Recombinant Alpaca Anti-Rabbit IgG monoclonal antibody, Alexa Fluor®647

Background B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease and the most common adult leukemia in western countries. IgVH mutational status distinguishes two major types of CLL, each associated with a different prognosis and survival. Sequencing identified NOTCH1 and SF3B1 as the two main recurrent mutations. We described a novel method to clarify how these mutations affect gene expression by finding small-scale signatures that predict the IgVH, NOTCH1 and SF3B1 mutations. We subsequently defined the biological pathways and correlation networks involved in disease development, with the potential goal of identifying new drugable targets. Methods We modeled a microarray dataset consisting of 48807 probes derived from 163 samples. The use of Fisher's ratio and fold change combined with feature elimination allowed us to identify the minimum number of genes with the highest predictive mutation power and, subsequently, we applied network and pathway analyses of these genes to identify their biological roles. Results The mutational status of the patients was accurately predicted (94-99%) using small-scale gene signatures: 13 genes for IgVH, 60 for NOTCH1 and 22 for SF3B1. LPL plays an important role in the case of the IgVH mutation, whereas MSI2, LTK, TFEC and CNTAP2 are involved in the NOTCH1 mutation, and RPL32 and PLAGL1 are involved in the SF3B1 mutation. Four high discriminatory genes (IGHG1, MYBL1, NRIP1 and RGS1) are common to these three mutations. The IL-4-mediated signaling events pathway appears to be involved as a common mechanism and suggests an important role of the immune response mechanisms and antigen presentation. Conclusions This retrospective analysis served to provide a deeper understanding of the effects of the different mutations in CLL disease progression, with the expectation that these findings will be clinically applied in the near future to the development of new drugs.

Breast cancer is the most common malignancy affecting women, but the heterogeneity of the condition is a significant obstacle to effective treatment. Triple negative breast cancers (TNBCs) do not express HER2 or the receptors for estrogen or progesterone, and so often have a poor prognosis. Tumor-infiltrating T cells have been well-characterized in TNBC, and increased numbers are associated with better outcomes; however, the potential roles of B cells and plasma cells have been large. Here, we conducted a retrospective correlative study on the expression of B cell/plasma cell-related genes, and the abundance and localization of B cells and plasma cells within TNBCs, and clinical outcome. We analyzed 269 TNBC samples and used immunohistochemistry to quantify tumor-infiltrating B cells and plasma cells, coupled with NanoString measurement of expression of immunoglobulin metagenes. Multivariate analysis revealed that patients bearing TNBCs with above median densities of CD38(+) plasma cells had significantly better disease-free survival (DFS) (HR = 0.44; 95% CI 0.26-0.77; p = 0.004) but not overall survival (OS), after adjusting for the effects of known prognostic factors. In contrast, TNBCs with higher immunoglobulin gene expression exhibited improved prognosis (OS p = 0.029 and DFS p = 0.005). The presence of B cells and plasma cells was positively correlated (p < 0.0001, R = 0.558), while immunoglobulin gene IGKC, IGHM, and IGHG1 mRNA expression correlated specifically with the density of CD38 plasma cells (IGKC p < 0.0001,R=0.647; IGHM p < 0.0001,R=0.580; IGHG1 p < 0.0001,R=0.655). Interestingly, after adjusting the multivariate analysis for the effect of intratumoral CD38(+) plasma cell density, the expression levels of all three genes lost significant prognostic value, suggesting a biologically important role of plasma cells. Last but not least, the addition of intratumoral CD38+ plasma cell density to clinicopathological features significantly increased the prognostic value for both DFS (Delta LR chi(2) = 17.28, p = 1.71E-08) and OS (Delta LR chi(2) = 10.03, p = 6.32E-08), compared to clinicopathological features alone. The best combination was achieved by integrating intratumoral CD38 plasma cell density and IGHG1 which conferred the best added prognostic value for DFS (Delta LR chi(2) = 27.38, p = 5.22E-10) and OS (Delta LR chi(2) = 21.29, p = 1.03E-08). Our results demonstrate that the role of plasma cells in TNBC warrants further study to elucidate the relationship between their infiltration of tumors and disease recurrence.