Variant Allele Frequency Threshold Research Articles

Clonal Hematopoiesis and Clinical Outcomes in Metastatic Castration-Resistant Prostate Cancer Patients Given Androgen Receptor Pathway Inhibitors (Alliance A031201).

Mutations in hematopoietic progenitor cells accumulate with age leading to clonal expansion, termed clonal hematopoiesis (CH). CH in the general population is associated with hematopoietic neoplasms and reduced overall survival (OS), predominantly through cardiovascular adverse events (CVAE). Because androgen receptor pathway inhibitors (ARPI) used in metastatic castration-resistant prostate cancer (mCRPC) are also associated with CVAEs and because CH negatively impacted survival in an advanced solid tumor cohort, we hypothesized that CH in mCRPC may be associated with increased CVAEs and inferior survival. A targeted DNA sequencing panel captured common CH mutations in pretreatment blood samples from 957 patients enrolled in Alliance A031201: a randomized trial of enzalutamide ± abiraterone/prednisone in the first-line mCRPC setting. The primary outcome was the impact of CH on OS; the secondary outcomes were progression-free survival (PFS) and CVAEs. Baseline comorbidities were similar by CH status. No differences in OS/progression-free survival were detected regardless of treatment arm or the variant allele frequency threshold used to define CH [primary: 2% (normal-CH, N-CH); exploratory: 0.5% (low-CH) and 10% (high-CH, H-CH)]. Patients with H-CH (7.2%) and TET2-mutated N-CH (6.0%) had greater odds of any CVAE (14.5% vs. 4.0%; P = 0.0004 and 12.3% vs. 4.2%; P = 0.010, respectively). More major CVAEs were observed in patients with H-CH (5.8% vs. 1.9%; P = 0.042) and N-CH (3.4% vs. 1.8%; P = 0.147). CH did not affect survival in patients with mCRPC treated with ARPIs in A031201. H-CH and TET2-mutated CH were associated with more CVAEs. These findings inform the risk/benefit discussion about ARPIs in mCRPC.

Association between CH mutation in TET2and solid cancer in elderly Asian women.

10585 Background: Clonal hematopoiesis (CH), defined by the expansion of mutated hematopoietic cells due to somatic mutations acquired during aging, is associated with increased age and inflammatory conditions. CH serves as a predictor for the risks of cancer and cardiovascular events, highlighting the importance of early detection of genetic alterations for proactive intervention. As Asian societies are experiencing rapid aging alongside genetic and environmental variations distinct from Western cohorts, we aimed to evaluate the predictive significance of CH in solid cancer in an Asian cohort. Methods: We conducted a nested case-control study within Singapore Longitudinal Ageing Study 1 (SLAS1; NCT03405675). Chinese females (aged 55.0-83.9), comprising 31 patients with solid cancers (breast, lung, colon) who developed cancer during the follow-up, and 29 controls, were randomly selected from the SLAS1. SLAS1 is a community-based longitudinal aging cohort study. Subjects aged 55 years or older, capable of self-ambulation, were recruited between 2003 and 2005 (n=2804) and were followed up in 2005-2007, 2008-2009, and 2018-2020. We linked SLAS1 with records from the Singapore National Death Registry to identify subjects with cancers as cause of death during this period (median follow-up period 17 years). Buffy coat samples taken at baseline were sequenced using the 42-gene target HemeMark panel (Lucence, Singapore) with a reported 0.1% limit of detection. Chi-square test and t-test were performed to compare the frequency of CH variants and the mean number of mutations per person in the case and control group. We evaluated multiple variant allele frequency (VAF) thresholds on an exploratory basis (0.1% to 2%). The relationship between CH mutations and the likelihood of developing cancer was investigated through a logistic regression model, considering CH mutation defined at various cutoff points. Results: CH was found in 29.0% (9/31) of cases and 37.9% (11/29) of controls, with common mutated genes being TET2 (8/31, 25.8%), ASXL1 (4/31, 12.9%), and DNMT3A (2/31, 6.5%) in case group (VAF threshold=0.5%). Multi-gene CH did not predict cancer at any threshold (with the lowest P value of 0.5858 corresponding to VAF 0.5), Genes correlating with increased cancer risk included TET2 with adjusting for age, which was notably significant at thresholds of 0.3% and above (VAF 0.3%, adjusted OR=2.25, 95% CI: 1.14-4.47, p=0.0201; 0.5%, adjusted OR=4.12, 95% CI: 2.00-8.88, p=0.0002). Conclusions: CH determined through multi-genes was not associated with solid cancers, but TET2 mutations at 0.3-0.5% VAF thresholds were more prevalent in cancer cases, as compared to the current 2% threshold used for calling CHIP. The finding in this nested case-control study, conducted within a cohort with a 17-year follow-up, implies a potential association between TET2 mutation and solid cancer. Further study is warranted on a larger cohort.

A novel strategy for detecting mutations from circulating tumor DNA with blood tissue only.

e20031 Background: Molecular Residual Disease (MRD) test detects tumor-specific mutations in circulating tumor DNA (ctDNA), which can indicate the risk of tumor recurrence. However, without tumor-specific mutations, detecting mutations in blood tissue alone is extremely challenging due to the low concentration of ctDNA in blood. To address this challenge, we proposed an innovative mutation filtering and calling strategy by simulating noise’s distribution and quality assessment to greatly boost the performance. Methods: We performed 406 pairs of MRD tests on both tumor and blood tissue of postoperative patients with mixed cancer types, including lung, gastrointestinal (GI), hepatobiliary, and pancreatic. Patients that possess one or more tumor-specific mutations in their blood tissue were classified as recurrence high-risk (n = 66), others were classified as recurrence low-risk (n = 340). All sequence data were analyzed using both traditional and proposed method. In the traditional method, VAF threshold, end-reads, multiple-align reads and population frequencies were used as filtering criteria. While in the proposed method, VAF threshold was excluded and two new parameters were introduced: The distribution of variant allele frequency (VAF) of sequencing noise, which simulated 42 healthy individuals’ background noise with conf > 0.995; The normalized proportion of supported unique and total variants, to assess the likelihood of mutations originated from ctDNA. Results: Among 406 patients of mixed tumor types, the traditional approach barely reached a sensitivity of 39.4% and a specificity of 89.1%, while our strategy achieved a sensitivity of 71.2% and a specificity of 80.9%. Particularly among the 92 patients with gastrointestinal tumor, by filtering specific noise caused by the MRD panel (e.g. multi-alignment error of MUC gene family), we further boost the sensitivity from 47.1% to 82.4% with a loss of specificity from 93.3% to 90.7%. Moreover, among 248 patients with lung tumors, the sensitivity and specificity between traditional approach and proposed method were 15.8%, 93.9%, and 52.6%, 90.0% respectively, which might be caused by the low recurrence rate in the patient cohort (19 out of 248). Conclusions: The novel strategy based on simulated noise distribution and read quality assessment surpasses the traditional threshold-based approaches in mutation detection, especially in deep sequencing like MRD tests. The findings of the study indicate that the recurrence monitoring strategy is applicable across various tumor types and exhibits superior performance in GI tumor, albeit with certain limitations. In conclusion, this study boosts the feasibility of MRD as a non-invasive alternative for tissue biopsy by providing a novel mutation filtering strategy, which further strengthen the potential of MRD testing in enhancing cancer patients’ long-term prognosis and treatment.

Tumor purity as confounder for the determination of tumor mutational burden (TMB).

e14656 Background: TMB determination is a prerequisite to stratify advanced cancer patients for pembrolizumab based on the histology-agnostic FDA approval. The accuracy of TMB measurement is challenged by low tumor purity resulting in a lower number of detected somatic variants for a fixed threshold of variant allele frequency (VAF) and a lower measured TMB level as a consequence. Methods: We derived a mathematical formula describing the influence of tumor purity on the VAF of somatic variants, in which the DNA copy number of the corresponding region of the tumor genome is a parameter. We simulated the effect of tumor purity in a cohort of 4,900 tumors from the TCGA project with tumor purity of at least 80%. TMB was calculated as the number of missense mutations with VAF greater or equal than 5%. Tumors were classified as TMB-high when harboring at least 199 missense mutations. This cutpoint corresponds to 10 mut/Mb as derived in the TMB bridging study. We compared tumors with a simulated tumor purity of 70%, 60%, ..., and 10% with tumors with a tumor purity of 80%, which served as reference. Results: The median of TMB in the pan-cancer cohort was reduced by 2%, 10%, and 32% for tumor purity of 60%, 40%, and 20% compared to the reference of 80%. For the 10% tumors with the worst estimation, TMB was reduced even by 12%, 35%, and 77% or more, respectively. In the pan-cancer cohort, the sensitivity to detect high TMB was 98%, 90%, and 62% for tumor purity of 60%, 40%, and 20%, respectively. For head and neck cancer, lung adenocarcinoma, lung squamous cell carcinoma, and cutaneous melanoma, the sensitivity to detect high TMB was 80%, 89%, 92%, and 91%, respectively, for tumors of tumor purity 40%. Conclusions: High tumor purity is critical for accurate determination of the TMB and to reach a high sensitivity concerning the selection of patients for immunotherapy. As shown by the simulations, the sensitivity to detect high TMB is close to 100% for tumor purity of at least 60%, while a substantial proportion of patients that could receive immunotherapy is missed for tumors with purity of 40% or less. Potential ways out of the TMB underestimation include increasing the sequencing coverage and lowering the VAF threshold for mutation calling or using clonal TMB with an adapted cutpoint instead of total TMB as biomarker for immunotherapy guidance.

Abstract 977: Germline BRCA1/2 mutations detected by circulating tumor DNA testing in breast cancer patients: A retrospective mutiinstitutional analysis

Abstract Background: The use of circulating tumor (ct) DNA testing allows to detect resistant and actionable somatic alterations and it could be associated with the incidental identification of germline mutations. An univocal variant allele frequency (VAF) threshold to distinguish germinal form somatic mutations has not been assessed yet. We aimed to correlate BRCA1/2 ctdNA detected mutations with germline mutational status to determine potential VAF cutoff. Materials and methods:We retrospectively analyzed the incidence of both somatic (s) and germinal (g) BRCA1/2 alterations in a multiinstitutional retrospective breast cancer (BC) cohort to assess the VAF threshold for the likelihood of germline mutations’ detection by ctDNA next-generation sequencing (Guardant 360) testing. Clinical variables were analyzed using descriptive analyses and receiving operating characteristic (ROC) curves were generated to determine the VAF cutoff. Results: Two hundred and fourteen patients (pts) with sBRCA1/2 mutations (including variant of uncertain significant and synonymous) detected by ctDNA testing, referred to the enrolling centers between January 2015 to May 2023 were included in the analysis. The median age at diagnosis was 50 years old (Interquartile range (IQR)43,5-61.5), 43% had a family history of cancer. Hormone receptor positive/HER2 negative was the most represented subtype (68%) followed by the HER2 positive (16%) and triple negative (16%) ones. At ctDNA baseline, 95.8% of pts had a metastatic disease; the main sites of metastases were bone (67.4%) and visceral (59.3%). Ninetyfour (43.9%) and 137 (64%) pts had a sBRCA1 and sBRCA2 alterations respectively, while 17 (7.9%) had a co-mutation in BRCA1 and BRCA2. The mean VAF value for sBRCA1 alterations was 10.1% (standard deviation (SD) 19.5%, range [0.04%-84.3%]) while for sBRCA2 alterations was 10.8% (SD 18.9%, range [0.21%-80.3%]). The germinal testing, performed per standard of care, was available for 100 pts (46.7%). Among these, 42 (42%) had at least a pathogenic variant detected. A gBRCA1 mutation was present in 11 (11%) pts while 25 (25%) had a gBRCA2 mutation. Comparing the somatic and germinal testing, 9/100 pts had a concordance for BRCA1 detection, the 2 discordant cases had both a low allele frequency sBRCA2 alterations; the concordance for BRCA2 detection was instead 100%. An optimal cutoff of 38.4% (AUC 0.98) for BRCA1 and 19.5% (AUC 0.96) for BRCA2 was assessed by ROC analysis as the likelihood of a germline meaning of a somatic mutation detected by ctDNA analysis. Conclusion: The identification of BRCA1/2 alterations in ctDNA could guide the use of germline testing. The VAF cutoff identified for the likelihood of a germinal mutation is lower than expected, suggesting that a wider population should be screened for germinal mutation with relevant impact on the therapeutic choices and family screening. Citation Format: Letizia Pontolillo, Carolina Reduzzi, Andrew A. Davis, Arielle J. Medford, Annika Putur, Lorenzo Gerratana, Katherine Clifton, Whitney L. Hensing, Marko Velimirovic, Surbhi Warrior, Mara S. Serafini, Eleonora Nicolò, Laura Munoz-Arcos, Jeannine Donahue, Charles S. Dai, Jennifer C. Keenan, Amir Behdad, William J. Gradishar, Diana Giannarelli, Emilio Bria, Cynthia X. Ma, Aditya Bardia, Massimo Cristofanilli. Germline BRCA1/2 mutations detected by circulating tumor DNA testing in breast cancer patients: A retrospective mutiinstitutional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 977.

TP53 Variant Allele Frequency and Therapy-Related Setting Independently Predict Survival in Myelodysplastic Syndromes with Del(5q)

Background: According to the international consensus classification (ICC), diagnosis of myelodysplastic syndromes (MDS) with del(5q) requires presence of i) del5q, alone or associated with no more than one other cytogenetic abnormality, exclusive of -7/del(7q), ii) <5% bone marrow (BM) and <2% circulating blasts, and iii) the absence of “multi-hit” TP53 ( Arber et al. Blood 2022;140: 1200). We have recently reported on TP53 variant allele frequency (VAF) ≥22% being the sole genetic predictor of inferior survival in MDS-del(5q) (Fleti. et al. AJH 2023;98:E76). In the current study, we examined the additional prognostic impact of therapy-related setting, in the context of TP53 VAF and other risk factors. Methods : The current study included patients with MDS-del(5q) who were seen at the Mayo Clinic or Moffitt Cancer Center, USA. Diagnosis of MDS-del(5q) and therapy-related qualification was according to ICC criteria ( Arber et al. Blood 2022;140: 1200), with the exception that patients with biallelic or multi-hit TP53 were included to allow unbiased analysis of impact on prognosis. Patient selection was based on availability of mutation information from next-generation sequencing (NGS). The latter was assessed either at diagnosis or at different time points during the disease course, but at least 3 months before leukemic transformation. Otherwise, all analyses, including estimation of overall and leukemia-free survival, were based on variables collected at time of initial diagnosis of MDS-del(5q). TP53 mutation VAF threshold of 22% was considered based on our previously published report cited above. Age cutoff at 67 years was determined by ROC analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: A total of 210 patients (median age 72 years; 65% females), meeting the above-stipulated criteria for MDS-del(5q), were considered: 172 primary and 38 therapy-related. Table 1 outlines clinical and laboratory characteristics obtained at the time of diagnosis: 42% were transfusion-dependent, 69% had hemoglobin level <10 g/dL, 30% platelet count <150 x 10(9)/L, and 15% carried additional cytogenetic abnormality. NGS data performed either at diagnosis or at least 3 months prior to leukemic transformation was available in 146 patients; the most frequent mutations were TP53 (21%), SF3B1 (16%), DNMT3A (12%), TET2 (12%), ASXL1 (10%), and JAK2 (7%) (Table 1). At a median follow-up of 3.9 years (range 0.002-18.5) for living patients, 95 (45%) deaths and 34 (16%) leukemic transformations were recorded; treatment undertaken included lenalidomide in 162 (77%) cases. NGS data-exclusive multivariable analysis that included all 210 patients identified therapy-related setting (p<0.01; HR 2.9; Figure 1a), age >67 years (p=0.01; HR 1.8), transfusion-dependency (p=0.04; HR 1.5), and platelet count <150 x 10(9)/L (p=0.03; HR 1.6), as independent predictors of inferior survival. The significant effect of age and platelet count was sustained when analysis was limited to patients with primary MDS-del(5q) but lost when applied to therapy-related disease. NGS data-inclusive multivariable analysis, which was applicable to the subset of 146 patients, identified, as risk factors, therapy-related setting (p<0.01; HR 2.3) and TP53 VAF ≥22% (p<0.01; HR 2.4; Figure 1b) while age >67 years sustained borderline significance (p=0.07; HR 1.8). Figure 1c depicts survival data based on the presence or absence of TP53 VAF ≥22% and therapy-related qualification; low-risk with no risk factors (N=112; median survival 11.7 years; 5/10-year survival 73%/52%) and high-risk with one or both risk factors (N=34; median survival 4 years; 5/10-year survival 42%/14%); leukemia-free survival was affected by TP53 VAF ≥22% (p<0.01) but not therapy-related setting (p=0.13). Conclusions : The current study highlights TP53 VAF and therapy-related setting as the most prominent risk factors for survival in MDS-del(5q) and proposes a simple risk model that distinguishes patients with a projected 10-year survival that exceeds 50%, in the absence of both risk factors, and plummets to 14%, in the presence of one of the two risk factors. Accordingly, allogeneic stem cell transplant might be deferred in the former instance while it should be pursued sooner than later, in the latter instance.

Venetoclax with decitabine or azacitidine in previously untreated TP53mut acute myeloid leukemia.

e19051 Background: Patients with del17p or TP53mut AML have a dismal prognosis marked by high relapse rates and short survival. The addition of venetoclax to a hypomethylating agent (HMA; decitabine or azacitidine) improved overall survival in AML compared to HMA monotherapy for non-induction candidates. However, data on the relative efficacy of venetoclax combination therapy in adverse-risk molecular groups remains sparse outside of clinical trials. The purpose of this study was to determine the efficacy of venetoclax combination therapy in patients with del17p or TP53mut AML, using HMA monotherapy as a comparison. Methods: We retrospectively analyzed 35 patients with del17p or TP53mut AML treated with venetoclax + HMA or HMA alone from January 1, 2013 to January 1, 2022 at VCU Massey Cancer Center. Patients were treated in the front-line setting and separated into two cohorts: those treated with venetoclax + HMA or HMA monotherapy. To reduce confounding effects, patients that received venetoclax following initiation of HMA monotherapy were excluded from the monotherapy arm. We recorded baseline patient and disease characteristics, cytogenetic risk, response, and survival. We used Fisher’s exact test for nominal data and Mann-Whitney for ordinal data. We analyzed survival by the Kaplan-Meier method and compared groups with the log-rank test. Results: We identified 35 patients with del17p or TP53mut AML treated with venetoclax + HMA or HMA monotherapy in the first line. Twenty-one (60.0%) patients were in the venetoclax combination arm, and 14 (40.0%) were in the monotherapy arm. There were no significant between-group differences in age (72 vs 74, p = 0.303), ECOG score (2 vs 1, p = 0.292), or Charlson Comorbidity Index score (6 vs 7, p = 0.211) between the combination and monotherapy arms, respectively. The median TP53mut variant allele frequency (VAF) was 35.5% in the venetoclax cohort and 42.2% in the HMA cohort ( p = 0.479). The composite complete remission (CCR) rate significantly favored the venetoclax arm at 40.0% (95% CI, 21.9 - 61.3), compared to 0% (95% CI, 0 - 27.8) in the monotherapy arm ( p = 0.029). Furthermore, the median overall survival significantly favored the venetoclax cohort at 6.0 months compared to 3.4 months in the HMA cohort ( p = 0.026). Strikingly, in the venetoclax combination arm, we identified that a TP53mut VAF threshold of less than 35.0% predicted superior survival at 11.8 months compared to 2.8 months for VAF of greater than 35% ( p = 0.035). The identification of a similar VAF threshold was limited in the monotherapy arm due to the availability of NGS data. Conclusions: The addition of venetoclax offers significantly superior response and survival compared to HMA monotherapy in patients with del17p or TP53mut AML. We identified a novel TP53mut VAF threshold of 35.0% that predicts superior survival with combination therapy. The clinical use of this threshold requires prospective validation.

Abstract A22: Complete mutation concordance with amplicon-based NGS between peripheral blood and bone marrow at 0.5% VAF threshold

Abstract Background: Acute myeloid leukemia (AML) is a hematological malignancy characterized by genomic heterogeneity throughout disease progression and between patients. Traditionally, both multiparameter flow cytometry (MFC) and next-generation sequencing (NGS) samples are collected via bone marrow (BM) aspiration, which is an invasive procedure with risk of infection. Peripheral blood (PB) has been demonstrated as a good substitute for BM but the concordance at different levels of NGS variant allele frequency (VAF) has not been defined. This study aims to demonstrate the relationship of NGS signals between PB and BM. Method: Genomic profiles of 60 matched samples of BM and PB were obtained retrospectively from 24 patients with de novo AML (n=17) or secondary AML (n=7). The samples were subjected to library preparation with a hematologic panel based on AmpliMark, an ultrasensitive amplicon-based NGS platform technology. The hematologic panel covers up to 45 genes common in myeloid and lymphoid neoplasms, with an established limit of detection of 0.1% VAF. Optimized bioinformatics pipeline and in-house sequencing noise removal algorithm were applied for variant selection. One of the secondary AML patients had a concurrent diagnosis of multiple myeloma; for the purposes of this analysis, the myeloma-associated variants were excluded. Results: 50 out of 60 matched BM and PB samples (83.3%) in the cohort were positive for at least one variant in either sample type. Complete or partial concordance of genetic profiles was achieved in 86.7% of the matched BM and PB samples. All samples obtained during diagnosis or relapse showed complete concordance, while all partial concordant and discordant profiles were sampled during post-treatment or remission stages. All BM samples with ≥1.5% abnormal cells detected by MFC demonstrated complete concordance between BM and PB genetic profiles. Analysis at the individual variant level (n=115) showed 78.6% of positive percent agreement (PPA) comparing PB to BM in variant detection. For variants detected at ≥0.5% and ≥0.1% VAF in the BM samples, 100% PPA and 86.4% PPA were achieved from matched PB samples, respectively. A strong correlation of VAF (r=0.98) was also demonstrated between variants detected in BM and PB. Conclusion: PB NGS is particularly sensitive for detection of myeloid specific mutations in de novo and secondary AMLs with complete concordance at VAFs ≥0.5%, when compared to BM. The high concordance and minimally invasive nature of PB sampling makes it a favorable option for diagnostic and monitoring of AML. Citation Format: Benjamin Lim, Chae Yin Cher, Jonathan Poh, Kao Chin Ngeow, Zi Yi Lim, Min-Han Tan. Complete mutation concordance with amplicon-based NGS between peripheral blood and bone marrow at 0.5% VAF threshold [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A22.

Properties of non-coding mutation hotspots as urinary biomarkers for bladder cancer detection

Mutations at specific hotspots in non-coding regions of ADGRG6, PLEKHS1, WDR74, TBC1D12 and LEPROTL1 frequently occur in bladder cancer (BC). These mutations could function as biomarkers for the non-invasive detection of BC but this remains largely unexplored. Massively-parallel sequencing of non-coding hotspots was applied to 884 urine cell pellet DNAs: 591 from haematuria clinic patients (165 BCs, 426 non-BCs) and 293 from non-muscle invasive BC surveillance patients (29 with recurrence). Urine samples from 142 non-BC haematuria clinic patients were used to optimise variant calling. Non-coding mutations are readily detectable in the urine of BC patients and undetectable, or present at much lower frequencies, in the absence of BC. The mutations can be used to detect incident BC with 66% sensitivity (95% CI 58–75) at 92% specificity (95% CI 88–95) and recurrent disease with 55% sensitivity (95% CI 36–74) at 85% specificity (95% CI 80–89%) using a 2% variant allele frequency threshold. In the NMIBC surveillance setting, the detection of non-coding mutations in urine in the absence of clinically detectable disease was associated with an increased relative risk of future recurrence (RR = 4.62 (95% CI 3.75–5.48)). As urinary biomarkers, non-coding hotspot mutations behave similarly to driver mutations in BC-associated genes and could be included in biomarker panels for BC detection.

Subclonal KIT D816V Mutations Are Prevalent in Chronic Myelomonocytic Leukemia and Correlate with Distinct Phenotypic Features

Background: Activating mutations in KIT are infrequently observed in myeloid neoplasms, but are a pathognomonic finding in systemic mastocytosis (SM) where the hotspot D816V variant is documented in 90-95% of patients. In SM, KIT variants may occur at a low variant allele frequency (VAF) below the limits of detection of most commercial next generation sequencing (NGS) assays necessitating digital droplet PCR (ddPCR) for accurate detection. Aggressive SM often presents concurrently with an associated hematologic neoplasm (SM-AHN). Chronic myelomonocytic leukemia (CMML) is the most frequently co-occurring hematologic malignancy, but KIT mutations are infrequently detected by conventional NGS. The limitations in detecting KIT variants by NGS and increased frequency of CMML in SM-AHN suggest that subclonal KIT mutations may be underrecognized in CMML. Further, identification may have prognostic and therapeutic implications given the emergence of KIT-targeted therapies in SM. Methods: A multicenter, retrospective study was performed to assess the prevalence of subclonal KIT D816V mutations in CMML patients and examine clinical implications. Eligible patients had pathologically confirmed CMML by WHO 2017 criteria, with available bone marrow (n=59) or peripheral blood (n=14) sample obtained prior to therapy. Samples underwent DNA sequencing using a 285-gene NGS panel (QIAseq), with a VAF threshold of 5% utilized for variant calling. KIT D816V mutations were assessed by ddPCR, with limit of detection of 0.03%. Clinical data was collected from baseline and follow up for phenotypic and prognostic comparisons. Fishers exact and Chi-square tests were used to compare categorical variables and the Mann-Whitney test for continuous variables. The Kaplan-Meier method was used to estimate overall survival (OS), with the log rank test used to compare survival between groups. The study was approved by institutional review boards at each institution. Results: A total of 73 patients were included with a median age of 71, and 66% were male. By WHO criteria, 50% were classified as CMML-0, 28% CMML-1, and 22% CMML-2; 59% of patients had proliferative CMML by FAB criteria. KIT D816V mutations were detected in 15 patients (21%) by ddPCR, compared to two (3%) by NGS. The median VAF of mutations detected by ddPCR was 0.06%, whereas the two variants detected by NGS had VAFs of 44.1% and 47.0%. Baseline characteristics of KIT wild-type (WT) and mutated (MUT) patients are detailed in Table 1. KIT MUT patients had lower white blood cell (p=0.007) and absolute monocyte counts (p=0.04), less frequent splenomegaly (p=0.05), more frequent karyotype abnormalities (p=0.14), and lower frequency of proliferative disease by FAB criteria (p=0.01). There was a median of 4 variants (range 0-7) detected per patient by NGS, and the most frequently mutated genes were TET2 (59%), SRSF2 (44%), ASXL1 (33%),RUNX1 (25%), and NRAS (25%). KIT MUT patients had a median of 3 total variants (range 0-6) detected by NGS compared to 4 (range 0-7) in KIT WT (p=0.63). There was a trend toward higher frequency of SF3B1 (23% vs. 5%, p=0.07) mutations in KIT MUT patients. Conversely, no mutations were observed in KRAS or CBL in KIT MUT patients, compared to 14% and 16% in KIT WT, respectively (p=0.34, p=0.20). When evaluating mutations in signaling pathways, there was a trend toward lower frequency in KIT MUT patients compared to WT (31% vs. 59%, p=0.12). With a median follow up of 48 months, median OS of the cohort was 30 months. KIT MUT patients demonstrated an OS of 27.5 months compared to 30 months in WT patients (HR 1.06, p=0.87). Transformation to AML was observed in 42% of KIT MUT patients compared to 29% of WT patients (p=0.49). A similar proportion of patients were treated with hypomethylating agents (HMAs), with a response rate of 14% in KIT MUT patients compared to 32% in WT (p=0.63), and complete remission rates of 0% vs. 27%, respectively. Conclusions: This study is the first to document the prevalence of subclonal KIT D816V mutations by ddPCR in CMML, revealing a high prevalence in comparison to detection by standard NGS assays. While sample size limited statistical comparisons, KIT mutations were associated with distinct phenotypic features, including less proliferative disease and poor response to HMAs. Given the efficacy of KIT inhibitors in SM-CMML, these findings could represent an avenue for targeted therapy in CMML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of an Ultrasensitive NGS-Based Blood Test for the Diagnosis of Early-Stage Lung Cancer: Sensitivity, a Hurdle Still Difficult to Overcome.

Simple SummaryCurrently, an accurate diagnosis of lung cancer relies on the microscopic examination of tissue biopsies. These samples can, however, only be obtained by invasive procedures. The aim of our study was to evaluate the use of a liquid biopsy for early-stage lung cancer detection in patients with a lung lesion on imaging. This approach would be particularly relevant for suspected lung lesions that are difficult to reach for a tissue-based diagnosis. Despite technical improvements for the use of liquid biopsy-based cell-free DNA analysis, its application for the detection of early-stage lung cancer is currently limited by sensitivity and a biological background of somatic variants.Diagnosis of lung cancer requires histological examination of a tissue sample, which in turn requires an invasive procedure that cannot always be obtained. Circulating tumor DNA can be reliably detected in blood samples of advanced-stage lung cancer patients and might also be a minimally invasive alternative for early-stage lung cancer detection. We wanted to explore the potential of targeted deep sequencing as a test for the diagnosis of early-stage lung cancer in combination with imaging. Mutation detection on cell-free DNA from pretreatment plasma samples of 51 patients with operable non-small cell lung cancer was performed and results were compared with 12 control patients undergoing surgery for a non-malignant lung lesion. By using a variant allele frequency threshold of 1%, somatic variants were detected in 23.5% of patients with a median variant allele fraction of 3.65%. By using this threshold, we could almost perfectly discriminate early-stage lung cancer patients from controls. Our study results are discussed in the light of those from other studies. Notwithstanding the potential of today’s techniques for the use of liquid biopsy-based cell-free DNA analysis, sensitivity of this application for early-stage lung cancer detection is currently limited by a biological background of somatic variants with low variant allele fraction.

Impact of Low-Burden TP53 Mutations in the Management of CLL.

In chronic lymphocytic leukemia (CLL), TP53 abnormalities are associated with reduced survival and resistance to chemoimmunotherapy (CIT). The recommended threshold to clinically report TP53 mutations is a matter of debate given that next-generation sequencing technologies can detect mutations with a limit of detection of approximately 1% with high confidence. However, the clinical impact of low-burden TP53 mutations with a variant allele frequency (VAF) of less than 10% remains unclear. Longitudinal analysis before and after fludarabine based on NGS sequencing demonstrated that low-burden TP53 mutations were present before the onset of treatment and expanded at relapse to become the predominant clone. Most studies evaluating the prognostic or predictive impact of low-burden TP53 mutations in untreated patients show that low-burden TP53 mutations have the same unfavorable prognostic impact as clonal defects. Moreover, studies designed to assess the predictive impact of low-burden TP53 mutations showed that TP53 mutations, irrespective of mutation burden, have an inferior impact on overall survival for CIT-treated patients. As low-burden and high-burden TP53 mutations have comparable clinical impacts, redefining the VAF threshold may have important implications for the clinical management of CLL.

TP53 Mutations with Low Variant Allele Frequency Predict Short Survival in Chronic Lymphocytic Leukemia.

In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear. Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort (n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials. In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort (n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases (n = 552) from the retrospective cohort, and the UK trials cohort. TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes.

Comparative Assessment of FLT3 Variant Allele Frequency By Capillary Electrophoresis and Next-Generation Sequencing in FLT3mut+ patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Who Received Gilteritinib Therapy

Comparative Assessment of FLT3 Variant Allele Frequency By Capillary Electrophoresis and Next-Generation Sequencing in FLT3mut+ patients with Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML) Who Received Gilteritinib Therapy

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions

BackgroundTargeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing.ResultsAll panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5–20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden.ConclusionThis comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

Clonal haematopoiesis in chronic ischaemic heart failure: prognostic role of clone size for DNMT3A- and TET2-driver gene mutations.

Somatic mutations of the epigenetic regulators DNMT3A and TET2 causing clonal expansion of haematopoietic cells (clonal haematopoiesis; CH) were shown to be associated with poor prognosis in chronic ischaemic heart failure (CHF). The aim of our analysis was to define a threshold of variant allele frequency (VAF) for the prognostic significance of CH in CHF. We analysed bone marrow and peripheral blood-derived cells from 419 patients with CHF by error-corrected amplicon sequencing. Cut-off VAFs were optimized by maximizing sensitivity plus specificity from a time-dependent receiver operating characteristic (ROC) curve analysis from censored data. 56.2% of patients were carriers of a DNMT3A- (N = 173) or a TET2- (N = 113) mutation with a VAF >0.5%, with 59 patients harbouring mutations in both genes. Survival ROC analyses revealed an optimized cut-off value of 0.73% for TET2- and 1.15% for DNMT3A-CH-driver mutations. Five-year-mortality was 18% in patients without any detected DNMT3A- or TET2 mutation (VAF < 0.5%), 29% with only one DNMT3A- or TET2-CH-driver mutations above the respective cut-off level and 42% in patients harbouring both DNMT3A- and TET2-CH-driver mutations above the respective cut-off levels. In carriers of a DNMT3A mutation with VAF ≥ 1.15%, 5-year mortality was 31%, compared with 18% mortality in those with VAF < 1.15% (P = 0.048). Likewise, in patients with TET2 mutations, 5-year mortality was 32% with VAF ≥ 0.73%, compared with 19% mortality with VAF < 0.73% (P = 0.029). The present study defines novel threshold levels for clone size caused by acquired somatic mutations in the CH-driver genes DNMT3A and TET2 that are associated with worse outcome in patients with CHF.

Next-Generation Sequencing in Non-BCR-ABLMechanisms Resistance in Patients with Chronic Myeloid Leukemia: Results of a Pilot Study

Introduction.In recent times, more and more data are available on the mechanisms of development of primary and secondary resistance to therapy with tyrosine kinase inhibitors (TKIs) in patients with CML. The use of next-generation sequencing (NGS) allows detecting mutations in theBCR-ABLkinase domain (KD) in some patients with resistance to TKI therapy. However, the reasons for resistance in patients without mutations in theBCR-ABLKD are still unclear. Aim.To determineBCR-ABL-independent molecular genetic markers of resistance to TKI therapy in patients with CML using NGS. Materials and methods.We examined blood samples from 15 patients with resistant CP CML (8 men and 7 women) aged 32 to 59 years (Me = 44 years). Resistance to TKI therapy was determined according to the ELN 2013 criteria. In this group: 2 patients received therapy with 2 TKIs, 8 patients with 3 TKIs, 3 patients - 4 TKIs, and 2 patients received 5 TKIs (Me = 3 TKIs). Cytogenetic test revealed trisomy 8 in 3 patients. One patient was diagnosed with the T315I mutation at once. The patients underwent NGS examination of the myeloid panel, which included 55 genes, with an average reading depth of 1000x using a MiSeq device (Illumina). The 2% threshold of variant allele frequency (VAF) was used. The clinical significance of mutations was evaluated using the COSMIC, ClinVar and OMIM databases. Results.By NGS we identified genetic aberrations in all patients: an average of 5 mutations (from 1 to 10 in one patient). A total of 77 mutations with unclear clinical significance were identified: 54 of them were missense mutations, 22 were synonymous mutations, and 1 frameshift mutation. The most common abnormalities were found in theNF1(10),TET2(8),ATRX(7), andSTAG2(6) genes. In 2 patients, the simultaneous presence of mutations in theATRXandNF1genes was found, they had a primary-resistant course of the disease, MMR was not achieved even on the 3rd and 4th lines of targeted therapy. Three patients were found to have simultaneous mutations in theNF1andSTAG2genes, including two patients who also had a primary-resistant course of the disease, and one of them showed resistance to all 5 TKIs. In 2 patients with the simultaneous presence of mutations in theATRXandTET2genes, resistance to 2 TKIs was registered; CCyR without MMR was achieved only with ponatinib. In 3 patients with singleSTAG2mutations, MMR was achieved on the third line of TKI. One patient with 3 mutations inTET2also achieved MMR only on the third line TKI. Mutations inTET2are more common in Ph-negative MPNs. In our study, mutations were most often detected in theNF1,ATRXandSTAG2genes. Although mutations in theNF1andATRXgenes have been described previously, they were found in responding patients with CP CML (Kim et. al. Blood 2017). In our study, mutations in these genes were found in therapy-resistant patients with an unfavorable course of the disease. TheNF1gene encodes a GTPase activating protein (GAP), which is involved in inhibiting the RAS/MAPK signal transduction pathway and is a tumor suppressor. Mutations in this gene are often associated with the development of CMML and JMML, resistance to therapy and decreased overall survival. TheATRXgene encodes a protein involved in chromatin remodeling, regulation of transcription, replication, and maintenance of telomere structure. TheSTAG2gene encodes a protein that is part of the cohesin complex. Possibly, abnormalities in these genes may belong to a clone of leukemic stem cells (Vetrie et. al. Nature 2020), cause their resistance to TKI,BCR-ABL-independent proliferative potential and their genetic instability. In addition, some of these mutations, possibly, are preleukemic - they could precede the emergence of the Ph-clone in such patients by the mechanism of clonal hematopoiesis of indeterminate potential and may cause an unfavorable prognosis. This allowed us to suggest the prognostic and therapeutic value of mutations in theNF1,ATRXandSTAG2genes in CP CML patients with resistance to TKI therapy. Conclusion.The obtained results of a pilot study of NGS use for predictingBCR-ABL-independent resistance to TKI therapy in patients with CML can serve as a basis for further research aimed at developing prognostic models and choosing a treatment method for resistance to targeted therapy. Disclosures Voloshin: Novartis:Honoraria, Speakers Bureau.Fominykh:Novartis:Honoraria, Speakers Bureau;Pfizer:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau.Shuvaev:Novartis:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau;Pfizer:Honoraria, Speakers Bureau.Martynkevich:Pfizer:Honoraria, Speakers Bureau;BMS:Honoraria, Speakers Bureau;Novartis:Honoraria, Speakers Bureau.

Robust assessment of tumor mutational burden in cytological specimens from lung cancer patients

ObjectivesTumor mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint inhibitor therapy. While the feasibility of TMB analysis on formalin-fixed paraffin-embedded (FFPE) samples has been thoroughly evaluated, only limited analyses have been performed on cytological samples, and no dedicated study has investigated concordance of TMB between different sample types. Here, we assessed TMB on matched histological and cytological samples from lung cancer patients and evaluated the accuracy of TMB estimation in these sample types. Materials and MethodsWe analyzed mutations and resulting TMB in FFPE samples and matched ethanol-fixed cytological smears (n = 12 matched pairs) by using a targeted next-generation sequencing assay (Oncomine™ Tumor Mutational Load). Two different variant allele frequency (VAF) thresholds were used to estimate TMB (VAF = 5% or 10%). ResultsAt 5% VAF threshold, 73% (107/147) of mutations were concordantly detected in matched histological and cytological samples. Discordant variants were mainly unique to FFPE samples (34/40 discordant variants) and mostly C:G > T:A transitions with low allelic frequency, likely indicating formalin fixation artifacts. Increasing the VAF threshold to 10% clearly increased the number of concordantly detected mutations in matched histological and cytological samples to 96% (100/106 mutations), and drastically reduced the number of FFPE-only mutations (from 34 to 4 mutations). In contrast, cytological samples showed consistent mutation count and TMB values at both VAF thresholds. Using FFPE samples, 2 out of 12 patients were classified as TMB-high at VAF cutoff of 5% but TMB-low at 10%, whereas cytological specimens allowed consistent patient classification independently from VAF cutoff. ConclusionOur results show that cytological smears provide more consistent TMB values due to high DNA quality and lack of formalin-fixation induced artifacts. Therefore, cytological samples should be the preferred sample type for robust TMB estimation.

Abstract 1335: Analytical evaluation of a targeted solid tumor next-generation sequencing assay

Abstract With the growing interest in personalized medicine for cancer patients, next generation sequencing (NGS) variant screening assays are becoming a powerful clinical tool for tumor classification. The AmpliSeqTM for Illumina Focus Panel is a targeted sequencing approach that interrogate 52 of the most common solid tumor genes for single nucleotide variants (SNVs), insertion/deletions (indels), and copy number variants (CNVs) in DNA and gene fusions in RNA concurrently. To evaluate the performance of the Focus assay, an analytical performance study was conducted. Using an automated workflow with high quality DNA reference samples, both intra-assay and inter-assay experiments resulted in high reproducibility (R2 &amp;gt;0.95) for detecting expected variants at various frequencies. Similarly, using high quality RNA reference samples, 100% of all expected fusions were detected in all experiments, demonstrating accuracy and precision of the Focus panel assay with high quality samples. To evaluate assay sensitivity, limit of detection (LOD) studies were carried out by titrating the nucleic acid input into the assay. Using a 5% variant allele frequency threshold (empirically established in the analysis pipeline), the assay detected all expected SNVs and indels across sample across sample input amounts. Using extracted DNA from a human cell line model (HL-60) with a confirmed MYC gene gain, results showed reliable MYC gain detection down to 4 copies in a diploid background. Similarly, extracted RNA from a confirmed EML4-ALK fusion positive cell line (H2228) diluted into background RNA showed 100% positive fusion calling down to 250pg or 133 copies. Finally, to understand the assay performance on clinically-relevant FFPE samples, somatic variant detection in 8 human cell lines and 68 total FFPE samples was evaluated. For FFPE DNA samples, the libraries averaged &amp;gt;90% on-target alignment and coverage uniformity with &amp;gt;3000x mean coverage across all samples. While FFPE RNA showed lower alignment for poor quality samples, the assay showed overall high positive and negative predictive value for variant and fusion calling. In summary, the AmpliSeqTM for Illumina Focus panel is an automation-compatible small panel NGS assay with robust performance to assess somatic variants in solid tumors. Citation Format: Jeff H. Tsai, Valerie Montel, Nathalie Mouttham. Analytical evaluation of a targeted solid tumor next-generation sequencing assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1335.

Laboratory cross-comparison of tumor BRCA1/2 analysis in a multicenter epithelial ovarian cancer series: The BORNEO GEICO60-0 study.

6055 Background: Epithelial ovarian cancer (EOC) identification of BRCA1 and BRCA2 mutations is usually carried out in germline, representing around 17% in high grade serous ovarian cancer (HGSOC) and further 5-7% are only identified in the tumor (somatic). The aim of this study was to identify in EOC tumor BRCA mutation frequency and inter-laboratory reproducibility using different Next-generation Sequencing (NGS) approaches. Methods: In an ambispective study design, a population of unselected consecutive non mucinous EOC was clinically annotated and Formalin-Fixed Paraffin-Embedded (FFPE) tumor BRCA1/2 mutation analysis was undertaken in two laboratories (Lab-1 and Lab-2) simultaneously. Both laboratories used their own validated NGS panels; variant allele frequency threshold was 5% for single nucleotide polymorphism and 10% for indels. Each laboratory classified variants into three categories based on ACMG criteria: non-mutated (class 1-2), Variants of Uncertain Significance (VUS: class 3) and likely pathogenic/pathogenic (class 4-5). Germline BRCA analysis was available according to local clinical practice or centralized in Lab-1 if histology was low grade. Results: Ninety FFPE samples were received, 8 had insufficient material to be analyzed in both laboratories and 6 cases were discarded due to tumor cellularity below 20% leaving 76 cases to be sequenced. The population had a median age of 58 (25-84) years, 87% (66/76) of HGSOC histology and 70% of advanced stages (III-IVB: 53) and 14.5% (11) germline BRCA mutations (3 with not available results). Lab-1 identified 17 class 4-5 mutations, 11 correspond to germline, 4 (5.3%) are just somatic and 2 have germline results not available yet. Lab-2 had one not valuable analysis and identified 16 class 4-5 mutations, 10 corresponding to germline and 4 somatic variants. Percentage of concordance between both laboratories was 96% (kappa coefficient 0.883; p value &lt; 0.0001). Three discordant out of 18 class 4-5 mutations included 2 undetected (VAF of 14.9% and 60.3% respectively) and one class 4 in Lab-2 classified as VUS in Lab-1 due to different interpretation criteria. Conclusions: The global BRCA mutation frequency in our series was 22.3% for Lab-1 and 21.0% for Lab-2. Concordance between tumor BRCA mutation analysis was high (96%). Nevertheless, further effort is required on harmonizing the technical and analytical aspects in tumor mutational analysis.