PAX5 P80R-mutated B-cell acute lymphoblastic leukemia with transformation to histiocytic sarcoma: clonal evolution assessment using NGS-based immunoglobulin clonality and mutation analysis.

Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.

Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences.

The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

Identification and characterization of collaborating oncogenes in compound mutant mice.

We have used proviral tagging in tumor-prone transgenic mice to identify collaborating oncogenes and genes contributing to tumor progression. This has yielded a series of oncogenes that could be assigned to different complementation groups in transformation: the myc, Pim, Bmi1, and Frat1 complementation groups. Frat1 is involved in tumor progression and appears to function in the Wnt signaling pathway. Overexpression of Fratl confers a growth advantage to transplanted tumor cells in vivo and to cells grown in vitro at high density. Frat1 might exert its activity by impairing the kinase activity of Gsk3beta, which is involved in the degradation of beta-catenin. Pim genes appear to act in tumor initiation and show strong synergism with myc in lymphomagenesis. Overexpression of Pim1 can also overcome some of the proliferative defects caused by defective interleukin signaling supporting a role of Pim1 in cell proliferation. We have applied proviral tagging in compound mutant Emu-myc/Pim1-/-/Pim2-/- mice to identify genes that can complement for the loss of Pim1 and Pim2 and, therefore, are able to synergize with c-myc in lymphomagenesis. A number of new as well as known genes have been found by this "complementation tagging." The latter included c-kit, Tp12, and cyclin D2, suggesting that Pim kinases might act upstream of or parallel to these known proto-oncogenes.

B-lineage transcription factors and cooperating gene lesions required for leukemia development.

Differentiation of hematopoietic stem cells into B lymphocytes requires the concerted action of specific transcription factors, such as RUNX1, IKZF1, E2A, EBF1 and PAX5. As key determinants of normal B-cell development, B-lineage transcription factors are frequently deregulated in hematological malignancies, such as B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and affected by either chromosomal translocations, gene deletions or point mutations. However, genetic aberrations in this developmental pathway are generally insufficient to induce BCP-ALL, and often complemented by genetic defects in cytokine receptors and tyrosine kinases (IL-7Rα, CRLF2, JAK2 and c-ABL1), transcriptional cofactors (TBL1XR1, CBP and BTG1), as well as the regulatory pathways that mediate cell-cycle control (pRB and INK4A/B). Here we provide a detailed overview of the genetic pathways that interact with these B-lineage specification factors, and describe how mutations affecting these master regulators together with cooperating lesions drive leukemia development.

IKZF1 deletions predict relapse in uniformly treated pediatric precursor B-ALL.

Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL) and is often difficult to predict. To explore the prognostic impact of recurrent DNA copy number abnormalities on relapse, we performed high-resolution genomic profiling of 34 paired diagnosis and relapse ALL samples. Recurrent lesions detected at diagnosis, including PAX5, CDKN2A and EBF1, were frequently absent at relapse, indicating that they represent secondary events that may be absent in the relapse-prone therapy-resistant progenitor cell. In contrast, deletions and nonsense mutations in IKZF1 (IKAROS) were highly enriched and consistently preserved at the time of relapse. A targeted copy number screen in an unselected cohort of 131 precursor B-ALL cases, enrolled in the dexamethasone-based Dutch Childhood Oncology Group treatment protocol ALL9, revealed that IKZF1 deletions are significantly associated with poor relapse-free and overall survival rates. Separate analysis of ALL9-treatment subgroups revealed that non-high-risk (NHR) patients with IKZF1 deletions exhibited a approximately 12-fold higher relative relapse rate than those without IKZF1 deletions. Consequently, IKZF1 deletion status allowed the prospective identification of 53% of the relapse-prone NHR-classified patients within this subgroup and, therefore, serves as one of the strongest predictors of relapse at the time of diagnosis with high potential for future risk stratification.

Characterization of pal-1, a common proviral insertion site in murine leukemia virus-induced lymphomas of c-myc and Pim-1 transgenic mice.

Insertional mutagenesis with Moloney murine leukemia virus (MoMLV) in c-myc and Pim-1 transgenic mice permits the identification of oncogenes that collaborate with the transgenes in lymphomagenesis. The recently identified common insertion site pal-1, in MoMLV-induced lymphomas, is located in a region in which several independent integration clusters are found: eis-1, gfi-1, and evi-5. Proviral insertions of MoMLV in the different integration clusters upregulate the transcriptional activity of the Gfi-1 gene, which is located within the pal-1 locus. The eis-1/pal-1/gfi-1/evi-5 locus serves as a target for MoMLV proviral insertions in pre-B-cell lymphomas of Emu-myc transgenic mice (20%) and in T-cell lymphomas of H-2K-myc (75%) and Emu-pim-1 (93%) transgenic mice. Many tumors overexpress both Gfi-1 as well as Myc and Pim gene family members, indicating that Gfi-1 collaborates with Myc and Pim in lymphomagenesis. Proviral integrations in the previously identified insertion site bmi-1 are, however, mutually exclusive with integrations in the eis-1/pal-1/gfi-1/evi-5 locus. This finding suggests that Bmi-1 and Gfi-1 belong to the same complementation group in lymphoid transformation.

Identification of cooperating oncogenes in E mu-myc transgenic mice by provirus tagging.

Mo-MLV infection of E mu-myc transgenic mice results in a dramatic acceleration of pre-B cell lymphomagenesis. We have used provirus tagging to identify genes that cooperate with the E mu-myc transgene in B cell transformation. Here we report on the identification of four loci, pim-1, bmi-1, pal-1, and bla-1, which are occupied by proviruses in 35%, 35%, 28%, and 14% of the tumors, respectively. bmi-1, pal-1, and bla-1 represent novel common proviral insertion sites. The bmi-1 gene encodes a 324 amino acid protein with a predominantly nuclear localization. bmi-1 is highly conserved in evolution and contains several motifs frequently found in transcriptional regulators, including a new putative zinc finger motif. No genes have yet been assigned to pal-1 and bla-1. The distribution of proviruses over the four common insertion sites suggests that provirus tagging can be used not only to identify the cooperating oncogenes but also to assign these genes to distinct complementation groups in tumorigenesis.

Bmi-1 collaborates with c-Myc in tumorigenesis by inhibiting c-Myc-induced apoptosis via INK4a/ARF.

The bmi-1 and myc oncogenes collaborate strongly in murine lymphomagenesis, but the basis for this collaboration was not understood. We recently identified the ink4a-ARF tumor suppressor locus as a critical downstream target of the Polycomb-group transcriptional repressor Bmi-1. Others have shown that part of Myc's ability to induce apoptosis depends on induction of p19arf. Here we demonstrate that down-regulation of ink4a-ARF by Bmi-1 underlies its ability to cooperate with Myc in tumorigenesis. Heterozygosity for bmi-1 inhibits lymphomagenesis in Emu-myc mice by enhancing c-Myc-induced apoptosis. We observe increased apoptosis in bmi-1(-/-) lymphoid organs, which can be rescued by deletion of ink4a-ARF or overexpression of bcl2. Furthermore, Bmi-1 collaborates with Myc in enhancing proliferation and transformation of primary embryo fibroblasts (MEFs) in an ink4a-ARF dependent manner, by prohibiting Myc-mediated induction of p19arf and apoptosis. We observe strong collaboration between the Emu-myc transgene and heterozygosity for ink4a-ARF, which is accompanied by loss of the wild-type ink4a-ARF allele and formation of highly aggressive B-cell lymphomas. Together, these results reinforce the critical role of Bmi-1 as a dose-dependent regulator of ink4a-ARF, which on its turn acts to prevent tumorigenesis on activation of oncogenes such as c-myc.