Molecular pathogenesis and prognostication of "low-grade'' and "high-grade" endometrial stromal sarcoma.

Endometrial stromal sarcomas (ESS) are a heterogeneous group of rare mesenchymal cancers. Considerable knowledge has been gained in recent years about the molecular characteristics of these cancers, which helps to classify them in a more meaningful manner leading to improved diagnosis, prognostication, and treatment. According to this classification, ESS is now grouped as low- or high-grade. ESS may have overlapping clinical presentation, morphology, and immunohistochemical profile. Their genetic characteristics allow subdivision of many of them depending on which pathogenetically important fusion genes they carry, but clearly much more needs to be unraveled in this regard. We here provide an overview of the molecular pathogenetic knowledge gained so far on low- and high-grade ESS.

MGMT promoter methylation is a rare epigenetic change in malignant effusions.

OBJECTIVE: The aim of this study was to analyse the promoter methylation status of the gene O6-methylguanine-DNA methyltransferase (MGMT) in malignant effusions, with focus on serous carcinoma. METHODS: Fresh-frozen cell pellets from 81 effusions (42 peritoneal, 38 pleural, one pericardial), consisting of 71 carcinomas of different origin (33 ovarian, 23 breast, six lung, five uterine corpus and four cervical carcinomas) and 10 malignant mesotheliomas, were analysed for MGMT methylation using pyrosequencing analysis. RESULTS: MGMT methylation at all four cytosine-guanine dinucleotide sites examined was detected in only 2/81 (2%) specimens, consisting of a high-grade serous carcinoma with high frequency of methylation, and a breast carcinoma with low methylation frequency. CONCLUSION: The findings in the present study suggest that MGMT methylation is a rare epigenetic change in malignant effusions of different origin.

RNA-sequencing identifies novel GREB1-NCOA2 fusion gene in a uterine sarcoma with the chromosomal translocation t(2;8)(p25;q13).

Sarcomas account for 3% of all uterine malignancies and many of them are characterized by acquired, specific fusion genes whose detection has increased pathogenetic knowledge and diagnostic precision. We describe a novel fusion gene, GREB1-NCOA2, detected by transcriptome sequencing and validated by reverse transcriptase polymerase chain reaction and Sanger sequencing in an undifferentiated uterine sarcoma. The chimeric transcript was an in-frame fusion between exon 3 of GREB1 and exon 15 of NCOA2. The fusion is reported here for the first time, but it involves the GREB1 gene, an important promoter of tumor growth and progression, and NCOA2 which is known to be involved in transcriptional regulation. The alteration and recombination of these genes played a role in the tumorigenesis and/or progression of this sarcoma.

Identification of novel cyclin gene fusion transcripts in endometrioid ovarian carcinomas.

Formation of fusion genes is pathogenetically crucial in many solid tumors. They are particularly characteristic of several mesenchymal tumors, but may also be found in epithelial neoplasms. Ovarian carcinomas, too, may harbor fusion genes but only few of these were found to be recurrent with a rate ranging from 0.5 to 5%. Because most attempts to find specific and recurrent fusion transcripts in ovarian carcinomas focused exclusively on high-grade serous carcinomas, the situation in the other carcinoma subgroups remains largely uninvestigated as far as fusion genes are concerned. We performed transcriptome sequencing on a series of 34 samples from ovarian tumors that included borderline, clear cell, mucinous, endometrioid, low-grade and high-grade serous carcinomas in search of fusion genes typical of these subtypes. We found a total of 24 novel fusion transcripts. The PCMTDI-CCNL2 fusion transcript, which involves a member of the cyclin family, was found recurrently involved but only in endometrioid carcinomas (4 of 18 tumors; 22%). We also found three additional fusion transcripts involving genes belonging to the cyclin family: ANXA5-CCNA2 and PDE4D-CCNB1 were detected in two endometrioid carcinomas, whereas CCNY-NRG4 was identified in a clear cell carcinoma. The recurrent involvement of CCNL2 in four fusions and of three other genes of the cyclin family in three additional transcripts hints that deregulation of cyclin genes is important in the pathogenesis of ovarian carcinomas in general but of endometrioid carcinomas particularly.

ZC3H7B-BCOR high-grade endometrial stromal sarcomas: a report of 17 cases of a newly defined entity.

High-grade endometrial stromal sarcoma likely encompasses underrecognized tumors harboring genetic abnormalities besides YWHAE-NUTM2 fusion. Triggered by three initial endometrial stromal sarcomas with ZC3H7B-BCOR fusion characterized by high-grade morphology and aggressive clinical behavior, we herein investigate the clinicopathologic features of this genetic subset by expanding the analysis to 17 such tumors. All of them occurred in adult women with a median age of 54 (range, 28-71) years. They were predominantly based in the endomyometrium and demonstrated tongue-like and/or pushing myometrial invasion. Most were uniformly cellular and displayed haphazard fascicles of spindle cells with mild to moderate nuclear atypia. Myxoid matrix was seen in 14 of 17 (82%) tumors, and collagen plaques were seen in 8 (47%). The mitotic index was ≥10 mitotic figures/10 high-power fields (HPFs) in 14 of 17 (82%) tumors with a median of 14.5 mitotic figures/10 HPFs. No foci of conventional or variant low-grade endometrial stromal sarcoma were seen. All tumors expressed CD10 with only limited or absent desmin, SMA and/or h-caldesmon staining. ER and PR expression in >5% of cells was seen in 4 of 12 (33%) tumors. Diffuse cyclin D1 and BCOR immunoreactivity was present in 7 of 8 (88%) and 7 of 14 (50%) tumors, respectively. Fluorescence in situ hybridization or targeted RNA sequencing confirmed ZC3H7B-BCOR fusion in all tumors, including four and two previously diagnosed as myxoid leiomyosarcoma and undifferentiated uterine sarcoma, respectively. Limited clinical data suggest that patients present at higher stage and have worse prognosis compared with published outcomes in low-grade endometrial stromal sarcoma. Tumors with ZC3H7B-BCOR fusion constitute a distinct group of endometrial stromal sarcomas with high-grade morphology that should be distinguished from other uterine mesenchymal neoplasms that may demonstrate myxoid morphology.

Fusion of the genes BRD8 and PHF1 in endometrial stromal sarcoma.

We present a new endometrial stromal sarcoma (ESS)-associated genomic rearrangement involving chromosome arms 5p and 6p and leading to the formation of a BRD8-PHF1 fusion gene. The PHF1 (PHD finger protein 1) gene, from 6p21, is known to be rearranged in ESS in a promiscuous way inasmuch as it has been shown to recombine with JAZF1, EPC1, MEAF6, and now also with BRD8, in tumors of this type. In all rearrangements of PHF1, including the present one, a recurrent theme is that the entire coding part of PHF1 constitutes the 3' end of the fusion. BRD8 (bromodomain containing 8) encodes a protein which is involved in regulation of protein acetylation and/or histone acetyl transferase activity. All the genetic fusions identified so far in ESS appear to recombine genes involved in transcriptional regulation, that is, polycomb group complex-mediated and aberrant methylation/acetylation genes. This adds to the likelihood that the new BRD8-PHF1 shares the same pathogenetic mechanism as the other ESS-specific rearrangements.

Involvement of DPP9 in gene fusions in serous ovarian carcinoma.

BACKGROUND: A fusion gene is a hybrid gene consisting of parts from two previously independent genes. Chromosomal rearrangements leading to gene breakage are frequent in high-grade serous ovarian carcinomas and have been reported as a common mechanism for inactivating tumor suppressor genes. However, no fusion genes have been repeatedly reported to be recurrent driver events in ovarian carcinogenesis. We combined genomic and transcriptomic information to identify novel fusion gene candidates and aberrantly expressed genes in ovarian carcinomas. METHODS: Examined were 19 previously karyotyped ovarian carcinomas (18 of the serous histotype and one undifferentiated). First, karyotypic aberrations were compared to fusion gene candidates identified by RNA sequencing (RNA-seq). In addition, we used exon-level gene expression microarrays as a screening tool to identify aberrantly expressed genes possibly involved in gene fusion events, and compared the findings to the RNA-seq data. RESULTS: We found a DPP9-PPP6R3 fusion transcript in one tumor showing a matching genomic 11;19-translocation. Another tumor had a rearrangement of DPP9 with PLIN3. Both rearrangements were associated with diminished expression of the 3' end of DPP9 corresponding to the breakpoints identified by RNA-seq. For the exon-level expression analysis, candidate fusion partner genes were ranked according to deviating expression compared to the median of the sample set. The results were collated with data obtained from the RNA-seq analysis. Several fusion candidates were identified, among them TMEM123-MMP27, ZBTB46-WFDC13, and PLXNB1-PRKAR2A, all of which led to stronger expression of the 3' genes. In view of our previous findings of nonrandom rearrangements of chromosome 19 in this cancer type, particular emphasis was given to changes of this chromosome and a DDA1-FAM129C fusion event was identified. CONCLUSIONS: We have identified novel fusion gene candidates in high-grade serous ovarian carcinoma. DPP9 was involved in two different fusion transcripts that both resulted in deregulated expression of the 3' end of the transcript and thus possible loss of the active domains in the DPP9 protein. The identified rearrangements might play a role in tumorigenesis or tumor progression.

Low frequency of ESRRA-C11orf20 fusion gene in ovarian carcinomas.

The identification of recurrent gene fusions in common epithelial cancers--for example, TMPRSS2/ERG in prostate cancer and EML4/ALK in nonsmall cell lung carcinomas--has raised the question of whether fusion genes are pathogenetically important also in ovarian carcinomas. The first recurrent fusion transcript in serous ovarian carcinomas was reported by Salzman et al. in 2011, who used deep paired-end sequencing to detect the fusion gene ESRRA-C11orf20 in 10 out of 67 (15%) serous ovarian carcinomas examined, a finding that holds great promise for our understanding of ovarian tumorigenesis as well as, potentially, for new treatment strategies. We wanted to test how frequent the ESRRA/C11orf20 fusion is in ovarian carcinomas of all subtypes, and therefore examined a series of 230 ovarian carcinomas of which 197 were of the serous subtype and 163 of the 197 were of stages III and IV--that is, the very same carcinoma subset where the fusion transcript had been found. We performed PCR and high-throughput sequencing analyses in search of the fusion transcript. We used the same primers described previously for the detection of the fusion and the same primer combination, but found no ESRRA/C11orf20 fusion in our series. A synthetic DNA plasmid containing the reported ESRRA/C11orf20 fusion was included as a positive control for our PCR experiments. Data from high-throughput sequencing of 23 ovarian carcinomas were screened in search of alternative partner(s) for the ESRRA and/or C11orf20 gene, but none was found. We conclude that the frequency of the ESRRA/C11orf20 gene fusion in serous ovarian carcinomas of stages III and IV must be considerable less than that reported previously (0/163 in our experience compared with 10/67 in the previous study). At the very least, it seems clear that the said fusion cannot be a common pathogenetic event in this tumor type.

Cytogenetic and molecular profile of endometrial stromal sarcoma.

Recent cytogenetic and molecular investigations have improved our understanding of endometrial stromal tumors, including sarcomas (ESS), and helped redefine their classification into more pathogenetically meaningful categories. Because much more can be gained through such studies, we add information on another 22 ESS examined by karyotyping, PCR analysis, expression array analysis, and transcriptome sequencing. In spite of the known preference for certain pathogenetic pathways, we found considerable genetic heterogeneity in high-grade (HG) as well as in low-grade (LG) ESS. Not all HG tumors showed a YWHAE-NUTM chimeric transcript and as many as six LGESS showed no hitherto known ESS-related fusions. Among the transcripts identified by transcriptome sequencing and verified by Sanger sequencing, new variants of ZC3H7-BCOR and its reciprocal BCOR-ZC3H7 were identified as was involvement of the CREBBP and MLLT4 genes (both well known leukemia-related genes) in two new fusions. FISH analysis identified a known EPC1-PHF1 fusion which led to the identification of a new variant at the molecular level. The fact that around 70 genes were found differentially expressed, by microarray analysis, when comparing LGESS showing ESS-related fusions with LGESS without such transcripts, underscores the biochemical importance of the observed genetic heterogeneity and hints that new subgroups/entities in LGESS still remain undiscovered. © 2016 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

Novel fusion genes and chimeric transcripts in ependymal tumors.

We have previously identified two ALK rearrangements in a subset of ependymal tumors using a combination of cytogenetic data and RNA sequencing. The aim of this study was to perform an unbiased search for fusion transcripts in our entire series of ependymal tumors. Fusion analysis was performed using the FusionCatcher algorithm on 12 RNA-sequenced ependymal tumors. Candidate transcripts were prioritized based on the software's filtering and manual visualization using the BLAST (Basic Local Alignment Search Tool) and BLAT (BLAST-like alignment tool) tools. Genomic and reverse transcriptase PCR with subsequent Sanger sequencing was used to validate the potential fusions. Fluorescent in situ hybridization (FISH) using locus-specific probes was also performed. A total of 841 candidate chimeric transcripts were identified in the 12 tumors, with an average of 49 unique candidate fusions per tumor. After algorithmic and manual filtering, the final list consisted of 24 potential fusion events. Raw RNA-seq read sequences and PCR validation supports two novel fusion genes: a reciprocal fusion gene involving UQCR10 and C1orf194 in an adult spinal ependymoma and a TSPAN4-CD151 fusion gene in a pediatric infratentorial anaplastic ependymoma. Our previously reported ALK rearrangements and the RELA and YAP1 fusions found in supratentorial ependymomas were until now the only known fusion genes present in ependymal tumors. The chimeric transcripts presented here are the first to be reported in infratentorial or spinal ependymomas. Further studies are required to characterize the genomic rearrangements causing these fusion genes, as well as the frequency and functional importance of the fusions. © 2016 Wiley Periodicals, Inc.

VOLIN and KJON-Two novel hyperdiploid myeloma cell lines.

Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines' chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology. © 2016 Wiley Periodicals, Inc.

Chromosome 19 rearrangements in ovarian carcinomas: zinc finger genes are particularly targeted.

Chromosome 19 is frequently rearranged in ovarian carcinomas, but the pathogenetic consequences of this are not clearly understood. We performed microarray gene expression analysis on 12 ovarian carcinomas that carry a rearranged chromosome 19 in their karyotype. These aberrant chromosomes have previously been microdissected and analyzed by array-based CGH. In the current study, we wanted to explore whether the genomic alterations thus detected correlated with changes in gene expression. The microarray gene expression analysis gave information on 407 genes mapping in gained genomic regions on chromosome 19, of which 92 showed association between DNA gain and upregulated expression. Of the genes showing this association, 39 (42%) showed gain in at least two samples. The majority of these 39 genes of interest (n = 24, 62%) encode zinc finger proteins, which otherwise make up only 15% of the approximately 1,400 genes on chromosome 19. The strongest association was found for ZNF223 which was upregulated in samples with genomic gain compared with samples without gain. We suggest that DNA copy number changes brought about by rearrangements of chromosome 19 contribute to ovarian carcinogenesis by leading to upregulation of ZNF223 and other zinc finger genes. © 2014 Wiley Periodicals, Inc.

Frequent translocations of 11q13.2 and 19p13.2 in ovarian cancer.

Aberrations of chromosome arm 19p in ovarian cancer were first described decades ago and have been confirmed in recent publications, which have focused on chromosome 11 as a translocation partner. Recently, genetic analysis of the ovarian cancer cell line SKOV3 revealed a rearrangement described as der(19)t(11;19)(q13.2;p13.2), which lead to a fusion protein containing parts of HOOK2 and frame shifted ACTN3 that had unknown functionality. To evaluate the frequency of these breakpoints, we used fluorescence in situ hybridization (FISH) probes flanking these genes for interphase analysis of ovarian cancer cells. We analyzed 49 primary cell cultures of ovarian cancers using FISH probes next to these breakpoints on chromosomes 11 and 19 defined in SKOV3. Co-localizations of the signals in interphase nuclei were considered to be positive fusions when the frequency was over the experimentally calculated cutoff of 24.3% (mean average value for normal ovary cells plus three times the standard deviation). Fusions between 11q13.2 and 19p13.2 were confirmed in 22 (45%) primary cell cultures of ovarian cancers. However, by PCR, the fusion originally described in SKOV3 was not detected in any of the primary cell cultures. Our results confirm other reports and show that these regions are very frequently involved in chromosomal rearrangements in ovarian cancer. Furthermore, they reveal a significant correlation (P = 0.023) of co-localized signals of 11q13.2 and 19p13.2 with low and intermediate grades in ovarian cancer.

Genomic profile of ovarian carcinomas.

BACKGROUND: It is known that all tumors studied in sufficient number to draw conclusions show characteristic/specific chromosomal rearrangements, and the identification of these chromosomes and the genes rearranged behind the aberrations may ultimately lead to a tailor-made therapy for each cancer patient. Knowledge about the acquired genomic aberrations of ovarian carcinomas is still unsatisfactory. METHODS: We cytogenetically analyzed 110 new cases of ovarian carcinoma of different histological subtypes using karyotyping of G-banded chromosomes and high-resolution comparative genomic hybridization. We first compared the aberration patterns identified by the two genomic screening techniques using the so-called "classical" pathological classification in which the carcinomas are grouped as tumors of types I and II. We also broke down our findings according to the more "modern" classification which groups the carcinomas in five different categories. RESULTS: The chromosomal breakpoints identified by karyotyping tended to cluster to 19p/q and to 11q, but no unquestionably recurrent rearrangement could be seen. Common imbalances were scored as gains from 1q, 3q, 7q, and 8q and losses from 17p, 19q, and 22q. Gain of material from 8q23 and losses from 19q and 22q have previously been described at high frequencies in bilateral and borderline ovarian carcinomas. The fact that they were present both in "precursor" lesions, i.e., borderline tumors, as well as in tumors of more advanced stages, i.e., carcinomas, highlights the possibility of an adenoma-carcinoma sequence in ovarian carcinogenesis. CONCLUSION: Based on the relatively simple genomic changes we identified in the low-grade serous carcinomas examined (n = 7) and which largely corresponded to the aberration pattern formerly identified in borderline tumors, one can interpret the cytogenetic data as supporting the view that the low-grade carcinomas represent a phenotypically more advanced stage of borderline tumors. Whether transition from low-grade to high-grade carcinoma also occurs, is a question about which the genomic data is still inconclusive.

RNA sequencing identifies fusion of the EWSR1 and YY1 genes in mesothelioma with t(14;22)(q32;q12).

Mesothelioma is a rare but very aggressive tumor derived from mesothelial cells. A number of often complex but nonrandom cytogenetic abnormalities have been found in these tumors, resulting in loss of chromosome bands 14q32 and 22q12 in more than 35% of the cases. In this study, we used RNA sequencing to search for fusion transcripts in a mesothelioma carrying a t(14;22)(q32;q12) as the sole chromosomal aberration and found an EWSR1-YY1 and its reciprocal YY1-EWSR1 fusion transcript. Screening 15 additional cases of mesothelioma from which we had RNA but no cytogenetic information, we identified one more tumor carrying an EWSR1-YY1 fusion gene but not the reciprocal YY1-EWSR1 transcript. RT-polymerase chain reaction and sequencing showed that in both cases exon 8 of EWSR1 (nucleotide 1,139, accession number NM_013986 version 3, former exon 7 in sequence with accession number X66899) was fused to exon 2 of YY1 (nucleotide 1,160, accession number NM_003403 version 3). The EWSR1 breakpoint in exon 8 in the EWSR1-YY1 chimeric transcript is similar to what is found in other fusions involving EWSR1 such as EWSR1-FLI1, EWSR1-DDIT3, and EWSR1-ATF1. The EWSR1-YY1-encoded protein is an abnormal transcription factor with the transactivation domain of EWSR1 and the DNA-binding domain of YY1. This is the first study to detect a specific fusion gene in mesothelioma (the reason how frequent the EWSR1-YY1 fusion is remains uncertain) and also the first time that direct involvement of YY1 in oncogenesis has been demonstrated.

Fusion of the ZC3H7B and BCOR genes in endometrial stromal sarcomas carrying an X;22-translocation.

Endometrial stromal sarcomas (ESS) are genetically heterogeneous uterine tumors in which a JAZF1-SUZ12 chimeric gene resulting from the chromosomal translocation t(7;17)(p15;q21) as well as PHF1 rearrangements (in chromosomal band 6p21) with formation of JAZF1-PHF1, EPC1-PHF1, and MEAF6-PHF1 chimeras have been described. Here, we investigated two ESS characterized cytogenetically by the presence of a der(22)t(X;22)(p11;q13). Whole transcriptome sequencing one of the tumors identified a ZC3H7-BCOR chimeric transcript. Reverse transciptase-PCR with the ZC3H7B forward and BCOR reverse primer combinations confirmed the presence of a ZC3H7-BCOR chimeric transcript in both ESS carrying a der(22)t(X;22) but not in a control ESS with t(1;6) and the MEAF6-PHF1 fusion. Sequencing of the amplified cDNA fragments showed that in both cases ESS exon 10 of ZC3H7B (from 22q13; accession number NM_017590 version 4) was fused to exon 8 of BCOR (from Xp11; accession number NM_001123385 version 1). Reciprocal multiple BCOR-ZC3H7B cDNA fragments were amplified in only one case suggesting that ZC3H7B-BCOR, on the der(22)t(X;22), is the pathogenetically important fusion gene. The putative ZC3H7B-BCOR protein would contain the tetratricopeptide repeats and LD motif from ZC3H7B and the AF9 binding site (1093-1233aa), the 3 ankyrin repeats (1410-1509 aa), and the NSPC1 binding site of BCOR. Although the presence of these motifs suggests various functions of the chimeric protein, it is possible that its most important role may be in epigenetic regulation. Whether or not the (patho)genetic subsets JAZF1-SUZ12, PHF1 rearrangements, and ZC3H7B-BCOR correspond to any phenotypic, let alone clinically important, differences in ESS remain unknown.

Genomic aberration patterns and expression profiles of squamous cell carcinomas of the vulva.

Little is known about the genomic abnormalities of squamous cell carcinomas (SCC) of the vulva and how they correlate with gene expression. We determined the genomic and expression profiles of 15 such SCC using karyotyping, DNA ploidy analysis, arrayCGH, and expression arrays. Four of the five cases with clonal chromosomal aberrations found by G-banding showed highly abnormal karyotypes with multiple rearrangements. The imbalances scored by arrayCGH mapped to different chromosomes with losses being more common than gains. Frequent losses were scored from 3p and 8p whereas gains were frequent from 3q and 8q (loss of 8p with concomitant gain of 8q mostly occurred via 8q isochromosome formation). This is the first study of vulvar tumors using arrayCGH, and some frequent imbalances could be defined precisely. Of particular note were the sometimes large, sometimes small deletions of 3p and 9p which had minute areas in 3p14 and 9p23 as minimal commonly deleted regions. FHIT (3p14) and PTPRD (9p23) are the only genes here. They were both lost in seven cases, including homozygous losses of PTPRD in four tumors. Using qPCR we could demonstrate deregulation of the FHIT gene in tumor cells. Hence, this gene is likely to play a pathogenetic role in vulvar SCC tumorigenesis. Expression array analyses also identified a number of other genes whose expression profile was altered. Notable among the downregulated genes were MAL (in 2q11), KRT4 (in 12q13), and OLFM4 (in 13q14), whereas upregulated genes included SPRR2G (in 1q21.3) and S100A7A (in 1q21.3).

Breakpoint characterization of the der(19)t(11;19)(q13;p13) in the ovarian cancer cell line SKOV-3.

About 20% of ovarian carcinomas show alterations of 19p13 and/or 19q13 in the form of added extra material whose origin often is from chromosome 11. Based on earlier spectral karyotype analysis of the ovarian cancer cell line SKOV-3, which shows an unbalanced translocation der(19)t(11;19), the aim of this study was to determine the precise breakpoints of that derivative chromosome. After rough delimitation of the breakpoints of microdissected derivative chromosomes by array analysis, we designed a matrix of primers spanning 11q13.2 and 19p13.2 detecting multiple amplicons on genomic and cDNA. Sequencing the amplicons, accurate localization of both breakpoints on both chromosomes was possible and we found that exon 14 of HOOK2 from chromosome 19 and exon 2 of ACTN3 from chromosome 11 were fused in the derivative chromosome. The breakpoint in the HOOK2 gene was in an intrinsic triplet of nucleic acids leading to a shift in the ACTN3 reading frame in the derivative chromosome. This frameshift alteration should give rise to an early stop codon causing a loss of function of ACTN3. Signals in two-dimensional Western blotting exactly match to calculated molecular mass and the isoelectric point of the fusion protein.

Genetic Characterization of Pediatric Mixed Phenotype Acute Leukemia (MPAL).

BACKGROUND/AIM: Mixed phenotype acute leukemia (MPAL) is a rare hematologic malignancy in which the leukemic cells cannot be assigned to any specific lineage. The lack of well-defined, pathogenetically relevant diagnostic criteria makes the clinical handling of MPAL patients challenging. We herein report the genetic findings in bone marrow cells from two pediatric MPAL patients. PATIENTS AND METHODS: Bone marrow cells were examined using G-banding, array comparative genomic hybridization, RNA sequencing, reverse transcription polymerase chain reaction, Sanger sequencing, and fluorescence in situ hybridization. RESULTS: In the first patient, the genetic analyses revealed structural aberrations of chromosomal bands 8p11, 10p11, 11q21, and 17p11, the chimeras MLLT10::PICALM and PICALM::MLLT10, and imbalances (gains/losses) on chromosomes 2, 4, 8, 13, and 21. A submicroscopic deletion in 21q was also found including the RUNX1 locus. In the second patient, there were structural aberrations of chromosome bands 1p32, 8p11, 12p13, 20p13, and 20q11, the chimeras ETV6::LEXM and NCOA6::ETV6, and imbalances on chromosomes 2, 8, 11, 12, 16, 19, X, and Y. CONCLUSION: The leukemic cells from both MPAL patients carried chromosome aberrations resulting in fusion genes as well as genomic imbalances resulting in gain and losses of many gene loci. The detected fusion genes probably represent the main leukemogenic events, although the gains and losses are also likely to play a role in leukemogenesis.

NUP214 fusion genes in acute leukemias: genetic characterization of rare cases.

Introduction: Alterations of the NUP214 gene (9q34) are recurrent in acute leukemias. Rearrangements of chromosomal band 9q34 targeting this locus can be karyotypically distinct, for example t(6;9)(p22;q34)/DEK::NUP214, or cryptic, in which case no visible change of 9q34 is seen by chromosome banding. Methods: We examined 9 cases of acute leukemia with NUP214 rearrangement by array Comparative Genomic Hybridization (aCGH), reverse-transcription polymerase chain reaction (RT-PCR), and cycle sequencing/Sanger sequencing to detect which fusion genes had been generated. Results: The chimeras DEK::NUP214, SET::NUP214, and NUP214::ABL1 were found, only the first of which can be readily detected by karyotyping. Discussion: The identification of a specific NUP214 rearrangement is fundamental in the management of these patients, i.e., AMLs with DEK::NUP214 are classified as an adverse risk group and might be considered for allogenic transplant. Genome- and/or transcriptome-based next generation sequencing (NGS) techniques can be used to screen for these fusions, but we hereby present an alternative, step-wise procedure to detect these rearrangements.