Assessing acute myeloid leukemia susceptibility in rearrangement-driven patients by DNA breakage at topoisomerase II and CCCTC-binding factor/cohesin binding sites.

An initiating DNA double strand break (DSB) event precedes the formation of cancer-driven chromosomal abnormalities, such as gene rearrangements. Therefore, measuring DNA breaks at rearrangement-participating regions can provide a unique tool to identify and characterize susceptible individuals. Here, we developed a highly sensitive and low-input DNA break mapping method, the first of its kind for patient samples. We then measured genome-wide DNA breakage in normal cells of acute myeloid leukemia (AML) patients with KMT2A (previously MLL) rearrangements, compared to that of nonfusion AML individuals, as a means to evaluate individual susceptibility to gene rearrangements. DNA breakage at the KMT2A gene region was significantly greater in fusion-driven remission individuals, as compared to nonfusion individuals. Moreover, we identified select topoisomerase II (TOP2)-sensitive and CCCTC-binding factor (CTCF)/cohesin-binding sites with preferential DNA breakage in fusion-driven patients. Importantly, measuring DSBs at these sites, in addition to the KMT2A gene region, provided greater predictive power when assessing individual break susceptibility. We also demonstrated that low-dose etoposide exposure further elevated DNA breakage at these regions in fusion-driven AML patients, but not in nonfusion patients, indicating that these sites are preferentially sensitive to TOP2 activity in fusion-driven AML patients. These results support that mapping of DSBs in patients enables discovery of novel break-prone regions and monitoring of individuals susceptible to chromosomal abnormalities, and thus cancer. This will build the foundation for early detection of cancer-susceptible individuals, as well as those preferentially susceptible to therapy-related malignancies caused by treatment with TOP2 poisons.

Prognostic value of minimal residual disease measured by fusion-gene transcript in infants with KMT2A-rearranged acute lymphoblastic leukaemia treated according to the MLL-Baby protocol.

The prognostic value of minimal residual disease (MRD) measured by fusion-gene transcript (FGT) detection was investigated in 76 infants (aged ≤1 year) with acute lymphoblastic leukaemia (ALL) with lysine methyltransferase 2A (KMT2A) rearrangements. Either at the end of induction or at later time-points, FGT-MRD-positivity was associated with poor outcome. FGT-MRD-positivity after first consolidation or first high-risk block detected 46·5% of infants with extremely poor outcome [disease-free survival (SE) 0·06 (0·06), cumulative incidence of relapse (SE) 0·91 (0·05)], which was also confirmed in multivariable analysis. Thus, FGT-MRD measurement at a single time-point clearly identifies infants with ALL who are curable with conventional chemotherapy and those who would benefit only from other treatment approaches.

The early achievement of measurable residual disease negativity in the treatment of adults with Philadelphia-negative B-cell acute lymphoblastic leukemia is a strong predictor for survival.

The minimal or measurable residual disease (MRD) status following induction/consolidation chemotherapy is an important prognostic endpoint in adult patients with newly diagnosed acute lymphoblastic leukemia (ALL). However, the optimal time-point (TP) of MRD assessment and its impact on outcome remains unclear. We analyzed 215 patients with newly diagnosed Philadelphia negative B-cell ALL who received intensive chemotherapy, and had available MRD assessment by multicolor flow cytometry at two separate TPs. The median time to first TP (1TP) and second TP (2TP) were 24 and 110 days, respectively. At 1TP, 148 patients (68%) were MRD negative and 67 (32%) were positive. Of the 148 patients with negative MRD at 1TP, 147 (99%) maintained it through 2TP. Patients who were MRD negative at both TPs, early MRD responders, had the 3-year event-free survival (EFS), and overall survival (OS) rates of 65% and 76%, respectively. Patients with improved MRD status from positive to negative, late MRD responders, had lower 3-year EFS and OS rates, 42% and 58%, respectively (P = .001). Multivariate analysis showed that KMT2A (MLL) rearrangement and MRD positivity at 1TP were the only factors correlated with worse OS. In conclusion, the earlier achievement of MRD negative remission is a stronger prognostic factor for survival.

A monocentric retrospective study of 138 therapy-related myeloid neoplasms.

As diagnosing therapy-related myeloid neoplasms (t-MN) is often challenging, we reviewed clinicopathological features of t-MN patients. Medical records of 138 patients, diagnosed with t-MN between 1995 and 2017, were reviewed. Of 138 patients, 80 had t-MDS, 53 t-AML, and 5 t-MDS/MPN (age, 22-88 years; median 64 years; male/female ratio, 0.8). The median latency time was 6 years and 5 months. Of 115 patients, 56 patients received cytotoxic-/radiotherapy for a solid tumor, 56 for hematological malignancy, and 3 for an auto-immune disorder, respectively. Another 21 patients had a combination of 2 disorders. Moreover, 2 patients had 3 previous malignancies. Breast cancer was the most prevalent tumor, followed by low-grade B non-Hodgkin lymphoma. Immunophenotyping and immunohistochemistry showed aberrant expression of B-, T-, or NK-cell markers in 21% and 6%, respectively. In 90% of the patients, dysplasia in ≥ 1 lineage was found. KMT2A fusion gene transcripts were seen in 5%. Cytogenetic analysis showed complex karyotypes (31%) and chromosome 5 and/or 7 abnormalities (40%). Almost 82% of the patients died and the median overall survival was about 1 year. Our study confirms that previous therapy for breast cancer is the most important cause of t-MN. KMT2A fusion genes are prevalent and complex karyotypes and/or chromosomes 5 and/or 7 abnormalities are common.

Mixed-Phenotype Acute Leukemia: Diagnostic Criteria and Pitfalls.

Mixed-phenotype acute leukemia (MPAL) is a heterogeneous category in the World Health Organization classification that comprises acute leukemias with discrete admixed populations of myeloid and lymphoid blasts ("bilineal") or with extensive coexpression of lymphoid and myeloid markers in a single blast population ("biphenotypic"). Flow cytometric findings suggestive of MPAL are often met with consternation by pathologists and oncologists alike, owing to unfamiliarity with the disease and uncertainty about how MPAL fits into established paradigms for treatment of acute leukemia. The purpose of this review is to explain the diagnostic criteria for MPAL, summarize its biological and clinical features, and address common diagnostic pitfalls of these unusual leukemias.

Reemergence of translocation t(11;19)(q23;p13.1) in the absence of clinically overt leukemia.

We report the case of a 10-year-old female with acute myeloid leukemia (AML) FAB M0 carrying a novel t(11;19)(q23;p13.1) MLL-ELL variant, in which intron 8 of MLL is fused to exon 6 of ELL. Complete remission, judged by morphology and cytogenetic analysis, was achieved after the conventional chemotherapy. Eight months after completion of therapy, the level of WT-1 in peripheral blood and the number of cells with the MLL-ELL fusion transcript resurged. However, the patient remained overtly healthy and the morphology in the bone-marrow smear was innocuous, with no sign of relapse or secondary leukemia. Without any evidence of relapse, the patient has been closely observed without any therapeutic intervention. For approximately 2 years after the completion of therapy, despite clonal proliferation of pre-leukemic cells with an MLL-ELL fusion gene, she has maintained complete remission. In this case, the rare variant form of MLL-ELL fusion that has been identified may be related to diminished leukemogenic capacity, resulting in the persistence of pre-leukemic status; an additional genetic abnormality may thus be necessary for full transformation of pre-leukemic cells.

Incidence of preleukemic fusion genes in healthy subjects.

The diagnostics of leukemia relies upon multi-parametric approach involving a number of different pathology disciplines such as flow cytometry, histopathology, cytogenetics and molecular genetics [fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR)]. Childhood leukemia is often determined by the presence of specific chromosomal translocation that entails the generation of preleukemic fusion genes (PFG). In the last two decades, several studies have reported observations that PFG are present in healthy population and not necessarily result in leukemia. The first such study by Limpens and colleagues on t(14/18)/ BCL2-JH [1] and next in line [2, 3] led to many questions regarding the significance of these chromosomal translocations in leukemogenesis. However, the data on the incidence of PFG are contradictive. This review aims to highlight the molecular genetic approaches used by various studies with regard to differences in diagnostics and incidence of PFG in healthy subjects. The focus is on the incidence and prevalence of the most common PFG such as TEL-AML1, MLL-AF4, BCR-ABL (p190), AML1-ETO, PML-RARA, and CBFB-MYH11 detected in umbilical cord blood, in neonatal blood spots (Guthrie cards (GC)), bone marrow, peripheral blood and tissues of amortized fetuses. We conclude that the incidence of PFG is significantly higher than incidence of leukemia and more sophisticated analysis of PFG in leukemogenic cell populations is warranted to relate the occurrence of PFG with leukemia. The emerging notion is that only those PFG may contribute to development of leukemia which arise in stem cells at specific time windows during development. Thus, screening of PFG in subpopulations of stem cells may be a challenge for assessment of predisposition to leukemia and for validation of cell transplant to minimize donor cell-derived leukemia.

Higher blood MLL1 mRNA and BDNF promoter IV on histone H3K4me3 levels in patients with schizophrenia.

We investigated the blood levels of mixed-lineage leukemia 1 (MLL1) mRNA and BDNF (brain derived neurotrophic factor) exon IV promoter on histone Histone 3 lysine 4 trimethylation (H3K4me3) in peripheral blood of patients with schizophrenia and controls. Over one year, 36 patients with schizophrenia and 32 controls were recruited. Psychiatric diagnoses were made based on DSM-IV criteria. Higher blood MLL1 mRNA and BDNF exon IV promoter on H3K4me3 levels were noted in patients with schizophrenia than in controls. The results showed that blood MLL1 mRNA and BDNF exon IV on H3K4me3 levels might be involved in the psychopathology of schizophrenia.

Clearance of minimal residual disease after allogeneic stem cell transplantation and the prediction of the clinical outcome of adult patients with high-risk acute lymphoblastic leukemia.

BACKGROUND AND OBJECTIVES: The molecular analysis of minimal residual disease (MRD) may provide information on the risk of recurrence in patients with acute lymphoblastic leukemia (ALL). The aim of this study was to correlate the kinetics of MRD clearance after allogeneic transplantation with the clinical outcome of adults with ALL. DESIGN AND METHODS: MRD was evaluated by real-time quantitative polymerase chain reaction (RQ-PCR) using probes derived from fusion chimeric genes (BCR/ABL and MLL/AF4) (n=22) or rearrangements of the T-cell receptor or immunoglobulin genes (n=21). Forty-three adult patients with ALL were studied to correlate the kinetics of MRD clearance before and after allogeneic hematopoietic stem cell transplantation. RESULTS: At 36 months, the overall survival of patients who underwent transplantation in hematologic remission (n= 37) was 80% for those who were PCR-negative before transplantation (n= 12) compared to 49% for PCR-positive patients (n= 25)(p=0.17). For the same patients the cumulative incidence of relapse was 0% and 46%, respectively (p=0.027). Moreover, the relapse rate of patients who were PCR-negative at day +100 after transplantation was remarkably low (7%) compared to that among patients who were PCR-positive (80%, p=0.0006). INTERPRETATION AND CONCLUSIONS: The kinetics of MRD clearance may help to identify patients at high risk of leukemia relapse after allogeneic stem cell transplantation. Patients not achieving an early molecular remission after transplantation require prompt and appropriate pre-emptive treatments such as infusions of donor lymphocytes or new experimental drugs.

Prenatal origin of childhood AML occurs less frequently than in childhood ALL.

BACKGROUND: While there is enough convincing evidence in childhood acute lymphoblastic leukemia (ALL), the data on the pre-natal origin in childhood acute myeloid leukemia (AML) are less comprehensive. Our study aimed to screen Guthrie cards (neonatal blood spots) of non-infant childhood AML and ALL patients for the presence of their respective leukemic markers. METHODS: We analysed Guthrie cards of 12 ALL patients aged 2-6 years using immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements (n = 15) and/or intronic breakpoints of TEL/AML1 fusion gene (n = 3). In AML patients (n = 13, age 1-14 years) PML/RARalpha (n = 4), CBFbeta/MYH11 (n = 3), AML1/ETO (n = 2), MLL/AF6 (n = 1), MLL/AF9 (n = 1) and MLL/AF10 (n = 1) fusion genes and/or internal tandem duplication of FLT3 gene (FLT3/ITD) (n = 2) were used as clonotypic markers. Assay sensitivity determined using serial dilutions of patient DNA into the DNA of a healthy donor allowed us to detect the pre-leukemic clone in Guthrie card providing 1-3 positive cells were present in the neonatal blood spot. RESULTS: In 3 patients with ALL (25%) we reproducibly detected their leukemic markers (Ig/TCR n = 2; TEL/AML1 n = 1) in the Guthrie card. We did not find patient-specific molecular markers in any patient with AML. CONCLUSION: In the largest cohort examined so far we used identical approach for the backtracking of non-infant childhood ALL and AML. Our data suggest that either the prenatal origin of AML is less frequent or the load of pre-leukemic cells is significantly lower at birth in AML compared to ALL cases.

The prognostic value of MLL-AF9 detection in patients with t(9;11)(p22;q23)-positive acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Translocation (9;11) is the most common t(11q23) in acute myeloid leukemia (AML). A considerable number of patients with this cytogenetic abnormality relapse and die of their disease. We evaluated the clinical significance of minimal residual disease (MRD) monitoring in t(9;11)(p22;q23)-positive AML patients using real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR) analysis. DESIGN AND METHODS: We identified 34 newly diagnosed patients with t(9;11)(p22;q23)-positive AML treated within three multicenter trials of the AML Study Group. MRD could be investigated by RQ-PCR in 19 patients during and after therapy. Because of the relatively low sensitivity of the RQ-PCR (10(-3) to 10(-4) at the cellular level), samples from RQ-PCR-negative patients were also analyzed by nested polymerase chain reaction (nPCR; sensitivity 10-4 to 10-5 at the cellular level). RESULTS: RQ-PCR monitoring revealed two groups of patients: group 1 (n=11) had negative RQ-PCR in all samples collected in hematologic complete remission whereas group 2 (n=8) had at least one positive RQ-PCR in samples collected in complete remission during therapy. Group 1 had a significantly lower cumulative incidence of relapse (p=0.004) and better overall survival (p=0.003) compared to group 2. nPCR did not add information to that gained from RQ-PCR. Molecular relapse was detected in two patients by RQ-PCR four and six weeks, respectively before hematologic relapse occurred. Quantitative MLL-AF9 levels at diagnosis or during and after therapy had no prognostic impact. INTERPRETATION AND CONCLUSIONS: Early achievement of sustained RQ-PCR negativity appears to be a prerequisite for long-term hematologic complete remission in t(9;11)-positive AML. Furthermore, RQ-PCR might be useful for early detection of relapse. Additional patients need to be studied to corroborate these findings.