Petter Brandal

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.

t(19;22)(q13;q12) Translocation leading to the novel fusion gene EWSR1‐ZNF444 in soft tissue myoepithelial carcinoma

Myoepithelial neoplasms of soft tissue have only recently been acknowledged as a separate diagnostic entity. To know based on histological appearance whether these tumors are benign or malignant is often difficult, and their tumorigenic mechanisms remain poorly understood. We report a myoepithelial carcinoma with an aberrant near‐diploid karyotype, 43∼47,XX,add(1)(p34)x2,add(3)(q27)x2,del(12)(q22),+add(18)(p11)x2,del(22)(q11),+r, found in cells cultured from a lung metastasis. The deletion in 22q led us to search by molecular cytogenetic means for possible EWSR1 rearrangements, and eventually a novel chimeric gene consisting of the 5′‐end of EWSR1 (22q12) and the 3′‐end of ZNF444 (19q13) was found. How the new fusion gene contributes to tumorigenesis is unknown, but the finding of an EWSR1 rearrangement suggests that this, possibly even the EWSR1‐ZNF444, is a defining pathogenetic feature of at least a subset of these tumors.

Genomic aberrations in mucinous tubular and spindle cell renal cell carcinomas.

Mucinous tubular and spindle cell carcinoma of the kidney is a new diagnostic entity. We present the pathologic and genomic characteristics of three such low-malignant tumors. Two of the tumors were found in women aged 19 and 52 years, the third tumor was found in an 80-year-old man, and the tumor stages were pT2N0MX, pT2NXMX, and pT1NXMX, respectively. Findings by immunohistochemistry were similar but not identical for the three cases; markers for both proximal and distal parts of the nephron were expressed in each tumor, a finding that is in agreement with data from previous studies. The Ki-67-labeling index was below 5 in all three cases. Two of the tumors were predominantly hypodiploid (DNA-indexes 0.77 and 0.80), whereas the third tumor was hypertriploid (1.57) as measured by DNA-image cytometry. From the latter tumor live cells were available making it possible to establish its karyotype: 62-70,XXX,+del(X)(q11),−1,+2,+4,−5,−6,+7,−8,−9,−10,−11,+12,−13,−14,−15,+16,+17,+18,−19,+20,+21,−22[cp15]. Interphase fluorescence in situ hybridization analyses with centromere-specific probes for chromosomes 1, 3, 4, 6, 7, 9, 10, 17, 18, 20, and X showed that the two hypodiploid tumors had disomic and monosomic chromosome populations, whereas the karyotyped, near-triploid tumor was dominated by trisomic chromosome populations. Comparative genomic hybridization analysis was normal for the karyotyped tumor but abnormal for the two others. We conclude that multiple numerical chromosome aberrations may be a feature of mucinous tubular and spindle cell carcinomas of the kidney, but beyond that no clear-cut karyotypic aberration pattern is so far discernible.

MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR

BackgroundMethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is a favorable prognostic factor in glioblastoma patients. However, reported methylation frequencies vary significantly partly due to lack of consensus in the choice of analytical method.MethodWe examined 35 low- and 99 high-grade gliomas using quantitative methylation specific PCR (qMSP) and pyrosequencing. Gene expression level of MGMT was analyzed by RT-PCR.ResultsWhen examined by qMSP, 26% of low-grade and 37% of high-grade gliomas were found to be methylated, whereas 97% of low-grade and 55% of high-grade gliomas were found methylated by pyrosequencing. The average MGMT gene expression level was significantly lower in the group of patients with a methylated promoter independent of method used for methylation detection. Primary glioblastoma patients with a methylated MGMT promoter (as evaluated by both methylation detection methods) had approximately 5 months longer median survival compared to patients with an unmethylated promoter (log-rank test; pyrosequencing P = .02, qMSP P = .06). One third of the analyzed samples had conflicting methylation results when comparing the data from the qMSP and pyrosequencing. The overall survival analysis shows that these patients have an intermediate prognosis between the groups with concordant MGMT promoter methylation results when comparing the two methods.ConclusionIn our opinion, MGMT promoter methylation analysis gives sufficient prognostic information to merit its inclusion in the standard management of patients with high-grade gliomas, and in this study pyrosequencing came across as the better analytical method.

Rearrangement of chromosomal region 8q11-13 in lipomatous tumours: correlation with lipoblastoma morphology.

Cytogenetics is of considerable value when diagnosing lipomatous tumours, as different tumour types have different more or less specific chromosomal abnormalities. One such entity is lipoblastoma, which is a benign lipomatous tumour that often exhibits rearrangements of chromosome bands 8q11–13, and the gene PLAG1 has been implicated as the target of these chromosomal changes. All lipomatous tumours karyotyped at the Norwegian Radium Hospital were reviewed, looking for rearrangements of 8q11–13. Five tumours exhibiting chromosomal abnormalities affecting this region were found. Only one of them was morphologically diagnosed as a lipoblastoma, two being classified as lipomas, one as a hibernoma, and one as a well‐differentiated liposarcoma. The two tumours successfully analysed with bacterial artificial chromosomes (BACs) covering the gene PLAG1 showed involvement of this gene in the rearrangement. The findings raise the question as to what extent the diagnosis lipoblastoma should be based on histopathological or cytogenetic/molecular data or a combination thereof. When karyotypic information from this series was combined with available literature data, it was found that the sensitivity of 8q11–13 rearrangements for diagnosing lipoblastomas when found in a lipomatous tumour was 77% and that the corresponding specificity was 98%. The validity of these calculations of the diagnostic information provided by the cytogenetic findings is, of course, totally dependent on the morphological diagnosis made in each case. Regardless of what the precise phenotypic diagnosis was, it is suggested that lipomatous tumours with 8q11–13 rearrangement constitute a distinct pathogenetic entity. When selective therapies tailor‐made against the specific pathogenetic rearrangement become available, it will become mandatory to pay more attention to the genetic constitution of the tumour cells than to their phenotypic appearance. Copyright

Genomic aberrations in 80 cases of primary glioblastoma multiforme: Pathogenetic heterogeneity and putative cytogenetic pathways.

Screening the whole glioblastoma multiforme (GBM) genome for aberrations is a good starting point when looking for molecular markers that could potentially stratify patients according to prognosis and optimal treatment. We investigated 80 primary untreated GBM using both G‐banding analysis and high‐resolution comparative genomic hybridization (HR‐CGH). Abnormal karyotypes were found in 83% of the tumors. The most common numerical chromosome aberrations were +7, −10, −13, −14, −15, +20, and −22. Structural abnormalities most commonly involved chromosomes 1 and 3, and the short arm of chromosome 9. HR‐CGH verified these findings and revealed additional frequent losses at 1p34‐36, 6q22‐27, and 19q12‐13 and gains of 3q26 and 12q13‐15. Although most karyotypes and gain/loss patterns were complex, there was also a distinct subset of tumors displaying simple karyotypic changes only. There was a statistically significant association between trisomy 7 and monosomy 10, and also between +7/−10 as putative primary aberrations and secondary losses of 1p, 9p, 13q, and 22q. The low number of tumors in the rarer histological tumor subgroups precludes definite conclusions, but there did not seem to be any clear‐cut cytogenetic‐pathological correlations, perhaps with the exception of ring chromosomes in giant cell glioblastomas. Our findings demonstrate that although GBM is a pathogenetically very heterogeneous group of diseases, distinct genomic aberration patterns exist.

Synchronous and metachronous skeletal osteosarcomas: The Norwegian Radium Hospital experience

Background. The purpose of this work was to study clinical and histopathological tumor characteristics of patients treated for synchronous or metachronous skeletal osteosarcoma at The Norwegian Radium Hospital from January 1, 1980 to January 1, 2008. Patients and methods. The hospital sarcoma database and patient records were reviewed to identify cases with synchronous or metachronous skeletal osteosarcoma. Patients with more than one skeletal lesion in the absence of pulmonary or other soft tissue tumor manifestations were included in the study, and histopathological slides from these tumors were reviewed. Results. Among a total of 297 registered osteosarcoma patients, six with synchronous (2.0%) and 10 with metachronous (3.4%) skeletal osteosarcomas were identified. All tumors were of high-grade malignancy. Treatment at the time of the first osteosarcoma diagnosis was in most cases wide resections and multi-agent chemotherapy according to international protocols, whereas the treatment for metachronous tumors was individualized and in general much less intensive. One patient was diagnosed with Li-Fraumeni syndrome, two other individuals may be suspected to have the same syndrome, and yet another patient had previously been treated for a bilateral retinoblastoma. Thirteen patients are dead, 11 from metastatic osteosarcoma, one from myelodysplastic syndrome, and one from wound infection and methotrexate-related nephrotoxicity; whereas three patients are still alive with no evidence of osteosarcoma. Conclusions. The prognosis for patients with synchronous and metachronous skeletal osteosarcoma is poor. However, because long-term survival is seen, aggressive treatment to selected cases, e.g., patients with an osteosarcoma predisposing syndrome and/or late occurring metachronous tumours, is justified. Revealing a possible clonal relationship between these tumors, e.g., by karyotyping, may be of interest for estimating prognosis and guide therapy intensiveness.

Genomic aberrations in diffuse low-grade gliomas.

The current classification of diffuse low‐grade gliomas is based mainly on histopathological criteria, which cannot accurately predict the highly variable clinical course observed in patients with such tumors. In an attempt to increase pathogenetic understanding of these tumors, we investigated 38 WHO Grade II astrocytomas, oligodendrogliomas, and oligoastrocytomas using a combination of G‐band chromosome analysis and high‐resolution comparative genomic hybridization (HR‐CGH). Abnormal karyotypes were found in 41% of tumors. Karyotypes of astrocytomas and oligodendrogliomas were near‐diploid whereas oligoastrocytomas also displayed near‐tetraploid clones. The most common aberrations were losses of chromosomes X, Y, 3, 4, 6, and 11 and gains of chromosomes 8 and 12. The only recurrent structural rearrangement was del(6)(q21). HR‐CGH analysis verified karyotyping findings but also revealed frequent losses at 1p, 17q, and 19q and gains of 7q, 10p, 11q, and 20p. Among the tumors were two gemistocytic astrocytomas, a subgroup of diffuse astrocytomas with a particular predisposition for progression but not studied cytogenetically before; one showed a near‐diploid, complex karyotype with structural aberrations of chromosomes 1, 3, and 11 whereas both displayed simple aberrations including loss of 11p by HR‐CGH. Our findings suggest that within diffuse low‐grade gliomas are genetically distinct entities that do not fit the currently used classification. In addition, tumors with complex chromosomal aberrations had a higher tendency for aggressive tumor behavior (shorter progression‐free survival) than tumors displaying simple aberrations only (P = 0.07). This could help identify genetic subsets of patients with low‐grade glioma who might benefit from early antineoplastic therapy.

Novel CSF1-S100A10 fusion gene and CSF1 transcript identified by RNA sequencing in tenosynovial giant cell tumors

RNA-sequencing was performed on three tenosynovial giant cell tumors (TSGCT) in an attempt to elicit more information on the mechanisms of CSF1 expression in this tumor type. A novel CSF1-S100A10 fusion gene was found in a TSGCT that carried the translocation t(1;1)(q21;p11) as the sole karyotypic abnormality. In this fusion gene, the part of CSF1 coding for the CSF1 protein (exons 1–8 in sequences with accession nos. NM_000757 and NM_172212) is fused to the 3′-part of S100A10. Since the stop codon TAG of CSF1 is present in it, the CSF1-S100A10 fusion gene’s predominant consequence seems to be the replacement of the 3′-untranslated region (UTR) of CSF1 (exon 9; nt 2092–4234 in sequence with accession no. NM_000757 or nt 2092–2772 in NM_172212) by the 3′-end of S100A10 (exon 3; nt 641–1055 in sequence with accession no. NM_002966). In the other two TSGCT, a novel CSF1 transcript was detected, the same in both tumors. Similar to the occurrence in the CSF1-S100A10 fusion gene, the novel CSF1 transcript 3′-UTR is replaced by a new exon located ∼48 kb downstream of CSF1 and 11 kb upstream of AHCYL1. Although only 3 TSGCT were available for study, the finding in all of them of a novel CSF1-S100A10 fusion gene or CSF1 transcript indicates the existence of a common pathogenetic theme in this tumor type: the replacement of the 3′-UTR of CSF1 with other sequences.

Genomic aberrations in pediatric gliomas and embryonal tumors

The pathogenesis of pediatric central nervous system tumors is poorly understood. To increase knowledge about the genetic mechanisms underlying these tumors, we performed genome‐wide screening of 17 pediatric gliomas and embryonal tumors combining G‐band karyotyping and array comparative genomic hybridization (aCGH). G‐banding revealed abnormal karyotypes in 56% of tumor samples (9 of 16; one failed in culture), whereas aCGH found copy number aberrations in all 13 tumors examined. Pilocytic astrocytomas (n = 3) showed normal karyotypes or nonrecurrent translocations by karyotyping but the well‐established recurrent gain of 7q34 and 19p13.3 by aCGH. Our series included one anaplastic oligoastrocytoma, a tumor type not previously characterized genomically in children, and one anaplastic neuroepithelial tumor (probably an oligoastrocytoma); both showed loss of chromosome 14 by G‐banding and structural aberrations of 6q and loss of 14q, 17p, and 22q by aCGH. Three of five supratentorial primitive neuroectodermal tumors showed aberrant karyotypes: two were near‐diploid with mainly structural changes and one was near‐triploid with several trisomies. aCGH confirmed these findings and revealed additional recurrent gains of 1q21‐44 and losses of 3p21, 3q26, and 8p23. We describe cytogenetically for the first time a cribriform neuroepithelial tumor, a recently identified variant of atypical teratoid/rhabdoid tumor with a favorable prognosis, which showed loss of 1p33, 4q13.2, 10p12.31, 10q11.22, and 22q by aCGH. This study indicates the existence of distinct cytogenetic patterns in pediatric gliomas and embryonal tumors; however, further studies of these rare tumors using a multimodal approach are required before their true genomic aberration pattern can be finally established.