Sarah T. Wilkinson

Increased MYC gene copy number correlates with increased mRNA levels in diffuse large B-cell lymphoma

Background Translocations involving the MYC gene and increased MYC mRNA levels are associated with poor outcome in diffuse large B-cell lymphoma. However, the presence of increased MYC gene copy number and/or polysomy of chromosome 8 have not been previously described. Design and Methods Utilizing dual color chromogenic in situ hybridization, we investigated MYC gene copy and chromosome 8 centromere numbers in 52 cases of diffuse large B-cell lymphoma. Cases were divided into those with “increased” or “not increased” MYC gene copy number for comparison with MYC mRNA levels, Ki-67 values, and survival. Results Increased MYC gene copy number was present in 38% of cases. Overall, the average MYC mRNA level was 2398 (range, 342 – 9783) and the percentage of nuclei positive for Ki-67 was 57.5% (range, 20–87%). Within the group with increased MYC copy number, the MYC mRNA values ranged from 816 to 5912 (average, 2843) and the Ki-67 values ranged from 23% to 83% (average, 57%). Within the group with not increased MYC copy number, MYC mRNA values ranged from 342 to 9783 (average, 2118) and the Ki-67 values ranged from 20% to 87% (average, 58%). There was a statistically significant relationship between increased MYC gene copy number and increased MYC mRNA (P=0.034) and a trend toward a relationship between increased mRNA and higher Ki-67 values. Conclusions This is the first report that low level copy number increases are common in diffuse large B-cell lymphoma and that these changes correlate with MYC mRNA in a statistically significant manner. MYC copy number changes are an additional possible molecular mechanism that may result in increased mRNA and, likely, high proliferation and poor outcome.

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.

Multimedia exposures to arsenic and lead for children near an inactive mine tailings and smelter site

Children living near contaminated mining waste areas may have high exposures to metals from the environment. This study investigates whether exposure to arsenic and lead is higher in children in a community near a legacy mine and smelter site in Arizona compared to children in other parts of the United States and the relationship of that exposure to the site. Arsenic and lead were measured in residential soil, house dust, tap water, urine, and toenail samples from 70 children in 34 households up to 7 miles from the site. Soil and house dust were sieved, digested, and analyzed via ICP-MS. Tap water and urine were analyzed without digestion, while toenails were washed, digested and analyzed. Blood lead was analyzed by an independent, certified laboratory. Spearman correlation coefficients were calculated between each environmental media and urine and toenails for arsenic and lead. Geometric mean arsenic (standard deviation) concentrations for each matrix were: 22.1 (2.59) ppm and 12.4 (2.27)ppm for soil and house dust (<63μm), 5.71 (6.55)ppb for tap water, 14.0 (2.01)μg/L for specific gravity-corrected total urinary arsenic, 0.543 (3.22)ppm for toenails. Soil and vacuumed dust lead concentrations were 16.9 (2.03)ppm and 21.6 (1.90) ppm. The majority of blood lead levels were below the limit of quantification. Arsenic and lead concentrations in soil and house dust decreased with distance from the site. Concentrations in soil, house dust, tap water, along with floor dust loading were significantly associated with toenail and urinary arsenic but not lead. Mixed models showed that soil and tap water best predicted urinary arsenic. In our study, despite being present in mine tailings at similar levels, internal lead exposure was not high, but arsenic exposure was of concern, particularly from soil and tap water. Naturally occurring sources may be an additional important contributor to exposures in certain legacy mining areas.

Home Water Treatment Habits and Effectiveness in a Rural Arizona Community

Drinking water quality in the United States (US) is among the safest in the world. However, many residents, often in rural areas, rely on unregulated private wells or small municipal utilities for water needs. These utilities may violate the Safe Drinking Water Act contaminant guidelines, often because they lack the required financial resources. Residents may use alternative water sources or install a home water treatment system. Despite increased home water treatment adoption, few studies have examined their use and effectiveness in the US. Our study addresses this knowledge gap by examining home water treatment in a rural Arizona community. Water samples were analyzed for metal(loid)s, and home treatment and demographic data were recorded in 31 homes. Approximately 42% of homes treated their water. Independent of source water quality, residents with higher income (OR = 1.25; 95%CI (1.00 – 1.64)) and education levels (OR = 1.49; 95%CI (1.12 – 2.12)) were more likely to treat their water. Some contaminant concentrations were effectively reduced with treatment, while some were not. We conclude that increased educational outreach on contaminant testing and treatment, especially to rural areas with endemic water contamination, would result in a greater public health impact while reducing rural health disparities.

Superoxide dismutase is regulated by LAMMER kinase in Drosophila and human cells

LAMMER kinases (also known as CDC-2-like or CLKs) are a family of dual specificity serine/threonine protein kinases that are found in all sequenced eukaryotic genomes. In the fission yeast, Schizosaccharomyces pombe, the LAMMER kinase gene, Lkh1, positively regulates the expression of the antioxidant defense genes, superoxide dismutase 1 (sod1+, CuZn-SOD) and catalase (ctt1+, CAT). We have shown that mutations in the Drosophila LAMMER kinase gene, Darkener of apricot (Doa), protect against the decrease in life span caused by the reactive oxygen species (ROS) generator paraquat, and at the same time show an increase in cytoplasmic (CuZn-Sod or SOD1) and mitochondrial superoxide dismutase (Mn-Sod or SOD2) protein levels and activity. The siRNA-mediated knock down of the human LAMMER kinase gene, CLK-1, in HeLa and MCF-7 human cell lines leads to an increase in both SOD1 activity and mRNA transcript levels. These data suggest that SOD1 is negatively regulated by LAMMER kinases in Drosophila and human cell lines and that this regulation may be conserved during evolution.

Mitochondrial Adaptations to Oxidative Stress Confer Resistance to Apoptosis in Lymphoma Cells

Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation to chronic oxidative stress can contribute to chemoresistance seen in lymphoma patients. Oxidative stress-resistant WEHI7.2 cell variants in a lymphoma tissue culture model exhibit a range of apoptosis sensitivities. We exploited this phenotype to test for mitochondrial changes affecting sensitivity to apoptosis in cells made resistant to oxidative stress. We identified impaired release of cytochrome c, and the intermembrane proteins adenylate kinase 2 and Smac/DIABLO, indicating inhibition of the pathway leading to permeabilization of the outer mitochondrial membrane. Blunting of a glucocorticoid-induced signal and intrinsic mitochondrial resistance to cytochrome c release contributed to both points of resistance. The level of Bcl-2 family members or a difference in Bim induction were not contributing factors. The extent of cardiolipin oxidation following dexamethasone treatment, however, did correlate with apoptosis resistance. The differences found in the variants were all proportionate to the degree of resistance to glucocorticoid treatment. We conclude that tolerance to oxidative stress leads to mitochondrial changes that confer resistance to apoptosis.

Lack and/or aberrant localization of major histocompatibility class II (MHCII) protein in plasmablastic lymphoma

Plasmablastic lymphoma (PBL) was recently reclassified as a distinct entity of mature B-cell neoplasm.1,2 However, the diagnostic distinction of PBL from diffuse large B-cell lymphoma (DLBCL) is still a common problem due to the lack of biomarkers for PBL.3 Recently, Montes-Moreno et al. published a study in Hematologica describing several PBL phenotypes that help to differentiate PBL from DLBCL.4 A major characteristic of these PBL cases (full and variant plasmablastic phenotypes) which distinguished them from conventional DLBCL was PRDM1/Blimp1 positivity.4 The authors further demonstrated that the rare acquisition of a partial, Blimp1 positive, plasmablastic phenotype in a minority of DLBCL cases was associated with poorer patient outcome.4 Blimp1 and MHC class II protein expression are inversely related as normal B cells enter the terminal differentiation program towards plasma cells. Given the expression of PRDM1/Blimp1 and poor outcome of PBL, Montes-Moreno et al. hypothesized that PBL cases would lack MHC class II expression.4 In DLBCL, Rimsza et al. previously showed that downregulation of the MHC class II mRNA and protein expression correlated with low numbers of tumor infiltrating CD8+ T cells and poor patient outcome, which may be likely due to a loss of immunosurveillance. 5-7 In the current study, in a collaborative effort, we extended the work carried out by Montes-Moreno et al. by analyzing MHC class II (HLA-DR) and CD8 expression using immunohistochemistry in 22 of the 35 PBL cases from the previously published case series.4 Due to tissue limitations, 13 cases were not analyzed. The area of tumor was identified by hematoxylin and eosin (HE obvious stromal and histiocytic cells excluded).5 HLA-DR staining intensity was semi-quantitatively scored: 0 = no staining; 1+ = faint partial staining; 2+ = complete or partial moderate staining; 3+ = complete strong staining. In addition, the HLA-DR staining pattern was qualitatively characterized as cell membrane (Figure 1C) or aberrant cytoplasmic (Figure 1D) as described previously,8 or as negative. Figure 1. Hematoxylin and eosin (H&E) staining shows (A) the typical morphology of PBL. (B) CD8 staining reveals the presence of one T cell in a tumor area. (C and D) Immunohistochemical staining of PBL cases with anti-HLA-DR demonstrates the typical membrane ... PBL cases with less than 10% of cells showing HLA-DR expression (cell membrane and aberrant cytoplasmic) were grouped together with the negative cases (“Low/Zero HLA-DR”). The 21 “Low/Zero HLA-DR” cases showed a median presence of 8.6% (±5 SD) CD8+ T cells (range 1.6-17%) (Figure 1E, Table 1). In one case, 60% of cells strongly expressed the aberrant cytoplasmic punctate pattern in the absence of membrane staining with an intensity of 3+. In this case, the CD8+ T-cell percentage was 15% (Figure 1E, Table 1). Table 1. HLA-DR expression and presence of CD8+ T cells in PBL cases (n=22). In the one case with more than 10% cells being positive, HLA-DR was exclusively localized in the cytoplasm with no surface expression. Within the “Low/Zero HLA-DR” group of PBL cases (Figure 1F, Table 2), 10 cases were HLA-DR negative with a median presence of 7% (±6 SD) CD8+ T cells (ranging 1-17%). In a further 2 cases of the “Low/Zero HLA-DR” group, a few cells (2% ±3 SD) expressed HLA-DR on the cell membrane with a median intensity of 2+ and showed a median presence of 9% (±4 SD) CD8+ T-cells (range 6-12%). In the remaining 9 cases of the “Low/Zero HLA-DR” group, 4% (±3 SD) of cells showed an aberrant cytoplasmic pattern in the absence of membrane staining with a median intensity of 2+ (±1 SD). The CD8+ T-cell percentages in the latter cases ranged from 3% to 17% with a median of 9.7% (±5 SD). Table 2. HLA-DR expression and presence of CD8+ T cells in the “Low/Zero” PBL group (n=21). Both the negative and aberrant cytoplasmic HLA-DR expression patterns were observed equally in all the plasmablastic phenotypes described previously.4 The PBL case series included 4 cases negative for Blimp1 which we also analyzed for HLA-DR expression:4 2 cases were negative for HLA-DR, and 2 expressed low levels of aberrant cytoplasmic HLA-DR (“Low/Zero HLA-DR”). The one PBL case with 60% of cells exclusively expressing cytoplasmic MHC class II showed the variant plasmablastic phenotype, being positive for Blimp1, MUM1 and CD38, weakly positive for CD20 and PAX5, and negative for XBP1s, CD138 and EBER. In summary, this study demonstrates the lack of MHC II protein expression on the surface membrane of most PBL cases, which is associated with only a modest decrease in CD8+ tumor infiltrating T cells. In contrast, our previous studies of this kind in DLBCL cases showed a much more pronounced decrease in CD8+ T-cell numbers in MHC II negative cases in which there was an average of 11% CD8+ T cells in the presence of MHC II cell membrane expression, but only 2.8% CD8+ T cells in the absence of MHC II protein cell membrane expression.5 The difference may be due to the low power of this study. The significance of the cytoplasmic localization of MHC class II protein is not yet clear, but may represent a stage of partial expression which is associated with an intermediate percentage of infiltrating T cells. The absence of MHC II protein expression may explain the poor outcome in PBL patients, and may serve as an additional diagnostic tool and as a biomarker for immunosurveillance.

Decreased major histocompatibility complex class II expression in diffuse large B-cell lymphoma does not correlate with CpG methylation of class II transactivator promoters III and IV

Diffuse large B-cell lymphoma (DLBCL) is a disease marked by heterogeneity in clinical presentation, morphology, and underlying biology. Importantly, this translates into variability of patient response to chemotherapy. While a majority of patients can be cured effectively with modern chemotherapy regimens, many present with or acquire resistance to treatment. Characterizing the mechanism(s) responsible for poor patient outcome is critical for identifying future targets to improve treatment.

Use of dust fall filters as passive samplers for metal concentrations in air for communities near contaminated mine tailings

Mine tailings are a source of metal exposures in many rural communities. Multiple air samples are necessary to assess the extent of exposures and factors contributing to these exposures. However, air sampling equipment is costly and requires trained personnel to obtain measurements, limiting the number of samples that can be collected. Simple, low-cost methods are needed to allow for increased sample collection. The objective of our study was to assess if dust fall filters can serve as passive air samplers and be used to characterize potential exposures in a community near contaminated mine tailings. We placed filters in cylinders, concurrently with active indoor air samplers, in 10 occupied homes. We calculated an estimated flow rate by dividing the mass on each dust fall filter by the bulk air concentration and the sampling duration. The mean estimated flow rate for dust fall filters was significantly different during sampling periods with precipitation. The estimated flow rate was used to estimate metal concentration in the air of these homes, as well as in 31 additional homes in another rural community impacted by contaminated mine tailings. The estimated air concentrations had a significant linear association with the measured air concentrations for beryllium, manganese and arsenic (p < 0.05), whose primary source in indoor air is resuspended soil from outdoors. In the second rural community, our estimated metal concentrations in air were comparable to active air sampling measurements taken previously. This passive air sampler is a simple low-cost method to assess potential exposures near contaminated mining sites.

Abstract 1769: Loss of major histocompatibility class II expression in diffuse large B-cell lymphoma may be related to stage of B-cell differentiation

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma diagnosed in the U.S. It displays marked heterogeneity, biologically and clinically, with patients demonstrating a wide range of responses to therapy. Major histocompatibility class II (MHCII) molecules are involved in antigen presentation and are vital to the immune system. Decreased MHCII expression is one of the normal changes seen as B-cells differentiate into mature, antibody-secreting plasma cells. This decrease occurs in concert with changes in other proteins and transcription factors which define the phenotype of a B- or plasma cell. Loss of MHCII expression in DLBCL patients is associated with extremely poor prognosis. In this study, we asked whether MHCII loss in DLBCL was associated with a more differentiated phenotype. We hypothesized that malignant cells from MHCII(-) DLBCL cases would be closer to the plasma cell end of the differentiation spectrum than MHCII(+) cases. We previously used a 40-case tissue microarray to demonstrate that although MHCII(-) DLBCL cases did not present a fully plasma cell differentiated phenotype, protein expression of the late B-cell marker, MUM1/IRF4, was inversely associated with MHCII, suggesting that MHCII loss may be associated with cases further along the differentiation continuum (Wilkinson, AACR 2009). Here, we built upon this work using immunohistochemistry (IHC) to score expression of a panel of B- and plasma cell markers. These included B-cell transcription factors Oct2, Bob 1, Pax5 and mature B-cell surface marker CD20 as well as plasma cell markers XBP1, BLIMP1, and CD138. IHC results were semi-quantitatively assessed by scoring both frequency and intensity of staining. The results were compared to MHCII expression as previously determined with gene expression profiling and IHC. Using this routine IHC assessment, the presence of plasma cell markers XBP1, BLIMP1, or CD138 did not correlate with low or absent expression of MHCII, while the B-cell markers were present in most cases. Thus, no clear evidence of a more differentiated phenotype was observed in MHCII(-) DLBCL. These findings could result from lack of association between loss of MHCII expression and plasma cell differentiation or the limitations of the methodology used. To further investigate this question, we have developed protocols with QdotsR (Life Technologies) to more accurately quantify expression of the plasma cell-associated proteins relative to MHCII. By using nanocrystals with narrow emission spectra coupled to monoclonal antibodies, multiple colors can be visualized and specifically measured in each cell. This technique may yield more accurate, quantitative data regarding protein expression of plasma cell markers and MHCII. The results of these studies should lead to a better understanding of MHCII expression in DLBCL with possible implications for therapeutic targeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1769.