Martin F. Fey

Human ΔNp73 regulates a dominant negative feedback loop for TAp73 and p53

Inactivation of the tumour suppressor p53 is the most common defect in cancer cells. p53 is a sequence specific transcription factor that is activated in response to various forms of genotoxic stress to induce cell cycle arrest and apoptosis. Induction of p53 is subjected to complex and strict control through several pathways, as it will often determine cellular fate. The p73 protein shares strong structural and functional similarities with p53 such as the potential to activate p53 responsive genes and the ability to induce apoptosis. In addition to alternative splicing at the carboxyl terminus which yields several p73 isoforms, a p73 variant lacking the N-terminal transactivation domain (ΔNp73) was described in mice. In this study, we report the cloning and characterisation of the human ΔNp73 isoforms, their regulation by p53 and their possible role in carcinogenesis. As in mice, human ΔNp73 lacks the transactivation domain and starts with an alternative exon (exon 3′). Its expression is driven by a second promoter located in a genomic region upstream of this exon, supporting the idea of two independently regulated proteins, derived from the same gene. As anticipated, ΔNp73 is capable of regulating TAp73 and p53 function since it is able to block their transactivation activity and their ability to induce apoptosis. Interestingly, expression of the ΔNp73 is strongly up-regulated by the TA isoforms and by p53, thus creating a feedback loop that tightly regulates the function of TAp73 and more importantly of p53. The regulation of ΔNp73 is exerted through a p53 responsive element located on the ΔN promoter. Expression of ΔNp73 not only regulates the function of p53 and TAp73 but also shuts off its own expression, once again finely regulating the whole system. Our data also suggest that increased expression of ΔNp73, functionally inactivating p53, could be involved in tumorogenesis. An extensive analysis of the expression pattern of ΔNp73 in primary tumours would clarify this issue.

Burdens and Benefits of Adjuvant Cyclophosphamide, Methotrexate, and Fluorouracil and Tamoxifen for Elderly Patients With Breast Cancer: The International Breast Cancer Study Group Trial VII

PURPOSE Information on the tolerability and efficacy of adjuvant chemoendocrine therapy for older women is limited. We studied these issues using the data collected as part of the International Breast Cancer Study Group Trial VII. PATIENTS AND METHODS Postmenopausal women with operable, node-positive breast cancer were randomized to receive either tamoxifen alone for 5 years (306 patients) or tamoxifen plus three consecutive cycles of classical cyclophosphamide (100 mg/m(2) orally days 1 to 14), methotrexate (40 mg/m(2) intravenous days 1 and 8), and fluorouracil (600 mg/m(2) intravenous days 1 and 8) every 28 days (CMF; 302 patients). The median follow-up was 8.0 years. RESULTS Among the 299 patients who received at least one dose of CMF, women 65 years of age or older (n = 76) had higher grades of toxicity compared with women less than 65 years old (n = 223) (P =.004). More women in the older age group compared with the younger women experienced grade 3 toxicity of any type (17% v 7%, respectively), grade 3 hematologic toxicity (9% v 5%, respectively), and grade 3 mucosal toxicity (4% v 1%, respectively). Older patients also received less than their expected CMF dose compared with younger postmenopausal women (P =.0008). The subjective burdens of treatment, however, were similar for younger and older patients based on quality-of-life measures (performance status, coping, physical well-being, mood, and appetite). For older patients, the 5-year disease-free survival (DFS) rates were 63% for CMF plus tamoxifen and 61% for tamoxifen alone (hazards ratio [HR], 1.00; 95% confidence interval [CI], 0.65 to 1.52; P =.99). For younger patients, the corresponding 5-year DFS rates were 61% and 53% (HR, 0.70; 95% CI, 0.53 to 0.91; P =.008), but the test for heterogeneity of CMF effect according to age group was not statistically significant. The reduced effectiveness of CMF among older women could not be attributed to dose reductions according to dose received. CONCLUSION CMF tolerability and effectiveness were both reduced for older patients compared with younger postmenopausal node-positive breast cancer patients who received tamoxifen for 5 years. The development and evaluation of less toxic and more effective chemotherapy regimens are required for high-risk elderly patients.

Risk Assessment in Patients with Acute Myeloid Leukemia and a Normal Karyotype

Purpose: The recognition of a number of leukemia-specific cytogenetic abnormalities and their role as independent prognostic factors have provided considerable insights into leukemia pathogenesis and have paved the way to adopt risk-adapted treatment. However, ∼50% of newly diagnosed acute myeloid leukemia (AML) have a normal karyotype. There has therefore been much interest in identifying molecular markers that could help to improve the prognostic stratification of patients with normal-karyotype AML. Experimental Design: Consecutive untreated AML patients (n = 67) from a single institution all with normal karyotype were analyzed for the presence of mutations in the myeloid transcription factor gene CEBPA (for CCAAT/enhancer binding protein-α), for internal tandem duplications (ITD) of the tyrosine kinase receptor gene FLT3 (for fms-like tyrosine kinase 3), and for expression of the BAALC gene (for brain and acute leukemia, cytoplasmic). Results: 17.9% of normal-karyotype AML had mutations in the CEBPA gene, and 28.4% had FLT3-ITD; 65.7% (44 of 67) had high BAALC expression and 34.3% (23 of 67) had low BAALC expression. Patients with CEBPA mutations had a very favorable course of their disease. Median disease-free survival (DFS) and overall survival (OS) were 33.5 and 45.5 months, respectively, compared with 10 (e.g., 12 months in patients without CEBPA mutations; P = 0.0017; P = 0.0007). AML patients with FLT3-ITD had significantly shorter median DFS (P = 0.0328) and OS (P = 0.0148) than patients without FLT3-ITD. High BAALC expression predicted for a shorter DFS (P = 0.0152) and OS (P = 0.0210) compared with AML with low BAALC expression; 53.7% of normal-karyotype AML had neither FLT3-ITD nor CEBPA mutations. We found that high BAALC expression in normal-karyotype AML with neither FLT3-ITD nor CEBPA mutations (18 of 67) indicates adverse prognosis for both DFS and OS (P = 0.0001; e.g., P = 0.0001) compared with the group with low BAALC expression and absent FLT3-ITD and CEBPA mutations (18 of 67). Thus, BAALC expression represents a novel prognostic marker particularly for normal-karyotype AML patients with neither FLT3-ITD nor CEBPA mutations. Conclusions: Assessment of CEBPA mutations, FLT3-ITD, and BAALC expression permits to split normal-karyotype AML into clinically distinct subgroups.

Prognostic impact of amenorrhoea after adjuvant chemotherapy in premenopausal breast cancer patients with axillary node involvement: results of the international Breast Cancer Study Group (IBCSG) trial VI

Adjuvant chemotherapy-induced amenorrhoea has been shown to be associated with reduced relapses and improved survival for premenopausal breast cancer patients. Amenorrhoea was, therefore, studied to define features of chemotherapy (i.e. duration and timing) and disease-related factors which are associated with its treatment effects. We reviewed data from IBCSG Trial VI, in which accrual was between July 1986 and April 1993. 1196 of the 1475 eligible patients (81%) were evaluable for this analysis. The median follow-up was 60 months. Women who experienced amenorrhoea had a significantly better disease-free survival (DFS) than those who did not (P = 0.0004), although the magnitude of the effect was reduced when adjusted for other prognostic factors (P = 0.09). The largest treatment effect associated with amenorrhoea was seen in patients assigned to receive only three initial CMF courses (5-yr DFS: 67% versus 49%, no amenorrhoea; hazard ratio, 0.55; 95% confidence interval, 0.38 to 0.81; P = 0.002). DFS differences between amenorrhoea categories were larger for patients with ER/PR positive tumours (hazard ratio, 0.65; 95% confidence interval, 0.53 to 0.80; P = 0.0001). Furthermore, patients whose menses returned after brief amenorrhoea had a DFS similar to those whose menses ceased and did not recover (hazard ratio, 1.10; 95% confidence interval, 0.75 to 1.62; P = 0.63). The effects associated with a permanent or temporary chemotherapy-induced amenorrhoea are especially significant for node-positive breast cancer patients who receive a suboptimal duration of CMF chemotherapy. Cessation of menses, even for a limited time period after diagnosis of breast cancer, might be beneficial and should be prospectively investigated, especially in patients with oestrogen receptor-positive primaries.

Effect of Single-Agent Rituximab Given at the Standard Schedule or As Prolonged Treatment in Patients With Mantle Cell Lymphoma: A Study of the Swiss Group for Clinical Cancer Research (SAKK)

PURPOSE To evaluate the effect of single-agent rituximab given at the standard or a prolonged schedule in patients with newly diagnosed, or refractory or relapsed mantle cell lymphoma (MCL). PATIENTS AND METHODS After induction treatment with the standard schedule (375 mg/m2 weekly x 4), patients who were responding or who had stable disease at week 12 from the start of treatment were randomly assigned to no further treatment (arm A) or prolonged rituximab administration (375 mg/m2) every 8 weeks for four times (arm B). RESULTS The trial enrolled 104 patients. After induction, clinical response was 27% with 2% complete responses. Among patients with detectable t(11;14)-positive cells in blood and bone marrow at baseline, four of 20, and one of 14, respectively, became polymerase chain-reaction-negative after induction. Anemia was the only adverse predictor of response in the multivariate analysis. After a median follow-up of 29 months, response rate and duration of response were not significantly different between the two schedules in 61 randomly assigned patients. Median event-free survival (EFS) was 6 months in arm A versus 12 months in arm B; the difference was not significant (P = .1). Prolonged treatment seemed to improve EFS in the subgroup of pretreated patients (5 months in arm A v 11 months in arm B; P = .04). Thirteen percent of patients in arm A and 9% in arm B presented with grade 3 to 4 hematologic toxicity. CONCLUSION Single-agent rituximab is active in MCL, but the addition of four single doses at 8-week intervals does not seem to significantly improve response rate, duration of response, or EFS after treatment with the standard schedule.

Induction of Genes Mediating Interferon-dependent Extracellular Trap Formation during Neutrophil Differentiation

Interferons (IFNs) are cytokines that possess potent anti-viral and immunoregulatory activities. In contrast, their potential role(s) in anti-bacterial defense and neutrophil activation mechanisms is less well explored. By comparing gene expression patterns between immature and mature human neutrophils, we obtained evidence that intracellular proteases and other anti-bacterial proteins are produced at earlier stages of maturation, whereas the genes for receptors and signaling molecules required for the release of these effector molecules are preferentially induced during terminal differentiation. For instance, mature neutrophils strongly expressed genes that increase their responses to type I and type II IFNs. Interestingly, granulocyte/macrophage colony-stimulating factor was identified as a repressor of IFN signaling components and consequently of IFN-responsive genes. Both IFN-α and IFN-γ induced strong tyrosine phosphorylation of STAT1 in mature but not in immature neutrophils. Functional in vitro studies suggested that IFNs act as priming factors on mature neutrophils, allowing the formation of extracellular traps upon subsequent stimulation with complement factor 5a (C5a). In contrast, both IFN-α and IFN-γ had only little capacity to prime immature cells in this system. Moreover, both IFNs did not have significant anti-proliferative effects on immature neutrophils. These data contribute to our understanding regarding changes of gene expression during neutrophil differentiation and IFN-mediated anti-bacterial defense mechanisms.

Long-term follow-up confirms the benefit of all-trans retinoic acid in acute promyelocytic leukemia

First results of a randomized trial (APL91 trial) and other randomized or non-randomized studies have shown that ATRA followed by chemotherapy significantly increased event-free survival (EFS) and survival, and decreased the incidence of relapse by comparison to chemotherapy alone in newly diagnosed APL. We present here long-term follow-up of the APL91 trial. In this trial, 101 patients had been randomized between ATRA followed by three courses of daunorubicin-AraC chemotherapy (ATRA group) and the same chemotherapy alone (chemotherapy group). Results were reanalyzed 73 months after closing of patient entry. Updated results of APL 91 trial found a Kaplan–Meier estimate of EFS and relapse rate at 4 years of 63% and 31% in the ATRA group, as compared to 17% and 78% in the chemotherapy group (P = 10−4 and relative risk 2.95, P = 10−4 and relative risk 3.68, respectively). Kaplan–Meier survival at 4 years was 76% in the ATRA group and 49% in the chemotherapy group (P = 0.026, relative risk 2.7). In the chemotherapy group, seven of the 27 relapses occurred after 18 months, but no relapse was seen after 43 months. In the ATRA group, four of the 17 relapses occurred after 18 months, including two late relapses (at 58 and 74 months). In the chemotherapy group, 23 of the 25 patients who relapsed achieved a second CR with ATRA, and the Kaplan–Meier estimate of second relapse was 40% at 30 months. In the ATRA group, the 10 patients who relapsed and were retreated with ATRA achieved a second CR. In conclusion, long-term results of APL91 trial confirm the superiority of the combination of ATRA and chemotherapy over chemotherapy alone in newly diagnosed APL, and that ATRA should be incorporated in the front-line treatment of APL.

Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.

High activity of sorafenib in FLT3-ITD-positive acute myeloid leukemia synergizes with allo-immune effects to induce sustained responses

Preliminary evidence suggests that the multikinase inhibitor sorafenib has clinical activity in FLT3-ITD-positive (FLT3-ITD) acute myeloid leukemia (AML). However, the quality and sustainability of achievable remissions and clinical variables that influence the outcome of sorafenib monotherapy are largely undefined. To address these questions, we evaluated sorafenib monotherapy in 65 FLT3-ITD AML patients treated at 23 centers. All but two patients had relapsed or were chemotherapy-refractory after a median of three prior chemotherapy cycles. Twenty-nine patients (45%) had undergone prior allogeneic stem cell transplantation (allo-SCT). The documented best responses were: hematological remission in 24 patients (37%), bone marrow remission in 5 patients (8%), complete remission (with and without normalization of peripheral blood counts) in 15 patients (23%) and molecular remission with undetectable FLT3-ITD mRNA in 10 patients (15%), respectively. Seventeen of the patients without prior allo-SCT (47%) developed sorafenib resistance after a median treatment duration of 136 days (range, 56–270 days). In contrast, allo-SCT patients developed sorafenib resistance less frequently (38%) and significantly later (197 days, range 38–225 days; P=0.03). Sustained remissions were seen exclusively in the allo-SCT cohort. Thus, sorafenib monotherapy has significant activity in FLT3-ITD AML and may synergize with allogeneic immune effects to induce durable remissions.

Additional chromosomal abnormalities in patients with acute promyelocytic leukaemia (APL) do not confer poor prognosis: results of APL 93 trial.

In spite of the recent improvement in the outcome of acute promyelocytic leukaemia (APL) with treatment combining all trans retinoic acid (ATRA) and chemotherapy (CT), some patients with this disease still have a poor outcome. The prognostic significance of chromosomal abnormalities in addition to t(15;17) in APL is uncertain. We examined the prognostic significance of secondary chromosomal changes in 292 patients included in a European trial who were treated with ATRA and CT. The incidence of chromosomal abnormalities in addition to t(15;17) was 26% and trisomy 8 was the most frequent secondary change (46% of the cases with secondary changes). No significant differences were seen with regard to age, sex, initial white blood cell count, % of circulating blasts, platelet count, fibrinogen level and incidence of microgranular variants between patients with or without additional rearrangements. Outcome was also similar between patients with t(15;17) alone and patients with t(15;17) and other clonal abnormalities for complete remission (92% vs. 93% respectively), event‐free survival at 2 years (76·1% vs. 78·1% respectively), relapse at 2 years (16·7% vs. 11·6% respectively) and overall survival at 2 years (79·9% vs. 79·5% respectively). Analysis according to the type of induction treatment (ATRA followed by CT or ATRA plus CT) or the type of maintenance treatment (with ATRA, low‐dose CT or both) also failed to show any difference between the two groups. Thus, in a large cohort of APL patients treated with ATRA and CT, additional chromosomal abnormalities had no impact on prognosis.