Emerging therapies for inv(16) AML.

The core binding factor composed of CBFß and RUNX subunits plays a critical role in most hematopoietic lineages and is deregulated in acute myeloid leukemia (AML). The fusion oncogene CBFß-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells and induces AML. This review focuses on novel insights regarding the molecular mechanisms involved in CBFß-SMMHC-driven leukemogenesis and recent advances in therapeutic approaches to target CBFß-SMMHC in inv(16) AML.

Characteristics and outcome of patients with acute myeloid leukaemia and t(8;16)(p11;p13): results from an International Collaborative Study.

In acute myeloid leukaemia (AML) t(8;16)(p11;p13)/MYST3-CREBBP is a very rare abnormality. Previous small series suggested poor outcome. We report on 59 patients with t(8;16) within an international, collaborative study. Median age was 52 (range: 16-75) years. AML was de novo in 58%, therapy-related (t-AML) in 37% and secondary after myelodysplastic syndrome (s-AML) in 5%. Cytogenetics revealed a complex karyotype in 43%. Besides MYST3-CREBBP, whole-genome sequencing on a subset of 10 patients revealed recurrent mutations in ASXL1, BRD3, FLT3, MLH1, POLG, TP53, SAMD4B (n = 3, each), EYS, KRTAP9-1 SPTBN5 (n = 4, each), RUNX1 and TET2 (n = 2, each). Complete remission after intensive chemotherapy was achieved in 84%. Median follow-up was 5·48 years; five-year survival rate was 17%. Patients with s-/t-AML (P = 0·01) and those with complex karyotype (P = 0·04) had an inferior prognosis. Allogeneic haematopoietic cell transplantation (allo-HCT) was performed in 21 (36%) patients, including 15 in first complete remission (CR1). Allo-HCT in CR1 significantly improved survival (P = 0·04); multivariable analysis revealed that allo-HCT in CR1 was effective in de novo AML but not in patients with s-AML/t-AML and less in patients exhibiting a complex karyotype. In summary, outcomes of patients with t(8;16) are dismal with chemotherapy, and may be substantially improved with allo-HCT performed in CR1.

ATMIN Is a Tumor Suppressor Gene in Lung Adenocarcinoma.

Tumor cells proliferate rapidly and thus are frequently subjected to replication stress and the risk of incomplete duplication of the genome. Fragile sites are replicated late, making them more vulnerable to damage when DNA replication fails to complete. Therefore, genomic alterations at fragile sites are commonly observed in tumors. FRA16D is one of the most common fragile sites in lung cancer, however, the nature of the tumor suppressor genes affected by FRA16D alterations has been controversial. Here, we show that the ATMIN gene, which encodes a cofactor required for activation of ATM kinase by replication stress, is located close to FRA16D and is commonly lost in lung adenocarcinoma. Low ATMIN expression was frequently observed in human lung adenocarcinoma tumors and was associated with reduced patient survival, suggesting that ATMIN functions as a tumor suppressor in lung adenocarcinoma. Heterozygous Atmin deletion significantly increased tumor cell proliferation, tumor burden, and tumor grade in the LSL-KRasG12D; Trp53 F/F (KP) mouse model of lung adenocarcinoma, identifying ATMIN as a haploinsufficient tumor suppressor. ATMIN-deficient KP lung tumor cells showed increased survival in response to replication stress and consequently accumulated DNA damage. Thus, our data identify ATMIN as a key gene affected by genomic deletions at FRA16D in lung adenocarcinoma. SIGNIFICANCE: These findings identify ATMIN as a tumor suppressor in LUAD; fragility at chr16q23 correlates with loss of ATMIN in human LUAD and deletion of Atmin increases tumor burden in a LUAD mouse model.

The impact of cytogenetics on duration of response and overall survival in patients with relapsed multiple myeloma (long-term follow-up results from BSBMT/UKMF Myeloma X Relapse [Intensive]): a randomised, open-label, phase 3 trial.

The Myeloma X trial (ISCRTN60123120) registered patients with relapsed multiple myeloma. Participants were randomised between salvage autologous stem cell transplantation (ASCT) or weekly cyclophosphamide following re-induction therapy. Cytogenetic analysis performed at trial registration defined t(4;14), t(14;16) and del(17p) as high-risk. The effect of cytogenetics on time to progression (TTP) and overall survival was investigated. At 76 months median follow-up, ASCT improved TTP compared to cyclophosphamide (19 months (95% confidence interval [95% CI] 16-26) vs. 11 months (9-12), hazard ratio [HR]: 0·40, 95% CI: 0·29-0·56, P < 0·001), on which the presence of any single high-risk lesion had a detrimental impact [likelihood ratio test (LRT): P = 0·011]. ASCT also improved OS [67 months (95% CI 59-not reached) vs. 55 months (44-67), HR: 0·64, 95% CI: 0·42-0·99, P = 0·0435], with evidence of a detrimental impact with MYC rearrangement (LRT: P = 0·021). Twenty-one (24·7%) cyclophosphamide patients received an ASCT post-trial, median OS was not reached (95% CI: 39-not reached) for these participants compared to 31 months (22-39), in those who did not receive a post-trial ASCT. The analysis further supports the benefit of salvage ASCT, which may still be beneficial after second relapse in surviving patients. There is evidence that this benefit reduces in cytogenetic high-risk patients, highlighting the need for targeted study in this patient group.

The oncofusion protein FUS-ERG targets key hematopoietic regulators and modulates the all-trans retinoic acid signaling pathway in t(16;21) acute myeloid leukemia.

The ETS transcription factor ERG has been implicated as a major regulator of both normal and aberrant hematopoiesis. In acute myeloid leukemias harboring t(16;21), ERG function is deregulated due to a fusion with FUS/TLS resulting in the expression of a FUS-ERG oncofusion protein. How this oncofusion protein deregulates the normal ERG transcription program is unclear. Here, we show that FUS-ERG acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS-ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR:RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-trans retinoic acid treatment of t(16;21) cells as well as FUS-ERG knockdown alleviate the myeloid-differentiation block. Together, the results suggest that FUS-ERG acts as a transcriptional repressor of the retinoic acid signaling pathway.

Modified cIg-FISH protocol for multiple myeloma in routine cytogenetic laboratory practice.

The International Myeloma Working Group recommends that fluorescence in situ hybridization (FISH) be performed on specifically identified plasma cells (PC). This is because chromosomal abnormalities are not frequently detected by traditional karyotyping due to the low proliferative rate of PC in multiple myeloma (MM). Conventional FISH enhances the sensitivity but lacks the specificity, as it does not distinguish PC from other hematopoetic cells. To fulfill this recommendation, PC need to be selected either by flow cytometry or immunomagnetic bead-based PC sorting or by concomitant labeling of the cytoplasmic immunoglobulin light chain, which allows for unambiguous identification. These techniques require expertise, time, and funding and are not easily incorporated into the routine workflow of the cytogenetic laboratory. We have modified and refined the technique using fixed cell pellets to achieve nicely separated and easily identifiable PC. With immunostaining and subsequent FISH (i.e., cytoplasmic immunoglobulin FISH, cIg-FISH), this technique can be easily incorporated into every cytogenetic laboratory. Twenty samples from patients with MM were subjected to routine FISH, cIg-FISH, and chromosomal karyotyping and the results were compared. Three FISH probes, which enabled detection of the t(4;14), t(14;16) and deletion of TP53, were used to validate this modified technique successfully.

KIT D816 mutation associates with adverse outcomes in core binding factor acute myeloid leukemia, especially in the subgroup with RUNX1/RUNX1T1 rearrangement.

Core binding factor (CBF)-positive acute myeloid leukemia (AML) presents a favorable prognosis, except for patients with KIT mutation, especially D816 mutation. The current retrospective study attempted to validate a prognostic role of KIT mutation in 121 Korean patients with CBF AML. The study patients consisted of 121 patients with CBF AML (82 patients with RUNX1/RUNX1T1 [67.8 %] and 39 patients with CBFB/MYH11 [32.2 %]) recruited from eight institutions in Korea. All patients received idarubicin plus cytarabine or behenoyl cytosine arabinoside 3 + 7 induction chemotherapy. The KIT gene mutation status was determined by direct sequencing analyses. A KIT mutation was detected in 32 cases (26.4 %) in our series of patients. The KIT mutation was most frequent in exon 17 (n = 18, 14.9 %; n = 16 with D816 mutation), followed by exon 8 (n = 10, 8.3 %). The presence of KIT D816 mutation was associated with adverse outcomes for the event-free survival (p = 0.03) and for the overall survival (p = 0.02). The unfavorable impact of D816 mutation was more prominent when the analysis was confined to the RUNX1/RUNX1T1 subtype. The KIT mutation was detected in 26.4 % of Korean patients with CBF AML. The KIT D816 mutation demonstrated an unfavorable prognostic implication, particularly in the RUNX1/RUNX1T1 subtype.

Secondary genetic lesions in acute myeloid leukemia with inv(16) or t(16;16): a study of the German-Austrian AML Study Group (AMLSG).

In this study, we evaluated the impact of secondary genetic lesions in acute myeloid leukemia (AML) with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11. We studied 176 patients, all enrolled on prospective treatment trials, for secondary chromosomal aberrations and mutations in N-/KRAS, KIT, FLT3, and JAK2 (V617F) genes. Most frequent chromosomal aberrations were trisomy 22 (18%) and trisomy 8 (16%). Overall, 84% of patients harbored at least 1 gene mutation, with RAS being affected in 53% (45% NRAS; 13% KRAS) of the cases, followed by KIT (37%) and FLT3 (17%; FLT3-TKD [14%], FLT3-ITD [5%]). None of the secondary genetic lesions influenced achievement of complete remission. In multivariable analyses, KIT mutation (hazard ratio [HR] = 1.67; P = .04], log(10)(WBC) (HR = 1.33; P = .02), and trisomy 22 (HR = 0.54; P = .08) were relevant factors for relapse-free survival; for overall survival, FLT3 mutation (HR = 2.56; P = .006), trisomy 22 (HR = 0.45; P = .07), trisomy 8 (HR = 2.26; P = .02), age (difference of 10 years, HR = 1.46; P = .01), and therapy-related AML (HR = 2.13; P = .14) revealed as prognostic factors. The adverse effects of KIT and FLT3 mutations were mainly attributed to exon 8 and tyrosine kinase domain mutations, respectively. Our large study emphasizes the impact of both secondary chromosomal aberrations as well as gene mutations for outcome in AML with inv(16)/t (16;16).

p210 BCR/ABL1 as a secondary change in a patient with acute myelomonocytic leukemia (M4Eo) with inv(16).

t(9;22) as a secondary change of inv(16) is a rare chromosome aberration in de novo acute myeloid leukemia (AML). Here, we report the case of a 31-year-old man with this rare abnormality. Karyotypic analysis showed a complex chromosome aberration:46,XY,der(8)t(8;10)(p23;q25),der(10)t(8;10)t(10;16)(p13;q22),der(16)inv(16)(p13q22)t(10;16)[4] and 46,XY,idem,t(9;22)(q34;q11)[6]. Fluorescence in situ hybridization detected both the CBFB and the BCR/ABL1 rearrangements. CBFB/MYH11 (A type) and BCR/ABL1 (b3a2) fusion transcripts were both detected by real-time quantitative RT-PCR. The patient was treated with standard AML chemotherapy and autologous peripheral blood stem cell transplantation. He also received imatinib (400 mg/day) during the chemotherapy intervals and after transplantation. Molecular remission was achieved at the beginning of the third chemotherapy and he remained in remission until the last follow-up (22 months after diagnosis). To our knowledge, this is the first reported case of de novo AML in which has p210(BCR/ABL1) occurred as a secondary change of inv(16).

Characteristics of translocation (16;16)(p13;q22) acute myeloid leukemia.

A subgroup of patients with core binding factor acute myeloid leukemias (AML) is characterized by the presence of the fusion gene CBFb-Myh11. At the cytogenetic level, most of these patients are identified by the presence of an inversion of chromosome 16 [inv(16)(p13q22)] and rarely by a translocation t(16;16)(p13;q22). The aim of this study is to describe the natural history of patients with t(16;16) [N = 6] treated at MD Anderson Cancer Center and compared them with a cohort of patients with inv(16)(p13q22) [n = 61]. In patients with t(16;16) the complete remission rate (CR) was 100% when treated with a combination of fludarabine and high-dose cytarabine. Median overall survival (OS) had not been achieved. There was no difference in response or OS or progression free survival between both groups. Presence of additional chromosomal abnormalities and molecular aberrations had no effect on prognosis. In conclusion, and consistent with previous reports, the natural history of patients with t(16:16)(p13;q22) is similar to that of classic patients with inv16 AML and therefore should be treated similarly.

Granulocytic sarcoma of abdomen in acute myeloid leukemia patient with inv(16) and t(6;17) abnormal chromosome: case report and review of literature.

Granulocytic sarcoma (GS) is composed of immature granulocytic precursors and is usually found in acute myeloid leukemia (AML) patients with t(8;21). Inv(16) is rarely associated with GS comparing with t(8;21) leukemia. Here we describe an abdominal GS patient in AML-M2 with acquired translocation between chromosomes 6 and 17 and inv (16). We have also summarized 20 reported GS cases with inv(16) and found that chloroma was most often found in abdominal lesions. Intestine maybe a tissue specific target for the expression of inv(16) leukemia. Complete physical examination and molecular diagnosis are necessary for AML patients to benefit from the diagnosis and therapeutic strategy.

De novo balanced translocation t (7;16) (p22.1; p11.2) associated with autistic disorder.

The high incidence of de novo chromosomal aberrations in a population of persons with autism suggests a causal relationship between certain chromosomal aberrations and the occurrence of isolated idiopathic autism. We report on the clinical and cytogenetic findings in a male patient with autism, no physical abnormalities and a de novo balanced (7;16)(p22.1;p16.2) translocation. G-banded chromosomes and fluorescent in situ hybridization (FISH) were used to examine the patient's karyotype as well as his parents'. FISH with specific RP11-BAC clones mapping near 7p22.1 and 16p11.2 was used to refine the location of the breakpoints. This is, in the best of our knowledge, the first report of an individual with autism and this specific chromosomal aberration.

Nuclear organisation in totipotent human nuclei and its relationship to chromosomal abnormality.

Studies of nuclear organisation, most commonly determining the nuclear location of chromosome territories and individual loci, have furthered our understanding of nuclear function, differentiation and disease. In this study, by examining eight loci on different chromosomes, we tested hypotheses that: (1) totipotent human blastomeres adopt a nuclear organisation akin to that of committed cells; (2) nuclear organisation is different in chromosomally abnormal blastomeres; and (3) human blastomeres adopt a ;chromocentre' pattern. Analysis of in vitro fertilisation (IVF) conceptuses permits valuable insight into the cell biology of totipotent human nuclei. Here, extrapolations from images of preimplantation genetic screening (PGS) cases were used to make comparisons between totipotent blastomeres and several committed cells, showing some differences and similarities. Comparisons between chromosomally abnormal nuclei and those with no detected abnormality (NDA) suggest that the former display a significant non-random pattern for all autosomal loci, but there is a less distinct, possibly random, pattern in 'NDA' nuclei. No evidence was found that the presence of an extra chromosome is accompanied by an altered nuclear location for that chromosome. Centromeric loci on chromosomes 15 and 16 normally seen at the nuclear periphery were mostly centrally located in aneuploid cells, providing some evidence of a 'chromocentre'; however, the chromosome-18 centromere was more peripheral, similar to committed cells. Our results provide clues to the nature of totipotency in human cells and might have future applications for preimplantation diagnosis and nuclear transfer.

Translocation-positive acute myeloid leukemia associated with valproic acid therapy.

Valproic acid is an effective anti-epileptic medication often used for long-term control of seizure disorders that has been implicated in hematological toxicities, including rare reports of myelodysplasia and acute leukemia. Here, we report a case of valproic acid-related leukemia-like syndrome with a t(8;16) chromosomal translocation. After discontinuing valproic acid, the hematological findings completely resolved.

A distinct epigenetic signature at targets of a leukemia protein.

BACKGROUND: Human myelogenous leukemia characterized by either the non random t(8; 21)(q22; q22) or t(16; 21)(q24; q22) chromosome translocations differ for both their biological and clinical features. Some of these features could be consequent to differential epigenetic transcriptional deregulation at AML1 targets imposed by AML1-MTG8 and AML1-MTG16, the fusion proteins deriving from the two translocations. Preliminary findings showing that these fusion proteins lead to transcriptional downregulation of AML1 targets, marked by repressive chromatin changes, would support this hypothesis. Here we show that combining conventional global gene expression arrays with the power of bioinformatic genomic survey of AML1-consensus sequences is an effective strategy to identify AML1 targets whose transcription is epigenetically downregulated by the leukemia-associated AML1-MTG16 protein. RESULTS: We interrogated mouse gene expression microarrays with probes generated either from 32D cells infected with a retroviral vector carrying AML1-MTG16 and unable of granulocyte differentiation and proliferation in response to the granulocyte colony stimulating factor (G-CSF), or from 32D cells infected with the cognate empty vector. From the analysis of differential gene expression alone (using as criteria a p value < 0.01 and an absolute fold change > 3), we were unable to conclude which of the 37 genes downregulated by AML1-MTG16 were, or not, direct AML1 targets. However, when we applied a bioinformatic approach to search for AML1-consensus sequences in the 10 Kb around the gene transcription start sites, we closed on 17 potential direct AML1 targets. By focusing on the most significantly downregulated genes, we found that both the AML1-consensus and the transcription start site chromatin regions were significantly marked by aberrant repressive histone tail changes. Further, the promoter of one of these genes, containing a CpG island, was aberrantly methylated. CONCLUSION: This study shows that a leukemia-associated fusion protein can impose a distinct epigenetic repressive signature at specific sites in the genome. These findings strengthen the conclusion that leukemia-specific oncoproteins can induce non-random epigenetic changes.

Prognostic significance of WT1 gene expression in pediatric acute myeloid leukemia.

BACKGROUND: The Wilms Tumor gene (WT1) encodes a transcription factor involved in kidney development and malignancy. WT1 expression in a subpopulation of early CD34+ cells has suggested its involvement in hematopoiesis. WT1 is aberrantly expressed in leukemias. High expression of WT1 at diagnosis has been associated with unfavorable prognosis in adult acute myeloid leukemia (AML). The prognostic relevance of WT1 expression in pediatric AML was evaluated in only one study, including 47 patients, which showed that very low levels of WT1 at presentation were associated with an excellent outcome. To test the validity of these findings we measured levels of WT1 in 41 newly diagnosed pediatric AML of the non-M3 FAB subtype. PROCEDURE: Patients were treated according to an AML-BFM 83-based protocol in a single institution. Mononucleated cells obtained from presentation BM aspirates were cryopreserved and later thawed and used for total RNA extraction and cDNA synthesis. The quantitative assessment of WT1 transcripts was made by real-time PCR (RQ-PCR). WT1 transcripts values were normalized with respect to the number of ABL transcripts. RESULTS: WT1 levels were significantly higher in patients bearing favorable chromosome abnormalities, t(8;21) and inv(16) (P = 0.002). Higher levels of WT1 expression were unexpectedly associated with a higher probability of overall survival by Cox regression analysis (P = 0.002). Multivariate regression analysis could not discriminate between the effects of WT1 and cytogenetics on survival. CONCLUSIONS: Higher WT1 expression was associated with favorable cytogenetics subtypes and accordingly with better outcome in children with AML in this study.

Myelodysplastic syndrome with an unbalanced 1;16 translocation in a case of early age of onset.

We present a 30-year-old male patient diagnosed with myelodysplastic syndrome (MDS). Chromosome analysis of the bone marrow aspirate revealed a derivative chromosome resulting from an unbalanced 1;16 translocation causing the gain of 1q and loss of 16q. Dual color fluorescence in situ hybridization demonstrated that the 1q;16p derivative chromosome contained centromeric material derived from chromosome 1, and accordingly the translocation was designated as a der (1)t(1;16)(p11;p11.1). Review of literature revealed that 1;16 translocation in MDS is nonrandom and is always described as unbalanced, resulting in trisomy 1q and monosomy 16q.