Diffusion MRI for following tumor modifications after neoadjuvant radiotherapy.

Neoadjuvant radiotherapy (NeoRT) improves tumor local control and tumor resection in many cancers. The timing between the end of the NeoRT and surgery is driven by the occurrence of side effects or the tumor downsizing. Some studies demonstrated that the timing of surgery and the RT schedule could influence tumor dissemination and subsequently patient overall survival. We demonstrated the impact of NeoRT on metastatic spreading in a Scid mice model. After an irradiation of 2x5gy, we show more metastasis in the lung when the mice are operated at day 4 compared to day 11. Here, our aim is to evaluate with functional MRI (fMRI) the impact of the radiation treatment on the tumor microenvironment and subsequently to identify non-invasive markers helping to determine the best timing to perform surgery for avoiding tumor spreading

Tumor modifications recorded with IVIM and DCE-MRI after Neoadjuvant radiotherapy.

Purpose or Objective Neoadjuvant radiotherapy (NeoRT) improves tumor local control and facilitates tumor resection in many cancers. We hypothesized anti-cancer treatments (i.e. radiotherapy) modify tumor microenvironment and could potentially impact distant metastases occurrence. Previously, we developed a pre-clinical model demonstrating an impact of NeoRT schedule and the timing of surgery on metastatic spreading (Leroi et al. Oncotarget 2015). Here, we aim to identify by fMRI noninvasive markers reflecting NeoRT related tumor microenvironment modifications that could predict the best timing for performing surgery and avoiding tumor spreading. Material and Methods To briefly delineate the NeoRT model, MDA-MB 231 tumor cells implanted in the flank of SCID mice were locally irradiated with 2x5Gy when tumor reached 100mm3 and then surgically removed at different time points. We performed fMRI, Diffusion Weighted (DW) and Dynamic Contract enhancement (DCE) – MRI, before RT and every 2 days between RT and surgery. We acquired 8 slices of 1 mm thickness and 0.5 mm gap with an “in plane voxel resolution” of 0.5 mm. For DW-MRI, we performed FSEMS (Fast Spin Echo MultiSlice) sequences, with 9 different Bvalue (from 40 to 1000) and B0. We performed IVIM (IntraVoxel Incoherent Motion) analysis to obtain information on intravascular diffusion, related to perfusion (F: perfusion factor) and subsequently tumor vessels perfusion. For DCE-MRI, we performed a T1 mapping with multiple TR and DCE acquisition with 200 repetitions of 3 sec each and gadolinium IV injection after 10 repetitions. We performed semi-quantitative analysis. We validated tumor perfusion by immunochemistry with injection of FITC-dextran IV 3 min before surgery and CD31 labelling. Human Ki67 was used for lung metastases labelling and quantification. Results After the tumor irradiation, we observed a significant and transient increase at day 6 (60% of the basal value (n=6, p<0,05)) of F and D* parameters related to perfusion. The other parameters of the DW-MRI, ADC and D presented no modifications. The sham irradiated tumors used as control showed no modifications of all fMRI parameters. At the same timing, 6 days post-radiotherapy, DCE-MRI significantly demonstrated a WhashinSlope (n=13, p<0,05) increase. Immunochemistry confirmed the increase of tumor perfusion when surgery is performed at day 6. The sham irradiated tumors never demonstrated such changes. Finally, when surgery is performed on tumor increased perfusion measured by fMRI, it demonstrated a burst of lung metastasis compared to the other timings. Conclusion We showed a significant difference in perfusion-related parameters with fMRI and immunochemistry at a specific time point after NeoRT. These modifications are correlated with an increase of metastasis spreading related to surgery procedure. These results open new perspectives in the personalized medicine and MRI guided surgery timing after NeoRT

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

peer reviewedEpithelial-to-mesenchymal transition (EMT) programs provide cancer cells with invasive and survival capacities that might favor metastatic dissemination. Whilst signaling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumor cells and the tumor microenvironment remains elusive. We aimed to identify EMT-regulated soluble factors that facilitate the recruitment of host cells in the tumor. 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, tumors presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumors with little or no EMT. Taken together, our results show that EMT programs trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favor cancer spread

Digenic Inheritance of Mutations in Homologous Recombination Genes in Cancer Patients

peer reviewedBackground/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.WALGEMED (WALlonia GEnomic MEDicine

Impact of neoadjuvant radiotherapy and the timing of surgery on the tumor microenvironment and metastatic spreading.

Neoadjuvant radiotherapy (NeoRT) is used in many types of cancers and aims at improving tumor local control and patient overall survival. The RT schedule and the timing of surgery are mostly empirically based on clinical experiences and side effect occurrence. However, a clinical study revealed that the timing of surgery following neoRT might be crucial for patient overall survival. Therefore, we hypothesized that RT may influence the tumor phenotype as well as the tumor microenvironment (TME) and consequently metastasis formation. As a “proof of concept”, we developed a pre-clinical model to study the impact of different RT schedules and timings of surgery on TME and metastatic dissemination and search for predicting markers thanks to metabolic analyses.To mimic neoRT used in clinic, we subcutaneously injected human cancer cells (MDA-MB-231) into the flank of SCID mice. When tumors reached an appropriate volume, we locally irradiated the primary tumor with different neoRT schedules (5x2Gy and 2x5Gy) inspired from clinical practice but adapted to mice. We surgically removed carefully tumors 4 or 11 days after the end of RT and kept the mice alive during 6 weeks for metastatic growth. Tumor samples were collected for extractions and histological analyses. Lungs were harvested at the end of the experiments for metastatic burden analysis. The occurrence of lung metastases was totally different according to the neoRT schedule and the time of surgery. After 2x5Gy, the size and the number of lung metastases were smaller when surgery was performed at 11 days after the end of RT, compared to 4 days. Inversely, in the 5x2Gy schedule, applying surgery at 4 days protected the mice against lung metastases compared to surgery at 11 days. These results demonstrate that the timing of surgery and RT schedules are both important factors that influence the formation of metastases. To decipher RT-induced TME modifications, we studied tumor features known to be implicated in tumor progression, such as hypoxia, blood vessel density, matrix metalloproteinases, adipocyte-like cells, inflammation. Although none of the observed RT-induced TME modifications was directly related to the metastatic burden, we pointed out a regulation of NK cell recruitment by neoRT, affecting metastatic dissemination. Our second aim was to identify predictive markers of the course of the disease for optimizing treatment. Thanks to nuclear magnetic resonance analysis and powerful statistical tools, the study of primary tumors at the time of surgery showed different metabolic profiles according to the RT schedule. Moreover, a correlation between this metabolic profile and metastatic burden was observed. Altogether, we provide, for the first time, experimental clues that neoadjuvant RT schedule and the timing of surgery influence the metastatic profile. Although researches are needed to elucidate the underlying mechanisms, our work, which draws the attention on therapy schedule, has clinical implication. Moreover, nuclear magnetic resonance and discriminant analyses showed an impact of neoRT on TME, which could be related to the metastatic profile, and are of interest to be studied in clinic