Use of germline genetic variability for prediction of chemoresistance and prognosis of breast cancer patients

The aim of our study was to set up a panel for targeted sequencing of chemoresistance genes and the main transcription factors driving their expression and to evaluate their predictive and prognostic value in breast cancer patients. Coding and regulatory regions of 509 genes, selected from PharmGKB and Phenopedia, were sequenced using massive parallel sequencing in blood DNA from 105 breast cancer patients in the testing phase. In total, 18,245 variants were identified of which 2565 were novel variants (without rs number in dbSNP build 150) in the testing phase. Variants with major allele frequency over 0.05 were further prioritized for validation phase based on a newly developed decision tree. Using emerging in silico tools and pharmacogenomic databases for functional predictions and associations with response to cytotoxic therapy or disease-free survival of patients, 55 putative variants were identified and used for validation in 805 patients with clinical follow up using KASP™ technology. In conclusion, associations of rs2227291, rs2293194, and rs4376673 (located in ATP7A, KCNAB1, and DFFB genes, respectively) with response to neoadjuvant cytotoxic therapy and rs1801160 in DPYD with disease-free survival of patients treated with cytotoxic drugs were validated and should be further functionally characterized. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.Czech Medical Council [15-25618A]; Charles University [GAUK 1776218

Effect of an Echinacea-Based Hot Drink Versus Oseltamivir in Influenza Treatment: A Randomized, Double-Blind, Double-Dummy, Multicenter, Noninferiority Clinical Trial

Background: Echinacea has antiviral activity against influenza viruses in vitro and has traditionally been used for treatment of colds and flu. Objectives: This randomized, double-blind, double-dummy, multicenter, controlled clinical trial compared a new echinacea formulation with the neuraminidase inhibitor oseltamivir, the gold standard treatment for influenza. Methods: Following informed consent, 473 patients with early influenza symptoms (≤48 hours) were recruited in primary care in the Czech Republic and randomized to either 5 days of oseltamivir followed by 5 days of placebo, or 10 days of an Echinacea purpurea-based formulation called Echinaforce Hotdrink (A. Vogel Bioforce AG, Roggwil, Switzerland). The proportion of recovered patients (influenza symptoms rated as absent or mild in the evening) was analyzed for noninferiority between treatment groups using a generalized Wilcoxon test with significance level α = 0.05 (2-sided) and using a CI approach in the per-protocol sample. Results: Recovery from illness was comparable in the 2 treatment groups at 1.5% versus 4.1% after 1 day, 50.2% versus 48.8% after 5 days, and 90.1% versus 84.8% after 10 days of treatment with Echinaforce Hotdrink and oseltamivir, respectively. Noninferiority was demonstrated for each day and overall (95% CI, 0.487–0.5265 by generalized Wilcoxon test). Very similar results were obtained in the group with virologically confirmed influenza virus infections and in a retrospective analysis during the peak influenza period. The incidence of complications was lower with Echinaforce Hotdrink than with oseltamivir (2.46% vs 6.45%; P = 0.076) and fewer adverse events (particularly nausea and vomiting) were observed with Echinaforce Hotdrink. Conclusions: Echinaforce Hotdrink is as effective as oseltamivir in the early treatment of clinically diagnosed and virologically confirmed influenza virus infections with a reduced risk of complications and adverse events. It appears to be an attractive treatment option, particularly suitable for self-care. Clinical trial identifier: Eudra-CT: 2010-021571-88. (Curr Ther Res Clin Exp. 2015; 77:66–72

Ideal marker for targeted axillary dissection (IMTAD): a prospective multicentre trial

Abstract Background Targeted axillary dissection (TAD) is an established method for axillary staging in patients with breast cancer after neoadjuvant chemotherapy (NAC). TAD consists of sentinel lymph node biopsy and initially pathological lymph node excision, which must be marked by a reliable marker before NAC. Methods The IMTAD study is a prospective multicentre trial comparing three localisation markers for lymph node localisation (clip + iodine seed, magnetic seed, carbon suspension) facilitating subsequent surgical excision in the form of TAD. The primary outcome was to prospectively compare the reliability, accuracy, and safety according to complication rate during marker implantation and detection and marker dislodgement. Results One hundred eighty-nine patients were included in the study—in 135 patients clip + iodine seed was used, in 30 patients magnetic seed and in 24 patients carbon suspension. The complication rate during the marker implantation and detection were not statistically significant between individual markers (p = 0.263; p = 0.117). Marker dislodgement was reported in 4 patients with clip + iodine seed localisation (3.0%), dislodgement did not occur in other localisation methods (p = 0.999). The false-negativity of sentinel lymph node (SLN) was observed in 8 patients, the false-negativity of targeted lymph nodes (TLN) wasn´t observed at all, the false-negativity rate (FNR) from the subcohort of ypN + patients for SLN is 9.6% and for TLN 0.0%. Conclusion The IMTAD study indicated, that clip + iodine seed, magnetic seed and carbon suspension are statistically comparable in terms of complications during marker implantation and detection and marker dislodgement proving their safety, accuracy, and reliability in TAD. The study confirmed, that the FNR of the TLN was lower than the FNR of the SLN proving that the TLN is a better marker for axillary lymph node status after NAC. Trial registration NCT04580251. Name of registry: Clinicaltrials.gov. Date of registration: 8.10.2020

SLC46A1 haplotype with predicted functional impact has prognostic value in breast carcinoma

Background and Objective: Membrane solute carrier transporters play an important role in the transport of a wide spectrum of substrates including anticancer drugs and cancer-related physiological substrates. This study aimed to assess the prognostic relevance of gene expression and genetic variability of selected solute carrier transporters in breast cancer. Methods: Gene expression was determined by quantitative real-time polymerase chain reaction. All SLC46A1 and SLCO1A2 exons and surrounding non-coding sequences in DNA extracted from the blood of patients with breast cancer (exploratory phase) were analyzed by next-generation sequencing technology. Common variants (minor allele frequency ≥ 5%) with in silico-predicted functional relevance were further analyzed in a large cohort of patients with breast cancer (n = 815) and their prognostic and predictive potential was estimated (validation phase). Results: A gene expression and bioinformatics analysis suggested SLC46A1 and SLCO1A2 to play a putative role in the prognosis of patients with breast cancer. In total, 135 genetic variants (20 novel) were identified in both genes in the exploratory phase. Of these variants, 130 were non-coding, three missense, and two synonymous. One common variant in SLCO1A2 and four variants in SLC46A1 were predicted to be pathogenic by in silico programs and subsequently validated. A SLC46A1 haplotype block composed of rs2239911-rs2239910-rs8079943 was significantly associated with ERBB2/HER2 status and disease-free survival of hormonally treated patients. Conclusions: This study revealed the prognostic value of a SLC46A1 haplotype block for breast cancer that should be further studied. © 2020, The Author(s), under exclusive licence to Springer Nature Switzerland AG part of Springer Nature.Ministry of Health of the Czech RepublicMinistry of Health, Czech Republic [17-28470A]; National Center of Medical Genomics [CZ.02.1.01/0.0/0.0/16_ 013/0001634]; Czech Ministry of Education, Youth and Sports INTERCOST [LTC19020]; Charles University project "Center of clinical and experimental liver surgery" [UNCE/MED/006]CZ.02.1.01/0.0/0.0/16_013/0001634; Univerzita Karlova v Praze, UK: UNCE/MED/006; Grantová Agentura, Univerzita Karlova, GA, UK; Ministerstvo Školství, Mládeže a Tělovýchovy, MŠMT: LTC19020; Ministerstvo Zdravotnictví Ceské Republiky, MZCR: 17-28470

Non-Coding Polymorphisms in Nucleotide Binding Domain 1 in <i>ABCC1</i> Gene Associate with Transcript Level and Survival of Patients with Breast Cancer

<div><p>Objectives</p><p>ATP-Binding Cassette (ABC) transporters may cause treatment failure by transporting of anticancer drugs outside of the tumor cells. Multidrug resistance-associated protein 1 coded by the <i>ABCC1</i> gene has recently been suggested as a potential prognostic marker in breast cancer patients. This study aimed to explore tagged haplotype covering nucleotide binding domain 1 of <i>ABCC1</i> in relation with corresponding transcript levels in tissues and clinical phenotype of breast cancer patients.</p><p>Methods</p><p>The distribution of twelve <i>ABCC1</i> polymorphisms was assessed by direct sequencing in peripheral blood DNA (n = 540).</p><p>Results</p><p>Tumors from carriers of the wild type genotype in rs35623 or rs35628 exhibited significantly lower levels of ABCC1 transcript than those from carriers of the minor allele (p = 0.003 and p = 0.004, respectively). The ABCC1 transcript levels significantly increased in the order CT-GT>CC-GT>CC-GG for the predicted rs35626-rs4148351 diplotype. Chemotherapy-treated patients carrying the T allele in rs4148353 had longer disease-free survival than those with the GG genotype (p = 0.043). On the other hand, hormonal therapy-treated patients with the AA genotype in rs35628 had significantly longer disease-free survival than carriers of the G allele (p = 0.012).</p><p>Conclusions</p><p>Taken together, our study shows that genetic variability in the nucleotide binding domain 1 has a significant impact on the ABCC1 transcript level in the target tissue and may modify survival of breast cancer patients.</p></div

Significant associations between DFS of patients with breast carcinoma and SNPs in <i>ABCC1</i>.

<p>Kaplan-Meier survival curves for patients treated by chemotherapy (A) and hormonal therapy (B) were analyzed by Breslow test. In part A, dashed line represents DFS of patients with the GG genotype in rs4148353, while solid line indicates that of patients with the T allele. In part B, dashed line represents DFS of patients with the G allele in rs35628 and solid line DFS of those with the AA genotype. All SNPs have been analyzed but to retain concise style only significant associations are reported.</p

Distribution of <i>ABCC1</i> SNPs and allele frequencies in breast cancer patients.

<p>Footnotes:</p><p>*NC = non-coding.</p>#<p>MAF = minor allele frequency.</p

Significant associations of <i>ABCC1</i> polymorphisms with expression levels.

<p>All SNPs and frequent diplotypes were analyzed but to retain concise style only significant associations are reported.</p><p>Footnotes:</p><p>*Analyzed by Mann-Whitney test. The higher is the rank the lower is the normalized expression ABCC1/reference genes.</p>#<p>Result passed FDR analysis for multiple testing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101740#pone.0101740-Benjamini1" target="_blank">[29]</a>.</p

Schematic representation of functional domains of ABCC1.

<p>Figure depicts functional domains of ABCC1 protein (A) and important structural motifs within NBD1 (B). Data modified from NCBI’s Conserved Domain Database (CDD) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101740#pone.0101740-MarchlerBauer1" target="_blank">[49]</a>.</p

Significant associations of <i>ABCC1</i> polymorphisms with clinical data.

<p>All SNPs and frequent diplotypes were analyzed but to retain concise style only significant associations are reported.</p><p>Footnotes:</p><p>*Analyzed by two-sided Fisher’s Exact test.</p>#<p>Result passed FDR analysis for multiple testing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101740#pone.0101740-Benjamini1" target="_blank">[29]</a>.</p