Influence of Gender on Radiosensitivity during Radiochemotherapy of Advanced Rectal Cancer

Gender is increasingly recognized as an important factor in medicine, although it has long been neglected in medical research in many areas. We have studied the influence of gender in advanced rectal cancer with a special focus on radiosensitivity. For this purpose, we studied a cohort of 495 men (84.1% ≥ T3, 63.6% N1, 17.6%, M1) and 215 women (84.2% ≥ T3, 56.7% N1, 22.8%, M1) who all suffered from advanced rectal cancer and were treated with radiochemotherapy. The energy deposited, DNA double-strand break (dsb) repair, occurrence of chromosomal aberrations, duration of therapy, tumor regression and tumor-infiltrating lymphocytes, laboratory parameters, quality of life and survival were assessed. The residual DNA dsb damage 24 h after irradiation in lymphocytes was identical in both sexes. Furthermore, chromosomal aberrations accurately reflecting radiosensitivity, were similar in both sexes. There were no gender-dependent differences in tumor regression, tumor-infiltrating lymphocytes and outcome indicating no differences in the radiosensitivity of cancer cells. The irradiated tumor volume in women was slightly lower than in men, related to body weight, no difference was observed. However, when the total energy deposited was calculated and related to the body weight, women were exposed to higher amounts of ionizing radiation. During radiochemotherapy, decreases in blood lymphocyte counts and albumin and several quality-of-life parameters such as nausea and vomiting, loss of appetite, and diarrhea were significantly worse in women. There is no difference in radiation sensitivity between men and women in both normal tissue and tumors. During radiochemotherapy, the quality of life deteriorates more in women than in men. However, women also recover quickly and there are no long-term differences in quality of life.

An assessment of cytotoxic potentiality of Aloe barbadensis on the root meristem cells of Trigonella foenum-graecum L.

The leaves of Aloe barbadensis are used in traditional and modern systems of medicine. The aim of this study was to evaluate the cytotoxic potentialities of aqueous solution of Aloe barbadensis leaves on Trigonella foenum-graecum root tip meristem cells using a cytogenetic approach. Treatments with various concentrations of Aloe barbadensis leaf extract to Trigonella foenum-graecum root meristem cells showed mito-inhibition and induced several chromosomal aberrations as chromsomal breakage, fragmentation, scattering, stickiness etc.

Prenatal Diagnosis and Genetic Analysis of 21q21.1–q21.2 Aberrations in Seven Chinese Pedigrees

Background: Chromosomal aberrations contribute to human phenotypic diversity and disease susceptibility, but it is difficult to assess their pathogenic effects in the clinic. Therefore, it is of great value to report new cases of chromosomal aberrations associated with normal phenotypes or clinical abnormalities.Methods: This was a retrospective analysis of seven pedigrees that carried 21q21.1–q21.2 aberrations. G-banding and single-nucleotide polymorphism array techniques were used to analyze chromosomal karyotypes and copy number variations in the fetuses and their family members.Results: All fetuses and their family members showed normal karyotypes in seven pedigrees. Here, it was revealed that six fetuses carried maternally inherited 21q21.1–q21.2 duplications, ranging from 1 to 2.7 Mb, but none of the mothers had an abnormal phenotype. In one fetus, an 8.7 Mb deletion of 21q21.1–q21.2 was found. An analysis of the pedigree showed that the deletion was also observed in the mother, brother, and maternal grandmother, but no abnormal phenotypes were found.Conclusion: This study identified 21q21.1–q21.2 aberrations in Chinese pedigrees. The carriers of 21q21.1–q21.2 duplications had no clinical consequences based on their phenotypes, and the 21q21.1–q21.2 deletion was transmitted through three generations of normal individuals. This provides benign clinical evidence for pathogenic assessment of 21q21.1–q21.2 duplication and deletion, which was considered a variant of uncertain significance and a likely pathogenic variant in previous reports.

Is Response to Genotoxic Stress Similar in Populations of African and European Ancestry? A Study of Dose-Response After in vitro Irradiation

Background: Exposure to genotoxic stress such as radiation is an important public health issue affecting a large population. The necessity of analyzing cytogenetic effects of such exposure is related to the need to estimate the associated risk. Cytogenetic biological dosimetry is based on the relationship between the absorbed dose and the frequency of scored chromosomal aberrations. The influence of confounding factors on radiation response is a topical issue. The role of ethnicity is unclear. Here, we compared the dose-response curves obtained after irradiation of circulating lymphocytes from healthy donors of African and European ancestry.Materials and Methods: Blood samples from six Africans living in Africa, five Africans living in Europe, and five Caucasians living in Europe were exposed to various doses (0–4 Gy) of X-rays at a dose-rate of 0.1 Gy/min using an X-RAD320 irradiator. A validated cohort composed of 14 healthy Africans living in three African countries was included and blood samples were irradiated using the same protocols. Blood lymphocytes were cultured for 48 h and chromosomal aberrations scored during the first mitosis by telomere and centromere staining. The distribution of dicentric chromosomes was determined and the Kruskal-Wallis test was used to compare the dose-response curves of the two populations.Results: No spontaneous dicentric chromosomes were detected in African donors, thus establishing a very low background of unstable chromosomal aberrations relative to the European population. There was a significant difference in the dose response curves between native African and European donors. At 4 Gy, African donors showed a significantly lower frequency of dicentric chromosomes (p = 8.65 10–17), centric rings (p = 4.0310–14), and resulting double-strand-breaks (DSB) (p = 1.32 10–18) than European donors. In addition, a significant difference was found between African donors living in Europe and Africans living in Africa.Conclusion: This is the first study to demonstrate the important role of ethnic and environmental factors that may epigenetically influence the response to irradiation. It will be necessary to establish country-of-origen-specific dose response curves to practice precise and adequate biological dosimetry. This work opens new perspective for the comparison of treatments based on genotoxic agents, such as irradiation.

Chromosome Folding Promotes Intrachromosomal Aberrations under Radiation- and Nuclease-Induced DNA Breakage

The long-standing question in radiation and cancer biology is how principles of chromosome organization impact the formation of chromosomal aberrations (CAs). To address this issue, we developed a physical modeling approach and analyzed high-throughput genomic data from chromosome conformation capture (Hi-C) and translocation sequencing (HTGTS) methods. Combining modeling of chromosome structure and of chromosomal aberrations induced by ionizing radiation (IR) and nuclease we made predictions which quantitatively correlated with key experimental findings in mouse chromosomes: chromosome contact maps, high frequency of cis-translocation breakpoints far outside of the site of nuclease-induced DNA double-strand breaks (DSBs), the distinct shape of breakpoint distribution in chromosomes with different 3D organizations. These correlations support the heteropolymer globule principle of chromosome organization in G1-arrested pro-B mouse cells. The joint analysis of Hi-C, HTGTS and physical modeling data offers mechanistic insight into how chromosome structure heterogeneity, globular folding and lesion dynamics drive IR-recurrent CAs. The results provide the biophysical and computational basis for the analysis of chromosome aberration landscape under IR and nuclease-induced DSBs.

Whole Genome Sequencing Identifies Non-KIT Mutations and Cytogenetic Aberrations in Systemic Mastocytosis but Has Limited Sensitivity for Detection of KIT D816V

Background: Systemic mastocytosis (SM) is a hematologic neoplasm characterized by the infiltration of clonal mast cells in the bone marrow or other extra-cutaneous organs. The clinical course varies between advanced and non-advanced (indolent and smoldering SM) forms of SM. The vast majority of patients harbor the activating D816V mutation in the KIT tyrosine kinase. Additional somatic mutations in other genes have been recognized as risk factors in SM. Cytogenetic aberrations are rarely found in SM but have been associated with advanced disease. Whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) have been described as an alternative to cytogenetics and targeted molecular genetic analysis in myeloid cancers. Aim: To assess the ability of WGS/WTS to detect cytogenetic aberrations and recurrent somatic mutations in SM. Methods: 120 patients (51 female, 69 male) diagnosed with SM were analyzed with WGS/WTS and results were compared with orthogonal data of KIT D816V PCR, targeted sequencing, and cytogenetics. 47 patients (39%) were diagnosed with advanced SM (1 mast cell leukemia, 3 aggressive SM, 43 SM with associated hematologic neoplasm). For WGS, 2x151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). BaseSpace's Tumor/Normal app v3 was used to call variants with Strelka Somatic Variant Caller v2.4.7 and structural variants (aberrations with >50bp in size) with Manta (v0.28.0). Genomic DNA from a mixture of multiple anonymous donors (Promega, Fitchburg, WI, USA) was used as normal. For WTS, 2x101 bp paired-end reads were produced with a median of 50 mio. reads per sample, aligned with STAR v2.5.0, and variants were called using Isaac Variant Caller v2.3.13. Results: WGS/WTS detected cytogenetic aberrations in 21% of patients: 2 patients displayed a complex aberrant karyotype, 3 balanced structural aberrations, 16 copy number alterations, and 6 copy number neutral losses of heterozygosity. Aberrations detected by chromosome banding analysis were also found by WGS in all but three patients (small clones with aberrations present in ≤20% of metaphases and <10% of interphase nuclei as determined by FISH). In contrast, WGS/WTS detected additional aberrations in 16 patients. The frequency of chromosomal aberrations detected by WGS/WTS was higher in advanced compared to non-advanced SM (34% vs. 12%, p<0.05). KIT D816V was detected by PCR in 98%, by WGS in 21% and by WTS in 46% of patients. The detection rate by WGS was significantly higher in advanced (36%) compared to non-advanced SM (12%, p<0.05) while no difference was observed for WTS (45% vs. 47%). Somatic mutations outside of KIT were analyzed within a subset of 121 genes recurrently mutated in hematologic neoplasms. 46% of patients showed non-KIT mutations with a median of 2 mutations per patient. Both frequency of non-KIT mutations as well as the median number of mutations per patient was higher in advanced (83%; n=3) compared to non-advanced SM (22%, n=1, p<0.05). Finally, we analyzed the impact of genetic aberrations on survival in our SM cohort. Patients were grouped according to the presence of chromosomal aberrations and gene mutations (non-KIT) as assessed by WGS/WTS. SM patients with both types of aberrations (n=16), one type of aberration (n=47; gene mutations only n=38; chromosomal aberrations only n=8), or no aberration but KIT D816V (n=57) showed significant differences in overall survival (p<0.05, Figure 1). Con clusions: WGS/WTS has limited sensitivity for detection of KIT D816V in SM. This finding can be explained by the low KIT D816V mutation burden typically found in bone marrow aspirates of SM patients. In line, we observed a slightly higher detection rate in advanced SM and in RNA-based WTS analysis. As WGS/WTS will be applied for the diagnostic workup of myeloid malignancies in the future and SM associated with other hematologic neoplasms may be overlooked if not specifically investigated, additional PCR-based techniques are still mandatory to rule out KIT D816V as a diagnostic criterion for SM. In contrast, WGS/WTS detects both chromosomal aberrations and additional gene mutations in patients with SM and can be used as an alternative to cytogenetics and targeted sequencing for risk assessment. In particular, the absence of genetic aberrations in WGS/WTS identifies SM patients with indolent course of the disease and favorable prognosis. Figure 1 Figure 1. Disclosures Hoermann: Novartis: Honoraria. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

Polycythemia Vera Patients Respond Better to Ropeginterferon Alfa-2b Than HU/BAT Irrespective of Pretreatment or Mutational Status; Results from 5 Years' Treatment in a Randomized, Controlled Setting in the PROUD-PV/Continuation-PV Trials

Introduction: Ropeginterferon alfa-2b (BESREMi®), a novel pegylated interferon with an extended half-life, was approved in Europe for treatment of patients with PV based on results from the phase 3 PROUD-PV and CONTINUATION-PV trials. Ropeginterferon alfa-2b treatment is recommended in hydroxyurea (HU) naïve patients as well as in those who have previously received HU. Therefore, treatment response was analyzed by prior HU treatment status, and the influence of baseline JAK2V617F allele burden and additional mutations - which may increase over time during non-disease modifying treatment - was explored. Methods: In PROUD-PV, patients aged ≥18 years, diagnosed with PV according to WHO 2008 criteria, and either cytoreduction-naïve or HU-pre-treated (for <3 years, without intolerance or resistance) were randomized 1:1 to receive ropeg or HU at individualized doses. After 12 months' treatment, patients could roll over into CONTINUATION-PV and patients in the HU arm were permitted to switch to best available treatment (BAT). After 5 years' treatment, complete hematologic response (CHR) and molecular response defined by modified ELN criteria were assessed in patients enrolled in the extension study CONTINUATION-PV (N=171). Sub-group analyses were performed by prior HU treatment, JAK2V617F allele burden category (≤50% or>50%), and in patients with available data (N=159), by the presence of non-driver mutations (TruSight™ Myeloid Panel, Illumina) or chromosomal aberrations (Affymetrix SNP6.0 array) at baseline. Results: After 5 years of treatment with ropeginterferon alfa-2b, high rates of CHR were sustained in both HU-naïve and HU pre-treated patients (53.1% and 61.3%, respectively), whereas in the control arm, the CHR rate was lower among HU pre-treated patients (36.0% compared to 48.0% for HU-naïve patients). Molecular response rates at 5 years in HU naïve and pre-treated patients were 71.4% and 64.5% respectively in the ropeginterferon alfa-2b arm and 26.0% versus 12.5% respectively in the control arm. Rates of adverse events (AEs), treatment-related AEs, serious AEs, and AEs leading to discontinuation were similar between the subgroups regardless of HU pre-treatment. Similar CHR rates were observed at 5 years irrespective of baseline JAK2V617F allele burden category (ropeginterferon alfa-2b arm: 57.1% versus 53.1% for allele burden ≤50% or >50%, respectively; control: 46.9% versus 38.5%, respectively). The molecular response rate in the ropeginterferon alfa-2b arm was higher among patients with baseline allele burden >50% (84.4% versus 61.3% for allele burden ≤50%); in the control arm there was no difference in molecular response rates between the allele burden subgroups (23.1% versus 20.8%, respectively). Of interest, the presence of non-driver mutations or chromosomal aberrations at baseline had no apparent influence on molecular response rates to ropeginterferon alfa-2b (64.5% compared with 70.7% in patients without these genetic abnormalities). Conclusion: High hematologic and molecular response rates in both HU-pretreated and HU-naïve patients and in those with more advanced JAK2V617F burden suggest that ropeginterferon alfa-2b is also a suitable treatment option in patients switching from HU. Disclosures Gisslinger: AOP Orphan Pharmaceuticals GmbH: Other: Personal fees, Research Funding; Novartis: Other: Personal fees, Research Funding; PharmaEssentia: Other: Personal fees; MyeloPro Diagnostics and Research: Other: Personal fees; Janssen-Cilag: Other: Personal fees; Roche: Other: Personal fees; Celgene: Other: Personal fees. Klade: AOP Orphan Pharmaceuticals GmbH: Current Employment. Pylypenko: Communal nonprofit enterprise "Cherkasy regional oncology dispensary of Cherkasy oblast council: Current Employment. Mayer: AOP Orphan Pharmaceuticals GmbH: Research Funding. Krejcy: AOP Orphan Pharmaceuticals GmbH: Current Employment. Empson: AOP Orphan Pharmaceuticals GmbH: Current Employment. Hasselbalch: Novartis, AOP Orphan: Consultancy, Other: Advisory Board. Kralovics: AOP Orphan Pharmaceuticals GmbH: Other: Personal fees; PharmaEssentia: Other: Personal fees; Qiagen: Other: Personal fees; Novartis: Other: Personal fees; MyeloPro Diagnostics and Research: Current holder of individual stocks in a privately-held company. Kiladjian: Novartis: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Other: Personal fees; Taiho Oncology, Inc.: Research Funding.

Evaluation of mutagenicity, genotoxicity and chronic toxicity of antiviral drug imidazolyl ethanamide pentandioic acid in in vitro and in vivo test systems

Introduction. The antiviral properties of imidazolyl ethanamide pentandioic acid (IPA), the active compound of the drug product, has been proven in various experimental models. However, the literature data on the toxicological properties of IPA are limited.Purpose. To evaluate mutagenic and genotoxic properties in in vitro and in vivo models, as well as to study the toxicity of IPA following chronic oral administration to rats and dogs.Materials and methods. Mutagenic and genotoxic properties of IPA were assessed using the Ames test, the test of chromosomal aberrations in human lymphocytes, and the micronucleus test in rats. The chronic toxicity of IPA was studied in Sprague Dawley rats and beagle dogs of both sexes, to which IPA was administered orally at doses of 30-300 mg/kg/day for 26 and 39 weeks, respectively.Results and discussion. In the Ames test, the addition of IPA up to the maximum dose (5000 mcg/plate) did not result in the increase in the number of revertant colonies. At a concentration of up to 5000 mcg/ml, IPA did not cause chromosomal aberrations in human leukocytes. At doses doses ≤ 2000 mg/kg, IPA did not increase the amount of micronuclei in the bone marrow of rats. In chronic experiments, animals tolerated the administration of IPA well: the dose without an observed effect (NOEL) for rats and dogs was 300 mg/kg/day.Conclusion. IPA did not show mutagenic and genotoxic properties in standard in vitro and in vivo tests. With chronic oral administration to rats and dogs, NOEL IPA equal to 300 mg/kg/day provided a systemic exposure that was 8-10 and 41-65 times higher than that in humans, respectively. The results obtained allow us to consider the safety profile of the prolonged use in humans as favorable.

Effluent from local palm oil mill refinery in Nigeria is excessively oily and potentially genotoxic

In spite of the fact that the informal, local mills in countries like Nigeria constitute a major portion of the palm oil refinery, adequate attention has not been paid to the quality of the palm oil mill effluent (POME) they generate. In this study, therefore, the physicochemical properties and genotoxic potential of POME generated by a local mill in Nigeria were investigated using the methods prescribed by the American Public Health Association and Allium cepa chromosomal assay, respectively. In addition to the presence of trace/toxic metals (Zn, Pb, Cd, Cr, Cu) and high biochemical oxygen demand, POME contained a very high oil and grease concentration of 10,500 mg L-1 as against the maximum limit of 25 mg L-1 prescribed in the Environmental Management Guideline for the Palm Oil Industry. Mitotic activities in A. cepa roots exposed to POME showed that the number of dividing cells and percentage mitotic index generally decreased with increasing POME concentrations. The major chromosomal aberrations induced by POME were sticky, C-mitosis, bridged anaphase, vagrant, and attached chromosomes. However, no chromosomal aberrations were observed in onion roots exposed to water (control). These results indicate that the local refinery from where the POME was obtained is inefficient at recovering oil from palm fibre. The effluent generated by the mill is also a potential pollutant capable of inducing genotoxic and other adverse effects. These results may be typical of many more local palm oil refineries who use mainly manual methods to extract oil from oil palm fruits.