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.

Genomic profile analysis of leiomyomas with bizarre nuclei and fumarate hydratase deficient leiomyomas: Strengths, weaknesses, and limitations of array-CGH interpretation.

A close relationship has been demonstrated between genomic complexity and clinical outcome in uterine smooth muscle tumors. We studied the genomic profiles by array-CGH of 28 fumarate hydratase deficient leiomyomas and 37 leiomyomas with bizarre nuclei (LMBN) from 64 patients. Follow-up was available for 46 patients (from three to 249 months, mean 87.3 months). All patients were alive without evidence of disease. For 51 array-CGH interpretable tumors the mean Genomic Index (GI) was 16.4 (median: 9.8; from 1 to 57.8), significantly lower than the mean GI in LMS (mean GI 51.8, p < 0.001). We described three groups: (1) a group with FH deletion (24/58) with low GI (mean GI: 11 vs. 22,4, p = 0.02), (2) a group with TP53 deletion (17/58) with higher GI (22.4 vs. 11 p = 0.02), and (3) a group without genomic events on FH or TP53 genes (17/58) (mean GI:18.3; from 1 to 57.8). Because none of these tumors recurred and none showed morphological features of LMS we concluded that GI at the cut-off of 10 was not applicable in these subtypes of LM. By integration of all those findings, a GI <10 in LMBN remains a valuable argument for benignity. Conversely, in LMBN a GI >10 or alteration in tumor suppressor genes, should not alone warrant a diagnosis of malignancy. Nine tumors were tested with Nanocind CINSARC® signature and all were classified in low risk of recurrence. We propose, based on our observations, a diagnostic approach of these challenging lesions.

Gynecological sarcomas, surgical management: primary, metastatic, and recurrent disease.

Adult-type gynecological soft tissue and visceral sarcomas are rare tumors, with an estimated incidence of 13% of all sarcomas and 4% of all gynecological malignancies. They most often develop in the uterus (83%), followed by the ovaries (8%), vulva and vagina (5%), and other gynecological organs (2%). The objective of this review is to provide an overview of the current management of gynecological sarcomas, according to international guidelines. The management of gynecological sarcomas should follow the recommendations for the management of soft tissue and visceral sarcomas. Centralizing cases in expert centers improves patient survival, both for the diagnostic phase and for multidisciplinary therapeutic management. In the case of pelvic soft tissue sarcomas, a radiological biopsy is essential before any surgical decision is taken. In the case of a myometrial tumour which may correspond to a sarcoma, if conservative surgery such as myomectomy or morcellation is planned, an ultrasound-guided biopsy with pathological analysis including comparative genomic hybridization analysis must be carried out. In all cases, en bloc surgery, without rupture, is mandatory. Many rare histological subtypes require specific surgical management.

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.

Molecular prognostication of uterine smooth muscle neoplasms: From CGH array to CINSARC signature and beyond.

Uterine leiomyoma and leiomyosarcoma are located at the ends of the spectrum of smooth muscle lesions. Leiomyosarcoma belongs to the complex genomic sarcomas characterized by complex karyotypes. In contrast, leiomyoma, has a low level of chromosomal complexity. The analysis of genomic profiles of uterine smooth muscle tumors shows that genomic complexity, which is an expression of chromosomal instability, correlates with the metastatic potential and malignity of tumors: the more genetically complex a smooth muscle tumor is, the more malignant is its progression. In uterine tumors with uncertain malignant potential, the assessment of genomic index by CGH array, that is, counting the genomic complexity of a tumor, allows tumors with a risk of recurrence such as leiomyosarcomas to be distinguished from benign tumors like leiomyomas. The prognosis of leiomyosarcoma is poor and the most powerful prognostic factor so far is stage, as the histologic grade is not informative. In the quest to find efficient molecular prognostic factors, the transcriptomic signature CINSARC Nanocind, a mirror of chromosomic complexity and instability, outperforms stage, in both overall and recurrence-free survival. Genomic index and the CINSARC signature will contribute to improving diagnoses, therapeutic strategies, and randomization in future clinical trials. The biological understanding of the links between the CINSARC signature and metastatic mechanisms may lead to the development of new drugs. Furthermore, ctDNA is a promising new technique to detect residual disease and early recurrence.

Relationships between highly recurrent tumor suppressor alterations in 489 leiomyosarcomas.

BACKGROUND: Leiomyosarcoma (LMS) is the most common soft tissue and uterine sarcoma, but no standard therapy is available for recurrent or metastatic LMS. TP53, p16/RB1, and PI3K/mTOR pathway dysregulations are recurrent events, and some LMS express estrogen receptor (ER) and/or progesterone receptor (PR). To characterize relationships between these pathway perturbations, the authors evaluated protein expression in soft tissue and uterine nonprimary leiomyosarcoma (np-LMS), including local recurrences and distant metastases. METHODS: TP53, RB1, p16, and PTEN expression aberrations were determined by immunohistochemistry (IHC) in tissue microarrays (TMAs) from 227 np-LMS and a comparison group of 262 primary leiomyosarcomas (p-LMS). Thirty-five of the np-LMS had a matched p-LMS specimen in the TMAs. Correlative studies included differentiation scoring, ER and PR IHC, and CDKN2A/p16 fluorescence in situ hybridization. RESULTS: Dysregulation of TP53, p16/RB1, and PTEN was demonstrated in 90%, 95%, and 41% of np-LMS, respectively. PTEN inactivation was more common in soft tissue np-LMS than uterine np-LMS (55% vs 31%; P = .0005). Moderate-strong ER expression was more common in uterine np-LMS than soft tissue np-LMS (50% vs 7%; P < .0001). Co-inactivation of TP53 and RB1 was found in 81% of np-LMS and was common in both soft tissue and uterine np-LMS (90% and 74%, respectively). RB1, p16, and PTEN aberrations were nearly always conserved in p-LMS and np-LMS from the same patients. CONCLUSIONS: These studies show that nearly all np-LMS have TP53 and/or RB1 aberrations. Therefore, therapies targeting cell cycle and DNA damage checkpoint vulnerabilities should be prioritized for evaluations in LMS.

Acquisition of cancer stem cell capacities after spontaneous cell fusion.

BACKGROUND: Cancer stem/Initiating cell (CS/IC) hypothesis argues that CS/ICs are responsible of tumour initiation, drug resistance, metastasis or disease relapse. Their detection in several cancers supports this concept. However, their origin is still misunderstood. Cell fusion is shown to take part in the formation of CS/ICs, i.e. fusion between mesenchymal stem cell and cancer cell. In a previous paper, we described that fusion leads to hybrids with metastatic capacity. This process triggered genomic rearrangements in hybrid cells together with increased metastasis development. Here, we hypothesize that cell fusion could be strong enough to provoke a cellular reprogramming and the acquisition of CS/IC properties, promoting metastasis formation. METHODS: After spontaneous cell fusion between E6E7 (IMR90 with the oncogenes E6 and E7) and RST (IMR90 fully transformed) cell lines, hybrid cells were selected by dual antibiotic selection. Cancer stem cells capacities were evaluated regarding capacity to form spheres, expression of stem cell markers and the presence of ALDHhigh cells. RESULTS: Our data show that after cell fusion, all hybrids contain a percentage of cells with CS/ICs properties, regarding. Importantly, we lastly showed that NANOG inhibition in H1 hybrid decreases this migration capacity while having no effect on the corresponding parental cells. CONCLUSIONS: Altogether these results indicate that the combination of CS/ICs properties and genomic rearrangement in hybrids is likely to be key to tumour progression.

Cell fusion enhances energy metabolism of mesenchymal tumor hybrid cells to sustain their proliferation and invasion.

BACKGROUND: Cell-to-cell fusion is emerging as a key element of the metastatic process in various cancer types. We recently showed that hybrids made from the spontaneous merging of pre-malignant (IMR90 E6E7, i.e. E6E7) and malignant (IMR90 E6E7 RST, i.e. RST) mesenchymal cells recapitulate the main features of human undifferentiated pleomorphic sarcoma (UPS), with a highly rearranged genome and increased spreading capacities. To better characterize the intrinsic properties of these hybrids, we investigated here their metabolic energy profile compared to their parents. RESULTS: Our results unveiled that hybrids harbored a Warburg-like metabolism, like their RST counterparts. However, hybrids displayed a much greater metabolic activity, enhancing glycolysis to proliferate. Interestingly, modifying the metabolic environmental conditions through the use of 5-aminoimidazole-4-carbox-amide-1-ß-D-ribofuranoside (AICAR), an activator of the 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), specifically reduced the growth of hybrids, and also abrogated the invasive capacity of hybrids displaying enhanced glycolysis. Furthermore, AICAR efficiently blocked the tumoral features related to the aggressiveness of human UPS cell lines. CONCLUSION: Altogether, our findings strongly suggest that hybrids rely on higher energy flux to proliferate and that a drug altering this metabolic equilibrium could impair their survival and be potentially considered as a novel therapeutic strategy.

Gastrointestinal stromal tumor enhancers support a transcription factor network predictive of clinical outcome.

Activating mutations in the KIT or PDGFRA receptor tyrosine kinases are hallmarks of gastrointestinal stromal tumor (GIST). The biological underpinnings of recurrence following resection or disease progression beyond kinase mutation are poorly understood. Utilizing chromatin immunoprecipitation with sequencing of tumor samples and cell lines, we describe the enhancer landscape of GIST, highlighting genes that reinforce and extend our understanding of these neoplasms. A group of core transcription factors can be distinguished from others unique to localized and metastatic disease. The transcription factor HAND1 emerges in metastatic disease, binds to established GIST-associated enhancers, and facilitates GIST cell proliferation and KIT gene expression. The pattern of transcription factor expression in primary tumors is predictive of metastasis-free survival in GIST patients. These results provide insight into the enhancer landscape and transcription factor network underlying GIST, and define a unique strategy for predicting clinical behavior of this disease.

LIX1 regulates YAP activity and controls gastrointestinal cancer cell plasticity.

Gastrointestinal stromal tumours (GISTs), the most common mesenchymal neoplasm of the gastrointestinal tract, result from deregulated proliferation of transformed KIT-positive interstitial cells of Cajal that share mesenchymal progenitors with smooth muscle cells. Despite the identification of selective KIT inhibitors, primary resistance and relapse remain a major concern. Moreover, most patients develop resistance partly through reactivation of KIT and its downstream signalling pathways. We previously identified the Limb Expression 1 (LIX1) gene as a unique marker of digestive mesenchyme immaturity. We also demonstrated that LIX1 regulates mesenchymal progenitor proliferation and differentiation by controlling the Hippo effector YAP1, which is constitutively activated in many sarcomas. Therefore, we wanted to determine LIX1 role in GIST development. We found that LIX1 is strongly up-regulated in GIST samples and this is associated with unfavourable prognosis. Moreover, LIX1 controls GIST cell proliferation in vitro and in vivo. Upon LIX1 inactivation in GIST cells, YAP1/TAZ activity is reduced, KIT (the GIST signature) is down-regulated, and cells acquire smooth muscle lineage features. Our data highlight LIX1 role in digestive mesenchyme-derived cell-fate decisions and identify this novel regulator as a target for drug design for GIST treatment by influencing its differentiation status.

Impact of preoperative treatment on the CINSARC prognostic signature: translational research results from a phase 1 trial of the German Interdisciplinary Sarcoma Group (GISG 03).

PURPOSE: CINSARC (Complexity INdex in SARComas) is a prognostic signature for soft tissue sarcoma that determines the risk for recurrence and may serve to guide the decision for adjuvant chemotherapy. The aim of this study was to compare the CINSARC signature of pre- and posttreatment biopsies of sarcoma patients treated within a phase I trial evaluating preoperative sunitinib and irradiation. METHODS: We retrieved 14 pairs of formalin-fixed paraffin-embedded blocks from pretreatment biopsies and posttreatment resection specimens and performed expression profiling of the 67 CINSARC signature genes. RESULTS: In 5/14 patients, both probes were unsuitable for expression analysis because there was no (vital) tissue left in biopsies or resection specimens. Comparing the CINSARC risk classification before and after treatment in the remaining patients, 2/9 shifted from a high- to a low-risk classification for metastatic disease after preoperative treatment with radiation therapy plus sunitinib and 7/9 pairs of pre- and posttreatment biopsies revealed identical results. CONCLUSION: Concurrent radiation therapy and sunitinib leads to diverging results of prognostic gene array testing in a relevant proportion of sarcoma patients. These changes may reflect tumor heterogeneity, local treatment effects, or prognostic changes of the disease. Caution is advised in the selection of samples and interpretation of test results.

Value of peri-operative chemotherapy in patients with CINSARC high-risk localized grade 1 or 2 soft tissue sarcoma: study protocol of the target selection phase III CHIC-STS trial.

BACKGROUND: The value of chemotherapy in soft tissue sarcoma (STS) remains controversial. Several expert teams consider that chemotherapy provides a survival advantage and should be proposed in high-risk (HR) patients. However, the lack of accuracy in identifying HR patients with conventional risk factors (large, deep, FNCLCC grade 3, extremity STS) is an issue that cannot be neglected. For example, while the FNCLCC grading system is a powerful tool, it has several limitations. CINSARC, a 67-gene signature, has proved to be an additional independent factor for predicting metastatic spread and outperforms histological grade. Regardless of FNCLCC grade, CINSARC stratifies patients into two separate prognostic groups: one with an excellent prognosis (low-risk (LR) CINSARC) and the other with a worse outcome (HR-CINSARC) in terms of metastatic relapse. Here we evaluate the role of chemotherapy in grade 1-2 STS patients with HR-CINSARC and assess the prognostic value of CINSARC in patients treated with standard of care. METHODS: CHIC is a parallel, randomized, open-label, multicenter study evaluating the effect on metastasis-free survival of adding perioperative chemotherapy to standard of care in patients with grade ½ STS sarcoma defined as HR by CINSARC. In this target selection design, 600 patients will be screened with CINSARC to randomize 250 HR-CINSARC patients between standard of care and standard of care plus chemotherapy (4 cycles of 3 weeks of intravenous chemotherapy with doxorubicin in combination with dacarbazine or ifosfamide according to histologic subtype). LR-CINSARC patients will be treated by standard of care according to the investigator. The primary endpoint is metastasis-free survival. Secondary endpoints include overall survival, disease-free survival and safety. Furthermore, the prognostic value of CINSARC will be evaluated by comparing LR-CINSARC patients to HR-CINSARC patients randomized in standard of care. DISCUSSION: CHIC is a prospective randomized phase III trial designed to comprehensively evaluate the benefit of chemotherapy in HR-CINSARC patients and to prospectively validate the prognostic value of CINSARC in grade ½ STS sarcoma patients. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT04307277 Date of registration: 13 March 2020.

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.

CINSARC signature as a prognostic marker for clinical outcome in sarcomas and beyond.

Prognostication is a key issue for sarcoma patients' care as it triggers the therapeutic approach including chemotherapy, which is still not standard for localized patients. Current prognostic evaluation, based on the FNCLCC grading system, has recently been improved by the CINSARC signature outperforming histology-based grading system by identifying high-risk patients in every grade, even in those considered as low. CINSARC is an expression-based signature related to mitosis and chromosome integrity with prognostic value in a wide range of cancers additional to sarcoma. First developed with frozen material, CINSARC is now coupled with NanoString technology allowing evaluation from FFPE blocks used in clinical practice. Consequently, CINSARC is currently evaluated in clinical trials with a dual objective of demonstrating the benefit of chemotherapy in sarcoma patients and testing its response prediction. Considering its overarching value in oncology, its development is welcome in any cancers where the prognostication needs to be improved.

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.

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.

Genome profiling is an efficient tool to avoid the STUMP classification of uterine smooth muscle lesions: a comprehensive array-genomic hybridization analysis of 77 tumors.

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 MYOCD gain (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.

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.

Expression and role of TYRO3 and AXL as potential therapeutical targets in leiomyosarcoma.

BACKGROUND: Leiomyosarcoma (LMS) are 15% of adult sarcomas and remain seldom curable in metastatic phase. The TAM receptors and their ligands are overexpressed or activated in multiple malignancies, including LMS. METHODS: The TAM receptor and ligand expression was evaluated in LMS cell lines and 358 sarcoma samples by either gene expression or immunohistochemistry. TYRO3 and AXL were knocked down. Crizotinib and foretinib were investigated in vitro. RESULTS: High expression of TYRO3 and AXL was detected in LMS cell lines. TYRO3 or AXL gene knockdown reduced cell proliferation/colony formation. Crizotinib and foretinib decreased TYRO3 and AXL phosphorylation, apoptosis, G2/arrest and reduced colony formation. Immunohistochemistry performed in 107 sarcomas showed higher expression of TYRO3 and GAS6 in LMS vs other sarcomas and nuclear TYRO3 only in LMS. Microarray gene expression performed in 251 sarcomas revealed significantly higher expression of TYRO3 and GAS6 in LMS than other sarcomas. Leiomyosarcoma patients with high expression of GAS6 or PROS1 present a significantly worse PFS. CONCLUSIONS: Leiomyosarcoma patients, especially those whom develop metastasis, express higher levels of TYRO3 and GAS6. Crizotinib and foretinib showed effective antitumour activity in LMS through TYRO3 and AXL deactivation indicating that clinical trials using TYRO3 and AXL inhibitors are warranted in advanced LMS.