Childhood Adult Acute Lymphoblastic Leukemia Research Articles

Interphase FISH Analysis Reveals High Frequence of Partial 6q Deletions in Adult Acute Lymphoblastic Leukemia (ALL) with Normal or Failed Karyotype.

Karyotypic analysis in adult ALL has provided relevant insights in the prognostic significance of chromosome abnormalities, which in turn become of potential importance for the design of risk-adapted therapeutic strategies for newly diagnosed cases. Among structural karyotypic changes, partial deletions of the long arm of chromosome 6 (6q) are reported to be between 4 and 6% of cases of adult ALL by conventional cytogenetics and depict a subset of patients with strict correlation with T-lineage ALL and an unfavorable prognostic likelihood. Recent studies at the molecular level in childhood ALL have shown 6q deletions in up to 30% of the investigated cases. With the aim of more precisely defining the exact incidence of 6q deletions in adult ALL, we started to investigate by interphase FISH patients with normal or failed karyotype included in the GIMEMA 0496 and 2000 protocols. These trials contemplated a central handling of bone marrow samples of the enrolled patients at presentation. We identified the following bacteria-derived artificial chromosome (BAC) clones with a known map location within the most common deleted region of 6q: 90G9 (6q13), 12B13 (6q15), 465M14 (6q21), 81H16 (6q26), 91016 (6q27). BAC clones were obtained from the Children's Hospital Oakland Research Institute (CHORI).Twenty-seven consecutive ALL patients have so far been analyzed by interphase FISH, 16 with T- and 11 with B-cell phenotype. Eleven cases had a normal karyotype and 16 a failed cytogenetic analysis. Among the cases with normal karyotype, 2 cases with T-cell phenotype showed a deletion in the 6q21 and 6q15 regions, whereas in the group with failed cytogenetics 1 case with B- and 3 cases with T-cell phenotype harbored deletions spanning respectively the 6q13-15 (2), 6q13-q21 and 6q15-21 regions. The median percentage of nuclei with 6q deletion was 30% (range 7–75%). T-ALL deleted cases showed the absence of the most frequent fusion genes detectable with molecular analysis, whereas a BCR-ABL rearrangement was identified in the only B-lineage ALL deleted case. Overall, partial 6q deletions were observed in 22% of the cases analyzed and in 31% of cases with T-cell phenotype. The present study shows that the probe set we have utilized is a useful tool for providing accurate cytogenetic diagnosis of partial 6q deletions and indicates that this abnormality is a frequent karyotypic change in adult ALL, particularly in T-lineage cases where it appears to represent the most relevant genetic anomaly. Since this abnormality is underestimated by conventional cytogenetic analysis, interphase FISH for 6q deletions should be included in the routine genetic screening of the ALL patients with failed or normal cytogenetics

Targeted therapy with monoclonal antibodies in acute lymphoblastic leukemia

To describe the current new targeted therapy with monoclonal and bispecific antibodies in adult acute lymphoblastic leukemia (ALL), to improve response rates and outcome. Blast cells in ALL express a variety of specific antigens, such as CD19, CD20, CD22, CD33 and CD52, and recently monoclonal antibodies (MoAbs) became available to target these antigens. The anti-CD20 MoAb rituximab has substantially improved the outcome in Burkitt lymphoma/leukemia, and is currently applied in de-novo B-precursor ALL. The MoAbs directed against CD22, linked to cytotoxic agents, either to calicheamicin (inotuzomab ozogamicin) or to plant or bacterial toxins (epratuzumab) are explored in refractory/relapsed childhood and adult ALL. Targeting CD19 is of great interest, as it is expressed in all B-lineage cells, including early precursors. The new bispecific antibody blinatumomab combines single chain antibodies to CD19 and CD3, and thereby T cells lyse the CD19 bearing B cells and is effective in patients with positive minimal residual disease (MRD) or refractory/relapsed ALL. Antibody therapy in ALL is very promising, with high rate of complete remission and MRD-negativity in advanced ALL. It is currently explored in de-novo ALL to establish the best setting in combination with chemotherapy or even as a monotherapy.

Design of the Hallmarq Trial, a Phase 3 Study of Vincristine Sulfate Liposome Injection (VSLI, Marqibo®) in Adults with Newly Diagnosed, Philadelphia Chromosome Negative Acute Lymphoblastic Leukemia

Abstract 4300 Background:Adult acute lymphoblastic leukemia (ALL) is a lethal and fulminant disease and one distinct from childhood ALL. There are approximately 2400 new cases per year in the US; approximately 80% of these will be Philadelphia chromosome (Ph) negative. Despite remarkable success in treatment of childhood and adolescent ALL, adult ALL patients are underserved by existing treatment options as reflected by the poor survival rates and extremely poor outcomes in the relapsed setting. Complete response (CR) rates in the front-line setting range from 90% in patients <30 years old to 57% in patients >60 years old and 3-year disease free survival ranges from 47% to 12% respectively. The unmet medical need in adult ALL is most pronounced in those age 60 and older. VSLI was recently granted accelerated FDA approval as a single-agent for the treatment of adults with advanced, relapsed and refractory Ph-negative ALL and has successfully completed a Phase 2 trial as a component of multi-agent therapy. Trial Design:HALLMARQ (NCT01439347) is a Phase 3, multi-national, randomized study to evaluate the substitution of VSLI for standard vincristine (VCR) in the induction, consolidation, and maintenance phases of combination chemotherapy in the treatment of patients ≥ 60 years old with newly diagnosed Ph-negative ALL. The chemotherapy “backbone” utilized in this trial is modified from the Cancer and Leukemia Group B 8811 protocol. Asparaginase product usage is optional. The total duration of protocol-specified therapy is up to 24 months. VCR is dosed at 1.4 mg/m2 with a per dose cap at 2 mg. VSLI is dosed at 2.25 mg/m2without a dose cap. A protocol-specified dose reduction algorithm applicable to both VSLI and VCR will be used to minimize Grade 3 neurotoxicity while also facilitating continued dosing. Growth factors (e.g., granulocyte colony-stimulating factor) and bowel regimens (e.g., daily stool softener) are recommended and will be prescribed according to investigator preference and institutional guidelines. The primary endpoint is overall survival (OS) and the study is powered for superiority. Secondary endpoints include remission rate, overall response rate, event-free survival, and safety and tolerability. Minimal residual disease (MRD) at multiple time points and pharmacokinetic parameters are being assessed. Conclusion:The HALLMARQ Trial will provide new insights into the frontline treatment of ALL in adults ≥60 years old as well as confirm the clinical benefit of VSLI monotherapy demonstrated in the Phase 2 RALLY Study that supported the initial product approval. HALLMARQ is based on a Special Protocol Assessment with the FDA and is currently enrolling.*On behalf of the HALLMARQ investigators. Disclosures:Deitcher:Talon Therapeutics: Employment, Equity Ownership. Silverman:Talon Therapeutics: Employment.

Prognostic value of cytogenetics in adult patients with Philadelphia-negative acute lymphoblastic leukemia

The prognostic value of cytogenetics in adult acute lymphoblastic leukemia (ALL) is not as established as in childhood ALL. We have analyzed the outcome and prognostic value of karyotype in 84 adults diagnosed with Philadelphia-negative ALL from a single institution that received induction chemotherapy and had successful karyotype performed. The most frequent finding was normal karyotype in 35 (42%) cases, followed by aneuploidies in 20 cases (24%) and t(4;11)(q21;q23)/MLL/AF4 in 5 (6%), and the remaining 24(27%) cases carried miscellaneous clonal abnormalities. The group of patients with t(4;11)(q21;q23)/MLL/AF4, hypodiploidy and low hyperdiploidy (less than 50 chromosomes) showed a worse outcome than those with normal karyotype and miscellaneous abnormalities in terms of overall survival (OS) (3 years OS; 47% vs. 13%, p = 0.014) and relapse-free survival (RFS) (3 years RFS; 44% vs. 27%, p = 0.005). Other cytogenetic prognostic classifications reported to date were tested in our series, but any was fully reproducible. In conclusion, karyotype is a useful tool for risk assessment in adult ALL. We have confirmed the bad prognosis of t(4;11)(q21;q23)/MLL/AF4 and hypodiploidy. Besides, low hyperdiploidy could also define a high-risk group of patients who might be candidates for more intensive treatment.

Osteonecrosis in childhood ALL

Kawedia et al report a 71% incidence of osteonecrosis (ON) in a cohort of children and young adult acute lymphoblastic leukemia (ALL) patients who underwent routine magnetic resonance imaging (MRI) screening at various time points on study. A majority of cases were asymptomatic. Older age, increased treatment intensity, low serum albumen, elevated lipid levels, and decreased dexamethasone clearance were risk factors associated with an increased incidence of osteonecrosis.

Pediatric-Type Therapy Including Lineage-Targeted Methotrexate to Improve Early Minimal Residual Disease Response and Survival In Adult Acute Lymphoblastic Leukemia (ALL): Interim Analysis of Northern Italy Leukemia Group Study 10/07

AbstractAbstract 2131 Background.Some studies have shown how the use of pediatric-type therapy (PDT) and minimal residual disease (MRD)-oriented programs could improve outcome of adult ALL. Study Design.We included PDT elements in a pilot trial started January, 2008. Protocol 10/07 consisted of standard induction and late reinduction courses plus 3 PDT blocks (modified after BFM therapy) alternating with 3 lineage-targeted Methotrexate (LTM) blocks (B-precursor: 2.5 g/m2; T-precursor: 5 g/m2; Ph+ or age >55 years: 1.5 g/m2). LTM targeted a Methotrexate plasma concentration of ∼35 and ∼65 micromol/L in B- and T-ALL, respectively, in line with a therapeutic concept developed at St. Jude's Hospital (Memphis, TN, USA) in childhood ALL. The program was integrated by (a) a randomised, radiation-free central nervous system (CNS) prophylaxis study comparing intrathecal Methotrexate, Ara-C, and Prednisone (×12) vs. liposomal Ara-C (DepoCyte 50 mg, ×6 [B-ALL]-8 [T-ALL]); (b) the molecular evaluation of MRD at pre-fixed treatment weeks (w), to optimise risk stratification and indications for allogeneic stem cell transplantation (allo-SCT); and (c) by concurrent imatinib in patients with Ph+ ALL. The whole chemotherapy plus MRD study sequence was as follows: prephase (Prednisone, Cyclophosphamide) → induction (Idarubicin, Vincristine, Asparaginase, Dexamethasone) + w4 MRD → PDT1 (Cyclophosphamide, Vincristine, Idarubicin, Dexamethasone, Ara-C, 6-mercaptopurine) → LTM1 (plus high-dose Ara-C) + w10 MRD → PDT2 → LTM2 (plus Asparaginase) + w16 MRD → PDT3 → LTM3 (plus HD Ara-C) + w22 MRD → reinduction (Idarubicin, Vincristine, Cyclophosphamide, Dexamethasone). CNS prophylaxis was administered only during PDT blocks, with a minimum interval >15 days from/to LTM cycles. Risk classes were standard (SR: pre-B and WBC<30; cortical T and WBC<100; CR at cycle 1) and high (HR: other subsets). Allo-SCT was prescribed as soon as possible to all patients with Ph/t(4;11)+ ALL or other highly adverse cytogenetics; with WBC >100, or CD1a-negative early or mature T phenotype; and to SR/HR patients with MRD >10-4 after LTM1 (w10) or detectable at any level after LTM3 (w22). Autologous SCT followed by maintenance was a suitable alternative when allo-SCT was not feasible. All patients not eligible to frontline allo-SCT and with low positive (<10-4) or negative MRD results were submitted to standard maintenance. Results.Seventy-five of 81 evaluable patients (median age 40 years [range 18–65], 54% male, 23% T-ALL, 60% HR, 20% Ph+) achieved CR (92.5%); 55 of them (73.5%) were alive in CR1 at time of interim analysis (April, 2010). Sixteen patients relapsed (21.5%) and four died in CR of therapy-related complications (one after allo-SCT and 3 aged >55 years after a PDT cycle). With a maximum follow-up slightly >2 years (27.5 mos.), projected overall survival (OS) at 1.5 years is 73% (95% CI 57%-83%), and disease-free survival (DFS) 66% (95% CI 52%-77%). The best results were observed in patients aged 55 years and less (n=65, OS 79%) and those with T-ALL (n=20, OS 86%). These findings correlated well with a favourable early MRD response in Ph-negative subsets: 49% of evaluable cases achieved a major MRD response (<10-4) at end of induction cycle (w4: MRD negative 6/13 T [46%] vs. 7/24 B [29%]; <10-4 2 T and 3 B; overall 61.5% T and 42% B), as did 64% of the patients after LTM1 (w10: MRD negative 11/15 T [73%] and 16/27 B [59%]; <10-4 2 T and 1 B; overall 87% T and 63% B). MRD results were predictive of CR durability. Pooled MRD data from all time-points (w4-22) indicated a probability of CR of 92% in 18 patients with all results <10-4 vs. 60% in 19 patients with at least one MRD value >10-4 (P=0.039). The flexible risk- and MRD-adapted SCT policy resulted in high early transplantability rate in patients with this indication (70% in T-ALL). Finally, the randomised CNS prophylaxis trial preliminarily confirmed the feasibility of intrathecal DepoCyte as planned (total randomised patients = 57). Conclusions.This regimen was highly active, yielding a >90% CR rate in unselected adults aged up to 65 years, with a major early MRD response of 63% in Ph-negative B-ALL and 87% in T-ALL. The toxicity observed after PDT blocks in a first group of patients aged >55 years required a reduction of PDT dose intensity in this age group. LTM therapy proved feasible, up to 5 g/m2 in T-ALL, and remarkable early OS/DFS rates were obtained in T-ALL and in unselected patients aged <55 years. Disclosures:Bassan: Mundipharma: Consultancy, Research Funding. Off Label Use: Study is devoted to assess feasibility/toxicity/value of intrathecal liposome-encapsulated cytarabine (DepoCyte) vs standard intrathecal therapy as prophylaxis of CNS recurrence in adult ALL.

Hematopoietic stem cell transplantation for adults with acute lymphoblastic leukemia

The long-term survival for patients with adult acute lymphoblastic leukemia (ALL) has not significantly changed over the past two decades and, as opposed to pediatric ALL, it is less than 40% despite high initial complete remission rate. These observations raise several important issues regarding the pathobiology of adult leukemic stem cells, the ability of adults to tolerate intensive treatment and the best postremission approach. Progress has been made in understanding the biology of adult ALL and its prognostic significance. The follow-up of minimal residual disease status, adopting protocols from childhood ALL to young adults and use of targeted therapy may improve long-term survival. Transplantation for standard-risk ALL in first remission proved to be beneficial in a large cooperative group study. Limited data on the use of alternative donor transplantation as well as reduced intensity protocols are now available for consideration in specific groups of patients. Data suggest that the best antileukemic treatment should be applied in first remission and type of treatment should be based on individualized risk stratification, while incorporating recent information on alternative donors and reduced intensity approaches to transplantation.

Transplantation in adult ALL

The value of the allogeneic graft-versus-leukemia effect in adult acute lymphoblastic leukemia (ALL) has now been conclusively demonstrated and confirmed. While this is true for adults in all age groups, it may not be the best clinical option for young adults for whom increasingly intensive pediatric protocols are clearly of benefit. On the other hand, there is potentially wider applicability of allogeneic donor transplantation for adults 25 to 45 years old, for whom matched unrelated donors may be as safe and effective as sibling donors, and for the patient older than 45 years for whom reduced-intensity conditioning may be a promising way forward. Since the treatment-related mortality of allogeneic transplantation remains significant, careful selection of patients is mandatory. Patients with the Philadelphia chromosome, those with t(4;11) and those with a complex karyotype remain transplant candidates, and allogeneic transplantation remains the best option for salvage, where achievable, in a remission beyond first. As in childhood ALL minimal residual disease studies may be extremely useful in predicting outcome and, therefore, strategy, but at present there are less definite data in adults. Clinical indications to harness the allogeneic effect will mature as the true value of pediatric protocols in adult patients and the safety and efficacy of a sibling, unrelated and reduced intensity transplant emerge in this disease.

Vincristine pharmacokinetics is related to clinical outcome in children with standard risk acute lymphoblastic leukemia

Vincristine is a key drug in the treatment of childhood and adult acute lymphoblastic leukemia (ALL), and many other childhood malignancies. Despite decades of wide clinical use, no data on the correlation between vincristine pharmacokinetics and long-term clinical outcome have been published. We here report clinical data (median follow-up time 10.5 years, range 7.3-12 years) for 86 children with B-cell precursor ALL, in whom vincristine kinetics were studied on treatment day 1. The median total plasma clearance was 429 and 331 ml/min per m(2) and the area under the plasma concentration-time curve (AUC) was 4.49 and 5.40 mg/l x min in relapse and non-relapse patients, respectively (not significant). In standard risk patients, where treatment depends more heavily on vincristine than in other subgroups, the relative risk (RR) of relapse was significantly increased for patients with clearance values above median (RR 5.2; P = 0.036), or AUC values below median (RR 5.8; P = 0.025). Our data suggest a relationship between the antileukemic effect and the systemic exposure of the drug, which warrants further studies.

The BCL-2 Antagonist ABT-737 Is Highly Effective on Primary Acute Lymphoblastic Leukemia Cells.

The treatment of adult acute lymphoblastic leukemia (ALL) remains unsatisfactory. A potential hope is now given to Philadelphia-positive cases by targeted treatment modalities. Among other pathways involved in cell proliferation, we have recently demonstrated (Blood 2007; 109:5473) the unfavorable role of ERK1/2 phosphorylation as an independent predictor of complete remission (CR) in adult ALL, suggesting the potential therapeutic value of other targeted therapies. The B-cell leukemia/lymphoma 2 (Bcl-2) family of proteins are important regulators of apoptosis and are frequently found aberrantly expressed, particularly in lymphoid malignancies. The role of Bcl-2 overexpression in tumorigenesis and chemoresistance prompted us to investigate whether the inhibition of the antiapoptotic function may result also in ALL in an attractive therapeutic strategy. In this study, we thus investigated the cell cycle and apoptotic effects of ABT-737 (kindly provided by Abbott Laboratories), a Bcl-2 (BH3) inhibitor, on both lymphoid leukemia cell lines and primary adult and childhood ALL cells. The lymphoid leukemia cell lines CEM and MOLT-4 were exposed to increasing concentrations of ABT-737 (from 0.1 to 1 μM) up to 72 hours. A dose- and time-dependent cell growth arrest and induction of apoptosis was found. In fact, measuring the subG0/1 peak at 48 hours, the levels of apoptosis increased in the CEM cell line from 14.1% (DMSO) to 34.4%, 64.5%, 86.5% and 98.6% at ABT-737 concentrations of 0.1, 0.25, 0.5 and 1 μM, respectively. Similarly, 48 hours of exposure to ABT-737 increased in MOLT-4 the Annexin V-positive cells from 7.2% to 64.2%. The effects of ABT-737 were then examined on primary blasts from 9 ALL patients (6 adults and 3 children). Bone marrow aspirates with a blast infiltration >70% were obtained at diagnosis from patients broadly characterized for clinical and biological parameters, as well as therapeutic response. ALL cells were cultured in vitro with ABT-737 (at increasing concentrations from 0.01 to 1 μM) for 24 hours. A significant decrease in viability was observed at 0.01 μM (p=0.008) with a remarkable dose-dependent increase of apoptosis. In fact, Annexin V-positive cells increased from a mean baseline value of 16.8% ± 8.8 to 43.6% ± 22.8 (p=0.04), 66% ± 21.3 (p=0.0001), 70.3% ± 26.9 (p=0.04), 74.6% ± 18.9 (p=0.03) and 76.2% ± 11.8 (p<0.0001) in the presence of ABT-737 at 0.01, 0.1, 0.25, 0.5 and 1 μM, respectively. A significant cell killing was demonstrated in all samples (9/9), including Ph-positive ALL. No significant cell cycle changes were instead detected even at higher concentration of ABT-737. In summary, our study shows for the first time a potent growth-inhibitory and pro-apoptotic activity of the Bcl-2 antagonist ABT-737, at nanomolar concentrations, on primary cells from adult and childhood ALL samples. These results prompt to further extend pre-clinical studies in the different biologically-defined subset of ALL and suggest a potential clinical development of a Bcl-2 family inhibitor in this disease.

Genome-Wide 500K SNP Array Analyses Reveal High Frequencies of Cryptic Genetic Abnormalities and Identify Recurrent Microdeletions Targeting Novel Genes in Adult Acute Lymphoblastic Leukemia.

The long-term disease-free survival rate of adult acute lymphoblastic leukemia (ALL) patients remains less than 40%, in contrast to pediatric cases where it approaches 80%. Furthermore, whereas genetic abnormalities are widely used in childhood ALL for assigning patients to prognostic risk groups, their use in adult ALL is generally restricted to the presence or absence of the t(9;22)/[BCR-ABL1]. Novel prognostic markers, allowing better treatment stratification, and new treatment targets are therefore much needed. We have investigated diagnostic samples from 45 adult ALL cases using genome-wide, high-resolution single nucleotide polymorphism (SNP) array analysis with the Affymetrix 10K, 250K Nsp, and 250K Sty chips, in total including more than 500,000 SNPs with a median inter-marker distance of <2.5 kb. This method, which has not previously been applied to adult ALL, enables the detection of genetic copy number abnormalities as well as uniparental disomies (UPDs) with a much higher resolution than cytogenetic and molecular genetic techniques. We detected 367 genetic abnormalities not corresponding to known copy number polymorphisms among the 45 cases. These comprised 94 copy number gains, 211 hemizygous deletions, 47 homozygous deletions, and 15 UPDs. All but three of the patients (93%) displayed one or more anomaly not detectable with standard genetic analyses. Most notably, we found high frequencies of deletions targeting the genes CDKN2A (P16) (21 cases; 47%), PAX5 (15 cases; 33%), IKZF1 (IKAROS) (8 cases; 18%), ETV6 (7 cases; 16%), RB1 (5 cases; 11%), and EBF1 (2 cases; 4.4%). Thirty-two cases (71%) harbored a deletion of at least one of these genes. CDKN2A deletions were homozygous in 17 cases and associated with partial UPDs in 5 cases. PAX5 deletions were always hemizygous and frequently large, including also CDKN2A in all but three patients. Notably, loss of IKZF1 was found in 5 of 10 (50%) t(9;22)/[BCR-ABL1]-positive ALL cases. Reverse transcriptase-PCR for this gene showed that some t(9;22)-positive cases without this deletion expressed a dominant-negative isoform of IKZF1, suggesting that different mechanisms for downregulation of IKZF1 occur and that this gene is frequently targeted in t(9;22)+ ALL. Furthermore, the SNP array analysis revealed novel recurrent deletions targeting the genes DLG2 (4 cases; 8.9%), LDOC1 (3 cases; 6.7%), ERBB4, and CDH13 (2 cases each; 4.4%). In conclusion, using high-resolution SNP arrays we detect a very high frequency of hidden genetic changes in adult ALL cases. Deletions, frequently cryptic, comprised 70% of the found abnormalities, suggesting that microdeletions are a characteristic feature of adult ALL. Most importantly, the analyses revealed recurrent genetic abnormalities in adult ALL targeting novel genes, not previously implicated in leukemogenesis. In addition to giving new insights into the leukemogenic process, these findings are likely to be clinically important, with the possibility of identifying new prognostic markers as well as future treatment targets in adult ALL.

Why and how to quantify minimal residual disease in acute lymphoblastic leukemia?

Several studies have demonstrated that monitoring of minimal residual disease (MRD) in childhood and adult acute lymphoblastic leukemia (ALL) significantly correlates with clinical outcome. MRD detection is particularly useful for evaluation of early treatment response and consequently for improved front-line therapy stratification. MRD information is also significant for children undergoing allogeneic hematopoietic stem cell transplantation and those with relapsed ALL. Currently, three highly specific and sensitive methodologies for MRD detection are available, namely multiparameter flow cytometric immunophenotyping, real-time quantitative polymerase chain reaction (RQ-PCR)-based detection of fusion gene transcripts or breakpoints, and RQ-PCR-based detection of clonal immunoglobulin and T-cell receptor gene rearrangements. In this review, characteristics, pitfalls, advantages and disadvantages of each MRD technique are critically discussed. The special emphasis is put on interlaboratory standardization, especially in view of the results obtained within the European collaborative BIOMED-1, BIOMED-2, and Europe Against Cancer projects and recent developments by European Study Group on MRD detection in ALL and EuroFlow Consortium. Standardized MRD techniques form the basis for stratification of patients into the risk groups in new treatment protocols mainly in childhood ALL. Only the results of these studies can answer the question whether MRD-based treatment intervention is associated with improved outcome.

Sensitivity and Applicability of Six-Color Flow Cytometry Is Comparable to ASO-Primer-Real-Time PCR (RQ-PCR) for Minimal Residual Disease (MRD) Monitoring in Adult Acute Lymphoblastic Leukemia (ALL) - A Comparative Analysis in 70 Patients from the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL).

Although the quantification of MRD by PCR or flow cytometry (FC) is of proven prognostic value in ALL, both methods were never compared in adults. Since 3- to 4-color FC showed equal or inferior sensitivity to PCR in childhood ALL, we herein prospectively compared six-color FC (6C-FC) at initial diagnosis and during early follow-up in adult ALL to RQ-PCR as reference method.Diagnostic samples from 70 consecutive ALL patients (pts) (22 T-, 48 B-lineage; 18 to 71 yrs) were screened by 14 different multiplex immungene consensus PCRs as published (Bruggemann et al., Blood, 2006) and simultaneously by 8 different 6C-FC combinations which included a total of 29 different antibodies. At least one of the consensus PCRs revealed monoclonality in 64/70 pts (91 %), whereas immunophenotypic aberrations could distinguish ALL from benign hematopoiesis in 67/70 cases (96 %). 6C-FC was positive in all PCR negative samples, while PCR was successful in all samples lacking immunophenotypic aberrations. The standardized 6C-FC method revealed a median of 4 immunophenotypic aberrations per pt (range 1 to 8). The most frequently observed aberrations in B-lineage ALL included CD58++ (76 %), CD11a++(50%), CD22++ (44 %), low CD38 (50%), and KORSA+ (35 %). Tdt+cytoplasmatic(cy)CD3+ hallmarked 86% of T-lineage ALL cases which were additionally characterized by CD7+ (100%), surface(s) CD3- (90%), CD99+ (52 %), and CD1a+ (38 %).Up to now, RQ-PCR assays have been established for 38 pts (median sensitivity 10−4; range 10−5 to 10−2) thus allowing for comparisons of MRD assessments to 6C-FC in 155 follow-up samples. 110/155 (71 %) samples were collected during the first 4 therapy months. 6C-FC marker combinations for follow-up always included CD34/CD10/CD19/CD45 in B-lineage and TdT/cyCD3/sCD3/CD5 in T-lineage ALL. Stainings were individualized by adding 2 pt-specific markers each to up to 3 tubes. A total of 155 follow-up specimens comprised 13 FC-/RQ-PCR+ (8 %), 4 FC+/RQ-PCR- (3 %), 88 concordantly negative (57 %), and 50 concordantly positive (32 %) samples. Due to extremely low MRD, in 11/13 FC-/RQ-PCR+ samples (85 %) the results of RQ-PCR were qualitatively positive only, but not accurately quantifiable.The minimum MRD level detectable by 6C-FC was 3x10−5, as proven by a concordantly positive RQ-PCR result. MRD levels obtained by both methods correlated well (Spearman r = 0.85; p < 0.0001) in follow-up samples that were positive both by RQ-PCR and by 6C-FC. The median ratio between 6C-FC and RQ-PCR MRD results was 0.48 (range 0.012 to 113). In 11% of samples the ratio between MRD levels obtained by the two methods differed more than tenfold.Our novel standardized 6C-FC approach is highly sensitive for MRD assessments in adult ALL. Our results suggest the applicability of 6C-FC in a multicenter setting, an excellent specificity, a good quantitative correlation and a similar sensitivity when compared to RQ-PCR. Longer follow-up and the inclusion of more samples in particular from later time points are required to decide whether or not the two methods are of comparable clinical significance.

Clinical manifestations and treatment of newly diagnosed acute lymphoblastic leukemia in adults

Adult acute lymphoblastic leukemia (ALL) is a heterogeneous disease with distinct biologic and prognostic subgroups. The treatment of adults with ALL has evolved largely from the therapy developed for childhood ALL and, despite differences across regimens, can be broadly classified as including induction, consolidation, maintenance, and central nervous system prophylaxis. Although there has been marked improvement in the outcomes for pediatric patients with ALL, the same success has not yet been realized for adult patients. Some of this difference can be attributed to a greater incidence of unfavorable cytogenetic subtypes in adults than in children. In addition, the ability to tolerate intensive regimens likely plays a role. This article reviews the classification, prognostic features, current treatment programs, and new advances as applied to adult patients with newly diagnosed ALL.

Measurement of Minimal Residual Disease (MRD) after Consolidation Therapy as a Means of Early Detection of Relapse in Adult Acute Lymphoblastic Leukemia (ALL).

Outcome for patients with adult ALL after relapse is poor, even if a second remission is achieved by salvage therapy and is consolidated by an allogeneic stem cell transplantation. There is considerable evidence from studies in childhood ALL and other hematologic malignancies that administration of salvage therapy in early molecular relapse may improve prognosis. We therefore investigated the value of continuous MRD-monitoring in prospectively monitored standard risk patients of the German Multicenter ALL-studies 06/99 and 07/03 during and after maintenance treatment beyond the first year of therapy. MRD was assessed by quantitative realtime PCR using clone-specific primers for the leukemia-specific Ig/TCR gene rearrangements. MRD high risk-patients (those with MRD > 10−4 at two successive time points after induction during the first year of therapy) are candidates for early therapy escalation according to the study protocol and were thus excluded from this analysis. Of the 108 remaining patients (77 male, 31 female), 30 (28%) became MRD-positive again after a median follow-up of 18 months after the end of consolidation treatment. Of these, 17 (57%) already relapsed. When only considering patients with MRD measurable within the quantitative range of the PCR 16/19 (84%) already relapsed. Of the MRD-negative patients only 5 of 78 (6%) have relapsed after a median follow up of 23 month after end of initial therapy. After conversion to MRD positivity, the median time to relapse was 9.5 months, with a median time to relapse of only 2.5 months once the patients' MRD was within the quantitative range of the PCR assay. Taken together, these data indicate that, if done at regular intervals, MRD-monitoring allows for accurate and timely identification of patients in need for treatment escalation in the vast majority of cases and may help to avoid overtreatment for those patients who are cured by conventional chemotherapy alone.

The presence of TEL/AML1 rearrangement and cryptic deletion of the TEL gene in adult acute lymphoblastic leukemia (ALL)

TEL/AML1 (also known as ETV6/RUNX1) rearrangement is the most frequent genetic change in childhood B-acute lymphoblastic leukemia (ALL) and is associated with a favorable prognosis. Its presence in adult ALL, however, has been questionable, and any association between TEL/AML1 rearrangement and clinical prognosis is unknown. To reveal the presence and incidence of the TEL/AML1 rearrangement in adult ALL, we applied fluorescence in situ hybridization (FISH). We conducted extra-signal, dual-color fluorescence in situ hybridization (ES-FISH) for TEL/AML1 rearrangement on bone marrow cells from 74 adult ALL patients and analyzed the survival time. We demonstrated the TEL/AML1 rearrangement in 2 patients (2.7%) among 74 patients with ALL. Of 74 adult ALL patients, 3 (4.0%) showed deletion of the TEL gene without TEL/AML1 rearrangement. The mean survival time of patients with TEL/AML1 +ALL and patients with cryptic TEL deletion was 33 and 5 months, respectively, whereas the mean survival time of 53 TEL/AML1 −ALL patients was 16.7 months. TEL/AML1 rearrangement is not unique in childhood ALL, and cryptic TEL deletion without TEL/AML1 rearrangement was more frequent than the TEL/AML1 rearrangement in adult ALL. We recommend TEL/AML1 FISH in adult ALL patients because conventional cytogenetic studies so far have yielded uninformative results.

The MTHFR C677T and A1298C Polymorphisms and Susceptibility to Childhood Acute Lymphoblastic Leukemia in Portugal

Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme in folate metabolism and in DNA methylation and synthesis. The role of two common polymorphisms at the MTHFR gene, C677T and A1298C, in the etiology of childhood or adult acute lymphoblastic leukemia (ALL) has been previously investigated. Although a protective effect of MTHFR*677T against ALL was systematically reported, the magnitude of the effect appeared to be influenced by population-specific gene-environmental interactions. The evidence of the role of MTHFR*1298C in ALL susceptibility was less consistent, emphasizing the need for enlarging molecular epidemiologic studies to independent trials from different populations. The authors analyzed in North Portugal the association between variations at the two MTHFR positions and risk of ALL by comparing genotypes and gene frequencies in 103 affected children with those in 111 healthy controls. None of the variations was found to significantly affect the risk of developing childhood ALL in North Portugal, and this finding per se is of relevance in further studies aimed at assessing the etiology of the pathology in this specific population. Despite the absence of statistical significance, these data revealed that the frequency of MTHFR*677T was lower in patients than in controls, a result that is congruent with other reports and with the functional model usually invoked to explain its ALL protective effect. Concerning MTHFR1298*C, this study failed to corroborate previous findings of decreased risk of ALL in the presence of the variant.

MLL-AF4 Positive Adult ALL Patients: VH6 Immunoglobulin Gene Rearrangements Predominate While FLT3 Mutations Are Rare.

Among patients with adult acute lymphoblastic leukemia (ALL) the t(4;11) translocation (resulting in the MLL-AF4 fusion gene) is a recurrent chromosomal abnormality occurring in approximately 10–15% of cases. It is associated with poor outcome and in the majority of cases stem cell transplantation is the only possible cure. The MLL-AF4 cases show a more immature phenotype than other subtypes frequently lacking Ig light chain and/or TCR gene rearrangements (Brumpt et al; Blood , 2000; 96:2254). We have previously observed that in adult ALL there is a bias towards the use of JH-proximal VH genes and in particular an over-usage of the VH6 gene segment, the most JH-proximal VH element. This is also common in MLL-AF4 patients while rarer in ALL carrying other chromosomal abnormalities (Mortuza et al, 2001; Blood , 97: 2716). VH6 gene usage is also predominant (8–12%) in adult ALL when compared to the mathematical expected VH6 gene usage (0.8%) or the usage observed in normal PB or in other B-cell malignancies, such as CLL (3.6%) and B cell lymphomas (0%).Finally, mutations of FLT3 are detected in a large proportion of acute myeloid leukemias and in a small proportion of childhood ALL carrying the MLL-AF4 fusion gene while it has seldom been observed in adult ALL with t(4;11).The aims of our study were twofold: 1) to expand on the initial observation that the VH6 gene rearrangement may be more frequent in adult patients carrying the t(4;11) translocation; 2). To assess the incidence of FLT3 mutations in an homogenous cohort of adult ALL patients carrying t(4;11) abnormality.Thirty-two cases of MLL-AF4 fusion positive adult ALL patients (17M/15F) were analysed in addition to the RS4;11 cell line. Pro-B phenotype (n=11)predominated. Age ranged between 22–55 yrs(median: 32 yrs). Patients had cytogenetic or molecular evidence of t(4;11). The WBC count at presentation was high (60–550x109/l; median: 220). Patients were tested for VH gene rearrangement using FRI and consensus-JH primers. Internal tandem duplication and codon 835–836 kinase mutations of the FLT3 were screened by DNA PCR analysis.Twenty-seven patients (84%) of the 32 tested carried a VH6 gene rearrangement. Of 36 IgH alleles, 27 (75%) were VH6 rearrangements. VH1 in 4 (11%) rearrangements was the second most common rearrangement while 3 were VH3 (8%) and 2 (5.5%) were VH4. Compared to our previous study (Mortuza et al, 2001; Blood , 97:2716) the incidence of VH6 IGH rearrangements in MLL positive cases represents a nine-fold increase (75% vs 9.4%) compared to non-MLL + cases. No Internal Tandem Duplication of the FLT3 gene was detected in the 31 cases tested, while one (3.6%) of the 28 cases analysed showed the recurrent mutation of the Asp835Tyr of the FLT3 gene. In conclusion, patients carrying the t(4;11) translocation show a very skewed pattern of IgH gene rearrangement restricted almost exclusively to the VH6 usage, in agreement with both the more immature B cell phenotype and observation in the mouse immune system. However, in t(4;11) adult ALL FLT3 is rarely found mutated and may therefore play a minor role in the poor outcome of this ALL subgroup.