Allogeneic Hematopoietic cell Transplantation Using Alemtuzumab in Asian Patients with Inborn Errors of Immunity.

Alemtuzumab is used with reduced-toxicity conditioning (RTC) in allogeneic hematopoietic cell transplantation (HCT), demonstrating efficacy and feasibility for patients with inborn errors of immunity (IEI) in Western countries; however, the clinical experience in Asian patients with IEI is limited. We retrospectively analyzed patients with IEI who underwent the first allogeneic HCT with alemtuzumab combined with RTC regimens in Japan. A total of 19 patients were included and followed up for a median of 18 months. The donors were haploidentical parents (n = 10), matched siblings (n = 2), and unrelated bone marrow donors (n = 7). Most patients received RTC regimens containing fludarabine and busulfan and were treated with 0.8 mg/kg alemtuzumab with intermediate timing. Eighteen patients survived and achieved stable engraftment, and no grade 3-4 acute graft-versus-host disease was observed. Viral infections were observed in 11 patients (58%) and 6 of them presented symptomatic. The median CD4+ T cell count was low at 6 months (241/µL) but improved at 1 year (577/µL) after HCT. Whole blood cells continued to exhibit > 80% donor type in most cases; however, 3/10 patients exhibited poor donor chimerism only among T cells and also showed undetectable levels of T-cell receptor recombination excision circles (TRECs) at 1 year post-HCT. This study demonstrated the efficacy and safety of alemtuzumab; however, patients frequently developed viral infections and slow reconstitution or low donor chimerism in T cells, emphasizing the importance of monitoring viral status and T-cell-specific chimerism. (238 < 250 words).

Histopathological maturation in juvenile xanthogranuloma: a blueberry muffin infant mimicking aleukemic leukemia cutis.

Juvenile xanthogranuloma (JXG) is usually identified by Touton giant cells, so their absence can complicate diagnosis. We encountered a case of non-typical neonatal JXG lacking Touton giant cells, which was difficult to differentiate from aleukemic leukemia cutis because of overlapping histopathological characteristics. A 1 month-old girl presented with a blueberry muffin rash and multiple 1-2 cm nodules within the subcutaneous and deeper soft tissues. Blood tests revealed pancytopenia. The initial nodule biopsy showed mononuclear cell infiltration, suggestive of mature monocytes or histiocytes, but no Touton giant cells. Bone marrow examination showed no evidence of leukemia. Despite worsening of the rash, pancytopenia, and weight gain over the following month, the results of the second biopsy remained consistent with the initial findings. Consequently, we provisionally diagnosed aleukemic leukemia cutis and initiated chemotherapy. After two courses of chemotherapy, the pancytopenia improved, but the nodules only partially regressed. A third biopsy of the nodule was performed to evaluate the histological response, and revealed Touton giant cells, confirming the diagnosis of JXG. In conclusion, distinguishing non-typical JXG from aleukemic leukemia cutis is challenging. This case highlights the importance of multiple biopsies and the potential for histopathological maturation.

A case of neurodegenerative Langerhans cell histiocytosis exhibiting high osteopontin in cerebrospinal fluid without specific radiological abnormalities.

Neurodegenerative Langerhans cell histiocytosis (ND-LCH) manifests several years after onset of LCH, with progressive neurological symptoms and characteristic brain imaging features. Although ND-LCH has a dismal neurological prognosis, distinct treatment strategies are not available owing to the unknown pathophysiology. We describe the case of a 6-year-old boy who developed left convergent strabismus four years after onset of multisystem LCH (MS-LCH). Although radiological imaging showed no abnormalities, the osteopontin level in the cerebrospinal fluid (CSF-OPN) was highly elevated without other abnormal CSF findings, leading to a diagnosis of ND-LCH. The patient received monthly intravenous immunoglobulin therapy for four years, without symptoms worsening. To investigate the relevance of OPN levels in LCH, we retrospectively analyzed serum and CSF OPN levels in eight LCH patients. Serum OPN levels were markedly elevated in the two MS-LCH patients with macrophage activation (400 and 445 ng/mL) compared to the other six patients (mean: 59 ng/mL). CSF-OPN levels were elevated in the ND-LCH patient (620 ng/mL) compared to the two patients with pituitary involvement (160 and 182 ng/mL), suggesting that the pathophysiology of ND-LCH reflects its inflammatory status. Analysis of CSF-OPN levels would be a useful tool to detect and treat ND-LCH.

L-asparaginase as an efficient salvage therapy for refractory acute myeloid leukemia with chromosome 7 abnormalities: a case series.

Acute myeloid leukemia (AML) with chromosome 7 abnormalities has a dismal prognosis due to a poor complete remission (CR) rate after induction chemotherapy. Although various salvage therapies for refractory AML have been developed for adults, few salvage therapies are available for children. Here, we report the cases of three patients with refractory AML with chromosome 7 abnormalities (Patient 1, with inv(3)(q21;3q26.2) and monosomy 7; Patient 2, with der(7)t(1;7)(?;q22); patient 3, with monosomy 7) who were successfully treated with L-asparaginase (L-ASP) as salvage therapy. All three patients achieved CR several weeks after L-ASP treatment, and two patients successfully underwent hematopoietic stem cell transplantation (HSCT). Patient 2 relapsed after the second HSCT in the form of an intracranial lesion, but achieved and sustained CR for 3 years with weekly L-ASP maintenance therapy. Immunohistochemical staining for asparagine synthetase (ASNS), whose gene is located at 7q21.3, was performed for each patient. The result was negative in all patients, which suggests that haploid 7q21.3 and other chromosome 7 abnormalities leading to haploinsufficiency of ASNS contribute to a high susceptibility to L-ASP. In conclusion, L-ASP is a promising salvage therapy for refractory AML with chromosome 7 abnormalities, which are associated with ASNS haploinsufficiency.

Protocol for a high titer of BaEV-Rless pseudotyped lentiviral vector: Focus on syncytium formation and detachment.

The development of hematopoietic stem cell (HSCs) gene therapy for DNA repair disorders, such as Fanconi anemia and Bloom syndrome, is challenging because of the induction of HSCs apoptosis by cytokine stimulation. Although the Baboon envelope pseudotyped lentiviral vector (BaEV-Rless-LV) has been reported as a non-stimulatory gene transfer tool, the virus titer of BaEV-Rless-LV is too low for use in clinical applications. Transfected 293 T cells with helper plasmids, including the BaEV-Rless plasmid, showed morphological changes, such as syncytium formation and detachment. To establish a novel protocol for producing a high titer of BaEV-Rless-LV, we optimized three aspects of a basic virus production protocol by focusing on modifying culture conditions and the use of reagents: the virus titer increased 3-fold when the amount of BaEV-Rless plasmid was increased 1.2-fold; the highest titer was obtained when the viral supernatant was harvested at 48-h post-transfection, despite complete syncytium formation and detachment of the 293 T cells; and the use of poly-L-lysine-coated culture plates to enhance the adhesion and proliferation of 293 T cells and prevent detachment doubled the titer. Collectively, our novel protocol resulted in a 10-fold titer increase compared to the basic protocol and may be useful in clinical applications for treating DNA repair disorders.

Pediatric inflammatory myofibroblastic tumor of the bladder with ALK-FN1 fusion successfully treated by alectinib.

An inflammatory myofibroblastic tumor (IMT) is a mesenchymal neoplasm characterized by the proliferation of myofibroblasts and inflammatory cell infiltration. Although radical resection is the only established treatment strategy for IMT, it can cause functional disorders when vital organs are affected. We describe a case of pediatric IMT of the bladder with FN1-ALK (fibronectin 1-anaplastic lymphoma kinase) fusion. Radical resection might lead to urinary disturbance due to the large tumor size at diagnosis. However, the tumor was successfully treated with alectinib, a second-generation ALK inhibitor, followed by transurethral resection of the bladder tumor without any complications.

Presence of identical B-cell clone in both cerebrospinal fluid and tumor tissue in a patient with opsoclonus-myoclonus syndrome associated with neuroblastoma.

Opsoclonus-myoclonus syndrome associated with neuroblastoma (OMS-NB) is a refractory paraneoplastic syndrome which often remain neurological sequelae, and detailed pathogenesis has remained elusive. We encountered a pediatric patient with OMS-NB treated by immunosuppressed therapy who showed anti-glutamate receptor δ2 antibody and increased B-cells in cerebrospinal fluid (CSF), and multiple lymphoid follicles containing abundant Bcells in tumor tissue. Unbiased B-cell receptor repertoire analysis revealed identical B-cell clone was identified as the dominant clone in both CSF and tumor tissue. These identical B-cell clone may contribute to the pathogenesis of OMS-NB. Our results could facilitate the establishment of pathogenesis-based treatment strategies for OMS-NB.

Refractory gastroduodenal ulcers: A rare complication with Bloom syndrome.

Bloom syndrome patients often develop severe gastrointestinal symptoms mainly caused by gastric tumors due to DNA repair disorder. Here, we report 31-year-old Bloom syndrome patient suffering persistent abdominal pain due to refractory gastroduodenal ulcers which required gastroduodenectomy. Various causes should be considered, and the accumulation of their reports is warranted.

Automation of hybridization and capture based next generation sequencing library preparation requires reduction of on-deck bead binding and heated wash temperatures.

Capture-based library preparation for next generation sequencing (NGS) offers a balance between sequencing depth and bioinformatics cost of analysis. Liquid handling automation enhances the reliability of the library preparation process by reducing sample-to-sample variation and substantially enhances throughput, particularly when it can be employed in a 'walk-away' fashion with limited hands-on interaction. This requires complex series of mixing and heating steps like those utilized in capture chemistries to happen on the liquid handler. While developing liquid handling automation for Integrated DNA Technologies (IDT) xGen Exome, Illumina TruSight Oncology 500, and Personal Genome Diagnostics (PGDx) elio Plasma Resolve chemistries on the PerkinElmer Sciclone liquid handler, we found that applying the capture temperatures recommended for manual library preparation results in low yield on automation. To restore the final library yield, we reduced bead binding and/or heated wash temperatures of the Peltier heaters on the liquid handlers by about 10°C. Since this applied across three unique capture-based chemistries, we consider this a generalizable principle of automating capture on the Sciclone. We hypothesize that this is driven by the very different thermodynamic environments represented by a sealed plate on a thermal cycler and a plate with a lid on a Peltier heater. This phenomenon should be considered when automating NGS library preparation on PerkinElmer Sciclone instruments.

Utility of 18F-FDG-PET for detecting acute lymphoblastic leukemia: a case series of pediatric acute lymphoblastic leukemia without hematological symptoms.

Acute leukemia is typically diagnosed from presenting features related to hematological symptoms, but certain patients present with prominent musculoskeletal pain without signs of hematological abnormality. We reviewed the medical records of 58 children diagnosed with acute lymphoblastic leukemia (ALL) at our hospital to evaluate initial features. Forty six of these patients had hematological symptoms, anemia, or hemorrhage (Group H), while 12 patients had prominent musculoskeletal pain without hematological symptoms (Group P). Diagnosis of leukemia took significantly more time for those 12 patients (Group H, 17.1 days; Group P, 48.5 days). In three of the 12 patients in Group P, localized abnormal imaging findings and unremarkable blood test results led to initial diagnoses of chronic recurrent multifocal osteomyelitis, bone fracture, and septic osteomyelitis. However, 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET) revealed multiple intense bone foci or systemic bone marrow uptake, leading to the diagnosis of ALL. A review of 18F-FDG-PET results from 23 patients with ALL who underwent a PET scan (Group H, n = 15; Group P, n = 8) showed multiple bone foci or systemic bone marrow uptake in all cases. In conclusion, lack of hematological symptoms in ALL patients can delay diagnosis, and 18F-FDG-PET is useful for diagnosing leukemia in such cases.

Common Oncogene Mutations and Novel SND1-BRAF Transcript Fusion in Lung Adenocarcinoma from Never Smokers.

Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%), and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1-9 of SND1 and exons 2 to 3' end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation, and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy.

Comparison of fluorescence in situ hybridization (FISH) and dual-ISH (DISH) in the determination of HER2 status in breast cancer.

The determination of HER2 amplification is critical to selecting appropriate patients for HER2 targeted therapy in breast cancer. Dual in situ hybridization (DISH), an alternative to fluorescence in situ hybridization (FISH) and immunohistochemistry, is now available. To compare the FISH and DISH methods, we tested 251 samples enriched for common or difficult-to-assess HER2 anomalies. Seven samples failed DISH testing. There was a 64% (156/244) concordance between FISH and DISH by anomaly (κ = 0.58, 95% confidence interval, 0.51-0.65; P < .0001) and an 83% (203/244) concordance by amplification status (κ = 0.58; 95% confidence interval, 0.47-0.69; P < .0001). DISH resulted in lower estimates of HER2/ centromere 17 ratios than FISH, and many cases that were equivocal with FISH were normal with DISH. DISH did not detect any case with coamplification of HER2 and centromere 17. Using a cohort of difficult specimens, we observed less than 95% concordance between FISH and DISH. DISH may underestimate the HER2/chromosome 17 ratio, or FISH may overestimate this ratio.

Tolerance to solar ultraviolet-B radiation in the citrus red mite, an upper surface user of host plant leaves.

Plant-dwelling mites are potentially exposed to solar ultraviolet-B (UVB) radiation that causes deleterious and often lethal effects, leading most mites to inhabit the lower (underside) leaf surfaces. However, in species of spider mite belonging to the Genus Panonychus, a substantial portion of individuals occur on upper leaf surfaces. We investigated whether the upper leaf surfaces of citrus trees are favorable for P. citri, and to what extent they are tolerant to UVB radiation. If eggs are not adequately protected from UVB damage, females may avoid ovipositing on the upper surfaces of sunny leaves. To test this, we conducted laboratory experiments using a UVB lamp, and semioutdoor manipulative experiments. As a result, P. citri eggs are tolerant to UVB. Field studies revealed that the ratio of eggs and adult females on upper leaf surfaces were larger for shaded than for sunny leaves. However, 64-89% of eggs hatched successfully even on sunny upper leaf surfaces. Nutritional evaluation revealed that whether on sunny or shaded leaves, in fecundity and juvenile development P. citri reaped the fitness benefits of upper leaf surfaces. Consequently, P. citri is tolerant to UVB damage, and inhabiting the upper surfaces of shaded leaves is advantageous to this mite.

Spectrum-specific damage and solar ultraviolet radiation avoidance in the two-spotted spider mite.

The spatial distribution of the two-spotted spider mite Tetranychus urticae Koch is biased toward the lower surfaces of leaves as compared with the upper leaf surfaces on their host plants. Because of the deleterious effects of solar ultraviolet (UV) irradiation, we hypothesized T. urticae remains on lower leaf surfaces as an adaptation to avoid solar UV radiation (UVR). We examined the effects of solar UVR components on females and tested whether spatial distribution was associated with solar UVR avoidance. Attenuation of solar UVR using UV opaque film increased fecundity and reduced the movement of females from the upper to the lower leaf surfaces. In contrast, diverting solar UVR to the lower leaf surface using a light reflection sheet caused the mites to move from the lower to the upper leaf surfaces; however, attenuated UV reflection did not, suggesting that they occupy the lower leaf surface to avoid solar UVR. In monochromatic UVR tests, no eggs hatched when placed under 280-300 nm radiation, whereas almost all eggs hatched at 320-360 nm. Adult females, however, did not avoid wavelengths of 280 and 300 nm, but avoided 320-340 nm. We conclude that T. urticae exploit UVA information to avoid ambient UVB radiation.

Toxicology of engineered nanomaterials - a review of carcinogenic potential.

Nanotechnology has considerable socioeconomic potential. Benefits afforded by engineered nanoparticles (NP: defined as being less than 100 nm in diameter) are expected to be significant in fields such as plastics, energy, electronics, aerospace and medicine. However, NPs are being introduced into the market without adequate assessment of their potential toxicities. It is urgently important to conduct risk assessment of commercial NPs and establish a framework enabling risk management which is not subordinate to their commercial production. An overview of currently available carcinogenicity risk evaluation results of NP materials raises serious questions as to their safety. NP sized titanium dioxide (nTiO(2)) and carbon black (nCB) are carcinogenic to the lung of female rats, and the tumors preferentially include squamous cell morphology. Carbon nanotubes (CNT) induce mesotheliomas when applied intraperitoneally in rats and mice. Data for Fullerenes are insufficient to evaluate carcinogenic risk. Sub-chronic toxicity data indicate that, in general, NPs form aggregates and agglomerates and cause foreign body reactions at their applied sites with inflammatory cell, including macrophage, infiltration. These findings are similar to the biological effects of asbestos, a potent carcinogen, and indicate that careful assessment of NPs is indispensable.

Advanced malignant solitary fibrous tumor in pelvis responding to radiation therapy.

Solitary fibrous tumor (SFT) is a rare spindle cell neoplasm that is benign in most cases. Although SFT was first recognized to arise only in the pleura, recent reports indicate that SFT can involve a wide range of anatomical sites. To date, 17 cases of pelvic SFT have been reported. Herein is reported a case of a 74-year-old woman with a giant malignant SFT in the pelvis. Along with massive invasion to adjacent organs and multiple lung metastases detected on radiography, biopsy from the tumor through the vaginal wall showed malignant looking spindle-cell neoplasm with increased cellularity, areas of necrosis, and high mitotic activity (5/10 high-power fields). Immunohistochemically, the tumor cells were diffusely and strongly positive for CD34, CD99, and bcl-2. Based on pathological features and clinical presentation, diagnosis of malignant SFT was made. The patient received systemic and the intra-arterial chemotherapy followed by whole pelvic radiation therapy (50 Gy). Initial chemotherapies failed to control the tumor. Afterwards, improvement was observed radiologically and pathologically in the 12 months' follow up after the radiation therapy. This is the first report related to therapeutic remarks on advanced malignant SFT.