Takayuki Takachi

A novel homozygous 8-base pair deletion mutation in the glycoprotein Ibα gene in a patient with Bernard-Soulier syndrome

We present a patient with Bernard–Soulier syndrome harboring a novel mutation. Flow cytometric analysis showed that the glycoprotein (GP) Ib/IX complex was absent from the platelet surface. By immunoblotting, GPIbα, GPIbβ and GPIX were not detected in the platelet lysates. Glycocalicin, the soluble GPIbα fragment, was also absent in the plasma. Genetic analysis revealed a novel homozygous 8-base pair deletion in the GPIbα gene, 1136_1143delTTCACATG, which was predicted to cause a frame shift and the addition of 13 altered amino acids followed by premature termination. No mutation was found in the coding sequence of the GPIbβ or GPIX genes. We demonstrated that the novel deletion mutation resulted in complete defectiveness of the GPIbα protein and null expression of the entire GPIb/IX complex, and was responsible for the Bernard–Soulier syndrome phenotype in this patient. Although the presence of a truncated GPIbα protein has been often documented, complete absence of the protein has been scarcely reported in Bernard–Soulier syndrome patients with a GPIbα mutation causing a premature stop codon. An underlying molecular mechanism to explain how the synthesis of a truncated protein is inhibited in selected cases remains to be elucidated.

Juvenile myelomonocytic leukemia with less aggressive clinical course and KRAS mutation.

To the Editor: An 11-month-old male presented with hepatosplenomegaly and juvenile xanthogranuloma on the face and the trunk. Laboratory findings included leukocytosis (16 10/L) with monocytosis (3 10/L) and 0% leukemic blasts, anemia (hemoglobin 8.6 g/dl) and thrombocytopenia (platelets 65 10/L). The level of fetal hemoglobin (HbF) was 2.5%. A bone marrow aspirate revealed <3% blasts with normal karyotype. Spontaneous colony formation in the absence of added hematopoietic growth factors were demonstrated in the peripheral blood cells. Single nucleotide substitution at codon 12 of the KRAS gene from GGT to GTT was detected in the bone marrow cells, while NRAS mutations at codon 12, 13, or 61 were not detected. Examination of PTPN11 mutation was also performed later and indicated no abnormalities. The diagnostic criteria for JMML were fulfilled. During 3 months of observation, leukocytosis improved spontaneously and anemia and thrombocytopenia did not progress. Treatment with 13-cis retinoic acid was started 9 months from diagnosis, Although white cell counts showed no significant change, platelet counts increased up to 150 10/L (Supplemental Fig. 1). Twenty-eight months later, his parents decided to discontinue 13-cis retinoic acid, mainly due to economical reasons. White cell counts gradually decreased to 80–90 10/L, and platelet counts were sustained with 120– 170 10/L for 5 years. Splenomegaly and peripheral blood spontaneous colony formation remained, although reduced in size or in number, respectively. JMML is a progressive and often rapidly fatal disease. HSCT is currently considered the only curative treatment for this disease, but treatment-related mortality and relapse rates remain high [1–3]. Although JMML is usually aggressive, some patients have been shown to have a prolonged and stable clinical course without HSCT [3]. Recently, prognostic impact of genetic abnormalities has been debated. Matsuda et al. [4] reported three patients with RAS Gly12Ser mutation showing spontaneous improvement of hematological abnormalities lasting for 2–4 years with neither intensive chemotherapy nor HSCT. On the other hand, Flotho et al. [5] reported six patients with RAS mutations other than RAS Gly12Ser who had long-term survival lasting for 4–21 years without HSCT. The present case had a KRAS Gly12Val mutation and has survived for 8 years after diagnosis without HSCT. Our case received 13-cis retinoic acid for 28 months, during which time platelet counts worsened and then continued to be elevated even after stopping the treatment. It is impossible to assess whether the improvement was due to the drug or whether it reflected the natural course of the disease. The optimal timing of HSCT for JMML is unknown. Therefore, differentiation-inducing or RAS-targeting therapeutics prior to HSCT might be justified for some patients who have mismatched donor. Specific genetic abnormalities, such as RAS mutation, might be a surrogate marker for identifying those who might respond to such treatments. Further studies are needed to elucidate whether specific genetic abnormalities correlate with clinical courses in JMML.

Human T-cell leukemia virus type 1 Tax oncoprotein represses the expression of the BCL11B tumor suppressor in T-cells

Human T‐cell leukemia virus type 1 (HTLV‐1) is the etiological agent of adult T cell leukemia (ATL), which is an aggressive form of T‐cell malignancy. HTLV‐1 oncoproteins, Tax and HBZ, play crucial roles in the immortalization of T‐cells and/or leukemogenesis by dysregulating the cellular functions in the host. Recent studies show that HTLV‐1‐infected T‐cells have reduced expression of the BCL11B tumor suppressor protein. In the present study, we explored whether Tax and/or HBZ play a role in downregulating BCL11B in HTLV‐1‐infected T‐cells. Lentiviral transduction of Tax in a human T‐cell line repressed the expression of BCL11B at both the protein and mRNA levels, whereas the transduction of HBZ had little effect on the expression. Tax mutants with a decreased activity for the NF‐κB, CREB or PDZ protein pathways still showed a reduced expression of the BCL11B protein, thereby implicating a different function of Tax in BCL11B downregulation. In addition, the HTLV‐2 Tax2 protein reduced the BCL11B protein expression in T‐cells. Seven HTLV‐1‐infected T‐cell lines, including three ATL‐derived cell lines, showed reduced BCL11B mRNA and protein expression relative to an uninfected T‐cell line, and the greatest reductions were in the cells expressing Tax. Collectively, these results indicate that Tax is responsible for suppressing BCL11B protein expression in HTLV‐1‐infected T‐cells; Tax‐mediated repression of BCL11B is another mechanism that Tax uses to promote oncogenesis of HTLV‐1‐infected T‐cells.

EFFECT OF CHARCOAL HEMOPERFUSION FOR REMOVAL OF PLASMA METHOTREXATE IN A PATIENT WITH ACUTE RENAL FAILURE

Strategies effective for accelerating methotrexate removal in delayed methotrexate excretion have not been universally accepted. The authors report a case of a 12-year-old girl with osteosarcoma who developed acute renal failure immediately after the first administration of high-dose methotrexate. Plasma methotrexate was effectively removed with repeated charcoal hemoperfusion in addition to plasma exchange and leucovorin rescue. Charcoal hemoperfusion was most effective for reducing plasma methotrexate with approximately 50% of methotrexate being reduced during each of the procedures. No rebound increase in MTX levels was observed. The patient received further therapy with other cancer drugs and has been well for 3.5 years.

A 2-year-old boy with a stage III yolk sac tumor occurring in an intra-abdominal retained testis.

A major complication of retained testes is an occurrence of malignancy later in life. We, herein, report the case of a 2-year-old boy who presented with a huge yolk sac tumor with retroperitoneal lymph nodes metastasis that originated in a left intra-abdominal undescended testis. Computed tomography and magnetic resonance imaging showed a huge round tumor connecting to the left retroperitoneal lymph nodes with metastasis extending from the left pelvic region to the left renal hilum. The serum alpha-fetoprotein level was 36,528 ng/mL. The right abdominal tumor appeared to be a giant testis that had strangulated at the neck of the cord. The tumor had ruptured at the side of the left pelvic lymph node metastasis, and a yolk sac tumor was diagnosed from a histologic analysis of the resected specimens. Postoperative PEB chemotherapy was effective, and a complete surgical resection of the tumor was performed 3 months after the initial laparotomy. The pathologic findings showed fibrous tissue without any tumor cells. The patient has been doing well for 18 months after the radical operation. This case might be a coincidental association of a yolk sac tumor occurring in an undescended testis, which thus caused a delay in making an accurate diagnosis.

Human T-cell leukemia virus type 1 (HTLV-1) Tax1 oncoprotein but not HTLV-2 Tax2 induces the expression of OX40 ligand by interacting with p52/p100 and RelB.

Human T-cell leukemia virus type 1 (HTLV-1) is a causative retrovirus of adult T-cell leukemia and HTLV-1-associated myelopathy. Unlike HTLV-1, the same group of retrovirus HTLV-2 has not been found to be associated with these diseases. HTLV-1 and HTLV-2 encode transforming proteins Tax1 and Tax2, and a few distinct activities of Tax1 from those of Tax2 have been proposed to contribute to the HTLV-1-specific pathogenesis of disease. One significant difference of Tax1 from Tax2 is the activation of transcription factor NF-κB2/p100/p52. We found that Tax1 but not Tax2 induces the expression of OX40 ligand (OX40L) in a human T-cell line. To induce the OX40L expression, Tax1 but not Tax2 was observed to interact with NF-κB2/p100/p52 and RelB and the distinct interaction activity was mediated by the Tax1 amino acid region of 225-232. In addition, Tax1 but not Tax2 or Tax1/225-232 interacted with p65, p50, and c-Rel; however, the interactions were much less than those noted with NF-κB2/p100/p52 and RelB. OX40L is a T-cell costimulatory molecule of the tumor necrosis factor family, and its signal plays a critical role in establishing adaptive immunity by inducing the polarized differentiation of T-cells to cells such as T helper type 2 and T follicular helper cells. Therefore, the present findings suggest that Tax1 might alter the immune response to HTLV-1 and/or differentiation of HTLV-1-infected T-cells via OX40L induction, thereby acting as a factor mediating the distinct phenotypes and pathogenesis of HTLV-1 from that of HTLV-2.

Refractory Infantile Spasms Associated With Mosaic Variegated Aneuploidy Syndrome

BACKGROUND Mosaic variegated aneuploidy syndrome (Online Mendelian Inheritance in Man 257300), or premature chromatid separation syndrome, is a rare cancer-prone disorder associated with an autosomal recessive trait related to BUB1B gene mutations. The risk of malignancy is high, with rhabdomyosarcoma, Wilms tumor, and leukemia reported in several cases. Clinical features also include prenatal-onset growth retardation, microcephaly, mild dysmorphism, feeding difficulty, hypotonia, seizures, and developmental delay. PATIENT A boy patient exhibited severe developmental delay, microcephaly, hypotonia, intractable seizures including infantile spasms with hypsarrhythmia at 6 months old, and Dandy-Walker malformation on magnetic resonance imaging. Seizures were refractory to conventional antiepileptics and treatment with adrenocorticotropic hormone. Wilms tumor and an unidentified intraorbital tumor also developed at 22 months old. RESULTS Chromosomal analysis showed multiple aneuploid cells, and premature chromatid separation was found in all chromosomes in 59.5% of 119 cells, indicating mosaic variegated aneuploidy syndrome. CONCLUSIONS The present case report demonstrates that mosaic variegated aneuploidy syndrome can be associated with developmental brain anomalies that lead to early-onset epileptic encephalopathy. Awareness of this disorder is important not only for proper diagnosis but also for genetic counseling of the family.

Reactive tonsillar enlargement with strong 18F-FDG uptake after chemotherapy for tonsillar diffuse large B-cell lymphoma.

We describe a 14-year-old boy who exhibited left palatine tonsillar enlargement after 6 cycles of aggressive chemotherapy for diffuse large B-cell lymphoma of the right palatine tonsil. The cervical computed tomography scan at 4 months after completion of chemotherapy revealed enlargement of the left palatine tonsil in addition to the thymus without any clinical symptoms. The 18F-fluorodeoxyglucose positron emission tomography indicated focal areas of strong 18F-fluorodeoxyglucose uptake in the left palatine tonsil. Histologic examination confirmed tonsillar hyperplasia with no evidence of recurrence. Reactive tonsillar hyperplasia after chemotherapy is rarely reported.

Lymphoblastic lymphoma with mature b‐cell immunophenotype and MLL‐AF9 in a child

To the Editor: A distinct subset of acute lymphoblastic leukemia (ALL) characterized by a unique triad of non-L3 morphology, mature B-cell immunophenotype, and MLL gene rearrangement has been reported, especially in infancy [1–3]. Although lymphoblastic lymphoma (LBL) and ALL share common morphological, biological, and molecular features, LBL with this triad has not been reported. A 3-year-old female presented with a tumor on the cheek. A biopsy was compatible with B-lineage LBL, with L1-morphology on the stamp preparation (Fig. 1). By flow cytometry, the blast cells were positive for CD19, CD22, and HLA-DR and negative for cytoplasmic CD3, terminal deoxytransferase (TdT), CD10, CD20, CD34, CD56, and myeloperoxidase. Notably, surface immunoglobulin M and l-chain were clonally positive. Because rearrangement of CMYC and IgH were not demonstrated by FISH, we then examined frozen-stored tumor tissue by RT-PCR which amplifies minor/major BCR-ABL, TEL-AML1, E2A-PBX1, SIL-TAL, MLL-AF4, MLL-AF6, MLL-AF9, and MLL-ENL. A MLL-AF9 fusion was detected. The presence of MLL rearrangement was further confirmed by FISH. By conventional karyotyping, we obtained only two metaphases displaying no abnormalities. The imaging studies revealed a small metastatic nodule in the liver. Neither central nervous system nor bone marrow invasion was demonstrated. The patient was diagnosed with LBL with the Murphy stage III disease. We administered ALL-type multidrug chemotherapy, resulting in a rapid and complete response. The patient has maintained complete remission for 2 years since the end of the therapy. LBL with a mature B-cell immunophenotype has been rarely reported. In 1987, Sheibani et al. [4] reported an elderly patient with precursor B-cell LBL that displayed surface light chain immunoglobulin restriction. In 1990, the authors reported an additional three cases with the same features and suggested that these cases formed a previously unrecognized variant of LBL [4,5]. Chromosomal karyotyping was not performed in the four cases. Only limited data are available for cytogenetic abnormalities in B-lineage LBL. To date, only 10 cases have been described with conventional karyotyping. In none of these, was there an 11q23 abnormality [6–9]. Recently, Shafer et al. [10] reported two cases with cutaneous LBL with an early precursor B-cell immunophenotype. In one of the cases, MLL gene rearrangement was demonstrated by FISH on sections of paraffin-embedded formalin-fixed tissue, although the partner gene that formed the fusion gene with MLL was not verified. In the reported cases with ALL harboring the triad, t(9;11)(p22;q23) was most frequently observed; there were 10 cases with this translocation among 11 cases with karyotyping data. Notably, FISH or spectral karyotyping disclosed MLL gene rearrangement in 8 out of 11 cases, while conventional karyotyping (e.g., G-banding) was not able to demonstrate any abnormalities [1–3]. These observations suggest the importance of molecular analysis together with conventional karyotyping for appropriately diagnosing ALL (or LBL) with this triad. We

Alveolar rhabdomyosarcoma after treatment of osteosarcoma

Secondary rhabdomyosarcoma (RMS) after treatment of osteosarcoma (OS) is rare. Reported here is the case of a metachronous RMS in the nasal cavity, developing 12 years after successful treatment of non‐metastatic OS. The patient was diagnosed as having OS of the femur at 2 years of age. Chemotherapy for OS included doxorubicin (cumulative dose, 488 mg/m2). No radiotherapy was given. There was no family history suggestive of cancer predisposition syndrome. At 14 years of age, alveolar RMS was diagnosed on histopathology. PAX3‐FKHR fusion transcripts were detected on reverse transcription–polymerase chain reaction. Germline TP53 mutation was not seen on standard DNA sequencing. The occurrence of secondary sarcomas, in the Childrens Cancer Survivor study conducted in North America, has been associated with high cumulative doses of anthracyclines, which may also have played a role in the development of RMS in the present case. In the future, novel molecular technologies might uncover genetic cancer predisposition in patients with metachronous cancers.