Clonal evolution in myelodysplastic syndromes.

Cancer development is a dynamic process during which the successive accumulation of mutations results in cells with increasingly malignant characteristics. Here, we show the clonal evolution pattern in myelodysplastic syndrome (MDS) patients receiving supportive care, with or without lenalidomide (follow-up 2.5-11 years). Whole-exome and targeted deep sequencing at multiple time points during the disease course reveals that both linear and branched evolutionary patterns occur with and without disease-modifying treatment. The application of disease-modifying therapy may create an evolutionary bottleneck after which more complex MDS, but also unrelated clones of haematopoietic cells, may emerge. In addition, subclones that acquired an additional mutation associated with treatment resistance (TP53) or disease progression (NRAS, KRAS) may be detected months before clinical changes become apparent. Monitoring the genetic landscape during the disease may help to guide treatment decisions.

Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial.

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

Expression of C-IAP1, C-IAP2 and SURVIVIN discriminates different types of lymphoid malignancies.

(De-)regulation of apoptosis plays an important role in normal and malignant lymphopoiesis. Apoptosis-regulating genes of the BCL-2 family and the recently identified inhibitors of apoptosis (IAP) family have been implicated in different types of non-Hodgkin lymphoma (NHL). To investigate whether expression of specific apoptosis-regulating genes correlated with different types of lymphoid malignancies, we measured the expression of five BCL-2 family genes, four IAP family genes and SMAC by real-time quantitative polymerase chain reaction in patient samples. In total, 137 samples from B- and T-cell acute lymphoblastic leukaemia (ALL), B-cell chronic lymphocytic leukaemia (CLL), six different NHL types and three control tissue types were analysed. The data were further analysed using cluster and discriminant analysis. Three specific expression patterns were identified for CLL, ALL and NHL respectively. CLL samples, as well as B-ALL and follicular lymphoma samples showed high similarity in the expression of these apoptosis-regulating genes and could be distinguished from each other and other diseases and controls. Discriminant analysis identified three members of the IAP family, C-IAP1, C-IAP2 and SURVIVIN, as the most informative genes to discriminate between these lymphoid malignancies.

Transcriptional repression of the Neurofibromatosis-1 tumor suppressor by the t(8;21) fusion protein.

Von Recklinghausen's disease is a relatively common familial genetic disorder characterized by inactivating mutations of the Neurofibromatosis-1 (NF1) gene that predisposes these patients to malignancies, including an increased risk for juvenile myelomonocytic leukemia. However, NF1 mutations are not common in acute myeloid leukemia (AML). Given that the RUNX1 transcription factor is the most common target for chromosomal translocations in acute leukemia, we asked if NF1 might be regulated by RUNX1. In reporter assays, RUNX1 activated the NF1 promoter and cooperated with C/EBPalpha and ETS2 to activate the NF1 promoter over 80-fold. Moreover, the t(8;21) fusion protein RUNX1-MTG8 (R/M), which represses RUNX1-regulated genes, actively repressed the NF1 promoter. R/M associated with the NF1 promoter in vivo and repressed endogenous NF1 gene expression. In addition, similar to loss of NF1, R/M expression enhanced the sensitivity of primary myeloid progenitor cells to granulocyte-macrophage colony-stimulating factor. Our results indicate that the NF1 tumor suppressor gene is a direct transcriptional target of RUNX1 and the t(8;21) fusion protein, suggesting that suppression of NF1 expression contributes to the molecular pathogenesis of AML.

Intratumoral administration of recombinant human interleukin 12 in head and neck squamous cell carcinoma patients elicits a T-helper 1 profile in the locoregional lymph nodes.

The objective of this Phase II study was to evaluate the pharmacodynamic and immune effects of intratumorally administered recombinant human interleukin-12 (IL-12) on regional lymph nodes, primary tumor, and peripheral blood. Ten previously untreated patients with head and neck squamous cell carcinoma were injected in the primary tumor two to three times, once/week, at two dose levels of 100 or 300 ng/kg, before surgery. We compared these patients with 20 control (non-IL-12-treated) patients. Toxicity was high, with unexpected dose-limiting toxicities at the 300 ng/kg dose level. Dose-dependent plasma IFN-gamma and IL-10 increments were detected. These cytokine levels were higher after the first injection than after the subsequent injections. A rapid, transient reduction in lymphocytes, monocytes, and all lymphocyte subsets, especially natural killer cells, was observed, due to a redistribution to the lymph nodes. In the enlarged lymph nodes of the IL-12-treated patients, a higher percentage of natural killer cells and a lower percentage of T-helper cells were found compared with control patients. The same pattern was detected in the infiltrate in the primary tumor. Real-time semiquantitative PCR analysis of peripheral blood mononuclear cells in the peripheral blood showed a transient decrease of T-bet mRNA. Interestingly, the peripheral blood mononuclear cells in the lymph nodes showed a 128-fold (mean) increase of IFN-gamma mRNA. A switch from the Th2 to a Th1 profile in the lymph nodes compared with the peripheral blood occurred in the IL-12-treated patients. In conclusion, in previously untreated head and neck squamous cell carcinoma patients, recombinant human IL-12 intratumorally showed dose-limiting toxicities at the dose level of 300 ng/kg and resulted in measurable immunological responses locoregionally at both dose levels.

Epigenetic regulation of tumor suppressors in t(8:21)-containing AML.

The t(8;21) is perhaps the most frequent chromosomal translocation associated with acute myeloid leukemia. The translocation creates a fusion protein that consists of the DNA binding domain of the RUNX1 transcription factor fused to the MTG8 transcriptional co-repressor to create a potent transcriptional repressor. Here, we discuss the possibility that the t(8;21) fusion protein represses tumor suppressors that regulate the RAS signaling pathway and the p53 oncogenic checkpoint.

Biodistribution and retention time of retrovirally labeled T lymphocytes in mice is strongly influenced by the culture period before infusion.

T lymphocytes used for adoptive immunotherapy are often cultured before transfer to generate sufficient amounts of effector cells with desired specificity. Modification of lymphocytes induced by in vitro activation and expansion may influence their potential effector capacity by altering the survival and trafficking patterns after transfer. In this report, the authors show that the culture period of T cells after ConA/IL-2 stimulation strongly influences the retention and tissue distribution of these cells after infusion into syngeneic C57BL/6 mice. Infused labeled cells that have been cultured for 3 days remained in the peripheral blood and organs in at least a ten-fold higher number than cells cultured for 8 days. In addition, cells cultured for 3 days preferentially migrate to lungs and liver shortly after infusion and subsequently to lymph nodes and spleen. Cells cultured for 8 days preferentially migrate to liver and can be hardly detected in lymph nodes. In contrast, labeled cells cultured for 3 days are predominantly present in lymph nodes starting from day 8 until day 28. We showed that accurate monitoring of transferred cells is feasible, which may contribute to understanding response to adoptive immunotherapy.

Abnormal WT1 expression in the CD34-negative compartment in myelodysplastic bone marrow.

In normal bone marrow, WT1 expression is restricted to CD34+ cells. We assessed WT1 mRNA expression levels with quantitative, real-time reverse transcription polymerase chain reaction in normal, myelodysplastic (MDS) and secondary acute myeloid leukaemia (sAML) bone marrow subfractions, based on differentiation status. The highest WT1 expression was observed in the primitive CD34+ rhodamine-123 (rho) dull cells, both in healthy donors and MDS or sAML patients. In contrast to normal CD34-negative bone marrow cells, WT1 was present in CD34-negative bone marrow cells in 12 out of 13 MDS patients and two sAML samples. Further analysis of this aberrant WT1 expression was performed in the CD34-negative subfractions of three MDS patients. In one of these, WT1 expression was found exclusively in the erythroid cells. This patient was completely transfusion dependent and showed morphological dyserythropoiesis. In another MDS patient, WT1 expression was found in a non-erythroid compartment. We conclude that abnormal WT1 expression may contribute to the disturbed differentiation of haematopoietic cells in MDS patients.

The t(8;21) fusion protein, AML1 ETO, specifically represses the transcription of the p14(ARF) tumor suppressor in acute myeloid leukemia.

The t(8;21) is one of the most frequent chromosomal translocations associated with acute leukemia. This translocation creates a fusion protein consisting of the acute myeloid leukemia-1 transcription factor and the eight-twenty-one corepressor (AML1 ETO), which represses transcription through AML1 (RUNX1) DNA binding sites and immortalizes hematopoietic progenitor cells. We have identified the p14(ARF) tumor suppressor, a mediator of the p53 oncogene checkpoint, as a direct transcriptional target of AML1 ETO. AML1 ETO repressed the p14(ARF) promoter and reduced endogenous levels of p14(ARF) expression in multiple cell types. In contrast, AML1 stimulated p14(ARF) expression and induced phenotypes consistent with cellular senescence. Chromatin immunoprecipitation assays demonstrated that AML1 ETO was specifically bound to the p14(ARF) promoter. In acute myeloid leukemia samples containing the t(8;21), levels of p14(ARF) mRNA were markedly lower when compared with other acute myeloid leukemias lacking this translocation. Repression of p14(ARF) may explain why p53 is not mutated in t(8;21)-containing leukemias and suggests that p14(ARF) is an important tumor suppressor in a large number of human leukemias.