Different Time Points Of Treatment Research Articles

Abstract PO3-23-08: Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib

Abstract Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib. Marie Fongaard1, Leonard Schmiester2, Salim Ghannoum1, Pål Marius Bjørnstad1, Tatjana Bosnjak3, Signe Meltzer Kleivbo3, Knut Selsås4, Stephanie Beate Geisler5, Kamilla Fjermeros5, Sameer Bhargava5, Manouchehr Seyedzadeh6, Unn-Cathrin Buvarp5, Aino Katri Rosenskiold5, Nam Thi Nguyen5, Torben Lüders7, Diether Lambrechts8, Marianne Lyngra9, Arnoldo Frigessi2, Vessela Kristensen1,10, Jürgen Geisler5,10,*, Xavier Tekpli1,* 1) Department of Medical Genetics, Oslo University Hospital, Oslo, Norway 2) University of Oslo, Oslo Centre for Biostatistics and Epidemiology (OCBE), University of Oslo, Norway 3) Novartis, Medical Affairs, Oslo, Norway 4) Department of Breast and Endocrine Surgery, Akershus University Hospital, Oslo, Norway 5) Department of Oncology, Akershus University Hospital, Oslo, Norway 6) Department of Radiology, Akershus University Hospital, Oslo, Norway 7) Department of Clinical Molecular Biology (EPIGEN), Akershus University Hospital, Oslo, Norway 8) Laboratory for Translational Genetics, Department of Human Genetics, KU Leuven, Leuven, Belgium 9) Department of Pathology, Akershus University Hospital, Oslo, Norway 10) University of Oslo, Institute of Clinical Medicine, Faculty of Medicine, Oslo, Norway< * Jointly supervised the work, shared corresponding authors Background The recent introduction of CDK4/6 inhibitors is regarded as a major improvement in the field of breast cancer therapy. A growing body of evidence suggests that CDK4/6 inhibitors may impact on the recruitment of immune cells within the tumor microenvironment during therapy. Further, CDK4/6 inhibitors may potentially contribute to the extinction or survival of specific cancer cell clones through evolutionary pressure exerted on breast tumors. Clinical trial The NEOLETRIB-study is a multicenter, single-arm, open-label phase II clinical trial. Patients with locally advanced, LUM-A/LUM-B breast cancer, characterized by cT3-cT4 tumors and/or cN2-3 lymph node involvement, receive ribociclib (600 mg daily, 21 days on and 7 days off) in combination with letrozole (2.5 mg daily) for a duration of 6 months in the neoadjuvant setting. Patients with large cT2 tumors are also allowed to participate. Aim of the study Using single-cell analyses at different time points of treatment, we aim at studying the evolutionary trajectories of cancer and immune cells in 30 patients participating in the NEOLETRIB-trial. Methods Tumor biopsies taken before treatment (baseline) and during treatment (day 21 of ribociclib cycles 1 and 6, respectively), underwent single-cell RNA (scRNA), T cell receptor (TCR), and B cell receptor (BCR) sequencing. The sequencing data was first processed using cellRanger and Seurat . Results Initial clustering of the scRNA enabled the identification of the main cell types in breast tumors, which included T/NK cells, B cells, epithelial cells, cancer-associated fibroblasts, myeloid cells and endothelial cells. Focusing on epithelial cells, we were distinguished cancer cells from normal epithelial cells using the InferCNV algorithm. Further, by estimating the copy number events, we determined the genetic background of clones resistant or sensitive to combination therapies for each patient, revealing the dynamics of tumor heterogeneity evolution during treatment. Additionally, we examined CD4, CD8 and NK cell types, identified different cell states within these cell types and inferred pseudotime and immune cell type differentiation trajectories. ConclusionsWe identified the cell types present in tumor biopsies from the NEOLETRIB trial. We characterized the effects of therapies on the evolution of cancer and immune cells and gain insights into the factors influencing sensitivity and resistance to the combination of letrozole and ribociclib. Citation Format: Marie Fongård, Leonard Schmiester, Salim Ghannoum, Marius Bjørnstad, Tatjana Bosnjak, Signe Kleivbo, Knut Selsås, Stephanie Geisler, Kamilla Fjermeros, Sameer Bhargava, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Aino Vuoriluoto, Nam Thi Nguyen, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. Cancer and immune cells evolutionary trajectories under treatment with the aromatase inhibitor letrozole and the CDK4/6 inhibitor ribociclib [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-08.

Abstract PO4-02-01: Characterization of immune and malignant cell evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen positive breast cancer

Abstract Background Aromatase inhibitors (AIs) are commonly used in the management of estrogen receptor-positive (ER+) breast cancer, as they effectively reduce estrogen levels which inhibits tumor growth and recurrence. In postmenopausal women with ER+ breast cancer, aromatase inhibitors are increasingly used in selected patients as neoadjuvant therapy to reduce the tumor burden before surgery and to improve overall treatment outcomes. Clinical trial The NEOLETEXE trial aimed to treat postmenopausal patients with locally advanced breast cancer. It was a neoadjuvant, randomized, open-label, intra-patient, cross-over, single-center phase II clinical trial. Most patients presented large T3/T4 tumors and/or N2/N3 involvement. Patients were randomly assigned to receive neoadjuvant therapy with either letrozole (2.5 mg daily) or exemestane (25 mg daily) for 3 months followed by a cross-over to the alternative AI for another 3 months followed by surgery. A total of 102 patients were enrolled in the NEOLETEXE trial. Aim of the study We aim at using single-cell analyses at different time points of treatment, to characterize evolutionary trajectories of cancer and immune cells in 23 patients from the NEOLETEXE trial. Methods Tumor biopsies taken before treatment (baseline) at crossover (after 3 months) and end of neoadjuvant therapy (after 6 months), were analyzed by single-cell RNA (scRNA), T cell receptor (TCR), and B cell receptor (BCR) sequencing. The data were first processed using cellRanger and the Seurat package. Results The main cell types found in tumors were identified through clustering of the scRNA data. We then focused on the epithelial cells to first distinguish between the normal and malignant epithelial cells using the InferCNV algorithm. For each patient, the analysis of inferred copy number events allowed to elucidate the genetic background of clones resistant or sensitive to aromatase inhibitors. This analysis unveiled the evolutionary dynamics of tumor heterogeneity during treatment. We next examined CD4, CD8, NK cells, and macrophages, using RNA velocity and diffusion pseudotime using CellRank and scVelo to delineate the differentiation trajectories of immune cell types. Conclusions We created a detailed map that provides a high-resolution view of the diverse cell types present in tumor biopsies from the NEOLETEXE trial. We characterize the impact of therapies on the evolutionary dynamics of both cancer and immune cells. Our analyzes sought insights into the underlying factors that contribute to the sensitivity and resistance to aromatase inhibitors. Citation Format: Leonard Schmiester, Salim Ghannoum, Marie Fongård, Marius Bjørnstad, Knut Selsås, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Torben Lüders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Vessela Kristensen, Jürgen Geisler, Xavier Tekpli. Characterization of immune and malignant cell evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-02-01.

Abstract LB394: Tumor evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen receptor positive breast cancer

Abstract BackgroundAromatase inhibitors (AIs) are commonly used in the management of estrogen receptor-positive (ER+) breast cancer as they effectively reduce estrogen levels which inhibits tumor growth and recurrence. Aromatase inhibitors are increasingly used in selected patients as neoadjuvant therapy. Clinical trialThe NEOLETEXE is a neoadjuvant, randomized, open-label, intra-patient, cross-over, single-center phase II clinical trial, aimed to treat postmenopausal patients with locally advanced breast cancer. Most patients presented large T3/T4 tumors and/or N2/N3 involvement. Patients were randomly assigned to neoadjuvant therapy with either letrozole (2.5 mg daily) or exemestane (25 mg daily) for 3 months followed by a cross-over to the alternative AI for another 3 months followed by surgery. A total of 102 patients were enrolled in the NEOLETEXE trial. Aim of the studyWe aimed to compare clones derived from bulk exome sequencing with single-cell analyses at DNA and RNA level from the same tumors at different time points of treatment in order to characterize evolutionary trajectories of cancer and immune cells in 23 patients at scRNA level and 16 patients at scDNA level from the NEOLETEXE trial. MethodsTumor biopsies taken before treatment (baseline) at crossover (after 3 months) and end of neoadjuvant therapy (after 6 months), were analyzed by single-cell DNA (MissionBio) and scRNA (10xGenomics), T cell receptor (TCR), and B cell receptor (BCR) sequencing. Results We employed the The Mission Bio Tapestri Platform, which enables targeted scDNAseq of nuclei isolated from fresh frozen tumor tissue using microfluid droplet technology. A custom panel of 497 amplicons covering 528 sequence variants was designed to study treatment response selected by the following criteria: 1) Up to 10 mutations from each sublonal clusters defined by analysing 32 samples (3 time points) from 11 NeoLetExe patients with whole exome sequencing (WES) data prioritizing mutations in genes part of the Cancer Gene Census (COSMIC), 2) known mutations covering all chromosomes to enable CNV analysis and 3) “Hand-picked” mutations with significance for breast cancer (e.g. hotspot ESR1 mutations). We performed SNV and CNV analysis and reconstruction of mutational lineages with user developed tools. On average, 81% of the clusters defined by WES was validated by scDNAseq and copy number profiles matching between the two data sets. Fishplot with variants defining cell clusters in WES using data from scDNAseq show clonal development during treatment.The scRNA analyses revealed cell types were identified through clustering of the scRNA data. We focused on the epithelial cells to first distinguish between the normal and malignant epithelial cells using the InferCNV algorithm. We next examined CD4, CD8, NK cells, and macrophages, using RNA velocity and diffusion pseudotime using CellRank and scVelo to delineate the differentiation trajectories of immune cell types. Conclusions We created a detailed map that provides a high-resolution view of the diverse clones and cell types present in tumor biopsies from the NEOLETEXE trial. We characterize the impact of therapies on the evolutionary dynamics of both cancer and immune cells. Our analyzes sought insights into the underlying factors that contribute to the sensitivity and resistance to aromatase inhibitors. Citation Format: Vessela N. Kristensen, Grethe I. Alnaes, Patrik Vernhoff, Leonard Schmiester, Salim Ghannoum, Marie Fongaard, Paal M. Bjornstad, Knut Selsaas, Stephanie Geisler, Manouchehr Seyedzadeh, Unn-Cathrin Buvarp, Torben Luders, Diether Lambrechts, Marianne Lyngra, Arnoldo Frigessi, Xavier Tekpli, Jurgen Geilser. Tumor evolution upon treatment pressure with aromatase inhibitors in locally advanced estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB394.

Clinical Study of San Wei Sheng Huo Decoction in the Treatment of Refractory Chemotherapy-Induced Thrombocytopenia

To investigate the efficacy and safety of treating refractory chemotherapy-induced thrombocytopenia (RCIT) with San Wei Sheng Huo Decoction (SWSHD) as the main formula. A retrospective study was conducted and the data of RCIT patients treated with SWSHD as the main formula were collected. Changes in peripheral blood platelet (PLT) levels at different time points of treatment were examined and the significant effective rate (SER) and effective rate (ER) were analyzed. We measured the increase in peripheral blood PLT count before and after treatment, analyzed the differences in PLT count increase for different degrees of RCIT treatment, and evaluated the safety of the treatment. A total of 35 cases of RCIT were included in the study. With SWSHD as the main treatment formula, the 2-week ER and SER were 74.29% and 14.29%, respectively, the 2-month ER and SER were 84.38% and 60.50, respectively, and the 1-year ER and SER were 92.31% and 80.77%, respectively. PLT count increased at all time points after treatment compared with that before treatment ( P<0.01). Subgroup analysis showed that, 2 months after treatment started, peripheral blood PLT counts increased by as much as 51.02×10 9L －1 in the severe RCIT group, higher than that of the moderate RCIT group at 36.58×10 9L －1 ( P<0.05), and the difference persisted until 1 year after the treatment. No obvious traditional Chinese medicine-related adverse reaction was observed during the treatment. SWSHD takes effect rapidly and its effect is long-lasting and stable. Furthermore, SWSHD has a more significant effect on severe RCIT.

Cytogenetic effects of magnevist and methotrexate on mice bone marrow

This study was established to investigate the cytogenetic effects of magnevist on mice bone marrow before and after methotrexate (MTX) 2.5 mg/kg. The negative control group was treated with 0.2 ml distilled water. Magnevist was given alone to the mice intraperitoneally with 0.6 mg/kg for 24 hrs and 48 hrs. Similarly, MTX was only intravenously injected with 2.5 mg/kg for 24 hrs and 48 hrs, which were considered as positive controls. Moreover, different combinations of magnevist/ MTX were given in terms of different time points of treatment. Results showed high induction (p&lt;0.05) of chromosomal aberrations (CAs) and micronuclei (MN) and reduction in mitotic index (MI) of following treatments: magnevist, MTX and magnevist/MTX combination, compared to corresponding control groups. Interestingly, the combination magnevist and MTX treatment demonstrated higher induction in all biological end points than other groups. We concluded that the high combination effects may belong to increase in the aggressiveness of MTX that enhanced by magnevist and vice versa.

Artificial immune system features added to breast cancer clinical data for machine learning (ML) applications

We here propose a new method of combining a mathematical model that describes a chemotherapy treatment for breast cancer with a machine-learning (ML) algorithm to increase performance in predicting tumor size using a five-step procedure. The first step involves modeling the chemotherapy treatment protocol using an analytical function. In the second step, the ML algorithm is trained to predict the tumor size based on clinico-pathological data and data obtained from magnetic resonance imaging results at different time points of treatment. In the third step, the model is solved according to adjustments made at the individual patient level based on the initial tumor size. In the fourth step, the important variables are extracted from the mathematical model solutions and inserted as added features. In the final step, we applied various ML algorithms on the merged data. Performance comparison among algorithms showed that the root mean square error of the linear regression decreased with the addition of the mathematical results, and the accuracy of prediction as well as the F1-scores increased with the addition of the mathematical model to the neural network. We established these results for four different cohorts of women at different ages with breast cancer who received chemotherapy treatment.

Correlation of the tumor mutational burden with the composition of the immune cell subpopulations in peripheral blood of triple-negative breast cancer patients undergoing neoadjuvant therapy with durvalumab: Results from the prospectively randomized GeparNuevo trial.

588 Background: The GeparNuevo trial is a randomized, double-blind, multi-center phase II trial of neoadjuvant therapy in patients with early-stage triple negative breast cancer (TNBC) investigating the role of durvalumab, an anti-PD-L1 antibody, which blocks PD-L1 binding to PD1 and CD80, in addition to standard chemotherapy with nab-Paclitaxel (nab-Pac) followed by Epirubicin plus Cyclophosphamid (EC; Loibl S et al. ASCO 2018). Since the tumor mutational burden (TMB) has been suggested to be associated with a better outcome of patients undergoing immunotherapy and an increased T cell response, we determined whether there exists a link between TMB and immune cell composition, frequency and function in patients of the GeparNuevo trial. Methods: In order to determine possible predictive and / or prognostic biomarkers, tumor biopsies taken at recruitment from 149 patients out of the 174 enrolled patients underwent deep sequencing in order to determine the TMB. In addition, for 120 patients blood samples were taken at recruitment and during different time points of treatment (after durvalumab pre-treatment, after Nab-Pac and at surgery after EC) and evaluated using multicolor flow cytometry by monitoring the absolute cell counts of T cells, B cells and NK cells as well as the frequency, composition and functionality of different immune cell populations. Results: The TMB of the GeparNuevo cohort was in line with published data with a mean of 1.8 mutations/MB (range 0.02 – 7.65), respectively. Preliminary evaluation demonstrated a significant correlation of TMB with blood parameters, in particular with subsets of CD8+ T cells. Interestingly, the data suggest a negative correlation of TMB with the frequency of effector cells while a positive correlation exists with the effector memory cells at recruitment. In depth analyses of a correlation with treatment arm and clinical responses are currently performed. Conclusions: Using this approach we hope to identify biomarkers, which will allow a better selection of TNBC patients undergoing specific immunotherapies. Clinical trial information: NCT02685059.

Abnormal B-Cell Subset and Blimp-1-Mediated Humoral Responses Associated With Visceral Leishmaniasis Pathogenesis.

B-cells have a spectrum of functions ranging from antibody production to antigen presentation and have additional vital roles in immune mechanisms. There is rudimentary knowledge about the role of B-cells in intracellular infections with contradictory findings. We explored the role of B-cell dysfunctions in visceral leishmaniasis (VL) pathogenesis in terms of the phenotypic and functional properties of B-cells during the course of disease. This study was performed on blood and splenic aspirates (SA) of VL cases pre- and post-treatment. Whole blood was used for flow cytometric studies for determining the profiles of B-cells at different time-points of treatment. Peripheral blood mononuclear cells were used for magnetic purification of B-cells, for transcriptional studies by real-time polymerase chain reaction (RT-PCR). Serum/plasma was used for direct agglutination test for determining parasite-specific antibodies and SA were used for scoring the presence of parasite by microscopic examination. Flow cytometric studies depicted decreased B-cell percentages during the entire course of disease and attainment of exhaustive phenotype with tissue-like memory cell markers, indicative of B-cell dysfunctions in VL. In addition, B-cells had compromised abilities of antigen processing and presentation and altered levels of B-lymphocyte-induced maturation protein-1 (Blimp-1). Blimp-1 expression goes hand in hand with B-cell maturation antigen and transmembrane activator and calcium modulator (TACI) and cyclophilin ligand interactor, suggestive of its role in promoting plasma cell survival and antibody production. Elevated level of VL-specific antibody titre was directly correlated with exhausted phenotype and also with disease severity during VL. This study indicated for impaired B-cell functions during chronic infection which may lead to pathological consequences in human VL.

Induced PD-1 Expression on Bone Marrow CD34+ Cells from MDS Patients Treated with 5-Azacitadine in Combination with Nivolumab and/or Ipilimumab

Introduction PD-1 is a negative costimulatory receptor on activated T lymphocytes which counters the activation signal provided by T cell receptor ligation. The expression of PD-1 on T lymphocytes can be dynamically modulated by DNA methylation. A recent study in solid tumors (Kleffel S et al, Cell 2015) suggested that PD-1 was not only expressed by immune cells, but also by a subpopulation of cancer cells, and the cancer intrinsic PD-1 can promote tumorigenesis. Our previous study (Yang H et al, Leukemia 2014) demonstrated an increased PD-1 mRNA expression in peripheral blood mononuclear cells from MDS patients under hypomethylating agent (HMA) treatment, but little is known about detailed PD-1 expression in MDS. PD-1 signaling has been reported to be involved in MDS pathogenesis and resistance mechanisms to HMAs (Yang H et al, Leukemia 2014). Therefore, the PD-1 pathway represents an attractive target and anti-PD-1 monoclonal antibodies are being increasingly used to study MDS. Precise understanding of PD-1 expression in MDS patients may allow for effective treatment.Methods and Human Specimens With this purpose, using multicolor flow cytometry analysis, we studied the PD-1 expression on bone marrow Lin-CD34+ cells (Lineage cells: CD2, CD3, CD4, CD7, CD10, CD11b, CD14, CD19, CD20, CD33, CD56 and CD235a) from 51 patients enrolled in a clinical trial 2014-0930. Based on 5-azacytidine treatment, these patients can be divided into two groups: group A included 29 patients (57%) (22 MDS, 5 CMML, 1 MDS/MPD and 1 AML) which were treated with 5-azacitidine in combination with ipilimumab (N=10), nivolumab (N=16) or ipilimumab + nivolumab (N=2), one patient treated with 5-azacytidine only was also included; group B included 22 patients (43%) (16 MDS, 4 CMML and 2 MDS/MPD) which did not receive 5-azacytidine treatment and were treated with ipilimumab (N=12), nivolumab (N=5) or ipilimumab + nivolumab (N=5) alone. Positive PD-1 expression was considered when the median fluorescence intensity (MFI) ratio between sample and FMO negative control was more than 2.Results In group A, 26 patients (90%) were previously untreated at the time of enrollment. PD-1 expression was not observed in 8 untreated baseline samples collected. We then performed the analysis in samples under different time points of treatment. PD-1 expression was observed in 11 patients (38%, 9 responders, 1 stable disease and 1 non-responder) in at least one time point during treatment. Interestingly, 9 (82%) of them were treated with 5-azacytidine in combination with nivolumab, the other two were treated with 5-azacytidine in combination ipilimumab and ipilimumab + nivolumab separately. PD-1 expression was observed in 56% of the patients under 5-azacytidine in combination with nivolumab treatment. Figure 1 is an example of PD-1 expression in Lin-CD34+ cells from patient treated with 5-azacytidine in combination with nivolumab.In group B, all patients except one (95%) were previously treated at the time of enrollment. PD-1 expression was observed in 2 out of 13 baseline samples collected, and the PD-1 expression was not detected on these two patients after follow-up treatment with ipilimumab. PD-1 expression was observed in two patients (9%, 1 responder, 1 stable disease) in at least one time point during treatment (1 ipilimumab, 1 ipilimumab + nivolumab).Taken together, PD-1 expression was significantly induced in patients treated with 5-azacytidine when compare to patients without 5-azacytidine treatment (38% vs 9%, p=0.023), especially in the group of patients treated with 5-azacytidine in combination with nivolumab (56% vs 10%, p=0.037, compare with 5-azacytidine in combination with ipilimumab). Induction of PD-1 expression may contribute to the response of the treatment (77% vs 46%, p=0.058).Conclusion Our study demonstrates that induced expression of PD-1 on MDS Lin-CD34+ cells may contribute to the response of MDS patients to treatment with 5-azacytidine in combination with nivolumab. PD-1 methylation as well as other biological functions related to this treatment need to be further studied. [Display omitted] DisclosuresColla:Abbvie: Research Funding. Daver:Novartis: Research Funding; Incyte: Research Funding; Otsuka: Consultancy; Daiichi-Sankyo: Research Funding; Sunesis: Consultancy; ImmunoGen: Consultancy; Karyopharm: Consultancy; Alexion: Consultancy; Incyte: Consultancy; Kiromic: Research Funding; Pfizer: Consultancy; ARIAD: Research Funding; Pfizer: Research Funding; Novartis: Consultancy; BMS: Research Funding; Karyopharm: Research Funding; Sunesis: Research Funding.

Treatment for patient with chronic postherpetic neuralgia

Fifty postherpetic neuralgia (PHN) patients with a course ≥3 years and the Pain Numeric Rating scale (NRS)≥5 were included in the analysis. All patients were treated with nerve block and subcutaneous injection in the affected area. The paravertebral nerve blocks were performed by injecting 5 ml lidocaine in the concentrations of 0.3%; the concentration of lidocaine for branch blockage and peripheral nerves was 1.0%, and that for subcutaneous injection was 0.3%, respectively; 2 mg dexamethasone and 0.5 mg vitamin B12 were added in the injections. The NRS scores and quality of life scores were documented before and after the treatment. The NRS score was significantly decreased at different time points of treatment (F=279.6, P<0.01). At the end of the treatment and at 1 month, 3months, 6months of follow-up, the scores were significantly lower than those before the treatment. All patients showed enormous improvement in the quality of life at 1, 3 and 6 months after treatment. The pain relief was excellent or good in 38 cases. The efficacy of treatment is associated with skin scar and allodynia. The study indicates that nerve block and subcutaneous injection has a satisfactory efficacy in treatment of chronic postherpetic neuralgia. Key words: Neuralgia, postherpetic; Nerve block; Injections, subcutaneous

Abstract 2840: Characterization of pazopanib resistance in renal cell carcinoma cell lines

Abstract BACKGROUND: Together with Sunitinib (Sun), Pazopanib (Paz) is considered as a gold standard in metastatic renal cell carcinoma treatment (RCC). Similarly to other tyrosine kinase inhibitors (TKI), development of resistance constitutes the main problem of Paz-based therapy. This warrants the in vivo investigations of the processes related to Paz resistance development. METHODS: Parental human RCC cell lines, 786-O/wt and CAKI-2/wt, were continuously treated with Paz at IC50 concentration and Paz-resistant 786-O and CAKI-2 cell subpopulations (786-O/res and CAKI-2/res) were developed. Parental and resistant cells were compared in terms of the malignant phenotype by WST-1 proliferation, cross-resistance to another TKI, migration wound healing assays and signaling pathways analysis by Western blot. MiRNA expression was analyzed using microarrays (Agilent Technologies). RESULTS: The in vitro analysis of RCC cells growth under Paz treatment revealed that this drug inhibited proliferation of parental 786-O and CAKI-2 cells with IC50 (inhibitory concentration of 50%) of 14 and 11 µmol/L, respectively. The continuous treatment with this drug resulted in the gradual resistance development in both tested cell lines. At the end of the period of continuous treatment (32 passages, 3 months) the resistance level of 786-O/res and CAKI-2/res cells was increased 2-fold compared with 786-O/wt and CAKI-2/wt. The analysis of cell motility revealed that the short-termed exposure to Paz retarded the migration of both parental and Paz-resistant 786-O and CAKI-2 cells. However, no differences in the motility rates of parental and resistant cells were observed. The analysis of the signaling pathways that might be involved in the resistance development revealed no changes in the expression of epithelial-to-mesenchymal transition (EMT) markers N-cadherin, vimentin and β-catenin between parental and Paz-resistant subpopulations of both cell lines. Cell response to Paz treatment differs between the tested parental RCC cell lines CAKI-2 and 786-O in terms of the expression of phosphorylated forms of the growth-promoting molecules of Akt-mTOR and MAPK-ERK pathways. A significant change in miRNA expression was found in both cell lines at different time points of treatment compared to parenteral cells. CONCLUSIONS: Our data suggest that the process of Paz resistance development is cell line specific. Akt-mTOR and MAPK-ERK signaling pathways seem to be related to Paz resistance development in RCC. Development of resistance is associated with specific miRNA alterations. Further studies have to analyze the functional effects of miRNAs on resistance and possible signaling pathways. Citation Format: Bozhena Vynnytska-Myronovska, Joana Heinzelmann, Sebastian Hölters, Michael Stoeckle, Kerstin Junker. Characterization of pazopanib resistance in renal cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2840.

Efficacy and safety of fenofibrate add-on therapy for patients with primary biliary cholangitis and a suboptimal response to UDCA.

primary biliary cholangitis (PBC) patients with a suboptimal response to ursodeoxycholic acid (UDCA) have a significantly worse survival rate. Fenofibrate has been shown to improve the short-term biochemical response in this group of patients. However, there is limited data available on the safety and efficacy of its long-term use, especially in patients with cirrhosis. Methods：in this retrospective cohort study, fenofibrate was given to PBC patients with a suboptimal response to at least 12 months of UDCA (13-15 mg/kg/d) therapy. Biochemistry data, GLOBE score and UK-PBC risk score at baseline and at different time points of treatment were compared. The safety profiles were also compared between cirrhotic and non-cirrhotic patient groups. Results：fenofibrate (200 mg/day) was given to 39 PBC patients with a suboptimal response to UDCA (15 cirrhotic and 24 non-cirrhotic patients). In the 26 patients who completed more than one year of combination therapy, the alkaline phosphatase (ALP) levels were 215 (185, 326) U/l, 122 (110, 202) U/l, 128 (106, 194) U/l, 124 (100, 181) U/l and 120 (82, 168) U/l, at baseline, three months, six months, 12 months and 24 months, respectively. All p values were < 0.01 when compared to baseline values. After two years of combination therapy, the UK-PBC risk score and GLOBE score did not significantly improve. The overall rates of adverse events were not significantly different between the cirrhotic and non-cirrhotic group. The elevation of liver enzymes was the most frequent side effect (n = 7), leading to a discontinuation in four patients. Furthermore, after two years of combination therapy, the serum creatinine levels and estimated glomerular filtration rates (eGFR) were significantly worse in both groups. fenofibrate add-on therapy could improve ALP and γ-GT levels in both non-cirrhotic and cirrhotic PBC patients with a suboptimal response to UDCA. However, patients need to be monitored carefully for a potential liver injury and nephrotoxicity.

The role of antioxidants in attenuation of Caenorhabditis elegans lethality on exposure to TiO2 and ZnO nanoparticles

The exponential increase in the usage of engineered nanoparticles (ENPs) has raised global concerns due to their potential toxicity and environmental impacts. Nano-TiO2 and nano-ZnO have been extensively used in various applications. Thus, there is a need for determining the toxic potentials of ENPs as well as, to develop the possible attenuation method for ENPs toxicity. Both in the in vitro and in vivo systems, exposure to the majority of ENPs have shown Reactive Oxygen Species (ROS) generation, which leads to oxidative stress mediated inflammation, genotoxicity, and cytotoxicity. Hence, with the rationale of determining easy and economical protection against ENPs exposure, the amelioration effect of the antioxidants (curcumin and vitamin-C) against the nano-TiO2 and nano-ZnO induced ROS and lethality were investigated in Caenorhabditis elegans. We not only employed pre-treatment and along with treatment approach, but also determined the effect of antioxidants at different time points of treatment. Our study revealed that both the antioxidants efficiently ameliorate nanoparticles induced ROS as well as lethality in worms. Further, the pretreatment approach was more effective than the along with treatment. Therefore, our study indicates the possibility of evading the nanotoxicity by incorporating curcumin and vitamin-C in everyday diet.

Dysregulation of Autophagy Contributes to Anal Carcinogenesis.

IntroductionAutophagy is an intracellular catabolic process that removes and recycles unnecessary/dysfunctional cellular components, contributing to cellular health and survival. Autophagy is a highly regulated cellular process that responds to several intracellular signals, many of which are deregulated by human papillomavirus (HPV) infection through the expression of HPV-encoded oncoproteins. This adaptive inhibitory response helps prevent viral clearance. A strong correlation remains between HPV infection and the development of squamous cell carcinoma (SCC) of the anus, particularly in HIV positive and other immunosuppressed patients. We hypothesize that autophagy is inhibited by HPV–encoded oncoproteins thereby promoting anal carcinogenesis (Fig 1).Materials and MethodsHPV16 transgenic mice (K14E6/E7) and non-transgenic mice (FVB/N), both of which do not spontaneously develop anal tumors, were treated topically with the chemical carcinogen, 7,12-Dimethylbenz[a]anthracene (DMBA), to induce anal cancer. The anuses at different time points of treatment (5, 10, 15 and 20 weeks) were analyzed using immunofluorescence (IF) for two key autophagy marker proteins (LC3β and p62) in addition to histological grading. The anuses from the K14E6/E7 mice were also analyzed for visual evidence of autophagic activity by electron microscopy (EM). To see if there was a correlation to humans, archival anal specimens were assessed histologically for grade of dysplasia and then analyzed for LC3β and p62 protein content. To more directly examine the effect of autophagic inhibition on anal carcinogenesis, nontransgenic mice that do not develop anal cancer with DMBA treatment were treated with a known pharmacologic inhibitor of autophagy, chloroquine, and examined for tumor development and analyzed by IF for autophagic proteins.ResultsHistologically, we observed the progression of normal anoderm to invasive SCC with DMBA treatment in K14E6/E7 mice but not in nontransgenic, syngeneic FVB/N background control mice. With the development of low-grade dysplasia in the K14E6/E7 mice, there was an increase in both punctate LC3β and p62 expression while EM revealed increased autophagosomes without evidence of autophagolysosomes. These observations are consistent with autophagy being inhibited at a later stage in the autophagic process. In contrast, in high-grade dysplasia and SCC in the DMBA-treated K14E6/E7 mice, there were decreased levels of p62 with a continued increase in punctate LC3β expression by IF, while autophagolysosomes were seen on EM, consistent with the process of autophagy proceeded to completion. Similar findings, including histological grade dependent changes in LC3β and p62 expression, were noted with human samples upon analysis of IF. Finally, with pharmacologic inhibition of autophagy in DMBA-treated, nontrangenic FVB/N mice, there was a significant increase in anal cancer development similar to that observed in DMBA- treated K14E6/E7 mice.ConclusionAutophagic dysregulation is noted early on in HPV-associated anal carcinogenesis (low-grade dysplasia), with normalization of the autophagic process arising in late stages of HPV-associated anal carcinogenesis (high-grade dysplasia and invasive carcinoma).

The Experience of Extended Bowel Resection in Individuals With a High Metachronous Colorectal Cancer Risk: A Qualitative Study.

To ascertain individual experiences of extended bowel resection as treatment for colorectal cancer (CRC) in those with a high metachronous CRC risk, including the self-reported adequacy of information received at different time points of treatment and recovery. . Qualitative. . Participants were recruited through the Australasian Colorectal Cancer Family Registry and two hospitals in Melbourne, Australia. . 18 individuals with a high metachronous CRC risk who had an extended bowel resection from 6-12 months ago. . Semistructured interviews. Data were analyzed thematically. . In most cases, the treating surgeon decided on the best option regarding surgical treatment. Participants felt well informed about the surgical procedure. Information related to surgical outcomes, recovery, and lifestyle adjustment from surgery was not always adequate. Many participants described ongoing worry about developing another cancer. . Patients undergoing an extended resection to reduce metachronous CRC risk require detailed information delivered at more than one time point and relating to several different aspects of the surgical procedure and its outcomes. . An increased emphasis should be given to the provision of patient information on surgical outcomes, recovery, and lifestyle adjustment. Colorectal nurses could provide support for some of the reported unmet needs.

Molecular cloning and expression analysis of WRKY transcription factor genes in Salvia miltiorrhiza.

BackgroundWRKY proteins comprise a large family of transcription factors and play important regulatory roles in plant development and defense response. The WRKY gene family in Salvia miltiorrhiza has not been characterized.ResultsA total of 61 SmWRKYs were cloned from S. miltiorrhiza. Multiple sequence alignment showed that SmWRKYs could be classified into 3 groups and 8 subgroups. Sequence features, the WRKY domain and other motifs of SmWRKYs are largely conserved with Arabidopsis AtWRKYs. Each group of WRKY domains contains characteristic conserved sequences, and group-specific motifs might attribute to functional divergence of WRKYs. A total of 17 pairs of orthologous SmWRKY and AtWRKY genes and 21 pairs of paralogous SmWRKY genes were identified. Maximum likelihood analysis showed that SmWRKYs had undergone strong selective pressure for adaptive evolution. Functional divergence analysis suggested that the SmWRKY subgroup genes and many paralogous SmWRKY gene pairs were divergent in functions. Various critical amino acids contributed to functional divergence among subgroups were detected. Of the 61 SmWRKYs, 22, 13, 4 and 1 were predominantly expressed in roots, stems, leaves, and flowers, respectively. The other 21 were mainly expressed in at least two tissues analyzed. In S. miltiorrhiza roots treated with MeJA, significant changes of gene expression were observed for 49 SmWRKYs, of which 26 were up-regulated, 18 were down-regulated, while the other 5 were either up-regulated or down-regulated at different time-points of treatment. Analysis of published RNA-seq data showed that 42 of the 61 identified SmWRKYs were yeast extract and Ag+-responsive. Through a systematic analysis, SmWRKYs potentially involved in tanshinone biosynthesis were predicted.ConclusionThese results provide insights into functional conservation and diversification of SmWRKYs and are useful information for further elucidating SmWRKY functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1411-x) contains supplementary material, which is available to authorized users.

Degree of Patient Pain, Complications, and Satisfaction after Root Canal Treatment or a Single Implant: A Preliminary Prospective Investigation

IntroductionDentists often face the choice between tooth retention with root canal treatment and tooth replacement with implant treatment. To date, there has not been a prospective clinical trial directly comparing nonsurgical root canal treatment and single delayed implant therapy with regard to the degree of preoperative and postoperative pain, complications, and patient satisfaction. MethodsTwenty-four patients had initial nonsurgical root canal treatment, and another 24 had single implant treatment in healed sites. Questionnaires were given at pretreatment, 7 days, 3 months, 6 months, and 12 months. ResultsAll patients completed the 12-month follow-up period. No significant difference in pain, complications, or overall satisfaction was noted between the 2 groups at any of the time points (P > .05). However, there were differences within each group between the time points. There was more pain at pretreatment for root canal treatment and 7-day post-treatment point for single implant treatment than any other time point. For complications, more were reported at the 7-day post-treatment point than any other time point for both groups. With overall satisfaction, there was no difference from any time point for either group. ConclusionsThe results of this study suggest that patients perceive both treatments with high degrees of satisfaction with minimal pain and discomfort. Differences were found at different time points of treatment and were related to the nature of the treatment itself. This information is useful to help patients with treatment decisions.

Hyperlipidemia and Hypothyroidism Among Metastatic Renal Cell Carcinoma Patients Taking Sunitinib Malate. Related or Unrelated Adverse Events?

BackgroundIn recent years, new-onset hypothyroidism was extensively reported in patients receiving sunitinib for malignancy. Effects of sunitinib on serum lipids are not described, however a hyperlipidemic state is commonly observed in hypothyroid patients. Here we report about the incidence and severity of hypercholesterolemia and hypertriglyceridemia in a cohort of patients receiving sunitinib for metastatic renal cell carcinoma. Patients and MethodsThyroid function tests, serum triglycerides, and cholesterol were prospectively evaluated in 39 consecutive metastatic renal cell carcinoma patients, who were receiving sunitinib as a first-line treatment. Incidence of hyperlipidemia, thyroid function impairment, and their possible relationship were investigated. ResultsThyroid function tests, serum cholesterol, and triglycerides were assessed at baseline and before the beginning of each sunitinib cycle. During treatment, median triglyceride levels increased up to 271.3 mg/dL, and median cholesterol increased up to 234.7 mg/dL (+113% and +22%, respectively). A hyperlipidemic state developed in 27 patients (69.2%) within a mean time of 1.8 six-week cycles (range, 1-5 cycles) and persisted during treatment. Hypothyroidism was observed in 20 patients (51.2%) and usually developed within 2.3 cycles. Because hypothyroidism and hyperlipidemia developed at different time points of treatment and among different patients, our results failed to demonstrate a correlation between these adverse events. ConclusionNew-onset hyperlipidemia was observed in an increased percentage of patients taking sunitinib. The mechanism of this side effect is still unclear. We recommend careful monitoring of serum lipid levels during sunitinib administration to recognize possible consequences, especially on cardiovascular health.

Ptprg and BCR/ABL1 Expression In CML Patients At Diagnosis and Upon TKI Treatment: Preliminary Results

BackgroundChronic Myeloid Leukemia (CML), is a biologically and clinically heterogeneous disease that originates in a pluripotent bone marrow stem cell. The hallmark of CML is the presence of an aberrant gene, BCR-ABL1, which encodes for a constitutively activated tyrosine kinase. Protein tyrosine phosphatases can negatively regulate ABL1 mediated signaling by dephosphorylating the kinase and/or its substrates and impaired phosphatase activity has been linked to several solid cancers and leukemias. We have previously reported the role of the receptor-type phosphatase PTPRG as tumor suppressor gene and demonstrated a significant decrease in the PTPRGexpression level in CML.Here we have investigated the expression of PTPRG gene in 32 CML patients aiming at the evaluation of the clinical relevance of the PTPRGdown regulation. MethodsAll 32 CML patients included in this study were diagnosed in chronic phase. Thirteen untreated patients diagnosed with philadelphia-negative myelod disorders were included in the study as control group. The study was approved by the Local Ethics Committee, and informed consent in accordance with declaration of Helsinki was obtained from each patient. The expression level of PTPRG gene was evaluated by a sybr green real-time RT-PCR assay in 2 samples from each patient, taken at diagnosis and following TKI treatment. The beta-Actin (ACTB) housekeeping gene was used for normalization. Results were validated using predesigned TaqMan quantitative RT-PCR assays for PTPRG (intracellular domain) and ABL1 genes. Quantitative RT-PCR was used to quantify BCR-ABL1 gene fusion transcripts according to the European Leukemia Net guidelines. Statistical analysis and comparisons were performed by Mann-Whitney's and Student paired T tests using the SPSS software. ResultsThe mean levels of PTPRG transcript, expressed as PTPRG/ACTB ratio, were significantly lower in CML samples at diagnosis compared to the non-CML control group (0,44%, range 0-0,37 vs 6,29%, range 0,09-52; p=0.020). Recovery of PTPRG mRNA expression at variable levels, ranging from 0.17 to 30%, was detected in 29/32 follow up samples, taken at different time points of treatment (from 3 months to 10 years). Differences in PTPRG gene expression levels between CML samples at diagnosis and after treatment were statistically significant (p=0,027). No statistically significant correlation was observed between PTPRG/ACTB and BCR-ABL/ABL1 ratios nor with deepness of molecular response or SOKAL and EUTOS scores, even though 2 of the 3 patients showing higher level of PTPRGmRNA at diagnosis than in the follow up sample showed resistance to TKI treatment. ConclusionsIn this study we confirm a down regulation of PTPRG in a high percentage of CML patients and show that its expression is restored upon treatment with TKIs. Deregulated expression of PTPRG phosphatase underline its role as a tumor suppressor gene in CML and highlights its potential use as a new bio-marker of disease potentially usable in association with BCR/ABL1, allowing for a better understanding of the molecular mechanism underlying the development of CML, and propose possible new avenues for therapeutic treatment. Disclosures:Yassin:qatar national research fund: Patents & Royalties, Research Funding.

Clinical and Patient-Reported Outcomes of Dasatinib Therapy in Patients with Imatinib-Resistant or -Intolerant Chronic Myeloid Leukemia

AbstractAbstract 4445Despite remarkable responses with imatinib in chronic phase chronic myeloid leukemia (CML-CP), there remains a proportion of patients who are resistant or intolerant to imatinib treatment. Dasatinib is effective second-line treatment for this patient population. Importantly, without effective therapeutic intervention, these patients inevitably progress to advanced phases of disease, have a short survival and declined health outcomes. This emphasizes the importance of patient-reported outcome assessment in these patients. Comprehensive information about the results of dasatinib therapy both in terms of clinical and patient-reported outcomes will be helpful in evaluation of risks/benefits of treatment in this patient population. We aimed to study response rates as well as quality of life (QoL) parameters and symptom profile in imatinib-resistant or -intolerant CML-CP patients receiving dasatinib.38 CML-CP patients resistant or -intolerant to imatinib were enrolled in the study (mean age - 50 years old, SD 14.5; range −22–79 years; male/female – 19/19). The median of disease duration was 5.7 years (1–13 years). The median duration of imatinib treatment - 39 months (5–101 months). 33 patients had resistance to imatinib treatment; 5 patients were intolerant to imatinib. Patients received dasatinib in the dose of 100 mg daily. Median follow-up was 12 months. For QoL assessment patients filled out the SF-36 and for symptom assessment – Comprehensive Symptom Profile in Chronic Myeloid Leukemia Patients (CSP Leuk-CML). The CSP Leuk-CML is developed to assess profile of 47 symptoms specific for patients with CML. To compare patient population with normative data the sample from population norm (PN) database adjusted to age and gender was used. For comparisons Mann-Whitney test was used. Symptom severity and percentages of patients with symptoms at moderate-to-severe (ratings3 5) levels was evaluated.High rates of hematologic (complete, 90%) and cytogenetic (major, 85%; complete, 35%) response were observed for the majority of patients at 6 months from the start of therapy. During observation period two patients died (at 2 and 6 months after therapy initiation), one patient discontinued treatment due to significant thrombocytopenia at 2 months, and one patient discontinued treatment due to disease progression at 6 months after therapy initiation. At base-line patients experienced impaired QoL as compared to population norms: the values for the majority of SF-36 scales were significantly lower than in control group (p<0.05). No QoL impairment was observed in 35% of patients. 25% of patients had either mild (Integral QoL index <25% decrease from a PN) or moderate (25–50% decrease from a PN) QoL impairment; other 20% of patients - severe QoL impairment (50–75% decrease from a PN), and 20% of patients - critical QoL impairment (>75% decrease from a PN). At base-line the majority of patients (96%) experienced fatigue; a half of them suffered from moderate-to-severe fatigue. 75% of patients experienced at least one moderate-to-severe symptom; more than 40% had more than 7 moderate-to-severe symptoms. At 12 months after dasatinib treatment 47% of patients experienced no QoL impairment; the number of patients with severe or critical QoL impairment slightly decreased (25% and 10%, respectively). At 12 months after the start of therapy QoL treatment response in terms of stabilization or improvement was registered in the majority of patients (81%). In nearly all patients symptom severity did not change or became lower at different time-points of treatment as compared with base-line. The number of patients with moderate-to-severe symptoms decreased while treatment.Thus, treatment with dasatinib is beneficial for imatinib-resistant or -intolerant CML-CP patients both in terms of clinical and patient-reported outcomes. Desirable clinical and patient-reported outcomes were registered for the majority of patients. Comprehensive evaluation of the results of second-line treatment of CML-CP allows to assess the risks and benefits of therapy both from physician's and patient's perspective. Disclosures:No relevant conflicts of interest to declare.