The expression level of BAALC-associated microRNA miR-3151 is an independent prognostic factor in younger patients with cytogenetic intermediate-risk acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease whose prognosis is mainly related to the biological risk conferred by cytogenetics and molecular profiling. In elderly patients (60 years) with normal karyotype AML miR-3151 have been identified as a prognostic factor. However, miR-3151 prognostic value has not been examined in younger AML patients. In the present work, we have studied miR-3151 alone and in combination with BAALC, its host gene, in a cohort of 181 younger intermediate-risk AML (IR-AML) patients. Patients with higher expression of miR-3151 had shorter overall survival (P=0.0025), shorter leukemia-free survival (P=0.026) and higher cumulative incidence of relapse (P=0.082). Moreover, in the multivariate analysis miR-3151 emerged as independent prognostic marker in both the overall series and within the unfavorable molecular prognostic category. Interestingly, the combined determination of both miR-3151 and BAALC improved this prognostic stratification, with patients with low levels of both parameters showing a better outcome compared with those patients harboring increased levels of one or both markers (P=0.003). In addition, we studied the microRNA expression profile associated with miR-3151 identifying a six-microRNA signature. In conclusion, the analysis of miR-3151 and BAALC expression may well contribute to an improved prognostic stratification of younger patients with IR-AML.

The lincRNA HOTAIRM1, located in the HOXA genomic region, is expressed in acute myeloid leukemia, impacts prognosis in patients in the intermediate-risk cytogenetic category, and is associated with a distinctive microRNA signature.

Long non-coding RNAs (lncRNAs) are deregulated in several tumors, although their role in acute myeloid leukemia (AML) is mostly unknown.We have examined the expression of the lncRNA HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in 241 AML patients. We have correlated HOTAIRM1 expression with a miRNA expression profile. We have also analyzed the prognostic value of HOTAIRM1 expression in 215 intermediate-risk AML (IR-AML) patients.The lowest expression level was observed in acute promyelocytic leukemia (P < 0.001) and the highest in t(6;9) AML (P = 0.005). In 215 IR-AML patients, high HOTAIRM1 expression was independently associated with shorter overall survival (OR:2.04;P = 0.001), shorter leukemia-free survival (OR:2.56; P < 0.001) and a higher cumulative incidence of relapse (OR:1.67; P = 0.046). Moreover, HOTAIRM1 maintained its independent prognostic value within the favorable molecular subgroup (OR: 3.43; P = 0.009). Interestingly, HOTAIRM1 was overexpressed in NPM1-mutated AML (P < 0.001) and within this group retained its prognostic value (OR: 2.21; P = 0.01). Moreover, HOTAIRM1 expression was associated with a specific 33-microRNA signature that included miR-196b (P < 0.001). miR-196b is located in the HOX genomic region and has previously been reported to have an independent prognostic value in AML. miR-196b and HOTAIRM1 in combination as a prognostic factor can classify patients as high-, intermediate-, or low-risk (5-year OS: 24% vs 42% vs 70%; P = 0.004).Determination of HOTAIRM1 level at diagnosis provided relevant prognostic information in IR-AML and allowed refinement of risk stratification based on common molecular markers. The prognostic information provided by HOTAIRM1 was strengthened when combined with miR-196b expression. Furthermore, HOTAIRM1 correlated with a 33-miRNA signature.

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

RHAMM-R3 peptide vaccination in patients with acute myeloid leukemia, myelodysplastic syndrome, and multiple myeloma elicits immunologic and clinical responses.

The receptor for hyaluronic acid-mediated motility (RHAMM) is an antigen eliciting both humoral and cellular immune responses in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM). We initiated a phase 1 clinical trial vaccinating 10 patients with R3 (ILSLELMKL), a highly immunogenic CD8(+) T-cell epitope peptide derived from RHAMM. In 7 of 10 patients, we detected an increase of CD8(+)/HLA-A2/RHAMM R3 tetramer(+)/CD45RA(+)/CCR7(-)/CD27(-)/CD28(-) effector T cells in accordance with an increase of R3-specific CD8(+) T cells in enzyme linked immunospot (ELISpot) assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Three of 6 patients with myeloid disorders (1/3 AML, 2/3 MDS) achieved clinical responses: one patient with AML and one with MDS showed a significant reduction of blasts in the bone marrow after the last vaccination. One patient with MDS no longer needed erythrocyte transfusions after 4 vaccinations. Two of 4 patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunologic and clinical responses, and therefore RHAMM constitutes a promising target for further immunotherapeutic approaches. This study is registered at http://ISRCTN.org as ISRCTN32763606 and is registered with EudraCT as 2005-001706-37.

5-Azacytidine to Treat Acute Myeloid Leukemia In Elderly or Frail Patients: A Phase II Study (SAKK 30/07).

Background: Acute Myeloid Leukemia (AML) in the elderly is notoriously difficult to treat and has a low remission rate with very few long term survivors when using standard treatment approaches. Azacytidine, a hypomethylating agent, has been shown to induce remission and prolong survival in patients with myelodysplastic syndromes; studying this approach to patients with AML is therefore warranted. We present results of an ongoing phase II trial treating elderly or frail AML patients with Azacytidine. Methods: AML elderly or frail patients, and therefore unfit for an intensive chemotherapy regimens, with a WHO performance status 3 were considered for this trial. Trial therapy consisted of 100mg/m2 of Azacytidine injected subcutaneously on 5 consecutive days every 28 days up to 6 cycles, stopping at 6 months if no hematological improvement achieved, or earlier in the case of progression or complications. Treatment was continued beyond 6 months in responding patients. Trial therapy was considered uninteresting if the response rate (CR + PR) within 6 months of therapy initiation was 15% or less and promising if 34% or more. Using the exact single-stage phase II design by A'Hern with a 5% significance level and 90% power, 43 patients were required: If 10 or fewer achieved a response within 6 months the trial therapy should not be considered for further investigation in its current format for this indication and patient population. Results: Between September 2008 and January 2010, 45 evaluable patients across 10 Swiss centers were accrued with a median follow-up of 7 months (range: 0 - 13). 27 (60%) were male, median age was 74 (range: 55 - 86) years and 35 (78.8%) had performance status 0-1. Patients had been excluded from more intensive chemotherapy regimens because of age (n = 37) or due to comorbidities or patient refusal (n=8). Five patients had therapy related AML. Patients received a median of 3 (range: 1 - 10) cycles. Treatment was stopped for not achieving a response by the 6th cycle in 2 patients and earlier in 26 patients (for disease progression in 5, toxicity in 3, patient refusal in 2, recurrent infections in 1, and death in 8). Seventeen patients remain on therapy. The median time spent in the hospital was 12 days (1 - 30) in 24/38 patients hospitalized during the first treatment cycle and 13 days (2 - 28) in 15/31 patients hospitalized during subsequent cycles. Adverse events of grade III or higher most frequently reported were constitutional or hematologic, i.e. fatigue in 5, febrile neutropenia in 8, infections in 6, dyspnea in 6, anemia in 3, neutropenia in 12 and thrombocytopenia in 10, hemorrhage in 2 and retinal detachment in 5. Based on available data on 38 patients, CR/CRi or hematologic improvement or stable disease within 6 months of trial registration was observed in a proportion of patients. Final and mature data, determining whether the predefined proportion of responding patients has been reached or not, will be presented at the conference. Up to now there were a total of 26 deaths. Median overall survival time was 5.7 months (95% CI: 3.1, 8.7). Conclusions: The current results of this slightly modified Azacytidine schedule demonstrate a feasible new therapy option for elderly or frail AML patients in an outpatient setting with moderate, mainly hematologic toxicity.

Mutated regions of nucleophosmin 1 elicit both CD4+ and CD8+ T-cell responses in patients with acute myeloid leukemia.

Mutations in the nucleophosmin gene (NPM1(mut)) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1(mut). In the present study, we were able to demonstrate both CD4(+) and CD8(+) T-cell responses against NPM1(mut). Ten peptides derived from wild-type NPM1 and NPM1(mut) were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr(51)-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4(+) T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1(mut) induced CD8(+) T-cell responses. A total of 33% of the NPM1(mut) AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4(+) T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8(+) and CD4(+) T cells. The results of the present study show that NPM1(mut) induces specific T-cell responses of CD4(+) and CD8(+) T cells and therefore is a promising target for specific immunotherapies in AML.

Clinical, molecular, and prognostic significance of WHO type inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and various other 3q abnormalities in acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) [inv(3)/t(3;3)] is recognized as a distinctive entity in the WHO classification. Risk assignment and clinical and genetic characterization of AML with chromosome 3q abnormalities other than inv(3)/t(3;3) remain largely unresolved. PATIENTS AND METHODS: Cytogenetics, molecular genetics, therapy response, and outcome analysis were performed in 6,515 newly diagnosed adult AML patients. Patients were treated on Dutch-Belgian Hemato-Oncology Cooperative Group/Swiss Group for Clinical Cancer Research (HOVON/SAKK; n = 3,501) and German-Austrian Acute Myeloid Leukemia Study Group (AMLSG; n = 3,014) protocols. EVI1 and MDS1/EVI1 expression was determined by real-time quantitative polymerase chain reaction. RESULTS: 3q abnormalities were detected in 4.4% of AML patients (288 of 6,515). Four distinct groups were defined: A: inv(3)/t(3;3), 32%; B: balanced t(3q26), 18%; C: balanced t(3q21), 7%; and D: other 3q abnormalities, 43%. Monosomy 7 was the most common additional aberration in groups (A), 66%; (B), 31%; and (D), 37%. N-RAS mutations and dissociate EVI1 versus MDS1/EVI1 overexpression were associated with inv(3)/t(3;3). Patients with inv(3)/t(3;3) and balanced t(3q21) at diagnosis presented with higher WBC and platelet counts. In multivariable analysis, only inv(3)/t(3;3), but not t(3q26) and t(3q21), predicted reduced relapse-free survival (hazard ratio [HR], 1.99; P < .001) and overall survival (HR, 1.4; P = .006). This adverse prognostic impact of inv(3)/t(3;3) was enhanced by additional monosomy 7. Group D 3q aberrant AML also had a poor outcome related to the coexistence of complex and/or monosomal karyotypes and cryptic inv(3)/t(3;3). CONCLUSION: Various categories of 3q abnormalities in AML can be distinguished according to their clinical, hematologic, and genetic features. AML with inv(3)/t(3;3) represents a distinctive subgroup with unfavorable prognosis.

Monosomal karyotype in acute myeloid leukemia: a better indicator of poor prognosis than a complex karyotype.

PURPOSE: To investigate the prognostic value of various cytogenetic components of a complex karyotype in acute myeloid leukemia (AML). PATIENTS AND METHODS: Cytogenetics and overall survival (OS) were analyzed in 1,975 AML patients age 15 to 60 years. RESULTS: Besides AML with normal cytogenetics (CN) and core binding factor (CBF) abnormalities, we distinguished 733 patients with cytogenetic abnormalities. Among the latter subgroup, loss of a single chromosome (n = 109) conferred negative prognostic impact (4-year OS, 12%; poor outcome). Loss of chromosome 7 was most common, but outcome of AML patients with single monosomy -7 (n = 63; 4-year OS, 13%) and other single autosomal monosomies (n = 46; 4-year OS, 12%) did not differ. Structural chromosomal abnormalities influenced prognosis only in association with a single autosomal monosomy (4-year OS, 4% for very poor v 24% for poor). We derived a monosomal karyotype (MK) as a predictor for very poor prognosis of AML that refers to two or more distinct autosomal chromosome monosomies (n = 116; 4-year OS, 3%) or one single autosomal monosomy in the presence of structural abnormalities (n = 68; 4-year OS, 4%). In direct comparisons, MK provides significantly better prognostic prediction than the traditionally defined complex karyotype, which considers any three or more or five or more clonal cytogenetic abnormalities, and also than various individual specific cytogenetic abnormalities (eg, del[5q], inv[3]/t[3;3]) associated with very poor outcome. CONCLUSION: MK enables (in addition to CN and CBF) the prognostic classification of two new aggregates of cytogenetically abnormal AML, the unfavorable risk MK-negative category (4-year OS, 26% +/- 2%) and the highly unfavorable risk MK-positive category (4-year OS, 4% +/- 1%).

Receptor activator for NF-κB ligand in acute myeloid leukemia: expression, function, and modulation of NK cell immunosurveillance.

The TNF family member receptor activator for NF-κB ligand (RANKL) and its receptors RANK and osteoprotegerin are key regulators of bone remodeling but also influence cellular functions of tumor and immune effector cells. In this work, we studied the involvement of RANK-RANKL interaction in NK cell-mediated immunosurveillance of acute myeloid leukemia (AML). Substantial levels of RANKL were found to be expressed on leukemia cells in 53 of 78 (68%) investigated patients. Signaling via RANKL into the leukemia cells stimulated their metabolic activity and induced the release of cytokines involved in AML pathophysiology. In addition, the immunomodulatory factors released by AML cells upon RANKL signaling impaired the anti-leukemia reactivity of NK cells and induced RANK expression, and NK cells of AML patients displayed significantly upregulated RANK expression compared with healthy controls. Treatment of AML cells with the clinically available RANKL Ab Denosumab resulted in enhanced NK cell anti-leukemia reactivity. This was due to both blockade of the release of NK-inhibitory factors by AML cells and prevention of RANK signaling into NK cells. The latter was found to directly impair NK anti-leukemia reactivity with a more pronounced effect on IFN-γ production compared with cytotoxicity. Together, our data unravel a previously unknown function of the RANK-RANKL molecule system in AML pathophysiology as well as NK cell function and suggest that neutralization of RANKL with therapeutic Abs may serve to reinforce NK cell reactivity in leukemia patients.

In situ RHAMM protein expression in acute myeloid leukemia blasts suggests poor overall survival.

Treatment options for patients with high-risk acute myeloid leukemia (AML) include high-dose chemotherapy regimens in combination with allogeneic hematopoietic stem cell transplantation, which takes advantage of the donor T-cell-mediated graft-versus-leukemia effect. Together with beneficial responses observed in assays targeted at leukemia-associated antigens (LAA), this encouraged research on cancer vaccines and adoptive cellular therapies in AML. The receptor for hyaluronic acid-mediated motility (RHAMM, CD168) was identified as one of the most promising LAA in AML. Thus far, little is known about in situ expression in leukemic bone marrow blasts or the prognostic role of RHAMM and its interaction partners in AML. We immunohistochemically analyzed the expression and prognostic significance of RHAMM on trephine bone marrow biopsies from 71 AML cases that had been evaluated for cytogenetics and presence of FLT3-internal tandem duplications and NPM1 mutations. Fifty-five patients (77%) were treated with curative intent, while 16 (23%) received the most appropriate supportive care. Twenty of 71 (28%) AML cases were considered RHAMM+. Receiver operating characteristic curves showed significant discriminatory power considering overall survival (OS) in AML patients treated curatively for RHAMM (p = 0.015). Multivariable analysis revealed that expression of RHAMM in >5% of leukemic blasts identifies a subgroup of curatively treated cases with adverse OS independent of failures to achieve complete remission. RHAMM not only represents a promising LAA with specific T-cell responses in AML but, if assessed in situ on blasts, also a probable prognostic factor.

Azacytidine for acute myeloid leukemia in elderly or frail patients: a phase II trial (SAKK 30/07).

Abstract This phase II trial treated elderly or frail patients with acute myeloid leukemia (AML) with single-agent subcutaneous azacytidine at 100 mg/m(2), on 5 of 28 days for up to six cycles. Treatment was stopped for lack of response, or continued to progression in responders. The primary endpoint was response within 6 months. A response rate ≥ 34% was considered a positive trial outcome. From September 2008 to April 2010, 45 patients from 10 centers (median age 74 [55-86] years) were accrued. Patients received four (1-21) cycles. Best response was complete response/complete response with incomplete recovery of neutrophils and/or platelets (CR/CRi) in eight (18%; 95% confidence interval [CI]: 8-32%.), 0 (0%) partial response (PR), seven (16%) hematologic improvement, 17 (38%) stable disease. Three non-responding patients stopped treatment after six cycles, 31 patients stopped early and 11 patients continued treatment for 8-21 cycles. Adverse events (grade ≥ III) were infections (n = 13), febrile neutropenia (n = 8), thrombocytopenia (n = 7), dyspnea (p = 6), bleeding (n = 5) and anemia (n = 4). Median overall survival was 6 months. Peripheral blood blast counts, grouped at 30%, had a borderline significant association with response (p = 0.07). This modified azacytidine schedule is feasible for elderly or frail patients with AML in an outpatient setting with moderate, mainly hematologic, toxicity and response in a proportion of patients, although the primary objective was not reached.

G-CSF-induced remission in two cases of acute myeloid leukemia.

We report on two elderly patients with newly diagnosed acute myeloid leukemia (AML) who were treated in palliative intention because of comorbidities and intermediate or poor risk cytogenetics. Both received G-CSF to reduce the risk of infection related to neutropenia. Interestingly, one patient achieved a full hematological remission and the other a peripheral remission with dramatic reduction of the bone marrow blast count. Although a direct therapeutic effect of myeloid growth factors seems to be unusual in AML, the use of G-CSF or GM-CSF may be recommended in patients such as elderly patients who are not suited for intensive chemotherapy.