Μg Of Genomic DNA Research Articles

An efficient qPCR assay for the quantification of human cells in preclinical animal models by targeting human specific DNA in the intron of BRCA1.

Precise quantification of grafted human cells in preclinical animal models such as non-human primates, rodents and rabbits is needed for the evaluations of the safety and efficacy of cell therapy. Quantitative PCR (qPCR) as a swift, sensitive and powerful assay is suitable for human cell quantification. However, it is a formidable challenge due to that the genome of non-human primates share more than 95% of similarity as human. In the present study, we developed a probe-based quantitative PCR (qPCR) assay for the quantification of human cells in preclinical animal models via targeting human specific DNA in the intron of BRCA1 (termed BRCA1-qPCR). The 5' and 3' end of BRCA1-qPCR probe was conjugated with FAM and non-fluorescent quencher-minor groove binder (NFQ-MGB), respectively. 1 µg of genomic DNA from human and preclinical animal models including rhesus monkeys, cynomolgus monkeys, New Zealand white rabbits, SD rats, C57BL/6 and BALB/c mice were used for determining the specificity and sensitivity of the BRCA1-qPCR assay. A calibration curve was generated by BRCA1-qPCR analysis of linearized plasmid containing targeted human specific DNA in BRCA1. The BRCA1-qPCR assay was validated by analysis of 0.003%, 0.03% and 0.3% of human leukocytes mixed within murine leukocytes. The BRCA1-qPCR assay detected human DNA rather than DNA from tested species. The amplification efficiency of the BRCA1-qPCR assay was 95.4% and the linearity of the calibration curve was R2 = 0.9997. The BRCA1-qPCR assay detected as low as 5 copies of human specific DNA and is efficient to specially amplify 30 pg human DNA in the presence of 1µg of genomic DNA from tested species, respectively. The BRCA1-qPCR assay was able to quantify as low as 0.003% of human cells within murine leukocytes. The BRCA1-qPCR assay is efficient for the quantification of human cells in preclinical animal models.

A Novel Universal CD7-Targeted CAR-T Cell Therapy for Relapsed or Refractory T-Cell Acute Lymphoblastic Leukemia and T-Cell Lymphoblastic Lymphoma

Background Universal CAR-T (UCAR-T) using gene-editing techniques has attracted significant attention due to its prompt availability for patients. To date, there are only a few clinical case reports of universal CAR-T therapy for T-cell malignancies. Here, we explored the efficacy and safety of CD7-targeted UCAR-T (RD13-01) cells for relapsed or refractory (R/R) T-cell acute lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) in a phase I clinical trial (NCT04620655). Methods RD13-01 is an allogeneic, healthy donor-derived, and CRISPR/Cas9-edited anti-CD7 CAR-T cell product. RD13-01 consists of a CAR with a CD7-binding single-chain variable fragment (scFv), a 4-1BB costimulatory domain, CD3ζ signaling domain, a γc intracellular domain, in addition to an NK inhibitory (NKi) ligand containing an EC1-EC2 extracellular domain of E-cadherin fused to a CD28 co-stimulatory domain. The CAR and the NKi ligand are linked by an F2A self-cleaving peptide. Intravenous fludarabine (25-30 mg/m2/d), cyclophosphamide (300mg/m2/d) and etoposide (100 mg/m2/d) were given to all patients on Day -6 to Day -3 prior to CD7 UCAR-T cell infusion. Results Between December 2020 and May 2022, 10 patients (7 males, 3 females), 7 with T-ALL and 3 with T-LBL were enrolled. Median patient age was 16.5 years (range: 2-27 years). Patients had a median of 4.5 (2-6) prior lines of therapies. Two patients relapsed from a previous allogeneic hematopoietic stem cell transplant (allo-HSCT) within 6 months. In addition, 3 patients who showed no response (NR) to prior autologous CD7 CAR-T therapy were also included. Patient characteristics are shown in Table 1. Four patients harbored high-risk genotypes including STIL-TAL1, TP53, BCR-ABL, and others. By morphology, the median bone marrow (BM) blasts at enrollment were 60% (4%-96.5%). Of the eight patients with peripheral blood (PB) blasts, the median blasts were 27% (9%-85%). At enrollment, 8 patients had extramedullary disease (EMD) including 3 with central nervous system (CNS) involvement (1 CNS-1, 2 CNS-3), 3 with diffuse involvement, and 3 had localized EMD. The median transfection efficiency of the RD13-01 product was 64.6% (range: 59.0%-91.2%). A single dose of CD7 UCAR-T cells were infused to the first 3 enrolled patients at 0.5-1×107 cells/kg. Subsequently, 6 patients received a medium dose of 2×107cells/kg, and 1 received a high dose of 4×107cells/kg. By the cut-off date of July 11, 2021, the median observation time was 153 days (range: 28-315 days). On day 28 post infusion, 8/10 (80%) patients achieved complete remission (CR) in BM/PB, and 7 of the 8 patients were minimal residual disease (MRD) negative. Notably, the 3 patients who had failed prior autologous CD7 CAR-T therapy achieved CR. The first enrolled 2 patients who received the low dose showed NR and withdrew on day 28. Among the 7 patients with EMD, 4 achieved EMD CR at the median day 30, 1 had progression disease (PD) and 2, with NR in BM, withdrew prior to EMD evaluation. With a median time of 47 days (40-67 days) following infusion, 6 patients in CR (including 1 MRD +CR) underwent consolidation allo-HSCT, and 4/6 remained progression-free longest up to 315 days. However, 2/6 relapsed without CD7 lost on day 186 and day 200, respectively, and subsequently died. In addition, 1 CR patient died from bacterial infection on day 35. One PD patient received salvage transplantation and died from relapse on day 150. Post infusion, 9/10 patients experienced grade I cytokine release syndrome (CRS) and 1/10 had grade 3 CRS. Only 1 patient with CNS-3 experienced grade 3 neurotoxicity. By qPCR, CD7 UCAR-T cells reached a median peak level of 1.43×106 copies /μg genomic DNA (1.11×105~4.33×106)copies /μg genomic DNA, which occurred on day 12 (7-24 days). The median peak of circulating CD7 UCAR-T was 47.6% (0.01%~83.9%) occurring on day 7 (6-19 days), as measured by flow cytometry (Figure.1). Conclusions Our phase I trial showed that the allogeneic "off-the-shelf” RD13-01 product was safe and dose-dependently effective in treating patients with heavily pretreated T-ALL/LBL, including those with EMD and prior allo-HSCT, those who already failed autologous CD7 CAR-T therapy, and who could not manufacture autologous CAR-T from the patient's own PB due to high blasts in PB and those with rapid disease progression. Long-term observation and more patients are needed to further evaluate the safety and efficacy of CD7 UCAR-T cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice

CRISPR tools can generate knockout and knock-in animal models easily, but the models can contain off-target genomic lesions or random insertions of donor DNAs. Simpler methods to identify off-target lesions and random insertions, using tail or earpiece DNA, are unavailable. We develop CRISPR-KRISPR (CRISPR-Knock-ins and Random Inserts Searching PRotocol), a method to identify both off-target lesions and random insertions. CRISPR-KRISPR uses as little as 3.4 μg of genomic DNA; thus, it can be easily incorporated as an additional step to genotype founder animals for further breeding.

Ultrasensitive Simultaneous Detection of Multiple Rare Modified Nucleosides as Promising Biomarkers in Low-Put Breast Cancer DNA Samples for Clinical Multi-Dimensional Diagnosis.

Early cancer diagnosis is essential for successful treatment and prognosis, and modified nucleosides have attracted widespread attention as a promising group of cancer biomarkers. However, analyzing these modified nucleosides with an extremely low abundance is a great challenge, especially analyzing multiple modified nucleosides with a different abundance simultaneously. In this work, an ultrasensitive quantification method based on chemical labeling, coupled with LC-MS/MS analysis, was established for the simultaneous quantification of 5hmdC, 5fdC, 5hmdU and 5fdU. Additionally, the contents of 5mdC and canonical nucleosides could be obtained at the same time. Upon derivatization, the detection sensitivities of 5hmdC, 5fdC, 5hmdU and 5fdU were dramatically enhanced by several hundred times. The established method was further applied to the simultaneous detection of nine nucleosides with different abundances in about 2 μg genomic DNA of breast tissues from 20 breast cancer patients. The DNA consumption was less than other overall reported quantification methods, thereby providing an opportunity to monitor rare, modified nucleosides in precious samples and biology processes that could not be investigated before. The contents of 5hmdC, 5hmdU and 5fdU in tumor tissues and normal tissues adjacent to the tumor were significantly changed, indicating that these three modified nucleosides may play certain roles in the formation and development of tumors and be potential cancer biomarkers. While the detection rates of 5hmdC, 5hmdU and 5fdU alone as a biomarker for breast cancer samples were 95%, 75% and 85%, respectively, by detecting these three cancer biomarkers simultaneously, two of the three were 100% consistent with the overall trend. Therefore, simultaneous detection of multiple cancer biomarkers in clinical samples greatly improved the accuracy of cancer diagnosis, indicating that our method has great application potential in clinical multidimensional diagnosis.

Whole-genome resequencing reveals domestication and signatures of selection in Ujimqin, Sunit, and Wu Ranke Mongolian sheep breeds.

ObjectiveThe current study aimed to perform whole-genome resequencing of Chinese indigenous Mongolian sheep breeds including Ujimqin, Sunit, and Wu Ranke sheep breeds (UJMQ, SNT, WRK) and deeply analyze genetic variation, population structure, domestication, and selection for domestication traits among these Mongolian sheep breeds.MethodsBlood samples were collected from a total of 60 individuals comprising 20 WRK, 20 UJMQ, and 20 SNT. For genome sequencing, about 1.5 μg of genomic DNA was used for library construction with an insert size of about 350 bp. Pair-end sequencing were performed on Illumina NovaSeq platform, with the read length of 150 bp at each end. We then investigated the domestication and signatures of selection in these sheep breeds.ResultsAccording to the population and demographic analyses, WRK and SNT populations were very similar, which were different from UJMQ populations. Genome wide association study identified 468 and 779 significant loci from SNT vs UJMQ, and UJMQ vs WRK, respectively. However, only 3 loci were identified from SNT vs WRK. Genomic comparison and selective sweep analysis among these sheep breeds suggested that genes associated with regulation of secretion, metabolic pathways including estrogen metabolism and amino acid metabolism, and neuron development have undergone strong selection during domestication.ConclusionOur findings will facilitate the understanding of Chinese indigenous Mongolian sheep breeds domestication and selection for complex traits and provide a valuable genomic resource for future studies of sheep and other domestic animal breeding.

Targeted Delivery for Cardiac Regeneration: Comparison of Intra-coronary Infusion and Intra-myocardial Injection in Porcine Hearts.

BackgroundThe optimal delivery route to enhance effectiveness of regenerative therapeutics to the human heart is poorly understood. Direct intra-myocardial (IM) injection is the gold standard, however, it is relatively invasive. We thus compared targeted IM against less invasive, catheter-based intra-coronary (IC) delivery to porcine myocardium for the acute retention of nanoparticles using cardiac magnetic resonance (CMR) imaging and viral vector transduction using qPCR.MethodsFerumoxytol iron oxide (IO) nanoparticles (5 ml) were administered to Yorkshire swine (n = 13) by: (1) IM via thoracotomy, (2) catheter-based IC balloon-occlusion (BO) with infusion into the distal left anterior descending (LAD) coronary artery, (3) IC perforated side-wall (SW) infusion into the LAD, or (4) non-selective IC via left main (LM) coronary artery infusion. Hearts were harvested and imaged using at 3T whole-body MRI scanner. In separate Yorkshire swine (n = 13), an adeno-associated virus (AAV) vector was similarly delivered, tissue harvested 4–6 weeks later, and viral DNA quantified from predefined areas at risk (apical LV/RV) vs. not at risk in a potential mid-LAD infarct model. Results were analyzed using pairwise Student's t-test.ResultsIM delivery yielded the highest IO retention (16.0 ± 4.6% of left ventricular volume). Of the IC approaches, BO showed the highest IO retention (8.7 ± 2.2% vs. SW = 5.5 ± 4.9% and LM = 0%) and yielded consistent uptake in the porcine distal LAD territory, including the apical septum, LV, and RV. IM delivery was limited to the apex and anterior wall, without septal retention. For the AAV delivery, the BO was most efficient in the at risk territory (Risk: BO = 6.0 × 10−9, IM = 1.4 × 10−9, LM = 3.2 × 10−10 viral copies per μg genomic DNA) while all delivery routes were comparable in the non-risk territory (BO = 1.7 × 10−9, IM = 8.9 × 10−10, LM = 1.2 × 10−9).ConclusionsDirect IM injection has the highest local retention, while IC delivery with balloon occlusion and distal infusion is the most effective IC delivery technique to target therapeutics to a heart territory most in risk from an infarct.

Anti-CD30 Chimeric Antigen Receptor T Cell Therapy for CD30+ Relapsed/Refractory Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma Patients

Chimeric antigen receptor T (CAR-T) cell therapy had shown remarkable efficacy in B-cell acute lymphoblastic leukemia and several B-cell lymphomas using CD19/22 antigen as target. Similarly, CD30 was found over-expressed on all stage of the cells in classical Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL). Given the encouraging clinical success achieved by CD30 monoclonal antibody brentuximab vedotin, targeting CD30 with CAR-T cells would be a promising therapeutic approach for these populations. Here we conducted an open-label, single-center and single-arm pilot study, aimed to evaluate the feasibility and safety of anti-CD30 CAR-T cell therapy relapsed/refractory in HL and ALCL patients. This trial is registered with ChiCTR, number ChiCTR-OPN-16009069.From August, 2016 to May, 2018, 4 HL and 2 ALCL patients with a median age of 26.5 years (range, 22-55 years) were enrolled in this study. Disease diagnosis and CD30expression was confirmed again by pathology and immunohistochemistry at enrollment. All the patients had relapsed or primary refractory disease, after at least two lines of systemic therapy. A pre-conditioning FC (fludarabin 25mg/m2, cyclophosphamide 20mg/kg for 3 days) regimen was give before CAR-T cell infusion. A total dose of 0.7-2.45×107 (median 1.175×107) per kg of CAR-T cells was infused over 3 to 7 consecutive days. The CAR transgene copy, representing CAR-T cells expansion, reached the peak at about 1 week after first administration, and then decreased. CAR transgene copy was still above 100 per μg genomic DNA in peripheral blood even 6 months after infusion. 5/6 (83.3%) patients had grade 0-1 cytokine release syndrome (CRS), with fever lower than 39℃ and duration less than 2 week. Levels of cytokines, including interleukin 6, ferritin and C-reactive protein were monitored. The maximum fold change of the cytokines from baseline was less than 10. No sign of neurotoxicity, bone marrow suppression or other adverse events were observed. The toxicity was generally tolerable, except for one HL patient, who experienced grade 4 CRS, eventually died from pneumorrhagia at the 20th day of infusion. The other 5 patients all achieved complete remission (CR) successfully. PET scan showed a CR status as early as the first month after infusion. Although relapse was observed in one HL patient 2 months after infusion, there were 3 of them remained CR state for over 1 year according to PET scan. The median progression free survival was 7 months.In conclusion, our work demonstrated that CD30 CAR-T cell therapy is efficient and safe for HL and ALCL patients, providing a new option in treating CD30+ relapsed/refractory lymphomas, while a lager study scale was further demanded in our trial to obtain more robust support. DisclosuresNo relevant conflicts of interest to declare.

A comprehensive molecular and morphological study of the effects of space flight on human capillary endothelial cells: sample quality assessment and preliminary results.

ESA (ESA ILSRA-2009-1026); ASI (contract no. 5681); Regione Toscana (POR FSE 2007-2013-FORTEC); Kayser Italia; Lions Club International, District 108LA, Toscana, Italy.

Monitoring of Parasite Kinetics in Indian Post-Kala-azar Dermal Leishmaniasis.

The potential reservoirs of leishmaniasis in South Asia include relapsed cases of visceral leishmaniasis (VL), patients with post-kala-azar dermal leishmaniasis (PKDL), and an asymptomatically infected population. Therefore, assessment of cure in terms of parasite clearance, early detection of PKDL, and asymptomatic VL are pivotal for ensuring elimination. This study aimed to monitor the efficacy of miltefosine and liposomal amphotericin B (LAmB) in PKDL based on parasite load. Patients with PKDL were recruited from the dermatology outpatient departments or during active field surveys. Skin biopsies were collected at disease presentation, immediately at the end of treatment, and 6 months later. The presence of parasite DNA was assessed by internal transcribed spacer-1 polymerase chain reaction, and quantified by amplification of parasite kinetoplastid DNA. At disease presentation (n = 184), the median parasite load was 5229 (interquartile range [IQR], 896-50898)/μg genomic DNA (gDNA). Miltefosine cleared the parasites to <10 in the macular (n = 17) and polymorphic (n = 21) variants, and remained so up to 6 months later (<10 parasites). LAmB reduced the parasite burden substantially in macular (n = 34; 2128 [IQR, 544-5763]/µg gDNA) and polymorphic PKDL (n = 36; 2541 [IQR, 650-9073]/µg gDNA). Importantly, in patients who returned 6 months later (n = 38), a resurgence of parasites was evident, as the parasites increased to 5665 (IQR, 1840-17067)/µg gDNA. This study established that quantifying parasite load is an effective approach for monitoring patients with PKDL, wherein miltefosine demonstrated near-total parasite clearance and resolution of symptoms. However, in cases treated with LAmB, the persistence of parasites suggested treatment inadequacy. This needs immediate redressal in view of the leishmaniasis elimination program targeted for 2020.

Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli.

To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose. The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R ) at 30°C, but have no effect on PR-kan R gene at 37°C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected. Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.

Preclinical Biodistribution and Safety Evaluation of a pbi-shRNA STMN1 Lipoplex after Subcutaneous Delivery.

Stathmin-1 (STMN1) is a microtubule-destabilizing protein which is overexpressed in cancer. Its overexpression is associated with poor prognosis and also serves as a predictive marker to taxane therapy. We have developed a proprietary bi-functional shRNA (bi-shRNA) platform to execute RNA interference (RNAi)-mediated gene silencing and a liposome-carrier complex to systemically deliver the pbi-shRNA plasmids. In vitro and in vivo testing demonstrated efficacy and specificity of pbi-shRNA plasmid in targeting STMN1 (Phadke, A. P., Jay, C. M., Wang, Z., Chen, S., Liu, S., Haddock, C., Kumar, P., Pappen, B. O., Rao, D. D., Templeton, N. S., et al. (2011). In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein. DNA Cell Biol. 30, 715-726.). Biodistribution and toxicology studies in bio-relevant Sprague Dawley rats with pbi-shRNA STMN1 lipoplex revealed that the plasmid DNA was delivered to a broad distribution of organs after a single subcutaneous injection. Specifically, plasmid was detected within the first week using QPCR (threshold 50 copies plasmid/1 µg genomic DNA) at the injection site, lung, spleen, blood, skin, ovary (limited), lymph nodes, and liver. It was not detected in the heart, testis or bone marrow. No plasmid was detected from any organ 30 days after injection. Treatment was well tolerated. Minimal inflammation/erythema was observed at the injection site. Circulating cytokine response was also examined by ELISA. The IL-6 levels were induced within 6 h then declined to the vehicle control level 72 h after the injection. TNFα induction was transiently observed 4 days after the DNA lipoplex treatment. In summary, the pbi-shRNA STMN1 lipoplex was well tolerated and displayed broad distribution after a single subcutaneous injection. The pre-clinical data has been filed to FDA and the pbi-shRNA STMN1 lipoplex is being investigated in a phase I clinical study.

Whole genome sequence analysis of BT-474 using complete Genomics' standard and long fragment read technologies.

BackgroundThe cell line BT-474 is a popular cell line for studying the biology of cancer and developing novel drugs. However, there is no complete, published genome sequence for this highly utilized scientific resource. In this study we sought to provide a comprehensive and useful data set for the scientific community by generating a whole genome sequence for BT-474.FindingsFive μg of genomic DNA, isolated from an early passage of the BT-474 cell line, was used to generate a whole genome sequence (114X coverage) using Complete Genomics’ standard sequencing process. To provide additional variant phasing and structural variation data we also processed and analyzed two separate libraries of 5 and 6 individual cells to depths of 99X and 87X, respectively, using Complete Genomics’ Long Fragment Read (LFR) technology.ConclusionsBT-474 is a highly aneuploid cell line with an extremely complex genome sequence. This ~300X total coverage genome sequence provides a more complete understanding of this highly utilized cell line at the genomic level.Electronic supplementary materialThe online version of this article (doi:10.1186/s13742-016-0113-x) contains supplementary material, which is available to authorized users.

The Presence of ASXL1 Mutations As Well As a Total Number of Myeloid Driver Mutations Higher Than Two Is Strongly Associated with the Diagnosis of Primary Myelofibrosis As Opposed to Essential Thrombocythemia

Introduction: Primary Myelofibrosis (PMF) and Essential Thrombocythemia (ET) are myeloproliferative neoplasms with similar genetic backgrounds. Both diseases are characterized, at the molecular level, by mutations in the genes JAK2, MPL and CALR. In addition recurring mutations is several other genes have been described in myeloid malignancies in general. Although the differential diagnosis between PMF and ET may be straight forward in most cases, there is a significant clinical and pathologic overlap between these two conditions, making the differential diagnosis difficult sometimes, mostly between early PMF and ET. With the goal of utilizing genomic information to better differentiate ET from PMF we decided to identify and compare all genomic alterations present in patients with ET and PMF, through whole exome / genome sequencing of paired granulocytes and skin.Methods: A total of 84 patients with either PMF (N=48) or ET (N=36) were analyzed. DNA was extracted from CD66b+ magnetic bead selected granulocytes (EasySep, Stem Cell Technologies) and matched skin biopsies with QiaAmp DNA Mini kit (Qiagen). Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). Tumor coverage was 150x and germline was 60x. The combined output of these 3 softwares was further filtered by in-house criteria in order to reduce false-positive calls (minimum coverage at both tumor/germline ≥8 reads; fraction of reads supporting alternate allele ≥5% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline >2). All JAK2 and CALR mutations were validated through Sanger sequencing. Validations of other somatic mutations are under way at this point. For this work, other myeloid driver mutations were defined as mutations occurring recurrently in myeloid malignancies in the medical literature, and in this cohort of patients these mutations were present in the following genes: ASXL1, ATM, CALR, CBL, CUX1, DNMT3A, EZH2, GATA2, GNAS, IDH1, IDH2, JAK2, MPL, NRAS, SH2B3, SF3B1, STAG2, TET2, NFE2, SMC3, SUZ12, PRPF8, SRSF2, U2AF1, TP53. Fisherxs exact test was used for statistical comparisons.Results: The most common mutated genes after JAK2 and CALR were ASXL1 (n=16), TET2 (n=9) and DNMT3A (n=9). After data analysis, the patients could be divided in 7 groups based on the genomic profile:A – JAK2 mutation as the single genetic abnormality (JAK2_Single) (N=24), B – JAK2 plus other myeloid driver mutations (JAK2_Plus) (N=25), C - CALR mutation as the single genetic abnormality (CALR_Single) (N=11), D – CALR plus other myeloid driver mutations (CALR_Plus) (N=9), E – MPL mutation (N=1), F – Triple negative without other myeloid driver mutations (TN_Single) (N=8), G – No JAK2, CALR or MPL (triple negative) but with other myeloid driver mutations (TN_plus) (N=6)1 – The presence of 3 or more total myeloid driver mutations was strongly associated with a diagnosis of PMF Table 1mut<3mut>2TE282PMF2521P= 0.00022 – The presence of ASXL1 mutations was strongly associated with a diagnosis of PMF Table 2ASXL1+ASXL1-TE135PMF1533P=0.0007In order to validate our findings in an independent cohort of patients, we performed the same analysis using data from 2 published studies that evaluated myeloid multi-gene panels in ET and PMF (Nangalia J, NEJM 2013) (Lundberg P, Blood, 2014). We pooled together all patients with ET (N=117) and PMF (N=56) from both studies and repeated the two previous analyses, that confirmed the previous results: Table 3mut<3mut>2TE1106PMF4214P=0.0005ASXL1+ASXL1-TE4113PMF1442P=3.9E-05Conclusions: We have demonstrated that ASXL1 mutations as well as a number of myeloid driver mutations higher than two is strongly associated with PMF. This information may be useful in the near future to improve the differential diagnosis between ET and PMF. DisclosuresNo relevant conflicts of interest to declare.

Genomic Profile of Patients with Triple Negative (JAK2, CALR and MPL) Essential Thrombocythemia and Primary Myelofibrosis

Introduction: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are myeloproliferative neoplasms (MPN) with similar driver mutations. The three hallmark molecular alterations in these diseases are JAK2, MPL and CALR mutations. Patients with myelodysplastic/myeloproliferative (MPN/ MDS) neoplasms such as refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) also present with the same hallmark genetic changes. Nevertheless, roughly 10% of these patients do not present mutations in neither of these genes. Recent data suggest that triple negative patients have a more aggressive clinical course. While the molecular alterations present in patients with MPN have been extensively studies, the genomic profile of triple negative TE/PMF/RARS-T has not been described. To better characterize these patients we performed whole exome / genome sequencing of paired granulocytes and skin from 15 triple negative MPN patientsMethods: A total of 15 patients with triple negative MPN or MPN/ MDS [PMF (N=6)/TE (N=8)/RARS-T (N=1)] were analyzed. DNA was extracted from CD66b+ magnetic bead selected granulocytes (EasySep, Stem Cell Technologies) and matched skin biopsies with QiaAmp DNA Mini kit (Qiagen).Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). Tumor coverage was 150x and germline coverage was 60x. The combined output of these 3 softwares was further filtered by in-house criteria in order to reduce false-positive calls (minimum coverage at both tumor/germline ≥8 reads; fraction of reads supporting alternate allele ≥5% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline >2). All JAK2 and CALR mutations were validated through Sanger sequencing. Validations of other somatic mutations are under way at this point.Results: First we asked whether other hematopoietic related genes could be responsible for the pathogenesis of the triple negative cases. With that goal we searched for high confidence mutations in genes that are mutated in at least 1% of patients with hematopoietic tumors on COSMIC (catalog of somatic mutations in cancer) database and also genes known to be recurrently mutated in myeloid malignancies. Only 6 out of 15 patients presented mutations in other myeloid related genes. The diagnosis of these patients were PMF=4, TE=2. The hematopoietic related genes mutated in these patients were: ASXL1 (n=4), CUX1 (n=3), NRAS (n=2) and ATM, CBL, CSFR3, CREBBP, DNMT3A, ETV6, EZH2, JARID2, MLL2, PHF6, SRSF2, STAG2, TET2, GNAS, U2AF1 (n=1).Noteworthy, we have found one known oncogenic mutation in CSFR3, an alteration supposed to be specific for chronic neutrophilic leukemia and atypical CML, in a patient with ET, and an oncogenic mutation in GNAS in a patient with PMF. In addition, the patient with RSRA-T had a putative oncogenic mutation in PTPN11Remarkably, the average number of hematopoietic related mutations in these patients was 5, significantly higher than the total number of mutations found in another cohort of patients with either JAK2 (average = 1.7) or CALR mutations (average = 1.9). Although our numbers are small, we may speculate that the high incidence of ASXL1 mutations (28%) associated with a high number of prognostically detrimental mutations can partially explain the worse outcomes associated with triple negative MPN. Noteworthy is also the high prevalence of CUX1 mutations in this subset of patients (21%) when compared to other myeloid malignancies in general.Regarding the other 9 patients for whom no hematopoietic mutations could be identified, 6 patients had ET, 2 patients PMF and one patient had RARS-T.Conclusion: We have shown that: i-patients with triple negative MPN are molecularly heterogeneous, with one group presenting a high number of hematopoietic related mutations, ii-the most common mutations present in these patients are ASXL1, CUX1 and NRAS, iii-The majority of these patients do not present mutations in hematopoietic related genes, what suggests that non-described molecular mechanisms are operating in these patients. DisclosuresNo relevant conflicts of interest to declare.

Determining lower limits of detection of digital PCR assays for cancer-related gene mutations

Digital PCR offers very high sensitivity compared to many other technologies for processing molecular detection assays. Herein, a process is outlined for determining the lower limit of detection (LoD) of two droplet-based digital PCR assays for point mutations of the epidermal growth factor receptor (EGFR) gene. Hydrolysis probe mutation-detection assays for EGFR p.L858R and p.T790M mutations were characterized in detail. Furthermore, sixteen additional cancer-related mutation assays were explored by the same approach. For the EGFR L8585R assay, the assay sensitivity is extremely good, and thus, the LoD is limited by the amount of amplifiable DNA that is analyzed. With 95% confidence limits, the LoD is one mutant in 180,000 wild-type molecules for the evaluation of 3.3μg of genomic DNA, and detection of one mutant molecule in over 4 million wild-type molecules was achieved when 70 million copies of DNA were processed. The measured false-positive rate for the EGFR L8585R assay is one in 14 million, which indicates the theoretical LoD if an unlimited amount of DNA is evaluated. For the EFGR T790M assay, the LoD is one mutant in 13,000 for analysis of a 3.3μg sample of genomic DNA, and the dPCR assay limit sensitivity approaches one mutant in 22,000 wild-type molecules.

Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics

Single-cell genomics is a powerful tool for exploring the genetic makeup of environmental microorganisms, the vast majority of which are difficult, if not impossible, to cultivate with current approaches. Here we present a comprehensive protocol for obtaining genomes from uncultivated environmental microbes via high-throughput single-cell isolation by FACS. The protocol encompasses the preservation and pretreatment of differing environmental samples, followed by the physical separation, lysis, whole-genome amplification and 16S rRNA-based identification of individual bacterial and archaeal cells. The described procedure can be performed with standard molecular biology equipment and a FACS machine. It takes <12 h of bench time over a 4-d time period, and it generates up to 1 μg of genomic DNA from an individual microbial cell, which is suitable for downstream applications such as PCR amplification and shotgun sequencing. The completeness of the recovered genomes varies, with an average of ∼50%.

Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation

Inheritance of DNA cytosine methylation pattern during successive cell division is mediated by maintenance DNA (cytosine-5) methyltransferase 1 (DNMT1). Lysine 142 of DNMT1 is methylated by the SET domain containing lysine methyltransferase 7 (SET7), leading to its degradation by proteasome. Here we show that PHD finger protein 20-like 1 (PHF20L1) regulates DNMT1 turnover in mammalian cells. Malignant brain tumor (MBT) domain of PHF20L1 binds to monomethylated lysine 142 on DNMT1 (DNMT1K142me1) and colocalizes at the perinucleolar space in a SET7-dependent manner. PHF20L1 knockdown by siRNA resulted in decreased amounts of DNMT1 on chromatin. Ubiquitination of DNMT1K142me1 was abolished by overexpression of PHF20L1, suggesting that its binding may block proteasomal degradation of DNMT1K142me1. Conversely, siRNA-mediated knockdown of PHF20L1 or incubation of a small molecule MBT domain binding inhibitor in cultured cells accelerated the proteasomal degradation of DNMT1. These results demonstrate that the MBT domain of PHF20L1 reads and controls enzyme levels of methylated DNMT1 in cells, thus representing a novel antagonist of DNMT1 degradation.

White spot syndrome virus (WSSV) and prevalence of disease resistance in a commercially cultured population of Penaeus monodon Fabricius, 1798 (Decapoda, Dendrobranchiata)

White spot disease (WSD) caused by the white spot syndrome virus (WSSV) is the leading cause for huge economic destruction in the shrimp aquaculture industry. The present study investigates the prevalence of WSSV and disease-resistant shrimps among a commercially cultured population of Penaeus monodon Fabricius, 1798. Results suggest that WSSV prevalence among 32 culture ponds affected by various diseases was 58.2% cumulatively in 2009 and 2010. Real-time PCR yielded 1.46 × 108, 1.94 × 106 and 3.6 × 104 copies of WSSV per μg of genomic DNA of shrimp in severe, moderate and low infected shrimps, successively. Among the shrimps collected, the prevalence of disease resistance was 31.4% and 37.1% consecutively in 2009 and 2010. It was found that 34% of the shrimps were disease resistant while considering both years. This study shows the importance of using disease-resistant seeds for better shrimp health management.

Whole-Exome Sequencing In Myelodysplastic Syndromes With 5q- and Normal Karyotype

IntroductionMyelodysplastic Syndromes (MDS) are a heterogeneous group of clonal myeloid stem cells disorders with high prevalence in the elderly characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and an increased risk of transformation to acute myeloid leukemia (AML). The karyotype is the clinical parameter with the strongest prognostic impact according the IPSS-R (Greenberg et al., 2012). The most frequent cytogenetic alteration is the chromosome 5q deletion (del[5q]) which as a single anomaly, confers a good prognosis and predicts an excellent response to lenalidomide. Whether other genetic abnormalities routinely cooperate with del(5q) is not known. Whole-exome sequencing (WES) is a powerful tool to identify somatic mutations in protein coding genes that might cooperate with del(5q).In order to better understand the genetic basis of MDS with del(5q), we performed whole-exome sequencing (assessing 334,378 exons) of tumor-normal paired samples from 21 MDS patients. Herein we describe the preliminary findings. The analysis is ongoing and the complete results will be presented in the meeting. MethodsA total of 21 patients with MDS (16 with del(5q) as a sole abnormality, 3 with del(5q) and additional alterations and 2 with normal karyotype) were included in our study. We examined a total of 25 tumor samples (21 diagnostic bone marrow (BM) samples with matched CD3+ cells as a controls, additional BM samples from 3 patients during lenalidomide treatment and 1 bone marrow sample from a del(5q) patient after AML progression). DNA was extracted from BM samples and from isolated peripheral blood CD3+ cells (magnetic-activated cell sorting (MACS), MiltenyiBiotec GmbH, Germany). The purity of CD3+ cells was assessed by FC 500 flow cytometer (Beckman Coulter, Hialeah, Fl, USA). Only DNA that fulfilled quality controls required by WES were submitted. For each diagnostic sample, we performed Conventional G-banding cytogenetics and fluorescence in situ hybridization (FISH, to confirm or dismiss 5q deletions).Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The captured and amplified exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Whole-exome sequencing data were analyzed using an in-house bioinformatics pipeline as previously reported. Somatic mutations identified as alterations present in tumor but not in the matched CD3+ sample were validated by Sanger sequencing. ResultsIn our preliminary analysis of WES from 12 patients (10 patients with 5q- and 2 patients with normal karyotype), a total of 249 non-silent somatic variant candidates were identified, of which 146 were confirmed as somatic mutations. Recurrent mutations were observed in three genes (ASXL1, NBPF10 and SF3B1) in 3 different patients. Seven genes (HRNR, JAK2, POTEG, MUC5B, PHLDA, TTN, ZNF717) were mutated in two patients. Mutations in several genes known to be mutated in MDS (ASXL1, JAK2, RUNX1, SF3B1, SRSF2 and TET2) were also identified. Patients with the 5q deletion had an average of 11 mutations whereas patients with normal karyotype had a higher mean (14.5). Mutated genes identified in both groups were HRNR, JAK2, MUC5B, NBPF10 and SF3B1. No mutations in TP53 were detected in this subset. Pathway analysis of the complete list of somatically mutated genes will be carried out once all 21 patients are analyzed. The four in-treatment samples will be examined from their matched diagnostic samples. ConclusionsWhole-exome sequencing of largely del(5q) MDS patient samples identified both known and previously unreported somatic mutations. Analysis of additional samples will allow a more complete description of the genes and pathways that may cooperate with del(5q) in the development and progression of MDS. AcknowledgmentsFinancial support: This work has been supported (in part) by a grant from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); Acción COST BM0801: European Genomics and Epigenomics Study on MDS and AML; Sociedad Española de Hematología y Hemoterapia (SEHH) and MDS Celgene. FootnotesRafael Bejar and Francesc Sole contributed equally. Disclosures:Díez-Campelo:Novartis and Celgene: Honoraria, Research Funding. Cañizo:Celgene Jansen-Cilag Arry Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sanchez:Celgene: Honoraria, Research Funding. Bejar:Genoptix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Solé:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Diagnostic and Prognostic Utility Of a 26-Gene Panel For Deep-Sequencing Mutation Analysis In Myeloid Malignancies

▪ IntroductionMolecular mutation analyses are performed in myeloid malignancies either in a stepwise procedure, i.e. one target gene after each other or are not performed at all, e.g. in low-risk MDS. A comprehensive pan-myeloid panel to simultaneously target mutations in 26 genes allows a comprehensive analysis with the perspective to detect disease defining mutations in the majority of patients. AimsTo test the utility of a pan-myeloid panel in routine diagnostics. MethodsWe developed sensitive next-generation deep-sequencing (NGS) assays comprising in total 26 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PHF6, RUNX1, SF3B1, SRSF2, TET2, TP53, U2AF1, and WT1. With the exception of RUNX1, which was sequenced on the 454 Life Sciences NGS platform (Branford, CT), all remainder genes were studied using a combination of a microdroplet-based assay (RainDance, Lexington, MA) and the MiSeq sequencing instrument (Illumina, San Diego, CA). The assay's turn-around time was less than 6 days, loading up to eight patients per sequencing run. In summary, 389 amplicons were designed with a median length of 206 bp (range 150-240 bp), representing a total target sequence of 78.15 kb. The sequencing library was constructed starting off 2.2 μg genomic DNA per patient, purified from isolated mononuclear cells. Using the 500 cycles sequencing-by-synthesis chemistry in median 7.644 millions of paired-end reads were generated per run. This resulted in a median coverage per gene of 7,626 reads (range 174-12,256). The lower limit of detection was set at a cut-off of 3%. ResultsThus far, 191 prospectively collected cases have been analyzed during routine operations. In all cases the assay was successfully performed. Mutations (range 0-7) have been found in 119/191 (62.3%) cases. The major disease categories were as follows: MDS (n=76), suspected MDS (n=28), MDS/MPN (n=10), reactive bone marrow conditions (n=46), AML (n=8), CML (n=3), other conditions (n=20).We first were interested to address the utility of the panel in MDS when the analysis was restricted to the five prognostically relevant predictors of poor overall survival according to Bejar et al. (N Engl J Med. 2011;364:2496-506), i.e. ASXL1, ETV6, EZH2, RUNX1, and TP53. In detail, 69 cases with MDS were studied and in 42.0% (29/69) of cases mutations had been detected in these five genes while 40 patients showed no mutation. Interestingly, upon extending the analysis to the remainder 21 genes, at least one more mutation was discovered in 72.5% (29/40) of these cases, thereby extending the number of cases with at least one mutation to 84.1% (58/69) of patients. Of note, in 65.5% (19/29) of these latter cases, spliceosome mutations occurred in a mutually exclusive manner (SRSF2, SF3B1, U2AF1), thus also detecting mutations conferring a favorable clinical outcome, i.e. SF3B1 alterations.We next studied in more detail 28 cases with suspected MDS according to cytomorphology, i.e. cases with dysplastic features not sufficient to diagnose MDS. When again in a first step the five predictors of poor overall survival according to Bejar et al. were analyzed, mutations in ASXL1, ETV6, EZH2, RUNX1, and TP53 were observed in 25.0% of cases (7/28). In the group of 75.0% (21/28) of samples with no mutations according to Bejar et al., 28.6% (6/21) of cases harbored a mutation in the group of the 21 remainder genes analyzed simultaneously in the gene panel assay. Thus, in total the number of cases with at least one mutation increased to 46.4% (13/28) of patients. Of note, 6 of the 13 suspected MDS cases with mutations had a normal karyotype. In summary, with respect to correlations between these two cohorts, we observed that morphologically confirmed MDS cases (n=69) showed a higher number of mutated genes compared to “suspected MDS” cases (n=28) (1.88 vs 0.71; p<0.001). ConclusionA pan-myeloid screening assay using NGS allows to address 26 relevant gene mutations in myeloid malignancies with diagnostic or prognostic impact. This approach is scalable and adoptable to accommodate the inclusion of novel gene targets according to the latest evidence from the literature. Importantly, given the broad spectrum of mutations in myeloid diseases covered by such a panel, mutations can be identified in the majority of patients and enable to support a more comprehensive classification in these complex diseases. Disclosures:Kohlmann:MLL Munich Leukemia Laboratory: Employment. Kuznia:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.