Early changes in gene expression profiles in AML patients during induction chemotherapy.

BACKGROUND: Elucidation of the genetic mechanisms underlying treatment response to standard induction chemotherapy in AML patients is warranted, in order to aid in risk-adapted treatment decisions as novel treatments are emerging. In this pilot study, we explored the treatment-induced expression patterns in a small cohort of AML patients by analyzing differential gene expression (DGE) over the first 2 days of induction chemotherapy. METHODS: Blood samples were collected from ten AML patients at baseline (before treatment initiation) and during the first 2 days of treatment (Day 1; approximately 24 h, and Day 2; approximately 48 h after treatment initiation, respectively) and RNA was extracted for subsequent RNA sequencing. DGE between time points were assessed by pairwise analysis using the R package edgeR version 3.18.1 in all patients as well as in relation to treatment response (complete remission, CR, vs non-complete remission, nCR). Ingenuity Pathway Analysis (Qiagen) software was used for pathway analysis and visualization. RESULTS: After initial data quality control, two patients were excluded from further analysis, resulting in a final cohort of eight patients with data from all three timepoints. DGE analysis demonstrated activation of pathways with genes directly or indirectly associated with NF-κB signaling. Significant activation of the NF-κB pathway was seen in 50% of the patients 2 days after treatment start, while iNOS pathway effects could be identified already after 1 day. nCR patients displayed activation of pathways associated with cell cycle progression, oncogenesis and anti-apoptotic behavior, including the STAT3 pathway and Salvage pathways of pyrimidine ribonucleotides. Notably, a significant induction of cytidine deaminase, an enzyme responsible for the deamination of Ara-C, could be observed between baseline and Day 2 in the nCR patients but not in patients achieving CR. CONCLUSIONS: In conclusion, we show that time-course analysis of gene expression represents a feasible approach to identify relevant pathways affected by standard induction chemotherapy in AML patients. This poses as a potential method for elucidating new drug targets and biomarkers for categorizing disease aggressiveness and evaluating treatment response. However, more studies on larger cohorts are warranted to elucidate the transcriptional basis for drug response.

The association of four genetic variants with myelosuppression in gemcitabine-treated Japanese is not evident in gemcitabine/carboplatin-treated Swedes.

Gemcitabine/carboplatin-induced myelosuppressive adverse drug reactions (ADRs) are clinical problems leading to patient suffering and dose alterations. There is a need for personalised medicine to improve treatment effects and patients' well-being. We tested four genetic variants, rs11141915, rs1901440, rs12046844 and rs11719165, previously suggested as potential biomarkers for gemcitabine-induced leukopenia/neutropenia in Japanese patients, in 213 Swedish gemcitabine/carboplatin-treated non-small cell lung cancer (NSCLC) patients. DNA was genotyped using TaqMan probes and real-time PCR. The relationships between the risk alleles and low toxicity (non-ADR: Common Terminology Criteria for Adverse Events [CTCAE] grades 0) or high toxicity (ADR: CTCAE grades 3-4) of platelets, leukocytes and neutrophils were evaluated using Fisher's exact test. The risk alleles did not correlate with myelosuppression, and the strongest borderline significance (not withstanding adjustment for multiple testing) was for rs1901440 (neutropenia, p = 0.043) and rs11719165 (leukopenia, p = 0.049) where the risk alleles trended towards lower toxicity, contrasting with previous study findings. Risk alleles and higher risk scores were more common among our patients. We conclude that the genetic variants do not apply to Swedish patients treated with gemcitabine/carboplatin. However, they can still be important in other populations and cohorts, especially in a gemcitabine monotherapy setting, where the causal genetic variation might influence myelosuppressive ADRs.

Activation of the µ-opioid receptor by alicyclic fentanyls: Changes from high potency full agonists to low potency partial agonists with increasing alicyclic substructure.

Fentanyl analogs represent an important group of new psychoactive substances and knowing their efficacy and potency might assist in interpreting observed concentrations. The potency of fentanyl analogs can be estimated from in vitro studies and can be used to establish structure-activity relationships. In this study, recombinant CHO-K1 cells (AequoScreen) expressing the human µ-opioid receptor were used to establish dose-response curves via luminescent analysis for cyclopropyl-, cyclobutyl-, cyclopentyl-, cyclohexyl-, and 2,2,3,3-tetramethylcyclopropylfentanyl (TMCPF), on three separate occasions, using eight different concentrations in an eight-fold serial dilution in triplicates starting at ~60 µM. Fentanyl was used as a full agonist reference while morphine and buprenorphine were included for comparison. Cyclopropylfentanyl (EC50 = 4.3 nM), cyclobutylfentanyl (EC50 = 6.2 nM), and cyclopentylfentanyl (EC50 = 13 nM) were full agonists slightly less potent than fentanyl (EC50 = 1.7 nM). Cyclohexylfentanyl (EC50 = 3.1 µM, efficacy 48%) and TMCPF (EC50 = 1.5 µM, efficacy 65%) were partial agonists less potent than morphine (EC50 = 430 nM). Based on the results, cyclopropyl-, cyclobutyl-, and cyclopentylfentanyl would be expected to induce intoxication or cause fatal poisonings at similar concentrations to fentanyl, while the toxic or fatal concentrations of cyclohexylfentanyl and TMCPF would be expected to be much higher.

Single-Cell RNA Sequencing of Hematopoietic Stem and Progenitor Cells Treated with Gemcitabine and Carboplatin.

Treatments that include gemcitabine and carboplatin induce dose-limiting myelosuppression. The understanding of how human bone marrow is affected on a transcriptional level leading to the development of myelosuppression is required for the implementation of personalized treatments in the future. In this study, we treated human hematopoietic stem and progenitor cells (HSPCs) harvested from a patient with chronic myelogenous leukemia (CML) with gemcitabine/carboplatin. Thereafter, scRNA-seq was performed to distinguish transcriptional effects induced by gemcitabine/carboplatin. Gene expression was calculated and evaluated among cells within and between samples compared to untreated cells. Cell cycle analysis showed that the treatments effectively decrease cell proliferation, indicated by the proportion of cells in the G2M-phase dropping from 35% in untreated cells to 14.3% in treated cells. Clustering and t-SNE showed that cells within samples and between treated and untreated samples were affected differently. Enrichment analysis of differentially expressed genes showed that the treatments influence KEGG pathways and Gene Ontologies related to myeloid cell proliferation/differentiation, immune response, cancer, and the cell cycle. The present study shows the feasibility of using scRNA-seq and chemotherapy-treated HSPCs to find genes, pathways, and biological processes affected among and between treated and untreated cells. This indicates the possible gains of using single-cell toxicity studies for personalized medicine.

ABCB1 single-nucleotide variants and survival in patients with glioblastoma treated with radiotherapy concomitant with temozolomide.

Standard treatment for glioblastoma (GBM) patients is surgery and radiochemotherapy (RCT) with temozolomide (TMZ). TMZ is a substrate for ABCB1, a transmembrane drug transporter. It has been suggested that survival for GBM patients receiving TMZ is influenced by different single-nucleotide variants (SNV) of ABCB1. We therefore examined SNV:s of ABCB1, namely 1199G>A, 1236C>T, 2677G>T/A, and 3435C>T and correlated to survival for GBM patients receiving RCT. In a pilot cohort (97 patients) a significant correlation to survival was found for SNV 1199G>A, with median OS for variant G/G patients being 18.2 months versus 11.5 months for A/G (p = 0.012). We found no correlation to survival for the other SNV:s. We then expanded the cohort to 179 patients (expanded cohort) and also included a confirmatory cohort (49 patients) focusing on SNV 1199G>A. Median OS for G/G versus A/G plus A/A was 15.7 and 11.5 months, respectively (p = 0.085) for the expanded cohort and 13.8 versus 16.8 months (p = 0.19) for the confirmatory. In conclusion, in patients with GBM receiving RCT with TMZ, no correlation with survival was found for the SNV:s 1236C>T, 2677G>T/A, and 3435C>T of ABCB1. Although the SNV 1199G>A might have some impact, a clinically significant role could not be confirmed.

Dynamics of vitellogenin mRNA expression during vitellogenesis in the banana shrimp Penaeus (Fenneropenaeus) merguiensis using real-time PCR.

An open reading frame (ORF) of vitellogenin (Vg) cDNA was amplified from the ovaries of the banana shrimp, Penaeus merguiensis. An examination of Vg-deduced amino acid sequence revealed the presence of cleavage sites at a consensus motif for subtilisin-like endoproteases prior to the N-terminal sequences of purified vitellin (Vt) subunits. A comparison of the primary structures of Vg molecules in decapod crustacean species revealed the existence of a common characteristic structure, and phylogenetic analysis reflected the current taxonomic classifications of crustaceans. A PCR product of 1.1 kb encoding the 3'-end of Vg cDNA was cloned from the hepatopancreas. Although its sequence was almost identical to that of the same region of the ovarian Vg, with only 18 nucleotide differences, analysis suggests that they have been subjected to natural selection, indicating that there may be two different, tissue-specific Vg genes in P. merguiensis. This is consistent with the different expression patterns of Vg mRNA, as determined by real-time PCR. Vg mRNA levels were maintained at low levels during the previtellogenic stage and they increased as vitellogenesis progressed to reach a peak at the early vitellogenic stage in the ovary or at the vitellogenic stage in the hepatopancreas, and thereafter, levels decreased. Expression of Vg mRNA was much higher in the ovary compared to the hepatopancreas at all stages of ovarian development, implying that the ovary is mainly responsible for Vt synthesis. These indicate that penaeids constitute a unique model for vitellogenesis, showing intraovarian gene expression and synthesis of yolk protein.