Mutant Allele Frequency Research Articles

Clinical sequencing uncovers the genomic characteristics and mutation spectrum of the 2018 African swine fever virus in Guangdong, China.

African swine fever (ASF) outbreak have caused tremendous economic loss to the pig industry in China since its emergence in August 2018. Previous studies revealed that many published sequences are not suitable for detailed analyses due to the lack of data regarding quality parameters and methodology, and outdated annotations. Thus, high-quality genomes of highly pathogenic strains that can be used as references for early Chinese ASF outbreaks are still lacking, and little is known about the features of intra-host variants of ASF virus (ASFV). In this study, a full genome sequencing of clinical samples from the first ASF outbreak in Guangdong in 2018 was performed using MGI (MGI Tech Co., Ltd., Shenzhen, China) and Nanopore sequencing platforms, followed by Sanger sequencing to verify the variations. With 22 sequencing corrections, we obtained a high-quality genome of one of the earliest virulent isolates, GZ201801_2. After proofreading, we improved (add or modify) the annotations of this isolate using the whole genome alignment with Georgia 2007/1. Based on the complete genome sequence, we constructed the methylation profiles of early ASFV strains in China and predicted the potential 5mC and 6mA methylation sites, which are likely involved in metabolism, transcription, and replication. Additionally, the intra-host single nucleotide variant distribution and mutant allele frequency in the clinical samples of early strain were determined for the first time and found a strong preference for A and T substitution mutation, non-synonymous mutations, and mutations that resulted in amino acid substitutions into Lysine. In conclusion, this study provides a high-quality genome sequence, updated genome annotation, methylation profile, and mutation spectrum of early ASFV strains in China, thereby providing a reference basis for further studies on the evolution, transmission, and virulence of ASFV.

Circulating Tumor DNA is Unreliable to Detect Somatic Gene Alterations in Gastrointestinal Peritoneal Carcinomatosis

IntroductionTumor agnostic circulating tumor DNA (ctDNA) is routinely used to guide treatment decisions in gastrointestinal (GI) cancers, especially metastatic cancers. The amount of ctDNA detected in plasma is affected by stage, tumor burden, and tumor vascularization. We hypothesized that peritoneal carcinomatosis (PC) is associated with lower ctDNA levels than other metastatic sites in GI cancers due to the plasma–peritoneal barrier.MethodsWe conducted a retrospective analysis of patients with stage II–IV GI cancers treated at our institution between 2015 and 2020 with available panel-based ctDNA results (Guardant 360TM). ctDNA analysis was performed on early and pretreatment samples. We compared the reported maximum variant allele frequency (mVAF) of somatic mutations across metastatic sites.ResultsOf the 279 patients with GI cancers (colorectal, upper GI, pancreaticobiliary), 212 had stage IV disease (PC: n = 61; visceral metastases: n = 138; other metastases: n = 13). Mean mVAF increased with increasing stages of disease (stage II: 3.6 ± 7; stage III: 6.4 ± 10; stage IV: 28.0 ± 51; p < 0.01). Among patients with stage IV disease, PC was associated with lower ctDNA levels independent of primary tumor site (PC only: 12.1%; PC+ visceral metastases: 26.8%; and visceral metastases only: 35.0%; p < 0.01). In a subset of patients (n = 27, matched pair analysis of genomic alterations (GAs) showed fewer GAs were detected in plasma compared with tissue.ConclusionsPC of GI origin is associated with significantly lower ctDNA levels compared with visceral metastasis. Caution is warranted when interpreting ctDNA results from patients with PC due to lower sensitivity for detecting actionable mutations.

A Prospective Validation Study of Lung Cancer Gene Panel Testing Using Cytological Specimens.

Simple SummaryThe gold standard for genetic panel test samples is considered to be tissue specimens. However, in clinical practice, tissue specimens are often unavailable. Therefore, using an amplicon-based high-sensitivity next-generation sequencing panel test capable of measuring eight druggable genes, we enrolled patients who underwent diagnostic procedures to evaluate analysis accuracy, nucleic acid yield, and quality using cytological specimens. Cytological specimens were suitable for both nucleic acid yield and specimen quality due to the ease of collection and processing. Cytological sample analysis detected gene mutations in 68.7% of lung adenocarcinomas, and these samples were consistent with companion diagnostic tests 99.5% of the time. Moreover, the allele frequency of gene mutations in cytological specimens showed a high correlation with tissue specimens. This is the first study to prospectively evaluate the feasibility of a lung cancer gene panel test using cytological specimens.Background: Genetic panel tests require sufficient tissue samples, and therefore, cannot always be performed. Although collecting cytological specimens is easier than tissue collection, there are no validation studies on the diagnostic accuracy of lung cancer gene panel tests using cytology samples. Methods: Using an amplicon-based high-sensitivity next-generation sequencing panel test capable of measuring eight druggable genes, we prospectively enrolled consecutive patients who underwent diagnostic procedures. We evaluated the analysis accuracy rate, nucleic acid yield, and the quality of cytological specimens under brushing, needle aspiration, and pleural effusion. We then compared these specimens with collected tissue samples. Results: In 163 prospectively enrolled cases, nucleic acid extraction and analysis accuracy was 100% in cases diagnosed with adenocarcinoma. Gene mutations were found in 68.7% of cases with 99.5% (95% CI: 98.2–99.9) concordance to companion diagnostics. The median DNA/RNA yield and DNA/RNA integrity number were 475/321 ng and 7.9/5.7, respectively. The correlation coefficient of the gene allele ratio in 64 cases compared with tissue samples was 0.711. Conclusion: The success of gene analysis using cytological specimens was high, and the yield and quality of the extracted nucleic acid were sufficient for panel analysis. Moreover, the allele frequency of gene mutations in cytological specimens showed high correlations with tissue specimens.

Bias and inconsistency in the estimation of tumour mutation burden

BackgroundTumour mutation burden (TMB), defined as the number of somatic mutations per megabase within the sequenced region in the tumour sample, has been used as a biomarker for predicting response to immune therapy. Several studies have been conducted to assess the utility of TMB for various cancer types; however, methods to measure TMB have not been adequately evaluated. In this study, we identified two sources of bias in current methods to calculate TMB.MethodsWe used simulated data to quantify the two sources of bias and their effect on TMB calculation, we down-sampled sequencing reads from exome sequencing datasets from TCGA to evaluate the consistency in TMB estimation across different sequencing depths. We analyzed data from ten cancer cohorts to investigate the relationship between inferred TMB and sequencing depth.ResultsWe found that TMB, estimated by counting the number of somatic mutations above a threshold frequency (typically 0.05), is not robust to sequencing depth. Furthermore, we show that, because only mutations with an observed frequency greater than the threshold are considered, the observed mutant allele frequency provides a biased estimate of the true frequency. This can result in substantial over-estimation of the TMB, when the cancer sample includes a large number of somatic mutations at low frequencies, and exacerbates the lack of robustness of TMB to variation in sequencing depth and tumour purity.ConclusionOur results demonstrate that care needs to be taken in the estimation of TMB to ensure that results are unbiased and consistent across studies and we suggest that accurate and robust estimation of TMB could be achieved using statistical models that estimate the full mutant allele frequency spectrum.

Characterization of the mutational landscape of high-grade serous ovarian tumors before and after neo-adjuvant chemotherapy (267)

Characterization of the mutational landscape of high-grade serous ovarian tumors before and after neo-adjuvant chemotherapy (267)

CRISPR-mediated knockout of nicotinic acetylcholine receptor (nAChR) α6 subunit confers high levels of resistance to spinosyns in Spodoptera frugiperda

Spinosyn insecticides (spinosad and spinetoram) have been widely used to control a number of agricultural pests including the fall armyworm, Spodoptera frugiperda. Mutations of the nicotinic acetylcholine receptor α6 subunit (nAChRα6) have been reported to confer high levels of resistance to spinosyns in several insect pests. Here we used CRISPR-mediated gene knockout to determine the involvement of S. frugiperda nAChRα6 (Sfα6) in spinosyns susceptibility. A Sfα6 knockout strain of S. frugiperda (Sfα6-KO) was established using dual single guide RNA (sgRNA) directed large fragment deletion with the CRISPR/Cas9 system. Sfα6-KO showed high levels of resistance to spinosad (307-fold) and spinetoram (517-fold) compared with the progenitor strain YJ-19, while no resistance was observed to emamectin benzoate, indoxacarb, chlorfenapyr, chlorantraniliprole and broflanilide. Genetic analyses confirmed that spinosad resistance in Sfα6-KO was autosomal, incompletely recessive and tightly linked to the edited deletion mutation of Sfα6. Our results provided in vivo functional evidence for Sfα6 as the major target of spinosyns against S. frugiperda, and demonstrated that disruption of Sfα6 causes high level resistance to spinosyns. Although no mutations of Sfα6 have yet been reported in any field populations of S. frugiperda, it is critical to develop F1 screens and/or DNA-based methods to detect and monitor the mutant allele frequencies of Sfα6 across global populations of S. frugiperda.

Somatic GJA4 gain-of-function mutation in orbital cavernous venous malformations

Orbital cavernous venous malformation (OCVM) is a sporadic vascular anomaly of uncertain etiology characterized by abnormally dilated vascular channels. Here, we identify a somatic missense mutation, c.121G > T (p.Gly41Cys) in GJA4, which encodes a transmembrane protein that is a component of gap junctions and hemichannels in the vascular system, in OCVM tissues from 25/26 (96.2%) individuals with OCVM. GJA4 expression was detected in OCVM tissue including endothelial cells and the stroma, through immunohistochemistry. Within OCVM tissue, the mutation allele frequency was higher in endothelial cell-enriched fractions obtained using magnetic-activated cell sorting. Whole-cell voltage clamp analysis in Xenopus oocytes revealed that GJA4 c.121G > T (p.Gly41Cys) is a gain-of-function mutation that leads to the formation of a hyperactive hemichannel. Overexpression of the mutant protein in human umbilical vein endothelial cells led to a loss of cellular integrity, which was rescued by carbenoxolone, a non-specific gap junction/hemichannel inhibitor. Our data suggest that GJA4 c.121G > T (p.Gly41Cys) is a potential driver gene mutation for OCVM. We propose that hyperactive hemichannel plays a role in the development of this vascular phenotype.

Increased blood-based intratumor heterogeneity (bITH) is associated with unfavorable outcomes of immune checkpoint inhibitors plus chemotherapy in non-small cell lung cancer

BackgroundThe combination of immune checkpoint inhibitors (ICIs) and chemotherapy has been the standard first-line treatment for advanced non-small cell lung cancer (NSCLC) patients with driver-gene negative. However, efficacy biomarkers for ICIs-based combination therapy are lacking. We aimed to identify potential factors associated with outcomes of ICIs plus chemotherapy at baseline and dynamic changes in peripheral blood.MethodsWe collected plasma samples of 51 advanced NSCLC patients without EGFR/ALK/ROS1 alteration at baseline and/or after two treatment cycles of ICIs plus chemotherapy. A blood-based intratumor heterogeneity (bITH) score was calculated based on the allele frequencies of somatic mutations using a 520-gene panel. bITH-up was defined as a ≥ 10% increase in bITH score from baseline, with a second confirmatory measurement after treatment.ResultsAt baseline, the number of metastatic organs and lung immune prognostic index (LIPI) were significantly associated with shorter progression-free survival (PFS) of ICIs plus chemotherapy, while bITH and other common molecular biomarkers, including ctDNA level, blood-based tumor mutational burden (bTMB), and PD-L1 expression, had no effect on PFS. LRP1B mutation at baseline was significantly associated with favorable outcomes to ICIs plus chemotherapy. There were 37 patients who had paired samples at baseline and after two cycles of treatment, with the median interval of 53 days. Intriguingly, patients with bITH-up had significant shorter PFS (HR, 4.92; 95% CI, 1.72–14.07; P = 0.001) and a lower durable clinical benefit rate (0 vs 41.38%, P = 0.036) than those with bITH-stable or down. Case studies indicated that bITH was promising to predict disease progression.ConclusionsThe present study is the first to report that increased bITH is associated with unfavorable outcomes of ICIs plus chemotherapy in advanced NSCLC patients.

CMDB: the comprehensive population genome variation database of China.

A high-quality genome variation database derived from a large-scale population is one of the most important infrastructures for genomics, clinical and translational medicine research. Here, we developed the Chinese Millionome Database (CMDB), a database that contains 9.04 million single nucleotide variants (SNV) with allele frequency information derived from low-coverage (0.06×-0.1×) whole-genome sequencing (WGS) data of 141 431 unrelated healthy Chinese individuals. These individuals were recruited from 31 out of the 34 administrative divisions in China, covering Han and 36 other ethnic minorities. CMDB, housing the WGS data of a multi-ethnic Chinese population featuring wide geographical distribution, has become the most representative and comprehensive Chinese population genome database to date. Researchers can quickly search for variant, gene or genomic regions to obtain the variant information, including mutation basic information, allele frequency, genic annotation and overview of frequencies in global populations. Furthermore, the CMDB also provides information on the association of the variants with a range of phenotypes, including height, BMI, maternal age and twin pregnancy. Based on these data, researchers can conduct meta-analysis of related phenotypes. CMDB is freely available at https://db.cngb.org/cmdb/.

IDH1 p.R132H ctDNA and D-2-hydroxyglutarate as CSF biomarkers in patients with IDH-mutant gliomas.

We aimed to evaluate IDH1 p.R132H mutation and 2-hydroxyglutarate (2HG) in cerebrospinal fluid (CSF) as biomarkers for patients with IDH-mutant gliomas. CSF was collected from patients with infiltrating glioma, and 2HG levels were measured by liquid chromatography-mass spectrometry. IDH1 p.R132H mutant allele frequency (MAF) in CSF-ctDNA was measured by digital droplet PCR (ddPCR). Tumor volume was measured from standard-of-care magnetic resonance images. The study included 48 patients, 6 with IDH-mutant and 42 with IDH-wildtype gliomas, and 57 samples, 9 from the patients with IDH-mutant and 48 from the patients with IDH-wildtype gliomas. ctDNA was detected in 7 of the 9 samples from patients with IDH-mutant glioma, and IDH1 p.R132H mutation was detected in 5 of the 7 samples. The MAF ranged from 0.3 to 39.95%. Total 2HG level, D-2HG level, and D/L-2HG ratio in CSF were significantly higher in patients with IDH-mutant gliomas than in patients with IDH-wildtype gliomas. D-2HG level and D/L-2HG ratio correlated with total tumor volume in patients with IDH-mutant gliomas but not in patients with IDH-wildtype gliomas. Our results suggest that detection of IDH1 p.R132H mutation by ddPCR and increased D-2HG level in CSF may help identify IDH-mutant gliomas. Our results also suggest that D-2HG level and D/L-2HG ratio correlate with tumor volume in patients with IDH-mutant gliomas. Further prospective studies with larger cohorts are needed to validate these findings.

ASSOCIATION BETWEEN VASCULAR ENDOTHELIAL GROWTH FACTOR GENE POLYMORPHISMS AND THE RISK OF PREECLAMPSIA IN IRAQI PREGNANT WOMEN

Background: Vascular endothelial growth factor (VEGF) is an essential factor for angiogenesis and plays important role in placental development. Objective: To investigate the association between VEGF single nucleotide polymorphisms (SNPs) +936 Cytosine/Thymine (C/T) and -634 Guanine/Cytosine (G/C) and preeclampsia risk. Methods: A total of 50 cases of pregnant women with preeclampsia and 50 healthy pregnant women (as a control) were involved in this case control study. Blood samples were collected form each woman and the Deoxyribonucleic acid (DNA) was extracted. Amplification of VEGF gene was done by conventional polymerase chain reaction (PCR) and then detection of SNPs-634 G/C and +936 C/T were carried out by restriction fragment length polymorphism PCR (PCR-RFLP). Results: The frequency of different genotypes of VEGF SNPs +936 C/T &amp;-634 G/C are in accordance with Hardy Weinberg equilibrium. VEGF polymorphism +936 C/T appeared in 3 genotypes after digestion with restriction enzymes Cytosine Cytosine (CC), Cytosine Thymine (C/T) &amp; Thymine Thymine (TT). VEGF SNP -634 G/C appeared in 3 genotypes after digestion with restriction enzymes; those were Guanine Guanine (GG), Guanine Cytosine (GC) and CC. The heterozygous genotype CT of polymorphism +936 C/T was more frequent among preeclamptic patients than the controls (26% versus 16%). Likewise, TT genotype was more frequent among preeclamptic patients (8% versus 2%) with no significant differences. At the allelic level, the difference was more prominent. The frequency of mutant allele (T allele) was much more frequent in preeclamptic patients than controls (21% versus 10%) with a statistically significant difference (p=0.049). Although CC genotype of polymorphism -634 G/C was more frequent among preeclamptic patients than controls (14% versus 10%), the difference was not significant (p=0.704). Likewise, there were no significant differences in allele frequency. Conclusion: The study suggested that the mutant T allele of VEGF +936 C/T polymorphism was associated with increased preeclampsia risk and disease development. Keywords: Vascular endothelial growth factor, polymorphisms, preeclampsia Citation: Khaleel MF, Al-Moayad HA. Association between vascular endothelial growth factor gene polymorphisms and the risk of preeclampsia in Iraqi pregnant women. Iraqi JMS. 2022; 20(1): 106-112. doi: 10.22578/IJMS.20.1.14

Molecular Epidemiological Survey for Degenerative Myelopathy in German Shepherd Dogs in Japan: Allele Frequency and Clinical Progression Rate.

Simple SummaryCanine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease caused by a DM-associated mutation (SOD1:c.118G>A, p.E40K) that commonly occurs in German Shepherd Dogs (GSDs). This study aimed to determine the mutant allele frequency in the Japanese GSD population and to analyze the clinical progression rate among GSDs with the homozygous mutant A/A genotype. The survey found 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%) among the 541 dogs examined, indicating that the mutant allele frequency was 0.220; the Japanese GSD population can be considered to be in Hardy–Weinberg equilibrium. Clinical analysis revealed that the clinical progression rate was particularly high (100%) among A/A dogs aged >10 years. Appropriate mating management is crucial for the management and prevention of DM in the Japanese GSD population.Canine degenerative myelopathy (DM) is an adult-onset, chronic, progressive neurodegenerative disease reported in multiple canine breeds, including the German Shepherd Dog (GSD). Clinical signs include progressive motor neuron paralysis, which begins in the pelvic limbs and eventually leads to respiratory distress, which may necessitate euthanasia. A common DM-associated mutation is a single nucleotide substitution that causes an amino acid substitution (c.118G>A, p.E40K) in the canine SOD1 gene. This SOD1 mutation and the clinical progression rate of A/A risk genotype in the Japanese GSD population have not been analyzed before. Therefore, the aim of this study was to determine the frequency of the mutated allele and analyze the clinical progression rate in the Japanese GSD population. We studied 541 GSDs registered with the Japanese German Shepherd Dog Registration Society between 2000 and 2019. Genotyping was performed using real-time PCR with DNA extracted from the hair roots of each dog. The study revealed 330 G/G dogs (61%), 184 G/A dogs (34%), and 27 A/A dogs (5%), indicating a frequency of the mutant allele of 0.220, which are in Hardy–Weinberg equilibrium. We analyzed the clinical signs in A/A dogs with an age limit of 10 years based on information obtained from the dogs’ owners. Of the seven A/A dogs older than 10 years, owners reported DM-related clinical signs, indicating a clinical progression rate of 100%. These results, further genotyping, and thorough clinical examinations of SOD1 A/A risk genotype will help control and prevent DM in the Japanese GSD population.

Quantitative assessment of copy number alterations by liquid biopsy for neuroblastoma.

Liquid biopsy, a method of detecting genomic alterations using blood specimens, has recently attracted attention as a noninvasive alternative to surgical tissue biopsy. We attempted quantitative analysis to detect amplification of MYCN (MYCNamp) and loss of heterozygosity at 11q (11qLOH), which are clinical requisites as prognostic factors of neuroblastoma (NB). In this study, cell-free DNA (cfDNA) was extracted from plasma samples from 24 NB patients at diagnosis. Copy numbers of MYCN and NAGK genes were quantitatively analyzed by droplet digital PCR (ddPCR). 11qLOH was also assessed by detecting allelic imbalances of heterozygous single nucleotide polymorphisms in the 11q region. The results obtained were compared to those of specimens from tumor tissues. The correlation coefficient of MYCN copy number of cfDNA and tumor DNA was 0.88 (p < 0.00001). 11qLOH was also accurately detected from cfDNA, except for one case with localized NB. Given the high accuracy of liquid biopsy, to investigate components of cfDNA, the proportion of tumor-derived DNA was estimated by examining the variant allele frequency of tumor-specific mutations in cfDNA. The proportion of tumor-derived DNA in cfDNA was 42.5% (range, 16.9%-55.9%), suggesting sufficient sensitivity of liquid biopsy for NB. In conclusion, MYCN copy number and 11qLOH could be quantitatively analyzed in plasma cfDNA by ddPCR assay. These results suggest that plasma cfDNA can be substituted for tumor DNA and can also be applied for comprehensive genomic profiling analysis.

PB2066: THE STUDY OF GSTP1 AND MDR1 GENES POLYMORPHISMS IN HODGKIN’S LYMPHOMA AND THEIR LINK WITH CHEMOTHERAPY RESPONSE.

Background: Glutathione-S-transferase P1 and ATP-dependent transporter P-glycoprotein are enzymes of the second phase of xenobiotic metabolism that participate in biotransformation of agents of standard protocol of Hodgkin’s Lymphoma chemotherapy. The polymorphism of the GSTP1 and MDR1 genes encoding these proteins causes changes in their activity, which can provide an individual patients’ response to chemotherapy. In this regard, the search for predictive genetic factors for the effectiveness of antitumor therapy is an important task. Aims: to demonstrate the association between polymorphic variants of GSTP1 (A313G, C341T) and MDR1 (C1236T, G2677T, C3435T) genes in patients with Hodgkin’s lymphoma. Methods: Overall, 77 Hodgkin’s lymphoma patients aged 18-74 years who were treated by BEACOPP and ABVD chemotherapy protocols in Oncohematological Departments of City Clinical Hospital №2 and Novosibisk State District clinical Hospital were enrolled in this study. The effectiveness of chemotherapy was evaluated after 4-6 cycles of chemotherapy in accordance with clinical guidelines. Depending on the achieved results of treatment, all patients were divided into two groups: patients with complete/partial remission and patients with progressive/refractory or relapsed lymphoma. Polymorphisms of GSTP1 (A313G, C341T) and MDR1 (C1236T, G2677T, C3435T) genes were defined by PCR method with real-time detection in the samples of peripheral blood in the Centre of Collective Usage «Proteomic analysis» FRC FTM. The odds ratio (OR) used to evaluate the link between genotypes with efficacy of chemotherapy. Results: We found the following frequencies of mutant alleles in Hodgkin’s Lymphoma patients: GSTP1313G – 31.17%, GSTP1 341T – 9.7%, MDR1 1236T – 50.00%, MDR1 2677T – 48.70%, MDR13435 ТОНН – 58.44%. Patient’s genotypes were similar to described genotypes of healthy Europeans. There was no significant difference in distribution of frequency alleles and GSTP1 A313G (χ2=0,89, p=0,34), GSTP1 C341T (χ2=0,11 R=0,74), MDR1 C1236T (χ2=0,01, p=0,93), MDR1 G2677T (χ2=0,37, p=0,54), MDR1 C3435T (χ2=0,01, р=0,97) genotypes between group of Hodgkin’s lymphoma patients with complete/partial remission and group with progressive/refractory or relapsed lymphoma. At the same time we found the significant correlation between GSTP1 A313G heterozygotes and formation of progressive/refractory and prolapsed course of disease (OR = 10.80; CI 95% = 1.23 – 496.52). There was no significant association between efficacy of chemotherapy and other studied genotypes, namely GSTP1 C341T, MDR1 C1236T, MDR1 G2677T, MDR1 C3435T genotypes. Summary/Conclusion: The observed correlation between GSTP1 A313G gene polymorphisms and formation progressive/refractory or prolapsed course of disease needs to be studied to provide safe and effective specific chemotherapy.

P1051: A PHASE 2 STUDY OF IMG-7289 (BOMEDEMSTAT) IN PATIENTS WITH ADVANCED MYELOFIBROSIS

Background: Lysine-specific demethylase-1 (LSD1) is an activity critical for the self-renewal of malignant myeloid cells and maturation of megakaryocytes, cells central to the pathogenesis of MF. Bomedemstat is an orally active LSD1 inhibitor that in mouse models ameliorated the hallmarks of MPNs and improved survival (Kleppe et al. 2015; Jutzi et al. 2018). Aims: IMG-7289-CTP-102 is an ongoing, global, open-label Phase 2 study evaluating bomedemstat dosed QD in MF patients (NCT03136185). Key eligibility criteria include patients intolerant, refractory, resistant, or inadequately controlled by approved therapy, and platelet count ≥100 x 109/L. Key objectives are safety and reduction of spleen volume (SVR) by MRI/CT and total symptoms scores (TSS) using the MPN-SAF instrument. Methods: Serial bone marrow (BM) biopsies and imaging studies are read centrally. 261 genes are serially sequenced to quantify changes in the allelic frequencies of mutations (MAF) and identify new mutations. The starting dose is 0.6 mg/kg/d titrating, as needed, to a platelet count of 50-75x109/L. Results: At 89 patients, the study is now fully enrolled: 46% primary MF, 33% post-essential thrombocythaemia-MF, 21% post-polycythaemia vera-MF. Median age is 68 (35-88) with 52% males. Prior treatment with ruxolitinib was reported in 83% (74/89); 46% had also received at least 1 additional treatment. 30% of patients (27/89) had received ≥1 RBC transfusion prior to dosing. By IPSS, 53% were high-risk, 40% int.-2, and 7% int.-1. At screening (N=103), sequencing to a mean depth of >1000 bp, JAK2 was mutated in 69%, CALR in 21%, MPL in 6%; 59% had ≥2 mutations of which 71% were high-molecular risk mutations in ASXL1, IDH1/2, EZH2, U2AF1, TP53 and/or SRSF2. At data cutoff (3 Feb 2022), the median duration of treatment is 28 weeks (2-131). Of patients for whom TSS data is available at 24 weeks in those with baseline values ≥20, 72% (18/25) had a reduction in TSS; 24% (6/25) reported a ≥50% reduction. Of patients evaluable for SVR (N=50), 64% had a reduction in spleen volume from baseline; in 28%, SVR was ≥20%. Of evaluable patients (N=41), 90% had stable (∆ <±1.0 g/dL) or improved (≥1.0 g/dL) hemoglobin. Of patients with BM fibrosis scoring post-baseline (N=52), 31% improved by 1 grade and 50% were stable. CCL5 and S100A8/A9 cytokines were elevated at baseline in 50% (16/32) and 78%, respectively; at Day 84, 81% and 68%, respectively, showed reductions of at least 10% and of those, 100% (CCL5) and 47% (S100A8/A9) normalized. In follow-up sequencing at around Week 24, of 60 mutant alleles in 32 patients, the mean MAF fell by 39% in 48%. JAK2 MAFs fell by 31% [SD 5%] in 46% (N=24); ASXL1 MAFs fell 40% in 71%. Clones with JAK2 and/or ASXL1 mutations were most affected. No new mutations have been identified and no patient has transformed to AML. The most common non-hematologic AEs reported by patients was dysgeusia in 36% (32/90) and diarrhoea in 34% (31/90). All dysgeusia events were grade 1/2 and 1 led to treatment discontinuation. Of 14 related SAEs, 4 were Grade 2, 9 Grade 3 and 1 Grade 4 (thrombocytopenia). Twenty-nine patients remain on bomedemstat. Early terminations due to AEs occurred in 18 (20%) patients (9 related to bomedemstat), and 13 discontinued for other reasons. There have been no safety signals, DLTs, or deaths related to drug. Summary/Conclusion: In patients with advanced MF, bomedemstat alone had an acceptable tolerability profile, relieved symptoms, reduced spleen volume and mutation burden while improving fibrosis and anemia without safety signals.

P1256: A NOVEL DROP-OFF DIGITAL PCR ASSAY FOR CXCR4 MUTATION SCREENING IN IGM GAMMOPATHIES: FIRST DATA FROM THE FONDAZIONE ITALIANA LINFOMI BIO-WM STUDY

Background: In IgM gammopathies: Waldenström’s Macroglobulinemia (WM) and IgM gammopathy of uncertain significance (IgM-MGUS), MYD88L265P and CXCR4S338X are the most frequent mutations, and both have clinical implications in WM. Recent studies demonstrated that, in unselected bone marrow (BM) samples, allele specific polymerase chain reaction (AS-qPCR) was superior in detecting CXCR4S338X compared to next generation sequencing, and digital PCR (dPCR) was more sensitive than AS-qPCR for MYD88L265P detection, highlighting the lack of a gold standard molecular method in WM diagnostics. Aims: The aim of the present study was to evaluate a new sensitive approach based on a drop-off dPCR assay for CXCR4S338X mutation detection in BM and peripheral blood (PB) samples from WM and IgM-MGUS patients, enrolled between 2018 and 2020 in the multicenter observational trial from the fondazione italiana linfomi (“BIO-WM”, NCT03521516), and to correlate the mutational status with clinical features. Methods: Genomic DNA from 240 patients (184 WM, 56 IgM-MGUS) was analyzed by dPCR: 51 BM-CD19 selected cells (CD19+), 189 BM and 239 PB unselected white blood cells (WBC). Allele frequency (AF) of CXCR4 mutations at p.S388 locus (CXCR4MUT) was detected by a drop-off dPCR assay, with a sensitivity of 0,001%. For all patients, MYD88L265P mutation data were also available. Results: Forty-one out of 184 WM patients (22%) showed CXCR4MUT in BM (median AF 1.6%, range: 0.14%-23.1%) while only 8 were positive also in PB (20% of the BM mutated cases, median AF: 0.6%, range: 0.23%-35.2%). Moreover, 39/41 (95%) were MYD88L265P, too. Conversely, only 5 out of 56 IgM-MGUS patients (9%) scored CXCR4MUT in BM (median AF 0.65%, range: 1.1%-0.2%) and none in PB: all patients were MYD88L265P, too. Interestingly, 38 BM WBC samples (36 WM, 2 IgM-MGUS) mutated for both CXCR4MUT and MYD88L265P showed correlation in mutational levels between the two mutations (R2=0.8). On the contrary, no correlation was observed between the two mutations in CD19+ cells (R2=0.06). Overall, clinical features of CXCR4MUT vs CXCR4WT patients did not differ significantly, except for lower hemoglobin levels (median 11.6 g/dl vs 12.8 g/dl, p=0.002) and higher serum IgM monoclonal component (median 1.76 g/dl vs 1 g/dl, p=0.016). Only 15/41 WM CXCR4MUT patients (37%) received immediate treatment at the time of trial enrollment (7 DRC, 3 R-Benda, 5 others: 2 single agent and 3 R-chemo based therapy). These cases showed higher disease and mutational burden compared to the 26 CXCR4MUT patients still in watch and wait (WW) at the time of the present analysis. Indeed, median histologic BM infiltration was 80% vs 30% (p<0.001), median IgM monoclonal component was 3.6 g/dl vs 1.6 g/dl (p=0.003) and median CXCR4MUT AF was 9.5% vs 0.8% (p<0.001), respectively. Finally, at the present the median follow-up (FU) of the trial is relatively short (34 months) and no statistically significant differences in outcome parameters were recorded between CXCR4MUT and CXCR4WT cases. Summary/Conclusion: We here describe a sensitive, easily applicable and standardizable approach for detection of the most common CXCR4 mutations on BM WBC, with no need for CD19+ selection. A lower mutational AF was observed in IgM-MGUS compared to WM and, similarly to what was previously reported for MYD88L265P, PB resulted suboptimal for CXCR4MUT detection. A statistically significant difference in CXCR4MUT level was observed between patients treated at the time of enrollment and those still in WW. However, a longer FU is needed to better clarify the clinical implications of CXCR4 mutations in our series.

P1048: MYF3001: A RANDOMIZED OPEN LABEL, PHASE 3 STUDY TO EVALUATE IMETELSTAT VERSUS BEST AVAILABLE THERAPY IN PATIENTS WITH INTERMEDIATE-2 OR HIGH-RISK MYELOFIBROSIS REFRACTORY TO JANUS KINASE INHIBITOR

Background: Myelofibrosis (MF) is a life-threatening myeloproliferative neoplasm. The Janus Kinase inhibitors (JAKi) ruxolitinib and fedratinib are the only FDA approved treatment options for MF. Despite benefits reported with ruxolitinib in the front-line setting, a high proportion of patients discontinue treatment (Abdelrahman 2015), and the median overall survival (OS) is 11-16 months (Kuykendall 2018; Newberry 2017; Schain 2019; Palandri 2019; Mascarenhas 2020), highlighting a great unmet need for pts non-responsive to a JAKi treatment. Imetelstat, a first-in-class telomerase inhibitor, has shown meaningful clinical improvement in IMbark, a Phase 2 study in patients with intermediate-2 or high-risk MF who have relapsed after or are refractory to JAKi (Mascarenhas JCO 2021). Treatment with 9.4 mg/kg imetelstat resulted in 32.2% symptom response (total symptom score [TSS] reduction ≥50%) at Week 24 and median overall survival (OS) of 29.9 months with overall study follow up of 27.4 months. Dose-dependent inhibition of telomerase with imetelstat resulted in on-target activity that correlated with clinical benefits; dose-dependent reduction in variant allele frequency of MF driver mutations indicated targeting of the underlying malignant clone. Aims: The Phase 2 results support continued study of imetelstat 9.4 mg/kg in a Phase 3 randomized controlled study, for which the study design is described here. Methods: Study MYF3001 (IMpactMF; NCT04576156) is an open label, randomized (2:1), multicenter, Phase 3 study of imetelstat compared with best available therapy (BAT) in ~320 patients with intermediate 2 or high-risk MF refractory to JAKi treatment. Patients will be randomized to receive imetelstat 9.4 mg/kg IV every 21 days or investigator selected BAT including hydroxyurea, thalidomide, interferon, danazol, hypomethylating agents, chemotherapy, or other non-JAKi containing therapy as appropriate). Eligible patients will be stratified based on a) Intermediate 2 or high-risk per Dynamic International Prognostic Scoring System; b) platelet count at entry (platelets ≥ 75 and < 150 x 109/L vs ≥ 150 x 109/L). Patients who meet progressive disease criteria and discontinue BAT may be eligible to crossover to imetelstat. Results: The primary endpoint is OS and one interim analysis is planned when approximately >71% of death events have occurred. Secondary endpoints include symptom and spleen response rates at week 24, progression-free survival, clinical response assessment per modified 2013 International Working Group - Myeloproliferative Neoplasms Research and Treatment criteria, time to and duration of response, reduction in degree of bone marrow fibrosis, safety, pharmacokinetics and patient-reported outcomes. Biomarkers and mutation analyses will be performed to evaluate the impact of imetelstat on reduction/depletion of malignant clones. (Figure 1) Image:Summary/Conclusion: Approximately 180 sites are planned in North and South America, Europe, Middle East, Australia and Asia. The study is open for enrollment.

P996: UTILITY OF KIT P.D816 IN MYELOID NEOPLASM WITHOUT DOCUMENTED SYSTEMIC MASTOCYTOSIS TO DETECT HIDDEN MAST CELLS IN BONE MARROW

Background:KIT p.D816 mutation is strongly associated with systemic mastocytosis (SM). Although it is not specific for SM, it is one of the diagnostic criteria for SM since its presence in the right morphologic context (i.e. presence of atypical mast cell clusters) is a strong support for neoplastic nature of mast cells. 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. Aims: Therefore, we wanted to assess if KIT mutations in this patient population are associated with unsuspected SM. Methods: We searched NGS result in our institution between 1/1/2013 and 9/30/2021 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). Results: A total of 49 patients who had KIT mutation and had available formalin-fixed paraffin embedded blocks were identified. Thirty-eight (77.6%) patients has acute myeloid leukemia (AML) and 6 patients had chronic myelomonocytic leukemia (CMML, 12.2%). The remaining patients had myelodysplastic syndrome (n=1), myelodysplastic/myeloproliferative neoplasm, unclassifiable (n=1), myeloproliferative neoplasm (MPN, n=1), AML in remission (n=1) and therapy-related myeloid neoplasm (n=1). A total of 41 patients had a single KIT mutation and 8 patients had 2 or more KIT mutations. KIT p.D816V mutation was the most common mutation (n=38, 64.4%), followed by D816Y (n=8, 13.6%), D816H (n=5, 8.5%), Y418_D419insFF (n=2, 3.4%), R815_D816insVL (n=1, 1.7%), S197L (n=1, 1.7%), K558E (n=1, 1.7%), D419del (n=1, 1.7%), N822K (n=1, 1.7%) and R815_D816insIPP (n=1, 1.7%). Immunohistochemical stains for CD117 and MST were performed in all 49 patients. The MST stain was more suitable since 10 patients (20%) had increased blasts with CD117 expression which obscured morphologic review. A total of 4 patient (8.2%) showed mast cell nodules where spindled shaped mast cells were present, meeting the criteria for SM. All four patients had KIT p.D816V mutation and had high mutant allelic frequency (~50%) except one patient (1%). These patients had MPN (n=1), AML (n=1), AML in remission (n=1) and CMML (n=1), respectively. One (2%) patient has increased (>10%) mast cells but scattered throughout the bone marrow space without nodules and mast cells were round-to-ovoid shape. Eleven patients (22.4%) showed rare scattered mast cells and 33 patients (67.3%) did not show mast cells at all. Summary/Conclusion: We discovered approximately 8% of patients who had myeloid neoplasms with unexpected KIT mutations are met for systemic mastocytosis after additional immunohistochemical studies. Our data support that application of additional immunohistochemical studies are recommended to identify underrecognized SM when KIT mutations are unexpectedly found by molecular assays.

Abstract 534: An ultrasensitive high-plex assay detecting 24 PIK3CA mutations using SAGAsafe® technology and 6-color Crystal Digital PCR™

Abstract Starting from a minimally invasive liquid biopsy sample such as a blood sample, one can determine genetic and biological properties of cancer by measuring circulating tumor DNA (ctDNA). ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. The focus of this study was to demonstrate the performance of the patented SAGAsafe digital PCR chemistry using 6-color Crystal Digital PCR, by adapting a SAGAsafe assay for a clinically relevant oncogene, PIK3CA. PIK3CA encodes the p110-alpha kinase and is the target of a recently approved drug, alpelisib, for metastatic breast cancer. By combining the power of the SAGAsafe technology with that of 6-color Crystal Digital PCR on the naica system®, we present a highly multiplexed, ultrasensitive proof-of-concept PIK3CA detection assay capable of detecting and quantifying 24 PIK3CA mutations in a single digital PCR reaction. By evaluating the sensitivity, specificity and precision of the high-plex 6-color PIK3CA digital PCR assay, we show that despite the complexity of the assay, the data were extremely clean resulting in highly accurate quantifications down to mutant allele frequencies (MAFs) in the 0.01% range. In addition, superb PIK3CA mutation detection specificity and sensitivity were observed with Limits of Blank ranging from 0.003% to &amp;lt;0.001% MAF, and Limits of Detection ranging from 0.009% to 0.002% MAF in a high background of wild-type DNA. Moreover, a high concordance was observed between Crystal Digital PCR PIK3CA results and NGS data on breast cancer biopsies. By joining 6-color Crystal Digital PCR and ultrasensitive SAGAsafe technology, high sensitivity and specificity can be maintained even in a complex multiplex digital PCR assay. This proof-of-concept assay sets the stage for the future development of ultrasensitive, highly multiplexed cancer detection panels to achieve better patient stratification and monitoring during therapy. Citation Format: Miguel Alcaide, Anthony M. George, Yilun Chen, Cécile Jovelet, Sahbi Ben Kilani, Lao H. Saal, Allison C. Mallory. An ultrasensitive high-plex assay detecting 24 PIK3CA mutations using SAGAsafe® technology and 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 534.

Abstract 5158: Detection of EGFR mutations in plasma cfDNA and paired CTCs of NSCLC patients before and after osimertinib therapy using crystal digital PCR

Abstract Background: Circulating tumor DNA (ctDNA) analysis has clinical utility in EGFR mutantNSCLC. Circulating tumor cells (CTCs) constitute a unique source of information at thecellular level. Digital PCR (dPCR) is a valuable tool for accurate and valid mutation analysisin liquid biopsy. The aim of the current study was to detect EGFR mutations in plasma cfDNA and paired CTC-fractions of NSCLC patients before the initiation of osimertinib therapy and at progression of disease using crystal digital PCR (cdPCR). Patients and Methods: Forty-eight patients with EGFR mutated lung adenocarcinomas resistant to 1st or 2nd generation EGFR TKIs were treated with osimertinib in the context of a multicenter Phase II clinical study (NCT02771314). 2 mL of plasma were used for cfDNA extraction using the IDXtract kit (ID-Solutions, France). Crystal dPCR (naica® system, StillaTechnologies, France) was used to quantify mutation allele frequencies (MAF) of EGFRmutations in 91 cfDNA samples before and after osimertinib. 80 identical plasma samples were also analyzed with the FDA-approved cobas® technology. In parallel, EGFR mutations were detected with cdPCR in 64 matched CTC-fractions enriched by Parsortix™ (ANGLE plc, UK).Quality control steps were followed both at the preanalytical and analytical level. Results: Direct comparison between cdPCR and cobas® revealed high concordance rates forall EGFR mutations; however, in case of T790M, cdPCR proved to be more sensitive. Patients with higher %MAFs at PD presented significantly lower PFS compared to those with lower levels or without EGFR mutations. Direct comparison of EGFR genotyping between primary tissue and baseline plasma cfDNA samples revealed high concordance rates, too. During EGFR mutation analysis in paired CTC-derived gDNA, 11 samples were found positive for EGFR mutations with %MAF ranging from 0.2 to 2.25%. Conclusions: Crystal dPCR combines the unique benefits of sensitivity and accuracy with the multiplexing capacity for the detection of multiple EGFR mutations in plasma cfDNA samples and CTCs. High concordance rates were observed between cdPCR and cobas® assay. For the first time, cdPCR was applied to detect EGFR mutations in CTC-derived gDNA of NSCLC patients under osimertinib. Discordance between plasma and CTC-fractions might be indicative of tumor heterogeneity and predictive for acquired resistance. Acknowledgements-The research presented was carried out within the framework of a Stavros NiarchosFoundation grant to the National and Kapodistrian University of Athens-The authors would like to thank Larry Ba (Stilla Technologies, France) and Daniel Henaff(ID-solutions, France) for kindly providing the naica® digital PCR system and the ID-solutionskits, respectively. Citation Format: Aliki Ntzifa, Athanasios Kotsakis, Vassilis Georgoulias, Evi Lianidou. Detection of EGFR mutations in plasma cfDNA and paired CTCs of NSCLC patients before and after osimertinib therapy using crystal digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5158.