AMeD syndrome is characterized by aplastic anemia, mental retardation, short stature, and microcephaly and is caused by digenic mutations in the aldehyde dehydrogenase 2 ( ALDH2) and alcohol dehydrogenase 5 ( ADH5) genes. We have successfully performed hematopoietic stem cell transplantation in two patients with AMeD syndrome and isochromosome 1q. AMeD syndrome with myelodysplastic syndrome or acute myeloblastic leukemia generally has a poor prognosis; however, early diagnosis may improve treatment response. Although the gain of 1q has been considered as a form of early clonal evolution in Fanconi anemia, it may be an equally important finding observed in AMeD syndrome.

Myeloproliferative disorder (MPD) is a rare complication in individuals with Noonan syndrome (NS) and an RIT1 pathogenic variant; only three cases have previously been reported. However, this condition can be life-threatening. In the present case, a neonate with a massive capillary leak and compromised general condition caused by aggressive MPD, we employed cytoreductive chemotherapy using low-dose cytarabine, which proved to be a valuable therapeutic intervention. For patients presenting with suspected MPD and exhibiting features associated with NS early in life, genetic testing that includes sequencing of RIT1 may confer significant benefits to diagnosis and disease management.

Pediatric erythroblastic transformation of JAK 2-mutated prefibrotic primary myelofibrosis with concurrent PHF6 mutationsTokiko Oshiro1, Satoru Hamada1, Sinobu Kiyuna1, Hideki Sakiyama1, Nobuyuki Hyakuna2, Tomoko Tamaki3, Hideki Muramatsu4, Koichi Nakanishi5Department of Pediatrics, University of Ryukyus HospitalOkinawa prefectural Red Cross Blood CenterDepartment of Pathology and Oncology, Graduate school of Medicine, University of RyukyusDepartment of Pediatrics, Graduate School of Medicine, University of NagoyaDepartment of Pediatrics, Graduate School of Medicine, University of The RyukyusCorrespondence: Satoru Hamada,Department of Pediatrics, Faculty of Medicine, University of Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0125, [email protected] the Editor: Primary myelofibrosis (PMF) is a rare condition in children. According to the 2016 World Health Organization (WHO), classification of myeloproliferative neoplasms (MPN), PMF is divided into prefibrotic PMF (pre-PMF) and overt fibrotic PMF1. Pre-PMF is the proliferation of predominantly abnormal megakaryocytes and minimal or no reticulin fibrosis. Therefore, the lack of fibrosis in the early phase of thrombocytosis can be misdiagnosed as essential thrombocythemia2. One significant complication of MPN is leukemic transformation (LT); however, only a few cases of PMF in children have been reported3. The clinical utility of the three driver mutations in JAK2 , CALR, and MPL has been shown, especially when JAK2 is central to the pathogenesis of the MPN phenotype4. Additional mutations in ASXL1, SRSF2, IDH1/2, or EZH2 have been shown5. PHF6is an X-linked tumor suppressor gene with a somatic mutation that causes an aggressive type of myeloid neoplasm6. Here, we report a case of pediatricJAK2 -mutated pre-PMF with concurrent PHF6 mutations that transformed into AML within a year of diagnosis.A 14-year-old boy with no medical history was admitted to our hospital with lumbago. Physical examination revealed splenomegaly (5 cm below the costal margins). A complete blood count showed a white blood cell (WBC) count of 5.8×109/L, neutrophil count of 31%, lymphocyte count of 36%, red blood cell count of 7.34×109/L, hemoglobin concentration of 140 g/L, and platelet count of 1010×109/L. The patient was diagnosed with essential thrombocythemia based on bone marrow findings, which showed hypercellularity (80-100%), increasing with separated circled-multinucleated megakaryocytes, hyper-segmented-megakaryocytes, atypical megakaryocytes, and micromegakaryocytes (Figure 1A), and was treated with anagrelide. At this time, there were no blasts or reticulin fibers. The patient had no karyotypic abnormalities. After written informed consent was obtained, target capture-based next generation sequencing (NGS) was performed on bone marrow DNA for the following genes: MPL, ASXL1, CBL, JAK3, EZH2, IDH1, IDH2, JAK1, PHF6, SF 3B1, TET2, TP53, U2AF1, JAK2, NRAS/KRAS and IKZF1 by previous described methods7. Among these mutations, JAK2 V617F with mutant allele percentage 4% and PHF6 p.Q121Xmutation with 64% were identified. In addition, we generated an MPN gene panel (JAK2 V617F, JAK2 exon12, MPL W515L, MPL W515K, CALR type1-5 ) using DNA microarray methods (SRL International Inc. Japan) and only the JAK2V617F mutation was identified. As the platelet count decreased, his symptoms became well-controlled. However, teardrop-shaped red blood cells and myeloblasts were observed in the peripheral blood six months later, and we performed a bone marrow biopsy. Results indicated hypercellularity (80-100%) with moderate fibrosis (MF grade 1; Figure 1B). Therefore, ruxolitinib was administered for myelofibrosis. Five months later, he showed elevated lactate dehydrogenase (LDH) levels and thrombocytopenia. Bone marrow aspiration revealed increased cellularity with predominant erythropoiesis and 40% erythroblasts (Figure 1C). Flow cytometric analyses revealed 14% glycophorin A and 90% CD34 positive blast cells. No reticulin fibrosis progression was observed. The cytogenetic analysis revealed a normal karyotype. The patient was diagnosed with acute erythroleukemia secondary to PMF. He underwent HLA haploidentical peripheral blood stem cell transplantation(haplo-HCT) from his mother, using post-transplantation cyclophosphamide (PT-Cy) for graft-versus-host disease (GVHD) prophylaxis. The conditioning regimen consisted of total body irradiation (12 Gy delivered in six fractions from days -8 to -6), fludarabine (30mg/m2 from days -5 to -2), and cytarabine (3,000 mg/m2×2 from days -5 to -4). GVHD prophylaxis consisted of high-dose PT-Cy (50 mg/kg intravenously on days 3 and 4) in combination with tacrolimus and mycophenolate mofetil from day 5 onward. Infused donor cells were 5.4×106/kg CD34 cells and 4.0×108/kg CD3 positive T cells. Engraftment occurred on day 21, and complete chimerism was achieved on day 33. He had several transplantation-related complications, including grade II acute GVHD (gut), which was treated with prednisolone; BK virus-associated hemorrhagic cystitis; and bronchiolitis obliterans syndrome (Supplementary Figure). He has been in complete remission for 7 years after transplantation. Considering that the bone marrow features are characterized by increasing cellularity with atypical megakaryocytes at clinical onset, the patient should first be diagnosed with pre-PMF.Our patient developed AML (FAB M6) 11 months after the diagnosis of pre-PMF. In terms of time to progression, median time (range) to progression was 11.8 years (7.9-15.7 years) in pre-PMF8. According to the Dynamic International Prognostic Scoring System (DIPSS) Plus score, our case was classified as low-risk. Candidate genes contributing to LT from MPN to AML have been identified, including TP53, TET2, ASXL1, EZH2, IDH1/2, RUNX1, U2AF1, NRAS/KRAS , and SRSF2 5. The adverse impact of molecular characteristics on survival in pre-PMF and overt PMF has been reported as a high mutation risk in EZH2, ASXL1, IDH1, IDH2 , and SRSF2 8. In our case, no additional somatic alterations were detected; however, a PHF6 mutation was identified. Somatic PHF6 mutations have been found in 2–3% of AML6, 9. The percentage of blasts in the bone marrow tends to be higher in patients with myeloid malignancies harboringPHF6 mutations6. AML with high PHF6expression levels than controls correlated with shorter overall survival10. Furthermore, increased PHF6 levels may be associated with CD34 positivity10. In a case series of MPN with increased fibrosis and blast crisis, 22 patients withPHF6 mutations in MPN were enriched11. Thus,PHF 6 mutations can contribute to myeloid leukemic transformation in JAK2 -mutated pre-PMF.AcknowledgementsWe are grateful to our patients and his family. And we would like to thank our colleagues for helpful discussion regarding this case.Conflict-of-interestThe authors declare that there is no conflict of interest.【References】(1) Tefferi A. Primary myelofibrosis: 2017 update on diagnosis, risk-stratification, and management. Am J Hematol. 2016;91(12):1262-1271.(2) Edahiro Y, Araki M, Inano T, Ito M, Morishita S, Misawa K, Fukuda Y, Imai M, Ohsaka A, Komatsu N. Clinical and molecular features of patients with prefibrotic primary myelofibrosis previously diagnosed as having essential thrombocythemia in Japan. Eur J Haematol. 2019;102(6):516-520.(3) DeLario MR, Sheehan AM, Ataya R, Bertuch AA, Vega C, Webb CR, Lopez-Terrada D, Venkateswaran L. Clinical, histopathologic, and genetic features of pediatric primary myelofibrosis–an entity different from adults. Am J Hematol. 2012;87(5):461-464.(4) Nangalia J, Green TR. The evolving genomic landscape of myeloproliferative neoplasms. Hematology Am Soc Hematol Educ Program. 2014;2014(1):287-296.(5) Andrew J. Dunbar, Raajit K. Rampal, Ross Levine; Leukemia secondary to myeloproliferative neoplasms. Blood 2020;136(1):61–70.(6) Mori T, Nagata Y, Makishima H, Sanada M, Shiozawa Y, Kon A, Yoshizato T, Sato-Otsubo A, Kataoka K, Shiraishi Y, Chiba K, Tanaka H, Ishiyama K, Miyawaki S, Mori H, Nakamaki T, Kihara R, Kiyoi H, Koeffler HP, Shih LY, Miyano S, Naoe T, Haferlach C, Kern W, Haferlach T, Ogawa S, Yoshida K. Somatic PHF6 mutations in 1760 cases with various myeloid neoplasms. Leukemia. 2016;30(11):2270-2273.(7) Muramatsu H, Okuno Y, Yoshida K, Shiraishi Y, Doisaki S, Narita A, Sakaguchi H, Kawashima N, Wang X, Xu Y, Chiba K, Tanaka H, Hama A, Sanada M, Takahashi Y, Kanno H, Yamaguchi H, Ohga S, Manabe A, Harigae H, Kunishima S, Ishii E, Kobayashi M, Koike K, Watanabe K, Ito E, Takata M, Yabe M, Ogawa S, Miyano S, Kojima S. Clinical utility of next-generation sequencing for inherited bone marrow failure syndromes. Genet Med. 2017;19(7):796-802.(8) Guglielmelli P, Pacilli A, Rotunno G, Rumi E, Rosti V, Delaini F, Maffioli M, Fanelli T, Pancrazzi A, Pietra D, Salmoiraghi S, Mannarelli C, Franci A, Paoli C, Rambaldi A, Passamonti F, Barosi G, Barbui T, Cazzola M, Vannucchi AM; AGIMM Group. Presentation and outcome of patients with 2016 WHO diagnosis of prefibrotic and overt primary myelofibrosis. Blood. 2017;129(24):3227-3236.(9) de Rooij JD, van den Heuvel-Eibrink MM, van de Rijdt NK, Verboon LJ, de Haas V, Trka J, Baruchel A, Reinhardt D, Pieters R, Fornerod M, Zwaan CM. PHF6 mutations in paediatric acute myeloid leukaemia. Br J Haematol. 2016;175(5):967-971.(10) Mousa NO, Gado M, Assem MM, Dawood KM, Osman A. Expression profiling of some Acute Myeloid Leukemia - associated markers to assess their diagnostic/prognostic potential. Genet Mol Biol. 2021;44(1):e20190268.(11) Kurzer JH, Weinberg OK. PHF6 Mutations in Hematologic Malignancies. Front Oncol. 2021;11:704471.Figure legendsFigure 1A. Bone marrow aspiration at first visit(day-370).Square (A) shows a separated circular multinucleated megakaryocyte, (B) a hypersegmented megakaryocyte, (C) an atypical megakaryocyte, and (D) a micromegakaryocyte.Figure 1B. Bone marrow biopsy on day-170.Silver impregnation shows moderate myelofibrosis. Arrows indicate reticulin fibers.Figure 1C. Bone marrow smear (1000×, May-Grunwald-Giemsa stain). The smear shows abnormal megakaryoblasts with round or oval nuclei, loose chromatin, agranular cytoplasm with blebs.Supplemental Figure 1. Clinical course of patients.A bone marrow biopsy on day-170 shows myelofibrosis, and bone marrow aspiration on day-30 shows erythroleukemia. aGVHD, acute graft-versus host disease; PSL, prednisolone; TBI, total body irradiation; FLU, fludarabine; Ara-C, cytarabine; CY, cyclophosphamide; MMF, mycophenolate mofetil; Haplo-SCT, haploidentical stem cell transplantation; BMA, bone marrow aspiration; BMB, bone marrow biopsy; BM, bone marrow.

We conducted a cross-sectional study using a questionnaire to explore the late effects in survivors of juvenile myelomonocytic leukemia (JMML). The attending pediatric hematologist oncologists completed the questionnaires. All survivors (N=30) had undergone allogeneic hematopoietic stem cell transplantation. Approximately 83% survivors showed more than one late effect. The identified late effects included endocrine, dental, skin, ophthalmologic, musculoskeletal, pulmonary, neurocognitive, and cardiovascular dysfunction. The prevalence of short stature and cardiovascular and kidney dysfunction was significantly elevated among survivors aged ≥18 years. Therefore, a multidisciplinary follow-up system for survivors of JMML is crucial.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene and is associated with increased susceptibility to cutaneous leiomyomas, uterine leiomyomas, and renal cell carcinoma (RCC). This report describes a seven-year-old male who developed a large right kidney tumor with multiple cystic lesions that contained enhanced solid components. Whole-exome sequencing identified his germline mutation in the FH gene and its loss of heterozygosity in the tumor. This was the youngest-onset case of HLRCC-associated RCC to date. This report may affect the starting age for future RCC-surveillance programs for patients with HLRCC.

Background: Patients with relapsed or refractory neuroblastoma have a poor prognosis; there are limited effective and safe rescue chemotherapies for these patients. Development of new chemotherapy regimens for these patients is a key imperative. Procedure: We retrospectively analyzed patients with refractory or relapsed neuroblastoma who received irinotecan, etoposide, and carboplatin (IREC) as a second-line treatment for neuroblastoma. We evaluated the therapeutic response, toxicity, and survival outcomes. We also assessed the impact of UGT1A1 gene polymorphisms, which are involved in irinotecan metabolism, on the outcomes and toxicity. Results: A total of 131 cycles of IREC were administered to 43 patients with a median of two cycles per patient (range, 1–10). All patients were classified as high-risk (International Neuroblastoma Risk Group). Seven patients had relapsed before IREC. One patient (2%) showed partial response and 37 patients (86%) developed stable disease (disease control rate: 88%). Grade IV neutropenia was observed in 127 cycles (97%), while ≥ grade III gastrointestinal toxicity was observed in 3 cycles (2%). There was no IREC-related mortality. The one-year overall survival and progression-free survival rates were 65% and 52%, respectively. Patients with UGT1A1 polymorphisms showed a higher frequency of grade IV neutropenia; however, there was no increase in treatment-related mortality or nonhematological toxicity in these patients. Patients with UGT1A1 gene polymorphisms showed better one-year survival rate than the wild type (80% vs. 44%, p = 0.012). Conclusions: This study suggests that IREC is well-tolerated by patients with UGT1A1 polymorphisms and is a promising second-line chemotherapy for refractory/relapsed neuroblastoma.

Background: Central venous catheters (CVCs) have been essential devices for the treatment of children with hematological and oncological disorders. Only few studies investigated the complications and selections of different types of CVCs in these pediatric patients. This study aimed to compare risk factors for unplanned removal of two commonly used CVCs, i.e., peripherally inserted central catheters (PICCs) and tunneled CVCs, and propose better device selection for the patient. Procedure: This retrospective, single center cohort analysis was conducted on pediatric patients with hematological and oncological disorders inserted with either a PICC or a tunneled CVC. Results: Between January 1, 2013, and December 31, 2015, 89 patients inserted with tunneled CVCs (total 21,395 catheter-days) and 84 with PICCs (total 9,177 catheter-days) were followed up until the catheter removal. The median duration of catheterization was 88 days in PICCs and 186 days in tunneled CVCs (p = 1.24×10-9). PICCs at the 3-month cumulative incidence of catheter occlusion (5.2% vs. 0%, p = 4.08×10-3) and total unplanned removal (29.0% vs 7.0%, p = 0.0316) were significantly higher, whereas no significant difference was observed in the cumulative incidence of central line-associated bloodstream infection (11.8% vs. 2.3%, p = 0.664). Multivariable analysis identified younger age (<2 years) (subdistribution hazard ratio [SHR], 2.29; 95% confidence interval [CI], 1.27–4.14) and PICCs (SHR, 2.73; 95% CI, 1.48–5.02) were independent risk factors for unplanned removal. Conclusion: Our results suggest that tunnel CVCs would be a preferred device for children with hematological and oncological disorders requiring long-term, intensive treatment.

Submission type: Correspondence and LettersAzacitidine as a bridge to allogeneic stem cell transplantation in a patient with juvenile myelomonocytic leukemia: a case reportAkira Takebayashi, MD1, Tsukasa Hori, MD, PhD1, Masaki Yamamoto, MD, PhD1, Takeshi Tsugawa, MD, PhD1, Keita Igarashi, MD1, Kotoe Iesato, MD, PhD1, Ryo Hamada, MD1, Hideki Muramatsu, MD, PhD2, Yukihiko Kawasaki, MD, PhD11Departments of Pediatrics, Sapporo Medical University School of Medicine, Sapporo, Japan2Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, JapanCorresponding author : Akira Takebayashi, MD, 291, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan. Phone: +81-11-611-2111; Fax: +81-11-611-0352; E-mail: [email protected] Words : JMML, juvenile myelomonocytic leukemia, azacitidine, bridge, bridging therapy, stem cell transplantationRunning title : Azacitidine as a bridging therapy to JMMLWords count : 488 words, 1 table.Conflict of interest statement: The authors have no conflict of interest to declare.To the Editor:Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic /myeloproliferative neoplasm in infants and toddlers. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative therapy for most patients, but no appropriate pre-HSCT bridging therapy has been established. Since Furlan1 reported the effectiveness of azacitidine in pre-HSCT JMML, several reports have been published. Here we report a case of JMML treated with azacitidine.A 6-month-old male presented with hepatosplenomegaly and thrombocytopenia. Laboratory examinations revealed monocytosis (8.5 × 109 /L), mild anemia (hemoglobin: 10.2 g/dL), thrombocytopenia (17 × 109/L), and 11.7% hemoglobin F. Myeloblasts were peripheral blood: 2.0%; bone marrow: 8.2%. Fluorescence in situ hybridization detected monosomy-7 cells. Molecular analysis found a somatic KRAS p.G13D mutation, and he was diagnosed with JMML.We planned an unrelated bone marrow transplantation (UR-BMT) because an HLA-matched related donor was unavailable, and used 6-mercaptoprine (6-MP) as a bridging therapy. However, he still needed frequent transfusions and his hepatosplenomegaly was exacerbated (TABLE 1). We therefore changed the bridging therapy from 6-MP to azacitidine (2.5 mg/kg, over 1 hour intravenously on 7 consecutive days, every 28 days; TABLE 1). Platelet transfusion frequency decreased and he became transfusion-independent after azacitidine Cycle 6. Peripheral blood myeloblasts disappeared after Cycle 6, and the hepatosplenomegaly had completely improved after Cycle 8. Monosomy-7 cells significantly decreased, but did not disappear until his UR-BMT. He maintained clinical partial remission and genetic stable disease after Cycle 1, evaluated with response criteria defined by C. M. Niemeyer et al. Adverse effects of azacitidine included febrile neutropenia after Cycle 1 and exacerbated thrombocytopenia during Cycles 1 and 2. However, he suffered no adverse effects after Cycle 3.UR-BMT from an HLA 7/8 matched donor was performed after azacitidine Cycle 8, when the patient was in partial remission. His conditioning regimen included busulfan, fludarabine and melphalan; a short course of methotrexate and tacrolimus was used as prophylaxis against for graft-versus-host disease. He remains in complete remission with complete donor chimerism at 27 months after UR-BMT.Azacitidine is a DNA-hypomethylating agent used for myelodysplastic syndrome in adults, but is not generally used for JMML. Cseh reported twelve patients with JMML treated with azacitidine,3of whom nine patients were treated with azacitidine before HSCT and five achieved partial or complete remission. The remaining four received HSCT during progressive disease after 1–4 cycles of azacitidine therapy. Although the optimal number of azacitidine cycles is unknown, continuing azacitidine therapy for six to eight cycles might be valuable if the patient can tolerate it.Another infant with JMML, somatic KRAS mutation and monosomy-7 who achieved sustained remission following azacitidine monotherapy had been reported,4 but identifying patients who can sustain remission without HSCT is not currently possible. Reportedly, DNA methylation patterns in JMML can be predictive for prognosis,5,6 but whether they can predict response to hypomethylating agents or not is unclear. Although azacitidine bridging therapy is effective for JMML patients, further studies are needed to clarify several questions about JMML treatment.

Microsatellites are a set of repeating base sequences of one to several bases in a chromosome. In general, mismatch repair (MMR) proteins correct the base mismatches that occur during DNA replication. However, tumor cells with deficient MMR function accumulate genetic mutations and cause changes in the repeat counts in microsatellite sites, and such a status is referred to as microsatellite instability (MSI)-high status. According to recent research, MSI-high status is associated with responsiveness to therapies with immune checkpoint inhibitors [1].MSI status has been well described in various adult solid tumors; for instance, one of the largest studies has demonstrated that 1188 (9.9%) of 12,019 patients exhibited an MSI-high signature in various types of tumors [2]. However, there are no sufficient investigations on the MSI status in pediatric solid tumors, except those on limited tumor subtypes, including glioblastoma and medulloblastoma [3, 4]. Herein, we investigated the MSI status in pediatric patients with various solid tumors who died due to the tumor and also evaluated the potential of immune checkpoint inhibitors in refractory pediatric solid tumors.From April 2000 to May 2019, a total of 334 pediatric patients with solid tumors were admitted to the Nagoya University Hospital (Table 1 ). Although the majority of patients survived, 74 (22%) died, including 68 due to relapse or refractory tumor, 4 due to pulmonary complications after stem cell transplantations, and 2 due to infection after chemotherapies. We retrospectively analyzed the formalin-fixed paraffin-embedded tumor tissues of 40 (54%) of the 74 patients who died to assess the MSI status (Supplemental Table 1 , Supplemental Figure 1 ) using five multiplexed markers for determining the MSI-high phenotype (BAT-25, BAT-26, MONO-27, NR-21, and NR-24) (Supplemental methods ). Results demonstrated that 36 cases were microsatellite-stable and none of the patients had an MSI-high status; however, this observation could not be confirmed for the remaining four patients because of poor sample quality.These results indicate that MSI-high status is rare in pediatric patients with solid tumors who die of the disease. Therefore, surveillance of MSI status in children with refractory/relapsed solid tumors might have a limited role in predicting the responsiveness to immune checkpoint inhibitors.