Neutrophil-derived APRIL concentrated in tumor lesions by proteoglycans correlates with human B-cell lymphoma aggressiveness.

A PRoliferation-Inducing TNF Ligand (APRIL) costimulates B-cell activation. When overexpressed in mice, APRIL induces B-cell neoplasia, reminiscent of human B-cell chronic lymphoid leukemia (B-CLL). We analyzed APRIL expression in situ in human non-Hodgkin lymphomas. APRIL up-regulation was only observed in high-grade B-cell lymphomas, diffuse large B-cell lymphoma (DLBCL), and Burkitt lymphoma (BL). Up-regulation was seen in 46% and 20% of DLBCL and BL, respectively. In DLBCL, neutrophils, constitutively producing APRIL and infiltrating the tumor tissue, were the main cellular source of APRIL. Rare DLBCL cases showed a predominance of histiocytes or mesenchymal cells as APRIL source. APRIL secreted by neutrophils accumulated on tumor cells via proteoglycan binding. In addition to proteoglycans, DLBCL tumor cells expressed the APRIL signaling receptor, TACI and/or BCMA, indicating that these tumor cells are fully equipped to respond to APRIL. A retrospective clinical analysis revealed a significant correlation between high expression of APRIL in tumor lesions and decreased overall patient survival rate. Hence, APRIL produced by inflammatory cells infiltrating lymphoma lesions may increase tumor aggressiveness and affect disease outcome.

Monitoring of NY-ESO-1 specific CD4+ T cells using molecularly defined MHC class II/His-tag-peptide tetramers.

MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4(+) T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/NY-ESO-1 tetramers, we could demonstrate that in DR52b(+) cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccine-induced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4(+) T cells, include central and transitional memory polyfunctional populations, and do not include CD4(+)CD25(+)CD127(-) regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.

Defining new criteria for selection of cell-based intestinal models using publicly available databases.

BACKGROUND: The criteria for choosing relevant cell lines among a vast panel of available intestinal-derived lines exhibiting a wide range of functional properties are still ill-defined. The objective of this study was, therefore, to establish objective criteria for choosing relevant cell lines to assess their appropriateness as tumor models as well as for drug absorption studies. RESULTS: We made use of publicly available expression signatures and cell based functional assays to delineate differences between various intestinal colon carcinoma cell lines and normal intestinal epithelium. We have compared a panel of intestinal cell lines with patient-derived normal and tumor epithelium and classified them according to traits relating to oncogenic pathway activity, epithelial-mesenchymal transition (EMT) and stemness, migratory properties, proliferative activity, transporter expression profiles and chemosensitivity. For example, SW480 represent an EMT-high, migratory phenotype and scored highest in terms of signatures associated to worse overall survival and higher risk of recurrence based on patient derived databases. On the other hand, differentiated HT29 and T84 cells showed gene expression patterns closest to tumor bulk derived cells. Regarding drug absorption, we confirmed that differentiated Caco-2 cells are the model of choice for active uptake studies in the small intestine. Regarding chemosensitivity we were unable to confirm a recently proposed association of chemo-resistance with EMT traits. However, a novel signature was identified through mining of NCI60 GI50 values that allowed to rank the panel of intestinal cell lines according to their drug responsiveness to commonly used chemotherapeutics. CONCLUSIONS: This study presents a straightforward strategy to exploit publicly available gene expression data to guide the choice of cell-based models. While this approach does not overcome the major limitations of such models, introducing a rank order of selected features may allow selecting model cell lines that are more adapted and pertinent to the addressed biological question.

Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and various other 3q abnormalities in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) [inv(3)/t(3;3)] is recognized as a distinctive entity in the WHO classification. Risk assignment and clinical and genetic characterization of AML with chromosome 3q abnormalities other than inv(3)/t(3;3) remain largely unresolved. PATIENTS AND METHODS: Cytogenetics, molecular genetics, therapy response, and outcome analysis were performed in 6,515 newly diagnosed adult AML patients. Patients were treated on Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research (HOVON/SAKK; n = 3,501) and German-Austrian Acute Myeloid Leukemia Study Group (AMLSG; n = 3,014) protocols. EVI1 and MDS1/EVI1 expression was determined by real-time quantitative polymerase chain reaction. RESULTS: 3q abnormalities were detected in 4.4% of AML patients (288 of 6,515). Four distinct groups were defined: A: inv(3)/t(3;3), 32%; B: balanced t(3q26), 18%; C: balanced t(3q21), 7%; and D: other 3q abnormalities, 43%. Monosomy 7 was the most common additional aberration in groups (A), 66%; (B), 31%; and (D), 37%. N-RAS mutations and dissociate EVI1 versus MDS1/EVI1 overexpression were associated with inv(3)/t(3;3). Patients with inv(3)/t(3;3) and balanced t(3q21) at diagnosis presented with higher WBC and platelet counts. In multivariable analysis, only inv(3)/t(3;3), but not t(3q26) and t(3q21), predicted reduced relapse-free survival (hazard ratio [HR], 1.99; P < .001) and overall survival (HR, 1.4; P = .006). This adverse prognostic impact of inv(3)/t(3;3) was enhanced by additional monosomy 7. Group D 3q aberrant AML also had a poor outcome related to the coexistence of complex and/or monosomal karyotypes and cryptic inv(3)/t(3;3). CONCLUSION: Various categories of 3q abnormalities in AML can be distinguished according to their clinical, hematologic, and genetic features. AML with inv(3)/t(3;3) represents a distinctive subgroup with unfavorable prognosis.

Cloning of an interferon-gamma regulated human melanoma-associated antigen: identity to the intercellular adhesion molecule ICAM-1.

The human melanoma-associated antigen identified by the monoclonal antibody (mAb) Me14-D12 is a cell surface protein whose expression is induced by interferon-gamma (IFN-gamma). We have recently reported the molecular cloning of a genomic probe specific for the gene and mRNA of this protein. By screening with the genomic probe, we have now isolated a full length 3.0 kb cDNA from a Raji cell line-derived lambda-gt10 library. Sequence analysis of this cDNA showed a 99.8% homology with the intercellular adhesion molecule-1 (ICAM-1). Mouse Ltk- cells stably transfected with the human cDNA clone were found to express the ICAM-1 antigenic determinants detected by mAb Me14-D12 and a reference anti-ICAM-1 mAb, as judged by surface immunofluorescence. Immunoprecipitation of surface-iodinated proteins with mAb Me14-D12 revealed the presence of a 90 kD molecule with identical mobility to ICAM-1. In addition, mAb Me14-D12 could inhibit the phorbolester-stimulated aggregation of U937 cells. The findings show that the human melanoma-associated Me14-D12 antigen is the adhesion molecule ICAM-1.

Adult-type rhabdomyosarcoma: analysis of 57 cases with clinicopathologic description, identification of 3 morphologic patterns and prognosis.

Adult-type rhabdomyosarcoma (RMS) has been classically defined as a pleomorphic sarcoma with desmin expression occurring in adult patients. To reevaluate this entity, we analyzed a series of 57 cases using immunohistochemistry for desmin, myogenin, alpha smooth muscle actin, h-caldesmon, pankeratin AE1/AE3, epithelial membrane antigen (EMA), S100 protein, CD34, MDM2, and CDK4. In this series, there were 36 men and 21 women aged from 22 to 87 years (median: 59). Tumors were mainly located in the lower limbs (27 cases), trunk wall (15 cases), and upper limbs (10 cases). Most tumors were deeply located (51/54) with a size from 1 to 30 cm (median: 8 cm). Cases were classified in 3 histologic categories: spindle cell RMS (25 cases), pleomorphic RMS (16 cases), and mixed type (16 cases). Forty-one tumors were grade 3 and 16 grade 2. Immunohistochemistry showed that every case was positive for desmin and myogenin. Alpha smooth muscle actin was positive in 21%, pankeratin AE1/AE3 in 20%, and CD34 in 13.2%. Treatment modalities and follow-up were available in 46 cases. Median follow-up was 60.9 months. Eight patients developed a local recurrence and 16 a distant metastasis with a 5-year overall survival rate of 52.6% and a 5-year metastasis-free survival of 62.9%. The only predictive factor for metastasis was histologic grade. In conclusion, adult-type RMS is a rare sarcoma occurring mainly in the extremities and trunk wall with 2 main histologic patterns, spindle cell, and pleomorphic patterns, which represent the end of the spectrum of a single entity.

Opposing roles for calcineurin and ATF3 in squamous skin cancer.

Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent. Here we report that genetic and pharmacological suppression of calcineurin/nuclear factor of activated T cells (NFAT) function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-ras(V12) (also known as Hras1)-expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53 (also known as TRP53)-dependent cancer cell senescence, thereby increasing tumorigenic potential. ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma.

A key element for the development of suitable anti-cancer drugs is the identification of cancer-specific enzymatic activities that can be therapeutically targeted. Mucosa-associated lymphoid tissue transformation protein 1 (MALT1) is a proto-oncogene that contributes to tumorigenesis in diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) subtype, the least curable subtype of DLBCL. Recent data suggest that MALT1 has proteolytic activity, but it is unknown whether this activity is relevant for tumor growth. Here we report that MALT1 is constitutively active in DLBCL lines of the ABC but not the GCB subtype. Inhibition of the MALT1 proteolytic activity led to reduced expression of growth factors and apoptosis inhibitors, and specifically affected the growth and survival of ABC DLBCL lines. These results demonstrate a key role for the proteolytic activity of MALT1 in DLBCL of the ABC subtype, and provide a rationale for the development of pharmacological inhibitors of MALT1 in DLBCL therapy.

Comprehensive molecular profiling of lung adenocarcinoma.

Adenocarcinoma of the lung is the leading cause of cancer death worldwide. Here we report molecular profiling of 230 resected lung adenocarcinomas using messenger RNA, microRNA and DNA sequencing integrated with copy number, methylation and proteomic analyses. High rates of somatic mutation were seen (mean 8.9 mutations per megabase). Eighteen genes were statistically significantly mutated, including RIT1 activating mutations and newly described loss-of-function MGA mutations which are mutually exclusive with focal MYC amplification. EGFR mutations were more frequent in female patients, whereas mutations in RBM10 were more common in males. Aberrations in NF1, MET, ERBB2 and RIT1 occurred in 13% of cases and were enriched in samples otherwise lacking an activated oncogene, suggesting a driver role for these events in certain tumours. DNA and mRNA sequence from the same tumour highlighted splicing alterations driven by somatic genomic changes, including exon 14 skipping in MET mRNA in 4% of cases. MAPK and PI(3)K pathway activity, when measured at the protein level, was explained by known mutations in only a fraction of cases, suggesting additional, unexplained mechanisms of pathway activation. These data establish a foundation for classification and further investigations of lung adenocarcinoma molecular pathogenesis.

Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1.

BACKGROUND: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis. MATERIALS AND METHODS: VEGFR1- and CD34-expressing cells of leukapheresis products were analyzed by flow cytometry. Alternatively spliced isoforms of VEGFR1 mRNA were quantified using reverse transcription PCR. RESULTS: Plasma concentrations of sVEGFR1 decreased after HDC, but significantly increased after ASCT. In the CAM assay, sera of patients elicited a proangiogenic effect before and after HDC, but a strong antiangiogenic response after ASCT, comparable to that of bevacizumab at therapeutic concentrations. LP contains high concentrations of sVEGFR1, and high density of VEGFR1(+) neutrophilic granulocytes, in which mRNA expression is shifted toward the soluble VEGFR1 isoform. CONCLUSION: Neutrophil-derived antiangiogenic sVEGFR1 within the LP may contribute to the therapeutic efficacy of ASCT.

Integrative molecular bioinformatics study of human adrenocortical tumors : microRNA, tissue-specific target prediction, and pathway analysis.

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR-mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT(miR-511) and dCT(miR-503) (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

Tenascin-W, a new marker of cancer stroma, is elevated in sera of colon and breast cancer patients.

Tenascins are extracellular matrix proteins present during the development of organisms as well as in pathological conditions. Tenascin-W, the fourth and last member of the tenascin family remains the least well-characterized one. Our study aimed to evaluate the potential significance of tenascin-W as cancer biomarker by monitoring its presence in the serum of colorectal and breast cancer patients and its expression in colorectal tumor tissues. To measure serum tenascin-W levels, a sensitive sandwich-ELISA was established. Mean tenascin-W concentration in sera of patients with nonmetastatic colorectal cancer at time of diagnosis was highly increased compared to that of healthy volunteers. A similar tendency was observed for tenascin-C in the same patient cohort. However, the increase was much more striking for tenascin-W. We also detected elevated tenascin-W levels in sera of breast cancer patients. Furthermore, we could show a prominent expression of tenascin-W in extracts from colorectal tumor tissues by immunoblot analysis, whereas tenascin-W was not detectable in the corresponding normal colon mucosa. To confirm the western blot results, we performed immunohistochemistry of frozen sections of the same patients as well as of an additional, independently chosen collection of colorectal cancer tissues. In all cases, similarly to tenascin-C, tenascin-W was detected in the tumor stroma. Our results reveal a clear association between elevated levels of tenascin-W and the presence of cancer. These results warrant further studies to evaluate the potential value of serum and tissue tenascin-W levels as diagnostic, prognostic or monitoring biomarker in colorectal, breast and possibly other solid cancers.

Asymmetric cancer cell division regulated by AKT.

Human tumors often contain slowly proliferating cancer cells that resist treatment, but we do not know precisely how these cells arise. We show that rapidly proliferating cancer cells can divide asymmetrically to produce slowly proliferating "G0-like" progeny that are enriched following chemotherapy in breast cancer patients. Asymmetric cancer cell division results from asymmetric suppression of AKT/PKB kinase signaling in one daughter cell during telophase of mitosis. Moreover, inhibition of AKT signaling with small-molecule drugs can induce asymmetric cancer cell division and the production of slow proliferators. Cancer cells therefore appear to continuously flux between symmetric and asymmetric division depending on the precise state of their AKT signaling network. This model may have significant implications for understanding how tumors grow, evade treatment, and recur.