ALBERT

Description: The NOTCH1 gene encodes a transmembrane receptor important for the T-lineage commitment. Activating NOTCH1 mutations had been found in approximately 56% of pediatric T-lymphoblastic leukemia (T-ALL) samples and were associated with a favourable outcome. Here the frequency and the association of NOTCH1 mutations to clinical features, as well as molecular markers were analyzed for the first time in a large cohort of adult T-ALL patients. Genomic DNA of pretreatment samples of 126 adult T-ALL patients enrolled on the GMALL protocols 05/93 and 06/99 were analyzed by direct sequencing for the presence of NOTCH1 mutations in the heterodimerization domain (HD-C, HD-N), the polypeptide enriched in proline, glutamate, serine and threonine (PEST; divided into PEST-1 and PEST-2) domain, and the transactivation domain (TAD). In addition, leukemic blasts were molecularly characterized for mRNA expression of HOX11, HOX11L2, ERG, and BAALC by real-time RT-PCR. Immunophenotyping was performed differentiating T-ALL into three subtypes (early, thymic, mature). NOTCH1 mutations were identified in 72 (57%) of the 126 patients including mutations in the HD (n=56), in the TAD (n=5), and in the PEST (n=20) domain. Eleven patients had more than one mutation, including 3 patients with HD and TAD, and 6 patients with coexisting HD and PEST mutations. T-ALL patients carrying mutations revealed similar clinical characteristics as compared to NOTCH1 wild type cases with respect to age, sex, CNS involvement, white blood count. Moreover, no differences were seen in the response to induction therapy or survival between NOTCH1 wild type and mutated cases (in the overall groups as well as in subgroups analyses). In the expression pattern of HOX11, HOX11L2, BAALC, or ERG there were no significant differences between the two NOTCH1 groups. Patients with mutated NOTCH1 showed a higher frequency of a thymic immunophenotype (65% vs. 39% for unmutated cases; P=0.013). Furthermore, a significant association was observed between the expression of specific surface antigens and the NOTCH1 genotype: NOTCH1 mutated cases were significantly associated with the expression of CD10 (P

Description: Background: Controversial data exist concerning the prognostic impact of the expression of the two orphan HOX genes TLX1 and TLX3 in pediatric and adult ALL. To further clarify this situation we have investigated adult T-ALL patients for the expression of the TLX1 and TLX3 oncogenes using Real Time quantitative PCR. Altogether 146 cases of adult T-lineage ALL, treated according to the German Multicenter ALL (GMALL) Therapy trial 5/93, were investigated, thus, representing the largest cohort of adult T-ALL patients investigated so far. Methods: Analysis was done with a Rotor Gene 3000 cycler. PCR primers and TaqMan probes were located in exon 1 and exon 2 of TLX1 and in exon 5 and 6 of TLX3. Cell lines HPB-ALL and ALL-SIL were used as positive controls and samples of healthy donors were used as negative controls. Patients: The mean age of the patients was 31 (16–63) ys. Thirty-seven patients (25.2%) had an early T-ALL, 33 (22.5%) had a thymic T-ALL and 77 (52.4%) had a mature T-ALL. Results: Fourty-six patients (43%) were TLX1-positive, 15 (10%) were TLX3-positive and 6 (4.1%) showed an expression of both oncogenes. The TLX1-expression was significantly associated with the immunophenotype: 19% (7/36) of early T, 18% (6/33) of mature T and 43% (33/77) of thymic T-ALL were TLX1-positive (p=0.0077). There was no significant association of the TLX3-positive cases with a certain immunophenotype: (8 (10%) thymic, 2 (6%) mature, 5 (14%) early) (p〉.05). There were no significant differences in CR rates between the TLX1- and TLX3-positive patients. The probabilities of survival in the 4 different subgroups were as follows: TLX1-positive/TLX3-negative 0.57 (N=40), TLX1-negative/TLX3-negative 0.37 (N=91), TLX1-negative/TLX3-positive 0.20 (N=9), TLX1-positive/TLX3-positive 0.00 (N=6) (p log rank = 0.01). The probabilities of continuous complete remission (CCR) in the 4 subgroups (129 eligible patients) were the following: TLX1-positive/TLX3-negative 0.71 (N=36), TLX1-negative/TLX3-negative 0.45 (N=80), TLX1-negative/TLX3-positive 0.20 (N=9), TLX1-positive/TLX3-positive 0.0 (N=4) (p log rank = 0.04). Within the three different immunophenotypic subgroups the expression of TLX1/TLX3 did not show a significant association with survival or CCR. Conclusions: The GMALL study group has shown previously that the immunologic subtypes of T-ALL are significantly associated with outcome, with poorer survival for early/mature T-ALL and better outcome for thymic T-ALL. The data presented here show that TLX1 expression is associated with a favourable immunophenotype and a better prognosis. TLX3 expression is not significantly associated with certain immunophenotypes and confers an inferior outcome both in remission duration and overall survival in adult ALL. The patients disclosing expression of both oncogenes showed the worst prognosis but comprised only a small number (6/147). These results suggest that TLX3-positive patients, particularly those with coexpression of TLX1, should be treated as a high risk group.

Description: Background: IKAROS (IKZF1) is a key transcription factor for B-cell development. The IKZF1 protein exerts its functions as a dimer (homodimer or heterodimer with other IKAROS family members). Various IKZF1 intragenic deletions have been identified as recurrent aberrations in acute lymphoblastic leukemia: those resulting in the loss of transcription of one allele or at least loss of the dimerization domain ("haploinsufficient", "non-functional") and those involving only the DNA-binding domain but retaining the dimerization domain of the transcription factor, leading to so-called "dominant negative" isoforms. Several studies in pediatric ALL have confirmed the negative prognostic impact of IKZF1 alterations although there is still no consensus if they are independent risk factors in a multivariate analysis. The situation in adult ALL is less clear and few larger studies have addressed this issue, in particular the prognostic impact of different types of IKZF1 alterations. Objectives: We investigated archived patient samples obtained in the context of the German Multicenter ALL Study Group (GMALL) therapy studies for different IKZF1 alterations to assess their prognostic implications and their molecular pattern. Methods: We used RT-PCR to determine the presence of aberrant IKZF1 transcript variants and additionally investigated all patients by DNA-based PCRs for the IKZF1 aberrations D4-7, D2-7, D4-8, D2-8. Rare transcript variants, such as D2-3, D2, D5-7 were molecularly characterized on the DNA level by specific PCRs. IKZF1 aberrations were quantified using gel densitometry and/or quantitative real time PCR such that clonal and subclonal aberrations could be distinguished. All chromosomal breakpoints were molecularly characterized. Results: In total, 482 B cell precursor ALL patients aged between 15 and 65 years were included in the analysis. Samples were obtained at primary diagnosis and tested BCR-ABL -negative. Other molecular aberrations were tested according to the GMALL standards: 39 MLL-AF4-positive, 4 MLL-ENL, 1 MLL-AF9, 30 TCF3-PBX1, 3 ETV6-RUNX1. The immunophenotypes were the following: 424 pre B/common, 58 pro B. One hundred and twenty-eight out of 482 (27%) patients carried at least one IKZF1 deletion, with 37 patients carrying more than one deletion. Altogether there were 175 IKZF1 deletions, 71 cases of D4-7, 47 with D2-7, 26 with D4-8, 19 with D2-3, 10 with D2-8, and 1 with D5-7 and D2 each. Taken together 56 patients (12%) carried only non-functional mutations while 50 (10%) had dominant-negative mutations only. There was a group of 22 patients with both types of mutations (5%). Evaluating the prognostic impact, we considered the effect of non-functional and dominant-negative mutations separately. Patients with a non-functional IKZF1 mutation had a reduced overall survival (OS at 5 ys 37% vs 59% in IKZF1-unmutated patients, N=78/404, p=0.001) while dominant-negative mutations had no effect on OS (OS at 5 ys 54% compared to 56%, N=72/410, p=0.95). However, solely present subclonal non-functional mutations did not affect survival (OS at 5 ys 59% compared to 59%, N=23/404). In the dominant-negative group, clonality had no influence on OS (p=0.56, N=45/73/351). Taken together, patients with clonal non-functional IKZF1 had a significantly worse OS compared to those without (0.28 vs 0.59, N=53/427, p95%) putative cryptic recombination sites were identified in the near vicinity thus underlining the causal role of the VDJ recombination machinery in the generation of these aberrations. Conclusions: We conducted a detailed and large-scale molecular analysis of intragenic IKZF1 deletions and identified clonal non-functional IKZF1 deletions as adverse prognostic factor in adult and adolescent B cell precursor ALL patients treated according to the GMALL protocols. Subclonal, or dominant negative IKZF1 mutations had no prognostic effect. It remains open for further analysis whether clonal non-functional IKZF1 deletions represent an independent prognostic factor in MRD-based risk stratification. Disclosures Wäsch: German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Description: The chromosomal translocation t(1;19) with fusion transcript E2A-PBX1 is the third most prevalent recurrent translocation in adult ALL (after t(9;22) and t(4;11)), accounting for about 3% of all cases. It’s prognostic impact in adults has been controversially discussed and it has been suggested that this aberration might define a high risk patient group. However, this would be in contrast to the situation in pediatric ALL, where E2A-PBX1/t(1;19) meanwhile defines a patient group with a better than average outcome. We have investigated retrospectively by RT-PCR 337 B-precursor BCR-ABL-/MLL-AF4-negative ALL patients enrolled in the GMALL 5/93 and 6/99 therapy trials for the presence of this mRNA fusion transcript. This represents the largest cohort of adult BCR-ABL-negative patients investigated for E2A-PBX1 by RT-PCR thus far. We assessed the clinical characteristics of the E2A-PBX1-positive patients and their overall survival. Results: Eighteen patients were E2A-PBX1-positive by RT-PCR (5.8% of the investigated group, roughly 3% of the total of all ALL patients). Fourteen had a pre B (cyIg+), and 4 a common immunophenotype. Twenty-two percent of all pre B ALL patients were E2A-PBX1-positive. The median age of the positive patients at diagnosis was 27 (15–60) years and the median leukocyte count was 36.700/μl (N=17) with 7 of 17 patients presenting with an initial leukocyte count 〉30.000/nl (59% vs 27% in the negative group, p=0.005). Compared to the E2A-PBX1-negative group (239 common, 49 pre B, 31 pro B, median age 32 years, median leukocyte count 11.900 (N=312) the E2A-PBX1-positive patients had a lower overall survival rate (0.33 vs. 0.36). The CR rate did not differ significantly between E2A-PBX1-positive and -negative patients. These results support the assumption that in contrast to the situation in pediatric ALL E2A-PBX1 defines a relatively unfavourable genetic subgroup in adult ALL. It is strongly correlated with high leukocyte count (〉30.000/μl) at diagnosis which is a highly significant poor prognostic factor in B-precursor ALL. It remains to be established whether E2A-PBX1 can be confirmed as an independent prognostic factor within standard risk B-precursor ALL. E2A-PBX1 may be useful to separate in so called standard risk ALL a patient group which may benefit from stem cell transplantation in first CR.

Description: Promotor demethylation of oncogenes has been associated with transcriptional activation in cancer cells. The proto-oncogene HOX11/TLX1 has been found to be aberrantly expressed in up to 30% of adult T-ALL patients. In few, a translocation between the HOX11 locus at 10q24 and the T-cell receptor locus has been identified. In the majority of cases the mechanism leading to HOX11 reactivation remains unclear. It had been proposed that an epigenetical modification by demethylation of the proximal HOX11 promotor could be responsible for an aberrant expression of HOX11. To test this hypothesis we have correlated the methylation status of CpG residues in the proximal HOX11 promotor with the gene expression status of HOX11 in adult T-ALL samples from the German Multicenter ALL Study (GMALL) 5/93 and 6/99. HOX11 expression was measured in 286 pretreatment peripheral blood and bone marrow blasts by comparative real-time RT-PCR as described previously. The methylation status was then randomly analyzed after bisulphite treatment by methylation-specific PCR (MSP) in 53 T-ALL samples with HOX11 expression (HOX11 positive) and 102 samples without HOX11 expression (HOX11 negative). Of the 150 analyzed patients only 4% of HOX11 positive patients (n=53) and 10% of HOX11 negative patients were methylated (M) in the analyzed promotor area. HOX11 negative patients were significantly more common associated with an unmethylated status (U) then HOX11 positive patients (57% vs 32%, p=0.003). The most prominent methylation phenotype in HOX11 positive patients compared to HOX11 negative samples was a mixed (MU) methylation status (55% vs. 27%, p=0.001). Interestingly, remission duration was significantly higher in pt. with the MU methylation status (M=49%, U=54% vs. MU=76%, p-logrank=0.0362). This translated also into a significant difference in the overall survival (M=55%, U=49%, MU=75%, p-logrank= 0.0202). However, in a multivariate analysis the methylation status could not be confirmed as an independent prognostic factor. The promotor-associated CpG methylation status was found to be remarkably heterogenous in the analyzed adult T-ALL patients with a predominantly unmethylated status in HOX11 negative samples and a mixed methylated/unmethylated picture in HOX11 positive samples. These findings contrast with our initial hypothesis that HOX11 expression is silenced in normal tissue by a promotor-associated CpG methylation and aberrantly reexpressed in leukemia cells by demethylation. However, various CpG residues associated with the HOX11 promotor might be of variable importance for the gene expression and intrinsic limitations of methylation-specific PCR might have influenced our analysis. Bisulphite sequencing techniques as well as the newly developed genome-wide cytosine methylation array could help overcome these issues. Understanding the role of promotor-associated methylation in HOX11 expression could clarify pathways in leukemogenesis and provide a valuable tool for better risk stratification in T-ALL.

Description: Introduction: Despite the recent identification of the Ph-like subgroup of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), a large number of BCP-ALL patients lack cytogenetic and molecular defined lesions. To get a higher resolution and a broader molecular view of relapsed BCP-ALL, we designed a multi-omics study to reveal age-overriding relapse-driving alterations that may unravel novel molecular targets. Methods: We studied 150 paired samples (initial diagnosis: ID; relapse: REL; complete remission: CR) from 50 patients without known translocations. The cohort consisted of 24 adult and 26 pediatric patients with minimal residual disease 〈 0.05 % at CR. All patients were treated in population based German study trials (GMALL, BFM). We examined the mutational and copy number status via exome sequencing, obtained expression profiles and fusion-genes via RNA-sequencing and the methylation status via Illumina Methylation Array. Results: With a lenient approach detecting drivers and passengers, we identified significantly more mutations in REL compared to ID samples (adult median: 52 vs 38; pediatric median: 39 vs 27). In addition, we detected 4 hypermutators (more than 100 mutations per sample), 2 were pediatric and 2 were adult samples, 3 of which were REL samples. The most recurrently mutated genes were KRAS (n=15), NRAS (n=15), TP53 (n=13), CDC27 (n=13), KMT2D (n=11), IKZF1 (n=11), CREBBP (n=10) and FLT3 (n=6; Figure 1), with mutations present in both age cohorts. NT5C2, SYK and CHD1 were exclusively mutated in the pediatric cohort with at least 3 mutations. NT5C2 was also specific for early REL. Of all REL mutations, 225 mutations (14%, mean: 4 mutations/patient) were sub-clonal (under 〈 5% mutation frequency) at ID. Copy number alterations (CNA) varied greatly among pediatric and adult samples: 6% of pediatric and 18% of adult samples had aneuploidies and or copy neutral loss of heterozygosity of whole chromosomes. Chromosomal aberrations at ID persisted at relapse (100 %). Particular targets of CNA affected well-described genes like CDKN2A, CDKN2B, PAX5 on chr9p. Genes preferentially subjected to homozygous deletions were VPREB1 (n=6), SH2B3 (n=4), and ETV6 (n=2). All SH3B2 deletions were found in pediatric samples. On the epi-genomic level, the principal component analysis of the most variable CG-sites revealed a stable methylation profile during the course of the disease. However, we found a clear separation into a smaller pediatric-dominated cluster (n=24; 20 pediatric, 4 adult) and a larger mixed-age cluster (n=76; Fig. 1, Cluster A). Differentially methylated regions, affecting a total of 269 genes, characterized the separation of the smaller cluster, henceforth called Methylation Deregulated (MDR) cluster. The samples of the MDR cluster showed also a distinct gene expression profile by RNA-seq supporting a tight connection between the methylation status and its transcriptional program. A subset of 97 genes was differentially expressed including MAPK and PDGFR genes as most prominently deregulated. Additionally we defined a MDR expression classifier comprising 30 genes (Fig. 1). On the mutational level, the MDR samples had 20 % fewer mutations (mean: 25.3) compared to the remaining samples (mean: 31.3) and fewer CNVs for the most frequently affected genes. Characterising the non-MDR samples, a third of those were categorized as Ph-like ALL using the 15 gene classifier in an unsupervised clustering; this signature also coincided with the presence of well-known fusion-genes (Fig. 1, Cluster B). The remaining samples were defined by chromosomal instability (CI; Fig. 1, Cluster C). In the CI cluster, mutations in epigenetic regulators were twice as frequent when compared to the remaining samples. Conclusions: We describe three distinct clusters in relapsed BCP-ALL, which are characterized by a different genetic alterations: a novel MDR cluster by distinct methylation changes, the Ph-like cluster by gene fusions and the CI cluster by chromosomal instability. The cluster assignment was stable over the course of the disease. All clusters occurred in pediatric and adult patients, with the methylation-driven cluster predominantly in pediatrics. The MDR cluster showed significantly fewer mutations and CNVs compared to the other two clusters. The MDR samples showed activation of the MAPK signaling pathway pointing to actionable therapeutic targets. Figure 1 Figure 1. Disclosures Gökbuget: Pfizer: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.

Description: Overexpression of BAALC is an adverse prognostic factor in adults with cytogenetically normal acute myeloid leukemia and T-cell acute lymphoblastic leukemia (ALL). Here, we analyzed the prognostic significance of BAALC in B-precursor ALL. BAALC MRNA expression was determined in 368 primary adult B-precursor ALL patients enrolled on the 06/99 and 07/03 GMALL trials. Patients were grouped into tertiles according to BAALC expression (T1-T3). Higher BAALC expression (T3 vs T2 vs T1) was associated with higher age (P 〈 .001), a higher white blood cell count (P = .008), CD34 (P = .001), BCR-ABL (P 〈 .001), and MLL-AF4 (P 〈 .001). Higher BAALC expression predicted primary therapy resistance in the overall cohort (P = .002) and in the BCR-ABL− and MLL-AF4− subgroup (P = .01). In BCR-ABL− and MLL-AF4− patients, higher BAALC expression was associated with a shorter overall survival (OS; 5-year OS: T3, 38%; T2, 52%; T1, 70%; P = .004) and independently predicted OS in multivariate models (P = .03). Gene-expression profiling revealed an up-regulation of stem cell markers and genes involved in chemoresistance (TSPAN7 and LYN) in the high BAALC group. Thus, high BAALC expression is associated with an immature, chemoresistant leukemic phenotype and identifies patients with inferior OS. Determination of BAALC might contribute to risk assessment of molecularly undefined adult B-precursor ALL.

Description: Persistence or recurrence of minimal residual disease (MRD) after chemotherapy results in clinical relapse in patients with acute lymphoblastic leukemia (ALL). In a phase 2 trial of B-lineage ALL patients with persistent or relapsed MRD, a T cell–engaging bispecific Ab construct induced an 80% MRD response rate. In the present study, we show that after a median follow-up of 33 months, the hematologic relapse-free survival of the entire evaluable study cohort of 20 patients was 61% (Kaplan-Meier estimate). The hema-tologic relapse-free survival rate of a subgroup of 9 patients who received allogeneic hematopoietic stem cell transplantation after blinatumomab treatment was 65% (Kaplan-Meier estimate). Of the subgroup of 6 Philadelphia chromosome–negative MRD responders with no further therapy after blinatumomab, 4 are in ongoing hematologic and molecular remission. We conclude that blinatumomab can induce long-lasting complete remission in B-lineage ALL patients with persistent or recurrent MRD. The original study and this follow-up study are registered at www.clinicaltrials.gov as NCT00198991 and NCT00198978, respectively.

Description: MLL translocations in adult B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) are largely restricted to the immature CD10− immunophenotypes. MLL-AF4 is known to be the most frequent fusion transcript, but the exact frequencies of MLL aberrations in CD10− adult BCP-ALL are unknown. We present a genetic characterization of 184 BCR-ABL− CD10− adult ALL cases (156 cyIg−, 28 cyIg+) diagnosed between 2001 and 2007 at the central diagnostic laboratory of the GMALL study group. Patient samples were investigated by RT-PCR for MLL-AF4, MLL-ENL, and MLL-AF9 and by long-distance inverse polymerase chain reaction, thus also allowing the identification of unknown MLL fusion partners at the genomic level. MLL-AF4 was detected in 101 (54.9%) and MLL-ENL in 11 (6.0%) cases. In addition, rare MLL fusion genes were found: 2 MLL-TET1 cases, not previously reported in ALL, 1 MLL-AF9, 1 MLL-PTD, a novel MLL-ACTN4, and an MLL-11q23 fusion. Chromosomal breakpoints were determined in all 118 positive cases, revealing 2 major breakpoint cluster regions in the MLL gene. Characteristic features of MLL+ patients were significantly lower CD10 expression, expression of the NG2 antigen, a higher white blood count at diagnosis, and female sex. Proposals are made for diagnostic assessment. The clinical studies are registered at http://www.clinicaltrials.gov as NCT00199056 and NCT00198991.

Description: Mesenchymal stromal cells (MSCs) are an essential cell type of the hematopoietic microenvironment. Concerns have been raised about the possibility that MSCs undergo malignant transformation. Several studies, including one from our own group, have shown the presence of cytogenetic abnormalities in MSCs from leukemia patients. The aim of the present study was to compare genetic aberrations in hematopoietic cells (HCs) and MSCs of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) patients. Cytogenetic aberrations were detected in HCs from 25 of 51 AML patients (49%) and 16 of 43 MDS patients (37%). Mutations of the FLT3 and NPM1 genes were detected in leukemic blasts in 12 (23%) and 8 (16%) AML patients, respectively. Chromosomal aberrations in MSCs were detected in 15 of 94 MDS/AML patients (16%). No chromosomal abnormalities were identified in MSCs of 36 healthy subjects. We demonstrate herein that MSCs have distinct genetic abnormalities compared with leukemic blasts. We also analyzed the main characteristics of patients with MSCs carrying chromosomal aberrations. In view of these data, the genetic alterations in MSCs may constitute a particular mechanism of leukemogenesis.