Gene Expression Profiling (GEP) of CD38-Expressing/Unmutated B-Cell Chronic Lymphocytic Leukemia (B-CLL) Cells by Using a Statistical Approach Suitable for Analysis of Unbalanced Datasets.
Authors: Gattei Valter , Benedetti Dania , Marconi Daniela , Zucchetto Antonella , Dal Michele , Bulian Pietro , Poeta Giovanni Del , Palermo Giuseppe , Steffan Agostino , Degan Massimo , Campanini Renato , Bomben Riccardo 
Published: October 2019

Abstract
B-CLL is a apparently homogeneous disease with variable clinical courses, which can be foreseen by the presence of mutated (M) or unmutated (UM) IgVH genes and the expression of prognostic markers, including CD38. Since a correlation between high CD38 and UM IgVH gene configuration has been described, we performed GEP to identify the gene signature of CD38+/UM B-CLLs. Purified (>95%) B-CLL cells from 44 cases were utilized for a dual-labeling GEP strategy (Operon Human Genome 2.1 OligoSet; 21,329 70mers) with pooled normal PB B-cells as common reference. 12 B-CLLs were UM (<2% IgVH mutations) and CD38pos (CD38>30% of B-CLL cells), while 32 were M (>2% IgVH mutations) and CD38neg (CD38<10% of B-CLL cells). To discover genes differentially expressed in the two categories and overcome the problem of unbalanced dataset, we applied an original bioinformatic approach called multi-SAM (Significance Analysis of Microarrays). This consists in reiterated applications of SAM analysis comparing the less populated CD38pos/UM class with 1,000 random samplings, each of 12 cases, from the CD38neg/M class. For each single application of SAM, a list of differentially expressed genes (p<10-3) was generated. At the end of 1,000 reiterations, each single gene was labeled with a 0-1,000 list score (LS) based on the times it was selected by multi-SAM as differentially expressed. A significant LS threshold>300 was determined by applying multi-SAM to 1,000 random comparisons of two mock-classes, each of 12 cases, from the same dataset. The final gene list was further shrunk by keeping only the genes with a median-log-difference (MLD) between the two categories exceeding the absolute value of 1; eventually, a list of 132 genes (44 down-regulated and 88 up-regulated in CD38pos/UM cases) was obtained. According to these analyses, CD38pos/UM B-CLLs overexpressed the following gene groups: i) genes related to lipid metabolism: mainly Lipoprotein Lipase (LS=744, MLD=2.05), but also low-density-lipoprotein receptor (LDLR) and LDLR-related-protein-5, these latter with a LS>300 but lower (0.7) MLDs. ii) genes related to cell-cell/cell-matrix interactions: CD49d/alpha4 integrin (LS=354, MLD=1.14), a molecule whose expression has already been correlated with CD38 in previous extensive surface antigen expression studies of ours; the C-C chemokines MIP-1alpha (a.k.a. CCL3; LS=660, MLD=1.46) and MIP-1beta (a.k.a. CCL4; LS=334, MLD=1.36); CD72 (low-affinity CD100 ligand; LS=523, MLD=1.06). iii) genes related to vescicle trafficking/cytoskeletron reorganization: septin-7 (LS 386, MLD=1.19) and septin-10 (LS=926, MLD=3.12); the spastic paraplegia-20 protein (a.k.a. spartin, LS=886, MLD=1.84); iii) Activation-Induced Cytidine Deaminase (AICD; LS=599, MLD=2.07), a gene preliminarly found as overexpressed in UM B-CLLs. Altogether, these genes, besides having clinical value as additional prognosticators, may be implied in several aspects of the functional cross-talk between CD38pos/UM B-CLL and neighbouring cells within the lymph node microenvironment, this interplay eventually affecting survival of tumor cells.