Sandra D. Bohling

Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkins lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38β-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.

Immunosuppressive regulatory T cells are associated with aggressive breast cancer phenotypes: a potential therapeutic target.

FoxP3 is a marker for immunosuppressive CD25+CD4+ regulatory T cells. These regulatory T cells are thought to play a role in inducing immune tolerance to antigens and may be selectively recruited by carcinomas. We investigated whether breast carcinomas had significant numbers of FoxP3-positive regulatory T cells by immunohistochemistry, and if their presence was associated with other prognostic factors, such as Nottingham grade, hormone receptor immunohistochemical profile, tumor size, or lymph node metastases. Ninety-seven needle core or excisional breast biopsies with invasive breast carcinoma diagnosed at the University of Washington were stained with antibodies to FoxP3, estrogen receptor, and Her2/neu. The numbers of FoxP3-positive cells present within the neoplastic epithelium, and immediately adjacent stroma were counted manually in three high-powered fields (HPFs; × 400) by two independent pathologists. The average scores were then correlated with the parameters of interest. A threshold of ≥15 FoxP3-positive cells/HPF was used to define a FoxP3-positive case in some analyses. Higher average numbers of FoxP3-positive cells present significantly correlated with higher Nottingham grade status (P=0.000229). In addition, the presence of significant numbers (≥15/HPF) of FoxP3-positive cells in breast carcinoma was positively associated with higher Nottingham grade (P=0.00002585). Higher average numbers of FoxP3-positive cells were also significantly associated with larger tumor size (>2.0 cm; P=0.012824) and trended toward an association with estrogen receptor negativity. Interestingly, ‘triple-negative’ (estrogen and progesterone receptor negative and Her2/neu negative) Nottingham grade III cases were also significantly associated with high numbers of FoxP3 cells. These results argue that regulatory T cells may play a role in inducing immune tolerance to higher grade, more aggressive breast carcinomas, and are a potential therapeutic target for these cancers.

PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number

Polymerase chain reaction (PCR)-based analysis for detecting immunoglobulin heavy chain gene (IgH) rearrangements in lymphoproliferative disorders is well established. The presence of one or two discrete bands is interpreted as a monoclonal proliferation, whereas a smear pattern represents a polyclonal population. Prompted by our observation of discrete bands in histologically reactive processes with a relative paucity of B cells, we sought to determine whether low numbers of B cells in biopsy specimens could artifactually produce pseudomonoclonal bands. We performed IgH PCR analysis on serially diluted DNA samples from 5 B cell non-Hodgkins lymphomas (B-NHLs), 5 reactive lymph nodes, 5 reactive tonsils and 10 microdissected germinal centers from a lymph node with follicular hyperplasia. We also assessed multiple aliquots of DNA samples from small biopsy specimens of reactive lymphocytic processes from the stomach (5 cases). PCR products were evaluated using high resolution agarose or polyacrylamide gels, and DNA sequencing was performed on IgH PCR products from two reactive germinal centers, which yielded monoclonal bands of identical size. All 5 B-NHLs harboring monoclonal B cell populations yielded single discrete bands, which were maintained in all dilutions. By contrast, all of the reactive lesions with polyclonal patterns at 50 ng/microl starting template concentration showed strong pseudomonoclonal bands at dilutions of 1:1,000 to 1:1,500 in placental DNA. Two of the microdissected reactive germinal centers that showed bands of identical size on duplicate reactions were proven to have different IgH sequences by sequencing. We conclude that specimens containing low numbers of polyclonal B cells may produce pseudomonoclonal bands on IgH PCR analysis. IgH PCR analysis should be performed on multiple aliquots of each DNA sample, and only samples that yield reproducible bands of identical size can be reliably interpreted as monoclonal.

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

Background: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. Methods: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). Results: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. Conclusions: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

Rapid Simultaneous Amplification and Detection of the MBR/JH Chromosomal Translocation by Fluorescence Melting Curve Analysis

Polymerase chain reaction (PCR) amplification and product analysis for the detection of chromosomal translocations, such as the t(14;18), has traditionally been a two-step process. PCR product detection has generally entailed gel electrophoresis and/or hybridization or sequencing for confirmation of assay specificity. Using a microvolume fluorimeter integrated with a thermal cycler and a PCR-compatible double-stranded DNA (dsDNA) binding fluorescent dye (SYBR Green I), we investigated the feasibility of simultaneous thermal amplification and detection of MBR/JH translocation products by fluorescence melting curve analysis. We analyzed DNA from 30 cases of lymphoproliferative disorders comprising 19 cases of previously documented MBR/JH-positive follicle center lymphoma and 11 reactive lymphadenopathies. The samples were coded and analyzed blindly for the presence of MBR/JH translocations by fluorescence melting curve analysis. We also performed dilutional assays using the MBR/JH-positive cell line SUDHL-6. Multiplex PCR for MBR/JH and beta-globin was used to simultaneously assess sample adequacy. All (100%) of the 19 cases previously determined to be MBR/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at approximately 90 degrees C by melting curve analysis after amplification. Fluorescence melting peaks obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T) showed melting temperatures (Tm) at 88.85+/-1.15 degrees C. In addition, multiplex assays using both MBR/JH and beta-globin primers yielded easily distinguishable fluorescence melting peaks at approximately 90 degrees C and 81.2 degrees C, respectively. Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 100-fold. Simultaneous amplification and fluorescence melting curve analysis is a simple, reliable, and sensitive method for the detection of MBR/JH translocations. The feasibility of specific PCR product detection without electrophoresis or utilization of expensive fluorescently labeled probes makes this method attractive for routine molecular diagnostics.

Microarray Analysis of B-Cell Lymphoma Cell Lines with the t(14;18)

The t(14;18) is the most common genetic alteration in follicular lymphoma, and is detectable in a subset of diffuse large B-cell lymphomas (DLBCL), resulting in over-expression of the anti-apoptotic protein BCL-2. Although the t(14;18)-induced over-expression of BCL-2 is an important step in lymphomagenesis, this aberration alone is not sufficient to produce malignant lymphoma. Further analysis of these tumors is needed to identify additional genes that might be involved in the genesis of follicular lymphoma and progression to DLBCL. To address this issue, we analyzed the gene expression profiles of four t(14;18)-positive cell lines and two t(11;14)-positive mantle-cell lymphoma cell lines using cDNA microarrays containing 4364 genes, and compared them to the genetic profile of phenotypically purified B-cells obtained from hyperplastic tonsils. A total of 137 genes were differentially expressed by approximately twofold or more in the t(14;18) cell lines relative to tonsillar B-cells. 68 genes were up-regulated, 69 genes were down-regulated, and approximately 20% of the differentially regulated genes had no known function. The up-regulated genes included a number of genes involved in the promotion of cellular proliferation and survival, as well as cell metabolism. Down-regulated genes included mediators of cell adhesion and negative regulators of cell activation and growth. Hierarchical clustering analysis separated the t(14;18) and mantle-cell lines into distinct groups based on their gene expression profiles. We confirmed the differential expression of approximately 80% of selected up- and down-regulated genes identified by microarray analysis by quantitative real-time fluorescence reverse transcriptase polymerase chain reaction (RT-PCR) analysis and/or immunoblotting. This study demonstrates the utility of cDNA microarray analysis for the assessment of global transcriptional changes that characterize t(14;18)-positive cell lines, and also for the identification of novel genes that could potentially contribute to the genesis and progression of non-Hodgkins lymphomas with this translocation.

Fluorescence PCR quantification of cyclin D1 expression.

We have used a continuous fluorescence monitoring method to assess cyclin D1 mRNA expression in a variety of hematological and non-hematological processes. We examined 14 cell lines, 11 reactive lymphoid tissues, and 57 primary hematopoietic neoplasms including mantle cell lymphoma (MCL) (n = 10), chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (n = 11), acute lymphoblastic leukemia/lymphoma (n = 15), follicular lymphoma (n = 6), peripheral T-cell lymphoma (PTCL) (n = 3), anaplastic large cell lymphoma (n = 3), hairy cell leukemia (n = 3), Burkitt lymphoma (n = 1), Burkitt-like lymphoma (n = 4), and plasmacytoma (n = 1) for the expression of cyclin D1 mRNA using fluorescently labeled sequence-specific hybridization probes. Fluorescence (F) was plotted against cycle (C) number over 45 cycles. The log-linear portion of the F versus C graph identified a fractional cycle number for threshold fluorescence. A beta-globin mRNA transcript with equivalent amplification efficiency to that of cyclin D1 was used for assessment of RNA integrity and normalization. In general, the MCLs demonstrated substantially higher levels of cyclin D1 mRNA than the other lymphoproliferative processes. Moderately high levels of cyclin D1 mRNA were detected in one PTCL. On average, the CLL/SLL cases showed cyclin D1 mRNA levels two to three orders of magnitude lower than observed in the MCLs. Cell lines derived from non-hematopoietic neoplasms such as fibrosarcoma, small cell carcinoma, and neuroblastoma showed comparable or higher levels of cyclin D1 mRNA than the MCLs. Our results indicate that quantitative real-time reverse transcription (RT) polymerase chain reaction is a simple, rapid, and accurate technique for assessing cyclin D1 expression, and while it is not specific, it can reliably be used in the distinction of MCL from CLL/SLL.

Gene expression profiling of cell lines derived from T-cell malignancies.

The expression profiles of eight cell lines derived from T‐cell malignancies were compared to CD4‐positive T‐cells using cDNA microarray technology. Unsupervised hierarchical clustering of 4364 genes demonstrated substantial heterogeneity resulting in four distinct groups. While no genes were found to be uniformly up‐ or down‐regulated across all cell lines, we observed 111 over‐expressed genes (greater than two‐fold) and 1118 down‐regulated genes (greater than two‐fold) in the lymphomas as a group when compared to CD4‐positive T‐cells. These included genes involved in cytokine signaling, cell adhesion, cytoskeletal elements, nuclear transcription factors, and known oncogenes and tumor suppressor genes. Quantitative fluorescent reverse transcription‐polymerase chain reaction analysis demonstrated 70% concordance with the microarray results. While freshly isolated malignant cells may differ in their individual expression patterns relative to established cell lines from the same diagnoses, we feel that the variety of different lymphocytic cell lines that we examined provides a representative picture of the molecular pathogenesis of T‐cell malignancies.

Follicular Lymphoma with Monocytoid B-Cell Proliferation: Molecular Assessment of the Clonal Relationship between the Follicular and Monocytoid B-Cell Components

Although a number of studies have recognized that follicular lymphomas may be accompanied by a prominent proliferation of monocytoid B-cells, the clonal relationship between these components has not been adequately assessed. Using laser capture microdissection, we isolated the follicular and monocytoid B-cell components from four well-characterized cases of follicular lymphoma with prominent monocytoid B-cells. DNA from each component was analyzed using polymerase chain reaction (PCR)-based methods to assess for clonal rearrangements of the immunoglobulin heavy chain gene (IgH) and for the presence of the bcl-2 gene major breakpoint region/joining region (MBR/JH) DNA fusion products by conventional PCR and fluorescence melting curve analysis. Evidence of clonal identity was established in the follicular and monocytoid B-cell components of three cases by demonstration of IgH gene rearrangements of identical size using IgH PCR, by comparison of complementarity determining region III (CDRIII) DNA sequences, or by detection of bcl-2 MBR/JH fusion products of identical size and/or melting temperature. Molecular analysis of the fourth case revealed a monoclonal and MBR/JH-positive follicular component accompanied by a polyclonal and MBR/JH-negative monocytoid B-cell proliferation. We conclude that the follicular and monocytoid B-cell components of this variant of follicular lymphoma are clonally identical in the majority of cases. However, in a minority of these cases, the monocytoid B-cell component is reactive. Larger studies that assess the prognostic significance of follicular lymphoma with monocytoid B-cells will benefit from molecular studies that assess the clonal relationship of both components.

Solution-Based Scanning for Single-Base Alterations Using a Double-Stranded DNA Binding Dye and Fluorescence-Melting Profiles

DNA molecules differing by as little as a single-base substitution have traditionally been distinguished by gel electrophoresis-based methodologies that exploit differences in the sequence-specific properties of double-stranded DNA (dsDNA) such as melting temperature and secondary conformational configuration. By comparison, solution-based fluorescence methods using sequence-specific probes are limited to detecting mutations restricted to very short segments of DNA ( approximately 20 bp). We describe a solution-based fluorescence method that discriminates between wild-type and mutant sequences using a dsDNA binding dye, and interrogates a region of >200 nucleotides. This method is based on melting theory and entails fluorescence monitoring of the melting temperatures of GC-clamped amplicons subjected to gradual and progressive thermal denaturation in the presence of a constant concentration of urea. Heterozygous samples are easily identified by the lower melting temperatures of the less thermodynamically stable heteroduplex mismatches from the wild-type:mutant DNA hybrids as compared to the more stable wild-type Watson-Crick duplexes. All of the four possible sets of mismatches (A.G/T.C, T.G/A.C, G.G/C.C, and T.T/A.A) represented in 17 heterozygous mutations distributed throughout the length of 20 different amplicons (104 to 212 bp), were distinguished from the wild-type by their altered melting profiles. This methodology is advantageous in that it obviates gel electrophoresis or labeled oligonucleotide probes. Significantly, it expands the region of interrogation for detection of single-base changes using fluorescence-based methods in solution, and is amenable for automation and adaptation to high-throughput systems.