Genetic profiles and clinical features in subcutaneous panniculitis-like T-cell lymphomas.

Subcutaneous panniculitis-like T-cell lymphoma (SPTCL) is a rare peripheral T-cell lymphoma characterized by cutaneous lesions and immunologic manifestations. The five-year survival rate of SPTCL has been reported to be over 80%, indicating a favorable prognosis. Recent studies have uncovered recurrent germline variants in HAVCR2, encoding an immunomodulator. In this study, we integrated whole-exome sequencing data from 60 samples collected from 36 SPTCL patients, encompassing six patients of our cohort and 30 patients of publicly available data. We identified 138 somatic mutations in skin tumors of 24 patients and HAVCR2 germline mutations in 23 of 29 patients. HAVCR2 p.Tyr82Cys mutations were identified in four of six Japanese patients. During the clinical courses of four patients, cyclophosphamide, hydroxydaunomycin, vincristine, and prednisone were administered to all patients, but it resulted in incomplete responses in all four patients. However, disease conditions of all patients remained stable with additional treatment, including autologous peripheral blood stem cell transplantation. Over a 7.5-year median follow-up, one patient developed autoimmune-related diseases, while one developed other hematological malignancy, resulting in death. To our knowledge, this is the first report of recurrent HAVCR2 germline mutations in Japanese patients, suggesting the necessity for long-term follow-up.

[Durable remission of T-cell prolymphocytic leukemia with CLEC16A::IL2 after allogeneic hematopoietic stem cell transplantation].

A 64-year-old woman presented with fine motor impairment in both hands. MRI revealed a contrast-enhanced lesion in the medulla oblongata. Lymphoid cells with abnormal blebs were observed and a CD4+/CD8+ double positive (DP) T cell population was detected by flow cytometry (FCM) in the bone marrow (BM) and the peripheral blood (PB). CLEC16A::IL2 fusion gene was identified by whole exome sequencing with DNA prepared from DP T cells. Clonal rearrangement of the T-cell receptor gene and expression of TCL1A protein were detected. This led to a diagnosis of T-cell prolymphocytic leukemia (T-PLL) with central nervous system (CNS) infiltration. Abnormal cells in BM and PB became undetectable on microscopy and FCM, and the CNS lesion disappeared on MRI after second-line therapy with alemtuzumab. Meanwhile, the CLEC16A::IL2 fusion mRNA remained detectable in PB. Allogeneic hematopoietic stem-cell transplantation was performed, and the fusion mRNA has now been undetectable for more than 5 years since transplantation. This is the first report of a T-PLL case with a CLEC16A::IL2 fusion gene.

[Waldenström macroglobulinemia complicated by peripheral neuropathy due to neural infiltration].

A 51-year-old man with the chief complaint of glove- and stocking-type dysesthesia for >3 years was diagnosed with Waldenström's macroglobulinemia (WM) based on IgM-type M-proteinemia, bone marrow infiltration of plasmacytoid B cells, multiple lymphadenopathies, and splenomegaly. A nerve conduction examination suggested demyelinating neuropathy. Serum anti-myelin-associated glycoprotein antibody was negative. Sural nerve biopsy showed myelin thinning, suggesting demyelination. Axonal damage and tumor cell infiltration in the intrafascicular epineurium were also observed. After chemotherapies with rituximab and bendamustine, M-proteinemia and lymphadenopathies disappeared. However, abnormalities in the nerve conduction examination and dysesthesia were only slightly alleviated. As articles describing patients with WM with peripheral nerve infiltration are limited, we report this case with a literature review.

[Acute myeloid leukemia harboring NUP98::DDX10].

NUP98::DDX10 is a rare fusion gene associated with acute myeloid leukemia (AML), for which the prognosis and indication for allogeneic hematopoietic stem cell transplantation are unknown. A 48-year-old woman was diagnosed with AML harboring NUP98::DDX10. The results of quantitative RT-PCR of the fusion mRNA as a minimal residual disease (MRD) marker guided the treatment. In August 2019, the patient achieved hematological remission following standard remission induction therapy with idarubicin and cytarabine. After four cycles of consolidation therapies, MRD was detected, and she underwent allogeneic stem cell transplantation in May 2020. As MRD persisted in June, the immunosuppressant was stopped and three cycles of azacitidine were administered. Despite this, a hematological relapse occurred in January 2021 that was resistant to high-dose cytarabine and an investigational agent. She died as a result of the disease's progression. Thus, a second thought should be given to the timing of transplantation, the bridging, and the intervention for relapse after transplantation. The cases must be accumulated.

Impact of immunophenotypic characteristics on genetic subgrouping in childhood acute lymphoblastic leukemia: Tokyo Children's Cancer Study Group (TCCSG) study L04-16.

Immunophenotyping was performed in 1044 consecutive childhood acute lymphoblastic leukemia (ALL) patients enrolled in the Tokyo Children's Cancer Study Group L04-16 trial, revealing novel findings associated with genetic abnormalities. In addition to TCF3-PBX1 and MEF2D fusions, the CD10(+) subtype of KMT2A-MLLT3-positive ALL frequently exhibited the cytoplasmic-µ(+) pre-B ALL immunophenotype. Although ETV6-RUNX1 was significantly correlated with myeloid antigen expression, more than half of patients expressed neither CD33 nor CD13, while the CD27(+) /CD44(-) immunophenotype was maintained. Expression of CD117 and CD56 in B-cell precursor-ALL was limited to certain subtypes including ETV6-RUNX1 and KMT2A-MLLT3. Besides BCR-ABL1, CRLF2, hyperdiploidy, and hypodiploidy, CD66c was also expressed in Ph-like kinase fusion-, PAX5 fusion-, and DUX4 fusion-positive ALL, but not in MEF2D fusion-positive ALL, indicating constant selectivity of CD66c expression. In T-ALL, SIL-TAL1-positive patients were likely to exhibit a more mature immunophenotype. Expression of CD21 and CD10 was not rare in T-ALL, while lack of CD28 was an additional feature of early T-cell precursor-ALL. Considering the immunophenotype as a prognostic maker, MEF2D fusion-positive ALL with CD5 expression may be associated with a poorer prognosis in comparison with those lacking CD5 expression. In cases with characteristic marker expression, the presence of certain fusion transcripts could be predicted accurately.

Effects of DNA Methylation on Leukemia Cell Proliferation.

BACKGROUND: In this study, we aimed to investigate the effect of DNA methyltransferase 1 gene (DNMT1) expression on leukemia cell proliferation. METHODS: Following stimulation with interferon-α (IFN-α) or methylation inhibitor for three days, we evaluated changes in the DNMT1 expression levels, cell proliferation activity, and Bcl-2-Associated X Protein (BAX) expression levels in the chronic myelogenous leukemia cell line K562 and the acute monocytic leukemia cell line THP-1. RESULTS: DNMT1 expression levels and cell proliferation activity decreased in K562 and THP-1 cells, whereas BAX expression levels increased. CONCLUSIONS: These results suggest that the enzymatic activity of DNMT1 promotes the proliferation of tumor cells and that tumor cell proliferation can be suppressed by inhibiting DNMT1 enzymatic activity. Furthermore, because DNA methylation is associated with apoptosis, a process critical to cell growth and injury in leukemia, assessing DNMT1expression levels might help in treatment decisions for leukemia patients.

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.

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.

Establishment of immunity against Epstein-Barr virus infection in a patient with CHARGE/complete DiGeorge syndrome after peripheral blood lymphocyte transfusion.

CHARGE syndrome is a rare congenital malformation syndrome which may share symptoms with DiGeorge syndrome. Complete DiGeorge syndrome (cDGS) is a severe form of DiGeorge syndrome, characterized by a CD3+ T-cell count of <50/mm3 due to athymia, and is fatal without immunologic intervention. We performed peripheral blood lymphocyte transfusion (PBLT) from an HLA-identical sibling without pretransplant conditioning in a CHARGE/cDGS patient with a novel CHD7 splice site mutation. Cyclosporine and short-term methotrexate were used for graft versus host disease (GVHD) prophylaxis, and neither acute nor chronic GVHD was observed. After PBLT, T-cell proliferative response to phytohemagglutinin and concanavalin A recovered, and intractable diarrhea improved. EBV infection, evidenced by a gradual increase in the viral genome copy number to a maximum of 2861 copies/µgDNA on day 42 after PBLT, resolved spontaneously. HLA A2402 restricted, EBV-specific CTLs were detected from peripheral blood on day 148, and EBV seroconversion was observed on day 181. Thus, EBV-specific immunity was successfully established by PBLT. Our results indicate that PBLT is a simple and effective therapy to reconstitute immune systems in CHARGE/DiGeorge syndrome.

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.

Influence of In Vitro Hemolysis on 80 Different Laboratory Tests.

BACKGROUND: In vitro hemolysis is probably the most common pre-analytic problem in laboratory medicine. However, it introduces variation into results in unknown ways. Therefore, the purpose of this study was to assess the quantitative effects of hemolysis on 80 different, routine laboratory tests. METHODS: We examined the ratio of hemolysis in our hospital from January 1 to March 31, 2015. Next, to study the effect of in vitro hemolysis of whole blood, we added lysed erythrocytes to pooled specimens of serum or plasma to give hemoglobin concentrations of 0.9 to 8.1 g/L and 2.8 to 14 g/L, respectively, and a rating by colorimetry of 0 to 4+ hemolyzed. Then, 80 different laboratory tests were determined with a Hitachi 7700 autoanalyzer for biochemical tests and with AIA 2000, Cobas 6000, and Lumipulus G1200 machines for other tests. RESULTS: Hemolysis occurred in a total in 8.6% of the specimens in our hospital. Significant correlations with the hemolysis ratio were observed in 43 of 80 laboratory tests. At apparent hemolysis, 11 test levels increased and 7 test levels decreased due to hemolysis. Among the 11 tests, potassium, aspartate aminotransferase, lactate dehydrogenase, thymol turbidity test (TTT), zinc sulfate turbidity test (ZTT), and hyaluronic acid tests showed proportional increases due to hemolysis. CONCLUSIONS: Hemolysis is a common problem for accuracy in many routine laboratory tests. Although the quantitative effects of hemolysis can only be roughly estimated in this report, the approximate extent of change in specific laboratory tests is useful for establishing a baseline for future hemolytic studies.

Imatinib-induced Severe Hepatitis in a 9-Year-old Girl With Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia.

Imatinib mesylate has dramatically improved the outcome of children with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph* ALL) and is now included as first-line therapy. Uncommon adverse effects of this drug for pediatric use, however, are largely unknown. We report the first case of a 9-year-old child who developed severe acute hepatitis with grade 4 transaminases and bilirubin elevation during imatinib treatment for Ph* ALL. Liver biopsy showed extensive lobular and pericentral necrosis of hepatocytes. Liver function recovered after discontinuing imatinib with a 4-week prednisolone. Extensive hepatic necrosis should be considered not only in adults but also in children under imatinib administration.

Sustained Appearance of Urinary Podocytes Suggests Poor Renal Prognosis in Kidney Transplant Patients with Focal Segmental Glomerulosclerosis: Case Reports and Review of Literature.

Recent studies have indicated that the detection of urinary podocytes holds major significance for focal segmental glomerulosclerosis (FSGS). We present two cases of FSGS after kidney transplantation, focusing on urinary podocytes. In Case 1, treatment led to incomplete remission with the reduction of urinary podocytes, and his renal function was preserved. Case 2, however, showed continuous increase in proteinuria with loss of renal function despite apheresis. Urinary podocytes remained high throughout. On the basis of this experience, we suggest the significance of the detection of urinary podocytes for determining renal prognosis in FSGS following renal allograft.

Clinical usefulness of the PAXgene™ bone marrow RNA system for stabilizing total RNA.

The collection of clinical samples, such as bone marrow (BM) and peripheral blood, is an important procedure for the extraction of the cellular RNA. It is essential to preserve the extracted RNA during and after the collection of clinical samples to ensure the accurate analysis of gene expression. To date, the PAXgene™ Blood RNA System has been proven useful for stabilizing RNA extracted from peripheral blood; however, a problem concerning the stability of the total RNA stored using the system has been identified. The PAXgene™ Bone Marrow RNA System (BM system) is a newly developed system, and its clinical usefulness as a stabilizer for the cellular RNA in BM and peripheral blood was investigated with respect to the quality of RNA extracted using this system. A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was carried out using total RNA extracted with the BM system, which showed that total RNA was more stable in the BM system than in the conventional system, indicating that the BM system can be applied to RT-PCR. The BM system enabled us to detect Wilms' tumor suppressor gene (WT1) more effectively than the conventional system. In conclusion, the BM system is clinically valuable for extracting and stabilizing total RNA of high quality.

Persistent low level Epstein-Barr virus DNAemia in childhood cancer survivors.

BACKGROUND: Clinical observation of Epstein-Barr virus (EBV) status has not documented in childhood cancer survivors (CCSs) sustaining long-term remission of malignant diseases. Thus, the aim of this study was to evaluate the EBV status in children with various malignant diseases after they completed their treatments. PATIENTS AND METHODS: Thirty consecutive children with various malignant diseases previously received treatment at the University of Tsukuba Hospital. Nine cases had acute lymphoblastic leukemia (ALL), 10 had solid tumors, 4 had lymphoma, 4 had CNS tumors, and 3 had acute myeloid leukemia (AML). EBV DNA in 328 whole blood samples were monitored by real-time QPCR for all cases after treatment. Clinical records and laboratory data were also reviewed. RESULTS: There were 6/30 (20%) cases with continuous detection of EBV DNA while there were 24/30 (80%) cases without continuous EBV DNA. EBV DNAemia was persistently observed in 4/9 (44.4%) cases with ALL and in 2/4 (50%) cases with lymphoma. Persistent EBV DNAemia can be observed for >5 years without any EBV associated symptoms or diseases. CONCLUSIONS: Childhood cancer survivors have persistent EBV DNAemia more frequently, which is thought to be observed in cases with ALL and lymphoma with higher tendency for >5 years after treatment. Persistent EBV DNAemia is frequent in CCSs aged 5-10 years. Any immunological alteration is speculative in a pathophysiology of persistent EBV DNAemia.

Acute monocytic leukemia with KMT2A::LASP1 developed 9 months after diagnosis of acute megakaryoblastic leukemia in a 2-year-old boy.

Acute myeloid leukemia (AML) is known as one of the subsequent malignant neoplasms that can develop after cancer treatment, but it is difficult to distinguish from relapse when the preceding cancer is leukemia. We report a 2-year-old boy who developed acute megakaryoblastic leukemia (AMKL, French-American-British classification [FAB]: M7) at 18 months of age and achieved complete remission with multi-agent chemotherapy without hematopoietic stem cell transplantation. Nine months after diagnosis and 4 months after completing treatment for AMKL, he developed acute monocytic leukemia (AMoL) with the KMT2A::LASP1 chimeric gene (FAB: M5b). The second complete remission was achieved using multi-agent chemotherapy and he underwent cord blood transplantation 4 months after AMoL was diagnosed. He is currently alive and disease free at 39 and 48 months since his AMoL and AMKL diagnoses, respectively. Retrospective analysis revealed that the KMT2A::LASP1 chimeric gene was detected 4 months after diagnosis of AMKL. Common somatic mutations were not detected in AMKL or AMoL and no germline pathogenic variants were detected. Since the patient's AMoL was different from his primary leukemia of AMKL in terms of morphological, genomic, and molecular analysis, we concluded that he developed a subsequent leukemia rather than a relapse of his primary leukemia.

Cardiac Tamponade as a Recurrence of Angioimmunoblastic T-Cell Lymphoma with the Detection of a p.Gly17Val RHOA Mutation in the Pericardial Effusion.

Angioimmunoblastic T-cell lymphoma (AITL) is an intractable type of T-cell lymphoma. We and others have identified that the p.Gly17Val RHOA mutation is specifically identified in AITL. We herein report a patient whose condition deteriorated, resulting from massive pericardial effusion one month after undergoing autologous transplantation for AITL. He was diagnosed with cardiac tamponade caused by AITL recurrence in the presence of the p.Gly17Val RHOA mutation as well as T-lineage cells with an aberrant immune-phenotype in the pericardial effusion. This case suggests that a precision medicine approach by detecting the presence of a p.Gly17Val RHOA mutation is useful for the management of AITL.

Focal segmental glomerulosclerosis as a complication of hepatitis B virus infection.

Human hepatitis B virus (HBV) is well known as a cause of membranous nephropathy (MN). While the association of HBV infection with MN is strong, data regarding its association with other glomerular diseases are conflicting. Here, we report a case of focal segmental glomerulosclerosis (FSGS) with HBV infection. In this case, we have found HBV-DNA in urinary podocytes by real-time PCR methods. After the administration of anti-viral therapy, FSGS improved, paralleling the decreased level of HBV-DNA in podocytes. The refractory FSGS induced by HBV could be effectively treated with appropriate anti-viral agents.