Longitudinal Plasma Samples Research Articles

Validation of Amphiregulin As a Monitoring Biomarker during Treatment of Life-Threatening Acute Gvhd: A Secondary Analysis of 2 Prospective Clinical Trials

Background: Patients with life-threatening acute graft-versus-host disease (aGVHD) often have severe symptoms related to organ/tissue damage, elevated blood biomarkers of aGVHD, or both. Monitoring such patients can be challenging due to unpredictable variations in symptoms that may not be related to aGVHD pathogenesis, such as dietary changes, medication side effects, or infections. In our previously published phase 1 study of urinary-derived human chorionic gonadotropin/epidermal growth factor (uhCG/EGF) for the treatment of life-threatening aGVHD (NCT02525029), we recognized amphiregulin (AREG) as a potential GVHD monitoring biomarker that is elevated at the start of treatment and decreases in patients who respond to treatment. Here we show the results of AREG as a monitoring biomarker in our larger uhCG/EGF treated cohort and validate them in an independent cohort of patients with steroid-refractory aGVHD receiving ruxolitinib as second-line therapy in the REACH1 study.Methods: Longitudinal plasma samples were cryopreserved at study baseline and on study Days 7, 14, 28, and 56 from patients enrolled in both NCT02525029 (N=51) and REACH1 (N=60). In samples collected during NCT02525029, AREG was measured using enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), and ST2 and REG3a were measured using a multiplex Luminex-based array (R&D Systems). Samples from REACH1 were analyzed for concentrations of AREG, ST2, and REG3a using the ProteinSimple Ella platform (Bio-Techne, San Jose, CA). Comparisons of biomarker concentrations between response groups were performed using nonparametric one-way analysis of variance (Kruskal-Wallis test). Analyses of change of biomarkers from baseline to subsequent study days were performed using nonparametric matched pairs analysis (Wilcoxon signed rank test). Biomarker cutoffs relevant for overall survival at study baseline were identified using recursive partitioning on the NCT02525029 dataset and tested in the REACH1 dataset.Results: In patients with a complete response (CR) at Day 28 to uhCG/EGF, AREG decreased 3-fold from baseline to Day 56 (mean, 98 vs 32 pg/mL, P=0.006, Figure 1A). AREG did not significantly change over time in patients with a partial response (PR) or no response (NR) to uhCG/EGF. Similarly, in patients with a Day 28 CR to ruxolitinib, AREG decreased 2.8-fold from baseline to Day 56 (mean, 174.7 vs 63.6 pg/mL, P=0.007, Figure 1B). AREG also decreased 2.0-fold over time in patients treated with ruxolitinib who had a PR to treatment (mean baseline 288.2 vs 146.1 pg/mL at Day 56, P=0.017) but no change in those with progressive disease (PD). ST2 declined with CR, but to a lesser extent, over time in patients with a CR in both studies (1.4-fold decrease in NCT02525029, P=0.04 and 2.2-fold decrease in REACH1, P=0.021). REG3a did not significantly change over time in responders in either clinical trial. Biomarker cutoffs associated with a rapidly fatal course were identified in NCT02525029 for AREG (>212 pg/mL associated with a median survival of 62 days, P=0.006, Figure 1C), ST2 (>292 ng/mL associated with a median survival of 239 days, P<0.001), and REG3a (>13.5 ng/mL associated with a median survival of 416 days, P=0.01). These cutoffs were not statistically significant in REACH1, although patients with a baseline AREG >212 pg/mL had a median survival of 165 days vs not reached (P=0.1, Figure 1D) in REACH1.Conclusions: Using samples collected during 2 prospective clinical trials on 2 different measurement platforms, we conclude that AREG is a useful longitudinal monitoring biomarker for patients with life-threatening aGVHD. AREG levels were generally higher in REACH1 than NCT02525029, which could reflect differences in assay, severity of illness, or both. Patients with a high baseline AREG are at high risk of early death and should be monitored on a consistent platform during serial assessments. [Display omitted] DisclosuresPratta: Incyte: Current Employment. Betts: Patent Disclosures: Patents & Royalties: B.C.B. holds a patent (WO2015120436A2) related to CD4+ T cell pSTAT3 as a marker and therapeutic target of acute GVHD. B.C.B. additionally holds a provisional patent (WO2017058950A1) related to the use of JAK inhibitors for rejection and GVHD prevention. . Galvin: Incyte: Current Employment. MacMillan: Equilium: Other: DSMB member; Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy. Weisdorf: Fate Therapeutics: Research Funding; Incyte: Research Funding. Holtan: Generon: Consultancy; Incyte: Consultancy, Research Funding. OffLabel Disclosure:uhCG/EGF has orphan drug designation for the treatment of acute GVHD

Proteomic Profiling of Hypoxia-Induced Changes in Cell-Derived Extracellular Matrix From Bladder Cancer Cell Lines

The extracellular matrix (ECM) is an important component of the tumor microenvironment with key roles in cancer development, metastasis, inflammation and treatment resistance. There are synergies between a cancerous ECM and hypoxia. Hypoxia is a predominant feature of advance bladder cancer. It influences ECM remodeling, and a cancerous ECM can also promote hypoxia signaling through HIF1. We hypothesized that proteomic profiling of the hypoxic-induced ECM will identify potential new biomarkers and therapeutic targets. Our first objective was to identify differentially expressed proteins in the ECM from cells cultured in hypoxia vs normoxia and compare with proteins found in plasma from patients undergoing radiotherapy.UMUC3, J82, RT4 and T24 bladder cancer cell lines were cultured under normoxic (21% O2) and hypoxic condition (0.2% O2). Cell cultures were decellularized with NH4OH, and the cell-derived ECM (CDM) proteins recovered. CDM samples were then in-gel trypsin digested and tandem mass spectrometry performed. Experiments were performed in biological triplicate. Hypoxia-induced cellular and CDM changes were validated by western blotting. In vitro potential biomarkers were validated comparing with LC-SWATH-MS data of longitudinal plasma samples from 10 bladder cancer patients undergoing radiotherapy.Our results showed a strong influence of hypoxia on bladder CDM. The abundance of 66 out of 186 detected ECM proteins changed significantly (P < 0.05) in response to hypoxia in at least one of the cells lines. The results also highlighted diversity of CDM composition in response to hypoxia as only one protein (angiopoetin-4) was up-regulated across all four cell lines. The 66 proteins segregated samples into hypoxic and normoxic groups when analyzed with PCA and heatmap clustering. Gene ontological analysis indicated that the hypoxia-induced changes in CDM proteins have functions related to ECM structure, cell adhesion binding, integrin binding and grow factor activity. LC-SWATH analysis showed that several of the CDM proteins e.g., TGFB1, ANGPTL4, GDF5 were detectable in plasma from bladder cancer patients.The CDM composition changes in response to hypoxia. The 66 proteins identified stratified bladder cells lines according to oxygenation status and several were identified in plasma samples from bladder cancer patients. This newly identified bladder hypoxic matrisome signature will be evaluated further in plasma samples from a bladder cancer cohort as a potential biomarker for patient stratification to identify those likely to benefit from hypoxia-modifying treatments.

Circulating metabolites in progression to islet autoimmunity and type 1 diabetes

Metabolic dysregulation may precede the onset of type 1 diabetes. However, these metabolic disturbances and their specific role in disease initiation remain poorly understood. In this study, we examined whether children who progress to type 1 diabetes have a circulatory polar metabolite profile distinct from that of children who later progress to islet autoimmunity but not type 1 diabetes and a matched control group. We analysed polar metabolites from 415 longitudinal plasma samples in a prospective cohort of children in three study groups: those who progressed to type 1 diabetes; those who seroconverted to one islet autoantibody but not to type 1 diabetes; and an antibody-negative control group. Metabolites were measured using two-dimensional GC high-speed time of flight MS. In early infancy, progression to type 1 diabetes was associated with downregulated amino acids, sugar derivatives and fatty acids, including catabolites of microbial origin, compared with the control group. Methionine remained persistently upregulated in those progressing to type 1 diabetes compared with the control group and those who seroconverted to one islet autoantibody. The appearance of islet autoantibodies was associated with decreased glutamic and aspartic acids. Our findings suggest that children who progress to type 1 diabetes have a unique metabolic profile, which is, however, altered with the appearance of islet autoantibodies. Our findings may assist with early prediction of the disease.

Plasma BRAF Mutation Detection for the Diagnostic and Monitoring Trajectory of Patients with LDH-High Stage IV Melanoma.

Simple SummaryFor patients with metastatic melanoma, a rapid BRAF mutation assessment is vital to reveal the treatment options per patient. Additionally, close monitoring of the disease during treatment is essential to allow for adjustments in the treatment strategy when needed. The aim of this prospective study was to confirm the clinical validity of circulating tumor DNA (ctDNA) for minimally invasive BRAF mutation testing and treatment monitoring of metastatic melanoma patients with elevated lactose dehydrogenase serum levels. We observed that ctDNA-based BRAF mutation testing was a reliable and minimally-invasive alternative to tissue-based testing in 98% of all patients and was 100% specific. The changes in ctDNA levels during therapy appeared helpful for disease monitoring and outperformed other blood-based biomarkers in predicting treatment response.For patients with newly diagnosed metastatic melanoma, rapid BRAF mutation (mBRAF) assessment is vital to promptly initiate systemic therapy. Additionally, blood-based biomarkers are desired to monitor and predict treatment response. Circulating tumor DNA (ctDNA) has shown great promise for minimally invasive mBRAF assessment and treatment monitoring, but validation studies are needed. This prospective study utilized longitudinal plasma samples at regular timepoints (0, 6, 12, 18 weeks) to address the clinical validity of ctDNA measurements in stage IV melanoma patients with elevated serum lactate dehydrogenase (LDH > 250U/L) starting first-line systemic treatment. Using droplet digital PCR, the plasma mBRAF abundance was assessed in 53 patients with a BRAFV600 tissue mutation. Plasma mBRAF was detected in 50/51 patients at baseline (98% sensitivity; median fraction abundance of 19.5%) and 0/17 controls (100% specificity). Patients in whom plasma mBRAF became undetectable during the first 12–18 weeks of treatment had a longer progression-free survival (30.2 vs. 4.0 months; p < 0.001) and cancer-specific survival (not reached vs. 10.2 months; p < 0.001) compared to patients with detectable mBRAF. The ctDNA dynamics outperformed LDH and S100 dynamics. These results confirm the clinical validity of ctDNA measurements as a minimally invasive biomarker for the diagnostic and monitoring trajectory of patients with LDH-high stage IV melanoma.

Plasma C-Reactive Protein and Pentraxin-3 Reference Intervals During Normal Pregnancy.

Although several biomarkers are available to monitor the acute phase response, the short pentraxin C-reactive protein (CRP) is dominating in clinical practice. The long pentraxin 3 (PTX3) is structurally and functionally related to CRP, but not liver-derived. In addition, increased levels of PTX3 have been linked to preeclampsia. Reference intervals are usually based on healthy blood donors. Several physiological and immunological alterations occur during normal pregnancy with subsequent potential effects on blood analytes. Hence, this study aims to determine pregnancy-specific reference intervals for CRP and PTX3. Longitudinal clinical data and blood plasma samples from the 1st, 2nd and 3rd trimester of 100 healthy, non-medicating, females aged 18–40 at the time-point of conception were available to us. High‐sensitivity CRP measurements were performed by turbidimetry and enzyme-linked immunosorbent assay (ELISA) was used to quantify PTX3. CRP and PTX3 levels followed each other during the first two trimesters and both increased during the third trimester. CRP showed a median of 4.12 mg/L in the third trimester, and were significantly higher compared to the first (median 2.39 mg/L, p<0.0001) and the second (median 2.44 mg/L, p=0.0006) trimesters. In the third trimester PTX3 levels reached a median of 7.70 µg/L, and were significantly higher compared to the first (median 3.33 µg/L, p<0.0001) and the second (median 3.70 µg/L, p<0.0001) trimesters. Plasma albumin was inversely correlated with CRP (rho=-0.27, p<0.0001), but not with PTX3. In conclusion, it is important to consider pregnancy-specific reference values as elevations of CRP and PTX3 during the later phase may occur in absence of infection.

Abstract 582: Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma

Abstract Driver mutations in CTNNB1 are a hallmark of hepatoblastoma (HB) and offer a common biomarker for a liquid biopsy approach that is based on the presence of CTNNB1 circulating tumor DNA (ctDNA). Initial results 1 showed an association of CTNNB1 ctDNA variant allele frequency (VAF) identified through digital droplet PCR (ddPCR) and the levels of serum Alpha-fetoprotein (AFP, current HB biomarker) throughout the course of treatment of three patients with HB. However, applying custom probe-designed ddPCR assays may pose challenges for real-time data at diagnosis. Our primary objective is to investigate the utility of a universal next-generation sequencing (NGS) assay to detect CTNNB1 ctDNA in patients with HB. Our secondary objective is to compare the levels of: (1) NGS ctDNA, (2) ddPCR ctDNA, (3) AFP, and verify if they correlate with tumor burden and treatment response. We developed and tested a custom NGS assay covering exons 2 to 4 of CTNNB1 for detection of somatic mutations in plasma. We used the QIASeq platform which: (1) is based on a single primer extension and, thus, captures both single nucleotide variants (SNV) and structural variants (SV), and (2) integrates unique molecular indexing (UMI), thus allowing determination of ctDNA levels from VAF. Our cohort included 18 patients, 13 with Sanger sequencing confirmed CTNNB1 somatic mutations in the matched tumors: 6 missense SNVs and 7 deletions ranging from 96 to 348 base pairs. Our NGS assay was able to accurately detect mutations in 9/13 (69%, 7 SV and 2 SNV) of the Sanger confirmed cases, and SV and missense SNV in 2/5 (40%) cases pending orthogonal Sanger confirmation. Available ddPCR data for 3 patients (8 samples) 1 and corresponding NGS data showed similar ctDNA levels (deviation ranging between 1.2-8.5%). NGS CTNNB1 variants were detected in samples taken at initial diagnosis in 8/9 (89%), following neoadjuvant chemotherapy in 4/9 (44%), post-operatively for fully resected localized disease in 0/4 (0%), and metastatic recurrence in 1/5 (20%), suggesting a positive correlation between CTNNB1 variants and tumor burden. AFP levels were available for 24/27 samples, with abnormal levels detected in 23 samples. The ctDNA levels determined from NGS did not correlate with AFP levels; however, longitudinal plasma samples (n=9) showed similar dynamics of both ctDNA and AFP, reflecting the response to treatment. To conclude, we show that ctDNA detectable with our universal HB NGS assay is a good surrogate marker of tumor burden and shows correlation with treatment response. SV, prevalent in approximately 50% of cases of HB, may be inadequately captured and thereby misrepresented in probe-capture NGS platforms but were highly detectable in our assay. Further work is needed to understand the interplay of both AFP and ctDNA in HB monitoring. Reference: 1. Kahana-Edwin S, McCowage G, Cain L, et al. Exploration of CTNNB1 ctDNA as a putative biomarker for hepatoblastoma. Pediatr Blood Cancer. 2020. doi:10.1002/pbc.28594 Citation Format: Smadar Kahana-Edwin, Andre E. Minoche, Lucy E. Cain, Geoffrey McCowage, Sarah E. Woodfield, Sanjeev A. Vasudevan, Sarah Kummerfeld, Jonathan Karpelowsky. Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma [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 582.

Abstract 540: Molecular response evaluation of patients with metastatic colorectal cancer using circulating tumor DNA

Abstract Introduction: Patients with colorectal cancer with unresectable and isolated liver metastases (CRLM) are mostly treated with chemotherapy and targeted treatments like anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapy. Clinical response evaluation of systemic treatment is performed by radiological CT imaging, which can detect disease progression. However, CT imaging does not always give information about the viability of the tumor tissue nor does it constitute the genomic changes of the tumor, i.e. development of sub-clones following the pressure of treatment. Cell-free circulating tumor DNA (ctDNA) derived from liquid biopsies is a minimally invasive biomarker that has great potential for tumor detection and is present in relatively high levels in the plasma of patients with CRLM. Liquid biopsy ctDNA allows for longitudinal follow-up and gives the possibility to track intratumor heterogeneity caused by different sub-clones without a repeated tumor biopsy. Here, we aim to assess the use of molecular profiling using serial liquid biopsies as a biomarker for treatment response evaluation. Methods: Tumor tissue obtained prior to treatment as well as longitudinal liquid biopsies were collected from patients with CRLM who participated in a prospective clinical trial with CRC and were treated with panitumumab and doublet chemotherapy. Liquid biopsy ctDNA was isolated and analyzed by targeted sequencing using a panel of 33-genes, allowing to map dynamic molecular changes during treatment. ctDNA sequencing results were corrected for germline variants and clonal hematopoiesis variants by using targeted sequencing data of patient-matched tumor tissue DNA and white blood cell (WBC) genomic DNA, respectively. ‘Molecular response' was defined as the elimination of more than 95% of ctDNA after treatment compared to the measurement before treatment initiation. Detection of disease progression by ctDNA was compared to CT imaging. Results: At present, 110 longitudinal plasma samples as well as WBC genomic DNA and tumor tissue DNA are analyzed from a cohort of 33 patients. Currently, we are expanding the number of patients and samples. Molecular responders to treatment showed a significantly longer overall survival than non-responders (median 51 vs 25 months; p=0.033; HR=3.7). In addition, significantly earlier detection of disease progression was observed using ctDNA compared to radiological imaging (median difference of 4.8 months; p=0.006). Conclusion: Serial plasma ctDNA analyses in patients with mCRC provide a minimally-invasive tool for longitudinal treatment response evaluation of dynamic genomic alterations and creates an opportunity for patient subset selection for possible adaptation of the treatment regimen. Citation Format: Iris van 't Erve, Jamie E. Medina, Alessandro Leal, Eniko Papp, Karen Bolhuis, John K. Simmons, Samuel Angiuoli, Cornelis J. Punt, Gerrit A. Meijer, Victor E. Velculescu, Remond J. Fijneman. Molecular response evaluation of patients with metastatic colorectal cancer using circulating tumor DNA [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 540.

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

Abstract Introduction: Two metrics are commonly used to quantify tumor-specific variants in cell-free DNA (cfDNA): the variant allele frequency (VAF), defined as the ratio of the number of variant alleles to wild-type alleles, and mean tumor molecules (MTM)/mL of plasma. The main difference between the two metrics is that molecular disease levels expressed in MTMs account for the total amount of cfDNA. Changes in cfDNA levels, which occur in response to cytotoxic therapies, surgery, and inflammation, have shown to impact the detection and quantification of tumor variants in plasma, leading to misinterpretation of the test results. Here we sought to explore the relationship between VAF and MTM and evaluate the performance of each metric in monitoring molecular residual disease. Methods: We have analyzed 6569 plasma samples from 3389 patients including patients with primary diagnosis of colorectal cancer (n = 2413), breast (n = 170), pancreatic (n = 132), esophageal (n = 86), lung (n = 60), liver (n = 54), other cancers (n = 474) across multiple settings. ctDNA was quantified using a personalized and tumor-informed (bespoke mPCR NGS) assay. We examined the correlation between MTM and VAF for all ctDNA positive plasma samples (n = 1970) from 1283 patients using log-log regression. For patients with longitudinal plasma time points, further analyses on dynamic changes in MTM and VAF was performed to assess the sample-level discrepancy between the two metrics. Furthermore, the absolute values and the dynamic changes in MTM and VAF levels were plotted and compared against clinical truth based on imaging results and patient outcomes. Results: Across all samples median cfDNA concentrations were 7.9 ng/mL (range: 1.3-381.6 ng/mL). Analysis of all ctDNA positive samples revealed a positive correlation between VAF and MTM, with R2 = 0.91 (slope: 0.86). From 3180 longitudinal plasma samples, we found 2.4% (76/3180) of samples, where changes relative to the previous time point were discordant between MTM and VAF. Interestingly, cfDNA concentrations in these samples were significantly altered. In general, cfDNA levels are representative of intrinsic cell turnover. While VAF is directly related to ctDNA, it is inversely related to cfDNA levels. Increased cfDNA levels were seen in the setting of active cytotoxic therapy, perioperatively, and in the setting of advanced disease, suggesting these settings may be prone for VAF to underestimate the true ctDNA burden. Here we provide evidence that failure of VAF to account for the dynamic changes in cfDNA concentration in plasma may result in misinterpretation of the patient's disease burden. This suggests that in certain indications MTM would be more clinically relevant than VAF. Conclusion: Although both VAF and MTM are highly correlated, our analysis shows when cfDNA levels are significantly altered, MTM/mL provides a more accurate measure of molecular disease burden. Citation Format: Antony Tin, Vasily Aushev, Ekaterina Kalashnikova, Raheleh Salari, Svetalana Shchegrova, Gordon Fehringer, Meenakshi Malhotra, Harini Ravi, Himanshu Sethi, Maxim Brevnov, Bernhard Zimmermann, Angel Rodriguez, Paul R. Billings, Alexey Aleshin. Correlation of variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors [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 569.

Lipidomics study of plasma from patients suggest that ALS and PLS are part of a continuum of motor neuron disorders

Motor neuron disorders (MND) include a group of pathologies that affect upper and/or lower motor neurons. Among them, amyotrophic lateral sclerosis (ALS) is characterized by progressive muscle weakness, with fatal outcomes only in a few years after diagnosis. On the other hand, primary lateral sclerosis (PLS), a more benign form of MND that only affects upper motor neurons, results in life-long progressive motor dysfunction. Although the outcomes are quite different, ALS and PLS present with similar symptoms at disease onset, to the degree that both disorders could be considered part of a continuum. These similarities and the lack of reliable biomarkers often result in delays in accurate diagnosis and/or treatment. In the nervous system, lipids exert a wide variety of functions, including roles in cell structure, synaptic transmission, and multiple metabolic processes. Thus, the study of the absolute and relative concentrations of a subset of lipids in human pathology can shed light into these cellular processes and unravel alterations in one or more pathways. In here, we report the lipid composition of longitudinal plasma samples from ALS and PLS patients initially, and after 2 years following enrollment in a clinical study. Our analysis revealed common aspects of these pathologies suggesting that, from the lipidomics point of view, PLS and ALS behave as part of a continuum of motor neuron disorders.

Broadly neutralizing antibody responses in the longitudinal primary HIV-1 infection Short Pulse Anti-Retroviral Therapy at Seroconversion cohort.

Objective:Development of immunogens that elicit an anti-HIV-1 broadly neutralizing antibody (bnAb) response will be a key step in the development of an effective HIV-1 vaccine. Although HIV-1 bnAb epitopes have been identified and mechanisms of action studied, current HIV-1 envelope-based immunogens do not elicit HIV-1 bnAbs in humans or animal models. A better understanding of how HIV-1 bnAbs arise during infection and the clinical factors associated with bnAb development may be critical for HIV-1 immunogen design efforts.Design and methods:Longitudinal plasma samples from the treatment-naive control arm of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) primary HIV-1 infection cohort were used in an HIV-1 pseudotype neutralization assay to measure the neutralization breadth, potency and specificity of bnAb responses over time.Results:In the SPARTAC cohort, development of plasma neutralization breadth and potency correlates with duration of HIV infection and high viral loads, and typically takes 3–4 years to arise. bnAb activity was mostly directed to one or two bnAb epitopes per donor and more than 60% of donors with the highest plasma neutralization having bnAbs targeted towards glycan-dependent epitopes.Conclusion:This study highlights the SPARTAC cohort as an important resource for more in-depth analysis of bnAb developmental pathways.

Liquid biopsy in extranodal NK/T-cell lymphoma: a prospective analysis of cell-free DNA genotyping and monitoring

Satisfactory tumor material is often hard to obtain for molecular analysis in extranodal natural killer (NK)/T-cell lymphoma (NKTCL) at present. However, the accuracy and utility of circulating cell-free DNA (cfDNA) genotyping have not been adequately assessed in NKTCL. We therefore performed targeted next-generation sequencing on tumor tissues and a series of longitudinal plasma samples prospectively collected from a cohort of high-risk NKTCL patients. Concordance of genotyping results of paired baseline tumor and cfDNA and the predictive value of dynamic cfDNA monitoring were evaluated. At baseline, 59 somatic variants in 31 genes were identified in tumor and/or plasma cfDNA among 19 out of 24 high-risk NKTCL patients (79.2%). Plasma cfDNA had a sensitivity of 72.4% for detection of somatic variants identified in tumor biopsies before treatment. Plasma cfDNA also allowed the identification of mutations that were undetectable in tumor biopsies. These results were also verified in a validation cohort of an additional 23 high-risk NKTCL patients. Furthermore, longitudinal analysis showed that patients with rapid clearance of NKTCL-related mutations from plasma had higher complete remission rates (80.0% vs 0%; P = .004) and more favorable survival (1-year progression-free survival [PFS] rate, 79.0% vs 20.0%; P = .002) compared with those with persisting or emerging mutations in plasma. In addition, low cfDNA concentration before treatment was associated with favorable survival outcome for patients with NKTCL (1-year PFS, 90.0% vs 36.4%; P = .012). In conclusion, cfDNA mirrors tumor biopsy for detection of genetic alterations in NKTCL and noninvasive dynamic plasma cfDNA monitoring might be a promising approach for tracking response and survival outcome for patients with NKTCL.

Ultra-sensitive detection and quantification of HPV DNA in the plasma of patients with oropharyngeal squamous cell carcinoma (OPSCC) enrolled in the OPTIMA 2 treatment de-escalation trial.

6048 Background: Human papillomavirus (HPV) infection is a primary factor driving the increasing incidence of OPSCC. As patients with HPV+ OPSCC show significantly improved treatment response and prognosis, there is an urgent need to de-escalate treatment of HPV+ OPSCC that optimizes oncologic control while minimizing treatment-related toxicity. Cell-free HPV DNA (cfHPV-DNA) from plasma specimens represents a promising noninvasive surrogate of disease burden in these patients. To enable cfHPV-DNA analysis as a strategy to monitor response to therapy and guide treatment de-escalation, we developed a highly sensitive assay for HPV16/18 detection and quantification in plasma, based on the SafeSEQ next-generation sequencing (NGS) technology. Methods: Longitudinal plasma samples were collected from patients with locoregional HPV+ OPSCC treated on our institutional de-escalation protocol of induction chemoimmunotherapy followed by risk/response stratified de-escalated locoregional therapy, OPTIMA 2 (NCT03107182). Neck CT or MRI was obtained for all patients at baseline and following induction chemoimmunotherapy; radiographic response to induction therapy was assessed per RECIST 1.1 criteria. cfHPV-DNA was quantified in plasma samples collected at baseline and at the end of induction therapy. Changes in cfHPV-DNA levels were correlated with radiographic response. Results: The SafeSEQ HPV assay demonstrates high analytical sensitivity, with ability to detect a single copy of HPV DNA. Replicate testing of contrived samples containing HPV 16/18 DNA at defined levels revealed robust quantitative detection across a dynamic range over 5 orders of magnitude. The assay showed a low level of background signal ( &lt; 0.04 copies per sample) across 20 healthy donor samples, indicating high specificity. In plasma samples collected at baseline from patients enrolled in OPTIMA 2, cfHPV-DNA was detected at levels ranging from 1 to &gt; 30,000 copies/ml. A high correlation was observed between dynamic changes in patients’ cfHPV-DNA levels and radiographic responses following induction therapy. Furthermore, in samples collected longitudinally during induction therapy, changes in cfHPV-DNA levels accurately tracked radiographic responses to therapy. Conclusions: We have developed a highly sensitive and specific cfHPV-DNA detection assay based on SafeSEQ NGS technology and have successfully applied it to monitor therapeutic response in HPV+ OPSCC patients. The assay exhibits robust quantitative detection of HPV across a broad range of levels, even when only a few copies are present, enabling high-resolution molecular monitoring. Prospective studies are underway to further evaluate the kinetics of cfHPV-DNA as a predictor of response to therapy in order to more precisely guide the management of patients with HPV+ OPSCC.

Circulating Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients.

SARS-CoV-2 proteins were measured in longitudinal plasma samples collected from 13 participants who received two doses of mRNA-1273 vaccine. 11 of 13 participants showed detectable levels of SARS-CoV-2 protein as early as day one after first vaccine injection. Clearance of detectable SARS-CoV-2 protein correlated with production of IgG and IgA.

TP53-Mutated Circulating Tumor DNA for Disease Monitoring in Lymphoma Patients after CAR T Cell Therapy.

Chimeric antigen receptor T (CAR T) cell immunotherapy has shown remarkable efficacy in non-Hodgkin’s lymphoma (NHL) patients. Minimal residual disease (MRD) monitoring in NHL is essential after CAR T cell therapy, which can be achieved by monitoring circulating tumor DNA (ctDNA). The mutation of TP53 in NHL has been suggested to be associated with a poor prognosis. However, whether TP53-mutated ctDNA can be used as a biomarker remains undetermined. In this study, a total of 40 patients with mutated TP53 who received CAR T cell treatment were analyzed, and specific probes targeting 29 different TP53 mutation sites in the 40 patients were designed and verified. Then, the presence of TP53-mutated ctDNA in longitudinal plasma samples was tracked by droplet digital PCR. Patients were stratified into two groups, favorable or unfavorable, based on their highest ctDNA level using a MAF cutoff of 3.15% according to the ROC curve. The unfavorable group had significantly worse PFS than the favorable group (p < 0.001). Our results suggest that patients with mutated TP53 with a favorable ctDNA profile in the first trimester have better prognostic outcomes than patients with an unfavorable profile, and ctDNA can be a reliable predictor of the subsequent clinical outcome.

SARS-CoV-2 RNAemia Predicts Clinical Deterioration and Extrapulmonary Complications from COVID-19.

BackgroundThe determinants of coronavirus disease 2019 (COVID-19) disease severity and extrapulmonary complications (EPCs) are poorly understood. We characterized relationships between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNAemia and disease severity, clinical deterioration, and specific EPCs.MethodsWe used quantitative and digital polymerase chain reaction (qPCR and dPCR) to quantify SARS-CoV-2 RNA from plasma in 191 patients presenting to the emergency department with COVID-19. We recorded patient symptoms, laboratory markers, and clinical outcomes, with a focus on oxygen requirements over time. We collected longitudinal plasma samples from a subset of patients. We characterized the role of RNAemia in predicting clinical severity and EPCs using elastic net regression.ResultsOf SARS-CoV-2–positive patients, 23.0% (44 of 191) had viral RNA detected in plasma by dPCR, compared with 1.4% (2 of 147) by qPCR. Most patients with serial measurements had undetectable RNAemia within 10 days of symptom onset, reached maximum clinical severity within 16 days, and symptom resolution within 33 days. Initially RNAemic patients were more likely to manifest severe disease (odds ratio, 6.72 [95% confidence interval, 2.45–19.79]), worsening of disease severity (2.43 [1.07–5.38]), and EPCs (2.81 [1.26–6.36]). RNA loads were correlated with maximum severity (r = 0.47 [95% confidence interval, .20–.67]).ConclusionsdPCR is more sensitive than qPCR for the detection of SARS-CoV-2 RNAemia, which is a robust predictor of eventual COVID-19 severity and oxygen requirements, as well as EPCs. Because many COVID-19 therapies are initiated on the basis of oxygen requirements, RNAemia on presentation might serve to direct early initiation of appropriate therapies for the patients most likely to deteriorate.

Circulating Tumor DNA Reflects Uveal Melanoma Responses to Protein Kinase C Inhibition.

Simple SummaryUveal melanoma (UM) is a rare cancer, with no effective standard systemic therapy in the metastatic setting. Over 95% of UM harbor activating driver mutations that can be detected in the circulation. In this study, circulating tumor DNA (ctDNA) was measured in 17 metastatic UM patients treated with protein kinase C inhibitor (PKCi)-based therapy. ctDNA predicted response to targeted therapy and increasing UM ctDNA preceded radiological progression with a lead-time of 4–10 weeks. Next generation sequencing (NGS) of ctDNA also identified prognostic and treatment resistance mutations. Longitudinal ctDNA monitoring is useful for monitoring disease response and progression in metastatic UM and is a valuable addition to adaptive clinical trial design.The prognosis for patients with UM is poor, and recent clinical trials have failed to prolong overall survival (OS) of these patients. Over 95% of UM harbor activating driver mutations, and this allows for the investigation of ctDNA. In this study, we investigated the value of ctDNA for adaptive clinical trial design in metastatic UM. Longitudinal plasma samples were analyzed for ctDNA in 17 metastatic UM patients treated with PKCi-based therapy in a phase 1 clinical trial setting. Plasma ctDNA was assessed using digital droplet PCR (ddPCR) and a custom melanoma gene panel for targeted next generation sequencing (NGS). Baseline ctDNA strongly correlated with baseline lactate dehydrogenase (LDH) (p < 0.001) and baseline disease burden (p = 0.002). Early during treatment (EDT) ctDNA accurately predicted patients with clinical benefit to PKCi using receiver operator characteristic (ROC) curves (AUC 0.84, [95% confidence interval 0.65–1.0, p = 0.026]). Longitudinal ctDNA assessment was informative for establishing clinical benefit and detecting disease progression with 7/8 (88%) of patients showing a rise in ctDNA and targeted NGS of ctDNA revealed putative resistance mechanisms prior to radiological progression. The inclusion of longitudinal ctDNA monitoring in metastatic UM can advance adaptive clinical trial design.

Significant compartment-specific impact of different RNA extraction methods and PCR assays on the sensitivity of hepatitis E virus detection.

RNA detection in plasma/stool is the gold-standard for diagnosis of hepatitis E virus (HEV) infection. The impact of viral extraction methods on HEV RNA detection is poorly investigated. We determined the limit of detection of the RealStar HEV RT-PCR V2.0 Kit (altona Diagnostics, RS) utilizing 3 RNA extraction methods (COBAS® AmpliPrep Total Nucleic Acid Isolation Kit, TNAi Roche; MagNA Pure 96 DNA, Viral NA SV Kit, MgP; QIAamp Viral RNA mini Kit Qiagen; VRK) in plasma and stool. The most sensitive method was evaluated in a total of 307 longitudinal samples of patients with HEV infection (acute=18/chronic=36) and compared to results with the former diagnostic standard of our centre (TNAi/FastTrack Diagnostic; FTD). The plasma-LOD was 49, 94 and 329IU/mL for extraction with MgP, VRK and TNAi respectively. In stool, the LOD was 21IU/mL, 528IU/mL and indefinable for extraction with TNAi, VRK and MgP respectively. Utilizing longitudinal patient plasma samples, MgP/RS revealed 56 HEV RNA-positive samples in 158 negative samples as determined by TNAi/FTD. In stool, from 37 HEV negative samples (TNAi/FTD), 15 were positive with TNAi/RS. At end of treatment, 8 out of 27 chronically infected patients were RNA positive with MgP/RS, while classified negative with TNAi/FTD. A relapse occurred in 3 of these patients. Different methods for RNA extraction and quantification have a significant, compartment-specific impact on the sensitivity of HEV detection. Knowledge about the favourable combinations of extraction and quantification has important implications for diagnosis and patients receiving antiviral therapy.

Abstract P809: A Comprehensive P75 Neurotrophin Receptor Gene Network and Pathway Analyses Identifying New Target Genes for Stroke Recovery

Introduction: Therapeutic interest into the neurotrophins resides in their ability to regulate the process of neuronal and synaptic regeneration following acute brain injuries such as intracerebral hemorrhage, subarachnoid hemorrhage and ischemic stroke. P75 neurotrophic receptor (p75NTR) is an important receptor for the role of neurotrophins in modulating brain plasticity and apoptosis. The current understanding of the role of p75NTR in cellular adaptation following pathological insults remains blurred. Methods: We identified p75NTR and related genes through extensive data mining of a PubMed literature search including published works related to p75NTR from the past 20 years. Bioinformatic network and pathway analyses of identified genes (n=235) were performed using ReactomeFIViz in Cytoscape based on the highly reliable Reactome functional interaction network algorithm. This approach merges interactions extracted from human curated pathways with predicted interactions from machine learning. Results: Genome-wide pathway analysis showed total of 16 enriched hierarchical clusters. A total of 278 enriched single pathways were also identified (p&lt;0.05, FDR corrected). Gene network analyses showed multiple known and new targets in the p75NTR gene network. This study provides a comprehensive analysis and investigation into the current knowledge of p75NTR signaling networks and pathways. Discussion: This study provides the largest comprehensive gene and functional network library of p75NTR and incorporates current knowledge using a large dataset approach that increases the overall understanding of complex p75NTR networks. These results suggest both new possible target genes for further investigation in p75NTR research, while also validating previously conducted research. These results can be used to generate novel hypotheses to gain a greater understanding of p75NTR in stroke. Future directions: We are currently sequencing miRNA libraries from ischemic and hemorrhagic stroke patients’ plasma with longitudinal plasma samples and clinical data. This data together with presented results will be used to test identified novel targets in p75NTR engineered models in order seek novel therapeutic strategies to increase recovery after stroke.

Antibody affinity maturation and plasma IgA associate with clinical outcome in hospitalized COVID-19 patients

Hospitalized COVID-19 patients often present with a large spectrum of clinical symptoms. There is a critical need to better understand the immune responses to SARS-CoV-2 that lead to either resolution or exacerbation of the clinical disease. Here, we examine longitudinal plasma samples from hospitalized COVID-19 patients with differential clinical outcome. We perform immune-repertoire analysis including cytokine, hACE2-receptor inhibition, neutralization titers, antibody epitope repertoire, antibody kinetics, antibody isotype and antibody affinity maturation against the SARS-CoV-2 prefusion spike protein. Fatal cases demonstrate high plasma levels of IL-6, IL-8, TNFα, and MCP-1, and sustained high percentage of IgA-binding antibodies to prefusion spike compared with non-ICU survivors. Disease resolution in non-ICU and ICU patients associates with antibody binding to the receptor binding motif and fusion peptide, and antibody affinity maturation to SARS-CoV-2 prefusion spike protein. Here, we provide insight into the immune parameters associated with clinical disease severity and disease-resolution outcome in hospitalized patients that could inform development of vaccine/therapeutics against COVID-19.

Liquid biopsy by combining 5-hydroxymethylcytosine signatures of plasma cell-free DNA and protein biomarkers for diagnosis and prognosis of hepatocellular carcinoma

BackgroundLiquid biopsy based on 5-hydroxymethylcytosine (5hmC) signatures of plasma cell-free DNA (cfDNA) originating from tumor cells provides a novel approach for early diagnosis in hepatocellular carcinoma (HCC). Here, we sought to develop a reliable model using cfDNA 5hmC signatures and protein biomarkers for diagnosis and prognosis of HCC.Patients and methodsWe carried out genome-wide 5hmC sequencing of cfDNA samples collected from 165 healthy volunteers, 62 liver cirrhosis (LC) patients and 135 HCC patients. A sensitive 5hmC diagnostic model was developed based on 5hmC signatures selected by sparse Partial Least Squares Discriminant Analysis and cross-validation to define the weighted diagnostic score (wd-score). Then, we combined protein biomarkers with the wd-score to build a more robust score (HCC score) by logistic regression.ResultsThe distribution pattern of differential 5hmC regions could clearly distinguish HCC patients, LC patients and healthy volunteers. The wd-score based on 64 5hmC signatures in cfDNA achieves 93.24% of area under the curve (AUC) to distinguish HCC patients from non-HCC patients, and the HCC score by combing protein biomarkers achieves 92.75% of AUC to distinguish HCC patients from LC patients. Meanwhile, the HCC score showed high capacity for screening high recurrence risk patients after receiving surgical resection, and appeared to be an independent indicator for both relapse-free survival (P = 0.00865) and overall survival (P = 0.000739). Furthermore, the values of the HCC score in patients' longitudinal plasma samples were positively associated with tumor burden dynamics during follow-up.ConclusionWe have developed and validated a novel non-invasive liquid biopsy strategy for HCC diagnosis, prognosis and surveillance during HCC progression.