Median Copy Research Articles

Droplet digital PCR (ddPCR) for the detection and quantification of Ureaplasma spp.

BackgroundUreaplasma spp. are associated with various infectious diseases in females, but there is still limited evidence regarding whether they are related to nonspecific cervicitis. The aim of this study was to develop and evaluate a digital droplet PCR (ddPCR) assay for the detection and quantification of Ureaplasma spp. in cervical swabs.MethodsA total of 267 non-specific cervicitis (NSC) patients and 195 asymptomatic females were included in this study. We produced standard curves for Ureaplasma spp. to evaluate the analytical performance of the ddPCR assay. Then, we detected and quantified the bacterial load of Ureaplasma spp. in cervical swabs.ResultsThe prevalences of U. parvum were 37.8% (101/267) and 29.7% (58/195), U. urealyticum were 9.0% (24/267) and 8.7% (17/195) in the NSC group and control group, respectively. In addition, the median copy number of U. parvum was 2.5 × 104 copies/ml (n = 101) in the NSC group and 9.2 × 103 copies/ml (n = 58) in the control group. The U. parvum load in the NSC group was significantly higher than that in the asymptomatic individuals (P < 0.001). whereas the median load of U. urealyticum was 8.4 × 103 copies/ml (n = 24) and 1.4 × 103 (n = 17) copies/ml in the two groups, respectively, , the difference was not statistically significant (P = 0.450).ConclusionsOur study is the first to develop a droplet digital PCR (ddPCR) method for the detection and quantification of Ureaplasma spp. in clinical samples, and the method has excellent analytical performance and a wide range of clinical application prospects.

Foscarnet Therapy for Pure Red Cell Aplasia Related to Human Parvovirus B19 Infection in Kidney Transplant Recipients: A Preliminary Exploration.

BackgroundParvovirus B19-associated pure red cell aplasia (PVB19-PRCA) is an uncommon but serious complication after kidney transplantation. Currently, intravenous immunoglobulin (IVIG) is preferred as the first-line treatment for PVB19-PRCA, but presents with disadvantages of disease recurrence and expensive cost. In this context, we propose that foscarnet therapy for kidney transplantation recipients (KTR) with PVB19-PRCA may be an alternative scenario. No related study has been reported, and we performed this study to assess the efficacy and safety of foscarnet for PVB19-associated PRCA in KTR.MethodsWe conducted a retrospective review of PVB19-PRCA in KTR at our center over 9-year period. The data on therapy and outcomes in all cases treated with foscarnet are detailed records and summarized.ResultsAmong our 68 patients, PVB19-PRCA was confirmed in 50 based on inclusion/exclusion criteria. All patients presented with refractory anemia and low reticulocyte percentage (<0.5%), the mean hemoglobin of patients was 79.8±12.6g/L at the time of PVB19-PRCA was identified. The median serum genome copy number of parvovirus B19 at diagnosis was 9.6 log10 copies per milliliter. A total of 11 patients received foscarnet therapy, of 10 patients responded well to the treatment and maintained no recurrence. But 1 patient had a poor response to foscarnet therapy. Except for this patient, the mean hemoglobin level gradually increased from 68.5±9.3 g/L to 73.2±8.8 g/L, and the mean percentage of reticulocytes steadily increased from 0.1±0.0% to 7.6±2.9% after foscarnet therapy. The median serum genome copy number of parvovirus B19 decreased from 9.8 log10 to 6.1 log10 copies per milliliter. There was no significant difference (P=0.61, 0.60) in serum creatinine and glomerular filtration rate before and after foscarnet treatment. At the latest follow-up, the mean hemoglobin was 131.5±12.5 g/L and the hemoglobin correction occurred in all patients.ConclusionFoscarnet therapy doesn't seem to be worse than IVIG for PVB19-PRCA in KTR, and it can be an alternative option.

Abstract 575: Enhancing cell-free DNA mutation detection with novel liquid-phase extraction

Abstract Background: Low yields of extracted cell-free DNA (cfDNA) from plasma limit further development of liquid biopsies for early-stage cancer diagnostics and cancer screening applications. Conventional nucleic acid extraction methods have limitations in capturing low levels of cfDNA and risk capturing high levels of high molecular weight contaminating genomic DNA. We developed a liquid-phase based DNA extraction method (PHASIFY™ ENRICH; Phase Scientific International Ltd., Kwun Tong, Hong Kong) that leverages aqueous two-phase systems (ATPS) to purify, concentrate, and enrich for cfDNA to improve mutant signal detection. Methods: The PHASIFY ATPS consists of unique formulations that are optimized to drive cfDNA into one liquid phase and plasma contaminants into the other, achieving simultaneous target isolation and concentration. In a previous study using the PHASIFY technique, we demonstrated improved cfDNA and mutant copy yield over solid-phase methods. We further developed an additional size-selection feature (PHASIFY ENRICH) to remove high molecular weight genomic DNA (&amp;gt;500 bp) and to reduce signal noise for molecular diagnostics. We compared cfDNA recovery and mutation detection from plasma samples extracted with the industry standard QIAamp Circulating Nucleic Acid kit (QCNA; Qiagen, Hilden, Germany) and PHASIFY ENRICH cfDNA extraction kit. According to the respective kit protocols, the cfDNA was extracted from plasma from patients with advanced cancers, including those known to shed low amounts of cfDNA into circulation (e.g., low grade serious ovarian, appendiceal, thyroid, and pancreatic cancer), with known tissue mutation status. Total DNA recovery was determined with the Quant-iT PicoGreen dsDNA Assay Kit. Mutation status was determined with probe-based Droplet Digital PCR (ddPCR; Bio-Rad, Hercules, CA). In a follow up study, we tested a similar cohort of plasma samples that were previously tested negative for cfDNA mutation status when using QCNA cfDNA extraction, despite known positive mutation status from corresponding tumor tissue genotyping. Results: In the head-to-head study against QCNA, 57 plasma samples from 23 unique patients with diverse advanced cancers with known tissue mutation status were used for cfDNA extraction. The PHASIFY ENRICH method resulted in a 153% increase in median mutant copy recovery compared to QCNA (P = 0.01 Wilcoxon Signed-Rank). In the follow up study, 47 plasma samples from 31 patients with diverse advanced cancers and prior cfDNA negative mutation status with tissue positive mutation status were used for extraction. Of the 47samples extracted by PHASIFY ENRICH, 9 samples resulted in a conversion to positive cfDNA mutation status compared to no mutant signals when previously using QCNA. Conclusion: Our results indicate that PHASIFY ENRICH provides improved cfDNA mutation detection by increasing cfDNA recovery and reducing contaminating genomic DNA background. Citation Format: Filip Janku, Helen Huang, David Y. Pereira, Masae Kobayashi, Steven G. Call, Kristen T. Woodbury, Felix Chao, Daniel R. Marshak, Ricky Y. Chiu. Enhancing cell-free DNA mutation detection with novel liquid-phase extraction [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 575.

Molecular Responses after Switching from a Standard-Dose Twice-Daily Nilotinib Regimen to a Reduced-Dose Once-Daily Schedule in Patients with Chronic Myeloid Leukemia: A Real Life Observational Study (NILO-RED)

IntroductionStandard dosing regimen for the BCR-ABL1 tyrosine kinase inhibitor (TKI) nilotinib in chronic myeloid leukemia (CML) is 300mg twice daily (BID) in the frontline setting and 400mg BID in the 2nd line setting or beyond. Patients (pts) are instructed to fast within 2 hours before and 1 hour after medication intake due to significant high-fat food effects on drug bioavailability. On-label dose adjustments to a lower-dose once-daily (QD) regimen are recommended solely in case of significant toxicity.Study goalThe primary aim of our observational two-center study was to ask whether nilotinib-treated adult CML pts were able to maintain major molecular responses (MMR) within 1 year after conversion to a more convenient nilotinib QD regimen at reduced doses, regardless of reasons for tapering nilotinib. Pts with minor transcripts or a history of blast crisis or allogeneic stem cell transplantation were excluded.PatientsEighty one pts in chronic phase were identified and included and results from the first 67 pts with a follow-up of at least 12 months after the switch to the once-daily dose-reduced nilotinib regimen are presented. Median age at study entry was 52 years (range: 19-82). There were 25 males (37.3%) and the Sokal score was low, intermediate, high or unknown in 43.3%, 37.3%, 13.4% and 6% of pts, respectively. All pts had major-type BCR-ABL1 transcripts. Nilotinib was started as 1st line TKI at 300mg BID in 68.6% of pts and as 2nd or subsequent line TKI at 400 or 300 mg BID in 31.4% of pts due to intolerance, warning, resistance or absence of a deep molecular response on prior therapy. Median duration of nilotinib prior switching to a reduced QD dosing was 29 months (range: 1-94) and median duration of MMR was 25 months (range: 3-212). Primary reason for the switch to a reduced QD nilotinib dosing was non-serious adverse events in 37.3% of pts and improvement in pts convenience in 62.7% of pts. Nilotinib was reduced down to 450mg QD, 400mg QD or 300mg QD in 86.6%, 10.4% and 3% of pts, respectively. A 2nd dose reduction from 450mg to 300mg QD was performed in 30 pts after a median time of 18 months (range: 4-37). The median duration of low-dose nilotinib QD was 24 months (5-54). No severe tolerance issues were encountered and no ischemic cardiovascular events occurred. Eleven pts stopped nilotinib due to treatment-free remission attempt (n=10) or persistent adverse event (n=1). All pts were alive at last follow-up.Evolution of molecular responsesAt baseline, the number of pts in MMR, MR4 and ≥MR4.5 was 9 (13.4%), 17 (25.4%) and 41 (61.2%), respectively and 32 pts (47.8%) had undetectable BCR-ABL1 transcripts. Median BCR-ABL1/ABL1 % IS was 0.006% (range: 0.0007-0.063) and median ABL1 copy number in pts with undetectable transcripts was 87259 (range: 36018-921339). Only 2 pts treated with 1st line nilotinib lost MMR 4 and 6 months after QD dose reduction of nilotinib with BCR-ABL1/ABL1 % IS at 0.13 and 0.11%, respectively. Both pts spontaneously regained MMR 4 months later without any treatment modification. Kaplan-Meier analysis of survival without unconfirmed MMR loss was 97% (95% CI: 92.9-100) at 12 months and analysis by molecular response category showed that none of the pts who were at least in MR4 at baseline lost MMR (Figure 1). At last follow-up on low-dose QD nilotinib, the number of pts in MMR, MR4 and ≥MR4.5 was 4 (6.3%), 11 (16.4%) and 52 (77.6%), respectively and 37 pts had undetectable transcripts (55.2%). Median BCR-ABL1/ABL1 % IS was 0.0035% (range: 0.00025-0.058) and median ABL1 copy number in pts with undetectable transcripts was 96000 (range: 23285-472000).Ten pts in long-lasting deep molecular response stopped nilotinib without further treatment after a median duration of TKI and of nilotinib of 60 months (range: 50-149) and 56 months (range: 42-87), respectively. Their follow-up after nilotinib discontinuation was 18 months (range: 5-33). Only 1 of them lost MMR and restarted therapy 5 months later while the others remained treatment-free.ConclusionSwitching to a nilotinib QD regimen at reduced doses as maintenance therapy after achievement of a MMR on standard-dose nilotinib BID schedule is feasible and safe in chronic phase CML pts regardless of prior treatment history and does not compromise the anti-leukemic efficacy of nilotinib. Our results may pave the way for clinical trials aiming at systematic avoidance of overtreatment and improvement of pts convenience in treatment with nilotinib. [Display omitted] DisclosuresRea:Pfizer: Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Honoraria. Cayuela:BMS: Honoraria; Novartis: Honoraria. Etienne:BMS: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Consultancy, Research Funding.

Use of Leflunomide for Treatment of Cytomegalovirus Infection in Recipients of Allogeneic Stem Cell Transplant

Background :Reactivation of cytomegalovirus (CMV) commonly occurs post allogeneic stem cell transplant (ASCT). The incidence is at least 40-50% in India, where most patients and donors are CMV seropositive. Ganciclovir, although the drug of choice, is expensive and associated with significant toxicities. Second line agents like foscarnet and cidofovir are not available in India leaving few therapeutic options. Leflunomide, an anti rheumatoid arthritis drug, acts by inhibiting virion assembly of CMV and has been effective in post renal transplant CMV infections. This drug is inexpensive and has a favourable toxicity profile. There have been only occasional case reports of leflunomide post ASCT. Here we report the efficacy of leflunomide in CMV reactivation in ASCT setting.Methods :All patients who underwent ASCT at our centre between April 2015 and February 2017 were included in this retrospective study. Serologic evaluation for CMV infections was done at baseline for all patients and donors. Conditioning regimens used were either myeloablative [total body irradiation (TBI)-cyclophosphamide (Cy)] or reduced intensity (fludarabine with either melphalan, or Cy or treosulfan +/- 2 gray TBI). Graft versus host disease (GVHD) prophylaxis used was cyclosporine or tacrolimus with methotrexate or mycofenolate mofetil for matched sibling donor (MSD) and matched unrelated donor (MUD) transplants. Post transplant Cy was used for haplo-identical transplants (HIT).Monitoring was done twice weekly using quantitative CMV DNA polymerase chain reaction (QPCR) on peripheral blood post transplant till day +120 or longer if patient was on steroids. Patients who were detected to have a positive QPCR greater than 500 copies/mL at 2 consecutive time points were started on pre-emptive therapy. Ganciclovir was the preferred agent for pre-emptive therapy, however in case of ongoing cytopenias, leflunomide was used. In case of clinical resistance to ganciclovir, leflunomide was added as second line. Leflunomide was started at a loading dose of 100 mg once a day for 3 days followed by daily maintenance dose of 20 mg till clearance of CMV (undetectable CMV DNA on QPCR at 2 consecutive time points) or intolerance. Failure of leflunomide was defined as > 1 log increase in CMV copy number at any time or if another anti CMV agent was added due to CMV disease or non-clearance. Invasive procedures such as broncho-alveolar lavage, colonoscopic biopsies, and liver biopsies were used to detect CMV disease, when clinically suspected.Results :Seventy patients underwent ASCT during this period. This included 47 MSD transplants, 18 HIT and 5 MUD transplants. There were 49 episodes of CMV reactivation in 43 patients (61%). The incidence was highest in HIT with 17 of 18 patients developing reactivation.Leflunomide was used in 24 (49%) of the 49 episodes of CMV reactivations. It was used as 1st line agent in 15 episodes and as 2nd line in 9. The median CMV copy number/ml was 0.82x 103 (range 0 to 13x103) on day of starting leflunomide. The median duration of use was 34 days (range 6 to 85 days). It was used pre-emptively in 20 episodes and as therapeutic (i.e. patient had end organ involvement) in 4. None of the patients had toxicity requiring stopping of the drug. Nine (38%) of the 24 CMV reactivation episodes cleared with leflunomide. When used as a 1st line agent, CMV infection cleared in 7 of the 15 episodes (46%) in comparison to ganciclovir as 1st line with which 12 (52%) of 23 episodes cleared.Seven of the 24 episodes had initial CMV burden more than 2 x 103 copies/mL. Leflunomide failed in all of these, as against clearance in 9 of the 17 episodes which had CMV less than 2 x 103 copies/mL. (p= 0.022). None of the patients who received leflunomide in therapeutic setting cleared CMV (0 of 4), compared to 9 of 20 with pre-emptive leflunomide. (p= NS).Conclusions:This is the largest study of using leflunomide for CMV reactivation in post ASCT setting to our knowledge. Leflunomide has considerable activity against CMV, however it is best used when CMV copies are less than 2000 /mL in pre-emptive setting. In therapeutic setting its role seems limited. A larger prospective study is warranted. DisclosuresNo relevant conflicts of interest to declare.

Strong correlation between urine and vaginal swab samples for bacterial vaginosis.

BackgroundVaginal swabs have been traditionally used for the diagnosis of bacterial vaginosis (BV). Currently, there are limited studies that have investigated the use of other sample types other than vaginal swabs for the detection of BV from South African populations. This study investigated whether urine can be used for the detection of BV-associated microorganisms in South African pregnant women.MethodsOne-hundred self-collected vaginal swabs and urine samples were obtained from women presenting for antenatal care at King Edward VIII Hospital in Durban. The BD MAX™ vaginal panel assay was used for diagnosing BV and droplet digital polymerase chain reaction was used to quantify Gardnerella vaginalis, Prevotella bivia, Atopobium vaginae and Lactobacillus crispatus. The absolute counts were determined on the QX200 Droplet Reader (Bio-Rad) using the QuantaSoft Software. Data analysis was performed with statistical computing software called R, version 3.6.1.ResultsMedian copy numbers obtained for G. vaginalis and P. bivia across urine and swabs in BV-positive samples were not significantly different (p = 0.134 and p = 0.652, respectively). This was confirmed by the correlation analysis that showed a good correlation between the two sample types (G. vaginalis [r = 0.63] and P. bivia [r = 0.50]). However, the data obtained for A. vaginae differed, and a weak correlation between urine and swabs was observed (r = 0.21). Bacterial vaginosis-negative samples had no significant difference in median copy numbers for L. crispatus across the urine and swabs (p = 0.062), and a good correlation between the sample types was noted (r = 0.71).ConclusionThis study highlights the appropriateness of urine for the detection of microorganisms associated with BV.

Phase I trial of fourth-generation chimeric antigen receptor T-cells targeting glypican-3 for advanced hepatocellular carcinoma.

4088 Background: GPC3 is highly expressed in hepatocellular carcinoma (HCC) and is a promising target for HCC immunotherapy. Earlier phase I trial results demonstrated that second-generation GPC3-specific chimeric antigen receptor (CAR-GPC3) T cells were well tolerated in advanced HCC patients and showed clinical benefits (Shi D, Clin Can Res, 2020). Further preclinical studies showed that CAR-GPC3 T cells combined with multi-tyrosine kinase inhibitors (TKIs) were more effective in killing GPC3+ HCC xenografts (Wu X, Mol Ther, 2019), and fourth-generation (4G) CAR-GPC3 T cells co-expressing a transcription factor more effectively killed GPC3+ HCC xenografts. Methods: In this single-arm, open-label, first-in-human phase I trial (NCT03980288), we investigated the safety and antitumor activity of autologous 4G-CAR-GPC3 T cells for GPC3+ heavily pretreated advanced HCC patients. Patients received 4G-CAR-GPC3 T cell infusion(s) after lymphodepletion pretreatment and 67% patients combined with TKIs. Adverse events (AEs) were graded by CTCAE 5.0. Cytokine release syndrome (CRS) was graded by American Society for Transplantation and Cellular Therapy criteria (2019). Tumor response was assessed by RECISTv1.1. Results: As of January 30th, 2021, 6 subjects with HBV-related metastatic HCC were enrolled. All had progressed on ≥ 2 lines of systemic therapy, with at least one TKI combined with anti-PD-1/PD-L1 immunotherapy or FOLFOX4 chemotherapy. After lymphodepletion, patients were treated with 1-2 cycles (totaling 2.5–5×108 CAR+ cells) of 4G-CAR-GPC3 T cell therapy. Among them, 1 patient received half dose of sorafenib and 3 patients received half dose of regorafenib along with cell infusion. No dose limiting toxicity (DLT) occurred. The maximum tolerated dose (MTD) was not observed. No patient withdrew from the trial due to AE. No treatment-related death or neurotoxicity occurred. The most common ≥ grade 3 AEs were hematological toxicity, mainly due to lymphodepletion, and patients recovered within 2 weeks after therapy. All patients developed CRS including 3 at grade 2 and 3 at grade 3. Patients recovered from CRS after tocilizumab therapy with corticosteroid (4 patients) or without. One patient achieved partial response and continued after 18 weeks. The objective response rate (ORR) and disease control rate (DCR) were 16.7% and 50%, respectively. The median progression free survival (mPFS) was 4.2 months. The median peak CAR-GPC3 copies were 5067 copies/ug gDNA, and CAR-GPC3 copies were detectable on day 28 ranging 113–2071 copies/ug gDNA. Conclusions: Our study is the first to report that 4G-CAR-GPC3 T cell therapy in combination with TKIs has a manageable safety profile while demonstrating potential antitumor activity for heavily pretreated advanced HCC patients; however, CRS should be diligently managed. Clinical trial information: NCT03980288.

Long-term follow-up results of a multicenter first-in-human study of the dual BCMA/CD19 Targeted FasT CAR-T GC012F for patients with relapsed/refractory multiple myeloma.

8014 Background: The dual CAR-T GC012F developed on the novel FasT CAR-T platform targeting B cell maturation antigen (BCMA), and CD19 was designed to improve depth of response and overall efficacy for CAR-T as therapy for Multiple Myeloma. Here, we present updated data for study (NCT04236011; NCT04182581) including additional pts treated. Methods: From October 2019 to July 2020, 19 heavily pretreated Relapsed/Refractory Multiple Myeloma (RRMM) pts (age 27-71) with a median of 5 prior lines (range 2-9) received a single infusion of GC012F. 94.7% (18/19) were high risk (HR- defined by mSMART), 5 pts had extramedullary disease, 1 pts presented with plasma cell leukemia, and 15/19 were refractory to last therapy. 4/19 pts had received prior anti- CD38, 18/19 pts prior IMiD. 18/19 pts were refractory to PI, 17 pts to IMiD, 3 pts being primary refractory. After lymphodepletion over 2-3 days (30 mg/m2/d, 300mg/ m2/d Flu/Cy) CAR-T cells were administered as single infusion at 3 dose levels: 1x105/kg (DL1) n=1, 2x105/kg (DL2) n=9 and 3x105/kg (DL3) n=9. Results: As of Jan 12, 2021 cut-off, 19 pts were evaluated for response. Overall response rate (ORR) was 94.7% - all responses VGPR or better (94.7% - 16/18 sCR, 2/18 VGPR) with all pts MRD- by flow cytometry (10-4-10-6) - earliest response d 28 post infusion. 100% of pts achieved a reduction of paraprotein, 18/19 a 100% reduction. Best response was MRD- sCR in 16/19 patients (84.2%). In DL3 (n=9) 4 additional pts were response evaluable for 6 mth follow-up: 100% (9/9) of pts achieved MRD-sCR as best response, 87.5% (7/8) of response evaluable pts maintained MRD-sCR at landmark analysis of 6 mths. At data cut off, the median time to follow up was 13.8 mths (6.1-16.4) – median duration of response not yet reached. Cytokine Release Syndrome (CRS) and ICANs were graded by ASBMT criteria: grade 1-2 n=16 (84.2%), grade 3 n=2 (10.5%). Median duration of CRS was 4 d (1-8 d). No grade 4/5 CRS or ICANS were observed. 2 deaths occurred on study and were assessed as not related to therapy – one reported previously; one patient was diagnosed with pseudomonas pneumonia on Day 52, refused life-saving treatment and passed. CAR-T median Tmax was 10 d (range 8-14d), median peak copy number (Cmax) was 127548 (16,011-374,346) copies /ug DNA with long duration of persistence of up to 60 weeks at time of data cut off. Patients continue to be monitored for safety and efficacy. Conclusions: BCMA-CD19 dual FasT CAR-T GC012F showed early, deep and durable responses with a high ORR (94.7% - VGPR and better) including a high MRD- sCR rate (DL3=100%, n=9) in high risk RRMM pts including those refractory to anti-CD38, PI and IMIDs with a favorable safety profile consistent with previous findings. BCMA-CD19 dual FasT CAR-T GC012F may present a new treatment approach for patients with RRMM including those with high-risk features refractory to standard of care. Clinical trial information: NCT04236011; NCT04182581.

Genomic profiling of esophageal squamous cell carcinoma to reveal actionable genetic alterations.

e16042 Background: Esophageal cancer is the eighth most common cancer in the world and more than half of global cases occur in China. Studies demonstrated that esophageal squamous cell carcinomas (ESCC) and esophageal adenocarcinomas (EAC) are two distinct disease entities. Due to the lack of effective therapies, the five-year survival rates of ESCC patients remain dismal. Therefore, there is an urgent need to establish a framework through genomic profiling to facilitate the development of precision therapies for ESCC. Methods: To characterize therapeutic targets in 118 Chinese ESCC patients, deep panel sequencing of 831 cancer genes (OncoPanscan, Genetronhealth) was performed on their tumor tissues and paired genomic DNA samples. Results: The most frequently mutated genes in our ESCC cohort were TP53 (97%), PIK3CA (19%), CDKN2A (18%), NOTCH1 (17%), KMT2D (15%), LRP1B (15%) NOTCH3 (15%) NFE2L2 (13%), and EP300 (12%). Consistent with previous reports, we found significantly elevated mutations in cancer-related genes including NOTCH1 (16.9%), NOTCH2 (3.2%), NOTCH3 (15.3%) and RB1 (9.3%). Importantly, 17.8% (21/118) patients in our cohort harbored the 11q13 amplicon ( CCND1, FGF3, FGF4 and FGF19). The median copy number was 8.19 (range 6.07-42.3). These patients can participate in clinical trials with FGFR inhibitor alone or in combination with CDK4/6 inhibitors. Additionally, we also observed frequent genetic alterations in the KEAP1 (Kelch-like ECH-associated protein 1)-NFE2L2 (nuclear factor erythroid 2 like 2)-CUL3 (cullin 3) pathway. 80% (12/15) of missense mutations in NFE2L2 were located at the KEAP1 binding domain of NRF2 protein. These mutations were either around the ETGE motif (D77G, E79Q, G81V/D and E82D) or the DLG motif (D27V, I28T, D29G, L30F, G31E, V32E, R34G). We also identified four missense mutations of KEAP1 and one alternation of CUL3 in splicing site. Taken together, 17% (20/118) of ESCC patients harbored mutations in the NFE2L2/KEAP1/CUL3 pathway, which may be eligible for clinical trials of glutaminase inhibitor telaglenastat. Two patients had high level of ERBB2 amplification which can be targeted with anti-HER2 therapy. Furthermore, 11.9% (14/118) patients carried activating PIK3CA mutations including N345K, E542K, E545K, M1043I and H1047R which may be targeted by PIK3CA inhibitor alpelisib. Lastly, patients with loss-of-function mutation in NF1 (n = 4), STK11 (n = 1) and PTEN (n = 3) can be respectively targeted with MEK inhibitor and mTOR inhibitor. Overall, 43% of patients in our ESCC cohort had actionable genetic mutations with corresponding precision therapy options. Conclusions: Our findings indicated that amplification of the 11q13 amplicon and dysfunction of the KEAP1-NRF2-CUL3 axis are the major driving events of ESCC. The results of genomic profiling can guide physicians to enroll a significant portion of ESCC patients into genomically matched clinical trials.

Evaluating a clinically validated circulating tumor HPV DNA assay in saliva as a proximal biomarker in HPV+ oropharyngeal squamous cell carcinoma.

6063 Background: HPV genomic DNA in plasma and saliva has been widely studied, however more recently, circulating tumor human papillomavirus DNA (ctHPVDNA) has emerged as a reliable biomarker for surveillance in HPV+ oropharyngeal squamous cell carcinoma (OPSCC). A commercial assay for this biomarker distinguishes tumor-derived viral DNA (tumor-tissue modified viral DNA or TTMV) from other non-cancer associated sources of HPV DNA. The use of this technology has been previously described in plasma, but its utility in saliva is currently unknown. Methods: A prospectively collected and banked biospecimen repository was used to identify 46 patients with HPV+ OPSCC with paired pre treatment plasma and saliva samples. All samples were assessed for DNA integrity and TTMV using a clinically validated ddPCR-based assay (NavDx™; Naveris Inc, Natick, MA) to measure TTMV for HPV-16, -18, -31, -33 and -35 from frozen plasma and saliva samples. Retrospective chart review was performed to collect clinical and pathological data. Graphpad was used for statistical analysis. Spearman’s r was used to correlate TTMV copies in saliva and plasma. Wilcoxon test was used to compare between sample types. Mann-Whitney test was used for categorical variables. Results: TTMV DNA was detectable in 43 of 46 plasma samples and in 44 of 46 saliva samples. One plasma sample failed quality control measures, one of each sample type had undetectable TTMV, and one of each type was indeterminate. Of 41 evaluable patients with paired samples, there were 38 (93%) males, 36 (88%) were stage I-II, 5 (12%) were stage III-IV (AJCC 8th, clinical staging), and 25 (61%) had a history of smoking with a median of 37.5 pack years. TTMV was significantly enriched in saliva compared to plasma (p&lt;0.0001), with median copy number 14,139 copies/ml (IQR=193,339.5) and 774.7 copies/ml (IQR=4,826.1), respectively. There was a significant positive correlation between plasma and saliva TTMV levels (r=0.344, p=0.028). There was no difference in overall stage for either specimen type. There was a trend in both sample types toward higher TTMV in patients with a history of smoking. Pack-year history was available for 38 (93%) patients in the final cohort. When grouping by pack-years, plasma TTMV approached significance (p=0.058) while high saliva TTMV was significantly associated with &gt;10 pack-year history (p=0.011). Conclusions: This is the first study to demonstrate successful quantification of tumor-tissue modified HPV DNA in saliva. Compared to plasma, pre treatment saliva samples demonstrated significantly higher levels of TTMV. TTMV distinguishes ctHPVDNA from other sources of HPV. These data highlight the potential use of TTMV detection in saliva for early detection of HPV+ OPSCC as well as its potential role in local surveillance after treatment. More research is needed to elucidate the effects of smoking on TTMV levels.

The Stable Matching Problem in TBEV Enzootic Circulation: How Important Is the Perfect Tick-Virus Match?

Tick-borne encephalitis virus (TBEV), like other arthropod-transmitted viruses, depends on specific vectors to complete its enzootic cycle. It has been long known that Ixodes ricinus ticks constitute the main vector for TBEV in Europe. In contrast to the wide distribution of the TBEV vector, the occurrence of TBEV transmission is focal and often restricted to a small parcel of land, whereas surrounding areas with seemingly similar habitat parameters are free of TBEV. Thus, the question arises which factors shape this focal distribution of TBEV in the natural habitat. To shed light on factors driving TBEV-focus formation, we used tick populations from two TBEV-foci in Lower Saxony and two TBEV-foci from Bavaria with their respective virus isolates as a showcase to analyze the impact of specific virus isolate-tick population relationships. Using artificial blood feeding and field-collected nymphal ticks as experimental means, our investigation showed that the probability of getting infected with the synonymous TBEV isolate as compared to the nonsynonymous TBEV isolate was elevated but significantly higher only in one of the four TBEV foci. More obviously, median viral RNA copy numbers were significantly higher in the synonymous virus–tick population pairings. These findings may present a hint for a coevolutionary adaptation of virus and tick populations.

Human herpesvirus 6 reactivation in unmanipulated haploidentical hematopoietic stem cell transplantation predicts the occurrence of grade II to IV acute graft-versus-host disease.

Human herpesvirus 6 (HHV-6) reactivation is relatively common after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the incidence of HHV-6 reactivation and the clinical outcomes following unmanipulated haploidentical HSCT (haplo-HSCT) remain unknown. We prospectively monitored blood HHV-6 DNA using real-time quantitative polymerase chain reaction weekly until day 100 post unmanipulated haplo-HSCT in patients with hematological malignancies. From November 2016 to March 2017, 102 patients (58 male and 44 female, median age 25(2-58) years old) were enrolled. Within 100days post-transplantation, 27 patients (27/136, 19.9%) developed HHV-6 viremia with a median onset time of 14 (7-98) days. The cumulative incidence of HHV-6 reactivation on day 100 post-HSCT was 25.5±4.3% in haplo-HSCT. The median HHV-6 copy number was 1.45×103 (5.48×102 -2.00×104 ) copies/ml. The HHV-6 viremia duration time was 7days in 23 patients, 14days in one patient and 21days in one patient. In multivariate analysis, prior HHV-6 reactivation was an independent risk factor for grade 2-4 graft-versus-host disease (GVHD). But it did not influence the overall survival (OS)(HR 1.624, 95%CI 0.768-3.432, P=.204), disease-free survival (DFS) (HR 1.640, 95%CI 0.799-3.367, P=.177) and non-relapse mortality (NRM) (HR 1.644, 95%CI 0.670-4.038, P=.278). The reactivation of HHV-6 after unmanipulated haploidentical transplantation predicts the occurrence of grade 2-4 a-GVHD, but it may not influence the overall survival (OS), disease-free survival (DFS) and non-relapse mortality (NRM).

Clinical Results of a Multicenter Study of the First-in-Human Dual BCMA and CD19 Targeted Novel Platform Fast CAR-T Cell Therapy for Patients with Relapsed/Refractory Multiple Myeloma

Introduction To further improve efficacy and duration of response of CAR-T therapy for Relapsed/Refractory Multiple Myeloma (R/R MM), we have designed a dual FasT CAR-T targeting both B cell maturation antigen (BCMA), a well-established MM target, and CD19, which is expressed on MM cells and their progenitors. Here we report early results from the first-in-human multicenter clinical study (NCT04236011; NCT04182581) to determine safety, pharmacokinetics (PK) and efficacy of BCMA-CD19-directed FasT CAR-T (GC012F) in patients with R/R MM. Methods The BCMA-CD19 dual CAR was constructed by linking BCMA and CD19 scFv, joined by a CD8 hinge, transmembrane domain, co-stimulatory domain and CD3z. Peripheral blood (PB) mononuclear cells were obtained by leukapheresis, T cells were isolated and CAR-T cells were manufactured (FasT CAR platform). From September 2019 to April 2020, we enrolled 16 heavily pretreated R/R MM patients (Age range 27-71), with a median of 5 prior lines of therapies (range 2-7), 93.8% (15/16) of these patients were high risk as defined by mSMART criteria, 5 had extramedullary disease. 4 out of 16 patients had received prior anti CD38 therapy, 93.8% (15/16) patients had received prior IMiD, all patients received at least 1 prior PI and corticosteroids with 3 patients being primary refractory to last therapy. Prior to CAR-T infusion patients received a conditioning regimen over 3 days of 30 mg/m2/d fludarabine and 300 mg/m2/d cyclophosphamide. CAR-T cells were administered in a single infusion at 3 dose levels 1x105/Kg (DL1) (1 patients), 2x105/Kg (DL2) (9 patients) and 3x105/Kg (DL3) (6 patients). Results As of July 17th 2020, all 16 patients were evaluable for response assessment, 15 out of 16 patients responded to treatment (ORR 93.8%) in all dose levels with the earliest response observed at day 28. Best response to date is MRD- CR/sCR in 9/16 patients (56.3%). In DL3 100% (6/6) of patients achieved sCR, 3 at data cut off had been confirmed by PET-CT. In all response evaluable patients, 78.6% (11/14) were MRD- by flow at month 1, and 100% at month 3 (11/11) and 6 (10/10) (sensitivity by flow cytometry measured at 10-4 in 7 patients, and at 10-6 in 9 patients tested by EuroFlow with at least 1.08x107 cells analyzed). At data cut off, the median follow up time was 7.3 months, the longest follow up was 10 months post infusion. CAR-T PK in PB was monitored by qPCR and flow cytometry. The CAR-T median proliferation peak was reached on Day10 (Day8-Day14), and the median peak copy number was 140,982 (16,011-374,346) copies /ug DNA. GC012F showed an acceptable safety profile with 14 out of 16 patients experiencing a cytokine release syndrome (CRS) grade 1-2 (n=14, 87.5%) and 2 grade 3 (n=2, 12.5%). The median duration of CRS was 4 days (1-8 days). No neurotoxicity of any grade was observed. One patient (DL2) presented with fever and died shortly after Day 78 of unknown cause during the COVID-19 Pandemic. Two patients had progression of extramedullary disease while achieving MRD negativity at month 1 and 3, respectively. At landmark analysis at 6 months, all patients in DL3 had achieved and maintained MRD- sCR including patients heavily pretreated including Daratumumab - among them 83.3% (5/6) patients in DL3 had high risk features according to mSMART criteria, and 5 out of 6 patients in DL3 were assessed by 10-6 Euroflow for MRD. The study is still enrolling patients and we will continue to be monitoring safety and efficacy including duration of response. Conclusion The data of BCMA-CD19 dual FasT CAR-T showed an early and high response rate with 93.8% ORR to date with a promising early high MRD-sCR rate in the highest dose level DL3 (100%) which was sustained with a median duration of follow up of 7.3 months at cut off. The data shows very promising activity of the BCMA-CD19 dual FasT CAR-T with a favorable safety profile in R/R MM patients. 93.8% (15/16) of the treated patients exhibited high risk features - a specifically difficult to treat patient population which remains a high unmet medical need in Multiple Myeloma. This data indicates that BCMA-CD19 dual FasT CAR-T (GC012F) may present an effective new treatment option for patients with R/R MM including those with high-risk features who failed multiple prior therapies including anti-CD38. The study is still ongoing and enrolling patients, we will update the results as they become available. Disclosures Zhao: Gracell Biotechnologies Ltd: Current Employment. Han:Gracell Biotechnologies Co., Ltd.: Current Employment. Chen:Gracell Biotechnologies Ltd: Current Employment. Xu:Gracell Biotechnologies Ltd: Current Employment. Zhang:Gracell Biotechnologies Ltd: Current Employment. He:Gracell Biotechnologies Co., Ltd.: Current Employment. Shi:Gracell Biotechnologies Ltd: Current Employment. Han:Gracell Biotechnologies Co., Ltd.: Current Employment. Ye:Gracell Biotechnologies Co., Ltd.: Current Employment. Wang:Gracell Biotechnologies Ltd: Current Employment. Liu:Gracell Biotechnologies Co., Ltd.: Current Employment. Shen:Gracell Biotechnologies Ltd: Current Employment. Cao:Gracell Biotechnologies Ltd: Current Employment. Sersch:Gracell Biotechnologies Co., Ltd.: Current Employment.

Cerebrospinal Fluid (CSF) Analysis of Tumor-Specific Cell-Free DNA (cfDNA) As a Diagnostic and Prognostic Tool for Central Nervous System (CNS) Invasion in Lymphoma

Background: CNS invasion in aggressive lymphoma (including diffuse large B-cell [DLBCL], double-hit [DHL], Burkitt [BL] and plasmablastic lymphoma [PBL]) remains a major diagnostic and therapeutic challenge. Parenchymal CNS invasion requires a brain biopsy to diagnose, and conventional CSF evaluations lack sensitivity. Furthermore, determining which patients (pts) without CNS disease at diagnosis might be at high risk of future CNS relapse, and thus would most benefit from aggressive CNS prophylaxis, is difficult. Predictors that use clinical variables (e.g. the CNS-IPI) can select a high-risk group with ~10% risk of CNS relapse, but ~50% of events occur in low/intermediate-risk groups. We evaluated a novel CSF assay that detects clonotype-specific cell-free DNA (cfDNA) by next-generation sequencing (NGS) combined with multiplex PCR (Chang T et al, BMC Cancer 2020). This assay can identify minimal residual disease (MRD) in the plasma of pts with DLCBL and can predict systemic relapse (Roschewski et al, Lancet Oncol 2015). Methods: To examine the diagnostic sensitivity of the NGS-MRD assay in the CSF, we prospectively collected CSF from 6 pts with lymphomas who had known parenchymal or leptomeningeal CNS invasion. We also tested stored DNA from historical CSF samples of 8 pts with CNS lymphoma who tested negative by conventional IGH-PCR. We then enrolled 19 pts with newly diagnosed high-risk lymphomas without CNS invasion to examine the prognostic value of the NGS-MRD assay in the CSF collected at diagnosis. In the two-step assay, tumor-specific clonotype sequences were first identified from genomic DNA in primary tumor specimens using NGS of rearranged IGK, IGH (VJ or DJ), and IGL loci. Tumor-specific clonotypes were then selected for tracking in the CSF (split into acellular CSF fluid and cell pellet) and in paired plasma samples. The assay quantitated copy numbers of each tracked sequence per mL of acellular CSF or per 106 cells in the pellet, as well as tumor clonotype frequency (%) per all B-cells. Results: The NGS-MRD detected tumor-specific clonotype in 100% of CSF samples from pts with known CNS lymphoma (n=6; Fig A), including pts with isolated parenchymal-only CNS disease that was not detected by CSF cytology, flow cytometry, or IGH-PCR. For pts with CNS parenchymal disease, median cfDNA copy count in the CSF fluid was 2 /mL (range, 0.4-929) and median clonotype frequency was 9.0% (range, 0.03-68.9%). For pts with leptomeningeal disease, these values were 2233 /mL (1.2-5620) and 37% (1.7-98.4%), respectively (Fig. B). Furthermore, the NGS-MRD assay detected tumor clonotypes in DNA isolates from 6 historical CSF samples (collected in 2014-2018) of pts with known CNS disease, in which lymphoma was not detectable by conventional IGH-PCR (2 additional historical samples failed DNA quality check). Among 19 newly diagnosed pts with no known CNS invasion (median age 57, 42% women, median LDH 352 IU/L), 12 (63%) had DLBCL with high risk of CNS relapse by virtue of high CNS-IPI or epidural tumor, and 7 had other high-risk histologies (2 DHL, 2 BL, HIV+ PBL, and 2 lymphoblastic leukemias). The CSF NGS-MRD assay was positive for tumor-specific clonotype in 8 pts (42%). Median cfDNA copy count in the CSF fluid was 3 /mL (range, 0.9-15.6) and median clonotype frequency was 18.6% (range, 1.4-28.5%). We observed no significant correlation between the amount of cfDNA and number of red cells /mL of CSF (a measure of potential CSF contamination by blood during collection procedure; Fig. C) or between the amount of cfDNA in plasma and in CSF (Fig. D). With median follow-up of 11 months, no pts with negative baseline NGS-MRD assay in CSF relapsed, whereas 2 of 10 with a positive assay had a CNS relapse (12-month incidence, 29%; Fig. E), despite both pts having negative CNS imaging, CSF cytology, flow cytometry, and IGH-PCR at diagnosis. Conclusions: In this proof-of-concept study, the NGS-MRD CSF assay showed 100% sensitivity for diagnosing intraparenchymal CNS invasion in aggressive lymphomas that were not detected in the CSF using conventional methods. In a prospective cohort of newly diagnosed pts without baseline CNS invasion but at high clinical risk for CNS relapse, the assay identified lymphoma-derived cfDNA in 42% of cases. The 100% sensitivity and 100% negative predictive value of the assay for subsequent CNS relapse warrants further prospective evaluation as a potential tool for personalizing CNS-directed prophylaxis. Disclosures Olszewski: Spectrum Pharmaceuticals: Research Funding; TG Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Genentech, Inc.: Research Funding. Mullins:Adaptive Biotechnologies: Current Employment, Other: shareholder.

Abstract 4743: Genetic characteristics of PanNETs, Rectal NETs, Thoracic NETs and its correlation with efficacy of chemotherapy

Abstract Background: Neuroendocrine tumors (NETs) are clinical heterogeneous. To date, no studies have focused on the genetic characteristics of NETs at different anatomical sites in the Chinese cohort. We aim to explore the genetic similarities or differences between NETs at different sites, and evaluate the predictive indicators of chemotherapy based on Temozolomide. Methods: The patients were from a prospective clinical study STEM (from a single institute, NCT03204032, NCT03204019). This cohort included 47 patients with NETs, including 14 Pancreatic NETs (PanNETs), 11 Rectal NETs, 14 Thoracic NETs, and 8 Others. Whole exome sequencing (WES) was performed. Results: A total of 76,057 somatic mutations were detected in 16,510 genes. The median tumor mutation burden (TMB) was 13.4, 10.5, 11.0 and 11.9 in PanNETs, Rectal NETs, Thoracic NETs and Others, respectively. After sequencing depth filtering, they were 3.5, 3.9, 3.3 and 3.6, respectively. Low intra-tumoral heterogeneity (ITH) was observed in NETs. The median ITH index was 2, 2.5, 2.5 and 2 in PanNETs, Rectal NETs, Thoracic NETs and Others, respectively; the median Shannon diversity index (SDI) was 0.23, 0.13, 0.23 and 0.24, respectively. The driver gene mutations varied in NETs at different sites. The recurrently mutated driver genes were MEN1, ATM, TSC2, PLXNB2, MUC6, AMER1 and USP9X for PanNETs; APC, APOB and FAT1 for Rectal NETs; KMT2B and CACNA1A for Thoracic NETs. Copy number analysis showed that copy number loss frequently occurred in all NETs. The median copy number variation burden (CNV burden) was 34.0, 10.9, 16.1 and 11.6 in PanNETs, Rectal NETs, Thoracic NETs and Others, respectively. The CNV burden of PanNETs was higher than others. Based on the results of CNVs, NETs at different sites could be distinguished by linear discriminant analysis (LDA). We analyzed the correlation between genetic characteristics and progression-free survival (PFS). As a result, NETs with higher TMB (&amp;gt;10 /Mb, P=0.043) or higher CNV burden (&amp;gt;20, P=0.047) showed statistically longer PFS. More patients should be included to find a correlation between genetic characteristics and PFS. Conclusions: NETs at different sites harbored different somatic driver mutations, and copy number loss was widespread in NETs. NETs with higher TMB or higher CNV burden showed statistically longer PFS. Citation Format: Yihebali Chi, Weili Liu, Lijie Zuo, Yuehua Wang, Weijing Cai, Susheng Shi, Yanying Ge, Rui Li, Lijie Song, Yiqi Yang, Zheng Liu, Xiaowu Dou, Hong Zhao. Genetic characteristics of PanNETs, Rectal NETs, Thoracic NETs and its correlation with efficacy of chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4743.

Non-amplification genetic alterations of HER2 gene in non-small cell lung carcinoma

AimsThe present study investigated the incidence and spectrum of human epidermal growth factor receptor 2 (HER2) mutations, associated clinicopathological characteristics and the co-occurrence of HER2 gene amplification in the HER2...

Association analysis of KMT2D copy number variation as a positional candidate for growth traits.

Copy number variations (CNVs) are an important source of genetic variation, which can affect a wide range of economic traits by diverse mechanisms. KMT2D (Lysine methyltransferase 2D) is an important positional candidate for growth traits. Quantitative trait loci (QTLs) with large effects on economically important traits cover the KMT2D gene. The KMT2D gene overlays a CNV within its exons, hence it was chosen as a crucial candidate gene to study the association between CNV and growth traits. Further, KMT2D, a major mammalian histone H3K4 mono-methyltransferase, plays a critical role in regulating development, differentiation, metabolism, and tumor suppression. Therefore, we proposed the hypothesis that KMT2D CNV may have phenotypic effects on sheep growth traits. In our study, KMT2D CNVs in three Chinese sheep breeds were detected by quantitative polymerase chain reaction (qPCR), and the loss copy was found to be the dominant genotype. Association analysis between growth traits and KMT2D CNV was also performed, which revealed that individuals with the median copy showed better performance than those with the loss copy in all three breeds. This research suggested that KMT2D CNV can be used as a promising marker for sheep molecular breeding.

FGFR2/3 genomic alterations (GA) in cell-free (cf)DNA from patients (pts) with advanced urothelial carcinoma (aUC).

565 Background: Erdafitinib is approved in pts with aUC with relevant FGFR2/3 GA. BLC2001 trials in pts with activating FGFR2/3 mutations reported 40% ORR to erdafitinib (49% for those with single nucleotide variants [SNVs] and 16% with fusions), 39% stable disease rate, and potentially reduced response to anti-PD-L1. Genomic profiling with plasma cfDNA next-generation sequencing (NGS) is increasingly used to identify targetable GA in pts with advanced solid tumors and presents a minimally invasive option for identification of FGFR2/3 GA. Methods: Genomic data from the Guardant360 database were queried from clinical results released from 10/19/15 - 8/28/19 for clinical samples submitted with diagnoses of aUC or related diagnoses (e.g. bladder cancer, renal pelvis carcinoma). All assays included FGFR2/3 fusions and complete sequencing of all critical exons harboring sensitizing FGFR2/3 SNVs. Results: 1349 results from 1096 unique pts were identified. Somatic GA were identified in 1192 tests (88%) from 997 pts. Fusions and/or nonsynonymous SNVs in FGFR2/3 were identified in 201 pts (20%); 141 pts (14%) had at least one characterized activating FGFR2/3 GA. Of 34 pts (3.4%) with FGFR3 fusions, partners included TACC3 (32), JAKMIP1 (1), and TNIP2 (1). Overall, most SNVs identified in FGFR3 were predicted to be activating (103/125, 82%) whereas in FGFR2 most were variants of uncertain significance (VUS; 62/72, 86%). Of 89 unique variants (59 in FGFR2, 30 in FGFR3), 19 (21%) were activating mutations (7 in FGFR2, 12 in FGFR3). The most common activating SNVs in FGFR3 were S249C (58 pts), Y373C (20) and R248C (10), and in FGFR2 was N549K (4). VUS in both genes were individually uncommon (no VUS recurring in &gt;3 pts). Median copy number-adjusted clonality of SNVs in FGFR3 was higher than those in FGFR2 (0.80 vs 0.20); this remained true when limiting to only characterized activating mutations (0.84 vs 0.17). Conclusions: cfDNA NGS analysis identifies fusions and a broad spectrum of SNVs in FGFR2/3, including heterogeneous subclonal mutations, at a rate similar to reported tissue testing. cfDNA is a minimally invasive option for pts with aUC to assess candidacy for erdafitinib and clinical trials.

Lenti-D Hematopoietic Stem Cell Gene Therapy Stabilizes Neurologic Function in Boys with Cerebral Adrenoleukodystrophy

Cerebral adrenoleukodystrophy (CALD) is a rare metabolic disorder in which rapid and progressive inflammatory demyelination leads to irreversible loss of neurologic function and death. There is no approved treatment for CALD, although Lenti-D Drug Product (DP) is in development as an autologous hematopoietic stem cell (HSC) gene therapy. We have previously shown promising results from the ALD-102 clinical study of the safety and efficacy of Lenti-D DP in boys ≤17 years old with CALD; based on data from 29 patients, Lenti-D DP was generally safe and led to stabilization of disease progression. The primary efficacy endpoint in ALD-102 is the proportion of patients who are alive and free of major functional disabilities (MFD) at Month 24. The primary safety endpoint is the proportion of patients who experience acute (≥Grade 2) or chronic graft-versus-host disease (GVHD) by Month 24. ALD-102 has completed enrollment, with 32 boys having received Lenti-D DP as of April 2019. The median DP cell dose was 11.4 (min - max, 5.0 - 20.1) × 106 CD34 cells/kg; median DP vector copy number was 1.2 (min - max, 0.5 - 2.7) copies/diploid genome. Median day of neutrophil and platelet engraftment was 13.0 days (min-max, 11.0 - 41.0) and 32.0 days (min - max, 16.0 - 60.0), respectively. The median follow-up time for the cohort of 32 patients is 21.2 months (min - max, 0.0 – 60.2). Fifteen patients have completed 24 months of follow-up in ALD-102 and continue to be free of major functional disabilities (MFD) through their last follow-up in a long-term extension study. Fourteen patients remain in ALD-102; the longest follow-up among these patients is 20.4 months. Two patients were withdrawn and referred for allo-HSCT before their Month 24 visit; another experienced early and rapid disease progression while on-study resulting in MFDs and death. All other Lenti-D DP-treated patients showed stabilization of neurologic function score at their last follow-up (stable NFS was defined as NFS ≤4 without a change of >3 from baseline). There have been no reports of graft failure, GVHD, or transplant-related mortality; recorded adverse events are consistent with myeloablative conditioning. There is no evidence of replication competent lentivirus or insertional oncogenesis. Lenti-D DP appears to stabilize neurologic disease progression and continues to show favorable safety with the longest follow-up at 60.2 months. Additional follow-up is ongoing to assess durability of effects and long-term safety.

Non-invasive Molecular Detection of Minimal Residual Disease in Papillary Thyroid Cancer Patients.

Background: Papillary thyroid cancer (PTC) is the most common type of thyroid malignancy. Serum thyroglobulin (Tg) levels are used to monitor PTC treatment response and recurrences however, in about 25% of the cases the sensitivity of this method is compromised due to either the presence of neutralizing anti-Tg antibodies (TgAb) or the absence of Tg in less differentiated tumors. Up to 80% of PTC tumors harbor the c.1799T>A hotspot mutation in the BRAF gene (BRAFV600E). Here, we assessed the potential use of plasma cell-free BRAFV600E mutant tumor DNA (ctDNA) levels in determining the minimal residual tumor status of PTC patients.Methods: Patients were classified as either having persistent disease (PD) or no evidence of disease (NED) based on clinicopathological assessments. Tumor BRAFV600E status was determined by both direct sequencing and digital PCR. Plasma total cell-free BRAFV600 wild type DNA (cfDNA) and ctDNA fractions circulating in the plasma of PTC patients were determined by an emulsion based-digital PCR and total ctDNA was quantified by 3D digital PCR. The total ctDNA levels (copies/ml) were then compared to patients' clinicopathological features.Results: About 74% (28/38) of tumors harbored the BRAFV600E mutation. Percent plasma ctDNA fractions for PD patients with BRAFV600E tumors ranged from 0 to 2.07%, whereas absolute plasma ctDNA copies ranged from 0 to 62 copies. The ctDNA levels accurately detected tumor burden of PTC patients whose tumors harbored BRAFV600E; median plasma ctDNA copy numbers were significantly higher (Wilcoxon test, p = 0.03) in patients with metastasis (MET) (20 copies/ml) compared to patients with non-metastatic (non-MET) tumors (1 copy/ml). The plasma ctDNA levels (copies/ml) accurately determined the disease status of PTC patients with sensitivity of 86% and specificity of 90% as compared to 78% sensitivity and 65% specificity determined by serum Tg levels (ng/ml) with areas under the curves (AUC) of 0.88 and 0.71, respectively. Intriguingly, plasma total cfDNA levels were significantly higher in patients with no evidence of residual disease (NED) compared to persistent disease (PD) patients.Conclusions: Our study supports the clinical applicability of plasma ctDNA as biomarker to determine the residual tumor status and tumor burden of PTC patients.