PDGFRß promotes oncogenic progression via STAT3/STAT5 hyperactivation in anaplastic large cell lymphoma.

BACKGROUND: Anaplastic large cell lymphoma (ALCL) is an aggressive non-Hodgkin T cell lymphoma commonly driven by NPM-ALK. AP-1 transcription factors, cJUN and JUNb, act as downstream effectors of NPM-ALK and transcriptionally regulate PDGFRß. Blocking PDGFRß kinase activity with imatinib effectively reduces tumor burden and prolongs survival, although the downstream molecular mechanisms remain elusive. METHODS AND RESULTS: In a transgenic mouse model that mimics PDGFRß-driven human ALCL in vivo, we identify PDGFRß as a driver of aggressive tumor growth. Mechanistically, PDGFRß induces the pro-survival factor Bcl-xL and the growth-enhancing cytokine IL-10 via STAT5 activation. CRISPR/Cas9 deletion of both STAT5 gene products, STAT5A and STAT5B, results in the significant impairment of cell viability compared to deletion of STAT5A, STAT5B or STAT3 alone. Moreover, combined blockade of STAT3/5 activity with a selective SH2 domain inhibitor, AC-4-130, effectively obstructs tumor development in vivo. CONCLUSIONS: We therefore propose PDGFRß as a novel biomarker and introduce PDGFRß-STAT3/5 signaling as an important axis in aggressive ALCL. Furthermore, we suggest that inhibition of PDGFRß or STAT3/5 improve existing therapies for both previously untreated and relapsed/refractory ALK+ ALCL patients.

Down-regulation of the Notch pathway mediated by a gamma-secretase inhibitor induces anti-tumour effects in mouse models of T-cell leukaemia.

BACKGROUND AND PURPOSE: gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy. EXPERIMENTAL APPROACH: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours. KEY RESULTS: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis. CONCLUSIONS AND IMPLICATIONS: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

Identification of additional cytogenetic and molecular genetic abnormalities in acute myeloid leukaemia with t(8;21)/AML1-ETO.

AML1-ETO collaborates with further genetic abnormalities to induce acute myeloid leukaemia (AML). We analysed 99 patients with an AML1-ETO rearrangement for additional aberrations. Frequent genetic abnormalities were, loss of a sex chromosome (56/99, 56.5%) and del(9)(q22) (24/99, 24.2%). The most frequent molecular aberrations were mutations of KITD816 (3/23, 13%) and NRAS (8/89, 8.9%). Further molecular abnormalities were FLT3 mutations (3/87, 3.4%), AML1 (1/26, 3.8%) and PU1 (1/14, 7.1%). MLL-PTD, KRAS and CEBPA mutations were not found. These clinical findings support the model that AML1-ETO collaborates with other genetic alterations, such as mutations of receptor tyrosine kinases, to induce AML.

DNA index of glial tumors in children. Correlation with tumor grade and prognosis.

BACKGROUND: Although DNA index (DI) has prognostic significance in a variety of pediatric malignancies, there are few data regarding its utility in central nervous system (CNS) tumors. We have previously shown that patients with hyperdiploid medulloblastoma have a significantly better survival than those whose tumors are diploid. Here, we examine the effect of DI and tumor grade on the progression free survival (PFS) of 57 patients with a variety of glial neoplasms. METHODS: DI was determined by flow cytometry on freshly obtained tumor tissue from the initial diagnostic specimens; a DI = 1.0 was defined as diploid (DIP), 1.0 < DI < 1.1 as near diploid (NDIP), and DI > 1.1 as hyperdiploid (HYP). Tumors were histologically graded according to the World Health Organization classification. RESULTS: There were 21 Grade I tumors, 20 Grade II, 8 Grade III, and 8 Grade IV. Among the 41 low grade tumors (Grade I-II), 39 were DIP or NDIP, and 2 were HYP. Among the 16 high grade tumors (Grade III-IV), 9 were DIP, 2 NDIP, and 5 HYP. The 4-year PFS of low grade tumors was 70% (standard deviation [SD] 12%) versus 8% (SD 7%) for high grade tumors. There was a significant correlation between low grade tumor histology and a DIP/NDIP DI (P = 0.015), and univariate analysis suggested improved PFS was associated with DIP/NDIP tumors (P = 0.05). However, DI did not remain a significant prognostic factor after being stratified by tumor grade (P = 0.87). CONCLUSIONS: Unlike medulloblastoma, DI is not an independent prognostic factor in pediatric glial tumors.

The retinoblastoma protein binds to a family of E2F transcription factors.

E2F is a transcription factor that helps regulate the expression of a number of genes that are important in cell proliferation. Recently, several laboratories have isolated a cDNA clone that encodes an E2F-like protein, known as E2F-1. Subsequent characterization of this protein showed that it had the properties of E2F, but it was difficult to account for all of the suggested E2F activities through the function of this one protein. Using low-stringency hybridization, we have isolated cDNA clones that encode two additional E2F-like proteins, called E2F-2 and E2F-3. The chromosomal locations of the genes for E2F-2 and E2F-3 were mapped to 1p36 and 6q22, respectfully, confirming their independence from E2F-1. However, the E2F-2 and E2F-3 proteins are closely related to E2F-1. Both E2F-2 and E2F-3 bound to wild-type but not mutant E2F recognition sites, and they bound specifically to the retinoblastoma protein in vivo. Finally, E2F-2 and E2F-3 were able to activate transcription of E2F-responsive genes in a manner that was dependent upon the presence of at least one functional E2F binding site. These observations suggest that the E2F activities described previously result from the combined action of a family of proteins.

Management of cytogenetic data in multi-center leukemia trials.

This paper provides methods for storing and retrieving cytogenetics data, which must reside jointly in a reference laboratory and in a data co-ordinating center. The major piece of data is the character string that uses the International System for Human Cytogenetic Nomenclature. Simple and complex data retrieval, using Statistical Analysis System (SAS) functions INDEX and SUBSTR, are illustrated. Use of the methods of this paper can save considerable costs in setting up cytogenetic data systems compared with other proposed systems which have come to the authors' attention.

Screening for neuroblastoma in North America. 2-year results from the Quebec Project.

The Quebec Neuroblastoma Screening Project was initiated to assess the clinical and biological aspects of screening infants for the presence of neuroblastoma in North America. All children born in the province of Quebec from May 1, 1989 to April 30, 1994 are eligible for participation. This report provides results from 22 months' accrual of infants who were screened using urine-saturated filter paper for determination of the catecholamine metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA). More than 157,000 infants have been screened to date at 3 weeks of age, representing 92% of the entire birth population of Quebec. Over 98,000 infants have been screened a second time at 6 months of age, which made up 76% of the Quebec birth cohort. After a two-stage initial screening, 340 (0.13%) infants (182 at 3 weeks and 158 at 6 months) required second laboratory examinations because of elevated levels of urinary VMA, HVA, or both. Twenty infants from the 3-week screening (0.01%) and nine from the 6-month screening (0.01%) were subsequently referred to one of four Quebec pediatric oncology centers for neuroblastoma evaluation. Seven of 20 children from the 3-week screening and two of nine children from the 6-month screening have been identified as having neuroblastoma. During the same period, 14 additional children in the birth cohort were diagnosed clinically with neuroblastoma; eight were diagnosed prior to screening at 3 weeks of age, three children had negative results at 3 weeks of age, two had negative results at 3 weeks and at 6 months of age, and one had never been screened.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1/ETO, with similarity to Drosophila segmentation gene, runt.

We have developed a restriction map of the chromosome 21 breakpoint region involved in t(8;21)(q22;q22.3) acute myelogenous leukemia (AML) and have isolated a genomic junction clone containing chromosome 8 and 21 material. Using probes from these regions, rearrangements have been identified in each of nine cases of t(8;21) AML examined. In addition, we have isolated cDNA clones from a t(8;21) AML cDNA library that contain fused sequences from chromosome 8 and 21. The chromosome 8 component, referred to as ETO (for eight twenty-one), is encoded over a large genomic region, as suggested by the analysis of corresponding yeast artificial chromosomes (YACs). The DNA sequence of the chromosome 21 portion of the fusion transcript is derived from the normal AML1 gene. A striking similarity (67% identity over 387 bp, with a corresponding 69% amino acid identity) was detected between AML1 and the Drosophila segmentation gene, runt. The critical consequence of the translocation is the juxtaposition of 5' sequences of AML1 to 3' sequences of ETO, oriented telomere to centromere on the der(8) chromosome.

Ploidy of lymphoblasts is the strongest predictor of treatment outcome in B-progenitor cell acute lymphoblastic leukemia of childhood: a Pediatric Oncology Group study.

PURPOSE: Using the technique of recursive partitioning and amalgamation analysis with verification, the Pediatric Oncology Group (POG) investigated the independent prognostic significance of previously published prognostic factors significantly associated with event-free survival (EFS) in B-progenitor cell acute lymphoblastic leukemia (ALL). PATIENTS AND METHODS: Age, leukocyte count, sex, immunophenotype (expression of cytoplasmic immunoglobulin [Ig] and of surface antigens CD10 and CD34), and DNA index (ratio of the flow cytometry-determined DNA content of leukemia cells to that of normal diploid cells) were the variables used in the evaluation of four antimetabolite-based chemotherapy regimens in 1,535 children with the newly diagnosed B-progenitor cell ALL between February 1986 and May 1990. RESULTS: There were three subgroups at widely different risks of treatment failure. A DNA index greater than 1.16 was the most prognostic feature. The final prognostic subgrouping was as follows: (1) DNA index greater than 1.16; (2) DNA index less than or equal to 1.16, age less than 11.0 years, and leukocyte count less than 50 x 10(9)/L; and (3) DNA index less than or equal to 1.16, (age greater than 11.0 years, and/or leukocyte count greater than 50 x 10(9)/L). These groups made up 20%, 53%, and 27% of the patients and had 4-year EFS rates (SE) of 90.1% (6.3%), 80.5% (5.1%), and 50.4% (7.6%), respectively. CONCLUSIONS: Use of the DNA index, leukocyte count, and age--data that are relatively inexpensive and simple to obtain--may be sufficient to stratify patients with B-progenitor cell ALL for risk-directed therapy. Patients at an extremely low risk of failing therapy (approximately 20% of cases in this study) can thus be identified and spared the toxic short-term and late effects of more intensive therapies that may be needed for children with less favorable clinical and biologic features.

Current results of studies of immunophenotype-, age- and leukocyte-based therapy for children with acute lymphoblastic leukemia. The Pediatric Oncology Group.

From February 1986 to January 1991 the Pediatric Oncology Group (POG) treated 2404 children or adolescents with acute lymphoblastic leukemia (ALL) on immunophenotype (T-, B-, Pre-B, or Early pre-B-cell), age, and leukocyte count based treatment protocols (ALinC 14, T-cell 3, B-cell and infant leukemia studies). The immunophenotypic subgroups comprised 78.9% B-precursor cell, 15.1% T-cell, 2.0% B-cell, and 4% infant ALL. Patients with B-progenitor cell ALL were stratified by age and leukocyte count and randomized to receive induction therapy comprised of vincristine, prednisone, and asparaginase with triple intrathecal chemotherapy (methotrexate, hydrocortisone, cytarabine), followed by intensification with moderate-dose MTX (Regimen A), moderate-dose MTX plus asparaginase (Regimen B), moderate-dose MTX plus cytarabine given early (Regimen C), or moderate-dose MTX plus cytarabine given over the first 16 months of therapy (Regimen D). Continuation therapy comprised mercaptopurine and methotrexate with vincristine plus prednisone pulses. Central nervous system preventive treatment was continued for two years. Patients with T-cell or B-cell ALL or infants less than 1 yr old were treated on individual very intensive multiagent therapy protocols. The 4-year event-free survival for all patients was 66.4% +/- 2.4%; B-precursor ALL approximately 72%, T-ALL approximately 50%, B-ALL approximately 60%, and infants less than 1 yr old approximately 16.5%. We conclude that about two-thirds of newly diagnosed children with ALL can be cured with this approach which spares the majority of children exposure to alkylating agents, anthracyclines, epipodophylotoxins, and irradiation, diminishing the risks of serious acute and late effects.

Increased adenosine deaminase (ADA) activity and a shift from ADA-dependent to ADA-independent phases during T-cell activation: a paradox.

During activation of WF rat splenic T-cells, a change occurs with respect to susceptibility to a toxic accumulation of adenosine or deoxyadenosine (dADO) in the presence of adenosine deaminase (ADA) blockade. Addition of nucleoside 1 hour after the initiation of a concanavalin A response in the presence of 2'deoxycoformycin (DCF) markedly inhibited the response, whereas delay of addition of the nucleoside for 24-48 hours resulted in minimal or no inhibition. Inhibition was not simply the result of prolonged incubation of cells in the presence of nucleoside and was apparently not attributable to an effect on proliferating cells. Addition of interleukin 2 (IL-2) to cultures containing DCF and dADO did not reverse the inhibitory effect, which suggests that IL-2-producing T-cells also were not the target of nucleoside toxicity. A twofold increase in ADA activity that occurred during T-cell activation was nonessential for the survival of mitogen-activated T-cells in the presence of toxic concentrations of dADO and did not account for an apparent increased resistance of these cells to nucleoside toxicity. These paradoxical observations prompted an analysis of ADA activity in various populations of activated T-cells enriched with cells in G0/G1, S, or G2+M cell-cycle phases, which indicated that increased ADA activity was not associated with a specific period during cell-cycle traverse, but, rather, coincided with cell enlargement in preparation for mitosis. In conclusion, either an early event in T-cell mitogenesis is highly susceptible to nucleoside toxicity or a mechanism independent of ADA is acquired during T-cell activation that allows proliferating T-cells to resist toxic concentrations of nucleoside.