Our contribution to the TCGA findings of a Comprehensive Breast Cancer Analysis

The Windber Research Institute is proud to be part of the just published online article in the journal Nature http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11412.html  (September 23^rd, 2012) by The Cancer Genome Atlas (TCGA) Network entitled “Comprehensive molecular portraits of human tumors.” Together with our military collaborators, the Walter Reed National Military Medical Center (WRNMMC), we provided breast tissue specimens, DNA and relevant donor data for this ground breaking research. A total of fifteen Tissue Source Sites were involved in this national scientific project sponsored by the National Institutes of Health (NIH). Our journey to such a success story was as expected not very easy. Our efforts through the years to acquire and maintain the very best quality tissue specimens paid off when we qualified to become tissue providers for TCGA. The strict qualification criteria of this project kept us on our toes. Our qualification rates were usually between 85% and 100%. We were highly commended by the Biospecimen Core Resource group (Nationwide Children’s Hospital Biospecimen Core) as they compared our performance with that of the other tissue source sites. All this attests to the hard work and dedication of our staff. Their constant and daily efforts to provide the highest level of efficiency and perfection made it possible for us to be part of this major scientific project.  As a small not for profit research institute established twelve years ago, we are proud to be in the same playing field with institutions such as Duke University, Mayo Clinic, MD Anderson Cancer Center, University of Pittsburgh and University of North Carolina at Chapel Hill to mention just a few of the other Tissue Source Sites for this project. 

In this project, 800 breast tumors were genetically characterized using multiple technological platforms. The study was able to characterize four major sub-types of breast cancer (HER-2 enriched, Luminal A, Luminal B, and Basal-like) as previously described and provided a more comprehensive understanding of the mechanism behind each type of breast cancer. Furthermore, the study identified molecular similarities between one sub-type of breast cancer and ovarian cancer. Charles Perou, PhD, the corresponding author says “This study has now provided a near complete framework for the genetic causes of breast cancer which will significantly impact clinical medicine in the coming years as these genetic markers are evaluated as possible markers of therapeutic responsiveness.” Dr. Perou is the May Goldman Shaw Distinguished professor of Molecular Oncology at the University of North Carolina at Chapel Hill and a member of UNC Lineberger Comprehensive Cancer Center.

The tissue bank at Windber will continue to engage in ground breaking research activities in the coming years. This will encourage our donors and help with new therapeutic targets for treatment to alleviate the sufferings of the many breast cancer patients out there. We are working for you!

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature11412.html

Summer Internship at a Biorepository

Matthew Masiello spent the summer interning with our tissue bank at Windber Research Institute. He will be pursuing a degree in International Relations from American University in Washington, DC this fall. 

By Matthew Masiello

As high school was coming to an end, I, as well as a multitude of my peers was on the look for summer jobs, searching for positions that would sustain us throughout the summer and hopefully be able to support maybe a fraction of the total cost of the college that was approaching in the fall. It just so happened that as my friends found themselves working in restaurants, manual labor, or totally bypassing the opportunity to work during the summer before their new scholarly life, I found myself walking into the doors at Windber Research Institute ready to begin my internship within the tissue bank.

The tissue bank at WRI provides the optimal isothermal freezer space for storing blood, tissue, and DNA samples. Over the past decade the tissue bank has gone from great to greater and to maintain such levels of efficiency and reliability certain procedures are instilled to monitor the isothermal freezers and their samples inside. So as an intern, my assignment was to perform a Quality Assurance project on the mass amount of DNA samples that the freezers contain, meaning that I was to sort through boxes of DNA, confirming specific sample locations and identification with the extensive software program that accounts for all samples. Up front, it was a task I was ready to embrace, however, I do suppose every job comes with its own fair share of difficulties and mine just happened to be enduring cold hands/fingertips due to handling samples kept at -80° Celsius or less and squinting through dry eyes because of my proximity to the dry ice that I used daily to perform my work. However, through determination and hand-warmers I accomplished my task and felt as though I was contributing to an already efficient system.

Another task I had the opportunity to explore was blood processing.  This procedure involves centrifuging, labeling, aliquoting and scanning the samples into a sample management software program.  Efficiency and organization were in place, common themes at the tissue bank. Honestly, this process was a humbling experience at the beginning of my time at WRI.  It would have never come across my mind if I had worked at any other sort of job that my hands resemble the San Andreas Fault when it came to using the pipette to draw from the sample tube. Truly, only at a tissue bank do you find what your actual natural abilities are. Thankfully, at the same rate I found myself truly enjoying the process as I was becoming better at handling the pipette and more and more efficient with the task at hand.

My internship at WRI’s tissue bank was not only scientifically satisfying, but also prepped me for what I should expect with any sort of occupation I might find myself in the future. Efficient management and clear-cut organization are key factors to any system that strives to be the best and both are clearly exemplified at the tissue bank, and I will guarantee that I remember my time here and use the skills and styles that I developed and apply them to any sort of work I find myself conducting in the approaching years.

When You Need to Re-evaluate Tissue pathology Data

When we receive pathology report for data entry, we usually review this to determine it matches the slide available before the information goes through the data entry step. If we therefore find that an  OCT.specimen has a diagnosis that does not correlate to the received participant information (for example we have tumor size 1.0 cm or greater; while OCT diagnosis – no tumor seen) the OCT block will be tracked and retrieved for re-evaluation. Deeper sections will becut for a second review. A record is kept of all the samples that we consider for deeper sections. This record shows the information received, along with the diagnosis of the first evaluation of the H&E slide. After the second evaluation is performed, the information is added to the record.

This re-evaluation process, as we call it, has been very helpful. The deeper cuts, in some cases, have revealed tumors making these samples suitable for certain research protocols thus increasing the number of material in our tissue bank that can be utilized for research.  This is always something that the research scientsist are happy about!

Researching Our Biospecimens

The tissue bank at the Windber Research Institute focuses on collecting and storing well annotated and high quality tissue and blood samples.  In order to demonstrate that the samples are of the highest quality, we do biospecimen research on samples in house to test different processing methods or storage conditions and determine which are best.  Pre-analytical variables that could affect downstream research processes that utilize DNA or RNA are studied in ongoing projects at our tissue bank.  High DNA/RNA concentration and good yield, integrity and purity are important in all experiments. 

            One such experiment is our biospecimen research project where we looked at the integrity and purity of RNA that was isolated from breast tissue collected from reductive mammoplasties.  Tissue specimens were processed by flash freezing or embedding in optimal cutting temperature (OCT) medium and either immediately stored at -80°C or kept at room temperature over varying time intervals.  RNA isolation was performed on all samples and the Nanodrop spectrophotometer was used to determine the concentration and purity of the RNA.  RNA integrity number was also determined using the Agilent Bioanaylzer.  Since laser capture microdissection is sometimes an integral part of tissue analysis, we also determined its effect on RNA integrity.  Quantitative Real-Time PCR was done for comparative measurement of the expression of four house keeping genes: B-actin, glyceraldehyde-3-phosphate dehydrogenase, cyclophilin A and porphobilinogen deaminase.  Results of this study can be viewed on the additional poster.

A Cut above the Rest

Laser Microdissection is an intermediate step in the preparation of tissue specimens for research.  This service is provided by our tissue bank and gives researchers the ability to obtain macromolecules (DNA, RNA and protein) from specific cell types of a tissue specimen.  There are three Microdissection Microscopes currently in the market- Arcturus Pixcell, Leica Lasermicrodissection ASLMD and the Zeiss PALM.  We currently use the Leica ASLMD system.

Our primary focus in Windber is the study of breast tissue which is highly heterogeneous with numerous cell types within the primary tumor and the surrounding tissue.  Laser microdissection has allowed us to isolate pure samples from both invasive and in situ tumors (with and without involvement within invasive tumors), as well as the microenvironment surrounding the tumors that can then be used for downstream analyses.

We have used Laser Microdissected tissue samples for a variety of protocols requiring the preparation of homogenous isolates of DNA, RNA and protein.  Because the quality of RNA extracted from tissues can be affected by sample preparation and processing, we have fine-tuned our procedures for processing these samples.  Our protocols include careful cleaning of all equipment with RNAse inhibitors, use of special stains with lower water content, and rapidly performing the isolation process.  The time to process each sample is kept to less than 30 minutes from the time the specimen is sectioned through staining, microdissection and placement into the cell lysis buffer.  Below are images of a ductal carcinoma in situ (DCIS) from which RNA was isolated from the Microdissected cells.  The image labeled (A) is an Hematoxylin and Eosin Stain; (B) is a representative section in the same area stained with the RNA staining procedure and image (C) is the DCIS after Microdissection.

We have published details of our protocols in the chapter titled- “Laser Microdissection for Gene Expression Profiling” in the book “Laser Capture Microdissection Methods and Protocols Series: Methods in Molecular Biology”, Vol 755, Second Edition, Humana Press. 



A



B









C



WRI in Bloom

 As I think about Windber Research Institute (WRI) in bloom this spring, I am reminded of the renewal and regrowth we experienced in 2005.  As our staff and research expanded, we outgrew the facility originally built for ten to fifteen people.

St. Mary's Church

In August 2005, Windber Research Institute moved to the location on the former site of St. Mary's Hungarian Roman Catholic Church. In an effort to honor the support from the Windber community, the arch of St. Mary's Church was preserved and incorporated into the design of the new facility.

Just as WRI has grown over the years, the tissue bank continues to blossom as well.  We have gone from our first batch of samples in 2001 when electronic records were scant, to our current status where electronic records abound…shipping manifests, databases, checklists, reports, automated monitoring, QA… we have it covered.  It is amazing to look back at where we were in our infancy and see how we have grown into a mature biorepository in 2012.  

Windber Research Institute

Just as spring brings new growth after the winter hibernation, exciting opportunities are upon us!  A most encouraging development is our new partnership with the U.S. Military Cancer Institute to handle their biobanking needs.  We recently expanded our tissue bank adding additional space and freezers to accommodate frozen tissue specimens from the U.S. Military Cancer Institute’s research program.  This contract serves as a vote of confidence in our leading edge facility and biobanking team and we are proud to be working with them.

Maintaining the Freezers 24/7

Preserving the integrity of our precious samples is a top priority in our tissue bank.  We recognize that if it were not for the generosity of the patients, this great resource would not exist.  To this end, daily checks are performed to ensure that everything is operating smoothly and within acceptable limits.

WRI’s Tissue Bank consists of ten liquid nitrogen freezers and five negative 80 freezers which are continuously monitored by an automated system. If a temperature falls out of range, the system will call staff from a designated call list so the necessary action can be taken.

In addition to the automated system, we also perform manual checks as a backup method.  Every day, two staff members perform AM and PM checks of the tissue bank.  The checklist includes room temperatures, freezer temperatures, nitrogen levels, oxygen levels and verification that freezers are locked.  During non-business hours, security staff walks through the facility and performs the same checks every four hours.

All checklists are double checked at the beginning of every week to look for possible trending patterns.  This process can help to give us a heads up of a possible problem in the future.  One such occasion was when we noticed liquid nitrogen levels rising instead of falling on one of the freezers (this parameter is not recorded on the automated system).  We did some troubleshooting and ended up needing to do a total thaw of the freezer to clear out an obstruction that was leading to the erroneous readings.

Maintaining the tissue bank is a 24 hour 7 day a week job, and every day many steps are taken to ensure the quality and integrity of our samples remain high.

Combing Through Samples to Eliminate Knots

Here at WRI, we have a growing inventory of Clinical Breast Care Project (CBCP) blood and tissue samples totaling 80,000 aliquots.  We have been collecting and storing samples since 2001.  Every sample donated is a valuable asset for our biomedical research activities.  We manage our inventory using quality standards so that we can provide high quality specimens to our research scientists.  Therefore our aim is to reduce errors to the barest minimum (as is humanly possible).   

To ensure these goals are met, we perform regular Sample Quality Assurance (SQA). One of our SQA was performed on CBCP samples collected prior to December 2007.  We predicted there would be some errors made in the early days of sample collection especially since we have modified our Standard Operating Procedures (SOP) over the years.  This SQA was the first that represented a time frame of this magnitude.

The project was initiated in August 2010 and completed in December 2011.  Boxes were physically pulled from the freezers and compared to sample records in our electronic databases.  It was not practical to check every individual sample so we formulated a plan to check 20% of blood samples per box and 100% of tissue samples per box.  Sample discrepancies were corrected and logged onto our Quality Assurance tracking database.  Paper and electronic records were kept during the SQA project to monitor progress and keep track of corrections.

The SQA project allowed us to check 19.7% of our total CBCP inventory.  We observed an error rate of 2.5%.The most common errors were from hand written information on sample tubes. We observed during this SQA exercise that errors were more common during the earlier years when we did not utilize a bar coding system. 

 We have since implemented a bar-coding system which has decreased data entry time and errors.  We also continually update and improve our SOP’s for sample collection, processing, and storage.  All inventory errors have so far been corrected. 

Biorepository……………Drug Discovery

Biorepositories and Drug Discovery go hand in hand.

The role of the specimen Donor in this relationship cannot be overestimated.

The flow chart above summarizes the inter-relationship between all of these entities.

A

Donor provides specimens which are procured by specialized entities (surgeons, pathologists, nurses, etc)

These specimens include body fluids and solid tissue.

B

These specimens are processed in the tissue bank in a variety of specialized ways to meet the research demands

C

Using a wide variety of technologies, the scientists utilize the processed products to generate data, which forms the core/lead for new drugs.

The tissue bank at Windber Research Institute is part of this cycle and we are constantly operating at the highest level of efficiency to ensure the success of new drug development projects.

The Ebb and Flow of Liquid Nitrogen through the Years



Portable Dewar from the "old" days



When the tissue bank was established, our liquid nitrogen freezers were attached to portable dewar tanks which had to be exchanged periodically.  In order to get the tanks into our building, they had to be wheeled up a rather long ramp.  This process was not easy and required more than one person to heave the tanks to their destination.  I imagine the architect did not take into consideration how much exertion would be required to move a large tank up a long hill… As we grew and moved into a new facility, we switched to two 1500 gallon stationary bulk tanks for our supply source.  We monitored the levels in the tanks daily and telephoned in our order every 2 weeks.



Filling bulk tank with liquid nitrogen



Several years later, we transitioned to one 3000 gallon tank. The  video below shows the venting of liquid nitrogen out of the tank into the atmosphere.  This had to be done before the unit was taken out of service.  If I had planned ahead, we would have had enough Dippin Dots for the entire department. The larger 3000 gallon tank offered several improvements.  Along with a cost savings, there are less frequent fill ups and a high-tech monitoring system.  The new tank is equipped with wireless telemetry allowing remote monitoring of the nitrogen level.  Deliveries of nitrogen are automatically scheduled without a call from us!



It’s in the details: Tracking Tissue Morphology

With thousands of OCT tissue samples stored over the last 10 years we have mastered our record keeping capabilities which provide information about OCT blocks as they move in and out of the freezers and information regarding the consumption of the OCT tissue specimens for our internal research activities (sample usage).  Throughout the years we have learned that tracking tissue morphology is critical because we are able to determine when an OCT block is no longer useful for research. With every slice of the frozen OCT tissue block, the morphology changes and it is important to know when this block is no longer a representation of the original material that was deposited in the tissue bank.

So, the first step in this Morphology Tracking process is to cut each OCT block, stain, date and archive each stained H&E slide as it arrives in the tissue bank.  When the sample is selected for research the archived H&E slide is pulled and reviewed by a designated pathologist and areas of interested annotated.  The sample is then used for research and a second H&E section is made after all the sections for research have been obtained.  This second H&E is reviewed to insure that all areas of interest for research still remain and the tissue morphology has not changed (from its original format) due to cutting deeper into the OCT tissue block. Detailed notes are made throughout these processes, which include notes on changes observed in tumor size which may indicate that the tumor is near exhaustion.  This process is repeated each time the sample is used for research and when the section fails to be a true representation of the original OCT block, the block is marked as “exhausted” and it is no longer available for research. 

What makes us “Tick?”

In any given establishment, many factors contribute to its success and well-being. In our tissue bank, one such factor that makes us “tick” is our staff. We strongly believe that we are where we are today due to the dedication and commitment of those who run the day-to-day activities of our tissue bank. We have been fortunate that over the past 10 operating years that our staff turnover has been very minimal, allowing most of us to witness the tissue bank grow and mature from the ground up. We have all learned through experience and enjoy the benefits of our hard work and dedication. While there are multiple units in the tissue bank, most staff members are cross-trained to handle multiple activities, allowing us to function efficiently.  Our staff is required to take the National Cancer Institutes (NCI) course on “Protecting Human Research Participants” so that we fully understand the rules and regulations governing the use of human subjects for research. We have a very personal attachment to our jobs. We recognize the patients' contributions and protect our specimens to ensure there is no wastage and that every single biospecimen is used to its fullest potential. We understand the mantra “garbage in garbage out” and we are committed to providing researchers with the highest grade quality biospecimens. This quality results in research that can be translated into the clinic. Through our long-term experience in tissue banking, our staff has grown into a highly skilled team that “ticks” like a well-oiled precision timepiece.

Welcome to the Windber Research Institute Tissue Banking blog.

Who are we?

We are a state of the art biorepository located in rural Pennsylvania established in 2001.  We house over 80,000 human specimens including blood, DNA, RNA and assorted tissue including breast, heart, kidney, lung, colon and reproductive tissues.  We collect, process, store and distribute these biospecimens to support scientific discovery.

Why are we here?

Our mission is to enhance the reliability of molecular research by providing high-quality biospecimens to the scientific community. Our experiences over the years have helped us gain valuable insight that has allowed us to achieve this goal.  We have built the repository from the ground up and we would like to share our experiences and knowledge we have gained along this journey. 

We strive to be an informative blog to help others in this industry.  We welcome the exchange of ideas as well as anecdotal stories and lessons learned (sometimes the hard way) along the road.

Here we come!

After over 10 years of operating a biorepository in a Research Institute, we are ready to share our experience with you by taking advantage of the available internet networking tools.  Stay tuned......................