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.     

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.     

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.     

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.     

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.     

Mutations found in cell‐free DNAs of patients with malignant lymphoma at remission can derive from clonal hematopoiesis

Cell‐free DNA (cfDNA) analysis to detect circulating tumor DNA has been focused on monitoring malignant lymphomas. However, clonal hematopoiesis of indeterminate potential (CHIP)‐associated mutations can also be detected by cfDNA analysis. Our aim is to investigate the origin of mutations detected in cfDNA among B‐cell lymphoma patients. MYD88/CD79B, DNMT3A, and TP53 were chosen as genes of interest, representing each of the following categories: lymphoma driver genes, CHIP‐related genes, and genes shared between lymphoma and CHIP. Seventy‐five B‐cell lymphoma patients were included in this retrospective study. Serum cfDNAs at time of complete metabolic response (CMR) were sequenced for TP53 (N = 75) and DNMT3A (N = 49). MYD88 p.L265P and CD79B p.Y196C/H mutations were analyzed in diffuse large B‐cell lymphoma (DLBCL) patients whose tumor samples were available (N = 29). Two and seven mutations in TP53 and DNMT3A, respectively, were detected in cfDNA at CMR. These mutations were detected in either bone marrow mononuclear cells (BMMC) or PBMC. Although four DNMT3A mutations were also detected in tumors, median variant allele frequencies in the tumors (<1.0%) were significantly lower than those in both BMMC (6.1%) and serum (5.2%) obtained before the therapy. Conversely, five MYD88 and three CD79B mutations detected in tumors were confirmed in cfDNA before therapy, but not in BMMC nor in cfDNA at CMR. Thus, all TP53 and DNMT3A mutations detected in cfDNA at remission seemed to originate from CHIP rather than from residual disease. Results of liquid biopsy should be carefully interpreted, especially in genes shared between lymphomas and CHIP.     

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.     

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.     

Barcode sequencing identifies resistant mechanisms to epidermal growth factor receptor inhibitors in circulating tumor DNA of lung cancer patients

Most patients with epidermal growth factor receptor (EGFR) mutation‐positive non‐small cell lung cancer (NSCLC) will inevitably develop acquired resistance induced by treatment with EGFR tyrosine kinase inhibitors (EGFR‐TKI). The mechanisms of resistance to EGFR‐TKI are multifactorial, and the detection of these mechanisms is critical for treatment choices in patients who have progressed after EGFR‐TKI therapy. We evaluated the feasibility of a molecular barcode method using next‐generation sequencing to detect multifactorial resistance mechanisms in circulating tumor DNA and compared the results with those obtained using other technologies. Plasma samples were collected from 25 EGFR mutation‐positive NSCLC patients after the development of EGFR‐TKI resistance. Somatic mutation profiles of these samples were assessed using two methods of next‐generation sequencing and droplet digital PCR (ddPCR). The positive rate for EGFR‐sensitizing mutations was 18/25 (72.0%) using ddPCR, 17/25 (68.0%) using amplicon sequencing, and 19/25 (76.0%) using molecular barcode sequencing. Rate of the EGFR T790M resistance mutation among patients with EGFR‐sensitizing mutations was shown to be 7/18 (38.9%) using ddPCR, 6/17 (35.3%) using amplicon sequencing, and 8/19 (42.1%) using molecular barcode sequencing. Copy number gain in the MET gene was detected in three cases using ddPCR. PIK3CA, KRAS and TP53 mutations were detected using amplicon sequencing. Molecular barcode sequencing detected PIK3CA, TP53, KRAS, and MAP2K1 mutations. Results of the three assays were comparable; however, in cell‐free DNA, molecular barcode sequencing detected mutations causing multifactorial resistance more sensitively than did the other assays.     

Disease‐specific mutations in mature lymphoid neoplasms: Recent advances

Mature lymphoid neoplasms (MLN) are clinically and pathologically more complex than precursor lymphoid neoplasms. Until recently, molecular characterization of MLN was mainly based on cytogenetics/fluorescence in situ hybridization, allele copy number, and mRNA expression, approaches that yielded scanty gene mutation information. Use of massive parallel sequencing technologies has changed this outcome, and now many gene mutations have been discovered. Some of these are considerably frequent in, and substantially specific to, distinct MLN subtypes, and occur at single or several hotspots. They include the V600E BRAF mutation in hairy cell leukemia, the L265P MYD88 mutation in Waldenström macroglobulinemia, the G17V RHOA mutation in angioimmunoblastic T‐cell lymphoma and peripheral T‐cell lymphoma, not otherwise specified, and the Y640F//D661Y/V/H/I//N647I STAT3 mutations in T‐cell large granular lymphocytic leukemia. Detecting these mutations is highly valuable in diagnosing MLN subtypes. Defining these mutations also sheds light on the molecular pathogenesis of MLN, furthering development of molecular targeting therapies. In this review, we focus on the disease‐specific gene mutations in MLN discovered by recent massive sequencing technologies.     

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。     

Mutational analysis of primary central nervous system lymphoma

Little is known about the genomic basis of primary central nervous system lymphoma (PCNSL) tumorigenesis. To investigate the mutational profile of PCNSL, we analyzed nine paired tumor and germline DNA samples from PCNSL patients by high throughput exome sequencing. Eight genes of interest have been further investigated by focused resequencing in 28 additional PCNSL tumors to better estimate their incidence. Our study identified recurrent somatic mutations in 37 genes, some involved in key signaling pathways such as NFKB, B cell differentiation and cell cycle control. Focused resequencing in the larger cohort revealed high mutation rates for genes already described as mutated in PCNSL such as MYD88 (38%), CD79B (30%), PIM1 (22%) and TBL1XR1 (19%) and for genes not previously reported to be involved in PCNSL tumorigenesis such as ETV6 (16%), IRF4 (14%), IRF2BP2 (11%) and EBF1 (11%). Of note, only 3 somatically acquired SNVs were annotated in the COSMIC database. Our results demonstrate a high genetic heterogeneity of PCNSL and mutational pattern similarities with extracerebral diffuse large B cell lymphomas, particularly of the activated B-cell (ABC) subtype, suggesting shared underlying biological mechanisms. The present study provides new insights into the mutational profile of PCNSL and potential targets for therapeutic strategies.     

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...     

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.     

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 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.     

Gene expression profiling of primary vitreoretinal lymphoma

The characteristics of tumor cells of primary vitreoretinal lymphoma (PVRL) have not been defined, although researches have shown that most cases are of diffuse large B‐cell lymphoma (DLBCL). To determine the subtype and biological characteristics of tumor cells of PVRL, we performed a gene expression profiling analysis. RNA was extracted from the vitreous fluid of 7 PVRL patients and from nodal samples of 10 DLBCL patients: 6 of germinal center B‐cell (GCB) type and 4 of activated B‐cell (ABC) type determined by Hans’ criteria. Six PVRL samples showed gene expression profiles that were similar to each other. The patterns were different from those of the ABC‐type nodular DLBCL but relatively close to those of the GCB‐type nodular DLBCL. Interestingly, all of the 6 examined PVRL samples had either MYD88^L265P or mutation in the immunoreceptor tyrosine‐based activation motif (ITAM) region of CD79B. Five PVRL patients with similar gene expression profiles were treated with a standardized regimen: intravitreal administration of methotrexate (MTX) followed by six courses of systemic high doses of MTX. As a result, 2 patients had CD79B mutations and showed early central nervous system (CNS) progression. Patients without CNS progression did not have this mutation. In conclusion, PVRL had unique genetic features: an expression pattern different from ABC‐type and relatively close to GCB‐type DLBCL. CD79B mutations showed potential to serve as prognostic markers for CNS progression.     

Clinical significance of the mutational landscape and fragmentation of circulating tumor DNA in renal cell carcinoma

Reliable biomarkers for renal cell carcinoma (RCC) have yet to be determined. Circulating tumor DNA (ctDNA) is an emerging resource to detect and monitor molecular characteristics of various tumors. The present study aims to clarify the clinical utility of ctDNA for RCC. Fifty‐three patients histologically diagnosed with clear cell RCC were enrolled. Targeted sequencing was carried out using plasma cell‐free DNA (cfDNA) and tumor DNA. We applied droplet digital PCR (ddPCR) to validate detected mutations. cfDNA fragment size was also evaluated using a microfluidics‐based platform and sequencing. Proportion of cfDNA fragments was defined as the ratio of small (50‐166 bp) to large (167‐250 bp) cfDNA fragments. Association of mutant allele frequency of ctDNA with clinical course was analyzed. Prognostic potential was evaluated using log‐rank test. A total of 38 mutations across 16 (30%) patients were identified from cfDNA, including mutations in TP53 (n = 6) and VHL (n = 5), and median mutant allele frequency of ctDNA was 10%. We designed specific ddPCR probes for 11 mutations and detected the same mutations in both cfDNA and tumor DNA. Positive ctDNA was significantly associated with a higher proportion of cfDNA fragments (P = .033), indicating RCC patients with ctDNA had shorter fragment sizes of cfDNA. Interestingly, the changes of mutant allele frequency in ctDNA concurrently correlated with clinical course. Positive ctDNA and fragmentation of cfDNA were significantly associated with poor cancer‐specific survival (P < .001, P = .011). In conclusion, our study shows the clinical utility of ctDNA status and cfDNA fragment size as biomarkers for prognosis and disease monitoring in RCC.     

Prognostic role of PIK3CA mutations of cell‐free DNA in early‐stage triple negative breast cancer

PIK3CA is an oncogene that encodes the p110α component of phosphatidylinositol 3‐kinase (PI3K); it is the second most frequently mutated gene following the TP53 gene. In the clinical setting, PIK3CA mutations may have favorable prognostic value for hormone receptor‐positive breast cancer patients and, during the past few years, PIK3CA mutations of cell‐free DNA (cfDNA) have attracted attention as a potential noninvasive biomarker of cancer. However, there are few reports on the clinical implications of PIK3CA mutations for TNBC patients. We investigated the PIK3CA major mutation status of cfDNA as a noninvasive biomarker of cancer using droplet digital polymerase chain reaction (ddPCR), which has high level sensitivity and specificity for cancer mutation, in early‐stage 49 triple negative breast cancer (TNBC) patients. A total of 12 (24.4%) of 49 patients had PIK3CA mutations of cfDNA. In a median follow up of 54.4 months, the presence of PIK3CA mutations of cfDNA had significant impacts on relapse‐free survival (RFS; P = 0.0072) and breast cancer‐specific survival (BCSS; P = 0.016), according to the log‐lank test. In a Cox proportional hazards model, the presence of PIK3CA mutations of cfDNA had significant prognostic value in the univariate and multivariate analysis. Additionally, the presence of PIK3CA mutations of cfDNA was significantly correlated with positive androgen receptor phosphorylated form depending on PI3K signaling pathway (pAR) which is independent favorable prognostic factors of TNBC. We demonstrated that the presence of PIK3CA major mutations of cfDNA could be a discriminatory predictor of RFS and BCSS in early‐stage TNBC patients and it was associated with PI3K pathway‐dependent AR phosphorylation.     

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.