Pretreatment Expression Levels Research Articles

The Immunogenomic Landscape of Peripheral High-Dose IL-2 Pharmacodynamics in Patients with Metastatic Renal Cell Carcinoma: A Benchmark for Next-Generation IL-2-Based Immunotherapies.

High-dose (HD) IL-2 was the first immuno-oncology agent approved for treating advanced renal cell carcinoma and metastatic melanoma, but its use was limited because of substantial toxicities. Multiple next-generation IL-2 agents are being developed to improve tolerability. However, a knowledge gap still exists for the genomic markers that define the target pharmacology for HD IL-2 itself. In this retrospective observational study, we collected PBMC samples from 23 patients with metastatic renal cell carcinoma who were treated with HD IL-2 between 2009 and 2015. We previously reported the results of flow cytometry analyses. In this study, we report the results of our RNA-sequencing immunogenomic survey, which was performed on bulk PBMC samples from immediately before (day 1), during (day 3), and after treatment (day 5) in cycle 1 and/or cycle 2 of the first course of HD IL-2. As part of a detailed analysis of immunogenomic response to HD IL-2 treatment, we analyzed the changes in individual genes and immune gene signatures. By day 3, most lymphoid cell types had transiently decreased, whereas myeloid transcripts increased. Although most genes and/or signatures generally returned to pretreatment expression levels by day 5, certain ones representative of B cell, NK cell, and T cell proliferation and effector functions continued to increase, along with B cell (but not T cell) oligoclonal expansion. Regulatory T cells progressively expanded during and after treatment. They showed strong negative correlation with myeloid effector cells. This detailed RNA-sequencing immunogenomic survey of IL-2 pharmacology complements results of prior flow cytometry analyses. These data provide valuable pharmacological context for assessing PBMC gene expression data from patients dosed with IL-2-related compounds that are currently in development.

Dynamic contrast-enhanced magnetic resonance imaging in cervical cancer: correlation between quantitative parameters and molecular markers hypoxia-inducible factors-1-alpha, vascular endothelial growth factor, and Ki-67

To investigate whether dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has the potential to non-invasively detect microenvironmental condition by quantitatively measuring blood perfusion, vessel wall permeability, and vascularity, and to elucidate the possible correlations between DCE-MRI quantitative parameters and the expression level of hypoxia, vascularity, and cell proliferation related molecular biomarkers. In this prospective single center clinical study, 58 patients diagnosed with cervical cancer underwent DCE-MRI before anticancer treatment were enrolled. Ktrans, Kep, Ve, and Vp were generated from Extended Toft's model. Then patients conducted colposcopy biopsy within 1 week after DCE-MRI. Pretreatment expression levels of HIF-1α, VEGF and Ki-67 were assessed and scored by immunohistochemistry on colposcopy obtained tumor specimens. In HIF-1α low-expression group, Ktrans (p=0.031) and Kep (p=0.012) values were significantly higher than the high-expression group. In VEGF high-expression group, Ktrans (p=0.044) and Ve values (p=0.021) were significantly higher than the low-expression group. In Ki-67 high-expression group, Ktrans (p=0.026) and Kep (p=0.033) were significantly higher than the low-expression group. Multiple linear regression analyses and Pearson correlation revealed that Ktrans independently negatively correlated with HIF-1α expression, Ve independently positively correlated with VEGF, and Kep independently positively correlated with Ki-67. The area under the ROC curves of Ktrans for HIF-1α, Ve for VEGF, and Kep for Ki-67 were 0.728, 0.743, 0.730, respectively. Our results suggest that DCE-MRI quantitative parameters could be potentially used as imaging markers for non-invasively detecting microenvironmental hypoxia, vascularity and proliferation in cervical cancer patients.

CML-313 Kinetics of CITED2 Gene Expression in Chronic Myeloid Leukemia Patients

In CML, BCR-ABL1 oncoprotein induces overexpression of STAT5 and its target genes for example CITED2 gene. This gene is involved in regulating the proliferation and quiescence states of leukemic stem cells (LSCs). Recently, studies reported that adding pioglitazone (PPARγ agonist approved for T2DM treatment) to imatinib may decrease the transcription of both STAT5 and CITED2 gene, eliminate the quiescent LSCs (resist TKIs), and ameliorate the patients' response. The primary objective is to clarify the value of combining pioglitazone with imatinib to downregulate the CITED2 gene aiming to eradicate the LSCs in CML treatment. In the context of a clinical trial (approved by the Ethical Committee of the Faculty of Medicine Mansoura University), the study group patients (N=26) were treated with combination therapy after having their consent. The planned follow-up time is 2 years. Hematology unit, Oncology Center, Mansoura University. The eligibility criteria for study group patients were de novo, Philadelphia+ve CML (chronic phase) aged 18-60 years old. Those patients were treated with imatinib 400 mg and pioglitazone 15 mg once daily for 6 months. Pre- and post-treatment samples (after 6 months) were collected to assess the expression levels of the CITED2 gene. Responders continued on the same TKI and their sequential BCR/ABL Q-PCR was monitored. The study proposed that this combination therapy can put more patients into deeper and faster molecular response via eliminating the LSCs. The study group included 15 males (57.7%) and 11 females (42.3%) with a mean age of 43.2 years. The BCR-ABL and CITED2 genes' pretreatment expression levels decreased significantly after 6 months of combined treatment (p<0.001 and p=0.005, respectively). Moreover, at 6 months 65.4% and 23% of the patients had response ≥ CCyR and ≥ MMR respectively. The most frequent complaints among patients were increased body weight and edema (p<0.001). Adding pioglitazone to imatinib improved the patients' response and downregulate the overexpressed CITED2 gene. A longer follow-up is planned and needed to confirm the safety and efficacy of the combination.

P711: KINETICS OF CITED2 GENE EXPRESSION IN CHRONIC MYELOID LEUKEMIA PATIENTS

Background: In chronic myeloid leukemia (CML), BCR-ABL1 oncoprotein induces overexpression of STAT5 and its target genes. Some of these genes are involved in regulating the leukemic stem cells (LSCs) cell cycle. CITED2 gene controls the balance between the proliferation and the quiescence states of the LSCs that resist tyrosine kinase inhibitors (TKIs). Recently, studies reported that adding peroxisome proliferator‐activated receptor γ agonists (approved for type 2 Diabetes mellitus treatment) to TKI therapy may decrease the transcription of STAT5 and its target genes, eliminate the quiescent LSCs and ameliorate the patients’ response. Aims: The aim of the study is to clarify the value of combining the glitazones with the TKIs in the treatment of denovo CML CP patients as regard the patients’ response and effect of this combination therapy on the expression of CITED2 gene. Methods: In a context of a clinical trial (approved by the ethical Committee of the Faculty of Medicine Mansoura University), eligible denovo patients of CML chronic phase (N=26) were treated with imatinib 400 mg combined and pioglitazone 15 mg (off label use) daily for 6 months after having their consent. Responders continued on same TKI and their sequential BCR/ABL Q-PCR was monitored. The non-responder patients were switched to another line and excluded. Pre and post-treatment samples were collected to assess the expression levels of CITED2 gene. Results: The study group included 15 males (57.7%) and 11 females (42.3%) with a mean age of 43.2 years. Their median BCR-ABL gene expression before treatment was 79% (range = 0.63-380) that decreased to 3.65% (range = 0.05-20) and 0.750 % (range = 0.002-46) on 3rd and 6th months post-treatment. Among the study group 100% of the patients achieved hematological response (HR) at 3 months. At 6 months 65.4% of the patients had response more than CCyR and 23% of them had MMR at 6 months respectively. The most frequent complains among patients were increase body weight and edema (p value < 0.001). The median of CITED2 gene pre-treatment and post-treatment expression levels were: 276.3 (range= 1-241221.7) and 2.6 (range= 0.006-86475.3) with a significant decrease in expression (p=0.005) after the combined treatment. Summary/Conclusion: Pioglitazone in combination with imatinib improved the patients’ response and downregulate the expression of the CITED2 overexpressed gene. Longer follow up is planned and needed to confirm whether it can effectively induce more deep molecular response among the patients or not.

Plasmodium falciparum Drug Resistance Genes pfmdr1 and pfcrt In Vivo Co-Expression During Artemether-Lumefantrine Therapy.

Background: Artemisinin-based combination therapies (ACTs) are the global mainstay treatment of uncomplicated Plasmodium falciparum infections. PfMDR1 and PfCRT are two transmembrane transporters, associated with sensitivity to several antimalarials, found in the parasite food vacuole. Herein, we explore if their relatedness extends to overlapping patterns of gene transcriptional activity before and during ACT administration. Methods: In a clinical trial performed in Tanzania, we explored the pfmdr1 and pfcrt transcription levels from 48 patients with uncomplicated P. falciparum malaria infections who underwent treatment with artemether-lumefantrine (AL). Samples analyzed were collected before treatment initiation and during the first 24 h of treatment. The frequency of PfMDR1 N86Y and PfCRT K76T was determined through PCR-RFLP or direct amplicon sequencing. Gene expression was analyzed by real-time quantitative PCR. Results: A wide range of pre-treatment expression levels was observed for both genes, approximately 10-fold for pfcrt and 50-fold for pfmdr1. In addition, a significant positive correlation demonstrates pfmdr1 and pfcrt co-expression. After AL treatment initiation, pfmdr1 and pfcrt maintained the positive co-expression correlation, with mild downregulation throughout the 24 h post-treatment. Additionally, a trend was observed for PfMDR1 N86 alleles and higher expression before treatment initiation. Conclusion: pfmdr1 and pfcrt showed significant co-expression patterns in vivo, which were generally maintained during ACT treatment. This observation points to relevant related roles in the normal parasite physiology, which seem essential to be maintained when the parasite is exposed to drug stress. In addition, keeping the simultaneous expression of both transporters might be advantageous for responding to the drug action.

Treatment outcomes in poor metabolizers of cytochrome P450 2A6 with concurrent chemoradiotherapy in locally advanced head- and neck-squamous cell carcinoma.

Human Cytochrome 2A6 (CYP2A6) is involved in the oxidative metabolism of the nicotine to the inactive cotinine. CYP2A6 is a primary enzyme in nicotine metabolism, the enzyme has been proposed as a novel target for smoking cessation. A total of 70 male patients of locally advanced head- and neck-squamous cell carcinoma confirmed by histopathological examination were enrolled in this study. All patients received concurrent chemoradiotherapy (total dose of 70 Gray in 35 fractions in 7 weeks with concurrent tablet capecitabine 1250 mg/m2/day). Response assessment was based on response evaluation criteria in solid tumor criteria. Total ribonucleic acid (RNA) was isolated from the whole blood of all patients by TRI REAGENT BD (SIGMA USA) followed by real-time polymerase chain reaction assay which was done in studying messenger RNA (mRNA) expression of Excision Repair Cross Complementation Group 1 in blood lymphocytes of patient. The most common stage prevalent was Stage IV A in 28 (56%) patients followed by Stage III in 16 (32%) patients. Out of 70, 20 (28.6%) patients defaulted for treatment, so the analysis was done in 56 patients. A total of 19 (34%) patients had a complete response (CR) and 17 (30%) patients had no response. In all the patients who had CR, posttreatment relative quantification (RQ) expression levels were high. Among nonresponders only three had higher RQ folds and the rest 14 had lower RQ folds. Posttreatment expression levels of CYP2A6 were found to be a better predictor for tumor response to the treatment than the pretreatment expression levels. Almost all the patients having higher RQ folds had CR and those having lower RQ folds had either no response or progressive disease on follow-up visits.

Amyotrophic lateral sclerosis transcriptomics reveals immunological effects of low-dose interleukin-2

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease causing upper and lower motor neuron loss and currently no effective disease-modifying treatment is available. A pathological feature of this disease is neuroinflammation, a mechanism which involves both CNS-resident and peripheral immune system cells. Regulatory T-cells are immune-suppressive agents known to be dramatically and progressively decreased in patients with amyotrophic lateral sclerosis. Low-dose interleukin-2 promotes regulatory T-cell expansion and was proposed as an immune-modulatory strategy for this disease. A randomized placebo-controlled pilot phase-II clinical trial called Immuno-Modulation in Amyotrophic Lateral Sclerosis was carried out to test safety and activity of low-dose interleukin-2 in 36 amyotrophic lateral sclerosis patients (NCT02059759). Participants were randomized to 1MIU, 2MIU-low-dose interleukin-2 or placebo and underwent one injection daily for 5 days every 28 days for three cycles. In this report, we describe the results of microarray gene expression profiling of trial participants' leukocyte population. We identified a dose-dependent increase in regulatory T-cell markers at the end of the treatment period. Longitudinal analysis revealed an alteration and inhibition of inflammatory pathways occurring promptly at the end of the first treatment cycle. These responses are less pronounced following the end of the third treatment cycle, although an activation of immune-regulatory pathways, involving regulatory T-cells and T helper 2 cells, was evident only after the last cycle. This indicates a cumulative effect of repeated low-dose interleukin-2 administration on regulatory T-cells. Our analysis suggested the existence of inter-individual variation amongst trial participants and we therefore classified patients into low, moderate and high-regulatory T-cell-responders. NanoString profiling revealed substantial baseline differences between participant immunological transcript expression profiles with the least responsive patients showing a more inflammatory-prone phenotype at the beginning of the trial. Finally, we identified two genes in which pre-treatment expression levels correlated with the magnitude of drug responsiveness. Therefore, we proposed a two-biomarker based regression model able to predict patient regulatory T-cell-response to low-dose interleukin-2. These findings and the application of this methodology could be particularly relevant for future precision medicine approaches to treat amyotrophic lateral sclerosis.

Study of HOTAIR LncRNA in AML patients in context to FLT3-ITD and NPM1 mutations status

BackgroundLong non-coding RNAs (LncRNAs) have recently been considered promising biomarkers for oncogenesis due to their epigenetic regulatory effects. HOTAIR is one of the oncogenic LncRNAs that was previously studied in different non-hematological malignancies. The current study set out to detect the expression level of HOTAIR LncRNA in AML patients concerning their clinical characteristics, laboratory data, FLT3-ITD, and NPM1 mutations, as well as treatment outcome. This study included quantitative detection of HOTAIR gene expression in 47 cases of AML using quantitative reverse transcription polymerase chain reaction, as well as NPM1 and FLT3-ITD genotyping.ResultsThe HOTAIR expression was significantly higher in AML patients 6.87 (0.001) than in normal controls 1.66 (0.004–6.82) (p 0.007). The HOTAIR expression level was affected by chemotherapy, and it was correlated to hemoglobin level (p 0.001), age, total leukocytic count (p 0.022), and NPM1 mutation (p 0.017). HOTAIR gene expression level showed a correlation to relapse-free survival in the study group (p 0.04).ConclusionHOTAIR is overexpressed in patients with acute myeloid leukemia (AML). HOTAIR pre-treatment and post-chemotherapy gene expression levels can predict chemosensitivity and relapse.

Association of expression of epigenetic molecular factors with DNA methylation and sensitivity to chemotherapeutic agents in cancer cell lines

BackgroundAltered DNA methylation patterns play important roles in cancer development and progression. We examined whether expression levels of genes directly or indirectly involved in DNA methylation and demethylation may be associated with response of cancer cell lines to chemotherapy treatment with a variety of antitumor agents.ResultsWe analyzed 72 genes encoding epigenetic factors directly or indirectly involved in DNA methylation and demethylation processes. We examined association of their pretreatment expression levels with methylation beta-values of individual DNA methylation probes, DNA methylation averaged within gene regions, and average epigenome-wide methylation levels. We analyzed data from 645 cancer cell lines and 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer datasets. We observed numerous correlations between expression of genes encoding epigenetic factors and response to chemotherapeutic agents. Expression of genes encoding a variety of epigenetic factors, including KDM2B, DNMT1, EHMT2, SETDB1, EZH2, APOBEC3G, and other genes, was correlated with response to multiple agents. DNA methylation of numerous target probes and gene regions was associated with expression of multiple genes encoding epigenetic factors, underscoring complex regulation of epigenome methylation by multiple intersecting molecular pathways. The genes whose expression was associated with methylation of multiple epigenome targets encode DNA methyltransferases, TET DNA methylcytosine dioxygenases, the methylated DNA-binding protein ZBTB38, KDM2B, SETDB1, and other molecular factors which are involved in diverse epigenetic processes affecting DNA methylation. While baseline DNA methylation of numerous epigenome targets was correlated with cell line response to antitumor agents, the complex relationships between the overlapping effects of each epigenetic factor on methylation of specific targets and the importance of such influences in tumor response to individual agents require further investigation.ConclusionsExpression of multiple genes encoding epigenetic factors is associated with drug response and with DNA methylation of numerous epigenome targets that may affect response to therapeutic agents. Our findings suggest complex and interconnected pathways regulating DNA methylation in the epigenome, which may both directly and indirectly affect response to chemotherapy.

Folate pathway gene expression in metastatic colorectal cancer patients treated with arfolitixorin/5-FU-based chemotherapy.

99 Background: Arfolitixorin is the natural, biologically active form of the marketed folates and is expected to be efficacious in a larger proportion of patients with less inter- and intra-individual variability compared with e.g. leucovorin. We have previously found a positive correlation between survival and expression of folate pathway genes in stage III/IV CRC treated with 5-fluorouracil/leucovorin (5-FU/LV). Low expression of folate-related genes may lead to poor response to 5-FU/LV-based treatment, since suboptimal transport and metabolization of LV yield insufficient active [6R]-5,10-methylenetetrahydrofolate and weak inhibition of the target enzyme thymidylate synthase (TYMS). The aim of the present study was to investigate possible confounders and biomarkers of arfolitixorin/5-FU-based treatment in relation to safety and response in a phase I/IIa metastatic colorectal cancer (mCRC) trial. Methods: ISO-CC-005 is a multi-center, phase I/IIa study in mCRC patients eligible for 5-FU/folate therapy alone or in combination with irinotecan or oxaliplatin ± bevacizumab. Patients were also treated with different doses of arfolitixorin as a single or double bolus. The study investigated safety and tolerability of arfolitixorin, and anti-tumor activity was evaluated by overall response rate (ORR) per RECIST v1.1 after 4 cycles of chemotherapy. RNA was prepared from FFPE tumor tissue, reverse transcribed and used for gene expression profiling. The following genes of interest were evaluated: ABCC3, MTHFD2, SLC46A1, SLC19A1, SLC25A32 and TYMS. An ANOVA test was used to rule out potential biases in the baseline expression levels of the genes and to assess the potential association with clinical response. Results: Eighty-one (77.1%) of 105 patients provided material for this analysis. A lower pre-treatment expression of TYMS was associated with clinical benefit (PR and SD; p = 0.021). No clear association was identified between the gene expression markers and the number of adverse events. Gender was not significantly associated with differences in gene expression. Conclusions: Low pre-treatment expression levels of TYMS were associated with clinical benefit (PR and SD) following treatment. Given the role of this gene in the folate metabolic pathways we plan to further assess its predictive potential on a larger cohort during our ongoing global phase III AGENT study. In parallel an assessment of the expression of the other candidate genes on specific patient sub-groups is currently ongoing. These studies will provide additional cues on the use of these genes as predictive markers for treatment outcome and their role in the mode of action of the drug. Clinical trial information: NCT02244632.

A high number of PD-L1+ CD14+ monocytes in peripheral blood is correlated with shorter survival in patients receiving immune checkpoint inhibitors.

Targeting of anti-programmed cell death protein-1 (PD-1) and anti-programmed death-ligand 1 (PD-L1) is a standard therapeutic strategy for various cancers. The aim of the present study was to investigate the prognostic effect of pretreatment PD-L1 expression levels in peripheral blood mononuclear cell (PBMC) subsets for patients with several cancer types receiving anti-PD-1 blockade therapies. Thirty-two patients undergoing anti-PD-L1 blockade therapy, including 15 with non-small cell lung cancer, 14 with gastric cancer, 1 with melanoma, 1 with parotid cancer, and 1 with bladder cancer, were recruited for the present study. PD-L1 expression levels in CD3+, CD4+, CD8+, CD45RA+ and CCR7+ T cells; CD20+ B cells; CD14+ and CD16+ monocytes were measured via flow cytometry before treatment. The percentages of PD-L1+ cells in respective PBMC subsets were compared with respect to different clinicopathological conditions and the association with overall survival (OS) was assessed. The percentages of PD-L1+ with CD3+, CD4+ and CD8+ T cells including naïve and memory T cell subsets, or CD20+ B cells during pretreatment were not markedly correlated with the OS of patients (p > 0.05); however, the percentage of the PD-L1+ CD14+ monocyte subset was significantly correlated with OS (p = 0.0426). Increase in pretreatment expression levels of PD-L1 on CD14+ monocytes is associated with the OS of patients treated with immune checkpoint inhibitors. Further evaluation of large sample size and each specific cancer type might clarify the predictive role of PBMC in patients.

Translesion synthesis genes POLI and REV1 in breast tumors and response to neoadjuvant chemotherapy.

e12642 Background: Translesion synthesis (TLS) of DNA by specialized polymerases (POLs) and REV1, a scaffold protein that recruits other TLS DNA POLs for nascent strand extension, experimentally enable tumor cells to replicate through DNA damage. TLS POLs are hypothesized to contribute to treatment resistance. Methods: We conducted a discovery gene expression study using NanoString technology with a bespoke codeset enriched for DNA damage response genes and RNA from 12 TN, 10 HER2+ and 29 ER+/HER2- breast cancer patients before neoadjuvant chemotherapy (NACT) and when available, in the matching residual tumor. Eleven patients achieved a pathologic complete response (pCR), 12 had &lt; 1 cm residual disease (partial responders) and 28 patients had &gt; 1cm residual disease (non-responders). RNA counts were normalized and log-transformed for differential expression analyses corrected for false discovery. Results: DNA polymerase Iota ( POLI) was the top overexpressed gene in the pretreatment biopsy of non-responders independent of tumor subtype (p corrected = 0.0002). Expression of REV1 was also significantly higher in non-responders and positively correlated with POLI (r = 0.577, p &lt; 0.0001). Despite a small sample size, pretreatment expression levels of POLI and REV1 were significantly higher in ER+ (n = 38) than ER- (n = 13) tumors (p = 0.0006). When restricted to ER+ tumors, POL1 and REV1 expression levels were significantly higher in pretreatment biopsy of non-responders (n = 25) than partial and complete responders (n = 13). For ER+ patients, response by median POL1/REV1 expression levels are shown in Table. Pre/post treatment POL1 and REV1 expression did not change in patients with residual disease. Conclusions: In discovery, two genes involved in TLS of DNA (POLI and REV1) were found overexpressed in pretreatment biopsies from breast cancers poorly responsive to NACT, independent of tumor subtype. Overexpression was more common among ER+ tumors, which exhibit a high inherent resistance to chemotherapy. If confirmed, expressed levels of POLI and REV1 may identify a subgroup of breast cancers resistant to NACT for which targeted inhibition of mutagenic TLS may be beneficial. [Table: see text]

Expression levels of blood platelet and C-reactive protein in patients with severely pneumonic and their predictive values for efficacy

This paper aims to detect the expression levels of blood platelet (PLT) and C-reactive protein (CRP) in severely pneumonic patients and analyze their correlation. For this purpose, eighty-one severely pneumonic patients were retrospectively selected as an observation group and 106 healthy people as a control group. Pretreatment and post-treatment expression levels of PLT and CRP, their predictive values for efficacy, and correlation of PLT, CRP, and PSI scores in observation group after treatment were analyzed. Before treatment, the expression level of PLT in the observation group was higher than the control group (P&lt; 0.05). In the observation group, the expression level of PLT after treatment was significantly lower than that before treatment (P&lt; 0.05). Before treatment, the expression level of CRP in the observation group was higher than the control group (P&lt; 0.05). In the observation group 1) the pretreatment PLT expression level was higher than that in the control group; 2) the post-treatment PLT expression level was significantly lower than that in the pretreatment one; 3) the pretreatment CRP expression level was higher than that in the control group; and 4) the post-treatment CRP expression level was significantly lower than the pretreatment one (All P-values&lt; 0.05). Based upon the efficacy, the observation group was divided into an effective group and an invalid group. The post-treatment expression levels of PLT and CRP in the effective group were lower than those in the invalid group (P&lt; 0.05). Based upon the ROC curve, the area under curves (AUC) of PLT, CRP, and joint detection were 0.843, 0.864, and 0.886, respectively. When the cut-off point was &gt; 0.579, the best specificity and sensitivity were 98.44 and 70.59%, respectively. According to the Pearson test, positive correlations existed between PLT and CRP, between PLT and PSI scores, and between CRP and PSI scores. In conclusion, the expression levels of PLT and CRP in severely pneumonic patients might be used to evaluate the efficacy and conducive to detection of the disease, which have high application values in clinic.

Abstract 4246: Association of transcriptional levels of folate-mediated one-carbon metabolism related genes in cancer cell lines with drug treatment response

Abstract Folate-mediated one-carbon metabolism (OCM) is essential for growth and survival of cancer cells. It affects biosynthesis of nucleotides and amino acids, regulation of redox status, methylation of nucleic acids and proteins, and genome maintenance. We investigated whether the response of cancer cells to antitumor therapy treatment may be partially influenced by variation in expression of one-carbon metabolism genes. We used publicly available gene expression data and drug response measures for 251 antitumor agents in 635 cancer cell lines with matching information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources. We examined whether pre-treatment expression levels of OCM-related genes were associated with drug response. Among the 34 OCM genes examined, expression of GART, TYMS, SHMT2, MTR, ALDH2, BHMT, MAT2B, MTHFD2, NNMT, and SLC46A1 showed modest correlations with response to treatment with a variety of antitumor agents. Higher expression levels of the SLC46A1 transporter gene were associated with resistance to multiple drugs, whereas elevated expression of GART, TYMS, SHMT2, MTR, BHMT, and MAT2B was associated with chemosensitivity to multiple antitumor agents. NNMT expression was bimodally distributed and showed different directions of association with various agents. Correlation of increased NNMT expression with sensitivity to dasatinib was validated in the NCI-60 cancer cell line panel. Expression of several OCM genes was strongly associated with expression of multiple components of drug target pathways. For example, expression of both TYMS and GART was strongly positively correlated with BRCA1 expression, NNMT expression was associated with expression of multiple drug target genes including EGFR and ABL2, and the expression of TYMS, DHFR, and SHMT1 was positively correlated with AURKB expression. Pretreatment expression levels of many OCM genes including DHFR, TYMS, ATIC, GART, AHCY, MTHFD1, SLC19A1, and multiple other genes were positively correlated with each other, suggesting their co-regulation. In contrast, NNMT expression was negatively correlated with expression of several other OCM genes. Further studies could investigate whether correlations of expression levels of individual OCM genes with drug response are related to specific metabolic roles of these genes or whether such associations may be due to the general increase in cellular growth and proliferation and tumor progression, which may affect sensitivity to cancer treatment. Citation Format: Julia Krushkal, Suleyman Vural, Dong-Joon Min. Association of transcriptional levels of folate-mediated one-carbon metabolism related genes in cancer cell lines with drug treatment response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4246.

Association of transcriptional levels of folate-mediated one-carbon metabolism-related genes in cancer cell lines with drug treatment response

Folate-mediated one-carbon metabolism is essential for growth and survival of cancer cells. We investigated whether the response of cancer cells to antitumor treatment may be partially influenced by variation in expression of one-carbon metabolism genes. We used cancer cell line information from the Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer resources to examine whether variation in pretreatment expression of one-carbon metabolism-related genes was associated with response to treatment. GART, TYMS, SHMT2, MTR, ALDH2, BHMT, MAT2B, MTHFD2, NNMT, and SLC46A1 showed modest statistically significant correlations with response to a variety of antitumor agents. Higher expression levels of SLC46A1 were associated with resistance to multiple agents, whereas elevated expression of GART, TYMS, SHMT2, MTR, BHMT, and MAT2B was associated with chemosensitivity to multiple drugs. NNMT expression was bimodally distributed and showed different directions of association with various agents. Correlation of increased NNMT expression with sensitivity to dasatinib was validated in the NCI-60 cancer cell line panel. Pretreatment expression levels were correlated among many one-carbon metabolism genes. Expression of several folate genes was strongly associated with expression of multiple components of drug target pathways. Molecular mechanisms underlying associations of one-carbon metabolism gene with drug response require further investigation.

Abstract P3-10-14: LIV-1 expression in primary breast cancers in the I-SPY 2 TRIAL

Abstract Background: LIV-1 is an estrogen-inducible gene that has been implicated in epidermal-to-mesenchymal transition (EMT) in preclinical models of progression and metastasis. Its expression is associated with node-positivity in breast cancer; and has been detected in a variety of cancer types, including estrogen receptor positive breast cancers. SGN-LIV1A is a novel antibody drug conjugate targeting LIV-1 that is currently being evaluated in the I-SPY 2 TRIAL. In this pilot study, we evaluated LIV-1 levels by IHC within HR/HER2/MammaPrint (MP) defined subtypes among patients screening for the I-SPY 2 TRIAL and its correlation to microarray assessed LIV-1 expression levels. Method: In a pilot study, LIV-1 IHC staining was performed by Quest Diagnostics on the pre-treatment samples of 38 patients screening for the I-SPY 2 TRIAL. Pre-treatment expression data generated on a custom Agilent 44K platform was also available. We summarized the LIV-1 H-Scores and percent (%)-positivity across the population and within HR/HER2/MP subtypes; and we assessed the Pearson correlation between LIV-1 H-Score and LIV-1 gene expression levels. In addition, we compared the pre-treatment LIV-1 expression levels within HR/HER2/MP subtypes across I-SPY 2 TRIAL patients from completed arms and their relevant controls (n=989) using ANOVA and post-hoc Tukey tests. Our statistics are descriptive rather than inferential; and does not take into account multiplicities of other biomarkers outside of this study. Results: Of the 38 patients evaluated, 37 have LIV-1 %-positivity &amp;gt; 0; and 18 (47%) have 100% LIV1 positivity. The median LIV-1 H-Score is 200; and 89% of patients (34/38) have moderate/high LIV-1 staining (with H-Score≥100). Of the 34 patients who proceeded onto the trial (and have known HR/HER2/MP status), 9 are triple negative, 19 are HR+HER2-, and 6 are HER2+. Due to our small sample size, we did not further subset the triple negative and HER2+ cases; but within the HR+HER2- patients, 10 are MP1 compared to 9 who are MP2 class. LIV1 H-Score appears highest within the HR+HER2-MP1 cases (median: 290), followed by the HER2+ (median: 216), then the HR+HER2-/MP2 (median: 155), and the TN (median: 120) subtype. LIV1 H-score is significantly correlated with LIV-1 mRNA expression levels (Rp=0.79, p&amp;lt;0.0001). Consistent with these observations, LIV-1 pre-treatment expression levels are significantly higher in the HR+HER2-MP1 group relative to all other HR/HER2/MP defined subtypes (Tukey HSD p &amp;lt; 0.0001) across the I-SPY 2 TRIAL population. The HR+HER2+MP1 group also have high LIV-1 expression levels. Conclusion: Our result suggest that although LIV-1 expression differs by subtype, it is expressed at a moderate/high level in the majority of patients. The good correlation between IHC and array-based LIV-1 expression levels enables us to leverage the entire existing I-SPY 2 dataset and confirm the high rates of LIV-1 expression across the I-SPY 2 population. Further studies to evaluate LIV-1 expression as a biomarker of response to LIV-1 targeting therapies for the neoadjuvant treatment of breast cancer are warranted and ongoing in I-SPY 2. Citation Format: Yau C, Brown-Swigart L, Asare S, I-SPY 2 TRIAL Consortium, Esserman L, van' t Veer L, Beckwith H, Forero A, Rugo H. LIV-1 expression in primary breast cancers in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-10-14.

Longitudinal Transcriptional Response of Glycosylation-Related Genes, Regulators, and Targets in Cancer Cell Lines Treated With 11 Antitumor Agents.

Cellular glycosylation processes are vital to cell functioning. In malignant cells, they are profoundly altered. We used time-course gene expression data from the NCI-60 cancer cell lines treated with 11 antitumor agents to analyze expression changes of genes involved in glycosylation pathways, genes encoding glycosylation targets or regulators, and members of cancer pathways affected by glycosylation. We also identified glycosylation genes for which pretreatment expression levels or changes after treatment were correlated with drug sensitivity. Their products are involved in N-glycosylation and O-glycosylation, fucosylation, biosynthesis of poly-N-acetyllactosamine, removal of misfolded proteins, binding to hyaluronic acid and other glycans, and cell adhesion. Tumor cell sensitivity to multiple agents was correlated with transcriptional response of C1GALT1C1, FUCA1, SDC1, MUC1; members of the MGAT, GALNT, B4GALT, B3GNT, MAN, and EDEM families; and other genes. These genes may be considered as potential candidates for drug targeting in combination therapy to enhance treatment response.

CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma

AbstractThe anti-CD38 monoclonal antibody daratumumab is well tolerated and has high single agent activity in heavily pretreated relapsed and refractory multiple myeloma (MM). However, not all patients respond, and many patients eventually develop progressive disease to daratumumab monotherapy. We therefore examined whether pretreatment expression levels of CD38 and complement-inhibitory proteins (CIPs) are associated with response and whether changes in expression of these proteins contribute to development of resistance. In a cohort of 102 patients treated with daratumumab monotherapy (16 mg/kg), we found that pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) compared with patients who achieved less than PR. However, cell surface expression of the CIPs, CD46, CD55, and CD59, was not associated with clinical response. In addition, CD38 expression was reduced in both bone marrow–localized and circulating MM cells, following the first daratumumab infusion. CD38 expression levels on MM cells increased again following daratumumab discontinuation. In contrast, CD55 and CD59 levels were significantly increased on MM cells only at the time of progression. All-trans retinoic acid increased CD38 levels and decreased CD55 and CD59 expression on MM cells from patients who developed daratumumab resistance, to approximately pretreatment values. This resulted in significant enhancement of daratumumab-mediated complement-dependent cytotoxicity. Together, these data demonstrate an important role for CD38 and CIP expression levels in daratumumab sensitivity and suggest that therapeutic combinations that alter CD38 and CIP expression levels should be investigated in the treatment of MM. These trials were registered at www.clinicaltrials.gov as #NCT00574288 (GEN501) and #NCT01985126 (SIRIUS).

Abstract P5-15-05: The expression of genes involved in cholesterol biosynthesis is heterogeneous in breast cancer and may predict sensitivity to statin treatment and clinical outcome

Abstract Background: Epidemiological evidence supports a protective role of cholesterol lowering drugs against breast cancer recurrence. Preclinical studies also support this role since statins can suppress breast cancer progression in various models. The response of breast cancer cells to statin treatment is however heterogeneous. We performed a comprehensive molecular characterisation of the anti-cancer mechanisms of statins in-vitro in view of identifying biomarkers of statin sensitivity in breast cancer. Methods: Breast cancer cell lines were treated with atorvastatin and the effects on cell proliferation were determined. A panel of cell lines displaying a diverse range of sensitivity to atorvastatin treatment were selected for gene expression microarray profiling following treatment. Differentially expressed genes were identified and subjected to gene ontology analysis and links to outcome following breast cancer diagnosis were explored. Results: Statin treatment elicited a more potent inhibition of proliferation in estrogen receptor (ER) negative cell lines. This was reflected on the whole genome transcriptional scale as the ER negative cells also displayed a more robust dysregulation of mRNA transcripts relative to the less-sensitive ER expressing cells. Down-regulation of DNA replication, regulation of cell cycle progression, and other proliferation-associated biological processes via mechanisms influencing the transcriptional activity of E2F was observed in the statin-sensitive cells. Importantly, cholesterol biosynthesis via the mevalonate pathway was upregulated in all cell lines following statin treatment. This effect was directly correlated with the pre-treatment expression levels of target genes involved in this biological process, suggesting a link with sensitivity to statin treatment. The expression of cholesterol biosynthesis genes was also found to be variable in primary breast tumors and a significant association was observed between decreased expression and an improved recurrence-free and overall survival, especially in ER positive tumors. Conclusions: These data suggest that the normal feedback regulation of the mevalonate pathway induced by statins may be compromised in a subset of breast cancer cells and may be exploited to predict breast cancer prognosis and sensitivity to statin treatment. Citation Format: Kimbung S, Feldt M, Bosch A, Borgquist S. The expression of genes involved in cholesterol biosynthesis is heterogeneous in breast cancer and may predict sensitivity to statin treatment and clinical outcome. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-15-05.

The BDNF Val66Met polymorphism and plasma brain-derived neurotrophic factor levels in Han Chinese heroin-dependent patients.

BDNF and its gene polymorphism may be important in synaptic plasticity and neuron survival, and may become a key target in the physiopathology of long-term heroin use. Thus, we investigated the relationships between brain-derived neurotrophic factor (BDNF) plasma concentrations and the BDNF Val66Met nucleotide polymorphism (SNP) in heroin-dependent patients. The pretreatment expression levels of plasma BDNF and the BDNF Val66Met SNP in 172 heroin-dependent patients and 102 healthy controls were checked. BDNF levels were significantly lower in patients (F = 52.28, p < 0.0001), but the distribution of the SNP was not significantly different. Nor were plasma BDNF levels significantly different between Met/Met, Met/Val, and Val/Val carriers in each group, which indicated that the BDNF Val66Met SNP did not affect plasma BDNF levels in our participants. In heroin-dependent patients, plasma BDNF levels were negatively correlated with the length of heroin dependency. Long-term (>15 years) users had significantly lower plasma BDNF levels than did short-term (<5 years) users. We conclude that plasma BDNF concentration in habitual heroin users are not affected by BDNF Val66Met gene variants, but by the length of the heroin dependency.