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Abstract Overexpression of the EPS15 Homology Domain containing 1 (EHD1) protein has been linked to tumorigenesis but whether its core function as a regulator of intracellular traffic of cell surface receptors plays a role in oncogenesis remains unknown. We establish that EHD1 is overexpressed in Ewing sarcoma (EWS), with high EHD1 mRNA expression specifying shorter patient survival. ShRNA-knockdown and CRISPR-knockout with mouse Ehd1 rescue established a requirement of EHD1 for tumorigenesis and metastasis. RTK antibody arrays identified IGF-1R as a target of EHD1 regulation in EWS. Mechanistically, we demonstrate a requirement of EHD1 for endocytic recycling and Golgi to plasma membrane traffic of IGF-1R to maintain its surface expression and downstream signaling. Conversely, EHD1 overexpression-dependent exaggerated oncogenic traits require IGF-1R expression and kinase activity. Our findings define the RTK traffic regulation as a proximal mechanism of EHD1 overexpression-dependent oncogenesis that impinges on IGF-1R in EWS, supporting the potential of IGF-1R and EHD1 co-targeting.

Abstract Background Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. Results To investigate if the mir-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. A CpG island in the promoter of the ANK1 host gene of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2′-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p and ANK1, indicating common epigenetic regulation in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. Conclusions miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma, and this knowledge contributes to the understanding of osteosarcoma biology.

Lung metastasis is the major cause of death in osteosarcoma patients. However, molecular mechanisms underlying this metastasis remain poorly understood. To identify key molecules related with pulmonary metastasis of pediatric osteosarcomas, we analyzed high‐throughput miRNA expression in a cohort of 11 primary tumors and 15 lung metastases. Results were further validated with an independent cohort of 10 primary tumors and 6 metastases. In parallel, we performed immunohistochemical analysis of activated signaling pathways in 36 primary osteosarcomas. Only phospho‐AKT associated with lower overall survival in primary tumors, supporting its role in osteosarcoma progression. CTNNB1 expression also associated with lower overall survival but was not strong enough to be considered an independent variable. Interestingly, miR‐200c was overexpressed in lung metastases, implicating an inhibitory feed‐back loop to PI3K‐AKT. Moreover, transfection of miR200c‐mimic in U2‐OS cells reduced phospho‐AKT levels but increased cellular migration and proliferation. Notably, miR‐200c expression strongly correlated with miR‐141 and with the osteogenic inhibitor miR‐375, all implicated in epithelial to mesenchymal transition. These findings contrast epithelial tumors where reduced miR‐200c expression promotes metastasis. Indeed, we noted that osteosarcoma cells in the lung also expressed the epithelial marker CDH1, revealing a change in their mesenchymal phenotype. We propose that miR‐200c upregulation occurs late in osteosarcoma progression to provide cells with an epithelial phenotype that facilitates their integration in the metastatic lung niche. Thus, our findings identify phospho‐AKT in the primary tumor and miR‐200c later during tumor progression as prognostic molecules and potential therapeutic targets to prevent progression and metastasis of pediatric osteosarcomas.

Objetivo: Caracecterizar morfológica e inmunohitoquimicamente el carcinoma apocrino mamamario. Materiales y métodos: Este estudio toma 24 casos diagnosticados como carcinomas apocrinos o con rasgos apocrinos, clasificándolos como puros y mixtos, reconociendo la presencia de componentes in situ asociados, para evaluar la expresión inmunohistoquímica del receptor de andrógenos (AR), receptor de estrógenos (ER), receptor de progesterona (PR), gross cistyc disease proteína (GCDFP-15), BCL2,KI67 y ERB-2 tanto en las áreas infiltrantes como intraepiteliales, estudiando adicionalmente la amplificación génica del ERB-2 en el cromosoma 17 por métodos de FISH. Resultados: Los tumores clasificados como puros correspondieron a 11 casos que expresan la siguiente positividad inmunohistoquímica en las áreas infiltrativas: AR (100%), ER (18%), PR (18%), GCDFP (63%), BCL2 (54%), KI67 (28%) y ERB-2(28%) con una positividad de GCDFP-15(100%) en la áreas in situ. Los tumores mixtos correspondientes a 13 casos mostraron la siguiente positividad inmunohistoquímica: AR (58%), ER (46%), PR (46%), GCDFP-15 (50%), BCL2 (33%), KI67 (58%) y ERB-2(16%), con una positividad de GCDFP-15 (100%) en las áreas in situ. Las áreas in situ expresan GCDFP-15 en todos los casos con una reducción de la expresión en las zonas de infiltración del mismo al 63 y 50% en los tumores puros y mixtos, respectivamente. Conclusiones: Los carcinomas mamarios apocrinos puros deben ser distinguidos de los mixtos mediante un examen morfológico detallado y por su perfil inmunohistoquímico (AR+, ER-, PR-/GCDFP+, BCL2+/-, KI67-, ERB2/-).

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 104 features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.

OBJECTIVE:Growing evidences indicate that the histone methyltransferase EZH2 (enhancer of zeste homolog 2) may be an appropriate therapeutic target in some tumors. Indeed, a high expression of EZH2 is correlated with poor prognosis and metastasis in many cancers. In addition, 3-Deazaneplanocin A (DZNep), an S-adenosyl-L homocysteine hydrolase inhibitor which induces EZH2 protein depletion, leads to cell death in several cancers and tumors. The aim of this study was to determine whether an epigenetic therapy targeting EZH2 with DZNep may be also efficient to treat chondrosarcomas. METHODS:EZH2 expression was determined by immunohistochemistry and western-blot. Chondrosarcoma cell line CH2879 was cultured in the presence of DZNep, and its growth and survival were evaluated by counting adherent cells periodically. Apoptosis was assayed by cell cycle analysis, Apo2.7 expression using flow cytometry, and by PARP cleavage using western-blot. Cell migration was assessed by wound healing assay. RESULTS:Chondrosarcomas (at least with high grade) highly express EZH2, at contrary to enchondromas or chondrocytes. In vitro, DZNep inhibits EZH2 protein expression, and subsequently reduces the trimethylation of lysine 27 on histone H3 (H3K27me3). Interestingly, DZNep induces cell death of chondrosarcoma cell lines by apoptosis, while it slightly reduces growth of normal chondrocytes. In addition, DZNep reduces cell migration. CONCLUSION:These results indicate that an epigenetic therapy that pharmacologically targets EZH2 via DZNep may constitute a novel approach to treat chondrosarcomas.

Abstract  Ewing’s sarcoma (ES)/peripheral neuroectodermal tumor (PNET) are malignant neoplasms affecting children and young adults. We performed a study to typify the histological diversity and evaluate antibodies that may offer diagnostic/prognostic support. In total, 415 cases of genetically confirmed paraffin-embedded ES/PNET were analyzed on whole sections and in tissue microarrays. This study confirms the structural heterogeneity of ES/PNET, distinguishing three major subtypes: conventional ES (280 cases); PNET (53 cases); and atypical ES/PNET (80), including large cells, vascular-like patterns, spindle pattern, and adamantinoma-like configuration. All cases presented positivity for at least three of the four tested antibodies (CD99, FLI1, HNK1, and CAV1). CAV1 appeared as a diagnostic immunomarker of ES/PNET being positive in CD99-negative cases. Hence, the immunohistochemical analysis confirmed the diagnostic value of all four antibodies, which together cover more than 99% of the tumors, independently of the histological variety. The univariate analysis for survival revealed atypical ES as the only histological parameter apparently associated with less favorable clinical outcome, particularly in the subgroup of patients treated with surgery. In conclusion, the diagnosis of atypical ES is a challenge for the pathologist and needs support from molecular techniques to perform an optimal differential diagnosis with other small round cell tumors. [ABSTRACT FROM AUTHOR]

Ubiquitin and telomerase immunohistochemical expression patterns in cervical cancer were compared with normal cervical tissue samples. Eighty-one cervical cancer cases and 22 normal exo-endocervical tissue were examined with polyclonal antibody for ubiquitin and 44G12 clone for telomerase using tissue microarrays. The results were interpreted using a semiquantitative scale The average age of patients was 50.67 years. The most frequent histological types were moderately differentiated epidermoid carcinoma (43.5%), according to the degree of differentiation, and endocervical adenocarcinoma (42.1%). Immunohistochemical findings were as follows: 98.7% of cervical cancers showed immunoexpression for ubiquitin and 52.6% for telomerase. Statistically significant differences were found in tumor immunoreactivity when compared with control tissue (p < 0.0007) for both biomarkers. There was no significant difference in biomarker expression at different histological types of tumors, although telomerase was less expressed in endocervical adenocarcinoma. Our findings confirm that abnormal immunoexpression pattern of ubiquitin and telomerase is common in HPV-positive cervical cancer, indicating the existence of an intense degradation of proteins, subsequent cellular immortalization and maintenance of the malignant phenotype. [ABSTRACT FROM AUTHOR]

Abstract  We aimed to detect truncated CCN3 protein variants in formalin-fixed paraffin-embedded samples of eight Wilms’ tumors using anti-K19M and novel domain-specific antibodies, anti-NH2, anti-NH3, anti-NH4, and anti-NH5 raised against C-terminal (CT) domain and modules 1, 2, 3, and 4 of the CCN3 protein, respectively. In Wilms’ tumors, all the domain antibodies except anti-NH4 exhibited both nuclear and cytoplasmic staining in blastema as well as primitive tubules. NH4 was detected only in the cytoplasm of tumor cells. Normal fetal kidneys revealed mainly cytoplasmic immunoreactivity for all antibodies in tubules and glomeruli, except for K19 and NH5, which showed some nuclear staining. Our data suggest expression of a truncated nuclear CCN3 variant lacking the thrombospondin type-1-like domain and cytoplasmic full-length CCN3 protein in Wilms’ tumor cells. In addition, normal fetal kidneys express mainly full-length protein mostly localized to cytoplasm. Truncated CCN3 protein in Wilms’ tumor cells may provide evidence for its tumorigenic role in these tumors. Uniform NH5 staining compared to variable expression of K19M indicates that using NH5 is a better approach for detecting the CT domain of CCN3 protein in archival specimens. Thus, the domain-specific antibodies represent valuable tools for detecting CCN3 protein variants in normal and neoplastic kidneys. [ABSTRACT FROM AUTHOR]

Abstract  In addition to clinical and biological factors, further valuable prognostic information in neuroblastoma (Schwannian stroma-poor) (NB) patients is provided by the histopathologic analysis and the application of the International Neuroblastoma Pathology Classification (INPC) system. The objective of this study was to assess the prognostic impact of the INPC classification in a series of NB (Schwannian stroma-poor) and its relation with other prognostic factors. One hundred eighty-two cases of NB were collected from the files of the Spanish Neuroblastoma Registry. Slides were reviewed, and NB cases were grouped into favorable and unfavorable categories according to INPC criteria, taking into account morphological features (mitosis–karyorrhexis index, histological subtype) and patient’s age at diagnosis. Other pathological [presence of calcifications, tissular components, and number of mitotic cells per 10 high-power field (HPF)], immunohistochemical (P-glycoprotein and Ki-67 protein expression) and genetic (MYCN amplification and chromosome 1p deletion) features were also studied. Statistical analyses of overall survival with Kaplan–Meier curves and a multivariate study using Cox regression were performed (40.3% of NBs were considered favorable and 59.7% unfavorable). Unfavorable NB showed a mean survival time of 57 months compared with 89 months in favorable cases. Advanced stage, more than ten mitoses per 10 HPF, Ki-67 expression in more than 30% of tumor cells, MYCN oncogene amplification and chromosome 1p deletion were observed more frequently in unfavorable NB. The Cox regression analysis demonstrated that clinical stage (International Neuroblastoma Staging System stage 4) and histological subtype (undifferentiated NB) were the most important factors that influence the overall survival (p<0.001). INPC classification results are major prognostic indicators in NB and should be considered in the therapeutic stratification of NB patients. [ABSTRACT FROM AUTHOR]

Abstract  This paper discusses the diversity of synovial sarcomas (SSs) [biphasic (BSS), monophasic fibrous (MFSS), and poorly differentiated (PDSS)] and tissue microarray (TMA) evaluation of the immunophenotypic and histological progression of SSs in nude mice using three TMAs comprising 11 primary SSs (8 MFSSs, 2 BSSs, and 1 PDSS) and their xenografts. BSS and MFSS progressively transformed to a similar undifferentiated phenotype with loss of glandular component in the xenografts. Epidermal growth factor receptor and SALL2 were expressed in primary tumors and xenografts. Enhanced bcl-2 and bax expression were noted in xenografts. Ki-67 overexpression in xenografts correlated with high mitotic index. Epithelial membrane antigen (EMA) and cytokeratin AE1/AE3 were detected in all original and xenografted SSs. Hierarchical clustering differentiated original MFSS and BSS, but their xenografts clustered together due to similar immunoexpression profile. Our study demonstrates definite phenotypic variability of BSS and MFSS in the xenografts. Differences in immunoexpression for various markers existed between primary tumor and xenografts but not between subtypes. Hierarchical clustering grouped TMA immunostaining data and confirmed immunophenotypic variability; however, it failed to reveal any immunophenotypic differences between SYT-SSX1 and SYT-SSX2 type tumors. Nonetheless, reverse-transcriptase–polymerase chain reaction detected SYT-SSX transcripts in all primary SSs and their xenografts, thereby demonstrating their genetic stability. [ABSTRACT FROM AUTHOR]

Anaplastic carcinoma of the thyroid gland (ACT) is a highly malignant tumor that is almost invariably associated with a fatal outcome. It demonstrates a variety of peculiar histological features, with squamoid, giant cell and spindle cell growth patterns. The spindle cell variant of ACT is usually indistinguishable from a true sarcoma and it can simulate fibrosarcoma, malignant fibrous histiocytoma (MFH), hemangiopericytoma and angiosarcoma or rhabdomyosarcoma. Although a rhabdomyosarcomatous appearance has sometimes been mentioned in the literature, true skeletal muscle differentiation has never been consistently proved. We report two cases of ACT with rhabdomyosarcomatous differentiation, as demonstrated by means of immunohistochemistry and electron microscopy. Both cases disclosed a very similar histological appearance, with a main population of small, pleomorphic, round-to-oval cells arranged in a storiform pattern, admixed with scattered pleomorphic giant cells, an image similar to that of the usual type of MFH. Stains for epithelial markers showed only few, scattered, weakly positive cells. Thyroglobulin and calcitonin were negative in tumor cells in both cases. On the contrary, positivity to vimentin was strong and generalized. Immunomarkers of muscular differentiation showed a consistent positivity. At the ultrastructural level, the cells disclosed the same spindle and pleomorphic morphology, with large, bizarre nuclei and cytoplasm with abundant mitochondria, rough endoplasmic reticulum, secretory granules and lipid droplets. There were also cells with wide cytoplasm filled with filamentous material, either of actin or myosin, as well as Z-band material. In conclusion, the cases reported here show a clear-cut rhabdomyosarcomatous differentiation of ACT, confirmed both immunohistochemically and ultrastructurally, a feature not previously reported in the literature. These findings may contribute to the broadening of the differentiation spectrum of this unusual neoplasm. [ABSTRACT FROM AUTHOR]

Previous studies have shown that during the period subsequent to the Chernobyl accident, increases in morbidity, aggressivity and proliferative activity of renal-cell carcinomas (RCCs) in Ukrainian patients were recognized. The present paper describes the molecular alterations of those tumor suppressor genes located on chromosome 9p21 ( INK4a/ARF locus and p15 INK4B) in 26 primary renal-cell epithelial tumors from patients with different degrees of radiation exposure after the Chernobyl accident in Ukraine. Radiometric measurement of Cesium 137 ( 137Cs) was conducted with 1-day urine from all patients before surgery. Our results demonstrate that RCCs from patients living in the radio-contaminated areas showed aberrant hypermethylation of p14 ARF and p16 INK4A genes, associated with increased p38MAPK, p14 ARF, mdm2, cyclinD1 and Ki67 protein expression levels. Present findings show the possibility that chronic long-term low-dose radiation activates the INK4a/ARF locus, targeted by activation of the p38MAPK cascade. These actions could lead to disruptions and loss of cell cycle checkpoints and, thereby, to cellular transformation. [ABSTRACT FROM AUTHOR]

We report, for the first time, mutations in the Alu repeat regions in the genome of kidney tumors induced by diethylstilbestrol in Syrian hamsters. Among the 66 loci amplified by 11 random primers, 28 loci exhibited insertions, deletions or losses or gains in intensity in the genome of kidney tumor tissues compared with normal kidney tissues from age-matched hamsters. Higher numbers of mutated Alu loci were observed in the tumors of old hamsters compared with young hamsters. In N-ethyl-N-nitrosourea- and diethylstilbestrol-treated hamsters deletion of a 0.59 kb locus amplified with primer OPC03 was observed in most of the female hamsters, but not in male hamsters. An insertion mutation of a 0.498 kb locus amplified with primer OPC03 was observed in 12 of 36 diethylstilbestrol-induced kidney tumors. The cloning and sequencing of the 0.498 kb locus amplified with primer OPC03 revealed that it had significant sequence similarity to the mouse RIKEN cDNA clone. These findings indicate that age, sex and co-exposure to N-ethyl-N-nitrosourea influence mutations in the Alu repeat sequences in the genome of diethylstilbestrol-induced kidney tumors in Syrian hamsters. Structural alterations in Alu repeats in critical target genes may be involved in diethylstilbestrol-induced carcinogenesis. [ABSTRACT FROM AUTHOR]

В останні роки в науковій пресі з'явилисm численні дані про молекулярну біологію раку молочної залози. У зв'язку з цим, гістологічна класифікація ВООЗ (2004 ) виявилась застарілою. Сьогодні більшість відомостей про прогноз, лікування та клінічний результат захворювання грунтується на результатах молекулярних і біологічних досліджень останніх років. Розглянуто сучасні підходи до питань гістопатологічної діагностики раку молочної залози на основі нових технологій. Обговорено рекомендації з імуногістохімічного тестування рецепторів естрогенів і прогестерону , а також рецепторів до епідермального фактора росту 2 (HERB2) у зразках пухлинної тканини. Запропоновано алгоритм оцінки отриманих даних, включаючи позитивні, спірні й негативні результати, що стосуються експресії білків HERB2 і ампліфікації гена. На основі характеру експресії генів, вивченого за технологією мікрочіпів з подальшою ієрархічної кластеризацією РНК нативної тканини, виділено нові генетичні профілі пухлини. Показано можливість застосування методів імуногістохімії в тканинах, залитих у парафін. Рекомендовано імуногістохімічну панель для діагностики наступних типів раку молочної залози: люмінальної А (Luminal А) , люмінальної В (Luminal В), базальноподібний і HER2/neu.Заключна частина роботи присвячена варіантам раку молочної залози з генетичною сприйнятливістю до BRCA1 і 2. Такі пухлини вкрай агресивні, характеризуються високим ступенем злоякісності і мають несприятливий прогноз. Проаналізовано нові підходи до терапії захворювання.

Ubiquitin and telomerase immunohistochemical expression patterns in cervical cancer were compared with normal cervical tissue samples. Eighty-one cervical cancer cases and 22 normal exo-endocervical tissue were examined with polyclonal antibody for ubiquitin and 44G12 clone for telomerase using tissue microarrays. The results were interpreted using a semiquantitative scale The average age of patients was 50.67 years. The most frequent histological types were moderately differentiated epidermoid carcinoma (43.5%), according to the degree of differentiation, and endocervical adenocarcinoma (42.1%). Immunohistochemical findings were as follows: 98.7% of cervical cancers showed immunoexpression for ubiquitin and 52.6% for telomerase. Statistically significant differences were found in tumor immunoreactivity when compared with control tissue (p < 0.0007) for both biomarkers. There was no significant difference in biomarker expression at different histological types of tumors, although telomerase was less expressed in endocervical adenocarcinoma. Our findings confirm that abnormal immunoexpression pattern of ubiquitin and telomerase is common in HPV-positive cervical cancer, indicating the existence of an intense degradation of proteins, subsequent cellular immortalization and maintenance of the malignant phenotype.