Immunohistochemical Detection of MYC-driven Diffuse Large B-Cell Lymphomas

Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. A small subset of DLBCLs has translocations involving the MYC locus and an additional group has a molecular signature resembling Burkitt lymphoma (mBL). Presently, identification of such cases by morphology is unreliable and relies on cytogenetic or complex molecular methods such as gene transcriptional profiling. Herein, we describe an immunohistochemical (IHC) method for identifying DLBCLs with increased MYC protein expression. We tested 77 cases of DLBCL and identified 15 cases with high MYC protein expression (nuclear staining in >50% of tumor cells). All MYC translocation positive cases had increased MYC protein expression by this IHC assay. In addition, gene set enrichment analysis (GSEA) of the DLBCL transcriptional profiles revealed that tumors with increased MYC protein expression (regardless of underlying MYC translocation status) had coordinate upregulation of MYC target genes, providing molecular confirmation of the IHC results. We then generated a molecular classifier derived from the MYC IHC results in our cases and employed it to successfully classify mBLs from two previously reported independent case series, providing additional confirmation that the MYC IHC results identify clinically important subsets of DLBCLs. Lastly, we found that DLBCLs with high MYC protein expression had inferior overall survival when treated with R-CHOP. In conclusion, the IHC method described herein can be used to readily identify the biologically and clinically distinct cases of MYC-driven DLBCL, which represent a clinically significant subset of DLBCL cases due to their inferior overall survival

Increased SYK activity is associated with unfavorable outcome among patients with acute myeloid leukemia.

Recent discoveries have led to the testing of novel targeted therapies for the treatment of acute myeloid leukemia (AML). To better inform the results of clinical trials, there is a need to identify and systematically assess biomarkers of response and pharmacodynamic markers of successful target engagement. Spleen tyrosine kinase (SYK) is a candidate therapeutic target in AML. Small-molecule inhibitors of SYK induce AML differentiation and impair leukemia progression in preclinical studies. However, tools to predict response to SYK inhibition and to routinely evaluate SYK activation in primary patient samples have been lacking. In this study we quantified phosphorylated SYK (P-SYK) in AML cell lines and establish that increasing levels of baseline P-SYK are correlated with an increasing sensitivity to small-molecule inhibitors targeting SYK. In addition, we found that pharmacological inhibition of SYK activity extinguishes P-SYK expression as detected by an immunohistochemical (IHC) test. Quantitative analysis of P-SYK expression by the IHC test in a series of 70 primary bone marrow biopsy specimens revealed a spectrum of P-SYK expression across AML cases and that high P-SYK expression is associated with unfavourable outcome independent of age, cytogenetics, and white blood cell count. This study thus establishes P-SYK as a critical biomarker in AML that identifies tumors sensitive to SYK inhibition, identifies an at-risk patient population, and allows for the monitoring of target inhibition during treatment

Traceability Issue in PM2.5 and PM10 Measurements

Nowadays particle size and mass concentration measurements are the important parameter of the ambient air quality standards of several countries. The regulatory limits of mass concentration of particulate matter (PM) for the size classes of PM2.5 and PM10, i.e., particle sizes of less than or equal to 2.5 and 10 mu m in aerodynamic diameter, respectively in air are defined on yearly and hourly time-weighted-average basis. However, these limits are different in different regulations of the countries. Both of the parameters relate with the human health, climate and other issues, therefore accurate and precise measurement of these parameters are very important. Despite this, so far not much work has progressed in national metrology institutes (NMIs) worldwide on calibration and traceability issue of PM measurements. In this paper in context of PM measurement traceability, we present systematically the (1) air quality regulation in different countries, (2) reference methods for size and mass measurements, (3) variation/error and limitations of PM measurements based on the current results in this study and previously published results, (4) current status of PM size and mass calibration facility, (5) expected uncertainty in PM measurements, (6) add-on uncertainty in other parameters of national ambient air quality standards due to PM measurements, (7) where does traceability of PM issue stand against other parameters of air quality standards and its impact on health and climate, (8) NMIs working on this issue, (9) status at Bureau International des Poids et Mesures (BIPM), France and (10) conclusion. The aim of this paper is to better understand the importance of international system of units (SI) traceability issue in PM measurements, so wherever and whenever it is measured, should be acceptable everywhere, and data should be comparable for improving air quality and thus the quality of life. Funding agencies should be aware of this issue, and accept the results from the principle investigators and team only when their results have the traceability link to SI. NMIs should make program to involve industries in gas and aerosol metrology work to fulfill the requirement of calibration and standards. The regulatory authorities/ministry should work together with NMIs to improve the data quality of ambient measurements. This will greatly help to better make the policies and decisions on the related impacts. These were also the ultimate goals of "one-day pre-AdMet workshop" organized at National Physical Laboratory, New Delhi, India on February 20th, 2013

Immunohistochemical detection of MYC in representative Primary DLBCLs.

<p>Photomicrographs of select tumors and reactive tissue stained for MYC (positive staining = brown nuclei). Positive control (Burkitt lymphoma with a confirmed <i>MYC</i> translocation) revealed uniform, intense staining in >90% of tumor cells (Burkitt). In contrast, reactive lymphoid tissue revealed variable staining in only 10% of normal lymphocyte nuclei (Tonsil). Representative images from DLBCL cases and associated percent MYC+ tumor nuclei: Case 1, 90% MYC+; Case 7, 70% MYC+; and Cases 35 and 38, 30% MYC+. MYC staining was exclusively nuclear in all cases under the described staining conditions.</p

Characteristics of R-CHOP treated Patient Cohort used for Survival Analysis.

1)<p>MYC High cases show positive staining for MYC in >50% of tumor nuclei; MYC Low cases show positive staining for MYC in ≤50% tumor nuclei.</p>2)<p>Cases classified as MYC IHC-High are associated with a higher IPI than cases classified as MYC IHC-Low (p = 0.06, Kruskal-Wallis test).</p

Comparison of MYC protein expression and <i>MYC</i> translocation status in cases of Primary <i>(de novo)</i> DLBCL;

<p>(<b>A</b>). Comparison of percent MYC positive tumor nuclei for Burkitt lymphoma, reactive tonsil and 56 primary DLBCLs with their corresponding <i>MYC</i> translocation status (bar graph; pink = <i>MYC</i> translocation, green = no <i>MYC</i> translocation, gray = not determined). Tumors with an IHC score of >50% are indicated in red at left. Tumors are arranged by IHC-determined percentage of MYC-positive nuclei. <b>Comparison of MYC protein expression and </b><b><i>MYC</i></b><b> translocation in cases of Secondary DLBCL;</b> (<b>B</b>). Comparison of percent MYC positive tumor nuclei for Burkitt lymphoma, reactive tonsil and 21 cases of recurrent or transformed DLBCLs with their corresponding <i>MYC</i> translocation status (bar graph; pink = <i>MYC</i> translocation, green = no <i>MYC</i> translocation). Tumors with an IHC score of >50% are indicated in red at left. Tumors are arranged by IHC-determined percentage of MYC-positive nuclei.</p

GSEA of MYC target genes in tumors characterized for MYC protein expression;

<p>(<b>A</b>). Gene Set Enrichment Analysis (GSEA) of all primary DLBCL cases reveals coordinate upregulation of MYC and MYC target genes (black vertical lines) in tumors with >50% MYC positive tumor nuclei (including <i>MYC</i> translocation-positive and –negative cases) (See Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033813#pone.0033813.s006" target="_blank">Methods S1</a> for additional details). <b>Gene Set Enrichment Analysis (GSEA) of MYC-target genes in </b><b><i>MYC</i></b><b> translocation–negative Primary DLBCL tumors;</b> (<b>B</b>) <i>MYC</i> translocation-negative primary DLBCL cases with >50% MYC positive tumor nuclei have coordinate upregulation of MYC and MYC target genes (black vertical lines).</p