Immunoparesis defined by heavy+light chain suppression is a novel marker of long-term outcomes in cardiac AL amyloidosis.

Cardiac involvement and presenting dFLC (difference between involved and uninvolved free light chains) are independent predictors of outcome in systemic AL amyloidosis. These markers have limited prognostic utility in patients surviving the initial months following diagnosis. Here we assessed immunoparesis, as determined by novel heavy+light chain (HLC) immunoassays, as a prognostic marker for survival in AL amyloidosis. HLC measurements identified immunoparesis of at least one immunoglobulin (Ig) isotype in 145 (85%) patients; and severe immunoparesis (≥2 Ig isotypes suppressed by >50% below normal levels) in 29 (17%) patients. Median overall survival (OS) on intention to treat (ITT) analysis was 26·2 months. In the ITT cohort, dFLC >180 mg/l was associated with shorter OS (P = 0·05); whereas HLC immunoparesis was not prognostic. On a landmark analysis of 127 patients alive at 6 months, presenting dFLC was not prognostic for OS (P = 0·33) and severe HLC immunoparesis trended towards poorer survival (20·2 vs. 42·8 months; P = 0·09). In the subset of patients with cardiac involvement, severe HLC immunoparesis conferred very poor outcome (median OS 8·8 vs. 29·9 months, P = 0·007). In conclusion, severe HLC immunoparesis is an independent marker of long-term poor prognosis in AL patients with cardiac involvement. The pathophysiological significance of this observation needs further study.

Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL.

The ataxia telangiectasia mutated (ATM) protein is the principal activator of the p53 protein in the response to DNA double-strand breaks. Mutations in the ATM gene have been previously found in B-cell chronic lymphocytic leukemias (B-CLLs) but their clinical significance is unknown. We analyzed 155 CLL tumors and found 12% with ATM mutations and 4% with TP53 mutations; 2 tumors contained mutations in both genes. Retrospective analysis on selected samples indicated that the ATM mutations were usually present at diagnosis. Compared with patients with wild-type ATM/TP53 genes, patients with ATM mutations had statistically significantly reduced overall and treatment-free survival. Although present in both IGVH mutation subgroups, ATM mutations were associated with unmutated IGVH genes and they provided independent prognostic information on multivariate analysis. Mutations in the ATM gene resulted in impaired in vitro DNA damage responses. Tumors with ATM mutations only partially correlated with tumors with loss of an ATM allele through an 11q deletion and, interestingly, those 11q-deleted tumors with a second wild-type ATM allele had a preserved DNA damage response. The majority of patients with ATM mutations were refractory to DNA damaging chemotherapeutic drugs and as such might benefit from therapies that bypass the ATM/p53 pathway.

Contribution of the ABC transporters Bcrp1 and Mdr1a/1b to the side population phenotype in mammary gland and bone marrow of mice.

The ability of cells to export Hoechst 33342 can be used to identify a subpopulation of cells (side population [SP]) with characteristics of stem cells in many tissues. The ATP-binding cassette transporters Bcrp1 (Abcg2) and Mdr1a/1b (Abcb1a/1b) have been implicated as being responsible for this phenotype. To further explore the involvement of these transporters in the SP phenotype, we have generated Bcrp1/Mdr1a/1b triple knockout mice and studied the effect of their absence on the SP in bone marrow and mammary gland. Whereas in bone marrow Bcrp1 was almost exclusively responsible for the SP, both transporters contributed to the SP phenotype in the mammary gland, where their combined absence resulted in a nearly complete loss of SP. Interestingly, bone marrow of Mdr1a/1b-/- mice frequently displayed an elevated SP, which was reversible by the Bcrp1 inhibitor Ko143, suggesting that Bcrp1 can compensate for the loss of Mdr1a/1b in bone marrow.

A novel CDK inhibitor, CYC202 (R-roscovitine), overcomes the defect in p53-dependent apoptosis in B-CLL by down-regulation of genes involved in transcription regulation and survival.

B-cell chronic lymphocytic leukemia (B-CLL) is a clinically variable disease where mutations in DNA damage response genes ATM or TP53 affect the response to standard therapeutic agents. The in vitro cytotoxicity of a novel cyclin-dependent kinase inhibitor, CYC202, was evaluated in 26 B-CLLs, 11 with mutations in either the ATM or TP53 genes, and compared with that induced by ionizing radiation and fludarabine. CYC202 induced apoptosis within 24 hours of treatment in all 26 analyzed tumor samples independently of ATM and TP53 gene status, whereas 6 of 26 B-CLLs, mostly ATM mutant, showed marked in vitro resistance to fludarabine-induced apoptosis. Compared with B-CLLs, normal T and B lymphocytes treated with CYC202 displayed reduced and delayed apoptosis. Using global gene expression profiling, we found that CYC202 caused a significant down-regulation of genes involved in regulation of transcription, translation, survival, and DNA repair. Furthermore, induction of apoptosis by CYC202 was preceded by inhibition of RNA polymerase II phosphorylation, leading to down-regulation of several prosurvival proteins. We conclude that CYC202 is a potent inducer of apoptosis in B-CLL regardless of the functional status of the p53 pathway, and may be considered as a therapeutic agent to improve the outcome of resistant B-CLL tumors.

Apoptotic resistance to ionizing radiation in pediatric B-precursor acute lymphoblastic leukemia frequently involves increased NF-kappaB survival pathway signaling.

To investigate possible causes of the variable response to treatment in pediatric B-precursor acute lymphoblastic leukemia (ALL) and to establish potential novel therapeutic targets, we used ionizing radiation (IR) exposure as a model of DNA damage formation to identify tumors with resistance to p53-dependent apoptosis. Twenty-one of 40 ALL tumors responded normally to IR, exhibiting accumulation of p53 and p21 proteins and cleavage of caspases 3, 7, and 9 and of PARP1. Nineteen tumors exhibited apoptotic resistance and lacked PARP1 and caspase cleavage; although 15 of these tumors had normal accumulation of p53 and p21 proteins, examples exhibited abnormal expression of TRAF5, TRAF6, and cIAP1 after IR, suggesting increased NF-kappaB prosurvival signaling as the mechanism of apoptotic resistance. The presence of a hyperactive PARP1 mutation in one tumor was consistent with such increased NF-kappaB activity. PARP1 inhibition restored p53-dependent apoptosis after IR in these leukemias by reducing NF-kappaB DNA binding and transcriptional activity. In the remaining 4 ALL tumors, apoptotic resistance was associated with a TP53 mutation or with defective activation of p53. We conclude that increased NF-kappaB prosurvival signaling is a frequent mechanism by which B-precursor ALL tumors develop apoptotic resistance to IR and that PARP1 inhibition may improve the DNA damage response of these leukemias.

Microarray analysis reveals that TP53- and ATM-mutant B-CLLs share a defect in activating proapoptotic responses after DNA damage but are distinguished by major differences in activating prosurvival responses.

The ATM/p53-dependent DNA damage response pathway plays an important role in the progression of lymphoid tumors. Inactivation of the ATM or TP53 gene is frequent in B-cell lymphocytic leukemia (B-CLL) and leads to aggressive disease. Although the ATM and p53 pathways overlap, they are not congruent, and it is unclear how the mechanism of tumor progression differs between ATM- and p53-deficient tumors. Using microarray analysis of ATM-mutant, TP53-mutant, and ATM/TP53 wild-type B-CLLs, we show that after exposure to DNA damage transcriptional responses are entirely dependent on ATM function. The p53 proapoptotic responses comprise only a part of ATM-regulated transcription; additionally, ATM regulates prosurvival responses independently of p53. Consequently, the greater severity of the TP53-mutant B-CLLs compared with ATM-mutant B-CLLs is consistent with the additive effect of defective apoptotic and elevated survival responses after DNA damage in these tumors. We also show that transcription expression profiles of ATM-deficient, TP53-deficient, and wild-type B-CLLs are indistinguishable before irradiation. Therefore, damage-induced transcriptional fingerprinting can be used to stratify tumors according to their biologic differences and simultaneously identify potential targets for treating refractory tumors.

Functional and molecular characterisation of mammary side population cells.

BACKGROUND: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown. METHODS: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP. RESULTS: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures. CONCLUSION: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.