Effect of additional cytogenetic abnormalities on survival in arsenic trioxide-treated acute promyelocytic leukemia.

Frontline arsenic trioxide (ATO)-based treatment regimens achieve high rates of long-term relapse-free survival in treating acute promyelocytic leukemia (APL) and form the current standard of care. Refining prognostic estimates for newly diagnosed patients treated with ATO-containing regimens remains important in continuing to improve outcomes and identify patients who achieve suboptimal outcomes. We performed a pooled analysis of exclusively ATO-treated patients at a single academic institution and from the ALLG APML4 and Alliance C9710 studies to determine the prognostic importance of additional cytogenetic abnormalities and/or complex karyotype. We demonstrated inferior event-free survival for patients harboring complex karyotype (hazard ratio [HR], 3.74; 95% confidence interval [CI], 1.63-8.56; P = .002), but not for patients harboring additional cytogenetic abnormalities (HR, 2.13; 95% CI, 0.78-5.82; P = .142). These data support a role for full karyotypic analysis of all patients with APL and indicate a need for novel treatment strategies to overcome the adverse effect of APL harboring complex karyotype.

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability.

BACKGROUND: The U937 cell line is widely employed as a research tool. It has a complex karyotype. A PICALM-MLLT10 fusion gene formed by the recurrent t(10;11) translocation is present, and the myeloid common deleted region at 20q12 has been lost from its near-triploid karyotype. We carried out a detailed investigation of U937 genome reorganisation including the chromosome 20 rearrangements and other complex rearrangements. RESULTS: SNP array, G-banding and Multicolour FISH identified chromosome segments resulting from unbalanced and balanced rearrangements. The organisation of the abnormal chromosomes containing these segments was then reconstructed with the strategic use of targeted metaphase FISH. This provided more accurate karyotype information for the evolving karyotype. Rearrangements involving the homologues of a chromosome pair could be differentiated in most instances. Centromere capture was demonstrated in an abnormal chromosome containing parts of chromosomes 16 and 20 which were stabilised by joining to a short section of chromosome containing an 11 centromere. This adds to the growing number of examples of centromere capture, which to date have a high incidence in complex karyotypes where the centromeres of the rearranged chromosomes are identified. There were two normal copies of one chromosome 20 homologue, and complex rearrangement of the other homologue including loss of the 20q12 common deleted region. This confirmed the previously reported loss of heterozygosity of this region in U937, and defined the rearrangements giving rise to this loss. CONCLUSIONS: Centromere capture, stabilising chromosomes pieced together from multiple segments, may be a common feature of complex karyotypes. However, it has only recently been recognised, as this requires deliberate identification of the centromeres of abnormal chromosomes. The approach presented here is invaluable for studying complex reorganised genomes such as those produced by chromothripsis, and provides a more complete picture than can be obtained by microarray, karyotyping or FISH studies alone. One major advantage of SNP arrays for this process is that the two homologues can usually be distinguished when there is more than one rearrangement of a chromosome pair. Tracking the fate of each homologue and of highly repetitive DNA regions such as centromeres helps build a picture of genome evolution. Centromere- and telomere-containing elements are important to deducing chromosome structure. This study confirms and highlights ongoing evolution in cultured cell lines.

Hemopoietic Cell Kinase amplification with Protein Tyrosine Phosphatase Receptor T depletion leads to polycythemia, aberrant marrow erythoid maturation, and splenomegaly.

Deletion of long arm of chromosome 20 [del(20q)] is the second most frequent recurrent chromosomal abnormality in hematological malignancies. It is detected in 10% of myeloproliferative neoplasms, 4-5% of myelodysplastic syndromes, and 1-2% of acute myeloid leukaemia. Recurrent, non-random occurrence of del(20q) indicates that it is a pathogenic driver in myeloid malignancies. Genetic mapping of patient samples has identified two regions of interest on 20q - the "Common Deleted Region" (CDR) and "Common Retained Region" (CRR), which was often amplified. We proposed that the CDR contained tumor suppressor gene(s) (TSG) and the CRR harbored oncogene(s); loss of a TSG together with over-expression of an oncogene favored development of myeloid malignancies. Protein Tyrosine Phosphatase Receptor T (PTPRT) and Hemopoietic cell kinase (HCK) were identified to be the likely candidate TSG and oncogene respectively. Retroviral transduction of HCK into PTPRT-null murine LKS+ stem and progenitor cells resulted in hyperproliferation in colony forming assays and hyperphosphorylation of intracellular STAT3. Furthermore, over half of the murine recipients of these transduced cells developed erythroid hyperplasia, polycythemia and splenomegaly at 12 months, although no leukemic phenotype was observed. The findings suggested that HCK amplification coupled with PTPRT loss in del(20q) leads to development of a myeloproliferative phenotype.

The Dicentric Chromosome dic(20;22) Is a Recurrent Abnormality in Myelodysplastic Syndromes and Is a Product of Telomere Fusion.

We describe a recurrent dicentric chromosome formed by telomere fusion between chromosome 20 and chromosome 22 in 4 cases of myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML). In particular, the presence of residual telomere sequences at the site of translocation in 3 of the 4 cases makes a compelling case for telomere fusion. This is the first description of a recurrent telomere fusion event in any malignant condition. The 20q subtelomeric region was retained in all 4 examples despite deletion of the 20q12 region closer to the centromere. The original dicentric chromosome in all 4 cases contained nucleolus organiser region material from the short arm of chromosome 22 and had also undergone secondary rearrangements that produced amplification of the common gained region on 20q. We propose that the sequence of events producing this chromosome abnormality is: degradation of the telomeres, formation of an unstable dicentric chromosome by 20q and 22p telomere fusion, breakage-fusion-bridge cycles causing copy number aberration between the centromeres, selection of cells with 20q12 deletion, and further selection of cells with 20q11.2 gain. The last 2 steps are driver events responsible for the abnormal chromosomes found in the malignant cells. Finding recurrent patterns in the complex genome reorganisation events that characterise poor-prognosis, complex-karyotype AML and MDS will help us understand the mechanisms and oncogenic driver mutations in these poorly understood malignancies.

Use of arsenic trioxide in remission induction and consolidation therapy for acute promyelocytic leukaemia in the Australasian Leukaemia and Lymphoma Group (ALLG) APML4 study: a non-randomised phase 2 trial.

BACKGROUND: Initial treatment of acute promyelocytic leukaemia traditionally involves tretinoin (all-trans retinoic acid) combined with anthracycline-based risk-adapted chemotherapy, with arsenic trioxide being the treatment of choice at relapse. To try to reduce the relapse rate, we combined arsenic trioxide with tretinoin and idarubicin in induction therapy, and used arsenic trioxide with tretinoin as consolidation therapy. METHODS: Patients with previously untreated genetically confirmed acute promyelocytic leukaemia were eligible for this study. Eligibilty also required Eastern Cooperative Oncology Group performance status 0-3, age older than 1 year, normal left ventricular ejection fraction, Q-Tc interval less than 500 ms, absence of serious comorbidity, and written informed consent. Patients with genetic variants of acute promyelocytic leukaemia (fusion of genes other than PML with RARA) were ineligible. Induction comprised 45 mg/m(2) oral tretinoin in four divided doses daily on days 1-36, 6-12 mg/m(2) intravenous idarubicin on days 2, 4, 6, and 8, adjusted for age, and 0·15 mg/kg intravenous arsenic trioxide once daily on days 9-36. Supportive therapy included blood products for protocol-specified haemostatic targets, and 1 mg/kg prednisone daily as prophylaxis against differentiation syndrome. Two consolidation cycles with tretinoin and arsenic trioxide were followed by maintenance therapy with oral tretinoin, 6-mercaptopurine, and methotrexate for 2 years. The primary endpoints of the study were freedom from relapse and early death (within 36 days of treatment start) and we assessed improvement compared with the 2 year interim results. To assess durability of remission we compared the primary endpoints and disease-free and overall survival at 5 years in APML4 with the 2 year interim APML4 data and the APML3 treatment protocol that excluded arsenic trioxide. This study is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12605000070639. FINDINGS: 124 patients were enrolled between Nov 10, 2004, and Sept 23, 2009, with data cutoff of March 15, 2012. Four (3%) patients died early. After a median follow-up of 4·2 years (IQR, 3·2-5·2), the 5 year freedom from relapse was 95% (95% CI 89-98), disease-free survival was 95% (89-98), event-free survival was 90% (83-94), and overall survival was 94% (89-97). The comparison with APML3 data showed that hazard ratios were 0·23 (95% CI 0·08-0·64, p=0·002) for freedom from relapse, 0·21 (0·07-0·59, p=0·001) for disease-free survival, 0·34 (0·16-0·69, p=0·002) for event-free survival, and 0·35 (0·14-0·91, p=0·02) for overall survival. INTERPRETATION: Incorporation of arsenic trioxide in initial therapy induction and consolidation for acute promyelocytic leukaemia reduced the risk of relapse when compared with historical controls. This improvement, together with a non-significant reduction in early deaths and absence of deaths in remission, translated into better event-free and overall survival. FUNDING: Phebra.

Monosomal karyotype predicts inferior survival independently of a complex karyotype in patients with myelodysplastic syndromes.

BACKGROUND: Conflicting data exist about the impact of a monosomal karyotype (MK) on overall survival (OS) for patients with myelodysplastic syndromes (MDSs) and particularly for those with a complex karyotype (CK). This study was aimed at determining whether an MK is associated with OS independently of the number of cytogenetic abnormalities (CAs) in a population-based MDS cohort. METHODS: Cancer registry data on incident MDS cases were linked with cytogenetic data and hospital administrative data from 2000 to 2010 for the Australian state of Victoria. RESULTS: Between 2000 and 2010, 1404 incident MDS cases with cytogenetic results were identified. A CK, defined as 3 or more abnormalities, was present in 126 (9%). A very complex karyotype (vCK), defined as 5 or more abnormalities, was present in 95 (7%). An MK was associated with worse OS in the whole cohort (median 6 vs 39 months, P < 0.001) including those with a coexisting CK (6 vs 17 months, P < 0.001) or vCK (6 vs 9 months, P = 0.02). After adjustments for the number of CAs, an MK remained independently associated with OS, although its effect size decreased with increasing cytogenetic complexity (hazard ratio for an MK, 4.81; 95% confidence interval, 3.08-7.52; hazard ratio for the number of CAs, 1.22; 95% confidence interval, 1.15-1.30; and hazard ratio for the interaction between an MK and CAs, 0.83; 95% confidence interval, 0.77-0.89). CONCLUSIONS: These results support the clinical utility of an MK as an independent predictor of adverse outcomes for MDS patients, even among CK and vCK groups, although its prognostic effect decreases with increasing cytogenetic complexity.

Health care workers in danger zones: a special report on safety and security in a changing environment.

INTRODUCTION: Violence against humanitarian health care workers and facilities in situations of armed conflict is a serious humanitarian problem. Targeting health care workers and destroying or looting medical facilities directly or indirectly impacts the delivery of emergency and life-saving medical assistance, often at a time when it is most needed. PROBLEM: Attacks may be intentional or unintentional and can take a range of forms from road blockades and check points which delay or block transport, to the direct targeting of hospitals, attacks against medical personnel, suppliers, patients, and armed entry into health facilities. Lack of access to vital health care services weakens the entire health system and exacerbates existing vulnerabilities, particularly among communities of women, children, the elderly, and the disabled, or anyone else in need of urgent or chronic care. Health care workers, especially local workers, are often the target. METHODS: This report reviews the work being spearheaded by the Red Cross and Red Crescent Movement on the Health Care in Danger initiative, which aims to strengthen the protections for health care workers and facilities in armed conflicts and ensure safe access for patients. This includes a review of internal reports generated from the expert workshops on a number of topics as well as a number of public sources documenting innovative coping mechanisms adopted by National Red Cross and Red Crescent Societies. The work of other organizations is also briefly examined. This is followed by a review of security mechanisms within the humanitarian sector to ensure the safety and security of health care personnel operating in armed conflicts. RESULTS: From the existing literature, a number of gaps have been identified with current security frameworks that need to be addressed to improve the safety of health care workers and ensure the protection and access of vulnerable populations requiring assistance. A way forward for policy, research, and practice is proposed for consideration. CONCLUSION: While there is work being done to improve conditions for health care personnel and patients, there need to be concerted actions to stigmatize attacks against workers, facilities, and patients to protect the neutrality of the medical mission.

Underestimation of myelodysplastic syndrome incidence by cancer registries: Results from a population-based data linkage study.

BACKGROUND: Myelodysplastic syndromes (MDS) appear to be underreported to cancer registries, with important implications for cancer and transfusion support service planning and delivery. Two population-based databases were linked to estimate MDS incidence more accurately. METHODS: Data from the statewide Victorian Cancer Registry (VCR) and Victorian Admitted Episode Dataset (VAED, capturing all inpatient admissions), in Australia, were linked. Incidence rates were calculated based on VCR reported cases and using additional MDS cases identified in VAED. Differences between reported and nonreported cases were assessed. A multivariate capture-recapture method was used to estimate missed cases. RESULTS: Between 2003 and 2010, 2692 cases were reported to VCR and an additional 1562 cases were identified in VAED. Annual incidence rate for those aged 65 years and older based on VCR was 44 per 100,000 (95% confidence interval [CI] = 43-45 per 100,000) and 68 per 100,000 (95% CI = 67-70 per 100,000) using both data sets. Cases not reported to VCR were more likely to have had previous malignancies recorded in VAED (23% versus 19%, P = .003) and to require red cell transfusion (59% versus 54%, P = .003). Using the multivariate model, an estimated 1292 cases were missed by both data sources: the re-estimate was 5546 (95% CI = 5438-5655) MDS cases, with an annual incidence in those aged 65 or older of 103 per 100,000 (95% CI = 100-106). CONCLUSIONS: This study reports a higher incidence of MDS using 2 data sources from a large and well-defined population than reported using cancer registry notifications alone.

Resistance to CDK2 inhibitors is associated with selection of polyploid cells in CCNE1-amplified ovarian cancer.

PURPOSE: Amplification of cyclin E1 (CCNE1) is associated with poor outcome in breast, lung, and other solid cancers, and is the most prominent structural variant associated with primary treatment failure in high-grade serous ovarian cancer (HGSC). We have previously shown that CCNE1-amplified tumors show amplicon-dependent sensitivity to CCNE1 suppression. Here, we explore targeting CDK2 as a novel therapeutic strategy in CCNE1-amplified cancers and mechanisms of resistance. EXPERIMENTAL DESIGN: We examined the effect of CDK2 suppression using RNA interference and small-molecule inhibitors in SK-OV-3, OVCAR-4, and OVCAR-3 ovarian cancer cell lines. To identify mechanisms of resistance, we derived multiple, independent resistant sublines of OVCAR-3 to CDK2 inhibitors. Resistant cells were extensively characterized by gene expression and copy number analysis, fluorescence-activated cell sorting profiling and conventional karyotyping. In addition, we explored the relationship between CCNE1 amplification and polyploidy using data from primary tumors. RESULTS: We validate CDK2 as a therapeutic target in CCNE1-amplified cells by showing selective sensitivity to suppression, either by gene knockdown or using small-molecule inhibitors. In addition, we identified two resistance mechanisms, one involving upregulation of CDK2 and another novel mechanism involving selection of polyploid cells from the pretreatment tumor population. Our analysis of genomic data shows that polyploidy is a feature of cancer genomes with CCNE1 amplification. CONCLUSIONS: These findings suggest that cyclinE1/CDK2 is an important therapeutic target in HGSC, but that resistance to CDK2 inhibitors may emerge due to upregulation of CDK2 target protein and through preexisting cellular polyploidy.

Chromothripsis under the microscope: a cytogenetic perspective of two cases of AML with catastrophic chromosome rearrangement.

Chromothripsis is a recently described phenomenon identified in cancer cells that produces catastrophic chromosome reorganization of one or a small number of chromosomes. It has been proposed that the multiple breakage events occur at a single point in time. Here we introduce the term anachromosome to describe an abnormal chromosome produced by chromothripsis. We report two cases of acute myeloid leukemia matching the description of chromothripsis that illustrate different aspects of this phenomenon from a cytogenetic perspective. Fluorescence in situ hybridization (FISH) analyses, including multicolor FISH and FISH for repeat elements that are not present on microarrays and that are resistant to sequencing, helped interpret the rearrangements but did not reveal their level of complexity. The anachromosomes conformed to the normal constraints of chromosome structure by including segments that provide two telomeres and a centromere. In patient samples, there are mixtures of cells with and without deletions. The deletion B allele frequencies for heterozygous loci in a mixture of cells with and without the deletions create a distinctive array pattern that is consistent with all the deletions in the anachromosomes having occurred concurrently. This evidence supporting the single-event hypothesis for chromothripsis has not previously been highlighted, to our knowledge. In the context of exploring mechanisms for chromosome shattering, we discuss a possible connection between chromosome pulverization and fragile sites. Understanding chromothripsis in the context of chromosome biology will help us identify its causes and consequences.

FISH detection of PML-RARA fusion in ins(15;17) acute promyelocytic leukaemia depends on probe size.

The diagnosis of acute promyelocytic leukaemia (APL) is usually confirmed by cytogenetics showing the characteristic t(15;17), but a minority of patients have a masked PML/RARA fusion. We report ten patients with APL and no evidence of the t(15;17), in whom the insertion of RARA into PML could not be demonstrated by initial FISH studies using a standard dual fusion probe but was readily identified using smaller probes. Given the need for rapid diagnosis of APL, it is important to be aware of the false negative rate for large PML/RARA FISH probes in the setting of masked rearrangements.

Genome organization and the role of centromeres in evolution of the erythroleukaemia cell line HEL.

BACKGROUND AND OBJECTIVES: The human erythroleukaemia (HEL) cell line has a highly rearranged genome. We matched whole chromosome analysis with cytogenomic microarray data to build a detailed description of these rearrangements. METHODOLOGY: We used a combination of single nucleotide polymorphism array and multiple fluorescence in situ hybridization approaches, and compared our array data with publicly available data for different sublines of HEL. B allele frequencies revealed the fate of each homologue for most chromosomes. RESULTS: At least two instances of the breakage-fusion-bridge cycle appear to have facilitated amplification of oncogenes and deletion of tumour suppressor genes. Because our study included centromere identification, we found that some abnormal chromosomes had centromeres that did not match the identity of the rest of the chromosome. CONCLUSIONS AND IMPLICATIONS: This study highlights the variety of complementary methods required to understand remodelling of the genome in cancer and uncover some of the mechanisms involved. We present evidence of centromere capture as a means of preserving broken chromosome segments. Testing for another highly repetitive DNA region, the nucleolus organizer region, helped identify the steps involved in chromosome 9 copy number aberrations. Increased use of techniques for identifying centromeres and other repetitive DNA regions will add to our understanding of genome remodelling and evolution. The pattern of chromosome 20 aberration in HEL supports an association of 20q11.21 amplification with erythroleukaemia (acute myeloid leukaemia subtype M6) in the context of 20q12 deletion. The differences between the karyotypes in different HEL sublines highlight the constantly evolving genomes of cultured cell lines.

Deletion(20q) as the sole abnormality in plasma cell myeloma is not associated with plasma cells as identified by cIg FISH.

Deletion of 20q is a common finding in myeloid disorders but it is also observed in plasma cell myeloma (PCM). As a del(20q) in a patient receiving treatment for myeloma may indicate therapy-related myelodysplastic syndrome (t-MDS), it is important to differentiate chromosome abnormalities associated with myeloma from those reflecting t-MDS. We performed fluorescence in situ hybridization (FISH) using a 20q12 probe (D20S108) in conjunction with cytoplasmic immunoglobulin (cIg) staining in 20 PCM cases with a del(20q) in order to confirm the cell type involved. Of the nine cases studied with a clone showing a del(20q) as the sole abnormality, 8 of 9 demonstrated loss of the D20S108 signals in non-plasma cells only and 5 of 9 had either a confirmed myeloid malignancy in addition to PCM or showed evidence of dysplastic changes in the marrow; however, of the 11 patients with a del(20q) within a complex PCM karyotype, 4 of 11 showed loss of the D20S108 signals in plasma cells only and 7 of 11 showed no significant loss in either plasma cells or non-plasma cells. Therefore, our results indicate that a del(20q) as the sole abnormality in PCM is present in non-plasma cells and, therefore, suggests the presence of an associated myeloid malignancy.

Hyperhaploid plasma cell myeloma.

Conventional cytogenetics shows chromosome abnormalities in one third of plasma cell myeloma (PCM) cases. These chromosome abnormalities can be used to divide PCM into two major aneuploidy groups: hyperdiploid PCM and non-hyperdiploid PCM. Hypodiploid PCM is associated with a poor prognosis relative to other ploidy groups. Hypodiploid karyotypes usually have a modal number of 40 or more. Near haploidy is a rare phenomenon in PCM. We present three cases of PCM with hyperhaploid karyotypes from our laboratory, and review three cases reported in the literature. All six cases had modal numbers ranging from 27 to 33. Two copies of chromosomes 3, 7, 9, 11, 15, 18 and 19 were present in all cases. Five of the six cases had two copies of chromosome 21 and four of the six cases had two copies of chromosome 5. The three patients studied at our laboratory had aggressive disease and a short survival but the small number of cases makes predicting outcome in this group difficult. The consistent pattern of cytogenetic abnormalities present in these cases suggests that hyperhaploidy may be a distinct cytogenetic entity in PCM.

Will a peripheral blood (PB) sample yield the same diagnostic and prognostic cytogenetic data as the concomitant bone marrow (BM) in myelodysplasia?

In patients with myelodysplastic syndromes (MDS), chromosome anomalies are detected by conventional cytogenetic studies (CCS) and/or interphase fluorescence in situ hybridization (FISH) of bone marrow (BM) samples and provide prognostic and diagnostic information, which can direct therapy. Whether peripheral blood (PB) can be substituted for bone marrow in these cases and can provide the same information remains unknown. Concurrent BM and PB specimens collected from 100 patients with recently diagnosed MDS were studied using both CCS and FISH. While 68% of BM samples showed an abnormal karyotype by CCS, only 31% of PB samples were abnormal by CCS. In 12% of patients, FISH and CCS were discordant due to the inability of the FISH panel to detect all possible abnormalities. However, only one case (1%) had a cryptic abnormality detected by FISH. BM and PB FISH were discordant in 3% of cases, most likely due to the smaller clone size in PB vs. BM. While PB should not be substituted for BM at diagnosis, it is a viable alternative for monitoring patients using the appropriate FISH probe(s).