The O-Linked N-Acetylglucosamine Containing Epitope H (O-GlcNAcH) is Upregulated in the Trophoblastic and Downregulated in the Fibroblastic Cells in Missed Miscarriage Human Chorionic Villi With Simple Hydropic Degeneration.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.

Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin ß4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling. Chronic treatment with sotorasib induced WNT expression and activated the WNT/ß-catenin signaling pathway. Thus, silencing both ITGB4 and ß-catenin significantly improved sotorasib sensitivity in tolerant, acquired, and inherently resistant cells. In addition, the proteasome inhibitor carfilzomib (CFZ) exhibited synergism with sotorasib by down-regulating ITGB4 and ß-catenin expression. Furthermore, adagrasib phenocopies the combination effect of sotorasib and CFZ by suppressing KRAS activity and inhibiting cell cycle progression in inherently resistant cells. Overall, our findings unveil previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and propose a promising treatment strategy to overcome resistance.

Erratum: A Non-genetic Mechanism Involving the Integrin ß4/Paxillin Axis Contributes to Chemoresistance in Lung Cancer.

[This corrects the article DOI: 10.1016/j.isci.2020.101496.].

The expression of the O-linked N-acetylglucosamine containing epitope H in the gemistocytic, pilocytic and subependymal giant cell astrocytomas.

The epitope H contains an O-linked N-acetylglucosamine residue in a specific conformation and/or environment recognized by the monoclonal antibody H. It has previously been shown that the epitope H is present in several types of normal and pathological cells and in several polypeptides. In normal human brains the epitope H is present mostly to a minority of fibrous astrocytes, whereas it is greatly up-regulated in reactive astrocytes and is increased in well differentiated fibrillary astrocytomas compared to anaplastic astrocytomas and glioblastomas. In this study the expression of the epitope H was investigated in thirty cases of gemistocytic (WHO grade II), pilocytic (WHO grade I), and subependymal giant cell (WHO grade I) astrocytomas using the mAbH with the indirect immunoperoxidase method. The ten cases of gemistocytic astrocytomas revealed an overall high expression pattern. The ten cases of pilocytic astrocytomas revealed a biphasic pattern of epitope H expression. The dense tumor areas composed of elongated pilocytic cells revealed high expression of the epitope H. The loose cystic tumor areas composed of stellate cells revealed low expression of the epitope H. The ten cases of subependynal giant cell astrocytomas occurring in tuberous sclerosis revealed an overall high expression pattern. This study shows that there is high expression of the epitope H in gemistocytic, pilocytic and subependymal giant cell astrocytomas. Collectively considering, the present and our previous data, it appears that there is a spectrum of the expression levels of the epitope H ranging from the high expression in the reactive astrocytes and low grade astrocytomas to the low/null expression in the normal astrocytes and glioblastomas.

Alternative lengthening of telomeres, ATRX loss and H3-K27M mutations in histologically defined pilocytic astrocytoma with anaplasia.

Anaplasia may be identified in a subset of tumors with a presumed pilocytic astrocytoma (PA) component or piloid features, which may be associated with aggressive behavior, but the biologic basis of this change remains unclear. Fifty-seven resections from 36 patients (23 M, 13 F, mean age 32 years, range 3-75) were included. A clinical diagnosis of NF1 was present in 8 (22%). Alternative lengthening of telomeres (ALT) was assessed by telomere-specific FISH and/or CISH. A combination of immunohistochemistry, DNA sequencing and FISH were used to study BRAF, ATRX, CDKN2A/p16, mutant IDH1 p.R132H and H3-K27M proteins. ALT was present in 25 (69%) cases and ATRX loss in 20 (57%), mostly in the expected association of ALT+/ATRX- (20/24, 83%) or ALT-/ATRX+ (11/11, 100%). BRAF duplication was present in 8 (of 26) (31%). H3-K27M was present in 5 of 32 (16%) cases, all with concurrent ATRX loss and ALT. ALT was also present in 9 (of 11) cases in the benign PA precursor, 7 of which also had ATRX loss in both the precursor and the anaplastic tumor. In a single pediatric case, ALT and ATRX loss developed in the anaplastic component only, and in another adult case, ALT was present in the PA-A component only, but ATRX was not tested. Features associated with worse prognosis included subtotal resection, adult vs. pediatric, presence of a PA precursor preceding a diagnosis of anaplasia, necrosis, presence of ALT and ATRX expression loss. ALT and ATRX loss, as well as alterations involving the MAPK pathway, are frequent in PA with anaplasia at the time of development of anaplasia or in their precursors. Additionally, a small subset of PA with anaplasia have H3-K27M mutations. These findings further support the concept that PA with anaplasia is a neoplasm with heterogeneous genetic features and alterations typical of both PA and diffuse gliomas.

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

BACKGROUND: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting. RESULTS: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells. CONCLUSION: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

Expression of the O-linked N-acetylglucosamine containing epitope H in normal myometrium and uterine smooth muscle cell tumors.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H (mAbH). We have previously shown that epitope H is present in more than one polypeptide and in various types of normal and pathological cells. In the present study, we focused on uterine smooth muscle cell tumors and their adjacent normal myometrium to gain further insight into the expression patterns of epitope H in human tissues. The indirect immunoperoxidase method was applied using the mAbH and the monoclonal anti-cytokeratin 8 antibody (AbCK8) in 50 cases of typical uterine leiomyomas and in five cases of uterine leiomyosarcomas, with four cases belonging to Group II A and one to Group III according to Bell et al. [6]. Western immunoblotting was applied using mAbH and AbCK8 in five cases of uterine leiomyomas and their adjacent myometrium. The main results were as follows: (1) epitope H showed intense immunohistochemical expression in 46% (23/50) and moderate expression in 54% (27/50) of uterine leiomyomas, (2) epitope H showed intense immunohistochemical expression in 40% (2/5) and moderate expression in 60% (3/5) of uterine leiomyosarcomas, (3) epitope H showed no difference in the immunohistochemical expression between leiomyomas and their adjacent myometrium and between leiomyosarcomas and their adjacent myometrium, (4) immunohistochemical expression of cytokeratin 8 was not detected in the normal and neoplastic smooth muscle cells, (5) Western immunoblotting showed that in the smooth muscle cells of the myometrium and leiomyomas, epitope H is localized in four polypeptides with molecular weights of 100, 61, 59, and 54 kDa, and (6) Western immunoblotting did not detect cytokeratin 8 in the normal and neoplastic smooth muscle cells. The present results indicate fluctuations of the epitope expression levels in uterine smooth muscle cell tumors and their adjacent myometrium. These fluctuations may be of interest for gaining insight into the pathogenesis of uterine smooth muscle cell tumors, since O-GlcNAc glycosylation is involved in cell cycle and apoptosis pathways and may modify proteins involved in oncogenesis (tumor suppressor proteins and oncoproteins) and proteins with important biological functions such as cytoskeletal proteins, transcription factors, and heat-shock proteins. Furthermore, the present results indicate that cytokeratin 8, without being present in the cells of the myometrium, leiomyomas and leiomyosarcomas, shares its epitope H, which contains its unique sugar O-N-acetylglucosamine residue, with four other unrelated polypeptides produced by the normal and neoplastic smooth muscle cells. This should be considered when using anti-cytokeratin 8 antibodies in immunohistochemistry against smooth muscle cell tumors to avoid false positive immunohistochemical results.

Glioblastoma in natalizumab-treated multiple sclerosis patients.

We present two natalizumab-treated multiple sclerosis patients who developed glioblastoma multiforme (GBM) with variable outcomes. One patient had an isocitrate dehydrogenase (IDH)-wildtype GBM with aggressive behavior, who declined treatment and died 13 weeks after symptoms onset. The other patient underwent resection of an IDH-mutant secondary GBM that arose from a previously diagnosed grade II astrocytoma. He is still alive 5 years after the diagnosis of GBM. JC virus was not detected in either case. Whether natalizumab played a role in the development of GBM in those patients deserves further investigation.

Nuclear localization of cytokeratin 8 and the O-linked N-acetylglucosamine-containing epitope H in epithelial cells of infiltrating ductal breast carcinomas: a combination of immunogold and EDTA regressive staining methods.

In a previous study, the authors have shown cytokeratin 8 (CK8) and epitope H ultrastructural localization in breast cancer cell nuclei. Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H. In this study, double immunogold labeling of CK8 and epitope H combined with the EDTA regressive staining method was applied in biopsy material from infiltrating ductal breast carcinomas and fibroadenomas, to localize both antigens in correlation to RNPs distribution in the nuclear subcompartments of cancer cells. CK8 and epitope H were localized mostly over condensed chromatin, whereas staining was weaker over interchromatin granule clusters and perichromatin fibers. These results revealed, the distribution of CK8 in the nucleus as MAR-binding protein, contributing in the organization of the nuclear DNA in the neoplastic cell, as well as the distribution of O-GlcNAc glycosylated polypeptides bearing the epitope H. The latter finding indicates that these polypeptides might play a significant role in the neoplastic behavior of breast cancer cells because they colocalize in the same nuclear subcompartments with proteins modified by O-GlcNAc, such as hnRNPs G and A1, RNA polymerase II, its transcription factors, and the oncogene product of c-myc. These proteins are known to participate in coordinated transcription/RNA processing events, contributing in the neoplastic behavior of breast cancer cells.

Pigmented ganglioglioma in a patient with chronic epilepsy and cortical dysplasia.

We report a rare case of a 22-year-old woman with biopsy-proven pigmented ganglioglioma. The patient initially underwent a right temporal lobectomy for intractable seizures at the age of 9 and remained seizure free for several years but subsequently developed complex partial seizures. Due to enhancement of a left mesial occipital lesion on preoperative MRI of the brain, the patient underwent a left subdural electrode placement and simultaneous biopsy of the left mesial occipital lesion. Biopsy results revealed a rare pigmented ganglioglioma, World Health Organization Grade I. The seizure focus was identified in the left mesial occipital lobe and the patient underwent tumor resection. An extensive literature search revealed that our patient is the fourth case of pigmented ganglioglioma described in the literature and was positive for BRAF V600E mutation by molecular studies.

Mitochondria-rich normal, metaplastic, and neoplastic cells show overexpression of the epitope H recognized by the monoclonal antibody H.

The epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by monoclonal antibody H (mAbH). We have previously shown that the epitope H is present in more than one polypeptide and in various types of normal and pathological cells. In the present study, we focused on mitochondria-rich normal, metaplastic, and neoplastic cells, prompted by our recent immuno-electron microscopy findings that mAbH clearly stains the mitochondria of breast epithelial cells in infiltrating ductal breast carcinomas and fibroadenomas. The indirect immunoperoxidase method was applied using the mAbH to investigate the distribution of the epitope H in mitochondria-rich normal cells and in metaplastic and neoplastic oncocytic cells. Immunohistochemical staining for the mAbH was observed in oxyphil cells of parathyroid glands, in striated duct cells of parotid glands, in urinary tubules of kidneys, in parietal cells of gastric body mucosa, in oxyphil cells of Hashimoto's thyroiditis, in epithelial cells of Warthin's tumors of the parotid gland, in neoplastic cells of oxyphil adenomas and carcinomas (Hürthle's tumors) of the thyroid gland, and in neoplastic cells of oncocytomas of the kidneys. The present study shows that the epitope H is strongly expressed in mitochondria-rich normal cells, as well as in metaplastic and neoplastic oncocytic cells, which are known to have cytoplasms packed with mitochondria. Since mAbH recognizes an O-GlcNAc residue, our findings indicate that O-GlcNAc-glycosylated polypeptides are present at mitochondria where the components of the respiratory chain and energy transduction are localized. These findings may be of interest for gaining insight into the histophysiology of mitochondria-rich normal cells and into the pathogenesis of oncocytic lesions, since O-GlcNAc glycosylation may modify proteins involved in oncogenesis such as tumor suppressor proteins and oncoproteins, as well as proteins with important biological functions such as cytoskeletal proteins, transcription factors, heat-shock proteins, and chromatin proteins.

Hyperdense suprasellar mass: An unusual radiological presentation of intracranial dermoid cyst.

We report an exceedingly rare patient with a hyperdense suprasellar dermoid cyst and a pertinent review of the literature. Intracranial dermoid tumors are rare congenital lesions of the brain that account for less than 1% of all intracranial tumors. Even though they are rare, typical CT scan and MRI features and location allow radiological diagnosis in the majority of patients. Radiologically, dermoid cysts typically present as low density masses on CT scan and are generally hyperintense on T1-weighted MRI sequences with variable signal on T2-weighted sequences. The recognition of atypical features can avoid diagnostic pitfalls and is clinically relevant for overall surgical management.

A 40-year-old male with an intraventricular tumor. Combined tanycytic ependymoma and subependymoma.

Combined tumors showing histologic features of both ependymoma and subependymoma have been described. In this report we present a case of combined tanycytic ependymoma with foci of subependymoma (WHO grade II), occurring in a 40 year-old male, which arose in the wall of the lateral ventricle. The tanycytic ependymoma component showed elongated fibrillary cells with a fascicular pattern of growth, while the subependymoma component showed clustered cell bodies surrounded by a fibrillary stroma with a microcystic appearance.We consider the present case to be an unusual example of tanycytic ependymoma; which to the best of our knowledge has not been associated with a subependymoma.

Immunophenotype of myxopapillary ependymomas.

Myxopapillary ependymoma (MPE) is a slow-growing tumor occurring almost exclusively in the region of conus medullaris, cauda equina, and filum terminale. On microscopic examination, some of these tumors show solid sheets of cells with an epithelioid morphology mimicking a metastatic carcinoma. Several immunohistochemical studies addressed this issue with discordant results. We report the immunohistochemical findings of 9 additional cases of MPE. From 2004 to 2011, a total of 9 cases of MPE were recorded in our surgical pathology files. The histologic material and clinical data were reviewed for each case. There were 6 female and 3 male patients. The ages ranged from 15 to 58 years (mean, 31 y). Eight cases were intradural, lumbosacral (L1-S1), and 1 case was located in the sacrum. All tumors expressed CD99 and GFAP (100%). Eight tumors were positive for CD56 (89%). All tumors (100%) expressed focally CKAE1/AE3. One tumor (11%) was focally positive for CK8/18 and CK7. D2-40 was focally positive in 1 case (11%). PLAP and AFP were both negative in all cases. Synaptophysin was focally positive in 1 case. NSE was positive in all cases. All tumors were negative for CK5/6, CK20, E-cadherin, and TTF-1. Our study shows that the vast majority of MPE are positive for CD99, CD56, and GFAP. In selective cases, especially when the material obtained for pathologic evaluation is scanty and the tumor displays epithelioid appearance, the diagnosis may be challenging owing to cytokeratin positivity suggesting metastatic carcinoma. However, the clinical and radiologic features in addition to the positivity for GFAP should prompt pathologists to consider MPE in the differential diagnosis of such cases. Interestingly, we found that MPE are positive for NSE, which suggests a neuroglial differentiation.

Use of the reamer-irrigator-aspirator may reduce tumor dissemination during intramedullary fixation of malignancies.

Intramedullary nail fixation is the treatment of choice for impending and pathologic fractures secondary to metastatic cancer; however, this procedure has been shown to cause systemic embolization of intramedullary contents. This article reports the use of the reamer-irrigator-aspirator (RIA) (Synthes, Paoli, Pennsylvania) instead of a standard femoral reamer to decrease tumor intravasation during femoral intramedullary nail fixation for impending or pathologic fractures.Twenty-one consecutive patients indicated for fixation of malignant femoral lesions were treated with intramedullary nail placement. The RIA was used for canal preparation, and solid reamings were collected and submitted for analysis by a single pathologist. The volume of each specimen was recorded, and representative samples were examined histologically to determine their percent tumor content. These data were then used to estimate the volume of tumor retrieved by the RIA in each case. The mean volume of reamings collected by the RIA was 75.0 cc per case (range, 23.4-196.0 cc), and the mean tumor content was 24.8% (range, 1.0%-60.0%). The mean estimated volume of tumor retrieved in each case was 16.7 cc (range, 0.35-36.0 cc). In 2 cases, the tip of the RIA dissociated from the device intraoperatively but was retrieved without adverse consequence to the patient. Use of the RIA in cases of femoral intramedullary nail fixation for pathologic lesions or fractures effectively retrieves variable amounts of intramedullary contents, including tumor. By preventing the systemic dissemination of malignant cells, this technique may reduce the risk of distant metastases.

Hypoxia upregulates the expression of the O-linked N-acetylglucosamine containing epitope H in human ependymal cells.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAc) residue in a specific conformation and/or environment recognized by mouse IgM monoclonal antibody H (mabH). Epitope H is present in several types of cells and in several polypeptides outside the CNS. Previous results have shown that in the adult human brains, epitope H is confined mostly to a minority of fibrous astrocytes, and it is greatly upregulated in the reactive astrocytes. Post-translational modification with O-GlcNAc occurs on many proteins involved in several cell processes, such as cell cycle progression, apoptosis, proteasome degradation pathways, and modulation of cellular function in response to nutrition and stress. Hypoxia is one of the major causes of cellular stress. Therefore, in this study, we used the mAbH and the indirect immunoperoxidase method to investigate the expression of epitope H in ependymal cells in brains of persons who died with signs of hypoxic encephalopathy. The results of the present study showed that practically all ependymal cells showed cytoplasmic staining for epitope H in supranuclear cytoplasm in the brain of two premature neonates and in ten infants who died with signs of hypoxic encephalopathy. However, the overwhelming majority of ependymal cells of the nine human embryos taken from legal abortions, ranging from 26 days until 13 weeks of gestational age, and of the ten infants' brains without any sign of hypoxic encephalopathy remained negative. Only occasionally did the ependymal cells show weak cytoplasmic staining in some foci. In addition, the reactive astrocytes in the hypoxic brains showed strong cytoplasmic staining, confirming previous results.