ALBERT

Description: This paper reports the results of tree-ring dating and accelerator mass spectrometry (AMS) wiggle-matching for wooden Buddhist statues stored at the Eungjindang Hall of Neunggasa Temple, South Korea. Among 23 statues, 10 were successfully dated by tree rings. The cutting date of logs used for the statues was determined as some time between late fall 1684 and early spring 1685 when the bark ring (AD 1684) completed latewood formation. The 95.4% confidence interval of a radiocarbon date (cal AD 1688–1713, 2 σ), which was obtained by wiggle-matching 7 samples of a statue, is similar to the dendro-date (AD 1684). A historical document recorded that the statues in the Eungjindang of Neunggasa were dedicated in July 1685. The dendro-date and written record indicate that Eungjindang statues were made within 3–8 months after log cutting. This seems rather short if we consider the period required for natural drying to avoid defects such as cracking and crooking.

Description: Introduction: Idiopathic erythrocytosis (IE) is characterized by a persistently elevated hemoglobin, equivocal erythropoietin (EPO) levels, absence of janus kinase 2 (JAK2) mutations suggestive of polycythemia vera (PV) and no secondary cause. One study used a targeted 21 gene next-generation sequencing panel and identified novel variants in known erythrocytosis-related genes as well as novel genes associated with the oxygen-sensing pathway. However, expanded sequencing of blood and matched tissue samples in a large ethnically diverse group of IE patients has not been performed. Methods: All patients signed informed consent to participate in an observational study approved by the Institutional Review Board; they provide blood and buccal mucosa samples at study entry and at 24-month follow-up. Patients were enrolled if JAK2 testing and a complete work up for secondary causes was negative. They were required to have hemoglobin levels greater than 16 g/dL on two occasions or greater than 15 g/dL if undergoing phlebotomy. Our initial sequencing of 20 IE patients was performed utilizing high resolution whole-exome sequencing of circulating blood samples (disease) at a mean coverage of 390x and matched normal (buccal) samples at mean coverage of 300x. To stratify samples by genetic ancestry, we performed a population stratification principle component analysis (PCA) and STRUCTURE using Ancestry Informative Markers derived from 1K Genome Phase1_v3 Exome database. The primary in-silico analysis was performed on the baseline samples from treatment-naïve patients. The whole-exome data was generated in accordance to GATK's best practices with same filters applied as described by Exome Aggregation Consortium. The additional downstream in-silico paired analysis was performed using MutSig2.0 (Mutation Significance) algorithm to determine significant mutations and GISTIC (The Genomic Identification of Significant Targets in Cancer) to identify the significant copy number events, IPA (Ingenuity Pathway Analysis) to determine pathways along with other computational . Results: Median age at baseline was 52 years (range 35-71). Six patients (30%) were female and 14 (70%) were male. Median values and ranges for laboratory parameters at baseline were as follows: WBC 6.6 x 109/L (5-9.7), Hgb 17 g/dL (15.5-19.8), Plt 218 x 109/L (86-374), and EPO level 9.8 IU/L (2-14.3). Three patients had a personal history of malignancy, including 2 with lymphoma. Two patients had a family history of myeloid malignancy (chronic myeloid leukemia and PV). Our ancestry analysis of initial 20 patients with IE identified 6 patients with high European percent ancestry (EUR), 1 patient with high Asian percent ancestry (EAS) and 13 patients with high percent Ad Mixed ancestry (AMR). In our cohort, 60% (12/20) of patients had been also diagnosed with a liver disorder (11 with fatty liver, 1 with cirrhosis) that was not significantly different across populations. We identified, on average, 42 non-silent somatic mutations (not present in the buccal samples) in whole blood across our cohort with no statistical difference (p=0.671) in mutation burden between ancestry groups or between patients with and without liver disease. Age, gender, and ethnicity were not associated with mutation burden. Utilizing MutSig algorithm, we identified a novel candidate gene, CHAF1A, with high mutation prevalence of 30% in patients with IE. Further analysis of mutation landscape identified somatic nonsilent mutations in 25 known oncogenes which were present in at least 10% of patients. Our mutation signatures in IE identified a significant association with failure of double stranded DNA repair. Only one patient had a mutation in TET2. Further analysis of copy number indicated copy number loss in genes such as SETD3 and GSH associated with chromatin assembly which may suggest alterations in chromatin assembly and changes in the epigenome. Our analysis also identified a high number of 9p and 13q gains in patients with IE. Conclusion: In this study, we utilized high-resolution next generation sequencing in association with comprehensive clinical annotation to determine potential molecular drivers of IE in a multi-ethnic population. We identified somatic mutations in a subset of patients which may represent clonal hematopoiesis. Long term follow up of outcomes in this cohort may clarify the significance of these mutations in the pathogenesis of IE. Disclosures No relevant conflicts of interest to declare.

Description: Background: Idiopathic erythrocytosis (IE) is an entity characterized by a persistently elevated hemoglobin, variable erythropoietin (EPO) level, absence of janus kinase 2 (JAK2) mutations suggestive of polycythemia vera (PV) and no identifiable secondary cause. Previous studies have compared IE to PV, showing a lower incidence of venous thrombosis and leukemic transformation in IE but similar incidence of arterial events. PV is known to be associated with constitutional symptoms and splenomegaly, while hereditary erythrocytosis can be associated with recurrent headaches and fatigue. A comprehensive assessment of clinical features including symptoms in IE has not been performed. Methods: Patients signed informed consent to participate in an observational study approved by the Institutional Review Board. Enrollment criteria included: age 18 years or older; hemoglobin level greater than 16 g/dL on two occasions at least 3 months apart or greater than 15 g/dL if undergoing phlebotomy; negative testing for JAK2 mutations; and negative work up for secondary causes of erythrocytosis. Baseline assessment included history and physical exam, vital signs, pulse oximetry, and body mass index. Baseline laboratory exams included a complete blood cell count, complete metabolic panel, C-reactive protein, iron panel, ferritin, hemoglobin A1C, erythropoietin level. Abdominal ultrasound was performed to evaluate for splenomegaly. The Myeloproliferative Symptom Assessment Form (MPN-SAF) was used to assess for the presence and severity of a broad range of symptoms that may be expected to occur in patients with IE. The MPN-SAF was administered at baseline and every 6 months thereafter. Results: 35 patients had data available for analysis. Patient characteristics are shown in Table 1. The most prevalent co-morbid conditions were those known to be associated with cardiovascular disease risk and metabolic syndrome, including hepatic steatosis identified on abdominal ultrasound in 63% of patients. Three (8.6%) patients had a history of venous or arterial thrombosis. Two (5.8%) patients had a history of lymphoma (NK/T-cell and Hodgkin). Three patients (8.6%) had a first-degree relative with a myeloproliferative neoplasm (chronic myelomonocytic leukemia, essential thrombocytosis and polycythemia vera) and one patient had a son with IE and history of stroke. 16 (46%) patients were taking aspirin and 11 (31%) had undergone phlebotomy within 3 months of study enrollment. Patients reported the following symptoms on the MPN-SAF at baseline: fatigue (77%), early satiety (57%), difficulty sleeping (57%), numbness/tingling (51%), headaches (49%), concentration problems (40%), itching (40%), bone pain (37%), night sweats (37%), depression (37%), abdominal pain (37%), abdominal discomfort (37%), inactivity (37%), problems with sexual desire/function (34%), dizziness/lightheadedness (31%), cough (26%), fever (17%), and unintentional weight loss (17%). Fatigue carried the highest average symptom intensity (3.77, SD 3.17). Discussion: In this study, we describe the clinical features associated with IE in a multiracial cohort. Patients with IE are frequently symptomatic and have a high incidence of hepatic steatosis by ultrasound. Disclosures No relevant conflicts of interest to declare.

Description: Introduction:Chimeric antigen receptor (CAR) T-cells are engineered T-cells that target specific antigens on tumor cell surfaces. CD19 directed CAR T-cells have demonstrated efficacy in B-cell lymphomas and B-cell acute lymphoblastic leukemia (B-ALL); however, patients are at risk for life-threatening complications including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Grade 3 ICANS and higher have been reported in 12-28% and Grade 3 CRS and higher in 1-23% in pivotal studies for B-cell lymphoma.1 The majority, if not all, patients with Grade 3 ICANS and CRS require intensive care unit (ICU) admission. The long-term outcomes of patients requiring ICU admission after CD19 directed CAR T-cells are unknown. Methods:Clinical data were retrospectively reviewed from consecutive patients with relapsed or refractory B-cell lymphomas and B-ALL treated with axicabtagene ciloleucel or tisagenlecleucel at a single institution from January 1, 2016 to July 1, 2020. Diagnosis of CRS and ICANS was based on the American Society for Transplantation and Cellular Therapy consensus grading.2 Demographics, medical co-morbidities, therapeutic interventions, relapse rate, and all-cause mortality were assessed. Results:Thirty-seven patients received CAR T-cells. Sixty-seven percent (n=25) of patients were male with a median age of 55 years (range 23 - 77). Eighty-nine percent of patients had large B cell lymphoma. Patients received a median of 3 prior therapies (range 1 - 6). The median IPI and ECOG scores at time of CAR T-cells administration were 3 and 1, respectively. Forty-three percent (n=16) of patients required ICU admission. The median length of ICU stay was 3.5 days (range 1 - 18). The median CRS and ICANS grades in patients requiring ICU admission were 2 and 3, compared to 2 and 0 in patients not requiring ICU admission, respectively. Thirty-one percent of ICU admitted patients had CRS grade 3 or above, and 61% had ICANS grade 3 or above. All 4 patients with B-ALL experienced grade 3 or above CRS and required ICU admission. Only 1 of the 37 patients (2.7%) patients died from CRS. Fifty-six percent of ICU patients experienced disease progression during follow-up, compared to 33% of non-ICU patients (OR 2.50, p = 0.20, 95% CI 0.55-12.11). Over a median follow up of 399 days, 56% of ICU patients died compared to 14% of non-ICU patients (OR 7.23, p = 0.01, 95% CI 1.32-54.27). The majority of deaths (58%) were due to disease progression. Conclusions:A significant number of patients who receive CD-19 direct CAR T-cells require ICU admission. Patients requiring ICU admission after CAR T-cell have a worse outcome and overall survival, compared to non-ICU patients. The mortality rate of ICU admitted patients is primarily driven by death from disease. Disclosures Tzachanis: MS:Research Funding;EUSA Pharma:Consultancy;Fate:Research Funding;Genetech:Research Funding;Gilead Sciences:Consultancy, Research Funding, Speakers Bureau;Incyte:Research Funding;Jazz Pharmaceuticals:Consultancy;Kyowa Kirin:Consultancy;Magenta:Consultancy;Takeda:Consultancy, Speakers Bureau.Goodman:Seattle Genetics:Consultancy;EUSA Pharma:Consultancy.

Description: Diffuse large B-cell lymphoma (DLBCL), a subtype of non-Hodgkin lymphoma, has a five-year survival rate of up to 70%; however, approximately half of patients will ultimately relapse and/or become refractory to therapy. In this clinical setting, autologous chimeric antigen receptor T-cell (CAR-T) therapy has been approved by the United States Food and Drug Administration. While these therapies show significant promise, methods that enable monitoring of therapeutic efficacy could be clinically useful. The use of cell-free DNA (cfDNA), also known as liquid biopsy, has been utilized as a noninvasive surrogate to tumor tissue for the identification of tumor-specific mutations and for monitoring treatment response across a number of cancer types. Here we present data that demonstrate the feasibility of using cfDNA to monitor response to CAR-T therapy in patients with refractory DLBCL. Whole blood was collected during conditioning chemotherapy prior to CAR-T administration, at the time of or shortly after administration, and throughout treatment for 12 patients. A total of 127 blood samples were collected and analyzed with a median of 10 samples (range 8-13) from each patient. For each sample, blood was separated using centrifugation to yield plasma and a buffy coat sample. Each plasma sample was subjected to cfDNA extraction using an automated, bead-based method. Extracted cfDNA was then converted to libraries for next generation sequencing and subsequently assayed with low-coverage (~0.4X) genome-wide sequencing. Copy number alteration (CNA) events were identified and characterized using analytical methods originally developed for noninvasive prenatal testing. To quantify the level of CNAs present in the plasma of cancer patients, we utilized the genomic instability number (GIN). The GIN is a metric intended to capture genome-wide autosomal deviation from empirically derived euploid dosage of the genome in circulation and is calculated as the absolute deviation of observed normalized sequencing read coverage from expected normalized read coverage summed across 50,034 autosomal segments. Cellular DNA from each buffy coat was also collected using a column-based extraction process and used as the template for quantification of the CAR-T construct utilized for the therapy. In this initial small cohort of 12 patients, the majority (8/12) were male and the median age was 52 years (range: 38-77). At the date of data censoring, four patients had an ongoing complete response, five had a complete or partial response but have since relapsed or are now deceased, one had a mixed response, and two had progressive disease. The GIN threshold (GIN = 170) has been utilized previously to identify patients with an aberrant CNA profile consistent with the presence of a tumor. When this threshold was applied, all 12 patients had an elevated GIN at the time of CAR-T administration. To determine whether a patient was progressing despite CAR-T therapy, the GIN values for each patient were evaluated throughout treatment (median=70 days; range=23-154 days). In all four patients with an ongoing complete response, the GIN value at the time point closest to the date of data censoring was below the threshold, suggesting the GIN was consistent with clinical response. Of the five patients that ultimately progressed after an initial PR or CR, four had evidence of progression based on the GIN. In one patient that had a mixed response but ultimately progressed, the progression could be observed using the GIN 66 days before clinical relapse was noted. Finally, two patients had no evidence of a clinical response; however, one of these patients had no detectable ctDNA after treatment and represented a second discordant result in this cohort. In addition to measuring cfDNA, the CAR construct was measured in the background of the total cellular DNA obtained from the buffy coat in those patients receiving Axi-Cel (n=11) using digital PCR. As expected, all 11 patients that received this CAR-T therapy showed no presence of the construct prior to CAR-T administration. After administration, the construct was detected in 4/11 baseline samples and all 11 patients showed the presence of the construct at some point during their treatment. Overall, these data describe a proof-of-concept for the use of multiple liquid biopsy technologies to monitor therapeutic response in DLBCL patients receiving CAR-T therapy. Disclosures Goodman: EUSA Pharma: Consultancy; Seattle Genetics: Consultancy. Holden:Laboratory Corporation of America: Current Employment. Fitzgerald:Laboratory Corporation of America: Current Employment. Almasri:Laboratory Corporation of America: Current Employment. McLennan:Laboratory Corporation of America: Current Employment. Eisenberg:Laboratory Corporation of America: Current Employment, Current equity holder in publicly-traded company; OmniSeq: Membership on an entity's Board of Directors or advisory committees. Tzachanis:MS: Research Funding; EUSA Pharma: Consultancy; Fate: Research Funding; Genetech: Research Funding; Gilead Sciences: Consultancy, Research Funding, Speakers Bureau; Incyte: Research Funding; Jazz Pharmaceuticals: Consultancy; Kyowa Kirin: Consultancy; Magenta: Consultancy; Takeda: Consultancy, Speakers Bureau. Jensen:Laboratory Corporation of America: Current Employment; PetDx: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Kurzrock:Turning Point Therapeutics: Consultancy; Foundation Medicine: Research Funding; Sequenom: Research Funding; Pfizer: Consultancy, Research Funding; Medimmune: Research Funding; Genentech: Research Funding; OmniSeq: Research Funding; Debiopharm: Research Funding; Incyte: Research Funding; Takeda: Research Funding; TopAlliance: Research Funding; Boehringer Ingelheim: Research Funding; CureMatch Inc: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; IDbyDNA: Current equity holder in private company; Roche: Consultancy; Actuate Therapeutics: Consultancy; Neomed: Consultancy; X Biotech: Consultancy; Guardant: Research Funding; Grifols: Research Funding; CureMetrix: Membership on an entity's Board of Directors or advisory committees; Bicara Therapeutics, Inc.: Consultancy; TD2/Volastra: Consultancy; Konica Minolta: Research Funding; Merck Serono: Research Funding.

Description: Introduction: The activity of immune checkpoint blockade including anti-cytotoxic T-lymphocyte associated protein-4 and anti-programmed cell death protein-1 monoclonal antibodies in hematologic malignancies is limited outside of classical Hodgkin lymphoma and primary mediastinal B-cell lymphoma. Tumor mutational burden (TMB), programmed death ligand-1 (PD-L1) expression, and microsatellite instability-high (MSI-H) are well-established biomarkers predicting response to checkpoint blockade in solid malignancies. In addition, tumors with high TMB, defined as ≥10 mutations/megabase (mut/Mb), and/or MSI-H are Food and Drug Administration (FDA) approved tissue agnostic biomarkers for treatment with pembrolizumab. The frequencies of high TMB, MSI-H, and expression pattern of PD-L1 across specific hematologic malignancies are undefined. Methods: Patients with hematologic malignancies who had next generation sequencing (NGS) performed by Foundation One Heme were identified. TMB and MSI were measured by NGS. TMB was classified as high if ≥10 mut/Mb and low if