Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation

Scientific Reports / Applied Precision Cancer Medicine in Neuro-Oncology

Brain tumours that are refractory to treatment have a poor prognosis and constitute a major challenge in offering effective treatment strategies. By targeting molecular alterations, precision cancer medicine may be a viable option for the treatment of brain tumours. In this retrospective analysis of our PCM platform, we describe the molecular profiling of primary brain tumours from 50 patients. Tumour samples of the patients were examined by a 161-gene next-generation sequencing panel, immunohistochemistry, and fluorescence in situ hybridization (FISH). We identified 103 molecular aberrations in 36 (72%) of the 50 patients. The predominant mutations were TP53 (14.6%), IDH1 (9.7%) and PIK3CA (6.8%). No mutations were detected in 14 (28%) of the 50 patients. IHC demonstrated frequent overexpression of EGFR and mTOR, in 38 (76%) and 35 (70%) patients, respectively. Overexpression of PDGFRa and PDGFRb were less common and detected in 16 and four patients, respectively. For 35 patients a targeted therapy was recommended. In our database, the majority of patients displayed mutations, against which targeted therapy could be offered. Based on our observations, PCM may be a feasible novel treatment approach in neuro-oncology.(VLID)492126

Diagnosis and immunophenotype of 188 pediatric lymphoblastic lymphomas treated within a randomized prospective trial: experiences and preliminary recommendations from the European childhood lymphoma pathology panel

The majority of lymphoblastic (precursor cell) neoplasms presents as leukemias. Consequently, the guidelines for lineage determination and subtyping of precursor cell neoplasms were primarily established for flow cytometry methods. Large-scale studies of nonleukemic lymphoblastic lymphomas are lacking so far. We analyzed a large series of pediatric patients with lymphoblastic lymphoma treated within a prospective randomized trial (the Euro-LB 02 study). Among 193 lymphomas, in which a detailed immunohistochemical analysis was carried out, there were several unusual and diagnostically challenging morphologic and immunophenotypical variants. These included 11 lymphomas with mixed phenotypes expressing markers of at least 2 hematopoietic lineages, 7 terminal deoxynucleotide transferase-negative lymphoblastic lymphomas, and 3 undifferentiated hematopoietic neoplasms that could not be assigned to any lineage with certainty. Our data indicate that World Health Organization guidelines for lineage determination and subtyping of precursor cell leukemia need to be adapted before they can be applied to immunohistochemical diagnosis of lymphoma. Using the experience from this cohort we suggest a resource-saving diagnostic staining panel for the immunohistochemical analysis of precursor cell neoplasms in formalin-fixed paraffin-embedded tissue

HSP27 and 70 expression in thymic epithelial tumors and benign thymic alterations : diagnostic, prognostic and physiologic implications

Thymic Epithelial Tumors (TETs), the most common tumors in the anterior mediastinum in adults, show a unique association with autoimmune Myasthenia Gravis (MG) and represent a multidisciplinary diagnostic and therapeutic challenge. Neither risk factors nor established biomarkers for TETs exist. Predictive and diagnostic markers are urgently needed. Heat shock proteins (HSPs) are upregulated in several malignancies promoting tumor cell survival and metastases. We performed immunohistochemical staining of HSP27 and 70 in patients with TETs (n=101) and patients with benign thymic alterations (n=24). Further, serum HSP27 and 70 concentrations were determined in patients with TETs (n=46), patients with benign thymic alterations (n=33) and volunteers (n=49) by using ELISA. HSPs were differentially expressed in histologic types and pathological tumor stages of TETs. Weak HSP tumor expression correlated with worse freedom from recurrence. Serum HSP concentrations were elevated in TETs and MG, correlated with clinical tumor stage and histologic subtype and decreased significantly after complete tumor resection. To conclude, we found HSP expression in the vast majority of TETs, in physiologic thymus and staining intensities in patients with TETs have been associated with prognosis. However, although interesting and promising the role of HSPs in TETs as diagnostic and prognostic or even therapeutic markers need to be further evaluated.(VLID)467571

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7

BACKGROUND: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. OBJECTIVE: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD METHODS: The anti‐basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). RESULTS: As assessed by serial section staining, 2D7(+) cells were found to co‐express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7(+ )bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7(+) cells/mm(2): CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm(2)). CONCLUSIONS: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti‐leukaemic treatment

Antibody opsonization enhances MAIT cell responsiveness to bacteria via a TNF-dependent mechanism

Mucosal-associated invariant T (MAIT) cells are an abundant human T cell subset with anti-microbial properties. They can respond to bacteria presented via antigen-presenting cells (APCs) such as macrophages, which present bacterially derived ligands from the riboflavin synthesis pathway on MR1. Moreover, MAIT cells are also highly responsive to cytokines which enhance and even substitute for TCR-mediated signaling. The mechanisms leading to an efficient presentation of bacteria to MAIT cells by APCs have not been fully elucidated. Here we showed that the monocytic cell line THP-1 and B cells activated MAIT cells differentially in response to E. coli. THP-1 cells were generally more potent in inducing IFNγ and IFNγ/TNF production by MAIT cells. Furthermore, THP-1 cells but not B cells produced TNF upon bacterial stimulation, which in turn supported IFNγ production by MAIT cells. Finally, we addressed the role of antibody-dependent opsonization of bacteria in the activation of MAIT cells using in vitro models. We found that opsonization had a substantial impact on downstream MAIT cell activation by monocytes. This was associated with enhanced activation of monocytes and increased TNF release. Importantly, this TNF acted in concert with other cytokines to drive MAIT cell activation. These data indicate both a significant interaction between adaptive and innate immunity in the response to bacteria, and an important role for TNF in MAIT cell triggering

Antibody opsonization enhances MAIT cell responsiveness to bacteria via a TNF-dependent mechanism

Mucosal-associated invariant T (MAIT) cells are an abundant human T cell subset with anti-microbial properties. They can respond to bacteria presented via antigen-presenting cells (APCs) such as macrophages, which present bacterially derived ligands from the riboflavin synthesis pathway on MR1. Moreover, MAIT cells are also highly responsive to cytokines which enhance and even substitute for TCR-mediated signaling. The mechanisms leading to an efficient presentation of bacteria to MAIT cells by APCs have not been fully elucidated. Here we showed that the monocytic cell line THP-1 and B cells activated MAIT cells differentially in response to E. coli. THP-1 cells were generally more potent in inducing IFNγ and IFNγ/TNF production by MAIT cells. Furthermore, THP-1 cells but not B cells produced TNF upon bacterial stimulation, which in turn supported IFNγ production by MAIT cells. Finally, we addressed the role of antibody-dependent opsonization of bacteria in the activation of MAIT cells using in vitro models. We found that opsonization had a substantial impact on downstream MAIT cell activation by monocytes. This was associated with enhanced activation of monocytes and increased TNF release. Importantly, this TNF acted in concert with other cytokines to drive MAIT cell activation. These data indicate both a significant interaction between adaptive and innate immunity in the response to bacteria, and an important role for TNF in MAIT cell triggering