Awesome

Automatic assessment of LNEN tumor proliferative active with Pathonet:

Supervised deep learning network dedicated to the detection of Ki-67 or PHH3 positive cells on immunostained whole slide images (WSI). This network is an adaptation of the Pathonet model for pulmonary neuroendocrine neoplasms (LNEN); it classifies cells according to two classes, either negative or positive to an immunomarker. This directory also allows the creation of spatial statistics based on graph construction, as proposed by Bullloni and colleagues See : Automated analysis of proliferating cells spatial organization predicts prognosis in lung neuroendocrine neoplasms, Cancers 2021

• Original article for the deep learning framework: F. Negahbani Pathonet, SCI REPORT 2021.
• Original code: https://github.com/SHIDCenter/PathoNet
• Method used for the automated measurements of Ki-67 and PHH3 indices in "Assessment of the current and emerging criteria for the histopathological classification of lung neuroendocrine tumours in the lungNENomics project." ESMO Open 2023 (under review)

Installation

• Clone this repository: tested on Python 3.6.13
• Install tensorflow-gpu: tested on v1.13.1
• Install keras tested on v2.2.4
• Install cudatoolkit tested on v10.0
• Install cudnn tested on v7.6.5
• Install networkx tested on v2.5.1
• Install opencv-python tested on v4.1.1.26
• Install scikit-image tested on 0.16.2
• Install scikit-learn tested on 0.24.2
• Install pillow tested on 8.4.0
• Install any version of pandas, numpy, matplotlib, scipy
• For simplicity FrEIA Flows: tested on the recent branch has already be cloned in this repository
• Other dependencies in environment.yml

Install all packages with this command:

$ conda env create -f environment.yml

Datasets

This method has been tested for 2 types of immunostained WSI:

• Ki-67:
  • Number of LNEN Ki-67 annotated tiles = 848 (5 patients)
  • To train Pathonet these tiles have been combined to the breast tumor annoted tiles, from the SHIDC-B-Ki-67 data set used in the original paper.
• PHH3:
  • Number of LNEN PHH3 annotated tiles = 2375 (21 patients)
• LNEN tiles have autotated semi-automatically using the QuPath software.

These two dataset are available on request from mathiane[at]iarc[dot]who[dot]int and will soon be available online.

Step 1: Tiles preprocessing

• Convert annotation files listing all cells on each tile, with their coordinates and class (positive or negative for an immunolabel) in .json format to matrices saved in .npy format.
• Command line:

python preprocessin.py --inputdir PPH3Dataset/train256 --outputdir PPH3DatasetPrepocessed/train256

Step 2: Training

• An example of the configurations used to train the model to automatically measure Ki-67 expression is given in configs/train_Ki67_LNEN.json; similarly an example for the detection of PHH3 positive cells is given in configs/train_PHH3_LNEN.json.
• The command below is used to train the model:

python train.py --configPath configs/train_Ki67_LNEN.json

• The trained model weights for Ki-67-stained LNEN WSIs is stored in the file CheckpointKi67/Pathonet_Ki67_for_LNEN.hdf5, and for PHH3-stained LNEN WSIs in CheckpointPHH3/CheckpointPHH3_70epochs.hdf5

Step 3: Test

• An example of the configurations used to test the model is given in configs/eval_Ki67_LNEN.json
• The command below is used to evaluate the model:

python evaluation.py --inputPath test256_LNENonly --configPath configs/eval_Ki67_LNEN.json 

Step 4: Optimize the cell detection threshold

• The post-processing pipeline applied after UNET uses two thresholds to establish which are the "true cells". We propose to optimize these thresholds for marker-positive and marker-negative cells using the eval_opt_thresholds.py script.
• The following command is used to run the script for channel 0, which is associated with cells detected as marker positive:

python eval_opt_thresholds.py --inputPath test256 --configPath configs/eval_Ki67_LNEN.json --dataname OptTh0_pos_cells.csv --minth 75 --minth 95 --channel 0

• Network performance is tested for all thresholds between minth and minth in steps of 5 units.
• Results are saved in the table specified by dataname.
• To optimize the threshold associated with cells detected as negative at the marker, specify --channel 1.

Step 5: Inference

• The infer.py script is used to run the model in inference mode.
• Command line

python infer.py --inputPath KI67_Tiles_256_256_40x/ --configPath configs/eval_Ki67_LNEN.json --outputPath Inference --save_numpy --visualization

• The inputPath folder must have the following structure:
  • inputPath
    • patient_ID
      • accept
        • patient_id_tiles_x_y.jpg
• The inference script saves the inference results for each tile in three formats:
  • json file (default)
  • npy numpy matrix if the --save_numpy argument is specified
  • jpg annotated image if the --visualization argument is specified
• The outputPath folder will have the same organization as the inputPath folder.

Step 6: Calculate spatial statistics:

Step 6.1: Create a table of detected cells within the tumour area

-In order to create the graph needed to compute the spatial statistics, we first need to create a table of detected cells within the tumour area, with their xy coordinates and class.

• For this we assume that the tumour has been previously segmented as described in TumorSegmentationCFlowAD github repository.
• Command line:

python table_of_cells_after_segmentation.py --inputdir ~/LNENWork/Ki67InferencePathonet --patient_id TNE1983 --segmentation_dir TumorSegmentation_Ki67

• The segmentation_dir should follow the following architecture:
  • segmentation_dir
    • patient_id
      • prediction_tumor_normal_{patient_id}.csv
• For example the table prediction_tumor_normal_TNE1983.csv contains the following information:

• The output table is stored in inputdir/patient_id/{patient_id}_cells_detected_segmented.csv and will contains the following information:

Note: In this table, the label 1 corresponds to a positive cell and 2 to a negative cell.

Step 6.2: Compute sptatial metrics according to graph theory

• • The graph_theory_analysis.py script can be used to create a graph of the positive cells detected by Pathonet by connecting all the cells in a 2000 micron^2 area, according to which global and local spatial statistics are calculated.
• Command line:

python graph_theory_analysis.py --rootdir /LNENWork/Ki67InferencePathonet --patient_id TNE1983

• This script generated the following output files in the rootdir/patient_id folder:
  • {patient_id}_2000_micron.gpickle: graph
  • {patient_id}_graph_2000_micron_global_features.json: global spatial statistics
  • {patient_id}_graph_2000_micron_local_features_segmented.csv: local spatial statistics