published: August 2020

e22177

Background: GISTs are rare tumors of the GI tract. In France, their incidence is estimated to be 9- 12/106 inhabitants/year. Imatinib (IM) has been approved to treat unresectable and/or metastatic Kit-positive GISTs since 2002 but information on routine use, safety and efficacy in unselected “real life” setting is lacking. An observational cohort (EPIGIST) in France was designed to provide data on survival, safety and treatment patterns and quality of life. Methods: EPIGIST is a nationwide multi-center, observational study on GIST patients (pts) treated with IM for the first time between the availability on the French Market and the end of the 2008. Centers were randomly selected in national files of oncologists, gastrointestinal surgeons and gastrointestinal specialists. The planned follow-up duration was three years. A case report form (CRF) had to be completed at inclusion and during each follow-up visits. Quality of life was assessed using QLQ-C30 and SF36 questionnaires. Results: 29 on 51 selected centers enrolled at least one pt and 127 pts were included (as of 12/2008), The median age of disease onset was 59 years (range 29 - 85) with 48% pts>50 years. 63% were symptomatic at diagnosis; 69% were fully active (grade 0 on the ECOG index). Primary tumors were most often gastric (34%), or from jejunum/ileum (24%). At diagnosis 84% of pts had a tumor size over 5cm. 64% of patients had surgery of the primary tumor before starting IM. For 86% of the pts, IM was given at an in initial dosage of 400 mg, 8% at 300 mg and 6% at 800mg. Compliance was superior to 90% for all pts. With a median follow-up of 1.72 years (CI95%: [1.08;1.95]), two-years overall survival from first treatment with IM was 88.1% (CI95%: [65.3%-96.3%]). Conclusions: EPIGIST is still an ongoing survey. Current results confirm previous published data on survival in GIST treated with IM in an unselected cohort of patients outside of a clinical trial.

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Translational cell study exploring the role of estrogen receptor β expression as a predictive and/or prognostic factor in breast cancer patients
Authors: Saji S. , Honma N. , Hirose M. , Hayashi S. , Kuroi K.
Published: August 2020

e22185

Background: We have reported that positive expression of Estrogen receptor β (ERβ) was associated with better prognosis in the early breast cancer patients treated with adjuvant tamoxifen monotherapy (J Clin Oncol. 2008). In addition, this was also true in the ERα-negative/PR-negative/Her-2 negative patients. We explored the biological impact of ERβ in breast cancer cell lines to determine whether these observations were due to its prognostic power or predictive power of response to the therapy. Methods: Since MCF-7 cell was ERβ-negative ERα-positive cell line, we established two stable clones of MCF-7 by introducing ERβ expression vector (β-clone 1, β-clone 2) as the model of ERβ-positive ERα-positive breast cancer. MDA-MB 231 cell was used as ERβ-positive triple-negative cell line. These cells were subjected to proliferation, expression and functional analysis. Results: In western blotting, both β-clone 1 and clone 2 showed decreased expression of PR and Her-2 than parent MCF-7, although there were no differences in ERα expression. Expression of ERβ decreased estradiol (E2) induced proliferation ability and rate of cells in S-phase cycle. PPT (ERα-specific agonist) and DPN (ERβ-specific agonist) did not show any difference in response, and IC 50 for 4 OH-tamoxifen and fulvestrant did not differ among MCF-7, β-clone 1 and clone 2 (0.05–0.1 μM). Whereas, cell death due to deprivation of E2 from 1nM to 1pM was more frequently observed in ERβ-expressing clones than in parent MCF-7 cell. These cell deaths did not involve standard apoptosis pathway with caspase-3/7 activation and PARP cleavage. E2, DPN and PPT did not affect the proliferation of ERβ-positive triple negative MDA-MB 231 cell, and IC 50 for 4-OH tamoxifen was too high (8 μM) to be achieved in clinical pharmacological dose. Conclusions: From our cell study, better prognosis of ERβ-positive breast cancer patient who treated with adjuvant tamoxifen is mainly due to its own favorable biological behavior. However, this prognostic impact may include the favorable response to the treatment, when we use estrogen-deprivation therapy such as aromatase inhibitors (AIs). Additional clinical study in AI users would be required to address this issue.

No significant financial relationships to disclose.