A microRNA Prognostic Signature in Patients with Diffuse Intrinsic Pontine Gliomas through Non-Invasive Liquid Biopsy.

Diffuse midline gliomas (DMGs) originate in the thalamus, brainstem, cerebellum and spine. This entity includes tumors that infiltrate the pons, called diffuse intrinsic pontine gliomas (DIPGs), with a rapid onset and devastating neurological symptoms. Since surgical removal in DIPGs is not feasible, the purpose of this study was to profile circulating miRNA expression in DIPG patients in an effort to identify a non-invasive prognostic signature with clinical impact. Using a high-throughput platform, miRNA expression was profiled in serum samples collected at the time of MRI diagnosis and prior to radiation and/or systemic therapy from 47 patients enrolled in clinical studies, combining nimotuzumab and vinorelbine with concomitant radiation. With progression-free survival as the primary endpoint, a semi-supervised learning approach was used to identify a signature that was also tested taking overall survival as the clinical endpoint. A signature comprising 13 circulating miRNAs was identified in the training set (n = 23) as being able to stratify patients by risk of disease progression (log-rank p = 0.00014; HR = 7.99, 95% CI 2.38-26.87). When challenged in a separate validation set (n = 24), it confirmed its ability to predict progression (log-rank p = 0.00026; HR = 5.51, 95% CI 2.03-14.9). The value of our signature was also confirmed when overall survival was considered (log-rank p = 0.0021, HR = 4.12, 95% CI 1.57-10.8). We have identified and validated a prognostic marker based on the expression of 13 circulating miRNAs that can shed light on a patient's risk of progression. This is the first demonstration of the usefulness of nucleic acids circulating in the blood as powerful, easy-to-assay molecular markers of disease status in DIPG. This study provides Class II evidence that a signature based on 13 circulating miRNAs is associated with the risk of disease progression.

Tumor Biomarkers for the Prediction of Distant Metastasis in Head and Neck Squamous Cell Carcinoma.

Distant metastases (DM) in head and neck squamous cell carcinoma (HNSCC) remain a challenge as treatment options are limited. To identify biomarkers predictive of DM in primary tumors (PT), gene expression profiling was performed in PT from patients who did, or did not develop DM (T-with and T-without, n = 25 and 24, respectively), and in matched DM. A total of 185 and 42 differentially expressed genes were identified in the T-with vs. T-without and the T-with vs. DM comparisons, respectively. The intersection between these two comparisons identified COX7A1 and TBX5 as common genes. In three independent datasets, both genes were able to significantly distinguish patients according to their DM-free survival. By functional biological analyses, the T-without group showed enrichment in immune-response pathways, whereas the T-with group showed an enrichment in B-plasma cells and Tregs. Increased enrichment of proliferation-related pathways was observed in the T-with group compared with that in the DM group. Further comparisons with/without DM are needed to confirm these data in order to improve clinical management of HNSCC.

Ovarian Cancer Translational Activity of the Multicenter Italian Trial in Ovarian Cancer (MITO) Group: Lessons Learned in 10 Years of Experience.

Ovarian cancer is the most lethal gynecological cancer, and despite years of research, with the exception of a BRCA mutation driving the use of PARP inhibitors, no new prognostic/predictive biomarkers are clinically available. Improvement in biomarker selection and validation may derive from the systematic inclusion of translational analyses into the design of clinical trials. In the era of personalized medicine, the prospective centralized collection of high-quality biological material, expert pathological revision, and association to well-controlled clinical data are important or even essential added values to clinical trials. Here, we present the academic experience of the MITO (Multicenter Italian Trial in Ovarian Cancer) group, including gynecologists, pathologists, oncologists, biostatisticians, and translational researchers, whose effort is dedicated to the care and basic/translational research of gynecologic cancer. In our ten years of experience, we have been able to collect and process, for translational analyses, formalin-fixed, paraffin-embedded blocks from more than one thousand ovarian cancer patients. Standard operating procedures for collection, shipping, and processing were developed and made available to MITO researchers through the coordinating center's web-based platform. Clinical data were collected through dedicated electronic case report forms hosted in a web-based electronic platform and stored in a central database at the trial's coordinating center, which performed all the analyses related to the proposed translational researches. During this time, we improved our strategies of block management from retrospective to prospective collection, up to the design of a prospective collection with a quality check for sample eligibility before patients' accrual. The final aim of our work is to share our experience by suggesting a guideline for the process of centralized collection, revision processing, and storing of formalin-fixed, paraffin-embedded blocks for translational purposes.

A functional gene expression analysis in epithelial sinonasal cancer: Biology and clinical relevance behind three histological subtypes.

Epithelial sinonasal cancers (SNCs) are rare diseases with overlapping morphological features and a dismal prognosis. We aimed to investigate the expression differences among the histological subtypes for discerning their molecular characteristics. We selected 47 SNCs: (i) 21 nonkeratinizing squamous cell carcinomas (NKSCCs), (ii) 13 sinonasal neuroendocrine cancers (SNECs), and (iii) 13 sinonasal undifferentiated cancers (SNUCs). Gene expression profiling was performed by DASL (cDNA-mediated annealing, selection, extension, and ligation) microarray analysis with internal validation by quantitative RT-PCR (RT-qPCR). Relevant molecular patterns were uncovered by sparse partial-least squares discriminant analysis (sPLS-DA), microenvironment cell type (xCell), CIBERSORT, and gene set enrichment (GSEA) analyses. The first two sPLS-DA components stratified samples by histological subtypes. xCell highlighted increased expression of immune components (CD8+ effector memory cells, in SNUC) and "other cells": keratinocytes and neurons in NKSCC and SNEC, respectively. Pathway enrichment was observed in NKSCC (six gene sets, proliferation related), SNEC (one gene set, pancreatic ß-cells), and SNUC (twenty gene sets, some of them immune-system related). Major neuroendocrine involvement was observed in all the SNEC samples. Our high-throughput analysis revealed a good diagnostic ability to differentiate NKSCC, SNEC, and SNUC, but indicated that the neuroendocrine pathway, typical and pathognomonic of SNEC is also present at lower expression levels in the other two histological subtypes. The different and specific profiles may be exploited for elucidating their biology and could help to identify prognostic and therapeutic opportunities.

Are Fusion Transcripts in Relapsed/Metastatic Head and Neck Cancer Patients Predictive of Response to Anti-EGFR Therapies?

Prediction of benefit from combined chemotherapy and the antiepidermal growth factor receptor cetuximab is a not yet solved question in head and neck squamous cell carcinoma (HNSCC). In a selected series of 14 long progression-free survival (PFS) and 26 short PFS patients by whole gene and microRNA expression analysis, we developed a model potentially predictive of cetuximab sensitivity. To better decipher the "omics" profile of our patients, we detected transcript fusions by RNA-seq through a Pan-Cancer panel targeting 1385 cancer genes. Twenty-seven different fusion transcripts, involving mRNA and long noncoding RNA (lncRNA), were identified. The majority of fusions (81%) were intrachromosomal, and 24 patients (60%) harbor at least one of them. The presence/absence of fusions and the presence of more than one fusion were not related to outcome, while the lncRNA-containing fusions resulted enriched in long PFS patients (P = 0.0027). The CD274-PDCD1LG2 fusion was present in 7/14 short PFS patients harboring fusions and was absent in long PFS patients (P = 0.0188). Among the short PFS patients, those harboring this fusion had the worst outcome (P = 0.0172) and increased K-RAS activation (P = 0.00147). The associations between HNSCC patient's outcome following cetuximab treatment and lncRNA-containing fusions or the CD274-PDCD1LG2 fusion deserve validation in prospective clinical trials.

Integrative miRNA-Gene Expression Analysis Enables Refinement of Associated Biology and Prediction of Response to Cetuximab in Head and Neck Squamous Cell Cancer.

This paper documents the process by which we, through gene and miRNA expression profiling of the same samples of head and neck squamous cell carcinomas (HNSCC) and an integrative miRNA-mRNA expression analysis, were able to identify candidate biomarkers of progression-free survival (PFS) in patients treated with cetuximab-based approaches. Through sparse partial least square-discriminant analysis (sPLS-DA) and supervised analysis, 36 miRNAs were identified in two components that clearly separated long- and short-PFS patients. Gene set enrichment analysis identified a significant correlation between the miRNA first-component and EGFR signaling, keratinocyte differentiation, and p53. Another significant correlation was identified between the second component and RAS, NOTCH, immune/inflammatory response, epithelial-mesenchymal transition (EMT), and angiogenesis pathways. Regularized canonical correlation analysis of sPLS-DA miRNA and gene data combined with the MAGIA2 web-tool highlighted 16 miRNAs and 84 genes that were interconnected in a total of 245 interactions. After feature selection by a smoothed t-statistic support vector machine, we identified three miRNAs and five genes in the miRNA-gene network whose expression result was the most relevant in predicting PFS (Area Under the Curve, AUC = 0.992). Overall, using a well-defined clinical setting and up-to-date bioinformatics tools, we are able to give the proof of principle that an integrative miRNA-mRNA expression could greatly contribute to the refinement of the biology behind a predictive model.

Applicability of Under Vacuum Fresh Tissue Sealing and Cooling to Omics Analysis of Tumor Tissues.

CONTEXT: Biobanks of frozen human normal and malignant tissues represent a valuable source for "omics" analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis strongly relies on the collection, handling, storage procedures, and quality control of fresh human tissue samples. OBJECTIVE: We tested whether under vacuum storage (UVS) effectively preserves tissues during the time between surgery and storage for "omics" analyses. DESIGN: Normal and matched tumor specimens, obtained from 16 breast, colon, or lung cancer patients and 5 independent mesenchymal tumors, were dissected within 20 minutes from surgical excision and divided in three to five aliquots; for each tissue sample, one aliquot was snap-frozen in liquid nitrogen (defined as baseline or T0 samples), and the other portions were sealed into plastic bags and kept at 4°C for 1, 24, 48, or 72 hours under vacuum and then frozen. The tissue and molecular preservation under vacuum was evaluated over time in terms of histomorphology, transcription (Illumina microarrays), protein (surface-enhanced laser desorption/ionization-time of flight/mass spectrometry and Western blot), and metabolic profile (nuclear magnetic resonance spectroscopy). RESULTS: Tissue morphology, Mib-1, and vimentin immunostaining were preserved over time without signs of tissue degradation. Principal variance component analysis showed that time of storage had a minimal effect on gene expression or the proteome, but affected the preservation of some metabolites to a greater extent. UVS did not impact the RNA and protein integrity or specific phosphorylation sites on mTOR and STAT3. Measurement of metabolites revealed pronounced changes after 1 hour of storage. CONCLUSIONS: Our results show that UVS can preserve tissue specimens for histological, transcriptomic, and proteomic examinations up to 48 hours and possibly longer, whereas it has limitations for metabolomic applications.

Functional Genomics Uncover the Biology behind the Responsiveness of Head and Neck Squamous Cell Cancer Patients to Cetuximab.

PURPOSE: To identify the tumor portrait of the minority of head and neck squamous cell carcinoma (HNSCC) patients with recurrent-metastatic (RM) disease who upon treatment with platinum-based chemotherapy plus cetuximab present a long-lasting response. EXPERIMENTAL DESIGN: The gene expression of pretreatment samples from 40 HNSCC-RM patients, divided in two groups [14 long-progression-free survival (PFS) and 26 short-PFS (median = 19 and 3 months, respectively)], was associated with PFS and was challenged against a dataset from metastatic colon cancer patients treated with cetuximab. For biologic analysis, we performed functional and subtype association using gene set enrichment analysis, associated biology across all currently available HNSCC signatures, and inferred drug sensitivity using data from the Cancer Genomic Project. RESULTS: The identified genomic profile exhibited a significant predictive value that was essentially confirmed in the single publicly available dataset of cetuximab-treated patients. The main divergence between long- and short-PFS groups was based on developmental/differentiation status. The long-PFS patients are characterized by basal subtype traits such as strong EGFR signaling phenotype and hypoxic differentiation, further validated by the significantly higher association with the hypoxia metagene. The short-PFS patients presented a strong activation of RAS signaling confirmed in an in vitro model of two isogenic HNSCC cell lines sensitive or resistant to cetuximab. The predicted drug sensitivity for all four EGFR inhibitors was higher in long- versus short-PFS patients (P range: <0.0022-1e-07). CONCLUSIONS: Our data uncover the biology behind response to platinum-based chemotherapy plus cetuximab in RM-HNSCC cancer and may have translational implications improving treatment selection. Clin Cancer Res; 22(15); 3961-70. ©2016 AACRSee related commentary by Chau and Hammerman, p. 3710.

Interleukin 21 Controls mRNA and MicroRNA Expression in CD40-Activated Chronic Lymphocytic Leukemia Cells.

Several factors support CLL cell survival in the microenvironment. Under different experimental conditions, IL21 can either induce apoptosis or promote CLL cell survival. To investigate mechanisms involved in the effects of IL21, we studied the ability of IL21 to modulate gene and miRNA expressions in CD40-activated CLL cells. IL21 was a major regulator of chemokine production in CLL cells and it modulated the expression of genes involved in cell movement, metabolism, survival and apoptosis. In particular, IL21 down-regulated the expression of the chemokine genes CCL4, CCL3, CCL3L1, CCL17, and CCL2, while it up-regulated the Th1-related CXCL9 and CXCL10. In addition, IL21 down-regulated the expression of genes encoding signaling molecules, such as CD40, DDR1 and PIK3CD. IL21 modulated a similar set of genes in CLL and normal B-cells (e.g. chemokine genes), whereas other genes, including MYC, TNF, E2F1, EGR2 and GAS-6, were regulated only in CLL cells. An integrated analysis of the miRNome and gene expression indicated that several miRNAs were under IL21 control and these could, in turn, influence the expression of potential target genes. We focused on hsa-miR-663b predicted to down-regulate several relevant genes. Transfection of hsa-miR-663b or its specific antagonist showed that this miRNA regulated CCL17, DDR1, PIK3CD and CD40 gene expression. Our data indicated that IL21 modulates the expression of genes mediating the crosstalk between CLL cells and their microenvironment and miRNAs may take part in this process.

miR-342 regulates BRCA1 expression through modulation of ID4 in breast cancer.

A miRNAs profiling on a group of familial and sporadic breast cancers showed that miRNA-342 was significantly associated with estrogen receptor (ER) levels. To investigate at functional level the role of miR-342 in the pathogenesis of breast cancer, we focused our attention on its "in silico" predicted putative target gene ID4, a transcription factor of the helix-loop-helix protein family whose expression is inversely correlated with that of ER. ID4 is expressed in breast cancer and can negatively regulate BRCA1 expression. Our results showed an inverse correlation between ID4 and miR-342 as well as between ID4 and BRCA1 expression. We functionally validated the interaction between ID4 and miR-342 in a reporter Luciferase system. Based on these findings, we hypothesized that regulation of ID4 mediated by miR-342 could be involved in the pathogenesis of breast cancer by downregulating BRCA1 expression. We functionally demonstrated the interactions between miR-342, ID4 and BRCA1 in a model provided by ER-negative MDA-MB-231 breast cancer cell line that presented high levels of ID4. Overexpression of miR-342 in these cells reduced ID4 and increased BRCA1 expression, supporting a possible role of this mechanism in breast cancer. In the ER-positive MCF7 and in the BRCA1-mutant HCC1937 cell lines miR-342 over-expression only reduced ID4. In the cohort of patients we studied, a correlation between miR-342 and BRCA1 expression was found in the ER-negative cases. As ER-negative cases were mainly BRCA1-mutant, we speculate that the mechanism we demonstrated could be involved in the decreased expression of BRCA1 frequently observed in non BRCA1-mutant breast cancers and could be implicated as a causal factor in part of the familial cases grouped in the heterogeneous class of non BRCA1 or BRCA2-mutant cases (BRCAx). To validate this hypothesis, the study should be extended to a larger cohort of ER-negative cases, including those belonging to the BRCAx class.

Comparison of microarray platforms for measuring differential microRNA expression in paired normal/cancer colon tissues.

BACKGROUND: Microarray technology applied to microRNA (miRNA) profiling is a promising tool in many research fields; nevertheless, independent studies characterizing the same pathology have often reported poorly overlapping results. miRNA analysis methods have only recently been systematically compared but only in few cases using clinical samples. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the inter-platform reproducibility of four miRNA microarray platforms (Agilent, Exiqon, Illumina, and Miltenyi), comparing nine paired tumor/normal colon tissues. The most concordant and selected discordant miRNAs were further studied by quantitative RT-PCR. Globally, a poor overlap among differentially expressed miRNAs identified by each platform was found. Nevertheless, for eight miRNAs high agreement in differential expression among the four platforms and comparability to qRT-PCR was observed. Furthermore, most of the miRNA sets identified by each platform are coherently enriched in data from the other platforms and the great majority of colon cancer associated miRNA sets derived from the literature were validated in our data, independently from the platform. Computational integration of miRNA and gene expression profiles suggested that anti-correlated predicted target genes of differentially expressed miRNAs are commonly enriched in cancer-related pathways and in genes involved in glycolysis and nutrient transport. CONCLUSIONS: Technical and analytical challenges in measuring miRNAs still remain and further research is required in order to increase consistency between different microarray-based methodologies. However, a better inter-platform agreement was found by looking at miRNA sets instead of single miRNAs and through a miRNAs - gene expression integration approach.

Identification of a chrXq27.3 microRNA cluster associated with early relapse in advanced stage ovarian cancer patients.

A major challenge in advanced-stage epithelial ovarian cancer (EOC) is prediction of chemoresistant relapse. Our aim was to identify a microRNA (miRNA) signature associated with early relapse in advanced-stage EOC patients. miRNA expression was assessed by microarray profiling in training (n = 55) and test (n = 30) sets selected on the basis of time to relapse (TTR), followed by internal quantitative reverse transcriptase-PCR validation on a set of 45 consecutive cases unselected for clinical response and external in silico validation on publicly available datasets. Thirty-two differentially expressed miRNAs in early vs. late relapsing patients were identified in the training set. In the test set, 8 of these, belonging to a cluster located on chrXq27.3, were down-modulated in early relapsing patients. Hierarchical clustering of the internal validation set according to chrXq27.3 miRNA expression associated low miRNA expression with shorter TTR (log-rank P=0.00074, HR 2.44). The cluster was an independent prognostic factor in both internal and external validation sets. Forced expression of chrXq27.3-cluster selected miRNAs in human EOC cellular models was associated to reduction of cell proliferation and increased sensitivity to cisplatin. The role of down-modulation of the chrXq27.3 miRNA cluster in early relapse of advanced-stage EOC patients and its association to a reduced sensitivity to chemotherapeutic treatments warrant further investigation.

Characterization of compound 584, an Abl kinase inhibitor with lasting effects.

BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl+ neoplastic cells.

Resistance to imatinib represents an important scientific and clinical issue in chronic myelogenous leukemia. In the present study, the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 proved to be an active inhibitor of Bcr-Abl in several chronic myelogenous leukemia cell lines and transfectants, with IC(50) values in the low nanomolar range, 1 to 2 logs lower than those obtained with imatinib. Cells expressing activated forms of KIT or platelet-derived growth factor receptor (PDGFR), two additional targets of imatinib, were unaffected by SKI-606, whereas activity was found against PIM2. SKI-606 retained activity in cells where resistance to imatinib was caused by BCR-ABL gene amplification and in three of four Bcr-Abl point mutants tested. In vivo experiments confirmed SKI-606 activity in models where resistance was not caused by mutations as well as in cells carrying the Y253F, E255K, and D276G mutations. Modeling considerations attribute the superior activity of SKI-606 to its ability to bind a conformation of Bcr-Abl different from imatinib.

Bcl-XL down-regulation suppresses the tumorigenic potential of NPM/ALK in vitro and in vivo.

Deregulated apoptosis is a common finding in tumorigenesis. The oncogenic tyrosine kinase nucleophosmin/anaplastic lymphoma kinase (NPM/ALK) delivers a strong survival signal in anaplastic large cell lymphomas (ALCLs). Although NPM/ALK activates multiple antiapoptotic pathways, the biologic relevance and therapeutic potential of more downstream apoptotic effectors are mostly unknown. In this report, the NPM/ALK-mediated induction of Bcl-XL (but not of Bcl-2) was identified in human ALCL-derived cells. NPM/ALK kinase activity was required to promote Bcl-XL expression and its protective effect on mitochondrial homeostasis. Down-regulation of Bcl-XL significantly reduced the antiapoptotic potential of NPM/ALK in both transformed murine Ba/F3 pro-B cells and human ALCL-derived KARPAS-299 cells. To elucidate the role of Bcl-XL in vivo, Ba/F3-NPM/ALK+ cells expressing a doxycycline (Dox)-inducible Bcl-XL antisense transgene (pTet-ON) were injected into nude mice. Doxycycline administration prevented a fatal systemic disease in 15 of 15 intravenously injected mice and the appearance of subcutaneous tumor xenografts in 9 of 12 mice; in vivo down-regulation of Bcl-XL was also documented. Our results show a pivotal role for Bcl-XL in ALK-mediated oncogenicity; a single protein placed downstream of a known oncogene can be crucial for the survival of neoplastic cells both in vitro and in vivo. Bcl-XL deserves further investigation as a possible therapeutic target in ALK+ ALCLs.

Constitutive activation of Jak2 contributes to proliferation and resistance to apoptosis in NPM/ALK-transformed cells.

OBJECTIVE: The t(2;5) translocation results in a 80-kDa oncogenic fusion protein consisting of NPM and the kinase domain of the tyrosine kinase ALK and is present in over half the cases of anaplastic large cell lymphoma (ALCL). NPM/ALK exerts its transforming potential via activation of multiple signaling pathways promoting growth factor independence and protection from apoptosis. Jak/Stat signaling is aberrantly activated in several human hematopoietic malignancies. We investigated the role of Jak2 in the context of NPM/ALK-mediated oncogenesis. MATERIALS AND METHODS: Constitutive tyrosine phosphorylation of Jak2 was analyzed by Jak2 immunoprecipitation and subsequent anti-phosphotyrosine Western blotting. NPM/ALK-transformed cells were treated with the Jak2 inhibitor AG490 or transfected with wild-type or dominant-negative Jak2 expression constructs to measure 3[H]-thymidine incorporation. Apoptosis was assessed by flow cytometric analysis of annexin V-stained cells. The effect of Jak2 on Stat5-dependent transcriptional activity was measured by beta-casein promoter-dependent luciferase expression. RESULTS: Jak2 was found to be constitutively tyrosine phosphorylated in ALCL cells and in NPM/ALK-transformed hematopoietic cells. Also, NPM/ALK was present in immunoprecipitates of Jak2. Inhibition of Jak2 led to a reduction of NPM/ALK-mediated proliferation and induced apoptosis. Stat5-dependent transcriptional activity was inhibited by transfection of NPM/ALK-transformed cells with a dominant-negative Jak2 expression construct or treatment with AG490. CONCLUSION: Constitutive activation of Jak2 constitutes a pro-proliferative, anti-apoptotic signaling pathway in NPM/ALK-transformed hematopoietic cells.

Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells.

Betulinic acid is a triterpene with selective cytotoxicity against melanoma, neuroectodermal and malignant brain tumor cell lines. In this study the betulinic acid activity was evaluated, in comparison with doxorubicin, on different human neoplastic and non-neoplastic cell lines and on proliferating normal lymphocytes. Growth inhibition was evident in all the neoplastic cell lines independently on p53 status and histotype. Antiproliferative activity of betulinic acid was related to a cytotoxic effect on two p53 wild-type and on one p53 mutant cell lines and to a cytostatic effect on one p53 mutant melanoma clone. At the same concentrations, normal cells were unaffected indicating a selective effect of this agent. A cytotoxic activity of doxorubicin was evident on all the tested systems. In vivo experiments, performed on one of these cell lines, confirmed the antineoplastic activity of this drug. These data support further preclinical studies of betulinic acid not confined to melanoma and neuroectodermal tumors independently of p53 status.