Therapeutic Management of Patients with FLT3 + Acute Myeloid Leukemia: Case Reports and Focus on Gilteritinib Monotherapy.

Acute myeloid leukemia is a malignant disorder of the bone marrow, characterized by differentiation, clonal expansion, and uncontrolled proliferation of malignant myeloid progenitor cells and by several molecular and genetic abnormalities. A mutation of FMS-like tyrosine kinase 3 gene can be observed in about one-third of cases of acute myeloid leukemia. Two FLT3 inhibitors are actually approved for FLT3 mutated acute myeloid leukemia: midostaurin, a multikinase first generation inhibitor with lower affinity for FLT3 binding, and gilteritinib fumarate, a potent second-generation inhibitor of both FLT3-ITD and TKD. Gilteritinib is a new effective and well-tolerated drug for patients with relapsing or refractory FLT3-positive acute myeloid leukemia. Thanks to its efficacy, low toxicity, its good manageability (oral formulation), this drug is suitable for all the patients, including elderly frail patient with concomitant therapies or pre-existing or underlying diseases, and can be used also in the outpatient setting, reducing risks and costs related to the hospitalization. We report and discuss seven cases of different patients with FLT3 positive acute myeloid leukemia successfully managed with gilteritinib in the real clinical practice.

INCB84344-201: Ponatinib and steroids in frontline therapy for unfit patients with Ph+ acute lymphoblastic leukemia.

Tyrosine kinase inhibitors have improved survival for patients with Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). However, prognosis for old or unfit patients remains poor. In the INCB84344-201 (formerly GIMEMA LAL 1811) prospective, multicenter, phase 2 trial, we tested the efficacy and safety of ponatinib plus prednisone in newly diagnosed patients with Ph+ ALL ≥60 years, or unfit for intensive chemotherapy and stem cell transplantation. Forty-four patients received oral ponatinib 45 mg/d for 48 weeks (core phase), with prednisone tapered to 60 mg/m2/d from days-14-29. Prophylactic intrathecal chemotherapy was administered monthly. Median age was 66.5 years (range, 26-85). The primary endpoint (complete hematologic response [CHR] at 24 weeks) was reached in 38/44 patients (86.4%); complete molecular response (CMR) in 18/44 patients (40.9%) at 24 weeks. 61.4% of patients completed the core phase. As of 24 April 2020, median event-free survival was 14.31 months (95% CI 9.30-22.31). Median overall survival and duration of CHR were not reached; median duration of CMR was 11.6 months. Most common treatment-emergent adverse events (TEAEs) were rash (36.4%), asthenia (22.7%), alanine transaminase increase (15.9%), erythema (15.9%), and γ-glutamyltransferase increase (15.9%). Cardiac and vascular TEAEs occurred in 29.5% (grade ≥3, 18.2%) and 27.3% (grade ≥3, 15.9%), respectively. Dose reductions, interruptions, and discontinuations due to TEAEs occurred in 43.2%, 43.2%, and 27.3% of patients, respectively; 5 patients had fatal TEAEs. Ponatinib and prednisone showed efficacy in unfit patients with Ph+ ALL; however, a lower ponatinib dose may be more appropriate in this population. This trial was registered at www.clinicaltrials.gov as #NCT01641107.

Log reduction of leukemic cells and minimal residual disease by flow cytometry represent effective predictors of clinical outcome in elderly patients with acute myeloid leukemia.

BACKGROUND: Nowadays minimal residual disease (MRD) and log-reduction of leukemic cells are poorly investigated in elderly patients with acute myeloid leukemia (AML) treated with hypometilating agents (HMAs). Studies focusing on MRD in elderly AML patients who received HMAs are scant and devoid of rigorous criteria for both enrollment and monitoring. Log-reduction has never been investigated in these patients. Thus, the purpose of our study was to compare the prognostic impact of MRD and log-reduction of leukemic cells at the optimal time of assessment in older AML patients. METHODS: Elderly patients who completed at least six cycles of HMAs and showed suitable leukemia-associated immunophenotypes (LAIPs) for the MRD and log-reduction assessment by flow cytometry were enrolled in the study. RESULTS: After comparing the times of assessment C4 (4-cycles) and C6 (6-cycles), C6 has been chosen as optimal. Patients who achieved MRD negativity or 2-log-reduction of leukemic cells at C6 had a significantly longer DFS. Particularly, results of 2-log-reduction were confirmed a multivariate analysis. Patients with MRD negativity or 2-log reduction of leukemic cells showed an improvement of their OS, although not significantly. CONCLUSIONS: Our data confirmed the predictive role of MRD and 2-log reduction also in older AML patients treated with HMAs.

Assessment of liver stiffness measurement and ultrasound findings change during inotuzumab ozogamicin cycles for relapsed or refractory acute lymphoblastic leukemia.

In adult patients, acute lymphoblastic leukemia (ALL) is a rare hematological cancer with a cure rate below 50% and frequent relapses. With traditional therapies, patients with relapsed or refractory (R/R) ALL have a survival that may be measured in months; in these patients, inotuzumab ozogamicin (IO) is an effective therapy. IO was linked to increased risk of veno-occlusive disease/sinusoid obstruction syndrome (VOD/SOS), liver injury, and various grade of liver-related complications during clinical trials and real-life settings; however, hepatologic monitoring protocol is not established in this population. In our institution, 21 patients who received IO (median of 6 doses of IO administered) for R/R ALL were prospectively followed for hepatologic surveillance, including clinical evaluation, ultrasonography, and liver stiffness measurement (LSM) biochemistry. After a median follow-up of 17.2 months, two SOS events were reported (both after allogeneic transplant) as IO potentially related clinically relevant adverse event. Mild alterations were reported in almost the totality of patients and moderate-severe liver biochemical alterations in a quarter of patients. Within biochemicals value, AST and ALP showed an augment related to IO administration. LSM linearly augmented for each IO course administered. Baseline LSM was related to liver-related changes, especially with the severity of portal hypertension (PH)-related complications. Pre-transplant LSM was higher in patients receiving IO when compared with a control cohort. PH-related complications were discovered in nearly 77% of patients, with clinically significant PH occurrence and development of ascites in 38% and 14%, respectively. This prospective experience constitutes the rationale to design a hepatologic monitoring program in patients receiving IO. LSM may be of pivotal importance in this program, constituting a rapid and effective screening that quantitatively correlates with liver alterations.

Safety profile and impact on survival of tyrosine kinase inhibitors versus conventional therapy in relapse or refractory FLT3 positive acute myeloid leukemia patients.

Relapsed or refractory (R/R) acute myeloid leukemia (AML) has a poor prognosis, and new therapies are a major clinical need. When mutated, FLT3 drives neoplastic cell proliferation. New drugs (i.e., tyrosine kinase inhibitors, TKIs) showed effectiveness in FLT3-AML and promise to change disease history and outcome. We evaluated the benefit conferred by TKIs in terms of survival, burden of complications and surrogate endpoint of quality of life in a retrospective cohort of 49 FLT3 positive, R/R AML patients. Patients who received TKIs were compared to those treated with conventional chemotherapy. Treatment with TKIs conferred a better OS and wea associated with a lower burden and severity of adverse events. Importantly, patients who received TKIs showed reduced time of hospitalization. In conclusion, treatment with TKI in R/R FLT3-AML was related to a better survival, less and milder AEs, and shorter hospitalization.

MEC (mitoxantrone, etoposide, and cytarabine) induces complete remission and is an effective bridge to transplant in acute myeloid leukemia.

INTRODUCTION: Clinical response and chemosensitivity of relapse or refractory AML patients were evaluated after rescue and bridge-to-transplant MEC (mitoxantrone, etoposide, and cytarabine) regimen. METHODS AND PATIENTS: Fifty-five consecutive AML patients were treated with MEC from 2009 to 2018. Chemosensitivity was evaluated by WT1 quantification. RESULTS: 27/55 patients (49.1%) had AML resistant to induction and 28/55 patients (50.9%) had AML relapse. 25/55 patients (45.5%) achieved a CR after one course of MEC, and 12 patients (21.8%) achieved WT1 negativity. In 12 patients, a second MEC was administered. Four out of 12 patients improved significantly their response with the 2nd MEC. MEC was an effective bridge to transplant, 32/55 patients (58.2%) received an allogenic stem cell transplant. Median overall survival (OS) from MEC was 455 days (95% CI 307-602 days.); patient with WT1 negative CR had the best OS (P<.000). CONCLUSION: WT1 is a useful marker of chemosensitivity after MEC as rescue and bridge-to-transplant therapy.

Identification of Two DNMT3A Mutations Compromising Protein Stability and Methylation Capacity in Acute Myeloid Leukemia.

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795 ∗ ; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.

Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia.

BACKGROUND: Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. METHODS: The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. RESULTS: We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. CONCLUSIONS: Our data suggest that WEE1 plays a role in ALL blast's survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL.

The clonal evolution of two distinct T315I-positive BCR-ABL1 subclones in a Philadelphia-positive acute lymphoblastic leukemia failing multiple lines of therapy: a case report.

BACKGROUND: The treatment of Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients who harbor the T315I BCR-ABL1 mutation or who have two or more mutations in the same BCR-ABL1 molecule is particularly challenging since first and second-generation Tyrosine Kinase Inhibitors (TKIs) are ineffective. Ponatinib, blinatumomab, chemotherapy and transplant are the currently available options in these cases. CASE PRESENTATION: We here report the case of a young Ph+ ALL patient who relapsed on front-line dasatinib therapy because of two independent T315I-positive subclones, resulting from different nucleotide substitutions -one of whom never reported previously- and where additional mutant clones outgrew and persisted despite ponatinib, transplant, blinatumomab and conventional chemotherapy. Deep Sequencing (DS) was used to dissect the complexity of BCR-ABL1 kinase domain (KD) mutation status and follow the kinetics of different mutant clones across the sequential therapeutic approaches. CONCLUSIONS: This case presents several peculiar and remarkable aspects: i) distinct clones may acquire the same amino acid substitution via different nucleotide changes; ii) the T315I mutation may arise also from an 'act' to 'atc' codon change; iii) the strategy of temporarily replacing TKI therapy with chemo or immunotherapy, in order to remove the selective pressure and deselect aggressive mutant clones, cannot always be expected to be effective; iv) BCR-ABL1-mutated sub-clones may persist at very low levels (undetectable even by Deep Sequencing) for long time and then outcompete BCR-ABL1-unmutated ones becoming dominant even in the absence of any TKI selective pressure.

Prexasertib, a Chk1/Chk2 inhibitor, increases the effectiveness of conventional therapy in B-/T- cell progenitor acute lymphoblastic leukemia.

During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Targeting the p53-MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients.

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL.

In vitro and in vivo single-agent efficacy of checkpoint kinase inhibition in acute lymphoblastic leukemia.

BACKGROUND: Although progress in children, in adults, ALL still carries a dismal outcome. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), a potent, selective ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). METHODS: The effectiveness of PF-00477736 as single agent in B-/T-ALL was evaluated in vitro and in vivo studies as a single agent. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B-/T-ALL cell lines. Finally, the action of PF-00477736 was assessed in vivo using leukemic mouse generated by a single administration of the tumorigenic agent N-ethyl-N-nitrosourea. RESULTS: Chk1 and Chk2 are overexpressed concomitant with the presence of genetic damage as suggested by the nuclear labeling for γ-H2A.X (Ser139) in 68 % of ALL patients. In human B- and T-ALL cell lines, inhibition of Chk1/2 as a single treatment strategy efficiently triggered the Chk1-Cdc25-Cdc2 pathway resulting in a dose- and time-dependent cytotoxicity, induction of apoptosis, and increased DNA damage. Moreover, treatment with PF-00477736 showed efficacy ex vivo in primary leukemic blasts separated from 14 adult ALL patients and in vivo in mice transplanted with T-ALL, arguing in favor of its future clinical evaluation in leukemia. CONCLUSIONS: In vitro, ex vivo, and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia, and they provide a strong rationale for its future clinical investigation.

Revealing very small FLT3 ITD mutated clones by ultra-deep sequencing analysis has important clinical implications in AML patients.

FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2-2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.

Rationale for a pediatric-inspired approach in the adolescent and young adult population with acute lymphoblastic leukemia, with a focus on asparaginase treatment.

In the last two decades great improvements have been made in the treatment of childhood acute lymphoblastic leukemia, with 5-year overall survival rates currently approaching almost 90%. In comparison, results reported in adolescents and young adults (AYAs) are relatively poor. In adults, results have improved, but are still lagging behind those obtained in children. Possible reasons for this different pattern of results include an increased incidence of unfavorable and a decreased incidence of favorable cytogenetic abnormalities in AYAs compared with children. Furthermore, in AYAs less intensive treatments (especially lower cumulative doses of drugs such as asparaginase, corticosteroids and methotrexate) and longer gaps between courses of chemotherapy are planned compared to those in children. However, although favorable results obtained in AYAs receiving pediatric protocols have been consistently reported in several international collaborative trials, physicians must also be aware of the specific toxicity pattern associated with increased success in AYAs, since an excess of toxicity may compromise overall treatment schedule intensity. Cooperative efforts between pediatric and adult hematologists in designing specific protocols for AYAs are warranted.

Posterior reversible encephalopathy syndrome in a B-cell acute lymphoblastic leukemia young adult patient treated with a pediatric-like chemotherapeutic schedule.

We report here the case of a young adult affected by pre B-cell acute lymphoblastic leukemia (ALL), who developed, during a pediatric-like chemotherapy consolidation schedule with high dosage of Methotrexate, a severe neurological toxicity. Clinical presentation and neuroimaging data were diagnostic for posterior reversible encephalopathy syndrome (PRES). A complete resolution was quickly obtained with medical blood pressure control and anticonvulsants administration. To the best of our knowledge, this is the first case of PRES described in the adult ALL setting. Currently, the clinical management of this aggressive disease is moving towards a pediatric-like approach also in adult patients, due to the better outcome reached with intensive chemotherapeutic regimens in children population. However, therapy-related toxicities have to be taken into account, since their onset may adversely affect patients' clinical outcome.

Use of a high sensitive nanofluidic array for the detection of rare copies of BCR-ABL1 transcript in patients with Philadelphia-positive acute lymphoblastic leukemia in complete response.

Monitoring of minimal residual disease (MRD) by quantification of BCR-ABL1 transcript levels has become a main part of the management of patients with BCR-ABL1-positive acute lymphoblastic leukemia (ALL) in treatment with tyrosine kinase inhibitors (TKIs). The failure to achieve molecular negativity shortly after starting TKI has been demonstrated to be predictive of relapse, suggesting that an accurate measurement of low BCR-ABL1 levels may have a role in preventing hematological relapse. Despite the big efforts made by many European laboratories within the European Study Group, at the time of writing a standardized procedure to quantify and express results is still missing for BCR-ABL1-positive ALL. In this study, in order to detect with high sensitivity low levels of BCR-ABL1 transcripts, we used a new technology and a new molecular approach based on microfluidic digital polymerase chain reaction (dPCR) using Taqman chemistry and we compared obtained results with those generated by the conventional method based on reverse transcriptase PCR reaction (RQ-PCR) for BCR-ABL1 and total ABL1, with TaqMan chemistry and with Applied Biosystems instrument. We demonstrated the dPCR is high-sensitive (able to detect a single copy of BCR-ABL1) and reliable (results are comparable to those obtained by BCR-ABL1 quantification with conventional technology), allowing an accurate monitoring of BCR-ABL1-positive ALL patients in complete remission.

CDKN2A/B alterations impair prognosis in adult BCR-ABL1-positive acute lymphoblastic leukemia patients.

PURPOSE: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors. PATIENTS AND METHODS: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1-positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis-relapse samples were further available and analyzed for 19 (19%) cases. RESULTS: CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P = 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P = 0.0206), disease free-survival (P = 0.0010), and cumulative incidence of relapse (P = 0.0014). CONCLUSIONS: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1-positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes.