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Although radiation therapy is one of the most significant treatment modalities for localized prostate cancer, the prognostic factors for biochemical recurrence (BCR) regarding the treatment plan are unclear. We aimed to develop a novel dosiomics-based prediction model for BCR in patients with prostate cancer and clarify the correlations between the dosimetric factors and BCR.This study included 489 patients with localized prostate cancer (BCR: 96; no-BCR: 393) who received intensity modulated radiation therapy. A total of 2475 dosiomic features were extracted from the dose distributions on the prostate, clinical target volume (CTV), and planning target volume. A prediction model for BCR was trained on a training cohort of 342 patients. The performance of this model was validated using the concordance index (C-index) in a validation cohort of 147 patients. Another model was constructed using clinical variables, dosimetric parameters, and radiomic features for comparisons. Kaplan-Meier curves with log-rank analysis were used to assess the univariate discrimination based on the predictive dosiomic features.The dosiomic feature derived from the CTV was significantly associated with BCR (hazard ratio, 0.73; 95% CI, 0.57-0.93; P = .01). Although the dosiomics model outperformed the dosimetric and radiomics models, it did not outperform the clinical model. The performance significantly improved by combining the clinical variables and dosiomic features (C-index: 0.67; 95% CI, 0.65-0.68; P.0001). The predictive dosiomic features were used to distinguish high-risk and low-risk patients (P.05).The dosiomic feature extracted from the CTV was significantly correlated with BCR in patients with prostate cancer, and the dosiomics model outperformed the model with conventional dose indices. Hence, new metrics for evaluating the quality of a treatment plan are warranted. Moreover, further research should be conducted to determine whether dosiomics can be incorporated in a clinical workflow or clinical trial.

Our previous study had shown that member 13 (Hspa13) of heat shock protein family A (Hsp70) promotes plasma cell (PC) production and antibody secretion. To further explore Hspa13 expression and function, we combined single-cell RNA-sequencing and antigen receptor lineage (BCR) analysis to characterize sheep red cell‒primed splenocytes. The single-cell transcriptional profiles revealed that Hspa13 is specifically and highly expressed in PCs. These results suggest that Hspa13 is a novel PC-specific marker. In terms of its function, we found that the CD19cre-mediated conditional knock-out (cKO) of Hspa13 reduced the expression of Ebi3 and IL-10 in PCs. Ebi3 and IL-10 are important factors in IL-4‒secreting type 2 helper T cell (Th2) activation and differentiation. As expected, we found that the Hspa13 cKO reduced IL‒4-expressing follicular helper T (Tfh2) cells. Finally, the single-cell antigen receptor analysis demonstrated that the Hspa13 cKO reduced the Aicda-mediated antibody class-switching recombination (CSR) and somatic hypermutation (SHM) in germinal centers (GCs) B cells. Altogether, the single-cell atlas of splenocytes revealed a critical indirect role for the novel PC-specific marker Hspa13 in CSR and SHM in GC B cells by promoting Ebi3 and IL-10 expression in PCs to induce IL-4-expressing Tfh2 cells. Further exploration of Hspa13 expression and function will provide valuable clues for how to use Hspa13 in the treatment of autoimmune diseases.

Myeloid/lymphoid neoplasm with t(8;22)(p11.2;q11.2)/BCR-FGFR1 is an extremely rare diagnosis, with few reported cases to date. In contrast to other FGFR1-partner rearrangements that are associated with chronic eosinophilic leukemia, acute myeloid leukemia, and/or lymphoblastic lymphoma, patients with BCR-FGFR1 have a myeloproliferative disorder that closely resembles chronic myeloid leukemia (CML). The current report describes a rare case of a 61 year old man with an atypical CML phenotype associated with t(8;22)(p11.2;q11.2)/BCR-FGFR1. A literature review is presented to enhance the awareness of this rare diagnostic entity.

Stringent regulation of IgE antibody production is critical for constraining allergic responses. This review discusses recent advances in understanding cell-intrinsic and extrinsic mechanisms that regulate the genesis and fate of IgE B cells. B cell-intrinsic regulation of IgE is orchestrated by the IgE B Cell Receptor (BCR). Through its antigen-independent signaling and low surface expression, the IgE BCR drives IgE B cells to differentiate into short-lived plasma cells and/or undergo apoptosis, restricting IgE-expressing cells from entering long-lived compartments. The pivotal extrinsic regulators of IgE responses are T follicular helper cells (T(FH)). T(FH) produce IL-4 and IL-21, which, respectively, are the major activating and inhibitory cytokines for IgE class-switching. Other newly identified T follicular subsets also contribute to IgE regulation. Although IgE responses are normally constrained, recent studies suggest that specific conditions can induce the formation of IgE responses with enhanced affinity or longevity, effectively ‘breaking the rules’ of IgE regulation.

Chronic myeloid leukemia (CML) has long been considered as a model of cancer caused by a single-driver genetic lesion (BCR/ABL1 rearrangement) that codes for a unique, gain-of-function, deregulated protein. However, in the last decade, high-throughput sequencing technologies have shed light on a more complex genetic landscape, in which additional mutations may be found in different disease phases, including diagnosis. These genetic lesions may even precede the occurrence of the Philadelphia (Ph) chromosome, pointing to an antecedent premalignant state of clonal hematopoiesis (CH) at least in some patients. Preliminary data support the hypothesis that the most frequent CH-associated mutations (DNMT3A, TET2, and ASXL1) may be associated with a risk of vascular event, but a definitive answer for this topic is still lacking. Moreover, several recent studies have linked a much more complex genetic background in chronic-phase CML, including signs of clonal evolution over time, with depth of treatment responses or with patient survival. In the present review, we address the current state of the art on age-related CH, its association with cardiovascular risk, and its pathophysiology; review the current knowledge on CH that precedes the acquisition of the Ph chromosome in CML patients; and discuss available evidence on the prognostic and predictive value of additional mutations in chronic-phase CML, either as a sign of clonal dynamics under treatment or as markers of an antecedent CH.

Targeting BCR and BCL-2 signaling is a widely used therapeutic strategy for chronic lymphocytic leukemia. C481S mutation decreases the covalent binding affinity of ibrutinib to BTK, resulting in reversible rather than irreversible inhibition. In addition to BTK, mutations in PLCG2 have been demonstrated to mediate acquired ibrutinib resistance. Venetoclax, a highly selective BCL2 inhibitor, has high affinity to the BH3-binding grove of BCL2. Mutation in BCL2 (Gly101Val) decreases the affinity of BCL2 for venetoclax and confers acquired resistance in cell lines and primary patient cells. This review discusses the common mechanisms of resistance to targeted therapies in chronic lymphocytic leukemia.

The finding that (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins IGs) are expressed in a significant fraction of chronic lymphocytic leukemia (CLL) highlighted the importance of antigen selection in disease pathogenesis. Subsets of patients sharing the same stereotyped BcR IG display consistent biological features and, at least for certain subsets, clinical presentation and outcome, including the response to particular treatment. On these grounds, BcR IG stereotypy emerges as a useful tool for dissecting the pronounced heterogeneity of CLL toward refining risk stratification and therapeutic management aligned with the principles of precision medicine.

Considering the critical roles of hsa-miR-155-5p participated in hematopoietic system, this study aims to clarify the possible pathogenesis of chronic myeloid leukemia (CML) induced by hsa-miR-155-5p.Three different strategies were employed, namely a network-based pipeline, a survival analysis and genetic screening method, and a simulation modeling approach, to assess the oncogenic role of hsa-miR-155-5p in CML. We identified new potential roles of hsa-miR-155-5p in CML, involving the BCR/ABL-mediated leukemogenesis through MAPK signaling. Several promising targets including E2F2, KRAS and FLI1 were screened as candidate diagnostic marker genes. The survival analysis revealed that mRNA expression of E2F2, KRAS and FLI1 was negatively correlated with hsa-miR-155-5p and these targets were significantly associated with poor overall survival. Furthermore, an overlap between CML-related genes and hsa-miR-155-5p target genes was revealed using competing endogenous RNA (ceRNA) networks analysis. Taken together, our results reveal the dynamic regulatory aspect of hsa-miR-155-5p as potential player in CML pathogenesis.

Pregnancy alters B cell development and function. B cell activation is initiated by antigens binding to the BCR leading to B cell survival, proliferation, antigen presentation and antibody production. We performed a genome-wide transcriptome profiling of splenic B cells from pregnant (P) and non-pregnant (NP) mice and identified 1136 genes exhibiting differential expression in B cells from P mice (625 up- and 511 down-regulated) compared to NP animals. In silico analysis showed that B cell activation through BCR seems to be lowered during pregnancy. RT-qPCR analysis confirmed these data. Additionally, B cells from pregnant women stimulated in vitro through BCR produced lower levels of inflammatory cytokines compared to non-pregnant women. Our results suggest that B cells acquire a state of hypo-responsiveness during gestation, probably as part of the maternal immune strategy for fetal tolerance but also open new avenues to understand why pregnant women are at highest risk for infections.

In the study of immunoglobulin V genes in fish genomes, we found that the species Gouania willdenowi does not possess any such regions, neither for the heavy chain nor for the light chains. Also, genes that code for the immunoglobulin constant regions were also not found. A detailed analysis of the chromosomal region of these genes revealed a deletion in the entire locus for regions of the heavy and light chains. These studies provide evidence that this species does not possess genes coding for immunoglobulins. Additionally, we found the genes that code for CD79a and CD79b protein molecules have also been deleted. Regions for the Tα/β lymphocyte receptors are present but the T γ/δ receptors were not found. In transcripts of two other Gobiesocidae species, Acystus sp. and Tomicodon sp., no antibody sequences could be detected, possibly indicating the absence of immunoglobulins in all species of this family.

We have recently identified a novel lymphocyte that is a dual expresser (DE) of TCRαβ and BCR. DEs in T1D patients are predominated by a public BCR clonotype (clone-x) that encodes a potent autoantigen that cross-activates insulin-reactive T cells. Betts and colleagues were able to detect DEs but alleged to not detect high DE frequency, clone-x, or similar clones in T1D patients. Unfortunately, the authors did not follow our methods and when they did, their flow cytometric data at two sites were conflicting. Moreover, contrary to their claim, we identified clones similar to clone-x in their data along with clones bearing the core motif (DTAMVYYFDYW). Additionally, their report of no increased usage of clone-x VH/DH genes by bulk B cells confirms rather than challenges our results. Finally, the authors failed to provide data verifying purity of their sorted DEs, making it difficult to draw reliable conclusion of their repertoire analysis. This Matters Arising Response paper addresses the Japp et al. (2021) Matters Arising paper, published concurrently in Cell.

Introduction The BCR/ABL1 fusion exists in several different isoforms, depending on the precise position of the t(9;22)(q34;q11) genomic breakpoints in each case. In chronic myeloid leukemia (CML), the two most common isoforms are known as e13a2 and e14a2, which are shorthand for mRNA fusions of BCR exon 13 or BCR exon 14, respectively, to ABL1 exon 2. Historically, these two fusions were referred to as b2a2 and b3a2, with b2 (BCR exon 13) and b3 (BCR exon 14) corresponding to the second and third exons within the classically-defined 5.8-kb major breakpoint cluster region (M-BCR) within BCR.1 Both e13a2 and e14a2 encode a 210-kDa BCR/ABL1 protein (p210), but e14a2 encodes a chimeric protein that is 25 amino acids larger than e13a2, corresponding to the 75 bp of BCR exon 14. At the genomic DNA level, the chromosome 22 breakpoint typically occurs in BCR intron 13 for e13a2 and BCR intron 14 for e14a2. Collectively, e13a2 and e14a2 BCR/ABL1 account for 98% of CML cases, with the majority of these expressing e14a2. Approximately 10% of cases express both isoforms.1,2 A large international survey of CML cases found small but significant differences in the relative prevalence of the two fusions in relation to age and gender, with e13a2 being more frequent in males and less common in the elderly.2

In the past decade, inhibition of B-cell receptor-associated (BCR) Bruton tyrosine kinase (BTK), phosphoinotiside-3 kinase (PI3K), and the pro-survival protein BCL2 using small molecules has led to a paradigm shift in CLL therapy.1,2 However, resistance to targeted agents is inevitable. In addition to acquiring target protein mutations, CLL cells may lose dependence on Bcl-2.3–6 Emerging data suggest that such cells may be susceptible to inhibition of alternate BCL2 family proteins, as well as signaling pathways that are activated in this setting.6

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) expressed in T cells may regulate immune responses in the gut. In addition to T cells, B cells are also an important population in the gut-associated lymphoid tissues that orchestrate mucosal homeostasis. However, the role of CEACAM1 in B cells has not been elucidated. We herein analyzed mature B cells to determine the functions of CEACAM1. Flow cytometry revealed high expression of CEACAM1 on B cells in secondary lymphoid tissues. Cytokine production induced by activation of B cell receptor (BCR) signaling was suppressed by CEACAM1 signaling in contrast to that associated with either Toll-like receptor 4 or CD40 signaling. Confocal microscopy revealed co-localization of CEACAM1 and BCR when activated with anti-Igμ F(ab’)2 fragment. Overexpression of CEACAM1 in a murine B cell line, A20, resulted in reduced expressions of activation surface markers with decreased Ca2+ influx after BCR signal activation. Overexpression of CEACAM1 suppressed BCR signal cascade in A20 cells in association with decreased spontaneous proliferation. Our results suggest that CEACAM1 can regulate BCR-mediated mature B cell activation in lymphoid tissues. Therefore, further studies of this molecule may lead to greater insights into the mechanisms of immune responses within peripheral tissues and the potential treatment of inflammatory diseases.

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory illness in children of less than 5 years of age which usually results in hospitalization or even in death. Vaccine development is hampered in consequence of a failed vaccine trial with fatalities in the 1960s. Even though research has been more focused on the RSV fusion protein in its pre-fusion conformation, maternal vaccination with post-fusion protein (post F) was considered as a promising vaccine strategy for passive immunization of babies, because post F preserves very potent neutralizing epitopes. We extensively analyzed post F-binding B cell receptor (BCR) repertoires of three vaccinees who received a post F-subunit vaccine in the context of a first-in-human, Phase 1, randomized, observer-blind, placebo-controlled clinical trial (ClinicalTrials.gov Identifier: NCT02298179). In order to compare the vaccine-induced BCR repertoires with BCR repertoires induced by natural infection, we also analyzed pre F- and post F-binding BCRs isolated from a healthy blood donor with relatively high F-binding memory B cell (MBC) frequencies. Analysis of the vaccine-induced repertoires revealed that preferentially VH4-encoded BCRs were expanded in response to vaccination. Estimation of antigen-driven selection further demonstrated that expanded BCRs accumulated positively selected replacement mutations which substantiated the hypothesis that post F-vaccination induces diversification of VH4-encoded BCRs in germinal centers. Comparison of the vaccine-induced BCR repertoires with clonally related pre and post F-binding BCRs of the healthy blood donor suggested that the vaccine expanded pre/post F cross-reactive MBCs. Interestingly, several vaccine-induced BCRs shared stereotypic VDJ gene junctions with known neutralizing Abs. Once expressed for functional characterization, the selected monoclonal Abs demonstrated the predicted neutralization activities in plaque reduction neutralization assays indicating that the post F-vaccine induced expansion of neutralizing BCRs.

The 2016 World Health Organization entity 'Myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2' encompasses a group of rare neoplasms that result from the formation of a fusion gene that leads to expression of an aberrant tyrosine kinase. This entity also contains variant JAK2 fusion partners, and detection of this defining event can be facilitated by various cytogenetic and molecular methods. Cryptic rearrangements of 9p24/JAK2 can be particularly challenging to identify. We describe the use of chromosomal microarray analysis (CMA), fluorescence in situ hybridization (FISH) with a probe for JAK2, and genomic mate pair analysis to describe a complex karyotype with a t(9;22) that produced a functional BCR-JAK2 fusion, leading to the appropriate diagnosis for the patient. This case highlights the importance of using an integrated genomic approach to fully define complex aberrations to assign proper diagnoses.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ(2) (PLCγ(2)). The PLCγ(2) variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ(2) variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ(2)-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca(2+)](i)), we show that various CLL-specific PLCγ(2) variants such as PLCγ(2)S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ(2) phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca(2+)](i). Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

Background Acute promyelocytic leukemia (APL) has a characteristic peculiar morphologic and genetic features as well as a more favorable outcome. We studied the differential effect of bcr-1 and bcr-3 isoforms of the promyelocytic leukemia/retinoic acid receptor alpha (PML-RARA) transcript together with FLT3 internal tandem duplication (FLT3-ITD) mutation status on the outcome of newly diagnosed de novo APL patients. Patients and Methods This cohort study included all patients diagnosed with APL at outpatient medical and pediatric oncology clinics of the National Cancer Institute, Cairo University, Cairo, Egypt, from May 2012 to January 2018. Results The study included 118 patients with APL, 71 adults (60.2%) and 47 children (39.8%). Median (range) age was 25 (1.5-70) years. Children had significantly higher total leukocyte count (≥10 × 109/L), disseminated intravascular coagulation (DIC), and thrombocytopenia ( Conclusion Age is an independent prognostic factor for APL. bcr-3 is significantly associated with FLT3-ITD in adults with APL. DIC and FLT3-ITD are adverse prognostic factors in children with APL. Despite children being at higher risk, outcome is better than in adults.

B cells express B-cell receptors (BCRs) which recognize antigen to trigger signaling cascades for B-cell activation and subsequent antibody production. BCR activation has a crucial influence on B-cell fate. How BCR is activated upon encountering antigen remains to be solved, although tremendous progresses have been achieved in the past few years. Here, we summarize the models that have been proposed to explain BCR activation, including the cross-linking model, the conformation-induced oligomerization model, the dissociation activation model, and the conformational change model. Especially, we elucidate the partially resolved structures of antibodies and/or BCRs by far and discusse how these current structural and further immunogenomic messages and more importantly the future studies may shed light on the explanation of BCR activation and the relevant diseases in the case of dysregulation.

Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. A second, commercially developed ddPCR assay for BCR-ABL1 was also evaluated. ABL1 and BCR-ABL1 transcript levels were similar with all assays, but the proportion of deep molecular responses was lower with ddPCR than with qPCR. The EAC ddPCR assay had a false-positive rate of 4% using a cutoff of three BCR-ABL1 copies per duplicate, compared with 2% without cutoff for the commercial ddPCR. The detection rate for molecular response 4.5 was 100, and a shift toward more minimal residual disease was seen in patient samples. In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.

miR-615-3p has previously been described as up-regulated in prostate cancer (PC) tissue samples compared with nonmalignant controls; however, its prognostic potential and functional role in PC remain largely unknown. In this study, we investigated the clinical and biological relevance of miR-615-3p in PC. The expression of miR-615-3p was measured in PC tissue specimens from 239 men who underwent radical prostatectomy (RP), and it was investigated if miR-615-3p could predict postoperative biochemical recurrence (BCR). These findings were subsequently validated in three independent RP cohorts (n = 222, n = 273, and n = 387) and functional overexpression studies conducted in PC cells (PC3M). High miR-615-3p expression was significantly associated with BCR in four independent PC patient cohorts (P < 0.05, log-rank test). In addition, high miR-615-3p expression was a significant predictor of PC-specific survival in univariate (hazard ratio, 3.75; P < 0.001) and multivariate (hazard ratio, 2.66; P = 0.008) analysis after adjustment for the Cancer of the Prostate Risk Assessment Post-Surgical (CAPRA-S) nomogram in a merged RP cohort (n = 734). Moreover, overexpression of miR-615-3p in PC cells (PC3M) significantly increased cell viability, proliferation, apoptosis, and migration. Together, our results suggest that miR-615-3p is a significant predictor of postoperative BCR and PC-specific survival and has oncogenic functions in PC cells.

Targeting the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) pathway represents a milestone in cancer therapy. However, the biologic features of diffuse large B-cell lymphoma (DLBCL) with PD-L1 expression remains unknown. We evaluated the correlation between pSYK and PD-L1 mRNA levels with RNAscope in situ hybridization and protein levels with immunohistochemistry in 108 cases of DLBCL, 25 of which featured loss of B-cell receptor (BCR), and investigated the effects of BCR signaling and MYC on PD-L1 mRNA and protein level with qPCR, immunoblotting and flow cytometery in DLBCL cell lines. PD-L1 amplification was detected with fluorescent in situ hybridization. Animal studies were applied to validate the in vitro findings. pSYK and MYC correlated with both PD-L1 mRNA and protein level. Genetic aberrations involving PD-L1 were rare in DLBCL. BCR signaling and MYC increased PD-L1 mRNA and protein expression. Inhibition of BCR signaling and BCR knockdown down-regulated PD-L1. DLBCL with a loss of loss of BCR showed low levels of PD-L1 mRNA and protein. PD-L1 was down-regulated by ibrutinib in a xenograft mouse model and correlated with slower tumor growth. In conclusion, this study demonstrates that DLBCL with PD-L1 expression features an activated B-cell receptor signal pathway, and that BCR inhibition and PD-L1 blockage may potentially synergize to targeting DLBCL.

As a continuation to our research, a series of novel Bcr-Abl inhibitors incorporated with 6-phenyl-1H-indazol-3-amine as hinge binding moiety (HBM) were developed based on confirmation analysis. Biological results indicated that these compounds exhibited an enhanced inhibition against Bcr-AblWT and Bcr-AblT315I in kinases assays, along with improved anti-proliferative activities in K562 cell assays. In particular, compound Y9 displayed comparable potency with that of imatinib. It potently inhibited Bcr-AblWT and Bcr-AblT315I kinases with IC50 of 0.043 μM and 0.17 μM, respectively. Furthermore, compound Y9 inhibited the proliferation of K562 and K562R cells with IC50 of 1.65 μM and 5.42 μM, respectively. Therefore, 6-phenyl-1H-indazol-3amine as HBM, combined with flexible linker, is a successful strategy contribute to research on T315I mutant resistance, and compound Y9 could be served as a starting point for further optimization.

BCR/ABLp210 fusion gene, the characteristic biomarker of chronic myelogenous leukemia (CML), contains two different transcription isoforms, e13a2 and e14a2, which lead to differences in the pathological features and response to targeted drug. At present, there is short of simple and fast technology to distinguish these two transcript isoforms. In this paper, RNA fusion-triggered rolling circle amplification (RF-RCA) strategy was developed to distinguish e13a2 and e14a2 transcripts directly from RNA extraction in one step. The simultaneous binding of dumbbell template and corresponding primer with target fused RNA can induce their proximal hybridization and trigger the RCA to produce lots of tandem repeat G-quadruplexes sequences for real time fluorescence readout with the interaction of Thioflavin T and G-quadruplex. The proposed strategy can detect as low as 0.1 aM target and discriminate e13a2 (0.01%) and e14a2 (0.1%) transcript isoforms directly from complex genomic RNA extraction, proving high sensitivity and specificity. Furthermore, the RF-RNA was successfully applied to analyze BCR/ABLp210 isoforms from clinical samples for accurately molecular subtyping and monitoring the response of imatinib treatment. The developed RF-RCA strategy presented an ultrasensitive, accurate and pragmatic toolbox to simple and rapid discriminate BCR/ABLp210 fusion isoforms for promoting clinical research and personalized treatment of CML.

Tumor intraductal carcinoma/cribriform architecture (IDC/C) is associated with an unfavorable prognosis and biochemical recurrence (BCR) in prostate cancer (PCa). Up to 70% of PCa patients are IDC/C-negative, but it is estimated that 20% of these cases still experience BCR. Thus, biomarkers for better detection of aggressive disease in IDC/C-negative patients are required.To investigate tumor-specific methylation of the transcription factor GBX2 as a novel prognosticator and predictor of BCR in PCa patients stratified by histopathologic features including IDC/C.Using genome-wide methylome profiling, we identified higher GBX2 methylation in grade group (GG) 4 tumors compared to GG1 (discovery cohort). The prognostic nature of GBX2 methylation was validated in silico using The Cancer Genome Atlas data (n=478) and a quantitative methylation assay for radical prostatectomy samples (n=254). Regulation of GBX2 methylation was investigated in prostate cells using methyl-CpG-binding domain sequencing and methylation analysis in functional knockouts of TET2, a key epigenetic player in prostate carcinogenesis.The association of GBX2 methylation with Gleason score (GS), pathologic stage (pT), IDC/C, and BCR was analyzed using Kruskal-Wallis and Mann-Whitney tests. Univariate and multivariate Cox regression analyses were used to predict BCR.GBX2 methylation was associated with GS (p0.05), pT (p0.01), and BCR (p0.05). GBX2 methylation (p=0.004), GS (p0.001), pT (p=0.012), and prostate-specific antigen (p=0.005) were independent predictors of BCR. Among IDC/C-negative patients, GBX2 methylation improved prediction of BCR (p=0.002). Loss of TET2 in prostate cells resulted in greater GBX2 methylation.We identified GBX2 methylation as a novel prognostic factor in PCa and an independent predictor of BCR. We demonstrated the additive value of GBX2 methylation in predicting BCR among IDC/C-negative patients and elucidated a novel TET2-mediated upstream epigenetic regulatory mechanism of GBX2.We identified GBX2 methylation as a promising prognostic biomarker that could improve the identification of prostate cancer patients at higher risk of biochemical recurrence.

Primary CNS lymphomas (PCNSL) are rare and poor prognosis diffuse large B-cell lymphomas. Because of the brain tumor environment and the restricted distribution of drugs in the CNS, specific PCNSL patient-derived orthotopic xenograft (PDOX) models are needed for preclinical research to improve the prognosis of PCNSL patients. PCNSL patient specimens (n = 6) were grafted in the caudate nucleus of immunodeficient nude mice with a 83% rate of success, while subcutaneous implantation in nude mice of human PCNSL sample did not generate lymphoma, supporting the role of the brain microenvironment in the PCNSL physiopathology. PDOXs showed diffuse infiltration of B-cell lymphoma cells in the brain parenchyma. Each model had a unique mutational signature for genes in the BCR and NF-κB pathways and retained the mutational profile of the primary tumor. The models can be stored as cryopreserved biobank. Human IL-10 levels measured in the plasma of PCNSL-PDOX mice showed to be a reliable tool to monitor the tumor burden. Treatment response could be measured after a short treatment with the targeted therapy ibrutinib. In summary, we established a panel of human PCNSL-PDOX models that capture the histological and molecular characteristics of the disease and that proved suitable for preclinical experiments. Our methods of generation and characterization will enable the generation of additional PDOX-PCNSL models, essential tools for cognitive and preclinical drug discovery.

Identification of BCR-ABL1 fusion gene amplification status is critically important in the effective management of chronic myelogenous leukemia (CML) patients. Earlier reports suggested that overexpression of BCR-ABL1 either through amplification of BCR-ABL1 fusion gene or by the up regulation of BCR-ABL1 transcript level might be an early phenomenon in the establishment of IM resistance and disease evolution in CML. In the current study, we performed dual color dual fusion locus specific BCR/ABL1 FISH analysis along with karyotype analysis using GTG banding (G-banding using trypsin and Giemsa) technique in 489 patients with different clinical stages of CML at diagnosis or during the course of the disease to unravel the spectrum of BCR-ABL1 fusion gene amplification status. Among the study group analyzed, it was found that prevalence of occurrence of BCR-ABL1 fusion gene amplification was significantly higher in advanced stages of disease and in IM resistant CML-CP patients when compared to initial stage of disease, de novo CML-CP. Cytogenetic and metaphase FISH characterization on our study samples revealed that BCR-ABL1 fusion gene amplification was occurred through the formation of extra copies Ph chromosomes and isoderived Ph chromosomes. Current study suggests that unrestrained activity of BCR-ABL1 played a vital role in resistance to targeted therapy and disease evolution in CML. In our study population, patients in progressive stage CML and in IM resistant CP with multiple copies of BCR-ABL1 fusion gene displayed a poor response to targeted treatment with IM. Hence, the early identification of BCR-ABL1 fusion gene amplification using FISH technique will lead to improved interventions and outcome in future CML patients.

Sjögren's syndrome (SS) is characterized by B cell hyperactivity documented by the production of plethora of autoantibodies and a strong tendency for NHL of B cell origin. Classical predictors of lymphoma have been already proposed and proved their validity, including clinical, serological and histopathologic biomarkers. The process of lymphomagenesis is multistep and encompasses mechanisms of antigen driven selection of the BCR with RF activity and various genetic contributors implicated in B cell proliferation, cell growth and cell cycle control, enhanced by a complex milieu of cytokines and trophic agents that are abundant within the inflammatory lesion of minor salivary glands of SS patients. Extensive efforts in the basic research field have revealed several novel biomarkers for lymphoma prediction while the major cellular and molecular mechanisms of evolutionary transition of B cells towards malignancy are under investigation. In this review, we present the current data regarding the newly proposed biomarkers for SS associated lymphoma prediction and a hypothetical model of lymphomagenesis based on the emerging data.

Aims Distinct types of PML-RARα hybrid transcripts viz bcr-1, bcr-2 and bcr-3 result from translocation between chromosomes 15 and 17 t(15;17) in Acute Promyelocytic Leukemia patients. We aimed to determine the frequencies of the PML-RARα transcripts and FLT3-ITD mutations in APL patients to evaluate their prognostic implications and also to analyze their impact on disease outcome. Main method RT-PCR and Rq-PCR were adopted for transcript typing and quantitation of PML-RARα transcripts while FLT3-ITD was detected by PCR in APL patients. Key findings PML-RARα bcr-1, bcr-2 and bcr-3 transcripts were found in 26, 3 and 16 cases respectively. 64.4% patients achieved complete remission, 22.2% expired early wherein majority of the cases expressed bcr-3 transcript (p = 0.03). 50% relapse rate was observed in patients with bcr-3 transcripts. Multivariate analysis showed expression of bcr-3 transcript associated with early death (p = 0.027) and increased relapse risk (P = 0.046). Patients expressing bcr-3 hybrid transcript showed lowest OS of 28.0 months (± 5.26) (p = 0.027). FLT3-ITD mutation was detected in 5 (11.1%) patients and presence of these mutations was not associated either with PML-RARα transcripts or with disease outcome. Significance bcr-3 transcript has a more lethal outcome and is also associated with frequent relapse risk in APL patients of our region.

PURPOSE/OBJECTIVE(S) Stereotactic body radiation therapy (SBRT) has emerged as a promising modality for the treatment of prostate cancer (PCa) and involves the delivery of ablative doses of radiation to the prostate. The patterns of failure at the time of biochemical recurrence (BCR) after SBRT remain unknown. The purpose of this study is to explore the development of distant metastasis (DM) following the diagnosis of BCR. MATERIALS/METHODS We queried a multi-institutional database of 2,430 patients who were prospectively treated with SBRT on single institution phase II trials or registries to identify patients who developed a BCR by the Phoenix criteria or by initiation of salvage therapy for PSA rise at a lower threshold. Patterns of distant failure were obtained for patients who had BCR, including presence and sites of DM. Multivariable analysis (MVA) using logistic regression was used to evaluate the association between multiple clinicopathologic and treatment variables and DM. RESULTS 150 patients with BCR were identified (6.2% of total). Of these, 19% presented with low-risk, 42% with intermediate-risk, and 39% with high-risk disease. The median PSA at BCR was 4.56, median time to BCR was 36 months, and median follow-up after BCR was 15.5 months. Sixty-seven patients (44.7%) had DM identified at or after the time of BCR. Among those with DM, 13 had M1a disease, 50 had M1b disease, 3 had M1c disease, and 1 had an unknown distribution of disease. Time to BCR was significantly shorter in patients with DM compared to those who did not have DM (28.1 vs. 41.4 months, P = 0.002) and initial PSA (iPSA) was significantly greater (11.0 vs. 8.0, P = 0.03). On MVA, Gleason Grade Group (GG) 4 (vs. GG 1, OR 3.76, P = 0.047) and higher SBRT dose (36.25 Gy vs. 35 Gy: OR 4.03, P = 0.02; 40 Gy vs. 35 Gy: OR 3.73, P = 0.03) were significantly associated with DM. CONCLUSION A substantial proportion of patients with BCR after SBRT for localized PCa ultimately develop DM, particularly those with higher iPSA and shorter time to BCR. The association with SBRT dose is likely reflective of the use of higher doses in more modern cohorts with higher-risk disease, while the association with GG likely reflects the more aggressive biology of these tumors at presentation. Further research into patterns of failure after SBRT is needed, especially in the context of more sensitive nuclear imaging. Early predictors of treatment failure are needed to facilitate timely treatment intensification.

Context: Ibrutinib inhibits BTK with the most typical response in CLL being partial remission (PR) with measurable minimal residual disease (MRD) in blood. Remission is usually maintained until development of genetically-driven resistance. Objective: Mechanism of adaptation and survival in MRD. Patients and Methods: Pre-treatment CLL cells and under-treatment MRD were systematically collected from 33 high-risk CLL patients. Samples were characterized by high-dimensional, single-cell flow cytometry; bulk genomic, transcriptomic, and chromatin accessibility profiling; and single-cell RNA-seq profiling. Results: The genetic composition of MRD remained constant across timepoints, indicating that ibrutinib did not have any impact in shaping its genomic diversity. Ibrutinib induced chromatin dynamics in MRD resulting in a predominantly closed state. Chromatin regions that became more accessible under ibrutinib were enriched of binding sites for transcription factor that lay downstream ERK signaling. Regions that turned into a less accessible state were enriched for the binding sites of TF emanating from the BCR. At the transcriptomic level, most of the differentially expressed genes between pre- and post-treatment samples were downregulated in MRD, while only a fraction was upregulated. IL4, NF-kB, and metabolism and proliferation signatures were downregulated in MRD, while MAPK and RAS signatures were upregulated. CLL spleen samples from TCL1 mice under ibrutinib treatment showed an upregulation of MAPK pathway genes compared to untreated mice. Single-cell transcriptomes revealed a distinct post-treatment cell population driven by MAPK activity that functionally pre-existed in a fraction of CLL cells of baseline samples before ibrutinib start. At the signaling level, ibrutinib had no effect on the integrity of RAS-BRAF-MAPK-ERK pathway protein expression upon crosslinking of the BCR with anti-IgM but not after stimulation of TLR9 or CD40. Functional validation on primary samples by western blotting shows that RAS/ERK can be activated by BCR stimulation, irrespective of ibrutinib treatment in ex vivo experiments. Pharmacological inhibition of MEK (trametinib) and ERK (ulixertinib) synergized with ibrutinib, leading to decreased cell viability. Conclusions: MRD under ibrutinib adapts its phenotype in an epigenetic way to maintain functional competence of BCR signaling via the MAPK pathway, which turned to be a vulnerability of MRD persisting under ibrutinib.

The advent of novel targeted therapies, specifically Bruton’s tyrosine kinase (BTK) inhibitors and the B-cell lymphoma 2 (BCL2) inhibitor, venetoclax, has revolutionized the treatment approach in the frontline setting for patients with chronic lymphocytic leukemia (CLL). By targeting BTK, a critical kinase in proximal B-cell receptor (BCR) signaling, this class of small molecule inhibitors impairs BCR-signaling and activation of NF-κB, and inhibits cell proliferation and migration.1,2 BCL2 is an antiapoptotic protein that is overexpressed in various B-cell cancers, including CLL.3 Venetoclax disrupts antiapoptotic signaling through BCL2, thereby inducing programmed cell death of CLL cells.4 Both of these options have significantly improved the efficacy of treatment in older patients in which efficacy of more traditional chemoimmunotherapy regimens was often hampered by increased toxicity limiting therapy. When embarking on initial therapy for patients with CLL and deciding between various options, it has become important to consider age, comorbidities, and functional condition as well as their disease specific features such as the results of their FISH and IGHV mutational status. Armed with this information, a provider can then review potential options of therapy with each patient focusing on both their disease specific features and comorbidities while taking into consideration their desire for continuous treatment versus time-limited therapy.

Checkpoint immunotherapy (CPI) has increased survival for some patients with advanced-stage bladder cancer (BCa). However, most patients do not respond. Here, we characterized the tumor and immune microenvironment in pre- and post-treatment tumors from the PURE01 neoadjuvant pembrolizumab immunotherapy trial, using a consolidative approach that combined transcriptional and genetic profiling with digital spatial profiling. We identify five distinctive genetic and transcriptomic programs and validate these in an independent neoadjuvant CPI trial to identify the features of response or resistance to CPI. By modeling the regulatory network, we identify the histone demethylase KDM5B as a repressor of tumor immune signaling pathways in one resistant subtype (S1, Luminal-excluded) and demonstrate that inhibition of KDM5B enhances immunogenicity in FGFR3-mutated BCa cells. Our study identifies signatures associated with response to CPI that can be used to molecularly stratify patients and suggests therapeutic alternatives for subtypes with poor response to neoadjuvant immunotherapy.

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease characterized by a defective adaptive immune receptor repertoire (AIRR) that fails to distinguish self from non-self antigens. The AIRR is vast, encompassing four T cell receptor (TCR) and three B cell receptor (BCR) chains, each of which displays an extraordinary amino acid sequence variability in the antigen-binding site. How the concerted action of T and B cell clones is associated with the development and clinical evolution of immune-mediated diseases is still not known. Using a new immunosequencing technology that allows the unbiased amplification of the seven receptor chains, we conducted an in-depth quantitative analysis of the seven-receptor chain variability in RA. Compared to healthy controls, the AIRR in RA was found to be characterized by a lower BCR diversity, the depletion of highly similar BCR clones, an isotype-specific signature as well as a skewed IGL chain and gene segment usage. A predictor based on quantitative multi-chain AIRR information was able to accurately predict disease, including the elusive seronegative subset of RA patients. AIRR features of the seven immune receptor chains were also different between patients with distinct clinically relevant phenotypes. Incorporating HLA variation data, we were able to identify the TCR clones that are specifically associated with the main disease risk variants. The longitudinal analysis of the AIRR revealed that treatment with Tumor Necrosis Factor (TNF) inhibitors selectively restores the diversity of B cell clones in RA patients by reducing the frequency of clones with a similar biochemical profile. The biochemical properties of the TNFi-modulated clones were also found to differ between responders and non-responders, supporting a different antigenic reactivity in the B cell compartment of these two groups of RA patients. Our comprehensive analysis of the TCR and BCR repertoire reveals a complex T and B cell architecture in RA, and provides the basis for precision medicine strategies based on the highly informative features of the adaptive immune response.

Germinal centres (GC) are sites of B-cell expansion and selection, which are essential for antibody affinity maturation. Compared to naive B-cells, GC B-cells have notable changes in their cell surface glycans. While these changes are routinely used to identify the GC, functional roles for these changes have yet to be ascribed. Detection of GCs by the antibody GL7 reflects a reduction in the glycan ligands for CD22, which is an inhibitory co-receptor of the B-cell receptor (BCR). To test a functional role for downregulated CD22 ligands in the GC, we generated a mouse model that maintains CD22 ligands on GC B-cells. With this model, we demonstrate that glycan remodeling plays a critical role in the maintenance of B-cells in the GC. Sustained expression of CD22 ligands induces higher levels of apoptosis in GC B-cells, resulting in reduced memory B-cell and plasma cell output as well as delayed affinity maturation of antibodies. The defect we observe in this model is dependent on CD22, highlighting that coordinated downregulation of CD22 ligands on B cells plays a critical function in the GC. Collectively, our study uncovers a crucial role for glycan remodeling and CD22 in B-cell fitness in the GC.

Background: Chronic myeloid leukemia (CML) is a reciprocal translocation disorder driven by a breakpoint cluster region (BCR)-Abelson leukemia virus (ABL) fusion gene that stimulates abnormal tyrosine kinase activity. Tyrosine kinase inhibitors (TKIs) are effective in the treatment of Philadelphia chromosome (Ph)+ CML patients. However, the appearance of TKI-resistant CML cells is a hurdle in CML treatment. Therefore, it is necessary to identify novel treatments that could target a different mechanism than that of tyrosine kinases. Methods: The study was designed to verify whether C-X-C chemokine receptor 2 (CXCR2) could be a novel target for TKI-resistant CML treatment. We examined CXCR2 ligands from CML patient samples and TKI-resistant CML cell lines. Then, we inhibited CXCR2 and examined the effects on cell proliferation and apoptosis using immunoblotting and flow cytometry. The CXCR2 inhibition effect was also confirmed using a mouse xenograft model with TKI-sensitive and -resistant CML cells. Findings: Interleukin 8 (IL-8), a CXCR2 ligand, was significantly increased in the bone marrow serum of initially diagnosed CML patients. CML cell lines expressed CXCR2, regardless of their sensitivity to TKIs. IL-8 stimulated CXCR2, mTOR, and c-Myc mRNA expression in CML cell lines. CXCR2 antagonists suppressed the proliferation of CML cells via cell cycle arrest in the G2/M phase. In addition, CXCR2 inhibition attenuated mTOR, c-Myc, and BCR-ABL expression, leading to CML cell apoptosis, irrespective of TKI responsiveness. Moreover, SB225002, a CXCR2 antagonist, caused higher cell death in CML cells than TKIs. Using a mouse xenograft model, we confirmed that SB225002 suppresses CML cells, with a prominent effect on TKI-resistant CML cells. Interpretation: Our findings demonstrate that IL-8 is a prognostic factor to the progress of CML. Inhibiting the CXCR2-mTOR-c-Myc cascade is a promising therapeutic strategy to overcome TKI-sensitive and -insensitive CML. Thus, CXCR2 blockade is a novel therapeutic strategy to treat CML, and SB225002, a commercially available CXCR2 antagonist, might be a drug candidate to treat TKI-resistant CML. Funding Statement: This study was supported by the Bio & Medical Technology Development Program of the National Research Foundation, which is funded by the Ministry of Science & ICT (2017M3A9C8060403). Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This study was approved by the internal review board of the Korea University Anam Hospital (IRB No. 2015AN0267).

Background: Long non-coding RNAs (lncRNAs) have been documented to be associated with genomic instability. Genomic instability-associated lncRNAs of prostate cancer (PCa) and their critical clinical implications remain to be understood. Method: A signature ( GILncSig ) was derived by genomic instability-associated lncRNAs to classify PCa patients into high-risk and low-risk group. The association of GILncSig and biochemical recurrence (BCR) is validated across three different datasets. The mutation burden and critical gene expression were compared between two risk groups. Findings: GILncSig separated patients into high-risk (n=121) group and low-risk (n=121) group in the train set. Patients with high GILncSig score suffered from more frequent BCR than those with low GILncSig score. The results were further validated in the test, the whole TCGA cohort, and different subgroups stratified by age and Gleason score (GS). High GILncSig risk score was significantly associated with high mutation burden and the low PTEN and CDK12 expression in PCa. The predictive performance of our BCR model outperformed other existing BCR models of PCa. The GILncSig could also predict BCR in the subgroup of patients with TP53 mutation or wild type. Interpretation: This study may provide a vital method to evaluate the impact of lncRNAs in genomic instability and new insights into the epigenetic mechanism of BCR. Funding: This work was supported by the National key research and development program of China (Grant No. 2017YFC0908003), National Natural Science Foundation of China (Grant No.81902578, 81974098, 8197032158), China Postdoctoral Science Foundation (2017M612971), Post-doctoral Science Research Foundation of Sichuan University (2020SCU12041). Declaration of Interests: The authors have no conflicts of interest to declare. Ethics Approval Statement: Due to the public availability of data in the TCGA database, there was no need to provide ethical consent.

Background: Biochemical recurrence (BCR) of prostate cancer (PCA) patient indicates poor prognosis and more complex treatment. However, the tools for predicting BCR remain controversial. This study performed a comprehensive analysis of gene expression and DNA methylation data to establish a BCR classifier for PCA. Methods: We analyzed DNA methylation data and mRNA expression datasets for over 1000 clinical samples from the TCGA cohort. We integrated RNA-sequencing and DNA methylation data to identify DNA methylation (DNAm) driven differentially expressed genes (DEGs). Then, these genes were determined to build a prognostic classifier by Kaplan-Meir analysis and 1000 times Cox LASSO regression with 10-fold CV. Furthermore, other datasets were utilized to validate externally. Findings: We identified a total of 65 DNA methylation-driven DEGs, and developed a four-DNAm-driven DEGs-based classifier which has been shown to accurately predict the BCR-free survival of patients in TCGA and validation sets. Additionally, multivariate Cox regression analysis determined the Classifier as an independent predictor of BCR (hazard ratio [HR] 0.426 [95% CI 0.194–0.936], P < 0.05). Then, a nomogram based on the Classifier and Gleason score was constructed, which show a predictive accuracy significantly higher than that of each variable alone (AUC at 1-, 3-, 5 years 0.701,0.799, 0.838). Interpretation: We identified and validated the BCR classifier of PCA constructing by four DEGs, suggesting this Classifier has the potential to guide treatment decisions for patients at differing risks of BCR. Funding Statement: This study was funded by the National Natural Science Foundation of China. Declaration of Interests: The authors declare no other competing interests.

Objective To evaluate the clinical significance of intraoperative bulbocavernosus reflex (BCR) during untethering surgery in predicting post-operative voiding function. Methods We conducted a retrospective review of pediatric patients who underwent untethering surgery with available intraoperative baseline BCR. BCR response during surgery was classified into loss or maintenance. Post-operative voiding function was determined as worsened or maintained based on history, postvoid residual urine measurement, and urodynamic study (UDS). Data regarding demographics, diagnosis, pre-operative voiding difficulty, re-untethering, syrinx, and abnormalities in electromyography were collected for analysis. Results We included 106 patients, with a mean age of 3.3 years, and 49 patients were male. BCR was lost in 15 patients during surgery and voiding function worsened in 14 patients after surgery. Lumbosacral lipoma was the most common diagnosis, and 16 patients were diagnosed with lipomyelomeningocele (LMMC). The sensitivity and specificity of intraoperative BCR for post-operative worsening of voiding function were 35.7%, and 88.5% at 6 months, respectively. The diagnosis of LMMC was statistically significant in a logistic regression analysis. The specificity of BCR at 6 months in patients with diagnosis other than LMMC was 93.4%, and intraoperative BCR was significant in a logistic regression analysis. Conclusions Intraoperative BCR during untethering could predict bladder function 6 months post-operatively with high specificity (88.5%), particularly in cases other than LMMC (93.4%), indicating that voiding function deterioration will not occur if intraoperative BCR is preserved. Significance Intraoperative BCR during untethering surgery is a useful tool to predict post-operative voiding outcome.

To screen the methylated genes for early diagnosis and biochemical recurrence (BCR) prediction in prostate cancer (PCa) patients.Differentially methylated CpG sites (DMCs) of PCa were screened out from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database. Combined with TCGA RNA sequencing data and clinical information, the DMCs associated genes with different expression and related to BCR were selected as candidate genes. Then, the expression level of the best candidate gene ZNF154 was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, the prognosis potential of the hypermethylation gene ZNF154 was assessed by Kaplan-Meier, univariate and multivariate cox regression analysis.A total of 87 candidate genes were screened out. Compared to benign prostate (BP) tissues, ZNF154 has three hypermethylation sites (cg03234186, cg12506930, cg26465391) in the promoter region in PCa tissues. qRT-PCR results showed that ZNF154 expression level was reduced in PCa tissues than in BP tissues (P = 0.004). Besides, the ZNF154 methylation level was negatively correlated with mRNA expression (r = -0.766, P 0.001), and was highly cancer-specific in PCa (area under the curves (AUCs) = 90.030%). In addition, Kaplan-Meier analysis showed ZNF154 methylation level was associated with BCR (P = 0.005), and ZNF154 could be an independent factor for BCR prediction in PCa by using univariate and multivariate cox regression analysis (P = 0.035, HR = 8.218).87 PCa specific genes were obtained. Further analysis gave the evidence that ZNF154 can be used as a specific maker for PCa diagnosis. Hypermethylation level of ZNF154 lead to gene expression inhibition and function loss, which contribute to the development and poor outcomes in PCa. In addition, the mean methylation level of ZNF154 can be used as an independent risk factor to predict BCR.

The complex genetic diversity ofchronic lymphocytic leukemia(CLL) makes it difficult to determine the effective and durable therapy beneficial to patients. During the several past years' significant insights in the biology of the disease and its treatment have been made, allowing for the identification of promising novel therapeutic agents. The investigation of signaling pathways to understand the biological character of CLL together with the development of molecular profiling is key in personalized approach in therapy for this disease. As it was already proven,maltotriose(M3) modified fourth generationpoly(propyleneimine)dendrimers(PPI-G4) modulateBCR, TRAILandWNTsignaling pathway gene expression in CLL cells and strongly influence their survival by inducingapoptosisand inhibiting proliferation. The aim of this study was to evaluate the influence of PPI-G4-M3 dendrimers on NFκB pathway gene expression in CLL (MEC-1) cells with 60 K microarray, as it is one of the major factors in thepathogenesisof B-cellneoplasms. The findings were compared with those obtained withFludarabine(FA) and the results indicate that PPI-G4-M3 dendrimers affect the expression of the examined genes and exert comparable effect on the CLL cells to FA. Dendrimers are one of the most potent groups of nanometer-sizedmacromoleculesfor closing the gap between the present ineffective treatment and the future effective personalized therapy due to their potential versatile biological properties

The majority of patients with TKI failure respond to HCT. However, the relapse risk remains high. This study has evaluated transplant outcomes in 223 CML patients with TKI failure due to resistance (n = 132) or intolerance (n = 29), as well as those that were TKI naïve/responding with advanced disease (n = 35) or with chronic phase (CP, n = 27). We studied outcomes according to post-transplant BCR-ABL transcript level within 3 months. With respect to transplant outcomes according to the post-transplant BCR/ABLtranscript level within 3 months, the group failing to achieve a 1.3 log reduction (n = 14, 12.4%) showed the highest relapse rate of 78.6% at 5 years, compared to 26.2% and 24.1% in the groups achieving 1.3-4.0 log reduction (n = 45, 39.8%), and ≥4.1 log reduction (n = 54, 47.8%) respectively (p 0.001). Multivariate analysis confirmed that the group failing to achieve a 1.3 log reduction had a 2.3-fold higher risk of death and 6.6 times higher risk of relapse. Poor overall survival after HCT was associated with advanced disease at diagnosis, but not disease status prior to HCT. Of 61 patients who relapsed after HCT, 47 were treated with post-transplant TKI therapy; those receiving TKI after loss of MR2 or MMR showed higher rates of response and survival compared to those receiving TKI after hematologic relapse (p 0.001). QPCR log reduction level within 3 months post transplantation is prognostic in this population.

B-cell linker protein (BLNK) is an adaptor protein that plays a crucial role in the B cell antigen receptor (BCR) signal pathway. To investigate the function of BLNK in teleost fish, we cloned a BLNK ortholog gene from the orange-spotted grouper (Epinephelus coioides). Homology analysis showed that the grouper BLNK (EcBLNK) had a 34%-77% amino acid identity in comparison to other vertebrates and shared the highest amino acid identity with BLNK from the Asian seabass Lates calcarifer. EcBLNK comprises an N-terminal SAM domain and a C-terminal B-cell linker SH2 domain. Ten tyrosine residues were well conserved between teleost fish and mammals. Tissue distribution analysis showed that EcBLNK was expressed mainly in immune organs and expression was at the highest level in head kidney. Co-localization of EcBLNK and EcCD79a was observed in transfected HEK293T cells. Overexpression of EcBLNK did not activate nuclear factor kappa-light-chain-enhancer of activated B cells. The protein level of EcBLNK in grouper head kidney leukocytes was increased by stimulation with lipopolysaccharide. In groupers infected with Cryptocaryon irritans, EcBLNK was regulated in the infected sites and the systemic organ which suggests that EcBLNK was activated in the immune response to parasite infection.

There is the first evidence of changes in the kinetics of B cell antigen receptor (BCR) internalisation of neoplastic cells in chronic lymphocytic leukemia (CLL) after the short-term and long-term administration of ibrutinib. We aimed to assess the influence of short-term and long-term ibrutinib treatment on the HLA-DR expression on CLL cells, T cells and monocytes. The immunophenotyping of CLL and immune cells in peripheral blood was performed on 16 high-risk CLL patients treated with ibrutinib. After early ibrutinib administration, the HLA-DR expression on CLL cells reduced (P = 0.032), accompanied by an increase in CLL cell counts in peripheral blood (P = 0.001). In vitro culturing of CLL cells with ibrutinib also revealed the reduction in the HLA-DR expression at protein and mRNA levels (P 0.01). The decrease in HLA-DR on CLL cells after the first month was followed by the gradual increase of its expression by the 12th month (P = 0.001). A one-month follow-up resulted in elevated absolute counts of CD4

The fundamental structure of eukaryotic cell plasma membrane is the phospholipid bilayer, which contains four major phospholipids. These phospholipids are asymmetrically distributed between the outer and inner leaflets. P4-ATPase flippase complexes play essential roles in ensuring this asymmetry. We found that conditional deletion of Tmem30a, the β subunit of P4-ATPase flippase complex, caused pancytopenia in mice. Tmem30a deficiency resulted in depletion of lineage-committed blood cells in the peripheral blood, spleen, and bone marrow. Ablation of Tmem30a also caused the depletion of hematopoietic stem cells (HSCs). HSC RNA sequencing results revealed that multiple biological processes and signal pathways were involved in the event, including mammalian target of rapamycin signaling, genes for HSC stemness, and genes responding to interferons. Our results also revealed that targeting Tmem30a signaling had therapeutic utility in BCR/ABL1-induced chronic myeloid leukemia.

Fast identification of BCR-ABL fusion genes is critical for precise diagnosis, risk stratification and therapy scheme selection in leukemia. More convenient methods are needed for quickly detection of the BCR-ABL fusion genes. Multiplex fluorescent reverse transcription quantitative real-time PCR (Multiplex RT-qPCR) methods are developed for detection of the at least 14 subtypes of BCR-ABL fusion genes in one tube at a time by using patients' bone marrow samples. The new Multiplex RT-qPCR method could quickly detect BCR-ABL fusion genes with sensitivity up to 10-106 copies. It can detect the fusion genes in patients' bone marrow samples containing any subtypes of the major bcr (M-bcr) e13a2, e14a2, e13a3 and e14a3, the minor bcr (m-bcr) e1a2 and e1a3, the micro bcr (μ-bcr) e19a2 and e19a3, and the nano bcr (n-bcr) e6a2 and e6a3. The specificity is comparable to the FISH methods. The cutoff for clinical diagnosis of BCR-ABL(+) is also determined by testing in clinical chronic myeloid leukemia samples. This is a new fast method with high sensitivity and specificity for clinical detection of BCR-ABL fusion genes. It will benefit the precise diagnosis, targeted therapy and minimal residual disease (MRD) monitoring in leukemia.

Efflux pumps (EPs) are an important mechanism responsible for extrusion of toxic compounds from the cell. Engineering of microbial cell EPs is a promising strategy in the production of biofuels and chemicals. In this study, we evaluate the ability of 44 native Escherichia coli EPs to enhance tolerance to short and medium carbon chain alcohols (SMCAs). Among EPs studied, MdtJ, Bcr, and MdtH and YdeA transported the majority of tested monohydroxy, dihydroxy, and trihydroxy alcohols, respectively. Overexpression of MdtJ, Bcr, MdtH and YdeA increased the tolerance of strains to SMCAs by 35%–50%. The efficiency of EPs for transporting SMCAs was verified by use of the 1, 2, 4-butanetriol (BT) synthesis system: where overexpression of MdtH and YdeA effectively increased the titers of BT by 31% and 18.5%, respectively. This work demonstrates that the innate EP system of E. coli is a resource which might be exploited to improve the synthesis of biofuels and chemicals.

Mechanistic target for rapamycin (mTOR) is a serine/threonine protein kinase that forms two distinct complexes mTORC1 and mTORC2, integrating mitogen and nutrient signals to regulate cell survival and proliferation; processes which are commonly deregulated in human cancers. mTORC1 and mTORC2 have divergent molecular associations and cellular functions: mTORC1 regulates in mRNA translation and protein synthesis, while mTORC2 is involved in the regulation of cellular survival and metabolism. Through AKT phosphorylation/activation, mTORC2 has also been reported to regulate cell migration. Recent attention has focused on the aberrant activation of the PI3K/mTOR pathway in B cell malignancies and there is growing evidence for its involvement in disease pathogenesis, due to its location downstream of other established novel drug targets that intercept B cell receptor (BCR) signals. Shared pharmacological features of BCR signal inhibitors include a striking "lymphocyte redistribution" effect whereby patients experience a sharp increase in lymphocyte count on initiation of therapy followed by a steady decline. Chronic lymphocytic leukemia (CLL) serves as a paradigm for migration studies as lymphocytes are among the most widely travelled cells in the body, a product of their role in immunological surveillance. The subversion of normal lymphocyte movement in CLL is being elucidated; this review aims to describe the migration impairment which occurs as part of the wider context of cancer cell migration defects, with a focus on the role of mTOR in mediating migration effects downstream of BCR ligation and other microenvironmental signals.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous diagnostic category with different molecular subtypes defined by distinct gene expression patterns and divergent mechanisms of oncogenic activation. Several studies have suggested an inferior survival for patients of the activated B-cell-like (ABC) versus the germinal center B-cell-like (GCB) DLBCL subtype which has led to increasing interest in investigating pharmacological inhibition of signaling pathways which contribute to lymphomagenesis and that are specifically utilized by ABC DLBCL cells. One of these signaling cascades is the B-cell receptor (BCR) pathway and several approaches in clinical trials to target this cascade have demonstrated promising therapeutic activity. This review discusses our current understanding of the role of BCR signaling in different DLBCL subtypes, including primary central nervous system lymphoma (PCNSL), a subgroup of DLBCL that is particularly dependent on BCR signaling. One specific aim of this review is to highlight novel approaches to therapeutically target BCR signaling in DLBCL.

INTRODUCTION AND OBJECTIVE:Biochemical recurrence (BCR) after radical prostatectomy (RP) for non-metastatic prostate cancer (PCa) leads to clinical progression only in a subpopulation. The aim of t...