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.

NKX3.1 Expression in Salivary Gland "Intraductal" Papillary Mucinous Neoplasm: A Low-Grade Subtype of Salivary Gland Mucinous Adenocarcinoma.

BACKGROUND: Salivary gland intraductal papillary mucinous neoplasm (SG IPMN) is a recently proposed entity characterized by a papillary-cystic proliferation of mucin-producing cells. Because of overlapping histologic features and a clonal AKT1 p.E17K variant, SG IPMN has been presumed to be a precursor or a low-grade subtype of mucinous adenocarcinoma. NKX3.1 is a tumor suppressor gene located on chromosome 8p and is a known immunohistochemical marker of prostate epithelium and mucinous acinar cells of the intraoral salivary glands. METHODS: We retrieved 12 SG IPMN cases, and performed histologic and genetic analysis. Given the association of SG IPMN with mucinous acinar cells, we also investigated the performance of NKX3.1 as a marker of this tumor entity. RESULTS: Diffuse and strong NKX3.1 expression was observed in all SG IPMN cases (12/12, 100%) as well as in normal mucinous acinar cells. In contrast, mucoepidermoid carcinoma and pancreatic IPMN cases as well as normal serous acinar cells were negative for NKX3.1. Genetically, 11 of 12 cases (92%) harbored an AKT1 p.E17K variant. A novel PTEN frameshift deletion (p.G36Dfs*18) was detected in the other single case. At least one of the histologic features implying malignant tumors, such as severe cellular atypia, brisk mitotic activity, high Ki-67 proliferating index, lymphovascular invasion, and lymph node metastasis, was detected in 6 SG IPMN cases (50%). CONCLUSION: The findings suggest that SG IPMN is a low-grade subtype of mucinous adenocarcinoma which may be derived from mucinous acinar cells of the minor salivary gland.

Comparison of clonal architecture between primary and immunodeficient mouse-engrafted acute myeloid leukemia cells.

Patient-derived xenografts (PDX) are widely used as human cancer models. Previous studies demonstrated clonal discordance between PDX and primary cells. However, in acute myeloid leukemia (AML)-PDX models, the significance of the clonal dynamics occurring in PDX remains unclear. By evaluating changes in the variant allele frequencies (VAF) of somatic mutations in serial samples of paired primary AML and their PDX bone marrow cells, we identify the skewing engraftment of relapsed or refractory (R/R) AML clones in 57% of PDX models generated from multiclonal AML cells at diagnosis, even if R/R clones are minor at <5% of VAF in patients. The event-free survival rate of patients whose AML cells successfully engraft in PDX models is consistently lower than that of patients with engraftment failure. We herein demonstrate that primary AML cells including potentially chemotherapy-resistant clones dominantly engraft in AML-PDX models and they enrich pre-existing treatment-resistant subclones.

Next-generation genome sequencing of a matched normal-tumor pair from a patient with intractable gestational choriocarcinoma: A case report.

Gestational choriocarcinoma is a gestational trophoblastic neoplasia (GTN) originating from trophoblastic cells with abnormal proliferation. Although chemotherapy is effective for treating this cancer, when patients develop chemoresistance, personalized treatment, such as the use of drugs matching their genomes, is required. The present report describes a case of intractable gestational choriocarcinoma identified using a next-generation sequencing (NGS)-based tumor panel. A 51-year-old woman was diagnosed with gestational choriocarcinoma via pathological and short tandem repeat analyses. The patient did not achieve remission despite many regimens of chemotherapy, including high-dose therapy with autologous peripheral blood stem cell transplantation. To identify drugs tailored to this particular choriocarcinoma, NGS was performed on the tumor of the patient, and the tumor genome was compared with that of the patient's blood sample using the NCC Oncopanel System. Consequently, 245 single nucleotide variants (SNVs) with a mean SNV allele frequency of 63.1% were identified. This high frequency was because the genome of the gestational choriocarcinoma contained part of the genome of the partner. Therefore, our experience of the present intractable case of choriocarcinoma suggested that matched normal-tumor pair analysis is not appropriate for treatment decisions in GTN cases. When using an NGS-based tumor panel to assess choriocarcinoma, researchers must consider whether the genomic DNA of the patient and their partner are involved in the GTN.

Identification of the novel deletion-type PML-RARA mutation associated with the retinoic acid resistance in acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) and arsenic trioxide (ATO) are essential for acute promyelocytic leukemia (APL) treatment. It has been reported that mutations in PML-RARA confer resistance to ATRA and ATO, and are associated with poor prognosis. Although most PML-RARA mutations were point mutations, we identified a novel seven amino acid deletion mutation (p.K227_T233del) in the RARA region of PML-RARA in a refractory APL patient. Here, we analyzed the evolution of the mutated clone and demonstrated the resistance of the mutated clone to retinoic acid (RA). Mutation analysis of PML-RARA was performed using samples from a chemotherapy- and ATRA-resistant APL patient, and the frequencies of mutated PML-RARA transcript were analyzed by targeted deep sequencing. To clarify the biological significance of the identified PML-RARA mutations, we analyzed the ATRA-induced differentiation and PML nuclear body formation in mutant PML-RARA-transduced HL-60 cells. At molecular relapse, the p.K227_T233del deletion and the p.R217S point-mutation in the RARA region of PML-RARA were identified, and their frequencies increased after re-induction therapy with another type of retinoiec acid (RA), tamibarotene. In deletion PML-RARA-transduced cells, the CD11b expression levels and NBT reducing ability were significantly decreased compared with control cells and the formation of PML nuclear bodies was rarely observed after RA treatment. These results indicate that this deletion mutation was closely associated with the disease progression during RA treatment.

Mutation analysis of therapy-related myeloid neoplasms.

We analyzed the genetic mutation status of 13 patients with therapy-related myeloid neoplasms (t-MN). Consistent with previous reports, t-MN cells preferentially acquired mutations in TP53 and epigenetic modifying genes, instead of mutations in tyrosine kinase and spliceosome genes. Furthermore, we compared the mutation status of three t-MN cells with each of the initial lymphoid malignant cells, and identified common mutations among t-MN and the initial malignant cells in two patients. In a patient who developed chronic myelomonocytic leukemia (CMML) after follicular lymphoma (FL), TET2 mutation was identified in both CMML and FL cells. Notably, the TET2 mutation was also identified in peripheral blood cells in the disease-free period with the same allelic frequency as CMML and FL cells, but not in a germ-line control, indicating that the TET2 mutation occurred somatically in the initiating clone for both malignant cells. On the other hand, a germ-line MYB mutation was identified in a patient who developed myelodysplastic syndromes (MDS) after FL. These results suggest that germ-line deposition and clonal hematopoiesis are closely associated with t-MN susceptibility; however, further analysis is necessary to clarify the mechanism required to provide the initiating clone with lineage commitment and clonal expansion.

[MonoMAC syndrome patient developing myelodysplastic syndrome following persistent EBV infection].

An 18-year-old man was diagnosed with Epstein-Barr virus (EBV) -associated hemophagocytic syndrome (HPS) and treated with prednisolone (PSL) at a previous hospital. During PSL tapering, the HPS symptoms reappeared, and the patient was referred to our hospital. Increased PSL improved the symptoms, but the EBV infection remained unresolved. At age 20, he was admitted to our hospital for newly developed pneumonia and diagnosed with myelodysplastic syndrome (refractory cytopenia with multilineage dysplasia) (MDS-RCMD; normal karyotype, IPSS: Int-1) by bone marrow examination. MDS remission was achieved following bone marrow transplantation from an unrelated donor, and he is currently alive without relapse. The patient's father had also been diagnosed with MDS when he was young and died from leukoencephalopathy at approximately 50 years old. These observations support a diagnosis of familial MDS. GATA2 mutation p.R230Hfs*44 was identified in both bone marrow and control cells (buccal swab) at MDS diagnosis, and he was diagnosed with monocytopenia and mycobacterial infection (MonoMAC) syndrome. Furthermore, an acquired STAG2 mutation (splicing site change, c.820-2A>G) in the bone marrow cells was also identified, which might contribute to MDS progression.

Recurrent alterations of the WW domain containing oxidoreductase gene spanning the common fragile site FRA16D in multiple myeloma and monoclonal gammopathy of undetermined significance.

The putative tumor suppressor gene WW domain containing oxidoreductase (WWOX) spans a common fragile site (CFS) on chromosome 16q23.3. CFSs are regions of profound genomic instability and sites for genomic deletions in cancer cells. Therefore, WWOX is structurally altered in diverse nonhematological cancer types. However, the function of WWOX in hematological tumor types, including multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS) remains unclear. WWOX expression and methylation in patients with MM, MGUS, or noninvasive lymphoma (control) were analyzed using reverse transcription- and methylation specific-polymerase chain reaction analysis. Variant WWOX transcripts were detected in 65 and 50% of patients with MM and MGUS, respectively, compared with 10% of controls. WWOX expression was higher in patients with MM, and WWOX promoter methylation was detected in 35% of patients with MM compared with 5% of patients with MGUS and 4% of controls. WWOX promoter methylation was significantly associated with shorter overall survival time of patients, in particular those with MM who were never treated with novel agents. Genomic alterations, including deletions and promoter methylation that affect WWOX expression occur early and may be involved in the pathogenesis, progression, and prognosis of MM.

Long non-coding RNA MALAT1 is an inducible stress response gene associated with extramedullary spread and poor prognosis of multiple myeloma.

Extramedullary myeloma (EMM) occurs when myeloma develops outside the bone marrow; it often develops after chemotherapy and is associated with the acquisition of chemo-resistance and a fatal course. The mechanisms underlying extramedullary spread have not yet been fully elucidated. MALAT1 is a highly abundantly and ubiquitously expressed long non-coding RNA that plays important roles in cancer metastasis. The aims of this study were to clarify the association of MALAT1 with EMM and to elucidate the underlying mechanism of EMM formation under chemotherapeutic pressure. MALAT1 expression was significantly higher in multiple myeloma (MM) than in monoclonal gammopathy of undetermined significance. Furthermore, MALAT1 expression was markedly higher in EMM compared with that in corresponding intramedullary myeloma cells. A higher MALAT1 level was associated with shorter overall and progression-free survival. MALAT1 expression level was positively correlated with expression of HSP90AA1, HSP90AB1 and HSP90B1 but not with TP53 expression. MALAT1 was significantly upregulated by bortezomib and doxorubicin. Considering the known functions of MALAT1, our results suggest that it acts as a stress response gene that is upregulated by chemotherapy, thereby linking chemotherapy to EMM formation. Elucidating the biological implication of long non-coding RNA contributes to deeper understanding concerning the pathogenesis and investigation of novel therapeutic targets for MM.

[Biomarkers of Leukemia ~Description of the Practical Use and Operation in the Clinical Laboratory~]-.

Genetic testing of hematological malignancy is-.indispensable to categorize and diagnose leukemia. The quantitation of fusion gene mRNA built up by chromosomal translocation including BCR-ABL1 (major, minor), RUNX1-RUNX1T1, and PML-RARA and detection of the JAK2 (V617F) mutant gene are performed by our- selves in our hospital. Efficient, practical use is necessary because the number of medical technologists is limited and numbers of tests are increasing annually. The detection of leukemic cells is important in hematological tests. In addition, experienced medical technologists can predict the existence of fusion mutant genes. In this report, we introduce our experience regarding the practical use and operation of biomarkers for leukemia. Medical technologists take advantage of peripheral blood tests for screening, such as the complete blood count (CBC), hemogram, fibrin and fibrinogen degradation products (FDP), and the quantitation of fusion gene mRNA, which offers a definitive diagnosis including BCR-ABL1, RUNX1-RUNX1T1, and PML-RARA, and genetic tests are performed efficiently. [Review].

A new staging system to predict prognosis of patients with multiple myeloma in an era of novel therapeutic agents.

Various prognostic markers for multiple myeloma (MM) have been identified, and stratification using these markers is considered important to optimize treatment strategies. The international staging system (ISS) is now a widely accepted prognostic staging system for MM patients; however, its validity is controversial in the era of new therapeutic regimens, since ISS had been established before introduction of new agents. We retrospectively reviewed prognostic factors in order to seek out an alternative staging system more suitably applied to MM patients treated with novel agents. We analyzed 178 newly diagnosed MM patients who received either conventional chemotherapy without novel agents (CT; n = 79) or chemotherapy with novel agents (NT; n = 99). Although median overall survival (OS) of patients treated with CT is significantly different depending on stages of ISS, ISS had no effect on OS among patients treated with NT. Meanwhile, we identified hemoglobin (Hb) and plasmacytoma as independent risk factors for OS in patients who received NT. Using these two parameters, we stratified NT patients into three stages; stage 1 (Hb≥10 g/dL and absence of plasmacytoma), stage 2 (not stage 1 or 3), and stage 3 (Hb <10 g/dL and presence of plasmacytoma). We found that there were significant differences in median OS among the three stages (8.13, 5.95, and 2.45 yr for stages 1, 2, and 3, respectively). This preliminary study suggests that this alternative staging system based on Hb and plasmacytoma is a simple and useful way to predict prognosis of MM patients in the novel agent era.

DNMT3B7 expression related to MENT expression and its promoter methylation in human lymphomas.

DNA methyltransferase (DNMT) 3B7 is the most expressed DNMT3B splice variant. It was reported that the loss of DNMT3B function led to overexpression of the MEthylated in Normal Thymocyes (MENT) and accelerated mouse lymphomagenesis. We investigated the DNMT3B7 expression and its relationship to MENT expression and promoter methylation in human lymphomas. DNMT3B7 and MENT expression were significantly (p<0.0001, p<0.01) higher in lymphomas than in non-malignant. Expression of DNMT3B7 and MENT were associated with MENT promoter hypomethylation. DNMT3B7 overexpression might interfere with the normal DNA methylation mechanism required for silencing the MENT proto-oncogene, and may accelerate human lymphomagenesis.