Francesca Micci

Consistent rearrangement of chromosomal band 6p21 with generation of fusion genes JAZF1/PHF1 and EPC1/PHF1 in endometrial stromal sarcoma

Endometrial stromal sarcomas (ESS) represent <10% of all uterine sarcomas. Cytogenetic data on this tumor type are limited to 32 cases, and the karyotypes are often complex, but the pattern of rearrangement is nevertheless clearly nonrandom with particularly frequent involvement of chromosome arms 6p and 7p. Recently, a specific translocation t(7;17)(p15;q21) leading to the fusion of two zinc finger genes, juxtaposed with another zinc finger (JAZF1) and joined to JAZF1 (JJAZ1), was described in a subset of ESS. We present three ESS whose karyotypes were without the disease-specific t(7;17) but instead showed rearrangement of chromosomal band 6p21, twice as an unbalanced t(6p;7p) and once as a three-way 6;10;10 translocation. All three tumors showed specific rearrangement of the PHD finger protein 1 (PHF1) gene, located in chromosomal band 6p21. In the two tumors with t(6;7), PHF1 was recombined with the JAZF1 gene from 7p15, leading to the formation of a JAZF1/PHF1 fusion gene. The third tumor showed a t(6p;10q;10p) as the sole karyotypic abnormality, leading to the fusion of PHF1 with another partner, the enhancer of polycomb (EPC1) gene from 10p11; EPC1 has hitherto not been associated with neoplasia. The PHF1 gene encodes a protein with two zinc finger motifs whose involvement in tumorigenesis and/or tumor progression has not been reported before, but its rearrangement clearly defines a new pathogenetic subgroup of ESS.

High‐resolution analysis of genetic stability of human adipose tissue stem cells cultured to senescence

The potential use of human mesenchymal stem cells for therapeutic applications implies large scale in vitro culture, increasing the probability of genetic instability and transformation. We examine here the incidence of unbalanced and balanced chromosome rearrangements in polyclonal and single cell‐derived cultures of human adipose stem cells to senescence. G‐banding karyotyping of the polyclonal cultures shows a normal karyotype. In addition, high‐resolution microarray‐based comparative genomic hybridization analyses relative to uncultured adipose stem cells from the same donors reveal overall genomic stability in long‐term (∼6 months) polyclonal and clonal culture. One adipose stem cell clone displayed minor deletions in gene‐rich telomeric and sub‐telomeric regions on three chromosomes in early passage. This however, was detected only in a sub‐population of cells that was subsequently spontaneously eliminated from the culture. Apparent pericentromeric instabilities are also occasionally detected in specific chromosomes. Our results indicate that clonal chromosomal aberrations may arise transiently in early passage adipose stem cells (ASC) cultures. Nonetheless, incidence of these aberrations seems to be negligible in the majority of long‐term ASC cultures, at least under the culture conditions used here.

DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB5, SCGB3A1, and CRABP1 as novel targets

BackgroundThe epigenetics of ovarian carcinogenesis remains poorly described. We have in the present study investigated the promoter methylation status of 13 genes in primary ovarian carcinomas (n = 52) and their in vitro models (n = 4; ES-2, OV-90, OVCAR-3, and SKOV-3) by methylation-specific polymerase chain reaction (MSP). Direct bisulphite sequencing analysis was used to confirm the methylation status of individual genes. The MSP results were compared with clinico- pathological features.ResultsEight out of the 13 genes were hypermethylated among the ovarian carcinomas, and altogether 40 of 52 tumours were methylated in one or more genes. Promoter hypermethylation of HOXA9, RASSF1A, APC, CDH13, HOXB5, SCGB3A1 (HIN-1), CRABP1, and MLH1 was found in 51% (26/51), 49% (23/47), 24% (12/51), 20% (10/51), 12% (6/52), 10% (5/52), 4% (2/48), and 2% (1/51) of the carcinomas, respectively, whereas ADAMTS1, MGMT, NR3C1, p14ARF, and p16INK 4awere unmethylated in all samples. The methylation frequencies of HOXA9 and SCGB3A1 were higher among relatively early-stage carcinomas (FIGO I-II) than among carcinomas of later stages (FIGO III-IV; P = 0.002, P = 0.020, respectively). The majority of the early-stage carcinomas were of the endometrioid histotype. Additionally, HOXA9 hypermethylation was more common in tumours from patients older than 60 years of age (15/21) than among those of younger age (11/30; P = 0.023). Finally, there was a significant difference in HOXA9 methylation frequency among the histological types (P = 0.007).ConclusionDNA hypermethylation of tumour suppressor genes seems to play an important role in ovarian carcinogenesis and HOXA9, HOXB5, SCGB3A1, and CRABP1 are identified as novel hypermethylated target genes in this tumour type.

Detection of a t(1;22)(q23;q12) translocation leading to an EWSR1-PBX1 fusion gene in a myoepithelioma.

Chromosome banding as well as molecular cytogenetic methods are of great help in the diagnosis of mesenchymal tumors. Myoepithelial neoplasms of soft tissue including myoepitheliomas, mixed tumors, and parachordomas are diagnoses that have been increasingly recognized the last few years. It is still debated which neoplasms should be included in these morphologically heterogeneous entities, and the boundaries between them are not clear‐cut. The pathogenetic mechanisms behind myoepithelial tumors are unknown. Only five parachordomas and one mixed tumor have previously been karyotyped, and nothing is known about their molecular genetic characteristics. We present a mesenchymal tumor classified as a myoepithelioma that had a balanced translocation t(1;22)(q23;q12) as the sole karyotypic change. A novel EWSR1‐PBX1 fusion gene consisting of exons 1–8 of the 5′‐end of EWSR1 and exons 5–9 of the 3′‐end of PBX1 was shown to result from the translocation. Both genes are known to be targeted also by other neoplasia‐specific translocations, PBX1 in acute lymphoblastic leukemia and EWSR1 in several solid tumors, most of which are malignant. Based on the structure of the novel fusion gene detected, its transforming mechanism is thought to be the same as for other fusion genes involving EWSR1 or PBX1.

Cytogenetic and molecular genetic analyses of endometrial stromal sarcoma: nonrandom involvement of chromosome arms 6p and 7p and confirmation of JAZF1/JJAZ1 gene fusion in t(7;17)

Endometrial stromal sarcomas (ESS) are rare neoplasms with the capacity both to invade the myometrium locally and to give rise to extrauterine metastases. Cytogenetic abnormalities have been reported in 22 cases of ESS, mostly involving rearrangements of chromosomes 6, 7, and 17. The most characteristic translocation of this tumor type, t(7;17)(p15 approximately p21;q12 approximately q21), was recently shown to generate a JAZF1/JJAZ1 fusion gene. We report three additional cases of ESS with abnormal karyotypes, whose interpretation was based on the combined analysis by conventional cytogenetics and cross-species color banding FISH (RxFISH). The combination of G-banding and RxFISH in every case gave additional information beyond that obtained by either technique alone, determining the identity of even complex inter- as well as intrachromosomal rearrangements. In one of the three tumors, a t(7;17) was seen; molecular genetic studies identified the JAZF1/JJAZ1 fusion gene in this case. Two tumors had aberrations that included structural changes of chromosome arms 6p and 7p. Evidently, karyotypic, and hence pathogenetic, heterogeneity exists for tumors classified as endometrial stromal sarcomas based on their phenotypic features.

Genomic Aberrations in Carcinomas of the Uterine Corpus

Endometrial carcinoma, the most common invasive neoplasm of the female genital tract, occurs either in a hormone‐related, less virulent form (type I) or in a hormone‐independent, more aggressive form (type II). Another cancer of the uterine corpus is carcinosarcoma, a biphasic or mixed epithelial–mesenchymal tumor, now classified as metaplastic carcinoma. We examined by karyotyping and comparative genomic hybridization a consecutive series of 67 endometrial carcinomas and 15 carcinosarcomas and compared the cytogenetic features of the different carcinoma subtypes. All three subtypes of uterine carcinoma had in common a nonrandom gain of material from 1q and 8q but differed from one another in other respects. Endometrial carcinomas of type I mostly presented gains from chromosome arms 1q and 8q and losses from Xp, 9p, 9q, 17p, 19p, and 19q, whereas endometrial carcinomas of type II showed a more complex imbalance picture, with gains from chromosome arms 1q, 2p, 3q, 5p, 6p, 7p, 8q, 10q, and 20q and losses from Xq, 5q, and 17p. The carcinosarcomas mostly showed gains of or from 1q, 5p, 8q, and 12q but losses from 9q, that is, they were much more similar to endometrial carcinomas in their pattern of acquired genomic changes than to sarcomas of the uterine corpus. It was also possible to identify different copy number changes among the different grades of type I carcinomas, between serous papillary and clear‐cell carcinomas of type II, as well as between homologous and heterologous carcinosarcomas. Specifically, type I adenocarcinomas that were highly differentiated mostly showed gains from 1q and 10p; those that were moderately differentiated showed gains from 1q, 7p, 7q, and 10q as well as losses from Xp, 9p, 9q, 17p, 19p, and 19q; whereas those poorly differentiated showed gains from 1q, 2p, 2q, 3q, 6p, 8q, and 20q but losses from Xp, Xq, 5q, 9p, 9q, 17p, and 17q. The serous papillary carcinomas showed gains from 1q, 2p, 2q, 3q, 5p, 6p, 6q, 7p, 8q, 18q, 20p, and 20q but losses from 17p, whereas the clear‐cell carcinomas showed gains from 3q, 7p, 8q, 10q, 16p, and 20q but losses from 6q. Finally, the homologous carcinosarcomas presented gains from 1p, 1q, 8q, 12q, and 17q as well as losses from 9q and 13q, whereas the heterologous tumors showed gains from 1q, 8p, and 8q. The reproducibility of the observed correlations between karyotypic aberration patterns and histological differentiation was underscored by the fact that those carcinosarcomas whose epithelial component resembled type I endometrial carcinomas also exhibiting a type I aberration profile, whereas carcinosarcomas with a type II carcinoma differentiation had karyotypic abnormalities similar to those of type II endometrial carcinomas.

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.

Novel Fusion of MYST/Esa1-Associated Factor 6 and PHF1 in Endometrial Stromal Sarcoma

Rearrangement of chromosome band 6p21 is recurrent in endometrial stromal sarcoma (ESS) and targets the PHF1 gene. So far, PHF1 was found to be the 3′ partner in the JAZF1-PHF1 and EPC1-PHF1 chimeras but since the 6p21 rearrangements involve also other chromosomal translocation partners, other PHF1-fusions seem likely. Here, we show that PHF1 is recombined with a novel fusion partner, MEAF6 from 1p34, in an ESS carrying a t(1;6)(p34;p21) translocation as the sole karyotypic anomaly. 5′-RACE, RT-PCR, and sequencing showed the presence of an MEAF6-PHF1 chimera in the tumor with exon 5 of MEAF6 being fused in-frame to exon 2 of PHF1 so that the entire PHF1 coding region becomes the 3′ terminal part of the MEAF6-PHF1 fusion. The predicted fusion protein is composed of 750 amino acids and contains the histone acetyltransferase subunit NuA4 domain of MEAF6 and the tudor, PHD zinc finger, and MTF2 domains of PHF1. Although the specific functions of the MEAF6 and PHF1 proteins and why they are targeted by a neoplasia-specific gene fusion are not directly apparent, it seems that rearrangement of genes involved in acetylation (EPC1, MEAF6) and methylation (PHF1), resulting in aberrant gene expression, is a common theme in ESS pathogenesis.

Extraskeletal myxoid chondrosarcoma : multimodal diagnosis and identification of a new cytogenetic subgroup characterized by t(9;17)(q22;q11)

Extraskeletal myxoid chondrosarcoma is a rare malignant soft tissue tumour that can be difficult to diagnose correctly, especially preoperatively. We describe four cases of extraskeletal myxoid chondrosarcoma of the extremities diagnosed by a multimodal approach. The cytological examination of fine-needle aspirates showed small and round, mildly pleomorphic cells lying in sheets and cords, but also dispersed within a myxoid and metachromatic intercellular substance. Histological, electron microscopic and immunocytochemical examination also yielded findings compatible with the diagnosis of extraskeletal myxoid chondrosarcoma. Cytogenetic analysis demonstrated a t(9;22)(q22;q12) in two tumours and a t(9;17)(q22;q11) in the third and fourth. The translocation t(9;22)(q22;q12) has been described repeatedly in extraskeletal myxoid chondrosarcoma but never in other tumours; hence, the detection of this pathognomonic chromosome abnormality in short-term cultured cells from fine-needle aspirates verified the diagnosis in two of the cases. The t(9;17)(q22;q11) found in the last two cases probably represents a new cytogenetic subgroup of extraskeletal myxoid chondrosarcoma as it, too, is unknown in other contexts. The multimodal approach taken in these four cases enabled a definite diagnosis of a rare malignant tumour whose cytological and histological features alone are usually not sufficiently distinct to rule out other differential diagnostic possibilities.

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 EWSR1such 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.