Differences Between CEBPA bZIP and TAD Mutations and Their Effect on Outcome-an Analysis in 4578 Patients with Acute Myeloid Leukemia

Mutations of the key myeloid transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) are found in 5-10% of patients with acute myeloid leukemia (AML). Two mutational clusters exist, in the aminoterminal transcription activation domains (TAD1 or 2) and in the basic leucine zipper domain (bZIP) located at the carboxyterminal-part of the protein. Biallelic mutations (biCEBPA) have been found to be associated with improved outcome and are now included as an independent entity in the WHO-classification. In contrast, monoallelic CEBPA-mutations (moCEBPA) do not appear to provide prognostic information. We characterized a large cohort of AML patients for CEBPA mutations and further analyzed the mutational spectrum of mono- and biallelic CEBPA-mutant AML patients to better understand potential differences in the biology of these groups. Patients and Methods: Patients (including all age groups) analyzed had a newly diagnosed AML and were registered in clinical protocols of the Study Alliance Leukemia (SAL)(AML96, AML2003 or AML60+, SORAML) or the SAL-register. Screening for CEBPA mutations was done using PCR and capillary electrophoresis. All identified CEBPA mutations were confirmed using conventional Sanger sequencing and the samples were further analyzed using next generation sequencing (Trusight Myeloid Panel, Illumina) for the presence of associated alterations. Results: In the 4578 patients analyzed, 228 (5%) with CEBPA-mutations were identified. An initial analysis revealed substantial clinical differences between the different mutation subtypes. Patients with biCEBPA (n=111) were significantly younger (median age 46 yrs) than wt-CEBPA patients (median 57 yrs; p<.001). Interestingly, single bZIP mutant patients (n=64) had a similar median age (50 yrs.) as biCEBPA, whereas single TAD mutant patients (n=53) were significantly older (median 63 yrs.). In addition, WBC counts, CD34 positivity as well as the history of prior MDS differed between the subgroups (single TAD mutant had significantly lower WBC counts, lower rate of CD34 positivity and had a higher rate of prior MDS than biCEBPA and single bZIP mutant patients). Along with this, the distribution of co-mutations differed significantly between the subgroups, especially GATA2 mutations were more common in biCEBPA and single bZIP mutant patients (37% and 34%, respectively) compared to only 3% (single TAD)(p=.001). A similar pattern was seen for mutations in DNMT3A (8% biCEBPA, 20% single bZIP vs. 36% single TAD; p=.001), and NPM1 (3% biCEBPA, 8% single bZIP, 32% single TAD; p<.001). In 2897 patients, the different CEBPA mutations were correlated with outcome. This analysis indicated a differential effect of the individual mutations on outcome, with an improved rate of complete remission (CR), overall survival (OS) and event free survival (EFS) for biCEBPA and single bZIP mutations in univariate and multivariate analyses (shown for OS in Figure 1a). Given the similarity of single bZIP and biCEBPA mutations, it appears reasonable to speculate on a common mechanistical background, since most of the biCEBPA mutants include a bZIP alteration. Recent experimental evidence generated by several groups indeed supports a specific role of these bZIP missense mutations. To address this in the clinical context, we regrouped patients with mutant CEBPA into patients with (n=157) or without bZIP mutations (n=71), irrespective of the biallelic status. As illustrated in Figure 1b, the bZIP mutant group had a significantly better OS, similar results were obtained for EFS and CR. In multivariate analysis, the presence of a bZIP mutation was the strongest indicator for achievement of CR (HR 7.5, 95% CI: 3-19; p<.001), and together with favorable cytogenetics the factor associated with best OS (HR: .48; 95% CI .36-.64; p<.001). In conclusion, our results obtained in one of the largest cohorts of AML patients analyzed for CEBPA mutations indicate that especially the presence of a missense bZIP mutation is associated with a favorable outcome in AML patients. These data point to substantial differences in prognostic implications of individual CEBPA mutations and support the major functional divergence of these alterations. If confirmed, these results might necessitate further refinement of their use in AML-classification. Disclosures Middeke: Sanofi: Honoraria. Platzbecker:Janssen-Cilag: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; TEVA Pharmaceutical Industries: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Thiede:AgenDix: Employment, Other: Ownership.

Short-Spindled Cell Haemangioblastoma with CD34 Expression: New Histopathological Variant or Just a Stochastic Cytological Singularity?

Haemangioblastomas are neoplasms of uncertain histogenesis with cellular and reticular variants advocated in current lore. Herein we describe an intriguing cerebellar specimen with unusual traits including spindle cell morphology and CD34 positivity. A thirty-nine-year old man had an infratentorial tumour discovered incidentally and resected three times. In all the instances, histopathological diagnosis was haemangioblastoma; nonetheless, he had neither physical stigmata nor family history of von Hippel-Lindau disease. By histology, the lesion was composed of areas of conventional stromal cells admixed with territories populated by short-spindled cells packed in lobules, sometimes giving the appearance ofgomitoli. Immunoperoxidase-coupled reactions confirmed the expression of inhibin A, neuron-specific enolase (NSE), PS100, and CD57 but also revealed focal immunolabeling for CD34, CD99, and FXIIIa. This case highlights the potential phenotypical diversity that can be found within these neoplasms. Rather than uncertain histogenesis, it may in fact reflect multiple lines of differentiation—histomimesis—prone to adopt unusual morpho- and immunophenotypes in a subset of haemangioblastomas.

CD34 Expression in the Stromal Cells of Alveolar Adenoma

The alveolar adenoma of the lung is a rare benign tumor characterized by a proliferation of both the alveolar epithelial cells and the mesenchymal septal cells. Immunohistochemically, the epithelial cells stain for cytokeratin (CK) AE1AE3, CK7, thyroid transcription factor 1 (TTF1), and surfactant apoprotein confirming the derivation by the type 2 pneumocytes. The stromal cells are negative for these markers but they show focally smooth muscle and muscle-specific actin positivity. We describe two cases that showed immunohistochemically a CD34 positivity of the mesenchymal septal cells. This aspect has been previously described in a two cases report, but not emphasized by the authors as a distinctive feature of the lesion. We consider this CD34 positivity as a marker of immaturity or stemness of the lesional septal spindle cells, that could be responsible of the different phenotypic and morphological profile of the interstitial cells, that could be, therefore, considered neoplastic and not reactive.

Insight Into T-ALL Leukaemia Initiating Cells, Clonal Evolution and Impact of Treatment In Vivo Using Immune-Compromised Mouse Model,

Abstract 3577 T-ALL constitutes 15% of ALL in children, requires more aggressive treatment, and exhibits a 5 year cure rate of around 75%. Relapsed T-ALL has a dismal salvage rate. Understanding the leukaemic stem cells responsible for clonal evolution and the emergence of treatment resistance may facilitate the treatment. We used immuno-compromised NOD/Shi-scid/IL-2Rγnull (NOG) mice to study in vivo engraftment kinetics and clonal evolution of a SIL-TAL+ primary T-ALL. Initially, we compared the pre-injection immunophenotype with that after primary transplantation. While 18% of the pre-injection cells were CD34+, 14% of human cells (hCD45+) at termination of the first passage were CD34+. Although the leukaemia-specific TCRγ gene rearrangement was stable throughout, cells following transplantation exhibited greater resistance to vincristine in vitro indicating the emergence of drug resistant cells. We next engrafted cells from the primary recipient into a second cohort of mice and evaluated the impact of vincristine in vivo on leukaemic subpopulations in different organs. Four weeks post-injection, vincristine treatment (1mg/kg once weekly by IV injection) was initiated. Control animals from this time exhibited increasing spleen weight compared with vincristine-treated mice whose spleen sizes decreased throughout treatment. One week following initiation of vincristine treatment, cells from the control animals predominantly comprised of more mature CD34-CD7+CD5+CD3- or CD34-CD7-CD5+CD3- cells in the BM, liver and spleen as well as small populations of more immature CD34+CD7-CD5-CD3-, CD34+CD7+CD5-CD3-, CD34+CD7+CD5+CD3- cells. An hCD34+CD34-CD7-CD5-CD3- subpopulation was also evident in all organs. In contrast, vincristine-treated mice revealed a reduction in the proportions of CD34-CD7+CD5+CD3- and CD34-CD7-CD5+CD3- cells in all organs, with a concomitant increase in CD34+CD7-CD5-CD3-, CD34+CD7+CD5-CD3- and CD34+CD7+CD5+CD3- as well as a dramatic increase in the proportion of hCD45+CD34-CD7-CD5-CD3- cells. After 4 weeks following treatment-initiation, cells from the control mice again significantly comprised CD34-CD7+CD5+CD3- and, to a greater proportion than week 1, CD34-CD7-CD5+CD3- cells. The proportion of hCD45+CD34-CD7-CD5-CD3- cells was also slightly elevated in all organs compared with week 1. The TCRγ gene rearrangement was easily detectable. In contrast, residual cells from the organs of vincristine-treated animals almost entirely comprised hCD45+CD34-CD7-CD5-CD3- and no TCRγ gene rearrangement could be detected, suggesting these residual cells were both drug resistant and very immature. Finally, we harvested cells from animals 6 weeks after initiation and 2 weeks after cessation of treatment which coincided with leukaemia presentation in control animals. At this time-point, cells from both the control and treated mice significantly comprised CD34-CD7+CD5+CD3-, CD34-CD7-CD5+CD3- (which was reduced in proportion compared with week 4), as well as an emergent CD34-CD7+CD5-CD3- population. The proportion of hCD45+CD34-CD7-CD5-CD3- cells was lower in all organs compared with week 4. Vincristine treated cells were, therefore, able to rapidly repopulate the organs following cessation of treatment. At the end of the second passage (untreated), immunophenotyping of human cells revealed a further dramatic reduction in CD34 positivity to <1% compared with 18% and 14% at pre-injection and end of first engraftment, respectively. There was also significant emergence of hCD45+CD34-CD7-CD5-CD3- cells. We are currently investigating the sensitivity of these cells to vincristine in vitro. Intriguingly, the concomitant loss of CD34 positivity, reduction in vincristine sensitivity and increase in hCD45+CD34-CD7-CD5-CD3- cells indicates the outgrowth of a very immature population of drug resistant leukaemia stem cells during serial transplantations in mice. We are currently characterising these hCD45+CD34-CD7-CD5-CD3- cells by immunophenotyping with additional stem cell markers, gene expression analysis, SIL-TAL+ PCR, and cell sorting to investigate their ability to engraft and reconstitute leukaemia in mice. We are addressing the possibility that they can give rise to relapse over a prolonged period following treatment cessation. These findings have important implications for the development of novel therapies for T-ALL. Disclosures: No relevant conflicts of interest to declare.

N-RasG12D induces features of stepwise transformation in preleukemic human umbilical cord blood cultures expressing the AML1-ETO fusion gene

AML1-ETO (AE) is a fusion product of t(8;21) observed in 40% French-American-British M2 type of acute myeloid leukemia (AML). Clinical data suggest that Ras mutation is a frequent cooperating event in t(8;21) AML. Whether constitutively active Ras promotes leukemogenesis on the t(8;21) background has not been demonstrated experimentally. Here, we retrovirally expressed N-RasG12D in AE-expressing human hematopoieticcells to investigate cooperativity. The AE/N-RasG12D cultures were cytokine-independent, enriched for CD34 positivity, and possessed increased colony-forming and replating abilities. N-RasG12D expression led to Bcl-2 up-regulation and reduced apoptosis. Ectopic Bcl-2 expression also resulted in enhanced colony-forming and replating abilities but was insufficient to sustain cytokine independence. AE/N-RasG12D cells were more sensitive to Bcl-2 inhibition with ABT-737 than parent AE cells. Enhanced engraftment of AE/N-RasG12D cells was observed on intrafemoral injection into immunodeficient mice, presumably because of improved survival in the bone marrow microenvironment. N-RasG12D promotes progression toward transfor-mation in AE-expressing cells, partially through up-regulating Bcl-2.