Wei-min Liu

Clinical Utility of Microarray-Based Gene Expression Profiling in the Diagnosis and Subclassification of Leukemia: Report From the International Microarray Innovations in Leukemia Study Group

PURPOSE The Microarray Innovations in Leukemia study assessed the clinical utility of gene expression profiling as a single test to subtype leukemias into conventional categories of myeloid and lymphoid malignancies. METHODS The investigation was performed in 11 laboratories across three continents and included 3,334 patients. An exploratory retrospective stage I study was designed for biomarker discovery and generated whole-genome expression profiles from 2,143 patients with leukemias and myelodysplastic syndromes. The gene expression profiling-based diagnostic accuracy was further validated in a prospective second study stage of an independent cohort of 1,191 patients. RESULTS On the basis of 2,096 samples, the stage I study achieved 92.2% classification accuracy for all 18 distinct classes investigated (median specificity of 99.7%). In a second cohort of 1,152 prospectively collected patients, a classification scheme reached 95.6% median sensitivity and 99.8% median specificity for 14 standard subtypes of acute leukemia (eight acute lymphoblastic leukemia and six acute myeloid leukemia classes, n = 693). In 29 (57%) of 51 discrepant cases, the microarray results had outperformed routine diagnostic methods. CONCLUSION Gene expression profiling is a robust technology for the diagnosis of hematologic malignancies with high accuracy. It may complement current diagnostic algorithms and could offer a reliable platform for patients who lack access to todays state-of-the-art diagnostic work-up. Our comprehensive gene expression data set will be submitted to the public domain to foster research focusing on the molecular understanding of leukemias.

MYC/BCL2 protein coexpression contributes to the inferior survival of activated B-cell subtype of diffuse large B-cell lymphoma and demonstrates high-risk gene expression signatures: a report from The International DLBCL Rituximab-CHOP Consortium Program

Diffuse large B-cell lymphoma (DLBCL) is stratified into prognostically favorable germinal center B-cell (GCB)-like and unfavorable activated B-cell (ABC)-like subtypes based on gene expression signatures. In this study, we analyzed 893 de novo DLBCL patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). We show that MYC/BCL2 protein coexpression occurred significantly more commonly in the ABC subtype. Patients with the ABC or GCB subtype of DLBCL had similar prognoses with MYC/BCL2 coexpression and without MYC/BCL2 coexpression. Consistent with the notion that the prognostic difference between the 2 subtypes is attributable to MYC/BCL2 coexpression, there is no difference in gene expression signatures between the 2 subtypes in the absence of MYC/BCL2 coexpression. DLBCL with MYC/BCL2 coexpression demonstrated a signature of marked downregulation of genes encoding extracellular matrix proteins, those involving matrix deposition/remodeling and cell adhesion, and upregulation of proliferation-associated genes. We conclude that MYC/BCL2 coexpression in DLBCL is associated with an aggressive clinical course, is more common in the ABC subtype, and contributes to the overall inferior prognosis of patients with ABC-DLBCL. In conclusion, the data suggest that MYC/BCL2 coexpression, rather than cell-of-origin classification, is a better predictor of prognosis in patients with DLBCL treated with R-CHOP.

Mutational profile and prognostic significance of TP53 in diffuse large B-cell lymphoma patients treated with R-CHOP: Report from an International DLBCL Rituximab-CHOP Consortium Program Study

TP53 mutation is an independent marker of poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL) treated with cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (CHOP) therapy. However, its prognostic value in the rituximab immunochemotherapy era remains undefined. In the present study of a large cohort of DLBCL patients treated with rituximab plus CHOP (R-CHOP), we show that those with TP53 mutations had worse overall and progression-free survival compared with those without. Unlike earlier studies of patients treated with CHOP, TP53 mutation has predictive value for R-CHOP-treated patients with either the germinal center B-cell or activated B-cell DLBCL subtypes. Furthermore, we identified the loop-sheet-helix and L3 motifs in the DNA-binding domain to be the most critical structures for maintaining p53 function. In contrast, TP53 deletion and loss of heterozygosity did not confer worse survival. If gene mutation data are not available, immunohistochemical analysis showing > 50% cells expressing p53 protein is a useful surrogate and was able to stratify patients with significantly different prognoses. We conclude that assessment of TP53 mutation status is important for stratifying R-CHOP-treated patients into distinct prognostic subsets and has significant value in the design of future therapeutic strategies.

CD30 expression defines a novel subgroup of diffuse large B-cell lymphoma with favorable prognosis and distinct gene expression signature: a report from the International DLBCL Rituximab-CHOP Consortium Program Study

CD30, originally identified as a cell-surface marker of Reed-Sternberg and Hodgkin cells of classical Hodgkin lymphoma, is also expressed by several types of non-Hodgkin lymphoma, including a subset of diffuse large B-cell lymphoma (DLBCL). However, the prognostic and biological importance of CD30 expression in DLBCL is unknown. Here we report that CD30 expression is a favorable prognostic factor in a cohort of 903 de novo DLBCL patients. CD30 was expressed in ∼14% of DLBCL patients. Patients with CD30(+) DLBCL had superior 5-year overall survival (CD30(+), 79% vs CD30(-), 59%; P = .001) and progression-free survival (P = .003). The favorable outcome of CD30 expression was maintained in both the germinal center B-cell and activated B-cell subtypes. Gene expression profiling revealed the upregulation of genes encoding negative regulators of nuclear factor κB activation and lymphocyte survival, and downregulation of genes encoding B-cell receptor signaling and proliferation, as well as prominent cytokine and stromal signatures in CD30(+) DLBCL patients, suggesting a distinct molecular basis for its favorable outcome. Given the superior prognostic value, unique gene expression signature, and significant value of CD30 as a therapeutic target for brentuximab vedotin in ongoing successful clinical trials, it seems appropriate to consider CD30(+) DLBCL as a distinct subgroup of DLBCL.

Patients with diffuse large B-cell lymphoma of germinal center origin with BCL2 translocations have poor outcome, irrespective of MYC status: a report from an International DLBCL rituximab-CHOP Consortium Program Study

Diffuse large B-cell lymphoma can be classified by gene expression profiling into germinal center and activated B-cell subtypes with different prognoses after rituximab-CHOP. The importance of previously recognized prognostic markers, such as Bcl-2 protein expression and BCL2 gene abnormalities, has been questioned in the new therapeutic era. We analyzed Bcl-2 protein expression, and BCL2 and MYC gene abnormalities by interphase fluorescence in situ hybridization in 327 patients with de novo disease treated with rituximab-CHOP. Isolated BCL2 and MYC rearrangements were not predictive of outcome in our patients as a whole, but only in those with the germinal center subtype of lymphoma. The prognostic relevance of isolated MYC rearrangements was weaker than that of BCL2 isolated translocations, but was probably limited by the rarity of the rearrangements. Seven of eight patients with double hit lymphoma had the germinal center subtype with poor outcome. The germinal center subtype patients with isolated BCL2 translocations had significantly worse outcome than the patients without BCL2 rearrangements (P=0.0002), and their outcome was similar to that of patients with the activated B-cell subtype (P=0.30), but not as bad as the outcome of patients with double hit lymphoma (P<0.0001). Bcl-2 protein overexpression was associated with inferior outcome in patients with germinal center subtype lymphoma, but multivariate analysis showed that this was dependent on BCL2 translocations. The gene expression profiling of patients with BCL2 rearrangements was unique, showing activation of pathways that were silent in the negative counterpart. BCL2 translocated germinal center subtype patients have worse prognosis after rituximab-CHOP, irrespective of MYC status, but the presence of combined gene breaks significantly overcomes the prognostic relevance of isolated lesions.

An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5‐d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r2 correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra‐laboratory reproducibility and with comparable quality and reliability.

Intraplatform Reproducibility and Technical Precision of Gene Expression Profiling in 4 Laboratories Investigating 160 Leukemia Samples: The DACH Study

BACKGROUND Gene expression profiling has the potential to offer consistent, objective diagnostic test results once a standardized protocol has been established. We investigated the robustness, precision, and reproducibility of microarray technology. METHODS One hundred sixty individual patient samples representing 11 subtypes of acute and chronic leukemias, myelodysplastic syndromes, and nonleukemia as a control group were centrally collected and diagnosed as part of the daily routine in the Munich Leukemia Laboratory. The custom AmpliChip Leukemia research microarray was used for technical analyses of quadruplicate mononuclear cell lysates in 4 different laboratories in Germany (D), Austria (A), and Switzerland (CH) (the DACH study). RESULTS Total-RNA preparations were successfully performed in 637 (99.5%) of 640 cases. Mean differences between pairs of laboratories in the total-RNA yield from the same sample ranged from 0.02 mug to 1.03 mug. Further processing produced 622 successful in vitro transcription reactions (97.6%); the mean differences between laboratories in the cRNA yield from the same sample ranged from 0.40 mug to 6.18 mug. After hybridization to microarrays, a mean of 47.6%, 46.5%, 46.2%, and 46.4% of probe sets were detected as present for the 4 laboratories, with mean signal-intensity scaling factors of 3.1, 3.7, 4.0, and 4.2, respectively. In unsupervised hierarchical cluster and principal component analyses, replicates from the same patient always clustered closely together, with no indications of any association between gene expression profiles due to different operators or laboratories. CONCLUSIONS Microarray analysis can be performed with high interlaboratory reproducibility and with comparable quality and high technical precision across laboratories.

New data on robustness of gene expression signatures in leukemia: comparison of three distinct total RNA preparation procedures

BackgroundMicroarray gene expression (MAGE) signatures allow insights into the transcriptional processes of leukemias and may evolve as a molecular diagnostic test. Introduction of MAGE into clinical practice of leukemia diagnosis will require comprehensive assessment of variation due to the methodologies. Here we systematically assessed the impact of three different total RNA isolation procedures on variation in expression data: method A: lysis of mononuclear cells, followed by lysate homogenization and RNA extraction; method B: organic solvent based RNA isolation, and method C: organic solvent based RNA isolation followed by purification.ResultsWe analyzed 27 pediatric acute leukemias representing nine distinct subtypes and show that method A yields better RNA quality, was associated with more differentially expressed genes between leukemia subtypes, demonstrated the lowest degree of variation between experiments, was more reproducible, and was characterized with a higher precision in technical replicates. Unsupervised and supervised analyses grouped leukemias according to lineage and clinical features in all three methods, thus underlining the robustness of MAGE to identify leukemia specific signatures.ConclusionThe signatures in the different subtypes of leukemias, regardless of the different extraction methods used, account for the biggest source of variation in the data. Lysis of mononuclear cells, followed by lysate homogenization and RNA extraction represents the optimum method for robust gene expression data and is thus recommended for obtaining robust classification results in microarray studies in acute leukemias.