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.     

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.     

淋巴浆细胞性淋巴瘤/华氏巨球蛋白血症10例临床病理学特征分析及MYD88基因突变检测

背景与目的：淋巴浆细胞性淋巴瘤/华氏巨球蛋白血症（lymphoplasmacytic lymphoma/Waldenström macroglobulinemia,LPL/WM）是罕见的B细胞惰性淋巴瘤,其诊断常需要与其他小B细胞性肿瘤作鉴别。本研究皆在探讨LPL/WM的临床病理学特点,并观察MYD88基因突变在此类肿瘤中的检出频率。方法：分析10例LPL/WM病例的临床资料、组织学形态和免疫组织化学表型,并以聚合酶链反应（polymerase chain reaction,PCR）扩增及直接测序法检测肿瘤标本MYD88基因的状态。结果：10例患者均为男性,中位年龄61岁。患者多表现为乏力和贫血,均有不同程度的IgM型免疫球蛋白血症和肿瘤骨髓累及。淋巴结活检标本光镜检查显示淋巴结结构部分存在,特别是可以见到开放的淋巴窦。肿瘤由数量不等的浆细胞、小淋巴细胞及浆样淋巴细胞组成。骨髓活检标本也可见到相同形态的细胞浸润。免疫组织化学染色显示,10例肿瘤细胞均呈CD20弥漫阳性并限制性表达免疫球蛋白轻链κ,6例瘤细胞表达CD23,2例部分瘤细胞表达CD5,未见病例表达CD10,Ki-67增殖指数在5%~30%之间。另外,2例LPL/WM在其病灶中尚可见到较多的IgG4阳性反应性浆细胞浸润。所有LPL/WM病例肿瘤组织均有MYD88 L265P突变检出,而对照组其他小B细胞性肿瘤均为阴性结果。结论：LPL/WM多具有典型的临床病理学形态,但偶亦可出现异常表型。MYD88 L265P作为LPL/WM的特征性遗传学特点,其检测的引入可使此类肿瘤的诊断和鉴别有更可靠的依据。     

Molecular and genetic biomarkers implemented from next-generation sequencing provide treatment insights in clinical practice for Waldenström macroglobulinemia

Waldenström macroglobulinemia (WM) is a distinct type of indolent lymphoplasmacytic lymphoma (LPL) with a high frequency of MYD88^L265P mutation. Treatment for WM/LPL is highly variable in clinic and ibrutinib (a Bruton tyrosine kinase inhibitor, BTKi) has become a new treatment option for WM. To investigate the clinical impact of genetic alterations in WM, we assembled a large cohort of 219 WMs and 12 LPLs dividing into two subcohorts: a training cohort, patients sequenced by a same targeted 29-gene next-generation sequencing (NGS) panel, and a validation cohort, patients sequenced by allele specific-PCR or other targeted NGS panels. In both training and validation subcohorts, MYD88^L265P and TP53 mutations showed favorable and adverse prognostic effects, respectively. CXCR4 nonsense/missense mutations (CXCR4^NS/MS), cytogenetic complex karyotypes, and a family history of lymphoma/leukemia in first-degree relatives were associated with significantly worse clinical outcomes only or more in the validation subcohort. We further investigated the efficacy of various treatments and interaction with genetic factors in the entire cohort. Upfront dexamethasone usage was associated with poorer clinical outcomes in patients who received non-proteasome-containing chemotherapy as first-line treatment independent of genetic factors. Maintenance rituximab was associated with better survival. Ibrutinib/BTKi showed potential benefit in relapsed/refractory patients and patients without CXCR4^NS/MS including those with TP53 mutations. In conclusion, genetic testing for MYD88^L265P, TP53, and CXCR4 mutations and cytogenetic analysis provide important information for prognosis prediction and therapy selection. The findings in these study are valuable for improving treatment decisions on therapies available for WM/LPL patients with integration of NGS in clinic.     

Genetic evidence implies that primary and relapsed tumors arise from common precursor cells in primary central nervous system lymphoma

Primary central nervous system lymphoma (PCNSL) is a rare subtype of lymphoma that arises within the brain or the eyes. PCNSL recurs within the central nervous system (CNS) in most relapsed cases, whereas extra‐CNS relapse is experienced in rare cases. The present study aimed at identifying the presence of common precursor cells (CPC) for primary intra‐ and relapsed extra‐CNS tumors, and further assessing the initiating events in bone marrow (BM). Targeted deep sequencing was carried out for five paired primary intra‐ and relapsed extra‐CNS tumors of PCNSL. Two to five mutations were shared by each pair of intra‐ and extra‐CNS tumors. In particular, MYD88 mutations, L265P in three and P258L in one, were shared by four pairs. Unique somatic mutations were observed in all five intra‐CNS tumors and in four out of five extra‐CNS tumors. Remarkably, IgH clones in the intra‐ and the extra‐CNS tumors in two pairs were distinct from each other, whereas one pair of tumors shared identical monoclonal IgH rearrangement. In a cohort of 23 PCNSL patients, L265P MYD88 mutations were examined in tumor‐free BM mononuclear cells (MNC) in which the PCNSL tumors had L265P MYD88 mutations. L265P MYD88 mutations were detected by a droplet digital PCR method in nine out of 23 bone marrow mononuclear cells. These results suggest that intra‐ and extra‐tumors are derived from CPC with MYD88 mutations in most PCNSL, arising either before or after IgH rearrangement. The initiating MYD88 mutations may occur during B‐cell differentiation in BM.     

Activation of TAK1 by MYD88 L265P drives malignant B-cell Growth in non-Hodgkin lymphoma

Massively parallel sequencing analyses have revealed a common mutation within the MYD88 gene (MYD88_L265P) occurring at high frequencies in many non-Hodgkin lymphomas (NHLs) including the rare lymphoplasmacytic lymphoma, Waldenström''s macroglobulinemia (WM). Using whole-exome sequencing, Sanger sequencing and allele-specific PCR, we validate the initial studies and detect the MYD88_L265P mutation in the tumor genome of 97% of WM patients analyzed (n=39). Due to the high frequency of MYD88 mutation in WM and other NHL, and its known effects on malignant B-cell survival, therapeutic targeting of MYD88 signaling pathways may be clinically useful. However, we are lacking a thorough characterization of the role of intermediary signaling proteins on the biology of MYD88_L265P-expressing B cells. We report here that MYD88_L265P signaling is constitutively active in both WM and diffuse large B-cell lymphoma cells leading to heightened MYD88_L265P, IRAK and TRAF6 oligomerization and NF-κB activation. Furthermore, we have identified the signaling protein, TAK1, to be an essential mediator of MYD88_L265P-driven signaling, cellular proliferation and cytokine secretion in malignant B cells. Our studies highlight the biological significance of MYD88_L265P in NHL and reveal TAK1 inhibition to be a potential therapeutic strategy for the treatment of WM and other diseases characterized by MYD88_L265P.     

Comprehensive Laboratory Diagnostic Workup for Patients with Suspected Intraocular Lymphoma including Flow Cytometry,Molecular Genetics and Cytopathology

Background: Intraocular lymphoma (IOL) presents a real challenge in daily diagnostics. Cyto- and/or histopathology of vitreous body represent the diagnostic cornerstones. Yet, false negative results remain common. Therefore, we analyzed the diagnostic significance of flow cytometry (FC) within the workup algorithm of IOL and compared its sensitivity with the results obtained from routine cytopathology and molecular genetics; Methods: Seven patients undergoing vitrectomy due to suspected IOL were investigated by FC and parallel cytopathology and, if available, digital droplet PCR (ddPCR) for MYD88 L265P; Results: Four out of seven patients were finally diagnosed with IOL. Among the IOL patients, cytopathology confirmed the presence of lymphoma cells in only two cases. In contrast, FC was positive for IOL in all four cases, and FC additionally confirmed the lack of IOL in the remaining patients. In IOL patients diagnosed by FC and with available ddPCR, the diagnosis of IOL was confirmed by the presence of the MYD88 L265P mutation in all three patients; Conclusions: The combination with FC was superior to cytopathology alone in the diagnostic work-up of IOL, and it showed an excellent correlation with ddPCR results. A comprehensive diagnostic panel consisting of cytopathology, FC and molecular genetics should be considered for the work-up of suspected IOL.     

弥漫性大B细胞淋巴瘤中MYD88基因突变及其临床病理相关性分析

背景与目的：MYD88基因在弥漫性大B细胞淋巴瘤（diffuse large B-cell lymphoma,DLBCL）中有一定突变率,但其临床病理相关性目前研究报道甚少。该研究旨在分析DLBCL中MYD88基因突变的发生率及与临床病理参数的相关性。方法：收集121例DLBCL患者的临床病理资料,采用免疫组织化学法分析其免疫表型,采用PCR扩增及直接测序法检测MYD88 L265P位点突变情况,采用统计学方法分析MYD88突变与各临床病理参数的相关性。结果：121例DLBCL患者中,38例（31.4%）检测到MYD88 L265P突变。其中男性50例,女性71例,患者性别与该基因突变没有相关性（P=0.609）。年龄≥60岁组MYD88突变率为40.3%（25/62）,显著高于<60岁组（13/59,22.0%）（P=0.030）。MYD88突变主要发生在结外部位,其中最常见的是乳腺（12/13,92.3%）、男性生殖系统（10/11,90.9%）、女性生殖系统（5/6,83.3%）及中枢神经系统（4/6,66.7%）;结外DLBCL中的MYD88突变率（35/98,35.7%）显著高于结内者（2/20,10%）（P=0.024）。Non-GCB型DLBCL中MYD88突变率为39.7%（25/63）,显著高于GCB亚型（10/55,18.2%）（P=0.010）;结外DLBCL组中MYD88突变与免疫分型的相关性更加显著（P=0.003）,而结内组中两者无相关性（P=0.776）。Bcl-2蛋白阳性组（30/77,39.0%）及MYC/Bcl-2蛋白双表达组（19/46,41.3%）中MYD88突变率分别高于Bcl-2阴性组（5/40,12.5%）及非双表达组（16/70,22.9%）（P=0.003和0.034）。Ki-67增殖活性与MYD88基因突变显著相关［高增殖活性组为38.8%（33/85）,低增殖活性组为6.3%（2/32）］（P<0.001）。该基因突变与MYC蛋白及CD5表达均无相关性（P=0.581和0.759）。结论：MYD88 L265P突变好发于年龄≥60岁、non-GCB起源及特殊结外部位（如乳腺、中枢神经系统及生殖系统等）的DLBCL中,且具有较高增殖指数及MYC/Bcl-2蛋白双表达率;其预后相关性有待积累更多病例进一步分析。MYD88基因突变有望为揭示DLBCL发病机制及靶向治疗提供新的理论依据。     

Detection of L265P MYD‐88 mutation in a series of clonal B‐cell lymphocytosis of marginal zone origin (CBL‐MZ)

Clonal B‐cell lymphocytosis of marginal zone origin (CBL‐MZ) is a recently described entity characterized by the presence of clonal B cells in the blood and/or bone marrow (BM) with morphologic and immunophenotypic features consistent with marginal zone derivation in otherwise healthy individuals. CBL‐MZ is commonly associated with paraproteinemia, usually immunoglobulin M (IgM), raising diagnostic difficulties from Waldenstrom macroglobulinemia (WM). The aim of the present study was to determine the presence of MYD‐88 L265P mutation in a well‐characterized series of CBL‐MZ to identify cases that may in fact represent WM. Fifty‐three CBL‐MZ cases were retrospectively evaluated. MYD‐88 L265P mutation was determined by allele‐specific polymerase chain reaction in blood and/or BM mononuclear cells. Almost half of the CBL‐MZ cases (49%) were associated with paraproteinemia mainly of the IgM type (65%). MYD‐88 L265P mutation was identified in 10 cases (19%). These cases may truly represent WM, whereas 43 cases (81%) are still classified as CBL‐MZ. Mutated cases were all associated with paraproteinemia compared with 37% of the nonmutated ones (P < .0001). In addition, mutated cases displayed more frequently CD38 and CD25 positivity (P = .002 and P = .005, respectively). Moreover, cases without paraproteinemia presented more frequently with lymphocytosis, irrespective of the presence of the MYD‐88 mutation (P = .02). The present study demonstrates that MYD‐88 L265P mutation may represent the only sensitive marker for the differentiation of CBL‐MZ from probable WM. However, further studies are warranted to better define the biological significance of MYD‐88 L265P mutation and to clarify whether the presence of the mutation establishes WM diagnosis or that it can also be present in borderline cases associated with paraproteinemia.     

伴MYD88 L265P突变的脾边缘区淋巴瘤临床特征分析

目的 分析伴MYD88 L265P突变的脾边缘区淋巴瘤(splenic marginal zone lymphoma,SMZL)患者的临床特征.方法 回顾性分析本中心SMZL患者队列,将MYD88 L265P突变型患者的临床特征与野生型以及华氏巨球蛋白血症(Waldenstr?m macroglobulinemia,W...     

Hemophagocytic lymphohistiocytosis as an onset of diffuse large B-cell lymphoma: A case report

A 53-year-old male presented with a 1-month history of hyperpyrexia. The clinical manifestations revealed hemophagocytic lymphohistiocytosis (HLH). Although a lymph node biopsy could not be obtained, a bone marrow biopsy revealed the activated B-cell subtype of diffuse large B-cell lymphoma (DLBCL). After being treated with HLH-1994 (dexamethasone and etoposide), a rituximab-containing chemotherapy and target agents involving bortezomib, the patient achieved remission. To understand the molecular profile of patient, next-generation sequencing and MYD88 L265P mutation examinations were performed, and the patient was determined to be positive for the MYD88 L265P mutation. Reports of DLBCL with plasmacytic differentiation and a MYD88 innate immune signal transduction adaptor L265P mutation concurrent with HLH are rare. Early recognition, precise diagnosis and timely therapy are pivotal in improving patient prognosis. Furthermore, molecular profiling enables researchers to develop potential therapies aimed at the activated NF-κB and endoplasmic reticulum stress signaling pathways. The present study highlights this pathogenesis and provides suggestions for further individualized therapeutics.     

Concurrent Waldenstrom’s Macroglobulinemia and Myelodysplastic Syndrome with a Sequent t(10;13)(p13;q22) Translocation

Myelodysplastic syndromes (MDS) and Waldenstrom’s macroglobulinemia (WM) are rarely synchronous. Ineffective myelopoiesis/hematopoiesis with clonal unilineage or multilineage dysplasia and cytopenias characterize MDS. Despite a myeloid origin, MDS can sometimes lead to decreased production, abnormal apoptosis or dysmaturation of B cells, and the development of lymphoma. WM includes bone marrow involvement by lymphoplasmacytic lymphoma (LPL) secreting monoclonal immunoglobulin M (IgM) with somatic mutation (L265P) of myeloid differentiation primary response 88 gene (MYD88) in 80–90%, or various mutations of C-terminal domain of the C-X-C chemokine receptor type 4 (CXCR4) gene in 20–40% of cases. A unique, progressive case of concurrent MDS and WM with several somatic mutations (some unreported before) and a novel balanced reciprocal translocation between chromosomes 10 and 13 is presented below.     

Characteristics of Waldenström Macroglobulinemia in Korean Patients According to Mutational Status of MYD88 and CXCR4: Analysis Using Ultra-Deep Sequencing

BackgroundLittle is known about the mutational frequency of myeloid differentiation factor 88 (MYD88) and C-X-C chemokine receptor type 4 (CXCR4) and the corresponding characteristics in Asian individuals afflicted with Waldenström macroglobulinemia (WM). We investigated the characteristics of WM according to mutational status of MYD88/CXCR4, and attempted to determine the lineage commitment among hematopoietic cells by MYD88^L265P single-cell sequencing on bone marrow (BM) smear slides.Materials and MethodsCXCR4 mutations (muts) were detected using ultra-deep sequencing using target capture. Mutational burden of MYD88 was assessed using real-time polymerase chain reaction. Single-cell sequencing for MYD88 was performed on lymphocytes, plasmacytoid lymphocytes, plasma cells, and neutrophils using laser microdissection.ResultsAmong 31 patients, the frequencies of MYD88/CXCR4 muts were as follows: MYD88 wild type (WT) CXCR4^WT (6 patients, 19.4%), MYD88^L265PCXCR4^WT (19 patients, 61.4%), MYD88^L265PCXCR4^mut (6 patients, 19.4%; 1 frameshift and 5 nonsense muts). Immunoglobulin M levels of MYD88^L265CXCR4^WT patients were significantly higher than those of MYD88^WTCXCR4^WT patients (P = .024). Tumor burden in BM was highest in patients with MYD88^L265PCXCR4^mut (82.0%), followed by MYD88^L265PCXCR4^WT (52.8%) and MYD88^WTCXCR4^WT (14.2%) (P < .001). The quantity of MYD88-mutated DNA tended to correlate with tumor burden in BM (correlation coefficient 0.647; P = .009). MYD88^L265P was detected in plasma cells, plasmacytoid lymphocytes, and lymphocytes but not neutrophils.ConclusionThe frequency of MYD88/CXCR4 muts in Korean and Caucasian patients with WM was similar, however 5 of the 6 CXCR4 muts were nonsense—a proportion higher than reported frequencies in Caucasian individuals. Ultra-deep sequencing was capable of detecting CXCR4 muts not detectable using Sanger sequencing, suggesting a possible replacement of the B-cell sorting.     

Whole-Exome Sequencing Revealed the Mutational Profiles of Primary Central Nervous System Lymphoma

BackgroundPrimary central nervous system lymphoma (PCNSL) is a highly aggressive type of extranodal non-Hodgkin lymphoma, of which approximately 90% of the cases are diffuse large B-cell lymphoma (DLBCL). In recent years, the incidence of PCNSL has significantly increased in women and older men. Although advanced treatments such as high-dose methotrexate (HD-MTX) and targeted agents have been introduced, the prognosis of these patients remains poorer than those with other forms of non-Hodgkin's lymphoma.MethodsTwelve cases of Chinese PCNSL were analyzed to detect their genetic alterations using whole-exome sequencing (WES). We identified 448 potential somatic single nucleotide variants (SNVs) with a median of 12 SNVs per PCNSL sample and 35 small indels with potentially protein-changing features in 9 PCNSL samples.ResultsWe found that myeloid differentiation factor 88 (MYD88) had the highest mutation frequency, which affected the activity of the nuclear factor-κB (NF-κB) pathway. PCNSL samples with low-density lipoprotein receptor-related protein 1B (LRP1B) mutations had a higher mutation rate than samples with wild-type LRP1B. Polycystic kidney and hepatic disease 1 (PKHD1), the causal gene of autosomal recessive polycystic kidney disease (ARPKD), was identified in 2 PCNSL cases and exhibited missense mutations. Pathway analysis revealed enrichment in pathways associated with central carbon metabolism in cancer, renal cell carcinoma, nicotine addiction, bladder cancer, and long-term depression.ConclusionsWES revealed significantly mutated genes associated with the molecular mechanisms of PCNSL, which could serve as therapeutic targets to improve patient outcomes.     

Noninvasive detection of microsatellite instability in patients with endometrial cancer

The analysis of mismatch repair proteins in solid tissue is the standard of care (SoC) for the microsatellite instability (MSI) characterization in endometrial cancer (EC). Uterine aspirates (UAs) or circulating-DNA (cfDNA) samples capture the intratumor heterogeneity and provide a more comprehensive and dynamic molecular diagnosis. Thus, MSI analysis by droplet-digital PCR (ddPCR) in UAs and cfDNA can provide a reliable tool to characterize and follow-up the disease. The UAs, paraffin-embedded tumor tissue (FFPE) and longitudinal plasma samples from a cohort of 90 EC patients were analyzed using ddPCR panel and compared to the SoC. A high concordance (96.67%) was obtained between the analysis of MSI markers in UAs and the SoC. Three discordant cases were validated as unstable by ddPCR on FFPE samples. Besides, a good overall concordance (70.27%) was obtained when comparing the performance of the ddPCR assay on UAs and cfDNA in high-risk tumors. Importantly, our results also evidenced the value of MSI analysis to monitor the disease evolution. MSI evaluation in minimally invasive samples shows great accuracy and sensitivity and provides a valuable tool for the molecular characterization and follow-up of endometrial tumors, opening new opportunities for personalized management of EC.     

Bing-Neel Syndrome: Update on the Diagnosis and Treatment

Bing-Neel syndrome (BNS) is a rare syndrome that occurs in patients with Waldenstrom macroglobulinemia and is characterized by lymphoplasmacytic infiltration into the leptomeningeal tissue and/or the central nervous system. It represents an extramedullary manifestation which may translate into various neurological symptoms. Accurate diagnosis of BNS can be established via histologic sampling and cerebrospinal fluid examination with molecular analysis of some genetic markers including immunoglobulin heavy locus rearrangements and MYD88 L265P mutation. The use of Bruton tyrosine kinase inhibitors such as ibrutinib resulted in promising outcomes. In this review, we discuss the pathophysiology, clinical manifestations, diagnostic characteristics, and an overview of the current treatment modalities of BNS.     

Lack of Associations between TLR9 and MYD88 Gene Polymorphisms and Risk of Chronic Lymphocytic Leukemia

Background: Genetic factors like single nucleotide polymorphisms (SNPs) may play an important role in the etiology of chronic lymphocytic leukemia (CLL). Mutations in Toll like receptor 9 (TLR9) and myeloid differentiation primary response 88 (MYD88) genes may lead to an abnormal immune response that may cause greater cell proliferation and thus alter an individual’s susceptibility to haematological malignancies including CLL. Objective: This work was designed to study any association of the TLR9 (rs2066807C/G and rs187084T/C) and MYD88 (L265P) single nucleotide polymorphism (SNPs) with risk of CLL in Egyptians. Materials and methods: One hundred patients with CLL and 100 healthy controls from the Egyptian population were genotyped by the polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) method. Results: With TLR9 rs2066807C/G the CC genotype was more frequent in both control and patient groups while for TLR9 rs187084T/C the TT genotype was most common. There were no significant associations with CLL risk. With MYD88 (L265P) only the TT genotype was detected. Conclusion: Our preliminary data suggest that polymorphisms in the TLR9 and MYD88 genes may not contribute to CLL susceptibility. To the best of our knowledge, this study is the first dealing with TLR9 and MYD88 gene polymorphisms in CLL patients. Further studies with larger sample size should be conducted to validate these results in the Egyptian population.     

Molecular profiling of Chinese R-CHOP treated DLBCL patients: Identifying a high-risk subgroup

Diffuse large B-cell lymphoma (DLBCL) is a clinically aggressive and heterogenous disease. Although most patients can be cured by immunochemotherapy, 30% to 40% patient will ultimately develop relapsed or refractory disease. Here, we investigated the molecular landscapes of patients with diverse responses to R-CHOP. We performed capture-based targeted sequencing on baseline samples of 105 DLBCL patients using a panel consisting of 112 lymphoma-related genes. Subsequently, 81 treatment-naïve patients with measurable disease and followed for over 1 year were included for survival analysis. Collectively, the most commonly seen mutations included IGH fusion (69%), PIM1(33%), MYD88 (29%), BCL2 (29%), TP53 (29%), CD79B (25%) and KMT2D (24%). Patients with TP53 mutations were more likely to have primary refractory disease (87.0% vs 50.0%, P = .009). For those with TP53 disruptive mutations, 91.7% patients were in the primary refractory group. Interestingly, BCL-2 somatic hypermutation was only seen in patients without primary refractory disease (P = .014). In multivariate analysis, BCL-2 amplification (hazard ratio [HR] = 2.94, P = .022), B2M mutation (HR = 2.99, P = .017) and TP53 mutation (HR = 3.19, P < .001) were independently associated with shorter time to progression (TTP). Furthermore, TP53 mutations was correlated with worse overall survival (P = .049). Next, we investigated mutation landscape in patients with wild-type (WT) TP53 (n = 58) and found that patients harboring MYD88 L265P had significantly inferior TTP than those with WT or non-265P (P = .046). Our study reveals the mutation spectrum of treatment-naive Chinese DLBCL patients. It also confirms the clinical significance of TP53 mutations and indicates the prognostic value of MYD88 L265P in TP53 WT patients.     

Droplet digital polymerase chain reaction assay and peptide nucleic acid‐locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T‐cell lymphoma

Angioimmunoblastic T‐cell lymphoma (AITL) is a subtype of nodal peripheral T‐cell lymphoma (PTCL). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next‐generation sequencing (NGS), droplet digital PCR (ddPCR) and peptide nucleic acid‐locked nucleic acid (PNA‐LNA) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA‐LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL198; c.[50G > T;51A > C], p.Gly17Val in PTCL216; and c.50G > A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and the PNA‐LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of ddPCR/PNA‐LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position.