Diagnostic Performance of Immunohistochemistry Compared to Molecular Techniques for Microsatellite Instability and p53 Mutation Detection in Endometrial Cancer.

Molecular algorithms may estimate the risk of recurrence and death for patients with endometrial cancer (EC) and may impact treatment decisions. To detect microsatellite instabilities (MSI) and p53 mutations, immunohistochemistry (IHC) and molecular techniques are used. To select the most appropriate method, and to have an accurate interpretation of their results, knowledge of the performance characteristics of these respective methods is essential. The objective of this study was to assess the diagnostic performance of IHC versus molecular techniques (gold standard). One hundred and thirty-two unselected EC patients were enrolled in this study. Agreement between the two diagnostic methods was assessed using Cohen's kappa coefficient. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) of the IHC were calculated. For MSI status, the sensitivity, specificity, PPV and NPV were 89.3%, 87.3%, 78.1% and 94.1%, respectively. Cohen's kappa coefficient was 0.74. For p53 status, the sensitivity, specificity, PPV, and NPV were 92.3%, 77.1%, 60.0% and 96.4%, respectively. Cohen's kappa coefficient was 0.59. For MSI status, IHC presented a substantial agreement with the polymerase chain reaction (PCR) approach. For the p53 status, the moderate agreement observed between IHC and next generation sequencing (NGS) methods implies that they cannot be used interchangeably.

Nanofitin as a New Molecular-Imaging Agent for the Diagnosis of Epidermal Growth Factor Receptor Over-Expressing Tumors.

Epidermal growth-factor receptor (EGFR) is involved in cell growth and proliferation and is over-expressed in malignant tissues. Although anti-EGFR-based immunotherapy became a standard of care for patients with EGFR-positive tumors, this strategy of addressing cancer tumors by targeting EGFR with monoclonal antibodies is less-developed for patient diagnostic and monitoring. Indeed, antibodies exhibit a slow blood clearance, which is detrimental for positron emission tomography (PET) imaging. New molecular probes are proposed to overcome such limitations for patient monitoring, making use of low-molecular-weight protein scaffolds as alternatives to antibodies, such as Nanofitins with better pharmacokinetic profiles. Anti-EGFR Nanofitin B10 was reformatted by genetic engineering to exhibit a unique cysteine moiety at its C-terminus, which allows the development of a fast and site-specific radiolabeling procedure with 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM). The in vivo tumor targeting and imaging profile of the anti-EGFR Cys-B10 Nanofitin was investigated in a double-tumor xenograft model by static small-animal PET at 2 h after tail-vein injection of the radiolabeled Nanofitin 18F-FBEM-Cys-B10. The image showed that the EGFR-positive tumor (A431) is clearly delineated in comparison to the EGFR-negative tumor (H520) with a significant tumor-to-background contrast. 18F-FBEM-Cys-B10 demonstrated a significantly higher retention in A431 tumors than in H520 tumors at 2.5 h post-injection with a A431-to-H520 uptake ratio of 2.53 ± 0.18 and a tumor-to-blood ratio of 4.55 ± 0.63. This study provides the first report of Nanofitin scaffold used as a targeted PET radiotracer for in vivo imaging of EGFR-positive tumor, with the anti-EGFR B10 Nanofitin used as proof-of-concept. The fast generation of specific Nanofitins via a fully in vitro selection process, together with the excellent imaging features of the Nanofitin scaffold, could facilitate the development of valuable PET-based companion diagnostics.

Soluble factors regulated by epithelial-mesenchymal transition mediate tumour angiogenesis and myeloid cell recruitment.

Epithelial-mesenchymal transition (EMT) programmes provide cancer cells with invasive and survival capacities that might favour metastatic dissemination. Whilst signalling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumour cells and the tumour microenvironment remains elusive. We aimed to identify EMT-regulated soluble factors that facilitate the recruitment of host cells in the tumour. Our findings indicate that EMT phenotypes relate to the induction of a panel of secreted mediators, namely IL-8, IL-6, sICAM-1, PAI-1 and GM-CSF, and implicate the EMT-transcription factor Snail as a regulator of this process. We further show that EMT-derived soluble factors are pro-angiogenic in vivo (in the mouse ear sponge assay), ex vivo (in the rat aortic ring assay) and in vitro (in a chemotaxis assay). Additionally, conditioned medium from EMT-positive cells stimulates the recruitment of myeloid cells. In a bank of 40 triple-negative breast cancers, tumours presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumours with little or no EMT. Taken together, our results show that EMT programmes trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favour cancer spread.

Digenic Inheritance of Mutations in Homologous Recombination Genes in Cancer Patients.

BACKGROUND/OBJECTIVES: BRCA1, BRCA2, ATM, and CHEK2 are known cancer predisposition genes (CPGs), but tumor risk in patients with simultaneous pathogenic variants (PVs) in CPGs remains largely unknown. In this study, we describe six patients from five families with multiple cancers who coinherited a combination of PVs in these genes. METHODS: PVs were identified using NGS DNA sequencing and were confirmed by Sanger. RESULTS: Families 1, 2, and 3 presented PVs in BRCA2 and ATM, family 4 in BRCA2 and BRCA1, and family 5 in BRCA2 and CHEK2. PVs were identified using NGS DNA sequencing and were confirmed by Sanger. The first family included patients with kidney, prostate, and breast cancer, in addition to pancreatic adenocarcinomas. In the second family, a female had breast cancer, while a male from the third family had prostate, gastric, and pancreatic cancer. The fourth family included a male with pancreatic cancer, and the fifth family a female with breast cancer. CONCLUSIONS: The early age of diagnosis and the development of multiple cancers in the reported patients indicate a very high risk of cancer in double-heterozygous patients associated with PVs in HR-related CPGs. Therefore, in families with patients who differ from other family members in terms of phenotype, age of diagnosis, or type of cancer, the cascade testing needs to include the study of other CPGs.

Case Report Series: Aggressive HR Deficient Colorectal Cancers Related to BRCA1 Pathogenic Germline Variants.

Objective: The link between BRCA1 and homologous recombination deficiency (HRD) in cancer has gained importance with the emergence of new targeted cancer treatments, while the available data on the role of the gene in colorectal cancer (CRC) remain contradictory. The aim of this case series was to elucidate the role of known pathogenic BRCA1 variants in the development of early-onset CRC. Design: Patients were evaluated using targeted next generation sequencing, exome sequencing and chromosomal microarray analysis of the paired germline and tumor samples. These results were used to calculate the HRD score and the frequency of mutational signatures in the tumors. Results: Three patients with metastatic CRC were heterozygous for a previously known BRCA1 nonsense variant. All tumors showed remarkably high HRD scores, and the HRD-related signature 3 had the second highest contribution to the somatic pattern of variant accumulation in the samples (23% in 1 and 2, and 13% in sample 3). Conclusions: A BRCA1 germline pathogenic variant can be involved in CRC development through HRD. Thus, BRCA1 testing should be considered in young patients with a personal history of microsatellite stable CRC as this could further allow a personalized treatment approach.

Tumor Microenvironment Modifications Recorded With IVIM Perfusion Analysis and DCE-MRI After Neoadjuvant Radiotherapy: A Preclinical Study.

PURPOSE: Neoadjuvant radiotherapy (NeoRT) improves tumor local control and facilitates tumor resection in many cancers. Some clinical studies demonstrated that both timing of surgery and RT schedule influence tumor dissemination, and subsequently patient overall survival. Previously, we developed a pre-clinical model demonstrating the impact of NeoRT schedule and timing of surgery on metastatic spreading. We report on the impact of NeoRT on tumor microenvironment by MRI. METHODS: According to our NeoRT model, MDA-MB 231 cells were implanted in the flank of SCID mice. Tumors were locally irradiated (PXI X-Rad SmART) with 2x5Gy and then surgically removed at different time points after RT. Diffusion-weighted (DW) and Dynamic contrast enhancement (DCE) MRI images were acquired before RT and every 2 days between RT and surgery. IntraVoxel Incoherent Motion (IVIM) analysis was used to obtain information on intravascular diffusion, related to perfusion (F: perfusion factor) and subsequently tumor vessels perfusion. For DCE-MRI, we performed semi-quantitative analyses. RESULTS: With this experimental model, a significant and transient increase of the perfusion factor F [50% of the basal value (n=16, p<0.005)] was observed on day 6 after irradiation as well as a significant increase of the WashinSlope with DCE-MRI at day 6 (n=13, p<0.05). Using immunohistochemistry, a significant increase of perfused vessels was highlighted, corresponding to the increase of perfusion in MRI at this same time point. Moreover, Tumor surgical resection during this peak of vascularization results in an increase of metastasis burden (n=10, p<0.05). CONCLUSION: Significant differences in perfusion-related parameters (F and WashinSlope) were observed on day 6 in a neoadjuvant radiotherapy model using SCID mice. These modifications are correlated with an increase of perfused vessels in histological analysis and also with an increase of metastasis spreading after the surgical procedure. This experimental observation could potentially result in a way to personalize treatment, by modulating the time of surgery guided on MRI functional data, especially tumor perfusion.

Radiolabelling of lipid-based nanocarriers with fluorine-18 for in vivo tracking by PET.

Organic nanoparticles made out of biodegradable and biocompatible materials have attracted increased attention in the therapeutic and diagnostic fields. In this study, we attempted to explore a new radiolabelling chelating free strategy for biodegradable sphingomyelin nanometric emulsions with fluorine-18 (18F), a radioisotope regularly used in clinic. [18F]fluoride was produced by the cyclotron and was incorporated into 4-[18F]fluorobenzamido-N-ethylmaleimide ([18F]FBEM), which was coupled next to the emulsions previously functionalized with a thiol group, via inclusion of either a thiol-PEG-lipid (SH-PEG12-C18), or a peptide-PEG-lipid (Cys-Pro-Ile-Glu-Asp-Arg-Pro-Met-Cys-PEG8-C18) derivative. Radiolabelled emulsions were obtained in a rapid and efficient fashion through facile-conjugated chemistry without the use of organic solvents, and characterized in terms of size, polydispersity, surface charge, pH, and osmolarity. PET imaging and biodistribution studies in BALB/c mice allowed obtaining the pharmacokinetics of the radiolabelled emulsions and determining the clearance pathways. Altogether, we confirmed the potential of this new technique for the radiolabelling of lipid-based drug nanosystems for application in PET imaging diagnosis.

Impacts of Ionizing Radiation on the Different Compartments of the Tumor Microenvironment.

Radiotherapy (RT) is one of the most important modalities for cancer treatment. For many years, the impact of RT on cancer cells has been extensively studied. Recently, the tumor microenvironment (TME) emerged as one of the key factors in therapy resistance. RT is known to influence and modify diverse components of the TME. Hence, we intent to review data from the literature on the impact of low and high single dose, as well as fractionated RT on host cells (endothelial cells, fibroblasts, immune and inflammatory cells) and the extracellular matrix. Optimizing the schedule of RT (i.e., dose per fraction) and other treatment modalities is a current challenge. A better understanding of the cascade of events and TME remodeling following RT would be helpful to design optimal treatment combination.

New role of osteopontin in DNA repair and impact on human glioblastoma radiosensitivity.

Glioblastoma (GBM) represents the most aggressive and common solid human brain tumor. We have recently demonstrated the importance of osteopontin (OPN) in the acquisition/maintenance of stemness characters and tumorigenicity of glioma initiating cells. Consultation of publicly available TCGA database indicated that high OPN expression correlated with poor survival in GBM patients. In this study, we explored the role of OPN in GBM radioresistance using an OPN-depletion strategy in U87-MG, U87-MG vIII and U251-MG human GBM cell lines. Clonogenic experiments showed that OPN-depleted GBM cells were sensitized to irradiation. In comet assays, these cells displayed higher amounts of unrepaired DNA fragments post-irradiation when compared to control. We next evaluated the phosphorylation of key markers of DNA double-strand break repair pathway. Activating phosphorylation of H2AX, ATM and 53BP1 was significantly decreased in OPN-deficient cells. The addition of recombinant OPN prior to irradiation rescued phospho-H2AX foci formation thus establishing a new link between DNA repair and OPN expression in GBM cells. Finally, OPN knockdown improved mice survival and induced a significant reduction of heterotopic human GBM xenograft when combined with radiotherapy. This study reveals a new function of OPN in DNA damage repair process post-irradiation thus further confirming its major role in GBM aggressive disease.

The timing of surgery after neoadjuvant radiotherapy influences tumor dissemination in a preclinical model.

Neoadjuvant radiotherapy (neoRT) used in cancer treatments aims at improving local tumor control and patient overall survival. The neoRT schedule and the timing of the surgical treatment (ST) are empirically based and influenced by the clinician's experience. The current study examines how the sequencing of neoRT and ST affects metastatic dissemination. In a breast carcinoma model, tumors were exposed to different neoRT schedules (2x5Gy or 5x2Gy) followed by surgery at day 4 or 11 post-RT. The impact on the tumor microenvironment and lung metastases was evaluated through immunohistochemical and flow cytometry analyses. After 2x5Gy, early ST (at day 4 post-RT) led to increased size and number of lung metastases as compared to ST performed at day 11. Inversely, after 5x2Gy neoRT, early ST protected the mice against lung metastases. This intriguing relationship between tumor aggressiveness and ST timing could not be explained by differences in classical parameters studied such as hypoxia, vessel density and matrix remodeling. The study of tumor-related inflammation and immunity reveals an increased circulating NK cell percentage following neoRT as compared to non irradiated mice. Then, radiation treatment and surgery were applied to tumor-bearing NOD/SCID mice. In the absence of NK cells, neoRT appears to increase lung metastatic dissemination as compared to non irradiated tumor-bearing mice. Altogether our data demonstrate that the neoRT schedule and the ST timing affect metastasis formation in a pre-clinical model and points out the potential role of NK cells. These findings highlight the importance to cautiously tailor the optimal window for ST following RT.