Supplementary Figure 1 from Genetic Polymorphisms in Vitamin D Receptor <i>VDR/RXRA</i> Influence the Likelihood of Colon Adenoma Recurrence

The presence of B cells in early stage non-small cell lung cancer (NSCLC) is associated with longer survival, however, the role these cells play in the generation and maintenance of anti-tumor immunity is unclear. B cells differentiate into a variety of subsets with differing characteristics and functions. To date, there is limited information on the specific B cell subsets found within NSCLC. To better understand the composition of the B cell populations found in NSCLC we have begun characterizing B cells in lung tumors and have detected a population of B cells that are CD79A+CD27−IgD−. These CD27−IgD− (double-negative) B cells have previously been characterized as unconventional memory B cells and have been detected in some autoimmune diseases and in the elderly population but have not been detected previously in tumor tissue.

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell-cell interaction.

Abstract Recent studies in human lung squamous cell carcinoma (SqCC) have shown that upregulation of B cell-associated genes strongly correlate with patient survival. More than any genetic factor (immunologic or otherwise) CD79A, a pan-B cell marker, was found to have the strongest predictive value. Expression of this marker was capable of identifying short (< 2 years) and long (> 6 years) term lung SqCC survivors, with greater than 80% accuracy. These data suggest that the presence of CD79A-expressing tumor-infiltrating B (TIL-B) cells may be contributing to patient outcome. The aim of this study was to characterize CD79A+ TIL-B cells in SqCC patient samples and evaluate their role in lung SqCC survival. We evaluated human lung cancer specimens from long and short term survivors for the presence of CD79A+ TIL-B cells by immunofluorescence and found that their presence directly correlates with survival time. Further analysis showed that CD79A expression found in tumors of long term survivors (>6 years) co-localizes with CD20 staining but not CD138 staining, indicating that the cells responsible for producing a survival signature are not plasma cells. These data indicate that CD79A+ TIL-B cells are contributing to the generation of an efficient immune response in lung SqCC and may be doing so through non-humoral functions. Citation Format: Sara Centuori, Samuel Kim, Cecil Gomes, Charles Putnam, David Mount, Linda Garland, Jesse Martinez. Tumor-infiltrating B cells are predictive of human lung squamous cell survival. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1299. doi:10.1158/1538-7445.AM2015-1299

Pearson correlation coefficient for expression analysis of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) demonstrated Aurora A and B are highly correlated with MYC in DLBCL and mantle cell lymphoma (MCL), while both Auroras correlate with BCL2 only in DLBCL. Auroras are up-regulated by MYC dysregulation with associated aneuploidy and resistance to microtubule targeted agents such as vincristine. Myc and Bcl2 are differentially expressed in U-2932, TMD-8, OCI-Ly10 and Granta-519, but only U-2932 cells over-express mutated p53. Alisertib [MLN8237 or M], a highly selective small molecule inhibitor of Aurora A kinase, was synergistic with vincristine [VCR] and rituximab [R] for inhibition of cell proliferation, abrogation of cell cycle checkpoints and enhanced apoptosis versus single agent or doublet therapy. A DLBCL (U-2932) mouse model showed tumor growth inhibition (TGI) of ∼ 10-20% (p = 0.001) for M, VCR and M-VCR respectively, while R alone showed ∼ 50% TGI (p = 0.001). M-R and VCR-R led to tumor regression [TR], but relapsed 10 days after discontinuing therapy. In contrast, M-VCR-R demonstrated TR with no relapse >40 days after stopping therapy with a Kaplan-Meier survival of 100%. Genes that are modulated by M-VCR-R (CENP-C, Auroras) play a role in centromere-kinetochore function in an attempt to maintain mitosis in the presence of synthetic lethality. Together, our data suggest that the interaction between alisertib plus VCR plus rituximab is synergistic and synthetic lethal in Myc and Bcl-2 co-expressing DLBCL. Alisertib plus vincristine plus rituximab [M-VCR-R] may represent a new strategy for DLBCL therapy.

Background: Numerous microarray based prognostic gene expression signatures of primary neoplasms have been published but often with little concurrence between studies, thus limiting their clinical utility. We describe a methodology using logistic regression, which circumvents limitations of conventional Kaplan Meier analysis. We applied this approach to a thrice-analyzed and published squamous cell carcinoma (SQCC) of the lung data set, with the objective of identifying gene expressions predictive of early death versus long survival in early-stage disease. A similar analysis was applied to a data set of triple negative breast carcinoma cases, which present similar clinical challenges.Methods: Important to our approach is the selection of homogenous patient groups for comparison. In the lung study, we selected two groups (including only stages I and II), equal in size, of earliest deaths and longest survivors. Genes varying at least four-fold were tested by logistic regression for accuracy of prediction (area under a ROC plot). The gene list was refined by applying two sliding-window analyses and by validations using a leave-one-out approach and model building with validation subsets. In the breast study, a similar logistic regression analysis was used after selecting appropriate cases for comparison.Results: A total of 8594 variable genes were tested for accuracy in predicting earliest deaths versus longest survivors in SQCC. After applying the two sliding window and the leave-one-out analyses, 24 prognostic genes were identified; most of them were B-cell related. When the same data set of stage I and II cases was analyzed using a conventional Kaplan Meier (KM) approach, we identified fewer immune-related genes among the most statistically significant hits; when stage III cases were included, most of the prognostic genes were missed. Interestingly, logistic regression analysis of the breast cancer data set identified many immune-related genes predictive of clinical outcome.Conclusions: Stratification of cases based on clinical data, careful selection of two groups for comparison, and the application of logistic regression analysis substantially improved predictive accuracy in comparison to conventional KM approaches. B cell-related genes dominated the list of prognostic genes in early stage SQCC of the lung and triple negative breast cancer.

The pathogenesis and drug resistance of symptomatic CLL patients involves genetic changes associated with the CLL clone as well as changes within the microenvironment. To further understand these processes, we compared early stage CLL to symptomatic late stage using gene expression and serum cytokine profiling to gain insight of the genetic and microenvironment changes associated with the most severe form of the disease. Patients were classified into low stage (Rai stage 0/I/II) and high stage (Rai stage III/IV). Gene expression profiles were obtained on pretreatment samples using the HG-U133A 2.0 Affymetrix platform. A comparison of low versus high stage CLL revealed a set of 21 genes differentially expressed genes. 15 genes were up regulated in the high stage compared to low stage while 6 genes were down regulated. Analysis of GO molecular function revealed 9 of 21 genes were involved in transcription factor activity. Serum cytokine profiles showed six cytokines to be significantly different in high stage patients. Two chemokines, SDF-1/CXCL12 and uPAR known to be involved in stem cell mobilization and homing were increased in serum of high stage patients. This study has identified therapeutic targets for symptomatic CLL patients.

Vascular endothelial growth factorB(VEGF-B) has recently been shown to be a promising novel neuroprotective agent for several neurodegenerative conditions. In the current study we extended previous work on neuroprotective potential for Parkinson's disease (PD) by testing an expanded dose range of VEGF-B (1 and 10 lg) and directly comparing both neuroprotective and neurorestorative effects of VEGFB in progressive unilateral 6-hydroxydopamine (6-OHDA) PD models to a single dose of glial cell line-derived neurotrophic factor (GDNF, 10 lg), that has been established by several groups as a standard in both preclinical PD models. In the amphetamine-induced rotational tests the treatment with 1 and 10 lg VEGF-B resulted in significantly improved motor function of 6-OHDA-lesioned rats compared to vehicle-treated 6-OHDA-lesioned rats in the neuroprotection paradigm. Both doses of VEGF-B caused an increase in tyrosine hydroxylase (TH)-positive cell and fiber count in the substantia nigra (SN) and striatum in the neuroprotective experiment. The effect size was comparable to the effects seen with GDNF. In the neurorestoration paradigm, VEGF-B injection had no significant effect in either the behavioral or the immunohistochemical analyses, whereas GDNF injection significantly improved the amphetamine-induced rotational behavior and reduced TH-positive neuronal cell loss in the SN. We also present a strong positive correlation (p = 1.9e-50) of the expression of VEGF-B with nuclearencoded mitochondrial genes involved in fatty acid metabolism in rat midbrain, pointing to the mitochondria as a site of action of VEGF-B. GDNF showed a positive correlation with nuclear-encoded mitochondrial genes that was not nearly as strong (p = 0.018). VEGF-B counteracted rotenoneinduced reduction of (a) fatty acid transport protein 1 and 4 levels and (b) both Akt protein and phosphorylation levels in SH-SY5Y cells. We further verified VEGF-B expression in the human SN pars compacta of healthy controls and PD patients, in neuronal cells that show co-expression with neuromelanin. These results have demonstrated that VEGF-B has potential as a neuroprotective agent for PD therapy and should be further investigated. (C) 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Background: Inflammatory breast cancer (IBC) is a rare, highly aggressive form of breast cancer.The mechanism of IBC carcinogenesis remains unknown.We sought to evaluate potential genetic risk factors for IBC and whether or not the IBC cell lines SUM149 and SUM190 demonstrated evidence of viral infection.Methods: We performed single nucleotide polymorphism (SNP) genotyping for 2 variants of the ribonuclease (RNase) L gene that have been correlated with the risk of prostate cancer due to a possible viral etiology.We evaluated dose-response to treatment with interferon-alpha (IFN-α); and assayed for evidence of the putative human mammary tumor virus (HMTV, which has been implicated in IBC) in SUM149 cells.A bioinformatic analysis was performed to evaluate expression of RNase L in IBC and non-IBC.Results: 2 of 2 IBC cell lines were homozygous for RNase L common missense variants 462 and 541; whereas 2 of 10 non-IBC cell lines were homozygous positive for the 462 variant (p= 0.09) and 0 of 10 non-IBC cell lines were homozygous positive for the 541 variant (p = 0.015).Our real-time polymerase chain reaction (RT-PCR) and Southern blot analysis for sequences of HMTV revealed no evidence of the putative viral genome. Conclusion:We discovered 2 SNPs in the RNase L gene that were homozygously present in IBC cell lines.The 462 variant was absent in non-IBC lines.Our discovery of these SNPs present in IBC cell lines suggests a possible biomarker for risk of IBC.We found no evidence of HMTV in SUM149 cells.A query of a panel of human IBC and non-IBC samples showed no difference in RNase L expression.Further studies of the RNase L 462 and 541 variants in IBC tissues are warranted to validate our in vitro findings.

Background. Adrenocortical carcinoma (ACC) is associated with poor survival rates. The objective of the study was to analyze ACC gene expression profiling data for prognostic biomarkers and therapeutic targets.Methods. We profiled 44 ACC and 4 normal adrenals on Affymetrix U133 Plus 2 expression microarrays. Pathway and transcriptional enrichment analysis was performed. Protein levels were determined by Western blot. Drug efficacy was assessed against ACC cell lines. Previously published expression datasets were analyzed for validation.Results. Pathway enrichment analysis identified marked dysregulation of cyclin-dependent kinases and mitosis. Overexpression of PTTG1, which encodes securin, a negative regulator of p53, was identified as a marker of poor survival. Median survival for patients with tumors expressing high PTTG1 levels (log2 ratio of PTTG1 to average beta-actin <-3.04) was 1.8 years compared with 9.0 years if tumors expressed lower levels of PTTG1 (P < .0001). Analysis of a previously published dataset confirmed the association of high PTTG1 expression with a poor prognosis. Treatment of 2 ACC cell lines with vorinostat decreased securin levels and inhibited cell growth (median inhibition concentrations of 1.69 mu mol/L and 0.891 mu mol/L, for SW-13 and H295R, respectively).Conclusion. Overexpression of PTTG1 is correlated with poor survival in ACC. PTTG1/securin is a prognostic biomarker and warrants investigation as a therapeutic target.

Background: Traditional drug discovery methods have a limited role in rare cancers. We hypothesized that molecular technology including gene expression profiling could expose novel targets for therapy in adrenocortical carcinoma (ACC), a rare and lethal cancer. SPARC (secreted protein acidic rich in cysteine) is an albumin-binding matrix-associated protein that is proposed to act as a mechanism for the increased efficacy of a nanoparticle albumin-bound preparation of the antimicrotubular drug Paclitaxel (nab-paclitaxel).Methods: The transcriptomes of 19 ACC tumors and 4 normal adrenal glands were profiled on Affymetrix U133 Plus2 expression microarrays to identify genes representing potential therapeutic targets. Immunohistochemical analysis for target proteins was performed on 10 ACC, 6 benign adenomas, and 1 normal adrenal gland. Agents known to inhibit selected targets were tested in comparison with mitotane in the 2 ACC cell lines (H295R and SW-13) in vitro and in mouse xenografts.Results: SPARC expression is increased in ACC samples by 1.56 +/- 0.44 (mu +/- SD) fold. Paclitaxel and nab-paclitaxel show in vitro inhibition of H295R and SW-13 cells at IC50 concentrations of 0.33 mu M and 0.0078 mu M for paclitaxel and 0.35 mu M and 0.0087 mu M for nab-paclitaxel compared with mitotane concentrations of 15.9 mu M and 46.4 mu M. In vivo nab-paclitaxel treatment shows a greater decrease in tumor weight in both xenograft models than mitotane.Conclusions: Biological insights garnered through expression profiling of ACC tumors suggest further investigation into the use of nab-paclitaxel for the treatment of ACC.

The complex, partially redundant gene regulatory architecture underlying vertebrate heart formation has been difficult to characterize. Here, we dissect the primary cardiac gene regulatory network in the invertebrate chordate, Ciona intestinalis. The Ciona heart progenitor lineage is first specified by Fibroblast Growth Factor/Map Kinase (FGF/MapK) activation of the transcription factor Ets1/2 (Ets). Through microarray analysis of sorted heart progenitor cells, we identified the complete set of primary genes upregulated by FGF/Ets shortly after heart progenitor emergence. Combinatorial sequence analysis of these co-regulated genes generated a hypothetical regulatory code consisting of Ets binding sites associated with a specific co-motif, ATTA. Through extensive reporter analysis, we confirmed the functional importance of the ATTA co-motif in primary heart progenitor gene regulation. We then used the Ets/ATTA combination motif to successfully predict a number of additional heart progenitor gene regulatory elements, including an intronic element driving expression of the core conserved cardiac transcription factor, GATAa. This work significantly advances our understanding of the Ciona heart gene network. Furthermore, this work has begun to elucidate the precise regulatory architecture underlying the conserved, primary role of FGF/Ets in chordate heart lineage specification.

Abstract Low circulating levels of vitamin D affect colorectal cancer risk. The biological actions of the hormonal form of vitamin D, 1,25(OH)2D3, are mediated by the vitamin D receptor (VDR), which heterodimerizes with retinoid X receptors (RXR). Using a single nucleotide polymorphism (SNP) tagging approach, we assessed the association between genetic variations in RXRA and VDR and odds of recurrent (metachronous) colorectal neoplasia in a pooled population of two studies. A total of 32 tag SNPs in RXRA and 42 in VDR were analyzed in 1,439 participants. A gene-level association was observed for RXRA and any (P = 0.04) or proximal (P = 0.03) metachronous neoplasia. No gene-level associations were observed for VDR, nor was any single SNP in VDR related to any metachronous adenoma after correction for multiple comparisons. In contrast, the association between RXRA SNP rs7861779 and proximal metachronous neoplasia was of borderline statistical significance [odds ratio (OR), 0.68; 95% confidence interval (95% CI), 0.53-0.86; unadjusted P = 0.001; adjusted P = 0.06], including when observed independently in each individual study. Haplotypes within linkage blocks of RXRA support an ∼30% reduction in odds of metachronous neoplasia arising in the proximal colon among carriers of specific haplotypes, which was strongest (ORproximal, 0.67; 95% CI, 0.52–0.86) for carriers of a CGGGCA haplotype (rs1805352, rs3132297, rs3132296, rs3118529, rs3118536, and rs7861779). Our results indicate that allelic variation in RXRA affects metachronous colorectal neoplasia, perhaps of particular importance in the development of proximal lesions. Cancer Res; 70(4); 1496–504

INTRODUCTIONA hidden Markov model (HMM) is a probabilistic model of a multiple sequence alignment (msa) of proteins. In the model, each column of symbols in the alignment is represented by a frequency distribution of the symbols (called a “state”), and insertions and deletions are represented by other states. One moves through the model along a particular path from state to state in a Markov chain (i.e., random choice of next move), trying to match a given sequence. The next matching symbol is chosen from each state, recording its probability (frequency) and also the probability of going to that state from a previous one (the transition probability). State and transition probabilities are multiplied to obtain a probability of the given sequence. The hidden nature of the HMM is due to the lack of information about the value of a specific state, which is instead represented by a probability distribution over all possible values. This article discusses the advantages and disadvantages of HMMs in msa and presents algorithms for calculating an HMM and the conditions for producing the best HMM.

It is difficult to find a global optimal alignment of more than two sequences (and, especially, more than three) that includes matches, mismatches, and gaps and that takes into account the degree of variation in all of the sequences at the same time. Thus, approximate methods are used, such as progressive global alignment, iterative global alignment, alignments based on locally conserved patterns found in the same order in the sequences, statistical methods that generate probabilistic models of the sequences, and multiple sequence alignments produced by graph-based methods. When 10 or more sequences are being compared, it is common to begin by determining sequence similarities between all pairs of sequences in the set. A variety of methods are then available to cluster the sequences into the most related groups or into a phylogenetic tree. This article discusses several of these methods and provides data that compare their utility under various conditions.

Sequence alignment editors enable the user to manually edit a multiple sequence alignment (msa) in order to obtain a more reasonable or expected alignment. Editors allow sequences to be reordered and/or modified using the computer's cut and paste commands. They are designed to accept various msa formats and to provide the output file in a suitable user-designated format. Sequence formatters provide various output formatting options, such as color and shading schemes to enhance visualization of residue alignments. The formatters can output files in Postscript, EPS, RTF, and other widely recognized formats, while accepting the standard input formats, such as MSF, ALN, and FASTA. This article introduces a number of sequence alignment editors and formatters, and provides links to sites where they can be found.

INTRODUCTION Maximum likelihood (ML) methods are especially useful for phylogenetic prediction when there is considerable variation among the sequences in the multiple sequence alignment (msa) to be analyzed. ML methods start with a simple model, in this case a model of rates of evolutionary change in nucleic acid or protein sequences and tree models that represent a pattern of evolutionary change, and then adjust the model until there is a best fit to the observed data. For phylogenetic analysis, the observed data are the observed sequence variations found within the columns of an msa. The ML method is similar to the maximum parsimony method in that the analysis is performed on each column of an msa.

INTRODUCTIONThe original Dayhoff percent accepted mutation (PAM) matrices were developed based on a small number of protein sequences and an evolutionary model of protein change. By extrapolating from the observed changes at small evolutionary distances to large ones, it was possible to establish a PAM250 scoring matrix for sequences that were highly divergent. Another approach to finding a scoring matrix for divergent sequences is to start with a more divergent set of sequences and produce a scoring matrix from the substitutions found in those less-related sequences. The blocks amino acid substitution matrices (BLOSUM) scoring matrices were prepared this way. This article explains how BLOSUM scoring matrices were created and how they can best be used.