cfDNA Analysis Research Articles

Utilizing Cell-Free Urinary and Plasma Tumor DNA to Predict Pathologic Stage at Radical Cystectomy

ObjectiveTo assess the ability of cell-free urinary and plasma tumor DNA (cfDNA) to predict pathologic stage at radical cystectomy for patients with clinical muscle-invasive bladder cancer. MethodsA total of 25 patients with clinical muscle-invasive bladder cancer were enrolled before undergoing radical cystectomy. Blood and urine were collected before surgery. The 600-gene PredicineATLAS panel was used to sequence blood buffy-coat germline DNA, plasma cfDNA, and urine cfDNA samples. Low-pass whole genome sequencing was performed on plasma- and urine-derived cfDNA. CfDNA tumor fraction (TF), genome-wide copy number burden (CNB), and estimated tumor mutational burden (TMB) were measured in both plasma and urine samples and their correlation with pathologic T-stage was examined. ResultsThree of 25 plasma samples had insufficient cfDNA. In 22 of 22 plasma samples and 24 of 25 urine samples, at least one nonsynonymous somatic variant was detected. Across the cohort, 44% of plasma variants were concordant with paired urine variants. The mean number of variants did not differ between noninvasive (< pT1/pN0) and invasive disease (≥ pT1 or N+) for both plasma (8 vs. 9.5 variants; P = 0.85) and urine (33.7 vs. 30 variants; P = 0.45). A strong correlation was observed between urine TF and urine CNB score within patients (rv = 0.92). Plasma TF (r = 0.38), urine TF (r = 0.21), and urine CNB score (r = 0.16) exhibited positive correlations with pT stage. Patients with carcinoma in situ (CIS) had higher mean urine TF and CNB scores ( P = 0.07 and P = 0.05, respectively). Plasma TF and CNB score did not correlate with the presence of CIS.ConclusionsCombining plasma- and urine-based cfDNA analysis may help identify patients with residual disease at radical, although we were unable to predict pathologic T-stage based on these metrics.The presence of CIS may contribute to greater urinary CNB and TF levels. Considering CIS in the analysis may improve the ability to correlate tumor metrics with pathologic stage. Low-pass whole genome sequencing–derived urinary CNB correlates strongly with urinary TF and may provide a less resource-intensive method for future longitudinal disease monitoring.

Diagnostics of inflammaging in relation to sarcopenia.

One of the theories about aging focuses on the immune response and relates to the activation of subclinical and chronic inflammation. This study was designed to investigate the relationship between inflammation and sarcopenia and to evaluate the influence of lifestyle on the inflammatory profile. Finally, therapeutic strategies to counteract the pathophysiological effect of skeletal muscle aging were also indicated. One hundred seventy-three individuals aged 71.5 ± 6.8 years were divided into two groups: sarcopenia and probable sarcopenia (n = 39) and no sarcopenia (n = 134). Sarcopenia was assessed according to the algorithm of the European Working Group on Sarcopenia in the older adults 2. C-reactive protein (CRP) (p = 0.011) and CRP/albumin ratio (p = 0.030) as well as IL-1β (p = 0.002), cfDNA (p < 0.001) and bilirubin levels (p = 0.002) were significantly higher in the sarcopenia group as opposed to the no sarcopenia group. No significant differences were observed between groups in the concentration of TNFα (p = 0.429) and IL-6 (p = 0.300). An inverse correlation was found between gait speed and cfDNA (rs = -0.234, p < 0.01) and IL-1β (rs = -0.263, p < 0.01). The ROC analysis of cfDNA, CRP, IL-1β and bilirubin ranged from 0.6 to 0.7, which confirms the association between sarcopenia and inflammatory mediators and indicates high clinical usefulness of cfDNA and bilirubin in sarcopenia prediction. We also indicated a link between inflammation and fitness level in the older adult thereby providing evidence that lifestyle exercise should be a key therapeutic strategy in sarcopenia prevention.

Machine Learning Application in cfDNA Analysis to Achieve Tumour Assessment

Breast cancer (BC) is the leading cause of cancer in women and the second leading cause of cancer-related death. Early and accurate screening of BC is a promising way of reducing the proportion of patients with advanced stages of BC. In recent years, the non-invasive test of tumour diagnosis by assessing the level of Plasma cell-free DNA (cfDNA) has become a research hotspot. Here, we demonstrate the use of random forest models to predict BC by evaluating the levels of 26 known breast cancer-related cfDNA methylation molecular markers (model-tested accuracy of 67.88%). Then, we improved the accuracy of the model to 71.52% by parameter optimization. In addition, considering that the diagnosis of BC is closely related to the health of every female, we have extended the project from scientific research to social investigation by carrying out a sample survey of Chinese college students to understand various perspectives on the application of artificial intelligence in the diagnosis of diseases. We found that the response was rather optimistic, while some participants showed concerns about the maturity of the technology and the disclosure of privacy. Therefore, future research should focus on the optimisation of the machine learning model, so as to effectively improve the accuracy of diagnosis and provide better pre-service for the population at risk of cancer.

P-171 Identification of aneuploid embryos using cell free DNA in embryo culture medium

Abstract Study question Can noninvasive preimplantation genetic testing (PGT) using cell free DNA from embryo culture medium discriminate embryonic aneuploidy status? Summary answer Aneuploid embryos in older patients can be identified by cell free DNA in embryo culture medium. What is known already There have been reports of non-invasive PGT by analysing cell free DNA (cfDNA) released from embryos into culture medium, and found to be highly correlated with the analysis results of Trophectoderm (TE)-biopsy (Carmen.Rubio, 2020).We have initiated in 2021, a preliminary research program for the clinical application of niPGT. In aneuploid embryos, we have found a tendency for a high concordance rate between cfDNA analysis and blastocyst analysis results, suggesting the usefulness of cfDNA analysis in terms of identifying aneuploid embryos.Subsequently, we examined the usefulness of cfDNA analysis by comparing the concordance rate with cfDNA analysis and TE biopsy of blastocysts. Study design, size, duration After thawing pronuclear-stage frozen embryos for which consent was obtained for research use, they were co-cultured until Day 4, and then individually cultured on in new dishes.40 embryos that reached morula on day 4 or day 5 and blastocyst on day 6 or day 7 were included. Participants/materials, setting, methods The biopsied TE, the biopsied blastocyst, and the cfDNA obtained after individual culture were all sent to an external provider (Igenomix: Valencia, Spain) for chromosomal aneuploidy analysis by NGS. The concordance rate of the analysis for blastocysts in cfDNA and TE was calculated by dividing them into euploidy and aneuploidy, and by age. Concordance between the results of the blastocyst analysis and the culture medium were assessed according to the standards of the inspection company. Main results and the role of chance For the blastocyst analysis results, the concordance rate with cfDNA was 76.5% (26/34) and the concordance rate with TE was 85.3% (29/34), showing no difference. For the euploid blastocysts analyzed, the matching rate with cfDNA was 61.1% (11/18) and with TE was 77.8% (14/18), showing no difference. Similarly, for aneuploid blastocysts, the concordance rate with cfDNA was 93.8% (15/16) and with TE was 93.8% (15/16), showing no difference. Detailed analysis of aneuploid blastocysts revealed that the concordance rate with cfDNA was 88.9% (8/9) and the concordance rate with TE was 88.9% (8/9) in women under 39 years of age. In addition, the concordance rate with cfDNA was 100.0% (7/7) with TE was 100.0% (7/7) in those aged 40 and over. (Fisher's exact test) Limitations, reasons for caution Concordance between cfDNA and blastocyst analyzes in euploid embryos was low, continued follow-up is considered necessary. Wider implications of the findings In a detailed analysis of aneuploid blastocysts aged 40 years or older, Similar to TE, cfDNA showed a high concordance rate with the results of blastocyst analysis, demonstrating that cfDNA provides sufficient accuracy for identifying aneuploid embryos in women over 40 years of age. Trial registration number not applicable

O-225 May embryonic cfDNA in the culture media play a role as a non-invasive assessment of aneuploidy in PGT

Abstract Non-invasive preimplantation genetic testing for aneuploidies (niPGT-A) in spent blastocyst media (SBM) is a promising technique that has the potential to revolutionize current preimplantation genetic testing procedures. This technique is based in the analysis of the cell-free DNA (cfDNA) released by the embryo during the latest stages of preimplantation development. It allows for the detection of chromosomal abnormalities in embryos without the need for invasive procedures that can harm the embryo (reviewed in Navarro-Sánchez et al., 2022). Our group among others has conducted different studies in this field, and the early clinical application of the cfDNA analysis in the culture medium has been proposed as a screening tool, giving a priority score for embryo transfer based on the media results (Rubio et al., 2019, 2020). In the final analysis of our multicenter study in day-6 and day-7 blastocysts, we have been able to compare the chromosomal results of the SBM with trophectoderm (TE) biopsies in 2,187 samples, and with the inner cell mass ICM in 230 blastocysts. In this study, the analysis of the cfDNA showed a concordance rate of 87% with the ICM biopsy, which is currently considered the gold standard for PGT-A. However, it is important to continue evaluating the factors that may affect the informativity and concordance rates of cfDNA-based assays, such as the culture day when medium is collected, contamination with external and/or cumulus cell DNA, and previous manipulation of the embryos. Multivariate analysis has shown that the longer the time in culture, the higher the informativity rate, and female age had the greatest impact on concordance rates with TE biopsies. Regarding technical aspects, the SBMs with higher number of reads after sequencing resulted in higher concordance rates, reflecting the importance of analysing samples with sufficient DNA quantity. Finally, the predictive value in terms of concordance with TE biopsies was higher for aneuploid blastocyst. No significant independent correlation was observed for embryo quality. The improvements in IVF lab protocols and the use of time-lapse and short incubation protocols for vitrified-thawed blastocysts are also noteworthy, as they may further increase the concordance rates of cfDNA-based assays with blastocysts. In fact, concordance in previously vitrified-thawed blastocysts as result as high as 90%. Regarding clinical outcomes, several groups have evaluated non-invasive PGT-A with transfer of euploid embryos showing good results. In a pilot study with only 7 SETs in couples with PGT-A or PGT-SR indication, 5 pregnancies were achieved resulting in 5 livebirths (Xu et al., 2016). In a second study, in couples with recurrent spontaneous abortion or repeated implantation failure, 50 transfers of euploid media were performed resulting in a 58% clinical pregnancy rate, with 27 healthy babies born (Fang et al., 2019). Similar results were described in good prognosis patients &amp;lt;38 years of age with ongoing pregnancy rates comparable to PGT-A (61.5%), and higher than conventional IVF or ICSI (48.5%) (Franco et al., 2021). Overall, the high concordance rates with trophectoderm biopsies and inner cell mass biopsies are encouraging, and the clinical outcomes with transfer of euploid embryos are promising. It is exciting to see the progress in non-invasive preimplantation genetic testing using cfDNA in culture medium, and it will be interesting to see how this technology evolves and its potential role as a screening or diagnostic tool in the future.

Plasma cell-free tumor DNA, PIK3CA and TP53 mutations predicted inferior endocrine-based treatment outcome in endocrine receptor-positive metastatic breast cancer

PurposeHow to factor both tumor burden and oncogenic genomic mutations as variables to predict the outcome of endocrine-based therapy (ET) in ER-positive/HER2-negative metastatic breast cancer patients (MBC) remains to be explored.MethodBlood samples prospectively collected from 163 ER-positive/HER2-negative female MBC patients, before ET, were used for cell-free tumor DNA (cfDNA) analysis. cfDNA was subjected to next-generation sequencing (NGS) to interrogate oncogenic PIK3CA hotspot and TP53 DNA-binding domain (DBD) mutations, including single nucleotide variants (SNVs) or small insertions and deletions (InDels). The variant calling threshold was set at 0.5%. Progression-free survival (PFS) was measured from the start of the ET treatment to the time of disease progression of the same treatment regimen.ResultsOverall, the median PFS was 8.3 months (95% CI 5.7–11.1 months). The median cfDNA was 38.5 ng (range 4.4–1935 ng). The proportion of patients with PIK3CA and TP53 alterations were 25.1 and 15.3%, respectively. Patients with high total cfDNA (HR 1.74, p = 0.003), PIK3CA mutation (HR 1.74, p = 0.007), and TP53 mutation (HR 1.64, p = 0.047) in liquid biopsy conferred worse outcome after ET. Even for patients with low tumor burden, the detrimental effect of PIK3CA or TP53 mutation remained significant (p < 0.001). For patients with either PIK3CA (p < 0.001) or TP53 mutation (p = 0.004), there was significant positive correlation between allele frequency (AF) and total cfDNA.ConclusionAfter adjustment of cfDNA level, PIK3CA and TP53 mutations observed in liquid biopsy exerted detrimental effects on the outcome of ET-based regimens. The AF of PIK3CA or TP53 may be a surrogate marker for PFS.

Identification of Somatic Mutations in Plasma Cell-Free DNA from Patients with Metastatic Oral Squamous Cell Carcinoma.

The accurate diagnosis and treatment of oral squamous cell carcinoma (OSCC) requires an understanding of its genomic alterations. Liquid biopsies, especially cell-free DNA (cfDNA) analysis, are a minimally invasive technique used for genomic profiling. We conducted comprehensive whole-exome sequencing (WES) of 50 paired OSCC cell-free plasma with whole blood samples using multiple mutation calling pipelines and filtering criteria. Integrative Genomics Viewer (IGV) was used to validate somatic mutations. Mutation burden and mutant genes were correlated to clinico-pathological parameters. The plasma mutation burden of cfDNA was significantly associated with clinical staging and distant metastasis status. The genes TTN, PLEC, SYNE1, and USH2A were most frequently mutated in OSCC, and known driver genes, including KMT2D, LRP1B, TRRAP, and FLNA, were also significantly and frequently mutated. Additionally, the novel mutated genes CCDC168, HMCN2, STARD9, and CRAMP1 were significantly and frequently present in patients with OSCC. The mutated genes most frequently found in patients with metastatic OSCC were RORC, SLC49A3, and NUMBL. Further analysis revealed that branched-chain amino acid (BCAA) catabolism, extracellular matrix-receptor interaction, and the hypoxia-related pathway were associated with OSCC prognosis. Choline metabolism in cancer, O-glycan biosynthesis, and protein processing in the endoplasmic reticulum pathway were associated with distant metastatic status. About 20% of tumors carried at least one aberrant event in BCAA catabolism signaling that could possibly be targeted by an approved therapeutic agent. We identified molecular-level OSCC that were correlated with etiology and prognosis while defining the landscape of major altered events of the OSCC plasma genome. These findings will be useful in the design of clinical trials for targeted therapies and the stratification of patients with OSCC according to therapeutic efficacy.

METB-07. MINIMALLY-INVASIVE DIAGNOSIS OF DIFFUSE LEPTOMENINGEAL GLIONEURONAL TUMOR (DLGNT) USING CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING IN PEDIATRIC AND YOUNG ADULT PATIENTS

Abstract Diffuse leptomeningeal glioneuronal tumor (DLGNT) presents with nodular leptomeningeal disease throughout the neuroaxis. Histology shows variable presence of a low density intraparenchymal lesion containing OLIG2-expressing neoplastic cells. Molecularly, DLGNT is characterized by the presence of KIAA1549::BRAF fusion and chromosome arm 1p deletion. Concomitant gain of chromosome arm 1q is associated with poor prognosis. Diagnosis by meningeal biopsy is often non-diagnostic and we hypothesized analysis of cell free DNA (cfDNA) from cerebrospinal fluid (CSF) could represent a superior diagnostic modality. We analyzed CSF samples from 7 patients, median age of 18 years, with DLGNT including matched germline DNA. MSK-IMPACT, a hybridization capture-based next-generation DNA sequencing panel, was utilized to analyze the DNA. Prior tissue biopsy was done in 5/7 patients. Of these, biopsy was non-diagnostic in 3 patients and positive for the KIAA1549:BRAF fusion in 2. Analysis of CSF cfDNA detected KIAA1549::BRAF fusion in 5/7 (71%) of patients, including all patients with prior non-diagnostic biopsy or tissue quantity insufficient for molecular testing. Analysis of CSF cfDNA copy number profiles revealed 1p-/1q+ in 6 patients and 1p- only in the remaining 1 patient, fully concordant with available prior tissue testing in all instances. In 2 patients with prior tissue-confirmed KIAA1549::BRAF fusion, the fusion was not detected in CSF cfDNA during/after treatment with a MEK inhibitor, but additional somatic alterations and DNA copy number profiles were similar to the prior biopsies. Additional somatic alterations were identified in ATRX, BCOR, RARA, STAT5A, SPOP, PPM1D, MED12, KMT2C, RECQL and SETD2, including likely oncogenic secondary driver alterations that have not been previously reported in DLGNT. Given the high molecular diagnostic yield and concordance with open biopsy, cfDNA CSF sequencing should be considered as a first line diagnostic maneuver for patients with MRI findings suspicious for DLGNT, followed by open biopsy only if CSF is non-diagnostic.

METB-05. NON-INVASIVE DIAGNOSIS OF BRAINSTEM GLIOMAS IN PEDIATRIC, ADOLESCENT, AND YOUNG ADULT PATIENTS THROUGH CEREBROSPINAL FLUID CELL-FREE DNA SEQUENCING

Abstract Intrinsic brainstem tumors arising in pediatric, adolescent, and young adult patients comprise a spectrum of entities, predominantly diffuse midline gliomas (DMG) and IDH mutant astrocytomas. Accurate molecular diagnosis is essential for prognostication and optimal therapy. Therapeutic considerations include inclusion (in IDH mutant) or exclusion (in H3K27M) of adjuvant Temozolomide post radiotherapy and use of molecular targeted therapy (IDH inhibitors). Surgical biopsy, however, is associated with increased risk of permanent neurological deficits and may yield insufficient or non-diagnostic tissue. We hypothesized that minimally-invasive “liquid biopsy” of cerebrospinal fluid (CSF) could represent a superior diagnostic modality in this patient population, employing molecular analysis of cell free DNA (cfDNA). We analyzed 44 CSF samples from 38 unique patients for recurrent driver mutations in brainstem gliomas, including H3K27M, IDH1, and IDH2. MSK-IMPACT, a NY state-authorized hybridization capture-based next-generation panel DNA sequencing assay, was used for analysis. Samples without a detectable driver mutation by MSK-IMPACT testing were further subjected to gene targeted testing by droplet digital PCR. In all, 10/44 (22.7%) samples had mutations detected by MSK-IMPACT using standard calling criteria and 13 of the remaining 34 samples (38.2%) had supporting evidence of a mutation based on manual review. Further testing by ddPCR was performed on a subset of cases (based on DNA availability) which confirmed 7 of the previous low-level mutations and uncovered 4 additional mutations. Overall, 27/44 (61.4%) of cases had detectable evidence of a mutation, and of the 23 patients without a known tissue diagnosis, a driver in the CSF was identified for 47.8% of patients (11/23). Minimally-invasive analysis of CSF cfDNA in patients with intrinsic brainstem tumors has a high diagnostic yield and may obviate the need for tissue biopsy in a majority of patients. Testing with high sensitivity assays is valuable to maximize the rate of detection.

The coming of age of liquid biopsy in neuro-oncology.

The clinical role of liquid biopsy in oncology is growing significantly. In gliomas and other brain tumours, targeted sequencing of cell-free DNA (cfDNA) from CSF may help differential diagnosis when surgery is not recommended and be more representative of tumour heterogeneity than surgical specimens, unveiling targetable genetic alterations. Given the invasive nature of lumbar puncture to obtain CSF, the quantitative analysis of cfDNA in plasma is a lively option for patient follow-up. Confounding factors may be represented by cfDNA variations due to concomitant pathologies (inflammatory diseases, seizures) or clonal haematopoiesis. Pilot studies suggest that methylome analysis of cfDNA from plasma and temporary opening of the blood-brain barrier by ultrasound have the potential to overcome some of these limitations. Together with this, an increased understanding of mechanisms modulating the shedding of cfDNA by the tumour may help to decrypt the meaning of cfDNA kinetics in blood or CSF.

Investigating the "Fetal Side" in Recurrent Pregnancy Loss: Reliability of Cell-Free DNA Testing in Detecting Chromosomal Abnormalities of Miscarriage Tissue.

(1) Background: The aim of our study is to evaluate whether cell-free DNA testing can overlap the genetic testing of miscarriage tissue in women with early pregnancy loss (EPL) and length of recurrent pregnancy loss (RPL); (2) Methods: We conducted a prospective cohort study at the Pregnancy Loss Unit of the Fondazione Policlinico Universitario A. Gemelli (IRCCS), Rome, Italy between May 2021 and March 2022. We included women with EPL and length of RPL. Gestational age was >9 weeks + 2 days and <12 weeks + 0 days of gestation corresponding to a crown rump length measurement of >25 and <54 mm. Women underwent both dilation and curettage for the collection of miscarriage tissue and for blood sample collection. Chromosomal microarray analysis (CMA) on miscarriage tissues was performed by oligo-nucleotide- and single nucleotide polymorphisms (SNP)-based comparative genomic hybridization (CGH+SNP). Maternal blood samples were analyzed by Illumina VeriSeq non-invasive prenatal testing (NIPT) to evaluate the cell-free fetal DNA (cfDNA) and the corresponding fetal fraction and the presence of genetic abnormalities; (3) Results: CMA on miscarriage tissues revealed chromosome aneuploidies in 6/10 cases (60%), consisting of trisomy 21 (5 cases) and monosomy X (one case). cfDNA analysis was able to identify all cases of trisomy 21. It failed to detect monosomy X. A large 7p14.1p12.2 deletion concomitant to trisomy 21 was, in one case, detected by cfDNA analysis but it was not confirmed by CMA on miscarriage tissue. (4) Conclusions: cfDNA largely reproduces the chromosomal abnormalities underlying spontaneous miscarriages. However, diagnostic sensitivity of cfDNA analysis is lower with respect to the CMA of miscarriage tissues. In considering the limitations when obtaining biological samples from aborted fetuses suitable for CMA or standard chromosome analysis, cfDNA analysis is a useful, although not exhaustive, tool for the chromosome diagnosis of both early and recurrent pregnancy loss.

All That Glitters in cfDNA Analysis Is Not Gold or Its Utility Is Completely Established Due to Graft Damage: A Critical Review in the Field of Transplantation.

In kidney transplantation, a biopsy is currently the gold standard for monitoring the transplanted organ. However, this is far from an ideal screening method given its invasive nature and the discomfort it can cause the patient. Large-scale studies in renal transplantation show that approximately 1% of biopsies generate major complications, with a risk of macroscopic hematuria greater than 3.5%. It would not be until 2011 that a method to detect donor-derived cell-free DNA (dd-cfDNA) employing digital PCR was devised based on analyzing the differences in SNPs between the donor and recipient. In addition, since the initial validation studies were carried out at the specific moments in which rejection was suspected, there is still not a good understanding of how dd-cfDNA levels naturally evolve post-transplant. In addition, various factors, both in the recipient and the donor, can influence dd-cfDNA levels and cause increases in the levels of dd-cfDNA themselves without suspicion of rejection. All that glitters in this technology is not gold; therefore, in this article, we discuss the current state of clinical studies, the benefits, and disadvantages.

Abstract B016: Liquid biopsy biomarker analysis of a phase II trial of sacituzumab govitecan in castrate resistant metastatic prostate cancer

Abstract Background: Sacituzumab Govitecan (SG) is an antibody-drug conjugate targeting Trop-2 and has been approved for use in breast and bladder cancers. Trop-2 has been shown to be overexpressed in multiple solid tumors including prostate cancer. Here we report an interim analysis of longitudinal liquid biopsies collected in a phase II clinical trial of SG to identify biomarkers of response and resistance. Methods: We collected longitudinal blood samples from 20 patients for circulating tumor cell (CTC) and cell-free DNA (cfDNA) analysis as part of an open label Phase II trial of SG in patients with mCRPC (ClinicalTrials.gov Identifier: NCT03725761). Eligibility criteria for the trial included rising PSA and radiographic progression on abiraterone acetate or enzalutamide in the mCRPC setting. Liquid biopsies were collected at baseline, C1D8, and then every 3 cycles until disease progression. cfDNA was sequenced using a custom 822 gene targeted capture panel (IDT). CTCs were isolated with antibodies to either EpCAM or Trop-2 followed by parallel enumeration, EpCAM and Trop-2 protein phenotyping and RNA extraction for AR/NEPC marker transcriptional profiling. Results: Although no PSA50 responses were observed, the 6-month rPFS rate was 45% (64% in chemotherapy naïve patients). For this analysis, these patients will be termed responding patients. The number of Trop-2-postive CTCs at baseline was significantly higher in responding (24.3 ±13.4 per 7.5 mL blood) than non-responding (10.0±3.8) patients. During treatment the number of Trop-2 positive CTCs in responders significantly decreased by C3D1 to 10.0 ±3.8. Interestingly, while the number of Trop-2 positive CTCs continued to decline to 2.8±1.99 cells/7.5 mL, EpCAM-positive CTCs increased from 12.5±10.52 cells/7.5 mL to 23.2±19.49 at end of trial. Gene expression analysis demonstrated high expression of genes specific to adenocarcinoma including AR-splice variants, TMPRSS2, KLK2 and KLK3 in the majority of patients, with conversion to AR-variant negative CTCs identified in responders. Preliminary cfDNA analysis identified one patient with a mutation acquired in the gene encoding topoisomerase 1 (TOP1) upon progression on SG, with additional analysis ongoing. Conclusions: Patients who responded to SG had a more Trop-2 positive CTCs with adenocarcinoma characteristics with expression of AR pathway genes. Longitudinal analysis of liquid biopsy biomarkers identified multiple mechanisms of acquired SG resistance. Decline in Trop2-CTC population with parallel expansion of EpCAM-CTC population in multiple patients suggests loss of Trop2 surface protein expression as resistance mechanism, while identification of a cfDNA TOP1 mutation in one patient at time of progression is consistent with acquired resistance to the topoisomerase inhibitor payload as has been reported in metastatic triple negative breast cancer. Citation Format: Jamie M. Sperger, Amy K. Taylor, Yue Shi, Charlotte N. Stahlfeld, Kyle Helzer, Matthew Bootsma, Katherine R. Kaufmann, Marina N. Sharifi, Christos E. Kyriakopoulos, Susan Slovin, Scott Tagawa, Scott Dehm, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis of a phase II trial of sacituzumab govitecan in castrate resistant metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B016.

Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas: a proof-of-concept study.

Analyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA). In 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up. A total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse. In summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation.

Gene body hypomethylation of pyroptosis-related genes NLRP7, NLRP2, and NLRP3 facilitate non-invasive surveillance of hepatocellular carcinoma.

Programmed cell death (PCD) resistance is a key driver of cancer occurrence and development. The prognostic relevance of PCD-related genes in hepatocellular carcinoma (HCC) has attracted considerable attention in recent years. However, there is still a lack of efforts to compare the methylation status of different types of PCD genes in HCC and their roles in its surveillance. The methylation status of genes related to pyroptosis, apoptosis, autophagy, necroptosis, ferroptosis, and cuproptosis was analyzed in tumor and non-tumor tissues from TCGA. Whole-genome bisulfite sequencing (WGBS) data of paired tumor tissue and buffy coat samples were used to filter the potential interference of blood leukocytes in cell-free DNA (cfDNA). The WGBS data of healthy individuals' and early-stage HCC patients' cfDNA were analyzed to evaluate the distinguishing ability. The average gene body methylation (gbDNAme) of pyroptosis-related genes (PRGs) was significantly altered in HCC tissues relative to normal tissues, and their distinguishing ability was higher compared to the other types of PCD-related genes. The gbDNAme of NLRP7, NLRP2, and NLRP3 was reflective of the hypomethylation in HCC tissues, and methylation levels of NLRP3 correlated positively with its expression level (r=0.51). The candidate hypomethylated PRGs could discriminate between early HCC patients and healthy controls in cfDNA analysis with high accuracy (area under the receiver operation curve, AUC=0.94). Furthermore, the hypomethylation of PRGs was associated with poor prognosis of HCC. Gene body hypomethylation of PRGs is a promising biomarker for early HCC detection, monitoring of tumor recurrence, and prognosis prediction.

Cell free DNA (cfDNA) assessment of esophagogastric (EG) cancer using MSK-ACCESS.

4036 Background: The incidence of EG cancer is rising, the treatment paradigm is increasingly complex, and the role of predictive biomarkers is critical in selecting optimal therapies. Therefore, longitudinal, non-invasive methods to characterize dynamic tumor molecular profiles, such as cfDNA, are needed. Methods: Patients with EG cancer receiving care at Memorial Sloan Kettering (MSK) had cfDNA analysis with an ultrasensitive capture based liquid biopsy for detection of somatic alterations in 129 cancer associated genes (MSK ACCESS). Tumor samples were analyzed with MSK-IMPACT, a capture based next generation sequencing (NGS) platform. Results: 100 patients with EG adenocarcinoma underwent cfDNA analysis to evaluate for molecular residual disease with MSK ACCESS. 85 (85%) had tissue NGS with MSK IMPACT. Median TMB was 5.3 mut/Mb (IQR 2.6-7.4) and median number of alterations per sample was 11 (IQR 7-17). 71% had TP53 mutations and 22% had an ERBB2 amplification (amp). 79 (79%) patients had cfDNA obtained prior to treatment (50 [63%] metastatic disease; 29 [37%] localized disease) and 21 (21%) at the time of progression. Alterations were detectable on cfDNA more frequently in patients with later stage disease (p=0.031) and fewer alterations per sample were detected compared to tissue NGS (mean difference 14, 95% CI 10-18, p&lt;0.001). The most common were TP53 (62%), CDKN2A (12%), PIK3CA (10%), ARID1A (10%), and ATM (10%). TP53 was detected with increasing frequency in patients with later stage disease (p=0.004). However, amplifications, which included ERBB2, MYC, EGFR, and FGFR2, were detected exclusively in those with stage IV disease (p=0.005) and cfDNA was less sensitive in detecting amplifications compared to tissue NGS (p&lt;0.001). This is notable as HER2 positivity is determined in part by ERBB2amp. 25 of 30 HER2+ patients had both tissue NGS and cfDNA. 19 (76%) had ERBB2amp on tissue NGS and only 8 (32%) had ERBB2amp on cfDNA (p=0.004). Additionally, copy number was lower on cfDNA compared to tissue NGS (mean difference 13, 95% CI 1.3-24.9, p=0.02) and IHC 3+ tumors were more likely to have ERBB2amp detected than IHC 2+ tumors (p=0.049). However, alterations along the MAPK, PI3K, WNT, and TGF-β pathways, which are associated with trastuzumab resistance and poor survival, were detected with similar frequency on both platforms (40 v 32%, p=0.77). Conclusions: Our findings in this large prospective analysis highlight the utility of cfDNA with MSK ACCESS as a complimentary tool for molecular characterization in patients with EG cancer. cfDNA in EG cancer. [Table: see text]

Prognostic value of TP53 mutation status in metastatic triple-negative and hormone receptor-positive breast cancer using cell-free DNA genomic profiling.

e13099 Background: TP53 is one of the most frequently mutated genes in metastatic breast cancer (MBC); however, its prognostic role remains undetermined. Plasma-based genotyping via cell-free DNA (cfDNA) is a noninvasive means of assessing the genomic landscape of MBC, accounting for heterogeneity between tumors. This study evaluated the association between TP53 alterations detected via cfDNA and clinical outcomes among patients with hormone receptor-positive HER2-negative breast cancer (HR+/HER2- BC) and triple-negative breast cancer (TNBC) in a metastatic setting. Methods: cfDNA analyses were performed on plasma samples collected from patients with TNBC and HR+/HER2- BC treated at the Massachusetts General Hospital Cancer Center and Washington University in St Louis using Guardant360, a 74-gene cfDNA sequencing assay. Progression-free survival (PFS) and overall survival (OS) were compared based on TP53 alteration status in cfDNA. Multivariable Cox regression was performed adjusting for age, de novo metastases, number of prior treatments, and immunotherapy. Results: TP53 alterations were found in 187 out of 540 patients with HR+/HER2- BC (34.6%) and 87 out of 117 patients with TNBC (74.4%). The most frequent co-existing mutations with TP53 mutations in TNBC were PIK3CA (27.6%), EGFR (26.4%), and CCNE1 (20.7%). For HR+/HER2- BC, the top co-existing mutations were PIK3CA (51.3%), ESR1 (30.5%), and EGFR (21.9%). Median PFS for patients with TNBC with and without TP53 mutations was 6.8 and 6.5 months, respectively (HR = 1.1, 95% CI 0.6-2.0, p = 0.66). Median OS for patients with TNBC with TP53 mutations was 14.2 months, whereas patients without TP53 mutations did not reach median OS (HR = 2.0, 95% CI 0.9-4.1, p = 0.08). Median PFS and OS for patients with HR+/HER2- BC were stratified by the type of therapy initiated immediately after cfDNA testing. Median PFS for patients who received endocrine therapy with and without TP53 mutations was 13.1 vs. 15.9 months, respectively (HR = 1.1, 95% CI 0.7-1.6, p = 0.78). Median OS with and without TP53 mutations was 40.6 vs. 55.8 months, respectively (HR = 1.4, 95% CI 0.8-2.3, p = 0.25). For patients with HR+/HER2- BC who received non-endocrine-based therapy following cfDNA testing, median PFS was 4.2 vs 5.6 months for those with and without TP53 mutations, respectively (HR = 1.0, 95% CI 0.6-1.7, p = 0.92). Median OS for those with TP53 mutations was 9.9 months and 17.1 months for those without (HR = 1.8, 95% CI 1-3.3, p = 0.05). Conclusions: TP53 alterations were detectable via cfDNA in patients with metastatic HR+/HER2- BC and TNBC and were associated with poor survival suggesting a potential prognostic role. The analysis did not filter out potential clonal hematopoiesis, a limitation of the study. Further study is warranted to determine the association of these mutations with outcomes in the setting of specific treatment regimens.

HERCULES: A prostate cancer (PC) sequencing panel for parallel analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs).

5036 Background: In metastatic PC, tracking genomic alterations over time and with treatment can predict disease outcomes, guide treatment, and identify novel therapeutic targets. Analysis of peripheral blood ctDNA offers a non-invasive liquid biopsy for genomic profiling, but ctDNA often comprises a small portion of total cell-free DNA (cfDNA) in the blood, requiring high depth of sequencing which may still miss PC-relevant variants. We and others have shown that single CTCs captured from the same blood sample yield additional PC-relevant genomic data when combined with ctDNA profiling, but currently there is no assay that can be applied uniformly to both analytes. Here, we present a new amplicon-based sequencing panel capable of detecting PC-relevant variants in both cfDNA and single CTCs. Methods: PC-relevant genes were curated by overlapping reviews of cBioPortal for cancer genomics, the catalogue of somatic mutations in cancer (COSMIC), and PC-relevant genomic literature. HERCULES targets 36 genes with 258 amplicons covering PC-associated mutations and copy number variations. AmpliSeq HD targeted sequencing technology was applied for analysis of both cfDNA and CTCs. Analytical validation was performed sequentially, first using AcroMetrix and Seracare DNA control samples, then single and pooled LNCap and PC3 PC cells and cfDNA, then 8 patient peripheral blood samples of matched plasma cfDNA and single CTCs captured using the RareCyte platform from participants in SWOG S1802, an ongoing CTEP/CIRB-approved phase 3 trial for men with metastatic PC. Results: In DNA control samples with allele frequencies of 1%, 0.25%, and 0.1%, HERCULES displayed sensitivity of 100%, 75%, and 50%, respectively. In matched single cells and cfDNA from cell lines, HERCULES detected 100% of database-annotated somatic variants in cfDNA and in pools of 2 or more cells, and it identified &gt;90% of variants in single cells. In 16 matched patient samples of cfDNA and single CTCs, with read coverage depths of &gt;50,000x for 20ng cfDNA and &gt;1,500x for single CTCs, HERCULES detected shared variants (mean N per sample = 6.1) as well as unique somatic variants (mean of 1.1 CTC variants not called in the cfDNA, and 4.1 cfDNA variants not called in the CTCs). Conclusions: HERCULES is, to our knowledge, the first PC-specific targeted sequencing panel capable of parallel genomic profiling of both ctDNA and CTCs. In our preliminary analytical validation, it detected somatic variants with high sensitivity in matched cfDNA and CTC samples. With additional validation, HERCULES has the potential to augment conventional ctDNA profiling by illuminating both cell-free and cellular genomic evolution during PC treatment and progression.

Fibroblast growth factor receptor 3 (FGFR3) alterations in PROOF 302: A phase III trial of infigratinib (BGJ398) as adjuvant therapy in patients (pts) with invasive urothelial carcinoma (UC).

4511 Background: Radical surgery with or without (neo)adjuvant cisplatin-based chemotherapy [(N)AC] is standard in fit pts with muscle invasive UC of bladder (UBC) or upper tract (UTUC). However, recurrence rates are high, underscoring the need for novel therapies. Since up to 70% of UTUC and 20% of UBC tumors were reported to harbor FGFR3 alterations, targeting this pathway is a rational approach. Infigratinib is a selective FGFR1–3 inhibitor with efficacy in advanced UC with FGFR3 alterations [Pal et al. 2018]. PROOF 302 investigated the efficacy and safety of adjuvant infigratinib vs placebo in pts with high-risk invasive UC. Methods: PROOF 302 was a global, randomized, double-blind, placebo-controlled, phase III trial for pts with high-risk invasive UTUC or UBC (≤15%) with FGFR3 alteration (i.e. mutation, gene fusion / translocation) with residual (≥ypT2 and/or ypN+) tumor after NAC, or pts ineligible for, or refusing, cisplatin-based AC, ≤120 days post-surgery. Pts were randomized 1:1 to infigratinib 125 mg or placebo daily on days 1–21 every 28 days for up to 52 weeks or until recurrence, unacceptable toxicity, or death. Primary endpoint: centrally reviewed disease-free survival (DFS). Secondary endpoints: investigator-assessed DFS, metastasis-free survival, overall survival, safety/tolerability. Exploratory endpoints: QOL, pharmacokinetics, genomic analysis of tumor and cfDNA. Assessment of genomic alterations was pursued using descriptive statistics. Results: We present the results of the genomic analysis of radical surgery tissues from pts screened for the trial using the FoundationOne platform. Out of 617 pts screened, 188 (30.5%) had alterations in FGFR 1-4 genes: 102/237 (43%) in UTUC, 85/369 (23%) in MIBC and 1/11 (9%) with unknown tumor origin. Of these 188 pts, median age 74 (32-90 years), 76% were male, 55% had UTUC, 44% had UBC. Genomic alterations are shown. In UTUC, FGFR3 had 56% single-nucleotide variations (SNV) and insertions/deletions (INDELS), 40% amplifications and 70% structural variants (SV). In UBC, FGFR3 had 44% SNV and INDELS, 60% amplifications and 30% SV. Conclusions: FGFR3 alterations were seen in only 19% of all pts screened for PROOF-302, a trial that was enriched for UTUC: 71/237 (30%) of UTUC and 48/369 (13%) of UBC. The trial was stopped early by the sponsor, but the genomic analysis provides insights into the prevalence of FGFR3 alterations; assessment of correlations with the primary/secondary endpoints is ongoing. The nature and frequency of co-occurring alterations in tissue samples is being investigated to help inform combination therapy strategies and putative resistance mechanisms. Clinical trial information: NCT04197986 . [Table: see text]

Harnessing olaparib, palbociclib, and endocrine therapy: A phase I/II trial of olaparib, palbociclib and fulvestrant in patients with BRCA mutation-associated, hormone receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (HOPE).

TPS1126 Background: Hormone receptor-positive (HR+) metastatic breast cancer (MBC) in patients (pts) with germline or somatic BRCA1 or BRCA2 (g/s BRCA1/2) mutations has dual drivers: hormone receptor signaling and homologous recombination deficiency. The PALOMA-3 trial showed superiority of fulvestrant (F) with palbociclib (P) compared to F alone. The OlympiAD trial showed the poly-ADP ribose polymerase inhibitor (PARPi) olaparib (O) to be superior to chemotherapy in BRCA1/2-associated MBC. However, treatment resistance inevitably develops; targeting dual therapeutic drivers concurrently may delay or circumvent resistance. O and P have overlapping hematologic toxicity and the safety of combined O, P and F is unknown. The tolerability of O + P is particularly relevant given the emergence of potentially less myelosuppressive PARPi. Methods: HOPE (NCT03685331) is a phase I/II trial to evaluate safety and efficacy of O, P and F in pts with g/s BRCA1/2-associated, HR+ MBC. Eligible pts have an ECOG performance status 0-1, measurable/evaluable breast cancer, any/no prior endocrine therapy, and 0-2 lines of chemotherapy for MBC. Prior platinum is allowed for curative intent if completed at least 12 months prior to MBC diagnosis, or for MBC if no progression during therapy. Prior PARPi and CDK4/6i are permitted on phase I, and are permitted during phase II if there was no progression on them. Treatment (28-day cycles) consists of: O orally twice daily continuously; F 500mg intramuscularly on day 1 of each cycle plus load; and P orally once daily on days 1-21. Phase I begins with a 28-day safety run-in of O and F alone. O and P doses are based on dose level (DL): DL 0 (starting level), O 300mg, P 75mg; DL 1, O 300mg, P 100mg; DL 2, O 300mg, P 125mg; DL-1, O 250mg, P 75mg and DL-2, O 200mg, P 75mg. For phase II, O and P will be dosed at MTD. Pts have tissue collection at baseline and peripheral blood cfDNA analysis at baseline, at progression, and at all scan timepoints (every 3 cycles). Phase I primary endpoint is MTD. A 3+3 dose escalation design is used with a 30% rate of dose limiting toxicity (DLT) deemed acceptable, and 6 patients treated at a dose for it to be declared MTD. A minimum of 2 and a maximum of 18 patients will be enrolled on the phase I. Phase II primary endpoint is PFS using Kaplan-Meier methods and secondary efficacy endpoints are objective response rate and 24-week clinical benefit rate. Phase II will evaluate 54 subjects to provide 80% power to detect an increase in PFS from 7 months with O monotherapy to 10 months. Exploratory objectives include examination of baseline tissue for PARPi predictive biomarkers and measures of tumor immunogenicity as well as serial serum evaluation for reversion mutations. Enrollment is ongoing. Clinical trial information: NCT03685331 .