variant allele frequency
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2022 ◽  
Author(s):  
Abhay Singh ◽  
Nuria Mencia-Trinchant ◽  
Elizabeth A. Griffiths ◽  
Alaa Altahan ◽  
Mahesh Swaminathan ◽  
...  

PURPOSE Hematologic toxic effects of peptide receptor radionuclide therapy (PRRT) can be permanent. Patients with underlying clonal hematopoiesis (CH) may be more inclined to develop hematologic toxicity after PRRT. However, this association remains understudied. MATERIALS AND METHODS We evaluated pre- and post-PRRT blood samples of patients with neuroendocrine tumors. After initial screening, 13 cases of interest were selected. Serial blood samples were obtained on 4 of 13 patients. Genomic DNA was analyzed using a 100-gene panel. A variant allele frequency cutoff of 1% was used to call CH. RESULT Sixty-two percent of patients had CH at baseline. Persistent cytopenias were noted in 64% (7 of 11) of the patients. Serial sample analysis demonstrated that PRRT exposure resulted in clonal expansion of mutant DNA damage response genes ( TP53, CHEK2, and PPM1D) and accompanying cytopenias in 75% (3 of 4) of the patients. One patient who had a normal baseline hemogram and developed persistent cytopenias after PRRT exposure showed expansion of mutant PPM1D (variant allele frequency increased to 20% after exposure from < 1% at baseline). In the other two patients, expansion of mutant TP53, CHEK2, and PPM1D clones was also noted along with cytopenia development. CONCLUSION The shifts in hematopoietic clonal dynamics in our study were accompanied by emergence and persistence of cytopenias. These cytopenias likely represent premalignant state, as PPM1D-, CHEK2-, and TP53-mutant clones by themselves carry a high risk for transformation to therapy-related myeloid neoplasms. Future studies should consider CH screening and longitudinal monitoring as a key risk mitigation strategy for patients with neuroendocrine tumors receiving PRRT.


2021 ◽  
Vol 11 (12) ◽  
Author(s):  
Paola Guglielmelli ◽  
Giuseppe G. Loscocco ◽  
Carmela Mannarelli ◽  
Elena Rossi ◽  
Francesco Mannelli ◽  
...  

AbstractArterial (AT) and venous (VT) thrombotic events are the most common complications in patients with polycythemia vera (PV) and are the leading causes of morbidity and mortality. In this regard, the impact of JAK2V617F variant allele frequency (VAF) is still debated. The purpose of the current study was to analyze the impact of JAK2V617F VAF in the context of other established risk factors for thrombosis in a total of 865 2016 WHO-defined PV patients utilizing two independent cohorts: University of Florence (n = 576) as a training cohort and Policlinico Gemelli, Catholic University, Rome (n = 289) as a validation cohort. In the training cohort VT free-survival was significantly shorter in the presence of a JAK2V617F VAF > 50% (HR 4; p < 0.0001), whereas no difference was found for AT (HR 0.9; p = 0.8). Multivariable analysis identified JAK2V617F VAF > 50% (HR 3.8, p = 0.001) and previous VT (HR 2.2; p = 0.04) as independent risk factors for future VT whereas diabetes (HR 2.4; p = 0.02), hyperlipidemia (HR 2.3; p = 0.01) and previous AT (HR 2; p = 0.04) were independent risk factors for future AT. Similarly, JAK2V617F VAF > 50% (HR 2.4; p = 0.01) and previous VT (HR 2.8; p = 0.005) were confirmed as independent predictors of future VT in the validation cohort. Impact of JAK2V617F VAF > 50% on VT was particularly significant in conventional low-risk patients, both in Florence (HR 10.6, p = 0.005) and Rome cohort (HR 4; p = 0.02). In conclusion, we identified JAK2V617F VAF > 50% as an independent strong predictor of VT, supporting that AT and VT are different entities which might require distinct management.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brooks A. Benard ◽  
Logan B. Leak ◽  
Armon Azizi ◽  
Daniel Thomas ◽  
Andrew J. Gentles ◽  
...  

AbstractThe impact of clonal heterogeneity on disease behavior or drug response in acute myeloid leukemia remains poorly understood. Using a cohort of 2,829 patients, we identify features of clonality associated with clinical features and drug sensitivities. High variant allele frequency for 7 mutations (including NRAS and TET2) associate with dismal prognosis; elevated GATA2 variant allele frequency correlates with better outcomes. Clinical features such as white blood cell count and blast percentage correlate with the subclonal abundance of mutations such as TP53 and IDH1. Furthermore, patients with cohesin mutations occurring before NPM1, or transcription factor mutations occurring before splicing factor mutations, show shorter survival. Surprisingly, a branched pattern of clonal evolution is associated with superior clinical outcomes. Finally, several mutations (including NRAS and IDH1) predict drug sensitivity based on their subclonal abundance. Together, these results demonstrate the importance of assessing clonal heterogeneity with implications for prognosis and actionable biomarkers for therapy.


2021 ◽  
Author(s):  
Michael William Drazer ◽  
Claire C Homan ◽  
Kai Yu ◽  
Marcela Cavalcante de Andrade Silva ◽  
Kelsey E. McNeely ◽  
...  

Currently, there are at least a dozen recognized hereditary hematopoietic malignancies (HHMs), some of which phenocopy others. Among these, three HHMs driven by germline mutations in ANKRD26, ETV6, or RUNX1 share a phenotype of thrombocytopenia, qualitative platelet defects, and an increased lifetime risk of hematopoietic malignancies (HMs). Prior work has demonstrated that RUNX1 germline mutation carriers experience an elevated lifetime risk (66%) for developing clonal hematopoiesis (CH) prior to age 50. Germline mutations in ANKRD26 or ETV6 phenocopy RUNX1 germline mutations, but no studies have focused on the risk of CH in individuals with germline mutations in ANKRD26 or ETV6. To determine the prevalence of CH in individuals with germline mutations in ANKRD26 or ETV6, we performed next generation sequencing on hematopoietic tissue from twelve individuals with either germline ANKRD26 or germline ETV6 mutations. Each patient had thrombocytopenia but had not developed HMs. Among the seven individuals with germline ANKRD26 mutations, one patient had a CH clone driven by a somatic SF3B1 mutation (p.Lys700Glu). This mutation increased from a variant allele frequency (VAF) of 9.4% at age 56 to 17.4% at age 60. None of the germline ETV6 mutation carriers had evidence of CH at the limits of detection of the NGS assay (5% VAF). Unlike individuals with germline mutations in RUNX1, no individuals under the age of 50 with germline mutations in ANKRD26 or ETV6 had detectable CH. This work demonstrates that ANKRD26 germline mutation carriers, but not ETV6 mutation carriers, experience elevated risk for CH.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3450-3450
Author(s):  
Mihee Kim ◽  
Taehyung Kim ◽  
Seo-Yeon Ahn ◽  
Sung-Hoon Jung ◽  
Ga-Young Song ◽  
...  

Abstract As interest in elderly Acute Myeloid Leukemia (AML) patients increases, American society of hematology (ASH) 2020 guidelines for treating newly diagnosed AML in older adults suggested diverse treatment options. The guidelines suggest using monotherapy over combination of hypomethylation agent (HMAs) with other agents in newly diagnosed AML in older adults due to similar efficacy and the potential for more toxicity. HMAs alone is still used widely as an alternative treatment for patients who cannot use venetoclax due to the high cost and poor performance score. If there are early predictors of responsiveness to Decitabine mono therapy, it will be helpful to decide whether to combine Novel agents. This retrospective cohort study from a single institution aimed to evaluate the prognostic significance of Variant allele frequency (VAF) changes in elderly patients after 4 th cycle of decitabine. Total 123 patients with elderly AML were eligible. 57 patients performed follow-up bone marrow biopsy and 49 patients were available of follow up targeted NGS samples from biopsy after 4th cycle of decitabine. To clarify the immortal timed bias, landmark analyses were performed with patients (n=84) who remained at least the median time to perform follow-up bone marrow biopsy after 4th cycle of decitabine treatment. 24 patients (54.5%, 24 of 44) showed more than 50% decrease of VAF after 4 th cycle of decitabine (figure 1a). DMNT3A, TET2, IDH1, IDH2, and SETBP1 and SMC1A showed less than 50% of the decreases of VAF. Patients with DNA methylation genes showed significantly reduced VAF less than 50% (figure 1b). A significant difference of ∆VAF was observed depending on CR status (p=0.021). The survival outcome of patients who showed more than 50% decrease of initial VAF after 4th cycle of decitabine was significantly better than that that with less than 50% decrease of VAF(1-year OS VAF decrease ≥ 50% (n=23), 75.0%; VAF decrease &lt; 50% (n=20), 38.5%; no mutation (n=12), 45.5%; not available of follow up targeted NGS sample (n=29), 16.6%; p &lt; 0.001, figure 2a). Mutations in DNMT3A, TET2, and ASXL1 (DTA genes) were detected in samples from 19 patients at diagnosis. After the exclusion of DTA mutations, the survival outcome improved prognostic risk stratification power of NGS-based MRD assessment in AML. The survival outcome of patients who showed more than 50% decrease of initial VAF after 4th cycle of decitabine was significantly better than that that with less than 50% decrease of VAF(1-year OS VAF decrease ≥ 50% (n=24), 75.0%; VAF decrease &lt; 50% (n=19), 35.1%; no mutation (n=12), 50.1%; not available of follow up targeted NGS sample (n=29), 16.6%; p&lt;0.001, figure 2b). In conclusion, more than 50% decrease of VAF was important negative prognostic factors by improving overall response rate and OS. In case of patients with older adults who received decitabine treatment, if follow up BM biopsy after 4 th cycles of decitabine treatment showed more than 50% reduction of VAF, it may suggest to maintain decitabine treatment. However, if VAF is reduced by less than 50% in follow up BM biopsy, the residual disease burden is considered for the selection of combination treatment to improve survival outcome. Figure 1 Figure 1. Disclosures Kim: Bristol-Meier Squibb: Research Funding; Paladin: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1828-1828
Author(s):  
Maher Albitar ◽  
Hong Zhang ◽  
Andrew L. Pecora ◽  
Andrew Ip ◽  
Andre H. Goy ◽  
...  

Abstract Introduction: Using next generation sequencing (NGS) in monitoring residual disease in patients with myeloid neoplasms is complicated by the significant heterogeneity in these diseases and the frequent presence of CHIP (clonal hematopoiesis of indeterminate potential) in patients with hematologic neoplasms on which these neoplasms arise. This is particularly relevant post hematopoietic stem cell transplant (HSCT). We explored the ability of using plasma cell-free DNA (cfDNA) in monitoring patients after HSCT and evaluated the potential of using liquid biopsy as a replacement for bone marrow biopsy. Method: cfDNA was isolated from 204 peripheral blood samples obtained from 75 patients, collected at various time points ranging from 27 days to 650 days (median 178 days) post-transplant. DNA from 102 bone marrow (BM) samples was extracted and sequenced using the same panel and approach as cfDNA. Diagnoses included 30 acute myeloid leukemia (AML), 2 chronic myelogenous leukemia (CML), 5 chronic myelomonocytic leukemia (CMML), 4 lymphoma, 10 myelodysplastic syndrome (MDS), 2 multiple myeloma (MM), 9 myeloproliferative neoplasm (MPN), 1 aplastic anemia, and 11 acute lymphoblastic leukemia. cfDNA was sequenced by NGS using 177 gene panel on Illumina platform. Single primer extension (SPE) approach with UMI was used. Sequencing depth was increased to more than 2000X after removing duplicates. Low-level mutations were confirmed by inspecting BAM file. Results: 156 cfDNA samples (76%) tested negative and 48 samples from 30 different patients were positive. The negative samples were collected from 28 days to 650 days post-transplant (median 277 days). The positive samples were collected from 27 days to 650 days post-transplant (median 188 days). One of these positive patients was in full clinical relapse at the time of testing. No negative patient who remained negative had clinical relapse. Five patients converted from negative to positive and 12 from positive to negative with subsequent testing. Three from the converted to positive patients developed clinical relapse. Patients who were positive without clinical relapse had median variant allele frequency (VAF) of 0.85% (range: 0.01-13.25) and typically one mutated gene. The mutated genes in this group were: JAK2, IDH2, ASXL1, TET2, DNMT3A, ASXL1, PTPN11, SF3B1, MPL, CEBPA1. Patients who had clinical relapse (#4) had median VAF of 16.33% (0.4%-57.63%) with multiple mutated genes. The mutated genes in this group were: TP53, FLT3, ASXL1, CEBPA, EZH1, NRAS, SETBP1, TET2. To evaluate relevance to BM testing, we compared BM samples with cfDNA samples collected within 120 days of each other. This showed 17 pairs with concordant negative results, 10 with concordant positive results, 5 pairs with positive by cfDNA but negative by BM cells, and one pair with positive by BM but negative by cfDNA. This BM positive sample was performed at 78 days after the cfDNA sample and showed mutation in DNMT3A gene at VAF of 0.63%. Four of the 5 pairs with positive cfDNA but negative BM were collected approximately 3 months after bone marrow and the 5th case was one month prior to BM sample. Conclusion: These data suggest that monitoring residual disease after HSCT using cfDNA and NGS is a reliable approach and may replace the need of bone marrow biopsy. However, low-level mutations should not be used as the sole criterion for determining relapse. Variant allele frequency and the mutated gene should be considered in evaluating actionable findings. Disclosures Pecora: Genetic testing cooperative: Membership on an entity's Board of Directors or advisory committees; Genetic testing cooperative: Other: equity investor. Rowley: ReAlta Life Sciences: Consultancy.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4675-4675
Author(s):  
Christina K Ferrone ◽  
Amy JM McNaughton ◽  
Iran Rashedi ◽  
Hubert Tsui ◽  
Michael J Rauh

Abstract The recognition of MDS is challenging in early stages, where diagnosis may rely solely upon morphological criteria for dysplasia, a non-specific finding prone to inter-observer variation. Patients with equivocal bone marrow (BM) findings may be discharged from Hematology clinics and lost to follow up, or subjected to serial, invasive BM investigations and diagnostic delays. We therefore aimed to demonstrate the importance of hematopathologist-triaged, targeted NGS in identifying clonal cytopenias of undetermined significance (CCUS) in cases where MDS diagnostic criteria are not met based on morphology or cytogenetic analysis. We explored this using three REB-approved cohorts. Our first cohort was retrospective with BM samples ranging from 2010-14, involving cases that were previously suspicious for but non-MDS diagnostic. This included 70 patients from Sunnybrook (SHSC) and Kingston Health Sciences Centres (KHSC): 16 age-matched controls (8 negative lymphoma staging, 8 non-MDS cytopenias); 18 suspicious for MDS; 20 MDS; and 16 MDS/MPN. DNA was extracted and NGS was performed using our custom 48-gene Ion Torrent AmpliSeq myeloid panel (ThermoFisher). We identified suspected mutations in 2/16 (13%) controls (i.e. CHIP), 12/18 (67%) suspicious cases, 17/20 (85%) MDS cases, and 16/16 (100%) MDS/MPN cases. The mean and median number of mutations per suspicious patient (respectively 0.89 and 1; most commonly in SF3B1, TET2, RUNX1, and ASXL1) were lower than MDS (1.85 and 2; p=0.011) and MDS/MPN (3.13 and 3; p&lt;0.0001). There was a significant difference in the average variant allele frequency (VAF) per patient (those with ≥1 mutation) between control and suspicious groups (p=0.022), however, there were no significant differences in the average VAF between suspicious, MDS, and MDS/MPN cases. Furthermore, of the 16 patients with BM suspicious for MDS, 7 went on to get MDS. 4 of these patients had at least 1 clinically relevant somatic variant, while 3 had none. Of those with at least 1 variant, 3 had IPSS-level cytopenias at the time, indicating that had their mutational status been known at the time of their assessment, they would have been diagnosed with the provisional CCUS entity (while the rest would be classified as CHIP). To supplement these findings, we are amassing a prospective cohort involving cases at SHSC where patients have either idiopathic cytopenias (ICUS), or confirmed MDS diagnoses with one or more previously non-diagnostic BM. To date, we have performed sequencing for 36 of these patients, including 23 ICUS and 13 diagnosed MDS cases. Of the ICUS cases, 10 (44%) had at least 1 variant (mean # variant/patient = 1, mean variant allele frequency (VAF) = 34.0%) consistent with CCUS, while 12/13 (92%) of MDS patients had at least 1 variant (mean # variants/patient = 2, mean VAF = 42.3%). These findings are consistent with CCUS being common in suspicious MDS cases, with similar clonal size but lesser mutational burden than diagnosed MDS. In addition to these preliminary findings, 15/36 patients have serial samples that we are currently processing for NGS (among other cases we are accruing to present at the ASH meeting). By exploring serial cases with molecular results pre- and post- MDS diagnosis, we aim to further elucidate which features of CCUS may predict progression to MDS. Finally, we assessed clonality in cases suspicious for myeloid malignancy in our existing prospective myeloid NGS cohort at KHSC (Ferrone et al, JMD 2021). In this cohort of 168 patients, when focusing on cytopenias yet to be diagnosed, 71 patients had suspected MDS, MPN, or MDS/MPN prior to NGS (completed using the Oncomine Myeloid Assay; ThermoFisher). 36/71 (51%) were found to have variants that indicate clonality. This facilitated diagnoses of either myeloid malignancies or pre-malignant states, with nine cases in total of ICUS resulting in the identification of variants that were non-diagnostic of MDS (mainly in TET2), but indicative of CHIP (n=2) or CCUS (n=7). Furthermore, for the limited number with available follow up data, we found no significant difference in survival between individuals with low-grade MDS (n=10) and CCUS (n=6) (p=0.457). This evidence is in keeping with recent findings that the clinical features of CCUS may be consistent with low-risk MDS, emphasizing the importance of closely monitoring these patients, and even the possibility of assessing and treating them similarly to those with low-risk MDS. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 11 ◽  
Author(s):  
Riccardo Moia ◽  
Micol Giulia Cittone ◽  
Paola Boggione ◽  
Giulia Francesca Manfredi ◽  
Chiara Favini ◽  
...  

A total of 63 myeloproliferative neoplasms [MPN; 9 polycythemia vera (PV), 32 essential thrombocythemia (ET), and 22 myelofibrosis (MF)] underwent spleen stiffness (SS) measurement by vibration-controlled transient elastography equipped with a novel spleen-dedicated module. Higher SS values significantly correlated with grade 2-3 bone marrow (BM) fibrosis (p=0.035), with hemoglobin level &lt;10 g/dl (p=0.014) and with white blood cells ≥10,000/μl (p=0.008). Median SS was significantly higher in MF patients compared to ET and PV (p=0.015). SS also correlated with higher JAK2 variant allele frequency (p=0.02). This study identifies SS as a potential noninvasive tool that reflects BM fibrosis and the mutational burden in MPN.


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