Wholistic approach: Transcriptomic analysis and beyond using archival material for molecular diagnosis.

Many neoplasms remain unclassified after histopathological examination, which requires further molecular analysis. To this regard, mesenchymal neoplasms are particularly challenging due to the combination of their rarity and the large number of subtypes, and many entities still lack robust diagnostic hallmarks. RNA transcriptomic profiles have proven to be a reliable basis for the classification of previously unclassified tumors and notably for mesenchymal neoplasms. Using exome-based RNA capture sequencing on more than 5000 samples of archival material (formalin-fixed, paraffin-embedded), the combination of expression profiles analyzes (including several clustering methods), fusion genes, and small nucleotide variations has been developed at the Centre Léon Bérard (CLB) in Lyon for the molecular diagnosis of challenging neoplasms and the discovery of new entities. The molecular basis of the technique, the protocol, and the bioinformatics algorithms used are described herein, as well as its advantages and limitations.

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.

Prediction of desmoid tumor progression using miRNA expression profiling.

Desmoid tumor is a rare connective tissue tumor with locoregional aggressiveness but unpredictable behavior. The miRNA profile was ascertained for 26 patients included in the Desminib phase II trial and an independent validation cohort of 15 patients. Predictive and prognostic supervised analysis on the Desminib cohort failed to identify miRNAs differentially expressed between progressive and non-progressive patients under imatinib treatment or between progressive and non-progressive patients after discontinuation of imatinib. However, an unsupervised hierarchical clustering of the Desminib cohort identified two groups (A and B) of 13 patients each, where only the number of previous lines of treatment before inclusion in the study differed significantly between the two groups. Time to progression after discontinuation of imatinib was longer in group B than in group A. Fifteen miRNAs were highly statistically differentially expressed between groups A and B, targeting more than 3000 genes, including AGO1, BCL2, CDK6, SMAD4, PTEN, CCND1, VEGFA, and RB1. These results were confirmed in the independent validation cohort: hierarchical clustering of these 15 miRNAs identified two groups, in which time to recurrence was statistically different (28.8 months vs 68.8 months). These results provide the first indication of the prognostic value of miRNA expression profiling with a possible direct impact on patient management. A more precise miRNA signature must now be determined to select patients who would not benefit from surgical resection of their tumor and who ought to be monitored without treatment.

Usefulness of an RNA extraction-free test for the multiplexed detection of ALK, ROS1, and RET Gene Fusions in Real Life FFPE Specimens of Non-Small Cell Lung Cancers.

BACKGROUND: ALK, ROS1 and RET rearrangements occur, respectively, in 5%, 2%, and 1% non-small cell lung cancers (NSCLC). ALK and ROS1 fusion proteins detection by immunohistochemistry (IHC) has been validated for rapid patient screening, but ROS1 fusions need to be confirmed by another technique and no RET IHC test is available for clinical use. RESEARCH DESIGN AND METHODS: We report herein the usefulness of the HTG EdgeSeq Assay, an RNA extraction-free test combining a quantitative nuclease protection assay with NGS, for the detection of ALK, ROS1 and RET fusions from 'real-life' small NSCLC samples. A total of 203 FFPE samples were collected from 11 centers. They included 143 rearranged NSCLC (87 ALK, 39 ROS1, 17 RET) and 60 ALK-ROS1-RET negative controls. RESULTS: The assay had a specificity of 98% and a sensitivity for ALK, ROS1 and RET fusions of 80%, 94% and 100% respectively. Among the 19 HTG-assay false negative samples, the preanalytical conditions were identified as the major factors impacting the assay efficiency. CONCLUSIONS: Overall, the HTG EdgeSeq assay offers comparable sensitivities and specificity than other RNA sequencing techniques, with the advantage that it can be used on very small and old samples collected multicentrically.

Micro-RNA profiles in osteosarcoma as a predictive tool for ifosfamide response.

Micro-RNAs (miRNA) are currently used as cancer biomarkers for hematological cancers and solid tumors. Osteosarcoma is the first primary malignant bone tumor, characterized by a complex genetic and resistance to conventional treatments. For this latter property, the median survival has not been improved since 1990 despite preoperative administration of chemotherapeutic agents. The prediction of tumor response before chemotherapy treatment would constitute a major progress for this pathology. We assessed in this study if miRNA profiling could surpass the current limitations for osteosarcoma diagnosis. We measured the miRNA expression in different osteosarcoma samples: (i) 27 osteosarcoma paraffin-embedded tumors from patients, (ii) human osteosarcoma cell lines, and (iii) tumors from a syngeneic rat osteosarcoma model, recapitulating human osteosarcoma. miRNA profiles were determined using microfluidic cards performing high-throughput TaqMan(®) -based PCR assays, called TaqMan(®) Low Density Arrays. Osteosarcoma of rat and human origins showed a miRNA signature, which could discriminate good from bad responders. In particular, we identified five discriminating miRNAs (miR-92a, miR-99b, miR-132, miR-193a-5p and miR-422a) in patient tumors, which could be easily transferable to diagnosis. These discriminating miRNAs, as well as those identified in rat, targeted the TGFß, the Wnt and the MAP kinase pathways. These results indicate that our platform constitutes a potent diagnostic tool to predict tumor sensitivity to a drug in attempt to better adapt treatment to tumor biological specificities and also to identify new potential therapeutic strategies.

miRNA Profiling: How to Bypass the Current Difficulties in the Diagnosis and Treatment of Sarcomas.

Sarcomas are divided into a group with specific alterations and a second presenting a complex karyotype, sometimes difficult to diagnose or with few therapeutic options available. We assessed if miRNA profiling by TaqMan low density arrays could predict the response of undifferentiated rhabdomyosarcoma (RMS) and osteosarcoma to treatment. We showed that miRNA signatures in response to a therapeutic agent (chemotherapy or the mTOR inhibitor RAD-001) were cell and drug specific on cell lines and a rat osteosarcoma model. This miRNA signature was related to cell or tumour sensitivity to this treatment and might be not due to chromosomal aberrations, as revealed by a CGH array analysis of rat tumours. Strikingly, miRNA profiling gave promising results for patient rhabdomyosarcoma, discriminating all types of RMS: (Pax+) or undifferentiated alveolar RMS as well as embryonal RMS. As highlighted by these results, miRNA profiling emerges as a potent molecular diagnostic tool for complex karyotype sarcomas.

SRF Fusions Other Than With RELA Expand the Molecular Definition of SRF-fused Perivascular Tumors.

Pericytic tumors encompass several entities sharing morphologic and immunohistochemical features. A subset of perivascular myoid tumors associated with the SRF-RELA fusion gene was previously described. Herein, we report a series of 13 tumors belonging to this group, in which we have identified new fusion genes by RNA-sequencing, thus expanding the molecular spectrum of this entity. All patients except 1 were children and infants. The tumors, frequently located in the head (n=8), had a mean size of 38 mm (range 10 to 150 mm) and were mostly (n=9) well-circumscribed. Exploration of the follow-up data (ranging from 3 to 68 mo) confirmed the benign behavior of these tumors. These neoplasms presented a spectrum of morphologies, ranging from perivascular patterns to myoid appearance. Tumor cells presented mitotic figures but without marked atypia. Some of these tumors could mimic sarcoma. The immunohistochemical profiles confirmed a pericytic differentiation with the expression of the smooth muscle actin and the h-caldesmon, as well as the frequent positivity for pan-cytokeratin. The molecular analysis identified the expected SRF-RELA fusion gene, in addition to other genetic alterations, all involving SRF fused to CITED1, CITED2, NFKBIE, or NCOA2. The detection of SRF-NCOA2 fusions in spindle cell rhabdomyosarcoma of the infant has previously been described, representing a risk of misdiagnosis, although the cases reported herein did not express MyoD1. Finally, clustering analyses confirmed that this group of SRF-fused perivascular myoid tumors forms a distinct entity, different from other perivascular tumors, spindle cell rhabdomyosarcomas of the infant, and smooth muscle tumors.

A spliced isoform of interleukin 6 mRNA produced by renal cell carcinoma encodes for an interleukin 6 inhibitor.

Interleukin 6 (IL-6) is a multifunctional and pleiotropic cytokine and in renal cell carcinoma (RCC), this cytokine exerts proinflammatory, immunosuppressive and growth stimulating properties. A spliced isoform of IL-6 mRNA has been described in peripheral blood mononuclear cells and encodes for a potential protein lacking IL-6 activity. In the present study, a novel spliced form of IL-6 mRNA was found detectable in RCC cell lines, normal renal cells, but not in other tumor cells. This splicing resulted in a frameshift and the generation of multiple stop codon in the spliced IL-6 mRNA. However, two ATG of the third IL-6 exon were identified as translation initiation sites and two truncated IL-6 (tIL-6) with the expected molecular weight were recovered from transfected cell supernatant. The cDNA of a spliced form of IL-6 mRNA detected in RCC lines was cloned, and expressed in a baculovirus expression vector. The functional properties of the tIL-6 were investigated and this protein blocked IL-6 bioactivity, including mitogenic activity on tumor cells. In conclusion, this spliced form of IL-6 mRNA detected in RCC encodes for a truncated IL-6 with IL-6 antagonist properties.

A novel regulation of PD-1 ligands on mesenchymal stromal cells through MMP-mediated proteolytic cleavage.

Whether fibroblasts regulate immune response is a crucial issue in the modulation of inflammatory responses. Herein, we demonstrate that foreskin fibroblasts (FFs) potently inhibit CD3+ T cell proliferation through a mechanism involving early apoptosis of activated T cells. Using blocking antibodies, we demonstrate that the inhibition of T cell proliferation occurs through cell-to-cell interactions implicating PD-1 receptor expressed on T cells and its ligands, PD-L1 and PD-L2, on fibroblasts. Dual PD-1 ligand neutralization is required to abrogate (i) binding of the PD-1-Fc fusion protein, (ii) early apoptosis of T cells, and (iii) inhibition of T cell proliferation. Of utmost importance, we provide the first evidence that PD-1 ligand expression is regulated through proteolytic cleavage by endogenous matrix metalloproteinases (MMPs) without transcriptional alteration during culture-time. Using (i) different purified enzymatic activities, (ii) MMP-specific inhibitors, and (iii) recombinant human MMP-9 and MMP-13, we demonstrated that in contrast to CD80/CD86, PD-L1 was selectively cleaved by MMP-13, while PD-L2 was sensitive to broader MMP activities. Their cleavage by exogenous MMP-9 and MMP-13 with loss of PD-1 binding domain resulted in the reversion of apoptotic signals on mitogen-activated CD3+ T cells. We suggest that MMP-dependent cleavage of PD-1 ligands on fibroblasts may limit their immunosuppressive capacity and thus contribute to the exacerbation of inflammation in tissues. In contrast, carcinoma-associated fibroblasts appear PD-1 ligand-depleted through MMP activity that may impair physical deletion of exhausted defective memory T cells through apoptosis and facilitate their regulatory functions. These observations should be considered when using the powerful PD-1/PD-L1 blocking immunotherapies.

Insulin-like growth factor type 1 receptor (IGF-1R) exclusive nuclear staining: a predictive biomarker for IGF-1R monoclonal antibody (Ab) therapy in sarcomas.

AIMS: A minority of patients with advanced sarcoma achieve prolonged progression free survival (PFS) with insulin growth factor type 1 receptor (IGF-1R) monoclonal antibody (Ab) therapy. A biomarker identifying those patients beforehand would be useful to select patients for the development of these agents. METHODS: This single centre series includes patients with unresectable or metastatic soft tissue sarcomas (STS), Ewing sarcoma (ES) and osteosarcoma treated with IGF-1R Ab (R1507, IMC-A12, SCH 717454 and CP-751.871) in the Centre Léon Bérard. Tumour samples were analysed by immunohistochemistry for expression of IGF-1R, insulin-like growth factor binding protein type 3 (IGFBP-3), Ki67, epidermal growth factor receptor (HER1) and human epidermal growth factor receptor 2 (HER2). Predictive factors for PFS and overall survival (OS) were investigated. RESULTS: All tumour samples had a positive IGF-1R immunostaining on 60% to 100% of tumour cells. IGFBP-3 immunostaining was observed in 12 (75%) samples with 5% to 100% of positive cells. IGF-1R immunostaining was nuclear (n=9, 56%), cytoplasmic (n=4, 25%), or nuclear +cytoplasmic (n=3, 19%). Neither IGFBP-3 expression, nor Ki67 was correlated to PFS. HER2 and HER1 staining were positive in 0 and 2 samples respectively (both primary resistant to IGF-1R Ab therapy). Exclusive intra-nuclear immunoreactivity for IGF-1R was significantly associated with a better PFS (p=0.01) and OS (p=0.007). CONCLUSION: Exclusive nuclear localisation of IGF-1R is an easily testable biomarker associated with a better PFS and OS for patients treated with IGF-1R Ab therapy. Nuclear localisation of IGF-1R in tumour cells might be a hallmark of pathway activation.