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For antigen recognition, lampreys use leucine-rich repeats (LRR) instead of immunoglobulin V-(D)-J domains to generate variable lymphocyte receptors (VLR) of three types: VLRA, VLRB, and VLRC. VLRB-bearing lymphocytes respond to immunization with proliferation and differentiation into plasmacytes that secrete multivalent VLRB antibodies. Here, we immunized lampreys with B cells from patients with chronic lymphocytic leukemia (CLL) to generate recombinant monoclonal VLRB antibodies, one of which, VLR39, was specific for the donor CLL cells. The target epitope of VLR39 was shown to be the complementarity determining region 3 (CDR3) of the heavy chain variable region (VH) of the B-cell receptor. Using this antibody to monitor the CLL donor after chemoimmunotherapy-induced remission, we detected VLR39+ B cells in the patient 51 months later, before significant increase in lymphocyte count or CD5+ B cells. This indication of reemergence of the leukemic clone was verified by VH sequencing. Lamprey antibodies can exhibit exquisite specificity for a protein epitope, a CLL signature VH CDR3 sequence in this case, and offer a rapid strategy for generating anti-idiotype antibodies for early detection of leukemia recurrence. Cancer Immunol Res; 1(4); 223–8. ©2013 AACR.

PDF file - 192K, Figure S1: Flow cytometric analysis of monoclonal VLR39 reactivity with different cell types and cell lines. Figure S2: Flow cytometric analysis of monoclonal VLR39 reactivity with CLL cells with different VH gene sets. Figure S3: CLL VH gene sequence analysis. Figure S4: IGHV-D-J sequence alignment of VLR39 sorted cells. Table S1: CLL VH gene families and HCDR3 sequences. Table S2: Detection of CLL recurrence by quantitative real-time

Distribution of IGHV4-34 CLL according to IGHV mutational status and subset membership.

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication.Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes.Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively).Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

Univariable and multivariable analysis for TTFT within M-CLL.

The global monkeypox outbreak in 2022 has severely affected the life and health of people. Currently, partial smallpox vaccines have been approved for monkeypox prevention. Considering the potential occupational health risks of monkeypox infection among healthcare workers (HCWs), this study explored the willingness of Chinese HCWs to receive the monkeypox vaccine and analyzed the factors influencing their decision. We conducted an online cross-sectional survey among HCWs of 10 Chinese hospitals from May 30th, 2022 to August 1st, 2022. Specifically, a self-report questionnaire was administered to evaluate the attitude and acceptance of HCWs toward the monkeypox vaccine, followed by a multivariate logistic regression analysis to determine the independent predictors of vaccination. The survey included 1032 participants, of whom 90.12% expressed their willingness for vaccination (vaccine hesitancy rate = 9.88%). Univariate analysis showed that 11 variables differed significantly between the vaccine acceptance and vaccine hesitancy groups. Multivariate logistic regression analysis demonstrated that the age of 30-40 years (odds ratio [OR] = 0.504, 95% confidence interval [CI]: 0.284-0.893, p = 0.019 vs. age of30 years old), working in a secondary hospital (OR = 0.449, 95% CI: 0.249-0.808, p = 0.019 vs. working in a tertiary hospital), considering vaccination necessary for controlling monkeypox infection (OR = 4.135, 95% CI: 2.109-8.106, p 0.001 vs. not considering it necessary), willingness to pay for the monkeypox vaccine (OR = 2.125, 95% CI: 1.206-3.745, p = 0.009 vs. no willingness to pay), considering implementation of mandatory vaccination necessary (OR = 1.990, 95% CI: 1.023-3.869, p = 0.043 vs. not considering it necessary), and recommending family members and friends to take the vaccine (OR = 13.847, 95% CI: 7.487-25.609, p 0.001 vs. not recommending) were crucial independent predictors of the willingness to receive monkeypox-related vaccination. This study evaluated the acceptance and hesitancy rates of Chinese HCWs toward the monkeypox vaccine and found that the willingness to receive vaccination was mainly correlated to age, hospital level, and attitude toward vaccination. Therefore, to promote vaccine absorption, we recommend expanding publicity, formulating reasonable policies, and improving the recognition of vaccines.

Objective The Coronavirus Disease 2019 (COVID-19) has brought severe damage to global health and socioeconomics. In China, traditional Chinese medicine (TCM) is the most important complementary and alternative medicine (CAM) and it has shown a beneficial role in the prevention and treatment of COVID-19. However, it is unknown whether patients are willing to accept TCM treatment. The objective of our study is to investigate the acceptance, attitude, and independent predictors of TCM among asymptomatic COVID-19 patients admitted to Shanghai fangcang hospital during the outbreak of the COVID-19 pandemic in Shanghai in 2022. Methods A cross-sectional study was conducted on asymptomatic COVID-19 patients in the largest fangcang hospital in Shanghai, China, from April 22, 2022, to May 25, 2022. Based on the literature review of previous similar studies, a self-report questionnaire was developed to assess the patients’ attitude and acceptance of TCM, and a multivariate logistic regression analysis was conducted to determine the independent predictors of TCM acceptance. Results A total of 1,121 patients completed the survey, of whom 91.53% were willing to accept CAM treatment whereas 8.65% of participants showed no willingness. Among those who were positive to receive CAM treatment, 70.51% of the participants were willing to be treated with Chinese herbal medicine, 62.79% with Chinese patent medicine, 34.96% with massage therapy, 33.20% with moxibustion treatment, and 29.00% with cupping therapy. There were significant differences in the 10 variables such as monthly income level, SARS-CoV-2 vaccination, and patients' cognition and attitude towards TCM among the patients who were willing or unwilling to accept TCM treatment. Multivariate logistic regression analysis revealed that the patients who have received two doses of COVID-19 vaccine (OR = 2.069, 95%CI: 1.029–4.162, P = 0.041 vs not received), understood the culture of TCM (OR = 2.293, 95%CI: 1.029–4.162, P = 0.014 vs not understood), thought TCM needs a longer time to exert efficacy (OR = 1.607, 95%CI: 0.849–3.034, P = 0.145 vs not thought), thought the treatment of TCM is safe (OR = 2.856, 95%CI: 1.334–6.112, P = 0.007 vs not thought), thought the treatment of TCM is effective (OR = 2.724, 95%CI: 1.249–5.940, P = 0.012 vs not thought), and those who informed their attending physician if using TCM for treatment (OR = 3.455, 95%CI:1.867–6.392, P P

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed “satellites,” were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL. Key Points: • In a series of 29 856 CLL patients, the incidence of BcR stereotypy peaked at 41%. • Higher-order relations exist between stereotyped subsets, particularly for those from U-CLL, for which satellite subsets were identified.

Aberrant destruction of the articular extracellular matrix (ECM) has been considered to be one of the pathological features of osteoarthritis (OA) which results in chondrocyte changes and articular cartilage degeneration. The MAPK signaling pathway serves a key role by releasing cartilage-degrading enzymes from OA chondrocytes. However, the use of MAPK inhibitors for OA is hindered by their potential long-term toxicity. Vicenin 3 is one of the major components of the Jian-Gu injection which is effective in the clinical treatment of OA. However, its potential impact on OA remain poorly understood. Therefore, the present study aimed to assess the effects of vicenin 3 on interleukin (IL)-1β-treated SW1353 chondrocytes, which mimic the microenvironment of OA. These chondrocytes were pretreated with vicenin 3 (0, 5 and 20 µM) for 1 h and subsequently stimulated with IL-1β (10 ng/ml) for 24 h. Nitric oxide (NO) production was measured using the Griess reaction, whereas the production of prostaglandin E2 (PGE2), matrix metalloproteinases (MMPs), A disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs), collagen type II and aggrecan were measured using ELISA. The mRNA expression of MMPs and ADAMTSs were measured using reverse transcription-quantitative PCR. The protein expression levels of MAPK were measured using western blotting. Vicenin 3 was found to significantly inhibit IL-1β-induced production of NO and PGE. Increments in the expression levels of MMP-1, MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5 induced by IL-1β, in addition to the IL-1β-induced degradation of collagen type II and aggrecan, were all reversed by vicenin 3 treatment. Furthermore, vicenin 3 suppressed IL-1β-stimulated MAPK activation, an effect that was similar to that exerted by SB203580, a well-known p38 MAPK inhibitor. In conclusion, vicenin 3 may confer therapeutic potential similar to that of the p38 MAPK inhibitor for the treatment of OA.

International audience; Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease exemplary of this process, and a model for neoplasms in general, we created transgenic mice overexpressing the enzyme, activation-induced deaminase (AID), whose normal function is to induce DNA mutations in B lymphocytes. AID allows normal B lymphocytes to develop more effective immunoglobulin (Ig)-mediated immunity, but also is able to mutate non-Ig genes, predisposing to cancer. In chronic lymphocytic leukemia (CLL), AID expression correlates with poor prognosis suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Em-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL-cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in non-Igs genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically-similar amino acid substitutions as in human CLL and lymphoma.Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease that exemplifies this process and is a model for neoplasms in general, we created transgenic mice overexpressing the enzyme activation-induced deaminase (AID), which has a normal function of inducing DNA mutations in B lymphocytes. AID not only allows normal B lymphocytes to develop more effective immunoglobulin-mediated immunity, but is also able to mutate nonimmunoglobulin genes, predisposing to cancer. In CLL, AID expression correlates with poor prognosis, suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eμ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in nonimmunoglobulin genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

Progression of chronic lymphocytic leukemia (CLL) results from the expansion of a small fraction of proliferating leukemic B cells. When comparing the global gene expression of recently divided CLL cells with that of previously divided cells, we found higher levels of genes involved in regulating gene expression. One of these was the oncogene Musashi 2 (MSI2), an RNA-binding protein that induces or represses translation. While there is an established role for MSI2 in normal and malignant stem cells, much less is known about its expression and role in CLL. Here we report for the first time ex vivo and in vitro experiments that MSI2 protein levels are higher in dividing and recently divided leukemic cells and that downregulating MSI2 expression or blocking its function eliminates primary human and murine CLL and mature myeloid cells. Notably, mature T cells and hematopoietic stem and progenitor cells are not affected. We also confirm that higher MSI2 levels correlate with poor outcome markers, shorter time-to-first-treatment, and overall survival. Thus, our data highlight an important role for MSI2 in CLL-cell survival and proliferation and associate MSI2 with poor prognosis in CLL patients. Collectively, these findings pinpoint MSI2 as a potentially valuable therapeutic target in CLL.

Deep eutectic solvents (DESs) are emerging as new extraction solvents for the extraction of bioactive compounds from natural plants. In this work, a total of thirteen choline chloride-based DESs were synthesized and evaluated for the simultaneous extraction of five bioactive flavone di-C-glycosides from Premna fulva Craib. DES-F, which was composed of choline chloride and 1,3-propanediol, showed the best extraction efficiency for the flavone di-C-glycosides, exceeding that of the conventional industrial solvent (40% ethanol). Subsequently, the extraction parameters for ultrasonic-assisted extraction (liquid-solid ratio, water content (%) in the selected DES, and extraction time) were optimized using response surface methodology. Furthermore, the microstructural alterations in the samples after extraction were evaluated using scanning electron microscopy. In addition, we further verified the recovery of flavone di-C-glycosides from the DESs using macroporous resins and evaluated the reusability of the DESs as solvents. The results indicated that DES-F was an efficient and sustainable solvent for the extraction of bioactive flavone di-C-glycosides from Premna fulva Craib. Therefore, this novel strategy may represent a sustainable approach for the extraction of bioactive products and could make phytochemistry more attractive and environmentally friendly.

Craniosynostosis (CRS) is a congenital abnormality deformity with a heterogenous genetic contribution. Previously, there are two attempts to collect genes that are genetically associated with craniosynostosis and some related syndromes with 57 (Twigg and Wilkie, 2015) and 39 (Goos and Mathijssen, 2019) genes identified, respectively. We expanded this list of craniosynostosis genes by adding another 17 genes with an updated literature search. These genes are shown to be more likely to be intolerant to functional mutations. Of these 113 craniosynostosis genes, 21 (19% vs. 1.5% baseline frequency) are cancer driver genes, a 14-fold enrichment. The cancer-craniosynostosis connection is further validated by an over-representation analysis of craniosynostosis genes in KEGG cancer pathway and several cancer related gene-sets. Many cancer-craniosynostosis overlapping genes participate in intracellular signaling pathways, which play a role in both development and cancer. This connection can be viewed from the oncogenesis recapitulates ontogenesis framework. Nineteen craniosynostosis genes are transcription factor genes (16.8% vs. 8.2% baseline), and craniosynostosis genes are also enriched in targets of certain transcription factors or micro RNAs.

Richter's transformation (RT) is a complication of chronic lymphocytic leukemia (CLL) characterized by the development of an aggressive lymphoma, typically diffuse large B cell lymphoma (DLBCL). RT is associated with high expression of activation-induced cytidine deaminase (AID) and aberrant somatic hypermutation of non-immunoglobulin genes. Current frontline therapy of CLL, ibrutinib, eliminates AID + CLL cells and alters tissue microenvironment to Th1-biased niches; however approximately 10-20% of CLL patients develop RT upon ibrutinib treatment. In addition, ibrutinib leads to a clinical response in only ~40% of RT patients. Thus, RT remains an important unmet challenge for CLL patients and the mechanism of RT development during ibrutinib treatment is poorly understood. To determine the contributions of AID and T cells in RT development, and to understand the efficacy of ibrutinib in the treatment of RT, we first created patient-derived xenografts (PDXs) with samples from three RT patients who were refractory to (immuno)chemotherapy but had not previously received ibrutinib. In each case, the B-lymphocytes were larger in size and expressed lower levels of CD5 but higher levels of activation markers compared with the CLL cell counterpart. In PDXs, ibrutinib treatment led to significantly less RT cells in spleen with percentage of inhibition ranging from 28-60% for all three cases. Notably, AID levels were similar in RT cells residing in spleen of ibrutinib-treated and control mice. The numbers of T cells were also similar, although Th1:Th2 ratios were significantly increased by ibrutinib (control vs ibrutinib group: 33% vs 42%, P=0.01). Interestingly, the growth and survival of RT cells located in bone marrow (BM) were not affected by ibrutinib. We then investigated the efficacy of ibrutinib in the prevention and treatment of RT in a novel strain of TCL-1 mice, TCL1/Igκ-AID mice, that have AID overexpressed solely in B lymphocytes. These mice develop accelerated CLL disease, have shorter overall survival (OS), and bear increased numbers of AID signature mutations in genes that are drivers in CLL and DLBCL. First we injected primary splenocytes from a TCL1/Igκ-AID mouse (A-31) into wild-type C57BL/6 mice. A week later, mice were treated with vehicle or ibrutinib continuously for 3 weeks (N=5 per group, duplicated experiments). At the end of treatment, RT cells were found in 25% of recipients in the control group and only in 10% of ibrutinib-treated recipients. Ibrutinib-treated animals had overall smaller spleens and lymph nodes (LNs) filled with significantly less CLL or RT cells. Importantly, the balance of Th1/Th2 cytokines was also significantly changed by ibrutinib (Th1/Th2 in control vs ibrutinib: 25% vs 47%, P=0.01). However, again RT cells in BMs of treated and control animals were similar. We then repeated the experiment to determine overall survival by giving the mice vehicle or ibrutinib continuously. Compared to the TCL1/Igκ-AID (A-31) donors, recipients treated with control vehicle developed more aggressive CLL with shorter OS. However, Ibrutinib did not change the OS; both control and ibrutinib treated recipients had similar OS with all the mice developed and died from RT eventually. Finally, to determine the efficacy of ibrutinib in the treatment of RT in TCL1/Igκ-AID mice, we sorted A-31-derived RT cells collected from LNs, and injected these into C57BL/6 mice. Mice then received treatment for 3 weeks. Notably, ibrutinib did not change the numbers or sizes of LNs or spleens. The numbers of RT cells at all sites, including BM, remained the same. In both groups, the number of T cells and the Th1:Th2 were not altered by ibrutinib. In summary, our data suggest that ibrutinib is somewhat effective against primary RT cells in the spleen in PDX models but not at all in the aggressive TCL1/IgK-AID mouse model. This might be because ibrutinib does not reduce AID levels in tissue-resident RT cells. Notably, although RT cells in spleens and LNs respond to ibrutinib, those residing in BM are resistant in both PDX and TCL1/IgK-AID models. This suggests different clones or different ibrutinib-responsiveness of RT cells in primary versus secondary lymphoid tissues. Overall, these results suggest that ibrutinib does not prevent or cure RT. Additionally, TCL1/Igκ-AID mice might provide a model to study the development, prevention, and treatment of RT without the influence of prior chemo- or other therapies. Disclosures Wirtz: Abbvie: Current Employment. Szafer-Glusman: AbbVie: Current Employment, Other: Stock or other ownership.

The 2D monolayer [Formula: see text]-SiP has a honeycomb lattice and an intrinsic indirect band gap. Herein, the density functional theory calculations are performed to modulate the electronic structure of 2D monolayer [Formula: see text]-SiP by applying strains. The band gap of monolayer [Formula: see text]-SiP is monotonously reduced by the strains. More interestingly, a direct band gap is more likely to be achieved by applying strains along the armchair direction than along the zigzag direction. Finally, 2D monolayer [Formula: see text]-SiP can possess a tunable direct band gap of 1.57–0.73 eV (HSE06) and considerable visible light absorption index, by applying compression strains of −6–−10% along the armchair direction. The work provides a route of modulating the electronic and optical properties of monolayer [Formula: see text]-SiP, which extends its application range for various fields such as electronic devices and solar energy conversion.

Amino acid replacement mutations in certain chronic lymphocytic leukemia (CLL) stereotyped B cell receptor (BCR) immunoglobulins (IGs) at defined positions within antigen-binding sites strongly imply antigen selection. Prime examples of this are CLL subset 4 BCR IGs using IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements. Conspicuously, and unlike most CLL IGs, subset 4 IGs do not bind apoptotic cells. By testing the (auto)antigenic reactivities of subset 4 IGs toward viable lymphoid-lineage cells and specific autoantigens typically bound by IGHV4-34+ IGs, we found that IGs from both subset 4 and non-subset 4 IGHV4-34-expressing CLL cases bound naïve B cells. However, only subset 4 IGs reacted with memory B cells. Furthermore, subset 4 IGs did not bind DNA nor i or I carbohydrate antigens that are common targets of IGHV4-34-utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively. Notably, we found that subset 4 IG binding to memory B lymphocytes depends on an aspartic acid at position 66 of FR3 in the rearranged IGKV2-30 gene; this amino acid residue is acquired by somatic mutation. Our findings illustrate the importance of positive and negative selection criteria for structural elements in CLL IGs and suggest that autoantigens driving normal B cells to become subset 4 CLL cells differ from those driving IGHV4-34+ B cells in other diseases.

Purpose: We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication. Experimental Design: In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34–expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes. Results: We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly (P < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). Conclusions: Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

Intraclonal subpopulations of circulating chronic lymphocytic leukemia (CLL) cells with different proliferative histories and reciprocal surface expression of CXCR4 and CD5 have been observed in the peripheral blood of CLL patients and named proliferative (PF), intermediate (IF), and resting (RF) cellular fractions. Here, we found that these intraclonal circulating fractions share persistent DNA methylation signatures largely associated with the mutation status of the immunoglobulin heavy chain locus (IGHV) and their origins from distinct stages of differentiation of antigen-experienced B cells. Increased leukemic birth rate, however, showed a very limited impact on DNA methylation of circulating CLL fractions independent of IGHV mutation status. Additionally, DNA methylation heterogeneity increased as leukemic cells advanced from PF to RF in the peripheral blood. This frequently co-occurred with heterochromatin hypomethylation and hypermethylation of Polycomb-repressed regions in the PF, suggesting accumulation of longevity-associated epigenetic features in recently born cells. On the other hand, transcriptional differences between paired intraclonal fractions confirmed their proliferative experience and further supported a linear advancement from PF to RF in the peripheral blood. Several of these differentially expressed genes showed unique associations with clinical outcome not evident in the bulk clone, supporting the pathological and therapeutic relevance of studying intraclonal CLL fractions. We conclude that independent methylation and transcriptional landscapes reflect both preexisting cell-of-origin fingerprints and more recently acquired hallmarks associated with the life cycle of circulating CLL cells.

A new C25 terpene consisting of C15 and C10 terpenes, sarcaglabrin A, and two new sesquiterpene dimers, sarcaglabrins B and C, were isolated from the aerial parts of a medicinal plant Sarcandra glabra used in Guangxi Zhuang Autonomous Region, China. Their structures were elucidated on the basis of detailed spectroscopic analyses including calculation of the ECD spectrum. Sarcaglabrin A was assigned as a conjugate of lindenane type sesquiterpene and ocimene type monoterpene, while sarcaglabrins B and C were elucidated to be lindenane sesquiterpene dimers. Their cytotoxicity against human cancer cell lines were evaluated.

Six new limonoids, munropins A−F (1–6), were isolated from the aerial parts of a Chinese medicinal plant, Munronia pinnata (Meliaceae). The structures of 1–6 were assigned by detailed analyses of their spectroscopic data. Munropins A (1) and B (2) are limonoids possessing a prieurianin skeleton with α,β-unsaturated γ-lactam moieties at C-17. Munropins C (3), D (4), and E (5) are also prieurianin type limonoids with an α,β-unsaturated γ-lactone moiety, an acetyl group, and an acetoxyacetyl group at C-17, respectively. Munropin F (6) was assigned as a nimbolinin type limonoid with a γ-hydroxy-α,β-unsaturated γ-lactone moiety.

The overall survival (OS) of patients with chronic lymphocytic leukemia (CLL) has improved over the last decades mainly due to advances in the understanding of the disease biology and the introduction of novel therapeutic approaches(1). In the present retrospective study we investigated trends in OS in subgroups of cases defined by genetic and immunogenetic features aiming at addressing the question whether advances in chemoimmunotherapy had a uniform impact across all CLL patients. We found that such advances have translated into prolonged OS in all prognostic subgroups examined except those carrying TP53 abnormalities, as expected, but also those assigned to stereotyped subsets #1 and #2, that are generally devoid of such gene aberrations. This latter finding, reported here for the first time, indicates the need for alternative treatment options for these patients.

Chronic lymphocytic leukemia (CLL) is a clonal disease of B lymphocytes manifesting as an absolute lymphocytosis in the blood. However, not all lymphocytoses are leukemic. In addition, first-degree relatives of CLL patients have an ~15 % chance of developing a precursor condition to CLL termed monoclonal B cell lymphocytosis (MBL), and distinguishing CLL and MBL B lymphocytes from normal B cell expansions can be a challenge. Therefore, we selected FMOD, CKAP4, PIK3C2B, LEF1, PFTK1, BCL-2, and GPM6a from a set of genes significantly differentially expressed in microarray analyses that compared CLL cells with normal B lymphocytes and used these to determine whether we could discriminate CLL and MBL cells from B cells of healthy controls. Analysis with receiver operating characteristics and Bayesian relevance determination demonstrated good concordance with all panel genes. Using a random forest classifier, the seven-gene panel reliably distinguished normal polyclonal B cell populations from expression patterns occurring in pre-CLL and CLL B cell populations with an error rate of 2 %. Using Bayesian learning, the expression levels of only two genes, FMOD and PIK3C2B, correctly distinguished 100 % of CLL and MBL cases from normal polyclonal and mono/oligoclonal B lymphocytes. Thus, this study sets forth effective computational approaches that distinguish MBL/CLL from normal B lymphocytes. The findings also support the concept that MBL is a CLL precursor.

Taking the design of Tai'an city square as an example, the paper analyzes different types of city square landscape design, elaborates on their commonness and characteristics, and makes a detailed study of the emerging city square to provide a solid theoretical foundation for the innovational design of new city squares in the new era .

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

Tumor-specific metabolic changes can reveal new therapeutic targets. Our findings implicate a supporting role for fatty acid metabolism in chronic lymphocytic leukemia (CLL) cell survival. Peroxisome proliferator–activated receptor (PPAR)-α, a major transcriptional regulator of fatty acid oxidation, was recently shown to be upregulated in CLL. To evaluate PPARα as a potential therapeutic target, we developed a highly selective, potent small molecule antagonist of PPARα, NXT629. NXT629 inhibited agonist-induced transcription of PPARα-regulated genes, demonstrating target engagement in CLL cells. Furthermore, NXT629 induced apoptosis of CLL cells even in the presence of a protective microenvironment. To mimic the proliferative lymphoid compartment of CLL, we examined the activity of NXT629 on CLL cells that were stimulated to proliferate in vitro. NXT629 reduced the number of leukemia cells undergoing cell division. In addition, in two xenograft mouse models of CLL (one a model for nondividing and one for dividing CLL), NXT629 reduced the number of viable CLL cells in vivo. Overall, these results suggest that fatty acid metabolism promotes survival and proliferation of primary CLL cells and that inhibiting PPARα gene regulation could be a new therapeutic approach to treating CLL.

Significance A pathogenetic role of NOTCH1 in chronic lymphocytic leukemia (CLL) has been implied by the presence of deregulating mutations in a relatively small fraction of cases. Our results now indicate that ∼50% of CLL cases devoid of mutations express the active form of NOTCH1 ICN1 (intracellular portion of NOTCH1), thus implicating a much broader role of this transcription factor in the disease. ICN1 + CLL cases display equivalent NOTCH1-dependent transcriptional responses regardless of the gene mutation status, indicating that the detection of ICN1 represents a reliable biomarker of NOTCH1 activation for diagnostic and therapeutic targeting. Finally, our results identify the NOTCH1-dependent transcriptional program in CLL cells, thus providing direct insights into the pathogenesis of a large fraction of CLL cases.

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water (2H2O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Array comparative genomic hybridization (aCGH) has yet to be fully leveraged in a prognostic setting in chronic lymphocytic leukemia (CLL). Genomic imbalance was assessed in 288 CLL specimens using a targeted array. Based on 20 aberrations in a hierarchical manner, all 228 treatment-naive specimens were classified into a group with poor outcome (20.6%) exhibiting at least one aberration that was univariately associated with adverse outcome (gain: 2p, 3q, 8q, 17q, loss: 7q, 8p, 11q, 17p, 18p), good outcome (32.5%) showing 13q14 loss without any of the other 10 aberrations (gain: 1p, 7p, 12, 18p, 18q, 19, loss: 4p, 5p, 6q, 7p) or intermediate outcome (remainder). The three groups were significantly separated with respect to time to first treatment and overall survival (p < 0.001), and validation of the stratification scheme was performed in two independent datasets. Gain of 3q and 8q, and 17p loss were determined to be independent unfavorable prognostic biomarkers. TP53, NOTCH1 and SF3B1 mutations correlated with the presence of one poor outcome aCGH marker, at a considerably higher frequency than when only considering poor risk aberrations routinely detected by fluorescence in situ hybridization (FISH). These data support genomic imbalance evaluation in CLL by aCGH to assist in risk stratification.