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.

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.

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.

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.

The paradox of 20q11.21 amplification in a subset of cases of myeloid malignancy with chromosome 20 deletion.

Deletion of the long arm of one chromosome 20 (del(20q)) is a well-recognized abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and is presumed to cause loss of a tumor suppressor gene at 20q12. In a previously published series of MDS and AML cases, which had lost this region via unbalanced translocation, around 40% of cases were shown to have additional copies of the chromosome 20 abnormalities, with resulting gain or amplification of the retained parts of chromosome 20, most often 20q11.2. We have used FISH and array comparative genomic hybridization, to define further the retained and amplified regions. We now report targeted amplification of 20q11.21 in four of the 22 cases selected for further study and in one new case. The shortest amplified region of 250 kb in a series of five patients with three to ten copies of the 20q11.21 region contained the complete HCK, TM9SF4, PLAGL2, and POFUT1 genes. By RT-PCR we have shown that there is correlation between amplification and increased expression of these four genes in most cases. Localized and high level amplification of the common 250 kb region is evidence for activation of an oncogene in this region in these MDS and AML cases. Cases with 20q11.21 amplification tended to have a high proportion of erythroblasts in the marrow, with two cases diagnosed as erythroleukemia (AML-M6). Chromosome sub-band 20q11.21 amplification may therefore prove to be a marker of a specific subset of AML/MDS with a significant erythroid component.

Conducting qualitative research on cervical cancer screening among diverse groups of immigrant women: research reflections: challenges and solutions.

OBJECTIVE: To explore the research lessons learned in the process of conducting qualitative research on cervical cancer screening perspectives among multiple ethnolinguistic groups of immigrant women and to provide guidance to family medicine researchers on methodologic and practical issues related to planning and conducting focus group research with multiple immigrant groups. DESIGN: Observations based on a qualitative study of 11 focus groups. SETTING: Hamilton, Ont. PARTICIPANTS: Women from 1 of 5 ethnolinguistic immigrant groups and Canadian-born women of low socioeconomic status. METHODS: We conducted 11 focus groups using interactive activities and tools to learn about women's views of cervical cancer screening, and we used our research team reflections, deliberate identification of preconceptions or potential biases, early and ongoing feedback from culturally representative field workers, postinterview debriefings, and research team debriefings as sources of information to inform the process of such qualitative research. MAIN FINDINGS: Our learnings pertain to 5 areas: forming effective research teams and community partnerships; culturally appropriate ways of accessing communities and recruiting participants; obtaining written informed consent; using sensitive or innovative data collection approaches; and managing budget and time requirements. Important elements included early involvement, recruitment, and training of ethnolinguistic field workers in focus group methodologies, and they were key to participant selection, participation, and effective groups. Research methods (eg, recruitment approaches, inclusion criteria) needed to be modified to accommodate cultural norms. Recruitment was slower than anticipated. Acquiring signed consent might also require extra time. Novel approaches within focus groups increased the likelihood of more rich discussion about sensitive topics. High costs of professional translation might challenge methodologic rigour (eg, back-translation). CONCLUSION: By employing flexible and innovative approaches and including members of the participating cultural groups in the research team, this project was successful in engaging multiple cultural groups in research. Our experiences can inform similar research by providing practical learning within the context of established qualitative methods.

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.

Secondary clonal cytogenetic abnormalities following successful treatment of acute promyelocytic leukemia.

To identify patients who developed secondary clonal cytogenetic aberrations (CCA) following therapy for acute promyelocytic leukemia (APL), we retrospectively analyzed cytogenetic results from 123 patients diagnosed with APL between 1995 and 2007, who had ongoing cytogenetic analysis undertaken in our laboratory. During follow-up for APL we identified 12 patients (9.8%) who developed CCA, not detected at diagnosis of APL and unrelated to their original APL karyotype. All patients had received all-trans retinoic acid (ATRA) and chemotherapy and were in complete remission for APL when secondary CCA were identified. The median latency period between diagnosis of APL and emergence of secondary CCA was 27.5 months (range: 2-54 months). To date, four patients with CCA have been diagnosed with therapy-related myelodysplastic syndrome (t-MDS)/acute myeloid leukemia (t-AML), giving a median t-MDS/AML free survival of 78 months, with follow-up ranging between 20 and 136 months from APL diagnosis. Three patients have died: two patients died of t-AML and another developed relapsed APL with persistence of his secondary clone but no diagnosis of t-MDS/AML and died from transplant-related complications. Two patients are alive with t-MDS. Seven patients with CCA are alive with no morphological evidence of MDS at the time of their last known follow-up; thus median survival has not been reached. The appearance of these abnormalities in the absence of morphological evidence of MDS in the majority of patients is unusual, and highlights the importance of continued cytogenetic follow-up in these patients. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

Abnormal cytogenetics in orbital lipoma.

Orbital lipoma is a rare entity with only a small number of cases previously described. The authors describe a case of orbital lipoma in a 56-year-old man, which was treated with surgical excision. Cytogenetic analysis of the lesion demonstrated abnormalities of chromosome 12, consistent with chromosomal abnormalities in lipomas found elsewhere in the body. Therefore, cytogenetic analysis may be useful to differentiate lipomatous tumors from normal orbital fat.

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.

AIDS-related plasmablastic lymphoma of the oral cavity associated with an IGH/MYC translocation--treatment with autologous stem-cell transplantation in a patient with severe haemophilia-A.

Plasmablastic lymphoma is an AIDS related lymphoma that continues to have a poor prognosis despite significant advances in the management of HIV and lymphoproliferative diseases. In part this has been due to limited insights into the biology of this disease and the molecular mechanisms of oncogenesis. To date molecular abnormalities have not been described in plasmablastic lymphoma, and its aggressive clinical behaviour has been difficult to understand. We describe the first reported cytogenetic abnormality in plasmablastic lymphoma, an IgH/MYC translocation. It is also the first description of autologous stem cell transplantation in a patient with severe haemophilia A.

Translocation (14;18)(q32;q21) in acute lymphoblastic leukemia: a study of 12 cases and review of the literature.

We present a series of 12 cases of de novo acute lymphoblastic leukemia (ALL) with translocation t(14;18)(q32;q21). The median age of patients at presentation was 65.5 years, and no patient presented with a past history or any clinical evidence of lymphoma. A Burkitt translocation was identified in 4 of the 12 cases by conventional cytogenetics but fluorescence in situ hybridization using a MYC probe identified a further three cases of MYC rearrangement: one with a cryptic t(8;14) involving the der(14)t(14;18), one showing MYC translocated onto a marker chromosome, and one associated with a t(8;9)(q24;p13) translocation. A review of the literature identified an extremely close association between the t(14;18) and the t(8;9), with the latter translocation found only in the presence of t(14;18). The present study confirms the previously reported dismal prognosis of t(14;18)-associated ALL.

Three adults with acute lymphoblastic leukemia and dic(7;9)(p11.2;p11).

We report the cases of three adults with acute lymphoblastic leukemia (ALL) who had a dic(7;9)(p11.2;p11) on the diagnostic bone marrow cytogenetic analysis. All three were males with B-ALL (aged 25, 38, and 48 years) who at presentation had 90-100% replacement of marrow with lymphoblasts. One patient died 23 months post induction therapy, which was 9 months post allogeneic stem cell transplantation (SCT); as of writing, the other two patients were in remission and well, one of them at 4 years after SCT and the other at 7.5 years without SCT. To our knowledge, these cases are the first reported in adult ALL with dic(7;9) and demonstrate a consistent phenotype, with good initial response to therapy but variable long-term outcome.

Cytogenetic and FISH techniques in myeloid malignancies.

Chromosome analysis is an essential part of the diagnostic testing of myeloid malignancies. Good chromosome preparations are essential for a complete cytogenetic analysis. This means plentiful metaphase spreads with well-spread crisply banded chromosomes. To achieve such a result, several variables, including the growth rate of the leukemic cells, are critical. The method described in this chapter has been extensively tested and should produce reasonable results from most cases. Fluorescence in situ hybridization (FISH) is less influenced by sample variation and as a result may be obtained from either metaphase spreads or interphase cells. Moreover, FISH is capable of describing chromosome rearrangements at the gene level, rather than at the gross level shown by conventional cytogenetics. It does not, however, provide information on genetic rearrangements other than at the specific target site of the probe used, unlike conventional cytogenetics. Thus, these two techniques complement each other and are both now essential elements of chromosome analysis.

Deletion of the derivative chromosome 9 in chronic myeloid leukemia.

With the development of fluorescence in situ hybridization (FISH), it was possible to detect the BCR-ABL fusion signal in both metaphase spreads and interphase cells of patients with chronic myeloid leukemia (CML). However, the use of FISH to detect residual disease in patients with CML post therapy was limited by the false positive rate using the early single fusion probes. Therefore, dual fusion probes that created a fusion signal on the derivative chromosome 9 in addition to the fusion sifnal on the Philadelphia chromosome or derivative chromosome 22 were developed. Using these second-generation probes, it was discovered that a significant proportion of CML cases has a sub-microscopic deletion at the site of the ABL-BCR fusion. This chapter outlines a testing strategy to identify deleltions of the derivative chromosome 9 and to use combinations of probes to identify residual disease in these cases.