Although, several molecular and cytogenetic lesions have emerged as potential prognostic indicators for CLL many disparities and confounding issues limit their clinical utility 
. For example, although primary resistance to fludarabine has been shown to occur in patients harboring p53 deletions, a recent study reported that treatment-naïve patients with p53 deletions exhibit clinical heterogeneity with some patients experiencing an indolent course 
. These published clinical studies suggest that there are underlying differences in CLL biology, which if understood, could provide more reliable prognostic information for individual patients.
The data in this study have highlighted a link between H2O2-induced changes in phosphorylation of signaling proteins downstream of the BCR and in vitro F-ara-A-mediated apoptosis in CLL B cells. Specifically, the data showed: (1) The sample cohort could be divided into two groups based on the size of a cell subset within each sample that was responsive to the reactive oxygen species H2O2 based on increased phosphorylation of p-Lyn, p-Syk, p-BLNK, p-PLCγ2 and p-Stat5. (2) In most cases in which the H2O2 responsive subpopulation was greater than 30%, a cell subset proficient for F-ara-A mediated apoptosis was seen in the same sample. (3) A mixture modeling metric was derived that was linked to the presence or absence of these observed cell subsets to apoptosis response. (4) AUC values for this model were above 0.75 for p-Lyn, p-Syk, p-BLNK, p-PLCγ2 and p-Stat5 and stratified patient samples according to their apoptotic pathway response.
Although the CLL sample cohort was obtained from CLL patients receiving different treatments, it was striking that their H2
response was able to segregate the samples into two groups. The concentration of H2
used in study was in the millimolar range and was chosen based on its ability to mediate a response in leukemic B cells but not in healthy B cells as previously reported 
. It is difficult to ascertain exact intracellular concentrations of H2
as its production tends to be localized either in the plasma membrane or in endosomes and inactivating antioxidant enzymes prevent indiscriminate oxidation of intracellular molecules by H2
is produced by NADPH Oxidase (Nox) enzymes that are activated by cell surface receptors including BCR 
. To function as an intracellular signaling molecule, H2
must be imported into the cytosol and reported intracellular concentrations range from micromolar to millimolar levels 
. As an intracellular second messenger, H2
results in amplification of receptor tyrosine kinase signaling by transiently and reversibly inactivating tyrosine phosphatases (PTPS) through reversible oxidation of the catalytic cysteine to sulfenic acid 
. A likely role for PTPs in both ligand-dependent and independent BCR signaling was revealed in several studies. In healthy B cells and follicular lymphoma H2
participates in anti-µ mediated signaling 
. Other reports showed that Syk was activated by pervanadate/H2
in the absence of BCR crosslinking 
That activated BCR signaling molecules, in the absence of ligand, play an important survival role in CLL and other B cell malignancies is substantiated by recent studies. One study showed that in CLL B cells where Lyn protein is over-expressed, its inhibition by small molecule inhibitors in vitro
in the absence of a BCR ligand, induced apoptosis 
. Corroborating these findings, in vitro
treatment of DLBCL and CLL cells with R406 a small molecule inhibitor of Syk (a substrate of Lyn) also induced apoptosis 
. More recently, a phase I/II clinical trial of fostamatinib disodium, an oral Syk inhibitor showed clinical activity in CLL and non-Hodgkins lymphoma 
. Another study showed a negative correlation between the expression of ZAP70 with the phosphorylation state of Syk and a positive correlation between p-Syk with p21cip
, a cell cycle inhibitor 
. Further insights into the relationship of phosphatase activity with BCR signaling molecules and apoptosis could be determined by experiments including specific tyrosine phosphatase inhibitors specifically targeting SHP-1 and/or CD45. A priori, such inhibitors would be predicted to promote CLL blast cell survival. Consistent with this hypothesis, ectopic expression of protein tyrosine phosphatase, PTPRO, (silenced in CLL by DNA methylation) increased growth inhibition in response to F-ara-A 
. In DLBCL, PTPROt was identified as a tumor suppressor with a role in tonic BCR signaling 
. Furthermore, additional studies will be required to determine whether lymphoid tyrosine phosphatase (Lyp) also known as PTPN22, whose expression was reported to be increased in CLL B cells, plays a role in the response of CLL B cells to therapeutic agents (Negro et al., Blood
(ASH Annual Meeting Abstracts) 2009 114: Abstract 800).
Although not definitively proven, the data in this study potentially support a mechanism whereby H2O2 through inhibition of tyrosine phosphatases relieves a negative feedback loop that results in activation of signaling proteins within the BCR network. Regardless of its exact mechanism of action, H2O2 was able to reveal differential signaling within CLL samples and these signaling differences appear to be associated with a signaling posture that either drives, or is driven by the ability of these cells to undergo apoptotic induction by, in this case F-ara-A.
In this study, SCNP analysis, combined with mixture modeling identified at least two phenotypes of CLL B cells based on their H2
– mediated response of signaling molecules (, (B), (C), ). Notably, some samples demonstrated simultaneous presence of both cell subsets, suggesting co-evolution of signaling phenotypes, a common precursor of these cell subsets, or a lineage relationship between the two subpopulations of cells (). Interestingly, and in contrast to studies where the presence of ZAP70 and unmutated IgVH
correlated with greater anti−μ-mediated-BCR signaling 
, the signaling responses described here were unrelated to the IgVH
mutational status or to ZAP70 expression and spanned a range of cytogenetic abnormalities. Although further studies are warranted to investigate this issue, it is important to note that the above studies 
were accomplished via indirect assay of total phospho-tyrosine on signaling proteins. In our study, we undertook direct assay of phosphorylation sites using antibodies directed against known, functional, epitopes on a per cell basis. Additionally, no associations were observed between SHP-1, SHP-2, CD22 or CD45 expression levels with H2
-mediated signaling (data not shown).
Although not a member of the canonical BCR signaling network, the increase seen in H2
–mediated p-Stat5 could be due to a bystander effect resulting from phosphatase inhibition with consequent increases in kinase activities for which Stat5 is a substrate. Interestingly, Sattler et al, showed the importance of H2
generation with consequent increases in p-Stat5 in several hematopoietic growth factor cascades in cell lines 
. A pivotal role was also demonstrated for activated Stat5 in hematopoietic stem cell self- renewal and expansion of multi-potential progenitors in myeloid disease 
. In addition, highly distinctive cytokine responses in Stat5 phosphorylation were reported in both normal and leukemic stem/progenitor cells 
. Furthermore, in a recent study phospholipase C-β3 was shown to be a tumor suppressor by acting as a scaffold for simultaneous interaction with p-Stat5 and SHP-1 and by doing so promoted the dephosphorylation of p-Stat5 
. Whether these mechanisms regulate p-Stat5 in CLL awaits further study.
The clinical complexity (and unpredictability) of CLL as well as the many components governing cell proliferation and survival mechanisms, suggest a diversity of mechanisms that give rise to CLL. Nonetheless, the current studies, although mechanistically incomplete demonstrate a convergence of signaling patterns in CLL that lead to a remarkably limited set of phenotypic cell signaling outcomes. This suggests that despite the underlying molecular and clinical heterogeneity that maintains cellular homeostasis in CLL, only a limited number of signaling pathway variations exist and these may be exploited for therapeutic benefit. Although the sample set was limited, the encouraging AUC values () endorse follow-up studies with expanded sample cohorts, both cryopreserved and fresh, to determine whether SCNP of individual samples can predict treatment outcome and stratify patients who might gain the most benefit from fludarabine-based treatment regimens.