Keiichiro Hattori

MYD88 (L265P) mutation is associated with an unfavourable outcome of primary central nervous system lymphoma.

Primary central nervous system lymphomas (PCNSL) are rare forms of diffuse large B cell lymphoma (DLBCL) that arise within the brain or the eyes. The current molecular classification of DLBCL distinguishes two main subtypes: activated B-cell-like (ABC)-lymphoma and germinal centre B-cell-like (GCB)-lymphoma (Zhang et al, 2013). PCNSL typically show an immunophenotype resembling that of ABC-DLBCL (Zhang et al, 2013). Somatic mutations in the myeloid differentiation primary response gene 88. (MYD88), CD79b molecule, immunoglobulin-associated beta (CD79B) and caspase recruitment domain family, member 11 (CARD11) genes were recently shown to control cell survival of ABC-DLBCL by promoting the nuclear factor (NF)jB signalling pathway (Zhang et al, 2013). The mutation frequencies of MYD88, CD79B and CARD11 are 15–30%, 17–23% and 8–18% in ABC-DLBCL (Zhang et al, 2013) and are reported to be 40–79%, 30~-44% and 11–30% in PCNSL, Indicating that they are strongly over-represented in PCNSL (Braggio et al, 2015; Yamada et al, 2015). In addition, several other gene mutations were more prevalent in PCNSL than in DLBCL, not otherwise specified; “pim-1 oncogene” (PIM1: 20–44%), “transducin (beta)-like 1 X-linked receptor 1” (TBL1XR1: 7–23%), “BTG family, member 2” (BTG2: 22– 30%), “PR domain containing 1, with ZNF domain” (PRDM1: 7–20%) and “tumor necrosis factor, alpha-induced protein 3” (TNFAIP3: 11–20%) (Braggio et al, 2015). Nonetheless, the clinical relevance of these mutations remains unclear, although several specific clinical parameters may predict the treatment outcome of PCNSL patients. These include altered mental activity and elevated creatinine clearance (CrCl) (Taoka et al, 2010); age >50 years and Karnofsky performance score <70 (Memorial Sloan-Kettering Cancer Center prognostic score; Abrey et al, 2006); age >75 years, increased serum lactate dehydrogenase level, cerebrospinal fluid protein concentration and lymphoma involvement of deep brain structures (International Extranodal Lymphoma Study Group prognostic scoring system; Abrey et al, 2006), were identified as prognostic factors. However, recent advances in genetic knowledge are not incorporated in any of these proposals. Hence, we conducted this study to elucidate the impact of gene mutations on clinical outcome of PCNSL. We analysed 42 human immunodeficiency virus (HIV)negative PCNSL patients who were treated with a modified version of the European Organization for Research and Treatment of Cancer protocol (Taoka et al, 2010) at the University of Tsukuba Hospital between March 2005 and May 2015. This protocol consisted of intermediate-dose methotrexate (MTX), lomustine, procarbazine, methylprednisolone and intrathecal chemotherapy with MTX and cytarabine (Table SI) (Taoka et al, 2010). All PCNSL patients in this study had DLBCL. GCB and ABC phenotypes were assessed by immunohistochemistory using anti-CD10, MUM1 and BCL6 antibodies. Targeted deep sequencing was performed by using Ion Ampliseq technology (Methods S1, Table SII). Clinical parameters were collected from the clinical record (Methods S1). Overall survival (OS) and progression-free survival (PFS) were estimated using the KaplanMeier method and compared by the Peto-Peto generalized Wilcoxon test. All data were analysed with EZR (http://www. jichi.ac.jp/saitama-sct/SaitamaHP.files/statmedEN.html). Multivariate analysis was performed with Cox regression model. Significance was set at P ≤ 0 05. The major clinical characteristics of the 42 patients are summarized in Table SIII. At least one mutation was detected in 38 out of 42 cases (90 4%). Notably, we found recurrent mutations in MYD88 (67%), CD79B (43%), PIM1 (69%), TBL1XR1 (24%), BTG2 (29%), PRDM1 (24%) and TNFAIP3 (12%) (Table SIV, Fig. S1) at frequencies similar to those previously reported (Braggio et al, 2015; Yamada et al, 2015). For the entire cohort, the median follow-up time was 26 months (range, 1–105 months), with 3-years OS and PFS of 46% and 26%, respectively, comparable to the survival rate of our previous cohort treated with the same protocol (Taoka et al, 2010). Univariate analysis showed that age >75 years (P = 0 0105) and altered mental activity (P = 0 0202) (Table SV) were significantly associated with inferior OS. In addition, MYD88 L265P mutation (P = 0 127, Fig. 1A), CrCl >90 ml/min (P = 0 124) and deep brain involvement (P = 0 0649) showed a tendency to be associated with inferior OS, although statistically non-significant (Table SV). When adjusted in a multivariate Cox regression analysis, MYD88 L265P mutation remained as a significant risk factor for death (hazard ratio (HR), 2 903; 95% confidence interval (CI), 1 013–8 323, P = 0 0474), together with altered mental activity (HR, 4 729; 95% CI, 1 522–14 7, P = 0 0072) (Table I). Regarding PFS, CrCl >90 ml/min (P = 0 0286) and altered mental activity (P = 0 0473) were significant factors for inferior PFS (Table SV). MYD88 L265P mutation (P = 0 0872, Fig. 1B) was not significantly associated, but showed a tendency to be

BCL6 locus is hypermethylated in angioimmunoblastic T-cell lymphoma.

BCL6, a master transcription factor for differentiation of follicular helper T (TFH) cells, is highly expressed in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphomas (PTCL) containing tumor cells with TFH features. TET2, encoding an epigenetic regulator, is frequently mutated in AITL/PTCL. We previously reported that Tet2 knockdown mice developed T-cell lymphomas with TFH features. Hypermethylation of the Bcl6 locus followed by BCL6 upregulation was thought to be the key event for lymphoma development in mice. The mechanisms by which BCL6 expression is upregulated in human AITL/PTCL, however, have not been elucidated. Here, we investigated the impact of TET2 mutations on methylation of BCL6 locus in human AITL/PTCL samples. Hypermethylation of the BCL6 locus was more frequent in PTCL samples than B-cell lymphoma samples (PTCL vs B-cell lymphomas: 9/42 vs 0/35). PTCL samples with TET2 mutations were more frequently hypermethylated than those without TET2 mutations (PTCL with TET2 mutations vs without mutations: 6/22 vs 0/17). BCL6 expression in hypermethylated samples was higher than that in low methylated samples. Deregulated BCL6 expression caused by hypermethylation and TET2 mutations may result in skewed TFH differentiation and eventually contribute to AITL/PTCL development in patients, as well as lymphoma development in Tet2 knockdown mice.

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.

Detection of the circulating tumor DNAs in angioimmunoblastic T- cell lymphoma

Recent genetic studies identified that the disease-specific G17V RHOA mutation, together with mutations in TET2, DNMT3A, and IDH2, is a hallmark of angioimmunoblastic T cell lymphomas (AITL). The diagnostic value of these mutations is now being investigated. Circulating tumor DNAs (ctDNAs) may offer a non-invasive testing for diagnosis and disease monitoring of cancers. To investigate whether these mutations are useful markers for ctDNAs in AITL and its related lymphomas, we performed targeted sequencing for TET2, RHOA, DNMT3A, and IDH2 in paired tumors and cell-free DNAs from 14 patients at diagnosis. Eighty-three percent of mutations detected in tumors were also observed in cell-free DNAs. During the disease course, mutations were detectable in cell-free DNAs in a refractory case, while they disappeared in a chemosensitive case. These data suggest that the disease-specific gene mutations serve as sensitive indicators for ctDNAs and may also be applicable for non-invasive monitoring of minimal residual diseases in AITL.

Blastic plasmacytoid dendritic cell neoplasm arising from clonal hematopoiesis

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare subtype of myeloid neoplasm. Clonal evolution in the development of BPDCN remains to be elucidated. In the present study, we examined clonal evolution in a case of BPDCN by analyzing the distribution of gene mutations in tumor cells and non-tumor blood cells. The p.D1129fs and p.K1005fs TET2 mutations, p.P95H SRSF2 mutation, and p.L287fs NPM1 mutation were identified in a skin tumor at diagnosis and peripheral blood mononuclear cells at relapse. Notably, the p.D1129fs TET2 and p.L287fs NPM1 mutations were observed only in tumor cells, while the p.K1005fs TET2 and p.P95H SRSF2 mutations were found in both tumor cells and non-tumor blood cells. Recent genetic studies have suggested that some blood cancers may originate from clonal hematopoiesis, harboring somatic mutations. In the present case, the data suggest that BPDCN originated from clonal hematopoiesis with the p.K1005fs TET2 and p.P95H SRSF2 mutations via acquisition of the additional p.D1129fs TET2 and p.L287fs NPM1 mutations.

Liquid biopsy for the identification of intravascular large B-cell lymphoma

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MyD88 Mutation in Elderly Predicts Poor Prognosis in Primary Central Nervous System Lymphoma: Multi-Institutional Analysis

BACKGROUNDnRecent genetic analysis of primary central nervous system lymphoma (PCNSL) showed that the MyD88 L265P mutation, which is related to NF-κB signaling, was a genetic hallmark for PCNSL; thus it could serve as a genetic marker for diagnosis and a potential target for molecular therapy. However, the role of the MyD88 mutation in PCNSL has not been defined. In this study, we investigated the role of the MyD88 mutation and clinical features of PCNSL-treated patients at several institutions to determine its significance as a prognostic factor.nnnMETHODSnForty-one PCNSL (diffuse large B-cell type) patients from 8 institutions were included in this study. Their median age was 68 years; median follow-up was 26.7 months; median overall survival was 26.7 months; and their 1-year, 3-year, and 5-year survival rates were 75.6%, 58.5%, and 43.9%, respectively. Deoxyribonucleic acid was extracted from frozen tissue, and the MyD88 L265P mutation was evaluated by polymerase chain reaction and direct sequencing.nnnRESULTSnThe MyD88 L265P mutation was found in 61.0% (25/41) of cases. Kaplan-Meier analysis revealed that neither MyD88 L265P mutation nor age >65 years alone significantly predicted overall survival relative to MyD88 wild type and age <65. The MyD88 L265P mutation was predominantly present in patients aged >65 years. Among age >65 patients, the MyD88 L265P mutation portended a worse overall survival compared with the MyD88 wild type (11.5 vs. 56.2 months P < 0.04).nnnCONCLUSIONnThe MyD88 L265P mutation predicted a poor prognosis in elderly PCNSL patients. A new tailor-made treatment strategy might be needed for these patients.

A single institutional retrospective evaluation for younger patients with primary central nervous lymphomas on a modified R-MPV regimen followed by radiotherapy and high dose cytarabine

We conducted a retrospective analysis of patients younger than 60 years (N = 10, median age 54.5) with newly diagnosed primary central nervous system lymphoma (PCNSL) at the University of Tsukuba Hospital from January 2008 to November 2016. All the patients were scheduled to receive a single regimen without registration to any clinical trials. This was based on a phase 2 study by Memorial Sloan-Kettering Cancer Center (MSKCC); induction chemotherapy with rituximab, methotrexate, procarbazine, and vincristine (R-MPV) (five to seven cycles), followed by whole-brain radiotherapy (rd-WBRT) (23.4 Gy) and two high-dose cytarabine (HD-AC) cycles as a consolidation. The median age was 54.5 years, and median follow up duration was 33.1 months. The 3-year overall survival (OS) and progression-free survival (PFS) were 69% (95% CI 31-89%) and 56% (95% CI 20-81%). The median OS and PFS were not reached, respectively. Acute and delayed toxicities were manageable. In particular, OS and PFS of seven patients who achieved CR by the R-MPV induction chemotherapy were significantly superb (3-year OS, 100%; 3-year PFS, 80%), implying that a large proportion of patients in CR after the completion of this treatment may achieve durable disease control. On the other hand, all of the three patients who had progressive disease during this treatment died of disease progression within 1 year after diagnosis without achieving CR. Identifying the patients having a risk of failure in the R-MPV induction chemotherapy is important, and may allow us to consider a potentially more effective regimen.

Random Skin Biopsies Before Brain Biopsy for Intravascular Large B-Cell Lymphoma

OBJECTIVEnTo evaluate effectiveness of random skin biopsies for intravascular large B-cell lymphoma (IVLBCL) with or without central nervous system (CNS) involvement.nnnMETHODSnData from 21 patients with suspected IVLBCL (7 with CNS involvement and 14 without CNS involvement) who underwent single (4 patients), double (1 patient), and random (16 patients) skin biopsies were retrospectively analyzed.nnnRESULTSnIVLBCL was diagnosed in 16 patients (including 6 with CNS involvement). Sensitivity, specificity, and positive predictive value of random skin biopsies were 75%, 100%, and 100%. Ratio of tumor-positive biopsy samples to plasma soluble interleukin-2 receptor (sIL-2R) values was significantly correlated in cases with data on both variables. sIL-2R values in the 6 tumor-negative skin samples (median, 1415 U/mL; range, 487-3200 U/mL) were significantly lower than in tumor-positive skin samples (median, 3550 U/mL; range, 595-8700 U/mL) with at least 1 skin specimen obtained. Mean ratio of tumor-positive biopsy samples in IVLBCL cases with low sIL-2R (<3000 U/mL) was only 45%, indicating a requirement for 3-site multiple sampling. No differences in median sIL-2 values between cases of IVLBCL with and without CNS involvement were found (2795 U/mL vs. 3550 U/mL). Steroids administered before diagnosis yielded false-negative results in 3 of 5 IVLBCL cases (all false-negative cases were IVLBCL with CNS involvement), whereas none of 11 IVLBCL cases without steroid administration yielded false-negative results.nnnCONCLUSIONSnRandom skin biopsies before brain biopsy are recommended in patients with suspected IVLBCL regardless of CNS involvement, but low sIL-2R values and steroids may yield false-negative results.

Clinical and biological significance of adamantinomatous craniopharyngioma with CTNNB1 mutation

OBJECTIVEThe Wnt/β-catenin signaling pathway is strongly implicated in the pathogenesis of adamantinomatous craniopharyngioma (adaCP). However, there is no evidence that the CTNNB1 mutation activates the target gene of Wnt/β-catenin signaling, and it is unknown whether it affects the tumorigenesis of adaCP. To assess the effect of the CTNNB1 mutation of adaCP, the authors analyzed the correlation between the mutation and clinical, radiological, pathological, and biological findings.METHODSBetween 2003 and 2015, 42 patients (24 male and 18 female, median age 42 years) with either papillary craniopharyngioma (papCP) or adaCP underwent tumor resection at the authors institution. BRAF V600E and CTNNB1 in papCP and adaCP samples were sequenced by next-generation sequencing and the Sanger method, and mRNA expression levels of Axin2 and BMP4 were evaluated by RT-PCR. Axin2, BMP4, β-catenin, and BRAF expression were evaluated by immunohistochemistry. Other data were collected from clinical reports.RESULTSThe BRAF V600E mutation was detected in all 10 cases of papCP (100%). CTNNB1 exon 3 mutations were detected in 21 of 31 (68%) cases of adaCP, excluding 1 case for which there were no available sequence data. The mRNA expression level of Axin2 was significantly higher in adaCPs with a CTNNB1 mutation than in those without (p < 0.05). The immunohistochemical findings of Axin2 and BMP4 did not correlate with CTNNB1 mutation positivity. When patients who received adjuvant radiation therapy were excluded, progression-free survival was shorter in the mutation-positive group than in the mutation-negative group (log-rank test, p = 0.031). Examination of clinical characteristics and immunohistochemical findings of adaCPs showed that there was no significant correlation between CTNNB1 mutation positivity and age, sex, tumor volume, gross-total resection, optic tract edema, calcification, or T1 signal intensity of cyst fluid on MRI, β-catenin, and MIB-1 index.CONCLUSIONSThese results raise the possibility that the CTNNB1 mutation in adaCP may be associated with disease recurrence, and genes related to the Wnt/β-catenin signaling pathway might represent a therapeutic target.