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The outcome of stage II–III colorectal cancer (CRC) is highly variable and therapeutic choice is currently based on TNM staging with a few additional biomarkers. However, studies show that some stage III patients have a better prognosis than some stage II patients. A promising consensus molecular (CMS) classification with prognostic relevance has been developed, but it is not used in daily practice. Our team developed CINSARC, a 67-gene expression prognostic signature, whose prognostic value has been demonstrated in many cancer types. It is applicable to formalin-fixed, paraffin-embedded (FFPE) blocks using NanoString® technology. We investigated whether it could predict outcome in stage II–III CRC. We established the CINSARC classification on the TCGA retrospective cohort comprising 297 stage II–III CRC patients using RNA sequencing and on a second independent cohort comprising 169 cases using NanoString® technology. We compared its recurrence-free and overall survival prognostic value with TNM staging and CMS classification. In the TCGA cohort, we showed that CINSARC significantly splits the population of stage II–III CRC into two groups with different progression-free interval (P= 1.68 × 10−2; HR = 1.87 [1.11–3.16]) and overall survival (P= 3.73 × 10−3; HR = 2.45 [1.31–4.59]) and is a strong prognostic factor in multivariate analysis, outperforming TNM staging and CMS classification. We validated these results in the second cohort by applying CINSARC on FFPE samples with Nanostring® technology. CINSARC is a ready-to-use tool with a robust independent prognostic value in stage II–III CRC.

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.

Les tumeurs desmoïdes dérivent de cellules souches mésenchymateuses et leur pathogénie est fortement liée à la cascade Wingless/Wnt où la dérégulation de la β-caténine joue un rôle majeur. Une mutation du gène CTNNB1 codant la β-caténine est retrouvée dans la majorité des cas de tumeurs desmoïdes sporadiques et des mutations constitutionnelles du gène APCont été décrites dans les formes héréditaires chez les patients atteints de polypose adénomateuse familiale (FAP). Les œstrogènes pourraient jouer également un rôle dans la pathogénie, une donnée à la base de l’utilisation de l’hormonothérapie. D’autres voies de signalisation ont été impliquées dans le développement des tumeurs desmoïdes comme les voies Notch, Hedgehog, JAK/STAT, PI3 Kinase/AKT et mTOR. Les métalloprotéases sont exprimées dans les tumeurs desmoïdes et jouent un rôle dans la capacité d’invasion. Le TGF-ß, en tant que facteur de croissance, stimule l’activité transcriptionnelle de la β-caténine. De futures études devront s’attacher à mieux décrire et comprendre l’environnement immunitaire des tumeurs desmoïdes. Une des difficultés majeures pour l’étude expérimentale des tumeurs desmoïdes est la quasi-absence de modèle préclinique que ce soit in vitro ou in vivo. C’est en partie pour cela que les interactions entre les différentes voies de signalisations ici présentées et leurs conséquences dans le développement des tumeurs desmoïdes sont encore mal connues.

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.

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 a uterine smooth muscle lesion is, in the majority of cases, straightforward. However, in a small number of cases, the morphological criteria used in such lesions cannot differentiate with certainty a benign from a malignant lesion and a diagnosis of smooth muscle tumor with uncertain malignant potential (STUMP) is made. Uterine leiomyosarcomas are often easy to diagnose but it is difficult or even impossible to identify a prognostic factor at the moment of the diagnosis with the exception of the stage. We hypothesize, for uterine smooth muscle lesions, that there is a gradient of genomic complexity that correlates to outcome. We first tested this hypothesis on STUMP lesions in a previous study and demonstrated that this 'gray category' could be split according to genomic index into two groups. A benign group, with a low to moderate alteration rate without recurrence and a malignant group, with a highly rearranged profile akin to uterine leiomyosarcomas. Here, we analyzed a large series of 77 uterine smooth muscle lesions (from 76 patients) morphologically classified as 19 leiomyomas, 14 STUMP and 44 leiomyosarcomas with clinicopathological and genomic correlations. We confirmed that genomic index with a cut-off=10 is a predictor of recurrence (P<0.0001) and with a cut-off=35 is a marker for poor overall survival (P=0.035). For the tumors confined to the uterus, stage as a prognostic factor was not useful in survival prediction. At stage I, among the tumors reclassified as molecular leiomyosarcomas (ie, genomic index ≥10), the poor prognostic markers were: 5p gain (overall survival P=0.0008), genomic index at cut-off=35 (overall survival P=0.0193), 13p loss including RB1(overall survival P=0.0096) and 17p gain including MYOCDgain (overall survival P=0.0425). Based on these findings (and the feasibility of genomic profiling by array-comparative genomic hybridization), genomic index, 5p and 17p gains prognostic value could be evaluated in future prospective chemotherapy trials.

ABSTRACTRNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

Secretory breast carcinoma (SBC) is a rare breast carcinoma with distinctive morphologic features and a recurrent specific chromosomal translocation t(12;15)(p13;q25), usually of low histologic grade and favorable prognosis. We describe the morphologic and genetic characteristics of 11 cases of SBC from 10 patients. Histologic and immunohistochemical analyses, fluorescence in situ hybridization using break-apart probes specific to ETV6on 12p13, reverse transcription polymerase chain reaction with in-house probes specific to the ETV6-NTRK3gene fusion, and DNA copy number variation by array comparative genomic hybridization analyses were performed on all cases. Seven cases were of low histologic grade, 3 were intermediate, and 1 had high-grade nuclear atypia, necrosis, and numerous mitoses. This patient had a fatal outcome. Five cases displayed low hormonal receptor expression, whereas the rest had basal-type immunoprofiles. All interpretable cases harbored an ETV6-NTRK3gene fusion by reverse transcription polymerase chain reaction and/or an ETV6rearrangement by fluorescence in situ hybridization, with duplication of the oncogenic derivative in 2 cases. Array comparative genomic hybridization analysis showed simplex genomic profiles. The 2 cases with ETV6-NTRK3duplication included a gain of 12p starting from the ETV6locus to the telomere, associated with a gain of the 15q from the centromere to NTRK3in 1 case, and in the other a normal profile up to NTRK3on 15q, and then a loss up to the telomere, suggesting loss of corresponding normal chromosome 15. These findings provide a novel insight into the morphologic and genetic spectrum of SBC, ranging from low-grade to high-grade histology, with occasional low hormonal receptor expression, simplex genomic profiles, and possible unfavorable course.

Heterologous differentiation in myxoid liposarcoma is a rare phenomenon. Few cases have been reported thus far, often without molecular assays, and the concept of “dedifferentiated” myxoid liposarcoma remains controversial. We describe a primary myxoid liposarcoma with chondroid and osseous components affecting the right thigh of a 27-year-old woman. We wondered whether these areas represented dedifferentiated components or simply metaplasia, and performed fluorescent in situ hybridization and array comparative genomic hybridization assays in the myxoid liposarcoma component and chondroid component. Fluorescent in situ hybridization analysis demonstrated a DDIT3gene rearrangement in both components; array comparative genomic hybridization analysis did not detect any gain or loss of DNA regions in both components. Our results, demonstrating that both components have the same molecular alterations, suggest that heterologous components seen in some myxoid liposarcomas reflect a metaplastic phenomenon and not a real dedifferentiation phenomenon, challenging the concept of “dedifferentiated” myxoid liposarcoma.

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.

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.

Sarcomas are rare, heterogenous, and often difficult to classify. A large proportion of sarcomas are associated with specific molecular genetic lesions such as translocations, mutations, and amplifications, which are helpful in the diagnosis of individual cases. However, the exact impact of molecular genetics on the final diagnosis of sarcomas is unknown. In this study, all soft tissue and visceral sarcomas arising in patients living in 3 European regions in 2 countries (representing 13 million inhabitants) were collected and reviewed during 2 consecutive years. A molecular analysis was performed for all suspicions of sarcomas with specific genetic lesions mutations of KITPDGFRAin gastrointestinal stromal tumors (GISTs), reciprocal translocation, or amplification of MDM2in atypical lipomatous tumors, well-differentiated liposarcoma-dedifferentiated liposarcoma (ALTWDLPS-DDLPS). To evaluate the impact of molecular tests, a premolecular analysis diagnosis was proposed with 3 categories of certainty: certain, probable, or possible. A molecular analysis was performed in 7631484 tumors corresponding to 295 cases in which GIST was suspected, 248 sarcomas with a suspicion of translocation, and 220 cases in which ALTWDLPS-DDLPS was suspected. Molecular analysis was found to be useful (confirms a probable diagnosis) in 11 (4) GISTs, 62 (26) suspicions of translocation, and 66 (31) suspicions of ALTWDLPS-DDLPS; and necessary (allows a possible diagnosis) in 2 (<1) GISTs, 31 (12) suspicions of translocation, and 19 (9) suspicions of ALTWDLPS-DDLPS. This study performed in an epidemiological setting demonstrates the significant impact of molecular analysis on the final sarcoma diagnosis and favors such an analysis on any tumor with a suspicion of a specific genomic abnormality and for which the diagnosis is uncertain.

Over the past decade, comprehensive genomic studies demonstrated that leiomyosarcomas and most of the tumors previously labeled as ‘malignant fibrous histiocytomas’ share complex karyotypes and genomic profiles, and can be referred to as ‘sarcomas with complex genomics’. We recently reported a series of 160 sarcomas with complex genomics such as leiomyosarcomas, myxofibrosarcomas, pleomorphic liposarcomas/rhabdomyosarcomas and undifferentiated pleomorphic sarcomas. These tumors present with a frequent loss of chromosome 10 region encompassing the tumor suppressor gene PTEN. In the present study, we assessed PTEN genomic level and protein expression in this large series of sarcomas with complex genomics, as well as activation of downstream pathways. PTEN partial genomic loss was observed in only 46% of tumors, especially in well-differentiated leiomyosarcomas, whereas up to 68% of these tumors demonstrate a loss of protein expression on western blot analysis. Specific discrepancies in PTEN immunohistochemical results suggested bias in this latter technique. PTEN mutations were rare, with only 4 point mutations in the 65 samples studied. Subsequent activation of AKT and mTOR pathways was only observed in 2 out of 3 of PTEN-deleted tumors. On the other hand, RICTOR, a major component of the mTOR complex 2, was significantly overexpressed in well-differentiated leiomyosarcomas. These results, confirmed on tissue micro-array immunohistochemical analysis of 459 sarcomas, could suggest a link between RICTOR overexpression and leiomyosarcomas oncogenesis. As therapeutics directed against the mTOR pathway are assessed in sarcomas, RICTOR overexpression in sarcomas and its links to therapeutic response need to be assessed.

The high mobility group A (HMGA2) gene encodes a protein that alters chromatin structure and regulates the transcription of many genes; it is implicated in both benign and malignant neoplasias, but its rearrangements are a feature of development of several mesenchymal tumors. Given its implication in these tumors and particularly adipocytic tumors, and the availability of antibodies usable on paraffin-embedded tissues, we evaluated the immunohistochemical expression of this gene in a series of 1052 mesenchymal tumors. The objective was to define the value and limitations of HMGA2 immunohistochemical expression for histotyping, and compare with molecular data reported in the literature. We thus analyzed 880 cases on tissue microarray and 182 cases on whole sections (211 adipocytic tumors, 628 sarcomas, 213 benign mesenchymal tumors, and 10 normal adipose tissues). A nuclear immunostaining was detected in 86% of conventional and intramuscular lipomas, in 86% of well-differentiated liposarcomas and in 67% of dedifferentiated liposarcomas, as opposed to 16% of other benign adipose tumors and to 15% of non-well-differentiated liposarcoma/dedifferentiated liposarcoma sarcomas. Among benign mesenchymal tumors and lesions, it was detected in 90% of nodular fasciitis and in 88% of benign fibrous histiocytomas with respective specificities of 85 and 100%, and in 90% of aggressive angiomyxoma, contrary to other vulvovaginal tumor types, which expressed HMGA2 only rarely. The normal adipose tissue was always negative for HMGA2. Although not specific, immunohistochemical detection of the HMGA2 protein is helpful for the distinction of normal adipose tissue from well-differentiated lesions, particularly on biopsy or on re-excision. It is less sensitive than MDM2/CDK4 for dedifferentiated liposarcomas diagnosis, but it appears more specific to distinguish dedifferentiated liposarcomas from other poorly differentiated sarcomas. Finally, and may be more importantly, HMGA2 is useful for the diagnosis of benign fibrous histiocytoma, nodular fasciitis and vulvovaginal benign mesenchymal tumors.

Alterations of the p53 pathway are among the most frequent aberrations observed in human cancers. We have performed an exhaustive analysis of TP53, p14, p15, and p16status in a large series of 143 soft tissue sarcomas, rare tumors accounting for around 1% of all adult cancers, with complex genetics. For this purpose, we performed genomic studies, combining sequencing, copy number assessment, and expression analyses. TP53mutations and deletions are more frequent in leiomyosarcomas than in undifferentiated pleomorphic sarcomas. Moreover, 50% of leiomyosarcomas present TP53biallelic inactivation, whereas most undifferentiated pleomorphic sarcomas retain one wild-type TP53allele (87.2%). The spectrum of mutations between these two groups of sarcomas is different, particularly with a higher rate of complex mutations in undifferentiated pleomorphic sarcomas. Most tumors without TP53alteration exhibit a deletion of p14and/or lack of mRNA expression, suggesting that p14loss could be an alternative genotype for direct TP53inactivation. Nevertheless, the fact that even in tumors altered for TP53, we could not detect p14 protein suggests that other p14 functions, independent of p53, could be implicated in sarcoma oncogenesis. In addition, both p15and p16are frequently codeleted or transcriptionally co-inhibited with p14, essentially in tumors with two wild-type TP53alleles. Conversely, in TP53-altered tumors, p15and p16are well expressed, a feature not incompatible with an oncogenic process.

Most Her2 testing guidelines recommend that all cases scoring Her2 2+ by immunohistochemistry should be analyzed by fluorescent in situhybridization (FISH) to determine HER2status to confirm eligibility for Trastuzumab therapy in breast cancer. The aim of our study was to determine HER2gene and chromosome 17 (CEN17) status in a series of 108 Her2 2+ consecutive cases and study the correlation between pathological characteristics of the tumors and HER2 amplification. Invasive breast cancers were tested by FISH using the Dako HER2FISH pharmDx® kit. The Her2 immunohistochemistry protocol was performed using the polyclonal AO485 antibody (Dako®) diluted to 1:1500. HER2and CEN17 status were correlated to tumor SBR grade, mitotic count, estrogen receptor, progesterone receptor status and percentage of Her2 immunohistochemistry-positive cells. Following Food and Drug Administration guidelines, ie, HER2/CEN17 ratio ≥2 and an HER2copy number >4, amplified cases were observed in 36 (33%) and 49 (45%) cases, respectively, and following American Society of Clinical Oncology/College of American Pathologists guidelines, ie, HER2/CEN17 ratio >2.2 and an HER2copy number >6, amplified cases represented 30 and 24% of the study population, respectively. Chromosome 17 polysomy (CEN17 >2.25) was observed in 39 (36%) tumors. Significant positive correlations were found between FISH HER2amplified cases and Her2 immunostaining >60% (P=1.1.10−5), SBR grade 3 (P=0.0001), nuclear atypia (P=0.03) and mitotic count (P=0.008). By multivariate analysis, Her2 immunostaining >60% (P<10−3) and SBR grade 3 (P<10−3) were independent factors predicting HER2amplification status irrespective to cutoff guidelines. All SBR grade 3 cases with more than 60% Her2+ cells had an HER2/CEN17 ratio ≥2, only one had a ratio ≤2.2. In our series of consecutive Her2 2+ cases, one-third demonstrated HER2amplification, and one-third had chromosome 17 polysomy. Pathological factors, in particular SBR grade 3 and more than 60% Her2+ cells, were significantly correlated with HER2amplification.

Superficial primitive Ewing's sarcomas are rare and have been reported to be of favorable prognosis compared to conventional deep-seated tumors. In the skin and subcutis, the diagnosis is often difficult, and performing molecular cytogenetic techniques may be helpful. We performed a retrospective analysis of 14 cases of superficial Ewing's sarcomas, all confirmed by molecular cytogenetics. Clinical, histological, immunohistochemical, molecular cytogenetic, therapeutic, and follow-up data are reported. There were 11 female and 3 male patients aged from 12 to 77 years (median: 17 years). Seven tumors occurred in the extremities, five in the trunk wall, and two in the head. Tumor size ranged from 1 to 5 cm (median, 3 cm). They were all small round-cell proliferations with a strong membranous positivity for CD99. Ewing's sarcoma translocations/fusion gene transcripts were detected in eight cases, both by FISH and reverse transcriptase (RT)–PCR. Four tumors were positive by RT–PCR alone (FISH not done in three cases and not interpretable in one case), and two cases were positive by FISH alone (RT–PCR not done). Surgical resection was performed in all patients. Chemotherapy was given in ten patients and radiotherapy in six. At last medical examination (median follow-up, 47 months), two patients who underwent surgical resection alone had died from the tumor. Our results confirm that superficial Ewing's sarcomas are of good prognosis. Given the difficulty of the diagnosis and the importance of an adapted treatment, a confirmation of the diagnosis by molecular or cytogenetic techniques is recommended when dealing with a superficial tumor.

Dedifferentiated and differentiated liposarcoma are characterized by 12q15 chromosomal amplification. Comparative genomic hybridization is a powerful tool able to detect DNA copy number changes in the genome. This technique has been widely used in frozen tumors and in some studies in paraffin-embedded tumors fixed with formalin. The purpose of this study was to demonstrate the ability of CGH to detect DNA copy number changes in the genome when the DNA was extracted from tissues fixed with Holland Bouin's fluid. Sixteen liposarcoma tumors both frozen and fixed in Holland Bouin's fluid were characterized by CGH. Eighty-one percent of the main chromosomal alterations detected in the frozen liposarcomas (amp 12q15, amp 6q23, amp 1p32, amp 16q22, 7, 8) were detected in the corresponding fixed tumors. The limitation of this technique when using Holland Bouin's fluid extracted DNA compared with formalin-extracted DNA was the yield of analyzable samples. Eighty-one percent of tumors fixed with Holland Bouin's fluid (13/16) were analyzable compared with 100 of formalin-fixed tumors (4/4). This study demonstrates that comparative genomic hybridization is a useful tool even if only fixed tissues (formalin and Holland Bouin's fluid tissues) are available, and that it allows more tumors to be analyzed in retrospective studies.

Chromatin-bound MDM2 regulates serine metabolism and nucleotide synthesis to sustain tumor growth in liposarcoma.