Liquid biopsy testing in metastatic or advanced breast cancer patients during the COVID-19 pandemic

Objective: During the COVID-19 pandemic, cancer patients had restricted access to standard of care tissue biopsy. Liquid biopsy assays using next generation sequencing technology provides a less invasive method for determining circulating tumour mutations (ctDNA) associated with targeted treatments or prognosis. As part of deploying technology to help cancer patients obtain molecular testing, a clinical program was initiated to offer liquid biopsy testing for Canadian patients with advanced or metastatic breast cancer. Method(s): Blood was drawn in two 10 mL StreckTM DNA BCTs and sent to the CAP/CLIA/DAP accredited Imagia Canexia Health laboratory for testing using the clinically validated Follow ItTM liquid biopsy assay. Plasma was isolated using a double spin protocol and plasma cell-free DNA (cfDNA) extracted using an optimized Promega Maxwell RSC method. Extracted cfDNA was amplified using the multiplex amplicon-based hotspot 30 or 38 gene panel and sequenced. An inhouse developed bioinformatics pipeline and reporting platform were used to identify pathogenic single nucleotide variants (SNVs), indels (insertions and deletions), and gene amplification. Included in the panel are genes associated with metastatic breast cancer: AKT1, BRAF, ERBB2, ESR1, KRAS, PIK3CA, TP53. Result(s): To identify biomarkers, 1214 metastatic or advanced breast cancer patient cfDNA samples were tested. There were 15 cases sent for repeat testing. We reported 48% of samples harboring pathogenic ctDNA mutations in TP53 (22%), PIK3CA (19%), ESR1 (18%), AKT1 (2%), ERBB2 (1.5%). Co-occurring variants were identified in samples with ESR1/PIK3CA as well as TP53/PIK3CA (both p-values <0.001). Interestingly, 29% of samples with mutated ESR1 harbored >= 2 ESR1 ctDNA mutations. In 56% of cases, previous molecular testing indicated the cancer subtype as hormone receptor (ER, PR) positive with/without HER2 negative status. In this specific subgroup, 49% harbored ctDNA mutations with 63% of those being PIK3CA and/or ESR1 mutations. Conclusion(s): A population of Canadian women with metastatic breast cancer were tested using a liquid biopsy gene panel during the COVID-19 pandemic for identification of biomarkers for targeted therapeutic options. Over 50% of the samples were identified as hormone positive, with greater than 60% harboring PIK3CA and ESR1 ctDNA mutations. Studies have shown that metastatic PIK3CA mutated ER-positive/HER2-negative tumors are predictive to respond to alpelisib therapy and have FDA and Health Canada approval. Additionally, ESR1 mutations are associated with acquired resistance to antiestrogen therapies, and interestingly we identified 29% of ESR1 mutated samples with multiple mutations possibly indicating resistance subclones. In future studies, longitudinal monitoring for presence of multiple targetable and resistance mutations could be utilized to predict or improve clinical management.

A Case Report Of The Effects Of Chemotherapy On Post-heart-transplant Gene Expression Profiling

Introduction: Malignancies are a major complication of heart transplant (HT). Noninvasive surveillance after HT using gene expression (GEP) profiling and donor derived cell free DNA (dd-cfDNA) are noninferior to biopsy and are widely utilized. The interpretation of % dd-cfDNA, is not well understood in malignancies with a conceptual increase in the recipient fraction. The effect of chemotherapy on GEP in the setting of post-HT surveillance has not been described to the best of our knowledge. Hypothesis: Induction of chemotherapy will cause global transcriptional reduction in GEP. Method(s): GEP was performed with AlloMap (AM, CareDx), which evaluates expression levels of 11 mononuclear cell genes, involved in lymphocyte activation, T-cell priming, cell migration, hematopoietic proliferation, steroid sensitivity, and platelet activation. Scores range from 0-40, higher scores have a stronger correlation with rejection. At our center a total of 995 draws were analyzed from 2019-2022. In parallel dd-cfDNA, which informs about graft injury was analyzed using AlloSure (AS, CareDx). Case Events: A 71-year-old male HT recipient for nonischemic cardiomyopathy and no rejection history was diagnosed with metastatic gastric adenocarcinoma at 16 months post-HT. Following diagnosis, mycophenolic acid was stopped, prednisone 5 mg was started, and tacrolimus trough goal was gradually lowered to 4-6 given infectious complications. Palliative chemotherapy with folinic acid, fluorouracil (5-FU), oxaliplatin (FOLFOX) was initiated at 18 months post-HT with planned dose reduction of oxaliplatin and holding of 5-FU bolus to reduce risk of myelosuppression given comorbidities. Oxaliplatin was stopped at 18 months post HT. Due to COVID he last received 5-FU at 33 months post-HT. Graft function remained stable and DSA negative. At 36 months post-HT, he developed a bowel obstruction without surgical options for interventions and expired shortly thereafter. Result(s): With initiation of prednisone and following chemotherapy there was a drastic decrease in AM scores (Fig. A). Steroid therapy led to an 18% decline in AM scores, the greatest decrease occurred with chemotherapy, with 67% decline from the mean when compared to all center patients (Fig B). Dd-cfDNA levels remained stable during the course aside from one early elevation. Conclusion(s): To the best of our knowledge this is the first published case on the effect of chemotherapy on GEP profiling in the setting of post-HT surveillance. This case advises caution when interpreting GEP in the setting of chemotherapy showing great reduction in GEP scores. While dd-cfDNA levels remained relatively stable after malignancy diagnosis and treatment initiation further studies will need to inform on the use of both GEP and dd-cfDNA in these patients.Copyright © 2022

Circulating cfDNA is an excellent marker of severity in Covid-19

Introduction: LDH is released by cytokine-mediated tissue damage, reflecting injury and possible organ dysfunction. It is associated with severity and mortality in patients with Covid-19. Circulating free DNA (cfDNA) is a marker of tissue damage, released by the nucleus and mitochondria after cell destruction. Recent studies have showed its possible role as a marker of tissue damage in Covid-19. Method(s): Prospective, observational, longitudinal study of 91 hospitalized patients with different degrees of severity. The evolution distinguished viral, early inflammatory and late inflammatory phases. Clinical data, immune cells, proinflammatory cytokine levels(TNF-alpha,IL-1beta,IL-8,IL-6,INF-gamma,IL-17A andG-CSF),serum inflammatory markers (CRP,PCT,D-dimer,ferritin) and tissue damage markers (LDH and cfDNA) were included. Result(s): LDH levels increased in parallel with severity, but did not discriminate mortality in the first sample and only showed significantly higher levels in critically ill patients. The IL-6/LDH correlation was close and significant in all degrees of severity and at all stages of the disease. The cfDNA value was higher in more severe and patients who died, and was the only biomarker that remained higher in these patients during the 3 phases of the disease. The multivariate study showed that cfDNA, and not IL-6, was an independent risk factor for critical status and ICU admission. Conclusion(s): cfDNA is an excellent marker of tissue damage, severity and mortality in Covid-19 disease, better than LDH. The IL-6/LDH association seems to reflect better timely inflammation, and cfDNA probably reflects better the extent of tissue damage.

Covid-19 and NETs

Introduction: Neutrophil extracellular traps (NETs) have recently been linked to an important role in the pathogenesis of Covid-19. Method(s): Prospective observational study of 91 hospitalized patients. We studied longitudinally the viral phase, early inflammatory and late, and the 4 most specific components of NETs: cell free-DNA (cfDNA), MPO-DNA and NE-DNA complexes and citrullinated Histone 3 (citH3). Result(s): We observed elevated levels vs controls of MPO-DNA and NE-DNA complexes and cfDNA at admission and in the 3 phases of the disease. CitH3 was elevated from the early inflammatory phase onwards. There was a significant correlation in survivors (r=0.798) and in all severity degrees between MPO and NE and between cfDNA and H3 cit (r=0.3), but not in the rest of combinations among the 4, nor in dead patients. We did not observe any correlation in any group between MPO or NE with citH3. There was an increase of only cfDNA levels in more severe patients. The area under the ROC curve for critical severity and mortality was high for cfDNA (0.7327 and 0.7482) and much poorer for the other 3 NETs markers. Conclusion(s): -We found evidence of neutrophil activation of NETs components in Covid-19, during the 3 phases of the disease, but without a clear relationship with severity and mortality. -cfDNA was related to severity and mortality, and its sources appeared to be more related to tissue damage than to NETs -The best correlation between them was MPO-NE, and these more neutrophil-specific markers reflect probably better NET formation. NETs role has maybe been overestimated using other less specific markers.

Impact of Sars-CoV-2 Vaccination on Donor-Derived Cell Free DNA Levels in Renal Transplant Recipients

Purpose: COVID-19 poses a disproportionate threat to renal transplant recipients (RTR), who are chronically immunosuppressed. Studies have indicated a 16% mortality rate compared to <5% for the general population. Effective vaccines (Pfizer, Moderna and, Johnson & Johnson) provided hope for protection against severe COVID-19 in this at-risk population. However, based on experience with vaccines against other viral infections, two primary concerns arose: 1) would the SARS-CoV-2 vaccines be effective in this population;2) could these vaccines provoke rejection? Methods: To address these questions, we tested serum creatinine, anti-SARS-CoV-2 S antibody (Roche ElecsysR), Donor Specific anti HLA Antibodies, other antibodies against SARS-CoV-2, other coronaviruses (LABScreenTMCOVID Plus, One Lambda), and donor derived cell free DNA (dd-cfDNA;fraction, absolute and total quantity, using the ProsperaTM Test, Natera, Inc.) in RTR at the time of vaccine doses 1 and 2 and 1, 3, and 6 months after the second dose. dd-cfDNA >=1% and 78 cp/ mL indicated an increased risk of rejection. 53 patients were consented and enrolled in the study. This study received IRB approval. Statistical analysis was performed using paired two-tailed student's t-test. Result(s): This preliminary analysis analyzed the impact of vaccination on dd-cfDNA levels in 31 RTR patients. This cohort was primarily female (67%) and of hispanic descent (48.3%) with a median age 55 years (range: 19-81). All but 1 patient received the Pfizer vaccination series. Mean time from transplant to vaccination 1 was 114.6 months (range: 10-359 months). Between vaccination 1 and 2, no patients had clinical suspicion of rejection, were hospitalized or underwent for-cause biopsy. No significant differences in dd-cfDNA or total cf-DNA levels were found by Prospera testing between vaccination 1 and 2. (Table 1). Between vaccination 1 and 2, one patient had an increase dd-cfDNA% above the normal range (0.14%, 2.37%), but absolute dd-cfDNA quantity remained in normal range (13.70 cp/mL, 66.08 cp/ mL). At the time of the vaccination 1, dd-cfDNA% was elevated in 2 patients. At vaccination 2, dd-cfDNA% had returned to the normal range for one patient (ddcfDNA quantity was normal for both vaccinations), while both dd-cfDNA% and quantity remained elevated in the other. Conclusion(s): Based on measurement of dd-cfDNA fraction, absolute quantity and total quantity with the Prospera test at vaccination 1 and 2, there was no evidence of SARS-CoV-2 vaccination-induced rejection.

Impact of the Covid-19 Pandemic on Longitudinal Molecular Surveillance in the KOAR Registry

Purpose: The SARS-CoV2 pandemic increased the complexity of delivering routine clinical care and laboratory services for immunosuppressed kidney transplant (KTx) recipients. We evaluated how the pandemic impacted adherence with scheduled laboratory draws among patients enrolled in the Kidney allograft Outcomes AlloSure Registry (KOAR, NCT03326076). Method(s): 1663 kidney transplant (KTx) recipients undergoing post-transplant surveillance using donor-derived cell-free DNA (dd-cfDNA, AlloSure, CareDx Inc.) were enrolled in KOAR between 2017 and 2021. Participating centers were free to individualize their surveillance strategies. We estimated adherence by using the pre-pandemic distribution of surveillance dd-cfDNA draws across participating sites to establish a baseline regimen, and then compared adherence before the pandemic (P1;through 1/2020) with two subsequent periods in 2020: P2 (2/2020 - 6/2020), coinciding with the first wave of infections, and P3 (7/2020 - 12/2020), which captures the bulk of the second and third waves in the US. Result(s): The distribution of surveillance dd-cfDNA draws at participating sites before COVID (P1) identified 7 peaks corresponding to draw points at or around months 1, 2, 3, 4, 6, 9, and 12 [Figure 1a]. Estimated adherence during P1 based on this regimen was 60.5%. Over the subsequent 5 months (P2), reflecting the early months of the pandemic, adherence declined to 50.5% (p < 0.01). After the expanded availability of mobile phlebotomy services in 7/2020 and despite rising SARS-CoV2 case counts and hospitalizations, adherence during P3 improved to 57.6% (p < 0.01 compared to P2, p = 0.1 compared to P1) [Figure 1b]. Conclusion(s): Our findings demonstrate that adherence to laboratory surveillance among transplant recipients enrolled in the KOAR registry declined in the early period of the SARS-CoV2 pandemic, however, a variety of adaptations in the latter half of 2020, including the widespread availability of remote phlebotomy for these patients, appears to have led to substantial improvements, with adherence approaching pre-pandemic levels.

Third Dose of SARS-CoV-2 mRNA Vaccines Significantly Increases Delta (B.1.617.2) Variant Anti-Spike Antibody Levels in Kidney Transplant Recipients

Purpose: Emerging SARS-CoV-2 variants may be associated with a higher risk of breakthrough infections compared to wild-type (WT) virus in kidney transplant recipients (KTRs). The purpose of this study was to evaluate antiviral immune responses against WT and Delta (B.1.617.2) variant of SARS-CoV-2 after 3 doses of SARS-CoV-2 mRNA vaccines in KTRs. Method(s): We conducted a multicenter prospective cohort study of adult KTRs who received 3 doses of BNT162b2 or mRNA-1273. Blood samples were collected from KTRs before and 4 weeks after the 3rd vaccine dose. Sera from pre-pandemic healthy controls (HCs) and pre-pandemic kidney transplant control patients (KCs) were used for comparison. A Luminex-based multiplex assay was used to measure anti-spike antibodies for the WT, Alpha, Beta, Gamma and Delta variants of SARSCoV- 2. A surrogate virus neutralization test was used to assess neutralization against the WT and Delta variant. Patients were also monitored for rejection using several non-invasive biomarkers. Result(s): 54 KTRs were enrolled in the study. The median age was 63, 44% were female and the median time post-transplantation was 42 months. 94% received BNT162b2 vaccine. After the 3rd vaccine dose, there was a significant increase in anti-spike antibody MFIs against the WT, Alpha, Beta, Gamma and Delta variants (Fig. 1A, p<0.0001 for all). For comparison, all pre-pandemic HCs and KCs had a negative result for anti-spike antibody levels (Fig. 1B). Prior to the 3rd vaccine dose, 29% of KTRs had anti-spike antibodies against the WT compared to only 2% against the Delta variant (Fig. 1C, p=0.0001). After the 3rd vaccine dose, 67% of KTRs had anti-spike antibodies against the WT compared to 25% against the Delta variant (p<0.0001, Fig. 1D). Differences between WT and other variants are shown in Figure 1C-D. After the 3rd vaccine dose, there was a 2.1-fold and 2.5-fold increase in the percentage of KTRs with neutralizing responses against the WT and Delta variant respectively (p<0.0001 for both). There was no significant change in serum creatinine, proteinuria, or donor-derived cell-free DNA levels after vaccination. No episodes of rejection occurred during follow-up. Conclusion(s): Two doses of SARS-CoV-2 mRNA vaccines in KTRs are associated with minimal anti-spike antibody response directed against the Delta variant of SARS-CoV-2. After the third dose, a quarter of KTRs developed anti-spike antibodies directed against the Delta variant of SARS-CoV-2.

Infection Impacts dd-cfDNA in Lung Transplant Recipients

Purpose: Infection is the leading cause of death within 1 year post lung transplant. Graft injury secondary to infection is affected by both source and organism. Donor derived cell-free DNA (dd-cfDNA) is a consistent marker of graft injury, but previously reported dd-cfDNA levels with infections have been inconsistent. We compared dd-cfDNA concentrations across different infection types. Method(s): We reviewed infections in lung transplant recipients (LTR) between 5/2019-6/2021 with paired dd-cfDNA at time of infection. All were confirmed infections (i.e. requiring therapy). Infection source (respiratory vs non-respiratory) and organism were collected. Samples were excluded if there was concurrent ACR, AMR or CLAD at time of dd-cfDNA. The primary endpoint was dd-cfDNA levels across cohorts. Result(s): Fifty paired samples from 20 LTR were identified;31 samples were excluded due to concurrent diagnoses. Infections included viral (n=18, 36%), bacterial (n=18, 36%), and fungal (n=10, 20%). Four cultures (8%) had multiple organisms. Most common within each group were CMV (n=4) and COVID (n=4) for viral, Pseudomonas aeruginosa (n=4) for bacterial, and Aspergillus (n=7) for fungal. Median dd-cfDNA was 1.30% in viral infections, 1.93% in bacterial, and 0.99% in fungal;respiratory infections (n=42) was 1.42% and 0.95% in non-respiratory (n=8). Conclusion(s): There was a statistically significant increase in dd-cfDNA between each infection compared to a normal cohort, but no statistical differences between infection groups. The trend towards significance of respiratory vs non-respiratory indicates that dd-cfDNA may be a useful marker of injury specific to the graft caused by infection. Further investigation with serial samples prior to and following treatment of the infection will be important to better understand this trend. (Figure Presented).

COURSE AND OUTCOMES OF COVID-19 INFECTIONS IN LUNG TRANSPLANT RECIPIENTS

SESSION TITLE: COVID-19 Infections: Issues During and After Hospitalization SESSION TYPE: Original Investigations PRESENTED ON: 10/17/2022 01:30 pm - 02:30 pm PURPOSE: It has been established that recipients of solid organ transplants have worse outcomes compared to the general population from COVID-19 infections. We sought to determine the course and outcomes of lung transplant recipients (LTR) with COVID-19 infections based on vaccination status and treatments. METHODS: We performed a retrospective study of all LTR from Inova Fairfax Hospital with COVID-19 infections. Infection was confirmed based on symptoms and testing from an urgent care, hospital, or home kit. Patients with presumed but unconfirmed COVID-19 infections were excluded. The study timeframe was the two-year period: 3/1/2020 - 2/28/2022. Data collected included patient demographics, transplant type, immunosuppression, immunization status, episodes of rejection, donor derived cell-free DNA (dd-cfDNA) values (where available), spirometric data, outpatient/inpatient treatments, hospitalization data, and outcomes including death, infections, and other complications. The severity of illness was based on the 8-point ordinal scale. RESULTS: There were 45 LTR who tested positive during the study period;22 male and 23 female, average age of 57 and mean time from transplant of 4 years. 11 of the patients were unvaccinated (UV), 2 partially vaccinated (PV), 11 vaccinated non-boosted (VNB), and 21 vaccinated and boosted (VB). In total, 34 (76%) LTR required hospitalization. Of those hospitalized: 7 UV, 1 PV, 11 VNB, and 15 FV. In addition, 7 of those hospitalized required intubation with only 1/7 surviving to discharge. Overall, 8/45 (17.8%) patients died from COVID-19: 3 UV, 1PV, 1 VNB, 3VB. Infectious complications included 3 cases of PCP, 1 empyema, and 1 reactivation of CMV. For individuals who had spirometry at least 2 weeks after diagnosis (n =25), FVC decreased in 17 LTR by an average of 0.17 L, the FEV1 decreased in 14 LTR by an average of 0.14 L. On repeat spirometry testing (n=14), FVC further decreased in 9 LTR by 0.25 L and the FEV1 further decreased in 7 LTR by 0.13 L. CONCLUSIONS: A large proportion of LTR with COVID-19 infections require hospitalization (76%) with a high associated mortality rate and a sustained lung function decline seen in many who survive. The high mortality was independent of vaccination status, likely reflecting the inability of LTR to mount an immune response. A high index of suspicion and monitoring for superimposed infections, especially PCP, appears prudent. The sustained decline in lung function raises the notion of COVID-19 as a precipitating factor for chronic lung allograft dysfunction (CLAD). CLINICAL IMPLICATIONS: LTR who contract COVID-19 infection represent a high-risk population, even in those fully vaccinated, with potential for hospitalization, death, loss of lung function, and infectious complications. In this population, new algorithms for immunosuppression, monitoring and treatments may help to improve outcomes. DISCLOSURES: No relevant relationships by Shambhu Aryal No relevant relationships by A. Whitney Brown, value=Honoraria Removed 04/03/2022 by A. Whitney Brown No relevant relationships by A. Whitney Brown, value=Honoraria Removed 04/03/2022 by A. Whitney Brown No relevant relationships by A. Whitney Brown, value=Consulting fee Removed 04/03/2022 by A. Whitney Brown No relevant relationships by Jessica Chun No relevant relationships by Meg Fregoso No relevant relationships by Vikramjit Khangoora Advisory Committee Member relationship with Boehringer Ingelheim Please note: 2019-2021 Added 04/03/2022 by Christopher King, value=Consulting fee Advisory Committee Member relationship with Actelion Please note: 2019-2022 Added 04/03/2022 by Christopher King, value=Consulting fee Advisory Committee Member relationship with United Therapeutics Please note: 2019-2022 Added 04/03/2022 by Christopher King, value=Consulting fee Speaker/Speaker's Bureau relationship with Actelion Please note: 2019-2022 Added 04/0 /2022 by Christopher King, value=Consulting fee Speaker/Speaker's Bureau relationship with United Therapeutics Please note: 2020-22 Added 04/03/2022 by Christopher King, value=Consulting fee Consultant relationship with Veracyte Please note: $1001 - $5000 by Steven Nathan, value=Honoraria Removed 03/29/2022 by Steven Nathan Consultant relationship with United Therapeutics Please note: $5001 - $20000 by Steven Nathan, value=Consulting fee Consultant relationship with Bellerophon Please note: $5001 - $20000 by Steven Nathan, value=Consulting fee Speaker/Speaker's Bureau relationship with Roche-Genentech Please note: $5001 - $20000 by Steven Nathan, value=Honoraria Speaker/Speaker's Bureau relationship with Boerhinger-Ingelheim Please note: $20001 - $100000 by Steven Nathan, value=Honoraria No relevant relationships by Alan Nyquist No relevant relationships by Michelle Schreffler Speaker/Speaker's Bureau relationship with United Therapeutics Please note: 2020-2022 Added 04/01/2022 by Oksana Shlobin, value=Consulting fee Speaker/Speaker's Bureau relationship with Bayer Please note: 2020-2022 Added 04/01/2022 by Oksana Shlobin, value=Honoraria Speaker/Speaker's Bureau relationship with Janssen&Janssen Please note: 2020-2022 Added 04/01/2022 by Oksana Shlobin, value=Honoraria Consultant relationship with Altavant Please note: 2020-2022 Added 04/01/2022 by Oksana Shlobin, value=Consulting fee Consultant relationship with Acceleron Please note: 2020-2022 Added 04/01/2022 by Oksana Shlobin, value=Honoraria Consultant relationship with United Therapeutics Please note: 2020-2022 Added 04/01/2022 by Oksana Shlobin, value=Honoraria No relevant relationships by Anju Singhal No relevant relationships by Christopher Thomas

A SPECTRUM OF CAUSES OF MOLECULAR INJURY IS DETECTED BY DONOR-DERIVED CELL-FREE DNA AFTER LUNG TRANSPLANT: PRELIMINARY EXPERIENCE

SESSION TITLE: Lung Transplantation: New Issues in 2022 SESSION TYPE: Rapid Fire Original Inv PRESENTED ON: 10/19/2022 11:15 am - 12:15 pm PURPOSE: Donor-derived cell-free DNA (dd-cfDNA) is a promising plasma analyte for surveillance of rejection and lung transplant (LT) injury. Herein we report our preliminary real-world experiences in concert with standard of practice (SOP) assessments. METHODS: We performed a prospective, cross-sectional, cohort study of a clinically available dd-cfDNA test (the Prospera™ test, Natera, Inc.) combined with SOP clinical assessments − spirometry, fiberoptic bronchoscopy (FOB), donor-specific HLA antibodies (DSA). Single LT dd-cfDNA results were corrected (2X) for lung mass before analysis. Clinical-pathologic cohorts were assigned based on ISHLT guidelines for acute cellular rejection (ACR), uncomplicated chronic lung allograft dysfunction (U-CLAD), and either CoVid-19 or Non-CoVid-19 allograft infection. We also compared median dd-cfDNA fractions between patients experiencing allograft dysfunction (AD) (defined by ΔFEV1≥ -10%) vs stability (STA) and stratified by DSA status. Groups were analyzed by Mann-Whitney (p<0.05) and data expressed as median with 25-75% interquartile range (IQR). RESULTS: A total of 54 plasma samples from 42 unique LT recipients (Single=6, Double=36) were collected at Spectrum Health between November 2021 and February 2022. Primary diagnoses included chronic obstructive pulmonary disease (n=7), interstitial lung disease (n=31), CoVid-19 related ARDS (n=2), CF (n=1) and PAH (n=1). Matching histopathology was available for 68% of dd-cfDNA samples. dd-cfDNA fraction trended 2-fold higher in patient with ACR (1.59%, IQR: 0.09-3.57;n=3) and U-CLAD (1.88%, IQR: 0.88-3.32;n=4) than STA (0.86%, IQR: 0.21-1.62;n=14) patients. Patients with CoVid-19 had significantly higher dd-cfDNA fraction (6.91%, IQR: 2.41-9.77;n=4) than both STA (p=0.035) and NON-CoVid-19 infection cohorts (p=0.049). Although no antibody-mediated rejection (AMR) events were observed, dd-cfDNA fraction was significantly elevated in DSA(+) patients (2.75%, IQR: 1.72-6.25;n=8, class I (4) and II (4)) vs DSA(-) (1.035%, 0.04-1.64;n=46) cohorts (p=0.011). A trend was noted with elevated dd-cfDNA with AD (1.58%, IQR: 0.74-3.62;n=17) vs AS (1.05%, 0.66-1.79;n=37) (p=0.29). CONCLUSIONS: Our preliminary experience is consistent with prior studies, suggesting elevated dd-cfDNA fraction during LT allograft rejection and specific types of infection, in particular, CoVid-19. Of interest, dd-cfDNA detected potential occult molecular injury associated with anti-HLA DSA. CLINICAL IMPLICATIONS: dd-cfDNA fraction assessment after LT represents a valuable clinical tool for clinical surveillance of organ transplant health. DISCLOSURES: Employee relationship with Veracyte, Inc Please note: 2 years by Sangeeta Bhorade, value=Salary Removed 04/03/2022 by Sangeeta Bhorade Employee relationship with Natera Inc Please note: 2/22/22- present Added 04/03/2022 by Sangeeta Bhorade, value=Salary Employee relationship with Natera Please note: 05/2021-present Added 04/04/2022 by Kathryn Crabtree, value=Salary research relationship with United Therapeutics Please note: 2016- ongoing by Reda Girgis, value=Grant/Research research relationship with Pfizer Please note: 2014-2020 by Reda Girgis, value=Grant/Research Speaker/Speaker's Bureau relationship with Boehringher Ingelheim Please note: 2016-ongoing by Reda Girgis, value=Honoraria Speaker/Speaker's Bureau relationship with Genentech Please note: 2016-ongoing by Reda Girgis, value=Honoraria no disclosure on file for Cameron Lawson;No relevant relationships by Edward Murphy Employee relationship with Natera, Inc. Please note: 2020- present by David Ross, value=Salary

A Case of Multifocal Necrotizing Blastomycosis Pneumonia and Acute Respiratory Distress Syndrome (ARDS) in an Adolescent with Type 1DiabetesMellitus

Introduction: Blastomyces species are thermally dimorphic fungi endemic to North America, especially areas bordering the Mississippi, Ohio and St. Lawrence rivers, and the Great Lakes. Blastomycosis infections are estimated to occur in 3-13% in the pediatric population. Pediatric literature for blastomycosis has been mostly limited to small studies and case series. Recent literature suggests increasing rates of infections, less morbidity and mortality as compared to adults, with asthma as the most common comorbid condition. Although pulmonary disease is the most common presentation, it rarely progresses to acute respiratory distress syndrome (ARDS). Case Description: A 17- year-old female, living in the Chicago area, and with type 1 diabetes mellitus and childhood asthma, presented to the emergency room with acute hypoxemic respiratory failure after 14 days of cough, dyspnea, chest pain, and fevers as high as 105°F. Her initial radiographic imaging revealed bilateral infiltrates and consolidations in the right middle and lower lobes. She was admitted to the step down unit for further care. A respiratory viral panel, including COVID-19 evaluation, was negative. She was started on low-flow nasal cannula, ceftriaxone, azithromycin, albuterol, and maintenance IV fluids. On hospital day 2, she was transferred to the pediatric intensive care unit for worsening respiratory distress and escalated to high-flow nasal cannula. She was treated empirically for presumed bacterial pneumonia with ceftriaxone (7-day course), azithromycin (5-day course), cefepime (5-day course), clindamycin (2-day course), and vancomycin (14-day course). Despite this treatment, repeat chest imaging showed worsening disease and she required escalation to BiPAP for progression of her ARDS and impending respiratory failure. Karius testing results indicated Blastomyces dermatitidis at low levels typically not clinically relevant. Sputum and bronchoalveolar lavage cultures demonstrated no significant pathogenic bacteria. Pathology exam of the biopsy obtained from bronchoscopy was consistent with Blastomyces. Urine antigen test was positive for both Blastomyces and Histoplasma. She clinically improved after initiating Amphotericin B lipid complex (6-day course), with transition to oral itraconazole and adjunctive therapy with IV methylprednisolone. She was discharged home after a 30-day hospital stay. Discussion: Pulmonary blastomycosis presents with a broad variety of signs and symptoms. Timely diagnosis is challenging. Pulmonary blastomycosis has no pathognomonic radiographic patterns. Severe acute pulmonary infection that fails to respond to antibacterial treatment should prompt investigation for fungal infection, including urine antigen tests for Histoplasma and Blastomyces, serum galactomannan, beta-1,3-D-glucan, and next-generation sequencing of microbial cell-free DNA (eg, Karius test). Close respiratory monitoring should occur in a pediatric intensive care unit. Conclusion: Blastomycosis is not typically in the initial differential diagnosis unless the patient has other clinical findings, fails to improve on antibacterial therapy, or has identified risk factors for exposure. Failure of prompt recognition is associated with poor outcomes, increased morbidity and mortality, increased length of hospital stay, and cost.

ENDOGENOUS REGULATION OF NEUTROPHIL EXTRACELLULAR TRAPS IN ACUTE ISCHAEMIC STROKE DUE TO VACCINE-INDUCED IMMUNE THROMBOTIC THROMBOCYTOPENIA

Background and aims: Vaccination is one of the most important countermeasures in the ongoing COVID19-pandemic. Pharmacovigilance concerns, however, emerged after very rare, but potentially disastrous thrombotic complications following vaccination with ChAdOx1. The underlying pathology was described as platelet factor 4 antibody mediated vaccine-induced immune thrombotic thrombocytopenia (VITT). Mechanisms of immunothrombosis including the regulation of neutrophil extracellular traps (NETs) might be of crucial importance in VITT. Methods: In this study, we had the exceptional opportunity to investigate blood and thrombus samples of a patient who suffered severe ischaemic stroke due to VITT undergoing successful mechanical thrombectomy (MT) in comparison to 13 control stroke patients with similar clinical characteristics without VITT. Cerebral thrombi were stained for complement factors, NETs-markers, DNase and LL-37 and were histologically assessed. In blood samples before MT and 7 days later, cell-free DNA, myeloperoxidase-histone complexes, myeloperoxidase activity, DNase activity, LL-37 and inflammatory cytokines were determined. Results: We identified NETs-markers in thrombi of all patients. The thrombus of the VITT-patient, however, exclusively revealed complement factors and high amounts of NETs-degrading DNase and LL-37, which protects NETs from degradation. In accordance, ex vivo and in vitro studies also implied a disturbed regulation of NETs in VITT-samples. Moreover, we found markedly pronounced temporal changes of specific circulating cytokines in the VITT-case. Conclusions: The results of this study suggest a disturbed endogenous regulation of NETs in VITT involving a pro-inflammatory response and fulminant clinical course. Further studies are warranted to confirm our results and to provide deeper insights into the causative mechanisms.

Intravenous Immunoglobulin but Not Dexamethasone Inhibits Neutrophil Pro-Inflammatory Activity While Maintaining Key Antimicrobial Functions

Rationale. Acute respiratory distress syndrome (ARDS) is a heterogeneous clinical disease. ARDS immunopathology due to lung infection involves an array of immune cells and the importance of granulocytes, and in particular neutrophils and neutrophil extracellular trap production (NETosis), has recently come to light. Despite over 20 well run, randomized, controlled trials, no specific therapies for ARDS are available and mortality remains high. Current treatments for ARDS are primarily limited to supportive therapies, including lung protective ventilation, and in certain situations, systemic steroid administration. Recently, clinical studies adding intravenous immunoglobulin (IVIG), an FDA approved drug, to standard ARDS therapy have shown faster recovery with less severe symptoms, suggesting a complementary beneficial effect, but the mechanism(s) remain unknown. Interestingly, previous in vitro studies found that IVIG can impair some inflammatory pathways in neutrophils. Our study assessed effects of IVIG with and without dexamethasone (a key glucocorticoid used in COVID-19 ARDS) in neutrophils ex vivo and in vivo in COVID-19 patients. Methods. Ex vivo treatment of neutrophils with IVIG or dexamethasone was conducted, followed by assessment of NETosis, oxidative burst and phagocytosis. Additionally, cell-free DNA was quantified in the blood of COVID-19 patients before and after treatment with IVIG. Ex vivo NETosis and plasma cell-free DNA was quantified using the QuantiT ™ PicoGreen™ dsDNA Assay Kit (Invitrogen). Oxidative burst was assessed by OxyBURST™ Green H2DCFDA, SE (Invitrogen) and phagocytosis of pHrodo™ Red S. aureus Bioparticles™ (Invitrogen) was quantified. Results. IVIG inhibits crucial neutrophil inflammatory pathways such as NETosis and oxidative burst while concomitantly enhancing phagocytic activity (Figure panels A-C). Notably, dexamethasone does not impact any of these critical pathways. Moreover, COVID-19 patients undergoing standard treatment plus IVIG had decreased cell-free DNA in the circulation 5 days after initiation of a 4 day treatment course, suggesting decreased NETs in circulation (Figure panel D) which possibly reverted at a later timepoint. Conclusion. Our data demonstrate potential targeted beneficial effects of IVIG in the context of neutrophil-mediated immunopathology. We demonstrate an ex vivo inhibitory effect of IVIG on pro-inflammatory pathways in neutrophils, which may lead to diminished immunopathology in disease states worsened by neutrophil-driven destruction. Based on the compelling evidence of the contribution of neutrophils to development and severity in ARDS, our evidence of IVIG impairing key pro-inflammatory functions in neutrophils (where dexamethasone does not) suggests a theoretical potential complementary beneficial effect of adding IVIG to standard treatment for infection induced ARDS although further research is needed.

Plasma Neutrophil Extracellular Trap Levels Correlate with Acute Respiratory Distress Syndrome Disease Severity

Rationale: Acute Respiratory Distress Syndrome (ARDS) is characterized by acute onset of hypoxic respiratory failure, which can be complicated by multi-organ dysfunction and death. ARDS results from inflammatory alveolar injury precipitated by direct or indirect lung injury. Neutrophils play a central role in the pathology of ARDS and release neutrophil extracellular traps (NETs) to trap and kill pathogens. Dysregulated NET formation, however, can cause inflammatory tissue damage and exacerbate acute lung injury as in COVID-19 associated ARDS. Whether detection of plasma NETs predicts outcomes in non-COVID-19 associated ARDs remains unknown. We hypothesized that plasma NET levels correlate directly with disease severity and mortality in non-COVID-19 ARDS patients. Methods: We obtained previously collected plasma samples from patients (n=200) with moderate to severe ARDS enrolled in the Re-evaluation of Systemic Early Neuromuscular Blockade (ROSE) trial at three different time points (admission, 24 hours, and 48 hours after admission) complete with clinical outcome data through 28 days after admission. We also examined age- and gender-matched healthy donor plasma (n=20). We assayed cell-free DNA levels via fluorescence and MPO-DNA complexes as a surrogate for NETs in each plasma sample. Clinical outcomes from ROSE trial participants were correlated with the quantification of NETs. We also assessed NET formation by neutrophils isolated from healthy adults following incubation with ARDS patient and healthy donor plasma samples using live cell imaging and confocal microscopy. Results: We demonstrated elevated plasma markers of NETs (cell-free DNA and MPO-DNA complexes) in ARDS plasma compared to healthy donor plasma. Deceased study participants demonstrated higher plasma NET levels on admission and at 48 hours as compared to ARDS survivors (admission: p = 0.0045 and 48 hours: p = 0.0050). Increased plasma NETs on admission, at 24 hours, and 48 hours also correlated with illness severity. Furthermore, ARDS plasma samples induced NET formation in vitro in neutrophils isolated from healthy donors while control plasma did not. Conclusion: NET formation is increased in plasma from patients with ARDS compared to healthy donor plasma, consistent with the inflammatory alveolar injury seen in ARDS. Additionally, plasma from ARDS patients induces NET formation in vitro in PMNs isolated from healthy adult donors. We speculate that exaggerated NET formation may serve as a novel biomarker for inflammatory lung injury in ARDS resulting from multiple etiologies and that strategies targeting NET formation may improve outcomes in ARDS.

Abstracts from the 54th European Society of Human Genetics (ESHG) Conference

The proceedings contain 1623 papers. The topics discussed include: the COVID-19 host genetics initiative - an international, open science effort to identify genetic risk factors for COVID19 severity and susceptibility;clinical implementation of RNA sequencing for Mendelian disease diagnostics;local gene co-expression measurements in single-cells highlight inter-individual specificity;a cross-disorder dosage sensitivity map of the human genome;biallelic ATG7 variants impair autophagy leading to neurological disease;epilepsy polygenic risk scores in >269k individuals with and without epilepsy;machine learning methods for prioritizing genetic variants;Mendel Lecture - Cell-free DNA in plasma: coming in different sizes and shapes;imaging the accessible genome at nanometer scale;retrotransposition in brain: does LINE activity in the central nervous system matter?;activation of transposons in neurological disorders;how to transfer genomic data internationally in compliance with the GDPR;mutational signatures of environmental agents and chemotherapeutics in human cellular models;and the art, science and practice of implementing genomics in clinical care.

Poke Not Prod: Improving Quality of Life Through Non-Invasive Rejection Surveillance for Heart Transplant Recipients

Purpose: Gene expression profiling (GEP) and donor-derived cell-free DNA (dd-cfDNA) provide effective non-invasive rejection surveillance for heart transplant (HT) recipients with a trend toward improved quality of life. During the COVID-19 pandemic, rejection monitoring and titration of mediations in HT patients was difficult due to limited health-care resources for endomyocardial biopsy (EMBx). This is the first Canadian study to assess non-invasive rejection surveillance in improving patient satisfaction and reducing anxiety during HT rejection screening. Methods: Adult HT recipients, at least 6 months post-transplant, were enrolled to have surveillance EMBx replaced by non-invasive rejection testing with GEP and dd-cfDNA. Patients with multiorgan transplant, dialysis, or high rejection risk (recent acute cellular rejection ≥ grade 2R, new graft dysfunction, or heart failure) were excluded. All patients completed the Medical Outcomes Study 12-item Short Form Health Survey (SF-12) and a patient satisfaction survey. Thematic analysis was performed for open-ended responses. Results: Out of 90 patients screened, 31 had their routine EMBx replaced by non-invasive rejection testing. Based on test results, 89% of EMBx were safely eliminated. On the SF-12, participants had a median physical health score of 43 (40-53) and mental health score of 53 (46-58) out of 100. Patients’ self-reported satisfaction was 90%. Median self-reported anxiety score prior to EMBx was 50 (10-71) versus 2.5 (0-7.5) out of 100 prior to GEP/dd-cfDNA. Four codes (“emotions” (pain, anxiety, fear), “time”, “biopsy”, “accuracy”) were used to uncover two themes of “Superiority to Biopsy” and “Mental or Physical Stress”. Patients described feeling much more satisfied and less emotionally distressed with the non-invasive screening compared to EMBx. HT patients reported less fear and anxiety, reduced pain, and enjoyed the simplicity of non-invasive testing. Conclusion: Non-invasive rejection surveillance screening can positively impact patients’ mental health. In this study, non-invasive rejection surveillance eliminated the recovery time and risk of an invasive procedure for HT recipients while reducing anxiety, improving patient satisfaction, and providing an alternative way to screen patients during a period of limited resources due to a global pandemic.

eP455: Fetal ultrasound presentation and neonatal diagnosis of Freeman-Sheldon syndrome in son of previously undiagnosed adult male

Background: Freeman-Sheldon syndrome [distal arthrogryposis type 2A (OMIM #193700), DA2A, Freeman-Burian syndrome] is a rare autosomal dominant multiple pterygium syndrome caused by alterations in MYH3. The phenotypic features, particularly of the face, are distinct and easily recognizable, and the diagnosis can be confirmed with molecular gene analysis. Fetal ultrasound imaging may provide important diagnostic clues to facilitate the diagnostic process. Informed consent and parental permission were provided by the parents. Case presentation: The infant’s mother presented for a Maternal Fetal Medicine genetic counseling telehealth appointment (due to COVID-19 pandemic restrictions) as a G7P2132, 32-year old female who had insulin-dependent diabetes and thrombocytosis. Her partner was a 24-year old male with a history of hearing loss, a V-shaped palate, and a lower lip cleft. Gestational age was 14 4/7 weeks and the indications were: increased nuchal translucency, paternal complex medical history, maternal G6PD heterozygote, and recurrent pregnancy loss. During the genetic counseling session, the following were addressed: 1) Maternal heterozygote status for G6PD indicated that if the fetus was male, there was a 50% chance he would be affected with G6PD-deficiency;2) Increased nuchal translucency on fetal ultrasound (US) with measurement at 98th percentile is associated with an increased risk of chromosomal abnormalities, microdeletion/duplications, and Noonan syndrome. The patient reportedly had low risk cell-free DNA but results were not available to the counselor at the time of consult. The option for additional genetic screening and diagnostic testing was declined;3) Three first trimester pregnancy losses with the father of this baby (FOB) were addressed, and parents deferred chromosome analyses at the time;4) Mother shared FOB’s complex history of bilateral sensorineural hearing loss, V-shaped cleft palate, lower lip cleft, and micrognathia. However, father was not present during the telehealth encounter. Mother was counseled regarding the possibility of an autosomal dominant condition with the potential risk to the pregnancy of up to 50%. It was recommended that the FOB have a clinical genetics evaluation, which could potentially provide a specific diagnosis and inform recurrence risk and management guidance. Follow-up MFM genetic counseling telephone visit occurred with the mother at 31 6/7 weeks gestation due to multiple congenital anomalies evident on fetal ultrasound. A 25 week fetal ultrasound revealed hypotelorism and a thickened nuchal translucency. A repeat study at 29 weeks revealed a V-shaped palate with a possible cleft, micrognathia, and midline mandibular cleft. FOB’s history was revisited. It was determined that he had 3 previous “no shows” to Genetics clinic appointments and did not pursue evaluation after the last counseling appointment. Again, it was emphasized that in order to best make a diagnosis for the family, an affected person would need to undergo a thorough evaluation, including medical and family history review, physical examination, and any indicated genetic testing. The parents were comfortable with the likelihood that the baby had the same condition as the father, but variable expressivity and broad range pf phenotypic presentation were explained. Recommendations for postnatal evaluation of the infant and pertinent genetic testing were provided. Consultative Genetics evaluation of the infant at 2 days of age revealed a short, broad forehead with supraorbital fullness leading to a horizontal brow indentation;mask-like facial appearance;hypotelorism;very deep set eyes with blepharophimosis;deep, creased nasal bridge;small, upturned nose with hypoplastic alae and narrow nares;microstomia with pursed lips;glossoptosis;micrognathia;2 deep vertical chin creases;short neck with excess nuchal skin;inverted and wide spaced nipples;clenched hands with 5th digits overlying 4th and 2nd overlying 3rd, bilaterally;bilateral vertical talus;2nd toes longer and overlying rd toes;clinodactyly of 4th and 5th toes bilaterally;and deep gluteal crease with no visible sinus. There were no evident contractures. The father has a complex history with no medical assessments prior to age 18. He reported that he did “not look like anyone else” in his family. He has a diagnosis of autistic spectrum disorder, a submucous cleft, vision issues, hearing loss necessitating a hearing aid on the left, and a history of cholesteatomas and of mastoidectomy. On brief examination, he had a mask-like face, blepharophimosis, left microphthalmia, left esotropia, narrowing of his midface, deep vertical crease on the mandibular region, microstomia, broad great toes, single flexor creases on the thumbs, and contracture of right thumb. Maxillofacial CT of the infant revealed hypoplastic mandibular body, ramus, and condyles bilaterally with micrognathia and retrognathia;hypoplastic maxilla bilaterally;and enophthalmos with retracted appearance of globes in the bony orbits bilaterally. Multiple facial bone abnormalities were seen, including microsomia, micrognathia, retrognathia, orbital hypotelorism and enophthalmos Genetic testing was performed via a custom Whole Exome Slice at GeneDx laboratories and included the MYH3 and TNNI2 genes. Results revealed a heterozygous pathogenic change in MYH3 (c.2015 G>A;p. R6724) consistent with the diagnosis of Freeman-Sheldon syndrome. Conclusion: The presentation of “midline mandibular cleft” on fetal ultrasound was the most specific prenatal finding. This is a very rare fetal finding. Thus, it should prompt further evaluation to assess for true clefting versus ridging or creasing. Additionally, targeted assessment for other findings or clinical clues for Freeman-Sheldon syndrome, such as contractures, “windmill vane” hand, and mouth size, could aid in the differential diagnosis considerations and the diagnostic process. Admittedly, these are position and quality dependent, and are challenging to assess even in ideal situations. The phenotype of the father was immediately recognizable. However, due to COVID-19 pandemic restrictions, prior to the infant’s birth, only telehealth visits were conducted and the father’s participation was by telephone. This limited the ability to narrow the differential diagnosis without visualization of his distinct phenotypic features. Finally, missed opportunities to diagnose the father prior to this pregnancy occurred. Many clinics send “no show” letters to referring providers and patients, as we do. Emphasizing the importance of diagnosis prior to pregnancy for individuals concerned about having a genetic disorder should be considered as part of the information shared in these letters.

PostCOVID effect on endothelial function in hypertensive patients: A new research opportunity.

SARS-CoV-2 is causing devastation both in human lives and economic resources. When the world seems to start overcoming the pandemics scourge, the threat of long-term complications of COVID-19 is rising. Reports show that some of these long-term effects may contribute to the main cause of morbimortality worldwide: the vascular diseases. Given the evidence of damage in the endothelial cells due to SARS-CoV-2 and that endothelial dysfunction precedes the development of arteriosclerosis, the authors propose to measure endothelial function around 6-12 months after acute disease in hypertensive patients, especially if they have other cardiovascular risk factors or overt vascular disease. The methods the authors propose are cost-effective and can be made available to any hypertension unit. These methods could be the "in vivo" assessment of endothelial function by flow mediated vasodilatation after ischemia by Laser-Doppler flowmetry and the measurement of plasma free circulating DNA and microparticles of endothelial origin.