bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular mechanism of in vitro pemetrexed sensitivity according to histological type in non-small cell lung cancer.
Authors: Yoshida Tsukihisa , Yano Tokujiro , Kohno Mikihiro , Haro Akira , Okamoto Tatsuro , Maehara Yoshihiko
Published: March 2020

e13090

Background: Pemetrexed (Alimta, ALM) is a new multitarget antifolate metabolite anticancer drug. Non-small cell lung cancer (NSCLC) is now treated with selective chemotherapy based on its classification as either non-squamous cell carcinoma or squamous cell carcinoma (SCC). This study was designed to investigate the relationship between in vitro chemosensitivity to ALM and both clinicopathological factors and the expressions of ALM-related enzymes and carrier molecule in NSCLC. Methods: Seventy NSCLC samples (56 adenocarcinoma and 14 SCC) were used. Chemosensitivity was assessed by the succinate dehydrogenase inhibition (SDI) test for three antimetabolic anticancer drugs, including ALM, 5-fluorouracil, gemcitabine. Samples showing more than the median of the succinate dehydrogenase (SD) activity, which reflects the cell viability, were defined as "Resistant" and samples showing less than the median were defined as "Sensitive." The mRNA expression levels of target enzymes of ALM (thymidylate synthase (TS), dihydrofolate reductase and glycinamide ribonucleotide formyltransferase), metabolic enzymes of ALM (γ-glutamyl hydrase (GGH) and folypolyglutamate synthase) and reduce folate carrier (RFC) were measured by reverse transcriptase polymerase chain reaction analysis to examine the influence on chemosensitivity to ALM. Results: Of 3 antimetabolic drugs, only ALM showed significant difference of chemosensitivity according to the histological type. SCC samples were more resistant to ALM than adenocarcinoma samples. In adenocarcinoma, no significant relationship between chemosensitivity to ALM and clinicopathological factors was observed. SCC samples showed higher expression of TS and lower expression of RFC than adenocarcinoma samples. In adenocarcinoma, only GGH was inversely correlated with chemosensitivity to ALM. Conclusions: The present study suggested that, in SCC, the expressions of TS and RFC were associated with the chemosensitivity to ALM. In adenocarcinoma, the expression of GGH was associated with the chemosensitivity to ALM.
Perceptions of clinical trials in gastrointestinal/hepatobiliary cancer patients: A comprehensive survey in a Korean tertiary hospital.
Authors: Park Lee Chun , Lee Jeeyun , Kim Hee Young , Choi Moon Ki , Hong Jung Yong , Park Silvia , Maeng Chi Hoon , Do Ingu , Park Se Hoon , Park Joonoh , Kang WonKi , Lim Hoyeong , Park Young Suk
Published: March 2020

e13094

Background: In the past few years, the volume of clinical trials has increased rapidly in East Asia, especially in Asian prevalent cancer types such as gastric cancer, hepatobiliary cancer. The actual degree of understanding or perceptions of clinical trials by cancer patients have been rarely studied in East Asian countries. Methods: Between July 1st 2011 and November 30th 2011, we conducted a prospective survey study regarding cancer patient’s awareness and willingness to participate a clinical trial. The questionnaire consisted of 21 questions based on an Index of Clinical Trial Understanding. Patients with gastrointestinal/hepatobiliary cancer who visited the Hemato-Oncology outpatient clinic at Samsung Medical Center and signed informed consent form were enrolled. The survey was conducted by a well-trained research nurse and statistical analysis was analyzed at the biostatistics core at SMC. Results: In this survey study, 1,000 patients were asked to participate and 675 patients consented to participate (67.5%). The awareness of clinical trial was substantially higher in patients with higher level of education (p<.001), married (p=.004), and higher economic status (p=.001). Nevertheless, willingness to participate the clinical trial was not significantly increased by higher level of education (p=.286), married (p=.685) and higher income (p=.310). The most common source for acquisition of clinical trial was attending physicians (52.0%) followed by mass media (36.6%), other patients (6.39%), internet (3.3%) and others (1.5%). The most influential factors for patient’s willingness to participate were physician’s opinion (n=181, 26.8%), limited treatment options (n=178, 26.4%), and expectations towards new anti-cancer drug (n=142, 21.0%). Patients were likely to refuse to participate a clinical trial due to unverified treatment modality (n=320, 47.4%) and negativity for clinical trials (n=193, 28.6%). Conclusions: We surveyed a large patient cohort to specifically inquire willingness and awareness of clinical trial in Asian-prevalent cancer types. Further correlative analyses with various variable will be presented at the meeting.
Digoxin exposure and prostate cancer mortality: A matched cohort study.
Authors: Flahavan Evelyn , Bennett Kathleen , Sharp Linda , Barron Thomas Ian
Published: March 2020

e13096

Background: Digoxin (DIG) exposure has been associated with reduced prostate cancer (PC) incidence in epidemiological studies. Preclinical data suggests that this anti-cancer effect is mediated through the inhibition of hypoxia-inducible factor 1-α by DIG. This retrospective cohort study examines associations between DIG exposure and mortality in men with PC. Methods: Men diagnosed with PC during 2001–2006 were identified from National Cancer Registry Ireland records and linked prescription claims data. Propensity scores for DIG exposure in the 90 days prior to PC diagnosis were estimated. DIG exposed and unexposed men were matched (1:1) within a calliper of 0.2 standard deviations of the propensity score logit, using greedy matching without replacement. Standardized differences were used to assess covariate balance (z-score <0.1) between matched cohorts. Hazard ratios (HR) for associations between DIG exposure and all-cause (ACM) or PC-specific (PCM) mortality were estimated using Cox proportional hazards models adjusted for age, comorbidity, tumour stage and grade. Categorical exposure-response analyses were carried out using tertiles of exposure (low, intermediate, high) in the 90 days pre-diagnosis. Results: 5734 PC cases were identified from the linked database. 395 cases received DIG in the 90 days pre-diagnosis, of which 391 were matched to unexposed controls. Matched covariate balance was acceptable. In adjusted analyses, DIG exposure was not associated with ACM (HR 1.06, 95% CI 0.88-1.27) but was associated with a small but non-significant reduction in the risk of PCM (HR 0.89, 95% CI 0.68-1.17). In the exposure-response analysis, DIG exposure in the highest tertile, but not in the intermediate or lower tertiles, was associated with a reduced risk of PCM approaching statistical significance (HR 0.69, 95% CI 0.47-1.01, p=0.059). Men in the high DIG exposure group received a supply of DIG for 98% of their eligible follow up in the year post diagnosis. Post diagnostic DIG exposure in the intermediate and lower tertiles was 94% and 80% respectively. Conclusions: DIG exposure was associated with a non-significant decrease in PCM. Stratification by exposure suggests the presence of an exposure-response relationship.
Phase I study of panobinostat (LBH589) and letrozole in post-menopausal women with metastatic breast cancer.
Authors: Tan Winston , Allred Jacob B , Moreno-Aspitia Alvaro , Northfelt Donald W. , Ingle James N. , Perez Edith A.
Published: March 2020

e13501

Background: Loss of estrogen receptor alpha gene expression has been associated with insensitivity to endocrine therapy in human breast cancer patients. Histone deacetylase (HDAC) inhibitors have recently been found to restore sensitivity to the estrogen receptor by modulation of the estrogen and progesterone receptors. This had been shown with both aromatase and tamoxifen refractory and in triple negative cell lines . We performed a Phase I study of the combination of LBH589 (panobinostat) and letrozole to evaluate safety and tolerability in patients with metastatic breast cancer prior to the performance of a phase II trial. Methods: We enrolled postmenopausal women with metastatic breast cancer, ECOG PS 0 or 1, ANC>1500/mm3, platelets>100,000/mm3, normal total bilirubin, and ALT/AST adequate laboratory tests were eligible. Letrozole dose was 2.5 mg/day orally. Dose of LBH589: Level 1, 20 mg orally three times weekly; Level 2, 30 mg orally three times a week. Results: 12 patients (dose level 1:6 patients, dose level 2: 6 patients) have been enrolled. 43 cycles of treatment have been given to these12 patients. Initial cohort of 3 patients at the 20 mg dose level had no dose limiting toxicity (DLT). At the 30 mg dose level 3/6 patients had DLT (thrombocytopenia grade 1: 2 pts; grade 3:1 pt; grade 4: 1 pt; and diarrhea grade 3: 1 pt. One pt at the 30 mg dose level has a confirmed partial response and remains on study after 6 cycles of treatment. Subsequent cohort of 3 patients had 1 dose DLT with doubling of the creatinine. The main DLT was thrombocytopenia in 3/6 pts at the 30 mg dose level. Conclusions: The recommended dose for phase II testing of LBH589 is 20 mg orally 3 times per week in combination with standard dose letrozole.
Automated quantitative analyses (AQUA) of cyclic-AMP-response-element-binding protein (CREB), phosphorylated CREB (pCREB), and ataxia-telangiectasia-mutated protein kinase (ATM) protein expression in patients (pts) undergoing thoracic radiation for locally advanced non-small cell lung cancer (NSCLC).
Authors: Chao Bo H. , Traynor Anne M. , Yang David T , Zhang Chong , Pier Thomas , Kim KyungMann , Rehrauer William M. , Cannon Donald Maurice , Cannon George M. , Bye Tyler , Tibbetts Randal S.
Published: March 2020

e13537

Background: Increased protein expression of CREB, a DNA damage-regulated transcription factor, has been associated with poor survival in advanced NSCLC. We have demonstrated that exposure of cancer cell lines to low dose ionizing radiation (IR) increased pCREB on a Ser133 residue. However, exposure to high dose IR correlated with pCREB on Ser121 by ATM, a master regulator of the cellular DNA damage response. This unusual bimodal response of CREB to IR, reflected by activation at low doses and attenuation at high doses, suggested that CREB may control cell fate decisions in response to DNA damage. We hypothesized that patterns of protein expression of CREB, pCREB Ser133, and ATM would predict treatment response in pts who have undergone radiotherapy for locally advanced NSCLC. Methods: Diagnostic tumor specimens were obtained from pts who underwent thoracic radiation in a clinical trial for locally advanced NSCLC. Protein expression of CREB, pCREB Ser133, and ATM was assessed by AQUA. Wilcoxon rank sum test was used to assess differences in protein expression. Univariate regression was conducted to evaluate protein expression and clinical outcomes. Kruskal Wallis test was used to assess protein expression and pneumonitis. Results: Sufficient tumor tissue was available for 35 of 79 pts enrolled. Best responses in 6 months included 12% CR, 73% PR, 9% SD, and 6% PD. Pre-treatment protein expression per AQUA analyses of CREB, pCREB Ser133, and ATM were not statistically associated with time to in-field progression, time to out-of-field progression, time to distant metastasis, best response, pneumonitis, or overall survival. Unexpectedly, increased expression of pCREB Ser133 was associated with durable anti-tumor response to radiation at a median duration of 14.6 months (p=0.03). Conclusions: Protein expression per AQUA of CREB, pCREB Ser133, and ATM did not confirm our hypotheses, possibly related to our limited sample size. Further analysis of the role of CREB in response to IR in cancer pts is ongoing.
Effect of short hairpin RNA targeting of fibroblast activation protein on tumor growth and angiogenesis in a 4T1 murine breast cancer model.
Authors: Cai Fan , Peng Feng , Wang Chunting , Li Zhiyong , Xian Shuang , Wei Yuquan
Published: March 2020

e13538

Background: Fibroblast activation protein(FAP) is an increased abnormal signal protein in the TME and has  both dipeptidyl peptidase and endopeptidase activity. FAP has been recognized to influence cancer growth and invasion, through its non- enzyme function or proteolytic activities in concert with other moleculars such as CD26, uPAR, β1-integrin, and MMP-1. Since FAP is over expression in epithelial cancers and absent from normal adult tissues, it has long been suggested to be a target of antitumor therapy. Methods: 21 BALB/C female mice implanted with 4T1 breast cancer were enrolled and randomly divided into three groups (n=7 per group) .The experiment group(E-group) was administered with shRNA targeting of FAP every 48 hours in 2 weeks by local administration, and the two control groups with HK or 5%GS. Tumor volumes and weights were monitored. Expression of FAP was detected by RT-PCR and immunohistochemistry staining. Collagen fiber, microvessel density and cell apoptosis were investigated. Results: The expression of FAP was reduced about 50% by shRNA targeting of FAP; the tumor volume and weight of the E-group showed significant reduction of 40% compared with the controls (p=0.001). Knockdown of FAP increased accumulation of disorganized collagen fibers; the MVD in control groups was about 4 times at least compared to the E-group (p<0.001) and apoptosis cells markedly increased 3 fold (p=0.0015). Conclusions: Knockdown of FAP by shRNA interference mediated inhibition of breast cancer growth, amelioration of the tumor microenvironment including increased accumulation of disorganized collagen, suppressed angiogenesis, and promotion of apoptosis. FAP contributes to the formation of TME and is a stimulative factor of breast cancer growth. Target of FAP may be a potential supplemental therapeutic method for breast cancer.
SYK expression in metastatic retinoblastoma.
Authors: Brennan Rachel Christine , Wilson Matthew W. , Dyer Michael A. , Bahrami Armita
Published: March 2020

e13544

Background: Retinoblastoma is an aggressive childhood cancer of the retina. While more than 90% of patients are cured when tumor is confined to the globe, for up to half of children in developing countries with advanced tumor at diagnosis, retinoblastoma remains a lethal disease. Recent whole-genome sequencing demonstrated that the retinoblastoma genome is relatively stable (Zhang et al). However, RB1 loss is associated with epigenetic deregulation of several cancer pathways. Significantly, the proto-oncogene SYK, spleen tyrosine kinase, which has no role in normal eye development, is upregulated in retinoblastoma and required for tumor cell survival (Zhang et al). Immunohistochemical (IHC) studies showed that SYK is ubiquitously overexpressed in intraocular retinoblastoma, whereas it is absent in normal retina. The purpose of this study is to investigate whether SYK overexpression persists in metastatic retinoblastoma. Methods: From 1962-2011, seven metastatic retinoblastoma tumor specimens (excluding bone marrow or cerebrospinal fluid) from six retinoblastoma patients were retrieved from the Pathology Archives in our institution. Morphology and supportive IHC, to include immunopositivity for synaptophysin and immunonegativity for cytokeratins, confirmed the diagnosis of metastatic retinoblastoma in each case. IHC for SYK was performed according to the previously published technique (Zhang et al). Results: Tumors occurred in two male and four female patients with seven metastatic retinoblastoma samples in scalp, lymph node, neck, ovary and bone (femur). By IHC, all seven metastatic retinoblastoma tumor samples were positive for SYK. Conclusions: This is the first report of SYK overexpression in metastatic retinoblastoma. Drugs targeting SYK are already in clinical trials for adults with leukemia and rheumatoid arthritis, and preclinical efficacy in vitro and in vivo has been established. Moving from bench to bedside with this epigenetically targeted agent warrants further investigation.
Reactivation of p53 mutant protein by PRIMA-1 and induction of apoptosis in pancreatic cancer cells.
Authors: Izetti Patricia , Hautefeuille Agnes , Abujamra Ana Lucia , Farias Caroline Brunetto de , Roesler Rafael , Macedo Gabriel de Souza , Alemar Barbara , Lenz Guido , Osvaldt Alessandro Bersch , Hainaut Pierre , Ashton-Prolla Patricia
Published: March 2020

e13546

Background: Inactivating TP53 mutations are common events in different types of cancers, including pancreatic adenocarcinoma, where it occurs in as much as 70% of cases. Accumulation of mutant p53 provides a molecular target that may be reactivated into a conformation capable of arresting tumor growth. The small molecule PRIMA-1 (p53 reactivation and induction of massive apoptosis) has been shown to selectively induce apoptosis in tumor cells by reactivating some p53 mutants, but no previous studies have investigated its effects in pancreatic cancer. Methods: PANC-1 (mutant TP53 R273H) and CAPAN-2 (wild-type TP53) pancreatic cell lines were used as in vitro models. We tested the effects of PRIMA-1 on cell viability (MTT assay), apoptosis (morphology, AnnexinV/FITC-FACS), cell cycle (BrdU incorporation) and expression of p53 regulated proteins by western blotting. As control of p53-dependent effects, PANC-1 cell lines were transfected with siRNA against TP53 (sip53). Results: PRIMA-1 selectively induced apoptosis in PANC-1 cells compared to CAPAN-2 cells and this effect was concomitant with an increase in the levels of MDM2, Bax and cleaved caspase-3 as detected by western blot analysis. Treatment with PRIMA-1 for 24h induced a 50% reduction in DNA synthesis whereas G2/M arrest was detected after 12h of treatment. p53 silencing in PANC-1 decreased the cytotoxicity of PRIMA-1, characterizing a p53-dependent effect. Finally, N-acetylcysteine completely blocked PRIMA-1-induced growth suppression and apoptosis, suggesting that PRIMA-1 exerts its effect at least in part via restoring redox-dependent effects to mutant p53. Conclusions: Our data indicate that PRIMA-1 induces apoptosis in TP53 mutant pancreatic cancer cells by promoting the re-activation of p53 and subsequent of proapoptotic signaling pathways, suggesting a possible mechanism for effective targeting of pancreatic cancer.
Genomics and proteomics-based discovery of novel cancer biomarkers and molecular targets for cell-permeable peptide/small molecule-based drugs.
Authors: Daigo Yataro , Takano Atsushi , Nakamura Yusuke
Published: March 2020

e13552

Background: Identification of cancer-specific oncoproteins is an effective approach to develop new diagnostics and therapeutics. Methods: We have established a strategy as follows to identify new oncoproteins, which can be applied as biomarkers and drug development; i) To identify genes overexpressed in 120 clinical lung cancers using the cDNA microarray representing 27,648 genes, ii) To verify the genes for their low expression in 23 normal tissues by northern-blotting, iii) To validate the clinicopathological significance of their protein expression by tissue microarray covering 262 cases of non-small cell lung cancers (NSCLCs), iv) To verify whether they are essential for the growth of cancer cells by siRNAs, v) To do immunoprecipitation assays and mass-spectrometric analysis to identify their interacting proteins in cancer cells, and screening of cell-permeable peptides that could inhibit the protein-protein interaction that is essential for carcinogenesis. Results: We identified 35 oncoproteins to be upregulated in the majority of lung cancers, and further selected CDCA5 (cell division cycle associated 5) as a good candidate. Tumor tissue microarray analysis of 262 NSCLC patients revealed that CDCA5 positivity was an independent prognostic factor. Suppression of CDCA5 expression with siRNAs inhibited the growth of lung cancer cells; concordantly, induction of exogenous expression of CDCA5 conferred growth-promoting activity in mammalian cells. We also found that extracellular signal-regulated kinase (ERK) kinase interacted with and phosphorylated CDCA5 at Serine 209 in vivo. Functional inhibition of the interaction between CDCA5 and ERK kinase by a cell-permeable peptide corresponding to a 20-amino-acid sequence part of CDCA5, which included the Serine 209 phosphorylation site by ERK, significantly reduced phosphorylation of CDCA5 and resulted in growth suppression of lung cancer cells. Conclusions: CDCA5 positivity should be useful as a novel prognostic biomarker in the clinic. Selective suppression of the ERK-CDCA5 pathway by cell-permeable peptide or small molecule-based drugs could be a promising strategy for cancer therapy.
Immunohistochemical expression of phospho-mTOR protein in breast carcinoma tissues and correlation with clinicopathologic parameters.
Authors: Michalaki Vasiliki , Frangou-Plemenou M , Kondi-Pafiti Agathi , Gennatas Konstantinos
Published: March 2020

e13556

Background: The mammalian target of rapamycin (m-TOR) kinase plays a central role in coordinating cell growth in response to mitogens, amino acid and energy sufficiency. The pathway leading to mTOR activation by these factors, is important for the regulation of protein synthesis and cell survival with implications in carcinogenesis. For these reasons, mTOR is currently under investigation as a potential target for anti-cancer therapy. The aim of this study was to determine the phospho-mTOR (p-mTOR) kinase expression in breast cancer tissues and the relationships with clinicopathological parameters of the breast tumors. Methods: We investigated the status of p-mTOR, in 140 breast carcinomas by immunohistochemistry. Western blot analysis was performed to verify the expression levels of p-mTOR. The relationship of protein expression with clinicopathological data and the correlations of protein expression levels were explored. Multiple logistic regression analyses with stepwise method was used in order to find independent factors associated wth p-mTOR. Results: 47.1% of the patients were p-mTOR positive. In univariate analyses a greater proportion of p-mTOR positive patients was found in those having progesterone receptors, in those hving higher grades of differentiation. When multiple analysis was conducted the only factor that was independently associated with p-mTOR kinase was the tumors grade of differentiation adjusted for age. Subjects with moderate grade of differentiation had greater likelihood for being p-mTOR positive compared to those with low grade of differentiation with odds ratio equal to 3.869 (95% CI: 1.35-11.04, p=0.012). Conclusions: The kinase p-mTOR was found in the neoplastic cells of almost half of the patients were enrolled. Overexpression of p-mTOR was correlated with well-to-moderately differentiated tumors (p<0.001) Our results indicate that dysregulation of the p-mTOR pathway may have an important role in the development of breast carcinoma, but not necessarily in the progression of the disease.
The roles of hypoxia, PTEN, and Rad51 in mediating metastatic prostate cancer cells' responses to PARP inhibitor and topoisomerase 1 inhibitor.
Authors: Zhang Jingsong , Wu Minghui , Wang Xue
Published: March 2020

e13564

Background: With the recent success of poly (ADP-ribose) polymerase inhibitor (PARPi) in the treatment of BRCA1 or BRCA2 mutated cancers, there is increasing interest to explore synthetic lethality in cancers with defective DNA repair pathways. Rad51 is an essential protein in the homologous recombination repair (HRR) of DNA double strand breaks. Previous studies with non metastatic prostate cancer (mCaP) cells have reported low Rad51 levels in cells with loss of PTEN or under hypoxia, which then led to their sensitivity to PARPi. Given intra tumor hypoxia and loss of PTEN is common in mCaP, we test PAPRi, ABT888 and DNA damaging topoisomerase I inhibitor, CPT11, either alone or in combination in mCaP preclinical models. Methods: mCaP cell lines with functional PTEN (DU145) and loss of PTEN (PC3) were grew under normoxia (21% O2) or hypoxia (0.2% O2). DNA damage, HRR, apoptosis were assessed with comet assay, western blot, immunofluorescence and flowcytometry. The regulation of RAD51 was studied with quantitative RT-PCR and RAD51 promoter reporter assay. PC3 xenograft was used for in vivo study. Results: Despite of its low levels of expression under hypoxia, up regulation of Rad51 was observed soon after treating hypoxic PC3 and Du145 cells with ABT888 or SN38, an active metabolite of CPT11. Such Rad51 up regulation led to less DNA damage and apoptosis under hypoxia compared to normoxia. Inhibiting RAD51 expression with siRNA overcame PC3 and Du145’s resistance to SN38. Furthermore, ABT888 enhanced the activities of SN38 as detected by clonogenic assay and flowcytometry under both normoxia and hypoxia. Consistent with the in vitro data, ABT888 by itself had limited anti-tumor activities despite the loss of PTEN in PC3 xenografts. The anti-tumor activity of single agent CPT11 was significantly improved with the ABT888 and CPT11 combination (P<0.008). Conclusions: neither loss of PTEN nor hypoxia sensitized mCaP cells to PARPi or DNA damaging drugs. Such resistance under hypoxia was at least partly due to up regulation of Rad51. Combining ABT888 with CPT11 overcame the resistance to CPT11 under hypoxia and enhanced its anti-tumor activities both in vitro and in vivo.
Effect of a combination of epigenetic agents on the malignant phenotype in models of T-cell lymphoma.
Authors: Marchi Enrica , Kalac Matko , Bongero Danielle , McIntosh Christine , Fogli Laura K , Scotto Luigi , Rossi Maura , Zinzani Pier Luigi , Pileri Stefano A , Piccaluga Pier Paolo , O'Connor Owen
Published: March 2020

e13569

Background: CHOP and CHOP-like chemotherapy are the most used regimens for the treatment of peripheral T-cell lymphomas (PTCLs) despite sub-optimal results. Histone deacetylase inhibitors (HDACIs) have shown class activity in PTCLs. The interaction between the HDACIs (depsipeptide (R), belinostat (B), vorinostat (V) and panobinostat (P)) and a DNMT inhibitor (decitabine (D) was investigated in vitro, in vivo and at the molecular level in T-cell lymphoma and leukemia cell lines (H9, HH, P12, PF-382). Methods: For cytotoxicity assays, luminescence cell viability assay was used (CellTiter-Glo). Drug:drug interactions were analyzed with relative risk ratios (RRR) based on the GraphPad software (RRR<1 defining synergism). Apoptosis was assessed by Yo-Pro-1 and propidium iodine followed by FACSCalibur acquisition. Gene expression profiling was analyzed using Illumina Human HT-12 v4 Expression BeadChip microarrays and Gene Spring Software for the analysis. Results: The IC50s for B, R, V, P, D and 5-Azacytidine alone were assessed at 24, 48 and 72 hours. In cytotoxicity assays the combination of D plus B, R, V or P at 72 hours showed synergism in all the cell lines (RRRs 0.0007-0.9). All the cell lines were treated with D, B or R for 72 hours and all the combinations showed significantly more apoptosis than the single drug exposures and controls (RRR < 1). In vivo, HH SCID beige mice were treated i.p. for 3 cycles with the vehicle solution, D or B or their combination at increasing dose. The combination cohort showed statistically significant tumor growth inhibition compared to all the other cohorts. Gene expression analysis revealed differentially expressed genes and modulated pathways for each of the single agent treatment and the combination. The effects of the two drugs were largely different (only 39 genes modified in common). Most of the effects induced by the single agent were maintained in the combination group. Interestingly, 944 genes were modulated uniquely by the combination treatment. Conclusions: The combination of a DNMTI and HDACIs is strongly synergistic in vitro, in vivo and at the molecular level in model of T-cell lymphoma and these data will constitute the basis for a phase I-II clinical trials.
Differential expression of biomarkers in gastrointestinal versus pulmonary carcinoid tumors.
Authors: Fong Erika , Peterson Gilbert , Basu Gargi D. , Blevins Richard , Ashfaq Raheela
Published: March 2020

e13571

Background: Carcinoid tumors are endocrine neoplasms that commonly arise in the GI and pulmonary system. About 12,000 cases of carcinoid tumor are diagnosed a year with a 5-year survival rate of 67.2% .The majority of the cases present with advance disease with limited treatment options,therefore evaluation of predictive markers for chemotherapeutic agents is highly desirable. We studied the biomarker expression in carcinoid tumors and evaluated any differences based on site of origin. Methods: By computer search we retrospectively identified all cases submitted to Caris Life Sciences with the diagnosis of “carcinoid”. The cases were classified by primary site into three groups: Pulmonary, GI tract (including esophagus, stomach, small bowel, colon, rectum, appendix and pancreas) and miscellaneous. Biomarker expression performed by IHC and scored semi quantitatively by a pathologist for SPARC mono, SPARC poly, Her2, TOPO2, TOPO1, PGP, MRP1, PTEN, TS, ERCC1, RRM1,MGMT, C-KIT, ER, PR and AR and SSTR2 and SSTR5 analysis by DNA Microarray were compared between the groups. Results: 146 carcinoid tumors were interrogated for biomarkers, 33 pulmonary, 97 GI and 16 miscellaneous (mediastinum, thymus, cervix, breast and unknown primary site). Due to small number of tumors in the miscellaneous category, analysis was restricted to pulmonary and GI carcinoid tumors only. Based on our analysis the pulmonary carcinoids differentially express Topo2, PGP, MGMT, CKIT and PR, while the GI carcinoids differentially express Topo1, RRM1 and ER and SSTR2 and SSTR5. There is equivalent expression of SPARC, MRP1, PTEN, TS, ERCC1 at both sites . All pulmonary and GI carcinoid tumors were negative for Her2. Conclusions: Based on this study it appears that there are differences in biomarker expression in GI and Pulmonary Carcinoid tumors. Treatment with irinotecan, gemcitabine, anti-estrogens and Octreotide may be more relevant in GI carcinoids based on molecular markers present while targets associated with response to anthracyclines, temozolomide, imatinib may be more commonly expressed in pulmonary carcinoid tumors. Further studies are needed to understand the underlying biology and molecular drivers in this group of tumors.
Synergistic activity of vertical combinations of agents targeting multiple steps along the RAF/MEK/ERK cascade as a therapeutic strategy in human tumors.
Authors: Cesta Incani Ursula , Del Curatolo Anais , Di Sanza Cristina , Falcone Italia , Cognetti Francesco , Ciuffreda Ludovica , Milella Michele
Published: March 2020

e13572

Background: BRAF-selective kinase inhibitors have potent antitumor effects in mutant BRAF(V600E) tumors; however, in BRAF-wt cells, they paradoxically activate MEK/ERK. In addition, MEK blockade may induce compensatory signaling through both upstream pathway elements (RAF) and parallel pathways (PI3K/AKT/mTOR). Methods: We set out to define molecular and functional effects of single and combined BRAF (GSK2118436A, BRAF-I) and MEK (GSK1120212B, MEK-I) inhibition, using WB analysis to dissect signaling and a fixed dose-ratio experimental design to assess functional synergism by conservative isobologram analysis. Results: In A549 lung adenocarcinoma (KRAS G12S), BRAF-I (10 μM) induces hyperphosphorylation of CRAF, MEK, ERK, and p90RSK, while MEK-I (10 nM), alone or in combination with BRAF-I, potently offsets MAPK activation. Combined BRAF-I and MEK-I suppress malignant growth and survival at 72 h with highly synergistic effects in the A549 (lung, KRAS G12S), H1299 (lung, NRAS), HCT116 (colon, KRAS G13D), and MIAPACA (pancreatic, KRAS G12V) models, with combination indexes (CI), ranging from 1.37 to 0.12. Conversely, in other lung cancer models (H460, Calu-1, Calu-3) the combination of BRAF-I and MEK-I produced modestly additive to highly antagonistic antitumor effects. In BRAF-mutant melanoma and colon carcinoma models (M14 and HT29), there was no paradoxical activation of the MEK/ERK module in response to BRAF-I and both BRAF-I and MEK-I had pronounced growth inhibitory effects as single agents, but were frankly antagonistic in combination. Similarly, the pan-RAF inhibitor RAF265 did not cause MAPK activation and did not result in synergistic growth inhibition when combined with the MEK-I in the A549 and MIAPACA cell lines. Conclusions: Overall, our data indicate that combined inhibition of multiple signaling elements along the RAF/MEK/ERK pathway results in strongly synergistic growth inhibition, particularly in tumors with RAS mutations. Additional studies to better define genetic determinants of sensitivity/resistance and molecular mechanisms of therapeutic synergism of combined BRAF-I and MEK-I are currently ongoing.
Dual endothelin-1/VEGFsp receptor (DEspR): Common target on tumor vascular endothelial cells (TVECs), tumor cells (TCs), and cancer stem cells (CSCs) in glioblastoma and pancreatic cancer.
Authors: Herrera Victoria L.M. , Tan Glaiza L.A. , Moran Ann Marie , Pasion Kristine A. , Decano Julius L , Ruiz-Opazo Nelson
Published: March 2020

e13574

Background: New therapies are needed for glioblastoma (GBM) and pancreatic adenocarcinoma (PCa), given their low survival rates. To address this mandate, we tested the hypothesis that inhibition of DEspR, as a common target on TVECs, TCs, and CSCs, can affect multiple key steps in metastasis: angiogenesis, invasiveness, anoikis resistance. Methods: Various analyses were performed: co-immunostaining of tumor tissue and cancer cell lines from PCa and GBM using anti-CD133 and anti-DEspR mAbs; in vitro anti-DEspR mAb inhibition of angiogenesis and TC invasiveness; isolation, functional validation, and DEspR immunostaining of CSCs from PCa (Panc1) and GBM (U87) cell lines; DEspR inhibition of Panc1- and U87-derived CSC tumorsphere formation and CSC-enriched xenograft tumor growth in nude rats respectively; and phosphoproteome analysis of DEspR signaling. Results: In PCa and GBM, we detected DEspR+ TVECs, TCs, and CD133+ putative CSCs in contrast to respective normal tissues. DEspR expression on TCs and TVECs were confirmed in PCa (Panc1) and GBM (U87) cell lines, and HUVECs undergoing angiogenesis, respectively. Dose-dependent anti-DEspR mAb inhibition decreased HUVEC angiogenesis and Panc1 invasiveness compared to isotype controls. DEspR+ expression was detected in functionally validated Panc1 and U87 CSCs exhibiting tumorsphere formation, increased tumorigenicity in nude rat xenograft models, and self-renewal from xenograft tumors. DEspR inhibition decreased tumorsphere formation and increased dead cell numbers, suggesting decreased anoikis resistance for both U87 and Panc1 CSCs. Phosphoproteome analysis detected DEspR signaling through phosphoproteins implicated in TVEC-TC crosstalk, angiogenesis, and anoikis resistance. DEspR inhibition decreased tumor growth of Panc1 and U87 CSC-enriched subcutaneous xenograft tumors in nude rat models. Conclusions: Data demonstrate DEspR as a common target on TVECs, TCs, and CSCs in PCa and GBM, whose inhibition provides a potential therapeutic strategy for simultaneous inhibition of multiple pro-malignancy steps: angiogenesis, invasiveness, and anoikis resistance.
Evaluation of the PI3K inhibitor BAY 80-6946 in hematologic malignancies.
Authors: Jeffers Michael , Dubowy Ronald L. , Lathia Chetan D. , Mallon Robert , Appleman Leonard Joseph , Ramanathan Ramesh K. , Patnaik Amita
Published: March 2020

e13576

Background: BAY 80-6946 is a new investigational compound that potently inhibits all PI3K Class I isoforms. BAY 80-6946 is under phase 1 clinical evaluation and PRs have been observed in solid tumors (ST) and follicular lymphoma (FL). The present analysis was performed to gain mechanistic insights into the activity of this compound in FL and potentially other hematological malignancies. Methods: Preclinical: The growth-inhibitory activity of BAY 80-6946 on hematological cancer cell lines (n= 32) was determined by the CellTiterGlo assay. GS-1101 (formerly called CAL-101) and cisplatin (CP) were also tested. Clinical: Specimens from subjects enrolled in a BAY 80-6946 Phase 1 trial MTD expansion were used. Levels of various cytokines and chemokines were determined by ELISA or multiplex immunoassay in plasma samples (n= 27) obtained prior to and during BAY 80-6946 treatment of 6 subjects (3 FL + 3 ST). Proteins examined included CXCL13 (involved in B-cell homing) and BAFF (involved in B-cell survival). Tumor tissue was used to determine PTEN expression via IHC and the mutational status of PTEN via sequencing. PIK3CA mutational status was evaluated in tumor tissue and plasma via BEAMing. Results: Preclinical: BAY 80-6946 was more potent on B-cell lymphomas and other hematological cancer cell lines than GS-1101 or CP (median IC50 in μM: BAY 80-6946= 0.49; GS-1101= 37; CP= 2.9). Clinical: Plasma levels of CXCL13 decreased and BAFF increased following BAY 80-6946 administration in all subjects examined (CXCL13 mean change: -58%; p= 0.004; BAFF mean change: +67%; p= 0.042). PTEN expression was lower in FL compared to ST. No mutations in PTEN or PIK3CA were identified in FL. Mutations in PIK3CA were identified in some ST. Conclusions: These preclinical results indicate that the observed early clinical activity of BAY 80-6946 in FL may be due to a direct anti-proliferative effect on malignant B-cells. The plasma chemokine/cytokine results support the possibility that the modulation of factors involved in B-cell homing and survival may play a mechanistic role in mediating this activity. Overall these results support the continued clinical evaluation of BAY 80-6946 in FL and other hematological malignancies.
Pharmacodynamic monitoring of DNA damage biomarkers in circulating tumor cells (CTCs) to assess the mechanism of action of the topoisomerase-1 (Topo1) inhibitor, NKTR-102.
Authors: Sukumaran Sujita , Neal Christopher , Woo Jacky , Melnikova Vladislava O. , Anderes Kenna Lynn , Brew Christine Taylor , Davis Darren W. , Hoch Ute
Published: March 2020

e13577

Background: Inhibitors of Topo1 abrogate DNA replication and induce apoptosis in tumor cells. Topo1 inhibitors are effective cancer therapeutics however improved pharmacokinetic profiles have been sought. NKTR-102 is a next generation topoisomerase I-inhibitor with a unique pharmacokinetic profile that provides continuous exposure of the active metabolite, SN38. NKTR-102 is in phase III clinical development for patients with metastatic breast cancer. Monitoring DNA damage response markers in CTCs may be an informative, minimally invasive approach to detect drug activity in patients. Objectives of this study are to: 1) develop a multiplex immunofluorescent assay for measuring DNA damage effects of SN38 in CTCs; 2) obtain data to determine whether measuring DNA damage biomarkers in CTCs isolated from blood of cancer patients treated with SN38 ex vivo could be applied clinically to identify the best responders to NKTR-102. Methods: Solid tumor cell lines were treated with SN38 for 24 hr. Expression levels of gH2AX, survivin, BRCA1, BRCA2, RAD51, ATM and TUNEL were measured by quantitative laser scanning cytometry (LSC).Cytotoxcity was confirmed by MTT assay. Blood samples from 3 cancer patients were treated ex vivo with SN38 and CTCs were recovered and analyzed for expression of DNA damage biomarkers by LSC. Results: Treatment of H460 and HCT116 cells with SN38 led to a dose-dependent increase in the level of protein expression and/or frequency of positive cells for all DNA damage markers. Ex vivo treatment of breast cancer patients' blood with SN38 for 24 hr induced a dose-dependent increase in the expression of ATM (7.8-fold) and RAD51(188-fold) in one patients’ blood sample. An increase in the percent TUNEL-positive cells (0-20% in untreated to 44-54% in drug-treated samples) was observed in 2 patients’ samples without affecting the total number of isolated CTCs. Conclusions: Quantitative multiplex immunofluorescent assays for monitoring changes in gH2AX, ATM, RAD51 protein expression and percent TUNEL positive CTCs have been developed using LSC. These assays have been incorporated into an ongoing phase III clinical study of NKTR-102.
Circulating VEGF levels as a biomarker to clinical benefit to PTC299, the first inhibitor of VEGF synthesis.
Authors: George Claude Paul , Luke Jason John , Winkelman J. , Cao Yen Thi Kim Hong , Shakes LaShaina , Donnelly Kerri , Darby Charles Harris , Davis Thomas W. , Elfring Gary L. , Schwartz Gary K.
Published: March 2020

e13588

Background: Solid tumor growth is dependent on angiogenesis, a process mediated primarily by vascular endothelial growth factor (VEGF). Currently approved anti-angiogenic agents sequester VEGF or block activation of VEGF receptors. Despite extensive investigations, no reliable biomarkers have been identified for these agents. PTC299 is an investigational oral small molecule that differs from existing therapies by inhibiting VEGF synthesis at the translational level while conserving physiologic VEGF expression. Methods: A phase 1b clinical trial is evaluating PTC299 monotherapy (36 pts) or in combination with docetaxel (10 pts) from 0.3 mg/kg to 600 mg bid in patients with advanced incurable cancer. Serum VEGF was assessed at baseline and during therapy. The maximum on‑treatment reduction in VEGF at any post-baseline visit was used for a stratified analysis. Decreases in VEGF, defined as <10% (low) and ≥10% (high), identified the strata. Duration of therapy was used as a surrogate for stable disease in a Kaplan‑Meier analysis. Results: The study enrolled 46 patients (20 males; 26 females; median age: 62 years). Patients with lower VEGF reductions while on therapy (<10%, n=12) had a median duration of stable disease of 5.3 weeks while patients with greater maximal VEGF reductions (≥10%, n=31) had significantly longer stable disease (median 12.0 weeks, log-rank p<0.001). VEGF decreases were greater in patients with higher baseline values. No Grade 3 or 4 hypertension, bleeding, or proteinuria occurred. One patient had an acute liver injury. Conclusions: Reductions in circulating VEGF are associated with prolonged duration of anti-angiogenic therapy and stable disease. Results are consistent with preclinical experiments and support the hypothesis that PTC299 does not affect physiologic VEGF but inhibits pathologic VEGF. In this study, oral administration of PTC299 as monotherapy or in combination with docetaxel therapy has been generally well tolerated at all doses tested.
Expression of type I insulin-like growth factor receptor (IGF1R) in tamoxifen-resistant cells: Implications for cotargeting of IGF1R and estrogen receptor (ER) in breast cancer.
Authors: Fagan Dedra H. , Uselman Ryan R. , Sachdev Deepali , Yee Douglas
Published: March 2020

e13590

Background: Crosstalk between the insulin-like growth factor (IGF) and estrogen receptor (ER) systems has been well defined in breast cancer. The majority of anti-IGF1R clinical trials are in estrogen receptor-positive patients who have progressed on prior endocrine therapy; early reports show no benefit for addition of IGF1R inhibitors to endocrine therapy in this setting. Methods: To examine the effectiveness of IGF1R inhibitors, tamoxifen-resistant (TamR) cells were generated in MCF-7L and T47D cells. IGF1R was inhibited by monoclonal antibody (dalotuzumab) or IGF1R/insulin receptor (IR) tyrosine kinase inhibitor (AEW541). Results: TamR cells had diminished levels of IGF1R with unchanged levels of IR. Further, TamR cells failed to respond to IGF-I-induced Akt activation, proliferation, and anchorage-independent growth while retaining responsiveness to both insulin and IGF-II. Dalotuzumab inhibited IGF-I-mediated Akt phosphorylation, proliferation, and anchorage-independent growth in parental cells, but had no effect on TamR cells. AEW541 with equal potency for the IGF1R and IR, inhibited IGF-I-, IGF-II-, and insulin-stimulated Akt phosphorylation, proliferation, and anchorage-independent growth in parental cells. Interestingly, AEW541 also inhibited insulin- and IGF-II-stimulated effects in TamR cells. MCF-7L xenografts from estrogen replete mice were inhibited by dalotuzumab. However, dalotuzumab add no additional benefit to tamoxifen in estrogen depleted mice. Xenograft tumors from tamoxifen treated mice had reduced levels of IGF1R mRNA. Conclusions: Because ER regulates IGF1R mRNA expression, inhibition of IGF1R does not add to tamoxifen growth inhibition of breast cancer cells. Furthermore, inhibition of IR may be necessary to manage tamoxifen resistant breast cancer. The failure of clinical trials of IGF1R inhibitors in endocrine resistant breast cancer could be explained by this crosstalk between the two growth regulatory receptors.
Serious adverse events among a population-based cohort of patients receiving first-line chemotherapy for metastatic colorectal cancer (mCRC).
Authors: Jang Raymond Woo-Jun , Enright Katherine , Booth Christopher M. , Chan Kelvin K. , Yun Lingsong , Krzyzanowska Monika K.
Published: March 2020

e14003

Background: Little is known about the toxicity of first line chemotherapy for mCRC in the general population. We sought to determine the proportion of patients who experienced at least one of the following: a serious toxicity (defined as an emergency department visit or hospitalization) or death within 30 days of receiving chemotherapy. Methods: All patients, age 18 or older, diagnosed with CRC from January 1 2007 to December 31 2009 in Ontario, Canada were identified using the Ontario Cancer Registry. Patient records were linked deterministically to multiple provincial healthcare databases to identify receipt of first-line chemotherapy for metastatic disease and to evaluate Emergency Department (ED) visits, hospitalizations, and deaths. An event was determined to be potentially treatment related if it occurred within 30 days of any cycle of chemotherapy. Results: The cohort contained 2359 patients. Mean age was 62 (range 19-89) and 59% were men. See table below. Conclusions: A significant proportion of patients visit the ED at some point during first line chemotherapy and many are hospitalized. A quarter of all patients had a hospital admission whereby an infectious complication was at least a contributing diagnosis. A significant minority of patients died within 30 days of receiving chemotherapy, raising concerns of either poor patient selection or severe treatment toxicity. [Table: see text]
Colorectal peritoneal carcinomatosis treated by completed surgery and systemic chemotherapy without HIPEC.
Authors: Becouarn Yves , Maziere Camille , Brouste Veronique , Desolneux Gregoire , Fonck Marianne , Bechade Dominique , Evrard Serge
Published: March 2020

e14017

Background: Cytoreductive peritoneal surgery (CPS) associated with hyperthermic peritoneal chemotherapy (HIPEC) has become the standard treatment for peritoneal carcinomatosis (PC) of colorectal origins. Nevertheless, if CPS efficacy is demonstrated, evidence supporting HIPEC role is lacking. Methods: Overall survival (OS), progression-free survival (PFS) and outcomes were analysed retrospectively for thirty consecutively included patients treated for colorectal PC with CPS and systemic chemotherapy only. Outcomes and morbidity of the treatment were compared to outcomes and morbidity in the literature following CPS + HIPEC treatments. Results: PCI indices ranged from 1-21 with a mean of 9. Median hospitalization duration was 15 days (range 5-51), and median intensive care unit stay was 1 day (range 0-7). There was no post-operative mortality. Seventeen complications were Grade 1 or 2 (Dindo & Clavien), and remaining 13 were Grade 3. Median follow up was 40.7 [95%CI: 15.1 – 66.3] months. Median OS was 30.7 months [95%CI: 12.65-48.66}]. OS estimates were 83% [95CI%: 63.9- 92.5] and 45% [95%CI: 23.7-64.4] at 1 year and 3 years respectively. Median PFS was 11.1 months [95%CI: 7.98-14.25]. PFS at one year was 43.2% [95%CI: 24.7-60.4]. Patients without any other metastases (n=17) had a median OS of 73.3 months [95%CI: 25.7-120.9], compared with median OS of 18.7 months [95%CI, 16.9-20.6] for patients with at least one liver or lung metastases (p=0.011). Conclusions: The results obtained for patients with colorectal PC treated by CPS and systemic chemotherapy compare favourably with series including HIPEC in the treatment. No mortality, shorter operation duration and hospitalization, less blood loss and transfusions, and no hematologic toxicity were observed.