Secretory carcinoma of the breast: a genetically defined carcinoma entity.

Secretory carcinomas (SBC) are characterized by their characteristic histomorphology and more favorable prognosis compared to invasive ductal carcinoma of usual type (IDC). On this basis, 13 SBCs are evaluated by molecular and immunohistochemical (IH) methods. 13 SBCs and 4 IDCs were analyzed for ETV6-NTRK3 gene fusion by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Fluorescence in situ Hybridization (FISH). 8 of 13 microdissected SBCs with evaluable DNA were evaluated for genetic alterations (GA) by comparative genomic hybridization (CGH). IH included estrogen-receptor (ER), progesterone-receptor (PR), Her-2/neu and Ki-67 (MIB-1) in all 13 cases. Molecular and immunohistochemical results in SBCs were compared with previous data regarding immunohistochemical and molecular characteristics of IDCs. 12 of 13 (92 %) SBC cases, but not IDCs expressed the ETV6-NTRK3 fusion gene which encodes a chimeric tyrosine kinase. Retroviral transfer of ETV6-NTRK3 (EN) into murine mammary epithelial cells resulted in transformed cells that readily formed epithelial tumors in nude mice. CGH revealed an average of 2.0 GAs (range 0-6), including recurrent gains of chromosome 8q and 1q and losses of 22q. Four SBCs were positive for ER and 2 were positive for PR. The mean MIB-1-labeling index was 11.4% (range: <1-34%). Her-2/ neu protein overexpression was detected in 1 case (score 3+). Compared to previous findings in IDCs, SBCs are characterized by the recurrent expression of ETV6-NTRK3 fusion gene, a relatively low number of GAs, low proliferative rate, infrequent Her-2/ neu protein overexpression and a lower rate of steroid hormone receptor expression. These results support the hypothesis that SBCs have immunohistochemical and genetic features that specifically distinguish them from IDCs.

Molecular detection of the ETV6-NTRK3 gene fusion differentiates congenital fibrosarcoma from other childhood spindle cell tumors.

Congenital fibrosarcoma (CFS) is a pediatric spindle cell tumor of the soft tissues that usually presents before the age of 2 years. Although these tumors display histologic features of malignancy and frequently recur, they have a relatively good prognosis and only rarely metastasize. CFS must therefore be differentiated from more aggressive spindle cell sarcomas that occur during childhood, particularly adult-type fibrosarcoma (ATFS), which can have an identical morphology. CFS must also be distinguished from benign but cellular fibroblastic lesions of the same age group, including infantile fibromatosis (IFB) and myofibromatosis (MFB). Unfortunately, standard pathologic examination often does not differentiate CFS from these other conditions. The authors recently identified a novel chromosomal translocation in CFS, t(12;15)(p13;q25), which gives rise to an ETV6-NTRK3 gene fusion. They subsequently developed reverse transcription-polymerase chain reaction (RT-PCR) assays that can detect ETV6-NTRK3 fusion transcripts in CFS frozen or paraffin-embedded tumor specimens. To confirm the use of this assay in the differential diagnosis of CFS, they have screened a larger series of childhood pediatric spindle cell lesions for ETV6-NTRK3 gene fusions, including 11 cases of CFS, 13 malignant spindle cell tumors (including ATFS), and 38 benign spindle cell tumors (including IFB and MFB). Of the 11 cases diagnosed as CFS, 10 showed the ETV6-NTRK3 gene fusion, whereas none of the 51 other malignant or benign spindle cell tumors demonstrated this fusion gene. They also compared their RT-PCR findings with those of conventional cytogenetics and with immunohistochemical detection of the ETV6-NTRK3 protein using antisera to NTRK3. They conclude that RT-PCR analysis is superior to these techniques for the detection of the ETV6-NTRK3 gene fusion in pediatric spindle cell tumors, and it is a reliable and specific modality for the diagnosis of CFS.

The ETV6-NTRK3 gene fusion encodes a chimeric protein tyrosine kinase that transforms NIH3T3 cells.

The congenital fibrosarcoma t(12;15)(p13;q25) rearrangement splices the ETV6 (TEL) gene on chromosome 12p13 in frame with the NTRK3 (TRKC) neurotrophin-3 receptor gene on chromosome 15q25. Resultant ETV6-NTRK3 fusion transcripts encode the helix - loop - helix (HLH) dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. We show here that ETV6-NTRK3 homodimerizes and is capable of forming heterodimers with wild-type ETV6. Moreover, ETV6-NTRK3 has PTK activity and is autophosphorylated on tyrosine residues. To determine if the fusion protein has transforming activity, NIH3T3 cells were infected with recombinant retroviral vectors carrying the full-length ETV6-NTRK3 cDNA. These cells exhibited a transformed phenotype, grew macroscopic colonies in soft agar, and formed tumors in severe combined immunodeficient (SCID) mice. We hypothesize that chimeric proteins mediate transformation by dysregulating NTRK3 signal transduction pathways via ligand-independent dimerization and PTK activation. To test this hypothesis, we expressed a series of ETV6-NTRK3 mutants in NIH3T3 cells and assessed their transformation activities. Deletion of the ETV6 HLH domain abolished dimer formation with either ETV6 or ETV6-NTRK3, and cells expressing this mutant protein were morphologically non-transformed and failed to grow in soft agar. An ATP-binding mutant failed to autophosphorylate and completely lacked transformation activity. Mutants of the three NTRK3 PTK activation-loop tyrosines had variable PTK activity but had limited to absent transformation activity. Of a series of signaling molecules well known to bind to wild-type NTRK3, only phospholipase-Cgamma (PLCgamma) associated with ETV6-NTRK3. However, a PTK active mutant unable to bind PLCgamma did not show defects in transformation activity. Our studies confirm that ETV6-NTRK3 is a transforming protein that requires both an intact dimerization domain and a functional PTK domain for transformation activity. Oncogene (2000) 19, 906 - 915.

ETV6-NTRK3 gene fusions and trisomy 11 establish a histogenetic link between mesoblastic nephroma and congenital fibrosarcoma.

Congenital mesoblastic nephroma (CMN) is an infantile spindle cell tumor of the kidney that is subdivided into "classical" and "cellular" forms based on the degree of cellularity and mitotic activity. The histogenesis of CMN remains obscure, but relationships to other pediatric renal neoplasms have been proposed. However, cellular CMN is virtually identical histologically to congenital fibrosarcoma (CFS), a malignant tumor of fibroblasts in children of the same age group. Moreover, cytogenetic studies have reported common trisomies in CFS and cellular CMN, particularly of chromosome 11. We show here that t(12;15)(p13;q25)-associated ETV6-NTRK3 gene fusions described in CFS are also present in cellular CMN. ETV6-NTRK3 chimeric transcripts were detected in 8 of 9 cellular CMNs and 2 of 2 mixed CMNs. In contrast, all of the four classical CMNs tested were negative, as were cases of Wilms' tumor and clear cell sarcoma of the kidney. Moreover, we found trisomy 11 only in cellular or mixed CMNs with the ETV6-NTRK3 gene fusion. Our studies indicate that classical and cellular CMN have different genetic features and support the concept that cellular CMN is histogenetically related to CFS. They also provide insight into potential mechanisms involved in the transformation of the classical into the cellular form of CMN.

Absence of detectable EWS/FLI1 expression after therapy-induced neural differentiation in Ewing sarcoma.

Ewing sarcoma and other peripheral primitive neuroectodermal tumors (pPNETs) display limited neural differentiation and are thought to have a neural crest origin Greater than 95% of these tumors share common t(11;22)(q24;q12) ort(21;22)(q22;q12) chromosomal translocations leading to ES/FLI1 or EWS/ERG gene fusions, respectively. The resulting chimeric oncoproteins seem to function as aberrant transcription factors. However, whether these molecules contribute to the limited neural differentiation observed in pPNETs or actually inhibit differentiation remains unclear. We report a Ewing sarcoma case from the forearm of a 10-year-old girl which expressed EWS/FLI1 fusion transcripts. The tumor was treated with surgery, chemotherapy, and local radiation, but residual tumor was detected within a year as a well-differentiated peripheral neural tumor lacking detectable EWS/FLI1 expression. Further studies suggested that the primary and residual tumors were clonally related. This association between apparent therapy-induced differentiation in Ewing sarcoma and absence of detectable fusion transcripts in the residual tumor provides presumptive evidence that EWS/FLI1 expression may inhibit differentiation in tumour cells.

A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma.

Congenital (or infantile) fibrosarcoma (CFS) is a malignant tumour of fibroblasts that occurs in patients aged two years or younger. CFS is unique among human sarcomas in that it has an excellent prognosis and very low metastatic rate. CFS is histologically identical to adult-type fibrosarcoma (ATFS); however, ATFS is an aggressive malignancy of adults and older children that has a poor prognosis. We report a novel recurrent t(12;15)(p13;q25) rearrangement in CFS that may underlie the distinctive biological properties of this tumour. By cloning the chromosome breakpoints, we show that the rearrangement fuses the ETV6 (also known as TEL) gene from 12p13 with the 15q25 NTRK3 neurotrophin-3 receptor gene (also known as TRKC). Analysis of mRNA revealed the expression of ETV6-NTRK3 chimaeric transcripts in all three CFS tumours analysed. These were not detected in ATFS or infantile fibromatosis (IFB), a histologically similar but benign fibroblastic proliferation occurring in the same age-group as CFS. ETV6-NTRK3 fusion transcripts encode the helix-loop-helix (HLH) protein dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. Our studies indicate that a chimaeric PTK is expressed in CFS and this may contribute to oncogenesis by dysregulation of NTRK3 signal transduction pathways. Moreover, ETV6-NTRK3 gene fusions provide a potential diagnostic marker for CFS.