Association of Minimal Residual Disease With Superior Survival Outcomes in Patients With Multiple Myeloma: A Meta-analysis.

IMPORTANCE: Numerous studies have evaluated the prognostic value of minimal residual disease (MRD) in patients with multiple myeloma (MM). Most studies were small and varied in terms of patient population, treatment, and MRD assessment methods. OBJECTIVE: To evaluate the utility of MRD detection in patients with newly diagnosed MM. DATA SOURCES: A Medline search was conducted for articles published in English between January 1990 and January 2016. STUDY SELECTION: Eligible studies reported MRD status and progression-free survival (PFS) or overall survival (OS) in 20 or more patients following treatment. Among 405 articles identified, 21 met the initial eligibility criteria and were included in the analysis. DATA EXTRACTION AND SYNTHESIS: Information on patient characteristics, treatment, MRD assessment, and outcomes were extracted using a standard form. MAIN OUTCOMES AND MEASURES: The impact of MRD status on PFS and OS was assessed by pooling data from relevant trials. Data were adjusted to allow for different proportions of patients with MRD in different studies, and analyzed using the Peto method. Forest plots were created based on Cox model analysis. Other prespecified research questions were addressed qualitatively. RESULTS: Fourteen studies (n = 1273) provided data on the impact of MRD on PFS, and 12 studies (n = 1100) on OS. Results were reported specifically in patients who had achieved conventional complete response (CR) in 5 studies for PFS (n = 574) and 6 studies for OS (n = 616). An MRD-negative status was associated with significantly better PFS overall (hazard ratio [HR], 0.41; 95% CI, 0.36-0.48; P < .001) and in studies specifically looking at CR patients (HR, 0.44; 95% CI, 0.34-0.56; P < .001). Overall survival was also favorable in MRD-negative patients overall (HR, 0.57; 95% CI, 0.46-0.71; P < .001) and in CR patients (HR, 0.47; 95% CI, 0.33-0.67; P < .001). Tests of heterogeneity found no significant differences among the studies for PFS and OS. CONCLUSIONS AND RELEVANCE: Minimal residual disease-negative status after treatment for newly diagnosed MM is associated with long-term survival. These findings provide quantitative evidence to support the integration of MRD assessment as an end point in clinical trials of MM.

The gene expression signature associated with TP53 mutation/deletion in chronic lymphocytic leukaemia is dominated by the under-expression of TP53 and other genes on chromosome 17p.

In chronic lymphocytic leukaemia (CLL), TP53 mutation and deletion are strongly associated with one another and with adverse clinical outcome. Mutant TP53 protein typically accumulates to high levels and has been reported to have transcriptional regulatory activity distinct from that of wild-type TP53. To investigate whether such an effect is relevant to CLL, carefully balanced primary CLL samples with or without TP53 mutation/deletion were compared for their gene expression profiles using high-density DNA microarrays. Ninety-six and eight differentially expressed genes were identified, respectively, using two alternative statistical approaches with different stringencies. None of the differentially expressed genes were known to be regulated by mutant TP53, and only four of the 67 under-expressed genes were known transcriptional targets of wild-type TP53. Significantly, both approaches showed that gene under-expression was the dominant feature of TP53-mutant CLL samples. Furthermore, a disproportionate number of the under-expressed genes were located on chromosome 17p, the most significant being TP53 itself. Together, these results indicate that any transcriptional regulatory effects of mutant TP53 in CLL cells are overshadowed by the under-expression of co-deleted TP53 and other genes on chromosome 17p. Our findings have implications for emerging therapeutic strategies that target mutant TP53.

A novel type of p53 pathway dysfunction in chronic lymphocytic leukemia resulting from two interacting single nucleotide polymorphisms within the p21 gene.

The ATM-p53 pathway plays an important role in the biology of chronic lymphocytic leukemia (CLL). Its functional integrity can be probed by exposing CLL cells to ionizing radiation (IR) and measuring levels of p53 protein and one of its transcriptional targets, the cyclin-dependent kinase inhibitor p21. We have previously identified two abnormal p53/p21 response patterns associated with inactivating mutations of TP53 and ATM, respectively. Here, we describe a third abnormal response pattern characterized by failure of p21 protein accumulation despite a normal p53 protein response. This so-called "type C" response was detected in 10.6% of unselected patients and was associated with resistance of CLL cells to p53-dependent killing by IR, with the clinically more aggressive variant of CLL characterized by unmutated immunoglobulin heavy-chain genes and with a single nucleotide polymorphism at codon 31 of the p21 gene in which Ser is replaced by Arg. CLL samples with this allelic variant displayed impaired IR-induced up-regulation of total p21 mRNA and did not express the Arg-encoding transcript, except in those cases harboring an additional single nucleotide polymorphism (T instead of C) in the 3'-untranslated region of the same p21 allele. Our data provide new insight into the importance of p21 in CLL biology.

B-cell receptor translocation to lipid rafts and associated signaling differ between prognostically important subgroups of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is a highly heterogeneous disease in which interaction of the malignant cells with antigen is thought to play a key role. Individual CLL-cell clones markedly differ in their ability to respond to B-cell receptor ligation, but the mechanism underlying the frequent hyporesponsiveness is incompletely understood. Our aim was to further clarify the extent and cause of the B-cell receptor signaling abnormality in CLL and to assign pathophysiologic relevance to the presence or absence of B-cell receptor responsiveness. We show that extracellular signal-regulated kinase-2 phosphorylation, intracellular Ca2+ increases, CD79a phosphorylation, and translocation of the B-cell receptor to lipid rafts in response to ligation with anti-immunoglobulin M (as a surrogate for antigen) are features of CLL cells with relatively unmutated VH genes (<5% deviation from germ line) and a poor prognosis. B-cell receptor stimulation in these cases also promoted cell survival. In clones with mutated VH genes (>5% deviation from germ line), surface immunoglobulin M ligation failed to induce receptor translocation to rafts or to prolong cell survival. This failure of receptor translocation observed in mutated CLL cells was associated with the constitutive exclusion of the B-cell receptor from rafts by a mechanism involving src-dependent interactions between the B-cell receptor and the actin cytoskeleton. We conclude that exposure to antigen promotes the survival of unmutated CLL clones, contributing to the poor prognosis of this group. In contrast, hyporesponsive mutated CLL clones may have developed into a stage where continuous exposure to antigen results in relative tolerance to antigenic stimulation mediated by the exclusion of the B-cell receptor from lipid rafts.

Basic fibroblast growth factor suppresses p53 activation in the neoplastic cells of a proportion of patients with chronic lymphocytic leukaemia.

p53 is the most frequently inactivated gene in human cancers, reflecting its pivotal role in maintaining genomic integrity. The present study was conducted to explore the possibility that tumour cells with no intrinsic defects of the p53 pathway might nevertheless acquire p53 dysfunction through extrinsic suppression of the pathway by microenvironmental factors. Neoplastic cells from patients with chronic lymphocytic leukaemia (CLL) were cultured in the presence or absence of basic fibroblast growth factor (bFGF) and exposed to ionizing radiation (IR) to induce p53 accumulation. bFGF is greatly increased in the plasma of CLL patients and can suppress p53 activation in some experimental models. IR induced a marked increase in p53 levels in 28 samples from 24 patients. bFGF inhibited IR-induced p53 accumulation to some extent in most of these samples and by more than 50% in seven samples from seven patients. Suppression of p53 activation by bFGF was frequently but not always accompanied by upregulation of the p53-inhibitory protein MDM2 and/or phosphorylation of MDM2 at serine 166, and was associated with impaired transcriptional activation of the p53 target gene p21. These observations provide the first demonstration in human cancer cells that the p53 pathway can be suppressed by factors in the tumour-cell microenvironment.

The ATR-p53 pathway is suppressed in noncycling normal and malignant lymphocytes.

Chronic lymphocytic leukaemia (CLL) results from the accumulation of apoptosis-resistant clonal B cells that are arrested in G0/G1, and is heterogeneous with respect to clinical outcome. An aggressive form of the disease is identified by an impaired p53 response to ionizing radiation (IR). This is associated with inactivating mutations of either p53 or ATM, a regulator of p53 activated by IR-induced DNA damage. Since other forms of DNA damage activate p53 via ATR, a kinase closely related to ATM, abnormalities of the ATR-p53 pathway also have the potential to result in p53 dysfunction. We therefore tested cases of CLL for abnormal p53 responses to ultraviolet irradiation (UVC), a known activator of ATR, to screen for additional forms of p53 dysfunction. CLL cells and normal peripheral blood mononuclear cell (PBMC) preparations (predominantly noncycling lymphocytes) were treated with UVC and assessed for p53 responses. In all of the CLL cases and PBMC preparations tested, we were unable to detect p53 accumulation, phosphorylation or transcriptional consequences in response to UVC-induced DNA damage. The most likely explanation for the absence of UVC-induced p53 activation in CLL and normal lymphocytes was that, in contrast to other cell types, the UVC-induced ATR pathway was inactive. This notion was confirmed by showing that ATR protein was absent or undetectable in all of the cases of CLL and normal PBMCs screened. This was an unexpected finding because ATR was thought to be essential for the viability of somatic cells and for normal human and murine embryonic development. An obvious difference between the cell lines used as positive controls for ATR antibodies and the CLL cells/PBMCs was that the former were actively cycling while the latter were quiescent. We therefore hypothesized that the ATR-p53 pathway is selectively downregulated in noncycling lymphocytes. To test this, we induced cycling in the T-cell fraction of PBMC preparations and demonstrated that ATR protein expression was restored. Furthermore, p53 was upregulated and phosphorylated in response to UVC in these cells. Our data support the conclusion that the ATR-p53 pathway is suppressed in noncycling lymphocytes via ATR downregulation. We tentatively suggest that this repressed DNA damage response may have evolved to protect quiescent lymphocytes from the potential for p53-dependent apoptosis in the face of some forms of endurable genotoxic stress. If this is the case, DNA repair and genome stability might be compromised in quiescent lymphocytes with potentially negative consequences.

V(H) gene usage differs in germline and mutated B-cell chronic lymphocytic leukemia.

BACKGROUND AND OBJECTIVES: Given the prognostic relevance that the identification of mutated and germline subgroups of chronic lymphocytic leukemia (CLL) has recently acquired we set out to analyze in depth individual VH gene usage rearrangements in patients with mutated and germline CLL. DESIGN AND METHODS: Using sequence analysis of FR1/JH polymerase chain reaction products, the VH immunoglobulin gene configuration was analyzed in 159 rearranged IgH alleles from 154 CLL patients. Having previously identified a spatial relationship between VH gene usage and JH proximity in patients with acute lymphocytic leukemia (ALL), we performed linear and Poisson regression analysis on patients with germline and mutated CLL against VH rearrangements from normal peripheral blood. RESULTS: Sequence analysis showed that 102 patients (64%) had mutated sequences (>2% DNA base pair changes) while 57 (36%) had germline sequences. The germline CLL group showed JH proximal overusage similar to that reported in ALL patients, while the mutated CLL group showed a pattern comparable to that of the control group (peripheral blood rearranged VH sequences). The CDR3 region was statistically longer in the patients with germline CLL than in those with mutated CLL. INTERPRETATION AND CONCLUSIONS: This study highlights differences in the VDJ profile in mutated and germline CLL, consistent with the suggestion that CLL comprises two subgroups. The interpretation of these differences is that the B-cell of CLL, particularly in the germline group, may derive from a pool that has been unable to follow or complete the normal pathway of B-cell differentiation.

Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia.

Established adverse prognostic factors in chronic lymphocytic leukemia (CLL) include CD38 expression, relative lack of IgV(H) mutation, and defects of the TP53 gene. However, disruption of the p53 pathway can occur through mechanisms other than TP53 mutation, and we have recently developed a simple screening test that detects p53 dysfunction due to mutation of the genes encoding either p53 or ATM, a kinase that regulates p53. The present study was conducted to examine the predictive value of this test and to establish the relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation. CLL cells from 71 patients were examined for IgV(H) mutation, CD38 expression, and p53 dysfunction (detected as an impaired p53/p21 response to ionizing radiation). Survival data obtained from 69 patients were analyzed according to each of these parameters. Relative lack of IgV(H) mutation (less than 5%; n = 45), CD38 positivity (antigen expressed on more than 20% of malignant cells; n = 19), and p53 dysfunction (n = 19) were independently confirmed as adverse prognostic factors. Intriguingly, all p53-dysfunctional patients and all but one of the CD38(+) patients had less [corrected] than 5% IgV(H) mutation. Moreover, patients with p53 dysfunction and/or CD38 positivity (n = 31) accounted for the short survival of the less mutated group. These findings indicate that the poor outcome associated with having less than 5% IgV(H) mutation may be due to the overrepresentation of high-risk patients with p53 dysfunction and/or CD38 positivity within this group, and that CD38(-) patients with functionally intact p53 may have a prolonged survival regardless of the extent of IgV(H) mutation.