Human Tissue Bank Research Articles

Banking of human ovarian tissue potentially contaminated by cancer cells: experimental model for study of cryo-stability of these cells.

Auto-transplantation of cryopreserved ovarian tissue for cancer survivors comes with the primary concern of the possible existence of cancer cells in the transplanting tissue. Lineal cancer cells are presented by industry in form of separated cells (suspensions). Data about experimental models for evaluation of a cryopreservation effect on viability of compacted lineal cancer cells is currently limited. This study aims to develop a suitable experimental model for cryobiological investigations of compacted cancer cells obtained after in vitro culture of a cell suspension. Suspended lineal breast cancer cells (ZR-75-1 and MDA-MB-231) were in vitro cultured in AIM V medium for formation of monolayer. Evaluation of the cell viability was performed by healing assay, transmembrane cell migration, invasion assay and immunofluorescent test of F-actin. It was established the possibility of formation of monolayer from viable cancer cells, scarification of monolayer of these cells and formation of compacted fragments. It is described also a behaviour of compacted cells during cryopreservation (saturation by permeable cryoprotectants, thawing and removal of cryoprotectants). The described method can be used for cryobiological investigations of lineal suspended cancer cells in compacted form as a model of tissues contaminated by malignant cells and solid tumors.

Development of a new reproductive tissue cryopreservation clinical service for children: the Oxford programme

PurposeThis article describes the development of a new reproductive tissue cryopreservation clinical service for children at high risk of infertility in the NHS during times of severe financial constraints in the health service.MethodA development plan with two phases was drawn up. Phase 1 restricted the service to childhood cancer patients referred to the Oxford Paediatric Oncology and Haematology Principle Treatment Centre. It was estimated that there would be 10 patients/year and used existing staff and facilities from paediatric oncology, surgery, anaesthetics radiology, pathology, psychology, teenage–young adult gynaecology, and an existing Human Tissue Authority tissue bank with a licence for storage of tissue under a Human Sector Licence. Phase 2 extended the service to include children and young adults across England, Wales and Ireland—patients from Scotland having access to a research programme in Edinburgh. The main challenge in phase 2 being resources and the need for patients to be able to be treated as close to home as safely as possible.ResultsThe Oxford team developed information resources and eligibility criteria based on published best practice, referral and treatment pathways, multidisciplinary team meetings, a network of third party sites, and a dedicated case management and database. As the programme expanded, the Oxford team was able to justify to management the need for a dedicated theatre list. Patient feedback through questionnaires, qualitative work conducted as part of a Ph.D. thesis as well as direct patient stories and interviews in TV, and radio features underpins the positive impact the programme has on patients and their families.ConclusionThe Oxford Reproductive Cryopreservation programme delivers fertility preservation treatment to children and young adults at high risk of infertility safely, effectively and as close to home as possible. The onward view is to apply for national funding for this programme for recognition and sustainability.

Improve the Rate of Bone Collection at Human Tissue Banks

Lack of familiarity with collection and inspection procedures, incorrect bone-packaging procedures, and unclear instructions for bone placement during storage are primary reasons for the resultant low bone collection pass rate of bone banks. Moreover, 8 cases / operations were directly affected by this problem, which caused bone-nail dislocations during the post-operative period that nearly caused medical disputes. The present project was designed to improve the pass rate of the bone of the human organ to 95%. Education and training programs were planned, visual-aid posters depicting standard procedures were produced, the repository was remarked and relabeled, and a regular audit system was established with the medical team. The pass rate for the collection of the bone of the human organ increased from 71.4% pre-intervention to 96% post-intervention. The project reduced patient complaints and raised the accuracy of the bone collection process.

The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease.

The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40min of their removal from the patient, and in less than 5-10min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180°C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.

Status quo of management of the human tissue banks in Taiwan.

As the technologies associated with transplantation and biological tissue engineering continue to advance, human cells and tissues form an integral part to the practice of regenerative medicine. The patient's use of tissues entails the risk of introducing, transmitting and spreading communicable diseases. To prevent such risk and to ensure that the human organs, tissues and cells remain intact and functional after being handled and processed, the transplanted tissues must be subject to good management standards through all stages of collection, screening, processing, storage and distribution as the safety of the users is of the utmost importance. On February 2009, the government of Taiwan promulgated the Regulations for Administration on Human Organ Bank that requires all human tissues banks to adhere to the Good Tissue Practice for Human Organ, Tissue and Cell in terms of establishment and operation in order to cope with the international management trend and the development and management need of the domestic industry. Six years have passed since the law became effective. This article seeks to introduce the current management mechanism and status quo of management of human tissue banks in Taiwan. We also conducted statistical analysis of the data relating to the tissue banks to identify potential risks and the room for improvement. The study concludes that human tissue banks in Taiwan are on the right track with their management practice, leading to a state of steady development and progress.

Cryopreserved Human Precision-Cut Lung Slices as a Bioassay for Live Tissue Banking. A Viability Study of Bronchodilation with Bitter-Taste Receptor Agonists.

Human precision-cut lung slices (hPCLSs) provide a unique ex vivo model for translational research. However, the limited and unpredictable availability of human lung tissue greatly impedes their use. Here, we demonstrate that cryopreservation of hPCLSs facilitates banking of live human lung tissue for routine use. Our results show that cryopreservation had little effect on overall cell viability and vital functions of immune cells, including phagocytes and T lymphocytes. In addition, airway contraction and relaxation in response to specific agonists and antagonists, respectively, were unchanged after cryopreservation. At the subcellular level, cryopreserved hPCLSs maintained Ca(2+)-dependent regulatory mechanisms for the control of airway smooth muscle cell contractility. To exemplify the use of cryopreserved hPCLSs in smooth muscle research, we provide evidence that bitter-taste receptor (TAS2R) agonists relax airways by blocking Ca(2+) oscillations in airway smooth muscle cells. In conclusion, the banking of cryopreserved hPCLSs provides a robust bioassay for translational research of lung physiology and disease.

わが国におけるヒト組織バンク- 健常人組織・細胞の必要性-

わが国におけるヒト組織バンク- 健常人組織・細胞の必要性-

Effect of miRNAs in lung cancer suppression and oncogenesis

AbstractMicroRNA (miRNAs) is a group of small non-coding RNAs. It is involved in multiple cellular processes including proliferation, development, metabolism, differentiation and apoptosis; many of which are linked to several pathological conditions, including cancer. Lung cancer is one of the leading causes of mortality in the world: in 2008 for example, there were 163,000 deaths as a result of lung cancer. Despite technologies emerging which provide the potential for novel targeted therapies and improved early diagnoses, the overall rate of five-year survival still remains at only 15%. One reason for this disappointing statistic is related to the presentation of the disease, and specifically a lack of markers for early detection. Notably, the expression of some miRNAs has been reported to be involved in the diagnosis, classification and even prognosis of lung cancer. Tumorsuppressive and oncogenic miRNAs were found in lung carcinogenesis and the biological functions of these miRNAs have been validated in transplantable lung cancer models and human paired normal-malignant lung tissue banks. Some of these tumor-suppressive and oncogenic miRNAs related to lung cancer will be reviewed here. This article will focus on emphasing miRNAs effectiveness as a biomarker in lung carcinogenesis and candidate pharmacology. Furthermore, how these findings improve our understanding of lung cancer biology and therapy will also be discussed.

40. Protocols that avoid fracturing in whole organs and solutions

Cryopreservation requires transformation of a biological material into a solid state for long-term stability. As a sample solidifies during cooling, mechanical stress accumulates due to changing temperature gradients and differential thermal contraction of heterogeneous sample components. If stress becomes larger than the material strength of the sample, fracturing will occur. The extraordinary low critical cooling rate of M22 vitrification solution (0.1 °C/min) has allowed our laboratory to study fracture avoidance during vitrification of multi-liter volumes of solution. A few general observations are notable. Rapid cooling through high sub-zero temperatures ( > 80 °C) is permissible and desirable to reduce risk of ice growth and cryoprotectant toxicity, but cooling should slow as the glass transition temperature (−123 °C) is approached to allow more time for stress relaxation. Glass containers facilitate fracturing by differential thermal contraction stress because vitrification solutions adhere to hydrophilic glass surfaces. Containers made from polyethylene or similar hydrophobic materials can eliminate this source of stress by allowing samples to contract away from container surfaces as they cool. Tissue permeated with cryoprotectant is generally more resistant to fracturing than the same volume of pure vitrification solution, making fracture-free vitrification of a pure solution a conservative test for suitability of a cooling protocol for fracture avoidance. Following these principles, our laboratory has routinely cooled M22-perfused rabbit kidneys to −135 °C without fracturing. Although not believed necessary for long-term storage, a vitrified kidney was also successfully recovered from liquid nitrogen immersion storage without fracturing using the following protocol: Standard perfusion and cooling to −135 °C, 75 min @ −130 °C, 60 min @ −160 °C, 120 min @ −188 °C, 13 days @ −196 °C (under liquid nitrogen), 90 min @ −185 °C, 90 min @ −155 °C, 18 h @ 135 °C, followed by standard warming, cryoprotectant removal, and transplantation with no hemorrhaging or other indication of fractures. For further exploration, a 200 mL bottle (greater than the mass of a human kidney) of 105% M22 was successfully cooled from ∼ 0 °C to liquid nitrogen temperature without fracturing using a simple convection cooling protocol: Placement in 110 °C vapor for 90 min, 170 °C vapor for 4 h, −186 °C vapor for 19 h, then immersion in liquid nitrogen (−196 °C) for 90 min prior to removal and observation that the solution had no fractures. Liquid nitrogen is far colder than necessary for long-term storage; −135 °C to −140 °C is likely satisfactory to arrest ice nucleation over time periods of clinical interest. These experiences suggest that fracture avoidance may be one of the easier problems of human tissue and organ banking. Rather than gross fractures, thermomechanical stress is a suspected cause of more subtle, but still-significant lesions that have sometimes been observed after transplantation of vitrified rabbit kidneys. Developing protocols for cooling as quickly as possible to minimize ice nucleation, while avoiding damaging thermal strain within tissue, is vital to the future success of organ banking. Finite element analysis computer models will be invaluable for this purpose.

Application of Quality Assurance Strategies in Diagnostics and Clinical Support Services in Iranian Hospitals.

Iran has a widespread diagnostics and clinical support services (DCSS) network that plays a crucial role in providing diagnostic and clinical support services to both inpatient and outpatient care. However, very little is known on the application of quality assurance (QA) policies in DCSS units. This study explores the extent of application of eleven QA strategies in DCSS units within Iranian hospitals and its association with hospital characteristics. A descriptive cross-sectional study was conducted in 2009/2010. Data were collected from 554 DCSS units among 84 hospitals. The average reported application rate for the QA strategies ranged from 57%-94% in the DCSS units. Most frequently reported were checking drugs expiration dates (94%), pharmacopoeia availability (92%), equipment calibration (87%) and identifying responsibilities (86%). Least reported was external auditing of the DCSS (57%). The clinical chemistry and microbiology laboratories (84%), pharmacies, blood bank services (83%) reported highest average application rates across all questioned QA strategies. Lowest application rates were reported in human tissue banks (50%). There was no significant difference between the reported application rates in DCSS in the general/specialized, teaching/research, nonteaching/research hospitals with the exception of pharmacies and radiology departments. They reported availability of a written QA plan significantly more often in research hospitals. Nearly all QA strategies were reported to be applied significantly more often in the DCSS of Social Security Organization (SSO) and private-for-profit hospitals than in governmental hospitals. There is still room for strengthening the managerial cycle of QA systems and accountability in the DCSS in Iranian hospitals. Getting feedback, change and learning through application of specific QA strategies (eg, external/internal audits) can be improved. Both the effectiveness of QA strategies in practice, and the application of these strategies in outpatient DCSS units require further policy attention.

Banking of human tissue toward the realization of regenerative medicine

Banking of human tissue toward the realization of regenerative medicine

<b>研究用既存ヒト試料(バンキング試料)の利用における一般同意等に関する組織提供患者の意識調査 ～再同意は必要か？～ </b>

<b>研究用既存ヒト試料(バンキング試料)の利用における一般同意等に関する組織提供患者の意識調査 ～再同意は必要か？～ </b>

Production of an osteoinductive demineralised bone matrix powder without the use of organic solvents

Demineralised bone matrix (DBM) is produced by grinding cortical bone into a powder, sieving the powder to obtain a desired size range and then demineralising the powder using acid. Protocols for the production of DBM powder have been published since 1965 and the powder can be used in lyophilised form or it can be mixed with a carrier to produce a paste or putty. The powder is generally produced from cortical bone which has been processed to remove blood, bone marrow and bone marrow components, including fat. Removal of fat is accomplished by incorporating incubation in an organic solvent, often chloroform, chloroform/methanol or acetone. The use of organic solvents in a clean room environment in a human tissue bank is problematic and involves operator exposure and the potential for the solvent to be trapped in air filters or recirculated throughout the clean room suite. Consequently, in this study, we have developed a cortical bone washing step which removes fat/lipid without the use of an organic solvent. Bone was prepared from six femoral shafts from three donors by dissecting soft tissue and bisecting the shaft, the shafts were then cut into ~9-10 cm lengths. These struts were then taken through a series of hot water washes at 56-59 °C, centrifugation and decontamination steps. Washed cortical struts were then lyophilised before being ground with a compressed air milling machine. The ground bone was sieved, demineralised, freeze-dried and terminally sterilised with a target dose of 25 kGy gamma irradiation. The DBM powder was evaluated for residual calcium content, in vitro cytotoxicity and osteoinductivity by implantation into the muscle of an athymic mouse. Data indicated that in addition to removing in excess of 97% DNA and extractable soluble protein, the washing protocol reduced lipid 10,000-fold. The processed bone was easily ground without clogging the grinder; the sterilised DBM powder was not cytotoxic but was osteoinductive in the animal model. Therefore, we have developed a method of producing osteoinductive DBM without the need to use organic solvents.

Lutein and Preterm Infants With Decreased Concentrations of Brain Carotenoids

Lutein and zeaxanthin are dietary carotenoids that may influence visual and cognitive development. The objective of this study was to provide the first data on distribution of carotenoids in the infant brain and compare concentrations in preterm and term infants. Voluntarily donated brain tissues from 30 infants who died during the first 1.5 years of life were obtained from the Eunice Kennedy Shriver National Institute of Child Health and Human Development Brain and Tissue Bank. Tissues (hippocampus and prefrontal, frontal, auditory, and occipital cortices) were extracted using standard lipid extraction procedures and analyzed using reverse-phase high-pressure liquid chromatography. Lutein, zeaxanthin, cryptoxanthin, and β-carotene were the major carotenoids found in the infant brain tissues. Lutein was the predominant carotenoid accounting for 59% of total carotenoids. Preterm infants (n = 8) had significantly lower concentrations of lutein, zeaxanthin, and cryptoxanthin in their brain compared with term infants (n = 22) despite similarity in postmenstrual age. Among formula-fed infants, preterm infants (n = 3) had lower concentrations of lutein and zeaxanthin compared with term infants (n = 5). Brain lutein concentrations were not different between breast milk-fed (n = 3) and formula-fed (n = 5) term decedents. In contrast, term decedents with measurable brain cryptoxanthin, a carotenoid that is inherently low in formula, had higher brain lutein, suggesting that the type of feeding is an important determinant of brain lutein concentrations. These data reveal preferential accumulation and maintenance of lutein in the infant brain despite underrepresentation in the typical infant diet. Further investigation on the impact of lutein on neural development in preterm infants is warranted.

Factors affecting total dissolved solids concentration of γ-ray-irradiated aqueous hexamethylenetetramine solution: A dosimetric study

A new γ-ray-radiation dosimetric system (TDS-HMTA), comprising a 'total dissolved solids (TDS)' meter and 0.02 M aqueous hexamethylenetetramine (HMTA) solution, is introduced for medical and biological applications. Gamma-ray radiolysis of aqueous HTMA solutions increases the concentrations (ppm) of TDS, which is measured by the TDS meter. The effects of HMTA concentration, absorbed radiation dose, absorbed dose rate, and storage time on the TDS concentration of irradiated HMTA solutions were studied. It was found that 0.02 M aqueous HMTA solution yields the highest sensitivity to γ-ray-radiation according to TDS concentration measurements. The effect of absorbed radiation dose was studied in the range 1.64–435.5 kGy. The TDS concentration increases linearly up to the maximum of the studied absorbed radiation dose range (R2 = 0.9965). The overall coefficient of variation (CV %) associated with TDS concentration measurements of 0.02 M HMTA solution as a function of absorbed dose was found to be 0.732%. The effect of dose rate on the TDS concentration was studied in the range 0.33–3.31 kGy/h. It was found, also, that the TDS concentration is relatively stable over a storage period of 144 h after irradiation with different doses. The tissue equivalency of 0.02 M aqueous HMTA solutions allow it to be used for radiation dose measurement during sterilization in human tissue banks. Therefore, this system (TDS–HMTA) could be considered as a promising candidate for γ-ray radiation dosimetry in technical, medical and research fields.

Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank

IntroductionOur efforts to prevent and treat breast cancer are significantly impeded by a lack of knowledge of the biology and developmental genetics of the normal mammary gland. In order to provide the specimens that will facilitate such an understanding, The Susan G. Komen for the Cure Tissue Bank at the IU Simon Cancer Center (KTB) was established. The KTB is, to our knowledge, the only biorepository in the world prospectively established to collect normal, healthy breast tissue from volunteer donors. As a first initiative toward a molecular understanding of the biology and developmental genetics of the normal mammary gland, the effect of the menstrual cycle and hormonal contraceptives on DNA expression in the normal breast epithelium was examined.MethodsUsing normal breast tissue from 20 premenopausal donors to KTB, the changes in the mRNA of the normal breast epithelium as a function of phase of the menstrual cycle and hormonal contraception were assayed using next-generation whole transcriptome sequencing (RNA-Seq).ResultsIn total, 255 genes representing 1.4% of all genes were deemed to have statistically significant differential expression between the two phases of the menstrual cycle. The overwhelming majority (221; 87%) of the genes have higher expression during the luteal phase. These data provide important insights into the processes occurring during each phase of the menstrual cycle. There was only a single gene significantly differentially expressed when comparing the epithelium of women using hormonal contraception to those in the luteal phase.ConclusionsWe have taken advantage of a unique research resource, the KTB, to complete the first-ever next-generation transcriptome sequencing of the epithelial compartment of 20 normal human breast specimens. This work has produced a comprehensive catalog of the differences in the expression of protein-coding genes as a function of the phase of the menstrual cycle. These data constitute the beginning of a reference data set of the normal mammary gland, which can be consulted for comparison with data developed from malignant specimens, or to mine the effects of the hormonal flux that occurs during the menstrual cycle.

Spatial cluster analysis of nanoscopically mapped serotonin receptors for classification of fixed brain tissue

We present a cluster spatial analysis method using nanoscopic dSTORM images to determine changes in protein cluster distributions within brain tissue. Such methods are suitable to investigate human brain tissue and will help to achieve a deeper understanding of brain disease along with aiding drug development. Human brain tissue samples are usually treated postmortem via standard fixation protocols, which are established in clinical laboratories. Therefore, our localization microscopy-based method was adapted to characterize protein density and protein cluster localization in samples fixed using different protocols followed by common fluorescent immunohistochemistry techniques. The localization microscopy allows nanoscopic mapping of serotonin 5-HT1A receptor groups within a two-dimensional image of a brain tissue slice. These nanoscopically mapped proteins can be confined to clusters by applying the proposed statistical spatial analysis. Selected features of such clusters were subsequently used to characterize and classify the tissue. Samples were obtained from different types of patients, fixed with different preparation methods, and finally stored in a human tissue bank. To verify the proposed method, samples of a cryopreserved healthy brain have been compared with epitope-retrieved and paraffin-fixed tissues. Furthermore, samples of healthy brain tissues were compared with data obtained from patients suffering from mental illnesses (e.g., major depressive disorder). Our work demonstrates the applicability of localization microscopy and image analysis methods for comparison and classification of human brain tissues at a nanoscopic level. Furthermore, the presented workflow marks a unique technological advance in the characterization of protein distributions in brain tissue sections.

Mesenchymal stem cell isolation from human umbilical cord tissue: understanding and minimizing variability in cell yield for process optimization.

Human tissue banks are a potential source of cellular material for the nascent cell-based therapy industry; umbilical cord (UC) tissue is increasingly privately banked in such facilities as a source of mesenchymal stem cells for future therapeutic use. However, early handling of UC tissue is relatively uncontrolled due to the clinical demands of the birth environment and subsequent transport logistics. It is therefore necessary to develop extraction methods that are robust to real-world operating conditions, rather than idealized operation. Cell yield, growth, and differentiation potential of UC tissue extracted cells was analyzed from tissue processed by explant and enzymatic digestion. Variability of cell yield extracted with the digestion method was significantly greater than with the explant method. This was primarily due to location within the cord tissue (higher yield from placental end) and time delay before tissue processing (substantially reduced yield with time). In contrast, extraction of cells by explant culture was more robust to these processing variables. All cells isolated showed comparable proliferative and differentiation functionality. In conclusion, given the challenge of tightly controlled operating conditions associated with isolation and shipping of UC tissue to banking facilities, explant extraction of cells offers a more robust and lower-variability extraction method than enzymatic digestion.

Professor Réal Lapointe: the impact of portal embolization on postoperative morbidity and mortality after liver resection.

Professor Real Lapointe (Figure 1) is the Director of General Surgery Division in University of Montreal, and Chief of Hepatobiliary and Pancreatic (HBP) Surgery and Liver Transplantation Department in Centre Hospitalier de l’Universite de Montreal (CHUM). As holder of the Roger Des Groseillers Chair in HBP Surgical Oncology, his clinical and translational research focuses on evaluating new drugs in HBP oncology and different surgical approaches for colorectal liver metastases, and on developing new biological markers from human tissue bank from patients with primary and secondary liver cancer (e.g., hepatic colorectal metastases), pancreatic and bile duct cancer.

Abstract 5597: Molecular classification of glioblastoma multiforme using isoform-level gene expression signatures.

Abstract Glioblastomamultiforme (GBM) is one of the most deadly cancers in adult and even with aggressive therapy prognosis is poor with a median survival of about 15 months. Recent large scale genomic studies have shed light on the molecular mechanisms of cancer and shown the existence of distinct molecular sub-groups of GBM whose identification is the key to develop specific therapeutic regimen for each GBM group. Since transcriptome studies, by us and others, have discovered that majority of genes produce multiple protein isoforms, which could be involved in multiple functional pathways, we hypothesized that the isoform-level expression profiling will generate better classification to identify themolecular sub-groups of GBM. To test this hypothesis, we performed isoform-level analysis of the exon array expression data for GBM patient samples from the TCGA data portal, and discovered that isoform-level analysis identifies 2.5 fold more differentially expressed transcript variants than differentially expressed genes captured by gene-level analysis, indicating that isoform-level expression profiling is more sensitive in identifying molecular changes among GBM patients. Next, we applied consensus non-negative matrix factorization (NMF) clustering method, based on isoform-level expression ofmost variable isoforms and effectively grouped the GBM samples into 4 sub-groups with significant (p=0.0103) survival differences between the groups. In contrast, though clustering based on gene-level expression produced four homogenous groups there was no significant survival difference among the sub-groups. Based on the prognostic value of the molecular sub-groups, our goal was to build a classifier that can assign each GBM patient a molecular sub-group.We compared the prediction accuracy of a gene based vs an isoform based classifier to identify sub-group and found that isoform based classifier is a better predictor (85%vs90%). Using the Random forest feature selection we have build a classifier based on the expression of 147 isoforms that is ∼91% accurate and have developed a high-throughput RT-qPCR assay to measure the expression of these discriminatory isoforms. We have successfully validated the classifier to identify the molecular sub-group in an independent cohort of GBM patient samples from the Human Brain Tumor Tissue bank at University of Pennsylvania.Our study has led to the development of a classification assay for GBM patient sub-grouping, which can be useful in developing targeted drug therapy for each sub-group, and suggests that isoform based expression analysis can lead to better molecular classification of cancer, a requirement for the quest of personalized therapy. Citation Format: Sharmistha Pal, Yingtao Bi, Lukasz Macyszyn, Louise C. Showe, Donald M. O'Rourke, Ramana V. Davuluri. Molecular classification of glioblastoma multiforme using isoform-level gene expression signatures. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5597. doi:10.1158/1538-7445.AM2013-5597 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.