Free DNA Research Articles

Abstract P5-05-01: Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer

Abstract Introduction: Identification of Molecular Residual Disease (MRD) in patients with breast cancer with circulating tumor DNA (ctDNA) presents a strategy to identify patients at high risk of relapse. Approaches that detect ctDNA at lower concentrations are required to increase sensitivity and improve on the lead time between ctDNA detection and clinical relapse. Here we present results using novel highly sensitive tumor-informed sequencing assays for ctDNA detection of MRD based on detection of multiple patient specific mutations in ctDNA. Methods: 62 stage II-III breast cancer patients (23 hormone receptor positive HER2 negative (HR+HER2-), 20 HER2+, 15 triple negative breast cancer (TNBC) and 4 unknown receptor status) enrolled in the ChemoNEAR sample collection study were included. All patients received neoadjuvant chemotherapy, followed up by surgery, with samples taken at diagnosis, and post-surgery every 3 months for the first two years, followed by every 6 months for up to five years. Tumor DNA from FFPE samples and germline was Whole Exome Sequenced to identify patient specific mutations and design anchored-multiplex PCR (AMP™) Personalized Cancer Monitoring (PCMTM) assays to track mutations in plasma. Cell free DNA was extracted from 613 plasma samples (median volume 4ml, range 0.5-4.5ml) and sequenced with PCMTM assays, with 37-177 variants (median 52) per panel, to a depth of 100,000x per locus. A proprietary algorithm was used to identify ctDNA. Results: At a median follow-up of 52.7 months post-surgery (range 15.3-96.4 months), ctDNA was detected in 25.8% (16/62) of patients, with detected ctDNA levels ranging from allele frequency (AF) of 0.01%, to 32.5%) (median 0.24% AF). Detection of ctDNA was associated with a high risk of future relapse (HR 65.4, 95% CI 14.5-293.7), with a median lead-time from ctDNA detection to clinical relapse of 13.7 months (range 3.9-58.9). MRD was identified in 76.9% (10/13) of patients who relapsed. ctDNA was detected prior to relapse in both patients with brain only relapse, but with a reduced lead time over clinical relapse (5.73 and 3.90 months), which was previously not achievable with digital PCR MRD-detection assays. Of patients with assessable baseline samples, 81% (39/48) had ctDNA detected. No patients with undetected ctDNA, or detectable ctDNA with AF< 0.1%, relapsed during follow-up, whereas ctDNA was detected at baseline in all 10 patients who relapsed during follow-up (p=0.1). Conclusions: PCMTM detected breast cancer relapse with a long lead-time over clinical relapse, and strong association with relapse free survival, an advancement over previously published data with digital PCR MRD detection. Prospective, interventional trials are now required to assess whether treatment on the basis of MRD detection improves outcome, including the TRAK ER Trial (NCT04985266). Citation Format: Isaac Garcia-Murillas, Giselle Walsh-Crestani, Edward Phillips, Rosalind Cutts, Sarah Hrebien, Kathryn Dunne, Kally Sidhu, Robert Daber, Amber C. Carter, Lorena De La Peña, Stephen Johnston, Alistair Ring, Simon Russell, Abigail Evans, Anthony Skene, Duncan Wheatley, Ian Smith, Nicholas Turner. Personalized Cancer Monitoring (PCM): a novel ctDNA tool to detect molecular residual disease in patients with early-stage breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-01.

Abstract P4-01-14: Changes in the Genomic Spectrum of Actionable Alterations in HER2 Negative Metastatic Breast Cancer in Serial Cell Free DNA (cfDNA) Analysis

Abstract Background: Serial cell free DNA (cfDNA) analysis in metastatic breast cancer (MBC) is a noninvasive method for tracking tumor evolution and the emergence of new somatic alterations through the course of the disease. The detection of new actionable alterations may impact treatment selection; thus, serial cfDNA collection is increasingly being performed in the clinic. However, it is not well-understood how often serial cfDNA testing identifies new actionable alterations. We evaluated changes in the genomic spectrum of actionable alterations in serial cfDNA analysis of HER2 negative (HER2-) MBC. Method: Patients with HER2- MBC who underwent plasma-based cfDNA testing (Guardant360, 74-gene assay) between February 2015 and February 2021 at an academic institution were included. A retrospective review of records was conducted to identify subtype, demographics, lines of therapy, and cfDNA results. At baseline cfDNA testing, all cfDNA alterations were considered new. For patients with serial draws, new cfDNA alterations in each draw, compared to previous draws, were quantified and characterized. The pathogenicity of new alterations was determined using the OncoKB precision oncology database. New pathogenic alterations were further classified as actionable alterations (AA) using the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Alterations that met the ESCAT I (alteration-drug match was associated with improved outcomes in clinical trials) or ESCAT II (alteration-drug match was associated with antitumor activity) criteria were considered as AA. Results: 344 patients with hormone receptor positive (HR+) MBC and 95 patients with triple negative (TN) MBC had a baseline cfDNA draw. Among the HR+ cohort, 139, 79 and 48 patients had 2nd, 3rd, and 4th serial cfDNA draws, respectively. Among the TN cohort, 33 and 18 patients had 2nd and 3rd serial cfDNA draws, respectively. Table 1 depicts the prevalence of AA found in each of these draws, as well as the median number of prior therapies at each point of collection and the mean time between serial draws. The median number of new genomic alterations was lower in subsequent draws compared to the baseline, regardless of subtype (HR+: 4 vs. 2, TN: 4 vs. 1.5-3, at baseline vs. subsequent draws, respectively). In the HR+ cohort the proportion of patients with new AA (ESCAT I/II) decreased from 63% at baseline to 27-33% in the 2nd-4th draws. While some of the new AA in subsequent draws were new actionable variants in the same genes that were known to be altered in previous draws (in 19%, 68% and 57% of patients with new AA in the 2nd, 3rd and 4th draws, respectively), the remainder were new AA in previously unaltered genes. In the TN cohort 25% of patients had new AA at baseline and this proportion of TN patients with new AA continued to decrease in subsequent draws (9% and 0% in the 2nd and 3rd draws, respectively). PIK3CA and ESR1 were the most frequent genes with new AA in the ER+ cohort, regardless of draw number. In the TN cohort, PIK3CA was the most frequent gene with new AA in the first draw, and in the second draw, new AA in PIK3CA, BRCA2, and ERBB2 were present at an equal frequency. Conclusion: While the proportion of patients with HER2- MBC identified to have new AA in serial cfDNA decreased with time for both HR+ and TNBC, new alterations continue to emerge, particularly for patients with HR+ MBC. Further research is needed to determine the impact of serial cfDNA testing on the selection of genotype-matched therapy and outcomes. Table 1. Citation Format: Yael Bar, Jennifer C. Keenan, Lianne Ryan, Dejan Juric, Jennifer Shin, Seth A. Wander, Laura M. Spring, Beverly Moy, Leif Ellisen, Steven J. Isakoff, Aditya Bardia, Neelima Vidula. Changes in the Genomic Spectrum of Actionable Alterations in HER2 Negative Metastatic Breast Cancer in Serial Cell Free DNA (cfDNA) Analysis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-01-14.

Liquid Biopsies for Circulating Tumor DNA Detection May Reveal Occult Hematologic Malignancies in Patients With Solid Tumors.

High-risk clonal hematopoiesis (CH) is frequently incidentally found in patients with solid tumors undergoing plasma cell-free DNA sequencing. Here, we aimed to determine if the incidental detection of high-risk CH by liquid biopsy may reveal occult hematologic malignancies in patients with solid tumors. Adult patients with advanced solid cancers enrolled in the Gustave Roussy Cancer Profiling study (ClinicalTrials.gov identifier: NCT04932525) underwent at least one liquid biopsy (FoundationOne Liquid CDx). Molecular reports were discussed within the Gustave Roussy Molecular Tumor Board (MTB). Potential CH alterations were observed, and patients referred to hematology consultation in the case of pathogenic mutations in JAK2, MPL, or MYD88, irrespective of the variant allele frequency (VAF), or in DNMT3A, TET2, ASXL1, IDH1, IDH2, SF3B1, or U2AF1 with VAF ≥ 10%, while also considering patient cancer-related prognosis. TP53 mutations were discussed case-by-case. Between March and October 2021, 1,416 patients were included. One hundred ten patients (7.7%) carried at least one high-risk CH mutation: DNMT3A (n = 32), JAK2 (n = 28), TET2 (n = 19), ASXL1 (n = 18), SF3B1 (n = 5), IDH1 (n = 4), IDH2 (n = 3), MPL (n = 3), and U2AF1 (n = 2). The MTB advised for hematologic consultation in 45 patients. Overall, 9 patients of 18 actually addressed had confirmed hematologic malignancies that were occult in six patients: two patients had myelodysplastic syndrome, two essential thrombocythemia, one a marginal lymphoma, and one a Waldenström macroglobulinemia. The other three patients were already followed up in hematology. The incidental findings of high-risk CH through liquid biopsy may trigger diagnostic hematologic tests and reveal an occult hematologic malignancy. Patients should have a multidisciplinary case-by-case evaluation.

CGAS-STING pathway as a potential trigger of immunosenescence and inflammaging.

Aging is associated with an increased incidence of autoimmune diseases, despite the progressive decline of immune responses (immunosenescence). This apparent paradox can be explained by the age-related chronic low-grade systemic inflammation (inflammaging) and progressive dysregulation of innate signaling. During cellular aging, there is an accumulation of damaged DNA in the cell's cytoplasm, which serves as ubiquitous danger-associated molecule, promptly recognized by DNA sensors. For instance, the free cytoplasmic DNA can be recognized, by DNA-sensing molecules like cGAS-STING (cyclic GMP-AMP synthase linked to a stimulator of interferon genes), triggering transcriptional factors involved in the secretion of pro-inflammatory mediators. However, the contribution of this pathway to the aging immune system remains largely unknown. Here, we highlight recent advances in understanding the biology of the cGAS-STING pathway, its influence on the senescence-associated secretory phenotype (SASP), and its modulation of the immune system during sterile inflammation. We propose that this important stress sensor of DNA damage is also a trigger of immunosenescence and inflammaging.

Versatile fluorescence detection of T4 PNK and mRNA based on unique DNA nanomachine amplification

The development of DNA nanomachines provides a new strategy for the detection of tumor markers. In this work, an intelligent three-dimensional (3D) DNA walking machine with polynucleotide kinase (PNK) activator was designed, which was coupled with unique nanomachine formed by DNA nanowire cascade amplification reaction for versatile fluorescence detection of T4 PNK activity and messenger RNA (mRNA). When PNK exists, the free DNA walker was formed by hydrolysis cleavage of exonuclease, then the fluorophore-labeled report probe on the Au nanoparticles (NPs) was sheared during cycling cleavage reaction, thus the fluorescence signal was recovered for detection of PNK. Moreover, the DNA nanowires were produced by rolling ring amplification, then target mRNA sequentially initiated interval hybridization of hairpin probes through DNA nanowire, thus realizing DNA cascade reaction (DCR) with high “on” signal of DNA nanomachine for mRNA assay. This developed novel fluorescence nanomachine reported a new assay method with promising application for versatile targets and showed great potential for molecular-target therapies, and clinic diagnostics.

Whole-Exome Sequencing and cfDNA Analysis Uncover Genetic Determinants of Melanoma Therapy Response in a Real-World Setting

Although several studies have explored the molecular landscape of metastatic melanoma, the genetic determinants of therapy resistance are still largely unknown. Here, we aimed to determine the contribution of whole-exome sequencing and circulating free DNA (cfDNA) analysis in predicting response to therapy in a consecutive real-world cohort of 36 patients, undergoing fresh tissue biopsy and followed during treatment. Although the underpowered sample size limited statistical analysis, samples from non-responders had higher copy number variations and mutations in melanoma driver genes compared to responders in the BRAF V600+ subset. In the BRAF V600- subset, Tumor Mutational Burden (TMB) was twice that in responders vs. non-responders. Genomic layout revealed commonly known and novel potential intrinsic/acquired resistance driver gene variants. Among these, RAC1, FBXW7, GNAQ mutations, and BRAF/PTEN amplification/deletion were present in 42% and 67% of patients, respectively. Both Loss of Heterozygosity (LOH) load and tumor ploidy were inversely associated with TMB. In immunotherapy-treated patients, samples from responders showed higher TMB and lower LOH and were more frequently diploid compared to non-responders. Secondary germline testing and cfDNA analysis proved their efficacy in finding germline predisposing variants carriers (8.3%) and following dynamic changes during treatment as a surrogate of tissue biopsy, respectively.

Non-invasive fetal genotyping for maternal alleles with droplet digital PCR: A comparative study of analytical approaches.

To develop a flexible droplet digital PCR (ddPCR) workflow to perform non-invasive prenatal diagnosis via relative mutation dosage (RMD) for maternal pathogenic variants with a range of inheritance patterns, and to compare the accuracy of multiple analytical approaches. Cell free DNA (cfDNA) was tested from 124 archived maternal plasma samples: 88 cases for sickle cell disease and 36 for rare Mendelian conditions. Three analytical methods were compared: sequential probability ratio testing (SPRT), Bayesian and z-score analyses. The SPRT, Bayesian and z-score analyses performed similarly well with correct prediction rates of 96%, 97% and 98%, respectively. However, there were high rates of inconclusive results for each cohort, particularly for z-score analysis which was 31% overall. Two samples were incorrectly classified by all three analytical methods; a false negative result predicted for a fetus affected with sickle cell disease and a false positive result predicting the presence of an X-linked IDS variant in an unaffected fetus. ddPCR can be applied to RMD for diverse conditions and inheritance patterns, but all methods carry a small risk of erroneous results. Further evaluation is required both to reduce the rate of inconclusive results and explore discordant results in more detail.

High-Throughput Exonuclease Assay Based on the Fluorescent Base Analogue 2-Aminopurine.

Exonucleases are essential enzymes that remove nucleotides from free DNA ends during DNA replication, DNA repair, and telomere maintenance. Due to their essential role, they are potential targets for novel anticancer and antimicrobial drugs but have so far been little exploited. Here, we present a simple and versatile real-time exonuclease assay based on 2-aminopurine, an intrinsically fluorescent nucleotide that is quenched by neighboring bases when embedded in DNA. We show that our assay is applicable to different eukaryotic and bacterial exonucleases acting on both 3' and 5' DNA ends over a wide range of protein activities and suitable for a high-throughput inhibitor screening campaign. Using our assay, we discover a novel inhibitor of the Mycobacterium tuberculosis PHP-exonuclease that is part of the replicative DNA polymerase DnaE1. Hence, our novel assay will be a useful tool for high-throughput screening for novel exonuclease inhibitors that may interfere with DNA replication or DNA maintenance.

Association of emergent neuroendocrine prostate cancer detected by liquid biopsies with survival and treatment resistance.

247 Background: The mainstay of therapy in metastatic prostate cancer is androgen receptor (AR) signaling inhibition. However, the emergence of early castration resistance or neuroendocrine transformation is associated with poor prognosis. Reliable biomarkers are needed to identify these patients and guide selection of clinical therapy. Methods: mRNA was isolated from EpCAM-positive circulating tumor cells (CTCs) isolated from patients with CSPC, CRPC, or NEPC to measure expression of KLK2, KLK3 (PSA), TMPRSS2, FOLH1 (PSMA), synaptophysin ( SYP), and chromogranin ( CHGA). Post-hoc retrospective analysis of an institutional review board–approved prospective cohort (N = 98) was performed to identify patterns of gene expression. Samples were considered AR+ if 3 of 4 AR pathway genes ( TMPRSS2, KLK2, KLK3, and FOLH1) were positive, and were considered NE+ if either or both SYP or CHGA were positive. Blood samples from two prospective clinical trials of men with mCRPC treated with abiraterone and enzalutamide, respectively, were analyzed to confirm results. Longitudinal samples were collected from 17 patients (6 NEPC and 11 Adenocarcinoma) and cell free DNA was isolated and sequenced using a novel targeted exon panel. Results: AR and/or NE positive patients were found to have a median overall survival (OS) of 8.58 months as compared to a median OS of 29.6 months in the AR and NE negative population (p<0.0001; HR=2.75 [1.60-4.56]). In the subset of castrate resistant prostate cancer (CRPC) patients, AR+ and/or NE+ patients (n=31) were found to have a median OS of 6.74 months vs 18.79 months in the AR- and NE- group (n=39) (p= 0.0009; HR 2.38 [1.36-4.18]). We also tested samples from a phase II ARSI trials with abiraterone and enzalutamide, respectively. None of the baseline samples from these two trials met the above criteria for NEPC (AR- NE+) on their CTCs or histologically. Three of 48 total patients were identified with expression in their baseline blood samples without loss of AR target gene expression (AR+/NE+). Preliminary analysis of this small patient cohort in comparison to patients who were NE- shows that NE+ patients had worse OS (HR= 5.5906 [1.143-27.36), as would be expected by patients with emerging neuroendocrine differentiation. Integrated ctDNA sequencing identified mutations in genes associated with NEPC. Conclusions: The expression of NE genes in liquid biopsies while retaining AR target gene expression is associated with worse OS and may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype. Early identification of these patients may improve therapeutic decisions and improved patient outcomes. Pairing genomic alteration with changes in gene expression may additionally offer the basis for a new mechanism to assess efficacy of novel therapeutics in future clinical trials.

CDK4/6 Inhibitors Overcome Endocrine ESR1 Mutation-Related Resistance in Metastatic Breast Cancer Patients

ESR1 mutations contribute to endocrine resistance and occur in a high percentage of hormone-receptor-positive (HR+) metastatic breast cancer (mBC) cases. Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) changed the treatment landscape of HR+ mBC, as they are able to overcome estrogen resistance. The present retrospective study investigates the clinical benefit of CDK4/6i in ESR1 mutant HR+ mBC patients treated with a CDK4/6i as first- or second-line therapy. Plasma was collected at baseline prior to CDK4/6i plus hormone therapy as a first- or second-line treatment. Circulating free DNA (cfDNA) was extracted from plasma, and ESR1 mutation analysis was performed on a ddPCR. Statistical analyses were performed to investigate the predictive power of ESR1 mutations and any association with clinical factors. A total of 42 patients with mBC treated with CDK4/6i plus endocrine therapy as first- (n = 35) or second-line (n = 7) were enrolled. Twenty-eight patients received hormonal therapy (AI or tamoxifen) in the adjuvant setting. ESR1 mutation status in blood was associated with shorter median disease-free survival (DFS) (30 vs. 110 months; p = 0.006). Multivariate analysis confirmed ESR1 mutations as independent factors of resistance in adjuvant hormone therapy. On the contrary, no difference in progression-free survival (PFS) was observed in the presence or absence of an ESR1 mutation in patients treated with CDK4/6i as first-line treatment (p = 0.29). No statistically significant correlation between the best response to CDK4/6i and ESR1 mutation was found (p = 0.46). This study indicates that the ESR1 mutation detected in cfDNA is an independent predictive factor of clinical recurrence in the adjuvant setting and that CDK4/6i can overcome ESR1-dependent resistance.

Co‐Assembly of Peptide with G‐Quadruplex DNA: A Strategic Approach to Develop Anticancer Therapeutics

AbstractG‐quadruplex (G4) is one of the non‐canonical nucleic acid structures that regulate multiple key biological processes. The formation of G4 hinders the progression of cancer. The molecular entities which can stabilize the G4 structure are very important to design anticancer therapeutics. Herein, it is described that a synthetic dendron‐like peptide, Cδ3‐(YYEE)‐E co‐assembles with G4 DNA in aqueous buffer containing Na+ ions. Various orthogonal biophysical studies have established the co‐assembly phenomenon. In silico, molecular docking studies also corroborate the results obtained from biophysical experiments. Importantly, the co‐assembly enhances the thermal stability of G4 DNA compared to free G4 DNA. The peptide inhibits the activity of telomerase enzyme which is found to be over‐expressed in most of the cancer cells and also down‐regulates the c‐Myc oncogenic expression. The peptide exhibits significant cytotoxic effect on cancer cells (HeLa and U2OS) compared to non‐cancer cells (HEK293) under similar experimental conditions. The study highlights the applicability of a synthetic bioactive peptide as a putative anticancer therapeutic.

New insights on partial trisomy 3q syndrome: de novo 3q27.1-q29 duplication in a newborn with pre and postnatal overgrowth and assisted reproductive conception

BackgroundDuplications of the long arm of chromosome 3 are rare, and associated to a well-defined contiguous gene syndrome known as partial trisomy 3q syndrome. It has been first described in 1966 by Falek et al., and since then around 100 patients have been reported. Clinical manifestations include characteristic facial dysmorphic features, microcephaly, hirsutism, congenital heart disease, genitourinary anomalies, hand and feet abnormalities, growth disturbances and intellectual disability. Most of cases are due to unbalanced translocations, inherited from a parent carrying a balanced aberration (reciprocal translocation or inversion), and rarely the genomic anomaly arises de novo. Very few studies report on the prenatal identification of such rearrangements.Case presentationHereby, we report on a newborn with a rare pure duplication of the long arm of chromosome 3. Noninvasive prenatal test (cell free fetal DNA analysis on maternal blood), performed for advanced parental age and use of assisted reproductive technique, evidenced a partial 3q trisomy. Then, invasive cytogenetic (standard and molecular) investigations, carried out through amniocentesis, confirmed and defined a 3q27.1-q29 duplication spanning 10.9 Mb, and including about 80 genes. Our patient showed clinical findings (typical facial dysmorphic features, esotropia, short neck, atrial septal defect, hepatomegaly, mild motor delay) compatible with partial trisomy 3q syndrome diagnosis, in addition to pre- and postnatal overgrowth.ConclusionsAdvanced parental age increases the probability of chromosomal and/or genomic anomalies, while ART that of epigenomic defects. Both conditions, thus, deserve more careful prenatal monitoring and screening/diagnostic investigations. Among the latter, cell free fetal DNA testing can detect large segmental aneuploidies, along with chromosomal abnormalities. It identified in our patient a wide 3q rearrangement, then confirmed and defined through invasive molecular cytogenetic analysis. Neonatologists and pediatricians must be aware of the potential risks associated to duplication syndromes. Therefore, they should offer to affected subjects an adequate management and early and careful follow-up. These may be able to guarantee to patients satisfactory growth and development profiles, prevent and/or limit neurodevelopmental disorders, and timely recognition of complications.

Mutant-RB1 circulating tumor DNA in the blood of unilateral retinoblastoma patients: What happens during enucleation surgery: A pilot study.

Cell free DNA (cfDNA) and circulating tumor cell free DNA (ctDNA) from blood (plasma) are increasingly being used in oncology for diagnosis, monitoring response, identifying cancer causing mutations and detecting recurrences. Circulating tumor RB1 DNA (ctDNA) is found in the blood (plasma) of retinoblastoma patients at diagnosis before instituting treatment (naïve). We investigated ctDNA in naïve unilateral patients before enucleation and during enucleation (6 patients/ 8 mutations with specimens collected 5-40 minutes from severing the optic nerve) In our cohort, following transection the optic nerve, ctDNA RB1 VAF was measurably lower than pre-enucleation levels within five minutes, 50% less within 15 minutes and 90% less by 40 minutes.

Making the undetectable detectable: Pushing limits of biomolecular recognition by ultra-sensitive multiplexed single-particle liposome readouts.

Biomolecular recognition is fundamental for all biological and chemical processes. However, current screening methods are often reliant on large robotic devices, require long running times and much manpower as well as excessive materials cost. Here I will present our recent work implementing multiple miniaturized screening assays for ultra-sensitive and high-throughput readout at the single-particle level of DNA-DNA recognition, sub-attoliter cargo delivery, transporter function and label-free DNA biosensing. I will focus on the single-particle combinatorial multiplexed liposome fusion mediated by DNA for parallelized multistep and non-deterministic fusion of individual sub-attoliter nanocontainers (SPARCLD). Our new toolbox allowed us to attain efficient (>93%) and leakage free stochastic fusion sequences for arrays of surface-tethered target liposomes with six freely diffusing populations of cargo liposomes, each functionalized with individual lipidated single-stranded DNA and fluorescently barcoded by a distinct ratio of chromophores. Because each lipid nanoparticle is an autonomous nanocontainer the stochastic fusion sequence resulted in massive number of distinct permutations of cargo delivery loadings and sequences. Real-time recordings and machine learning analysis allowed the rapid classification of >16,000 fusions and 566 distinct fusion sequences within hours. The high-density arrays of surface-tethered target nanocontainers (∼42,000 containers per mm2) offers entire combinatorial multiplex screens using only picograms of material and is implanted for screening transporter function and label free DNA biosensing.

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

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

A retrospective study of circulating tumor DNA for minimal residual disease detection in the management of colorectal cancer.

227 Background: Circulating tumor DNA (ctDNA) consists of small fragments of DNA released into the plasma from cancer cells. Tumor informed ctDNA testing by whole exome sequencing of the primary tumor coupled with multiplex polymerase chain reaction (PCR)-based next generation sequencing of cell free DNA in serum, can detect and quantify ctDNA, allowing for detection of minimal residual disease (MRD). Methods: We conducted a retrospective analysis of 120 patients with colorectal cancer who had at least one tumor-informed ctDNA MRD assay completed between May 6, 2019, and July 1, 2022, at Mayo Clinic, Rochester. Disease recurrence was defined as radiographic evidence consistent with recurrence with or without biopsy confirmation. Data are presented descriptively, including number of ctDNA assays completed, ctDNA positivity prior to disease recurrence, and ctDNA concordance with carcinoembryonic antigen (CEA). The study protocol was exempt after review by the Mayo Clinic Institutional Review Board. Results: One-hundred twenty patients were included in the study with median age at initial disease diagnosis of 67.0 years. Sixty-four percent were male and 94% were white. At time of initial diagnosis, 10 had stage I, 23 stage II, 60 stage III, and 25 stage IV disease. In 120 patients, 476 ctDNA assays were performed, and 110 recurrences were observed among 62 patients. There were 39 instances in which ctDNA was obtained prior to clinical detection of recurrence and of these, at least one ctDNA assay was positive in 28 cases (72%). CEA was obtained prior to recurrence in 88 instances and was elevated in only 39 cases (44%). Among 37 instances where both ctDNA and CEA testing were obtained prior to recurrence, ctDNA was positive in 27 cases (73%) while CEA was positive in 14 cases (38%). ctDNA was positive in 83% of recurrences involving liver and 62.5% of recurrences involving only the lungs. ctDNA and CEA positivity are shown by location of recurrence. ctDNA and CEA were discordant in 44% of instances in which the assays were completed within 2-weeks of each other. Results of a ctDNA assay impacted management decisions in 16% of cases. Conclusions: ctDNA detected 72% of recurrences with higher rates of detection in liver versus lung metastasis. ctDNA performed superiorly to serum CEA in detection of recurrence, suggesting a role in disease surveillance. However, ctDNA impacted disease management in only 16% of patients, suggesting need for better patient selection and further study for ctDNA-based disease management strategy (more details will be reported). [Table: see text]

Enhanced Single-Strand Breaks of a Nucleic Acid by Gold Nanoparticles under X-ray Irradiation

The hydroxyl radical concentration-dependent yield of single-strand breaks (SSBs), obtained through correction of scavenging and hindrance effects caused by gold nanoparticles (AuNPs), for fluorophore- and quencher-labeled DNA on AuNPs was 10 times that of free DNA based on fluorescence measurements of X-ray-irradiated DNA on AuNPs. By comparing the fluorescence data that revealed the number of SSBs with the results of mass spectrometry measurements that detected the total damage to DNA, we found that SSBs dominated DNA damage for DNA on AuNPs whereas non-SSB damage dominated for free DNA. The yield of RNA SSBs under X-ray irradiation was similar to that of DNA in the presence of AuNPs, whereas free RNA was more resistive to radiation than DNA. These results indicated the enhanced SSBs were likely catalyzed through the conversion from nucleobase damage to SSBs by AuNPs. The outcome of this work impacts materials and environmental science, sensing, nanotechnology, biology, and medicine.

Identification of GNAS Variants in Circulating Cell-Free DNA from Patients with Fibrous Dysplasia/McCune Albright Syndrome.

Fibrous dysplasia/McCune-Albright syndrome (FD/MAS) is a rare mosaic bone and endocrine disorder. Although most variants affect the GNAS R201 codon, obtaining a genetic diagnosis is difficult because not all cells harbor the variant, and an invasive biopsy may be required. We explored the presence of GNAS p.R201 variants in blood circulating cell free DNA (ccfDNA) using sensitive techniques of digital droplet polymerase chain reaction (PCR) (ddPCR) and competitive allele-specific TaqMan PCR (castPCR) in an effort to improve the genetic diagnosis of FD/MAS. We isolated ccfDNA from the plasma of 66 patients with a wide range of disease severity and performed both ddPCR and castPCR mutation analysis to search for GNAS p.R201H or R201C variants. We detected R201 variants in ccfDNA samples of 41 of 66 (62.1%) patients by either castPCR or ddPCR, and 45 of 66 (68.2%) of patients if the techniques were combined. Variant detection was more likely in patients with more severe disease. Skeletal disease burden score (SBS) was significantly higher in patients who had detectable variants, and SBS was a predictor of variant allele frequency. By ddPCR analysis, patients aged ≤30 years had higher detection rates, and higher variant allele frequencies, independent of disease burden. We detected variant DNA in only one patient with monostotic FD by ddPCR only. In summary, we have demonstrated that ccfDNA containing variant GNAS can be isolated from the plasma of patients with FD/MAS and that ddPCR and castPCR methods have similar variant detection rates. This methodology represents an important potential advancement in diagnosis for patients with FD/MAS, especially those younger than 30 years or with more severe disease. Published 2023. This article is a U.S. Government work and is in the public domain in the USA.

Midgut Volvulus Adds a Murine, Neutrophil-Driven Model of Septic Condition to the Experimental Toolbox

Severe infections that culminate in sepsis are associated with high morbidity and mortality. Despite continuous efforts in basis science and clinical research, evidence based-therapy is mostly limited to basic causal and supportive measures. Adjuvant therapies often remain without clear evidence. The objective of this study was to evaluate the septic volvulus ischemia-reperfusion model in comparison to two already established models and the role of neutrophil extacellular traps (NETs) in this model. The technique of the murine model of midgut volvulus was optimized and was compared to two established models of murine sepsis, namely cecal ligation and puncture (CLP) and intra-peritoneal (i.p.) injection of lipopolysaccharide (LPS). Midgut volvulus for 15 min caused a comparable mortality (38%) as CLP (55%) and peritoneal LPS injection (25%) at 48 h. While oxidative stress was comparable, levels of circulating free DNA (cfDNA), and splenic/hepatic and pulmonary translocation of bacteria were decreased and increased, respectively at 48 h. DNases were increased compared to the established models. Proteomic analysis revealed an upregulation of systemic Epo, IL-1b, Prdx5, Parp1, Ccl2 and IL-6 at 48 h in comparison to the healthy controls. Midgut volvulus is a stable and physiological model for sepsis. Depending on the duration and subsequent tissue damage, it represents a combination of ischemia-reperfusion injury and hyperinflammation.

Can circulating cell free DNA be a promising marker in ovarian cancer? – a genome-scale profiling study in a single institution

BackgroundCell-free DNA (cfDNA) is emerging as a potential biomarker for the detection of ovarian cancer (OC). Recently, we reported a method based upon cfDNA whole-genome sequencing data including the nucleosome distribution (nucleosome footprinting NF), terminal signature sequence (motif), DNA fragmentation (fragment), and copy number variation (CNV).In the present study, we explored whether multiomics early screening technology in cfDNA can be applied for early screening of ovarian cancer.MethodsFifty-nine patients with OC and 100 healthy controls were included in this prospective study. Cell-free DNA was extracted from plasma and analyzed by low-pass whole-genome sequencing. Genomic features were obtained for all samples of the cohort, including copy number variation (CNV), 5’-end motifs, fragmentation profiles, and nucleosome footprinting (NF). An integrated scoring system termed the OC score was developed based on the performance of these four features.ResultsAll four features showed diagnostic potential for OC. Based on the unique genome features of cfDNA, the OC score has high accuracy in distinguishing OC patients from healthy controls (AUC 97.7%; sensitivity 94.7%; specificity 98.0%) as a new comprehensive diagnostic method for OC. The OC score showed a gradual trend from healthy controls to OC patients with different stages, especially for early OC monitoring of concern, which achieved a satisfactory sensitivity (85.7%) at a high specificity.ConclusionsThis is the first study evaluating the potential of cell-free DNA for the diagnosis of primary OC using multidimensional early screening technology. We present a promising method to increase the accuracy of prediction in patients with OC.