AimsWe aimed to evaluate the performances of the Idylla GeneFusion Assay (IGFA) designed to detect, in a single, rapid and fully automated assay, ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 gene fusions ...and MET exon 14 skipping in cancer samples.MethodsBased on a set of tumours enriched in cases with gene fusions, we applied the IGFA to tumour areas of various sizes and tumour cell contents. IGFA results were compared with those obtained with other methods (immunohistochemistry, fluorescent in situ hybridisation, DNA and RNA next-generation sequencing).ResultsWe selected 68 tumours: 49 cases with known gene fusions (8 ALK, 8 ROS1, 5 RET, 7 NTRK1, 3 NTRK2 and 6 NTRK3 ones) or MET exon 14 skipping mutations (12 cases) and 19 cases with no fusion and no MET mutation. We performed 128 IGFA tests on distinct tissue areas. The global sensitivity and specificity of the IGFA were, respectively, 62.82% and 99.2% with variations between molecular targets and tissue areas. Of note, 72.5% sensitivity and 98.79% specificity were obtained in 37 tissue areas fulfilling the manufacturer’s recommendations (ie, at least 10% of tumour cells in at least 20 mm² of tissue area). The rate of non-conclusive results was higher in small samples with low percentages of tumour cells.ConclusionsThe IGFA could contribute to the rapid detection of targetable gene fusions and mutations, especially in context of rapidly growing cancers requiring urgent therapeutic choices.
The presence of circulating tumor cells (CTCs) and CTC clusters, also known as tumor microemboli, in biological fluids has long been described. Intensive research on single CTCs has made a ...significant contribution in understanding tumor invasion, metastasis tropism, and intra-tumor heterogeneity. Moreover, their being minimally invasive biomarkers has positioned them for diagnosis, prognosis, and recurrence monitoring tools. Initially, CTC clusters were out of focus, but major recent advances in the knowledge of their biogenesis and dissemination reposition them as critical actors in the pathophysiology of cancer, especially metastasis. Increasing evidence suggests that "united" CTCs, organized in clusters, resist better and carry stronger metastatic capacities than "divided" single CTCs. This review gathers recent insight on CTC cluster origin and dissemination. We will focus on their distinct molecular package necessary to resist multiple cell deaths that all circulating cells normally face. We will describe the molecular basis of their increased metastatic potential as compared to single CTCs. We will consider their clinical relevance as prognostic biomarkers. Finally, we will propose future directions for research and clinical applications in this promising topic in cancer.
Abstract
Sézary Syndrome (SS) is a rare aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis, and a circulating atypical CD4 + T-cell clonal population. The ...diversity of Sézary cell (SC) phenotype and genotype may reflect either plasticity or heterogeneity, which was difficult to evaluate dynamically until the achievement of long-term SC expansion. Therefore, we developed six defined culture conditions allowing for the expansion of SC defined by their phenotype and monoclonality in four of seven SS cases. Engraftment of SC through the intrafemoral route into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(
tm1Wjll
)/SzJ (NSG) mice was achieved in 2 of 14 SS cases. Secondary xenograft by percutaneous injection mimicked most of the features of SS with dermal infiltration, epidermotropism, and blood spreading. These models also allowed assessing the intra-individual heterogeneity of patient SC. Subclones sharing the same TCR gene rearrangement evolved independently according to culture conditions and/or after xenografting. This clonal selection was associated with some immunophenotypic plasticity and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed us to develop eight new SC lines derived from four different patients. These lines represent the cell of origin diversity of SC and provide new tools to evaluate their functional hallmarks and response to therapy.
Targetable kinase fusions are extremely rare (<1%) in colorectal cancers (CRCs), making their diagnosis challenging and often underinvestigated. They have been shown particularly frequently among ...MSI-High, BRAF/KRAS/NRAS wild-type CRCs with MLH1 loss (MLH1loss MSI-High wild-type).
We searched for NTRK1, NTRK2, NTRK3, ALK, ROS1, BRAF, RET, and NRG1 kinase fusions in CRCs using methods easy-to-implement in pathology laboratories: immunohistochemistry (IHC), fluorescent in situ hybridization (FISH), and fully automated real-time PCR targeted analyses. RNA-sequencing analyses were used for confirmation.
Among 84 selected MLH1 deficient (IHC) CRCs cases, MLH1loss MSI-High wild-type CRCs consisted first in 19 cases after Idylla™ analyses and finally in 18 cases (21%) after RNA-sequencing (detection of one additional KRASG12D mutation). FISH (and when relevant, IHC) analyses concluded in 5 NTRK1, 3 NTRK3, 1 ALK, 2 BRAF, and 2 RET FISH positive tumors. ALK and NTRK1 rearranged tumors were IHC positive, but pan-TRK IHC was negative in the 3 NTRK3 FISH positive tumors. RNA-sequencing analyses confirmed 12 of 13 fusions with only one false positive RET FISH result. Finally, 12/18 (67%) of MLH1loss MSI-High wild-type CRCs contained targetable kinase fusions.
Our study demonstrates the feasibility, but also the cost-effectiveness, of a multistep but rapid diagnostic strategy based on nonsequencing methods to identify rare and targetable kinase fusions in patients with advanced CRCs, as well as the high prevalence of these kinase fusions in MLH1loss MSI-High wild-type CRCs. Nevertheless, confirmatory RNA-sequencing analyses are necessary in case of low FISH positive nuclei percentage to rule out FISH false-positive results.
•Gene fusions are very rare (<1%) in colorectal cancers (CRCs) but more frequent in MSI-High RAS-BRAF wild-type CRCs.•Among 84 CRCs with MLH1 loss, including 18 RAS-BRAF wild-type cases, we diagnosed 12 (66.7%) gene fusions.•Tumors with MLH1 loss, microsatellite instability and no RAS-BRAF mutation must be tested for targetable gene fusions.•NTRK1, NTRK3, ALK, RET, and BRAF gene fusions diagnosis is attainable in CRCs using fluorescent in situ hybridization (FISH).•Low percentage FISH results must be confirmed using RNA-sequencing analyses.
To determine whether the mutational profile of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) is unique by comparison with other diffuse large B-cell lymphoma subtypes, we ...analyzed a total cohort of 20 PCLBCL-LT patients by using next-generation sequencing with a lymphoma panel designed for diffuse large B-cell lymphoma. We also analyzed 12 pairs of tumor and control DNA samples by whole-exome sequencing, which led us to perform resequencing of three selected genes not included in the lymphoma panel: TBL1XR1, KLHL6, and IKZF3. Our study clearly identifies an original mutational landscape of PCLBCL-LT with a very restricted set of highly recurrent mutations (>40%) involving MYD88 (p.L265P variant), PIM1, and CD79B. Other genes involved in B-cell signaling, NF-κB activation, or DNA modeling were found altered, notably TBL1XR1 (33%), MYC (26%) CREBBP (26%), and IRF4 (21%) or HIST1H1E (41%). MYD88L265P variant was associated with copy number variations or copy neutral loss of heterozygosity in 60% of patients. The most frequent genetic losses involved CDKN2A/2B, TNFAIP3/A20, PRDM1, TCF3, and CIITA. Together, these results show that PCLBCL-LT exhibits a unique mutational landscape, combining highly recurrent hotspot mutations in genes involved in NF-kB and B-cell signaling pathways, which provides a rationale for using selective inhibitors of the B-cell receptor.
IMPORTANCE: The activating mutation of MYD88 L265P is a frequent feature of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT), reported in up to 69% of the cases. Whether patients ...with MYD88 mutation display specific clinical and evolutive features has not been evaluated. OBJECTIVE: To identify the clinical characteristics associated with MYD88 mutation, confirm its high prevalence, and evaluate its effect on prognosis in patients with PCLBCL-LT. DESIGN, SETTING, AND PARTICIPANTS: A retrospective multicenter study was conducted using the medical records of patients from dermatology departments belonging to the French Study Group for Cutaneous Lymphomas. Sixty-one patients with a diagnosis of PCLBCL-LT made between 1988 and 2010 who were available for molecular study were included. Of these, 58 patients displaying interpretable results constituted the study group. Median follow-up was 33 months, and 39 patients (67%) were monitored until death. MAIN OUTCOMES AND MEASURES: Clinical features (age, sex, number of skin lesions, tumor stage, and location as leg vs elsewhere), MYD88 mutation (allele-specific TaqMan polymerase chain reaction assay), treatment regimen, and outcome were recorded. Baseline characteristics and outcome were compared according to the status of MYD88. RESULTS: The median age of the patients was 79 years, and 59% were female. Skin lesions were located on the leg in 76% of the cases. Thirty-four of 58 patients (59%) harbored the MYD88 L265P mutation. Patients had similar clinical characteristics at presentation regardless of their MYD88 status, except that those harboring the MYD88 mutation were older (P = .006) and had more frequent involvement of the leg (P = .008). Patients harboring the MYD88 mutation had 3- and 5-year–specific survival rates of 65.7% and 60.2% vs 85.4% and 71.7% in patients with the wild-type allele. The MYD88 mutation was significantly associated with shorter disease-specific survival in univariate (P = .03) and multivariate (odds ratio, 3.01; 95% CI, 1.03-8.78; P = .04) analysis. There was no significant difference between the groups in their treatment regimens. Considering overall survival, in univariate (P = .002) and multivariate (odds ratio, 2.94; 95% CI, 1.18-7.30; P = .02) analysis, MYD88 L265P mutation was an independent adverse prognostic factor. CONCLUSIONS AND RELEVANCE: This study confirms the high prevalence of MYD88 L265P mutation in PCLBCL-LT and shows its association with shorter survival. The clinical effect of MYD88 mutation activating the nuclear factor-κB pathway supports the use of targeted therapies at the time of relapse after conventional therapies.
Lymphomas represent a heterogeneous group of cancers characterized by clonal lymphoproliferation. Over the past decades, frequent epigenetic dysregulations have been identified in hematologic ...malignancies including lymphomas. Many of these impairments occur in genes with established roles and well-known functions in the regulation and maintenance of the epigenome. In hematopoietic cells, these dysfunctions can result in abnormal DNA methylation, erroneous chromatin state and/or altered miRNA expression, affecting many different cellular functions. Nowadays, it is evident that epigenetic dysregulations in lymphoid neoplasms are mainly caused by genetic alterations in genes encoding for enzymes responsible for histone or chromatin modifications. We summarize herein the recent epigenetic modifiers findings in lymphomas. We focus also on the most commonly mutated epigenetic regulators and emphasize on actual epigenetic therapies.
BRAF inhibitors have demonstrated improvement of overall survival in patients with metastatic melanoma and BRAF(V600) mutations. In order to evaluate BRAF tumor heterogeneity between primary and ...metastatic site, we have evaluated the performance of immunohistochemistry (IHC) with an anti-BRAF(V600E) antibody in both localization by comparison with high resolution melting analysis followed by Sanger sequencing in a parallel blinded study. A total of 230 samples distributed as primary melanoma (n = 88) and different types of metastatic samples (n = 142) were studied in 99 patients with advanced or metastatic melanoma (stage III or IV). The prevalence of each BRAF mutation was c.1799T>A, BRAF(V600E) (45.2%), c.1799_1800TG>AA, BRAF(V600E2) (3.0%), c.1798_1799GT>AA, BRAF(V600K) (3.0%), c.1801 A>G, BRAF(K601E) (1.3%), c.1789_1790CT>TC, BRAF(L597S) (0.4%), c.1780G>A, BRAF(D594N) (0.9%) respectively. IHC was positive in 109/112 samples harboring BRAF(V600E/E2) mutations and negative in other cases. The cytoplasmic staining was either strongly positive in tumor cells of BRAF(V600E) mutated cases. It appeared strong brown, different from the vesicular grey cytoplasmic pigmentation of melanophages. Concordance between the two techniques was 96.4%. Sensitivity of IHC for detecting the BRAF(V600E/E2) mutations was 97.3%, while specificity was 100%. Both our IHC and molecular study demonstrated homogeneity between primary and metastatic sites for BRAF status in melanoma. This study also provides evidence that IHC may be a cost-effective first-line method for BRAF(V600E) detection. Thereafter, molecular techniques should be used in negative, ambiguous or non-contributive cases.
Cutaneous T‐cell lymphomas (CTCLs) are telomerase‐positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT ...re‐expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re‐expression. We analyzed hTERT promoter methylation status in CTCL cells compared with healthy cells. Gene‐specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from −650 to −150 bp and a hypomethylated proximal region from −150 to +150 bp. Interestingly, the hypermethylated region matches with the recently named TERT hypermethylated oncogenic region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect of THOR on two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment and a DNA methyltransferase inhibitor (DNMTi) 5‐azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.
Cutaneous T‐cell lymphomas (CTCLs) represent a group of lymphoproliferative disorders arising from the skin. CTCLs are characterized by hTERT gene expression despite the lack of hTERT amplifications or rearrangements. Here, we investigated hTERT promoter methylation and associated TERT hypermethylated oncogenic region (THOR) with hTERT reactivation in CTCL. Additionally, we evaluated THOR methylation and hTERT expression after treatment with epigenetic drugs. Altogether, our study offers a better understanding of the response to epigenetic drugs in patients with CTCL.
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