ALBERT

Description: TLX1 is a homeodomain transcription factor generally associated with a favorable outcome in T-cell acute lymphoblastic leukemia (T-ALL). However, the molecular mechanisms of TLX1 deregulation remain unclear and various transcript levels in the absence of 10q24 abnormalities have been reported. A reproducible and accurate delineation of TLX1+ T-ALL will be necessary for proper therapeutic stratification. We have studied 264 unselected T-ALLs (171 adults and 93 children) and show that T-ALLs expressing high levels of TLX1 (n = 35, 13%), defined as a real-time quantitative polymerase chain reaction (RQ-PCR) level of TLX1 greater than 1.00 ABL, form a homogeneous oncogenic group, based on their uniform stage of maturation arrest and oncogenetic and transcriptional profiles. Furthermore, TLX1-high T-ALLs harbor molecular TLX1 locus abnormalities in the majority (31/33), a proportion largely underestimated by standard karyotypic screening. T-ALLs expressing TLX1 at lower levels (n = 57, 22%) do not share these characteristics. Prognostic analysis within the adult LALA94 and GRAALL03 prospective protocols demonstrate a better event-free survival (P = .035) and a marked trend for longer overall survival (P = .059) for TLX1-high T-ALLs, while the expression of lower levels of TLX1 does not impact on prognosis. We propose that TLX1+ T-ALLs be defined as cases expressing TLX1/ABL ratios greater than 1 and/or demonstrating TLX1 rearrangement. Therapeutic modification should be considered for those patients.

Description: Adoptive immunotherapy based on the injection of allogenic cytotoxic T-lymphocytes (CTL) during or after bone marrow transplant (BMT) has established itself as a potent anti-neoplastic treatment for several malignancies. However this approach is limited by the occurence of graft versus host disease (GVH). Using a previously described murine adoptive immunotherapy model where donor and recipient are mismatched for a single dominant minor histocompatibility antigen (H7a), and where a powerful anti-neoplastic effect is seen without GVH, we sought to determine what rendered cancer cells more vulnerable than normal cells to immune attack. B10.H7a mice were lethally irradiated and reconstituted with B10.H7b (H7a negative) T-depleted bone marrow. On the day of transplant, these mice received a B16.F10 (H7a positive) melanoma challenge. Adoptive transfer of splenocytes obtained from B10.H7b mice previously immunized with B10.H7a splenocytes was performed on day 7 post BMT. Following transfer of those splenocytes containing primed anti-H7a CTL, neither GVH nor vitiligo was noted in recipients despite the fact that H7a is expressed in all tissues and organs. 50% of treated mice, versus 0% of controls rejected the tumor and survived 100 days. Overall survival was increased to 80% when adoptive transfer was carried on day 3 post BMT. The injection of anti-H7b CTL had no effect on melanoma growth. Thus, the anti-tumor activity was T-cell receptor recognition dependent and not a mere bystander effect. In treated mice but not in controls, tumor histology and flow cytometry revealed important CTL infiltration (80% of those CTL being MHC-H7a tetramer positive), increased expression of MHC class I molecules (MHC I) at melanoma cell surface, expression of Rae-1 (an NKG2D ligand), tumor necrosis and decreased angiogenesis. Importantly, normal skin in treated or control animals showed no increased expression of MHC I or Rae-1. B16.F10 melanoma cells express almost no MHC I, and no Rae-1 when cultured in vitro. Co-incubation of B16.F10 cells with INFγ leads to increased MHC I expression but no induction of Rae-1 expression, implying that at least a second factor is present in vivo to account for the expression of this stress ligand. An additional role for INFγ was evidenced when anti-H7a CTL were injected in INFγ receptor knock-out recipients. The angiostatic effect noted after anti-H7a CTL injection was abrogated and no mice were cured. Thus, INFγ-mediated angiostasis on the tumor stroma was crucial for the inhibition of cancer progression. Conclusion: in our model, the differential immune susceptibility of tumor versus normal cells appears to stem from the fact that neoplastic cells are induced to express more MHC I and stress ligands such as Rae-1. Those can respectively increase target antigen density at the cell surface and mediate CTL co-stimulation or cytotoxicity, through NKG2D receptors. Strategies exploiting stress ligand induction as well as the effect of INFγ on MHC expression and angiostasis may contribute to successfully separate anti-tumor from GVH effects.

Description: The TLX1 gene (10q24) codes for a homeodomain transcription factor not expressed in normal T lymphopoiesis. It’s expression as evaluated by RQ-PCR has been reported in 20–50% of T-ALLs while standard cytogenetic identifies TLX1 translocations to T cell receptor (TCR) genes in only 4–14% of T-ALLs. Accordingly, the stringency of the association between TLX1-TCR translocations and TLX1 overexpression has been questioned. To our knowledge, no systematic search for 10q24 translocations by FISH or molecular means has been conducted. Published T-ALL series demonstrate that a proportion of samples express TLX1 at very high levels while the remaining express TLX1 at either low or undetectable levels. In the absence of a uniform threshold, the biological significance of TLX1 levels of expression requires more exploration as it might account for the discrepancies regarding the prognosis impact of TLX1 expression and allow better T-ALL stratification. We have studied 224 unselected T-ALLs (147 adults and 77 children). Levels of TLX1 expression, assessed by RQ-PCR, were correlated to immunophenotypic, oncogenetic and molecular cytogenetic analysis. Thirty-two T-ALLs (14%, 27 adults and 5 children) expressed TLX1 at high levels (TLX1-high group) with a median level (normalised with ABL) of 746%. Fourty-nine samples (22%, 36 adults and 13 children) expressed TLX1 at lower levels (TLX1-low group) with a median level of 0,1% (range: 0,002%–11%). Caryotypes were available for 144 samples and demonstrated a 10q24 abnormality in only 11 cases, all within the TLX1-high group. Ongoing molecular analysis (FISH and LM-PCR) have revealed 17 additional 10q24 translocations/abnormalities among the TLX1-high samples with available material. Among those 17 samples, 12 had a representative caryotype without 10q24 abnormalities. In contrast, the 10q24 locus was normal in all 6 TLX1-low T-ALLs tested by FISH. Oncogenetic data show that HOX11-high T-ALLs form a homogeneous group, without coexisting TLX3, CALM-AF10 or TAL1 deregulation while TLX1-low and TLX1-negative T-ALLs often express either TLX3, CALM-AF10 or TAL1. Immunophenotypic data show that TLX1-high T-ALLs correspond to a β-selection stage of maturation arrest while TLX1-low T-ALLs show heterogeneous stages of maturation arrest: 20 immature(IM), 17 IMβ/pre-αβ, 5 TCRαβ and 6 TCRγδ. The respective prognosis of T-ALLs in correlation with their TLX1 expression level is currently being analysed. We conclude that virtually all T-ALLs expressing TLX1 at high level bear a 10q24 translocation and form a distinct homogeneous oncogenic group. Caryotype analysis largely underestimates 10q24 translocations. A uniform definition of TLX1 expression positivity has impact on T-ALL stratification in clinical protocols. Based on cytogenetic, immunophenotypic and oncogenetic arguments, we propose a definition of TLX1(+) T-ALLs restricted to T-ALLs expressing high levels (〉100% ABL) of TLX1 and/or having a genetic abnormality implicating TLX1. T-ALLs expressing low levels of TLX1 do not share these characteristics and their clinical relevance should be assessed separately.

Description: Despite many recent successes in the treatment of cancer, the development of chemoresistance in many of the initially responding patients, and primary resistance in others, remains a major impediment in therapy development. Our studies provide evidence for a novel mechanism underlying drug resistance: Gli1 dependent drug glucuronidation. While carrying out a Phase II clinical trial of targeting the eukaryotic translation initiation factor eIF4E with ribavirin in M4/M5 subtypes of AML, we observed that all responding patients eventually became clinically and molecularly resistant. To understand the cause of this resistance, we generated ribavirin resistant cell lines. In these models, ribavirin no longer targeted eIF4E activity or impaired growth, and importantly, the ability of ribavirin to bind eIF4E was severely impaired. However, the eIF4E gene was not mutated and its protein levels were not altered. The cell lines could be divided into two groups: type I with a defect in drug uptake and type II with a normal uptake. In type I resistant cells, we observed a substantial reduction in levels of Adenosine Kinase (ADK) an enzyme that catalyzes the rate limiting step in the metabolic activation of ribavirin allowing its retention in the cells. We used RNA Sequencing to examine the molecular underpinnings of type II resistance. Our data revealed a drastic increase in the levels of Gli1. In stably overexpressing cells, Gli1 was sufficient to produce the same resistance phenotype that we observed for type II cell models, both molecularly and at the level of cell growth. In addition, Gli1 overexpression correlated with the loss of drug-to-target interaction, as observed by our eIF4E immunoprecipitation studies using 3H-Ribavirin, similarly to the resistant cell lines. Conversely, Gli1 knockdown in type II cells or its pharmacological inhibition with the FDA approved Gli1 inhibitor GDC0449/Vismodegib, restored the eIF4E-ribavirin interaction and re-sensitized these cells to ribavirin. Our subsequent studies revealed a close correlation between Gli1 expression and the protein levels of the UGT1A glucuronosyl transferase enzymes involved in phase II drug metabolism whereby xenobiotics or metabolites are modified by the addition of a sugar, glucuronic acid. Given these findings, we examined whether the loss of the eIF4E interaction in resistant cells was due to the glucuronidation of ribavirin. Using 13C/12C ribavirin and mass spectrometry, we observed glucuronidated forms of ribavirin in resistant cells and cells overexpressing Gli1 but not in parental cells and that ribavirin is glucuronidated on its triazole ring which binds eIF4E. Treatment of cells with the Gli1 inhibitor GDC0449 reduces UGT1A levels, and correlates with reduced levels of ribavirin-glucuronides and the re-emergence of ribavirin-eIF4E complexes. We further hypothesized that the type II resistant cells could be resistant to other drugs. We observe that our ribavirin resistant cell lines are also resistant to the cornerstone of AML therapy, cytarabine. GDC0449/Vismodegib treatment reverts resistance to cytarabine in these cells. Preliminary studies indicate that these cells are also resistant to azacytidine and cisplatin. This is particularly striking as these cells were never exposed to these compounds. Thus, this could represent a novel form of multi-drug resistance. To establish the clinical relevance of our findings to patients in our AML ribavirin trial, we examined features of type I and type II resistance. Out of 10 patient samples available for evaluation, all six responding patient specimens showed elevated Gli-1 mRNA levels, up to 26 fold, upon relapse relative to levels during response. For most, the ratio of Gli1 during response relative to at relapse was about 2-4 fold with some patients up to 10 fold. For the two patients examined that did not respond, both had highly elevated Gli-1 levels prior to treatment relative to healthy individuals, and this was not lowered after 28 days of ribavirin treatment. We also noted elevated UGT1A protein levels upon relapse in our patient population. Type I resistance was observed in only two patients whereas Gli1 and UGT1A were dysregulated at relapse in all patients examined. In summary, we identified a novel form of drug resistance: Gli1 dependent drug glucuronidation. Treatment with Gli1 inhibitors appears to be a promising avenue for overcoming this form of drug resistance. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4305 Background Treatment options are limited for refractory acute leukemia (AL) patients. Few data are available on the potential utility vs futility of a third course of induction chemotherapy. We have reviewed the characteristics and outcome of 35 patients treated with a third intensive induction course for refractory AL at our institution and other centers affiliated to the Quebec Leukemia Cell Bank (BCLQ). Methods and patients Adult AL patients who never achieved complete remission (CR) or who relapsed within 6 months after 2 consecutive induction courses and who received a third line intensive induction course between 2000 and 2010 were included in this study. Clinical, laboratory and treatment data were collected from hospital and/or BCLQ files. Outcomes were measured after the third induction course. Primary outcomes were achievement of CR/CRi, overall survival (OS) at 12 months and event-free survival (EFS), defined as the percentage of patients alive and leukemia free at 12 months. Secondary outcomes included median OS, median duration of remission for those obtaining CR and treatment related mortality (TRM) associated with the third induction course. OS was estimated using the Kaplan-Meier method and survival curves were compared using the Log-rank test. Alive patients were censored at their last follow-up. Results Thirty five patients met the inclusion criteria. The median age was 43 years old (20–62). Thirty one patients (89%) were diagnosed with AML and 4 patients (11%) with ALL or acute leukemia of ambiguous lineage. The median white blood cell count at diagnosis was 23 × 109/L (0.7–223). For AML patients, cytogenetics was favourable in 1 patient (3%), intermediate in 17 (55%) and adverse in 13 (42%). None of the 6 patients with normal karyotype for whom molecular characterisation was performed had a favourable genotype defined as NPM1 mutated without FLT3-ITD mutation. The patient with favourable karyotype had a variant t(8;21)(q22;q22) translocation without KIT mutation. Eighteen patients (51%) were primarily refractory to prior therapy and 17 (49%) had relapsed within 6 months of CR. The third line induction regimen consisted of cyclophosphamide and etoposide in 11 patients (31%), high dose cytarabine-based regimen in 10 (29%), mitoxantrone and etoposide in 7 (20%), fludarabine, cytarabine and idarubicin in 5 (14%) and other regimen in 2(6%). Patients with sustained remission after their third induction course were followed for a minimum of 12 months except for one patient alive and leukemia free at 6 months. Patients who were not in sustained remission have all been followed until death or transition to palliative care. CR after the third induction course was achieved in 6 patients (17%). At 12 months, OS was 11% and EFS was 6%. After the third induction, median OS was 5 months and median duration of remission was 6 months. Among 20 patients treated in our institution, the third induction TRM was 15% and median duration of hospitalisation, excluding patients who died in aplasia, was 38 days (20–70). Median OS was higher in the intermediate than in the adverse cytogenetics group (8 vs 2 mo, p=0.0008, figure 1) and in patients who achieved CR after third induction vs no CR (12 vs 4 mo, p=0.03). There was no difference in median OS between primary refractory and relapsed patients (8 vs 4 months, p=0.52). Allogeneic stem cell transplantation (SCT) was performed in 10 patients (29%) of whom 4 (40%) were in CR before transplantation. The only two long term survivors were in the allogeneic SCT group. Their percentages of blasts at transplant were 3 and 69 %. Conclusion Only two refractory patients are long term survivors and they share several features: non adverse cytogenetics, young age (20 and 23) and they underwent allogeneic SCT. Adverse cytogenetics impinges negatively on outcome, even in this highly refractory group of subjects. The prognosis of AL patients with persistent disease after 2 induction courses is extremely poor and represents a major unmet therapeutic need. Refractory patients with adverse cytogenetics were unsalvageable in this series. The best approach for patients not in CR after 2 inductions probably remains allogeneic SCT, when possible. We consider that in patients who cannot be offered an allogeneic SCT, administration of a third course of conventional chemotherapy is practically useless. These patients should be considered candidates for investigational treatments. Disclosures: Bergeron: Merck: Research Funding; Roche: Honoraria; Celgene: Research Funding.