Search

Background/aims Postoperative adhesions may induce adverse outcomes in patients. Adhesion formation is initiated by fibrin accumulation at the surgical site which is followed by local neutrophilia and the establishment of neutrophil extracellular traps (NET). Previous reports have suggested that the preventive efficacy of reagents designed to reduce postoperative adhesion is inversely correlated with neutrophilia and NET production. Antithrombin (AT) is a natural inhibitor of thrombin, a key factor in coagulation. Here, we evaluate whether treatment with AT and/or NET inhibitors prevent or reduce postoperative adhesion formation in mice. Methods Mice were treated with AT and/or NET inhibitors before and/or after cecum cauterization and their adhesion scores were evaluated on day 7 post-operation. Immunochemistry/ immunofluorescence analyses were also performed and we used GSK484, an inhibitor of peptidyl arginine deiminase 4 (PAD4), as the NET inhibitor. Results AT or GSK484 partially rescued postoperative adhesion formation in mice. AT prevented thrombin-induced plasminogen activator inhibitor 1 and interleukin-6 expression in mesothelial cells in vitro. However, AT could not prevent neutrophilia or NETs formation around the injured serosa. Finally, we investigated a combination of AT and a PAD4 inhibitor and found that this could inhibit almost all adhesion formation in these animals. Since AT-inactivating proteases are liberated following NET release, they might dampen the biological action of the AT treatment. This suggests that NET inhibitors might allow AT to exert its full action in the surgically injured serosa. Conclusion Combined treatment with AT and GSK484 may effectively attenuate postoperative adhesion production in mice.

Lymphangioleiomyomatosis (LAM) is a rare destructive lung disease characterized by multiple thin-walled pulmonary cysts. The currently proposed diagnostic algorithm emphasizes the characteristic cystic appearance on high-resolution computed tomography (HRCT) so uncommon HRCT appearances present challenges to establishing the proper LAM diagnosis. The objective of this study is to accrue uncommon chest HRCT appearances, determine frequencies in both tuberous sclerosis complex (TSC)-associated LAM (TSC-LAM) and sporadic LAM (S-LAM) patients. 311 females referred to our hospital, including 272 S-LAM patients (mean age 39.2 years) and 39 TSC-LAM patients (mean age 38.3 years), were retrospectively evaluated. We found 2 types of radiologic findings likely to make HRCT cyst appearance atypical: characteristics of the cyst itself and uncommon findings in addition to cysts. We found that approximately 80% of LAM patients, whether TSC-associated or sporadic, showed typical HRCT appearance with mild to severe cystic destruction. The remaining 20% displayed unusual profiles in cyst appearance as well as additional findings aside from cyst: the former includes large cyst, thickened walls, and irregularly shaped whereas the latter includes ground glass attenuation and diffuse noncalcified nodules. It is important to be aware of various radiologic findings that make HRCT cystic appearance atypical of LAM.

Background/aims Although adhesion formation is a frequent adverse event following intraperitoneal surgery, efficient prophylactic interventions have not yet been established. We recently reported that blockade of interleukin (IL)-6 prevented postoperative adhesion after cecum cauterization. Intriguingly, this intervention dampened tumor necrosis factor (TNF) induction in the injured serosa. Herein, we addressed whether TNF might be a key target and, if so, how TNF blockade rescued adhesion formation. Methods Mice were administered an anti-TNF biologic (etanercept) on days -2 and -1 before and upon cecal cauterization. The adhesion scores were evaluated at day 7 postoperatively. Histological alterations were examined by immunochemistry/immunofluorescence studies. We incubated human neutrophils and mesothelial cell line cells with recombinant TNF in the presence of etanercept and measured transcript levels of cytokines and chemokines by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Results Etanercept rescued mice from adhesion formation, accompanied by a robust reduction of neutrophilia in the injured serosa. Immunofluorescence revealed a substantial formation of neutrophil extracellular traps (NETs) with the potential to induce tissue damage and profibrotic responses. In contrast, the etanercept-treated mice lacked NET formation. In addition, etanercept inhibited TNF-induced IL-6, TNF, and neutrophil-recruiting chemokines in neutrophils and mesothelial cells, a major cellular source of myofibroblasts in the adhesion band. Conclusion Prophylactic administration of etanercept might be a potential strategy for preventing postoperative adhesion formation.

Aims Pleomorphic adenoma gene 1 (PLAG1) rearrangement is well known in pleomorphic adenoma (PA), which is histologically characterised by admixed epithelial and mesenchymal components. Multiple fusion variants of PLAG1 and HMGA2 have been reported; currently, however, little is known regarding the clinicopathological impacts of these fusion types METHODS AND RESULTS: We examined the PLAG1- and HMGA2-related fusion status in 105 PAs and 11 cases of carcinoma ex PAs (CXPA) arising from salivary glands and lacrimal glands to elucidate their correlation to the clinicopathological factors. Forty cases harboured PLAG1 fusion genes: CTNNB1-PLAG1 in 22 cases, CHCHD7-PLAG1 in 14 cases and LIFR-PLAG1 in four cases. Only two cases possessed HMGA2 fusion genes. The mean age of LIFR-PLAG1-positive cases was significantly higher than that of CTNNB1-PLAG1- and CHCHD7-PLAG1-positive cases (P = 0.0358). PAs located in the submandibular gland demonstrated CTNNB1-PLAG1 fusion at a significantly higher rate than other fusions (P = 0.0109). Histologically, PLAG1 fusion-positive cases exhibited chondroid formation and plasmacytoid features more commonly (P = 0.043, P = 0.015, respectively) and myxoid abundant feature less frequently (P = 0.031) than PLAG1 fusion-negative cases. For CXPAs, four CTNNB1-PLAG1 fusions were detected in two salivary duct carcinomas and two myoepithelial carcinomas. Ductal formation was observed frequently (90.9%) in residual PA. Conclusions The presence of PLAG1 fusion was associated with specific histological features in PA. Detecting the PLAG1 fusion gene and searching residual ductal formation in salivary gland malignant tumours with extensive hyalinisation could be useful for diagnosis.

Lymphangioleiomyomatosis (LAM) is a rare pulmonary disease characterised by the proliferation of smooth muscle-like cells (LAM cells), and an abundance of lymphatic vessels in LAM lesions. Studies reported that vascular endothelial growth factor-D (VEGF-D) secreted by LAM cells contributes to LAM-associated lymphangiogenesis, however, the precise mechanisms of lymphangiogenesis and characteristics of lymphatic endothelial cells (LECs) in LAM lesions have not yet been elucidated. In this study, human primary-cultured LECs were obtained both from LAM-affected lung tissues (LAM-LECs) and normal lung tissues (control LECs) using fluorescence-activated cell sorting (FACS). We found that LAM-LECs had significantly higher ability of proliferation and migration compared to control LECs. VEGF-D significantly promoted migration of LECs but not proliferation of LECs in vitro. cDNA microarray and FACS analysis revealed the expression of vascular endothelial growth factor receptor (VEGFR)-3 and integrin α9 were elevated in LAM-LECs. Inhibition of VEGFR-3 suppressed proliferation and migration of LECs, and blockade of integrin α9 reduced VEGF-D-induced migration of LECs. Our data uncovered the distinct features of LAM-associated LECs, increased proliferation and migration, which may be due to higher expression of VEGFR-3 and integrin α9. Furthermore, we also found VEGF-D/VEGFR-3 and VEGF-D/ integrin α9 signaling play an important role in LAM-associated lymphangiogenesis.

Birt–Hogg–Dubé syndrome (BHDS), an autosomal dominant inheritance disease caused by folliculin (FLCN) mutations, is associated with lung cysts and spontaneous pneumothorax. The possibility of FLCN haploinsufficiency in pleural mesothelial cells (PMCs) contributing to development of pneumothorax has not yet been clarified. Electron microscopy revealed exposed intercellular boundaries between PMCs on visceral pleura and decreased electron density around the adherens junctions in BHDS. To characterize cellular function of PMCs in BHDS patients (BHDS-PMCs), during surgery for pneumothorax, we established the flow cytometry-based methods of isolating high-purity PMCs from pleural lavage fluid. BHDS-PMCs showed impaired cell attachment and a significant decrease in proliferation and migration, but a significant increase in apoptosis compared with PMCs from primary spontaneous pneumothorax (PSP) patients (PSP-PMCs). Microarray analysis using isolated PMCs revealed a significant alteration in the expression of genes belonging to Gene Ontology terms “cell-cell adhesion junction” and “cell adhesion molecule binding”. Gene set enrichment analysis demonstrated that CDH1, encoding E-cadherin, was identified in the down-regulated leading edge of a plot in BHDS-PMCs. AMPK and LKB1 activation were significantly impaired in BHDS-PMCs compared with PSP-PMCs. Our findings indicate that FLCN haploinsufficiency may affect the E-cadherin-LKB1-AMPK axis and lead to abnormal cellular function in BHDS-PMCs.

The term "apocryphal" has been applied to a broad range of medieval Slavonic texts. Many of them were composed in the Judeo-Hellenistic literary tradition and brought into the Slavic lands, forming a particular textual corpus abundant in a variety of contents and narrative styles. However, there is also a group of pieces regarded as Slavonic apocrypha but whose origin is unclear. The Dream of King Jehoash, a very short story written in Old Slavonic, is one of such texts, copies of which were mostly circulated from the 13th to the 18th century in Russia. This paper compares nine copies of the Dream, including the oldest one, analyzes linguistic and structural features of them, and presents the early transmission pattern of copies. Based on a particular expression reminiscent of the one found in The Song of Songs, the author concludes that the Dream was a Slavonic creation.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor characterized by rapid progression. The mechanisms that lead to a shift from initial therapeutic sensitivity to ultimate therapeutic resistance are poorly understood. Although the SCLC genomic landscape led to the discovery of promising agents targeting genetic alterations that were already under investigation, results have been disappointing. Achievements in targeted therapeutics have not been observed for over 30 years. Therefore, the underlying disease biology and novel targets urgently require a better understanding. Epigenetic regulation is deeply involved in the cellular plasticity that could shift tumor cells to the malignant phenotype. We have focused on a histone modifier, LSD1, that is overexpressed in SCLC and is a potent therapeutic target. Interestingly, the LSD1 splice variant LSD1+8a, the expression of which has been reported to be restricted to neural tissue, was detected and was involved in the expression of neuroendocrine marker genes in SCLC cell lines. Cells with high expression of LSD1+8a were resistant to CDDP and LSD1 inhibitor. Moreover, suppression of LSD1+8a inhibited cell proliferation, indicating that LSD1+8a could play a critical role in SCLC. These findings suggest that LSD1+8a should be considered a novel therapeutic target in SCLC., Highlights • LSD1+8a, which was reported to be restricted to neural tissue, is expressed in SCLC. • LSD1+8a is involved in the expression of neuroendocrine marker genes. • SCLC cell lines with high expression of LSD1+8a were resistant to chemotherapy.

This paper attempts to uncover the textual relationships between Croatian manuscripts of the Story of Akir the Wise and other South Slavonic copies of the same text. The Story of Akir the Wise, an apocryphal text originating in the ancient Middle East earlier than 500 B.C., was translated into Church Slavonic, probably in the 12th or the 13th century. The story was disseminated mostly among the Orthodox Slavs, but was also transmitted to the Catholic Slavs in Croatia. The South Slavonic copies, although outnumbered by the Russian ones, include the oldest extant manuscript preserved at the Savina Monastery in Montenegro. The question of the Slavonic archetype of the Story is still open because of the absence of a Greek recension. In Croatia, three copies have been preserved in Glagolitic, Cyrillic, and Latin scripts. This paper treats the South Slavonic copies of the Story, composed from the 14th to the 17th century inside and outside Croatia, and points out some textual features connecting the Croatian copies with other Cyrillic copies composed in Serbia and Bulgaria. Based on text-critical analysis, it is argued that the Croatian copies have a common source, which is a descendent of another older source that appeared in the Slavia Orthodoxa; some Serbian and Bulgarian copies also derived from that source. The paper also argues that the scribes of the Story not only copied their source texts but furthermore intentionally engaged in editing their texts in accordance with the language practices and social environment within which they worked., Priča o Akiru premudrom, apokrifna priča koja potječe s antičkoga Bliskog istoka iz vremena prije 500. g. pr. Kr., prevedena je na crkvenoslavenski jezik najvjerojatnije u 12. ili 13. stoljeću. Priča je bila raširena pretežno među pravoslavnim Slavenima, većinom u Rusiji, no ipak je prispjela i do Hrvatske, u Dalmaciju. Najstariji prijepis sačuvan je na Balkanu, što sugerira da je prvi prijevod nastao na južnoslavenskom području, premda konačno rješenje pitanja o nastanku slavenskoga prijevoda još i danas ostaje otvoreno, zbog nepostojanja grčke verzije. U Hrvatskoj je priča poznata po trima prijepisima, napisanim na glagoljskom, ćiriličnom i latiničnom pismu. U radu se predstavlja tekstualni odnos južnoslavenskih prijepisa Priče, nastalih između 14. i 17. stoljeća, te se iznosi dosada neprimijećena povezanost triju hrvatskih prijepisa s dvama rukopisima očuvanima u Srbiji i Bugarskoj. Na temelju analiza jezičnih i sadržajnih značajka južnoslavenskih prijepisa Priče prikazuje se postanak različitih redakcija hrvatskih i drugih južnoslavenskih prijepisa. Ističe se kakva je pri tome bila uloga prepisivača, koji nisu samo prepisali, nego su preradili prethodni tekst u skladu s jezičnom praksom i društvenim uvjetimau kojima su djelovali.

This report describes two patients with sporadic lymphangioleiomyomatosis complicated by protein-losing enteropathy (PLE). Imaging studies indicated retroperitoneal lymphangioleiomyomas and abnormalities of the adjacent digestive tract. Endoscopic mucosal biopsy revealed colonic lymphangiectasia in one patient; whereas the site in the other patient was intestinal. Treatment with sirolimus led to the complete resolution of PLE within several months; additionally, marked shrinkage was observed in the lymphangioleiomyomas of both cases. These findings suggest that colonic or intestinal lymphatic congestion due to neighboring lymphangioleiomyomas was the mechanism for the development of PLE. At the time of writing this report, the beneficial effect of sirolimus has lasted for more than 3 years.

Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease entailing cystic destruction of the lungs and progressive respiratory failure. LAM lungs are histologically characterized by the proliferation of smooth muscle-like cells (LAM cells) and an abundance of lymphatic vessels. To elucidate the pathophysiological processes of LAM, cell-type-specific analyses are required. However, no method exists for isolating the individual types of cells in LAM lesions. Therefore, we established a fluorescence-activated cell sorting (FACS)-based method for the direct isolation of LAM cells and other various cellular components from LAM-affected lung tissue. We obtained LAM-affected lung tissue from resections or transplant recipients and prepared single-cell suspensions. FACS, immunohistochemical, and molecular analysis were used cooperatively to isolate HMB45-positive LAM cells with tuberous sclerosis complex ( TSC) 2 loss of heterozygosity (LOH). Using a combination of antibodies against an epithelial cell adhesion molecule (EpCAM) and podoplanin, we fractionated CD45-negative lung cells into three groups: lymphatic endothelial cells (LEC) (EpCAM−/podoplaninhi subset), alveolar type II cells (EpCAMhi/podoplanin− subset), and mesenchymal cells (EpCAM−/podoplanin−/low subset). During subsequent analysis of HMB45 expression, as a LAM-specific marker, we clearly identified LAM cells in the mesenchymal cell population. We then discovered that CD90+/CD34− cells in the mesenchymal cell population are not only positive for HBM45 but also had TSC2 LOH. These isolated cells were viable and subsequently amenable to cell culture. This method enables us to isolate LAM cells and other cellular components, including LAM-associated LEC, from LAM-affected lung tissues, providing new research opportunities in this field.

Background: Lymphangioleiomyomatosis (LAM) is a tumor consisting of benign-looking neoplastic cells, but its wretched clinical outcome often resembles a malignant disease. LAM cell clusters (LCCs), unique microstructures commonly found in LAM-associated chylous effusion, are aggregates of LAM cells rimmed by lymphatic endothelium. LCCs seem to be crucial participants in the dissemination and progression of LAM. Methods and Results: LCCs isolated from LAM-associated chylous effusion were embedded in a three-dimensional (3-D) culture gels, and then placed in a humidified CO(2) incubator. During serial observations of their morphological changes, we found tube formations with lymphatic endothelial cells when LCCs settled side by side in 3-D gels. On the other hand, when LCCs were embedded separately enough to be isolated at their initial sites of settlement in the gels, each of LCCs gradually broke down, leaving a “cyst-like” hole after 7 days at the site where LCCs initially resided. Finally, we demonstrated that “cyst-like” hole formation in 3-D gels was inhibited with treatment with doxycycline or recombinant human VEGF receptor-3. Conclusions: We consider that our observation of this sequence in vitro illustrates how LCCs behave in vivo while enacting their important role in the progression of LAM. Our results indicate that the 3-D gel culture system for LCCs is a useful tool for exploring the effects of new therapeutic drugs under conditions when LCCs are constantly available.

Lymphangioleiomyomatosis (LAM) (MIM #606690) is a rare lung disorder leading to respiratory failure associated with progressive cystic destruction due to the proliferation and infiltration of abnormal smooth muscle-like cells (LAM cells). LAM can occur alone (sporadic LAM, S-LAM) or combined with tuberous sclerosis complex (TSC-LAM). TSC is caused by a germline heterozygous mutation in either TSC1 or TSC2, and TSC-LAM is thought to occur as a result of a somatic mutation (second hit) in addition to a germline mutation in TSC1 or TSC2 (first hit). S-LAM is also thought to occur under the two-hit model involving a somatic mutation and/or loss of heterozygosity in TSC2. To identify TSC1 or TSC2 changes in S-LAM patients, the two genes were analyzed by deep next-generation sequencing (NGS) using genomic DNA from blood leukocytes (n = 9), LAM tissue from lung (n = 7), LAM cultured cells (n = 4), or LAM cell clusters (n = 1). We identified nine somatic mutations in six of nine S-LAM patients (67 %) with mutant allele frequencies of 1.7-46.2 %. Three of these six patients (50 %) showed two different TSC2 mutations with allele frequencies of 1.7-28.7 %. Furthermore, at least five mutations with low prevalence (

Birt-Hogg-Dubé syndrome (BHDS) is an autosomal dominant cancer syndrome caused by a germline mutation of the folliculin (FLCN) gene. Previous studies have suggested that truncated mutant folliculin proteins generated by disease causing FLCN mutations may retain partial functionality and contribute to disease phenotype. A 38-year-old Russian man presented with a left renal tumor. He underwent a left radical nephrectomy and histological examination confirmed the diagnosis of chromophobe renal cell carcinoma. He had papulae on his face suggestive of fibrofolliculomas, and pulmonary cysts on his computed tomography of the chest. He had a family history of skin manifestations. Genetic analysis identified a genomic deletion including the putative promoter region of FLCN exon 1 in the germline, and the second hit on the remaining wild-type FLCN in the renal carcinoma cells, which is expected to cause the complete lack of folliculin protein. Immunohistochemistry with the use of anti-folliculin antibody showed no antibody-binding on chromophobe renal carcinoma cells. These findings suggest that the decreased FLCN expression itself without producing mutated folliculin proteins can be at risk for developing clinical manifestations of BHDS: fibrofolliculomas, lung cysts, and tumorigenesis in the kidneys. This sheds light on the pathogenesis of BHDS and the role of FLCN as a tumor suppressor gene.

Twenty-four surgically resected, gallbladder pyloric gland adenomas (GB-PGAs) were examined and their features were compared with the reported features of stomach, duodenum, and pancreatic PGAs to better understand GB-PGAs. Clinical information on background gallbladder lesions and histologic data, including tumor grade, existence of squamoid morules, intratumoral cholesterosis, and intracytoplasmic mucins were collected. Immunohistochemical staining for MUC2, MUC5AC, MUC6, CDX2, pepsinogen I, p53, and MIB-1/nuclear β-catenin were evaluated. Targeted mutational analyses of KRAS exon2, GNAS exon 7, and CTNNB1 exon 3 were conducted. We found that 29.2% of the GB-PGAs were histologically high-grade dysplasias/carcinomas; 70.8% were low grade; and 20.8% and 33.3% contained squamoid morules and intratumoral cholesterosis, respectively. In addition, 45.8% and 54.2% of GB-PGAs were mucin-rich and mucin-poor types, respectively. Immunohistochemically, MUC6 was diffusely positive in all GB-PGAs; MUC2, MUC5AC, and CDX2 were only focally positive, and no pepsinogen-I positive cells were observed. Nuclear β-catenin accumulation was observed in all cases; however, the ratio varied among cases. Mucin-poor types were significantly associated with high histologic grade dysplasias/carcinomas and high nuclear β-catenin labeling indices. Mutational analyses identified CTNNB1 mutations in 100% of GB-PGAs (21/21), KRAS in 4.2% (1/23), and GNAS in 0% (0/22). The present study clarified the unique histologic features, phenotypic differentiation, and molecular statuses frequently associated with GB-PGAs. Altogether, our data suggest that tumorigenesis of GB-PGA is distinct from that of stomach, duodenum, and pancreatic PGAs.

Here, we present a case of non-small cell lung carcinoma (NSCLC) with widespread and strong nuclear immunopositivity for both thyroid transcription factor-1 (TTF-1) and ΔNp63/p40 (p40). Double immunofluorescence for TTF-1 and p40 showed coexpression of both markers in the tumor cells. Furthermore, PTEN (pHis123Asp) and TP53 (pVal272Leu) mutations were identified as possible mitogenic driver mutations by next-generation sequencing. To the best of our knowledge, this is the first case of NSCLC harboring concurrent PTEN and TP53 mutations with widespread and strong coexpression of TTF-1 and p40, which has been confirmed in the resected specimen, and only the second documented case of NSCLC with TTF-1 and p40 diffuse coexpression in the carcinoma cells from the same individual. Our case illustrates the possibility that poorly differentiated NSCLCs with widespread and strong nuclear positivity for TTF-1 and p40 may be an underrecognized and new entity.

This paper deals with a special verb form prefixed with uz- in Croatian. Uz-prefixed present forms from imperfective verbs, known as a functional equivalent to the second future, are rarely used in contemporary Croatian, despite the inclusion of this phenomenon in standard grammars. Based on dialectal as well as diachronic evidence, this paper defines the use of the uz-prefixed present as a remnant of old Štokavian features. Further, connecting this form to the old Czech vz- future, the author argues that it originated from a particular usage of the prefix *vъz- in old Slavic dialects, which originally marked the perfective aspect but became by reanalysis a future marker. The Czech prefix po- used to form the future tense of motion verbs is considered as indicative; similar to the Czech po-, the uz-prefixed present is characterized as a synthetic future imperfective form, but one that is only available in the subordinate clause whose matrix has future reference., Rad je posvećen posebnom glagolskom obliku s prefiksom uz- u hrvatskom jeziku. Riječ je o prezentskom obliku glagola s prefiksalnim morfemom uz- koji se smatra funkcionalnim ekvivalentom futura drugog, no rijetko se upotrebljava u suvremenom jeziku. Na osnovi dijalektnih i povijesnih podataka, utvrđuje se da je ovaj oblik ostatak stare štokavske jezične osobine. Razmatrajući analogni prezentski oblik s prefiksalnim elementom vz- u staročeškom jeziku, nastanak toga oblika autorica povezuje s funkcijom prefiksa uz- koji se prvobitno upotrebljavao u tvorbi svršenih glagola, ali je bio reanaliziran kao oznaka budućega vremena. Raspravlja se i o morfološkom statusu toga oblika kao o sintetičkom obliku futura glagola nesvršenoga vida, analogna obliku budućega vremena nesvršenih glagola kretanja koji se u suvremenome češkom jeziku tvore s prefiksom po-.

Aims To characterize the pathological features of pulmonary cysts, and to elucidate the possible mechanism of cyst formation in the lungs of patients with Birt–Hogg–Dube syndrome (BHDS), a tumour suppressor gene syndrome, using histological and morphometric analyses. Methods and results We evaluated 229 lung cysts from 50 patients with BHDS and 117 from 34 patients with primary spontaneous pneumothorax (PSP) for their number, size, location and absence or presence of inflammation. The BHDS cysts abutted on interlobular septa (88.2%) and had intracystic septa (13.6%) or protruding venules (39.5%) without cell proliferation or inflammation. The frequencies of these histological characteristics differed significantly from those seen in the lungs of patients with PSP (P

Basaloid squamous cell carcinoma (BSCC) of the esophagus is a rare variant of typical squamous cell carcinoma (SCC) associated with poor survival. A characteristic feature is nuclear accumulation of β-catenin, without a mutation of the gene. We studied the methylation status of Wnt antagonist genes, such as secreted frizzled-related protein (sFRP) gene family members, Wnt inhibitory factor-1 (WIF-1), Dickkopf-1 (Dkk-1), and human Dapper protein-1 (HDPR-1), and alterations of the APC, Axin1, and Axin2 genes in 30 cases of esophageal BSCC. β-catenin and sFRP (sFRP-1, sFRP-2, sFRP-4, sFRP-5) protein expression was examined by immunohistochemistry. APC, Axin1, and Axin2 gene mutations were detected in 3, 2, and 2 cases, respectively, and 6 cases (20 %) harbored at least 1 alteration in these genes. Methylation of the sFRP-2 promoter region was observed in all cases, and methylation was frequent in sFRP-1 and sFRP-5, but infrequent in Dkk-1, WIF-1, sFRP-4, and HDPR-1. sFRP-2 expression was almost completely absent in 25 cases (83 %), consistent with the methylation status. Nuclear accumulation of β-catenin was observed in all cases. sFRP-5 expression was associated with a low nuclear β-catenin labeling index. These results show that sFRP-2 is a target gene of hypermethylation in esophageal BSCC and suggest that sFRP-2 might contribute to BSCC tumorigenesis through the Wnt/β-catenin signaling pathway.

We report a 17-year-old male with a histopathologic diagnosis of lymphangioleiomyomatosis after surgery for a pneumothorax. In general, lymphangioleiomyomatosis has been considered a female-specific disease. However, there are a few lymphangioleiomyomatosis cases reported in males, and our patient is the youngest case reported. Spontaneous pneumothorax occurs most commonly in males in their late teens and early twenties. Histopathologic diagnosis cannot always be performed in young males with pneumothorax. However, simple diagnosis should be avoided, and lymphangioleiomyomatosis should be considered as an underlying disease. This remarkable case provides new and valuable clinical insights into young male pneumothorax.

Babingtonite, Ca2Fe2+Fe3+[Si5O14(OH)] (Z = 2, space group $$ P\overline{1} $$ ) from Yakuki mine (Japan), Gronsjoberget (Sweden), Kandivali Quarry (India), Baveno Quarry (Italy), Brastad Mine (Norway), and Kouragahana (Japan), and manganbabingtonite, Ca2(Mn2+, Fe2+)Fe3+[Si5O14(OH)], from Iron Cap mine (USA) were studied using electron-microprobe analysis (EMPA), 57Fe Mossbauer analysis and single-crystal X-ray diffraction methods to determine the cation distribution at M1 and M2 and to analyze its effect on the crystal structure of babingtonite. Although all studied babingtonite crystals are relatively homogeneous, chemical zonation due to mainly Fe ↔ Mn substitution is observed in manganbabingtonite. Mossbauer spectra consist of two doublets with isomer shift (I.S.) = 1.16–1.22 mm/s and quadrupole splitting (Q.S.) = 2.33–2.50 mm/s and with I.S. = 0.38–0.42 mm/s and Q.S. = 0.82–0.90 mm/s, assigned to Fe2+ and Fe3+ at the M1 and M2 octahedral sites, respectively. The determined ratio of Fe2+/total Fe in manganbabingtonite (0.26) was smaller than that in the others (0.35–0.44) because of high Mn2+ content instead of Fe2+. The unit-cell parameters of babingtonite are a = 7.466–7.478, b = 11.624–11.642, c = 6.681–6.690 A, α = 91.53–91.59, β = 93.86–93.94, γ = 104.20–104.34o, and V = 560.2–562.3 A3, and those of manganbabingtonite are a = 7.4967(3), b = 11.6632(4), c = 6.7014(2) A, α = 91.602(2), β = 93.989(2), γ = 104.574(3)o, and V =565.09(5) A3. Structural refinements converged to R 1 values of 1.64–3.16 %. The distance was lengthened due to the substitution of large octahedral cations such as Mn2+ for Fe2+. The increase of the M1-O8, M1-O8’ and M1-O13 lengths with mean ionic radii is slightly more pronounced than of the other M1-Oi lengths. The lengthened M1-O13 distance leads the positive correlation between Si5-O15-Si1 angle and M1-O13 distance. The increase of Si2-O3-Si1 and Si5-O12-Si4 angles due to the increase of mean ionic radius of M2 is also observed.

Recently, several studies have reported that dysfunctions in protein phosphatase 2A (PP2A) caused by alterations in protein phosphatase 2 regulatory subunit A, alpha (PPP2R1A) are responsible for tumorigenesis and tumor progression in several types of cancers. The impact of PPP2R1A mutations remains unknown in gastrointestinal stromal tumors (GISTs), although mutations in KIT and PDGFRA, which result in constitutive activation of the receptor tyrosine kinase pathway, are important in GIST tumorigenesis. In this study, we performed mutation analysis of PPP2R1A to examine the frequency of PPP2R1A mutations and their clinicopathological correlation in 94 GIST cases. In addition, we performed an in vitro analysis to investigate the effects of PPP2R1A mutations on cell proliferation and kinase phosphorylation in GIST cells. Seventeen GIST cases (18%) harbored mutations in PPP2R1A. All but one of these 17 cases harbored a KIT, PDGFRA, HRAS, NRAS, or KRAS mutation as the oncogenic driver mutation, and the remaining case was immunohistochemically negative for succinate dehydrogenase B (SDHB). Multivariate analysis showed that larger tumor size, higher mitotic rate, and PPP2R1A mutation are independent prognostic factors for overall survival; however, PPP2R1A mutation was not an independent prognostic factor for disease-free survival. The transduction of GIST cells with mutant PPP2R1A induced an accelerated growth rate via increased phosphorylation of Akt1/2, ERK1/2, and WNK1, a kinase associated with angiogenesis. In addition, the transduction of GIST cells with mutant PPP2R1A caused increased c-kit phosphorylation, suggesting that c-kit is also a target of PP2A, reinforcing the tumorigenic capabilities of c-kit. Furthermore, the transducing GIST cells with wild-type PP2A dephosphorylated mutant c-kit. This study provides a new insight into the biology of GISTs and their phosphatase activity, and activated PP2A could be a therapeutic target in GISTs.

Multifocal micronodular pneumocyte hyperplasia is a rare pulmonary manifestation of tuberous sclerosis complex (TSC) that is a tumor suppressor gene disorder characterized by many hamartomas. A purported mechanism of hamartomatous proliferation in TSC is constitutive activation of the mammalian target of rapamycin (mTOR) signaling pathway dysregulated by a functional loss of TSC genes. Although multifocal micronodular pneumocyte hyperplasia develops locally as self-limited, benign lesions, it is morphologically similar to the preinvasive lesion of pneumocytes that characterize atypical adenomatous hyperplasia or bronchioloalveolar carcinoma. Frequently both conditions include a loss of heterozygosity on TSC. The goal of this study was to determine whether multifocal micronodular pneumocyte hyperplasia is neoplastic. Loss of heterozygosity on TSC genes and immunohistochemistry for mTOR-related proteins (phospho-mTOR, phospho-p70S6K, phospho-S6, and phospho-Akt) were analyzed in 42 lesions: 16 multifocal micronodular pneumocyte hyperplasia (7 patients with TSC, 1 TSC not confirmed), 14 atypical adenomatous hyperplasia, and 12 bronchioloalveolar carcinoma (9 and 12 patients, respectively). The results showed that at least one of two multifocal micronodular pneumocyte hyperplasia lesions from each patient had loss of heterozygosity on TSC1 or TSC2 (15 or 50%) and were frequently immunopositive for phospho-mTOR (88%), phospho-p70S6K (100%), and phospho-S6 (100%) but not phospho-Akt (14%), an upstream regulatory protein of mTOR. Loss of heterozygosity of TSC was found in the preinvasive lesions of pneumocytes, equal to or less than multifocal micronodular pneumocyte hyperplasia. In contrast, phospho-Akt was expressed in the preinvasive lesions of pneumocytes more frequently than multifocal micronodular pneumocyte hyperplasia, but the other mTOR-related proteins were less frequently expressed in the former than in the latter. These outcomes suggest that functional loss of TSCs and consequent hyperphosphorylation of mTOR-related proteins in multifocal micronodular pneumocyte hyperplasia may cause its benign neoplastic proliferation of pneumocytes.

This Correspondence addresses the use of LAM and LAM-derived cells in Issaka et al. (Am J Pathol 2009,175: 1410–1420)

Lymphangioleiomyomatosis (LAM) is a rare neoplastic disease in which abnormal smooth muscle-like cells (LAM cells) proliferate in the lungs and along the axial lymphatic systems, including the lymph nodes and thoracic ducts. LAM cells are transformed due to loss-of-function type mutations of either the TSC1 or TSC2 tumor suppressor genes. The pathological features include the proliferation of benign-looking LAM cells and the existence of abundant lymphatic vessels that are associated with clinical conditions such as chyle leakage. LAM cells produce potent lymphangiogenic growth factors (VEGF-C and VEGF-D) and the lymphatic vessel density within LAM lesions correlates with the histologic severity of LAM. The serum VEGF-D level increases in LAM, especially in patients with lymphatic involvement. LAM cell clusters (LCCs), which are postulated pathologically to be generated by lymphangiogenesis-mediated fragmentation and subsequent shedding into the lymphatic circulation, are observed in both chylous effusion and LAM-associated lymphatics within LAM tissue specimens. The identification of LCCs in chylous effusion together with the characteristic clinical manifestations can therefore be an alternative for a lung biopsy if LAM patients are complicated with chylous effusion.LAM appears to be a disease involving a dysfunction of the lymphatic system and a fascinating model of tumor dissemination that is exclusively lymphangitic. LAM-associated lymphangiogenesis that mediates the shedding of LCCs seems to play a central role in the dissemination of LAM cells and progression in LAM and it may also be a potential therapeutic target as well as the dysregulated mTOR signaling pathway.

A 30-year-old Japanese woman with lymphangioleiomyomatosis (LAM) developed a left chylothorax and chylous ascites. A pleuro-peritoneal communication was confirmed by a scintigram with (99)mTc-labeled macroaggregated-albumin injected into the peritoneal cavity. Video-assisted thoracic surgery revealed a protruding papillary lesion on the left diaphragm. Chyle was oozing into the pleural cavity through this lesion. Histopathological analyses demonstrated that the protrusion was a diaphragmatic LAM lesion and that LAM-associated lymphangiogenesis enabled communication between the pleural and peritoneal cavities through lymphatic vessels. This case demonstrated a new mechanism for chylous pleural effusion in LAM and illustrates the significance of LAM-associated lymphangiogenesis.

Objectives Transforming growth factor beta (TGF-beta) is a dominant mediator of pancreatic fibrosis. The objective of this study was to identify cellular sources of TGF-beta mRNA and compare the results with previous immunohistochemical/in situ hybridization studies. Methods In situ hybridization of TGF-beta was conducted for 9 human tissues of chronic obstructive pancreatitis (COP) and 2 control specimens. By classifying these 9 COP tissues into 3 fibrosis phases by the amount of fibrotic space, histopathologic changes were examined for each fibrosis phase. Whether or not TGF-beta-positive cells were closely distributed to fibrosis was also investigated in control and COP cases. Results Three cases were categorized in early, intermediate, and advanced stages of fibrosis. Transforming growth factor beta mRNA was identified for a part of small duct epithelia, that is, intercalated ductule cells, centroacinar cells, and/or metaplastic ductal structures adjacent to acinar cells. The number of TGF-beta-positive cells was greater in COP cases than in controls. In controls and in the early stage of fibrosis, no fibrosis was seen near TGF-beta-positive cells. Conclusions Small duct epithelia are the main cellular sources of TGF-beta in COP, and many of them may be working for COP fibrosis either directly or indirectly.

A 37-year-old woman presented with a cough and discomfort in the chest. Computed tomography revealed the right pleural effusion and a number of cysts in the lungs. Thoracentesis revealed LAM cell clusters (LCC) in chylous pleural effusion, confirmed by immunocytochemical examinations showing that the cells at the center of cluster were LAM cells positive for alpha-smooth muscle actin and HMB45 and the outer layer was lymphatic endothelium cells. When LCC were cultured in vitro, the loss of heterozygosity of TSC2 markers was detected. This case illustrates that LAM can be diagnosed by the identification of LCC without an invasive biopsy if complicated with chylous effusion.

Recently, the impact of telomere dysregulation on malignant progression has been reported in many cancers. A few studies have examined TERT promoter mutations in gastrointestinal stromal tumors (GISTs). Irregular telomerase activation can be maintained by TERT hot spot alterations and alternative lengthening of telomeres (ALT) characterized by inactivation of either the alpha-thalassemia/mental retardation syndrome X-linked (ATRX) or death domain–associated protein (DAXX). To elucidate the clinicopathological impact of telomere dysregulation in GISTs, we examined 92 cases of GISTs for TERT promoter hot spot mutations along with immunohistochemical analysis of ATRX and DAXX expression, and compared these findings with the clinicopathological features. Univariate clinicopathological analysis revealed that tumor site, smaller tumor size, presence of necrosis, higher mitotic rate (>5/50 high-power fields) and risk classification were prognostic factors for either disease-free survival or overall survival. Two of 92 informative cases (2.2 %) were found to have heterozygous TERT promoter mutations (C228T), and these mutations occurred in a low-risk and a high-risk tumor, respectively. On immunohistochemical analysis for ATRX and DAXX, 16 (17.4 %) and 3 (3.3 %) of 92 cases showed loss of expression of ATRX and DAXX, respectively. Loss of expression of ATRX and DAXX were mutually exclusive except for one case. TERT promoter mutations were also mutually exclusive of the ALT phenotype. Telomere dysregulation was not associated with patient survival; however, telomere dysregulation was frequently observed in tumors of extra-gastric origin, which have an adverse outcome compared to those of gastric origin. Electronic supplementary material The online version of this article (doi:10.1186/s40064-015-1606-2) contains supplementary material, which is available to authorized users.

Basaloid squamous cell carcinoma (BSCC) is a rare and poorly differentiated variant of typical squamous cell carcinoma, and is characterised in part by activation of the Wnt signalling pathway. We previously demonstrated that constitutive activation of the Wnt signalling pathway by epigenetic silencing of secreted frizzled-related protein 4 (SFRP4) is observed in this tumour. Increasing evidence shows that the Wnt signalling pathway cross-talks with other developmental pathways, including the Hedgehog (HH) pathway. The HH pathway is stimulated by inactivating mutations of PTCH1, which have a well-described oncogenic role in basal cell carcinoma (BCC) of the skin. We employed polymerase chain reaction followed by direct sequencing to detect inactivating mutations of PTCH1 using archival tissue samples of 30 oesophageal BSCCs. The frequency of PTCH1 mutation was compared to that of Wnt component genes that we reported previously. We found PTCH1 mutations in 53.3% (16/30) of cases, revealing T1195S as a hotspot mutation. This frequency is quite high for cancers other than BCC of the skin, and PTCH1 mutations were almost mutually exclusive with mutations in APC, Axin1 and Axin2. Considering the fact that activation of Wnt signalling via down-regulation of APC and SFRP5 due to promoter methylation is observed in BCC of the skin, Wnt signalling activation in oesophageal BSCC might be a secondary effect of the PTCH1-inactivating mutations. These findings suggest that the HH and Wnt pathways coordinately contribute to tumourigenesis in oesophageal BSCC. Furthermore, this study provides a potential therapeutic application for HH pathway inhibitors in oesophageal BSCC with highly malignant potential.

Hermansky-Pudlak syndrome (HPS) is a hereditary disorder involving the sorting processes of intracellular organelles such as lysosomes of reticuloendothelial cells. Pale ear (ep) mouse is known to have the HPS1 gene mutation, which is seen in patients with HPS and pulmonary fibrosis. As pulmonary fibrosis is not spontaneously observed in ep mice, we hypothesized that external stimuli are necessary for the genetic predisposition of its development. We used silica as the external stimulus to induce the alveolar macrophage-mediated inflammatory response and evaluated the pathological changes of the lung and biochemical analysis of collagenolytic lysosomal enzymes cathepsins L and B in ep mice. Treatment with silica induced the following: persistent accumulation of activated macrophages; delayed clearance of silica from alveolar spaces; and increased collagen fibers in alveolar tissues, which were shown with trichrome staining in ep mice. The comparison of bronchoalveolar lavage cells between the naïve ep and control mice revealed: decreased enzymatic activities but increased antigenic levels of cathepsins L and B, resulting in significantly lower ratios of activity to antigen; increased ceroid deposits and cathepsin L antigens in lysosomes; and no abnormal forms of cathepsins were detected. After silica instillation, activities of cathepsin L in the ep mice increased but ratios of activity to antigen were still significantly low. These phenomena induced by silica suggest that external stimuli bring forth fibrogenesis in the animal models or humans that have HPS1 gene mutation.

Aims: We investigated a rare case of small vessel vasculitis (SVV) limited to pleuropulmonary manifestations, possibly induced by endotoxin, to determine the activation of immuno-mediated cells and endothelia in the pleuropulmonary circulation. Methods and results: A 44-year-old man with a high fever was X-rayed, revealing bilateral pleural effusion and atelectasis in the chest. His laboratory data were within normal limits except for a high white blood cell count and a high C-reactive protein level. Autoantibodies including anti-neutrophil cytoplasmic antibody were negative. Endotoxin was detected in his sera, but repeated cultures of sputa, urine, blood and the pleural effusion were negative for bacteria. Video-assisted thoracic surgery was performed and lung and parietal pleura specimens were obtained. Histology showed arterioles or small arteries infiltrated by monocytes or neutrophils with fibrinoid necrosis and acute or chronic venulitis. A diagnosis of SVV in the lung and pleura was made. Immunohistochemistry revealed that interleukin (IL)-1β was expressed in monocytes and vascular cell adhesion molecule (VCAM)-1 on endothelial cells in the vasculitic lesions in the lung. Conclusions: Endotoxin possibly induced the inflammation in this apparently unique case of pleuropulmonary small vessel vasculitis. Immunohistochemistry revealed the expression of IL-1β and VCAM-1 which may have caused activation of monocytes and endothelial cells within the vasculitic lesions.

An asymptomatic patient with exogenous lipoid pneumonia (ELP) due to silent aspiration of liquid paraffin ingested as a lubricant was diagnosed by bronchoalveolar lavage (BAL). BAL fluid separated into oily upper phase and lower aqueous phase spontaneously. Microscopic analysis of BAL cells revealed the presence of lipid-laden alveolar macrophages. Classic histochemical staining and electron microscope examination indicated that neutral lipid was dominant but phospholipid was also present in the lipid-laden macrophages. Together with the history of ingestion of liquid paraffin, we identified that the ingested liquid paraffin was the origin of the neutral lipid in the lipid-laden macrophages observed in the BAL fluid.

Solitary fibrous tumors (SFTs), initially observed in the pleura, were later found to develop in almost any extrapleural site. Dedifferentiation within SFTs was characterized only recently. We report a case of dedifferentiated SFT arising within the pelvis of a 70-year-old Japanese woman. Macroscopically, the resected tumor measured 17 × 17 × 13 cm. Histologically, the tumor displayed distinct heterologous osteosarcomatous and chondrosarcomatous components on a background of conventional SFT. Immunohistochemistry uncovered a loss of CD34 expression in the dedifferentiated area, whereas the nuclear expression of signal transducer and activator of transcription-6 (STAT6) and NGFI-A-binding protein 2 (NAB2) was maintained in both components. The p53 mutation 158 CGC > CAC (A158H) was found only in the dedifferentiated component. Furthermore, a fusion gene of NAB2(exon6)-STAT6(exon18) was detected in both the conventional and dedifferentiated components. The patient died of the disease 4 months after surgery. This case identifies a possible role of p53 dysfunction in the dedifferentiation process of SFT as reported in other sarcomas.

Background Human papillomavirus-associated oropharyngeal carcinoma (HPV-OPC) is clinicopathologically distinct entity from the HPV-unassociated one (nHPV-OPC). This study aimed to determine the relationship between histological subtypes of OPC and HPV status for Japanese cases and to identify histological structures of HPV-OPC. Methods 66 OPC cases were categorized into conventional squamous cell carcinoma (SCC) and the variants. Conventional SCC was subcategorized into keratinizing (KSCC), non-keratinizing (NKSCC), and hybrid SCC (HSCC). HPV status of all cases was determined using p16-immunohistochemistry and HPV-DNA ISH. Results Two histological subtypes, NKSCC and HSCC, tended to be HPV-OPC and KSCC tended to be nHPV-OPC with statistical significance. Two histological structures, abrupt keratinization, defined in the text, and comedo-necrosis among non-maturing tumor island, were observed for 58.1% and 38.7% of HPV-OPC, and tended to exist for HPV-OPC with statistical significance. Conclusions This study showed the association of NKSCC/HSCC with HPV-OPC in Japanese cases, and two histological structures, abrupt keratinization and comedo-necrosis among non-maturing island, were considered characteristic histological features of HPV-OPC. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1816432541113073.