T-lineage 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.

Abstract 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

Value of Immunophenotype in Intensively Treated Adult Acute Lymphoblastic Leukemia: Cancer and Leukemia Group B Study 8364

Value of Immunophenotype in Intensively Treated Adult Acute Lymphoblastic Leukemia: Cancer and Leukemia Group B Study 8364

Value of immunophenotype in intensively treated adult acute lymphoblastic leukemia: cancer and leukemia Group B study 8364.

The prognostic value of immunophenotype in adult acute lymphoblastic leukemia (ALL) has varied based on the methods used, surface markers studied, and therapy administered. From April 1991 to September 1996, samples of leukemic marrow or blood from 259 eligible and evaluable adult ALL patients entering dose-intensive Cancer and Leukemia Group B (CALGB) front-line treatment protocols were prospectively studied for immunophenotypic classification by multiparameter flow cytometry (MFC) in a central laboratory. A B-lineage (B-LIN) phenotype was expressed in 79% of cases, with one third coexpressing myeloid antigens. A T-lineage (T-LIN) phenotype was expressed in 17% of cases, with one quarter coexpressing myeloid antigens. Since the advent of more intensive CALGB therapy which incorporated cyclophosphamide and the early use of L-asparaginase into the backbone of daunorubicin, vincristine and prednisone, together with central nervous system prophylaxis for adult ALL, no significant differences in response rates, remission duration, or survival have been seen in those patients coexpressing myeloid antigens. The T-LIN phenotype was associated with younger age (P =.01), a higher male to female ratio (P =.01), higher white blood cell count (P =.001) and hemoglobin (P <.001) levels, presence of a mediastinal mass (P <. 001), and longer survival (P =.01) and disease-free survival (DFS) (P =.01) when compared to patients with a B-LIN phenotype. The 3-year probability of survival and DFS (95% confidence interval [CI]) of T-LIN adult ALL was 0.62 (0.46 to 0.76) and 0.62 (0.44 to 0. 77), respectively. Comparatively, the 3-year probability of survival and DFS (95% CI) of B-LIN adult ALL was 0.42 (0.35 to 0.50) and 0.39 (0.31 to 0.47), respectively. The number of T markers expressed in T-LIN ALL cases was shown to have prognostic significance. In particular, patients expressing six or more markers compared with patients expressing three or fewer markers had longer DFS (P =.003) and survival (P =.004). The presence of the Philadelphia chromosome was significantly associated with B-LIN ALL cases which coexpressed CD19(+), CD34(+), and CD10(+) (49%; P =.003), whereas the majority of t(4;11) cases were CD19(+), CD34(+) but CD10(-). The knowledge gained from this study of MFC of a large number of patients will permit a reduction in the number of antigens to be evaluated in future studies. Overall, this should lead to cost savings without loss of valuable information. A rational approach for future studies would be to use four-color flow cytometry (instead of the current three-color) to help further streamline the study of immunophenotype of adult ALL by MFC.

Array Based Comparative Genomic Hybridization Detects Copy Number Alterations in 80.3% of Adult Acute Lymphoblastic Leukemia (ALL) with Normal Karyotype or Failed Chromosome Banding Analysis and Identifies a Subset with Only Submicroscopic Alterations Associated with Favorable Outcome

Abstract Background: Based on chromosome banding analyses (CBA) more than 70% of ALL harbor chromosome abnormalities. These include balanced translocations and a large spectrum of deletions and gains. In up to 10% of ALL CBA fails. In approximately 20% of cases no chromosome abnormalities are detected. This might be due to the fact that ALL blasts failed to proliferate in vitro, to the submicroscopic size of alterations or to a truly normal karyotype. Aim: To characterize the subset of ALL with normal karyotype or failed CBA using array based comparative genomic hybridization (aCGH) and to evaluate whether this technique can provide relevant information in the diagnostic work-up and prognostication of ALL. Patients and Methods: Out of a total of 757 adult ALL patients (age 18.0-91.4 yrs, median 52.5 yrs) analyzed at diagnosis between 2005 and 2014 we selected a subset of 190 cases with normal karyotype (n=144; 75.8%) or failure of CBA (less than 11 analyzable metaphases without clonal chromosome abnormalities, n=46; 24.2%). All cases were analyzed by aCGH (12 x 270 K microarray slides, Roche Nimblegen, Madison, WI). In addition, data on FISH or PCR for BCR-ABL1 and MLL rearrangements was available in 170/190 pts. Results: 92 cases were classified as B-lineage ALL, 46 as T-lineage ALL and 14 showed a Burkitt phenotype. For 38 pts no data on ALL immunophenotype was available. Out of 170 pts investigated by FISH or PCR 21 (12.4%) pts were positive for BCR-ABL1 and 3 (1.8%) pts showed an MLL rearrangement. All 14 pts with a Burkitt phenotype showed a MYC-rearrangement by FISH. In 12 cases (6.3%) aCGH analyses failed due to poor DNA quality. By aCGH 143/178 (80.3%) pts harbored an aberrant karyotype while only 35 showed no copy number alteration (19.7%). 785 copy number alterations were observed in 143 pts (mean 5.5 per case; range: 1-47). 292 were whole chromosome gains (n=164) or losses (n=128) while 493 were alterations affecting certain chromosome regions. Losses of chromosomal regions were more common than gains (333 vs 160). 253 gains and 222 losses, i.e. 60.5% of all affected chromosomes/chromosomal regions, were larger than 10 Mbp. This size is above the resolution of CBA and thus we concluded that these aberrations were missed by CBA due to lack of proliferation of ALL blasts in vitro. 239 losses and 71 gains were smaller than 10 Mbp and thus are not detectable by CBA. In 40 pts (22.5%) only submicroscopic alterations were detected. Most frequent alterations observed by aCGH were: Losses of 9p21 (CDKN2A) (n=58; 32.6%), 6q (n=21; 11.8%), 13q14 (RB1) (n=21; 11.8%), 7q34 (TCRB) (n=21; 11.8%), 12p13 (ETV6) (n=14; 7.9%), 7p12 (IKZF1) (n=13; 7.3%), 14q32 (IGH) (n=8; 4.5%), 5q33 (EBF1) (n=7; 3.9%), 10q23 (GRID1, PTEN) (n=6; 3.4%), 3p (n=6; 3.4%) as well as gains of 1q (n=14; 7.9%). Deletions of 5q33, 6q, 7p12, 7q34, 9p21, 10q23, 12p13 and 13q14 were observed in both B- and T-cell precursor ALL, respectively. In contrast, losses and gains of whole chromosomes, gains of 1q and 14q32 deletions were only detected in pts with B-cell precursor ALL. In the subset of 40 pts harboring only submicroscopic abnormalities the most frequently affected regions were loss of 9p21 (CDKN2A) (40.0%), 14q32 (IGH) (10.0%), 7q34 (TRBV) (10.%), 13q14 (RB1; RCBTB2) (7.5%), 21q22 (KCNJ15) (7.5%). No recurrent gain was identified. Clinical follow-up data was available for 95 pts. Based on aCGH 12 cases with a typical pattern of chromosome losses characteristic for the ALL subset with a low hypodiploid karyotype were detected. As has been described previously for this group they showed a dismal outcome with a median OS of only 5.3 months. The relative risk for death compared to all others amounted to 4.5 (p=0.047). There was a tendency for better OS in patients showing only submicroscopic abnormalities by aCGH, OS at 3 years was 83.6% compared to 60.5% in all others (p=0.09). Conclusions: 80.3% of ALL with normal karyotype in chromosome banding analysis or failed cytogenetics habor copy number alterations detectable by array CGH. The pattern of lost and gained chromosomal regions is comparable to the alterations most frequently occurring in ALL. 12 patients (6.7%) with a characteristic low hypodiploid karyotype were detected, who showed the known poor outcome. A novel subset of 22.5% of pts was identified showing submicroscopic copy number changes only and a favorable outcome. Thus, nearly a third of the target population can be further classified by aCGH for better prognostication. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

T Cell Exhaustion and Downregulation of Cytotoxic NK Cells – an Immune Escape Mechanism in Adult Acute Lymphoblastic Leukemia

Abstract Adult acute lymphoblastic leukemia (ALL) remains a therapeutic challenge with less than 40% long term survival. There is growing evidence that malignant diseases exert an “immune editing” effect which blocks antitumor immunity and permits tumor growth through immune evasion. Such tumor escape represents an obstacle for anticancer immunotherapy. In ALL such immune escape mechanisms are not well characterized. We therefore profiled cellular immunity in ALL, by characterizing the subsets of T cells, regulatory T cells (Treg), natural killers (NK) cells and γd T cells, using various functional markers including T cell exhaustion and NK cell activating or inhibitory molecules. Forty ALL patients were included in the study. The median age was 39 y (range, 18-75). Thirty-six presented with B-lineage ALL and 4 with T-lineage ALL. Mononuclear cells were isolated from blood (n=19) or bone marrow (n=21) at the onset of leukemia or at relapse. The median infiltration of blasts was 85% (range 24-96%). Healthy donor peripheral blood (n=12) and bone marrow (n=9), from age and gender matched population, were simultaneously analyzed as controls. Extra-and intra cellular staining were performed using using antibodies directed against CD3, CD4, CD8, CD45, CD45, CD45RA, CD45RO, CCR7, CD95, CD27, CD19, CD14, CD127, CD25, Foxp3, Helios, αβTCR, HLA-DR, CD117, CD20, CD10, CD22, CD34, LAG3, PD1, PDL1, CD56, NKG2A, NKG2C, NKG2D, KIR2DL1, KIR2DL3, CD57, CD33, CD11b, CD15, CD38 and CD24. Data were acquired on a BD LSRFORTESSA flow cytometer. The expression of programmed cell death 1 (PD-1, CD279) receptor on CD8+T cells was significantly increased in blood and bone marrow of ALL patients compared to healthy donors (p&lt;0.0001 and p=0.004, respectively) (Fig. 1). Focusing on the different subsets, CD8+ effector memory T cells significantly over-expressed PD-1 in blood and bone marrow of ALL patients compared to healthy donors (p=0.008 and p=0.04, respectively). Moreover, there was a significant positive correlation between PD-1 expression on CD8+ effector memory T cells and blast infiltration (R2=0.23, 95%CI 0.026-0.76, p=0.04). Expression of the co-inhibitory receptor lymphocyte-activation gene 3 (LAG-3, CD223) was similar in ALL patients compared to healthy donors. A significantly higher frequency of T regulators (CD25+, CD127 low, Foxp3+) was found in bone marrow microenvironment in ALL patients (4.3% versus 1.6%, p=0.02). Concerning γd T cells, frequency was similar in blood and bone marrow of ALL patients compared with healthy donors. There was a significantly lower frequency of CD56dimNKG2A+KIR-CD57- (p=0.02) in the bone marrow of ALL patients indicating a maturation arrest. Interestingly, expression of the activating receptor NKG2D which plays an important role in triggering the NK cell–mediated tumor cell lysis was significantly reduced in NK cells of ALL patients while no difference in NK cell expression of NKG2C was found(Fig. 2). Adult patients with ALL show evidence of immune-editing of T cells and NK cells. This global immunosuppressive mechanism may contribute to the eventual escape of ALL from immune control. PD-1, overexpression, described in acute myeloid leukemia and chronic myeloid leukemia has been implicated in T-cell exhaustion and subsequent tumor immune evasion. Our data suggests similar immune escape mechanisms pertain in ALL. Effective antileukemia immunotherapy will require targeting one or more of these immunosuppressive pathways to achieve optimum results. Disclosures Fathi: Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

Reappraisal Of The p16INK4A/Prb Pathway and Telomerase Activity, Markers Of Cell Activation and Aging, In Adult Acute Lymphoblastic Leukemia: A Graall Study

Abstract Introduction The treatment of Adult Acute Lymphoblastic Leukemia (ALL) has improved by the use of pediatric-like approaches, erasing the poor prognostic impact of numerous variables. New markers are however still needed to identify poor prognosis patients in prospective trials. The p16INK4A/CDK4-6/pRb pathway and telomerase activity (TA), markers of cell activation and aging, were analyzed in 175 adult ALLs, treated between November 2003 and January 2007 according to the GRAALL/GRAAPH trials, to investigate their prognostic value in this context. Methods The cohort comprised 105 males and 70 females aged between 16 and 59 years (median: 36). Immunophenotype by flow-cytometry allowed for the characterization of 123 B- and 52 T-lineage ALLs, 163 of them being subdivided according to EGIL criteria. Cytogenetic and/or molecular analyses allowed for the detection of BCR-ABL, MLL-AF4 or E2A-PBX1 fusion transcripts, NOTCH1 mutations in T-ALLs, and IKAROS deletions in BCR-ABL-negative B-ALLs. In all cases, cell samples were obtained at diagnosis, before any treatment, from bone marrow aspiration after the patients provided informed consent. Flow cytometric analysis of the DNA content was performed for evaluation of the percentages of cells in S/G2/M phases of the cell cycle. Cell cycle regulatory proteins were examined in 135 samples, by western blot. The TA assay was performed on 156 samples according to a telomeric repeat amplification protocol. Furthermore, in vitro analyses of the p16INK4A/CDK4-6/pRb pathway and TA were carried out in normal peripheral blood lymphocytes before and after stimulation and during lymphocyte long-term culture. Results The p16INK4A/CDK4-6/pRb pathway and TA were analyzed according to the immunological phenotype and molecular characteristics of ALLs. Leukocytosis (p&lt;0.0001), proliferating blast cell percentage (p=0.004), CDK6 (p = 0.011), and pRb phosphorylation (p-pRb) (p = 0.003) were significantly higher in T-ALLs compared to B-ALLs, while p16INK4A expression was significantly higher in the latter (p = 0.002). Enhanced p16INK4A significantly correlated with B- and T-maturation (p = 0.03) and MLL rearrangement (p &lt; 0.003). Among sub-groups defined by clinical or biological data, the most significant relationships between the p16INK4A/CDK4-CDK6/pRb pathway or TA and prognosis were observed for BCR-ABL1+ ALLs (31 cases). An above median expression of CDK4 was related to shorter disease-free survival (DFS) (p = 0.031) and overall survival (OS) when taking into account either death due to any cause (p = 0.005), or only disease-related death (p = 0.018). In spite of the small size of this series, this prognostic value remained when the analysis was restricted to patients who did not receive allogeneic stem-cell transplantation (non-Allo SCT) for DFS (p = 0.034), OS (p = 0.019) or disease-related OS (p = 0.05). A shorter DFS was also associated with the CDK4/6 mediated-phosphorylation of pRb (p-pRb/pRb ratio &gt; 0) (p = 0.042), paradoxically, with high expression levels of p16INK4A (p = 0.029), and also with above median TA (p=0.040). Patients with the highest TA (over quartile 75) showed a significantly shorter OS (p = 0.018) especially when considering only disease-related death (p = 0.007). The poor prognostic value of above median TA for OS (p = 0.026) and disease–related OS (p = 0.009) was also confirmed in the non-Allo SCT group. Considering that IKAROS is deleted in most of BCR-ABL1+ ALLs and since survival was similar in BCR-ABL1+ ALLs and BCR-ABL1-/IKAROSdel B-ALLs, these two groups of patients were pooled for analysis (42 cases). A shorter DFS was linked to pRb phosphorylation (p = 0.036), while both a shorter DFS (p = 0.026) and OS (p = 0.055) were noted for patients with high TA. In vitro analyses in normal lymphocytes demonstrated that increased expression of p16INK4A, CDK4, p-pRb and TA is related to cell activation, suggesting that ALL blasts with these criteria could be in an activated stage. Conclusions These data bring new perspectives to the biological characterization of ALLs and associate a poor prognosis in BCR-ABL1+ ALLs with enhanced cell activation. Additional investigations could focus, in a prospective series, on the analysis of the cell activation markers described here and on the development of new therapeutic strategies by proposing the association of lymphocyte activation inhibitors. Disclosures: No relevant conflicts of interest to declare.

The prognostic significance of minimal residual disease in adult Egyptian patients with precursor acute lymphoblastic leukemia

Minimal residual disease (MRD) studies in adult acute lymphoblastic leukemia (ALL) give highly significant prognostic information superior to other standard criteria as age, gender and total leucocytic count (TLC) in distinguishing patients at high and low risk of relapse. We aimed to determine the value of MRD monitoring by flowcytometry (FCM) in predicting outcome in adult Precursor ALL patients. Bone marrow (BM) samples were analyzed by 4-color FCM collected at diagnosis and after induction therapy (MRD1) to correlate MRD positivity with disease free survival (DFS) and overall survival (OS). Study included 57 adult ALL patients (44 males and 13 females) with a median age of 22 years (18-49). DFS showed no significant difference with age, gender and initial TLC (p=0.838, 0.888 and 0.743, respectively). Cumulative DFS at 2 years was 34% for B-lineage ALL (n: 35) and 57% for T-lineage ALL (n: 18) (p = 0.057). Cumulative DFS at 2 years was 7% for MRD1 positive (high risk, HR) versus 57% for MRD1 negative patients (Low risk, LR) (p < 0.001). Cumulative DFS at 2 years was 29% for HR patients (n: 26) versus 55% for LR (n: 27) according to GMALL classification (p = 0.064). Cumulative OS did not differ according to age, gender and TLC (p = 0.526, 0.594 and 0.513, respectively). Cumulative OS at 2 years was 36% for B ALL (n: 39) versus 77% for TALL (n: 18) (p = 0.016) and was 49% for Philadelphia chromosome (Ph) negative patients versus 0% for Ph-positive patients (p < 0.001). Regarding MRD1, OS at 2 years was 18% for MRD1 HR (n: 17) versus 65% for MRD1 LR (n: 38) (p < 0.001). OS was 35% for high-risk patients (n: 30) and 62% for low-risk patients (n: 27) classified according to GMALL risk stratification (p = 0.017). MRD by FCM is a strong independent predictor of outcome in terms of DFS and OS and is a powerful informative parameter in guiding individual treatment in ALL patients.

Novel agents and biomarkers for acute lymphoid leukemia.

New genetic markers for adult acute lymphoblastic leukemia (ALL) have been found to have prognostic impact, such as the lymphoid transcription factor gene IKZF1 alterations, which are associated with a high rate of leukemic relapse in B-ALL. Although complete remission rates by induction chemotherapy in ALL are now high, the long-term survival is still disappointing. Improvements in the survival outcome of ALL have been observed in young adults as a result of the use of pediatric inspired regimens and the broadening of the number of patients who are eligible for allogeneic SCT. Development of new and less toxic agents also provide promise to improve the outcome in adult ALL, such as tyrosine kinase inhibitors in Ph-positive ALL, rituximab in CD20-positive disease, blinatumomab in precursor B-ALL and nelarabine in T-lineage ALL. Challenges for the future are to implement genomic profiling into the clinical setting to guide risk stratification and providing novel targets for tailored therapies.

Long-Term Outcomes of TBI-Based Myeloablative SCT In 292 Consecutive Adults with Ph-Negative ALL: Similar Outcomes for Transplantation Using Related Donor, Well-Matched Unrelated Donor, or Partially Matched Unrelated Donor Sources

Abstract Abstract 2355 Background: The graft-versus-leukemia effect in adult acute lymphoblastic leukemia (ALL) has now been definitely confirmed from the ‘matched related donor (RD) versus no donor' comparisons. However, no definite conclusions can be extracted from these data as to whether or not there is a survival advantage to RD-stem cell transplantation (SCT) over other therapeutic modalities for both high-risk and standard-risk patients with Philadelphia (Ph)-negative ALL. In addition, ‘RD versus no donor' approach is becoming outmoded, as in many studies those previously in a ‘no donor' category are now undergoing unrelated donor (URD)-SCT. Aims: We report long-term outcomes of total body irradiation-based myeloablative SCT in 292 consecutive adults with Ph-negative ALL who received transplants at our center between 1995 and 2008 (median follow-up of survivors, 85 months). This study focused on following questions: (1) How different are the outcomes of SCT according to the donor sources? (2) Which factors are important to determination of transplantation outcome? (3) Which URD should be chosen? (4) Is there a role of autologous (AUTO)-SCT plus maintenance chemotherapy? Methods: Median age was 25 years (range, 15–63 years). Overall, 237 (81.2%) of 292 patients had one or more high-risk features, including adverse cytogenetics [t(4;11), t(8;14), complex (&gt;=5 abnormalities), Ho-Tr], older age (&gt;=35 years), high leukocyte counts (&gt;=30×109/L for B-ALL, &gt;=100×109/L for T-ALL), or delayed first complete remission (CR1; &gt;28 days). Two hundred and forty-one patients (82.5%) were transplanted in CR1; 22 (7.6%) in CR2; and 29 (9.9%) in advanced status. URD sources were classified as well-matched (WM), partially matched (PM), and mismatched (MM) based on a new proposed guideline from the NMDP-CIBMTR. Donor sources were RD (n=132), WM-URD (n=30), PM-URD (n=19), MM-URD (n=19), and AUTO (n=92). All patients and donors provided written informed consent, and the treatment protocol was approved by the institutional review board of The Catholic University of Korea. Results: Cumulative incidence of relapse at 5 years was 48.5% for AUTO versus 32.6% for RD, 19.4% for WM-URD, 32.3% for PM-URD, and 51.0% for MM-URD (RR, 2.70; 95% CI, 1.65 to 4.42; p&lt;0.001). In multivariate analyses, other factors associated with higher relapse risk included transplantation in CR2 or later (p&lt;0.001), T-lineage ALL (p=0.020), and adverse cytogenetics (p=0.038). Cumulative incidence of non-relapse mortality (NRM) at 5 years was 40.5% for MM-URD versus 19.6% for SD, 20.3% for WM-URD, 15.8% for PM-URD, and 9.8% for AUTO (RR, 3.09; 95% CI, 1.32 to 7.25; p=0.010). Patients older than 35 years had higher NRM (p=0.007). As a result, disease-free survival (DFS) at 5 years was inferior using AUTO (46.1%; RR, 1.69; 95% CI, 1.14 to 2.51; p=0.010) or MM-URD (26.3%; RR, 2.03; 95% CI, 1.05 to 3.95; p=0.036), compared to RD sources, while DFS from all other donor sources was approximately equivalent (53.5% for RD, 63.3% for WM-URD, and 57.0% for PM-URD). Transplantation in CR2 or later (p&lt;0.001), older age (p=0.020), and adverse cytogenetics (p=0.041) were associated with poorer DFS. In a pairwise comparison of outcomes between RD-SCT and AUTO-SCT for patients in CR1, the inferiority of AUTO-SCT was observed, particularly in high-risk patients. Conversely, in standard-risk patients, AUTO-SCT yielded comparable outcomes to RD-SCT. Summary/Conclusions: Our long-term data suggest that outcomes are similar for transplantation using SD, WM-URD, or PM-URD sources, and these may be considered the best donor sources for adults with Ph-negative ALL, especially for those with high-risk features. Disclosures: No relevant conflicts of interest to declare.

Status of Minimal Residual Disease Determines Outcome of Autologous HSCT in Adults with High-Risk Acute Lymphoblastic Leukemia.

Abstract Abstract 2321 Poster Board II-298 The role of autologous hematopoietic stem cell transplantation (autoHSCT) in the treatment of adult acute lymphoblastic leukemia (ALL) is a subject of controversies as several prospective studies failed to prove its advantage over maintenance chemotherapy. Those studies, however, did not take into account the status of minimal residual disease (MRD), which is now recognized a potent predictor for relapse among patients treated with conventional-dose chemotherapy. The goal of this analysis was to determine the impact of MRD on outcome of autoHSCT. Data on 123 autoHSCT recipients collected from 6 study groups cooperating in the European Leukemia Net were analyzed. Median age of 77 B-lineage and 46 T-lineage high-risk ALL patients was 31 (16-59) years. Ph+ ALL was recognized in 20 cases. All patients were in first complete remission (CR) lasting 6 (1.5-22) months. Peripheral blood was used as a source of stem cells in 67 patients whereas bone marrow, in 56 cases. Conditioning was based on chemotherapy alone (n=76) or total body irradiation (n=47). MRD was evaluated in bone marrow with the use of either multiparametric flow cytometry (n=79) or molecular techniques (n=44). MRD level of 0.1% bone marrow cells was used as a cut-off point for the purpose of this study. At the time of autoHSCT MRD was &amp;0.1% in 93 patients and ≧0.1% in 30 cases. With the median follow up of 5 years, the probability of leukemia-free survival (LFS) at 5 years for the whole group equaled 48% (+/-5). Three patients died of transplantation-related complications. The LFS rate was significantly higher for patients with the MRD level at transplantation &amp;0.1% compared to those with MRD ≧0.1% (57% vs. 19%, p=0.0002). The difference was particularly pronounced for peripheral blood HSCT (66% vs. 20%, p=0.0006) and for T-lineage ALL (62% vs. 8%, p=0.001). In a multivariate analysis adjusted for other potential prognostic factors (age, CR duration, Ph+ ALL, immunophenotype, source of stem cells, type of conditioning), the MRD status &amp;0.1% remained the only independent factor associated with increased LFS (HR=2.5, p=0.0009). CONCLUSIONS: MRD status is the most important predictor for LFS after autoHSCT in adults with ALL. More than half of patients with high risk disease and low MRD level at the time of transplantation may be cured. This observation may contribute to re-evaluation of the role of autoHSCT in the therapy of adult ALL. Disclosures: No relevant conflicts of interest to declare.

Expression of interleukin 15 in primary adult acute lymphoblastic leukemia

Interleukin-15 (IL-15) has been associated with the growth, survival and biological behavior of leukemic cells and response to therapy. We determined the expression of IL-15 in lymphoblasts and evaluated its potential impact on the outcome in adult acute lymphoblastic leukemia (ALL). Between June 1999 and June 2006, ALL samples were collected from 87 adult patients before initiation of antineoplastic therapy. These patients were enrolled in the German Multicenter Acute Lymphoblastic Leukemia June 1999 and July 2003 study trials. The expression of IL-15 in leukemic cells was analyzed by real-time polymerase chain reaction. The expression of IL-15 correlated with the immunophenotype: T-lineage ALL had a more than 4-fold higher IL-15 mRNA expression as compared with B-cell precursor (BCP)-ALL (P < .001). Patients with BCR-ABL(+)-BCP-ALL had lower IL-15 expression compared with BCR-ABL(-)-BCP-ALL (P = .041). Furthermore, higher expression of IL-15 was associated with mediastinal (P = .001) and lymph node infiltration (P = .051), but not with hepatomegaly and splenomegaly. Notably, high IL-15 expression in BCP-ALL was associated with an inferior relapse-free survival (RFS) at 5 years (0.17 +/- 0.13 vs 0.47 +/- 0.13) (P = .008), but there was no impact on overall survival (P = .249). Differential expression of IL-15 in adult ALL at diagnosis was associated with clinical features and outcome, in particular, RFS. It remains to be evaluated whether IL-15 might be a relevant therapy target, or might be used for risk stratification.

High Expression of IL-15 Correlates with Disease Recurrence of Adult B-Lineage Acute Lymphoblastic Leukemia

Abstract Cytokines are involved in the response to therapy and resistance of leukemic cells as well as their biological behaviour. Interleukin (IL)-15 has been shown to enhance the proliferation of both normal and malignant lymphocytes, thus suggesting a potential role in leukemogenesis and disease progression. In this study, we analyzed the expression of IL-15 in leukemic cells from 87 patients with adult acute lymphoblastic leukemia (ALL) at initial diagnosis. Expression of IL-15 was measured by real-time RT-PCR and normalized to the internal standard b-actin. mRNA levels were correlated with protein expression by western blot analysis. We found that expression of IL-15 correlated significantly with immunophenotype: T-lineage ALL (n=23) had a more than 4-fold higher IL-15 mRNA expression as compared to B-cell precursor (BCP)-ALL (n=64, P&amp;lt;0.001). IL- 15 expression was higher in common and pre-B as compared to pro-B-ALL, and CD1+ cortical T-ALL expressed IL-15 much higher than pre-T or mature T-ALL (median values: 0.152, 0.025, and 0.026, respectively). Patients with BCR-ABL+ BCP-ALL (n=18) had lower IL-15 expression compared to BCR-ABL− BCP-ALL (n=38, P=0.041). Furthermore, lower expression values of IL-15 were significantly associated with central nervous system involvement (P&amp;lt;0.001). In contrast, higher expression of IL-15 was significantly correlated with mediastinal (P=0.001) and lymph node infiltration (P=0.051), but not with hepatomegaly and splenomegaly. Notably, high IL-15 expression in BCP-ALL correlated strongly with inferior probability of relapse free survival (pRFS) at five years: pRFS 0.17 ± 0.13 (IL-15 expression &amp;gt; median, n=27) versus 0.47 ± 0.13 (&amp;lt; median, n=37) (P=0.008). However, only a weak correlation of the probability of overall survival (pOS) and IL-15 expression was observed (pOS: 0.41 ± 0.11 versus 0 ± 0, P=0.188). In conclusion, differential expression of IL-15 in adult ALL at diagnosis was associated with clinical features and outcome, in particular relapse free survival. It remains to be evaluated whether IL-15 might be a relevant therapy target, or might be used for risk stratification.

Prophylaxis and therapy of central nervous system involvement in adult acute lymphoblastic leukemia: recommendations of the Polish Adult Leukemia Group

The central nervous system (CNS) is one of the most frequent extramedullary locations of adult acute lymphoblastic leukemia (ALL), affecting approximately 5% of patients at diagnosis. T-lineage ALL, high initial leukocyte counts and mediastinal involvement are the predisposing factors. In case of relapse, if no prophylaxis was administered, the rate of CNS involvement reaches 30-50%. As the prognosis of patients with isolated or mixed CNS relapse is particularly poor, adequate prophylaxis seems critical. The treatment comprises intrathecal cytostatics, cranial and spinal cord irradiation, as well as systemic chemotherapy including agents penetrating to the CNS. This strategy allows a reduction in CNS relapses to less than 5% of cases. Compliance to the prophylactic protocols should be one of the principles in the treatment of adult ALL.

Central Nervous System Involvement in Adult Acute Lymphoblastic Leukemia (ALL) at Diagnosis and/or at First Relapse: Results from the GET-LALA Group.

Abstract Outcome of adult ALL with central nervous system (CNS) involvement is not clearly defined. We studied 104 patients presenting with CNS involvement at diagnosis among 1493 patients (7%) included into the LALA-87 or LALA-94 trials, and 109 patients (9% of first remitters) presenting CNS disease at the time of first relapse among the 709 relapsing patients (15%) included initially in these trials. Treatment of patients presenting CNS involvement at diagnosis consisted in initial chemotherapy completed by 18 double or triple intrathecal injections associated with 15 to 20 Gy cranial irradiation, followed when possible by intensification by allogeneic or autologous stem cell transplantation (SCT). At diagnosis, 43 patients (41%) presenting with CNS involvement had T-lineage ALL, 53 (51%) had B-lineage ALL (of whom 9 were diagnosed as Philadelphia (Ph) chromosome positive ALL), 8 had undifferentiated ALL or unknown immunophenotype. Eighty-seven of 104 (84%) patients with CNS involvement at diagnosis achieved complete remission (CR). Fifty-three patients underwent SCT (25 allogeneic SCT from matched related or unrelated donor, 28 autologous SCT). Overall survival at 7 years was 34% in those with CNS involvement at diagnosis versus 29% (p = NS) for those without. DFS at 7 years was 35% versus 28% (p = NS). There were no significant differences between patients with CNS involvement and those without CNS involvement regarding T lineage ALL, B lineage ALL (including or not Ph ALL). There were also no significant differences regarding patients who underwent transplantation as consolidation intensification, while in patients receiving only chemotherapy patients without initial CNS involvement had a better outcome (p = 0.01). Among the 709 patients with primary relapse, 66 patients (61%) presented a CNS relapse combined with bone marrow relapse, whereas 17 relapses (15%) and 26 relapses (24%) were CNS relapses combined with another extramedullary relapse or isolated CNS relapses respectively. Median time to relapse was 6.7 months (range, 1–62) in patients with CNS relapse versus 11.2 months (1.7–111) in relapsing patients without CNS involvement. Eleven patients (10%) with CNS relapse had CNS involvement at diagnosis, while 98 patients were diagnosed with CNS disease only at the time of first relapse. Overall, 38 out of 109 patients with CNS relapse (35%) achieved CR. The median OS was 6.3 months. Outcome was similar in terms of CR proportion and OS in relapsing patients without CNS involvement. The 2-year OS rates did not show any difference among patients with CNS relapse who had CNS involvement at diagnosis and those with CNS disease only diagnosed at the time of first relapse.Overall, CNS leukemia in adult ALL is uncommon at diagnosis. Patients have a similar outcome than those who did not present with CNS involvement. However, patients benefit from intensification therapy by autologous or allogeneic SCT. CNS leukemia at first relapse are also uncommon but probably underestimated. Outcome is particularly poor as this of all adult ALL in first relapse.

Treatment Strategies for Patients with Relapsed Acute Lymphoblastic Leukemia: Allogeneic Stem Cell Transplantation Is Effective without Prior Reinduction Chemotherapy.

Abstract Introduction: The outcome of salvage chemotherapy in refractory or relapsed adult acute lymphoblastic leukemia (ALL) is poor. In this situation allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative therapy, but it is not clear whether reinduction chemotherapy prior to HSCT is required to achieve long-term remissions. Methods: Here, we present a retrospective analysis of 65 patients (median age 29 years, 17–54) who received allogeneic HSCT for refractory or relapsed ALL from 1995 until 2006 (1st relapse n=50, 2nd or higher relapse n=7, primary refractory n=8, B-lineage ALL n=50, T-lineage ALL n=15). 22/65 patients were transplanted without reinduction chemotherapy prior to HSCT (median blasts in the bone marrow 30%, 7–90), whereas 43/65 patients received reinduction. 23 out of these 43 patients achieved CR2, but 21/43 patients were refractory (median blasts 70%, 10–95). Patients received standard high-dose (n=61) or reduced intensity conditioning (RIC) (n=4) and HLA-matched (n=58) or -mismatched (n=7) HSCT from related (n=26) or unrelated (n=39) donors. Results: Median follow-up of the surviving patients was 53 months (2–130). Overall survival (OS) of the whole cohort at 6 months, 1, 3 and 5 years was 61%, 40%, 27% and 24%, respectively. Deaths were due to relapse (n=29), GVHD (n=8), infections (n=7) or other causes (n=4). Chronic GVHD (cGVHD) occurred in 28/53 patients (53%) who survived more than 100 days after HSCT. Patients who developed cGVHD had significantly better outcome than patients without cGVHD (p&lt;0.0001) with OS at 6 months, 1 year, 3 years and 5 years being 88% vs. 58%, 72% vs. 25%, 55% vs. 10% and 47% vs. 10%, respectively. There was a trend towards improved OS for the cohort which did not receive reinduction chemotherapy compared to the cohort which received reinduction (p=0.06) with OS at 6 months, 1, 3 and 5 years being 64% vs. 60%, 55% vs. 31%, 45% vs. 17% and 36% vs. 14%, respectively. Importantly, in the reinduction cohort there were no differences in OS for the subgroups of patients transplanted in CR2 or transplanted with active disease. Deaths in the reinduction cohort were mainly due to relapse (64% vs. 50%), suggesting that reinduction therapy might have contributed to the selection of therapy-resistant leukemia clones. In further univariate analyses risk group at diagnosis, number of relapses, time-to-relapse, donor type and ALL lineage did not have significant impact on OS. Conclusions: Patients who undergo allogeneic HSCT for relapsed ALL can achieve long-term survival, which is projected at about 24% at 5 years. The development of cGVHD leads to increased OS, whereas risk group at diagnosis, number of relapses, time-to-relapse, donor type and ALL lineage do not have significant impact. Importantly, application of reinduction chemotherapy prior to HSCT does not improve outcome, even if a second complete remission is achieved. These results demonstrate the effectiveness of HSCT without prior disease control and again demonstrate the importance of graft-versus-leukemia-reactions in ALL.

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).

Abstract 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 &lt; 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.