Assessment of 2-Year Storage Conditions on Protein, RNA, and DNA in Unstained Human Tissue Sections, Including a Novel Multiplex Digital Gene Expression Profiling Method with Implications for Biobanking.

Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often stored for years before being used. Previous studies have evaluated the effects of time, temperature, humidity, and inert gases on preservation of USS; however, no study has examined all four variables together. Methods: In the current work, we prospectively and blindly assessed time points from 0 to 24 months, room versus refrigerated temperature, and presence of a desiccant and/or nitrogen atmosphere on a variety of benign and malignant tissues from North America and Africa. End points included immunohistochemistry (IHC), in situ hybridization (ISH), extracted RNA and DNA quantity and quality, and messenger RNA performance in a novel, multiplexed digital gene expression profiling assay of both housekeeping and tumor-specific genes. Results: We found that using current methods of antigen retrieval, staining, and extraction, the end points of IHC, ISH, RNA, and DNA were well preserved under the various conditions tested, with implications that pre-embedding factors contribute to variability in subsequent tissue integrity. We also document that spectrophotometric estimations of nucleic acid concentrations were in general estimated to be higher than with fluorimetric methods, which may be pertinent to end assay development. We further describe a new multiplex assay, the PlexSet digital gene expression assay, suitable for evaluating RNA quality in FFPE tissues. Conclusion: Altogether, these results may provide helpful guidance with regard to approaches for long-term storage conditions for USS.

Recommendations for Tissue Microarray Construction and Quality Assurance.

Tissue microarrays (TMAs) are important tools to conserve precious tissue resources from increasingly smaller biopsies and to control experimental costs and variation across sample sets. The quality assurance assessment of TMA materials created at centralized biobanks has not been standardized. Herein, we outline 2 processes for the construction of TMAs ("recipient block" and "tape" methods) and the associated preconstruction quality control measures (pathology review, protein and RNA assessment, map creation, and storage conditions) developed by the AIDS Cancer Specimen Resource (ACSR) Network's Science and Technology Core. These steps provide a suggested framework for quality assessment that allows end-users, receiving materials from tissue banks, confidence in their experimental results.

Enhanced DNA repair and genomic stability identify a novel HIV-related diffuse large B-cell lymphoma signature.

Diffuse large B-cell lymphoma (DLBCL) is up to 17-fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (-) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(-) germinal center B-cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression (CCNA2, CCNB1, CDC25A, E2F1), DNA replication (MCM2, MCM4, MCM7) and DNA damage repair, including eight Fanconi anemia genes (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT/UBE2T, FANCV/MAD2L2), were significantly increased in HIV(+) GCB-DLBCL tumors compared to HIV(-) tumors. In contrast, genes associated with cell cycle inhibition (CDKN1A, CDKN1B) as well as apoptosis regulating BCL2 family members (BCL2, BAX, BIM, BMF, PUMA) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB-DLBCL tumors have fewer copy number variations than their HIV(-) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB-DLBCL is a distinct molecular malignancy from HIV(-) GCB-DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.

Incorporation of Digital Gene Expression Profiling for Cell-of-Origin Determination (Lymph2Cx Testing) into the Routine Work-Up of Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphomas (DLBCL) represent a clinically heterogeneous group of lymphomas that are classified together based on similarities in morphology and immunophenotype. Gene expression profiling further classifies DLBCL into distinct molecular subgroups based on cell-of-origin (COO), including Germinal Center B-cell type, Activated B-cell type, and Unclassified type. COO assignment of DLBCL has important biological and prognostic significance, as well as emerging therapeutic implications. Herein, we describe the first clinical validation of a digital gene expression profiling assay (Lymph2Cx) to perform COO assignment in the routine work-up of DLBCL using formalin-fixed paraffin-embedded (FFPE) tissue sections and describe the results of 90 consecutive DLBCL cases analyzed prospectively by a College of American Pathologists/Clinical Laboratory Improvement Amendments (CAP/CLIA)-certified clinical molecular diagnostics laboratory.

Molecular classification of primary mediastinal large B-cell lymphoma using routinely available tissue specimens.

Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression-based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx's utility for routine diagnostic purposes and therapeutic decision making.

A phase II study of belinostat (PXD101) in relapsed and refractory aggressive B-cell lymphomas: SWOG S0520.

Recent advances in diffuse large B-cell lymphomas (DLBCL) have underscored the importance of tumor microenvironment in escaping host anti-tumor responses. One mechanism is loss of major histocompatibility Class II antigens (MHCII) associated with decreased tumor infiltrating T lymphocytes (TIL) and poor survival. Transcription of MHCII is controlled by CIITA which in turn is regulated by histone acetylation. In this study, we hypothesized that HDAC inhibition with belinostat increases MHCII, CIITA expression, TIL and improves patient outcomes. Primary objective was evaluation of toxicity and response. Twenty-two patients were enrolled for the study. Belinostat was well tolerated with mild toxicity. Two partial responses were observed at 5, 13 months after registration for an overall response rate (ORR) (95% CI) of 10.5% (1.3-33.1%), and three patients had stable disease for 4.7, 42.3+, and 68.4 + months with minimum 3-year follow-up. Included correlative studies support the hypothesis and serve as the basis for SWOG S0806 combining vorinostat with R-CHOP.

Determining cell-of-origin subtypes of diffuse large B-cell lymphoma using gene expression in formalin-fixed paraffin-embedded tissue.

The assignment of diffuse large B-cell lymphoma into cell-of-origin (COO) groups is becoming increasingly important with the emergence of novel therapies that have selective biological activity in germinal center B cell-like or activated B cell-like groups. The Lymphoma/Leukemia Molecular Profiling Project's Lymph2Cx assay is a parsimonious digital gene expression (NanoString)-based test for COO assignment in formalin-fixed paraffin-embedded tissue (FFPET). The 20-gene assay was trained using 51 FFPET biopsies; the locked assay was then validated using an independent cohort of 68 FFPET biopsies. Comparisons were made with COO assignment using the original COO model on matched frozen tissue. In the validation cohort, the assay was accurate, with only 1 case with definitive COO being incorrectly assigned, and robust, with >95% concordance of COO assignment between 2 independent laboratories. These qualities, along with the rapid turnaround time, make Lymph2Cx attractive for implementation in clinical trials and, ultimately, patient management.

A model of sensitivity and resistance to histone deacetylase inhibitors in diffuse large B cell lymphoma: Role of cyclin-dependent kinase inhibitors.

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G 2/M arrest and apoptosis and resistance by reversible G 1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G 1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

The copper chelator ATN-224 induces peroxynitrite-dependent cell death in hematological malignancies.

Chemoresistance due to oxidative stress resistance or upregulation of Bcl-2 contributes to poor outcome in the treatment of hematological malignancies. In this study, we utilize the copper-chelator drug ATN-224 (choline tetrathiomolybdate) to induce cell death in oxidative stress-resistant cells and cells overexpressing Bcl-2 by modulating the cellular redox environment and causing mitochondrial dysfunction. ATN-224 treatment decreases superoxide dismutase 1 (SOD1) activity, increases intracellular oxidants, and induces peroxynitrite-dependent cell death. ATN-224 also targets the mitochondria, decreasing both cytochrome c oxidase (CcOX) activity and mitochondrial membrane potential. The concentration of ATN-224 required to induce cell death is proportional to SOD1 levels, but independent of Bcl-2 status. In combination with doxorubicin, ATN-224 enhances cell death. In primary B-cell acute lymphoblastic leukemia patient samples, ATN-224 decreases the viable cell number. Our findings suggest that ATN-224's dual targeting of SOD1 and CcOX is a promising approach for treatment of hematological malignancies either as an adjuvant or as a single agent.

Partial plasma cell differentiation as a mechanism of lost major histocompatibility complex class II expression in diffuse large B-cell lymphoma.

Loss of major histocompatibility complex class II (MHC II) expression is associated with poor patient outcome in diffuse large B-cell lymphoma (DLBCL). As MHC II molecules are lost with plasmacytic differentiation in normal cells, we asked whether MHC II loss in DLBCL is associated with an altered differentiation state. We used gene expression profiling, quantum dots, and immunohistochemistry to study the relationship between MHC II and plasma cell markers in DLBCL and plasmablastic lymphoma (PBL). Results demonstrate that MHC II(-) DLBCL immunophenotypically overlap with PBL and demonstrate an inverse correlation between MHC II and plasma cell markers MUM1, PRDM1/Blimp1, and XBP1s. In addition, MHC II expression is significantly higher in germinal center-DLBCL than activated B cell-DLBCL. A minor subset of cases with an unusual pattern of mislocalized punctate MHC II staining and intermediate levels of mRNA is also described. Finally, we show that PBL is negative for MHC II. The results imply a spectrum of MHC II expression that is more frequently diminished in tumors derived from B cells at the later stages of differentiation (with complete loss in PBL). Our observations provide a possible unifying concept that may contribute to the poor outcome reported in all MHC II(-) B-cell tumors.

Vascular endothelial growth factor receptor-1 and receptor-2 initiate a phosphatidylinositide 3-kinase-dependent clonogenic response in acute myeloid leukemia cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) interacts with two high-affinity receptor tyrosine kinases (RTK) on vascular endothelium to initiate complementary but disparate biologic responses. We previously reported that acute myeloid leukemia (AML) cells express VEGF and one or both VEGF-A receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). To evaluate receptor-selective trophic response to VEGF-A in AML cells, we investigated receptor-specific ligand activation responsible for VEGF-initiated clonogenic response. MATERIALS AND METHODS: Using KG1 (VEGFR-1+/VEGFR-2+) and HL60 (VEGFR-1+) cells with differential VEGF receptor display, we investigated ligand-induced clonogenic response and receptor-initiated signaling after stimulation with VEGF-A, the VEGFR-1 selective ligand placental growth factor (PlGF), or receptor-specific antibody agonists. RESULTS: Recombinant human (rhu)-VEGF increased S-phase fraction and stimulated colony formation in both KG1 and HL60 cells. Ligation of VEGFR-1 or VEGFR-2 with receptor-specific antibody agonists triggered equivalent and concentration-dependent stimulation of colony recovery in KG1 cells, whereas clonogenic response in HL60 cells was restricted to VEGFR-1 activation by antibody or PlGF. In serum-deprived KG1 and HL60 cells, rhu-VEGF stimulated rapid and sustained phosphorylation of Akt/PKB that was inhibited by the phosphatidyl inositol 3-kinase (PI3-K) kinase inhibitor wortmannin. Preincubation with wortmannin inhibited VEGF-induced colony formation in a concentration-dependent fashion. rhu-VEGF-induced clonogenic response and Akt phosphorylation was abolished by the VEGF-RTK inhibitor SU-5416 at concentrations greater than 10 microM, whereas MEK inhibition by PD98059 (1 and 10 microM) was ineffective. In vivo suppression of Akt phosphorylation was confirmed in myeloblast lysates from three patients with advanced myeloid malignancies treated with SU5416. CONCLUSION: These data indicate that VEGF interaction with either VEGFR-1 or VEGFR-2 initiates a clonogenic response in AML cells that is PI3-kinase dependent. RTK inhibitors with broad specificity for angiogenic receptors represent novel therapeutics that merit further clinical investigation in AML.