Developing Molecular Signatures for Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is a clonal malignancy of mature B cells that displays a great clinical heterogeneity, with many patients having an indolent disease that will not require intervention for many years, while others present an aggressive and symptomatic leukemia requiring immediate treatment. Although there is no cure for CLL, the disease is treatable and current standard chemotherapy regimens have been shown to prolong survival. Recent advances in our understanding of the biology of CLL have led to the identification of numerous cellular and molecular markers with potential diagnostic, prognostic and therapeutic significance. We have used the recently developed digital multiplexed gene-expression technique (DMGE) to analyze a cohort of 30 CLL patients for the presence of specific genes with known diagnostic and prognostic potential. Starting from a set of 290 genes we were able to develop a molecular signature, based on the analysis of 13 genes, which allows distinguishing CLL from normal peripheral blood and from normal B cells, and a second signature based on 24 genes, which distinguishes mutated from unmutated cases (LymphCLL Mut). A third classifier (LymphCLL Diag), based on a 44-gene signature, distinguished CLL cases from a series of other B-cell chronic lymphoproliferative disorders (n = 51). While the methodology presented here has the potential to provide a "ready to use" classification tool in routine diagnostics and clinical trials, application to larger sample numbers are still needed and should provide further insights about its robustness and utility in clinical practice

Evaluation of paroxysmal nocturnal hemoglobinuria screening by flow cytometry through multicentric interlaboratory comparison in four countries.

OBJECTIVES: Paroxysmal nocturnal hemoglobinuria (PNH) is currently diagnosed by flow cytometry; although highly sensitive, its interpretation and reporting appear as critical as its technique. Thus, we developed a quality control scheme for the French-speaking region based on the international recommendations for PNH screening. METHODS: After a topical workshop, we proposed a 1-year, two-step survey program to any volunteering French-speaking clinical laboratory. The first survey consisted of sending raw data files to evaluate gating and the interpretation strategy of each center. The second stipulated sending fresh whole-blood samples to evaluate the whole process and its practice. RESULTS: Forty-nine participants from voluntary centers returned results for each of the two successive surveys. On virtual survey, 27% reported false-positive PNH created by immature granulocytes, whereas the minor PNH clone was not detected by 9%. On fresh survey, 63% of centers used at least the same six-color combination (CD24, CD14, CD33, CD15, CD45, and fluorescent aerolysin), and nearly 70% of participants were able to perform a sensitivity test less than 0.1% on neutrophils. All participants detected the major PNH clone, yet 16% returned false-positive results for the non-PNH clone case. CONCLUSIONS: We succeeded in rallying numerous French-speaking clinical laboratories for both surveys and in harmonizing the technical practice by highlighting common pitfalls

Hairy cell leukemia: a specific 17-gene expression signature points to new targets for therapy

Background: Hairy cell leukemia (HCL) is a rare chronic B cell malignancy, characterized by infiltration of bone marrow, blood and spleen by typical "hairy cells" that bear the BRAFV600E mutation. However, in addition to the intrinsic activation of the MAP kinase pathway as a consequence of the BRAFV600E mutation, the potential participation of other signaling pathways to the pathophysiology of the disease remains unclear as the precise origin of the malignant hairy B cells. Materials and methods: Using mRNA gene expression profiling based on the Nanostring technology and the analysis of 290 genes with crucial roles in B cell lymphomas, we defined a 17 gene expression signature specific for HCL. Results: Separate analysis of samples from classical and variant forms of hairy cell leukemia showed almost similar mRNA expression profiles apart from overexpression in vHCL of the immune checkpoints CD274 and PDCD1LG2 and underexpression of FAS. Our results point to a post-germinal memory B cell origin and in some samples to the activation of the non-canonical NF-κB pathway. Conclusions: This study provides a better understanding of the pathogenesis of HCL and describes new and potential targets for treatment approaches and guidance for studies in the molecular mechanisms of HCL.</p

LDOC1 expression in CLL samples.

<p>CLL samples were separated into those highly expressing LDOC1 and those with absent/low expression. Listed are those genes that fulfill the following criteria: >2-fold change in expression between LDOC1 pos samples compared to LDOC1 neg samples, with a p-value < 0.05 or >2-fold change in expression between LDOC1 neg samples compared to LDOC1 pos samples, with a p-value < 0.05.</p><p>LDOC1 expression in CLL samples.</p

Correlation between mRNA and protein expression.

<p><b>(A)</b> Correlation between CD38 protein expression, as measured by flow cytometry (% positive CLL B cells) and CD38 mRNA counts, as measured by the nCounter (arbitrary units). (B) Quantification of CD38 and ZAP70 mRNA counts in normal peripheral blood (PB), pure B cells, and in CLL B cells. CLL B cells were analyzed for ZAP70 and for CD38 expression by flow cytometry and then grouped for the mRNA determination, according to presence or absence of these two antigens. (C) Immunoglobulin light chain ratios in 30 CLL patient samples (rhombi) and in normal B cells (triangles: normal PB samples; circles: pure B cell samples). The mean +/- 2SD interval for ratios from polyclonal normal B cells and normal PB is shown (mean 0.89; SD = 0.22; small dots).</p

Analysis of the expression of 290 genes in normal PB, pure B cell and CLL samples.

<p>(A) Venn diagram of genes expressed preferentially in the different sample groups (normal PB, n = 5; pure B cells, n = 4; and CLL samples, n = 30). Genes were considered preferentially expressed by one sample group, if they showed an expression level ≥50 counts and a ≥2-fold difference in expression levels between the 2 groups, with a p-value ≤0.05. (B) Samples from PB (n = 5), B cell samples (n = 4), and samples from CLL patients (n = 30) were analyzed by PCA, based on the results of the differential expression of 290 genes. (C) PCA analysis on the same samples as in (b), but using a restricted set of 44 genes relevant for this purpose according to their differential expression in CLL, B cells and normal blood. (D) Heat map of normal PB, pure B cells, and CLL samples, analyzed with thirteen genes overexpressed homogenously (CV<5%) in all CLL samples compared to normal PB and pure B cells. Unsupervised analysis shows a perfect clustering of the CLL samples compared to the normal samples.</p