Li–Fraumeni syndrome (LFS) is a genetic predisposition to cancer development at young age.1, 2, 3 The underlying genetic defect is usually a germline mutation in the TP53 gene or mutations in genes encoding p53-regulatory enzymes.3 Common clinical characteristics of these patients include: (i) childhood malignancies; (ii) typical malignancies like soft tissue sarcomas, osteosarcomas, brain tumors and adrenocortical tumors; and (iii) several family members with typical malignancies or other malignancies diagnosed before the age of 60.
Somatic TP53 mutations are uncommon in acute myelogenous leukemia (AML)4 and AML is an uncommon malignancy in LFS.1 Here, we report a patient with LFS and adult AML. The patient had a germ-line TP53 mutation and the AML cells showed multiple cytogenetic abnormalities. She was treated with the histone deacetylase (HDAC) inhibitor valproic acid in combination with all-trans retinoic acid (ATRA) and theophyllamine. The patient was included in a clinical study after written informed consent and the study was approved by the local Ethics Committee.
The patient was a 51-year-old female with a history of breast cancer, three soft tissue sarcomas and anal carcinoma. Her first malignancy was a sarcoma diagnosed at the age of 28 years. Two of the sarcomas were treated with surgery alone, one sarcoma with surgery+chemotherapy and the breast cancer with surgery+irradiation+chemotherapy. Anal carcinoma was diagnosed at the age of 48 years and treated with surgery, multiple liver metastasis were later detected and she was treated with repeated cycles of oxilaplatin, 5-fluorouracil and folic acid. The patient's oldest son had a rhabdomyosarcoma and died from acute leukemia when he was 12 years old. Both the patient and her son had the same TP53 mutation in exon 8, codon 290 (base substitution CGC → CTC, amino-acid substitution Arg → Leu).
Leukemia was diagnosed at the age of 51 and was characterized by (i) AML-M0 with membrane molecule phenotype CD11c−CD13+CD14−CD15−CD33+CD34+CD45+ CD117+; (ii) no genetic Flt3 abnormalities, (iii) multiple cytogenetic abnormalities with karyotype 44–46, XX, del(2)(q2?), del(4)(q21?), del5(5)(q21?), i(17)(q10) +21[cp17]. The patient was treated with ATRA 22.5 mg/m2 twice daily days 1–14 and from day 3 also valproic acid (serum levels 179–365 μ M) and theophyllamine (serum levels 49-64 μ M). During treatment hemoglobin levels and platelet counts (range 178–233 × 109/l) were stable. Peripheral blood blast counts showed an initial decline from 30.4 to 23.1 × 109/l on day 7. This was accompanied by signs of chromatin condensation in the leukemia cells. However, from day 21 a marked increase in peripheral blood blast counts were observed and valproic acid/theophyllamine were stopped due to progressive disease.
We first investigated the expression of p53 protein isolated from enriched AML cells (i) before therapy; (ii) on day 3 following treatment with ATRA alone; and (iii) on days 7 and 11 when she received ATRA in combination with valproic acid and theophyllamine. Wild-type p53 is usually detected in two distinct isoforms with alternating expression in native human AML cells (Figure 1a). In contrast, only minor amounts of p53 could be detected in the leukemia cells of this patient before and during therapy (Figure 1b). Previous studies suggest that the Li–Fraumeni-associated p53 mutants induce a trans-dominant loss of function effect on the wild-type protein.2 Our present results demonstrate that this is true also in AML, and the low levels were not significantly altered during treatment. Thus, the present AML disease should be regarded as a functional p53 knockout, suggesting that biological effects of treatment are p53 independent.
We also investigated the expression of the p53-family proteins p63 and p73 before and during treatment (Figure 2). No p63 was observed before or during therapy, while a minor decrease in p73 was observed. The p21 protein was not expressed before or during treatment.
Furthermore, we investigated the membrane molecule phenotype for AML cells isolated before therapy, after 2 days of ATRA treatment (day 3) and after combination therapy with ATRA+theophyllamin+valproic acid (days 7 and 10). The leukemia cells showed a gradual decrease in CD71 and an increase in CD11b during treatment (Figure 1c). In contrast, no major alterations were observed for CD13, CD14, CD15, CD33, CD34, CD64 and CD117 (data not shown). Previous studies in the U-937 cell line suggest that ATRA may contribute to the effects on CD11b and CD71,5 but studies in native human AML cells do not support this.6 We conclude that it seems unlikely that these effects are caused by ATRA alone.
The phosphorylation status of intracellular mediators (Stat1, Stat3, Stat5, Stat6, Erk1/2, Akt, p38, CREB) can influence on AML disease progression.7 We compared phosphorylation status of these proteins from cells isolated before and after 10 days of treatment. The most striking effects were decreased mean fluorescence intensity (MFI) of Stat1 and Stat6, and increased MFI of CREB (Figure 1d). We then investigated the effect of combination therapy on mediator phosphorylation after exposure to exogenous cytokines. Dual AML cell populations were detected when we examined the phosphoresponse of Stat1 to IFNγ, the Stat3 response to G-CSF and the Stat5 response to IFNγ, and GM-CSF (Figure 1e). For all these responses we observed a gradual reduction/disappearance of the low-fluorescent subset during treatment. The responses to SFD-1 and Flt3L were not altered for Stat1/3/5. The phosphoresponses of Stat6, Erk1/2, Akt, CREB and p38 were not altered for any of the 5 cytokines during therapy. Thus, the combination therapy has p53-independent effects both on the basal phosphorylation level and phosphoresponsiveness of several intracellular mediators in native human AML cells. Previous studies in the U-937 cell line suggest that ATRA can increase Stat1 phosphorylation,8 but the more complex effects observed in our patient are unlikely to be caused by ATRA alone. Furthermore, the increased basal phosphorylation of CREB is probably caused by increased cAMP levels due to theophyllamine treatment.9
The AML cells showed no spontaneous in vitro proliferation. Detectable proliferation was observed in response to IL3, Flt3L and GM-CSF and this was not altered during treatment. Proliferation in response to lineage-associated growth factors (G-CSF, M-CSF, Tpo, Epo) was not observed for any sample.
The use of HDAC inhibitors in AML therapy is now considered, and the first clinical studies suggest that this therapeutic strategy can improve peripheral blood cell counts and even induce hematological remission.10 HDACs deacetylate not only histones but also many other nuclear and cytoplasmic proteins including p53, heat shock protein 90, and several transcription factors and structural proteins. Thus, other mechanisms than altered histone acetylation may contribute to the effects of HDAC inhibitors on malignant cells, and p53-mediated effects may then be important. In fact, it has recently been reported that HDAC inhibitors may contribute to selective cancer therapy by depletion of mutant p53 to restore wild type p53 function in tumors.11
Valproic acid has been used for HDAC inhibition in several studies. The biological effects of HDAC inhibitors have been suggested to affect p53 through at least two different mechanisms: (i) altered histone acetylation with altered TP53 gene expression; and (ii) altered acetylation of the p53 protein.10 In the present patient, we combined valproic acid with ATRA and theophyllamine. ATRA is used for differentiation induction in acute promyelocytic leukemia and can also induce biological alterations in other forms of AML. Theophyllamine seems to inhibit AML cell proliferation and increase sensitivity to ATRA through the increase of intracellular cAMP levels.9 On the basis of our present results, we conclude that combination of targeted therapy has p53 independent effects on signal transduction and membrane molecule expression of malignant cells, and these therapeutic approaches should be further investigated in AML as well as in other malignancies with mutational or functional p53 inactivation.
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The work was supported by the Norwegian Cancer Society, the Research Council of Norway and the Bergen Translational Research Grant Program.
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Ånensen, N., Skavland, J., Stapnes, C. et al. Acute myelogenous leukemia in a patient with Li–Fraumeni syndrome treated with valproic acid, theophyllamine and all-trans retinoic acid: a case report. Leukemia 20, 734–736 (2006). https://doi.org/10.1038/sj.leu.2404117
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