Florence Pedeutour

Structure of the supernumerary ring and giant rod chromosomes in adipose tissue tumors

Supernumerary ring or giant rod marker chromosomes are a characteristic of well‐differentiated liposarcomas (WDLPS) and atypical lipomas (ALP) and are often observed as the sole cytogenetic abnormality, but are rare in lipomas. Using a combination of different methods, we extensively investigated the structure and composition of rings and giant rods in a series of 17 WDLPS‐ALP samples and three intra‐ or intermuscular lipomas (IMLP), revealing a unique combination of particular features strikingly related to these tumors. Although the rings and rods displayed in vitro and in vivo stability, the presence of alpha‐satellites could not be detected on these supernumerary structures. Comparative genomic hybridization analysis, in combination with fluorescence in situ hybridization, identified the chromosomal regions contributing to the formation of these chromosomes: in WDLPS‐ALP, all carried amplifications of 12q14–15 and the MDM2 gene, with variable other noncontiguous regions. In the three IMLP, the rings consistently carried amplifications of 12q15–21 and 1q21, but increased copies of MDM2 were found in only one case. Other genes located more proximal in 12q14–15 were amplified in several WDLPS‐ALP, but showed a normal copy number in IMLP. Furthermore, the immunohistochemical expression of the MDM2 protein was detected in most (12/14) WDLPS‐ALP, in 1–30% of the cells, but never in IMLP. These supernumerary chromosomes represent a peculiar kind of amplification structure, midway between double minute chromosomes and homogeneously staining regions, but the mechanisms underlying the formation of these structures remain obscure. Genes Chromosomes Cancer 24:30–41, 1999.

Identification of novel fusion partners of ALK, the anaplastic lymphoma kinase, in anaplastic large‐cell lymphoma and inflammatory myofibroblastic tumor

ALK‐positive anaplastic large‐cell lymphoma (ALCL) has been recognized as a distinct type of lymphoma in the heterogeneous group of T/Null‐ALCL. While most of the ALK‐positive ALCL (ALKomas) are characterized by the presence of the NPM‐ALK fusion protein, the product of the t(2;5)(p23;q35), 10–20% of ALKomas contain variant ALK fusions, including ATIC‐ALK, TFG‐ALK, CLTC‐ALK (previously designated CLTCL‐ALK), TMP3‐ALK, and MSN‐ALK. TMP3‐ALK and TMP4‐ALK fusions also have been detected in inflammatory myofibroblastic tumors (IMTs), making clear that aberrations of the ALK gene are not associated exclusively with the pathogenesis of ALK‐positive ALCL. Here we report results of molecular studies on two lymphoma cases and one IMT case with variant rearrangements of ALK. Our study led to the detection of the CLTC‐ALK fusion in an ALCL case and to the identification of two novel fusion partners of ALK: ALO17 (KIAA1618), a gene with unknown function, which was fused to ALK in an ALCL case with a t(2;17)(p23;q25), and CARS, encoding the cysteinyl‐tRNA synthetase, which was fused to ALK in an IMT case with a t(2;11;2)(p23;p15;q31). These results confirm the recurrent involvement of ALK in IMT and further demonstrate the diversity of ALK fusion partners, with the ability to homodimerize as a common characteristic.

Genetics of dermatofibrosarcoma protuberans family of tumors: from ring chromosomes to tyrosine kinase inhibitor treatment.

Dermatofibrosarcoma protuberans (DP) is a rare, slow‐growing, infiltrating dermal neoplasm of intermediate malignancy, made up of spindle‐shaped tumor cells often positive for CD34. The preferred treatment is wide surgical excision with pathologically negative margins. At the cytogenetic level, DP cells are characterized by either supernumerary ring chromosomes, which have been shown by using fluorescence in situ hybridization techniques to be derived from chromosome 22 and to contain low‐level amplified sequences from 17q22‐qter and 22q10–q13.1, or t(17;22), that are most often unbalanced. Both the rings and linear der(22) contain a specific fusion of COL1A1 with PDGFB. Similar to other tumors, the COL1A1‐PDGFB fusion is occasionally cryptic, associated with complex chromosomal rearrangements. Although rings have been mainly observed in adults, translocations have been reported in all pediatric cases. DP is therefore a unique example of a tumor in which (i) the same molecular event occurs either on rings or linear translocation derivatives, (ii) the chromosomal abnormalities display an age‐related pattern, and (iii) the presence of the specific fusion gene is associated with the gain of chromosomal segments, probably taking advantage of gene dosage effects. In all DP cases that underwent molecular investigations, the breakpoint localization in PDGFB was found to be remarkably constant, placing exon 2 under the control of the COL1A1 promoter. In contrast, the COL1A1 breakpoint was found to be variably located within the exons of the α‐helical coding region (exons 6–49). No preferential COL1A1 breakpoint and no correlation between the breakpoint location and the age of the patient or any clinical or histological particularity have been described. The COL1A1‐PDGFB fusion is detectable by multiplex RT‐PCR with a combination of forward primers designed from a variety of COL1A1 exons and one reverse primer from PDGFB exon 2. Recent studies have determined the molecular identity of “classical” DP, giant cell fibroblastoma, Bednar tumor, adult superficial fibrosarcoma, and the granular cell variant of DP. In approximately 8% of DP cases, the COL1A1‐PDGFB fusion is not found, suggesting that genes other than COL1A1 or PDGFB might be involved in a subset of cases. It has been proposed that PDGFB acts as a mitogen in DP cells by autocrine stimulation of the PDGF receptor. It is encouraging that inhibitory effects of the PDGF receptor tyrosine kinase antagonist imatinib mesylate have been demonstrated in vivo; such targeted therapies might be warranted in the near future for treatment of the few DP cases not manageable by surgery.

Translocation-positive low-grade fibromyxoid sarcoma: clinicopathologic and molecular analysis of a series expanding the morphologic spectrum and suggesting potential relationship to sclerosing epithelioid fibrosarcoma: a study from the French Sarcoma Group.

Low-grade fibromyxoid sarcomas (LGFMS) bear either the t(7,16) (q32-34;p11) or t(11,16) (p11;p11) translocations, resulting in FUS-CREB3L2 or FUS-CREB3L1 fusions, respectively. Heretofore, fusion transcripts were mainly detected in frozen tissues, using reverse transcription-polymerase chain reaction. In this study, we aimed to develop a reliable method to detect these in paraffin-embedded tissues, and to examine the clinicopathologic characteristics of a series of translocation-positive LGFMS. Sixty-three neoplasms with typical morphologic features of LGFMS and 66 non-LGFMS tumors selected for their resemblance to LGFMS (LGFMS-like tumors) were examined. RNA of sufficient quality could be extracted from 111/129 (86%) cases (59 LGFMS, 52 non-LGFMS). Of all, 48/59 (sensitivity, 81%) LGFMS contained detectable transcripts (45 FUS-CREB3L2, 3 FUS-CREB3L1). Most relevant clinicopathologic features of fusion-positive LGFMS included predominance in lower extremities (22/48; thigh: 13/48), deep situation (46/48), and occasional presence of unusual histologic features, for example, hypercellular areas (16/48), foci of epithelioid cells (13/48), and giant rosettes (6/48). Most tumors expressed EMA (41/45), at least focally, CD99 (38/41) and bcl-2 (36/41) while being essentially negative for CD34 (2/45), mdm2 (1/41), smooth muscle actin (1/45), S100 protein (0/46), desmin (0/44), h-caldesmon (0/42), keratins (0/44), and CD117 (0/40). Eleven presumed LGFMS were fusion negative. Of all, 7/52 non-LGMFS neoplasms contained FUS-CREB3L2 transcripts, of which 4 had been diagnosed as sclerosing epithelioid fibrosarcoma. In conclusion, FUS-CREB3L1/L2 fusion transcripts can be detected in paraffin-embedded LGFMS in a sensitive manner, using reverse transcription-polymerase chain reaction. Most fusion-positive LGFMS are EMA-positive and CD34/S100/smooth muscle actin negative. The presence of epithelioid cells and fusion transcripts in both LGFMS and a subset of sclerosing epithelioid fibrosarcoma suggest that these neoplasms might be related.

Detection of MDM2-CDK4 amplification by fluorescence in situ hybridization in 200 paraffin-embedded tumor samples: utility in diagnosing adipocytic lesions and comparison with immunohistochemistry and real-time PCR.

Atypical lipomatous tumor/well-differentiated liposarcomas and dedifferentiated liposarcomas are characterized by the amplification of MDM2 and CDK4 genes. To evaluate the accuracy of fluorescence in situ hybridization (FISH) analysis in the differential diagnosis of adipose tissue tumors, we investigated MDM2-CDK4 status by FISH, real-time polymerase chain reaction (PCR) [quantitative PCR (Q-PCR)] and immunohistochemistry (IHC) in a series of 200 adipose tumors. First, we evaluated MDM2-CDK4 amplification and expression in a series of 94 well-defined adipose tissue tumors. Results showed that FISH was interpretable in 45 of 50 cases (90%), and was more specific and sensitive than Q-PCR and IHC. We then used the same techniques as complementary diagnostic tools in a series of 106 adipose and soft tissue tumors of unclear diagnosis to distinguish between (i) lipomas and atypical lipomatous tumor/well-differentiated liposarcomas, (ii) malignant undifferentiated tumors and dedifferentiated liposarcomas, and (iii) a variety of benign tumors and liposarcomas. Our results indicate that although helpful, IHC alone is often insufficient to solve diagnostic problems. FISH and Q-PCR methods gave concordant results and were equally informative in most cases. However, the proportion of noninterpretable cases was slightly higher with FISH than with Q-PCR. When tumor cells represented a minor component of the tumor tissue, such as with inflammatory tumors, FISH was more powerful than Q-PCR by allowing visualization of individual cells. In conclusion, we recommend that the evaluation of MDM2-CDK4 amplification using FISH or Q-PCR be used to supplement IHC analysis when diagnosis of adipose tissue tumors is not possible based on clinical and histologic information alone.

Translocation, t(17;22)(q22;q13), in dermatofibrosarcoma protuberans: a new tumor-associated chromosome rearrangement

A translocation, t(17;22)(q22;q13), was identified in two cases of dermatofibrosarcoma protuberans (DP). They bring to four the number of DP cases characterized by an identical t(17;22)(q22;q13), which can be considered as a new tumor-associated chromosome rearrangement. To date, this translocation has been found only in DP and its juvenile form, giant-cell fibroblastoma. This finding has two major consequences. First, it casts light on the development and significance in DP of ring chromosomes which consistently harbor sequences derived from chromosomes 17 and 22. Second, the identification of this new chromosome marker, and eventually of the underlying molecular rearrangement, should help to classify DP, a soft-tissue tumor of still uncertain cell origin. In addition, it could be used to differentiate DP from truly benign or malignant entities, in order that this tumor of intermediate malignancy could be adequately managed.

Dermatofibrosarcoma protuberans, giant cell fibroblastoma, and hybrid lesions in children: clinicopathologic comparative analysis of 28 cases with molecular data--a study from the French Federation of Cancer Centers Sarcoma Group.

The clinicopathologic and immunohistochemical features of 28 dermatofibrosarcoma protuberans (DFSP), giant cell fibroblastomas (GCFs), and hybrid lesions occurring in children are presented, including molecular data for seven of them. There were 19 pure adult-type DFSP (9 male and 10 female patients aged between a few days [neonate] and 13 years, median 7 years), 5 pure GCF (all males aged from 2 to 8 years, median 4 years), and 4 hybrid tumors (all males aged from 1 to 4 years, median 2.5 years). Tumor locations in pure adult-type DFSP included the trunk (6) and lower (11) and upper (2) limbs. Pure GCFs were observed on the trunk (4) and knee (1), and hybrid lesions on the trunk (2) and lower (1) and upper (1) extremities. Tumor size (n = 20) ranged from 0.6 to 5 cm (median 2 cm). Histologically, pure DFSP presented as monotonous and infiltrative, low-grade, dermal/hypodermal storiform spindle cell proliferations, sparing adnexal structures. GCF showed a dense fibrous to myxoid matrix containing slender wavy spindle cells and multinucleated giant stromal cells often lining angiectoid spaces. Hybrid lesions showed varying combinations of DFSP and GCF areas. Mitotic activity ranged from 1 to 3 mitoses per 10 high power fields. All tumors were diffusely positive for vimentin and CD34 but negative for smooth muscle actin, desmin, epithelial membrane antigen, and cytokeratins; one pure adult-type DFSP was also S-100 protein positive; <1% of nuclei were Ki67 (Mib-1) positive. One karyotyped adult-type DFSP showed an unbalanced t(17;22) (q22;q13) translocation. Multiplex RT-PCR analysis and sequencing of PCR products in seven cases showed COL1A1-PDGFB gene fusion transcripts in two pure DFSP, two pure GCFs, and one hybrid lesion. Results were uncertain in one pure GCF; one adult-type DFSP was negative. Treatment procedures were known for 27 patients, consisting of 16 wide excisions and 11 marginal excisions. Follow-up information on 15 widely excised tumors (median 24 months; range 5–144 months) showed no recurrence. Five of six marginally excised lesions with available follow up recurred 2 months to 6 years (median 2 years) after initial surgery; all but one were cured by wide reexcision. None of the tumors metastasized. In conclusion, this study emphasizes 1) the occurrence of adult-type DFSP in children, 2) the close relationship between DFSP and GCF clinically, histologically, and molecularly, 3) the excellent prognostic of these lesions if widely excised, and 4) the diagnostic usefulness of RT-PCR analyses in detecting the COL1A1-PDGFB gene fusion transcripts resulting from the t(17;22) (q22;q13) in paraffin-embedded tissues.

Pseudosarcomatous myofibroblastic proliferations of the bladder: a clinicopathologic study of 42 cases.

Inflammatory pseudotumor or pseudosarcomatous fibromyxoid tumor and postoperative spindle cell nodule of the bladder are unusual lesions of uncertain pathogenesis which share overlapping, if not identical, histologic features. We present our experience with 42 cases, the largest series to date, to study the etio-pathogenesis, histologic features, biologic behavior and relationship to “inflammatory myofibroblastic tumor” of childhood. Patients ranged in age from 7 to 77 years (mean 47 y) and males predominated (3.2:1). Most patients presented with hematuria (31/42). Common associations were smoking (10/30) and previous instrumentation or surgery (9/42). The clinicopathologic features of patients having or not having prior instrumentation were identical. Grossly the lesions were polypoid or nodular and involved any portion of bladder wall, most commonly the dome (9/27) and measured 1 to 10 cm (mean 4 cm). They were composed of spindled and stellate cells arranged in a myxoid background with numerous inflammatory cells. Myxoid hypocellular areas were more pronounced near the mucosal surface with greater cellularity and a fascicular arrangement in the deep aspect of the lesion. “Atypical” features included mitotic activity (0 to 20/10 HPF; mean 2/10 HPF; median 1/10 HPF; none atypical), necrosis (22/42), and extension into muscularis propria (28/32) or perivesicular fat (3/8). Lesions were positive for cytokeratin (31/33), SMA (23/34), desmin (21/35), and Alk-1 protein (12/26). FISH confirmed the Alk-1 translocation in 4/6 cases. Treatment included transurethral resection (30/42), partial cystectomy (9/42), and total cystectomy (3/42). Initial diagnostic error resulted in radiotherapy and chemotherapy in 3 patients. Follow-up was available in 28 patients. (range 3to 93 mo; median 25 mo). Three patients developed recurrences, but none had metastases. Because the clinicopathologic features of lesions associated with and without instrumentation were similar and inseparable, we believe they are essentially the same entity, and propose the term pseudosarcomatous myofibroblastic proliferation. The preponderance of evidence which includes the extravesical growth, local recurrence, and Alk-1 gene translocation in some cases suggests perhaps a neoplastic process with limited growth potential. Even in the face of atypical histologic features (muscle invasion and necrosis) the prognosis is excellent. Despite the Alk-1 gene translocation, there continues to be sufficient evidence for regarding these as distinct from the so-called inflammatory myofibroblastic tumor of childhood.

HMGA2 is the partner of MDM2 in well-differentiated and dedifferentiated liposarcomas whereas CDK4 belongs to a distinct inconsistent amplicon

Data concerning the fine structure of the 12q13‐15 amplicon which contains MDM2 and CDK4 in well‐differentiated and dedifferentiated liposarcomas (WDLPS/DDLPS) are scarce. We investigated a series of 38 WDLPS/DDLPS using fluorescence in situ hybridization analysis with 17 probes encompassing the 12q13‐15 region. In addition, using quantitative RT‐PCR we studied the expression of MDM2, CDK4, DDIT3 (CHOP/GADD153), DYRK2, HMGA2, TSPAN31 and YEATS4 (GAS41) in 11 cases. We showed that CDK4 (12q14.1) belonged to a distinct amplicon than MDM2 (12q15). There was no continuity in the amplified sequences between MDM2 and CDK4. Moreover, while MDM2 was amplified and overexpressed in all cases, CDK4 was not amplified or overexpressed in 13% of cases. The centromeric border of the CDK4 amplicon was located immediately downstream the 5′ end of DDIT3, a gene known for being involved in myxoid liposarcoma translocations. DDIT3 was amplified in 3 cases and overexpressed in 9 cases. The overexpression of DDIT3 was correlated to the CDK4 amplification and not to its own amplification status. This suggested that the CDK4 amplicon, as well as the overexpression of DDIT3, might be generated by the disruption of a fragile region in 5′ DDIT3. HMGA2 was always amplified and rearranged indicating that it plays a central role in WDLPS/DDLPS. HMGA2 rearrangement frequently resulted in a loss of the 3′ end region that is a binding site for let‐7. We also found a frequent amplification and overexpression of YEATS4, an oncogene that inactivates P53, suggesting that YEATS4 might play an important role together with MDM2 in WDLPS/DDLPS oncogenesis.

Well-differentiated and dedifferentiated liposarcomas.

Atypical lipomatous tumor or well-differentiated liposarcoma (ALT-WDLPS) and dedifferentiated liposarcoma (DDLPS) share the same basic genetic abnormality characterized by a simple genomic profile with a 12q14–15 amplification involving MDM2 gene. These tumors are the most frequent LPS. This paper reviews the molecular pathology, general clinical and imaging features, histopathology, new diagnostic tools, and prognosis of ALT-WDLPS and DDLPS.