Mutant Allele Frequency Research Articles

TP53 Mutation Status in Myelodysplastic Neoplasm and Acute Myeloid Leukemia: Impact of Reclassification Based on the 5th WHO and International Consensus Classification Criteria: A Korean Multicenter Study.

TP53 mutations are associated with poor prognosis in myelodysplastic neoplasm (MDS) and AML. The updated 5th WHO classification and International Consensus Classification (ICC) categorize TP53-mutated MDS and AML as unique entities. We conducted a multicenter study in Korea to investigate the characteristics of TP53-mutated MDS and AML, focusing on diagnostic aspects based on updated classifications. This study included patients aged ≥ 18 yrs who were diagnosed as having MDS (N=1,244) or AML (N=2,115) at six institutions. The results of bone marrow examination, cytogenetic studies, and targeted next-generation sequencing, including TP53, were collected and analyzed. TP53 mutations were detected in 9.3% and 9.2% of patients with MDS and AML, respectively. Missense mutation was the most common, with hotspot codons R248/R273/G245/Y220/R175/C238 accounting for 25.4% of TP53 mutations. Ten percent of patients had multiple TP53 mutations, and 78.4% had a complex karyotype. The median variant allele frequency (VAF) of TP53 mutations was 41.5%, with a notable difference according to the presence of a complex karyotype. According to the 5th WHO classification and ICC, the multi-hit TP53 mutation criteria were met in 58.6% and 75% of MDS patients, respectively, and the primary determinants were a TP53 VAF >50% for the 5th WHO classification and the presence of a complex karyotype for the ICC. Collectively, we elucidated the molecular genetic characteristics of patients with TP53-mutated MDS and AML, highlighting key factors in applying TP53 mutation-related criteria in updated classifications, which will aid in establishing diagnostic strategies.

Predictive genomic and transcriptomic analysis on endoscopic ultrasound-guided fine needle aspiration materials from primary pancreatic adenocarcinoma: a prospective multicentre study

We apply endoscopic ultrasound-guided fine needle aspiration biopsy to cytopathologically diagnose and sample nucleic acids from primary tumours regardless of the disease stage. 397 patients with proven pancreatic adenocarcinoma were included and followed up in a multicentre prospective study. DNA and mRNA were extracted from materials of primary tumours obtained by endoscopic ultrasound-guided fine needle aspiration biopsy and analysed using targeted deep sequencing and RNAseq respectively. The variant allele frequency of the KRAS mutation was used to evaluate the tumour cellularity, ranging from 15 to 20% in all cells, regardless of the tumour stage. The molecular profile of metastatic primary tumours significantly differed from other types of tumours, more frequently having TP53 mutations (p=0.0002), less frequently having RNF43 mutations, and possessing more basal-like mRNA component (p=0.001). Molecular markers associated with improved overall survival were: mutations in homologous recombination deficiency genes in patients who received first-line platinum-based chemotherapy (p=0.025) and wild-type TP53 gene in patients with locally advanced tumours who received radio-chemotherapy (p=0.01). The GemPred transcriptomic profile was associated with a significantly better overall survival in patients with locally advanced or metastatic pancreatic cancer who received a gemcitabine-based first-line treatment (p=0.019). The combination of genomic and transcriptomic analyses of primary pancreatic tumours enables us to distinguish metastatic tumours from other tumour types. Our molecular strategy may assist in predicting overall survival outcomes for platinum or gemcitabine-based chemotherapies, as well as radio-chemotherapy. Institut National Du Cancer (BCB INCa_7294), CHU of Toulouse, Inserm and Ligue Nationale Contre le Cancer (CIT program).

Clinical features and search for genetic determinants of postprandial hypoglycemia.

To test whether postprandial hypoglycaemia is an extreme and repeatable phenotype of glucose metabolism. Secondly, we explored the genetic determinants of this phenotype. We conducted this study using data from Pinggu Metabolic Disease Study database (n = 3,345). We selected subjects after an oral glucose tolerance test (OGTT) (2 h, glucose <3 mmol/L_ and compared clinical features with those of normal glucose tolerance (NGT). We additionally selected 75 subjects as super-healthy control group. Whole-exome sequencing (WES) was performed on postprandial hypoglycaemic and super-healthy controls. We also evaluated several candidate genes believed to be important in pancreatic hypoglycaemia. We found 13 participants (0.39%) had an OGTT 2 h glucose <3 mmol/L. Ten patients were men (76.9%). All 13 participants had insulin > 3 uU/mL when postprandial blood glucose levels were <3 mmol/L. WES analysis identified one gene, paternally expressed 3 (PEG3), which had three rare mutations in four patients (30.8%). Minor allele frequencies (MAF) of rare PEG3 mutations were significantly higher in subjects with postprandial hypoglycaemia than in super-healthy controls. Among all four subjects with PEG3 gene mutations, 71.4% were men, and their body mass index (BMI) was significantly lower than that of the NGT. Postprandial hypoglycaemia is an extreme and reproducible phenotype in the general population. PEG3 mutations may represent a potential genetic aetiology for postprandial hypoglycaemia. Further research with larger and more diverse populations and a broader genetic focus is needed to understand the genetic basis of postprandial hypoglycaemia.

A Prospective Observational Study on Analyzing Lung Cancer Gene Mutation Variant Allele Frequency (VAF) and Its Correlation with Treatment Efficacy

A few studies have reported on the variability of the variant allele frequency (VAF) of gene mutations and the relationship between VAF and the therapeutic effect of molecular-targeted drugs. This joint study was conducted from May 2020 to January 2022 between St. Marianna University School of Medicine and the DNA Chip Research Institute. Cytology samples were used to verify the usefulness of a lung cancer compact panel test, which is a high-sensitivity next-generation sequencing (NGS) gene panel test. We analyzed the distribution of VAF and the duration of the initial molecular-targeted drug administration in patients with advanced-stage epidermal growth factor receptor (EGFR) mutations. Of the 196 patients diagnosed with non-small cell carcinoma (NSCLC), gene mutations were detected in 114 (58.2%) cases and in 68.7% of patients with adenocarcinomas (an increased detection rate). A VAF of 30% or more for DNA mutations was confirmed in 35 patients (33.7%), 10% to &lt;30% in 38 (36.5%), and &lt;10% in 31 (29.8%), including 18 patients (17.3%) with &lt;6%, which is considered the lower limit of the LOD for other companion diagnostic kits. EGFR mutations were detected in 59 patients (30%), of whom 35 were in an advanced stage and received molecular-targeted drugs as their initial treatment. Groups A and B comprised patients with a VAF of ≥10% and &lt;10%, respectively, with 27 patients (median VAF 30.6%, range 11.4–77%) in group A and 8 (median VAF 5.4%, range 0.1–8.5%) in group B. The duration of the administration of molecular-targeted drugs was 17 months (range 3–42 months) in group A and 14 months (range 1–45 months) in group B, with no statistically significant difference between the two groups (p-value = 0.7). Twenty-seven percent of patients had a VAF &lt; 10%. Even in patients with a low VAF for gene mutations, sufficient therapeutic effects were observed with molecular-targeted drugs, suggesting the importance of detecting mutations using a highly sensitive method at initial diagnosis. Further prospective validation studies of a larger number of cases are warranted.

18-Years of single-centre DNA testing in over 7000 index cases with inherited retinal dystrophies and optic neuropathies

Inherited retinal dystrophies (IRDs) and inherited optic neuropathies (IONs) are characterized by distinct genetic causes and molecular mechanisms that can lead to varying degrees of visual impairment. The discovery of pathogenic variants in numerous genes associated with these conditions has deepened our understanding of the molecular pathways that influence both vision and disease manifestation and may ultimately lead to novel therapeutic approaches. Over the past 18 years, our DNA diagnostics unit has been performing genetic testing on patients suspected of having IRD or ION, using state-of-the-art mutation detection technologies that are continuously updated. This report presents a retrospective analysis of genetic data from 6237 IRD and 780 ION patients. Out of these, 3054 IRD patients (49.0%) and 211 ION patients (27.1%) received a definitive molecular diagnosis, with disease-causing variants identified in 139 different genes. The genes most implicated in disease pathologies are ABCA4, accounting for 23.8% of all IRD/ION index cases, followed by BEST1 (7.8%), USH2A (6.2%), PRPH2 (5.7%), RPGR (5.6%), RS1 (5.5%), OPA1 (4.3%), and RHO (3.1%). Our study has compiled the most extensive dataset in combined IRD/ION diagnostics to date and offers valuable insights into the frequencies of mutant alleles and the efficiency of mutation detection in various inherited retinal conditions.

Intramuscular hemangioma capillary type with HRAS mutation: Expanding the molecular genetic spectrum with an emphasis on overlap with arteriovenous malformations and distinct from infantile hemangioma

AimsIntramuscular hemangioma capillary type (IHCT) is a rare entity that refers to fast-flow vascular lesions. This study aims to elucidate the relationships between clinicopathological, radiological, and molecular characteristics in IHCT patients. Methods and resultsWe reviewed all IHCT cases which were treated surgically in our pathology database from 2014 to 2023. Ten cases were analyzed via next-generation sequencing (NGS) and Sanger sequencing. The cohort consisted of 10 patients (6 males, 4 females) with a median age of 18 years (range: 1–37). Disease lesions were located in the trunk (n = 4), upper extremity (n = 2), lower extremity (n = 2), shoulder (n = 1), and neck (n = 1). IHCT is most commonly a progressively increasing painless mass. Histopathologically, all lesions exhibited aggregates, lobules, and anastomosing cords of capillary-type vessels separating or infiltrating the skeletal muscles. Four cases exhibited irregularly dilated vessels with thick walls, such as arteriovenous malformations (AVM) in the lesion's periphery. MRI findings commonly demonstrated a well-delineated, homogeneous mass. Somatic mutations were detected in seven of the ten IHCT cases. Four cases harbored mutations in MAP2K1 (p.Q58_E62del, p.K57_G61del, p.K57 N), two cases harbored mutations in KRAS (p.Q61R and p.L56V, p.G13R), and one case harbored a mutation in HRAS (p.D69_Q70insRWYSAMRD). Mutant allele frequencies detected by sequencing ranged from 9.98% to 15.97%. ConclusionsThe hemodynamic and molecular genetic phenotypes of IHCT closely resemble those observed in AVMs. Newly identified KRAS missense mutations, including cases with coexisting mutation types, and HRAS insertion mutations offer valuable insights into the genetic basis of vascular anomalies. These findings may also present potential targets for the development of novel pharmacotherapeutic interventions.

Strong association of single nucleotide polymorphisms in BRCA1, ATM, and CHEK2 with breast cancer susceptibility in a sub-population of Iranian women.

Breast cancer (BC) is the most prevalent malignancy in females worldwide. Mutations in the DNA repair pathway genes contribute to a significant increase in BC risk. The present study aimed to assess the frequency of polymorphisms in BRCA1, ATM, and CHEK2 genes and their association with BC susceptibility in the Kurdish population from the West of Iran. In the present case-control study, the distribution of single nucleotide polymorphisms (SNPs) in CHEK2 (rs17879961), ATM (rs28904921), and BRCA1 (rs80357906, rs1555576855, rs1555576858, and rs397509247) genes were investigated in 335 BC cases and 354 healthy-matched controls by Taqman allelic discrimination assay. The chi-square goodness-of-fit test was employed for the assessment of Hardy-Weinberg Equation. Relative risk and odds ratios were calculated based on the Koopman asymptotic score and the Baptista-Pike method, respectively. Also, the sensitivity and specificity of each polymorphism were assessed using the Wilson-Brown test and a P-value < 0.05 indicating significant differences between the two groups in all assessments. Data showed there was a strong association between rs397509247 (OR = 7.53, 95% CI 1.88-90.91, p = 0.004), rs1555576858 (OR = 10.53, 95% CI 0.01-0.51, p = 0.0005), and rs80357906 (OR = 6.33, 95% CI 0.05-0.043, p < 0.0001) in BRCA1 gene and rs17879961 (OR = 3.52, 95% CI 0.084-0.946, p = 0.02) in CHEK2 gene, with BC risk in the population of interest. Among these, rs28904921 in ATM gene demonstrated the strongest association (OR = 72.66, 95% CI 0.007-0.214, p < 0.0001). This suggests that these SNPs, particularly rs28904921, are significantly associated with an increased risk of BC in the studied population. Our results indicated that BRCA1, ATM, and CHEK2 polymorphisms have a high frequency in the Iranian breast cancer population, with some mutant allele frequencies being much higher than those reported in other populations. We have also provided a simple, multiplex, rapid, and accurate genotyping assay that is useful in clinical settings.

Evaluation of blood MSI burden dynamics to trace immune checkpoint inhibitor therapy efficacy through the course of treatment.

Analysis of serial liquid biopsy (LB) samples has been found to be a promising approach for the monitoring of tumor dynamics in the course of therapy for patients with colorectal cancer (CRC). Currently, somatic mutations are used for tracing the dynamics of the tumor via LB. However, the analysis of the dynamic changes in the molecular signatures such as microsatellite instability (MSI) is not currently used. We hypothesized that changes in blood MSI burden (bMSI) could be registered using serial LB sampling in the course of immune checkpoint inhibitors (ICI), and that its changes could potentially correlate with treatment outcomes. We report the preliminary findings of the observational trial launched to study (NCT06414304) the dynamics of bMSI in 9 MSI-positive CRC patients receiving ICI. NGS-based MSI testing was performed on both pre-treatment FFPE and serial LB samples. For patients who had detectable bMSI burden in any of the LB samples (n = 8, 89%), median bMSI was 1.4% (range, 0.01-40%). Among patients with detectable MSI in available FFPE samples, median MSI burden was 29.3% (range, 10-40%). bMSI detected in baseline LB and FFPE samples were positively correlated (Pearson's R 0.47). Maximal variant allele frequencies of driver mutations observed in LB were also positively correlated with bMSI burden (Pearson's R 0.7). Patients who had clinical benefit had undetectable bMSI burden at follow-up. Our results provide the rationale for further validation of bMSI as a predictive biomarker of ICI in MSI-positive patients.

Clinical utility of ctDNA by amplicon based next generation sequencing in first line non small cell lung cancer patients.

The assessment of ctDNA has emerged as a minimally invasive avenue for molecular diagnosis and real-time tracking of tumor progression in NSCLC. However, the evaluation of ctDNA by amplicon-based NGS has been not endorsed by all the healthcare systems and remains to be fully integrated into clinical routine practice. To compare tissue single-gene with plasma multiplexed testing, we retrospectively evaluated 120 plasma samples from 12 consecutive patients with advanced non-squamous NSCLC who were part of a prospective study enrolling treatment-naïve patients and in which tissue samples were evaluated using a single-gene testing approach. While the plasma ctDNA detection of EGFR and BRAF mutations had an acceptable level of concordance with the archival tissue (85%), discordance was seen in all the patients in whom ALK alterations were only detected in tissue samples. Among six responders and six non-responders, early ctDNA mutant allelic frequency (MAF) reduction seemed to predict radiologic responses and longer survival, whereas increasing MAF values with the emergence of co-mutations like BRAFV600E, KRASG12V or TP53M237I seemed to be an early indicator of molecular and radiologic progression. This report using an amplicon-based NGS assay on ctDNA underscores the real-life need for plasma and tissue genotyping as complementary tools in the diagnostic and therapeutic decision-making process.

A Novel AKT1, ERBB2, ESR1, KRAS, PIK3CA, and TP53 NGS Assay: A Non-Invasive Tool to Monitor Resistance Mechanisms to Hormonal Therapy and CDK4/6 Inhibitors.

Background: Patients with hormone receptor-positive (HR+)/HER2- metastatic breast cancer (mBC) generally receive hormonal therapy (HT) combined with CDK4/6 inhibitors (CDK4/6i). Despite this treatment, resistance mechanisms to CDK4/6i emerge and the majority of these patients experience disease progression (PD). This highlight the necessity to uncover the resistance mechanism to CDK4/6i through the identification of specific biomarkers. The primary objective is to assess the accuracy and feasibility of a novel multi-gene target panel NGS assay on circulating tumor DNA (ctDNA) to detect molecular alterations of AKT1, ERBB2, ESR1, KRAS, PIK3CA, and TP53 genes in women with BC undergoing HT plus CDK4/6i treatment. Secondarily, the study aims to explore the relationship between genomic profiling and clinical outcomes. Materials and Methods: Plasma samples were collected from 16 patients diagnosed with advanced/locally advanced HR+/HER2- BC at 2 time points: T0 (baseline) and at T1 (3 months after CDK4/6i treatment). Starting from 2 mL of plasma, ctDNA was isolated and libraries were set up using the Plasma-SeqSensei (PQS)® Breast Cancer IVD Kit, sequenced on Nextseq 550 and analyzed using the Plasma-SeqSensei™ IVD Software®. Results: Among the five patients who presented PD, three had PIK3CA mutations and, of these, two showed a higher mutant allele frequency (MAF) at T1. In three patients with stable disease and in eight patients with partial response, the MAF of the detected alterations decreased dramatically or disappeared during CDK4/6i treatment. Conclusions: Based on our findings, the liquid biopsy analysis using the PQS panel seems to be both feasible and accurate, demonstrating a strong sensitivity in detecting mutations. This exploratory analysis of the clinical outcome associated to the mutational status of patients highlights the potential of molecular analysis on liquid biopsy for disease monitoring, although further validation with a larger patient cohort is necessary to confirm these preliminary observations.

Post-surgery sequelae unrelated to disease progression and chemotherapy revealed in follow-up of patients with stage III colon cancer

We studied the poorly-known dynamics of circulating DNA (cir-nDNA), as monitored prospectively over an extended post-surgery period, in patients with cancer. On patients with stage III colon cancer (N=120), using personalised molecular tags we carried out the prospective, multicenter, blinded cohort study of the post-surgery serial analysis of cir-nDNA concentration. 74 patients were included and 357 plasma samples tested. During post-operative follow-up, the patients' median cir-nDNA concentration was greater (P<0.0001 in the [43-364 days range]) than both the median value in healthy individuals and the pre-surgery value. These cir-nDNA levels were highly associated with NETs markers (P-value associating MPO and cir-nDNA, and NE and cir-nDNA are 6.6 x 10-17, and 1.9 x 10-7), in accordance with previous reports which indicate that cir-nDNA are NETs by-products. Unexpectedly, in 34 out of 50 patients we found that NETs continued to be formed for an extended duration post-surgery, even in patients without disease progression. Given that this phenomenon was observed in patients without adjuvant CT, and in patients >18 months post-surgery, the data suggest that the persistence of NETs formation is not due to the adjuvant CT. (1), Given the inter-patient heterogeneity, the post-surgery cir-nDNA level cannot be considered a reliable value, and caution must be exercised when determining mutation allele frequency or the mutation status; and (2), specific studies must be undertaken to investigate the possible clinical impact of the persistent, low-grade inflammation resulting from elevated NETs levels, such as observed in these post-surgery patients, given that such levels are known to potentially induce adverse cardiovascular or thrombotic events. This work was supported by the H2020 European ERA-NET grant on Translational Cancer Research (TRANSCAN-2).

Genetic screening of newborns for deafness over 11 years in Beijing, China: More infants could benefit from an expanded program.

Genetic screening of newborns for deafness plays an important role in elucidating the etiology of deafness, diagnosing it early, and intervening in it. Genetic screening of newborns has been conducted for 11 years in Beijing. It started with a chip to screen for 9 variants of 4 genes in 2012; the chip screened for 15 variants of those genes in 2018, and it now screens for 23 variants of those genes. In the current study, a comparative analysis of three screening protocols and follow-up for infants with pathogenic variants was performed. The rates of detection and hearing test results of infants with pathogenic variants were analyzed. Subjects were 493,821 infants born at 122 maternal and child care centers in Beijing from April 2012 to August 2023. Positivity increased from 4.599% for the chip to screen for 9 variants to 4.971% for the chip to screen for 15 variants, and further to 11.489% for the chip to screen for 23 variants. The carrier frequency of the GJB2 gene increased from 2.489% for the chip to screen for 9 variants and 2.422% for the chip to screen for 15 variants to 9.055% for the chip to screen for 23 variants. The carrier frequency of the SLC26A4 gene increased from 1.621% for the chip to screen for 9 variants to 2.015% for the chip to screen for 15 variants and then to 2.151% for the chip to screen for 23 variants. According to the chip to screen for 9 variants and the chip to screen for 15 variants, the most frequent mutant allele was c.235delC. According to the chip to screen for 23 variants, the most frequent mutant allele was c.109G>A. The chip to screen for 15 variants was used to screen 66.67% (14/21) of newborns with biallelic variants in the SLC26A4 gene for newly added mutations. The chip to screen for 23 variants was used to screen 92.98% (53/57) of newborns with biallelic variants in the GJB2 gene (52 cases were biallelic c.109G>A) and 25% (1/4) of newborns with biallelic variants in the SLC26A4 gene for newly added mutations. Among the infants with pathogenic variants (biallelic variants in GJB2 or SLC26A4), 20.66% (25/121) currently have normal hearing. In addition, 34.62% (9/26) of newborns who passed the hearing screening were diagnosed with hearing loss. Findings indicate that a growing number of newborns have benefited, and especially in the early identification of potential late-onset hearing loss, as the number of screening sites has increased. Conducting long-term audiological monitoring for biallelic variants in individuals with normal hearing is of paramount significance.

A phase I/II study of nanoliposomal irinotecan and carboplatin in patients with advanced or metastatic, poorly differentiated gastroenteropancreatic neuroendocrine carcinoma characterized by tissue and liquid next-generation sequencing.

52 Background: For locally advanced or metastatic gastroenteropancreatic neuroendocrine carcinomas (GEP-NECs), standard chemotherapy typically incorporates etoposide or irinotecan plus platinum agents. Herein, we initiate a phase I/II trial to apply Nal-IRI in combination with carboplatin as the 1st line treatment in patients with GEP-NECs to define the dose-limiting toxicity (DLT) and recommended phase II dose (RP2D). Methods: In the phase I part, traditional 3+3 design was applied. Enrolled patients would receive escalating dose of 60 (level -1), 80 (starting dose, level 0) or 100 (level 1) mg/m2 salt-form nal-IRI plus carboplatin AUC=4 with maximum total dose of 600 mg on D1, every 21 days as a cycle. DLTs were evaluated during the first one cycle of treatment with tumor assessment every 6 weeks. Prophylaxis G-CSF was not allowed in the first cycle Paired testing of tissue (TBx) and liquid biopsy (LBx) was done by Illumina TSO500 platform before treatment. The second LBx would be repeated for confirmed partial responders when progression. Results: Totally 6 GEP-NEC patients with primary sites of colon in three, and each one in esophagus, ampulla of Vater and pancreas were enrolled to level 0. One patient had DLT of grade 4 neutropenia lasting for 3 days even under salvage G-CSF. Treatment-related adverse events showed grade 3/4 neutropenia (33.4%), grade 2 anemia (16.7%), grade 3 fatigue (16.7%), and grade 2 skin rash (16.7%). Four patients achieved confirmed partial response and 2 patients got stable disease, with the objective response rate of 66.7% (95% confidence interval: 22%-96%). After the data and safety monitoring board meeting, level 0 was decided as RP2D but not escalation to level 1 because of acceptable toxicity profiles and promising efficacies. Comprehensive genomic profiling showed that driver mutations were identified in three cases including two harboring BRAFV600E mutation and one with KRASG12D mutation. The other three cases lacking driver mutation showed frequent copy number alteration including 1 with MDM2 amplification (copy number 27) and a wild-type TP53. Homologous recombination deficiency (HRD), as determined by GIS score, was identified in a colon GEP-NEC who remained to be HRD-positive at progression but a higher TMB (from 4.7 to 10.2 Muts/Mb) and a higher BRAFV600E mutation allele frequency (from 24.1% to 43.6%) in tumor tissue, compared to the baseline profiling. Conclusions: Based on current data, the RP2D is 80 mg/m2 of nal-IRI (salt-form) and carboplatin of AUC=4, every 3 weeks. The extension phase II study in GEP-NEC is ongoing. Clinical trial information: NCT05385861 .

Associative study of maternal genetic variations with preeclampsia in Russian population: SNP-SNP interactions and haplotypes association

Preeclampsia is a pregnancy-associated hypertensive pathology that affects maternal and fetal quality of life. It was linked to several environmental and genetic factors including genetic polymorphisms. This study aimed to study the association, SNP-SNP interactions and haplotypes associations of thrombophilia, folate cycle and hypertension maternal genetic variations with preeclampsia risk in Russian women from Rostov region. 53 pregnant women diagnosed with preeclampsia were compared with 3108 women with relatively healthy pregnancy. DNA was extracted from blood samples and real time allele specific PCR was used for genotyping. MDR analysis was used for SNP-SNP interactions study. According to our data, F5(G1691A) and ITGB3(T1565C) mutant homozygotes were associated with higher risk of preeclampsia. MTRR (A66G) mutant allele (G) was shown as significant preeclampsia risk factor. ADD1 (G1378T) showed significantly higher frequency of mutant allele (T) and mutant homozygote genotype (TT) in preeclampsia patients comparing to control group. MDR showed that this polymorphism has the higher entropy among targeted variations which was confirmed by binary haplotypes association study. We have also examined linkage disequilibrium showing two haploblocks in between the studied genetic variations included MTHFR (C677T) and MTHFR (A1298C) block and AGT (T704C) with AGT (C521T) block. Results suggest several genetic variations and haplotypes as perspective maternal marker for preeclampsia which will contribute to improve prognosis tools and accordingly treatment and prevention possibilities.

Liquid Biopsy and 18F-FDG PET/CT Derived Parameters as Predictive Factors of Osimertinib Treatment in Advanced EGFR-Mutated NSCLC

ObjectivesDespite the outstanding results achieved by osimertinib for the treatment of advanced EGFR-mutated NSCLC, the development of resistance is almost inevitable. While molecular mechanism responsible for osimertinib resistance are being mostly revealed, the definition of predictive biomarkers is crucial in order to identify patients at higher risk of progression and optimize treatment strategy. Materials and MethodsThis is a prospective single-center study aimed to assess the potential role of liquid biopsy and 18F-FDG PET/CT derived metabolic parameters as noninvasive predictive biomarkers of osimertinib outcomes in advanced EGFR-mutated NSCLC patients. Patients underwent blood samples for ctDNA analysis at baseline, after 15 days and 1 month (t1) of osimertinib. 18F-FDG PET/CT was performed at baseline and after 1 month of osimertinib. ResultsSeventy-two advanced EGFR-mutated NSCLC patients treated with osimertinib in first (n = 63) and in second-line (n = 9) were prospectively enrolled. Baseline positive shedding status was significantly associated with a shorter progression-free survival (PFS) (9.5 vs. 29.2 months, P = .031). The achievement of an early metabolic response (MR) after 1 month of treatment led to a significantly improved PFS (16.8 vs. 5.5 months, P = .038) and OS (35.2 vs. 15.3 months, P = .047). Early MR was significantly correlated with subsequent radiologic response (P = .010). All 18F-FDG PET/CT baseline parameters were significantly related to baseline EGFR activating mutation allele frequency. Both clearance and no detection of EGFR at t1 were significantly associated with MR (P = .001 and P = .004, respectively). ConclusionMolecular and 18F-FDG PET/CT derived metabolic parameters might represent a useful tool to predict osimertinib outcome in advanced EGFR-mutated NSCLC patients.

Positive correlation between Bax and Bcl-2 gene polymorphisms with the risk of endometriosis: A case-control study.

Endometriosis is a chronic, gynecological disorder, and the disease's pathogenesis is still debatable. Genes related to apoptosis have been revealed to be deregulated in endometriosis. This study investigates the relationship between polymorphic variants of Bax -248G A and Bcl-2 -938C A promoter regions with endometriosis risk in an Iranian population. In this case-control study, the polymorphisms of Bax -248G A and Bcl-2 -938C A promoter regions were analyzed in 127 Iranian cases and 125 controls who were referred to Ali-ibn-Abi Taleb Educational hospital, Zahedan, Iran between May 2022 and February 2023. The genotypic analysis was performed for all the subjects using the polymerase chain reaction-restriction fragment length polymorphism method. The frequencies of mutant allele A carriers and the A allele of Bax -248G A polymorphism showed about 2-fold significant increase of endometriosis risk (p = 0.04; p = 0.01, respectively). The frequencies of the mutant genotype AA and A allele carriers of Bcl-2 -938C A polymorphism were approximately 4 and 2.5-fold higher in endometriosis compared to the control women, which were highly significant (p 0.001). Moreover, the allele A frequency of Bcl-2 -938C A was associated with a 2-fold higher risk of endometriosis (p 0.001). Furthermore, the combination effects of these 2 single nucleotide polymorphisms showed that women with Bax -248G A GGand Bcl-2 -938C A AA variant alleles were associated with about 5 times higher risk of endometriosis (p 0.001). Notably, a significant difference was observed in mutant allele distribution between minimal/mild (stage I and II) and moderate/severe (stage III and IV) women with endometriosis disease. The results of our study provide evidence that Bcl-2 -938C A and Bax -248G A single nucleotide polymorphisms might be associated with the risk of endometriosis.

Liquid biopsy in brain tumors: moving on, slowly.

Due to limited access to the tumor, there is an obvious clinical potential for liquid biopsy in patients with primary brain tumors. Here, we review current approaches, present limitations to be dealt with, and new promising data that may impact the field. The value of circulating tumor cell-free DNA (ctDNA) in the cerebrospinal fluid (CSF) for the noninvasive diagnosis of primary brain tumors has been confirmed in several reports. The detection of ctDNA in the peripheral blood is desirable for patient follow-up but requires ultrasensitive methods to identify low mutant allelic frequencies. Digital PCR approaches and targeted gene panels have been used to identify recurrent hotspot mutations and copy number variations (CNVs) from CSF or plasma. Tumor classification from circulating methylomes in plasma has been actively pursued, although the need of advanced bioinformatics currently hampers clinical application. The use of focused ultrasounds to open the blood-brain barrier may represent a way to enrich of ctDNA the peripheral blood and enhance plasma-based liquid biopsy. Monitoring CNVs and hotspot mutations by liquid biopsy is a promising tool to detect minimal residual disease and strengthen response assessment in patients with primary brain tumors. Novel methods to increase the relative and/or absolute amount of ctDNA can improve the clinical potential of plasma-based liquid biopsies.

Optimization of Tumor Dissection Procedures Leads to Measurable Improvement in the Quality of Molecular Testing

Molecular tests have an inherent limit of detection (LOD) and, therefore, require samples with sufficiently high percentages of neoplastic cells. Many laboratories use tissue dissection; however, optimal procedures for dissection and quality assurance measures have not been established. In this study, several modifications to tissue dissection procedures and workflow were introduced over 4 years. Each modification resulted in a significant improvement in one or more quality assurance measures. The review of materials following dissection resulted in a 90% reduction in KRAS mutations below the stated LOD (P=0.004). Mutation allele frequencies correlated best with estimated tumor percentages for pathologists with more experience in this process. The direct marking of unstained slides, use of a stereomicroscope, validation of extraction from diagnostic slides, and use of a robust, targeted next-generation sequencing platform all resulted in reduction of quantity not sufficient specimens from 20% to 25% to nearly 0%, without a significant increase in test failures or mutations below the LOD. These data indicate that post-dissection review of unstained slides and monitoring quantity not sufficient rate, test failure rate, and mutation allele frequencies are important tumor dissection quality assurance measures that should be considered by laboratories performing tissue dissections. The amendments to tissue dissection procedures enacted during this study resulted in a measurable improvement in the quality and reliability of this process based on these metrics.

Utility of KIT Mutations in Myeloid Neoplasms Without Documented Systemic Mastocytosis to Detect Hidden Mast Cells in Bone Marrow

BackgroundKIT p.D816 mutation is strongly associated with systemic mastocytosis (SM). Next-generation sequencing (NGS) is now routinely performed in almost all bone marrow sample and KIT mutations are detected from patients who are not known or suspected to have SM. Therefore, we wanted to assess if KIT mutations in this patient population are associated with unsuspected SM. MethodsWe searched NGS result in our institution with positive result for KIT mutation from patients with known/suspected myeloid neoplasms. Patients with previously documented history of systemic mastocytosis were excluded. Bone marrow biopsies from patients with KIT mutation were assessed with immunohistochemical stains for CD117 and mast cell tryptase (MST). ResultsBone marrow biopsies were assessed with immunohistochemical stains for CD117 and mast cell tryptase (n = 49). Most patients had acute myeloid leukemia (AML, n = 38) or chronic myelomonocytic leukemia (CMML, n = 6). Immunohistochemical stains for CD117 and tryptase were performed in all 49 patients. A total of 4 patients (8.2%) showed mast cell nodules where spindled shaped mast cells were present, meeting the WHO criteria for SM. All four patients had KIT p.D816V mutation and had high mutant allelic frequency (∼ 50%) except one patient (1%). ConclusionWe discovered approximately 8% of patients who had myeloid neoplasms with unexpected KIT mutations fulfilled the diagnostic criteria for systemic mastocytosis after additional immunohistochemical studies. Our data support that application of additional immunohistochemical studies is recommended to identify underrecognized SM when KIT mutations are found by molecular assays.

Plasmodium falciparum dhps and dhfr markers of resistance to sulfadoxine–pyrimethamine five years (2016–2020) after the implementation of seasonal malaria chemoprevention in Cameroon

Background Antimalarial drug resistance is a major challenge in the fight against malaria. Cameroon implemented seasonal malaria chemoprevention (SMC) with sulfadoxine–pyrimethamine and amodiaquine (SPAQ) to over 1.5 million children aged 3–59 months from 2016, raising concerns whether drug pressure may lead to a selection of known parasite resistance mutations. This study aimed at assessing the profiles of plasmodium falciparum dihydrofolate reductase (DHFR) and plasmodium falciparum dihydropteroate synthase (DHPS) gene mutations that encode enzyme targeting SP before and 5 years after the introduction of SMC in the northern part of Cameroon. Methods Dried blood spots were prepared from symptomatic P. falciparum-positive children prior to SPAQ administration in 2016 and after the SMC round of 2020. DNA was extracted using the Chelex-100 method, and dhfr and dhps mutations were determined after a nested polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique and agarose gel electrophoresis. Results 405 children with acute uncomplicated malaria were recruited. Of 405 samples, 201/405 (49.63%) were collected in 2016 and 204/405 (50.37%) were collected in 2020. High levels of mutant alleles S108N, C59R, N51I of dhfr were obtained both in 2016 and 2020 (174 (100%), 166 (95.4%), 131 (75.3%)); (140 (99.4%), 131 (92.2%), 114 (80.3%)) while the frequency of dhps mutant alleles in the A437G and K540E loci stood at 93 (51.9%) and 6 (3.4%) in 2016 and 73 (52.5%) and 4 (2.8%) in 2020, respectively. The quintuple resistant haplotype IRNGE was found in two (1.1%) and one (0.7%) in 2016 and 2020, respectively. No significant difference was observed in the frequency of the studied mutations between the two time points, although we noted a rise in the resistance conferring haplotype IRNG in 2020. Conclusions Continuous monitoring is recommended to preempt the widespread occurrence of high-grade resistance bearing parasites in the northern regions of Cameroon.