cfDNA Analysis Research Articles

Ultrasensitive Detection of Stage I Lung Adenocarcinoma Using Cell-Free DNA Breakpoint Motif Profiling: A Prospective Cohort Study

Background: Early diagnosis benefits lung cancer patients with higher survival, but most patients are diagnosed after metastasis. Although cell-free DNA (cfDNA) analysis holds promise for early detection, the current sensitivity in early-stage lung cancer is unsatisfying. We aimed to establish a predictive model using cfDNA fragmentomics for high-sensitivity detection of invasive Stage I lung adenocarcinoma (ADC). Methods: Stage I lung ADC patients from three medical centers were enrolled whose plasma cfDNA samples were profiled by whole-genome sequencing (WGS). Multiple cfDNA fragmentomic features and machine learning models were compared in the training cohort (Center I) to build a predictive model. Its performance was assessed in an internal (Center I) and external (Center II/III) validation cohort. Findings: 292 patients were enrolled in the study. We constructed a logistic regression model using cfDNA 6bp breakpoint-motif feature in the training cohort (cancer: 150, healthy: 115). The model yielded high sensitivity (98·0%) and specificity (94·7%) in the internal validation cohort [cancer: 102, healthy: 75, Area Under the Curve (AUC): 0·985], while a 92·5% sensitivity and a 90·0% specificity were achieved in the external validation cohort (cancer: 40, healthy: 40, AUC: 0·954). The model exhibited an exceptional ability for identifying early-stage (100% sensitivity for minimally invasive adenocarcinoma, MIA) and <1 cm (92·9%-97·7% sensitivity) tumors. Furthermore, its predictive power remained high when reducing sequencing depth to 0·5× (AUC: 0·977 and 0·931 for the two validation cohorts). Interpretation: We established an ultrasensitive assay for detecting Stage I lung adenocarcinoma with early-stage, small-size tumors using the cfDNA breakpoint-motif feature. Funding: National Natural Science Foundation of China (82002451), Institutional Fundamental Research Funds (2018PT32033), Ministry of Education, Innovation Team Development Project (IRT- 17R10), Beijing Hope Run Special Fund of Cancer Foundation of China (LC2019B15). Declaration of Interest: XC, RL, HB, XW, and YS are employees of Nanjing Geneseeq Technology Inc., China. All other authors have declared no conflicts of interest. Ethical Approval: All study protocols were approved by the ethics committee of each hospital and following the Good Clinical Practice Guidelines of the International Conference on Harmonization.

Cell‐free DNA testing of maternal blood in screening for trisomies in twin pregnancy: updated cohort study at 10–14 weeks and meta‐analysis

ABSTRACTObjectiveTo expand the limited knowledge on cell‐free DNA (cfDNA) analysis of maternal blood for trisomies 21, 18 and 13 in twin pregnancy by updating the data from The Fetal Medicine Foundation (FMF) on prospective first‐trimester screening and those identified in a systematic review of the literature.MethodsThe FMF data were derived from prospective screening for trisomies 21, 18 and 13 in twin pregnancies at 10 + 0 to 14 + 1 weeks' gestation using the Harmony® prenatal test. A search of MEDLINE, EMBASE, CENTRAL (The Cochrane Library), ClinicalTrials.gov and the International Clinical Trials Registry Platform (World Health Organization) was carried out to identify all peer‐reviewed publications on clinical validation or implementation of maternal cfDNA testing for trisomies 21, 18 and 13 in twin pregnancy, irrespective of gestational age at testing, in which data on pregnancy outcome were provided for at least 85% of the study population. Meta‐analysis was performed using the FMF data and data from the studies identified by the literature search. This review was registered in the PROSPERO international database for systematic reviewsResultsIn the FMF study, cfDNA testing was carried out in 1442 twin pregnancies and a result was obtained, after first or second sampling, in 1367 (94.8%) cases. In 93.1% (1272/1367) of cases, there was prenatal or postnatal karyotyping or birth of phenotypically normal babies; 95 cases were excluded from further analysis either because the pregnancy ended in termination, miscarriage or stillbirth with no known karyotype (n = 56) or there was loss to follow‐up (n = 39). In the 1272 pregnancies included in the study, there were 20 cases with trisomy 21, 10 with trisomy 18, two with trisomy 13 and 1240 without trisomy 21, 18 or 13. The cfDNA test classified correctly 19 (95.0%) of the 20 cases of trisomy 21, nine (90.0%) of the 10 cases of trisomy 18, one (50.0%) of the two cases of trisomy 13 and 1235 (99.6%) of the 1240 cases without any of the three trisomies. The literature search identified 12 relevant studies, excluding our papers because their data are included in the current study. In the combined populations of our study and the 12 studies identified by the literature search, there were 137 trisomy‐21 and 7507 non‐trisomy‐21 twin pregnancies; the pooled weighted detection rate (DR) and false‐positive rate (FPR) were 99.0% (95% CI, 92.0–99.9%) and 0.02% (95% CI, 0.001–0.43%), respectively. In the combined total of 50 cases of trisomy 18 and 6840 non‐trisomy‐18 pregnancies, the pooled weighted DR and FPR were 92.8% (95% CI, 77.6–98.0%) and 0.01% (95% CI, 0.00–0.44%), respectively. In the combined total of 11 cases of trisomy 13 and 6290 non‐trisomy‐13 pregnancies, the pooled weighted DR and FPR were 94.7% (95% CI, 9.14–99.97%) and 0.10% (95% CI, 0.03–0.39%), respectively.ConclusionsIn twin pregnancy, the reported DR of trisomy 21 by cfDNA testing is high, but lower than that in singleton pregnancy, whereas the FPR appears to be equally low. The number of cases of trisomy 18 and more so trisomy 13 was too small for accurate assessment of the predictive performance of the cfDNA test. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Optimization of Preanalytical Variables for cfDNA Processing and Detection of ctDNA in Archival Plasma Samples.

DNA released from cells into the peripheral blood is known as cell-free DNA (cfDNA), representing a promising noninvasive source of biomarkers that could be utilized to manage Diffuse Large B-Cell Lymphoma (DLBCL), among other diseases. The procedure for purification and handling of cfDNA is not yet standardized, and various preanalytical variables may affect the yield and analysis of cfDNA, including the purification kits, blood collection tubes, and centrifugation regime. Therefore, we aimed to investigate the impact of these preanalytical variables on the yield of cfDNA by comparing three different purification kits DNeasy Blood & Tissue Kit (Qiagen), QIAamp Circulating Nucleic Acid Kit (Qiagen), and Quick-cfDNA Serum & Plasma Kit (Zymo Research). Two blood collection tubes (BCTs), EDTA-K2 and Cell-Free DNA (Streck), stored at four different time points before plasma was separated and cfDNA purified, were compared, and for EDTA tubes, two centrifugation regimes at 2000 × g and 3000 × g were tested. Additionally, we have tested the utility of long-term archival blood samples from DLBCL patients to detect circulating tumor DNA (ctDNA). We observed a higher cfDNA yield using the QIAamp Circulating Nucleic Acid Kit (Qiagen) purification kit, as well as a higher cfDNA yield when blood samples were collected in EDTA BCTs, with a centrifuge regime at 2000 × g. Moreover, ctDNA detection was feasible from archival plasma samples with a median storage time of nine years.

Abstract 598: Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer

Abstract Background: Combination therapy with dabrafenib + trametinib demonstrated robust activity in patients (pts) with BRAFV600E-mutant advanced non-small cell lung cancer (NSCLC), but its resistance mechanisms are poorly known. Liquid biopsy components such as circulating tumor cells (CTCs) and cell-free (cf) tumor DNA can provide a comprehensive genomic picture of tumor content. Molecular profiling of single CTCs from pts with BRAF-V600Emutant NSCLC was performed to carry out a pilot study to identify resistance mutations at failure to dabrafenib + trametinib and to compare the mutations detected on CTCs to the mutations found on cfDNA and tumor biopsies. Patients and Methods: Eight pts with advanced BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib were prospectively enrolled between Jul 2018 and Mar 2019 at Gustave Roussy (IDRCB2008-A00585-50). Bloods samples were collected. Matched tissue-cfDNA and CTCs were available in 3 pts and matched tissue-CTCs for 4 pts. Single CTC isolation strategy included RosetteSep enrichment, immunofluorescent staining (Hoechst/CD45/cytokeratins) and fluorescence activated cell-sorting. The process to identify CTC mutations included Ampli1 whole-genome amplification, quality controls, multiplex targeted PCR with the Ampli1 CHPCustomBeta cancer panel developed by (Menarini Silicon Biosystems) and next-generation sequencing (NGS). The cfDNA was analyzed using InVisionFirst-Lung. Tissue samples were analyzed using targeted NGS in the MATCH-R trial (Recondo G; NPJ Precis Oncol 2020). Results: Single CTCs were isolated from 7 pts. As baseline characteristics, the median age was 66 years, 5 (71%) were smoker; all the pts with adenocarcinoma histology. Most of the pts received dabrafenib + trametinib as 2nd line (86%). The median of CTCs isolated by patient was 20 (8-28). A wide spectrum of mutations in CTCs was observed at treatment failure that were involved in the main cancer pathways, including MAPK (n=1; NRAS), tyrosine kinase receptors (n=5; EGFR, ALK, FLT3, HER2,…), signal transduction (n=4; IDH1, EZH2,⋯), and DNA repair (n=2; AKT1, ATM,⋯). In the same CTC, several mutations were observed in 5/7 patients, commonly involving more than one cancer pathways. A higher degree of mutational diversity was observed in CTCs compared to tumor tissue biopsies and cfDNA. In the 3 patients with an available tumor/liquid biopsy, only 1 shared mutations between CTCs and matched tumor and cfDNA. Conclusion: Single CTC profiling reveals a wide spectrum of therapeutic resistance mutations not detected by other analyses in pts with BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib. Integration of single CTC sequencing to tumor and cfDNA analysis, provides important perspectives to assess heterogeneous resistance mechanisms and to guide precision medicine in BRAFV600E- NSCLC. Citation Format: Laura Mezquita, Marianne Oulhen, Agathe Aberlenc, Marc Deloger, Aurélie Honoré, Marianna Garonzi, Genny Buson, Claudio Forcato, Yann Lecluse, Mihaela Aldea, Maud NgoCamus, Claudio Nicotra, Karen Howarth, Ludovic Lacroix, Luc Friboulet, Benjamin Besse, Nicolò Manaresi, David Planchard, Françoise Farace. Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 598.

Radiogenomic Analysis of Locally Advanced Lung Cancer Based on CT Imaging and Intratreatment Changes in Cell-Free DNA.

The radiologic appearance of locally advanced lung cancer may be linked to molecular changes of the disease during treatment, but characteristics of this phenomenon are poorly understood. Radiomics, liquid biopsy of cell-free DNA (cfDNA), and next-generation sequencing of circulating tumor DNA (ctDNA) encode tumor-specific radiogenomic expression patterns that can be probed to study this problem. Preliminary findings are reported from a radiogenomic analysis of CT imaging, cfDNA, and ctDNA in 24 patients (median age, 64 years; range, 49-74 years) with stage III lung cancer undergoing chemoradiation on a prospective pilot study (NCT00921739) between September 2009 and September 2014. Unsupervised clustering of radiomic signatures resulted in two clusters that were associated with ctDNA TP53 mutations (P = .03) and changes in cfDNA concentration after 2 weeks of chemoradiation (P = .02). The radiomic features dissimilarity (hazard ratio [HR] = 0.56; P = .05), joint entropy (HR = 0.56; P = .04), sum entropy (HR = 0.53; P = .02), and normalized inverse difference (HR = 1.77; P = .05) were associated with overall survival. These results suggest heterogeneous and low-attenuating disease without a detectable ctDNA TP53 mutation was associated with early surges of cfDNA concentration in response to therapy and a generally better prognosis. Keywords: CT-Quantitative, Radiation Therapy, Lung, Computer Applications-3D, Oncology, Tumor Response, Outcomes Analysis Clinical trial registration no. NCT00921739 Supplemental material is available for this article. © RSNA, 2021.

Abstract 638: Enhancing the molecular diagnosis, prognosis, and treatment monitoring of retinoblastoma using cell free DNA in aqueous humor

Abstract Purpose: All previous studies of retinoblastoma (RB) aqueous humor (AH) analysis for tumor-derived cell-free DNA (cfDNA) have focused on sampling AH from eyes undergoing therapy; results from AH liquid biopsy at the time of diagnosis have not yet been published. Herein, we detail the diagnostic and prognostic genomic biomarkers found in the AH cfDNA from 7 RB eyes at diagnosis, each with longitudinal evaluation of over 12 months in follow-up. Methods: Subjects included 7 eyes of 6 RB patients who underwent AH sampling at diagnosis. CfDNA from each AH sample was isolated and sequenced to assess genome-wide somatic copy number alterations (SCNAs), followed by targeted resequencing and mutation detection using a custom hybridization panel for RB1 and MYCN. Results were compared to peripheral blood RB1 testing and matched tumor samples, when available. Tumor fraction (TFx) was calculated using ichorCNA. Results: Five of 7 diagnostic AH samples contained highly recurrent RB SCNAs, 4 with large scale alterations and 1 with a focal RB1 gene deletion. Mutational analysis of AH cfDNA identified pathogenic somatic variants in 5 diagnostic AH samples with high variant allele frequency, while the remaining 2 diagnostic AH samples had either a high TFx from SCNAs or a focal MYCN amplification. Taken together, somatic tumoral genomic information was detected in all 7 diagnostic AH samples. The 2 eyes that required enucleation had poor prognostic biomarkers (chromosome 6p gain and MYCN amplification) present at the time of diagnosis. TFx from longitudinal AH sampling corresponded to treatment response over time in all cases for which sequential AH samples were available. Conclusions: This study demonstrates that AH sampling at diagnosis is both feasible and safe. Molecular profiling of AH provides a plethora of diagnostic and prognostic information from only a single 0.1 mL AH sample. 1 Citation Format: Liya Xu, Mary E. Kim, Ashley Polski, Rishvanth K. Prabakar, Chen-Ching Peng, Patricia Chévez-Barrios, Jonathan W. Kim, Rachana Shah, Rima Jubran, Peter Kuhn, David Cobrinik, James Hicks, Jesse L. Berry. Enhancing the molecular diagnosis, prognosis, and treatment monitoring of retinoblastoma using cell free DNA in aqueous humor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 638.

Abstract LB043: Efficacy of Lorlatinib in Treatment-Naïve Patients (pts) With ALK-Positive Advanced Non-Small Cell Lung Cancer (NSCLC) in Relation to EML4-ALK Variant Type and ALK Mutations

Abstract BACKGROUND: Lorlatinib, a 3rd generation ALK tyrosine kinase inhibitor, has shown overall and intracranial activity in ALK+ advanced NSCLC. In the randomized, multicenter, phase 3 study in pts with previously untreated ALK+ advanced NSCLC (CROWN; NCT03052608), lorlatinib resulted in a statistically significant and clinically meaningful improvement in progression-free survival (PFS) vs crizotinib.1 To identify molecular correlates of response, we performed molecular profiling of circulating free DNA (cfDNA) and tumor tissue. METHODS: Plasma and tumor tissue samples were available from 130 and 118 pts in the lorlatinib arm, and from 125 and 104 pts in the crizotinib arm, respectively. Plasma DNA was analyzed for ALK fusions and mutations by next-generation sequencing (NGS; Guardant360, Guardant Health, Inc., Redwood City, CA, USA); tumor tissue DNA was analyzed with an ALK-mutation focused NGS panel (MolecularMD, Portland, OR, USA). Objective response rate (ORR), duration of response (DOR), and PFS were evaluated by blinded independent central review according to EML4-ALK variant type and ALK resistance mutation status. RESULTS: At screening, 19 ALK missense mutations and 1 deletion were detected in plasma of 11 pts (5 and 6 in the lorlatinib and crizotinib arms, respectively). Most pts harbored 1 mutation, but 3 pts harbored ≥3 mutations. ALK fusions were detected in plasma of 48% of pts. EML4-ALK variants 1, 2, and 3 were detected in 14.6%, 5.4%, and 13.8% and in 20.0%, 1.6%, and 16.8% of pts, in the lorlatinib and crizotinib arms, respectively. Variants 4, 5, 7, and 8 were detected in 11.5% and 7.2% of pts, and less frequent fusion partners in 1.5% and 3.2% of pts, respectively. ORRs were generally higher in the lorlatinib arm vs the crizotinib arm, regardless of variant subtypes and/or mutational status, with no striking differences between EML4-ALK variants 1, 2, and 3 (range, 72% to 86% for lorlatinib and 50% to 76% for crizotinib). Median DOR and PFS were not reached for variants 1 and 3 in the lorlatinib arm, and ranged from 5.7 to 6.5 months, and from 7.4 to 7.6 months in the crizotinib arm, respectively. CONCLUSION: Pts with untreated ALK+ advanced NSCLC had higher ORRs and potentially longer DOR and PFS across predefined biomarker subgroups when treated with lorlatinib compared with crizotinib in a phase 3 CROWN study. Based on pretreatment cfDNA analysis, lorlatinib led to similar clinical benefit regardless of the type of EML4-ALK variant or presence of ALK kinase mutations. REFERENCE: 1. Shaw AT, et al. N Engl J Med. 2020;383:2018-2029. Citation Format: Alessandra Bearz, Jean-François Martini, Jacek Jassem, Sang-We Kim, Gee-Chen Chang, Alice Shaw, Deborah Shepard, Elisa Dall'O', Anna Polli, Holger Thurm, Gerard Zalcman, Maria Rosario Garcia Campelo, Konstantin Penkov, Hidetoshi Hayashi, Benjamin J. Solomon. Efficacy of Lorlatinib in Treatment-Naïve Patients (pts) With ALK-Positive Advanced Non-Small Cell Lung Cancer (NSCLC) in Relation to EML4-ALK Variant Type and ALK Mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB043.

Non-invasive prenatal testing in the management of twin pregnancies.

Twin pregnancies are common and associated with pregnancy complications and adverse outcomes. Prenatal clinical management is intensive and has been hampered by inferior screening and less acceptable invasive testing. For aneuploidy screening, meta‐analyses show that non‐invasive prenatal testing (NIPT) through analysis of cell‐free DNA (cf‐DNA) is superior to serum and ultrasound‐based tests. The positive predictive value for NIPT is driven strongly by the discriminatory power of the assay and only secondarily by the prior risk. Uncertainties in a priori risks for aneuploidies in twin pregnancies are therefore of lesser importance with NIPT. Additional information on zygosity can be obtained using NIPT. Establishing zygosity can be helpful when chorionicity was not reliably established early in pregnancy or where the there is a concern for one versus two affected fetuses. In dizygotic twin pregnancies, individual fetal fractions can be measured to ensure that both values are satisfactory. Vanishing twins can be identified by NIPT. Although clinical utility of routinely detecting vanishing twins has not yet been demonstrated, there are individual cases where cf‐DNA analysis could be helpful in explaining unusual clinical or laboratory observations. We conclude that cf‐DNA analysis and ultrasound have synergistic roles in the management of multiple gestational pregnancies.

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

Targeted-Capture Sequencing Approach to V(D)J Rearrangement, Immunoglobulin (Ig) Translocation and Mutation Detection in Multiple Myeloma (MM): Potential Applications in Minimal Residual Disease (MRD) Analysis and Comparison to Multiparameter Flow Cytometry (MFC) in the Myeloma Canada Research Network (MCRN) 001 Study with Intravenous Busulfan and Melphalan (BuMel) Conditioning Followed By Lenalidomide Maintenance in Newly Diagnosed Multiple Myeloma (NDMM)

Combined Genotype Analysis of Tumor and Cell-Free DNA By Ultra-Deep Targeted Sequencing: Correlation with PET-Scan Imaging in a Prospective Cohort of Diffuse Large B-Cell Lymphoma Patients

The emerging concept of liquid biopsy in diffuse large B-cell lymphoma (DLBCL) is based on the release of Cell-free DNA (cfDNA) into the blood by tumor cells undergoing apoptosis or necrosis that can be used for the identification of mutations (Bohers et al., Haematologica 2015 ; Rossi et al., Blood 2017). cfDNA theoretically can give therefore information regarding potentially actionable mutations, clonal evolution, and genetic mechanisms of resistance. To determine the clinical relevance of cfDNA, we assessed prospectively by Ultra-deep targeted next generation sequencing (NGS) the basal DLBCL genetic pattern, its modification upon treatment using plasma cfDNA analysis and its correlation with PET scan imaging (clinical.gov number: NCT02339805).MethodsThirty consecutive untreated DLBCL or primary mediastinal B-cell lymphoma (PMBL) were enrolled and followed after rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) or R-CHOP-like chemotherapies. Mutational status of a 34-genes lymphopanel (CIITA, B2M, TNFRSF14, CD58, NOTCH1, NOTCH2, MFHAS1, XPO1, MYC, CDKN2A/B, FOXO1, TP53, GNA13, BCL2, TNFAIP3, MYD88, PIM1, CARD11, IRF4, PRDM1, EZH2, KMT2D, EP300, MEF2B, CREBBP, BRAF, CD79A/B, ITPKB, TCF3, ID3, SOCS1 and STAT6) was assessed in both tumor (genomic DNA, gDNA) and cfDNA at the time of diagnosis using AmpliSeq®/PGM® technologies (Dubois et al., Clinical Cancer Research 2016) . Longitudinal analysis of plasma samples collected at mid-treatment (C4) and at the end of treatment or at relapse/progression was performed and correlated with PET scan characteristics obtained at baseline (Metabolic tumor volume, MTV), mid and end-treatment (Delta SUV and Deauville score). To increase the NGS sensitivity and for cost-effectiveness reasons, only mutations detected at baseline were monitored during the follow-up. Variant analysis was performed using an in-house generated bioinformatics pipeline.ResultsPatient clinical features were as follow: IPI: 0-1, n =8; IPI: 2-3, n=14; IPI: 4-5, n =8; median age was 64 y; bone marrow involvement: 0/25 patients; elevated LDH: 12/30; Fifteen patients were sub-classified as GCB, 9 as ABC/non GCB and 3 as PMBL. cfDNA was available for the 30 patients at the time of diagnosis. Mean plasma concentration was 74 ng/mL (14.6 - 433) and sequencing was carried out with a mean depth of 3750x. cfDNA mutations were identified in 20 patients (67%) and were consistent with the pattern usually observed in the different subtypes of DLBCL. gDNA mutations from tissue biopsies (11 frozen/14 FFPE) were found in 22/25 (88%) cases. In 3/22 cases for whom gDNA analysis failed, an informative mutational pattern was finally successfully obtained in matched cfDNA. Variant allelic frequency (VAF) was lower in cfDNA (mean 15%) compared to tumor DNA (mean 33%). Finally, among the 22 tumor/plasma matched samples, 8 patients had no detectable tumoral cfDNA, whereas 4 patients displayed additional mutations in plasma compared to tumor. At the time of diagnosis, a high [cfDNA] correlated with the presence of tumoral circulating DNA and cfDNA mutations were observed in all patients with elevated LDH. MTV correlated with the highest concentrations of cfDNA and VAF rate. Moreover, 3 / 4 patients presenting additional mutations in cfDNA, as compared to gDNA, had a high MTV (> 2000cm3), suggesting that cfDNA mutations reflect more accurately tumor heterogeneity than gDNA analysis. After R-CHOP/ R-CHOP-like regimens, a rapid clearance of cfDNA mutations was observed in 13/17 cases. Conversely, among the 4/17 patients whose basal DLBCL mutations did not disappear at mid-treatment, 3 were in partial response according to Deauville score, including 2 with a delta SUVmax < 70%. One patient in partial remission had progressive disease 6 months after the end of the treatment and still harboured tumoral cfDNA, as shown by a persistent detectable TP53 mutation.ConclusionOur study demonstrates that cfDNA genotyping of DLBCL is an accurate genotyping tool and represents a real-time and non-invasive approach to tracking treatment-resistant clones. It highlights the major interest of the liquid biopsy in the context of bulky tumors where cfDNA is more representative than gDNA by capturing the entire tumoral mutation profile. cfDNA analysis represents therefore a complementary approach to TEP scan imaging at baseline and during follow-up for the management of DLBCL. DisclosuresStamatoullas:Takeda: Consultancy; Celgene Corporation: Honoraria. Tilly:Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Immunogen: Honoraria; Gilead: Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria. Jardin:Roche: Honoraria; celgen: Honoraria; Janssen: Honoraria.

Liquid Biopsy for the Identification of Intravascular Large B-Cell Lymphoma

[Backgrounds] Intravascular large B cell lymphoma (IVLBCL) is a rare subtype of extranodal diffuse large B-cell lymphoma (DLBCL) that features lymphoma cells lodged in lumina of small vessels. Because of the lack of mass formation, diagnosis by tumor biopsy is often difficult, and thus, cell-free DNA (cfDNA) analysis might be useful for assisting diagnosis of IVLBCL. In this study, we first aimed at investigating the prevalence of mutations in genes involved in the B-cell receptor, Toll-like receptor/interleukin-1 receptor, and nuclear factor kappa B pathways. We then evaluated the liquid biopsies for detection of hot-spot mutations by droplet digital polymerase chain reaction (ddPCR).[Patients and Methods] Twenty-seven IVLBCL patients whose archived samples were available were included in this study. The patients were diagnosed as IVLBCL from January 2005 to May 2017 in University of Tsukuba Hospital, JA Toride Medical Center, and Kameda Medical Center. The study was approved by the review board in each institution, and conducted according to the Declaration of Helsinki. Tissue-derived DNA (tdDNA) was extracted from formalin-fixed paraffin-embedded specimens and/or cryo-preserved bone marrow samples, in which the lymphoma cell infiltration had been pathologically confirmed. cfDNA was extracted from 1 ml of serum or plasma obtained prior to chemotherapy. We performed targeted sequence for 8 genes (B2M, BTG2, CARD11, CD79B, MYD88, PIM1, PRDM1, and TNFAIP3) in paired tdDNA and cfDNA from 9 IVLBCL subjects by Ion Torrent Personal Genome Machine (Thermo Fisher Scientific). MYD88 L265P (c.794T>C) mutational analysis was performed by ddPCR assays (BioRad) for 46 samples in total collected from 27 subjects. Statistical analysis was performed with EZR version 1.3.2. Paired t-test was used to compare variant allele frequencies (VAFs) in paired samples.[Results] All cases were categorized as the non-GCB by Hans classification. Random skin biopsies (RSB) and bone marrow biopsies (BMB) were performed in 21 and 27 patients. The diagnostic yield of RSB was 81.0% (17 of 21). Initial BMB detected lymphoma cells in 48.1% (13/27) patients, but repeated BMB increased successful detection up to 66.7% (18/27). tdDNA was available in 26 out of 27 subjects, and in 9 of which paired cfDNA was also available (Figure 1). In one subject, only cfDNA but not tdDNA was available. Targeted sequencing for 8 genes demonstrated at least one mutation in CD79B, MYD88, PIM1, or PRDM1 in both tdDNA and cfDNA, or only in cfDNA, in 8 of 9 subjects (Figure 2). We identified 3 missense CD79B mutations in 6 (66.7%; Y196C in 2, Y196H in 3, and L199P in 1). All MYD88 mutations were L265P, which were found in 5 of 9 (55.6%). Together, either Y196 CD79B or L265P MYD88 mutation was found in 7 of 9 (77.8%). PIM1 mutations and PRDM1 mutations were seen each in 3 (33.3%). VAFs were higher in cfDNA than in tdDNA (mean, 45.6% vs. 3.7%; P=0.0006, Figure 3). Four mutations in 3 patients were detected only in cfDNA. On the other hand, all variants detected in tdDNA were confirmed in cfDNA. ddPCR assay for the L265P MYD88 mutation with all 46 samples including tdDNA and cfDNA revealed the L265P MYD88 mutation in 24/46 (52.2%) samples from 16/27 (59.3%) IVLBCL patients. Furthermore, sequential analysis of cfDNA was performed in one of the MYD88 -mutated patients. The L265P MYD88 mutation was detected (VAF=30%) in cfDNA collected 17 weeks before the diagnosis of IVLBCL, whereas the mutation was not detected in DNA derived from the bone marrow sample that was collected at the same timing and resulted in the negative pathological finding.[Conclusion] Mutations in MYD88 and CD79B are frequent in IVLBCL . Targeted sequencing suggested that tumor cell-derived DNA is abundant in serum/plasma. Liquid biopsy to detect L265P MYD88 and Y196 CD79B may provide a powerful tool for approaching diagnosis of IVLBCL. [Display omitted] DisclosuresChiba:Nippon Shinyaku: Honoraria, Research Funding.

DETERMINANTS OF RESISTANCE TO ENGINEERED T‐CELL THERAPIES TARGETING CD19 IN LYMPHOMA

Introduction: Anti-CD19 chimeric antigen receptor (CAR19) T-cells have activity in patients with relapsed/refractory large B-cell lymphoma (rrLBCL), but over half of patients ultimately relapse. We applied cell-free DNA (cfDNA) analysis to patients receiving Axicabtagene Ciloleucel (axi-cel) to identify determinants of resistance and characterize molecular thresholds predictive of treatment failure. Methods: We developed a novel hybrid-capture approach allowing evaluation of both circulating tumor-derived DNA (ctDNA) and CAR19-derived cfDNA (Fig 1A). We applied this to 381 plasma, tumor, and germline DNA samples from 64 rrLBCL patients, including prior to and during treatment and at relapse. We evaluated all samples for somatic alterations across 246 genes, as well as quantitative levels of ctDNA and CAR19-cfDNA. Results: The median follow-up for the cohort was 12.5 months, and 55% (35/64) progressed after axi-cel. We identified 100.5 mutations/case, which was similar to a cohort of 136 untreated DLBCL patients (P>0.8). Notable differences included more alterations in TP53 (P = 0.02), EP300 (P = 0.02), SETD1B (P = 0.04), ARID5B (P = 0.04), BTK (P = 0.04), CD79A (P = 0.03), CXCR4 (P = 0.03) and RHOA (P = 0.03) in rrDLBCL, and fewer alterations in CD79B (P = 0.04) and PIM1 (P = 0.03). When considering ctDNA quantity, both pre- and on-treatment levels were prognostic for PFS, with higher levels correlating with adverse outcome (Pre-LD [HR = 1.5, CI = 1.1-1.9], Day 0 [HR = 1.6, CI = 1.2-2.3], Day +7 [HR = 1.5, CI = 1.1 – 2.0], Day +28 [HR = 1.7, CI = 1.4-2.2]; Fig 1C). In contrast, higher CAR19-cfDNA levels at Day +7 were associated with favorable outcome (HR 0.52, CI 0.32-0.87). CAR19-cfDNA was also correlated with CAR19 T-cell levels by flow cytometry (Pearson r = 0.7, P < 0.001; Fig 1B). When we assessed the effect of specific mutations on outcomes, we observed recurrent emergence and clonal selection of variants at relapse, including mutations in CD19, PAX5 and TP53 (Fig 1D). Finally, mutations in multiple genes were identified as being prognostic for outcome, including CD58, PAX5 and IRF8 (Fig 1E). Notably these include both immune-mediated and target-mediated putative mechanisms of resistance. For example, CD58 encodes a costimulatory T-cell molecule which has been implicated in CAR19 resistance (Majzner et al., Blood 2020), and PAX5 and IRF8 encode transcriptional regulatory elements that are central to B-cell differentiation and phenotype. Conclusions: Baseline and interim ctDNA and CAR19-cfDNA measurements have prognostic significance in LBCL patients being treated with CAR19 T-cells. Additionally, genomic alterations in several genes, including CD19, CD58, PAX5 and IRF8 are associated with inferior outcomes, and thus represent candidate resistance mechanisms that warrant further study with the goal of improving future generations of CAR T-cell therapy. (A) Overview of experimental schema (B) Relationship between CAR19 T-cell qualification by cfDNA and flow cytometry (C) Results of univariate cox proportional hazards for EFS for indicated variables. (D) Clonal selection of mutations in specific genes in patients experiencing relapse shown as a volcano plo. Mutated genes under significant positive selection are shown on the right in red; size of dot proportional to number of mutations (also shown in parentheses). (E) Left: Recurrently mutated genes in patients receiving axi-cel therapy,stratified by progression vs. non- progression following CAR19 therapy. Right: effect of mutations in given gene on EFS (hazard ration from proportional hazard model); significant values (P < 0.05) shown in green. EFS, event-free survival; HR, hazard-ratio; ctDNA, circulating tumor DNA; cfDNA, cell-free DNA; CAR19, anti-CD19 chimeric antigen receptor T-cell; pre-lymphodepletion; SNV, single nucleotide variant Keywords: Genomics, Epigenomics, and Other -Omics, Cellulartherapies, Liquid biopsy Conflicts of interests pertinent to the abstract D. M Kurtz Consultant or advisory role: Roche, Genentech, Foresight Diagnostics Stock ownership: Foresight Diagnostics M. S Khodadoust Research funding: Corvus Pharmaceuticals R. Majzner Consultant or advisory role: Xyphos Inc, Lyell Immunopharma C. L Mackall Consultant or advisory role: Lyell, Nektar, PACT, Bryologyx, Vor, Roche, Adaptimmune, Glaxo-Smith-Kline, Allogene, Apricity Health, Unum Therapeutics Stock ownership: Lyell, Allogene, Apricity Health, Unum Therapeutics Research funding: Obsidian M. Diehn Consultant or advisory role: Roche, Foresight Diagnostics, Quanticell, Novartis, Astra Zeneca, BioNTech Stock ownership: Foresight Diagnostics, D. M Miklos Consultant or advisory role: Miltenyi Biotech, Allogene, Kite-Gilead, BMS, Celgene, Juno, Novartis, Adaptive Biotechnologies, Precision Bioscience Research funding: Becton Dickinson, Kite-Gilead, Novartis A. A Alizadeh Consultant or advisory role: Foresight Diagnostics, Genentech, Janssen, Pharmacyclics, Gilead, Celgene, Chugai, Roche Stock ownership: Foresight Diagnostics Research funding: Pfizer. AACR-ICML JOINT SESSION: ARTIFICIAL INTELLIGENCE IN LYMPHOMA DIAGNOSIS AND TREATMENT

Diagnostic and Prognostic Value of Circulating Cell-Free DNA for Cholangiocarcinoma.

The analysis of cfDNA has been applied as a liquid biopsy in several malignancies. However, its value in the diagnosis and prognosis of cholangiocarcinoma (CCA) have not been well defined. We aimed to investigate the diagnostic and prognostic values of cfDNA level and tumor-specific mutation in circulating DNA (ctDNA) in CCA. The plasma cfDNA levels from 62 CCA patients, 33 benign biliary disease (BBD) patients and 30 normal controls were quantified by fluorescent assay. Targeted probe-based sequencing of 60 genes was applied for mutation profiling in 10 ctDNA samples and their corresponding treatment-naïve tissues. cfDNA levels in CCA were significantly higher than those in BBD and normal controls. We found that cfDNA levels at 0.2175 and 0.3388 ng/µL significantly discriminated CCA from healthy controls and BBD with 88.7 and 82.3% sensitivity and 96.7 and 57.6% specificity, respectively. cfDNA levels showed superior diagnostic efficacy in detecting CCA compared to CEA and CA19-9. ARID1A (30%), PBRM1 (30%), MTOR (30%), and FGFR3 (30%) mutations were the most common. Using nine frequently mutated genes in the ctDNA samples, the diagnostic accuracy of cfDNA sequencing was 90.8%, with 96.7% average sensitivity and 72.4% specificity. This study supports the use of cfDNA as a diagnosis and prognostic biomarker for CCA.

Phase Ib/II study of capecitabine 7/7 schedule with neratinib in patients with HER2-positive metastatic breast cancer (MBC).

e15016 Background: Neratinib (N) is a potent irreversible inhibitor of HER1, HER2, and HER4. Capecitabine (X) at optimal dose of 7 days on and 7 days off schedule (7/7) is well-tolerated with low rates of ≥ grade (G3) diarrhea. Methods: We conducted phase Ib/II study of N with X (7/7) with loperamide and colestipol prophylaxis in patients (pts) with pretreated HER2+ MBC (NCT03377387). Eligible pts had normal left ventricular ejection fraction (≥ 50%), any and &lt; 4 prior chemotherapy-based treatments in phase Ib and II, respectively. Primary endpoint of phase Ib is maximum tolerated dose (MTD) and phase II is response rate. Secondary endpoints are safety, tolerability, and progression-free survival. Exploratory endpoint is to quantify cell-free DNA (cfDNA), correlating with response. Phase Ib follows traditional 3+3 design with 4 dose levels. In phase II, if &gt; 3/9 respond, study is expanded to 24. If &gt; 10/24 respond, study is deemed successful. Results: As of 2-4-2021, 10 pts were enrolled in phase Ib. 4 pts were treated at dose level 1 (X at 1500 mg BID at 7/7 and N at 240 mg daily); 2/4 pts experienced with G3 diarrhea during cycle 1. Six pts were treated at dose level -1 (X at 1000 mg BID 7/7 and N at 240 mg daily); 1 (17%) developed G3 diarrhea. The MTD is X at 1000 mg BID 7/7 and N at 240 mg daily. Twenty-two of 24 pts have been enrolled in phase II. Of 22 pts, data show 6 with partial response, 8 with stable disease, 3 with progressive disease, and 5 have not been assessed radiographically. Overall, 6/22 (27.3%) and 1/22 (4.5%) had all G and G3 diarrhea, respectively. Other significant toxicities at MTD included G2 hand foot syndrome (n = 1, 4.5%), G1 fatigue (n = 1, 4.5%) and G1 nausea (n = 4, 18%). Conclusions: The MTD is X at 1000 mg BID at 7/7 and N at 240 mg daily. This combination is safe and well tolerated with G3 diarrhea rate of 4.5%, which is significantly lower than the X 14/7 schedule in NALA study. The phase II portion of the study is near completion and updated result will be presented. Analysis of cfDNA, to correlate with response for phase II portion, is ongoing. Clinical trial information: NCT03377387.

Cell-free circulating tumor DNA (cfDNA) analysis of advanced thymic epithelial tumors (TETs).

8577 Background: Thymic epithelial tumors (TETs) are rare tumors originating from the epithelial cells of the thymus. Thymomas tend to be slowly growing, whereas thymic carcinomas are more aggressive and often metastasize wildly. TETs have a very low tumor mutational burden (TMB). cfDNA has been used in several tumor types to describe the molecular characteristics and select treatment options, especially in absence of tissue availability. There is no information on the cfDNA detected in TETs. The purpose of this study was to identify common genomic alterations occurring in circulating tumor DNA (ctDNA) in patients with advanced TETs, detected using a cfDNA assay. Methods: We retrospectively evaluated 157 TET samples from the Guardant Health database between November 2017 – November 2020. The cfDNA analysis interrogated single nucleotide variants (SNV), fusions, indels and copy number variations (CNV) of up to 83 genes using a commercially available liquid biopsy assay (Guardant360; Guardant Health, Redwood City, CA) . We evaluated the frequency of genomic alterations based on diagnosis, age, and sex. Results: In this cohort, 66% of the patients had thymic carcinoma and 34% had thymoma. The median age was 60 years, and 59% of patients were male. 126 patients (80%) of this cohort had ≥1 somatic alteration detected. The most prevalent mutations detected are TP53 (55%), KIT (13%), EGFR (12%), BRCA2 (11%), PIK3CA (10%), ARID1A (10%), ATM (10%), KRAS (9%), APC (9%), and BRAF (9%). Mutations were more commonly observed in thymic carcinomas than thymomas, but statistical significance was not reached due to the small sample size. Frequencies of the observed genomic alterations are shown in the table below. Conclusions: This study confirms that advanced stage TETs shed tumor DNA into the circulation that can be picked up in the majority of patients, using a solid tumor platform, despite the low TMB typically observed in these tumors. This assay can potentially be used to monitor response to therapy. A more targeted gene panel, enriched for genes commonly mutated in TETs (e.g. GTF2I, BAP1, CYLD) might provide further insights in the future in the management of TETs.[Table: see text]

Clinical outcomes for plasma-based comprehensive genomic profiling versus tissue testing in advanced lung adenocarcinoma.

9027 Background: Somatic genomic testing is recommended by numerous expert guidelines to inform targeted therapy treatment for patients with advanced lung adenocarcinoma (aLUAD). The NILE study was a prospective observational study that demonstrated non-inferiority of cell-free circulating tumor DNA (cfDNA)-based tumor genotyping compared to tissue-based genotyping to find targetable genomic alterations in patients with newly diagnosed aLUAD. As the cohort has matured, clinical outcomes data can now be reported. Methods: This prospective, multicenter North American study (NCT03615443) enrolled patients with previously untreated aLUAD who had standard of care (SOC) tissue genotyping performed and concurrent comprehensive cfDNA analysis using the commercially available Guardant360 assay (Guardant Health, Redwood City, CA). After 12 months of study enrollment, objective response rates, disease control rate, and time to treatment data were collected for patients with targetable genomic alterations, as defined by NCCN guidelines, who were treated with physician’s choice of therapy. Results: Among 282 patients on the study, 89 (31.6%) had an actionable biomarker detected by tissue (21.3%) and/or cfDNA (27.3%) analysis. Sixty-one (68.5%) of these patients were treated with an FDA-approved targeted therapy guided by somatic genotyping results ( EGFR, ALK, ROS1). Thirty-three patients were eligible for clinical response evaluation and demonstrated an objective response rate of 58% and disease control rate of 94%. Twenty-five (76%) achieved a durable response &gt; 6 months; 17 (52%) achieved a durable response &gt; 12 months. Patients responded to targeted therapy regardless of the variant allele frequency of the target alteration. The time to treatment (TtT) was significantly faster for cfDNA-informed biomarker detection as compared to tissue genotyping (median 18 vs 31 days, respectively; p = 0.0008). Conclusions: This is the first prospective community-based study to find that cfDNA detects guideline-recommended biomarkers at a rate similar to tissue genotyping, and therapeutic outcomes based on plasma-based comprehensive genomic profiling are comparable to published tissue-based targeted therapy clinical outcomes. The NILE study complements and confirms findings in the prospective FLAURA and SLLIP studies, which exclusively enrolled at academic sites. Clinical trial information: NCT03615443.

Interim results of PATHFINDER, a clinical use study using a methylation-based multi-cancer early detection test.

3010 Background: PATHFINDER (NCT04241796) is an interventional, prospective study evaluating implementation of a blood-based multi-cancer early detection (MCED) test that uses targeted methylation-based cfDNA analysis to detect multiple cancer types and simultaneously predict cancer signal origin (CSO). We present a prespecified interim analysis of PATHFINDER evaluating an MCED test in a clinical setting. Methods: Participants (pts; ≥50y) were enrolled into 2 risk cohorts: non-elevated and elevated (smoking history, prior cancer [ &gt; 3y post treatment], or genetic predisposition). MCED test results (cancer signal detected/not detected) were returned to investigators; pts with a signal detected also received a CSO prediction and underwent further diagnostic testing by their medical team. The primary objective was to assess the extent of diagnostic testing needed to achieve diagnostic resolution (eg, time to resolution, number/type of tests). Secondary endpoints included positive predictive value (PPV) and a measure of test satisfaction (following diagnostic resolution [signal detected] and post test [signal not detected]). Results: PATHFINDER consented 6796 pts before closing accrual on 12/4/20; as of October 6, 2020, 4086 consented, 4047 enrolled, and 4033 analyzable pts were included in the interim analysis (62.4% female, 92.1% white). Two study-related adverse events (anxiety of mild severity) were reported. Cancer signal was detected in 1.5% (62/4033) of pts; 40/62 reached diagnostic resolution to date. Kaplan-Meier estimate of median time to resolution was 78 (95% CI, 54-151) days. Among 40 pts that reached diagnostic resolution, ≥1 imaging test was performed in 93% (37/40); ≥1 invasive procedure was performed in 72% (13/18) versus 18% (4/22) of pts with diagnostic resolution of cancer versus no cancer, respectively. Based on results to date, PPV was 45% (95% CI, 30.7-60.2%; 18/40). Of 18 cancer diagnoses, 11 were solid tumors (3 stage IV, 6 stages I-III, 1 metastatic recurrence, 1 missing stage), and 7 were hematologic malignancies (1 stage IV, 4 stages I-III, 2 without AJCC stage). Accuracy of the top CSO prediction in true positives was 82.4% (95% CI, 59.0-93.8%; 14/17). Most pts were satisfied with the test (43.7% extremely satisfied, 30.7% very satisfied, 14.6% satisfied). Signal detection rate and test satisfaction were similar in the 2 risk cohorts; PPV tended to be higher in the elevated risk cohort, as expected. Conclusions: An interim analysis of this return of results study demonstrated promising MCED test results. Of 40 pts achieving diagnostic resolution, nearly half had a diagnostic workup confirming cancer; CSO was predicted with high accuracy for detected cancers. Taken together with the rarity of adverse events and high test satisfaction, these results support the feasibility of clinical implementation. Full enrollment cohort data will be available at the meeting. Clinical trial information: NCT04241796.

TRIO-US B-12 TALENT: Phase II neoadjuvant trial evaluating trastuzumab deruxtecan with or without anastrozole for HER2-low, HR+ early stage breast cancer.

TPS603 Background: Although patients with hormone receptor-positive (HR+)/HER2-negative breast cancer (BC) frequently respond clinically to neoadjuvant treatment, fewer than 10% achieve a pathologic complete response (pCR) with standard chemotherapy or endocrine therapy, even in combination with targeted agents such as CDK4/6 inhibitors. Thus, finding more effective therapies for this disease remains an area of unmet need. HER2 amplification is a known driver of endocrine resistance and HER2 protein may be expressed at a low level (IHC 1+ or 2+) in up to 60% of HR+ BC. Trastuzumab deruxtecan (DS-8201a, T-DXd) is a novel HER2-targeting antibody drug conjugate (ADC) that is FDA approved for HER2-positive metastatic BC and has demonstrated promising clinical efficacy in HER2-low BC with an objective response rate of ̃37%. The aim of TALENT (TRIO-US B-12) is to evaluate the clinical activity and toxicity of neoadjuvant T-DXd either alone or in combination with endocrine therapy in patients with HR+/HER2-low early BC. Methods: TRIO-US B-12 TALENT (NCT04553770) is an ongoing randomized, multicenter, open-label, two-stage, phase II neoadjuvant trial for participants with early stage, HR+, HER2-low expressing (1+ or 2+ by IHC) BC. Eligible participants include men and women with previously untreated, operable invasive BC greater than 2.0 cm (cT2) in size. Pts with recurrent or metastatic BC, or inflammatory BC are excluded. Pts are randomized 1:1 to receive six cycles of T-DXd (5.4 mg/kg IV q21 days) either alone or in combination with anastrozole (1 mg PO QD). Men and pre/peri menopausal women randomized to the anastrozole arm also receive standard of care GnRH agonist. Stratification factors include HER2 expression (1+ or 2+) and menopausal status. Tumor tissue is taken at baseline, cycle 1 day 17-21, and at surgery. Blood samples are taken at four time points for biomarker analysis. The primary endpoint is pCR rate (breast and lymph node) at definitive surgery. In stage I, 58 participants will be randomized (29/arm). If &gt;2 participants in an arm achieve pCR, that arm will expand (stage II) to enroll an additional 15 participants (total of 44/arm). A pCR rate of &gt;10% (5/44) would be considered favorable, warranting further evaluation of the treatment in a larger trial. Other endpoints include safety, changes in Ki67 expression, Residual Cancer Burden index, biomarker analysis (including serial cfDNA analysis), and health-related quality of life. As of January 2021, four participants have enrolled. Conclusions: To our knowledge this is the only ongoing study evaluating T-DXd with or without endocrine therapy for HR+, HER2-low breast cancer in the neoadjuvant setting. The study will shed light on clinical activity and biomarkers, which may guide larger confirmatory studies for patients with HR+, HER2-low early breast cancer. Clinical trial information: NCT04553770.

Double Drop-Off Droplet Digital PCR: A Novel, Versatile Tool for Mutation Screening and Residual Disease Monitoring in Acute Myeloid Leukemia Using Cellular or Cell-Free DNA

In acute myeloid leukemia (AML), somatic gene mutations are important prognostic markers and increasingly constitute therapeutic targets. Therefore, robust, sensitive, and fast diagnostic assays are needed. Current techniques for mutation screening and quantification, including next-generation sequencing and quantitative PCR, each have weaknesses that leave a need for novel diagnostic tools. We established double drop-off digital droplet PCR (DDO-ddPCR) assays for gene mutations in NPM1, IDH2, and NRAS, which can detect and quantify diverse alterations at two nearby hotspot regions present in these genes. These assays can be used for mutation screening as well as quantification and sequential monitoring. The assays were validated against next-generation sequencing and existing ddPCR assays and achieved high concordance with an overall sensitivity comparable to conventional digital PCR. In addition, the feasibility of detecting and monitoring genetic alterations in peripheral blood cell-free DNA (cfDNA) of patients with AML by DDO-ddPCR was studied. cfDNA analysis was found to have similar sensitivity compared to quantitative PCR-based analysis of peripheral blood. Finally, the cfDNA-based digital PCR in several clinical scenarios was found to be useful in long-term monitoring of target-specific therapy, early response assessment during induction chemotherapy, and identification of mutations in patients with extramedullary disease. Thus, DDO-ddPCR-based cfDNA analysis may complement existing genetic tools for diagnosis and disease monitoring in AML.

Monitoring epidermal growth factor receptor C797S mutation in Japanese non-small cell lung cancer patients with serial cell-free DNA evaluation using digital droplet PCR.

Osimertinib is a third‐generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR‐TKI) that is effective in treating both naïve and T790M‐mutated EGFR‐TKI‐resistant non–small cell lung cancer patients. The EGFR C797S mutation is the major osimertinib resistance mechanism. The present study monitored the EGFR C797S mutation during osimertinib treatment in Japanese patients using droplet digital PCR (ddPCR). In our first cohort, C797S detection was validated with tumor specimens and/or plasma samples from 26 patients using ddPCR with custom‐designed probes detecting and discriminating T790M and C797S in cis and trans positions. In our second cohort, 18 patients with EGFR‐T790M who were going to start osimertinib were analyzed using ddPCR by collecting the plasma samples every month from the beginning of the course of osimertinib. In the first cohort, C797S was detected in 15.4% of patients. C797S and T790M in cis and trans positions were distinguished using ddPCR. In the second cohort, serial cfDNA evaluation revealed that the rate of EGFR mutation changes with disease state. Increases of EGFR mutation were detected, including C797S several months before the diagnosis of disease progression. As with the first cohort, C797S and T790M in cis and trans position were distinguished by ddPCR at disease progression. Coincidentally, in the first cohort, next generation sequencing detected NRAS Q61K mutation and the resistance with NRAS Q61K mutation was overcome by trametinib. In the second cohort, serial cfDNA analysis was useful for evaluating bone oligo‐progression and local radiation therapy.