Name: generation of comprehensive thoracic oncology database - tool for translational research
Uploaded: 2011-01-22T17:15:00
Duration: 11 min 18 s
Description: Watch this Scientific Journal Video about Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research at JoVE.com

This work was supported by NIH grants 5R01CA100750-07, 5R01CA125541-04, 3R01CA125541-03S1, 5R01CA129501-03, 3R01CA129501-02S1 to RS

1. University Clinical Research Protocols:
<ol>
<li>Two university protocols were developed for the purposes of this initiative. The first protocol allows for the prospective procurement of tissues from lung cancer, esophageal cancer, carcinoid tumor, thymoma, and mesothelioma patients. The protocol also allows blood and other bodily fluids to be collected from patients for biomarker studies. The protocol permits researchers to obtain clinical information from the patient source of such specimens via chart abstraction and to store both specimen and clinical data in a protected database.</li>
<li>The second protocol is similar to the first except that it allows researchers to access previously banked tissues and other samples obtained during the course of a patient's cancer diagnosis and treatment. Patients who have consented to surgery at the University of Chicago and who have signed this protocol are eligible.</li>
</ol>
2. Clinical Data Collection Protocol:
<ol>
<li>Patients being seen at the University of Chicago Medical Center for treatment of the aforementioned malignancies were included in this protocol.</li>
<li>Eligible patients were identified by their treating oncologist and were consented by a member of the clinical team trained in the two protocols.</li>
<li>Once consented, a patient's medical history information was obtained via chart abstraction and was entered into the database by a member of the clinical support team.</li>
</ol>
3. Specimen Collection Protocol:
Tissue samples
<ol>
<li>Tissues containing a known or suspected malignancy obtained during a patient's standard clinical care via biopsy or surgery were included in this protocol. No additional tissue, outside of what was necessary for a patient's diagnostic workup, was obtained.</li>
<li>After surgical removal of the sample necessary for the preparation of permanent sections, the residual tissue was placed on ice.</li>
<li>A tissue procurement technician transported the residual sample on ice to the pathology department.</li>
<li>Residual tissues were weighed, measured, transferred to storage, labeled, and properly documented according to the standard operating procedure.</li>
<li>Long-term storage of the specimens was maintained in a -80&deg;C freezer in the pathology department.</li>
<li>In order to access tissue samples from patients who had already undergone surgery for a thoracic malignancy, lists of patients maintained by collaborating surgeons and radiation oncologists were referenced. In this manner patients of interest were identified. Their tumor tissue samples could be retrieved from the department of pathology if consent was obtained.</li>
</ol>
Blood samples
<ol start="7">
<li>During clinically indicated blood draws, the protocol also allowed for an additional two to six tubes of blood in a 5 mL green top (Heparin) tube, one 10mL purple top tube (EDTA germline DNA) and one 10 mL red top (Serum) tube of blood.</li>
<li>Up to six samples at different time points were taken, with the date of the blood draw annotated.</li>
<li>Blood samples were centrifuged at 2000 rpm for 10 minutes.</li>
<li>Plasma and serum components were aliquoted in cryovial tubes in 1 mL portions.</li>
<li>For the collection of white blood cells, the upper 1-2 mL of the interface/red blood cell fraction were resuspended with 1-2 mL of cell preservation medium (MEM EBS media + 10% fetal cal serum+ 5% DMSO).</li>
<li>All samples were frozen slowly in an isopropyl freezing box at 70-80&deg;C for 16-24 hours then transferred to a -70&deg;C to -80&deg;C storage box.</li>
<li>All samples were labeled with a bar-coded unique identifier and appropriately documented in the sample procurement form by a lab technician.</li>
</ol>
Other body fluids:
<ol start="14">
<li>Fluid not collected for clinical use could be collected and stored under this protocol. Sputum samples were collected and sent for cytology. Sputum samples were stored on ice at 4&deg;C while being transferred.</li>
<li>Sputum samples were then transferred to 15 mL Falcon tubes and centrifuged at 1400 rpm for 10 minutes.</li>
<li>The supernatant was aliquoted in 4 mL portions in 6mL cyrovials. Cyrovials were placed in a freezer, on dry ice, or in an isopropyl freezing box to be subsequently frozen to a -70&deg;C to -80&deg;C. After 16-24 hours, samples were transferred to a -70&deg;C to -80&deg;C storage box.</li>
<li>All samples were labeled with a bar-coded unique identifier and appropriately documented in the sample procurement form by a lab technician.</li>
</ol>
4. Building the Informatics Infrastructure:
<ol>
<li>After evaluating a number of database management programs, Microsoft Access was selected as the program to house clinical and laboratory data for the Thoracic Oncology Program Database Project based on its operability and ability to link related sets of data.</li>
<li>A team of clinicians, basic science researchers, a biostatistician, and a bioinformatics expert was convened to identify variables of interest to capture within the database.</li>
<li>The team identified data elements related to patient demographics, cancer characterization, epidemiological factors, and specimen annotation based on the needs of thoracic oncology researchers and referring to standards set regarding Common Data Elements (CDEs) by the National Cancer Institute.</li>
<li>The team developed a coding scheme to make data analyzable for research purposes. When possible, data was coded using numeric variables so as to reduce the amount of free-text entered into the database.</li>
<li>Variables of interest were divided among seven different tables within Microsoft Access to capture varied aspects of related information.</li>
</ol>
5. Designing the Contents of Each Table:
<ol>
<li>Seven primary tables were created: 1) Patients table, 2) Sample Data table, 3)TMA table, 4) DNA Specimens table, 5) Chest Oncology Conference table, 6) Cell Line table, and the 7) C. elegans table.</li>
<li>The Patients table was designed as the only table within the database to house clinically relevant information about the patients, their cancer, their clinical course, their risk factors, and their outcome (Fig 1). The intent of this particular design was to limit redundancy within the database.</li>
<li>The Sample Data table links pathological specimens to their patient sources. Each sample is given a sample pathology number and this number is related to the patient's medical record number. Multiple specimens may be obtained from each individual and so the table also includes information about the date the specimen was obtained and the type of specimen collected: initial tumor, recurrent tumor, or autopsy specimen.</li>
<li>The TMA table has been used thus far to capture protein expression data for 63 unique proteins. A TMA can be used to characterize the distribution of differentially expressed proteins in tumor and non-tumor tissue by using antibodies to localize proteins (Fig 2). Protein expression is represented by a score of 0, 1, 2, or 3 based on the pathologist's impression of the intensity and percentage of TMA staining. 
When percent staining is measured, a score of 0 indicates no staining, 1 indicates less than 11% staining, 2 indicates less than 50% staining, and 3 indicates greater than 50% staining. When intensity is measured, a score is assigned based on the relative amount of staining, also on a scale of 0 to 3. In some cases, IHC slides are also scanned at high resolution and staining intensity is quantified by Automated Cellular Imaging System (ACIS) imaging software in parallel to the pathologist's scoring. However, for both techniques, a higher score indicates greater protein expression. 
In addition, the TMA table annotates the location of the TMA punch for future reference. Furthermore, the database includes the source of the tissue (tumor, normal, lymph node, metastatic tissue), the location within the sample (center, edge), the histology of the cancer, and the medical record number to link this information back to the Patients table.</li>
<li>The DNA Specimens table lists all the DNA stored within the laboratory. DNA samples are linked to their patient source via the patient's medical record number. Basic information about the specimen is captured to describe the source of the sample, including the location and the histology of the tumor. The purpose of this table is to describe the genetic alterations which have been characterized within the specimen using polymerase chain reaction, standard DNA sequencing, and mutational analyses. 
The table also captures variables such as amino acid changes, nucleotide changes, homozygosity, synonymity, and the gene in which the mutation has occurred. Examples of genes which have been studied include Paxillin, cCbl, EGFR, p53, KRAS, cMet, and EphB4. The investigator who has characterized the mutations is also listed.</li>
<li>The fifth table within the database is the Chest Oncology Conference table. The chest oncology conference is a weekly meeting of medical oncologists, thoracic surgeons, pathologists, radiologists, radiation oncologists, and other members of the thoracic oncology clinical team who meet to develop coordinated patient treatment plans. The purpose of this table is to list the patients who have been discussed at the conference as part of their standard of care. Information regarding the availability of pathological specimens for each patient is also captured in this table.</li>
<li>The sixth table is the Cell Lines table. This is a free standing table within the database because it is not linked to any of the other tables. It describes the cell lines that have been utilized in the laboratory for research purposes. The table captures the amino acid changes, nucleotide changes, the homozygosity and synonymity of mutations, and the location of mutations within cell line DNA.</li>
<li>The seventh table is the C. elegans table and it is also a free standing table. This table lists orthologous receptor tyrosine kinases and includes the needle score, which is a measure of similarity with human proteins.</li>
</ol>
6. Establishing Relationships Among Tables:
<ol>
<li>Each table is assigned a primary key, which serves as a unique identifier for each entry within the table. By definition, a unique identifier value cannot be repeated. For example, within the Patients table, the primary key is the medical record number since a MRN can only signify one unique individual. Within the Sample Data table, the primary key is the sample pathology (SP) number. As the TMA and DNA Specimens tables did not have unique identifiers, a dummy number was established as the primary key. The TMA and DNA tables were then linked to the other tables using the MRN and the SP numbers, respectively. This ensures that each TMA punch and DNA specimen are linked back to both specific information on the sample and clinical information from the donating patient.</li>
<li>In Microsoft Access, individual tables are linked via their primary keys such that logical relationships are established (Fig 3). These relationships are necessary to generate queries where data is gathered from multiple tables.</li>
</ol>
7. Querying:
<ol>
<li>Performing a query of related sets of data is relatively simple within Microsoft Access. A query can be designed by selecting the "query design option" under the create tab.</li>
<li>Tables including the fields of interest are selected and displayed.</li>
<li>Variables from the tables of interest are selected and, if necessary, can be filtered based on the researcher's criteria of interest (Fig 4).</li>
<li>The query can then be run, resulting in the desired fields listed in spreadsheet form.</li>
</ol>
8. Exporting Data:
<ol>
<li>Once a query has been generated, the data can be exported. While most researchers prefer the data to be in the form of a Microsoft Excel spreadsheet, data can be exported to a number of other programs using the export menu under the "external data" tab. Data can be saved using the appropriate file extension.</li>
<li>When the data is exported for statistical purposes, a prescribed set of variables are included in the export so that a comprehensive analysis can be done to control for covariates.</li>
</ol>
9. Importing Data:
<ol>
<li>Importing the data requires a perfect match between the format of the imported data and the format of the table in Access. The variables of interest within the table to import must have the same names as the Access table. The spelling must be exact and there cannot be spaces where spaces do not exist in the Access table.</li>
<li>Once symmetry between the two tables is attained, the user has the ability to use an append or update query to import the data into Access. An append query will allow the user to add new rows of data into the Access database. For example, if information is available about patients that were not already in the database, an append query could be used to add those patients. However, if new data is available on patients or specimens that need to be modified, an update query must be performed on those entries.</li>
</ol>
10. Updating the Database:
<ol>
<li>Members of the database project have prescribed roles in ensuring that the database is kept current. One full-time employee is tasked with regularly populating and updating the database with clinical information based on patients who have been consented in clinic.</li>
<li>Another full time employee, serving as the data manager, is charged with obtaining laboratory data as it becomes available and inputting this information into the database with an update or append query.</li>
<li>Research assistants who are trained in the protocol are responsible for performing a systematic update of the database every six months to obtain the most recent data available. This is particularly important for fields such as vital status and date of last contact, as these fields have a direct impact on survival analyses performed on the data.</li>
</ol>
11. Access to the Database:
<ol>
<li>The database is only accessible to individuals who are HIPAA compliant and are included under the IRB protocol. Access is further reduced to individuals trained in Microsoft Access and who have direct responsibilities for updating or modifying the data.</li>
<li>Researchers who have contributed to the database may request information from the database from the data manager, but they themselves are not permitted to access the data directly.</li>
<li>The data manager provides de-identified information to the requesting researchers by removing variables such as the medical record number and the patient's name when generating an export query.</li>
</ol> 
12. Representative Results:
A researcher may be interested in knowing the clinical significance of over-expression of the protein Paxillin in non-small cell lung cancer. As this researcher has generated a great deal of TMA data in the database for Paxillin, the data manager approves the researcher's request to access clinical information to correlate with the laboratory data. The data manager runs a query where he combines both the Patients Table and the TMA Table. Variables of interest from the Patients table include the patient's date of birth, their race, the histology of their cancer, the stage of their cancer, their date of diagnosis, their vital status, their date of death, and their date of last contact. Using these variables, such as age at diagnosis and stage, important confounders can be accounted for and controlled. From the TMA table, important information such as the tumor type and the protein expression can be ascertained.

As the two tables are linked via the medical record number, patient information from individuals whose tumors have been studied for Paxillin expression are included in the output. The results can be filtered so that only patients with non-small cell lung cancer are displayed. The results can be further refined based on the needs of the researcher.

These results can be exported for primary data analysis by the biostatistician and the results are then shared with the researcher.

Project home page: Access Database template and Standard Operating Procedure are available at: 
<a href="http://www.ibridgenetwork.org/uctech/salgia-thoracic-oncology-access-template">http://www.ibridgenetwork.org/uctech/salgia-thoracic-oncology-access-template</a>
License: Freely available for academic and non-profit use.
Restrictions to use by non-academics: Commercial users require a license. For questions regarding commercial uses, please contact The University of Chicago's Office of Technology and Intellectual Property (UChicagoTech) at (773) 702-1692 or <a href="http://www.tech.uchicago.edu">www.tech.uchicago.edu</a>
 <img src="/files/ftp_upload/2414/2414fig1.jpg" alt="Figure 1" /> 
Figure 1. A screenshot of the Access Database depicting a section of the Patients Table.
 <img src="/files/ftp_upload/2414/2414fig2.jpg" alt="Figure 2" /> 
Figure 2. Schematic depicting a tissue microarray (TMA)2
 <img src="/files/ftp_upload/2414/2414fig3.jpg" alt="Figure 3" /> 
Figure 3. Screenshot depicting relationships established among tables within the Access database. Tables are linked via primary keys.
 <img src="/files/ftp_upload/2414/2414fig4.jpg" alt="Figure 4" /> 
Figure 4. Sample query for Paxillin mutation, TMA results, and clinical variables.

<ol>
	<li>Adamski, J., Finnegan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Perspectives+on+Microsoft+Office+Access.">New Perspectives on Microsoft Office Access.</a> , (2007).</li><li>Giltnane, J., Rimm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technology+Insight:+Identification+of+biomarkers+with+tissue+microarray+technology.">Technology Insight: Identification of biomarkers with tissue microarray technology.</a> Nat Clin Pract Oncol. 1, 104-111 (2004).</li></ol>

No conflicts of interest declared.

<table border="1">
 <tbody>
 <tr>
 <td>Material Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf</td>
 <td> 
 </td>
 </tr>
 <tr>
 <td>Conical centrifuge tube</td>
 <td>Falcon</td>
 <td>518-PG</td>
 </tr>
 <tr>
 <td>Minimum essential medium eagle (MEM)</td>
 <td>Sigma</td>
 <td>M4655-500ML</td>
 </tr>
 <tr>
 <td>Fetal Calf Serum</td>
 <td>Cellgro FBS HI</td>
 <td>MTT35011CV</td>
 </tr>
 <tr>
 <td>Dimethyl Sulfoxide (DMSO)</td>
 <td>American Bioanalytical </td>
 <td>AB03091</td>
 </tr>
 <tr>
 <td>BD Vacutainer Serum Tubes</td>
 <td>Fischer Scientific </td>
 <td>367815</td>
 </tr>
 </tbody>
</table>

generation of comprehensive thoracic oncology database - tool for translational research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

Watch this Scientific Journal Video about Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research at JoVE.com

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

generation-comprehensive-thoracic-oncology-database-tool-for

Research

JoVE Journal

Medicine

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma and devastating tissue loss. Despite favorable and highly encouraging early and intermediate functional outcomes, rejection of the highly immunogenic skin component of a VCA and potential adverse effects of chronic multi-drug immunosuppression continue to hamper widespread clinical application of VCA. Therefore, research in this novel field needs to focus on translational studies related to unique immunologic features of VCA and to develop novel immunomodulatory strategies for immunomodulation and tolerance induction following VCA without the need for long term immunosuppression.
This article describes a reliable and reproducible translational large animal model of VCA that is comprised of an osteomyocutaneous flap in a MHC-defined swine heterotopic hind limb allotransplantation. Briefly, a well-vascularized skin paddle is identified in the anteromedial thigh region using near infrared laser angiography. The underlying muscles, knee joint, distal femur, and proximal tibia are harvested on a femoral vascular pedicle. This allograft can be considered both a VCA and a vascularized bone marrow transplant with its unique immune privileged features. The graft is transplanted to a subcutaneous abdominal pocket in the recipient animal with a skin component exteriorized to the dorsolateral region for immune monitoring.
Three surgical teams work simultaneously in a well-coordinated manner to reduce anesthesia and ischemia times, thereby improving efficiency of this model and reducing potential confounders in experimental protocols. This model serves as the groundwork for future therapeutic strategies aimed at reducing and potentially eliminating the need for chronic multi-drug immunosuppression in VCA.

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in VCA.

Vascularized Composite Allotransplantation (VCA) such as hand and face transplants represent a viable treatment option for complex musculoskeletal trauma ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients&#039; Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Database-guided Flow-cytometry for Evaluation of Bone Marrow Myeloid Cell Maturation

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow cytometry (MFC) for myeloid disease diagnosis in France. The protocol presented here was agreed-upon and applied between September 2013 and November 2015 in six French diagnostic laboratories (University Hospitals of Saint-Etienne, Grenoble, Clermont-Ferrand, Nice, and Lille and Institut Paoli-Calmettes in Marseille) and allowed the standardization of bone marrow sample preparation and data acquisition. Three maturation databases were developed for neutrophil, monocytic, and erythroid lineages with bone marrow from "healthy" donor individuals (individuals without any evidence of a hematopoietic disease). A robust method of analysis for each myeloid lineage should be applicable for routine diagnostic use. New cases can be analyzed in the same manner and compared against the usual databases. Thus, quantitative and qualitative phenotypic abnormalities can be identified and those above 2SD compared with data of normal bone marrow samples should be considered indicative of pathology. The major limitation is the higher variability between the data achieved using the monoclonal antibodies obtained with the methods based on hybridoma technologies and currently used in clinical diagnosis. Setting criteria for technical validation of the data acquired may help improve the utility of MFC for MDS diagnostics. The establishment of these criteria requires analysis against a database. The reduction of investigator subjectivity in data analysis is an important advantage of this method.

The MDS diagnosis is difficult in the absence of morphological criteria or non-informative cytogenetics. MFC could help refine the MDS diagnostic process. To become useful for clinical practice, the MFC analysis must be based on parameters with sufficient specificity and sensitivity, and data should be reproducible between different operators.

A working group initiated within the French Cytometry Association (AFC) was developed in order to harmonize the application of multiparameter flow ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. <span style="font-size: 11pt; line-height: 115%; font-family: Arial, sans-serif; color: rgb(41, 43, 49); background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand ...