Additional Copies Of Chromosome Research Articles

Additional copies of 1q negatively impact the outcome of multiple myeloma patients and induce transcriptomic deregulation in malignant plasma cells

Additional copies of chromosome 1 long arm (1q) are frequently found in multiple myeloma (MM) and predict high-risk disease. Available data suggest a different outcome and biology of patients with amplification (Amp1q, ≥4 copies of 1q) vs. gain (Gain1q, 3 copies of 1q) of 1q. We evaluated the impact of Amp1q/Gain1q on the outcome of newly diagnosed MM patients enrolled in the FORTE trial (NCT02203643). Among 400 patients with available 1q data, 52 (13%) had Amp1q and 129 (32%) Gain1q. After a median follow-up of 62 months, median progression-free survival (PFS) was 21.2 months in the Amp1q group, 54.9 months in Gain1q, and not reached (NR) in Normal 1q. PFS was significantly hampered by the presence of Amp1q (HR 3.34 vs. Normal 1q, P < 0.0001; HR 1.99 vs. Gain1q, P = 0.0008). Patients with Gain1q had also a significantly shorter PFS compared with Normal 1q (HR 1.68, P = 0.0031). Concomitant poor prognostic factors or the failure to achieve MRD negativity predicted a median PFS < 12 months in Amp1q patients. Carfilzomib–lenalidomide–dexamethasone plus autologous stem cell transplantation treatment improved the adverse effect of Gain1q but not Amp1q. Transcriptomic data showed that additional 1q copies were associated with deregulation in apoptosis signaling, p38 MAPK signaling, and Myc-related genes.

SPECTRUM OF CYTOGENETIC ABNORMALITIES IN PEDIATRIC PATIENTS WITH ACUTE MYELOID LEUKEMIA

Objective: To determine the frequency and types of cytogenetic abnormalities in pediatric Acute Myeloid Leukemia. Material and Methods: This cross-sectional study was conducted from January 2021 to October 2021 in Hematology department of the CHUGHTAI Institute of Pathology in Lahore Pakistan. Total 60 patients who were newly diagnosed with Acute myeloid leukemia in CHUGHTAI Institute of Pathology and were also referred from Children Hospital Lahore were included. Patients under the age of 16 years and from both gender were included. Informed consent was taken from patient’s guardian/parents. Patients had their bone marrow aspirates processed for standard G-banding and their karyotypes were examined using a cyto-vision system, where the number of chromosomes was counted and examined for any changes, such as damaged, missing, rearranged, or additional copies of chromosomes. Morphology, immunophenotyping of aspirate, and trephine biopsy are used to make the diagnosis of AML. Data was entered in SPSS version 23.0. Result: There were total 60 patients included in the study. The mean age was 10.2+ 3.2 years. There were n=32(53.3%) males and n=28(46.6%) female. Normal karyotype was observed in n=36(60%) patients, abnormal were seen in n=14(23.3%) and unsuccessful was shown in n=10(16.6%) patients. Favorable cytogenetic results were seen in n=6 (10%), intermediate in n=41 (68.3%), and unfavorable in n=4 (6.6%) cases. t(8;21) (q22:q22) and t(15;17)(q24;q21) were found in 8.3% and 1.6% of our study population respectively which have favorable prognosis . One of our patients had complex cytogenetic abnormalities, including [del5p, del7q, del11q-17+] which shows poor prognosis. Conclusion: The study discovered that 14 (23.3%) of the total pediatric patients with acute myeloid leukemia showed aberrant cytogenetic abnormalities. Chromosomal abnormalities should be identified as early as possible since they can be used for AML risk stratification and prediction of prognosis. Keywords: Acute myeloid leukemia, Complex cytogenetic, Cyto-vision, Karyotype, Morphology, Pediatric

What We Should Not Forget about Down Syndrome

Down syndrome is the foremost common genetic cause of intellectual disability. The additional copy of chromosome 21 confers potential changes in virtually all organ systems, including the brain, neck structures, and spine. Neuroradiologists should be aware of the multitude of imaging findings in patients with Down syndrome to correctly identify and diagnose life-altering conditions associated with this syndrome. In particular, the high prevalence of age-related cognitive decline and dementia stands out more clearly in recent decades due to the notable increase in these individuals' survival. Although the early and timely diagnosis of cognitive decline in patients with varying degrees of intellectual disability has not been an easy task from the clinical point of view, anatomic and functional brain studies have shown an essential role because they allow the early recognition of abnormalities that precede the cognitive decline. Furthermore, the similarities and differences in neuropathologic, genetic, and imaging aspects in patients with Down syndrome have allowed extrapolation for a better understanding of the mechanisms linked to Alzheimer disease development.Learning Objective: To review and systematize the distinctive characteristics and abnormalities of the head and neck, vertebral column, and CNS present in Down syndrome

Origin of a second malignancy harboring isochromosome 12p [i(12p)] in patients with history of testicular germ cell tumor (TGCT).

412 Background: The cumulative incidence of subsequent solid malignant neoplasm (SMN) after treatment of TGCT is about 10% over a 25-year period. i(12p) has been used to define tumor of germ cell origin. We evaluated the prevalence of i(12p) on various subsequent SMN in patients with a history of TGCT. Methods: From the Tumor Registry database at MD Anderson, we identified 655 TGCT patients who developed a second (third or fourth) malignancy between January 1990 and September 2018. Of those 114 SMN from the GI tract, GU system, chest, brain, sarcomas or melanomas between 2011 and 2017, 43 had tumor tissue available at our institution and of sufficient quality for fluorescence in situ hybridization (FISH) of i(12p). Results: Our cohort comprised 43 patients, excluding 2 cases (#6 and 7) whose SMN tested positive and their non-tumor controls negative for i(12p). Diagnosis of primary TGCT and SMN: age, mean 41 yrs (range 20-68 yrs) and 59 yrs (range 34-86 yrs), respectively, and interval of 18 yrs (range 0-57 yrs). Of those 43 cases (5 did not hybridize), 5 (11%) harbored i(12p), while 16 (37%) had additional copies of chromosome 12 without apparent gain of 12p. Conclusions: Prevalence of i(12p) on subsequent SMN in our patient cohort with a history of TGCT was 11%. Detection of i(12p) in those patients with TGCT in general and with nonseminomas in particular suggests somatic transformation, emphasizes importance of a complete surgical resection of residual teratomatous tumor after chemotherapy, and implicates a stem-cell origin and nature (e.g., plasticity, heterogeneity, dormancy) of cancer. *deceased; Rx – initial treatments; S – seminoma; NS – nonseminoma; BEP – bleomycin, etoposide, cisplatin; CEB – carboplatin, etoposide, bleomycin; XRT – radiation therapy. Groot HJ, Lubberts S, de Wit R, et al. Risk of solid cancer after treatment of testicular germ cell cancer in the platinum era. J Clin Oncol 2018;36:2504-13.

Altered development of dopaminergic neurons differentiated from stem cells from human exfoliated deciduous teeth of a patient with Down syndrome

BackgroundDown syndrome (DS) is a common developmental disorder resulting from the presence of an additional copy of chromosome 21. Abnormalities in dopamine signaling are suggested to be involved in cognitive dysfunction, one of the symptoms of DS, but the pathophysiological mechanism has not been fully elucidated at the cellular level. Stem cells from human exfoliated deciduous teeth (SHED) can be prepared from the dental pulp of primary teeth. Importantly, SHED can be collected noninvasively, have multipotency, and differentiate into dopaminergic neurons (DN). Therefore, we examined dopamine signaling in DS at the cellular level by isolating SHED from a patient with DS, differentiating the cells into DN, and examining development and function of DN.MethodsHere, SHED were prepared from a normal participant (Ctrl-SHED) and a patient with DS (DS-SHED). Initial experiments were performed to confirm the morphological, chromosomal, and stem cell characteristics of both SHED populations. Next, Ctrl-SHED and DS-SHED were differentiated into DN and morphological analysis of DN was examined by immunostaining. Functional analysis of DN was performed by measuring extracellular dopamine levels under basal and glutamate-stimulated conditions. In addition, expression of molecules involved in dopamine homeostasis was examined by quantitative real-time polymerase chain reaction and immunostaining. Statistical analysis was performed using two-tailed Student’s t-tests.ResultsCompared with Ctrl-SHED, DS-SHED showed decreased expression of nestin, a neural stem-cell marker. Further, DS-SHED differentiated into DN (DS-DN) exhibiting decreased neurite outgrowth and branching compared with Ctrl-DN. In addition, DS-DN dopamine secretion was lower than Ctrl-DN dopamine secretion. Moreover, aberrant expression of molecules involved in dopaminergic homeostasis was observed in DS-DN.ConclusionsOur results suggest that there was developmental abnormality and DN malfunction in the DS-SHED donor in this study. In the future, to clarify the detailed mechanism of dopamine-signal abnormality due to DN developmental and functional abnormalities in DS, it is necessary to increase the number of patients for analysis. Non-invasively harvested SHED may be very useful in the analysis of DS pathology.

Cytogenetic pattern profiling in cases of Acute Lymphoblastic Leukemia in pediatric age group

IntroductionAcute lymphoblastic leukemia (ALL) comprises about 70–80% of childhood leukemia. The present work was undertaken to study the spectrum of chromosomal abnormalities in North Indian population in haematologically confirmed pediatric ALL patients using bone marrow aspirates. MethodsBone marrow aspirates (0.6ml) after adding 15ml RPMI medium were divided into three parts for immediate culture, 24h culture and 48h culture method, were incubated according to their respective time duration and karyotyping was done. ResultsOut of 20 cases results were obtained in 14 cases. Out of these 9 cases (64.2%) in present study belonged to hypodiploid group. Trisomy was found in 3 (21.42%) cases and polyploidy in 1 (7.1%) case. Three year old male patient showed translocation t (21; 4) with deletion of long arm of chromosome 5 and absence of 7, 11, 12 and Y chromosomes. 4 Year old male patient showed translocation involving chromosome 13 with absence of chromosomes 7, 10, 11 and 12.5year old male patient showed one dicenteric 5 chromosome with additional copies of chromosomes 6, 8, 9, 21 and 22. DiscussionNumerical and structural chromosomal abnormalities found in Acute Lymphoblastic Leukemia have prognostic significance. Review of world literature shows that there is geographical variation in ploidy pattern of ALL. Our findings will help to play a key role in risk stratification and treatment protocols considering the genetic diversity of pediatric ALL in North Indian population.

Down Syndrome Mosaism in Samples of Iraqi Patients

The Down syndrome (DS) is the well-known trisomy, which is caused by additional copy ofchromosome 21.There are three types of DS. First, fully trisomy (47,XY,+21or 47,XX,+21).Second, translocation DS which result as translocation between chromosome 14 and21(46,XY,trans (14:21) or (46,XX,trans (14:21)). And the third type the mosaic DS that two celllines present in the individual. Mostly, studies indicate that frequency of each type 95%, 4% and1% respectively. Our study aims to estimate the frequency of each of the three types of DSchromosomal abnormalities in Iraqi samples. Chromosomal analysis using G-band technique wasperformed for 200 Down syndrome cases and 168 of their parents (whenever there were mosaicDS cases their parents were submitted chromosomal analysis). Fifty-seven percentage of caseswere fully Trisomy, forty-three percentage of them were mosaic DS, and no translocation patternwas recorded. The maternal ages were between 25-45 for the mosaic DS mothers. According tothis study, the frequency of mosaic DS was varied. It seems more investigations need to be donefor larger number of DS, and the impact of environmental changes in last decades need to bestudied more to be sure of its role in increasing of the proportion of this type of Down syndrome.

Clonal Progression Including Evolution Of An iAMP21 Chromosome In Xenograft Models Of BCP-ALL

Insight into the role of recurrent genomic changes arising in leukemia has depended largely on the functional analysis of cell lines and transgenic animals. However not all acquired abnormalities are represented in available cell lines and a few are too complex to be faithfully engineered in animal models. One such abnormality is intrachromosomal amplification of chromosome 21 (iAMP21), a heterogeneous cytogenetic rearrangement, with a distinct clinical profile, occurring in 2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The most highly amplified segment of iAMP21 varies in size and copy number but has always included a 5.1 Mb common region of amplification (CRA). Regions flanking the most highly amplified segment have profiles that may be step-like, or more complex combinations of lower level amplification, normal copy number and deletion. Abnormalities at other chromosomal locations such as deletions of IKZF1, CDKN2A/B or RB1 and rearrangements activating CRLF2 co-occur with iAMP21 but have never been shown to precede its formation.To investigate clonal evolution and to provide a resource for future functional studies, we established xenografts from three cases of iAMP21 BCP-ALL in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Signs of disease were seen between 6 and 8 months after intrafemoral injection and autopsy revealed marked splenomegaly in all cases. Cells isolated from the bone marrow and spleen, were positive for human B cell markers CD19 and CD10. Disease onset was more rapid (3-6 months) when primografts were transplanted and cells from these secondary mice were used to establish leukemia in a third generation. Whole body imaging of mice transplanted with xenograft cells, transduced with a lentivirus expressing luciferase, revealed the rapid spread of disease from the injection site to the contralateral femur, sternum, pelvis, ribs, spine and skull, with the spleen becoming the major site of engraftment at late stage disease.Comparison of SNP 6.0 array profiles of presentation and xenografts samples revealed several examples of post-transplant genomic progression which, with the exception of a biallelic deletion of the CDKN2A/B locus, had not previously been reported in iAMP21 patients. Additional copies of chromosomes 9 and 12 in patient samples were lost in xenografts resulting in copy number neutral loss of heterozygosity (CNN-LOH) for chromosome 12 but not 9. Analysis of further primary iAMP21 BCP-ALL cases identified two with CNN-LOH for part of the long arm of chromosome 12, suggesting involvement of an imprinted locus in this region.iAMP21 chromosomes were retained in all xenografts, supporting evidence that it is a primary abnormality. However, as these rearrangements have been considered to be stable once formed, we were surprised to observe acquisition of a non–contiguous deletion of chromosome 21 in one secondary mouse and all of its tertiary recipients. The deletion was proximal to the CRA and resulted in extension of pre-existing haploinsufficient regions by 6.6 Mb. Evolution of this iAMP21 chromosome was confirmed by fluorescence in-situ hybridisation (FISH) using probes targeting the deletion, with loss of signal observed in all cells from mice carrying the deletion but in no cells from the presentation sample or unaffected mice. Although we cannot rule out that the deletion arose in response to adaptive pressure specific to the xeno-environment, opportunities to observe similar progression in patients have been limited because FISH analysis has generally targeted only the CRA and few paired presentation/relapse samples have been analysed by genomic array. While it remains likely that overrepresentation of oncogenes within the CRA acts as a primary driver of iAMP21 BCP-ALL, the additional deletion may highlight the position of tumour suppressor genes involved in leukemia progression. As these patients are treated as a single clinical entity, an important implication of these data is that the position and extent of deletions in iAMP21 rearrangements may influence response to therapy.In conclusion, xenografts of iAMP21 BCP-ALL closely resemble human primary disease and will make valuable pre-clinical models for basic and translational research. As models for the study of clonal evolution they support previous evidence that iAMP21 is a primary abnormality and have revealed novel forms of clonal progression. Disclosures:No relevant conflicts of interest to declare.

Abstract C111: Pediatric Preclinical Testing Program (PPTP): Evaluation of the MEK1/2 inhibitor AZD6244 against juvenile pilocytic astrocytoma (JPA) xenografts

Abstract Background: AZD6244 (ARRY-142886) is a potent small molecule inhibitor of MEK1/2 that is in phase 2 clinical development. Against the PPTP in vivo tumor panels, AZD6244 induced significant differences in EFS distribution in 10 of 37 (27%) solid tumor models and 0 of 6 acute lymphoblastic leukemia (ALL) models, but there were no objective responses. Recently, molecular characterization of JPAs has shown tandem duplication producing a novel fusion gene (KIAA1549-BRAF) that lacks the BRAF regulatory domain and leads to constitutive activation of BRAF. Activating point mutations in BRAF are less frequent in JPA, accounting for only 5 percent of cases. AZD6244 was evaluated against two JPA xenografts, BT-35 and BT-40, that are used for secondary testing by the PPTP. Methods: AZD6244 was dissolved in 0.5% hydroxypropyl methyl cellulose, 0.1% Polysorbate 80 and administered p.o., using a twice daily schedule (excepting weekends, for which a once daily schedule was used) for 6 weeks at a dose of 100 or 75 mg/kg. Tumors were followed for an additional 6 weeks. Three measures of antitumor activity were used: 1) response criteria modeled after the clinical setting; 2) treated to control (T/C) tumor volume at day 21; and 3) a time to event (4-fold increase in tumor volume) measure based on the median EFS of treated and control lines (intermediate activity required EFS T/C &amp;gt; 2, and high activity additionally required a net reduction in median tumor volume at the end of the experiment). Phosphorylation of ERK1/2, as a surrogate for in vivo inhibition of MEK1/2 was determined by immunoblotting. Genomic DNA from BT-35 and BT-40 was screened for BRAF mutations with primers designed to amplify exons 1–18. Purified BRAF BAC DNA was labeled with digoxigenin-11-dUTP (Roche Molecular Biochemicals, Indianapolis, IN) by nick translation and used for FISH analysis. Results: Both JPA models have additional copies of chromosome 7, but lack evidence of focal duplication of BRAF. Sequencing showed that BRAF is wild type in BT-35 but that BT-40 harbors an activating mutation [V600E]. Pharmacodynamic studies indicated that AZD6244 administered at 100 mg/kg twice-daily completely inhibited ERK1/2 phosphorylation. AZD6244 was evaluated against two JPA xenografts; BT-40 xenografts completely regressed on treatment with AZD6244, but re-grew subsequent to stopping therapy. In contrast, BT-35 xenografts progressed on AZD6244 treatment. Conclusions: The PPTP previously demonstrated that AZD6244 had limited in vivo activity against the pediatric preclinical models evaluated, despite inhibition of MEK1/2 activity. However, AZD6244 was highly active at the same dose and schedule against BT-40 JPA xenografts that harbor constitutively activated BRAF. The BT-40 xenograft model may be valuable for developing rational combinations of molecularly-targeted agents against JPAs with BRAF activation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C111.

Cytogenetics of long‐term survivors of ETV6‐RUNX1 fusion positive acute lymphoblastic leukemia

This study describes the cytogenetics of 33 children with ETV6-RUNX1 positive acute lymphoblastic leukemia (ALL) who had been in continuous complete remission for a minimum of 8.8 years [median event-free survival (EFS) 10.9 years]. The results were compared with a published series of 16 fusion positive patients treated on the same childhood ALL trial, who had relapsed (median EFS, 2.3 years). Interphase fluorescence in situ hybridization (FISH) at diagnosis showed deletion of the second ETV6 signal from all fusion positive cells in 45% of the long-term survivors but in none of the relapsed patients, whereas patients with mixed populations with retained or lost second signals were more frequent among those who had relapsed (69%) than the long-term survivors (21%). Interphase populations with two fusion signals in 18% of the long-term survivors and 31% of relapsed patients were smaller in the long-term survivors (median, 4% of total cells) than in the relapsed patients (median, 84%). The additional copy of chromosome 21 in 30% of long-term survivors and in 69% of relapsed patients was a derived chromosome 21 in 20% and 55% of patients, respectively. Metaphase FISH for 26 long-term survivors and 15 relapsed patients revealed complex karyotypes in both groups. Variant translocations involved different chromosome arms between the long-term survivors and relapsed patients. It appears that the two groups have some distinguishing cytogenetic features at the time of diagnosis, which may provide pointers to relapse that are worthy of more detailed study.

The spectrum of cytogenetic changes in the colorectal polyps with different histologic structure

A cytogenetic study on colorectal polyps with different histological structures from 54 patients was carried out. Diploid karyotypes dominated in these polyps. Abnormal karyotypes were found in 17 (31.5%) polyps: the most frequent anomalies were in adenomas, in hamartomatous polyps they were absent. The typical features of the karyotypes were numeric changes, such as additional copies of chromosomes 13, 7, 20, 18 and 16 in some combinations. The polyps with high-grade dysplasia in all cases had abnormal karyotypes. One adenoma with dysplastic changes had specific chromosomal anomalies, such as additional copies of chromosomes 13, 18, and 20; this adenoma localized near adenocarcinoma. Unlike the previous studies of the mucous membrane of intestine from the patients suffering from ulcerative colitis and Cronh’s disease, we have studied the peculiarities of the karyotype exactly in inflammatory polyps, and found the chromosomal anomalies in 21.4% of cases.

Collaboration between Activating Mutations in JAK2 and Trisomy 21 in the Acute Lymphoblastic Leukemias of Down Syndrome (DS).

Additional copies of chromosome (chr) 21 are the most common chromosomal aneuploidy in childhood leukemia. Patients with DS have a markedly increased risk for both AML and ALL. Thus trisomy 21 is leukemogenic. DS-AML is uniquely characterized by an acquired mutation in the chr X gene GATA1. The existence of a similar unique collaborating mutation in DS-ALL has been postulated but remained elusive. To identify such mutations, we performed a large mutational screen in 81 diagnostic samples of B cell precursor DS-ALL, stored in central labs of 9 European childhood ALL protocols (representing more than 8000 samples of childhood ALL). Mutations in JAK2 were identified in 16 (19.7%) DS-ALL patients. The mutations are different from those observed in myeloproliferative disorders. In fifteen patients a point mutation resulted in substitution of the conserved Arginine at position 683. In one patient an in-frame insertion caused displacement of the same amino-acid. The mutations cause constitutive activation of JAK2 and are predicted to disrupt a critical interaction between the pseudokinase and the SH2 domains. The mutations are acquired as they do not exist in remission samples. The mutated RNA is expressed. They are unique to DS-ALL as screening of over 300 non DS-ALL, as well as DS-AMKL, has revealed only one instance with a similar mutation. Strikingly, that patient had an ALL with an iso21q abnormality! The clinical presentation and survival data of DS-ALL patients with and without JAK2 mutation is currently analyzed and will be presented in the meeting. Our data demonstrate that novel mutations of JAK2 cooperate with trisomy 21 in at least 20% of ALLs in Down Syndrome. Thus it seems that, like DS-AML, at least 20% of DS-ALLs are different from sporadic childhood leukemias and characterized by unique acquired mutations in genes located outside chr 21 (GATA1 on the chr X and JAK2 on chr 9). Beyond the obvious therapeutic implications, these observations raise the hypothesis that JAK2 is important in B cell development and that constitutive activation of JAK2 in B cell precursors provides a survival advantage in the presence of a germline trisomy 21.

Artificially Introduced Aneuploid Chromosomes Assume a Conserved Position in Colon Cancer Cells

BackgroundChromosomal aneuploidy is a defining feature of carcinomas. For instance, in colon cancer, an additional copy of Chromosome 7 is not only observed in early pre-malignant polyps, but is faithfully maintained throughout progression to metastasis. These copy number changes show a positive correlation with average transcript levels of resident genes. An independent line of research has also established that specific chromosomes occupy a well conserved 3D position within the interphase nucleus.Methodology/Principal FindingsWe investigated whether cancer-specific aneuploid chromosomes assume a 3D-position similar to that of its endogenous homologues, which would suggest a possible correlation with transcriptional activity. Using 3D-FISH and confocal laser scanning microscopy, we show that Chromosomes 7, 18, or 19 introduced via microcell-mediated chromosome transfer into the parental diploid colon cancer cell line DLD-1 maintain their conserved position in the interphase nucleus.ConclusionsOur data is therefore consistent with the model that each chromosome has an associated zip code (possibly gene density) that determines its nuclear localization. Whether the nuclear localization determines or is determined by the transcriptional activity of resident genes has yet to be ascertained.

Identification of Specific Transcriptional Patterns Associated with Hyperdiploidy in Multiple Myeloma.

Karyotypic instability is strongly associated with multiple myeloma (MM). According to the chromosome number pattern, two major groups are recognized: hyperdiploid (H) tumors, associated with recurrent trisomies involving non-random chromosomes (3, 5, 7, 9, 11, 15, 19 and 21); and non hyperdiploid (NH) tumors associated with hypodiploid, pseudodiploid or near-tetraploid karyotypes. MM patients are approximately equally distributed between the two categories; notably, the most recurrent IGH translocations and chromosome 13 deletion appear to be prevalently associated with NH-MM, whereas recent evidences have suggested that H-MM correlates with a favorable prognosis. To molecularly characterize these two genetic categories, we performed a gene expression profiling analysis on 66 newly-diagnosed MM, characterized by FISH analyses for IGH translocations, 13q14 deletions and additional copies of chromosomes 1, 11 and 19. The ploidy status was investigated by combining two recently proposed FISH approaches (Wuilleme S. et al., 2004; Chng W.J. et al., 2005). The gene expression profiles of highly purified MM plasma cells have been generated by means of high-density oligonucleotide arrays (Affymetrix GeneChip U133A) and subsequently analyzed using unsupervised and supervised approaches (two-dimensional hierarchical clustering and SAM, respectively). The differential expression of 229 genes distinguished the 28 H-MM from the 38 NH-MM cases. The 208 upregulated genes in H-MM mapped mainly on the chromosomes involved in hyperdiploidy, while a significant percentage (29%) of the 21 genes upregulated in NH-MM were localized on 16q. The identified transcripts have been further validated on a publicly available gene expression dataset of an independent cohort of 64 MM patients (Carrasco et al., 2005). Notably, the global classification rate for the 64 cases resulted of 81%, confirming the validity of the identified transcriptional fingerprint. A functional analysis revealed a significant fraction of genes involved in protein biosynthesis (38%), transcriptional machinery and oxidative phosphorylation. Furthermore, an integrative genomic approach using a model-free statistical method (LAP, locally adaptive statistical procedure) supported these findings, allowing the identification in H-MM of globally upregulated regions on the chromosomes 3, 5, 9, 15 and 19, along with the downregulation of a region on 16q arm. Remarkably, two sub-groups are clearly distinguishable within H-MM group, one associated with chromosome 11 gain and the other showing 1q gain and chromosome 13 deletion. A supervised analysis of the H-11 vs H-13/1 patients identified 57 differentially expressed genes. Eleven of the 18 genes up-regulated in the H-11 group mapped to chromosome 11, whereas 21 of the 39 genes up-regulated in the H-13/1 group mapped to the 1q region. Notably, CCND2 resulted the most significantly upregulated gene in H-13/1 group. Our data reinforce the importance of combining cytogenetics and gene expression approaches for a better definition of the genetic alterations in MM and provide a molecular and genomic framework for dissection of disease pathogenesis and clinical management.

The identification of chromosome abnormalities associated with the invasive phenotype of uveal melanoma in vitro

Tumour cell cultures are often highly heterogeneous, containing sub-populations of cells with differing characteristics. To identify chromosome abnormalities that are associated with the invasive phenotype, we isolated highly invasive uveal melanoma cell populations using the Transwell assay. Using this invasion assay, invasive sub-populations of primary uveal melanoma short-term cultures, and an established cell line, were specifically isolated. A series of sequential assays were undertaken to enrich the invasive population, and the enhanced invasive ability was confirmed by Transwell invasion assay. Chromosome abnormalities in invasive and parental cells were identified by karyotyping and confirmed by comparative genome hybridisation. Invasive sub-populations of uveal melanoma cells were isolated from 3 uveal melanoma short term cultures and a uveal melanoma cell line. In all cases, invasive sub-populations had either acquired additional chromosome abnormalities to those present in the parental cell line, or other abnormalities present in the parental lines were lost. In the established cell line (SOM 157), invasive cells were characterised by widespread chromosomal instability, frequent telomere associations and additional copies of chromosome 20. The invasive phenotype of SOM 196 associated with the presence of a derivative chromosome 5, der(5)t(5;11)(q35;q12) whilst a translocation t(17;20)(q12;q13) was predominant amongst non-invasive cells. In two additional cultures, deletions on chromosome 6q were associated with reduced invasive ability. In conclusion, highly invasive populations of uveal melanoma cells demonstrate chromosomal abnormalities that differ from non-invasive cells. These include chromosome instability and abnormalities of chromosome 20, observations echoing those seen in metastatic uveal melanoma.

Rapid detection of trisomy 21 by homologous gene quantitative PCR (HGQ-PCR).

Down's syndrome results from the production of three copies of chromosome 21 within a cell. We have devised a method termed the homologous gene quantitative polymerase chain reaction (HGQ-PCR), which uses one pair of primers and which can directly identify the additional copy of chromosome 21 by simultaneously amplifying two highly homologous genes of the human liver-type phosphofructokinase located on chromosome 21 (PFKL-CH21) and the human muscle-type phosphofructokinase located on chromosome 1 (PFKM-CH1) for self-detecting determination. On analysis of 34 cases of Down's syndrome, including two cases of unbalanced translocation 46, XY, der (14; 21) (q10; q10), + 21, and 100 normal individuals, the relative ratio of the PFKM-CH1/PFKL-CH21 product was 1.33 +/- 0.323 (mean +/- SD) and 0.40 +/- 0.16 (mean +/- SD) for disomy DNA and trisomy DNA, respectively. The difference between these two groups was highly significant (P < 0.001). These results indicate that this quantitative method is practical and may be used for the prenatal diagnosis of Down's syndrome caused by trisomy 21.

Cytogenetic analysis of intestinal polyps in polyposis syndromes: Comparison with sporadic colorectal adenomas

Few cytogenetic studies of polyps from patients with polyposis syndromes have been reported. We studied 27 colonic adenomatous polyps from familial adenomatous polyposis (FAP), two polyps of the small bowel from Peutz-Jeghers syndrome (PJS), and four colorectal juvenile polyps from juvenile polyposis syndrome (JPS). The karyotypic results were compared with 32 sporadic colorectal adenomatous polyps. Nineteen colorectal adenomas had abnormal karyotypes; of these, five were from patients with FAP and 14 were sporadic adenomas. Numerical changes were the most frequent change (14 adenomas); additional copies of chromosome 7 (eight adenomas) and 13 (seven adenomas) occurred most often and were present in both FAP and sporadic adenomas. Only five adenomas, all sporadic, had structural chromosome abnormalities. Normal karyotypes were obtained from 32 adenomas, and chromosome counts but not karyotypes were obtained from eight polyps owing to poor chromosome morphology. The JPS and PJS polyps had normal karyotypes. These data indicate that adenomas from patients with FAP tend to have fewer structural abnormalities than sporadic adenomas and that numerical abnormalities are the most common chromosome abnormality in both FAP and sporadic polyps and suggest that the mechanism which causes loss of heterozygosity (LOH) in the adenoma to carcinoma sequence operates on a level below that of the whole chromosome.

Correlation of secondary cytogenetic abnormalities with histologic appearance in non-Hodgkin's lymphomas bearing t(14;18)(q32;q21).

Successful cytogenetic studies were performed on 69 biopsies from 64 patients with non-Hodgkin's lymphoma bearing a t(14;18)(q32;q21) translocation. This translocation appears to be a primary abnormality associated with the development of certain B-cell non-Hodgkin's lymphomas. We correlated the occurrence of secondary abnormalities, in addition to the t(14;18)(q32;q21), with histologic subtype to test the hypothesis that secondary abnormalities correlate with more aggressive histologic appearance. A large number of secondary abnormalities were identified, the most frequent being additional copies of chromosomes 7 (30%), 12 (22%), 18 (22%), 20 (16%), or 21 (14%), deletion of a portion of the long arm of chromosome 6 (17%), and either an additional chromosome 17 or an isochromosome for the long arm of chromosome 17 (13%). An extra chromosome 7 was highly associated with a diffuse histologic pattern; it was present in 52% of patients with a diffuse pattern and in only 15% of those with a follicular pattern (P = .002). A weaker association with a diffuse growth pattern was found for the addition of chromosome 17 or an i(17q); it was found in 24% of patients with a diffuse pattern and only 5% of those with a follicular pattern (P = .05). No other significant correlations between secondary chromosome abnormalities and histologic subtype were identified. Although the explanation for this association is not clear, it appears that patients with B-cell non-Hodgkin's lymphomas bearing the t(14;18)(q32;q21) translocation which also have an additional chromosome 7 are likely to exhibit a diffuse growth pattern.