Corrigendum to: Comparison of variant allele frequency and number of mutant molecules as units of measurement for circulating tumor DNA

e21057 Background: The advances in deep sequencing technologies makes circulating tumor DNA (ctDNA) detection in preoperative plasma a more and more significant indicator in early stage cancers. In particular, it allows us to learn a lot about tumor progression before surgery. Plasma variant allele frequency (VAF) of preoperative ctDNA mutations had been verified to correlate with tumor sizes in early lung cancers. Identification of lymph node (LN) metastasis status before surgery in patients with lung cancer is valuable for prognosis and treatment strategy decisions. To evaluate whether ctDNA predict the LN metastasis status, we conducted this analysis. Methods: Preoperative plasma and the matched leukocyte before treatment were collected from 38 early stage lung cancer patients, in which 8 cases with lymph node metastasis. 150-gene panel sequencing were performed on the specimens and the sequencing data were used as primary cohort. Multivariable logistic regression analysis was used to develop a prediction model based on the DNA and other clinical characteristics. The performance of the model was assessed by its calibration, discrimination and clinical usefulness in the public TRACERx 100 early lung cancers cohort. Results: The proportion of high variant allele frequency (HAF proportion) of mutations was significantly associated with LN status (p < 0.05, for both primary and validation cohorts). A prediction model was developed and predictors included the HAF proportion, smoking status and tumor size. The C-index of the model was 0.419 in primary cohort. In external validation cohort, the AUCs for LN positive was 0.678. Conclusions: Based on pan-gene panel deep sequencing, a big proportion of high variant allele frequency mutations in a single patient indicates advanced disease progression, which may reflect in LN involvement status. A logistic regression model was constructed using ctDNA and clinical characteristics. It showed considerable performance in the prediction of LN metastasis in early lung cancers.

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18 New method of assessing tumor heterogeneity utilizing both circulating tumor DNA and tissue DNA to predict the response to immunotherapy

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0018 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A18-A18

Author(s):

Jaeyoun Choi ◽

Myungwoo Nam ◽

Stanislav Fridland ◽

Jinyoung Hwang ◽

Chan Mi Jung ◽

...

Keyword(s):

Lung Cancer ◽

Immune Response ◽

Allele Frequency ◽

Scoring System ◽

Tumor Heterogeneity ◽

Metastatic Breast ◽

Circulating Tumor Dna ◽

List Type ◽

Multiple Biopsies ◽

Tumor Dna

BackgroundTumor heterogeneity assessment may help predict response to immunotherapy. In melanoma mouse models, tumor heterogeneity impaired immune response.1 In addition, among lung cancer patients receiving immunotherapy, the high clonal neoantigen group had favorable survival and outcomes.2 Ideal methods of quantifying tumor heterogeneity are multiple biopsies or autopsy. However, these are not feasible in routine clinical practice. Circulating tumor DNA (ctDNA) is emerging as an alternative. Here, we reviewed the current state of tumor heterogeneity quantification from ctDNA. Furthermore, we propose a new tumor heterogeneity index(THI) based on our own scoring system, utilizing both ctDNA and tissue DNA.MethodsSystematic literature search on Pubmed was conducted up to August 18, 2020. A scoring system and THI were theoretically derived.ResultsTwo studies suggested their own methods of assessing tumor heterogeneity. One suggested clustering mutations with Pyclone,3 and the other suggested using the ratio of allele frequency (AF) to the maximum somatic allele frequency (MSAF).4 According to the former, the mutations in the highest cellular prevalence cluster can be defined as clonal mutations. According to the latter, the mutations with AF/MSAF<10% can be defined as subclonal mutations. To date, there have been no studies on utilizing both ctDNA and tissue DNA simultaneously to quantify tumor heterogeneity. We hypothesize that a mutation found in only one of either ctDNA or tissue DNA has a higher chance of being subclonal.We suggest a scoring system based on the previously mentioned methods to estimate the probability for a mutant allele to be subclonal. Adding up the points that correspond to the conditions results in a subclonality score (table 1). In a given ctDNA, the number of alleles with a subclonality score greater than or equal to 2 divided by the total number of alleles is defined as blood THI (bTHI) (figure 1). We can repeat the same calculation in a given tissue DNA for tissue THI (tTHI) (figure 2). Finally, we define composite THI (cTHI) as the mean of bTHI and tTHI.Abstract 18 Table 1Subclonality scoreAbstract 18 Figure 1Hypothetical distribution of all alleles found in ctDNA bTHI = the number of alleles with a subclonality score greater than or equal to 2/the total number of alleles found in ctDNA = 10/20 =50%Abstract 18 Figure 2Hypothetical distribution of all alleles found in tissue DNA tTHI= the number of alleles with a subclonality score greater than or equal to 2/the total number of alleles found in tissue DNA = 16/40 = 40% cTHI= (bTHI + tTHI)/2 = 45%ConclusionsTumor heterogeneity is becoming an important biomarker for predicting response to immunotherapy. Because autopsy and multiple biopsies are not feasible, utilizing both ctDNA and tissue DNA is the most comprehensive and practical approach. Therefore, we propose cTHI, for the first time, as a quantification measure of tumor heterogeneity.ReferencesWolf Y, Bartok O. UVB-Induced Tumor Heterogeneity Diminishes Immune Response in Melanoma. Cell 2019;179:219–235.McGranahan N, Swanton C. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade. Science 2016;351:1463–1469.Ma F, Guan Y. Assessing tumor heterogeneity using ctDNA to predict and monitor therapeutic response in metastatic breast cancer. Int J Cancer 2020;146:1359–1368.Liu Z, Xie Z. Presence of allele frequency heterogeneity defined by ctDNA profiling predicts unfavorable overall survival of NSCLC. Transl Lung Cancer Res 2019;8:1045–1050.

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Examination of the predicted prevalence of Gitelman syndrome by ethnicity based on genome databases

Scientific Reports ◽

10.1038/s41598-021-95521-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Atsushi Kondo ◽

China Nagano ◽

Shinya Ishiko ◽

Takashi Omori ◽

Yuya Aoto ◽

...

Keyword(s):

Allele Frequency ◽

Carrier Frequency ◽

Japanese Population ◽

Autosomal Recessive ◽

Disease Prevalence ◽

Gitelman Syndrome ◽

Variant Allele ◽

Variant Allele Frequency ◽

Genome Databases ◽

Heterozygous Carriers

AbstractGitelman syndrome is an autosomal recessive inherited salt-losing tubulopathy. It has a prevalence of around 1 in 40,000 people, and heterozygous carriers are estimated at approximately 1%, although the exact prevalence is unknown. We estimated the predicted prevalence of Gitelman syndrome based on multiple genome databases, HGVD and jMorp for the Japanese population and gnomAD for other ethnicities, and included all 274 pathogenic missense or nonsense variants registered in HGMD Professional. The frequencies of all these alleles were summed to calculate the total variant allele frequency in SLC12A3. The carrier frequency and the disease prevalence were assumed to be twice and the square of the total allele frequency, respectively, according to the Hardy–Weinberg principle. In the Japanese population, the total carrier frequencies were 0.0948 (9.5%) and 0.0868 (8.7%) and the calculated prevalence was 0.00225 (2.3 in 1000 people) and 0.00188 (1.9 in 1000 people) in HGVD and jMorp, respectively. Other ethnicities showed a prevalence varying from 0.000012 to 0.00083. These findings indicate that the prevalence of Gitelman syndrome in the Japanese population is higher than expected and that some other ethnicities also have a higher prevalence than has previously been considered.

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Abstract 569: Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors

10.1158/1538-7445.am2021-569 ◽

2021 ◽

Author(s):

Antony Tin ◽

Vasily Aushev ◽

Ekaterina Kalashnikova ◽

Raheleh Salari ◽

Svetalana Shchegrova ◽

...

Keyword(s):

Solid Tumors ◽

Allele Frequency ◽

Tumor Burden ◽

Variant Allele ◽

Variant Allele Frequency

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Direct quantification of EGFR variant allele frequency in cell-free DNA using a microfluidic-free digital droplet PCR assay

Methods in Cell Biology - Microfluidics in Cell Biology Part C: Microfluidics for Cellular and Subcellular Analysis ◽

10.1016/bs.mcb.2018.10.002 ◽

2018 ◽

pp. 119-131 ◽

Cited By ~ 4

Author(s):

Benjamin Demaree ◽

Daniel Weisgerber ◽

Ata Dolatmoradi ◽

Makiko Hatori ◽

Adam R. Abate

Keyword(s):

Allele Frequency ◽

Variant Allele ◽

Variant Allele Frequency ◽

Pcr Assay ◽

Cell Free Dna ◽

Digital Droplet Pcr ◽

Free Dna ◽

Droplet Pcr ◽

Direct Quantification

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Variant Allele Frequency

10.32388/85cu2j ◽

2020 ◽

Author(s):

Keyword(s):

Allele Frequency ◽

Variant Allele ◽

Variant Allele Frequency

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Serial Monitoring of Circulating Tumor DNA by Next-Generation Gene Sequencing as a Biomarker of Response and Survival in Patients With Advanced NSCLC Receiving Pembrolizumab-Based Therapy

JCO Precision Oncology ◽

10.1200/po.20.00321 ◽

2021 ◽

pp. 510-524

Author(s):

Jeffrey C. Thompson ◽

Erica L. Carpenter ◽

Benjamin A. Silva ◽

Jamie Rosenstein ◽

Austin L. Chien ◽

...

Keyword(s):

Overall Survival ◽

Progression Free Survival ◽

Circulating Tumor Dna ◽

Variant Allele ◽

Molecular Response ◽

Next Generation ◽

Free Survival ◽

Variant Allele Fraction ◽

Tumor Dna ◽

Allele Fraction

PURPOSE Although the majority of patients with metastatic non–small-cell lung cancer (mNSCLC) lacking a detectable targetable mutation will receive pembrolizumab-based therapy in the frontline setting, predicting which patients will experience a durable clinical benefit (DCB) remains challenging. MATERIALS AND METHODS Patients with mNSCLC receiving pembrolizumab monotherapy or in combination with chemotherapy underwent a 74-gene next-generation sequencing panel on blood samples obtained at baseline and at 9 weeks. The change in circulating tumor DNA levels on-therapy (molecular response) was quantified using a ratio calculation with response defined by a > 50% decrease in mean variant allele fraction. Patient response was assessed using RECIST 1.1; DCB was defined as complete or partial response or stable disease that lasted > 6 months. Progression-free survival and overall survival were recorded. RESULTS Among 67 patients, 51 (76.1%) had > 1 variant detected at a variant allele fraction > 0.3% and thus were eligible for calculation of molecular response from paired baseline and 9-week samples. Molecular response values were significantly lower in patients with an objective radiologic response (log mean 1.25% v 27.7%, P < .001). Patients achieving a DCB had significantly lower molecular response values compared to patients with no durable benefit (log mean 3.5% v 49.4%, P < .001). Molecular responders had significantly longer progression-free survival (hazard ratio, 0.25; 95% CI, 0.13 to 0.50) and overall survival (hazard ratio, 0.27; 95% CI, 0.12 to 0.64) compared with molecular nonresponders. CONCLUSION Molecular response assessment using circulating tumor DNA may serve as a noninvasive, on-therapy predictor of response to pembrolizumab-based therapy in addition to standard of care imaging in mNSCLC. This strategy requires validation in independent prospective studies.

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Genotyping of circulating tumor DNA in cholangiocarcinoma reveals diagnostic and prognostic information

Scientific Reports ◽

10.1038/s41598-019-49860-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 9

Author(s):

T. J. Ettrich ◽

D. Schwerdel ◽

A. Dolnik ◽

F. Beuter ◽

T. J. Blätte ◽

...

Keyword(s):

Deep Sequencing ◽

Tumor Tissue ◽

Circulating Tumor Dna ◽

Therapeutic Strategies ◽

Variant Allele Frequency ◽

Prognostic Information ◽

Driver Genes ◽

Tumor Load ◽

Molecular Landscape ◽

Tumor Dna

Abstract Diagnosis of Cholangiocarcinoma (CCA) is difficult, thus a noninvasive approach towards (i) assessing and (ii) monitoring the tumor-specific mutational profile is desirable to improve diagnosis and tailor treatment. Tumor tissue and corresponding ctDNA samples were collected from patients with CCA prior to and during chemotherapy and were subjected to deep sequencing of 15 genes frequently mutated in CCA. A set of ctDNA samples was also submitted for 710 gene oncopanel sequencing to identify progression signatures. The blood/tissue concordance was 74% overall and 92% for intrahepatic tumors only. Variant allele frequency (VAF) in ctDNA correlated with tumor load and in the group of intrahepatic CCA with PFS. 63% of therapy naive patients had their mutational profile changed during chemotherapy. A set of 76 potential progression driver genes was identified among 710 candidates. The molecular landscape of CCA is accessible via ctDNA. This could be helpful to facilitate diagnosis and personalize and adapt therapeutic strategies.

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