A transgenic mouse model demonstrating the oncogenic role of mutations in the polycomb-group gene EZH2 in lymphomagenesis.

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

TOMM20 as a Potential Prognostic Biomarker in Chordoma: Results From a High-Volume, Single-Center Study.

OBJECTIVES: As few large studies identify correlative biomarkers in chordoma, our objective was to use our large, single-center chordoma tumor bank to identify novel signaling pathways. METHODS: Clinical and pathologic data for 73 patients with chordoma were retrospectively collected. Tumor microarrays were built from 61 archived chordoma specimens; immunohistochemistry for TOMM20, TIGAR, and MCT1 were performed; and semiquantitative analysis of staining intensity and percentage of positive tumor cells was performed. Average composite scores of MCT1, TIGAR, and TOMM20 expression were compared by disease status and anatomic location. RESULTS: Higher expression of TOMM20 was seen in recurrent and metastatic chordomas compared with primary lesions. Comparing composite scores of primary lesions in patients with primary disease only vs those with recurrent disease showed that TIGAR and TOMM20 expressions are significantly higher in primary lesions, followed by a history of recurrence. A TOMM20 composite score of greater than or equal to 3 significantly decreased overall survival (hazard ratio [HR], 5.83) and recurrence-free survival (HR, 8.95). CONCLUSIONS: Identifying novel signaling pathways that promote chordoma growth and recurrence is critical for developing targeted therapy for chordoma. TOMM20 may be a biomarker associated with chordoma disease progression.

Comprehensive Evaluation and Unique Morphologic Features of Nonsteroidal Anti-Inflammatory Drug (NSAID) Enteropathy in the Terminal Ileum.

Differentiating NSAID enteropathy from Crohn disease can be challenging on terminal ileum biopsy. It is important to distinguish these two entities for management. In this study, clinical, radiographic, endoscopic, and histologic features of 30 patients diagnosed with NSAID enteropathy and 30 patients diagnosed with Crohn disease on terminal ileal biopsy were compared. None of the patients in the NSAID cohort demonstrated significant changes on imaging performed prior to biopsy. Depending on disease severity, patients with Crohn disease showed imaging findings ranging from minimal changes to significant bowel wall and/or luminal changes. Endoscopically, erythema, erosions, and/or ulcers in the terminal ileum were observed in a majority of NSAID cases. Patients with active Crohn disease were noted to have erythema, ulcers, strictures, and/or visible inflammation throughout the distal ileum and colon. Histologically, at least 80% of cases in each cohort showed ulceration/erosion and cryptitis/crypt abscesses. The Crohn disease cohort had significantly higher degree of lymphoplasmacytic inflammation in the lamina propria compared to the NSAID cohort. In contrast, a significantly higher percentage of the NSAID cohort had gland/crypt atrophy. A decrease in number of Paneth cells was found in the NSAID cohort. Decreased lamina propria lymphoplasmacytic inflammation was the most specific and the presence of gland atrophy the most sensitive feature favoring NSAID enteropathy. In summary, the combination of gland/crypt/Paneth cell atrophy with decreased lamina propria lymphoplasmacytic inflammation can aid in differentiating NSAID enteropathy from Crohn disease in terminal ileal biopsies; however, correlation with clinical, radiographic, and endoscopic findings is still required.

Association of bullous pemphigoid and comorbid health conditions: a case-control study.

BACKGROUND: Bullous pemphigoid is an autoimmune skin disease characterized by the formation of blisters between the epidermis and dermis. Comorbidities of pemphigoid have not been well-described. Identification of comorbidities associated with pemphigoid is important to decrease morbidity and mortality. OBJECTIVE: To identify the comorbid health conditions of bullous pemphigoid. METHODS: This was a case-control study of 91 cases of pemphigoid verified by clinical and laboratory diagnosis and 546 age- and sex-matched controls with complete follow-up at a large metropolitan quaternary care medical center. RESULTS: The average age of bullous pemphigoid patients was 76 years and 53% of patients were female. Forty-eight (53%) of the BP patients had a history of inpatient hospitalization, of which 22 (24.2%) were hospitalized for either previously undiagnosed BP or an exacerbation of BP. Bullous pemphigoid was significantly associated with hypertension [adjusted odds ratio (95% confidence interval)]: [2.03 (1.24-3.32)], diabetes mellitus [2.59 (1.60-4.19)], chronic kidney disease [2.29 (1.19-4.40)], end-stage renal disease [3.82 (1.48-9.85)], basal cell carcinoma of the skin [6.00 (1.94-18.6)], and obstructive sleep apnea [5.23 (2.45-11.19)]. 78% of BP patients used at least one systemic immunosuppressant. There was no significant association between treatments for pemphigoid and any of the comorbidities. CONCLUSIONS: Bullous pemphigoid patients need screening for comorbid health conditions even though treatment options do not seem to be associated with these comorbidities.

Rasagiline improves learning and memory in young healthy rats.

The effect of rasagiline on learning and memory in Lister-Hooded rats was investigated in this study. Two cognitive tests were used: a 24-h temporal deficit novel object recognition test and a modified water maze task. Rasagiline (0.3 and 1 mg/kg) was administered subcutaneously 15 min before the cognitive tests. In a novel object recognition test, rasagiline treatment enhanced object recognition memory. A small effect was observed with 0.3 mg/kg rasagiline; at 1 mg/kg, rasagiline-treated animals spent twice as much time exploring the novel object. On the water maze test, the use of an on-demand platform allowed adjustment of the difficulty of this spatial learning task. This enabled the detection of a small positive effect of rasagiline (1 mg/kg) on spatial learning, which was not observed in earlier reports. For the first time, our study has showed the procognitive effect of rasagiline in young healthy rats. On the basis of these findings, a monoamine oxidase-B inhibitor would seem to be a potential symptomatic treatment for cognitive impairments affecting patients with neurodegenerative disorders.

Malignant melanoma arising in a primary mediastinal germ cell tumor.

Primary mediastinal germ cell tumors with somatic malignancies are rare. We report a case of a 34-year old man with melanoma arising in a primary mediastinal mixed germ cell tumor. On initial biopsy, the patient was found to have a germ cell tumor containing yolk sac and embryonal components only. After chemotherapy, histopathological evaluation of the residual tumor in the wide local resection specimen revealed a mature teratoma with melanoma. Molecular studies demonstrated that the residual germ cell tumor harbored KIT and NRAS mutations associated with malignant melanoma.

Evaluation of objective and subjective treatment outcomes in orthodontic cases treated with extraction of a mandibular incisor.

OBJECTIVES: To analyze changes in occlusal characteristics following mandibular incisor extractions (MIE), to determine the usefulness of wax setups in treatment planning MIE cases and to compare the pre- and posttreatment dental attractiveness between MIE cases and nonextraction (NE) controls. MATERIALS AND METHODS: The Peer Assessment Rating (PAR) Index was used to score pre- and posttreatment dental casts of MIE cases (n = 14) and matched NE controls (n = 14). Occlusal characteristics were evaluated on diagnostic wax setups and posttreatment casts. Attractiveness of pre- and posttreatment cases judged on intraoral photographs of cases (n = 6) and controls (n = 6) were rated by 76 dental students and 10 laypeople using visual analogue scales (VAS). RESULTS: The difference in PAR score reduction (%) between the MIE and NE groups was not significant. Between the wax setup and posttreatment casts, there were moderate correlations in overjet, overbite, and right canine classification. There was no significant difference in pre- and posttreatment change in VAS scores (%) for attractiveness between the MIE (49.8 ± 4.3 [S.E.]) and control groups (40.8 ± 4.3 [S.E.]). However, there was a significant difference (P = .000) between the observer groups. CONCLUSIONS: There were no significant differences in the treatment outcomes of orthodontic cases treated with MIE or NE, indicating that MIE is a valid treatment option. A wax setup is moderately correlated with posttreatment results. Both laypeople and dental students rated posttreatment dental attractiveness higher than pretreatment in MIE and NE groups. Dental students tended to be more critical than laypeople in their ratings.

The desmoplakin-intermediate filament linkage regulates cell mechanics.

The translation of mechanical forces into biochemical signals plays a central role in guiding normal physiological processes during tissue development and homeostasis. Interfering with this process contributes to cardiovascular disease, cancer progression, and inherited disorders. The actin-based cytoskeleton and its associated adherens junctions are well-established contributors to mechanosensing and transduction machinery; however, the role of the desmosome-intermediate filament (DSM-IF) network is poorly understood in this context. Because a force balance among different cytoskeletal systems is important to maintain normal tissue function, knowing the relative contributions of these structurally integrated systems to cell mechanics is critical. Here we modulated the interaction between DSMs and IFs using mutant forms of desmoplakin, the protein bridging these structures. Using micropillar arrays and atomic force microscopy, we demonstrate that strengthening the DSM-IF interaction increases cell-substrate and cell-cell forces and cell stiffness both in cell pairs and sheets of cells. In contrast, disrupting the interaction leads to a decrease in these forces. These alterations in cell mechanics are abrogated when the actin cytoskeleton is dismantled. These data suggest that the tissue-specific variability in DSM-IF network composition provides an opportunity to differentially regulate tissue mechanics by balancing and tuning forces among cytoskeletal systems.

An Oncofetal Glycosaminoglycan Modification Provides Therapeutic Access to Cisplatin-resistant Bladder Cancer.

BACKGROUND: Although cisplatin-based neoadjuvant chemotherapy (NAC) improves survival of unselected patients with muscle-invasive bladder cancer (MIBC), only a minority responds to therapy and chemoresistance remains a major challenge in this disease setting. OBJECTIVE: To investigate the clinical significance of oncofetal chondroitin sulfate (ofCS) glycosaminoglycan chains in cisplatin-resistant MIBC and to evaluate these as targets for second-line therapy. DESIGN, SETTING, AND PARTICIPANTS: An ofCS-binding recombinant VAR2CSA protein derived from the malaria parasite Plasmodium falciparum (rVAR2) was used as an in situ, in vitro, and in vivo ofCS-targeting reagent in cisplatin-resistant MIBC. The ofCS expression landscape was analyzed in two independent cohorts of matched pre- and post-NAC-treated MIBC patients. INTERVENTION: An rVAR2 protein armed with cytotoxic hemiasterlin compounds (rVAR2 drug conjugate [VDC] 886) was evaluated as a novel therapeutic strategy in a xenograft model of cisplatin-resistant MIBC. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Antineoplastic effects of targeting ofCS. RESULTS AND LIMITATIONS: In situ, ofCS was significantly overexpressed in residual tumors after NAC in two independent patient cohorts (p<0.02). Global gene-expression profiling and biochemical analysis of primary tumors and cell lines revealed syndican-1 and chondroitin sulfate proteoglycan 4 as ofCS-modified proteoglycans in MIBC. In vitro, ofCS was expressed on all MIBC cell lines tested, and VDC886 eliminated these cells in the low-nanomolar IC50 concentration range. In vivo, VDC886 effectively retarded growth of chemoresistant orthotopic bladder cancer xenografts and prolonged survival (p=0.005). The use of cisplatin only for the generation of chemoresistant xenografts are limitations of our animal model design. CONCLUSIONS: Targeting ofCS provides a promising second-line treatment strategy in cisplatin-resistant MIBC. PATIENT SUMMARY: Cisplatin-resistant bladder cancer overexpresses particular sugar chains compared with chemotherapy-naïve bladder cancer. Using a recombinant protein from the malaria parasite Plasmodium falciparum, we can target these sugar chains, and our results showed a significant antitumor effect in cisplatin-resistant bladder cancer. This novel treatment paradigm provides therapeutic access to bladder cancers not responding to cisplatin.

Oncofetal Chondroitin Sulfate Glycosaminoglycans Are Key Players in Integrin Signaling and Tumor Cell Motility.

Many tumors express proteoglycans modified with oncofetal chondroitin sulfate glycosaminoglycan chains (ofCS), which are normally restricted to the placenta. However, the role of ofCS in cancer is largely unknown. The function of ofCS in cancer was analyzed using the recombinant ofCS-binding VAR2CSA protein (rVAR2) derived from the malaria parasite, Plasmodium falciparum We demonstrate that ofCS plays a key role in tumor cell motility by affecting canonical integrin signaling pathways. Binding of rVAR2 to tumor cells inhibited the interaction of cells with extracellular matrix (ECM) components, which correlated with decreased phosphorylation of Src kinase. Moreover, rVAR2 binding decreased migration, invasion, and anchorage-independent growth of tumor cells in vitro Mass spectrometry of ofCS-modified proteoglycan complexes affinity purified from tumor cell lines on rVAR2 columns revealed an overrepresentation of proteins involved in cell motility and integrin signaling, such as integrin-ß1 (ITGB1) and integrin-α4 (ITGA4). Saturating concentrations of rVAR2 inhibited downstream integrin signaling, which was mimicked by knockdown of the core chondroitin sulfate synthesis enzymes ß-1,3-glucuronyltransferase 1 (B3GAT1) and chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1). The ofCS modification was highly expressed in both human and murine metastatic lesions in situ and preincubation or early intravenous treatment of tumor cells with rVAR2 inhibited seeding and spreading of tumor cells in mice. This was associated with a significant increase in survival of the animals. These data functionally link ofCS modifications with cancer cell motility and further highlights ofCS as a novel therapeutic cancer target. IMPLICATIONS: The cancer-specific expression of ofCS aids in metastatic phenotypes and is a candidate target for therapy. Mol Cancer Res; 14(12); 1288-99. ©2016 AACR.

Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein.

Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.

Hypoxia influences protein transport and epigenetic repression of CHO cell cultures in shake flasks.

Shake flasks and bench-top bioreactors are widely used for cell culture process development, however, culture performances significantly differ between them. In order to apply the results received from small-scale cultures to production scale, it is important to understand the mechanisms underlying the differences between various culture systems. This study analyzes the expression patterns of Chinese hamster ovary (CHO) cells producing IgG-fusion protein B0 cultured in shake flasks and 5-L bench-top bioreactors by CHO-specific DNA microarrays. The data show that hypoxia was present in shake flask cultures but not in controlled, bench-top bioreactors. Hypoxic conditions appeared to be associated with epigenetic repression resulting in decreased cell culture performance and protein productivity, which is also present during large-scale bioreactor operations due to oxygen gradients. High protein productivity was associated with increased cellular machinery for protein transport and secretion in conjunction with decreased epigenetic repression in bench-top bioreactor cultivation. Metal ions could improve cell growth and protein production under hypoxia and this condition could be mimicked in small-scale bioreactors to facilitate cell culture process scale-up.

Identification and validation of a novel mature microRNA encoded by the Merkel cell polyomavirus in human Merkel cell carcinomas.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is present in approximately 80% of human Merkel cell carcinomas (MCCs). A previous in silico prediction suggested MCPyV encodes a microRNA (miRNA) that may regulate cellular and viral genes. OBJECTIVES: To determine the presence and prevalence of a putative MCPyV-encoded miRNA in human MCC tumors. STUDY DESIGN: Over 30 million small RNAs from 7 cryopreserved MCC tumors and 1 perilesional sample were sequenced. 45 additional MCC tumors were examined for expression of an MCPyV-encoded mature miRNA by reverse transcription real-time PCR. RESULTS: An MCPyV-encoded mature miRNA, "MCV-miR-M1-5p", was detected by direct sequencing in 2 of 3 MCPyV-positive MCC tumors. Although a precursor miRNA, MCV-miR-M1, had been predicted in silico and studied in vitro by Seo et al., no MCPyV-encoded miRNAs have been directly detected in human tissues. Importantly, the mature sequence of MCV-miR-M1 found in vivo was identical in all 79 reads obtained but differed from the in silico predicted mature miRNA by a 2-nucleotide shift, resulting in a distinct seed region and a different set of predicted target genes. This mature miRNA was detected by real-time PCR in 50% of MCPyV-positive MCCs (n = 38) and in 0% of MCPyV-negative MCCs (n = 13). CONCLUSIONS: MCV-miR-M1-5p is expressed at low levels in 50% of MCPyV-positive MCCs. This virus-encoded miRNA is predicted to target genes that may play a role in promoting immune evasion and regulating viral DNA replication.

Association of Merkel cell polyomavirus-specific antibodies with Merkel cell carcinoma.

BACKGROUND: Merkel cell polyomavirus (MCPyV) has been detected in approximately 75% of patients with the rare skin cancer Merkel cell carcinoma. We investigated the prevalence of antibodies against MCPyV in the general population and the association between these antibodies and Merkel cell carcinoma. METHODS: Multiplex antibody-binding assays were used to assess levels of antibodies against polyomaviruses in plasma. MCPyV VP1 antibody levels were determined in plasma from 41 patients with Merkel cell carcinoma and 76 matched control subjects. MCPyV DNA was detected in tumor tissue specimens by quantitative polymerase chain reaction. Seroprevalence of polyomavirus-specific antibodies was determined in 451 control subjects. MCPyV strain-specific antibody recognition was investigated by replacing coding sequences from MCPyV strain 350 with those from MCPyV strain w162. RESULTS: We found that 36 (88%) of 41 patients with Merkel cell carcinoma carried antibodies against VP1 from MCPyV w162 compared with 40 (53%) of the 76 control subjects (odds ratio adjusted for age and sex = 6.6, 95% confidence interval [CI] = 2.3 to 18.8). MCPyV DNA was detectable in 24 (77%) of the 31 Merkel cell carcinoma tumors available, with 22 (92%) of these 24 patients also carrying antibodies against MCPyV. Among 451 control subjects from the general population, prevalence of antibodies against human polyomaviruses was 92% (95% CI = 89% to 94%) for BK virus, 45% (95% CI = 40% to 50%) for JC virus, 98% (95% CI = 96% to 99%) for WU polyomavirus, 90% (95% CI = 87% to 93%) for KI polyomavirus, and 59% (95% CI = 55% to 64%) for MCPyV. Few case patients had reactivity against MCPyV strain 350; however, indistinguishable reactivities were found with VP1 from strain 350 carrying a double mutation (residues 288 and 316) and VP1 from strain w162. CONCLUSION: Infection with MCPyV is common in the general population. MCPyV, but not other human polyomaviruses, appears to be associated with Merkel cell carcinoma.