Abstract
Background/Aim: Mesotheliomas are tumors similar to, and probably derived from, mesothelial cells. They carry acquired chromosomal rearrangements, deletions affecting CDKN2A, pathogenetic polymorphisms in NF2, and fusion genes which often contain the promiscuous EWSR1, FUS, and ALK as partner genes. Here, we report the cytogenomic results on two peritoneal mesotheliomas. Materials and Methods: Both tumors were examined using G-banding with karyotyping and array comparative genomic hybridization (aCGH). One of them was further investigated with RNA sequencing, reverse transcription polymerase chain reaction (RT-PCR), Sanger sequencing, and fluorescence in situ hybridization (FISH). Results: In the first mesothelioma, the karyotype was 25∼26,X,+5,+7,+20[cp4]/50∼52,idemx2[cp7]/46,XX[2]. aCGH detected gains of chromosomes 5, 7, and 20 with retained heterozygosity on these chromosomes. In the second tumor, the karyotype was 46,XX,inv(10)(p11q25)[7]/46,XX[3]. aCGH did not detect any gains or losses and showed heterozygosity for all chromosomes. RNA sequencing, RT-PCR/Sanger sequencing, and FISH showed that the inv(10) fused MAP3K8 from 10p11 with ABLIM1 from 10q25. The MAP3K8::ABLIM1 chimera lacked exon 9 of MAP3K8. Conclusion: Our data, together with information on previously described mesotheliomas, illustrate two pathogenetic mechanisms in peritoneal mesothelioma: One pathway is characterized by hyperhaploidy, but with retained disomies for chromosomes 5, 7, and 20; this may be particularly prevalent in biphasic mesotheliomas. The second pathway is characterized by rearrangements of MAP3K8 from which exon 9 of MAP3K8 is lost. The absence of exon 9 from oncogenetically rearranged MAP3K8 is a common theme in thyroid carcinoma, lung cancer, and spitzoid as well as other melanoma subtypes.
- Peritoneal mesothelioma
- biphasic mesothelioma
- hyperhaploidy
- disomic chromosomes
- inv(10)(p11q25)
- MAP3K8::ABLIM1 chimeric gene
- exon 9 of MAP3K8
Mesotheliomas are rare, mostly malignant tumors similar to, and probably derived from, mesothelial cells, i.e., the cells lining the body’s serous cavities (1-3). More than 80% of mesotheliomas are developed in the wall lining of the pleural cavity, 7% in the peritoneum, 1% in the pericardium, and less than 1% in the tunica vaginalis (4, 5). The three histological types, epithelioid, sarcomatoid, and biphasic (the latter subtype has both epithelioid and sarcomatous differentiation), account for 50-60%, 10%, and 30-40% of mesotheliomas, respectively (3), and influence disease prognosis (6, 7). Malignant mesothelioma is strongly associated with exposure to asbestos (8-11). Other etiological factors are genetic predisposition (12-14), radiation exposure, and viral infection that alone or together with asbestos exposure can cause malignant mesotheliomas (15, 16).
Mesotheliomas carry acquired genetic aberrations (17). Cytogenetic analysis of 131 tumors has shown that most mesotheliomas have complex karyotypes (18). Comparative genomic hybridization, loss of heterozygosity, and fluorescence in situ hybridization (FISH) studies have detected nonrandom gains and losses of chromosomal regions (19-21). Deletions of or from chromosome arms 1p, 3p, 6q, 9p, 15q, and 22q are frequent, homozygous deletions have been found to be the main mechanism affecting p16/CDKN2A (on 9p21), and inactivating mutations coupled with allelic loss were found to involve the NF2 locus (on 22q12) (17, 22).
Next generation sequencing (also called high throughput sequencing, deep sequencing etcetera) techniques were shown to be a valuable tool in detecting acquired genetic alterations in mesothelioma (23-26) and RNA sequencing was found to be a powerful tool for detection of fusion genes in cancer (27, 28). Using sequential combination of karyotyping and RNA-sequencing (27), our group detected an EWSR1::YY1 chimera and its reciprocal YY1::EWSR1 fusion transcript in a malignant mesothelioma carrying a t(14;22)(q32;q12) chromosomal translocation (29). In the same study, we identified another malignant mesothelioma carrying an EWSR1::YY1 fusion gene and concluded that a subgroup of malignant mesotheliomas is indeed characterized by EWSR1::YY1 (29). Two years later and using the same sequential methodology, we found novel TNS3::MAP3K3 and ZFPM2::ELF5 fusion genes in a multicystic mesothelioma with t(7;17)(p12;q23) and t(8;11)(q23;p13) (30). Our early findings and conclusions were later confirmed by other investigators (31-37). The reported fusion genes often contain the promiscuous EWSR1 and FUS as 5′-end partners (33, 37) and the likewise promiscuous ALK as the 3′-partner gene (34, 35). In addition, some of the fusion genes found in mesotheliomas are also present in the neoplastic cells of other tumors. For example, the EWSR1::ATF1 and FUS::ATF1 fusion genes are found in clear cell sarcomas and angiomatoid fibrous histiocytomas (38-42) whereas EWSR1::NR4A3 is found in extraskeletal myxoid chondrosarcoma (43-45). Here, we report our genetic findings on two additional peritoneal mesotheliomas with characteristic genetic profiles.
Materials and Methods
Ethics statement. The study was approved by the Regional Committee for Medical and Health Research Ethics, South-East Norway (S-07474a, REK Sør-Øst). Written informed consent was obtained from patients prior to publication of the case details. The Ethics Committee’s approval included a review of the consent procedure. All patient information has been de-identified.
Case 1. A 42-year-old woman underwent right-sided ileocolectomy after examination of a preoperative needle biopsy had revealed a clear cell, vascular, tumor-suspect lesion. An abdominal tumor measuring 10×9×6 cm was found. Microscopic evaluation revealed a highly cellular tumor, in some areas with dilated vascular spaces (Figure 1A). In some parts, it was made up of spindle cells with overt atypia forming vessel-like spaces (Figure 1B). Vascular formations lined by less atypical cells were seen in other areas (Figure 1C) and in other still, epithelioid cells with a clear cytoplasm and low-grade atypia predominated (Figure 1D). Immunohistochemistry (IHC) showed expression of pan-cytokeratin (AE1/AE3) and partial expression of CK5/6, calretinin (Figure 1E), and podoplanin (D2-40) in the epithelioid areas. KIT (CD117), EMA, WT1, PECAM1 (CD31), SMA, desmin, ERG, CD34, DOG1, PAX8, SALL4, SF1, PMEL (HMB45), MLANA (melan A), TNFRSF8 (CD30), synaptophysin, CLDN4 (claudin-4), and STAT6 were negative. BAP1 was retained. The differential diagnosis was between mesothelioma and hemangio-pericytoma. The tumor was sent for consultation to Massachusetts General Hospital (Boston, MA, USA) where the diagnosis of biphasic mesothelioma was favored.
Pathological findings in case 1. (A) Hematoxylin and eosin staining showing a highly cellular tumor with dilated vascular spaces in some areas. (B) Part of the tumor consisted of spindle cells with overt atypia forming vessel-like spaces. (C) Part of the tumor with vascular formations lined by cells with a lesser degree of atypia. (D) Areas with cells of epithelioid morphology, clear cytoplasm, and low-grade atypia. (E) Immunohistochemistry showing expression of calretinin.
Case 2. A 25-year-old woman was diagnosed with a papillary well-differential benign mesothelial tumor based on examination of a preoperative needle biopsy. She subsequently underwent left-sided salpingo-oophorectomy, omentectomy, appendectomy, and excision of various peritoneal lesions. A papillary tumor measuring 9×5×4 cm was found on the surface of the left ovary. Microscopic examination of the ovarian tumor showed cells with characteristic mesothelial morphology, predominantly in areas of papillary growth (Figure 2A and B) but also in foci with solid growth (Figure 2C), forcing a modification of the diagnosis to malignant mesothelioma. Tumor tissue was additionally found in two preoperative peritoneal biopsies. IHC of them showed expression of calretinin (Figure 2D), WT1 (Figure 2E), and podoplanin (D2-40) (Figure 2F) but loss of desmin confirming the malignant diagnosis. EMA staining was negative. BAP1 was retained.
Pathological findings in case 2. (A) Hematoxylin and eosin staining showing cells with characteristic mesothelial morphology, predominantly in areas of papillary growth. (B) Higher magnification of the area shown in A. (C) Cells with characteristic mesothelial morphology in foci with solid growth. (D) Immunohistochemistry showing expression of calretinin. (E) Immunohistochemistry showing expression of WT1. (F) Immunohistochemistry showing expression of D2-40.
G-banding and karyotyping. Samples from the surgically removed tumors were short-term cultured as described elsewhere (46, 47). In brief, a part of resected specimens was minced with scalpels into 1-2 mm fragments and enzymatically disaggregated with collagenase II (Worthington, Freehold, NJ, USA). The resulting cells were cultured, harvested, and processed for cytogenetic examination (46, 47). Chromosome preparations were G-banded with Wright’s stain (Sigma Aldrich, St Louis, MO, USA) and examined. Metaphases were analyzed and karyograms prepared using the CytoVision computer-assisted karyotyping system (Leica Biosystems, Newcastle upon Tyne, UK). The karyotypes were described according to the International System for Human Cytogenomic Nomenclature (48).
Genomic DNA extraction and array comparative genomic hybridization (aCGH) analysis. aCGH was performed using the CytoSure array products (Oxford Gene Technology, Begbroke, Oxfordshire, UK) following the company’s protocols (49). The array combines long-oligo probes for copy number variant (CNV) detection alongside single nucleotide polymorphism (SNP) probes for identification of loss-of-heterozygosity (LOH). Genomic DNA was extracted using the Maxwell RSC instrument System and the Maxwell RSC Tissue DNA Kit (Promega, Madison, WI, USA) and the concentration was measured with a Quantus fluorometer (Promega). The reference DNA was Promega’s human genomic DNA (Promega). The slides (CytoSure Cancer +SNP array) were scanned in an Agilent SureScan Dx microarray scanner using Agilent Feature Extraction Software (version 12.1.1.1). Data were analyzed using the CytoSure Interpret analysis software (version 4.9.40). Apart from copy number variation (CNV), the software detects allelic imbalance and loss-of-heterozygosity based on the B-allele frequency methodology (50). The software annotations are based on human genome build 19.
Total RNA extraction and RNA sequencing. Total RNA was extracted using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) and the QiaCube automated purification system (Qiagen) and the concentration was measured with the QIAxpert microfluidic UV/VIS spectrophotometer (Qiagen). One μg of total RNA was sent to the Genomics Core Facility (http://genomics.no/oslo/) at the Norwegian Radium Hospital, Oslo University Hospital for high-throughput paired-end RNA-sequencing. The software FusionCatcher was used to detect possible fusion transcripts (51, 52).
Reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing analyses. cDNA was synthesized from one μg of total RNA using the iScript Advanced cDNA Synthesis Kit for RT-qPCR according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). cDNA corresponding to 20 ng total RNA was used as template in subsequent PCR assays. The quality of the cDNA synthesis was assessed by amplification of a cDNA fragment of the ABL protooncogene 1, non-receptor tyrosine kinase (ABL1) gene using the primer combination ABL1-91F1/ABL1-404R1 (53). In order to confirm the existence of the chimeric fusion, RT-PCR and Sanger sequencing analyses were performed using the Direct Cycle Sequencing Kit according to the company’s recommendations (ThermoFisher Scientific, Waltham, MA, USA). The primers were M13For-MAP3K8-1577F1: TGTAAAACGACGGCCAGT CCC AAG AGC CGC AGA CCT ACT AA and M13Rev-ABLIM1-407R1: CAGGAAACA GCTATGACC GAA ATG TTT GGT CTG GAC CCG AA.
Sequencing was run on the Applied Biosystems SeqStudio Genetic Analyzer system (ThermoFisher Scientific). The Basic Local Alignment Search Tool (BLAST) was used to compare the sequences obtained by Sanger sequencing with the NCBI reference sequences NM_005204.4 of the mitogen-activated protein kinase 8 (MAP3K8), transcript variant 1, and NM_002313.7 of actin binding LIM protein 1 (ABLIM1), transcript variant 1 (54). The BLAST-like alignment tool (BLAT) and the human genome browser at UCSC were also used to map the sequences on the Human GRCh37/hg19 assembly (55, 56).
Fluorescence in situ hybridization (FISH). FISH analysis was performed using in-house prepared probes as previously described (57, 58). The probes were made from commercially available bacterial artificial chromosomes (BAC) purchased from the BACPAC Resource Center operated by BACPAC Genomics (Emeryville, CA, USA). BAC clones retrieved from the RPCI-11 Human BAC library were selected according to physical and genetic mapping data on p and q arms of chromosome 10 (see below) as reported on the Human Genome Browser at the University of California, Santa Cruz browser on Human GRCH38/hg38 (56). In addition, FISH mapping of the clones on normal controls was performed to confirm their chromosomal location. The probe for the MAP3K8 gene was BAC RP11-350D11 (accession numbers AQ530804.1, AQ530801.1, AZ516988.1, and AZ516986.1) mapping to 10p11.23 at position chr10:30346217-30536207. The probe for the ABLIM1 gene was composed from BAC clones RP11-317F20 (accession numbers AQ535184.1, AQ535181.1, and AL133384.12) and RP11-620D7 (accession numbers AQ404856 and AQ401659) mapping to 10q25.3 at positions chr10:114366439-114534062 and chr10:114698913-114887429, respectively. DNA was extracted and probes were labelled and hybridized as previously described (57, 58). The probe for the MAP3K8 gene was labelled with Texas Red-5-dUTP (PerkinElmer, Boston, MA, USA) to obtain a red signal whereas the probe for the ABLIM1 gene was labelled with fluorescein-12-dUTP (PerkinElmer) to obtain a green signal. Chromosome preparations were counterstained with 0.2 μg/ml DAPI and overlaid with a 24×50 mm2 coverslip. Fluorescent signals were captured and analyzed using the CytoVision system (Applied Imaging, Newcastle, UK).
Results
Both mesotheliomas had clonal chromosomal abnormalities in their neoplastic cells. Two karyotypically related clones were found in the first mesothelioma: one hyperhaploid with 25∼26 chromosomes without any structural aberrations (Figure 3A) and its duplicated version, a hyperdiploid clone with 50∼52 chromosomes (Figure 3B). The karyotype was 25∼26,X,+ 5,+7,+20[cp4]/50∼52,idemx2[cp7]/46,XX[2] (Figure 3). A single pericentric inversion involving bands 10p11 and 10q25 was found in seven out of 10 metaphase spreads from the second peritoneal mesothelioma corresponding to the karyotype 46,XX,inv(10)(p11q25)[7]/46,XX[3] (Figure 4).
Cytogenetic analysis of case 1. (A) Karyogram from the hyperhaploid clone with karyotype 26,X,+5,+7,+20. (B) Karyogram showing a duplicated clone with the karyotype 52,XX,+5,+5,+7,+7,+20,+20.
Cytogenetic analysis of case 2. Partial karyogram showing pericentric inv(10)(p11q25) together with the normal chromosome 10. Arrows indicate breakpoints.
In the mesothelioma with hyperhaploidy aCGH detected gains of chromosomes 5, 7, and 20 (data not shown) with retained heterozygosity on these chromosomes (Table I). In the second peritoneal mesothelioma with inv(10)(p11q25), aCGH did not detect any gains or losses (data not shown) and showed heterozygosity for all chromosomes (Table I).
Homozygosity observed for each chromosome in the hyperhaploid mesothelioma (case 1) and the mesothelioma carrying inv(10)(p11q25) (case 2).
Analysis of the RNA sequencing data using the FusionCatcher software detected three MAP3K8::ABLIM1 and one ABLIM1::MAP3K8 chimeric transcript in the only tumor examined using these techniques, the mesothelioma with inv(10)(p11q25) (Table II).
The MAP3K8::ABLIM1 and ABLIM1::MAP3K8 fusion transcripts detected in peritoneal mesothelioma carrying inv(10)(p11q25) after analysis of RNA sequencing data with FusionCatcher. Exons are based on the reference sequences NM_005204.4 for MAP3K8 and NM_002313.7 for ABLIM1.
RT-PCR/cycle (Sanger) sequencing verified the presence of the MAP3K8::ABLIM1 chimeric transcript in which exon 8 of MAP3K8 (nt 1722 of sequence with accession number NM_005204.4) fused to exon 2 of ABLIM1 (nt 320 of sequence with accession number NM_002313.7) (Figure 5A). No other chimeric transcripts were examined by RT-PCR/cycle (Sanger) sequencing.
Sanger sequencing and fluorescence in situ hybridization (FISH) of peritoneal mesothelioma currying inv(10)(p11q25). (A) Partial chromatogram showing the fusion point of exon 8 of MAP3K8 with exon 2 of ABLIM1. (B) Ideogram of chromosome 10 showing the position of the genes MAP3K8 and ABLIM1. (C) FISH on a metaphase spread showing yellow fusion signals on the p and q arms of inv(10)(p11q25) together with red (MAP3K8) and green (ABLIM1) signals on the normal chromosome 10.
FISH analysis on metaphase spreads using in-house prepared MAP3K8 and ABLIM1 probes on chromosome bands 10p11 and 10q25, respectively (Figure 5B), showed fusion (yellow) signals on both the p and q arm of the inv(10)(p11q25) chromosome whereas a red (MAP3K8) and a green (ABLIM1) signal were seen on the normal chromosome 10 (Figure 5C).
Discussion
Near-haploid karyotypes are rare, reported in less than 1% of cytogenetically investigated neoplastic lesions (18, 59). They may occur in both hematological malignancies and solid tumors (18, 59-63). In the mesothelioma context, a near-haploid karyotype was first reported in a biphasic peritoneal mesothelioma (63), and subsequent studies have shown that biphasic mesotheliomas are indeed genetically characterized by genomic near-haploidization (64-66). Chromosomes 5 and 7 always remain disomic in these karyotypes and half of the reported cases also have two copies of chromosome 20 (63-66). Thus, the karyotype of the present biphasic mesothelioma - 25∼26,X,+5,+7,+20[cp4]/55∼56,idemx2[cp7]/46,XX[2] - is in full agreement with the previously established picture of biphasic mesothelioma karyotypes (63-66).
Disomy for chromosomes 5, 7, and 20 has also been reported to be a feature of hyperhaploid chondrosarcomas (59) and inflammatory leiomyosarcomas (67). Using B allele frequency methodology (50) on the hyperhaploid tumor of the present study, we found that the disomic chromosomes 5, 7, and 20 retained heterozygosity. This agrees with previous observations that in tumors with near-haploid karyotypes, the disomic chromosomes are maternal and paternal (67-69). Hyperhaploidy may affect thousands of genes and may result in massive loss of heterozygosity. In inflammatory leiomyosarcoma, which is cytogenetically characterized by hyperhaploidy, disomic chromosomes were found to have higher gene expression levels than monosomic chromosomes (67). The mechanisms whereby hyperhaploidy contributes to tumorigenesis, are unknown, however, although disturbed gene dosage balance may play a role in the development of biphasic mesothelioma as well as other hyperhaploid tumor types (70, 71).
The MAP3K8 gene has nine exons (NCBI reference sequence: NM_005204.4) and codes for a cytoplasmic protein that is a member of the serine/threonine protein kinase family and activates both the MAP and JNK kinase pathways (72-74). Truncations of MAP3K8 were found in thyroid carcinoma (75, 76) and lung cancer (77) whereas both truncations and fusion genes of MAP3K8 were found in spitzoid melanomas (78) and other melanoma subtypes (79-82). In all tumors with MAP3K8 rearrangements, lack of exon 9 was a common pathogenetic theme (76). Exon 9 of MAP3K8 codes for the last 43 amino acids of the MAP3K8 protein (position 425-467 in reference sequence NP_005195.2: DSSCTGSTEESEMLKRQRSLY IDLGALAGYFNLVRGPPTLEYG). This part of the protein is a kinase repression domain, has a degradation signal (degron) between positions 435 and 457 (SEMLKRQRSLYIDLGA LAGYFNL), and a portion of the PEST sequence (DSSCTG STEESEML) which acts as a signal for MAP3K8 degradation (83). Absence of the last 43 amino acids results in stabilization of the truncated MAP3K8 protein, higher kinase activity, and higher oncogenic capacity (83, 84). Truncated MAP3K8 protein was indeed found to have much higher transformation activity than does wild-type MAP3K8 (75-78, 85).
ABLIM1 encodes a protein that binds to actin filaments and mediates interactions between actin and cytoplasmic targets (86). This protein, together with the proteins encoded by ABLIM2 (on 4p16) and ABLIM3 (on 5q32), constitute the actin-binding LIM (ABLIM) protein family (86-89). At the N-terminal end, ABLIM proteins contain four LIM zinc-binding domains, followed by an ABLIM anchor superfamily domain and a Villin headpiece domain at the C-terminal end (86-89).
In the peritoneal mesothelioma with inv(10)(p11q25), based on the reference sequences NM_005204.4/NP_005195.2 for MAP3K8 and NM_002313.7/NP_002304.3 for ABLIM1, the MAP3K8::ABLIM1 chimera lacks exon 9 of MAP3K8, a fusion gene feature similar to all previously reported MAP3K8 rearrangements (see above). The MAP3K8::ABLIM1 is predicted to code for a 1121 amino acid long chimeric protein containing the catalytic domain of the Serine/Threonine kinase of MAP3K8 and all functional domains of ABLIM1. The exact cellular role of the chimeric protein is unknown; for that, functional studies are required.
In conclusion, we present the genetic analyses of two mesotheliomas that have developed via alternative pathogenetic mechanisms. The dominating feature of the first was karyotypic hyperhaploidy, but with disomies of chromosomes 5,7, and 20, a pattern that appears to be characteristic of biphasic mesotheliomas. The second displayed a MAP3K8::ABLIM1 fusion gene from which exon 9 of MAP3K8 was missing. This pathogenetic mechanism is described for the first time in mesothelioma but appears to be a common theme for MAP3K8-rearrangements in other tumor types.
Footnotes
Conflicts of Interest
The Authors declare that they have no potential conflicts of interest.
Authors’ Contributions
IP designed and supervised the research, performed molecular genetic experiments, bioinformatics analyses, and wrote the manuscript. KA performed molecular genetic experiments, fluorescence in situ hybridization, and interpreted the data. MB performed molecular genetic experiments. LG performed the cytogenetic analysis. BD performed the pathological examination. ML-I performed the pathological examination. FM evaluated the data. SH assisted with experimental design and writing of the manuscript. All Authors read and approved of the final manuscript.
- Received May 1, 2023.
- Revision received May 22, 2023.
- Accepted June 15, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
