Lack of Association of the NPAS2 Gene Ala394Thr Polymorphism (rs2305160:G>A) with Risk of Chronic Lymphocytic Leukemia

Background: NPAS2 is a product of the circadian clock gene. It acts as a putative tumor suppressor by playing an important role in DNA damage responses, cell cycle control and apoptosis. Chronic lymphocytic leukemia (CLL) appears to be an apoptosis related disorder and alteration in the NPAS2 gene might thereforebe directly involved in the etiology of CLL. Here, the Ala394Thr polymorphism (rs2305160:G>A) in the NPAS2 gene was genotyped and melatonin concentrations were measured in a total of seventy-four individuals, including thirty-seven CLL cases and an equal number of age- and sex-matched healthy controls in order to examine the effect of NPAS2 polymorphism and melatonin concentrations on CLL risk in a Pakistani population. Materials and Methods: Genotyping of rs2305160:G>A polymorphism at NPAS2 locus was carried out by amplificationrefractory mutation system-polymerase chain reaction (ARMS-PCR). Melatonin concentrations were determined by enzyme linked immunosorbent assay (ELISA). Statistical analysis was performed using Statistical Packagefor Social Sciences software. Results: Our results demonstrated no association of the variant Thr genotypes (Ala/ Thr and Thr/Thr) with risk of CLL. Similarly, no association of rs2305160 with CLL was observed in either females or males after stratification of study population on a gender basis. Moreover, when the subjects with CLL were further stratified into shift-workers and non-shift-workers, no association of rs2305160 with CLL was seen in either case. However, significantly low serum melatonin levels were observed in CLL patients as compared to healthy subjects (p<0.05). Also, lower melatonin levels were seen in shift-workers as compared to non-shift-workers (p<0.05). There was no significant difference (p>0.05) in the melatonin levels across NPAS2 genotypes in all subjects, subjects with CLL who were either shift workers or non-shift-workers. General Linear Model (GLM) univariate analysis revealed no significant association (p>0.05) of the rs2305160 polymorphism of the NPAS2 gene with melatonin levels in any of the groups. Conclusions: While low melatonin levels and shift-work can be considered as one of the risk factors for CLL, the NPAS2 rs2305160 polymorphism does not appear to have any association with risk of CLL in our Pakistani population.     

Liquid biopsy of cerebrospinal fluid for MYD88 L265P mutation is useful for diagnosis of central nervous system lymphoma

The current standard of diagnosing central nervous system (CNS) lymphoma is stereotactic biopsy, however the procedure has a risk of surgical complication. Liquid biopsy of the CSF is a less invasive, non-surgical method that can be used for diagnosing CNS lymphoma. In this study, we established a clinically applicable protocol for determining mutations in MYD88 in the CSF of patients with CNS lymphoma. CSF was collected prior to the start of chemotherapy from 42 patients with CNS lymphoma and matched tumor specimens. Mutations in MYD88 in 33 tumor samples were identified using pyrosequencing. Using 10 ng each of cellular DNA and cell-free DNA (cfDNA) extracted from the CSF, the MYD88 L265P mutation was detected using digital PCR. The conditions to judge mutation were rigorously determined. The median Target/Total value of cases with MYD88 mutations in the tumors was 5.1% in cellular DNA and 22.0% in cfDNA. The criteria to judge mutation were then determined, with a Target/Total value of 0.25% as the cutoff. When MYD88 mutations were determined based on these criteria, the sensitivity and specificity were 92.2% and 100%, respectively, with cellular DNA; and the sensitivity and specificity were 100% with cfDNA. Therefore, the DNA yield, mutated allele fraction, and accuracy were significantly higher in cfDNA compared with that in cellular DNA. Taken together, this study highlights the importance of detecting the MYD88 L265P mutation in cfDNA of the CSF for diagnosing CNS lymphoma using digital PCR, a highly accurate and clinically applicable method.     

The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid

The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well‐known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited.     

TP53 Gene 72 Arg/Pro (rs1042522) Single Nucleotide Polymorphism Contribute to Increase the Risk of B-Chronic Lymphocytic Leukemia in the Sudanese Population

Objective: This study aimed at exploring the association of TP53 72Arg/Pro polymorphism and Risk of ChronicLymphocytic Leukemia and to assess the correlation between TP53 72Arg/Pro polymorphism and clinical parameter,hematological profile and some biological prognostic markers among Sudanese patients with chronic lymphocyticleukemia. Methods: A case-control study was conducted in Khartoum state, Sudan, during the period from April 2017 toApril 2018, involved 110 B-CLL patients and 80 healthy volunteers as a control group. Physical examination, CompleteBlood Count and Immunophenotype were performed in all patients to confirm the diagnosis. Clinical staging such asRai and Binet were studied. CD38 and ZAP70 were performed by Flow Cytometry. Blood samples were collected fromall participants; DNA was extracted by using ANALYTIKJENA Blood DNA Extraction Kit (Germany) and analyzedTP53 codon 72Arg/Pro Polymorphism by using AS-PCR. The statistical analysis was performed using SPSS version23.0 software (Chicago, IL, USA). Results: the Arg/Pro was the most frequent genotype in B-CLL patients(50%),followed by Arg/Arg (25.5%) and Pro/Pro (24.5%), whereas in healthy control group Arg/Pro was the most frequent(47.5%), followed by Arg/Arg (45%) and Pro/Pro (7.5%). Our data indicate a higher frequency of homozygous Pro/Pro in the B-CLL patients as compared to controls with an OR of 4.01 for the Pro/Pro genotype and lower frequencyof Arg/Arg genotype in CLL patients as compared to controls with an OR of .42 for the Arg/Arg genotype. Also, thePro allele showed higher risk than Arg allele (P value=0.000, OR 2.23, 95% CI=1.45-3.41). No significant associationbetween gender, clinical staging systems (Rai, Binet), biological prognostic markers (CD38 expression or ZAP70expression), and TP53 codon 72Arg/Pro polymorphisms, except Arg/Arg genotype tended to be associated with youngerage (P =0.04). Conclusion: Our data suggested that Pro/Pro genotype contribute to increased susceptibility to B-ChronicLymphocytic Leukemia risk in our population tenfold higher than those had Arg/Arg genotype     

Infections in Chronic Lymphocytic Leukemia: Risk Factors, and Impact on Survival, and Treatment

Patients with chronic lymphocytic leukemia (CLL) are at an increasing risk of infectious morbidity and mortality. Infections are generally due to bacteria and influenced by the degree of hypogammaglobulinemia; although, in more advanced stages of disease they may also be contributed by neutropenia due to bone marrow infiltration and/or cytotoxic therapy. Furthermore, defect in cell-mediated immunity appears to be a predisposing factor to infections in patients treated with newer purine analogues. Controversies surrounding the pathogenesis of infectious complications in CLL raise several questions on their management. Patients with advanced disease who receive cytotoxic therapy might qualify for antibacterial prophylaxis. Intravenous immunoglobulin (IVIG), although of scientific interest, may be of little relevance at the present time. The new growth factors should be tested in well-designed prospective studies.     

Associations Between TLR9 Polymorphisms and Cancer Risk: Evidence from an Updated Meta-analysis of 25,685 Subjects

A meta-analysis incorporating 34 case-control studies from 19 articles involving 12,197 cases and 13,488controls was conducted to assess the effects of three genetic variants of Toll-like receptor 9 (TLR9): rs187084,rs352140, and rs5743836. Studies on associations between TLR9 polymorphisms and cancer risk weresystematically searched in electronic databases. The reported odds ratios (OR) and 95% confidence intervals(CI) were pooled to assess the strength of any associations. The results showed that the rs187084 polymorphismwas significantly associated with an increased risk of cancer (CC vs TC+TT: OR=1.14, 95% CI=1.02-1.28),specifically cervical cancer (C vs T: OR=1.19, 95% CI=1.05-1.34; TC vs TT: OR=1.32, 95% CI=1.10-1.58; CCvs TT: OR=1.31, 95% CI= 1.03-1.68; CC+TC vs TT: OR=1.32, 95% CI=1.11-1.56), and that this association wassignificantly positive in Caucasians (CC vs. TC+TT: OR=1.18, 95% CI=1.01-1.38). The rs352140 polymorphismhad a protective effect on breast cancer (GA vs GG: OR=0.77, 95% CI=0.66-0.89), whereas the rs5743836polymorphism was likely protective for digestive system cancers (CC+TC vs TT: OR=0.81, 95% CI=0.66-0.98).In conclusion, our results suggest that the rs187084 polymorphism may be associated with an elevated cancerrisk, whereas polymorphisms of rs352140 and rs5743836 may play protective roles in the development of breastand digestive system cancers, respectively. From the results of this meta-analysis further large-scale case-controlstudies are warranted to verify associations between TLR9 polymorphisms and cancer.     

Clinical significance of disease‐specific MYD88 mutations in circulating DNA in primary central nervous system lymphoma

Recent sequencing studies demonstrated the MYD88 L265P mutation in more than 70% of primary central nervous system lymphomas (PCNSL), and the clinical significance of this mutation has been proposed as diagnostic and prognostic markers in PCNSL. In contrast, mutational analyses using cell‐free DNAs have been reported in a variety of systemic lymphomas. To investigate how sensitively the MYD88 L265P mutation can be identified in cell‐free DNA from PCNSL patients, we carried out droplet digital PCR (ddPCR) and targeted deep sequencing (TDS) in 14 consecutive PCNSL patients from whom paired tumor‐derived DNA and cell‐free DNA was available at diagnosis. The MYD88 L265P mutation was found in tumor‐derived DNA from all 14 patients (14/14, 100%). In contrast, among 14 cell‐free DNAs evaluated by ddPCR (14/14) and TDS (13/14), the MYD88 L265P mutation was detected in eight out of 14 (ddPCR) and in 0 out of 13 (TDS) samples, implying dependence on the detection method. After chemotherapy, the MYD88 L265P mutation in cell‐free DNAs was traced in five patients; unexpectedly, the mutations disappeared after chemotherapy was given, and they remained undetectable in all patients. These observations suggest that ddPCR can sensitively detect the MYD88 L265P mutation in cell‐free DNA and could be used as non‐invasive diagnostics, but may not be applicable for monitoring minimal residual diseases in PCNSL.     

Rapid detection of the MYD88 L265P mutation for pre- and intra-operative diagnosis of primary central nervous system lymphoma

The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is a disease-specific mutation of primary central nervous system lymphoma (PCNSL) among the central nervous system tumors. Accordingly, this mutation is considered a reliable diagnostic molecular marker of PCNSL. As the intra-operative diagnosis of PCNSL is sometimes difficult to achieve using histological examinations alone, intra-operative detection of the MYD88 L265P mutation could be effective for the accurate diagnosis of PCNSL. Herein, we aimed to develop a novel rapid genotyping system (GeneSoC) using real-time polymerase chain reaction (PCR) based on microfluidic thermal cycling technology. This real-time PCR system shortened the analysis time, which enabled the detection of the MYD88 L265P mutation within 15 min. Rapid detection of the MYD88 L265P mutation was performed intra-operatively using GeneSoC in 24 consecutive cases with suspected malignant brain tumors, including 10 cases with suspected PCNSL before surgery. The MYD88 L265P mutation was detected in eight cases in which tumors were pathologically diagnosed as PCNSL after the operation, while wild-type MYD88 was detected in 16 cases. Although two of the 16 cases with wild-type MYD88 were pathologically diagnosed as PCNSL after the operation, MYD88 L265P could be detected in all eight PCNSL cases harboring MYD88 L265P. The MYD88 L265P mutation could also be detected using cell-free DNA derived from the cerebrospinal fluid of two PCNSL cases. Detection of the MYD88 L265P mutation using GeneSoC might not only improve the accuracy of intra-operative diagnosis of PCNSL but also help the future pre-operative diagnosis through liquid biopsy of cerebrospinal fluid.     

Aberrant expression of MYD88 via RNA-controlling CNOT4 and EXOSC3 in colonic mucosa impacts generation of colonic cancer

In 2020, the worldwide incidence and mortality of colorectal cancer (CRC) were third and second, respectively. As the 5-y survival rate is low when CRC is diagnosed at an advanced stage, a reliable method to predict CRC susceptibility is important for preventing the onset and development and improving the prognosis of CRC. Therefore, we focused on the normal colonic mucosa to investigate changes in gene expression that may induce subsequent genetic alterations that induce malignant transformation. Comprehensive gene expression profiling in the normal mucosa adjacent to colon cancer (CC) compared with tissue from non-colon cancer patients was performed. PCR arrays and qRT-PCR revealed that the expression of 5 genes involved in the immune response, including MYD88, was increased in the normal mucosa of CC patients. The expression levels of MYD88 were strikingly increased in precancerous normal mucosa specimens, which harbored no somatic mutations, as shown by immunohistochemistry. Microarray analysis identified 2 novel RNA-controlling molecules, EXOSC3 and CNOT4, that were significantly upregulated in the normal mucosa of CC patients and were clearly visualized in the nuclei. Forced expression of EXOSC3 and CNOT4 in human colonic epithelial cells increased the expression of IFNGR1, MYD88, NFκBIA, and STAT3 and activated ERK1/2 and JNK in 293T cells. Taken together, these results suggested that, in the inflamed mucosa, EXOSC3- and CNOT4-mediated RNA stabilization, including that of MYD88, may trigger the development of cancer and can serve as a potential predictive marker and innovative treatment to control cancer development.     

Associations between AT-rich Interactive Domain 5B gene Polymorphisms and Risk of Childhood Acute Lymphoblastic Leukemia: a Meta-analysis

Previous genome-wide association studies (GWAS) have implicated several single nucleotide polymorphisms (SNPs) in the AT-rich interactive domain 5B (ARID5B) gene with childhood acute lymphoblastic leukemia (ALL). However, replicated studies reported some inconsistent results in different populations. Using meta-analysis, we here aimed to clarify the nature of the genetic risks contributed by the two polymorphisms (rs10994982, rs7089424) for developing childhood ALL. Through searches of PubMed, EMBASE, and manually searchingrelevant references, a total of 14 articles with 16 independent studies were included. Odds ratios (ORs) with 95% confidence intervals (95%CI) were calculated to assess the associations. Both SNPs rs10994982 and rs7089424showed significant associations with childhood ALL risk in all genetic models after Bonferroni correction. Furthermore, subtype analyses of B-lineage ALL provided strong evidence that SNP rs10994982 is highly associated with the risk of developing B-hyperdiploid ALL. These results indicate that SNPs rs10994982 andrs7089424 are indeed significantly associated with increased risk of childhood ALL.     

初治弥漫大B细胞淋巴瘤患者MYD88基因突变的临床特征及预后分析

目的:探讨MYD88基因突变对初治弥漫大B细胞淋巴瘤(diffuse large B-cell lymphoma,DLBCL)的临床特征及预后的影响.方法:收集2013年1月至2015年1月空军军医大学第二附属医院血液科的74例初治DLBCL患者,回顾分析MYD88突变组(MYD88mut)和MYD88野生组(MYD8...     

Angiogenesis in B-Cell Chronic Lymphocytic Leukemia: Methods of Study, Clinical Significance and Prognostic Implications

Recent studies indicate that angiogenesis may be involved in the pathogenesis of certain hematological malignancies. However as far as B-cell chronic lymphocytic leukemia (CLL) is concerned, current data dealing with the evaluation of bone marrow (BM) microvessel density, a marker of angiogenesis grade, do not as yet provide definitive results. It is now clear that the mRNA isoforms VEGF121 and VEGF 165 are expressed by B-CLL cells. In addition, low cellular and high serum levels of VEGF correlated with a poor clinical outcome. Although these data do not as yet show that angiogenesis is essential for B-CLL, it may indeed be relevant in the leukemic process so characteristic of this diseases.     

Prognostic and Predictive Significance of Smudge Cell Percentage on Routine Blood Smear in Chronic Lymphocytic Leukemia

Introduction/BackgroundSmudge cells are ruptured lymphocytes present on routine blood smears of chronic lymphocytic leukemia (CLL) patients. We evaluated prognostic and predictive significance of smudge cell percentage on a blood smear in CLL patients.Materials and MethodsWe calculated smudge cell percentages (ratio of smudged to intact cells plus smudged lymphocytes) on archived blood smears of 222 untreated CLL patients registered at Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi over the past 12 years.ResultsThe male:female ratio was 3:1, and median age 60 (range, 28-90) years. Median absolute lymphocyte count was 42 × 10⁹/L. The median smudge cell percentage was 29.6% (range, 4%-79%). We found no correlation of proportion of smudge cells with age, sex, lymphocyte count, organomegaly, or response to therapy, although there was a significant correlation with the Rai stage at diagnosis. Median smudge cell percentage in stage 0 and I was 33% (range, 12%-79%), in stage II 31% (range, 12%-61%), and stage III and IV 21% (range, 4%-51%) (P < .001). Patients with ≤ 30% smudge cells had a shorter median progression-free period (PFP) of 30 months compared with patients who had more than 30% smudge cells (PFP, 45 months; P = .01). The 5-year survival rate was 51% for patients with 30% or fewer smudge cells, and it was 81% for patients with more than 30% smudge cells (P < .001) at a median follow-up of 3.5 years.ConclusionSimple and inexpensive detection of smudge cells on routine blood smears seems useful in predicting progression-free and overall survival in CLL patients and might be beneficial in countries with limited resources.     

Expression of TLR9 within human glioblastoma

Immunostimulating oligonucleotides containing CpG motifs (CpG-ODN) have shown promising antitumor activity in preclinical glioma models. CpG motifs are specifically recognized by the Toll-like receptor 9 (TLR9), mainly expressed in plasmacytoid dendritic cells (pDCs) and B cells. Expression of TLR9 within human glioma samples has not been investigated. As CpG-ODN is currently under clinical trials in glioma patients, we investigated whether TLR9 is expressed at the RNA levels in a series of 37 human glioblastomas (GBM) by quantitative PCR. TLR9 expression was detected at variable levels, which might suggest that some patients are more likely to benefit from treatment with CpG-ODN than others. No significant relationships between TLR9 expression and age, sex, tumor location, lymphocytes infiltration, oligodendroglial components or survival were found. TLR9 is unlikely to be expressed by tumor cells as no TLR9 expression was detected in pure human GBM xenografts. Immunocytochemistry studies showed TLR9 expression in some macrophages/microglial cells. The expression of TLR9 within human GBM strengthens the rationale for the utilization of CpG-ODN in this disease.     

Meta-analysis of Associations Between four Polymorphisms in the Matrix Metalloproteinases Gene and Gastric Cancer Risk

Background: Matrix metalloproteinases (MMPs) play important roles in pathogenesis and developmentof cancer. Recently, many studies have show associations between polymorphisms in the promoter regions ofMMPs and risk of gastric cancer. The present meta-analysis was conducted in order to investigate the potentialassociation between four polymorphisms in the MMP gene and gastric cancer risk. Methods: A computerizedliterature search was conducted in databases of Med-line, Embase, Science Citation Index and PubMed till June2013 for any MMP genetic association study of gastric cancer. Odds ratios (ORs) and 95 % confidence intervals(CIs) were estimated for each gene under dominant and recessive models, and heterogeneity between studies wasassessed using the Q test and I2 value. Overall and subgroup analyses according to ethnicity were carried out withStata 12.0. Results: 14 reports covering 8,146 patients (2,980 in the case group and 5,166 in the control group)were included in the present meta-analysis. We found that the MMP-7 (-181A>G) polymorphism increased thegastric cancer risk in therecessive model (GG vs. AA/AG, OR=1.768, 95% CI =1.153-2.712). For MMP2 −1306C>T, MMP1-1607 1G/2G, and MMP9−1 562 C>T, there were no associations between these polymorphisms andthe risk of gastric cancer under dominant or recessive models. Conclusion: This meta-analysis suggested thatthe MMP7-181 A>G polymorphism may contribute to gastric cancer susceptibility. More studies are needed,especially in Europeans, in the future.     

Evidence for and Against Green Tea and Turmeric in the Management of Chronic Lymphocytic Leukemia

Complementary and alternative medicine (CAM) is a diverse group of medical and health care systems, practices, and products that are not generally considered part of conventional medicine. Chronic lymphocytic leukemia (CLL) is the most common leukemia diagnosed in the western hemisphere, and 16.5% to 66% of patients have reported using CAM. Most patients use spiritual/mind–body techniques and high doses of vitamins and herbs (most commonly polyphenols, including teas). We have reviewed the reported data on green tea and turmeric use in CLL patients.     

Upregulation of Galectin-9 and PD-L1 Immune Checkpoints Molecules in Patients with Chronic Lymphocytic Leukemia

Background: Deviation of host immune response by engagement of inhibitory receptors is one of the well-known mechanisms of tumor cells for immune evasion and survival. PD-1/PD-L1 and Tim-3/Gal-9 axes are two major pathways in this area which their contribution has been documented in a variety of malignancies. In this study, Gal-9 and PD-L1 expression was investigated in leukemic cells from patients with Chronic Lymphocytic Leukemia (CLL). Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 25 untreated CLL patients and 15 sex- and age-matched healthy controls. CLL patients were classified into different clinical stages based on the Rai staging system. Total RNA was extracted from all samples and applied for cDNA synthesis. Relative expression of Gal-9 and PD-L1 mRNA was determined by Real-Time PCR using β-actin as a housekeeping gene. Results: Gal-9 and PD-L1 mRNA was significantly more expressed in CLL patients compared to healthy controls (ppatients in advanced clinical stages showed higher expression of Gal-9 and PD-L1 in comparison to patients in early clinical stages (pConclusion: Our promising results regarding over-expression of Gal-9 and PD-L1 in CLL patients call future complementary studies to more evaluate and confirm these pathways for immunotherapy approaches of this malignancy. Upregulation of both Gal-9 and PD-L1 in CLL patients with advanced clinical stages introduces them as useful prognostic biomarkers for disease progression.     

SOHO State of the Art Updates and Next Questions: Clonal Evolution in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is an indolent disease with a long-lasting clinical course, with indication for treatment only when symptomatic. Its clinical heterogeneity is widely reported, with some patients requiring treatment soon after diagnosis because of development of cytopenia or bulky lymphadenopathy, and others showing a stable or a slowly progressive disease not requiring treatment for decades. Longitudinal sampling of peripheral blood, with accessible tumor cells and circulating tumor DNA, enabled the analysis of disease growing dynamics and the characterization of clonal evolution. Here we describe the main known features of CLL genomics and its shaping upon treatment, which can lead to progression, treatment refractoriness, or transformation into an aggressive lymphoma.