Clonal Gene Research Articles

Comparison of Two Quantitative PCR–Based Assays for Detection of Minimal Residual Disease in B-Precursor Acute Lymphoblastic Leukemia Harboring Three Major Fusion Transcripts

Two quantitative PCR (qPCR)-based methods, for clonal immunoglobulin or T-cell receptor gene (Ig/TCR) rearrangements and for fusion transcripts, are widely used for the measurement of minimal residual disease (MRD) in patients with B-precursor acute lymphoblastic leukemia (ALL). MRD of bone marrow samples from 165 patients carrying the three major fusion transcripts, including 74 BCR-ABL1, 54 ETV6-RUNX1, and 37 TCF3-PBX1, was analyzed by using the two qPCR-based methods. The correlation coefficient of both methods was good for TCF3-PBX1 (R2=0.8088) and BCR-ABL1 (R2=0.8094) ALL and moderate for ETV6-RUNX1 (R2=0.5972). The concordance was perfect for TCF3-PBX1 ALL (97.2%), substantially concordant for ETV6-RUNX1 ALL (87.1%), and only moderate for BCR-ABL1 ALL (70.6%). The discordant MRD, positive for only one method with a difference greater than one log, was found in 4 of 93 samples (4.3%) with ETV6-RUNX1, 31 of 245 samples (12.7%) with BCR-ABL1, and none of TCF3-PBX1 ALL. None of the eight non-transplanted patients with BCR-ABL1-MRD (+)/Ig/TCR-MRD (-) with a median follow-up time of 73.5 months had hematologic relapses. Our study showed an excellent MRD concordance between the two qPCR-based methods in TCF3-PBX1 ALL, whereas qPCR for Ig/TCR is more reliable in BCR-ABL1 ALL.

Treatment of indolent cutaneous T-cell lymphoma with hypofractionated radiation therapy in three cats.

Feline indolent cutaneous T-cell lymphoma (ICL) is an uncommon neoplastic disease. There is currently no consensus on treatment recommendations for ICL. To report the clinical outcome of three cats with ICL treated with hypofractionated electron-beam radiotherapy (RT). Three privately owned cats with ICL. Medical records and client surveys were reviewed. A diagnosis of probable ICL was based on history, clinical presentation and histopathological findings, and confirmed using CD3 immunohistochemical analysis and PCR for antigen receptor gene rearrangement (PARR). All cats were treated with hypofractionated RT (four fractions of 8Gy). All cats presented with skin lesions characterised by erythema and alopecia that were refractory to previous treatment with systemic glucocorticoids. Before hypofractionated RT treatment, lesions were histologically described as having diffuse infiltration of the dermis with CD3+ T cells. Molecular clonality analysis revealed clonal T-cell receptor gamma gene rearrangement. After RT, two cats showed histological improvement defined by decreased infiltration of lymphocytes, with cellular infiltrate present only in the deeper dermis; one cat had near complete histological resolution of lesions with only minimal residual lymphocytes. One cat was determined to have a complete clinical response while the other showed partial responses. No acute adverse effects of radiation were observed; chronic effects included leukotrichia, partial alopecia and mild fibrosis. All clients reported improvement in quality of life for their cats. Clinical and histological improvement in these cats suggests that hypofractionated RT can be a useful treatment modality for cats with ICL.

Next-Generation Sequencing–Based Clonality Assessment of Ig Gene Rearrangements: A Multicenter Validation Study by EuroClonality-NGS

Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

Comparison of Genetic Profiling of Primary Central Nervous System (CNS) Lymphoma before and after Extra-CNS Relapse

IntroductionLymphomas of the central nervous system (CNS), including primary CNS lymphoma (PCNSL), have recently been focused on as promising targets for immune checkpoint blockade therapies. However, approximately one-third of patients with PCNSL are resistant to initial chemotherapies, and almost all patients will eventually develop recurrence or disease progression. During the clinical course, systemic extra-CNS lesions are observed in 3% of relapsed or refractory (r/r) PCNSL. To elucidate the mechanism of this uncommon recurrence suggestive of tumor oncogenic evolution and overcoming the immune privilege, we performed comprehensive genomic analysis comparing primary with systemic r/r PCNSL using next-generation sequencing approaches.MethodsPatients at our institute with an episode of both PCNSL and extra-CNS relapse or disease progression with a pathological diagnosis of diffuse large B-cell lymphoma were screened. Genomic DNA was extracted from primary, recurrent, or refractory tumors, and corresponding germline samples. The samples were either formalin-fixed, paraffin-embedded (FFPE) or fresh frozen tissue if available. Clonal immunoglobulin gene rearrangements were detected using the IdentiClone™ IGH Gene Clonality Assay (Invivoscribe Technologies). DNA sequencing was performed on the Ion Torrent Proton™ platform after targeted amplification with the customized panel designed using the Ion AmpliSeq Designer™ tool (Life Technologies). This panel consists of whole exons of 64 genes associated with lymphoid malignancies, including those involved with the NFκB pathway, B-cell differentiation, epigenetic regulation of transcription, immune surveillance, and tumor suppression. Single nucleotide variations (SNVs), short insertions and deletions (InDels), and copy number variations were called using the Biomedical Genomics Workbench (Qiagen) after filtering and annotation using public variant databases such as dbSNP, COSMIC, and GRCh37 hg19. From these SNVs and InDels, somatic mutations were defined as non-synonymous, with a minimal coverage of 100, and with a frequency of subtracting germline reads from tumor reads of 10% or more. To confirm targeted DNA sequencing findings, we performed Sanger sequencing. To determine the cell-of-origin (COO), immunohistochemistry (IHC) using the Hans algorithm was performed. The expression of HLA class I, HLA class II, PD-L1, and PD-L2 was also assessed with IHC.ResultsSix patients who were initially treated for PCNSL and who either relapsed (n = 5) or progressed (n = 1) were recruited. The relapse sites were the ileum (n = 2), testis with para-aortic lymph node (n = 1), intraabdominal lymph node (n = 1), and nasal sinuses (n = 1). The remaining refractory PCNSL was a patient who showed progression in multiple organs including the CNS with systemic lymph nodes. Only one relapsed patient was positive for HLA class I by IHC, with stronger staining seen at the relapsed site compared with the primary CNS site. Targeted DNA sequencing detected somatic PIM1 mutations in all primary and r/r tumors (Figure); nonsense mutations were detected in 50% of primary tumors and 67% of r/r ones. Compared to primary tumors, PIM1 was the most common gained mutation in r/r ones, and additionally gained somatic mutations such as CD79B, MLL2 and CD58 were observed. Partial loss of CIITA and the partial gain of NFκB pathway-related genes such as MALT1 and CARD11 were also detected. Somatic mutations were observed at a frequency of 1% in the germline sample of one patient (case 1 in Figure).ConclusionsOur study suggested that the evolution of mutations in PCNSL enabled systemic disease progression with a breakthrough of immune privilege. These findings were characterized not only by an immunological overpowering but also by the dysregulation of B-cell proliferation signaling, suggesting that loss of function of PIM1 had occurred. [Display omitted] DisclosuresToyoda:Chugai: Honoraria. Narita:Chugai, MSD, Eisai Co. Ltd., Nobelpharma Co.: Honoraria; Abbvie: Consultancy; Nihon Medi-Physics Co., Daiichi-Sankyo Co, Ono Pharmaceutical Co., AbbVie GK: Research Funding. Maruyama:Chugai, Kyowa Hakko Kirin, Ono, Celgene, Janssen, Eisai, Mundipharma, Takeda: Honoraria; Dai-ichi Sankyo, Chugai, Kyowa Hakko Kirin, Ono, Celgene, Janssen, GSK, Eisai, Mundipharma, Takeda, AbbVie, MSD, Sanofi, Pfizer, Otsuka, Novartis, Solasia, Zenyaku: Research Funding. Tobinai:Daiichi Sankyo Co., Ltd: Consultancy, Honoraria; Servier: Research Funding; Eisai: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; HUYA Bioscience: Honoraria; GlaxoSmithKline: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; AbbVie: Research Funding; Takeda: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria; Mundipharma: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding. Kobayashi:Pfizer, Ohtsuka, Astellas, Ariad: Research Funding.

A Spic/BCLAF1 Complex Suppresses Pre-B Cell Receptor Signaling in Response to RAG-Mediated DNA Breaks

B cell development is carefully orchestrated to generate a diverse antibody repertoire while preventing malignant transformation. At early developmental stages, the pre-B cell receptor (pre-BCR) is an essential regulator of B cell maturation, coordinating both clonal expansion and immunoglobulin light chain gene (Igl) assembly. Igl gene rearrangement proceeds through V(D)J recombination, which requires the generation of DNA double-stranded breaks (DSBs) and the subsequent joining of two distant DNA segments. The DSBs necessary for Igl gene rearrangements are generated by the RAG1 and RAG2 proteins, which form the RAG endonuclease. Once RAG DSBs are generated, cells must be prevented from re-entering cell cycle where DSBs can be aberrantly repaired as deleterious translocations. Thus, temporal segregation of pre-BCR driven proliferation and Igl gene assembly is essential for normal maturation and prevention of errors that could lead to leukemic transformation. The pre-BCR signals through the SYK tyrosine kinase to both drive proliferation and initiate Igl gene rearrangement. Unopposed activation of the pre-BCR, particularly increased SYK activity, blocks B cell development and supports leukemic transformation. Furthermore, pre-B leukemic blasts depend on SYK for proliferation and survival. Tight regulation of pre-BCR signaling is essential for enforcing normal maturation and suppressing leukemic transformation.We previously showed that pre-BCR signaling is inhibited by signals from RAG DSBs. Specifically, RAG DSBs trigger ATM-dependent induction of SPIC, an ETS family transcriptional repressor with significant homology to PU.1 and SPIB. SPIC inhibits expression of the SYK tyrosine kinase resulting in suppression of pre-BCR signaling, inhibition of pre-B cell proliferation, and limitation of additional RAG DSB generation. In order to determine the mechanism of SPIC-mediated gene regulation and to determine the role of SPIC in early B cell development, we compared SPIC and PU.1 binding throughout the genome in early B cells. Chromatin-immunoprecipitation followed by sequencing (ChIP-seq) identified shared binding sites for SPIC and PU.1 throughout the genome in pre-B cells suggesting that both transcription factors regulate a similar cohort of genes. Interestingly, binding of SPIC to the Syk promoter and the Igl(kappa) enhancer results in reduction of PU.1 binding at these sites and suppression of transcription of the respective genes. These results demonstrate that SPIC antagonizes PU.1 function to repress pre-BCR signaling in early B cells.SPIC shares a DNA binding domain motif with the other ETS-family transcription factors, PU.1 and SPIB, but has a unique transactivation domain, which could recruit transcriptional repressors to gene regulatory regions. To determine the unique binding partners of SPIC and PU.1, we generated pre-B cells expressing tetracycline-inducible FLAG-SPIC and FLAG-PU.1. The tagged proteins were purified from nuclei using tandem affinity purification. Mass spectrometry of the precipitated samples identified BCLAF1 (Bcl2 associated transcription factor 1) as a unique SPIC interactor. In contrast, IRF4 binds to PU.1 but not to SPIC. In pre-B cells, BCLAF1 binds to regulatory regions of a similar set of genes as SPIC. Consistent with this, BCLAF1 binds to the Syk promoter only in cells with RAG DSBs, which correlates with binding of SPIC and loss of PU.1 at this site. RNAi-mediated knockdown of BCLAF1 results in increased Syk mRNA expression. However, loss of BCLAF1 does not affect SPIC occupancy at the Syk promoter. Thus, SPIC binds DNA but only represses transcription if BCLAF1 is also recruited. Collectively, our findings reveal that, in response to RAG DSBs, SPIC and BCLAF1 cooperate to antagonize PU.1 function and regulate expression of a broad genetic program that modulates pre-BCR signaling to enforce normal B cell maturation. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Minimal Residual Disease (MRD) and MRD-Based Treatment Decisions in Ph/BCR-ABL Negative Adult Acute Lymphoblastic Leukemia (ALL): Experience from the German Multicenter Study Group for Adult ALL (GMALL)

Abstract Detectable MRD during chemotherapy is considered to be the most important prognostic factor in ALL. Pts with molecular failure (MolF) have a poorer overall survival (OS), inferior remission duration (RD) and poorer prognosis even after stem cell transplantation (SCT) compared to pts with molecular CR (MolCR) (Gokbuget et al, Blood 2012: 120(9): 1868). Little is known however on the outcome of pts with MRD results not fitting in the above categories e.g. MRD low positive and on the impact of additional treatments in pts with MolF. We report an expanded cohort of pts treated according to protocol 07/03 (within the trial or in GMALL registry). MRD was tested by real-time PCR of clonal TCR and Ig gene rearrangements in a reference laboratory (Kiel). The analysis refers to molecular response in ‘wk 16‘ after induction/1st consolidation and to molecular relapse (MolR) later than wk 16 (all definitions see table). The GMALL recommended referral of MolF and MolR pts to SCT in CR1. The median age of 2061 pts with Ph-neg ALL (1484 trial, N=577 registry) was 34 (16-65) yrs. Pts 625 (59%)/1857 pts had a MolCR and196 (18%) MolF. In addition 7% were MRDneg and 16% MRDpos - both not fulfilling the criteria of MolCR or MolF (definitions see table). Results for OS at 5 yrs (MolCR vs MolF vs MRDneg vs MRDpos) were 83% vs 43% vs 78% vs 68% resp. and for RD 80% vs 38% vs 76% vs 56% resp. Differences were significant (p In 196 MolF pts OS and RD from wk16 onward was 43% and 38% at 5 yrs resp. 121 (62%) MolF pts received SCT in ongoing 1st CR. The SCT rate in MolF pts increased from 54% (2003-2009) to 72% (2010-2016)(p=.008). OS and RD for non-SCT vs SCT pts was 28% vs 53% (p Out of 196 MolF pts, 19 relapsed shortly after wk 16, 2 pts were withdrawn, 1 pt died in CR and data were missing in 6 pts. Of the remaining 168 pts, 52 received immediate SCT (31%), whereas 115 pts received consolidation. Data on molecular response after the next 1-3 cycles or after SCT were available for 142 pts. In B-prec ALL (N=95) and T-ALL (N=47) the MolCR rate was 34% and 43% resp. for non-SCT approaches, whereas 75% and 71% resp. achieved a Mol CR after immediate SCT (table). However, the OS with immediate SCT vs later SCT was 34% (N=52) vs 63% (N=69) at 5 yrs resp (p=.002) and the corresp. RD was 42% vs 66% (p=.001) resp. Furthermore, pts with next consolidation leading to molCR had a superior OS (76% at 5yrs) vs those who did not (44% at 5yrs) (p=.02). A similar difference was seen for RD (60% vs 36% resp.; p=.08). In addition 102 pts with MolR during/after chemo were observed (median time to MolR: 18 mo). OS and RD from MolR onwards were 51% and 35% at 5 yrs. 38 pts were transplanted in ongoing 1st CR. OS w/o and with SCT was 37% vs 71% (p=.001) resp. with corresp. RD of 5% vs 70% (p Important conclusions for future MRD based therapy can be drawn from this large adult ALL cohort: 1) a similar poor prognostic impact of MolF and MolR is underlined; 2) it is demonstrated that pts with any detectable MRD (MRDpos) have an intermediate prognosis and require regular follow-up MRD testing; 3) SCT offers an advantage in MolF and MolR pts, however 4) evidence was provided that later SCT (i.e. in better molecular status) could be superior to immediate SCT; 5) compliance with MRD testing and SCT in MolF increased over time; 6) many MolF pts relapsed quickly; 7) additional chemotherapy offered limited chances to achieve a MolCR, but 8) promising MolCR rates were observed for targeted therapies. For future optimisation of MRD based therapy, logistics for decisions have been implemented earlier in the GMALL trial 08 in order to avoid rapid relapses. A first stratification takes place already after induction and targeted therapies are recommended in all MolF pts before SCT. Download : Download high-res image (209KB) Download : Download full-size image Disclosures Rollig: Janssen: Research Funding; Bayer: Research Funding. Kneba: AbbVie: Consultancy, Honoraria, Other: travel support, Research Funding; F. Hoffmann-LaRoche: Consultancy, Honoraria, Other: travel support, Research Funding; Gilead: Consultancy, Honoraria, Other: travel support, Research Funding; Janssen-Cilag: Consultancy, Honoraria, Other: travel support, Research Funding; Mundipharma: Consultancy, Honoraria, Other: travel support, Research Funding.

Germline Mutations in CTLA4 and Spectrum of Bone Marrow Features Overlapping with Aplastic Anemia and Large Granular Lymphocyte Lymphocytosis

Introduction: CTLA4 haploinsufficiency due to germline heterozygous mutations in the cytotoxic T-lymphocyte antigen-4 gene (CTLA4) results in severe systemic immune dysregulation; infiltrative lymphocytic lesions of lungs (e.g. lymphocytic pneumonitis), GI tract (e.g.lymphocytic colitis), and CNS; and a spectrum of autoimmunity, immunodeficiency and peripheral blood cytopenias. We describe the hematologic, bone marrow (BM), and flow cytometry (FC) features of CTLA4 haploinsufficiency of a cohort of 14 patients, which show overlapping morphologic features with acquired aplastic anemia (acAA), and large granular lymphocyte lymphocytosis (LGL) in a subset of patients. We compared the findings to those in a separate cohort of 20 age matched patients diagnosed with acAA. There were significant differences between acAA and CTLA4 deficiency based on analysis of BM lymphocyte subsets and marrow lymphocyte infiltration patterns.Materials and Methods: Peripheral blood (PB), BM and FC analysis of patients with germline mutations in CTLA4, and acAA was performed along with review of clinical, molecular and laboratory data. Statistical comparisons were made using Mann Whitney test, with p < 0.05 for significance.Results: Median age at initial evaluation for patients with CTLA4 haploinsufficiency was 23.5 years (range 11-71 years), with a male-to-female ratio of 11:3. 11 out of 14 patients had cytopenias. Of those with cytopenias 2/11 had pancytopenia, 4/11 had bicytopenia and 5/11 had unilineage cytopenia. Hypogammaglobulinemia was common (13/14). In some cases cytopenias were recognized as autoimmune in etiology with immune thrombocytopenic purpura and autoimmune hemolytic anemia, among the most common hematologic manifestations. Three patients met criteria for a diagnosis of moderate AA, 1 for severe AA, and 1 for LGL. Overall bone marrow biopsies were hypocellular for age (10/14) with a subset (29% [4/14]) showing severe trilineage hypoplasia with ≤20% cellularity. Atypical interstitial CD8-positive T-cell infiltrates with prominent non-paratrabecular CD4-positive T-cell aggregates were evident by immunohistochemistry in most cases (10/14), accompanied by a near absence of B-cells. T-cell clonality was assessed by T-cell receptor (TCR) gamma PCR and showed abnormal oligoclonal T-cell populations in 3/8, and one case with a clonal T-cell receptor gene rearrangement. B-cell IgH PCR reactions were polyclonal. In all cases, cytogenetic studies were either negative or showed only minor abnormalities (i.e. loss of Y chromosome). Given the morphologic overlap of a subset of CTLA4 cases with acAA, we compared bone marrow flow cytometry analysis of 8 CTLA4 cases with 20 acAA cases. CTLA4 patients showed >5 fold reduction in the median percentage of B-cells in comparison to acAA (p=0.0005). While acAA cases demonstrated relative preservation the B-cell compartment with intact B-cell maturation sequence; in contrast CTLA4 cases showed abnormal B-cell maturation patterns in half of cases characterized by few detectable early B-cell precursors, and progressive loss of intermediate and mature forms. T-cell populations in CTLA4 showed inverted CD4:CD8 ratios with relatively increased CD57+ T-cells, and atypical dim expression of CD20 on T-cells. Morphologically, when the markedly hypocellular CTLA4 cases were compared to acAA on core biopsies, little difference in marrow cellularity was seen; however, the CTLA4 cases showed multiple large atypical T-cell aggregates and near total absence of B-cells, which was not seen in acAA; acAA cases showed scattered background T-cells and B-cells.Conclusions: CTLA-4 deficiency presents with cytopenias, autoimmunity, and immunodeficiency with a subset of patients that have features overlapping with bone marrow failure, specifically AA and LGL. Herein we demonstrate a combination of abnormal BM findings, previously unreported, comprising hypocellularity, CD8-positive interstitial T-cell infiltrates with atypical CD4-positive T-cell aggregates, marked B-cell lymphopenia, increased LGLs and abnormal T-cell clonality patterns by PCR. These features may help identify patients harboring CTLA4 mutations for early detection and optimal therapeutic management. DisclosuresYoung:Novartis/GSK to institute: Research Funding. Townsley:Novartis/GSK to institute: Research Funding.

Dynamics of extended-spectrum cephalosporin-resistant Escherichia coli in pig farms: A longitudinal study

ObjectivesPoint prevalence estimates of extended-spectrum cephalosporin-resistant Escherichia coli (ESC-R-Ec) are important surveillance measures but may not uncover the ESC-R-Ec dynamics within pig farms. A longitudinal study was therefore performed by sampling individual pigs, pig farmers and the environment. MethodsOn average, 30 (range 10–46) piglets of 31 Swiss farms were sampled during the suckling, weaning and fattening stages (n= 2437 samples). In addition, stool from pig farmers and environmental samples were obtained and metadata collected by questionnaires. ESC-R-Ec was identified by routine culture, and clonal relationships and resistance genes were derived from whole genome sequencing data. ResultsWorking on pig farms was not associated with an increased prevalence of ESC-R-Ec in humans. ESC-R-Ec prevalence significantly decreased from 6.2% to 3.9% and 1.8% for the suckling, weaned and fattening pigs, respectively (P < 0.001). Within the 57 ESC-R-positive suckling piglets, persisting carriage was detected in 25 animals at two consecutive time points and one animal at three consecutive time points. Clonal spread (n=7 farms, 22.6%) and horizontal gene transfer (n=1 farm, 3%) within pigs but not between humans and animals was detected. Liquid manure (n=10 samples, 16.7%) was identified as the major environmental reservoir of ESC-R-Ec in the pig farm environment. ConclusionsPig farming practices like all-in-all-out systems, but not antimicrobial usage, were associated with reduced risk of ESC-R-Ec at the farm level. As carriage duration is normally short within the individual pigs, the risk of recolonisation and clonal spread of ESC-R-Ec might be reduced by applying appropriate decontamination strategies.

Activity of plazomicin against carbapenem-intermediate or -resistant Escherichia coli isolates from the United States and international sites in relation to clonal background, resistance genes, co-resistance, and region.

Emerging carbapenem resistance in Escherichia coli, including sequence type 131 (ST131), threatens therapeutic efficacy. Plazomicin (PLZ), a semisynthetic aminoglycoside approved by the FDA in 2018, overcomes the most common aminoglycoside resistance mechanisms and maintains activity against many carbapenem-intermediate or -resistant (CIR) E. coli strains. To assess plazomicin susceptibility among CIR E. coli in relation to region and multiple bacterial characteristics. We determined broth microdilution MICs for plazomicin and 11 comparators against 343 CIR clinical E. coli isolates, then compared susceptibility results by bacterial characteristics and region. The collection comprised 203 US isolates (2002-17) and 141 isolates from 17 countries in Europe, Latin America, and the Asia-West Pacific region (2003-17). Isolates were characterized for phylogenetic group, resistance-associated sequence types (STs) and subsets thereof, and relevant β-lactamase-encoding genes. Plazomicin exhibited the highest percentage susceptible (89%) after tigecycline (99%). The percentage susceptible to plazomicin varied significantly by phylogroup (63%, group B1; versus >93%, others) and ST131 subclone (92%, H30Rx; versus 87%-89%, H30R1 and non-H30), but not ST. It also varied by resistance genotype [higher with Klebsiella pneumoniae carbapenemase (KPC), lower with metallo-β-lactamases], global region [highest for Latin America (94%), lowest for Asia-West Pacific (69%)], and US region (80%, South, versus 96%-100%, others). Although reduced susceptibility to comparators often predicted reduced susceptibility to plazomicin, even among comparator-intermediate or -resistant isolates the plazomicin-susceptible fraction was ≥77%, except for amikacin (53%). The likely utility of plazomicin against CIR E. coli is high overall, but varies with region and multiple bacterial characteristics.

Serial ctDNA profiling in patients with metastatic prostate cancer undergoing treatment with radium-223.

e17018 Background: Assessment of the anti-tumor response to radium-223 (Ra-223) treatment is difficult in individuals. PSA levels seldom fall and imaging of existing bone metastases is imprecise. Circulating tumor DNA (ctDNA) is a non-invasive marker of prostate tumor DNA, with static measurements demonstrating high concordance rates with tumor DNA. Serial monitoring of ctDNA levels may provide insights into therapy response, targetable mutations, and progression of disease. We report changes in ctDNA profiles and clinical associations of 10 patients who had serial ctDNA profiles during Ra-223 treatment. Methods: Serial ctDNA analyses of mutations (73 genes) and amplifications (18 genes) were performed via a commercial provider (Guardant 360; Guardant Health). Assays were performed before treatment, after the third Ra-223 dose, and after the final Ra-223 dose. ctDNA data (minor allele frequencies for mutations, copy number for amplifications) were reviewed to identify clonal mutations with at least 50% change in highest allelic frequency or 30% change in copy number during treatment. Clinical data (performance status (PS), PSA, pain score, prescribed oral morphine equivalents (OME), imaging, progression free survival (PFS), and overall survival) were gathered via chart review. Clinical progression was defined by an increase in at least 2 bone lesions on bone scan, progression of soft tissue disease by RECIST, worsening PS, or at least 20% increase in OME. Results: Three of 10 patients had a reduction in the proportion of mutated or amplified tumor DNA as measured by ctDNA profiling, after receiving Ra-223 (response group). Median PFS defined by time to PSA rise of at least 25% in this group was 120 days (95% CI 83 to 237) compared to 29 days (95% CI 21 to 145) in the group without a reduction (no response group). Median clinical PFS defined as above was 266 days (95% CI 109 to 291) in the response group compared to 83 days (95% CI 33 to 182) in the no response group. There was no difference in baseline pain score between the two groups, though median baseline PSA was higher in the no-response group (97.14 compared to 20.05). Reductions in allelic frequency were noted in several mutated genes, including AR, ARID1A, CDK12, and TP53(G245C), during Ra-223 therapy of subjects in the response group. Decreases in copy number were also seen in several amplified genes, including AR, CCNE1, CDK6, FGFR1. In the no-response group, 4/7 patients had an increase in AR amplification; 3/7 patients had an increase in variant allele fraction of TP53 mutations. In the response group of 3 patients who had a reduction in clonal mutations, none had an increase in the AR amplification. Conclusions: A reduction in proportion of clonal mutations or gene amplification measured in ctDNA may be associated with a longer PFS in patients treated with Ra-223. Mutations in the TP53 gene and amplification of AR gene were associated with shorter PFS.

IRG-4mC: Neural Network Based Tool for Identification of DNA 4mC Sites in Rosaceae Genome

DNA N4-Methylcytosine is a genetic modification process which has an essential role in changing different biological processes such as DNA conformation, DNA replication, DNA stability, cell development and structural alteration in DNA. Due to its negative effects, it is important to identify the modified 4mC sites. Further, methylcytosine may develop anywhere at cytosine residue, however, clonal gene expression patterns are most likely transmitted just for cytosine residues in strand-symmetrical sequences. For this reason many different experiments are introduced but they proved not to be viable choice due to time limitation and high expenses. Therefore, to date there is still need for an efficient computational method to deal with 4mC sites identification. Keeping it in mind, in this research we have proposed an efficient model for Fragaria vesca (F. vesca) and Rosa chinensis (R. chinensis) genome. The proposed iRG-4mC tool is developed based on neural network architecture with two encoding schemes to identify the 4mC sites. The iRG-4mC predictor outperformed the existing state-of-the-art computational model by an accuracy difference of 9.95% on F. vesca (training dataset), 8.7% on R. chinesis (training dataset), 6.2% on F. vesca (independent dataset) and 10.6% on R. chinesis (independent dataset). We have also established a webserver which is freely accessible for the research community.

Imaging cell lineage with a synthetic digital recording system.

During multicellular development, spatial position and lineage history play powerful roles in controlling cell fate decisions. Using a serine integrase-based recording system, we engineered cells to record lineage information in a format that can be read out in situ. The system, termed integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR), allowed in situ reconstruction of lineage relationships in cultured mouse cells and flies. intMEMOIR uses an array of independent three-state genetic memory elements that can recombine stochastically and irreversibly, allowing up to 59,049 distinct digital states. It reconstructed lineage trees in stem cells and enabled simultaneous analysis of single-cell clonal history, spatial position, and gene expression in Drosophila brain sections. These results establish a foundation for microscopy-readable lineage recording and analysis in diverse systems.

Perspective: sensitive detection of residual lymphoproliferative disease by NGS and clonal rearrangements—how low can you go?

Malignant lymphoproliferative disorders collectively constitute a large fraction of the hematological cancers, ranging from indolent to highly aggressive neoplasms. Being a diagnostically important hallmark, clonal gene rearrangements of the immunoglobulins enable the detection of residual disease in the clinical course of patients down to a minute fraction of malignant cells. The introduction of next-generation sequencing (NGS) has provided unprecedented assay specificity, with a sensitivity matching that of polymerase chain reaction-based measurable residual disease (MRD) detection down to the 10-6 level. Although reaching 10-6 to 10-7 is theoretically feasible, employing a sufficient amount of DNA and sequencing coverage is placed in the perspective of the practical challenges when relying on clinical samples in contrast to controlled serial dilutions. As we discuss, the randomness of subsampling must be taken into account to accommodate the sensitivity threshold-in terms of both the required number of cells and sequencing coverage. As a substantial part of the reviewed studies do not state the depth of coverage or even amount of DNA in some cases, we call for increased transparency to enable critical assessment of the MRD assays for clinical implementation and feasibility.

In-utero exposure to zidovudine-containing antiretroviral therapy and clonal hematopoiesis in HIV-exposed uninfected newborns.

Zidovudine (ZDV) has been extensively used in pregnant women to prevent vertical transmission of HIV but few studies have evaluated potential mutagenic effects of ZDV during fetal development. Our study investigated clonal hematopoiesis in HIV-exposed uninfected (HEU) newborns, 94 of whom were ZDV-exposed and 91 antiretroviral therapy (ART)-unexposed and matched for potential confounding factors. Utilizing high depth sequencing and genotyping arrays, we comprehensively examined blood samples collected during the first week after birth for potential clonal hematopoiesis associated with fetal ZDV exposure, including clonal single nucleotide variants (SNVs), small insertions and deletions (indels), and large structural copy number or copy neutral alterations. We observed no statistically significant difference in the number of SNVs and indels per person in ZDV-exposed children (adjusted ratio [95% confidence interval, CI] for expected number of mutations = 0.79 [0.50--1.22], P = 0.3), and no difference in the number of large structural alterations. Mutations in common clonal hematopoiesis driver genes were not found in the study population. Mutational signature analyses on SNVs detected no novel signatures unique to the ZDV-exposed children and the mutational profiles were similar between the two groups. Our results suggest that clonal hematopoiesis at levels detectable in our study is not strongly influenced by in-utero ZDV exposure; however, additional follow-up studies are needed to further evaluate the safety and potential long-term impacts of in-utero ZDV exposure in HEU children as well as better investigate genomic aberrations occurring late in pregnancy.

Clonal Clusters, Molecular Resistance Mechanisms and Virulence Factors of Gram-Negative Bacteria Isolated from Chronic Wounds in Ghana.

Wound infections are common medical problems in sub-Saharan Africa but data on the molecular epidemiology are rare. Within this study we assessed the clonal lineages, resistance genes and virulence factors of Gram-negative bacteria isolated from Ghanaian patients with chronic wounds. From a previous study, 49 Pseudomonas aeruginosa, 21 Klebsiella pneumoniae complex members and 12 Escherichia coli were subjected to whole genome sequencing. Sequence analysis indicated high clonal diversity with only nine P. aeruginosa clusters comprising two strains each and one E. coli cluster comprising three strains with high phylogenetic relationship suggesting nosocomial transmission. Acquired beta-lactamase genes were observed in some isolates next to a broad spectrum of additional genetic resistance determinants. Phenotypical expression of extended-spectrum beta-lactamase activity in the Enterobacterales was associated with blaCTX-M-15 genes, which are frequent in Ghana. Frequently recorded virulence genes comprised genes related to invasion and iron-uptake in E. coli, genes related to adherence, iron-uptake, secretion systems and antiphagocytosis in P. aeruginosa and genes related to adherence, biofilm formation, immune evasion, iron-uptake and secretion systems in K. pneumonia complex. In summary, the study provides a piece in the puzzle of the molecular epidemiology of Gram-negative bacteria in chronic wounds in rural Ghana.

Histological Comparison Of Ground Section, Decalcified Section and Resin Embedded Section Of Bone - A Review

In Ground section there is no need to remove the mineral, so that the bone in the section contains both the mineral and the collagen that form most of the bone structure. Resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. It gives better nuclear staining and reduces hydrolysis but may damage the tissue. In decalcified section inorganic substance is dissolved and the organic substance remains while in ground section organic substance is burnt and inorganic substance remains. This review is done to compare the ground section, decalcified section and resin embedded section of the bone to determine the better processing for microscopical study of the bone.

Clonotypic Features of Rearranged Immunoglobulin Genes Yield Personalized Biomarkers for Minimal Residual Disease Monitoring in Multiple Myeloma.

Due to improved treatment, more patients with multiple myeloma (MM) reach a state of minimal residual disease (MRD). Different strategies for MM MRD monitoring include flow cytometry, allele-specific oligonucleotide-quantitative PCR, next-generation sequencing, and mass spectrometry (MS). The last 3 methods rely on the presence and the stability of a unique immunoglobulin fingerprint derived from the clonal plasma cell population. For MS-MRD monitoring it is imperative that MS-compatible clonotypic M-protein peptides are identified. To support implementation of molecular MRD techniques, we studied the presence and stability of these clonotypic features in the CoMMpass database. An analysis pipeline based on MiXCR and HIGH-VQUEST was constructed to identify clonal molecular fingerprints and their clonotypic peptides based on transcriptomic datasets. To determine the stability of the clonal fingerprints, we compared the clonal fingerprints during disease progression for each patient. The analysis pipeline to establish the clonal fingerprint and MS-suitable clonotypic peptides was successfully validated in MM cell lines. In a cohort of 609 patients with MM, we demonstrated that the most abundant clone harbored a unique clonal molecular fingerprint and that multiple unique clonotypic peptides compatible with MS measurements could be identified for all patients. Furthermore, the clonal immunoglobulin gene fingerprints of both the light and heavy chain remained stable during MM disease progression. Our data support the use of the clonal immunoglobulin gene fingerprints in patients with MM as a suitable MRD target for MS-MRD analyses.

T-lymphoblastic leukemia/lymphoma with interfollicular growth pattern and Castleman-like morphologic features

Indolent T-lymphoblastic proliferation (iT-LBP) is a recently recognized benign pathologic condition commonly associated with Castleman disease. The lymph node of this condition shows preserved lymphoid architecture with proliferation of nonclonal T-lymphoblasts in the interfollicular area. iT-LBP is a rare condition and a diagnosis of exclusion. As a rule, it requires sophisticated techniques to exclude T-lymphoblastic leukemia/lymphoma (T-LBL/L). We present an unusual T-LBL/L in a lymph node mimicking iT-LBP. The case showed preserved nodal architecture, morphologic features of hyaline vascular Castleman disease, proliferation of T-lymphoblasts in the interfollicular area, and no clonal T-cell receptor gene rearrangement. The follow-up lymph node specimen excised 6 months later showed full-blown lymphoblastic lymphoma with an abnormal karyotype, near-early-T-precursor phenotype, partial CD19 expression, and SETD2 mutation.

CD3−CD4+ Lymphocytic Variant Hypereosinophilic Syndrome: Diagnostic Tools Revisited

Identification of patients with lymphocytic variant hypereosinophilic syndrome (L-HES) is challenging, and has important prognostic and therapeutic implications. This study was undertaken to assess diagnostic tools for L-HES and to develop evidence-based diagnostic recommendations. Biomarkers of T-cell-driven disease were compared between patients with L-HES versus idiopathic HES (I-HES) variants. Those performed routinely (serum immunoglobulin levels, T-cell phenotyping, T-cell receptor [TCR] gene rearrangement patterns) were collected from medical files, whereas others were prospectively assessed on stored blood samples (serum CCL17/thymus and activation regulated chemokine [TARC] levels, invitro cytokine production). This study included 48 patients with I-HES and 20 with L-HES associated with a CD3-CD4+ T-cell subset, including 7 with less than 5% aberrant cells. Neither increased serum immunoglobulin levels nor clonal TCR gene rearrangements were sufficiently sensitive or specific for L-HES. In contrast, systematically enhanced expression of the T-cell surface antigens CD2, CD5, CD45RO, and CD95 by these cells allowed for accurate detection by flow cytometry. Serum CCL17/TARC levels were significantly higher in patients with L-HES compared with those with I-HES, and a threshold of 3000 pg/mL allowed for detection of all subjects with L-HES with 75% specificity. Quantification of intracytoplasmic cytokine production by flow cytometry is the most reliable method for detection of enhanced type 2 cytokine expression, most notably for IL-4 and IL-13. Adapting the standard of procedure for T-cell phenotyping in patients with unexplained hypereosinophilia is currently the most reliable means of identifying those with CD3-CD4+ L-HES.

Liquid Biopsy Cell-free DNA Biomarkers in Patients With Oligometastatic Colorectal Cancer Treated by Ablative Radiotherapy.

To investigate the usefulness of cell-free DNA (cfDNA) in patients with oligometastasis. This study included oligometastatic colorectal cancer (CRC) patients who underwent ablative irradiation using stereotactic body radiotherapy or proton beam therapy for metastatic lesions at a single institution. cfDNA was purified from the plasma of pretreated patients and gene mutations were analyzed by next-generation sequencing. Progression-free survival (PFS) was statistically compared according to gene mutation, clonality or allele frequency. A total of 20 patients were analyzed. Mutations were detected in the following genes; TP53 (45%), APC (40%), KRAS (15%), PIK3CA (15%), NF1 (5%), BRCA1 (5%), ERBB2 (5%), FBXW7 (5%), KIT (10%), and HRAS (10%). Patients with multi-clonality of gene mutation showed tendency for poor PFS (p=0.07). Among 7 patients whose metastatic site was the lung, those with no cfDNA detected had significantly better PFS than those with cfDNA (p=0.02). cfDNA profiles could be predictive tools for early recurrence of oligometastatic CRC patients after ablative radiotherapy.