Exosomal Thomsen-Friedenreich Glycoantigen: A New Liquid Biopsy Biomarker for Lung and Breast Cancer Diagnoses.

Exosomes are nanosized extracellular vesicles released by cells to transport biomolecules such as proteins and RNAs for intercellular communication. Exosomes play important roles in cancer development and metastasis; therefore, they have emerged as potential liquid biopsy biomarkers for cancer screening, diagnosis, and management. Many exosome cargos, including proteins, RNAs, and lipids, have been extensively investigated as biomarkers for cancer liquid biopsy. However, carbohydrates, an important type of biomolecule, have not yet been explored for this purpose. In this study, we reported a new exosomal carbohydrate biomarker, α-linked Thomsen-Friedenreich glycoantigen (TF-Ag-α; Galß1-3GalNAc-α). To translate our discovery into clinical settings, we developed a surface plasmon resonance-based assay which utilized a unique mAb, JAA-F11, with high specificity to measure the levels of exosomal TF-Ag-α in blood. To the best of our knowledge, we are the first to demonstrate that exosomes carry TF-Ag-α. We detected exosomal TF-Ag-α in as low as 10 µL serum samples from patients with cancer, but in contrast, levels were negligible in those from normal controls. With a total of 233 patients with cancer and normal controls, we showed that exosomal TF-Ag-α detected lung cancer (n = 60) and breast cancer (n = 95) from normal controls (n = 78) with ≥95% and ≥97% accuracy, respectively. These results demonstrated that exosomal TF-Ag-α is a potential liquid biopsy biomarker for cancer diagnosis. SIGNIFICANCE: Exosomes or small extracellular vesicles have emerged as potent biomarkers of cancer liquid biopsy. We discovered a new exosomal carbohydrate marker, TF-Ag-α (Galß1-3GalNAc-α), and showed that exosomal TF-Ag-α detected both lung and breast cancers with >95% accuracy. Our findings demonstrated that exosomal TF-Ag-α is a promising liquid biopsy biomarker for cancer screening and early detection.

Spontaneously ruptured endometriomas presenting with symptoms and imaging findings worrisome for carcinomatosis: A case report.

INTRODUCTION AND IMPORTANCE: Endometriomas are the most common presenting subtype of endometriosis. Although most endometriomas are asymptomatic, patients can rarely present acutely with spontaneous rupture causing diffuse peritonitis and severe systemic inflammatory response. CASE PRESENTATION: Here we describe a case of ruptured endometriomas in a 26-year-old nulligravid female with a history of heavy menses, progressive abdominal distension, and a recent urinary tract infection. The patient presented to the emergency department with upper abdominal pain radiating to her back with associated nausea. Computed tomography (CT) scan demonstrated diffuse ascites with a large, multilobulated, and multicystic septated mass arising in the right pelvis and extending into the lower abdomen. Findings were concerning for peritoneal carcinomatosis and the patient was admitted for evaluation. She developed progressive signs of sepsis and was emergently brought to the operating room for surgical exploration on hospital day (HD) number two. She was found to have ruptured pelvic cysts arising from both ovaries with diffuse contamination of the abdomen by cyst contents and bilateral salpingo-oophorectomy (BSO) was performed. Final pathology demonstrated benign bilateral endometriomas. CLINICAL DISCUSSION: Endometrioma rupture is extremely rare and imaging findings may appear to represent disseminated peritoneal malignancy. CT findings demonstrating a pelvic mass with concurrent ascites should raise clinical suspicion for ruptured endometrioma, particularly in younger patients. CONCLUSION: Prompt surgical exploration and complete resection of pathologic tissue may be necessary for diagnosis and treatment in some patients with clinical deterioration related to perforated endometriomas. Combined oral contraceptives are recommended in the postoperative period.

Delivery of anti-microRNA-21 by lung-targeted liposomes for pulmonary fibrosis treatment.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder with a low survival rate. Pulmonary fibrosis is one of the complications of COVID-19 and has a high prevalence in COVID-19 patients. Currently, no effective therapies other than lung transplantation are available to cure IPF and post-COVID-19 pulmonary fibrosis. MicroRNAs are small non-coding RNAs that mediate the development and progression of pulmonary fibrosis, thus making them potent drug candidates for this serious disease. MicroRNA-21 (miR-21) promotes not only the differentiation of fibroblasts to myofibroblasts but also epithelial-mesenchymal transition, both of which have been proposed as fundamental processes in pulmonary fibrosis development. Delivery of anti-miR-21 to block the miR-21-associated fibrogenic pathways represents a promising therapy for pulmonary fibrosis. However, microRNA treatment is challenged by quick degradation of RNA in blood, poor cellular uptake, and off-target effects. To overcome these challenges, we developed a lung-targeted, cationic liposome formulation to encapsulate anti-miR-21, enhance its delivery efficiency, and improve the therapeutic efficacy. We optimized the liposome formulation and demonstrated the anti-fibrotic effects using both in vitro and in vivo lung fibrosis models. Our results showed that anti-miR-21 delivered by cationic liposomes suppressed myofibroblast differentiation, reduced the synthesis of extracellular matrix, and inhibited fibrosis progression.

Ruptured granulosa cell tumor of the ovary presenting with catastrophic intra-abdominal hemorrhage: A case report.

INTRODUCTION AND IMPORTANCE: Adult granulosa cell tumor (GCT) is a rare stromal cell neoplasm that most often arises from the ovary. Presenting symptoms are related to external compression of adjacent structures (mass effect) or secretion of hormones such as estrogen. Patients most commonly present with irregular menstruation, postmenopausal bleeding, and abdominal pain. Prolonged estrogen exposure can contribute to endometrial adenocarcinoma development in untreated patients. The highly vascular nature of GCTs can lead to hemorrhagic rupture in rare cases. PRESENTATION OF CASE: We describe a case of adult GCT in a 44-year-old female with a history of irregular menstrual bleeding and anemia. The patient presented with shortness of breath and abdominal pain. Computed tomography (CT) scan demonstrated possible hemorrhagic ascites of unclear etiology and a pelvic mass. The patient was brought to the operating room in hemorrhagic shock for surgical exploration where she was found to have active bleeding of a ruptured ovarian tumor for which she underwent left salpingo-oophorectomy. Postoperative course was unremarkable, and pathology demonstrated ruptured GCT. CLINICAL DISCUSSION: Although rare, ovarian tumors can present with massive bleeding following rupture. Granulosa cell tumors are surreptitious as they grow slowly, and symptoms such as distention, abdominal pain, and irregular vaginal bleeding are nonspecific. CONCLUSION: CT findings demonstrating a pelvic mass in the setting of spontaneous intra-abdominal bleeding should raise clinical suspicion, particularly in patients with histories of menstrual abnormalities. Patients with suspected intra-abdominal hemorrhage due to any cause are best treated by prompt surgical exploration and aggressive resuscitation.

Core needle biopsy wash as a tool for acquiring additional diagnostic material for laboratory testing.

AIMS: To determine if a simple prewash step added to the processing workflow of tissue procurement by a core needle biopsy device will recover enough cells to expand the laboratory testing armamentarium. METHODS: Tissue was obtained from unfixed resection specimens using a core needle device and washed in a buffered solution before fixation. This creates a liquid aliquot from which dislodged cells can be kept and separated from the tissue specimen, the latter of which can then undergo traditional formalin-fixed, paraffin-embedded processing. RESULTS: Cells dislodged from the tissue during the biopsy procedure are recoverable, are representative of the tissue section and of sufficient quantities for additional laboratory testing. CONCLUSIONS: The core needle biopsy wash is an under-recognised and underutilised approach to extending the diagnostic capabilities of the limited amount of targeted material obtained during this common procedure. The ability to recover supplemental amounts of diagnostic material yields great potential as a substrate for a multitude of current and developing laboratory assays.

An iOS App to Expedite the Evaluation of Immunohistochemistry Control Tissue.

Control tissues play a vital role in diagnostic immunohistochemistry. They serve to document that the appropriate antibody was used, on the correct control tissue, and run on optimized conditions. As part of the evolving process of standardization in diagnostic immunohistochemistry, specific tissues have been identified based on agreement by experts in this field capable of serving as the benchmark(s) for several antibodies. These tissues are recommended based on known and predictable levels of strong, weak, and no expression of the antigen being queried. These tissues can be used for positive and negative control purposes, respectively, and are regarded as primary positive and/or negative external control tissue. If some of these tissues are not present in sufficient numbers in a laboratory's archive for daily use, they can still be used as the basis to evaluate other tissues that are not as well characterized and chosen to serve as secondary and external positive controls. In this manner, either the former or latter can function as external positive and negative control tissue for the quality assurance of immunohistochemistry done in a laboratory. The use of the selected tissues may be applicable to the detection of several different antigens by a number of separate antibodies, with differences in the staining of specific cells or the localization in staining within those cells. However, the amount of information needed to be familiar with to render a correct interpretation of the control tissue may prove daunting. One means of dealing with this problem would be to create a document capable of serving as a reference guide. Traditional types of references, however, may suffer from issues related to convenience, updating, and mobility. Herein we describe a mobile device application (app) created to serve as a reference for control block tissues. This app can capably house and easily retrieve all the relevant information on all the antibodies and their respective control tissues in a laboratories test menu, thus enabling the use of standardized tissues as control material and spreading the ability to perform immunohistochemical quality control to individual pathologists.

Quantitative proteomic profiling of paired cancerous and normal colon epithelial cells isolated freshly from colorectal cancer patients.

PURPOSE: The heterogeneous structure in tumor tissues from colorectal cancer (CRC) patients excludes an informative comparison between tumors and adjacent normal tissues. Here, we develop and apply a strategy to compare paired cancerous (CEC) versus normal (NEC) epithelial cells enriched from patients and discover potential biomarkers and therapeutic targets for CRC. EXPERIMENTAL DESIGN: CEC and NEC cells are respectively isolated from five different tumor and normal locations in the resected colon tissue from each patient (N = 12 patients) using an optimized epithelial cell adhesion molecule (EpCAM)-based enrichment approach. An ion current-based quantitative method is employed to perform comparative proteomic analysis for each patient. RESULTS: A total of 458 altered proteins that are common among >75% of patients are observed and selected for further investigation. Besides known findings such as deregulation of mitochondrial function, tricarboxylic acid cycle, and RNA post-transcriptional modification, functional analysis further revealed RAN signaling pathway, small nucleolar ribonucleoproteins (snoRNPs), and infection by RNA viruses are altered in CEC cells. A selection of the altered proteins of interest is validated by immunohistochemistry analyses. CONCLUSION AND CLINICAL RELEVANCE: The informative comparison between matched CEC and NEC enhances our understanding of molecular mechanisms of CRC development and provides biomarker candidates and new pathways for therapeutic intervention.

Front-end genomics: using an alternative approach for the recovery of high-quality DNA from core needle biopsies.

AIMS: Determine whether a simple prewash step will provide adequate amounts of high-quality DNA from core needle biopsies for molecular sequencing studies. METHODS: The quantitative and qualitative metrics of DNA recovered from core needle biopsies processed either by 1) formalin fixation and paraffin embedding (FFPE), 2) cells recovered after the core needle biopsy was washed, and 3) frozen sections of the core needle biopsy tissue were evaluated and compared to one another. RESULTS: Fairly equivalent amounts of DNA can be obtained from cells recovered from a prewash step relative to the FFPE and frozen section samples. The number of amplifiable DNA in the wash sample was greater than that from the FFPE samples. The average molecular size of DNA in the wash sample was greater than that of both the FFPE and frozen samples. CONCLUSIONS: Although more starting material in terms of the number of cells was present in both the FFPE and frozen section samples than the wash samples, equivalent to better results were obtained from the latter with regard to quality. This approach may be a means to better aliquot the diminutive amounts of tissue associated with core needle biopsies, allowing dissociated cells to be dedicated for molecular studies while keeping the tissue intact for morphological studies.

Core Needle Biopsy Wash Optimization: Enabling Specimen Integrity for both Cytological and Histological Evaluation.

BACKGROUND: The recovery of cells after washing core needle biopsies represents an under-utilized approach to extend the diagnostic capacity of these diminutive specimens. Recovery of these cells can be dedicated for molecular studies so that the biopsy itself can be used apropos for its intended purpose, diagnosis. Non-enzymatic and enzymatic reagents have the potential to increase the number of cells dissociating from the tissue core, but can also negatively impact the quality of the tissue itself. MATERIALS AND METHODS: Three different means (phosphate-buffered saline, a non-enzymatic and an enzymatic solution) were used to wash core needle biopsies. The washed cells were recovered by traditional preparatory methods and evaluated for cellularity and cytomorphology. The post-washed cores were processed by formalin fixation, paraffin embedding and evaluated for integrity and morphological quality. RESULTS: The enzymatic solution damaged both the cytological and tissue specimens, while the saline and non-enzymatic process allowed for the comparable recovery of cells and tissue for evaluation. CONCLUSION: Adequate numbers of cells are dissociated from the tissue core when needle biopsies are washed. The recovery and preservation of cells and tissue for morphological interpretation was optimal when solutions devoid of enzymes were used for washing.

Maximizing derivable information from cytologic specimens for pathologic and molecular diagnostics.

INTRODUCTION: The advent of precision medicine will increase the demand for molecular testing on patient tumor specimens. Cytology specimens have been shown to be ideal substrates for molecular testing, but their often paucicellular nature can lead to conflicts in prioritizing sample management. A microfluidic platform was investigated to determine whether cytologic and molecular data could be procured from the same cells, obviating the need for partitioning a sample by multiplexing it instead. MATERIALS AND METHODS: Cytology samples were created from a tissue source, stained with a supravital dye, and enriched using immunomagnetic beads. These cells and the attached immunomagnetic beads were then run through a microfluidic channel, temporarily immobilized for cytologic examination, and then recovered. The cytologic characteristics of these cells was compared with cells from the same source prepared by conventional cytologic preparatory means. DNA was extracted from the cells recovered from the microfluidic channel and the nature of their integrity was assessed. RESULTS: Cytologic features between cells run in a microfluidic channel and prepared by conventional means were similar. The DNA recovered from the cells run through the microfluidic channel was of high molecular weight. CONCLUSIONS: Microfluidics enables multiplex testing of cytologic specimens, allowing for cytology-based diagnostic examination and recovery of high-quality DNA. This approach will be of particular benefit for cytology specimens that are paucicellular and will need molecular testing.

Conditional prerequisites for microchannel cytologic analysis on wet mount (fluid-based) biopsies.

BACKGROUND: Advanced capabilities in genomic sequencing developed in the research sector will soon enter the clinical arena. Issues such as the proportioning of patient specimen material for traditional bright-field microscopic evaluation or dedication for molecular analysis will intensify, particularly in situations of small core biopsies. Microfluidics appears aptly suited as a platform capable of allowing traditional cytologic diagnostics and downstream molecular analysis from the same specimen. However, clarification is needed to determine that forces which act on cells in a fluidic environment do not drastically alter their cytologic features. METHODS: Cells were processed for flow-through in a microfluidic channel and evaluated qualitatively and quantitatively for alterations due to fluid-shear stress or anoikis. RESULTS: Processing caused separation of cells from cohesive clusters to smaller groups and individual cells, leading to greater variation in parameters associated with the nucleus in nontumor cells but no significant change in tumor cells. These differences were most readily apparent by quantitative measures, and to a lesser extent, qualitative evaluation. Time-dependent processing played a larger role in cytologic alteration than fluid-shear stress for nontumor cells. CONCLUSIONS: Passage of cells through a microfluidic channel is a feasible approach that can be integrated into future platforms intent on integrating cytologic assessment of cells with recovery of the same cells for downstream assays.

ERBB2 juxtamembrane domain (trastuzumab binding site) gene mutation is a rare event in invasive breast cancers overexpressing the ERBB2 gene.

The recent development of targeted therapies using monoclonal antibodies has added new dimensions to breast cancer treatment. Trastuzumab has been added to the regimens that contain chemotherapeutic agents, which has improved the clinical outcomes of patients in both the adjuvant and metastatic settings. However, trastuzumab resistance, both de novo and acquired, continues to be problematic. There have been scattered studies reporting ERBB2 gene mutation, but nothing is currently known about the ERBB2 binding site mutations. In the current study, we examined the ERBB2 juxtamembrane domain trastuzumab binding site for mutations in invasive breast cancers overexpressing ERBB2. Pure tumor cells of 54 breast cancer patients were procured using laser capture microdissection. Two polymerase chain reaction primer pairs were designed to amplify the trastuzumab binding site sequence. The polymerase chain reaction product was sequenced. Standard clinicopathological data were recorded. For the 54 patients, there was one (2%) case that showed missense point mutation in exon 17 (H559A). There were nine patients treated with trastuzumab in the metastatic setting, none of which had gene mutation. Therefore, we conclude that ERBB2 juxtamembrane domain (trastuzumab binding site) gene mutation is a rare event in breast cancer. Although it is unclear whether this substitution would result in trastuzumab target therapy resistance, this would not account for the relatively high frequency of this resistance encountered clinically.

Immunohistochemical detection of arginine methylated proteins (MeRP) in archival tissues.

AIMS: To (i) determine whether methylarginine-specific antibodies can be employed for standard immunohistochemical analysis of paraffin-embedded tissues, (ii) analyse methylarginine expression in normal and neoplastic tissues and (iii) correlate methylarginine expression with that of protein arginine methyltransferase (PRMT1), the predominant cellular arginine methyltransferase. METHODS AND RESULTS: Immunohistochemistry of normal and cancer tissues was performed utilizing three commercial polyclonal antibodies: anti-methylarginine-specific antibody (anti-mRG) raised against a methylarginine peptide, Control antibody (anti-RG), a control antiserum raised against a corresponding arginine peptide without any methylated residues and anti-PRMT1. Nuclear and/or cytoplasmic methylarginine expression was detected in all keratinized and non-keratinized epithelia. A preliminary survey of a series of thyroid, pancreatic, colonic and gastric cancers identified a different pattern of methylarginine expression in comparison with normal tissue. A correlation between methylarginine staining and PRMT1 expression was found in all normal and cancer tissues analysed. CONCLUSION: Methylarginine-specific antibodies are capable of recognizing methylarginine proteins (MeRP) in paraffin-embedded tissues. Methylarginine proteins are expressed widely and show differences in subcellular localization in various organs and neoplastic conditions. The efficient detection of methylproteins by standard immunohistochemistry provides a new tool to investigate the role of methylarginine proteins (MeRP) in biological processes including carcinogenesis.

Breast carcinoma with amplified HER2: a gene expression signature specific for trastuzumab resistance and poor prognosis.

Recent trials have shown remarkable efficacy from combined trastuzumab and chemotherapy in the adjuvant setting of breast cancer. In spite of these successes, refractory breast cancer has emerged as a clinically problematic outcome for a subset of patients managed this way. In an effort to clarify and optimize the treatment regimens for breast cancer patients who are candidates to receive trastuzumab, we sought to analyze whether a distinctive genetic signature could be characterized that would reliably predict the treatment outcome. The ability to predict who will respond and who will become refractory to this agent will allow for improved, rational clinical management of these patients and further stratify the personalized nature of this treatment regimen. In this study, 41 consecutive cases of breast carcinoma with well-documented amplification of the human epidermal growth factor receptor-2 gene and corresponding banked fresh-frozen tissue were identified and divided into two separate groups based on whether they received trastuzumab or not. The first group consisted of 12 patients who had received trastuzumab in the adjuvant setting, of which three later experienced tumor recurrence. The second group consisted of 10 patients not treated with trastuzumab, of which 6 were later found to have recurrence. Differentially expressed genetic profiles were determined using human genome-wide Illumina Bead Microarrays. The differentially expressed genes for non-recurrence vs recurrence in the trastuzumab-treated group were distinct from those in the same comparison group in the untreated group. Differential expression of key genes identified in this study might offer an insight into a possible mechanism of trastuzumab resistance in breast carcinoma, and may emerge as potential predictive biomarkers indicative of trastuzumab resistance.

Normal colon epithelium: a dataset for the analysis of gene expression and alternative splicing events in colon disease.

BACKGROUND: Studies using microarray analysis of colorectal cancer have been generally beleaguered by the lack of a normal cell population of the same lineage as the tumor cell. One of the main objectives of this study was to generate a reference gene expression data set for normal colonic epithelium which can be used in comparisons with diseased tissues, as well as to provide a dataset that could be used as a baseline for studies in alternative splicing. RESULTS: We present a dependable expression reference data set for non-neoplastic colonic epithelial cells. An enriched population of fresh colon epithelial cells were obtained from non-neoplastic, colectomy specimens and analyzed using Affymetrix GeneChip EXON 1.0 ST arrays. For demonstration purposes, we have compared the data derived from these cells to a publically available set of tumor and matched normal colon data. This analysis allowed an assessment of global gene expression alterations and demonstrated that adjacent normal tissues, with a high degree of cellular heterogeneity, are not always representative of normal cells for comparison to tumors which arise from the colon epithelium. We also examined alternative splicing events in tumors compared to normal colon epithelial cells. CONCLUSIONS: The findings from this study represent the first comprehensive expression profile for non-neoplastic colonic epithelial cells reported. Our analysis of splice variants illustrate that this is a very labor intensive procedure, requiring vigilant examination of the data. It is projected that the contribution of this set of data derived from pure colonic epithelial cells will enhance studies in colon-related disease and offer a vital baseline for studies aimed at elucidating the mechanisms of alternative splicing.

Importance of cell-procurement methods in transforming personalized cancer treatment from concept to reality.

The much-anticipated promise of personalized cancer care is to deliver therapies best suited for a patient based on the knowledge of that individual's genetics and tumor characteristics. This transformative approach will require many changes in the scientific and medical community, one of the most fundamental being the direct study of human tissue biospecimens. Biospecimens will be integral to the elucidation of biomarkers that will help identify and serve as potential diagnostic, prognostic and therapeutic targets in cancer. Despite the vast repositories of fixed-tissue biospecimens that are in existence, a number of flaws exist that hinder their reliable use as instruments from which to enable personalized cancer research and clinical care. A new view of biospecimen worth in the future will mandate that the molecules within its cells are reflective of their in vivo state, and not altered by external variables introduced during the excision and processing of the biospecimen. Research on biospecimen collection is a legitimate field of study that will be necessary for personalized cancer care to become a reality.

A comparative analysis of two tissue procurement approaches for the genomic profiling of clinical colorectal cancer samples.

BACKGROUND: The basis for personalized medicine is the creation of a repository of knowledge about the genetic alterations involved in disease processes. Integral to achieving this goal is the querying of well-preserved, high-quality human tissue samples. Making these findings relevant involves the interrogation of large numbers of samples. The pace with which changes have occurred versus the potential pace with which changes can occur may be indicative of problems associated with traditional approaches on collecting biospecimens. Therefore, transforming personalized medicine from concept to reality may require an alternative approach in the field of tissue specimen procurement. MATERIALS AND METHODS: "Exfoliation and Enrichment" (EE), a recently described rapid and cost-effective approach for procuring cells, was utilized and assessed relative to a more traditional but temporally and economically comparable approach. Material from the same tumor sample, one collected by EE but the other frozen, were procured, the DNA extracted, and the samples analyzed at the global and gene-specific level by array comparative genomic hybridization and quantitative polymerase chain reaction, respectively. RESULTS: Both approaches resulted in the rapid procurement and retrieval of well-preserved cells and nucleic acids. The presence of "contaminating" normal cells in the more traditional approach masked the significance of genetic gains and losses, findings that were more readily apparent from the material derived by the EE method. CONCLUSION: The EE approach represents a cost-effective alternative to traditional cell-procurement methods that results in the generation of superior genomic data.

Universal Reference RNA is not a representative normal sample for oligonucleotide microarray studies.

Translational research has been defined as the scientific study using human material that will ultimately generate patient specific data. A major caveat in human directed study is the availability of high quality and quantities of patient derived homogeneous cells for analysis. Whereas there exist sources for which tumor tissue and blood samples can be made available, the same cannot be said for normal tissue. The absence of normal control tissue has led to the creation of pooled cell lines and tissues for purchase known as "reference RNA". Although initially created for purposes of standardization, the difficulty associated with acquiring normal tissue has led some investigators to use sources of universal pooled RNA for comparative analysis with clinical tissue specimens. In order to study the effects of using Universal Reference RNA on expression profiling experiments we have evaluated the performance of universal RNA compared to RNA obtained from a purified population of colon epithelial cells in defining a set of altered transcripts in colon cancer.

An exfoliation and enrichment strategy results in improved transcriptional profiles when compared to matched formalin fixed samples.

BACKGROUND: Identifying the influence formalin fixation has on RNA integrity and recovery from clinical tissue specimens is integral to determining the utility of using archival tissue blocks in future molecular studies. For clinical material, the current gold standard is unfixed tissue that has been snap frozen. Fixed and frozen tissue however, both require laser capture microdissection to select for a specific cell population to study. The recent development of a sampling method capable of obtaining a viable, enriched cell population represents an alternative option in procuring cells from clinical material for molecular research purposes. The expression profiles of cells obtained by using this procurement approach, in conjunction with the profiles from cells laser capture microdissected from frozen tissue sections, were compared to the expression profiles from formalin fixed cells to determine the influence fixation has on expression profiles in clinical material. METHODS: Triplicate samples of non-neoplastic colonic epithelial cells were recovered from a hemicolectomy specimen using three different procurement methods from the same originating site: 1) an exfoliation and enrichment strategy 2) laser capture microdissection from formalin fixed tissue and 3) laser capture microdissection from frozen tissue. Parameters currently in use to assess RNA integrity were utilized to assess the quality of recovered RNA. Additionally, an expression microarray was performed on each sample to assess the influence each procurement technique had on RNA recovery and degradation. RESULTS: The exfoliation/enrichment strategy was quantitatively and qualitatively superior to tissue that was formalin fixed. Fixation negatively influenced the expression profile of the formalin fixed group compared to both the frozen and exfoliated/enrichment groups. CONCLUSION: The exfoliation/enrichment technique represents a superior alternative in tissue procurement and RNA recovery relative to formalin fixed tissue. None of the deleterious effects associated with formalin fixation are encountered in the exfoliated/enriched samples because of the absence of its use in this protocol. The exfoliation/enrichment technique also represents an economical alternative that will yield comparable results to cells enriched by laser capture microdissection from frozen tissue sections.