Paola Panetta

Toward Optimization of Postremission Therapy for Residual Disease–Positive Patients With Acute Myeloid Leukemia

PURPOSE Despite the identification of several baseline prognostic indicators, the outcome of patients with acute myeloid leukemia (AML) is generally heterogeneous. The effects of autologous (AuSCT) or allogeneic stem-cell transplantation (SCT) are still under evaluation. Minimal residual disease (MRD) states may be essential for assigning patients to therapy-dependent risk categories. PATIENTS AND METHODS By multiparametric flow cytometry, we assessed the levels of MRD in 142 patients with AML who achieved complete remission after intensive chemotherapy. RESULTS A level of 3.5 x 10(-4) residual leukemia cells (RLCs) after consolidation therapy was established to identify MRD-negative and MRD-positive cases, with 5-year relapse-free survival (RFS) rates of 60% and 16%, respectively (P < .0001) and overall survival (OS) rates of 62% and 23%, respectively (P = .0001). Of patients (n = 77) who underwent a transplantation procedure (56 AuSCT and 21 SCT procedures); 42 patients (55%) were MRD positive (28 patients who underwent AuSCT and 14 patients who underwent SCT) and 35 patients (45%) were MRD negative (28 patients who underwent AuSCT and seven who underwent SCT). MRD-negative patients had a favorable prognosis, with only eight (22%) of 35 patients experiencing relapse, whereas 29 (69%) of 42 MRD-positive patients experienced relapse (P < .0001). In this high-risk group of 42 patients, we observed that 23 (82%) of 28 of those who underwent AuSCT experienced relapse, whereas six (43%) of 14 who underwent SCT experienced relapse (P = .014). Patients who underwent SCT also had a higher likelihood of RFS (47% v 14%). CONCLUSION A threshold of 3.5 x 10(-4) RLCs postconsolidation is critical for predicting disease outcome. MRD-negative patients have a good outcome regardless of the type of transplant they receive. In the MRD-positive group, AuSCT does not improve prognosis and SCT represents the primary option.

Cytogenetic and molecular diagnostic characterization combined to postconsolidation minimal residual disease assessment by flow cytometry improves risk stratification in adult acute myeloid leukemia

A total of 143 adult acute myeloid leukemia (AML) patients with available karyotype (K) and FLT3 gene mutational status were assessed for minimal residual disease (MRD) by flow cytometry. Twenty-two (16%) patients had favorable, 115 (80%) intermediate, and 6 (4%) poor risk K; 19 of 129 (15%) carried FLT3-ITD mutation. Considering postconsolidation MRD status, patients with good/intermediate-risk K who were MRD(-) had 4-year relapse-free survival (RFS) of 70% and 63%, and overall survival (OS) of 84% and 67%, respectively. Patients with good- and intermediate-risk K who were MRD(+) had 4-year RFS of 15% and 17%, and OS of 38% and 23%, respectively (P < .001 for all comparisons). FLT3 wild-type patients achieving an MRD(-) status, had a better outcome than those who remained MRD(+) (4-year RFS, 54% vs 17% P < .001; OS, 60% vs 23%, P = .002). Such an approach redefined cytogenetic/genetic categories in 2 groups: (1) low-risk, including good/intermediate K-MRD(-) with 4-year RFS and OS of 58% and 73%, respectively; and (2) high risk, including poor-risk K, FLT3-ITD mutated cases, good/intermediate K-MRD(+) categories, with RFS and OS of 22% and 17%, respectively (P < .001 for all comparisons). In AML, the integrated evaluation of baseline prognosticators and MRD improves risk-assessment and optimizes postremission therapy.

Chromosomal Aberration of the 11q23 Locus in Acute Leukemia and Frequency of MLL Gene Translocation Results in 378 Adult Patients

Structural abnormality of the 11q23 band (11q23+) bearing the MLL gene translocation (MLL+) is a recurrent chromosome change observed in 3% to 7% of acute lymphoblastic leukemias and in 3% to 4% of acute myeloblastic leukemias. The resolution of conventional cytogenetics (CC) in detecting 11q23 rearrangement is limited when the translocative partner has a telomeric location; furthermore, CC can barely discriminate between true 11q23+/MLL+ and rearrangements clustering within the 11q22~25 region without MLL involvement (MLL–). We characterized a series of 378 consecutive patients with adult acute leukemia by using CC, fluorescence in situ hybridization (FISH), and multiplex karyotyping (MFISH) analysis. Our aim was to define the frequency of cryptic MLL+ cases and the frequency of MLL+ within 11q22~25+ cases. As expected, FISH was more sensitive than CC in detecting MLL+ cases, but rather unexpectedly, 9 (45%) of 20 patients with 11q22~25+ were MLL–. A better characterization of 11q22~25+/MLL– leukemias is relevant for the identification of new, recurrent translocations. Moreover, these cases should be readily distinguishable from 11q23+/MLL+ cases. We recommend that karyotypic analysis always be complemented by molecular or FISH methods to unravel MLL rearrangements.

Chromosomal Aberration of the 11q23 Locus in Acute Leukemia and Frequency of MLL Gene Translocation

Structural abnormality of the 11q23 band (11q23+) bearing the MLL gene translocation (MLL+) is a recurrent chromosome change observed in 3% to 7% of acute lymphoblastic leukemias and in 3% to 4% of acute myeloblastic leukemias. The resolution of conventional cytogenetics (CC) in detecting 11q23 rearrangement is limited when the translocative partner has a telomeric location; furthermore, CC can barely discriminate between true 11q23+/MLL+ and rearrangements clustering within the 11q22 to approximately 25 region without MLL involvement (MLL-). We characterized a series of 378 consecutive patients with adult acute leukemia by using CC, fluorescence in situ hybridization (FISH), and multiplex karyotyping (M-FISH) analysis. Our aim was to define the frequency of cryptic MLL+ cases and the frequency of MLL+ within 11q22 to approximately 25+ cases. As expected, FISH was more sensitive than CC in detecting MLL+ cases, but rather unexpectedly, 9 (45%) of 20 patients with 11q22 to approximately 25+ were MLL-. A better characterization of 11q22 to approximately 25+/MLL- leukemias is relevant for the identification of new, recurrent translocations. Moreover, these cases should be readily distinguishable from 11q23+/MLL+ cases. We recommend that karyotypic analysis always be complemented by molecular or FISH methods to unravel MLL rearrangements.

BRCA1, PARP1 and γH2AX in acute myeloid leukemia: Role as biomarkers of response to the PARP inhibitor olaparib

Olaparib (AZD-2281, Ku-0059436) is an orally bioavailable and well-tolerated poly(ADP-ribose) polymerase (PARP) inhibitor currently under investigation in patients with solid tumors. To study the clinical potential of olaparib as a single-agent for the treatment of acute myeloid leukemia (AML) patients, we analyzed the in vitro sensitivity of AML cell lines and primary blasts. Clinically achievable concentrations of olaparib were able to induce cell death in the majority of primary AML case samples (88%) and tested cell lines. At these concentrations, olaparib preferentially killed leukemic blasts sparing normal lymphocytes derived from the same patient and did not substantially affect the viability of normal bone marrow and CD34-enriched peripheral blood cells obtained from healthy donors. Most primary AML analyzed were characterized by low BRCA1 mRNA level and undetectable protein expression that likely contributed to explain their sensitivity to olaparib. Noteworthy, while PARP1 over-expression was detected in blasts not responsive to olaparib, phosphorylation of the histone H2AFX (γH2AX) was associated with drug sensitivity. As to genetic features of tested cases the highest sensitivity was shown by a patient carrying a 11q23 deletion. The high sensitivity of AML blasts and the identification of biomarkers potentially able to predict response and/or resistance may foster further investigation of olaparib monotherapy for AML patients unfit to conventional chemotherapy.

Clinical Relevance of Minimal Residual Disease Detection in Adult Acute Myeloid Leukemia

We have used flow cytometry to quantify minimal residual disease (MRD) in 63 patients with acute myeloid leukemia (AML). No significant correlation was found between the level of MRD after induction and disease outcome. After consolidation, a threshold of 3.5 x 10(-4) residual leukemic cells divided the 57 evaluable patients into two distinct groups: the MRDCons(+) and the MRDCons(-) group, with a relapse rate of 81% (22/27) and 27% (8/30), respectively (p = 0.000035). Although not correlated with prognosis, the level of MRD after induction course affected the degree of cytoreduction achieved with consolidation. In fact, the patients who entered a MRDCons(-) status had a median number of leukemic residual cells of 1.8 x 10(-4) after induction; at the same stage, the bone marrow of patients who were in a MRDCons(+) condition harbored a median level of 1.7 x 10(-3) malignant residual cells (p = 0.00073). The MRDCons(+) status also correlated significantly with poor/intermediate risk cytogenetics, MDR1 phenotype, short duration of overall survival, and relapse-free survival (p = 0.024, 0.021, 0.00001, and 0.00001, respectively). In multivariate analysis, the MRDCons(+) status was associated with a high probability of relapse (p < 0.00026) and short duration of relapse free survival (p = 0.008). Stem cell transplantation did not seem to alter the prognostic impact of high levels of MRD after consolidation: within the MRDCons(+) group, the relapse rate after transplant was 78%. Thus, a MRD > or = 3.5 x 10(-4) leukemic cells at the end of consolidation strongly predicts relapse, and is significantly associated with MDR1-positive phenotype and intermediate/unfavorable cytogenetics.

Identification of emerging FLT3 ITD-positive clones during clinical remission and kinetics of disease relapse in acute myeloid leukaemia with mutated nucleophosmin.

FLT3 internal tandem duplication (ITD) mutations are frequently detected at diagnosis in cytogenetically normal acute myeloid leukaemia (CN‐AML) and predict unfavourable outcome. FLT3 ITD is an unstable aberration and may be lost or acquired at relapse. Recent whole genome sequencing studies have suggested that FLT3 ITD+ve AML relapse may evolve from small subclones undetectable at diagnosis by routine polymerase chain reaction (PCR). We developed a patient‐specific real‐time quantitative‐PCR (RQ‐PCR) to implement FLT3 ITD detection in six AML patients whose blasts carried wild‐type FLT3 at diagnosis and who relapsed with FLT3 ITD by routine PCR. Patient‐specific forward primers were designed after cloning and sequencing the FLT3 ITD in each case. The assay allowed retrospective detection of FLT3 ITD in diagnostic samples of 4/6 cases and to establish the kinetics of clonal evolution preceding relapse. After conventional chemotherapy, all patients had early relapse despite having been classified as NPM1+ve/FLT3 ITD−ve at presentation, with shorter remissions being observed in four patients re‐classified as FLT3 ITD+ve by the new assay. Notably, FLT3 ITD clone became detectable by conventional PCR in three patients tested during remission after initial treatment. Our data underscore the need of identifying low FLT3 ITD levels, which are probably associated with relapse in otherwise good prognosis CN‐AML.

New reciprocal translocation t(6;10) (q27;q11) associated with idiopathic myelofibrosis and eosinophilia

Idiopathic myelofibrosis (IM), is a chronic myeloproliferative disorder (MPD) characterised by marrow fibrosis, extramedullary haematopoiesis and a leuco-erythroblastic picture of the peripheral blood. Cytogenetic data of IM is scarce: no specific karyotypic anomalies have been yet described. Trisomy 1q, del(13q), del(20q) and trisomy 8, appear in two-thirds of the cases with chromosome aberrations. We report on a 41-year-old patient diagnosed with IM associated with eosinophilia, bearing a novel translocation t(6;10)(q27;q11) as the sole chromosome anomaly. The patient, progressed to AML-M5a within 18 months from diagnosis. Recently new specific chromosomal translocations have been described in chronic MPD. These findings have allowed the classification of new syndromes with defined molecular abnormalities. The case we describe, because of the peculiar clinical features and the association with a previously unreported chromosomal translocation, might be a noteworthy addition.

The genotype nucleophosmin mutated and FLT3-ITD negative is characterized by high bax/bcl-2 ratio and favourable outcome in acute myeloid leukaemia

Nucleophosmin gene (NPM1) mutations characterize acute myeloid leukaemia (AML) with normal karyotype and frequently co‐exist with FLT3 internal tandem duplications (ITD). We evaluated bcl‐2, bax, NPM1 and FLT3‐ITD in 222 AML patients. Bax/bcl‐2 ratio >0·35 and NPM1 without FLT3‐ITD were significantly associated (P = 0·0001). NPM1‐mutated (mt)/FLT3‐ITD negative patients showed a higher complete remission (CR) rate (90%, P = 0·0002) and a longer overall survival (OS, P = 0·00007). NPM1‐mt/FLT3‐ITD negative plus bax/bcl‐2 > 0·35 subset showed a very high CR rate (96%), very long OS (P = 0·00005) and disease‐free survival (P = 0·004). The favourable prognosis of NPM1‐mt/FLT3‐ITD negative patients might be explained by a higher bax/bcl‐2 ratio.

Multidimensional Flow Cytometry for Detection of Minimal Residual Disease in Acute Myeloid Leukemia

The term minimal residual disease (MRD) describes the situation in which, after chemotherapy for acute leukemia (AL), a morphologically normal bone marrow (BM) can still harbor a relevant amount of residual malignant cells. Several techniques are now amenable to investigate MRD, and all together they have designated a new era in which a re-definition of the current criteria of complete remission (CR) is required. Depending upon the measured level of MRD we can distinguish a variety of clinical situations ranging from a potentially cured disease to short-term remission. In the context of this spectrum of conditions there would be room for different therapeutic strategies ranging from no further therapy to pre-emptive therapy to treat early relapses (immunologic and/or molecular relapses). This review will focus on the state of art of MRD detection in acute myeloid leukemia (AML) using multidimensional flow cytometry (MFC), and will cover the laboratory and clinical aspects of this approach.