Cervical lymphoepithelioma-like carcinoma with deficient mismatch repair and loss of SMARCA4/BRG1: a case report and five related cases.

BACKGROUND: We encountered a cervical lymphoepithelial carcinoma (LEC) possessing a predominantly solid architecture with deficient mismatch repair (dMMR) and loss of expression of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex subunit. This is the first case report of LEC with dMMR and loss of SWI/SNF complex subunit. CASE PRESENTATION: A 34-year-old woman presented at our hospital with menstrual irregularities and abnormal vaginal bleeding. Magnetic resonance imaging revealed an exophytic mass in the posterior uterine cervix. Biopsy specimens confirmed squamous cell carcinoma with a 2018 International Federation of Gynecology and Obstetrics (FIGO) uterine cervical cancer stage of IB2. In a subsequent conization specimen, the tumor appeared exophytic. Microscopically, the tumor cells formed a predominant solid architecture. Abundant lymphocytic infiltration was observed. The pathological diagnosis indicated human papillomavirus (HPV)-associated squamous cell carcinoma with LEC pattern and pT1b2. Immunohistochemically, high programmed death-ligand 1 (PD-L1) expression, dMMR, and loss of the switch/sucrose non-fermentable family-related, matrix-associated, actin-dependent regulator of chromatin subfamily member 4 (SMARCA4)/BRG1, an SWI/SNF complex subunit, were observed. The patient underwent a radical hysterectomy and is alive without disease one year and five months later. Our analysis of five additional LEC cases revealed a consistent association with high-risk HPV and elevated PD-L1 expression. In addition to the present case, another patient exhibited dMMR. The SWI/SNF complex was retained except in the present case. The prognosis was favorable in all cases. CONCLUSIONS: This unique case of LEC with dMMR suggests a distinct clinical entity with potential immunotherapy implications. Analysis of the other five LEC cases revealed that LEC was immune hot, and immune checkpoint inhibitors may be effective. The two dMMR cases showed loss of MLH1 and PMS2 expressions, and prominently high tumor PD-L1 expression. In those cases, dMMR might have contributed to the morphological characteristics of LEC.

One-step generation of mice with gene editing by Tol2 transposon-dependent gRNA delivery.

Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.

Detection of Balenine in Mouse Plasma after Administration of Opah-Derived Balenine by HPLC with PITC Pre-Column Derivatization.

We examined the absorption of balenine (Bal) in mouse blood after the administration of a high-purity Bal prepared from opah muscle. Using HPLC with phenyl isothiocyanate pre-column derivatization, we successfully isolated imidazole peptides and their constituents. We detected Bal and 3-methylhistidine (3-Me-His) in mouse blood 1 h after the administration of opah-derived Bal. The concentrations of Bal and 3-Me-His significantly increased to 128.27 and 69.09 nmol/mL in plasma, respectively, but were undetectable in control and carnosine (Car)-administrated mice. In contrast, ß-alanine and histidine did not increase in mouse plasma 1 h after the administration of Car and opah-derived Bal. The present study is the first report on the absorption of food-derived Bal in mouse blood and serves as a pilot study for future clinical trials.

Generation of a tendon-like tissue from human iPS cells.

Tendons and ligaments are essential connective tissues that connect the muscle and bone. Their recovery from injuries is known to be poor, highlighting the crucial need for an effective therapy. A few reports have described the development of artificial ligaments with sufficient strength from human cells. In this study, we successfully generated a tendon-like tissue (bio-tendon) using human induced pluripotent stem cells (iPSCs). We first differentiated human iPSCs into mesenchymal stem cells (iPSC-MSCs) and transfected them with Mohawk (Mkx) to obtain Mkx-iPSC-MSCs, which were applied to a newly designed chamber with a mechanical stretch incubation system. The embedded Mkx-iPSC-MSCs created bio-tendons and exhibited an aligned extracellular matrix structure. Transplantation of the bio-tendons into a mouse Achilles tendon rupture model showed host-derived cell infiltration with improved histological score and biomechanical properties. Taken together, the bio-tendon generated in this study has potential clinical applications for tendon/ligament-related injuries and diseases.

Cytological features of BCOR-CCNB3 sarcoma: Comparison with Ewing sarcoma and synovial sarcoma.

INTRODUCTION: BCOR-CCNB3 sarcoma (BCS) is one of the histological types classified as an undifferentiated small round cell sarcoma of bone and soft tissue. This sarcoma frequently develops in males under 20 years of age. Histologically, a delicate capillary network has been reported as a conspicuous finding. In this study, the cytological findings of BCS were observed in two cases of primary lesions and one case of a lung metastatic lesion. The cytological findings of BCS were compared with its histological mimics, and the characteristic findings of BCS were examined. METHODS: Three cases of BCS were studied, and a cytological comparison was performed with 8 cases of Ewing sarcoma (ES) and 10 cases of synovial sarcoma (SS; monophasic type: 7 cases, biphasic type: 2 cases, poorly differentiated: 1 case). RESULTS: In all BCS cases, small clusters with thin and delicate vascular cores and tiny vascular fragments were conspicuous. In ES and SS cases, although small clusters with vascular cores were observed, the vascular cores were thicker than in BCS, and no tiny vascular fragments appeared in most cases. Cytomorphological differences of tumour cells were also observed among BCS, ES, and SS. Predominantly rounded nuclei with fine chromatin and inconspicuous nucleoli can be cytological clues for BCS. CONCLUSIONS: BCS shows characteristic cytological findings that make the diagnosis of BCS more likely than that of ES and SS. Cytological evaluation is a useful tool for appropriate differential diagnosis that leads to a more accurate final diagnosis and rapid treatment.

Isolation of balenine from opah (Lampris megalopsis) muscle and comparison of antioxidant and iron-chelating activities with other major imidazole dipeptides.

Balenine (Bal) in opah muscle was extracted using hot water and purified by ion-exchange chromatography and recrystallization to provide 41 g of over 95% pure Bal from 1 kg of opah muscle. The structure of purified Bal was identical to that of an authentic Bal standard by NMR analysis. The antioxidant (ORAC and HORAC values) and Fe(II) ion-chelating abilities of purified Bal were examined by comparison with two major imidazole dipeptides, carnosine (Car) and anserine (Ans). Opah-derived Bal showed significantly higher ORAC and HORAC values and Fe(II) ion-chelating ability at 0.3 mM. In silico molecular simulation revealed that Bal and Car formed hydrogen bonds between the hydrogen atom of the imidazole imino group and the carboxyl carbonyl oxygen, whereas Ans did not. The proposed method for extracting and purifying Bal from opah muscle suggests that opah can be utilized as a functional food or Bal resource.

Mkx regulates the orthodontic tooth movement via osteoclast induction.

INTRODUCTION: The periodontal ligament (PDL) plays an important role in orthodontic tooth movement; however, the underlying molecular mechanism remains unclear. We have previously reported that the Mohawk homeobox (Mkx), a tendon-specific transcription factor, is expressed in the PDL and regulates its homeostasis. MATERIALS AND METHODS: In the present study, we examined the role of Mkx in orthodontic tooth movement via bone remodeling induced by mechanical stimulation in Mkx-deficient rats, which are widely used as experimental animals for orthodontic force application. Orthodontic tooth movement of the maxillary first molar was performed in 7-week-old male Mkx-deficient rats (n = 4) and wild-type Wistar rats (n = 4) using coil springs for 14 days. Hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to evaluate morphological changes and osteoclasts. Furthermore, changes in the expression of receptor activator nuclear factor-kappa B ligand (RANKL) were demonstrated using immunostaining. RESULTS: The amount of tooth movement was significantly lower in Mkx-deficient rats than in wild-type rats. The number of TRAP-positive cells was suppressed in Mkx-deficient rats on the compression side. CONCLUSION: Orthodontic tooth movement experiments in Mkx-deficient rats suggested that Mkx is involved in osteoclast induction at the alveolar bone surface on the compression side. This study reveals the possibility that Mkx plays a mechanosensory role in orthodontic tooth movement by inducing RANKL expression and osteoclastogenesis.

Primary thymic adenocarcinoma with an aggressive clinical course: An autopsy case showing signet ring cell-like features.

Thymic adenocarcinoma is an extremely rare neoplasm, and little is known about its pathogenesis and clinical characteristics. A 52-year-old man presented to our clinic with severe dyspnea. At initial presentation, massive carcinomatous pleuritis and pericarditis were observed, and a lobulated mass in the anterior mediastinum was found on computed tomography. Cytological examination revealed adenocarcinoma accompanied by signet ring cells; however, his tumor showed aggressive growth without any possibility of treatment, and he died as a result of cancer progression within one month of admission. An autopsy confirmed thymic adenocarcinoma showing various histological features including mucinous, signet ring cell-like, and trabecular features. Immunohistochemically, the tumor cells were positive for cytokeratin (CK) (AE1/AE3) but negative for TTF-1. In addition, some tumor cells were positive for CD5 and KIT. Further examination revealed that tumor cells of the nonmucinous type were positive for CK7, and negative for CK20 and caudal-type homeobox 2 (CDX2). The tumor cells with mucinous and signet ring-like features were positive for CK20 and CDX2 and negative for CK7, indicating enteric differentiation. In particular, tumor cells with signet ring cell-like features indicated widespread lymphangitic carcinomatosis and pulmonary tumor thrombotic microangiopathy (PTTM). The presence of signet ring cell-like features with enteric differentiation is suggestive of a fulminant clinical course due to widespread lymphangiosis carcinomatosa and PTTM in patients with thymic adenocarcinoma. KEY POINTS: Thymic adenocarcinoma is an extremely rare neoplasm. Histological features of thymic adenocarcinoma include mucinous, signet ring cell-like, and trabecular features. Tumor cells with signet ring cell-like features indicate widespread lymphangitic carcinomatosis and pulmonary tumor thrombotic microangiopathy. The presence of signet ring cell-like features with enteric differentiation is associated with a fulminant clinical course.

In vitro Neo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System.

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

Female-specific gene regulation in malaria parasites by an AP2-family transcription factor.

The malaria gametocyte, the gamete precursor, is the essential stage for malaria transmission to the mosquito vector. In the vertebrate host's blood, it develops into a mature male or female capable of transforming into a gamete in the mosquito blood meal. Despite the importance of this stage in the malaria life cycle, the genetic regulation of gametocyte development is poorly understood. In particular, transcription factors involved in sex-specific gene expression have not been identified. In this paper, we report that an AP2-family transcription factor, AP2-FG, is responsible for female-specific gene regulation. AP2-FG expression in Plasmodium berghei was observed exclusively in female gametocytes, in the beginning of 4-6 h before sexual dimorphism manifests in developing gametocytes. AP2-FG disruption resulted in the arrest of female maturation, but did not affect the development of males. Chromatin immunoprecipitation sequencing analysis suggested that AP2-FG directly regulates over 700 genes. Its targets include genes for female gametocyte-specific functions, such as gametogenesis, fertilization and zygote development. AP2-FG binding to target gene promoters was associated with a 10 bp sequence motif. These results indicate that AP2-FG plays a role in the differentiation of early gametocytes to mature females by governing a female-specific gene expression repertoire.

Organizing Pneumonia after Nivolumab Treatment in a Patient with Pathologically Proven Idiopathic Pulmonary Fibrosis.

Acute exacerbation of pre-existing interstitial lung disease (ILD) associated with systemic anticancer therapy is recognized as a life-threatening adverse event of lung cancer treatment. Programmed cell death 1 (PD-1) checkpoint inhibitors, such as nivolumab, often induce pneumonitis in patients with cancer; however, the tolerance and safety of nivolumab for advanced lung cancer with ILD are unclear. We report a 72-year-old patient with lung cancer with pathologically proven idiopathic pulmonary fibrosis who was treated with nivolumab. She demonstrated pneumonitis with an organized pneumonia (OP) pattern, but no acute exacerbation of ILD featuring a diffuse alveolar damage (DAD) pattern. She was successfully treated with corticosteroid therapy, and maintained good disease control after the discontinuation of nivolumab. She also showed pseudoprogression of the primary tumor, implying infiltration of T-cells into the lung. These findings suggest that T-cell activation by nivolumab treatment might not be directly associated with acute ILD exacerbation, and that treatable OP might be a major pulmonary complication of nivolumab in patients with pre-existing ILD, similar to patients without underlying ILD.

Ablation of miR-146b in mice causes hematopoietic malignancy.

Excessive and constitutive activation of nuclear factor-κB (NF-κB) leads to abnormal cell proliferation and differentiation, leading to the development of malignant tumors, including lymphoma. MicroRNA 146a (miR-146a) and miR-146b, both of which carry an identical seed sequence, have been shown to contribute to inflammatory diseases and tumors by suppressing the expression of key molecules required for NF-κB activation. However, the functional and physiological differences between miR-146a and miR-146b in disease onset have not been fully elucidated. In this study, we generated miR-146b-knockout (KO) and miR-146a-KO mice by genome editing and found that both strains developed hematopoietic malignancies such as B-cell lymphoma and acute myeloid leukemia during aging. However, the B-cell lymphomas observed in miR-146a- and miR-146b-KO mice were histologically different in their morphology, and the malignancy rate is lower in miR-146b mice than miR-146a mice. Upon mitogenic stimulation, the expression of miR-146a and miR-146b was increased, but miR-146b expression was lower than that of miR-146a. Using a previously developed screening system for microRNA targets, we observed that miR-146a and miR-146b could target the same mRNAs, including TRAF6, and inhibit subsequent NF-κB activity. Consistent with these findings, both miR-146a- and miR-146b-KO B cells showed a high proliferative capacity. Taken together, sustained NF-κB activation in miR-146b KO mice could lead to the development of hematopoietic malignancy with aging.

Pembrolizumab and salvage chemotherapy in EGFR T790M-positive non-small-cell lung cancer with high PD-L1 expression.

Immuno-checkpoint inhibitors (ICI) have become an effective treatment option for non-small-cell lung cancer patients. However, ICI therapy was reported to be less effective in patients with epidermal growth factor receptor (EGFR) mutations than in those with wild-type EGFR. We report here that an non-small-cell lung cancer patient with the EGFR mutant T790M showed a programmed cell death ligand 1 (PD-L1) expression level that increased from <25% to >90% after eighth-line osimertinib therapy. He was treated with pembrolizumab as a ninth-line treatment, and attained stable disease. After the pembrolizumab therapy, he was treated with gemcitabine, which produced a good response despite being the 10th-line treatment. We should consider administering ICI and chemotherapy even to EGFR mutant patients after failure of EGFR tyrosine kinase inhibitor, especially in cases with high PD-LI expression.

Combinatorial CRISPR/Cas9 Approach to Elucidate a Far-Upstream Enhancer Complex for Tissue-Specific Sox9 Expression.

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

MET amplification in endometrial cancers with clear-cell carcinoma components.

Endometrial clear-cell carcinoma (ECC) is relatively rare. The expression of diagnostic markers in this disease is similar to that of clear-cell carcinoma, but the molecular carcinogenic events and therapeutic targets are mostly unknown. MET gene amplification has been reported in various cancers, including ovarian clear-cell carcinomas; however, the MET gene status has not previously been examined in ECC. We performed real-time quantitative PCR (QPCR) and fluorescence in situ hybridization (FISH) to analyze the MET gene statuses of 12 ECC cases. We found MET amplifications in two cases (2/12; 16.7%) by both methods. Of the 12 cases, 9 were pure clear-cell carcinomas, and 3 were mixed types that included mixes with endometrioid carcinomas in 2 cases, and the remaining case was a heterologous-type carcinosarcoma that primarily consisted of a clear-cell carcinoma component and a scarce chondrosarcoma component. Both of the MET amplification cases were mixed; one contained endometrioid features, and the other chondrosarcoma features. This is the first report to analyze the statuses of the MET gene in ECCs, and the two mixed cases exhibited amplifications that are shared with ovarian clear-cell carcinomas. Further studies with larger numbers of cases are necessary to reveal the relationship between ECC and MET amplification.

A case of peritoneal malignant mesothelioma following radiation therapy for cervical cancer.

The present study presents a case of peritoneal malignant mesothelioma (PMM) following radiation therapy for cervical cancer. A 34-year-old Japanese woman, without asbestos exposure, was referred to the Department of Gynecologic Oncology, Saitama Medical University International Medical Center due to a cervical mass, and was diagnosed with cervical squamous cell carcinoma (SCC). The serum levels of tumor markers, including SCC antigen and cancer antigen 125 (CA125) were 229.0 ng/ml and 54.4 U/ml, respectively. The patient underwent concurrent chemoradiotherapy (CCRT), and a complete response was achieved. After 54 months, ascites was found at the rectouterine pouch, but peritoneal cytology suggested reactive mesothelial cell. After 62 months of CCRT, magnetic resonance imaging revealed masses in both the salpinges. The serum levels of SCC and CA125 were 0.9 ng/ml and 506.1 U/ml, respectively. Following this, left salpingectomy and peritoneal biopsy were performed laparoscopically. Histologic examination revealed atypical mesothelial cells with no continuity of background tubal epithelium. Immunohistochemistry showed positive staining for calretinin, thrombomodulin, mesothelin and glucose transporter 1. Based on these findings, the patient was diagnosed with PMM epithelioid type and underwent systemic chemotherapy; stable disease status has been obtained for 3 months. This case demonstrates the possibility of PMM occurrence within 10 years after radiotherapy, and indicates the importance of histological and immunohistochemical examination, particularly in cases of an atypical tumorigenesis pattern from the primary cancer.

Erratum: Pre-operative evaluation of circulating KL-6 levels as a biomarker for epithelial ovarian carcinoma and its correlation with tumor MUC1 expression.

[This corrects the article DOI: 10.3892/ol.2017.6254.].

Pre-operative evaluation of circulating KL-6 levels as a biomarker for epithelial ovarian carcinoma and its correlation with tumor MUC1 expression.

Krebs von den Lungen-6 (KL-6), a mucinous sialylated sugar chain on human mucin-1 glycoprotein (MUC1), is a diagnostic marker for interstitial lung diseases. Furthermore, elevated serum KL-6 levels have been observed in certain malignant tumor types of epithelial origin. The expression of MUC1 has been observed in patients with epithelial ovarian cancer (EOC) and is considered a potential therapeutic target. In the present study, KL-6 serum levels were investigated in patients clinically suspected of having malignant ovarian tumors. A total of 219 patients were enrolled in the study, which analyzed their serum KL-6 levels in addition to tumor expression of MUC1 using immunohistochemistry. High serum KL-6 levels were predominantly observed in patients with EOC, and did not occur in patients with benign or borderline tumors. The level of serum KL-6 was highly correlated with tumor stage, grade and histological type, and demonstrated superior sensitivity for the detection of ovarian cancer compared with that of serum cancer antigen 125. High serum KL-6 was significantly associated with shorter progression-free survival. In addition, tumor MUC1 expression status was significantly correlated with serum KL-6 levels. These data suggest that serum KL-6 may be a useful, non-invasive biomarker surrogate for tumor MUC1 expression in future clinical trials of MUC1-targeted therapy.

Correlation Between Tumor Mesothelin Expression and Serum Mesothelin in Patients with Epithelial Ovarian Carcinoma: A Potential Noninvasive Biomarker for Mesothelin-targeted Therapy.

BACKGROUND: The cell surface glycoprotein mesothelin is highly expressed in several malignant diseases. Normal mesothelin expression is limited to mesothelial cells lining the pleura, peritoneum, and pericardium, making it a biomarker and an attractive target for cancer therapy. METHODS: We investigated tumor mesothelin expression and serum mesothelin levels in patients with epithelial ovarian cancer or borderline tumors. In total, 161 patients selected from a previous prospective study were analyzed for tumor mesothelin expression using immunohistochemistry and serum mesothelin expression using enzyme-linked immunosorbent assay. RESULTS: Eighty-eight (68.8%) epithelial ovarian cancers and eight (24.2%) borderline tumors showed high mesothelin expression, which was associated with shorter progression-free and overall survival. The tumor mesothelin expression status was moderately correlated with serum mesothelin levels in epithelial ovarian cancer patients. Based on receiver operating characteristic analysis, a serum mesothelin level above 2.20 nM predicted high tumor mesothelin expression in epithelial ovarian cancer patients (area under the curve = 0.81). In 45 patients with recurrent epithelial ovarian cancer, we observed relatively lower levels of serum mesothelin, compared to the level at the primary diagnosis. We also tracked the change in the serum mesothelin level during the course of second-line chemotherapy and found a discrepancy between the clinical response and the serum mesothelin change in some patients, which suggested tumor heterogeneity among the tumor cells with or without mesothelin expression. CONCLUSION: Serum mesothelin may be a useful noninvasive biomarker surrogate for tumor mesothelin expression in future clinical trials for mesothelin-targeted therapy.

Regulation of visual Wulst cell responsiveness by imprinting causes stimulus-specific activation of rostral cells.

Imprinting behaviour in chicks can be induced exclusively during a short period after hatching. During this period, visual information on the imprinting stimulus is conveyed to the visual Wulst (VW) in the telencephalon, which corresponds to the visual cortex of mammals, and then to the memory-storing region known as the intermediate medial mesopallium. These two regions are indispensable for imprinting. We previously showed that imprinting training altered the response pattern of the VW to the imprinting stimulus; however, the precise distribution of cells and the mechanism involved with this altered response remains unclear. Here we showed that a specific population of rostral VW cells responded to the imprinting stimulus by analysing the subcellular localization of Arc/arg3.1 transcripts in VW cells. GABAergic parvalbumin (PV) cells are abundant in the dorsal region of this area, and imprinting training doubled the number of activated PV-positive neurons. An injection of bicuculline, a GABA(A) receptor antagonist, in the dorsal VW disturbed the rostral distribution of responsive cells and thus resulted in a lack of imprinting. These results suggest that activated PV cells restrict VW cells response to dorsal area to form a specific imprinting pathway.