Vitamin C Deficiency in Patients With Acute Myeloid Leukemia.

Vitamin C has been shown to play a significant role in suppressing progression of leukemia through epigenetic mechanisms. We aimed to study the role of vitamin C in acute myeloid leukemia (AML) biology and clinical course. To this purpose, the plasma levels of vitamin C at diagnosis in 62 patients with AML (including 5 cases with acute promyelocytic leukemia, APL),7 with myelodysplastic syndrome (MDS), and in 15 healthy donors (HDs) were studied. As controls, vitamins A and E levels were analysed. Expression of the main vitamin C transporters and of the TET2 enzyme were investigated by a specific RQ-PCR while cytoplasmic vitamin C concentration and its uptake were studied in mononuclear cells (MNCs), lymphocytes and blast cells purified from AML samples, and MNCs isolated from HDs. There were no significant differences in vitamin A and E serum levels between patients and HDs. Conversely, vitamin C concentration was significantly lower in AML as compared to HDs (p<0.0001), inversely correlated with peripheral blast-counts (p=0.029), significantly increased at the time of complete remission (CR) (p=0.04) and further decreased in resistant disease (p=0.002). Expression of the main vitamin C transporters SLC23A2, SLC2A1 and SLC2A3 was also significantly reduced in AML compared to HDs. In this line, cytoplasmic vitamin C levels were also significantly lower in AML-MNCs versus HDs, and in sorted blasts compared to normal lymphocytes in individual patients. No association was found between vitamin C plasma levels and the mutation profile of AML patients, as well as when considering cytogenetics or 2017 ELN risk stratification groups. Finally, vitamin C levels did not play a predictive role for overall or relapse-free survival. In conclusion, our study shows that vitamin C levels are significantly decreased in patients with AML at the time of initial diagnosis, further decrease during disease progression and return to normal upon achievement of CR. Correspondingly, low intracellular levels may mirror increased vitamin C metabolic consumption in proliferating AML cells.

Mutational profile of ZBTB16-RARA-positive acute myeloid leukemia.

BACKGROUND: The ZBTB16-RARA fusion gene, resulting from the reciprocal translocation between ZBTB16 on chromosome 11 and RARA genes on chromosome 17 [t(11;17)(q23;q21)], is rarely observed in acute myeloid leukemia (AML), and accounts for about 1% of retinoic acid receptor-α (RARA) rearrangements. AML with this rare translocation shows unusual bone marrow (BM) morphology, with intermediate aspects between acute promyelocytic leukemia (APL) and AML with maturation. Patients may have a high incidence of disseminated intravascular coagulation at diagnosis, are poorly responsive to all-trans retinoic acid (ATRA) and arsenic tryoxyde, and are reported to have an overall poor prognosis. AIMS: The mutational profile of ZBTB16-RARA rearranged AML has not been described so far. MATERIALS AND METHODS: We performed targeted next-generation sequencing of 24 myeloid genes in BM diagnostic samples from seven ZBTB16-RARA+AML, 103 non-RARA rearranged AML, and 46 APL. The seven ZBTB16-RARA-positive patients were then screened for additional mutations using whole exome sequencing (n = 3) or an extended cancer panel including 409 genes (n = 4). RESULTS: ZBTB16-RARA+AML showed an intermediate number of mutations per patient and involvement of different genes, as compared to APL and other AMLs. In particular, we found a high incidence of ARID1A mutations in ZBTB16-RARA+AML (five of seven cases, 71%). Mutations in ARID2 and SMARCA4, other tumor suppressor genes also belonging to SWI/SNF chromatin remodeling complexes, were also identified in one case (14%). DISCUSSION AND CONCLUSION: Our data suggest the association of mutations of the ARID1A gene and of the other members of the SWI/SNF chromatin remodeling complexes with ZBTB16-RARA+AMLs, where they may support the peculiar disease phenotype.

Early intracranial haemorrhages in acute promyelocytic leukaemia: analysis of neuroradiological and clinico-biological parameters.

Acute promyelocytic leukaemia (APL) represents a modern success of precision medicine. However, fatalities occurring within the first 30 days of induction treatment, in particular intracranial haemorrhage (ICH), remain the main causes of death. We studied the clinico-biological characteristics of 13 patients with APL who experienced ICH. Compared to 85 patients without this complication, patients with ICH were older and more frequently had high-risk APL. Moreover, positivity for the 'swirl' sign at neuroradiological imaging and hydrocephalus were predictors of a fatal outcome, together with lower fibrinogen, prolonged international normalized ratio (INR) and higher lactate dehydrogenase levels.

Cardiovascular events in cancer survivors.

Malignant disease and its treatment carry huge burdens for patients. Some are immediate, in that the disease itself presents as a life threatening event, or the treatment may result in immediate and devastating toxicity. More often the treatment of cancer is associated with more subtle or late events, yet these may impact the quality of life for cancer survivors in a variety of ways. In addition to the physical sequelae of cancer or its treatment, cancer survivors often experience consequences in the form of social or mental incapacity. Session III of this Colloquium on Cardio-Oncology focuses on some of these concerns, both from the perspective of health care providers who strive to minimize the burdens, but also from the viewpoint of the patient him or herself who must deal with the price that must often be paid for increased survival or cure.

Mutational landscape of patients with acute promyelocytic leukemia at diagnosis and relapse.

Despite the high probability of cure of patients with acute promyelocytic leukemia (APL), mechanisms of relapse are still largely unclear. Mutational profiling at diagnosis and/or relapse may help to identify APL patients needing frequent molecular monitoring and early treatment intervention. Using an NGS approach including a 31 myeloid gene-panel, we tested BM samples of 44 APLs at the time of diagnosis, and of 31 at relapse. Mutations in PML and RARA genes were studied using a customized-NGS-RNA panel. Patients relapsing after ATRA-chemotherapy rarely had additional mutations (P = .009). In patients relapsing after ATRA/ATO, the PML gene was a preferential mutation target. We then evaluated the predictive value of mutations at APL diagnosis. A median of two mutations was detectable in 9/11 patients who later relapsed, vs one mutation in 21/33 patients who remained in CCR (P = .0032). This corresponded to a significantly lower risk of relapse in patients with one or less mutations (HR 0.046; 95% CI 0.011-0.197; P < .0001). NGS-analysis at the time of APL diagnosis may inform treatment decisions, including alternative treatments for cases with an unfavorable mutation profile.

Acute Myeloid Leukemia with Concomitant BCR-ABL and NPM1 Mutations.

We present a case report of a patient with acute myeloid leukemia (AML) characterized by the simultaneous presence of nucleophosmin 1 (NPM1) mutation and the breakpoint cluster region-Abelson (BCR-ABL) fusion oncogene. Our findings emphasize the importance of routinely including BCR-ABL in the diagnostic workup of AML in order to offer to the patients the most appropriate risk category and treatment options.

A rare BCR-ABL1 transcript in Philadelphia-positive acute myeloid leukemia: case report and literature review.

BACKGROUND: Philadelphia (Ph) chromosome results from the reciprocal translocation t(9;22)(q34.1;q11.2) and is diagnostic for chronic myeloid leukemia (CML). However, this translocation is also found in acute lymphoid leukemia (ALL), as well as in rare cases of acute myeloid leukemias (AML). Most patients with CML harbor either the e13a2 or the e14a2 BCR-ABL fusion product, while a small subset of the cases expresses e1a2 or e19a2 transcripts. Moreover, several atypical BCR-ABL1 transcripts, beside the most common e1a2, e13a2 and e14a2, have been described, mainly in patients with CML. However, ALL and de novo AML may also carry BCR-ABL1 atypical transcripts which will confer a poor prognosis. CASE PRESENTATION: A 78-years old male was admitted at our hospital with clinical and laboratory features allowing to make the diagnosis of AML. No evidence of a preceding CML (splenomegaly or basophilia) was found. The karyotype on G-banded metaphases was 46,XY, t(9;22)(q34;q11). While the molecular analysis was ongoing, the patient started treatment based on hydroxyurea followed by 5-aza-2'-deoxycytidine. The molecular biology analysis revealed the simultaneous presence of the common p190 e1a2 and the rare e6a2 isoforms. Because of persistent pancytopenia and presence of blasts, according to the molecular data, he was then switched to tyrosine kinase inhibitors (TKIs) treatment. Nevertheless, after 2 months, the patient was still refractory to second line treatment dying because of a pulmonary infection. CONCLUSION: The atypical p190 e6a2 transcript seems to be associated in AML with aggressive disease. TKI therapy alone does not seem to control the disease. Prompt observations on these patients carrying rare BCR-ABL1 transcripts may help to establish optimal treatment approaches on these aggressive BCR-ABL1 phenotypes in different setting of patients.

Identification and monitoring of atypical PML/RARA fusion transcripts in acute promyelocytic leukemia.

Once the diagnostic suspicion of acute promyelocytic leukemia (APL) has been raised, international guidelines recommend prompt initiation of tailored therapy and supportive care, while awaiting for genetic confirmation of the diagnosis, and the identification of the specific PML/RARA isoform by reverse transcriptase polymerase chain reaction (RT-PCR). Depending on the PML break point, usually located within intron 6, exon 6, or intron 3, different PML/RARA transcript isoforms may be generated, that is, long (bcr1), variant (bcr2), and short (bcr3), respectively. We report here the characterization of three APL cases harboring atypical PML/RARA transcripts, which were not clearly detectable after standard RT-PCR amplification. In all three cases, clinical, morphological, and immunophenotypic features were consistent with APL. Direct sequencing allowed the identification of atypical break points within the PML and RARA genes. Then, we designed a patient-specific quantitative real-time PCR for the atypical transcripts, which allowed for specific quantitative evaluation of minimal residual disease (MRD) during follow-up. Despite the rarity of APL cases with an atypical PML/RARA fusion, our study indicates that an integrated laboratory approach, employing several diagnostic techniques is crucial to timely diagnose APL. This approach allows prompt initiation of specific targeted treatment and reliable MRD monitoring in atypical APL cases.

Molecular remission as a therapeutic objective in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a subtype of acute leukemia characterized by a unique t(15;17) translocation generating the PML/RARA fusion gene and hybrid oncoprotein. Besides its critical role in leukemogenesis, this genetic aberration serves as a disease-specific biomarker for rapid diagnosis and monitoring of minimal residual disease (MRD). Moreover, PML/RARA is specifically targeted by All-trans retinoic acid (ATRA) and arsenic trioxide (ATO), two agents that synergistically act to induce degradation of the oncoprotein. Large clinical studies including two randomized trials conducted in newly diagnosed APL patients have shown that the ATRA-ATO combination is superior to conventional ATRA and chemotherapy both in terms of efficacy and safety. Preliminary studies using oral formulations of arsenic and ATRA suggest that oral arsenic is as effective and manageable as intravenous ATO. Following early retrospective studies indicating the prognostic relevance of PML/RARA monitoring, several prospective studies were conducted in large cohorts of APL patients enrolled in clinical trials with the aim of better assessing the prognostic value of longitudinal PCR testing. The results consistently showed that molecular remission (defined as negativization of the PCR test for PML/RARA) correlates with a significantly decreased risk of relapse, whereas persistence of PCR positivity for PML/RARA after consolidation or conversion from negative to positive during follow-up is strongly associated with hematologic relapse. Based on these data, various groups started using pre-emptive salvage therapy for patients who persisted PCR-positive after frontline consolidation or converted from negative to positive PCR during follow-up. Finally, several expert panels have recommended that molecular remission should be considered a therapeutic objective in APL, and molecular response has been adopted as a study endpoint in modern clinical trials.

Prolonged treatment with arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA) for relapsed acute promyelocytic leukemia previously treated with ATRA and chemotherapy.

Prolonged therapy with all-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) is highly effective in newly diagnosed acute promyelocytic leukemia (APL) but there is limited data on the efficacy of this regimen in the relapse setting. We report here on 22 APL patients treated with prolonged ATRA-ATO therapy at the time of disease relapse. Twenty patients obtained molecular complete remission (CRm) after 2 cycles (90%). Of these, two patients underwent hematopoietic stem cell transplant (HSCT) while the remaining proceeded to receive additional cycles (up to a total of 5) of ATRA-ATO. With a median follow-up of 58 months from the time of relapse (range: 21-128 months), the 4-year OS probability was 0.85 (95% CI 0.61-0.94), DFS was 0.74 (95% CI 0.49-0.88), and EFS 0.68 (95% CI 0.45-0.83). Two patients were resistant to ATRA-ATO salvage and five relapsed at a median of 19 months. Of these, four died due to progressive disease while three relapsed achieved a new CRm after further salvage therapy. This experience confirms the potentially curative effect of prolonged ATRA-ATO therapy in relapsed APL, especially in patients with long first complete remission.

The forkhead box C1 (FOXC1) transcription factor is downregulated in acute promyelocytic leukemia.

Forkhead box (FOX) genes encode transcription factors, which regulate embryogenesis and play an important role in hematopoietic differentiation and in mesenchymal niche maintenance. Overexpression of the family member FOXC1 has been reported in solid tumors and acute myeloid leukemia (AML). We studied FOXC1 expression and function in acute promyelocytic leukemia (APL) and normal hematopoietic progenitors. FOXC1 mRNA and protein levels were significantly lower in primary marrow samples from 27 APL patients, as compared to samples obtained from 27 patients with other AML subtypes, and 5 normal CD34+ hematopoietic cells. FOXC1 expression significantly increased in APL samples at the time of remission following consolidation treatment. In cell lines overexpressing PML-RARA, and in the NB4 t(15;17)-positive cell line, FOXC1 expression was lower than in other non-APL cell lines, and increased following treatment with all-trans retinoic acid (ATRA), due to functional binding of ATRA to the FOXC1 promoter region. Reduced FOXC1 expression was also associated to DNA hypermethylation of the +354 to +568 FOXC1 region, both in primary APL, and in NB4 cells. Treatment of NB4 cells with decitabine demethylated FOXC1 and upregulated its expression. Our findings indicate that FOXC1 is consistently repressed in APL due to hypermethylation and the presence of the PML-RARA rearrangement. A potential role of hypomethylating treatment in advanced APL remains to be established.

Treatment of Low-Blast Count AML using Hypomethylating Agents.

In 2002, the WHO classification reduced the proportion of blasts in the bone marrow (BM) necessary for the diagnosis of acute myeloid leukemia (AML) from 30% to 20%, eliminating the RAEB-t subtype of myelodysplastic syndromes (MDS). However, this AML subtype, defined as low-blast count AML (LBC-AML, with 20-30% BM-blasts) is characterized by peculiar features, as increased frequency in elderly individuals and after cytotoxic treatment for a different primary disease (therapy-related), poor-risk cytogenetics, lower white blood cell counts, and less frequent mutations of NPM1 and FLT3 genes. The clinical course of this entity is often similar to MDS with 10-19% BM-blasts. The hypomethylating agents azacitidine and decitabine have been shown to induce responses and prolong survival both in MDS and LBC-AML. The role of these agents has also been demonstrated in AML with >30% BM-blasts, particularly in patients with poor-risk cytogenetics and in AML with myelodysplasia-related changes. Most recent studies are evaluating strategies to improve outcome, including combinations of hypomethylating agents with immune-response checkpoint inhibitors, which have a role in cancer immune surveillance. Efforts are also ongoing to identify mutations which may predict response and survival in these patients.