Upfront DPYD Genotyping and Toxicity Associated with Fluoropyrimidine-Based Concurrent Chemoradiotherapy for Oropharyngeal Carcinomas: A Work in Progress

Simple SummaryThe combination of carboplatin and 5-fluorouracil (5-FU) is effective when used concurrently with radiotherapy for locoregionally advanced oropharyngeal carcinomas. DPYD polymorphisms can be associated with an increased risk of severe toxicity to fluoropyrimidines. Upfront screening for the DPYD*2A allele has been available in the province of Québec, Canada, since March 2017. This study aimed to determine the effect of upfront genotyping on the incidence of grade ≥3 toxicities. We included 181 patients in the analysis. Extended screening for three supplemental at-risk DPYD variants was also retrospectively performed in August 2019. The DPYD*2A, c.2846A>T and c.1236G>A polymorphisms were associated with an increased risk of grade ≥3 toxicity to 5-FU. Upfront DPYD genotyping can thus identify patients in whom 5-FU-related toxicity should be avoided.AbstractBackground: 5-FU-based chemoradiotherapy (CRT) could be associated with severe treatment-related toxicities in patients harboring at-risk DPYD polymorphisms. Methods: The studied population included consecutive patients with locoregionally advanced oropharyngeal carcinoma treated with carboplatin and 5-FU-based CRT one year before and after the implementation of upfront DPYD*2A genotyping. We aimed to determine the effect of DPYD genotyping on grade ≥3 toxicities. Results: 181 patients were analyzed (87 patients before and 94 patients following DPYD*2A screening). Of the patients, 91% (n = 86) were prospectively genotyped for the DPYD*2A allele. Of those screened, 2% (n = 2/87) demonstrated a heterozygous DPYD*2A mutation. Extended genotyping of DPYD*2A-negative patients later allowed for the retrospective identification of six additional patients with alternative DPYD variants (two c.2846A>T and four c.1236G>A mutations). Grade ≥3 toxicities occurred in 71% of the patients before DPYD*2A screening versus 62% following upfront genotyping (p = 0.18). When retrospectively analyzing additional non-DPYD*2A variants, the relative risks for mucositis (RR 2.36 [1.39–2.13], p = 0.0063), dysphagia (RR 2.89 [1.20–5.11], p = 0.019), and aspiration pneumonia (RR 13 [2.42–61.5)], p = 0.00065) were all significantly increased. Conclusion: The DPYD*2A, c.2846A>T, and c.1236G>A polymorphisms are associated with an increased risk of grade ≥3 toxicity to 5-FU. Upfront DPYD genotyping can identify patients in whom 5-FU-related toxicity should be avoided.     

A method for the amplification of chemically induced transformation in C3H/10T1/2 clone 8 cells: its use as a potential screening assay

A method has been developed by which to amplify expression of phenotypic transformation of C3H/10T1/2 clone 8 mouse embryo cells not otherwise observed in the standard transformation assay. The expression of transformed foci was amplified by subcultivating chemically treated target cells after they had reached confluence and replating them at subconfluent cell densities. Conditions leading to the expression of the highest numbers of transformed foci include a) a cell seeding density for chemical treatment of 1 X 10(4) cells/dish, b) subculture 4 weeks after treatment, and c) replating cells at a density of 2 X 10(5) cells/-dish. Agents capable of inducing transformation in the standard assay (e.g., 4,4'-bis(dimethylamino)benzophenone, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and others) also yielded transformation in the replating assay. The more marginal transforming activities of chemicals such as ethyl methanesulfonate, 7-(bromomethyl)-12-methylbenz[a]anthracene, and N-methyl-N'-nitro-N-nitrosoguanidine were enhanced by the amplification procedure. Compounds that failed to elicit focal transformation in the standard assay (e.g., dibenz[a,h]anthracene, Tris(2,3-dibromopropyl) phosphate, lead acetate, benzidine, propyleneimine, N-hydroxy-2-fluorenylacetamide, and numerous other compounds of various chemical classes) induced significant levels of phenotypic transformation upon amplification. Noncarcinogens (e.g., phenanthrene, anthracene, 2-aminobiphenyl, cycloheximide, and others) failed to cause significant phenotypic transformation even when cells were replated. To further enhance the applicability of this new replating system, an exogenous source of metabolic activation was added: a 9,000 X g supernatant from Aroclor 1254-induced rat hepatic S-9. This activation system was found a) to be only minimally cytotoxic by itself and b) to be able to mediate NADPH-dependent, dose-dependent toxicity, and transformation by activating the procarcinogens dimethylnitrosamine, 2-naphthylamine, 2-aminoanthracene, and aflatoxin B1. With the use of this revised assay, 14 coded and 23 model compounds were tested. Agreement with in vivo results was observed to be over 85%. The marked sensitivity and discriminatory ability of this revised assay procedure suggest its usefulness as a screen for potential carcinogens of diverse chemical structure.     

Correlation of Infants'Brain and Behavior Response to Temporal Changes in Visual Stimulation

This research was designed to test the hypothesis that a direct, parametric relationship exists between infant attention and the occipital visual evoked potential (VEP) induced by temporal frequency of visual pattern stimulation. Infants (13-weeks-old; N=32) were shown identical checkerboard patterns modulated at different rates of temporal change in a paired-comparison design and durations of orienting responses were measured. Another group of infants (13-weeks-old; N=14) were shown a series of checkerboard patterns, each modulated at a different temporal rate, and the midline occipital brain response was recorded. The results showed that behavioral attention and the signal strength/sec of the VEP are both significantly related to temporal frequency of stimulation. Furthermore, when plotted to the log₂ of temporal rate, both behavioral and neurophysiological response functions were best described by inverted U-shaped curves with similar maxima (4.8 Hz vs 5.8 Hz). These data taken together indicate that infants’visual attention and occipital brain response to temporal frequency covary and that a common neurophysiological mechanism may be involved.     

Effect of 3-aminobenzamide on the induction of toxicity and transformation by ethyl methanesulfonate and methylcholanthrene in BALB/3T3 cells

3-Aminobenzamide, a potent inhibitor of nuclear poly ADP-ribosylsynthetase, was tested for its ability to alter the toxic and/ortransforming effects of ethyl methanesulfonate, methyl methanesulfonateand 3-methylcholanthrene in BALB/3T3 clone A31-1 cells. 3-Aminobenzamideenhanced the toxic effects of both ethyl methanesulfonate andmethyl methanesulfonate in a dose dependent manner, but hadminimal effects on 3-methylcholanthrene induced toxicity. Similarly,3-aminobenzamide greatly enhanced ethyl methanesulfonate inducedtransformation while failing to enhance the transformation ofBALB/3T3 clone A31-1 cells by 3-methylcholanthrene. These resultsstress the importance of poly ADP-ribosyl synthetase in repairof DNA damage and the chemical induction of transformation invitro.     

Effects of 3-aminobenzamide on the induction of morphologic transformation by diverse compounds Balb/3T3 cells in vitro

When cells were exposed simultaneously for a 24-h period tothe poly(ADP-ribose) synthetase inhibitor 3-aminobenz-amide(3AB) (1 or 3 mM) plus aflatoxin B₁ (AfB₁), no increase in toxicityand limited enhancement of transformation frequency (<2 x)was observed. Similarly, simultaneous treatment of cell with3AB plus methylcholanthrene (MCA) had limited effects, slightlydecreasing both toxicity and transformation. In contrast, simultaneoustreatment with non-toxic doses of 3AB together with the alkylatingagents N-methyl-N'-N-nitro-nitrosoguanidine (MNNG) or ethylmethane-sulfonate (EMS) resulted in substantial enhancementof the toxicity and transforming effects of both short-chainalkylating agents. When EMS and varying doses of 3AB (0.1–3mM) were administered simultaneously for 24 h, increasing levelsof 3AB were found to cause a dose-dependent enhancement in toxicityand transformation. To explore the relationship of MNNG- and3AB-induced effects, two further experiments were performed.First, cells were treated with MNNG plus 3AB for varying lengthsof time (4, 24, 72 h). Although exposure for as little as 4h enhanced toxicity and transformation, these effects were evenmore profound following 24 or 72 h exposure. Second, cells wereexposed to 3AB for varing times prior to or after MNNG exposure.Under these conditions the addition of 3AB up to 6 h post MNNGexposure caused profound enhancement of toxicity and transformation,whereas addition of 3AB 24 h post exposure had minimal effects.Thus the co-carcinogenic effect of 3AB is agent-specific, time-specificand dose-dependent.     

Morphological transformation of BALB/3T3 cells by various procarcinogens in the presence of a rat liver S-9 activation system

An Aroclor-induced rat hepatic S-9 metabolic activation system was incorporated into the BALB/3T3 cell transformation assay to increase its sensitivity to a wide range of procarcinogens. S-9 was prepared from Aroclor 1254-induced (500 mg/kg) Fischer 344 rats. Cyclophosphamide, dimethylnitrosamine, 2-aminofluorene, and 2-naphthylamine were metabolized to reactive forms capable of inducing both dose-dependent toxicity and morphological transformation of BALB/3T3 cells. Treatments without an exogenous metabolic activation system were nontoxic and nontransforming. Adaptation of this commonly used exogenous metabolic activation system to BALB/3T3 cells will allow detection of the transforming potential of procarcinogens which test negative in a standard assay.     

Rapid structural alterations of the active zone lead to sustained changes in neurotransmitter release

The likelihood with which an action potential elicits neurotransmitter release, the release probability (p_r), is an important component of synaptic strength. Regulatory mechanisms controlling several steps of synaptic vesicle (SV) exocytosis may affect p_r, yet their relative importance in determining p_r and eliciting temporal changes in neurotransmitter release at individual synapses is largely unknown. We have investigated whether the size of the active zone cytomatrix is a major determinant of p_r and whether changes in its size lead to corresponding alterations in neurotransmitter release. We have used a fluorescent sensor of SV exocytosis, synaptophysin-pHluorin, to measure p_r at individual synapses with high accuracy and employed a fluorescently labeled cytomatrix protein, Bassoon, to quantify the amount of active zone cytomatrix present at these synapses. We find that, for synapses made by a visually identified presynaptic neuron, p_r is indeed strongly correlated with the amount of active zone cytomatrix present at the presynaptic specialization. Intriguingly, active zone cytomatrices are frequently subject to synapse-specific changes in size on a time scale of minutes. These spontaneous alterations in active zone size are associated with corresponding changes in neurotransmitter release. Our results suggest that the size of the active zone cytomatrix has a large influence on the reliability of synaptic transmission. Furthermore, they implicate mechanisms leading to rapid structural alterations at active zones in synapse-specific forms of plasticity.