Marta Brunetti

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.

Genomic imbalances are involved in miR-30c and let-7a deregulation in ovarian tumors: implications for HMGA2 expression

The High-mobility group AT-hook 2 protein (HMGA2) is involved in different processes during tumorigenesis. High expression levels of HMGA2 are found in various types of cancer, with recent studies highlighting the important role of miRNAs in the regulation of HMGA2 expression. We report a study of 155 ovarian tumors (30 sex-cord stromal tumors, 22 borderline tumors, and 103 carcinomas) analyzed for HMGA2 expression as well as the expression of two miRNAs targeting this gene, let-7a and miR-30c. We also evaluated the expression of the fragile histidine triad (FHIT) and lin28 homologues (LIN28A/B) genes which are known to be an enhancer of miR-30c expression and a repressor of let-7a, respectively. HMGA2 was found expressed at high levels in most samples analyzed, with clear cell carcinomas as the only exception. let-7a and miR-30c were highly deregulated in all tumor types. LIN28A and FHIT were found overexpressed in all examined tumor types. The chromosomal imbalances that might lead to loss of the genes expressing let-7a and miR-30c could be evaluated on the basis of previously generated karyotypic and high resolution comparative genomic hybridization (CGH) data on 103 tumors. 76% of the samples with an imbalanced genome had at least one chromosomal aberration leading to a deletion of a miRNA cluster for let-7a and miR-30c. FISH using locus specific probes for these clusters validate the aberrations at the gene level. Our study shows that genomic imbalances are involved in miR-30c and let-7a deregulation. One can reasonably assume that dysregulation of these miRNAs is a cause leading to HMGA2 upregulation in ovarian tumors.

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.

Expressions of miR-30c and let-7a are inversely correlated with HMGA2 expression in squamous cell carcinoma of the vulva

Malignant tumors of the vulva, most of them squamous cell carcinomas, account for only 5% of cancers of the female genital tract. Though little is known about the genetic features of these tumors, the Fragile Histidine Triad (FHIT) and High Mobility Group AT-hook 2 (HMGA2) genes were found deregulated. We wanted to gain more knowledge about the expression of HMGA2-related miRNAs such as miR-30c and let-7a, and whether a correlation exists between the expression of FHIT and HMGA2, in this tumor type. An inverse correlation was found in-as-much as HMGA2 was highly expressed (mean fold change 8.8) whereas miR30c and let-7a were both downregulated (mean fold change -3.9 and -2.3, respectively). The consistent overexpression of HMGA2 found in all tumors adds to the likelihood that this gene is of importance in SCC pathogenesis. Moreover, we came to the conclusion that miRNAs may be the cause of the deregulation of HMGA2. Our results also show that SCC of the vulva presents a characteristic molecular pattern with FHIT being downregulated whereas HMGA2 is upregulated.

Identification of an EPC2-PHF1 fusion transcript in low-grade endometrial stromal sarcoma

Recurrent chromosomal translocations leading to gene fusion formation have been described in uterine sarcomas, including low-grade endometrial stromal sarcoma (LG-ESS). Involvement of the PHF1 gene in chromosomal rearrangements targeting band 6p21 has been found in LG-ESS with different partners from JAZF1 mapping in 7p15, to EPC1 from 10p11, MEAF6 from 1p34, and BRD8 from 5q31. In the present study, RNA sequencing of a LG-ESS showed a novel recombination of PHF1 with the Enhancer of Polycomb homolog 2 (EPC2). RT-PCR followed by Sanger sequencing and FISH analysis confirmed the EPC2-PHF1 fusion transcript.

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.

Genetic heterogeneity in leiomyomas of deep soft tissue

Leiomyoma of deep soft tissue is a rare type of benign smooth muscle tumor that mostly occurs in the retroperitoneum or abdominal cavity of women, and about which very little genetic information exists. In the present study, eight leiomyomas of deep soft tissue were genetically analyzed. G-banding showed that three tumors carried rearrangements of the long arm of chromosome 12, three others had 8q rearrangements, the 7th tumor had deletion of the long arm of chromosome 7, del(7)(q22), and the 8th had aberrations of chromosome bands 3q21∼23 and 11q21∼22. The target genes of the 12q and 8q aberrations were HMGA2 and PLAG1, respectively. In the leiomyomas with 12q rearrangements, both HMGA2 and PLAG1 were expressed whereas in the tumors with 8q aberrations, only PLAG1 was expressed. In the cases without 12q or 8q aberrations, the expression of HMGA2 was very low and PLAG1 was expressed only in the case with del(7)(q22). All eight leiomyomas of deep soft tissue expressed MED12 but none of them had mutation in exon 2 of that gene. In two tumors with 12q rearrangements, RPSAP52 on 12q14.3 was fused with non-coding RNA (accession number XR_944195) from 14q32.2 or ZFP36L1 from14q24.1. In a tumor with inv(12), exon 3 of HMGA2 was fused to a sequence in intron 1 of the CRADD gene from 12q22. The present data together with those of our two previous studies in which the fusions KAT6B-KANSL1 and EWSR1-PBX3 were described in two retroperitoneal leiomyomas carrying a t(10;17)(q22;q21) and a t(9;22)(q33;q12) translocation, respectively, show that leiomyomas of deep soft tissue are genetically heterogenous but have marked similarities to uterine leiomyomas.

The microRNA miR-192/215 family is upregulated in mucinous ovarian carcinomas

Different microRNAs are dysregulated in ovarian cancer where some of them have proved to be valid biomarkers. miRNA profiling analyses have shown that the different histotypes of ovarian carcinoma display differential expression of specific miRNAs. In the present study, we used miRNA-sequencing and Real-Time qPCR to detect the expression levels of miRNAs belonging to the miRNA-192/215 family, namely miR-192, miR-194, and miR-215, in different types of ovarian neoplasia, finding that miR-192, miR-194, and miR-215 were upregulated in ovarian carcinomas of the mucinous subtype, but downregulated in other types of carcinoma and in sex cord-stromal tumors. The expression of the said miRNAs was 6-fold higher in mucinous tumors compared to the other histotypes making them candidates for a possible role as diagnostic biomarkers.

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

Recurrent fusion transcripts in squamous cell carcinomas of the vulva

Juxtaposition of two different genes or gene parts due to chromosomal rearrangement is a well-known neoplasia-associated pathogenetic mechanism. The detection and characterization of such tumorigenic fusions is of great importance both research-wise, diagnostically because they may be specific for distinct tumor entities, and because they may serve as therapeutic targets for antioncogenic drugs that interact directly with the molecular changes responsible for neoplastic transformation. At present, more than 10,000 fusion transcripts have been reported in different types of neoplasia, with one tenth of them being identified in squamous cell carcinomas (SCC) of different locations. No recurrent fusion gene has to date been identified in SCC of the vulva. We performed high-throughput paired-end RNA-sequencing of 12 vulvar SCC and found two recurrent fusions with the STIP1-CREB3L1 and ZDHHC5-GPR137 being present in two tumors each. The transcripts were detected only in the tumor samples, not in normal vulvar tissue from healthy donors used as control. The CREB3L1 and ZDHHC5 genes encode proteins involved in transcription suggesting that the chimeras may alter downstream events in their respective pathways. Expression analysis of the CREB3L1 gene showed the presence of two distinct groups of tumors, one having fusion and downregulation of the gene and the other showing upregulation of CREB3L1.