Lisa M Rimsza

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• Albero, R., Bea, S., Campo, E., Capdevila, C., Castellano, G., Clot, G., Demajo, S., Enjuanes, A., Gine, E., Jaffe, E. S., Jares, P., Lopez-guillermo, A., Nadeu, F., Navarro, A., Ott, G., Pinyol, M., Rimsza, L. M., Rosenwald, A., Scott, D. W., & Staudt, L. M. (2021). A Cyclin D1-Dependent Transcriptional Program Predicts Clinical Outcome in Mantle Cell Lymphoma.. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(1), 213-225. doi:10.1158/1078-0432.ccr-20-2868
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  Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation leading to cyclin D1 overexpression. Cyclin D1 is a major cell-cycle regulator and also regulates transcription, but the impact of cyclin D1-mediated transcriptional dysregulation on MCL pathogenesis remains poorly understood. The aim of this study was to define a cyclin D1-dependent gene expression program and analyze its prognostic value..We integrated genome-wide expression analysis of cyclin D1-silenced and overexpressing cells with cyclin D1 chromatin-binding profiles to identify a cyclin D1-dependent transcriptional program in MCL cells. We analyzed this gene program in two MCL series of peripheral blood samples (n = 53) and lymphoid tissues (n = 106) to determine its biological and clinical relevance. We then obtained a simplified signature of this program and evaluated a third series of peripheral blood MCL samples (n = 81) by NanoString gene expression profiling to validate our findings..We identified a cyclin D1-dependent transcriptional program composed of 295 genes that were mainly involved in cell-cycle control. The cyclin D1-dependent gene program was overexpressed in MCL tumors directly proportional to cyclin D1 levels. High expression of this program conferred an adverse prognosis with significant shorter overall survival of the patients. These observations were validated in an independent cohort of patients using a simplified 37-gene cyclin D1 signature. The cyclin D1-dependent transcriptional program was also present in multiple myeloma and breast tumors with cyclin D1 overexpression..We identified a cyclin D1-dependent transcriptional program that is overexpressed in MCL and predicts clinical outcome.
• Glinsmann-gibson, B., Larsen, B., Maguire, A., Mcgrath, M., Ramsower, C., Rimsza, L., Wisner, L., & Zellner, K. (2021). 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.. Biopreservation and biobanking, 20(6), 473-484. doi:10.1089/bio.2021.0023
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  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.
• Balasubramanian, S., Byrum, S. D., Chavez, E. A., Hung, S. S., Jenjaroenpun, P., Kendrick, S., Rimsza, L. M., Shponka, V., Steidl, C., Tannahill, D., Wongsurawat, T., & Xu, Y. (2020). Activation-induced cytidine deaminase localizes to G-quadruplex motifs at mutation hotspots in lymphoma.. NAR cancer, 2(4), zcaa029. doi:10.1093/narcan/zcaa029
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  Diffuse large B-cell lymphoma (DLBCL) is a molecularly heterogeneous group of malignancies with frequent genetic abnormalities. G-quadruplex (G4) DNA structures may facilitate this genomic instability through association with activation-induced cytidine deaminase (AID), an antibody diversification enzyme implicated in mutation of oncogenes in B-cell lymphomas. Chromatin immunoprecipitation sequencing analyses in this study revealed that AID hotspots in both activated B cells and lymphoma cells in vitro were highly enriched for G4 elements. A representative set of these targeted sequences was validated for characteristic, stable G4 structure formation including previously unknown G4s in lymphoma-associated genes, CBFA2T3, SPIB, BCL6, HLA-DRB5 and MEF2C, along with the established BCL2 and MYC structures. Frequent genome-wide G4 formation was also detected for the first time in DLBCL patient-derived tissues using BG4, a structure-specific G4 antibody. Tumors with greater staining were more likely to have concurrent BCL2 and MYC oncogene amplification and BCL2 mutations. Ninety-seven percent of the BCL2 mutations occurred within G4 sites that overlapped with AID binding. G4 localization at sites of mutation, and within aggressive DLBCL tumors harboring amplified BCL2 and MYC, supports a role for G4 structures in events that lead to a loss of genomic integrity, a critical step in B-cell lymphomagenesis.
• Rimsza, L. M., Connors, J. M., Farinha, P., Gascoyne, R. D., Kendrick, S., Persky, D. O., Puvvada, S. D., Schmelz, M., Scott, D. W., Sehn, L. H., Slack, G. W., & Tan, K. L. (2017). Aberrant cytoplasmic expression of MHCII confers worse progression free survival in diffuse large B-cell lymphoma.. Virchows Archiv : an international journal of pathology, 470(1), 113-117. doi:10.1007/s00428-016-2041-7
• Rimsza, L. M., Bernstein, S. H., Fisher, R. I., Friedberg, J. W., Glinsmann-gibson, B., Leblanc, M., Li, H., Maddox, A. M., Miller, T. P., Persky, D. O., Puvvada, S. D., Schmelz, M., & Smith, S. M. (2016). A phase II study of belinostat (PXD101) in relapsed and refractory aggressive B-cell lymphomas: SWOG S0520.. Leukemia & lymphoma, 57(10), 2359-69. doi:10.3109/10428194.2015.1135431
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  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.
• Rimsza, L. M., Baldwin, H. S., Lee, K., Martin, G. R., Scholl, F. A., & Tome, M. E. (2014). 322 - Dysregulation of Catalase and Glutathione Peroxidase1 Impacts Diffuse Large B-Cell Lymphoma (DLBCL) Patient Survival. Free Radical Biology and Medicine, 76, S134. doi:10.1016/j.freeradbiomed.2014.10.219
• Scott, D. W., Wright, G. W., Williams, M., Lih, J., Jaffe, E. S., Rosenwald, A., Campo, E., Chan, W. C., Connors, J. M., Braziel, R. M., Ott, G., Delabie, J., Weisenburger, D. D., Cook, J. R., Greiner, T. C., Fu, K., Walsh, W., Gascoyne, R. D., Staudt, L. M., , Rimsza, L. M., et al. (2014). Accurate Diagnosis of Aggressive B Cell Non-Hodgkin Lymphomas Using Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissues. Blood, 124(21), 3016-3016. doi:10.1182/blood.v124.21.3016.3016
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  ![Graphic][1] Background: Currently, diagnosis of aggressive B cell non-Hodgkin lymphomas (agg-B-NHL) uses a varying combination of morphology, immunophenotyping, cytogenetics, and/or other molecular techniques resulting in a fragmented, confusing diagnostic system. We sought to develop a multi-analyte gene expression signature assay that could consolidate the diagnostic process into a single platform to improve standardization and accuracy. Methods: We used formalin-fixed, paraffin-embedded tissue biopsies, qualified by an expert Hematopathology review panel, tumor content of ≥60%, and confirmed B cell immunophenotype. Diagnostic categories included diffuse large B cell lymphoma (DLBCL) including the activated B cell-like (ABC), germinal center B cell-like (GCB) subtypes, unclassifiable (UNC) DLBCL, primary mediastinal B cell lymphoma (PMBCL), Burkitt lymphoma (BL), and mantle cell lymphoma (MCL). Using our previous GEP data, diagnostic signatures, nCounter system (Nanostring, Seattle, WA), and employing published procedures (Scott et al, Blood 2014); we designed probes to 800 genes with utility in distinguishing between these pathological entities. The training cohort comprised 107 unique cases, whose FFPET biopsies were independently assayed at the Molecular Characterization Laboratory, Frederick National Laboratory for Cancer Research (Frederick, MD) and the Centre for Lymphoid Cancer, BC Cancer Agency (Vancouver, BC). The resulting algorithm was locked down and applied to an independent cohort of 199 cases. The nucleic acids from FFPET biopsies from these cases were extracted and run across the two independent laboratories with 83 cases run at both laboratories to assess inter-laboratory performance. The “gold standard” by which the Nanostring classification was compared was based on Affymetrix gene expression profiling of matched frozen biopsies in the cases of ABC, GCB, and UNC DLBCL (Lenz et al. NEJM 2008) and on the pathological diagnosis by the Hematopathology review panel in the cases of BL, MCL, and PMBCL. The use of human tissues and clinical data for this study was approved by the University of Arizona Institutional Review Board in accordance with the Declaration of Helsinki. Results: The final locked algorithm consisted of 297 gene probes including 47 housekeeping genes. Thirty-six cases from the training cohort were run again on the new lot of Nanostring code set to allow for cross code set calibration of the assay. The laboratory procedure and algorithm, together termed the "Lymph5Cx" test, consists of a hierarchical series of pair-wise comparisons. In the independent validation set, 257/282 assays (91.1%) yielded gene expression data of sufficient quality (total of 185 of the 199 cases). A classification summary is given in the Table below. In this cohort, 136 cases (82%) were correctly assigned while 11 cases (6.6%) were assigned incorrect diagnoses as follows: 6 BL assigned to GCB, 1 GCB labeled a PMBCL, and 4 PMBCL assigned to DLBCL subtypes. The Lymph5Cx test included categories of indeterminate results between two diagnostic entities and were declared borderline, as indicated in the Table. The agreement between the 2 laboratory sites was 71/72 (99%) of cases that yielded adequate gene expression data at both sites. Conclusions: The Lymph5Cx test was robust and able to discriminate the often clinically difficult diagnostic categories of agg-B-NHL using a single methodology for cases with histologic and immunophenotypic features of an agg-B-NHL. Misclassification errors were low, suggesting that this test would be useful adjunct to current diagnostic methods. In addition, targetable pathways, as well as genes associated with known prognostic signatures in DLBCL (stromal) and MCL (proliferation) were quantified. Investigation into these latter aspects is on-going. Gene expression signature assays have become a useful clinical and research tool in the on-going area of precision therapeutics based on highly-defined molecular entities. | | # cases | % accurate | % borderline | % error | | ---- | ------- | ---------- | ------------ | ------- | | ABC | 26 | 76.9% | 23.1% | 0.0% | | GCB | 27 | 88.9% | 7.4% | 3.7% | | BL | 48 | 68.8% | 19.8% | 11.5% | | PMBL | 30 | 80.0% | 6.7% | 13.3% | | MCL | 34 | 100.0% | 0.0% | 0.0% | Table Disclosures Scott: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Wright: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Williams: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Lih: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Jaffe: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Rosenwald: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Campo: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Chan: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Connors: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Smeland: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Braziel: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Ott: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Delabie: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Weisenburger: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Cook: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Greiner: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Fu: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Walsh: Nanostring: The author is a potential inventor on a patent application using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Gascoyne: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Staudt: Nanostring: The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. Rimsza: Nanostring: Research Funding, The author is a potential inventor on a patent applicaiton using Nanostring technology for a different assay, which has been licensed from the NIH by Nanostring Patents & Royalties. [1]: /embed/inline-graphic-2.gif
• Bernstein, S. H., Burack, R., Cebula, E., Epner, E. M., Fisher, R. I., Leblanc, M., Miller, T. P., Rimsza, L. M., & Unger, J. M. (2013). A phase II multicenter trial of hyperCVAD MTX/Ara-C and rituximab in patients with previously untreated mantle cell lymphoma; SWOG 0213.. Annals of oncology : official journal of the European Society for Medical Oncology, 24(6), 1587-93. doi:10.1093/annonc/mdt070
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  Rituximab-hyper-CVAD alternating with rituximab-high-dose methotrexate and cytarabine is a commonly utilized regimen in the United States for mantle cell lymphoma (MCL) based on phase II single institutional data. To confirm the clinical efficacy of this regimen and determine its feasibility in a multicenter study that includes both academic and community-based practices, a phase II study of this regimen was conducted by SWOG..Forty-nine patients with advanced stage, previously untreated MCL were eligible. The median age was 57.4 years (35-69.8 years)..Nineteen patients (39%) did not complete the full scheduled course of treatment due to toxicity. There was one treatment-related death and two cases of secondary myelodysplastic syndrome (MDS). There were 10 episodes of grade 3 febrile neutropenia, 19 episodes of grade 3 and 1 episode of grade 4 infection. With a median follow-up of 4.8 years, the median progression-free survival was 4.8 years (5.5 years for those ≤ 65 years) and the median overall survival (OS) was 6.8 years..Although this regimen is toxic, it is active for patients ≤ 65 years of age and can be given both at academic centers and in experienced community centers.
• Huang, X., Meng, B., Iqbal, J., Ding, B. B., Perry, A. M., Cao, W., Smith, L. M., Bi, C., Jiang, C., Greiner, T. C., Weisenburger, D. D., Rosenwald, A., Ott, G., Delabie, J., Campo, E., Braziel, R. M., Gascoyne, R. D., Cook, J. R., Tubbs, R. R., , Jaffe, E. S., et al. (2013). Activation of the STAT3 signaling pathway is associated with poor survival in diffuse large B-cell lymphoma treated with R-CHOP.. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 31(36), 4520-8. doi:10.1200/jco.2012.45.6004
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  We previously reported that constitutive STAT3 activation is a prominent feature of the activated B-cell subtype of diffuse large B-cell lymphomas (ABC-DLBCL). In this study, we investigated whether STAT3 activation can risk stratify patients with DLBCL..By an immunohistochemical method, we investigated phosphotyrosine STAT3 (PY-STAT3) expression from 185 patients with DLBCL treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone). Cell line-based siRNA experiments were also performed to generate an 11-gene, PY-STAT3 activation signature, which was used to study a previously published cohort of 222 patients with DLBCL. The STAT3 activation status determined by these two methods and by STAT3 mRNA levels were then correlated with survival..PY-STAT3 was detected in 37% of DLBCL and enriched in ABC-DLBCL cases (P = .03). PY-STAT3 positivity significantly correlated with poor overall survival (OS; P = .01) and event-free survival (EFS; P = .006). Similar observations were made for high levels of STAT3 mRNA. In multivariable analysis, PY-STAT3 status (P = .02), International Prognostic Index (P = .02), and BCL2 expression (P = .046) were independent prognosticators of OS in this cohort. Among the cell-of-origin subgroups, PY-STAT3 was associated with poor EFS among non-germinal center B-cell DLBCL cases only (P = .027). Similarly, the 11-gene STAT3 activation signature correlated with poor survival in the entire DLBCL cohort (OS, P < .001; EFS, P < .001) as well as the ABC-DLBCL subgroup (OS, P = .029; EFS, P = .025)..STAT3 activation correlated with poor survival in patients with DLBCL treated with R-CHOP, especially those with tumors of the ABC-DLBCL subtype.
• Rimsza, L. M., Braziel, R. M., Cheson, B. D., Czuczman, M. S., Dakhil, S. R., Fisher, R. I., Friedberg, J. W., Gopal, A. K., Kaminski, M. S., Leblanc, M., Maloney, D. G., Miller, T. P., Press, O. W., Spier, C. M., & Unger, J. M. (2013). A comparative analysis of prognostic factor models for follicular lymphoma based on a phase III trial of CHOP-rituximab versus CHOP + 131iodine--tositumomab.. Clinical cancer research : an official journal of the American Association for Cancer Research, 19(23), 6624-32. doi:10.1158/1078-0432.ccr-13-1120
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  There is currently no consensus on optimal frontline therapy for patients with follicular lymphoma. We analyzed a phase III randomized intergroup trial comparing six cycles of CHOP-R (cyclophosphamide-Adriamycin-vincristine-prednisone (Oncovin)-rituximab) with six cycles of CHOP followed by iodine-131 tositumomab radioimmunotherapy (RIT) to assess whether any subsets benefited more from one treatment or the other, and to compare three prognostic models..We conducted univariate and multivariate Cox regression analyses of 532 patients enrolled on this trial and compared the prognostic value of the FLIPI (follicular lymphoma international prognostic index), FLIPI2, and LDH + β2M (lactate dehydrogenase + β2-microglobulin) models..Outcomes were excellent, but not statistically different between the two study arms [5-year progression-free survival (PFS) of 60% with CHOP-R and 66% with CHOP-RIT (P = 0.11); 5-year overall survival (OS) of 92% with CHOP-R and 86% with CHOP-RIT (P = 0.08); overall response rate of 84% for both arms]. The only factor found to potentially predict the impact of treatment was serum β2M; among patients with normal β2M, CHOP-RIT patients had better PFS compared with CHOP-R patients, whereas among patients with high serum β2M, PFS by arm was similar (interaction P value = 0.02)..All three prognostic models (FLIPI, FLIPI2, and LDH + β2M) predicted both PFS and OS well, though the LDH + β2M model is easiest to apply and identified an especially poor risk subset. In an exploratory analysis using the latter model, there was a statistically significant trend suggesting that low-risk patients had superior observed PFS if treated with CHOP-RIT, whereas high-risk patients had a better PFS with CHOP-R.
• Rimsza, L. M., Bernstein, S. H., Fisher, R. I., Goldman, B. H., Miller, T. P., & Persky, D. O. (2012). A phase II study of PXD101 (belinostat) in relapsed and refractory aggressive B-cell lymphomas (rel/ref ABCL): SWOG S0520.. Journal of Clinical Oncology, 30(15_suppl), e18536-e18536. doi:10.1200/jco.2012.30.15_suppl.e18536
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  e18536 Background: The mechanism of action of histone deacetylase inhibitors (HDACI) in lymphomas is unknown. Loss of major histocompatibility Class II antigens (MHC II) in diffuse large B-cell lymphomas (DLBCL) is associated with decreased tumor infiltrating T lymphocytes (TIL) and poor survival. Transcription of MHC II is controlled by CIITA, which is itself regulated by histone acetylation, We hypothesized that PXD101 (belinostat), an HDACI, would increase MHC II expression in tumor cells, enhance immunosurveillance and improve outcome. Methods: Theprimary objective was to evaluate response rate and toxicity of PXD101 in patients (pts) with rel/ref ABCL with up to 5 prior chemotherapy regimens. Secondary objectives were to estimate the 6-month progression-free survival (PFS) and to assess MHC II and TIL. In a two-stage design, if at least 1 response was observed in the first 20 pts, another 20 pts would be accrued. PXD101 was administered at 1000 mg/m2 IV days 1-5 of 21-day cycle for up to 2 years. Res...
• Rimsza, L. M., Schwartz, M., Chan, W. C., Jaffe, E. S., Gascoyne, R. D., Campo, E., Rosenwald, A., Ott, G., Cook, J. R., Tubbs, R. R., Braziel, R. M., Delabie, J., Miller, T. P., Staudt, L. M., & Wright, G. E. (2011). Accurate classification of diffuse large B-cell lymphoma into germinal center and activated B-cell subtypes using a nuclease protection assay on formalin-fixed, paraffin-embedded tissues.. Clinical cancer research : an official journal of the American Association for Cancer Research, 17(11), 3727-32. doi:10.1158/1078-0432.ccr-10-2573
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  Classification of diffuse large B-cell lymphoma (DLBCL) into cell-of-origin (COO) subtypes based on gene expression profiles has well-established prognostic value. These subtypes, termed germinal center B cell (GCB) and activated B cell (ABC) also have different genetic alterations and overexpression of different pathways that may serve as therapeutic targets. Thus, accurate classification is essential for analysis of clinical trial results and planning new trials by using targeted agents. The current standard for COO classification uses gene expression profiling (GEP) of snap frozen tissues, and a Bayesian predictor algorithm. However, this is generally not feasible. In this study, we investigated whether the qNPA technique could be used for accurate classification of COO by using formalin-fixed, paraffin-embedded (FFPE) tissues. We analyzed expression levels of 14 genes in 121 cases of R-CHOP-treated DLBCL that had previously undergone GEP by using the Affymetrix U133 Plus 2.0 microarray and had matching FFPE blocks. Results were evaluated by using the previously published algorithm with a leave-one-out cross-validation approach. These results were compared with COO classification based on frozen tissue GEP profiles. For each case, a probability statistic was generated indicating the likelihood that the classification by using qNPA was accurate. When data were dichotomized into GCB or non-GCB, overall accuracy was 92%. The qNPA technique accurately categorized DLBCL into GCB and ABC subtypes, as defined by GEP. This approach is quantifiable, applicable to FFPE tissues with no technical failures, and has potential for significant impact on DLBCL research and clinical trial development.
• Wilkinson, S. T., Fernandez, D. R., Vanpatten, K. A., Glinsmann-gibson, B. J., Grogan, T. M., & Rimsza, L. M. (2010). Abstract 1769: Loss of major histocompatibility class II expression in diffuse large B-cell lymphoma may be related to stage of B-cell differentiation. Cancer Research, 70(8_Supplement), 1769-1769. doi:10.1158/1538-7445.am10-1769
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  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.
• O'riain, C., O'shea, D. M., Yang, Y., Gribben, J. G., Yeboah-afari, J., Bhaw-rosun, L., Fleischmann, C., Mein, C. A., Crook, T., Smith, P., Kelly, G., Rosenwald, A., Ott, G., Campo, E., Rimsza, L. M., Smeland, E. B., Chan, W. C., Gascoyne, R. D., Reimer, S., , Braziel, R. M., et al. (2009). Array-based DNA methylation profiling in follicular lymphoma.. Leukemia, 23(10), 1858-66. doi:10.1038/leu.2009.114
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  Quantitative methylation profiling was performed using the Illumina GoldenGate Assay in untreated follicular lymphoma (FL) (164), paired pre- and post-transformation FL (20), benign haematopoietic (24) samples and purified B and T cells from two FL cases. Methylation values allowed separation of untreated FL samples from controls with one exception, based primarily on tumour-specific gains of methylation typically occurring within CpG islands. Genes that are targets for epigenetic repression in stem cells by Polycomb Repressor Complex 2 were significantly over-represented among hypermethylated genes. Methylation profiles were conserved in sequential FL and t-FL biopsies, suggesting that widespread methylation represents an early event in lymphomagenesis and may not contribute substantially to transformation. A significant (P
• Rimsza, L. M., Braziel, R. M., Campo, E., Chan, W. C., Cook, J. R., Delabie, J., Fisher, R. I., Gascoyne, R. D., Jaffe, E. S., Miller, T. P., Ott, G., Rosenwald, A., Schwartz, M., Staudt, L. M., Tubbs, R. R., & Wright, G. E. (2009). Accurate Classification of Diffuse Large B Cell Lymphoma Into Germinal Center and Activated B Cell Subtypes Using a Nuclease Protection Assay On Formalin Fixed Paraffin Embedded Tissue: A Study From the Lymphoma and Leukemia Molecular Profiling Project.. Blood, 114(22), 620-620. doi:10.1182/blood.v114.22.620.620
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  Abstract 620 Classification of DLBCL into cell-of-origin (COO) subtypes based on gene expression profiles has well-established prognostic value. These subtypes, termed Germinal Center B cell (GCB) and Activated B cell (ABC) also have different genetic alterations and over-expression of different pathways that may serve as therapeutic targets. Thus, accurate classification is essential for analysis of clinical trial results and planning new trials using targeted agents. The gold standard for COO classification uses gene expression profiling (GEP) of snap frozen tissues, and a Bayesian predictor algorithm utilizing the expression levels of 14 key genes (G. Wright et al PNAS 2003). An immunohistochemistry (IHC) classification scheme by C. Hans et al (Blood 2004), based on 3 antibodies, is widely used as a substitute for GEP classification, however does not completely correlate with GEP. We recently described a qNPA assay (ArrayPlate R , High ThroughPut Genomics, Tucson, AZ) with excellent correlation between frozen and formalin fixed paraffin embedded (FFPE) tissues (R. Roberts et al, Lab Invest 2007). In this study, we investigated whether this technique could be used for accurate classification of COO using FFPE tissues. We expanded the previous gene probe repertoire of the DLBCL-ArrayPlate R assay to include the 14 genes (represented by 17 probe sets) most pertinent to COO classification. 52 cases of R-CHOP treated DLBCL that had undergone GEP using the Affymetrix U133 Plus 2.0 microarray and had matching FFPE blocks were analyzed with qNPA in duplicate. The genes included CD10, LRMP, CCND2, ITPKB, PIM1, IL16, IRF4, FUT8, BCL6, PTPN1, LM02, CD39, MYBL1, IGHM . Results were evaluated using the previously published algorithm with a leave-one-out cross validation scheme to classify cases into GCB or ABC subtypes. These results were compared to COO classification based on frozen tissue GEP profiles. All 14 genes in all 52 cases were successfully analyzed with no missing data points. For each case, a probability statistic was generated indicating the likelihood that the classification using qNPA was accurate. Of the 54 cases, 25 were GCB, 27 were ABC and 4 were unclassifiable by GEP. Of the GCB cases, 23/25 (92%) were classified correctly by qNPA with a confidence cut-off of >0.9 and 25/25 (100%) classified correctly with a confidence cut-off of >0.8. Of the ABC cases, 25/27 (93%) were correctly classified as ABC using qNPA with a confidence cut-off of >0.9 and 27/27 (100%) classified correctly with a confidence cut-off of >0.8. In summary, the qNPA technique accurately categorized DLBCL into GCB and ABC subtypes, as defined by GEP. There were no technical difficulties with any of the pathological materials although they were collected retrospectively from a variety of institutions and countries with different fixation methods. This approach represents a substantial improvement over previously published IHC methods and is applicable to FFPE tissues, therefore overcoming the need for snap frozen materials. This technically robust classification method has potential to have a significant impact on future DLBCL research and clinical trial development. Disclosures: Rimsza: High Throughput Genomics: HTG provided the assays at no charge to Dr. Rimsza9s lab. Schwartz: High Throughput Genomics: Employment. Gascoyne: Roche Canada: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding.
• Stopeck, A. T., Rimsza, L. M., Bellamy, W. T., Fisher, R. I., Iannone, M., Leblanc, M., Miller, T. P., Persky, D. O., & Unger, J. M. (2009). A phase II trial of single agent bevacizumab in patients with relapsed, aggressive non-Hodgkin lymphoma: Southwest oncology group study S0108.. Leukemia & lymphoma, 50(5), 728-35. doi:10.1080/10428190902856808
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  This is the first report of the Southwest oncology group phase II trial of single agent bevacizumab in patients with relapsed, aggressive non-Hodgkin lymphoma (NHL). Fifty-two patients in first or second relapse with diffuse large B-cell or mantle cell lymphoma were enrolled. Patients were treated with bevacizumab at 10 mg/kg every 2 weeks. Therapy was well tolerated with no unexpected toxicities observed. Six-month progression-free survival (PFS) was 16% with a response rate of 2% and median duration of response or stable disease of 5.2 months (range 3.5-72.7). Vascular endothelial growth factor A (VEGF) and VEGF receptor expression was observed in 70% and 65% of specimens, respectively. In an exploratory subgroup analysis, baseline urine VEGF and plasma vascular cell adhesion molecule-1 (VCAM) levels correlated with survival. Prolonged PFS in several patients as well as biomarker studies suggest the VEGF pathway plays an important role in aggressive NHL. Clinical trials combining active chemotherapy regimens with VEGF targeted agents are currently in progress.
• Wilkinson, S. T., Bob, R., Stein, H., Schwartz, M., Braziel, R. M., Gascoyne, R. D., & Rimsza, L. M. (2009). A Comparison of Gene Expression Pattern in Major Histocompatibility Class II-Low Diffuse Large B-Cell Lymphoma with Plasmablastic Lymphoma.. Blood, 114(22), 1941-1941. doi:10.1182/blood.v114.22.1941.1941
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  Abstract 1941 Poster Board I-964 Multiple studies have repeatedly shown that loss of MHC II expression correlates with poor patient prognosis in diffuse large B-cell lymphoma (DLBCL). Major histocompatibility complex class II (MHCII) molecules present peptides for antigen recognition and are important for the adaptive immune response. Loss of MHCII expression is also one of the changes seen during normal B-cell differentiation into plasma cells. Plasmablastic lymphoma (PBL) is another B-cell lymphoma characterized by a proliferation of large B-cells with a plasma cell immunophenotype and very poor prognosis. In this study, we questioned whether DLBCL cases that have low MHCII expression have a similar gene expression pattern to PBL. Unstained cuts from formalin-fixed, paraffin-embedded tissue blocks of 101 DLBCL and 76 PBL cases were analyzed for gene expression using a quantitative nuclease protection assay (qNPA, ArrayPlate R ). The 42 genes on the array were previously identified as B-cell lineage-related or prognostically important in DLBCL. DLBCL cases were divided into low [MHCII(-)] and high [MHCII(+)] MHC II expression using a 20% cutoff for expression of HLA-DRB by qNPA, as previously described (L Rimsza et al, Blood 2008). Genes that differed significantly between lymphoma types were determined using the Partek Genomics Suite R software, using ANOVA tests with a false discovery rate of 0.05. Thirty of the 42 genes on the array (71%) were differentially expressed between DLBCL as a whole and PBL. As expected from the literature, the PBL cases had less expression of B-cell antigen, MHCII, and germinal center-related genes as compared to DLBCL. Of these 30 genes, 29 were also different between MHCII(+) and PBL. In contrast, only 21 genes of the 42 on the array (50%) were differentially expressed between MHCII(-) and PBL, indicating a less dissimilar expression pattern between these two sets of cases. Of the 21 genes, two were uniquely different between MHCII(-) and PBL. Both of these, FN1 and CTGF , are found in the extracellular matrix and were low in the MHCII(-) cases. This finding, that the MHCII(-) cases are similar, but not identical to PBL, agrees with our previous immunohistochemistry studies suggesting MHCII(-) cases may be invoking selected mechanisms of differentiation (S Wilkinson et al, AACR Annual Meeting 2009, #2712). Our findings confirm the hypothesis that MHCII(-) DLBCL have a more plasma cell-like expression pattern than MHCII(+) DLBCL. These findings may have implications for pathogenesis and treatment. Disclosures: Schwartz: High Throughput Genomics: Employment. Gascoyne: Roche Canada: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Rimsza: High Throughput Genomics: Memorandum of understanding with HTG to run qNPA assay at no cost..
• Rimsza, L. M., Briehl, M. M., Grogan, T. M., Johnson, D. B., Miller, T. P., Oberley, L. W., Roberts, R. A., & Tome, M. E. (2005). A redox signature score identifies diffuse large B-cell lymphoma patients with a poor prognosis.. Blood, 106(10), 3594-601. doi:10.1182/blood-2005-02-0487
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  Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease in which approximately 40% of the patients respond well to current chemotherapy, but the prognosis for the other 60% is poor. The Leukemia/Lymphoma Molecular Profiling Project (LLMPP) used microarray technology to define a molecular profile for each of 240 patients with DLBCL and develop a molecular outcome predictor score that accurately predicted patient survival. Data from our laboratory and others suggest that alterations in antioxidant defense enzyme levels and redox environment can be oncogenic and affect the response to glucocorticoid treatment, one of the components of combination chemotherapy regimens for lymphoma. The goal of the current study was to reanalyze the LLMPP microarray data to determine whether the levels of antioxidant defense enzymes and redox proteins were correlated with prognosis in DLBCL. We found that patients with DLBCL with the worst prognosis, according to the outcome predictor score, had decreased expression of catalase, glutathione peroxidase, manganese superoxide dismutase, and VDUP1, a protein that inhibits thioredoxin activity. The data suggest that the patients with the worst prognosis combine a decrease in antioxidant defense enzyme expression with an increase in thioredoxin system function (the redox signature score).
• Christensen, R., Calhoun, D., & Rimsza, L. (2000). A practical approach to evaluating and treating neutropenia in the neonatal intensive care unit. Clinics in Perinatology, 27(3). doi:10.1016/s0095-5108(05)70040-3
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  Neutropenia is a relatively common problem in the NICU, recognized in as many as 8% of patients at some time during their hospital stay. In most instances, neutropenia among NICU patients is of short duration and has little influence on outcome. In other cases it is prolonged and severe, and constitutes a serious antimicrobial defense deficiency. When a neonatologist discovers a low blood neutrophil count, choices must be made regarding further evaluation and treatment. The authors hope that the information provided in this article is useful in making these choices.
• Sola, M., Rimsza, L., & Christensen, R. (1999). A bone marrow biopsy technique suitable for use in neonates. British Journal of Haematology, 107(2). doi:10.1046/j.1365-2141.1999.01712.x
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  Thrombocytopenia and neutropenia are common among neonates in intensive care units. Bone marrow aspirations are sometimes performed as part of their evaluation. However, marrow biopsies have not been reported from living neonates. Since architecture and cellularity cannot generally be accurately assessed from marrow aspirates, we devised a biopsy technique which we successfully applied to five cytopenic neonates (three with severe persistent thrombocytopenia and two with idiopathic neutropenia). This technique used a 19 gauge, half-inch Osgood needle to obtain bone marrow clots from the tibias of small preterm neonates which enabled the assessment of marrow cellularity and architecture. On the basis of our initial experience we have ceased using the traditional bone marrow aspiration technique in neonates and now use this technique exclusively.