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A higher level of tumor-infiltrating lymphocytes (TILs) is associated with better prognosis in breast cancer patients. Adoptive transfer of lymphocytes coupled with conventional therapies has appealed to many clinicians and investigators as an effective treatment strategy for cancer patients, which necessitates efficient activation and expansion of cytotoxic T lymphocytes precisely targeting cancer cells. To comprehensively understand composition of TILs and to provide a grounding in adoptive T cell therapy, we analyzed the T cell receptor (TCR) repertoires in ex vivo–expanded TILs from nine breast cancer patients via next-generation sequencing. For the three of them, TCR repertoires of TILs gathered after the initial culture during 2 weeks were additionally analyzed and compared to those of TILs that underwent ex vivo rapid expansion procedure (REP). Diversity of TCR repertoire was variable among the patients. V/J segment usage in the clonotypes was similar among patients, with variable distribution of read counts for each V/J segment. The top 50% of most frequently observed VJ combinations was present in > 80% of the total clonotypes. Compared with TCGA data, the samples contained a similar amount of recurrent CDR3 sequences, but clonotype expansion was variable among the samples. In terms of clinicopathologic factor, presence of in vitro reactivity among triple-negative breast cancer cases seemed to be related to lower Shannon’s index, but p value was not statistically significant. In addition, the proportion of CD45RO+ cells out of CD8+ T cells were negatively correlated with Shannon’s diversity index for both TCRα and TCRβ chains (p = 0.010) via Spearman test. In this study, we identified a heterogeneous pattern of expanded T cell clones and stable usage of V/J segments in ex vivo–expanded TILs from breast cancer patients. Further large-scale studies are requisite to elucidate the clinical significance of TCR repertoires.

Phelan-McDermid syndrome (PMS) is a 22q13.3 deletion syndrome. Most PMS patients show global developmental delay and some of them suffer from developmental regression. The deleted region contains ARSA, which is responsible for metachromatic leukodystrophy (MLD). Here we report an extremely rare case of PMS characterized by unusual, rapidly progressive developmental regression due to additional pathogenic mutation in ARSA. Considering the 1 in 100 chance of an MLD carrier, co-occurrence of PMS and MLD in a patient is possible if either parent carries a heterozygous ARSA mutation. Therefore, MLD should be ruled out in PMS patients with severe neurological phenotype.

Background The genetic features and management of lateralized overgrowth have been elusive. This study was performed to investigate the causative genes and outcomes of alpelisib-treated patients with lateralized overgrowth. Methods Fourteen patients with lateralized overgrowth were enrolled. Clinical features and whole-body magnetic resonance imaging (WB-MRI) findings were evaluated. Deep sequencing with a customized 143 gene panel of affected tissue and peripheral white blood cells was performed. Propranolol was administered and clinical outcomes were examined. The PIK3CA inhibitor alpelisib was administered via a managed access program. Results Genetic testing identified PIK3CA (n = 7, 50%), KRAS (n = 2, 14.3%), PTEN (n = 1, 7.1%), MAP2K3 (n = 1, 7.1%), GNAQ (n = 1, 7.1%), TBC1D4 (n = 1, 7.1%), and TEK (n = 1, 7.1%) mutations. Propranolol was administered in 12 patients, and 7 experienced relief of symptoms. Alpelisib was administered in two volunteers with a PIK3CA mutation, and the WB-MRI after 1 year of treatment showed a reduction in proliferated masses. Conclusions Deep sequencing identified diverse genetic features of lateralized overgrowth. Targeted therapy with a PIK3CA inhibitor would be the primary therapeutic strategy for PIK3CA-related overgrowth syndrome.

Background: The expression of major histocompatibility complex class I (MHC I) has previously been reported to be negatively associated with estrogen receptor (ER) expression. Furthermore, the expression of MHC I, the level of tumor-infiltrating lymphocytes (TILs), and the expression of interferon (IFN) mediator MxA are positively associated with one another in human breast cancers. This study aimed to investigate the mechanisms of association of MHC I with estrogen and IFN signaling. Methods: The human leukocyte antigen (HLA)-ABC protein expression was analyzed in breast cancer cell lines. The expressions of HLA-A and MxA mRNAs were analyzed in MCF-7 cells in Gene Expression Omnibus (GEO) data. ER and HLA-ABC expressions and TIL levels in tumor tissue were also analyzed in ER+/ human epidermal growth factor receptor 2 (HER2)- breast cancer patients who randomly received either neoadjuvant chemotherapy or estrogen modulator treatment following surgical resection. Results: HLA-ABC protein expression was decreased after β-estradiol treatment or hESR-GFP transfection and increased after fulvestrant or IFN-γ treatment in breast cancer cell lines. In GEO data, HLA-A and MxA expression was increased after ESR1 shRNA transfection. When comparing the two patient groups, ER Allred score was significantly lower and the HLA-ABC expression and TIL levels were significantly higher in the estrogen modulator treated group than the chemotherapy treated group. Conclusion: MHC I expression and TIL levels might be affected by ER pathway modulation and IFN treatment. Further studies elucidating the mechanism of MHC I regulation could suggest a way to boost TIL influx in cancer in a clinical setting.

BackgroundAmbroxol (ABX) has been suggested as an augmentative pharmacological agent for neuronopathic Gaucher disease (nGD). This study assessed the long-term safety and efficacy of combined therapy with high-dose ABX and enzyme replacement therapy (ERT) in nGD.MethodsABX+ERT therapy was administered for 4.5 years in four patients with nGD. ABX was initiated at a dose of 1.5 mg/kg/day, and the dose was escalated up to 27 mg/kg/day. The target plasma level was 10 µmol/L or less. The changes in glucocerebrosidase activity, biochemical, safety and neurocognitive findings were assessed.ResultsEnhanced residual GCcase activity was observed in all patients, as evidenced in both in vitro and in vivo studies. During the first 2 years of study with ABX (up to 21 mg/kg/day), mean seizure frequencies and neurocognitive function worsened. After ABX dosage was increased up to 27 mg/kg/day of ABX, its trough plasma concentration was 3.2–8.8 µmol/L. Drug-to-drug interaction, especially with antiepileptic drug significantly affected the pharmacokinetic parameters of ABX. Importantly, at 27 mg/kg/day of ABX, the seizure frequencies markedly decreased from the baseline, and the neurocognitive function was improved. In addition, Lyso-Gb1, a biomarker for the severity and progression of GD, was normalised in all patients. High-dose ABX was well-tolerated with no severe adverse events.ConclusionsLong-term treatment with high-dose ABX+ERT was safe and might help to arrest the progression of the neurological manifestations in GD.

Lesch-Nyhan syndrome (LNS) is an X-linked recessive disorder caused by mutations in the HPRT1 gene. The clinical features and mutation spectrum of 26 Korean LNS patients from 23 unrelated families were retrospectively reviewed. The HPRT1 gene was analyzed by direct sequencing of genomic DNA. The median age at diagnosis was 2.3 years (range, 4 months-22.6 years) and the initial presenting features included developmental delay, orange colored urine, and self-injurious behaviors. Most patients were wheelchair-bound and suffered from urinary complications and neurologic problems such as self-mutilation and developmental delay. Twenty different mutations in HPRT1 were identified among 23 independent pedigrees, including six novel mutations. The most common mutation type was truncating mutations including nonsense and frameshift mutations (45%). Large deletions in the HPRT1 gene were identified in exon 1, exons 5-6, exons 1-9, and at chr X:134,459,540-134,467,241 (7702 bp) including the 5'-untranslated region, exon 1, and a portion of intron 1. In conclusion, this study describes the phenotypic spectrum of LNS and has identified 20 mutations from 23 Korean families, including six novel mutations in Korean patients with LNS.

(1) Background: Pancreatic cancer is a high devastating disease with the lowest survival rate among all common cancers due to difficulties in early diagnosis. The purpose of this study was to identify and characterize the distinct subset of blood cell population elevated in peripheral blood mononuclear cells (PBMC) of pancreatic cancer to evaluate the potential markers for diagnosis of pancreatic cancer, (2) Methods: We analyzed differential gene expression in PBMC from normal individuals and pancreatic cancer patients utilizing transcriptome analysis. Flow cytometry analysis was applied to identify the discrete subset of interleukin-7 receptor (IL-7R) expressing cells in these cells. The expression of IL-7R during tumorigenesis was determined in syngeneic mouse model of pancreatic cancer in vivo, (3) Results: PBMC from pancreatic cancer patients expressed elevated IL-7R mRNA compared to healthy control individuals. IL-7R expressing cells rapidly appeared from the early stages of the onset of tumor formation in syngeneic pancreatic cancer mouse model in vivo. The discrete subset of IL-7R positive cells mainly consist of naïve T, central memory T, and effector memory T cells, (4) Conclusions: Taken together, our present findings suggest that pancreatic cancer patients expressed higher level of IL-7R expression in PBMC that rapidly emerged from the onset of early pancreatic tumor formation in vivo than normal individuals. Thus, it can be used as a novel biological marker for early events of pancreatic cancer development.

Schaaf-Yang syndrome (SYS) is a recently identified disorder caused by a loss-of-function mutation in a maternally imprinted gene, MAGEL2, at 15q11.2q13. Due to its extreme rarity and wide range of clinical severity, clinical suspicion is difficult for a physician. In the current study, its frequency among the Korean pediatric patients with developmental delay (DD) or intellectual disability (ID) was assessed. As the first report of Korean patients with SYS, our study aims to increase the awareness of this condition among the physicians taking care of the pediatric patients with DD/ID and hypotonia. The patients diagnosed with SYS by whole-exome sequencing (WES) among the 460 Korean pediatric patients with DD/ID were included, and their clinical and molecular features were reviewed. Four patients (0.9%) were diagnosed with SYS. Profound DD (4 patients), multiple anomalies including joint contractures and facial dysmorphism (4 patients), generalized hypotonia (3 patients), and severe respiratory difficulty requiring mechanical ventilation (3 patients) were noted in most cases, similar to those in previous reports. Sleep apnea (2 patients), autistic features (2 patients), a high grade of gastroesophageal reflux (1 patient), and seizures (1 patient) were found as well. A total of 3 different truncating MAGEL2 mutations were identified. A previously-reported mutation, to be the most common one, c.1996dupC, was found in 2 patients. The other 2 mutations, c.2217delC and c.3449_3450delTT were novel mutations. As MAGEL2 is maternally imprinted, 2 patients had inherited the MAGEL2 mutation from their respective healthy fathers. SYS is an extremely rare cause of DD/ID. However, hypotonia, joint contractures, profound DD/ID and facial dysmorphism are the suggestive clinical features for SYS. As a maternally imprinted disorder, it should be reminded that SYS may be inherited in form of a mutation from a healthy father.

Background Gaucher disease (GD) is caused by a deficiency of β-glucocerebrosidase, encoded by GBA. Haplotype analyses previously demonstrated founder effects for particular GBA mutations in Ashkenazi Jewish and French-Canadian populations. This study aimed to investigate the clinical characteristics and mutation spectrum of GBA in Korean GD patients and to identify founder effect of GBA p.G85E in non-neuronopathic GD patients. Results The study cohort included 62 GD patients from 58 unrelated families. Among them, 18 patients from 17 families harbored the p.G85E mutation. Haplotype analysis was performed for 9 probands and their parents for whom DNA samples were available. In 58 unrelated probands, the GBA mutation p.L483P was the most common (30/116 alleles, 26%), followed by p.G85E (16%), p.F252I (13%), and p.R296Q (9%). The median age at diagnosis of the 18 patients harboring the p.G85E mutation was 3.8 (range 1.2–57) years. No patients developed neurological symptoms during follow-up periods of 2.2–20.3 (median 13.9) years. The size of the shared haplotype containing GBA p.G85E was 732 kbp, leading to an estimated age of 3075 years. Conclusion The GBA p.G85E mutation, which appears to be neuroprotective despite producing distinctive visceromegaly and skeletal symptoms, exhibited a potential founder effect in Korean GD patients.

Purpose In the presence of interferon, proteasome subunits are replaced by their inducible counterparts to form an immunoproteasome (IP) plays a key role in generation of antigenic peptides presented by MHC class I molecules, leading to elicitation of a T cell‒mediated immune response. Although the roles of IP in other cancers, and inflammatory diseases have been extensively studied, its significance in breast cancer is unclear. Materials and Methods We investigated the expression of LMP7, an IP subunit, and its relationship with immune system components in two breast cancer cohorts. Results In 668 consecutive breast cancer cohort, 40% of tumors showed high level of LMP7 expression, and tumors with high expression of LMP7 had more tumor-infiltrating lymphocytes (TILs) in each subtype of breast cancer. In another cohort of 681 triple-negative breast cancer patients cohort, the expression of LMP7 in tumor cells was significantly correlated with the amount of TILs and the expression of interferon-associated molecules (MxA [p < 0.001] and PKR [p < 0.001]), endoplasmic reticulum stress-associated molecules (PERK [p=0.012], p-eIF2a [p=0.001], and XBP1 [p < 0.001]), and damage-associated molecular patterns (HMGN1 [p < 0.001] and HMGB1 [p < 0.001]). Patients with higher LMP7 expression had better disease-free survival outcomes than those with no or low expression in the positive lymph node metastasis group (p=0.041). Conclusion Close association between the TIL levels and LMP7 expression in breast cancer indicates that better antigen presentation through greater LMP7 expression might be associated with more TILs.

Adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs) has been successful in treating a considerable proportion of patients with metastatic melanoma. In addition, some patients with several other solid tumors were recently reported to have benefited clinically from such ACT. However, it remains unclear whether ACT using TILs is broadly applicable in breast cancer, the most common cancer in women. In this study, the utility of TILs as an ACT source in breast cancers was explored by deriving TILs from a large number of breast cancer samples and assessing their biological potentials. We successfully expanded TILs ex vivo under a standard TIL culture condition from over 100 breast cancer samples, including all breast cancer subtypes. We also found that the information about the percentage of TIL and presence of tertiary lymphoid structure in the tumor tissues could be useful for estimating the number of obtainable TILs after ex vivo culture. The ex vivo expanded TILs contained a considerable level of central memory phenotype T cells (about 20%), and a large proportion of TIL samples were reactive to autologous tumor cells in vitro. Furthermore, the in vitro tumor-reactive autologous TILs could also function in vivo in a xenograft mouse model implanted with the primary tumor tissue. Collectively, these results strongly indicate that ACT using ex vivo expanded autologous TILs is a feasible option in treating patients with breast cancer.

PURPOSE Congenital hypopituitarism is caused by mutations in pituitary transcription factors involved in the development of the hypothalamic-pituitary axis. Mutation frequencies of genes involved in congenital hypopituitarism are extremely low and vary substantially between ethnicities. This study was undertaken to compare the clinical, endocrinological, and radiological features of patients with an isolated growth hormone deficiency (IGHD) or combined pituitary hormone deficiency (CPHD). MATERIALS AND METHODS This study included 27 patients with sporadic IGHD and CPHD. A mutation analysis of the POU1F1, PROP1, LHX3, LHX4, and HESX1 genes was performed using genomic DNA from peripheral blood leukocytes. RESULTS IGHD and CPHD were observed in 4 and 23 patients, respectively. Mean age at diagnosis was 8.28±7.25 years for IGHD and 13.48±10.46 years for CPHD (p=0.37). Serum insulin-like growth factor-1 and peak growth hormone (GH) levels following GH stimulation tests were significantly lower in patients with CPHD than in those with IGHD (p

Glutamine metabolism is emerging as one aspect of dysregulated metabolism of tumors. Triple-negative breast cancer (TNBC) cells are glutamine dependent, whereas luminal-type cells tend to be glutamine independent. Therefore, TNBC patients might benefit from therapies targeting glutamine metabolism. To investigate the clinical significance of glutamine metabolism, we examined expression and prognostic significance of glutaminase in tumor cells and tumor-infiltrating lymphocytes (TILs) in TNBC. We retrieved 658 surgically resected TNBCs and analyzed glutaminase expression in tumor cells and TILs by immunohistochemical staining. Glutaminase expression was observed in 237 cases (36.0%) in tumor cells and 104 cases (15.5%) in TILs. Although glutaminase expression in tumor cells was significantly associated with a low level of TILs (p = 0.018), glutaminase expression in TILs was significantly higher in cases with a high level of TILs (p = 0.031). Glutaminase expression in tumor cells was significantly associated with poor disease-free survival in patients with lymph node metastasis and high levels of TILs (p = 0.020). In addition, it was an independent poor prognostic factor (hazard ratio = 10.643, 95% confidence interval = 1.999-56.668; p = 0.006). Glutaminase expression in tumor cells was observed in a subset of TNBC patients. It was significantly associated with a low level of TILs and poor disease-free survival in TNBCs presenting with lymph node metastasis and high levels of TILs.

Identification of tumor antigens that induce cytotoxic T lymphocytes (CTLs) is crucial for cancer-vaccine development. Despite their predictive ability, current algorithmic approaches and human leukocyte antigen (HLA)-peptidomic analysis allow limited selectivity. Here, we optimized a method to rapidly screen and identify highly immunogenic epitopes that trigger CTL responses. We used a combined application of this method involving immune-specific signature analysis and HLA-associated peptidomics using samples from six patients with triple-negative breast cancer (TNBC) in order to select immunogenic HLA epitopes for in vitro testing. Additionally, we applied high-throughput imaging at the single-cell level in order to confirm the immunoreactivity of the selected peptides. The results indicated that this method enabled identification of promising CTL peptides capable of inducing antitumor immunity. This platform combining high-resolution computational analysis, HLA-peptidomics, and high-throughput immunogenicity testing allowed rapid and robust identification of highly immunogenic epitopes and represents a powerful technique for cancer-vaccine development.

Neurofibromatosis type 1 (NF1) is caused by heterozygous mutation in the NF1 gene. NF1 is one of the most common human genetic diseases. However, the overall genotype–phenotype correlation has not been known, due to a wide spectrum of genotypic and phenotypic heterogeneity. Here we describe the detailed clinical and genetic features of 427 Korean NF1 patients from 389 unrelated families. Long range PCR and sequencing of genomic DNA with multiplex ligation-dependent probe amplification analysis identified 250 different NF1 mutations in 363 families (93%), including 94 novel mutations. With an emphasis on phenotypes requiring medical attention (classified and termed: NF1+), we investigated the correlation of NF1+ and mutation types. NF1+ was more prevalent in patients with truncating/splicing mutations and large deletions than in those with missense mutations (59.6%, 64.3% vs. 36.6%, p = 0.001). This difference was especially significant in the patients younger than age 19 years. The number of items in NF1+ was a higher in the former groups (0.95 ± 0.06, 1.18 ± 0.20 vs. 0.56 ± 0.10, p = 0.002). These results suggest that mutation types are associated not only with higher prevalence of severe phenotypes in NF1 but also with their earlier onset.

Complement factor H (CFH) is a fluid phase regulator of complement proteins and functions to prevent complement attack and immune surveillance. CFH is known to inactivate therapeutic antibody-dependent complement-mediated cellular cytotoxicity. We found that CFH was highly expressed in human lung cancer cells and tissues. To investigate mechanisms of CFH upregulation, we searched for a CFH transcription factor and its regulatory factors. First, signal transducer and activator of transcription 4 (STAT4) expression patterns coincided with CFH expression patterns in lung cancer tissues. Knockdown of STAT4 led to decreased CFH secretion from lung cancer cells. STAT4 bound directly to the CFH promoter, as demonstrated by luciferase reporter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay, suggesting that STAT4 is a transcription factor for CFH. In addition, a low level of suppressors of cytokine signaling (SOCS)-1/3, a Janus kinase (JAK) inhibitor, was observed in lung cancer cells and its transfection decreased CFH protein levels and promoter activity. Unexpectedly, the low level of SOCS-1/3 was not due to epigenetic silencing. Instead, differential methylation was found on the regulatory region of STAT4 between normal and lung cancer cells. In conclusion, our results demonstrated that CFH is upregulated by constitutive activation of STAT4, which is accounted for by SOCS silencing in lung cancer cells.

// Sun-Young Kong 2, 3, 4 , Kwang-Soo Kim 1 , Jiewan Kim 1 , Min Kyeong Kim 2 , Ki Hong Lee 2 , Je-Yong Lee 1 , Nuri Oh 1 , Ji-In Park 1 , Ji-Hoon Park 1 , Sun-Hee Heo 5 , Sung Han Shim 1 , Dong Ryul Lee 1 , Keun Pil Kim 6 , Kyung-Soon Park 1 1 Department of Biomedical Science, College of Life Science, CHA University, Seoul, Korea 2 Department of System Cancer Science, Graduate School of Cancer Science and Policy, National Cancer Center, Seoul, Korea 3 Translational Epidemiology Branch, Research Institute, National Cancer Center, Seoul, Korea 4 Department of Laboratory Medicine, Center for Diagnostic Oncology, Hospital, National Cancer Center, Seoul, Korea 5 Biomedical Research Center, Asan Institute for Science and Department of Pathology, Asan Medical Center, University of Ulsan, College of Medicine, Ulsan, Korea 6 Department of Life Science, Chung-Ang University, Seoul, Korea Correspondence to: Kyung-Soon Park, email: kspark@cha.ac.kr Keywords: ELK3, GATA3, metastasis, migration, invasion Received: February 02, 2016 Accepted: August 13, 2016 Published: August 19, 2016 ABSTRACT Triple-negative breast cancer is a highly aggressive tumor subtype that lacks effective therapeutic targets. Here, we show that ELK3 is overexpressed in a subset of breast cancers, in particular basal-like and normal-like/claudin-low cell lines. Suppression of ELK3 in MDA-MB-231 cells led to transdifferentiation from an invasive mesenchymal phenotype to a non-invasive epithelial phenotype both in vitro and in vivo . Suppression of ELK3 resulted in extensive changes in genome expression profiles. Among these, GATA3, a master suppressor of metastasis, was epigenetically activated. Also, suppression of GATA3 led to the restoration of migration and invasion. These results suggest that the ELK3-GATA3 axis is a major pathway that promotes metastasis of MDA-MB-231 cells.

Objectives: We tried to describe cellular immune response (tertiary lymphoid structures (TLSs), lymphoid aggregates, tumor infiltrating lymphocytes (TILs)) in neoplastic microenvironment of ductal carcinoma in situ (DCIS) with or without associated microinvasion. Methods: The histopathologic parameters of 177 DCIS and 27 DCIS with microinvasion were evaluated. We determined number of ducts involved by DCIS, and calculated percentage of these ducts surrounded by TLSs. TILs were quantitated in 27 microinvasive cases. Results: Tumors having higher percentage of DCIS ducts associated with TLSs had higher incidence of microinvasion ( P

// Hee Jin Lee 1,* , In Hye Song 1,* , In Ah Park 1 , Sun-Hee Heo 1 , Young-Ae Kim 1 , Jin-Hee Ahn 2 and Gyungyub Gong 1 1 Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 2 Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea * These authors have contributed equally to this work Correspondence to: Gyungyub Gong, email: // Keywords : breast carcinoma; tumor-infiltrating lymphocytes; major histocompatibility complex I; human leukocyte antigen; Pathology Section Received : December 14, 2015 Accepted : April 03, 2016 Published : April 18, 2016 Abstract Tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have a strong prognostic and predictive significance. However, the mechanism of TIL influx in TNBC is unclear. Expression of major histocompatibility complex class I (MHC I) on the tumor cell is essential for the effective killing of tumor by cytotoxic TILs. In our current study, human leukocyte antigen (HLA) expression was inversely correlated with estrogen receptor (ER) expression in normal and cancerous breast tissue and positively correlated with TILs in breast cancer. The ER score was inversely correlated with TILs in breast cancer. HLA-A and CD8B gene expression was negatively correlated with ESR1 and positively correlated with interferon-associated gene expression in The Cancer Genome Atlas (TCGA) data. Negative correlation between ESR1 and HLA and positive correlation between interferon-associated and HLA gene expression were also confirmed in Cancer Cell Line Encyclopedia (CCLE) data. Taken together, our data suggest that a lower expression of HLA in luminal-type tumors might be associated with low level of TILs in those tumors. Further investigation of the mechanism of higher HLA expression and TIL influx in TNBC may help to boost the host immune response.

Interferons (IFNs) play an important role in tumor-immune system interactions. As one of the main mediators of IFNs, myxovirus resistance A (MxA) is upregulated in various cancers. However, the exact role of MxA in breast cancer is not fully understood. As part of the immune response to tumors, tumor-infiltrating lymphocytes (TILs) have prognostic significance in breast cancer. The aim of our present study was to examine the relationship between MxA and immune system components, including the amount of TILs and human leukocyte antigen (HLA) expression, in breast cancer. TILs, MxA expression, HLA intensity, and clinicopathological factors were retrospectively analyzed in 688 patients with primary breast cancer between 1993 and 1998 and in 705 patients with triple-negative breast cancer (TNBC) between 2004 and 2011. MxA expression was higher in TNBC tumors than in other subtypes. High MxA levels were associated with a higher histologic grade, abundant TILs, and stronger HLA-ABC expression in both the TNBC subtype within the consecutive breast cancer cohort and the validation TNBC cohort. MxA expression showed a significant positive correlation with TILs, the number of CD8(+) cells, and the number of CD69(+) cells in the validation TNBC cohort. High MxA levels and abundant TILs were found to be independent prognostic factors for disease-free survival in patients with TNBC. These results indicate that MxA expression is closely related to TILs in TNBC and, along with TILs, provides prognostic information after chemotherapy in patients with TNBC.

BACKGROUND Most triple-negative breast cancers (TNBCs) have a high histologic grade, are associated with high endoplasmic stress, and possess a high frequency of TP53 mutations. TP53 missense mutations lead to the production of mutant p53 protein and usually show high levels of p53 protein expression. Tumor-infiltrating lymphocytes (TILs) accumulate as part of the anti-tumor immune response and have a strong prognostic and predictive significance in TNBC. We aimed to elucidate the association between p53 expression and the amount of TILs in TNBC. METHODS In 678 TNBC patients, we evaluated TIL levels and expression of endoplasmic stress molecules. Immunohistochemical examination of p53 protein expression was categorized into three groups: no, low, and high expression. RESULTS No, low, and high p53 expression was identified in 44.1% (n = 299), 20.1% (n = 136), and 35.8% (n = 243) of patients, respectively. Patients with high p53 expression showed high histologic grade (p < .001), high TIL levels (p = .009), and high expression of endoplasmic reticulum stress-associated molecules (p-eIF2a, p = .013; XBP1, p = .007), compared to patients with low p53 expression. There was no significant difference in disease-free (p = .406) or overall survival rates (p = .444) among the three p53 expression groups. CONCLUSIONS High p53 expression is associated with increased expression of endoplasmic reticulum stress molecules and TIL influx.

Background: Tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) are prognostic markers in triple-negative breast cancer (TNBC). Our study analyzed the relationship between cluster of differentiation (CD)11c-positive dendritic cells (DCs) and TILs and TLSs to elucidate mechanisms of TIL influx. Materials and Methods: Immunohistochemical staining for CD4, CD8, and CD11c in tissue microarrays from 681 patients with TNBC was performed. The proportions of TILs and TLSs were reviewed. Two additional TNBC gene expression datasets were used. Results: CD11c expression showed a significantly positive correlation with the level of TILs and the number of CD4(+) and CD8(+) T-cells, as well as an abundance of TLSs. CD11C gene expression was also significantly correlated with expression of CD4, CD8, and genes related to TLSs in both datasets. Conclusion: We demonstrated a strong correlation of CD11c expression, which represents DCs, with TILs and TLSs in TNBC. Further investigation is warranted to identify therapeutic modalities that facilitate recruitment and activation of DCs.

The level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures are significant prognostic and predictive factors in primary breast cancer. However, the understanding about differences in tumor-infiltrating lymphocytes and tertiary lymphoid structures at various metastatic sites or between primary breast tumors and metastatic sites is limited. A total of 335 cases of metastatic breast cancer from four metastatic sites (lung, liver, brain, and ovary) were included. We analyzed the percentages of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in the primary and metastatic sites. The mean level of tumor-infiltrating lymphocytes in the lung metastases was higher than in the liver, brain, ovary, and matched primary tumors, while metastatic tumors of the liver and brain showed lower levels of tumor-infiltrating lymphocytes than primary tumors. Tertiary lymphoid structures were only found in the lung and liver, and in cases of brain metastases the change of tertiary lymphoid structures from present to absent significantly affected the level of tumor-infiltrating lymphocytes in metastases compared with that in matched primary tumors. Patients with a lower histological grade, hormone receptor positivity in primary tumors and metastases, a lower level of tumor-infiltrating lymphocytes and absence of tertiary lymphoid structures in primary tumors, a higher level of tumor-infiltrating lymphocytes and presence of tertiary lymphoid structures in metastases, and lung metastases showed significantly better overall survival. Our results showed that metastatic breast tumors in the lung had more tumor-infiltrating lymphocytes than did tumors at other sites and matched primary tumors. In addition, the presence of tertiary lymphoid structures in metastatic sites is a critical factor for the level of tumor-infiltrating lymphocytes.

Background Fatty acid oxidation disorders (FAODs) include more than 15 distinct disorders with variable clinical manifestations. After the introduction of newborn screening using tandem mass spectrometry, early identification of FAODs became feasible. This study describes the clinical, biochemical and molecular characteristics of FAODs patients detected by newborn screening (NBS) compared with those of 9 patients with symptomatic presentations. Methods Clinical and genetic features of FAODs patients diagnosed by NBS and by symptomatic presentations were reviewed. Results Fourteen patients were diagnosed with FAODs by NBS at the age of 54.8 ± 4.8 days: 5 with very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, 5 with medium chain acyl-CoA dehydrogenase (MCAD) deficiency, 1 with primary carnitine deficiency, 1 with carnitine palmitoyltransferase 1A (CPT1A) deficiency, 1 with long-chain 3-hydroxyacyl-CoA dehydrogenase or mitochondrial trifunctional protein (LCAHD/MTP) deficiency, and 1 with short chain acyl-CoA dehydrogenase (SCAD) deficiency. Three patients with VLCAD or LCHAD/MTP deficiency developed recurrent rhabdomyolysis or cardiomyopathy, and one patient died of cardiomyopathy. The other 10 patients remained neurodevelopmentally normal and asymptomatic during the follow-up. In 8 patients with symptomatic presentation, FAODs manifested as LCHAD/MTP deficiencies by recurrent rhabdomyolysis or cadiomyopathy (6 patients), and VLCAD deficiency by cardiomyopathy (1 patient), and CPT1A deficiency by hepatic failure (1 patient). Two patients with LCHAD/MTP deficiencies died due to severe cardiomyopathy in the neonatal period, and developmental disability was noted in CPT1A deficiency (1 patient). Conclusions NBS helped to identify the broad spectrum of FAODs and introduce early intervention to improve the clinical outcome of each patient. However, severe clinical manifestations developed in some patients, indicating that careful, life-long observation is warranted in all FAODs patients.

ELK3 is a member of the Ets family of transcription factors. Its expression is associated with angiogenesis, vasculogenesis, and chondrogenesis. ELK3 inhibits endothelial migration and tube formation through the regulation of MT1-MMP transcription. This study assessed the function of ELK3 in breast cancer (BC) cells by comparing its expression between basal and luminal cells in silico and in vitro. In silico analysis showed that ELK3 expression was higher in the more aggressive basal BC cells than in luminal BC cells. Similarly, in vitro analysis showed that ELK3 mRNA and protein expression was higher in basal BC cells than in normal cells and luminal BC cells. To investigate whether ELK3 regulates basal cell migration or invasion, knockdown was achieved by siRNA in the basal BC cell line MDA-MB-231. Inhibition of ELK3 expression decreased cell migration and invasion and downregulated MT1-MMP, the expression of which is positively correlated with tumor cell invasion. In silico analysis revealed that ELK3 expression was associated with that of MT1-MMP in several BC cell lines (0.98 Pearson correlation coefficient). Though MT1-MMP expression was upregulated upon ELK3 nuclear translocation, ELK3 did not directly bind to the 1.3-kb promoter region of the MT1-MMP gene. These results suggest that ELK3 plays a positive role in the metastasis of BC cells by indirectly regulating MT1-MMP expression.

Background: Ambroxol (ABX) has been suggested as an augmentative pharmacological agent for neuronopathic Gaucher disease (nGD). This study assessed the long-term safety and efficacy of combined therapy with high-dose ABX and enzyme replacement therapy (ERT) in nGD. Methods: Four patients with nGD were enrolled. ABX ERT therapy was administered for 4·5 years. Ambroxol was initiated at a dose of 1·5 mg/kg/day, and the dose was escalated up to 27 mg/kg/day. The changes in glucocerebrosidase (GBA) activity, biochemical, safety, and neurocognitive findings were assessed. Findings: Enhanced residual GBA activity was observed in all patients, as evidenced in both in vitro and in vivo studies. Mean seizure frequencies markedly decreased from the baseline, and the neurocognitive function was maintained. Lyso-Gb1, a biomarker for the severity and progression of GD, gradually decreased in all patients. High-dose ABX was well-tolerated with no severe adverse events. Interpretation: Long-term treatment with high-dose ABX ERT was safe and shows promise for arresting the progression of the neurological manifestations in GD. Clinical Trial Number: The study protocol was registered at the Clinical Research Information Service, Korea Centers for Disease Control and Prevention, Ministry of Health and Welfare (Republic of Korea) (no. KCT0003218). Funding Statement: This research was supported in part by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (NRF- 2015K1A4A3046807, NRF-2016M3A9B4915706 and NRF-2018M3A9H1078335) and by ISU ABXIS, Gyeonggi-do, Korea. Declaration of Interests: "None declared." Ethics Approval Statement: The study protocol was approved by the Institutional Review Board and the Ministry of Food and Drug Safety, Korea (no. 30449).

Nonimmune hydrops fetalis is the most severe clinical manifestation of lysosomal storage diseases (LSDs). Around 14 different LSDs have been accounted for as 1–15% of the cause of nonimmune hydrops fetalis. We report a Korean infant affected by an extremely rare but severe form of sialic acid storage disease. The patient presented with nonimmune hydrops fetalis, dysmorphic facial features, hepatosplenomegaly, and dysostosis multiplex and died at 39 days of age due to persistent pulmonary hypertension. LSD was suspected based on the presence of diffuse vacuolation of syncytiotrophoblast, villous stromal cells, and intermediate trophoblast in placental biopsy. Increased excretion of urinary free sialic acid was detected by liquid chromatography-tandem mass spectrometry. The patient was compound heterozygous of the c.908G>A (p.Trp303Ter) and the splicing mutation c.1259+5G>T (IVS9+5 G>T) in the SLC17A5 gene.

Supplemental Digital Content is available in the text, Fabry disease is a rare X-linked lysosomal storage disorder caused by an α-galactosidase A deficiency. The progressive accumulation of globotriaosylceramide (GL-3) results in life-threatening complications, including renal, cardiac, and cerebrovascular diseases. This study investigated the phenotypic and molecular spectra of GLA mutations in Korean patients with Fabry disease using a nationwide survey. This study included 94 patients from 46 independent pedigrees: 38 adult males, 46 symptomatic females, and 10 pediatric males. Each diagnosis was based on an enzyme assay and GLA gene mutation analysis. The mean age at presentation was 24 years (range, 5–65 years); however, the diagnoses were delayed by 21 ± 19 years after the onset of symptoms. Those patients with late-onset Fabry disease were diagnosed by family screening or milder symptoms at a later age. Forty different mutations were identified: 20 missense (50%), 10 nonsense (25%), 8 frameshift (20%), and 2 splice site (5%) mutations. Five of them were novel. IVS4+919G>A (c.936+919 G>A) was not detected among the 6505 alleles via newborn screening using dried blood spots. Enzyme replacement therapy (ERT) was performed in all the males and pediatric patients, whereas 75% of the symptomatic females underwent ERT for 4.2 ± 3.6 years. This study described the demographic data, wide clinical spectrum of phenotypes, and GLA mutation spectrum of Fabry disease in Korea. Most of the patients had classical Fabry disease, with a 4 times higher incidence than that of late-onset Fabry disease, indicating an underdiagnosis of mild, late-onset Fabry disease.

Background Fabry disease is characterised by the progressive accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids in vascular endothelial cells. Enzyme replacement therapy (ERT) clears this accumulation. We analysed plasma proteome profiles before and after ERT to characterise its molecular pathology. Methods Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time of flight tandem mass spectrometry (MALDI-TOF MS) and tandem mass spectrometry (MS/MS) were done using plasma samples before and after ERT in eight patients with classical Fabry disease Results After short-term ERT (4–12 months), the levels of 15 plasma proteins involved in inflammation, oxidative and ischaemic injury, or complement activation were reduced significantly. Among them, β-actin (ACTB), inactivated complement C3b (iC3b), and C4B were elevated significantly in pre-ERT Fabry disease plasma compared with control plasma. After longer-term ERT (46–96 months), iC3b levels gradually decreased, whereas the levels of other proteins varied. The gradual reduction of iC3b was comparable to that of Gb3 levels. In addition, iC3b increased significantly in pre-ERT Fabry disease mouse plasma, and C3 deposits were notable in renal tissues of pre-enzyme replacement therapy patients. Conclusion These results indicated that C3-mediated complement activation might be altered in Fabry disease and ERT might promote its stabilisation.

Plasma chitotriosidase activity is used for diagnosis and monitoring of Gaucher disease. However, homozygous duplication of a 24 bp region in exon 10 of the chitotriosidase gene (CHIT1) abolishes enzyme activity, limiting its use as a biomarker in Gaucher disease. This study investigates the allele frequency of the 24 bp duplication, in both the general Korean population and in patients with Gaucher disease. Fifteen Korean patients with Gaucher disease and 231 Korean normal individuals were enrolled. Genotyping was performed to identify the 24 bp duplication in exon 10 of CHIT1 using DNA extracted from peripheral leukocytes or dried blood spots. Two patients with Gaucher disease (13.3%) had normal plasma chitotriosidase activity, and carried a homozygous 24 bp duplication of exon 10 of the CHIT1 gene. Nine patients were heterozygote carriers (60.0%). Of the normal 231 Korean individuals, heterozygous duplication was detected in 109 individuals (47.2%) and homozygous duplication in 75 (32.5%). The allele frequency was 56.1% (95% confidence interval, 49.4-62.7%). The frequency of the 24 bp duplication was remarkably high in both Korean patients with Gaucher disease and in the normal population, limiting the efficacy of chitotriosidase as a biomarker in Gaucher disease in Korea. New biomarkers are required that consider the genetic characteristics of different populations.

1,3-Dinitrobenzene (DNB) is an industrial intermediate and testicular toxicant that has been shown to target Sertoli cells. The mechanism of action of DNB in the testis, however, is unclear. To investigate global alterations in gene or protein expression during testicular toxicity, testes from rats treated orally with DNB were subjected to microarray and two-dimensional gel electrophoresis (2-DE) analyses. Histopathological abnormalities were detected in the testes of the DNB-treated rats. Microarray analysis revealed that, during early testicular toxicity, several genes involved in apoptosis, germ cell/Sertoli cell junction, and tight junction signaling pathways were differentially expressed. Based on 2-DE analysis, 36 protein spots showing significantly different expression during early testicular toxicity were selected and identified. Network analysis of the identified proteins revealed that these proteins are associated with cellular development or reproductive system diseases. Collectively, these data will help clarify the molecular mechanism underlying testicular toxicity in DNB-exposed rats.

Theranostic agents present a promising clinical approach for cancer detection and treatment. We herein introduce a microbubble and liposome complex (MB-Lipo) developed for ultrasound (US) imaging and activation. The MB-Lipo particles have a hybrid structure consisting of a MB complexed with multiple Lipos. The MB components are used to generate high echo signals in US imaging, while the Lipos serve as a versatile carrier of therapeutic materials. MB-Lipo allows high contrast US imaging of tumor sites. More importantly, the application of high acoustic pressure bursts MBs, which releases therapeutic Lipos and further enhances their intracellular delivery through sonoporation effect. Both imaging and drug release could thus be achieved by a single US modality, enabling in situ treatment guided by real-time imaging. The MB-Lipo system was applied to specifically deliver anti-cancer drug and genes to tumor cells, which showed enhanced therapeutic effect. We also demonstrate the clinical potential of MB-Lipo by imaging and treating tumor in vivo.

Background/Aims: This study aimed to clarify the frequency, phenotypes, and molecular spectrum of DUOX2, TPO, TSHR, and TG mutations in patients with congenital hypothyroidism (CH) with enlarged or normal-sized eutopic thyroid glands. Methods: The study cohort included 43 subjects from 41 unrelated families who had CH with eutopic thyroid glands. Mutation analyses of DUOX2, TPO, and TSHR were performed. The functional capacities of novel missense variants of DUOX2 were verified by measuring H2O2 generation in vitro. Results: Of the 43 subjects, 23 (53.5%) had sequence variants in at least one gene. Twelve different DUOX2 variants, including seven novel variants, were identified in 20 subjects. A functional analysis of the DUOX2 variants revealed that most variants, other than p.G206V and p.H678R, caused a significant reduction in H2O2 generation. Therefore, 15 subjects harbored functionally deleterious DUOX2 variants. Of these, 5 subjects had transient CH, and 10 were found to have permanent CH. Sequence variants in TSHR were identified in 5 subjects. One of the 43 subjects (2.3%) had sequence variants in two different genes. Conclusions:DUOX2 variants are a relatively common cause of CH with normal-sized or enlarged eutopic thyroid glands. Variable phenotypes were associated with partial loss of the functional activity of DUOX2 variants.

Tcea3 is present in high concentrations in mouse embryonic stem cells (mESCs) and functions to activate Lefty1, a negative regulator of Nodal signaling. The Nodal pathway has numerous biological activities, including mesoderm induction and patterning in early embryogenesis. Here, we demonstrate that the suppression of Tcea3 in mESCs shifts the cells from pluripotency into enhanced mesoderm development. Vascular endothelial growth factor A (VEGFA) and VEGFC, major transcription factors that regulate vasculogenesis, are activated in Tcea3 knocked down (Tcea3 KD) mESCs. Moreover, differentiating Tcea3 KD mESCs have perturbed gene expression profiles with suppressed ectoderm and activated mesoderm lineage markers. Most early differentiating Tcea3 KD cells expressed Brachyury-T, a mesoderm marker, whereas control cells did not express the gene. Finally, development of chimeric embryos that included Tcea3 KD mESCs was perturbed.

In this study we developed a textbook for middle school mathematics, especially focusing on the 1st grade, based on storytelling, did experimental lessons using the developed textbook, and surveyed the responses of students to the lesson by three kinds of questionnaire and teacher interview. The results of this study can serve as basic data for other researches about storytelling-related education in school mathematics.

Type 1 citrullinemia (CTLN1) often presents as a hyperammonemic encephalopathy in the neonatal period, but it can also develop in the late-infantile period and in adults. In addition, some patients can be identified in the presymptomatic period by neonatal or family member screening. In this study, twenty Korean patients with CTLN1 (19 families) were examined; fourteen patients with neonatal-onset, three with late-onset, and three that were identified presymptomatically. The 13 patients with hyperammonemic encephalopathy received continuous venovenous hemofiltration (CVVH) or peritoneal dialysis (PD). Although the hyperammonemia was relieved more effectively in the six patients on CVVH than the seven on PD, most of these patients suffered from severe neurologic deficits. Recurrent hyperammonemic episodes (7 pts, 35%), recurrent and reversible acute hepatic dysfunction (5 pts, 25%), and focal cerebral infarction (2 pts, 10%) were noted. The neonates with hyperammonemic encephalopathy had extensive brain injuries at the onset of hyperammonemia, followed by encephalomalacia and brain atrophy at quite an early age. Genetic testing for the ASS1 gene revealed a different mutation spectrum from those of other ethnicities; Three common mutations, c.421-2A>G (37.8%), c.1128-6_1188dup67 (18.9%), and p.Gly324Ser (16.2%), accounted for 73% of the mutations. The poor outcome was expected in patients with the peak ammonia level at onset over 600μmol/L, whose proportion was higher in the neonatal presentation group than in the presymptomatic/late presentation group. Our findings add to the current understanding of the ethnic diversity of CTLN1 from both clinical and genetic perspectives.

Disorders of sex development (DSD) vary phenotypically and are caused by a number of genetic etiologies. This study investigated the genetic etiology of DSD patients using targeted exome sequencing of 67 known DSD-associated genes in humans. This study included 37 patients with 46, XY DSD and seven patients with 46, XX DSD. We identified known pathogenic mutations or deletion in nine (20.5%) patients in the AR, CYP17A1, SRD5A1, and DMRT1/2 genes. Novel variants were identified in nine patients (20.5%) in the AR, ATRX, CYP17A1, CHD7, MAP3K1, NR5A1, and WWOX genes. Among them, four patients harbored pathogenic or likely pathogenic variants, while the remaining five patients (11.4%) had variants of uncertain significance. We were able to make a genetic diagnosis in 29.5% of patients with pathogenic or likely pathogenic mutations. Targeted exome sequencing is an efficient tool to improve the diagnostic yield of DSD, despite its phenotypic and genetic heterogeneity.

// In Ah Park 1, * , Sun-Hee Heo 1, 2, * , In Hye Song 1 , Young-Ae Kim 1, 2 , Hye Seon Park 1, 2 , Won Seon Bang 1, 2 , Suk Young Park 1, 2 , Jeong-Hyon Jo 3 , Hee Jin Lee 1, ** , Gyungyub Gong 1, ** 1 Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 2 Asan Center for Cancer Genome Discovery, Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 3 Department of Pathology, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea * Authors share co-first authorship ** Authors share co-corresponding authorship Correspondence to: Hee Jin Lee, email: backlila@gmail.com Gyungyub Gong, email: gygong@amc.seoul.kr Keywords: breast carcinoma, tumor-infiltrating lymphocytes, endoplasmic reticulum, high-mobility group protein Received: April 23, 2016 Accepted: July 19, 2016 Published: August 2, 2016 ABSTRACT Background: Although the prognostic and predictive significance of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) have been shown, the cause of the TIL influx is unclear. Here, we investigated whether extracellular secretion of HMGN1 is associated with TIL influx, as well as increased endoplasmic reticulum stress (ERS), in human TNBC. Methods: We reviewed the slides of 767 patients with TNBC and evaluated the TIL levels. We also assessed the expression of HMGs and several ERS-associated molecules using immunohistochemical staining. Western blot analysis of human TNBC cell lines and pharmacological ERS inducers was used to determine if HMGN1 migrates from the nucleus to the extracellular space in response to ERS. Results: On immunohistochemical staining, either higher nuclear or cytoplasmic expression of both HMGB1 and HMGN1 was significantly associated with ERS. TILs showed a positive correlation with the cytoplasmic expression of the HMGs. Western blot analysis of TNBC cell lines showed that ERS induction resulted in the secretion of HMG proteins. Conclusions: This is the first study to elucidate the associations among ERS, secretion of HMGs, and degree of TILs in TNBCs. Understanding the mechanisms of TIL influx will help in the development of effective immunotherapeutic agents for TNBC.