Pre-therapeutic dihydropyridimidine dehydrogenase (DPD) deficiency screening: Impact on fluoropyrimidine dose reduction at the second chemotherapy cycle and on early severe toxicity.

3096 Background: DPD deficiency screening before fluoropyrimidine is a matter of debate. To avoid lethal toxicity, French authorities impose DPD screening before fluoropyrimidine-based chemotherapy by dosing uracilemia since April 2019. Methods: We have included all consecutive adult patients receiving 5-fluorouracil (5-FU) or capecitabin from April 2019 to January 2020 in 6 cancer centers. During the study period, different methods for screening had been applied: DPYD complete sequencing, phenotype (uracilemia and/or dihydrouracilemia/uracilemia ratio - UH2/U -) or both. All sceening tests were conducted in the same laboratory. Association between the method of DPD screening and fluoropyrimidine dose reduction at second chemotherapy cycle or on severe ≥grade 3 early toxicity (between first and second cycle) was evaluated using multivariate logistic regression. Concordance between genotype and phenotype for DPD deficiency was explored using Cohen Kappa test. Results: We included 597 patients, the median age was 63 (range, 55-77). The most prevalent cancers were digestive (68.3%), head and neck (19.4%) and breast (9.2%). 12.3% of patients received capecitabine and 87.3% received polychemotherapy. DPD deficiency screening was done for most of patients (n=519, 86.9%). DPD screening method consisted in full sequencing of DPYP (n=41; 7.9%), phenotype analysis (n=44, 8.5%) or both (n=424, 83.6%). We did not identify any complete DPD deficiency. Uracilemia was dosed for 467 patients, the median was 6.5 ng/mL and for 21 patients (4.5%) uracilemia was > 16 ng/mL and/or UH2/U <6, suggesting DPD deficiency. Severe early toxicities were observed for 82 patients (14%), with two patients presenting grade 5 toxicity. Overall DPD screening and method of DPD screening were not associated with fluoropyrimidine dose reduction at second cycle or early severe toxicity. In multivariate analysis, the only predictor for fluoropyrimidine reduction at second cycle (n = 125 patients) was polychemotherapy (OR=2.8; p=0.012). Kappa between uracilemia and UH2/U was 0.23 (poor concordance). Kappa between DPYP sequencing and uracilemia or UH2/U was 0.09 (very poor concordance). Conclusions: No DPD deficiency screening method was associated with dose adaptation at second cycle or early severe toxicity. The optimal strategy for DPD screening requires further clinical evaluation.

The Effect of Direct Oral Anticoagulants on Antithrombin Activity Testing Is Abolished by DOAC-Stop in Venous Thromboembolism Patients

Context.— Direct oral anticoagulants (DOACs) may cause false negative results of antithrombin (AT) deficiency screening. Objective.— To evaluate the impact of DOAC-Stop, an agent reversing in vitro effects of DOACs, on AT testing in anticoagulated patients. Design.— We assessed 130 venous thromboembolism patients aged 46.7 ± 13.5 years. Blood samples were collected 2 to 27 hours after DOAC intake from 49 patients on rivaroxaban, 54 on apixaban, and 27 on dabigatran. Antithrombin activity was assessed using the activated factor X (FXa)–based and the activated factor II (FIIa)–based method twice, before and after DOAC-Stop treatment, together with plasma DOAC levels using coagulometric assays. Results.— The use of DOAC-Stop did not influence AT activity measured using the FIIa-based assay, whereas there was a marked decrease in AT activity determined using the FXa-based assay (ΔAT = 16.9%; 95% CI, 12.9%–19.1%). The AT-FIIa assay revealed decreased AT level (&lt;79%) in all 10 (7.7%) genetically confirmed AT-deficient patients treated with rivaroxaban or apixaban (n = 5 each), whereas the AT-FXa assay showed decreased AT activity (&lt;83%) in 2 subjects on rivaroxaban and 1 on apixaban with low plasma DOAC concentrations (&lt;90 ng/mL). After DOAC-Stop median AT-FXa activity lowered from 83.5% (interquartile range, 66%–143%) to 65.5% (interquartile range, 57%–75%; P = .005; ΔAT = 18%) in AT-deficient patients, without any false negative results. The ΔAT in the FXa-based assay correlated with rivaroxaban and apixaban concentrations in the AT-deficient patients (r = 0.99, P &lt; .001). Conclusions.— Application of DOAC-Stop enables reliable evaluation of AT deficiency screening in patients taking rivaroxaban or apixaban and tested using the FXa-based method.

Geographical Accessibility to Glucose-6-Phosphate Dioxygenase Deficiency Point-of-Care Testing for Antenatal Care in Ghana

Background: Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency screening test is essential for malaria treatment, control, and elimination programs. G6PD deficient individuals are at high risk of severe hemolysis when given anti-malarial drugs such as primaquine, quinine, other sulphonamide-containing medicines, and chloroquine, which has recently been shown to be potent for the treatment of coronavirus disease (COVID-19). We evaluated the geographical accessibility to POC testing for G6PD deficiency in Ghana, a malaria-endemic country. Methods: We obtained the geographic information of 100 randomly sampled clinics previously included in a cross-sectional survey. We also obtained the geolocated data of all public hospitals providing G6PD deficiency testing services in the region. Using ArcGIS 10.5, we quantified geographical access to G6PD deficiency screening test and identified clinics as well as visualize locations with poor access for targeted improvement. The travel time was estimated using an assumed speed of 20 km per hour. Findings: Of the 100 clinics, 58% were Community-based Health Planning and Services facilities, and 42% were sub-district health centers. The majority (92%) were Ghana Health Service facilities, and the remaining 8% were Christian Health Association of Ghana facilities. Access to G6PD deficiency screening test was varied across the districts, and G6PD deficiency screening test was available in all eight public hospitals. This implies that the health facility-to-population ratio for G6PD deficiency testing service was approximately 1:159,210 (8/1,273,677) population. The spatial analysis quantified the current mean distance to a G6PD deficiency testing service from all locations in the region to be 34 ± 14 km, and travel time (68 ± 27 min). The estimated mean distance from a clinic to a district hospital for G6PD deficiency testing services was 15 ± 11 km, and travel time (46 ± 33 min). Conclusion: Access to POC testing for G6PD deficiency in Ghana was poor. Given the challenges associated with G6PD deficiency, it would be essential to improve access to G6PD deficiency POC testing to facilitate administration of sulphadoxine-pyrimethamine to pregnant women, full implementation of the malaria control program in Ghana, and treatment of COVID-19 patients with chloroquine in malaria-endemic countries. To enable the World Health Organization include appropriate G6PD POC diagnostic tests in its list of essential in-vitro diagnostics for use in resource-limited settings, we recommend a wider evaluation of available POC diagnostic tests for G6PD deficiency, particularly in malaria-endemic countries.

Molecular Basis of CYP19A1 Deficiency in a 46,XX Patient With R550W Mutation in POR: Expanding the PORD Phenotype

Context Mutations in cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We report a novel R550W mutation in POR identified in a 46,XX patient with signs of aromatase deficiency. Objective Analysis of aromatase deficiency from the R550W mutation in POR. Design, setting, and patient Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C &gt; T/p.R550W in POR. Wild-type and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children’s Hospital, Bern, Switzerland. Main outcome measure and Results POR-R550W showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity, and CYP17A1 as well as CYP21A2 activities were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions Pathological effects due to POR-R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.

Molecular Basis of CYP19A1 Deficiency in a 46, XX Patient with R550W Mutation in POR: Expanding the PORD Phenotype

Context: Mutations in Cytochrome P450 oxidoreductase (POR) cause a form of congenital adrenal hyperplasia (CAH). We are reporting a novel R550W mutation in POR identified in a 46, XX patient with signs of aromatase deficiency. Objective: Analysis of aromatase deficiency from R550W mutation in POR. Design, Setting, and Patient: Both the child and the mother had signs of virilization. Ultrasound revealed the presence of uterus and ovaries. No defects in CYP19A1 were found, but further analysis with a targeted Disorders of Sexual Development NGS panel (DSDSeq.V1, 111 genes) on a NextSeq (Illumina) platform in Madrid and Barcelona, Spain, revealed compound heterozygous mutations c.73_74delCT/p.L25FfsTer93 and c.1648C&gt;T/p.R550W in POR. WT and R550W POR were produced as recombinant proteins and tested with multiple cytochrome P450 enzymes at University Children&rsquo;s Hospital, Bern, Switzerland. Main Outcome Measure and Results: R550W POR showed 41% of the WT activity in cytochrome c and 7.7% activity for reduction of MTT. Assays of CYP19A1 showed a severe loss of activity and CYP17A1, as well as CYP21A2 activities, were also lost by more than 95%. Loss of CYP2C9, CYP2C19, and CYP3A4 activities was observed for the R550W-POR. Predicted adverse effect on aromatase activity as well as a reduction in binding of NADPH was confirmed. Conclusions: Pathological effects due to POR R550W were identified, expanding the knowledge of molecular pathways associated with aromatase deficiency. Screening of the POR gene may provide a diagnosis in CAH without defects in genes for steroid metabolizing enzymes.