Detection Of MYD88 L265P Mutation Research Articles

Blood and cerebrospinal fluid biomarkers in neuro-oncology.

The purpose of this review is to discuss the value of blood and CSF biomarkers in primary CNS tumors. Several analytes can be assessed with liquid biopsy techniques, including circulating tumor cells, circulating cell-free tumor DNA, circulating cell-free RNA, circulating proteins and metabolites, extracellular vesicles and tumor-educated platelets. Among diffuse gliomas of the adult, ctDNA in blood or CSF has represented the most used analyte, with the detection of molecular alterations such as MGMT promoter, PTEN, EGFRVIII, TERT promoter mutation and IDH R132H mutation. In general, CSF is enriched for ctDNA as compared with plasma. The use of MRI-guided focused ultrasounds to disrupt the blood-brain barrier could enhance the level of biomarkers in both blood and CSF. The detection of MYD88 L265P mutation with digital droplet PCR and the detection of ctDNA with next generation sequencing represent the best tools to diagnose and monitoring CNS lymphomas under treatment. In meningiomas, the low concentration of ctDNA is a limiting factor for the detection of driver mutations, such as NF2, AKTs, SMO, KLF4, TRAF7, SMARCB1, SMARCE1, PTEN, and TERT; an alternative approach could be the isolation of ctDNA through circulating extracellular vesicles. Liquid biopsies are being used extensively for diagnosis and surveillance of diffuse midline gliomas, in particular with the detection of the driver mutation H3K27M. Last, specific methylome patterns in CSF may allow the distinction of glioblastomas from CNS lymphomas or meningiomas. This review summarizes the current knowledge and future perspectives of liquid biopsy of blood and CSF for diagnosis and monitoring of primary CNS tumors.

PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay: A highly sensitive method for detection of MYD88 L265P mutation.

Agarose gel-based conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays are currently used for sensitive detection and quantification of MYD88 L265P mutation. Visual inspection of an agarose gel can often be ambiguous. We propose a new allele-specific quantification PCR (AS-qPCR) assay, PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay, that uses Intercalating Nucleic Acid (INA®) technology for increased affinity and specificity. This study compares PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay with conventional AS-PCR. We included a total of 102 peripheral and bone marrow blood samples from 94 patients with a lymphoproliferative disorder. Droplet digital PCR (ddPCR) was used as a third method in case of discrepancy. A positive percent agreement of 100% (95% CI 0.92-1.0) and a negative percent agreement of 98% (95% CI 0.90-1.0) were found between the conventional AS-PCR and the AS-qPCR methods. Including the ddPCR results to validate the discrepant cases, the sensitivity and specificity of PlentiPlex™ MYD88 Waldenström lymphoma qPCR Assay were 1.0 (95% CI 0.97-1.0) and 1.0 (95% CI 0.96-1.0), respectively. Our data demonstrate that PlentiPlex™ MYD88 Waldenström lymphoma qPCR assay is a fast, highly sensitive, and specific method for the detection of MYD88 L265P compared with conventional AS-PCR.

Lymphoplasmacytic lymphoma: a clinicopathological and prognostic analysis of 27 cases

Objective: To study the clinical manifestations, pathologic features, diagnosis and differential diagnosis, treatment and prognosis of lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia (LPL/WM). Methods: Twenty-seven cases of LPL from January 2016 to December 2020 at Guangdong Provincial People's Hospital were collected. The clinical data, histomorphology, immunophenotype, MYD88 L265P mutation, treatment and prognosis were analyzed retrospectively. Results: There were 19 males and 8 female patients, with median age of 63 years. The most common initial symptoms were fatigue related to anemia. Bone marrow was involved in all cases, lymphadenopathy was seen in 11 cases and splenomegaly in 10 cases. Monoclonal IgM type protein was detected in 25 cases, meeting the diagnostic criteria of WM. Microscopically, bone marrow and lymph nodes were infiltrated by small lymphocytes, plasmacytoid lymphocytes or plasma cells. The cells expressed pan B-cell markers and showed immunoglobulin light chain restriction. There was no expression of CD5, and low expression of CD23 and CD10; Ki-67 index was usually low. The positive rate of MYD88 L265P mutation was 73.9% (17/23). Most of the patients were treated with rituximab combined with alkylating agents, nucleoside analogues or immunomodulators, and the few patients with relapse or progression were treated with Ibutinib. During the 3-168 months' follow-up period, recurrence or progression were seen in nine cases. Thrombocytopenia, elevated β2-microglobulin and high-risk group were associated with recurrence or progression of the disease (P<0.05). The overall survival (OS) and progression-free survival (PFS) of the high-risk patients were significantly lower than those of the low-medium risk patients (P<0.05). Conclusions: LPL/WM is an exclusive diagnosis; the detection of MYD88 L265P mutation has high diagnostic value, but it is not specific. These cases should be assessed comprehensively for their clinical manifestation, serum IgM protein level and immunophenotype. The overall prognosis of LPL/WM is good, but there are still a small number of high-risk patients with rapid progress, and so the symptomatic patients should be diagnosed accurately and treated in a timely manner.

ML-7 Liquid biopsy for MYD88 mutation in cerebrospinal fluid in patients with suspected primary CNS lymphoma

Background: Treatment intervention for central nervous system lymphoma (CNSL) requires pathological diagnosis by surgical biopsy. However, there are some cases in which the risk of surgery is high due to age, comorbidities, localization of lesions, etc. We are developing a CNSL diagnostic method based on the detection of MYD88 L265P mutation by digital PCR (dPCR) using CSF-DNA, and a high accuracy with a sensitivity of 92.9% and a specificity of 100% has been reported. Here, we report two cases with suspected brain stem CNSL, whose treatment strategy was determined by integrated clinico-laboratory information including neurological presentations, imaging, and the result of liquid biopsy. Result: Case 1. A 63-year-old woman visited our hospital with a complaint of right hemiplegia, which deteriorated in two months. MR images revealed a contrast-enhancing lesion in the left midbrain-ventral pons, suggesting CNSL. Biopsy was not considered because of its location, while dPCR using CSF-DNA showed a cluster of MYD88 mutation signals. Based on these work-ups, she was treated with high-dose methotrexate-based chemotherapy, resulting in a complete response with marked improvement of symptoms. Case 2. An 83-year-old man was referred for a history of diplopia and ataxic gait lasting for a month. MR images revealed an invasive lesion on his right midbrain-dorsal pons. Biopsy was declined due to the location, and liquid biopsy using CSF-DNA was performed to assist the diagnosis. In the first test, the CSF-DNA yield was too insufficient to determine the mutation signal by dPCR. The second dPCR using sufficient amount of CSF-DNA resulted in the Target/Total value of 0.049% which was lower than the threshold, suggesting the absence of MYD88 mutation. The patient underwent radiation therapy accordingly.Conclusions: CSF MYD88 mutation analysis by dPCR may have clinical utility and requires sufficient amount of CSF-DNA for exclusion of noise signals.

Detection of MYD88 L265P Mutation By Next Generation Deep Sequencing in Peripheral Blood Mononuclear Cells of Waldenstrom Macroguloblinemia and IgM Monoclonal Gammopathy of Undetermined Significance

Introduction: Whole genome sequencing has recently revealed MYD88 L265P somatic mutations in Waldenstrom macroguloblinemia (WM) tumor cells. Allele-specific polymerase chain reaction (AS-PCR) using bone marrow (BM) aspirate and peripheral blood (PB) is the most frequently used technique to detect the specific mutation. In search for a non-invasive, simple, and more sensitive detection of MYD88 L265P mutation, we performed next generation deep sequencing (NGS) technique using unselected peripheral blood mononuclear cells (PBMCs) collected from WM and IgM monoclonal gammopathy of undetermined significance (IgM MGUS) patients. The main purpose of this study is to assess the clinical implication of MYD88 L265P mutation burden detected by NGS technique using unselected PBMCs.Patients and methods: The study was approved from the Institutional Review Board. After informed consent was obtained in accordance with the Declaration of Helsinki, PBMCs were collected from 38 patients with WM and 20 patients with IgM MGUS. The median time from diagnosis to PBMC collection was 2.2 years (range, 0-25 years). Twenty-nine (76.3%) of WM patients were previously treated. The overall response (CR+PR+MR) rate at the time of analysis was 75.8% (22/29). Genomic DNA was extracted from unselected PBMCs and 300 ng of DNA was used for NGS analysis. Variation rates of MYD88 at the nucleotide position corresponding to L265P were measured with an Illumina MiSeq DNA sequencer by molecular barcoding technology essentially as previously reported (Nature Protocols 12, 664:2017). PCR products were sequenced on a MiSeq DNA sequencer with a Reagent kit v2. for 300 cycles. Serial dilution assessment was carried out to investigate the sensitivity of the method by analyzing the DNA of BM sample from a MYD88 mutation positive patient diluted in DNA from a healthy control.Results: Dilution assessment demonstrated that NGS technique was detectable to a sensitivity of 0.02%, ×5 higher than previously reported sensitivity by AS-PCR. MYD88 L265P mutation was detected from 6/9 (66.7%), 8/29 (27.6%), and 11/20 (55%) in patients with untreated WM, previously treated WM, and IgM MGUS, respectively. Among the patients who were positive forMYD88 L265P mutations, the median percentage of mutant allele relative to WT was 3.0% (0.14-32.3%), 0.24% (0.02-33.8%), and 0.55% (0.06-2.85%), in untreated WM, previously treated WM, and IgM MGUS, respectively. Patients with Untreated WM and those with lymphadenopathy showed a trend toward higher mutant allele burden in PBMCs, but the difference was not statistically significant. Among 26 previously treated WM patients, those with at least minimal response showed significantly lower detection rate of MYD88 L265P mutation (13.6 vs. 75.0%, P=0.02) and mutant allele burden (median, 0.00% vs. 0.23%, P=0.01), compared to patients with less than minimal response.Conclusion: This study is the first to report NGS analysis of MYD88 L265P mutation using unselected PBMCs of WM and IgM MGUS patients. The sensitivity for MYD88 L265P mutation in unselected PBMCs from IgM MGUS patients was comparable with that detected by AS-PCR using CD19-selected PB cells (Leukemia, 2014;28:1698-704). Although its specificity is in need for further investigations, NGS technique successfully detected the mutation carried by minimal tumor cells included in unselected PBMCs with high sensitivity as AS-PCR using CD19-selected PB cells. The mutation detection rate of previously treated WM patients was lower than that of untreated patients in our study and previously reported cohort; however, this in turn may potentially indicate that NGS detection of MYD88 L265P mutation in PBMC be clinically utilized in assessing treatment response by monitoring tumor burden during patients' clinical course. DisclosuresNo relevant conflicts of interest to declare.

Serial Detection of MYD88 L265P Mutation in the Aqueous Humor of a Patient with Vitreoretinal Lymphoma for Disease Monitoring

ABSTRACT Purpose To report a case of a patient whose MYD88 mutation disappeared from the aqueous humor following treatment with intravitreal methotrexate. Methods A retrospective review of clinical, histopathological and imaging records. Results A 49-year-old woman presented with bilateral primary vitreoretinal lymphoma confirmed by molecular and next-generation sequencing studies on vitreous biopsy samples. Initially, the MYD88 L265P mutation was detected in aqueous samples of both eyes. Serial testing for MYD88 L265P mutations performed on aqueous samples collected at the time of the weekly intravitreal methotrexate injections showed the mutation ceased to be detected after four weekly injections in the non-vitrectomized right eye and after two weekly injections in the vitrectomized left eye. Clinical improvement accompanied the negativization of the mutation in both eyes. Conclusion We present a case that demonstrates the possible utilization of serial testing for the MYD88 L265P mutation as a tool for monitoring disease course in vitreoretinal lymphoma.

Detection of MYD88 L265P mutation by next-generation deep sequencing in peripheral blood mononuclear cells of Waldenström's macroglobulinemia and IgM monoclonal gammopathy of undetermined significance.

We investigated the feasibility of using next-generation sequencing (NGS) technique using molecular barcoding technology to detect MYD88 L265P mutation in unselected peripheral blood mononuclear cells (PBMCs) in 52 patients with Waldenström’s macroglobulinemia [1] and 21 patients with IgM-monoclonal gammopathy of undetermined significance (MGUS). The NGS technique successfully detected the MYD88 L265P in unselected PBMCs at a sensitivity of 0.02%, which was ×5 higher than that of AS-PCR. All the results between paired BM and PB samples from 2 IgM MGUS and 4 untreated WM patients matched completely. MYD88 L265P mutation was detected in 14/21 (66.7%), 14/19 (73.7%), and 10/33 (30.3%) with the median mutant allele burden of 0.36% (range, 0.06–2.85%), 0.48% (range, 0.02–32.3%), and 0.16% (range, 0.02–33.8%), in IgM-MGUS, untreated WM, and previously treated WM, respectively. Multiple linear regression analysis identified an absolute peripheral lymphocyte count as the positive predictor of PB mutant allele burden (R2 = 0,72, P<0.0001). Our non-invasive, simple NGS method has the potential to detect MYD88 L265P mutations in PBMCs of IgM MGUS and WM patients, which may especially utilized for monitoring minimal residual tumor burden after treatment.

PS1325 IDENTIFICATION AND TARGETING OF MOLECULAR SIGNATURES OF HYPOMETHYLATING AGENT RESISTANCE IN MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA

Background: Waldenström macroglobulinemia (WM) is a subset of lymphoplasmacytic lymphoma (LPL) with IgM paraprotein. Genetic hallmark of WM is MYD88 L265P mutation which is important for its diagnosis and pathophysiology of this disease. Other genetic changes including CXCR4 mutation, which is associated with WHIM syndrome, are reported to be common in WM. Whereas comprehensive information on genetic changes of LPL with other paraprotein like IgG is scarce. Formalin-fixed paraffin-embedded (FFPE) samples are available for genomic studies, but it contains abundant non-tumor cells. Aims: In this report, we aim to evaluate, first, genetic difference between WM and LPL with IgG paraprotein (IgG type) using targeted next-generation sequencing (NGS) from FFPE DNA and second, the usefulness of allele-specific oligonucleotide (ASO) PCR and peptide nucleic acid (PNA) PCR clamp method compared with next-generation sequencing. Methods: Twenty patients with pathologically confirmed LPL (10 of WMs and 10 of IgG type LPLs) were subjected to this study after informed consent. There were include 8 DNA extracted from bone marrow (BM) FFPE samples and 12 DNA extracted from BM cells. MYD88 L265P was detected by ASO-PCR. CXCR4 mutations were detected by PNA PCR clamp method with 18-mer PNA, which enables to detect the most frequently mutated amino acid: S338. Using the amplification products from that, detected mutations were confirmed by Sanger sequencing. We also tried detecting CXCR4 mutations by only Sanger sequencing. The mutations in 12 genes were detected by targeted sequencing on coding sequence with NGS. Mann-Whitney U test and Chi-squared test were used for statistical analysis. This study was approved by the research ethics committee of Gunma University Hospital. Results: Using NGS, we found the somatic mutations in WMs and the IgG type LPLs: MYD88 (4 vs. 5), CXCR4 (3 vs. 2), NOTCH2 (0 vs. 1), ARID1A (9 vs. 8), RAG2 (5 vs. 5), KMT2D (10 vs. 10), TP53 (0 vs. 0), MYBBP1A (0 vs. 1), CD79B (0 vs. 0), PRDM1 (0 vs. 1), CD274 (0 vs. 0), PDCD1LG2 (2 vs. 0); there were no statistical differences, suggesting genetic similarity between these two subsets of LPL, IgM type and IgG type. MYD88 L265P was found in 9/10 (90%) of WM cases and 6/10 (60%) of the IgG type by ASO-PCR (p = 0.40); However, it was found only in 4/10 (40%) of WM cases and 5/10 (50%) of the IgG type by NGS (p = 0.05). In addition, A260 V was found in 1 of IgG type LPL case. All MYD88 L265P mutations detected by NGS were also detected by ASO-PCR. CXCR4 mutations were found in 1/9 (11%) of WM cases (T318 frameshift) and 2/9 (22%) of IgG type (T318 frameshift and S338∗) by PNA PCR clamp method (p = 0.22). Whereas, these were found in 3/10 (30%) of WM cases and 2/10 (20%) of IgG type by NGS (p = 0.67). NGS enabled to detect the three CXCR4 mutations detected by PNA PCR clamp method and the mutations in outside of target area of PNA (E345 frameshift and L305F). Summary/Conclusion: Our NGS analysis suggest genetic similarity between two subsets of LPL, WM and IgG type. ASO-PCR is superior sensitive to NGS in detection of MYD88 L265P mutation, and PNA PCR clamp method enabled to detect the CXCR4 mutations in FFPE samples even if they were the frameshift mutations. Meanwhile, NGS analysis uncovered mutations other than ASO-PCR and PNA PCR clamp method targets.

Bing Neel Syndrome in a Previously Untreated Patient With Waldenström's Macroglobulinemia: Contribution of MYD88 L265P Mutation on Cerebrospinal Fluid

Bing Neel Syndrome (BNS) is defined as direct central nervous system involvement of Waldenstrom’s macroglobulinemia. BNS is usually a late event, although an incidence of 30% to 36% has been described in large series of previously untreated patients. A wide variety of clinical manifestations and radiologic findings for BNS have been reported in published data, depending on the site and type of infiltration. In addition to the radiologic findings, the diagnostic approach includes lymphoplasmacytic cell quantitation and flow cytometric analysis of the cerebrospinal fluid (CSF). Recently, the evaluation of MYD88 L265P mutation in the CSF has been proposed as a possible diagnostic biomarker for BNS. We describe the case of a 58-year-old patient with BNS. The detection of MYD88 L265P mutation in the CSF contributed to the diagnosis and to the sequential monitoring of minimal residual disease. In the future, the use of CSF sequential molecular monitoring could play an important role in treatment decisions.

MYD88 L265P and CXCR4 mutations in lymphoplasmacytic lymphoma identify cases with high disease activity.

Recurrent mutations in MYD88 have been identified in >90% of lymphoplasmacytic lymphoma (LPL). Recently, WHIM (warts, hypogammaglobulinaemia, infections, myelokathexis) syndrome-like mutations in CXCR4 have been described in 28% of LPL cases, and seem to impact clinical presentation and response to therapy. We investigated the presence of the MYD88 L265P mutation in 90 decalcified, formalin-fixed, paraffin-embedded (FFPE) bone marrow (BM) biopsies, including 51 cases of LPL, 14 cases of B-cell chronic lymphocytic leukaemia (CLL), 13 cases of marginal zone lymphoma (MZL) and 12 normal controls. In addition, the C-terminal domain of CXCR4 was sequenced in LPL cases. MYD88 L265P was found in 49/51 (96%) LPL cases and in 1/13 (7·6%) MZL (splenic type), whereas all CLL samples remained negative. The two MYD88 wild type LPL cases were associated with cold agglutinin disease. Mutations in CXCR4 were detected in 17/47 (36·2%) LPL cases, which showed a higher extent of BM infiltration and lower leucocyte counts (P=0·02), haemoglobin (P=0·05) and platelet counts (P=0·01). In conclusion the detection of MYD88 L265P mutation in FFPE samples is reliable and useful for subtyping small B-cell lymphomas in BM biopsies. In addition, the presence of CXCR4 mutations identifies a subgroup of LPL patients with higher disease activity.

Development of high-resolution melting analysis for the detection of the MYD88 L265P mutation

ObjectiveRecurrent L265P mutation of myeloid differentiation primary response gene 88 (MYD88) has been identified in a proportion of diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia. The present study aimed to establish a rapid, sensitive, and reliable method using high-resolution melting analysis (HRMA) to detect MYD88 L265P mutation in DLBCL. Designs and methodsThe sensitivity of HRMA in the detection of MYD88 L265P mutation was evaluated. MYD88 L265P mutation was further screened in 120 patients with DLBCL. The results of HRMA were validated by direct DNA sequencing. ResultsFor the detection of MYD88 L265P mutation, the reproducible maximal sensitivity of HRMA was 5% higher than that obtained by direct DNA sequencing (25%). Heterozygous MYD88 L265P mutations were identified in 11 (9.2%) DLBCL cases, all of which were diagnosed as non-germinal-center B cell (non-GCB) DLBCL. ConclusionsThe HRMA assay is a rapid, sensitive, reliable, and high-throughput method to screen MYD88 L265P mutation and could be used in clinical diagnostic laboratories.