Frequent genetic alterations in immune checkpoint-related genes in intravascular large B-cell lymphoma.

Intravascular large B-cell lymphoma (IVLBCL) is a unique type of extranodal lymphoma characterized by selective growth of tumor cells in small vessels without lymphadenopathy. Greater understanding of the molecular pathogenesis of IVLBCL is hampered by the paucity of lymphoma cells in biopsy specimens, creating a limitation in obtaining sufficient tumor materials. To uncover the genetic landscape of IVLBCL, we performed whole-exome sequencing (WES) of 21 patients with IVLBCL using plasma-derived cell-free DNA (cfDNA) (n = 18), patient-derived xenograft tumors (n = 4), and tumor DNA from bone marrow (BM) mononuclear cells (n = 2). The concentration of cfDNA in IVLBCL was significantly higher than that in diffuse large B-cell lymphoma (DLBCL) (P < .0001) and healthy donors (P = .0053), allowing us to perform WES; most mutations detected in BM tumor DNA were successfully captured in cfDNA and xenograft. IVLBCL showed a high frequency of genetic lesions characteristic of activated B-cell-type DLBCL, with the former showing conspicuously higher frequencies (compared with nodal DLBCL) of mutations in MYD88 (57%), CD79B (67%), SETD1B (57%), and HLA-B (57%). We also found that 8 IVLBCL (38%) harbored rearrangements of programmed cell death 1 ligand 1 and 2 (PD-L1/PD-L2) involving the 3' untranslated region; such rearrangements are implicated in immune evasion via PD-L1/PD-L2 overexpression. Our data demonstrate the utility of cfDNA and imply important roles for immune evasion in IVLBCL pathogenesis and PD-1/PD-L1/PD-L2 blockade in therapeutics for IVLBCL.

Clinical utility of target capture-based panel sequencing in hematological malignancies: A multicenter feasibility study.

Although next-generation sequencing-based panel testing is well practiced in the field of cancer medicine for the identification of target molecules in solid tumors, the clinical utility and clinical issues surrounding panel testing in hematological malignancies have yet to be fully evaluated. We conducted a multicenter prospective clinical sequencing study to verify the feasibility of a panel test for hematological tumors, including acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma. Out of 96 eligible patients, 79 patients (82%) showed potentially actionable findings, based on the clinical sequencing assays. We identified that genetic alterations with a strong clinical significance were found at a higher frequency in terms of diagnosis (n = 60; 63%) and prognosis (n = 61; 64%) than in terms of therapy (n = 8; 8%). Three patients who harbored a germline mutation in either DDX41 (n = 2) or BRCA2 (n = 1) were provided with genetic counseling. At 6 mo after sequencing, clinical actions based on the diagnostic (n = 5) or prognostic (n = 3) findings were reported, but no patients were enrolled in a clinical trial or received targeted therapies based on the sequencing results. These results suggest that panel testing for hematological malignancies would be feasible given the availability of useful diagnostic and prognostic information. This study is registered with the UMIN Clinical Trial Registry (UMIN000029879, multiple myeloma; UMIN000031343, adult acute myeloid leukemia; UMIN000033144, diffuse large B-cell lymphoma; and UMIN000034243, childhood leukemia).

Truncated RUNX1 Generated by the Fusion of RUNX1 to Antisense GRIK2 via a Cryptic Chromosome Translocation Enhances Sensitivity to Granulocyte Colony-Stimulating Factor.

Fusions of the Runt-related transcription factor 1 (RUNX1) with different partner genes have been associated with various hematological disorders. Interestingly, the C-terminally truncated form of RUNX1 and RUNX1 fusion proteins are similarly considered important contributors to leukemogenesis. Here, we describe a 59-year-old male patient who was initially diagnosed with acute myeloid leukemia, inv(16)(p13;q22)/CBFB-MYH11 (FAB classification M4Eo). He achieved complete remission and negative CBFB-MYH11 status with daunorubicin/cytarabine combination chemotherapy but relapsed 3 years later. Cytogenetic analysis of relapsed leukemia cells revealed CBFB-MYH11 negativity and complex chromosomal abnormalities without inv(16)(p13;q22). RNA-seq identified the glutamate receptor, ionotropic, kinase 2 (GRIK2) gene on 6q16 as a novel fusion partner for RUNX1 in this case. Specifically, the fusion of RUNX1 to the GRIK2 antisense strand (RUNX1-GRIK2as) generated multiple missplicing transcripts. Because extremely low levels of wild-type GRIK2 were detected in leukemia cells, RUNX1-GRIK2as was thought to drive the pathogenesis associated with the RUNX1-GRIK2 fusion. The truncated RUNX1 generated from RUNX1-GRIK2as induced the expression of the granulocyte colony-stimulating factor (G-CSF) receptor on 32D myeloid leukemia cells and enhanced proliferation in response to G-CSF. In summary, the RUNX1-GRIK2as fusion emphasizes the importance of aberrantly truncated RUNX1 in leukemogenesis.

Pyruvate secreted from patient-derived cancer-associated fibroblasts supports survival of primary lymphoma cells.

Cancer-associated fibroblasts (CAF) are a key component in the tumor microenvironment and play functional roles in tumor metastasis and resistance to chemotherapies. We have previously reported that CAF isolated from lymphoma samples increase anaerobic glycolysis and decrease intracellular production of reactive oxygen species, promoting the survival of tumor cells. Herein, we analyzed the mechanisms underlying this support of tumor-cell survival by CAF. As direct contact between lymphoma cells and CAF was not indispensable to survival support, we identified that the humoral factor pyruvate was significantly secreted by CAF. Moreover, survival of lymphoma cells was promoted by the presence of pyruvate, and this promotion was canceled by inhibition of monocarboxylate transporters. Metabolome analysis of lymphoma cells in coculture with CAF demonstrated that intermediates in the citric acid cycle were significantly increased, indicating that tumor cells produced energy by aerobic metabolism. These findings indicate that energy production in lymphoma cells is regulated in coordination not only with anaerobic glycolysis, but also with aerobic metabolism termed the reverse-Warburg effect, involving the secretion of pyruvate from CAF resulting in increased use of the citric acid cycle in lymphoma cells.

Fcγ receptors: genetic variation, function, and disease.

Fcγ receptors (FcγRs) are key immune receptors responsible for the effective control of both humoral and innate immunity and are central to maintaining the balance between generating appropriate responses to infection and preventing autoimmunity. When this balance is lost, pathology results in increased susceptibility to cancer, autoimmunity, and infection. In contrast, optimal FcγR engagement facilitates effective disease resolution and response to monoclonal antibody immunotherapy. The underlying genetics of the FcγR gene family are a central component of this careful balance. Complex in humans and generated through ancestral duplication events, here we review the evolution of the gene family in mammals, the potential importance of copy number, and functionally relevant single nucleotide polymorphisms, as well as discussing current approaches and limitations when exploring genetic variation in this region.

Phase II study of intrabone single unit cord blood transplantation for hematological malignancies.

The outcomes of cord blood transplantation with non-irradiated reduced-intensity conditioning for hematological malignancies need to be improved because of graft failure and delayed engraftment. Intrabone infusion of cord blood cells has the potential to resolve the problems. In this phase II study, 21 adult patients with hematological malignancy received intrabone transplantation of serological HLA-A, B, and DR ≥4/6 matched single cord blood with a median number of cryopreserved total nucleated cells of 2.7 × 107 /kg (range, 2.0-4.9 × 107 /kg) following non-irradiated fludarabine-based reduced-intensity conditioning. Short-term methotrexate and tacrolimus were given as graft-versus-host disease prophylaxis, and granulocyte colony-stimulating factor was given after transplantation. No severe adverse events related to intrabone injection were observed. The cumulative incidences of neutrophils ≥0.5 × 109 /L, reticulocytes ≥1%, and platelets ≥20 × 109 /L recoveries were 76.2%, 71.4%, and 76.2%, respectively, with median time to recoveries of 17, 28, and 32 days after transplantation, respectively. The probability of survival with neutrophil engraftment on day 60 was 71.4%, and overall survival at 1 year after transplantation was 52.4%. The incidences of grade II-IV and III-IV acute graft-versus-host disease were 44% and 19%, respectively, with no cases of chronic graft-versus-host disease. The present study showed the safety of direct intrabone infusion of cord blood. Further analysis is required to confirm the efficacy of intrabone single cord blood transplantation with non-irradiated reduced-intensity conditioning for adult patients with hematological malignancy. This study was registered with UMIN-CTR, number 000000865.

Malignant lymphoma with cardiac involvement.

Malignant lymphoma with cardiac involvement is difficult to diagnose and treatment selection decisions can be challenging, because patients usually present with atypical disease involvement and the incidence is low. Herein, we describe the clinical characteristics and courses of three non-Hodgkin lymphoma patients showing cardiac involvement. All three patients were male, ages 32, 74 and 64 years. All three patients had presented with cardiac involvement mainly in the right heart system. We promptly performed needle biopsies for patients 1 and 3, and open-heart biopsy for patient 2, which showed PMBL for patient 1, DLBCL for patients 2 and 3. Since we were concerned regarding possible transient exacerbation of heart failure or the occurrence of fatal arrhythmia, we chose to start with relatively low dose chemotherapeutic interventions or pre-phase steroid therapy. After one course of chemotherapy or pre-phase steroid therapy, symptoms associated with heart failure almost completely subsided, and we further administered full-dose chemotherapy thereafter, resulting in complete responses in 2 cases. This case series demonstrates that malignant lymphoma with cardiac involvement is a treatable disease, despite widespread involvement. Furthermore, rapid and appropriate diagnostic imaging and biopsy are important when this disease is suspected.

Peripheral blood cell-free DNA is an alternative tumor DNA source reflecting disease status in myelodysplastic syndromes.

Genetic alterations in myelodysplastic syndromes (MDS) are critical for pathogenesis. We previously showed that peripheral blood cell-free DNA (PBcfDNA) may be more sensitive for genetic/epigenetic analyses than whole bone marrow (BM) cells and mononuclear cells in peripheral blood (PB). Here we analyzed the detailed features of PBcfDNA and its utility in genetic analyses in MDS. The plasma-PBcfDNA concentration in MDS and related diseases (N = 33) was significantly higher than that in healthy donors (N = 14; P = 0.041) and in International Prognostic Scoring System higher-risk groups than that in lower-risk groups (P = 0.034). The concentration of plasma-/serum-PBcfDNA was significantly correlated with the serum lactate dehydrogenase level (both P < 0.0001) and the blast cell count in PB (P = 0.034 and 0.025, respectively). One nanogram of PBcfDNA was sufficient for one assay of Sanger sequencing using optimized primer sets to amplify approximately 160-bp PCR products. PBcfDNA (approximately 50 ng) can also be utilized for targeted sequencing. Almost all mutations detected in BM-DNA were also detected using corresponding PBcfDNA. Analyses using serially harvested PBcfDNA from an RAEB-2 patient showed that the somatic mutations and a single nucleotide polymorphism that were detected before allogeneic transplantation were undetectable after transplantation, indicating that PBcfDNA likely comes from MDS clones that reflect the disease status. PBcfDNA may be a safer and easier alternative to obtain tumor DNA in MDS.

Inhibitory FcγRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity.

A major feature that distinguishes type I from type II anti-CD20 monoclonal antibodies (mAbs) and reduces their therapeutic efficacy is the tendency to internalize from the cell surface. We have shown previously that the extent of internalization correlates with the capacity of type I mAb to simultaneously engage both CD20 and the inhibitory Fcγ receptor, FcγRIIb, in a bipolar configuration. Here, we investigated whether mAbs directed at other B-cell surface receptors also engaged FcγRIIb and whether this interaction promoted internalization. Most mAbs engaged and activated FcγRIIb, with the strength of activation related to the level of mAb bound to the cell surface. However, engagement did not affect internalization of most mAb-ligated receptors, either in cell lines or primary chronic lymphocytic leukemia cells with the exception of CD19 and CD38. Furthermore, at high cell concentrations/density both cis and trans interactions between cell-surface bound mAb and FcγRIIb were evident, but trans interactions did not inhibit type I anti-CD20 mAb-mediated internalization. These data identify that FcγRIIb is engaged by many mAbs in both cis and trans configurations, triggering its activation, but that internalization via FcγRIIb occurs for only a select subset. These findings have implications when designing new antibody-based therapeutics.

De novo diffuse large B-cell lymphoma with a CD20 immunohistochemistry-positive and flow cytometry-negative phenotype: molecular mechanisms and correlation with rituximab sensitivity.

CD20 is expressed in most B-cell lymphomas and is a critical molecular target of rituximab. Some B-cell lymphomas show aberrant CD20 expression, and rituximab use in these patients is controversial. Here we show both the molecular mechanisms and the clinical significance of de novo diffuse large B-cell lymphomas (DLBCL) that show a CD20 immunohistochemistry (IHC)-positive and flow cytometry (FCM)- negative (IHC[+]/FCM[-]) phenotype. Both IHC and FCM using anti-CD20 antibodies L26 and B1, respectively, were analyzed in 37 of the 106 cases of de novo DLBCL; 8 (22%) of these cases were CD79a(+)/CD20(+) with IHC and CD19(+)/CD20(-) with FCM. CD20 (MS4A1) mRNA expression was significantly lower in IHC(+)/FCM(-) cells than in IHC(+)/FCM(+) cells (P = 0.0005). No genetic mutations were detected in MS4A1 promoter and coding regions. Rituximab-mediated cytotoxicity in the CDC assay using IHC(+)/FCM(-) primary cells was significantly lower than in IHC(+)/FCM(+) cells (P < 0.05); however, partial effectiveness was confirmed. FCM using rituximab detected CD20 more efficiently than B1. No significant difference was observed between IHC(+)/FCM(-) and IHC(+)/FCM(+) patients in overall survival (P = 0.664). Thus, lower expression of CD20 mRNA is critical for the CD20 IHC(+)/FCM(-) phenotype. Lower CD20 expression with FCM does not rule out rituximab use in these patients if expression is confirmed with IHC. FCM using rituximab may be more informative than B1 for predicting rituximab effectiveness in IHC(+)/FCM(-) cases.

Utility of cerebrospinal fluid liquid biopsy in distinguishing CNS lymphoma from cerebrospinal infectious/demyelinating diseases.

BACKGROUND: Distinguishing between central nervous system lymphoma (CNSL) and CNS infectious and/or demyelinating diseases, although clinically important, is sometimes difficult even using imaging strategies and conventional cerebrospinal fluid (CSF) analyses. To determine whether detection of genetic mutations enables differentiation between these diseases and the early detection of CNSL, we performed mutational analysis using CSF liquid biopsy technique. METHODS: In this study, we extracted cell-free DNA from the CSF (CSF-cfDNA) of CNSL (N = 10), CNS infectious disease (N = 10), and demyelinating disease (N = 10) patients, and performed quantitative mutational analysis by droplet-digital PCR. Conventional analyses were also performed using peripheral blood and CSF to confirm the characteristics of each disease. RESULTS: Blood hemoglobin and albumin levels were significantly lower in CNSL than CNS infectious and demyelinating diseases, CSF cell counts were significantly higher in infectious diseases than CNSL and demyelinating diseases, and CSF-cfDNA concentrations were significantly higher in infectious diseases than CNSL and demyelinating diseases. Mutation analysis using CSF-cfDNA detected MYD88L265P and CD79Y196 mutations in 60% of CNSLs each, with either mutation detected in 80% of cases. Mutual existence of both mutations was identified in 40% of cases. These mutations were not detected in either infectious or demyelinating diseases, and the sensitivity and specificity of detecting either MYD88/CD79B mutations in CNSL were 80% and 100%, respectively. In the four cases biopsied, the median time from collecting CSF with the detected mutations to definitive diagnosis by conventional methods was 22.5 days (range, 18-93 days). CONCLUSIONS: These results suggest that mutation analysis using CSF-cfDNA might be useful for differentiating CNSL from CNS infectious/demyelinating diseases and for early detection of CNSL, even in cases where brain biopsy is difficult to perform.

Safety and antitumor activity of copanlisib in Japanese patients with relapsed/refractory indolent non-Hodgkin lymphoma: a phase Ib/II study.

The safety, efficacy, and pharmacokinetics of copanlisib were evaluated in this phase Ib/II study in Japanese patients with relapsed/refractory indolent non-Hodgkin lymphoma (NHL). The primary endpoint was safety at the recommended dose; efficacy endpoints included objective response rate (ORR), progression-free survival (PFS), and overall survival. In phase Ib, patients received copanlisib 45 mg intravenously on days 1, 8, and 15 of a 28-day cycle, and when tolerated, consecutive patients received copanlisib 60 mg. As no dose-limiting toxicities occurred at the 45 mg (n = 3) or 60 mg (n = 7) dose in phase Ib, the recommended dose for Japanese patients was determined to be 60 mg, and this dose was used in phase II (n = 15). Although all patients experienced at least one treatment-emergent adverse event (TEAE), with hyperglycemia being the most common AE, no AE-related deaths were reported. The ORR was 68.0% (17/25 patients), median PFS was 302 (95% CI 231-484) days, and the duration of response was 330 (range 65-659) days. The pharmacokinetic properties of copanlisib were similar between Japanese and non-Japanese patients. Overall, copanlisib 60 mg had an acceptable safety profile and showed promising antitumor activity in Japanese patients with relapsed/refractory indolent NHL.

Using peripheral blood circulating DNAs to detect CpG global methylation status and genetic mutations in patients with myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a hematopoietic stem cell disorder. Several genetic/epigenetic abnormalities are deeply associated with the pathogenesis of MDS. Although bone marrow (BM) aspiration is a common strategy to obtain MDS cells for evaluating their genetic/epigenetic abnormalities, BM aspiration is difficult to perform repeatedly to obtain serial samples because of pain and safety concerns. Here, we report that circulating cell-free DNAs from plasma and serum of patients with MDS can be used to detect genetic/epigenetic abnormalities. The plasma DNA concentration was found to be relatively high in patients with higher blast cell counts in BM, and accumulation of DNA fragments from mono-/di-nucleosomes was confirmed. Using serial peripheral blood (PB) samples from patients treated with hypomethylating agents, global methylation analysis using bisulfite pyrosequencing was performed at the specific CpG sites of the LINE-1 promoter. The results confirmed a decrease of the methylation percentage after treatment with azacitidine (days 3-9) using DNAs from plasma, serum, and PB mono-nuclear cells (PBMNC). Plasma DNA tends to show more rapid change at days 3 and 6 compared with serum DNA and PBMNC. Furthermore, the TET2 gene mutation in DNAs from plasma, serum, and BM cells was quantitated by pyrosequencing analysis. The existence ratio of mutated genes in plasma and serum DNA showed almost equivalent level with that in the CD34+/38- stem cell population in BM. These data suggest that genetic/epigenetic analyses using PB circulating DNA can be a safer and painless alternative to using BM cells.

Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation.

A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of bone marrow transplantation is presented. Only the expression of HLA-B51, which was a mismatched HLA locus in the graft-versus-host direction, was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. All CTL clones, that were isolated from the recipient's blood when acute graft-versus-host disease developed, recognized the mismatched B(∗)51:01 molecule in a peptide-dependent manner. The pre-transplant leukemia blasts were lysed by CTL clones, whereas the post-transplant leukemia blasts were not lysed by any CTL clones. The IFN-γ ELISPOT assay revealed that B(∗)51:01-reactive T lymphocytes accounted for the majority of the total alloreactive T lymphocytes in the blood just before leukemia relapse. These data suggest that immune escape of leukemia blasts from CTL pressure toward a certain HLA molecule can lead to clinical relapse after bone marrow transplantation.

Single-nucleotide Fcγ receptor polymorphisms do not impact obinutuzumab/rituximab outcome in patients with lymphoma.

Single-nucleotide polymorphisms (SNPs) have been shown to influence Fcγ receptor (FcγR) affinity and activity, but their effect on treatment response is unclear. We assessed their importance in the efficacy of obinutuzumab or rituximab combined with chemotherapy in untreated advanced follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) in the GALLIUM (www.clinicaltrials.gov #NCT01332968) and GOYA (#NCT01287741) trials, respectively. Genomic DNA was extracted from patients enrolled in GALLIUM (n = 1202) and GOYA (n = 1418). Key germline SNPs, FCGR2A R131H (rs1801274), FCGR3A F158V (rs396991), and FCGR2B I232T (rs1050501), were genotyped and assessed for their impact on investigator-assessed progression-free survival (PFS). In both cohorts there was no prognostic effect of FCGR2A or FCGR3A. In FL, FCGR2B was associated with favorable PFS in univariate and multivariate analyses comparing I232T with I232I, with a more modest association for rituximab-treated (univariate: hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.54-1.14; P = .21) vs obinutuzumab-treated patients (HR, 0.56; 95% CI, 0.34-0.91; P = .02). Comparing T232T with I232I, an association was found for obinutuzumab (univariate: HR, 2.76; 95% CI, 1.02-7.5; P = .0459). Neither observation retained significance after multiple-test adjustment. FCGR2B was associated with poorer PFS in multivariate analyses comparing T232T with I232I in rituximab- but not obinutuzumab-treated patients with DLBCL (HR, 4.40; 95% CI, 1.71-11.32; P = .002; multiple-test-adjusted P = .03); however, this genotype was rare (n = 13). This study shows that FcγR genotype is not associated with response to rituximab/obinutuzumab plus chemotherapy in treatment-naive patients with advanced FL or DLBCL.

Dasatinib-induced Reversible Demyelinating Peripheral Neuropathy and Successful Conversion to Nilotinib in Chronic Myelogenous Leukemia.

Dasatinib, a tyrosine kinase inhibitor, is commonly used in the treatment of chronic myelogenous leukemia. A rare side effect is peripheral neuropathy. A 54-year-old woman experienced gradually accelerated dysesthesia and hypoesthesia in her extremities, 2 months following treatment with dasatinib. Nerve conduction studies revealed a prolonged conduction velocity with temporal dispersion, indicating demyelinating peripheral neuropathy. After changing dasatinib to nilotinib, both her clinical symptoms and electrophysiological data gradually improved. We herein report the findings of this case with a review of the pertinent literature.