Nuclear-receptor-mediated telomere insertion leads to genome instability in ALT cancers.

The breakage-fusion-bridge cycle is a classical mechanism of telomere-driven genome instability in which dysfunctional telomeres are fused to other chromosomal extremities, creating dicentric chromosomes that eventually break at mitosis. Here, we uncover a distinct pathway of telomere-driven genome instability, specifically occurring in cells that maintain telomeres with the alternative lengthening of telomeres mechanism. We show that, in these cells, telomeric DNA is added to multiple discrete sites throughout the genome, corresponding to regions regulated by NR2C/F transcription factors. These proteins drive local telomere DNA addition by recruiting telomeric chromatin. This mechanism, which we name targeted telomere insertion (TTI), generates potential common fragile sites that destabilize the genome. We propose that TTI driven by NR2C/F proteins contributes to the formation of complex karyotypes in ALT tumors.

CINSARC signature outperforms gold-standard TNM staging and consensus molecular subtypes for clinical outcome in stage II-III colorectal carcinoma.

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.

Solid-type adenoid cystic carcinoma of the breast, a distinct molecular entity enriched in NOTCH and CREBBP mutations.

Adenoid cystic carcinoma (ACC) of the breast with a predominant solid pattern is difficult to diagnose with certainty and differentiate from more common triple-negative breast cancers (TNBCs) of basal-phenotype. To better characterize solid ACC, we performed a clinical, morphological, immunohistochemical, and molecular comparative analysis of 33 ACCs of the breast comprising 17 solid variant ACCs and 16 conventional ACCs. Solid ACCs displayed basaloid morphology with an exclusive or predominant epithelial cell population associated with decreased myoepithelial differentiation, while demonstrating MYB protein overexpression similar to the more common type of ACC. Strong and diffuse MYB expression by immunochemistry was observed in 14/17 (82%) of solid ACCs while MYB rearrangements were detected by break apart fluorescence in situ hybridization (FISH) in only 3/16 (19%) of solid ACCs. Conversely, weak MYB immunohistochemical expression was observed in only 7/204 (3%) of TNBC. Solid ACCs displayed a transcriptomic profile distinct from conventional ACCs with 549 genes showing a highly significant differential expression between conventional and solid ACC [false discovery rate (FDR) < 0.01; log2FC > |1|]. EnrichR and Kegg Pathway analyses identified PI3K-Akt and focal adhesion signaling pathways as significantly overexpressed in conventional ACCs compared with solid ACCs which significantly overexpressed the nitrogen metabolism pathway. CREBBP mutations and NOTCH activating gene mutations were only present in solid ACCs, concerning 5/16 (31%) of cases for each gene. Tumors with NOTCH activating mutations displayed a strong diffuse nuclear NICD1 staining, an established marker of Notch pathway activation. Solid ACCs also differed from basal-type TNBC, with fewer TP53 mutations and a more stable genomic profile on array comparative genomic hybridization (CGH). In summary, solid-type ACC of the breast is a distinct molecular entity within the ACC family and is different from common basal-type TNBC. MYB is a diagnostically useful biomarker of solid ACC and NOTCH could be a novel potential therapeutic target in 30% of cases.

Developmental and cancer-associated plasticity of DNA replication preferentially targets GC-poor, lowly expressed and late-replicating regions.

The spatiotemporal program of metazoan DNA replication is regulated during development and altered in cancers. We have generated novel OK-seq, Repli-seq and RNA-seq data to compare the DNA replication and gene expression programs of twelve cancer and non-cancer human cell types. Changes in replication fork directionality (RFD) determined by OK-seq are widespread but more frequent within GC-poor isochores and largely disconnected from transcription changes. Cancer cell RFD profiles cluster with non-cancer cells of similar developmental origin but not with different cancer types. Importantly, recurrent RFD changes are detected in specific tumour progression pathways. Using a model for establishment and early progression of chronic myeloid leukemia (CML), we identify 1027 replication initiation zones (IZs) that progressively change efficiency during long-term expression of the BCR-ABL1 oncogene, being twice more often downregulated than upregulated. Prolonged expression of BCR-ABL1 results in targeting of new IZs and accentuation of previous efficiency changes. Targeted IZs are predominantly located in GC-poor, late replicating gene deserts and frequently silenced in late CML. Prolonged expression of BCR-ABL1 results in massive deletion of GC-poor, late replicating DNA sequences enriched in origin silencing events. We conclude that BCR-ABL1 expression progressively affects replication and stability of GC-poor, late-replicating regions during CML progression.

GREB1-CTNNB1 fusion transcript detected by RNA-sequencing in a uterine tumor resembling ovarian sex cord tumor (UTROSCT): A novel CTNNB1 rearrangement.

Mutations of CTNNB1 have been implicated in tumorigenesis in many organs. However, tumors harboring a CTNNB1 translocation are extremely rare and this translocation has never been reported in a uterine mesenchymal neoplasm. We report a novel translocation t(2;3)(p25;p22) involving the GREB1 (intron 8) and CTNNB1 (exon 3) in a uterine tumor resembling ovarian sex cord tumor (UTROSCT), which exhibited extrauterine metastasis. The translocation detected by RNA-sequencing was validated by RT-PCR, and resulted in nuclear expression of ß-catenin. Juxtapositioning with GREB1, which is overexpressed in response to estrogens, resulted in overexpression of a truncated and hypophosphorylated nuclear ß-catenin in the primary and recurrent tumors. This accumulation of nuclear ß-catenin results in a constitutive activation of the Wnt/ß-catenin signaling pathway with a major oncogenic effect. The CTNNB1 gene fusion, promoted by an estrogen-responsive gene (GREB1), could be a potential driver of tumorigenesis in this case and a therapeutic target with adapted inhibitors. RT-PCR and immunohistochemistry performed on 11 additional UTROSCTs showed no CTNNB1 fusion transcript or nuclear ß-catenin immunoreactivity.

Leiomyosarcomas: whole genome sequencing for a whole biology characterization.

PURPOSE OF REVIEW: Leiomyosarcoma (LMS) is among the more aggressive sarcomas and still suffers from the lack of efficient systemic treatment after, or before, surgery. During the last decades, one provider of therapeutic improvement has been the targeting of genome alterations. Efforts have thus been done to apply next-generation sequencing approaches to those tumours to decipher their oncogenesis and find out such targets. RECENT FINDINGS: Sequencing performed so far, based on exome, mostly confirmed that p53 and RB1 are the two main pathways altered in LMS oncogenesis. There are few point mutations in LMS genome, which is mainly characterized by numerous chromosomal rearrangements. Data from whole genome sequencing are now mandatory to decipher mechanisms triggering chromosomal instability and mutational process. SUMMARY: Although each LMS appears to have quite private genetic alterations leading to oncogenesis, it is likely that the altered biological pathways are relatively homogeneous within each of the LMS subgroups. Understanding this oncogenesis, thanks to integrated approaches involving whole genome and transcriptome sequencing together with functional and clinical characterizations will certainly give us the keys to relevant and effective new therapeutic approaches.

Uterine and vaginal sarcomas resembling fibrosarcoma: a clinicopathological and molecular analysis of 13 cases showing common NTRK-rearrangements and the description of a COL1A1-PDGFB fusion novel to uterine neoplasms.

Mesenchymal neoplasms of the uterus (corpus and cervix) encompass a heterogeneous group of tumors with differing morphologies, immunophenotypes and molecular alterations. With the advent of modern molecular techniques, such as next generation sequencing, newly defined genetic abnormalities are being reported in this group of neoplasms. Herein we report the clinicopathological and molecular features of a series of 13 spindle cell sarcomas of the uterus and vagina (10 cervix, 2 uterine corpus, 1 vagina) with morphology resembling fibrosarcoma. After targeted RNA-sequencing, dual FISH fusion and array-CGH analysis, 7 of 13 tumors exhibited NTRK rearrangements (6 TPM3-NTRK1 and 1 EML4-NTRK3) and 3 a COL1A1-PDGFB fusion; in the other 3 neoplasms, all of which were positive with S100 (2 diffuse, 1 focal), we identified no rearrangement. All the NTRK fusion-positive sarcomas were located in the cervix and exhibited diffuse staining with Trk while all the other neoplasms were negative. CD34 was diffusely positive in all 3 of the COL1A1-PDGFB fusion sarcomas. The latter molecular abnormality is identical to that commonly found in dermatofibrosarcoma protuberans and has not been reported previously in uterine mesenchymal neoplasms. We suggest that uterine sarcomas with a morphology resembling fibrosarcoma (and in which leiomyosarcoma and the known molecularly confirmed high-grade endometrial stromal sarcomas have been excluded) can be divided into 3 groups:- an NTRK fusion group, a COL1A1-PDGFB fusion group and a group containing neither of these molecular abnormalities which, on the basis of positive staining with S100, could be tentatively classified as malignant peripheral nerve sheath tumor, although additional molecular studies may identify specific genetic alterations necessitating a nomenclature change. We suggest a diagnostic algorithm when reporting such neoplasms. Identification of these newly described fusion-associated sarcomas is important given the potential for targeted treatments.

Genomic and transcriptomic comparison of post-radiation versus sporadic sarcomas.

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 CDKN2A and 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 MYC amplifications (96%) and KDR variants (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.

Alternative PDGFD rearrangements in dermatofibrosarcomas protuberans without PDGFB fusions.

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-PDGFB fusion 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-PDGFB fusion was present in 40% of cases (n = 8/20), and subsequently confirmed with a COL1A1 break-apart FISH probe in all but one case (n = 7/8). 55% of cases (n = 11/20) displayed novel PDGFD rearrangements; PDGFD being 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-PDGFD cases displayed fibrosarcomatous transformation and homozygous deletions of CDKN2A at 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-PDGFB fusions can be cytogenetically cryptic on FISH testing in a subset of cases, thereby representing a diagnostic pitfall that pathologists should be aware of.

ETV4 is a useful marker for the diagnosis of CIC-rearranged undifferentiated round-cell sarcomas: a study of 127 cases including mimicking lesions.

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.

Fluorescence in situ hybridization analysis is a helpful test for the diagnosis of dermatofibrosarcoma protuberans.

Cytogenetically, most dermatofibrosarcoma protuberans are characterized by chromosomal rearrangements resulting in the collagen type-1 alpha 1 (COL1A1)-platelet-derived growth factor ß (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.

Smooth muscle differentiation identifies two classes of poorly differentiated pleomorphic sarcomas with distinct outcome.

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.

Transgelin is a novel marker of smooth muscle differentiation that improves diagnostic accuracy of leiomyosarcomas: a comparative immunohistochemical reappraisal of myogenic markers in 900 soft tissue tumors.

Immunohistochemical use of myogenic markers serves to define smooth or skeletal muscle differentiation in soft tissue tumors. Establishing smooth muscle differentiation in malignant lesions can be challenging in some cases. We immunohistochemically examined 900 soft tissue tumors selected from the French Sarcoma Group's archived tissue collection, which contains a large number of leiomyosarcomas. The four most widely used smooth muscle diagnostic markers were evaluated (smooth muscle actin, desmin, h-caldesmon and calponin), and compared with a novel marker, transgelin. The diagnostic performance of each marker was statistically assessed in terms of sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV) and accuracy (A), in leiomyosarcomas versus all other sarcomas including gastrointestinal stromal tumors (GIST), and second in leiomyosarcomas versus specific tumor types. In leiomyosarcomas versus all other sarcomas including GIST, transgelin emerged as the best diagnostic marker (Se: 83%, Sp: 82%, PPV: 67%, NPV: 92%, A: 83%), compared with smooth muscle actin (Se: 75%, Sp: 83, PPV: 66%, NPV: 89%, A: 81%), desmin (Se: 45%, Sp: 88%, PPV: 62%, NPV: 79%, A: 75%), h-caldesmon (Se: 50%, Sp: 90%, PPV: 67%, NPV: 81%, A: 78%) and calponin (Se: 76%, Sp: 70, PPV: 52%, NPV: 87%, A: 71%). In leiomyosarcomas compared with other specific tumor types such as undifferentiated pleomorphic sarcoma and myxofibrosarcoma, the accuracy for transgelin varied from 80 to 87% whereas it was lower for all other markers (between 51 and 80%). These results indicate that transgelin could be used in practice as an additional marker useful for decision making, especially in those tumors with incomplete immunophenotypes.

Comprehensive Immune Profiling Unveils a Subset of Leiomyosarcoma with "Hot" Tumor Immune Microenvironment.

Purpose: To investigate the immune biomarker in Leiomyosarcoma (LMS), which is rare and recognized as an immune cold cancer showing a poor response rate (<10%) to immune checkpoint inhibitors (ICIs). However, durable response and clinical benefit to ICIs has been observed in a few cases of LMS, including, but not only, LMS with tertiary lymphoid structure (TLS) structures. Patients and methods: We used comprehensive transcriptomic profiling and a deconvolution method extracted from RNA-sequencing gene expression data in two independent LMS cohorts, the International Cancer Genome Consortium (ICGC, N = 146) and The Cancer Genome Atlas (TCGA, N = 75), to explore tumor immune microenvironment (TIME) in LMS. Results: Unsupervised clustering analysis using the previously validated two methods, 90-gene signature and Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT), identified immune hot (I-H) and immune high (I-Hi) LMS, respectively, in the ICGC cohort. Similarly, immune active groups (T-H, T-Hi) were identified in the TCGA cohort using these two methods. These immune active ("hot") clusters were significantly associated, but not completely overlapping, with several validated immune signatures such as sarcoma immune class (SIC) classification and TLS score, T cell inflamed signature (TIS) score, immune infiltration score (IIS), and macrophage score (M1/M2), with more patients identified by our clustering as potentially immune hot. Conclusions: Comprehensive immune profiling revealed a subset of LMS with a distinct active ("hot") TIME, consistently associated with several validated immune signatures in other cancers. This suggests that the methodologies that we used in this study warrant further validation and development, which can potentially help refine our current immune biomarkers to select the right LMS patients for ICIs in clinical trials.

Analysis of the Peritumoral Tissue Unveils Cellular Changes Associated with a High Risk of Recurrence.

BACKGROUND: The management of soft-tissue sarcoma (STS) relies on a multidisciplinary approach involving specialized oncological surgery combined with other adjuvant therapies to achieve optimal local disease control. Purpose and Results: Genomic and transcriptomic pseudocapsules of 20 prospective sarcomas were analyzed and revealed to be correlated with a higher risk of recurrence after surgery. CONCLUSIONS: A peritumoral environment that has been remodeled and infiltrated by M2 macrophages, and is less expressive of healthy tissue, would pose a significant risk of relapse and require more aggressive treatment strategies.

Distinct Cellular Origins and Differentiation Process Account for Distinct Oncogenic and Clinical Behaviors of Leiomyosarcomas.

In leiomyosarcoma (LMS), a very aggressive disease, a relatively transcriptionally uniform subgroup of well-differentiated tumors has been described and is associated with poor survival. The question raised how differentiation and tumor progression, two apparently antagonist processes, coexist and allow tumor malignancy. We first identified the most transcriptionally homogeneous LMS subgroup in three independent cohorts, which we named 'hLMS'. The integration of multi-omics data and functional analysis suggests that hLMS originate from vascular smooth muscle cells and show that hLMS transcriptional program reflects both modulations of smooth muscle contraction activity controlled by MYOCD/SRF regulatory network and activation of the cell cycle activity controlled by E2F/RB1 pathway. We propose that the phenotypic plasticity of vascular smooth muscle cells coupled with MYOCD/SRF pathway amplification, essential for hLMS survival, concomitant with PTEN absence and RB1 alteration, could explain how hLMS balance this uncommon interplay between differentiation and aggressiveness.

From PTEN loss of expression to RICTOR role in smooth muscle differentiation: complex involvement of the mTOR pathway in leiomyosarcomas and pleomorphic sarcomas.

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.

New insights in sarcoma oncogenesis: a comprehensive analysis of a large series of 160 soft tissue sarcomas with complex genomics.

Adult soft tissue sarcomas (STS) are rare tumours of mesenchymal lineage. Based on cytogenetic and comparative genomic hybridization (CGH) data, they can be divided into 'STS with simple genomics', displaying a characteristic genetic alteration, and 'STS with complex genomics' (SCG), where multiple genomic alterations occur. This latter group is mostly composed of leiomyosarcomas (LMS) and pleiomorphic undifferentiated tumours previously labelled as 'malignant fibrous histiocytomas' (MFH), corresponding in fact to myxofibrosarcomas (MFS), pleiomorphic liposarcomas/rhabdomyosarcomas (P-LPS, P-RMS), and undifferentiated pleiomorphic sarcomas (UPS). Their pathobiology is still not well understood, leading to challenges in diagnosis and therapeutic management. We report here a comprehensive study encompassing array-CGH and transcriptome analysis data of a large series of 160 SCG. Non-supervised clustering of transcriptome data led to the identification of five groups of tumours, one of them (group A) corresponding to well-differentiated LMS and the other four (B-E) to 'MFH' and poorly differentiated LMS. Welch analysis of transcriptome data in these groups allowed us to retrieve several genes of potential interest. Among them, RB1 alteration is a constant thread in SCG, often associated with RBL2 loss. PTEN tumour suppressor deletion would also stand out as a major recurrent event, especially in groups A, C, and D. The WNT canonical pathway could be potentially involved, as demonstrated by up-regulation of one of its inhibitors, DKK1, in groups D and E, whereas DKK1 is significantly down-regulated in groups A, B, and C. These data suggest a very complex interplay between pathways downstream of PTEN and the WNT canonical pathway, providing new hints about SCG pathobiology and their potential therapeutic targets.

The CDKN2A/CDKN2B/CDK4/CCND1 pathway is pivotal in well-differentiated and dedifferentiated liposarcoma oncogenesis: an analysis of 104 tumors.

The MDM2 and CDK4 genes are the main targets of chromosome 12 amplification in well-differentiated and dedifferentiated liposarcomas. Nevertheless, around 10% of these tumors do not amplify CDK4. To find substitutive alterations of CDK4 amplification, we analyzed a large series of liposarcomas by array-CGH, real-time genomic PCR, gene expression array, and real-time RT-PCR. We demonstrate that an alteration in the CDKN2A/CDKN2B/CDK4/CCND1 pathway is present in almost all cases without CDK4 amplification, thereby confirming the pivotal role of this pathway in liposarcoma oncogenesis. Moreover, we show that cell cycle and differentiation are driven by a subtle and complex balance between members of this pathway. Finally, we demonstrate that in tumors without amplification/overexpression of CDK4, the chromosome 1q21-1q23 region is a preferential partner of chromosome 12 amplicon, suggesting that the mechanism of amplification is slightly different in this group of tumors.