Telomere Length Recovery: A Strong Predictor of Overall Survival in Acute Promyelocytic Leukemia.

Telomeres are the capping ends of chromosomes that protect the loss of genetic material and prevent chromosomal instability. In human tissue-specific stem/progenitor cells, telomere length (TL) is maintained by the telomerase complex, which consists of a reverse transcriptase catalytic subunit (TERT) and an RNA template (TERC). Very short telomeres and loss-of-function mutations in the TERT and TERC genes have been reported in acute myeloid leukemia, but the role of telomeres in acute promyelocytic leukemia (APL) has not been well established. We report the results for a large cohort of 187 PML/RARα-positive APL patients. No germline mutations in the TERT or TERC genes were identified. Codon 279 and 1062 TERT polymorphisms were present at a frequency similar to that in the general population. TL measured in blood or marrow mononuclear cells at diagnosis was significantly shorter in the APL patients than in healthy volunteers, and shorter telomeres at diagnosis were significantly associated with high-risk disease. For patients who achieved complete remission, the median increase in TL from diagnosis to remission (delta TL) was 2.0 kilobase (kb), and we found delta TL to be the most powerful predictor of overall survival when compared with well-established risk factors for poor outcomes in APL.

Dynamics of leukemia stem-like cell extinction in acute promyelocytic leukemia.

Many tumors are believed to be maintained by a small number of cancer stem-like cells, where cure is thought to require eradication of this cell population. In this study, we investigated the dynamics of acute promyelocytic leukemia (APL) before and during therapy with regard to disease initiation, progression, and therapeutic response. This investigation used a mathematical model of hematopoiesis and a dataset derived from the North American Intergroup Study INT0129. The known phenotypic constraints of APL could be explained by a combination of differentiation blockade of PML-RARα-positive cells and suppression of normal hematopoiesis. All-trans retinoic acid (ATRA) neutralizes the differentiation block and decreases the proliferation rate of leukemic stem cells in vivo. Prolonged ATRA treatment after chemotherapy can cure patients with APL by eliminating the stem-like cell population over the course of approximately one year. To our knowledge, this study offers the first estimate of the average duration of therapy that is required to eliminate stem-like cancer cells from a human tumor, with the potential for the refinement of treatment strategies to better manage human malignancy.

Arsenic trioxide in front-line therapy of acute promyelocytic leukemia (C9710): prognostic significance of FLT3 mutations and complex karyotype.

The addition of arsenic trioxide (ATO) to frontline therapy of acute promyelocytic leukemia (APL) has been shown to result in significant improvements in disease-free survival (DFS). FLT3 mutations are frequently observed in APL, but its prognostic significance remains unclear. We analyzed 245 newly diagnosed adult patients with APL treated on intergroup trial C9710 and evaluated previously defined biological and prognostic factors and their relationship to FLT3 mutations and to additional karyotypic abnormalities. FLT3 mutations were found in 48% of patients, including 31% with an internal tandem duplication (FLT3-ITD), 14% with a point mutation (FLT3-D835) and 2% with both mutations. The FLT3-ITD mutant level was uniformly low, < 0.5. Neither FLT3 mutation had an impact on remission rate, induction death rate, DFS or overall survival (OS). The addition of ATO consolidation improved outcomes regardless of FLT3 mutation type or level, initial white blood cell count, PML-RARA isoform type or transcript level. The presence of a complex karyotype was strongly associated with an inferior OS independently of post-remission treatment. In conclusion, the addition of ATO to frontline therapy overcomes the impact of previously described adverse prognostic factors including FLT3 mutations. However, complex karyotype is strongly associated with an inferior OS despite ATO therapy.

All-trans retinoic acid and late relapses in acute promyelocytic leukemia: very long-term follow-up of the North American Intergroup Study I0129.

We report a long-term follow-up (median 11.8 years) of the First North American Intergroup Study. 379 patients were randomized to induction with ATRA or to chemotherapy. All complete responders (CR) received consolidation chemotherapy, then randomized to 1 year ATRA or observation. 245 patients received ATRA sometime during the study: 195 (80%) achieved a CR. Nine (4.6%) relapsed late (>3 years from CR), the last occurred after 4.6 years; 7 of them were still alive after 5.5-15 years. In APL patients, late relapses are uncommon, and those who sustain CR >5 years can be considered cured.

KLF6 loss of function in human prostate cancer progression is implicated in resistance to androgen deprivation.

Inactivation of the transcription factor/tumor suppressor Krüppel-like factor 6 (KLF6) has been described in prostate cancer (PC). This study investigated the prevalence and significance of KLF6 exon 2 mutations and splice variants (SVs) in different stages of human PC progression. By using laser-capture microdissection and recombinant clone isolation of DNA sequences to enhance sensitivity, base changes were found in 20 (24.7%) of 81 PC tissues versus 1 (4%) of 25 normal prostate tissues (P = 0.02). Of 26 base changes, 54% produced nonsynonymous mutations. Only three mutations had driver characteristics (PCs, 4%; NPs, 0%). By using microdissection of fresh-frozen tissues and recombinant isolation of RNA sequences, SVs were found in 39 (75%) of 52 PCs and in 10 (45%) of 22 NPs (P = 0.01). Sixteen different SVs, including 13 unique SVs, were identified that used cryptic splicing sites and encoded nonfunctional KLF6 proteins. PCs that had survived hormone (androgen)-deprivation therapy (n = 21) had a significantly higher (P < 0.05) incidence, number, and expression level of nonfunctional SVs than either NPs (n = 22) or hormone-naïve PCs (n = 25). Forced expression of nonfunctional SVs conferred a survival advantage of androgen-dependent LNCaP cells under castration-simulated culture conditions. Together, these data suggest that decreased availability of functional KLF6 contributes to clinical PC progression. This decrease arises infrequently by somatic mutation and more commonly by the acquisition of SVs that provide a survival advantage under castrate conditions, enabling resistance to hormone therapy.

Treatment-influenced associations of PML-RARα mutations, FLT3 mutations, and additional chromosome abnormalities in relapsed acute promyelocytic leukemia.

Mutations in the all-trans retinoic acid (ATRA)-targeted ligand binding domain of PML-RARα (PRα/LBD+) have been implicated in the passive selection of ATRA-resistant acute promyelocytic leukemia clones leading to disease relapse. Among 45 relapse patients from the ATRA/chemotherapy arm of intergroup protocol C9710, 18 patients harbored PRα/LBD+ (40%), 7 of whom (39%) relapsed Off-ATRA selection pressure, suggesting a possible active role of PRα/LBD+. Of 41 relapse patients coanalyzed, 15 (37%) had FMS-related tyrosine kinase 3 internal tandem duplication mutations (FLT3-ITD+), which were differentially associated with PRα/LBD+ depending on ATRA treatment status at relapse: positively, On-ATRA; negatively, Off-ATRA. Thirteen of 21 patients (62%) had additional chromosome abnormalities (ACAs); all coanalyzed PRα/LBD mutant patients who relapsed off-ATRA (n = 5) had associated ACA. After relapse Off-ATRA, ACA and FLT3-ITD+ were negatively associated and were oppositely associated with presenting white blood count and PML-RARα type: ACA, low, L-isoform; FLT3-ITD+, high, S-isoform. These exploratory results suggest that differing PRα/LBD+ activities may interact with FLT3-ITD+ or ACA, that FLT3-ITD+ and ACA are associated with different intrinsic disease progression pathways manifest at relapse Off-ATRA, and that these different pathways may be short-circuited by ATRA-selectable defects at relapse On-ATRA. ACA and certain PRα/LBD+ were also associated with reduced postrelapse survival.

Prognostic relevance of integrated genetic profiling in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML. METHODS: We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients. RESULTS: We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67). CONCLUSIONS: We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.).

Arsenic trioxide improves event-free and overall survival for adults with acute promyelocytic leukemia: North American Leukemia Intergroup Study C9710.

Arsenic trioxide (As(2)O(3)) is a highly effective treatment for patients with relapsed acute promyelocytic leukemia (APL); its role as consolidation treatment for patients in first remission has not been defined. We randomized 481 patients (age ≥ 15 years) with untreated APL to either a standard induction regimen of tretinoin, cytarabine, and daunorubicin, followed by 2 courses of consolidation therapy with tretinoin plus daunorubicin, or to the same induction and consolidation regimen plus two 25-day courses of As(2)O(3) consolidation immediately after induction. After consolidation, patients were randomly assigned to one year of maintenance therapy with either tretinoin alone or in combination with methotrexate and mercaptopurine. Ninety percent of patients on each arm achieved remission and were eligible to receive their assigned consolidation therapy. Event-free survival, the primary end point, was significantly better for patients assigned to receive As(2)O(3) consolidation, 80% compared with 63% at 3 years (stratified log-rank test, P < .0001). Survival, a secondary end point, was better in the As(2)O(3) arm, 86% compared with 81% at 3 years (P = .059). Disease-free survival, a secondary end point, was significantly better in the As(2)O(3) arm, 90% compared with 70% at 3 years (P < .0001). The addition of As(2)O(3) consolidation to standard induction and consolidation therapy significantly improves event-free and disease-free survival in adults with newly diagnosed APL. This trial was registered at clinicaltrials.gov (NCT00003934).

Single cycle of arsenic trioxide-based consolidation chemotherapy spares anthracycline exposure in the primary management of acute promyelocytic leukemia.

PURPOSE Event-free survival following all-trans-retinoic acid (ATRA) -based therapy for acute promyelocytic leukemia (APL) averages 70% at 5 years. While arsenic trioxide (ATO) can induce remissions in 95% of relapsed patients, few studies have addressed the integration of ATO into the primary management of APL. This study examines the efficacy of a single cycle of ATO-based consolidation therapy in a treatment regimen designed to decrease exposure to other cytotoxic agents. PATIENTS AND METHODS After induction with ATRA and daunorubicin (DRN), untreated patients with APL received 3 days of cytarabine and DRN followed by 30 doses of ATO beginning on day 8. Molecular remitters received 2 years of risk-based maintenance therapy. Results Forty-one of 45 patients receiving induction therapy achieved remission; four patients died (one before treatment was initiated). Thirty-seven patients received consolidation and maintenance; of these one patient relapsed (CNS) and one died in remission during maintenance therapy (hepatic sickle cell crisis). With a median follow-up of 2.7 years, estimated disease-free survival was 90%; overall survival for all patients was 88%. Despite a total anthracycline dose of only 360 mg/m(2), cardiac ejection fraction decreased by > or = 20% in 20% of patients. CONCLUSION These data, combined with other recent studies using ATO in the primary management of APL, demonstrate the important role that ATO can play in the primary management of this curable disease. Future studies should continue to focus on reducing the toxicity of treatment without increasing the relapse rate.

A phase II trial of arsenic trioxide for relapsed and refractory acute lymphoblastic leukemia.

We designed a phase II trial of arsenic trioxide (AT) for the treatment of relapsed and refractory acute lymphoblastic leukemia (ALL). The dose administered was 0.25 mg/kg/day intravenously for 5-7 days per week for up to 60 days. Of 11 patients eligible, eight had B-cell and three T-cell ALL and two were Philadelphia chromosome-positive. The median duration of therapy was 21 days (range 7-28). One patient died of an infection. There were no responses. Ten patients have died. The median survival was 3.2 months (range 1.2-4.1). We conclude that AT is not active in the treatment of ALL.

Molecular load of pathologically occult metastases in pelvic lymph nodes is an independent prognostic marker of biochemical failure after localized prostate cancer treatment.

PURPOSE: Thirty percent of patients treated with curative intent for localized prostate cancer (PC) experience biochemical recurrence (BCR) with rising serum prostate-specific antigen (sPSA), and of these, approximately 50% succumb to progressive disease. More discriminatory staging procedures are needed to identify occult micrometastases that spawn BCR. PATIENTS AND METHODS: PSA mRNA copies in pathologically normal pelvic lymph nodes (N0-PLN) from 341 localized PC patients were quantified by real-time reverse-transcriptase polymerase chain reaction. Based on comparisons with normal lymph nodes and PLN with metastases and on normalization to 5 x 10(6) glyceraldehyde-3'-phosphate dehydrogenase mRNA copies, normalized PSA copies (PSA-N) and a threshold of PSA-N 100 or more were selected for continuous and categorical multivariate analyses of biochemical failure-free survival (BFFS) compared with established risk factors. RESULTS: At median follow-up of 4 years, the BFFS of patients with PSA-N 100 or more versus PSA-N less than 100 was 55% and 77% (P = .0002), respectively. The effect was greatest for sPSA greater than 20 ng/mL, 25% versus 60% (P = .014), Gleason score 8 or higher, 21% versus 66% (P = .0002), stage T3c, 18% versus 64% (P = .001), and high-risk group (50% v 72%; P = .05). By continuous analysis PSA-N was an independent prognostic marker for BCR (P = .049) with a hazard ratio of 1.25 (95% CI, 1.001 to 1.57). By categorical analysis, PSA-N 100 or more was an independent variable (P = .021) with a relative risk of 1.98 (95% CI, 1.11 to 3.55) for BCR compared with PSA-N less than 100. CONCLUSION: PSA-N 100 or more is a new, independent molecular staging criterion for localized PC that identifies high-risk group patients with clinically relevant occult micrometastases in N0-PLN, who may benefit from additional therapy to prevent BCR.

Phase II study of all-trans retinoic acid in the accelerated phase or early blastic phase of chronic myeloid leukemia: a study of the Eastern Cooperative Oncology Group (E1993).

The aims of this study were to evaluate the safety and efficacy of all-trans retinoic acid (ATRA) in the treatment of the accelerated and blastic phase of chronic myeloid leukemia (CML) and to evaluate in vitro correlates of biological activity. ATRA was administered in an intermittent schedule to patients with CML in the accelerated or blastic phases for a 6 week induction period, which was continued if there was evidence of clinical response or stable disease. If the patient was progressing at 6 weeks, interferon-alpha could be added to the ATRA. Laboratory correlative studies were performed prior to treatment and at intervals during treatment to evaluate effects on maturation and differentiation, and on CML progenitor cell growth by assessment of colony-forming cells (CFC). Eighteen patients were enrolled. There was 1 complete response, 1 partial response and 2 with hematological improvement. A fifth patient was stable on ATRA and interferon for several months. Laboratory data for the responders demonstrated high sensitivity of primary CFC to ATRA prior to treatment and low serial CFC counts on ATRA therapy. ATRA demonstrated clinical and hematological activity in 5 of 18 patients with the accelerated phase of CML, and there was evidence of a biological effect in laboratory studies of 3 of the 5 patients' progenitor cells. Combination therapy with other differentiating agents may be useful in this disease.

Quantitative real-time RT-PCR analysis of PML-RAR alpha mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129.

The potential prognostic value of quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR [qrtPCR]) measurements of PML-RAR alpha mRNA in acute promyelocytic leukemia was retrospectively assessed before treatment and at 3 posttreatment intervals in 123 patients on intergroup protocol 0129. The primary measure was the PML-RAR alpha(GAPDH) normalized quotient (NQ), that is, PML-RAR alpha mRNA copies divided by glyceraldehyde-3'-phosphate dehydrogenase (GAPDH) mRNA copies. Only samples with more than 2.5 x 10(5) copies of the housekeeping gene GAPDH mRNA (detection sensitivity exceeding 10(4)) were considered NQ evaluable. With RNA from low-density selected cells, paired peripheral blood (PB) and bone marrow samples (n = 140) had comparable NQs (P <.001). Before treatment, high NQ was associated with short-form PML-RAR alpha (P <.001), but not with white blood cell count or clinical outcome. Following treatment, NQ was lower in all-trans retinoic acid-induced complete remission (CR) than chemotherapy-induced CR (P =.018) and at first test after consolidation chemotherapy (P =.037). After consolidation chemotherapy, patients with NQ exceeding 10(-5) had 4.1-fold increased relapse risk (P =.008); however, 73% of patients who experienced relapse had NQ lower than 10(-5). In the follow-up period (FUP), any NQ exceeding 10(-5) and 10(-6) had 17.5-fold and 7.6-fold increased relapse risk, respectively (P <.001), while no gradation of relapse risk (approximately 18%) could be identified at NQ lower than 10(-6), including NQ(-). These results indicate that qrtPCR monitoring of PML-RAR alpha NQ can identify patients at high risk of relapse and suggest that clinically practical PB NQ monitoring at more frequent FUP intervals may improve predictive accuracy for relapse or continuing CR in patients with persistent, fluctuating minimal residual disease levels.

Variant-type PML-RAR(alpha) fusion transcript in acute promyelocytic leukemia: use of a cryptic coding sequence from intron 2 of the RAR(alpha) gene and identification of a new clinical subtype resistant to retinoic acid therapy.

The physiologic actions of retinoic acids (RAs) are mediated through RA receptors (RARs) and retinoid X receptors (RXRs). The RAR(alpha) gene has drawn particular attention because it is the common target in all chromosomal translocations in acute promyelocytic leukemia (APL), a unique model in cancer research that responds to the effect of RA. In the great majority of patients with APL, RAR(alpha) is fused to the PML gene as a result of the t(15;17) translocation. Three distinct types of PML-RAR(alpha) transcripts, long (L), short (S), and variant (V), were identified. The V-type is characterized by truncation of exon 6 of PML and in some cases by the insertion of a variable "spacer" sequence between the truncated PML and RAR(alpha) mRNA fusion partners, although the precise mechanisms underlying formation of the V-type transcript remain unclear. To get further insights into the molecular basis of the t(15;17), we sequenced the entire genomic DNA region of RAR(alpha). Of note, all previously reported "spacer" sequences in V-type transcripts were found in intron 2 of the RAR(alpha) gene and most of these sequences were flanked by gt splice donor sites. In most cases, these "cryptic" coding sequences maintained the ORF of the chimeric transcript. Interestingly, two cases with a relatively long spacer sequence showed APL cellular and clinical resistance to RA treatment. In these cases, the aberrant V-type PML-RAR(alpha) protein displayed increased affinity to the nuclear corepressor protein SMRT, providing further evidence that RA exerts the therapeutic effect on APL through modulation of the RAR-corepressor interaction. Finally, among patients with the L- or S-type PML-RAR(alpha) fusion transcript, some consensus motifs were identified at the hotspots of the chromosome 17q breakpoints within intron 2 of RAR(alpha), strengthening the importance of this intron in the molecular pathogenesis of APL.

Frequent mutations in the ligand-binding domain of PML-RARalpha after multiple relapses of acute promyelocytic leukemia: analysis for functional relationship to response to all-trans retinoic acid and histone deacetylase inhibitors in vitro and in vivo.

This study identified missense mutations in the ligand binding domain of the oncoprotein PML-RARalpha in 5 of 8 patients with acute promyelocytic leukemia (APL) with 2 or more relapses and 2 or more previous courses of all-trans retinoic acid (RA)-containing therapy. Four mutations were novel (Lys207Asn, Gly289Arg, Arg294Trp, and Pro407Ser), whereas one had been previously identified (Arg272Gln; normal RARalpha1 codon assignment). Five patients were treated with repeat RA plus phenylbutyrate (PB), a histone deacetylase inhibitor, and one patient experienced a prolonged clinical remission. Of the 5 RA + PB-treated patients, 4 had PML-RARalpha mutations. The Gly289Arg mutation in the clinical responder produced the most defective PML-RARalpha function in the presence of RA with or without sodium butyrate (NaB) or trichostatin A. Relapse APL cells from this patient failed to differentiate in response to RA but partially differentiated in response to NaB alone, which was augmented by RA. In contrast, NaB alone had no differentiation effect on APL cells from another mutant case (Pro407Ser) but enhanced differentiation induced by RA. These results indicate that PML-RARalpha mutations occurred with high frequency after multiple RA treatment relapses, indicate that the functional potential of PML-RARalpha was not correlated with clinical response to RA + PB treatment, and suggest that the response to RA + PB therapy in one patient was related to the ability of PB to circumvent the blocked RA-regulated gene response pathway.