Variant Allele Fraction Research Articles

Circulating tumor DNA–based genomic landscape of KRAS wild-type pancreatic adenocarcinoma.

747 Background: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer deaths. KRAS is a primary oncogenic driver in the majority of PDAC patients. KRAS wild-type (wt) PDAC (~10%) is a molecularly heterogeneous subgroup that may harbor alternate drivers and targetable alterations. In this study, we sought to characterize the circulating tumor DNA (ctDNA) landscape of alterations in PDAC patients harboring KRAS mutations ( KRAS mut) compared to KRAS wt. Methods: We analyzed ctDNA samples from 2000 patients ( N= 1000 each for KRAS mut and KRAS wt) collected prospectively between 2019-2022 using a 74-83 gene next generation sequencing panel (Guardant360). KRAS mutation not detected was used as a proxy for KRAS wt. To limit bias from low tumor shed, samples were excluded if they did not have maximum variant allele fraction (VAF) > 0.34% and if the only maximum VAF > 0.34% was within putative germline range (40-60%). Statistical analyses were performed using Fisher’s exact test or t-test. Results: No significant gender differences were noted between KRAS wt and KRAS mut patients. Median age was 72yo in KRAS wt and 69yo in KRAS mut patients. Overall, KRAS wt patients had higher frequency of alterations in ATM, BRAF, ERBB2, FGFR2, JAK3, MET and NOTCH1 compared to KRAS mut (all p < 0.05). Meanwhile, KRAS wt patients had lower frequency of alterations in CDKN2A, SMAD4, CDK6, ARID1A, and TP53 compared to KRAS mut (all p < 0.05). The most frequently mutated gene in KRAS wt PDAC was TP53 (46%), followed by ATM (26%) and EGFR (11%). The most common currently targetable alterations identified in KRAS wt patients were ATM (26%), BRCA1/2 (12%), EGFR (11%; 70% mutations, 30% amplifications), FGFR1/2 (9%), BRAF (8%; 91% mutations, 9% amplifications), PIK3CA (7%), MET (7%; 80% mutations, 20% amplifications) and ERBB2 (5.8%; 79% mutations, 21% amplifications) among others. Median tumor mutational burden for KRAS wt patients was higher than KRAS mut (10.0 vs. 7.77 mut/Mb, p = 0.035). There were no significant differences in rates of MSI-H between KRAS wt and KRAS mut patients (1.9% vs. 1.3%, p = 0.37). KRAS wt patients harbored higher number of oncogenic gene fusions compared to KRAS mut (1.6% vs 0.2%, p = 0.0013). The most common fusion partners among KRAS wt patients included FGFR2 (0.4%), BRAF (0.4%), ALK (0.2%), NTRK (0.2%), RET (0.2%), FGFR3 (0.1%) and EGFR (0.1%). Potential germline variants (pathogenic/likely pathogenic) were detected in both KRAS wt and mut patients, most commonly in the DNA-damage repair genes, including ATM (3.5%), BRCA2 (1.8%) and BRCA1 (0.6%). Conclusions: Targetable alterations and oncogenic rearrangements are enriched in KRAS wt PDAC compared to KRAS mut. These analyses provide additional therapeutic options and may improve outcomes for KRAS wt patients, warranting blood-based ctDNA genomic profiling in PDAC, especially when a tissue biopsy is not feasible or sufficient for comprehensive genomic profiling.

Use of circulating tumor DNA (ctDNA) for early assessment of treatment response in patients with advanced colorectal cancer (aCRC): A real-world (RW) analysis.

246 Background: Data suggests that changes in ctDNA quantity correlate with response to therapy in patients with advanced solid malignancies. However, there is little consistency on how to calculate and interpret such changes. Here, we apply a clinically-validated molecular response algorithm to a RW cohort of patients with aCRC to further evaluate its ability to assess treatment outcomes. Methods: We queried the Guardant INFORM database, which comprises aggregated commercial payer health claims and de-identified records from patients with comprehensive ctDNA testing via Guardant360 (G360) from September 2018-March 2022. Patients with aCRC who received G360 within 15 weeks prior to new treatment initiation (any line of therapy) and a second test 3-15 weeks after treatment initiation were retrospectively evaluated using the G360 Response algorithm. Cox proportional hazards (CPH) were used for RW time to next treatment (TTNT), time to treatment discontinuation (TTD), and overall survival (rwOS) analyses. Patients categorized as molecular responders had a >50% decrease in mean variant allele fraction (VAF) ratio from pre-treatment to on-treatment. Gender, age, line of therapy (LOT) and comorbidities were included as covariates. Median TTNT, TTD, and rwOS were calculated by Kaplan Meier. Results: Of 185 aCRC patients with eligible MR results, 65% received chemotherapy +/- VEGF, 21% received regimens containing anti-EGFR monoclonal antibodies, and 14% received other therapies. 43% of aCRC patients were classified as molecular responders, 42% were non-responders, and 15% were not evaluable by the algorithm due to no/low ctDNA at one or both timepoints. 16% of patients cleared their ctDNA on treatment (i.e., ctDNA became undetectable). Molecular responders had significantly longer TTNT (median 10.1 months vs 6.1 months; HR p < 0.005), TTD (median 5.2 Months vs 3.9 months, HR p=0.041), and rwOS (not reached vs 17.8 months, HR p=0.017). Conclusions: Patients with aCRC classified as molecular responders, as calculated by this algorithm, had prolonged time on treatment and overall survival. Compared to tumor markers, ctDNA has a short half-life, which can allow for early response assessment, as shown by our study. These findings are relevant for clinical care, with future potential to allow for adaptive clinical trial design. [Table: see text]

Genomic landscape of acquired resistance to targeted therapies in metastatic colorectal cancer (mCRC).

190 Background: Therapies targeting oncogenic driver mutations have radically altered the treatment paradigm for mCRC and improved outcomes. New selective inhibitors aim to expand the portion of mCRC with targetable alterations. However, these drugs are limited by a relatively short duration of response before the emergence of acquired resistance. We hypothesized that both the putative resistance mechanism (mutations (MUT) versus copy number alterations (CNA)) and the clonality of the emergent mutations may influence time to resistance. Methods: We screened the MSKCC IMPACT dataset of 5,403 MSS (microsatellite stable) CRC patient samples (3,704 primary sequenced, 1669 metastatic site sequenced) for patients (pts) who had been treated with targeted therapies against EGFR, BRAF, KRAS G12C, or HER2 with tissue or liquid biopsy samples profiled at progression. Clinicopathological features and acquired genomic changes emerging at resistance were assessed. Results: 42 pts were identified with targeted oncogenic drivers consisting of BRAF V600E (17 pts), EGFR (12 pts), KRAS G12C (11 pts), and HER2 (2 pts). Progression specimens analyzed consisted of tissue biopsies in 17 pts (40%) and circulating free DNA (cfDNA) in 21 pts (50%), 4 pts had both tissue and cfDNA analyzed at progression. Median time on targeted therapy (TOT) was 7.1 months (95% CI: 5.6-8.5). Putative resistance alterations were identified in 33 pts (79%) (18 cfDNA, 11 tissue, 4 both cfDNA and tissue) and consisted of MUT alone in 30%, MUT+CNA or rearrangement 70%. Concurrent PIK3CA mutations in pre-treatment tissues were identified in 17 pts (40%) and did not associate with TOT (p=0.68); acquired PI3K pathway alterations were identified in 5 pts (12%), including 2 pts who had baseline tumor PIK3CA mutation. Number of putative resistance alterations ranged from 0-13, with multiple resistance alterations identified in 18 pts (15 ctDNA, 3 tissue), and these could be categorized into three groups: one alteration (15/33; 45%), 2-4 alterations (8/33; 24%), and ≥5 alterations (10/33; 30%). Evaluating TOT by type of alteration: MUT only: 9.8 months, MUT+ CNA+/- rearrangement: 6.6 months (p=0.0079). Evaluating TOT by number of alterations: >4 alterations: 6.1 months versus 1-4 alterations: 8.9 months (p=0.012), or 1 alteration: 8.0 months versus >1 alteration: 6.7months (p= 0.5). There was no significant difference in TOT when evaluating by clonality of mutations, where subclonal was defined as <5% of highest variant allelic fraction (clonal vs subclonal, 9.7 vs 6.7 months, p= 0.09). Fusions identified at time of resistance (involving BRAF in 2 pts, MET in 1 pt, RET in 1 pt) occurred with ≥5 alterations in 3 of 4 pts. Conclusions: Presence of CNA and ≥5 new alterations at resistance were associated with shorter TOT, and there was a trend for subclonal alterations and shorter TOT. New approaches that target underlying mechanisms for these changes may extend TOT.

Sequencing of genes of drug response in tumor DNA and implications for precision medicine in cancer patients.

Tumor DNA sequencing is becoming standard-of-care for patient treatment decisions. We evaluated genotype concordance between tumor DNAand genomic DNA from blood and catalogued functional effects of somatic mutations in 21 drug response genes in 752 solid tumor patients. Using a threshold of 10% difference between tumor and blood DNA variant allele fraction (VAF), concordance for heterogenous genotype calls was 78%and increased to 97.5% using a 30% VAFthreshold. Somatic mutations were observed in all 21 drug response genes, and 44% of patients had at least one somatic mutation in these genes. In tumor DNA, eight patients had a frameshift mutation in CYP2C8, which metabolizes taxanes. Overall, somatic copy number losses were more frequent than gains, including for CYP2C19 and CYP2D6 which had the most frequent copy number losses. However, copy number gains in TPMT were more than four times as common as losses. Seven % of patients had copy number gains in ABCB1, a multidrug resistance transporter of anti-cancer agents. These results demonstrate tumor-only DNA sequencing might not be reliable to call germline genotypes of drug response variants.

Parental mosaicism detection and preimplantation genetic testing in families with multiple transmissions of de novo mutations

BackgroundDe novo mutations (DNMs) are linked with many severe early-onset disorders ranging from rare congenital malformation to intellectual disability. Conventionally, DNMs are considered to have an estimated recurrence rate of...

Biallelic TET2 mutations and canonical ASXL1 mutations are frequent and cooccur in Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): An institutional experience and review of literature.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is recurrently mutated in epigenetic pathway genes. We studied the myeloid-related genetic mutations in a cohort of five patients with BPDCN and one paired relapse case at our institution and identified a high frequency of biallelic TET2 and canonical ASXL1 (c.1934dupG) mutations. The number of cases is small, but the variant allele fraction (VAF) sums of the TET2 mutations, as well as the persistence of TET2 mutations in a case of relapsed BPDCN, suggest an ancestral/founder nature of TET2 clones in the cases. Further literature review shows a high frequency of biallelic TET2 mutations in reported cases of BPDCN.

Mosaic variants detectable in blood extend the clinicogenetic spectrum of GLI3-related hypothalamic hamartoma

PurposeHypothalamic hamartoma (HH) can be syndromic (eg, Pallister-Hall syndrome [PHS], HH, and mesoaxial polydactyly) or nonsyndromic. Most PHS cases have germline variants in GLI3, but a minority remain unresolved. Some nonsyndromic HH cases have GLI3 mosaic variants in the brain. PHS and nonsyndromic HH are regarded as 2 separate GLI3-related disorders, clinically and genetically. Here, we searched for mosaic variants in unsolved cases. MethodsHigh-depth exome sequencing was performed on leukocyte-derived DNA in 1 unsolved PHS and 25 nonsyndromic HH cases. We searched for mosaic variants in GLI3 and other HH-associated genes. Mosaic variants were confirmed by droplet-digital polymerase chain reaction. ResultsThe PHS case had a GLI3 stop-gain variant c.2845G>T; p.(Glu949Ter) at 6.9% variant allele fraction (VAF). Two nonsyndromic cases had GLI3 variants—a stop-gain (c.2639C>A; p.(Ser880Ter), VAF 3.7%) and a frameshift (c.3326_3330del; p.(Glu1109AlafsTer18), VAF 7.8%). The nonsyndromic patient with 3.7% VAF in blood had 35.8% VAF in HH tissue. He had a vestigial extra digit removed adjacent to his left fifth finger. ConclusionGLI3 mosaicism is associated with a phenotypic spectrum from PHS to HH with subtle extra PHS features, to isolated nonsyndromic HH. High-depth sequencing permits detection of low-level mosaicism, which is an important cause of both syndromic and nonsyndromic HH.

Nuclease Enrichment and qPCR Detection of Rare Nucleotide Variants.

The emergence of circulating DNA analysis in blood during the past decade has responded to the need for noninvasive alternatives to classical tissue biopsies. This has coincided with the development of techniques that allow the detection of low-frequency allele variants in clinical samples that typically carry very low amounts of fragmented DNA, such as plasma or FFPE samples. Enrichment of rare variants by nuclease-assisted mutant allele enrichment with overlapping probes (NaME-PrO) enables a more sensitive detection of mutations in tissue biopsy samples alongside standard qPCR detection assays. Such sensitivity is normally achieved by other more complex PCR methods, such as TaqMan qPCR and digital droplet PCR (ddPCR). Here we describe a workflow of mutation-specific nuclease-based enrichment combined with a SYBR Green real-time quantitative PCR detection method that provides comparable results to ddPCR. Using a PIK3CA mutation as an example, this combined workflow enables detection and accurate prediction of initial variant allele fraction in samples with a low mutant allele frequency (<1%) and could be applied flexibly to detect other mutations of interest.

Frequent somatic mosaicism in T lymphocyte subsets in individuals with and without multiple sclerosis.

Somatic variants are variations in an individual's genome acquired after the zygotic stadium and result from mitotic errors or not (fully) repaired DNA damage. To investigate whether somatic mosaicism in T lymphocyte subsets is enriched early in multiple sclerosis (MS). We identified somatic variants with variant allele fractions ≥1% across the whole exome in CD4+ and CD8+ T lymphocytes of 21 treatment-naive MS patients with <5 years of disease duration and 16 partially age-matched healthy controls. We investigated the known somatic STAT3 variant p.Y640F in peripheral blood in a larger cohort of 446 MS patients and 259 controls. All subjects carried 1-142 variants in CD4+ or CD8+ T lymphocytes. Variants were more common, more abundant, and increased with age in CD8+ T lymphocytes. Somatic variants were common in the genes DNMT3A and especially STAT3. Overall, the presence or abundance of somatic variants, including the STAT3 p.Y640F variant, did not differ between MS patients and controls. Somatic variation in T lymphocyte subsets is widespread in both control individuals and MS patients. Somatic mosaicism in T lymphocyte subsets is not enriched in early MS and thus unlikely to contribute to MS risk, but future research needs to address whether a subset of variants influences disease susceptibility.

Detection of mosaic variants using genome sequencing in a large pediatric cohort.

The increased use of next-generation sequencing has expanded our understanding of the involvement and prevalence of mosaicism in genetic disorders. We describe a total of eleven cases: nine in which mosaic variants detected by genome sequencing (GS) and/or targeted gene panels (TGPs) were considered to be causative for the proband's phenotype, and two of apparent parental mosaicism. Variants were identified in the following genes: PHACTR1, SCN8A, KCNT1, CDKL5, NEXMIF, CUX1, TSC2, GABRB2, and SMARCB1. In addition, we identified one large duplication including three genes, UBE3A, GABRB3, and MAGEL2, and one large deletion including deletion of ARFGAP1, EEF1A2, CHRNA4, and KCNQ2. All patients were enrolled in the NYCKidSeq study, a research program studying the communication of genomic information in clinical care, as well as the clinical utility and diagnostic yield of GS for children with suspected genetic disorders in diverse populations in New York City. We observed variability in the correlation between reported variant allele fraction and the severity of the patient's phenotype, although we were not able to determine the mosaicism percentage in clinically relevant tissue(s). Although our study was not sufficiently powered to assess differences in mosaicism detection between the two testing modalities, we saw a trend toward better detection by GS as compared with TGP testing. This case series supports the importance of mosaicism in childhood-onset genetic conditions and informs guidelines for laboratory and clinical interpretation of mosaic variants detected by GS.

A Novel Approach to Detect IDH Point Mutations in Gliomas Using Nanopore Sequencing: Test Validation for the Clinical Laboratory

The use of standard next-generation sequencing technologies to detect key mutations in IDH genes for glioma diagnosis imposes several challenges, including high capital cost and turnaround delays associated with the need for batch testing. For both glioma testing and testing in other tumor types where highly specific mutation identification is required, the high-throughput nature of next-generation sequencing limits the feasibility of using it as a primary approach in clinical laboratories. We hypothesized that third-generation nanopore sequencing by Oxford Nanopore Technologies has the capability to overcome these limitations. This study aimed to develop and validate a nanopore-based IDH mutation detection assay for clinical practice using glioma formalin-fixed, paraffin-embedded (FFPE) tissue. Glioma FFPE (n=66) samples with confirmed IDH gene mutational status were sequenced on the MinION device using an amplicon-based approach. All cases were concordant when compared with the reference results. Limit of blank and limit of detection for the variant allele fraction were 1.5% and 3.3%, respectively, at 500× read depth per gene. Total sequencing cost per sample was CAD$50 to CAD$134 with results being available in 9 to 15 hours. These findings demonstrate that nanopore-sequencing technology can be leveraged to develop low-cost, high-performance clinical sequencing-based assays with quick turnaround times to support the detection of targeted mutations in FFPE tumor tissue.

A Phase II Single-arm Study of Palbociclib in Patients With HER2-positive Breast Cancer With Brain Metastases and Analysis of ctDNA in Patients With Active Brain Metastases

IntroductionPalbociclib is highly efficacious and well tolerated in hormone-receptor positive (HR+) metastatic breast cancer (BC) but its activity for HER2+ BC with brain metastases (BM) is unknown. MethodsIn a single-arm phase II study we evaluated palbociclib with trastuzumab for patients with HER2+ MBC and BM. The primary endpoint was BM response rate. Circulating tumor DNA (ctDNA) was evaluated at baseline, and in a subset of patients at cycle 3 and progression. We also retrospectively identified additional patients with metastatic BC, active BM, and a ctDNA assessment prior to therapy for BM. ResultsTwelve patients with HER2+ MBC were enrolled, 4 with HR+ and 8 with HR- disease. No responses were seen. Best response was stable disease for 6 patients and progressive disease for 6 patients. The median PFS was 2.2 months, interquartile range (IQR) was 1.56 to 3.63 months. The median OS was 13.1 months and IQR was 9.4 to 23.8 months The CNS was the primary site of progression for all patients. The median variant allele fraction (VAF) of the dominant variant in each patient was 0.18% (interquartile range [IQR] 0.12%-0.47%) with a median number of somatic alterations of 1. We additionally evaluated ctDNA results from 26 patients with BC and active BM, among whom the median VAF was 11.8% (IQR 3.9%-27.3%) with a median number of alterations was 6 (IQR 4-9). Notably, progressive systemic disease was significantly less frequent in the trial cohort compared with additional retrospectively identified patients (8% vs. 81%). ConclusionPalbociclib did not demonstrate activity in HER2+ MBC with BM. Patients with progressive BM but stable, responding, or absent systemic disease have low VAF and number of alterations detected by ctDNA analysis from blood.

A comparative analysis of RAS variants in patients with disorders of somatic mosaicism

RAS genes (HRAS, KRAS, and NRAS) are commonly mutated genes in cancer, and activating RAS variants are also found in disorders of somatic mosaicism (DoSM). A survey of the mutational spectrum of RAS variants in DoSM has not been performed. A total of 938 individuals with suspected DoSM underwent high-sensitivity clinical NGS-based testing. We investigated the mutational spectrum and genotype-phenotype associations of mosaic RAS variants. Here we present a series of individuals with DoSM with RAS variants. Classic hotspots, including Gly12, Gly13, and Gln61 constituted the majority of RAS variants observed in DoSM. Further, we present 12 individuals with HRAS and KRAS in-frame duplications/insertions (dup/ins) variants in the switch II domain. Among the 18.3% of individuals with RAS in-frame dup/ins variants, clinical findings were mainly associated with vascular malformations. Hotspots were associated with a broad phenotypic spectrum, including vascular tumors, vascular malformations, nevoid proliferations, segmental overgrowth, digital anomalies, and combinations. The median age at testing for individuals with in-frame dup/ins variants was older, and the variant allele fraction was lower compared to those with mosaic RAS hotspots. Our work provides insight into the allelic and clinical heterogeneity of mosaic RAS variants in non-malignant conditions.

CASMART, a one-step CRISPR Cas12a-mediated isothermal amplification for rapid and high-resolution digital detection of rare mutant alleles

Convenient, ultrasensitive, and accurate detection of rare variants is essential for early cancer diagnosis and precision medicine, however, despite years of efforts, tools that have all these qualities remain elusive. Here, we developed a one-step CRISPR/Cas12a-based digital diagnostic platform for accurately quantifying mutant alleles, referred to as the CRISPR ASsoaciated Mutation Allele Rapid Test (CASMART). The platform accurately quantifies the variant allele frequency of EGFR L858R within 1 h at 42 °C and can detect mutant targets as low as 0.3 copies/μL (0.498 aM) in mock multiplex cfDNA samples. We further investigated the applicability of CASMART using human genomic samples with confirmed EGFR L858R mutations previously measured variant allele frequency by next-generation sequencing. Comparison across platforms revealed equivalent detection performance (Pearson's correlation coefficient, R2 = 0.9208) and high quantification accuracy for mutation allele frequency (intraclass correlation coefficient = 0.959). Our one-step approach enables easy and accurate variant allele frequency measurement of rare mutant alleles without PCR instrumentation, while the assay time was reduced by approximately half compared to the digital PCR with the shortest turnaround. The CASMART is an alternative to conventional single nucleotide polymorphism detection methods with great potential as a next-generation biosensor for rapidly quantifying the variant allele fraction, especially in resource-limited settings.

Relative infectivity of the SARS-CoV-2 Omicron variant in human alveolar cells.

With the continuous emergence of highly transmissible SARS-CoV-2 variants, the comparison of their infectivity has become a critical issue for public health. However, a direct assessment of the viral characteristic has been challenging because of the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing to facilitate the evaluation. In a proof-of-concept study with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our system demonstrates that the Omicron variant is 5- to 7-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the initial stage of infection. To our knowledge, for the first time, this study measures the relative infectivity of the Omicron variant under multiple virus co-infection and provides new experimental procedures that can be applied to monitor emerging viral variants.

High prevalence of low-allele-fraction somatic mutations in STAT3 in peripheral blood CD8+ cells in multiple sclerosis patients and controls.

Somatic mutations have a central role in cancer, but there are also a few rare autoimmune diseases in which somatic mutations play a major role. We have recently shown that nonsynonymous somatic mutations with low allele fractions are preferentially detectable in CD8+ cells and that the STAT3 gene is a promising target for screening. Here, we analyzed somatic mutations in the STAT3 SH2 domain in peripheral blood CD8+ cells in a set of 94 multiple sclerosis (MS) patients and 99 matched controls. PCR amplicons targeting the exons 20 and 21 of STAT3 were prepared and sequenced using the Illumina MiSeq instrument with 2x300bp reads. We designed a novel variant calling method, optimized for large number of samples, high sequencing depth (>25,000x) and small target genomic area. Overall, we discovered 64 STAT3 somatic mutations in the 193 donors, of which 63 were non-synonymous and 77% have been previously reported in cancer or lymphoproliferative disease. The overall median variant allele fraction was 0.065% (range 0.007-1.2%), without significant difference between MS and controls (p = 0.82). There were 26 (28%) MS patients vs. 24 (24%) controls with mutations (p = 0.62). Two or more mutations were found in 9 MS patients vs. 2 controls (p = 0.03, pcorr = 0.12). Carriership of mutations associated with older age and lower neutrophil counts. These results demonstrate that STAT3 SH2 domain is a hotspot for somatic mutations in CD8+ cells with a prevalence of 26% among the participants. There were no significant differences in the mutation prevalences between MS patients and controls. Further research is needed to elucidate the role of antigenic stimuli in the expansion of the mutant clones. Furthermore, the high discovered prevalence of STAT3 somatic mutations makes it feasible to analyze these mutations directly in tissue-infiltrating CD8+ cells in autoimmune diseases.

Arcch: Improving the Performance of Clonal Hematopoiesis Variant Calling and Interpretation Using a Consensus Based Approach

Introduction: The acquisition of somatic mutations in hematopoietic stem and progenitor stem cells with resultant clonal expansion is known as clonal hematopoiesis (CH). CH is linked to a higher risk of hematologic malignancies and other adverse health outcomes. The prevalence of CH is dependent on sequencing technique. With ultra-high-depth sequencing, CH can be detected in most adults. However, CH variant calling at very low variant allele fractions (VAF) is challenging due to difficulty in distinguishing low frequency CH mutations from sequencing artifacts. Here we develop and validate a novel CH variant calling approach that combines ensemble based variant calling with advanced artifact filtering and outperforms commonly used somatic variant callers. Methods: We sequenced a tumor-normal dilution series from six AML patients and 27 technical controls with a targeted panel utilizing unique molecular indexes that included nine common CH genes (DNMT3A, TET2, ASXL1, TP53, PPM1D, JAK2, SF3B1, SRSF2) with a mean unique coverage of 18,268x. In total we identified 32 mutations in these six samples validated by an independent sequencing panel performed for the purpose of clinical testing. The variant allele fraction (VAF) in the resulting tumor-dilution samples ranged from 0.1%-50% VAF. Results: For higher VAF mutations (>5%), Mutect2, Lofreq, and Vardict showed reasonable sensitivity (range: 0.70-1.00) and positive predictive values (PPV) (range: 0.80-1.00). However, at lower VAF, PPV was poor; for mutations between 0.1-0.4% VAF, the PPV ranged between 0.01-0.22. We applied additional filters based on sequencing quality, depth, regional complexity, and an empiric estimate of sequencing error at the position of a given called variant using technical controls. Taking the consensus of three callers and including our additional filters, we not only retained sensitivity and PPV for higher VAF variants above 1% (0.92 sensitivity, 0.95 PPV) but improved the PPV with retained sensitivity for variants between 1-0.1% VAF (0.91 sensitivity, 0.83 PPV). Applying our consensus approach to blood samples drawn from 31 healthy individuals sequenced using the same targeted panel with variants validated using an orthogonal approach, we saw excellent sensitivity and PPV across a wide range of VAFs (Sensitivity 0.99 [>1% VAF], 0.92 [0.2-1% VAF], and 0.82 [0.1-0.2% VAF]; PPV 1.00 [>1% VAF], 0.98 [0.2-1% VAF], 0.97 [0.2-0.1% VAF]). We compared our consensus calling and advanced filtering approach to a machine learning model. A decision-tree-based ensemble machine learning algorithm, XGBoost, was trained on the tumor-dilution samples with validation using 31 blood samples with orthogonal sequencing. The variant caller output (allele counts, flags, etc.), additional false positive filtering metrics, and statistical results from the technical controls were used as features. Sensitivity and PPV was high for mutations above 1% VAF (0.97 sensitivity and 0.82 PPV) with the performance declining for lower VAF mutations (0.72 sensitivity and 0.65 PPV). Thus, a machine learning approach did not outperform consensus calling with advanced artifact filtering. Finally, we developed a custom annotation pipeline for CH variant pathogenicity classification incorporating previously reported CH mutations in large cohorts, cancer somatic mutation frequency databases and curated cancer driver knowledge databases. Conclusion: We developed ArCCH, an advanced artifact filtering and consensus CH calling pipeline that includes custom annotation features to facilitate CH pathogenicity determination. Our results show that our consensus approach with advanced error correction filters substantially improves the performance of CH variant calling beyond commonly used single variant callers. This end-to-end flexible workflow package is publicly available on Terra and will facilitate future CH studies.

The Effect of Clonal Hematopoiesis of Indeterminate Potential (CHIP) and Aspirin on Clinical Outcomes in the Healthy Elderly: A Sub-Study of the Aspirin in Reducing Events in the Elderly (ASPREE) Randomized Controlled Trial

Background Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is a risk factor for blood cancer, cardiovascular disease (CVD) and other diseases. Recent experimental evidence suggests a causal link between CHIP and CVD, however it is not known whether individuals with CHIP are more likely to benefit from preventive interventions for CVD, such as low-dose aspirin. ASPREE was a double-blind, randomized, placebo-controlled trial comparing 100mg aspirin with placebo on disability-free survival (DFS, composed of death, dementia and physical disability) in initially healthy participants aged ≥70y (≥65y for US minorities of Black and Hispanic background). We measured CHIP in serial blood samples of 11,298 ASPREE participants at baseline and/or year 3 (Y3), and assessed incidence of CHIP and its association with various clinical outcomes. We also examined whether randomization to aspirin had a differential effect on clinical outcomes according to the presence of CHIP at study entry. Methods CHIP was measured in 19 CHIP-associated genes using a custom targeted amplicon-based NGS assay, designed to have a sensitivity of 0.5-2% variant allele fraction (VAF). CHIP was defined as a VAF ≥ 2%. We evaluated the association of CHIP at baseline and Y3, defined as a categorical variable (VAF< 2%, VAF 2-10%, VAF >10%), with clinical outcomes, including DFS, all-cause mortality, cancer and major adverse cardiovascular events (MACE, composed of fatal coronary heart disease, non-fatal myocardial infarction and ischemic stroke) using Y3-landmark Cox proportional hazards models. Risk factors for incident CHIP at Y3 were identified using multivariable logistic regression. Treatment heterogeneity of aspirin was assessed in CHIP subgroups overall and by the most common genes. Results CHIP was present in 3,345/9,926 (34%) participants at baseline and in 3,685/9,812 (39%) at Y3. Of the 5,612 participants without CHIP at baseline who had a Y3 sample, 1,195 (25%) developed CHIP by Y3 (incidence 7% per year). Risk factors for incident CHIP included cancer history (odds ratio [OR] (95% CI) 1.20 [1.02-1.41] P=0.031), and family history of blood cancer (OR 1.50 [1.16-1.93] P=0.002). With regards to clinical outcomes, the presence of CHIP at >10% VAF at baseline (but not VAF 2-10%) was associated with increased all-cause mortality (adjusted [aHR] [95% CI] 1.53 [1.23-1.90] P<0.001). This increased risk appeared to be driven by increased cancer-related mortality (baseline aHR 1.62, [1.19-2.22] p=0.002) more than cardiovascular-related mortality (baseline aHR 1.34 [0.84-2.12] p=0.21). CHIP at >10% at baseline and Y3 was also associated with increased risk of blood cancer (HR 2.48 [1.59-3.88] and 3.24 [1.98-5.30], both P<0.001) and other cancers at Y3 (HR 1.31 [1.02-1.68] P=0.033). After adjustment for potential confounders, this association remained significant for blood cancers (aHR 2.29 [1.44-3.62] p<0.001) but not non-blood cancers (aHR 1.21 [0.98 -1.48] P=0.07). There was no evidence of an association between CHIP at 2-10% and >10% with MACE (aHR 0.91 [0.74-1.11] P=0.34 and aHR 0.73 [0.50-1.07] P=0.11, respectively). There was no evidence of a differential effect of aspirin on DFS according to presence of CHIP at study entry (CHIP HR 0.91 [0.77-1.07], no-CHIP HR 0.88 [0.70-1.10], test for heterogeneity P=0.83) or on MACE (CHIP HR 1.23 [0.85-1.80], no-CHIP HR 0.80 [0.62-1.03] heterogeneity P=0.06) - Figure 1. There was no subgroup difference for the effect of aspirin on incident cancer or cancer-related mortality. Participants with detectable JAK2V617F (VAF > 0.5%, n=123) had worse outcomes if randomized to aspirin, including the primary outcome of DFS (Fig 1). There was no differential effect of aspirin in the JAK2V617F subgroup for MACE (Fig 1). Conclusions This is the largest primary CVD prevention trial to evaluate the intervention effect according to presence of CHIP in healthy elderly. We found CHIP at >10% VAF was associated with increased all-cause mortality, which appeared to be driven largely by increased cancer-related mortality. There was no evidence that participants with CHIP were more likely to benefit from aspirin, including for MACE. Furthermore, aspirin appeared to reduce DFS for individuals with JAK2V617F mutation, although sub-group numbers were small. Our results suggest aspirin should not be used for primary prevention in older individuals with CHIP, including JAK2V617F. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK2V617F Molecular Response to Ruxolitinib in Patients with PV and ET Is Associated with Lower Risk of Progression to Secondary Myelofibrosis

Introduction. Ruxolitinib (RUX) is approved for patients (pts) with Polycythemia Vera (PV) who are refractory or intolerant to hydroxycarbamide, based on composite endpoint of spleen volume reduction and Hct control (RESPONSE-1 trial) and Hct control (RESPONSE-2). Conversely, studies in pts with Essential Thrombocythemia (ET) were inconclusive, and the drug is not approved in that indication. Aim. The aim of current study was to analyze changes of JAK2V617F variant allele fraction (VAF) in PV and ET pts who were long-term treated with RUX, and the clinical correlates and outcome of molecular responses. Patients and Methods. The study included 65 PV and 12 ET pts; the latter had been enrolled in phase-2 studies and were still on RUX owing to continuing clinical benefits. Median time from diagnosis to RUX was 5.9y (0.6-24y), age 58y (25-87y), 50.6% were male, history of thrombosis in 21 pts (27%). A JAK2V617F mutation was found in 75 pts (97.4%), JAK2ex12 and MPLW515L in 1 pt each. At circa-annual intervals, JAK2V617F VAF was measured in granulocytes by digital PCR or highly sensitive (threshold <0.01%) RTQ-PCR. MPLW515L was measured by digital PCR. IWG-MRT criteria were used for clinical and molecular response assignment. The latter included partial (PMR: a >50% VAF reduction) and complete (CMR; JAK2V617F undetectable) molecular response, confirmed at >1y. We also considered a deep molecular response (DMR) as a confirmed VAF<2%. The nonparametric Wilcoxon rank-sum test, Kaplan-Meier estimate of survival and log-rank test were used as appropriate. Results. Median FU on RUX was 8.8y (1.3-14.1). The median RUX dose was 20 mg die (5-50mg). RUX was discontinued in 23 pts for AE (n=7, 9.1%), disease progression (n= 12, 15.6%), refractoriness/loss of response (n=3, 3.8%) or pt decision (n=1, 1.3%), after a median of 5.0y (0.8-13y). While on RUX, there were 3 thrombosis and 4 bleedings. Progression to secondary MF (sMF) occurred in 24 pts (31.1%) after a median of 6.0y (2-11.5); of these, 22 (92%) were PV, accounting for 34% of all PV compared to 16.6% of ET. Three pts (3.9%) transformed to AML after 9.2y (1.3-13.7y) and died, and 2 pts (2.6%) died after evolution to sMF while on RUX. A IWG-MRT response was reached in 92% for Hct, 59% for platelet count, 73% for leukocyte count, 77% for spleen volume (<50% palpatory). The median JAK2V617F VAF was 47% (21-98%) and 68% (15-99%) at diagnosis and RUX start, respectively. The overall mean VAF decrease was 64.6%+27.5%. A PMR was observed in 33 pts (44.2%), after a median time of 3.3y (1-7.6y), 51% and 50% of PV and ET; the baseline JAK2V617F VAF was 61.3±27.7% for PV and 32.3±10.4% for ET. A CMR (confirmed) was declared in 5 JAK2V617F mutated pts (6.5%; median baseline VAF 44.7±36.8%) of which 3 were PV, and in the MPLW515L mutated ET pt (baseline VAF 33%). A DMR was confirmed in 9 pts (11.7%; median baseline VAF 30.5±16.0), of which 6 were PV. Median time to CMR and DMR was 4.6y (1.1-7.6y) and 5.0y (2.1-12.1y), respectively. Mean duration of CM + DMR was 8y (7-12y). There was no correlation between any category of molecular response and response of Hct, platelets and spleen length, while normalization of leukocytes occurred more frequently in pts with PMR (93.8% vs 62.5%, P=0.02). Of the pts who progressed to sMF, 21 of 24 pts (87.5%) had not attained an at least PMR (P<0.0001). The HR of sMF evolution was 19.9 (95%CI, 2.4-166.7) for those who did not obtain any molecular response and 3.56 (95%CI, 0.37-34.6) for those who obtained PMR, using CMR+DMR as reference (P<0.0001) (Figure 1). By ROC analysis, a cutoff level of <60% JAK2V617F VAF at baseline (involving 35 pts, 45.5% of the entire population) was associated with a significantly greater likelihood to obtain CMR+DMR (37.1% vs 2.4%; P<0.0001) as well as PMR (60% vs 38.2%, P=0.01). All 3 pts who transformed to AML had not obtained any molecular response. Discussion. In this study, 18% of PV and ET pts receiving RUX developed sustained JAK2V617F complete plus deep molecular response (JAK2V617<2%) over long term treatment, that were associated with lower risk of progression to secondary MF; also pts attaining a PMR had reduced probability to develop sMF compared to those who did not show any changes in JAK2V617 VAF, overall reinforcing that attainment of molecular response may be a surrogate of disease modification. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genetic Markers and Front Line FCR/BR Vs. Rve, Gve and Give Treatment - Outcome Results from the CLL13/GAIA Trial

BACKGROUND: Genomic aberrations, IGHV mutation status and recurrent mutations in genes such as TP53 are established prognostic factors in CLL in the context of chemoimmunotherapy (CIT). For time limited treatment with venetoclax in combination with Rituximab (RVe), Obinutuzumab (GVe) and Obinutuzumab and Ibrutinib (GIVe) the role of these factors is less-well established. METHODS: The phase 3 GAIA/CLL13 trial compared 3 different time-limited venetoclax-based combinations against CIT in fit, treatment-naïve patients with CLL without del(17p) or TP53 mutations detected via Sanger sequencing. Patients were randomized to receive CIT (FCR for patients ≤65y; BR for patients >65y), RVe, GVe or GIVe. Co-primary endpoints were PFS and MRD in peripheral blood at month 15 via 4-colour flow. Genomic aberrations were assessed at enrolment by FISH, IGHV by sequencing with a threshold of 98% homology and mutations via amplicon-based targeted NGS for TP53, NOTCH1, SF3B1, MYD88, KRAS, NRAS, BRAF, EGR2, NFKBIE, RPS15, XPO1 and BIRC3 in 913 of 926 patients (98.6%) of the full trial cohort. RESULTS: The incidences of genomic aberrations based on the hierarchical model were del(11q) 17.5%, +12 16.2%, mono-allelic del(13q) 44.6% and normal karyotype 21.7%. IGHV was unmutated (U-IGHV) in 55.9% and mutated (M-IGHV) in 41.0% of patients (ITT). Subset#2 accounted for 50 patients equally distributed among U-IGHV and M-IGHV. The incidence of gene mutations was SF3B1 22.6%, NOTCH1 20.5% (exon 34 and 3'UTR), NFKBIE 13.6%, BIRC3 11.8%, BRAF/KRAS together 9.6%, XPO1 7.9%, EGR2 5.6%, RPS15 5.5% and MYD88 4.2%. Minor mutations with a variant allele fraction (VAF) of 1.5% to 10% were frequent in BIRC3 and NFKBIE (59.6% and 56.0% of all variants, respectively), while minor mutations in TP53 not previously detected by Sanger sequencing were rare (2.0%). Undetectable MRD (uMRD) was not associated with any genetic subgroup in the pooled analysis of GVe/RVe/GIVe. Single arm analysis revealed NOTCH1 mutation associated with lower rate of uMRD with GVe (74.4% vs 88.6%, p=0.02) and higher uMRD rate in +12 cases with RVe (73.5% vs. 52.2% in del(13q), p=0.03). With CIT a significantly lower uMRD rate was found in U-IGHV (42.0% vs. 66.3% in M-IGHV, p<0.01) and del(11q) (39.0% vs. 58.4% in del(13q), p=0.04) while mutated MYD88 associated with a higher uMRD rate (92.3% vs 50.2%, p=0.02). At a median follow-up of 38.8 months, there were 188 PFS events in the ITT population. For CIT U-IGHV (HR 4.47, p<0.01), del(11q) (HR 2.68, p<0.01) and mutated NOTCH1 with VAF>10% (HR 2.01, p=0.01) associated significantly with shorter PFS in univariate analysis. In the combined analysis for RVe/GVe/GIVe U-IGHV (HR 2.29, p<0.01), mutations in NOTCH1 (HR 1.76, p<0.01) and BRAF/RAS (HR 2.45, p<0.01) were associated with significantly shorter PFS and del(13q) (HR 0.67, p=0.03) with longer PFS. A separate analysis for each venetoclax arm identified mutated NOTCH1 (GVe: HR 2.2 p=0.04 and GIVe: HR 2.2, p=0.05), BIRC3 (GIVe: HR 3.6 p<0.01), BRAF/KRAS/NRAS (RVe: HR 3.6, p<0.01 and GIVe: HR 3.7, p<0.01) NFKBIE (GVe: HR 3.0, p<0.01), del(13q) (GVe: HR 0.4, p=0.01) and U-IGHV (GVe: HR 2.6, p=0.03 and GIVe: HR 2.7, p=0.03) as prognostic factors for PFS. Subset 2 was not associated with shorter PFS, also not among M-IGHV patients. In multivariable analysis including all clinical, laboratory and genetic markers significantly associated with PFS, treatment with GVe and GIVe were independent favorable factors (HR 0.42 and 0.33, respectively, both p<0.01) and U-IGHV (HR 2.43, p<0.01), and mutated NOTCH1 (HR 1.46, p=0.03) were confirmed as independent prognostic factors. In a separate multivariable analysis only for CIT, del(11q) (HR 1.89, p=0.03), U-IGHV (HR 3.08, p<0.01), and mutated NOTCH1 >10% VAF (HR 2.12, p=0.01) were verified as independent adverse genetic risk factors. For the combined RVe/GVe/GIVe group, mutated BRAF/RAS (HR 1.87, p=0.01), U-IGHV (HR 1.85, p<0.01), and mutated NOTCH1 (HR 1.54, p=0.04) were confirmed as independent adverse prognostic factors. CONCLUSIONS: Early results from CLL13/GAIA show association of gene mutations with lower rate for uMRD and shorter PFS after CIT or venetoclax-based therapy. IGHV and NOTCH1 mutation retain their prognostic role with both treatment types. Mutations in the RAF/RAS pathway (9.6% overall) were associated with shorter PFS with venetoclax-based therapy, while with CIT an impact on outcome is not apparent at present. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal