Exploration of Tumor Biopsy Gene Signatures to Understand the Role of the Tumor Microenvironment in Outcomes to Lisocabtagene Maraleucel.

In the TRANSCEND NHL 001 study, 53% of patients with relapsed/refractory large B-cell lymphoma (LBCL) treated with lisocabtagene maraleucel (liso-cel) achieved a complete response (CR). To determine characteristics of patients who did and did not achieve a CR, we examined the tumor biology and microenvironment from lymph node tumor biopsies. LBCL biopsies from liso-cel-treated patients were taken pretreatment and ∼11 days posttreatment for RNA sequencing (RNA-seq) and multiplex immunofluorescence (mIF). We analyzed gene expression data from pretreatment biopsies (N = 78) to identify gene sets enriched in patients who achieved a CR to those with progressive disease. Pretreatment biopsies from month-3 CR patients displayed higher expression levels of T-cell and stroma-associated genes, and lower expression of cell-cycle genes. To interpret whether LBCL samples were "follicular lymphoma (FL)-like," we constructed an independent gene expression signature and found that patients with a higher "FL-like" gene expression score had longer progression-free survival (PFS). Cell of origin was not associated with response or PFS, but double-hit gene expression was associated with shorter PFS. The day 11 posttreatment samples (RNA-seq, N = 73; mIF, N = 53) had higher levels of chimeric antigen receptor (CAR) T-cell densities and CAR gene expression, general immune infiltration, and immune activation in patients with CR. Further, the majority of T cells in the day 11 samples were endogenous. Gene expression signatures in liso-cel-treated patients with LBCL can inform the development of combination therapies and next-generation CAR T-cell therapies.

Transcriptional profiling by sequencing of oropharyngeal cancer.

OBJECTIVE: To compare full transcriptome expression levels of matched tumor and normal samples from patients with oropharyngeal carcinoma stratified by known tumor etiologic factors. PATIENTS AND METHODS: Full transcriptome sequencing was analyzed for 10 matched tumor and normal tissue samples from patients with previously untreated oropharyngeal carcinoma. Transcriptomes were analyzed using massively parallel messenger RNA sequencing and validated using the NanoString nCounter system. Global gene expression levels were compared in samples grouped by smoking status and human papillomavirus status. This study was completed between June 10, 2010, and June 30, 2011. RESULTS: Global gene expression analysis indicated tumor tissue from former smokers grouped more closely to the never smokers than the current smokers. Pathway analysis revealed alterations in the expression of genes involved in the p53 DNA damage-repair pathway, including CHEK2 and ATR, which display patterns of increased expression that is associated with human papillomavirus-negative current smokers rather than former or never smokers. CONCLUSION: These findings support the application of messenger RNA sequencing technology as an important clinical tool for more accurately stratifying patients based on individual tumor biology with the goal of improving our understanding of tumor prognosis and treatment response, ultimately leading to individualized patient care strategies.

Transcriptome analysis of the medulla tissue from cattle in response to bovine spongiform encephalopathy using digital gene expression tag profiling.

Bovine spongiform encephalopathy (BSE) is a transmissible, fatal neurodegenerative disorder of cattle produced by prions. The use of excessive parallel sequencing for comparison of gene expression in bovine control and infected tissues may help to elucidate the molecular mechanisms associated with this disease. In this study, tag profiling Solexa sequencing was used for transcriptome analysis of bovine brain tissues. Replicate libraries were prepared from mRNA isolated from control and infected (challenged with 100 g of BSE-infected brain) medulla tissues 45 mo after infection. For each library, 5-6 million sequence reads were generated and approximately 67-70% of the reads were mapped against the Bovine Genome database to approximately 13,700-14,120 transcripts (each having at least one read). About 42-47% of the total reads mapped uniquely. Using the GeneSifter software package, 190 differentially expressed (DE) genes were identified (>2.0-fold change, p < .01): 73 upregulated and 117 downregulated. Seventy-nine DE genes had functions described in the Gene Ontology (GO) database and 16 DE genes were involved in 38 different pathways described in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Digital analysis expression by tag profiling may be a powerful approach to comprehensive transcriptome analysis to identify changes associated with disease progression, leading to a better understanding of the underlying mechanism of pathogenesis of BSE.

Standardizing the next generation of bioinformatics software development with BioHDF (HDF5).

Next Generation Sequencing technologies are limited by the lack of standard bioinformatics infrastructures that can reduce data storage, increase data processing performance, and integrate diverse information. HDF technologies address these requirements and have a long history of use in data-intensive science communities. They include general data file formats, libraries, and tools for working with the data. Compared to emerging standards, such as the SAM/BAM formats, HDF5-based systems demonstrate significantly better scalability, can support multiple indexes, store multiple data types, and are self-describing. For these reasons, HDF5 and its BioHDF extension are well suited for implementing data models to support the next generation of bioinformatics applications.

Analyzing gene expression data from microarray and next-generation dna sequencing transcriptome profiling assays using GeneSifter analysis edition.

Transcription profiling with microarrays has become a standard procedure for comparing the levels of gene expression between pairs of samples, or multiple samples following different experimental treatments. New technologies, collectively known as next-generation DNA sequencing methods, are also starting to be used for transcriptome analysis. These technologies, with their low background, large capacity for data collection, and dynamic range, provide a powerful and complementary tool to the assays that formerly relied on microarrays. In this chapter, we describe two protocols for working with microarray data from pairs of samples and samples treated with multiple conditions, and discuss alternative protocols for carrying out similar analyses with next-generation DNA sequencing data from two different instrument platforms (Illumina GA and Applied Biosystems SOLiD).

Transcriptional profiling of human cavernosal endothelial cells reveals distinctive cell adhesion phenotype and role for claudin 11 in vascular barrier function.

To determine specific molecular features of endothelial cells (ECs) relevant to the physiological process of penile erection we compared gene expression of human EC derived from corpus cavernosum of men with and without erectile dysfunction (HCCEC) to coronary artery (HCAEC) and umbilical vein (HUVEC) using Affymetrix GeneChip microarrays and GeneSifter software. Genes differentially expressed across samples were partitioned around medoids to identify genes with highest expression in HCCEC. A total of 190 genes/transcripts were highly expressed only in HCCEC. Gene Ontology classification indicated cavernosal enrichment in genes related to cell adhesion, extracellular matrix, pattern specification and organogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed high expression of genes relating to ECM-receptor interaction, focal adhesions, and cytokine-cytokine receptor interaction. Real-time PCR confirmed expression differences in cadherins 2 and 11, claudin 11 (CLDN11), desmoplakin, and versican. CLDN11, a component of tight junctions not previously described in ECs, was highly expressed only in HCCEC and its knockdown by siRNA significantly reduced transendothelial electrical resistance in HCCEC. Overall, cavernosal ECs exhibited a transcriptional profile encoding matrix and adhesion proteins that regulate structural and functional characteristics of blood vessels. Contribution of the tight junction protein CLDN11 to barrier function in endothelial cells is novel and may reflect hemodynamic requirements of the corpus cavernosum.

The microarray data analysis process: from raw data to biological significance.

Despite advances in microarray technology that have led to increased reproducibility and substantial reductions in the cost of microarrays, the successful use of this technology is still elusive for many researchers, and microarray data analysis in particular presents a substantial bottleneck for many biomedical researchers. There are many reasons for this, including the expense of and a lack of adequate training in the use of analysis software. An additional reason is that microarray data analysis has largely been treated in the past as a set of separate steps, with the majority of emphasis being placed on statistical analysis and visualization of the data. For many biomedical researchers determining the biological significance of the data has been the greatest challenge and in the last several years more emphasis has been placed on this aspect of the analysis process. Despite this broadening of the scope of analysis there are still several aspects of the process that continue to be neglected, including additional related and interdependent aspects, such as experimental design, data accessibility, and platform selection. Though not traditionally thought of as integral to the data analysis process, these factors have profound effects on the analysis process. This article will discuss the importance of these additional aspects, as well as statistical analysis and determination of biological significance of microarray data. A summary of currently available software options will also be presented with a focus on the aspects discussed.

Dendritic-cell-associated C-type lectin 2 (DCAL-2) alters dendritic-cell maturation and cytokine production.

Dendritic-cell (DC)-associated C-type lectin receptors (CLRs) take up antigens to present to T cells and regulate DC functions. DCAL-2 is a CLR with a cytosolic immunoreceptor tyrosine-based inhibitory motif (ITIM), which is restricted to immature DCs (iDCs), monocytes, and CD1a+ DCs. Cross-linking DCAL-2 on iDCs induced protein tyrosine phosphorylation and MAPK activation as well as receptor internalization. To test if DCAL-2 is involved in DC maturation and cytokine expression, we stimulated iDCs with anti-DCAL-2 mAb with or without LPS, zymosan, or CD40L. While anti-DCAL-2 did not induce iDCs to mature, it did up-regulate CCR7 expression and IL-6 and IL-10 production. DCAL-2 signals augmented DC maturation induced by LPS or zymosan, increasing both CCR7 and DC-LAMP expression. Of interest, DCAL-2 ligation had the opposite effects on TLR versus CD40L signaling: anti-DCAL-2 suppressed TLR-induced IL-12 expression, but significantly enhanced CD40L-induced IL-12 production. DCAL-2 ligation also suppressed the ability of TLR-matured DCs to induce IFN-gamma-secreting Th1 cells but augmented the capacity of CD40L-matured DCs to polarize naive T cells into Th1 cells. Thus, DCAL-2 may program DCs differently depending on whether DCs are signaled via TLRs or by T cells. DCAL-2 may be a potential immunotherapeutic target for modulating autoimmune diseases or for developing vaccines.

Caspase activity is required for stimulated B lymphocytes to enter the cell cycle.

Following activation with proliferative stimuli, including ligation of CD40, dense human tonsillar B cells (>98% cells in G(0)) have increased cleavage and activation of caspase-8 and -6 accompanied by decreased caspase-3 activation and apoptosis. Proliferation was blocked by either a broad specificity caspase inhibitor or inhibitors selective for caspase-6 or caspase-8. In contrast, an inhibitor selective for caspase-3 was without effect. Furthermore, induction of cyclin D and cyclin-dependent kinase 4 mRNA and protein was blocked upon inhibition of caspase-6, but not caspase-3. Thus, caspase-6-like activity is required for quiescent B cells to increase the expression of genes required for entry into G(1). In support of this model, the transcriptional suppressor special AT-rich sequence-binding protein 1, a preferred caspase-6 substrate, was cleaved upon B cell stimulation. Caspase activity was not required for all signaling events, as caspase inhibitors did not affect the phosphorylation of p42/44 mitogen-activated protein kinase, the expression of the survival factor cellular inhibitor of apoptosis 2, or the production of IL-6 by stimulated G(0) B cells. These findings suggest a mechanism by which caspase-6 may selectively allow entry of quiescent B cells into the cell cycle.

Dendritic cell-associated lectin-1: a novel dendritic cell-associated, C-type lectin-like molecule enhances T cell secretion of IL-4.

We have characterized dendritic cell (DC)-associated lectin-1 (DCAL-1), a novel, type II, transmembrane, C-type lectin-like protein. DCAL-1 has restricted expression in hemopoietic cells, in particular, DCs and B cells, but T cells and monocytes do not express it. The DCAL-1 locus is within a cluster of C-type lectin-like loci on human chromosome 12p12-13 just 3' to the CD69 locus. The consensus sequence of the DCAL-1 gene was confirmed by RACE-PCR; however, based on sequence alignment with genomic DNA and with various human expressed sequence tags, we predict that DCAL-1 has two splice variants. C-type lectins share a common sequence motif of 14 invariable and 18 highly conserved aa residues known as the carbohydrate recognition domain. DCAL-1, however, is missing three of the cysteine residues required to form the standard carbohydrate recognition domain. DCAL-1 mRNA and protein expression are increased upon the differentiation of monocytes to CD1a(+) DCs. B cells also express high levels of DCAL-1 on their cell surface. Using a DCAL-1 fusion protein we identified a population of CD4(+) CD45RA(+) T cells that express DCAL-1 ligand. Coincubation with soluble DCAL-1 enhanced the proliferation of CD4(+) T cells in response to CD3 ligation and significantly increased IL-4 secretion. In contrast, coincubation with soluble DC-specific ICAM-3-grabbing nonintegrin (CD209) fusion protein as a control had no effect on CD4(+) T cell proliferation or IL-4 and IFN-gamma secretion. Therefore, the function of DCAL-1 on DCs and B cells may act as a T cell costimulatory molecule, which skews CD4(+) T cells toward a Th2 response by enhancing their secretion of IL-4.