We intended to evaluate diagnostic utility of a targeted gene sequencing by using next generation sequencing (NGS) panel in patients with intractable early-onset epilepsy (EOE) and find the efficient analytical step for increasing the diagnosis rate. We assessed 74 patients with EOE whose seizures started before 3 years of age using a customized NGS panel that included 172 genes. Single nucleotide variants (SNVs) and exonic and chromosomal copy number variations (CNVs) were intensively examined with our customized pipeline and crosschecked with commercial or pre-built software. Variants were filtered and prioritized by in-depth clinical review, and finally classified according to the American College of Medical Genetics and Genomics guidelines. Each case was further discussed in a monthly consensus meeting that included the participation of all laboratory personnel, bioinformaticians, geneticists, and clinicians. The NGS panel identified 28 patients (37.8%) with genetic abnormalities; 25 patients had pathogenic or likely pathogenic SNVs in 17 genes including SXTBP1 (n = 3), CDKL5 (n = 2), KCNQ2 (n = 2), SCN1A (n = 2), SYNGAP1 (n = 2), GNAO1 (n = 2), KCNT1 (n = 2), BRAT1, WWOX, ZEB2, CHD2, PRICKLE2, COL4A1, DNM1, SCN8A, MECP2, SLC9A6 (n = 1). The other 3 patients had pathogenic CNVs (2 duplications and 1 deletion) with varying sizes (from 2.5 Mb to 12 Mb). The overall diagnostic yield was 37.8% after following our step-by-step approach for clinical consensus. NGS is a useful diagnostic tool with great utility for patients with EOE. Diagnostic yields can be maximized with a standardized and team-based approach.

Oxidative stress is an imbalance between free radicals and antioxidant molecules that can play an important role in the pathogenesis of iron‐deficiency anemia (IDA). The aim of this study was to investigate oxidative status in patients with IDA and alteration of oxidative status after iron treatment. Thirty‐three female patients with IDA and 25 healthy controls were included in this study. Oxidant and total antioxidant capacity were determined using free oxygen radicals test and free oxygen radicals defence (Form CR 3000, Callegari, Parma, Italy). Catalase activity was measured by spectrophotometer using a commercially available kit (Bioxytech Catalase‐520, OxisResearch, Portland, OR). Oxidant activity in patients with IDA was significantly higher than controls (P<0.05), while total antioxidant and catalase activity were significantly lower (P0.05). In conclusion, our data indicate that blood reactive oxygen species was lower and total antioxidant and catalase activity were higher after rather than before treatment in patients with IDA. The results of our study support the higher oxidative stress hypothesis in IDA; however, due to the limited number of cases included, more studies may be required to confirm the results. J. Clin. Lab. Anal. 23:319–323, 2009.

Circulating tumor DNA (ctDNA) may aid in personalizing ovarian cancer therapeutic options. Here, we aimed to assess the clinical utility of serial ctDNA testing using tumor-naïve, small-sized next-generation sequencing (NGS) panels. A total of 296 patients, including 201 with ovarian cancer and 95 with benign or borderline disease, were enrolled. Samples were collected at baseline (initial diagnosis or surgery) and every 3 months after that, resulting in a total of 811 blood samples. Patients received adjuvant therapy based on the current standard of care. Cell-free DNA was extracted and sequenced using an NGS panel of 9 genes: TP53, BRCA1, BRCA2, ARID1A, CCNE1, KRAS, MYC, PIK3CA, and PTEN. Pathogenic somatic mutations were identified in 69.2% (139/201) of patients with ovarian cancer at baseline but not in those with benign or borderline disease. Detection of ctDNA at baseline and/or at 6 months follow-up was predictive of progression-free survival (PFS). PFS was significantly poorer in patients with detectable pathogenic mutations at baseline that persisted at follow-up than in patients that converted from having detectable ctDNA at baseline to being undetectable at follow-up; survival did not differ between patients without pathogenic ctDNA mutations in baseline or follow-up samples and those that converted from ctDNA positive to negative. Disease recurrence was also detected earlier with ctDNA than with conventional radiologic assessment or CA125 monitoring. These findings demonstrate that serial ctDNA testing could effectively monitor patients and detect minimal residual disease, facilitating early detection of disease progression and tailoring of adjuvant therapies for ovarian cancer treatment. Significance: In ovarian cancer, serial circulating tumor DNA testing is a highly predictive marker of patient survival, with a significantly improved recurrence detection lead time compared with conventional monitoring tools.

Purpose: Patient-specific molecular alterations leading to PARP inhibitor (PARPi) resistance are relatively unexplored. In this study, we analyzed serially collected circulating tumor DNA (ctDNA) from patients with BRCA1/2 mutations who received PARPis to investigate the resistance mechanisms and their significance in postprogression treatment response and survival. Experimental Design: Patients were prospectively enrolled between January 2018 and December 2021 (NCT05458973). Whole-blood samples were obtained before PARPi administration and serially every 3 months until progression. ctDNA was extracted from the samples and sequenced with a 531-gene panel; gene sets for each resistance mechanism were curated. Results: Fifty-four patients were included in this analysis. Mutation profiles of genes in pre-PARPi samples indicating a high tumor mutational burden and alterations in genes associated with replication fork stabilization and drug efflux were associated with poor progression-free survival on PARPis. BRCA hypomorphism and reversion were found in 1 and 3 patients, respectively. Among 29 patients with matched samples, mutational heterogeneity increased postprogression on PARPis, showing at least one postspecific mutation in 89.7% of the patients. These mutations indicate non-exclusive acquired resistance mechanisms—homologous recombination repair restoration (28%), replication fork stability (34%), upregulated survival pathway (41%), target loss (10%), and drug efflux (3%). We observed poor progression-free survival with subsequent chemotherapy in patients with homologous recombination repair restoration (P = 0.003) and those with the simultaneous involvement of two or more resistance mechanisms (P = 0.040). Conclusions: Analysis of serial ctDNAs highlighted multiple acquired resistance mechanisms, providing valuable insights for improving postprogression treatment and survival.