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PubMed2006

Unravelling the proteome of formalin-fixed paraffin-embedded tissue.

Biofluid detectable biomarkers that originate at the site of diseased tissues would be advantageous, in that, they may provide mechanistic information concerning the manifestation and progression of the disease. Unfortunately, tissue biopsies are precious samples that can generally be acquired in small amounts due to the invasive nature of the sample collection. One of the foundations of pathological diagnosis for decades has been from formalin-fixed paraffin-embedded (FFPE) tissues, of which a vast archive exists worldwide. These tissues have also been widely used for immunohistochemistry and in situ hybridization studies examining for expression of specific proteins or transcripts. Unfortunately, the ability to analyse FFPE tissues using mass spectrometry (MS) has been essentially non-existent until recently. In this review, methods that allow the extraction of peptides from FFPE tissues and their proteomic analysis using MS are described. The ability to identify the proteins extracted from FFPE tissues allows comparative analyses that enable the potential discovery of novel biomarkers at the site of the diseased tissue.

Animals↗

PubMed2006

Direct quantification of gene expression in homogenates of formalin-fixed, paraffin-embedded tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.

DNA↗

PubMed2005

A practical approach to the clinical diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour and other small round cell tumours sharing EWS rearrangement using new fluorescence in situ hybridisation probes for EWSR1 on formalin fixed, paraffin wax embedded tissue.

BACKGROUND: Over 90% of Ewing's sarcoma/primitive neuroectodermal tumour (ES/PNET) cases have the t(11;22) chromosomal rearrangement, which is also found in other small round cell tumours, including desmoplastic small round cell tumour (DSRCT) and clear cell sarcoma (CCS). Although this rearrangement can be analysed by fluorescence in situ hybridisation (FISH) using routinely formalin fixed, paraffin wax embedded (FFPE) tissues when fresh or frozen tissues are not available, a sensitive and convenient detection method is needed for routine clinical diagnosis. AIMS: To investigate the usefulness of newly developed probes for detecting EWS rearrangement resulting from chromosomal translocations using FISH and FFPE tissue in the clinical diagnosis of ES/PNET, DSRCT, and CCS. METHODS: Sixteen ES/PNETs, six DSRCTs, and six CCSs were studied. Three poorly differentiated synovial sarcomas, three alveolar rhabdomyosarcomas, and three neuroblastomas served as negative controls. Interphase FISH analysis was performed on FFPE tissue sections with a commercially available EWSR1 (22q12) dual colour, breakapart rearrangement probe. RESULTS: One fused signal and one split signal of orange and green, demonstrating rearrangement of the EWS gene, was detected in 14 of 16 ES/PNETs, all six DRSCTs, and five of six CCSs, but not in the negative controls. CONCLUSIONS: Interphase FISH using this newly developed probe is sensitive and specific for detecting the EWS gene on FFPE tissues and is of value in the routine clinical diagnosis of ES/PNET, DSRCT, and CCS.

Adolescent↗

PubMed2005

Molecular diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor in formalin-fixed paraffin-embedded tissues by RT-PCR and fluorescence in situ hybridization.

Recent studies have suggested that formalin-fixed paraffin-embedded (FFPE) tissues can be used for molecular analyses by fluorescence in situ hybridization (FISH) and RT-PCR. We analyzed 18 cases of ES/PNET for the t(11;22)(q24;q12) and t(21;22)(q22;q12) fusion transcripts by RT-PCR and analyzed for EWS translocation by interphase FISH with a dual color fusion probe to compare these two approaches directly. RT-PCR detected 13 (72%) EWS-FLI-1 fusions (type I=10, type II=3) and 2 (11%) EWS-ERG fusions. Three cases could not be evaluated because the housekeeping gene phosphoglycerate kinase (internal mRNA control) was not amplified. FISH was diagnostic in 15 of 18 cases (83%). There were three discordant cases between RT-PCR and FISH (concordance of 83%). Using a combination of RT-PCR and FISH, the results were complementary. One advantage of RT-PCR analysis was that subtypes of EWS translocation could be determined specifically (type I, type II and ERG). These findings indicate that because of the difficulties and limitations associated with the molecular analysis of FFPE tissues, a combination of RT-PCR and FISH may be a better approach to enhance the sensitivity and accuracy of detecting ES/PNET translocations in FFPE tissues with suboptimally preserved nucleic acids.

Base Sequence↗

PubMed2006

Mass spectrometric analysis of formalin-fixed paraffin-embedded tissue: unlocking the proteome within.

The predominance of tissues stored worldwide in hospitals and clinical laboratories exist in formalin-fixed paraffin-embedded (FFPE) blocks that are generated by simple and well-established protocols. Although generation of FFPE tissues has facilitated their characterization by such techniques as histopathology, they have proven refractory to biomarker discovery investigations using state-of-the-art MS-based proteomic methodologies. Very recently new methods have been developed that enable proteins extracted from FFPE tissues to be analyzed by MS. This review will highlight and discuss those efforts that have led to this exciting recent progress. Although these developments are quite new, the ability to conduct MS-based proteomic analyses of FFPE tissues opens heretofore intractable clinical samples for discovery-based biomarker research.

Cryopreservation↗

PubMedDate not supplied

Detection of chicken anaemia virus DNA from formalin-fixed tissues by polymerase chain reaction.

Chicken anaemia virus (CAV) DNA was detectable from various samples such as cell-free virus, infected MDCC-MSB1 cells, unfixed liver homogenates, formalin-fixed liver homogenate or formalin-fixed paraffin-embedded (FFPE) tissues from experimental or field infected chicks using PCR assay. The detection limit of the first PCR assay was 1 infected cell or 10(-1.5) TCID50 of cell-free virus (strain A2). The nested PCR assay increased the sensitivity 10- or 100-fold. CAV DNA was detectable in the other 14 Japanese strains isolated from 1976 to 1994 by the PCR assay. All the amplified products were digested with BglII, HindIII, PstI and SacI. These results suggest that the region amplified was highly conserved among the strains. The nested PCR assay was very sensitive. However, CAV DNA was detectable in most field samples using the first PCR assay. Therefore, the nested PCR assay may not always be necessary. In contrast, the nested PCR assay was necessary to detect CAV DNA in FFPE tissues or formalin-fixed material. Use of the PCR assay in CAV DNA detection from FFPE tissues may be most valuable in diagnosis of diseases caused by or associated with CAV, because it allows detection of both microscopic lesions and CAV DNA.

Animals↗

PubMed2006

Protein extraction from formalin-fixed, paraffin-embedded tissue sections: quality evaluation by mass spectrometry.

A satisfactory protocol of protein extraction has been established based on the heat-induced antigen retrieval (AR) technique widely applied in immunohistochemistry for archival formalin-fixed, paraffin-embedded (FFPE) tissue sections. Based on AR, an initial serial experiment to identify an optimal protocol of heat-induced protein extraction was carried out using FFPE mouse tissues. The optimal protocol for extraction of proteins was then performed on an archival FFPE tissue of human renal carcinoma. FFPE sections were boiled in a retrieval solution of Tris-HCl containing 2% SDS, followed by incubation. Fresh tissue taken from the same case of renal carcinoma was processed for extraction of proteins by a conventional method using radioimmunoprecipitation assay solution, to compare the efficiency of protein extraction from FFPE tissue sections with extraction from fresh tissue. As a control, further sections of the same FFPE sample were processed by the same procedure without heating treatment. Evaluation of the quality of protein extracted from FFPE tissue was done using gel electrophoresis and mass spectrometry, showing most identified proteins extracted from FFPE tissue sections were overlapped with those extracted from fresh tissue.

Animals↗

PubMed2004

Primary central nervous system posttransplant lymphoproliferative disorders.

Posttransplant lymphoproliferative disorders (PTLDs) represent a spectrum ranging from Epstein-Barr virus (EBV)-driven polyclonal lymphoid proliferations to EBV+ or EBV- malignant lymphomas. Central nervous system (CNS) PTLDs have not been characterized fully. We reviewed the clinical, radiologic, and pathologic features of 12 primary CNS PTLDs to define them more precisely. Patients included 10 males and 2 females (median age, 43.4 years) who were recipients of kidney (n = 5), liver (n = 2), heart (n = 2), peripheral blood stem cells (n = 2), or bone marrow (n = 1). All received immunosuppressive therapy. CNS symptoms developed 3 to 131 months (mean, 31 months) after transplantation. By neuroimaging, most showed multiple (3 to 9) intra-axial, contrast-enhancing lesions. Histologic sections showed marked expansion of perivascular spaces by large, cytologically malignant lymphoid cells that were CD45+, CD20+, EBV+ and showed light chain restriction or immunoglobulin gene rearrangement. In distinction to PTLDs in other organ systems, CNS PTLDs were uniformly high-grade lymphomas that fulfilled the World Health Organization criteria for monomorphic PTLDs. Extremely short survival periods were noted for each CNS PTLD that followed peripheral blood stem cell transplantation. Survival of others with CNS PTLD varied; some lived more than 2 years.

Adult↗

PubMed1999

Multiplex polymerase chain reaction for the detection of mycobacterial DNA in cases of tuberculosis and sarcoidosis.

The aims of the present study were: (1) to design a sensitive and specific polymerase chain reaction-based method that would allow detection of most common human typical and atypical mycobacterial strains and (2) to apply the method on formalin-fixed paraffin-embedded (FFPE) tissue and sputum samples from patients with clinicopathological evidence of tuberculosis and sarcoidosis. Three sets of primers were selected. The first detects specifically members of the Mycobacterium tuberculosis (M. tuberculosis) complex, amplifying a 243 bp fragment of the gene encoding the immunogenic protein MPB 64, whereas the second traces members of the Mycobacterium avium (M. avium) complex producing a 91 bp fragment of the IS1110 element. The third pair of primers is specific for slow-growing mycobacteria, amplifying a 383 bp region of the 65 kDa mycobacterial antigen gene. Our multiplex polymerase chain reaction assay identified mycobacterial DNA of 10(-3) colony-forming units (CFU)/mL from sputum samples, 10(-5) CFU/mL from FFPE tissue samples and 10(-6) CFU/mL from pure broth cultures. By performing the method on 75 FFPE tissue samples with histological and clinical evidence of tuberculosis and 300 sputum specimens from patients suspected of tuberculosis, we found 38 M. tuberculosis complex, 7 M. avium complex, and 14 slow-growing mycobacteria positive samples in the first case and in the second we found 95 M. tuberculosis complex, 21 M. avium complex, and 35 slow-growing mycobacteria positive samples. The sensitivity of the assay was significantly higher than that of Ziehl-Neelsen and in some cases higher than culture, especially when applied on atypical mycobacteria. In addition, 25 cases histologically and clinically characterized as sarcoidosis were investigated for mycobacterial DNA sequences and in nine of these, DNA corresponding to M. tuberculosis complex was detected. The method described can be applied directly on FFPE and sputum samples and allows not only the detection of mycobacterial DNA, but also an assessment concerning the species involved.

DNA, Bacterial↗

PubMed2006

[Clinicopathologic and genetic studies of desmoid-type fibromatosis].

OBJECTIVE: To study the clinicopathological and genetic features of desmoid-type fibromatosis, and to investigate the feasibility of detecting trisomy 8 in formalin fixed, paraffin embedded (FFPE) tissue by fluorescence in-situ hybridization (FISH). METHODS: A total of 96 cases were included in this study. All patients had clinical information. Histopathologic and immunohistochemical evaluations were available in 69 cases, and ultrastructural evaluation was done in 2 cases of desmoid-type fibromatosis. FFPE tissue sections were available in 20 tumors for the trisomy 8 detection by FISH. RESULTS: There were 20 male and 76 female patients with ages ranging from 8 to 86 years (mean 35.3 years). Clinically, there were 44 extra-abdominal tumors, 28 abdominal wall tumors and 23 intra-abdominal lesions mostly involving the mesentery. Most cases presented with nodular or funicular masses with white firm cut surfaces, measuring 0.6 to 24.0 cm (mean 8.4 cm) in size. Histologically, desmoid-type fibromatoses showed longitudinal fascicles of spindle fibroblasts and myofibroblasts in a predominantly collagenous background. The tumor cells stained positive for vimentin, alpha-smooth muscle actin, desmin, and beta-catenin (47.8%, 33/69). Ultrastructurally, most tumor cells had features of fibroblasts, including rich endoplasmic reticulum and Golgi apparatus. Some tumor cells were myofibroblast-like cells exhibiting intercellular junctions, fibronexous junctions and stress fibers. Trisomy 8 was detected in 6 of 20 cases of desmoid-type fibromatosis including 5 of the 8 recurrent tumors but only one of the 12 primary tumors. The latter tumor also recurred three years later. CONCLUSIONS: Desmoid-type fibromatosis is an intermediate (locally aggressive) tumor that occurs predominantly in young females. The lesion consists of fibroblasts and myofibroblasts with the latter showing characteristic features including stress fibers and fibronexous junctions. Trisomy 8 can be detected in FFPE tissue by FISH, and its presence serves as a useful predictor of tumor recurrence and may define a subtype of desmoid-type fibromatosis with high recurrence rate.

Actins↗

PubMed2006

Isolation and comparative characterization of Ki-67 equivalent antibodies from the HuCAL phage display library.

It has been shown that a repetitive motif with the sequence FKEL(F) within the Ki-67 antigen (pKi-67) serves as an epitope for the Ki-67 antibody and equivalent clones. However, no direct correlation between reactivity towards Ki-67 epitopes and reactivity in formalin-fixed paraffin-embedded (FFPE) tissue could be found. In this study our aim was the isolation and characterization of new monoclonal Ki-67 equivalent antibodies in an in vitro approach. To select pKi-67 reactive phage antibodies, we used a large naive Fab-phage library (Human Combinatorial Antibody Library; HuCAL). We implemented a panning strategy against two different overlapping peptides, both containing the 'FKELF' epitope. ELISA screening of randomly picked phage antibody clones after the third selection round yielded six highly reactive clones against the 'FKELF' epitope, of which five were found to be reactive in FFPE tissue, showing a Ki-67 equivalent staining pattern. Substitutional epitope analysis on peptide arrays of the new recombinant pKi-67 binders and of the established murine clones Ki-67, Mib-1 and Mib-5 were carried out to compare their fine specificities. The results suggest that the lysine residue in the epitope is critical for recognition of Ki-67 antigen in FFPE tissue.

Amino Acid Sequence↗

PubMed2001

The suitability of DNA extracted from formalin-fixed, paraffin-embedded tissues for double differential polymerase chain reaction analysis.

Formalin-fixed, paraffin-embedded (FFPE) tissues are one of the popular sources of diagnostic materials, the easiest to store and transport. They are often used as the source of nucleic acids for retrospective molecular analyses based on DNA amplification by polymerase chain reaction (PCR). However, it is known that nucleic acids from paraffin-embedded tissues are much worse templates than those recovered from fresh tissues. It is exceptionally important in a quantitative analysis, including double differential PCR (ddPCR). Therefore, a pilot study comparing quantity and quality of DNA extracted with various paraffin removal and DNA isolation procedures from FFPE tissues was conducted. Furthermore, the suitability of DNA isolated with optimized procedure for the assessment of erbB-2 average gene copy number (AGCN) was checked. Specimens for comparison of extraction and isolation procedures were generated from the same human normal thyroid tissue embedded in paraffin to eliminate variabilities in tissue processing and sample size. Three procedures of paraffin removal and three procedures of DNA extraction from deparaffinized tissue were compared. Only one procedure provided DNA, which was efficiently amplified in ddPCR. The material obtained with this optimized procedure was used to check the precision of ddPCR by evaluation of AGCN of erbB-2 oncogene. Low variability of obtained results close to expected AGCN value (AGCN=1) indicates high reproducibility of the method, as well as its high accuracy, if the normal value of erbB-2 AGCN in the examined tissue is assumed.

DNA↗

PubMed2006

Limited tissue fixation times and whole genomic amplification do not impact array CGH profiles.

BACKGROUND: Array comparative genomic hybridisation (CGH) is a powerful method for the genetic analysis of lesional and normal tissues to identify genomic imbalances associated with malignancies. However, the use of this technique with DNA extracted from archival formalin fixed, paraffin embedded (FFPE) tissue specimens, the most widely available resource for retrospective studies, is subject to quantitative and qualitative limitations. In this report, the suitability and integrity of the DNA extracted from FFPE MCF7 breast cancer cells fixed for different periods of time for array CGH applications were examined. RESULTS: Using our established cDNA microarray protocol in conjunction with whole genome amplification methods, the genetic profiles of freshly harvested MCF7 cells and their matched FFPE counterparts were analysed. Congruent profiles between FFPE MCF7 cells and their fresh counterpart and between amplified and non-amplified FFPE MCF7 cells were observed. Our results demonstrate that formalin fixation of <20 hours has no significant adverse effect on the integrity of DNA for array CGH studies. CONCLUSIONS: Our findings attest to the fidelity of our array CGH methods to effectively examine material recovered from FFPE tissue specimens for microarray applications. This in turn has great potential to identify novel diagnostic and prognostic markers for human disease.

Breast Neoplasms↗

PubMed1996

Method to make paraffin-embedded breast and lymph tissue mimic fresh tissue in DNA analysis.

Determination of DNA ploidy has been found to be of diagnostic and prognostic value with regard to many solid tumors. Flow cytometric analysis of DNA ploidy is dependent on the binding of fluorescent dyes to DNA. Preserving cell morphology by fixing the tissue in formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA. This distortion of DNA content measurement can cause inaccuracies in DNA-ploidy determinations of formalin-fixed tissue specimens and precludes the use of appropriate DNA standards. Therefore, it has been impossible to determine accurately the DNA ploidy of formalin-fixed, paraffin-embedded (FFPE) tissues. Using formalin-fixed cells as a model for FFPE cells, we developed a thermal treatment method to reverse the effect of formalin on the binding of propidium iodide to DNA. Applying this approach to the preparation of FFPE lymph node and breast tissue for DNA analysis, we have developed a method that makes the binding of PI to the DNA of FFPE tissue mimic that of fresh tissue. Following dewaxing, rehydration, and trypsin treatment, the FFPE tissue, resuspended in PBS, was heated to 75 degrees C for 90 min to restore the PI binding to that of fresh cells. This method makes it possible to use fresh, DNA-diploid cells as an internal control and, thus, determine more accurately the DNA ploidy of tumors preserved in formalin and paraffin.

Aneuploidy↗

PubMed2004

The HOPE-technique permits Northern blot and microarray analyses in paraffin-embedded tissues.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

Blotting, Northern↗

PubMed1993

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

Base Sequence↗

PubMed2006

Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy.

BACKGROUND: Traditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets. METHODS: We evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE) tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp. RESULTS: The DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens. CONCLUSION: The DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available.

Journal Article↗

PubMed2002

Rapid and accurate detection of monoclonal immunoglobulin heavy chain gene rearrangement by DNA melting curve analysis in the LightCycler System.

The detection of immunoglobulin heavy chain gene rearrangement (IgH-R) is a standard tool for distinguishing polyclonal from monoclonal B-cell populations. Current DNA-based polymerase chain reactions (PCR) strategies can diagnose monoclonal IgH-R either by measuring the length of the amplicon or by detecting gel mobility variations owing to sequence-dependent conformational changes. However, amplification and analysis remain sequential operations usually requiring manual transfer. We have developed a novel PCR strategy for detecting monoclonal IgH-R that monitors fluorescence of the specific double-stranded DNA binding dye SYBR Green I during melting curve analysis using the LightCycler System. We compared polyacrylamide gel electrophoresis (PAGE) versus melting curve analysis in 130 clinical DNA samples from formalin-fixed, paraffin-embedded (FFPE) tissues (mostly skin biopsies) of 128 patients. The identical FR3 primers were used to amplify the IgH variable region for both analytic techniques. We detected IgH-R in 24 DNA samples from FFPE tissue of 22 patients. Melting curve analysis, compared to PAGE, revealed no false negative and no false positive results, yielding both sensitivity and specificity equal to 100%. We also compared Southern blot analysis versus melting curve analysis in 23 clinical DNA samples from fresh-frozen lymph nodes of 23 patients. We detected IgH-R by melting curve analysis in 7 DNA samples from fresh-frozen lymph nodes. Melting curve analysis, compared to Southern blot analysis, revealed sensitivity equal to 58.3% (7 of 12) and specificity equal to 100% (11 of 11). We conclude that continuous fluorescence monitoring of PCR products with DNA melting curve analysis can rapidly and reproducibly distinguish polyclonal from monoclonal B-cell populations.

Adult↗