Raport stiintific anual privind implementarea proiectului in perioada ianuarie – decembrie 2015

Clinical and Biomathematical Modeling of Vascular Changes Following

Anti-Angiogenic Therapy in Advanced Colorectal Carcinoma Activity report for January, 2015 - December 2015

Project Director: Dr. Gabriel Gruionu ABSTRACT: Colorectal carcinoma (CRC) represents an important health burden, being the third leading cause of cancer death in the world. The cancer-stromal cell interaction contributes directly to tumor growth and metastasis by creating an imbalance of positive and negative growth factors and increased microvascular density via angiogenesis. Recently, advances in chemotherapy and biological therapies targeting the angiogenic growth factors offered additional hope for treatment but the exact biological mechanisms are still not clear. Therefore, the long-term goal of the proposed research is to gain improved understanding of the mechanisms by which tumor microvascular networks (MVN) respond to chemotherapy and/or anti-angiogenic therapy. To address this, the first objective is to map the MVN and cellular components of normal colorectal tissue and CRC tumors before and after chemotherapy and/or anti-angiogenic treatment in CRC patients. Changes in morphometric and hemodynamic parameters of MVN will be observed with a novel combination of minimally invasive ultrasound and confocal imaging techniques (CE-EUS and CLE) correlated with the type and dosage of therapy. The second objective is to develop computer simulations of blood flow and structural adaptation of the MVN in CRC tumors. The predictions of the model will be compared with the observed changes in the MVN structure following chemo- and anti-angiogenic treatment. The project will have a significant scientific and social impact leading to a better categorization and prediction of patient’s response to treatment before exposure to chemotherapy or surgery. Specific activity calendar:

Activity A 1.1 Set-up of a structured database and definition of the experimental models Progress: A complex database was set up on a dedicated computer and back up to an external hard drive to include all the identifying information (age, ID, location, sex), imaging procedure, biopsy location, diagnosis, treatment dosage and final evaluation. Since 2014 10 new patients were enrolled in the study for the contrast ultrasound study and were added to the existing database. The details about the study are described under A2.1 update below. A1.2. Protocol based inclusion of patients (CE-EUS and CLE) and data sampling. Progress: Up to date, over 60 patients were enrolled in the study (age 49-76 years). The consent forms that every patient has to sign before the beginning of the study were attached to the previous report.

M1-3 M4-6 M7-9 M10-11 M12-14 M15-17 M18-20 M21-23 M24-26 M27-29 M30-32 M33-35

A 3.1 Development of the mathematical model of normal and untreated CNC MVN

A 4.2 Forecasting angiogenesis during and after antiangiogenic treatment

A 1.1 Set-up of a structured database and definition of the experimental models

A 1.2 Protocol-based inclusion of patients (CE-EUS & CLE), tissue sampling

A 2.1 Pathological and immunohistochemical analysis of biopsy samples

A 2.3 Mathematical model of the anti-angiogenic treated MVN

For CLE examination we use the dedicated system (EC-3870 CIFK, Pentax, Tokyo, Japan), which has a miniature confocal microscope integrated into the distal tip of a conventional flexible endoscope. The endomicroscope uses a laser bean with an excitation wavelength of 488 nm and a maximum laser power output at the surface of the targeted tissue of ≤1mW. This results in optical sections of 475x475µm2 with a lateral resolution of 0.7 µm for a 7 µm thick slice (z-axis). CLE imaging is performed on fresh biopsies of both normal mucosa and colorectal cancer tissue obtained during lower GI endoscopic procedures. The biopsies undergo a staining protocol, including incubation (1h, 37°C, 1:10 dilution) with Alexa-Fluor 488 labeled anti-CD31 antibodies. Scanning is performed with the biopsies on histology glass slides, placed in direct and gentle contact with the distal tip of the confocal laser endomicroscope. Consecutive images are captured and digitally stored on the system’s hard drive as grey-scale images (250-300 for each biopsy sample) for later download and processing. The imaging procedures are backed by immunohistochemical studies of corresponding biopsies for evaluating tumor vascular patterns and performing a thorough morphometric analysis. Compared to the previous 2012 article published in PLoS One, the new article also published in 2014 in PLoS One introduced new morphometric parameters and demonstrated for the first time that tumor vessels are not actually more tortuous than the normal tissue vessels (see abstract below) [1]. A2.1. Pathological and immunohistological analysis of biopsies. Progress: We continued the analysis of the biopsies using new and improved techniques for detecting tumor vasculature. We have identified new markers which are more specific to tumor endothelium than the traditional markers. We use CD105 to identify the vessels which are new and specific to tumors vs. the host vessels. Experimental Protocol

Subjects

Tissue specimens from ten patients between the ages of 45–70 years old, with histologically proven rectal cancer, undergoing surgical resection at the Department of Surgery from Emergency County Hospital of Craiova, were collected during colonoscopy. The patient population contained stage II-III (according to AJCC staging system) rectal adenocarcinomas without metastatic spread and it is part of a cohort described before. Fresh tissues from these patients were immediately processed for both CLE and immunohistochemistry assessment. The study was conducted according to the Code of Ethics of the World Medical Association (Declaration of Helsinki, 1964, as revised in 2004) and approved by the local Ethics Committee. All the patients included read and accepted the written informed consent prior to study entry.

Confocal laser endomicroscopy

The biopsy samples obtained from standard colonoscopy (CFQ160ZL, Olympus,Tokyo, Japan) were processed following a standardized protocol. During the endoscopic procedure, for every patient, several biopsies were taken from tumor, avoiding the ulcerated areas as much as possible, as well as from macroscopically normal surrounding tissue samples. The biopsies were immersed immediately in 10% neutral buffered formalin for histopathological analysis, as well as in saline solution for the ex-vivo immunohistochemical processing. Samples from saline solution were thoroughly washed and incubated for one hour in the dark, at 37°C, with Alexa-Fluor 488-labeled anti-CD31 (PECAM) antibody (mouse anti-human IgG1, Exbio, Prague, Czech Republic) or respectively FITC-labeled anti-CD105/Endoglin antibody (mouse anti-human IgG2a, Exbio), diluted as 1:15 and 1:5 in saline with 1% bovine serum albumin (BSA, Sigma-Aldrich, Munich, Germany). Afterwards, the excess antibodies were washed away in saline and the samples were immediately visualized in confocal laser endomicroscopy imaging to assess the microvascularization ex vivo up to a maximum depth of 250 µm. Confocal laser endomicroscopy images were acquired using Pentax EC-3870 CIFK, Tokyo, Japan, a dedicated endomicroscopy system with an excitation wavelength of 488 nm and with a maximum laser power output of ≤1 mW at the surface of the tissue.

To assess both endothelial markers more accurately, we used the color overlay function in the ImageJ image processing software (National Institutes of Health, USA). This software was used to obtain the Z projection of the confocal serial images from each biopsy sample previously combined into stacks. Thus, on the acquired images, Z-stacks and 3D reconstruction was performed, then the vascular density and the vessel diameters were measured within two 50x475 µm rectangular regions of interest centered in the middle of each image in the horizontal and vertical direction.

The results of the study were published by our group in the World Journal of Gastrointestinal Oncology in 2015: “Tumor neoangiogenesis detection by confocal laser endomicroscopy and anti-CD105 antibody: Pilot study” [1]. Abstract AIM: To evaluate neoangiogenesis in patients with colon cancer by two fluorescently labeled antibodies on fresh biopsy samples imaged with confocal laser endomicroscopy (CLE). METHODS: CLE is an imaging technique for gastrointestinal endoscopy providing in vivo microscopy at subcellular resolution. An important question in validating tumor angiogenesis is what proportion of the tumor vascular network is represented by pre-existing parent tissue vessels and newly formed vessels. CD105 (endoglin) represents a proliferation-associated endothelial cell adhesion molecule. In contrast to pan-endothelial markers, such as CD31, CD105 is preferentially expressed in activated endothelial cells that participate in neovascularization. Thus, we evaluated CD105 and CD31 expression from samples of ten patients with primary rectal adenocarcinoma, using a dedicated endomicroscopy system. A imaging software was used to obtain the Z projection of the confocal serial images from each biopsy sample previously combined into stacks. Vascular density and vessel diameters were measured within two 50 µm x 475 µm rectangular regions of interest centered in the middle of each image in the horizontal and vertical direction. The results were averaged over all the patients and were expressed as the mean ± SE. RESULTS: The use of an anti-CD105 antibody was found to be suitable for the detection of blood vessels in colon cancer. Whereas anti-CD31 antibodies stained blood vessels in both normal and pathologic colon equally, CD105 expression was observed primarily in malignant lesions, with little or no expression in the vessels of the normal mucosa (244.21 ± 130.7 vessels/mm3 in only four patients). The average diameter of anti-CD105 stained vessels was 10.97 ± 0.6 µm in tumor tissue, and the vessel density was 2787.40 ± 134.8 vessels/mm3. When using the anti-CD31 antibody, the average diameter of vessels in the normal colon tissue was 7.67 ± 0.5 µm and the vessel density was 3191.60 ± 387.8 vessels/mm3, while in the tumors we obtained an average diameter of 10.88 ± 0.8 µm and a vessel density of 4707.30 ± 448.85 vessels/mm3. Thus, there were more vessels stained with CD31 than CD105 (P < 0.05). The average vessel diameter was similar for both CD31 and CD105 staining. A qualitative comparison between CLE vs immunohistochemistry lead to similar results. CONCLUSION: Specific imaging and quantification of tumor microvessels are feasible in human rectal cancer using CLE examination and CD105 immunostaining of fresh tissue samples. Another study with the aim of evaluating tumor vascularity in CRC by CE-EUS with time intensity curve (TIC) analysis backed by assessment of molecular markers of angiogenesis. The results of the study were presented at the Digestive Disease Week conference in Washington, DC and submitted for publication in the Gastrointestinal Endoscopy journal which is indexed ISI with an impact factor of 5.37. Study protocol: Inclusion criteria: Patients who were diagnosed with primary colon and rectal tumors, age 18-90 years old, men or women, signed informed consent for EUS with contrast- enhancement and tissue sampling. Exclusion criteria: prior treatment with chemo-radiotherapy, failure to provide informed consent and severe coagulopathy.

The EUS local staging and low mechanical index CE-EUS (MI 0.2) was performed using a radial EUS scope (EG-3670URK, Pentax, Hamburg, Germany) coupled with a Hitachi Preirus US system (Hitachi Medical Corp, Tokyo, Japan). For contrast we used a bolus injection 4.8 ml SonoVue® (Bracco. Italy). Sixty seconds video sequences were recorded on the embedded HDD of the ultrasound system for later analysis. A dedicated software was used for offline TIC analysis of the recorded video sequences (VueBox™,Bracco) TIC parameters are: PE - peak enhancement, RT - rise time, mTT - mean transit time, TTP - time to peak, AUC - area under the curve, a.u. arbitrary units. A sample of the results is shown Figure 1 with PE = 3.58 a.u. and AUC = 34.39 a.u.

Fig. 1. TIC measurements. The conclusions of the study were that the low mechanical index CE-EUS examination and TIC analysis enable the evaluation of tumour angiogenesis in CRC. Of all TIC parameters, PE and AUC may predict prognosis for CRC patients. Further studies are necessary for establishing the role of CE-EUS in the evaluation of CRC as well as for identifying an optimal combination of functional and molecular markers of prognostic significance. Activity 3.1. Development of the mathematical model for the normal and treated CRC MVN (colorectal cancer microvascular network). Aim of the study: To develop computer simulations of blood flow and structural adaptation of the MVN in CRC tumors. Progress: We are currently working on a mathematical model of incomplete networks as tumor vascular networks from biopsies are incomplete (Figure 2). For incomplete networks we are using a novel mathematical modeling procedure which uses an annealing function method, which helps us simulate the blood flow when the boundary conditions are not know. We are using a model of a normal microvascular network from the dorsal skinfold chamber to develop the mathematical model of the simulated annealing

method (SAM). Briefly, SAM finds a global minimum for an error function, E (in this case the number of segments with reversed flow) with many local minima. To implement this method, we have chosen an initial distribution of intravascular pressures in each vessel segment of the network. We match the initial direction of the flow according to the experimental observations. The boundary segment pressure is varied randomly:

PA=120- ΔPA(1-rand);PV=2+ΔPV*rand

WhereΔPA=ΔPV=50mmHg;

The “wrong” solution is accepted with a probability 0.0015*exp(-(E(n)-E(n-1))/T); where n= the annealingmethod iteration number and T= 10,000 is an arbitrary parameter called the temperature of the coolingfunction.Theannealingmethodalgorithmwasrunsuccessively5,000times.TheresultoftheSAMapplicationisadistributionofintravascularpressurethatcorrespondstotheobservedflowdirection.Asimilarfunctionisimplementedseparatelyforthevenouscirculation(Figure2).

a b c

Figure 2. An incomplete arterial and venous vascular network (a), the mathematical simulation of the network (b) and the annealing function simulation including the blood flow values (c). The results of this study will be submitted to publication to an ISI ranked journal. A similar approach will be used for the vascular networks of the colorectal tumors obtained from the experimental part of the grant. Activity 4.1 Forecasting the angiogenesis before and after the angiogenesis treatment The mathematical approach for forecasting the malignant potential of a vascular network is using the morphometric characteristics of the tumour vasculature to generate several parameters such as the fractal dimension, which could enhance the diagnostic of tumour vs. normal tissues based on the vasculature. Briefly, the images generated during eCLE examinations are stored for offline processing. We only use images without procedural artifacts such as bowel movement or slippage of particulate matter (bubbles, fecal debris). We process the images using a computer aided diagnosis (CAD) module of a proprietary medical imaging system (NAVICAD) developed with the Matlab programming software (Matlab, The MathWorks Inc. USA). The CAD application includes image processing functions, a module for fractal analysis, grey-level co-occurrence matrix (GLCM) computation module, an anatomical feature identification module based on Marching Squares and linear interpolation methods. A two-layer neural network integrates the image analysis parameters (fractal dimension, lacunarity, contrast correlation, energy, homogeneity, and feature number) derived from the imaging processing step and automatically generates a diagnosis. The application diagram is presented in Figure 3.

Figure 3. Diagram of the diagnosis algorithm.

An example of how the algorithm works is presented in Figure 4. Several parameters of the morphometric analysis are presented: Fractal dimension, Lacunarity, Contrast, Correlation; Energy; Homogeneity; Features number.

Figure 4. a. Normal colon mucosa with round shaped crypts (blue circle), situated at relatively equal distance one from another, dark goblet cells (yellow circles), and narrow and regular blood vessels surrounding the crypts (red arrows). b. Normal colon mucosa image processed: Fractal dimension=1.732; Lacunarity=0.13; Contrast=0.26; Correlation=0.97; Energy=0.24; Homogeneity=0.89; Features No=14. The completion of the study will be result in a publication in an ISI ranked journal. Bibliography: 1. Ciocâlteu A, Săftoiu A, Pirici D, Georgescu CV, Cârţână T, Gheonea DI, Gruionu LG, Cristea CG,

Gruionu G. Tumor neoangiogenesis detection by confocal laser endomicroscopy and anti-CD105 antibody: Pilot study. World J Gastrointest Oncol 2015; 7(11): 361-368.

2. Cârțână ET, Streata I, Nicoli E, Uscatu D, Ciocalteu AM, Cherciu IF, Gheonea DI, Georgescu CV, Ioana MI, Gruionu G, Saftoiu A. Evaluation of Tumour Angiogenesis in Colorectal Cancer Based on Quantitative Contrast-Enhanced Endoscopic Ultrasonography and Molecular Analysis. Digestive Disease Week, Washington, DC. Gastrointest Endosc 2015;81:AB175.

3. Elena-Tatiana Cârţână, Dan Ionuţ Gheonea, Irina Florina Cherciu, Alexandru Bărbălan, Ioana Streață, Mihai Ioana, Daniel Pirici, Claudia-Valentina Georgescu, Valeriu Șurlin, Gabriel Gruionu, Adrian Săftoiu. Quantitative contrast-enhanced endoscopic ultrasound correlated with molecular markers of tumour angiogenesis for the evaluation of colorectal cancer patients. Gastrointestinal Endoscopy (ISI indexed, impact factor 5.37) (under review).

Project Director: Conf. Dr. Gabriel Gruionu