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Giant cell tumors (GCTs) with high mobility group AT-Hook 2 (HMGA2)::nuclear receptor corepressor 2 (NCOR2) fusion are rare mesenchymal tumors of controversial nosology, which have been anecdotally reported to respond to CSFR1 inhibitors. Here, we performed a comprehensive study of 6 GCTs with HMGA2::NCOR2fusion and explored their relationship with other giant cell-rich neoplasms. Tumors occurred in 4 females and 2 males ranging in age from 17 to 32 years old (median 24). Three lesions originated in subcutaneous soft tissue and 3 in bone. Tumor size ranged from 20 to 33 mm (median 27 mm). The lesions had a nodular/multinodular architecture and were composed of sheets of mononuclear “histiocytoid” cells with uniform nuclei intermingled with multinucleated giant cells. Mitotic activity was low and nuclear atypia and metaplastic bone were absent. Variable findings included necrosis, cystic degeneration, lymphocytic infiltrate (sometimes forming nodules), and xanthogranulomatous inflammation. On immunohistochemistry, all cases focally expressed pan-keratin and were negative with SATB2 and H3.3G34W. Whole RNA-sequencing was performed in all cases of GCT with HMGA2::NCOR2fusion and a subset of giant cell-rich tumors (tenosynovial-GCT, n = 19 and “wild-type” GCT of soft tissue, n = 9). Hierarchical clustering of RNA-sequencing data showed that GCT with HMGA2::NCOR2fusion formed a single cluster, independent of the other 2 entities. Methylome profiling showed similar results, but the distinction from “wild-type” GCT of soft tissue was less flagrant. Gene expression analysis showed similar levels of expression of the CSF1/CSFR1 axis between GCT with HMGA2::NCOR2fusion and tenosynovial-GCT, supporting their potential sensitivity to CSFR1 inhibitors. Clinical follow-up was available for 5 patients (range: 10 to 64 mo; median 32 mo). Three patients (60%) experienced local recurrences, whereas none had distant metastases or died of disease. Overall, our study confirms and expands previous knowledge on GCT with HMGA2::NCOR2fusion and supports its inclusion as an independent entity.

Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare soft tissue tumor with a predilection for the distal extremities and a tendency for local recurrence. Morphologically, MIFS consists of spindle and bizarre epithelioid cells resembling virocytes embedded in a fibrous to myxoid stroma with an abundant inflammatory infiltrate. Importantly, the molecular landscape of MIFS is wide and includes: VGLL3amplification, BRAFfusion/amplification and OGA/TGFBR3rearrangements. In this study, we describe a variant of MIFS showing a frequent nodular configuration associated with necrosis and recurrent YAP1::MAML2fusions. The cohort consisted of 7 patients (4 females and 3 males) ranging in age from 21 to 71 years (median: 47 years). Two tumors (28%) occurred in acral locations while the remaining cases were more widely distributed (thigh, n= 2; arm, n= 1; neck; n= 1; chest-wall, n= 1). Tumor size ranged from 10 to 38 mm (median: 20 mm). Histologically, lesions frequently presented as nodules with central areas of necrosis, and were predominantly composed of sheets of epithelioid cells with large vesicular nuclei and prominent nucleoli (Reed-Sternberg-like cells or virocytes). The stroma was mostly fibrous and showed a polymorphous inflammatory infiltrate. Myxoid stromal changes were focally seen in one case, and pseudolipoblasts were absent. The immunophenotype was nonspecific, with only pan-keratin (AE1-AE3) and cyclin D1 expression in a subset of cases. RNA-Sequencing detected YAP1::MAML2fusions in 3/7 cases; aCGH showed no significant gene copy number variations in 4 tested cases, and FISH analysis showed no VGLL3amplification in 1 tested case. Follow-up was available for 6 cases, ranging from 7 to 63 months (median: 42 months). Local recurrence and metastasis were not seen and one tumor showed spontaneous regression following initial biopsy. In conclusion, we describe a novel variant of MIFS with distinctive clinicopathological and molecular features for which we propose the term “nodular necrotizing” MIFS.

Myxoinflammatory fibroblastic sarcoma (MIFS) is a rare soft tissue tumor with a predilection for the distal extremities and a tendency for local recurrence. Morphologically, MIFS consists of spindle and bizarre epithelioid cells resembling virocytes embedded in a fibrous to myxoid stroma with an abundant inflammatory infiltrate. Importantly, the molecular landscape of MIFS is wide and includes: VGLL3amplification, BRAFfusion/amplification and OGA/TGFBR3rearrangements. In this study, we describe a variant of MIFS showing a frequent nodular configuration associated with necrosis and recurrent YAP1::MAML2fusions. The cohort consisted of 7 patients (4 females and 3 males) ranging in age from 21 to 71 years (median: 47 years). Two tumors (28%) occurred in acral locations while the remaining cases were more widely distributed (thigh, n= 2; arm, n= 1; neck; n= 1; chest-wall, n= 1). Tumor size ranged from 10 to 38 mm (median: 20 mm). Histologically, lesions frequently presented as nodules with central areas of necrosis, and were predominantly composed of sheets of epithelioid cells with large vesicular nuclei and prominent nucleoli (Reed-Sternberg-like cells or virocytes). The stroma was mostly fibrous and showed a polymorphous inflammatory infiltrate. Myxoid stromal changes were focally seen in one case, and pseudolipoblasts were absent. The immunophenotype was nonspecific, with only pan-keratin (AE1-AE3) and cyclin D1 expression in a subset of cases. RNA-Sequencing detected YAP1::MAML2fusions in 3/7 cases; aCGH showed no significant gene copy number variations in 4 tested cases, and FISH analysis showed no VGLL3amplification in 1 tested case. Follow-up was available for 6 cases, ranging from 7 to 63 months (median: 42 months). Local recurrence and metastasis were not seen and one tumor showed spontaneous regression following initial biopsy. In conclusion, we describe a novel variant of MIFS with distinctive clinicopathological and molecular features for which we propose the term “nodular necrotizing” MIFS.

NFATc2-rearranged sarcomas (NFATc2-Sarcomas) are infrequent round cell tumors characterized by EWSR1-NFATc2fusions and FUS-NFATc2fusions. Although our knowledge on these neoplasms has increased recently, novel diagnostic tools and more comprehensive series are still needed. Here, we describe the features of a series of seven molecularly confirmed NFATc2-Sarcomas (EWSR1-NFATc2, n= 4; FUS-NFATc2, n= 3) and demonstrate the utility of AGGRECAN immunohistochemistry for their identification. Patients were four males and three females, ranging in age from 19 to 66 years (median: 33). All were primary bone tumors (femur, n= 4; tibia, n= 2; ilium, n= 1), frequently infiltrating the surrounding soft tissues. Treatment often consisted of neoadjuvant chemotherapy and surgery. Follow-up was available for six patients (median 18 months, range 5–102 months), three patients died of disease and four patients are currently alive. Histologically, tumors consisted of monotonous round cells growing in lobules and sheets in variable amounts of fibrous to myxoid stroma. Other findings included spindle cells, corded and trabecular architecture, nuclear pleomorphism, cartilaginous differentiation, and osteoid-like matrix. Histological response to neoadjuvant chemotherapy was poor in all resection specimens available for review (n= 4). Tumors were diffusely positive for AGGRECAN and CD99 (7/7), and a subset expressed Pan-Keratin (AE1-AE3; 3/6), S100 (2/6), BCOR (2/6), ETV-4 (2/5), WT1 (2/6), and ERG (2/5). Desmin, NKX3-1, and SATB2 were negative (0/6). Diffuse AGGRECAN staining was also seen in 8/129 round cell sarcomas used for comparison, including mesenchymal chondrosarcoma (7/26) and CIC-sarcoma (1/26). Array-CGH showed complex karyotypes with recurrent deletions of tumor suppressor genes (CDKN2A/B, TUSC7, and DMD)in three FUS-NFATC2cases and a simpler profile without homozygous losses in one EWSR1-NFATc2case. Segmental chromosomal gains covering the loci of the fusion genes were detected in both variants. Overall, our study confirms and expands previous observations on NFATc2-sarcomas and supports that AGGRECAN is a useful biomarker of these tumors.

NFATc2-rearranged sarcomas (NFATc2-Sarcomas) are infrequent round cell tumors characterized by EWSR1-NFATc2fusions and FUS-NFATc2fusions. Although our knowledge on these neoplasms has increased recently, novel diagnostic tools and more comprehensive series are still needed. Here, we describe the features of a series of seven molecularly confirmed NFATc2-Sarcomas (EWSR1-NFATc2, n?=?4; FUS-NFATc2, n?=?3) and demonstrate the utility of AGGRECAN immunohistochemistry for their identification. Patients were four males and three females, ranging in age from 19 to 66 years (median: 33). All were primary bone tumors (femur, n?=?4; tibia, n?=?2; ilium, n?=?1), frequently infiltrating the surrounding soft tissues. Treatment often consisted of neoadjuvant chemotherapy and surgery. Follow-up was available for six patients (median 18 months, range 5–102 months), three patients died of disease and four patients are currently alive. Histologically, tumors consisted of monotonous round cells growing in lobules and sheets in variable amounts of fibrous to myxoid stroma. Other findings included spindle cells, corded and trabecular architecture, nuclear pleomorphism, cartilaginous differentiation, and osteoid-like matrix. Histological response to neoadjuvant chemotherapy was poor in all resection specimens available for review (n?=?4). Tumors were diffusely positive for AGGRECAN and CD99 (7/7), and a subset expressed Pan-Keratin (AE1-AE3; 3/6), S100 (2/6), BCOR (2/6), ETV-4 (2/5), WT1 (2/6), and ERG (2/5). Desmin, NKX3-1, and SATB2 were negative (0/6). Diffuse AGGRECAN staining was also seen in 8/129 round cell sarcomas used for comparison, including mesenchymal chondrosarcoma (7/26) and CIC-sarcoma (1/26). Array-CGH showed complex karyotypes with recurrent deletions of tumor suppressor genes (CDKN2A/B, TUSC7, and DMD)in three FUS-NFATC2cases and a simpler profile without homozygous losses in one EWSR1-NFATc2case. Segmental chromosomal gains covering the loci of the fusion genes were detected in both variants. Overall, our study confirms and expands previous observations on NFATc2-sarcomas and supports that AGGRECAN is a useful biomarker of these tumors.

Supplemental Digital Content is available in the text.Desmoid fibromatosis (DF) is a rare, locally aggressive, nonmetastasizing fibroblastic/myofibroblastic tumor with a tendency to recur and an unpredictable clinical course. A “wait-and-see” policy is the new standard of care. DF are characterized by activating alterations of the wnt/β-catenin pathway: CTNNB1or adenomatous polyposis coli gene (APC) mutations (these mutations being mutually exclusive). Desmoid-type fibromatosis of the breast (DFB) is rare with an incidence of 0.2% of breast tumors. The diagnosis of DFB is difficult, as it must be distinguished from metaplastic carcinoma and other spindle cell lesions. Sequencing of 128 DFB identified a lower rate of CTNNB1mutations using Sanger (65.6%) or Sanger+next-generation sequencing (77.7%) and a higher rate of APCmutations (11.8%) than in all-site DF. By excluding patients with familial adenomatous polyposis (n=2), the rate of APCmutations in DFB was high (10.7%). The distribution of CTNNB1mutations in DFB was different from all-site DF, with a higher rate of T41A (68.9%), a lower rate of S45F (5.7%), and a similar rate of S45T (12.6%). By combining the 2 molecular techniques in a 2-step manner (Sanger, then next-generation sequencing), we increased the detection rate of CTNNB1mutations and lowered the rate of wild-type tumors from 34.4% to 9.8%, therefore improving the diagnosis of DFB. The identification of the exon 3 CTNNB1mutation in breast spindle cell lesions is a highly specific tool for the diagnosis of DFB, in addition to extensive immunohistochemical analysis. Our study also underlines the importance of APCin DFB tumorigenesis. These findings have significant implications for patient care and management.

Cell fusion in tumor progression mostly refers to the merging of a cancer cell with a cell that has migration and immune escape capabilities such as macrophages. Here we show that spontaneous hybrids made from the fusion of transformed mesenchymal cells with partners from the same lineage undergo nonrecurrent large-scale genomic rearrangements, leading to the creation of highly aneuploid cells with novel phenotypic traits, including metastatic spreading capabilities. Moreover, in contrast to their parents, hybrids were the only cells able to recapitulate in vivo all features of human pleomorphic sarcomas, a rare and genetically complex mesenchymal tumor. Hybrid tumors not only displayed specific mesenchymal markers, but also combined a complex genetic profile with a highly metastatic behavior, like their human counterparts. Finally, we provide evidence that patient-derived pleomorphic sarcoma cells are inclined to spontaneous cell fusion. The resulting hybrids also gain in aggressiveness, exhibiting superior growth capacity in mouse models. Altogether, these results indicate that cell fusion has the potential to promote cancer progression by increasing growth and/or metastatic capacities, regardless of the nature of the companion cell. Moreover, such events likely occur upon sarcoma development, paving the way for better understanding of the biology, and aggressiveness of these tumors.

IMPORTANCE: Active therapeutic combinations, such as trabectedin and radiotherapy, offer potentially higher dimensional response in second-line treatment of advanced soft-tissue sarcomas. Dimensional response can be relevant both for symptom relief and for survival. OBJECTIVE: To assess the combined use of trabectedin and radiotherapy in treating patients with progressing metastatic soft-tissue sarcomas. DESIGN, SETTING, AND PARTICIPANTS: Phase 1 of this nonrandomized clinical trial followed the classic 3 + 3 design, with planned radiotherapy at a fixed dose of 30 Gy (3 Gy/d for 10 days) and infusion of trabectedin at 1.3 mg/m2 as the starting dose, 1.5 mg/m2 as dose level +1, and 1.1 mg/m2 as dose level –1. Phase 2 followed the Simon optimal 2-stage design. Allowing for type I and II errors of 10%, treatment success was defined as an overall response rate of 35%. This study was conducted in 9 sarcoma referral centers in Spain, France, and Italy from April 13, 2015, to November 20, 2018. Adult patients with progressing metastatic soft-tissue sarcoma and having undergone at least 1 previous line of systemic therapy were enrolled. In phase 2, patients fitting inclusion criteria and receiving at least 1 cycle of trabectedin and the radiotherapy regimen constituted the per-protocol population; those receiving at least 1 cycle of trabectedin, the safety population. INTERVENTIONS: Trabectedin was administered every 3 weeks in a 24-hour infusion. Radiotherapy was required to start within 1 hour after completion of the first trabectedin infusion (cycle 1, day 2). MAIN OUTCOMES AND MEASURES: The dose-limiting toxic effects of trabectedin (phase 1) and the overall response rate (phase 2) with use of trabectedin plus irradiation in metastatic soft-tissue sarcomas. RESULTS: Eighteen patients (11 of whom were male) were enrolled in phase 1, and 27 other patients (14 of whom were female) were enrolled in phase 2. The median ages of those enrolled in phases 1 and 2 were 42 (range, 23-74) years and 51 (range, 27-73) years, respectively. In phase 1, dose-limiting toxic effects included grade 4 neutropenia lasting more than 5 days in 1 patient at the starting dose level and a grade 4 alanine aminotransferase level increase in 1 of 6 patients at the +1 dose level. In phase 2, among 25 patients with evaluable data, the overall response rate was 72% (95% CI, 53%-91%) for local assessment and 60% (95% CI, 39%-81%) for central assessment. CONCLUSIONS AND RELEVANCE: The findings of this study suggest that the recommended dose of trabectedin for use in combination with this irradiation regimen is 1.5 mg/m2. The trial met its primary end point, with a high overall response rate that indicates the potential of this combination therapy for achieving substantial tumor shrinkage beyond first-line systemic therapy in patients with metastatic, progressing soft-tissue sarcomas. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02275286

Rhabdomyosarcomas with TFCP2fusions represent an emerging subtype of tumors, initially discovered by RNA-sequencing. We report herein the clinicopathological, transcriptional, and genomic features of a series of 14 cases. Cases were retrospectively and prospectively recruited and studied by immunohistochemistry (MYF4, MYOD1, S100, AE1/E3, ALK), fluorescence in situ hybridization with TFCP2break-apart probe (n= 10/14), array-comparative genomic hybridization (Agilent), whole RNA-sequencing (Truseq Exome, Illumina), or anchored multiplex PCR-based targeted next-generation sequencing (Archer® FusionPlex® Sarcoma kit). Patient’s age ranged between 11 and 86 years, including 5 pediatric cases. Tumors were located in the bone (n= 12/14) and soft tissue (n= 2/14). Most bone tumors invaded surrounding soft tissue. Craniofacial bones were over-represented (n= 8/12). Median survival was 8 months and five patients are currently alive with a median follow-up of 20 months. Most tumors displayed a mixed spindle cell and epithelioid pattern with frequent vesicular nuclei. All tumors expressed keratins and showed a rhabdomyogenic phenotype (defined as expression of MYF4 and/or MYOD1). ALK was overexpressed in all but three cases without underlying ALKfusion on break-apart FISH (n= 5) nor next-generation sequencing (n= 14). ALKupregulation was frequently associated with an internal deletion at genomic level. TFCP2was fused in 5′ either to EWSR1(n= 6) or FUS(n= 8). EWSR1was involved in both soft tissue cases. FISH with TFCP2break-apart probe was positive in all tested cases (n= 8), including one case with unbalanced signal. On array-CGH, all tested tumors displayed complex genetic profiles with genomic indexes ranging from 13 to 107.55 and recurrent CDKN2Adeletions. FET-TFCP2rhabdomyosarcomas clustered together and distinctly from other rhabdomyosarcomas subgroups. Altogether, our data confirm and expand the spectrum of the new family of FET-TFCP2rhabdomyosarcomas, which are associated with a predilection for the craniofacial bones, an aggressive course, and recurrent pathological features. Their association with ALK overexpression might represent a therapeutic vulnerability.

Rhabdomyosarcomas with TFCP2fusions represent an emerging subtype of tumors, initially discovered by RNA-sequencing. We report herein the clinicopathological, transcriptional, and genomic features of a series of 14 cases. Cases were retrospectively and prospectively recruited and studied by immunohistochemistry (MYF4, MYOD1, S100, AE1/E3, ALK), fluorescence in situ hybridization with TFCP2break-apart probe (n= 10/14), array-comparative genomic hybridization (Agilent), whole RNA-sequencing (Truseq Exome, Illumina), or anchored multiplex PCR-based targeted next-generation sequencing (Archer® FusionPlex® Sarcoma kit). Patient's age ranged between 11 and 86 years, including 5 pediatric cases. Tumors were located in the bone (n= 12/14) and soft tissue (n= 2/14). Most bone tumors invaded surrounding soft tissue. Craniofacial bones were over-represented (n= 8/12). Median survival was 8 months and five patients are currently alive with a median follow-up of 20 months. Most tumors displayed a mixed spindle cell and epithelioid pattern with frequent vesicular nuclei. All tumors expressed keratins and showed a rhabdomyogenic phenotype (defined as expression of MYF4 and/or MYOD1). ALK was overexpressed in all but three cases without underlying ALKfusion on break-apart FISH (n= 5) nor next-generation sequencing (n= 14). ALKupregulation was frequently associated with an internal deletion at genomic level. TFCP2was fused in 5′ either to EWSR1(n= 6) or FUS(n= 8). EWSR1was involved in both soft tissue cases. FISH with TFCP2break-apart probe was positive in all tested cases (n= 8), including one case with unbalanced signal. On array-CGH, all tested tumors displayed complex genetic profiles with genomic indexes ranging from 13 to 107.55 and recurrent CDKN2Adeletions. FET-TFCP2rhabdomyosarcomas clustered together and distinctly from other rhabdomyosarcomas subgroups. Altogether, our data confirm and expand the spectrum of the new family of FET-TFCP2rhabdomyosarcomas, which are associated with a predilection for the craniofacial bones, an aggressive course, and recurrent pathological features. Their association with ALK overexpression might represent a therapeutic vulnerability.

Post-radiation sarcomas are rare secondary cancers arising from radiation therapies. To date, few genetic specificities have been described for such malignancies and the oncogenesis of sarcomas with complex genetics (both sporadic and post-radiation) remains largely misunderstood. We performed genomic and transcriptomic analyses on 77 post-radiation sarcomas using DNA-array and RNA sequencing. Consequently, we were able to investigate changes in copy number variations, transcriptome profiling, fusion gene expression, and mutational landscapes. We compare these data to a reference cohort of 93 sporadic sarcomas. At genomic level, similar chromosomal complexity was observed both in post-radiation and sporadic sarcomas with complex genetics. We found more frequent CDKN2Aand CDKN2B(coding for p14/p16 and p15 proteins, respectively; at 9p21.3) losses in post-radiation (71%) than in sporadic tumors (39%; P= 6.92e−3). Among all detected fusion genes and punctual variations, few specificities were observed between these groups and such alterations are not able to drive a strong and specific oncogenesis. Recurrent MYCamplifications (96%) and KDRvariants (8%) were detected in post-radiation angiosarcomas, in agreement with the literature. Transcriptomic analysis of such angiosarcomas revealed two distinct groups harboring different genomic imbalances (in particular gains of 17q24.2-17qter) with different clinical courses according to patient's vital status. Differential gene expression analysis permitted to focus on the immune response as a potential actor to tumor aggressiveness. Histochemistry validated a lower inflammation and lower immune infiltrate at tumor periphery for highly aggressive angiosarcomas. Our results provide new genomic and transcriptomic information about post-radiation sarcomas. The techniques we used (RNA-seq and DNA-arrays) did not highlight major differences in sarcomas with complex genetics depending on the radiation context, revealing similar patterns of transcriptomic profiles and chromosomal copy number variations. Additional characterizations, particularly whole genome sequencing, could measure changes in DNA following radiation therapy in such malignancies and may precise their oncogenesis.

Post-radiation sarcomas are rare secondary cancers arising from radiation therapies. To date, few genetic specificities have been described for such malignancies and the oncogenesis of sarcomas with complex genetics (both sporadic and post-radiation) remains largely misunderstood. We performed genomic and transcriptomic analyses on 77 post-radiation sarcomas using DNA-array and RNA sequencing. Consequently, we were able to investigate changes in copy number variations, transcriptome profiling, fusion gene expression, and mutational landscapes. We compare these data to a reference cohort of 93 sporadic sarcomas. At genomic level, similar chromosomal complexity was observed both in post-radiation and sporadic sarcomas with complex genetics. We found more frequent CDKN2Aand CDKN2B(coding for p14/p16 and p15 proteins, respectively; at 9p21.3) losses in post-radiation (71%) than in sporadic tumors (39%; P= 6.92e−3). Among all detected fusion genes and punctual variations, few specificities were observed between these groups and such alterations are not able to drive a strong and specific oncogenesis. Recurrent MYCamplifications (96%) and KDRvariants (8%) were detected in post-radiation angiosarcomas, in agreement with the literature. Transcriptomic analysis of such angiosarcomas revealed two distinct groups harboring different genomic imbalances (in particular gains of 17q24.2-17qter) with different clinical courses according to patient’s vital status. Differential gene expression analysis permitted to focus on the immune response as a potential actor to tumor aggressiveness. Histochemistry validated a lower inflammation and lower immune infiltrate at tumor periphery for highly aggressive angiosarcomas. Our results provide new genomic and transcriptomic information about post-radiation sarcomas. The techniques we used (RNA-seq and DNA-arrays) did not highlight major differences in sarcomas with complex genetics depending on the radiation context, revealing similar patterns of transcriptomic profiles and chromosomal copy number variations. Additional characterizations, particularly whole genome sequencing, could measure changes in DNA following radiation therapy in such malignancies and may precise their oncogenesis.

Oncogenesis is considered to result from chromosomal instability, in addition to oncogene and tumor-suppressor alterations. Intermediate to aneuploidy and chromosomal instability, genome doubling is a frequent event in tumor development but the mechanisms driving tetraploidization and its impact remain unexplored. Cell fusion, one of the pathways to tetraploidy, is a physiological process involved in mesenchymal cell differentiation. Besides simple genome doubling, cell fusion results in the merging of two different genomes that can be destabilized upon proliferation. By testing whether cell fusion is involved in mesenchymal oncogenesis, we provide evidence that it induces genomic instability and mediates tumor initiation. After a latency period, the tumor emerges with the cells most suited for its development. Furthermore, hybrid tumor genomes were stabilized after this selection process and were very close to those of human pleomorphic mesenchymal tumors. Thus genome restructuring triggered by cell fusion may account for the chromosomal instability involved in oncogenesis.

Biphenotypic sinonasal sarcoma (BSNS) is a locally aggressive tumor occurring in the sinonasal region. It harbors both myogenic and neural differentiation and is characterized by PAX3rearrangement with MAML3as the most frequent fusion partner, but the partner of PAX3remains unidentified in a subset of cases. About 70 cases have been reported so far. In this study, we report a series of 41 cases with clinical, pathologic, and molecular description. Twenty-five (61%) patients were female individuals, and the median age was 49 years. Tumors arose predominantly in the nasal cavity and ethmoidal sinuses. Local recurrences occurred in 8 cases of the 25 (32%). Histologic features were characteristic of BSNS, with 5 cases showing focal rhabdomyoblastic differentiation. Immunohistochemistry showed a constant positivity of S100 protein and PAX3 and negativity of SOX10. MyoD1 was focally positive in 91% of cases, whereas only 20% were positive for myogenin. Molecular analysis showed a PAX3-MAML3transcript in 37 cases (90%). RNA sequencing was performed in the 4 negative cases for PAX3-MAML3fusion, and it showed that 1 case harbored a PAX3-FOXO1fusion, as previously described in the literature, and 2 novel fusions: PAX3-WWTR1fusion in 2 cases and PAX3-NCOA2fusion in 1 case. RNA sequencing results were confirmed by fluorescence in situ hybridization, reverse transcription-polymerase chain reaction, and Sanger sequencing. The PAX3-NCOA2-positive case showed focal rhabdomyoblastic differentiation. In conclusion, we report 2 novel fusions (PAX3-WWTR1and PAX3-NCOA2) in BSNS and show that MyoD1 is more sensitive than myogenin for demonstrating myogenic differentiation in this tumor.

Supplemental Digital Content is available in the text.SMARCA4-deficient thoracic sarcoma (SMARCA4-DTS) is a recently described entity with an aggressive clinical course and specific genetic alterations of the BAF chromatin remodeling complex. In the present study, we reviewed the clinical and pathologic features of 30 cases of SMARCA4-DTS, discussed its main differential diagnoses and the challenging diagnostic scenarios that the average pathologist may face. In addition, we tested the specificity of the “SMARCA4-DTS immunohistochemical signature” (co-loss of SMARCA4 and SMARCA2 with overexpression of SOX2) in a large cohort of intrathoracic malignancies. Patients ranged from 28 to 90 years of age (median: 48 y), with a marked male predominance (male:female=9:1) and they were usually smokers. Tumors were generally large compressive masses located in the mediastinum (n=13), pleura (n=5), lung (n=2) or in 2 or more of these topographies (n=10). Treatment strategies were varied, including 1 case treated with EZH2 inhibitors. Median overall survival was 6 months. Histologically, tumors were poorly differentiated frequently showing rhabdoid features. A subset of cases showed a focal myxoid stroma (7%, n=2/30) and rare cases displayed a previously unreported pattern simulating desmoplastic small round cell tumors (7%, n=2/30). Making a diagnosis was challenging when dealing with biopsy material from massively necrotic tumors and in this setting the expression of SOX2, CD34, and SALL4 proved useful. All tested cases displayed concomitant loss of SMARCA4 and SMARCA2 and most tumors expressed epithelial markers (Pan-keratin or EMA) (n=29/30), SOX2 (n=26/27), and CD34 (n=17/27). SMARCB1 expression was retained in all cases (23/23). SALL4 and Claudin-4 were expressed in a subset of cases (n=7/21 and 2/19, respectively). TTF-1 and P63 were focally expressed in 1 case each. P40 and NUT were not expressed (0/23 and 0/20, respectively) The SMARCA4-DTS immunohistochemical signature was both sensitive and specific, with only a subset of small cell carcinoma of the ovary hypercalcemic type showing overlapping phenotypes. Our study confirms and expands the specific features of SMARCA4-DTS, emphasizing the fact that they can be straightforwardly identified by pathologists.

This article focuses on pleomorphic sarcomas, which are malignant mesenchymal tumors with complex genetic background at the root of their morphologic pleomorphism. They are poorly differentiated tumors that may retain different lines of differentiation, sometimes correlating with clinicopathological or prognostic features. Accurate diagnosis in this group of tumors relies on adequate sampling due to their heterogeneity and assessment with both microscopy and large panels of immunohistochemistry. Molecular analyses have a limited role in their diagnosis as opposed to translocation-related sarcomas but may provide theranostic and important prognostic information in the future.

Supplemental Digital Content is available in the text.CIC-fused sarcomas represent an emerging family of tumors, for long connected to the Ewing family group of tumors, but underlined by distinct CICfusions with different partners. 3′ Fusion partners include DUX4, FOXO4, and, as recently emphasized, NUTM1.In this study, we report the clinicopathologic and molecular features of a series of 6 CIC-NUTM1sarcomas. Mean age at diagnosis was 6 years (2 to 27 y), and 4 patients were male individuals. Primary tumors were located in the central nervous system (n=3), paravertebral soft tissue and epidural spaces (n=1, each), and lung (n=1). Median overall survival was 17.5 months (7 to 37 mo), and all but one patient died of disease. All tumors displayed classic features of CIC-DUX4 sarcomas with round cell to epithelioid microscopic appearance. Most tumors expressed ETV4 and NUTM1 (n=5/6 and 6/6, respectively), whereas WT1cter was positive in only 2 cases. All tested tumors were positive for break-apart fluorescence in situ hybridization for CICand NUTM1. Apart from CICor NUTM1genomic breakpoints, no other recurrent copy number alteration was seen on genomic profiles. Fusion transcripts were identified by RNA-sequencing on either formalin-fixed paraffin-embedded or frozen material. CICand NUTM1breakpoints were located between exons 16 and 20 and exons 2 and 5, respectively. Altogether, CIC-NUTM1 sarcomas represent a new molecular variant of CIC-fused sarcomas with a predilection for the central nervous system and younger pediatric population. Its phenotype may be confused with NUT carcinomas.

Dermatofibrosarcoma protuberans is underlined by recurrent collagen type I alpha 1 chain-platelet-derived growth factor B chain(COL1A1-PDGFB) fusions but ~ 4% of typical dermatofibrosarcoma protuberans remain negative for this translocation in routine molecular screening. We investigated a series of 21 cases not associated with the pathognomonic COL1A1-PDGFBfusion on routine fluorescence in situ hybridization (FISH) testing. All cases displayed morphological and clinical features consistent with the diagnosis of dermatofibrosarcoma protuberans. RNA-sequencing analysis was successful in 20 cases. The classical COL1A1-PDGFBfusion was present in 40% of cases (n= 8/20), and subsequently confirmed with a COL1A1break-apart FISH probe in all but one case (n= 7/8). 55% of cases (n= 11/20) displayed novel PDGFDrearrangements; PDGFDbeing fused either to the 5′ part of COL6A3(2q37.3) (n= 9/11) or EMILIN2(18p11) (n= 2/11). All rearrangements led to in-frame fusion transcripts and were confirmed at genomic level by FISH and/or array-comparative genomic hybridization. PDGFD-rearranged dermatofibrosarcoma protuberans presented clinical outcomes similar to typical dermatofibrosarcoma protuberans. Notably, the two EMILIN2-PDGFDcases displayed fibrosarcomatous transformation and homozygous deletions of CDKN2Aat genomic level. We report the first recurrent molecular variant of dermatofibrosarcoma protuberans involving PDGFD, which functionally mimic bona fide COL1A1-PDGFB fusions, leading presumably to a similar autocrine loop-stimulating PDGFRB. This study also emphasizes that COL1A1-PDGFBfusions can be cytogenetically cryptic on FISH testing in a subset of cases, thereby representing a diagnostic pitfall that pathologists should be aware of.

Supplemental Digital Content is available in the text.CIC-rearranged sarcomas rarely occur in visceral organs including the kidney. The most common fusion partner with CICis the DUX4gene, but variant fusion partners have also been reported. Herein, we describe the clinicopathologic features and comprehensive molecular profiling of 4 cases of primary renal CIC-rearranged sarcomas. All cases occurred in females, age range 13 to 82 years and included 3 resections and 1 needle biopsy specimen. There was a tendency for development of metastatic disease predominantly to the lungs and poor disease outcome despite different treatment strategies. Histologically, variable round cell (20% to 100%), spindle cell (0% to 80%), and rhabdoid morphologies (0% to 20%) were seen. By immunohistochemistry diffuse WT1 nuclear (2 to 3+, ∼90%) labeling was present in 1 case, with cytoplasmic staining in the others (3+, 40% to 75%). CD99 was focally positive in all 4 cases (≤10%); 1 case each was diffusely positive for c-myc (2 to 3+, ∼90%) and ETV4 (3+, ∼90%); 1 case was focally positive for c-myc (2+, ∼5%) and calretinin (2+, ∼5%); and all cases were negative for cytokeratin and NKX2.2. CICrearrangement by fluorescence in situ hybridization was present in the 3 cases tested. Comprehensive genomic profiling (CGP) of 3 cases revealed a CIC-DUX4fusion in 2 cases, and 1 CIC-NUTM1fusion. All 4 CIC-rearranged renal sarcomas had low mutation burden, and except HLA-Aand MLLmutations lacked genomic alterations in other oncogenic drivers. Material from the needle biopsy was insufficient for CGP but that case was positive with the DUX4 immunohistochemical stain as were the 2 CIC-DUX4tumors. In conclusion, CIC-rearranged sarcomas rarely occur in the kidney with a tendency for poor outcome and in this series we illustrate an example with CIC-NUTM1fusion, an emerging variant, at a visceral site. Testing by fluorescence in situ hybridization or CGP is optimal to avoid missing cases that harbor variant fusion partners.

Dermatofibrosarcoma protuberans is underlined by recurrent collagen type I alpha 1 chain-platelet-derived growth factor B chain(COL1A1-PDGFB) fusions but ~ 4% of typical dermatofibrosarcoma protuberans remain negative for this translocation in routine molecular screening. We investigated a series of 21 cases not associated with the pathognomonic COL1A1-PDGFBfusion on routine fluorescence in situ hybridization (FISH) testing. All cases displayed morphological and clinical features consistent with the diagnosis of dermatofibrosarcoma protuberans. RNA-sequencing analysis was successful in 20 cases. The classical COL1A1-PDGFBfusion was present in 40% of cases (n= 8/20), and subsequently confirmed with a COL1A1break-apart FISH probe in all but one case (n= 7/8). 55% of cases (n= 11/20) displayed novel PDGFDrearrangements; PDGFDbeing fused either to the 5′ part of COL6A3(2q37.3) (n= 9/11) or EMILIN2(18p11) (n= 2/11). All rearrangements led to in-frame fusion transcripts and were confirmed at genomic level by FISH and/or array-comparative genomic hybridization. PDGFD-rearranged dermatofibrosarcoma protuberans presented clinical outcomes similar to typical dermatofibrosarcoma protuberans. Notably, the two EMILIN2-PDGFDcases displayed fibrosarcomatous transformation and homozygous deletions of CDKN2Aat genomic level. We report the first recurrent molecular variant of dermatofibrosarcoma protuberans involving PDGFD, which functionally mimic bona fide COL1A1-PDGFB fusions, leading presumably to a similar autocrine loop-stimulating PDGFRB. This study also emphasizes that COL1A1-PDGFBfusions can be cytogenetically cryptic on FISH testing in a subset of cases, thereby representing a diagnostic pitfall that pathologists should be aware of.

The diagnosis of malignant peripheral nerve sheath tumor remains challenging, especially in the sporadic setting. Malignant peripheral nerve sheath tumor is a rare malignancy, and owing to the lack of specific histological criteria, immunohistochemical and molecular diagnostic markers, several differential diagnoses must be considered, in particular melanoma. Recently, inactivation of the polycomb repressive complex 2 (PRC2), induced by inactivating mutations in two of its critical constituents SUZ12 and EED, was reported in a large subset of malignant peripheral nerve sheath tumors. Homozygous PRC2 inactivation induces complete loss of trimethylation at lysine 27 of histone 3 (H3K27me3). Recent studies suggest that complete loss of H3K27me3 is highly specific for malignant peripheral nerve sheath tumor and may be a useful immunohistochemical diagnostic marker. Therefore, to determine the specificity of the complete loss of H3K27me3 expression in the context of the differential diagnosis of malignant peripheral nerve sheath tumor from melanoma (its major potential mimic), we performed H3K27me3 immunohistochemistry in a pathologically and genetically well-characterized cohort of primary (neurofibromatosis type 1 (NF1), radiation-associated and sporadic context) malignant peripheral nerve sheath tumors (n=122) and in a cohort or primary (desmoplastic) and metastatic melanomas (n=265). In total, 88 (72%) malignant peripheral nerve sheath tumors, including 46 (71%) NF1-associated, 4 (100%) radiation-associated, and 38 (72%) sporadic tumors, showed complete loss of H3K27me3. We observed increased loss of H3K27me3 with increasing histological grade. Interestingly, we found complete loss of H3K27me3 in 37% (n=98) of all melanomas, including 25% (n=9) of primary desmoplastic melanomas. Moreover, partial loss ('mosaic' pattern) was observed in 23 (19%) of all malignant peripheral nerve sheath tumors and in 136 (51%) of all melanomas. Complete loss of H3K27me3 detected by immunohistochemistry is not specific for malignant peripheral nerve sheath tumor and cannot be used safely when distinguishing malignant peripheral nerve sheath tumor from melanoma.

The diagnosis of malignant peripheral nerve sheath tumor remains challenging, especially in the sporadic setting. Malignant peripheral nerve sheath tumor is a rare malignancy, and owing to the lack of specific histological criteria, immunohistochemical and molecular diagnostic markers, several differential diagnoses must be considered, in particular melanoma. Recently, inactivation of the polycomb repressive complex 2 (PRC2), induced by inactivating mutations in two of its critical constituents SUZ12and EED, was reported in a large subset of malignant peripheral nerve sheath tumors. Homozygous PRC2 inactivation induces complete loss of trimethylation at lysine 27 of histone 3 (H3K27me3). Recent studies suggest that complete loss of H3K27me3 is highly specific for malignant peripheral nerve sheath tumor and may be a useful immunohistochemical diagnostic marker. Therefore, to determine the specificity of the complete loss of H3K27me3 expression in the context of the differential diagnosis of malignant peripheral nerve sheath tumor from melanoma (its major potential mimic), we performed H3K27me3 immunohistochemistry in a pathologically and genetically well-characterized cohort of primary (neurofibromatosis type 1 (NF1), radiation-associated and sporadic context) malignant peripheral nerve sheath tumors (n=122) and in a cohort or primary (desmoplastic) and metastatic melanomas (n=265). In total, 88 (72%) malignant peripheral nerve sheath tumors, including 46 (71%) NF1-associated, 4 (100%) radiation-associated, and 38 (72%) sporadic tumors, showed complete loss of H3K27me3. We observed increased loss of H3K27me3 with increasing histological grade. Interestingly, we found complete loss of H3K27me3 in 37% (n=98) of all melanomas, including 25% (n=9) of primary desmoplastic melanomas. Moreover, partial loss ('mosaic' pattern) was observed in 23 (19%) of all malignant peripheral nerve sheath tumors and in 136 (51%) of all melanomas. Complete loss of H3K27me3 detected by immunohistochemistry is not specific for malignant peripheral nerve sheath tumor and cannot be used safely when distinguishing malignant peripheral nerve sheath tumor from melanoma.

This article focuses on families of round cell sarcomas other than classical Ewing sarcomas. Until recently, these tumors were referred to as so-called Ewing-like tumors, as they morphologically resemble Ewing sarcomas but are negative for canonical fusion transcripts of Ewing sarcomas involving gene members of the ETS family of transcription factors. Clinicopathologic and molecular evidence has dramatically influenced the diagnostic approach of these tumors in recent years. Molecular data that support these sarcoma subtypes are biologically distinct from those of Ewing sarcomas, thereby advocating discarding the all-embracing and confusing terminology of “Ewing-like tumors.”

Subsets of primitive round-cell sarcomas remain difficult to diagnose and classify. Among these is a rare round-cell sarcoma that harbors a CIC gene rearrangement known as CIC-rearranged undifferentiated round-cell sarcoma, which is most commonly fused to the DUX4 gene. Owing to its aggressive clinical behavior and potential therapeutic implications, accurate identification of this novel soft tissue sarcoma is necessary. Definitive diagnosis requires molecular confirmation, but only a few centers are as yet able to perform this test. Several studies have shown that PEA3 subfamily genes, notably ETV4 (belonging to the family of ETS transcription factors), are upregulated in CIC-rearranged undifferentiated round-cell sarcomas. We performed a detailed immunohistochemical analysis to investigate ETV4 expression in CIC-rearranged undifferentiated round-cell sarcomas and their potential mimics (especially Ewing sarcomas). The study cohort included 17 cases of CIC-rearranged undifferentiated round-cell sarcomas, and 110 tumors that morphologically mimic CIC-rearranged undifferentiated round-cell sarcomas: 43 Ewing sarcomas, 25 alveolar rhabdomyosarcomas, 20 poorly differentiated round-cell synovial sarcomas, 10 desmoplastic round-cell tumors, 5 BCOR-CCNB3 sarcomas, 5 lymphoblastic lymphomas, and 2 rhabdoid tumors. All CIC-rearranged undifferentiated round-cell sarcomas (on core needle biopsies and open biopsies) were ETV4-positive with a strong diffuse nuclear pattern. Among the other 110 tumors, only six cases (four Ewing sarcomas, one alveolar rhabdomyosarcoma, and one desmoplastic round-cell tumor) showed focal (<5% of tumor cells) and very weak nuclear expression of ETV4; all other tumors were completely negative for ETV4. We conclude that systematic immunohistochemical analysis of ETV4 makes it possible to diagnose undifferentiated round-cell sarcomas (with no molecular markers for sarcoma-associated translocation) such as CIC-rearranged undifferentiated round-cell sarcoma.

Subsets of primitive round-cell sarcomas remain difficult to diagnose and classify. Among these is a rare round-cell sarcoma that harbors a CICgene rearrangement known as CIC-rearranged undifferentiated round-cell sarcoma, which is most commonly fused to the DUX4gene. Owing to its aggressive clinical behavior and potential therapeutic implications, accurate identification of this novel soft tissue sarcoma is necessary. Definitive diagnosis requires molecular confirmation, but only a few centers are as yet able to perform this test. Several studies have shown that PEA3subfamily genes, notably ETV4(belonging to the family of ETStranscription factors), are upregulated in CIC-rearranged undifferentiated round-cell sarcomas. We performed a detailed immunohistochemical analysis to investigate ETV4 expression in CIC-rearranged undifferentiated round-cell sarcomas and their potential mimics (especially Ewing sarcomas). The study cohort included 17 cases of CIC-rearranged undifferentiated round-cell sarcomas, and 110 tumors that morphologically mimic CIC-rearranged undifferentiated round-cell sarcomas: 43 Ewing sarcomas, 25 alveolar rhabdomyosarcomas, 20 poorly differentiated round-cell synovial sarcomas, 10 desmoplastic round-cell tumors, 5 BCOR-CCNB3sarcomas, 5 lymphoblastic lymphomas, and 2 rhabdoid tumors. All CIC-rearranged undifferentiated round-cell sarcomas (on core needle biopsies and open biopsies) were ETV4-positive with a strong diffuse nuclear pattern. Among the other 110 tumors, only six cases (four Ewing sarcomas, one alveolar rhabdomyosarcoma, and one desmoplastic round-cell tumor) showed focal (<5% of tumor cells) and very weak nuclear expression of ETV4; all other tumors were completely negative for ETV4. We conclude that systematic immunohistochemical analysis of ETV4 makes it possible to diagnose undifferentiated round-cell sarcomas (with no molecular markers for sarcoma-associated translocation) such as CIC-rearranged undifferentiated round-cell sarcoma.

Supplemental Digital Content is available in the text.An accurate histopathologic diagnosis is essential for an adequate treatment of soft tissue sarcomas. The diagnosis of malignant peripheral nerve sheath tumor (MPNST) can be complex, particularly outside the neurofibromatosis type 1 (NF1) context. MPNST is a rare malignancy, and due to the lack of specific histologic criteria, several differential diagnoses must be considered. A total of 350 patients diagnosed with MPNST (from 1990 to 2013) were retrieved from the French sarcoma network (RRePS) and the Conticabase (Connective Tissue Cancer Network database). Tumor samples were available for 160 cases (45.2%). Pathology review, immunohistochemistry (IHC), and molecular analysis (when dealing with a monomorphic sarcoma) were systematically performed. Patient, tumor, and treatment characteristics were evaluated to identify prognostic factors for the definitive primary MPNST (n=106) cohort. Twenty-nine tumors (18.1%) initially diagnosed as MPNST were reclassified on the basis of histologic review, IHC, and molecular analysis. Patients with NF1 disease comprised 64% of the remaining cohort. The 5-year overall survival for patients from the entire cohort was 47%, 34.8% for NF1 patients, and 68.5% for patients without NF1 disease, making NF1 syndrome an independent poor prognostic factor of survival. Positive margins and lack of radiation therapy were independent predictors of local recurrence. The Fédération Nationale des Centres de Lutte Contre le Cancer tumor grade was an independent prognostic indicator of metastasis. Given the therapeutic implications of a misdiagnosis, the systematic pathology review, IHC, and molecular analysis (when dealing with monomorphic sarcoma) strategy allowed reclassification of 20% of cases, mainly the sporadic MPNSTs.

Les sarcomes sont des tumeurs rares et hétérogènes et l'amélioration des connaissances et de la prise en charge des patients nécessitent la mise en commun des données et matériels biologiques. La base nationale sarcome rassemble dans un entrepôt de données environ 60 000 patients avec des informations cliniques, anatomopathologiques, moléculaires, thérapeutiques et évolutives ainsi que les données sur les échantillons disponibles et les pratiques médicales. Cette mise en commun au sein de réseaux nationaux structurés constitue un outil majeur pour le Groupe Sarcome Français.

Lipoblastoma-like tumor (LLT) is a benign soft tissue tumor demonstrating mixed morphologic features of lipoblastoma, myxoid liposarcoma and spindle cell lipoma, but lacking genetic alterations associated with those tumors. LLT was originally thought to be specific to the vulva but has since been reported in the paratesticular region. The morphologic features of LLT overlap with those of “fibrosarcoma-like lipomatous neoplasm” (FLLN), a rare, indolent adipocytic neoplasm considered by some to form part of the spectrum of atypical spindle cell and pleomorphic lipomatous tumor (ASCPLT). We compared the morphologic, immunohistochemical and genetic features of 23 tumors previously classified as LLT (n=17) and FLLN (n=6).

HMGA2, CDK4, and JUNgenes have been described as frequently coamplified with MDM2in atypical lipomatous tumor, well-differentiated liposarcoma, and dedifferentiated liposarcoma. We studied the frequency of amplification of these genes in a series of 48 dedifferentiated liposarcomas and 68 atypical lipomatous tumors/well-differentiated liposarcomas. We correlated their amplification status with clinicopathological features and outcomes. Histologically, both CDK4(P=0.007) and JUN(P=0.005) amplifications were associated with dedifferentiated liposarcoma, whereas amplification of the proximal parts of HMGA2(5′-untranslated region (UTR) and exons 1–3) was associated with atypical lipomatous tumor/well-differentiated liposarcoma (P=0.01). CDK4amplification was associated with axial tumors. Amplification of 5′-UTR and exons 1–3 of HMGA2was associated with primary status and grade 1. Shorter overall survival was correlated with: age >64 years (P=0.03), chemotherapy used in first intent (P<0.001), no surgery (P=0.003), grade 3 (P<0.001), distant metastasis (P<0.001), node involvement (P=0.006), and CDK4amplification (P=0.07). In multivariate analysis, distant metastasis (HR=8.8) and grade 3 (HR=18.2) were associated with shorter overall survival. A shorter recurrence-free survival was associated with dedifferentiated liposarcoma (P<0.001), grade 3 (P<0.001), node involvement (P<0.001), distant metastasis (P=0.02), recurrent status (P=0.009), axial location (P=0.001), and with molecular features such as CDK4(P=0.05) and JUNamplification (P=0.07). Amplification of 5′-UTR and exons 1–3 (P=0.08) and 3′-UTR (P=0.01) of HMGA2were associated with longer recurrence-free survival. Distant metastasis was associated with shorter recurrence-free survival (HR=5.8) in multivariate analysis. Dedifferentiated liposarcoma type was associated with axial location, grade 3 and recurrent status. In conclusion, we showed that the amplification of HMGA2was associated with the atypical lipomatous tumor/well-differentiated liposarcoma histological type and a good prognosis, whereas CDK4and JUNamplifications were associated with dedifferentiated liposarcoma histology and a bad prognosis. In addition, we also provided the first description of the molecular evolution of a well-differentiated liposarcoma into four successive dedifferentiated liposarcoma relapses, which was consistent with our general observations.

HMGA2, CDK4, and JUN genes have been described as frequently coamplified with MDM2 in atypical lipomatous tumor, well-differentiated liposarcoma, and dedifferentiated liposarcoma. We studied the frequency of amplification of these genes in a series of 48 dedifferentiated liposarcomas and 68 atypical lipomatous tumors/well-differentiated liposarcomas. We correlated their amplification status with clinicopathological features and outcomes. Histologically, both CDK4 (P=0.007) and JUN (P=0.005) amplifications were associated with dedifferentiated liposarcoma, whereas amplification of the proximal parts of HMGA2 (5′-untranslated region (UTR) and exons 1–3) was associated with atypical lipomatous tumor/well-differentiated liposarcoma (P=0.01). CDK4 amplification was associated with axial tumors. Amplification of 5′-UTR and exons 1–3 of HMGA2 was associated with primary status and grade 1. Shorter overall survival was correlated with: age >64 years (P=0.03), chemotherapy used in first intent (P<0.001), no surgery (P=0.003), grade 3 (P<0.001), distant metastasis (P<0.001), node involvement (P=0.006), and CDK4 amplification (P=0.07). In multivariate analysis, distant metastasis (HR=8.8) and grade 3 (HR=18.2) were associated with shorter overall survival. A shorter recurrence-free survival was associated with dedifferentiated liposarcoma (P<0.001), grade 3 (P<0.001), node involvement (P<0.001), distant metastasis (P=0.02), recurrent status (P=0.009), axial location (P=0.001), and with molecular features such as CDK4 (P=0.05) and JUN amplification (P=0.07). Amplification of 5′-UTR and exons 1–3 (P=0.08) and 3′-UTR (P=0.01) of HMGA2 were associated with longer recurrence-free survival. Distant metastasis was associated with shorter recurrence-free survival (HR=5.8) in multivariate analysis. Dedifferentiated liposarcoma type was associated with axial location, grade 3 and recurrent status. In conclusion, we showed that the amplification of HMGA2 was associated with the atypical lipomatous tumor/well-differentiated liposarcoma histological type and a good prognosis, whereas CDK4 and JUN amplifications were associated with dedifferentiated liposarcoma histology and a bad prognosis. In addition, we also provided the first description of the molecular evolution of a well-differentiated liposarcoma into four successive dedifferentiated liposarcoma relapses, which was consistent with our general observations.

Ovarian small cell carcinoma of the hypercalcemic type (SCCOHT)/ovarian rhabdoid tumor is a rare and highly malignant tumor that typically occurs in young women. Up until now the diagnosis has been made on the basis of morphology without any specific immunohistochemical (IHC) markers. However, several authors have shown recently that SCCOHTs are characterized by inactivation of the SMARCA4gene (encoding the BRG1 protein) resulting in a loss of BRG1 protein expression in IHC. We evaluated BRG1 and INI1 expression in 12 SCCOHTs and in a series of 122 tumors that could mimic SCCOHT morphologically: 9 juvenile granulosa cell tumors, 47 adult granulosa cell tumors, 33 high-grade ovarian serous carcinomas, 9 desmoplastic round cell tumors, 13 Ewing sarcomas (5 from the pelvis and 8 from soft tissues), 1 round cell sarcoma associated with CIC-DUX4translocation from soft tissue (thigh), 1 case of high-grade endometrial stromal sarcoma of the ovary, and 9 melanomas. Forty-four adult granulosa cell tumors were interpretable by IHC. All 12 SCCOHTs were devoid of BRG1 expression and expressed INI1. All other interpretable 119 tumors showed BRG1 nuclear positivity, with variable staining proportions, ranging from 10% to 100% of positive cells (mean: 77%, median: 80%), variable intensities (weak: 5%, moderate: 37%, strong: 58%), and distributions: diffuse in 82 cases (70%) and heterogenous in 36 cases (30%). BRG1 positivity was heterogenous in desmoplastic round cell tumors and adult granulosa cell tumors. Overall, BRG1 is a useful diagnostic marker in SCCOHT, showing the absence of expression in SCCOHT. Nevertheless, the possible heterogeneity and the variable intensity of this staining warrant caution in the interpretation of BRG1 staining in biopsy specimens.

Cytogenetically, most dermatofibrosarcoma protuberans are characterized by chromosomal rearrangements resulting in the collagen type-1 alpha 1 (COL1A1)–platelet-derived growth factor β (PDGFB) fusion gene. This abnormality can be detected by fluorescence in situ hybridization (FISH) analysis in routine practice. The aim of this study was to evaluate the role of the FISH analysis in the diagnosis of dermatofibrosarcoma protuberans. A FISH analysis was prospectively and systematically performed on a series of 448 consecutive tumor specimens. All cases were reviewed by two independent pathologists and classified in three categories according to the probability of a DFSP diagnosis before molecular analyses. Cases were classified as certain when dermatofibrosarcoma protuberans was the only possible diagnosis. Those cases for which dermatofibrosarcoma protuberans remained the first diagnosis, but other differential diagnosis existed, were regarded as probable. When dermatofibrosarcoma protuberans was considered a differential diagnosis, they were labeled as possible. The final diagnosis was supported by clinicopathological findings and results of FISH analyses. Immunohistochemical analysis of CD34 was systematically performed, and additional markers when necessary. The cases (n=37) with a non-interpretable FISH were excluded. For the 185 certain tumors specimens: 178 (96%) FISH analyses showed a PDGFB/COL1A1 rearrangement, 7 (4%) were negative. For the 114 probable tumors specimens: 104 (91%) FISH analyses were positive and 10 (9%) were negative leading to a new diagnosis in 8 cases. For the 112 possible cases: 91 (81%) FISH analyses were negative and 21 (19%) were positive. Of the 21 cases, initial diagnoses included unclassified sarcoma, myxofibrosarcoma, dermatofibroma, reactive lesion, solitary fibrous tumor, perineurioma, benign nerve sheath tumor, and undifferentiated spindle cell tumor without malignant evidence. FISH analysis has been helpful for confirming the diagnosis of dermatofibrosarcoma protuberans in 25% (104/411) of cases and necessary for the diagnosis of dermatofibrosarcoma protuberans in 5% (21/411) of cases.

Cytogenetically, most dermatofibrosarcoma protuberans are characterized by chromosomal rearrangements resulting in the collagen type-1 alpha 1 (COL1A1)–platelet-derived growth factor β(PDGFB) fusion gene. This abnormality can be detected by fluorescence in situhybridization (FISH) analysis in routine practice. The aim of this study was to evaluate the role of the FISH analysis in the diagnosis of dermatofibrosarcoma protuberans. A FISH analysis was prospectively and systematically performed on a series of 448 consecutive tumor specimens. All cases were reviewed by two independent pathologists and classified in three categories according to the probability of a DFSP diagnosis before molecular analyses. Cases were classified as certain when dermatofibrosarcoma protuberans was the only possible diagnosis. Those cases for which dermatofibrosarcoma protuberans remained the first diagnosis, but other differential diagnosis existed, were regarded as probable. When dermatofibrosarcoma protuberans was considered a differential diagnosis, they were labeled as possible. The final diagnosis was supported by clinicopathological findings and results of FISH analyses. Immunohistochemical analysis of CD34 was systematically performed, and additional markers when necessary. The cases (n=37) with a non-interpretable FISH were excluded. For the 185 certain tumors specimens: 178 (96%) FISH analyses showed a PDGFB/COL1A1rearrangement, 7 (4%) were negative. For the 114 probable tumors specimens: 104 (91%) FISH analyses were positive and 10 (9%) were negative leading to a new diagnosis in 8 cases. For the 112 possible cases: 91 (81%) FISH analyses were negative and 21 (19%) were positive. Of the 21 cases, initial diagnoses included unclassified sarcoma, myxofibrosarcoma, dermatofibroma, reactive lesion, solitary fibrous tumor, perineurioma, benign nerve sheath tumor, and undifferentiated spindle cell tumor without malignant evidence. FISH analysis has been helpful for confirming the diagnosis of dermatofibrosarcoma protuberans in 25% (104/411) of cases and necessary for the diagnosis of dermatofibrosarcoma protuberans in 5% (21/411) of cases.

The clinical relevance of accurately diagnosing pleomorphic sarcomas has been shown, especially in cases of undifferentiated pleomorphic sarcomas with myogenic differentiation, which appear significantly more aggressive. To establish a new smooth muscle differentiation classification and to test its prognostic value, 412 sarcomas with complex genetics were examined by immunohistochemistry using four smooth muscle markers (calponin, h-caldesmon, transgelin and smooth muscle actin). Two tumor categories were first defined: tumors with positivity for all four markers and tumors with no or incomplete phenotypes. Multivariate analysis demonstrated that this classification method exhibited the strongest prognostic value compared with other prognostic factors, including histological classification. Secondly, incomplete or absent smooth muscle phenotype tumor group was then divided into subgroups by summing for each tumor the labeling intensities of all four markers for each tumors. A subgroup of tumors with an incomplete but strong smooth muscle differentiation phenotype presenting an intermediate metastatic risk was thus identified. Collectively, our results show that the smooth muscle differentiation classification method may be a useful diagnostic tool as well as a relevant prognostic tool for undifferentiated pleomorphic sarcomas.

The clinical relevance of accurately diagnosing pleomorphic sarcomas has been shown, especially in cases of undifferentiated pleomorphic sarcomas with myogenic differentiation, which appear significantly more aggressive. To establish a new smooth muscle differentiation classification and to test its prognostic value, 412 sarcomas with complex genetics were examined by immunohistochemistry using four smooth muscle markers (calponin, h-caldesmon, transgelin and smooth muscle actin). Two tumor categories were first defined: tumors with positivity for all four markers and tumors with no or incomplete phenotypes. Multivariate analysis demonstrated that this classification method exhibited the strongest prognostic value compared with other prognostic factors, including histological classification. Secondly, incomplete or absent smooth muscle phenotype tumor group was then divided into subgroups by summing for each tumor the labeling intensities of all four markers for each tumors. A subgroup of tumors with an incomplete but strong smooth muscle differentiation phenotype presenting an intermediate metastatic risk was thus identified. Collectively, our results show that the smooth muscle differentiation classification method may be a useful diagnostic tool as well as a relevant prognostic tool for undifferentiated pleomorphic sarcomas.

We report novel molecular and pathologic features of sarcomas involving the heart. Intimal sarcoma appears as the most frequent primary cardiac sarcoma within the largest described series of 100 primary cardiac sarcomas. Immunohistochemical analysis, fluorescence in situ hybridization, real-time polymerase chain reaction, and array-comparative genomic hybridization were performed on materials from 65 women and 35 men, aged 18 to 82 years (mean 50 y), retrieved from the French Departments of Pathology, between 1977 and early 2013. Right and left heart was involved in 44 and 56 cases, respectively. There were 42 intimal sarcomas, 26 angiosarcomas, 22 undifferentiated sarcomas, 7 synovial sarcomas, 2 leiomyosarcomas, and 1 peripheral neuroectodermal tumor. All but 1 angiosarcomas originated from the right heart, whereas 83 of the intimal sarcomas and 72 of the undifferentiated sarcomas were from the left heart. MDM2 overexpression was immunohistochemically observed in all intimal sarcomas, as well as in 10 of the 22 undifferentiated sarcomas and in 5 of the 26 angiosarcomas. MDM2amplification was only demonstrated in intimal sarcomas. Genomic analysis showed a complex profile, with recurrent 12q13-14 amplicon involving MDM2, 4q12 amplicon involving KITand PDGFRA, 7p12 gain involving EGFR, and 9p21 deletion targeting CDKN2A. Immunohistochemical detection of MDM2 overexpression can easily detect intimal sarcoma, provided that molecular aberration is proved. As resections are limited to the left atrium, this histologic subtype could benefit from therapies targeting PDGFRAor MDM2.

Dedifferentiated liposarcoma (DDLPS) has been defined as a tumor composed of well-differentiated liposarcoma associated with a nonlipogenic undifferentiated sarcoma and is genetically characterized by a 12q13-15 amplicon with MDM2amplification. Some peripheral (extremities, trunk wall, headneck) undifferentiated pleomorphic sarcomas (UPS) without areas of well-differentiated liposarcoma present an MDM2amplification. We addressed whether they are true DDLPS or not. We compared the clinical data, histologic data, MDM2 status (immunohistochemistry IHC, fluorescence in situ hybridization FISH), genomic profile (array comparative genomic hybridization), and follow-up of 19 patients with peripheral UPS with MDM2amplification and 62 with peripheral conventional DDLPS retrieved from the French sarcoma network (RRePS) and the Conticabase (Connective Tissue Cancer Network database). For a control cohort, we described 153 patients from the Conticabase, with peripheral UPS without expression of MDM2 by IHC. By IHC, tumor cells were positive for MDM2 in 59 conventional DDLPS and in all UPS with MDM2amplification. FISH analysis andor quantitative polymerase chain reaction showed amplification of MDM2in 54 conventional DDLPS and in all UPS with MDM2amplification. The 2-year overall survival rates of UPS with MDM2amplification, conventional DDLPS, and UPS without expression of MDM2 were 93.3, 90.7, and 73.9, respectively. Such similarities in the clinical characteristics, morphology, genomic profile, and follow-up of peripheral UPS with MDM2amplification and peripheral conventional DDLPS strongly suggest that peripheral UPS with MDM2amplification are in fact DDLPS. Faced with histologic diagnosis of UPS, a systematic IHC evaluation of MDM2 allows a selection of cases for FISH analysis permitting the diagnosis of DDLPS.

Several histological grading systems for soft tissue sarcomas have been described since the early 1980s. Their main objective is to select patients for adjuvant chemotherapy. Two histological grading systems are used in daily practice, the National Cancer Institute (NCI) and the French Federation of Cancer Centers Sarcoma Group (FNCLCC) systems. They have been devised by combining histological parameters: number of mitoses per high-power field, the presence of necrosis, cellular and nuclear morphology and the degree of cellularity for the NCI grading; and tumour differentiation, mitotic index and extent of necrosis for the French system. Histological grading is far more appropriate to assess the risk of metastasis. However, several limitations prevent its use: grade cannot be applied to all histological types, its reproducibility is not perfect, a three-grade system generates an intermediate grade with undetermined prognosis, and finally the core needle biopsy, now widely used for the diagnosis of soft tissue sarcoma, is not the best sample to assess the grade. The development of molecular grading in addition to histological grading probably represents the next step. Molecular signatures based on quantitative evaluation of chromosomal complexity such as CINSARC (complexity index in sarcomas) appear as a strong independent predictive factor for metastasis in several types of sarcoma, and even in several other types of cancer. When they can be instituted in daily practice on formalin fixed, paraffin embedded material, molecular signatures will not only provide information on risk of metastasis, but also better understanding of cancer development, response or resistance to evaluated drugs, and potential targets for future treatments.

Extracardiac rhabdomyomas, which currently are classified into fetal, adult, and genital types, are rare. We have identified 7 cases of a distinct morphologic variant of rhabdomyoma that affects mainly young men in the paratesticular region, seen in consultation between 2001 and 2011. The 7 male patients were adults (median age 24 y) and presented with tumors in paratesticular soft tissue (4 left-sided, 3 right-sided). Grossly, the median tumor size was 4.5 cm (range, 2.0 to 12 cm), and lesions were well circumscribed with a uniform tan-white cut surface. Microscopically, these rhabdomyomas were characterized by bundles of large well-differentiated skeletal muscle cells with copious eosinophilic cytoplasm that were variably round, polygonal, and occasionally strap shaped. The tumor cells were set in a dense hyalinized collagenous stroma, often with adjacent prominent lymphoplasmacytic aggregates. Tumor cells had round, occasionally vesicular, nuclei (sometimes binucleate or multinucleate) with small or inconspicuous nucleoli. All tumors lacked nuclear atypia and necrosis. Mitotic activity was virtually absent, although 1 tumor showed a count of 1 per 50 HPF. All tumors were diffusely positive for desmin, 44 were diffusely positive for fast myosin, and 11 examined was positive for myf-4. All patients were treated by local excision (5 with positive margins). Four patients with known follow-up data had no evidence of tumor recurrence or disease progression (median follow-up time 8.5 mo). The clinical course as determined thus far is benign, similar to other types of rhabdomyoma. However, this rare paratesticular subset of rhabdomyomas appears to be morphologically distinct from rhabdomyomas at other locations and appears to represent a separate variant.

Endometrial stromal sarcomas represent the second most common mesenchymal uterine tumor. The 2003 WHO classification distinguishes low-grade and undifferentiated endometrial stromal sarcomas with different prognoses. Endometrial stromal sarcomas are a genetically heterogeneous group of sarcomas harboring different cytogenetic anomalies. Recently, a fusion between the YWHAEand FAM22A/Bgenes subsequent to a t(10;17) (q22;p13) has been described in endometrial sarcomas with high-grade histology. We examined YWHAErearrangements by FISH break-apart and RT-PCR in a series of 27 undifferentiated uterine stromal sarcoma without JAZF1rearrangements. Immunohistochemistry (IHC) was carried out with a panel of antibodies (estrogen (ER) and progesterone (PR) receptors, CD10, Cyclin D1, β-catenin, p53, and Ki-67). We identified a subgroup of endometrial sarcomas with high-grade histology and uniform morphology harboring YWHAErearrangements. FISH break-apart was interpretable in 20 cases (74%). Twelve cases (60%) showed <10% of tumor cells with a YWHAErearrangement, 4 cases (20%) showed between 10 and ≤20%, and 4 (20%) >20%. RT-PCR was tested on 24/27 cases (88%) and 19 cases were interpretable (79%). Five cases (26%) showed a specific fusion transcript YWHAE–FAM22A/Bsequence. The best concordance rate between FISH and RT-PCR (94%) was obtained with the threshold of 20% of cells with a YWHAErearrangement. The YWHAE-rearranged cases showed high-grade morphology with uniform appearance, spindle or round epithelioid cells, low ER and PR, CD10 expression, and a high and diffuse positivity for Cyclin D1, p53, and nuclear β-catenin negativity. Cyclin D1 was the most sensitive marker for high-grade endometrial sarcomas with YWHAErearrangement. All undifferentiated uterine sarcomas with pleomorphic appearances did not harbor any YWHAErearrangements, except for one case. Overall, for endometrial sarcoma cases with high-grade morphology we recommend to test for YWHAErearrangements by FISH break-apart, a cost- and time-efficient method, and to complete the investigation by RT-PCR in borderline cases.