Circulating hsa-miR-5096 predicts 18F-FDG PET/CT positivity and modulates somatostatin receptor 2 expression: a novel miR-based assay for pancreatic neuroendocrine tumors

Gastro-entero-pancreatic neuroendocrine tumors (GEP-NETs) are rare diseases encompassing pancreatic (PanNETs) and ileal NETs (SINETs), characterized by heterogeneous somatostatin receptors (SSTRs) expression. Treatments for inoperable GEP-NETs are limited, and SSTR-targeted Peptide Receptor Radionuclide Therapy (PRRT) achieves variable responses. Prognostic biomarkers for the management of GEP-NET patients are required. 18F-FDG uptake is a prognostic indicator of aggressiveness in GEP-NETs. This study aims to identify circulating and measurable prognostic miRNAs associated with 18F-FDG-PET/CT status, higher risk and lower response to PRRT. Methods Whole miRNOme NGS profiling was conducted on plasma samples obtained from well-differentiated advanced, metastatic, inoperable G1, G2 and G3 GEP-NET patients enrolled in the non-randomized LUX (NCT02736500) and LUNET (NCT02489604) clinical trials prior to PRRT (screening set, n= 24). Differential expression analysis was performed between 18F-FDG positive (n=12) and negative (n=12) patients. Validation was conducted by Real Time quantitative PCR in two distinct well-differentiated GEP-NET validation cohorts, considering the primary site of origin (PanNETs n=38 and SINETs n=30). The Cox regression was applied to assess independent clinical parameters and imaging for progression-free survival (PFS) in PanNETs. In situ RNA hybridization combined with immunohistochemistry was performed to simultaneously detect miR and protein expression in the same tissue specimens. This novel semi-automated miR-protein protocol was applied in PanNET FFPE specimens (n=9). In vitro functional experiments were performed in PanNET models. Results While no miRNAs emerged to be deregulated in SINETs, hsa-miR-5096, hsa-let-7i-3p and hsa-miR-4311 were found to correlate with 18F-FDG-PET/CT in PanNETs (p-value:<0.005). Statistical analysis has shown that, hsa-miR-5096 can predict 6-month PFS (p-value:<0.001) and 12-month Overall Survival upon PRRT treatment (p-value:<0.05), as well as identify 18F-FDG-PET/CT positive PanNETs with worse prognosis after PRRT (p-value:<0.005). In addition, hsa-miR-5096 inversely correlated with both SSTR2 expression in PanNET tissue and with the 68Gallium-DOTATOC captation values (p-value:<0.05), and accordingly it was able to decrease SSTR2 when ectopically expressed in PanNET cells (p-value:<0.01). Conclusions hsa-miR-5096 well performs as a biomarker for 18F-FDG-PET/CT and as independent predictor of PFS. Moreover, exosome-mediated delivery of hsa-miR-5096 may promote SSTR2 heterogeneity and thus resistance to PRRT.


INSTALLATION
There are two ways to use the AND-Tool:  Source code: For who has a MATLAB licence, we provide the source code. In this case, it is enough to download the source code and extract the files from "ANDTool_v#.zip" to a custom folder. Then, 4 start MATLAB and select the folder containing the AND-Tool files. Upon typing "START_GUI" in the MATLAB Command Window the AND-Tool GUI will pop-up.
 Win-64 Standalone Version: To install and use the software from the standalone version you need the Administrator rights of the computer.
(b) You need an Internet connection active.
(c) Double-click on the file: "MyAppInstaller_web.exe" and follow the steps.
(d) Once the software is installed, to run the software: Double-click on the "AND_v_*_*_.exe" file, typically automatically generated in the "application" folder created in the selected path of the installation folder.

USAGE
The AND-Tool's GUI is designed for automatically analysing microscopy 2D images representing cells with nuclei stained using a nuclear-signal-of-interest, typically a DAB dye. The images considered are those acquired with a widefield microscope with a 20x objective and an embedded RGB camera, but adjusting the parameters is possible to analyse also images acquired using different objective lens..
The tool requires as input the: plus, the:  FastRed channel (i.e. mono-channel gray-level images)  FastBlue channel (i.e. mono-channel gray-level images)  nuclear-signal-of-interest (i.e. DAB) channel (mono-channel gray-level images) of each image to be analysed. Precisely, the tool requires to set in the first four fields of the GUI the path of the "RGB", "FastRed", "FastBlue" and "DAB" folders, containing the images to be analysed.
"RGB" image folder will be analysed. Accordingly, if your interest is to analyse a single specific image, include just that one in the folder set as "RGB" image folder.

IMAGE PRE-PROCESSING
 Vignetting correction: To improve the results is better to pre-process the original RGB images correcting for vignetting. One way is to use Fiji and run: "ImageJ/Fiji" -> "Process" -> "Subtract Background…" Note: set the "Rolling Ball Radius [pixels]" approximatively to the radius of the objects of interest.
 From "RGB" to "FastRed FastBlue DAB": as we wrote, the AND-Tool is designed for analysing RGB images acquired using a widefield microscope embedded with an RGB camera. To convert an RGB image into the 3 channels FastRed, FastBlue and DAB, you can use the Fiji function: "ImageJ/Fiji" -> "Image" -> "Colour Deconvolution" -> "FastRed FastBlue DAB" 6 07. GETTING STARTED AND-Tool is designed for automatically analysing microscopy 2D images representing cells with nuclei stained using a nuclear-signal-of-interest (e.g. DAB) dye.
The tool requires the as input the original RGB images plus, the FastRed channel, FastBlue channel, DAB channel of each image to be analysed. Accordingly, first of all, all the original RGB images are converted into the triple FastRed, FastBlue, and DAB channels using the "Colour Deconvolution" Fiji function.
All the images are then loaded and converted from the tool into 8-bits (i.e. intensity values from 0 to 255).
The tool then subdivides each image in ROIs defined using the FastRed channel and 2 thresholds defined by the user (hereafter named Th1 and Th1):  the "Dark-Red" ROIs, with intensity values of the FastRed channel between 0 and Th1  the "Light-Pink" ROIs, with intensity values of the FastRed channel between Th1 and Th2  the "White" ROIs, with intensity values of the FastRed channel between Th2 and 255.
Note that the ROIs are then smoothed with some morphological operations.
The nuclei are then segmented using the FastBlue channel. After that, the nuclear-signal-of-interest channel (typically the DAB images) is used to detect the nuclei positive to the dye of interest. Note that nuclei positive for the nuclear-signal-of-interest, but not for the FastBlue channel, will be considered as nuclei even if not present in the list of nuclei detected using the FastBlue channel. Furthermore, the images typically considered are those acquired with a widefield microscope with a 20x objective and an embedded RGB camera with a good quality nuclear-signal-of-interest channel, but adjusting the parameters is possible to analyse also images acquired using different objective lens and images with absent, faint and strong intensity of the nuclear-signal-of-interest channel (to understand better how to use the AND-Tool see the example in the following sections).
Finally, AND-Tool computes statistics, for instance the single nuclei intensity and frequency of nuclei in the different ROIs and saves as output a table reporting for each nucleus the mean intensity value.

GUI'S PARAMETERS
What follows is the list of parameters that can be set using the AND-Tool's GUI:  "RGB images -path": to define the absolute path of the folder containing the RGB images (i.e. RGBtype images) to be processed.
 "FastRed images -path": to define the absolute path of the folder containing the FastRed images (i.e. mono-channel gray-level images typically obtained using the Colour Deconvolution plugin of Fiji) to be processed.
 "FastBlue images -path": to define the absolute path of the folder containing the FastBlue images (i.e. mono-channel gray-level images typically obtained using the Colour Deconvolution plugin of Fiji) to be processed.
 "CHANNEL-OF-INTEREST images -path": to define the absolute path of the folder containing the nuclear-signal-of-interest images (typically, the DAB channel in mono-channel gray-level image format typically obtained using the Colour Deconvolution plugin of Fiji) to be processed.
 "Output Folder -path": to define the absolute path of the folder then used from the tool to save the output tables and images.
 "Th1 Red": Threshold value (Th1) value defining the highest level for the "Dark-Red" ROIs when analysing the FastRed images. The value must be a positive value between 1 and 255.
 "Th2 Pink": Threshold value (Th2) value defining the highest level for the "Light-Pink" ROIs when analysing the FastRed images. The value must be a positive value between 1 and 255 and must be higher than Th1.
 "Th3 Foreground Intensity": Threshold value (Th3) defining the maximum intensity value to be considered when defining the nuclei in the nuclear-signal-of-interest images. All pixels with original intensity value higher than this threshold are excluded. The value must be a positive value between 1 and 255.
 "Small ROI": Threshold value defining the smaller number of pixels considered when defining the red, pink and white ROIs. The value must be a positive value between 1 and 100000 pixels.
 "Small Nuclei": Threshold value defining the smaller number of pixels considered when detecting the nuclei. The value must be a positive value between 1 and 100000 pixels.
 "Large Nuclei": Threshold value defining the maximum number of pixels considered when detecting the nuclei. The value must be a positive value between 1 and 100000.
 "Channel selected for intensity statistics": Image from which to extract the intensity value considered for each segmented nucleus. Currently, two options are available: (a) "Intensity values extracted from the original RGB images gray converted"; (b) "Intensity values extracted from the single FastRed-FastBlue-Other channels".

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If the first option is selected, a gray conversion of the original RGB images will be used to extract the intensity value. If the second option is selected, the FastBlue images are used for extracting the intensity value of the blue nuclei, and the nuclear-signal-of-interest images are used to extract the intensity value for the other nuclei.
 "Visualize output": If enabled, the temporary images obtained during computation are visualised during the processing of the data.
 "Nuclei channel intensity level": To adjust the threshold between background and foreground of the nuclear-signal-of-interest channel (i.e. DAB images): (a) "Faint (but present) signal" enables a series of filters to increase the threshold between background and foreground and consider more bright spots as foreground; (b) "Strong or absent signal" (default) considers as foreground just the darker spots of the nuclearsignal-of-interest images.

NOTE OF USAGE
What follows is the general scheme of usage of the AND-Tool: For instance, in case of a strong nuclear-signal-of-interest: 9 RGB DAB or an absent nuclear-signal-of-interest: RGB DAB run the AND-Tool simply by setting the "Nuclei channel intensity level" to "Strong or absent signal".
In case of a faint nuclear-signal-of-interest: RGB DAB 10 run the AND-Tool by setting the "Nuclei channel intensity level" to "Faint (but present) signal" and carefully set Th3 to a level appropriate to exclude from the foreground the pixels with an high intensity value (otherwise wrongly considered as a correct nuclear signal).
In case of a RGB images presenting positive nuclei, showing a strong dark intensity for the nuclear-signal-ofinterest, but a practically absent signal in the specific nuclear-signal-of-interest images probably for problem occurred when using the Colour Deconvolution plug-in, a possible solution is to set for the "CHANNEL-OF-INTEREST images -path" parameter the folder of the original "RGB images" (that internally will be automatically converted in grey-levels when used for evaluating the nuclear-signal-of-interest) and then carefully set Th3 to a level appropriate to exclude from the foreground the pixels with an high intensity value (otherwise wrongly considered as a correct nuclear signal).

CONTACTS
By reading this manual you got a more complete overview of the various analysis options offered by the AND-Tool. Please, also visit the AND-Tool website: http://sourceforge.net/p/andtool, and If you need 11 further information or you have special requests, remember that we are open to collaborations! In that case contact Filippo Piccinini at: filippo.piccinini85@gmail.com