A quantification method of somatic mutations in normal tissues and their accumulation in pediatric patients with chemotherapy.

Somatic mutations are accumulated in normal human tissues with aging and exposure to carcinogens. If we can accurately count any passenger mutations in any single DNA molecule, since their quantity is much larger than driver mutations, we can sensitively detect mutation accumulation in polyclonal normal tissues. Duplex sequencing, which tags both DNA strands in one DNA molecule, enables accurate count of such mutations, but requires a very large number of sequencing reads for each single sample of human-genome size. Here, we reduced the genome size to 1/90 using the BamHI restriction enzyme and established a cost-effective pipeline. The enzymatically cleaved and optimal sequencing (EcoSeq) method was able to count somatic mutations in a single DNA molecule with a sensitivity of as low as 3 × 10-8 per base pair (bp), as assessed by measuring artificially prepared mutations. Taking advantages of EcoSeq, we analyzed normal peripheral blood cells of pediatric sarcoma patients who received chemotherapy (n = 10) and those who did not (n = 10). The former had a mutation frequency of 31.2 ± 13.4 × 10-8 per base pair while the latter had 9.0 ± 4.5 × 10-8 per base pair (P < 0.001). The increase in mutation frequency was confirmed by analysis of the same patients before and after chemotherapy, and increased mutation frequencies persisted 46 to 64 mo after chemotherapy, indicating that the mutation accumulation constitutes a risk of secondary leukemia. EcoSeq has the potential to reveal accumulation of somatic mutations and exposure to environmental factors in any DNA samples and will contribute to cancer risk estimation.

Morphologic Features of Invasion in Lung Adenocarcinoma: Diagnostic Pitfalls.

Reproducibility of pulmonary invasive adenocarcinoma diagnosis is poor when applying the World Health Organization (WHO) classification. In this article, we aimed first to explain by 3-dimensional morphology why simple pattern recognition induces pitfalls for the assessment of invasion as applied in the current WHO classification of pulmonary adenocarcinomas. The underlying iatrogenic-induced morphologic alterations in collapsed adenocarcinoma in situ overlap with criteria for invasive adenocarcinoma. Pitfalls in seemingly acinar and papillary carcinoma are addressed with additional cytokeratin 7 and elastin stains. In addition, we provide more stringent criteria for a better reproducible and likely generalizable classification.

High-efficiency EGFR genotyping using cell-free DNA in bronchial washing fluid.

BACKGROUND: EGFR mutation testing is required for treatment of lung adenocarcinoma using epidermal growth factor receptor-tyrosine kinase inhibitor. However, the amounts of tumor tissue or tumor cells obtained by bronchoscopy are often insufficient. Bronchial washing fluid, obtained by lavage with saline after tumor biopsy or brushing, and the supernatant of bronchial washing fluid are thought to contain cell-free DNA that would be potentially applicable for EGFR testing. METHODS: From among patients with suspected adenocarcinoma or non-small cell lung carcinoma diagnosed from biopsy or surgical specimens at the University of Tsukuba Hospital between 2015 and 2019, cell-free DNAs from 80 specimens of supernatant of bronchial washing fluid (50 with EGFR mutation and 30 with wild type EGFR) and 8 blood serum samples were examined for EGFR mutation using droplet digital PCR. RESULTS: Among the 50 patients harboring EGFR mutation, the rate of positivity for cell-free DNA extracted from supernatant of bronchial washing fluid was 80% (40/50). In nine of the EGFR mutation-positive cases, tumor cells were not detected by either biopsy or cytology, but the mutation was detected in four cases (4/9, 44%). Comparison of the cell-free DNA mutation detection rate between supernatant of bronchial washing fluid and blood serum in six cases showed that mutations were detected from the former in all cases (6/6, 100%), but from the latter in only one case (1/6, 17%). CONCLUSIONS: Using supernatant of bronchial washing fluid samples, the detection rate of EGFR mutation was high, and EGFR mutations were detectable even when no tumor cells had been detectable by biopsy or cytology. Supernatant of bronchial washing fluid might be an effective sample source for EGFR mutation testing.

High expression of eukaryotic elongation factor 1-alpha-2 in lung adenocarcinoma is associated with poor prognosis.

Eukaryotic elongation factor 1 alpha 2 (eEF1A2) encodes an isoform of the alpha subunit of the elongation factor 1 complex and is responsible for the enzymatic delivery of aminoacyl tRNA to the ribosome. Our proteomic analysis has identified eEF1A2 as one of the proteins expressed during malignant progression from adenocarcinoma in situ (AIS) to early invasive lung adenocarcinoma. The expression level of eEF1A2 in 175 lung adenocarcinomas was examined by immunohistochemical staining in relation to patient prognosis and clinicopathological factors. Quantitative PCR analysis and fluorescence in situ hybridization (FISH) were performed to evaluate the amplification of the eEF1A2 gene. Relatively high expression of eEF1A2 was observed in invasive adenocarcinoma (39/144 cases) relative to minimally invasive adenocarcinoma (1/10 cases) or AIS (0/21 cases). Among invasive adenocarcinomas, solid-type adenocarcinoma (15/32 cases, 47%) showed higher expression than other histological subtypes (23/92, 25%). Patients with eEF1A2-positive tumors had a significantly poorer prognosis than those with eEF1A2-negative tumors. Of the five tumors that were eEF1A2-positive, two cases showed amplified genomic eEF1A2 DNA, which was confirmed by both qPCR and FISH. These findings indicate that eEF1A2 overexpression occurs in the course of malignant transformation of lung adenocarcinomas and is partly due to eEF1A2 gene amplification.

Elastin in pulmonary pathology: relevance in tumours with a lepidic or papillary appearance. A comprehensive understanding from a morphological viewpoint.

Elastin and collagen are the main components of the lung connective tissue network, and together provide the lung with elasticity and tensile strength. In pulmonary pathology, elastin staining is used to variable extents in different countries. These uses include evaluation of the pleura in staging, and the distinction of invasion from collapse of alveoli after surgery (iatrogenic collapse). In the latter, elastin staining is used to highlight distorted but pre-existing alveolar architecture from true invasion. In addition to variable levels of use and experience, the interpretation of elastin staining in some adenocarcinomas leads to interpretative differences between collapsed lepidic patterns and true papillary patterns. This review aims to summarise the existing data on the use of elastin staining in pulmonary pathology, on the basis of literature data and morphological characteristics. The effect of iatrogenic collapse and the interpretation of elastin staining in pulmonary adenocarcinomas is discussed in detail, especially for the distinction between lepidic patterns and papillary carcinoma.

Mutational landscape of primary breast angiosarcoma with repeated resection and recurrence over a 15-year period: A case report.

Angiosarcoma is a rare malignant tumor derived from vascular endothelial cells and has a poor prognosis. We have experienced a case of multiple breast angiosarcoma for which multiple resections had been performed during the course of its progression over a period of more than 15 years, allowing comprehensive genetic mutation analysis. Somatic mutations in several cancer-related genes were detected, but no previously reported driver gene mutations of angiosarcoma were evident. Several germline mutations associated with malignancy, such as single nucleotide polymorphisms in Fibroblast Growth Factor Receptor 4 (FGFR4) (p.Gly388Arg, rs351855), Kinase Insert Domain Receptor (KDR) (Gln472His, rs1870377) and tumor protein p53 (TP53) (p.Pro72Arg, rs1042522) were detected. Common signatures and genetic mutations were scarce in the tumor samples subjected to genetic mutational analysis. These findings suggested that this case was very probably a multiprimary angiosarcoma.

Aberrant OCIAD2 demethylation in lung adenocarcinoma is associated with outcome.

Overexpression of OCIAD2 in lung adenocarcinoma has already been reported in several research articles, but the molecular mechanism involved remains unknown. Promoter CpG methylation is a representative form of epigenetic gene regulation, and a considerable number of tumor suppressor genes show hypermethylation in many cancers. In contrast, promoter CpG hypomethylation causes oncogene overexpression, resulting in carcinogenesis and malignant progression. In the present study, we investigated the CpG methylation and expression status of OCIAD2 using tumor tissues and adjacent normal tissues from seven cases of lung adenocarcinoma. We also examined the relationship between CpG methylation status and outcome in 58 patients with adenocarcinoma. Pyrosequencing showed that CpG sites in OCIAD2 promoter regions were more frequently demethylated in tumor tissues than in adjacent normal tissues, and reverse transcription-quantitative polymerase chain reaction revealed overexpression of OCIAD2 in lung adenocarcinoma. There was a correlation between OCIAD2 CpG demethylation and the level of mRNA expression, and statistical analysis showed that CpG hypomethylation of OCIAD2 was associated with poor outcomes. Our results suggest that overexpression of OCIAD2 might be caused mainly by CpG hypomethylation and that OCIAD2 methylation status might be a useful prognostic indicator in lung adenocarcinoma.

Social Capital and Post-traumatic Stress Disorder among Heavy Rainfall and Flood Victims in Japan.

This study examined the relationship between cognitive/structural social capital and post-traumatic stress disorder (PTSD) among victims of heavy rain and flood. Participants were individuals aged≥18 years affected by the July 2018 heavy rainfall in the cities of Kurashiki and Soja, Japan, and living in temporary housing. We distributed five copies of a questionnaire to 1,991 households and received responses from 1,927 individuals (907 men, 1,008 women, 12 respondents of unspecified sex) in 1,029 households (51.7%). We estimated odds ratios (ORs) and 95% confidence intervals (CIs) for associations between high (vs. low) social capital and PTSD or other outcomes. After covariate adjustment, the odds of having PTSD were lower in participants with high cognitive social capital than those with low cognitive social capital (OR=0.346, 95%CI: 0.263-0.456). Elderly women with higher structural social capital tended to have lower PTSD odds than those with lower structural social capital (OR=0.671, 95%CI: 0.431-1.046). The opposite pattern was observed for elderly men (OR=1.315, 95%CI: 0.792-2.183). Cognitive social capital is a protective factor that may reduce PTSD or promote a favorable PTSD prognosis after heavy rainfall and flood events. The associations between structural social capital and PTSD differ by age and sex.

Mechanomics Biomarker for Cancer Cells Unidentifiable through Morphology and Elastic Modulus.

Cellular mechanical properties are potential cancer biomarkers used for objective cytology to replace the current subjective method relying on cytomorphology. However, heterogeneity among intra/intercellular mechanics and the interplay between cytoskeletal prestress and elastic modulus obscured the difference detectable between malignant and benign cells. In this work, we collected high density nanoscale prestress and elastic modulus data from a single cell by AFM indentation to generate a cellular mechanome. Such high dimensional mechanome data was used to train a malignancy classifier through machine learning. The classifier was tested on 340 single cells of various origins, malignancy, and degrees of similarity in morphology and elastic modulus. The classifier showed instrument-independent robustness and classification accuracy of 89% with an AUC-ROC value of 93%. A signal-to-noise ratio 8 times that of the human-cytologist-based morphological method was also demonstrated, in differentiating precancerous hyperplasia cells from normal cells derived from the same lung cancer patient.

Gene expression profiles of the original tumors influence the generation of PDX models of lung squamous cell carcinoma.

Patient-derived xenograft (PDX) murine models are employed for preclinical research on cancers, including non-small cell lung cancers (NSCLCs). Even though lung squamous cell carcinomas (LUSCs) show the highest engraftment rate among NSCLCs, half of them nevertheless show PDX failure in immunodeficient mice. Here, using immunohistochemistry and RNA sequencing, we evaluated the distinct immunohistochemical and gene expression profiles of resected LUSCs that showed successful engraftment. Among various LUSCs, including the basal, classical, secretory, and primitive subtypes, those in the non-engrafting (NEG) group showed gene expression profiles similar to the pure secretory subtype with positivity for CK7, whereas those in the engrafting (EG) group were similar to the mixed secretory subtype with positivity for p63. Pathway analysis of 295 genes that demonstrated significant differences in expression between NEG and EG tumors revealed that the former had enriched expression of genes related to the immune system, whereas the latter had enriched expression of genes related to the cell cycle and DNA replication. Interestingly, NEG tumors showed higher infiltration of B cells (CD19+) and follicular dendritic cells (CD23+) in lymph follicles than EG tumors. Taken together, these findings suggest that the PDX cancer model of LUSC represents only a certain population of LUSCs and that CD19- and CD23-positive tumor-infiltrating immune cells in the original tumors may negatively influence PDX engraftment in immunodeficient mice.

Ovarian carcinoma immunoreactive antigen domain 2 controls mitochondrial apoptosis in lung adenocarcinoma.

Ovarian carcinoma immunoreactive antigen domain 2 (OCIAD2) has been reported to show significantly higher expression in invasive lung adenocarcinoma than in lung adenocarcinoma in situ, and its abnormal expression is associated with poorer prognosis of the patients. However, the cellular function of OCIAD2 in this tumor remains poorly understood. In the present study, we first validated that OCIAD2 showed higher expression in human lung adenocarcinoma tissues or cell lines than in normal lung tissue or immortalized normal bronchial epithelial cells. OCIAD2 was localized predominantly at the mitochondrial membrane in lung adenocarcinoma cells. Interestingly, suppression of OCIAD2 led to loss of mitochondrial structure and a reduction in the number of mitochondria. Moreover, OCIAD2 suppression led to downregulation of cellular growth, proliferation, migration, and invasion, and upregulation of mitochondria-related apoptosis. We also showed that OCIAD2 suppression induced a decrease in mitochondrial membrane potential and release of cytochrome c. Transcriptional profiling using RNA sequencing revealed a total of 137 genes whose expression was commonly altered after OCIAD2 knockdown in three lung adenocarcinoma cell lines (A549, HCC827, and PC9). Pathway enrichment analysis of those genes demonstrated significant enrichment in apoptotic signaling or endoplasmic reticulum (ER) stress pathways. Our data suggest that OCIAD2 inhibits the mitochondria-initiated apoptosis and thus promotes the survival of lung cancer cells. Therefore, OCIAD2 may be an effective target for treatment of lung adenocarcinoma.

ECT2 promotes lung adenocarcinoma progression through extracellular matrix dynamics and focal adhesion signaling.

Lung adenocarcinoma (LAC) is the most prevalent form of lung cancer. Epithelial cell transforming sequence 2 (ECT2) is a guanine nucleotide exchange factor that has been implicated in oncogenic and malignant phenotypes of LAC. Here, we identified an oncogenic role of ECT2 in the extracellular matrix (ECM) dynamics of LAC cells. We showed that suppression of ECT2 decreased adhesion and spreading of LAC cells on ECM components. Morphologically, ECT2-depleted cells exhibited a rounded shape and cytoskeletal changes. Examination of transcriptional changes by RNA sequencing revealed a total of 1569 and 828 genes whose expressions were altered (absolute fold change and a difference of >2 fold) in response to suppression of ECT2 in two LAC cells (Calu-3 and NCI-H2342), respectively, along with 298 genes that were common to both cell lines. Functional enrichment analysis of common genes demonstrated a significant enrichment of focal adhesions. In accord with this observation, we found that ECT2 suppression decreased the expression level of proteins involved in focal adhesion signaling including focal adhesion kinase (FAK), Crk, integrin ß1, paxillin, and p130Cas. FAK knockdown leads to impaired cell proliferation, adhesion, and spreading of LAC cells. Moreover, in LAC cells, ECT2 binds to and stabilizes FAK and is associated with the formation of the focal adhesions. Our findings provide new insights into the underlying role of ECT2 in cell-ECM dynamics during LAC progression and suggest that ECT2 could be a promising therapeutic avenue for lung cancer.

Tet2 deficiency in immune cells exacerbates tumor progression by increasing angiogenesis in a lung cancer model.

Immune cells harboring somatic mutations reportedly infiltrate cancer tissues in patients with solid cancers and accompanying clonal hematopoiesis. Loss-of-function TET2 mutations are frequently observed in clonal hematopoiesis in solid cancers. Here, using a mouse lung cancer model, we evaluated the activity of Tet2-deficient immune cells in tumor tissues. Myeloid-specific Tet2 deficiency enhanced tumor growth in mice relative to that seen in controls. Single-cell sequencing analysis of immune cells infiltrating tumors showed relatively high expression of S100a8/S100a9 in Tet2-deficient myeloid subclusters. In turn, treatment with S100a8/S100a9 promoted Vegfa production by cancer cells, leading to a marked increase in the tumor vasculature in Tet2-deficient mice relative to controls. Finally, treatment of Tet2-deficient mice with an antibody against Emmprin, a known S100a8/S100a9 receptor, suppressed tumor growth. These data suggest that immune cells derived from TET2-mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis and may provide a novel therapeutic target for lung cancer patients with TET2-mutated clonal hematopoiesis.

Lysosome-associated membrane protein 3 misexpression in salivary glands induces a Sjögren's syndrome-like phenotype in mice.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune sialadenitis with unknown aetiology. Although extensive research implicated an abnormal immune response associated with lymphocytes, an initiating event mediated by salivary gland epithelial cell (SGEC) abnormalities causing activation is poorly characterised. Transcriptome studies have suggested alternations in lysosomal function are associated with SS, but a cause and effect linkage has not been established. In this study, we demonstrated that altered lysosome activity in SGECs by expression of lysosome-associated membrane protein 3 (LAMP3) can initiate an autoimmune response with autoantibody production and salivary dysfunction similar to SS. METHODS: Retroductal cannulation of the submandibular salivary glands with an adeno-associated virus serotype 2 vector encoding LAMP3 was used to establish a model system. Pilocarpine-stimulated salivary flow and the presence of autoantibodies were assessed at several time points post-cannulation. Salivary glands from the mice were evaluated using RNAseq and histologically. RESULTS: Following LAMP3 expression, saliva flow was significantly decreased and serum anti-Ro/SSA and La/SSB antibodies could be detected in the treated mice. Mechanistically, LAMP3 expression increased apoptosis in SGECs and decreased protein expression related to saliva secretion. Analysis of RNAseq data suggested altered lysosomal function in the transduced SGECs, and that the cellular changes can chemoattract immune cells into the salivary glands. Immune cells were activated via toll-like receptors by damage-associated molecular patterns released from LAMP3-expressing SGECs. CONCLUSIONS: These results show a critical role for lysosomal trafficking in the development of SS and establish a causal relationship between LAMP3 misexpression and the development of SS.

Immunomodulatory Mechanism of Acyclic Nucleoside Phosphates in Treatment of Hepatitis B Virus Infection.

BACKGROUND AND AIMS: Current treatment with nucleos(t)ide analogs (NUCs) safely controls the replication of hepatitis B virus (HBV) and improves prognosis in patients with HBV. However, the inability to completely clear HBV is problematic, and novel therapies are desired. It has been believed that all NUCs have similar functions to inhibit HBV reverse transcriptase. However, our recent findings that only acyclic nucleoside phosphonates (ANPs; adefovir dipivoxil and tenofovir disoproxil fumarate) had an additional effect of inducing interferon (IFN)-λ3 in the gastrointestinal tract suggests that ANPs are not only distinct from nucleoside analogs (lamivudine and entecavir) in their structures but also in their functions. Because enteric lipopolysaccharide (LPS) can cross the intestine and affect peripheral blood mononuclear cells (PBMCs), we hypothesized that orally administered ANPs could have further additional effects to modulate LPS-mediated cytokine profile in PBMCs. APPROACH AND RESULTS: This study showed that pretreatment of PBMCs, from either healthy volunteers or patients with HBV, with ANPs inhibited LPS-mediated interleukin (IL)-10 production, which reciprocally induced IL-12p70 and tumor necrosis factor-α production in a dose-dependent manner. Furthermore, the combination of IFN-α and ANPs synergistically enhanced LPS-mediated IL-12p70 production in PBMCs. Mechanistic analyses revealed that cellular metabolites of ANPs directly bound the Akt protein, inhibiting its translocation to the plasma membrane, thereby impairing Akt phosphorylation. Therefore, pretreatment of PBMCs with ANPs impairs LPS-mediated IL-10 production. CONCLUSIONS: Among NUCs, only ANPs have an additional pharmacological effect modulating LPS-mediated cytokine production, which is expected to produce favorable immune responses toward HBV elimination. This additional immunomodulation by ANPs in PBMCs, as well as IFN-λ3 induction in the gastrointestinal tract, provides insights into HBV treatment.

Comprehensive genomic profiles of small cell lung cancer.

We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechanism of Rb1 deregulation. Thus, loss of the tumour suppressors TP53 and RB1 is obligatory in SCLC. We discovered somatic genomic rearrangements of TP73 that create an oncogenic version of this gene, TP73Δex2/3. In rare cases, SCLC tumours exhibited kinase gene mutations, providing a possible therapeutic opportunity for individual patients. Finally, we observed inactivating mutations in NOTCH family genes in 25% of human SCLC. Accordingly, activation of Notch signalling in a pre-clinical SCLC mouse model strikingly reduced the number of tumours and extended the survival of the mutant mice. Furthermore, neuroendocrine gene expression was abrogated by Notch activity in SCLC cells. This first comprehensive study of somatic genome alterations in SCLC uncovers several key biological processes and identifies candidate therapeutic targets in this highly lethal form of cancer.

An autopsy case of aortic dissection due to giant cell arteritis.

Giant cell arteritis (GCA) is a systemic vasculitis affecting mainly large and medium-sized arteries. GCA sometimes involves the aorta and its major branches and causes aortic dissection as a rare complication. We have experienced an autopsy case of aortic dissection due to GCA. The patient was an 87-year-old Japanese woman with Stanford type A aortic dissection who died 7 days after admission. Two years previously she had been diagnosed as having abdominal aortic aneurysm and undergone endovascular aneurysm repair (EVAR). Although she had no characteristic symptoms of GCA, autopsy revealed marked granulomatous inflammation in the dissected area and coronary arteries. Active arteritis was evident not only in the arteries of the upper extremity but also those in the lower extremity. Granulomatous inflammation was not evident in the aneurysm. The aortic dissection might have been an initial manifestation of GCA. We report the regions of GCA extension and its histology in detail.

High expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinoma is associated with tumor invasion and poor prognosis.

The expression of Ras-specific guanine nucleotide-releasing factor 2 (RasGRF2) in lung adenocarcinomas was examined using immunohistochemistry in relation to clinicopathological characteristics and prognosis. In comparison to low expression, high expression of RasGRF2 was more closely associated with poor prognosis. Interestingly, expression of phosphorylated epithelial cell transforming 2 (pECT2), which - like RasGRF2 - is also a guanine-nucleotide exchange factor, was also associated with prognosis, and patients with high expression of both RasGRF2 and pECT2 had a much poorer outcome than those who were negative for both.

Phosphorylation-dependent Akt-Inversin interaction at the basal body of primary cilia.

A primary cilium is a microtubule-based sensory organelle that plays an important role in human development and disease. However, regulation of Akt in cilia and its role in ciliary development has not been demonstrated. Using yeast two-hybrid screening, we demonstrate that Inversin (INVS) interacts with Akt. Mutation in the INVS gene causes nephronophthisis type II (NPHP2), an autosomal recessive chronic tubulointerstitial nephropathy. Co-immunoprecipitation assays show that Akt interacts with INVS via the C-terminus. In vitro kinase assays demonstrate that Akt phosphorylates INVS at amino acids 864-866 that are required not only for Akt interaction, but also for INVS dimerization. Co-localization of INVS and phosphorylated form of Akt at the basal body is augmented by PDGF-AA Akt-null MEF cells as well as siRNA-mediated inhibition of Akt attenuated ciliary growth, which was reversed by Akt reintroduction. Mutant phosphodead- or NPHP2-related truncated INVS, which lack Akt phosphorylation sites, suppress cell growth and exhibit distorted lumen formation and misalignment of spindle axis during cell division. Further studies will be required for elucidating functional interactions of Akt-INVS at the primary cilia for identifying the molecular mechanisms underlying NPHP2.

Urgent Proton Beam Therapy With Interinstitutional Transfer for Patients With Intracranial Rhabdomyosarcoma: Report of 3 Cases.

A number of cases have been reported in recent years regarding the use of proton beam therapy to mitigate adverse events affecting important cranial organs in cases of rhabdomyosarcoma at parameningeal sites. However, few reports have described the use of proton beam therapy as urgent radiotherapy for parameningeal rhabdomyosarcoma with intracranial extension. We treated 3 patients diagnosed with parameningeal rhabdomyosarcoma extending into the cranium who were assessed at other hospitals as suitable for urgent radiotherapy and transferred to our hospital for proton beam therapy. These patients comprised 2 boys and 1 girl 6 to 12 years of age at diagnosis, and proton beam therapy was started on days 5, 11, and 23 after diagnosis, respectively. Patients with parameningeal rhabdomyosarcoma extending into the cranium can be transferred to institutions equipped to perform proton beam therapy. To minimize the interval to starting therapy, medical information should be shared with institutions capable of providing such therapy as soon as the possibility of intracranial soft-tissue sarcoma is recognized. Proton beam therapy is 1 option for radiotherapy in cases of intracranial rhabdomyosarcoma.