ABSTRACT
Pan-Trk immunohistochemistry has been described as a screening test for the detection of NTRK fusions in a broad spectrum of tumor types. However, pan-Trk testing in the clinical setting may be limited by many factors, including analytical parameters such as clones, platforms, and protocols used. This study aimed to harmonize pan-Trk testing using various clones and immunohistochemical (IHC) platforms and to evaluate the level of analytical variability across pathology laboratories. We developed several IHC pan-Trk assays using clones EPR17341 (Abcam) and A7H6R (Cell Signaling Technology) on Ventana/Roche, Agilent, and Leica platforms. To compare them, we sent unstained sections of a tissue microarray containing 9 cases with NTRK3 fusions to participating laboratories, to perform staining on Ventana/Roche (10 centers), Agilent (4 centers), and Leica (3 centers) platforms. A ready-to-use pan-Trk IVD assay (Ventana/Roche) was also performed in 3 centers. All slides were centrally and blindly reviewed for the percentage of stained tumor cells. Laboratory-developed tests with clone EPR17341 were able to detect pan-Trk protein expression in all cases, whereas lower rates of positivity were observed with clone A7H6R. Moderate to strong variability of the positive cases rate was observed with both antibodies in each IHC platforms type and each of the positivity cut points evaluated (≥1%, ≥10%, and ≥50% of stained tumor cells). The rate of false-negative cases was lower when pan-Trk staining was assessed with the lowest positivity threshold (≥1%). In conclusion, most evaluated pan-Trk IHC laboratory-developed tests were able to detect NTRK3-fusion proteins; however, a significant analytical variability was observed between antibodies, platforms, and centers.
Subject(s)
Biomarkers, Tumor , Receptor, trkA , Humans , Immunohistochemistry , Biomarkers, Tumor/genetics , Oncogene Proteins, Fusion/metabolismABSTRACT
As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.
Subject(s)
Breast Neoplasms , rhoA GTP-Binding Protein , rhoC GTP-Binding Protein , Cell Transformation, Neoplastic , Female , Guanosine Triphosphate , Humans , rhoA GTP-Binding Protein/metabolism , rhoC GTP-Binding Protein/metabolismABSTRACT
Breast cancer (BC) remains the primary cause of death from cancer among women worldwide. Cholesterol-5,6-epoxide (5,6-EC) metabolism is deregulated in BC but the molecular origin of this is unknown. Here, we have identified an oncometabolism downstream of 5,6-EC that promotes BC progression independently of estrogen receptor α expression. We show that cholesterol epoxide hydrolase (ChEH) metabolizes 5,6-EC into cholestane-3ß,5α,6ß-triol, which is transformed into the oncometabolite 6-oxo-cholestan-3ß,5α-diol (OCDO) by 11ß-hydroxysteroid-dehydrogenase-type-2 (11ßHSD2). 11ßHSD2 is known to regulate glucocorticoid metabolism by converting active cortisol into inactive cortisone. ChEH inhibition and 11ßHSD2 silencing inhibited OCDO production and tumor growth. Patient BC samples showed significant increased OCDO levels and greater ChEH and 11ßHSD2 protein expression compared with normal tissues. The analysis of several human BC mRNA databases indicated that 11ßHSD2 and ChEH overexpression correlated with a higher risk of patient death, highlighting that the biosynthetic pathway producing OCDO is of major importance to BC pathology. OCDO stimulates BC cell growth by binding to the glucocorticoid receptor (GR), the nuclear receptor of endogenous cortisol. Interestingly, high GR expression or activation correlates with poor therapeutic response or prognosis in many solid tumors, including BC. Targeting the enzymes involved in cholesterol epoxide and glucocorticoid metabolism or GR may be novel strategies to prevent and treat BC.
Subject(s)
Breast Neoplasms/metabolism , Carcinogens/metabolism , Cholesterol/metabolism , Receptors, Glucocorticoid/metabolism , 11-beta-Hydroxysteroid Dehydrogenase Type 2/metabolism , Animals , Cell Line , Cell Line, Tumor , Cholesterol/analogs & derivatives , Epoxide Hydrolases/metabolism , Estrogen Receptor alpha/metabolism , Female , HEK293 Cells , Humans , MCF-7 Cells , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , RNA, Messenger/metabolismABSTRACT
An increasing number of sarcomas displaying a primitive, monomorphic spindle cell phenotype have been shown to harbor recurrent gene fusions, including biphenotypic sinonasal sarcoma (SNS). Occurring in the sinonasal area of middle-aged patients, SNS is a locally aggressive tumor harboring in 90% of cases recurrent gene fusions involving the PAX3 gene, in which the chimeric transcription factor induces an aberrant dual myogenic and neural phenotype. Here, we report an unusual oropharyngeal monomorphic spindle cell sarcoma in a 53-year-old man that revealed a novel RREB1-MKL2 gene fusion by RNA sequencing with the Illumina TruSight RNA Fusion Panel. The gene fusion was validated by RT-PCR. Although the tumor location is unusual (but head and neck seated), most of the other clinical, morphologic, immunophenotypic (focal combined expression of S100 protein, SMA, desmin, and myogenin) and oncogenic data suggest that this biphenotypic "oropharyngeal" sarcoma is closely related to the biphenotypic SNS spectrum. Notably, the RREB1-MKL2 chimeric transcription factor encoded by this fusion gene produced an increase in MKL2 expression, which regulates both neural and myogenic differentiation, mimicking the crucial role of PAX3 reported in SNS oncogenesis. NGS and especially RNA sequencing may be used to identify new candidate fusion oncogenes in soft tissue tumors, which would help in updating the existing classification. In turn, this would lead to better therapeutic management of patients.
Subject(s)
DNA-Binding Proteins/genetics , Paranasal Sinus Neoplasms/genetics , Transcription Factors/genetics , Biomarkers, Tumor/genetics , DNA-Binding Proteins/metabolism , Gene Fusion , Humans , Male , Middle Aged , Oncogene Fusion , Oncogene Proteins, Fusion/genetics , Oropharyngeal Neoplasms/genetics , Sarcoma/genetics , Soft Tissue Neoplasms/genetics , Transcription Factors/metabolismABSTRACT
AIMS: The diagnosis of malignant peripheral nerve sheath tumour (MPNST) may be challenging, especially in the sporadic setting. Owing to the lack of specific histological criteria, immunohistochemical and molecular diagnostic markers, several differential diagnoses must be considered, especially melanoma. Indeed, although S100 protein usually stains melanoma, other melanocytic markers are often negative, especially in spindle cell/desmoplastic types. This pattern of immunoreactivity resembles that of some nerve-derived tumours such as MPNST. Owing to their different clinical behaviours and therapeutic implications, accurate identification of these two different tumours is crucial. METHODS AND RESULTS: S100, SOX10, KBA62, MITF, HMB45, Melan-A, tyrosinase PNL2 and BRAF-V600E immunostaining was performed in a pathologically and genetically well-characterised cohort of primary MPNST (n = 124), including 66 (53%) NF1-associated tumours. Sox10 and KBA62 expression were found, respectively, in 102 (84%) and in 101 (83%) MPNST, whereas S100 was expressed in 64 cases (52%). We observed an increased loss of S100 with increasing histological grade (P = 0.0052). We found Melan-A expression in 14% (n = 17) of all MPNST, occurring in 82% (n = 14) of cases in an NF1 context. Six per cent (n = 8) of MPNST showed tyrosinase positivity, including seven (87%) NF1-associated. MITF expression was found in 10 (8%) MPNST. None expressed PNL2, HMB45 or BRAF-V600E. CONCLUSION: MPNST (in NF1 and a sporadic setting) can quite often be positive for Melan-A, tyrosinase and MITF. Pathologists should be cognisant of these exceptions to prevent confusion with melanoma.
Subject(s)
Biomarkers, Tumor/metabolism , Melanocytes/metabolism , Melanoma/diagnosis , Neurofibrosarcoma/diagnosis , Diagnosis, Differential , Female , Humans , MART-1 Antigen/metabolism , Male , Melanocytes/pathology , Melanoma/metabolism , Melanoma/pathology , Monophenol Monooxygenase/metabolism , Neurofibrosarcoma/metabolism , Neurofibrosarcoma/pathology , Proto-Oncogene Proteins B-raf/metabolism , S100 Proteins/metabolism , SOXE Transcription Factors/metabolismABSTRACT
BACKGROUND: To date, all studies conducted on breast cancer diagnosis have focused on the expression of the full-length 66-kDa estrogen receptor alpha (ERα66). However, much less attention has been paid to a shorter 46-kDa isoform (ERα46), devoid of the N-terminal region containing the transactivation function AF-1. Here, we investigated the expression levels of ERα46 in breast tumors in relation to tumor grade and size, and examined the mechanism of its generation and its specificities of coregulatory binding and its functional activities. METHODS: Using approaches combining immunohistochemistry, Western blotting, and proteomics, antibodies allowing ERα46 detection were identified and the expression levels of ERα46 were quantified in 116 ERα-positive human breast tumors. ERα46 expression upon cellular stress was studied, and coregulator bindings, transcriptional, and proliferative response were determined to both ERα isoforms. RESULTS: ERα46 was expressed in over 70% of breast tumors at variable levels which sometimes were more abundant than ERα66, especially in differentiated, lower-grade, and smaller-sized tumors. We also found that ERα46 can be generated via internal ribosome entry site-mediated translation in the context of endoplasmic reticulum stress. The binding affinities of both unliganded and fully-activated receptors towards co-regulator peptides revealed that the respective potencies of ERα46 and ERα66 differ significantly, contributing to the differential transcriptional activity of target genes to 17ß estradiol (E2). Finally, increasing amounts of ERα46 decrease the proliferation rate of MCF7 tumor cells in response to E2. CONCLUSIONS: We found that, besides the full-length ERα66, the overlooked ERα46 isoform is also expressed in a majority of breast tumors. This finding highlights the importance of the choice of antibodies used for the diagnosis of breast cancer, which are able or not to detect the ERα46 isoform. In addition, since the function of both ERα isoforms differs, this work underlines the need to develop new technologies in order to discriminate ERα66 and ERα46 expression in breast cancer diagnosis which could have potential clinical relevance.
Subject(s)
Breast Neoplasms/genetics , Estrogen Receptor alpha/genetics , Gene Expression Regulation, Neoplastic , Alternative Splicing , Breast Neoplasms/diagnosis , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cluster Analysis , Endoplasmic Reticulum Stress , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunohistochemistry , Protein Binding , Protein Biosynthesis , Protein Isoforms , Proteome , Proteomics/methods , Retrospective StudiesABSTRACT
BACKGROUND: CD70 is a costimulatory molecule of the tumour necrosis factor family expressed in activated immune cells and some solid tumours. In lymphocytes CD70 triggers T cell-mediated cytotoxicity and mitogen-activated protein kinase phosphorylation. METHODS: We evaluated the expression of CD70 in biopsies and melanoma cell lines. Using melanoma cell lines positive or not for CD70, we analysed CD70 function on melanoma progression. RESULTS: We report CD70 expression in human melanoma cell lines and tumour cells from melanoma biopsies. This expression was observed in 95% of primary melanomas but only 37% of metastases. Both monomeric and trimeric forms of CD70 were detected in tumour cell membrane fractions, whereas cytoplasmic fractions contained almost exclusively monomeric CD70. In vitro and in vivo experiments demonstrated that CD70 expression inhibited melanoma cell migration, invasion and pulmonary metastasis implantation independently of the tumour immune microenvironment. Increasing the levels of the trimeric form of CD70 through monoclonal antibody binding led to an increase in CD70+ melanoma cell invasiveness through MAPK pathway activation, RhoE overexpression, ROCK1 and MYPT1 phosphorylation decrease, and stress fibres and focal adhesions disappearance. CONCLUSIONS: Our results describe a new non-immunological function of melanoma-expressed CD70, which involves melanoma invasiveness through MAPK pathway, RhoE and cytoskeletal modulation.
Subject(s)
CD27 Ligand/physiology , Melanoma/pathology , Animals , CD27 Ligand/analysis , Cell Line, Tumor , Cell Movement , Cytoskeleton/physiology , Female , Humans , MAP Kinase Signaling System/physiology , Mice , Mice, Inbred C57BL , Neoplasm Invasiveness , Neoplasm Metastasis , rho GTP-Binding Proteins/physiology , rho-Associated Kinases/physiologyABSTRACT
This study tested the sensitivity and specificity of VE1 antibody raised against BRAFV600E protein, on 189 melanoma samples, compared with molecular testing. In addition, the therapeutic response to BRAF inhibitors was analysed in 27 patients, according to staining intensity (scored from weak to strong) and pattern (homogeneous or heterogeneous). BRAFV600E status during melanoma progression was evaluated in a cohort of 54 patients with at least paired-samples. High sensitivity (98.6%) and specificity (97.7%) of VE1 were confirmed. During melanoma progression different samples showed concordant phenotypes. Heterogeneous VE1 staining was observed in 28.5% of cases, and progression-free survival was higher in patients with tumour samples displaying such staining. These findings suggest that only VE1-negative tumours would be genotyped to detect other BRAFV600 mutations, and that either primary melanoma or metastasis can be tested using immunohistochemistry, according to the material available.
Subject(s)
Melanoma/genetics , Mutation/genetics , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/genetics , Antibodies, Monoclonal , Biomarkers, Tumor/analysis , DNA Mutational Analysis , Disease Progression , Genotype , Humans , Immunohistochemistry , Melanoma/drug therapy , Melanoma/pathology , Neoplasm Metastasis , Phenotype , Retrospective Studies , Sensitivity and Specificity , Skin Neoplasms/drug therapy , Skin Neoplasms/pathologyABSTRACT
Blood vessels and tumor angiogenesis are generally associated with tumor growth and poor clinical outcome of cancer patients. However, we recently discovered that some blood vessels present within the tumor microenvironment can be associated with favorable prognosis. These vessels, designated tumor high endothelial venules (HEVs), appear to facilitate tumor destruction by allowing high levels of lymphocyte infiltration into tumors. In this study, we investigated the mechanisms regulating HEV blood vessels in human breast cancer. We found that lymphotoxin ß was overexpressed in tumors containing high densities of HEVs (HEV(high)) and correlated to DC-LAMP, a marker of mature DCs. DCs were the main producers of lymphotoxin ß in freshly resected HEV(high) breast tumor samples, and the density of DC-LAMP(+) DCs clusters was strongly correlated with the density of tumor HEVs, T and B cell infiltration, and favorable clinical outcome in a retrospective cohort of 146 primary invasive breast cancer patients. Densities of tumor HEVs and DC-LAMP(+) DCs were strongly reduced during breast cancer progression from in situ carcinoma to invasive carcinoma, suggesting that loss of tumor HEVs is a critical step during breast cancer progression. Finally, an increase in the infiltration of regulatory T cells was observed in HEV(high) breast tumors, indicating that tumor HEVs can develop in the presence of regulatory T cells. Together, our results support a key role for DCs and DC-derived lymphotoxin in the formation of tumor HEVs. These findings are important because novel therapeutic strategies based on the modulation of tumor HEVs could have a major impact on clinical outcome of cancer patients.
Subject(s)
Breast Neoplasms/blood supply , Carcinoma, Ductal, Breast/blood supply , Dendritic Cells/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphotoxin-beta/biosynthesis , Neovascularization, Pathologic/immunology , Venules/pathology , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/pathology , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/immunology , Carcinoma, Ductal, Breast/pathology , Carcinoma, Intraductal, Noninfiltrating/blood supply , Carcinoma, Intraductal, Noninfiltrating/immunology , Carcinoma, Intraductal, Noninfiltrating/pathology , Dendritic Cells/metabolism , Dendritic Cells/pathology , Disease Progression , Endothelium, Vascular/ultrastructure , Female , Humans , Lysosomal-Associated Membrane Protein 3/analysis , Neoplasm Proteins/analysis , Retrospective Studies , T-Lymphocytes, Regulatory/immunology , Treatment Outcome , Tumor Microenvironment , Venules/immunologyABSTRACT
BACKGROUND: Sentinel lymph node biopsy (SLNB) is the most sensitive procedure for assessing nodal status in patients with primary melanoma. OBJECTIVE: To evaluate the predictive ability of usual primary melanoma prognosis factors of detecting sentinel lymph node (SLN) metastasis in patients with melanoma. PATIENTS AND METHODS: A cohort of 612 consecutive patients presenting with primary skin melanoma who underwent a SLNB was evaluated. Assessment of the determinants of SLN metastasis was based on general linear model analysis. The model performance was studied using the concordance statistic and the net reclassification index. The calibration was estimated using the Hosmer-Lemeshow test. RESULTS: The discrimination ability did not differ significantly between Breslow thickness (0.57), Clark index (0.61), ulceration (0.57) and histological subtype (0.55). Clark index, ulceration and Breslow thickness were all significant and independent determinants of SLN metastasis. The predictive ability of the final model was 0.657. CONCLUSION: Breslow thickness, Clark index and ulceration are independent predictors of a SLN metastasis.
Subject(s)
Lymph Nodes/pathology , Melanoma/pathology , Melanoma/secondary , Sentinel Lymph Node Biopsy/statistics & numerical data , Skin Neoplasms/pathology , Skin Ulcer/pathology , Adult , Aged , Cohort Studies , Confidence Intervals , Disease-Free Survival , Female , France , Humans , Lymphatic Metastasis/pathology , Male , Melanoma/mortality , Middle Aged , Neoplasm Invasiveness/pathology , Neoplasm Staging , Predictive Value of Tests , Prognosis , Regression Analysis , Risk Assessment , Skin Neoplasms/mortality , Survival AnalysisSubject(s)
Adenocarcinoma/secondary , Neoplasms, Unknown Primary/pathology , Peritoneal Neoplasms/secondary , Adenocarcinoma/chemistry , Adenocarcinoma/diagnosis , Adenocarcinoma/pathology , Aged, 80 and over , Algorithms , Biomarkers, Tumor , Female , Humans , Neoplasm Proteins/analysis , Organ Specificity , Peritoneal Neoplasms/chemistry , Peritoneal Neoplasms/diagnosis , Peritoneal Neoplasms/pathologySubject(s)
Neoplasms, Unknown Primary/pathology , Sarcoma, Synovial/secondary , Adenocarcinoma/diagnosis , Adult , Biomarkers, Tumor , Biopsy , Diagnosis, Differential , Female , Humans , Keratin-7/analysis , Liposarcoma/diagnosis , Magnetic Resonance Imaging , Neoplasm Proteins/analysis , Neoplasm Proteins/genetics , Neoplasms, Unknown Primary/chemistry , Neoplasms, Unknown Primary/diagnosis , Neoplasms, Unknown Primary/genetics , Proto-Oncogene Proteins/genetics , Repressor Proteins/genetics , Sarcoma, Synovial/chemistry , Sarcoma, Synovial/diagnosis , Sarcoma, Synovial/geneticsSubject(s)
Carcinoma/secondary , Neoplasms, Unknown Primary/pathology , Pathologists , Physician's Role , Algorithms , Biomarkers, Tumor , Carcinoma/chemistry , Carcinoma/diagnosis , Carcinoma/pathology , Humans , Immunohistochemistry , Immunophenotyping , Neoplasms, Unknown Primary/chemistry , Neoplasms, Unknown Primary/diagnosisABSTRACT
BACKGROUND: Using adaptive radiotherapy (ART), to determine objective clinical criteria that identify extremity soft tissue sarcoma (ESTS) patients requiring adaptation of their preoperative radiotherapy (RT) plan. PATIENTS AND METHODS: We included 17 patients with a lower extremity ESTS treated between 2019 and 2021 with preoperative RT, using helicoidal intensity-modulated RT (IMRT) tomotherapy, before surgical resection. We collected clinical, tumor parameters and treatment data. Repositioning was ascertained by daily Megavoltage computed tomography (MVCT) imaging. Using the PreciseART technology we retrospectively manually delineated at least one MVCT for each patient per week and recorded volume and dosimetric parameters. A greater than 5% change between target volume and planned target volume (PTV) dosimetric coverage from the initial planning CT scan to at least one MVCT was defined as clinically significant. RESULTS: All 17 patients experienced significant tumor volume changes during treatment; 7 tumors grew (41%) and 10 shrank (59%). Three patients (18%), all undifferentiated pleomorphic sarcomas (UPS) with increased volume changes, experienced significant reductions in tumor dose coverage. Seven patients required a plan adaptation, as determined by practical criteria applied in our departmental practice. Among these patients, only one ultimately experienced a significant change in PTV coverage. Three patients had a PTV decrease of coverage. Among them, 2 did not receive plan adaptation according our criteria. None of the patients with decreased tumor volumes had reduced target volume coverage. Monitoring volume variations by estimating gross tumor volume (GTV) on MVCT, in addition to axial and sagittal linear tumor dimensions, appeared to be most effective for detecting reductions in PTV coverage throughout treatment. CONCLUSIONS: Variations in ESTS volume are evident during preoperative RT, but significant dosimetric variations are rare. Specific attention should be paid to grade 2-3 UPSs during the first 2 weeks of treatment. In the absence of dedicated software in routine clinical practice, monitoring of tumor volume changes by estimating GTV may represent a useful strategy for identifying patients whose treatment needs to be replanned.
Subject(s)
Sarcoma , Soft Tissue Neoplasms , Humans , Radiotherapy Dosage , Tumor Burden , Retrospective Studies , Radiotherapy Planning, Computer-Assisted/methods , Sarcoma/diagnostic imaging , Sarcoma/radiotherapy , Sarcoma/surgery , Soft Tissue Neoplasms/diagnostic imaging , Soft Tissue Neoplasms/radiotherapy , Soft Tissue Neoplasms/surgery , Extremities/diagnostic imaging , Extremities/pathologyABSTRACT
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.
ABSTRACT
Desmoid tumors are benign monoclonal fibroblastic or myofibroblastic neoplasms, characterized by local invasiveness and high rates of recurrence. Desmoid tumors must be distinguished from benign fibroblastic and myofibroblastic lesions, as well as from low-grade sarcoma, which can appear histologically similar to desmoid tumors. This differential diagnosis can be very difficult, especially when diagnosis is based on a core needle biopsy. On the molecular level, most sporadic desmoid tumors are associated with mutations of the ß-catenin gene (CTNNB1). A minority of desmoid tumors are associated with Gardner syndrome and mutations of the familial adenomatous polyposis gene. We identified the common CTNNB1 mutations associated with sporadic desmoid tumors by direct sequencing: in (i) 260 cases of typical desmoid tumors; and (ii) in 191 cases of spindle cell lesions, which can morphologically 'mimic' desmoid tumors. Formalin-fixed paraffin-embedded tissues were obtained via core needle biopsy (n=150) or open biopsy/surgical excision (n=301). Only 16 cases (4%) were not analyzable (Bouin's fixed tissue). CTNNB1 mutations were observed in 223 of 254 (88%) of sporadic desmoid tumors. No CTNNB1 mutations were detected in all other lesions (n=175) studied. CTNNB1 sequencing can be easily and reliably done using tissues obtained via core needle biopsy. Detection of CTNNB1 mutations in formalin-fixed paraffin-embedded tissues among spindle cell lesions is proposed as a specific diagnostic tool for the diagnosis of desmoid tumors. This result has significant implications for patient care and management.
Subject(s)
Fibromatosis, Abdominal/diagnosis , Fibromatosis, Aggressive/diagnosis , Mutation , beta Catenin/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , DNA Mutational Analysis , Diagnosis, Differential , Female , Fibromatosis, Abdominal/genetics , Fibromatosis, Abdominal/surgery , Fibromatosis, Aggressive/genetics , Fibromatosis, Aggressive/surgery , Formaldehyde , Humans , Infant , Male , Middle Aged , Paraffin Embedding , Sarcoma/diagnosis , Tissue Fixation , Young AdultABSTRACT
The somatostatin receptor subtype 2 (sst2) behaves as a tumor suppressor when expressed and stimulated by its ligand somatostatin in pancreatic cancer. We reveal a mechanism underlying oncosuppressive action of sst2, whereby this inhibitory receptor upregulates the expression of the secreted angioinhibitory factor thrombospondin-1 (TSP-1), as demonstrated in exocrine BxPC-3 and endocrine BON pancreatic cancer cells. The sst2-dependent upregulation of TSP-1 occurs through the inhibition of the PI3K pathway. It depends on transcriptional and translational events, involving a previously undescribed IRES in the 5'-UTR of TSP-1 mRNA. Chick chorioallantoic membrane was used as an in vivo model to demonstrate that TSP-1 is a critical effector of the inhibitory role of sst2 on the neoangiogenesis and oncogenesis induced by pancreatic cancer cells. TSP-1 reduced in vitro tubulogenesis of endothelial cells when grown in conditioned medium from pancreatic cancer cells expressing sst2, as compared to those expressing the control vector. TSP-1 inhibited tumor cell-induced neoangiogenesis by directly sequestering the proangiogenic factor VEGF, and inactivating the angiogenesis initiated by VEGFR2 phosphorylation in endothelial cells. Using human pancreatic tissue-microarrays, the expression of both sst2 and TSP-1 was shown to be correlated during the pancreatic neoplastic program. Both proteins are nearly undetectable in normal exocrine pancreas and in most invasive cancer lesions, but their expression is strikingly upregulated in most preinvasive cancer-adjacent lesions. The upregulation of both sst2 and TSP-1 tumor suppressors may function as an early negative feedback to restrain pancreatic carcinogenesis.
Subject(s)
Pancreatic Neoplasms/physiopathology , Receptors, Somatostatin/physiology , Thrombospondin 1/physiology , Tumor Suppressor Proteins/physiology , Animals , Cell Line, Tumor , Chick Embryo , Gene Expression Profiling , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/prevention & control , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/genetics , Phosphatidylinositol 3-Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Neoplasm/genetics , RNA, Neoplasm/metabolism , Receptors, Somatostatin/genetics , Thrombospondin 1/genetics , Transplantation, Heterologous , Tumor Suppressor Proteins/genetics , Up-Regulation , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Inhibitor of apoptosis proteins (IAPs) regulate apoptosis and modulate NF-κB signaling thereby driving expression of genes involved in immune/inflammatory responses. The orally available IAP antagonist Debio 1143 has potential to enhance tumor response to chemoradiotherapy and/or immunotherapy. Patients with pre-operative squamous cell carcinomas of the head and neck (SCCHN) received: Debio 1143 monotherapy (200 mg/day [D]1-15 +/- 2); Debio 1143 (200 mg/day D1-15 +/- 2) plus cisplatin (40 mg/m2 D 1 and 8); cisplatin alone (40 mg/m2 D 1 and 8; EudraCT: 2014-004655-31). Pharmacokinetic/pharmacodynamic effects were assessed in plasma and resected tumors. Primary end point; effect of Debio 1143 on cellular IAP-1 (cIAP-1). Levels of cIAP-1/-2, X-linked inhibitor of apoptosis protein (XIAP), tumor infiltrating lymphocytes (TILs), including CD8+ T cells, programmed cell death protein 1 (PD-1), PD-ligand 1 (PD-L1), and gene expression were also analyzed. Twenty-three of 26 patients completed treatment. In the Debio 1143 monotherapy cohort (n = 13), mean tumor concentrations of Debio 1143 were 18-fold (maximum 55.2-fold) greater than in plasma, exceeding the half-maximal inhibitory concentration for cIAPs and XIAP by 100 to 1000-fold, with significant engagement/degradation of cIAP-1 (p < 0.05). Overall, levels of CD8+ TILs, PD-1, and PD-L1 positive immune cells increased significantly (p < 0.05) following Debio 1143 treatment. Changes were observed in the expression of genes related to NF-κB signaling. Treatments were well-tolerated. Debio 1143 penetrated SCCHN tumors, engaged cIAP-1, and induced immune inflammatory changes in the tumor microenvironment. Based on the mode of action demonstrated here and in previous studies, these data support future combinations of Debio 1143 with immune-checkpoint agents.
Subject(s)
Inhibitor of Apoptosis Proteins/pharmacology , Inhibitor of Apoptosis Proteins/pharmacokinetics , Squamous Cell Carcinoma of Head and Neck/drug therapy , Tumor Microenvironment/drug effects , Clinical Trials as Topic , Cohort Studies , Humans , Inhibitor of Apoptosis Proteins/administration & dosage , PharmacogeneticsABSTRACT
The receptor tyrosine kinase VEGFR-3 plays a crucial role in cancer-induced angiogenesis and lymphangiogenesis, promoting tumor development and metastasis. Here, we report the novel VEGFR-3 inhibitor EVT801 that presents a more selective and less toxic profile than two major inhibitors of VEGFRs (i.e., sorafenib and pazopanib). As monotherapy, EVT801 showed a potent antitumor effect in VEGFR-3-positive tumors, and in tumors with VEGFR-3-positive microenvironments. EVT801 suppressed VEGF-C-induced human endothelial cell proliferation in vitro and tumor (lymph)angiogenesis in different tumor mouse models. In addition to reduced tumor growth, EVT801 decreased tumor hypoxia, favored sustained tumor blood vessel homogenization (i.e., leaving fewer and overall larger vessels), and reduced important immunosuppressive cytokines (CCL4, CCL5) and myeloid-derived suppressor cells (MDSC) in circulation. Furthermore, in carcinoma mouse models, the combination of EVT801 with immune checkpoint therapy (ICT) yielded superior outcomes to either single treatment. Moreover, tumor growth inhibition was inversely correlated with levels of CCL4, CCL5, and MDSCs after treatment with EVT801, either alone or combined with ICT. Taken together, EVT801 represents a promising anti(lymph)angiogenic drug for improving ICT response rates in patients with VEGFR-3 positive tumors. Significance: The VEGFR-3 inhibitor EVT801 demonstrates superior selectivity and toxicity profile than other VEGFR-3 tyrosine kinase inhibitors. EVT801 showed potent antitumor effects in VEGFR-3-positive tumors, and tumors with VEGFR-3-positive microenvironments through blood vessel homogenization, and reduction of tumor hypoxia and limited immunosuppression. EVT801 increases immune checkpoint inhibitors' antitumor effects.
Subject(s)
Neoplasms , Vascular Endothelial Growth Factor Receptor-3 , Humans , Mice , Animals , Vascular Endothelial Growth Factor Receptor-3/therapeutic use , Neovascularization, Pathologic/drug therapy , Neoplasms/drug therapy , Angiogenesis Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Immunotherapy , Tumor MicroenvironmentABSTRACT
The clinical use of cytotoxic agents is plagued by systemic toxicity. We report a novel approach that seeks to design a "combi-molecule" to behave as an alkylating agent on its own and to undergo acid-catalyzed conversion to two bioactive species at a pH range akin to that of a tumor microenvironment: an AL530 prototype was synthesized and we studied its ability to release a chlorambucil analogue (CBL-A) plus a potent mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor (PD98059) at different pHs in buffered solutions, plasma and tumors. Its potency was compared in vitro with CBL+PD98059 (SRB assay) and in vivo in a xenograft model. Its target modulation was studied by western blotting and immunohistochemistry. AL530 released PD98059+CBL-A at mild acidic pH and in vitro was fivefold more potent than CBL and three-to-fivefold more potent than CBL+PD98059. In vivo it released high levels of PD98059 in tumors with a tumor/plasma ratio of five. It induced γ-H2AX phosphorylation and blocked pErk1,2, indirectly indicating its ability to damage DNA and modulate MEK. It induced significant tumor delay and less toxicity at unachievable doses for CBL and CBL+PD98059. We demonstrated the feasibility of a pH-labile combi-molecule capable of delivering high MEK inhibitor concentration in tumors, damaging DNA therein, and inducing tumor growth delay.