Peter Nørgaard

Transforming growth factor β and cancer

During the past two decades considerable data have accumulated on the molecular processes by which the cell phenotype is determined. The discovery of the polypeptide growth factors and the large number of scientific reports published on these are a major part of this research. A new concept of autocrine and paracrine action was formulated when observations on the topographical distribution of the growth factors in developing and adult tissues were compared to their effects on cell growth, cell differentiation, cell-cell and cell-matrix interaction. Thus, the growth factors are believed mainly to exert their effects in an autocrine (cells expressing both the growth factor and its receptor) and paracrine (different adjacent cell type expressing either the growth factor or its receptor) fashion in contrast to the endocrine mode of action of the classical hormones (I 1. The growth factors have been divided into two major classes which are complementary with respect to their effects on cell growth; growth stimulatory-mitogenic factors e.g. epidermal growth factor (EGF)-and growth inhibitory factors, e.g. transforming growth factor p (TGFP). As with other attempts to label biology, exceptions exist, but generally TGFB is considered a potent growth-suppressor of ectodermally derived cells. In addition, TGFP has been shown to affect cell differentiation and cell adhesion in a number of different cell types. A complex network of interacting regulators of cell metabolism such as growth factors, steroid hormones and cell adhesion molecules has been outlined and reports of newly discovered interactions are constantly adding to the complexity of this network. Most studies of TGFP-mediated functions have been performed in vitro, whereas

Double-hit BCL2/MYC translocations in a consecutive cohort of patients with large B-cell lymphoma - a single centre's experience.

Concurrent BCL2 and MYC translocations, so called double hit (DH), are a rare finding in large B‐cell lymphoma (LBCL). Based on data from retrospective series, DH has been correlated with aggressive clinical behaviour and poor outcome. We conducted a consecutive study of DH incidence and correlation with pathologic and clinical characteristics, including response to Rituximab‐containing chemotherapy and survival, in an unselected cohort of patients with LBCL. Translocations involving BCL2 and MYC loci were examined with fluorescent in situ hybridization (FISH) in 157 patients with diffuse large B‐cell lymphoma (DLBCL) or B‐cell lymphoma, unclassifiable, with features intermediate between diffuse large B‐cell lymphoma and Burkitt lymphoma (BCLU). The incidence of DH was 11% in the total cohort, 7% of primary LBCL and 21% of transformed LBCL. DH lymphomas were all GCB immunophenotype and were more often BCLU. No clinical characteristics were correlated with the presence of DH, which also had no impact on overall response rate (ORR), relapse rate or overall survival (OS). However, sub‐stratification of DH lymphomas by FISH indicated a possible inferior survival related to immunoglobulin MYC translocation partner gene. Screening of patients with BCLU and DLBCL of GCB type for DH BCL2/MYC translocation including MYC translocation partner gene may provide important prognostic information.

Inactivation of the transforming growth factor β type II receptor in human small cell lung cancer cell lines

SummaryTransforming growth factor β (TGF-β) exerts a growth inhibitory effect on many cell types through binding to two types of receptors, the type I and II receptors. Resistance to TGF-β due to lack of type II receptor (RII) has been described in some cancer types including small cell lung cancer (SCLC). The purpose of this study was to examine the cause of absent RII expression in SCLC cell lines. Northern blot analysis showed that RII RNA expression was very weak in 16 of 21 cell lines. To investigate if the absence of RII transcript was due to mutations, we screened the poly-A tract for mutations, but no mutations were detected. Additional screening for mutations of the RII gene revealed a GG to TT base substitution in one cell line, which did not express RII. This mutation generates a stop codon resulting in predicted synthesis of a truncated RII of 219 amino acids. The nature of the mutation, which has not previously been observed in RII, has been linked to exposure to benzo[a]-pyrene, a component of cigarette smoke. Since RII has been mapped to chromosome 3p22 and nearby loci are often hypermethylated in SCLC, it was examined whether the lack of RII expression was due to hypermethylation. Southern blot analysis of the RII promoter did not show altered methylation patterns. The restriction endonuclease pattern of the RII gene was altered in two SCLC cell lines when digested with Sma1. However, treatment with 5-aza-2′-deoxycytidine did not induce expression of RII mRNA. Our results indicate that in SCLC lack of RII mRNA is not commonly due to mutations and inactivation of RII transcription was not due to hypermethylation of the RII promoter or gene. Thus, these data show that in most cases of the SCLC cell lines, the RII gene and promoter is intact in spite of absent RII expression. However, the nature of the mutation found could suggest that it was caused by cigarette smoking.

MYC translocation partner gene determines survival of patients with large B-cell lymphoma with MYC- or double-hit MYC/BCL2 translocations

In large B‐cell lymphoma (LBCL) MYC‐ and MYC/BCL2 double‐hit (DH) translocations have been associated with inferior survival. We hypothesised that the negative prognostic impact of MYC translocation was determined by an immunoglobulin MYC translocation partner gene (IG‐MYC), as opposed to a non‐immunoglobulin partner gene (nonIG‐MYC). In a prospective, unselected cohort of 237 LBCL patients MYC and BCL2 translocations were identified by Flourescent in situ hybridisation (FISH) with split probes. MYC translocation partner gene was identified by IGH/MYC fusion probes and/or kappa/lambda split probes. Clinical data were collected from patient files. MYC translocation was identified in 28/225 patients. IG‐MYC translocation partner gene was identified in 12/24 patients. DH translocation was identified in 23/228 patients. IG‐MYC translocation partner gene was identified in 9/19 DH patients. Neither MYC‐nor DH translocation showed correlation with survival. However, MYC translocation with IG‐MYC translocation partner gene was associated with worse OS compared with both MYC translocation with nonIG‐MYC translocation partner gene (P = 0.02) as well as absence of MYC translocation (P = 0.03). In patients with DH a similar, however, stronger correlation was seen (P = 0.003 and P = 0.0004 respectively). MYC – or DH translocation with nonIG‐MYC translocation partner gene was not associated with worse overall survival (P = 0.2 and P = 0.3 respectively). Most patients received Rituximab (86%) and CHOP/CHOP‐like chemotherapy regimes (81%). We suggest that prognostic stratification of LBCL patients by MYC and/or DH translocations should include identification of MYC translocation partner gene because approximately half of the cases harbour nonIG‐MYC translocation partner genes with no or minor influence on survival.

Growth suppression by transforming growth factor beta 1 of human small-cell lung cancer cell lines is associated with expression of the type II receptor.

Nine human small-cell lung cancer cell lines were treated with transforming growth factor beta 1 (TGF-beta 1). Seven of the cell lines expressed receptors for transforming growth factor beta (TGF-beta-r) in different combinations between the three human subtypes I, II and III, and two were receptor negative. Growth suppression was induced by TGF-beta 1 exclusively in the five cell lines expressing the type II receptor. For the first time growth suppression by TGF-beta 1 of a cell line expressing the type II receptor without coexpression of the type I receptor is reported. No effect on growth was observed in two cell lines expressing only type III receptor and in TGF-beta-r negative cell lines. In two cell lines expressing all three receptor types, growth suppression was accompanied by morphological changes. To evaluate the possible involvement of the retinoblastoma protein (pRb) in mediating the growth-suppressive effect of TGF-beta 1, the expression of functional pRb, as characterised by nuclear localisation, was examined by immunocytochemistry. Nuclear association of pRb was only seen in two of the five TGF-beta 1-responsive cell lines. These results indicate that in SCLC pRb is not required for mediation of TGF-beta 1-induced growth suppression.

Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines.

In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII.

Glucocorticoids and histone deacetylase inhibitors cooperate to block the invasiveness of basal-like breast cancer cells through novel mechanisms

Aggressive cancers often express E-cadherin in cytoplasmic vesicles rather than on the plasma membrane and this may contribute to the invasive phenotype of these tumors. Therapeutic strategies are not currently available that restore the anti-invasive function of E-cadherin in cancers. MDA-MB-231 cells are a frequently used model of invasive triple-negative breast cancer, and these cells express low levels of E-cadherin that is mislocalized to cytoplasmic vesicles. MDA-MB-231 cell lines stably expressing wild-type E-cadherin or E-cadherin fused to glutathione S-transferase or green fluorescent protein were used as experimental systems to probe the mechanisms responsible for cytoplasmic E-cadherin localization in invasive cancers. Although E-cadherin expression partly reduced cell invasion in vitro, E-cadherin was largely localized to the cytoplasm and did not block the invasiveness of the corresponding orthotopic xenograft tumors. Further studies indicated that the glucocorticoid dexamethasone and the highly potent class I histone deacetylase (HDAC) inhibitor largazole cooperated to induce E-cadherin localization to the plasma membrane in triple-negative breast cancers, and to suppress cellular invasion in vitro. Dexamethasone blocked the production of the cleaved form of the CDCP1 (that is, CUB domain-containing protein 1) protein (cCDCP1) previously implicated in the pro-invasive activities of CDCP1 by upregulating the serine protease inhibitor plasminogen activator inhibitor-1. E-cadherin preferentially associated with cCDCP1 compared with the full-length form. In contrast, largazole did not influence CDCP1 cleavage, but increased the association of E-cadherin with γ-catenin. This effect on E-cadherin/γ-catenin complexes was shared with the nonisoform selective HDAC inhibitors trichostatin A (TSA) and vorinostat (suberoylanilide hydroxamic acid, SAHA), although largazole upregulated endogenous E-cadherin levels more strongly than TSA. These results demonstrate that glucocorticoids and HDAC inhibitors, both of which are currently in clinical use, cooperate to suppress the invasiveness of breast cancer cells through novel, complementary mechanisms that converge on E-cadherin.

Genetic and Functional Drivers of Diffuse Large B Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole-exome sequencing and transcriptome sequencing in a cohort of 1,001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Successful treatment with Ipilimumab and Interleukin-2 in two patients with metastatic melanoma and systemic autoimmune disease

Abstract Two patients were treated with immunotherapy for metastatic malignant melanoma (MM) despite suffering from systemic autoimmune disease, i.e., ulcerative colitis (UC) and Behcets disease (BD), respectively. Both patients benefitted from the treatment. The patient with UC achieved partial remission of all measurable parameters after treatment with Ipilimumab, while the patient with BD achieved a complete remission of MM after treatment with Interleukin-2 (IL-2) and Interferon-α (IFN-α). Moreover, no aggravation of symptoms related to the autoimmune diseases was seen during treatment, in contrast, clinical indications of improvement were observed. These two cases illustrate that the presence of autoimmune disease does not necessarily predict increased autoimmune toxicity in connection with immunotherapy. They also raise the question of whether autoimmune disease should continue to be an absolute exclusion criterion for treatment of MM with immunotherapy. Consequently, given the poor prognosis of refractory MM, immunotherapies need to be taken into consideration even in cases of autoimmune comorbidity due to the potential long-term benefit that these therapies offer to MM patients.

Serum YKL-40 concentrations in newly diagnosed multiple myeloma patients and YKL-40 expression in malignant plasma cells

Abstract:  Objectives: A potential role in cancer biology is suggested for YKL‐40 (CHI3L1, HC gp‐39). The purpose of this study was to evaluate the clinical value of serum YKL‐40 (sYKL‐40) in multiple myeloma (MM) and to examine YKL‐40 expression in malignant plasma cells (MM PCs). Methods: sYKL‐40 was measured by enzyme‐linked immunosorbent assay (ELISA) in 82 patients with newly diagnosed MM. YKL‐40 expression in immunophenotypically defined plasma cells was investigated by double‐labelled immunohistochemistry in 21 MM patients and by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) in cDNA archives generated by global RT‐PCR in seven controls, 14 patients with monoclonal gammopathy of undetermined significance (MGUS), 45 MM patients, nine patients with extramedullary myeloma (exMM), and seven human myeloma cell lines (HMCLs). Results: sYKL‐40 was elevated above a constructed reference range for healthy controls in 29% of the patients investigated. Patients with elevated sYKL‐40 had reduced overall survival and event‐free survival when compared to patients with normal sYKL‐40, but sYKL‐40 level was defeated by β2‐microglobulin in the multivariate analyses. Intramedullary MM PCs lacked significant expression of YKL‐40, but high levels of YKL‐40 expression were seen in extramedullary MM PCs from one exMM patient and in six HMCLs. Further investigations of other bone marrow (BM) cells showed YKL‐40 expression in megakaryocytes, neutrophils and adherent cells from long‐term BM cultures. Conclusions: In newly diagnosed MM‐patients, a sYKL‐40 elevated above the reference range predicts a poor clinical outcome, and YKL‐40 is expressed by other BM cells than MM PCs. At this point, routine measurements of sYKL‐40 are not warranted, but YKL‐40 should be considered as a potential player in the pathophysiology of MM.