LDOC1, A NOVEL BIOMARKER OF PROGNOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA

In chronic lymphocytic leukemia (CLL), one of the best predictors of outcome is the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes. Patients whose CLL cells have unmutated IGHV genes have a median survival of 8 years; those with mutated IGHV genes have a median survival of 25 years. To identify new prognostic biomarkers and molecular targets for therapy in untreated CLL patients, we reanalyzed the raw data from four published gene expression profiling microarray studies. Of 88 candidate biomarkers associated with IGHV somatic mutation status, we identified LDOC1 (Leucine Zipper, Down-regulated in Cancer 1), as one of the most significantly differentially expressed genes that distinguished mutated from unmutated CLL cases. LDOC1 is a putative transcription factor of unknown function in B-cell development and CLL pathophysiology. Using a highly sensitive quantitative RT-PCR (QRT-PCR) assay, we confirmed that LDOC1 mRNA was dramatically down-regulated in mutated compared to unmutated CLL cases. Expression of LDOC1 mRNA was also vii strongly associated with other markers of poor prognosis, including ZAP70 protein and cytogenetic abnormalities of poor prognosis (deletions of chromosomes 6q21, 11q23, and 17p13.1, and trisomy 12). CLL cases positive for LDOC1 mRNA had significantly shorter overall survival than negative cases. Moreover, in a multivariate model, LDOC1 mRNA expression predicted overall survival better than IGHV mutation status or ZAP70 protein, among the best markers of prognosis in CLL. We also discovered LDOC1S, a new LDOC1 splice variant. Using isoform-specific QRT-PCR assays that we developed, we found that both isoforms were expressed in normal B cells (naïve > memory), unmutated CLL cells, and in B-cell non-Hodgkin lymphomas with unmutated IGHV genes. To investigate pathways in which LDOC1 is involved, we knocked down LDOC1 in HeLa cells and performed global gene expression profiling. GFI1 (Growth Factor-Independent 1) emerged as a significantly up-regulated gene in both HeLa cells and CLL cells that expressed high levels of LDOC1. GFI1 oncoprotein is implicated in hematopoietic stem cell maintenance, lymphocyte development, and lymphomagenesis. Our findings indicate that LDOC1 mRNA is an excellent biomarker of overall survival in CLL, and may contribute to B-cell differentiation and malignant transformation.

Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

Improved relapse-free survival after autologous stem cell transplantation does not translate into better quality of life in chronic lymphocytic leukemia: lessons from the randomized European Society for Blood and Marrow Transplantation-Intergroup study

In chronic lymphocytic leukemia (CLL) medical progress is driven by clinical studies with relapse-free survival (RFS) as the primary endpoint. The randomized EBMT-Intergroup trial compared high-dose therapy and autologous stem cell transplantation (ASCT) to observation and demonstrated a substantial improvement of RFS without showing improved overall survival for the transplant arm. Here we report quality of life (QoL) information of the first 3 years following randomization from that study. The main objective was to assess the impact of treatment on QoL over time. Two secondary analyses were performed to further investigate the impact of ASCT and relapse on QoL. In the primary analysis, we demonstrate an adverse impact of ASCT on QoL which was largest at 4 months and continued throughout the first year after randomization. Further, we demonstrated a sustained adverse impact of relapse on QoL which worsened over time. Despite better disease control by ASCT the side effects thus turned the net effect towards inferior QoL in the first year and comparable QoL in the following 2 years after randomization. This study emphasizes the importance of information concerning QoL impacts when patients are counseled about treatments aimed at improving RFS in the absence of a survival benefit.

Shorter telomeres correlate with an increase in the number of uniparental disomies in patients with chronic lymphocytic leukemia.

This study investigated the correlation of the extent of chromosomal aberrations including uniparental disomies (UPDs) by SNP-chip analysis and FISH to telomere length in 46 patients with CLL. CLL harboring high risk aberrations, i.e. deletions of 11q22-23 or 17p13, had significantly shorter telomeres (higher ΔTL) compared to patients with CLL without such abnormalities. Patients with high chromosomal aberration rates had a worse overall survival compared to cases with lower aberration rates. Interestingly, however, an increase was found in the number of UPDs with shorter telomeres. These findings support the idea that telomeres in CLL cells play a role in the overall chromosome stability and could be involved in the occurrence of UPDs.

Molecular mechanisms of apoptosis induction in chronic lymphocytic leukemia

CLL is the most common adult leukemia in the Western World, yet very little is known about the biology of this disease. CLL cells have very high levels of NF-κB activity. Factors such as CD40 ligation and phorbol ester treatment induce NF-κB activity and also prevent apoptosis. Previous data from our laboratory demonstrated that MG-132, a proteasome inhibitor, blocked NF-κB activation and promoted apoptosis in CLL cells. These data suggested to us that NF-κB mediates survival in CLL. We examined NF-κB activity using two different chemotherapeutic agents, PS-341 and arsenic trioxide. PS-341, a proteasome inhibitor blocked NF-κB in CLL cells. This however, did not correlate with cell death. Resistant patient isolates displayed delayed Smac/DIABLO release in comparison to cytochrome c release. This suggests that IAPs are contributing to CLL cell survival and drug-resistance. Arsenic trioxide did not block NF-κB activity at therapeutic doses. However it was a potent inducer of apoptosis in CLL cells. We identified a novel mechanism by which arsenic induces increases in mitochondrial calcium to induce cytochrome c release and initiate apoptosis. Both PS-341 and arsenic trioxide are currently in Phase II clinical trials at M.D. Anderson Cancer Center. We conclude that NF-κB is not critical for PS-341 or arsenic trioxide-mediated cell death. ^

Chronic lymphocytic leukemia: A study of the changing patterns of the natural history, clinical factors associated with prognosis, and risk of second cancers

A retrospective cohort study was conducted among 1542 patients diagnosed with CLL between 1970 and 2001 at the M. D. Anderson Cancer Center (MDACC). Changes in clinical characteristics and the impact of CLL on life expectancy were assessed across three decades (1970–2001) and the role of clinical factors on prognosis of CLL were evaluated among patients diagnosed between 1985 and 2001 using Kaplan-Meier and Cox proportional hazards method. Among 1485 CLL patients diagnosed from 1970 to 2001, patients in the recent cohort (1985–2001) were diagnosed at a younger age and an earlier stage compared to the earliest cohort (1970–1984). There was a 44% reduction in mortality among patients diagnosed in 1985–1995 compared to those diagnosed in 1970–1984 after adjusting for age, sex and Rai stage among patients who ever received treatment. There was an overall 11 years (5 years for stage 0) loss of life expectancy among 1485 patients compared with the expected life expectancy based on the age-, sex- and race-matched US general population, with a 43% decrease in the 10-year survival rate. Abnormal cytogenetics was associated with shorter progression-free (PF) survival after adjusting for age, sex, Rai stage and beta-2 microglobulin (beta-2M); whereas, older age, abnormal cytogenetics and a higher beta-2M level were adverse predictors for overall survival. No increased risk of second cancer overall was observed, however, patients who received treatment for CLL had an elevated risk of developing AML and HD. Two out of three patients who developed AML were treated with alkylating agents. In conclusion, CLL patients had improved survival over time. The identification of clinical predictors of PF/overall survival has important clinical significance. Close surveillance of the development of second cancer is critical to improve the quality of life of long-term survivors. ^

The effects of proteasome inhibition in chronic lymphocytic leukemia and prostate cancer

The use of proteasome inhibitors in cancer has received much attention with the recent FDA approval of bortezomib (Velcade/PS-341). However, in the chronic lymphocytic leukemia (CLL) clinical trial, bortezomib was not as effective as it was in vitro. Accordingly, results in prostate cancer were not remarkable, although regression of lymphadenopathy was observed. This response was also seen in CLL. ^ The proteasome degrades ∼80% of intracellular proteins. Although specific pathways affected by proteasome inhibitors are known, there are still unidentified mechanisms by which they induce apoptosis. The efficacy and mechanism of action of the reversible proteasome inhibitor bortezomib were compared to the novel irreversible inhibitor NPI-0052 in this study, and their mechanisms of action in CLL and prostate cancer were examined. ^ NPI-0052 inhibited proteasome activity and induced apoptosis with more rapid kinetics than bortezomib in CLL. Inhibition of proteasome activity with NPI-0052 was also more durable. Interestingly, bortezomib is cleared from the serum within 15min, which is insufficient time for bortezomib to effectively inhibit the proteasome. However, only 5min exposure was needed for NPI-0052 to produce maximal proteasome inhibition. The data suggest that bortezomib's slow kinetics and reversible nature limit its potential in vivo and the use of NPI-0052 should be considered. ^ In examining the mechanism(s) by which bortezomib and NPI-0052 induce apoptosis in CLL, both were found to elicit the ER stress pathway. A stromal cell co-culture system prevented apoptosis induced by both proteasome inhibitors, suggesting that if such factors in vivo were responsible for reducing bortezomib's efficacy, NPI-0052 would not prove useful either. Finally, Lyn, a Src family kinase (SFK), was decreased in response to bortezomib and NPI-0052 and correlated with apoptosis induction in CLL and prostate cancer. Both proteasome inhibitors specifically targeted Lyn rather than SFKs in general. ^ SFKs are overexpressed in cancer and involved in cell signaling, survival, and metastasis. In prostate cancer cells, both proteasome inhibition and Lyn-silencing significantly inhibited migration. Preliminary evidence also suggested that Lyn downregulation decreases invasion potential. Together, these data suggest that proteasome inhibitors are potential candidates for anti-metastasic therapy and further investigation is warranted for the use of Lyn-targeted therapy to treat metastases. ^

CHARACTERIZATION OF TCL1-Tg:P53-/- MICE THAT RESEMBLE HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P-DELETION

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United Statesand Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. In order to understand the underneath mechanism in vivo, I have recently generated mice with Eu-TCL1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. The Eu-TCL1-Tg:p53-/- mice developed CLL disease at 3-4 months, significantly earlier than the parental Eu-TCL1-Tg mice that developed CLL disease at 8-12 months. Flow cytometry analysis showed that the CD5+/ IgM+ cell population appeared in the peritoneal cavity, bone marrow, and the spleens of Eu-TCL1-Tg:p53-/- mice significantly earlier than that of the parental Eu-TCL1-Tg mice. Massive infiltration and accumulation of leukemia cells were found in the spleen and peritoneal cavity. In vitro study showed that the leukemia cells isolated from the Eu-TCL1-Tg:p53-/- mice were more resistant to fludarabine treatment than the leukemia cells isolated from spleens of Eu-TCL1-Tg mice. Interestingly, TUNEL assay revealed that there was higher apoptotic cell death found in the Eu-TCL1-Tg spleen tissue compared to the spleens of the Eu-TCL1-Tg:p53-/- mice, suggesting that the loss of p53 compromises the apoptotic process in vivo, and this might in part explain the drug resistant phenotype of CLL cells with 17p-deletion. In the present study, we further demonstrated that the p53 deficiency in the TCL1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family especially miR-30d in leukemia lymphocytes from the Eu-TCL1-Tg:p53-/- mice. Such down-regulation of those microRNAs and up-regulation of Mcl-1 were also found in primary leukemia cells from CLL patients with 17p deletion. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eu-TCL1-Tg:p53-/- mice relative to Eu-TCL1-Tg mice. Exposure of leukemia cells isolated from Eu-TCL1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis & p53/miR30d-EZH2 may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53.

Aberrant B cell receptor signaling from B29 (Igβ, CD79b) gene mutations of chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) B cells characteristically exhibit low or undetectable surface B cell receptor (BCR) and diminished responses to BCR-mediated signaling. These features suggest that CLL cells may have sustained mutations affecting one or more of the BCR proteins required for receptor surface assembly and signal transduction. Loss of expression and mutations in the critical BCR protein B29 (Igβ, CD79b), are prevalent in CLL and could produce the hallmark features of these leukemic B cells. Because patient CLL cells are intractable to manipulation, we developed a model system to analyze B29 mutations. Jurkat T cells stably expressing μ, κ, and mb1 efficiently assembled a functional BCR when infected with recombinant vaccinia virus bearing wild-type B29. In contrast, a B29 CLL mutant protein truncated in the transmembrane domain did not associate with μ or mb1 at the cell surface. Another B29 CLL mutant lacking the C-terminal immunoreceptor tyrosine activation motif tyrosine and distal residues brought the receptor to the surface as well as wild-type B29 but showed significant impairment in anti-IgM-stimulated signaling events including mitogen-activated protein kinase activation. These findings demonstrate that B29 mutations previously identified in CLL patients can affect BCR-dependent signaling and may contribute to the unresponsive B cell phenotype in CLL. Finally, the features of the B29 mutations in CLL predict that they may be generated by somatic hypermutation.

Loss of PKR Activity in Chronic Lymphocytic Leukemia

There are a number of observations that suggest the dsRNA-activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full-length PKR is present but not active in 21 of 28 patient samples from B-cell chronic lymphocytic leukemia (B-CLL). PKR from these patients was unable to auto-activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58(IPK). We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B-CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL. (C) 2004 Wiley-Liss, Inc.

Biallelic ATM inactivation significantly reduces survival in patients treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 trial.

PURPOSE: The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. PATIENTS AND METHODS: We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. RESULTS: We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). CONCLUSION: The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

CLLU1 expression has prognostic value in chronic lymphocytic leukemia after first-line therapy in younger patients and in those with mutated IGHV genes.

CLLU1, located at chromosome 12q22, encodes a transcript specific to chronic lymphocytic leukemia and has potential prognostic value. We assessed the value of CLLU1 expression in the LRF CLL4 randomized trial. Samples from 515 patients with chronic lymphocytic leukemia were collected immediately before the start of treatment. After RNA extraction and cDNA synthesis, CLLU1 expression was assessed by quantitative polymerase chain reaction. In total, 247 and 268 samples were identified as having low and high CLLU1 expression, respectively. The median follow-up was 88 months. High CLLU1 expression was significantly correlated with unmutated IGHV genes, ZAP-70 and CD38 positivity, and absence of 13q deletion (all r>0.2, P

ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia.

Recent evidence suggests that - in addition to 17p deletion - TP53 mutation is an independent prognostic factor in chronic lymphocytic leukemia (CLL). Data from retrospective analyses and prospective clinical trials show that ∼5% of untreated CLL patients with treatment indication have a TP53 mutation in the absence of 17p deletion. These patients have a poor response and reduced progression-free survival and overall survival with standard treatment approaches. These data suggest that TP53 mutation testing warrants integration into current diagnostic work up of patients with CLL. There are a number of assays to detect TP53 mutations, which have respective advantages and shortcomings. Direct Sanger sequencing of exons 4-9 can be recommended as a suitable test to identify TP53 mutations for centers with limited experience with alternative screening methods. Recommendations are provided on standard operating procedures, quality control, reporting and interpretation. Patients with treatment indications should be investigated for TP53 mutations in addition to the work-up recommended by the International workshop on CLL guidelines. Patients with TP53 mutation may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens can yield a substantial proportion of complete responses, although of short duration. Ideally, patients should be treated within clinical trials exploring new therapeutic agents.